Innate and Adaptive Immune System Crosstalk in Parkinson's Disease Restricted; Files Only

Kline, Elizabeth (Summer 2019)

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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


3.1     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


4.1     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. 


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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