calpain and 4-hydroxy-2-nonenal

Retinal degenerative diseases, such as retinitis pigmentosa, are characterized by night blindness and peripheral vision loss caused by the slowly progressive loss of photoreceptor cells. A comprehensive molecular mechanism of the photoreceptor cell death remains unclear. We previously reported that heat shock protein 70 (HSP70), which has a protective effect on neuronal cells, was cleaved by a calcium-dependent protease, calpain, in N-methyl-N-nitrosourea (MNU)-treated mice retina. Carbonylated HSP70 is much more vulnerable than noncarbonylated HSP70 to calpain cleavage. However, it was not known whether protein carbonylation occurs in MNU-treated mice retina. In this study, we clearly show protein carbonylation-dependent photoreceptor cell death induced by MNU in mice. Therefore, protein carbonylation and subsequent calpain-dependent cleavage of HSP70 are key events in MNU-mediated photoreceptor cell death. Our data provide a comprehensive molecular mechanism of the photoreceptor cell death.

Topics: Aldehydes; Animals; Calpain; Cell Death; Disease Models, Animal; Eye Proteins; HSP70 Heat-Shock Proteins; Injections, Intraperitoneal; Male; Methylnitrosourea; Mice; Mice, Inbred C57BL; Models, Molecular; Oxidative Stress; Protein Carbonylation; Retina; Retinal Degeneration; Retinitis Pigmentosa

The endocochlear potential in the inner ear is essential for hearing ability, and maintained by various K(+) transport apparatuses including Na(+), K(+)-ATPase and gap junction-mediated intercellular communication (GJ-IC) in the lateral wall structures of the cochlea. Noise-induced hearing loss is known at least in part due to disruption of GJ-IC resulting from an oxidative stress-induced decrease in connexins (Cxs) level in the lateral wall structures. The purpose of this study was to investigate, using primary cultures of fibrocytes from the cochlear spiral ligament of mice, the mechanism underlying GJ-IC disruption induced by 4-hydroxynonenal (4-HNE), which is formed as a mediator of oxidative stress. An exposure to 4-HNE produced the following events: i.e., an increase in 4-HNE-adducted proteins; a decrease in the protein levels of Cx43, β-catenin, and Cx43/β-catenin complex along with intracellular translocation of this complex from the cell membrane to the cytoplasm; enhanced calpain-dependent degradation of endogenous α-fodrin; and disruption of GJ-IC. The 4-HNE-induced decrease in these protein levels and disruption of GJ-IC were most completely abolished by the calpain inhibitor PD150606. Taken together, our data suggest that 4-HNE disrupted GJ-IC through calpain-mediated degradation of Cx43 and β-catenin in primary cultures of fibrocytes derived from the cochlear spiral ligament.

Topics: Acrylates; Aldehydes; Animals; beta Catenin; Calpain; Carrier Proteins; Cell Communication; Cell Membrane; Cells, Cultured; Connexin 43; Cytoplasm; Gap Junctions; Male; Mice, Inbred Strains; Microfilament Proteins; Oxidative Stress; Proteolysis; Spiral Ligament of Cochlea

Previous workers have demonstrated that controlled mechanical ventilation results in diaphragm inactivity and elicits a rapid development of diaphragm weakness as a result of both contractile dysfunction and fiber atrophy. Limited data exist regarding the impact of pressure support ventilation, a commonly used mode of mechanical ventilation-that permits partial mechanical activity of the diaphragm-on diaphragm structure and function. We carried out the present study to test the hypothesis that high-level pressure support ventilation decreases the diaphragm pathology associated with CMV.. Sprague-Dawley rats were randomly assigned to one of the following five groups:1) control (no mechanical ventilation); 2) 12 hrs of controlled mechanical ventilation (12CMV); 3) 18 hrs of controlled mechanical ventilation (18CMV); 4) 12 hrs of pressure support ventilation (12PSV); or 5) 18 hrs of pressure support ventilation (18PSV).. We carried out the following measurements on diaphragm specimens: 4-hydroxynonenal-a marker of oxidative stress, active caspase-3 (casp-3), active calpain-1 (calp-1), fiber type cross-sectional area, and specific force (sp F). Compared with the control, both 12PSV and 18PSV promoted a significant decrement in diaphragmatic specific force production, but to a lesser degree than 12CMV and 18CMV. Furthermore, 12CMV, 18PSV, and 18CMV resulted in significant atrophy in all diaphragm fiber types as well as significant increases in a biomarker of oxidative stress (4-hydroxynonenal) and increased proteolytic activity (20S proteasome, calpain-1, and caspase-3). Furthermore, although no inspiratory effort occurs during controlled mechanical ventilation, it was observed that pressure support ventilation resulted in large decrement, approximately 96%, in inspiratory effort compared with spontaneously breathing animals.. High levels of prolonged pressure support ventilation promote diaphragmatic atrophy and contractile dysfunction. Furthermore, similar to controlled mechanical ventilation, pressure support ventilation-induced diaphragmatic atrophy and weakness are associated with both diaphragmatic oxidative stress and protease activation.

Topics: Aldehydes; Animals; Calpain; Caspase 3; Cytokines; Diaphragm; Interactive Ventilatory Support; Muscle Contraction; Muscular Atrophy; Oxidative Stress; Proteasome Endopeptidase Complex; Rats; Rats, Sprague-Dawley; Respiration, Artificial

Prolonged mechanical ventilation (MV) results in diaphragmatic weakness due to fiber atrophy and contractile dysfunction. Recent work reveals that activation of the proteases calpain and caspase-3 is required for MV-induced diaphragmatic atrophy and contractile dysfunction. However, the mechanism(s) responsible for activation of these proteases remains unknown. To address this issue, we tested the hypothesis that oxidative stress is essential for the activation of calpain and caspase-3 in the diaphragm during MV. Cause-and-effect was established by prevention of MV-induced diaphragmatic oxidative stress using the antioxidant Trolox. Treatment of animals with Trolox prevented MV-induced protein oxidation and lipid peroxidation in the diaphragm. Importantly, the Trolox-mediated protection from MV-induced oxidative stress prevented the activation of calpain and caspase-3 in the diaphragm during MV. Furthermore, the avoidance of MV-induced oxidative stress not only averted the activation of these proteases but also rescued the diaphragm from MV-induced diaphragmatic myofiber atrophy and contractile dysfunction. Collectively, these findings support the prediction that oxidative stress is required for MV-induced activation of calpain and caspase-3 in the diaphragm and are consistent with the concept that antioxidant therapy can retard MV-induced diaphragmatic weakness.

Topics: Aldehydes; Animals; Antioxidants; Atrophy; Calpain; Caspase 3; Chromans; Diaphragm; Electric Stimulation; Enzyme Activation; Female; Isometric Contraction; Lipid Peroxidation; Microfilament Proteins; Muscle Weakness; Oxidative Stress; Protein Carbonylation; Rats; Rats, Sprague-Dawley; Respiration, Artificial; Vesicular Transport Proteins

Peroxynitrite (PON, ONOO(-)), formed by nitric oxide synthase-generated nitric oxide radical ( NO) and superoxide radical (O(2) (-)), is a crucial player in post-traumatic oxidative damage. In the present study, we determined the spatial and temporal characteristics of PON-derived oxidative damage after a moderate contusion injury in rats. Our results showed that 3-nitrotyrosine (3-NT), a specific marker for PON, rapidly accumulated at early time points (1 and 3 h) and a significant increase compared with sham rats was sustained to 1 week after injury. Additionally, there was a coincident and maintained increase in the levels of protein oxidation-related protein carbonyl and lipid peroxidation-derived 4-hydroxynonenal (4-HNE). The peak increases of 3-NT and 4-HNE were observed at 24 h post-injury. In our immunohistochemical results, the co-localization of 3-NT and 4-HNE results indicates that PON is involved in lipid peroxidative as well as protein nitrative damage. One of the consequences of oxidative damage is an exacerbation of intracellular calcium overload, which activates the cysteine protease calpain leading to the degradation of several cellular targets including cytoskeletal protein (alpha-spectrin). Western blot analysis of alpha-spectrin breakdown products showed that the 145-kDa fragments of alpha-spectrin, which are specifically generated by calpain, were significantly increased as soon as 1 h following injury although the peak increase did not occur until 72 h post-injury. The later activation of calpain is most likely linked to PON-mediated secondary oxidative impairment of calcium homeostasis. Scavengers of PON, or its derived free radical species, may provide an improved antioxidant neuroprotective approach for the treatment of post-traumatic oxidative damage in the injured spinal cord.

Topics: Aldehydes; Animals; Biomarkers; Calcium Signaling; Calpain; Disease Progression; Female; Free Radical Scavengers; Free Radicals; Lipid Peroxidation; Nerve Degeneration; Nitric Oxide; Oxidative Stress; Peptide Fragments; Peroxynitrous Acid; Rats; Rats, Sprague-Dawley; Spectrin; Spinal Cord Injuries; Time Factors; Tyrosine; Up-Regulation

We assessed the temporal and spatial characteristics of PN-induced oxidative damage and its relationship to calpain-mediated cytoskeletal degradation and neurodegeneration in a severe unilateral controlled cortical impact (CCI) traumatic brain injury (TBI) model. Quantitative temporal time course studies were performed to measure two oxidative damage markers: 3-nitrotyrosine (3NT) and 4-hydroxynonenal (4HNE) at 30 min, 1, 3, 6, 12, 24, 48, 72 h and 7 days after injury in ipsilateral cortex of young adult male CF-1 mice. Secondly, the time course of Ca(++)-activated, calpain-mediated proteolysis was also analyzed using quantitative western-blot measurement of breakdown products of the cytoskeletal protein alpha-spectrin. Finally, the time course of neurodegeneration was examined using de Olmos silver staining. Both oxidative damage markers increased in cortical tissue immediately after injury (30 min) and elevated for the first 3-6 h before returning to baseline. In the immunostaining study, the PN-selective marker, 3NT, and the lipid peroxidation marker, 4HNE, were intense and overlapping in the injured cortical tissue. alpha-Spectrin breakdown products, which were used as biomarker for calpain-mediated cytoskeletal degradation, were also increased after injury, but the time course lagged behind the peak of oxidative damage and did not reach its maximum until 24 h post-injury. In turn, cytoskeletal degradation preceded the peak of neurodegeneration which occurred at 48 h post-injury. These studies have led us to the hypothesis that PN-mediated oxidative damage is an early event that contributes to a compromise of Ca(++) homeostatic mechanisms which causes a massive Ca(++) overload and calpain activation which is a final common pathway that results in post-traumatic neurodegeneration.

Topics: Aldehydes; Animals; Brain; Brain Injuries; Calcium; Calpain; Cerebral Cortex; Cytoskeleton; Lipid Peroxidation; Male; Mice; Mice, Inbred Strains; Nerve Degeneration; Nerve Tissue Proteins; Nitrates; Oxidative Stress; Peroxynitrous Acid; Spectrin; Time Factors; Tissue Distribution; Tyrosine

Studies have shown that the lipid peroxidation by-product, 4-hydroxynonenal (HNE), is involved in many pathological events in several neurodegenerative diseases. A number of signaling pathways mediating HNE-induced cell death in the brain have been proposed. However, the exact mechanism remains unknown. In the present study, we have examined the effects of HNE on cultured primary cortical neurons and found that HNE treatment leads to cell death via apoptosis. Both the caspase and calpain proteolytic systems were activated. There were also increased levels of phospho-p53 and cell cycle-related proteins. Gene transcription was further studied using microarray analysis. Results showed that majority of the genes associated with cell cycle regulation, response to stress, and signal transduction were differentially expressed. The various categories of differentially-expressed genes suggested that there are other parallel pathways regulating HNE-induced neuronal apoptosis. Collectively, these might help to elucidate similar molecular mechanisms involved during cell death in neurodegenerative diseases.

Topics: Acetylcysteine; Aldehydes; Animals; Apoptosis; Calpain; Caspases; Cell Cycle Proteins; Cell Death; Cell Survival; Cells, Cultured; Cerebral Cortex; Cytoskeleton; Free Radical Scavengers; Gene Expression Regulation; Genes, p53; Mice; Microarray Analysis; Neurons; Oxidative Stress; Signal Transduction; Transcription, Genetic; Ubiquitin