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Brain, Behavior, and Immunity
Brain, Behavior, and Immunity
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Article history: The most common Parkinson’s disease (PD) mutation is the gain-of-function LRRK2 G2019S variant,
Received 27 March 2017 which has also been linked to inflammatory disease states. Yet, little is known of the role of G2019S in
Received in revised form 7 August 2017 PD related complex behavioral or immune/hormonal processes in response to inflammatory/toxicant
Accepted 2 September 2017
challenges. Hence, we characterized the behavioral, neuroendocrine-immune and central monoaminergic
Available online 8 September 2017
responses in G2019S overexpressing mutants following systemic interferon-gamma (IFN-c) or
lipopolysaccharide (LPS) administration. Although LPS markedly (and IFN-c modestly in some cases)
Keywords:
increased cytokine and corticosterone levels, while inducing pronounced sickness and home-cage activity
Parkinson’s disease
Non-motor symptoms
deficits, the G2019S mutation had no effect on these parameters. No differences were observed with
Depression regards to brain microglia with the acute LPS injection, regardless of genotype. Nor did the G2019S muta-
Inflammation tion influence neurotransmitter levels within the medial prefrontal cortex or paraventricular nucleus of
Neuroendocrine the hypothalamus. However, the LRRK2 G2019S transgenic mice did have altered monoamine levels
Neurotransmission within the striatum and hippocampus. Indeed, G2019S mice had altered basal levels and turnover of
dopamine within the striatum, along with changes in hippocampal serotonin and norepinephrine activity
in response to LPS and IFN-c. The present findings suggest the importance of murine G2019S in hip-
pocampal and striatal neurotransmission, but that the transgene didn’t appear to be involved in func-
tional behavioral and stress-like hormonal and cytokine changes provoked by inflammatory insults.
Ó 2017 Published by Elsevier Inc.
https://doi.org/10.1016/j.bbi.2017.09.002
0889-1591/Ó 2017 Published by Elsevier Inc.
D. Litteljohn et al. / Brain, Behavior, and Immunity 67 (2018) 246–256 247
2006; Hakimi et al., 2011), and its expression was reported to be acclimated to the home-cage activity testing room for 16 h. Com-
increased by the inflammatory agents, interferon (IFN)-c or mencing at 0830 h on the day of experimentation and at 10 min
lipopolysaccharide (LPS) (Thévenet et al., 2011; Moehle et al., intervals, mice were individually removed from their grouped
2012; Kuss et al., 2014). Likewise, LRRK2 has been repeatedly enclosures and singly housed in fresh cages; as before, food and
linked to inflammatory aspects of PD, including microglial activa- water was provided ad libitum. Immediately upon cage transfer,
tion and peripheral immune cell alterations (Brockmann et al., mice were administered a single intraperitoneal injection, in a vol-
2017; Ma et al., 2016; Russo et al., 2015; Moehle et al., 2015; ume of 0.3 ml, of a) a PBS solution containing 0.1% BSA (Sigma
Kim et al., 2012; Lopez de Maturana et al., 2014). Accordingly, Aldrich) (i.e., the vehicle); b) recombinant murine IFN-c
LRRK2 likely contributes to the regulation of inflammatory pro- (25000IU, R&D Systems; reconstituted in 0.1% BSA); or c) LPS
cesses (Russo et al., 2014; Russo et al., 2015; Puccini et al., 2015), (10 lg, L8274: E. coli serotype O26:B6, Sigma Aldrich; in 0.1%
which in turn, can promote brain pathology. BSA). Drug doses were chosen based on earlier reports indicating
Although several reports suggest a pre-existing dopamine (DA) that systemic administration of IFN-c or LPS at these or similar
deficit in the absence of neuronal degeneration in LRRK2 G2019S doses influenced stress hormone levels, central monoamine activ-
mutant mice (Liu et al., 2015; Garcia-Miralles et al., 2015; Chou ity and behaviour (Crnic and Segall, 1992; Lacosta et al., 1999; Gibb
et al., 2014; Li et al., 2010; Melrose et al., 2010), little is known et al., 2008).
regarding the impact of a relevant inflammatory challenge, such
as LPS or IFN-c, which we have previously found to have marked 2.3. Behavioral analyses
systemic stressor effects (Litteljohn et al., 2017; Hayley et al.,
2008). Nor is it known whether G2019S mutant mice have altered Over the ensuing 90-min period, spontaneous locomotor activ-
stressor reactivity, as indicated by corticoid and cytokine ity was assessed with a Micromax infrared beam-break apparatus
responses, as well as neurochemical changes in non-motor stressor positioned exterior to the home-cage (AccuScan Instruments,
sensitive brain regions. To this end, we presently found that over- Columbus, OH). Sickness ratings were recorded for each mouse at
expression of the G2019S mutation did not influence LPS or IFN-c 75 and 90 min post-injection. Per the method of Hayley et al.
induced acute behavioral, cytokine or hormonal responses, but did (1999), sickness behaviors were scored on a 3-point scale (where
alter hippocampal and striatal monoamine levels and turnover. 0 = no symptom, 1 = one symptom present, 2 = two symptoms pre-
Brain microglia were also unaffected by the G2019S mutation. sents, 3 = three or more symptoms) with respect to curled body
However, the acute systemic LPS injection itself was not sufficient posture, ptosis (drooping eyelids), piloerection, lethargy, and over-
to induce signs of microgliosis, so it remains to be determined all non-responsiveness. This procedure was previously found to
whether the genotype might play a role with more severe inflam- provide less than 10% variability between raters blind to the treat-
matory insults. Together, these data further add to the complex ment mice received and was highly correlated with other methods
phenotype related to LRRK2 and suggest its importance in certain of scoring sickness (e.g. assessing severity of each symptom inde-
aspects of central monoamine signaling, but curiously not acute pendently) (Gandhi et al., 2007). All scores were obtained by the
behavioral, cytokine or stress-hormone responses to inflammatory same blind rater.
insults.
2.4. Brain dissection technique
the homogenizing solution in which they were initially frozen 200 mL of 1° antibodies at 1:2000 antibody to primary dilution
(0.1 mM Na2EDTA, 0.3 M monochloroacetic acid and 10% methanol solution (50 mL: 2500 mL NGS, 1500 mL Triton-X, 7.5 mL 2% bovine
in HPLC-grade water, with DHBA as an internal standard). Protein serum albumin (BSA), 38.5 mL 0.1 M PBS) and incubated overnight.
levels were determined with the Pierce BCA Protein Assay Kit Sections were then further washed and then placed in 200 mL of
(Thermo Scientific 23225). Homogenized samples were cen- 1:1000 2° antibody solution for Iba-1 (Alex Fluor 647 goat anti-
trifuged (12000 rpm for 3 min at 4 °C), after which 50 ll of super- rabbit IgG, ThermoFisher, A21246), and 1:500 for TH (Alex Fluor
natant was injected, at a flow rate of 1 ml/min, into the automated 350 goat anti-mouse IgG, ThermoFisher, A11068) for 3 h at room
HPLC system (Agilent 1100) with electrochemical detector (DEC- temperature. Sections were cover-slipped using fluoromount
ADE II SDC, Antec) and ZORBAX Eclipse XDB-C8 columns (Agilent:
4.6 mm inner diameter, 150 mm length, 5 mm particle size; ther-
mostated at 40 °C); the oxidation potential was maintained at
0.60 V. The mobile phase comprised: 90 mM sodium phosphate
monobasic, 1.7 mM 1-Octanesulfonic acid, 50 mM EDTA, 10% ace-
tonitrile, 50 mM citric acid (monohydrate), 5 mM KCL, and HPLC-
grade water. Monoamine and metabolite concentrations were
expressed relative to the protein content of the samples, and final
results presented as ng/mg protein.
(Sigma-Aldrich, St. Louis, MO) and were covered to avoid photo- 3. Results
bleaching.
Microglia density and ‘‘activation state” were assessed within 3.1. LRRK2 G2019S overexpression did not modify LPS-induced
the substantia nigra and striatum by well trained blind raters. changes in sickness, motor behaviour, corticosterone or cytokine levels
Specifically, overall density or area covered in these brain regions
was calculated using the standard readily available Image-J soft- The repeated measures ANOVA for home-cage locomotor activ-
ware program. Secondly, microglial morphology was assessed as ity yielded a significant main effect of Treatment (F2, 36 = 21.93,
an index for their activational state. Using our previously estab- p < 0.0001). The follow-up tests revealed that LPS suppressed
lished and well validated qualitative rating scale, we assessed home-cage activity in WT and G2019S mice alike (p < 0.05 relative
Iba-1 stained cells in terms of several parameters on a 0–3 scale: to mice treated with vehicle or IFN-c). Indeed, as shown in Fig. 1,
where 0 reflects the lowest state of activation, wherein microglia LPS provoked a greater than 60% reduction in home-cage activity
display highly ramified, thin processes with a small soma and 3 in both genotypes. Although IFN-c slightly suppressed home-
equates to the most activate state in which mostly all cells show cage (by 23%) activity in the G2019S animals, this effect was not
thick rounded soma and no or few processes. significant (p > 0.05; Fig. 1).
As for sickness behaviour, the repeated measures ANOVA
2.9. Statistical analyses detected a significant main effect of Treatment (F2, 36 = 379.05,
p < 0.0001). As shown in Fig. 1, this was attributable to the robust
The monoamine, cytokine and corticosterone data were ana- sickness-inducing effect of LPS, irrespective of genotype (p < 0.05
lyzed by 2 3 ANOVAs followed where appropriate by Tukey- relative to mice treated with vehicle or IFN-c). Indeed, there were
Kramer post hoc tests; i.e., for Genotype Treatment interactions no signs of sickness in non-LPS treated mice, whereas the endo-
and Treatment main effects absent a significant disordinal interac- toxin provoked obvious curled body posture and/or ptosis and
tion (a = 0.05). Plasma cytokine concentrations were transformed piloerection.
by natural logarithm (ln) to improve normality and homoscedas- Paralleling the behavioral findings, plasma corticosterone con-
ticity. Two-factor repeated measures ANOVAs were used to ana- centrations were significantly increased (4-fold) among the
lyze the behaviors. Similarly, a 2 (saline vs LPS) 2 (WT vs LPS-treated animals irrespective of LRRK2 G2019S overexpression
G2019S) ANOVA was used to assess glial immunereactivity. Data (F2, 36 = 72.68, p < 0.0001) (Fig. 1). IFN-c did not significantly
were evaluated using a StatView (version 6.0) statistical software impact corticosterone levels, despite a slight basal reduction (30%
package and visualized with GraphPad Prism 6 (La Jolla, CA). relative to WT mice) in G2019S mice.
Fig. 2. LPS and IFN-c increased circulating cytokine levels irrespective of LRRK2 G2019S overexpression. In both the GS-Tg and non-Tg mice, LPS increased the natural log-
transformed (ln) plasma levels of TNF-a, MCP-1, IL-6, and IL-10 (a–d); there were no significant between group differences in IL-1b (e). In addition, both MCP-1 (b) and IL-6 (c)
were augmented by IFN-c among mice of either genotype. Data are shown as mean ± SEM. ap < 0.05 relative to vehicle-treated and IFN-c-treated mice; bp < 0.05 relative to
vehicle-treated and LPS-treated mice.
250 D. Litteljohn et al. / Brain, Behavior, and Immunity 67 (2018) 246–256
3.2. LRRK2 G2019S overexpression did alter basal and LPS and IFN-c
induced striatal dopamine, as well as hippocampal NE and 5-HT levels
3.3. G2019S did not modify LPS or IFN-g induced monoamine changes
in PFC, PVN or locus coruleus
Fig. 3. Dopaminergic activity within the dorsal striatum: influence of LPS, IFN-c
and LRRK2 G2019S overexpression. GS-Tg mice had significantly lower overall DA
Within the hypothalamic PVN and the locus coruleus, LPS but levels, compared to their wild type (Non-Tg) littermates. Also, IFN- c administration
not IFN-c treatment provoked changes in monoamines, all of significantly reduced DA levels in the wild type animals only. In contrast, IFN- c
which were independent of LRRK2 G2019S overexpression. Specif- reduced levels of the metabolite, DOPAC, only within the GS-Tg mice. Finally,
ically, the multiple separate ANOVAs revealed a significant main dopaminergic turnover (DOPAC/DA ratio) was significantly augmented in wild type
mice but diminished in GS-Tg animals.
effect of Treatment for PVN 5-HIAA (F2, 29 = 4.23, p < 0.05); locus
coruleus NE (F2, 35 = 5.15, p < 0.05); and locus coruleus MHPG (F2,
35 = 4.60, p < 0.05). The follow-up tests indicated that LPS dimin- robust striatal amine changes and their biological significance
ished NE concentrations (p < 0.05), whilst significantly enhancing remains to be determined.
5-HIAA and MHPG accumulation within these respective brain
regions (p < 0.05).
While there were no significant differences in NE levels within 3.4. G2019S had no effect on microglial reactivity
the PFC, MHPG was markedly augmented by LPS treatment (F2,
36 = 5.19, p < 0.05) (Fig. 5). Similarly, both PFC 5-HT and 5-HIAA As depicted in Fig. 6, there was no evidence that the peripheral
were increased overall in response to the endotoxin treatment LPS injection provoked neuroinflammatory changes, nor was there
(Fs2, 36 = 3.73 and 5.55, p < 0.05 and 0.01). As shown in Fig. 5, the any affect of the G2019S mutation. At least with respect to the
G2019S genotype did not influence these monoamine changes. morphology and number of microglia stained with the Iba-1 anti-
Again, these effects were relatively modest compared to the more body there were no differences between the treatment groups
D. Litteljohn et al. / Brain, Behavior, and Immunity 67 (2018) 246–256 251
Fig. 4. Noradrenergic and serotonergic activity within the dorsal hippocampus: influence of LPS, IFN-c and LRRK2 G2019S overexpression. Within the dorsal hippocampus
(HC), NE concentrations were significantly increased following IFN-c administration in the GS-Tg and non-Tg mice alike (a). As well, compared to their non-Tg littermates, the
GS-Tg mice had elevated overall levels of dorsal hippocampal NE (a), 5-HT (c) and 5-HIAA (d). There were no between-group differences in the levels of MHPG (b). Data are
shown as mean ± SEM. bp < 0.05 relative to vehicle-treated mice; cp < 0.05 relative to non-Tg mice.
F2, 24 < 1. Indeed, Iba-1+ microglial density and morphological rat- LPS or IFN- c challenge. However, the LRRK2 G2019S mice did
ings were unaffected by LPS or genotype. Fig. 6 shows Iba-1 stain- show dramatic basal reductions in striatal DA and elevated hip-
ing in the substantia nigra (adjacent to co-stained TH+ dopamine pocampal norepinephrine and serotonin levels. Striatal DA turn-
neurons) and similarly, no differences whatsoever were observed over was also reduced in G2019S mice in response to LPS and
within the striatum (data not shown). Indeed, virtually all Iba-1 IFN- c. In contrast, other brain regions assessed, including the
cells displayed a similar morphology, characterized by small soma PFC, PVN and locus coruleus, displayed LPS or IFN- c induced neu-
with delicate, fine ramified projections. rochemical changes in the absence of any effect of genotype, sug-
gesting that the impact of the mutation is highly brain region
4. Discussion specific.
The blunted DA turnover in G2019S mice with IFN- c treatment
Substantial evidence points to a role for inflammation in the is particularly intriguing in light of the fact that IFN- c null mice
genesis of PD and its co-morbid features (Litteljohn and Hayley, were completely resistant to the neurodegenerative and neuroin-
2012; Moehle et al., 2012; Mangano and Hayley, 2009; Nagatsu flammatory impact of MPTP or paraquat (Mount et al., 2007;
and Sawada, 2005; Vila et al., 2001; Joers et al., 2016). LRRK2 is Mangano et al., 2011), as well as many of the behavioral responses
an ideal candidate gene that could act as a mediator of inflamma- to chronic stress (Litteljohn et al., 2014). Indeed, our present data
tory processes triggered by environmental insults. Indeed, LRRK2 is suggest that in the face of already reduced basal DA levels, the
found glia and immune cells and has critical roles in innate and G2019S overexpression further diminishes striatal DA utilization
adaptive immunity (Lee et al., 2015; Gardet et al., 2010). Yet, the (as indicated by a drop in the metabolite, DOPAC) in response to
existing data assessing the common G2019S mutation is confusing IFN- c. Also, IFN- c itself is know to be a potent inducer of
at best. Presently, we demonstrate that G2019S transgenic mice endogenous LRRK2 (Gardet et al., 2010), but it is unclear as to
over-expressing the murine form of the mutant gene display a phe- how the cytokine might affect the mutant form of the gene. Since
notype that is restricted to specific neurotransmitter changes and the present mice express elevated expression of the G2019S trans-
not general stressor or inflammatory processes (at least with gene in the context of background levels of the existing wild type
regards to corticosterone, microglia and cytokine levels) or sick- gene, it might be that the mutant and wild type forms of G2019S
ness and motor behavioral impairments induced by acute systemic compete with each other or interact in unexpected ways with
LPS or IFN- c treatment. regards to IFN- c neurochemical modulation.
As summarized in Table 1, the G2019S mutation had no influ- The selective serotonin and noradrenergic hippocampal
ence on the hormonal, cytokine and behavioral effects of acute changes presently observed in G2019S mice may hold importance
252 D. Litteljohn et al. / Brain, Behavior, and Immunity 67 (2018) 246–256
Fig. 5. Noradrenergic, serotonergic and dopaminergic activity within the medial prefrontal cortex: influence of LPS, IFN-c and LRRK2 G2019S overexpression. Within the PFC,
LPS significantly increased the levels of MHPG, 5-HT, 5-HIAA, DOPAC, and HVA (b, c, d, f, g). LRRK2 G2019S overexpression attenuated the LPS-induced increase in DOPAC
levels (f) and was itself seen to augment HVA concentrations overall (g). In contrast to the LPS treatment, IFN-c did not influence PFC monoaminergic neurotransmission
among mice of either genotype. Data are shown as mean ± SEM. ap < 0.05 relative to vehicle-treated controls; cp < 0.05 relative to non-Tg mice; *p < 0.05 relative to non-Tg
controls.
for co-morbid depressive or cognitive symptoms often observed in inflammatory cytokines. Similarly, Lopez de Maturana et al.
PD. Indeed, our own LPS and paraquat treatment models of PD pro- (2014) failed to detect a cytokine- or- COX-2-modifying influence
duced marked hippocampal neurochemical changes that were cor- of LRRK2 G2019S in LPS-stimulated fibroblasts and Gardet et al.
related with cognitive and depressive-like behaviors (Rudyk et al., (2010) did not find a difference in NF-jB activation in HEK293 cells
2015; Litteljohn et al., 2009). Moreover, LRRK2 G2019S mutation expressing LRRK2 G2019S.
carriers were reported to show lowered cognitive performance We also found that G2019S did not obviously influence brain
(Thaler et al., 2012) and an increased risk of premorbid affective microglia (at least within the substantia nigra and striatum;
disorders (Shanker et al., 2011), suggesting that the presence of regions obviously involved in PD and its comorbidities and that
the mutation has widespread functional effects. are normally sensitive to various stressors or toxicants). Of course,
Given that LRRK2 interacts with, and phosphorylates various the systemic LPS injection itself also did not affect microglia and
exocytic and endocytic proteins in the presynaptic compartment this was likely due to the relatively short sampling time (after
(Shin et al., 2008; Matta et al., 2012; Yun et al., 2013; Cirnaru 90 min) and route (ip.) of administration. In fact, we previously
et al., 2014; Parisiadou et al., 2014; Piccoli et al., 2011, 2014; found that even with central (intra-nigral) infusion of LPS, it still
Belluzzi et al., 2016), it might influence neurotransmission by mod- took two days to observed marked microglial activation these mice
ulating synaptic vesicle exo-endocytosis (Migheli et al., 2013; were sensitized to the neurodegenerative impact of subsequent
Arranz et al., 2015; Belluzzi et al., 2012). Alternatively, the paraquat exposure (Mangano and Hayley, 2009). It remains to be
G2019S mutation might have had developmental effects, possibly determined whether the G2019S mutation might have a role in
involving structural alterations and dendritic arbors and spine den- such an obvious central LPS induced neuroinflammatory microen-
sity or involving changes in intra-cellular protein trafficking that vironment. That said, we were presently more interested in
could influence neurotransmission. whether G2019S was important for basic acute inflammatory/
Some evidence suggests that the G2019S mutation promotes stressor responses that might be relevant for PD comorbidity,
exaggerated inflammatory responses (Moehle et al., 2015; rather than characterizing its role in neuroinflammatory provoked
Gloeckner et al., 2009; Kim et al., 2012). Yet, consistent with our neurodegeneration. Indeed, several papers have already explored
lack of an effect on LPS induced cytokines (TNF-a, MCP-1, IL-6, G2019S with regards to neurodegeneration and the findings have
IL-10), Moehle et al. (2015) reported that GS2019S mice displayed been negative or modest at best. However, this is the first report
unaltered blood chemistry and that LPS-stimulated primary to assess the G2019S mutation with regards to the stressor-like
G2019S macrophages did not produce elevated levels of pro- effects of LPS.
D. Litteljohn et al. / Brain, Behavior, and Immunity 67 (2018) 246–256 253
Fig. 6. Microglial reactivity was assessed using the Iba-1 marker (red) in wild type (WT) and G2019S mutants in response to LPS or saline treatment. Co-immunofluorescence
for dopamine neurons using tyrosine hydroxylase (TH; green) was also included for easy demarcation of the substantia nigra. Panels A-D show TH + dopamine neurons in the
top left corner (green) with adjacent Iba-1 + microglial cells (red) covering the entire nigra and adjacent regions. Indeed, Iba-1 + cells were uniformly spaced with no
difference in density between the saline treated WT and G2019S mutants (panels A and C, respectively), nor between the LPS treated WT or G2019S mutants (panels B and D,
respectively). Similarly, panels E and F (respectively) depict higher magnification Iba-1 immunofluorescence for saline and LPS treated WT mice. Clearly, there were no
differences in microglial morphology ratings between these, nor were there any differences in the similarly treated G2019S mutants (not shown). Virtually all microglia
displayed delicate, small soma with fine ramified processes.
As is commonly observed, LPS presently induced robust sick- entirely replaces the wild type form of the gene, and hence, isolates
ness behavior and plasma corticosterone elevations that paralleled the role of G2019S alone (and presumably at typical levels of
its cytokine alterations. In contrast, we found that IFN- c treatment expression). Indeed, most curiously, it was recently reported that
had no behavioral or corticoid effects and only modestly elevated G2019S knockin mice exhibited a basal hyper-kinetic phenotype
MCP-1 and IL-6. Since, the G2019S genotype did not have an affect (Longo et al., 2014).
on any of these outcomes, it appears that the neurochemical In summary, our data are consistent with very specific neuro-
changes related to the G2019S genotype were likely not secondary chemical deficits in G2019S mice and not widespread toxicity,
to any widespread toxicity or inflammatory effects that might be stressor or inflammatory changes. Conceivably, such neurochemi-
related to the mutation. cal changes could contribute to both the primary motor and the
It is important to note that the presently used mice expressed co-morbid non-motor features often evident in PD. Although
the murine LRRK2 G2019S mutation, as opposed the human form. we did not find gross behavioral deficits in these mice, there may
Liu et al. (2015), who used the same mice as the current study, have been more subtle behavioral effects that were missed. Yet,
found no behavioral deficits or neuronal loss in aged G2019S mice. it was clear that the G2019S overexpression did not at all influence
They did however report basal reductions of dopamine levels the typical acute behavioral and inflammatory response induced
within the striatum (Liu et al., 2015), which is consistent with by LPS. Our future studies will assess whether G2019S knockin
our present findings. Also of tremendous importance is the fact mice that lack any wild type LRRK2 expression are similar to the
that our mice bear not only the mutant G2019S transgene, but also currently used transgenic overexpressing mice in terms of
the wild type form of the LRRK2 gene. Thus, we are essentially responses to inflammatory challenges. It will also be of interest
looking at the combined effects of 6–8-fold over-expression of to determine whether advanced age may be a vulnerability factor
the mutant G2019S transgene in the context of the normal expres- in G2019S mice and if multiple hits with inflammatory and
sion of wild type LRRK2. This differs from some recent studies chemical toxins (e.g. paraquat) might collectively produce a more
using the knockin form of G2019S, in which the mutant form comprehensive PD-like phenotype.
254 D. Litteljohn et al. / Brain, Behavior, and Immunity 67 (2018) 246–256
Table 1
Summary of significant experimental findings.
Author contributions SNARE complex disassembling rate. Mol. Neurodegener. 11 (1), 1. https://doi.
org/10.1186/s13024-015-0066-z.
Brockmann, K., Schulte, C., Schneiderhan-Marra, N., Apel, A., Pont-Sunyer, C., Vilas,
Conceived and designed the experiments: DL, SH. Performed D., Ruiz-Martinez, J., Langkamp, M., Corvol, J.C., Cormier, F., Knorpp, T., Joos, T.O.,
the experiments: DL, SR, CR, ZD. Contributed reagents/materials/- Bernard, A., Gasser, T., Marras, C., Schüle, B., Aasly, J.O., Foroud, T., Marti-Masso,
J.F., Brice, A., Tolosa, E., Berg, D., Maetzler, W., 2017. Inflammatory profile
analysis tools: SH, HA, DL. Analyzed the data and wrote the paper:
discriminates clinical subtypes in LRRK2-associated Parkinson’s disease. Eur. J.
DL, SH. Neurol. 24 (2). 427 e6.
Chen, H., Zhao, E.J., Zhang, W., Lu, Y., Liu, R., Huang, X., Ciesielski-Jones, A.J., Justice,
Acknowledgments M.A., Cousins, D.S., Peddada, S., 2015. Meta-analyses on prevalence of selected
Parkinson’s nonmotor symptoms before and after diagnosis. Transl.
Neurodegener. 4 (1), 1. https://doi.org/10.1186/2047-9158-4-1.
Thanks to Dr. Jerzy Kulczycki (HPLC and RIA analyses) and Chou, J.S., Chen, C.Y., Chen, Y.L., Weng, Y.H., Yeh, T.H., Lu, C.S., Chang, Y.M., Wang, H.
Marzena Sieczkos (cytokine multiplex analysis) for their expert L., 2014. (G2019S) LRRK2 causes early-phase dysfunction of SNpc dopaminergic
neurons and impairment of corticostriatal long-term depression in the PD
technical assistance, and to Farah Wahbeh for help with
transgenic mouse. Neurobiol. Dis. 68, 190–199. https://doi.org/10.1016/j.
biospecimen collection. This research was supported by funds to nbd.2014.04.021.
D. Litteljohn [Canadian Institutes of Health Research (CIHR) and Cirnaru, M.D., Marte, A., Belluzzi, E., Russo, I., Gabrielli, M., Longo, F., Arcuri, L.,
Ontario Graduate Scholarship (OGS)] and S. Hayley (CIHR). CLINT Murru, L., Bubacco, L., Matteoli, M., Fedele, E., Sala, C., Passafaro, M., Morari, M.,
Greggio, E., Onofri, F., Piccoli, G., 2014. LRRK2 kinase activity regulates synaptic
Investigators include: Earl Brown, Derrick Gibbings, Shawn Hayley, vesicle trafficking and neurotransmitter release through modulation of LRRK2
David Park, Dana C. Philpott, John D. Rioux, Michael Schlossmacher, macro-molecular complex. Front. Mol. Neurosci. 27 (7), 49. https://doi.org/
and Erwin Schurr. 10.3389/fnmol.2014.00049.
Cookson, M.R., 2015. LRRK2 pathways leading to neurodegeneration. Curr. Neurol.
Neurosci. Rep. 15 (7), 42. https://doi.org/10.1007/s11910-015-0564-y.
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