Apoptosis In Neurodegenerative Disorders

越来越多的证据表明，在一些与年龄相关的疾病中，神经元可能通过一种称为细胞凋亡的程序性细胞死亡形式而死亡。在LNS的主要努力是旨在确定可能触发神经退行性疾病中的神经元凋亡的因素，以及负责执行细胞死亡程序的特定分子级联。-肌萎缩性侧索硬化症（ALS）的新凋亡机制：前列腺凋亡反应-4 （Par-4）是一种在死亡结构域中含有亮氨酸拉链结构域的蛋白，在前列腺癌细胞和海马神经元中被诱导进行凋亡上调。我们在ALS患者的腰椎脊髓样本中发现了更高的Par-4水平，并表明Par-4增加了运动神经元对氧化和兴奋毒性损伤的脆弱性。在其他研究中，我们发现ALS患者和转基因小鼠模型（Cu/Zn-SOD突变小鼠）脊髓鞘脂和胆固醇代谢异常，鞘磷脂、神经酰胺和胆固醇酯水平升高；在ALS小鼠中，这些异常先于临床表型。在ALS患者和小鼠中，氧化应激的增加与脂质改变有关，培养的运动神经元暴露于氧化应激会增加鞘磷脂、神经酰胺和胆固醇酯的积累。神经鞘脂合成的药理抑制可防止神经酰胺、神经鞘磷脂和胆固醇酯的积累，并保护运动神经元免受氧化和兴奋毒性损伤引起的死亡。这些发现提示鞘脂代谢的改变在ALS的发病机制中起关键作用。转录因子NF-kappaB和神经元凋亡：我们发现抑制内质网（ER） d-肌醇1,4,5-三磷酸（IP(3)）门控的钙释放导致培养的大鼠皮质神经元中NF-kappaB dna结合活性降低。当IP(3)受体被阻断时，NF-kappaB对肿瘤坏死因子- α和谷氨酸反应的激活被完全取消，并且tnf - kappab对thapsigargin处理导致ER钙消耗的激活也被IP(3)受体阻断而降低。我们使用无细胞系统进一步研究了IP(3)受体激活和NF-kappaB活性之间的关系。从成年大鼠大脑皮层中分离出富含内质网的微粒体，重悬，并用诱导或抑制内质网钙释放的药物处理。然后将它们重新离心，并将上清加入从同一来源组织分离的细胞质提取物中。我们发现微粒体在激活IP(3)受体或抑制ER Ca(2+)- atp酶时释放nf - kappab刺激信号，但对ryanodine没有反应。对完整细胞和无细胞制剂的研究表明，内质网释放的信号不是钙，而是对热和胰蛋白酶敏感的。我们的数据表明，神经元中组成型和诱导型NF-kappaB结合活性的主要组成部分都需要IP(3)受体的激活，并且内质网腔内钙水平的降低会触发内质网释放扩散性NF-kappaB激活信号。中风中神经元凋亡的机制：中风是长期残疾的主要原因，其严重程度与屈服于缺血性损伤的神经元数量直接相关。我们发现短暂局灶性脑缺血导致野生型小鼠酸性鞘磷脂酶（ASMase）活性、神经酰胺水平和炎症细胞因子的产生大幅增加，而缺乏ASMase的小鼠则没有。在缺乏ASMase的小鼠和用阻断神经酰胺产生的药物治疗的小鼠中，脑组织损伤程度降低，行为结果改善。我们发现，小鼠给予二氮氧化物（线粒体atp敏感钾通道的激活剂）后，大脑中动脉永久性闭塞后皮质梗死面积大幅减少（60%至70%）。二氮氧化合物可减少缺血皮层半暗区神经元凋亡，增加星形胶质细胞存活和活化。二氮氧化物的神经保护作用被5-羟基癸酸酯（线粒体atp敏感钾通道的选择性拮抗剂）所废除。对培养海马神经元的研究表明，二氮氧化物使线粒体去极化，阻止细胞色素c的释放，并保护细胞免受星孢素和化学缺氧诱导的死亡。在凋亡损伤的神经元中，二氮氧化物增加Bcl2水平，抑制Bax与线粒体的关联，表明线粒体atp敏感钾通道的激活可能通过差异调节促凋亡和抗凋亡蛋白来稳定线粒体功能。总的来说，这些数据表明线粒体atp敏感钾通道在缺血条件下调节神经元存活中起关键作用，并确定激活线粒体atp敏感钾通道的药物作为中风和相关神经退行性疾病的潜在治疗药物。DNA损伤反应和神经元凋亡：DNA损伤已被记录在神经退行性疾病中，包括阿尔茨海默病、帕金森病和中风。我们发现DNA损伤发生在受AD和PD相关条件的培养神经元中，包括暴露于淀粉样蛋白-肽和线粒体毒素。当培养的神经元或成年小鼠接受叶酸治疗时，DNA损伤减轻，而同型半胱氨酸则增加DNA损伤。另外的数据表明，同型半胱氨酸会损害神经元修复受损DNA的能力。DNA依赖性蛋白激酶（DNA- pk）参与V(D)J重组和DNA双链断裂修复，可能在DNA损伤引起的细胞死亡中发挥作用。我们发现，培养的缺乏DNA-PK活性的严重联合免疫缺陷（scid）小鼠海马神经元对暴露于拓扑异构酶抑制剂、淀粉样蛋白肽（A β）和谷氨酸诱导的凋亡高度敏感。在卡因酸诱导的癫痫发作后，成年scid小鼠海马CA1和CA3神经元的易感性也出现了类似的增加。我们的研究结果表明，在神经系统疾病可能发生的情况下，DNA-PK活性对神经元的存活很重要。神经发生中的细胞凋亡：神经祖细胞（NPC）可以增殖，分化为神经元或神经胶质细胞，或经历一种称为细胞凋亡的程序性死亡形式。虽然鼻咽癌的死亡发生在神经系统发育和成人中，但其潜在的机制尚不清楚。我们发现一氧化氮可以通过激活p38 MAP激酶、聚（adp -核糖）聚合酶和caspase-3的机制诱导C17.2 NPC死亡。在相关研究中，我们发现脑源性神经营养因子对神经干细胞凋亡具有抑制作用。在另一项研究中，我们发现Numb以一种异构体特异性的方式调节细胞对神经营养因子诱导的分化和细胞生存依赖性的敏感性。含有短PTB的麻木亚型增强了对NGF的分化反应，并通过依赖于内质网储存钙释放的机制增强了对NGF戒断的凋亡反应。这些发现表明，Numb对神经营养因子反应的异构体特异性调节可能在神经系统的发育和可塑性中发挥重要作用。

Increasing evidence suggests that neurons may die by a form of programmed cell death called apoptosis in several age-related disorders. A major effort in the LNS is aimed at identifying the factors that may trigger neuronal apoptosis in neurodegenerative disorders, as well as the specific molecular cascades responsible for executing the cell death program. --Novel Apoptotic Mechanisms in Amyotrophic Lateral Sclerosis (ALS): Prostate apoptosis response-4 (Par-4), a protein containing a leucine zipper domain within a death domain, is up-regulated in prostate cancer cells and hippocampal neurons induced to undergo apoptosis. We have found higher Par-4 levels in lumbar spinal cord samples from patients with ALS, and have shown that Par-4 increases the vulnerability of motor neurons to oxidative and excitotoxic insults. In other studies we have identified abnormalities in sphingolipid and cholesterol metabolism in the spinal cords of ALS patients and in a transgenic mouse model (Cu/Zn-SOD mutant mice), which manifest increased levels of sphingomyelin, ceramides and cholesterol esters; in the ALS mice these abnormalities precede the clinical phenotype. In ALS patients and mice increased oxidative stress occurs in association with the lipid alterations, and exposure of cultured motor neurons to oxidative stress increases the accumulation of sphingomyelin, ceramides and cholesterol esters. Pharmacological inhibition of sphingolipid synthesis prevents accumulation of ceramides, sphingomyelin and cholesterol esters and protects motor neurons against death induced by oxidative and excitotoxic insults. These findings suggest a pivotal role for altered sphingolipid metabolism in the pathogenesis of ALS. --The Transcription Factor NF-kappaB and Neuronal Apoptosis: We have discovered that inhibition of endoplasmic reticulum (ER) d-myo-inositol 1,4,5-trisphosphate (IP(3))-gated calcium release caused decreased basal NF-kappaB DNA-binding activity in cultured rat cortical neurons. Activation of NF-kappaB in response to tumor necrosis factor-alpha and glutamate was completely abolished when IP(3) receptors were blocked, and NF-kappaB activation in response to depletion of ER calcium by thapsigargin treatment was also decreased by IP(3) receptor blockade. We further investigated the relationship between IP(3) receptor activation and NF-kappaB activity using a cell-free system. Microsomes enriched in the ER were isolated from adult rat cerebral cortex, resuspended, and treated with agents that induce or inhibit ER calcium release. They were then recentrifuged, and the supernatant was added to cytoplasmic extract isolated from the same source tissue. We found that microsomes released an NF-kappaB-stimulating signal in response to activation of IP(3) receptors or inhibition of the ER Ca(2+)-ATPase, but not in response to ryanodine. Studies of intact cells and cell-free preparations indicated that the signal released from the ER was not calcium and was heat- and trypsin-sensitive. Our data suggest that activation of IP(3) receptors is required for a major component of both constitutive and inducible NF-kappaB binding activity in neurons and that decreasing ER intraluminal calcium levels triggers release of a diffusible NF-kappaB-activating signal from the ER. --Mechanisms of Neuronal Apoptosis in Stroke: Stroke is a major cause of long-term disability, the severity of which is directly related to the numbers of neurons that succumb to the ischemic insult. We found that transient focal cerebral ischemia induces large increases in acidic sphingomyelinase (ASMase) activity, ceramide levels, and production of inflammatory cytokines in wild-type mice, but not in mice lacking ASMase. The extent of brain tissue damage is decreased and behavioral outcome improved in mice lacking ASMase and in mice treated with a drug that blocks ceramide production. We have found that mice given diazoxide, an activator of mitochondrial ATP-sensitive potassium channels, exhibited a large (60% to 70%) decrease in cortical infarct size after permanent occlusion of the middle cerebral artery. Diazoxide decreases neuronal apoptosis and increases astrocyte survival and activation in the penumbral region of the ischemic cortex. The neuroprotective effect of diazoxide is abolished by 5-hydroxydecanoate, a selective antagonist of mitochondrial ATP-sensitive potassium channels. Studies of cultured hippocampal neurons reveal that diazoxide depolarizes mitochondria, prevents cytochrome c release, and protects cells against death induced by staurosporine and chemical hypoxia. Diazoxide increased the levels of Bcl2 and inhibited the association of Bax with mitochondria in neurons exposed to an apoptotic insult, suggesting that activation of mitochondrial ATP-sensitive potassium channels may stabilize mitochondrial function by differentially modulating proapoptotic and antiapoptotic proteins. Collectively, the data suggest that mitochondrial ATP-sensitive potassium channels play a key role in modulating neuronal survival under ischemic conditions, and identify agents that activate mitochondrial ATP-sensitive potassium channels as potential therapeutics for stroke and related neurodegenerative conditions. --DNA Damage Responses and Neuronal Apoptosis: DNA damage has been documented in neurodegenerative disorders including AD, PD and stroke. We have found that DNA damage occurs in cultured neurons subjected to conditions relevant to AD and PD including exposure to amyloid beta-peptide and mitochondrial toxins. The DNA damage is reduced when cultured neurons or adult mice are treated with folic acid, and the DNA damage is increased by homocysteine. Additional data suggest that homocysteine impairs the ability of the neurons to repair damaged DNA. DNA-dependent protein kinase (DNA-PK) is involved in V(D)J recombination and DNA double strand break repair, and may play a role in cell death induced by DNA damage. We found that cultured hippocampal neurons from severe combined immunodeficient (scid) mice which lack DNA-PK activity are hypersensitive to apoptosis induced by exposure to topoisomerase inhibitors, amyloid beta peptide (A beta) and glutamate. A similar increased vulnerability of hippocampal CA1 and CA3 neurons was observed in adult scid mice after kainate-induced seizures. Our results suggest that DNA-PK activity is important for neuron survival under conditions that may occur in neurological disorders. --Apoptosis in Neurogenesis: Neural progenitor cells (NPC) can proliferate, differentiate into neurons or glial cells, or undergo a form of programmed cell death called apoptosis. Although death of NPC occurs during development of the nervous system and in the adult, the underlying mechanisms are unknown. We discovered that nitric oxide can induce death of C17.2 NPC by a mechanism requiring activation of p38 MAP kinase, poly(ADP-ribose) polymerase and caspase-3. In related studies we have found that brain-derived neurotrophic factor prevents apoptosis of neural stem cells.. In another study we have found that Numb regulates the sensitivity of cells to neurotrophin-induced differentiation and cell survival dependency in an isoform-specific manner. Numb isoforms containing a short PTB enhance the differentiation response to NGF, and enhance apoptosis in response to NGF withdrawal by a mechanism dependent upon release of calcium from endoplasmic reticulum stores. These findings suggest that isoform-specific modulation of neurotrophin responses by Numb may play important roles in the development and plasticity of the nervous system.