Yanling Yin

Phosphorylation of p38 MAPK mediates hypoxic preconditioning-induced neuroprotection against cerebral ischemic injury via mitochondria translocation of Bcl-xL in mice

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.

Hypoxic preconditioning induced neuroprotection against cerebral ischemic injuries and its cPKCγ-mediated molecular mechanism.

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.

Effects of insulin-like growth factor 1 on synaptic excitability in cultured rat hippocampal neurons

Insulin-like growth factor 1 (IGF-1) has important functions in the brain, including metabolic, neurotrophic, neuromodulatory and neuroendocrine actions, and it also prevents beta amyloid-induced death of hippocampal neurons. However, its functions in the synaptic excitability remain uncertain. Here we investigated the effects of IGF-1 on synaptic excitability in cultured rat hippocampal neurons using whole-cell patch clamp recordings. Incubation the hippocampal neurons with different concentrations of IGF-1 for 24 h or 30 min significantly increased the frequency of spontaneous excitatory postsynaptic currents (sEPSCs), but had no effect on the frequency of miniature EPSCs (mEPSCs) and spontaneous inhibitory postsynaptic currents (sIPSCs). The mean amplitudes, rise, and decay kinetics of sEPSCs, mEPSCs, and sIPSCs were not significantly affected by IGF-1, indicating that IGF-1 increased the probability of neurotransmitter release but did not modulate postsynaptic receptors. The effects of IGF-1 were mediated by mitogen-activated protein kinase (MAPK). IGF-1 activated the ERK1/2 signaling pathway in cultured hippocampal neurons, and the inhibitor PD98059 blocked the enhancement of sEPSCs induced by IGF-1. These results demonstrated the regulatory function of IGF-1 on synaptic excitability in hippocampal neurons and its underlying signaling mechanism.

Effects of insulin‐like growth factor‐1 on okadaic acid‐induced apoptosis in SH‐SY5Y cells

The effects of insulin‐like growth factor‐1 (IGF‐1) on the cytotoxicity and apoptosis induced by okadaic acid (OA) in SH‐SY5Y cells were investigated. Cell viability was measured using the MTT (3‐(4,5‐dimethylthiazolyl‐2)‐2,‐5‐diphenyltetrazolium bromide) assay. Early and late apoptosis/necrosis were analyzed by flow cytometry using Annexin V and propidium iodide (PI) double‐staining. Caspase‐3 activation was detected by Western blot analysis. Preincubation with IGF‐1 for 24 h prevented cytotoxicity induced by 40 nM OA given for 24 h, and the MTT value significantly increased. Incubation with 20 nM OA for 24 h caused a marked increase in the percentage of early apoptotic and late apoptotic/necrotic cells, which was not dependent on the activation of caspase‐3. OA‐induced apoptosis was significantly decreased by pretreatment with 10 ng/ml of IGF‐1 for 24 h. The results supported the hypothesis that IGF‐1 may be useful in the treatment of Alzheimers disease.

Effects of insulin-like growth factor 1 on voltage-gated ion channels in cultured rat hippocampal neurons

Insulin-like growth factor 1 (IGF-1) has important functions in the brain, including metabolic, neurotrophic, neuromodulatory, and neuroendocrine actions, and it is also prevents amyloid beta-induced death of hippocampal neurons. However, its functions on the voltage-gated ion channels in hippocampus remain uncertain. In the present study, we investigated the effects of IGF-1 on voltage-gated potassium, sodium, and calcium channels in the cultured rat hippocampal neurons using the whole-cell patch clamp recordings. Following incubation with different doses of IGF-1 for 24 h, a block of the peak transient A-type K+ currents amplitude (IC50: 4.425 ng/ml, Hill coefficient: 0.621) was observed. In addition, after the application of IGF-1, the amplitude of high-voltage activated Ca2+ currents significantly increased but activation kinetics did not significantly alter (V1/2: -33.45 +/- 1.32 mV, k = 6.16 +/- 1.05) compared to control conditions (V1/2: -33.19 +/- 2.28 mV, k = 7.26 +/- 1.71). However, the amplitude of Na+, K+, and low-voltage activated Ca2+ currents was not affected by the application of IGF-1. These data suggest that IGF-1 inhibits transient A-type K+ currents and enhances high-voltage-activated Ca2+ currents, but has no effects on Na+ and low-voltage-activated Ca2+ currents.

A role of insulin-like growth factor 1 in β amyloid-induced disinhibition of hippocampal neurons

In the present study we investigated the effects of beta amyloid (Abeta) on inhibitory synaptic transmission in the cultured hippocampal neurons using whole-cell patch-clamp recordings and immunocytochemistry, and examined the role of insulin-like growth factor 1 (IGF-1). Incubation with 4 microM Abeta25-35 for 24 h significantly decreased the frequency of spontaneous inhibitory postsynaptic currents (sIPSCs), but had no effect on the mean amplitude. Pretreatment with 10 ng/ml IGF-1 for 24h prior to Abeta25-35 exposure blocked Abeta-induced disinhibition of hippocampal neurons. The frequency and mean amplitude of miniature IPSC (mIPSCs) were not significantly affected by Abeta. The rise and decay kinetics of sIPSCs and mIPSCs were similar for the control and Abeta25-35-treated hippocampal neurons. Immunocytochemistry showed no changes in the ratio of gamma-aminobutyric acid (GABA) positive cells subsequent to treatment with Abeta, or IGF-1. Together these data suggest that Abeta-induced the disinhibition in cultured hippocampal neurons, whereas IGF-1 could block this effect.

MicroRNA-378 Alleviates Cerebral Ischemic Injury by Negatively Regulating Apoptosis Executioner Caspase-3

miRNAs have been linked to many human diseases, including ischemic stroke, and are being pursued as clinical diagnostics and therapeutic targets. Among the aberrantly expressed miRNAs in our previous report using large-scale microarray screening, the downregulation of miR-378 in the peri-infarct region of middle cerebral artery occluded (MCAO) mice can be reversed by hypoxic preconditioning (HPC). In this study, the role of miR-378 in the ischemic injury was further explored. We found that miR-378 levels significantly decreased in N2A cells following oxygen-glucose deprivation (OGD) treatment. Overexpression of miR-378 significantly enhanced cell viability, decreased TUNEL-positive cells and the immunoreactivity of cleaved-caspase-3. Conversely, downregulation of miR-378 aggravated OGD-induced apoptosis and ischemic injury. By using bioinformatic algorithms, we discovered that miR-378 may directly bind to the predicted 3′-untranslated region (UTR) of Caspase-3 gene. The protein level of caspase-3 increased significantly upon OGD treatment, and can be downregulated by pri-miR-378 transfection. The luciferase reporter assay confirmed the binding of miR-378 to the 3′-UTR of Caspase-3 mRNA and repressed its translation. In addition, miR-378 agomir decreased cleaved-caspase-3 ratio, reduced infarct volume and neural cell death induced by MCAO. Furthermore, caspase-3 knockdown could reverse anti-miR-378 mediated neuronal injury. Taken together, our data demonstrated that miR-378 attenuated ischemic injury by negatively regulating the apoptosis executioner, caspase-3, providing a potential therapeutic target for ischemic stroke.

Nitric oxide-mediated pathways and its role in the degenerative diseases.

Nitric oxide (NO) is a relatively short-lived inorganic free radical, which can be produced by different types of cells in multi-cellular organisms. This diffusible messenger functions as either an effector or a second messenger in many intercellular communications or intracellular signaling pathways. NO becomes noxious if it is produced in excess. These effects are mainly mediated by the reactivity of NO with various reactive oxygen species, which can be countered by antioxidant enzymes. In addition, NO can directly modify biological molecules via S-nitrosylation and lead to altered signaling responses. Accumulating evidence suggests that NO has a double-edged role in a dose-dependent, cell-type specific, and biological milieu-dependent way. In the present review, we summarized the synthesis and signaling pathway of NO, and especially focused on its involvement in biological processes, such as endoplasmic reticulum stress, apoptosis and autophagy. Besides, we discussed the functions of NO in the nervous system and its potential role in neurodegenerative diseases. We proposed the target on NO may shed light on the treatment of the related diseases.

Autophagy-ERK1/2-Involved Disinhibition of Hippocampal Neurons Contributes to the Pre-Synaptic Toxicity Induced by Aβ42 Exposure

Alzheimers disease (AD) is a progressive neurodegenerative disease and the most frequent cause of progressive cognitive decline in the elderly population. To date, there is still no effective treatment for AD, requiring more underlying mechanisms. In the present study, we investigated the effects of Aβ42 on the inhibitory synaptic transmission in the cultured hippocampal neurons, and explored the possible mechanism. The frequency, but not amplitude, of miniature inhibitory post-synaptic currents was significantly suppressed by Aβ42, indicating that Aβ42 played its role in inhibitory transmitter release at the pre-synaptic sites. Aβ42 had no effect on miniature excitatory post-synaptic currents, suggesting GABAergic synapses are more susceptible to Aβ42 exposure. However, the number of GABAergic neurons or synapses was not influenced, suggesting the corresponding stage may be a preclinical one. The effect of Aβ42 can be mimicked by PD98059 (an inhibitor of ERK1/2) and blocked by curcumin (an activator of MEK), which reveals Aβ-involved influence is via the decreased phosphorylation of MAPK-ERK1/2. In addition, synaptophysin is confirmed to be a downstream protein of MAPK-ERK1/2 at the pre-synaptic site. At the same time, suppressed autophagy was observed after Aβ42 exposure, and the activation of autophagy increased pERK1/2 level and salvaged the disinhibition of hippocampal neurons. These data suggest that diminished GABAergic tone likely starts from the preclinical stage of AD, so some GABAergic stress test may be effective for identifying cognitively normal elder adults. Strategies against the dysfunction of autophagy should be adopted in the early stage of AD because of its initial effects.