Chronic oral administration of P. gingivalis induces microglial activation and degeneration of dopaminergic neurons possibly through increase in gut permeability and peripheral IL-17A in LRRK2 R1441G mice

doi:10.21203/rs.3.rs-34883/v1

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Chronic oral administration of P. gingivalis induces microglial activation and degeneration of dopaminergic neurons possibly through increase in gut permeability and peripheral IL-17A in LRRK2 R1441G mice

https://doi.org/10.21203/rs.3.rs-34883/v1

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Journal Publication

published 19 Nov, 2020

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Background

The R1441G mutation in the leucine-rich repeat kinase 2 (LRRK2) gene results in late-onset Parkinson’s disease (PD). Peripheral inflammation and gut microbiota are closely associated with the pathogenesis of PD. Chronic periodontitis is a common type of peripheral inflammation, which is associated with PD. Porphyromonas gingivalis (Pg), the most common bacterium causing chronic periodontitis, can cause alteration of gut microbiota. It is not known whether Pg-induced dysbiosis plays a role in the pathophysiology of PD.

Methods

In this study, live Pg were orally administrated to animals, three times a week for one month. Pg-derived lipopolysaccharide (LPS) was used to stimulate peripheral blood mononuclear cells in vitro. The effects of oral Pg administration on the gut and brain were evaluated through behaviors, morphology, and cytokine expression.

Results

Dopaminergic neurons in the substantia nigra were reduced and activated microglial cells were increased in R1441G mice given oral Pg. In addition, an increase in mRNA expression of tumor necrosis factor (TNF-α) and interleukin-1 β (IL-1β) as well as protein level of α-synuclein together with a decrease in zonula occludens-1 (Zo-1) were detected in the colon in Pg-treated R1441G mice. Furthermore, serum interleukin-17A (IL-17A) and brain IL-17 receptor A (IL-17RA) were increased in Pg-treated R1441G mice.

Conclusions

These findings suggest that LRRK2 causes gut leakage and further mediates peripheral IL-17A response in Pg-treated animals. We, thus, put forward the hypothesis that IL-17A in the serum may result in activation of the IL-17A-IL-17RA axis that aggravates dysfunction of dopaminergic neurons and provokes microglial activation in LRRK2 R1441G mice.

Neurobiology of Disease

chronic periodontitis

Parkinson’s disease

dopaminergic neurons

R144G LRRK2

IL-17-IL-17RA axis

Parkinson’s disease (PD) is the second most common neurodegenerative disease that results in a progressive movement disorder characterized by slowness, rigidity, gait difficulty, and rest tremors [1]. Degeneration of dopaminergic neurons in the substantia nigra pars compacta (SNpc) is one of the pathological hallmarks of PD [2–3]. Although the exact cause of PD remains poorly understood, it is generally believed that complex interactions between genetic and environmental factors contribute its development.

Leucine-rich repeat kinase 2 (LRRK2) mutants are the most common genetic factors in the pathogenesis of PD [4]. Substantial evidence suggests that mutant LRRK2 strongly activates brain immune cells, which in turn mediate neurodegeneration through neuroinflammation [5–6]. Interestingly, LRRK2 has been also linked to several systemic inflammatory diseases, such as inflammatory bowel disease and leprosy [7–8]. Activation of LRRK2, however, has been reported to induce opposite effects in the brain and the periphery. For example, activation of LRRK2 protects against infection in the gut, but causes neurodegeneration in the brain [9–10].

Recently, chronic systemic inflammatory diseases have been linked to the risk of developing PD. Periodontal disease is a common chronic inflammatory disease and is associated with PD [11–13]. Interestingly, Porphyromonas gingivalis (Pg), the major periodontal pathogen, induces dysbiosis of gut microbiota [14–15].

The relationship between intestinal function disorder and PD has attracted much attention [16–17]. Until now, the link between the two diseases was based only on motor disturbances caused by PD, which could lead to progression of periodontal disease [12–13]. However, whether periodontal disease can have an influence on initiation and progression of PD through the intestinal pathway and the underlying mechanism remain unclear.

Recent studies suggest that peripheral lymphocytes may play a central role in the pathophysiology of PD [18]. For example, interleukin-17A (IL-17A) level was significantly increased in the serum of patients with PD [19–21]. Furthermore, IL-17A could induce human induced pluripotent stem cell-derived midbrain neuronal cell death, possibly through IL-17 receptor A (IL-17RA) [19–20]. IL-17A is mainly driven by Th17 lymphocytes. Interestingly, Th17 cells have been linked to several immune-related diseases, including periodontal disease [22–23]. Th17 cells are also essential for normal defense against gut pathogens [24].

Therefore, we hypothesized that oral Pg might induce peripheral inflammatory responses leading to degeneration of dopaminergic neurons through the gut in LRRK2 R1441G mice.

Animals

All animal procedures were performed according to the Guide for the Care and Use of Laboratory Animals of Sun Yat-sen University (Guangzhou, China). All animals were housed in a specific pathogen‑free facility with a 12:12 h light/dark cycle, ad libitum food and water. In this study, 3- to 4-month-old FVB/NJ, and FVB/N-Tg (LRRK2*R1441G)135Cjli/J mice were purchased from Jackson Laboratory (Bar Harbor, ME, USA) and crossed in the Guangdong Laboratory Animals Monitoring Institute (Guangzhou, China). At one month, all littermates were genotyped. Genotyping was done by polymerase chain reaction (PCR) of tail DNA using a protocol from Jackson Laboratory. A total of 40 mice were used in this study and assigned to four groups: FVB/N + carboxymethyl cellulose (F + C), FVB/N + Pg (F + Pg), R1441G + C, and R1441G + Pg.

Pg cultures and administration

Pg was cultured in broth (Brian Heart Infusion, L-cysteine hydrochloride monohydrate, yeast extract, and chloroproto-ferriheme, Sigma-Aldrich, St. Louis, Missouri, USA). After that, Pg was placed in an anaerobic container for 48 h at 37°C. A total of 10⁹ colony-forming units of live Pg was suspended in 0.1 ml phosphate-buffered saline (PBS) with 2% carboxymethyl cellulose (CMC) (Sigma-Aldrich), and given to each mouse by gavage three times a week for about a month, as described previously [14-15]. The control group was administered 0.1 ml PBS with 2% CMC without Pg. After administration, all mice were allowed to eat and drink ad libitum.

Behavioral tests

Rotarod test

Animals were placed on an accelerating rotarod (Xin Ruan, Shanghai, China) with an accelerated speed of 4–40 rpm for 5 min, and the latency to fall was recorded each time. Animals were tested three times a day for three consecutive days, allowing for two days of training and acclimatization. A resting time of at least 30 min was given between trials. The results are presented as the average of the three times.

Open field

Animals were placed in the chamber (45 × 45 × 45 cm) with a video camera (Xin Ruan, Shanghai, China). Every mouse was carefully placed in the center of the chamber and allowed to freely explore the chamber. Animals were tested for two consecutive days, allowing for one day of training and acclimatization. The movement of mice was filmed and analyzed automatically for 10 min.

Immunofluorescence

Brain tissue and colon were removed, fixed, and dehydrated to further process for immunofluorescence. After blocking for 1 h at room temperature, brain sections were incubated with primary antibodies overnight at 4°C. The primary antibodies used in this study were tyrosine hydroxylase (TH) (MAB318, Millipore, Bedford, MA, USA), allograft inflammatory factor 1 (Iba1) IgG (019-19741, Wako, Japan), cleaved active caspase-3 (9661, Cell Signaling Technology, Danvers, MA, USA), LRRK2 (MJFF2 [c41-2]) (ab133474, Abcam, Cambridge, UK), MAP2 lgG (ab32454, Abcam), Iba1 lgG (MA5-27726, Thermo Fisher Scientific, Waltham, MA, USA) and IL-17RA lgG (ab180904, Abcam). Subsequently, the sections were incubated with Alexa 488 or 555-conjugated secondary antibodies (4408, 4413, Cell Signaling Technology) for another 1 h at room temperature. Finally, sections were viewed under a Nikon microscope (Japan). The numbers and density of target cells were measured using ImageJ v1.51 software.

Western blot

The brain was cut into sections in the mold. The colon and the SN tissue were homogenized in radioimmunoprecipitation assay buffer (Thermo Scientific) with phenylmethanesulfonyl fluoride (1:100) and phosphatase inhibitors (Roche, Basel, Switzerland) in an ultrasonic disintegrator. Homogenates were incubated on ice for 30 min and centrifuged at 12,000 rpm for 25 min at 4°C. The protein concentration was determined using the Pierce BCA Protein Assay Kit (Thermo Scientific). Equal quantities of proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes. The membranes were blocked with 5% non-fat dry milk or bovine serum albumin for 1 h at room temperature and incubated with primary antibody overnight at 4°C. The primary antibodies used were LRRK2 (MJFF2 [c41-2]) lgG (ab133474, Abcam), phospho-LRRK2 ^S935 lgG (ab133450, Abcam), MAP2 lgG (ab32454, Abcam), cleaved active caspase-3 lgG (BF0711, Affinity, China), and IL-17RA lgG (ab180904, Abcam). α-tubulin was used as a loading control. After incubating with anti-rabbit or anti-mouse secondary antibodies (7074, 7076, Cell Signaling Technology) for 1 h, the bands were visualized using the electrochemiluminescence detection reagents (Millipore) on an Amersham Imager 600 ( Amersham Biosciences, USA). The relative density of protein was analyzed by ImageJ v1.51 software.

Peripheral blood mononuclear cell(PBMC) cultures and stimulation

Spleens were mechanically disrupted and filtered through a 40 μm cell-strainer (Falcon, BD Biosciences, Durham, NC, USA) and isolated by Ficoll-Hypaque (Tianjin HaoYang Biological Manufacture Co, Ltd, Tianjin, China) density gradient centrifugation to procure PBMCs, according to the manufacturer’s instructions. The cells (2 × 10⁶/ml) were suspended in complete roswell park memorial institute 1640 medium and stimulated for 24 and 48 h with or without Pg-LPS (1 μg/ml, 2 μg/ml, 4 μg/ml, 8 μg/ml; SMB00610, Sigma-Aldrich) in the presence of anti-CD3 mAb and anti-CD28 mAb (553057, 553294, BD Biosciences) in round-bottomed 96-well plates, (200 μl/well) at 37°C and 5% CO₂.

Enzyme-linked immunosorbent assay (ELISA)

The levels of IL-17A in serum from Pg-treatment mice and supernatant from Pg-LPS-stimulated PBMCs were measured with an ELISA kit (88-7371, BioLegend, CA, USA). ELISA assays were performed according to the manufacturer’s instructions. Data were collected by an ELISA reader under a wavelength of 450 nm. The results are shown as the mean readings from triplicate wells.

Quantitative real-time polymerase chain reaction (RT-qPCR)

Total RNA from large intestines was extracted using TRI Reagent (Invitrogen, Carlsbad, CA, USA) and was quantified using a NanoDrop 2000 (Thermo Fisher Scientific). cDNA was synthesized with Novoscript^® Plus All-in-one-1^st Strand cDNA Synthesis SuperMix (Novoprotein, Shanghai, China), according to the manufacturer’s instructions. This cDNA was subsequently used for RT-qPCR analysis using specific validated primers (Takara, Japan) and SYBR qPCR Supermix Plus (Novoprotein) in eight straight tubes in the StepOnePlus instrument (Thermo Fisher Scientific). StepOnePlus^TMsoftware (Thermo Fisher Scientific) was used to analyze the standards and carry out the quantification. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA was used as the normalizing gene. The mRNA levels for each gene were expressed as 2 -^ΔΔCt, denoting fold-change. Primer sequences were: GAPDH (forward) 5¢-TCAATGAAGGGGTCGTTGAT-3¢, (reverse) 5¢-CGTCCCGTAGACAAAATGGT-3¢; interleukin-1β (IL-1β) (forward) 5¢-TGCCACCTTTTGACAGTGATG-3¢, (reverse) 5¢-ATACTGCCTGCCTGAAGCTC-3¢. tumor necrosis factor α (TNF-α) (forward) 5¢-GACGTGGAACTGGCAGAAGAG-3¢, (reverse) 5¢-TTGGTGGTTTGTGAGTGTGAG-3¢; and zonula occludens-1 (Zo-1) (forward) 5¢- AGCGAATGTCTAAACCTGGG-3¢, (reverse) 5¢- TCCAACTTGAGCATACACAGG-3¢.

Statistical analysis

Data were analyzed with one-way ANOVA with Tukey's multiple comparisons test using GraphPad Prism 6.0 software. The results are expressed as the mean ± SEM. Statistical significance was set at P < 0.05.

1. Oral Pg induced dopaminergic neuronal degeneration in the SNpc of mutant LRRK2 mice

To examine whether oral Pg can induce dopaminergic neuronal degeneration, Pg was administrated orally to FVBN mice and LRRK2 R1441G transgenic mice for a month. Immunofluorescence TH staining was used to examine loss of SNpc dopaminergic cells. We found that there was a significant loss of TH + neurons in the SNpc in R1441G mice, but not in FVBN mice (Fig. 1a). Confocal immunofluorescence imaging revealed active caspase-3 in the cytoplasm and cell nucleus of TH + SNpc dopaminergic neurons of LRRK2 R1441G mice, but not FVBN mice (Fig. 1b). Immunoblot further confirmed that protein level of cleaved active caspase-3 was greatly increased in the SN of LRRK2 R1441G mice after Pg treatment compared to FVBN mice (Fig. 1c). In addition, LRRK2 R1441G mice exhibited a significant reduction in the immunofluorescence intensity of SNpc MAP2 + dendrite (Fig. 1d), which was accompanied by the reduction in MAP2 + protein level (Fig. 1e). In contrast, the immunofluorescence intensity of SNpc MAP2 + dendrite was not significantly altered in FVBN + Pg. To further determine whether inflammation-induced neuronal loss is mutation specific, Pg was administered to mice overexpressing human wild-type LRRK2 (WT-OX). We found that there were no significant differences in TH + number and expression levels of MAP2 + protein between Pg-treated WT-OX and WT-OX mice (Supplementary Figs. 1a, b), thereby suggesting that oral Pg-induced neurodegeneration was mutant LRRK2-dependent. Meanwhile, there were no significant differences in the rotarod and open field tests among these groups (Supplementary Fig. 1c).

2. Oral Pg increased microglial activation in the SNpc of mutant LRRK2 mice

Over-activation of microglia has been linked to neurodegeneration in PD [25–26]. In the present study, there was a significant increase in the number of activated Iba1-positive microglia in the SNpc in R1441G mice compared to FVBN and WT-OX mice, one month following treatment with oral Pg (Fig. 2a, Supplementary Fig. 2a).

3. Oral Pg increased LRRK2 activation in the SN of R1441G mice

Mutant LRRK2 has been implicated in neuronal cell death and microglial inflammatory response of SNpc [5, 10]. In this study, both LRRK2 and LRRK2^p935 were significantly increased in the SN of Pg-treated R1441G mice compared to Pg-treated FVBN mice (Fig. 3a, b). Although LRRK2 protein expression was also increased in the SN of Pg-treated WT-OX mice, LRRK2^p935 was not altered in WT-OX after Pg treatment (Supplementary Fig. 2c). Double immunofluorescence staining using anti-LRKK2, anti-TH, and anti-Iba1 was performed to visualize the co-localization of LRRK2 in SNpc dopaminergic neurons and microglia. Consistent with western blots, the immunosignal of LRRK2 was evident in Pg-treated R1441G mice. In addition, LRRK2 was partially co-localized with TH + neurons and Iba1 + microglia (Figs. 3c, d ).

4. Mutant LRRK2 exacerbated Pg-induced peripheral IL-17A secretion and IL-17RA upregulation

IL-17A has been linked to the activation of LRRK2 [27]. We first examined the serum levels of IL-17A in animals receiving either CMC or Pg. There was no significant difference in IL-17A between FVBN and R1441G mice following CMC treatment (Fig. 4a). However, serum IL-17A was significantly increased in R1441G mice compared to FVBN mice following oral administration of Pg (Fig. 4a). Consistently, IL-17A was significantly increased in the supernatant of PBMCs from R1441G mice stimulated by Pg-derived LPS compared with FVBN mice (Fig. 4b). However, IL-17A protein level remained unchanged in the SN (Supplementary Fig. 2d). Instead, IL-17RA protein level was elevated in the SN of R1441G mice with Pg compared with FVBN mice (Fig. 4c). In addition, IL-17RA was co-localized with TH in the SN of R1441G mice with Pg (Fig. 4d)

5. Pg-treatment increased the accumulation of α-synuclein in neurons of the colon and induced activation of LRRK2

Emerging evidence suggests that α-synuclein accumulates in neurons of the gut prior to the brain in PD [17, 28]. Although histological analysis revealed normal morphology of the colon and small intestine in all groups (Supplementary Fig. 3a), we found that the α-synuclein in the myenteric plexus of the colon was higher in Pg-treated R1441G mice than in control mice (Fig. 5a). There was no detectable α-synuclein in the brain and small intestine (data not shown). Furthermore, immunoblot analysis demonstrated a significant increase in LRRK2 and LRRK2⁹³⁵ protein levels in the colon of Pg-treated R1441G mice compared to each of the other three groups of mice. Besides, oral administration of Pg led to a significant increase in mRNA expression of TNF-a, IL-1β, and Zo-1 in the colon of R1441G mice, but not FVBN mice.

LRRK2 is highly expressed in immune cells and mutation of LRRK2 has been linked to both intestinal inflammatory disease and PD [5, 29–30]. In this study, we investigated the contribution of oral Pg to the pathogenesis of mutant LRRK2-PD in LRRK2 (R1441G) mice. Although oral Pg induced a mild inflammatory response in the intestine, it caused a significant loss of dopaminergic neurons and profound microglial activation in the SNpc. In addition, oral Pg resulted in an IL-17A immune response in the periphery and upregulation of IL-17RA protein level of dopaminergic neurons, thereby suggesting that the interaction between IL-17A with IL-17RA may be responsible for neurodegeneration and neuroinflammation in LRRK2-associated PD. Furthermore, these oral Pg-mediated harmful effects are accompanied by an increase in LRRK2⁹³⁵ expression, which suggests a critical role of LRRK2 kinase in Pg-induced neuropathogenesis in LRRK2-associated PD.

Systemic inflammation has been shown to induce dopaminergic neuronal death through activation of LRRK2 [6]. We consistently found that dopaminergic degeneration was evident in R1441G animals following Pg administration. This event is mediated by aberrant LRRK2 kinase, as evidenced by an increase in LRRK2⁹³⁵ expression in both the brain and colon. Interestingly, Pg-induced expression of LRRK2⁹³⁵ led to profound activation of microglia, whereas the gut morphology remained intact in R1441G mice. Thus, it is intriguing how oral Pg induces neuroinflammation in the brain. In PD, α-synuclein is considered to play a pivotal role in brain-gut-microbiota axis interactions [28, 31]. Gut inflammation induces expression of α-synuclein and the latter travels along with the vague nerve to initiate the process of α-synuclein misfolding in the brain, which leads to neuroinflammation [32]. Previously, LRRK2 activity has been shown to enhance expression of α-synuclein [33–34]. Pg-mediated LRRK2 activation consistently induced expression of α-synuclein in the colon of R1441G mice. However, α-synuclein levels in the brain were not significantly different between FVBN and R1441G mice, thereby suggesting that α-synuclein may not be responsible for activation of microglia. This is in contrast to a recent observation that injection of α-synuclein fibrils in the gut mediates the spread of pathologic α-synuclein in the brain via the vagus nerve [35]. Use of different animal models may explain the discrepancy between our study and the previous study. Indeed, α-synuclein fibrils can effectively seed the formation of Lewy body-like inclusions due to their high aggregation propensity. Alternatively, gut-mediated systemic inflammation may induce brain inflammation via circulating cytokines [32, 36]. In the periphery, it has been reported that activation of LRRK2 in monocytes and macrophages modulates proinflammatory responses, such as IL-1β and TNF-a, which in turn lead to loss of the epithelial barrier function [5, 9, 37–38]. We consistently found that oral Pg induced LRRK2 activation and reduced epithelial barrier protein Zo-1. Consequently, loss of Zo-1 may evoke the release of Pg-derived LPS into the blood stream. Elevated IL-17A level has been reported in the serum from patients with PD [19–20]. Emerging evidence indicates that IL-17A can induce neuroinflammation in animal models and PD patients [39–42]. IL-17A level was consistently increased in the serum of R1441G mice with oral Pg, as well as Pg-derived LPS-stimulated PBMC from R1441G mice. Peripheral IL-17A has been reported to disrupt and cross the blood-brain-barrier [42]. However, neither IL-17A nor Th17 cells could be detected in the brain. Thus, peripheral IL-17A may be responsible for neurodegeneration. IL-17RA is required for the biological activity of IL-17A [43]. In our study, we found that IL-17RA was increased in the dopaminergic neurons of the SN. Several studies have shown that IL-17 could trigger neuronal death through IL-17RA [19]. Thus, it is likely that serum IL-17A may mediate neuronal death through interaction with IL-17RA in Pg-induced R1441G mice. Interestingly, IL-17A has been reported to mediate dopaminergic neurons degeneration via IL-17RA in microglia in a previous study [41]. However, we did not detect any expression of IL-17RA in microglia, although reactive microglia were evident in Pg-induced R1441G mice. The differing results may be due to the use of different models in the experiments.

Microglia in a state of heightened reactivity have a vital role in PD [44]. Evidence suggests that mutant LRRK2 may enhance microglial process outgrowth and inflammatory response leading to chronic damage of dopaminergic neurons [45–46]. Our study consistently showed a significant increase in Iba1 number in R1441G LRRK2 mice. Besides, our study showed LRRK2 co-localization in microglia and neurons. We, therefore, put forward the hypothesis that activation of the IL-17A-IL-17RA axis aggravates dysfunction of dopaminergic neurons and induces microglial activation in R1441G LRRK2 animals.

Our results indicate that activation of LRRK2 may have an important role in gut barrier leakage and IL-17A production, and subsequently trigger neuronal death through the IL-17A-IL-17RA axis. In conclusion, our results elucidate the role of the brain-gut axis in the pathophysiology of Parkinson’s disease.

LRRK2

Leucine rich repeat kinase 2;

Porphyromonas gingivalis;

LPS

Lipopolysaccharide;

IBA-1

Allograft inflammatory factor 1;

IL-17A

Interleukin-17A;

IL-1β

Interleukin-1β;

Zo-1

Zonula occludens-1;

Parkinson’s disease;

SNpc

Substantia Nigra pars compacta;

Tyrosine hydroxylase;

TNF-α

Tumor necrosis factor α;

CMC

Carboxymethyl cellulose;

PBMC

Peripheral blood mononuclear cell;

GAPDH

Glyceraldehyde-3-phosphate dehydrogenase;

WT-OX

Overexpressing human wild-type;

PCR

Polymerase chain reaction;

PBS

Phosphate-buffered saline;

CMC

Carboxymethyl cellulose;

Ethical Approval and Consent to participate

All protocols were performed according to the Guide for the Care and Use of Laboratory Animals of Sun Yat-sen University (Guangzhou, China) Committee

Consent for publication

Not applicable.

Availability of data and materials

All data generated or analysed during this study are included in this published article [and its supplementary information files].

Competing interests

The authors declare that they have no competing interests.

Funding

This work was supported by the Grants from the National Key Research and Development Program of China，Stem Cell and Translational Research (No. 2017YFA0105104)；the National Natural Science Foundation of China (No. 8187375, No.81671102)；Guangdong provincial science and technology plan project (No. 2016B030230002); the Southern China International Cooperation Base for Early Intervention and Functional Rehabilitation of Neurological Diseases (2015B050501003), Guangdong Provincial Engineering Center For Major Neurological Disease Treatment, Guangdong Provincial Translational Medicine Innovation Platform for Diagnosis and Treatment of Major Neurological Disease, Guangdong Provincial Clinical Research Center for Neurological Diseases.

Authors' contributions

YKF, YWP and ZP figured and planned the experiments. YKF, QLW, FYL, HJY, YWF, GL, XJL , SHL , YCL and YZ implemented the experiments. YKF, QLW, HJY and YWF analyzed the data. YKF and ZP wrote the paper. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Zhong Pei

Acknowledgements

We thank International Science Editing ( http://www.internationalscienceediting.com ) for editing this manuscript.

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Chronic oral administration of P. gingivalis induces microglial activation and degeneration of dopaminergic neurons possibly through increase in gut permeability and peripheral IL-17A in LRRK2 R1441G mice

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