LRRK2 pathogenic mutants change their enzymatic activities and the increased kinase activity induces p21(CIP1/Waf1) expression in neurons.

Abstract

Parkinson’s disease (PD) is a progressive neurodegenerative disease of which primary symptoms are impaired motor abilities, including resting tremor, rigidity, and bradykinesia. In PD patients, the loss of dopaminergic neuron in substantia nigra pars compacta (SNc) is a major pathological cause. The Leucine-rich repeat kinase2 (LRRK2) is known as one of causative-genes of familial PD patients. LRRK2 contains GTPase and kinase domains and their enzymatic activities are altered by pathogenic mutations. G2019S LRRK2 mutation is the most prevalent and studied mutation and enhance its kinase activity. In this paper, I investigated enzymatic activities of the pathogenic LRRK2 mutants, I2020T and G2385R in vitro and in living cells. I found I2020T and G2385R LRRK2 mutants harbored significantly increased GTPase activity than WT and G2019S mutants. The GTPase activity of R1441C mutants was decreased as previously reports, confirming validity of my GTPase assays. Kinase activity of I2020T showed significant increase than WT in vitro but not in transfected HEK-293T. G2385R mutants showed decreased kinase activity than WT both in transfected HEK-293T and in vitro, but the statistical significance was not observed in vitro.

LRRK2 interacts with, and phosphorylates several substrates which can modulate signal transduction and cell survival. Tumor suppressor p53 is known as a major transcription factor of apoptosis and cell cycle arrest. Phosphorylation of p53 is considered as a critical step for its activation status. LRRK2–related PD is reported as a late-onset disease and PD is an ageing-related disease. The p53-p21 pathway is the most common pathway for the senescence. Chronic senescence is also reported as a cause of ageing-related disease. My study revealed that LRRK2 phosphorylates p53 in vitro kinase assay. LRRK2 had been reported to phosphorylate TXR site. The mutation study suggested that T304 and T377 are sites of phosphorylation by LRRK2. In differentiated SH-SY5Y cells, p53 interacts with LRRK2 and decreased LRRK2 kinase activity by shLRRK2 transfection or treatment with LRRK2 kinase inhibitor reduced TXR phosphorylation of p53. Overexpression of G2019S LRRK2 by transient transfection showed increased phosphorylation of TXR sites on p53. Reduced LRRK2 kinase activity or G2019S LRRK2 expression showed decreased or increased translocalization of p53 to nucleus, respectively. Phospho-mimic (T304/377D) p53 also showed increased nucleus localization than that of WT p53. Reduced LRRK2 kinase experiments showed lower expression of p21 than normal differentiated SH-SY5Y or rat primary neuron. Increased LRRK2 kinase activity and phosphomimic p53 experiments also showed inducing p21 expression in differentiated SH-SY5Y, rat primary neuron. In addition, increased expression of p21 was observed in G2019S transgenic mouse mid brain. Moreover, induced p21 expression by ectopic p21, or expression of G2019S LRRK2 or phosphomimic p53 also promoted cytotoxicity in differentiated SH-SY5Y or rat primary neuron. The regulation mechanism between the GTPase and kinase activities of LRRK2 may be different from the classical relationship between GTPase and kinase. Besides, altered LRRK2 enzymatic activities would contribute to distinct pathogenesis of PD. And the increased phosphorylation of p53 by G2019S LRRK2 pathogenic mutants induces the p21 expression, leading to cytotoxic response via p21 expressing-senescence. This may be one of LRRK2-mediated PD pathogenesis mechanisms.