benzyloxycarbonylleucyl-leucyl-leucine-aldehyde and Nerve-Degeneration

Dysfunction of the proteasome system has been suggested to be implicated in neuronal degeneration. Modulation of KATP channels appears to affect the viability of neuronal cells exposed to toxic insults. However, the effect of KATP channel blockers on the neuronal cell death mediated by proteasome inhibition has not been studied. The present study investigated the effect of KATP channel blockers on proteasome inhibitor-induced apoptosis in differentiated PC12 cells and SH-SY5Y cells. 5-Hydroxydecanoate (a selective KATP channel blocker) and glibenclamide (a cell surface and mitochondrial KATP channel inhibitor) reduced the proteasome inhibitor-induced apoptosis. Addition of the KATP channel blockers attenuated the proteasome inhibitor-induced changes in the levels of apoptosis-related proteins, the loss of the mitochondrial transmembrane potential, the increase in the formation of reactive oxygen species and the depletion of glutathione in both cell lines. The results show that KATP channel blockers may attenuate proteasome inhibitor-induced apoptosis in PC12 cells by suppressing activation of the mitochondrial pathway and of the caspase-8- and Bid-dependent pathways. The preventive effect appears to be associated with the inhibition of the formation of reactive oxygen species and the depletion of glutathione. KATP channel blockade appears to prevent proteasome inhibition-induced neuronal cell death.

Topics: Animals; Apoptosis; Apoptosis Regulatory Proteins; Decanoic Acids; Dose-Response Relationship, Drug; Drug Therapy, Combination; Glutathione; Glyburide; Humans; Hydroxy Acids; KATP Channels; Leupeptins; Mitochondria; Nerve Degeneration; Neurogenesis; Neurons; Oxidative Stress; PC12 Cells; Potassium Channel Blockers; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Rats; Reactive Oxygen Species; Signal Transduction

The proteins' ubiquitination and their further degradation by proteasomes are crucial for cell cycle regulation, transcription and DNA replication, inflammatory response, and apoptosis. Proteasome inhibitors have recently become considered as a promising method in cancer and inflammatory disease therapy. In this study, utilizing the rat model, we try to establish the influence of proteasome inhibitor MG-132: (1) on the basis of spontaneous and evoked locomotor activity and (2) on the condition of nigrostriatal projections eight weeks after MG-132 intraperitoneal administration. We also discuss the current status of knowledge about intraperitoneal administration of MG-132, a laboratory method that is being used more and more. Our results revealed a lack of motor abnormalities, but significant loss (20%) of substantia nigra pars compacta dopaminergic neurons after systemic MG-132 administration. This loss was accompanied by a corresponding decrease (8%) of density of dopaminergic terminals in dorsolateral striatum. Moreover, evidence of very limited but ongoing fibre degeneration within the dorsal striatum suggests that MG-132 severely disturbed the nigrostriatal pathway. In summary, intraperitoneal application of proteasome inhibitor MG-132, despite the encouraging results of experimental treatment and prevention of many pathological processes, should be used with caution because of the potential adverse effects on the structure of the central nervous system, especially elements of the nigrostriatal pathway.

Topics: Animals; Immunohistochemistry; Injections, Intraperitoneal; Leupeptins; Male; Motor Activity; Nerve Degeneration; Proteasome Inhibitors; Rats; Rats, Wistar; Substantia Nigra

Infrasound is a kind of environmental noise. It can evoke biological resonance in organismic tissues including the central nervous system (CNS), causing displacement and distortion of cellular architectures. Several studies have revealed that certain intensity infrasound can impair normal functions of the brain, but the underlying mechanisms still remain largely unknown. Growing evidence has demonstrated that axonal degeneration is responsible for a variety of CNS dysfunctions. To explore whether neuronal axons are affected under infrasonic insults, we exposed cultured hippocampal neurons to infrasound with a frequency of 16 Hz and a pressure level of 130 dB for 1h, and examined the morphological and molecular changes of neuronal axons by immunocytochemistry and Western blotting, respectively. Our results showed that infrasound exposure significantly resulted in axonal degeneration of cultured hippocampal neurons, which was relatively independent of neuronal cell death. This infrasound-induced axonal degeneration can be significantly blocked by Ca²⁺ chelator EGTA and Rho kinase inhibitor Fasudil, but not by proteasome inhibitor MG132. Moreover, calcium imaging and RhoA activation assays revealed a great enhancement of Ca²⁺ influx within axons and RhoA activation after infrasound exposure, respectively. Depletion of Ca²⁺ by EGTA markedly inhibited this Ca²⁺ influx and attenuated RhoA activation as well. Thus, our findings revealed that axonal degeneration may be one of the important mechanisms underlying infrasound-induced CNS impairment, and Ca²⁺ influx pathway is likely implicated in the process.

Topics: Animals; Axons; Calcium; Calcium Signaling; Cells, Cultured; Egtazic Acid; Leupeptins; Nerve Degeneration; Noise; Rats; Rats, Sprague-Dawley

Defects in the ubiquitin-proteasome system have been related to aging and the development of neurodegenerative disease, although the effects of deficient proteasome activity during early postnatal development are poorly understood. Accordingly, we have assessed how proteasome dysfunction during early postnatal development, induced by administering proteasome inhibitors daily during the first 10 days of life, affects the behaviour of adult mice. We found that this regime of exposure to the proteasome inhibitors MG132 or lactacystin did not produce significant behavioural or morphological changes in the first 15 days of life. However, towards the end of the treatment with proteasome inhibitors, there was a loss of mitochondrial markers and activity, and an increase in DNA oxidation. On reaching adulthood, the memory of mice that were injected with proteasome inhibitors postnatally was impaired in hippocampal and amygdala-dependent tasks, and they suffered motor dysfunction and imbalance. These behavioural deficiencies were correlated with neuronal loss in the hippocampus, amygdala and brainstem, and with diminished adult neurogenesis. Accordingly, impairing proteasome activity at early postnatal ages appears to cause morphological and behavioural alterations in adult mice that resemble those associated with certain neurodegenerative diseases and/or syndromes of mental retardation.

Topics: Amygdala; Animals; Animals, Newborn; Ataxia; Biomarkers; Brain; Cognition Disorders; Depression; Disease Models, Animal; DNA; Dopaminergic Neurons; Enzyme Inhibitors; Exploratory Behavior; Hippocampus; Leupeptins; Memory; Mice; Mitochondria; Motor Activity; Nerve Degeneration; Nervous System; Oxidation-Reduction; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Ubiquitinated Proteins

The accumulation of misfolded and unfolded proteins in endoplasmic reticulum (ER) induces ER stress, activating the unfolded protein response (UPR). Recent evidence has suggested the relationship between UPR and dopaminergic neuronal cell death in Parkinson's disease (PD); however, it remains unclear whether it makes sense to modulate UPR, to mitigate the progression of PD. In this study, we investigated a role of the IRE1 alpha-XBP1 pathway in the survival of dopaminergic cells, under stress induced by PD-related insults. The exogenous expression of the active-form XBP1 (XBP1s) protein had protective effects against cell death induced by 1-methyl-4-phenylpyridinium (MPP+) and proteasome inhibitors. Moreover, adenoviral XBP1s expression significantly suppressed the degeneration of dopaminergic neurons in the mouse model of PD, as induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). These results demonstrate that the enhancement of XBP1 could be a novel PD therapeutic strategy.

Topics: 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine; 1-Methyl-4-phenylpyridinium; Acetylcysteine; Animals; Cell Death; Cell Line; Cell Survival; Cysteine Proteinase Inhibitors; DNA-Binding Proteins; Dopamine; Endoplasmic Reticulum; Endoribonucleases; Humans; Leupeptins; Male; Membrane Proteins; Mice; Mice, Inbred C57BL; Nerve Degeneration; Neurons; Parkinsonian Disorders; Protein Serine-Threonine Kinases; Regulatory Factor X Transcription Factors; RNA, Messenger; Signal Transduction; Stress, Physiological; Transcription Factors; X-Box Binding Protein 1

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

Impairment in ubiquitin-proteasome system (UPS) has recently been implicated in Parkinson's disease, as demonstrated by reduced proteasomal activities, protein aggregation and mutation of several genes associated with UPS. However, experimental studies with proteasome inhibitors failed to yield consensus regarding the effect of proteasome inhibition on dopaminergic degeneration. In this study, we systematically examined the effect of the proteasome inhibitor MG-132 on dopaminergic degeneration in cell culture and animal models of Parkinson's disease. Exposure of immortalized dopaminergic neuronal cells (N27) to low doses of MG-132 (2-10 microM) resulted in dose- and time-dependent cytotoxicity. Further, exposure to MG-132 (5 microM) for 10 min led to dramatic reduction of proteasomal activity (>70%) accompanied by a rapid accumulation of ubiquitinated proteins in these cells. MG-132 treatment also induced increases in caspase-3 activity in a time-dependent manner, with significant activation occurring between 90 and 150 min. We also noted a 12-fold increase in DNA fragmentation in MG-132 treated N27 cells. Similarly, primary mesencephalic neurons exposed to 5 microM MG-132 also induced >60% loss of TH positive neurons but only a minimal loss of non-dopaminergic cells. Stereotaxic injection of MG-132 (0.4 microg in 4 microl) into the substantia nigra compacta (SNc) in C57 black mice resulted in significant depletion of ipisilateral striatal dopamine and DOPAC content as compared to the vehicle-injected contralateral control sides. Also, we observed a significant decrease in the number of TH positive neurons in the substantia nigra of MG-132-injected compared to the vehicle-injected sites. Collectively, these results demonstrate that the proteasomal inhibitor MG-132 induces dopamine depletion and nigral dopaminergic degeneration in both cell culture and animal models, and suggest that proteasomal dysfunction may promote nigral dopaminergic degeneration in Parkinson's disease.

Topics: Analysis of Variance; Animals; Caspase 3; Caspases; Cell Death; Cells, Cultured; Cysteine Proteinase Inhibitors; Disease Models, Animal; DNA Fragmentation; Dopamine; Dose-Response Relationship, Drug; Embryo, Mammalian; Immunohistochemistry; Leupeptins; Mesencephalon; Mice; Mice, Inbred C57BL; Nerve Degeneration; Neurons; Neurotransmitter Agents; Proteasome Inhibitors; Rats; Tyrosine 3-Monooxygenase

Changes in prion protein (PrP) folding are associated with fatal neurodegenerative disorders, but the neurotoxic species is unknown. Like other proteins that traffic through the endoplasmic reticulum, misfolded PrP is retrograde transported to the cytosol for degradation by proteasomes. Accumulation of even small amounts of cytosolic PrP was strongly neurotoxic in cultured cells and transgenic mice. Mice developed normally but acquired severe ataxia, with cerebellar degeneration and gliosis. This establishes a mechanism for converting wild-type PrP to a highly neurotoxic species that is distinct from the self-propagating PrP(Sc) isoform and suggests a potential common framework for seemingly diverse PrP neurodegenerative disorders.

Topics: Animals; Apoptosis; Brain; Cell Survival; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Cytosol; Glycosylation; In Situ Nick-End Labeling; Leupeptins; Membrane Proteins; Mice; Mice, Transgenic; Multienzyme Complexes; Nerve Degeneration; Neurons; Presenilin-1; Prion Diseases; Prions; Promoter Regions, Genetic; Proteasome Endopeptidase Complex; Protein Conformation; Protein Folding; Protein Transport; PrPSc Proteins; Transfection; Tumor Cells, Cultured