As of yet, pharmacological treatments of stroke are only met with mediocre results, which are either ineffective or confounded by adverse effects, thus calling for a better understanding of endogenous neuroprotective mechanism. Previously, we have demonstrated that the translocated activation of conventional protein kinase Cγ (cPKCγ) is involved in the development of cerebral hypoxic preconditioning (HPC), one of the most profound neuroprotective strategies. This study was designed to substantiate the role of cPKCγ and its signaling molecules in HPC-induced neuroprotection against subsequent middle cerebral artery occlusion (MCAO)-induced permanent cerebral ischemic injuries. The effects of HPC and cPKCγ on cerebral ischemic injuries were studied by observing the changes in neurological deficits, infarct volume and neural cell apoptosis. cPKCγ membrane translocation (activation) and its interacting protein synapsin in the ischemic brain were examined by Western blot analysis. Proteomic approaches were employed to identify the cPKCγ-interacting proteins. We found that HPC could markedly attenuate MCAO-induced brain injuries and the decrease of cPKCγ membrane translocation, but cPKCγ inhibitor Go6983 could block HPC-induced neuroprotection. Among the 41 identified cPKCγ-interacting proteins, 17 up- and 6 down-regulated proteins were observed in cytosol or particulate fraction during HPC. In addition, the up-regulated synapsin could reciprocally co-precipitate with cPKCγ both in cytosol and particulate fractions, and Go6983 abolished HPC-induced inhibition on synapsin dephosphorylation in ischemic core and peri-infarct region (penumbra). This study is the first to report multiple cPKCγ-interacting proteins in HPC mouse brain and suggested that cPKCγ signaling molecules, especially the cPKCγ-synapsin pathway, might be responsible for HPC-induced neuroprotection against cerebral ischemic injuries of mice.
"This apparent discrepancy may be explained by the concentrations used in the two studies (20 μM vs 3 μM in the present study). These findings are of particular interest, since it is known that cytosolic PKCγ interacts with synapsin, a protein associated with synaptic vesicle release, which also binds with high affinity to actin, an essential component of the architecture of the cytoskeleton (Zhang et al., 2011). PKCγ can regulate cytoskeleton assembly (Rosenberg and Ravid, 2006) and has been reported to be actively involved in glutamate receptor trafficking, suggesting an important role for this kinase in synaptic development and plasticity (Patten and Ali, 2009). "
[Show abstract][Hide abstract] ABSTRACT: Polyphenols such as epigallocatechin gallate (EGCG) and resveratrol have received a great deal of attention because they may contribute to the purported neuroprotective action of the regular consumption of green tea and red wine. Many studies, including those published by our group, suggest that this protective action includes their abilities to prevent the neurotoxic effects of beta-amyloid, a protein whose accumulation likely plays a pivotal role in Alzheimer's disease. Moreover, the scavenging activities of polyphenols on reactive oxygen species and their inhibitory action of cyclooxygenase likely explain, at least in part, their antioxidant and anti-inflammatory activities. Besides these well-documented properties, the modulatory action of these polyphenols on intracellular signaling pathways related to cell death/survival (e.g., protein kinase C, PKC) has yet to be investigated in detail. Using rat hippocampal neuronal cells, we aimed to investigate here the effects of EGCG and resveratrol on cell death induced by GF 109203X, a selective inhibitor of PKC. The MTT/resazurin and spectrin assays indicated that EGCG and resveratrol protected against GF 109203X-induced cell death and cytoskeleton degeneration, with a maximal effect at 1 and 3 μM, respectively. Moreover, immunofluorescence data revealed that cells treated with these polyphenols increased PKC gamma (γ) activation and promoted neuronal interconnections. Finally, we found that the protective effects of both polyphenols on the cytoskeleton and synaptic plasticity were mediated by the PKCγ subunit. Taken together, the results suggest that PKC, and more specifically its γ subunit, plays a critical role in the protective action of EGCG and resveratrol on neuronal integrity.
"Mice exposed to progressive hypoxia for four times were designated as HPC group (35.6 ± 1.3 min for tolerant time, 3.8 ± 0.4% for ending oxygen concentration), and mice placed in open jars for the same times were used as normoxic control. MCAO-induced focal cerebral ischemia mouse model was conducted as described previously (Chen et al. 2007; Bu et al. 2011; Zhang et al. 2011). After anesthetization with pentobarbital sodium (0.06 g/kg i.p.), the left common carotid artery and ipsilateral external carotid artery were exposed and ligated through a ventral midline neck incision. "
[Show abstract][Hide abstract] ABSTRACT: J. Neurochem. (2012) 120, 830–841.
We previously reported the involvement of conventional protein kinase C (cPKC) βII, γ, novel PKC (nPKC) ε and their interacting proteins in hypoxic pre-conditioning (HPC)-induced neuroprotection. In this study, the large-scale miRNA microarrays and bioinformatics analysis were used to determine the differentially expressed miRNAs and their PKC-isoform specific gene network in mouse brain after HPC and 6 h middle cerebral artery occlusion (MCAO). We found 4 up-regulated and 13 down-regulated miRNAs in the cortex of HPC mice, 26 increased and 39 decreased gene expressions of miRNAs in the peri-infarct region of 6 h MCAO mice, and 11 up-regulated and 22 down-regulated miRNAs in the peri-infarct region of HPC and 6 h MCAO mice. Based on Diff Score, 19 differentially expressed miRNAs were identified in HPC and 6 h MCAO mouse brain. Then the miRNA-gene-network of 19 specified miRNAs target genes of cPKCβII, γ and nPKCε-interacting protein was predicted by using bioinformatics analysis of genome databases. Furthermore, the down-regulated miR-615-3p during HPC had a detrimental effect on the oxygen-glucose deprivation (OGD)-induced N2A cell injury. These results suggested that the identified 19 miRNAs, notably miR-615-3p, might target these genes of cPKCβII, γ and nPKCε-interacting proteins involved in HPC-induced neuroprotection.
Journal of Neurochemistry 12/2011; 120(5):830-41. DOI:10.1111/j.1471-4159.2011.07624.x · 4.28 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Hypoxic preconditioning (HPC) initiates intracellular signaling pathway to provide protection, but the role of p38 mitogen-activated protein kinase (p38 MAPK) in HPC-induced neuroprotection against cerebral ischemic injuries is a matter of debate. In this study, we found that HPC could reduce 6h middle cerebral artery occlusion (MCAO)-induced infarct volume, edema ratio and cell apoptosis, as well as enhancing the up-regulated p38 MAPK phosphorylation (P-p38 MAPK) levels in the peri-infarct region of mice after 6h MCAO. However, intracerebroventricular injection of p38 MAPK inhibitor SB203580 abolished this HPC-induced neuroprotection. HPC significantly increased the translocation of anti-apoptotic Bcl-2-related protein Bcl-xL from the cytosol to the mitochondria in the peri-infarct region of MCAO mice. Interestingly, the results of reciprocal immunoprecipitation showed that Bcl-xL and P-p38 MAPK were coimmunoprecipitated reciprocally only in the peri-infarct region of HPC and MCAO treated mice, while Bcl-xL and total p38 (T-p38 MAPK), not P-p38 MAPK, could be coimmunoprecipited by each other in the brain of normal control mice. In addition, we found SB203580 significantly decreased P-p38 MAPK levels, and inhibited HPC-induced mitochondria translocation of Bcl-xL in the brain of HPC and MCAO treated mice. Taken together, our findings suggested that P-p38 MAPK mediates HPC-induced neuroprotection against cerebral ischemic injury via mitochondria translocation of Bcl-xL, which might be a key anti-cell apoptotic mechanism of HPC.
Brain research 03/2013; 1503:78-88. DOI:10.1016/j.brainres.2013.01.051 · 2.84 Impact Factor
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