benzyloxycarbonylleucyl-leucyl-leucine-aldehyde and Parkinson-Disease

Parkinson's disease (PD) is a well-known age-related neurodegenerative disease. Considering the vital importance of disease modeling based on reprogramming technology, we adopted direct reprogramming to human-induced neuronal progenitor cells (hiNPCs) for in vitro assessment of potential therapeutics. In this study, we investigated the neuroprotective effects of cryptotanshinone (CTN), which has been reported to have antioxidant properties, through PD patient-derived hiNPCs (PD-iNPCs) model with induced oxidative stress and cell death by the proteasome inhibitor MG132. A cytotoxicity assay showed that CTN possesses anti-apoptotic properties in PD-hiNPCs. CTN treatment significantly reduced cellular apoptosis through mitochondrial restoration, such as the reduction in mitochondrial reactive oxygen species and increments of mitochondrial membrane potential. These effects of CTN are mediated via the nuclear factor erythroid 2-related factor 2 (NRF2) pathway in PD-hiNPCs. Consequently, CTN could be a potential antioxidant reagent for preventing disease-related pathological phenotypes of PD.

Topics: Case-Control Studies; Cell Line; Cellular Reprogramming; Gene Expression Regulation; Humans; Induced Pluripotent Stem Cells; Leupeptins; Mitochondria; Neuroprotective Agents; Oxidative Stress; Parkinson Disease; Phenanthrenes

Mutations in PARK2 (or parkin) are responsible for 50% of cases of autosomal-recessive juvenile-onset Parkinson's disease (PD). To date, 21 alternative splice variants of the human gene have been cloned. Yet most studies have focused on the full-length protein, whereas the spectrum of the parkin isoforms expressed in PD has never been investigated. In this study, the role of parkin proteins in PD neurodegeneration was explored for the first time by analyzing their expression profile in an in vitro model of PD. To do so, undifferentiated and all-trans-retinoic-acid (RA)-differentiated SH-SY5Y cells (which thereby acquire a PD-like phenotype) were exposed to PD-mimicking neurotoxins: 1-methyl-4-phenylpyridinium (MPP

Topics: 1-Methyl-4-phenylpyridinium; Alternative Splicing; Carbonyl Cyanide m-Chlorophenyl Hydrazone; Cell Differentiation; Cell Line; Cell Survival; Down-Regulation; Humans; In Vitro Techniques; Leupeptins; Models, Biological; Neurotoxins; Oxidopamine; Parkinson Disease; Protein Isoforms; Signal Transduction; Tretinoin; Ubiquitin-Protein Ligases

Loss-of-function in the Parkin protein is thought to play a part in causing neuronal cell death in patients with Parkinson's disease. This study explores the effect of Parkin degradation, via the overexpression of nucleus accumbens 1 (NAC1), on cell viability. It was found that NAC1 and Parkin are co-localized within the cell and interact with one another, leading to a decrease in Parkin levels. Moreover, NAC1 down-regulates Parkin by presenting it for ubiquitin-dependent proteasome degradation, which causes a decrease in proteasomal activity in neuronal cells. Consequently, this decrease in proteasomal activity leads to an increase in the cells' susceptibility to proteasome inhibition-induced toxicity. It was also found that Parkin and NAC1 are key proteins found to be present mainly in the cytoplasm and are co-localized in neurons of Parkinson's disease patients. Interestingly, mutation in the POZ/BTB domain (Q23L) of NAC1 disrupts the co-localization and interaction of NAC1 with Parkin and it further abrogates the proteasome inhibition-induced toxicity. We further observed that co-transfection of the mutant form of NAC1 with Parkin reversed the proteasome activity and 20S proteasome protein levels. These results indicate a novel interaction between NAC1 and Parkin that leads to neuronal cell death, a main characteristic in Parkinson's disease.

Topics: Aged, 80 and over; Animals; Brain; Cell Line, Transformed; Central Nervous System; Cysteine Proteinase Inhibitors; Cytoplasm; Down-Regulation; Glutathione Transferase; Humans; Immunoprecipitation; Leupeptins; Male; Mice; Nerve Tissue Proteins; Neurons; Parkinson Disease; Proteasome Endopeptidase Complex; Repressor Proteins; Ubiquitin-Protein Ligases

PINK1 is a mitochondrial kinase proposed to have a role in the pathogenesis of Parkinson's disease through the regulation of mitophagy. Here, we show that the PINK1 main cleavage product, PINK152, after being generated inside mitochondria, can exit these organelles and localize to the cytosol, where it is not only destined for degradation by the proteasome but binds to Parkin. The interaction of cytosolic PINK1 with Parkin represses Parkin translocation to the mitochondria and subsequent mitophagy. Our work therefore highlights the existence of two cellular pools of PINK1 that have different effects on Parkin translocation and mitophagy.

Topics: Cytosol; HEK293 Cells; HeLa Cells; Humans; Leupeptins; Mitochondria; Mitochondrial Membranes; Mitophagy; Parkinson Disease; Proteasome Inhibitors; Protein Binding; Protein Interaction Domains and Motifs; Protein Kinases; Protein Transport; Proteolysis; Ubiquitin-Protein Ligases; Valinomycin

Mutations in the PARK2 gene are associated with an autosomal recessive form of juvenile parkinsonism (AR-JP). These mutations affect parkin solubility and impair its E3 ligase activity, leading to a toxic accumulation of proteins within susceptible neurons that results in a slow but progressive neuronal degeneration and cell death. Here, we report that RTP801/REDD1, a pro-apoptotic negative regulator of survival kinases mTOR and Akt, is one of such parkin substrates. We observed that parkin knockdown elevated RTP801 in sympathetic neurons and neuronal PC12 cells, whereas ectopic parkin enhanced RTP801 poly-ubiquitination and proteasomal degradation. In parkin knockout mouse brains and in human fibroblasts from AR-JP patients with parkin mutations, RTP801 levels were elevated. Moreover, in human postmortem PD brains with mutated parkin, nigral neurons were highly positive for RTP801. Further consistent with the idea that RTP801 is a substrate for parkin, the two endogenous proteins interacted in reciprocal co-immunoprecipitates of cell lysates. A potential physiological role for parkin-mediated RTP801 degradation is indicated by observations that parkin protects neuronal cells from death caused by RTP801 overexpression by mediating its degradation, whereas parkin knockdown exacerbates such death. Similarly, parkin knockdown enhanced RTP801 induction in neuronal cells exposed to the Parkinson's disease mimetic 6-hydroxydopamine and increased sensitivity to this toxin. This response to parkin loss of function appeared to be mediated by RTP801 as it was abolished by RTP801 knockdown. Taken together these results indicate that RTP801 is a novel parkin substrate that may contribute to neurodegeneration caused by loss of parkin expression or activity.

Topics: Animals; Apoptosis; Brain; HEK293 Cells; Humans; Leupeptins; Mice; Mice, Knockout; Neurons; Oxidopamine; Parkinson Disease; PC12 Cells; Proteasome Endopeptidase Complex; Protein Binding; Proteolysis; Rats; Repressor Proteins; RNA, Small Interfering; Transcription Factors; Ubiquitin-Protein Ligases; Ubiquitination

Studies of Parkinson's disease (PD) have been hindered by lack of access to affected human dopaminergic (DA) neurons. Here, we report generation of induced pluripotent stem cells that carry the p.G2019S mutation (G2019S-iPSCs) in the Leucine-Rich Repeat Kinase-2 (LRRK2) gene, the most common PD-related mutation, and their differentiation into DA neurons. The high penetrance of the LRRK2 mutation and its clinical resemblance to sporadic PD suggest that these cells could provide a valuable platform for disease analysis and drug development. We found that DA neurons derived from G2019S-iPSCs showed increased expression of key oxidative stress-response genes and α-synuclein protein. The mutant neurons were also more sensitive to caspase-3 activation and cell death caused by exposure to stress agents, such as hydrogen peroxide, MG-132, and 6-hydroxydopamine, than control DA neurons. This enhanced stress sensitivity is consistent with existing understanding of early PD phenotypes and represents a potential therapeutic target.

Topics: Amides; Amino Acid Substitution; Animals; Cell Death; Cell Differentiation; Dopamine; Female; Humans; Hydrogen Peroxide; Induced Pluripotent Stem Cells; Leucine-Rich Repeat Serine-Threonine Protein Kinase-2; Leupeptins; Mesencephalon; Mice; Middle Aged; Mutation; Neurons; Oxidative Stress; Oxidopamine; Parkinson Disease; Phenotype; Protein Serine-Threonine Kinases; Pyridines; rho-Associated Kinases

Leucine-rich repeat kinase 2 (LRRK2) is the causal gene for autosomal dominant familial Parkinson's disease. We have previously reported a novel molecular feature characteristic to I2020T mutant LRRK2: higher susceptibility to post-translational degradation than the wild-type LRRK2. In the present study, we demonstrated that the protective effect of I2020T LRRK2 against hydrogen peroxide-induced apoptosis was impaired in comparison with the wild-type molecule. When the intracellular level of the protein had been allowed to recover by treatment with proteolysis inhibitors, the protective effect of I2020T LRRK2 against apoptosis was increased. We further confirmed that a decrease in the intracellular protein level of WT LRRK2 by knocking down resulted in a reduction of protectivity against apoptosis. These results suggest that higher susceptibility of I2020T mutant LRRK2 to intracellular degradation than the wild-type molecule may be one of the mechanisms involved in the neurodegeneration associated with this LRRK2 mutation.

Topics: Acetylcysteine; Apoptosis; Chloroquine; Gene Knockdown Techniques; Humans; Hydrogen Peroxide; Leucine-Rich Repeat Serine-Threonine Protein Kinase-2; Leupeptins; Lysosomes; Parkinson Disease; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Protein Serine-Threonine Kinases

Abnormal iron accumulation is linked to a variety of neurological disorders and may contribute to the progressive damage seen in these diseases. The biochemical processes responsible for iron accumulation are not known but are likely to entail alteration in transport into injured brain areas. The major transport protein responsible for uptake of iron is divalent metal transporter 1 (DMT1) and recent studies demonstrate that the 1B species is regulated post-translationally by degradation via the proteasomal pathway. As reported in this paper, the E3 ligase, parkin, when over-expressed in SH-SY5Y cells, results in a decrease in 1B-DMT1 isoforms and also a significant reduction in manganese transport and toxicity. Incubating cells over-expressing parkin with the proteasomal inhibitor, MG-132, restores 1B-DMT1 levels emphasizing that the observed changes are caused by degradation via the proteasomal pathway. Expression of the 1B species of DMT1 was also shown to be elevated in human lymphocytes containing a homozygous deletion of exon 4 of parkin and in brains of parkin knockout animals. Immunoprecipitation and immunofluorescent studies confirm that parkin co-localizes with DMT1 in SH-SY5Y cells transfected with wild-type parkin. These results demonstrate that parkin is the E3 ligase responsible for ubiquitination of the 1B species of DMT1.

Topics: Adult; Animals; B-Lymphocytes; Cation Transport Proteins; Cell Death; Cells, Cultured; Chlorides; Cysteine Proteinase Inhibitors; Exons; Gene Expression Regulation; Humans; Immunoprecipitation; Leupeptins; Male; Manganese; Manganese Compounds; Mice; Mice, Knockout; Mutation; Neuroblastoma; Parkinson Disease; Proteasome Endopeptidase Complex; Protein Isoforms; Transfection; Ubiquitin-Protein Ligases

Parkinson's disease (PD) is a devastating neurodegenerative disease characterized by a distinct set of motor symptoms. Loss-of-function mutations in PTEN-induced kinase 1 (PINK1) or parkin have been linked to early-onset autosomal recessive forms of familial PD. We have recently shown that parkin (an E3 ubiquitin ligase) and PINK1 (a serine/threonine kinase) affect one other's stability, solubility, and tendency to form cytoprotective aggresomes (Um et al., 2009). Here we validated the functional relevance of this mutual interaction under pathologic PD conditions, by investigating the changes of expression and solubility of these factors in response to PD-linked toxins. Consistent with our previous cell culture data, exposure of human dopaminergic neuroblastoma SH-SY5Y cells to PD-linked toxins (1-methyl-4-phenylpyridinium ion, 6-hydroxydopamine, or MG132) reduced Nonidet P-40-soluble parkin levels and induced PINK1 accumulation. Consistent with our previous findings from parkin knockout mice, rat models of PD (6-hydroxydopamine-, rotenone-, or MG132-induced PD) were also associated with an increase in soluble and insoluble PINK1 levels as well as enhanced formation of parkin aggregates. These findings suggest that both PINK1 and parkin play important roles in regulating the formation of Lewy bodies during the pathogenesis of sporadic and familial PD.

Topics: 1-Methyl-4-phenylpyridinium; Animals; Brain; Cell Line; Disease Models, Animal; Humans; Leupeptins; Lewy Bodies; Mice; Oxidopamine; Parkinson Disease; Protein Kinases; Rats; Solubility; Ubiquitin-Protein Ligases

Mutations in the PTEN-induced putative kinase 1 (PINK1), a mitochondrial serine-threonine kinase, and Parkin, an E3 ubiquitin ligase, are associated with autosomal-recessive forms of Parkinson disease (PD). Both are involved in the maintenance of mitochondrial integrity and protection from multiple stressors. Recently, Parkin was demonstrated to be recruited to impaired mitochondria in a PINK1-dependent manner, where it triggers mitophagy. Using primary human dermal fibroblasts originating from PD patients with various PINK1 mutations, we showed at the endogenous level that (i) PINK1 regulates the stress-induced decrease of endogenous Parkin; (ii) mitochondrially localized PINK1 mediates the stress-induced mitochondrial translocation of Parkin; (iii) endogenous PINK1 is stabilized on depolarized mitochondria; and (iv) mitochondrial accumulation of full-length PINK1 is sufficient but not necessary for the stress-induced loss of Parkin signal and its mitochondrial translocation. Furthermore, we showed that different stressors, depolarizing or non-depolarizing, led to the same effect on detectable Parkin levels and its mitochondrial targeting. Although this effect on Parkin was independent of the mitochondrial membrane potential, we demonstrate a differential effect of depolarizing versus non-depolarizing stressors on endogenous levels of PINK1. Our study shows the necessity to introduce an environmental factor, i.e. stress, to visualize the differences in the interaction of PINK1 and Parkin in mutants versus controls. Establishing human fibroblasts as a suitable model for studying this interaction, we extend data from animal and other cellular models and provide experimental evidence for the generally held notion of PD as a condition with a combined genetic and environmental etiology.

Topics: Blotting, Western; Cell Line; Cells, Cultured; Cysteine Proteinase Inhibitors; Cytosol; Fibroblasts; Humans; Hydrogen Peroxide; Ionophores; Leupeptins; Mitochondria; Mutation; Oligopeptides; Oxidants; Parkinson Disease; Protein Kinases; Protein Transport; RNA Interference; Transfection; Ubiquitin-Protein Ligases; Valinomycin

α-Synuclein (a-Syn) is a major component of fibrillar aggregates in Lewy bodies (LBs), a characteristic hallmark of Parkinson disease. Almost 90% of a-Syn deposited in LBs is phosphorylated at Ser-129. However, the role of Ser-129-phosphorylated a-Syn in the biogenesis of LBs remains unclear. Here, we investigated the metabolism of Ser-129-phosphorylated a-Syn. In SH-SY5Y cells, inhibition of protein phosphatase 2A/1 by okadaic acid, and inhibition of the proteasome pathway by MG132 or lactacystin accumulated Ser-129-phosphorylated a-Syn. However, these inhibitions did not alter the amounts of total a-Syn within the observation time. Inhibition of the autophagy-lysosome pathway by 3-methyladenine or chloroquine accumulated Ser-129-phosphorylated a-Syn in parallel to total a-Syn during longer incubations. Experiments using cycloheximide showed that Ser-129-phosphorylated a-Syn diminished rapidly (t(½) = 54.9 ± 6.4 min), in contrast to the stably expressed total a-Syn. The short half-life of Ser-129-phosphorylated a-Syn was blocked by MG132 to a greater extent than okadaic acid. In rat primary cortical neurons, either MG132, lactacystin, or okadaic acid accumulated Ser-129-phosphorylated a-Syn. Additionally, we did not find that phosphorylated a-Syn was ubiquitinated in the presence of proteasome inhibitors. These data show that Ser-129-phosphorylated a-Syn is targeted to the proteasome pathway in a ubiquitin-independent manner, in addition to undergoing dephosphorylation. The proteasome pathway may play a role in the biogenesis of Ser-129-phosphorylated a-Syn-rich LBs.

Topics: Acetylcysteine; alpha-Synuclein; Animals; Cell Line, Tumor; Cerebral Cortex; Cycloheximide; Cysteine Proteinase Inhibitors; Humans; Leupeptins; Lewy Bodies; Neurons; Okadaic Acid; Parkinson Disease; Phosphorylation; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Protein Phosphatase 1; Protein Phosphatase 2; Protein Synthesis Inhibitors; Rabbits; Ubiquitin

Mutations in PARK7 DJ-1 have been associated with autosomal-recessive early-onset Parkinson's disease (PD). This gene encodes for an atypical peroxiredoxin-like peroxidase that may act as a regulator of transcription and a redox-dependent chaperone. Although large gene deletions have been associated with a loss-of-function phenotype, the pathogenic mechanism of several missense mutations is less clear. By performing a yeast two-hybrid screening from a human fetal brain library, we identified TRAF and TNF receptor-associated protein (TTRAP), an ubiquitin-binding domain-containing protein, as a novel DJ-1 interactor, which was able to bind the PD-associated mutations M26I and L166P more strongly than wild type. TTRAP protected neuroblastoma cells from apoptosis induced by proteasome impairment. In these conditions, endogenous TTRAP relocalized to a detergent-insoluble fraction and formed cytoplasmic aggresome-like structures. Interestingly, both DJ-1 mutants blocked the TTRAP protective activity unmasking a c-jun N-terminal kinase (JNK)- and p38-MAPK (mitogen-activated protein kinase)-mediated apoptosis. These results suggest an active role of DJ-1 missense mutants in the control of cell death and position TTRAP as a new player in the arena of neurodegeneration.

Topics: Antineoplastic Agents; Apoptosis; Brain Neoplasms; Cell Line; DNA-Binding Proteins; Dopamine; Enzyme Activation; Humans; Inclusion Bodies; Intracellular Signaling Peptides and Proteins; JNK Mitogen-Activated Protein Kinases; Leupeptins; Mutation, Missense; Neuroblastoma; Nuclear Proteins; Oncogene Proteins; Oxidative Stress; p38 Mitogen-Activated Protein Kinases; Parkinson Disease; Phosphoric Diester Hydrolases; Protein Binding; Protein Deglycase DJ-1; Substantia Nigra; Transcription Factors; Two-Hybrid System Techniques

Spinocerebellar ataxia type 8 (SCA8) involves bidirectional expression of CUG (ATXN8OS) and CAG (ATXN8) expansion transcripts. The pathogenesis of SCA8 is complex and the spectrum of clinical presentations is broad. In the present study, we assessed the SCA8 repeat size ranges in Taiwanese Parkinson's disease, Alzheimer's disease and atypical parkinsonism and investigated the genetic variation modulating ATXN8 expression. Thirteen large SCA8 alleles and a novel ATXN8 -62 G/A promoter SNP were found. There is a significant difference in the proportion of the individuals carrying SCA8 larger alleles in atypical parkinsonism (P = 0.044) as compared to that in the control subjects. In lymphoblastoid cells carrying SCA8 large alleles, treatment of MG-132 or staurosporine significantly increases the cell death or caspase 3 activity. Although expressed at low steady-state, ATXN8 expression level is significantly higher (P = 0.012) in cells with SCA8 large alleles than that of the control cells. The ATXN8 transcriptional activity was significantly higher in the luciferase reporter construct containing the -62G allele than that containing the -62A allele in both neuroblastoma and embryonic kidney cells. Therefore, our preliminary results suggest that ATXN8 gene -62 G/A polymorphism may be functional in modulating ATXN8 expression.

Topics: Adult; Aged; Aged, 80 and over; Alzheimer Disease; Base Sequence; Case-Control Studies; Cell Line; Central Nervous System Diseases; DNA Primers; Female; Gene Expression; Humans; Leupeptins; Lymphocytes; Male; Microfilament Proteins; Middle Aged; Nerve Tissue Proteins; Parkinson Disease; Parkinsonian Disorders; Polymorphism, Single Nucleotide; Pyrimidines; RNA, Long Noncoding; RNA, Untranslated; Spinocerebellar Ataxias; Staurosporine; Sulfonamides; Trinucleotide Repeat Expansion

This study demonstrated that DA and its oxidative metabolites: H2O2 and aminochrome (AM), cyclized DA quinones, could all directly inhibit proteasome activity. DA and AM, especially AM, could induce intensive and irreversible proteasome inhibition, whereas proteasome inhibition induced by H2O2 was weaker and GSH reversible. It was concluded that DA induced irreversible proteasome inhibition via DA-derived quinones, rather than through small molecular weight ROS. The AM was also more toxic than H2O2 to dopaminergic MN9D cells. Furthermore the cytotoxicity and proteasome inhibition induced by DA, AM and H2O2 could be abrogated by GSH, ascorbic acid (AA), Vitamin E, SOD (superoxidase dismutase) or CAT (catalase) with different profiles. Only GSH was potent to abrogate DA, AM or H2O2-induced cell toxicity and proteasome inhibition, as well as to reverse H2O2-induced proteosome inhibition. Therefore, therapeutic strategies to increase GSH level or to use GSH substitutes should function to control PD onset and development.

Topics: Animals; Arachidonic Acid; Catalase; Cell Line; Chymotrypsin; Dopamine; Glutathione; Humans; Hydrogen Peroxide; Indolequinones; Kinetics; Leupeptins; Mice; Oxidative Stress; Parkinson Disease; Protease Inhibitors; Proteasome Inhibitors; Superoxide Dismutase; Vitamin E

In Parkinson's disease (PD), dopaminergic (DA) neurons in the substantia nigra (SN, A9) are particularly vulnerable, compared to adjacent DA neurons within the ventral tegmental area (VTA, A10). Here, we show that in rat and human, one RAB3 isoform, RAB3B, has higher expression levels in A10 compared to A9 neurons. RAB3 is a monomeric GTPase protein that is highly enriched in synaptic vesicles and is involved in synaptic vesicle trafficking and synaptic transmission, disturbances of which have been implicated in several neurodegenerative diseases, including PD. These findings prompted us to further investigate the biology and neuroprotective capacity of RAB3B both in vitro and in vivo. RAB3B overexpression in human dopaminergic BE (2)-M17 cells increased neurotransmitter content, [(3)H] dopamine uptake, and levels of presynaptic proteins. AAV-mediated RAB3B overexpression in A9 DA neurons of the rat SN increased striatal dopamine content, number and size of synaptic vesicles, and levels of the presynaptic proteins, confirming in vitro findings. Measurement of extracellular DOPAC, a dopamine metabolite, following l-DOPA injection supported a role for RAB3B in enhancing the dopamine storage capacity of synaptic terminals. RAB3B overexpression in BE (2)-M17 cells was protective against toxins that simulate aspects of PD in vitro, including an oxidative stressor 6-hydroxydopamine (6-OHDA) and a proteasome inhibitor MG-132. Furthermore, RAB3B overexpression in rat SN both protected A9 DA neurons and resulted in behavioral improvement in a 6-OHDA retrograde lesion model of PD. These results suggest that RAB3B improves dopamine handling and storage capacity at presynaptic terminals, and confers protection to vulnerable DA neurons.

Topics: 3,4-Dihydroxyphenylacetic Acid; Animals; Cell Line; Corpus Striatum; Dopamine; Female; Gene Expression; Gene Expression Profiling; Humans; In Vitro Techniques; Leupeptins; Levodopa; Models, Neurological; Oxidopamine; Parkinson Disease; Parkinsonian Disorders; Presynaptic Terminals; rab3 GTP-Binding Proteins; Rats; Rats, Sprague-Dawley; RNA, Messenger; Substantia Nigra; Synaptic Vesicles; Ventral Tegmental Area

Parkinson's disease (PD) is a neurologic disorder characterized by dopaminergic cell death in the substantia nigra. PD pathogenesis involves mitochondrial dysfunction, proteasome impairment, and alpha-synuclein aggregation, insults that may be especially toxic to oxidatively stressed cells including dopaminergic neurons. The enzyme methionine sulfoxide reductase A (MsrA) plays a critical role in the antioxidant response by repairing methionine-oxidized proteins and by participating in cycles of methionine oxidation and reduction that have the net effect of consuming reactive oxygen species. Here, we show that MsrA suppresses dopaminergic cell death and protein aggregation induced by the complex I inhibitor rotenone or mutant alpha-synuclein, but not by the proteasome inhibitor MG132. By comparing the effects of MsrA and the small-molecule antioxidants N-acetylcysteine and vitamin E, we provide evidence that MsrA protects against PD-related stresses primarily via methionine sulfoxide repair rather than by scavenging reactive oxygen species. We also demonstrate that MsrA efficiently reduces oxidized methionine residues in recombinant alpha-synuclein. These findings suggest that enhancing MsrA function may be a reasonable therapeutic strategy in PD.

Topics: Acetylcysteine; alpha-Synuclein; Animals; Antioxidants; Blotting, Western; Cell Death; Cells, Cultured; Cysteine Proteinase Inhibitors; Dopamine; Humans; Leupeptins; Mesencephalon; Methionine Sulfoxide Reductases; Mice; Neurons; Oxidation-Reduction; Oxidoreductases; Parkinson Disease; Rats; Rotenone; Uncoupling Agents; Vitamin E

This study demonstrated that dopaminergic MN9D and PC12 cells were more vulnerable than non-dopaminergic N2A cells to the challenge by proteasome inhibitor MG132, which could be alleviated by reductants and alpha-methyl tyrosine (alpha-MT), a specific tyrosine hydroxylase inhibitor. Furthermore, challenging non-dopaminergic N2A cells with exogenous DA could aggravate MG132-induced cell viability decrease, which could be abrogated by reductants but not by alpha-MT. It was observed that alpha-MT could decrease endogenous DA content in dopaminergic MN9D and PC12 cells while N2A cells could take in exogenous DA into cytosol. The endogenous DA in dopaminergic cells was demonstrated to inhibit proteasome activity in the cells and further sensitize the proteasome to MG132 inhibition. In addition, the endogenous DA was also implicated for the increased level of lipid peroxidation and ubiquitinated proteins as well as inclusion bodies formation when non-dopaminergic cells were challenged with exogenous DA. Taken together it is proposed that endogenous DA in dopaminergic neurons could promote selective dopaminergic neurodegeneration, especially under the conditions of exopathic or idiopathic defects of ubiquitin-proteasome system (UPS), which may be abolished by reductant remedy.

Topics: alpha-Methyltyrosine; Animals; Cell Survival; Cysteine Proteinase Inhibitors; Dopamine; Leupeptins; Malondialdehyde; Mice; Neurodegenerative Diseases; Neurons; Parkinson Disease; PC12 Cells; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Rats; Tetrazolium Salts; Thiazoles

The cause of selective dopaminergic neuronal degeneration in Parkinson disease has still not been resolved, but it has been hypothesized that oxidative stress and the ubiquitin-proteasome system are important in the pathogenesis. In this report, we investigated the effect of proteasome inhibition on oxidative stress-induced cytotoxicity in PC12 cells, an in vitro model of Parkinson disease. Treatment with proteasome inhibitors provided significant protection against toxicity by 6-hydroxydopamine and H(2)O(2) in a concentration-dependent manner. The measurement of intracellular reactive oxygen species using 2',7'-dichlorofluorescein diacetate demonstrated that lactacystin, a proteasome inhibitor, significantly reduced 6-hydroxydopamineand H(2)O(2)-induced reactive oxygen species production. Proteasome inhibitors elevated the amount of glutathione and phosphorylated p38 mitogen-activated protein kinase (MAPK) prior to glutathione elevation. The treatment with lactacystin induced the nuclear translocation of NF-E2-related factor 2 (Nrf2) and increased the level of mRNA for gamma-glutamylcysteine synthetase, a rate-limiting enzyme in glutathione synthesis. Furthermore, SB203580, an inhibitor of p38 MAPK, abolished glutathione elevation and cytoprotection by lactacystin. These data suggest that proteasome inhibition afforded cytoprotection against oxidative stress by the elevation of glutathione content, and its elevation was mediated by p38 MAPK phosphorylation.

Topics: Acetylcysteine; Active Transport, Cell Nucleus; Adrenergic Agents; Animals; Cell Nucleus; Cysteine Proteinase Inhibitors; Dose-Response Relationship, Drug; Glutathione; Hydrogen Peroxide; Imidazoles; Models, Biological; NF-E2 Transcription Factor; Oxidants; Oxidative Stress; Oxidopamine; p38 Mitogen-Activated Protein Kinases; Parkinson Disease; PC12 Cells; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Pyridines; Rats; Reactive Oxygen Species; Ubiquitin

Mutations in DJ-1 lead to early onset Parkinson's disease (PD). The aim of this study was to elucidate further the underlying mechanisms leading to neuronal cell death in DJ-1 deficiency in vivo and determine whether the observed cell loss could be prevented pharmacologically. Inactivation of DJ-1 in zebrafish, Danio rerio, resulted in loss of dopaminergic neurons after exposure to hydrogen peroxide and the proteasome inhibitor MG132. DJ-1 knockdown by itself already resulted in increased p53 and Bax expression levels prior to toxin exposure without marked neuronal cell death, suggesting subthreshold activation of cell death pathways in DJ-1 deficiency. Proteasome inhibition led to a further increase of p53 and Bax expression with widespread neuronal cell death. Pharmacological p53 inhibition either before or during MG132 exposure in vivo prevented dopaminergic neuronal cell death in both cases. Simultaneous knockdown of DJ-1 and the negative p53 regulator mdm2 led to dopaminergic neuronal cell death even without toxin exposure, further implicating involvement of p53 in DJ-1 deficiency-mediated neuronal cell loss. Our study demonstrates the utility of zebrafish as a new animal model to study PD gene defects and suggests that modulation of downstream mechanisms, such as p53 inhibition, may be of therapeutic benefit.

Topics: Animals; Animals, Genetically Modified; bcl-2-Associated X Protein; Cell Death; Disease Models, Animal; Embryo, Mammalian; Embryo, Nonmammalian; Gene Expression Regulation, Developmental; Humans; Hydrogen Peroxide; In Situ Hybridization; In Situ Nick-End Labeling; Leupeptins; Nerve Tissue Proteins; Neurons; Neurotoxins; Parkinson Disease; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Tumor Suppressor Protein p53; Tyrosine 3-Monooxygenase; Zebrafish; Zebrafish Proteins

Dopamine (DA) and its metabolites have been implicated in the pathogenesis of Parkinson's disease. DA can produce reactive-oxygen species and DA-derived quinones such as aminochrome can induce proteasomal inhibition. We therefore examined the ability of DA and MG132 to induce apoptosis and proteasomal inhibition in N27 rat dopaminergic cells. DA (0-500 micromol/L, 0-24 h) and MG132 (0-5 micromol/L, 0-24 h) treated N27 cells resulted in time- and concentration-dependent apoptosis. To better define DA and MG132-induced apoptosis, the activation of initiator caspases 2 and caspase 9 and the executioner caspase 3 was investigated. Activation of caspase 2, caspase 9, and caspase 3 occurred early and prior to cell death. In addition, N-acetylcysteine (NAC) blocked DA but not MG132-induced apoptosis and mitochondrial membrane potential loss. NAC can react with both reactive-oxygen and quinoid metabolites and its inhibitory activity suggests a role for reactive species in DA-induced apoptosis. Proteasomal inhibition was detected after DA treatment in N27 cells which occurred prior to cell death and was abrogated by NAC. Our results implicate DA-derived reactive species in proteasomal inhibition and caspase-dependent apoptosis in N27 cells. The ability of endogenous DA-derived metabolites to induce proteasomal inhibition and apoptosis may contribute to the selective loss of dopaminergic neurons in Parkinson's disease.

Topics: Acetylcysteine; Animals; Apoptosis; Caspases; Cell Line, Transformed; Cysteine Proteinase Inhibitors; Dopamine; Dose-Response Relationship, Drug; Enzyme Activation; Leupeptins; Mesencephalon; Nerve Degeneration; Neurons; Oxidative Stress; Parkinson Disease; Proteasome Endopeptidase Complex; Rats; Reactive Oxygen Species; Substantia Nigra

Relative neuronal vulnerability is a universal yet poorly understood feature of neurodegenerative diseases. In Parkinson's disease, dopaminergic (DA) neurons in the substantia nigra (SN) (A9) are particularly vulnerable, whereas adjacent DA neurons within the ventral tegmental area (A10) are essentially spared. Our previous laser capture microdissection and microarray study (Chung et al., 2005) demonstrated that molecular differences between these DA neurons may underlie their differential vulnerability. Here we show that G-substrate, an endogenous inhibitor of Ser/Thr protein phosphatases, exhibits higher expression in A10 compared with A9 DA neurons in both rodent and human midbrain. Overexpression of G-substrate protected dopaminergic BE(2)-M17 cells against toxins, including 6-OHDA and MG-132 (carbobenzoxy-L-leucyl- L-leucyl-L-leucinal), whereas RNA interference (RNAi)-mediated knockdown of endogenous G-substrate increased their vulnerability to these toxins. G-substrate reduced 6-OHDA-mediated protein phosphatase 2A (PP2A) activation in vitro and increased phosphorylated levels of PP2A targets including Akt, glycogen synthase kinase 3beta, and extracellular signal-regulated kinase 2 but not p38. RNAi to Akt diminished the protective effect of G-substrate against 6-OHDA. In vivo, lentiviral delivery of G-substrate to the rat SN increased baseline levels of phosphorylated Akt and protected A9 DA neurons from 6-OHDA-induced toxicity. These results suggest that inherent differences in the levels of G-substrate contribute to the differential vulnerability of DA neurons and that enhancing G-substrate levels may be a neuroprotective strategy for the vulnerable A9 (SN) DA neurons in Parkinson's disease.

Topics: Animals; Disease Models, Animal; Female; Humans; Leupeptins; Mice; Nerve Tissue Proteins; Oxidopamine; Parkinson Disease; Phosphoprotein Phosphatases; Protein Phosphatase 2; Rats; Rats, Sprague-Dawley; RNA Interference

Following our identification of PTEN-induced putative kinase 1 (PINK1) gene mutations in PARK6-linked Parkinson's disease (PD), we have recently reported that PINK1 protein localizes to Lewy bodies (LBs) in PD brains. We have used a cellular model system of LBs, namely induction of aggresomes, to determine how a mitochondrial protein, such as PINK1, can localize to aggregates. Using specific polyclonal antibodies, we firstly demonstrated that human PINK1 was cleaved and localized to mitochondria. We demonstrated that, on proteasome inhibition with MG-132, PINK1 and other mitochondrial proteins localized to aggresomes. Ultrastructural studies revealed that the mechanism was linked to the recruitment of intact mitochondria to the aggresome. Fractionation studies of lysates showed that PINK1 cleavage was enhanced by proteasomal stress in vitro and correlated with increased expression of the processed PINK1 protein in PD brain. These observations provide valuable insights into the mechanisms of LB formation in PD that should lead to a better understanding of PD pathogenesis.

Topics: Animals; Blotting, Western; Brain; Cell Line; Cloning, Molecular; Cricetinae; Cysteine Proteinase Inhibitors; Enzyme Inhibitors; Fluorescent Antibody Technique; Green Fluorescent Proteins; Humans; Leupeptins; Microscopy, Immunoelectron; Mitochondria; Mutant Proteins; Nerve Tissue Proteins; Parkinson Disease; Proteasome Endopeptidase Complex; Protein Kinases; Stress, Physiological; Transfection

Proteasomal dysfunction and alpha-synuclein (alpha-syn) have both been implicated in the pathogenesis of Parkinson's disease (PD). However, the functional relationship between these two remains elusive. Here we show that in human neuroblastoma cells, novel variants of alpha-syn with molecular weights of 22-25 kDa were induced after washout of the reversible proteasome inhibitors. Induction of these variants seemed to be a specific response to proteasome dysfunction, because the treatment and washout of other protease inhibitor or mitochondrial inhibitor did not induce these variants. Importantly, PD-linked alpha-syn mutations have effects on the formation of these variants. Recently, O-linked glycosylation and monoubiquitylation of alpha-syn have been reported. Despite the similarity in molecular weights, biochemical properties of our variants suggest that they are unrelated with such modifications. Taken together, these results suggest that alpha-syn is regulated by the specific functional state of the proteasomes, and PD-linked mutations may affect this regulation.

Topics: alpha-Synuclein; Glycosylation; Humans; Leupeptins; Parkinson Disease; Peptide Fragments; Proteasome Endopeptidase Complex; Recovery of Function; Ubiquitin

The formation of inclusion bodies in dopaminergic neurons is associated with the pathogenesis of Parkinson's disease. In order to clarify the role of dopamine/L-dopa in the formation of protein aggregates, we investigated dopamine/L-dopa-related aggregation using an experimental inclusion model. The inhibition of tyrosine hydroxylase (TH) by alpha-methyltyrosine dramatically decreased MG132-induced aggregate formation. In addition, the inhibition of TH caused the upregulation of proteasomes in cultured cells and the dopamine/L-dopa induced non-enzymatic polymerization of ubiquitin. This inhibition did not affect cell viability. These results suggest that dopamine/L-dopa might enhance aggregate formation, and that intracellular aggregates may not be toxic to cells.

Topics: alpha-Methyltyrosine; Animals; Cathepsin B; Cell Survival; Dopamine; Leupeptins; Levodopa; Parkinson Disease; PC12 Cells; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Rats; Tyrosine 3-Monooxygenase; Ubiquitin

Parkinson's disease (PD) is pathologically characterized by dopaminergic (DA) cell death and the presence of Lewy bodies (LB) in the brain. alpha-Synuclein (alpha-syn) and ubiquitin (Ub) are the major components of LB, however, the process of their accumulation and their relationship to DA cell loss has not yet been resolved. Now, in this journal, Inden et al. showed the protective effect of proteasome inhibitors (PSI) on DA cell death in the rat PD model using 6-hydroxyl dopamine (6-OHDA). Co-administration of PSI, lactacystin, or MG-132 significantly prevented the nigral degeneration and apomorphine-induced rotational asymmetry of the model with increased appearance of alpha-syn- and Ub-positive inclusions in the substantia nigra. This study indicates that in their model, accelerated formation of inclusions via proteasome inhibition protects against DA cell death. Previous literature linked the impairments or inhibitions of the ubiquitin-proteasome system (UPS) and DA cell death. However, this report implies that the relationship between the UPS and the pathogenesis of PD may be more complex than we thought.

Topics: Acetylcysteine; Animals; Apoptosis; Cysteine Proteinase Inhibitors; Dopamine; Humans; Leupeptins; Lewy Bodies; Neurotoxins; Oxidopamine; Parkinson Disease; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Protective Agents; Rats; Substantia Nigra

Mutations in the PARK7/DJ-1 gene cause autosomal-recessive Parkinson's disease. In some patients the gene is deleted. The molecular basis of disease in patients with point mutations is less obvious. We have investigated the molecular properties of [L166P]DJ-1 and the novel variant [E64D]DJ-1. When transfected into non-neuronal and neuronal cell lines, steady-state expression levels of [L166P]DJ-1 were dramatically lower than wild-type [WT]DJ-1 and [E64D]DJ-1. Cycloheximide and pulse-chase experiments revealed that the decreased expression levels of [L166P]DJ-1 were because of accelerated protein turnover. Proteasomal degradation was not the major pathway of DJ-1 breakdown because treatment with the proteasome inhibitor MG-132 caused only minimal accumulation of DJ-1, even of the very unstable [L166P]DJ-1 mutant. Because of the structural resemblance of DJ-1 with bacterial cysteine proteases, we considered an autoproteolytic mechanism. However, neither pharmacological inhibition nor site-directed mutagenesis of the putative active site residue Cys-106 stabilized DJ-1. To gain further insight into the structural defects of DJ-1 mutants, human [WT]DJ-1 and both mutants were expressed in Escherichia coli. As in eukaryotic cells, expression levels of [L166P]DJ-1 were dramatically reduced compared with [WT]DJ-1 and [E64D]DJ-1. Circular dichroism spectrometry revealed that the solution structures of [WT]DJ-1 and [E64D]DJ-1 are rich in beta-strand and alpha-helix conformation. Alpha-helices were more susceptible to thermal denaturation than the beta-sheet, and [WT]DJ-1 was more flexible in this regard than [E64D]DJ-1. Thus, structural defects of [E64D]DJ-1 only become apparent upon denaturing conditions, whereas the L166P mutation causes a drastic defect that leads to excessive degradation.

Topics: Cell Line; Circular Dichroism; Cycloheximide; Cysteine; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Enzyme Inhibitors; Escherichia coli; Gene Deletion; Humans; Intracellular Signaling Peptides and Proteins; Leupeptins; Mass Spectrometry; Multienzyme Complexes; Mutagenesis, Site-Directed; Mutation; Neurons; Oncogene Proteins; Parkinson Disease; Point Mutation; Proteasome Endopeptidase Complex; Protein Conformation; Protein Deglycase DJ-1; Protein Folding; Protein Structure, Secondary; Protein Synthesis Inhibitors; Reverse Transcriptase Polymerase Chain Reaction; Temperature; Time Factors

Parkinson's disease (PD) is characterized by loss of dopamine neurons in the substantia nigra and the presence of cytoplasmic inclusions known as Lewy bodies (LBs). Mutations in parkin cause autosomal recessive juvenile parkinsonism (AR-JP) that is distinct from sporadic PD by the general absence of LBs. Several studies have reported that parkin is present in LBs of sporadic PD but the role of parkin in LB formation is unclear. Aggresomes are perinuclear aggregates representing intracellular deposition of misfolded protein. LBs and aggresomes have been reported to share a common biogenesis. We have investigated the role of parkin in aggresome formation. In human SH-SY5Y neuroblastoma cells we observe that endogenous parkin is present in aggresomes induced by a variety of stresses including dopamine, proteosome inhibition and a pro-apoptopic stimulus. We show that vimentin is invariably collapsed around the aggresome but that the detection of ubiquitin is variable depending on the stress. We show that cells that stably over-express human wild-type parkin form fewer aggresomes upon stress compared to cells that express vector alone whereas over-expression of AR-JP causing mutants of parkin have no effect on stress-induced aggresome formation. Finally, we show that the prevention of aggresome formation by over-expression of wild-type parkin is not always associated with a beneficial effect on neuronal survival. Our findings suggest that parkin is important for aggresome formation in human neuronal cells and may lead to a better understanding of the biogenesis of LBs in sporadic PD.

Topics: Blotting, Western; Cell Survival; Dopamine; Gene Expression Regulation; Humans; Inclusion Bodies; Leupeptins; Microscopy, Fluorescence; Microtubule-Organizing Center; Parkinson Disease; Staurosporine; Tubulin; Tumor Cells, Cultured; Ubiquitin; Ubiquitin-Protein Ligases; Vimentin

Dysfunction of the ubiquitin-proteasome system (UPS) has been implicated in Parkinson's disease (PD) and other neurodegenerative disorders. We have investigated the effect of UPS inhibition on the metabolism of alpha-synuclein (SYN) and parkin, two proteins genetically and histopathologically associated to PD. Pharmacological inhibition of proteasome induced accumulation of both parkin and SYN in transfected PC12 cells. We found that this effect was caused by increased protein synthesis rather than impairment of protein degradation, suggesting that inhibition of the UPS might lead to non-specific up-regulation of cytomegalovirus (CMV)-driven transcription. To investigate whether endogenous parkin and SYN can be substrate of the UPS, untransfected PC12 cells and primary mesencephalic neurones were exposed to proteasome inhibitors, and parkin and SYN expression was evaluated at both protein and mRNA level. Under these conditions, we found that proteasome inhibitors did not affect the level of endogenous parkin and SYN. However, we confirmed that dopaminergic neurones were selectively vulnerable to the toxicity of proteasome inhibitors. Our results indicate that studies involving the use of proteasome inhibitors, particularly those in which proteins are expressed from a heterologous promoter, are subjected to potential artefacts that need to be considered for the interpretation of the role of UPS in PD pathogenesis.

Topics: alpha-Synuclein; Animals; Cell Aggregation; Cysteine Endopeptidases; Humans; Leupeptins; Mesencephalon; Multienzyme Complexes; Nerve Tissue Proteins; Parkinson Disease; PC12 Cells; Proteasome Endopeptidase Complex; Rats; Rats, Sprague-Dawley; RNA, Messenger; Synucleins; Transfection; Ubiquitin-Protein Ligases

Parkinson's disease (PD) is a neurodegenerative disorder characterized by degeneration of dopaminergic neurons in the substantia nigra. We previously mapped a locus for a rare familial form of PD to chromosome 1p36 (PARK6). Here we show that mutations in PINK1 (PTEN-induced kinase 1) are associated with PARK6. We have identified two homozygous mutations affecting the PINK1 kinase domain in three consanguineous PARK6 families: a truncating nonsense mutation and a missense mutation at a highly conserved amino acid. Cell culture studies suggest that PINK1 is mitochondrially located and may exert a protective effect on the cell that is abrogated by the mutations, resulting in increased susceptibility to cellular stress. These data provide a direct molecular link between mitochondria and the pathogenesis of PD.

Topics: Amino Acid Sequence; Animals; Apoptosis; Cell Line, Tumor; Codon, Nonsense; COS Cells; Exons; Humans; Leupeptins; Membrane Potentials; Mitochondria; Molecular Sequence Data; Mutation; Mutation, Missense; Neurons; Oxidative Stress; Parkinson Disease; Protein Kinases; Protein Structure, Tertiary; Transfection

Formation of intracellular inclusion bodies due to defects in the protein degradation machinery is associated with the pathogenesis of neurodegenerative diseases. Sequestosomal protein p62/A170/ZIP, which is an oxidative stress-related protein and a ubiquitin-binding protein, is a component protein of Lewy bodies that are observed in patients with Parkinson's disease. The association of p62 with poly-ubiquitinated proteins may be an important step in the formation of intracellular protein aggregates like Lewy bodies. To study the role of p62 in the formation of protein aggregates in PC12 cells, we monitored the intracellular localizations of p62 and ubiquitinated proteins and the levels of both components during treatment with MG132, a proteasome inhibitor. In the early stage of aggregate formation, p62 did not always co-localize with ubiquitin. In contrast, these proteins were always co-localized in later stages. After the treatment of the cells with MG132, we found that the expression level of p62 increased due to the transcriptional activation of the gene and that higher molecular sizes of p62, corresponding to mono- and di-ubiquitinated formes, were also formed. Both the transcriptional inhibitor actinomycin D and an antisense oligonucleotide of p62 inhibited the MG132-mediated increase of p62, the sequestration of ubiquitinated proteins, and the enlargement of the aggregates. Furthermore, p62-positive aggregates were observed primarily in surviving cells. Together, these results suggest that p62 plays an important role in the protection of cells from the toxicity of misfolded proteins by enhancing aggregate formation especially in the later stages.

Topics: Animals; Cell Aggregation; Heat-Shock Proteins; Humans; Leupeptins; Lewy Bodies; Male; Middle Aged; Parkinson Disease; PC12 Cells; Rats; Sequestosome-1 Protein; Transcriptional Activation

Impairment of the ubiquitin/proteasome system has been proposed to play a role in neurodegenerative disorders such as Alzheimer and Parkinson diseases. Although recent studies confirmed that some disease-related proteins block proteasomal degradation, and despite the existence of excellent animal models of both diseases, in vivo data about the system are lacking. We have developed a model for in vivo analysis of the ubiquitin/proteasome system by generating mouse strains transgenic for a green fluorescent protein (GFP) reporter carrying a constitutively active degradation signal. Administration of proteasome inhibitors to the transgenic animals resulted in a substantial accumulation of GFP in multiple tissues, confirming the in vivo functionality of the reporter. Moreover, accumulation of the reporter was induced in primary neurons by UBB+1, an aberrant ubiquitin found in Alzheimer disease. These transgenic animals provide a tool for monitoring the status of the ubiquitin/proteasome system in physiologic or pathologic conditions.

Topics: Alzheimer Disease; Animals; Boronic Acids; Cells, Cultured; Cysteine Endopeptidases; Fibroblasts; Leupeptins; Mice; Mice, Inbred C57BL; Mice, Transgenic; Microscopy, Fluorescence; Models, Animal; Multienzyme Complexes; Myocytes, Cardiac; Neurodegenerative Diseases; Neurons; Oligopeptides; Organ Specificity; Parkinson Disease; Proteasome Endopeptidase Complex; Recombinant Fusion Proteins; Tissue Distribution; Ubiquitin

Association between protein inclusions and neurodegenerative diseases, including Parkinson's and Alzheimer's diseases, and polyglutamine disorders, has been widely documented. Although ubiquitin is conjugated to many of these aggregated proteins, the 26S proteasome does not efficiently degrade them. Mutations in the ubiquitin-protein ligase Parkin are associated with autosomal recessive juvenile Parkinsonism. Although Parkin-positive inclusions are not detected in brains of autosomal recessive juvenile Parkinsonism patients, Parkin is found in Lewy bodies in sporadic disease. This suggests that loss of Parkin ligase activity via mutation, or sequestration to Lewy bodies, is a contributory factor to sporadic disease onset. We now demonstrate that decreased proteasomal activity causes formation of large, noncytotoxic inclusions within the cytoplasm of both neuronal and nonneuronal cells overexpressing Parkin. This is not a general phenomenon as there is an absence of similar inclusions when HHARI, a structural homolog of Parkin, is overexpressed. The inclusions colocalize with ubiquitin and with proteasomes. Furthermore, Parkin inclusions colocalize with gamma-tubulin, acetylated alpha-tubulin, and cause redistribution of vimentin, suggesting aggresome-like properties. Our data imply that lower proteasomal activity, previously observed in brain tissue of Parkinson's disease patients, leads to Parkin accumulation and a concomitant reduction in ligase activity, thereby promoting Lewy body formation.

Topics: Animals; Carrier Proteins; Chlorocebus aethiops; Cloning, Molecular; COS Cells; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Humans; Hydrogen Peroxide; Inclusion Bodies; Leupeptins; Lewy Body Disease; Microscopy, Confocal; Multienzyme Complexes; Neurons; Osmotic Pressure; Oxidative Stress; Parkinson Disease; Proteasome Endopeptidase Complex; Sorbitol; Tubulin; Tumor Cells, Cultured; Tunicamycin; Ubiquitin; Ubiquitin-Protein Ligases; Vimentin

The discovery of mutations in the gene for alpha-synuclein in familial Parkinson's disease (PD) has led to an increased interest in this pre-synaptic protein. Synphilin-1, a potential synuclein-binding protein, was cloned using yeast two-hybrid assays. The function of synphilin-1 is currently unknown, although it has been reported to be present along with alpha-synuclein in Lewy bodies in PD. In the present study, we monitored synphilin-1 aggregation directly using fusion proteins of synphilin-1 and green fluorescent protein (EGFP). Transfection of synphilin-EGFP fusion proteins formed cytoplasmic inclusions in HEK293 cells. Although these inclusions overlapped with the distribution of alpha-synuclein, they were unlike Lewy bodies in that they were not eosinophilic, and instead were membrane-bound, lipid-rich cytoplasmic inclusions.

Topics: Amino Acid Sequence; Carrier Proteins; Cell Line; Cloning, Molecular; Cysteine Endopeptidases; Embryo, Mammalian; Genes, Reporter; Green Fluorescent Proteins; Humans; Inclusion Bodies; Kidney; Leupeptins; Lewy Bodies; Luminescent Proteins; Molecular Sequence Data; Multienzyme Complexes; Nerve Tissue Proteins; Parkinson Disease; Point Mutation; Protease Inhibitors; Proteasome Endopeptidase Complex; Recombinant Fusion Proteins; Transfection; Two-Hybrid System Techniques

One of the hallmarks of Parkinson's disease (PD) is pathological structure, termed Lewy body, containing inclusions of ubiquitinated proteins in the dopaminergic neurons in the substantia nigra. The mechanism leading to the formation of these aggregates is unclear, although it has been shown that mutations in alpha-synuclein or in the ubiquitin-related enzyme UCH-L1 might induce such protein aggregation. We, therefore, examined the possible role of 6-hydroxydopamine (6-OHDA), a dopaminergic neurotoxin used in PD experimental models, in causing protein degradation and its association with the ubiquitin system. Using antiubiquitin antibodies we found that exposure of SH-SY5Y neuroblastoma and PC-12 cell lines to 6-OHDA increased the levels of free ubiquitin and ubiquitin-conjugated proteins, in a dose-dependent manner. Furthermore, metabolic labeling with 35S-methionine, demonstrated that 6-OHDA markedly increased protein degradation, as indicated by the secretion of protein metabolites to the medium. Inhibition of the proteasome activity by the specific inhibitor MG132, attenuated the protein degradation induced by 6-OHDA and potentiated its toxicity. Administration of the antioxidant N-acetylcysteine to the 6-OHDA-treated cells, increased cell survival and reduced protein degradation. In conclusion, our findings suggest that 6-OHDA toxicity is associated with protein degradation and ubiquitin-proteasome system activation.

Topics: Acetylcysteine; Animals; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Dose-Response Relationship, Drug; Free Radical Scavengers; Humans; Leupeptins; Lewy Bodies; Methionine; Models, Neurological; Multienzyme Complexes; Nerve Tissue Proteins; Neurons; Oxidopamine; Parkinson Disease; PC12 Cells; Proteasome Endopeptidase Complex; Rats; Substantia Nigra; Sulfur Radioisotopes; Sympatholytics; Ubiquitins

Mutations in the parkin gene cause autosomal recessive juvenile parkinsonism. Immunostaining of substantia nigra sections from sporadic Parkinson's disease (PD) cases shows that Parkin accumulates in axonal spheroids and in some Lewy bodies. Because ubiquitin is a major component of Lewy bodies and axonal spheroids, we investigated whether Parkin is metabolized via the ubiquitin/proteosomal pathway. Treatment of BE-M17 neuroblastoma cells with the proteosomal inhibitor, MG132, produced a band corresponding to di-ubiquitinated Parkin that was apparent by immunoblot using two different anti-Parkin antibodies. This higher mol. wt band also co-immunoprecipitated with Parkin. These data suggest that Parkin plays a role in the pathophysiology of sporadic PD, and that Parkin is a substrate for ubiquitination that is degraded by the proteosomal complex.

Topics: Aged; Antibodies; Cysteine Endopeptidases; Humans; Immunoblotting; Immunohistochemistry; Leupeptins; Ligases; Middle Aged; Multienzyme Complexes; Parkinson Disease; Precipitin Tests; Proteasome Endopeptidase Complex; Reference Values; Substantia Nigra; Substrate Specificity; Tumor Cells, Cultured; Ubiquitin-Protein Ligases; Ubiquitins