Adoptive Autophagy Activation: a Much-Needed Remedy Against Chemical Induced Neurotoxicity/Developmental Neurotoxicity

The profound significance of autophagy as a cell survival mechanism under conditions of metabolic stress is a well-proven fact. Nearly a decade-long research in this area has led scientists to unearth various roles played by autophagy other than just being an auto cell death mechanism. It is implicated as a vital cell survival pathway for clearance of all the aberrant cellular materials in case of cellular injury, metastasis, disease states, cellular stress, neurodegeneration and so on. In this review, we emphasise the critical role of autophagy in the environmental stressors-induced neurotoxicity and its therapeutic implications for the same. We also attempt to shed some light on the possible protective role of autophagy in developmental neurotoxicity (DNT) which is a rapidly growing health issue of the human population at large and hence a point of rising concern amongst researchers. The intimate association between DNT and neurodegenerative disorders strongly indicates towards adopting autophagy activation as a much-needed remedy for DNT.     

STAT3 Regulates MMP3 in Heme-Induced Endothelial Cell Apoptosis

Background

We have previously reported that free Heme generated during experimental cerebral malaria (ECM) in mice, is central to the pathogenesis of fatal ECM. Heme-induced up-regulation of STAT3 and CXCL10 promotes whereas up-regulation of HO-1 prevents brain tissue damage in ECM. We have previously demonstrated that Heme is involved in the induction of apoptosis in vascular endothelial cells. In the present study, we further tested the hypothesis that Heme reduces blood-brain barrier integrity during ECM by induction of apoptosis of brain vascular endothelial cells through STAT3 and its target gene matrix metalloproteinase three (MMP3) signaling.

Methods

Genes associated with the JAK/STAT3 signaling pathway induced upon stimulation by Heme treatment, were assessed using real time RT² Profile PCR arrays. A human MMP3 promoter was cloned into a luciferase reporter plasmid, pMMP3, and its activity was examined following exposure to Heme treatment by a luciferase reporter gene assay. In order to determine whether activated nuclear protein STAT3 binds to the MMP3 promoter and regulates MMP3 gene, we conducted a ChIP analysis using Heme-treated and untreated human brain microvascular endothelial cells (HBVEC), and determined mRNA and protein expression levels of MMP3 using qRT-PCR and Western blot. Apoptosis in HBVEC treated with Heme was evaluated by MTT and TUNEL assay.

Results

The results show that (1) Heme activates a variety of JAK/STAT3 downstream pathways in HBVEC. STAT3 targeted genes such as MMP3 and C/EBPb (Apoptosis-related genes), are up regulated in HBVEC treated with Heme. (2) Heme-induced HBVEC apoptosis via activation of STAT3 as well as its downstream signaling molecule MMP3 and upregulation of CXCL10 and HO-1 expressions. (3) Phosphorylated STAT3 binds to the MMP3 promoter in HBVEC cells, STAT3 transcribed MMP3 and induced MMP3 protein expression in HBVEC cells.

Conclusions

Activated STAT3 binds to the MMP3 promoter region and regulates MMP3 in Heme-induced endothelial cell apoptosis.     

SIPAR和STAT3相互作用并对其信号通路进行负调控

STAT3 对细胞的生长、存活、增殖以及机体的发育都具有重要的作用 . STAT3 基因的失活可以引起多种疾病,而 STAT3 基因的过度激活又可以引发很多癌症 . 为了对 STAT3 信号通路的调控做进一步的研究,采用酵母双杂交的方法,以 STAT3 蛋白全长作为诱饵蛋白,筛选了小鼠 7 天的胚胎文库 . 得到了与 STAT3 相互作用的一个功能未知的蛋白质,将其命名为 SIPAR (Stat3 interacting protein as a repressor). 免疫染色的实验结果表明, SIPAR 是一种在核内分布为主,细胞质中也有少许分布的蛋白质 . 荧光酶活性测定结果表明 SIPAR 对 STAT3 的转录活性具有抑制作用 . 斑马鱼注射实验说明 SIPAR 基因表达严重影响了斑马鱼的正常发育 .     

Resveratrol Protects Against Neurotoxicity Induced by Kainic Acid

Increased oxidative stress has been implicated in the mechanisms of excitotoxicity in hippocampus induced by kainic acid (KA), an excitatory glutamate receptor agonist. Resveratrol, a polyphenolic antioxidant compound enriched in grape, is regarded as an important ingredient in red wine to offer cardiovascular and neural protective effects. This study was designed to investigate whether resveratrol treatment may ameliorate neuronal death after KA administration. Adult Sprague Dawley male rats were treated with KA (8 mg/kg) daily for 5 days and another group was treated similarly with KA plus resveratrol (30 mg/kg/day). Three hr after the last treatment protocol, animals were sacrificed, and brain sections were obtained for histochemical and immunohistochemical identification of neurons, astrocytes and microglial cells. After KA administration, significant neuronal death and activation of astrocytes and microglial cells were observed in the hippocampal CA1, CA3 and polymorphic layer (hilar) of the dentate gyrus (DG) (P < 0.001). The KA-induced hippocampal neuronal damage was significantly attenuated by treatment with resveratrol (P < 0.001). Resveratrol also suppressed KA-induced activation of astrocytes and microglial cells. Since increased oxidative stress is a key factor for KA-induced neurotoxicity, this study demonstrated the ability of resveratrol to act as free radical scavenger to protect against neuronal damage caused by excitotoxic insults.Special issue dedicated to Dr. Lawrence F. Eng.     

SOCS3通过JNK和STAT3信号通路调控AKT

蛋白激酶B(AKT),在细胞存活、代谢、迁移和侵袭等信号通路中起着重要的调控作用。细胞信号转导抑制因子3(SOCS3)主要参与酪氨酸蛋白激酶(JAK)/信号传导子和转录激活子3(STAT3)传导途径的负反馈调节,可能参与AKT的磷酸化,进而调控肿瘤的发生。根据SOCS3蛋白的生物学特性和AKT信号通路的激活途径,综述了SOCS3在AKT信号通路中的调控作用,以期针对SOCS3靶向AKT信号通路进行抗肿瘤研究,为肿瘤的治疗提供一种新的思路。     

消癌平诱导caspase-3活化动态过程的实时监测

消癌平是从萝摩科(Asclepiadaceae)植物通关藤(Marsdenia tenacissima)根部提取的药物,已经广泛应用于癌症和多种炎症的临床治疗,并且具有较好的疗效。将消癌平注射液与细胞培养液按照1∶1的比例混合后共同培养人肺腺癌(ASTC-a-1)细胞,通过CCK-8方法检测发现消癌平能够显著抑制细胞的增长。然后利用荧光共聚焦扫描显微镜和荧光共振能量转移(fluorescence resonance energy transfer,FRET)技术在稳定表达了SCAT3质粒的单个ASTC-a-1细胞中实时动态监测消癌平处理后SCAT3的空间分布以及SCAT3被caspase-3切割的动态过程。实验结果表明:消癌平处理后20 min中SCAT3开始向细胞核以及细胞膜部位转移,约在100min左右SCAT3被迅速切割。     

Role of Gap Junction Protein Connexin43 in Astrogliosis Induced by Brain Injury

Astrogliosis is a process that involves morphological and biochemical changes associated with astrocyte activation in response to cell damage in the brain. The upregulation of intermediate filament proteins including glial fibrillary acidic protein (GFAP), nestin and vimentin are often used as indicators for astrogliosis. Although connexin43 (Cx43), a channel protein widely expressed in adult astrocytes, exhibits enhanced immunoreactivity in the peri-lesion region, its role in astrogliosis is still unclear. Here, we correlated the temporal and spatial expression of Cx43 to the activation of astrocytes and microglia in response to an acute needle stab wound in vivo. We found large numbers of microglia devoid of Cx43 in the needle wound at 3 days post injury (dpi) while reactive astrocytes expressing Cx43 were present in the peripheral zone surrounding the injury site. A redistribution of Cx43 to the needle site, corresponding to the increased presence of GFAP-positive reactive astrocytes in the region, was only apparent from 6 dpi and sustained until at least 15 dpi. Interestingly, the extent of microglial activation and subsequent astrogliosis in the brain of Cx43 knockout mice was significantly larger than those of wild type, suggesting that Cx43 expression limits the degree of microgliosis. Although Cx43 is not essential for astrogliosis and microglial activation induced by a needle injury, our results demonstrate that Cx43 is a useful marker for injury induced astrogliosis due to its enhanced expression specifically within a small region of the lesion for an extended period. As a channel protein, Cx43 is a potential in vivo diagnostic tool of asymptomatic brain injury.     

Lymphocyte Activation Gene-3 (LAG-3) Negatively Regulates Environmentally-Induced Autoimmunity

Environmental factors including drugs, mineral oils and heavy metals such as lead, gold and mercury are triggers of autoimmune diseases in animal models or even in occupationally exposed humans. After exposure to subtoxic levels of mercury (Hg), genetically susceptible strains of mice develop an autoimmune disease characterized by the production of highly specific anti-nucleolar autoantibodies, hyperglobulinemia and nephritis. However, mice can be tolerized to the disease by a single low dose administration of Hg. Lymphocyte Activation Gene-3 (LAG-3) is a CD4-related, MHC-class II binding molecule expressed on activated T cells and NK cells which maintains lymphocyte homeostatic balance via various inhibitory mechanisms. In our model, administration of anti-LAG-3 monoclonal antibody broke tolerance to Hg resulting in autoantibody production and an increase in serum IgE level. In addition, LAG-3-deficient B6.SJL mice not only had increased susceptibility to Hg-induced autoimmunity but were also unresponsive to tolerance induction. Conversely, adoptive transfer of wild-type CD4⁺ T cells was able to partially rescue LAG-3-deficient mice from the autoimmune disease. Further, in LAG-3-deficient mice, mercury elicited higher amounts of IL-6, IL-4 and IFN-γ, cytokines known to play a critical role in mercury-induced autoimmunity. Therefore, we conclude that LAG-3 exerts an important regulatory effect on autoimmunity elicited by a common environmental pollutant.     

Activation of Protein Kinase C by Trimethyltin: Relevance to Neurotoxicity

Abstract: The differentiated PC12 cell neuronal model was used to determine the effect of trimethyltin (TMT) on protein kinase C (PKC). Cells treated with 5–20 µ M TMT showed a partial and sustained PKC translocation within 30 min and persisted over a 24-h period. TMT treatment was accompanied by a low level of PKC down-regulation over 24 h, which was small compared with that produced by phorbol esters. Confocal imaging of differentiated PC12 cells showed that PKC translocates to the plasma membrane and the translocation is blocked by the PKC inhibitor chelerythrine (1 µ M ). Phorbol myristate-induced PKC down-regulation or inhibition with chelerythrine provided protection against TMT-induced cytotoxicity. It was concluded that TMT-induced PKC translocation and activation contribute to the cytotoxicity of TMT in differentiated PC12 cells.     

甲胺磷诱发母鸡迟发性神经毒性模型

以成年鸡为实验动物,对甲胺磷原药诱发鸡的迟发性神经毒性进行了研究。检测了甲胺磷对鸡脑神经病靶标酯酶（NTE）的体外抑制情况以及急性中毒后期鸡神经组织NTE活性变化。毒性试验发现,甲胺磷对京白母鸡（Gallus domestics）的毒性主要表现为急性中毒,未见有明显的迟发性神经毒性症状。结果表明,本实验未能通过经口及经皮两种染毒途径在母鸡上建立甲胺磷诱发的迟发性神经毒性模型。     

Crocin Inhibits Apoptosis and Astrogliosis of Hippocampus Neurons Against Methamphetamine Neurotoxicity via Antioxidant and Anti-inflammatory Mechanisms

Methamphetamine (METH) is a stimulant drug, which can cause neurotoxicity and increase the risk of neurodegenerative disorders. The mechanisms of acute METH intoxication comprise intra-neuronal events including oxidative stress, dopamine oxidation, and excitotoxicity. According to recent studies, crocin protects neurons by functioning as an anti-oxidant, anti-inflammatory, and anti-apoptotic compound. Accordingly, this study aimed to determine if crocin can protect against METH-induced neurotoxicity. Seventy-two male Wistar rats that weighed 260–300 g were randomly allocated to six groups of control (n?=?12), crocin 90 mg/kg group (n?=?12), METH (n?=?12), METH?+?crocin 30 mg/kg (n?=?12), METH?+?crocin 60 mg/kg (n?=?12), and METH?+?crocin 90 mg/kg (n?=?12). METH neurotoxicity was induced by 40 mg/kg of METH in four injections (e.g., 4?×?10 mg/kg q. 2 h, IP). Crocin was intraperitoneally (IP) injected at 30 min, 24 h, and 48 h after the final injection of METH. Seven days after METH injection, the rats’ brains were removed for biochemical assessment using the ELISA technique, and immunohistochemistry staining was used for caspase-3 and glial fibrillary acidic protein (GFAP) detection. Crocin treatment could significantly increase superoxide dismutase (P?<?0.05) and glutathione (P?<?0.01) levels and reduce malondialdehyde and TNF-α in comparison with the METH group (P?<?0.05). Moreover, crocin could significantly decline the level of caspase-3 and GFAP-positive cells in the CA1 region (P?<?0.01). According to the results, crocin exerts neuroprotective effects on METH neurotoxicity via the inhibition of apoptosis and neuroinflammation.     

Cyclooxygenase Regulates Angiogenesis Induced by Colon Cancer Cells

To explore the role of cyclooxygenase (COX) in endothelial cell migration and angiogenesis, we have used two in vitro model systems involving coculture of endothelial cells with colon carcinoma cells. COX-2-overexpressing cells produce prostaglandins, proangiogenic factors, and stimulate both endothelial migration and tube formation, while control cells have little activity. The effect is inhibited by antibodies to combinations of angiogenic factors, by NS-398 (a selective COX-2 inhibitor), and by aspirin. NS-398 does not inhibit production of angiogenic factors or angiogenesis induced by COX-2-negative cells. Treatment of endothelial cells with aspirin or a COX-1 antisense oligonucleotide inhibits COX-1 activity/expression and suppresses tube formation. Cyclooxygenase regulates colon carcinoma-induced angiogenesis by two mechanisms: COX-2 can modulate production of angiogenic factors by colon cancer cells, while COX-1 regulates angiogenesis in endothelial cells.