Study protocol of the DUtch PARkinson Cohort (DUPARC):a prospective, observational study of de novo Parkinson's disease patients for the identification and validation of biomarkers for Parkinson's disease subtypes, progression and pathophysiology

BACKGROUND: Parkinson's Disease (PD) is a heterogeneous, progressive neurodegenerative disorder which is characterized by a variety of motor and non-motor symptoms. To date, no disease modifying treatment for PD exists. Here, the study protocol of the Dutch Parkinson Cohort (DUPARC) is described. DUPARC is a longitudinal cohort study aimed at deeply phenotyping de novo PD patients who are treatment-naïve at baseline, to discover and validate biomarkers for PD progression, subtypes and pathophysiology. METHODS/DESIGN: DUPARC is a prospective cohort study in which 150 de novo PD subjects will be recruited through a collaborative network of PD treating neurologists in the northern part of the Netherlands (Parkinson Platform Northern Netherlands, PPNN). Participants will receive follow-up assessments after 1 year and 3 years, with the intention of an extended follow-up with 3 year intervals. Subjects are extensively characterized to primarily assess objectives within three major domains of PD: cognition, gastrointestinal function and vision. This includes brain magnetic resonance imaging (MRI); brain cholinergic PET-imaging with fluoroethoxybenzovesamicol (FEOBV-PET); brain dopaminergic PET-imaging with fluorodopa (FDOPA-PET); detailed neuropsychological assessments, covering all cognitive domains; gut microbiome composition; intestinal wall permeability; optical coherence tomography (OCT); genotyping; motor and non-motor symptoms; overall clinical status and lifestyle factors, including a dietary assessment; storage of blood and feces for additional analyses of inflammation and metabolic parameters. Since the start of the inclusion, at the end of 2017, over 100 PD subjects with a confirmed dopaminergic deficit on FDOPA-PET have been included. DISCUSSION: DUPARC is the first study to combine data within, but not limited to, the non-motor domains of cognition, gastrointestinal function and vision in PD subjects over time. As a de novo PD cohort, with treatment naïve subjects at baseline, DUPARC provides a unique opportunity for biomarker discovery and validation without the possible confounding influences of dopaminergic medication. TRIAL REGISTRATION: NCT04180865; registered retrospectively, November 28th 2019

The role of the gut and the gastrointestinal microbiome in Parkinson’s disease

INTRODUCTION: Parkinson’s disease (PD) is a disabling and progressive neurodegenerative disorder that is increasing in prevalence with the aging and urbanisation of the global population. The mechanisms underlying PD pathogenesis and progression are incompletely understood. Improved clinical recognition of early and prodromal non-motor symptoms (NMS), namely gastrointestinal (GI) dysfunction, has focused research over the last two decades on the roles of the gut. More recently, the influences of the microbiota-gut-brain-axis (MGBA) in the development and progression of PD have become an intensive area of research. Studies have demonstrated an association between the GM and a variety of PD-related characteristics, identifying important impacts on levodopa metabolism by certain microbiota. Importantly, the effect of device-assisted therapies (DATs) on the GM and the robustness of microbiota compositional differences between PD patients and household controls (HCs) has not been well defined. The aims of this thesis were to 1) investigate GI dysfunction and nutritional patterns in PD, 2) determine if the GM is a biomarker of PD, and 3) investigate the temporal stability of the GM in PD patients receiving standard therapies and those initiating DATs. METHODS: 103 PD patients and 81 HCs were recruited and participants with PD were considered in two sub-cohorts; 1) PD patients initiating DAT; either Deep Brain Stimulation (DBS) (n=10), or levodopa-carbidopa intestinal gel (LCIG) (n=11), who had GM sampling from stool at -2, 0, 2 and 4 weeks around initiation of DAT and baseline, 6 and 12 months following DAT initiation, 2) 82 PD patients receiving standard PD therapies, who had GM sampling from stool at baseline, 6 and 12 months. Validated PD questionnaire metadata ascertaining motor characteristics and NMS, as well as nutritional data in the form of a Food Frequency Questionnaire, were collected for all participants at baseline, 6 and 12 months. Total DNA was isolated from stool before sequencing the V3-V4 region of 16S rRNA. Relative bacterial abundances, diversity measures, compositional differences and clinical-microbiome associations were determined, as well as developing predictive modelling to identify PD patients and assess disease progression. RESULTS: PD patients reported more prevalent and severe GI dysfunction, especially constipation, which was almost three-times more common compared to HC subjects, (78.6% vs 28.4%, p<0.001). PD patients had a higher intake of total carbohydrates (279 g/day vs 232 g/day; p=0.034), which was largely attributable to an increased daily sugar intake (153 g/day vs 119 g/day; p=0.003), particularly of free sugars (61 g/day vs 41 g/day; p=0.001). Significant GM compositional differences across several taxonomic levels were apparent between PD patients and HCs and associated with a number of PD motor and NMS features, as well as certain therapies. Predictive models to distinguish PD from HCs were developed considering global GM profiles, achieving an area under the curve (AUC) of 0.71, which was improved by addition of data on carbohydrate intake (AUC 0.74). Longitudinal analysis demonstrated persistent underrepresentation of known short-chain fatty acid producing bacteria in PD patients, particularly those concerned with butyrate production; Butyricicoccus, Fusicatenibacter, Lachnospiraceae ND3007 group and Erysipelotrichaceae UCG−003. Taxa differences observed over the short-term (four week) sampling period around DAT (DBS and LCIG) initiation, were not sustained at 6 and 12 months. Despite this, persistent longer-term overrepresentation of Prevotella was observed after DBS initiation, and a trend was found that was suggestive of overrepresentation of Roseburia after LCIG initiation. These results suggest that there may be variable shorter and longer-term DBS and LCIG influences on the GM, which are complex and multifactorial. PD progression analysis did not identify distinct persisting GM compositional differences between faster and slower progressing patients, although predictive modelling was strengthened by the consideration of nutritional data, specifically protein intake, and improved the predictive capacity for PD progression. CONCLUSION: This thesis demonstrates that there are numerous clinically significant associations between the gut, GM and PD. GI dysfunction is common, and carbohydrate nutritional intake appears to be different from the general population in PD. Persistent alterations of GM composition in PD compared to HCs were found. These findings provide support for the existence of disturbances of gut homeostatic pathways, which may disrupt intestinal barrier permeability and lead to gut leakiness, in the pathogenesis of PD. This thesis also highlights the potential to use the GM in the identification of PD and the characterisation of disease progression

Bacterial Butyrate in Parkinson's Disease Is Linked to Epigenetic Changes and Depressive Symptoms

Background The gut microbiome and its metabolites can impact brain health and are altered in Parkinson's disease (PD) patients. It has been recently demonstrated that PD patients have reduced fecal levels of the potent epigenetic modulator butyrate and its bacterial producers. Objectives Here, we investigate whether the changes in the gut microbiome and associated metabolites are related to PD symptoms and epigenetic markers in leucocytes and neurons. Methods Stool, whole blood samples, and clinical data were collected from 55 PD patients and 55 controls. We performed DNA methylation analysis on whole blood samples and analyzed the results in relation to fecal short-chain fatty acid concentrations and microbiota composition. In another cohort, prefrontal cortex neurons were isolated from control and PD brains. We identified genome-wide DNA methylation by targeted bisulfite sequencing. Results We show that lower fecal butyrate and reduced counts of genera Roseburia, Romboutsia, and Prevotella are related to depressive symptoms in PD patients. Genes containing butyrate-associated methylation sites include PD risk genes and significantly overlap with sites epigenetically altered in PD blood leucocytes, predominantly neutrophils, and in brain neurons, relative to controls. Moreover, butyrate-associated methylated-DNA regions in PD overlap with those altered in gastrointestinal (GI), autoimmune, and psychiatric diseases. Conclusions Decreased levels of bacterially produced butyrate are related to epigenetic changes in leucocytes and neurons from PD patients and to the severity of their depressive symptoms. PD shares common butyrate-dependent epigenetic changes with certain GI and psychiatric disorders, which could be relevant for their epidemiological relation. (c) 2022 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder SocietyPeer reviewe

Deciphering the role of microbially-derived metabolites on the microbiota-gut-brain axis

The trillions of microbial organisms residing in the gut, microbiota, are now recognized as major modulators of physiology and health, quickly becoming one of the most exciting emerging areas in neuroscience. Preclinical and clinical research alike suggests that the metabolites produced by these gut microbes modulate brain, behavior and disease. Short-chain fatty acids, tryptophan metabolites and bile acids are promising targets for new microbiome-based therapies. But, little is known about their mechanisms. To this end, the second chapter of the thesis collates 278 studies relating to the human microbiota-gut-brain axis, identifying trends and technical/bioinformatics limitations. These studies across different disorders of the brain as well as healthy human behavioral functions. Then a 35 of these studies was reanalyzed with an up-to-date bioinformatics pipeline. New tools, mainly the gut-brain modules provide a predictive framework for identifying whether these gut microbial metabolic pathways are dysregulated in brain diseases and disorders. We uncovered evidence of disease-related alterations in microbial metabolic pathways in Alzheimer’s Disease, schizophrenia, anxiety and depression. Previous human studies suggest that astrocyte immunity and metabolism is affected by short-chain fatty acids. Thus we grew primary male and female mouse astrocyte cultures, treating them with acetate, butyrate and propionate. Butyrate treatment (0 – 25μM) increased gene expression of Bdnf and Pgc1-α expression, implicating histone-deacetylase inhibitor pathways only in female cells. Acetate (0 – 1500 μM) positively correlated with Ahr and Gfap expression in males, suggesting an immune modulatory role. These findings show a novel sex-dependent impact of acetate and butyrate, but not propionate on astrocyte gene expression. These studies increase understanding of microbial metabolites and how they might impact the brain. It also provides guidance to improve and direct future investigations aimed at identifying the mechanisms of other metabolites

The effect of intestinal inflammation and enteric nervous system deregulation in the pathogenesis of Parkinsonian syndrome

Increasing evidence from epidemiological studies, retrospective studies, clinical observations as well as pre-clinical in-vitro and in-vivo data suggest that Parkinson’s disease involves both the enteric and central nervous system. Notably intestinal dysfunction along with anosmia is one of the most common symptoms observed even decades before PD diagnosis. In order to understand how intestinal inflammation affects the enteric nervous system in the context of PD, we conducted multiple studies including a transgenic mouse model of PD (MitoPark), a mouse model of chronic exposure to environmental toxin manganese (Mn) as well as a known mouse model of intestinal inflammation-the dextran sodium sulfate (DSS) mouse model of colitis. PD and indeed many neurological disorders are gradually being recognized as multi-faceted maladies involving genetic predisposition and environmental triggers. Mn exposure has been implicated in environmentally-linked PD as evidenced by epidemiological studies done on humans exposed to Mn during mining, welding metals, and dry battery manufacturing. With the prevalence of high Mn content in the groundwater in many regions of US, we sought to elucidate the effect of chronic low-dose exposure to Mn on the GI tract. We found that Mn does affect the ENS, particularly the enteric glial cells (EGC) inducing mitochondrial dysfunction and ultimately cell death. In an in vivo model of chronic Mn exposure, we observed that even a low dose of Mn was sufficient to decrease GI motility, alter the gut microbiome population as well as GI metabolism. MitoPark mice that mimic the adult-onset and progressive nature of PD showed mild intestinal inflammation and spatial differences in gastrointestinal (GI) motility. Curiously, these mice have higher motility in the small intestine but lower in the large intestine. Moreover, exposure to an environmental toxin potentiated cell death in the colon with 12-week-old MitoPark having increased proapoptotic protein bax compared to non-exposed transgenic controls. In older MitoPark mice, the ENS showed presence of inflammation with increased expression of pro-inflammatory factors – inducible nitric oxide (iNOS) and tumor necrosis factor alpha (TNFα). We also found similar results in mice afflicted with DSS-induced colitis. These mice showed intestinal inflammation as well as immune cell infiltrations. Intriguingly, the lumbosacral region of the spinal cord and the substantia nigra region in the brain also showed higher proinflammatory (TNFα, iNOS and IL-1β) transcripts. Taken together, the data suggests that intestinal inflammation- caused either by a genetic predisposition or exposure to environmental toxin- can induce neurochemical changes in the ENS and consequently changes in the CNS via the gut-brain axis

Interplay between gut bacteria and Parkinson’s disease medication

Parkinson’s disease (PD) is a neurodegenerative disorder, which affects approximately 6 million individuals worldwide. The main pathologic feature observed in PD patients is the abnormal aggregation of protein and loss of dopaminergic neurons in the midbrain, resulting in motor deficits. Levodopa remains the “golden” standard treatment to restore the absence of dopamine in the brain. Although the start of levodopa treatment has an optimal efficacy, the progression of the disease causes a high variability in the efficacy of levodopa treatment among patients resulting in an unstable and unpredictable clinical response; motor-fluctuations. Besides motor deficits, PD patients also experience various non-motor symptoms such as gastrointestinal dysfunction. In this thesis, we showed that gut bacteria can contribute to the reduction of levodopa availability in the blood-circulation and that they can metabolize the unabsorbed residues of levodopa to various products that alter the gut motility. Furthermore, we showed that the most commonly used PD medications per se may affect the small intestinal motility, the main site of drug absorption, thereby altering the microbiota composition. Such events will potentially create a vicious cycle among the microbiota, PD medication, and gastrointestinal function, and urges for consideration of PD medication and gastrointestinal function when assessing alterations in the PD-associated microbiota. Finally, determining the clinical impact of gut bacteria on PD medication will help reduce the factors contributing to compromised levodopa bioavailability and the unwarranted side effects that result potentially in and from increased treatment regimen

Gateway to the gut : alterations in saliva in inflammatory bowel disease

Inflammatory bowel disease (IBD), which consists of Crohn’s disease and ulcerative colitis, is a chronic immune-mediated disease thought to result from genetic and environmental interaction which influence the commensal flora to trigger an inappropriate mucosal immune response. IBD primarily affects the intestines but is not restricted to them. Extraintestinal manifestations are frequently observed within the oral cavity. This thesis aimed to investigate different mediators of inflammation within the intestines and oral cavity as a reflection of defective immune responses in IBD. In our first study, we investigated the intestinal localization of macrophage growth factors IL-34 and CSF-1 and their involvement in IBD. IL-34 and CSF-1 demonstrated distinct expression patterns in the human intestine and were significantly elevated in human and experimental IBD. Infiltrating cells of the lamina propria and intestinal epithelial cells expressed IL-34, regulated by TNF-α through the NF-κB pathway. As a result, the newly discovered growth factor IL-34 was proposed as a new modulator of IBD. The remaining part of this thesis investigated the expression of inflammatory proteins in saliva in relation to IBD. The second study aimed to analyze calprotectin, an established fecal marker of IBD, for the first time in saliva of IBD patients. We found that calprotectin was significantly elevated in saliva of IBD patients, particularly in CD and most prominently in newly diagnosed CD patients. This opened up for new hypotheses in the oral-gut connection in IBD and supported the notion that the oral cavity may contain early evidence of intestinal inflammation. In a third study, we compared the profile of 92 known inflammatory proteins in saliva and serum of IBD patients. The salivary and circulatory inflammatory profiles were similar but reflected different aspects of IBD activity. Several serum proteins were significantly altered in IBD patients compared to controls, whereas IL-6 and MMP-10 – proteins involved in the pathogenesis of IBD and its extraintestinal manifestations – were significantly elevated in stimulated saliva of IBD patients, providing additional proof of subclinical inflammatory mimicry of intestinal disease by the oral cavity. In the final study, we confirmed our previous findings related to elevated salivary calprotectin in IBD and showed that the concentrations were not significantly affected by oral disease. Moreover, we investigated potential sources of salivary calprotectin and showed that neutrophils isolated from saliva express calprotectin, demonstrate reduced CD11b expression in IBD patients, but share a similar ability to secrete calprotectin. In conclusion, the work presented in this thesis highlights the aberrant immune responses associated to IBD and provides proof that such mechanisms can be reflected by the oral cavity