An investigation on the pathogenic mechanism of mutant PINK1-induced hereditary Parkinson's disease

Parkinson's disease (PD) affects ~ 1 % of the population above the age of 60 and is the most common neurodegenerative motor disorder. Recent studies indicated that numerous missense or truncating mutations of phosphatase and tesin homologue (PTEN)-induced kinase 1 (PINK1) gene are implicated in the pathogenesis of familial type 6 of Parkinson’s disease (PARK6) and that PINK1 is the second most frequent causative gene in early-onset Parkinson’s disease (PD)). PINK1 is believed to exert a neuroprotective effect on dopaminergic neurons in substantia nigra pars compacta (SNpc). PINK1 mutations associated with PARK6 could cause the loss of neuroprotective function and resulting degeneration of SNpc dopaminergic neurons and parkinsonism. The elucidation of molecular mechanism underlying PINK1 neuroprotective function is essential for understanding the pathogenesis of PARK6 caused by PINK1 mutations. PINK1 mutations have also been found in patients affected with sporadic early-onset parkinsonism. PARK6 and sporadic PD patients exhibit similar clinical features. Therefore, it is very likely that common molecular mechanisms are involved in the pathogenesis of both PARK6 and sporadic PD. Therefore, elucidation of the pathogenic mechanism underlying PARK6 is expected to shed a light on the molecular pathogenesis of more common sporadic PD. In the present study, we studied molecular mechanisms underlying the PINK1 neuroprotective function and pathogenic mechanism of PARK6 by performing the following investigations: (1) Wild-type PINK1 is believed to produce the neuroprotective and anti-apoptotic effects. In contrast, PARK6 mutant PINK1 loses its anti-apoptotic effect and causes the apoptotic death of SNpc dopaminergic neurons. To test this hypothesis, we investigated molecular mechanisms underlying wild-type PINK1-induced anti-apoptotic effect and the loss of anti-apoptotic function caused by PARK6 mutations. (2) PINK1 protein is mainly expressed in the mitochondria and that mitochondrial expression is necessary for PINK1-mediated protective effect. The Ser/Thr kinase domain is the only functional domain in PINK1 protein. Therefore, PINK1 functions as a mitochondrial Ser/Thr protein kinase and exerts its neuroprotective effect on SNpc dopaminergic neurons by phosphorylating unknown substrates in the mitochondria. In the present study, proteomic analysis was performed to identify mitochondrial PINK1 substrate(s). (3) Although in vitro cellular model has been widely used to study pathogenic mechanisms of neurological disorders, important findings observed from in vitro cellular model have to be corroborated by in vivo animal model. Furthermore, better understanding the molecular basis of mutant PINK1-induced degeneration of SNpc dopaminergic neurons in vivo is essential for the development of effective therapy for PARK6 or sporadic PD. The autosomal recessive inheritance mode indicates a loss-of-function caused by PINK1 mutations is involved in the pathogenesis of PARK6. In the present study, PINK1 knockout mice are prepared to investigate the molecular pathogenesis of PARK6 and physiological functions of PINK1 in vivo. In the first year of this project, our results indicated that overexpression of wild-type PINK1 blocked mitochondrial release of apoptogenic cytochrome-c, caspase-3 activation and apoptotic cell death induced by proteasome inhibitor MG132. N-terminal truncated PINK1(N35), which lacks mitochondrial localization sequence, did not block MG132-induced cytochrome-c release and cytotoxicity. Despite mitochondrial expression, PARK6 mutant (E240K), (H271Q), (G309D), (L347P), (E417G) and C-terminal truncated (C145) PINK1 failed to inhibit MG132-induced cytochrome-c release and caspase-3 activation. Overexpression of wild-type PINK1 blocked cytochrome-c release and cell death caused by atractyloside, which opens mitochondrial permeability transition pore (mPTP). PARK6 PINK1 mutants failed to inhibit atractyloside-induced cytochrome-c release. These results suggest that PINK1 exerts anti-apoptotic effect by inhibiting the opening of mPTP and that PARK6 mutant PINK1 loses its ability to prevent mPTP opening and cytochrome-c release. To identify putative mitochondrial substrates of PINK1, phosphorylated proteins of mitochondrial fraction prepared from control or PINK1-expressing cells were enriched by using phosphoprotein purification kit and then subjected to two-dimensional difference gel electrophoresis. Subsequently, phosphorylated protein spots upregulated in mitochondrial fraction of PINK1-expressing cells were excised, digested and subjected to peptide mass fingerprinting with the aid of MALDI-TOF mass spectrometry. We’ve successfully identified beta-subunit of mitochondrial ATP synthase, mitochondrial heat shock protein 60 (mthsp60) and mitochondrial heat shock protein 70 (mthsp70), which play important roles in mitochondrial functions, as substrates of PINK1-mediated phosphorylation. In the present study, mice lacking PINK1 expression are prepared to investigate the molecular pathogenesis of PARK6 and physiological functions of PINK1 in vivo. Linearized PINK1 knockout target vector was transfected into mouse ES cells. Subsequently, ES cells with corrected homologous recombination were injected into blastocysts. We have successfully obtained several heterozygous PINK1-deficient mice, which are then bred to generate homozygous PINK1 knockout mice. In the second and third year of this project, the following experiments will be performed: (1) Wild-type PINK1 is likely to inhibit the opening of mPTP and exerts its anti-apoptotic effect by phosphorylating component(s) of mPTP, such as voltage-dependent anion channel (VDAC) and adenine nucleotide translocator (ANT). On the other hand, PARK6 mutant PINK1 fails to phosphorylate subunit(s) of mPTP and block the opening of mPTP, leading to the loss of anti-apoptotic effect. Further in vitro and in vivo phosphorylation assays are performed to test the importance of mPTP phosphorylation in PINK1-mediated anti-apoptotic and neuroprotective effects. (2) Proteomic analysis identified beta-subunit of mitochondrial ATP synthase (ATPase-beta), mitochondrial heat shock protein 60 (mthsp60) and mitochondrial heat shock protein 70 (mthsp70) as substrates of PINK1-mediated phosphorylation. Further studies will be performed to investigation the functional importance of PINK1-mediated phosphorylation of ATPase-beta, mthsp60 or mthsp70. (3) PINK1 knockout mice are used to investigate molecular mechanisms underlying the PINK1 neuroprotective function and pathogenesis of PARK6.

Project ID:PG9704-0398
External Project ID:NHRI-EX97-9619NI