M.D. Cirnaru
Università degli Studi di Milano, 2016-01-11

Parkinson’s disease (PD) is a common neurodegenerative disease clinically characterized by bradykinesia, rigidity and resting tremor. PD is characterized pathologically by the degeneration of nigrostriatal dopaminergic neurons and the presence of Lewy bodies containing a small protein, alpha-synuclein. Mutations in Leucine-rich repeat kinase 2 gene (LRRK2) are associated with familial and sporadic Parkinson’s disease (PD). LRRK2 is a complex protein that consists of multiple domains executing several functions, including GTP hydrolysis, kinase activity, and protein binding. There are many single nucleotide alterations covering LRRK2's functional domains, but the main missense mutations that clearly segregate with PD in large family studies, cluster within the enzymatic domains. The G2019S mutation falls in the activation loop of the kinase domain generating a 2-fold increase in LRRK2 kinase activity. Although patients with LRRK2 mutations usually respond to levodopa therapy, this treatment is only symptomatic and it does not cure the cause of the disease. In particular, LRRK2 mutations lead to neuronal cell death and toxic protein aggregates and LRRK2 kinase activity seems to be responsible for the observed neurotoxicity. Our previous research pointed out that LRRK2 acts at the presynaptic site where interacts with synaptic vesicles (SV) and presynaptic proteins together with which it controls SV trafficking in a kinase depended manner. Our recent data indicate NSF not only as an interactor but also as a substrate for LRRK2 kinase activity in vitro. In the present work we analyze how LRRK2 increased kinase activity conferred by the PD related G2019S mutation influences the neuronal functions. We investigated whether G2019S mutation might affect presynaptic function in short term and substrate clearance in long term. Next, we evaluated the feasibility of two potential therapeutic strategies: the first implies the use of LRRK2 kinase inhibitors while the second focuses on treatment ameliorating protein degradation via induction of autophagy. By dynamic studies of SV release in cultured neurons of human LRRK2 G2019S (GS) overexpressing mice and in breed wild-type mice, we found that the increase of LRRK2 kinase activity positively correlates with an increase in the endocytosis rate of the SV. Moreover, we report also an impairment in the complexity of the neuronal tree of the GS neurons that depends on both increased protein level and kinase activity. We recently reported that LRRK2 phosphorylates NSF at threonine 645 inducing an increase in NSF’s ATP hydroxylation rate that determine an increase in the SNARE disassembly. Moreover, we found that aged GS mice show aberrant NSF protein accumulation and motor as well as cognitive impairment. We report also that the chronic treatment with trehalose, an autophagy inducing molecule, partially recovered the motor phenotype and NSF aggregation proposing it as an interesting therapeutic strategy.

diritti: info:eu-repo/semantics/openAccess
In relazione con info:eu-repo/semantics/altIdentifier/hdl/2434/353078
coordinatore: A. Corsini ; relatore: F. Gardoni; correlatore: G. Piccoli
Settore BIO/14 - - Farmacologia

Tesi di dottorato. | Lingua: Inglese. | Paese: | BID: TD18002328