The Role of Immune Mechanisms in Aging and Neurodegeneration Open Access

Kannarkat, George Thomachan (2015)

Permanent URL: https://etd.library.emory.edu/concern/etds/q237hr99x?locale=en
Published

Abstract

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
Figures and Tables
Fig 1: The Braak Hypothesis proposes a pattern for progression of disease pathology 25
Fig 2: Biosynthesis of peptide-MHC complexes 26
Fig 3: Model of MHCII mediated antigen presentation to CD4 T cells 26
Fig 4: RGS10 expression increases in peripheral B cells, monocytes/
macrophages, and granulocytes and decreases in Iba1+ cells in
the brain with age 46
Fig 5: Loss of RGS10 alters the immune cell repertoire in the brain but has little effect on immune cell frequency and number in the periphery of young mice 47
Fig 6: Loss of RGS10 alters B cell, monocyte/macrophage, and CD4+ T cell frequency and number in the periphery but not in the brain of
aged mice 48
Fig 7: Loss of RGS10 does not alter serum cytokine levels but decreases IL-6 levels in the CSF in aged mice 49
Fig 8: RGS10 expression does not change with age in the ventral midbrain or striatum and does not alter TH expression in the ventral midbrain in young or aged mice 50
Fig 9: Loss of RGS10 does not alter mRNA expression for TH, Parkin, or Nrf2 in the ventral midbrain 51
Fig 10: The levels of nigrostriatal DA and metabolites are independent of RGS10 expression 52
Fig 11: The loss of RGS10 alters immune cell chemotaxis in a cell-type specific manner to many immunologically relevant chemokines 65
Fig 12: The loss of RGS10 increases the recruitment of CD11b+CD11c- myeloid cells 3 days after induction of thioglycollate-induced peritonitis 66
Fig 13: The loss of RGS10 dampens the increase in CD45-low monocyte/microglia number in response to intracranial LPS/IFNg 67
Fig 14: Decreased RGS10 expression is associated with age in CD8+ T cells and CD16+ monocytes in PD patients and decreased RGS10 expression in CD16+ monocytes relative to healthy controls 68
Fig 15: Gating Strategy for Flow Cytometry Analysis 91
Fig 16: The high-risk rs3129882 GG genotype is associated with an increased level of MHC-II expression in B cells and monocytes and with increased inducibility of surface HLA-DQ expression 92
Fig 17: The high-risk rs3129882 GG genotype is associated with increased baseline expression and inducibility of MHC-II mRNA 93
Fig 18: The rs3129882 high-risk genotype is associated with increased plasma CCL-3 levels in PD patients with the high-risk rs3129882 GG genotype but not with altered frequencies of B cells and monocytes in the peripheral blood 95
Fig 19: Model depicting the association of the rs3129882 SNP with altered MHC-II expression on APCs and the potential for skewing the adaptive immune response and the predicted effects on vulnerable neuronal populations affected in PD 101
Fig 20: Esfenvalerate and rotenone but not permethrin modulate the IFNg mediated induction of MHC-II in THP-1 cells 110
Fig 21: Pyrethroids dampen the LPS-induced expression of costimulatory molecules on the surface of THP-1 cells 111
Fig 22: Pyrethroids alter the IFNg induced secretion of cytokine in THP-1
cells 112
Fig 23: Pyrethroids increase rate of Jurkat T cell line proliferation 113
Table 1: Genetic polymorphisms associated with sporadic forms of PD 25
Table 2: RT-PCR Primers 89
Table 3: Characteristics of MHC-II Expression Study Population 90
Table 4: General characteristics of PEG study population of European ancestry, n=962 (patients=465, controls=497) 96
Table 5: HLA-DRA rs3129882 marginal effects in PEG population, n=962 (patients=465, controls=497) 96
Table 6: Interaction, main, and joint effect estimates between HLA rs3129882 and pyrethroid exposure in PEG study population of European ancestry, using both an additive genetic model and AA vs GG; n=962
(patients=465, controls=497) 97
Table 7: Clinical characteristics of PEG PD patients of European ancestry, across follow-up exams by HLA rs3129882 genotype (AA vs GG) 98
Table 8: The direction of association of cis-eQTL level with the rs3129882 genotype depends on ethnicity 100

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