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Rab10 Phosphorylation Detection by LRRK2 Activity Using SDS-PAGE with a Phosphate-binding Tag
Published on: December 14, 2017
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Cellular and subcellular localization of Rab10 and phospho-T73 Rab10 in the mouse and human brain
Vijay Singh1, Marissa A Menard1, Geidy E Serrano2
1Center for Neurodegeneration and Experimental Therapeutics, University of Alabama at Birmingham, Birmingham, AL, 35294, USA.
Acta Neuropathologica Communications
|December 19, 2023
Summary
Mutations in Leucine-rich repeat kinase 2 (LRRK2) cause Parkinson's disease. Phosphorylated Rab10 (pRab10) is found in all brain cells but concentrates at presynaptic terminals, suggesting a role in neuronal dysfunction.
Area of Science:
- Neuroscience
- Cell Biology
- Genetics
Background:
- Autosomal dominant mutations in Leucine-rich repeat kinase 2 (LRRK2) are a genetic cause of Parkinson's disease (PD).
- The G2019S-LRRK2 mutation elevates LRRK2 kinase activity, leading to substrate hyper-phosphorylation.
- Rab10, a substrate of LRRK2, is phosphorylated at Threonine 73 (pRab10) and is implicated in neurodegenerative diseases like PD and Alzheimer's.
Purpose of the Study:
- To determine the cellular and subcellular localization of Rab10 and pRab10 in the brain.
- To understand the functional role of Rab10 and its post-translational modifications in neurodegeneration.
- To validate antibodies for detecting pRab10 in neural tissues.
Main Methods:
- Antibody specificity for pRab10 was confirmed using Rab10 antisense oligonucleotides and Rab10 knockout neurons.
- Tyramide signal amplification was employed to enhance weak immunofluorescence signals of pRab10.
- Immunofluorescence and colocalization studies were performed in mouse and human brain tissues.
Main Results:
- Rab10 and pRab10 were detected in various brain regions (cortex, striatum, substantia nigra) and cell types (neurons, astrocytes, microglia, oligodendrocytes).
- While Rab10 localized to the endoplasmic reticulum, lysosomes, and Golgi, pRab10 did not show significant colocalization with these organelles.
- pRab10 significantly overlapped with presynaptic terminal markers, including alpha-synuclein, in both mouse and human cortex.
Conclusions:
- Rab10 and pRab10 are ubiquitously expressed in the brain, but pRab10 is specifically enriched at neuronal presynaptic terminals.
- Increased LRRK2 kinase activity in PD may alter Rab10-mediated membrane trafficking at presynaptic terminals.
- These findings highlight a potential mechanism for LRRK2-associated neurodegeneration involving presynaptic dysfunction.
