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We previously reported that conventional protein kinase C (c PKC)β participated in hypoxic preconditioning-induced neuroprotection against cerebral ischemic injury, and collapsin response-mediated protein 2 ( CRMP2) was identified as a c PKCβ interacting protein. In this study, we explored the regulation of CRMP2 phosphorylation and proteolysis by c PKCβ, and their role in ischemic injury of oxygen-glucose deprivation ( OGD)-treated cortical neurons and brains of mice with middle cerebral artery occlusion-induced ischemic stroke. The results demonstrated that c PKCβ-mediated CRMP2 phosphorylation via the c PKCβ-selective activator 12-deoxyphorbol 13-phenylacetate 20-acetate ( DOPPA) and inhibition of calpain-mediated CRMP2 proteolysis by calpeptin and a fusing peptide containing TAT peptide and the calpain cleavage site of CRMP2 ( TAT- CRMP2) protected neurons against OGD-induced cell death through inhibiting CRMP2 proteolysis in cultured cortical neurons. The OGD-induced nuclear translocation of the CRMP2 breakdown product was inhibited by DOPPA, calpeptin, and TAT- CRMP2 in cortical neurons. In addition, both c PKCβ activation and CRMP2 proteolysis inhibition by hypoxic preconditioning and intracerebroventricular injections of DOPPA, calpeptin, and TAT- CRMP2 improved the neurological deficit in addition to reducing the infarct volume and proportions of cells with pyknotic nuclei in the peri-infact region of mice with ischemic stroke. These results suggested that c PKCβ modulates CRMP2 phosphorylation and proteolysis, and c PKCβ activation alleviates ischemic injury in the cultured cortical neurons and brains of mice with ischemic stroke through inhibiting CRMP2 proteolysis by phosphorylation. [ABSTRACT FROM AUTHOR]

Synapsins as a family of presynaptic terminal phosphoprotein participates in neuronal development, but their role in the synaptic plasticity of visual cortex is unclear. In this study, the impact of monocular deprivation (MD) on dynamic changes of isoform-specific protein expression and site 1 phosphorylation of synapsins in visual cortex of the postnatal mice were observed by using the technique of Western blot analysis. The results showed that the total (T-) protein levels of synapsins including the isoform of Ia/b, IIa/b and IIIa were about 21-26 % of adult level in visual cortex of mice at postnatal 7 days (P7), and then the T-synapsin Ia/b and IIb could quickly reach adult level at P35. However, the T-synapsin IIa and IIIa increased more slowly (71-74 % at P35), and then kept increasing in the visual cortex of mice at P60. Unlike to the changes of T-synapsins, the level of phosphorylated (P-) synapsin Ia/b (not IIa/b and IIIa) at site 1 increased with development to the highest level at P21, and then decreased rapidly to a low level in visual cortex of mice at P35-60. In addition, we found that the levels of P-synapsin Ia/b increased significantly in left visual cortex of P28 and P35 (not P21 and P42) mice with 1-week MD of right eye; and no significant changes of T-synapsins were observed in both left and right sides of visual cortex in P21-42 mice with MD treatment. These results suggested that the isoform-specific protein expression and site-1 phosphorylation of synapsins might play a different role in the synaptic plasticity of visual cortex, and MD delays the dynamic changes of phosphorylated synapsin Ia/b at site-1 in contralateral visual cortex of juvenile mice. [ABSTRACT FROM AUTHOR]

Collapsin response mediator protein 2 (CRMP2) is a brain-specific multifunctional adaptor protein involved in neuronal polarity and axonal guidance. Our previous results showed CRMP2 may be involved in the hypoxic preconditioning and ischemic injury, but the mechanism was not clear. This study explored whether CRMP2 was involved in NMDA-induced neural death, and the possible mechanism. Western blot analysis demonstrated that NMDA reduced the phosphorylation of CRMP2 and inspired the cleavage of CRMP2. Also, it was detected that NMDA treatment did not affect the phosphorylation of CRMP2 in early stage (<6 h). Over-expression of CRMP2 aggravated the NMDA-induced injury, suggesting the vital role of CRMP2 in excitotoxicity. Tat-CRMP2 was designed to provide the cleavage site of calpain. Thiazolyl blue tetrazolium bromide assay, Hoechst33342/Propidium Iodide staining and Western blot assay showed that Tat-CRMP2 pretreatment increased cell viability compared with the control group against NMDA exposure by decreasing the cleavage of CRMP2. In conclusion, these studies indicated that cleavage of CRMP2 plays an important role involved in the NMDA-induced injury. The cleavage of CRMP2 may be a promising target for excitatory amino acid-related ischemic and hypoxic injury. [ABSTRACT FROM AUTHOR]

Abstract: Neurogranin/RC3 (Ng/rodent cortex-enriched mRNA clone #3), a postsynaptic neuronal protein kinase C (PKC) substrate, binds calmodulin (CaM) at low Ca2+ levels. Neurotransmitters triggering influx calcium induce neurogranin phosphorylation by PKC in physiological or pathophysiological conditions. Phosphorylated Ng reduces the affinity of Ng to bind CaM, which may affect the activities of calmodulin-dependent downstream enzymes, such as nitric oxide synthase (NOS), CaM-dependent protein kinase II (CaMKII) and adenylate cyclase (AC). These protein enzymes have been reported to play key roles in the development of ischemic/hypoxic preconditioning (I/HPC). We previously demonstrated that activation of cPKCβII and γ isoforms may be involved in the early phase of cerebral hypoxic preconditioning. However, as a substrate of PKC, the role of Ng in the onset of cerebral hypoxic preconditioning is unknown. In this study, we examined the effects of repetitive hypoxic exposure on the status of Ng phosphorylation in the cortex and hippocampus of mice. Using Western blot analysis, we found that the levels of Ng phosphorylation in the cortex and hippocampus of the hypoxic group of mice increased significantly from that of the normoxic group (p <0.05). These results suggest that neurogranin protein may be involved in the development of cerebral hypoxic preconditioning. [Copyright &y& Elsevier]

Graphical abstract Highlights • cPKCγ could protect neurons against ischemic injury in vitro and in vivo. • cPKCγ knockout aggravated OGD-induced neurite damage and P-synapsin I decrease. • cPKCγ could interact with synapsin I in the cerebral cortex of mice. Abstract Conventional protein kinase C (cPKC)γ and synapsin Ia/b have been implicated in the development of ischemic stroke, but their relationships and functions are unclear. In the present study, the oxygen-glucose deprivation (OGD)-induced ischemic insult in primary cultured cortical neurons in vitro and middle cerebral artery occlusion (MCAO)-induced ischemic stroke model in vivo were used to elucidate the function of cPKCγ and its modulation on synapsin Ia/b phosphorylation in ischemic stroke. We found that cPKCγ knockout significantly increased the infarct volume of mice after 1 h MCAO/72 h reperfusion by using triphenyltetrazolium chloride (TTC) staining. In the primarily cultured cortical neurons, cPKCγ knockout also aggravated the OGD-induced cell death and morphological damage of neurites, while cPKCγ restoration could alleviate the ischemic injury. Among the five phosphorylation sites of synapsin Ia/b, only the phosphorylation levels of Ser549 and 553 could be modulated by cPKCγ in neurons following 0.5 h OGD/24 h reoxygenation. In addition, we found that cPKCγ and synapsin Ia/b could be reciprocally co-immunoprecipitated in the cerebral cortex of MCAO mice. Taken together, we proposed that cPKCγ alleviates ischemic injury through modulating Ser549/553- synapsin Ia/b phosphorylation in neurons of mice. [ABSTRACT FROM AUTHOR]

Background Thousands of infants and children are undergoing anesthesia around the world every day. But impacts of anesthetics on the developing neural system remain unclear yet. Previous evidence showed that anesthesia might affect the developing neural system. Thus, early-life anesthesia becomes a critical issue in clinical pediatric practice. Hence, propofol, a short-acting and widely applied intravenous anesthetic, has been gaining focus upon neonatal anesthesia. Methods Fifty-four male C57BL/6J mice were randomly divided into following three groups: group D6 intraperitoneally (i.p.) injected propofol (100 mg/kg body weight) once a day from postnatal day 6 (P6) to P11, group D1 administrated propofol (100 mg/kg, i.p.) at P6 solely and administrated normal saline (10 ml/kg, i.p.) from P7 to P11, and group N treated with normal saline (10 ml/kg, i.p.) from P6 to P11 as the control ( n = 18 per group). Then, at P28, nine mice were collected randomly from each group for NR2B membrane translocation and phosphorylation analysis, and the rest half in each group were assigned to perform Morris water maze tests from P28 to P35. Results Results showed that total protein expression levels of NR2B increased ( p < .001) while its membrane translocation decreased ( p < .001, n = 9 per group) in the hippocampus but not in the prefrontal cortex of neonatal mice after repeated propofol administration. Phosphorylation levels of NR2B at serine 1303 (D1: p < .05; D6: p < .001, n = 9 per group) and serine 1480 (D1: p < .01, D6: p < .001, n = 9 per group) increased significantly as well in the hippocampus compared with group N. In addition, memory deficits ( p < .05, n = 9 per group) were observed in Morris water maze tests of group D6 mice. Conclusions These results suggested that propofol exposure downregulates NR2B membrane translocation and causes spatial memory deficits, with a mediated increased NR2B protein expression and phosphorylation at Ser1303/1480 residues in the hippocampus of neonatal mice. [ABSTRACT FROM AUTHOR]

We and others previously reported that the pro-inflammatory cytokine tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and IL-6 significantly accumulate with age in mouse lung. This is accompanied by elevated phosphorylation of p38. Here, we further investigate whether aging affects activation of p38 signaling and the inflammatory reaction after exposure to lipopolysaccharide (LPS) in the lungs of mice in vivo and humans ex vivo. The data showed that activation of p38 peaked at 0.5 h and then rapidly declined in young (2-month-old) mouse lung, after intranasal inhalation challenge with LPS. In contract, activation of p38 peaked at 24 h and was sustained longer in aged (20-month-old) mice. As well as altered p38, activations of its upstream activator MKK and downstream substrate NF-κB were also changed in the lungs of aged mice, which corresponded with the absence in the early phase but delayed increases in concentrations of TNF-α, IL-1β and IL-6. Consistent with the above observations in mice, similar patterns of p38 signaling also occurred in human lungs. Compared with younger lungs from adult–middle aged subjects, the activation of p38, MKK and NF-κB, as well as the production of pro-inflammatory cytokines were significantly increased in the lungs of older subjects ex vivo. Exposure of human lung cells to LPS induced rapid activation of p38, MKK and NF-κB in these cells from adult–middle aged subjects, but not older subjects, with increases in the production of the pro-inflammatory cytokines. The LPS-induced rapid activation in the lung cells from adult–middle aged subjects occurred as early as 0.25 h after exposure, and then declined. Compared with adult–middle aged subjects, the LPS exposure did not induce marked changes in the early phase, either in the activation of p38, MKK and NF-κB, or in the production of TNF-α, IL-1β or IL-6 in the lung cells from older subjects. In contrast, these changes occurred relatively late, peaked at 16 h and were sustained longer in the lungs of older subjects. These data support the hypothesis that the sustained activation of the p38 signaling pathway at baseline and the absence in the early phase but delayed of p38 signaling pathway response to LPS in the elderly may play important roles in increased susceptibility of aged lungs to inflammatory injury. [ABSTRACT FROM AUTHOR]

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. [Copyright &y& Elsevier]

Abstract: Activation of p38 mitogen-activated protein kinase (p38 MAPK) has been implicated as a mechanism of ischemia/hypoxia-induced cerebral injury. The current study was designed to explore the involvement of p38 MAPK in the development of cerebral hypoxic preconditioning (HPC) by observing the changes in dual phosphorylation (p-p38 MAPK) at threonine180 and tyrosine182 sites, protein expression, and cellular distribution of p-p38 MAPK in the brain of HPC mice. We found that the p-p38 MAPK levels, not protein expression, increased significantly (p <0.05) in the regions of frontal cortex, hippocampus, and hypothalamus of mice in response to repetitive hypoxic exposure (H1–H6, n =6 for each group) when compared to values of the control normoxic group (H0, n =6) using Western blot analysis. Similar results were also confirmed by an immunostaining study of the p-p38 MAPK location in the frontal cortex, hippocampus, and hypothalamus of mice from HPC groups. To further define the cell type of p-p38 MAPK positive cells, we used a double-labeled immunofluorescent staining method to co-localize p-p38 MAPK with neurofilaments heavy chain (NF-H, neuron-specific marker), S100 (astrocyte-specific marker), and CD11b (microglia-specific maker), respectively. We found that the increased p-p38 MAPK occurred in microglia of cortex and hippocampus, as well as in neurons of hypothalamus of HPC mice. These results suggest that the cell type-specific activation of p38 MAPK in the specific brain regions might contribute to the development of cerebral HPC mechanism in mice. [Copyright &y& Elsevier]

Abstract: Accumulated studies have suggested that mitogen-activated protein kinase (MAPK) play a pivotal role in the development of cerebral hypoxic preconditioning (HPC). By using our “auto-hypoxia”-induced HPC mouse model, we have reported increased phosphorylation level of p38 MAPK, and decreased phosphorylation and protein expression levels of extracellular signal regulated kinases 1/2 (ERK1/2) in the brain of HPC mice. In the current study, we investigated the involvement of c-Jun N-terminal kinase (JNK) in the brain of HPC mice. By using Western blot analysis, we found that the phosphorylation levels of JNK at Thr183 and Tyr185 sites (phospho-Thr183/Tyr185 JNK), but not its protein expression, increased significantly (p <0.05, n =6 for each group) both in the hippocampus and frontal cortex of early (H1–H4) and delayed (H5 and H6) HPC mice than that of the normoxic group (H0, n =6). Similarly, enhanced phospho-Thr183/Tyr185 JNK was also observed by immunostaining in the hippocampus and frontal cortex of mice following series of hypoxic exposures (H3 and H6). In addition, we found that phospho-Thr183/Tyr185 JNK predominantly co-localized with a neuron-specific protein, neurogranin, in both the hippocampus and frontal cortex of HPC mice (H3) by using double-labeled immunofluorescence. These results suggest that the increased neuron-specific phosphorylation of JNK at Thr183/Tyr185, not protein expression, might be involved in the development of cerebral HPC of mice. [Copyright &y& Elsevier]

Abstract: Accumulated reports have suggested that activation of protein kinase C (PKC) isoforms may involve the activation of extracellular signal-regulated kinases 1/2 (ERK1/2) in the neuronal response to hypoxic stimuli. We have previously demonstrated that the membrane translocation or activation of conventional PKC (cPKC) βII, γ and novel PKC (nPKC) ɛ are increased in the early phase of cerebral hypoxic preconditioning in mice. However, the role of ERK1/2 in the development of cerebral hypoxic preconditioning is unclear. In the current study, we used Western blot analysis to investigate the effects of repetitive hypoxic exposure (H0–H6, n =6 for each group) on the levels of phosphorylation and protein expression of ERK1/2 in the frontal cortex and the whole hippocampus of mice. We found that the levels of phosphorylated ERK1/2, not protein expression of ERK1/2, decreased significantly in both cortex and hippocampus of the early hypoxic preconditioned mice (H1–H4), when compared to that of the normoxic group (p <0.05). In addition, a significant decrease (p <0.05) in the ERK1/2 protein expression, not the phosphorylated form of ERK1/2, was found both in the frontal cortex and hippocampus of mice followed hypoxia with previous hypoxia (H5 and H6). These results suggest that the decreased phosphorylation and downregulation of protein expression of ERK1/2 might be involved in the development of hypoxic preconditioning. [Copyright &y& Elsevier]

Abstract: Cerebral ischemic/hypoxic preconditioning (I/HPC) is a phenomenon of endogenous protection that renders the brain tolerant to sustained ischemia/hypoxia. This profound protection induced by I/HPC makes it an attractive target for developing potential clinical therapeutic approaches. However, the molecular mechanism of I/HPC is unclear. Cyclic AMP (cAMP) response element binding protein (CREB), a selective nuclear transcriptional factor, plays a key role in the neuronal functions. Phosphorylation of CREB on Ser-133 may facilitate its transcriptional activity in response to various stresses. In the current study, we observed the changes in CREB phosphorylation (Ser-133) and protein expression in the brain of auto-hypoxia-induced HPC mice by using Western blot analysis. We found that the levels of phosphorylated CREB (Ser-133), but not protein expression of CREB, increased significantly (p <0.05) in the hippocampus and the frontal cortex of mice after repetitive hypoxic exposure (H2–H4, n =6 for each group), when compared to that of the normoxic (H0, n =6) or hypoxic exposure once group (H1, n =6). In addition, a significant enhancement (p <0.05) of CREB phosphorylation (Ser-133) could also be found in the nuclear extracts from the whole hippocampus of hypoxic preconditioned mice (H2–H4, n =6 for each group). These results suggest that the phosphorylation of CREB might be involved in the development of cerebral hypoxic preconditioning. [Copyright &y& Elsevier]