1. Microbiology and Infectious Disease
  2. Neuroscience
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A gut bacterial amyloid promotes α-synuclein aggregation and motor impairment in mice

  1. Timothy R Sampson  Is a corresponding author
  2. Collin Challis
  3. Neha Jain
  4. Anastasiya Moiseyenko
  5. Mark S Ladinsky
  6. Gauri G Shastri
  7. Taren Thron
  8. Brittany D Needham
  9. Istvan Horvath
  10. Justine W Debelius
  11. Stefan Janssen
  12. Rob Knight
  13. Pernilla Wittung-Stafshede
  14. Viviana Gradinaru
  15. Matthew Chapman
  16. Sarkis K Mazmanian  Is a corresponding author
  1. California Institute of Technology, United States
  2. University of Michigan, United States
  3. Chalmers University of Technology, Sweden
  4. University of California, San Diego, United States
Research Article
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Cite this article as: eLife 2020;9:e53111 doi: 10.7554/eLife.53111

Abstract

Amyloids are a class of protein with unique self-aggregation properties, and their aberrant accumulation can lead to cellular dysfunctions associated with neurodegenerative diseases. While genetic and environmental factors can influence amyloid formation, molecular triggers and/or facilitators are not well defined. Growing evidence suggests that non-identical amyloid proteins may accelerate reciprocal amyloid aggregation in a prion-like fashion. While humans encode ~30 amyloidogenic proteins, the gut microbiome also produces functional amyloids. For example, curli are cell surface amyloid proteins abundantly expressed by certain gut bacteria. In mice overexpressing the human amyloid α-synuclein (αSyn), we reveal that colonization with curli-producing Escherichia coli promotes αSyn pathology in the gut and the brain. Curli expression is required for E. coli to exacerbate αSyn-induced behavioral deficits, including intestinal and motor impairments. Purified curli subunits accelerate αSyn aggregation in biochemical assays, while oral treatment of mice with a gut-restricted amyloid inhibitor prevents curli-mediated acceleration of pathology and behavioral abnormalities. We propose that exposure to microbial amyloids in the gastrointestinal tract can accelerate αSyn aggregation and disease in the gut and the brain.

Introduction

The accumulation and aggregation of amyloid proteins occurs during many neurodegenerative diseases. Synucleinopathies are one family of amyloid disease, which includes Parkinson’s disease (PD), Lewy Body disease (LBD), and Multiple System Atrophy (MSA). Central to their pathogenesis is the accumulation of the neuronal protein α-Synuclein (αSyn) into insoluble amyloid aggregations, which ultimately leads to inflammation and neuronal dysfunction (Jucker and Walker, 2013; Brettschneider et al., 2015). αSyn aggregation can contribute to the death of dopaminergic neurons in specific brain regions, resulting in motor symptoms (Poewe et al., 2017). Clinical and epidemiological data suggest that accumulation of αSyn aggregates may first occur at peripheral sites, such as the olfactory epithelium or gastrointestinal (GI) tract, before spreading to the brain (Braak et al., 2003). Individuals diagnosed with various synucleinopathies often display constipation and other GI dysfunctions years prior to the onset of movement dysfunction (Verbaan et al., 2007; Colosimo, 2011; Engen et al., 2017; Mertsalmi et al., 2017; Sakakibara et al., 2019). Experimental evidence exists for a prion-like spread of αSyn aggregates (Woerman et al., 2015), including propagation from the gut to the brain via the vagus nerve and/or spinal cord in rodent models (Holmqvist et al., 2014; Uemura et al., 2018; Van Den Berge et al., 2019). In humans, recent epidemiological studies suggest an association between truncal vagotomy and appendectomy with a decreased risk of PD (Svensson et al., 2015; Liu et al., 2017; Killinger et al., 2018), and in increased risk of comorbidity with inflammatory bowel disease (IBD) (Hui et al., 2018; Peter et al., 2018). While a role for protein aggregation and/or inflammation in the gut represents an emerging area of research in synucleinopathies, the GI tract has been implicated in other neurological disorders such as autism spectrum disorder, depression, anxiety and Alzheimer’s disease (Vuong et al., 2017).

The gut is colonized with a complex microbiome that impacts development and function of the immune, metabolic and nervous systems (Fung et al., 2017). Enterobacteriaceae, highly prevalent within the gut of humans, can produce functional amyloid proteins termed curli (Tursi and Tükel, 2018). Curli fibers are formed by bacterial secretion of an unfolded amyloid, CsgA, that subsequently aggregates extracellularly to form biofilms, mediate adhesion to epithelial cells, and are involved in bacteriophage defense (Tursi and Tükel, 2018; Vidakovic et al., 2018). Exposure to curli not only modulates host inflammatory responses within the intestinal tract and periphery (Gallo et al., 2015; Chen et al., 2016; Tursi and Tükel, 2018), but oral administration of curli-producing bacteria can also increase production and aggregation of the amyloid protein αSyn in aged rats and nematodes (Chen et al., 2016). Biochemical studies demonstrate that native, bacterial chaperones of curli are capable of transiently interacting with αSyn and modulating its aggregation (Chorell et al., 2015; Evans et al., 2015). Interestingly, diverse human amyloid proteins including αSyn, amyloid beta (Aβ), cellular prion protein (PrPC), and Tau can accelerate the amyloidogenesis of heterologous mammalian amyloid proteins (Clinton et al., 2010; Brettschneider et al., 2015; Katorcha et al., 2017). Lesions containing mixed human amyloids have been observed in neurodegenerative brains (Rahimi and Kovacs, 2014; Spires-Jones et al., 2017), implicating interactions between different amyloidogenic proteins in resulting pathology. Accordingly, we wondered whether a bacterial amyloid protein can contribute to heterologous aggregation of mammalian αSyn in the gut and the brain, leading to synucleinopathy-related behaviors.

Herein, we reveal that mono-colonization of αSyn-overexpressing mice with curli-producing Escherichia coli exacerbates motor impairment and GI dysfunction, and promotes αSyn aggregation and inflammation in the gut and brain. Enrichment of curli-producing E. coli to mice harboring a healthy human microbiome is sufficient to aggravate αSyn-dependent pathophysiology. The purified amyloidogenic subunit of curli fibers (CsgA) is sufficient to accelerate αSyn aggregation during in vitro biochemical assays and pathophysiology in mice following intra-intestinal administration, while variants of CsgA that are unable to form amyloids have no effect on αSyn aggregation. Oral treatment of mice with a gut-restricted amyloid inhibitor reduces csgA expression in the gut, limits αSyn aggregation in the brain, and alleviates GI and motor deficits in mice that overexpress αSyn. These data provide novel insights into a trans-kingdom interaction between the gut microbiome and mammalian amyloids, and suggest the possibility that carriage of particular bacterial taxa may be a factor that can exacerbate neurologic disease.

Results and discussion

Mono-colonization with curli-producing gut bacteria enhances αSyn pathophysiology

We previously identified that depletion of the microbiome reduces pathophysiology in Thy1-αSyn mice (alpha-synuclein overexpressing; ASO mice) (Sampson et al., 2016), which overexpress wild-type human αSyn. Due to accumulation and aggregation of neuronal αSyn, mice display increased neuroinflammation, GI dysfunction, and progressive motor abnormalities (Rockenstein et al., 2002; Chesselet et al., 2012; Wang et al., 2012) that are relevant in the study of synucleinopathies. Prior findings in germ-free Thy1-αSyn mice suggest that an unidentified member(s) of the gut microbiome may be pathogenic in this mouse model (Sampson et al., 2016). Interestingly, increased colonization and mucosal association with Enterobacteriaceae, such as E. coli, have been reported in individuals with PD compared to healthy controls (Forsyth et al., 2011), as well as a positive association of Enterobacteriaceae abundance with disease severity (Scheperjans et al., 2015; Li et al., 2017).

To establish whether E. coli promotes αSyn-dependent motor dysfunction, we mono-associated germ-free (GF) wild-type and ASO mice with the curli-producing E. coli strain MC4100, or Bacteroides fragilis strain NCTC9343 and segmented filamentous bacteria (SFB), which do not produce curli. E. coli exacerbated the αSyn-dependent motor defects in ASO animals across a battery of tests, compared to the other taxa (Figure 1A–F and Figure 1—figure supplement 1A–E). To determine the contribution of curli amyloids, we compared mice mono-colonized with wild-type E. coli (WT) to those mono-colonized with an isogenic mutant lacking genes encoding the curli biosynthesis machinery (ΔcsgBAC) (Wang and Chapman, 2008). Evaluation of coordinated motor function revealed that colonization with the curli-deficient strain did not elicit robust motor impairment (Figure 1A–F). We did not observe curli-dependent alterations to colonization levels or mucosal association, despite detecting csgA expression (Figure 1—figure supplement 1F–I). In addition, the curli-deficient mutant did not display alterations to lipopolysaccharide potency or structure (Figure 1—figure supplement 1J,K). Thus, curli-producing bacteria are capable of enhancing motor deficits in ASO mice.

Figure 1 with 3 supplements see all
Mono-colonization with curli-producing gut bacteria enhances αSyn pathophysiology germ-free wild-type BDF1 (WT) and Thy1-αSyn (ASO) mice were mono-colonized with wild-type E. coli MC4100 (WT) or an isogenic curli-deficient strain (ΔcsgBAC) at 5–6 weeks of age.

Motor function was assessed at 12–13 weeks of age by quantifying (A), beam traversal time, (B), pole descent time, (C), nasal adhesive removal time, (D), hindlimb clasping score, (E), wirehang time. (F), Principal component analysis of compiled motor scores from tests in (A–E). Quantification of (G, H) pS129αSyn and (I) proteinase K–resistant αSyn by immunofluorescence microscopy in the substantia nigra and midbrain. n = 13–16 (A–F), n = 4.5 (G–I). Points represent individuals, bars represent the mean and standard error. Data analyzed by one-way ANOVA with Tukey post-hoc test (A–E), and two-tailed t-test for (G–I). *p≤0.05; **p≤0.01; ***p≤0.001; ****p≤0.0001. Motor data are compiled from three independent cohorts.

ASO mice colonized with curli-producing bacteria displayed increased αSyn fibril reactivity and detergent-insoluble αSyn in the midbrain compared to mice with the ΔcsgBAC strain, despite similar transgene production (Figure 1—figure supplement 2A–D,F,G). Histological and western blot analysis revealed increased phospho-serine129 αSyn (pS129-αSyn) deposition in the substantia nigra (SN) of ASO mice, indicative of pathological αSyn aggregation, (Figure 1G,H, and Figure 1—figure supplement 2E,H,J). Further, mice colonized with WT E. coli show increased proteinase K-resistant αSyn inclusions in the midbrain (Figure 1I and Figure 1—figure supplement 2I), with little alteration to pS129-αSyn in the frontal cortex (Figure 1—figure supplement 2K,L). Additionally, we observed increased pS129-αSyn in the proximal large intestine, and elevated αSyn fibril reactivity in the duodenum and proximal large intestine of ASO mice mono-colonized with curli-producing E. coli (Figure 1—figure supplement 2M–P). Previous research has shown that synuclein pathology, neuroinflammation, and motor defects occur at early ages in the ASO mouse model without concomitant loss of striatal dopamine, tyrosine hydroxylase positive (TH+) neurons in the midbrain, or loss of neurons in the myenteric plexus (Fleming et al., 2004; Lam et al., 2011; Chesselet et al., 2012; Wang et al., 2012). Consistent with these prior observations, we do not observe decreases in total striatal dopamine, midbrain TH expression, TH+ neurons, nor myenteric PGP9.5+ neurons, under the colonization conditions and ages assessed, suggesting pathology and motor deficits are independent of dopamine or neuron loss (Figure 1, figure supplement S2Q-S).

Mono-colonization of ASO mice with curli-producing E. coli resulted in increased expression of the proinflammatory cytokines interleukin 6 (IL-6) and tumor necrosis factor alpha (TNFα) in brain-derived CD11b+ cells, and increased cytokine production in the midbrain and striatum (Figure 1—figure supplement 3A–C). In addition, Iba+ microglia morphologies indicated reduced activation in ASO mice colonized with the curli-deficient strain (Figure 1—figure supplement 3D–G). Multiplexed ELISA analysis revealed increased cytokine and chemokine production in colonic tissue of mice mono-colonized with curli-producing E. coli, irrespective of genotype (Figure 1—figure supplement 3H,J,K), and no noteworthy changes to serum cytokines of ASO mice based on colonization status (Figure 1—figure supplement 3I). These data reveal that a bacterial amyloid from the gut microbiome can exacerbate pathology and inflammation, in both the gut and brain, in mice that overexpress αSyn.

Curli biosynthesis within a complex microbiome contributes to motor and GI deficits

To explore host-microbiome interactions in a more natural context, we tested whether introduction of curli-producing bacteria to a healthy human microbiota is sufficient to enhance αSyn-dependent pathophysiology. GF ASO mice were transplanted with fecal microbiota from a human donor predicted to contain low levels of csgA, as indicated by PICRUSt analysis following 16S rRNA sequencing (Figure 2—figure supplement 1A). This single microbiome was supplemented with either WT E. coli or the ΔcsgBAC strain. Both strains reached similar abundances in the feces (Figure 2—figure supplement 1B,C), while csgA expression and amyloid production appeared only in mice colonized with WT bacteria (Figure 2—figure supplement 1C–E). Mice harboring a microbiota containing WT E. coli displayed significantly impaired motor and GI performance compared to animals with a complex microbiota plus the curli-deficient strain (Figure 2A–G). Moreover, enrichment of curli-producing bacteria resulted in increased αSyn fibril reactivity in the midbrain and elevated pS129-αSyn deposition in the SN, without changes to the number of TH+ neurons (Figure 2H and Figure 2—figure supplement 1F–J). Morphometric analysis of microglia demonstrated concomitant changes indicative of inflammatory status in the midbrain (Figure 2—figure supplement 1K–N). Therefore, in the context of a complex human microbiota, curli-producing E. coli are sufficient to modulate αSyn-mediated pathophysiology.

Figure 2 with 1 supplement see all
Curli biosynthesis within a complex microbiome contributes to motor and GI deficits Germ-free Thy1-αSyn (ASO) mice were colonized with fecal microbes derived from healthy human at 5–6 weeks of age, and concurrently supplemented with either wild-type E. coli MC4100 (+WT) or a curli-deficient strain (+ΔcsgBAC).

Motor function was tested longitudinally at 12, 15, and 20 weeks of age in the (A) beam traversal, (B) pole descent, (C) adhesive removal, (D) hindlimb clasping score, (E) wirehang tests. (F) Principal component analysis of compiled motor scores from tests in (A–E). (G) Fecal output over a 15-min period observed at week 21 of age. Quantification of (H) pS129αSyn by immunofluorescence microscopy in the substantia nigra. n = 12 (A–F), n = 6 (G), n = 4 (H). Data points represent individuals, bars represent the mean and standard error. Time courses analyzed by two-way ANOVA, with Sidak post-hoc test for between group comparisons indicated above individual time points, and brackets indicating significance between colonization status. Data in (H) analyzed two-tailed t-test. *p≤0.05; **p≤0.01; ***p≤0.001; ****p≤0.0001. Motor data are compiled from two independent cohorts.

The bacterial amyloid protein, CsgA, accelerates αSyn fibrilization

Emerging data suggest that non-orthologous amyloid proteins can accelerate heterologous aggregation (Clinton et al., 2010; Brettschneider et al., 2015; Katorcha et al., 2017). We therefore tested whether CsgA can directly impact aggregation of αSyn. Purified monomeric CsgA accelerated production of αSyn aggregates during in vitro biochemical amyloid assays (Figure 3A–C and Figure 3—figure supplement 1A), even at concentrations below which CsgA self-aggregates (Figure 3D). The αSyn fibrils purified from these reactions maintained an ability to accelerate αSyn amyloidogenesis (Figure 3—figure supplement 1B,C), suggesting that CsgA-induced αSyn aggregates are propagation-competent, similar to other amyloids (Spires-Jones et al., 2017). Surprisingly, we could not detect a direct interaction between purified CsgA and αSyn monomers by surface plasmon resonance (Figure 3—figure supplement 1D), perhaps indicating transient interactions or requirements for prior oligomerization of either or both proteins. Unlike αSyn, Tau aggregation was not accelerated by CsgA (Figure 3—figure supplement 1E–G). Native amyloid-forming properties of CsgA are required, as a non-amyloidogenic form of CsgA (CsgA:Q49A/N54A/Q139A/N144A; ‘SlowGo’ Wang and Chapman, 2008) did not augment αSyn aggregation in vitro (Figure 3—figure supplement 2A–C). To investigate the dependence of these amyloidogenic residues in vivo, we mono-colonized ASO mice with E. coli producing either wild-type CsgA or CsgA:SlowGo proteins (Wang and Chapman, 2008). Motor performance in ASO mice harboring SlowGo-producing E. coli was less severe in comparison to mice colonized with WT bacteria (Figure 3—figure supplement 2D–H), although not to the extent of the ΔcsgBAC strain, qualitatively (see Figure 1).

Figure 3 with 3 supplements see all
The bacterial amyloid protein, CsgA, accelerates αSyn fibrilization In vitro biophysical analysis with purified αSyn and CsgA proteins.

(A) Aggregation as measured by Thioflavin T fluorescence over time during αSyn amyloid formation alone or in the presence of CsgA monomers (25:1 molar ratio). Time to reach (B) exponential fibrilization/lag phase and (C) half-maximum from reactions in (A). (D) Representative transmission electron micrographs of αSyn or CsgA alone, or in combination, at 0 and 60 hr post-aggregation. n = 3 (A–C). Bars represent the mean and standard error. Data are analyzed by two-tailed, t-test. **p≤0.01; ***p≤0.001.

Next, a peptide spanning the aggregation domain of CsgA or a non-amyloidogenic version (N122A) (Tükel et al., 2009) were injected directly into the intestinal wall of SPF ASO mice. Intra-intestinal delivery of amyloidogenic CsgA peptide, but not the mutant peptide, resulted in progressive motor and GI dysfunction (Figure 3—figure supplement 3A–G). Furthermore, increased αSyn fibrils were detected in the midbrains of amyloidogenic peptide-injected animals (Figure 3—figure supplement 3H). We conclude that gut exposure to a bacterial amyloid is sufficient to exacerbate motor deficits and αSyn brain pathology in this mouse model, in a manner dependent on CsgA aggregation.

Curli-driven pathophysiology in mice requires functional amyloid formation

Epigallocatechin gallate (EGCG) is a plant-derived, dietary polyphenol that physically inhibits amyloid formation, including αSyn aggregation (Bieschke et al., 2010). EGCG is also capable of blocking CsgA amyloidogenesis and represses csgA transcript expression in E. coli through activation of specific stress response pathways within the bacterial cell (Serra et al., 2016). We reveal here that EGCG treatment did not impair E. coli growth in culture, but significantly reduced biofilm formation, a process dependent on the production and assembly of curli (Figure 4—figure supplement 1A,B) (Tursi and Tükel, 2018). Additionally, EGCG also inhibited CsgA-accelerated αSyn amyloid formation during in vitro biochemical aggregation assays (Figure 4A). Notably, EGCG remains largely gut-restricted in rodents and humans and is not readily bioavailable in circulation or brain tissues following oral administration (Lambert et al., 2003; Lin et al., 2007; Cai et al., 2018). Oral treatment of wild-type E. coli mono-colonized ASO mice with EGCG did not affect fecal E. coli abundance, but resulted in decreased csgA production (Figure 4B and Figure 4—figure supplement 1C). Assessment of motor performance revealed that EGCG improved both motor and GI defects exacerbated by curli-producing E. coli in ASO mice (Figure 4C–I and Figure 4—figure supplement 1D–I). In addition, oral EGCG administration reduced αSyn aggregation and pS129-αSyn deposition in both the striatum and midbrain (Figure 4J and Figure 4—figure supplement 1J–M). Furthermore, microglia morphological changes were limited in EGCG-treated mice (Figure 4—figure supplement 1O–R). In addition to its ability to directly inhibit aggregation of bacterial and host-derived amyloids, EGCG has antioxidant and anti-inflammatory activities (Li et al., 2004) that may also contribute to the rescue of CsgA-accelerated, αSyn-dependent pathophysiology. Collectively, these data reveal that curli is a specific bacterial structure that can accelerate mammalian amyloid aggregation in vitro and in vivo.

Figure 4 with 1 supplement see all
Curli-driven pathophysiology in mice requires functional amyloid formation.

(A), Representative in vitro αSyn aggregation measured by Thioflavin T fluorescence during αSyn amyloid formation alone or in the presence of CsgA (25:1 molar ratio), with and without EGCG (50 μM) treatment. (B–H) Germ-free Thy1-αSyn mice (ASO) were mono-colonized with WT E. coli at 5–6 weeks of age, and given water alone (Vehicle: Veh) or treated with EGCG ad lib in drinking water (+EGCG). (B) RNA was extracted from fecal pellets and csgA expression quantified by qRT-PCR, relative to rrsA. Motor function was assessed at 15–16 weeks of age by quantifying (C) beam traversal time, (D) pole descent time, (E) nasal adhesive removal time, (F) hindlimb clasping score, (G) wirehang tests. (H) Principal component analysis of compiled motor scores from tests in (C–G). (I) Fecal output over a 15 min period. (J) Quantification of insoluble αSyn fibrils in the ventral midbrain by dot blot assay. n = 8 (B); n = 10–11 (C–G); n = 4–5 (I, J). Points represent individuals, bars represent the mean and standard error. Data analyzed by two-tailed Mann-Whitney for B–G, (J) and two-way ANOVA with Sidek’s post-hoc test for I with between group comparisons indicated above individual time points, and brackets indicating significance between treatment status. *p≤0.05; **p≤0.01; ***p≤0.001; ****p≤0.0001. Motor data compiled from two independent cohorts.

Conclusion

The majority of synucleinopathy incidences are idiopathic, with multifactorial and complex risks that contribute to disease initiation and/or progression (Ritz et al., 2016; Soldner et al., 2016; Johnson et al., 2019). Our findings reveal that the bacterial amyloid CsgA can accelerate αSyn aggregation and enhance motor abnormalities in mice that are genetically predisposed to αSyn pathology. Curli production in the gut of non-susceptible mice does not impair motor performance at the timepoints examined, suggesting that this microbial trigger is not sufficient, but rather requires additional predisposing factors to promote disease outcomes. Further, inhibiting amyloid formation in the gut correlates with improvements in curli-induced behaviors and pathology. Our study does not reconcile how curli production within the gastrointestinal tract ultimately manifests αSyn aggregation in the brain. Experimental evidence exists for a prion-like spread of αSyn aggregates (Woerman et al., 2015), including propagation from the gut to the midbrain via the vagus nerve and/or spinal cord in rodent models (Uemura et al., 2018; Kim et al., 2019; Van Den Berge et al., 2019), although evidence for sustained propagation from the GI tract to structures outside the brainstem in primates is currently lacking (Manfredsson et al., 2018). Epidemiological studies suggest an association between full truncal vagotomy with a decreased risk of PD (Svensson et al., 2015). Intriguingly, there appears to be a positive correlation between inflammatory bowel disease and neurodegenerative disease (Hui et al., 2018; Peter et al., 2018). In experimental models, induction of GI inflammation, for instance through LPS or dextran sodium sulfate administration, is sufficient to induce αSyn pathology in the CNS (Choi et al., 2018; Kishimoto et al., 2019; Perez-Pardo et al., 2019). Aggregates of both CsgA and αSyn are capable of signaling through the innate immune receptor Toll-like receptor 2 (TLR2) leading to increased inflammatory responses (Tükel et al., 2009; Poewe et al., 2017). In fact, inhibiting TLR2 in αSyn-based mouse models leads to improved pathology and motor outcomes (Kim et al., 2015; Kim et al., 2018). Our data demonstrate curli-dependent increases of inflammatory markers in both the intestine and brain, as well as acceleration of αSyn pathology, but do not determine which of these may be the primary driver of curli-mediated pathophysiology. Disentangling these complex relationships in future studies will define how pathologic signals from bacterial amyloids are transduced from the gut to the brain, via transneuronal spread of αSyn aggregates and/or an inflammatory cascade, and mechanisms of gut-to-brain signaling that contribute to motor impairment.

Synucleinopathy is linked to marked changes to the structure of the microbiome. Numerous studies have reported microbiome differences in persons with PD compared to unaffected individuals (Hasegawa et al., 2015; Keshavarzian et al., 2015; Scheperjans et al., 2015; Unger et al., 2016; Bedarf et al., 2017; Hill-Burns et al., 2017; Hopfner et al., 2017; Li et al., 2017; Barichella et al., 2019; Lin et al., 2018); however, examination of the gut microbiome during other synucleinopathies, such as MSA or LBD are still in their infancy (Engen et al., 2017). Some reports have identified the family Enterobacteriaceae as enriched in PD compared to unaffected individuals (Unger et al., 2016; Li et al., 2017; Barichella et al., 2019). In those studies which performed correlation analysis between disease severity and microbiome composition, enrichment of Enterobacteriaceae is observed to be positively associated with worsened motor symptoms (Scheperjans et al., 2015; Li et al., 2017). To our knowledge, no study to date has identified differential abundance of csgA or curli-encoding genes specifically in human incidences, and only a single study to date has utilized metagenomic sequencing of the gut microbiome during PD (Bedarf et al., 2017). Despite recent advances defining microbiome changes in neurodegenerative diseases, a contributing role by gut bacteria to synucleinopathies in humans remains correlative (reviewed in Sampson, 2019). In experimental models, however, transplant of fecal microbiomes from PD patients into ASO mice results in greater motor deficits than microbiomes from healthy controls (Sampson et al., 2016), suggesting functional consequences to changes in gut microbial composition.

Although our findings herein are limited to a single transgenic animal mouse model, and similar pathologies are observed in aged rats and nematodes (Chen et al., 2016), it is possible that these observations are model specific. Non-amyloid contributions from diverse gut bacteria are also likely to occur and influence neurodegenerative outcomes. For instance, enrichment of the gut bacterium Proteus mirabilis, or intestinal administration of its purified LPS, impairs the dopaminergic system of mice and increases susceptibility to neurotoxins (Choi et al., 2018). Infection with Gram-negative bacteria, including Citrobacter sp. (a taxonomic relative of E. coli), can trigger a pathological process leading to neurodegenerative immune responses in the brain and loss of midbrain dopamine production in a PD-relevant mouse model (Matheoud et al., 2019). In addition, specific microbiome-derived metabolites promote microglia maturation (Erny et al., 2015) and enhance αSyn-dependent pathology in germ-free mice (Sampson et al., 2016). Conversely, specific microbial metabolites (Blacher et al., 2019) or fecal microbiome transplants (Sun et al., 2018) may provide protection from neurodegenerative insults.

While CsgA is one example of bacterially-produced amyloid, microbial amyloid formation in general may influence physiological processes with outcomes relevant for neurodegenerative disease (Friedland and Chapman, 2017). For example, the Pseudomonas sp. functional amyloid, FapC, may also accelerate αSyn aggregation in vitro (Christensen et al., 2019), and bacterial chaperones of CsgA modulate αSyn amyloidogenesis (Evans et al., 2015). More broadly, CsgA is observed to accelerate amyloid β aggregation in vitro (Hartman et al., 2013; Perov et al., 2019), as well as the disease-associated amyloids, islet amyloid polypeptide (IAPP) and semen enhancer of viral infection (SEVI) in vitro (Hartman et al., 2013), and serum amyloid A in mice (Lundmark et al., 2005). Emerging evidence in diverse animal models suggests that gut bacteria may modulate amyloid-driven diseases, not only in synucleinopathy as we and others describe, but Alzheimer’s disease (Harach et al., 2017; Dodiya et al., 2019) and amyotrophic lateral sclerosis (ALS) as well (Blacher et al., 2019), providing justification for future human studies and revealing possible new targets for interventions that may prevent, slow, or halt amyloid formation and neurodegenerative disease.

Materials and methods

Key resources table
Reagent type
(species) or
resource
DesignationSource or
reference
IdentifiersAdditional
information
Strain, strain background (Mus musculus)Thy1- alpha-synuclein (Line 61), BDF1 backgroundRockenstein et al., 2002ASO (alpha-synuclein over-expressing)With permission, UCSD
Strain, strain background (Escherichia coli)Str. K12, MC4100Zhou et al., 2013WT (wild-type)
Strain, strain background (Escherichia coli)Str. K12, MC4100:ΔcsgBACZhou et al., 2013ΔcsgBAC
Strain (Escherichia coli)Str. K12, MC4100: CsgA:Q49A/N54A/Q139A/N144AWang and Chapman, 2008SlowGo
AntibodyAnti-alpha synuclein, mouse monoclonalBDCat#: 610787(1:1000)
AntibodyAnti-pS129 alpha-synuclein, rabbit monoclonalAbCamCat#: Ab51253(1:1000)
AntibodyAnti-tyrosine hydroxylase, mouse monoclonalMilliporeCat#: MAB318(1:1000)
AntibodyAnti-Iba1, rabbitWakoCat#: 019–19741(1:1000)
AntibodyAnti-aggregated alpha-synuclein, rabbit polyclonalAbCamCat#: MJFR-14-6-4-2(1:1000)
AntibodyAnti-mouse IgG-546, goat polyclonalLife TechnologiesCat#: A-11003(1:1000)
AntibodyAnti-rabbit IgG-546, goat polyclonalLife TechnologiesCat#: A-11010(1:1000)
AntibodyAnti-mouse IgG-488, goat polyclonalLife TechnologiesCat#: A-11001(1:1000)
AntibodyAnti-rabbit IgG-HRP, goat polyclonalCell SignalingCat#: 7074(1:1000)
Peptide, recombinant proteinCsgA (N’-QYGGNN-C’)Bio-Synthesis, Inc; Tükel et al., 2009CsgA
Peptide, recombinant proteinN122A (N’QYGGNA-C’)Bio-Synthesis, Inc; Tükel et al., 2009N122A
Sequence-based reagent16S rRNA:
5’-TCCTACGGGAGGCAGCAGT-3’ and
5’-GGACTACCAGGGTATCTAATCCTGTT-3’
IDTqPCR primerPrimerBank
Sequence-based reagentrrsA:
5’-AGTGATAAACTGGAGGAGGTG-3‘ and
5’-GGACTACGACGCACTTTATGAG-3’
IDTqPCR primerPrimerBank
Sequence-based reagentcsgA:
5’-ATGACGGTTAAACAGTTCGG-3’ and
5’-AGGAGTTAGATGCAGTCTGG-3’
IDTqPCR primerPrimerBank
Sequence-based reagentgapdh:
5’-CATGGCCTTCCGTGTTCCTA-3’ and
5’-CCTGCTTCACCACCTTCTTGAT-3’
IDTqPCR primerPrimerBank
Sequence-based reagentil6:
5’-TAGTCCTTCCTACCCCAATTTCC-3’ and
5’-TTGGTCCTTAGCCACTCCTTC-3’,
IDTqPCR primerPrimerBank
Sequence-based reagentTH:
5’-CCAAGGTTCATTGGACGGC-3’ and
5’-CTCTCCTCGAATACCACAGCC-3’
IDTqPCR primerPrimerBank
Sequence-based reagenttnfa;
5’-CCCTCACACTCAGATCATCTTCT-3’ and
5’-GCTACGACAGTGGGCTACAG-3’
IDTqPCR primerPrimerBank

Animals

Male wild-type and Thy1-αSyn mice (Line 61, with permission from University of California, San Diego) were generated as described previously (Rockenstein et al., 2002; Chesselet et al., 2012; Sampson et al., 2016), through breeding female BDF1 background Thy1-αSyn mice to male BDF1 offspring generated via crossing female C57BL/6 with DBA/2 males (Charles River, Hollister, CA), and refreshing breeding pairs ~ 6 months. Germ-free (GF) Thy1-αSyn breeding pairs were generated via caesarian section and cross-fostered by GF Swiss-Webster dams. Conventionally colonized and mono-colonized animals were housed in autoclaved, ventilated, microisolator caging. GF animals were housed in open-top caging within flexible film isolators and maintained microbiologically sterile, confirmed via 16S rRNA PCR from fecal-derived DNA and culture of fecal pellets on Brucella blood agar or tryptic soy blood agar (Teknova, Hollister, CA) under anaerobic and aerobic conditions, respectively. Mono-colonized animals received ~108 cfu of the indicated bacterial strains in ~100 μL sodium bicarbonate buffer (5% w/v) at 5–6 weeks of age. Human microbiome colonized animals received ~100 μL 0.1 g/mL fecal extract from previously sampled healthy human donor (ENA Accession: PRJEB17694; Sample #: 10483.donor2.HC; MMA_008 [Sampson et al., 2016], California Institute of Technology’s Institutional Review Board #15–0568- exempt), at 5–6 weeks of age. For mice mono-colonized with E. coli containing plasmid vectors for CsgA and CsgA:SlowGo, drinking water was supplemented with 50 μg/mL kanamycin (Sigma Aldrich, St Louis, MO). Human microbiome colonized mice were associated with WT or ΔcsgBAC mutant E. coli at time of microbiota transplant. For epigallocatechin gallate (EGCG) treatment, animals received filter-sterilized EGCG (Sigma Aldrich) at 200 μg/mL in drinking water ad libitum beginning at 5–6 weeks of age. All animals received autoclaved food (LabDiet Laboratory Autoclavable Diet 5010, St Louis, MO) and water ad libitum, were maintained on the same 12 hr light-dark cycle, and housed in the same room of the facility. All animal husbandry and experiments were approved by the California Institute of Technology’s Institutional Animal Care and Use Committee (IACUC), through protocol #1707.

Motor function assessment

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Motor function was assessed similarly to previous studies (Fleming et al., 2004; Sampson et al., 2016), between hours 7 and 9 of the light phase, within a biosafety cabinet. Beam traversal and pole descent were performed first, followed by fecal output measurement. One day later, wirehang, adhesive removal and hindlimb scoring was performed. Beam traversal- A 1 m plexiglass beam consisting of four segments of 0.25 m in length (Stark’s Plastics, Forest Park, OH) was constructed with consecutively thinner widths of 3.5, 2.5, 1.5, and 0.5 cm, with 1 cm overhangs placed 1 cm below the surface of the beam and the narrowest end placed into home cage. Animals were trained for two consecutive days before the first testing. On the first day of training, animals received one trial with the home cage positioned close to the widest segment, where the animal was placed, and guided the animals forward along the narrowing beam. Animals received two more trials with limited or no assistance to encourage forward movement and stability on the beam. On the seccond day of training, animals were given three trials to traverse the beam, with little to no assistance. On the third day, animals were timed over three trials to traverse from the widest segment to the home cage. Time was measured from the placement of the animal’s forelimbs onto the second segment until a forelimb reached the home cage. Score was averaged over three trials. Pole descent- A 0.5 m long pole, 1 cm in diameter, wrapped with non-adhesive shelf liner to facilitate the animals grip, was placed into the home cage, with animals removed and placed into fresh housing. Animals received 2 days of training to descend from the top of the pole and into the home cage. On the first day, animals received three trials to descend the pole. First animals were placed head-down ~1/3 the distance above the base, the second trial from ~2/3 the height, and on the third trial animals were placed at the top of the pole, head-down. The second day of training, animals were tasked with descending, head-down, from the top of the pole, three times. On the day of testing, animals were placed head-down on the top of the pole and timed beginning when the experimenter released the animal and ended when one hind-limb was placed on the pole base. Score was averaged over three trials. Adhesive removal- ¼” round adhesive labels (Avery, Glendale, CA) were placed on the nasal bridge. Animals were placed into their home cage (with cage mates removed into separate cage) and timed to completely remove the sticker. Animals were recorded over two trials, and averaged. Hindlimb clasping reflex scoring- Animals were gently lifted by the mid-section of the tail and observed over ~5–10 s (Zhang et al., 2014). Animals were assigned a score of 0, 1, 2, or 3. 0 was scored to animals that freely moved both their limbs and extended them outwards. A 1 was assigned to animals which clasped one hindlimb inward for the duration of the restraint or if both legs exhibited partial inward clasping. 2 was given if both legs clasped inward for the majority of the observation, but still exhibited some flexibility. A score of 3 was assigned if animals displayed complete rigidity of hindlimbs that immediately clasped inward and exhibited no signs of flexibility. For animals in Figure 3—figure supplement 2G, animals were scored twice on 2 consecutive days and the score averaged, the remainder were scored once. Wirehang- Animals were placed in the center of a 30 cm by 30 cm screen with 1 cm wide mesh. The screen was inverted head-over-tail and placed on supports ~ 40 cm above an open cage with deep bedding. Animals were timed until they released their grip or remained for 60 s, and the score from two trials averaged. Fecal Output- Animals were removed from their home cages and placed into a 12 cm x 25 cm translucent cylinder. Fecal pellets were counted every 5 min, cumulative over 15 min. Principal component analysis- PCA was performed using ClustVis web tool, with default settings and reversed axis for display (Metsalu and Vilo, 2015).

Bacterial strains, manipulations, and characterizations

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Escherichia coli K12 str. MC4100 and the previously characterized isogenic deletion mutant of the csgBAC operon were cultured aerobically in YESCA media at 37°C (Zhou et al., 2013). Biofilm assays were performed via crystal violet staining of static culture at room temperature in YESCA as described previously (Zhou et al., 2013). Congo red staining was performed on YESCA agar following 2–3 days growth at room temperature as described previously (Zhou et al., 2013). Fecal bacterial DNA was isolated using QuickDNA Fecal/Soil Microbe Miniprep (Zymo Research, Irvine, CA). Fecal bacterial RNA was isolated using PowerMicrobiome RNA Isolation kit (MoBio, Carlsbad, CA) and cDNA generated via iScript cDNA Synthesis kit (BioRad, Hercules, CA). qPCR was performed with SybrGreen master mix (Applied Biosystems, Foster City, CA) on an AB7900ht instrument using the following primers: 16 s rRNA-5’-TCCTACGGGAGGCAGCAGT-3’ and 5’-GGACTACCAGGGTATCTAATCCTGTT-3’; rrsA-5’-AGTGATAAACTGGAGGAGGTG- ‘and 5’-GGACTACGACGCACTTTATGAG-3’; csgA- 5’-ATGACGGTTAAACAGTTCGG-3’ and 5’-AGGAGTTAGATGCAGTCTGG-3’. Bacteroides fragilis str. NTCT9343 was cultured anaerobically in brain-heart infusion (BHI) broth at 37°C, and colonized into GF animals via oral gavage of ~108 cfu in 100 μL sodium bicarbonate buffer (5% w/v) at 5–6 weeks of age. Segmented filamentous bacteria were colonized via bedding transfer and co-housing within a mono-associated gnotobiotic isolator. Colonization was confirmed by PCR for SFB 16 s RNA. For growth curves, E. coli was first grown aerobically at 37°C overnight in BHI broth, and subcultured at 1:400 in BHI containing indicated concentrations of EGCG, in a 200 μL volume in a 96-well plate. Plates were incubated at 37°C aerobically, with orbital shaking in a BioTek Cytation 5 plate reader, and the optical density at 600 nm measured every hour. Endotoxin content was measured following overnight growth at 37°C, aerobically in BHI using the Pierce LAL Chromogenic Endotoxin kit (ThermoFisher, Pittsburgh, PA) according to manufacturer’s instructions. LPS was stained from lysates derived from identically grown bacterial cultures following separation on a 4–20% SDS-PAGE gel with Pro-Q Emerald 300 LPS stain kit (ThermoFisher), according to manufacturer’s instructions.

α-Synuclein aggregation assays

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Thioflavin T (ThT) assays-Freshly purified CsgA (both wild-type and ‘SlowGo’) and αSyn were diluted in 50 mM KPi (pH 7.3) to the molar concentrations indicated in each experiment. Samples were incubated in 96-well, black, flat bottom plates at 37°C with 20 μM ThT and 100 mM NaCl under continuous shaking conditions, along with a 1 mm glass bead for homogenous mixing. The ThT fluorescence intensity was recorded in 30 min interval using a Tecan plate reader (excitation: 438 nm; emission: 495 nm; cut-off: 475 nm). TauK18 aggregation assay- 50 µM of TauK18 fragment with heparin (TauK18:Heparin 4:1) was incubated with or without CsgA (2 µM) in 14 mM MES buffer pH 6.8 at 37°C with continuous shaking in 96-well, black, flat bottom plates. 2 mm beads and 20 µM ThT was added to all the wells. The ThT fluorescence was monitored with previously mentioned parameters after 30 min in FLUOstar omega plate reader (BMG Labtech). Transmission electron microscopy- Aliquots of CsgA and αSyn reactions were taken at indicated timepoints. Five microliters of sample was applied on glow-discharged carbon-coated grids, incubated for 1 min and washed with MilliQ water before staining with 1% uranyl acetate. Samples were imaged on Jeol electron microscope (JEOL-1400 plus). Circular dichroism spectroscopy- αSyn alone or with CsgA (in 20 mM KPi pH 7.3) were analyzed using a Jasco J-810 spectropolarimeter from 190 nm to 250 nm at 25°C immediately after purification and at indicated timepoints.

Synuclein pathology and inflammatory responses

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CD11b enrichment- Animals were sedated with pentobarbital and perfused; whole brains were homogenized in PBS via passage through a 100 μm mesh filter, myelin debris were removed using magnetic separation with Myelin Removal Beads (Miltenyi Biotec, San Diego, CA), and subsequently CD11b positive enrichment performed similarly, via Microglia Microbeads (Miltenyi Biotec), according to manufacturer’s instructions. Cells were immediately lysed in Trizol and RNA extracted with the DirectZol RNA extraction (Zymo Research). cDNA and qPCR performed as described above with primers- gapdh: 5’-CATGGCCTTCCGTGTTCCTA-3’ and 5’-CCTGCTTCACCACCTTCTTGAT-3’; il6: 5’-TAGTCCTTCCTACCCCAATTTCC-3’ and 5’-TTGGTCCTTAGCCACTCCTTC-3’, tyrosine hydroxylase: 5’-CCAAGGTTCATTGGACGGC-3’ and 5’-CTCTCCTCGAATACCACAGCC-3’; and tnfa; 5’-CCCTCACACTCAGATCATCTTCT-3’ and 5’-gctacgacagtgggctacag-3’. Synuclein imaging- Perfused whole brains were dissected and fixed with 4% (w/v) paraformaldehyde. 50 μm sections were generated via vibratome. For proteinase-K resistant staining, free-floating sections were treated briefly with 5 μg/mL proteinase K (NEB, Ipswich, MA) and stained with anti-alpha synuclein (1:1000 mouse; #610787 BD, Franklin Lakes, NJ), pS129-synuclein staining utilized anti-pS129syn (1:1000 rabbit; #ab51253 AbCam, Cambridge, UK) and anti-tyrosine hydroxylase (1:1000 mouse; MAB318, Millipore, Burlington, MA). Sections were stained with secondary anti-mouse IgG-AF488 (1:1000, Life Technologies, Carlsbad, CA), anti-rabbit IgG-546 (1:1000, Life Technologies) and Neurotrace (Life Technologies), mounted with ProFade Diamond DAPI (Life Technologies) and imaged with a 20x objective on a Zeiss LSM800 confocal microscope. Sections corresponding to ~1500 μm from midline were counted manually for TH+ cell bodies and pS129syn puncta in ImageJ software. Microglia reconstructions- Microglia were imaged and analyzed as previously described (Erny et al., 2015; Sampson et al., 2016). Thin sections prepared as above were stained with anti-Iba1 (1:1000 rabbit, Wako, Richmond, VA) and anti-rabbit IgG-AF546 (1:1000, Life Technologies). Z-stacks were imaged at 1 μm steps and analyzed using Imaris software. ELISAs- For TNFα and IL6 (eBioscience, San Diego, CA), αSyn (ThermoFisher), and dopamine (Eagle Biosciences, Nashua, NH) tissue homogenates were prepared in RIPA buffer containing protease inhibitor (ThermoFisher), and ELISA performed according to manufacturer’s instructions with 100 μg of tissue. Multiplexed intestine (100 μg) and serum cytokine levels were measured on a Bio-Plex 200 using the Bio-Plex Mouse Cytokine 23-plex Assay (Biorad) according to manufacturer’s instructions. Dot blot- Tissue homogenates were spotted onto 0.45 μm nitrocellulose membranes, in 1 μg/μL aliquots. Membranes were blocked with 5% dry skim milk in Tris-buffered saline and stained with anti-aggregated alpha-synuclein antibody (1:1000, MJFR-14-6-4-2, rabbit, AbCam) and anti-rabbit IgG-HRP (1:1000, Cell Signaling Technology, Danvers, MA), and detected with Clarity chemiluminescence substrate (BioRad) on a BioRad GelDoc XR and densitometry performed.

Intestinal injections

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Synthetic hexapeptides of CsgA (Nterm-QYGGNN-Cterm) and the non-amyloidogenic mutant N122A (Nterm-QYGGNA-Cterm) were purchased from Bio-Synthesis Inc (Lewisville, TX). Mice were anesthetized with isoflurane (1–4% vol/vol) and kept on a self-regulating heating pad throughout the procedure. A 10 µl Hamilton syringe fitted with a 36-gauge beveled needle was loaded with 30 µg of either hexapeptide in saline (9 µl total). Each animal was injected in three locations (both sides of pyloric antrum and duodenum [0.5 cm past the pyloric sphincter]; 3 µl each location) by inserting the needle tip bevel facing up into the intestine wall at a 15° angle. After injection, the abdominal muscle/peritoneal layer and skin were sutured separately, then mice returned to home cages. Mice were injected subcutaneously with buprenorphine SR (1 mg/kg) and monitored for 3 days for normal food and water consumption.

Statistics and data availability

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Data were analyzed in GraphPad Prism, with the analysis indicated in each figure legend. Statistical output and numerical source data presented within the manuscript are available within the supplementary information included with this manuscript.

References

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Decision letter

  1. Isaac M Chiu
    Reviewing Editor; Harvard Medical School, United States
  2. Wendy S Garrett
    Senior Editor; Harvard T.H. Chan School of Public Health, United States
  3. Michel Desjardins
    Reviewer

In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.

Acceptance summary:

This is an exciting study demonstrating how gut bacteria and associated curli proteins can promote α-synuclein aggregation and pathology both in the gut and in the brain that lead to behavioral deficits in mice. There is increasing interest in how the gut microbiome and pathogens can influence the spread and development of synucleinopathies such as Parkinson's disease. This study opens up questions of whether dysregulation of bacterial strains that produce curli or other similar proteins are associated with synucleinopathies in humans. Thank you for contributing your study.

Decision letter after peer review:

Thank you for submitting your article "A gut bacterial amyloid promotes α-synuclein aggregation and motor impairment in mice" for consideration by eLife. Your article has been reviewed by three peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the evaluation has been overseen by a Reviewing Editor and Wendy Garrett as the Senior Editor. The following individuals involved in review of your submission have agreed to reveal their identity: Michel Desjardins (Reviewer #3).

The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.

Summary:

In this manuscript, Sampson et al. present evidence that bacteria in the gut can lead to the emergence of motor impairment in a PD-susceptible mouse model through the action of a bacterial amyloid protein. They examined whether a bacterially produced amyloid protein, curli, could contribute to the aggregation of mammalian αSyn, leading to PD-related neurological dysfunction. The manuscript is well written and concise. The authors used a "reductionist" approach and demonstrated that amyloid-producing bacteria induce PD-like symptoms in α-synuclein overexpressing mice (ASO). They then went on to show that the curli proteins (bacterial amyloid) has a similar effect when introduced in the gut. These data further support the emerging concept that peripheral events in the gut contributes to the pathophysiological process leading to PD. The study is well focused and built around complimentary sets of experiments with microorganisms and their protein products. Behavioral analyses, as well as characterization of the modification of dopaminergic neurons in the brain are provided. Their data indicates that motor impairment can occur without a significant loss of TH neurons in the substantia nigra but a decrease of dopamine in the striatum (as shown in a recent article by Matheoud et al., 2019). Finally, they used epigallocatechin gallate (EGCG) to block curli's ability to form biofilms and found that it blocked CsgA- accelerated αSyn formation.

All reviewers were overall enthusiastic about the findings presented in this study. However, they agree that it would be important for the authors to show that the motor impairment in their model can be reversed by L-DOPA treatment. Better clarity also about presented data (immunoblots, ENS analysis) would strengthen the manuscript.

Essential revisions:

1) If the behavioral findings are due to exacerbation of PD-like motor phenotype as the authors propose, then the authors should be able provide behavioral data showing that L-DOPA alleviates or reduces the behavioral abnormalities.

2) There seems to be a paucity of pathologic αSyn in TH neurons and the biochemical evidence is weak. It would help if the authors showed their full-length immunoblots of αSyn from the soluble versus insoluble fractions.

3) For neuropathology in the gut, can there be quantitative analysis of enteric neurons? The myenteric plexus was stained for α-synuclein and PGP9.5, but the numbers of enteric neurons were not quantified. Is there loss of neurons in the proximal large intestine?

4) A better explanation of how curli exacerbates the motor phenotype would help. Is it through enhancing inflammation, affecting DA metabolism or affecting other areas that express high levels of α-synuclein in these transgenic mice? Is there a correlation between aggregate burden or inflammation and behavior?

[Editors' note: further revisions were suggested prior to acceptance, as described below.]

Thank you for resubmitting your work entitled "A gut bacterial amyloid promotes α-synuclein aggregation and motor impairment in mice" for further consideration by eLife. Your revised article has been evaluated by Wendy Garrett (Senior Editor) and a Reviewing Editor.

The manuscript has been improved but there are some remaining issues that need to be addressed before acceptance, as outlined below:

All reviewers agree that improvements have been made including additional data for quantification of neurons in the gut myenteric plexus, and revisions to the text.

However, there is a major point that must be addressed with necessary revisions to the text to tone down the relevance of their animal model and findings in relation to PD given the inability to reverse the phenotype with L-DOPA in this model (Point #1). An additional point is raised concerning the full immunoblot (Point #2).

1) The authors cited prior published work that the behavioral deficits in the Thy1-αSyn mice are not responsive to L-DOPA and as such argue that this experiment will not provide useful information. While we agree with the authors that in light of these prior published works, L-DOPA experiments are not likely to provide useful information. However, the fact that L-DOPA does not have an effect on the Thy1-αSyn mice indicates that the authors' claims that behavioral findings are due to exacerbation of PD-like motor phenotypes is specious. Since L-DOPA does not rescue the phenotype, while we understand their arguments, this animal model is not reflective of PD. Yet the authors continue to make statements throughout the revised paper that the motor deficits are consistent with the hallmarks of PD. For instance, the Abstract states "Escherichia coli exacerbates hallmark disease features such as motor impairment and αSyn pathology in the gut and brain."

The authors need to therefore take a more balanced approach and present the Thy1-αSyn mice as a model of αSyn pathophysiology with accompanying behavior deficits and they should not make claims regarding motor impairment and the link to PD. In other words, the Thy1-αSyn mice represent a model of αSyn induced behavioral abnormalities only and claims regarding PD should be removed from the paper.

2) We could not easily find the requested full immunoblot of soluble vs. insoluble α-synuclein fractionations. It's currently in an embedded tab of an excel file: is there a way to post this on the actual figure instead of as a source data file (Figure 1—figure supplement 2D)?

https://doi.org/10.7554/eLife.53111.sa1

Author response

Essential revisions:

1) If the behavioral findings are due to exacerbation of PD-like motor phenotype as the authors propose, then the authors should be able provide behavioral data showing that L-DOPA alleviates or reduces the behavioral abnormalities.

In the Thy1-αSyn mouse model, loss of dopamine production and degeneration of dopaminergic neurons occurs at 14 months of age (Chesselet et al., 2012), which is significantly later than the appearance of motor dysfunction (2-4 months of age; Fleming et al., 2004), neuroinflammation (1 month of age; Watson et al., 2012), and non-motor symptoms (including gastrointestinal motility at 2-3 months of age; Wang et al., 2012). As described in our study and previous reports (Chesselet et al., 2012; Lam et al., 2011), there is no notable loss of striatal dopamine (Figure 1—figure supplement 2Q) or loss of tyrosine hydroxylase expression (Figure 1—figure supplement 2R) at the time points used throughout our work. Therefore, our data are in complete agreement with published literature for a lack of dopamine dependence at early stages of disease, strongly suggest that dopamine replacement therapy would not have benefits in mice at the ages tested. In fact, this experiment has been performed in SPF mice, which are somewhat different than our colonization paradigms, showing that L-DOPA treatment paradoxically results in slightly increased motor impairment in the Thy1-αSyn mouse model at early ages (Fleming et al., 2006). The reason for this previous observation still remains unknown.

While we are technically able to perform the L-DOPA treatment studies and had previously considered them, the experiment would take over 14 months to complete and we feel it would not provide useful information (based on the published data referenced above and our observations in Figure 1—figure supplement 2Q, R). Accordingly, we make no claims about the role dopamine on motor phenotypes, as the focus of our study is identification of a bacterial pathway that can promote αSyn aggregation and pathophysiology in Thy1-αSyn mice, rather than a better understanding of the mouse model itself.

Aggregation of αSyn is central to both Parkinson’s disease and the pathophysiology of this mouse model. Prior studies have modulated αSyn aggregation through small molecules in this mouse model and have demonstrated motor phenotypes can be limited when αSyn is inhibited (Richter et al., 2017; Wrasidlo et al., 2016), or enhanced when αSyn aggregation is accelerated in vivo (Pokrzywa et al., 2017). Exploring the molecular pathways into how aggregation leads to motor phenotypes would require a separate dedicated study.

We hope the referees and editors agree that the investment in time and effort for L-DOPA treatment experiments outweigh their expected value to the current study. We have done our best to not over-interpret our findings and have restricted discussion to results directly supported by data, namely the discovery that curli from the gut microbiome are sufficient to induce αSyn aggregation and motor phenotypes in the widely-used Thy1-αSyn model of synucleinopathy. In future studies, we plan to test whether curli play a role in other mouse models of disease.

2) There seems to be a paucity of pathologic αSyn in TH neurons and the biochemical evidence is weak. It would help if the authors showed their full-length immunoblots of αSyn from the soluble versus insoluble fractions.

We have included the requested full immunoblot of soluble vs. insoluble α-synuclein fractionations (Figure 1—figure supplement 2D) in Figure 1—source data 1. Thank you for this suggestion.

3) For neuropathology in the gut, can there be quantitative analysis of enteric neurons? The myenteric plexus was stained for α-synuclein and PGP9.5, but the numbers of enteric neurons were not quantified. Is there loss of neurons in the proximal large intestine?

This has been previously investigated, and the Thy1-αSyn mouse does not show degeneration of any neurons in the myenteric plexus (Wang et al., 2012). Similarly, in our hands, we do not observe striking differences in the number of neurons per ganglion in the myenteric plexus under these colonization conditions (new Figure 1—figure supplement 2M). We thank the referees for this suggestion.

4) A better explanation of how curli exacerbates the motor phenotype would help. Is it through enhancing inflammation or affecting DA metabolism or affecting other areas that express high levels of α-synuclein in these transgenic mice? Is there a correlation between aggregate burden or inflammation and behavior?

This is indeed a critical point and we have addressed the various ways curli may impact motor symptoms in the manuscript (through modulation of inflammation and/or through acceleration of αSyn aggregation), though disentangling the pathways downstream of curli-mediated αSyn aggregation is not trivial. It is important to consider that αSyn aggregates activate the same innate immune receptors as CsgA (Kim et al., 2013; Tukel et al., 2005), and that inflammation can increase the propensity of αSyn to aggregate (reviewed in Lema Tome et al., 2013). Therefore, αSyn amyloid formation and inflammatory processes are intrinsically linked, and likely both involved in the disease process in ways that are currently unknown for any Syn-based animal model.

We have carefully revised the text in the discussion to unambiguously state that our results do not distinguish between inflammatory or amyloid-mediated outcomes. Further, we are actively addressing this topic by generating new mouse lines lacking specific inflammatory, αSyn aggregation or dopamine pathways to explore how curli exacerbate motor phenotypes.

[Editors' note: further revisions were suggested prior to acceptance, as described below.]

[…]

However, there is a major point that must be addressed with necessary revisions to the text to tone down the relevance of their animal model and findings in relation to PD given the inability to reverse the phenotype with L-DOPA in this model (Point #1). An additional point is raised concerning the full immunoblot (Point #2). Please read below:

1) The authors cited prior published work that the behavioral deficits in the Thy1-αSyn mice are not responsive to L-DOPA and as such argue that this experiment will not provide useful information. While we agree with the authors that in light of these prior published works, L-DOPA experiments are not likely to provide useful information. However, the fact that L-DOPA does not have an effect on the Thy1-αSyn mice indicates that the authors' claims that behavioral findings are due to exacerbation of PD-like motor phenotypes is specious. Since L-DOPA does not rescue the phenotype, while we understand their arguments, this animal model is not reflective of PD. Yet the authors continue to make statements throughout the revised paper that the motor deficits are consistent with the hallmarks of PD. For instance, the Abstract states "Escherichia coli exacerbates hallmark disease features such as motor impairment and αSyn pathology in the gut and brain." The authors need to therefore take a more balanced approach and present the Thy1-αSyn mice as a model of αSyn pathophysiology with accompanying behavior deficits and they should not make claims regarding motor impairment and the link to PD. In other words, the Thy1-αSyn mice represent a model of αSyn induced behavioral abnormalities only and claims regarding PD should be removed from the paper.

We appreciate the comments and agree that eliminating any extrapolation from the mouse model to Parkinson’s disease is appropriate, and have done so. However, it is important to consider that while L-DOPA responsiveness is a necessary component of PD, other pathological processes are occurring prior to- and concurrent with- loss of dopamine neurons (e.g., αSynaccumulation, mitochondrial dysfunction, impaired proteostasis). Thy1-αSynand other mouse models allow exploration into other synucleinopathy-associated pathologies.

Based on the editorial feedback, we have carefully revised the manuscript to present the mouse model in a more balanced fashion. Specifically, we have removed almost all mention of PD in the introduction and Results sections, and importantly no longer refer to the characteristics of Thy1-αSynmice as “PD-like”. Text previously focused on PD is now more broadly framed in the context of synucleinopathy and we discuss the mouse model as being relevant for understanding physiological impact of αSynaccumulation. The few instances where we retain mention of PD reflects the fact that there is simply much more research on PD and the gut microbiome than other related disorders. Accordingly, most if not all use of “PD” is confined to referencing published literature, mostly in the Discussion section, while being careful to not give the impression that we are modeling PD in our study.

2) We could not easily find the requested full immunoblot of soluble vs. insoluble α-synuclein fractionations. It's currently in an embedded tab of an excel file: is there a way to post this on the actual figure instead of as a source data file (Figure 1—figure supplement 2D)?

We have added text to the figure legend to state explicitly that full length immunoblots are available in the associated source data file, as it would take considerable effort to reformat an existing figure. If the manuscript is accepted, the source data files are easily accessed with a hyperlink for each figure in the formatted text.

https://doi.org/10.7554/eLife.53111.sa2

Article and author information

Author details

  1. Timothy R Sampson

    Division of Biology & Biological Engineering, California Institute of Technology, Pasadena, United States
    Present address
    Department of Physiology, Emory University School of Medicine, Atlanta, United States
    Contribution
    Conceptualization, Investigation, Visualization
    For correspondence
    trsamps@emory.edu
    Competing interests
    has intellectual property pending in relationship to the content of this manuscript, US Patent App. 15/893,456 and 16/302,321.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-2486-8766
  2. Collin Challis

    Division of Biology & Biological Engineering, California Institute of Technology, Pasadena, United States
    Contribution
    Investigation, Visualization
    Contributed equally with
    Neha Jain
    Competing interests
    No competing interests declared
  3. Neha Jain

    Department of Molecular, Cellular, and Developmental Biology, University of Michigan, Ann Arbor, United States
    Present address
    Department of Bioscience and Bioengineering, Indian Institute of Technology, Jodhpur, India
    Contribution
    Investigation, Visualization
    Contributed equally with
    Collin Challis
    Competing interests
    No competing interests declared
  4. Anastasiya Moiseyenko

    Division of Biology & Biological Engineering, California Institute of Technology, Pasadena, United States
    Contribution
    Investigation, Visualization
    Competing interests
    No competing interests declared
  5. Mark S Ladinsky

    Division of Biology & Biological Engineering, California Institute of Technology, Pasadena, United States
    Contribution
    Investigation, Visualization
    Competing interests
    No competing interests declared
  6. Gauri G Shastri

    Division of Biology & Biological Engineering, California Institute of Technology, Pasadena, United States
    Contribution
    Investigation
    Competing interests
    No competing interests declared
  7. Taren Thron

    Division of Biology & Biological Engineering, California Institute of Technology, Pasadena, United States
    Contribution
    Resources, Investigation
    Competing interests
    No competing interests declared
  8. Brittany D Needham

    Division of Biology & Biological Engineering, California Institute of Technology, Pasadena, United States
    Contribution
    Investigation
    Competing interests
    No competing interests declared
  9. Istvan Horvath

    Department of Biology and Biological Engineering, Chalmers University of Technology, Gothenburg, Sweden
    Contribution
    Investigation, Visualization
    Competing interests
    No competing interests declared
  10. Justine W Debelius

    Department of Pediatrics, University of California, San Diego, San Diego, United States
    Present address
    Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Stockholm, Sweden
    Contribution
    Formal analysis
    Competing interests
    No competing interests declared
  11. Stefan Janssen

    Department of Pediatrics, University of California, San Diego, San Diego, United States
    Present address
    Algorithmic Bioinformatics, Justus-Liebig-University Giessen, Giessen, Germany
    Contribution
    Formal analysis
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-0955-0589
  12. Rob Knight

    1. Department of Pediatrics, University of California, San Diego, San Diego, United States
    2. Department of Computer Science and Engineering, University of California, San Diego, San Diego, United States
    Contribution
    Supervision
    Competing interests
    No competing interests declared
  13. Pernilla Wittung-Stafshede

    Department of Biology and Biological Engineering, Chalmers University of Technology, Gothenburg, Sweden
    Contribution
    Conceptualization, Supervision
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-1058-1964
  14. Viviana Gradinaru

    Division of Biology & Biological Engineering, California Institute of Technology, Pasadena, United States
    Contribution
    Supervision
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-5868-348X
  15. Matthew Chapman

    Department of Molecular, Cellular, and Developmental Biology, University of Michigan, Ann Arbor, United States
    Contribution
    Conceptualization, Supervision
    Competing interests
    a member of the Scientific Advisory Board of Axial Biotherapeutics.
  16. Sarkis K Mazmanian

    Division of Biology & Biological Engineering, California Institute of Technology, Pasadena, United States
    Contribution
    Conceptualization, Supervision
    For correspondence
    sarkis@caltech.edu
    Competing interests
    has financial interest in Axial Biotherapeutics. Has intellectual property pending in relationship to the content of this manuscript, US Patent App. 15/893,456 and 16/302,321.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-2713-1513

Funding

Larry L. Hillblom Foundation

  • Timothy R Sampson

Heritage Medical Research Institute

  • Viviana Gradinaru
  • Sarkis K Mazmanian

Knut och Alice Wallenbergs Stiftelse

  • Pernilla Wittung-Stafshede

Swedish Research Council

  • Pernilla Wittung-Stafshede

National Institute on Aging (AG054101)

  • Collin Challis

National Institute of General Medical Sciences (GM118651)

  • Matthew Chapman

National Institute of Neurological Disorders and Stroke (NS085910)

  • Sarkis K Mazmanian

Department of Defense (PD160030)

  • Sarkis K Mazmanian

Axial Biotherapeutics

  • Sarkis K Mazmanian

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Acknowledgements

We thank Drs. E Hsiao, M Sampson, S Campbell, D Yamashita, D Donabedian, and members of the SKM laboratory for helpful critiques and review of this manuscript. We are grateful to J Paramo, S Estrada, K Ly and the Caltech OLAR staff for animal care husbandry, and Y Garcia-Flores (Caltech) for technical support. Electron micrographs of amyloids were generated at the Microscopy and Imaging Analysis Laboratory Core at the University of Michigan. Fluorescent imaging and image analysis were performed in the Caltech Biological Imaging Facility, with the support of the Caltech Beckman Institute and the Arnold and Mabel Beckman Foundation. TRS was a Larry L Hillblom Foundation postdoctoral fellow. This project was supported by funds from the Heritage Medical Research Institute to VG and SKM; the Knut and Alice Wallenberg Foundation and Swedish Research Council to PW-S; the National Institutes of Health grants AG054101 (CC), GM118651 (MRC) and NS085910 (SKM); Axial Biotherapeutics to SKM; and the Department of Defense grant PD160030 to SKM.

Ethics

Animal experimentation: All animal husbandry and experiments were approved by the California Institute of Technology's Institutional Animal Care and Use Committee (IACUC) under protocol #1707.

Senior Editor

  1. Wendy S Garrett, Harvard T.H. Chan School of Public Health, United States

Reviewing Editor

  1. Isaac M Chiu, Harvard Medical School, United States

Reviewer

  1. Michel Desjardins

Publication history

  1. Received: October 28, 2019
  2. Accepted: January 23, 2020
  3. Version of Record published: February 11, 2020 (version 1)

Copyright

© 2020, Sampson et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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