www.nature.

com/npjparkd

ARTICLE OPEN

LRRK2 levels in immune cells are increased in Parkinson’s

disease
D. A. Cook1, G. T. Kannarkat1, A. F. Cintron1, Laura M. Butkovich1, Kyle B. Fraser2, J. Chang1, N. Grigoryan1, S. A. Factor3, Andrew B. West2,
J. M. Boss4 and M. G. Tansey1

Mutations associated with leucine-rich repeat kinase 2 are the most common known cause of Parkinson’s disease. The known
expression of leucine-rich repeat kinase 2 in immune cells and its negative regulatory function of nuclear factor of activated T cells
implicates leucine-rich repeat kinase 2 in the development of the inflammatory environment characteristic of Parkinson’s disease.
The aim of this study was to determine the expression pattern of leucine-rich repeat kinase 2 in immune cell subsets and correlate it
with the immunophenotype of cells from Parkinson’s disease and healthy subjects. For immunophenotyping, blood cells from 40
Parkinson’s disease patients and 32 age and environment matched-healthy control subjects were analyzed by flow cytometry.
Multiplexed immunoassays were used to measure cytokine output of stimulated cells. Leucine-rich repeat kinase 2 expression was
increased in B cells (p = 0.0095), T cells (p = 0.029), and CD16+ monocytes (p = 0.01) of Parkinson’s disease patients compared to
healthy controls. Leucine-rich repeat kinase 2 induction was also increased in monocytes and dividing T cells in Parkinson’s disease
patients compared to healthy controls. In addition, Parkinson’s disease patient monocytes secreted more inflammatory cytokines
compared to healthy control, and cytokine expression positively correlated with leucine-rich repeat kinase 2 expression in T cells
from Parkinson’s disease but not healthy controls. Finally, the regulatory surface protein that limits T-cell activation signals, CTLA-4
(cytotoxic T-lymphocyte-associated protein 4), was decreased in Parkinson’s disease compared to HC in T cells (p = 0.029). In sum,
these findings suggest that leucine-rich repeat kinase 2 has a regulatory role in immune cells and Parkinson’s disease. Functionally,
the positive correlations between leucine-rich repeat kinase 2 expression levels in T-cell subsets, cytokine expression and secretion,
and T-cell activation states suggest that targeting leucine-rich repeat kinase 2 with therapeutic interventions could have direct
effects on immune cell function.
npj Parkinson’s Disease (2017)3:11 ; doi:10.1038/s41531-017-0010-8

INTRODUCTION domain8 and a serine/threonine kinase domain.9 There are also

Parkinson’s disease (PD) is a progressive age-related movement multiple protein-interacting domains, including a leucine-rich
disorder. The histopathological features of PD include degenera- repeat domain, a C-terminal WD40 repeat domain, and armadillo
tion of dopaminergic neurons in the substantia nigra pars and ankyrin repeat domains.6, 7 Given the multiple, highly diverse
compacta (SNpc) and the presence of Lewy bodies (neuronal enzymatic and protein interacting domains, it is likely that LRRK2
inclusions of aggregated α-synuclein and other ubiquitinated may have different binding partners in different cell types. In
proteins). Despite decades of extensive study, the etiology of the support of this, LRRK2 has been shown in vitro to influence
sporadic form of PD remains unclear. Thus, the development of regulation of autophagy, macroautophagy,10 ceramide metabo-
new treatments and therapeutics has been slow-paced. In 2004, lism,11 neurite outgrowth, vesicular trafficking, cytoskeletal com-
multiple labs identified mutations in the leucine-rich repeat kinase ponents, and cell signaling pathways involving nuclear factor of
2 (LRRK2) gene as causative for a dominantly inherited form of PD, activated T cells (NFAT), Wnt, and nuclear factor-κB.10, 12–17
leading to an exciting new pathway for researchers to pursue.1, 2 Multiple mutations and normal genetic variations in the LRRK2
Mutations have also been found in sporadic cases at rates varying gene have been associated with disease.18 The six most common
from 0.3–41% depending on the country of origin and ethnicity of pathogenic mutations in LRRK2 associated with PD19 reside in the
the population studied.3 Due to similarities in the clinical GTPase and kinase domains.20, 21 The most prevalent mutation is
presentation of LRRK2-associated PD and idiopathic PD,3–5 the the G2019S mutation in the kinase domain.3 Although mutations
study of LRRK2 function has the potential to offer new insight into in LRRK2 only account for 1–2% of all PD cases, they are
the mechanisms underlying sporadic PD etiology. particularly prevalent in individuals of Ashkenazi Jewish (29.7%)
The LRRK2 gene is large, containing 51 exons that code for a
and North African Arab ancestry (41%) (ref. 3). The penetrance of
2527-amino acid protein of large molecular weight (~286 kDa)
the most common LRRK2 mutation (G2019S) ranges from 28% at
with several different functional and protein-interacting domains.6, 7
Enzymatic domains include a ROC (Ras of complex) GTPase 54 years of age to 74% at 79 years of age, suggesting that genetic

1
Department of Physiology, Emory University School of Medicine, Atlanta, GA, USA; 2Department of Neurology, Center for Neurodegeneration and Experimental Therapeutics,
University of Alabama at Birmingham, Birmingham, AL, USA; 3Department of Neurology and Movement Disorders Center, Emory University School of Medicine, Atlanta, GA, USA
and 4Department of Microbiology and Immunology, Emory University School of Medicine, Atlanta, GA, USA
Correspondence: M. G. Tansey (malu.tansey@emory.edu)

Received: 11 October 2015 Revised: 26 January 2017 Accepted: 2 February 2017

Published in partnership with the Parkinson’s Disease Foundation

 LRRK2 levels in PD immune cells
D A Cook et al.
2
and environmental modifiers influence lifetime risk for PD in KO mice (Supplementary Fig. S1b). In addition, there is a minimal
individuals with these mutations. shift in the histograms for the PBMCs from the mouse WT LRRK2-
Importantly, LRRK2 is not only expressed in neurons but is also or mouse G2019S-LRRK2-overexpressing BAC transgenic mouse
expressed in cells of both the innate and adaptive immune lines indicating lack of recognition of mouse LRRK2 via flow
system.22, 23 Interestingly, LRRK2 polymorphisms have been cytometry (Supplementary Fig. S1b).
associated with Crohn’s disease, an autoimmune inflammatory To validate the specificity of the LRRK2 antibody for human
bowel disease, and leprosy, an infection caused by Mycobacterium LRRK2 using flow cytometry applications, we performed experi-
leprae, supporting a link to immune function.24, 25 LRRK2 is also a ments in which induction of the human LRRK2 protein was
member of the receptor interacting protein kinase family, which confirmed in both a human monocytic cell line (THP-1) and
are proteins that detect and respond to cellular stress by primary human monocytes using both western blot and flow
regulating cell death and activation of the immune system.26, 27 cytometry under the same experimental conditions. A single
Pro-inflammatory signals, such as interferon-γ (IFN-γ),23, 28, 29 immunoreactive band was detectable in immunoblots with the
lipopolysaccharide (LPS),22, 30 and interleukin (IL)-1β (ref. 17) have c41-2 LRRK2 antibody in T cells, B cells, and monocytes isolated
been shown to increase LRRK2 expression. Specifically, in CD14+ from human peripheral blood (Supplementary Fig. S1a). Next,
macrophages, CD3+ T cells, and CD19+ B cells, increases in naive THP-1 cells were found to express low amounts of LRRK2
LRRK2 following IFN-γ stimulation have been observed.23, 28, 29 protein. Upon treatment with PMA, the cells differentiate into
Also, IFN-γ stimulation was shown to increase LRRK2 mRNA and macrophages and following stimulation with IFN-γ, their expres-
protein expression in the non-classical CD14+CD16+ monocyte sion of LRRK2 protein can be shown to increase by western blot
population.28 Pharmacological inhibition of LRRK2 with multiple (Fig. 1a, b). Importantly, this increase is accompanied by an
inhibitors resulted in decreased CD14, CD16, and major histo- increase in the LRRK2 signal by flow cytometry (Fig. 1c) using the
compatibility complex (MHC-II) expression, suggesting that LRRK2 c41-2 LRRK2 antibody and either of two different secondary
may play a significant role in the activation of monocytes via antibodies (Alexa-Fluor647 or FITC). In addition, human primary
IFN-γ.28 In addition, use of LRRK2 inhibitors results in decreased monocytes isolated from peripheral blood were found to express
LRRK2 protein expression after 24 h exposure in peripheral blood detectable levels of human LRRK2 protein by western blotting
mononuclear cells (PBMCs).31 Recently, it was reported that with c41-2 (Fig. 1a, b) and by flow cytometry with the same LRRK2
increased expression of LRRK2 in monocytes following IFN-γ antibody (Fig. 1c). As was seen in the cell lines, the levels of LRRK2
stimulation occurs via a mechanism involving extracellular signal- in the primary cells could be increased further by stimulation with
related kinase 5 (ERK5) signaling.29 IFN-γ as measured by western blot (Fig. 1a, b) and flow cytometry
Despite a growing wealth of evidence that LRRK2 is enriched in (Fig. 1c).
both innate and adaptive immune cells,26, 27 to date the majority
of studies involving LRRK2 and its associated mutations have
mainly been assessed for their effects on neuronal function. Given PD is associated with increased LRRK2 expression in innate and
that increased inflammation in the periphery and the brain has adaptive immune cells
been associated with the pathophysiology of PD,32–36 it is possible To investigate if LRRK2 levels differ between specific immune cell
that LRRK2 may play a role in this process and serve as a regulator populations and between healthy individuals and those with PD,
of inflammatory and immune responses that influence risk for immunophenotyping of peripheral blood was performed in cells
age-related degeneration and risk for PD. Based on the current from PD and age-matched healthy control (HC) subjects. LRRK2
literature, we hypothesized that LRRK2 expression is increased in expression has been reported in both monocytes and B cells,22, 23
cells from PD patients, causing a dysregulation of function and however, there are conflicting reports as to whether LRRK2 is
activation in cells of both the innate and adaptive immune system. expressed in T cells.22, 28 To date, the most commonly used
To test this, we investigated the extent to which various peripheral approach to ascertain LRRK2 expression levels has been western
immune cell types express LRRK2 and inflammatory cytokines blotting and quantitative reverse transcriptase-polymerase chain
under resting conditions and during immune activation in healthy reaction. Differences between antibodies and oligonucleotide
individuals and in patients with sporadic PD. primers could account for discrepancies between assays and labs.
We found that LRRK2 protein levels, reported as median
fluorescence intensity (MFI), were increased in CD16+ monocytes,
T cells, and B cells from PD patients compared to HC (Fig. 2a, b).
RESULTS
Importantly, the increase in LRRK2 is not a reflection of overall
Validation of Abcam c41-2 LRRK2 antibody for detection of human increases in protein content in immune cells in PD subjects vs. HCs
LRRK2 by flow cytometry as other cell-specific markers such as CD19, CD14, and 4-1BB were
The c41-2 Abcam LRRK2 (MJFF2) antibody is a rabbit polyclonal not significantly different between HC and PD subjects (Supple-
antibody used in the studies herein to detect human LRRK2 mentary Fig. S2).
protein in peripheral immune cell populations. Because the LRRK2 Given the diverse functions associated with T-cell subsets, we
antibody was not conjugated to a fluorophore, a fluorescein determined whether the increased LRRK2 levels in the PD group
isothiocyanate (FITC)-conjugated secondary anti-rabbit IgG anti- were altered in all subsets or only specific subsets. The data were
body was used for the flow cytometry studies. Although the stratified by CD8+ effector subsets and CD4+ effector, helper, and
manufacturer notes that c41-2 is cross-reactive with mouse LRRK2 regulatory subsets (Supplementary Table 3). We found no
protein in western blotting and histological applications, c41-2 significant difference in LRRK2 expression between different T-
only detects mouse LRRK2 when it is highly overexpressed as in cell subsets, but its levels are globally increased in all subsets in PD
the mouse WT-LRRK2- or mouse G2019S-LRRK2-overexpressing patients compared to HC subjects (Fig. 2c–e). One small exception
BAC transgenic lines (Supplementary Fig. S1a). In our hands, c41-2 is the CD4+ Teff subset; when comparing the PD and HC subjects
displays minimal cross-reactivity with the mouse LRRK2 protein the p-value was 0.051. In our study cohort, patients with PD had
relative to the human protein in peripheral blood immune cells increased LRRK2 expression in T cells, B cells and a pro-
under conditions for western blotting of sodium dodecyl sulfate inflammatory subset of monocytes (CD16+).
polyacrylamide gel electrophoresis. Consistent with this, the flow In addition to protein expression levels in immune cells, we
signal from endogenous mouse LRRK2 protein in PBMCs from assessed the frequencies of immune cell populations in our
C57BL/6J mice is indistinguishable from that of PBMCs from LRRK2 subjects. No significant differences between PD and HC in the

npj Parkinson’s Disease (2017) 11 Published in partnership with the Parkinson’s Disease Foundation
 LRRK2 levels in PD immune cells
D A Cook et al.
3
a THP-1 Human Monocytes
b LRRK2
THP-1 +PMA IFNγ γ 0.8
*

Densitometric Units
Relative to GAPDH
250kDa LRRK2 0.6

0.4 ***

0.2

0.0
THP-1 THP-1 Human Human
+ PMA/IFNγ Monocytes Monocytes
38kDa GAPDH + IFNγ

Secondary Only Secondary Only Secondary Only

THP-1 THP-1 Human

Monocytes

THP-1 THP-1 Human

+ PMA/IFN + PMA/IFN Monocytes
+ IFN

FITC
APC APC

Fig. 1 Antibody validation for detection of human LRRK2 protein by flow cytometry. a, b Increases in human LRRK2 protein in PMA
differentiated/IFN-γ-stimulated THP-1 human monocytic cell line, IFN-γ-stimulated human monocytes from peripheral blood are detectable by
western blot with the c41-2 LRRK2 antibody. Western blot analysis of LRRK2 levels in naive THP-1 (n = 3) and PMA-differentiated and IFN-γ-
stimulated THP-1 cells (n = 3) compared to human primary monocytes from peripheral blood (n = 3). c Increases in human LRRK2 protein
in permeabilized PMA differentiated/IFN-γ-stimulated THP-1 cells are detectable by flow cytometry with the c41-2 LRRK2 antibody and a FITC-
conjugated secondary antibody (1:10,000) or an Alexa 647-conjugated secondary (1:10,000)

frequencies of monocytes or B cells were observed (Supplemen- leukocyte antigen (HLA), is the antigen-presenting-molecule
tary Fig. S3a–b), but PD patients had a significantly decreased expressed on cells such as monocytes that activates CD4+ T cells
T-cell frequency compared to HC (Supplementary Fig. S3a). and is upregulated following an inflammatory stimulus. In
Consistent with what has been previously reported, it appears humans, there are three different isotypes, HLA-DR, -DQ, and
that this decrease is due strictly to the CD4+ subset and not to -DP, encoded by the MHC-II locus.39 To determine if there were
CD8+ T-cell frequencies37 (Supplementary Fig. S3c–e). In addition, any alterations in antigen presentation in patients with PD, we
there was a positive correlation between the expression level of assessed expression levels of HLA-DR and -DQ. Both PD and HC
LRRK2 and frequency of monocytes in PD patients (Fig. 3a). There groups displayed induction of HLA-DR and -DQ proteins after IFN-
was increased LRRK2 expression in subjects with higher frequen- γ stimulation with 80–90% of cells being HLA-DR/-DQ double
cies of CD14+ monocytes. Although not reaching significance, positive (Fig. 4b). Monocytes from both HC subjects and PD
there was a trend towards the correlations between LRRK2 patients continued to upregulate HLA-DQ over time (Fig. 4c). Both
expression and CD14+ frequencies between PD and HC being groups also upregulated HLA-DR over time; however, HC subjects
distinctly different (Fig. 3a). Other correlations between LRRK2 downregulated HLA-DR after 72 h while monocytes from PD
level and immune cell frequency were not statistically significant patients retained expression (Fig. 4d). Furthermore, after 18 h of
between PD and HC (Fig. 3b–d). In summary, LRRK2 expression is stimulation, LRRK2 levels were positively correlated with HLA-DR
increased in lymphocytes and inflammatory monocytes from PD MFI (Fig. 4e) in monocytes of PD subjects and negatively
patients and the levels of LRRK2 positively correlate with the correlated with HLA-DQ MFI (Fig. 4f) in monocytes from HC
frequency of monocytes in PD patients. patients. In summary, LRRK2 protein expression was induced in
monocytes from both groups after stimulation with IFN-γ at both
18 and 72 h post-stimulation. However, LRRK2 expression was
LRRK2 expression is induced by inflammatory stimuli in both PD positively correlated with MHC-II induction in PD patients and
and HC monocytes but shows opposite correlation with MHC-II negatively correlated in HC subjects.
induction in PD vs. HC subjects
There are several reports indicating that LRRK2 expression in LRRK2 induction is slower in T cells compared to monocytes in
immune cells increases following inflammatory stimuli such as both PD and HC subjects
IFN-γ or microbial components like LPS.22, 23, 30, 38 We sought to To determine the timing of LRRK2 induction following T-cell
replicate these data in monocytes and explore whether this activation, T cells were stimulated with anti-CD3/CD28-coated
paradigm held true in T cells. Cells were plated immediately beads and IL-2 (a cytokine necessary for T-cell activation and
following isolation from peripheral blood. Monocytes were proliferation) for 18 or 72 h. Stimulation for 18 h was not sufficient
stimulated with IFN-γ for 18 or 72 h. After18 h of stimulation, to induce increases in LRRK2; however, LRRK2 levels after 72 h of
LRRK2 expression in monocytes was not significantly increased stimulation were significantly increased in both CD4+ and CD8+
(Fig. 4a). However, by 72 h after stimulation, LRRK2 levels in T cells in PD and HC groups. There were no significant differences
monocytes were significantly increased relative to baseline in both between the means of LRRK2 expression in CD4+ T cells in PD and
PD and HC populations (Fig. 4a). MHC-II, also known as human HC after 72 h stimulation (Fig. 5a).

Published in partnership with the Parkinson’s Disease Foundation npj Parkinson’s Disease (2017) 11
 LRRK2 levels in PD immune cells
D A Cook et al.
4
HC PD

a Immune Cells b Monocytes

15000 15000
*

LRRK2 MFI

LRRK2 MFI
10000 10000

** *
5000 5000

0 0
Mono B Cells T Cells CD16+ CD14+

c T Cells d Th and Treg Subsets

5000 3000 * * * *
* *
4000

LRRK2 MFI
LRRK2 MFI

2000
3000

2000
1000
1000

0 0
CD4+ CD8+ Th1 Th2 Th17 Treg

e Teff Subsets
5000
n.s.
* * * ** ** ** **
4000
LRRK2 MFI

3000

2000

1000

0
Tcm Naive Tem Teff Tcm Naive Tem Teff
CD4+ CD8+
Fig. 2 LRRK2 expression in T cells, B cells, and a subset of monocytes is increased in PD patients compared to matched HC subjects. a LRRK2
median fluorescence intensity (MFI) in monocytes (t(64) = 1.57, p = 0.122, HC n = 30, PD n = 36), B cells (t(47) = 3.02, p = 0.004, HC n = 21, PD
n = 28), and T cells (t(63) = 2.35, p = 0.022, HC n = 29, PD n = 36), b a subset of monocytes (t(44) = 2.68, p = 0.01, HC n = 17, PD n = 29) c–e and
T-cell subsets (CD4+, t(61) = 2.17, p = 0.034, HC n = 29, PD n = 34; CD8+, t(65) = 2.39, p = 0.02 HC n = 31, PD n = 36; T helper HC n = 27, PD
n = 34: Th1, t(59) = 2.49, p = 0.016; Th2, t(59) = 2.41, p = 0.019; Th17, t(59) = 2.24, p = 0.029; Treg, t(55) = 2.06, p = 0.044 HC n = 26, PD
n = 31; CD4+ effector subsets HC n = 26, PD n = 33: Tcm t(57) = 2.08, p = 0.043; Naive t(57) = 2.18, p = 0.033; Tem t(57) = 2.02, p = 0.049; Teff,
t(57) = 1.99, p = 0.051; CD8+ effector subsets HC n = 22, PD n = 33: Tcm t(53) = 2.95, p = 0.005; Naive t(53) = 3.16, p = 0.003; Tem t(53) = 2.81,
p = 0.007; Teff t(53) = 2.70, p = 0.009) was determined by flow cytometry staining of total peripheral blood mononuclear cells. Means were
plotted with standard error of the mean. Two-tailed Student’s t-test between HC and PD was used to test for significance. *p < 0.05, **p < 0.01

PD is associated with higher LRRK2 induction in proliferating CD4+ T cells was not significantly different between the two
T cells compared to HCs groups, the LRRK2 MFI in CD8+ T cells was higher in PD patients
To determine if LRRK2 may be involved in regulation of T-cell than in HC subjects at every percent of proliferated cells
proliferation, cells were stained with CellTrace Violet, a cell (Supplementary Fig. S4b).
proliferation dye, and analyzed via flow cytometry. Typically, after
72 h of stimulation T cells will have divided between three and The T-cell activation marker CTLA4 is significantly decreased in
four times. To simplify this analysis, each cell division was T cells of PD patients following stimulation
analyzed individually for LRRK2 expression. We found that LRRK2 In addition to proliferation, we also assessed potential differences
was upregulated only in the early dividing cells in both PD in activation markers normally upregulated following stimulation
and HC groups (Fig. 5b). In CD8+ T cells, dividing cells from PD in T cells. If LRRK2 has a role in regulating processes involved in
patients had significantly higher levels of LRRK2 compared to immune cell activation, increased expression levels of
dividing cells from HC subjects (Fig. 5b). Importantly, no LRRK2 should correlate with differential activation of T cells. We
differences were observed in the percent of CD4+ or CD8+ T-cell looked at two common activation markers:CTLA-4, a negative
proliferation or percent of T cells that divided three times between regulator of activation, and 4-1BB, a receptor that amplifies T-cell
PD and HC subjects (Supplementary Fig. S4a). Although the activation by inducing secretion of IL-2. CTLA-4 competes with
relationship between LRRK2 MFI and percentage of proliferated CD28 to bind CD80/86 on antigen-presenting cells; but while

npj Parkinson’s Disease (2017) 11 Published in partnership with the Parkinson’s Disease Foundation
 LRRK2 levels in PD immune cells
D A Cook et al.
5

Fig. 3 LRRK2 expression in monocytes positively correlates with monocyte frequency in PD patients but not in HC subjects. Frequency of
a monocytes (HC R2(30) = 0.001, p = 0.88; PD R2(36) = 0.125, p = 0.034); F(1, 62) = 3.23, p = 0.077), b CD16+ monocytes (HC R2(17) = 0.004, p =
0.822, PD R2(29) = 0.006, p = 0.684; F(1,42) = 0.148, p = 0.703), c B cells (HC R2(23) = 0.002, p = 0.860, PD R2(28) = 0.003, p = 0.773; F(1, 45) =
0.092, p = 0.763) and d T cells (HC R2(29) = 0.045, p = 0.267, PD R2(37) = 0.061, p = 0.146; F(1, 61) = 0.117, p = 0.734) as a percentage of total
PBMCs determined by flow cytometry staining were plotted vs. LRRK2 median fluorescence intensity (MFI). Means were plotted with standard
error of the mean. ANCOVA was performed to assess differences of slopes between HC and PD. Linear regression was used to assess individual
correlations of slopes of HC and PD. Significance was set at p < 0.05

CD28 sends an activating signal when bound, CTLA-4 sends an secreted significantly more TNF compared to cells from HC
inhibitory signal when bound. After the 72 h stimulation, both PD subjects (Fig. 6d). Interestingly, at 72 h, IL-12p70 was the only
and HC T cells upregulated 4-1BB (Supplementary Fig. S2) and cytokine secreted at higher levels in PD T cells compared to HC
CTLA-4, however, CTLA-4 levels in CD8+ T cells of PD patients were T cells (Fig. 6d). In summary, several immune cell subsets from PD
significantly lower than those in HCs (Fig. 5c). patients secreted higher cytokines upon stimulation compared to
immune cells from HC subjects.
Immune cells from PD patients display similar cellular cytokine
expression but increased pro-inflammatory cytokine secretion
We wanted to further explore a potential role for LRRK2 in LRRK2 expression is positively correlated with cytokine expression
immune cell activation. Following stimulation, T cells secrete in PD patients
cytokines to further activate the immune cell response.40 To PD patients had higher LRRK2 expression in cells with a higher
investigate the hypothesis that LRRK2 expression affects cytokine amount of intracellular cytokine levels (Fig. 7a, b). T cells from PD
production, we used intracellular cytokine staining (ICS) to patients displayed an association between LRRK2 protein levels
measure cytokine expression in a cell type-specific manner as and cytokine expression (Fig. 7a, b). LRRK2 protein levels positively
well as multiplexed immunoassays to measure cytokine secretion correlated with IFN-γ, TNF, and IL-2 expression in T cells from PD
into the culture media. Cytokine measurements were performed patients; however, only IFN-γ expression in CD4+ T cells correlated
after 18 h of stimulation. Prior to harvest, cells were treated with with LRRK2 protein levels in HC subjects (Fig. 7a). In addition,
Brefeldin A, a compound that inhibits protein transport causing when comparing the two subject groups for interaction, only the
vesicle accumulation at the golgi complex/endoplasmic reticu- correlation between IL-2 expression in CD8+ T cells and LRRK2 MFI
lum.41 Cells were stained to measure total intracellular protein displayed significantly different slopes (Fig. 7b). In summary,
expression of IFN-γ, tumor necrosis factor (TNF), and IL-2. Protein LRRK2 levels positively correlated with cytokine expression and
levels of all cytokines were increased with stimulation, but no secretion in PD but not HC subjects.
significant differences were detected between PD and HC groups
(Fig. 6a, b).
Multiplexed immunoassays were performed on conditioned DISCUSSION
media from the 18 h-stimulated monocyte and 18- and 72 h- Recent new discoveries in immune cells have suggested a
stimulated T-cell samples to measure the levels of cytokines potential link for LRRK2 to the regulation of the immune system
secreted into the conditioned media. For 18 h-stimulated mono- and modulation of inflammatory responses. Herein, we demon-
cytes, cytokine secretion was significantly increased in the PD strate that LRRK2 is expressed in both innate and adaptive human
group for IL-12p70, IL-13, IL-1β, IL-2, IL-4, IL-6, and TNF (Fig. 6c). In immune cells and is expressed at higher levels in the immune cells
18 h-stimulated T cells, no significant differences were detected in of patients with late-onset PD compared to age-matched HC
levels of secreted IFN-γ and IL-2, but T cells from PD patients individuals. In addition, LRRK2 is induced in both human

Published in partnership with the Parkinson’s Disease Foundation npj Parkinson’s Disease (2017) 11
 LRRK2 levels in PD immune cells
D A Cook et al.
6

Fig. 4 The IFNγ-stimulated increase in LRRK2 protein in PD and HC monocytes displays similar kinetics but the correlation between LRRK2
levels and MHC-II expression is different for PD vs. HC. a LRRK2 induction (HC p = 0.0006, PD p < 0.0001), b %DR+DQ+ monocytes (18 and 72 h
HC p < 0.0001, PD p < 0.0001), c HLA-DQ induction (18 h HC p = 0.0001, PD p = 0.0042; 72 h HC p < 0.0001, PD p < 0.0001), and d HLA-DR
induction (18 h HC p < 0.0001, PD p < 0.0001; 72 h HC p = 0.0049, PD p < 0.0001) with or without 100 U/mL interferon-γ stimulation for 18 (HC
n = 25, PD n = 22) or 72 h (HC n = 10, PD n = 11) in paramagnetically, positively sorted monocytes was measured by flow cytometry. Means
were plotted with standard error of the mean. Two-way ANOVA with Sidak’s multiple comparisons post-hoc test was used to test for
significance. *p < 0.05, **p < 0.01, ***p < 0.001. e -DR median fluorescence intensity (MFI) (HC R2(25) = 0.013, p = 0.580, PD R2(22) = 0.195, p =
0.040; F(1,43) = 3.16, p = 0.083) and f -DQ MFI (HC R2(25) = 0.193, p = 0.028, PD R2(22) = 0.193, p = 0.540; F(1, 43) = 4.24, p = 0.046) were
measured using flow cytometry and plotted against LRRK2 MFI. ANCOVA was performed to assess differences of slopes between HC and PD.
Linear regression was used to assess individual correlations of slopes of HC and PD. Significance was set at p < 0.05

monocytes and T cells following an inflammatory stimulus; and in typically display increased cytokine secretion and greater antigen
PD patients there is a positive correlation between LRRK2 levels, presentation.42 Therefore, the finding that LRRK2 levels are
MHC-II induction, cytokine expression and secretion levels, and notably increased in CD16+ monocytes of PD patients may have
dampened expression of the T-cell inhibitory factor CTLA-4. Given functional relevance to disease risk or progression given that it
the established role of LRRK2 as a potential negative regulator of was recently reported that IFN-γ induces more LRRK2 in this
NFAT,16 the differences we observed between PD patients and HC subset of monocytes compared to classical CD16— monocytes.28
subjects suggest that LRRK2 may be important in the regulation of In addition, it has been shown that circulating monocytes are
both innate and adaptive immune cells within the context of PD. dysregulated in PD, displaying a hyperactive inflammatory
Of note, within our study population and similar to other study phenotype.43 The monocyte data taken together with the
populations, the PD group was predominantly male while the HC increased LRRK2 expression in T cells and B cells suggest that
group was predominantly female as they were generally the following an inflammatory challenge, individuals with PD will
spouses and caregivers of the PD patients. To ensure that the mount an exacerbated inflammatory response compared to
differences we observed were not due to this unequal sex healthy individuals that could lead to sustained immune activation
distribution, results were stratified by sex but did not affect the and acceleration of disease progression.
differences observed between PD and HC groups. In support for the role of LRRK2 in immune cell regulation, we
The increased levels of LRRK2 associated with PD suggest that found that LRRK2 protein expression increases in human immune
the protein is contributing to disease pathogenesis. CD14+CD16+ cells following inflammatory challenges. Thereafter, LRRK2 levels
monocytes are considered to represent a non-classical monocyte continue to increase over time in monocytes. Monocytes are
population that is characteristically more pro-inflammatory and antigen-presenting cells that are critical to the activation of the

npj Parkinson’s Disease (2017) 11 Published in partnership with the Parkinson’s Disease Foundation
 LRRK2 levels in PD immune cells
D A Cook et al.
7
HC PD
a CD4+ T Cells CD8+ T Cells
n.s.
10000 6000 * ***
***
8000 *
LRRK2 MFI 6000
4000

4000
2000
2000

0 0
0 18 72 h 0 18 72 h 0 18 72 h 0 18 72 h

b 8000 8000

6000 6000
LRRK2 MFI

4000 4000

**
2000 2000

0 0
No Div Div 1 Div 2 Div 3 Div 4 No Div Div 1 Div 2 Div 3 Div 4

c 1500 1500

***
**
CTLA4 MFI

1000 *** 1000

*** ***

500 500

0 0
Unstim Stim Unstim Stim Unstim Stim Unstim Stim

Fig. 5 The anti-CD3/CD28-stimulated increase in LRRK2 protein in T cells is slower than in monocytes and similar in PD and HC subjects but T-
cell proliferation is associated with greater increases in LRRK2 protein and dampened expression of the negative regulator of T-cell activation
CTLA4 in PD subjects. LRRK2 median fluorescence intensity (MFI) of a stimulated cells (HC 18 h n = 14, 72 h n = 16, p = 0.019; PD 18 h n = 22,
72 h n = 20, p < 0.0001; HC to PD at 72 h, p = 0.0809), b proliferating cells (CellTrace Violet) (p = 0.0045, HC n = 12, PD n = 11) and c CTLA-4 MFI
(HC n = 16, PD n = 20, CD8+p = 0.0034) were measured using flow cytometry following stimulation of paramagnetically, positively selected
T cells with 30 U/mL IL-2 and anti-CD3/CD28 stimulation beads for 72 h. Means were plotted with standard error of the mean. Two-way
ANOVA with Sidak’s multiple comparisons post-hoc test was used to test for significance. *p < 0.05, **p < 0.01, ***p < 0.001

adaptive immune response by upregulating MHC-II (HLA-DR/-DQ) that synergizes with environmental exposures to increase risk for
proteins loaded with antigens that activate CD4+ T cells. Although PD.45 Although the LRRK2 and MHC-II loci are encoded on
we observed no differences in the overall extent of MHC-II different chromosomes, perhaps the mechanism of altered
induction between the PD and HC groups, in HC subjects, LRRK2 antigen presentation increases susceptibility for sporadic PD.
expression is negatively correlated with HLA-DQ expression Previous studies have declared LRRK2 absent or undetectable in
whereas in PD patients, LRRK2 expression is positively correlated T-cell populations,28 however, the data presented here definitively
with HLA-DR expression. These data suggest that LRRK2 could be demonstrate inducible expression of LRRK2 not just in CD3+
regulating the antigen presentation function of human mono- T cells, but also in all functional T-cell subsets. There were no
cytes, and that such regulation is altered in PD patients. Studies significant differences between baseline levels of LRRK2 in subsets
have shown that inhibition of LRRK2 kinase activity with the of T cells; however, LRRK2 levels were increased in all of the T cells
LRRK2-IN1 inhibitor decreased expression of CD14, CD16, and of PD patients relative to those of HC subjects. Given that LRRK2
MHC-II in monocytes.28 However, due to the reported off-target has been shown to be a negative regulator of NFAT in HEK293
effects of LRRK2-IN1, specifically ERK5 inhibition,44 it is possible T cells,16 and NFAT is a necessary transcription factor for T-cell
that this decrease cannot be attributed wholly to LRRK2 kinase activation, we hypothesize that LRRK2 could also serve as a
activity. Alternatively, it is possible that LRRK2 localizes and negative regulator of T-cell activation via interaction with NFAT.
associates with binding partners involved in regulation of antigen Induction of LRRK2 was also seen in T cells, but was delayed until
presentation and that LRRK2 kinase activity is not required for 72 h and only seen in dividing T cells. PD patients also had greater
such interactions. In addition, recent studies suggest that antigen upregulation of LRRK2 in these dividing T cells, raising the
presentation is altered in a subset of individuals with PD in interesting possibility that LRRK2 is essential for T-cell division or
association with a non-coding SNP (rs 3129882) in the MHC-II locus acting in some sort of regulatory capacity during T-cell division.

Published in partnership with the Parkinson’s Disease Foundation npj Parkinson’s Disease (2017) 11
 LRRK2 levels in PD immune cells
D A Cook et al.
8

Fig. 6 Monocytes and T cells from PD patients display increased cellular expression and secretion of pro-inflammatory cytokines in
association with higher LRRK2 protein expression. a, b Cytokine expression measured through intracellular cytokine staining measured with
flow cytometry (HC n = 13, PD n = 22), c multiplexed immunoassay on conditioned media from 18 h stimulated monocytes (HC n = 9, PD n =
10) and d 18 h (HC n = 7, PD n = 8, p = 0.0154) and 72 h (HC n = 7, PD n = 8, p = 0.0001) stimulated T cells. Means were plotted with standard
error of the mean. Two-way ANOVA with Sidak’s multiple comparisons post-hoc test was used to test for significance. *p < 0.05, **p < 0.01,
***p < 0.001

Markers of T-cell activation such as CTLA-4 and 4-1BB are good by NFAT1 binding to the proximal promoter and is decreased
indicators of proper immune response and regulation. Immune when NFAT is inhibited.46 Therefore, our findings suggest that in
cells upregulate these markers following stimulation to keep the healthy individuals LRRK2 is a negative regulator of T-cell
inflammatory response in check. Interestingly, T cells from PD activation. Specifically, the increase in LRRK2 induced by stimula-
patients have an impaired capacity to upregulate CTLA-4 tion acts to limit NFAT-dependent transcription, as evidenced by
compared to those from HCs. CTLA-4 transcription is controlled reductions in expression of CTLA-4. In PD patients, alterations in

npj Parkinson’s Disease (2017) 11 Published in partnership with the Parkinson’s Disease Foundation
 LRRK2 levels in PD immune cells
D A Cook et al.
9

Fig. 7 LRRK2 protein in stimulated CD4+ and CD8+ T cells from PD patients displays significant positive correlations with cytokine-expression.
Fold change in percent of cytokine secretion (IFN-γ, TNF, and IL-2) by a CD4+ T cells (IFN-γ: PD R2(20) = 0.339, p = 0.002, HC R2(13) = 0.307, p =
0.049; F(1, 29) = 0.106, p = 0.747;TNF: PD R2(20) = 0.421, p = 0.002, HC R2(13) = 0.028, p = 0.585; F(1, 29) = 3.63, p = 0.067; IL-2: PD R2(20) = 0.293,
p = 0.0014, HC R2(13) = 0.044, p = 0.494; F(1, 29) = 2.19, p = 0.150) and b CD8+ T cells (IFN-γ: PD R2(22) = 0.400, p = 0.002, HC R2(14) = 0.040, p =
0.494; F(1, 32) = 2.04, p = 0.163; TNF: PD R2(22) = 0.398, p = 0.002, HC R2(14) = 0.031, p = 0.549; F(1, 32) = 2.65, p = 0.113; IL-2: PD R2(22) = 0.430,
p = 0.001, HC R2(14) = 0.007, p = 0.780; F(1, 32) = 5.46, p = 0.026) relative to unstimulated cells plotted against LRRK2 median fluorescence
intensity (MFI) of stimulated cells. ANCOVA was performed to assess differences of slopes between HC and PD. Linear regression was used to
assess individual correlations of slopes of HC and PD. Significance was set at p < 0.05

LRRK2 function may translate into poor regulation of T-cell states suggest that targeting LRRK2 with therapeutic interventions
responses and result in a pro-inflammatory environment char- is likely to have direct effects on immune cell function—whether
acteristic of PD. this affords benefit or untoward bystander effects remains to be
Cytokine secretion by T cells is a necessary step for promoting determined and is prerequisite to advancing LRRK2 kinase
the immune response, but prolonged secretion and/or increased inhibitors to clinical trials.
cytokine levels are hallmarks of inflammatory disease. In the CSF
and nigrostriatal regions of PD brains examined at autopsy, the
levels of pro-inflammatory cytokines such as IL-1β, TNF, IFN-γ, and MATERIALS AND METHODS
IL-6 were increased compared to those of age-matched HC Human subjects
subjects.47–49 In the present studies, monocytes and T cells from PD patients (40) and age-matched HCs (HC) (32) subjects were recruited
PD patients were shown to secrete more pro-inflammatory through the Immune System and Neurological Disease (ISND) Institutional
cytokines than T cells from healthy individuals. In addition, both Review Board (IRB)-approved research protocol at the Emory Movement
CD4+ and CD8+ T cells of PD patients expressing higher levels of Disorders Clinic and community outreach events. Subjects were excluded
inflammatory cytokines also expressed higher levels of based on age (younger than 50 and over 85 years of age), known familial
LRRK2 suggesting that LRRK2 protein expression is associated PD mutations and/or other known neurological, chronic or recent
with the inflammatory response characteristically seen in this infections, or autoimmune comorbidities. Subjects were genotyped for
neurodegenerative disease. Taken together, our findings suggest the G2019S LRRK2 mutation (LifeTechnologies #4351378, Grand Island,
that LRRK2 levels in peripheral T cells may serve as a biomarker for NY).
diagnosis and monitoring disease progression in PD patients. During recruitment, a confidential family history and environmental
questionnaire was used to assess history of disease and inflammation/
The significance of ourfindings are threefold. First, the selective
immune-relevant environmental exposures and comorbidities. Caffeine,
induction of LRRK2 in pro-inflammatory monocytes and its
nonsteroidal anti-inflammatory drug, and nicotine exposure was calculated
potential regulatory role in antigen presentation is a crucial step as milligram-years, milligram-years, and pack-years, respectively. The study
in activation of the immune response and merits further populations were balanced with respect to risk factors for PD,50–53
exploration. Second, defining the kinetics of LRRK2 expression including age, smoking, nonsteroidal anti-inflammatory drug use, caffeine
and its regulation in activated T cells is a critical first step toward intake, and rs3129882 (HLA-DRA SNP) genotype (Supplementary Table 1).
understanding the role that LRRK2 plays in the adaptive immune Study population was not sex-matched: the PD group was predominantly
system and its potential link to PD pathogenesis. Third, the male while HC subjects tended to be more female as they were generally
positive correlations between LRRK2 expression levels in T-cell the caregivers; however, when the results are stratified by sex, sex does not
subsets, cytokine expression and secretion, and T-cell activation account for the differences observed. Clinical severity of PD symptoms was

Published in partnership with the Parkinson’s Disease Foundation npj Parkinson’s Disease (2017) 11
 LRRK2 levels in PD immune cells
D A Cook et al.
10
also assessed using Hoehn and Yahr ratings and part II and III of the Unified (Sigma Aldrich, St Louis, MO, USA). Following transfer, the membrane
Parkinson’s Disease Rating Scale. was cut below the pre-stained 75 kDa molecular weight and incubated in
5% milk blocking solution in 1x Tris-buffered saline containing 0.1%
Human PBMC isolation, stimulation, and purification Tween-20 (Sigma Aldrich, St Louis, MO, USA) for 1 h. The corresponding
membrane sections were incubated overnight in blocking solution
Isolation, stimulation, and purification of peripheral immune cells was
containing primary antibody targeting either LRRK2 (MJFF c41-2, 1:5000;
performed as previously published in Kannarkat et al.45. Briefly, PBMCs
were isolated from whole blood using density centrifugation with Ficoll- Abcam, Cambridge, UK) or β-actin (sc-47778 β-Actin (C4) HRP 1:10,000,
Paque (GE Healthcare, Uppsala, Sweden). Monocytes and T cells were Santa Cruz Biotechnology) at 4 °C. Following three 5-min washes in 1x TBS
isolated from PBMCs using positive selection columns with anti-CD14 and with 0.1% Tween-20 with agitation, the LRRK2 probed membrane section
anti-CD3 paramagnetic beads, respectively (Miltenyi Biotec, Bergisch was incubated overnight in donkey anti-rabbit horseradish peroxidase
Gladbach, Germany). The isolated cell fraction was processed for flow conjugated secondary antibody (1:5000; Jackson ImmunoResearch, West
cytometry as follows. Monocytes and T cells were plated for stimulation in Grove, PA, USA) in blocking solution at 4 °C. Membranes were washed 3 ×
a 12-well plate at a density of 1x106 cells per well. Monocytes were plated 5 min in 1x TBS with 0.1% Tween-20, and imaged using Luminata
for 18 or 72 h with or without 5 ng/mL IFN-γ (PeproTech, Rocky Hill, NJ, Crescendo Chemiluminescent Substrate (Thermo Scientific).
USA). T cells were stimulated for 18 or 72 h with or without anti-CD3/CD28
beads (1:1 cells:beads) (Dynabeads® Life Technologies, Grand Island, NY,
Flow cytometry analysis
USA) and 30 U/mL recombinant human IL-2 (Biolegend, San Diego, CA,
USA). To stain for flow cytometry, 5 × 105 cells per well were washed once
with PBS and incubated for 30 min at 4 °C with LIVE/DEAD Fixable Red
(Life Technologies). Cells were incubated in 1x FACS buffer (1% bovine
Mouse PBMC isolation serum albumin, 0.1% sodium azide, and 1 mM EDTA) for 15 min at 37 °C
The following mouse strains were obtained from Jackson labs:B6.Cg-Tg with anti-human CCR7:phycoerythrin. Cells were washed and incubated for
(Lrrk2*G2019S)2Yue/J (#012467), B6.Cg-Tg(Lrrk2)6Yue/J (#012466), C57BL/ 20 min at 4 °C with surface antibodies detailed in Supplementary Table 2.
6J (#000664), and C57BL/6-Lrrk2tm1Mjfa/J (#012444). Blood from each Cells were intracellularly stained using the Fixation/Permeabilization Stain
mouse (200 µL) was collected in an EDTA vacutainer tube (BD Biosciences) Kit per manufacturer’s protocol (eBiosciences, San Diego, CA, USA). ICS
via cheek bleed. Blood was incubated in the dark for 10 min at room was performed on T cells stimulated for 18 h. Cells were incubated with
temperature with 1x RBC lysis buffer (Biolegend) to lyse red blood cells. 5 µg/mL Brefeldin A (Biolegend) for 7 h prior to harvest. After harvest, cells
Cells were pelleted and resuspended in phosphate buffered saline (PBS) for
were processed according to protocols detailed above. Cells were run
flow cytometry processing or lysed in sample buffer for western blot as
immediately on an LSRII instrument (BD Biosciences, Franklin Lakes, NJ,
detailed below.
USA). Supra Rainbow Spherobeads (SpheroTech, Lake Forest, IL, USA) and
OneComp Beads (eBiosciences) were used to set voltages and compensa-
Human THP-1 monocytic cell culture and differentiation tion settings between cytometry runs. Analysis of flow cytometry data was
THP-1 cells (ATCC #TIB-202) were maintained in RPMI1640 medium with performed using FlowJo Software v10.X (Ashland, OR, USA). Gates were set
2 mM L-glutamine adjusted to contain 1.5 g/L sodium bicarbonate, 4.5 g/L according to Supplementary Fig. S5. Because the LRRK2 antibody is
glucose, 10 mM HEPES, and 1 mM sodium pyruvate and supplemented unconjugated, a secondary antibody only control condition was used to
with 10% fetal bovine serum. THP-1 cells were terminally differentiated determine nonspecific binding and select for positively stained popula-
into macrophages by exposure to 100 nM of phorbol 12-myristate tions. Conjugated isotype control antibodies were used for the rest of the
12- acetate (PMA) (Sigma P-8139) for 72 h. Terminal differentiation was cellular protein markers stained. Fluorescence minus one controls were
confirmed as cells become adherent. Stimulation with 200 U/ml IFN-γ for used to account for fluorescence spillover between channels. Protein
18 h was performed to further increase LRRK2 protein levels. Trypsin was expression levels are reported as MFI.
used to lift the differentiated cells prior to use in western blotting or flow
cytometry. For flow cytometry, cells were permeabilized and stained
intracellularly with Abcam c41-2 Rb anti-LRRK2 antibody (1:50) and either a Cell proliferation assays
FITC-conjugated secondary (1:10,000) or an AlexaFluor-647-conjugated To assess T-cell proliferation, cells were stained with CellTrace Violet (Life
secondary (1:10,000). Technologies) according to the manufacturer’s protocol and plated for 72 h
with anti-CD3/CD28 Dynabeads® and 30 U/mL recombinant human IL-2.
Western blotting Unstimulated cells were harvested at 18 h post-plating as cells do not
THP-1 cells and human monocytes were lysed in 4x sample buffer (Bio-Rad, remain viable for 72 h without stimulation. At harvest, Dynabeads® were
Hercules, CA, USA) and heated at 80 °C for 5 min proteins were then removed using magnetic separation, washed, and stained according to the
separated in a 12% polyacrylamide gel (Bio-Rad) by gel electrophoresis and above flow analysis protocol.
transferred to a 45 µm polyvinylidene difluoride (PVDF) membrane (Sigma
Aldrich, St Louis, MO, USA). Following transfer, the membrane was cut Meso scale discovery multiplexed immunoassays
along the pre-stained standard band indicating 100 kDa molecular weight Conditioned media from plated cells was collected during cell harvest and
and incubated in a 5% skim milk blocking solution in 1x tris-buffered saline
stored at -80 °C until sample analysis. Media analyte levels were measured
containing 0.1% Tween-20 (TBST) (Sigma Aldrich). The corresponding
in duplicate using 3-plex (IL-2, TNFα, and IFN-γ) and 10-plex (IFN-γ, IL-10,
membrane sections were incubated overnight in blocking solution
containing a primary antibody targeting either GAPDH (SC-31915 1:1000; IL-12p70, IL-13, IL-1β, IL-2, IL-4, IL-6, IL-8, and TNFα) plates on a Sector 2400
Santa Cruz Biotechnology, Dallas, TX, USA) or LRRK2 (MJFF c41-2, 1:5000; instrument (Meso Scale Discovery, Rockville, MD, USA).
Abcam, Cambridge, UK) at 4 °C. Following three 5-min washes in 0.1% TBST
with agitation, membrane sections were incubated for 1 h at room Statistical analyses
temperature in either goat anti-rabbit (LRRK2) or rabbit anti-goat (GAPDH) A two-tailed Student’s t-test was used to make comparisons between HC
horseradish peroxidase conjugated secondary antibodies (1:1000; Jackson
subjects and PD patients in the immunophenotyping studies. A two-way
ImmunoResearch, West Grove, PA, USA) in blocking solution. Membranes
analysis of variance (ANOVA) followed by Sidak’s multiple comparisons
were again washed in 0.1% TBST, and imaged using 1:1 dilute SuperSignal
West FemtoChemiluminescent Substrate (Thermo Scientific, Waltham, MA, post-hoc test was used to compare baseline characteristics of the study
USA). Band intensity was determined using ImageStudio Software (Li-Cor population and inducibility of immune response following IFN-γ stimula-
Biosciences, Lincoln, NE, USA). LRRK2 expression (~286 kDa) has been tion in monocytes or anti-CD3/CD28 stimulation in T cells. Data was plotted
normalized to corresponding GAPDH (Fig. 1b; ~37 kDa). with means and standard error of the mean. Analysis of covariance
Mouse PBMCs and T cells, B cells, and monocytes isolated directly (ANCOVA) was used to assess differences in slopes of correlations between
from human blood were lysed in 1x Laemli buffer (40 mM NaF, 5% HC and PD. Linear regressions were performed to assess correlations of
dithiothreitol, 1x phosphatase inhibitors, 1x protease inhibitors). Proteins individual slopes of HC and PD. All statistical tests used are indicated in the
were separated in a 7.5% polyacrylamide gel (Bio-Rad, Hercules, CA, USA) figure legends. Graphpad Prism Version 6.05 (Prism, La Jolla, CA, USA)
by gel electrophoresis and transferred to 45 µM PVDF membrane software was used to perform all statistical analyses.

npj Parkinson’s Disease (2017) 11 Published in partnership with the Parkinson’s Disease Foundation
 LRRK2 levels in PD immune cells
D A Cook et al.
11
Human subjects research approval 16. Liu, Z. et al. The kinase LRRK2 is a regulator of the transcription factor NFAT that
All procedures involving human subjects were approved by the IRB of modulates the severity of inflammatory bowel disease. Nat. Immunol. 12,
Emory University in Atlanta, Georgia before study commenced. All 1063–1070 (2011).
participants provided written informed consent and the terms and risks 17. Hongge, L., Kexin, G., Xiaojie, M., Nian, X. & Jinsha, H. The role of LRRK2 in the
of the study were thoroughly explained before inclusion in the study. regulation of monocyte adhesion to endothelial cells. J. Mol. Neurosci. 55,
233–239 (2015).
18. Brice, A. Genetics of Parkinson's disease: LRRK2 on the rise. Brain 128(Pt 12),
ACKNOWLEDGEMENTS 2760–2762 (2005).
We thank members of the Boss and Tansey laboratories for helpful discussions. We 19. Rudenko, I. N. & Cookson, M. R. Heterogeneity of leucine-rich repeat kinase 2
also thank E. Sperin and K. Tansey for assistance with blood collection, and mutations: genetics, mechanisms and therapeutic implications. Neurotherapeutics
V. Herzberg for assistance with biostatistical analyses. Thanks to the Michael J. Fox 11, 738–750 (2014).
Foundation for Parkinson’s Research, American Parkinson’s Disease Association, 20. Rideout, H. J. & Stefanis, L. The neurobiology of LRRK2 and its role in the
Wilkin’s Parkinson’s Foundation, and Emory Udall Center of Excellence for Parkinson’s pathogenesis of Parkinson's disease. Neurochem. Res. 39, 576–592 (2014).
Research for facilitating study recruitment. Financial support for the authors was 21. Greggio, E. & Cookson, M. R. Leucine-rich repeat kinase 2 mutations and Par-
provided by the Michael J. Fox Foundation for Parkinson’s Research LRRK2 in kinson's disease: three questions. ASN Neuro. 1, AN20090007 (2009).
Idiopathic PD program (MGT, JMB), 1R21NS084647 from the National Institutes of 22. Hakimi, M. et al. Parkinson's disease-linked LRRK2 is expressed in circulating and
Health/National Institute of Neurological Disorders and Stroke (M.G.T, J.M.B.), the tissue immune cells and upregulated following recognition of microbial struc-
Sartain Lanier Family Foundation (S.A.F.), pre-doctoral training grant support 5T32 tures. J. Neural. Transm. 118, 795–808 (2011).
AI007610 from the National Institutes of Health/National Institute of Allergy and 23. Gardet, A. et al. LRRK2 is involved in the IFN-gamma response and host response
Infectious Disease (D.A.C.), NRSA award 1F31NS081830-01A1 from the National to pathogens. J. Immunol. 185, 5577–5585 (2010).
Institute of Neurologic Disorders and Stroke (G.T.K.), and post-doctoral training grant 24. Barrett, J. C. et al. Genome-wide association defines more than
support 5T32 ES 12870-12 from the National Institute of Environmental Health 30 distinct susceptibility loci for Crohn's disease. Nat. Genet. 40, 955–962
Sciences (A.F.C.), and NRSA award 1F31NS098673-01 from the National Institute of (2008).
Neurologic Disorders and Stroke (L.M.B.). This study was also supported in part by the 25. Zhang, F. R. et al. Genomewide association study of leprosy. N. Eng. J. Med. 361,
Emory Multiplexed Immunoassay Core (EMIC), which is subsidized by the Emory 2609–2618 (2009).
University School of Medicine and is one of the Emory Integrated Core Facilities. 26. Meylan, E. & Tschopp, J. The RIP kinases: crucial integrators of cellular stress. TIBS
Additional support was provided by the National Center for Advancing Translational 30, 151–159 (2005).
Sciences of the National Institutes of Health under Award Number UL1TR000454. The 27. Zhang, D., Lin, J. & Han, J. Receptor-interacting protein (RIP) kinase family. Cell.
content is solely the responsibility of the authors and does not necessarily reflect the Mol. Immunol. 7, 243–249 (2010).
official views of the National Institutes of Health. 28. Thevenet, J., Pescini-Gobert, R., Hooft van Huijsduijnen, R., Wiessner, C. & Sagot, Y.
J. Regulation of LRRK2 expression points to a functional role in human monocyte
maturation. PLoS ONE 6, e21519 (2011).
COMPETING INTERESTS 29. Kuss, M., Adamopoulou, E. & Kahle, P. J. Interferon-gamma induces leucine-rich
The authors declare no competing interests. repeat kinase LRRK2 via extracellular signal-regulated kinase ERK5 in macro-
phages. J. Neurochem. 129, 980–987 (2014).
30. Moehle, M. S. et al. LRRK2 inhibition attenuates microglial inflammatory
REFERENCES responses. J. Neurosci. 32, 1602–1611 (2012).
1. Paisan-Ruiz, C. et al. Cloning of the gene containing mutations that cause PARK8- 31. Perera, G., Ranola, M., Rowe, D. B., Halliday, G. M. & Dzamko, N. Inhibitor treatment
linked Parkinson's disease. Neuron 44, 595–600 (2004). of peripheral mononuclear cells from Parkinson’s disease patients further vali-
2. Zimprich, A. et al. Mutations in LRRK2 cause autosomal-dominant parkinsonism dates LRRK2 dephosphorylation as a pharmacodynamic biomarker. Sci. Rep. 6,
with pleomorphic pathology. Neuron 44, 601–607 (2004). 31391 (2016).
3. Thaler, A., Ash, E., Gan-Or, Z., Orr-Urtreger, A. & Giladi, N. The LRRK2 G2019S 32. Russo, I., Bubacco, L. & Greggio, E. LRRK2 and neuroinflammation: partners in
mutation as the cause of Parkinson's disease in Ashkenazi Jews. J. Neural. Transm. crime in Parkinson's disease? J. Neuroinflammation 11, 52 (2014).
116, 1473–1482 (2009). 33. Pradhan, S. & Andreasson, K. Commentary: progressive inflammation as a con-
4. Aasly, J. O. et al. Clinical features of LRRK2-associated Parkinson's disease in tributing factor to early development of Parkinson's disease. Exp. Neurol. 241,
central Norway. Ann. Neurol. 57, 762–765 (2005). 148–155 (2013).
5. Haugarvoll, K. et al. Lrrk2 R1441C parkinsonism is clinically similar to sporadic 34. Neumann, H., Kotter, M. R. & Franklin, R. J. M. Debris clearance by microglia: an
Parkinson disease. Neurology 70, 1456–1460 (2008). essential link between degeneration and regeneration. Brain 132, 288–295
6. Mata, I. F., Wedemeyer, W. J., Farrer, M. J., Taylor, J. P. & Gallo, K. A. LRRK2 in (2009).
Parkinson's disease: protein domains and functional insights. Trends Neurosci. 29, 35. McGeer, P. L., Itagaki, S., Boyes, B. E. & McGeer, E. G. Reactive microglia are
286–293 (2009). positive for HLA-DR in the substantia nigra of Parkinson's and Alzheimer's disease
7. Funayama, M. et al. A new locus for Parkinson's disease (PARK8) maps to chro- brains. Neurol. 38, 1285–1291 (1988).
mosome 12p11.2-q13.1. Ann. Neurol. 51, 296–301 (2002). 36. Whitton, P. S. Inflammation as a causative factor in the aetiology of Parkinson's
8. Guo, L. et al. The Parkinson's disease-associated protein, leucine-rich repeat disease. British. J. Pharmacol. 150, 963–976 (2007).
kinase 2 (LRRK2), is an authentic GTPase that stimulates kinase activity. Exp. Cell 37. Saunders, J. A. et al. CD4+ regulatory and effector/memory T cell subsets profile
Res. 313, 3658–3670 (2007). motor dysfunction in Parkinson's disease. J. Neuroimmune Pharmacol 7, 927–938
9. Gilsbach, B. K. et al. Roco kinase structures give insights into the mechanism of (2012).
Parkinson disease-related leucine-rich-repeat kinase 2 mutations. Proc. Natl Acad. 38. Gillardon, F., Schmid, R. & Draheim, H. Parkinson's disease-linked leucine-rich
Sci. USA 109, 10322–10327 (2007). repeat kinase 2(R1441G) mutation increases proinflammatory cytokine release
10. Manzoni, C. et al. mTOR independent regulation of macroautophagy by Leucine from activated primary microglial cells and resultant neurotoxicity. Neurosci. 208,
Rich Repeat Kinase 2 via Beclin-1. Sci. Rep. 6, 35106 (2016). 41–48 (2012).
11. Ferrazza, R. et al. LRRK2 deficiency impacts ceramide metabolism in brain. 39. Waldburger, J. M. et al. Lessons from the bare lymphocyte syndrome: molecular
Biochem. Biophys. Res. Commun. 478, 1141–1146 (2016). mechanisms regulating MHC class II expression. Immunol. Rev. 178, 148–165
12. Plowey, E. D., Cherra, S. J., Liu, Y. J. & Chu, C. T. Role of autophagy in (2000).
G2019S-LRRK2-associated neurite shortening in differentiated SH-SY5Y cells. 40. Guermonprez, P., Valladeau, J., Zitvogel, L., Thery, C. & Amigorena, S. Antigen
J. Neurochem. 105, 1048–1056 (2008). presentation and T cell stimulation by dendritic cells. Annu. Rev. Immunol. 20,
13. Schapansky, J., Nardozzi, J. D., Felizia, F. & LaVoie, M. J. Membrane recruitment of 621–667 (2002).
endogenous LRRK2 precedes its potent regulation of autophagy. Hum. Genet. 23, 41. Chardin, P. & McCormick, F. Brefeldin A: the advantage of being uncompetitive.
4201–4214 (2014). Cell 97, 153–155 (1999).
14. Ramonet, D. et al. Dopaminergic neuronal loss, reduced neurite complexity and 42. Ziegler-Heitbrock, L. The CD14+ CD16+ blood monocytes: their role in infection
autophagic abnormalities in transgenic mice expressing G2019S mutant LRRK2. and inflammation. J. Leukoc. Biol. 81, 584–592 (2007).
PLoS ONE 6, e18568 (2011). 43. Grozdanov, V. et al. Inflammatory dysregulation of blood monocytes in Parkin-
15. Berwick, D. C. & Harvey, K. LRRK2 functions as a Wnt signaling scaffold, bridging son's disease patients. Acta Neuropathol. 128, 651–663 (2014).
cytosolic proteins and membrane-localized LRP6. Hum. Genet. 21, 4966–4979 44. Deng, X. et al. Characterization of a selective inhibitor of the Parkinson's disease
(2012). kinase LRRK2. Nat. Chem. Biol. 7, 203–205 (2011).

Published in partnership with the Parkinson’s Disease Foundation npj Parkinson’s Disease (2017) 11
 LRRK2 levels in PD immune cells
D A Cook et al.
12
45. Kannarkat, G. T. et al. Common genetic variant association with altered HLA 52. Hernán, M. A., Takkouche, B., Caamaño-Isorna, F. & Gestal-Otero, J. J. A meta-
expression, synergy with pyrethroid exposure, and risk for Parkinson’s disease: an analysis of coffee drinking, cigarette smoking, and the risk of Parkinson's disease.
observational and case–control study. NPJ Parkinsons Dis. 1, 15002 (2015). Ann. Neurol. 52, 276–284 (2002).
46. Gibson, H. M. et al. Induction of the CTLA-4 Gene in human lymphocytes is depen- 53. Chen, H. et al. Nonsteroidal anti-inflammatory drugs and the risk of parkinson
dent on NFAT binding the proximal promoter. J. Immunol. 179, 3831–3840 (2007). disease. Arch. Neurol. 60, 1059–1064 (2003).
47. Mount, M. P. et al. Involvement of interferon-gamma in microglial-mediated loss
of dopaminergic neurons. J. Neurosci. 27, 3328–3337 (2007).
48. Mogi, M. et al. Interleukin-1 beta, interleukin-6, epidermal growth factor and This work is licensed under a Creative Commons Attribution 4.0
transforming growth factor-alpha are elevated in the brain from parkinsonian International License. The images or other third party material in this
patients. Neurosci. Lett. 180, 147–150 (1994). article are included in the article’s Creative Commons license, unless indicated
49. Blum-Degen, D. et al. Interleukin-1 beta and interleukin-6 are elevated in the otherwise in the credit line; if the material is not included under the Creative Commons
cerebrospinal fluid of Alzheimer's and de novo Parkinson's disease patients. license, users will need to obtain permission from the license holder to reproduce the
Neurosci. Lett. 202, 17–20 (1995). material. To view a copy of this license, visit http://creativecommons.org/licenses/by/
50. Chen, H. et al. Smoking duration, intensity, and risk of Parkinson disease. 4.0/
Neurology 74, 878–884 (2010).
51. Gagne, J. J. & Power, M. C. Anti-inflammatory drugs and risk of Parkinson disease:
a meta-analysis. Neurology 74, 995–1002 (2010). © The Author(s) 2017

Supplementary Information accompanies the paper on the npj Parkinson’s Disease website (doi:10.1038/s41531-017-0010-8).

npj Parkinson’s Disease (2017) 11 Published in partnership with the Parkinson’s Disease Foundation