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Parkinson’s disease: Are gut microbes involved?

2020; American Physiological Society; Volume: 319; Issue: 5 Linguagem: Inglês

10.1152/ajpgi.00058.2020

1522-1547

Yogesh Bhattarai, Purna Kashyap,

Botulinum Toxin and Related Neurological Disorders

ThemeMicrobiome and Host InteractionsParkinson's disease: Are gut microbes involved?Yogesh Bhattarai and Purna C. KashyapYogesh BhattaraiEnteric Neuroscience Program, Mayo Clinic, Rochester, Minnesota and Purna C. KashyapEnteric Neuroscience Program, Mayo Clinic, Rochester, MinnesotaPublished Online:20 Oct 2020https://doi.org/10.1152/ajpgi.00058.2020This is the final version - click for previous versionMoreSectionsPDF (721 KB)Download PDFDownload PDFPlus ToolsExport citationAdd to favoritesGet permissionsTrack citations ShareShare onFacebookTwitterLinkedInEmail AbstractDownload figureDownload PowerPointParkinson's disease (PD) is a common neurodegenerative disorder characterized by motor and gastrointestinal (GI) deficits. Despite its prevalence, the pathophysiology of PD is not well understood. Recent studies highlight the role of gut microbiota in neurological disorders. In this review, we summarize the potential role of gut microbiota in the pathophysiology of PD. We first describe how gut microbiota can be influenced by factors predisposing individuals to PD, such as environmental toxins, aging, and host genetics. We then highlight the effect of gut microbiota on mechanisms implicated in the pathophysiology of PD, including disrupted microbiota gut brain axis (GBA), barrier dysfunction, and immune dysfunction. It is too early to connect the dots between gut microbiota and PD to establish causation, and experiments focused on investigating interrelationship between gut microbiota and associated metabolites on GBA, barrier dysfunction, and immune activation will be crucial to fill in the gaps.INTRODUCTIONParkinson's disease (PD) is a commonly occurring progressive neurodegenerative disorder characterized by both motor (16) and nonmotor deficits, including olfactory deficits, cognitive decline, and gastrointestinal (GI) deficits (134). GI dysfunction is a prevalent nonmotor deficit in PD, which affects up to 60–80% of patient population and can be accompanied by alterations in the gut microbiota (1, 82, 102). The known role of gut microbiota alterations in GI dysfunction along with alterations described in PD patients has brought to attention the potential role of gut microbiota in pathophysiology of PD (1, 23, 41, 50). There is now accumulating evidence that links gut microbiota to central nervous system (CNS) diseases such as PD (17, 95, 123). In this review, we have examined in detail, the available evidence of GI dysfunctions and gut microbiota alterations in PD patients. Upon examining these studies, we believe that microbiota plays a central role in etiology of PD, primarily through mechanism that involve modulation of GI epithelial barrier integrity, immune function, and the microbiota gut-brain axis, which will be described in detail in the later part of the review (84, 108).GI DYSFUNCTION IS PREVALENT IN PD PATIENTSGI dysfunctions including abnormal salivation, nausea, dysphagia, altered gastric emptying, constipation, and defecatory dysfunction, are ubiquitous in PD patients (31). These GI abnormalities are a common nonmotor symptom of PD and may precede the onset of motor symptoms by several years, adding significantly to the healthcare burden and disrupted quality of life in PD patients (1, 82, 102, 119). The high prevalence (∼80%) and early onset of these GI dysfunctions in PD certainly raises a question regarding the role of GI tract in the pathogenesis of PD. In a large population-based study of more than 6,000 patients without PD that enrolled in the "Honolulu Heart Program," Abott et al. (1) found that patients who suffered from constipation (i.e., <1 bowel movement/day) had fourfold greater risk of developing PD in the future. Subsequent autopsy on 245 of these constipated subjects, with no clinical signs of parkinsonism and dementia showed higher incidental Lewy body (α-synuclein aggregates) formation in the substantia nigra, suggesting a possible link between delayed GI transit and PD (2).In addition to GI symptoms, physiological defects, such as disrupted intestinal permeability and altered immune activation, have also been observed in PD (see Fig. 1). Clairembault et al. (29) reported nearly 50% lower occludin expression in lysates of colon biopsies of PD patients, and Perez Pardo et al. (98) observed a reduction in the average intensity of ZO-1 immunolabeling in sigmoid colon biopsies from PD patients. Together, these studies indicate potential disruption of mechanisms that regulate epithelial paracellular permeability in PD patients. Schwiertz et al. (113) observed a simultaneous increase in markers of intestinal inflammation (fecal calprotectin) and disruption of the intestinal barrier (fecal zonulin and α-1-antitrypsin) in PD patients compared with age-matched controls. Since disruption in intestinal barrier function and intestinal inflammation are characteristics feature of inflammatory bowel disease (IBD), it is not surprising that three separate studies by Lin et al. (75), Weimers et al. (132), and Villumsen et al. (128) in distinct population cohorts all show an association between IBD and increased risk of PD later in life. Although it is tempting to speculate on the basis of these studies that risk of PD would be lower in patients that undergo colectomy, paucity of clinical data in IBD patients and controversial results in patients with other GI conditions make it difficult to evaluate whether this is, indeed, the case. Recently published studies that evaluated risk of PD in patients who underwent surgical procedures, such as colectomy and appendectomy for various GI conditions, including malignant neoplasms, benign tumors, noninfective inflammation, or disease of the appendix, found that the risk of developing PD increased in some cases, while decreased in others (66, 87, 120). These contradictory findings suggest that specific modulation of colonic factors, rather than full thickness colonic resection, is necessary to decrease risk of PD. Further studies are required to assess the role of colectomy in IBD patients and determine how colonic factors, such as microbiota, barrier function, and intestinal inflammation precisely play a role in PD pathophysiology. This will help us understand whether increased occurrence of GI-related physiological defects is a potential risk factor for PD or simply PD-related pathologies that precede motor symptoms.Fig. 1.Figure highlights gut microbiota-associated mechanistic pathways that regulate gastrointestinal and motor dysfunction in Parkinson's disease. Environmental toxins consumption alters gut microbiota composition and leads to disruption in epithelial barrier (1), alteration in immune activation (2), and disrupted microbiota-gut-brain axis communication through vagal and nonvagal pathways (3) to cause gastrointestinal and motor dysfunction in Parkinson's disease. TLRs, Toll-like receptors; ZO-1, zona occludins 1.Download figureDownload PowerPointGUT MICROBIOTA AND ITS METABOLITES ARE ALTERED IN PD PATIENTSSeveral studies have examined alteration in gut microbiota composition in PD patients, but as observed in other disease states, the results are heterogenous in terms of differences in specific taxa (Table 1). There are several factors that contribute to such variability, including lack of standardization in sample collection and sequencing techniques (V3-V4, V4, or V4 and V5), differences in study design, sample size, geographical diversity of patient population, and heterogeneous nature of PD (73, 103).Table 1. Gut microbiota alterations in Parkinson's diseaseStudy NumberStudy (Ref.)ParticipantsSample UsedDetection MethodSequence RegionOverall Changes (α/β Diversity)Increased in PDDecreased in PDGI Deficits?1.Aho et al. (2019) (5)Control: 64PD: 64Stool16S rRNAV3-V4α diversity no changeβ diversity no changeBifidobacteriumPrevotella (G), Roseburia (G),2.Bedarf et al. (2017) (12)Control: 28PD: 31StoolMetagenomic shotgun analysisIllumina Hiseq4000α diversity no changeβ diversity differentVerrucomicrobiaceae (F), Firmicutes (F), Akkermansia (G)Prevotellaceae (F), Erysipelotrichaceae (F), Prevotella (G), Eubacterium (G)No constipation3.Hasegawa et al. (2015) (49)Control: 36PD: 52Stool16S or 23S rRNA(qRT-PCR)Lactobacillus (G)Species: Clostridium coccoides group, Clostridium leptum subgroup, Bacteroides fragilis group4.Heintz-Buschart et al. (2018) (53)Control: 78PD: 76Stool and nasal wash samples16S and 18S rRNAV4α diversity no changeβ diversity differentVerrucomicrobia (P), Verrucomicrobia (O), Verrucomicrobiaceae (F), Akkermansia (G)5.Hill-Burns et al. (2017) (55)Control: 130PD: 197Stool16S rRNAα diversity no changeβ diversity differentBifidobacteriaceae (F), Lactobacillaceae (F), Tissierellaceae (F), Christensenellaceae (F), Verrucomicrobiaceae (F), Bifidobacterium (G), Lactobacillus (G), Akkermansia (G)Lachnospiraceae (F), Pasteurellaceae (F), Blautia (G), Roseburia (G), Faecalibacterium (G)6.Hopfner et al. (2017) (56)Control: 29PD: 29Stool16S rRNAV1, V2α diversity no changeβ diversity differentBarnesiellaceae (F), Enterococcaceae (F), Lactobacillaceae (F)7.Keshavarzian et al. (2015) (63)Control: 34PD: 38Sigmoidmucosal biopsies and stool sampleshigh‐throughput rRNA sequencingV4α diversity increase in PD β diversity differentBacteroidetes (P), Proteobacteria (P), Verrucomicrobia (P), Clostridiaceae (F), Oscillospira (G), Akkermansia (G)Firmicutes (P), Lachnospiraceae (F), Coprobacillaceae (F), Blautia (G), Coprococcus (G), Dorea (G), Roseburia (G)8.Li et al. (2017) (72)Control: 14PD: 24Stool16S rRNAV3, V4, V5Actinobacteria (P), Proteobacteria (P), Enterobacteriaceae (F), Streptococcaceae (F), Veillonellaceae (F), Acidaminococcus (G), Acinetobacter (G), Enterococcus (G), Escherichia-shigella (G), Megamonas (G), Megasphaera (G), proteus (G), Streptococcus (G)Bacteroidetes (P), Pasteurellaceae (F), Blautia (G), Faecalibacterium (G), Ruminococcus (G)9.Li C et al., 2019 (71)Control: 48PD: 51Stool16S rRNAV4α diversity no changereduced β-diversity in PD patientsAkkermansiaLactobacillus10.Lin et al. (2018) (73)Control: 75PD: 45Stool16S rRNAV4α diversity no changeβ diversity differentEubacteriaceae (F), Bifidobacteriaceae (F), Aerococcaceae (F), Desulfovibrionaceae (F)Tenericutes (P), Euryarchaeota (P), Firmicutes (P), Streptococcaceae (F), Methylobacteriaceae (F), Comamonadaceae (F), Halomonadaceae (F), Hyphomonadaceae (F), Brucellaceae (F), Xanthomonadaceae (F), Lachnospiraceae (F), Actinomycetaceae (F), Sphingomonadaceae (F), Pasteurellaceae (F), Micrococcaceae (F), Intrasporangiaceae (F), Methanobacteriaceae (F), Idiomarinaceae (F), Brevibacteriaceae (F), Gemellaceae (F)Constipation11.Lin et al. 2019 (74)Control: 77PD: 80Stool16S rRNAV3-V4Parabacteroides, Verrucomicrobia (P), Akkermansia, Butyricimonas, Veillonella, Odoribacter, Mucispirillum, Bilophila, Enterococcus, and LactobacillusPrevotella (G)Constipation12.Petrov et al. (2017) (99)Control: 66PD: 89Stool16S rRNAV3-V4α diversity decreaseβ diversity differentChristensenella (G), Catabacter (G), Lactobacillus (G), Oscillospira (G), Bifidobacterium (G)Dorea (G), Bacteroides (G), Prevotella (G), Faecalibacterium (G)13.Qian et al. (2018) (104)Control: 45 PD: 45Stool16S rRNAV3-V4α diversity increaseβ diversity differentClostridium IV (G), Aquabacterium (G), Holdemania (G), Sphingomonas (G), Clostridium XVIII (G), Butyricicoccus (G), Anaerotruncus (G)Constipation14.Scheperjans et al. (2015) (112)Control: 72,PD: 72Stool16S rRNAV1-V3α diversity no changeβ diversity differentLactobacillaceae (F), Verrucomicrobiaceae (F), Bradyrhizobiaceae (F), Clostridiales (F), Incertae Sedis IV (F)Prevotellaceae (F)Constipation15.Unger et al. (2016) (126)Control: 34PD: 34StoolqPCR using bacterial primersEnterobacteriaceae (F), Bifidobacterium (G)Bacteroidetes (P), Prevotellaceae (F, descriptively reduced), Lactobacillaceae (F), Enterococcaceae, Species: Faecalibacterium prausnitziiConstipationPD, Parkinson's disease; qPCR, quantitative PCR.A consistent finding among studies has been an increase in members of Enterobacteriaceae family and Helicobacter spp. (1.5–3-fold increase in H. pylori in gastric biopsies) (20, 85), as well as an increase in Lactobacillus and Akkermansia (see 18, 20, 27, 43, 57, 99, 112). This is particularly interesting as an increased abundance in members of Enterobacteriaceae family and H. pylori has been associated to severity of postural instability, gait difficulty, and overall worsening of motor functions in PD (57, 107, 112). Furthermore, eradication of H. pylori has been shown to improve absorption of levodopa resulting in better response of motor function to treatment (85). Increase in members of Lactobacillaceae has been associated with reduced concentrations of ghrelin in PD; a gut brain peptide implicated in upregulation of intestinal tight junction proteins (28), stimulation of intestinal motility (115), and maintenance and protection of normal nigrostriatal dopamine function (7, 125). Akkermansia on the other hand has been shown to use mucus as a carbon source and degrade the colonic mucus barrier (42), which might disrupt intestinal permeability and increase pathogen susceptibility causing inflammatory condition on the intestinal wall (114). In addition to the above gut microbial changes, PD patients also display reduced abundance of prominent short-chain fatty acid producers. Strains of Faecalibacterium spp., Blautia spp., Prevotella spp, and Roseburia spp, that are common butyrate producers are more consistently reduced in PD patients (126). Butyrate has been shown to exert barrier protective, anti-inflammatory role in the GI tract (8, 97) and prevent dopaminergic neuron degeneration and subsequent bradykinesia in mice (78). Together, the observed changes in gut microbiota in PD and their association with motor and nonmotor deficits, suggests that altered gut microbiota may play a role in PD (27, 57, 58, 85, 112).In an attempt to identify potential link between gut microbiota and PD, we will next examine factors that contribute to alterations in gut microbiota, and then discuss how gut microbiota changes could contribute to PD.FACTORS THAT CONTRIBUTE TO GUT MICROBIOTA ALTERATIONS IN PDHost GeneticsHost genetics differences have often been linked to differences in microbiome composition (46, 143). Using next generation sequencing approach to analyze fecal bacterial composition (V4 region of 16S rDNA), Goodrich et al. (46) showed that microbiota are more similar within twin pairs compared with unrelated individuals. Among various microbial taxa, studies show that taxa particularly from Firmicutes and Proteobacteria, including members of Christensenellaceae family are the most heritable taxon in humans (25, 45, 131). Several genome-wide association studies or quantitative trait locus (QTL) mapping studies have also successfully identified specific host genes and single nucleotide polymorphisms in the host genome that contribute to the variation of these microbial taxa (21, 32, 124). These studies show that host genetics plays a significant role on determining dominant gut microbiota composition.In addition to assessing the influence of host genetics on gut microbiota, studies have also been separately conducted to assess the role of host genetics in etiology of PD. Studies suggest that PD is modestly heritable with heritability estimate of around 30% (133). Higher percentage concordance for subclinical striatal dopaminergic dysfunction in sporadic and late-onset PD suggest that host genetics particularly contribute to striatal dopaminergic dysfunction, a primary cause for PD (101). Although these studies individually show that host genetics plays an important role in regulating gut microbiota composition and the pathology of the PD, it remains unclear what precise genetic changes in PD help drive specific changes in microbiota and whether these genetic changes induced microbiota alteration drive PD phenotype.Earlier studies have successfully associated specific genetic changes in GI diseases to change in specific microbial species (45, 67, 116). However, whether such association between individual genes and gut microbiota exists in PD is not known. In a minority of patients (10–15% of clinically diagnosed cases) where PD is inheritable (Familial PD), mutation in one of the eight genes responsible for aberrant protein formation and disrupted mitochondrial homeostasis are often responsible. They include SNCA (also known as PARK1, a gene that encodes for α-synuclein, a major component of Lewy bodies), PARK2 [also known as PRKN; parkin, a ubiquitin protein ligase involved in the degradation of abnormal proteins by the proteasome (86)], PARK5 (UCHL1; ubiquitin carboxy-terminal hydrolase L1, a gene responsible for generating the ubiquitin monomer), PARK6 (PINK1; encodes for putative serine-threonine kinase), PARK7 (also known as DJ-1; encodes for protein/nucleic acid deglycase DJ-1), PARK8 (LRRK2; encodes for Leucine-rich repeat kinase 2 enzyme), PARK11 (also known as GIGYF2; encodes for GRB10-interacting GYF protein-2), and NR4A2 (encodes for nuclear receptor subfamily 4, group A, member 2 protein) (79a). Recent studies have shown that specific genetic changes in the absence of gut microbiota cannot fully recapitulate the symptom severity seen in PD (3, 110). Using a α-synuclein (SNCA), a presynaptic nerve terminal protein, overexpressing germ-free and conventionally raised specific pathogen-free mice, Sampson et al. (110) showed that germ-free α-synuclein overexpressing mice did not exhibit PD phenotype compared with conventionally raised mice. Given that exposure to bacterial endotoxin, LPS is required to trigger persistent neuroinflammation and generate structurally distinct α-synuclein fibril structure involved in synucleinopathies and neurodegeneration (37, 51, 64), it seems likely that specific genetic mutation though necessary, might not be sufficient and potentially requires gut microbiota interaction to regulate symptom severity and disease outcome in genetic model of PD.Environmental ToxinsEnvironmental toxins from different chemical classes can alter both the microbial composition and the metabolic activity of the gut bacteria (reviewed in Refs. 30 and 80). A majority of PD cases are idiopathic and likely caused by exposure to environmental toxins, including herbicides and pesticides, such as rotenone, paraquat, organic solvents, and heavy metals, including vanadium and manganese (4, 70, 122). A recent meta-analysis of data that investigated prospective cohort and case-controlled epidemiological studies found that PD was associated with farming, and the risk of developing PD was increased by exposure to environmental toxins (100). Recent studies have hypothesized that an initial target of these environmental toxin consumption/exposure in PD could be the gut microbiota (55, 137). Hill-Burns et al. (55) found that the gut bacteria responsible for degrading some of these environmental toxins, such as atrazine (herbicide), and naphthalene (insect repellent), are altered in individuals with PD compared with patients that were prescribed PDmedication. Byperforming longitudinal analysis of 16S rRNA gene sequencing of fecal microbiome, Yang et al. (137) showed that three weeks of chronic rotenone administration, at a dose found commonly in pesticides, caused fecal microbiota alterations, mitochondrial disruption, and dopaminergic neuronal loss, along with behavioral and neuropathological features of PD. A direct association between environmental toxin and PD was also shown in a case-controlled prospective study, which assessed the health effects of lifetime exposure to rotenone and paraquat in farmers and found that exposure to these pesticides increases the risk of PD by 2.5-fold (122). Together, these data suggest that common environmental toxins and pesticides lead to behavioral and neuropathological symptoms of PD, via mechanisms that potentially involve gut microbiota alterations and disruption in mitochondrial activity, a cellular organelle that is also evolutionarily a descendant of endosymbiotic bacteria (130). Characteristic microbiota changes after rotenone administration include decreased bacterial diversity and increase in Firmicutes/Bacteroidetes ratio (137), a dysbiotic condition also observed in several other diseases, including colorectal cancer (9), hypertension (136), Type 2 diabetes (T2D), obesity (68), and inflammatory bowel disease (IBD) (9). The similarity in observed microbial changes postrotenone administration with various other diseases suggests that these gut microbial changes that occur in PD are common microbial marker of various chronic diseases. This observation raises two important questions: 1) how do microbiota change after toxin exposure specifically translate to PD, as opposed to other chronic diseases, and 2) is reversing these microbial changes sufficient to reverse the course of PD pathogenesis after toxin administration?Although these studies certainly suggest that chronic microbiota changes after toxin exposure might be involved in idiopathic PD, future studies using gnotobiotic mice model is necessary to enhance our current mechanistic understanding regarding how gut microbes are able to transform/metabolize different classes of environmental toxins and xenobiotics and alter their pharmacokinetic and pharmacodynamic properties to cause PD. It is also important to carefully study the effects of environmental toxins associated with PD in both conventional and gnotobiotic germ-free animal models to understand the relationship between environmental toxin and microbiota and use this knowledge to open new avenues of treatment options for PD patients.AgingBesides, environmental toxins and genetics, aging is the most important factor that contributes to alterations in gut microbiota and PD. Recent studies have indicated clear differences in gut microbiota composition among infants, toddlers, adults, and the elderly (18, 91). Using high-throughput sequencing of the 16S rRNA gene (amplicons derived from the V3-V4 region), Odamaki et al. (91) investigated the sequential changes in fecal microbiota composition samples from 367 healthy Japanese subjects between the ages of 0 and 104 yr. They found that the transition from infant to centenarian was accompanied by distinctive coabundance group (CAG; identifies species that are phylogenetically and/or functionally related on the basis of gene abundance) dominance at different stages of life. Significant abundance of Bifidobacterium coabundance groups (CAGs) was observed in infants and children; Lachnospiraceae CAGs were observed in adults; Eubacterium and Clostridiaceae CAGs were observed in the elderly, and Enterobacteriaceae CAGs were observed in both infants and the elderly (91). In addition to Odamaki et al. (91), Biagi et al. (18) from Northern Italy also consistently observed an increase in the relative abundance of Proteobacteria, a major phylum of facultative anaerobic gram-negative bacteria, which include several members, including Escherichia, Pseudomonas, Salmonella, Vibrio, Helicobacter, Yersinia, and Legionellales in subjects that were over 70 yr of age. Although it is still not clear whether these aging-associated changes in gut microbiota increase susceptibility to PD, these studies do, however, suggest that changes in bacterial composition with age consistently favor an increase in pathogenic species with age irrespective of geographical location. Pathogenic species from Enterobacteriaceae CAGs and from Proteobacteria phylum, such as Helicobacter, have been associated with neurodegenerative diseases, including PD (summarized in review in Ref. 43). Future studies need to investigate the contribution of aging-associated factors such as dietary modification, life style changes, and weakened immune function on gut microbiota that could lead to changes in gut microbiota composition. This will help determine whether it is "normal" age-related microbial changes seen in a "healthy" individual or whether it is external factors that promote microbial changes at an older age, which increases PD susceptibility in aging individuals.MECHANISMS THAT LINK ALTERED GUT MICROBIOTA TO PDDisrupted Barrier FunctionThe GI epithelial barrier comprises a thick mucus layer aloft a monolayer of epithelial cells that are interconnected through a system of junction proteins (Fig. 1). The proper maintenance of epithelial barrier integrity is crucial to prevent translocation of pathogenic luminal bacteria and bacterial metabolites that acts as a mediator of diseases, all the while providing regulatory signals to induce immune tolerance toward commensal microbes. Studies show that gut microbes play an important role in maintaining the epithelial barrier, particularly through butyrate production, which modulates expression of mucin-associated genes in goblet cells and regulates expression and distribution of epithelial tight junction proteins (54). Thus, it is not surprising that altered gut microbial composition, as observed in various GI and CNS diseases, including IBD (44), stress-related psychiatric disorders (61), autoimmune disorders (33), and PD (109) is more often accompanied by disrupted barrier function and increased mucosal colonization of adherent and invasive, pathogenic species in many of these diseases (36, 138).Although a disrupted intestinal barrier is a common denominator implicated in various GI and CNS diseases besides PD, the specific changes in gut microbiota and the microbial pathways that give rise to disrupted intestinal permeability needs to be investigated in the specific context of a PD. Gut microbiota changes as seen in PD can disrupt epithelial permeability through one of three ways, which include, increase in microbial enterotoxin production, increased luminal endotoxin expression, and decreased butyrate production (19). Helicobacterpylori, a gram-negative pathogen, more commonly found in PD patients and aged individuals, for example, produces enterotoxin, such as the vacuolating cytotoxin (VacA), and cytotoxin-associated gene A (CagA) and directly releases them on to the host epithelium to modulate expression of tight junction proteins, including claudin-1 and ZO-1 and disrupt intestinal permeability (35, 39, 85). Additionally, increased mucosal exposure to bacterial endotoxin (lipopolysaccharide, LPS) as observed in early "Hoehn & Yahr Stage II" PD patients (36) could also cause epithelial barrier disruption by downregulating tight junction occludin and ZO-1 mRNA expression (13), and by directly inducing phosphorylation of the 20-kDa myosin light chain (MLC20) (140). Finally, loss of butyrate producing probiotic strain, as seen in PD microbiota, could lead to disruption in epithelial barrier permeability through decreased mucus production and secretion, and by impairing proper localization of tight junction proteins assembly (76, 97, 106).Although it is becoming increasingly clear that altered gut microbiota can contribute to disrupted intestinal barrier permeability in PD through the mechanisms discussed above, how this leads to PD is not well understood. Recent studies show that disrupted intestinal permeability strongly correlates to increased luminal endotoxin translocation and expression of α-synuclein aggregates in the intestine of both humans and a mouse model of PD (36, 62). This is particularly interesting given the biophysical characteristics of α-synuclein as an antimicrobial peptide within the GI tract (10, 96). Overexpression of α-synuclein as a result of microbial infection or endotoxin induced epithelial damage has also been hypothesized as potential mechanism which leads to formation of α-synuclein aggregates in the ENS and subsequent α-synuclein trafficking to the CNS (10, 62). Future studies are necessary to understand the underlying molecular mechanisms of α-synuclein aggregation after barrier dysfunction, as well as its role on gut microbiota, and test whether prevention of barrier disruption through manipulation of gut microbes is sufficient to prevent gut α-synuclein pathology and thwart PD progression.Altered Immune ActivationThe GI tract holds the largest number of immune cells in the body, which includes the intestinal epithelial cells, macrophages, dendritic cells, B cells, and regulatory T cells. Intestinal epithelial cells, in particular, not only form a physical barrier for the gut microbes but are also dynamically active immune cells (83) that express pattern recognition receptors on the epithelial surface. Subsets of intestinal epithelial cells, especially the Paneth cells and microfold (M) cells, sample the microbe-laden luminal environment by recognizing conserved bacterial motifs and participate in downstream immune regulation by producing relevant antimicrobial peptides and proinflammatory cytokines, and by delivering bacterial antigens to dendritic cells (79, 92). Gut microbial interaction with the intestinal epithelial cells is, therefore, a crucial contributor to immune development and a potent regulator of homeostatic immune response (83).A mechanistic example of gut microbiota influencing host immunity to induce PD phenotype is through elevated gram negative bacteria expressing LPS in PD patients (47), which acts on the Toll-like receptors (TLRs) present in the epithelial, immune, and nerve cells of the GI tract to modulate innate immune response (79). Dysregulation of LPS-TLR signaling has, thus, been simultaneously implicated in both intestinal inflammation and PD pathogenesis. Perez-Pardo et al. (98), using TLR4-knockout (KO) mice, observed that while rotenone causes dysbiosis in both TLR4-KO and wild-type (WT) mice, TLR4-KO mice had decreased intestinal and motor dysfunction, lower intestinal inflammation, and motor neuron degeneration relative to WT TLR4-expressing mice. These data show that peripheral immune function regulated, at least in part, b