Apoptosis In Neurodegenerative Disorders

在阿尔茨海默病、帕金森病和亨廷顿病等神经退行性疾病中，神经元可能会因一种称为细胞凋亡的程序性细胞死亡而死亡。 神经科学实验室细胞和分子神经科学部分的一项主要工作旨在确定神经退行性疾病中细胞凋亡的触发因素，以及如何通过针对细胞凋亡过程中的特定分子事件来预防神经元变性。 对培养神经元的研究表明，谷氨酸受体的激活可引起 DNA 短暂损伤，由于钙介导的 DNA 修复蛋白 APE1 上调，这种损伤可迅速修复。 此外，我们还发现了一种线粒体解偶联蛋白（UCP4），它可以通过抑制氧化应激和稳定细胞钙稳态的机制来保护中风和阿尔茨海默病相关模型中的神经元。 我们还确定了脑源性神经营养因子（BDNF）在防止海马干细胞产生的神经元凋亡中的作用，这一发现表明有可能增加大脑替换丢失和受损神经元的能力。 在其他研究中，我们发现新生的神经元对 DNA 损伤诱导的细胞凋亡高度敏感，因为它们的端粒酶和端粒相关蛋白 TRF2 水平较低。临床前研究表明，静脉注射免疫球蛋白和γ-分泌酶抑制剂对中风模型有效。 最近，我们发现神经元表达几种 Toll 样受体，并提供了证据表明其中两种受体（TLR2 和 TLR4）的激活可以在阿尔茨海默病和中风的细胞培养物和动物模型中引发细胞凋亡。 在其他研究中，我们揭示了质膜氧化还原酶在衰老和神经退行性疾病实验模型中保护神经元免受凋亡的重要作用。 我们之前表明，在亨廷顿舞蹈病模型小鼠（亨廷顿舞蹈病小鼠）中，热量限制可以改善亨廷顿舞蹈病（HD）的发病机制并减缓疾病进展。我们现在报道，Sirt1（热量限制的有益代谢作用的调节剂）的过度表达可以保护神经元免受突变体 HTT 毒性，而 Sirt1 的减少会加剧突变体 HTT 毒性。 Sirt1 的过度表达可改善亨廷顿病小鼠的运动功能、减少脑萎缩并减轻突变 HTT 介导的代谢异常。进一步的机制研究表明，Sirt1 可以防止突变体 HTT 诱导的 BDNF 浓度及其受体 TrkB 信号传导的下降，并恢复纹状体中的多巴胺浓度。在亨廷顿病细胞模型中，Sirt1 脱乙酰酶活性是 Sirt1 介导的神经保护所必需的。值得注意的是，我们发现突变型 HTT 与 Sirt1 相互作用并抑制 Sirt1 脱乙酰酶活性，从而导致 Sirt1 底物（例如叉头盒 O3A (Foxo3a)）过度乙酰化，从而抑制其促生存功能。 Sirt1 的过度表达可以抵消突变体 HTT 诱导的脱乙酰酶缺陷，增强 Foxo3a 的脱乙酰化并促进细胞存活。 这些发现表明 Sirt1 在哺乳动物 HD 模型中具有神经保护作用，并为 HD 神经保护策略的开发开辟了新途径。 真核延伸因子 2 (eEF-2) 是蛋白质翻译机制的重要调节因子，它控制核糖体沿着 mRNA 的运动。 eEF-2 的活性受细胞能量状态和营养可用性的变化以及磷酸化和单 ADP 核糖基化等翻译后修饰的调节。然而，神经元细胞应激条件下调节蛋白质翻译的机制尚不清楚。在这里，我们发现，当大鼠海马神经元经历氧化应激（暴露于氢过氧化枯烯引起的脂质过氧化；CH）时，eEF-2 过度磷酸化和核糖基化，导致翻译活性降低。 eEF-2 的降解需要钙蛋白酶的蛋白水解活性，并且伴随着 eEF-2 在核区室中的积累。天然形式和磷酸化形式的 eEF-2 的亚细胞定位分别受 CRM1 和 14.3.3 的影响。在海马神经元中，p53 与非磷酸化（活性）eEF-2 相互作用，但与其磷酸化形式不相互作用。 p53-eEF-2 复合物存在于细胞质和细胞核中，当神经元经历氧化应激时，其丰度会增加。活性 eEF-2 的核定位取决于其与 p53 的相互作用，因为缺乏 p53 的细胞在核区室中含有较少的活性 eEF-2。海马神经元中 eEF-2 的过度表达导致接触 CH 后 eEF-2 的核水平增加并减少细胞死亡。我们的结果揭示了控制 eEF-2 差异亚细胞定位和活性状态的新分子机制，可能会影响氧化应激升高期间神经元的生存状态 核因子E2相关因子2（Nrf2）/抗氧化反应元件（ARE）途径是参与神经保护的重要细胞应激反应途径。我们之前筛选了几种天然植物化学物质，并确定白花丹素是 Nrf2/ARE 通路的新型激活剂，可以保护神经元免受缺血性损伤。在这里，我们将研究扩展到白花丹素的天然和合成衍生物。我们发现 5,8-dimethoxy-1,4-naphthoquinone (naphthazarin) 是 Nrf2/ARE 通路的有效激活剂，上调原代神经元和胶质细胞培养物中 Nrf2 驱动基因的表达，并保护神经元免受谷氨酸诱导的兴奋性毒性。 神经元是终末分化细胞，具有高代谢率和多种与其未分化前体不同的生物学特性。先前的研究表明，核苷酸切除 DNA 修复在有丝分裂后肌肉细胞和神经元中下调。在这里，我们表征了未分化和分化的人类神经细胞的 DNA 损伤敏感性和碱基切除 DNA 修复 (BER) 能力。结果表明，未分化的人 SH-SY5Y 神经母细胞瘤细胞对氧化损伤的敏感性低于分化细胞，部分原因是它们具有强大的 BER 能力，而这种能力在有丝分裂后神经元中严重减弱。分化细胞中 BER 活性的降低与关键长斑 BER 成分、瓣状核酸内切酶-1、增殖细胞核抗原和连接酶 I 的蛋白质水平降低相关。因此，由于增殖神经祖细胞具有更高的 BER 能力，与其神经元分化的后代相比，增殖神经祖细胞在修复 DNA 损伤方面更有效。在一项相关研究中，我们发现缺乏 NEIL1 的小鼠在水迷宫测试中表现出记忆保留受损，但在运动表现、焦虑或恐惧调节测试中没有异常。 NEIL1 缺陷会导致中风局灶性缺血/再灌注模型中脑损伤增加和功能结果缺陷。在缺血大脑的同侧和未受应激的老年 NEIL1 缺陷小鼠的线粒体裂解物中，含有 5-羟基尿嘧啶的气泡基质的切口能力较低。这些结果表明 NEIL1 在学习和记忆以及保护神经元免受缺血性损伤方面发挥着重要作用。

In neurodegenerative disorders such as Alzheimers, Parkinsons and Huntingtons diseases, neurons may die by a form of programmed cell death called apoptosis. A major effort in the Cellular and Molecular Neurosciences section of the Laboratory of Neurosciences is aimed at establishing what triggers apoptosis in neurodegenerative disorders and how neuronal degeneration might be prevented by targeting specific molecular events in the process of apoptosis. Studies of cultured neurons demonstrated that activation of glutamate receptors can induce a transient damage to DNA which is rapidly repaired as the result of calcium-mediated upregulation of the DNA repair protein APE1. In addition, we have identified a mitochondrial uncoupling protein (UCP4) that can protect neurons in models relevant to stroke and Alzheimers disease by a mechanism involving suppression of oxidative stress and stabilization of cellular calcium homeostasis. We have also established roles for brain-derived neurotrophic factor (BDNF) in preventing the apoptosis of neurons produced from stem cells in the hippocampus, a finding that suggests the possibility of increasing the capacity of the brain to replace lost and damaged neurons. In other studies we have found that newly generated neurons are highly sensitive to DNA damage-induced apoptosis because they have low levels of telomerase and the telomere-associated protein TRF2. Preclinical studies have shown that intravenous immunoglobulin and gamma-secretase inhibotors are effective in stroke models. More recently, we have shown that neurons express several toll-like receptors, and have provided evidence that activation of two of these receptors (TLR2 and TLR4) can trigger apoptosis in cell culture and animal models of Alzheimer's disease and stroke. In other studies we have revealed important roles for plasma membrane redox enzymes in protecting neurons against apoptosis in experimental models of aging and neurodegenerative disorders. We previously showed that calorie restriction ameliorated Huntington's disease (HD) pathogenesis and slowed disease progression in mice that model Huntington's disease (Huntington's disease mice). We now report that overexpression of Sirt1, a mediator of the beneficial metabolic effects of calorie restriction, protects neurons against mutant HTT toxicity, whereas reduction of Sirt1 exacerbates mutant HTT toxicity. Overexpression of Sirt1 improves motor function, reduces brain atrophy and attenuates mutant-HTT-mediated metabolic abnormalities in Huntington's disease mice. Further mechanistic studies suggested that Sirt1 prevents the mutant-HTT-induced decline in BDNF concentrations and the signaling of its receptor, TrkB, and restores dopamine concentrations in the striatum. Sirt1 deacetylase activity is required for Sirt1-mediated neuroprotection in Huntington's disease cell models. Notably, we show that mutant HTT interacts with Sirt1 and inhibits Sirt1 deacetylase activity, which results in hyperacetylation of Sirt1 substrates such as forkhead box O3A (Foxo3a), thereby inhibiting its pro-survival function. Overexpression of Sirt1 counteracts the mutant-HTT-induced deacetylase deficit, enhances the deacetylation of Foxo3a and facilitates cell survival. These findings show a neuroprotective role for Sirt1 in mammalian HD models and open new avenues for the development of neuroprotective strategies in HD. Eukaryotic elongation factor 2 (eEF-2) is an important regulator of the protein translation machinery whereby it controls the movement of the ribosome along the mRNA. The activity of eEF-2 is regulated by changes in cellular energy status and nutrient availability and by posttranslational modifications such as phosphorylation and mono-ADP-ribosylation. However, the mechanisms regulating protein translation under conditions of cellular stress in neurons are unknown. Here we show that when rat hippocampal neurons experience oxidative stress (lipid peroxidation induced by exposure to cumene hydroperoxide; CH), eEF-2 is hyperphosphorylated and ribosylated, resulting in reduced translational activity. The degradation of eEF-2 requires calpain proteolytic activity and is accompanied by accumulation of eEF-2 in the nuclear compartment. The subcellular localization of both native and phosphorylated forms of eEF-2 is influenced by CRM1 and 14.3.3, respectively. In hippocampal neurons p53 interacts with nonphosphorylated (active) eEF-2, but not with its phosphorylated form. The p53-eEF-2 complexes are present in cytoplasm and nucleus, and their abundance increases when neurons experience oxidative stress. The nuclear localization of active eEF-2 depends upon its interaction with p53, as cells lacking p53 contain less active eEF-2 in the nuclear compartment. Overexpression of eEF-2 in hippocampal neurons results in increased nuclear levels of eEF-2 and decreased cell death after exposure to CH. Our results reveal novel molecular mechanisms controlling the differential subcellular localization and activity state of eEF-2 that may influence the survival status of neurons during periods of elevated oxidative stress Nuclear factor E2-related factor 2 (Nrf2)/antioxidant response element (ARE) pathway is an important cellular stress response pathway involved in neuroprotection. We previously screened several natural phytochemicals and identified plumbagin as a novel activator of the Nrf2/ARE pathway that can protect neurons against ischemic injury. Here we extended our studies to natural and synthetic derivatives of plumbagin. We found that 5,8-dimethoxy-1,4-naphthoquinone (naphthazarin) is a potent activator of the Nrf2/ARE pathway, up-regulates the expression of Nrf2-driven genes in primary neuronal and glial cultures, and protects neurons against glutamate-induced excitotoxicity. Neurons are terminally differentiated cells with a high rate of metabolism and multiple biological properties distinct from their undifferentiated precursors. Previous studies showed that nucleotide excision DNA repair is downregulated in postmitotic muscle cells and neurons. Here, we characterize DNA damage susceptibility and base excision DNA repair (BER) capacity in undifferentiated and differentiated human neural cells. The results show that undifferentiated human SH-SY5Y neuroblastoma cells are less sensitive to oxidative damage than their differentiated counterparts, in part because they have robust BER capacity, which is heavily attenuated in postmitotic neurons. The reduction in BER activity in differentiated cells correlates with diminished protein levels of key long patch BER components, flap endonuclease-1, proliferating cell nuclear antigen, and ligase I. Thus, because of their higher BER capacity, proliferative neural progenitor cells are more efficient at repairing DNA damage compared with their neuronally differentiated progeny. In a related study we found that mice lacking NEIL1 exhibit impaired memory retention in a water maze test, but no abnormalities in tests of motor performance, anxiety, or fear conditioning. NEIL1 deficiency results in increased brain damage and a defective functional outcome in a focal ischemia/reperfusion model of stroke. The incision capacity on a 5-hydroxyuracil-containing bubble substrate was lower in the ipsilateral side of ischemic brains and in the mitochondrial lysates of unstressed old NEIL1-deficient mice. These results indicate that NEIL1 plays an important role in learning and memory and in protection of neurons against ischemic injury.