research and perspectives in neurosciences

Fondation Ipsen

Editor
Yves Christen, Fondation Ipsen, Paris (France)

Editorial Board
Albert Aguayo, McGill University, Montreal (Canada)
Philippe Ascher, Ecole Normale Supérieure, Paris (France)
Alain Berthoz, Collège de France, CNRS UPR 2, Paris (France)
Jean-Marie Besson, INSERM U 161, Paris (France)
Emilio Bizzi, Massachusetts Institute of Technology, Boston (USA)
Anders Bjorklund, University of Lund (Sweden)
Floyd Bloom, Scripps Clinic and Research Foundation, La Jolla (USA)
Joël Bockaert,Centre CNRS-INSERM de Pharmacologie Endocrinologie,
Montpellier (France)
Pierre Buser, Institut des Neurosciences, Paris (France)
Jean-Pierre Changeux, Collège de France, Institut Pasteur, Paris (France)
Carl Cotman, University of California, Irvine (USA)
Steven Dunnett, University of Cambridge, Cambridge (UK)
George Fink, Medical Research Council, Edingburgh (UK)
Fred Gage, Salk Institute, La Jolla (USA)
Jacques Glowinski, Collège de France, Paris (France)
Claude Kordon, INSERM U 159, Paris (France)
Michel Lacour, CNRS URA 372, Marseille (France)
Michel Le Moal, INSERM U 259, Bordeaux (France)
Gary Lynch, University of California, Irvine (USA)
Brenda Milner, McGill University, Montreal (Canada)
John Olney, Washington University Medical School, Saint Louis (USA)
Alain Privat, INSERM U 336, Montpellier (France)
Allen Roses, Duke University Medical Center, Durham (USA)
Constantino Sotelo, INSERM U 106, Paris (France)
Jean-Didier Vincent, Institut Alfred Fessard, CNRS, Gif-sur-Yvette (France)
Bruno Will, Centre de Neurochimie du CNRS/INSERM U 44,
Strasbourg (France)
Fred Gage Yves Christen (Eds.)

Retrotransposition,
Diversity and
the Brain
With 31 Figures, 21 in color

123
Gage, Fred H., Ph.D.
Laboratory of Genetics
The Salk Institute for Biological Studies
10010 North Torrey Pines Road
La Jolla, CA 92037
USA
e-mail: gage@salk.edu

Christen, Yves, Ph.D.

Fondation IPSEN
Pour la Recherche Thérapeutique
24, rue Erlanger
75781 Paris Cedex 16
France
e-mail: yves.christen@ipsen.com

ISSN 1861-2253
ISBN 978-3-540-74965-3 Springer Berlin Heidelberg New York

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Preface

The human brain is remarkably complex, permitting sophisticated behavioural reper-

toires, such as languages, tool use, self-awareness, symbolic thought, cultural learning
and consciousness. Each human being is different, due in part to the uniqueness of
the neuronal heterogeneity and interconnections in our brains. Brain complexity and
neuronal diversity are strongly related. The diversity of single neurons provides the
underpinnings for how neuronal circuits operate. How and when neuronal diversity is
generated, both in embryonic and adult neurogenesis, remain unknown.
In the immune system, the highly diverse array of antigen receptors can be at-
tributed to the stochastic nature of the recombination process in somatic precursor
cells, causing permanent changes in DNA and gene expression. This diverse population
is then the target of selective processes that favor the correct antigen-receptor match
and eliminate those with inadequate specificities, accounting for the rapid kinetics and
immense diversity observed in vivo. Evidence for a possible similarity between the
nervous and immune systems came from studies with mice deficient in DNA double
strand break (DSB) repair. Lessons learned from the discovery of the mechanism for
diversity in the immune system may be useful to the investigation of the mechanism
of diversity in neurons.
Retroelements are ancient mobile DNA found in most organisms. Long dismissed
as useless, selfish or “junk” DNA, they were thought to be mere intracellular parasites
from our distant evolutionary past. Together with their mutant relatives, L1 sequences
constitute almost 50% of the mammalian genome. L1s can retrotranspose in a defined
window of the neuronal differentiation, changing the genetic information in single
neurons in a “random” fashion, allowing the brain to develop in distinct different ways.
Such strategy contributes to expand the number of functionally distinct neurons that
could be produced from a given stem cell gene pool. This characteristic of variety
and flexibility may contribute to the uniqueness of an individual brain, even between
genetically identical twins. These mobile elements may be part of conserved core pro-
cess responsible for evoking facilitated complex non-random phenotypical variation
on which selection may act. A detailed understanding of the basic mechanisms of L1
activity may shed light on one possible mechanism for generating neural diversity.
This Fondation IPSEN Colloque Médecine et Recherche was devoted to the inter-
face between the complexity of brain organization and function, the mechanisms for
generating diversity and genetic mobility. The goal was to expand the current limits of
research in neurobiology not only to the benefit of those interested in the cellular and
molecular processes but also for the understanding of high-level cognitive functions
and the understanding of complex mental diseases.

Fred Gage
Yves Christen
Table of Contents

Telomeres and Telomerase in Human Health and Disease

J. Lin, E.S. Epel, E.H. Blackburn . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

Molecular and Circuit Mechanisms for Hippocampal Learning

S. Tonegawa, T.J. McHugh . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13

Retrotransposons – Natural and Synthetic

J.D. Boeke, W. An, L. Dai, E.S. Davis, J.S. Han, K.A. O’Donnell, L.Z. Scheifele,
S.J. Wheelan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21

Ancient Retrotransposons as Possible Remnants of the Primitive RNP World

R. Ivanyi-Nagy, J.-L. Darlix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33

Human Diversity and L1 Retrotransposon Biology: Creation of New Genes

and Individual Variation in Retrotransposition Potential
H.H. Kazazian, Jr., M.d.C. Seleme, D.V. Babushok, D.M. Ostertag, M.R. Vetter,
P.K. Mandal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43

From the “RNA World” to Brain Complexity: Generation of Diversity

A.R. Muotri, M.C.N. Marchetto, F.H. Gage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53

Endogenous Retroviruses and Human Neuropsychiatric Disorders

R.H. Yolken, H. Karlsson, I. Bossis, L. Asp, F. Dickerson, C. Nellåker, M. Elashoff,
E. Rubalcaba, R.P. Viscidi . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65

Is Psychosis Due to Retroviral/Retrotransposon Integration Close

to the Cerebral Dominance Gene?
T.J. Crow, J.S. Close, H.-S. Kim, M.T. Ross . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87

Microcephalies and DNA Repair

E.C. Gilmore, C.A. Walsh . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109

Subject Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121

List of Contributors

Asp, Linnéa
Department of Neuroscience, Karolinska Institutet, Retzius v 8, 17177 Stockholm,
Sweden

Babushok, D.V.
Department of Genetics, University of Pennsylvania School of Medicine, Philadelphia,
PA 19104, USA

Blackburn, Elizabeth H.
Biochemistry and Biophysics, University of California, San Francisco, CA 94158, USA

Boeke, Jef D.
High Throughput Biology Center, Johns Hopkins University School of Medicine, Bal-
timore, MD 21205, USA

Bossis, Ioannis
Stanley Laboratory of Developmental Neurovirology, Johns Hopkins University School
of Medicine, Baltimore, MD, USA

Close, J.S.
Sane Powic, Warneford Hospital, Oxford, OX3 7JX, USA

Crow, Timothy J.
Sane Powic, Warneford Hospital, Oxford, OX3 7JX, UK

Dai, Lixin
High Throughput Biology Center, Johns Hopkins University School of Medicine, Bal-
timore, MD 21205, USA

Darlix, Jean-Luc
LaboRetro, Unité de Virologie humaine INSERM, IFR128, ENS Lyon 46 allée d’Italie,
69364 Lyon, France

Davis, Edward S.
High Throughput Biology Center, Johns Hopkins University School of Medicine, Bal-
timore, MD 21205, USA
X List of Contributors

Dickerson, Faith
Sheppard Pratt Health System, Baltimore, MD, USA

Elashoff, Michael
Stanley Medical Research Institute, Chevy Chase, MD, USA

Epel, Elissa S.
Department of Psychiatry, UCSF Health Psychology Program, San Francisco, CA 94143,
USA

Gage, Fred H.
Laboratory of Genetics, The Salk Institute for Biological Studies, 10010 North Torrey
Pines Road, La Jolla, CA 92037, USA

Gilmore, Edward C.
Department of Neurology, Beth Israel Deaconess Medical Center, Child Neurology,
Massachusetts General Hospital, MA, USA

Han, Jeffrey S.
High Throughput Biology Center, Johns Hopkins University School of Medicine, Bal-
timore, MD 21205, USA

Ivanyi-Nagy, Roland
LaboRetro, Unité de Virologie humaine INSERM, IFR128, ENS Lyon 46 allée d’Italie,
69364 Lyon, France

Karlsson, Håkan
Department of Neuroscience, Karolinska Institutet, Retzius v 8, 17177 Stockholm,
Sweden

Kazazian, H.H., Jr.

Department of Genetics, University of Pennsylvania School of Medicine, Philadelphia,
PA 19104, USA

Kim, Heui-Soo
Section of Biological Systems, College of Natural Science, Pusan National, University,
San 30, Changjeon Dong, Pusan 609-735, South Korea

Lin, Jue
Biochemistry and Biophysics, University of California, San Francisco, CA 94158-2517,
USA

Mandal, P.K.
Department of Genetics, University of Pennsylvania School of Medicine, Philadelphia,
PA 19104, USA
 List of Contributors XI

Marchetto, Maria C.N.

Laboratory of Genetics, The Salk Institute for Biological Studies, 10010 North Torrey
Pines Road, La Jolla, CA 92037, USA

McHugh, Thomas J.
The Picower Institute for Learning & Memory, RIKEN-MIT Neuroscience Research
Center, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge
MA 02139-4307, USA

Muotri, Alysson R.
Laboratory of Genetics, The Salk Institute for Biological Studies, 10010 North Torrey
Pines Road, La Jolla, CA 92037, USA

Nellåker, Christoffer
Department of Neuroscience, Karolinska Institutet, Retzius v 8, 17177 Stockholm,
Sweden

O’Donnell, Kathryn A.
High Throughput Biology Center, Johns Hopkins University School of Medicine, Bal-
timore, MD 21205, USA

Ostertag, D.M.
Department of Genetics, University of Pennsylvania School of Medicine, Philadelphia,
PA 19104, USA

Ross, M.T.
X Chromosome Group, Wellcome Trust Sanger Institute, Wellcome Trust Genome
Campus, Hinxton, Cambridge CB10 1SA, USA

Rubalcaba, Elizabeth
Stanley Laboratory of Developmental Neurovirology, Johns Hopkins University School
of Medicine, Baltimore, MD, USA

Scheifele, Lisa Z.
High Throughput Biology Center, Johns Hopkins University School of Medicine, Bal-
timore, MD 21205, USA

Seleme, M.d.C.
Department of Genetics, University of Pennsylvania School of Medicine, Philadelphia,
PA 19104, USA

Tonegawa, Susumu
The Picower Institute for Learning & Memory, RIKEN-MIT Neuroscience Research
Center, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge
MA 02139-4307, USA
XII List of Contributors

Vetter, M.R.
Department of Genetics, University of Pennsylvania School of Medicine, Philadelphia,
PA 19104, USA

Viscidi, Raphael P.
Stanley Laboratory of Developmental Neurovirology, Johns Hopkins University School
of Medicine, Baltimore, MD, USA

Walsh, Christopher A.
Department of Neurology, Beth Israel Deaconess Medical Center, Division of Genetics,
Children’s Hospital Boston, Howard Hughes Medical Institute, Boston, MA, USA

Wenfeng, An
High Throughput Biology Center, Johns Hopkins University School of Medicine, Bal-
timore, MD 21205, USA

Wheelan, Sarah J.
High Throughput Biology Center, Johns Hopkins University School of Medicine, Bal-
timore, MD 21205, USA

Yolken, Robert H.
Stanley Laboratory of Developmental Neurovirology, Johns Hopkins University School
of Medicine, Baltimore, MD, USA
Telomeres and Telomerase in Human Health and Disease

Jue Lin1 , Elissa S. Epel2 , and Elizabeth H. Blackburn1

Introduction

Telomeres cap chromosome ends and help protect the genome. Telomere maintenance
consists of an integrated cellular system for telomere homeostasis that includes telom-
erase, which replenishes telomeric DNA lost from chromosomal termini. Telomerase,
with its highly specialized reverse transcriptase action, is therefore essential for ge-
nomic stability and long-term cell division. The activity of telomerase in human cells is
kept under a complex set of controls that include developmental, cell type-specific and
environmental modulators. We have reported that chronic psychological stress in peo-
ple leads to lower telomerase and shorter telomeres. From these and other studies, the
emerging overall pattern is that telomerase insufficiency is associated with conditions,
syndromes and diseases that can shorten human life.

Telomeres

Telomeres are DNA-protein complexes at the ends of eukaryotic chromosomes that

are essential for genomic stability. The telomeric complexes prevent the ends of lin-
ear chromosomes from being recognized as broken ends, which would otherwise
elicit inappropriate DNA damage responses with potentially deleterious consequences
(Blackburn 2001). Telomeric DNA sequences are lost after each cell division due to
incomplete replication by conventional DNA polymerases. Such progressive loss of
telomeric sequences – due to incomplete replication of DNA, and potentially also
from nuclease action on telomeric termini – leads to replicative senescence of dividing
cells.

Telomerase: a Specialized Cellular Reverse Transcriptase Essential

for Continued Cell Renewal

This end-replication problem is solved by the cellular enzyme telomerase. Telomerase,

a specialized ribonucleoprotein reverse transcriptase, synthesizes telomeric DNA, thus

1 Biochemistry and Biophysics, University of California, San Francisco, CA 94158-2517, USA

e-mail: elizabeth.blackburn@ucsf.edu
2 Department of Psychiatry, UCSF Health Psychology Program, San Francisco, CA 94143, USA

Gage et al.
Retrotransposition, Diversity and the Brain
© Springer-Verlag Berlin Heidelberg 2008
2 J. Lin, E.S. Epel, E.H. Blackburn

Fig. 1. A simplified diagram depicting human telomerase acting to elongate a chromosomal DNA
end. Deoxyribonucleoside triphosphate substrates (with base identities indicated in purple) are
added, templated by the RNA bases shown in blue. The core protein component hTERT is shown
in red and the essential telomerase RNA component hTER in blue

counteracting the losses of telomere sequence (Greider and Blackburn 1985). For this
purpose, telomerase uses its integral RNA molecule as the template to synthesize
telomeric sequence DNA (Greider and Blackburn 1987; 1989). The core telomerase
complex contains two subunits that are essential for its catalytic function: the protein
catalytic subunit (hTERT in humans; Nakamura et al. 1997) and the RNA component
(hTER, hTR or hTERC in humans; Greider and Blackburn 1989; Feng et al. 1995; Counter
et al. 1997; Lingner et al. 1997; Fig. 1). Like the reverse transcriptase (RT) of the human
retroelement LINE-1 (Piskareva and Schmatchenko 2006), telomerase lacks an RNase
H activity. Also like the human LINE element reverse transcriptase (Kulpa and Moran
2006), telomerase copies the RNA that is within the same telomerase RNP complex as the
protein RT subunit, not an RNA template added in trans. As discussed below, in humans,
telomerase activity is regulated during development and by different environmental
and physiological factors (Aisner et al. 2002; Cong et al. 2002; Forsyth et al. 2002).

The Evolution of Telomerase

Telomerase is found throughout eukaryotes ranging from those in deep branches in the
eukaryotic phylogeny (for example, Giardia and Trypanosoma) to protozoans, fungi,
plants, and the invertebrate and vertebrate metazoans. Hence telomerase is likely to
have been present early in the eukaryotic lineage or even at the very onset of that lineage.
But where did telomerase come from? How is it related to other reverse transcriptases,
such as those of retroelements (Fig. 2A)? Telomerase is conserved in multiple ways:
in being an RNP enzyme, in having domains of TERT conserved in addition to its
RT domain, and in having a conserved core structure of the telomerase RNA moiety
(Fig. 2B). Two different types of models have been considered for the evolution of
telomerase. These models, described next, are not mutually exclusive.

The “Catalytic RNA to Telomerase RNP” Model

The first model, which will be called the “RNA to telomerase RNP” model, was put forth
based on studies of the telomerase RNA component, the first of the core components to
 Telomeres and Telomerase in Human Health and Disease 3

Fig. 2. A. A typical retroelement reverse transcriptase (oval) and its long template RNA (black
line). B. The conserved core structure of telomerase RNA; note that only a limited region of the
RNA is used as a template for DNA synthesis. Top: TER of yeasts and ciliates; bottom, TER of
vertebrates (Lin et al. 2004)

be identified and functionally and structurally analyzed (Blackburn 1993, 1999). This
model was inspired by the discovery that RNA can act as a biological catalyst, as it does
in the case of self-splicing introns, RNAse P and the ribosome, and possibly in general
pre-mRNA splicing. Catalytic RNAs have been constructed with templated nucleic acid
polymerization properties. Hence, one model is that telomerase might have evolved
from an ancestral catalytic RNA that acquired, at some point in its evolution, a protein
component that took over RT catalytic function from the RNA.
The RNA to telomerase RNP model was proposed because, although it was clear that
protein is an essential part of telomerase and contains catalytic site amino acid residues,
certain small mutations of telomerase RNA residues often caused drastic effects on
telomerase function in vivo and in vitro. Striking examples have been observed in
multiple eukaryotes’ telomerases over several years of research. They include small
base substitutions that led to quantitatively large and RNA mutant-specific effects
on the rates of dNTP misincorporation, template slippage and mis-alignment on the
template (reviewed in Blackburn 1999; Lin et al. 2004). In some cases, even single base
substitutions led to deleterious and large effects on the enzyme reaction. In addition,
the telomeric DNA bases, even those that do not base-pair with the template, interact
with the TERT moiety of telomerase, and such interaction can have a large effect on the
catalytic reaction rate itself. This effect occurs at a step other than the DNA or dNTP
substrate binding steps, or the product release step of the polymerization reaction (Lee
and Blackburn 1993; Lee et al. 1993). Together these results point to a close involvement,
4 J. Lin, E.S. Epel, E.H. Blackburn

at a minimum, of the RNA in the ability of the telomerase RNP to carry out its reaction.
This functionality of telomerase RNA is in addition to its clear function as a template. In
summary, such results suggest that interactions involving the telomerase RNA within
the telomerase RNP can greatly influence the course of its polymerization and intrinsic
hydrolysis reactions.

The “Retroelement RT to Telomerase” Model

A second model for the evolution of telomerase is based on a phylogenetic comparison

of the TERTs in relation to eukaryotic evolution. Sequence alignments of TERTs with
Group II intron, retroelement and retroviral RTs indicate that the TERT RT domain is
most closely related to the RTs of Group II introns (Nakamura et al. 1997; Malik et al.
2000)
These alignments of amino acid sequences point to the model that the telomerase
TERT moiety evolved from a Group II RT (Malik et al. 1997). A recent version of this
model (Koonin 2006) posits that, at the time of the Archea-Eukarya split in evolution,
Group II introns spread into eukaryotes (with this invasion of Group II introns itself
possibly promoting that split), and that telomerase evolved when the ensuing chromo-
some fragmentation selected for a Group II RT-derived enzyme that could add DNA to
chromosome ends. Thus, in this model, an ancestral Group II RT protein evolved into
telomerase.
To complete any account of the inferred origin of telomerase, we must consider
how it acquired, for its now specialized DNA addition role, the built-in template that
is included within the larger telomerase RNA molecule. As described above, in the
“catalytic RNA to telomerase RNP” model, an ancestral, originally catalytically com-
petent telomerase RNA acquired the RT that then diverged into Group II RTs and into
the TERT moiety of telomerase. In the alternative “retroelement RT to telomerase”
model, this ancestral Group II intron RT, which had a conserved active site containing
metal-coordinating aspartates mediating catalysis, acquired an RNA. One conceivable
partial reconciliation of the two classes of models is that the telomerase RNA that
was acquired by a common RT protein ancestor of Group II introns and TERT was
derived from a ribozyme that had properties particularly suited to the role of adding
tandem short repeats specifically to telomeres. Such properties could include its tight
and specific binding to the TERT so that there was no dissociation of the template or its
DNA product from the RT after each round of copying the short template. Consistent
with this idea, much of the conserved core structure of telomerase RNA is involved in
its specific binding to the TERT protein in a way that promotes optimal and specific
template usage. This model leaves open the question of whether a direct ancestor of
telomerase RNA ever had any nucleic acid polymerization catalytic capability.

Control of telomerase in human cells

Although the controls of telomerase activity are many-faceted and complex, some gen-
eralizations may be made. Telomerase activity is high in mammalian embryonic stages
 Telomeres and Telomerase in Human Health and Disease 5

but is decreased later in life (Wright et al. 1996). Indeed, the majority – although not
all – of human somatic cell types have undetectable or very low telomerase activity.
However, the importance of telomerase, albeit at low levels, is becoming increasingly
evident in multiple human cell types, such as resting white blood cells and fibrob-
lasts. As a result of critically short telomeres resulting from the long-term insufficiency
of telomerase for telomere maintenance, such cells can enter replication senescence
(Harley et al. 1990). Telomere shortening was thus thought of as an unopposed mitotic
clock that counts the number of divisions a cell is able to go through before senescence
(Harley et al. 1990). However, although low, telomerase activity is expressed in a highly
regulated manner in some somatic cells. For example, during lymphocyte development,
differentiation and activation, telomerase activity is high in early stages of T and B cell
development, but the activity is decreased at later stages and in resting cells, although
it can be measured with suitable quantitative methods (Epel et al. 2004, 2006). These
findings imply that, in any cells with even low telomerase, the rate of telomere shorten-
ing can be modulated by, among other factors, the telomerase activity that counteracts
such shortening. With respect to the brain, formerly thought to have no telomerase and
to be essentially comprised of postmitotic cells, we have found low telomerase activity
in rodent hippocampus that includes stem and neural progenitor cells and have also
detected low telomerase activity in primary (that is, non-transformed) human neurons
in culture (J.L. and E.H.B, unpublished observations). Dividing brain stem cells have
been recently reported in human adults and presumably these cells will also contain
telomerase activity.
Multiple studies on aspects of telomerase control in cultured human cells have
been done (see, as representative examples, Endoh et al. 2005; Ritz et al. 2005). The
levels of telomerase core components TERT and TER, and of enzymatic activity, are
controlled by transcriptional control. Cis-acting elements in the TERT and TER pro-
moters include both positive and negative controlling elements. In addition, various
post-transcriptional control mechanisms resulting in regulation of telomerase activity
have been described for certain mammalian cells. However, much research remains to
be done to understand fully the control of telomerase expression and its activity in any
tissue, let alone in the mammalian or human brain.

Other Roles of Telomerase Besides Making Telomeric DNA Longer

Evidence is building for a cellular response to telomerase status independent of its role
in polymerizing telomeric DNA. Experimental telomerase upregulation in the mouse
has been shown to confer proliferation properties on hair follicle stem cells but not
on their progeny cells. Such experimental over-expression of the telomerase protein
TERT, even in mice genetically deleted for the RNA component of telomerase (which
therefore lack any telomerase enzymatic activity), specifically causes these stem cells
to proliferate excessively (Sarin et al. 2005). This result showed that TERT can exert
effects in vivo independent of its role in telomeric DNA polymerization. In cancer
cells, which have high telomerase activity levels, partially knocking down telomerase
RNA – by RNAi or ribozyme administration – rapidly caused the cells to change
their properties, including gene expression profiles and morphology, even though they
6 J. Lin, E.S. Epel, E.H. Blackburn

continued to divide (Li et al. 2005; Bagheri et al. 2006). Yeast and mammalian cells
can maintain telomeres and quite successful cell growth rates even when telomerase
is genetically deleted, through recombination-based pathways that, in essence, patch
together telomeric tracts onto shortened telomeres through “borrowing’ from other
chromosomal telomeric tracts’ DNA ends (Lundblad and Blackburn 1993). However,
in yeast cells under such a telomerase-independent telomere maintenance regime,
a sustained genome-wide expression response resembling an environmental stress
response was observed, despite the fact that these cells seemed to be growing well
(Nautiyal et al. 2002). There is also evidence for telomerase components in cells that
are not dividing: TERT protein has been reported to be expressed in postmitotic
hippocampal neurons even though telomerase enzymatic activity was not detected
(Fu et al. 2000). Along with other hints (reviewed in Blackburn 2001, 2005), these
findings point to possible functions for telomerase beyond its crucial and better-
known function of maintaining telomere length in dividing cells. Thus the control of
telomerase expression and activity is of great interest for all cells, including the stem
cells of the brain and their dividing as well as postmitotic progeny.

Telomere Maintenance, Human Aging and Aging-Related Diseases

Research in the past two decades points to a link between organismal aging and
aging-related diseases and cellular senescence caused by telomere shortening. Several
lines of evidence strongly suggest that the resulting telomere dysfunction could have
a causal role in some aging and aging-related diseases. White blood cells [leukocytes,
or peripheral blood mononuclear cells (PBMCS)] are the most readily available source
of normal human cells in which to measure telomere length or telomerase activity
directly. Numerous clinical studies link short telomere length in white blood cells
with aging-related disease or preclinical conditions of diseases. A short list of these
conditions includes increased mortality from cardiovascular disease and infectious
disease (Cawthon et al. 2003), heart disease (Starr et al. 2006; Brouilette et al. 2007)
including coronary atherosclerosis (Samani et al. 2001), premature myocardial infarc-
tion and stroke (Brouilette et al. 2003; Fitzpatrick et al. 2007), vascular dementia (von
Zglinicki et al. 2000), hypertension with carotid atherosclerosis (Benetos et al. 2004),
age-related calcific aortic stenosis (Kurz et al. 2004), increased pulse pressure (Jeanclos
et al. 2000) and stress (Epel et al. 2004), obesity and smoking (Valdes et al. 2005),
osteoarthritis (Zhai et al. 2006), Alzheimer’s disease (Panossian et al. 2003; Zhang et al.
2003), and insulin resistance, a preclinical condition for diabetes (Gardner et al. 2005;
Adaikalakoteswari et al. 2007).
Finally, the strongest evidence suggesting a direct role of telomerase and telomere
maintenance in aging and aging-related diseases came from study of the form of a rare
human genetic disease, dyskeratosis congenita, caused by haploinsufficiency of telom-
erase activity due to mutations in hTER (Dokal and Vulliamy 2003). Dyskerotosis con-
genita patients with hTER or hTERT mutations have shorter telomeres and lower telom-
erase activity (Marrone et al. 2005). Patients die of eventual failure of the hematopoietic
system, supporting the idea that premature senescence of the hematopoietic cells is
one of the underlying causes of mortality (Marrone et al. 2005).
 Telomeres and Telomerase in Human Health and Disease 7

Chronic Psychological Stress, Telomerase, Aging

and Aging-Related Diseases

Interestingly, cardiovascular diseases, neurodegenerative disease and immune dys-

function are aging-related diseases and are also stress-related diseases. Numerous
epidemiological studies have shown that chronic stress leads to a poor health profile
and to increased rates of stress-related diseases, including diabetes, cardiovascular dis-
eases, mental illness and dampened immune functions (Raikkonen et al. 1996; Sapolsky
1996; Biondi and Zannino 1997; Kendler et al. 1999; Charney and Manji 2004; McEwen
2004; Rosengren et al. 2004; Yusuf et al. 2004; Glaser and Kiecolt-Glaser 2005; Lupien
et al. 2005; Das and O’Keefe 2006; Shors 2006). Since telomere length is affected by
telomerase activity, we tested whether telomerase activity in PBMCs might be affected
by quantifiable measures of chronic psychological stress (Epel et al. 2004). We dis-
covered that chronic stress is associated with at least two markers of cellular aging:
notably, shorter telomere length and lower telomerase. We have reported, for the care-
fully controlled cohort of apparently healthy women aged between 20 and 50, that the
number of years of chronic life stress, as well as perception of life stress, is related to
lower telomerase activity and excessive telomere shortness in white blood cells. In the
same cohort of women, shorter telomere length was also related to greater excretion of
stress hormones (epinephrine, norepinephrine and cortisol) and lower telomerase was

Fig. 3. A new connection between psychological stress, telomerase activity and human disease
8 J. Lin, E.S. Epel, E.H. Blackburn

related to more epinephrine excretion, over a 12-hour night time period (Epel et al.
2006). These findings suggested that stress arousal might be one of the mediators in
the relation between psychological stress and cellular aging (Epel et al. 2006). Previ-
ous animal studies have shown that telomerase can also play a role in cardiovascular
disease pathobiology, but the relationship had not been examined in humans until
now. We found that women who had lower telomerase activity also had higher levels
of risk for cardiovascular disease, as represented by a cluster of symptoms called the
Metabolic Syndrome. Specifically, low telomerase (but not, in this relatively young co-
hort of women, telomere length) was associated with greater abdominal adiposity and
higher blood pressure, cholesterol and blood sugars (Epel et al. 2006). These findings
suggested for the first time that low telomerase in white blood cells may serve as a proxy
of disease risk, possibly before telomere shortening occurs. We also found that women
with low white blood cell telomerase (below the mean) responded to a standardized
laboratory stressor with a decrease in vagal tone (heart rate variability). (Epel et al.
2006). This type of decrease is generally an indicator of less healthy cardiac function.
Such responses to laboratory stress tend to have some traitlike characteristics (i.e., sta-
bility over time). Thus, we infer that habitually responding to stressful situations with
this more malignant cardiovascular reactivity profile is linked to lower white blood cell
telomerase. This work uncovered provocative new links between psychological stress
arousal, impaired telomere maintenance and risk of heart disease.

The Implications of Reverse Transcription –

Telomerase and Retrotransposition – in an Individual Human Life
Telomerase has evolved into an indispensable enzyme for the continued division of
eukaryotic cells, and hence it plays an essential role in eukaryotic life cycles, including
in every human life span. This fact is strikingly and starkly illustrated by the haplo-
insufficiency for telomerase in humans described above: individuals with a mutation
of the telomerase RNA gene that renders that allele non-functional die (apparently
from exhaustion of stem cells or progenitor cells) before they can reach old age, even
though their other telomerase RNA gene allele encodes a functional copy of the gene.
Although McClintock proposed some decades ago that movement of mobile elements
might be harnessed for developmental purposes (Fedoroff and Botstein 1992), until
recently there has been little clear evidence for whether any transposons, including
retroelements, might play a required role within one organism’s lifetime. Hence, in
contrast to telomerase’s reverse transcription action in vivo, the reverse transcription
associated with movement of retroelements had been thought to play roles that would
manifest only over evolutionary time frames, including roles in the diversification of
genomes and gene families (Yohn et al. 2005). In other words, the essential nature of the
reverse transcriptase action of telomerase throughout life had been thought to distin-
guish it from the reverse transcription events mediated by other reverse transcriptases,
including those of retroelements.
The discovery by Gage and collaborators (Muotri et al. 2005; Muotri and Gage 2006)
that certain neuronal stem cell progeny (neural progenitor) cells undergo cell-type spe-
cific retroelement mobilization refocuses interest on the potential of retrotransposition
 Telomeres and Telomerase in Human Health and Disease 9

for playing roles in any one human life. In the brain of each individual organism, the
genomic alterations resulting from retroelement movements have the potential for
a range of slightly differing genome readouts, not only in these somatic neural cells
themselves but also in their cell division offspring. This discovery opens up the possi-
bility that, in an individual’s brain, function may be influenced by its unique history of
retroelement movement events.
While retroelements are activated specifically in specific brain cells in the mouse,
they do not apparently move actively in cells in general. Thus both telomerase and
retroelement transcription have in common the feature that they are kept under tight
downregulation control in mammalian cells. It will be of interest to see whether any
transcriptional or other expression controls are shared between the telomerase reverse
transcriptase and the reverse transcriptase of the retroelements mobilized in mouse
brain stem cells. Human and mouse telomerase RNA and TERT are each regulated,
at the transcriptional and post-transcriptional levels, by positive and negative control
pathways (although the transcriptional control varies somewhat between these two
species). Mammalian retroelement transcription is also controlled by a multiplicity of
cell- and developmental stage-specific factors (for example, see Yu et al. 2001; Yang et al.
2003; Lavie et al. 2004; Xu and Blackburn 2004; Muckenfuss et al. 2006). Inspection of
the known transcriptional control factors for human telomerase does not yet suggest
any elements in common with those for the retroelements. However, as the control of
each type of RT is complex and not fully worked out to date, there exists the possibility
of shared controls that could be relevant for brain stem cell progeny functions. Further
investigation needs to be done to follow up the provocative hint that a feature common
to both these reverse transcriptases may be activation in stem cells or their immediate
progeny.

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Molecular and Circuit Mechanisms
for Hippocampal Learning

Susumu Tonegawa1 and Thomas J. McHugh1

The hippocampus is crucial for the formation of memories of facts and episodes
(Scoville and Milner 1957; Jarrard 1993; Squire et al. 2004; Burgess et al. 2002). In
storing the contents of a specific episode, the hippocampus must rapidly form and
maintain representations of the temporal and spatial relationship of events and keep
these representations distinct, allowing similar episodes to be distinguished, a property
termed pattern separation. Furthermore, because specific episodes are rarely replicated
in full, the hippocampus must be capable of using partial cues to retrieve previously
stored patterns of representations, a phenomenon referred to as pattern completion.
Based primarily on the anatomy (Fig. 1) and physiology of the hippocampus and its
associated cortical structures, computational neuroscientists have suggested specific
hippocampal subregions and circuits that may subserve these mnemonic requirements.
These are the feedforward pathway from the entorhinal cortex (EC) to the dentate
gyrus (DG) and on to CA3 for pattern separation, and the recurrent and highly plastic
connections in CA3 for pattern completion (Marr 1971; McClelland and Goddard 1996;
McNaughton and Nadel 1990; O’Reilly and McClelland 1994).

CA3 NMDA Receptors for Pattern Completion

CA3 pyramidal cells receive excitatory inputs from three sources: the mossy fibers
of the DG granule cells (GC), the perforant path axons of the stellate cells in the
superficial layers of the EC, and the recurrent collaterals (RC) of the CA3 pyramidal
cells and, in return, provide output to CA1 pyramidal cells via Schaffer Collaterals
(SC). The prominence of these RCs has led to suggestions that CA3 might engage these
connections to serve as an associative memory network. Associative networks, in which
memories are stored through modification of synaptic strength within the network,
are capable of retrieving entire memory patterns from partial or degraded inputs
(pattern completion; Marr 1971; Gardner-Medwin 1976; Hopfield 1982; McNaughton
and Morris 1987; Rolls 1989; Hasselmo et al. 1995).
We set out to obtain evidence for this hypothesis by targeting the knockout of
the NR1 gene, coding for the essential subunit of NMDA receptors, to postnatal CA3
pyramidal cells. Use of the Cre-loxP recombination system, in which the expression

1 The Picower Institute for Learning & Memory, RIKEN-MIT Neuroscience Research Center,
Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge MA 02139-4307,
USA, e-mail: tonegawa@mit.edu

Gage et al.
Retrotransposition, Diversity and the Brain
© Springer-Verlag Berlin Heidelberg 2008
14 S. Tonegawa, T.J. McHugh

Fig. 1. Hippocampal excitatory pathway. PP = Perforant Path; MF = Mossy Fiber; RC = Recurrent

Collaterals; SC = Schaffer Collaterals; TA = Temporoammonic Path. The feedforward pathway
from entorhinal cortext to dentate gyrus and to CA3 is hypothesized to play a role in pattern
separation while the RC in CA3 is hypothesized to be important for pattern completion

of the transgenic Cre gene was driven by the transcription-regulating elements of the
KA-1 gene, permitted us to obtain such a cell type-specific knockout mouse (CA3-
NR1 KO; Nakazawa et al. 2002). In situ hybridization, immunohistochemistry, and
hippocampal slice electrophysiology data confirmed that the knockout is restricted to
postnatal CA3 pyramidal cells.
The CA3-NR1 KO mice, in contrast to CA1-NR1 KO mice (Tsien et al. 1996; McHugh
et al. 1996), were normal in the standard hidden platform version of the Morris water
maze task (Fig. 2). However, when the probe test was conducted under the conditions
where only one of the four major visual cues (partial cue condition) was available after
the training was performed with the four cues (full cue condition), the mutant mice
exhibited a deficit compared to the control littermates (Fig. 2). These data indicate that
the mutant mice are capable of acquiring this spatial memory and also retrieving it as
long as the full set of cues are provided during the recall phase. However, the mutants
are impaired in retrieving the memory using a partial set of cues (only one of the four
major visual cues), conditions that are sufficient for recall in the control mice. These
data suggest that the CA3-NR1 mice suffer from a specific impairment in a pattern
completion-mediated recall.
This phenotype of the mutants observed at the behavioral level was corroborated
at the level of neuronal ensemble activities in the CA1 area, which was shown by in vivo
recording of CA1 pyramidal cells with the tetrode recording technique. The CA3-NR1
KO mice exhibited compact place fields that were indistinguishable from those of the
 Molecular and Circuit Mechanisms for Hippocampal Learning 15

Fig. 2. The CA3-NR1 KO mice are defective in pattern completion. The mutants and control
littermates (floxed) went through 12 day-long training in the hidden platform version of the
Morris watermaze task under the full cue conditions (four visual cues surrounding the pool).
The probe trial conducted on the 13th day under the same full cue condition indicated the mutant
is normal in the acquisition and retrieval of the spatial memory under these conditions. However,
the memory retrieval by the mutants was substantially diminished compared to the control mice
in the probe trial conducted the next day (14th day) under the partial cue condition (only one of
the four cues was available during this probe trial)

control mice under the familiar full cue conditions (four major visual cues; Fig. 3).
When these mice were transferred to a home cage and after several hours returned to
the same recording box with the full set of cues, the mutant place cells were reactivated
as well as the control place cells. However, when the mice were returned to the recording
box with a partial set of cues (only one of the four major visual cues), the extent of the
reactivation of the place cells by the mutants was significantly diminished compared
to the control mice (Fig. 3).
Thus, both behavioral and in vivo physiological data strongly support the hy-
pothesis that the NMDA receptors in the CA3 pyramidal cells, and probably synaptic
plasticity at the CA3-CA3 recurrent synapses, play a crucial role in pattern completion
in the hippocampus.

DG NMDA Receptors for Rapid Pattern Separation

The key data that support the hypothesis that the feedforward EC → DG → CA3
pathway may be responsible in pattern separation are that 1) the number of DG GCs is
substantially greater than the numbers of EC layer II stellate cells and CA3 pyramidal
cells, 2) the connection between DG and CA3 is two orders of magnitude more sparse
than the connections between other regions, including EC and DG, and 3) the DG
GC spiking activity is lower compared to other regions. It is therefore possible that
relatively overlapping memory engrams present in EC are separated (orthogonalized)
16 S. Tonegawa, T.J. McHugh

Fig. 3. CA1 place cells are reactivatable under full cue condition but not under partial cue
condition in CA3-NR1 KO mice. The CA3-NR1 KO mice formed compact CA1 place fields
in a familiar environment under full cue conditions. Upon a reexposure these place cells were
reactivated well under full cue conditions (four cues), but only poorly under partial cue conditions
(one cue)

as the information is transferred through the EC → DG → CA3 pathway. Since NMDA

receptors in DG GCs are expected to modulate the activity of DG GCs in an experience-
dependent manner, it is possible that we may see a deficit in an experience-dependent
pattern separation in mutant mice in which the NR1 gene knockout is targeted to
postnatal GCs.
We generated such NR1 knockout mice (DG-NR1 KO mice) fortuitously by employ-
ing the transcriptional regulatory elements of the proopiomelanocortin (POMC) gene
as the driver of the Cre expression and crossing the Cre transgenic mice with the same
“floxed” NR1 gene mice that we previously used for the CA3 (Nakazawa et al. 2002) and
CA1 (Tsien et al. 1996; McHugh et al. 1996) studies (McHugh et al. 2007). Again, in situ
hybridization in these mice, immunohistochemistry, and synaptic electrophysiology
confirmed that the NR1 knockout is well restricted to postnatal DG GCs.
The performance of the DG-NR1 KO mice was normal in the standard Morris
water maze task as well as in the standard contextual fear conditioning. However, in an
incremental context discrimination fear conditioning task, the mutant mice exhibited
a deficit in the early phase of the trials, although their ability to discriminate the contexts
developed slowly to the normal level as the trials were repeated. Thus, the mutant mice
were normal in spatial and contextual learning per se but had a problem in being able to
rapidly distinguish similar contexts with just a few trials, which the control littermates
accomplished with no problem. These results suggest that the NMDA receptors in
DG GCs and probably NMDA receptor-dependent synaptic plasticity at the perforant
path-DG GC synapses play an important role in fast (with one or two trials) pattern
separation. However, the fact that the mutant mice can catch up to the control mice with
more trials suggests that the hardwiring in the EC → DG → CA3 pathways permits
slow, multitrial-dependent acquisition of pattern separation.
To detect a pattern separation deficit of the DG-NR1 KO mice at the neuronal
ensemble activity level, we recorded with the tetrode technique the spiking activities in
 Molecular and Circuit Mechanisms for Hippocampal Learning 17

CA1 and CA3 as the mice explored two distinct contexts (low-walled white circular box
vs. black square box) at the same site in the same room. Earlier studies with normal rats
had shown that, under these conditions, individual pyramidal cells in CA1 exhibited
similar firing rates in the two contexts whereas those in CA3 displayed context-specific
firing rates (Leutgreb et al. 2005). Thus, in the latter case, there was a “remapping” of
the firing rates as the animals were shifted from one context to the other. Like rats, our
control mice showed significant rate remapping in CA3 but no remapping in CA1. In
contrast, the DG-NR1 KO mice exhibited a significant deficit in rate remapping in CA3
but no remapping in CA1. These results corroborate the behavioral deficit of contextual
discrimination and reinforce the conclusion that DG NMDA receptors play a role in
rapid pattern separation.

CA1 for Novelty Detection?

Our earlier study, carried out by applying the same interdisciplinary strategy to CA1
pyramidal cells, demonstrated that a knockout of NMDA receptors in this “outpost”
of the excitatory hippocampal trisynaptic pathway leads to a severe impairment in
hippocampus-dependent learning tests, such as the Morris water maze and trace
fear conditioning (Tsien et al. 1996; McHugh et al. 1996). This finding is in con-
trast to the knockout of the same NR1 gene in CA3 or DG, but it is not surpris-
ing because, in the CA1-NR1 KO mice, the NMDA receptors are knocked out not
only at the SC-CA1 synapses, the most downstream of the trisynaptic pathway, but
also at the temporoammonic (TA) path-CA1 synapses, an integral part of the direct
EC→CA1→(subiculum)→EC pathway. There has been a suggestion that inputs from
these two circuits (trisynaptic and temporoammonic) are “compared” at CA1 to gener-
ate a “novelty signal” that may be necessary to convert the hippocampus to a “learning
mode” (Fig. 4; Vinogradova 2001; Lisman 2005). After all, we may learn something
when we encounter novelty whereas we cannot learn from something we already know.
To address this postulated function of CA1, we need a new genetic manipulation tech-
nique that will allow us to block the SC input to CA1 specifically while keeping the TA
input intact or vice versa. Such a technique is under development.
The genetic technology that permits a cell-type specific and postnatal knockout
of a gene (such as the NR1 gene) and multidisciplinary analyses of these conditional
mutant mice are allowing us to test a number of hypotheses regarding the distinct func-
tions of hippocampal subregions and their circuits in various aspects of hippocampus-
dependent learning and memory. In the future, this general strategy could be extended
to brain systems and circuits outside of the hippocampus to uncover mechanisms
underlying memory and other cognitive functions.

Acknowledgements. We wish to thank many people who participated in the work outlined in
this short monograph. Many of them are co-authors of the original research papers from our
laboratory. Special thanks go to Kazu Nakazawa, Michael Fanselow, Matt Jones, Matt Wilson and
Nina Balthasar. The work was supported by NIH grants P50-MH 58880 and RO1-MH78821.
18 S. Tonegawa, T.J. McHugh

Fig. 4. Distinct mnemonic functions of hippocampal excitatory circuits. The cell type-restricted
knockout technology revealed that the NMDA receptors in CA3 pyramidal cells and DG granule
cells are important for pattern completion and pattern separation, respectively. The CA3 NMDA
receptors also play a role in rapid encoding of one trial/experience memory (Nakazawa et al. 2003).
It is hypothesized that CA1 pyramidal cells may compare the SC input which may be loaded with
previously acquired memory information and the TA input which conveys on-line sensory input
and, thereby, provides novelty/familiarity signal to the downstream areas like subiculum. The
novelty signal is thought to be important to convert the hippocampal to a “learning mode”

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Retrotransposons – Natural and Synthetic

Jef D. Boeke1 , Wenfeng An1 , Lixin Dai1 , Edward S. Davis1 , Jeffrey S. Han1 , Kathryn A.
O’Donnell1 , Lisa Z. Scheifele1 , and Sarah J. Wheelan1

Transposable elements are ubiquitous among sequenced genomes. The host genomes
roughly subdivide into two types: 1) streamlined, that is, small, with little space between
genes and lacking large introns, or 2) bulky, with lots of space between genes and
many large introns. Most microorganisms, along with selected vertebrates like the
pufferfish, fall into the first class, whereas mammals and most plants fall into the
second class. As can be seen from Fig. 1, transposable element abundance mirrors
the genome type of the host, with mobile elements comprising half or more of many
of these bulky genomes! Mobile elements are of two basic types: DNA transposons,
which predominantly mobilize via a cut and paste mechanism, and retrotransposons,
which move by a copy and paste mechanism involving reverse transcription of an
RNA intermediate (Fig. 1 right panel; Curcio and Derbyshire 2003). Retrotransposons
are found in virtually all eukaryotes, from yeast (Kim et al. 1998) to human (Lander

Fig. 1. Left panel shows the phylogenetic tree of life as determined by rDNA sequence alignments.
Selected organisms are shown, along with the fraction of their genome made up of mobile
elements as pie charts. On the right is the basic information flow used in the retrotransposition
process

1 High Throughput Biology Center, Johns Hopkins University School of Medicine, Baltimore,
MD 21205, USA, e-mail: jboeke@jhmi.edu

Gage et al.
Retrotransposition, Diversity and the Brain
© Springer-Verlag Berlin Heidelberg 2008
22 Jef D. Boeke et al.

et al. 2001). Remarkably, in a yeast cell, the number of retrotransposon copies can be
changed rather dramatically without a major impact on the phenotype of the host.
The change in copy number can be seen using a new tiling array technique by which
it is possible to comprehensively map the unique genomic regions adjacent to all
transposable element copies probed (Fig. 2; Wheelan et al. 2006). The ability of yeast
strains to tolerate very high copy numbers of transposons is due in part to the fact
that, in yeast, most insertions are targeted to non-essential genomic regions, even
though most of the genome is protein-coding (Chalker and Sandmeyer 1990; Devine
and Boeke 1996; Ji et al. 1993; Zou et al. 1996). This property and many others suggest
that retrotransposons are highly coevolved with their hosts.
L1 retrotransposons or LINE-1s are ubiquitous mammalian mobile elements. Each
mammalian species’ genome is littered with copies of an L1 species that has coevolved
with its genome (Gibbs et al. 2004; Kirkness et al. 2003; Lander et al. 2001; Waterston
et al. 2002). L1 elements directly make up about 17% of our genome and are responsible

Fig. 2. Mapping transposon insertion sites. Genomic regions adjacent to transposons are PCR
amplified and then identified by hybridization to a tiling array. Positive hybridization controls
produce a visible “TY” signal. Because features on the array are ordered by chromosomal location,
hybridization to adjacent features can be used to identify insertion sites in a wild-type yeast
strain (A) or a strain with high transposon copy number (B)
 Retrotransposons – Natural and Synthetic 23

for at least a third of our DNA by weight because they provide the molecular machinery
for mobilizing not only their own sequences but also the highly abundant Alu sequences
(Dewannieux et al. 2003), as well as the less abundant processed pseudogenes (Esnault
et al. 2000). The latter “retrotranscripts” are simply cellular mRNAs that have been
reverse transcribed by the L1 machinery and inserted into the genome, very much like
L1 itself is inserted. Retrotransposons move in the genome via a replicative process
(Fig. 3). After being transcribed into a full length RNA by host RNA polymerase, the
RNA can be translated to produce two proteins, ORF1p and ORF2p. Together with
the RNA, these form a ribonucleoprotein (RNP) complex (Martin 1991), which is
imported into the nucleus. ORF2p has endonuclease (Feng et al. 1996) and reverse
transcriptase (Mathias et al. 1991) functions essential for retrotransposition (Moran
et al. 1996). The endonuclease selects and cleaves the target site (Cost and Boeke 1998),
and the RNA is ultimately reverse transcribed to make a new retrotransposon copy,
a process known as target-primed reverse transcription (TPRT; Fig. 4; Luan et al.
1993).
In somatic and tissue culture cells, L1 expression and hence transposition appear to
be tightly regulated transcriptionally, and so the promoter that drives this expression

Fig. 3. Replicative cycle of L1 elements. A “donor” element (blue band on chromosome) is

transcribed in the nucleus. The RNA (red waved line) is exported to the cytoplasm, where it is
translated into ORF1 (yellow spheres) and ORF2 (blue sphere) proteins. The ribonucleoprotein
(RNP) complex is imported into the nucleus and used as the machinery to drive target-primed
reverse transcription (TPRT)/integration of a new copy of the element at a new locus (red band
on chromosome)
24 Jef D. Boeke et al.

Fig. 4. Mechanism of
LINE-1 integration by
TPRT. The endonuclease
(EN) domain of ORF2
creates a single-strand
nick in the target DNA.
The L1 RNA anneals with
the DNA and ORF2’s
reverse transcriptase (RT)
activity uses the target
DNA’s 3’-OH to prime
synthesis of first strand
cDNA

has been an object of considerable interest and scrutiny. Interestingly, although L1

elements in primates and rodents encode relatively similar proteins (percentage of
amino acid identity ranges from 20% at the N-terminus of ORF1 to >60% in ORF2),
the promoters not only lack sequence homology entirely but also have very different
structures (Fig. 5). Most mouse L1 promoters (in the A, F, TF and GF subfamilies of
mouse L1 elements), like those of other rodent L1s, are made up of a series of tandem
repeats of ∼200 bp, called monomers, followed by a short non-monomeric region
(Goodier et al. 2001; Padgett et al. 1988). Both subfamilies are relatively ancient and
most members are inactive. TF is a young, expanding subfamily containing ∼3 000
full-length members and ∼1 800 of them are active. GF is the most recently discovered
subfamily that contains ∼400 active elements. Both the TF and GF monomer are
70% identical to F-type monomer but differ from each other by 33%. In addition to
the differences among monomer sequences, the numbers of monomer repeats and
monomer lengths vary among individual element copies. In contrast, the human L1
promoter sequence in transpositionally and transcriptionally active (Ta) elements is

Fig. 5. Comparison of mouse and human L1 promoters. The 5’ UTR region of most mouse
L1 contains several tandem repeats (monomer) in the length of ∼200 bp. Each blue ar-
row represents a monomer sequence. The 5’ UTR of human L1 contains a ∼900 bp, non-
repetitive region (yellow arrow) that drives the transcription of L1 element. Black arrow de-
notes the first open reading frame of L1 (ORF1) and fine line arrow indicates the transcription
direction
 Retrotransposons – Natural and Synthetic 25

about 900 bp long, nonrepetitive and well-conserved in length, and it contains all of
the elements required for transcription downstream of the transcription start site
(Swergold 1990).

Selfish Gene?

In his book “The Selfish Gene,” Richard Dawkins outlines the idea that evolution is
driven at the level of individual genes. There is no more compelling example of this than
mobile genetic elements like retrotransposons, to which host genomes/organisms are

Fig. 6. Synthetic mouse L1 is much more active for retrotransposition than native mouse and
human L1 elements. Retrotransposition assay was performed in HeLa cells for native mouse
L1, synthetic mouse L1 and native human L1 elements, all of which were tagged with an
intron-interrupted neomycin resistance gene reporter. L1 function is scored as the number
of G418-resistant colonies because only when L1 completes one round of retrotransposition
does a cell become G418-resistant. Cells were diluted at ratios as indicated prior to G418
selection
26 Jef D. Boeke et al.

nothing more than “bags of genes” to exploit (Dawkins 1976). Like virtually all trans-
posable elements found in metazoans, L1 element transposition was until relatively
recently thought to be entirely germ-line specific, as predicted from strict “selfish gene”
theory. However, recent findings indicate that L1s are highly active transcriptionally in
mouse neuronal progenitor cells, and engineered human elements retrotranspose in
mouse brain in a neuron-specific manner (Muotri et al. 2005).

Fully Synthetic Retrotransposons Are Highly Active

L1 retrotransposons are potential tools for in vivo mutagenesis; however, native L1 ele-
ments are relatively inactive transpositionally in mice. To this end, we have constructed
a synthetic L1 element, referred to as ORFeus, consisting of two synonymously recoded
open reading frames (Han and Boeke 2004). The sequence is based on a native mouse
L1 element sequence, L1spa (Mulhardt et al. 1994) and can be controlled by either
generic (e.g., CMV or CAG promoter) or native L1 5’ end transcriptional control se-
quences. Such donor element constructs can be marked by a transposition indicator
gene, which is inserted in the antisense orientation relative to the transcription of the
ORFeus element. The reporter, either neo or gfp, is interrupted by an intron in the same
sense as the ORFeus donor element. In this way, the donor element does not express
the reporter because its coding region is disrupted by an inverse intron, but upon
retrotransposition, the intron is removed during the RNA step and an active reporter
gene is generated.

Fig. 7. Estimating germ-line insertion frequency by Southern blot analysis. The top left panel
is a schematic of the 10-copy ORFeus donor transgene concatemer with a detailed view of the
structure of a single-copy transgene under the regulation of CAG promoter and marked by
an intron-disrupted GFP reporter cassette driven by its own promoter and polyadenylation
site. The position of the Southern probe is indicated. The right panel is a Southern blot for
nine N2 progeny mice from breeding their F1 transgenic parent (the first lane) to a wild type
mouse
 Retrotransposons – Natural and Synthetic 27

Using a neo intron removal assay, ORFeus was found to be ∼200-fold more ac-
tive for retrotransposition in cell culture than native mouse L1 elements and was
even more active than the most active human elements studied previously (Fig. 6).
To study ORFeus activity in vivo, we developed transgenic mouse models in which
ORFeus expression was controlled by the constitutively active heterologous CAG pro-
moter, and we measured ORFeus retrotransposition activity both in germ-line and
somatic tissues (An et al. 2006). Germ-line retrotransposition frequencies resulting
in 0.3–0.4 insertions per animal were seen among progeny of ORFeus donor element
heterozygotes, as determined by Southern blotting (Fig. 7). This germ-line retro-
transposition frequency compares favorably with previously observed retrotranspo-
sition frequencies with native elements driven by heterologous promoters (Babushok
et al. 2006). Interestingly, we also observed somatic transposition events in 100% of
these ORFeus donor-containing animals, and many different insertions were readily
recovered from each animal using a modified inverse PCR protocol. Modeling ex-
ercises suggest that the numbers of somatic insertions per animal could be as high
as millions, suggesting that these animals could provide important new models for
cancer, as has recently been reported for the Sleeping Beauty DNA transposon (Col-
lier et al. 2005; Dupuy et al. 2005). Somatic retrotransposition was observed in all
tissues tested, including brain, but was not particularly elevated in any specific tis-
sue in these mice driven by the CAG promoter. Nearly 200 insertions were precisely
mapped, and their distribution in the mouse genome appeared random relative to
transcription units and GC content (Fig. 8). Constitutive ORFeus may be extraordi-
narily useful for in vivo mouse mutagenesis. Gene traps are being developed for these
purposes.

Fig. 8. Chromosomal distribution of mapped insertions. A total of 171 mappable insertions were
charted to mouse genome build 36 (short black lines to the right of individual chromosomes).
The approximate position of the donor concatemer on chromosome is marked (green asterisk),
which was located by fluorescent in situ hybridization (shown in the insert). Insert: Metaphase
spreads of splenocytes from donor-containing mice were probed with fluorescently labeled full-
length transgene cDNA probe (green) and subsequently with a whole-chromosome paint probe
for chromosome 7 (red). Chromosomes were counterstained with DAPI (blue)
28 Jef D. Boeke et al.

Neural-specific Retrotransposition – Could it be Conserved

from Rodents to Primates?

L1 elements in mouse and human, like most metazoan retrotransposons, show evidence
for germ-line-specific expression (Branciforte and Martin 1994; Ergun et al. 2004; Trel-
ogan and Martin 1995). There is evidence that native rodent L1s are active in neural
progenitor cells stimulated to differentiate in response to FGF-2 and are upregulated
transcriptionally. On this basis, Muotri et al. (2005) introduced a human L1 (driven
by a human L1 promoter) marked with a retrotransposition indicator gene into such
cells and into transgenic mice. Interestingly, when the cells were differentiated in tissue
culture into astrocytes, glia and neurons, retrotransposition of the human constructs
was only seen in those cells that differentiated into neuron-like cells. In some of these
cases, insertion of the new retrotransposons was into transcriptionally active target
genes in the differentiating neurons. Furthermore, significant retrotranspositional ac-
tivity of this element (as inferred from GFP staining) was observed in a wide variety
of neuronal cells in the brains of these mice. These results can be interpreted to sug-
gest that the highly divergent promoters of primate and rodent LINEs, as well as the
divergent proteins encoded by these elements, might be under genetic selection for
retrotranspositional activity in the brain. Not only are these promoters highly diver-
gent structurally, but there is also good reason to believe they have an independent
genetic origin. The “promoter capture” model (Khan et al. 2006) posits that, as the
host inactivates L1 promoters by various mechanisms, L1s can capture novel cellular
promoters by TPRT followed by incomplete reverse transcription (Fig. 9). This would
then put the element under control of a new promoter. The divergent structures of
primate and rodent elements support the idea that at least one such event occurred
between rodent and primate lineages. The hypothesis that the promoters are indepen-
dently derived yet retain germ-line- and neuron-specific activities could be tested by

Fig. 9. Promoter capture model. L1 may capture cellular promoters during evolution by trans-
posing a partially truncated element. During the TPRT reaction, the reverse transcription of L1
RNA may extend through ORF1 but fail to copy its own promoter. If this incomplete element is
inserted downstream of a cellular promoter, then the L1 might capture this sequence as its own
novel promoter
 Retrotransposons – Natural and Synthetic 29

examining the retrotranspositional activity of native mouse elements or ORFeus driven

by the native mouse L1 promoter. Such experiments are in progress (collaboration with
A. Muotri and F. Gage).

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Ancient Retrotransposons as Possible Remnants
of the Primitive RNP World

Roland Ivanyi-Nagy1 and Jean-Luc Darlix1

Summary

A recently discovered common trait of all eukaryotic organisms is a high prevalence

of mobile genetic elements in their genome. From yeast to man, transposable ele-
ments are mostly represented by retroelements, which account for about half of the
genome content in mammals and replicate by means of an obligatory RNA inter-
mediate. According to the “RNA world” hypothesis, one step of the “prebiotic life”
would correspond to populations of small RNA molecules forming increasingly com-
plex RNA networks generating and piling up information. To reach this organiza-
tional level, RNA molecules probably needed to interact with small basic peptides,
thus forming the backbone of ribonucleoparticles (RNPs). Aggregation and conden-
sation of diverse RNA populations by basic peptides can generate arrays of large
RNPs in which a drastic increase in RNA concentration leads to a crowding phe-
nomenon facilitating RNA-RNA, RNA-peptide and peptide-peptide interactions and
biochemical reactions. In addition, small basic peptides can have critical properties,
known as RNA chaperoning, whereby the most stable structure of RNA molecules is
rapidly reached and RNA-RNA annealing reactions can take place in a highly dy-
namic fashion. Thus, RNA chaperoning provided by basic peptides within highly
crowded RNA populations may actively create novel information essential for the
basic mechanisms of life. Simple retroelements from yeast to man encode such sim-
ple basic peptides with the RNA binding and chaperoning properties that are needed
for their replication. Here we will briefly review RNA chaperones of retroelements
and their mode of action, whereby these intrinsically flexible proteins can inter-
act with many different partners to establish networks required for the replication
of retroelements. We will also consider some mechanisms thought to regulate am-
plification of retroelements to control their invasion of and damage to eukaryotic
genomes.

Simple Retrotransposons Compose Half of our Genome

Intensive genome sequencing has revealed that the retrotransposon class of retroele-
ments can account for nearly half of the genome content in mammals (International

1 LaboRetro, Unité de Virologie humaine INSERM, IFR128, ENS Lyon 46 allée d’Italie,
69364 Lyon, France
e-mail:jldarlix@ens-lyon.fr

Gage et al.
Retrotransposition, Diversity and the Brain
© Springer-Verlag Berlin Heidelberg 2008
34 R. Ivanyi-Nagy, J.-L. Darlix

Human Genome Sequencing Consortium 2001; Mouse Genome Sequencing Consor-

tium 2002). Retroelements, and notably retrotransposons, are viewed as very ancient
genetic structures because they are ubiquitous in eukaryotes, from yeast to human
(Kazazian 2004). Replicating retroelements are small mobile genetic entities formed of
protein coding sequences, or open reading frames (ORF), flanked by terminal repeats.
Retroelements have been classified into retrotransposons with long terminal repeats
(LTR retrotransposons) such as TYs of yeast, or retrotransposons without LTR (non-
LTR retrotransposons) such as LINEs (Ostertag and Kazazian 2001; Kazazian 2004),
and retroviruses that encompass the widespread simple gamma-retroviruses, notably
the exogenous and endogenous murine leukemia viruses (MLV) and the family of
complex lentiviruses best represented by the human immunodeficiency viruses (HIV)
and simian immunodeficiency viruses (SIV)(Goff 2007; Balvay et al. 2007).
Retrotransposons and retroviruses share a similar genetic organization, firstly
ORF1 or gag encoding the major structural core protein(s), and secondly ORF2
or pol coding for the enzymes that are the RNA/DNA-dependent DNA polymerase
known as reverse transcriptase (RT) with RNaseH activity, and the integrase or
endonuclease (IN/EN; Ostertag and Kazazian 2001; Kazazian 2004). In addition,
LTR-retrotransposons and retroviruses code for a protease (PR) in pol. Replication-
competent retroviruses also contain a third ORF, the envelope (env), which is necessary
for the production of infectious virions and their dissemination in a horizontal fashion
within animal populations and eventually between different populations (Goff 2007;
Balvay et al. 2007).

The RNA/RNP World and Retroelements

RNA molecules are central players in all forms of life, from viroids to cells. Ma-
jor classes of RNA molecules present in cells include transfer RNAs (tRNAs), small
nuclear, nucleolar and cytoplasmic RNAs (sn-, sno- and scRNAs), ribosomal RNAs
(rRNAs), pre- and messenger RNA (mRNAs) coding for proteins, and lastly, the re-
cently discovered very large family of microRNAs, which includes small temporal RNAs
(stRNAs), small interfering RNAs (siRNAs), and Piwi-interacting RNAs (piRNAs) that
regulate the expression and stability of mRNAs (Lee and Ambros 2001; Lagos-Quintana
et al. 2001; O’Driscoll 2006; Lau et al. 2006). These diverse RNAs act in a coordinated,
highly regulated fashion as parts of complex RNA-RNA and RNA-protein interaction
networks, regulating the flow of information from DNA to proteins. In addition, ri-
bozymes – RNA molecules with catalytic activity – are considered to be vestiges of
the hypothetical prebiotic RNA world (Gilbert 1986), when both information storage
and chemical catalysis were carried out by RNA. As already pointed out, retroviral
and retrotransposon RNAs are diverse and abundant, with a major impact on eu-
karyotic genome formation and dynamics (Kazazian 2004). Viroid RNAs are unusual
because they are circular and single-stranded and contain a large number of modified
nucleotides (Symons 1997).
In most instances RNAs are single-stranded and thus correspond to flexible macro-
molecules that can adopt a wide variety of alternative conformations. However, in many
cases only one, well-defined structure is thought to be biologically active, which, besides
folding kinetics and thermodynamics, is also influenced by interacting partner(s). At
 Ancient Retrotransposons as Possible Remnants of the Primitive RNP World 35

the same time, a large fraction of an RNA population can be trapped in incorrect struc-
tures, highlighting the “RNA folding problem” in a dynamic biological environment
(Russell et al. 2002). Therefore, RNA folding must be assisted so that functionally rele-
vant conformations can be rapidly reached. In addition, assistance must be provided
so that possible RNA-RNA interactions can rapidly take place, leading to the formation
of a network of interacting, functionally active molecules.
Replication of retroelements takes place in a very small, compact ribonucleoparticle
(RNP) structure, where the single-stranded RNA is converted into a double-stranded
DNA by RT, assisted by a basic RNA-binding peptide encoded by the retroelement.
This unique basic peptide is encoded by ORF1 (Gag in retroviruses and certain LTR-
retrotransposons), and is called nucleocapsid protein (NC; Darlix et al. 2007). In ad-
dition to their nucleic acid binding ability, retroviral NC and retrotransposon NC-like
peptides have in common potent RNA condensing and chaperoning properties (Cristo-
fari and Darlix 2002).
According to the RNA world scenario, RNA replicators constituted a prebiotic
form of “life,” serving at the same time as genetic material and catalysts, predating the
appearance of templated protein synthesis (Gilbert 1986). Indeed, ribozymes in extant
organisms and artificial catalytic RNA molecules testify to the catalytic versatility
and capacity of RNA (e.g., Hager et al. 1996; Johnston et al. 2001; Spirin 2002; Muller
2006; Chen et al. 2007). However, the length of self-replicating RNA molecules and
thus the complexity of the RNA world were greatly limited by the inherent low fidelity
of (protein-unassisted) RNA replication, resulting in a lethal mutational load over
a certain “genome” length (error threshold; Eigen 1971). Small RNA chaperone-like
peptides, synthesized under prebiotic conditions in an untemplated manner, may have
relaxed these limitations by stabilizing and folding RNA molecules and facilitating
RNA-RNA interactions (Poole et al. 1998). In agreement with the proposed primeval
origin of RNA chaperones, extant proteins with RNA chaperone activity frequently
contain low amino acid complexity regions and they may function in the absence
of a well-defined structure (see below; Tompa and Csermely 2004; Ivanyi-Nagy et al.
2005). In addition, even short unstructured peptides may have potent RNA chaperone
activities (Ivanyi-Nagy and Darlix, unpublished data).
A second major increase in information content and complexity at the dawn of life
could have occurred in an RNA world by the invention of reverse transcription, followed
by the takeover of information storage by the more stable DNA. Given that retroelements
are ancient, highly repetitive and dispersed genetic entities in all eukaryotic genomes,
and that they replicate by reverse transcription of RNA into DNA, they have been
suggested to be early players in the formation of complex DNA genomes (Forterre
2005; Koonin et al. 2006). As discussed below, RNA chaperones probably played an
important role in this process, too.

A New World of Disorder

RNA chaperone proteins do not share a characteristic domain organization, specific

motif or signature sequence that could provide insights into the mechanism of RNA
structural rearrangements directed by them. Indeed, while some retroelement contain
one or two copies of a specific RNA-binding motif, called CCHC-type zinc finger
36 R. Ivanyi-Nagy, J.-L. Darlix

(Cx2 Cx4 Hx4 C), others – like TY1 or Gypsy – lack a well-defined, consensus RNA-
binding motif (Fig. 1; Covey 1986; Cristofari et al. 2002; Gabus et al. 2006). Despite
the absence of considerable sequence homology, NC or NC-like peptides are all rich in
basic amino acids and proline, and they frequently contain low amino acid complexity
regions, a combination of features that results in a highly flexible conformation (Dunker
et al. 2001; Fig. 1). Such proteins and peptides are now referred to as intrinsically
unstructured proteins (IUPs).
Based on the abundance of relatively long, continuous disordered fragments in RNA
chaperones, Tompa and Csermely (2004) recently proposed an elegant model to account
for the ATP-independent action of these proteins. According to their “entropy exchange
model,” cyclic RNA binding and release of the RNA chaperone, coupled with a reciprocal
entropy transfer process between the RNA and the protein, would provide the driving
force for rapid RNA structural rearrangements (Fig. 2). Based on that model, RNA
chaperones contact RNA in a (partly) unstructured state (Fig. 2, step 1). RNA binding
induces local disorder-to-order transition (folding) of the protein, accompanied by
the partial melting of the RNA secondary structure (step 2). The energy required for
RNA structure destabilization is thus covered by an entropy transfer associated with

Fig. 1. Domain organization and intrinsic disorder in retroelement Gag proteins. Disordered
regions in retrovirus and retrotransposon Gag proteins were predicted using the DisProt VL3-H
predictor (http://www.ist.temple.edu/disprot/predictor.php). GenBank ac-
cession numbers are the following: Ty3 Gag Q12173; Ty1 Gag AAA66937; Gypsy ORF1p AAA70218;
HIV-1 Gag NP_057850; MoMuLV Gag AAC82566; and LINE-1 ORF1p AAA67726. An amino acid
with a disorder score above 0.5 is considered to be in a disordered environment; one below 0.5
is considered to be ordered. The predicted disorder is illustrated by a color scale, with highly
flexible segments in red and well-folded domains in green. Basic and acidic amino acid residues
are indicated by dark blue and mauve symbols, respectively. Zn stands for the CCHC-type zinc
finger motif that is required for proper virion formation and genomic RNA replication in simple
(i.e., MLVs) and complex retroviruses (i.e., HIV-1; reviewed in Darlix et al. 2007). Note that
the NC peptidic regions (in dark blue) are small and flexible, in particular those of the ancient
retrotransposons TY1, TY3 and Gypsy
 Ancient Retrotransposons as Possible Remnants of the Primitive RNP World 37

RNA binding. Finally, the RNA chaperone is released from the partially melted RNA,
which is free to resume a conformational search (step 3). Successive cycles of transient
binding, accompanied by disorder-to-order transitions, lead to the formation of the
most stable RNA secondary structure (Fig. 2).

Fig. 2. The entropy exchange model of RNA chaperone function. The entropy exchange model of
Tompa and Csermely (2004) is illustrated here by the example of NCp7-mediated HIV-1 genomic
RNA dimerization. In the absence of NCp7, the dimer initiation sequence (DIS) of HIV-1 forms
a kissing-loop dimer structure. Upon binding of the highly flexible protein (step 1), the RNA
structure is destabilized (step 2), and adopts the more stable, extended dimer conformation
(step 3). See more detailed explanation of the model in the text. The figure is not drawn to scale.
PDB entries 1JU1, 2F4X, and 1ESK were used for figure drawing
38 R. Ivanyi-Nagy, J.-L. Darlix

In addition to providing a mechanistic basis for chaperone function, intrinsic

disorder may confer numerous additional advantages for RNA chaperones of retroele-
ments. Flexibility of the peptide chain allows high-affinity interactions with multiple,
structurally diverse substrate molecules (one-to-many signaling; Kriwacki et al. 1996;
Dunker et al. 1998). In this way, disordered peptidic segments may play a central
role in the organization and dynamics of RNA interaction networks, including cel-
lular macromolecular ensembles such as hnRNP’s, spliceosomes and ribosomes, by
acting as major functional units of hub proteins (Dunker et al. 2005; Ivanyi-Nagy et al.
2005).
Indeed, the NC chaperone of widespread MLVs – and probably all NCs and
NC-like peptides of retroelements – orchestrates viral RNA replication and virus
biogenesis and is at the same time a major component of the virion core struc-
ture (Darlix et al. 2007). These multiple functions appear to be mediated via in-
teractions with a wide array of protein and nucleic acid partners, such as the
retroelement RNA, tRNAs, RT, integrase (IN) and restriction factors (Darlix et al.
1995, 2007; Ivanyi-Nagy et al. 2005; Fig. 3). Furthermore, conformational flexibil-
ity is advantageous for NC-oligomer formation, necessary for RNP-core assembly
(Namba 2001) and stabilization by condensation, and it is essential during the
multistep conversion of the RNA into cDNA by RT (Morellet et al. 1994; Darlix
et al. 2007).

RNA Chaperones and the Regulation of Retrotransposon Replication

Given that retroelements can replicate at fairly high levels, their unregulated and con-
tinuous replication would create genetic instability in eukaryotes by damaging the
genome via different mechanisms, such as insertion mutagenesis, gene inactivation, or
chromosome rearrangement by homologous recombination between copies of retro-
transposon sequences (Ostertag and Kazazian 2001). A genome-wide impact of retro-
transposons has also been proposed to affect gene transcription in higher eukaryotes
(Han et al. 2004).
However, retrotransposition appears to be tightly regulated in a cumulative mode,
first at the transcriptional level, whereby transcription is either silenced by DNA methy-
lation or halted, at least in part, by cis-acting retrotransposon sequences (Han and
Boeke 2004), and also by host-encoded restriction factors and small RNAs (O’Donnel
and Boeke 2007).
More recently, cytidine deaminases have been found to restrict retrotransposition
of endogenous retroviruses and retrotransposons (Esnault et al. 2005; Schumacher
et al. 2005; Muckenfuss et al. 2006; Stenglein and Harris 2006; Chiu et al. 2006). By
analogy with HIV-1 restriction mediated by APOBEC3G (Harris et al. 2003; Mariani
et al. 2003), this family of enzymes seems to operate by deaminating C residues in
the newly made cDNA and thus through interaction with the reverse transcription
RNA-NC-RT complex (Fig. 3). Another negative regulation provided by the APOBEC
enzymes would be via specific RNPs interacting with the retroelement core (Chiu
et al. 2006; Stenglein and Harris 2006; Gallois-Montbrun et al. 2007; Holmes et al.
2007).
 Ancient Retrotransposons as Possible Remnants of the Primitive RNP World 39

Fig. 3. Network of NC chaperone-directed interactions in retroelements. The simple scheme

illustrates: (1) formation of the RNP or Core structure of the Retroelement (RTE) upon binding
of NC molecules to the the homologous RTE RNA (see arrows); (2) core formation and con-
densation induce molecular crowding and provide protection to the structure; (3) recruitment
of two RTE RNA molecules by NC (arrows), which chaperones RNA dimerization, as shown for
TYs and retroviruses; (4) packaging of tRNAs that serve as primers for cDNA synthesis by RT
chaperoned by NC; (5) interactions between RTE core structure and RNPs containing APOBECs
can potentially change C > U in the RTE RNA and in the newly made cDNA, causing an efficient
restriction of RTE replication and dissemination as well as microdiversity; (6) the prion protein
might also interfere with the process of cDNA synthesis and more generally with the assembly of
retroviral particles; (7) reverse transcription of the dimeric RNA – or pseudodiploid RNA – can
generate RTE diversity by means of recombination reactions; (8) RNAs other than RTE can be
packaged in trans and reverse transcribed, as shown for mouse sarcoma and leukemia viruses;
(9) Integration of the newly made cDNA copy is mediated by the integrase (IN) or endonuclease
enzyme, which can result in mutational insertions or promoter activation with an impact on the
transcriptional pattern of the host genome (transcriptome)

Furthermore, the prion protein, PrP, may well be a restriction factor acting during
core assembly. PrP resembles retroelement NC since it has potent RNA binding and
chaperoning properties (Gabus et al. 2001a,b). Upon binding to the retroelement RNA,
PrP chaperones its dimerization in vitro, resulting in the formation of highly compact
RNPs (Gabus et al. 2001b). In the context of retrovirus-infected cells, PrP is recruited
into newly formed viral particles and at the same time inhibits virion production and
infectivity (Leblanc et al. 2004, 2006). It remains to be shown whether PrP also has
a negative impact on retrotransposition.
40 R. Ivanyi-Nagy, J.-L. Darlix

Conclusions

RNA chaperones of retroelements are basic peptides lacking well-defined structured

motifs. This finding has led to their classification as intrinsically unstructured or
disordered proteins (IUPs), a property that confers on them the ability to inter-
act with many different partners and thus to accomplish a wide array of functions,
as demonstrated for retroviral NCs (Darlix et al. 1995, 2007). In the context of the
disorder-to-order transition, they act by assisting in the folding of RNA molecules.
Given the hypothesis that prebiotic life is represented by the RNA world and that the
disorder-to-order transition is necessary for such a creative process, RNA chaperones
of retroelements are central players since they assist the folding of RNA molecules,
cause a crowding phenomenon of RNA molecules in the interior of very small volumes
and provide assistance to multiple RNA-RNA interactions required for retroelement
replication, such as TY1 of yeast and mammalian retroviruses (Cristofari et al. 2000,
2002; Darlix et al. 2007) .
Since retrotransposons are ancient, we might then surmise that RNA chaperones
of retroelements are the origin of the more sophisticated, large cellular nucleic acid
chaperones – such as p53, YB1 and FMRP - that are abundant proteins found in all living
organisms, where they are essential in chromosome maintenance, DNA transcription,
RNA splicing, trafficking, translation and degradation (Cristofari and Darlix 2002;
Schroeder et al. 2004).

Acknowledgements. Work in LaboRetro is supported by ANRS, CNRS, INSERM, EC (TRIoH

consortium) and Sidaction. RIN is supported by an ANRS fellowship.

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Human Diversity and L1 Retrotransposon Biology:
Creation of New Genes and Individual Variation
in Retrotransposition Potential

H.H. Kazazian, Jr.1 , M.d.C. Seleme1 , D.V. Babushok1 , D.M. Ostertag1 , and M.R. Vetter1 ,
and P.K. Mandal1

The L1 retrotransposon is a major component of mammalian genomes and has molded

them throughout evolution in many ways, thereby expanding the possibilities for
human diversity. In this paper, we discuss one further mechanism by which L1 can alter
the genome, namely the retrotransposition of a transcript involving sequences from
two adjacent genes to form a new gene. In addition, a small fraction of L1 elements in the
human genome is still actively retrotransposing, but there are little data on the extent
of variation in retrotransposition potential among different individual human beings.
Here we present evidence for considerable individual variation in L1 retrotransposition
capability, a finding that has significant implications for the role of retrotransposition
in present-day human neurological diversity.

Introduction

Roughly 500 000 mostly truncated copies of L1 retrotransposons occupy the human
genome, of which about 6 000 are 6 kb or full length (Lander et al. 2001; Fig. 1). These
full-length L1s have a 5’UTR containing both sense and antisense internal promoters
(Swergold 1990; Speek 2001), a 1 kb ORF1 that encodes an RNA-binding protein with
nucleic acid chaperone activity (Martin and Bushman 2001), a 4 kb ORF2 that encodes
endonuclease activity (Feng et al. 1996), reverse transcriptase activity (Mathias et al.
1991), and a zinc knuckle domain (Fanning and Singer 1987). ORF2 is followed by
a short 3’ UTR and a poly A tail. Even though the complete functions of the ORFs are
still unknown, both are critical for the retrotransposition process (Moran et al. 1996).
Upon insertion of an L1 by target-primed reverse transcription (TPRT; Luan et al.
1993) on genomic DNA, the element is surrounded by 6–20 bp target site duplications
of genomic sequence derived from the insertion site (Singer et al. 1993; Scott et al. 1987).

Fig. 1. Structure of a full-

length 6 kb human L1
retrotransposon. See text
for details
1 Department of Genetics, University of Pennsylvania School of Medicine, Philadelphia,
PA 19104, USA, e-mail: kazazian@mail.med.upenn.edu

Gage et al.
Retrotransposition, Diversity and the Brain
© Springer-Verlag Berlin Heidelberg 2008
44 Kazazian et al.

L1s have contributed to genome development in a number of ways (Kazazian 2004).

They are able to not only insert themselves (Ostertag and Kazazian 2001) but they also
drive the insertion of Alu elements (Batzer and Deininger 2002), SVA elements (Ostertag
et al. 2003; Wang et al. 2005), and processed pseudogenes (Esnault et al. 2000; Wei et al.
2001). Insertions of these other elements account for another 12% of the human genome
(Zhang et al. 2003). Among additional ways that L1s have altered the genome are the 3’
transduction of sequences from one site to another (Moran et al. 1999; Goodier et al.
2000; Pickeral et al. 2000), endonuclease-independent insertion (Morrish et al. 2001),
formation of new exons (Gotea and Makalowski 2006), homologous recombination
between repeat sequences (Deininger and Batzer 1999; Jurka 2004), and chimeric
insertions involving small RNAs (Buzdin et al., 2003).
In the average human genome, roughly 100 L1s are still active, but the bulk of this
retrotransposition capability resides in a small subset of these elements (Brouha et al.
2003). Here we first discuss another way in which L1 contributes to genome development
and then the extent of individual variability in retrotransposition capability.

A New Mechanism for Gene Creation

Retrotransposition of mRNA to form processed pseudogenes is well known and has

accounted for over 8,000 mRNA copies in the human genome (Esnault et al. 2000; Zhang
et al. 2003). About 5% of these processed pseudogenes are transcribed due to serendip-
itous use of an external promoter (Zheng et al. 2005). At least one retrotransposed
mRNA copy has landed in an intron during primate evolution and been incorporated
into a fusion gene (Sayah et al., 2004). We have recently found another type of retro-
transposed mRNA. In this case, the RNA is derived from a readthrough transcript
connecting the RNA of a lipid kinase gene (gene 1-PIP5K1A) and a ubiquitin-binding
protein gene (gene 2-S5a) located 6 kb apart. RNA splicing occurred between exon
13 of gene 1 and exon 2 of gene 2 (Fig. 2; Akiva et al. 2005; Babushok et al. 2007).
The chimeric transcript was then retrotransposed into a new chromosomal site, and
we call this gene S5aL for S5a-like. This event occurred roughly 17 million years ago,
since this retrotransposed sequence is present in the genome of human beings, chimps,
gorillas, and orangutans, but not in the rhesus genome. The splicing event maintained
the reading frame such that translation occurs through to the stop codon of gene 2.

Fig. 2. Creation of a new gene by aberrant splicing and retrotransposition. (A) Neighboring 15-
exon PIP5K1A and 10-exon S5a genes on chr 1 are spliced to form PIP5K1A, S5a, and PIP5K1A-S5a
TIC mRNAs. Black rectangles, exons; curved lines, splicing. (B) PIP5K1A-S5a TIC was retrotrans-
posed by L1 to create S5aL gene on chr 10. TSD, target site duplication; pA, A-rich repeat. Regions
corresponding to PIP5K1A and S5a exons are shown as white and gray rectangles, respectively
 Human Diversity and L1 Retrotransposon 45

S5aL is transcribed specifically in the testes in both humans and chimpanzees and
is post-transcriptionally repressed by two independent mechanisms in these primate
lineages. In humans, a nucleotide deletion within 50 nucleotides 3’ of the initiation AUG
codon of gene 1 knocks out translation. In the chimpanzee, the reading frame is intact,
but translation is repressed by an unknown mechanism. Strong positive selection on
S5aL has led to its rapid divergence from the parental genes PIP5K1A and S5a, forming
a chimeric protein with a distinct cellular localization and minimal lipid kinase activity,
but significant affinity for cellular ubiquitinated proteins.
Thus, S5aL is a tightly regulated, testes-specific, novel ubiquitin-binding protein
that was formed by an unusual exon-shuffling mechanism in hominoid primates and
may represent an example of rapid evolution of male reproductive genes that drives
reproductive isolation and species divergence. To our knowledge, this is the only known
example of an exon-shuffled, retrotransposed gene involving the mRNA of two genes
in close proximity to each other. This fusion gene was found through a search of the
human working draft sequence looking specifically for fusion genes; in a similar search
of the mouse genome sequence, no such fusion genes were found. Whether any similar
chimeric genes are present in other organisms is unknown at this time.

Extensive Individual Variation in Retrotransposon Capability

Contributes to Human Genomic Diversity

Muotri et al. (2005) have hypothesized that human diversity in behavior and learning
may be due in part to retrotransposition of L1 elements during neuronal development.
They have found that human L1s can retrotranspose in neuronal precursors in cell cul-
ture and in cells contributing to various areas of the brain during mouse development.
We had previously shown that the bulk of retrotransposition capability present in the
human genome working draft is present in only a handful of L1 elements (Brouha et al.
2003). Since the extent to which human diversity may be controlled by retrotranspo-
sition depends heavily on the number and activity of active L1s in individual human
genomes (in addition to unknown host factors), we decided to determine the extent
of variation in retrotransposition capability for those six highly active or “hot” L1s
among individual human beings. To carry out this analysis, we determined the number
of copies of each element in each genome (homozygous present, heterozygous present,
or absent), the nucleotide polymorphisms in each element, and the retrotransposition
activity of each allele of the various elements (Seleme et al. 2006).
Of the six “hot” L1s that we had found in the database, we could not analyze three
because two were extremely rare in human genomes and one was in highly repetitive
DNA. Thus, our analysis centered on the remaining three “hot” L1s that we called
L1A, L1B, and L1C. We first determined the presence/absence of polymorphism of
the three L1s in 161 to 206 haploid genomes. Although the gene frequencies varied
from population to population, none of the elements departed significantly from the
Hardy-Weinberg equilibrium in any population. Overall, the frequencies of L1A, L1B
and L1C were 0.19, 0.46, and 0.46, respectively. Since all three of these L1s belong to the
youngest subfamily of L1s (Ta-1), it was not surprising that they are relatively recent
additions to the human genome and still have fairly low gene frequencies.
46 Kazazian et al.

We then obtained the complete sequence on L1A in 35 of 40 genomes containing this

L1, on L1B in 59 of 67 genomes, and on L1C in 78 of 96 genomes. We found a previously
uncharacterized allele in every five genomes for L1A and in every three genomes for
L1B and L1C. L1A had 17 polymorphic sites among nine alleles, whereas L1B and L1C
had 19 and 26 polymorphic sites among 18 and 26 alleles, respectively. Among the
unexpected findings were four different nonsense mutations, two in a common allele
of L1A and two in separate alleles of L1C.

Allelic Variation in Retrotransposition Capability:

Hot and Cool Alleles

We tested the retrotransposition capability of 46 of the 52 alleles of L1A, L1B, and L1C
in our cell culture assay. For the three elements, 22 of 46 tested alleles had 25% or
greater retrotransposition activity compared with the reference L1 (L1RP ) and were
called hot. However, the remaining 24 alleles (5 of 8 L1A, 3 of 16 L1B, and 16 of 22 L1C)
had activity <25% of L1RP and were cool. Of all loci containing hot L1s, 33% (57 of 170)
were cool.

L1 Retrotransposition Potential in Individuals and Populations

The number of possible genotypes in an individual for a locus with n alleles is n(n+1)/ 2
or 36 for L1A, 171 for L1B, and 351 for L1C with 8, 18, and 26 alleles, respectively. Because
we designated certain activity categories and there were only three or four activity
categories identified per locus, the many possible genotypes reduced to only 6, 10, and
9 possible activity phenotypes, respectively. Of those possible phenotypes, 66%, 80%,
and 44% per locus corresponded to hot L1 phenotypes, defined as having a bi-allelic
activity >25% that of L1RP . The remaining phenotypes with bi-allelic activity <25%
of L1RP were defined as cool L1 phenotypes. After assigning the activity values to the
allelic variants that each individual carried at L1A, L1B, and L1C loci, we observed that
only 11% (9 of 80), 44% (35 of 79) and 45% (36 of 80) of phenotypes per locus were hot.
For each individual, we added the activity values per locus to obtain the total L1
activity potential (three L1s combined). Figure 3 shows the wide distribution in L1
activity potentials per individual (from 0% to >300%) in the four populations that
we sampled. Of the 80 individuals studied, one was excluded because we could not
isolate his L1B element. For the remaining 79 individuals, 18% did not have a total L1
phenotype that was hot, 56% had a hot phenotype between 25% and 200%, and 26%
had a very hot phenotype that was >200%. Thus, the data suggest that nearly half of
individuals fall at the extremes of distribution of retrotransposition capability of these
elements, suggesting that individuals vary significantly in their risk of a new insertion
during meiosis or during development of their offspring.
To obtain an overall L1 activity potential per population, we added the values from
all individuals in a population and divided by the number of individuals (Fig. 4). We
tested whether the different populations were statistically different in their overall L1
activity potential. There was a >2-fold difference between the relative activity potential
 Human Diversity and L1 Retrotransposon 47

Fig. 3. Combined retrotransposition potential of three hot L1s/individual in four populations.

From 26% (African) to 55% (South American) of individuals per population have a unique L1
activity potential. White, black, and hatched bars represent individuals lacking a hot L1 phenotype
(<25%), having an intermediate L1 activity, and having a high L1 activity (>200%), respectively.
∗ The African distribution is based on 19 individuals

Fig. 4. Average retrotransposition potential of 3 hot L1s in four populations. The total retro-
transposition potential of L1A, L1B, and L1C for each individual was divided by the number
of individuals in the population to determine the average retrotransposition potential in each
population. The means of the four populations are significantly different by an ANOVA test
(p = 0.036)

of the highest (South Americans, 180%) and the lowest group (Asians, 81%). The
hypothesis that all population means are equal was marginally rejected by an ANOVA
test (P = 0.036) with South American and African means differing from those of Asians
48 Kazazian et al.

and Europeans. Note that the variation in L1 activity potential among individuals within
populations is much larger (0–300%) than that among different populations (81–180%),
a result consistent with other human population studies.

A Model for the Evolution of New Insertions and Implications

for the Role of Retrotransposition in Human Diversity
A major conclusion of the human population genetic study - that hot and cool alleles
of active L1s produce extensive individual variation in retrotransposition capability
rests on the proposition that L1 activity in cell culture mirrors L1 activity in vivo
(Moran et al. 1996). It is clear that L1 expression in vivo depends upon a number of
factors not evaluated in the cell culture assay, including chromatin status, presence of
appropriate transcription factors in the appropriate cell type, and DNA methylation,
among others. As a first approximation we asked whether the genomic region into
which the element is inserted allows its expression. L1A is located within an intron
of a gene that is expressed in at least some tissues. For L1B, presence/absence data
in three male genomes analyzed suggest that it may have retrotransposed carrying
a 3’ transduced sequence. L1C does not reside within or close to known or predicted
genes, so it is not known whether this element is expressed. Although we have no proof
that any of the three hot L1s – L1A, L1B, or L1C – is expressed in vivo, four of the
hot L1s – three in this study and the disease-producing hot L1, LRE1 – had common
alleles that demonstrated highly variable retrotransposition activity in cell culture.
These data suggest that the great bulk of hot L1s that are responsible for most in vivo
retrotransposition have both hot and cool alleles.
From this large study of alleles of young L1s currently expanding in the human
genome, we suggest a model for how full-length L1 insertions (about 35% of the total),
evolve in a population (Fig. 5). This work and the fact that nearly all present-day,
disease-causing L1 insertions are hot suggest that new insertions in a population are
derived nearly exclusively from hot L1s. Later, as a new insertion increases in gene
frequency through genetic drift, it also acquires random mutations, some of which
reduce its retrotransposition potential from hot to cool, which is the status of the three
elements we have studied. As the gene frequency of the L1 increases toward fixation,
alleles continue to accumulate mutations that render them either cool or dead for
subsequent retrotransposition.
The substantial individual variation in retrotransposition capability that we have
found for the three hot elements studied raises an important question: would this degree
of individual variation stand if other hot L1s present in the population were included
in the analysis? In other words, do L1A, L1B, and L1C combined constitute a significant
fraction of the hot L1 activity in world populations? We have used our data and those of
Boissinot et al. (2004) to estimate that L1A, L1B, and L1C may account for at least 1/ 3 of
the common hot L1 activity. After other hot L1s are studied in the individuals studied
here and in others, we predict that the proportion of individuals at the extremes of the
distribution of retrotransposition capability will decrease somewhat, but the difference
in retrotransposition potential of individuals at those extremes will increase. Thus,
we conclude that individual variation in retrotransposition activity is an important
contributor to human genetic diversity.
 Human Diversity and L1 Retrotransposon 49

Fig. 5. Model of the evolution of an L1 insertion in a population. Data presented here and evidence
that hot L1s account for most new insertions (Brouha et al. 2003) suggest that new insertions are
derived from hot L1s. Data on alleles of L1A, L1B, L1C, and LRE1 indicate that, after a hot L1
reaches an intermediate gene frequency in the population, it has a significant proportion of cool
alleles. As an L1 approaches fixation, mutations produce cool alleles and dead alleles. Shaded box
= L1 insertion in chromosomes (lines). Black dots = mutations

This conclusion has important implications for the role of retrotransposition in

producing neurological diversity among human beings. First of all, it is difficult to con-
sider retrotransposition as an important evolutionary mechanism to produce diversity
when such a high percentage of the total L1 activity in an individual (at least in cell
culture) is contributed by a very small number of very active L1s (a handful or less).
That is not to say that L1 elements never had a significant role in human diversity. It is
possible that the number of highly active L1s per individual was much greater millions
of years ago, and subsequently decreased due to mutation and population bottlenecks.
Second, if retrotransposition potential is very low in some individuals and very high in
others, it is likely that retrotransposition is an unstable mechanism for effecting change
in behavior or learning. Another population bottleneck could eliminate it entirely. We
conclude that although retrotransposition is an interesting and exciting possibility for
creating diversity, it is unlikely to be a major factor. On the other hand, surprises are
what keep biology interesting, and we are prepared to be surprised!
50 Kazazian et al.

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From the “RNA World” to Brain Complexity:
Generation of Diversity

Alysson R. Muotri1 , Maria C.N. Marchetto1 , and Fred H. Gage1

“To many neuroscientists one pyramidal cell is just like another.

I, on the contrary, believe that it is important to
distinguish the many types (and probably subtypes) of
pyramidal cells. One can often see that two pyramidal
cells look quite different.”
Francis Crick
In The Impact of Molecular Biology on Neuroscience (1999)
“I believe there is little reason to question
the presence of innate systems that are
able to restructure a genome.”
Barbara McClintock
In The Dynamic Genome: Barbara McClintocks’s Ideas
in the Century of Genetics (1992)

Summary

The recent finding that LINE-1 (Long Interspersed Nucleotide Elements-1, or L1)
retroelements are active in somatic neuronal progenitor cells has provided a potential
additional mechanism for generating neuronal diversification. L1 retrotransposition
in the nervous system challenges the idea of static neuronal genomes, adding a new
element for neuronal plasticity. Long dismissed as selfish or “junk” DNA, retroele-
ments are thought to be intracellular parasites from our distant evolutionary past.
Together with their mutated relatives, retroelement sequences constitute 45% of the
mammalian genome, with L1 alone representing 20%. The fact that L1 can retrotrans-
pose in a defined window of neuronal differentiation, changing the genetic information
in single neurons in an arbitrary fashion, could allow the brain to develop in distinctly
different ways. These characteristics of variety and flexibility may contribute to the
uniqueness of an individual brain. However, the extent of the impact of L1 on the
neuronal genome is unknown. In this chapter we will discuss the potential influence of
L1 retrotransposition during brain development and the evolutionary pressures that
may have selected this unexpected machinery of diversity in neuronal precursor cells.
The characterization of somatic neuronal diversification will not only be relevant for
the understanding of brain complexity and neuronal organization but may also shed
1 Laboratory of Genetics, The Salk Institute for Biological Studies, 10010 North Torrey Pines
Road, La Jolla, CA 92037, USA, e-mail: gage@salk.edu

Gage et al.
Retrotransposition, Diversity and the Brain
© Springer-Verlag Berlin Heidelberg 2008
54 A.R. Muotri, M.C.N. Marchetto, F.H. Gage

light on the differences in cognitive abilities, personality traits and many psychiatric
conditions observed in humans.

Introduction

There are several ways to study brain complexity. Perhaps the broadest view is to analyze
the brain’s action or its consequences. However, one could take a physiological approach
and investigate how different regions of the brain produce a specific task. Others may
try to understand the organization and rules of neuronal networks, or how the neural
cells are connected to each other through synapses, in a systematic manner. Then,
there are the cellular and molecular views. For those views, the ultimate characteristics
of a single neuron are present inside the cell and how genes and molecules react to
outside stimuli, from the environment or from the interaction with other cells. This
reductionist approach will certainly not explain how the brain works, but may provide
the necessary tools for understanding how the different levels of organization co-exist
to generate perception, action, feelings, memories and thoughts.
It has been known for more than a century, through the work of Camillo Golgi
and Santiago Ramon y Cajal, that neurons are specialized cells with a huge diversity
of shapes and connections. It is estimated that the human brain contains more than
10 000 different morphological types of neurons. However, neuronal diversity cannot
be defined only by morphology or anatomic position. Similar cells, located at the same
brain region, may have distinct electrophysiological properties and unique connection
within other neurons. Moreover, neurons are extremely plastic cells, allowing extraor-
dinary response upon micro and macro environmental stimulation. Any attempt to
understand how the brain works must take into account this huge neuronal diversity.
Such diversity is likely the reason why each one of us is unique; even genetically iden-
tical twins have different preferences or opinions. But the fundamental mechanisms
by which neural stem cells produce such a variety of neuronal types are slowly being
revealed.
In contrast to the single mechanism for the production of antibodies (VDJ re-
combination) in the immune system, several molecular mechanisms contribute to
the generation of neuronal diversity (Muotri and Gage 2006). Those mechanisms not
only act on the DNA, but also act on the RNA and protein levels, allowing epigenetic
modifications to take place. Among these mechanisms, alternative splicing, promoter
usage, alternate polyadenylation, RNA editing and post-translation modifications are
all part of the genetic tool box present in neuronal precursor cells. However, even
such a repertoire is not enough to justify the observed constellation of neuronal types.
New mechanisms are likely to be uncovered. We anticipate that novel strategies for the
neuronal diversity contribution are hidden in non-coding regions of the genome (Cao
et al. 2006; Muotri and Gage 2006). We have recently shown that an engineered human
L1 element can retrotranspose in neuronal precursor cells, changing neuron-related
gene expression, which, in turn, can influence neuronal cell fate in vitro (Muotri et al.
2005). Moreover, because L1 retrotransposition can also occur in the CNS neuropro-
genitor throughout embryo development and in the adult brain of transgenic mice, this
unexpected mechanism may contribute to neuronal diversity.
 From the “RNA World” to Brain Complexity: Generation of Diversity 55

The L1 retrotransposition causes a neuronal genetic mosaicism, i.e., the presence

of more than one genetically distinct neuronal type. Such mosaicism might be unde-
tectable unless closely inspected. In fact, genetic mosaicism is frequently overlooked
or interpreted as normal variation caused by stochastic developmental factors or the
unequal influence of the environment. However, depending on the mosaic nature,
frequency, environmental cues, and tissues of origin, even subtle alterations in gene
expression can contribute to detectable phenotypic alterations in the organism. Nor-
mal processes, such as aging, the generation of immune diversity, and the phenotypic
variability between monozygotic twins (such as schizophrenia) can be due to somatic
genetic mosaicism (Dipple and McCabe 2000; Machin 1996; Vijg 2000). The stochastic
nature of retrotransposon activity, and the large number of genes that this process
may affect, could produce an ample spectrum of neuronal diversity, which may affect
behavior, cognition and disease risk.

Silencing and Activation of L1 Retrotransposons

L1 retrotransposons can threaten the structure and regulate the expression of the
genome in different ways, such as creating new splicing forms, promoter activation,
skipping exons or gene inactivation among others (Gilbert et al. 2005; Kazazian 2004).
Such a variety of strategies makes L1 retrotransposons the most creative force shaping
the genomes throughout evolution.
Deleterious retrotransposition events in the germ line or in early development have
resulted in a variety of genetic disorders, and a somatic L1 retrotransposition in man has
resulted in a sporadic case of colon cancer (Kazazian 1998; Miki et al. 1992; Ostertag and
Kazazian 2001). In plants and other organisms in which transposition is not restricted
to the germ line, somatic activity of transposable elements provides the opportunity
for a phenotypic variability that can sometimes be stunning with regard to individual
genome flexibility (Lisch 2002). In contrast, retrotransposons are often assumed to be
silenced in mammalian somatic tissues. This assumption is based on several arguments.
First, there is no detectable level of retrotransposon expression in most somatic tissues.
However, only a few tissues have been subjected to meticulous analysis, including
subtype cell differences. Second, the somatic silencing of L1s fits well in the “selfish”
DNA hypothesis, where the mobile elements exist merely to propagate themselves,
so there is no reason to transpose in somatic cells. Finally, there is a clear detection
bias of somatic retrotransposition, since only visible mutants, usually leading to human
diseases, such as cancer, are detectable (Kazazian 2001; Kazazian et al. 1988). The lack of
experimental data and the paucity of natural evidence for somatic L1 retrotransposition
have led to the view that L1 activity is restricted to early embryonic and germ line
cells, suggesting that intrinsic factors may be present or absent in certain cell types
responsible for transposition (Mathias and Scott 1993; Prak et al. 2003). Nonetheless,
retrotransposon silencing could be physiologically attenuated. DNA methylation is
likely the most effective and global strategy against retrotransposon mobility (Yoder
et al. 1997). Accordingly, DNA methyltransferase-1 (Dmnt1)-deficient mouse embryos
have much higher levels of IAP (Intracisternal A-particle) retrotransposon transcripts
than their wild-type littermates (Walsh et al. 1998). Repression of retrotransposition
is removed under definite conditions during a specific developmental window. One
56 A.R. Muotri, M.C.N. Marchetto, F.H. Gage

example is the specific induction of IAP elements in the stem cells of the male germ
line at undifferentiated stages when they are de-methylated, leading to the hypothesis
that a similar mechanism may be found in somatic tissues.
One useful approach to track somatic retrotransposition is the analysis of the
L1-EGFP transgenic animal (Muotri et al. 2005; Prak et al. 2003). These mice were
engineered to carry an active L1 retrotransposon with an EGFP indicator cassette that
only expresses EGFP after retrotransposition and de novo insertions (Fig. 1). Because
the assay uses the strong and ubiquitous CMV promoter, it is expected to express EGFP
in a large spectrum of somatic cells, if retrotransposition indeed occurs. Obviously, the
system will not detect truncated or silenced insertions of the reporter cassette. Of the
several somatic tissues analyzed by immunohistochemistry, brain tissue was the only
tissue where EGFP expression was detected, specifically in neurons (Muotri et al. 2005).
The in vitro cellular assay indicated that L1 retrotransposition actually happened in
precursor cells rather than postmitotic neurons. Therefore, neuronal precursor cells
might have a greater frequency of L1 retrotransposition than other cell types and/or
this finding might be due to the long life of neurons, in contrast to the continuous
renewal of other cell types. Either way, the presence of EGFP-positive cells indicates
that somatic L1 silencing is incomplete in the brain. This observation suggests that
L1 retrotransposons might be activated in neuronal precursor cells and the resultant
retrotransposition events could alter the expression of neuronal genes. Such a mech-
anism could, presumably, generate a large spectrum of genetically distinct neurons,
adding to the great neuronal variation that is currently observed in the adult CNS. L1
activation is likely regulated by host factors in equilibrium: too much L1 retrotranspo-
sition can cause cell damage and induce the cells to die (Haoudi et al. 2004); too little
can limit neuronal diversity. The identification of neuronal host factors responsible
for L1 repression and/or activation will be extremely important to understanding how
retrotransposition is regulated.
L1 expression is dependent on the activation of its own 5’UTR sequence, which acts
as a promoter. The human L1 5’UTR is about 1 kb long, harboring one YY1-binding site
that is required for proper transcriptional initiation (Athanikar et al. 2004; Swergold
1990), two Sox (sex determining region of Y-chromosome, SRY, related HMG-box)
binding sites (Tchenio et al. 2000) and a runt-domain transcription factor 3 (RUNX3)
binding site (Yang et al. 2003). Interestingly, none of these factors is germ-cell spe-
cific, suggesting the presence of other, unknown factors. Sox proteins are expressed in
a variety of tissues, including neural stem cells (NSCs) and testis (Wegner 1999). The
lack of Sox2 allowed activation of neuronal genes and differentiation, suggesting that
Sox2 may function as a repressor of differentiation in NSCs (Graham et al. 2003). We
demonstrated that a decrease in Sox2 expression during the early stages of neuronal
differentiation is correlated with an increase in both L1 transcriptional activity and
retrotransposition (Muotri et al. 2005). We propose that L1 retrotransposons are si-
lenced in NSCs due to Sox2-mediated transcriptional repression. Down-regulation of
Sox2 accompanies chromatin modifications, such as DNA de-methylation and histone
acetylation, which may trigger neuronal differentiation (Fig. 2). Such a mechanism
preserves genetic stability in NSCs but allows instability to happen in neuronally com-
mitted cells.
 From the “RNA World” to Brain Complexity: Generation of Diversity 57

Fig. 1. Detection of L1 retrotransposition in the brains of transgenic mice. The structure of the
L1RP -EGFP transgene is indicated at the top of the figure. The retrotransposition-competent
human L1 (L1RP ) contains a 5’ untranslated region (UTR) that harbors an internal promoter,
two open reading frames (ORF1 and ORF2; not drawn to scale), and a 3’ UTR that ends in
a poly (A) tail. The EGFP retrotransposition indicator cassette consists of a backward copy of the
EGFP gene whose expression is controlled by the human cytomegalovirus major immediate early
promoter (pCMV) and the herpes simplex virus thymidine kinase polyadenylation sequence
(pA). This arrangement ensures that EGFP expression will only become activated upon L1
retrotransposition. The black arrows indicate PCR primers flanking the intron present in the
EGFP gene
58 A.R. Muotri, M.C.N. Marchetto, F.H. Gage

Fig. 2. A model for generation of neuronal diversity by L1 retrotransposition. In neural stem

cells, Sox2 expression is correlated with a repression of L1 retrotransposons and neuronal genes.
During early phases of neuronal differentiation, there is a reduction in the expression of Sox2
and other neuronal stem cell genes. As a result, L1 transcription can be activated, allowing
subsequent retrotransposition into neuronal genes such as for the Psd-93 gene. The resulting
retrotransposition events can alter gene expression, which, in turn, can influence the phenotype
of the resulting cell. The functional variability in gene expression induced by L1 retrotransposition
could also contribute, in principle, to the high cell death rate observed in adult neurogenesis,
where only a few newly born neurons successfully integrate into the pre-existing neuronal network
 From the “RNA World” to Brain Complexity: Generation of Diversity 59

L1 Targets in Neuronal Progenitor Cells

To cause a significant impact on neuronal genomes, new L1 insertions must target

important regulatory regions or genes that are being expressed at the moment of neu-
roblast differentiation. It is likely that only the combination of multiple L1 events, and
not an eventual catastrophic insertion in single neurons, will be ultimately responsible
for any change in the neuronal network. But L1 retrotransposition is a dangerous situ-
ation for the cell, since L1 insertions can hit essential genes that may induce cell death
or even target oncogenes, leading to a neoplastic transformation.
Despite the low number of examples, the sequence data from target insertional
sites in rat neuroblasts were often close to or inside neuron-associated genes (Muotri
et al. 2005). Even with a small sample, two L1 insertions were located in the same
gene, indicating that the integration process might not be completely random. Some
of these target genes included an olfactory receptor, ion channel-associated genes and
a cadherin receptor (Muotri et al. 2005). An L1 insertion in the promoter region of
the Psd-93 gene, encoding a post-synaptic density protein involved in different aspect
of synapse formation, significantly increased gene expression level and, consequently,
accelerated neuronal maturation in culture. Despite the fact that randomness seems to
be the best way for L1 to survive during evolution when they are active in germ cells,
somatic insertions might be controlled by local microvariations in DNA chromatin
structure that depend on different host factors in specific subsets of cell types. Thus,
we propose that L1 insertions in the nervous system are somehow guided to specific
gene targets. In a similar way, the yeast Ty1 transposon is highly nonrandom in vivo,
being preferentially inserted upstream of tRNA genes (Bachman et al. 2004; Devine
and Boeke 1996).
In the L1-EGFP transgenic mice, we followed the retrotransposition of a single
human L1 element and retrotransposition was detected by EGFP expression. However,
the indicator cassette did not reflect a direct measurement of the 3 000 estimated en-
dogenous active L1 retrotransposons (DeBerardinis et al. 1998; Goodier et al. 2001).
Moreover, as pointed out before, the L1 retrotransposition assay did not report EGFP-
truncated or silenced insertions. Additionally, it certainly did not account for the
indirect, in trans, L1-mediated insertions of Alus, retrotransposition-defective L1s,
and other non-autonomous RNAs. Virtually any RNA molecule can be subject to retro-
transposition if hijacked by L1 machinery. Thus, every single developing neuron can
potentially carry L1-mediated events, and if part of the resultant insertions occurs
in genes expressed during neuronal development, altering gene expression, then it is
possible that brain development could be significantly affected by L1 retrotransposi-
tion. It has been proposed that stochastic gene expression might be a fundamental
part of development and differentiation and, where it is advantageous, these stochastic
patterns are retained in the adult organism (Fiering et al. 2000). We speculate that these
new L1 retrotransposition events are stably integrated into the genomes of individual
neurons during the entire life of the organism. These insertions then act in a stochastic
fashion, working as “controlling elements,” fine-tuning to increase the probability that
genes will be differentially transcribed. The model is consistent with neuroblast differ-
entiation, in which similar cells are subjected to the same environmental stimuli but
do not respond uniformly. Thus, new insertions in neurons represent genomic “scars”
60 A.R. Muotri, M.C.N. Marchetto, F.H. Gage

that may have the potential to influence the fate of the resultant cells and, consequently,
the function of the neuronal network.
The study of the human L1 5’UTR promoter during neuronal differentiation re-
vealed that L1 activation occurs in the initial stages of cell differentiation. That is
exactly the same time that several neuronal genes, such as NeuroD1, are upregulated
and several cell cycle genes are downregulated (Hsieh et al. 2004; Zhao and Gage 2002).
Additionally, the strong anti-mitotic small modulatory NRSE dsRNA, responsible for
the neuronal fate of NSCs, is expressed in initial steps of differentiation, activating
several NRSE-containing neuron-specific genes and stopping the cell cycle (Kuwabara
et al. 2004). These data suggest that there is an orchestrated regulation during neu-
ronal differentiation, avoiding an eventual cell transformation. Such an idea conforms
with the low incidence of neuroblastomas (Zhu and Parada 2002) but does not ex-
clude the possibility that an abnormal L1 retrotransposition might lead to a neoplastic
transformation in CNS cells.
Taken together, a specific regulation of L1 retrotransposon activity that takes into
account its “non-random” neuronal insertion and a specific window of time during
cell differentiation may turn a potentially harmful phenomenon into a useful one.
The problem now, as with most novel scientific debates, is one of quantification and
significance. Future technologies for single-cell endogenous L1 activity assays will bring
new insights into the problem. Moreover, the generation of three-dimensional brain
mapping depicting the occurrence of L1 retrotransposition will allow the visualization
of preferential target neuronal subtypes. The comparison of normal brains with brains
where L1 activity is misregulated will provide the structural organization for the design
of algorithms that predict eventual retrotransposition-affected neuronal networks or
systems.

Evolutionary Consequences of L1 Impact in Neuronal Genomes

One of the most remarkable findings from the sequencing of the human genome is
that retrotransposable elements make up a significant portion of the human DNA
(Deininger et al. 2003). Based on reverse transcriptase (RT) phylogeny, L1 elements are
most closely related to the group II introns of mitochondria and eubacteria (Cavalier-
Smith 1991; Xiong and Eickbush 1990). These studies revealed that the RT enzyme is
extremely old and that retroelements can be viewed as relics or molecular fossils of
the first primitive replication systems in the progenote. The origin of retroelements
possibly traces back to the conversion of RNA-based systems, the “RNA World” (Orgel,
2004), to modern “DNA-based” systems. Current models suggest that these mobile
introns of eubacteria were transmitted to eukaryotes during the initial fusion of the
eubacterial and archaebacterial genomes or during the symbiosis that gave rise to
the mitochondria, generating the modern-day spliceosomal introns (Zimmerly et al.
1995). Further acquisition of an endonuclease enzyme and a promoter sequence cer-
tainly represented important steps in the evolution of L1 retrotransposons, providing
autonomy for L1s to insert into many locations throughout the genome.
The apparent lack of obvious function of retroelements in the genome suggests
that transposable elements are “selfish DNA,” acting as parasites in the genome to
 From the “RNA World” to Brain Complexity: Generation of Diversity 61

propagate themselves. This idea has long puzzled scientists and inspired the con-
cept of “junk DNA” to illustrate the idea that such sequences were mere evolutionary
remnants (Doolittle and Sapienza 1980; Orgel and Crick 1980). However, the recog-
nition that retrotransposons can actively reshape the genome is slowly challenging
this terminology. Moreover, the mammalian genome has suffered waves of transpo-
son bombardment, but the constant, single lineage of L1 history reveals that active
L1 were never absent from mammals’ genomes during evolution, suggesting an in-
extricable link between L1 and their hosts (Furano et al. 2004). The relationship be-
tween transposons and their hosts is probably not entirely antagonistic, as several
host genes have a high degree of homology to one or more transposable elements.
Evidence in the literature points to a somatic function for L1 transcripts, involving
cell proliferation (Kuo et al. 1998), differentiation (Mangiacasale et al. 2003) and early
embryo development (Pittoggi et al. 2003). Moreover, it is difficult to reconcile why
the genome would need so many copies of retrotransposons and whether this ex-
pansion has any correlation with retrotransposition itself. The restricted activity of
retrotransposons in germ or early embryonic cells apparently fits well with the “self-
ish DNA” concept, since new insertions will be passed to the next generations, but
somatic insertions pose a conundrum. According to the symbiotic theory, it is advan-
tageous to any transposable element to promote host mating, securing the propagation
of the “master” elements to the next generations. From this perspective, it is not
surprising that advantageous insertional events in the brain, resulting in the better
(cultural and social) fitness of the individual organism, also can contribute to the host
mating.
The evolution of the CNS provided a notable selective advantage, as information
about the environment could be processed rapidly and would allow organisms to more
readily meet the challenges of ever-changing environmental conditions. Moreover, epi-
genetic modification allowed the non-genetic transfer of information or transmission
of “culture” at an unprecedented magnitude. Such specialization is highly dependent
on the cognitive levels acquired by the species that are directly linked to the complexity
of the neuronal network. Therefore, the advantages gained by retaining the mecha-
nisms for somatic retrotransposition may outweigh the cost of a less plastic nervous
system. In fact, such a strategy expands the number of functionally distinct neurons
that could be produced from a given stem cell gene pool (Muotri and Gage 2006). This
characteristic of variety and flexibility may contribute to the uniqueness of an individ-
ual brain, even between genetically identical twins. Mobile elements in the brain may
be part of the conserved core process responsible for evoking the facilitated, complex,
non-random phenotypical variation on which selection may act. It is remarkable to
imagine that the brain is a consequence of ancient retrotransposition in eukaryotic
cells. Such a possibility has not been considered before, but it was suggested to us by
the experimental results.
The identification of L1 elements as potential creative somatic shapers of transcrip-
tional complexity in neuronal genomes may be an important phenomenon for develop-
mental neurosciences. The hypothesis that L1 activity is responsible for “fine-tuning”
neuronal wiring waves requires the merger of different fields and may consequently
open new ways of considering individual differences and the neuronal correlates of
human cognition. Rigorous experimental proof of this model will require attenuation
of retrotransposition activity from the mammalian genome and comparing their be-
62 A.R. Muotri, M.C.N. Marchetto, F.H. Gage

havior to that of wild type animals. Nonetheless, the experimental approach presents
a major methodological challenge for molecular biologists, since a canonical single-
gene knockout strategy is no longer suitable. On the other hand, the study of abnormal
activation of L1 retrotransposition in the brain may elucidate complex neurological
syndromes, permitting an understanding of diseases at a different level.

Acknowledgements. A.R.M is supported by the Rett Syndrome Research Foundation, M.C.N.M.

is supported by the George E. Hewitt Foundation for Medical Research and F.H.G. is supported by
the Lookout Fund and the National Institutes of Health: National Institute on Aging and National
Institute of Neurological Disease and Stroke.

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Endogenous Retroviruses
and Human Neuropsychiatric Disorders

Robert H Yolken2 , Håkan Karlsson1 , Ioannis Bossis2 , Linnéa Asp1 , Faith Dickerson3 ,
Christoffer Nellåker1 , Michael Elashoff 4 , Elizabeth Rubalcaba2 , and Raphael P. Viscidi2

Summary

Schizophrenia and related disorders are devastating human neuropsychiatric disorders

of complex etiology. Epidemiological and family studies indicate both genetic and
environmental contributions to disease etiology. We propose that the altered expression
of endogenous retroviruses such as HERV-W contribute to some cases of schizophrenia.
We present both theoretical considerations and experimental evidence supporting
this association. The further study of endogenous retroviral expression within the
central nervous system might lead to new methods for the diagnosis and treatment of
schizophrenia and related disorders.

Introduction

Schizophrenia is a pervasive neuropsychiatric disorder with worldwide prevalence. The

characteristic features of schizophrenia are “positive” symptoms of hallucinations or
delusions that are often accompanied by “negative” symptoms, such as emotional with-
drawal or amotivation. There is a great deal of individual variation in the symptoms of
schizophrenia, its clinical course, and response to medication. Pharmacological treat-
ments may reduce the severity of symptoms, but their effects are typically incomplete.
The effects of schizophrenia are devastating for the affected individuals, their families,
and society at large. In most areas of the world, schizophrenia is a major cause of
premature death, homelessness, and suicide (Jablensky 2000). Because schizophrenia
generally has the onset of symptoms in early adulthood and persists for life, it is associ-
ated with a high burden of disease (Rossler et al. 2005). According to the World Health
Organization, schizophrenia is the eighth leading cause of lost life years worldwide
in individuals 15–44 years of age (WHO World Health Report 2001). Schizophrenia is
also associated with a number of non-psychiatric medical conditions, such as heart
disease (Davidson 2002), diabetes (Zimmet 2005), metabolic syndrome (Meyer et al.
2005), and other autoimmune disorders (Eaton et al. 2006). Schizophrenia also shares
epidemiologic and phenotypic features with other serious neuropsychiatric disorders,

1 Department of Neuroscience, Karolinska Institutet, Retzius v 8, 17177 Stockholm, Sweden

2 Stanley Laboratory of Developmental Neurovirology, Johns Hopkins University School of
Medicine, Baltimore, MD, USA, e-mail: yolken@mail.jhmi.edu
3 Sheppard Pratt Health System, Baltimore, MD, USA
4 Stanley Medical Research Institute, Chevy Chase, MD, USA

Gage et al.
Retrotransposition, Diversity and the Brain
© Springer-Verlag Berlin Heidelberg 2008
66 R.H. Yolken et al.

including bipolar disorder (Maier et al. 2005; Moller 2003). There are currently no reli-
able laboratory diagnostic assays for the diagnosis of schizophrenia, the determination
of disease progression or the monitoring of response to medications. Consequently,
there is a pressing need for an increased understanding of the etiopathogenesis of
schizophrenia and for the development of new methods for diagnosis and therapy.
Current evidence suggests that schizophrenia is the result of both genetic and
environmental factors. Family and adoption studies have indicated a strong genetic
contribution to schizophrenia risk. Individuals who have a first-degree relative with
schizophrenia have an approximately 10-fold increased risk of schizophrenia as com-
pared to the general population. Individuals who have a monozygotic twin with
schizophrenia have an approximately 50-fold increased risk (Maki et al. 2005). How-
ever, it is notable that many individuals with schizophrenia do not have a first-degree
relative with schizophrenia and that, on a population basis, having a first-degree rel-
ative with schizophrenia does not contribute greatly to the attributable risk for this
disorder (Mortensen et al. 1999).
Based on the familial association with schizophrenia, a large number of studies
have attempted to find disease-associated genes. These studies have employed a wide
variety of genetic techniques, including family-based linkage analysis (Hovatti et al.
1998), transmission disequilibrium (Li et al. 2001), sib-pair analysis (Schwab et al.
2005), and case-control studies of single nucleotide polymorphisms (Chen et al. 2004).
Such studies have identified a large number of genetic regions and specific genes that
appear to be associated with increased risk of schizophrenia in some populations.
However, to date no genes of major effect (relative risk or odds ratios (>2.0)) have
been found for schizophrenia or the related disorders (Delisi and Fleischhaker 2007).
The genes that have been found generally have lower relative risks, are inconsistent
among populations, and are shared among psychiatric diagnoses (Gilbody et al. 2007).
Attempts to correlate results in different populations have also been hampered by
complex haplotype structure leading, in some cases, to the overestimation of the
concordance of genetic findings across populations (Mutsuddi 2006).
Because of the limitations of genetic studies, there has also been interest in the
identification of environmental factors that might modify gene expression. The role
of environmental factors is consistent with epidemiological studies indicating vari-
ation in the rate of schizophrenia in terms of season of birth, year of birth, urban
birth, and location within a relatively small geographic area (Kirkbride et al. 2006).
Increased risk of schizophrenia has also been associated with complications of preg-
nancy, particularly pre-eclampsia, which has been associated with a 2.5-fold risk of
increased schizophrenia in the offspring (Dalman et al. 1999). It is of note that these
epidemiological factors point to events occurring early in life. These exposures are thus
consistent with the concept that changes in the brain in early life may be associated
with abnormalities occurring in adolescence or young adulthood. The reasons for the
expression of the symptoms of schizophrenia in late adolescence or early adulthood
despite exposures in early life are not known with certainty, but they might be related
to neurodevelopmental changes in brain structure occurring during childhood and
adolescence (Lewis and Levitt 2002), the effects of hormonal activation (Stevens 2002),
the long-term consequences of the immune response to infection (Wright et al. 1993)
or the reactivation of latent infectious agents (Torrey 1988).
 Endogenous Retroviruses and Human Neuropsychiatric Disorders 67

While there are a number of environmental factors that might contribute to the
risk of schizophrenia, most research has been directed at the possible role of infectious
agents. In light of the above findings, studies of specific infections have focused on ones
which might occur in pregnancy or early life. Specific infectious agents that have been
associated with increased risk of schizophrenia following exposure in early life include
Toxoplasma gondii (Mortensen et al. 2006), herpes simplex virus type 2 (Buka et al.
2001), rubella virus (Brown and Susser 2002), influenza viruses (Brown et al. 2004),
enteroviruses (Rantakallio et al. 1997), and agents that cause bacterial meningitis
(Abrahao et al. 2005). In addition, increased levels of antibodies to infectious agents
have been found in adults with schizophrenia as compared to controls in different
populations. These agents include Toxoplasma gondii, cytomegalovirus, Epstein Barr
virus (Behr et al. 2006; Delisi et al. 1986; Leweke et al. 2004) Borna disease virus (Bode
et al. 2001) and Borrelia burgdorferi (Fallon and Nields 1994). In addition, DNA from
chlamydial organisms has been found in the blood of individuals with schizophrenia in
increased levels as compared to controls (Fellerhoff et al. 2007). Herpes simplex virus
Type 1, while not associated with an increased risk of schizophrenia, has been associated
with cognitive impairment and structural alterations in the brains of individuals with
schizophrenia (Dickerson et al. 2003; Prasad et al. 2007).
Numerous other agents have been associated with acute psychotic events similar to
those that occur in individuals with schizophrenia. These include human immunodefi-
ciency virus type 1 (Koutsilieri et al. 2002; Atlas et al. 2007), tick-borne encephalitis virus
(Chlabicz et al. 1996), hepatitis B virus (Weber et al. 1994), Taenia solium (Meza et al.
2005), plasmodia (Tilluckdharry et al. 1996; Alao and Dewan 2001) and other parasitic
agents (Singh and Singh 2000) capable of infecting the central nervous system (CNS).
The most consistent association between a specific infectious agent and risk of
schizophrenia has been with the apicomplexan organism, Toxoplasma gondii. A recent
meta-analysis of 23 independent studies from different areas of the world found that
serological evidence of Toxoplasma infection is associated with an increased risk of
schizophrenia with an odds ratio of approximately 2.7 (Torrey et al. 2007). Toxoplasma
antibodies in pregnancy have also been associated with increased risk of schizophrenia
in later life in two independent studies (Mortensen et al. 2006; Brown et al. 2005). It
is of note that Toxoplasma infection also causes altered behavior in animal model
systems and that this behavior can be normalized by treatment with anti-Toxoplasma
medications (Webster et al. 2006).
Increased levels of cytomegalovirus infection have also been found to be associ-
ated with schizophrenia in several different adult populations (Leweke et al. 2004).
Cytomegalovirus DNA has also been identified in the brain tissue of a small number of
individuals with schizophrenia (Moises et al. 1988). The associations between the other
infectious agents and the risk of schizophrenia have been more variable with relatively
low odds ratios and differences across populations.

Endogenous Retroviruses and Schizophrenia:

Theoretical Considerations
Human endogenous retroviruses (HERV) are assumed to be remnants of ancient retro-
viral infections of our ancestors’ germ-line cells. HERV sequences constitute approx-
68 R.H. Yolken et al.

imately 3–8% of the human genome and can be classified into at least 31 families.
Tissue-specific hybridization patterns to arrays of sequences representative of different
HERV families was recently reported, indicating a discrete and diversified regulation of
their transcriptional activities. Based on the evolving understanding of both endoge-
nous retroviruses and schizophrenia, we have postulated that endogenous retroviruses
contribute to the etiopathogenesis of some cases of this disorder. The specific hypothe-
ses are as follows:
1. Some cases of schizophrenia are related to the aberrant expression of endogenous
retroviruses within the CNS.
2. Individual differences in the response to endogenous retrovirus expression are
related to
a) polymorphisms within the endogenous retroviruses and adjacent genomic
sequences;
b) exposure to infectious agents and other environmental factors relating to
endogenous retrovirus interaction in utero and in later life; and
c) genetic variation in small molecule transport molecules within the CNS, some
of which also function as retrovirus receptors.

Findings relating to the epidemiology and pathogenesis consistent with a possible

role for endogenous retroviruses include the following:

1. the widespread distribution of endogenous retroviruses in the human genome is

consistent with the linkage of schizophrenia risk to many different genomic regions
and complex haplotype structure (Mutsuddi et al. 2006; Delisi and Fleischhaker
2007);
2. the fact that endogenous retroviruses can be activated by infecting viruses or
protozoa is consistent with the role of these infectious agents in the pathogen-
esis of schizophrenia, as discussed above. Specifically, endogenous retroviruses
have been shown to be activated by Toxoplasma gondii (Frank et al. 2006) and
cytomegaloviruses (Nelson et al. 1999), as well as by other human herpes viruses
(Christensen et al. 2007; Hsiao et al. 2006);
3. aberrant expression of human endogenous retroviruses has been associated with
autoimmune disorders (Anthony et al. 2006; Colmegna and Garry 2006), which
are also common in individuals with schizophrenia (Strous and Shoenfeld 2006);
4. human endogenous retroviruses are widely expressed in the human placenta dur-
ing fetal development, a critical time for the action of environmental factors in
schizophrenia (Malassine et al. 2005; Rote et al. 2004). Of particular interest is
the possible relationship between HERV expression and preeclampsia (Lee et al.
2001), in light of the relationship between preeclampsia and subsequent risk of
schizophrenia;
5. many endogenous retroviruses are active in the human CNS (Weis et al. 2007a)
and are differentially expressed in neuroinflammatory disorders (Anthony et al.
2006; Mameli et al. 2007); and
6. some endogenous retroviruses employ neuroactive molecules as receptors. For
example, the receptor for HERV-W is the ASCT1/ASCT2 family of neutral amino
acid transporters, which are responsible for the transport of excitatory amino acids
within the CNS (Marin et al. 2003; Lavillette et al. 2002). Levels of ASCT1 have been
 Endogenous Retroviruses and Human Neuropsychiatric Disorders 69

found to differ in the brains of individuals with schizophrenia as compared to

controls (Weis et al. 2007b).

It is of note that long interspersed nuclear elements (LINEs) and other retroelements
fulfill some, but not all, of these criteria relating to an elevated risk of developing
schizophrenia.
This review will present both previously published and new data relating to the
potential role of endogenous retroviruses in the etiopathogenesis of schizophrenia and
related disorders. This review will focus largely on the HERV-W family of retroviruses,
since this group has been the subject of the largest number of studies relating to
schizophrenia etiopathogenesis.

Altered HERV-W Expression in Schizophrenia

Using degenerate primers directed towards the retroviral pol gene (Tuke et al. 1997),
we investigated cerebrospinal fluids (CSFs) obtained from patients during their first
hospitalization for schizophrenia or schizoaffective disorder for the presence of retro-
viral RNA. Approximately one third of the samples tested positive by polymerase chain
reaction (PCR). Sequencing of each of these products verified that they were of retro-
viral origin and similar to endogenous retroviruses present in the human genome
(HERV; Karlsson et al. 2001). The majority of the sequences showed a high degree
of similarity to sequences previously identified in the CSF of patients suffering from
multiple sclerosis (MS), denoted MS-associated retrovirus (MSRV; Perron et al. 1997).
Probes derived from MSRV sequences were subsequently used to identify a novel fam-
ily of HERV denoted HERV-W (Blond et al. 1999). Sequences similar to HERV-W were
reported to be differentially present in the serum of MS patients (Garson et al. 1998)
as well as in plasma from recent-onset schizophrenia patients (Karlsson et al. 2004) as
compared to control individuals. More recently, Huang and coworkers (2006) detected
retroviral RNA, as well as antibody reactivity, in one third of patients with recent-onset
schizophrenia but not in control individuals. These sequences were also of endogenous
origin with the highest reported similarity to ERV9.
Based on these findings, we have directed additional studies toward the analysis
of HERV-W. According to Pavlicek et al. (2002), the human genome contains in ex-
cess of 600 HERV-W elements, the majority of which are long terminal repeat regions
(LTR) lacking internal sequence (gag, pol and env genes). The remaining elements
have been classified into two major categories: a total of 77 retroelements with provi-
ral structure containing intact LTRs and complete or partial internal sequences; and
149 pseudoelements with internal sequences, lacking the regulatory U3 region of the
5’-LTR and the U5 region of the 3’-LTR. Structurally these copies resemble retroviral
mRNAs and are thought to originate from LINE-mediated reverse transcription of such
mRNAs. The remaining elements were grouped together in a third category based on
the absence of the diagnostic regions (Pavlicek et al. 2002). Since these latter groups
lack regulatory promoter regions, they were suggested to be non-transcribed (Costas
2002; Pavlicek et al. 2002). Whether this is actually the case has, however, not been
investigated. Indeed, very little information exists regarding the expression and tran-
scriptional control of individual HERV-W elements. This gap in knowledge is in part
70 R.H. Yolken et al.

explained by the notion that such non-coding RNAs represent transcriptional noise and
therefore lack biological relevance but also by the methodological challenges associated
with studies of transcripts from large numbers of closely related sequences. Although
large-scale studies of transcripts from the different HERV families have been conducted
by means of hybridization to arrays of representative sequences (Seifarth et al. 2005)
or by use of degenerate primers and probes in real-time PCR assays (Forsman et al.
2005), the individual elements are not resolved by such methods.

Identification of the Source of HERV-W Transcripts

In a recent report, we used sequence-dependent variations in melting temperatures of

double-stranded real-time PCR products for studying expression of HERV-W elements
(Nellaker et al. 2006). This approach has recently been employed for strain identification
in clinical and veterinary virology (Pham et al. 2005; Waku-Kouomou et al. 2006), typing
of mycoplasma strains (Harasawa et al. 2005), genotyping of HLA variants (Graziano
et al. 2005), detection of translocations in cancers (Bohling et al. 1999) and scanning
for single nucleotide polymorphisms (Germer and Higuchi 1999).
By this approach, in combination with extensive sequencing of the different prod-
ucts, we reported cell line-specific transcription patterns of genomic regions harboring
HERV-W elements during base-line conditions. We also reported that not only herpes
simplex type 1 virus, but also influenza A virus infection of different human cell lines
results in significantly increased levels of such transcripts. In each of the different cell-
lines, the virus infection induced a unique response in terms of transactivated elements
(see Fig. 1). Mapping of these elements indicated a basal and regulated expression of
not only elements with a proviral structure but also elements lacking the regulatory

Fig. 1. Frequency distri-

bution plot of melting
temperatures represent-
ing variations in sequence
of amplified HERV W gag
sequences. Sequences am-
plified from the cell lines
investigated; CCF-STTG1,
293F and U937 were com-
pared between control
cells and cells exposed to
influenza A/WSN/33 virus
or serum deprivation.
Temperature ranges indi-
cate discrete temperature
categories representing
distinct sequences
 Endogenous Retroviruses and Human Neuropsychiatric Disorders 71

LTR regions. Serum deprivation induced a similar, but not identical, response in all
the cell lines investigated. Taken together, these findings suggest the existence of in-
trinsic mechanisms governing the expression of genomic regions harboring HERV-W
elements that can be further explored by methods of sufficient discriminatory power.
To further improve on the resolution of the analysis of melting temperatures de-
scribed above, we developed a molecular beacon to control for the inherent variations
in temperature in the heat-block of most thermocyclers. Used in combination with an
algorithm for the identification of the normalized melting temperature, we reported
an improved resolution of melting temperature analyses on the ABI Prism 7000 system
(Applied Biosystems) by approximately three-fold and elimination of the systematic
errors introduced by the instrument (Nellaker et al. 2007). We are currently applying
this method along with element-specific assays (Yao et al. 2006) to examine the ge-
nomic origin and functional significance of the differentially expressed elements in
individuals with schizophrenia.

Syncytin

One HERV-W element on chromosome 7q21 contains an intact env gene encoding an
envelope protein that has been denoted syncytin that has a proposed role in placenta
biogenesis (Blond et al. 2000; Mi et al. 2000). This proviral element, in the ERVWE1
locus, appears to be a bona fide gene domesticated during human evolution (Mallet et
al. 2004). Syncytin is expressed at the feto-maternal interface (Malassine et al. 2005),
an area characterized by immunological conflict between mother and fetus. Elsewhere,
expression of syncytin is very limited during normal conditions. Interestingly, aberrant
expression of this particular env gene has previously been associated with preeclampsia
(Chen et al. 2006; Keith et al. 2002; Knerr et al. 2002; Lee et al. 2001), multiple sclerosis
and motor neuron disease (Oluwole et al. 2007).
Studies of postmortem brains have indicated that individuals with schizophrenia
and related disorders have altered levels of the HERV-W gag protein (Weis et al. 2007a)
and of the HERV-W receptor neutral amino acid transporter 1 (ASCT1; Weis et al.
2007b) within several different brain regions as compared to controls. The levels of
HERV-W transcripts have also been found to be increased in some individuals with
these disorders (Yolken et al. 2000) but not others (Frank et al. 2005).
The study of the control of HERV-W expression has also been evaluated in cell
lines. In our experimental study on human cell lines, transcription of the env gene
encoding syncytin was strongly induced by influenza A virus infection. Studies on the
transcriptional regulation of syncytin have identified basal promoter activity in the U3
region of the 5’-LTR as well as enhancer activity in flanking genomic DNA upstream
of the LTR (Prudhomme et al. 2004). In this upstream regulatory region, binding sites
for several different transcription factors have been identified, including binding sites
for the transcription factor, glial cells missing (Gcm) 1. Overexpression of Gcm1 in
cells of placental origin has also been reported to induce fusion and elevated levels
of syncytin transcripts suggesting a functional role of Gcm1 in the transcriptional
regulation of syncytin. This role is also supported by the very restricted expression of
Gcm1, detectable only in the human placenta and adult parathyroid.
72 R.H. Yolken et al.

Glial cells missing

Gcm1 is the mammalian analog of the Gcm identified in Drosophila. Gcm mutant
flies were reported to lack glial cells. Subsequent studies identified Gcm as a binary
switch in the developing CNS, determining glial or neuronal fate of neural precursor
cells (reviewed in Jones 2005). Experimental studies in mice (Iwasaki et al. 2003) and
chickens (Soustelle et al. 2007) suggest that, in higher order species, the role of Gcm
in CNS development may be conserved and that vertebrate Gcm may determine glial
and/or neuronal fate depending on the cellular context (Soustelle et al. 2007). Whether
or not syncytin is also expressed during certain stages of human CNS development has
not been studied. Dupressoir and coworkers (2005) reported on the identification of two
retroviral env genes containing intact open reading frames in the mouse genome. These
were reported to be expressed at high levels only in the placenta and were suggested to
constitute functional murine analogs of human syncytin. These genes were therefore
denoted syncytins A and B. Based on our findings regarding transactivation of HERV-
W elements by influenza A virus, including that encoding syncytin (Nellaker et al.
2006), we investigated if one or both of the proposed mouse analogs would respond
similarly. We found syncytin B, but not syncytin A, to be greatly induced by the virus
infection in NIH-3T3 cells as well as in primary hippocampal neurons and glia (Asp
et al 2007). If regulated by mechanisms analogous to those observed in human cells,
a concomitant transcriptional up-regulation of mouse GCMa would be expected in
such infected cultures. Indeed, elevated levels of transcripts encoding mouse GCMa
were induced in all three cell systems following virus infection as well as in vivo during
the acute stages of a CNS infection with a mouse-adapted neurotropic strain (WSN/33)
of influenza A virus. Cloning and overexpression of GCMa did induce expression of
syncytin B, but not syncytin A, in NIH-3T3 cells, suggesting that the mouse syncytin B
is a functional analog to human syncytin. Our observation of readily detectable levels
of transcripts encoding GCMa and syncytin A and B in fetal brains supports the notion
that Gcm, and perhaps also syncytin B, may play a role during development of the
mammalian brain.

Antibodies to Retroviral Proteins in Individuals

with Psychiatric Disorders
The measurement of antibodies in the blood of individuals with psychiatric disor-
ders remains an important tool for the study of the role of potential antigens in the
etiopathogenesis of these disorders. In a preliminary study, we showed that individuals
with recent onset schizophrenia have increased levels of antibodies to purified viri-
ons derived from several group D retroviruses, including Mason-Pfizer monkey virus
(MPMV), baboon endogenous virus (BaEV) and simian retrovirus type 5 (SRV-5; Lille-
hoj et al. 2000). However, the specificity of the reactivity to cell culture-derived virions
is difficult to document with certainty. For this reason, we adapted methods for the
cloning and expression of retroviral proteins in a manner that facilitates the accurate
measurement of antibodies in large numbers of clinical samples. Initial testing was
performed using antigens derived from simian retrovirus type 1 based on the results
of the preliminary study.
 Endogenous Retroviruses and Human Neuropsychiatric Disorders 73

GST fusion proteins of the SRV1 gag and env were expressed in insect cells using
recombinant baculovirus. The SRV1 gag and env (excluding the signal peptide) coding
sequences were artificially synthesized (GenScript Corporation, Piscataway, NJ) and
codon-modified for optimal expression in insect cells. The genes were designed to
introduce BamH1 and EcoR1 restriction sites at the 5’ and 3’ ends, respectively, of
the coding sequence and cloned into the corresponding restriction sites of the bac-
ulovirus transfer vector pAcSecG2T (BD Biosciences, San Jose, CA) downstream of
the polyhedrin promoter and the GST open reading frame. An initial recombinant
baculovirus stock was generated by co-transfection of Spodoptera frugiperda sf9 insect
cells with the transfer vector and the ProEasy linear baculovirus DNA (AB Vector,
San Diego, CA) by using Cellfectin (Invitrogen) according to the procedure suggested
by the manufacturer of ProEasy. Large-scale production of recombinant proteins was
performed as previously described (Viscidi et al. 2003). Briefly, approximately 5 × 108
Trichoplusia ni (High Five) cells (Invitrogen, Carlsbad, CA) were infected with 2.5 ml
of a high-titer recombinant baculovirus stock in 20 ml of Ex-Cell 400 medium (JRH
Biosciences, Lenexa, KS) for 60 min at room temperature with periodic inversion.
Aliquots of infected cells (1 × 108 ) were grown as adherent cultures in tissue culture
plates (245 by 245 mm; Nunc, Naperville, IL) in a volume of 100 ml of Ex-Cell 400
medium supplemented with gentamicin (10 g/ml). After 96 h of incubation at 27◦ C, the
cells were harvested from the plates by scraping and were collected by centrifugation
at 2 000 rpm (Sorvall FH18/250 rotor) for 5 min. The cells were lysed in PBS containing
0.5% NP-40 and a cocktail of protease inhibitors (Roche). After brief sonication, clar-
ified cell lysates were obtained with high-speed centrifugation (15 000 xg). The final
protein concentration of the clarified lysates was adjusted to 3 mg/ml with PBS 0.05%
Tween-20 and aliquots stored at −70◦ C.
The GST-tagged proteins were bound to casein-coated microtiter plate as outlined
in Fig 2. Preparation of glutathione-casein and GST capture ELISA was performed as
previously described (Sehr et al. 2001) with minor modifications. In brief, casein (Sigma
Chemical Company, St Louis) at a concentration of 5 mg/ml in (PBS) was incubated for
15 min at room temperature (RT) with 0.4 mM N-ethylmaleimide (NEM; Sigma). Sub-
sequently, 4 mM sulfosuccinimidyl 4-[p-maleimidophenyl]butyrate (SSMBP; Pierce,
Rockford, IL) was added as crosslinker and the reaction proceeded for 30 min at
RT. Free SSMBP and NEM were separated from casein by size exclusion chromatog-
raphy on PD10 columns (Pharmacia). The protein fraction was then supplemented
with 10 mM glutathione (Sigma) and the coupling reaction was executed for 1 h at RT.
The glutathione-casein was separated from unbound glutathione by gel filtration with
PD10, using PBS as buffer, and was stored at −20◦ C in small aliquots.
Polysorb plastic plates, 96 wells (Nunc), were coated overnight at 4◦ C with
200 ng/well of glutathione casein in 50 mM carbonate buffer, pH 9.6. Thereafter, wells
were washed once with PBS 0.05% Tween 20 and incubated for 1 h at 37◦ C with 200 μl
of blocking buffer (0.2% casein, 0.5% polyvinyl alcohol, 0.5% polyvinylopyrolidone,
0.05% Tween 20 in PBS). Subsequently, the plates were incubated for 2 h at RT with the
cleared lysates from the recombinant baculovirus-infected insect cells overexpressing
GST-Gag and env proteins diluted in blocking buffer to 0.3 mg/ml of total protein.
Human sera were diluted 1/ 100 in blocking buffer and incubated on the ELISA plates
for 1 hr at 37◦ C. Bound human antibodies were detected by incubating with goat anti-
human IgG antibody conjugated to HRP (1/ 4000 dilution in blocking buffer) for 1 h at
74 R.H. Yolken et al.

Fig. 2. Outline of method

used for the binding of
the GST fusion proteins
to the microtiter plate and
the performance of the
enzyme immunoassays
described in the text.
(Derived from Sehr et al.
2001)

37◦ C. Bound reactivity was visualized by incubation with ABTS Peroxidase Substrate
(KPL, Gaithesburg, MD) for 10 min at RT. The enzyme reaction was stopped by adding
100 μl of 0.5% SDS and the absorbance was measured at 405 nm. All washing steps to
remove unbound reagents were performed with PBS containing 0.05% (v/ v) Tween 20
in an automated plate washer. Reactivity of the human sera to lysates from baculovirus-
infected insect cells expressing the GST protein alone was also measured using the same
ELISA procedure described above. For each sample, an adjusted optical density was
calculated by subtracting the value generated by reactivity with baculovirus proteins
generated without an insert from the value generated by the baculovirus-expressed
SRV-1 gag and SRV-1 envelope respectively. For each case sample, positivity was de-
fined as yielding an adjusted optical density greater than the 90th percentile of samples
from health controls run on the same mictotiter plate.
We employed the system described above to measure the levels of antibodies
to baculovirus cloned simian retrovirus type 1 gag and env proteins in individuals
with recent onset psychosis as well as individuals with established schizophrenia,
bipolar disorder, and controls. The study population consisted of 110 individuals with
recent onset psychosis, 319 individuals with established schizophrenia, 124 individuals
with established bipolar disorder and 199 control individuals without a psychiatric
disorder. The methods for the recruitment and evaluation of these individuals have
been previously described (Dickerson et al. 2003, 2004, 2007). As depicted in Fig. 3, we
found an increased level of antibodies to both envelope and gag proteins in individuals
with the psychiatric disorder, particularly those with recent onset psychosis (odds
ratio for individuals with antibodies to both SRV-1 gag and SRV-1 env 5.04, 95%
confidence interval 1.79–14.26, p = 0.002). It is notable that the antibody levels were
 Endogenous Retroviruses and Human Neuropsychiatric Disorders 75

Fig. 3. Reactivity to SRV-1 gag and env individuals with psychiatric disorders and controls SRV-1
gag and SRV-1 envelope proteins were cloned and expressed in baculovirus as described in the
text. The adjusted odds ratios were calculated using multiple logistic regression including age,
race, and gender in the regression equation

higher for individuals with recent onset psychosis than for individuals with established
disorder. This finding, which is consistent with the previous antibody and PCR-based
studies described above, suggests that the greatest amount of immune stimulation is
occurring near the time of the onset of disease symptoms and decreases later in the
course of the disease. The antigenic origin of the antibodies reactive to SRV-1 gag and
SRV-1 envelope protein in the individuals with psychiatric disorders is not known with
certainty. Possible sources of antigenic stimulation include HERV-W syncytin, which
shares a substantial amount of homology to SRV-1 envelope protein, other endogenous
human retroviruses, or exogenous group D retroviruses, which have been shown to
infect humans on rare occasions (Morozov et al. 1996). The specific antigens recognized
by the antibodies generated by individuals with psychiatric disorders are the subjects
of ongoing studies.

Polymorphisms in HERV K18

The association between HERVs and human psychiatric disorders may also be mediated
by genetic polymorphisms in HERV coding sequences. We determined the relationship
between polymorphisms in the envelope gene endogenous retrovirus HERV K18 and
the risk of schizophrenia. This endogenous retrovirus was selected since it is located on
chromosome 1q22-23, a region that has been shown to be associated with schizophrenia
76 R.H. Yolken et al.

in some linkage studies but for which a specific genetic association has not been found.
In addition, this element has been associated with diabetes in some populations,
perhaps related to its location in the first intron of CD48, a member of the SLAM
immunoglobulin supergene family central to the activity of natural killer cells (Messmer
et al. 2006). We first sequenced the HERV K18 envelope gene in brain samples obtained
postmortem from 83 individuals (Torrey et al. 2000). We identified a G/A polymorphism
at position 86204 in GenBank sequence AL121985.13 from chromosome 1 in DNA from
three of the 28 brains from individuals who had schizophrenia, but not in any of the
55 other DNA brain samples. This polymorphism, which results in an amino acid
change from isoleucine to valine in the HERV K18 envelope, was in complete linkage
disequilibrium with a synonymous C/T polymorphism located at nucleotide 85144
of sequence AL 121985, which is also within the HERV K18 envelope (Fig. 4). We
thus defined individuals with the homozygous A/A polymorphism at nucleotide 86204
and the homozygous T/T polymorphisms at nucleotide 85144 as having a high-risk
HERV K18 haplotype and developed a real time PCR assay for the detection of these
polymorphisms in additional brain samples. To date, we have found this high-risk
haplotype in the DNA from 16 of 126 (12.7%) individuals with schizophrenia but in
only 1 of 84 (1.2%) control individuals (p < 0.02 adjusted for age, race, and gender).
On the other hand, this haplotype was only found in 3 of 97 (3.1%) individuals with
affective disorders. This rate did not differ significantly from that of controls.

Fig. 4. Association between the HERV K18 high-risk haplotype and frontal cortex expression
of two isoforms of CD48. The bars represent the mean and 95% confidence intervals of the
expression in the frontal cortex of the 4-exon isoform of CD48 (left) and the 2-exon isoform
(right) in individuals with the high-risk haplotype of HERV K18 on chromosome 1q22 (High
Risk) and individuals who do not have this haplotype (Not high risk). The arrows indicate the
location of the polymorphisms that define this haplotype within the env gene of Herv K18. The
arrows are at nucleotides 85144 and 86204 of GenBank sequence AL 121985.13. The exon numbers
of CD48 are indicated; coding regions are shown in blue. Note that the Herv K18 retroelement is
oriented antisense to the coding of the CD84
 Endogenous Retroviruses and Human Neuropsychiatric Disorders 77

We also examined the association between this high-risk haplotype and gene
expression in the frontal cortex of 105 post-mortem brains that had been analyzed
by microarray analysis (Elashoff et al. 2007). We found a strong association between
the high-risk haplotype and the decreased expression of an alternate-spliced form of
CD48 (expression ratio = 0.82, 95% confidence interval 0.76–0.87, p < 0.001). On the
other hand, there was no association between the high-risk haplotype and expression
of the fully spiced form of CD48 (Fig. 4). The high-risk haplotype was also associated
with altered brain frontal cortex expression of genes in a number of different gene
ontology pathways related to the immune response. Overall, of the 359 genes in the
immune response gene ontology pathway on which we had data, there were 42 that
showed a significantly increased association with this haplotype and one that showed
a significant decrease (defined as p<0.01; Table 1). These findings are of interest in
light of studies indicating a high rate of immune dysregulation in individuals with
schizophrenia (Strous and Shoenfeld 2006).
Our studies indicate that a haplotype defined by polymorphisms on HERV K18
in chromosome 1q22-23 may be associated with an increased risk of schizophrenia
in individuals who die with this disease. While modest, this odds ratio is higher than
the odds ratios that have been found for other genetic alterations associated with
schizophrenia. This risk genotype was not associated with other psychiatric diseases
such as bipolar disorder, indicating a degree of specificity for schizophrenia. The
mechanism by which this polymorphism increases the risk of schizophrenia is likely to
be associated with the altered transcription of CD48, with subsequent changes in the
immune response to antigens. The specific pathways by which these alterations lead to
schizophrenia, as well as the role of these pathways in the autoimmune disorders that
are associated with schizophrenia, should be the subjects of additional investigations.
It is notable that the polymorphisms identified in this study and others within HERV
K18 on chromosome 1 are not included in several of the panels used for “whole
genome” association studies, due to their repetitive nature and the fact that they are
not amenable to hybridization methods that measure small target regions. Associations
between complex disorders and these regions would thus not be likely to be detected
by these methods. The ability to detect genetic risks for complex disorders thus might
be markedly enhanced by the development and utilization of methods capable of
identifying associations with endogenous retroviruses and other repetitive regions
throughout the human genome.

Conclusion

Despite intensive research efforts performed over many years in laboratories around
the world, the etiopathogenesis of schizophrenia remains unclear. We postulate that
detailed study of the role of endogenous retroviruses will contribute to a better un-
derstanding of this disease, as well as of other complex disorders of the human CNS.
Future studies will include more detailed genetic analyses of polymorphisms in HERV
regions of the human genome as well as more detailed genomic and proteomic analyses
of HERV gene expression. It is notable that many of the tools that are being developed
for genetic, genomic, and proteomic analyses largely exclude repetitive regions of the
genome, such as those that contain HERVs. It is thus imperative that techniques be
 78

Table 1. Genes with altered brain frontal cortex expression associated with the high-risk HERV K18 haplotype
Locus- Gene Gene name Ratio Standard P Value
link ID Symbol Error
3077 HFE Hemochromatosis 1.15 0.03 < 0.00001
3119 HLA-DQB1 Major histocompatibility complex, class II, DQ beta 1 1.12 0.03 >0.00001
R.H. Yolken et al.

3507 IGHM Immunoglobulin heavy constant mu 1.16 0.04 <0.00001

3107 HLA-C Major histocompatibility, complex, class 1, C 1.29 0.06 (<0.00001
6892 TAPBP TAP binding protein (tapasin) 1.13 0.03 0.00004
3105 HLA-A Major histocompatibility complex, class I, A 1.17 0.05 0.00011
8741 TNFSF13 Tumor necrosis factor (ligand) superfamily, member 13 1.15 0.05 0.00013
10859 LILRB1 Leukocyte immunoglobulin-like receptor, subfamily B (with TM and ITIM) 1.06 0.02 0.00019
7940 LST1 Leukocyte specific transcript 1 1.12 0.04 0.00021
3140 MR1 Major histocompatibility complex, class I-related 1.10 0.04 0.00040
3603 IL 16 Interleukin 16 (lymphocyte chemoattractant factor) 1.15 0.05 0.00045
5871 MAP4K2 Mitogen-activated protein kinase kinase kinase kinase 2 1.09 0.02 0.00045
63940 GPSM3 G-protein signaling modulator 3 (AGS3-like, C. elegans) 1.18 0.05 0.00045
25939 SAMHD1 SAM domain and HD domain 1 1.22 0.06 0.00064
2495 FTH1 Ferritin, heavy polypeptide 1 1.15 0.05 0.00078
2204 FCAR Fc fragment of IgA, receptor for 1.07 0.03 0.00070
355 TNFRSF6 Tumor necrosis factor receptor superfamily, member 6 1.12 0.05 0.00080
3806 KIR2DS1 Killer cell immunoglobulin-like receptor, two domains, short cytoplasmic 1.08 0.03 0.00142
4851 NOTCH1 Notch homolog 1, translocation-associated (Drosophila) 1.29 0.09 0.00170
10225 CD96 CD96 antigen 1.07 0.02 0.00200
3106 HLA-B Major histocompatibility complex, class 1, B 1.24 0.09 0.00215
10410 IFITM3 Interferon induced transmembrane protein 3 (1-8U) 1.42 0.12 0.00218
5393 PMSCL1 Polymyositis/scleroderma autoantigen 1, 75kDa 1.15 0.06 0.00244
9567 GTPBP1 GTP binding protein 1 1.09 0.04 0.00267
Table 1. (continued)
Locus- Gene Gene name Ratio Standard P Value
link ID Symbol Error
8519 IFITM1 Interferon induced transmembrane protein 1 (9-27) 1.24 0.09 0.00280
3429 IF127 Interferon, alpha-inducible protein 27 1.17 0.06 0.00284
3113 HLA-DPA1 Major histocompatibility complex, class II, DP alpha 1 1.14 0.06 0.00299
11025 LILRB3 Leukocyte immunoglobulin-like receptor, subfamily B (with Tm and ITIM) 1.10 0.05 0.00356
5989 RFX1 Regulatory factor X, 1 (influences HLA class II expression) 1.03 0.01 0.00395
2213 FCGR2B Fc fragment of IgG, low affinity IIb, receptor for (CD32) 1.08 0.04 0.00407
9466 IL27RA Interleukin 27 receptor, alpha 1.12 0.05 0.00411
3903 LAIR1 Leukocyte-associated Ig-like receptor 1 1.06 0.02 0.00416
714 C1QG Complement component 1, q subcomponent, gamma polypeptide 1.76 0.10 0.00432
4938 OAS1 2’,5’-oligoadenylate synthetase 1, 40/46kDa 1.11 0.05 0.00557
970 TNFSF7 Tumor necrosis factor (ligand) superfamily, member 7 1.12 0.05 0.00624
966 CD59 CD59 antigen P18-20 (antigen identified by monoclonal antibodies 1.09 0.04 0.00630
16.3A5, EJ16, EJ3, EL32 and G344)
2533 FYB FYN binding protein (FYB-120/130) 1.12 0.06 0.00650
80329 ULBP1 UL16ding protein 1 1.25 0.08 0.00700
972 CD74 CD74 antigen (invariant polypeptide of major histocompatibility complex) 1.17 0.07 0.00731
3570 IL6R Interleukin 6 receptor 1.14 0.06 0.00754
3811 KIR3DL1 Killer cell immunoglobulin-like receptor, three domains, long cytoplasmic 1.08 0.04 0.00833
9437 NCR1 Natural cytotoxicity triggering receptor 1 0.92 0.04 0.00919
6387 CXCL 12 Chemokine (C-X-C motif) ligand 12 (stromal cell-derived factor 1) 1.08 0.04 0.00959
Endogenous Retroviruses and Human Neuropsychiatric Disorders
79
80 R.H. Yolken et al.

developed that will allow for the inclusion of HERVs and other repetitive sequences in
the large-scale genetic analyses directed at identifying risk factors for schizophrenia.
The studies presented above indicate that only with the inclusion of HERV regions will
complex disorders such as schizophrenia be understood and new methods for disease
diagnosis, prevention, and treatment be developed.

Acknowledgements. This work was supported by grants from the Stanley Medical Research
Institute. We thank Ms Ann Cusic for her assistance in the preparation of this manuscript.

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Is Psychosis Due to Retroviral/Retrotransposon
Integration Close to the Cerebral Dominance Gene?

T.J. Crow1 , J.S. Close1 , H.-S. Kim2 , and M.T. Ross3

Summary
Scope is now limited for an environmental theory of the origins of psychosis. The last
serious candidate – infection by an exogenous virus – is ruled out by the observation
that, when two siblings become ill, they do so at the same age and not at the same time.
An evolutionary theory is required to explain the persistence of disadvantageous vari-
ation across human populations. The hypothesis has been proposed that the variation
associated with psychosis arises in relation to the speciation event - the genetic change
that initiated Homo sapiens as a species and postulated to have introduced cerebral
asymmetry (“the torque”) as the basis of language. There are strong grounds for be-
lieving the asymmetry gene to be in the X-Y homologous class. The leading candidate
is the ProtocadherinX/Y gene pair generated by a duplication from Xq21.3 to Yp that
occurred 4.5–6.0 million years ago. This gene has been subject to accelerated evolution
with 16 amino acid changing substitutions in PCDHY and, significantly, five changes in
PCDHX. Here we investigate the modified viral theory that psychosis is due to a retro-
viral insertion close to the cerebral dominance gene by examining retroviral sequences
in relation to PCDHX/Y within the homologous region, and retro elements that differ
between the homologous blocks, i.e., ones that might represent new insertions. Al-
though large numbers of such elements were found, we did not identify a particular
element that was likely to have caused changes in the expression of either PCDHX or
PCDHY.We conclude that psychosis is probably not due to retroviral insertion close
to the cerebral dominance gene and that the variance in gene expression arises from
some other mechanism, for example, epigenetic modification in meiosis.

Introduction
Psychosis (schizophrenia and manic depressive illness) constitutes a spectrum of ill-
nesses with an onset in early, middle and sometimes late adult life that occurs across
populations at a relatively high prevalence – 2–3% lifetime expectation. The commonly
held view that these disorders are multifactorial in origin and that a number of etio-
logical agents, each of small effect, have been identified is little more than a confession
1 SANE POWIC, Warneford Hospital, Oxford, OX3 7JX, UK, e-mail: tim.crow@psych.ox.ac.uk
2 Section of Biological Systems, College of Natural Science, Pusan National University, San 30,
Changjeon Dong, Pusan 609-735, South Korea
3 X Chromosome Group, Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus,
Hinxton, Cambridge CB10 1SA, UK

Gage et al.
Retrotransposition, Diversity and the Brain
© Springer-Verlag Berlin Heidelberg 2008
88 TJ Crow et al.

of ignorance. A genetic component is undoubted but remains ill-defined in Mendelian

terms. First-degree relatives are at approximately 10% risk of illness compared to 1% for
each of the main syndromes, e.g., schizophrenia or manic depressive illness, narrowly
defined. But a simple genetic theory cannot account for 1) the shortfall from 100% in
concordance in monozygotic twin-pairs, 2) onset in adult life, and 3) continued high
prevalence against the selective effects of a reduction in fertility. To explain these facts,
a gene-environment interaction is often invoked, but there is a dearth of environmental
agents to which an etiological role can plausibly be attributed. Psychogenic trauma has
fallen by the wayside from the failure of its advocates to specify the nature of the trauma
or to construct a plausible account of its psychodynamic effects. Other categories of
environmental agents can be reduced to toxins, infection and disturbances of immu-
nity. Of these, a toxic factor appears unlikely in view of the widespread geographical
distribution. Immunity has been invoked but never substantially documented. Thus
the category of infection remains a salient possibility.
The concept that schizophrenia might be horizontally transmitted has a long
history (Baillarger 1857; Hofbauer 1864; Wollenberg 1889), although only more recently
in a microbial context. It has been argued (Crow 1983) that some findings in family
studies are compatible with the notion that, in addition to a genetic predisposition,
what matters is proximity to an individual who already has the disease. Consistent with
this theory are the following findings:
1. Concordance rates are higher in dizygotic twins than in their siblings (Fischer
1973), although these relatives share genes to the same extent: because they are the
same age. twins may be in closer contact than siblings.
2. Concordance in relatives is greater in same-sex than opposite-sex pairs (Rosenthal
1962), but this effect occurs only within the family (i.e., first-degree relatives –
Penrose 1942); again it may be argued that same-sex pairs of relatives are in closer
physical proximity.
3. In monozygotic twins, the second member of the pair was reported at increased
risk after the onset of the illness in the first, and the increase was confined to pairs
who were together at this time (Abe 1969).

These arguments were placed in the context of other evidence consistent with a viral
etiology:

1. Coincidence of psychosis with infectious disease, particularly influenza

Menninger (1926) reported a series of 175 cases of post-influenzal psychosis and noted
that the initial clinical picture had the “unmistakable schizophrenic stigmas includ-
ing intrapsychic ataxia, emotional ideational spitting, incoherence, stereotypies and
other bizarre expressions” and that these symptoms were not conspicuously different
from those seen in the usual types of schizophrenic illness. He concluded that the
schizophrenic syndrome i) was the relatively most frequent post-influenzal psychosis,
ii) occurred with or without predisposition or hereditary taint, and iii) in most cases
terminated in complete recovery. But he modified his view and later wrote, “I think
I said seven years ago that influenza caused psychosis. I have changed my mind”
(Menninger 1928). Others pursued the viral hypothesis with greater tenacity. Goodall
 Is Psychosis Due to Retro-Element Integration? 89

(1932), in the wake of the encephalitis lethargica epidemic that followed and may
well have been related (Ravenholt and Foege 1982) to the 1918 influenza epidemic
noted that, “there are observers who consider that there is no essential difference
between psychotic disturbance connected with encephalitis (post-encephalitic) and
those met with in states covered by the description schizophrenia” and went on to
argue that, “epidemic encephalitis, with the somatic disorders which accompany it,
may be a virus disease and similarly caused perhaps are the schizophrenic states which
resemble them.” Jelliffe (1927), Hendrick (1928), and McCowan and Cook (1928) also
related some cases to the 1918 epidemic, which was almost certainly due to influenza
A (H1N1), but the association appears not to have occurred or to have been documented
subsequently.

2. Epidemiology

Seasonality:
Consistent with a viral etiology are seasonal changes. Data have been presented that
there is an excess of onsets of both mania and schizophrenia in the early summer
months (Hare and Walter 1978), expressed in relation to admissions for other types
of illness, and that there is a season-of-birth effect: individuals who later develop the
disease are more likely (by 4–8%) to have been born in the months of winter and early
spring than at other times of the year (Torrey and Petersen 1977). Similar effects are
seen for mania (Hare and Walter 1978) as for schizophrenia.
Opposing these assumptions are the arguments of Lewis (1989) that the season-
of-birth effect is due to “age incidence:” the fact that those who are born in the early
months of the year are at greater risk (and more exposure) than those born in the
later months when the data on subsequent incidence are collected by calendar year.
This argument has not been effectively eliminated, and the prediction that the season-
of-birth effect would reverse in the southern hemisphere if due to a climatic factor is
unclear.

Temporal Geographical Variations:

A challenge has been issued against the common assumption that the incidence of
schizophrenia is uniform with respect to time, and approximately similar in the various
populations of the world. Hare (1983) presented evidence on the basis of the increase
in hospital facilities that incidence of the disease increased in the nineteenth century.
Both Hare (1983) and Torrey (1980) have drawn attention to the relative scarcity
before 1800 of case descriptions that, with reasonable confidence, can be classified as
schizophrenia.
These arguments were coupled with evidence for significant variations in incidence
across populations (Torrey 1980; McGrath 2006). The following considerations are
relevant to claims that incidence varies over time and space:

1. In a review of the classical literature, Roccatagliata (1991) wrote that “we are
forced to recognise a substantial identity between the ancient and the modern
approach. There seemed to be no remarkable differences between the nosology
of schizophrenic disturbances proposed by the APA Diagnostic and Statistical
90 TJ Crow et al.

Manual of Mental Disorders on the one hand and classical nosology on the other ...
From a scientific viewpoint this confirms the existence above all of interpretive
models and natural psychiatric entities that have been modified only marginally by
historical and social development.” Such findings are paralleled by relative stability
of the clinical picture over time when records are available from recent centuries
(Turner 1992; Nixon and Doody 2005).
2. When systematically sought for, it appears that the patterns of illness described
as schizophrenia or manic depressive psychosis are relatively constant across
populations. Thus, for example, Murphy (1976), who examined psychological
abnormality in the Eskimo, the Yoruba in Nigeria and populations of Swe-
den and Canada reached the conclusion that “rather than being simply vio-
lations of the social norms of particular groups, as a labelling theory sug-
gests, symptoms of mental illness are manifestations of a type of affliction
shared by virtually all mankind.” The conclusion is borne out by studies in
relatively isolated and primitive populations. Thus in an investigation of the
prevalence of schizophrenia in Botswana, Ben-Tovim and Cushnie (1986) con-
cluded that prevalence was “within the range generally reported for indus-
trial communities. Remote village life in Botswana appears to offer no pro-
tection against the development of schizophrenia.” Similarly in a compari-
son of colored and black individuals of Bantu, Negroid and Khoisan origin
with different language and cultural backgrounds, Maslowski et al. (1998) con-
cluded that “core symptoms remained basically the same” across groups, but
the content of positive symptoms varied somewhat with culture and “no sta-
tistically significant differences in the presentation of negative symptoms were
found.”
3. In those cases where apparently unusual rates of schizophrenia have been ob-
served, eg Ireland, subsequent investigation has cast doubt on the exception-
ality. In an investigation of the incidence and prevalence in Ireland, Cabot
(1990) concluded that “cultural and genetic hypotheses have been advanced to
explain figures without a critical examination of the studies at the basis of
this claim ... and hypotheses are reviewed to show that their conclusions are
equivocal and ungeneralisable ... most often due to the method of case col-
lection and inconclusive across cultural comparison. It is hoped that Ireland
will not henceforth be considered a high prevalence area for schizophrenia
without more reliable research”. Similarly Ni Nuallain (1990) concluded that
the apparent higher rates for Ireland were due to “continued hospitalisation
of symptomatically recovered cases that has given rise to the mistaken impres-
sion that the prevalence of schizophrenia is unduly high in Ireland ... The work
reported here indicates substantial differences between results of case ascer-
tainment by hospital admission compared with those arrived at by standard-
ised interview diagnostic techniques”. Thus considerable caution is indicated
in reaching the conclusions suggested by McGrath (2005) that there are sub-
stantial and unaccounted for variations in incidence of psychosis across popula-
tions.
 Is Psychosis Due to Retro-Element Integration? 91

3. Direct Investigations of the Viral Hypothesis

Three sources of evidence are advanced as consistent with a viral aetiology:

1. Serum and antibody titers: Elevated CSF serum ratios of antibodies to cy-
tomegalovirus (Albrecht et al. 1980; Torrey et al. 1982) and two other herpes
viruses have been reported, but the most systematic studies (e.g., King et al. 1985)
do not show obvious excesses.
2. Cell culture techniques: In a search for known or unrecognized viruses, cere-
brospinal fluid (CSF) from about one third of patients with schizophrenia was
found to induce a cytopathic effect in human embryonic fibroblast cultures (Tyrrell
et al. 1979). The effect was not passaged but was prevented by filters of 50 nm pore
size; it was thought that the effect might reflect the presence of a small RNA virus.
Further investigation showed that the effect was also seen with CSF from some
patients with affective disorder and miscellaneous neurological conditions (Tay-
lor et al. 1985) and that it is not prevented by protein synthesis or nucleic acid
synthesis inhibition. Although these findings were initially reported as consistent
with the presence of a virus, the later findings made this unlikely, although they
might reflect release into the CSF of a toxic factor associated with neural damage.
3. Transmission experiments: CSF inducing cytopathic effects from patients with
schizophrenia was injected intracerebrally into mice and hamsters without signifi-
cant effects, but when marmosets were injected, behavioral changes were observed
over the next 2 12 years: the injected animals appeared less active. These experiments
were subsequently repeated without the same findings being obtained (Baker et al.
1989).

A Continuum of Psychosis

One of the problems for those who maintain that there are significant variations in
the incidence of either schizophrenia or manic depressive illness over time is that
the boundaries are unclear. Schizo-affective disorders are common and genuinely
intermediate between the more typical schizophrenic and manic depressive syndromes.
The WHO Ten Country Study of incidence and manifestations of schizophrenia
(Jablensky et al. 1992) reached the conclusion that “schizophrenic illnesses are ubiqui-
tous, appear with similar incidence in different cultures, and have clinical features that
are more remarkable by their similarity across cultures than by their differences.” Simi-
lar conclusions have been drawn concerning affective illness by Weissman et al. (1996).
Parallel to these are the findings of population surveys (van-Os et al. 1997). Symptoms
comparable to those seen in the typical syndromes are present in relatively high fre-
quency in the general population and have the same correlates. Thus it is possible that
we are dealing with continua of variation rather than discrete disease states.
Following extension of the contagion hypothesis to the case of siblings who both
develop psychosis, a test of horizontal transmission was devised. If transmission occurs
from one predisposed (e.g., genetically) sibling to another or if both encounter an
exogenous agent at the same time, illness onset will be expected to be earlier in the
younger than in the older sibling, i.e., onsets will occur at the same time and not at the
92 TJ Crow et al.

same age. While the initial observation suggested that this might be the case (Crow and
Done 1986), it was soon appreciated that an artefact enters the collection of such data
as follows: if information is collected at the time of onset of illness in one sibling, then
one is likely to hear about younger siblings who have already become ill but not those
who become ill later. The artefact can be removed by exclusion of the data to elder
sibling ill first pairs. When this is done the apparent bias disappears and the conclusion
is clear: onsets of illness in pairs of siblings occur on average at the same age and not
at the same time. This conclusion is awkward, not only for a contagion hypothesis but
also for any theory that invokes an environmental agent as relevant to onset of illness.

A New Hypothesis: Retroviruses and Proto-Oncogenes

Confronted with these findings. it appeared that a radical reorientation was required.
The genetic component appeared more substantial and an environmental precipitant
was apparently excluded, at least in the majority of cases.
Nevertheless some attractions of the viral hypothesis remained. It had the potential
to explain deleterious outcome and perhaps even the brain changes that were by this
time well documented in imaging (Johnstone et al. 1978) and post-mortem (Brown
et al. 1986) studies and also the sometimes relatively abrupt onset and episodicity.
Affective illnesses are often unequivocally episodic in nature and the same is true to
a lesser extent of those at the schizophrenic end of the spectrum. What could account
for this apparent lability if the basic predisposition was genetic?
A new finding also had to be accommodated:the brain changes, including particu-
larly ventricular enlargement, were at least in part lateralized. In a post-mortem study
(Brown et al. 1986), thinning of the parahippocampal gyrus was observed in those who
had suffered from schizophrenic psychoses relative to those with affective illness that
was selective to the left side (side-by-diagnosis interaction, p<0.02). The hypothesis
was proposed (Crow 1984) that: “Schizophrenia (and perhaps manic depressive psy-
chosis) is due to infection with a virus that becomes integrated in the genome sometimes
to be passed from one generation to the next..
The hypothesis took its origin in work on retroviruses (Weiss 1978), the discovery
of the enzyme reverse transcriptase (Baltimore 1969; Temin and Mizutani 1970) and to
models in which viral RNA was reverse transcribed into the host genome in the form
of a provirus (Lwoff 1965).
In its initial formulation, the hypothesis was related particularly to familial cases
and to the season-of-birth effect. However, neither of these restrictions appears nec-
essary. The hypothesis was thought also to have to address the problem of low con-
cordance rates (36–58%) in cases of monozygotic twins, and the necessity for genomic
integration of a virus at a consistent location in the human genome to produce the con-
sistencies (e.g., age of onset, brain changes) seen in psychosis. These difficulties were
resolved by the further hypothesis that the retroviral sequence integrates at a particular
locus, and this integration was predicted to be a factor associated with the growth of
the brain. Particular emphasis was laid on laterality. The hypothesis thus predicted that
the retrovirus had a particular affinity for the growth factor for cerebral asymmetry.
 Is Psychosis Due to Retro-Element Integration? 93

Thus the hypothesis was formulated: “The cerebral asymmetries underlying later-
ality are established by the trophic effects of a proto-oncogene, and that the retrovirus
responsible for psychosis interacts with the cellular oncogene to elicit enhanced activity,
which may sometimes be destructive.”
Inherent in this formulation was the notion that psychosis was human-specific and
perhaps related to the evolution of Homo sapiens.
In 1987 the hypothesis was modified as follows: “(it) is proposed that psychosis
results from aberrations of the genetic programme for the development of cerebral asym-
metries, these asymmetries being a particular feature of human evolution contributing
to the capacity for communication and social interaction. The development of cerebral
asymmetries is postulated to depend upon a specific interaction between a genetic se-
quence which has potential autonomy (e.g., a retrovirus or other mobile genetic element)
and a growth factor or proto-oncogene. The interaction ... and the persistence of the
psychoses (and their variation of form) is viewed as an unfortunate by-product of this
hotspot of genetic change” (Crow 1987).

Familial Prion Disease as a Paradigm for Transmissible

Neurodevelopmental Aberrations
Parallel to and interacting with this line of thought were our investigations of familial
prion disease, the Gerstmann-Sträussler syndrome (Baker et al. 1990; Masters et al.
1981). Marmosets inoculated intracerebrally with brain tissue from a woman with
Gerstmann-Sträussler syndrome developed an encephalopathy indistinguishable from
that seen in marmosets inoculated with brain tissue from a typical case of Creutzfeldt-
Jakob disease. It was also concluded that “As in Huntington’s disease, in the pedigree
of the patient with Gerstmann-Sträussler syndrome women who subsequently develop
the illness had increased fecundity. That the pathogen in human transmissible dementia
may arise from a sequence (which itself sometimes confers the selective advantage)
located within the human genome” (Owen et al. 1989).
With the identification of the prion gene by Stanley Prusiner, it became possible
to investigate sequence variation in relation to disease. The first mutation to cause
a neuropsychiatric syndrome was an insertion in the gene that tracked with the disease
within a family subsequently demonstrated to have extra repeats in the octapeptide
repeat sequence (Owen et al. 1989; Poulter et al. 1992). Another family was found to
have a single base pair change at amino acid 102, and this change was shown to be
significantly linked with disease within two families (Hsiao et al. 1989).
Thus a disease with a Mendelian pattern of transmission and an age of onset
apparently influenced by developmental factors can be shown to be due to an element
integrated in the human genome that also acts as an autonomous infectious agent.
Could this be a paradigm for psychosis?

The Central Paradox and the Origin of Modern Homo sapiens

The question was first formulated by Huxley et al. (1964) “If schizophrenia is genetic
in origin why are the genes not selected out?” There is no doubt that schizophrenia is
94 TJ Crow et al.

associated with a substantial fertility disadvantage, and the same is true of the affective
component of the spectrum (Stevens 1969). These questions can be put alongside
another question - how old is the genetic variation? – in the context of the Out of Africa
hypothesis of modern Homo sapiens (Stringer and McKie 1996), which argues that our
species arose some 100 000–150 000 years ago in Africa and subsequently expanded to
occupy five continents.
What explains the success of our species and its propensity to psychosis? Concern-
ing the latter, if one has variation that is present at this point in time and seeks its
origin, that clearly cannot be before the separation of the populations of the world, i.e.,
before the diaspora out of Africa, and this takes us close to the origin, i.e the speciation
event. The question of Huxley et al. is reopened. The answer they (Huxley et al. 1964)
gave is clearly wrong, as Kuttner et al. (1967) pointed out – there is no real evidence that
the balancing advantage they sought is associated with resistance to wound shock or
stress as they suggested. Nor does it make sense to postulate a balancing advantage that
is unrelated to the disadvantage, i.e., manifest in behavior and relating to the nervous
system. Rather, as Kuttner et al. proposed, one should seek some characteristic of the
species, e.g., complex social ability, intelligence or the capacity for language. These
faculties are clearly related but the most specific is surely language. It is language that is
most characteristic of the species and it is also this ability that can be related to aspects
of neural function.
Who had an idea of the nature of the speciation event? Curiously it seems to have
been Paul Broca (1877), the discoverer of language lateralization in the frontal lobes,
who wrote in a festschrift for Armand de Fleury, a colleague, “Man is, of all the animals,
the one whose brain in the normal state is the most asymmetrical. He is also the one
who possesses the most acquired faculties. Among these faculties ... the faculty of
articulate language holds pride of place. It is this that distinguishes us most clearly
from the animals.”
The evidence is that Paul Broca was right. Although it is controversial, two studies
in particular support the anatomical thesis. In a magnetic resonance imaging study,
Gilissen (2001) observed that the torque, the putative defining characteristic of the
human brain (Yakovlev and Rakic 1966), is absent in the brain of the chimpanzee.
Buxhoeveden et al. (2001) found that the asymmetries of the minicolumn structure
of the planum temporale, demonstrated to be asymmetrical to the left in a majority
of individuals by Geschwind and Levitsky (1968), are not present in the brains of the
chimpanzee and rhesus monkey. Parallel to this finding is evidence that directional
handedness on a population basis, a possible correlate of lateralization of the brain and
the capacity for language, is not present in the chimpanzee (Marchant and McGrew
1996; Provins et al. 1982).

Asymmetry as a Determinant of Human Ability

Annett (2002) has consistently maintained that cerebral asymmetry on a population

basis is the characteristic that defines Homo sapiens and provides the basis for language
and also that the genetics of asymmetry is the major determinant of human ability.
The evidence from the National Child Development Study (Crow et al. 1998)
strongly supports these contentions. In this cohort, degrees of lateralization were
 Is Psychosis Due to Retro-Element Integration? 95

assessed in terms of relative hand skill: the ability to tick squares with the right or
the left hand in one minute. Those who were strongly lateralized were at an advantage
with respect to those who were close to ambidexterity (the point of “hemispheric inde-
cision”) in verbal and non-verbal ability, reading and mathematical skills. For verbal
ability, there was a substantial sex difference in favor of females, but the relationship to
laterality was the same in the two sexes. The effect could not be attributed to a general
disadvantage of those with poor hand skill as, when plotted as a function of left against
right, verbal ability was found to be significantly impaired in those with good hand
skill but lacking lateralization (Leask and Crow 2001).
These findings have recently been replicated by Peters et al. (2006) in the large
(n = 255 000 subjects) internet survey conducted for the BBC. Those who described
themselves as ambidextrous were at a disadvantage relative to those who were more
strongly right- or left-handed for spatial and also verbal ability, with males having
an advantage for the former and females for the latter. It appears that the genetics of
lateralization is the major factor in the evolution of the human brain.

Schizophrenia as an Anomaly of Development

of Cerebral Asymmetry
In the meantime, evidence has accumulated that the structural changes in psychosis
are located in the cerebral cortex and relate to aspects of asymmetry. The form of the
asymmetry – from right frontal to left occipital, described as cerebral torque – has
implications for pathophysiology. Aspects of anatomical asymmetry are deviant in
individuals who suffer from psychosis (see Crow 1990, 1997b; Esiri and Crow 2002),
and there is evidence from a study of handedness in childhood that such individuals
are lateralizing less, or more slowly (Crow et al. 1996; Leask and Crow 2005), than the
population as a whole. In post-mortem studies, the anatomical changes appear to be
more posterior in the brain; losses or reversals of asymmetry have been detected in
fusiform, parahippocampal (McDonald et al. 2000), and superior temporal (Highley
et al. 1999a) gyri. A curious feature of these findings is that, although the loss or reversal
is present in both sexes, the relationship to age of onset differs: greater anomaly of
asymmetry is related to earlier age of onset in females but later age of onset in males.
Other sex differences have been detected. Density of fibers in the corpus callosum
was greater in females than in males, consistent with the generalization that connectiv-
ity is inversely related to degree of asymmetry (Highley et al. 1999b). In patients with
psychosis, fiber density was reduced relative to female controls whereas in males it was
increased relative to male controls. Consistent with these findings, in a magnetic reso-
nance imaging (MRI) study (Highley et al. 2003) white matter in the occipito-temporo-
parietal regions was greater in females than in males, whereas in female patients it was
reduced and in male patients increased by 50% relative to same-sex controls.
Thus there are morphological changes in the brain in psychosis that are sex-
dependent and may relate to the sex difference in age of onset: onsets are earlier in males
and earlier onsets are generally associated with poorer outcome and preponderance
of negative symptoms (affective flattening and poverty of speech). Conversely, with
increasing age of onset, the proportion of females increases and the form of the illness
is more likely to be paranoid, i.e., delusional. An obvious hypothesis of the sex difference
96 TJ Crow et al.

in age of onset is that it is developmental, but this argument encounters the difficulty
that the difference is in the wrong direction: brain size (Kretschmann et al. 1979) and
verbal ability (Crow et al. 1998) develop faster in females than in males, yet onset of
psychosis is earlier in males.
In frontal regions, no gross asymmetry and no change in gyral volume was detected
in schizophrenia (Highley et al. 2001), but in the density of cells in the cortex (in area
9), there was an asymmetry (greater cell density) to the left in controls and loss or
reversal of this asymmetry in patients (Cullen et al. 2006).

XY homology and the Xq21.3/Yp translocation

The most fundamental prediction of the asymmetry hypothesis is that the genetics
of psychosis is the genetics of the speciation of Homo sapiens (Crow 1997a); in other
words, the genetics of asymmetry is conceived as the species-defining characteristic.
To approach such predictions, it is necessary that the cerebral dominance gene or right
shift factor be identified.
A clue comes from sex chromosome aneuploidies. Individuals who lack an X chro-
mosome (XO, Turner’s syndrome) have non-dominant hemisphere (spatial) deficits on
cognitive testing. Individuals with an extra X (XXY, Klinefelter’s and XXX syndromes)
have verbal or dominant hemisphere deficits (Table 1). A possible explanation is that
an asymmetry determinant is present on the X chromosome. But then the question
arises of why males who only have one X chromosome do not have spatial deficits such
as are seen in Turner’s syndrome. The answer must be that the copy of the gene on
the X chromosome is complemented by a copy on the Y, i.e., that the gene is in the
X/Y homologous class and that the copy on the X is protected from inactivation on the
inactive X (Crow 1993). A hormonal explanation will not account for the similarity of
the changes in XXY individuals, who are male, and XXX individuals, who are female.
The case that the gene is present also on the Y chromosome is strongly reinforced by
the verbal deficits/delays that are observed in XYY individuals (Geerts et al. 2003).
The hypothesis is further strengthened by evidence that Turner’s and Klinefelter’s
syndrome individuals have corresponding deviations in anatomical asymmetry (Rezaie
et al. 2004) and by the demonstration of a same-sex concordance effect: the tendency for
handedness and sex to be associated above chance expectation, which is the hallmark
of X-Y linkage (Corballis et al. 1996). A role for an X-Y homologous gene is consistent
with the presence of a sex difference: brain growth is faster (Kretschmann et al. 1979)
and lateralization is stronger (Crow et al. 1998) in females.

Table 1. Neuropsychological impairments associated with sex chromosome aneuploidies

XX XY XO XXY XXX XYY
Normal Normal Turner’s Klinefelter’s
female male syndrome syndrome
Number of sex 2 2 1 3 3 3
chromosomes
Verbal ability normal normal normal delayed delayed delayed
Spatial ability normal normal decreased normal normal normal
 Is Psychosis Due to Retro-Element Integration? 97

Where might such a gene be located? A major chromosomal rearrangement

took place in the course of hominin evolution. Two regions on the human Y chro-
mosome short arm share homology with a single region on the human X chro-
mosome long arm (Xq21.3; Page et al. 1984; Lambson et al. 1992; Sargent et al.
1996). These homologies were created by the duplication of a 3.9 Mb contigu-
ous block of sequences from a chimpanzee hominin-precursor X chromosome to
the Y chromosome short arm, and the transposed block was subsequently split
by a paracentric inversion (by a recombination, presently undated, of LINE-1 ele-
ments; Schwartz et al. 1998; Skaletsky et al. 2003) to give two blocks of homology
in Yp11.2, Fig. 1). Genes within this region are therefore present on both the X
and Y chromosomes in H. sapiens but on the X alone in other great apes and pri-
mates.

Fig. 1. Regions of homol-

ogy between the X and Y
chromosomes according
to Affara et al (1996). The
block marked in red with
a sub-band in blue arose
4.5 to 6 million years ago
by a duplication from
Xq21.3 and Yp11.2 that,
it is argued here, initiated
the hominin lineage
98 TJ Crow et al.

Why were these Xq21.3/Yp11.2 homologous block sequences retained on Yp?

Three genes are known to be expressed within the transposed block: PABPC5, a poly
(A)-binding protein whose Y gametologue has been lost during hominin evolution;
TGIF2LX and Y (homeobox-containing genes with testis-specific expression), and Pro-
tocadherinX (PCDH11X) and ProtocadherinY (PCDH11Y). PCDH11X and Y (each
comprising seven extracellular cadherin motifs, a short transmembrane domain, and
an intracellular cytoplasmic tail), which code for cell surface adhesion molecules of the
cadherin superfamily, are of note because both forms of the gene have been retained
and are highly expressed in fetal and adult brain (Yoshida and Sugano 1999; Blanco
et al. 2000) including the germinal layer of the cortex (T.H. Priddle, personal commu-
nication). The protein products of this gene pair are thus expected to play a role in
intercellular communication, perhaps acting as axonal guidance factors and influenc-
ing the connectivity of the cerebral cortex. These genes may thus have been subject
to selective pressure relating to one or more brain characteristics during hominin
evolution (Williams et al. 2006).

Xq21.3/Yp as a Test Bed for Retrotransposon Insertion

As a result of the duplicative translocation, we have a block of the X chromosome that

is ancient in mammalian evolution and a duplicate block on the Y chromosome. Genes
and sequences within the latter thus evolve free from the selective pressures that apply
to the former. Thus genetic changes in the two regions acquire particular significance,
first with respect to the pressures leading to the maintenance of the block on the Y, and
second as an index of rates of change of sequences on the X and Y chromosomes.
There have been major rearrangements within the Y homologous block, including
4 deletions and a paracentric inversion (Fig. 2). One deletion (see Fig. 2) removed
the sequence of PABPC5 from the Y chromosome. This gene is therefore presumably
irrelevant to the selective pressures maintaining the Y sequences. A second deletion
removed exons 7 and 8 (both short and free of motifs) in the PCDH11Y sequence.
However, the gene remains in frame and is expressed in the brain.
The TGIFLX/Y gene pair is expressed in the testis. TGIFLX has been subject to
positive selection but this did not change in hominin evolution, and it is possible
that TGIFLY is inactivated by a frame shift mutation. This leaves the Protocadherin
PCDH11X/Y gene pair as the salient target of the selective pressure maintaining the
block on the Y. The fact that there are changes in the PCDH11X as well as the PCDH11Y
sequence (see page 99) can be adduced as further evidence that PCDH11Y is active.
The structure of Protocadherin 11X is represented in Fig. 3. Like other Protocad-
herins it has ectodomain repeats, in this case seven, a transmembrane domain and
a cytoplasmic domain that includes a potential beta-catenin binding site and a binding
site for protein phosphatase 1A, as well as a dodecapeptide repeat of obscure functional
specificity that is unique to this gene. The beta-catenin binding site is of interest in
view of the observations of Chen and Walsh (2003) that, if an extra beta-catenin gene
is inserted in the genome to be expressed in the brain of the mouse, then gyrification
of the cortex occurs. Thus it appears that some characteristics of the human cortex can
be provoked in the mouse by beta-catenin. The protein phosphatase 1A gene identifies
regions of the spines that make contact with incoming axons.
 Is Psychosis Due to Retro-Element Integration? 99
Fig. 2. Homologous blocks juxtaposed on
X and Y to show the gene content with
deletions marked a–d on the Y, deletion
a removing exons 7 to 8 of ProtocadherinY
(PCDHY) with the subsequent exons re-
maining in frame and expressed in fetal
and adult brain. TGIFLX and Y, TG inter-
acting factor-like gene on X and Y; PABPC5,
polyadenine binding protein C5

Since the translocation 6 million years ago, there have been 15 non-synonymous,
i.e., amino acid changing, substitutions on the Y chromosome. This might be expected,
as the new homologous region on the Y chromosome is in a sense redundant with
respect to expression of the gene on the X. But of particular interest is the fact there have
also been five amino acid changing substitutions in the X sequence, which previously
had been conserved in mammalian evolution. What can have accounted for this change
in the hominin lineage? It seems that these changes must be a response to the presence
of, and change within, the PCDHY sequence. In other words the PCDHX gene is
responding to the fact that it now has a partner, PCDHY, with which to interact.
Interestingly, three of these changes are located together within ectodomain 5 and
include an arginine to cysteine substitution on the surface of the molecule. This seems
likely to be reactive and therefore to signify significant structural change.
We have therefore a gene pair that is specific to hominins and has been subject to
change in the hominin lineage. The question arises whether any retroviral or retro-
element insertions could be relevant to the sequence of events in the evolution of H.
sapiens. It is possible to examine this relevance because of the duplication of the Xq21.3
100 TJ Crow et al.

Fig. 3. Diagram representing the structure of the Protocadherin11X protein with seven extracel-
lular repeats, a trans membrane domain, a lysine-rich region, possible beta-catenin and protein
phosphatase 1a binding sites, CM-2 motif and a dodecapeptide repeat region in exon 11 of
unknown function specific to this gene (From Skaletsky et al. 2003)

(using the software described by Smit, AFA, Hubley, R and Green, P. RepeatMasker
Open-3.0.1996–2004 http://www.repeatmasker.org/).
First we searched for retroviral sequences, focusing on HERV (human endogenous
retrovirus) sequences, within the Xq21.3/Yp11.2 homologous region. Seven such se-
quences were identified (Fig. 4a): three HERV-L elements, labelled A1-A3 and four
HERV-H elements, labelled H1–H4.
Six of these are conserved but the HERVHX3 sequence was removed from the
Yp11.2 homologous block by a deletion that also removed exons 7 and 8 of the PCDHY
sequence while leaving the gene in frame and expressed in the brain.
However, this event seems to be unrelated to the presence of the human endoge-
nous retroviral sequence because the whole sequence is within the deleted block and
apparently cannot have played a role in this rearrangement (Fig. 4b).
We next examined the presence of other retro-elements within the region using the
RepeatMasker programme (Smit, AFA, Hubley, R and Green, P. RepeatMasker Open-
3.0. 1996–2004 http://www.repeatmasker.org/). A number of elements are
present in the X sequence that are not present in the Y, and some of these presumably
represent insertions that occurred after the duplication event. The additions include
a number of LINE and Alu elements, an LF SINE element and three MIR sequences
(these latter are unlikely to represent de novo insertions since they became extinct with
 Is Psychosis Due to Retro-Element Integration? 101

Fig. 4. (a) Retroviral sequences in relation to PCDHX and PCDHY gene structure within the
Xq21.3/Yp11.2 region of homology. The retroviral sequences are homologous with the exception
that the sequence of HERV-HX-3 on the Y was removed by the deletion that also removed exons
7 and 8 from the PCDHY sequence. (b) The location of the HERV-HX-3 sequence within the
deletion and its relation to the exon structure of PCDHY

L2 elements). Conversely, a number of retro-elements (including L1HS sequences)

are present in PCDH11Y that are absent from the X. Barring the possibility, yet to
be investigated, that a homologous insertion has been deleted from one sequence
102 TJ Crow et al.

Fig. 5. Retro-elements within the PCDH11XY gene pair that lack homologues on the correspond-
ing chromosome

and remains on the other, one must assume that these are new insertions in the Y
sequence. They include two tigger elements, a number of LINEs L1 and Alu sequences,
as represented in Fig. 5.
All these sequences are included in the region between exons 1 and 11 of PCDHX
and PCDHY, respectively. While it is possible to imagine a complex scenario whereby
these elements might have a role in determining alternative splice forms, it is difficult
to see how such elements might affect expression of the whole protein. A simple
interpretation is that these are stochastic events that happened to have occurred within
this gene pair without functional consequence.
More relevant is what happens in the 5’ promoter region of the gene. Here we
compared the pattern of insertions in the 200 kb 5’ to the first exon. Some SINE
elements and DNA repeats are present within this region but these seem to be identical
in the X and the Y sequences (Fig. 6). There are some repeat sequences 100 kb upstream
from the start of the coding sequences and there is a large deletion from Y that is 40 kb
upstream of the start, but these distances are such that the changes seem unlikely to be
related to differences in gene expression. Although we have not conducted expression
studies, we think it likely that there have been no significant insertions in the sequence
that could have acted differentially on PCDHX or PCDHY.
In conclusion, we can probably exclude that the PCDHX/Y gene pair has been
subject to a specific retroviral or retro-element insertion in the course of hominin
 Is Psychosis Due to Retro-Element Integration? 103

Fig. 6. Retro-elements in the promoter region 5’ to the PCDH11XY gene pair are homologous
on the X and Y, indicating an absence of evidence of novel insertions that might have influenced
gene expression

evolution that selectively affected the expression of one or other of the two genes.
Although one cannot exclude a more complex scenario – for example, that one or more
of the elements within the gene itself or the HERV-H3 retroviral element might have
some influence on pairing of the two gene sequences in male meiosis and thereby exert
an influence on the expression of the gene from the inactive X chromosome – it seems
that the hypothesis that an insertion in relation to the cerebral dominance gene, if the
PCDHX/Y gene pair indeed is the correct candidate, initiated the specification of Homo
Sapiens, can be excluded.

Summary

1. As Broca and Annett have supposed, cerebral asymmetry appears to be the

characteristic that defines the human brain and accounts for the capacity for
language.
2. Evidence from sex chromosome aneuploidies suggests strongly that the gene for
asymmetry (the cerebral dominance gene) is present on both the X and the Y
chromosomes.
3. Lateralization is a major determinant of verbal, non-verbal and other human abil-
ities.
104 TJ Crow et al.

4. A duplication that occurred close to the chimpanzee hominin separation at 6

MYA created a region of homology between the X chromosome long-arm and Y
chromosome short-arm.
5. Within this region, the PCDH11X/Y gene pair has been identified that codes for
two cell surface adhesion factors that could act as axonal guidance agents.
6. There have been a number of alterations, including a deletion that removed exons
7 and 8 of PCDHY, 15 amino acid changes in the Y sequence and five in the X
sequence, that could have differentially affected the function of the two genes and
thus could account for a sex difference such as is seen in verbal ability and the rate
of brain growth.
7. One HERV-H element within the Y sequence has been deleted in the course of
hominin evolution.
8. A number of retro-elements are present in the X and Y sequences that are absent
from the homologous gene, but these are unlikely to have affected gene func-
tion.
9. No retro-element differences in the promoter regions of PCDHX and PCDHY
have been identified within the 35 kb 5’ to the gene. Although expression
studies have not been conducted, it seems that differences in PCDHX and
PCDHY gene expression are unlikely to be secondary to de novo retroviral or
retrotransposon insertion close to this candidate gene-pair for cerebral domi-
nance.

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Microcephalies and DNA Repair

Edward C. Gilmore1,2 and Christopher A. Walsh1,3

Head circumferences that are significantly smaller than normal are generally associ-
ated with lower intelligence, showing the relationship between intelligence and brain
size (Dolk 1991). Some of the factors that lead to normal brain size are beginning
to be understood by the study of genes that lead to small brains when disrupted.
Some of the genes that cause microcephaly are integral to DNA repair. The rela-
tionships between different mechanisms of microcephaly are just beginning to be
understood. Interestingly, there are potential relationships between the various mech-
anisms.

Microcephaly

The many etiologies of microcephaly generally can be categorized as environmental

or genetic. Genetic causes of microcephaly include both degenerative and static dis-
eases. Degenerative diseases present from shortly after birth to much later in life, with
progressive worsening of symptoms. In contrast, abnormalities of primary brain de-
velopment are present at birth. The clinical symptoms usually do not worsen over time.
The abnormalities in brain development that lead to microcephaly will be the focus of
this chapter.
The cerebral cortex makes up the largest structure of the human brain and is
always affected in microcephaly. Even though most brain volume is made up of
neuropil (glia, dendrites, etc.), the number of neurons determines the amount of
neuropil present. As a consequence, the number of neurons is an important deter-
minant of brain size. Knowing the factors regulating cerebral cortical cell number
is critical to understanding the many causes of microcephaly. Because murine cere-
bral cortical development is best understood, it will be presented as a model, though
higher mammals likely use similar mechanisms (Caviness et al. 1995). Proper regu-
lation of this process is important since most cerebral cortex neurons can be born
only during the normal neurogenesis process (Spalding et al. 2005; Bhardwaj et al.
2006).

1 Department of Neurology, Beth Israel Deaconess Medical Center,

e-mail: ecgilmore@partners.org
2 Child Neurology, Massachusetts General Hospital
3 Division of Genetics, Children’s Hospital Boston, Howard Hughes Medical Institute, Boston,
MA, USA

Gage et al.
Retrotransposition, Diversity and the Brain
© Springer-Verlag Berlin Heidelberg 2008
110 E.C. Gilmore et al.

Model of Cerebral Cortical Development

The murine cerebral cortex develops in the second half of gestation from a single
layer of pseudostratified epithelium to a six-layered structure from embryonic day
10 (E10) to around birth. When neurons differentiate, they migrate away from the
ventricular zone, next to the ventricular surface, towards the pial surface and past any
previously born neurons to the superficial regions of the cortical plate. This process
results in a cortex that has the earliest born cortical neurons residing in the deepest
portions of the cerebral cortex whereas the latest born reside superficially. There are
also functional differences between deep and superficial neurons, with the deepest
neurons projecting to thalamic regions and below whereas more superficial neurons
project to other regions of cerebral cortex. Prior to neurogenesis, the founding pool
of precursor cells in the ventricular zone appears to be regulated via programmed
cell death, as shown by the targeted deletion of genes required for apoptosis resulting
in enlargement of the cerebral cortex from excessive precursor cells, not from lack of
apoptosis later in development (Cecconi et al. 1998; Hakem et al. 1998; Kuida et al. 1998;
Yoshida et al. 1998). The cell cycle length of dividing cells is critical because slower
cell cycles result in fewer cell divisions within a given period of time. The length of
neurogenesis determines how many cell cycles can occur.
The differentiating-to-proliferating ratios of ventricular precursors are also impor-
tant to cerebral cortical size. As cells divide, there are three potential fates for the two
daughter cells: two precursors, one precursor and one neuron, or two neurons. As neu-
ronal precursors divide, the fates of the two precursors or two differentiated neurons
can be thought of as symmetric in that both cells have the same fate. In contrast, when
daughter cells are one differentiated neuron and one precursor, they can be thought of
as asymmetric because the fates of the two cells are different. These choices are tightly
controlled because if too many cells left the proliferating pool to differentiate early
in cortical development, there would be fewer cells in later portions of neurogenesis,
resulting in microcephaly (see Fig. 1B). Since the timing of differentiation determines
laminar position and phenotype (McConnell and Kaznowski 1991), too many neu-
rons differentiating early would leave too many early-born, deep neurons and too few
later-born, superficial neurons.
Each factor that determines the number of neurons, progenitor pool size, cell cycle
length, and differentiating/proliferating ratio can be affected by diseases causing mi-
crocephaly. Not surprisingly, chromosome number, growth factors, and transcription
factors can play an important role in each of these events (Haydar et al. 2000; Hodge
et al. 2004; Roy et al. 2004; Yun et al. 2004). Differentiation/proliferation ratios are likely
disrupted in many microcephaly vera patients.

Specific Microcephalies

Microcephaly vera (true microcephaly) is a group of autosomal recessive diseases of ab-

normal brain development that result in mental retardation but not other neurological
abnormalities (Woods et al. 2005). There are six known genetic loci that result in mi-
crocephaly vera (MCPH1-6); four of these have been cloned, but there are undoubtedly
others. MCPH5/ASPM, MCPH3/CDK5RAP2, and MCPH6/CENPJ encode proteins that
 Microcephalies and DNA Repair 111

Fig. 1. Models of normal development and microcephaly. Prior to cerebral corticogenesis at E9,
cell division produces more precursors via symmetric division (blue cells separating in the plane
of the ventricular surface) whereas apoptosis (crossed-out cells) eliminates some, establishing
the proper number of cerebral cortical precursors. (A) During normal development early in
corticogenesis at E13, many symmetrically dividing ventricular zone cells produce two additional
precursors (blue cells). However, asymmetric cell division (cleave plane away from ventricular
surface) produces one neuron that will eventually reside in deep regions of cortex (green) and
one precursor. Differentiating neurons migrate (elongated cell with arrowhead) to the superficial
regions of the cortical plate. Late in corticogenesis at E16, fewer mitoses produce additional
precursors, and more neurons (tan circles) are produced. Differentiating neurons migrate past
earlier born neurons to superficial regions of the cortical plate. (B) During early cortical plate
stage at E13, asymmetric division occurs too frequently, resulting in too many early-born, deep
layer neurons and too few precursors within the ventricular zone. Too few neuroblasts later in
development at E16 result in too few late-born, superficial neurons within the cortical plate since
they are produced at this time, with microcephaly as a result. (C) Mutations in NHEJ genes
result in cell death of differentiated neurons (crossed-out cells) that can be in either the cortical
plate or deeper near the ventricular zone. This process results in microcephaly because too few
neurons are present due to apoptosis after differentiation. (D) Mutations in XRCC2, which recent
studies have shown are required for homologous recombination, appear to result in cell death of
precursor cells and not of differentiated cells. Too few cortical neurons are present because too
few precursors are present

likely associate with spindle poles/centrosomes (Bond et al. 2005; Kouprina et al. 2005).
These genes potentially play a critical role in determining if neuronal precursors con-
tinue to divide or differentiate. In Drosophila, cellular polarity determines neuronal fate
via asymmetric division of cellular components (see Wodarz 2005 for review). There is
112 E.C. Gilmore et al.

evidence that similar mechanisms play a role in vertebrates as well (Chenn and Walsh
2002; Gotz and Huttner 2005). Mutations in a spindle-associated protein can cause
diminished cerebral cortical sizes through changing differentiation-to-proliferation
ratios (Feng and Walsh 2004; see Fig. 1B for model). However, the actual mechanisms
leading to these microcephalies remain to be definitively determined.
The function of the other identified microcephaly vera gene, MCPH1/micro-
cephalin, is potentially different. Mutations in microcephalin result in abnormalities
in chromosome condensation (Trimborn et al. 2004). In addition, the microcephalin
gene contains multiple BRCA1 C-terminal domains. These domains share homology
with proteins involved in cell cycle control and DNA repair. Microcephalin, also known
as BRIT1, appears to play a role in the response to DNA repair (Xu et al. 2004; Lin et al.
2005; Alderton et al. 2006); which could indicate that the function of microcephalin is
similar to other genes that result in microcephaly and is required for DNA repair.

DNA Repair and Neuronal Development

Damage to DNA can result in multiple abnormalities, including cancer, from activation
of oncogenes, disruption of cell cycle inhibitors, or defects in apoptotic pathways.
Neurons terminally differentiate, never to divide again. However, they are still subject
to DNA-damaging agents, such as oxidative stress, radiation, and other insults. This
susceptibility is especially important for humans since most, if not all, cortical neurons
may be present at the time of birth (Au and Fishell 2006). If DNA is not repaired
properly, the cells may undergo apoptosis or there could be defects in gene products.
Neurological phenotypes that result from abnormalities in DNA repair genes fall
into two groups. Some cause neurodegenerative diseases and others cause abnormal-
ities in neuronal development. These two phenotypes are not mutually exclusive, but
there are potentially useful distinctions. Those that cause neurodegeneration are not
the focus of this chapter and will only be discussed briefly to contrast with the devel-
opmental mutations.
Most of the neurological degenerative diseases that involve DNA repair revolve
around damage to the cerebellum and result in ataxia, a movement disorder. The best
characterized of these diseases is ataxia telangiectasia (A-T), resulting from mutations
in the ATM gene (Savitsky et al. 1995). Patients with A-T have progressive ataxia,
abnormal eye movements, dermal blood vessel proliferation, cancer predisposition
and immunodeficiency (Chun and Gatti 2004). A clinically similar disorder is known
as A-T-like disease and has similar clinical features. It is caused by mutations in the
gene MRE11 (Stewart et al. 1999). ATM is a protein that exists very early in the signaling
cascade that responds to DNA damage, in double-stranded DNA in particular. MRE11
is a component of MRN complex that is thought to help activate ATM and it coordinates
downstream signaling effects of ATM as well (Uziel et al. 2003; Taylor et al. 2004). Other
ataxia syndromes, ataxia with oculomotor apraxia (AOA) 1 and spinocerebellar ataxia
with neuropathy, are associated with mutations in the genes for aprataxin and tyrosyl-
DNA phosphodiesterase 1 (TDP1; Moreira et al. 2001; Takashima et al. 2002). Aprataxin
helps release adenosine mono-phosphate bound to 5’ phosphates that can occur during
ligation of broken DNA ends (Ahel et al. 2006; Rass et al. 2007). TDP1 is involved in
single-stranded DNA repair, and its mechanism of action could be related to helping
 Microcephalies and DNA Repair 113

release topoisomerase that is covalently attached to DNA (El-Khamisy et al. 2005).

Cockayne syndrome is associated with spasticity, mental retardation, and hearing loss
that seems to affect the white matter (axon tracts) more than the gray matter (neurons;
Rapin et al. 2000). It results from abnormalities in the single-stranded DNA repair
pathway nucleotide excision repair (Fousteri et al. 2006). Xeroderma pigmentosum is
characterized by photosensitivity, and cancer can also result in neuronal degeneration
leading to microcephaly. Xeroderma pigmentosum has been associated with eight
different genes involved in nucleotide-excision repair (Lehmann 2003).
In contrast to the degenerative diseases in which neuronal tissues form normally
and eventually die, abnormalities in neuronal production characterize the develop-
mental diseases. A number of DNA repair genes are required for normal neuronal de-
velopment. Like microcephaly vera described above, too few neurons result in smaller
brains. Nijmegen breakage syndrome is associated with microcephaly at birth, mental
retardation, cancer, and immunodeficiency (Digweed and Sperling 2004). This syn-
drome results from mutations in the NBS1 gene (Carney et al. 1998; Matsuura et al.
1998; Varon et al. 1998). NBS1 is also part of the MRN complex that helps activate ATM
and also acts downstream of ATM activation (see Kobayashi et al. 2004 for review).
Seckel syndrome has primary dwarfism with even more profound microcephaly than
is proportional to the body (Shanske et al. 1997). It has multiple linkage groups, but one
form is caused by mutations in the ATM- and RAD3-related gene, ATR. ATR functions
in a manner similar to ATM and helps activate cascades that respond to DNA damage
(Niida and Nakanishi 2006). Seckle syndrome patients do not have cancer predispo-
sition or immunodeficiency. Another disease characterized by microcephaly, mental
retardation and immunodeficiency is associated with mutations in the DSB repair gene
cernunnos (Buck et al. 2006). Cerunnos is involved in non-homologous end joining
(NHEJ), which repairs double-stranded DNA breaks. NHEJ brings two damaged ends
together, sometimes removing nucleotides before ligation and often resulting in imper-
fect repairs (Lees-Miller and Meek 2003). It can be used during all parts of the cell cycle
(Rothkamm et al. 2003). Homologous recombination (HR) uses a replicated strand of
DNA as a template for repair of double-strand breaks and does not introduce errors
(Sonoda et al. 2006). HR can only be used in late S and G2 phases of cell replication
since it requires a copied strand (Rothkamm et al. 2003).

Double-Stranded Break Repair and Microcephaly

NHEJ repair has a critical role during murine neuronal development as well. Targeted
disruption of several genes required for NHEJ interrupts lymphocyte development and
DNA repair and causes neuronal death whereas all other tissues are apparently spared.
Disruption of Ligase IV, XRCC4, Ku70/Ku80, and some mutations in DNA-dependent
kinase (DNA-PK) results in partial or severe neuronal cell death around the time of
terminal differentiation in mice (Barnes et al. 1998; Frank et al. 1998; Gao et al. 1998;
Gu et al. 2000; Vemuri et al. 2001). Disruption of ATM (a DNA damage sensor) or p53
(a downstream DNA damage mediator that helps regulate between cell cycle arrest and
apoptosis) along with ligase IV or p53 with XRCC4 prevents neuronal death during
development (Frank et al. 2000; Gao et al. 2000; Lee et al. 2000; Sekiguchi et al. 2001).
Interestingly, deletion of ATM in addition to Ku70, Ku80 or DNA-PK results in early
114 E.C. Gilmore et al.

embryonic lethality for unknown reasons (Gurley and Kemp 2001; Sekiguchi et al.
2001). Human mutations in Ligase IV have also been found to result in microcephaly
and immunodeficiency, supporting the relevance of murine findings in understanding
human development (O’Driscoll et al. 2001).
HR is also critical for murine brain development. Targeted disruption of XRCC2,
a gene involved in HR, results in apoptosis within the developing brain (Deans et al.
2000). Although these mice do not survive birth and fewer embryos than expected
are present late in gestation, these authors found that the dying cells appeared to be
differentiated neurons. However, a more recent analysis with conditional deletion of
XRCC2 using a neuron-specific Cre found that dying cells appeared to be proliferative,
not post-mitotic (Orii et al. 2006). The reason for the difference in findings is not clear
at this point. Dividing precursors may rely upon HR to repair DNA damage while they
are still proliferative since HR has the advantage that it is not error prone. However,
once neurons differentiate, HR is no longer an option since HR requires a copied strand.
Therefore, NHEJ must be used to repair double-stranded DNA breaks in differentiated
neurons, which could explain why most dying cells in NHEJ mutants appear to be
differentiated neurons.
For the purpose of this chapter, we have separated DNA repair genes into those
that affect primary brain development from those that cause neurodegeneration. In-
terestingly, defects that either cause neuronal cell death during development (ligase VI,
XRCC4, Ku70, Ku80, DNA-PK, NBS) or microcephaly (Seckles, cernunnos, ligase VI,
Nijmegen breakage syndrome) are associated with double-stranded DNA break repair.
Many mutations that result in neuronal degeneration are in genes that are critical for
single-stranded DNA repair (ataxia with oculomotor apraxia 1, spinocerebellar ataxia
with neuropathy, cockayne syndrome, and xeroderma pigmentosum). However, the
genes mutated in A-T and A-T-like disorder play a role in double-stranded DNA re-
pair. In fact, NBS1 (Nijmegen breakage syndrome) and MRE11 (A-T-like disorder) are
within the same complex upstream of ATM and ATR but may have different functions in
downstream signaling. The reason underlying the phenotypic differences of ATM/ATR
and NBS1/MRE11 mutations remains a mystery. Part of this underlying mystery is the
requirement for DNA repair in the first place (see below).

Integrating Models of Microcephaly

Different models for the development of microcephaly can be created from the data
presented so far (Fig. 1). Microcephaly can develop from failure to produce enough neu-
rons, which occurs when precursors cannot perform enough cell cycles or when ratios
of differentiating-to-proliferating cells favor differentiation too early. MCPH5/ASPM,
MCPH3/CDK5RAP2, and MCPH6/CENPJ fall into this category. Alternatively, the cel-
lular death in differentiated neurons or precursors that results from mutations in DNA
repair proteins will also result in microcephaly.
Data also suggest that the processes of DNA repair and neuronal symmet-
ric/asymmetric division could be related. As discussed above, microcephalin poten-
tially is involved in DNA repair and chromosome condensation, which could mean
that microcephalin is involved in both models of microcephaly, DNA repair and cell
proliferation. In addition, the identification of PAR3 as a protein that can work in
 Microcephalies and DNA Repair 115

conjunction with the NHEJ pathways suggests intriguing ties between DNA repair and
cell polarity establishment (Fang et al. 2007). PAR3 is known to be involved in control-
ling asymmetric division in primitive organisms and establishing neuronal polarity in
mammals (Joberty et al. 2000; Lin et al. 2000; Shi et al. 2003; Nishimura et al. 2004).
However, PAR3 also is associated with the NHEJ proteins Ku70/80 in normal conditions
and DNA-PK after irradiation. In addition, reduction of PAR3 causes cells to be more
susceptible to irradiation (Fang et al. 2007). The fact that PAR3 has a role both in
polarity and DNA repair could mean that DNA repair intrinsically is associated with
neuronal differentiation related to asymmetric division. Alternatively, evolution may
be merely resourceful in using the same factors for distinct pathways, both involving
DNA, either in repairing DNA or translocating nuclei into a differentiated cell.

Sources of DNA Breaks During Development

The vast diversity found among neurons has long led to speculation that DNA rear-
rangements could play a role in neuronal development as it does in lymphoid cells. For
instance, the restricted expression of one or several olfactory receptors in an individual
olfactory neuron out of approximately one thousand present in the genome (Zhang and
Firestein 2002; Niimura and Nei 2003) was a candidate for DNA rearrangement. Some
hypothesized a regulatory mechanism for olfactory neurons that was analogous to
immunoglobulin and T-cell receptor development. The expression of RAG1 in the ner-
vous system further fueled speculation (Chun et al. 1991). However, targeted deletion
in RAG1 did not result in any neuronal abnormalities (Mombaerts et al. 1992). In fact,
olfactory neurons can be used for somatic nuclear transfer to clone mice with normal
expression of olfactory receptors, indicating that there are no permanent changes to
the genome in these cells (Eggan et al. 2004; Li et al. 2004). However, when cortical plate
neurons were used as the source for nuclei in similar experiments, they performed
poorly. Nuclei derived from the ventricular zone were 10 times more efficient at pro-
ducing clones than differentiated neurons from the cortical plate (Yamazaki et al. 2001).
The reason for the reduction in the cortical neurons totipotency remains unclear.
A potential mechanism for DNA damage during development is oxidation. Evidence
of excessive oxidative damage can be found in neurons deficient in NHEJ repair proteins
(Karanjawala et al. 2002; Narasimhaiah et al. 2005). The source of oxidative damage is
speculated to be high energy metabolism during neuronal development associated with
rapid proliferation. However, cell cycle lengths in embryonic cerebral cortex (9.5 hrs)
are not dramatically faster than they are in the heart (13.4 hrs) at E10 in the rat. In
addition, cell cycle times increase further into cerebral cortical development while cells
become more susceptible to defects in NHEJ (Mirkes et al. 1989; Barnes et al. 1998;
Frank et al. 1998; Gao et al. 1998; Gu et al. 2000; Vemuri et al. 2001; Nowakowski et al.
2002). However, differentiating cells must rely upon NHEJ to repair double-stranded
breaks as discussed above, which may explain why neurons become more susceptible
during development. In addition, neurons have a period of naturally occurring cell
death whereas cardiac myocytes do not. This period of naturally occurring neuronal
death matches the number of afferents to targets and prunes neurons that do not make
proper connections (Oppenheim 1991). Therefore, neurons may be more susceptible
to DNA damage because of priming for programmed cell death.
116 E.C. Gilmore et al.

Another source of double-stranded breaks that could require repair during neu-
ronal development is the movement of L1 retrotransposons (Muotri et al. 2005). The
biology and functions of L1 retrotransposons are a main focus of this symposium and
do not need to be extensively detailed. Briefly, L1 retrotransposons appear to be able
to take advantage of DNA damage to find loci to integrate into (Rudin and Thompson
2001; Hagan et al. 2003). In addition, abnormalities in double-stranded break repair
factors can facilitate movement of retrotransposons (Morrish et al. 2002). However,
other retrotransposons may have difficulty moving in the absence of double-stranded
DNA break repair (Downs and Jackson 1999; Izsvak et al. 2004). The movement of
retrotransposons in developing neurons may require the machinery of DNA repair and
their movement may be facilitated by other sources of DNA damage.
The requirements for proper neuronal development are beginning to be understood
by study of both human and mouse mutations that result in microcephaly. The cellular
processes that lead to deficient numbers can be caused by either failure to produce
enough cells or cell death from failure to repair DNA. The relationship between these
seemingly unrelated processes is interesting but remains circumstantial. Increasing
neuronal diversity through movement of L1 retrotransposons or other mechanisms
associated with DNA repair is intriguing but is not well defined. However, DNA repair
is clearly integral to neuronal development, and understanding why that is so is likely
to be beneficial for understanding human disease.

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Subject Index

Antibodies (diversity of) 54 LINE-1 2, 22–28, 34, 43–62, 69, 116

Antibodies to retroviral proteins in psychiatric – Evolutionary consequences of L1 impact in
disorders 72–76 neuronal genomes 60–62
Asymmetry as a determinant of human ability – Mechanism of LINE-1 integration 24
94, 95 – Replicative cycle of LINE-1 23

Brain complexity 53–64 Manic depressive illness 87, 88, 92

McClintock B. 8, 53
Catalytic RNA 2–4, 34, 35 Memory 13–18
Cerebral asymmetries 92–96 Microcephaly and DNA repair 109–120
Cerebral cortical development 109–112 Mobile element 8, 55
Cerebral dominance gene 87–108 Multiple sclerosis 69
Crick F. 53
Cytomegalovirus 67 Neural stem cell 8, 9, 56–58
Neuronal progenitor 53, 58–60
Dawkins R. 25 Neuropsychiatric disorders 65–108
Diversity (Generation of) 53–64 NMDA receptor 13–17
DNA breaks during development 115, 116 Novelty detection 17
DNA repair and neuronal development 112,
113 Pattern completion 13–15
Double-stranded break repair and Pattern separation 15–17
microcephaly 113, 114
Phylogenetic tree of life and rDNA sequence
alignments 21
Entropy exchange model 36
Polymorphisms in HERV K18 76–80
Gag protein 36 Prebiotic world 34, 40
Gene creation 44, 45 Primitive RNA world 33–40
Glial cell missing (Gcm) 71, 72 Prion PrP 39, 93
Protocadherin X/Y 87, 98
Herpes simplex virus 67 Proto-oncogene 92, 93
Hippocampal learning (molecular and circuit Psychosis 65–108
mechanisms for) 13–18 – Continuum of 91, 92
HIV 36, 67 – Season of birth effect 92
Human diversity and L1 retrotransposon
biology 43–52 RNA chaperone 35–40, 43
Human endogenous retrovirus 67–80 RNA world and brain complexity 53–64
Human evolution 87, 92–94 RNA world hypothesis 33
Retrotransposition 8, 9
Influenza virus 67, 72, 88, 89 – Neural-specific retrotransposition 28
Retrotransposons 21–62, 98–103
Junk DNA 53, 61 – Ancient retrotransposons as possible
remnants of the primitive RNA world
Language lateralization 94 33–42
122 Subject Index

– Cancer and 28, 55 – Temporal geographical variations 89, 90

– Creation of new genes and 43–52 Selfish gene 25, 26, 53, 55
– Human diversity and 43–52 Sox protein 56–58
– Mapping transposon insertion site 22, 27 Syncitin 71, 72
– Natural and synthetic 21–32
– Neural-specific 28 Telomerase 1–12
– Psychosis and 87–108 – Control of 4–5
– Replication 37–39 – Evolution of 2
– Silencing and activation of 55–58 – Reverse transcription and 8, 9
– Xq21.3/Yp and 98–103 – Roles of 5, 6
Retrovirus 34, 40, 65–108 – Stress and 7, 8
Reverse transcriptase 1–3, 8, 9, 24, 43, 92
Telomeres and aging 6–8
Schizophrenia 65–108 Telomere and stress 7, 8
– As an anomaly of development 95, 96 Telomeres and telomerases in human health
– Environmental theory of 87, 88 and disease 1–12
– Epidemiology 89, 90 Toxoplasma 67, 68
– Genetic predisposition of 66, 88, 91–94
– Infectious disease and 88, 89 Xq21.3/Yp translocation 96–103
– Seasonality of 89 XY homology 96–98
List of volumes published in the series
“Research and Perspectives in Neurosciences”

P. Ascher, D.W. Choi, Y. Christen (Eds.) (1991) Glutamate, Cell Death and Memory,
ISBN 3-540-54134-9
F.H. Gage, Y. Christen (Eds.) (1992) Gene Transfer and Therapy in the Nervous System,
ISBN 3-540-55889-6
A.-M. Thierry, J. Glowinski, P.S. Goldman-Rakic, Y. Christen (Eds.) (1994) Motor and
Cognitive Functions of the Prefrontal Cortex, ISBN 3-540-57128-0
G. Buzsáki, R. Llinás, W. Singer, A Berthoz, Y. Christen (Eds.) (1994) Temporal Coding
in the Brain, ISBN 3-540-58074-3
A.R. Damasio, H. Damasio, Y. Christen (eds.) (1996) Neurobiology of Decision-Making,
ISBN 978-3-540-60143-2
F.H. Gage, Y. Christen (Eds.) (1997) Isolation, Characterization and Utilization of Stem
Cells, ISBN 978-3-540-26253-4
A.M. Galaburda, Y. Christen (Eds.) (1997) Normal and Abnormal Development of the
Cortex
J. Grafman, Y. Christen (Ed.) (1999) Neuronal Plasticity: Building a Bridge from the
Laboratory to the Clinic, ISBN 978-3-540-64357-9
P. Patterson, C. Kordon, Y. Christen (Eds.) (1999) Neuro-Immune Interactions in Neu-
rology and Psychiatric Disorders, ISBN 978-3-540-66013-2
C.E. Henderson, D. Green, J. Mariani, Y. Christen (Eds.) (2001) Neuronal Death by
Accident or by Design, ISBN 978-3-540-41777-4
J. Mallet, Y. Christen (Eds.) (2003) Neurosciences at the Postgenomic Era, ISBN 978-3-
540-00194-2
F.H. Gage, A. Björklund, A. Prochiantz, Y. Christen (Eds.) (2004) Stem Cells in the
Nervous System: Functional and Clinical Implications, ISBN 978-3-540-20558-6
J.-P. Changeux, A.R. Damasio, W. Singer, Y. Christen (Eds.) (2005) Neurobiology of
Human Values, ISBN 978-3-540-60143-2
B. Bontempi, A.J. Silva, Y. Christen (Eds.) (2007) Memories: Molecules and Circuits,
ISBN 978-3-540-45698-8