Homer1a attenuates glutamate-induced oxidative injury in HT-22 cells through regulation of store-operated calcium entry.

Calcium disequilibrium is extensively involved in oxidative stress-induced neuronal injury. Although Homer1a is known to regulate several neuronal calcium pathways, its effects on, or its exact relationship with, oxidative stress-induced neuronal injury has not yet been fully elucidated. We found that Homer1a protected HT-22 cells from glutamate-induced oxidative stress injury by inhibiting final-phase intracellular calcium overload and mitochondrial oxidative stress. In these cells, stromal interactive molecule 1 (STIM1) puncta, but not the protein level, was significantly increased after glutamate treatment. Store-operated calcium entry (SOCE) inhibitors and cells in which a key component of SOCE (STIM1) was knocked out were used as glutamate-induced oxidative stress injury models. Both models demonstrated significant improvement of HT-22 cell survival after glutamate treatment. Additionally, increased Homer1a protein levels significantly inhibited SOCE and decreased the association of STIM1-Orai1 triggered by glutamate. These results suggest that up-regulation of Homer1a can protect HT-22 cells from glutamate-induced oxidative injury by disrupting the STIM1-Oria1 association, and then by inhibiting the SOCE-mediated final-phrase calcium overload. Thus, regulation of Homer1a, either alone or in conjunction with SOCE inhibition, may serve as key therapeutic interventional targets for neurological diseases in which oxidative stress is involved in the etiology or progression of the disease.

Downregulation of STIM2 improves neuronal survival after traumatic brain injury by alleviating calcium overload and mitochondrial dysfunction.

Although store-operated calcium entry (SOCE) has been implicated in several neurological disorders, the exact mechanism for its role in traumatic brain injury (TBI) has not been elucidated. In this study, we found that TBI upregulated the expression of a calcium sensor protein called stromal interactive molecule 2 (STIM2); however, the levels of its homologue, STIM1, were unaffected. Both STIM1 and STIM2 are crucial components of SOCE, both in vivo and in vitro. Using shRNA, we discovered that downregulation of STIM2, but not STIM1, significantly improved neuronal survival in both an in vitro and in vivo model of TBI, decreasing neuronal apoptosis, and preserving neurological function. This neuroprotection was associated with alleviating TBI-induced calcium overload and preserving mitochondrial function. Additionally, downregulation of STIM2 not only inhibited Ca(2+) release from the endoplasmic reticulum (ER), but also reduced SOCE-mediated Ca(2+) influx, decreased mitochondrial Ca(2+), restored mitochondrial morphology and improved mitochondrial function, including MMP maintenance, ROS production and ATP synthesis. These results indicate that inhibition of STIM2 can protect neurons from TBI by inhibiting calcium overload and preserving mitochondrial function. This suggests that STIM2 might be an effective interventional target for TBI.

Unplanned Reoperations in Neurosurgical Patients Due to Postoperative Bleeding: A Single-Center Experience and Literature Review.

The aim of this study is to investigate the incidence of unplanned reoperations from all causes due to bleeding in neurosurgical patients. The medical records of patients who received neurosurgical procedures at our hospital were retrospectively reviewed and data of patients who received reoperations were extracted and summarized. A literature review was conducted of the Medline, Cochrane, EMBASE, and Google Scholar databases up to November 2013. The main outcome measure was the rate of unplanned reoperations due to bleeding. At our hospital, 68 patients with a mean age of 41.5 ±â€Š21.5 years (range, 7 months to 76 years) received an unplanned reoperation. More than 70% of the patients were older than 18 years, 64.7% were males, and 94.1% had cranial surgery. Almost 60% of the patients received >1 blood transfusion (58.8%) after the first surgery. Of the 68 patients, 35 (51.5%) received a second operation due to bleeding. Univariate logistic regression analysis only showed that an increasing time interval between the first and second surgery was associated with a decreased chance of the reoperation being performed due to bleeding (odds ratio [OR] = 0.843, 95% confidence interval [CI]: 0.720-0.987; P = .033). Of 229 studies identified, 5 retrospective reports with a total of 1375 patients were included in the analysis. The rate of reoperations for bleeding in the 5 studies ranged from 4.2% to 31.5%. Employing measures to reduce postoperative bleeding may help reduce the rate of unplanned neurosurgical reoperations.

Sirt3 protects cortical neurons against oxidative stress via regulating mitochondrial Ca2+ and mitochondrial biogenesis.

Oxidative stress is a well-established event in the pathology of several neurobiological diseases. Sirt3 is a nicotinamide adenine nucleotide (NAD+)-dependent protein deacetylase that regulates mitochondrial function and metabolism in response to caloric restriction and stress. This study aims to investigate the role of Sirt3 in H2O2 induced oxidative neuronal injury in primary cultured rat cortical neurons. We found that H2O2 treatment significantly increased the expression of Sirt3 in a time-dependent manner at both mRNA and protein levels. Knockdown of Sirt3 with a specific small interfering RNA (siRNA) exacerbated H2O2-induced neuronal injury, whereas overexpression of Sirt3 by lentivirus transfection inhibited H2O2-induced neuronal damage reduced the generation of reactive oxygen species (ROS), and increased the activities of endogenous antioxidant enzymes. In addition, the intra-mitochondrial Ca2+ overload, but not cytosolic Ca2+ increase after H2O2 treatment, was strongly attenuated after Sirt3 overexpression. Overexpression of Sirt3 also increased the content of mitochondrial DNA (mtDNA) and the expression of mitochondrial biogenesis related transcription factors. All these results suggest that Sirt3 acts as a prosurvival factor playing an essential role to protect cortical neurons under H2O2 induced oxidative stress, possibly through regulating mitochondrial Ca2+ homeostasis and mitochondrial biogenesis.

Sirt3 attenuates hydrogen peroxide-induced oxidative stress through the preservation of mitochondrial function in HT22 cells.

Sirtuins (Sirt) are a family of phylogenetically conserved nicotinamide adenine nucleotide (NAD(+))-dependent protein deacetylases, among which Sirt3 resides primarily in the mitochondria and serves as a stress responsive deacetylase, playing a role in protecting cells from damage under stress conditions. The present study aimed to investigate the role of Sirt3 in hydrogen peroxide (H(2)O(2))-induced oxidative neuronal injury in HT22 mouse hippocampal cells. Treatment with H(2)O(2) increased the expression of Sirt3 in a dose- and time-dependent manner, and the knockdown of Sirt3 using specific small interfering RNA (siRNA) exacerbated the H(2)O(2)-induced neuronal injury. The overexpression of Sirt3 induced by lentiviral transfection significantly reduced the generation of reactive oxygen species (ROS) and lipid peroxidation following injury, whereas the activities of endogenous antioxidant enzymes were not affected. Further experiments revealed that the H(2)O(2)-induced inhibition of mitochondrial complex activity and adenosine triphosphate (ATP) synthesis, the decrease in mitochondrial Ca(2+) buffering capacity and mitochondrial swelling were all partly reversed by Sirt3. Furthermore, the overexpression of Sirt3 attenuated the release of cytochrome c, the increase in the Bax/Bcl-2 ratio, as well as caspase-9/caspase-3 activity induced by H(2)O(2), and eventually inhibited apoptotic neuronal cell death. These results suggest that Sirt3 acts as a prosurvival factor, playing an essential role in protecting HT22 cells under H(2)O(2)-induced oxidative stress, possibly by inhibiting ROS accumulation and the activation of the mitochondrial apoptotic pathway.

Activation of mGluR5 attenuates NMDA-induced neurotoxicity through disruption of the NMDAR-PSD-95 complex and preservation of mitochondrial function in differentiated PC12 cells.

Glutamate-mediated toxicity is implicated in various neuropathologic conditions, and activation of ionotropic and metabotropic glutamate receptors is considered to be the most important mechanism. It has been reported that pharmacological saturation of metabotropic glutamate receptors (mGluRs) can facilitate N-methyl-D-aspartate receptor (NMDAR) related signaling cascades, but the mechanism leading to mGluR-NMDAR interactions in excitotoxic neuronal injury has remained unidentified. In the present study, we investigated the role of mGluR5 in the regulation of N-methyl-D-aspartate (NMDA)-induced excitotoxicity in differentiated PC12 cells. We found that activation of mGluR5 with the specific agonist R,S-2-chloro-5-hydroxyphenylglycine (CHPG) increased cell viability and inhibited lactate dehydrogenase (LDH) release in a dose-dependent manner. CHPG also inhibited an increase in the Bax/Bcl-2 ratio, attenuated cleavage of caspase-9 and caspase-3, and reduced apoptotic cell death after NMDA treatment. The NMDA-induced mitochondrial dysfunction, as indicated by mitochondrial reactive oxygen species (ROS) generation, collapse of mitochondrial membrane potential (MMP), and cytochrome c release, was also partly prevented by CHPG treatment. Furthermore, CHPG blocked the NMDA-induced interaction of NMDAR with postsynaptic density protein-95 (PSD-95), but had no effects on intracellular calcium concentrations. All these results indicated that activation of mGluR5 protects differentiated PC12 cells from NMDA-induced neuronal excitotoxicity by disrupting NMDAR-PSD-95 interaction, which might be an ideal target for investigating therapeutic strategies in various neurological diseases where excitotoxicity may contribute to their pathology.

MicroRNA-34a induces apoptosis in the human glioma cell line, A172, through enhanced ROS production and NOX2 expression.

BACKGROUND: MicroRNA is a type of non-coding small RNA involved in regulating genes and signaling pathways through incomplete complementation with target genes. Recent research supports key roles of miRNA in the formation and development of human glioma. METHODS: The relative quantity of miR-34a was initially determined in human glioma A172 cells and glioma tissues. Next, we analyzed the impact of miR-34a on A172 cell viability with the MTT assay. The effects of miR-34a overexpression on apoptosis were confirmed with flow cytometry and Hoechst staining experiments. We further defined the target genes of miR-34a using immunofluorescence and Western blot. RESULTS: MiR-34a expression was significantly reduced in human glioma A172 cells and glioma tissue, compared with normal glial cells and tissue samples. Our MTT data suggest that up-regulation of miR-34a inhibits cell viability while suppression of miR-34a enhances cell viability. Flow cytometry and Hoechst staining results revealed increased rates of apoptosis in A172 human glioma cells overexpressing miR-34a. Using immunofluorescence and Western blot analyses, we identified NOX2 as a target of miR-34a in A172 cells. CONCLUSION: MiR-34a serves as a tumor suppressor in human glioma mainly by decreasing NOX2 expression.

Blockade of SOCE protects HT22 cells from hydrogen peroxide-induced apoptosis.

Oxidative stress is an established event in the pathology of neurobiological diseases. Previous studies indicated that store-operated Ca(2+) entry (SOCE) has been involved in oxidative stress. The present study was carried out to investigate the effects of SOCE inhibition on neuronal oxidative stress injury induced by hydrogen peroxide (H2O2) in HT22 cells, a murine hippocampal neuronal model. H2O2 insult induced significant intracellular Ca(2+) overload, mitochondrial dysfunction and cell viability decrease. Inhibition of SOCE by pharmacological inhibitor and STIM1 RNAi significantly alleviated intracellular Ca(2+) overload, restored the mitochondrial membrane potential (MMP), decreased cytochrome C release and eventually inhibited H2O2-induced cell apoptosis. These findings suggest that SOCE inhibition exhibited neuroprotection against oxidative stress induced by H2O2 and SOCE might be a useful therapeutic target in neurobiological disorders.

Homer1 knockdown protects dopamine neurons through regulating calcium homeostasis in an in vitro model of Parkinson's disease.

Homer1 protein is an important scaffold protein at postsynaptic density and has been demonstrated to play a central role in calcium signaling in the central nervous system. The aim of this study was to investigate the effects of Homer1 knockdown on MPP(+) induced neuronal injury in cultured dopamine (DA) neurons. We found that down-regulating Homer1 expression with specific small interfering RNA (siRNA) significantly suppressed LDH release, reduced Propidium iodide (PI) or Hoechst staining, increased the number of tyrosine hydroxylase (TH) positive cells and DA uptake, and attenuated apoptotic and necrotic cell death after MPP(+) injury. Homer1 knockdown decreased intracellular reactive oxygen species (ROS) generation through inhibition of intracellular calcium overload, but did not affect the endogenous antioxidant enzyme activities. Calcium imaging was used to examine the changes of intracellular Ca(2+) concentration ([Ca(2+)]cyt) and Ca(2+) in endoplasmic reticulum (ER) ([Ca(2+)]ER), and the results showed that Homer1 siRNA transfection attenuated ER Ca(2+) release up to 120min after MPP(+) injury. Furthermore, decrease of [Ca(2+)]cyt induced by Homer1 knockdown in MPP(+) treated neurons was further enhanced by NMDA receptor antagonists MK-801 and AP-5, but not canonical transient receptor potential (TRPC) channel antagonist SKF-96365. l-type calcium antagonist isradipine but not nimodipine further inhibited intracellular calcium overload after MPP(+) insult in Homer1 down-regulated neurons. These results suggest that Homer1 knockdown has protective effects against neuronal injury in in vitro PD model by reducing calcium overload mediated ROS generation, and this protection may be dependent at least in part on the regulatory effects on the function of calcium channels in both plasma membrane and ER.