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Brain injury-induced proteolysis is reduced in a novel calpastatin-overexpressing transgenic mouse.

ABSTRACT: The calpain family of calcium-dependent proteases has been implicated in a variety of diseases and neurodegenerative pathologies. Prolonged activation of calpains results in proteolysis of numerous cellular substrates including cytoskeletal components and membrane receptors, contributing to cell demise despite coincident expression of calpastatin, the specific inhibitor of calpains. Pharmacological and gene-knockout strategies have targeted calpains to determine their contribution to neurodegenerative pathology; however, limitations associated with treatment paradigms, drug specificity, and genetic disruptions have produced inconsistent results and complicated interpretation. Specific, targeted calpain inhibition achieved by enhancing endogenous calpastatin levels offers unique advantages in studying pathological calpain activation. We have characterized a novel calpastatin-overexpressing transgenic mouse model, demonstrating a substantial increase in calpastatin expression within nervous system and peripheral tissues and associated reduction in protease activity. Experimental activation of calpains via traumatic brain injury resulted in cleavage of ?-spectrin, collapsin response mediator protein-2, and voltage-gated sodium channel, critical proteins for the maintenance of neuronal structure and function. Calpastatin overexpression significantly attenuated calpain-mediated proteolysis of these selected substrates acutely following severe controlled cortical impact injury, but with no effect on acute hippocampal neurodegeneration. Augmenting calpastatin levels may be an effective method for calpain inhibition in traumatic brain injury and neurodegenerative disorders.

SUBMITTER: Schoch KM

PROVIDER: S-EPMC3676438 | biostudies-literature | 2013 Jun

REPOSITORIES: biostudies-literature

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Brain injury-induced proteolysis is reduced in a novel calpastatin-overexpressing transgenic mouse.

Schoch Kathleen M KM von Reyn Catherine R CR Bian Jifeng J Telling Glenn C GC Meaney David F DF Saatman Kathryn E KE

Journal of neurochemistry 20130203 6

The calpain family of calcium-dependent proteases has been implicated in a variety of diseases and neurodegenerative pathologies. Prolonged activation of calpains results in proteolysis of numerous cellular substrates including cytoskeletal components and membrane receptors, contributing to cell demise despite coincident expression of calpastatin, the specific inhibitor of calpains. Pharmacological and gene-knockout strategies have targeted calpains to determine their contribution to neurodegene ...[more]

PMID: 23305291

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Project description:BackgroundHeat-shock protein B1 (HSPB1) is among the most well-known and versatile member of the evolutionarily conserved family of small heat-shock proteins. It has been implicated to serve a neuroprotective role against various neurological disorders via its modulatory activity on inflammation, yet its exact role in neuroinflammation is poorly understood. In order to shed light on the exact mechanism of inflammation modulation by HSPB1, we investigated the effect of HSPB1 on neuroinflammatory processes in an in vivo and in vitro model of acute brain injury.MethodsIn this study, we used a transgenic mouse strain overexpressing the human HSPB1 protein. In the in vivo experiments, 7-day-old transgenic and wild-type mice were treated with ethanol. Apoptotic cells were detected using TUNEL assay. The mRNA and protein levels of cytokines and glial cell markers were examined using RT-PCR and immunohistochemistry in the brain. We also established primary neuronal, astrocyte, and microglial cultures which were subjected to cytokine and ethanol treatments. TNFα and hHSPB1 levels were measured from the supernates by ELISA, and intracellular hHSPB1 expression was analyzed using fluorescent immunohistochemistry.ResultsFollowing ethanol treatment, the brains of hHSPB1-overexpressing mice showed a significantly higher mRNA level of pro-inflammatory cytokines (Tnf, Il1b), microglia (Cd68, Arg1), and astrocyte (Gfap) markers compared to wild-type brains. Microglial activation, and 1 week later, reactive astrogliosis was higher in certain brain areas of ethanol-treated transgenic mice compared to those of wild-types. Despite the remarkably high expression of pro-apoptotic Tnf, hHSPB1-overexpressing mice did not exhibit higher level of apoptosis. Our data suggest that intracellular hHSPB1, showing the highest level in primary astrocytes, was responsible for the inflammation-regulating effects. Microglia cells were the main source of TNFα in our model. Microglia isolated from hHSPB1-overexpressing mice showed a significantly higher release of TNFα compared to wild-type cells under inflammatory conditions.ConclusionsOur work provides novel in vivo evidence that hHSPB1 overexpression has a regulating effect on acute neuroinflammation by intensifying the expression of pro-inflammatory cytokines and enhancing glial cell activation, but not increasing neuronal apoptosis. These results suggest that hHSPB1 may play a complex role in the modulation of the ethanol-induced neuroinflammatory response.

Project description:BACKGROUND: The pepper fruit is the second most consumed vegetable worldwide. However, low temperature affects the vegetative development and reproduction of the pepper, resulting in economic losses. To identify cold-related genes regulated by abscisic acid (ABA) in pepper seedlings, cDNA representational difference analysis was previously performed using a suppression subtractive hybridization method. One of the genes cloned from the subtraction was homologous to Solanum tuberosum MBF1 (StMBF1) encoding the coactivator multiprotein bridging factor 1. Here, we have characterized this StMBF1 homolog (named CaMBF1) from Capsicum annuum and investigated its role in abiotic stress tolerance. RESULTS: Tissue expression profile analysis using quantitative RT-PCR showed that CaMBF1 was expressed in all tested tissues, and high-level expression was detected in the flowers and seeds. The expression of CaMBF1 in pepper seedlings was dramatically suppressed by exogenously supplied salicylic acid, high salt, osmotic and heavy metal stresses. Constitutive overexpression of CaMBF1 in Arabidopsis aggravated the visible symptoms of leaf damage and the electrolyte leakage of cell damage caused by cold stress in seedlings. Furthermore, the expression of RD29A, ERD15, KIN1, and RD22 in the transgenic plants was lower than that in the wild-type plants. On the other hand, seed germination, cotyledon greening and lateral root formation were more severely influenced by salt stress in transgenic lines compared with wild-type plants, indicating that CaMBF1-overexpressing Arabidopsis plants were hypersensitive to salt stress. CONCLUSIONS: Overexpression of CaMBF1 in Arabidopsis displayed reduced tolerance to cold and high salt stress during seed germination and post-germination stages. CaMBF1 transgenic Arabidopsis may reduce stress tolerance by downregulating stress-responsive genes to aggravate the leaf damage caused by cold stress. CaMBF1 may be useful for genetic engineering of novel pepper cultivars in the future.

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Brain injury-induced proteolysis is reduced in a novel calpastatin-overexpressing transgenic mouse.

Publications

Brain injury-induced proteolysis is reduced in a novel calpastatin-overexpressing transgenic mouse.

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