Objective/Rationale:
Autosomal dominant mutations in LRRK2 have been identified as a common cause for late-onset PD. Two of the most frequent PD-causing mutations, G2019S and R1441C, occur within the respective kinase and GTPase domains of LRRK2, and they are associated with enhanced LRRK2 kinase activity and neurotoxicity in vitro. We propose to use the nematode C. elegans as an experimental organism to model the effect of mutant LRRK2 on dopaminergic neurodegeneration and locomotor dysfunction, and to assess the critical role of kinase and GTPase domains in LRRK2-induced phenotype.
Project Description:
C. elegans has been used extensively to model various neurodegenerative diseases due to highly conserved neural pathways between the invertebrate and mammals. We have generated several transgenic C. elegans lines carrying the LRRK2 gene encoding the wild-type R1441C and G2019S proteins in dopaminergic neurons. Preliminary results suggest that overexpression of LRRK2 causes neurodegeneration and locomotor dysfunction, a phenotype that is more severe in animals carrying mutant LRRK2 than those with wild-type LRRK2. We will generate and characterize additional C. elegans models that carry kinase-dead or GTPase-dead mutations in the LRRK2 gene to determine whether kinase and GTPase activities of LRRK2 are required for mutant LRRK2-linked pathologic phenotype. We will also examine whether human LRRK2 is functionally conserved with LRK-1, the sole C. elegans homolog of LRRK2, in regulating neuronal function in vivo.
Relevance to Diagnosis/Treatment of Parkinson’s Disease:
The LRRK2 C. elegans transgenic lines, once characterized and validated as proposed, may serve as valuable in vivo models of PD for deciphering mechanisms underlying LRRK2-linked PD pathogenesis. In addition, C. elegans is amenable to common screening platforms for drug discovery. The LRRK2 C. elegans models of PD have the potential to be useful tools to facilitate the screening of compounds toward the development of PD therapeutics.
Anticipated Outcome:
We anticipate that our work will be able to address whether LRRK2 enzymatic activity is directly responsible for the LRRK2-induced phenotype in animal models. The successful development of LRRK2 C. elegans models will result in a valuable resource for the mechanistic studies of the role of LRRK2 in neuronal function and pathogenesis of PD, as well as an in vivo system for testing pharmacologic and therapeutic intervention for LRRK2-associated phenotype.
Final Outcome
We have established LRRK2-linked PD models in C. elegans that recapitulates several key features of PD, including DA neurodegeneration, and DA-specific locomotive dysfunction. The pathological phenotype induced by LRRK2 hyperactivation is reversible by treatment with exogenous DA. In contrast, expression of the GTP binding defective mutant, K1347A, or knockout of the C. elegans LRRK2 homolog, LRK-1, prevents the LRRK2-induced neurodegeneration and behavioral abnormalities. Our findings provide strong support for the critical role of GTPase/kinase activity in LRRK2-linked pathologies. These invertebrate models will be useful for studying pathogenesis of PD and for development of potential therapeutics for the disease. We have also found that knockout of C. elegans lrk-1 gene attenuates the LRRK2-induced neurodegenerative and behavioral phenotype, and may also confer increased resistance to heat shock and extended life span. Additional studies have expanded the scope of this project including the generation of pan-neuronal LRRK2 transgenic worm lines and the pilot experiments demonstrating the effect of a putative LRRK2 inhibitor in vivo using our C. elegans LRRK2 model of PD.