benzyloxycarbonylvalyl-alanyl-aspartyl-fluoromethyl-ketone and Parkinson-Disease

Topics: 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine; Amino Acid Chloromethyl Ketones; Animals; Apoptosis Inducing Factor; Caspase 1; Caspase 7; Cell Death; Cell Line, Tumor; Disease Models, Animal; Dopaminergic Neurons; Down-Regulation; Enzyme Activation; Humans; Mice, Knockout; Models, Biological; Motor Activity; Parkinson Disease; Poly(ADP-ribose) Polymerases

Environmental exposure to neurotoxic metals through various sources including exposure to welding fumes has been linked to an increased incidence of Parkinson's disease (PD). Welding fumes contain many different metals including vanadium typically present as particulates containing vanadium pentoxide (V2O5). However, possible neurotoxic effects of this metal oxide on dopaminergic neuronal cells are not well studied. In the present study, we characterized vanadium-induced oxidative stress-dependent cellular events in cell culture models of PD. V2O5 was neurotoxic to dopaminergic neuronal cells including primary nigral dopaminergic neurons and the EC50 was determined to be 37 microM in N27 dopaminergic neuronal cell model. The neurotoxic effect was accompanied by a time-dependent uptake of vanadium and upregulation of metal transporter proteins Tf and DMT1 in N27 cells. Additionally, vanadium resulted in a threefold increase in reactive oxygen species generation, followed by release of mitochondrial cytochrome c into cytoplasm and subsequent activation of caspase-9 (>fourfold) and caspase-3 (>ninefold). Interestingly, vanadium exposure induced proteolytic cleavage of native protein kinase Cdelta (PKCdelta, 72-74 kDa) to yield a 41 kDa catalytically active fragment resulting in a persistent increase in PKCdelta kinase activity. Co-treatment with pan-caspase inhibitor Z-VAD-FMK significantly blocked vanadium-induced PKCdelta proteolytic activation, indicating that caspases mediate PKCdelta cleavage. Also, co-treatment with Z-VAD-FMK almost completely inhibited V2O5-induced DNA fragmentation. Furthermore, PKCdelta knockdown using siRNA protected N27 cells from V2O5-induced apoptotic cell death. Collectively, these results demonstrate that vanadium can exert neurotoxic effects in dopaminergic neuronal cells via caspase-3-dependent PKCdelta cleavage, suggesting that metal exposure may promote nigral dopaminergic degeneration.

Topics: Amino Acid Chloromethyl Ketones; Animals; Apoptosis; Caspase 3; Caspase 9; Cation Transport Proteins; Cell Line; Cell Survival; Cysteine Proteinase Inhibitors; Cytochromes c; DNA Fragmentation; Dopamine; Dose-Response Relationship, Drug; Environmental Pollutants; Inhibitory Concentration 50; Mesencephalon; Mitochondria; Neurons; Neurotoxicity Syndromes; Oxidative Stress; Parkinson Disease; Protein Kinase C-delta; Rats; Reactive Oxygen Species; RNA Interference; Signal Transduction; Time Factors; Transferrin; Vanadium Compounds

Lewy bodies (LBs), which are the hallmark pathologic features of Parkinson's disease and of dementia with LBs, have several morphologic and molecular similarities to aggresomes. Whether such cytoplasmic inclusions contribute to neuronal death or protect cells from the toxic effects of misfolded proteins remains controversial. In this report, the role of aggresomes in cell viability was addressed in the context of over-expressing alpha-synuclein and its interacting partner synphilin-1 using engineered 293T cells. Inhibition of proteasome activity elicited the formation of juxtanuclear aggregates with characteristics of aggresomes including immunoreactivity for vimentin, gamma-tubulin, ubiquitin, proteasome subunit, and hsp70. As expected from the properties of aggresomes, the microtubule disrupting agents, vinblastin and nocodazole, markedly prevented the formation of these inclusions. Similar to LBs, the phosphorylated form of alpha-synuclein co-localized in these synphilin-1-containing aggresomes. Although the caspase inhibitor z-VAD-fmk significantly reduced the number of apoptotic cells, it had no impact on the percentage of aggresome-positive cells. Finally, quantitative analysis revealed aggresomes in 60% of nonapoptotic cells but only in 10% of apoptotic cells. Additionally, alpha-synuclein-induced apoptosis was not coupled with increased prevalence of aggresome-bearing cells. Taken together, these observations indicate a disconnection between aggresome formation and apoptosis, and support a protective role for these inclusions from the toxicity associated with the combined over-expression of alpha-synuclein and synphilin-1.

Topics: alpha-Synuclein; Amino Acid Chloromethyl Ketones; Apoptosis; Carrier Proteins; Cell Line; Cell Survival; Cysteine Proteinase Inhibitors; Humans; Inclusion Bodies; Lewy Bodies; Lewy Body Disease; Macromolecular Substances; Microtubules; Nerve Tissue Proteins; Neurons; Neuroprotective Agents; Nocodazole; Parkinson Disease; Recombinant Proteins; Synucleins; Transfection; Vinblastine

Parkinson's disease is a neurodegenerative disorder associated with selective loss of the dopaminergic neurons in the substantia nigra. We have previously shown that tetrahydrobiopterin (BH4), the obligatory cofactor for dopamine synthesis, exerts selective toxicity on dopamine-producing cells. In the present study we determined, both in vitro and in vivo, whether the cell death induced by this endogenous molecule involves apoptosis, resembling that which occurs in Parkinson's disease. Transmission electron microscopic analysis revealed that the dopamine-producing CATH.a cells underwent ultrastructural changes typical of apoptosis, such as cell shrinkage and chromatin condensation, upon exposure to BH4. The BH4 treatment also caused intranuclear DNA fragmentation as determined by TUNEL staining. A similar phenomenon also occurred in vivo, as the nigral cells became TUNEL-positive upon injection of BH4 into the substantia nigra. The BH4-induced CATH.a cell death seemed to involve macromolecule synthesis because cycloheximide and actinomycin D had protective effects. Concurrent treatment with the caspase inhibitor Z-VAD-FMK also suppressed cell death. BH4 treatment led to increases in the ratio of Bax/Bcl-x(L) mRNA and protein levels. Ca(2+) seemed to play a role in BH4-induced cell death, because BH4 caused an increase in Ca(2+) uptake and the intracellular Ca(2+) release blocker dantrolene, intracellular Ca(2+) chelator BAPTA/AM, and extracellular Ca(2+) chelator EGTA each attenuated the toxicity. These data provide evidence that the dopaminergic cell death induced by BH4 involves apoptosis and suggest relevance of this cell death to degeneration of the dopaminergic system in Parkinson's disease.

Topics: Amino Acid Chloromethyl Ketones; Animals; Apoptosis; Biopterins; Calcium; Cell Death; Cell Line; Cysteine Proteinase Inhibitors; DNA Fragmentation; Dopamine; Female; In Situ Nick-End Labeling; Mice; Neurons; Parkinson Disease; Rats; Rats, Sprague-Dawley

Parkinson's disease is characterized by a loss of dopaminergic nigrostriatal neurons. This neuronal loss is mimicked by the neurotoxin 1-methyl-4-phenylpyridinium (MPP+). MPP+ toxicity is mediated through inhibition of mitochondrial complex I, decreasing ATP production, and upregulation of oxygen radicals. There is evidence that the cell death induced by MPP+ is apoptotic and that inhibition of caspases may be neuroprotective. In primary cultures of rat mesencephalic dopaminergic neurons, MPP+ treatment decreased the number of surviving dopaminergic neurons in the cultures and the ability of the neurons to take up [3H]dopamine ([3H]DA). Caspase inhibition using the broad-spectrum inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (zVAD-fmk) spared MPP+-treated dopaminergic neurons and increased somatic size. There was a partial restoration of neurite length in zVAD-fmk-treated cultures, but little restoration of [3H]DA uptake. Peptide inhibitors of caspases 2, 3, and 9, but not of caspase 1, caused significant neuroprotection. Two novel caspase inhibitors were tested for neuroprotection, a broad spectrum inhibitor and a selective caspase 3 inhibitor; both inhibitors increased survival to >90% of control. No neuroprotection was observed with an inactive control compound. MPP+ treatment caused chromatin condensation in dopaminergic neurons and increased expression of activated caspase 3. Inhibition of caspases with either zVAD-fmk or a selective caspase 3 inhibitor decreased the number of apoptotic profiles, but not expression of the active caspase. We conclude that MPP+ toxicity in primary dopaminergic neurons involves activation of a pathway terminating in caspase 3 activation, but that other mechanisms may underlie the neurite loss.

Topics: 1-Methyl-4-phenylpyridinium; Amino Acid Chloromethyl Ketones; Animals; Caspase 3; Caspase Inhibitors; Caspases; Cell Survival; Cells, Cultured; Dopamine; Dose-Response Relationship, Drug; Enzyme Inhibitors; Mesencephalon; Neurites; Neurons; Neuroprotective Agents; Parkinson Disease; Rats; Rats, Sprague-Dawley; Tyrosine 3-Monooxygenase

Nerve growth factor (NGF) mediates a variety of nerve cell actions through receptor tyrosine kinase TrkA. It has been revealed that the Akt pathway contributes to the prevention of apoptosis. It is thought that Parkinson's disease involves apoptosis, and NGF prevents apoptosis in an in vivo model system. However, there is no evidence that the Akt pathway helps to prevent parkinsonism. Here, we report that NGF prevents apoptosis induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in PC12 cells as an in vitro model system of parkinsonism and that this survival effect diminishes on addition of LY294002, a specific inhibitor of phosphatidylinositol 3-kinase. Immunocytochemical analysis revealed that 1 mM MPTP-treated cells or dominant negative Akt-expressing cells, to which were added NGF and MPTP, undergo apoptosis. Moreover, the caspase-3-like activity is increased by addition of MPTP or MPTP with NGF and LY294002. The importance of another signal pathway is shown by PD98059, a specific inhibitor of MAP kinase (MAPK) kinase, but PD98059 does not alter the survival effect in this model system. These results indicate that the Akt pathway helps to prevent parkinsonism by suppressing caspase-3-like activity, but the MAPK pathway is not involved in the NGF-dependent survival enhancing effect in this model system.

Topics: 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine; Amino Acid Chloromethyl Ketones; Amino Acid Substitution; Animals; Apoptosis; Caspase 3; Caspases; Chromones; Coumarins; Cysteine Proteinase Inhibitors; Dopamine Agents; Drug Evaluation, Preclinical; Enzyme Inhibitors; Flavonoids; Genes, Dominant; MAP Kinase Signaling System; Mitogen-Activated Protein Kinases; Morpholines; Mutagenesis, Site-Directed; Neoplasm Proteins; Nerve Growth Factor; Oligopeptides; Parkinson Disease; Parkinsonian Disorders; PC12 Cells; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Phosphoserine; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Rats; Recombinant Fusion Proteins; Signal Transduction; Transfection

There is growing evidence that apoptotic mechanisms underlie the neurodegeneration leading to Parkinson's disease. 1-Methyl-4-phenylpyridinium ion (MPP(+)), the active metabolite of the parkinsonism-inducing drug MPTP, induced apoptosis in cultures of human SH-SY5Y neuroblastoma cells. Nuclear fragmentation, DNA laddering, and a 20% decrease in viability were seen after a 4-day incubation with 5 microM MPP(+). Cell viability decreased by 40% at 100 microM MPP(+), but the degree of apoptosis was not correlatively increased. The MPP(+)-induced apoptosis was completely prevented by the broad caspase inhibitor zVAD.fmk but not by the caspase-8 inhibitor IETD.fmk. Furthermore, MPP(+) had no effect on the levels of Fas or Fas-L, suggesting lack of activation of the Fas-L/Fas/caspase-8 pathway of apoptosis. There was no evidence of mitochondrial dysfunction at 5 microM MPP(+): No differences were seen in transmembrane potential or in cytochrome c release from controls. At 100 microM MPP(+), the mitochondrial potential decreased, and cytoplasmic cytochrome c and caspase-9 activation increased slightly. At both low and high concentrations of MPP(+), VDVADase and DEVDase activities increased. We conclude that MPP(+) can induce caspase-mediated apoptosis, which is prevented by caspase inhibition, at concentrations lower than those needed to trigger mitochondrial dysfunction and closer to those found in the brains of MPTP-treated animals.

Topics: 1-Methyl-4-phenylpyridinium; Amino Acid Chloromethyl Ketones; Apoptosis; Caspase 8; Caspase 9; Caspase Inhibitors; Caspases; Cyclosporine; Cysteine Proteinase Inhibitors; Cytochrome c Group; DNA Fragmentation; Dose-Response Relationship, Drug; Fas Ligand Protein; fas Receptor; Humans; Membrane Glycoproteins; Membrane Potentials; Mitochondria; Neoplasm Proteins; Nerve Tissue Proteins; Neuroblastoma; Oligopeptides; Osmolar Concentration; Parkinson Disease; Tumor Cells, Cultured

Proteasomal dysfunction has been recently implicated in the pathogenesis of several neurodegenerative diseases, including Parkinson's disease and diffuse Lewy body disease. We have developed an in vitro model of proteasomal dysfunction by applying pharmacological inhibitors of the proteasome, lactacystin or ZIE[O-tBu]-A-leucinal (PSI), to dopaminergic PC12 cells. Proteasomal inhibition caused a dose-dependent increase in death of both naive and neuronally differentiated PC12 cells, which could be prevented by caspase inhibition or CPT-cAMP. A percentage of the surviving cells contained discrete cytoplasmic ubiquitinated inclusions, some of which also contained synuclein-1, the rat homologue of human alpha-synuclein. However the total level of synuclein-1 was not altered by proteasomal inhibition. The ubiquitinated inclusions were present only within surviving cells, and their number was increased if cell death was prevented. We have thus replicated, in this model system, the two cardinal pathological features of Lewy body diseases, neuronal death and the formation of cytoplasmic ubiquitinated inclusions. Our findings suggest that inclusion body formation and cell death may be dissociated from one another.

Topics: Acetylcysteine; alpha-Synuclein; Amino Acid Chloromethyl Ketones; Animals; Apoptosis; Cell Differentiation; Cyclic AMP; Cysteine Endopeptidases; Immunoblotting; Immunohistochemistry; Inclusion Bodies; Lewy Body Disease; Multienzyme Complexes; Nerve Tissue Proteins; Neuroprotective Agents; Oligopeptides; Parkinson Disease; PC12 Cells; Protease Inhibitors; Proteasome Endopeptidase Complex; Rats; Synucleins; Ubiquitins