alpha-synuclein and linsidomine

Parkinson's disease (PD) is a progressive, chronic disease characterized by dyskinesia, rigidity, instability, and tremors. The disease is defined by the presence of Lewy bodies, which primarily consist of aggregated α-synuclein protein, and is accompanied by the loss of monoaminergic neurons. Current therapeutic strategies only give symptomatic relief of motor impairment and do not address the underlying neurodegeneration. Hence, we have identified Cu(II)(atsm) as a potential therapeutic for PD. Drug administration to four different animal models of PD resulted in improved motor and cognition function, rescued nigral cell loss, and improved dopamine metabolism. In vitro, this compound is able to inhibit the effects of peroxynitrite-driven toxicity, including the formation of nitrated α-synuclein oligomers. Our results show that Cu(II)(atsm) is effective in reversing parkinsonian defects in animal models and has the potential to be a successful treatment of PD.

Topics: alpha-Synuclein; Animals; Cell Line, Tumor; Cognition; Coordination Complexes; Disease Models, Animal; Humans; Male; Mice; Mice, Inbred C57BL; Molsidomine; Motor Activity; Neuroprotective Agents; Organometallic Compounds; Parkinson Disease; Positron-Emission Tomography; Radiopharmaceuticals; Rats; Rats, Sprague-Dawley; Thiosemicarbazones

Parkinson's disease (PD) is a progressive neurodegenerative movement disorder characterized by selective loss of dopaminergic neurons and the presence of Lewy bodies. Alpha-synuclein is a major component of Lewy bodies in sporadic PD, and genetic alterations in alpha-synuclein cause autosomal-dominant hereditary PD. The pathogenesis of PD remains incompletely understood, but it appears to involve both genetic susceptibility and environmental factors. Here we investigated the effect of alpha-synuclein expression on cell susceptibility to proteasome inhibition, oxidative and nitrative stresses by using a PC 12-Tet-off regulatory system. We found that inducible expression of A30P or A53T mutant alpha-synuclein decreased the proteasome activity, increased intracellular ROS levels, and enhanced lactacystin- and H2O2-induced cell death. Furthermore, 3-nitrotyrosine levels increased in cells expressing alpha-synuclein, and further increased after Sin-1 (a NO donor) treatment compared with untreated or treated non-induced cells. Expression of alpha-synuclein (mutant more than wild type) significantly enhances Sin-1 toxicity. These results indicate that genetic mutations in alpha-synuclein may increase neuronal vulnerability to cellular stress in aging and PD pathogenesis.

Topics: alpha-Synuclein; Animals; Cell Death; Dose-Response Relationship, Drug; Genetic Predisposition to Disease; Hydrogen Peroxide; Molsidomine; Mutation; Parkinson Disease; PC12 Cells; Rats; Reactive Oxygen Species

Parkinson's disease (PD) is a multifactorial disease caused by both genetic and environmental factors. Alpha-synuclein is of particular interest in PD since it is a major component of Lewy bodies and mutations in the alpha-synuclein gene were identified in familial PD. Oxidative stress and proteasomal dysfunction are implicated in the pathogenesis of PD but their interactions as well as their effect on aggregates formation are not yet clear. We therefore examined the roles of oxidative stress and proteasomal inhibition on protein aggregates induction in naïve and neuronally differentiated neuroblastoma SH-SY5Y cells. Neuroblastoma cells were stably transfected with wild type (WT) and A53T mutant alpha-synuclein. Naïve and transfected cells were exposed to oxidative stress induced by rotenone, SIN-I, FeCl(2,) and to proteasomal inhibition by lactacystin. Proteasomal inhibition caused a dose-dependent decrease in viability and induced protein aggregates formation containing alpha-synuclein and ubiquitin. Proteasomal inhibition induced significantly increased alpha-synuclein aggregation in cells expressing mutant alpha-synuclein. Exposure to reactive oxygen species (ROS) combined with proteasomal inhibition increased aggregates formation. Inclusion body formation and cell death of differentiated neuroblastoma cells overexpressing alpha-synuclein can serve as a valuable model for elucidating the molecular components that cause neurodegeneration in PD as well as evaluating pharmacological interventions.

Topics: alpha-Synuclein; Benzothiazoles; Cell Differentiation; Cell Line, Tumor; Cytoplasm; Ferrous Compounds; Humans; Immunohistochemistry; Molsidomine; Mutation; Neuroblastoma; Oxidative Stress; Parkinson Disease; Proteasome Inhibitors; Reactive Oxygen Species; Rotenone; Thiazoles; Ubiquitin

Increased accumulation of alpha-synuclein is associated with certain neurodegenerative diseases including Parkinson's disease (PD) and Alzheimer's disease (AD). One mechanism of alpha-synuclein-induced toxicity involves increased oxidative stress. It was unknown whether neurons overexpressing alpha-synuclein would exhibit increased sensitivity to hydrogen peroxide (H(2)O(2)) or 3-morpholinosydnonimine (SIN-1; a nitrous oxide donor). To study this, we developed a murine neuroblastoma (NB) cell line that overexpresses wild-type human alpha-synuclein (NBP2-PN54) under the control of the cytomegalovirus (CMV) promoter using a retroviral vector. Human alpha-synuclein mRNA and protein were readily detectable in NBP2-PN54 cells. Results showed that differentiated NBP2-PN54 cells exhibited decreased viability in comparison to differentiated vector (NBP2-PN1) and parent (NBP2) control cells. These cells also exhibited increased sensitivity to PGE(2), H(2)O(2) and SIN-1. Because of involvement of proteasome inhibition in neurodegeneration, we also investigated whether treatment of differentiated NBP2-PN54 cells with PGE(2), H(2)O(2) or SIN-1 inhibits proteasome activity. Results showed that H(2)O(2) and SIN-1 inhibited proteasome activity, but PGE(2) did not. These results suggest that overexpression of alpha-synuclein not only participates directly in degeneration of neurons, but it also increases the vulnerability of neurons to other potential neurotoxins.

Topics: alpha-Synuclein; Animals; Cell Differentiation; Cell Survival; Dinoprostone; Dose-Response Relationship, Drug; Humans; Hydrogen Peroxide; Mice; Molsidomine; Nerve Tissue Proteins; Neuroblastoma; Nitric Oxide Donors; Synucleins; Transfection; Tumor Cells, Cultured

Parkinson's disease is characterized by a progressive loss of dopaminergic neurons in the substantia nigra zona compacta, and in other subcortical nuclei associated with a widespread occurrence of Lewy bodies. The causes of cell death in Parkinson's disease are still poorly understood, but a defect in mitochondrial oxidative phosphorylation and enhanced oxidative stress have been proposed. We have examined 3-morpholinosydnonimine (SIN-1)-induced apoptosis in control and metallothionein-overexpressing dopaminergic neurons, with a primary objective to determine the neuroprotective potential of metallothionein against peroxynitrite-induced neurodegeneration in Parkinson's disease. SIN-1 induced lipid peroxidation and triggered plasma membrane blebbing. In addition, it caused DNA fragmentation, alpha-synuclein induction, and intramitochondrial accumulation of metal ions (copper, iron, zinc, and calcium), and enhanced the synthesis of 8-hydroxy-2-deoxyguanosine. Furthermore, it down-regulated the expression of Bcl-2 and poly(ADP-ribose) polymerase, but up-regulated the expression of caspase-3 and Bax in dopaminergic (SK-N-SH) neurons. SIN-1 induced apoptosis in aging mitochondrial genome knockout cells, alpha-synuclein-transfected cells, metallothionein double-knockout cells, and caspase-3-overexpressed dopaminergic neurons. SIN-1-induced changes were attenuated with selegiline or in metallothionein-transgenic striatal fetal stem cells. SIN-1-induced oxidation of dopamine to dihydroxyphenylacetaldehyde was attenuated in metallothionein-transgenic fetal stem cells and in cells transfected with a mitochondrial genome, and enhanced in aging mitochondrial genome knockout cells, in metallothionein double-knockout cells and caspase-3 gene-overexpressing dopaminergic neurons. Selegiline, melatonin, ubiquinone, and metallothionein suppressed SIN-1-induced down-regulation of a mitochondrial genome and up-regulation of caspase-3 as determined by reverse transcription-polymerase chain reaction. The synthesis of mitochondrial 8-hydroxy-2-deoxyguanosine and apoptosis-inducing factors were increased following exposure to 1-methyl-4-phenylpyridinium ion or rotenone. Pretreatment with selegiline or metallothionein suppressed 1-methyl-4-phenylpyridinium ion-, 6-hydroxydopamine-, and rotenone-induced increases in mitochondrial 8-hydroxy-2-deoxyguanosine accumulation. Transfection of aging mitochondrial genome knockout neurons with mitochondrial genome encoding complex-1 or mela

Topics: alpha-Synuclein; Animals; Apoptosis; Caspase 3; Caspases; Cell Line, Tumor; Corpus Striatum; Deoxyadenosines; Dopamine; Gene Expression Regulation; Humans; Lipid Peroxidation; Metallothionein; Metals; Mice; Mice, Inbred C57BL; Mice, Knockout; Mitochondria; Molsidomine; Nerve Tissue Proteins; Neurons; Neuroprotective Agents; Oxidative Stress; Selegiline; Stem Cells; Synucleins; Transcriptional Activation