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 second year of this project, we have successfully generated homozygous PINK1 knockout mice. Mouse PINK1 gene contains eight exons and is approximately 13 kb long. To generate PINK1 knockout mice, homologous recombination was targeted on the exon 1, which contains the start codon. The deletion of exon 1 is expected to result in the absence of functional PINK1 mRNA and PINK1 protein expression. Linearized PINK1 knockout target vector containing lacZ-neomycin resistance reporter/selection cassette was transfected into mouse ES cells. Southern blot analysis was performed to select correctly targeted ES cell lines, in which the exon 1 of PINK1 gene is absent. We have obtained two clones of ES cells with corrected homologous recombination, and these cell lines were injected into blastocysts and used for the generation of chimeric mice, which were then mated with C57BL/6J mice and bred to obtain F1 heterozygous mutant mice. Subsequently, F2 homozygous PINK1 knockout mice were obtained by interbreeding of F1 heterozygous mice. Southern blot analysis and PCR genotyping were performed to confirm the germline transmission of targeted allele in heterozygous PINK1+/- and homozygous PINK1-/- mice. The successful generation of homozygous PINK1-/- mice was further confirmed by Northern blot analysis. In contrast to wild-type or PINK1+/- mouse, an expected 2.3 kb PINK1 mRNA was absent in the brain of homozygous PINK1-/- mouse. RT-PCR analysis also demonstrated that in contrast to wild-type mouse, PINK1 mRNA was absent in the brain or heart of PINK1-/- mouse. It was hypothesized that PNIK1 exerts neuroprotective and anti-apoptotic effects on substantia nigra (SN) dopaminergic cells and that the absence of PINK1 expression lead to the degeneration of SN dopaminergic neurons and resulting parkinsonism. Various behavioral tests were used to evaluate the motor functions of wild-type or PINK1-/- mice of vary ages. Rotarod test indicated that balance and motor coordination functions of 9-month-old PINK1-/- mice were normal. Spontaneous locomotor activity of PINK1-deficient mice at 9 months of age was similar to that of wild-type mice. The pole test also indicated that the motor performance of 9-month-old PINK1-/- mice was not significantly different from that of wild-type mice. Immunohistochemical staining using anti-tyrosine hydroxylase antibody demonstrated that number and morphology of dopaminergic neurons in the substantia nigra of PINK1-deficient mice at 9 months of age were similar to those of wild-type mice. It is possible that unlike early-onset human PARK6 patients, PINK1 deficiency-induced neurodegeneration of SNpc dopaminergic neurons and resulting motor dysfunction in mice has a slower time course. Further study is required to investigate possible neurological phenotypes of PINK1-/- mice with an older age. The absence of PARK6 parkinsonism phenotypes in 9-month-old PINK1-null mice could result from several possible mechanisms, including the redundancy of mitochondrial Ser/Thr protein kinases, the absence of appropriate environmental triggers, compensation or species-dependent PINK1 function. Better understanding of molecular mechanism(s) responsible for the absence of neurological phenotypes in 9-month-old PINK1-/- mice could advance our knowledge of PD pathophysiology. The absence of PARK6 phenotypes in PINK1-deficient mice at 9 months of age suggests that knockin mouse model expressing PARK6 mutant PINK1 should also be prepared as in vivo PARK6 animal model. Multiple lines of evidence indicated that PINK1 plays an important role in regulating mitochondrial functions. Previous studies showed that inactivation of PINK1 gene in Drosophila caused defects in mitochondrial function and morphology. Knockdown of PINK1 expression in zebrafish also induced mitochondrial dysfunction and an increase in reactive oxygen species (ROS) production. Our recent investigation also demonstrated that wild-type PINK1 produced anti-apoptotic effect by blocking mitochondrial release of cytochrome-c and subsequent caspase-3 activation. Therefore, absence of PINK1 expression is likely to cause mitochondrial dysfunction of substantia nigra (SN) dopaminergic neurons. In the present study, we tested this hypothesis by measuring mitochondrial membrane potential (Δm) of substantia nigra neurons prepared from wild-type or PINK1-/- mice.Live cell imaging of potential sensitive dye TMRM fluorescence was performed to visualize hyperpolarized mitochondrial membrane potential (Δm) of cultured SN dopaminergic neurons and non-dopaminergic neurons prepared from control or PINK1-deficient mice. Compared to cultured wild-type SN dopaminergic cells, TMRM fluorescent intensity and Δm were significantly reduced in PINK1-deficienct SN dopaminergic neurons. A significant reduction in TMRM fluorescent signal and depolarized Δm was also observed from SN non-dopaminergic cells of PINK1-/- mice. These results indicate that PINK1 deficiency causes a loss of mitochondrial membrane potential of substantia nigra neurons. Mitochondria are the major intracellular site of free radical formation. Reactive oxygen species (ROS) formation in cultured SN dopaminergic neurons was measured by visualizing fluorescent imaging of cell-permeant ROS indicator DCF. Compared to wild-type SN dopaminergic cells, the basal ROS level was significantly increased in PINK1-deficient SN dopaminergic neurons. We also compared oxidative stressor H2O2-induced ROS production in cultured SN dopaminergic neurons of wild-type or PINK1-/- mice. H2O2-induced DCF fluorescence and ROS formation is greatly augmented in putative SN dopaminergic neurons of PINK1-/- mice. Our results indicate that PINK1 is required for maintaining mitochondrial membrane potential, which is the driving force behind oxidative phosphorylation and ion transportation. PINK1 deficiency-induced loss of mitochondrial membrane potential is expected to cause mitochondrial dysfunction. Basal ROS level and ROS formation induced oxidative stressor H2O2 were significantly increased in cultured SN dopaminergic neurons of PINK1-null mice, suggesting that absence of PINK1 expression causes oxidative stress in SN dopaminergic neurons. Our results provide the first evidence that lack of functional PINK1 expression in SN dopaminergic neurons causes mitochondrial dysfunction and elevated oxidative stress, which subsequently results in neuronal dysfunction and death of SN dopaminergic cells. Wild-type PINK1 is believed to function as a mitochondrial Ser/Thr protein kinase, suggesting that PINK1 maintains mitochondrial membrane potential, prevents ROS formation and exerts its neuroprotective effects by phosphorylating mitochondrial proteins. Our previous study identified -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 with the aid of MALDI-TOF mass spectrometry. In the third year of this project, the following experiments will be performed: (1) Proteomic analysis identified -subunit of mitochondrial ATP synthase (ATPase-), mitochondrial heat shock protein 60 (mthsp60) and mitochondrial heat shock protein 70 (mthsp70) as substrates of PINK1-mediated phosphorylation. Further studies are performed to investigation the functional importance of PINK1-mediated phosphorylation of ATPase-mthsp60 or mthsp70. (2) Our study provides the evidence that PINK1 deficiency-induced mitochondrial dysfunction and enhanced oxidative stress is involved in PARK6 pathogenesis. SN dopaminergic neurons of PINK1-null mice are further used to investigate molecular mechanisms by PINK1 deficiency causes the loss of mitochondrial membrane potential and increased ROS production. (3) In addition to PINK1 knockout mice, knockin mice expressing PARK6 mutant (G309D) or (L347P) PINK1 are generated to investigate molecular mechanisms underlying PINK1 neuroprotective function and pathogenesis of PARK6.

Project ID:PG9804-0589
External Project ID:NHRI-EX98-9619NI