Gene-Environment Interplay in Parkinson’s Disease Pathogenesis: The Role of Gastrointestinal Inflammation and Regulator of G-protein Signaling 10 in the Gut-Brain Axis Restricted; Files Only

Houser, Madelyn (Summer 2019)

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The etiology of Parkinson’s disease is unknown, but the prevalence of gastrointestinal problems in patients even before the onset of the characteristic motor impairments has prompted theories that pathology could develop initially in the gut and progress to the brain. The gastrointestinal abnormalities associated with Parkinson’s disease could reflect the presence of chronic intestinal inflammation. If so, then inflammatory immune responses could link gastrointestinal symptoms with the neuroinflammation and neuropathology observed in Parkinson’s disease.

In a cohort of human subjects, we confirmed greater incidence of intestinal disease and digestive problems in Parkinson’s disease patients compared to controls. We sought to determine whether indications of intestinal inflammation could be detected in these patients but found that levels of immune-related factors in stool were strongly influenced by factors including sex, body mass index, smoking history, and probiotics use. When these variables were accounted for, we identified elevated levels of four proinflammatory molecules – IL-1α, CXCL8, IL-1β, and CRP – in stool from Parkinson’s Disease patients.

To assess experimentally whether intestinal inflammation could exert pathological effects on the brain, we induced colitis in a mouse model and evaluated peripheral and neuroinflammation as well as neuron health in regions of the brain affected in Parkinson’s disease. We also sought to determine how the effects of colitis might interact with sex, genetic predisposition to hyperinflammatory responses, and exposure to an additional neurotoxic insult. We discovered that in male mice, colitis promoted sustained inflammation and indications of CD8+ T cell infiltration in the brain and perturbed dopaminergic neuronal activity without causing dopamine depletion. Colitis also augmented the impact of a known neurotoxicant in both sexes. These effects were particularly pronounced in mice that lacked the Regulator of G-Protein Signaling 10, which increased baseline intestinal inflammation and colitis severity.

This research confirms that intestinal inflammation is present in Parkinson’s disease and that it has the potential to impact dopaminergic neurons in the brain and, when combined with other risk factors, to produce parkinsonian neuropathology. Controlling chronic gut inflammation could be an intervention that reduces the risk for the development of clinical manifestations of Parkinson’s disease.

Table of Contents

Chapter 1: Introduction 1

1.1 Intestinal inflammation could promote the development of Parkinson’s disease 1

1.1a Mechanisms of intestinal modulation of CNS activity 1

1.1b Clinical features of Parkinson’s disease 7

1.1c Intestinal involvement in Parkinson’s disease 10

1.1d Model of gut-originating, inflammation-driven Parkinson’s disease pathogenesis 19

1.2 Regulator of G-Protein Signaling 10 25

1.2a RGS10 biochemistry 25

1.2b RGS10 in osteoclasts 26

1.2c RGS10 in cancer 28

1.2d RSG10 in neurotransmission 29

1.2e RGS10 in neuroinflammation and Parkinson’s Disease 30

1.2f RGS10 in peripheral immune cells and inflammation 32

1.2g RGS10 in aging 35

1.3 References 38

Chapter 2: Stool immune profiles evince gastrointestinal inflammation in Parkinson’s disease 77

2.1 Introduction 77

2.2 Methods 79

2.2a Subjects 79

2.2b Processing of stool 80

2.2c Multiplexed immunoassays 80

2.2d Statistical analysis 81

2.3 Results 82

2.3a Increased incidence of psychological and gastrointestinal symptoms in Parkinson’s disease patients and decreased coffee and alcohol consumption 82

2.3b Significantly higher levels of select stool analytes in PD patients 83

2.3c Sex significantly impacts stool analyte levels 84

2.3d BMI, smoking history, and probiotic usage strongly influence stool analyte levels 84

2.3e PD-associated stool inflammatory profile emerges when other factors are accounted for 85

2.3f PD-associated GI inflammation does not emerge only in advanced disease 85

2.4 Discussion 86

2.5 Figures and Tables 92

2.6 References 108

Chapter 3: Experimental colitis mimics intestinal inflammatory features of Parkinson’s disease and promotes sustained T cell-associated midbrain neuroinflammation and parkinsonian neuropathology 118

3.1 Introduction 118

3.2 Methods 121

3.2a Human subjects 121

3.2b Mouse procedures 122

3.2c RNA and protein isolation 124

3.2d Western blot 125

3.2e Quantitative PCR 125

3.2f HPLC 126

3.2g Multiplexed immunoassay 126

3.2h Flow cytometry 127

3.2i Immunostaining 127

3.2j Statistics 129

3.2k Study approval 130

3.3 Results 130

3.3a RGS10 deficiency induces intestinal inflammation and dysfunction 130

3.3b Inflammatory features of DSS colitis are consistent with those observed in colon tissue from Parkinson’s disease patients 131

3.3c Colitis and RGS10 deficiency perturb nigrostriatal dopaminergic systems and augment effects of MPTP 132

3.3d Increased activity of TH following colitis prevents dopamine deficiency 133

3.3e RGS10 deficiency impacts peripheral blood immune cell populations 134

3.3f Colitis and RGS10 deficiency promote sustained CD8+ T cell-associated inflammation in the brain 135

3.3g CD8+ T cell-associated inflammation in the SNpc is associated with striatal TH levels in males but not females 136

3.4 Discussion 137

3.5 Figures and Tables 147

3.6 References 164

Chapter 4: Discussion, Conclusions, and Future Directions 177

4.2 References 187

Figures and Tables

Table 1: No differences in levels of immune or angiogenesis factors in stool from non-household and household controls 92

Table 2: No differences between non-household and household controls in numerous demographic and health factors and practices 93-94

Table 3: Increased incidence of psychological and gastrointestinal symptoms in PD patients and decreased coffee and alcohol consumption 95

Table 4: No significant associations between PD duration and levels of stool immune and angiogenesis factors 96

Table 5: Comparison of levels of immune and angiogenesis factors in stool from PD patients and controls 97

Table 6: Comparison of levels of immune and angiogenesis factors in stool from PD patients and their spouses separated by sex 98

Table 7: Comparison of levels of immune and angiogenesis factors in stool from female and male subjects 99

Table 8: Association between PD status and levels of stool immune and angiogenesis factors when accounting for potential confounders or effect modifiers 100

Table 9: No significant associations between PD duration and levels of stool immune and angiogenesis factors 101

Table 10: Criteria for calculation of disease activity index for colitis 147

Table 11: Antibodies used in this study 148

Table 12: qPCR primers used in this study 149

Figure 1: Model of gut-originating, inflammation-driven PD pathogenesis 24

Figure 2: Levels of select stool analytes elevated in female PD patients but not male 102

Figure 3: Levels of nine chemokines and angiogenesis factors decrease with increasing BMI 103

Figure 4: Levels of seven cytokines and one vascular factor are reduced in subjects with a history of smoking 104

Figure 5: Levels of IL-6 decrease with increasing coffee consumption 105

Figure 6: Levels of eleven immune factors elevated in stool of subjects taking probiotics with especially strong effects in Parkinson’s disease (PD) patients 106

Figure 7: PD patients and controls differ in associations between subject age and levels of stool analytes 107

Figure 8: Mouse Experiment Design 150

Figure 9: Gating strategy for mouse peripheral blood mononuclear cells 151

Figure 10: Random forest models are accurate representations of the datasets 152

Figure 11: RGS10 deficiency induces intestinal inflammation and dysfunction and increases sensitivity to DSS colitis 153

Figure 12: Neurons and lymphoid cells do not express RGS10 in the murine colon 154

Figure 13: Colonic inflammation is present in PD patients and in mice with RGS10 deficiency and DSS colitis 155

Figure 14: DSS colitis and RGS10 deficiency impact nigrostriatal dopamine pathways and increase susceptibility to MPTP 156

Figure 15: Levels of factors regulating dopamine production, packaging, and reuptake are significantly correlated 157

Figure 16: Increased tyrosine hydroxylase activity prevents dopamine deficiency after colitis 158

Figure 17: RGS10 deficiency impacts circulating non-classical monocytes and T cells, and colitis augments these effects 159

Figure 18: Minimal impact of genotype or treatment on plasma cytokines at experiment endpoint 160

Figure 19: No differences in Iba1 protein levels among experimental groups 161

Figure 20: Colitis and RGS10 deficiency result in CD8+ T cell infiltration and elevated Ifng expression in the substantia nigra pars compacta 162

Figure 21: Factors most highly associated with striatal TH levels differ by sex 163

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