Funded Studies
Objective/Rationale:
The presynaptic alteration seen in several LRRK2 disease model suggest that LRRK2 might influence synaptic function through effects on presynaptic proteins. The common G2019S mutation consistently increases LRRK2 kinase activity in vitro suggesting that altered activity is linked to disease. Given that synaptic vesicles (SV) cycle is plastically shaped by kinases and phosphates, we aim to investigate if mutated LRRK2 alters SVs trafficking via phosphorylation of presynaptic proteins.
Project Description:
Notwithstanding the strong afford already spent by different laboratories in order to identify LRRK2 substrates, up to now no targets have been identified and validated. An intriguing possibility is that targets of LRRK2 are hidden among LRRK2 interactors. Therefore, we will perform highly specific in vitro assay focusing on a selected panel of presynaptic phosphoproteins we have described as LRRK2 interactors. LRRK2 kinase activity may perturb LRRK2 protein conformation and supramolecular organization, thus we plan to characterize biochemically the interaction between SV and LRRK2 upon alteration of LRRK2 endogenous kinase activity. A proper SV trafficking is instrumental for correct synaptic transmission and neuron functionality. Thus, we will characterize the impact of LRRK2 kinase activity on SV trafficking and synaptic transmission. The main model will consist in primary neuron cultures from wild-type pre-clinical models where LRRK2 kinase activity will be altered by pharmacological ablation; a combination of imaging techniques will allow us to analyze SV cycle.
Relevance to Diagnosis/Treatment of Parkinson’s Disease:
Independent evidences correlate LRRK2 to SV trafficking and interestingly, an increased dopamine turnover has been described also in LRRK2 mutation carriers. Thus, defects of SV trafficking in dopaminergic neurons might severely contribute to Parkinson’s Disease (PD) progression. Our aims are not only to provide critical information about the physiological role of LRRK2, but also to pave the way to understand the implication of LRRK2 during PD progression and to evaluate the potential feasibility of pharmacological approaches targeting LRRK2 kinase activity.
Anticipated Outcome:
The first goal is to define the amino acid motif, specifically phosphorylated by LRRK2 within at least one of the putative target proteins. The second goal is to determine how kinase activity modifies LRRK2 binding properties towards SV and SV associated proteins. The third expected result is to determine the specific impact of LRRK2 kinase activity on presynaptic SV release and to identify the role of LRRK2 at the presynaptic site.