An Investigation on the Pathogenic Mechanism of Mutant Lrrk2-Induced Parkinson$S Disease Using Transgenic Mouse Model.

Parkinson』s disease (PD) affects ~ 1 % of the population above the age of 60 and is the most common neurodegenerative motor disorder. Motor dysfunction of PD patients stems from degeneration of dopaminergic neurons in substantia nigra pars compacta (SNpc) that give rise to nigrostriatal pathway and the resulting impairment of dopaminergic neurotransmission in the striatum, which is required for normal motor function. The majority (~90 %) of PD cases is sporadic, and Mendelian familial forms of PD account for ~10 % of PD patients. Patients with familial type 8 of Parkinson』s disease (PARK8) exhibit late-onset parkinsonism symptoms and autosomal dominant inheritance. Recent studies identified missense mutations of leucine-rich repeat kinase 2 (LRRK2) as the cause of PARK8. LRRK2 is the most frequent causative gene in late-onset autosomal dominant PD. Furthermore, LRRK2 mutations are also detected in a significant fraction of sporadic PD patients. Especially, polymorphic (G2385R) LRRK2 mutation is the first identified genetic risk factor for sporadic PD patients of Chinese ethnicity. Late-onset clinical features of PD patients with LRRK2 mutation are similar to those of sporadic PD patients, suggesting that common molecular mechanisms are involved in the pathogenesis of both PARK8 and sporadic PD. Therefore, a better understanding of cellular and molecular mechanisms underlying mutant LRRK2-induced parkinsonism is expected to unravel the pathogenic mechanism of PARK8 and sporadic PD. In the present study, in vivo PD animal model is prepared by generating transgenic mice expressing mutant LRRK2. Subsequently, the following experiments are performed to investigate cellular and molecular mechanisms of mutant LRRK2-induced neurotoxicity and degeneration of nigrostriatal system using mutant LRRK2 transgenic mice: (1) LRRK2 mutation is believed to cause PD by directly inducing degeneration of SNpc dopaminergic neurons. However, a high level of LRRK2 expression is found in the striatum, and substantia nigra expresses a low level of LRRK2. Thus, it is possible that mutant LRRK2 causes neurotoxicity in dopaminoceptive striatum and indirectly causes degeneration of SNpc dopaminergic neurons. In the present study, we will determine the action site of mutant LRRK2-induced neurotoxicity in the nigrostriatal system of mutant LRRK2 transgenic mice. (2) Mitochondrial dysfunction and resulting activation of mitochondria-mediated apoptotic pathway are involved in degeneration of SNpc dopaminergic neurons in PD patients.Wild-type LRRK2 is expressed in the outer membrane of mitochondria and believed to play an important role in mitochondrial function. Thus, we will test the hypothesis that mutant LRRK2 causes mitochondrial dysfunction of SNpc dopaminergic or striatal medium spiny neurons and neuronal death by activating mitochondria-mediated apoptotic pathway. (3) Wild-type LRRK2 is believed to regulate neurotransmitter release and intracellular Ca2+ homeostasis of SNpc dopaminergic or striatal neurons. Therefore, whole-cell patch-clamp recordings and [Ca2+]i imaging using brain slices are performed to test the possibility that mutant LRRK2 causes the dysfunction of SNpc dopaminergic or striatal medium spiny neurons by altering synaptic responses, synaptic plasticity and intracellular Ca2+ dynamics, leading to an impaired motor function of nigrostriatal system and resulting parkinsonism. (4) LRRK2 belongs to the mitogen-activated protein kinase kinase kinase (MAPKKK) subfamily. It has been shown that PARK8 mutations including R1441C and G2019S resulted in augmented kinase activity of LRRK2 and that enhanced kinase activity of mutant LRRK2 mediated cytotoxicity. Thus, identification of substrate(s) of LRRK2-mediated phosphorylation is required to understand physiological function of LRRK2 and molecular mechanism of mutant LRRK2-induced parkinsonism. In the present study, proteomic analysis is performed to identify putative substrates of wild-type and mutant LRRK2. Our study should shed light on the pathogenic mechanism of PARK8 and sporadic PD and physiological function(s) of LRRK2 in the brain. The results obtained from the present investigation could also lead to the development of possible therapeutic strategies for PD. Keywords：Parkinson』s disease; Leucine-rich repeat kinase 2; PARK8; substantia nigra dopaminergic neurons; striatal medium spiny neurons.

Project ID:PA9706-1120
External Project ID:NSC97-3112-B182-005