Delineating Avenues for a Novel Disease Modifying Therapy for Parkinson's Disease Using Advanced Fluorescence Microscopy Techniques.

Sanstrum, Bethany J.

Delineating Avenues for a Novel Disease Modifying Therapy for Parkinson's Disease Using Advanced Fluorescence Microscopy Techniques.

Files

2018-08-phd-sanstrum.pdf (95.79 MB)

Date

2018-08

Authors

Sanstrum, Bethany J.

Department

Cell & Molecular Biology

Abstract

The most common mutation linked to Parkinson’s disease (PD) is the G2019S mutation of the leucine rich repeat kinase 2 (LRRK2) gene. LRRK2 is kinase active as a dimer and is involved in cellular maintenance through regulation of endocytosis and vesicle trafficking. The G2019S mutation has been shown to increase the kinase activity two-fold. I utilized advanced fluorescence microscopy techniques in live differentiated human neuroblastoma (SH-SY5Y) cells transfected with WT LRRK2-EGFP (WT) or G2019S LRRK2-EGFP (G2019S) to determine how the G2019S mutation affects the dynamics of LRRK2-EGFP within the cell and alters its association with substrates, Rab7 and EndophilinA1 (EndoA1). On the plasma membrane, G2019S expressing cells contained significantly higher levels of dimer species and large protein aggregates compared to WT expressing cells. G2019S was also found to have significantly higher levels of self-association and protein aggregation in the cytosol. These findings suggest that the G2019S variant of LRRK2 has an increased propensity to self-associate in the cytosol and on the plasma membrane. I also found alterations to LRRK2 substrate interactions with Rab7 and EndoA1, which likely affect the function of these proteins. Furthermore, because LRRK2 kinase inhibitors have been the focus of recent PD drug development, I also treated cells with well-studied inhibitors, MLi2, LRRK2-IN1, GNE-0877, and GNE-7915, to determine their effects on the intracellular dynamics of LRRK2-EGFP. Treatment of transfected cells with LRRK2 kinase inhibitors showed a significant increase in LRRK2 aggregation in the cytosol and at the plasma membrane. The effect that these aggregates have on cell function could affect their use as clinical treatments and warrants further study. Ultimately, these experiments provide vital information about the dynamics of LRRK2 and the effect of G2019S mutation and kinase inhibitor treatment in a novel context.

Keywords

Fluorescence Microscopy, Parkinson’s disease, LRRK2, EndophilinA1, Rab Proteins

URI

http://hdl.handle.net/10125/62242

Rights

All UHM dissertations and theses are protected by copyright. They may be viewed from this source for any purpose, but reproduction or distribution in any format is prohibited without written permission from the copyright owner.

Collections

Ph.D. - Cell and Molecular Biology

Full item page

Email libraryada-l@lists.hawaii.edu if you need this content in ADA-compliant format.