Brain, Behavior, and Immunity 67 (2018) 246–256

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Brain, Behavior, and Immunity

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Full-length Article

The impact of murine LRRK2 G2019S transgene overexpression on acute

responses to inflammatory challenge
Darcy Litteljohn, Chris Rudyk, Zach Dwyer, Kyle Farmer, Teresa Fortin, Shawn Hayley ⇑, Canadian Lrrk2 in
Inflammation Team (CLINT) 1
Department of Neuroscience, Carleton University, Ottawa, Ontario, Canada

a r t i c l e i n f o a b s t r a c t

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.

1. Introduction PD (Cookson, 2015). The most frequent of these mutations is the

Motor deficits are the most recognizable features of Parkinson’s
disease (PD) but are often coupled with anxiety, depression and
cognitive disturbances (Chen et al., 2015; Schneider and Obeso,
2015). Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene,
which encodes a large multimeric protein whose enzymatic core
comprises Roc-COR tandem GTPase and serine/threonine kinase
domains, have been linked to both familial and sporadic forms of
Abbreviations: 5-HIAA, 5-hydroxyindole acetic acid; 5-HT, serotonin; ANOVA,
analysis of variance; BLA, basolateral amygdala; DA, dopamine; DOPAC, 3,4-
dihydroxyphenylacetic acid; EDTA, ethylenediaminetetraacetic acid; HC, hip-
pocampus; HPLC, high-performance liquid chromatography; HVA, homovanillic
acid; IFN-c, interferon-c; KO, knockout; LC, locus coeruleus; LRRK2, leucine-rich
repeat kinase-2; MHPG, 3-methoxy-4-hydroexyphenylglycol; NE, norepinephrine;
PD, Parkinson’s disease; PVN, paraventricular nucleus of the hypothalamus.
⇑ Corresponding author at: 1125 Colonel By Drive, Ottawa, Ontario, K1S 5B6,
Canada.
E-mail address: shawn_hayley@carleton.ca (S. Hayley).
1
Membership of the Canadian LRRK2 in Inflammation Team (CLINT) is listed in the
Acknowledgments.
gain-of-function G2019S substitution (glycine-to-serine substitu-
tion at position 2019), which causes increased LRRK2 kinase activ-
ity (West et al., 2005; Smith et al., 2006).
Although LRRK2-associated PD generally resembles idiopathic
disease (e.g., Healy et al., 2008), the G2019S mutation may cause
more frequent, severe and/or earlier-occurring neuropsychiatric
symptoms (Goldwurm et al., 2006; Belarbi et al., 2010; Johansen
et al., 2011; Marras et al., 2011; Mirelman et al., 2015; Thaler
et al., 2016). Accordingly, LRRK2 G2019S overexpressing mice dis-
played impaired hippocampal neurogenesis (Winner et al., 2011;
Sweet et al., 2015), together with age-dependent cognitive and
affective behavioral deficits (Melrose et al., 2010; Volta et al.,
2015).
LRRK2 is expressed in several brain regions, including the sub-
stantia nigra pars compacta (SNc), dorsal striatum, hippocampus,
prefrontal cortex (PFC), hypothalamus, and locus coeruleus
(Simón-Sánchez et al., 2006; Taymans et al., 2006; Higashi et al.,
2007). It is also highly expressed in immune cells, including mono-
cytes, B-lymphocytes, macrophages and microglia (Miklossy et al.,

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
2006; Hakimi et al., 2011), and its expression was reported to be
increased by the inflammatory agents, interferon (IFN)-c or
lipopolysaccharide (LPS) (Thévenet et al., 2011; Moehle et al.,
2012; Kuss et al., 2014). Likewise, LRRK2 has been repeatedly
linked to inflammatory aspects of PD, including microglial activa-
tion and peripheral immune cell alterations (Brockmann et al.,
2017; Ma et al., 2016; Russo et al., 2015; Moehle et al., 2015;
Kim et al., 2012; Lopez de Maturana et al., 2014). Accordingly,
LRRK2 likely contributes to the regulation of inflammatory pro-
cesses (Russo et al., 2014; Russo et al., 2015; Puccini et al., 2015),
which in turn, can promote brain pathology.
Although several reports suggest a pre-existing dopamine (DA)
deficit in the absence of neuronal degeneration in LRRK2 G2019S
mutant mice (Liu et al., 2015; Garcia-Miralles et al., 2015; Chou
et al., 2014; Li et al., 2010; Melrose et al., 2010), little is known
regarding the impact of a relevant inflammatory challenge, such
as LPS or IFN-c, which we have previously found to have marked
systemic stressor effects (Litteljohn et al., 2017; Hayley et al.,
2008). Nor is it known whether G2019S mutant mice have altered
stressor reactivity, as indicated by corticoid and cytokine
responses, as well as neurochemical changes in non-motor stressor
sensitive brain regions. To this end, we presently found that over-
expression of the G2019S mutation did not influence LPS or IFN-c
induced acute behavioral, cytokine or hormonal responses, but did
alter hippocampal and striatal monoamine levels and turnover.
Brain microglia were also unaffected by the G2019S mutation.
However, the acute systemic LPS injection itself was not sufficient
to induce signs of microgliosis, so it remains to be determined
whether the genotype might play a role with more severe inflam-
matory insults. Together, these data further add to the complex
phenotype related to LRRK2 and suggest its importance in certain
aspects of central monoamine signaling, but curiously not acute
behavioral, cytokine or stress-hormone responses to inflammatory
insults.
2. Materials and methods
2.1. Animals
Development of the bacterial artificial chromosome G2019S
transgenic mouse, which overexpresses the mouse LRRK2
G2019S mutant protein 6–8-fold greater than normal, has been
described elsewhere (Li et al., 2010). These mice were obtained
from Jackson Laboratory (012467) and were bred in house with
female C57BL/6J wild-type (WT) mice producing a generation of
hemizygous G2019S-LRRK2 mice and WT mice. Male G2019S and
WT littermates (aged 8 weeks) were used. Animals were shipped
to our facility in same-sex and same-litter groupings of at least 2
per container. Upon arrival, the mice were housed in microisolator
cages in same-litter groups of 2 or 3 and maintained on a 12-h
light/dark cycle with lights on at 0800 h. A diet of standard labora-
tory mouse chow (Harlan Laboratories, WI) and water was pro-
vided ad libitum and room temperature maintained at 21 °C.
All animals were acclimated to the vivarium for a one-week period
prior to the initiation of procedures. All experimental procedures
were approved by the Carleton University Committee for Animal
Care and complied with the guidelines set out by the Canadian
Council for the Use and Care of Animals in Research. Animals were
12–15 weeks old at the time of experimentation.
2.2. Procedure
Mice were randomly assigned to one of six experimental condi-
tions (n = 7), as provided for by our 2 (Genotype: G2019S vs WT) 
3 (Treatment: vehicle, IFN-c, LPS) factorial design. All mice were
247
acclimated to the home-cage activity testing room for 16 h. Com-
mencing at 0830 h on the day of experimentation and at 10 min
intervals, mice were individually removed from their grouped
enclosures and singly housed in fresh cages; as before, food and
water was provided ad libitum. Immediately upon cage transfer,
mice were administered a single intraperitoneal injection, in a vol-
ume of 0.3 ml, of a) a PBS solution containing 0.1% BSA (Sigma
Aldrich) (i.e., the vehicle); b) recombinant murine IFN-c
(25000IU, R&D Systems; reconstituted in 0.1% BSA); or c) LPS
(10 lg, L8274: E. coli serotype O26:B6, Sigma Aldrich; in 0.1%
BSA). Drug doses were chosen based on earlier reports indicating
that systemic administration of IFN-c or LPS at these or similar
doses influenced stress hormone levels, central monoamine activ-
ity and behaviour (Crnic and Segall, 1992; Lacosta et al., 1999; Gibb
et al., 2008).
2.3. Behavioral analyses
Over the ensuing 90-min period, spontaneous locomotor activ-
ity was assessed with a Micromax infrared beam-break apparatus
positioned exterior to the home-cage (AccuScan Instruments,
Columbus, OH). Sickness ratings were recorded for each mouse at
75 and 90 min post-injection. Per the method of Hayley et al.
(1999), sickness behaviors were scored on a 3-point scale (where
0 = no symptom, 1 = one symptom present, 2 = two symptoms pre-
sents, 3 = three or more symptoms) with respect to curled body
posture, ptosis (drooping eyelids), piloerection, lethargy, and over-
all non-responsiveness. This procedure was previously found to
provide less than 10% variability between raters blind to the treat-
ment mice received and was highly correlated with other methods
of scoring sickness (e.g. assessing severity of each symptom inde-
pendently) (Gandhi et al., 2007). All scores were obtained by the
same blind rater.
2.4. Brain dissection technique
At 90-min post-injection, mice were sacrificed by rapid decapi-
tation and blood and brain tissue collected for later biochemical
analysis. In order to minimize the effects of diurnal variations,
experiments were carried out between the hours of 08:30 and
12:30. Brains were excised and sectioned into sequential coronal
slices using razor blades and a chilled stainless steel microdissect-
ing matrix with adjacent slots spaced 0.5 mm apart. Hollow biopsy
needles were used to collect the paraventricular nucleus of the
hypothalamus (PVN) and locus coruleus. The dorsal striatum, med-
ial prefrontal cortex (PFC) and dorsal hippocampus were excised
using chilled razor blades. Each of these stressor-sensitive brain
areas has been shown to express LRRK2 (though only moderately
in the locus coruleus) (Taymans et al., 2006; Higashi et al., 2007)
and all are implicated in depression and related non-motor PD
symptoms (Fanselow and Dong, 2010; Engeln et al., 2015). Tissue
samples were taken with reference to the mouse brain atlas of
Franklin and Paxinos (1997). Samples were snap frozen in homog-
enizing solution (see below) and stored at 80 °C until determina-
tion of central monoamine and metabolite levels using high
performance liquid chromatography (HPLC).
2.5. HPLC determination of central amine and metabolite
concentrations
Regional brain levels of norepinephrine (NE), serotonin (5-HT)),
and their primary metabolites 3,4-dihydroxyphenylacetic acid
(MHPG), 5-hydroxyindoleacetic acid (5-HIAA), respectively, were
determined by HPLC in keeping with previously reported methods
(Liu et al., 2014). Tissue punches were homogenized by ultrasonic
disruption (Sonic Dismembrator Model 100, Thermo Scientific) in
248 D. Litteljohn et al. / Brain, Behavior, and Immunity 67 (2018) 246–256

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.

2.6. Plasma corticosterone assay

At the time of decapitation, trunk blood was collected in tubes

containing 10 lg EDTA. Samples were centrifuged (3000g for
8 min) and the plasma removed and stored in aliquots at 80 °C
for later corticosterone determination with commercially available
radioimmunoassay kits (ICN Biomedicals, CA, USA). Samples were
assayed in duplicate within a single run to control for inter-assay
variability. Separate plasma aliquots were used for the cytokine
determinations.

2.7. Plasma cytokine quantification

Circulating levels of 5 different cytokines, namely tumor necro-

sis factor-alpha (TNF-a), monocyte chemoattractant protein-1
(MCP-1/CCL2) and the interleukins (IL) IL-1b, IL-6 and IL-10, were
determined by multiplex analysis using the Luminex 100
suspension-based bead array system (Luminex Corp., Austin, TX)
and a custom multiple cytokine detection kit (MILLIPLEX MAP
Mouse Cytokine/Chemokine Kit, Millipore, Cat. #MPXMCYTO-
70K). IFN-g, GM-CSF and IL-4 were also assayed but found to be
below the limit of detection. The assay was performed according
to the kit manufacturer’s instructions (see www.millipore.com/
userguides) and, unless otherwise indicated, all reagents were pro-
vided in the multiplex kit. Samples were diluted by a factor of 2.
Measurements falling below or above the assay’s working range
for a given analyte were assigned a value of one-half the lower
limit of quantitation or twice the upper limit of quantitation,
respectively. Samples from each of the 6 treatment groups were
run in duplicate and the data averaged prior to statistical analysis.

2.8. Microglial Iba-1 immunohistochemistry

A separate cohort of mice were perfused 90 min following the

saline or LPS injection with 4% PFA in 0.1 M PBS and post-fixed
for a further 24 h, followed by two days in 10% sucrose and then
transferred to a 30% sucrose solution. Brains were kept at 4 °C until
flash freezing followed by cryostat sectioning, with 20 lm sections
collected in well plates containing 1 mL 0.1 M PBS with 0.01% azide
and stored at 4 °C until fluorescent staining.
For fluorescent microscopy, sections were co-stained with ion-
ized calcium binding adaptor molecule 1 (Iba-1) (rabbit anti-
mouse, Abcam) and tyrosine hydroxylase (TH) (Immunostar,
22941). Iba-1 labels microglia, whereas TH staining labels dopa- Fig. 1. LRRK2 G2019S overexpression failed to modify LPS-induced changes in
mine neurons. Sections were rinsed 3  5 min with 10 mM PBS behaviour and plasma corticosterone. The LPS treatment markedly suppressed
home-cage activity (a), induced sickness behaviour (b), and raised plasma
and then blocked for 30 min using 250 mL blocking solution per corticosterone concentrations (c) in GS-Tg and non-Tg mice alike. Data are shown
well plate (50 mL: 2500 mL normal goat serum (NGS), 1500 mL as mean ± SEM. The behavioral data were averaged across time. ap < 0.05 relative to
10% Triton-X, 46 mL 10 mM PBS). Blocker was replaced with all other mice.
 D. Litteljohn et al. / Brain, Behavior, and Immunity 67 (2018) 246–256 249

(Sigma-Aldrich, St. Louis, MO) and were covered to avoid photo-
bleaching.
Microglia density and ‘‘activation state” were assessed within
the substantia nigra and striatum by well trained blind raters.
Specifically, overall density or area covered in these brain regions
was calculated using the standard readily available Image-J soft-
ware program. Secondly, microglial morphology was assessed as
an index for their activational state. Using our previously estab-
lished and well validated qualitative rating scale, we assessed
Iba-1 stained cells in terms of several parameters on a 0–3 scale:
where 0 reflects the lowest state of activation, wherein microglia
display highly ramified, thin processes with a small soma and 3
equates to the most activate state in which mostly all cells show
thick rounded soma and no or few processes.
2.9. Statistical analyses
The monoamine, cytokine and corticosterone data were ana-
lyzed by 2  3 ANOVAs followed where appropriate by Tukey-
Kramer post hoc tests; i.e., for Genotype  Treatment interactions
and Treatment main effects absent a significant disordinal interac-
tion (a = 0.05). Plasma cytokine concentrations were transformed
by natural logarithm (ln) to improve normality and homoscedas-
ticity. Two-factor repeated measures ANOVAs were used to ana-
lyze the behaviors. Similarly, a 2 (saline vs LPS)  2 (WT vs
G2019S) ANOVA was used to assess glial immunereactivity. Data
were evaluated using a StatView (version 6.0) statistical software
package and visualized with GraphPad Prism 6 (La Jolla, CA).
3. Results
3.1. LRRK2 G2019S overexpression did not modify LPS-induced
changes in sickness, motor behaviour, corticosterone or cytokine levels
The repeated measures ANOVA for home-cage locomotor activ-
ity yielded a significant main effect of Treatment (F2, 36 = 21.93,
p < 0.0001). The follow-up tests revealed that LPS suppressed
home-cage activity in WT and G2019S mice alike (p < 0.05 relative
to mice treated with vehicle or IFN-c). Indeed, as shown in Fig. 1,
LPS provoked a greater than 60% reduction in home-cage activity
in both genotypes. Although IFN-c slightly suppressed home-
cage (by 23%) activity in the G2019S animals, this effect was not
significant (p > 0.05; Fig. 1).
As for sickness behaviour, the repeated measures ANOVA
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
sickness-inducing effect of LPS, irrespective of genotype (p < 0.05
relative to mice treated with vehicle or IFN-c). Indeed, there were
no signs of sickness in non-LPS treated mice, whereas the endo-
toxin provoked obvious curled body posture and/or ptosis and
piloerection.
Paralleling the behavioral findings, plasma corticosterone con-
centrations were significantly increased (4-fold) among the
LPS-treated animals irrespective of LRRK2 G2019S overexpression
(F2, 36 = 72.68, p < 0.0001) (Fig. 1). IFN-c did not significantly
impact corticosterone levels, despite a slight basal reduction (30%
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

The multiple separate ANOVAs revealed a significant main

effect of Treatment on 4 of cytokines assayed: TNF-a (F2,
36 = 223.94, p < 0.000), MCP-1 (F2, 36 = 188.06, p < 0.0001), IL-6 (F2,
36 = 70.34, p < 0.0001), and IL-10 (F2, 36 = 99.53, p < 0.0001). IL-1b
was not significantly affected. IFN-c, GM-CSF and IL-4 were below
the limit of reliable detection in this assay so were not included in
any analyses. As shown in Fig. 2, LPS increased all of the cytokines,
whereas MCP-1 and IL-6 were also increased by IFN-c and geno-
type again had no significant influence (p < 0.05). Indeed, LPS pro-
voked an 3 to 4-fold elevation in these cytokines, relative to
vehicle treatment.

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

The ANOVA for striatal DA revealed a significant Genotype x

Treatment interaction (F2, 34 = 13.65, p < 0.0001). The follow up
comparisons showed that the G2019S mutants had dramatically
lower overall DA concentrations (60% reduction) compared to their
wild type counterparts (p < 0.050). Moreover, acute IFN- c treat-
ment significantly reduced striatal DA to a similar degree in wild
type (p < 0.05) but had no effect in G2019S mutant mice (Fig. 3).
The primary DA metabolite, DOPAC, significantly varied as a func-
tion of the immune treatment (F2, 34 = 5.51, p < 0.05); yet as shown
in Fig. 3, the IFN- c treatment reduced DOPAC (by 40%) only in
the G2019S mutants (p < 0.05) and not wild type mice. Finally,
the striatal DOPAC/DA (as a crude measure of turnover) revealed
a significant Genotype x Treatment interaction (F2, 34 = 16.06,
p < 0.0001). Indeed, while IFN- c increased the DOPAC/DA ration
in wild type mice, the opposite effect (a reduction) was observed
in G2019S transgenic mice (p < 0.05). Thus, G2019S mutants had
basal striatal DA reductions, coupled with altered dopaminergic
utilization in response to IFN- c challenge, relative to their wild
type littermates.
As depicted in Fig. 4, LRRK2 G2019S overexpression also mark-
edly influenced NE and 5-HT activity in the dorsal hippocampus.
Indeed, the multiple separate ANOVAs revealed that the G2019S
mice, irrespective of LPS or IFN-c treatment, had significantly
higher levels of NE, 5-HT and 5-HIAA relative to their WT litter-
mates (Fs1, 36 = 16.16, 8.57 and 15.72; p < 0.001, 0.01 and 0.001).
Additionally, IFN- c augmented dorsal hippocampal NE concentra-
tions among mice of either genotype (F2, 36 = 7.78, p < 0.05) (Fig. 3).
All these effects were very modest amounting to 20–30%
elevations.

3.3. G2019S did not modify LPS or IFN-g induced monoamine changes
in PFC, PVN or locus coruleus
Within the hypothalamic PVN and the locus coruleus, LPS but
not IFN-c treatment provoked changes in monoamines, all of
which were independent of LRRK2 G2019S overexpression. Specif-
ically, the multiple separate ANOVAs revealed a significant main
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-
ished NE concentrations (p < 0.05), whilst significantly enhancing
5-HIAA and MHPG accumulation within these respective brain
regions (p < 0.05).
While there were no significant differences in NE levels within
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
were increased overall in response to the endotoxin treatment
(Fs2, 36 = 3.73 and 5.55, p < 0.05 and 0.01). As shown in Fig. 5, the
G2019S genotype did not influence these monoamine changes.
Again, these effects were relatively modest compared to the more
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
levels, compared to their wild type (Non-Tg) littermates. Also, IFN- c administration
significantly reduced DA levels in the wild type animals only. In contrast, IFN- c
reduced levels of the metabolite, DOPAC, only within the GS-Tg mice. Finally,
dopaminergic turnover (DOPAC/DA ratio) was significantly augmented in wild type
mice but diminished in GS-Tg animals.
robust striatal amine changes and their biological significance
remains to be determined.
3.4. G2019S had no effect on microglial reactivity
As depicted in Fig. 6, there was no evidence that the peripheral
LPS injection provoked neuroinflammatory changes, nor was there
any affect of the G2019S mutation. At least with respect to the
morphology and number of microglia stained with the Iba-1 anti-
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-
ings were unaffected by LPS or genotype. Fig. 6 shows Iba-1 stain-
ing in the substantia nigra (adjacent to co-stained TH+ dopamine
neurons) and similarly, no differences whatsoever were observed
within the striatum (data not shown). Indeed, virtually all Iba-1
cells displayed a similar morphology, characterized by small soma
with delicate, fine ramified projections.
4. Discussion
Substantial evidence points to a role for inflammation in the
genesis of PD and its co-morbid features (Litteljohn and Hayley,
2012; Moehle et al., 2012; Mangano and Hayley, 2009; Nagatsu
and Sawada, 2005; Vila et al., 2001; Joers et al., 2016). LRRK2 is
an ideal candidate gene that could act as a mediator of inflamma-
tory processes triggered by environmental insults. Indeed, LRRK2 is
found glia and immune cells and has critical roles in innate and
adaptive immunity (Lee et al., 2015; Gardet et al., 2010). Yet, the
existing data assessing the common G2019S mutation is confusing
at best. Presently, we demonstrate that G2019S transgenic mice
over-expressing the murine form of the mutant gene display a phe-
notype that is restricted to specific neurotransmitter changes and
not general stressor or inflammatory processes (at least with
regards to corticosterone, microglia and cytokine levels) or sick-
ness and motor behavioral impairments induced by acute systemic
LPS or IFN- c treatment.
As summarized in Table 1, the G2019S mutation had no influ-
ence on the hormonal, cytokine and behavioral effects of acute
LPS or IFN- c challenge. However, the LRRK2 G2019S mice did
show dramatic basal reductions in striatal DA and elevated hip-
pocampal norepinephrine and serotonin levels. Striatal DA turn-
over was also reduced in G2019S mice in response to LPS and
IFN- c. In contrast, other brain regions assessed, including the
PFC, PVN and locus coruleus, displayed LPS or IFN- c induced neu-
rochemical changes in the absence of any effect of genotype, sug-
gesting that the impact of the mutation is highly brain region
specific.
The blunted DA turnover in G2019S mice with IFN- c treatment
is particularly intriguing in light of the fact that IFN- c null mice
were completely resistant to the neurodegenerative and neuroin-
flammatory impact of MPTP or paraquat (Mount et al., 2007;
Mangano et al., 2011), as well as many of the behavioral responses
to chronic stress (Litteljohn et al., 2014). Indeed, our present data
suggest that in the face of already reduced basal DA levels, the
G2019S overexpression further diminishes striatal DA utilization
(as indicated by a drop in the metabolite, DOPAC) in response to
IFN- c. Also, IFN- c itself is know to be a potent inducer of
endogenous LRRK2 (Gardet et al., 2010), but it is unclear as to
how the cytokine might affect the mutant form of the gene. Since
the present mice express elevated expression of the G2019S trans-
gene in the context of background levels of the existing wild type
gene, it might be that the mutant and wild type forms of G2019S
compete with each other or interact in unexpected ways with
regards to IFN- c neurochemical modulation.
The selective serotonin and noradrenergic hippocampal
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
PD. Indeed, our own LPS and paraquat treatment models of PD pro-
duced marked hippocampal neurochemical changes that were cor-
related with cognitive and depressive-like behaviors (Rudyk et al.,
2015; Litteljohn et al., 2009). Moreover, LRRK2 G2019S mutation
carriers were reported to show lowered cognitive performance
(Thaler et al., 2012) and an increased risk of premorbid affective
disorders (Shanker et al., 2011), suggesting that the presence of
the mutation has widespread functional effects.
Given that LRRK2 interacts with, and phosphorylates various
exocytic and endocytic proteins in the presynaptic compartment
(Shin et al., 2008; Matta et al., 2012; Yun et al., 2013; Cirnaru
et al., 2014; Parisiadou et al., 2014; Piccoli et al., 2011, 2014;
Belluzzi et al., 2016), it might influence neurotransmission by mod-
ulating synaptic vesicle exo-endocytosis (Migheli et al., 2013;
Arranz et al., 2015; Belluzzi et al., 2012). Alternatively, the
G2019S mutation might have had developmental effects, possibly
involving structural alterations and dendritic arbors and spine den-
sity or involving changes in intra-cellular protein trafficking that
could influence neurotransmission.
Some evidence suggests that the G2019S mutation promotes
exaggerated inflammatory responses (Moehle et al., 2015;
Gloeckner et al., 2009; Kim et al., 2012). Yet, consistent with our
lack of an effect on LPS induced cytokines (TNF-a, MCP-1, IL-6,
IL-10), Moehle et al. (2015) reported that GS2019S mice displayed
unaltered blood chemistry and that LPS-stimulated primary
G2019S macrophages did not produce elevated levels of pro-
inflammatory cytokines. Similarly, Lopez de Maturana et al.
(2014) failed to detect a cytokine- or- COX-2-modifying influence
of LRRK2 G2019S in LPS-stimulated fibroblasts and Gardet et al.
(2010) did not find a difference in NF-jB activation in HEK293 cells
expressing LRRK2 G2019S.
We also found that G2019S did not obviously influence brain
microglia (at least within the substantia nigra and striatum;
regions obviously involved in PD and its comorbidities and that
are normally sensitive to various stressors or toxicants). Of course,
the systemic LPS injection itself also did not affect microglia and
this was likely due to the relatively short sampling time (after
90 min) and route (ip.) of administration. In fact, we previously
found that even with central (intra-nigral) infusion of LPS, it still
took two days to observed marked microglial activation these mice
were sensitized to the neurodegenerative impact of subsequent
paraquat exposure (Mangano and Hayley, 2009). It remains to be
determined whether the G2019S mutation might have a role in
such an obvious central LPS induced neuroinflammatory microen-
vironment. That said, we were presently more interested in
whether G2019S was important for basic acute inflammatory/
stressor responses that might be relevant for PD comorbidity,
rather than characterizing its role in neuroinflammatory provoked
neurodegeneration. Indeed, several papers have already explored
G2019S with regards to neurodegeneration and the findings have
been negative or modest at best. However, this is the first report
to assess the G2019S mutation with regards to the stressor-like
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-
ness behavior and plasma corticosterone elevations that paralleled
its cytokine alterations. In contrast, we found that IFN- c treatment
had no behavioral or corticoid effects and only modestly elevated
MCP-1 and IL-6. Since, the G2019S genotype did not have an affect
on any of these outcomes, it appears that the neurochemical
changes related to the G2019S genotype were likely not secondary
to any widespread toxicity or inflammatory effects that might be
related to the mutation.
It is important to note that the presently used mice expressed
the murine LRRK2 G2019S mutation, as opposed the human form.
Liu et al. (2015), who used the same mice as the current study,
found no behavioral deficits or neuronal loss in aged G2019S mice.
They did however report basal reductions of dopamine levels
within the striatum (Liu et al., 2015), which is consistent with
our present findings. Also of tremendous importance is the fact
that our mice bear not only the mutant G2019S transgene, but also
the wild type form of the LRRK2 gene. Thus, we are essentially
looking at the combined effects of 6–8-fold over-expression of
the mutant G2019S transgene in the context of the normal expres-
sion of wild type LRRK2. This differs from some recent studies
using the knockin form of G2019S, in which the mutant form
entirely replaces the wild type form of the gene, and hence, isolates
the role of G2019S alone (and presumably at typical levels of
expression). Indeed, most curiously, it was recently reported that
G2019S knockin mice exhibited a basal hyper-kinetic phenotype
(Longo et al., 2014).
In summary, our data are consistent with very specific neuro-
chemical deficits in G2019S mice and not widespread toxicity,
stressor or inflammatory changes. Conceivably, such neurochemi-
cal changes could contribute to both the primary motor and the
co-morbid non-motor features often evident in PD. Although
we did not find gross behavioral deficits in these mice, there may
have been more subtle behavioral effects that were missed. Yet,
it was clear that the G2019S overexpression did not at all influence
the typical acute behavioral and inflammatory response induced
by LPS. Our future studies will assess whether G2019S knockin
mice that lack any wild type LRRK2 expression are similar to the
currently used transgenic overexpressing mice in terms of
responses to inflammatory challenges. It will also be of interest
to determine whether advanced age may be a vulnerability factor
in G2019S mice and if multiple hits with inflammatory and
chemical toxins (e.g. paraquat) might collectively produce a more
comprehensive PD-like phenotype.
254 D. Litteljohn et al. / Brain, Behavior, and Immunity 67 (2018) 246–256

Table 1
Summary of significant experimental findings.

Experimental endpoint ANOVA main effects and interactions

Main effect of Genotype Main effect of Treatment Genotype  Treatment interaction
LPS-induced D IFN-induced D
Behaviour
Home-cage activitya 
Sickness 
Blood plasma
Corticosterone 
TNF-a 
IL-6b  
IL-1b 
MCP-1  
IL-10 
Striatum
DA 
DOPAC 
DO/DA 
PVN
5-HIAA 
Locus coruleus
NE 
MHPG 
Hippocampus
NE  
5-HT 
5-HIAA 
PFC
MHPG 
5-HT 
5-HIAA 

Author contributions
Conceived and designed the experiments: DL, SH. Performed
the experiments: DL, SR, CR, ZD. Contributed reagents/materials/-
analysis tools: SH, HA, DL. Analyzed the data and wrote the paper:
DL, SH.
Acknowledgments
Thanks to Dr. Jerzy Kulczycki (HPLC and RIA analyses) and
Marzena Sieczkos (cytokine multiplex analysis) for their expert
technical assistance, and to Farah Wahbeh for help with
biospecimen collection. This research was supported by funds to
D. Litteljohn [Canadian Institutes of Health Research (CIHR) and
Ontario Graduate Scholarship (OGS)] and S. Hayley (CIHR). CLINT
Investigators include: Earl Brown, Derrick Gibbings, Shawn Hayley,
David Park, Dana C. Philpott, John D. Rioux, Michael Schlossmacher,
and Erwin Schurr.
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