Experiments in HEK-293 cells

The end-stage pathology of Parkinson’s disease (PD) involves the loss of dopamine-producing neurons in the substantial nigra compacta, but synaptic deregulation of these neurons begins much earlier in the disease process.  Understanding the mechanisms of axonal deficits may provide opportunities for early therapeutic intervention, yet they remain largely unknown. Given that increased kinase activity is an accepted pathogenic mechanism for the action of mutant LRRK2, our data in mice and findings in human showing higher LRRK2 levels in vulnerable dopamine neurons suggest that the aberrant phosphorylation of downstream targets in these neuronal populations may contribute to presynaptic dysfunctions. To investigate the molecular mechanisms by which the LRRK2 kinase activity may mediate presynaptic defects in dopamine neurons, we performed a phosphoproteomics screen using striatal synaptosomes from Lrrk2G2019S mice with or without LRRK2 kinase inhibitor treatment. We identified Rab3 proteins as differentially phosphorylated presynaptic LRRK2 substrates, consistent with previous reports indicating that Rab proteins serve as robust LRRK2 substrates across various cell types. Rab3 proteins are known to play a role in presynaptic vesicle trafficking events, and we found Rab3a and Rab3c to be amongst the most highly expressed proteins in our vulnerable dopamine neuron-specific proteome dataset. We demonstrated that LRRK2 phosphorylates these Rab3 proteins in synaptosomes from the SNc and striatum. Our unbiased MS data using mouse brain extracts indicate that increased Rab3 phosphorylation diminishes its interaction with Rim1 and Rim2 proteins. This is only observed with phosphorylated Rab3 but not with overexpressed phosphomimetic Rab3 constructs, widely used to probe consequences of altered phosphorylation across several studies.