2021 Workshop: Neurodegenerative Diseases in the Gut-Brain Axis—Parkinson's Disease

Abstract

Parkinson's disease (PD) is the second most common neurodegenerative disease, after Alzheimer disease, with an estimated annual cost in the United States of $52 billion.1Marras C. Beck J.C. Bower J.H. et al.Prevalence of Parkinson’s disease across North America.NPJ Parkinsons Dis. 2018; 4: 21Crossref PubMed Scopus (424) Google Scholar It is also the fastest growing neurologic disease, estimated to reach 13,000,000 patients by 2040. For many decades, PD research focused on the brain; however, PD symptoms are not limited to motor dysfunction, and patients frequently exhibit nonmotor problems that have a significant impact on their quality of life. Notably, patients who develop PD often have a history of bowel issues, predominantly constipation and gastroparesis (slow gastric emptying). In fact, gastrointestinal (GI) symptoms may be present decades before the appearance of motor symptoms,1Marras C. Beck J.C. Bower J.H. et al.Prevalence of Parkinson’s disease across North America.NPJ Parkinsons Dis. 2018; 4: 21Crossref PubMed Scopus (424) Google Scholar, 2Braak H. Rub U. Gai W.P. et al.Idiopathic Parkinson’s disease: possible routes by which vulnerable neuronal types may be subject to neuroinvasion by an unknown pathogen.J Neural Transm (Vienna). 2003; 110: 517-536Crossref PubMed Scopus (1035) Google Scholar, 3Kline E.M. Houser M.C. Herrick M.K. et al.Genetic and environmental factors in Parkinson’s disease converge on immune function and inflammation.Mov Disord. 2021; 36: 25-36Crossref PubMed Scopus (37) Google Scholar suggesting that GI dysfunction is an early manifestation of the disease. This has provided support for the novel concept proposed by Braak et al2Braak H. Rub U. Gai W.P. et al.Idiopathic Parkinson’s disease: possible routes by which vulnerable neuronal types may be subject to neuroinvasion by an unknown pathogen.J Neural Transm (Vienna). 2003; 110: 517-536Crossref PubMed Scopus (1035) Google Scholar in 2003 that PD may originate in the gut rather than the brain and the more recent theory that the GI tract is be a major source of inflammation contributing to neurodegeneration in PD.3Kline E.M. Houser M.C. Herrick M.K. et al.Genetic and environmental factors in Parkinson’s disease converge on immune function and inflammation.Mov Disord. 2021; 36: 25-36Crossref PubMed Scopus (37) Google Scholar Thus, a better understanding of the role of the GI tract in the initiation and propagation of PD pathology could lead to the identification of GI-based targets for all stages of the disease. From September 30 through October 1, 2021, a public workshop was convened by the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), in collaboration with the National Institute of Neurological Disorders and Stroke (NINDS) and National Institute of Environmental Health Sciences (NIEHS), to identify and discuss issues related to the involvement of the GI tract in PD, including its role in the etiology and manifestation of symptoms associated with the disease. The workshop objectives included the following topics:•coordinating care of motor symptoms and nonmotor GI dysfunction in patients with PD,•evaluating gut-brain communication in neurodegenerative disorders,•addressing the gaps in knowledge regarding changes in enteric sensory processes and the structure and function of the enteric nervous system in PD,•assessing the potential of the GI tract as a source of biomarkers and novel therapeutic targets for early PD,•facilitating cross-talk among brain-gut investigators to identify collaborative opportunities, and•defining areas of common interests in brain-gut research among the NIDDK, NINDS, and NIEHS. This workshop summary includes an overview of the sessions that were in the workshop, a table (Table 1) summarizing identified gaps and potential future directions, and a figure (Figure 1) summarizing the key elements in the involvement of the gut-brain axis in PD.Table 1Summary of Breakout Group Discussions Identifying the Gaps, Opportunities, and Resources Needed to Advance the FieldGroup membersKey discussion pointsGaps/opportunitiesResources requiredGroup 1:Michael CamilleriFrank HamiltonLaren BeckerAli KeshavarzianAnthony LangMeenakshi RaoThyagarajan SubramanianThe burden of GI dysfunctione in PD1.What is the role of GI symptoms in PD pathology?2.Can the nonmotor symptoms (prodromal phase) be leveraged for PD detection?3.What is needed to increase the opportunities for data/resource sharing across foundations and international/national institutes?1.GI dysfunction in PDa.Gender/sex differences in gut symptomsb.Onset of symptoms in PD patients, covert GI dysfunctionc.Differences in prodromal (nonmotor) phase in early vs late onset of diseased.More GI testing (eg, barrier function, histopathology)e.Biomarkers for prodromal GI dysfunction/disease progression/disease severity/University of Pennsylvania Smell Identification Testf.Relationship of α-SYN to disease burdeng.IBD and PD riskh.Comorbidity with mental healthi.Gut inflammation and PD symptoms2.Establish additional and leverage existing cohorts with early-stage patients, including collaborations with international institutes3.Include consideration of health disparities and environmental impact1.Partner with pharmaceutical industry to advance promising therapeutic approaches2.Add relevant GI measures to and collect biospecimens from existing and novel PD cohorts3.Establish and leverage PD registries4.Collect longitudinal GI samples from patient cohortsGroup 2:Fredric Manfredsson Kirsteen BrowningJon HollanderJames GalliganTim GreenamyreJeffrey KordowerKara MargolisShanthi SrinivasanParkinson's disease initiation: top down or bottom up?1.Is pathology in the ENS related to the cause or the result of CNS pathology (directionality)?2.Are GI symptoms due to early brain or early gut effects?1.Hyperfocus on α-SYN—why does it aggregate in specific cells?a.Microbiome as cause or effect in PD GI dysfunctionb.l-dopa/diet/genetic effects2.Microbial products3.Autonomic dysfunction—directionalitya.Same or different pathways for direction of transmissionb.Sympathetic nervous system—sacral spinal cordc.Nigral/GI dysfunction vs motor dysfunctiond.Affected areas outside GI tract—bladder, pancreas4.Lymphatics/liver—role in route of transmission of α-SYN5.Visceral pain6.Animal modelsa.Emergence of nonmotor symptomsb.Validity and applicability to humans1.NHP availability2.Validity of animal models3.Improved communication across siloed research fieldsGroup 3:Gary MaweTerez Shea-DonohueArt BeyderIssac ChiuBrain GulbransenBryan KillingerMalu TanseyLaura Volpicelli-DaleyMechanisms of gut dysfunction related to the ENS1.How does gut dysfunction in PD inform us of possible changes in the ENS and ENS circuitry?2.What are the potential mechanisms of these changes?3.Does PD affect gut sensation?4.What other cell types could be involved (eg, glia, immune cells, ICC)?1.α-SYN in the ENSa.Physiologic functionb.Cell types (neurons, glia, epithelial cells, EECs, SIP)i.Markers for glial subtypesii.Specific changes in subtypesiii.Communication with CNSc.Processes in folding, misfolding, and aggregation and effects on cell functiond.Aging2.ENS and ENS circuitry dysfunctiona.Determine whether and where ENS cell loss (neurodegeneration) occurs in PDb.Dopaminergic neuron function—physiologyc.Nitrergic/cholinergic (other) susceptibility in PDd.Sensatione.Neuroimmune interactionsf.Cell types involved (immune and other epithelial cells that have immune function)g.Neurogenic inflammation—calprotectin1.More translational animal models2.Validated biomarkers3.Links between animal and human pathologyGroup 4:John WileyPatricia GreenwelFaranak FattahiMadhu GroverPurna KashyapRoger LiddleEamonn QuigleyLorenz StuderDiagnostic and therapeutic potential of the gut in Parkinson's disease1.What data bases, biospecimens or other are needed to be in place to allow patient screening/diagnosis/phenotyping or to validate future cellular biomarkers?2.What would be needed for cellular and clinical phenotypes that would facilitate early diagnosis and/or stratification of PD subpopulations?3.What’s the future for GI-focused interventions?4.Are there potential initial GI relevant targets?1.Improved databasesa.Longitudinal samples relevant to both neurology and GIb.Improved coordination of care of motor and nonmotor manifestations, particularly recognition of prodromal GI symptomsc.Standardizations of protocols for sample collection/development of patient questionnairesd.Comparison of PD patients with and without GI symptoms2.Multidisciplinary approach (multiomics)—consortia3.Use of AI approaches4.PD therapy effects on gut function/microbiome/inflammation5.Identified therapeutic targetsa.Barrier functionb.Microbiome6.Potential gut-related therapeuticsa.Vagal nerve stimulationb.Probioticsc.Immunomodulation (TNF)d.Enteric α-SYNe.Prokinetics1.Improved animal- and cell-based models2.Access to PD patients with well characterized GI symptoms3.Foster team-based approaches using expertise at PD centers and through global collaborationNOTE. Bolded names indicate breakout group leaders and facilitators.AI, artificial intelligence; IBD, inflammatory bowel disease; ICC, interstitial cells of Cajal; NHP, non-human primate; SIP, smooth muscle–ICC–PDGFRα+ cells; TNF, tumor necrosis factor. Open table in a new tab NOTE. Bolded names indicate breakout group leaders and facilitators. AI, artificial intelligence; IBD, inflammatory bowel disease; ICC, interstitial cells of Cajal; NHP, non-human primate; SIP, smooth muscle–ICC–PDGFRα+ cells; TNF, tumor necrosis factor. The objective of this session was to review the epidemiology and risk factors of PD and its GI manifestations, as well as the involvement of the enteric nervous system (ENS) in PD. Although the underlying cause for a vast majority of cases is unknown, several environmental (such as pesticide exposure and rural living) and genetic factors can contribute to the development of PD. Epidemiologic studies have found an association of PD in humans with pesticide exposure, including the neurotoxins paraquat and rotenone,4Tanner C.M. Kamel F. Ross G.W. et al.Rotenone, paraquat, and Parkinson’s disease.Environ Health Perspect. 2011; 119: 866-872Crossref PubMed Scopus (890) Google Scholar both of which are absorbed by the gut and involve the dopamine transporter and the organic cation transporter-35Rappold P.M. Cui M. Chesser A.S. et al.Paraquat neurotoxicity is mediated by the dopamine transporter and organic cation transporter-3.Proc Natl Acad Sci U S A. 2011; 108: 20766-20771Crossref PubMed Scopus (134) Google Scholar on nerves and immune cells. Evident also is the socioeconomic impact of GI manifestations of PD, including social embarrassment, emotional distress, decreased quality of life of both patients and their caregivers, and significant economic burden in the form of direct medical costs as well as indirect and nonmedical costs. GI manifestations constitute important nonmotor, autonomic, or sensory symptoms that may also be prodromal and may facilitate early diagnosis. Retrospective studies have documented the occurrence of GI manifestations before motor symptoms, as summarized in Table 2. Prodromal GI symptoms may reflect important nonmotor, autonomic, or sensory changes and could facilitate early diagnosis. Dysfunctions of autonomic nerves, including the ENS, are key in the control of GI manifestations, and toxicant-mediated deficits in enteric neurons are reported in animal models of PD.6Natale G. Kastsiushenka O. Fulceri F. et al.MPTP-induced parkinsonism extends to a subclass of TH-positive neurons in the gut.Brain Res. 2010; 1355: 195-206Crossref PubMed Scopus (65) Google Scholar An extensive review documenting diverse, though not always replicated, abnormalities in the human ENS in PD has also been published recently.7Natale G. Ryskalin L. Morucci G. et al.The baseline structure of the enteric nervous system and its role in Parkinson’s disease.Life (Basel). 2021; 11: 732PubMed Google ScholarTable 2Comparison of Prevalence of Gastrointestinal Symptoms in Patients With Parkinson's Disease in Different SeriesSiteSingle center in the United StatesMulticenter internationalMulticenter in ItalySingle center in ArgentinaReference number23Edwards L.L. Pfeiffer R.F. Quigley E.M. et al.Gastrointestinal symptoms in Parkinson's disease.Mov Disord. 1991; 6: 151-156Crossref PubMed Scopus (317) Google Scholar24Chaudhuri K.R. Martinez-Martin P. Schapira A.H. et al.International multicenter pilot study of the first comprehensive self-completed nonmotor symptoms questionnaire for Parkinson's disease: the NMSQuest study.Mov Disord. 2006; 21: 916-923Crossref PubMed Scopus (787) Google Scholar25Martinez-Martin P. Schapira A.H. Stocchi F. et al.Prevalence of nonmotor symptoms in Parkinson's disease in an international setting; study using nonmotor symptoms questionnaire in 545 patients.Mov Disord. 2007; 22: 1623-1629Crossref PubMed Scopus (413) Google Scholar26Barone P. Antonini A. Colosimo C. et al.The PRIAMO study: A multicenter assessment of nonmotor symptoms and their impact on quality of life in Parkinson's disease.Mov Disord. 2009; 24: 1641-1649Crossref PubMed Scopus (1020) Google Scholar27Cersosimo M.G. Raina G.B. Pecci C. et al.Gastrointestinal manifestations in Parkinson's disease: prevalence and occurrence before motor symptoms.J Neurol. 2013; 260: 1332-1338Crossref PubMed Scopus (188) Google ScholarReferenceMov Disord 1991;6:151–156Mov Disord 2006;21:916–923Mov Disord 2007;22:1623–1629Mov Disord 2009;24:1641–1649J Neurol 2013;260:1332–1338PMID205700616547944175466691951401423263478Patients/control individuals, n94/50123/96545/no control individuals1,072/no control individuals129/120Evaluation toolSurvey of GISNMS QUESTNMS QUESTSemistructured interview on NMSStructured questionnaire of GI symptomsTaste/smell, %Not assessed2628.95Not assessedNot assessedDry mouth, %Not assessedNot assessedNot assessedNot assessed57.4Drooling, dribbling, %70.23541.5231.149.6Dysphagia, swallow problems, %52.123.628.3816.120.2Nausea, vomiting, %24.48.114.319.79.3Heartburn, %NS vs control individualsNot assessedNot assessedNot assessed34.1Bloating, %NS vs control individualsNot assessedNot assessedNot assessed35.1Constipation, %28.746.752.4824.653.6Defecatory dysfunction, %65.927.6 NS29.911.461.2Fecal incontinence, %Not assessed4.9 NS8.210.8Not assessedNOTE. Non-motor symptoms (NMS) QUEST is a 30-item screening questionnaire for Parkinson's disease.GIS, gastrointestinal symptoms; NS, not significant. Open table in a new tab NOTE. Non-motor symptoms (NMS) QUEST is a 30-item screening questionnaire for Parkinson's disease. GIS, gastrointestinal symptoms; NS, not significant. Prospective follow-up studies illustrate that the severity of dysphagia, salivary drooling, and defecatory disorders parallel the progression of motor symptoms in PD (based on Hoehn and Yahr stage). Indeed, the severity of dysphagia is associated with hospitalizations and mortality in PD. Among the GI symptoms in PD, constipation has the most rapidly progressive severity and frequency over 18 months. Diagnostic tests are available to diagnose motor dysfunction of the esophagus, stomach, colon, and defecatory mechanisms. Treatments for the GI complications, which may be aggravated by anti-PD therapy, are generally suboptimal, and the GI dysfunction may affect the response to PD treatment because of altered pharmacokinetics of orally administered medications. These keynote presentations underscored the concept that several converging factors likely contribute to the development of PD and provided the background and framework for the subsequent scientific sessions. One of the most active research areas in PD focuses on the mechanisms of communication between the brain and gut. These investigations involve this interrelationship as it relates to central nervous system (CNS) pathology and neurodegeneration, as well as defining the role of peripheral vs central pathology in GI dysfunction. There is now strong support for a functional neural circuitry link between the GI tract and areas of the CNS that are typically associated with PD. For example, a direct connection between substantia nigra pars compacta (SNc) dopaminergic neurons, the dorsal motor nucleus of the vagus (DMV), and vagal enteric innervation has been demonstrated using tracing studies. Importantly, acute modulation of SNc neurons confers an immediate effect on GI function, supporting the idea that the substantia nigra (SN) (via the vagus nerve) provides an ongoing tonic drive to enhance the tone throughout the GI tract. However, it is also important to consider the bidirectionality of the gut-brain axis in the pathogenesis of PD and consideration of other neural as well as nonneural pathways. There is considerable interest in the potential spread of α-synuclein (α-SYN) pathology between the ENS to the CNS, where the GI system and olfactory tracts may serve as important conduits for the transfer of injurious substances, specifically α-SYN, the chief component of Lewy pathology in the CNS and ENS. Indeed, an intriguing epidemiologic observation is that truncal vagotomy is associated with lower population evidence of PD.8Liu B. Fang F. Pedersen N.L. et al.Vagotomy and Parkinson disease: a Swedish register-based matched-cohort study.Neurology. 2017; 88: 1996-2002Crossref PubMed Scopus (241) Google Scholar Similarly, in the systemic paraquat/lectin model, SNc degeneration, as a result of ENS α-SYN pathology, was dependent on intact vagal connectivity. Such results are consistent with the Braak hypothesis of the etiology of PD. Interestingly, in the latter example, functional inhibition of nigral projections to the ENS prevented the spread of pathology, suggesting that the mechanism(s) underlying this spread is more complex than mere anatomic connectivity. Although the role of α-SYN in neurodegeneration is unknown, alteration to α-SYN homeostasis is considered a key event in disease etiology. It is important to note, however, that α-SYN is also expressed in myenteric neurons in healthy individuals, begging the question as to what precipitates pathologic changes. Nonetheless, it was emphasized that the literature presents divergent results as it relates to the relationship between ENS and CNS pathology, where recent articles have shown very robust pathology after peripheral inoculations of pathogenic α-SYN in a mouse model,9Kim S. Kwon S.-H. Kam T.I. et al.Transneuronal propagation of pathologic α-synuclein from the gut to the brain models Parkinson’s disease.Neuron. 2019; 103: 627-641Abstract Full Text Full Text PDF PubMed Scopus (566) Google Scholar whereas other published and unpublished studies have failed to confirm these findings.10Arotcarena M.L. Dovero S. Prigent A. et al.Bidirectional gut-to-brain and brain-to-gut propagation of synucleinopathy in non-human primates.Brain. 2020; 143: 1462-1475Crossref PubMed Scopus (84) Google Scholar,11Manfredsson F.P. Luk K.C. Benskey M.J. et al.Induction of alpha-synuclein pathology in the enteric nervous system of the rat and non-human primate results in gastrointestinal dysmotility and transient CNS pathology.Neurobiol Dis. 2018; 112: 106-118Crossref PubMed Scopus (97) Google Scholar For example, a recent study from the Bezard group demonstrated putamenal α-SYN pathology after the administration of α-SYN to the gut in a nonhuman primate model, but with no detectable α-SYN pathology in the DMV, suggesting an alternate route of pathologic spread.10Arotcarena M.L. Dovero S. Prigent A. et al.Bidirectional gut-to-brain and brain-to-gut propagation of synucleinopathy in non-human primates.Brain. 2020; 143: 1462-1475Crossref PubMed Scopus (84) Google Scholar One such alternative to the “vagal highway” of disease propagation is the idea that intestinal inflammation may drive PD pathogenesis and progression via a humoral process. There is evidence for both central and peripheral inflammation in various stages of PD, with markers of inflammation (eg, tumor necrosis factor) being present in cerebral spinal fluid and blood. This concept is supported by reports that proinflammatory cytokines are detected in stool from PD patients; however, their presence in stool is not correlated with plasma makers. Thus, the overarching hypothesis describes some form of gut-environment-aging interplay that may be triggered by dysbiosis, irritable bowel syndrome, infection, or inflammation,3Kline E.M. Houser M.C. Herrick M.K. et al.Genetic and environmental factors in Parkinson’s disease converge on immune function and inflammation.Mov Disord. 2021; 36: 25-36Crossref PubMed Scopus (37) Google Scholar in which the immune system is the arbiter. With aging, the capacity of the immune system to protect dissipates and, in turn, leads to a chronic inflammatory state and innate immune dysfunction that may drive PD pathogenesis. Despite the traction of the Braak hypothesis,12Rivera L.R. Poole D.P. Thacker M. et al.The involvement of nitric oxide synthase neurons in enteric neuropathies.Neurogastroenterol Motil. 2011; 23: 980-988Crossref PubMed Scopus (123) Google Scholar the brain-gut axis is a route of bidirectional communication, and there is evidence in support of both gut-brain and brain-gut etiologies for PD. This has led to potentially defining subsets of PD based on progression (eg, brain first vs periphery first). Moreover, one must also consider the alternative threshold theory, which posits that concurrent pathologies of α-SYN in the brain and the gut, because of differing abilities of generating compensatory mechanism, can lead to varying symptomatic onsets and, thus, provide the impression of the temporal progression of disease.13Engelender S. Isacson O. The threshold theory for Parkinson’s disease.Trends Neurosci. 2017; 40: 4-14Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar There is considerable interest in the role of the ENS in the pathogenesis of PD as well as the impact of PD on the ability of the ENS to coordinate secretomotor function. In addition, there are sensory disturbances in PD, but little is known of changes in the cells and mechanisms involved in enteric sensation, which are critical to the control of gut function. Enteric neurons are chronically exposed to mechanical and chemical stressors throughout life, which may increase susceptibility to stress and damage. Although there are inconsistent findings about which enteric neurons are affected by PD and whether there is de facto ENS neurodegeneration in PD, many GI motility disorders (gastroparesis, aging, diabetes) are associated with loss of enteric nitrergic neurons and aberrations in mitochondrial structure/mitophagy and/or pyroptosis.14Yarandi S.S. Srinivasan S. Diabetic gastrointestinal motility disorders and the role of enteric nervous system: current status and future directions.Neurogastroenterol Motil. 2014; 26: 611-624Crossref PubMed Scopus (113) Google Scholar The ENS develops as neural crest cells migrate and colonize the developing gut tube. Modeling/studying enteric neural crest migration may be a suitable means by which human physiology/pathophysiology can be studied and/or therapeutic targets identified for disorders involving the ENS such as PD. Because of its ability to recapitulate many developmental aspects in culture and its amenability to high-throughput drug discovery, leveraging human pluripotent stem cell differentiation may be an efficient means by which human ENS development can be studied.15Chng S.H. Pachnis V. Enteric nervous system: lessons from neurogenesis for reverse engineering and disease modelling and treatment.Curr Opin Pharmacol. 2020; 50: 100-106Crossref PubMed Scopus (9) Google Scholar In the past decade, it has become abundantly clear that enteric glia are actively involved in the control of regulated functions of the GI tract, including motility and secretion, and they are also playing a role in pathologic conditions.16Seguella L. Gulbransen B.D. Enteric glial biology, intercellular signalling and roles in gastrointestinal disease.Nat Rev Gastroenterol Hepatol. 2021; 18: 571-587Crossref PubMed Scopus (55) Google Scholar In the intestines, brain-gut crosstalk produces inflammation that may drive microglial activation and, although little is known about enteric glial involvement in PD, valuable clues could be derived from the CNS literature. The SN, for example, has a lower abundance of astrocytes and higher abundance of microglia compared to other brain regions. In the early stages of PD, microglia are a mixed population of pro- and anti-inflammatory phenotypes, which allows sampling of the environment and homeostatic regulation. Astrocytes are mobilized/activated to help clear α-SYN and up-regulate neuroprotective pathways to preserve neurons/neuronal function. As the disease progresses, however, microglia lose their capacity for tissue defense and repair and shift to a proinflammatory neurotoxic state. As a result, astrocytes lose support, and dopaminergic neurons become more vulnerable as a form of maladaptive plasticity. Aging has also been shown to enrich PD-related genes in microglia and astrocytes, as well as alter lipid handling and lysosome function, mitochondrial health, and inflammatory responses. The GI mucosal barrier is lined by epithelial barrier composed of highly organized, multiple diverse cell types that arise from the self-sustaining stem cell pool. Patients with PD display disruptions in epithelial barrier function and more gut inflammation, and they have increased levels of proinflammatory cytokines.17van IJzendoorn S.C.D. Derkinderen P. The intestinal barrier in Parkinson’s disease: current state of knowledge.J Parkinsons Dis. 2019; 9: S323-S329Crossref PubMed Scopus (37) Google Scholar Similarly, dysbiosis and a leaky gut lead to inflammation and changes in glial fibrillary acidic protein (with a potential role for glial PD genes), causing a change in enteric glia function, leading enteric neurodegeneration/neuroplasticity/immune modulation. As described, disruptions in the glial cell population are observed in the CNS, and if this is also occurring in the gut, it could contribute to the disruptions in barrier function. The peripheral trigger of ENS PD pathology may originate via entry of a pathogen that crosses the GI mucosal barrier, inducing α-SYN misfolding and aggregation. Enteroendocrine cells (EECs) could play an important role in the response to environmental toxins and, thus, act as a first falling domino in the pathogenesis of PD. EECs, which have been likened to taste buds of the GI tract, make up approximately 1% of the epithelial cell population, and they can be further subdivided by their signaling type/hormone content. They are electrically excitable, which makes them energy demanding and sensitive to environmental toxins present in the lumen; can be activated selectively by luminal contents via mechanical stimulation; and express chemoreceptors, including those that detect nutrients and bacterial products. Notably, EECs are high expressers of α-SYN, and communicate with a variety of cell types, including glial cells, other epithelial cells, immune cells, and both intrinsic and extrinsic primary afferent neurons. Killinger and Labrie18Killinger B. Labrie V. The appendix in Parkinson’s disease: from vestigial remnant to vital organ?.J Parkinsons Dis. 2019; 9: S345-S358Crossref PubMed Scopus (15) Google Scholar investigated the vermiform appendix as a possible player in the development of gut-derived PD. The appendix is a unique immune-enriched appendage that has been hypothesized to serve as an incubator of sorts for the microbiome that can recolonize the gut microbiota after major disruptions that occur in conditions such as secretory diarrhea. The appendix may be involved in disease spread through both indirect (eg, immune surveillance leading to community-based toxicity) and direct mechanisms, including the seeding and promotion of α-SYN aggregation in response to environmental insults, which then spreads to the CNS. In support of this scenario, α-SYN pathology has been identified within mucosal macrophages of the appendix, possibly indicating the accumulation of pathologic aggregates, and global inactivation or suppression of autophagy/lysosomal function within human PD appendix may enhance α-SYN pathology. Given the multiple potential etiologies of PD, the extensive bilateral connections between the gut and the brain, and the continued exposure of the GI tract to mechanical, chemical, and environmental stressors throughout life, it seems likely that the ENS plays a significant role in both gut-brain and brain-gut etiologies of PD. This section highlights potential emerging targets and treatments for PD beyond α-SYN as a biomarker and therapeutic target and historic approaches to enhance CNS dopamine levels. There is broad interest in characterizing the role of the microbiome (bacteria, fungi, protozoans, viruses, and possibly prions) and intestinal barrier dysfunction mentioned previously in the early pathophysiology of PD. Enthusiasm for this approach is based on the temporal relationship between GI and somatic symptoms. Specific areas of interest include an assessment of whether dysbiosis is present in patients newly diagnosed with PD and identifying individuals who are at increased risk of developing PD based on multiomics assessment of genetic, epigenetic, and metabolomic profiling. Key questions to be answered include the following:•determine whether PD is associated with an overall reduction in microbial diversity favoring an increase in the abundance of microbial species associated with intestinal barrier dysfunction,•assess whether activation of the gut immune system, that is, an increase in proinflammatory cytokines and/or decrease in anti-inflammatory cytokines, is present in individuals who develop PD; and•examine whether microbial dysbiosis is a consequence or driver of intestinal barrier dysfunction and activation of the gut immune system. Thus, does activation of the gut immune system precede the development of epithelial barrier dysfunction, or does dysbiosis precede the development of barrier dysfunction and activation of the gut immune system?19Bhattarai Y. Si J. Pu M. et al.Role of gut microbiota in regulating gastrointestinal dysfunction and motor symptoms in a mouse model of Parkinson’s disease.Gut Microbes. 2021; 131866974Crossref Scopus (28) Google Scholar,20Sampson T.R. Debelius J.W. Thron T. et al.Gut microbiota regulate motor deficits and neuroinflammation in a model of Parkinson’s disease.Cell. 2016; 167: 1469-1480Abstract Full Text Full Text PDF PubMed Scopus (1768) Google Scholar Potential interventions could include the use of targeted probiotics, prebiotics, and possibly fecal microbial transplantation, which could have potentially protective and/or restorative effects that promote a healthy intestinal barrier. Alternatively, supplementing the diet with specific amino acids such as l-glutamine may have beneficial anti-inflammatory and/or trophic effects on the integrity of the gut barrier. If specific proinflammatory pathways such as tumor necrosis factor-α are activated in PD, targeted interventions, including current anti-inflammatory therapies, may be useful. Although there is emerging interest in applications of cell-based and neuroprotective interventions to mitigate the effects of PD in the ENS, most novel therapies have focused on slowing the progression of or reversing CNS motor symptoms. CNS cell and gene therapy approaches aimed at protecting dopamine neurons or enhancing dopamine tone have been extensively evaluated in clinical trials. For example, fetal mesencephalic or autologous stem cell grafts or virally mediated gene therapy have been used to improve dopaminergic tone in the striatum. Conversely, gene therapy with neuroprotective compounds, such as glial cell-derived neurotrophic factor and neurturin, have also been tested in patients. Although the outcomes of such clinical trials have been mixed, they provide a backdrop for analogous approaches aimed at protecting ENS neurons and restoring their function in disease. The use of fetal tissue in the treatment of PD has well-described obstacles and concerns, including the ethical concern related to the acquisition of fetal tissue, the limited availability of such tissue, and the observation that grafted tissue from one individual shows variable rejection when implanted in the brain of another individual. These obstacles motivated interest in developing alternate approaches to generating stem cells, via somatic nuclear transfer or by using transcription factors to reprogram cells to induced pluripotent stem cells.21Kim T.W. Koo S.Y. Studer L. Pluripotent stem cell therapies for Parkinson disease: present challenges and future opportunities.Front Cell Dev Biol. 2020; 8: 729Crossref PubMed Scopus (43) Google Scholar It appears that somatic cells taken from a patient and implanted later as induced dopamine neurons (immune- matched) may fare better than nonmatched or partially matched cells. Harnessing the therapeutic potential of the transplantation of neurons or programmed induced pluripotent stem cells to treat neuropathology is a shared interest among CNS and ENS neuroscientists. Neurotrophins such as GDNF and brain-derived neurotrophic factor exert similar effects in the ENS. Challenges remain, including validating selectivity and durability of the intervention for dopaminergic neurons, particularly when translating promising animal studies to clinical trials in humans.22Chmielarz P. Saarma M. Neurotrophic factors for disease-modifying treatments of Parkinson's disease: gaps between basic science and clinical studies.Pharmacol Rep. 2020; 72: 1195-1217Crossref PubMed Scopus (19) Google Scholar In summary, although there continues to be keen interest in the development of new symptomatic treatments, disease-modifying drugs, strategies to replace or protect CNS dopamine neurons, innovative drug delivery systems, and novel surgical interventions for patients with PD, much of the discussion at this NIH workshop was devoted to the gut-brain axis and PD focused on the potential role of microbial dysbiosis in the early pathophysiology of PD, including activation of gut mucosal proinflammatory cascades and the relationship to intestinal barrier dysfunction. Improved understanding of the role of the gut-brain axis in PD may lead to gut-focused therapeutic strategies to slow progression or, possibly, reverse PD in the future. The following individuals, in addition to the coauthors, served as speakers, moderators, and/or breakout session participants: Anthony Lang, Timothy Greenamyre, James Galligan, Laura Volpicelli-Daley, Jeffrey Kordower, Malú Tansey, Faranak Fattahi, Meenakshi Rao, Bryan Killinger, Rodger Liddle, Arthur Beyder, Brian Gulbransen, Shanthi Srinivasan, Eamonn Quigley, Ali Keshavarzian, Purna Kashyap, and Lorenz Studer. The invited discussants were Laren Becker, Issac Chiu, Madhu Grover, Kara Margolis, and Tyagajaran Subramanian. BioRender was used to produce the summary figure.