Pathogenic Parkinson's Disease Mutations of LRRK2 Show Altered Golgi-Endosome Vesicle Trafficking

Abstract

Mutations within the LRRK2 (Leucine Rich Repeat Kinase 2) gene represent the most common genetic alterations linked to sporadic and familial Parkinson’s disease (PD). The majority of these mutations localize within the enzymatic core of the LRRK2 protein, with biochemical characterization of these constructs shown to have altered GTPase and kinase activity. In other research findings, LRRK2 interacts with distinct membrane structures, including caveolin, the endosome, and the Golgi apparatus. The identification that LRRK2 kinase activity is targeting a subset of vesicle trafficking proteins (e.g., Rabs and Endophilin) has established a potential biological function towards vesicle maturation. However, there is a significant deficit of direct observation of LRRK2 being targeted to and interacting with membrane proteins. Therefore, this thesis focused on establishing a system wherein LRRK2 complex formation on vesicles could be tracked in live cells, thus providing an online readout of the LRRK2 function in real-time. Herein this work demonstrates that fluorescence fluctuation microscopy, specifically cross-correlation Raster Imaging Correlation Spectroscopy, can provide a quantitative measurement of LRRK2 interaction with Rab proteins within live cells. These results were exciting as they were nearly identical to values previous reports using traditional biochemicalmethods. Furthermore, the application of these live-cell methods helped differentiate LRRK2-Rab interactions and vesicles formation with cells expressing a PD variant of LRRK2. The most striking readout was our ability to isolate targeted trafficking of LRRK2 bound vesicles and time-dependent interaction with cellular structures. Our findings establish that nanoscale microscopy can provide a unique look into LRRK2 cellular activity, which is not accessible with other experimental techniques. Hence, this might open the pathway in LRRK2 mediated neurodegeneration towards a promising future in PD research.