Induction Of Neuronal Apoptosis Research Articles

Nuclear translocation of annexin 1 following oxygen-glucose deprivation-reperfusion induces apoptosis by regulating Bid expression via p53 binding.

Previous data have suggested that the nuclear translocation of annexin 1 (ANXA1) is involved in neuronal apoptosis after ischemic stroke. As the mechanism and function of ANXA1 nuclear migration remain unclear, it is important to clarify how ANXA1 performs its role as an apoptosis ‘regulator' in the nucleus. Here we report that importazole (IPZ), an importin β (Impβ)-specific inhibitor, decreased ANXA1 nuclear accumulation and reduced the rate of neuronal death induced by nuclear ANXA1 migration after oxygen-glucose deprivation–reoxygenation (OGD/R). Notably, ANXA1 interacted with the Bid (BH3-interacting-domain death agonist) promoter directly; however; this interaction could be partially blocked by the p53 inhibitor pifithrin-α (PFT-α). Accordingly, ANXA1 was shown to interact with p53 in the nucleus and this interaction was enhanced following OGD/R. A luciferase reporter assay revealed that ANXA1 was involved in the regulation of p53-mediated transcriptional activation after OGD/R. Consistent with this finding, the nuclear translocation of ANXA1 after OGD/R upregulated the expression of Bid, which was impeded by IPZ, ANXA1 shRNA, or PFT-α. Finally, cell-survival testing demonstrated that silencing ANXA1 could improve the rate of cell survival and decrease the expression of both cleaved caspase-3 and cleaved poly(ADP-ribose) polymerase. These data suggested that Impβ-dependent nuclear ANXA1 migration participates in the OGD/R-dependent induction of neuronal apoptosis. ANXA1 interacts with p53 and promotes p53 transcriptional activity, which in turn regulates Bid expression. Silencing ANXA1 decreases the expression of Bid and suppresses caspase-3 pathway activation, thus improving cell survival after OGD/R. This study provides a novel mechanism whereby ANXA1 regulates apoptosis, suggesting the potential for a previously unidentified treatment strategy in minimizing apoptosis after OGD/R.

The role of myocardin-related transcription factor-A in Aβ25–35 induced neuron apoptosis and synapse injury

Myocardin-related transcription factor-A (MRTF-A) highly expressed in brain has been demonstrated to promote neuronal survival via regulating the transcription of related target genes as a powerful co-activator of serum response factor (SRF). However, the role of MRTF-A in Alzheimer's disease (AD) is still unclear. Here, we showed that MRTF-A was significantly downregulated in cortex of the Aβ25–35-induced AD rats, which played a key role in Aβ25–35 induced cerebral neuronal degeneration in vitro. Bilateral intracerebroventricular injection of Aβ25–35 caused significantly MRTF–A expression decline in cortex of rats, along with significant neuron apoptosis and plasticity damage. In vitro, transfection of MRTF-A into primary cultured cortical neurons prevented Aβ25–35 induced neuronal apoptosis and synapses injury. And luciferase reporter assay determined that MRTF-A could bind to and enhance the transactivity of the Mcl-1 (Myeloid cell leukemia-1) and Arc (activity-regulated cytoskeletal-associated protein) promoters by activating the key CArG box element. These data demonstrated that the decreasing of endogenous MRTF-A expression might contribute to the development of AD, whereas the upregulation MRTF-A in neurons could effectively reduce Aβ25–35 induced synapse injury and cell apoptosis. And the underlying mechanism might be partially due to MRTF-A-mediated the transcription and expression of Mcl-1 and Arc by triggering the CArG box.

Effects of emulsified isoflurane on apoptosis in human neuroblastoma cells: the role of c-Jun N-terminal kinase

Objective To investigate the effects of emulsified isoflurane on apoptosis in human neuroblastoma cells and the role of c-Jun N-terminal kinase(JNK)in it. Methods The human neuroblastoma SHSY-5Y cells were seeded in 96-well plates or dishes and then randomly divided into 8 groups using a random number table: control group(group C, n=24), different concentrations of lipid emulsion groups(LE1 groups[n=24], LE2 group[n=24] and LE3 group[n=72]), different concentrations of emulsified isoflurane groups(EI1 group[n=24], EI2 group[n=24]and EI3 group[n=72]), and emulsified isoflurane + JNK inhibitor SP600125 group(group EI-SP, n=24). At 24 h after the cells were plated, in LE1-3 groups, 30% lipid emulsion was added to the culture medium with the final concentrations of 0.395 6, 0.791 2 and 1.582 4 μl/ml, respectively; in EI1-3 groups, 8% emulsified isoflurane was added with the final concentrations of 0.56, 1.12 and 2.24 mmol/L, respectively; in group EI-SP, emulsified isoflurane was added with the final concentration of 1.12 mmol/L, and SP600125 was added at 1 h before addition of emulsified isoflurane with the final concentration of 10 μmol/L; the cells were cultured normally in group C. At 6, 12 and 24 h of incubation in EI3 and LE3 groups, and at 24 h of incubation or culture in the other groups, the morphology of cells was detected, the cell viability was measured using methyl thiazolyl tetrazolium assay, and the expression of JNK, phosphorylated JNK(p-JNK)and cytochrome c(Cyt c)was detected by Western blot. Results Compared with group C, the cell viability was significantly decreased, and the expression of p-JNK and Cyt c was significantly up-regulated at 24 h of incubation in group EI2 and at 12 and 24 h of incubation in group EI3, the cell viability was significantly decreased(P 0.05). Compared with group EI1, the cell viability was significantly decreased, and the expression of p-JNK and Cyt c was significantly up-regulated at 24 h of incubation in EI2, 3 groups(P 0.05). Conclusion High concentrations of emulsified isoflurane can induce apoptosis in neurons only when applied for a long time, while low concentrations do not have the effect when applied for a short time.The mechanism by which emulsified isoflurane induces neuronal apoptosis is related to activation of JNK pathway. Key words: Isoflurane; Fat emulsions, intravenous; Neurons; Apoptosis; JNK mitogen-activated protein kinases

Crosstalk between Ca2+ signaling and mitochondrial H2O2 is required for rotenone inhibition of mTOR signaling pathway leading to neuronal apoptosis.

Rotenone, a neurotoxic pesticide, induces loss of dopaminergic neurons related to Parkinson's disease. Previous studies have shown that rotenone induces neuronal apoptosis partly by triggering hydrogen peroxide (H2O2)-dependent suppression of mTOR pathway. However, the underlying mechanism is not fully understood. Here, we show that rotenone elevates intracellular free calcium ion ([Ca2+]i) level, and activates CaMKII, resulting in inhibition of mTOR signaling and induction of neuronal apoptosis. Chelating [Ca2+]i with BAPTA/AM, preventing extracellular Ca2+ influx using EGTA, inhibiting CaMKII with KN93, or silencing CaMKII significantly attenuated rotenone-induced H2O2 production, mTOR inhibition, and cell death. Interestingly, using TTFA, antimycin A, catalase or Mito-TEMPO, we found that rotenone-induced mitochondrial H2O2 also in turn elevated [Ca2+]i level, thereby stimulating CaMKII, leading to inhibition of mTOR pathway and induction of neuronal apoptosis. Expression of wild type mTOR or constitutively active S6K1, or silencing 4E-BP1 strengthened the inhibitory effects of catalase, Mito-TEMPO, BAPTA/AM or EGTA on rotenone-induced [Ca2+]i elevation, CaMKII phosphorylation and neuronal apoptosis. Together, the results indicate that the crosstalk between Ca2+ signaling and mitochondrial H2O2 is required for rotenone inhibition of mTOR-mediated S6K1 and 4E-BP1 pathways. Our findings suggest that how to control over-elevation of intracellular Ca2+ and overproduction of mitochondrial H2O2 may be a new approach to deal with the neurotoxicity of rotenone.

Hyperbaric Oxygen Treatment Protects Against Spinal Cord Injury by Inhibiting Endoplasmic Reticulum Stress in Rats.

Experimental animal study of treatment of SCI. To explore whether HBO treatment protects against secondary SCI by inhibiting the ER stress response. SCI is a neurological disorder that can severely limit the execution of the simplest day-to-day functions. ER stress plays an important role in the induction of neuronal apoptosis after SCI. HBO treatment can alleviate secondary injury and benefit neurological recovery after SCI, but the effect of HBO on ER stress response after SCI is yet to be characterized. The spinal cord of rats was injured via T10 laminectomy. Experimental animals were randomly assigned to 1 of 3 groups: sham-operated, SCI, and SCI+HBO. Each group was analyzed 1, 2, 3, 7, and 14 days after injury. Neurological recovery was evaluated using the Basso-Beattie-Bresnahan (BBB) scoring system and the TdT-mediated dUTP nick-end labeling reaction was carried out to visualize apoptotic cells. The expression of ER stress-related factors was evaluated by immunohistochemical, western blot, and real-time reverse transcription-polymerase chain reaction analyses. SCI-induced apoptosis and an increase in the levels of CCAAT-enhancer-binding protein homologous protein (CHOP), and caspase-12 and caspase-3 compared with the sham-operated group. HBO treatment decreased CHOP and caspase-12 and caspase-3 expression as well as apoptosis compared with the SCI group. In addition, BBB scores were improved in the SCI+HBO relative to the SCI group at 7 and 14 days. These results suggest that HBO treatment alleviates secondary injury to the spinal cord by inhibiting ER stress induced apoptosis, thereby promoting the recovery of neurological function. N/A.

MiR-133b-5p regulates the expression of the heat shock protein 70 during rat neuronal cell apoptosis induced by the gp120 V3 loop peptide.

Neuronal cell dysfunction and apoptosis, the main causes of HIV-associated dementia, and its underlying mechanism are important unsolved health problems. Many research reports suggest that miRNAs regulate HIV-1-induced apoptosis. We used the HIV-1 gp120 V3 Loop peptide to induce primary rat cortical neurons apoptosis. Next, we used a microRNA microarray to identify the significant changes of miRNA in the rat cortical neurons treated with the gp120 V3 loop peptide. We used western blot and real-time PCR to measure the regulation of heat shock protein 70 by rno-miR-133b-5p. In response to the gp120 V3 loop peptide treatment, rat cortical neurons exhibited 11 up-regulated and 21 down-regulated miRNAs. We further examined miR-133b-5p, a microRNA that was up-regulated more than 118-fold. In addition, both HSP70 mRNA and protein expression were dose-dependent in rats cortical neurons treated with gp120 V3 loop peptide for 48 hr. MiR-133b-5p could regulate heat shock protein 70 (HSP70) at both transcription and translation levels. Rno-miR-133b-5p might be less significant for the gp120 V3 loop peptide induced neuron apoptosis. Thus, we discovered a potential new target for the regulation of HIV-1 gp120- induced apoptosis.

Fluoride-Induced Neuron Apoptosis and Expressions of Inflammatory Factors by Activating Microglia in Rat Brain.

Excessive exposure to fluoride results in structural and functional damages to the central nervous system (CNS), and neurotoxicity of fluoride may be associated with neurodegenerative changes. Chronic microglial activation appears to cause neuronal damage through producing proinflammatory cytokines and is involved in many neurodegenerative disorders. It is not known about effects on microglia of fluoride. In the present study, healthy adult Wistar rats were exposed to 60 and 120ppm fluoride in drinking water for 10weeks, and control rats received deionized water. After 10weeks, rats were sacrificed under anesthesia then apoptosis in neuron and inflammatory factors secreted by microglia were determined. We found that apoptosis of neurons in fluoride-treated rat brain increased and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL)-positive immunofluorescence increased with increasing fluoride concentrations. Bax protein expression increased and Bcl-2 protein expression decreased in fluoride-treated rat brain compared with that of the control rat brain. The microglia in the hippocampus and cortex of fluoride-treated rats were activated by immunostaining with OX-42, a marker of activated microglia, and OX-42-positive microglia cells were more abundant in the hippocampus than in the cortex. The levels of IL-1β and IL-6 protein expression in OX-42-labeled microglial cells were significantly increased in the cortex and hippocampus of rats exposed to fluoride, and TNF-α immunoreactivity in microglial cells of the hippocampus was significantly higher in the 120ppm fluoride-treated group than that in the control group. Our results indicate that fluoride induced neuron apoptosis and expressions of inflammatory factors by activating microglia in rat brain.

Increased calcineurin expression after pilocarpine-induced status epilepticus is associated with brain focal edema and astrogliosis

Calcineurin plays an important role in the development of neuronal excitability, modulation of receptor's function and induction of apoptosis in neurons. It has been established in kindling models that status epilepticus induces brain focal edema and astrocyte activation. However, the role of calcineurin in brain focal edema and astrocyte activation in status epilepticus has not been fully understood. In this study, we employed a model of lithium-pilocarpine-induced status epilepticus and detected calcineurin expression in hippocampus by immunoblotting, brain focal edema by non-invasive magnetic resonance imaging (MRI-7T) and astrocyte expression by immunohistochemistry. We found that the brain focal edema was seen at 24 h after status epilepticus, and astrocyte expression was obviously seen at 7 d after status epilepticus. Meanwhile, calcineurin expression was seen at24 h and retained to 7 d after status epilepticus. A FK506, a calcineurin inhibitor, remarkably suppressed the status epilepticus-induced brain focal edema and astrocyte expression. Our data suggested that calcineurin overexpression plays a very important role in brain focal edema and astrocyte expression. Therefore, calcineurin may be a novel candidate for brain focal edema occurring and intracellular trigger of astrogliosis in status epilepticus.

Cholinergic synaptic transmissions were altered after single sevoflurane exposure in Drosophila pupa.

Purpose. Sevoflurane, one of the most used general anesthetics, is widely used in clinical practice all over the world. Previous studies indicated that sevoflurane could induce neuron apoptosis and neural deficit causing query in the safety of anesthesia using sevoflurane. The present study was designed to investigate the effects of sevoflurane on electrophysiology in Drosophila pupa whose excitatory neurotransmitter is acetylcholine early after sevoflurane exposure using whole brain recording technique. Methods. Wide types of Drosophila (canton-s flies) were allocated to control and sevoflurane groups randomly. Sevoflurane groups (1% sevoflurane; 2% sevoflurane; 3% sevoflurane) were exposed to sevoflurane and the exposure lasted 5 hours, respectively. All flies were subjected to electrophysiology experiment using patch clamp 24 hours after exposure. Results. The results showed that, 24 hours after sevoflurane exposure, frequency but not the amplitude of miniature excitatory postsynaptic currents (mEPSCs) was significantly reduced (P < 0.05). Furthermore, we explored the underlying mechanism and found that calcium currents density, which partially regulated the frequency of mEPSCs, was significantly reduced after sevoflurane exposure (P < 0.05). Conclusions. All these suggested that sevoflurane could alter the mEPSCs that are related to synaptic plasticity partially through modulating calcium channel early after sevoflurane exposure.

The α7 nAChR selective agonists as drug candidates for Alzheimer's disease.

The nicotinic acetylcholine receptors (nAChRs) are ion channels distribute in the central or peripheral nervous system. They are receptors of the neurotransmitter acetylcholine and activation of them by agonists mediates synaptic transmission in the neuron and muscle contraction in the neuromuscular junction. Current studies reveal relationship between the nAChRs and the learning and memory as well as cognation deficit in various neurological disorders such as Alzheimer's disease, Parkinson's disease, schizophrenia and drug addiction. There are various subtypes in the nAChR family and the α7 nAChR is one of the most abundant subtypes in the brain. The α7 nAChR is significantly reduced in the patients of Alzheimer's disease and is believed to interact with the Aβ amyloid. Aβ amyloid is co-localized with α7 nAChR in the senile plaque and interaction between them induces neuron apoptosis and reduction of the α7 nAChR expression. Treatment with α7 agonist in vivo shows its neuron protective and procognation properties and significantly improves the learning and memory ability of the animal models. Therefore, the α7 nAChR agonists are excellent drug candidates for Alzheimer's disease and we summarized here the current agonists that have selectivity of the α7 nAChR over the other nAChR, introduced recent molecular modeling works trying to explain the molecular mechanism of their selectivity and described the design of novel allosteric modulators in our lab.

The two-pore domain potassium channel KCNK5 deteriorates outcome in ischemic neurodegeneration.

Potassium channels can fulfill both beneficial and detrimental roles in neuronal damage during ischemic stroke. Earlier studies have characterized a neuroprotective role of the two-pore domain potassium channels KCNK2 (TREK1) and KCNK3 (TASK1). Protective neuronal hyperpolarization and prevention of intracellular Ca(2+) overload and glutamate excitotoxicity were suggested to be the underlying mechanisms. We here identify an unexpected role for the related KCNK5 channel in a mouse model of transient middle cerebral artery occlusion (tMCAO). KCNK5 is strongly upregulated on neurons upon cerebral ischemia, where it is most likely involved in the induction of neuronal apoptosis. Hypoxic conditions elevated neuronal expression levels of KCNK5 in acute brain slices and primary isolated neuronal cell cultures. In agreement, KCNK5 knockout mice had significantly reduced infarct volumes and improved neurologic function 24 h after 60 min of tMCAO and this protective effect was preserved at later stages of infarct development. KCNK5 deficiency resulted in a significantly reduced number of apoptotic neurons, a downregulation of pro-apoptotic and upregulation of anti-apoptotic factors. Results of adoptive transfer experiments of wild-type and Kcnk5 (-/-) immune cells into Rag1 (-/-) mice prior to tMCAO exclude a major role of KCNK5 in poststroke inflammatory reactions. In summary, KCNK5 expression is induced on neurons under ischemic conditions where it most likely exerts pro-apoptotic effects. Hence, pharmacological blockade of KCNK5 might have therapeutic potential in preventing ischemic neurodegeneration.

Lead induces COX-2 expression in glial cells in a NFAT-dependent, AP-1/NFκB-independent manner.

Epidemiologic studies have provided solid evidence for the neurotoxic effect of lead for decades of years. In view of the fact that children are more vulnerable to the neurotoxicity of lead, lead exposure has been an urgent public health concern. The modes of action of lead neurotoxic effects include disturbance of neurotransmitter storage and release, damage of mitochondria, as well as induction of apoptosis in neurons, cerebrovascular endothelial cells, astroglia and oligodendroglia. Our studies here, from a novel point of view, demonstrates that lead specifically caused induction of COX-2, a well known inflammatory mediator in neurons and glia cells. Furthermore, we revealed that COX-2 was induced by lead in a transcription-dependent manner, which relayed on transcription factor NFAT, rather than AP-1 and NFκB, in glial cells. Considering the important functions of COX-2 in mediation of inflammation reaction and oxidative stress, our studies here provide a mechanistic insight into the understanding of lead-associated inflammatory neurotoxicity effect via activation of pro-inflammatory NFAT3/COX-2 axis.

Following OGD/R, annexin 1 nuclear translocation and subsequent induction of apoptosis in neurons are assisted by myosin IIA in a TRPM7 kinase-dependent manner.

Transient receptor potential cation channel, subfamily M, member 7 (TRPM7) is a bifunctional channel protein that contains an α-kinase domain at its C-terminal. Previous studies have indicated that oxygen-glucose deprivation/reoxygenation (OGD/R) induces neuronal apoptosis via TRPM7. Annexin 1 and myosin IIA have been identified as TRPM7 kinase substrates; however, the role of annexin 1 in OGD/R-induced neuron apoptosis remains unclear. Here, we report that OGD/R induces nuclear translocation of annexin 1 in primary cultured neurons. Interestingly, ablation of the TRPM7 kinase or a point mutation in Ser(5) interferes with TRPM7 kinase-annexin 1 binding, decreasing annexin 1 nuclear translocation, and thereby reducing neuronal apoptosis. Furthermore, mutation of Arg(205), which intercepts annexin 1-formyl peptide receptor binding, also decreased annexin 1 nuclear translocation. Coimmunoprecipitation indicated that annexin 1 is moved as cargo through the cytoplasm by myosin IIA. However, inhibiting myosin IIA can decrease annexin 1 nuclear translocation. Moreover, blocking myosin IIA function by antagonist injection into the lateral ventricle was found to improve learning and memory in rats after middle cerebral artery occlusion and could also improve cell viability after OGD/R. Last, we determined that the annexin 1-myosin IIA complex is recognized and translocated by the importin α/β heterodimer. Therefore, TRPM7 kinase modulates OGD/R-induced neuronal apoptosis via annexin 1 carried by myosin IIA, while nuclear formyl peptide receptor (FPR)-annexin 1 binding and importin β are involved in nuclear translocation.

Ras Homolog Enriched in the Brain is Linked to Retinal Ganglion Cell Apoptosis after Light Injury in Rats

Ras homolog enriched in the brain (Rheb) is a small GTPase of the Ras family. It has been confirmed that Rheb activation not only regulates cell growth and migration but also induces neuron apoptosis after toxic stimuli. However, the function of Rheb in the retina is still not fully understood. To find out whether Rheb was involved in retinal neuron death, the expression profile of Rheb in light-damaged retinal ganglion cells (RGCs) of adult rats was investigated. Western blotting showed the expression of Rheb was significantly upregulated in the injured retina. Rheb was mainly detected in apoptotic RGCs by using double immunofluorescent staining. Active caspase-3 was upregulated and co-labeled with Rheb. Meanwhile, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) showed that Rheb-positive RGCs underwent apoptosis after light exposure, which suggested that Rheb might be relevant to RGC apoptosis following phototoxicity. Furthermore, Western blotting and immunofluorescence showed that the expression profiles of CyclinD1 and cyclin-dependent kinase 4 (CDK4) were parallel with that of Rheb in a time-space dependent manner. Based on this study, it is speculated that Rheb might play an important role in physiological and pathological process in light-induced retina damage, which might provide a potential therapeutic avenue of retinal degeneration.

Telomerase activity-independent function of telomerase reverse transcriptase is involved in acrylamide-induced neuron damage

Polyacrylamide is used widely in industry, and its decomposition product, acrylamide (ACR), readily finds its way into commonly consumed cosmetics and baked and fried foods. ACR exerts potent neurotoxic effects in human and animal models. Telomerase reverse transcriptase (TERT), the catalytic subunit of telomerase, traditionally has been considered to play an important role in maintaining telomere length. Emerging evidence has shown, however, that TERT plays an important role in neuroprotection by inhibiting apoptosis and excitotoxicity, and by promoting angiogenesis, neuronal survival and neurogenesis, which are closely related to the telomere-independent functions of TERT. We investigated whether and how the TERT pathway is involved in ACR induced neurotoxicity in rat cortical neurons. We found that ACR 1) significantly reduced the viability of cortical neurons as measured by MTT assay, 2) induced neuron apoptosis as revealed by FITC-conjugated Annexin V/PI double staining and flow cytometry (FACS) analysis, 3) elevated expression of cleaved caspase-3, and 4) decreased bcl-2 expression of cortical neurons. ACR also increased intracellular ROS levels in cortical neurons, increased MDA levels and reduced GSH, SOD and GSH-Px levels in mitochondria in a dose-dependent manner. We found that TERT expression in mitochondria was increased by ACR at concentrations of 2.5 and 5.0 mM, but TERT expression was decreased by 10 mM ACR. Telomerase activity, however, was undetectable in rat cortical neurons. Our results suggest that the TERT pathway is involved in ACR induced apoptosis of cortical neurons. TERT also may exert its neuroprotective role in a telomerase activity-independent way, especially in mitochondria.

17β-Estradiol protects cerebellar granule cells against β-amyloid-induced toxicity via the apoptotic mitochondrial pathway

Alzheimer's disease (AD) is a well-studied neurodegenerative disorder; nevertheless, significant therapeutic agents for the pharmacological treatment of this neuropathology are unavailable to date. The toxicity of amyloid β-peptide (Aβ) has been implicated as a critical cause in the development of AD, and Aβ-amyloid-induced toxicity is typically associated with apoptosis. Here, we investigated the effect of 17β-estradiol (E2) on Aβ-induced toxicity in cerebellar granule cells (CGCs). Our data showed a significant induction of apoptosis in neurons treated with Aβ, and the addition of E2 reduced this effect. In addition, E2 reduced the Aβ-induced up-regulation of Bax and down-regulation of Bcl-xL, and inhibited the subsequent mitochondrial release of cytochrome c and activation of caspase-3. Moreover, E2 inhibited Aβ-induced c-Jun N-terminal protein kinase (JNK) activation. Taken together, these findings indicate that E2 protects against Aβ-induced apoptosis in neuronal cells by preventing mitochondrial dysfunction and interfering with the JNK signalling cascade.

Protective Effect of Geranylgeranylacetone against Methamphetamine-Induced Neurotoxicity in Rat Pheochromocytoma Cells

Background: Methamphetamine is a central nervous system stimulant and is one of the agents most commonly abused by illicit drug users which could induce neuron apoptosis when it is used repeatedly and overdosed. Our previous study demonstrated that geranylgeranylacetone (GGA) was an inducer of thioredoxin-1 (Trx-1) and heat shock protein 70 (Hsp70), which played a cytoprotective role against neurotoxicity. Methods: Using the MTT assay, we detected the effect of GGA on cell viability by methamphetamine in rat pheochromocytoma (PC12) cells. Tyrosine hydroxylase, Trx-1, Hsp70, procaspase-9, procaspase-12 and procaspase-3 expression were examined by Western blot analysis. We also detected enzymatic activities of caspase-3 and caspase-9. Results: We found that GGA protected PC12 cells from apoptosis caused by methamphetamine. Furthermore, GGA reversed the decreases in Trx-1 and Hsp70 by methamphetamine, and prevented the methamphetamine-induced decreases in procaspase-9 and procaspase-3. On the other hand, GGA prevented the methamphetamine-induced increases in the enzymatic activity of caspase-9 and caspase-3. Procaspase-12 was not changed by any treatment. Conclusions: These results indicate that GGA protects PC12 cells from methamphetamine-induced toxicity by increasing Trx-1 and Hsp70 and by preventing mitochondria pathway-mediated apoptosis. In summary, GGA may be used as a therapy for neurotoxicity induced by methamphetamine.

MicroRNA‐124 Protects Neurons Against Apoptosis in Cerebral Ischemic Stroke

To explore the role and underlying mechanism of miR-124 in stroke. miR-124 expression was determined by real-time PCR. The effect of miR-124 on infarct area was assessed in middle cerebral artery occlusion (MCAO) mice. The influence of miR-124 on oxygen and glucose deprivation (OGD) induced neuron apoptosis and death was examined by immunofluorescence. The effect of miR-124 on apoptosis-related proteins was determined by Western blot. The level of miR-124 is significantly increased in ischemic penumbra as compared with that in nonischemic area of MACO mice. Brain tissue of stroke-prone spontaneously hypertensive rats (SHR-SP) also showed higher level of miR-124 as compared with that of spontaneously hypertensive rats (SHR). Consistently, OGD treatment obviously increased miR-124 level in primary neurons. In vivo, miR-124 overexpression significantly decreased, while miR-124 knockdown significantly increased, the infarct area of MCAO mice. In vitro, gain or loss of miR-124 function resulted in reduced or increased neuron apoptosis and death induced by OGD, and increased or reduced antiapoptosis protein, Bcl-2 and Bcl-xl, respectively. miR-124 plays a neurons-protective role via apoptosis-inhibiting pathway in ischemic stroke.

Activation of mitochondria-mediated apoptotic pathway in tri-ortho-cresyl phosphate-induced delayed neuropathy

Previous studies suggest that abnormal neurons death has been implicated in organophosphate-induced delayed neuropathy (OPIDN). However, the precise mechanism of neuronal death in OPIDN remains largely unknown. In this study, adult hens were treated with a dosage of 750mg/kg tri-ortho-cresyl phosphate (TOCP) by gavage, and then sacrificed on the time-points of 1, 5, 10, and 21days after dosing TOCP, respectively. The apoptotic change of spinal cord neurons induced by TOCP was examined, and the role of mitochondria-mediated apoptosis of neurons during OPIDN was investigated. TUNEL assays showed that apoptotic neurons in hen spinal cords began to appear on day 5 following TOCP exposure. Immunohistochemistry and western blot analysis revealed a translocation of cytochrome C from mitochondria to cytoplasm after dosing TOCP. Moreover, the level of Bcl-2, Bcl-xl, Pro-caspase3 and Pro-caspase9 in hen spinal cord was significantly decreased, whereas that of Bax and cleaved-PARP was significantly elevated. Taken together, these findings indicate that the administration of TOCP can induce neuron apoptosis in hen spinal cords, which might be mediated by the activation of mitochondrial apoptotic pathway.

Neonatal exposure to antiepileptic drugs disrupts striatal synaptic development

Drug exposure during critical periods of brain development may adversely affect nervous system function, posing a challenge for treating infants. This is of particular concern for treating neonatal seizures, as early life exposure to drugs such as phenobarbital is associated with adverse neurological outcomes in patients and induction of neuronal apoptosis in animal models. The functional significance of the preclinical neurotoxicity has been questioned due to the absence of evidence for functional impairment associated with drug-induced developmental apoptosis. We used patch-clamp recordings to examine functional synaptic maturation in striatal medium spiny neurons from neonatal rats exposed to antiepileptic drugs with proapoptotic action (phenobarbital, phenytoin, lamotrigine) and without proapoptotic action (levetiracetam). Phenobarbital-exposed rats were also assessed for reversal learning at weaning. Recordings from control animals revealed increased inhibitory and excitatory synaptic connectivity between postnatal day (P)10 and P18. This maturation was absent in rats exposed at P7 to a single dose of phenobarbital, phenytoin, or lamotrigine. Additionally, phenobarbital exposure impaired striatal-mediated behavior on P25. Neuroprotective pretreatment with melatonin, which prevents drug-induced neurodevelopmental apoptosis, prevented the drug-induced disruption in maturation. Levetiracetam was found not to disrupt synaptic development. Our results provide the first evidence that exposure to antiepileptic drugs during a sensitive postnatal period impairs physiological maturation of synapses in neurons that survive the initial drug insult. These findings suggest a mechanism by which early life exposure to antiepileptic drugs can impact cognitive and behavioral outcomes, underscoring the need to identify therapies that control seizures without compromising synaptic maturation.