The Role of Immune Mechanisms in Aging and Neurodegeneration Open Access

Kannarkat, George Thomachan (2015)

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In recent years, it has become increasingly clear that inflammation is a key driver of neurodegenerative pathology that can synergize with other factors such as aging and neuronal dysfunction. Two particularly interesting pathways in the immune system conferring disease risk are regulation of G-protein signaling (RGS) and antigen presentation. Specifically, the RGS10 protein is a negative regulator through its ability to accelerate deactivation of Gai or Gaq molecules and it has been implicated in age-related macular degeneration. The loss of RGS10 in a mouse leads to increased dopaminergic neuronal vulnerability to inflammation and dysregulated immune responses. In this work, it was demonstrated that there are alterations in immune cell populations but not dopaminergic neuron homeostasis with age in RGS10-/- mice. Furthermore, it was shown that loss of RGS10 alters immune cell chemotaxis. Because of these roles for RGS10, it could prove to be an interesting target for therapy in treating neurodegenerative disease. Antigen presentation has been implicated as a risk factor for Parkinson's disease through many genome-wide association studies identifying polymorphisms in the Major Histocompatibility Complex (MHC)-II locus. Herein, it was determined that a polymorphism in the HLA-DRA gene conferring higher risk for Parkinson's disease was associated with increased expression of MHC-II molecules as well as inducibility of these molecules in response to IFN-g. Furthermore, it was found that people with the high risk polymorphism who were exposed to the commonly used class of insecticides, pyrethroids, were at increased risk for Parkinson's disease compared to the low risk polymorphism demonstrating a gene-environment interaction between pyrethroids and the MHC-II locus. This class of insecticides were shown to have immunomodulatory properties suggesting that pyrethroids synergize with antigen presentation to dysregulate immune responses that put people at risk for neurodegeneration. Both regulation of G-protein signaling and antigen presentation have key roles in regulating immune responses that influence risk for neurodegenerative disease. The targeting of these processes for treatment of neurodegeneration has not been explored. It will be critical to understand and describe the role of the immune system in promoting and complementing other pathogenic processes intrinsic to neurons.

Table of Contents

Chapter 1: Introduction 1

1.1) RGS10 has an important role in aging of the immune system 1

1.1a) Function of RGS proteins 1

1.1b) RGS proteins have important roles in regulating immune cell function 2

1.1c) RGS proteins have a multifaceted role in aging 2

1.1d) RGS10 regulates neuroimmune interactions 3

1.2) Inflammation as a driver of PD Pathogenesis 5

1.2a) Etiology and Pathogenesis of PD 5

1.2b) Etiology of Sporadic Parkinson's Disease 6

1.2c) Evidence for Inflammation and Role for Innate Immunity in PD 8

1.2d) Engagement of Adaptive Immunity: Microglial Activation and MHC in PD 12

1.2e) Adaptive Immunity (I): T Lymphocytes in PD 14

1.2f) Adaptive Immunity (II): Antibodies and B Lymphocytes in PD 15

1.2g) Antigen Presentation as an Etiologic Factor for PD 22

1.3) Figures 25

Chapter 2: Age-related changes in Regulator of G-protein Signaling (RGS)-10 expression in peripheral and central immune cells may influence risk for age-related degeneration 28

2.1) Introduction 28

2.2) Materials and Methods 30

2.2a) Animals 30

2.2b) Flow Cytometry 30

2.2c) Immunofluorescence and Image Quantitation 31

2.2d) Cerebrospinal fluid (CSF) and Serum collection 32

2.2e) Multiplexed ELISAs 33

2.2f) Immune Cell Isolation from Adult Mouse Brain 33

2.2g) Western Blot Analysis 33

2.2h) Quantitative Real-time RT-PCR (QPCR) 34

2.2i) Dopamine Metabolism Measurement 34

2.2j) Statistical analysis 35

2.3) Results 36

2.3a) RGS10 expression in B cells, monocytes, and granulocytes is increased with age while microglial RG10 expression decreases 36

2.3b) Loss of RGS10 has minimal effect on frequency and number of peripheral immune cell subsets but does alter immune cell frequencies in the brain in young mice 37

2.3c) Loss of RGS10 alters B cell, M0, and CD4+ T cell frequency and number in the periphery of but not in the brains of aged mice 37

2.3d) Loss of RGS10 does not alter serum cytokine levels, but is associated with loss of age-related increase in levels of IL-6 in the cerebrospinal fluid 38

2.3e) RGS10 and Tyrosine Hydroxylase protein expression does not change with age in the ventral midbrain or striatum 38

2.3f) Loss of RGS10 does not alter tyrosine hydroxylase, Parkin, or Nrf2 mRNA expression in the ventral midbrain in young or aged mice 39

2.3g) Loss of RGS10 does not alter dopamine metabolism in the nigrostriatal pathway 40

2.4) Discussion 41

2.5) Figures 46

Chapter 3: Regulator of G-Protein Signaling 10 modulates immune cell chemotaxis in neuroinflammation 54

3.1) Introduction 54

3.2) Materials and Methods 55

3.2a) Animals 55

3.2b) Flow Cytometry 56

3.2c) Immune Cell Isolation from Adult Mouse Brain 57

3.2d) Boyden Chamber Assay for Chemotaxis 57

3.2e) Intracranial Administration of LPS/IFN-γ 58

3.2f) Thioglycollate-induced Peritonitis Model 58

3.2g) Human Subject Recruitment 58

3.2h) Isolation of Human Peripheral Blood Mononuclear Cells 59

3.2i) Statistical analysis 59

3.3) Results 59

3.3a) Lack of RGS10 alters chemotaxis in a cell-specific and chemokine-dependent manner in mouse PBMCs 59

3.3b) Loss of RGS10 alters immune cell recruitment to in vivo models of inflammation 60

3.3c) Age-dependent decreases in RGS10 expression are present in CD8+ T cells and CD16+ monocytes from Parkinson's disease patients 61

3.4) Discussion 61

3.5) Figures 64

Chapter 4: Common Genetic Variant Association with Altered HLA Expression, Synergy with Pyrethroid Exposure, and Risk for Parkinson's Disease: An Observational and Case-Control Study 69

4.1) Introduction 69

4.2) Materials and Methods 71

4.2a) MHC-II Expression Cohort Subject Recruitment 71

4.2b) Peripheral Blood Mononuclear Cell (PBMC) Isolation, Sorting, and Stimulation 72

4.2c) RNA Isolation, cDNA synthesis, and RT-PCR 72

4.2d) Flow Cytometry Analysis 73

4.2e) Mesoscale Discovery Multiplex ELISA 74

4.2f) Genevar Analysis 74

4.2g) Pesticide Exposure Cohort and Epidemiological Methods 74

4.2h) Statistical Analyses 77

4.2i) Study approval 78

4.3) Results 78

4.3a) MHC-II Expression Study Population 78

4.3b) The rs3129882 GG genotype is associated with increased surface MHC-II expression 78

4.3c) The rs3129882 GG genotype is associated with increased IFNg inducibility of HLA-DQ expression 79

4.3d) The rs3129882 GG genotype is associated with increased baseline expression and IFNg inducibility of MHC-II mRNA 80

4.3e) The rs3129882 high-risk genotype is associated with increased plasma CCL-3 (MIP-1a) levels in PD patients but not with altered frequencies of B cells and monocytes in the peripheral blood 81

4.3f) Pyrethroid exposure and the high-risk rs3129882 genotype increases odds for PD 82

4.3g) Genetic variation associated with ethnicity can reverse allelic rs3129882 association of MHC-II expression changes 83

4.4) Discussion 84

4.5) Figures 89

Chapter 5: The commonly used class of insecticides, pyrethroids, have acute immunomodulatory effects that impact mechanisms of antigen presentation 102

5.1) Introduction 102

5.2) Materials and Methods 103

5.2a) Cell Culture 103

5.2b) RNA Isolation, cDNA synthesis, and RT-PCR 104

5.2c) Flow Cytometry 104

5.2d) Multiplex Enzyme-linked Immunoassay 105

5.2e) Carboxyfluorescein Succinimidyl Ester (CFSE) Labeling 105

5.2f) Pesticide Handling 106

5.2g) Statistical Analyses 106

5.3) Results 106

5.3a) Rotenone and esfenvalerate alter induction of MHC-II mRNA in response to IFN-g 106

5.3b) Pesticides dampen the induction of costimulatory molecules 107

5.3c) Pesticides dysregulate cytokine secretion 107

5.3d) Pesticides increase rate of T cell proliferation 108

5.4) Discussion 108

5.5) Figures 110

Chapter 6: Future Directions and Applications to Human Disease and Therapy 114

6.1) Introduction 114

6.2) Future Directions 115

6.3) Immunomodulatory Therapies Targeting RGS Proteins 117

6.4) Immunomodulatory Therapies in PD 118

6.5) Conclusions 122

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