Proteasome inhibition by MG-132 protects against deltamethrin-induced apoptosis in rat hippocampus.

AIMS: Deltamethrin (DM), a type II synthetic pyrethroid insecticide, is widely used in agriculture and home pest control. The evaluation of their toxic effects is of major concern to public health. However, the molecular mechanism of DM-induced neurodegenerative disease is still far from clear. This study was designed to investigate the potential role of ubiquitin proteasome system (UPS) in DM-induced neurotoxicity where the proteasome inhibitor MG-132 could mitigate the neurotoxic effects. MAIN METHODS: Male Sprague-Dawley rats were divided into two batches. The first batch of rats was administrated with a single dose of DM (12.5 mg/kg) by intraperitoneal injections (i.p.) and the animals were then euthanized at 5, 24, and 48 h post injection. The second batch was treated as follow: control group, DM (12.5 mg/kg) groups for 24 h, MG-132 (0.5 mg/kg, i.p.) 2 h plus DM 24 h group, and MG-132 alone group. Ubiqutinatied proteins, DNA damage and apoptosis were investigated. KEY FINDINGS: DM treatment induced the ubiquitinated proteins expression with the peaks at 5 h. Moreover, DM increased DNA damage, early apoptotic rate, the expression level of Cleaved Caspase-3, caspase-3 activity and decreased the expression level of Bcl-2 at DM 24 h group. Compared to DM 24 h group, MG-132 pretreatment significantly down-regulated ubiquitinated proteins, lowered the DNA damage and apoptosis by decreasing Caspase-3 and increasing Bcl-2 expression. SIGNIFICANCE: These results indicate that MG-132 effectively alleviates DM-induced DNA damage and apoptosis by inhibiting ubiquitinated proteins. UPS may play a role in DM-induced neurodegenerative disorders.

Methamphetamine-induced neurotoxicity linked to ubiquitin-proteasome system dysfunction and autophagy-related changes that can be modulated by protein kinase C delta in dopaminergic neuronal cells.

A compromised protein degradation machinery has been implicated in methamphetamine (MA)-induced neurodegeneration. However, the signaling mechanisms that induce autophagy and ubiquitin-proteasome system (UPS) dysfunction are not well understood. The present study investigates the contributions of protein kinase C delta (PKCδ)-mediated signaling events in MA-induced autophagy, UPS dysfunction, and cell death. Using an in vitro mesencephalic dopaminergic cell culture model, we demonstrate that MA-induced early induction of autophagy is associated with reduction in proteasomal function and concomitant dissipation of mitochondrial membrane potential (MMP), followed by significantly increased PKCδ activation, caspase-3 activation, accumulation of ubiquitin-positive aggregates and microtubule-associated light chain-3 (LC3-II) levels. Interestingly, siRNA-mediated knockdown of PKCδ or overexpression of cleavage-resistant mutant of PKCδ dramatically reduced MA-induced autophagy, proteasomal function, and associated accumulation of ubiquitinated protein aggregates, which closely paralleled cell survival. Importantly, when autophagy was inhibited either pharmacologically (3-MA) or genetically (siRNA-mediated silencing of LC3), the dopaminergic cells became sensitized to MA-induced apoptosis through caspase-3 activation. Conversely, overexpression of LC3 partially protected against MA-induced apoptotic cell death, suggesting a neuroprotective role for autophagy in MA-induced neurotoxicity. Notably, rat striatal tissue isolated from MA-treated rats also exhibited elevated LC3-II, ubiquitinated protein levels, and PKCδ cleavage. Taken together, our data demonstrate that MA-induced autophagy serves as an adaptive strategy for inhibiting mitochondria-mediated apoptotic cell death and degradation of aggregated proteins. Our results also suggest that the sustained activation of PKCδ leads to UPS dysfunction, resulting in the activation of caspase-3-mediated apoptotic cell death in the nigrostriatal dopaminergic system.

Diminishing HDACs by drugs or mutations promotes normal or abnormal sister chromatid separation by affecting APC/C and adherin.

Histone acetyltransferases (HATs) and histone deacetylases (HDACs) play important roles in cell regulation, including cell cycle progression, although their precise role in mitotic progression remains elusive. To address this issue, the effects of HDAC inhibition were examined upon a variety of mitotic mutants of the fission yeast Schizosaccharomyces pombe, which contains three HDACs that are sensitive to trichostatin A (TSA) and are similar to human HDACs. Here it is shown that HDACs are implicated in sister chromatid cohesion and separation. A mutant of the cohesin loader Mis4 (adherin) was hypersensitive to TSA and synthetically lethal with HDAC deletion mutations. TSA treatment of mis4 mutant cells decreased chromatin-bound cohesins in the chromosome arm region. By contrast, HDAC inhibitors and clr6 HDAC mutations rescued temperature sensitive (ts) phenotypes of the mutants of the ubiquitin ligase complex anaphase-promoting complex/cyclosome (APC/C), which display metaphase arrest. This suppression coincided with facilitated complex formation of APC/C. Moreover, our mass spectrometry analysis showed that an APC/C subunit, Cut23/APC8, is acetylated. HATs and HDACs might directly target adherin and APC/C to ensure proper chromosome segregation, and anti-tumour effects of HDAC inhibitors could be attributed to this deregulation.

Increased activity of the ubiquitin-proteasome system in patients with symptomatic carotid disease is associated with enhanced inflammation and may destabilize the atherosclerotic plaque: effects of rosiglitazone treatment.

OBJECTIVES: We evaluated ubiquitin-proteasome activity in carotid plaques of asymptomatic and symptomatic patients and the effect of rosiglitazone, a peroxisome proliferator-activated receptor-gamma activator, in symptomatic plaques. BACKGROUND: The role of the ubiquitin-proteasome system, the major pathway for non-lysosomal intracellular protein degradation in eucaryotic cells, in the progression of atherosclerotic plaque to instability is unclear. METHODS: Plaques were obtained from 40 symptomatic and 38 asymptomatic patients undergoing carotid endarterectomy. Symptomatic patients received 8 mg rosiglitazone (n = 20) or placebo (n = 20) for 4 months before scheduled endarterectomy. Plaques were analyzed for macrophages (CD68), T-lymphocytes (CD3), inflammatory cells (HLA-DR), ubiquitin-proteasome activity, nuclear factor kappa B (NFkB), inhibitory kappa B (IkB)-beta, nitrotyrosine, matrix metalloproteinase (MMP)-9, and collagen content (immunohistochemistry and enzyme-linked immunosorbent assay). RESULTS: Compared with asymptomatic plaques, symptomatic plaques had more macrophages, T-lymphocytes, and HLA-DR+ cells (p < 0.001); more ubiquitin-proteasome activity and NFkB (p < 0.001); and more markers of oxidative stress (nitrotyrosine and O2- production) and MMP-9 (p < 0.01) along with a lesser collagen content and IkB-beta levels (p < 0.001). Compared with placebo-treated plaques, rosiglitazone-treated symptomatic plaques presented fewer inflammatory cells (p < 0.01); less ubiquitin, proteasome 20S, and NFkB (p < 0.01); less nitrotyrosine and O2- production (p<0.01); and greater collagen content (p<0.01), indicating a more stable plaque phenotype. CONCLUSIONS: Ubiquitin-proteasome overactivity is associated with enhanced inflammatory reaction in symptomatic plaques. The inhibition of ubiquitin-proteasome activity in lesions of symptomatic patients by rosiglitazone is associated with plaque stabilization, possibly by down-regulating NFkB-mediated inflammatory pathways.

Morphine modulation of the ubiquitin-proteasome complex is neuroprotective.

BACKGROUND: Over the past several decades, there is a growing need for the development of neuroprotective compounds, e.g, those that can prevent neural death. It was proposed that nitric oxide (NO), when induced by morphine, would produce neuroprotection in a human neuroblastoma cell line when tested concomitantly with compounds that produce intracellular oxidative stress and neuroinflammation. MATERIAL/METHODS: NO involvement in intracellular protein degradation controlled by the ubiquitin-proteasome complex was examined. Experiments were performed examining the following: a) neural cell viability and morphology; b) gene specific mRNA levels via semi-quantitative RT-PCR; c) protein levels via Western blotting; d) enzymatic activity via fluorogenic substrate-cleaving assays; and lastly, NO release via the Apollo 4000 real-time amperometric detector. RESULTS: Morphine induces the production of NO in human neuroblastoma cells, which can be blocked by naloxone and the cNOS inhibitor L-NAME. Rotenone, which induces oxidative stress and increases the expression of the proteasomal catalytic X subunit, causes the cells to die and morphine inhibits this process via NO. Rotenone also increases the activity of the 20S proteasome, whereas morphine alone or in the presence of rotenone caused a decrease in the activity of the 20S proteasome. Morphine decreases the expression of the immunoproteasome catalytic subunit LMP7 in response to inflammatory stimulation, demonstrating that morphine's neuroprotective action does not apply to only oxidative stress. Morphine significantly increases free ubiquitin, suggesting that morphine is inducing neuroprotection by reducing the amount of oxidized proteins targeted for degradation. CONCLUSIONS: Significant neuroprotection on the cellular and molecular levels was demonstrated and serves as a foundation for future work concerning the development of novel ligands for morphine's mu3 opiate receptor in an effort to prevent cellular death associated with neurodegenerative diseases.

New and emerging pharmacological targets for neuropathic pain.

Increasing knowledge of the molecular consequences of nerve injury and the availability of genome databases has greatly increased the range of potential targets for the pharmacological management of neuropathic pain. Controlling neuronal sensitization and the associated alterations in gene expression, protein modification, and neuronal excitability is the key to managing neuropathic pain. Control of neuronal sensitization can occur through inhibition of nerve injury-associated production of cytokines, activation of glial cells, modulation of potassium channel subtypes, mitogen-activated protein kinases, the ubiquitin-proteasome system, or the protection and amplification of spinal cord dorsal horn inhibitory systems. These new and already established targets promise unparalleled opportunities for the prevention, management, and resolution of persistent pain states following nerve injury.