Parkinson's Disease (PD) is a late-onset neurodegenerative disease characterized by progressive motor dysfunction caused by a loss of dopaminergic neurons for which there is no known cure. Among the most common genetic causes of PD are mutations in the leucine-rich repeat kinase 2 gene (LRRK2), encoding a multi-domain protein with kinase activity. The LRRK2 G2019S mutation causes hyperactivity of the kinase domain and is the most frequent LRRK2 mutation in patients with familial PD, though its role in PD pathology remains unclear. Preliminary data from the lab of our collaborator, Dr. David Park, demonstrated through a genetic screen in Drosophila melanogaster that the deletion of rbp9 encoding an RNA-binding protein prevented pathology induced by PD-relevant mutations in the LRRK2 kinase domain. The neuronal homolog of RBP9 in humans is HuD, a member of the Hu family of RNA-binding proteins that regulates the expression of many transcripts involved in neuronal development, plasticity, and survival. In addition, HuD has been shown to modify the age-at-onset or risk of developing PD. Here, we studied the effect of LRRK2 on the post-transcriptional regulation of mRNAs bound by HuD in the context of PD. Our findings showed that HuD is a substrate for LRRK2 phosphorylation in vitro, and that LRRK2 G2019S hyperphosphorylates HuD. We demonstrated that LRRK2 kinase activity is required for the binding of several transcripts by HuD that encode PD-relevant proteins such as α-synuclein and neuronal survival factor BDNF. Our findings in human neuroblastoma cells indicated that LRRK2 regulates the protein levels of HuD mRNA targets α-synuclein and BDNF in a mechanism that can by modified by HuD. Finally, we showed that the combination of HuD knockout with LRRK2 G2019S expression in mice rescues aberrant expression of HuD targets in mice with only the LRRK2 G2019S mutation or the knockout of HuD alone. Together, our findings demonstrate that LRRK2 affects the post-transcriptional regulation of HuD-bound mRNAs, and suggest the use of HuD as a potential therapeutic target in patients with PD caused by the LRRK2 G2019S mutation.

Indiana University-Purdue University Indianapolis (IUPUI) / Mutations in the gene encoding for leucine rich repeats kinase 2 (LRRK2) are commonly found in Parkinson’s disease. Recently, we found that the disease-associated point mutations at residue R1441 in the G domain (ROC) of LRRK2 resulted in perturbation of its GTPase activity. In this study, we compare the biochemical and biophysical properties of the ROC domain of LRRK2 carrying the PD-associated mutations at residue R1441 with those of the wild-type. We found that the disease-associated mutations (R1441C/G/H) showed marked quaternary structure compared to wild-type, in that the latter existed in solution in both monomeric and dimeric conformations dynamically regulated by GDP/GTP binding state, while we detected only monomeric conformation for three disease-associated mutants. To understand the structural basis for this plasticity and the activity reduction in the mutants, we solved a 1.6 Å crystal structure of the wild type ROC that shows a stable dimeric conformation in which the switch motifs and inter-switch regions mediate extensive interactions at the dimer interface. Residue R1441, where PD-associated mutations occur, forms exquisite interactions at the interface, thus suggesting a critical role of this residue in maintaining a dynamic dimer-monomer interconversion and conformational flexibility of the switch motifs. Consistently, substituting R1441 for other arbitrary mutations (R1441K/S/T) lead to similar perturbation of GTPase activity and dimerization defects as observed in the disease-associated mutants. Locking the ROC domain in either dimeric or monomeric conformations by engineered disulfide bond alters the binding affinity to GTP (but not GDP) and significantly reduce GTPase activity, thus suggesting that the dynamic dimer-monomer interconversion and conformational plasticity are essential for ROC function as a molecular switch modulating the kinase activity of LRRK2.