Innate and Adaptive Immune System Crosstalk in Parkinson's Disease Restricted; Files Only
Kline, Elizabeth (Summer 2019)
Abstract
Parkinson’s disease is a neurodegenerative movement disorder characterized by degeneration of dopaminergic neurons in the substantia nigra and aggregation of a-synuclein. The cause of Parkinson’s disease (PD) is unknown, although genetic and environmental risk factors have been identified. The signaling pathways downstream of antigen presentation and mixed lineage kinases regulate a multitude of neuronal and immune functions and are of interest in PD.
Mixed lineage kinases (MLKs) are expressed by both neuronal and glial cells. Because they are upstream of cell death pathways, previous work focused on inhibition as a possible therapeutic strategy. In this work, a neuroprotection study is presented in which specific inhibition of mixed lineage kinase 3 was tested in a neurotoxin model of PD in mice. It was found that the mixed lineage kinase 3 (MLK3) inhibitor did not interfere with the neurotoxin, was able to cross the blood-brain barrier, and did inhibit phosphorylation of an MLK3 target (JNK). By targeting MLK3 with the inhibitor CLFB-1134, protection of dopaminergic cell bodies and terminals was achieved, although striatal levels of dopamine were not restored to the level observed in control animals.
Antigen presentation via the protein major histocompatibility complex class II (MHCII) is suspected to contribute to PD pathology based on genome-wide association studies, post-mortem histology, and evidence from animal models of PD. Here it is reported that mice with peripheral myeloid deletion of MHCII display changes in T cell frequencies and maintain tyrosine hydroxylase expression in the striatum following nigral human a-synuclein expression. The pesticide cypermethrin, a member of the pyrethroid family known to increase risk for PD in humans, did not robustly exacerbate the inflammation or degeneration caused by human a-synuclein expression. This work also describes an investigation of the effects of an MHCII single nucleotide polymorphism genotype on T cell subsets in PD patients.
MLK3 kinase function and MHCII antigen presentation regulate key neuronal and immune functions that appear to influence the neurodegenerative process in PD. Anti-inflammatory therapeutic interventions that attempt to diminish MHCII expression and inhibition of MLK3 represent different approaches to preserve nigrostriatal dopamine and prevent the progression of degeneration.
Table of Contents
1. INTRODUCTION 1
1.1 Genetic and environmental factors converge to disrupt immune function and increase risk for Parkinson’s disease. 1
1.1a) Parkinson’s disease background 2
1.1b) Brain inflammation in PD and animal models of PD 4
1.1c) PD immunophenotype 13
1.1d) PD genetic factors with known immune system effects 21
1.1e) PD environmental risk factors with known immune system effects 34
1.1f) PD gene-environment interactions with known effects on the immune system 43
1.1g) Conclusion 45
1.2 Mixed-lineage kinase 3 46
2. THE SECOND GENERATION MIXED-LINEAGE KINASE-3 (MLK3) INHIBITOR CLFB-1134 PROTECTS AGAINST NEUROTOXIN-INDUCED NIGRAL DOPAMINERGIC NEURON LOSS 50
2.1 Introduction 50
2.2 Materials and Methods 52
2.2a) Screening pharmacokinetics in mice and rats 52
2.2b) 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine studies 52
2.2c) High performance liquid chromatography of striatal neurochemistry 56
2.2d) Phospho-JNK and total JNK immunoassay 56
2.2e) Western blot 57
2.2f) Histology and stereology 58
2.2g) Statistics 59
2.3 Results 59
2.3a) CLFB-1134 displays a favorable pharmacokinetic profile with high brain penetrance in rats and mice 59
2.3b) MLK3 inhibition in vivo with CLFB-1134 does not interfere with MPTP metabolism to MPP+ 63
2.3c) In vivo administration of CLFB-1134 engages the target MLK3 and results in attenuated JNK phosphorylation. 65
2.3d) CLFB-1134 protects against MPTP-induced loss of striatal dopaminergic terminals 67
2.3e) MLK3 inhibition in vivo with CLFB-1134 did not protect against MPTP-induced striatal DA depletion 69
2.3f) MLK3 inhibition in vivo with CLFB-1134 protects against MPTP-induced nigral dopaminergic degeneration. 69
2.4 Discussion 72
3. PERIPHERAL MYELOID CELL MHCII EXPRESSION IN MICE AFFECTS IMMUNOPHENOTYPE AND HUMAN WT a-SYNUCLEIN-INDUCED NEURODEGENERATION AND INFLAMMATION 77
3.1 Introduction 77
3.2 Materials and Methods 82
3.2a) Mice 82
3.2b) Virus and stereotaxic injection 83
3.2c) Immunofluorescence 84
3.2d) Western blot 85
3.2e) Immune cell isolation and flow cytometry 86
3.2f) Mesoscale Discovery multiplex enzyme linked immunosorbent assay 88
3.2g) Quantitative real time polymerase chain reaction 89
3.2h) Statistics 90
3.3 Results 90
3.3a) Verification of MHCII deletion in peripheral myeloid cells in LysMCre+ IAbfl/fl mice 90
3.3b) Immunophenotyping of LysMCre±IAbfl/f mouse peripheral blood mononuclear cells 97
3.3c) Immunophenotyping of LysMCre±IAbfl/f following 3 weeks of cypermethrin treatment 100
3.3d) T cell subsets in the peripheral blood and deep cervical lymph nodes of LysMCre±IAbfl/f mice 103
3.3e) T cell subsets of LysMCre±IAbfl/ mice following 3 weeks of cypermethrin treatment 111
3.3f) LysMCre-IAbfl/f mice do not differ in plasma levels of inflammatory cytokines 4 months post-rAAV2/9 human WT a-synuclein injection 116
3.3g) Cypermethrin treatment increases plasma IL-1b in LysMCre+I-Abfl/fl mice 118
3.3h) Demonstration of human a-synuclein expression in the SN following viral vector injection 121
3.3i) Inflammatory gene expression in the striatum 4 months after nigral rAAV2/9 human WT a-synuclein injection 124
3.3j) Assessment of striatal dopaminergic terminals 4 months after rAAV2/9 human WT WT a-synuclein injection 128
3.3k) Short-term cypermethrin exposure does not decrease tyrosine hydroxylase or dopamine transporter protein expression, even in the presence of human WT a-synuclein 131
3.4 Discussion 133
4. CHARACTERIZATION OF T CELL SUBSETS ASSOCIATED WITH rs3129882 GENOTYPE IN PARKINSON’S DISEASE PATIENTS AND CONTROLS 143
4.1 Introduction 143
4.2 Materials and Methods 146
4.2a) Cohort subject recruitment and genotyping 146
4.2b) Peripheral blood mononuclear cell isolation, cryopreservation, and thaw 147
4.2c) Flow cytometry 147
4.2d) Study approval 148
4.2e) Statistics 148
4.3 Results 148
4.3a) Characteristics of human study population 148
4.3b) High-risk rs3129882 genotype is associated with decreased number of Tregs, and interactions between SNP and PD affect effector memory subset counts 150
4.4 Discussion 159
5. CONCLUSIONS 162
APPENDIX 168
Antibodies used in Chapters 2-4 168
List of Figures and Tables
Figure 1. Working model of innate and adaptive immune crosstalk via MHCII in PD 33
Figure 2. CLFB-1134 does not interfere with MPTP metabolism 64
Figure 3. In vivo target engagement by CLFB-1134 66
Figure 4. CLFB-1134 protects against MPTP-induced loss of striatal TH 68
Figure 5. CLFB-1134 does not protect against MPTP-induced striatal DA depletion 70
Figure 6. CLFB-1134 protects against MPTP-induced nigral dopaminergic cell loss 71
Figure 7. Flow cytometry gating strategies for mouse splenocytes and mouse peritoneal immune cells 92-93
Figure 8. MHCII deletion in peripheral myeloid cells of naïve LysMCre+I-Abfl/fl mice 94
Figure 9. Flow cytometry gating strategies for mouse PBMC general immune cell types and PBMC and DCLN T cell subsets 95-96
Figure 10. MHCII deletion in LysMCre+I-Abfl/fl PBMCs 98-99
Figure 11. No difference in MHCII+ cell frequency or number between LysMCre-I-Abfl/fl and LysMCre+I-Abfl/fl PBMCs following 3 weeks of cypermethrin or vehicle treatment. 101-102
Figure 12. CD4+ T cell subsets are affected by LysMCre-mediated deletion of MHCII 104-107
Figure 13. The frequency of effector memory CD4+ T cells in mouse deep cervical lymph nodes is affected by LysMCre-mediated deletion of MHCII 108-110
Figure 14. Cypermethrin and LysMCre genotype affect PBMC T cell frequencies 112-114
Figure 15. Cypermethrin and LysMCre affect the frequency of total T cells and central memory CD4s in the deep cervical lymph nodes 114-115
Figure 16. Plasma cytokine and chemokine levels do not differ between LysMCre- and LysMCre+I-Abfl/fl mice 117-118
Figure 17. Plasma IL-1b levels increase with cypermethrin treatment in LysMCre+I-Abfl/fl mice 119-120
Figure 18. Targeting of rAAV2/9 human WT a-synuclein to the mouse substantia nigra 122
Figure 19. Targeting of rAAV2/5 human WT a-synuclein to the mouse substantia nigra 123
Figure 20. Inflammatory gene expression in the striatum 4 months after nigral rAAV2/9 human WT a-synuclein injection 125-127
Figure 21. Tyrosine hydroxylase and dopamine transporter protein expression are decreased ipsilateral to rAAV2/9 human WT a-synuclein injection in LysMCre-I-Abfl/fl striatum.
129-130
Figure 22. Short-term cypermethrin exposure does not decrease tyrosine hydroxylase or dopamine transporter protein expression, even in the presence of human WT a-synuclein 132
Figure 23. Human T cell subset gating strategy 151
Figure 24. High-risk rs3129882 genotype is associated with decreased number of Tregs, and interactions between SNP genotype and PD affect effector memory counts. 152-158
Table 1. Experimental groups for MPTP studies 55
Table 2. CLFB-1134 pharmacokinetic properties in mice and rats 60-62
Table 3. Primers for mouse striatal qPCR 89
Table 4. Characteristics of human study population 149
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