All ETDs from UAB

Advisory Committee Chair

Andrew B West

Advisory Committee Members

Talene A Yacoubian

Mathew S Goldberg

Jeremy H Herskowitz

Richard E Kennedy

Document Type

Dissertation

Date of Award

2019

Degree Name by School

Doctor of Philosophy (PhD) Heersink School of Medicine

Abstract

Parkinson disease (PD) is the second most common neurodegenerative disorder with no reliable biochemical biomarkers for disease prediction or progression, nor disease-modifying treatments to slow the relentless progression. Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are known to increase LRRK2 kinase activity and increase the risk for late-onset PD. In this thesis, I discovered that LRRK2 is secreted into exosomes in urine and CSF, where LRRK2 kinase activity, reflected by autophosphorylation at pS1292 site, is preserved and reflective of cytosolic LRRK2 levels. In a cohort of biosamples from LRRK2 mutation carriers and matched controls, with and without PD, LRRK2 autophosphorylation in urine exosomes was significantly higher in male LRRK2 mutation carriers with PD and lower in female LRRK2 mutation carriers with PD. These results indicate that urine exosomes show promise in predicting PD susceptibility in LRRK2 mutation carriers. In contrast, LRRK2 autophosphorylation levels in CSF exosomes were ten times higher than urine from the same subject collected at the same time and was saturated in different groups of patients irrespective of LRRK2 mutation status. Further, I found that total LRRK2 levels were not correlated between urine and CSF exosomes from the same participants, suggesting LRRK2 function and kinase activity differs between the brain and periphery. In sum, these results highlight the functional effects of LRRK2 mutations in mediating LRRK2 kinase activity and how these activities appear much higher in the brain than the periphery. Future studies may elucidate the mechanisms that govern LRRK2 activity in the brain and use the assays developed here to identify therapeutic strategies that may lower LRRK2 kinase activity. Although the pathogenesis of PD remains unclear, evidence from numerous lines of experimental investigations points towards defects in the endo-lysosomal pathway. Extracellular vesicles (EVs) are a subset of vesicles originating from the endo-lysosomal system and carry cytosolic proteins out of the cell and into easily accessible biofluids like urine. I characterized urine EV profiles using mass spectrometry whole-proteomic approaches and discovered a clear enrichment of proteins linked to neurological disease pathways and endo-lysosome function. Of those proteins I found to be stable over time in subject samples and with little inter-individual variability in different subjects, two proteins were identified Synaptosomal-associated protein 23 (SNAP23) and calbindin with 88% prediction success for PD diagnosis. A larger replication study using an orthogonal assay to measure SNAP23 and calbindin demonstrated these proteins together had a 76% prediction success in PD diagnosis. Overall, our data suggest that urinary EVs might serve as an accessible biomarker for neurodegenerative diseases like PD, with particular promise for SNAP23 and calbindin. Future studies are required to determine how SNAP23 and calbindin levels change over time in both health and disease states. These results have broadened the understanding and potential for LRRK2 and other novel biomarkers for PD and related disorders.

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