Regulation of parkin and PINK1 by neddylation.

Neddylation is a posttranslational modification that plays important roles in regulating protein structure and function by covalently conjugating NEDD8, an ubiquitin-like small molecule, to the substrate. Here, we report that Parkinson's disease (PD)-related parkin and PINK1 are NEDD8 conjugated. Neddylation of parkin and PINK1 results in increased E3 ligase activity of parkin and selective stabilization of the 55 kDa PINK1 fragment. Expression of dAPP-BP1, a NEDD8 activation enzyme subunit, in Drosophila suppresses abnormalities induced by dPINK1 RNAi. PD neurotoxin MPP(+) inhibits neddylation of both parkin and PINK1. NEDD8 immunoreactivity is associated with Lewy bodies in midbrain dopaminergic neurons of PD patients. Together, these results suggest that parkin and PINK1 are regulated by neddylation and that impaired NEDD8 modification of these proteins likely contributes to PD pathogenesis.

Detection of protein ubiquitination.

Ubiquitination, the covalent attachment of the polypeptide ubiquitin to target proteins, is a key posttranslational modification carried out by a set of three enzymes. They include ubiquitin-activating enzyme E1, ubiquitin-conjugating enzyme E2, and ubiquitin ligase E3. Unlike to E1 and E2, E3 ubiquitin ligases display substrate specificity. On the other hand, numerous deubiquitylating enzymes have roles in processing polyubiquitinated proteins. Ubiquitination can result in change of protein stability, cellular localization, and biological activity. Mutations of genes involved in the ubiquitination/deubiquitination pathway or altered ubiquitin system function are associated with many different human diseases such as various types of cancer, neurodegeneration, and metabolic disorders. The detection of altered or normal ubiquitination of target proteins may provide a better understanding on the pathogenesis of these diseases. Here, we describe protocols to detect protein ubiquitination in cultured cells in vivo and test tubes in vitro. These protocols are also useful to detect other ubiquitin-like small molecule modification such as sumolyation and neddylation.

Parkin, PINK1, and DJ-1 form a ubiquitin E3 ligase complex promoting unfolded protein degradation.

Mutations in PARKIN, pten-induced putative kinase 1 (PINK1), and DJ-1 are individually linked to autosomal recessive early-onset familial forms of Parkinson disease (PD). Although mutations in these genes lead to the same disease state, the functional relationships between them and how their respective disease-associated mutations cause PD are largely unknown. Here, we show that Parkin, PINK1, and DJ-1 formed a complex (termed PPD complex) to promote ubiquitination and degradation of Parkin substrates, including Parkin itself and Synphilin-1 in neuroblastoma cells and human brain lysates. Genetic ablation of either Pink1 or Dj-1 resulted in reduced ubiquitination of endogenous Parkin as well as decreased degradation and increased accumulation of aberrantly expressed Parkin substrates. Expression of PINK1 enhanced Parkin-mediated degradation of heat shock-induced misfolded protein. In contrast, PD-pathogenic Parkin and PINK1 mutations showed reduced ability to promote degradation of Parkin substrates. This study identified a functional ubiquitin E3 ligase complex consisting of PD-associated Parkin, PINK1, and DJ-1 to promote degradation of un-/misfolded proteins and suggests that their PD-pathogenic mutations impair E3 ligase activity of the complex, which may constitute a mechanism underlying PD pathogenesis.

Cystamine and cysteamine prevent 3-NP-induced mitochondrial depolarization of Huntington's disease knock-in striatal cells.

Abstract Cystamine significantly improved motor deficits and extended survival in mouse models of Huntington's disease (HD); however, the precise mechanism(s) by which cystamine and the related compound cysteamine are beneficial remain to be elucidated. Using clonal striatal cell lines from wild-type (STHdhQ7/HdhQ7) and mutant huntingtin knock-in (STHdhQ111/HdhQ111) mice, we have tested the hypothesis that cystamine and cysteamine could be beneficial by preventing the depolarization of mitochondria in cell cultures. Treatment with 3-nitroproprionic acid (3-NP), a mitochondrial complex II inhibitor, induces mitochondrial depolarization and cell death of mutant HD striatal cells but not of wild-type cells. The 3-NP-mediated decrease in the mitochondrial membrane potential was attenuated by 50 microm cystamine and completely inhibited by 250 microm cystamine. Similar results were obtained using cysteamine (50-500 microm). In addition, both cystamine and cysteamine significantly attenuated the 3-NP-induced cell death. Treatment of mutant HD striatal cells with 3-NP resulted in a robust decrease in the cellular and mitochondrial levels of glutathione (GSH) compared with cells exposed to the vehicle alone. Pre-treatment of the cells with cystamine and cysteamine completely prevented the 3-NP-mediated decrease in cellular and mitochondrial GSH levels. Incubation with L-buthionine (S,R) sulfoximine (BSO) 250 microm in combination with cystamine (250 microm) or cysteamine (250 microm) prior to being treated with 3-NP completely prevented the beneficial effects of cystamine and cysteamine on the 3-NP-mediated mitochondrial depolarization. These results demonstrate that cystamine and cysteamine prevent the 3-NP-induced mitochondrial depolarization of HD striatal cell cultures.

HD CAG repeat implicates a dominant property of huntingtin in mitochondrial energy metabolism.

The 'expanded' HD CAG repeat that causes Huntington's disease (HD) encodes a polyglutamine tract in huntingtin, which first targets the death of medium-sized spiny striatal neurons. Mitochondrial energetics, related to N-methyl-d-aspartate (NMDA) Ca2+-signaling, has long been implicated in this neuronal specificity, implying an integral role for huntingtin in mitochondrial energy metabolism. As a genetic test of this hypothesis, we have looked for a relationship between the length of the HD CAG repeat, expressed in endogenous huntingtin, and mitochondrial ATP production. In STHdhQ111 knock-in striatal cells, a juvenile onset HD CAG repeat was associated with low mitochondrial ATP and decreased mitochondrial ADP-uptake. This metabolic inhibition was associated with enhanced Ca2+-influx through NMDA receptors, which when blocked resulted in increased cellular [ATP/ADP]. We then evaluated [ATP/ADP] in 40 human lymphoblastoid cell lines, bearing non-HD CAG lengths (9-34 units) or HD-causing alleles (35-70 units). This analysis revealed an inverse association with the longer of the two allelic HD CAG repeats in both the non-HD and HD ranges. Thus, the polyglutamine tract in huntingtin appears to regulate mitochondrial ADP-phosphorylation in a Ca2+-dependent process that fulfills the genetic criteria for the HD trigger of pathogenesis, and it thereby determines a fundamental biological parameter--cellular energy status, which may contribute to the exquisite vulnerability of striatal neurons in HD. Moreover, the evidence that this polymorphism can determine energy status in the non-HD range suggests that it should be tested as a potential physiological modifier in both health and disease.

Increased glutathione levels in cortical and striatal mitochondria of the R6/2 Huntington's disease mouse model.

Huntington's disease (HD) is a progressive neurodegenerative disease characterized by a severe neuronal loss that occurs primarily in the neostriatum. It has been postulated that mitochondria dysfunction and oxidative stress may play significant roles in the etiology of the disease. Indeed, markers of oxidative stress damage have been detected in the brains of HD patients and in mouse models of HD. In this study, we evaluate the changes in the levels of the potent, endogenous antioxidant glutathione and enzymes involved in its metabolism or recycling in the cortex and striatum of an extensively studied HD mouse model (R6/2). In both cortex and striatum, the levels of cellular glutathione were not significantly different in the R6/2 mice when compared with littermate wild type controls. Remarkably, the levels of glutathione were significantly increased in mitochondria isolated from the cortex and striatum of R6/2 mice when compared with wild type control mice. This specific increase in the levels of glutathione in mitochondria suggests that a compensatory mechanism is induced in the R6/2 mice to protect against an increase in oxidative stress in mitochondria.

Mutant huntingtin directly increases susceptibility of mitochondria to the calcium-induced permeability transition and cytochrome c release.

Huntington's disease (HD) is initiated by an abnormally expanded polyglutamine stretch in the huntingtin protein, conferring a novel property on the protein that leads to the loss of striatal neurons. Defects in mitochondrial function have been implicated in the pathogenesis of HD. Here, we have examined the hypothesis that the mutant huntingtin protein may directly interact with the mitochondrion and affect its function. In human neuroblastoma cells and clonal striatal cells established from HdhQ7 (wild-type) and HdhQ111 (mutant) homozygote mouse knock-in embryos, huntingtin was present in a purified mitochondrial fraction. Subfractionation of the mitochondria and limited trypsin digestion of the organelle demonstrated that huntingtin was associated with the outer mitochondrial membrane. We further demonstrated that a recombinant truncated mutant huntingtin protein, but not a wild-type, directly induced mitochondrial permeability transition (MPT) pore opening in isolated mouse liver mitochondria, an effect that was prevented completely by cyclosporin A (CSA) and ATP. Importantly, the mutant huntingtin protein significantly decreased the Ca2+ threshold necessary to trigger MPT pore opening. We found a similar increased susceptibility to the calcium-induced MPT in liver mitochondria isolated from a knock-in HD mouse model. The mutant huntingtin protein-induced MPT pore opening was accompanied by a significant release of cytochrome c, an effect completely inhibited by CSA. These findings suggest that the development of specific MPT inhibitors may be an interesting therapeutic avenue to delay the onset of HD.