LRRK2 Function in Immune Cells and Contribution to Parkinson’s Disease Open Access

Cook, Darcie (Fall 2017)

Permanent URL: https://etd.library.emory.edu/concern/etds/m039k489b?locale=en
Published

Abstract

Leucine rich repeat kinase 2 (LRRK2) is a large protein with a GTPase and kinase domain and several protein-interacting domains. Mutations in the lrrk2 gene, specifically the enzymatic domains, are one of the most common causes of autosomal dominant Parkinson’s disease (PD). LRRK2 has been reported as a regulator of many cellular pathways, including inflammatory signaling, cytoskeletal maintenance, and autophagy. LRRK2 expression is enriched in cells of the immune system (CD4+ and CD8+ T cells, CD14+ monocytes, and CD19+ B cells), but its function in the immune system and its effects on age-related immunosenescence are as yet unknown. Its regulatory function of nuclear factor of activated T cells (NFAT) and nuclear factor kappa B (NF-κB) implicates LRRK2 in the development of the inflammatory environment characteristic of PD. Mutations or polymorphisms in the lrrk2 gene are also associated with several other immunological diseases, including Crohn’s disease and increased risk for leprosy infection, indicating that LRRK2 plays an important role in proper immune function.

The aim of this study was to determine the expression pattern of LRRK2 in immune cell subsets and correlate it with the immunophenotype of cells from PD and healthy subjects. In this study, it was shown that LRRK2 expression was increased in B cells, T cells, and CD16+ monocytes of PD patients compared to healthy controls (HC). LRRK2 induction was also increased in monocytes and dividing T cells in PD patients compared to HCs. In addition, PD patient monocytes secreted more inflammatory cytokines compared to HC, and cytokine expression positively correlated with LRRK2 expression in T cells from PD but not HCs. Finally, the regulatory surface protein that limits T cell activation signals, CTLA-4 (cytotoxic T-lymphocyte-associated protein 4), was decreased in PD compared to HC in T cells. Studies involving G2019S-LRRK2 overexpressing and wild type LRRK2 overexpressing mice were performed to assess contribution of mutant LRRK2 to dysregulation of inflammatory pathways. While data are still preliminary, results implicate gain-of-function kinase mutations in dysregulation of both NFAT and NF-κB signaling. In sum, these findings suggest that LRRK2 has a regulatory role in immune cells and PD. Functionally, the positive correlations between LRRK2 expression levels in T cell subsets, cytokine expression and secretion, and T cell activation states strongly suggest that targeting LRRK2 with therapeutic interventions is likely to have direct effects on immune cell function.

Table of Contents

 

Chapter 1: Introduction. 10

 

1.1 The Aging Immune System, Neuroinflammation, and Neurodegeneration. 10

 

1.1a Immunosenescence and Inflammaging. 10

 

1.1b Innate Immune Aging and Dysfunction. 10

 

1.1c Adaptive Immune Aging and Dysfunction. 10

 

1.1d Inflammation in the Brain and Neurodegeneration. 10

 

1.1e Parkinson’s Disease: Etiology, Pathogenesis, and Inflammation. 10

 

1.1f Conclusion. 10

 

1.2 Leucine Rich Repeat Kinase 2, Parkinson’s Disease, and Immune Function. 10

 

1.2a LRRK2 Structure and Function. 10

 

1.2b LRRK2 Expression. 10

 

1.2c LRRK2 Mutations, Polymorphisms, and Disease. 10

 

1.2d LRRK2 Regulation of the Immune System.. 10

 

1.2e LRRK2 Kinase/Small Molecule Inhibitors and Antisense Oligonucleotides (ASO) 10

 

1.2f Conclusion. 10

 

1.3 Thesis Aims. 10

 

1.4 Figures. 10

 

Chapter 2: LRRK2 Levels in Immune Cells Are Increased in Parkinson’s Disease. 10

 

2.1 Introduction. 10

 

2.2 Materials and Methods. 10

 

2.2a Human subjects. 10

 

2.2b Human Peripheral Blood Mononuclear Cell (PBMC) isolation, stimulation, and purification. 10

 

2.2c Mouse PBMC isolation. 10

 

2.2d Human THP-1 Monocytic Cell Culture and Differentiation. 10

 

2.2e Western Blotting. 10

 

2.2f Flow Cytometry Analysis. 10

 

2.2g Cell Proliferation Assays. 10

 

2.2h MesoScale Discovery multiplexed immunoassays. 10

 

2.2i Statistical analyses. 10

 

2.2j Human subjects research approval 10

 

2.3 Results. 10

 

2.3a Validation of Abcam c41-2 LRRK2 antibody for detection of human LRRK2 by flow cytometry. 10

 

2.3b PD is associated with increased LRRK2 expression in innate and adaptive immune cells. 10

 

2.3c LRRK2 expression is induced by inflammatory stimuli in both PD and HC monocytes but shows opposite correlation with MHC-II induction in PD versus HC subjects. 10

 

2.3d LRRK2 induction is slower in T cells compared to monocytes in both PD and HC subjects. 10

 

2.3e Parkinson’s disease is associated with higher LRRK2 induction in proliferating T cells compared to healthy controls. 10

 

2.3f The T cell activation marker CTLA4 is significantly decreased in T cells of PD patients following stimulation. 10

 

2.3g Immune cells from PD patients display similar cellular cytokine expression but increased pro-inflammatory cytokine secretion. 10

 

2.3h LRRK2 expression is positively correlated with cytokine expression in PD patients. 10

 

2.4 Discussion. 10

 

2.5 Figures. 10

 

Chapter 3. Assessing contribution of LRRK2 kinase activity to immune cell activation via NFAT and NF-κB.. 10

 

3.1 Introduction. 10

 

3.2 Materials and Methods. 10

 

3.2a Luciferase Assays. 10

 

3.2b Mouse PBMC and splenocyte isolation. 10

 

3.3c Cellular Fractionation. 10

 

3.2d PBMC and Splenocyte Stimulation. 10

 

3.2e Western Blot 10

 

3.2f ImageStream®.. 10

 

3.2g PFE360 Drug and Vehicle Preparation. 10

 

3.3 Results. 10

 

3.3a Optimization of PFE360 for in vitro kinase inhibition studies. 10

 

3.3a G2019S increases NFAT activity in a transfected T cell line. 10

 

3.3b LRRK2 Wild type overexpressing mice have increased phosphorylation. 10

 

3.3c Optimizing Fractionation for NFAT1 Translocation Studies. 10

 

3.3d Optimizing ImageStream® to Quantify Nuclear Translocation of NFAT.. 10

 

3.3e Gain-of-function G2019S LRRK2 mutation has increased basal levels of NF-kB signaling proteins. 10

 

3.4 Discussion. 10

 

3.5 Figures. 10

 

Chapter 4: Future Directions and Implications for Clinical Treatment 10

 

4.1 Introduction. 10

 

4.2 Future Directions. 10

 

4.3 LRRK2 Contribution to Parkinson’s Disease. 10

 

4.4 Conclusions. 10

 

REFERENCES CITED.. 10

 

Appendix I: Common Genetic Variant Association with Altered HLA Expression, Synergy with Pyrethroid Exposure, and Risk for Parkinson’s Disease: An Observational and Case-Control Study. 10

 

AI.1 Introduction. 10

 

AI.2 Methods. 10

 

AI.2a MHC-II Expression Cohort Subject Recruitment 10

 

AI.2b Peripheral Blood Mononuclear Cell (PBMC) Isolation, Sorting, and Stimulation. 10

 

AI.2c RNA Isolation, cDNA synthesis, and RT-PCR.. 10

 

AI.2d Flow Cytometry Analysis. 10

 

AI.2.e Mesoscale Discovery Multiplex ELISA.. 10

 

AI.2.f Genevar Analysis. 10

 

AI.2g Pesticide Exposure Cohort and Epidemiological Methods. 10

 

AI.2h Statistical Analyses. 10

 

AI.2i Study approval 10

 

AI.3 Results. 10

 

AI.3a MHC-II Expression Study Population. 10

 

AI.3b The rs3129882 GG genotype is associated with increased surface MHC-II expression. 10

 

AI.3c The rs3129882 GG genotype is associated with increased IFN-g inducibility of HLA-DQ expression. 10

 

AI.3d The rs3129882 GG genotype is associated with increased baseline expression and IFN-g inducibility of MHC-II mRNA.. 10

 

AI.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. 10

 

AI.3f Pyrethroid exposure and the high-risk rs3129882 genotype increases odds for PD.. 10

 

AI.3g Genetic variation associated with ethnicity can reverse allelic rs3129882 association of MHC-II expression changes. 10

 

AI.4 Discussion. 10

 

AI.5 Figures. 10

 

Appendix II: Other Unpublished Data. 10

 

AII.1 Immunophenotyping of Human G2019S LRRK2 Subjects*. 10

 

AII.2 LRRK2 expression by rs3129882 genotype. 10

 

AII.3 Figures. 10

 

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