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Updated: Jul 31, 2025
Rab10 Phosphorylation Detection by LRRK2 Activity Using SDS-PAGE with a Phosphate-binding Tag
Published on: December 14, 2017
The LRRK2 kinase substrates Rab8a and Rab10 contribute complementary but distinct disease-relevant phenotypes in
Adamantios Mamais1, Anwesha Sanyal2, Austin Fajfer1
1Center for Translational Research in Neurodegenerative Disease, Department of Neurology, University of Florida, Gainesville, Florida, USA.
Abstract:
Mutations in the LRRK2 gene cause familial Parkinson's disease presenting with pleomorphic neuropathology that can involve α-synuclein or tau accumulation. LRRK2 mutations are thought to converge toward a pathogenic increase in LRRK2 kinase activity. A subset of small Rab GTPases have been identified as LRRK2 substrates, with LRRK2-dependent phosphorylation resulting in Rab inactivation. We used CRISPR/Cas9 genome editing to generate a novel series of isogenic iPSC lines deficient in the two most well validated LRRK2 substrates, Rab8a and Rab10, from two independent, deeply phenotyped healthy control lines. Thorough characterization of NGN2-induced neurons revealed divergent effects of Rab8a and Rab10 deficiency on lysosomal pH, LAMP1 association with Golgi, α-synuclein insolubility and tau phosphorylation, while parallel effects on lysosomal numbers and Golgi clustering were observed. Our data demonstrate largely antagonistic effects of genetic Rab8a or Rab10 inactivation which provide discrete insight into the pathologic features of their biochemical inactivation by pathogenic LRRK2 mutation.
Insights
Mutations in Leucine-Rich Repeat Kinase 2 (LRRK2) cause Parkinson's disease. Inactivating LRRK2 substrates Rab8a and Rab10 in neurons revealed distinct effects on cellular pathways, offering new insights into LRRK2-related neurodegeneration.
Area of Science:
- Neuroscience
- Genetics
- Cell Biology
Background:
- Mutations in the Leucine-Rich Repeat Kinase 2 (LRRK2) gene are a significant cause of familial Parkinson's disease.
- Pathogenic LRRK2 mutations are associated with increased kinase activity, impacting cellular processes.
- LRRK2 substrates, including Rab GTPases like Rab8a and Rab10, are implicated in neurodegenerative pathways.
Approach:
- CRISPR/Cas9 genome editing was employed to create isogenic induced pluripotent stem cell (iPSC) lines lacking functional Rab8a and Rab10.
- These engineered cell lines were differentiated into NGN2-induced neurons for detailed cellular analysis.
- Two independent, well-characterized healthy control lines were used to ensure reproducibility.
Key Points:
- Rab8a and Rab10 deficiency exhibited divergent impacts on neuronal lysosomal pH, alpha-synuclein insolubility, and tau phosphorylation.
- Both Rab8a and Rab10 inactivation led to parallel alterations in lysosomal number and Golgi clustering.
- The study highlights largely antagonistic effects between Rab8a and Rab10 inactivation.
Conclusions:
- Genetic inactivation of Rab8a or Rab10 in neurons provides discrete insights into Parkinson's disease pathology linked to LRRK2.
- These findings suggest distinct roles for Rab8a and Rab10 in cellular homeostasis relevant to LRRK2 neuropathology.
- Understanding these divergent effects is crucial for deciphering the complex mechanisms of LRRK2-driven neurodegeneration.
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