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Gut-seeded α-synuclein fibrils promote gut dysfunction and brain pathology specifically in aged mice

Nature Neuroscience volume 23, pages 327–336 (2020)Cite this article

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Abstract

Parkinson’s disease is a synucleinopathy that is characterized by motor dysfunction, death of midbrain dopaminergic neurons and accumulation of α-synuclein (α-Syn) aggregates. Evidence suggests that α-Syn aggregation can originate in peripheral tissues and progress to the brain via autonomic fibers. We tested this by inoculating the duodenal wall of mice with α-Syn preformed fibrils. Following inoculation, we observed gastrointestinal deficits and physiological changes to the enteric nervous system. Using the AAV-PHP.S capsid to target the lysosomal enzyme glucocerebrosidase for peripheral gene transfer, we found that α-Syn pathology is reduced due to the increased expression of this protein. Lastly, inoculation of α-Syn fibrils in aged mice, but not younger mice, resulted in progression of α-Syn histopathology to the midbrain and subsequent motor defects. Our results characterize peripheral synucleinopathy in prodromal Parkinson’s disease and explore cellular mechanisms for the gut-to-brain progression of α-Syn pathology.

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Fig. 1: Inoculation of the duodenum with α-Syn PFF affects the ENS and GI function.
Fig. 2: Pathologic α-Syn disrupts GCase and GI function and is ameliorated by GBA1 peripheral gene transfer.
Fig. 3: ENS neurotransmission is disrupted by pathologic α-Syn and restored by GBA1 gene transfer.
Fig. 4: Inoculating the duodenum of aged mice with α-Syn PFF promotes progression of p-α-Syn to the brain.

Data availability

No datasets were generated or analyzed during the current study. The data that support the findings of this study are available from the corresponding author upon request.

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Acknowledgements

We thank C. Bedbrook for help with calcium imaging and K. Beadle and E. Mackey for additional technical support. This work was supported by the following grants to V.G.: NIH Director’s New Innovator IDP20D017782-01 and PECASE; NIH/NIA R01AG047664-01; NIH BRAIN 1U01NS090577; the Heritage Medical Research Institute; the Pew Charitable Trust; the Rogers Fellowship for Parkinson’s Research and the CZI Neurodegeneration Challenge Network. C.C. was supported by NIH/NIA F32AG054101. S.K.M. was supported by NIH/NINDS R01NS085910. V.G. and S.K.M. were supported by Department of Defense grant W81XWH-17-1-0588. L.A.V.-D. was supported by NINDS R01NS102257 and Morris K. Udall Centers of Excellence for Parkinson’s Disease Research P50NS108675. T.R.S was supported by the Larry L. Hillblom Foundation. B.B.Y. was supported by NIH/NIGMS 5T32GM007616 and the Caltech Center for Environmental Microbial Interactions (CEMI).

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Author notes

  1. These authors contributed equally: Acacia Hori, Timothy R. Sampson.

Authors and Affiliations

  1. Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA, USA

    Collin Challis, Acacia Hori, Timothy R. Sampson, Bryan B. Yoo, Rosemary C. Challis, Sarkis K. Mazmanian & Viviana Gradinaru

  2. Department of Physiology, Emory University School of Medicine, Atlanta, GA, USA

    Timothy R. Sampson & Adam M. Hamilton

  3. Center for Neurodegeneration and Experimental Therapeutics, University of Alabama at Birmingham, Birmingham, AL, USA

    Laura A. Volpicelli-Daley

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Contributions

C.C. and V.G. conceptualized the study and developed the research plan. C.C., V.G. and L.A.V.-D. designed the study. L.A.V.-D. generated the α-Syn PFFs and α-Syn monomers. C.C. performed the animal surgeries, tissue clearing, histology, calcium imaging and retro-orbital viral injections. C.C., A.H. and T.R.S. performed the behavior experiments. C.C., B.B.Y. and R.C.C. performed virus production, purification and verification. C.C. and T.R.S. performed protein analyses. C.C., B.B.Y. and R.C.C. performed confocal imaging. A.M.H. and T.R.S. performed RNA extraction and quantitative PCR analyses. C.C. performed data analyses. S.K.M. provided key reagents and methods. C.C. and V.G. wrote the manuscript. All authors contributed to discussion. V.G. supervised all the work.

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Correspondence to Viviana Gradinaru.

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Extended data

Extended Data Fig. 1 Effect of αSyn pathology on GI health and cytokine expression.

a-d, GI function in WT, ASO, and aged mice is characterized by fecal pellet production (a; WT vs. ASO **p = 0.022, ASO vs. Aged **p = 0.0075), fecal pellet weight (b; WT vs. ASO ****p < 0.0001, WT vs. Aged ***p = 0.0009, ASO vs. Aged ****p < 0.0001), proportion fecal water weight (c; WT vs. ASO ****p < 0.0001, ASO vs. Aged ****p < 0.0001), and whole gut fecal transit time (d; WT vs. ASO ****p < 0.0001, ASO vs. Aged **p = 0.0038). e, Animal weight (WT vs. Aged ****p < 0.0001, ASO vs. Aged ****p < 0.0001). f, qPCR analysis of GBA1 expression in duodenum. Dots represent average of technical triplicates from a single animal. g, Densitometry analysis of cytokine panel from duodenal lysates after inoculation with α-Syn PFF or α-Syn monomer 7 dpi (Fractalkine **p = 0.0030, IL-1a **p = 0.0070, IL-6 *p = 0.0415, IL-7 * p = 0.0400, MCP-1 *p = 0.0416, MCSF *p = 0.0467, MIG *p = 0.0412, TECK *p = 0.0365, TIMP-2 *p = 0.0400). h, Representative cytokine panels detected by chemiluminescence. i, Representative Western blot immunostained for IL-6 and densitometry analysis normalized to WT integrated densities (WT vs. PFF 60 dpi *p = 0.0327, 60 dpi PFF vs. monomer *p = 0.0480, WT vs. ASO *p = 0.0304). Data depicted are mean ± s.e.m. P values were determined by one-way ANOVA (a-f,i). The following n values represents number of independent animals used for statistical evaluation: e1a-c, WT = 42, ASO = 20, Aged = 19; e1d, WT = 13, ASO = 9, Aged = 12; e1e, WT = 42, ASO = 20, Aged = 19; e1f, WT = 3, ASO = 4, Aged = 4; e1g, monomer and PFF = 4 each; e1i, all conditions = 4 each.

Source data

Extended Data Fig. 2 Visualization of the duodenal ENS and cell proliferation.

a, Maximum intensity projection confocal image of duodenum whole-mount stained for PGP9.5 (neurons) and GFAP (EGCs). Higher magnification of dashed box shown in Fig. 1g. Scale bar, 200 µm. b, Virtual cross-section of the duodenal wall after optical clearing distinguishes myenteric and submucosal plexuses. Intestinal crypt layer is marked in blue. Scale bar, 100 µm. c, DAPI nuclear labeling visualizes the intestinal crypt layer used for histological analysis of the ENS (top). High-magnification of dashed box shows individual crypts (bottom). Scale bars, 100 µm. Experimental images for (a-c) were obtained from 8 independent mice, with similar results obtained. d, Quantification of number of myenteric neurons per crypt (top) or myenteric EGCs per crypt (bottom; WT vs. PFF 60 dpi *p = 0.0329, WT vs. PFF 120 dpi *p = 0.0232), e, Immunohistochemical labeling of EdU detects recently proliferated extraganglionic (open arrows) or myenteric ganglion cells (closed arrows). EGCs are co-labeled with GFAP. Scale bar, 20 µm. f, Quantification of total EdU+ cells per crypt (Monomer vs. PFF *p = 0.0071). Data depicted is mean ± s.e.m. P values were determined by One-Way ANOVA (d) or one-tailed Student’s t-test (f). The following n values represents number of independent animals used for statistical evaluation: e2d enteric neurons per crypt, WT = 6, PFF 0 dpi = 6, PFF 7 dpi = 6, PFF 21 dpi = 6, PFF 60 dpi = 6, PFF 120 dpi = 5, monomer conditions = 5 each; e2d EGCs per crypt, WT = 4, all PFF = 5 each, monomer 7 dpi = 4, monomer 60 dpi = 5; e2f, monomer = 4, PFF = 5.

Source data

Extended Data Fig. 3 Inoculation of duodenum with αSyn PFF promotes formation of αSyn filaments.

a, Representative dot blot images of duodenal homogenates immunostained with an aggregation-specific α-Syn antibody (α-Syn filament) show an increase after α-Syn PFF inoculation and in ASO mice. b, Densitometry analysis of α-Syn filament dot blots of duodenal homogenates normalized to pre-injection WT mice integrated densities (int. den.) (WT vs. ASO **p = 0.0092). c, Representative dot blot image of controls immunostained with α-Syn filament antibody: 50 ng BSA, 50 ng and 100 ng α-Syn monomer, 50 ng and 100 ng α-Syn PFF. d, Densitometry analysis of control blots normalized to 50 ng BSA integrated densities (BSA vs. PFF 50ng **p = 0.0080, BSA vs. PFF 100ng ***p = 0.0009, monomer 50ng vs. PFF 50ng *p = 0.0178, monomer 100ng vs. PFF 100ng **p = 0.0078). Data depicted is mean ± s.e.m. P values were determined by one-way ANOVA (a, d). The following n values represents number of independent animals used for statistical evaluation: e3b, WT = 8, all PFF = 5 each, all monomer = 4 each, ASO = 8, Aged = 5; e3d, BSA = 4, all other groups = 3 each.

Source data

Extended Data Fig. 4 GBA1 gene transfer increases GCase function and improves GI health.

a-d, Characterization of GI function before and after peripheral GBA1 gene transfer by quantifying fecal pellet production (b), fecal pellet weight (c; ASO 0 vs. 60 dpvi *p = 0.0265), proportion fecal water weight (d), and whole gut fecal transit time (e). e-h, Characterization of GI function before and after peripheral EGFP gene transfer by quantifying fecal pellet production (e), fecal pellet weight (f), proportion fecal water weight (g), and whole gut fecal transit time (h; 0 dpi WT vs. ASO **p = 0.0085, 60 dpi WT vs. ASO *p = 0.0293). Data depicted are mean ± s.e.m. P values were determined by two-way ANOVA (a-h). The following n values represents number of independent animals used for statistical evaluation: e4a-d, for 0 / 7 / 21 / 60 dpi: WT = 17 / 12 / 11 / 8, ASO = 13 / 12 / 11 / 10; e4e-h, all conditions = 4 each.

Source data

Extended Data Fig. 5 ENS neurotransmission is disrupted by αSyn pathology and restored by GBA1 gene transfer.

a, Quantification of average peak percent change in fluorescence and area under the curve after photostimulation pulse for jRGECO1a+-only or jRGECO1a+/ChR2+ duodenal neurons before and after inoculation (all ****p < 0.0001). b, Quantification of average peak percent change in fluorescence and area under the curve after photostimulation pulse for jRGECO1a+-only or jRGECO1a+/ChR2+ duodenal neurons before and after systemic delivery of AAV-PHP.S::ihSyn:GBA1 (Peak ΔF/F jRGECO1a+ 0 dpvi WT vs. ASO ****p < 0.0001, 7 dpvi WT vs. ASO ****p < 0.0001; Peak ΔF/F jRGECO1a+/ChR2+ 0 dpvi WT vs. ASO ****p < 0.0001, 7 dpvi WT vs. ASO ***p = 0.0006; AUC jRGECO1a+ 0 dpvi WT vs. ASO ****p < 0.0001, 7 dpvi WT vs. ASO **p = 0.0031; AUC jRGECO1a+/ChR2+ 0 dpi WT vs. ASO *p = 0.0398). Data depicted are mean ± s.e.m. P values were determined by two-way ANOVA. The following n values represents number of independent animals used for statistical evaluation: e5a, 0 dpi = 3, PFF 7 dpi = 4, PFF 60 dpi = 3, monomer 7 dpi = 3, monomer 60 dpi = 3; e5b, all conditions = 3.

Source data

Extended Data Fig. 6 Inoculation of duodenum with αSyn PFF in adult mice does not progress αSyn pathology to the nodose ganglion.

a, Maximum intensity 3D projection confocal image of a whole nodose ganglion from an α-Syn PFF inoculated WT mouse 60 dpi. Ganglion was PACT cleared and immunolabeled for PGP9.5 (neurons), S129P, and DAPI (nuclei). Experimental images were obtained from 14 independent mice, with similar results obtained. Scale bars, 100 µm. Z-stack depth, 500 µm. b, Maximum intensity 3D projection confocal image of a nodose ganglion from a WT mouse. Experimental images were obtained from 5 independent mice, with similar results obtained. Scale bar, 100 µm. Z-stack depth, 450 µm. c, Quantification of S129P+ nodose ganglion neurons (WT vs. ASO **p = 0.0011, monomer 7 dpi vs. ASO **p = 0.0027, monomer 60 dpi vs. ASO *p = 0.0176, ASO vs. Aged *p = 0.0323). Data depicted is mean ± s.e.m. P values were determined by one-way ANOVA. The following n values represents number of independent animals used for statistical evaluation: e6c, WT = 5, ASO = 5, Aged = 5, for 7 / 60 / 120 dpi, PFF = 5 / 5 / 4, monomer = 4 / 3 / 0.

Source data

Extended Data Fig. 7 Brain αSyn pathology observed in ASO mice but not after inoculation of duodenum with αSyn PFF in adult mice.

a-b, Maximum intensity projection confocal images of the brainstem (approximately −7.76 mm from Bregma) from an α-Syn PFF-inoculated 8 week old WT mouse 60 dpi (a) and an ASO mouse (b), immunolabeled for cholinergic neurons (ChAT) and S129P. Scale bars, 500 μm. Experimental images for (a-b) were obtained from 3 independent mice, with similar results obtained. c-d, Maximum intensity projection confocal images of the midbrain (approximately −3.64 mm from Bregma) from an α-Syn PFF-inoculated WT mouse 60 dpi (c) and an ASO mouse (d), immunolabeled for dopaminergic neurons (TH), S129P, and nuclei (DAPI). Scale bars: 500 μm. Experimental images for (c-d) were obtained from 3 independent mice, with similar results obtained. e-f, Densitometry analysis of S129P signal in the DMV (e; WT vs. ASO ***p = 0.0003, PFF 60 dpi vs. ASO ***p = 0.0007) and SNc (f; WT vs. ASO **p = 0.0011, PFF 60 dpi vs. ASO **p = 0.0014) per unit area, normalized to WT. Data depicted is mean ± s.e.m. P values were determined by one-way ANOVA (a, d). The following n values represents number of independent animals used for statistical evaluation: e7e-f, all conditions = 3 each.

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Extended Data Fig. 8 Duodenal inoculation with αSyn PFF in adult mice produces only mild sensorimotor deficits.

a-f, Sensorimotor behaviors after inoculation were quantified using average time to turn and descend a pole (a; PFF 0 vs. 60 dpi **p = 0.0012, 90 dpi PFF vs. monomer *p = 0.0265), average time before falling during the inverted wire-hang paradigm (b; PFF 0 vs. 60 dpi *p = 0.0306, PFF 0 vs. 120 dpi *p = 0.0285), average time to remove adhesive from nasal bridge (c; PFF 0 vs. 90 dpi **p = 0.0014, PFF 0 vs. 120 dpi *p = 0.0285, 60 dpi PFF vs. monomer *p = 0.0193, 90 dpi PFF vs. monomer *p = 0.0342), average weightlifting paradigm score (d), average time to cross a narrowing beam (e), and total number of slips incurred during 3 trials of the narrowing beam paradigm (f; 60 dpi PFF vs. BSA **p = 0.0023). Boxplots represent median, interquartile range, and 1.5x the interquartile range. g, Hot plate test did not reveal any changes in nociception at 60 dpi. Data depicted is mean ± s.e.m. P values were determined by two-way ANOVA (a-f) or one-way ANOVA (g). The following n values represents number of independent animals used for statistical evaluation: e8a-c, for 0 / 7 / 21 / 60 / 90 / 120 dpi, PFF = 16 / 14 / 14 / 11 / 9 / 8, monomer = 9 / 9 / 9 / 8 / 8 / 8, BSA = 17 / 16 / 11 / 9 / 7 / 7; e8d, for 0 / 7 / 21 / 60 / 90 / 120 dpi, PFF = 8 / 8 / 12 / 8 / 8 / 8, monomer = 9 / 9 / 9 / 8 / 8 / 8, BSA = 17 / 16 / 11 / 9 / 7 / 7; e8e-f, for 0 / 7 / 21 / 60 / 90 / 120 dpi, PFF = 8 / 8 / 10 / 7 / 6 / 6, monomer = 9 / 9 / 9 / 8 / 8 / 8, BSA = 17 / 16 / 11 / 9 / 7 / 7; e8g, baseline = 10, monomer = 4, PFF = 6.

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Extended Data Fig. 9 Duodenal αSyn PFF inoculation in aged mice does not result in SNc dopaminergic cell loss.

a-c, Representative images of the midbrain immunostained for dopaminergic neurons (TH), S129P, and nuclei (DAPI) from aged mice pre-inoculation (a), α-Syn PFF-inoculated aged mice 120 dpi, and α-Syn-monomer inoculated aged mice 120 dpi. Scale bars, 500 μm. Experimental images for (a-c) were obtained from 12 independent mice, with similar results obtained. d, Quantification of dopaminergic cell density within the boundary of the SNc as determined by TH+ signal (example shown in [a]). Data depicted is mean ± s.e.m. P values were determined by one-way ANOVA. The following n values represents number of independent animals used for statistical evaluation: e9d, all conditions = 4.

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Supplementary information

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Supplementary Tables 1 and 2.

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Supplementary Video 1

Representative movie of optogenetic stimulation and calcium imaging of the duodenal enteric nervous system. Raw grayscale movie collecting jRGECO1a signal from duodenal enteric neurons in response to photostimulation of ChR2-EYFP by a 473-nm pulse (indicated in frames with the word “light” at the top right). For demonstrative purposes, the movie shows a stimulation train of 5 pulses, 10 s apart.

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Challis, C., Hori, A., Sampson, T.R. et al. Gut-seeded α-synuclein fibrils promote gut dysfunction and brain pathology specifically in aged mice. Nat Neurosci 23, 327–336 (2020). https://doi.org/10.1038/s41593-020-0589-7

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