6-羟基褪黑素保护神经细胞抗缺血再灌注损伤的作用机制

目的探讨6-羟基褪黑素的神经保护作用及作用机理。方法体外培养神经母细胞瘤N2a细胞，缺糖、缺血清和缺氧（OGSD）90min，再正常培养不同时间，同时加入6-羟基褪黑素，观察细胞生存能力（MTT法）、线粒体跨膜电位变化（荧光标记）、线粒体细胞色素C释放（western blot）、caspase3活性（荧光底物）以及细胞内活性氧（荧光标记）的产生。结果6-羟基褪黑素抑制细胞色素C释放，抑制caspase3激活，清除细胞内活性氧，稳定线粒体跨膜电位。结论6-羟基褪黑素通过直接抗氧化作用和抑制线粒体凋亡途径促进神经细胞生存。     

线粒体自噬在褪黑素抗缺血再灌注损伤中的作用

目的初步探讨线粒体自体吞噬在褪黑素抗缺血再灌注损伤中的作用。方法体外培养N2a细胞,模拟缺血再灌注,加入褪黑素(melatonin,Mel),用DNA琼脂糖凝胶电泳和Caspase3活性测定分析细胞凋亡情况,并采用激光共聚焦分析线粒体自噬现象。结果(1)Mel能抑制缺血再灌注介导的N2a细胞caspase3的活性,并减轻N2a细胞DNA的片段化;(2)N2a细胞缺血90min再灌注12h,未观察到线粒体自噬现象,而加入Mel的N2a有大量的线粒体自噬现象。结论线粒体自噬可能是褪黑素抗缺血再灌注损伤的机制之一。     

体外模拟缺血再灌注诱导神经细胞线粒体功能改变的研究

目的观察线粒体功能失调在缺血再灌注后神经细胞凋亡中的作用.方法(1)采用体外培养神经母细胞瘤细胞株N2a,模拟缺血再灌注(先缺氧缺营养90 min,然后正常培养不同时间);(2)采用琼脂糖凝胶电泳检测细胞凋亡情况;(3)通过MTT法、细胞色素C释放和跨膜压的改变判断线粒体的功能;(4)Cas-pase 3活性测定采用水解其可见光底物.结果(1)N2a细胞缺血再灌注12 h即出现明显DNA片段化,24h更明显;(2)线粒体琥珀酸脱氢酶活性缺血再灌注24 h明显降低;跨膜电位在缺血再灌注1 h先短暂下降,3 h明显升高,6 h后降低,以后再没回升.缺血再灌注3 h细胞色素C开始释放,6 h达到高峰,并持续到24 h;(3)Caspase 3活性在缺血再灌注10 h升高,24 h达到高峰.线粒体通透性转换孔抑制剂cyclosporine A只能抑制部分caspase 3的活性和DNA片段化改变,而caspase 8抑制剂虽不能完全抑制caspase 3的活性,但能完全抑制DNA的片段化.结论缺血再灌注诱导神经细胞凋亡存在caspase 3依赖性和非依赖性两条途径,线粒体功能失调在缺血再灌注引起的N2a细胞凋亡过程中可能起到凋亡早期的启动和随后的信号放大作用.     

褪黑素在缺血再灌注引起神经元凋亡的保护作用

目的探讨模拟缺血再灌注引起神经元凋亡的途径和褪黑素(m elaton in,MT)抗凋亡的作用机理。方法建立原代培养大鼠小脑颗粒细胞的体外模拟缺血(Oxygen G lucose Deprivation,OGD)再灌注模型,测定培养液LDH活性和细胞DNA琼脂糖凝胶电泳;利用Rhodam ine123和激光共聚焦显微镜观察线粒体膜电位的变化;ELISA检测细胞浆中细胞色素C水平;用同样指标观察MT对其损伤的保护作用。结果在体外模拟缺血再灌注模型中培养液LDH活性增加(P<0.05),琼脂糖凝胶电泳DNA出现梯状条带,线粒体膜电位明显降低,细胞浆中细胞色素C含量增加(P<0.05),MT对上述现象有明显的抑制作用。结论(1)缺血再灌注引起的神经元凋亡机理之一是通过线粒体凋亡途径;(2)MT可通过阻止线粒体凋亡途径而保护模拟缺血再灌注诱导小脑颗粒细胞的凋亡。     

线粒体在缺血再灌注继发的神经元细胞凋亡中的作用

目的 观察线粒体功能失调在缺血再灌后神经元凋亡中的作用。方法(1)采用体外培养神经母细胞瘤细胞株N2a细胞，体外模拟缺血再灌注(缺氧90min，然后再灌不同时间)；(2)采用琼脂糖凝胶电泳检测细胞凋亡情况；(3)MTT法、细胞色素c释放和跨膜压的改变判断线粒体的功能；(4)caspase-3活性测定采用水解其可见光底物。结果(1)N2a细胞缺血再灌12h即出现明显DNA片段化，24h更明显；(2)线粒体酶活性降低．跨膜电位在缺氧再灌1h先短暂下降再升高然后又明显降低。细胞色素c缺氧再灌3h开始下降，6h达到高峰．持续到24h；(3)caspase-3活性在缺氧再灌10h升高，24h达到高峰，线粒体通透性转换孔形成抑制剂CsA只能抑制部分caspasc-3的活性和DNA片段化改变；而caspase-8抑制剂虽不能完全抑制caspase-3的活性但能完全抑制DNA的片段化。结论N2a凋亡尚存在有caspase-3依赖性和非依赖性两条途径线粒体功能失调在缺氧再灌引起的N2a细胞凋亡过程中可能起到凋亡早期的启动和随后的信号放大作用。     

褪黑素通过抑制线粒体通透性转换孔开放保护缺氧缺糖SHSY5Y细胞

目的观察褪黑素对体外缺氧缺糖SHSY 5Y细胞的保护作用及线粒体通透性转换孔开放对该保护作用的影响。方法体外培养SHSY 5Y神经母细胞瘤株,以缺氧缺糖(OGD)、褪黑素(10μm o l/L)伴或不伴苍术苷(atracty los ide)(20μm o l/L)处理,以M TT检测SHSY 5Y细胞活性,应用ca lce in-AM、HE t和fluo-3/AM的荧光强度测定OGD后SHSY 5Y细胞线粒体通透性转换孔(M TPT)开放、细胞内超氧阴离子和钙离子的浓度。结果褪黑素能提高OGD后SHSY 5Y细胞的活性,这种神经保护作用可被苍术苷阻断。褪黑素降低OGD后ca lce in-AM、HE t和fluo-3/AM的荧光强度。结论褪黑素通过抑制SHSY 5Y细胞M TPT开放,来降低细胞内超氧阴离子的浓度,进而抑制细胞内钙超载,从而达到保护SHSY 5Y细胞对抗OGD的细胞损害的作用。     

褪黑素调节DNA甲基化相关酶减轻大鼠脑缺血再灌注损伤

目的 观察褪黑素(Mel)对脑缺血再灌注损伤(CIRI)大鼠神经细胞凋亡的影响，并从DNA甲基化相关酶的角度探讨其分子机制。方法 健康成年雄性Sprague-Dawley大鼠35只，随机分为：假手术组(Sham组，n=5)、模型组(CIRI组，n=15)、褪黑素组(Mel组，n=15)。CIRI及Mel组大鼠采用改良的Zea-Longa线栓法建立大鼠CIRI模型，Sham组仅分离颈总动脉。Mel组于造模前后30 min经腹腔注射Mel（5 mg/kg体质量），Sham组于造模前后注射等量的生理盐水。CIRI后24 h，行苏木素－伊红(HE)染色观察大鼠缺血侧大脑皮质神经细胞形态学的变化；CIRI后6 h、24 h及72 h，TUNEL染色法观察Mel对大鼠缺血侧大脑皮质凋亡细胞的影响；免疫组织化学染色法观察Mel对大鼠缺血侧大脑皮质DNA甲基化转移酶1(DNMT1)、DNA甲基化转移酶3a(DNMT3a)蛋白表达的影响。结果 HE染色结果发现，Sham组大鼠大脑皮质神经细胞排列整齐、形态规则；CIRI组大鼠缺血侧大脑皮质神经细胞排列紊乱、形态不规则；Mel组大鼠缺血侧大脑皮质神经细胞排列较整齐、形态较规则。TUNEL染色结果显示，CIRI后6 h、24 h、72 h，与CIRI组相比，Mel组大鼠缺血侧大脑皮质TUNEL⁺细胞数量减少，差异均具有统计学意义(P<0.01)。免疫组织化学染色结果显示，CIRI后6 h、24 h及72 h，Mel组大鼠DNMT1⁺、DNMT3a⁺细胞数量较CIRI组减少，差异均具有统计学意义(P<0.01)。结论 Mel可抑制CIRI大鼠神经细胞凋亡，发挥神经保护作用，其机制可能与Mel降低DNMT1、DNMT3a蛋白表达，下调CIRI后DNA甲基化水平有关。 [引用格式:国际神经病学神经外科学杂志, 2021, 48(4): 333-338.]     

褪黑素对癫痫状态大鼠海马GAD、GABA-T活性和GABA、Glu含量的影响

目的从神经递质方面研究褪黑素(melatonin,Mel)抗癫痫作用的生化机制。方法采用匹罗卡品(pilocarpine,PILO)诱发大鼠癫痫持续状态(status epilepticus,SE)模型,观察大鼠SE后4个时相点即6、48、72h和7 d海马γ-氨基丁酸、谷氨酸含量和谷氨酸脱羧酶、γ-氨基丁酸转氨酶活性的变化,以及Mel对其变化的影响。结果PILO诱导大鼠SE后6 h~7 d海马谷氨酸含量显著升高,γ-氨基丁酸含量和谷氨酸脱羧酶活性显著降低,与对照组比较差异有统计学意义(P<0.05),γ-氨基丁酸转氨酶活性在SE后72 h才显著下降(P<0.05);给予Mel的大鼠在SE后6 h~7 d,海马γ-氨基丁酸含量和谷氨酸脱羧酶活性均显著高于仅予PILO处理的大鼠(P<0.05),而谷氨酸含量和γ-氨基丁酸转氨酶活性与仅予PILO处理的大鼠比较差异无统计学意义(P>0.05)。结论Mel通过增强SE大鼠海马谷氨酸脱羧酶活性及γ-氨基丁酸合成发挥抗癫痫作用。     

褪黑素抗衰老机制及其与缺血性卒中的关系

褪黑素具有显著抗衰老作用,其主要机制包括自由基清除和线粒体保护,褪黑素水平的下降可能是人体衰老的表现。此外,研究证实褪黑素具有降脂、降压和减重作用,可预防和延缓动脉粥样硬化进展,改善脑缺血再灌注损伤,在缺血性卒中领域具有良好的应用前景。本文对褪黑素抗衰老、预防卒中相关危险因素和改善卒中预后等方面的作用机制和应用前景进行总结。     

褪黑素对脑出血大鼠神经细胞mtDNA缺失及线粒体转录因子A表达的影响

目的观察褪黑素对脑出血大鼠神经细胞mtDNA缺失、线粒体转录因子A(mtTFA)表达的影响。方法 70只SD大鼠随机分为正常对照组、假手术组、脑出血模型组(模型组)、褪黑素治疗组(褪黑素组),组内随机分为12 h、1 d、2 d、4 d、7 d共5个时间点,应用修正神经功能缺损评分进行神经功能评估;应用荧光实时定量PCR技术对神经细胞mtTFA mRNA转录水平以及mtDNA缺失率进行定量分析;透射电镜技术观察神经元线粒体超微结构。结果脑出血后1 d~7 d褪黑素组神经功能评分低于模型组(P0.05);脑出血后12 h~2 d时褪黑素组mtTFA mRNA表达显著高于模型组(P0.05);褪黑素组mtDNA4834 bp缺失率低于模型组(P0.05);模型组神经元线粒体肿胀、空泡化、数量减少,褪黑素组线粒体超微结构病理改变轻微。结论褪黑素能够上调脑出血大鼠神经细胞mtTFA表达,减少mtDNA4834 bp缺失,褪黑素可能通过保护线粒体功能从而发挥神经保护作用。     

Neuroprotective effect of melatonin against ischemia/reperfusion-induced neuronal apoptosis in mouse cerebellum

BACKGROUND: Some experiments have demonstrated that melatonin (N-aceyl-5-methoxytryptamine, Mel) has antioxidation. However, whether it has neuroprotective effect in the ischemia/reperfusion injury of central nervous system is unclear. OBJECTIVE: To observe the protective effect of Mel on ischemia/reperfusion-induced cerebellar neuronal apoptosis of rats, and the action mechanism. DESIGN: Controlled observation experiment. SETTING: Department of Biochemistry and Molecular Biology, Tongji Medical College, Huazhong University of Science and Technology. MATERIALS: Eight Sprague-Dawley rats aged 7–8 days and weighing 10–12 g were provided by Medical Experimental Animal Center, Tongji Medical College,Huazhong University of Science and Technology. Anti-cytochrome C monoclonal antibody was purchased from R & D Company; 7-dichlorodihydrofluorescein diacetate(DCFH-DA), rhodamine 123 and Mel were purchased from Sigma Company (USA). Lactate dehydrogenase (LDH) kit was purchased from Nanjing Jiancheng Bioengineering Institute. METHODS: This experiment was carried out in the laboratory for Department of Biochemistry and Molecule Biology, Tongji Medical College between October 2002 and March 2004. Cerebellar neurons of rats were cultured in vitro. After oxygen-glucose deprivation (OGD) for 90 minutes, 1×10–4,1×10–6, 1×10–9 mol/L Mel was added, respectively, namely high-, middle-, and low-concentration Mel groups. Cells, which were cultured by OGD, served as model group, and control group, in which OGD intervention was omitted, was set. ①Cytochrome C level of mitochondrial cells in each group was detected by ELISA method. ②LDH activity in the cell culture fluid was measured, and cell membrane permeability change was analyzed. The cells in the Mel group with the lowest LDH activity served as Mel treatment group, i.e. cells were cultured with OGD, and then Mel was added; Meanwhile, Mel prevention group was set, i.e. Mel was added before OGD. Intervention was not changed in the model group and control group. ③ DNA level was analyzed and cell apoptosis was observed by agarose gel electrophoresis(AGE). ④Mitochondrial transmembrane potential of cells, and apoptotic way in each group were analyzed by confocal laser scanning microscopy. MAIN OUTCOME MEASURES: ①Mitochondrial cytochrome C level of cerebellar nerve cells. ②LDH activity of cerebellar nerve cells. ③ DNA AGE results. ④Mitochondrial transmembrane potential change. RESULTS: ①Mitochondrial cytochrome C level of cerebellar nerve cells: cytochrome C was obviously released at 6 hours of OGD-reperfusion. Mel inhibited the release of cytochrome C in dose-dependent manner. ②LDH activity of cerebellar nerve cells: LDH activity (A value) was significantly lower in the high- and middle-concentration Mel groups than in the model group (P < 0.05). LDH activity (A value) in the low-concentration Mel group was 0.415 0±0.012 9, indicating that Mel could decrease LDH activity of OGD-treated cell supernatant and promote membrane stablization in dose-dependent manner. ③AGE results of DNA: 1×10–9 mol/L was considered as the best concentration of melatonin. Cell DNA was extracted for AGE. Results presented typical ladder shape, indicating apoptosis appeared, while apoptosis was lessened in the Mel treatment group and Mel prevention group.④Mitochondrial transmembrane potential change: Experimental results showed that green fluorescein was evenly distributed in cerebellar granule cells cultured normally, and the axons of neurons were very clear. The body of neurons was condensed and the axons disappeared after cerebellar granule cells undergoing OGD injury. Mel could completely reverse the effect of OGD. CONCLUSION: Mel can enhance cerebellar neuronal membrane stabilization of rats in dose-dependent manner, and suppress OGD-induced apoptosis of cerebellar granule cells by preventing against mitochondrial apoptosis.     

Cyanide enhancement of dopamine-induced apoptosis in mesencephalic cells involves mitochondrial dysfunction and oxidative stress

Dopamine (DA)-induced neurotoxicity is potentiated when cellular metabolism is compromised. Since cyanide is a neurotoxin that produces mitochondrial dysfunction and stimulates intracellular generation of reactive oxygen species (ROS), KCN was used to study DA-induced apoptosis in primary cultured mesencephalon cells. Treatment of neurons with DA (300 microM) for 24h produced apoptosis as determined by TUNEL staining, DNA fragmentation and increased caspase activity. Pretreatment with KCN (100 microM) 30min prior to DA increased the number of cells undergoing apoptosis. When added to the cells alone, this concentration of KCN did not induce apoptosis. DA stimulated intracellular generation of ROS, and treatment with KCN enhanced ROS generation. Treatment of cells with glutathione or uric acid (antioxidants/scavengers) attenuated both the increase in ROS generation and the apoptosis, demonstrating that ROS are initiators of the cytotoxicity. Studies on the sequence of events mediating the response showed that DA-induced depolarization of the mitochondrial membrane was dependent on ROS generation and KCN enhanced this action of DA. Following changes in mitochondrial membrane potential, cytochrome c was released from mitochondria, leading to caspase activation and eventually cell death. These results demonstrate that oxidative stress and mitochondrial dysfunction are initiators of DA-induced apoptosis. Subsequent cytochrome c release activates the caspase effector component of apoptosis. Cyanide potentiates the neurotoxicity of DA by enhancing the generation of ROS and impairing mitochondrial function.     

Caspase-dependent and -independent death of camptothecin-treated embryonic cortical neurons.

This study investigates the mechanisms underlying death of cultured embryonic cortical neurons exposed to the DNA-damaging agent camptothecin and in particular the interdependence of the roles of cyclin-dependent kinases (Cdks), caspases, and mitochondrial function. Camptothecin evokes rapid neuronal death that exhibits nuclear features of apoptosis. This death is accompanied by loss of cytochrome c and mitochondrial transmembrane potential as well as by induction of caspase-3-like activity and caspase-2 processing. The Cdk inhibitor flavopiridol provides long-term rescue from death and prevents loss of cytochrome c and mitochondrial transmembrane potential as well as caspase activation and processing. General caspase inhibitors rescue neurons from this rapid apoptotic death but do not prevent them from undergoing delayed death in which nuclear features of apoptosis are absent. Moreover, the caspase inhibitors do not affect early cytochrome c release and delay but do not prevent the loss of transmembrane potential. Agents that directly disrupt mitochondrial function without inducing cytochrome c release lead to a caspase-independent death. These observations favor a model in which (1) DNA damage leads to Cdk activation, which lies upstream of release of cytochrome c and caspase activation; (2) cytochrome c release is caspase-independent and may occur upstream of caspase activation; (3) early apoptotic death requires caspases; and (4) delayed nonapoptotic death that occurs in the presence of caspase inhibitors is a consequence of prolonged loss of mitochondrial function. These findings shed light on the mechanisms by which DNA damage kills neurons and raise questions regarding the general utility of caspase inhibitors as neurotherapeutic agents.     

Time course assessment of methylmercury effects on C6 glioma cells: submicromolar concentrations induce oxidative DNA damage and apoptosis

Organic mercury is a well-known neurotoxicant although its mechanism of action has not been fully clarified. In addition to a direct effect on neurons, much experimental evidence supports an involvement of the glial component. We assessed methylmercury hydroxide (MeHgOH) toxicity in a glial model, C6 glioma cells, exposed in the 10(-5)-10(-8) M range. The time course of the effects was studied by time-lapse confocal microscopy and supplemented with biochemical data. We have monitored cell viability and proliferation rate, reactive oxygen species (ROS), mitochondrial transmembrane potential, DNA oxidation, energetic metabolism and modalities of cell death. The earliest effect was a measurable ROS generation followed by oxidative DNA damage paralleled by a partial mitochondrial depolarization. The effect on cell viability was dose dependent. TUNEL, caspase activity and real-time morphological observation of calcein-loaded cells showed that apoptosis was the only detectable mode of cell death within this concentration range. N-acetyl-cysteine (NAC) or reduced glutathione (GSH) completely prevent the apoptotic effect of MeHgOH. The lowest effective MeHgOH concentration was 10(-7) M for ROS and DNA OH-adducts generation. The effect of submicromolar concentrations of MeHgOH on C6 cells could be relevant in the developmental neurotoxicity caused by low dose, long-term exposures, such as those of food origin. In addition, we have shown that the same concentrations are effective in the induction of DNA oxidative damage, with further potential pathogenetic implications.     

Protein synthesis-dependent but Bcl-2-independent cytochrome C release in zinc depletion-induced neuronal apoptosis

Previously, we reported that chelation of intracellular zinc with N, N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN)-induced macromolecule synthesis-dependent apoptosis of cultured cortical neurons. According to the current theory of apoptosis, release of mitochondrial cytochrome C into the cytosol is required for caspase activation. In the present study, we examined whether cytochrome C release is dependent on macromolecule synthesis. Exposure of cortical cultures to 2 microM TPEN for 24 hr induced apoptosis as previously described. Fluorescence immunocytochemical staining as well as immunoblots of cell extracts revealed the release of cytochrome C into the cytosol 18-20 hr after the exposure onset. The cytochrome C release was completely blocked by the addition of cycloheximide or actinomycin D. Addition of the caspase inhibitor zVAD-fmk did not attenuate the cytochrome C release, whereas it blocked TPEN-induced apoptosis. Because Bcl-2 has been shown to block cytochrome C release potently, we exposed human neuroblastoma cells (SH-SY5Y) to TPEN. Whereas Bcl-2 overexpression completely blocked both cytochrome C release and apoptosis induced by staurosporine, it attenuated neither induced by TPEN. The present results suggest that, in neurons, macromolecule synthesis inhibitors act upstream of cytochrome C release to block apoptosis and that, in addition to the classical Bcl-2 sensitive pathway, there may exist a Bcl-2-insensitive pathway for cytochrome C release.     

Calcium and reactive oxygen species mediate staurosporine-induced mitochondrial dysfunction and apoptosis in PC12 cells

The bacterial alkaloid staurosporine is widely employed as an inducer of apoptosis in many cell types including neurons. The intracellular cascades that mediate staurosporine-induced apoptosis are largely unknown. Exposure of cultured PC12 cells to staurosporine resulted in a rapid (min) and prolonged (1–6 hr) elevation of intracellular free calcium levels [Ca²⁺]_i, accumulation of mitochondrial reactive oxygen species (ROS), and decreased mitochondrial 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction (1–4 hr). These early events were followed by membrane lipid peroxidation, loss of mitochondrial transmembrane potential, and nuclear apoptotic changes. Treatment of cells with serum or nerve growth factor within 1–2 hr of staurosporine exposure resulted in recovery of [Ca²⁺]_i and ROS levels, and rescued the cells from apoptosis. The increased [Ca²⁺]_i and ROS production were required for staurosporine-induced apoptosis because the intracellular calcium chelator BAPTA and uric acid (an agent that scavenges peroxynitrite) each protected cells against apoptosis. The caspase inhibitor zVAD-fmk and the anti-apoptotic gene product Bcl-2 prevented the sustained [Ca²⁺]_i increase and ROS accumulation induced by staurosporine indicating that caspases act very early in the apoptotic process. Our data indicate that a [Ca²⁺]_i increase is an early and critical event in staurosporine-induced apoptosis that engages a cell death pathway involving ROS production, oxidative stress, and mitochondrial dysfunction. J. Neurosci. Res. 51:293–308, 1998. © 1998 Wiley-Liss, Inc.     

Anti-death properties of TNF against metabolic poisoning: mitochondrial stabilization by MnSOD.

The cytokine tumor necrosis factor (TNF) is toxic to some mitotic cells, but protects cultured neurons from a variety of insults by mechanisms that are unclear. Pretreatment of neurons or astrocytes with TNF caused significant increases in MnSOD activity, and also significantly attenuated 3-nitropropionic acid (3-NP) induced superoxide accumulation and loss of mitochondrial transmembrane potential. In oligodendrocytes, however, MnSOD activity was not increased, and 3-NP toxicity was unaffected by TNF. Genetically engineered PC6 cells that overexpress MnSOD also were resistant to 3-NP-induced damage. TNF pretreatment and MnSOD overexpression prevented 3-NP induced apoptosis, and shifted the mode of death from necrosis to apoptosis in response to high levels of 3-NP. Mitochondria isolated from either MnSOD overexpressing PC6 cells or TNF-treated neurons maintained resistance to 3-NP-induced loss of transmembrane potential and calcium homeostasis, and showed attenuated release of caspase activators. Overall, these results indicate that MnSOD activity directly stabilizes mitochondrial transmembrane potential and calcium buffering ability, thereby increasing the threshold for lethal injury. Additional studies showed that levels of oxidative stress and striatal lesion size following 3-NP administration in vivo are increased in mice lacking TNF receptors.     

Mitochondrial involvement in nitric oxide-induced cellular death in cortical neurons in culture

Nitric oxide (NO) is an unstable molecule with physiological and pathological properties. In brain, NO acts as a modulator of neurotransmission as well as a protector against neuronal death from several death stimuli. However, beside this protector effect, high NO concentrations produce neuronal death by a mechanism in which the caspase pathway is implicated. In this work, we demonstrate that in cortical neurons the NO toxicity is mediated by mitochondrial dysfunction. SNAP, an NO donor, induces apoptosis in these cells because it 1) increases the p53 and 2) induces cytochrome c release and activation of caspase-9 and caspase-3. SNAP also induces necrosis, through 1) breakdown of the mitochondrial membrane potential, 2) ATP decrease, 3) ROS formation, and 4) LDH and ATP release, indicative of oxidative stress and death by necrosis. To sum up, in cortical neurons, high NO concentrations produced cellular death by both an apoptotic and a necrotic mechanism in which the mitochondria are implicated.