Hypoxia-induced deactivation of NGF-mediated ERK1/2 signaling in hippocampal cells: neuroprotection by acetyl-L-carnitine

Cellular and molecular pathways underlying hypoxic neurotoxicity and cell death are multifaceted and complex. Although many potentially neuroprotective agents have been investigated, the protection conferred is often inadequate, resulting in their insufficient clinical utility. In light of the above, we investigated the therapeutic potential and mechanism of action of acetyl-L-carnitine (ALCAR) in protecting hippocampal neurons from hypoxia-induced neurotoxicity and cellular death. Results showed decreased viability of hippocampal cells when exposed to hypoxia (3% O(2)) for 48 hr along with concomitant membrane depolarization, adenosine triphosphate depletion, DNA fragmentation, accentuated free radical production, and lactate dehydrogenase activity. Pretreatment with ALCAR significantly attenuated hypoxia-induced cytotoxicity in a dose-dependent manner and improved cellular glutathione levels and cytochrome c oxidase activity compared with normoxic controls. Supplementation of ALCAR also prevented apoptosis by down-regulating caspase-3 levels, cytochrome c release, and p-Bcl-2 expression. A decrease in nerve growth factor (NGF) was observed in hypoxic stress despite increased phosphorylation of ERK1/2 (extracellular signal-related kinase) and its downstream effector, Elk-1. Supplementation of ALCAR, on the other hand, up-regulated NGF and tyrosine kinase A expression along with concomitant increase in ERK1/2 phosphorylation, thus enhancing cell survival. ALCAR therefore provides neuroprotection by stabilizing mitochondrial membrane, restoring the cholinergic transmission, and more importantly, it stimulates NGF receptors, thus triggering cell survival pathway via ERK phosphorylation. Therefore, ALCAR may be useful as an effective therapeutic agent for hypoxic stress and associated neurodegenerative diseases.     

Increased phospholipase D2 activity during hypoxia-induced death of PC12 cells: its possible anti-apoptotic role

During hypoxic incubation (1% O2) of PC12 cells, the PLD activity was transiently increased within 12h, followed by a gradual decrease. In the in vitro assay, the increased PLD activity was independent of GTPgammaS required for PLD1 or of oleic acid for PLD(OA), suggesting the activation of PLD2. The level of PLD2 protein showed no change up to 12h but a gradual decrease after 24 h. Pretreatment of cells with S. chromofuscus PLD resulted in inhibition of hypoxia-induced apoptotic cell death. In contrast, 1-butanol, but not 2-butanol, potentiated cell death. Moreover, the number of apoptotic cells significantly reduced in PC12 cells over-expressing PLD2. These results raise the possibility that PLD2 activation may play an anti-apoptotic role in hypoxia-induced cell death.     

Inhibitory effect of zinc on hypoxic HIF-1 activation in astrocytes

Hypoxia-inducible factor-1 (HIF-1) regulates the expression of neuroprotective genes such as erythropoietin (EPO). We investigated the mechanism by which zinc, an excitotoxin-like metal, regulates HIF-1 under hypoxic conditions in astrocytes. In hypoxic LN215 cells, HIF-1alpha stabilized and accumulated in the nucleus, resulting in an increase in its DNA-binding activity to the EPO enhancer. Zinc inhibited hypoxia-induced increases in HIF-1 DNA-binding activity and the HIF-1-dependent mRNA expression of EPO. Zinc did not affect hypoxic stabilization of HIF-1alpha. Nuclear migration of HIF-1alpha upon hypoxia was reduced by zinc. Complete blockade of hypoxia-induced assembly of HIF-1alpha-HIF-1beta complex was observed after treatment of zinc. These findings suggest that zinc hampers hypoxia-stimulated HIF-1 activation in astrocytes by inhibiting nuclear HIF-1alpha translocation and subsequently disrupting HIF-1 heterodimerization.     

乌司他丁对缺氧诱导PC12细胞神经损伤的保护作用研究

目的 探讨乌司他丁对缺氧诱导PC12细胞神经损伤的作用及分子机制。方法 PC12细胞分为对照组、缺氧组、缺氧+乌司他丁低、中、高剂量组、缺氧+miR-NC组、缺氧+miR-190组、缺氧+乌司他丁+anti-miR-NC组、缺氧+乌司他丁+anti-miR-190组。检测培养液中乳酸脱氢酶（LDH）漏出率及细胞中超氧化物歧化酶（SOD）活性、丙二醛（MDA）含量;流式细胞术检测细胞凋亡;蛋白质印迹法（Western blotting）检测蛋白表达;实时荧光定量聚合酶链反应（qRT-PCR）检测miR-190表达水平。结果 与缺氧组比较,缺氧+乌司他丁低、中、高剂量组PC12细胞中LDH漏出率降低,SOD活性升高,MDA含量降低,细胞凋亡率降低,Bcl-2相对表达量升高,Bax相对表达量降低,miR-190相对表达量升高,且呈剂量依赖性（均P<0.05）。miR-190过表达后,LDH漏出率降低,SOD活性升高,MDA含量降低,细胞凋亡率降低,Bcl-2相对表达量升高,Bax相对表达量降低（均P<0.05）。抑制miR-190表达能逆转乌司他丁对缺氧诱导的PC12细胞损伤的作用。结论 乌司他丁可能通过上调miR-190表达对缺氧诱导PC12细胞神经损伤起保护作用。     

Protective effects of sesamin and sesamolin on hypoxic neuronal and PC12 cells

Reactive oxygen species (ROS) are important mediators of a variety of pathological processes, including inflammation and ischemic injury. The neuroprotective effects of sesame antioxidants, sesamin and sesamolin, against hypoxia or H2O2-induced cell injury were evaluated by cell viability or lactate dehydrogenase (LDH) activity. Sesamin and sesamolin reduced LDH release of PC12 cells under hypoxia or H2O2-stress in a dose-dependent manner. Dichlorofluorescein (DCF)-sensitive ROS production was induced in PC12 cells by hypoxia or H2O2-stress but was diminished in the presence of sesamin and sesamolin. We evaluated further the role of mitogen-activated protein kinases (MAPKs) and caspase-3 in hypoxia-induced PC12 cell death. Extracellular signal-regulated protein kinase (ERK) 1, c-jun N-terminal kinase (JNK), and p38 MAPKs of signaling pathways were activated during hypoxia. We found that the inhibition of MAPKs and caspase-3 by sesamin and sesamolin correlated well with the reduction in LDH release under hypoxia. Furthermore, the hypoxia-induced apoptotic-like cell death in cultured cortical cells as detected by a fluorescent DNA binding dye was reduced significantly by sesamin and sesamolin. Taken together, these results suggest that the protective effect of sesamin and sesamolin on hypoxic neuronal and PC12 cells might be related to suppression of ROS generation and MAPK activation.     

Role of low-affinity p75 receptor in nerve growth factor-inducible growth arrest of PC12 cells

Mutant PC12 cell clones (PC84 cells) were obtained by transfection with nerve growth factor (NGF) cDNA. These cells secreted active NGF, extended short processes, and proliferated faster than the parental PC12 cells. These features are of great interest because the parental PC12 cells cease proliferation and extend long processes when transfected with NGF cDNA. PC84 cells expressed a high level of acetylcholinesterase activity and neurofilament M, which indicates that PC84 cells were differentiated. The inhibition of TrkA by K252a diminished the short processes of PC84 cells but had no effect on their fast proliferation. The expression level of TrkA in PC84 cells was comparable to that in PC12 cells; whereas that of another NGF receptor, p75, was significantly lower. These data suggest that the decrease of p75 contributed to the continuous growth of PC84 cells, which was confirmed by suppressing p75 activity of PC12 cells with the antisense oligonucleotide of p75 or with anti-p75 neutralizing antibody. The treated cells did not cease proliferation in the presence of NGF and extended short processes. Our results suggest that NGF signaling via TrkA affects the differentiation characteristics of PC12 cells but that an additional signaling via p75 is necessary for the growth arrest of the cells.     

Expression of the urokinase plasminogen activator receptor is transiently required during "priming" of PC12 cells in nerve growth factor-directed cellular differentiation

We previously identified the urokinase plasminogen activator receptor (UPAR) as a gene induced by nerve growth factor (NGF), but not by epidermal growth factor (EGF), in PC12 cells (Farias-Eisner et al. [2000] J. Neurosci. 20:230-239). Antisense oligonucleotides for the UPAR mRNA or an antibody directed against UPAR protein, added simultaneously with NGF, block NGF-induced morphological and biochemical differentiation of PC12 cells. In this report, we show that anti-UPAR antibody blocks morphological differentiation and the expression of two NGF-specific secondary response genes, collagenase-1 and transin, in PC12 cells only during the first 2 hr following NGF exposure. These data suggest that induced UPAR expression is required only over a short period of time following exposure to NGF for the differentiation program in PC12 cells to proceed. For two models of "primed" PC12 cells, we found that UPAR expression and function are not required for NGF-induced differentiation. UPAR and the secondary response genes collagenase-1 and transin are not induced in "primed" PC12 cells in response to NGF, and anti-UPAR antibody does not block morphological differentiation in these cells. Our data suggests that UPAR is required only transiently during the "priming" of PC12 cells in NGF-induced PC12 cell differentiation.     

Similarities and Discrepancies in the Signaling Pathway for Nerve Growth Factor in an Insulin Producing Cell Line and a Neural Crest-Derived Cell Line

Like neuronal cells, insulin producing cells (beta cells) possess nerve growth factor (NGF) binding sites and express mRNA coding for the low- and high-affinity NGF receptors, p75^NGFR and Trk-A respectively. Although the role of NGF on neuronal cells is well documented, its function on beta cells is still unknown. As a first step towards the elucidation of the role of NGF on beta cells, we have characterized both types of NGF receptors on INS-1 cells, a beta cell line derived from a rat insulinorna and studied some early post-receptor events by comparing the signaling pathway of NGF in those cells and in PC12 cells, a well characterized NGF-responsive cell line. By polymerase chain reaction, immunocytochemistry, cross-linking and Western blot analysis, we clearly demonstrated that Trk-A and p75^NGFR, the two NGF receptors expressed in INS-1 cells and PC12 cells are similar. Moreover, upon NGF treatment, Trk-A is phosphorylated on tyrosine residues in both cell types in the same dose- and time-dependent manner. These data clearly demonstrate that the first step of NGF signal transduction is similar in PC12 and INS-1 cells. Although early responsive genes like NGFI-A and c-fos are induced in both cell types upon NGF treatment, the induction of c-jun expression is restricted to PC12 cells. Furthermore, the expression of late responsive genes, such as vgf and transin, which are induced by NGF in PC12 cells, are not induced in INS-1 cells. In conclusion, although the initial steps of NGF signal transduction are similar in PC12 and INS-1 cells, some of the later differ. These dissimilarities could suggest that NGF plays different roles in neuronal and pancreatic beta cells.     

Characterization of the neurotrophic interaction between nerve growth factor and secreted alpha-amyloid precursor protein

The expression and secretion of amyloid precursor protein (beta APP) is increased in rat cerebral cortices that have been denervated by subcortical lesions of the nucleus basalis of Meynert. The physiological role of the secreted beta APP in response to this injury has not been established. We have previously shown that secreted beta APP produced by alpha-secretase activity (sAPP(alpha)) potentiates the neuritogenic activity of nerve growth factor (NGF) in vitro on naive PC12 cells. In this investigation, we have further characterized the neurotrophic interaction of NGF and sAPP(alpha) using differentiated PC12 cells and rat primary cortical neurons. NGF required the expression of beta APP to maintain a neuronal phenotype. Reduction of endogenous beta APP expression by introduction of antisense oligonucleotides in the presence of NGF resulted in loss of neurites from differentiated PC12 cells but no apparent cell death. Addition of exogenous sAPP(alpha) (60--200 pM) potentiated the protective activity of NGF in serum-deprived differentiated PC12 cells as determined by retention of neurites and cell viability. In addition, exogenous sAPP(alpha) increased neuron viability in both short-term (3 days) cortical neuron cultures grown in the absence of serum and in long-term (9 days) cultures grown with serum. Disruption of the insulin signaling pathway by reduction of IRS-1 expression inhibited the ability of sAPP(alpha) to potentiate neurotrophic activity. These observations suggest that sAPP(alpha) acts as an injury-induced neurotrophic factor that interacts with NGF to enhance neuronal viability using the insulin signaling pathway.     

MicroRNA-433 Inhibits the Proliferation and Migration of HUVECs and Neurons by Targeting Hypoxia-Inducible Factor 1 Alpha

Emerging evidence has demonstrated an important role of microRNAs (miRNAs) in the pathogenesis of cerebral infarction. In the present study, a down-regulation of microRNA-433 (miR-433) is identified in hypoxia-induced human umbilical vein vascular endothelial cells (HUVECs) as well as in rat neurons, and is found to be negatively regulated cell proliferation and migration. Moreover, the expression of miR-433 is inversely correlated with the expression of hypoxia-inducible factor 1 alpha (HIF-1α), which has been shown to play critical role in responding to hypoxia conditions. Overexpression or knockdown of miR-433 responsively alters both mRNA and protein levels of HIF-1α and its downstream genes, vascular endothelial growth factor, Glut-1, and Angpt2. In a luciferase reporter system, miR-433 down-regulates the luciferase activity of HIF-1α 3′-UTR, and these effects are abolished by a mutation in the putative miR-433-binding site. Further investigation confirms that knockdown of HIF-1α blocked the stimulatory effect of anti-miR-433, while overexpression of HIF-1α reversed the inhibitory effects of pre-miR-433 on proliferation and migration of HUVEC and neurons. Taken together, our findings indicate that miR-433 plays an important role in response to hypoxia, inhibiting HUVEC and neuron proliferation and migration by down-regulating HIF-1α.     

PC12细胞缺氧后细胞凋亡与DNA损伤相关基因表达关系的研究

目的 探讨PC12细胞缺氧后细胞凋亡与DNA损伤的关系。方法 采用TUNEL法,结合应用流式细胞术观察PC12细胞缺氧培养不同时间点细胞凋亡现象,以及DNA损伤相关基因表达的改变。结果 在缺氧0.5h开始出现凋亡细胞,引时P53、P21^waf1/cip1蛋白表达开始增高。至缺氧1h凋亡细胞达高峰,此时P53、P21蛋白表达最高（P＜0.05）。至缺氧6－12h,则以坏死为主,此时以上基因表达均减弱。结论 PC12细胞在缺氧早期（0.5-2h）主要出现以凋亡为主的细胞死亡,伴有DNA损伤相关基因表达的动脉改变,提示缺氧后PC12细胞凋亡部分是由于DNA损伤严重、损伤不能及时修复所致。     

Role of nerve growth factor in oxidant-antioxidant balance and neuronal injury. I. Stimulation of hydrogen peroxide resistance

The nerve growth factor protein (NGF) regulates neuronal cell death during the development of embryonic sensory and sympathetic neurons in the peripheral nervous system (PNS). NGF protects the rat pheochromocytoma line PC12, a useful model of NGF responsive peripheral neurons, from hydrogen peroxide, which interacts with ferrous iron to generate hydroxyl radicals. Exogenous catalase provides protection, whereas superoxide dismutase (SOD) has no effect on neuronal survival when PC12 cells are challenged with hydrogen peroxide. NGF treatment of PC12 cells increases the activity of catalase. NGF protection from hydrogen peroxide is partially abolished by aminotriazole (Az), a low molecular weight catalase inhibitor. Taken together, these data are consistent with the hypothesis that NGF protects from peroxidative events and consequent cell death via an induction of free radical detoxifying mechanisms, such as catalase activity.