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Intestinal infection triggers Parkinson’s disease-like symptoms in Pink1−/− mice

Abstract

Parkinson’s disease is a neurodegenerative disorder with motor symptoms linked to the loss of dopaminergic neurons in the substantia nigra compacta. Although the mechanisms that trigger the loss of dopaminergic neurons are unclear, mitochondrial dysfunction and inflammation are thought to have key roles1,2. An early-onset form of Parkinson’s disease is associated with mutations in the PINK1 kinase and PRKN ubiquitin ligase genes3. PINK1 and Parkin (encoded by PRKN) are involved in the clearance of damaged mitochondria in cultured cells4, but recent evidence obtained using knockout and knockin mouse models have led to contradictory results regarding the contributions of PINK1 and Parkin to mitophagy in vivo5,6,7,8. It has previously been shown that PINK1 and Parkin have a key role in adaptive immunity by repressing presentation of mitochondrial antigens9, which suggests that autoimmune mechanisms participate in the aetiology of Parkinson’s disease. Here we show that intestinal infection with Gram-negative bacteria in Pink1−/− mice engages mitochondrial antigen presentation and autoimmune mechanisms that elicit the establishment of cytotoxic mitochondria-specific CD8+ T cells in the periphery and in the brain. Notably, these mice show a sharp decrease in the density of dopaminergic axonal varicosities in the striatum and are affected by motor impairment that is reversed after treatment with l-DOPA. These data support the idea that PINK1 is a repressor of the immune system, and provide a pathophysiological model in which intestinal infection acts as a triggering event in Parkinson’s disease, which highlights the relevance of the gut–brain axis in the disease10.

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Fig. 1: Gram-negative infection induces MitAP in vitro.
Fig. 2: C. rodentium infection induces MitAP and the establishment of cytotoxic mitochondria-specific CD8+ T cells in Pink1−/− mice.
Fig. 3: C. rodentium infection induces anti-OGDH T cell infiltration into the central nervous system.
Fig. 4: C. rodentium infection induces motor impairment and a loss of dopaminergic axonal varicosities in the striatum of Pink1−/− mice.

Data availability

The data supporting the findings of this study are available within the paper and its Supplementary Information files.

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Acknowledgements

M.D. and H.M.M. received a grant from the Michael J. Fox Foundation. The authors thank M. Guérin for technical support. L.-E.T. was supported by a grant from the Brain Canada Foundation, the Krembil Foundation, the CIHR (grant MOP106556), Parkinson Canada and the Henri and Berenice Kaufmann Foundation. A.-M.P. was supported by the Finnish Parkinson’s Disease Foundation. C.D. was supported by a studentship from Parkinson Canada. S.G. was supported by CIHR grants (MOP 133580 and PJT-162406) and NSERC (RGPIN-2014-05119). T.C. was supported by studentships from CIHR and Healthy Brains for Healthy Lives initiative.

Author information

Authors and Affiliations

Authors

Contributions

D.M. and T.C. helped conceive and perform most experiments. A.V., A.-M.P. and L.R. performed brain and neuron analyses and behavioural tests. C.D., A.L., M.-J.B. and L.Z. helped with the mice and some experiments. H.M.M., S.G., L.-E.T. and M.D. conceived the experiments, analysed the results and supervised the project. D.M., T.C., H.M.M., S.G., L.-E.T. and M.D. wrote the manuscript. D.M. and T.C. contributed equally. Figure 1a–e was performed by T.C. Image analysis (Fig. 1f, g) was performed by D.M. (D.M. and T.C. for Fig. 1h). In vivo infections were performed by L.Z. Faecal counts and spleen index (Fig. 2a, b) were performed by T.C. MitAP and CD8+ ELISPOT in Fig. 2c–f were performed by D.M. and T.C. All FACS analyses were performed by D.M. and A.L.C. Central nervous system tissue was obtained by A.-M.P. ELISPOT were performed by D.M. and T.C. For Fig. 3f–h, neuron cultures were made by M.-J.B. and C.D. FACS and MitAP assays for Fig. 3f–h were performed by D.M. Neuron cultures (Fig. 3i) were performed by L.R. and A.V. Co-culture experiments were performed by L.R., A.V. and D.M., and cell viability analyses were performed by L.R. and A.V. (Fig. 3i). Behavioural tests (Fig. 4a–c) were performed by D.M., T.C. and L.R. Perfusions (Fig. 4) were performed by A.V. TH staining and neuronal counts (Fig. 4d–f) were performed by A.-M.P. and A.V. Western blots were performed by T.C. and A.F. Bacterial counts and ELISAs (Extended Data Fig. 2a, d, e) were performed by T.C. Pathology scoring (Extended Data Fig. 2b, c) was performed by R.C. Blood cytokine analyses were performed by A.-M.P. Infection phenotyping (Extended Data Fig. 4) was performed by D.M., T.C., A.F., A.L., L.Z. and S.G. FACS staining and analysis for Extended Data Fig. 5 were performed by D.M. and A.L.C. Experiments in Extended Data Fig. 6 were performed by D.M. Quantification of T cell numbers (Extended Data Fig. 7) was performed by D.M. FACS staining and analysis (Extended Data Fig. 8) were performed by D.M. and A.L.C. Co-culture experiments for Extended Data Fig. 9 were performed by L.R. Behaviour experiments for Extended Data Fig. 10 were performed by D.M., T.C. and L.R., and perfusions were made by A.V. TH staining and neuron counts were performed by A.V. and A.-M.P. Experiments in Extended Data Fig. 10e were performed by A.-M.P. ELISPOT analysis (Extended Data Fig. 10f) was performed by D.M. and T.C. Videos were performed by T.C.

Corresponding authors

Correspondence to Heidi M. McBride, Samantha Gruenheid, Louis-Eric Trudeau or Michel Desjardins.

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Extended data figures and tables

Extended Data Fig. 1 EPEC infection results in the selective degradation of mitochondrial proteins.

RAW 264.7 macrophages were either infected with EPEC at a multiplicity of infection of 1 for 1, 2, 4 or 6 h or treated with 20 µM CCCP for 8 or 24 h alongside control (Ctl) cells. Total cell lysates were collected for all conditions, quantified, separated via SDS–PAGE, and probed for mitochondrial markers and LC3 via western blot. Data are representative of two independent experiments.

Extended Data Fig. 2 C. rodentium induces mild colonic pathology and inflammation in both wild-type and Pink1−/− mice.

a, The distal colon was collected at 13 d.p.i. and assessed for colon-associated C. rodentium in infected wild-type and Pink1−/− mice via serial dilution and plating on MacConkey agar. Data representative of two independent experiments. b, Analysis of colonic epithelial hyperplasia via measurement of crypt heights from haematoxylin and eosin (H&E)-stained colon sections. Data represents individual crypt measurements from five wild-type mice and six Pink1−/− mice. Data representative of two independent experiments. c, Tissue pathology scores from H&E-stained distal colon sections from infected wild-type and Pink1−/− mice. d, e, Protein quantification of faecal S100A8/A9 (d) and lipocalin-2 (e) from control and infected wild-type and Pink1−/− mice. Data pooled from two independent experiments. P values were determined by two-tailed Mann–Whitney unpaired t-test at a 95% confidence interval (a, b), two-way ANOVA at a 95% confidence interval (c), or two-way ANOVA at a 95% confidence interval with Tukey’s multiple comparison’s test. Data are mean ± s.e.m.

Source Data

Extended Data Fig. 3 C. rodentium infection induces a mild increase in systemic pro-inflammatory cytokines in both wild-type and Pink1−/− mice.

ad, Mice were infected with C. rodentium and serum was collected at 13 d.p.i. for ELISA analysis of the pro-inflammatory cytokines IL-6 (a), IL-17 (b), IP-10 (c) and TNF (also known as TNFα) (d). All data pooled from two independent experiments. P values were determined by two-way ANOVA at a 95% confidence interval. Data are mean ± s.e.m.

Source Data

Extended Data Fig. 4 C. rodentium infection parameters are similar in wild-type and Pink1−/− mice.

a, C. rodentium faecal burden in wild-type and Pink1−/− mice at 4, 8 and 12 d.p.i. Data pooled from three independent experiments. b, c, Body weight (b) and colon index (c) of control and infected wild-type Pink1−/− mice at 13 d.p.i. Data pooled from two independent experiments. d, e, Body weight (d) and spleen index (e) of uninfected and infected wild-type and Pink1−/− mice serially infected 4 times, 28 days apart, measured at 12 months after initial infection in a single cohort of mice. Data in a denote median values; data in bd are mean ± s.e.m.

Source Data

Extended Data Fig. 5 C. rodentium infection induces a similar CD8+ T cell immune response in both wild-type and Pink1−/− mice.

Mice were infected with C. rodentium and CD8+ T cell markers were assessed at 13 d.p.i. in the spleen. a, b, Flow cytometry was used to quantify the frequency of CD3+CD8+CD107a+ (a) and CD3+CD8+GrB+ (cytotoxic CD8+ T cells) (b) in control and infected wild-type and Pink1−/− mice. P values determined by two-way ANOVA at a 95% confidence interval with Tukey’s multiple comparison’s test. Data are mean ± s.e.m.

Source Data

Extended Data Fig. 6 Intraperitoneal injection of LPS induces anti-OGDH CD8+ T cells in Pink1−/− mice.

Wild-type and Pink1−/− littermates were injected with 1 mg kg−1 of LPS intraperitoneally 4 times, once a week starting at 6 weeks of age. Spleens were obtained seven days after the last treatment. The presence of anti-OGDH CD8+ T cells was tested for via IFNγ ELISPOT by replacement of peptides in MHC class I complexes on splenocytes with OGDH peptides, control gB peptides or vehicle (water), and plated with splenic T cells on IFNγ detection plates. PMA peptide-loading controls to assess the levels of MHC class I expression in each group are shown in the inset. Data are representative of three independent experiments performed in triplicate. Significance was determined by two-way ANOVA at a 95% confidence interval with Tukey’s multiple comparison’s test. Data are mean ± s.e.m.

Source Data

Extended Data Fig. 7 C. rodentium infection induces OGDH-specific CD8+ T cells in Pink1−/− mice.

Flow cytometry and spleen numeration were used to quantify OGDH-specific CD8+ T cells previously identified by their expression of IFNγ after stimulation with OGDH peptides. Analysis was done on both control and infected wild-type and Pink1−/− mice. Data are representative of three independent experiments. P values were determined by two-way ANOVA at a 95% confidence interval with Tukey’s multiple comparison’s test. Data are mean ± s.e.m.

Source Data

Extended Data Fig. 8 C.-rodentium-induced monocyte and T cell infiltration into the central nervous system occurs in both wild-type and Pink1−/− mice.

Brain and spinal cord tissue were assessed for haematopoietic cells in both control and infected wild-type and Pink1−/− mice. a, b, Absolute number of monocytes (a) or T cells (b) at 13 and 28 d.p.i. were calculated using true count beads. Data pooled from two independent experiments. Data are mean ± s.e.m.

Source Data

Extended Data Fig. 9 TH+ neurons are susceptible to damage from 2C cells after treatment with LPS and IFNγ.

Quantification (left) and representative images (right) of the cytotoxic assay in which neurons from Pink1−/− mice were treated with LPS, IFNγ or left untreated (control) and co-cultured with or without OGDH-specific CD8+ T cells isolated from 2C mice for two days. Exogenous addition of OGDH peptides (last column) was used as a positive control. Dopaminergic neurons were identified by TH staining, and damage was determined by the presence of swollen cell bodies and the shortening of dendrites. Data represent measurements from individual coverslips and are representative of three independent experiments. P values were determined by one-way ANOVA at a 95% confidence interval with Tukey’s multiple comparison’s test. Data are mean ± s.e.m.

Source Data

Extended Data Fig. 10 C.-rodentium-induced anti-mitochondrial CD8+ T cells and loss of locomotor function are transient.

a, Actimetry tests and grip strength of mice at 12 m.p.i. Data are from a single cohort of mice. b, Quantification of TH+ neurons in the ventral tegmental area after stereological analyses. Data pooled from two independent experiments. c, Representative images of TH staining in the dorsal striatum at 12 m.p.i. Data are from a single cohort. d, Quantification of TH+ neurons in the dorsal and ventral striatum after stereological analyses. Representative of a single cohort. e, Quantification of dopamine transporter (DAT) signal density in the dorsal and ventral striatum at 6 m.p.i. Data accumulated from mice from b. Data pooled from two independent experiments. f, Quantification of OGDH-specific T cells from wild-type and Pink1−/− mice at 13 d.p.i., 6 m.p.i. and 12 m.p.i. Data at 13 d.p.i. are representative of three independent experiments; data at 6 m.p.i. are representative of two independent experiments; data at 12 m.p.i. are from a single cohort of mice. P values determined by two-way ANOVA at a 95% confidence interval with Tukey’s multiple comparison’s test. Data are mean ± s.e.m.

Source Data

Supplementary information

Supplementary Figure

Supplementary Figure 1: Source western blot images. Samples were quantified and normalized prior to loading onto 4 separate gels. All 4 gels were loaded from the same tube simultaneously with 20 µg of protein. Ladder was loaded on the left of the samples once and twice on the right of the samples, and molecular weights indicated with a pen. Boxes indicate where the image was cropped for manuscript.

Reporting Summary

Supplementary Video 1: Pink1-/- mice develop an observable motor impairment during serial infection with C. rodentium.

WT and Pink1-/- mice were serially infected with C. rodentium four times over the course of four months and monitored for the development of a motor impairment. Four months after the initial infection, Pink1-/- mice (right video panel) developed an observable motor impairment compared to WT mice (left video panel) with regards to movement speed, splayed legs, decreased hind leg movement, and decreased tendency to perform vertical episodes.

Supplementary Video 2: L-DOPA treatment rescues the T-turn impairment observed in Pink1-/- mice serially infected with C. rodentium.

Pink1-/- mice serially infected with C. rodentium showed a decreased ability to T-turn (invert 180° when placed upright on a pole) (left video panel). Treatment of these mice with L-DOPA reverted the T-turn impairment (right video panel).

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Matheoud, D., Cannon, T., Voisin, A. et al. Intestinal infection triggers Parkinson’s disease-like symptoms in Pink1−/− mice. Nature 571, 565–569 (2019). https://doi.org/10.1038/s41586-019-1405-y

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