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Updated: Jul 8, 2025
Rab10 Phosphorylation Detection by LRRK2 Activity Using SDS-PAGE with a Phosphate-binding Tag
Published on: December 14, 2017
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.
Abstract:
Autosomal dominant pathogenic mutations in Leucine-rich repeat kinase 2 (LRRK2) cause Parkinson's disease (PD). The most common mutation, G2019S-LRRK2, increases the kinase activity of LRRK2 causing hyper-phosphorylation of its substrates. One of these substrates, Rab10, is phosphorylated at a conserved Thr73 residue (pRab10), and is one of the most abundant LRRK2 Rab GTPases expressed in various tissues. The involvement of Rab10 in neurodegenerative disease, including both PD and Alzheimer's disease makes pinpointing the cellular and subcellular localization of Rab10 and pRab10 in the brain an important step in understanding its functional role, and how post-translational modifications could impact function. To establish the specificity of antibodies to the phosphorylated form of Rab10 (pRab10), Rab10 specific antisense oligonucleotides were intraventricularly injected into the brains of mice. Further, Rab10 knock out induced neurons, differentiated from human induced pluripotent stem cells were used to test the pRab10 antibody specificity. To amplify the weak immunofluorescence signal of pRab10, tyramide signal amplification was utilized. Rab10 and pRab10 were expressed in the cortex, striatum and the substantia nigra pars compacta. Immunofluorescence for pRab10 was increased in G2019S-LRRK2 knockin mice. Neurons, astrocytes, microglia and oligodendrocytes all showed Rab10 and pRab10 expression. While Rab10 colocalized with endoplasmic reticulum, lysosome and trans-Golgi network markers, pRab10 did not localize to these organelles. However, pRab10, did overlap with markers of the presynaptic terminal in both mouse and human cortex, including α-synuclein. Results from this study suggest Rab10 and pRab10 are expressed in all brain areas and cell types tested in this study, but pRab10 is enriched at the presynaptic terminal. As Rab10 is a LRRK2 kinase substrate, increased kinase activity of G2019S-LRRK2 in PD may affect Rab10 mediated membrane trafficking at the presynaptic terminal in neurons in disease.
Insights
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.
