The Parkinson's disease (PD) causative PINK1 gene encodes a mitochondrial protein kinase called PTEN-induced kinase 1 (PINK1). The autosomal recessive pattern of inheritance of PINK1 mutations suggests that PINK1 is neuroprotective and therefore loss of PINK1 function causes PD. Indeed, overexpression of PINK1 protects neuroblastoma cells from undergoing neurotoxin-induced apoptosis. As a protein kinase, PINK1 presumably exerts its neuroprotective effect by phosphorylating specific mitochondrial proteins and in turn modulating their functions. Towards elucidation of the neuroprotective mechanism of PINK1, we employed the baculovirus-infected insect cell system to express the recombinant protein consisting of the PINK1 kinase domain either alone [PINK1(KD)] or with the PINK1 C-terminal tail [PINK1(KD+T)]. Both recombinant enzymes preferentially phosphorylate the artificial substrate histone H1 exclusively at serine and threonine residues, demonstrating that PINK1 is indeed a protein serine/threonine kinase. Introduction of the PD-associated mutations, G386A and G409V significantly reduces PINK1(KD) kinase activity. Since Gly-386 and Gly-409 reside in the conserved activation segment of the kinase domain, the results suggest that the activation segment is a regulatory switch governing PINK1 kinase activity. We also demonstrate that PINK1(KD+T) is approximately 6-fold more active than PINK1(KD). Thus, in addition to the activation segment, the C-terminal tail also contains regulatory motifs capable of governing PINK1 kinase activity. Finally, the availability of active recombinant PINK1 proteins permits future studies to search for mitochondrial proteins that are preferentially phosphorylated by PINK1. As these proteins are likely physiological substrates of PINK1, their identification will shed light on the mechanism of pathogenesis of PD.

Parkinson's disease (PD) is a common neurodegenerative disorder characterized by selective loss of dopaminergic neurons in the substantia nigra and formation of intracellular inclusions known as Lewy bodies in remaining cells. Despite recent development in PD research, rigorous biomarkers for pre-symptomatic diagnosis of PD are not available. Elucidation of the molecular basis of PD pathogenesis will facilitate the search of these biomarkers to allow early treatment. In addition to common sporadic PD, there exist rare familial forms of Parkinsonism, usually characterized by earlier age of onset and, in some cases, absence of typical Lewy body Abbreviations used: HtrA2, high temperature requirement serine protease 2; IKK, IjBa kinase; JNK, c-Jun NH 2 -terminal kinase; NF-jB, nuclear factor-jB; PanK2, pantothenate kinase 2; PD, Parkinson's disease; PI3-kinase, phosphatidylinositol-3 kinase; PINK1, PTEN-induced kinase 1; PTEN, phosphatase and tensin homologue deleted on chromosone 10; PTP, mitochondrial permeability transition pore; ROS, reactive oxygen species; siRNA, small interfering RNA; TIM, translocase of the inner membrane; TRAF, tumor necrosis factor receptor-associated factor; TRAP, tumor necrosis factor receptor-associated protein. AbstractMutations in PTEN-induced kinase 1 (PINK1) gene cause PARK6 familial Parkinsonism. To decipher the role of PINK1 in pathogenesis of Parkinson's disease (PD), researchers need to identify protein substrates of PINK1 kinase activity that govern neuronal survival, and establish whether aberrant regulation and inactivation of PINK1 contribute to both familial Parkinsonism and idiopathic PD. These studies should take into account the several unique structural and functional features of PINK1. First PINK1 is a rare example of a protein kinase with a predicted mitochondrial-targeting sequence and a possible resident mitochondrial function. Second, bioinformatic analysis reveals unique insert regions within the kinase domain that are potentially involved in regulation of kinase activity, substrate selectivity and stability of PINK1. Third, the C-terminal region contains functional motifs governing kinase activity and substrate selectivity. Fourth, accumulating evidence suggests that PINK1 interacts with other signaling proteins implicated in PD pathogenesis and mitochondrial dysfunction. The most prominent examples are the E3 ubiquitin ligase Parkin, the mitochondrial protease high temperature requirement serine protease 2 and the mitochondrial chaperone tumor necrosis factor receptor-associated protein 1. How PINK1 may regulate these proteins to maintain neuronal survival is unclear. This review describes the unique structural features of PINK1 and their possible roles in governing mitochondrial import, processing, kinase activity, substrate selectivity and stability of PINK1. Based upon the findings of previous studies of PINK1 function in cell lines and animal models, we propose a model on the neuroprotective mechanism of PINK1. This model may serve as a conceptual framework...

Mutations of the phosphatase and tensin homolog (PTEN)-induced kinase 1 (PINK1) gene can cause early-onset familial Parkinson disease (PD). PINK1 encodes a neuroprotective protein kinase localized at the mitochondria, and its involvement in regulating mitochondrial dynamics, trafficking, structure, and function is well documented. Owing to the lack of information on structure and biochemical properties for PINK1, exactly how PINK1 exerts its neuroprotective function and how the PD-causative mutations impact on PINK1 structure and function remain unclear. As an approach to address these questions, we conducted bioinformatic analyses of the mitochondrial targeting, the transmembrane, and kinase domains of PINK1 to predict the motifs governing its regulation and function. Our report sheds light on how PINK1 is targeted to the mitochondria and how PINK1 is cleaved by mitochondrial peptidases. Moreover, it includes a potential optimal phosphorylation sequence preferred by the PINK1 kinase domain. On the basis of the results of our analyses, we predict how the PD-causative mutations affect processing of PINK1 in the mitochondria, PINK1 kinase activity, and substrate specificity. In summary, our results provide a conceptual framework for future investigation of the structural and biochemical basis of regulation and the neuroprotective mechanism of PINK1.

obtained from neuritin-overexpression mice was no longer reduced. Consequently, the recognition memory test of AAV-neuritin mice showed a significant increase in recognition index when compared to AAV-control mice. Collectively, our study provided evidence for the association between ELF MFs exposure, impairment of recognition memory, and resulting changes in hippocampal dendritic spine density. Furthermore, overexpression of neuritin has the ability to prevent the ELF MFs-exposure-induced effect by increasing the hippocampal spine density.

Musculo-skeletal Diseases i a hazard to the use of the rAAV vector were identified. In conclusion, the study subjects had characteristics representative of a population with exudative AMD and are appropriate for enrollment in a safety study for rAAV.sFlt-1.