Elemental And Structural Organization Of Neurons And Glia

神经元和神经胶质细胞的元素和结构组织

• 批准号: 8342197
• 负责人: S BRIAN Andrews
• 金额: $ 129.93万
• 依托单位: NATIONAL INSTITUTE OF NEUROLOGICAL DISORDERS AND STROKE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8342197
• 关键词: Accounting; Alzheimer' s Disease; BCL2 gene; Bipolar Disorder; Borderline Personality Disorder; Bronchopulmonary Dysplasia; Calcium; Calcium Channel; Calcium Signaling; Cations; Cell Death; Cell physiology; Cells; Computer Simulation; Disease; Endoplasmic Reticulum; Energy-Filtering Transmission Electron Microscopy; Event; Functional disorder; Gene Expression; Glucose; Glutamate Receptor; Glutamates; Goals; Hippocampus (Brain); Homeostasis; Huntington Disease; Image; Imaging technology; Injury; Ischemia; Maps; Measurement; Mediating; Mitochondria; Mitochondrial Swelling; Modeling; Molecular; N-Methyl-D-Aspartate Receptors; N-Methylaspartate; Neurodegenerative Disorders; Neuroglia; Neuronal Injury; Neurons; Organelles; Outcome; Oxygen; Parkinson Disease; Pathway interactions; Patients; Physiology; Play; Precipitation; Proteins; Receptor Activation; Recovery; Relative (related person); Resolution; Risk; Role; Route; Signal Transduction; Single Nucleotide Polymorphism; Stimulus; Stroke; Techniques; Time; Toxic effect; Variant; Work; Zinc; base; deprivation; excitotoxicity; interest; mitochondrial dysfunction; novel; novel therapeutics; prevent; receptor; research study; voltage

The NMDA subtype of the glutamate receptor plays important and diverse roles in CNS function. Mitochondrial calcium (Ca2+) overload and subsequent dysfunction, due to excessive Ca2+ entry through glutamate over-activated NMDA receptors (NMDARs), are crucial early events in excitotoxic injury. However, the generally disappointing patient outcomes for anti-excitotoxic therapies targeted to NMDARs strongly suggest that factors beyond the activation of these receptors are at play. This information prompts searches for other important Ca2+-dependent injury pathways. Recent progress and the Specific Aims of ongoing work are summarized next. Aim #1: To determine whether NMDAR activation is selectively toxic or whether equivalent calcium loading through voltage-gated calcium channels could be equally toxic. Although NMDARs clearly play the preeminent role in excitotoxic Ca2+ loading, evidence from our lab and others indicates that alternative routes of Ca2+ entry, for example, through voltage-gated calcium channels (VGCCs), can contribute significantly to toxicity in developmentally mature neurons. Thus, we find that in most neurons in hippocampal and cortical cultures even maximal VGCC activation does not promote significant cell death because it induces much lower calcium elevations than does toxic NMDAR activation. In a small subset of neurons, however, depolarization evokes much stronger calcium elevations, approaching those induced by toxic NMDA. These neurons are characterized by elevated expression of VGCCs, which leads to enhanced Ca2+ loading, mitochondrial dysfunction and cell death. The results indicate that neuronal vulnerability tracks the extent of Ca2+ loading, thereby reinforcing the idea that toxicity does not explicitly depend on the route of Ca2+ entry. Aim #2: To elucidate the cellular basis for the loss of calcium homeostatis in Bipolar Disorder. Bipolar Disorder is characterized by altered intracellular calcium homeostasis. The molecular mechanisms underlying this abnormality have been variously proposed to involve dysfunctions in endoplasmic reticulum (ER) and/or mitochondria calcium transport, potentially mediated by Bcl-2, a key protein that interacts with these organelles to regulate Ca2+ signaling. This study examined the effects of the Bcl-2 gene single nucleotide polymorphism (SNP) rs956572 on intracellular Ca2+ dynamics in patients with Bipolar Disorder. We observed increased basal cytosolic Ca2+ levels in risk variant AA and a general inverse correlation with Bcl-2 levels across all SNP variants. Measurements of intraluminal ER Ca2+ concentrations and release rates showed that Bcl-2 exerts its effect in variant AA cells by directly targeting ER Ca2+ release through the InsP3 receptor. The results reveal a critical mechanism by which abnormal Bcl-2 gene expression in a BPD-associated SNP variant leads to dysfunctional calcium dynamics. Aim #3: To determine the role of zinc in glutamate excitotoxicity and oxygen-glucose deprivation. Elevation of cellular zinc (Zn2+) concentrations following transient ischemia contributes to neuronal injury, but the mechanism(s) of Zn2+ toxicity remain unclear. Recent experiments reveal that Zn2+ can enter neurons via VGCCs, and that both Ca2+ and Zn2+ accumulate in mitochondria and contribute to mitochondrial swelling. Zinc co-precipitation with calcium within mitochondria, as well as zinc-induced mitochondrial swelling in Ca2+-free medium, suggest the possibility that mitochondrial dysfunction may be an early step in the mechanism of Zn2+ toxicity. We plan to further evaluate the relative contribution of Zn2+ and Ca2+ to mitochondrial dysfunction during glutamate-induced toxicity, with the near-term goal of understanding the interplay between the two cations. Aim #4: To apply a novel imaging technology one that quantitatively maps intracellular calcium at the single organelle level -- to elucidate the role of intramitochondrial calcium precipitate formation in Ca2+ signaling. Recently we have collaboratively developed an energy filtering transmission electron microscopy (EFTEM) technique capable of acquiring quantitative, megapixel images of intracellular calcium with subcellular resolution. This approach is being used to study in sympathetic neurons the functional significance of the intramitochondrial calcium-rich precipitates that form during depolarization-induced mitochondrial Ca2+ accumulation and persist for many minutes after repolarization. This issue is important because sustained post-stimulus cytosolic Ca2+ elevations contemporaneous with repolarization-induced mitochondrial Ca2+ release critically regulate many cell functions like gene expression, but the factors that control the time course of elevated Ca2+ are controversial. EFTEM calcium maps now indicate that the duration of mitochondrial Ca2+ release after repolarization is much longer than predicted by standard computational models. We find that this prolongation can be explained by considering the aforementioned mitochondrial calcium precipitates in an expanded model. In the current iteration of the model, discharge of the precipitates maintains mitochondrial Ca2+ release at a rate that appropriately accounts for the time course of Ca2+ recovery.

谷氨酸受体的NMDA亚型在中枢神经系统功能中起着重要而多样的作用。由于谷氨酸过度激活的NMDA受体（NMDARs）导致的线粒体钙（Ca 2+）超载和随后的功能障碍是兴奋性毒性损伤的关键早期事件。然而，靶向NMDAR的抗兴奋性毒性疗法的患者结局普遍令人失望，这强烈表明这些受体激活以外的因素在起作用。这一信息提示搜索其他重要的Ca 2+依赖性损伤途径。下面总结了最近的进展和正在进行的工作的具体目标。 目标一：确定NMDAR激活是否具有选择性毒性或通过电压门控钙通道的等效钙负荷是否具有同等毒性。 虽然NMDAR在兴奋性毒性Ca 2+负荷中发挥着突出的作用，但我们实验室和其他实验室的证据表明，Ca 2+进入的替代途径，例如通过电压门控钙通道（VGCC），可以显著促进发育成熟神经元的毒性。因此，我们发现，在大多数神经元在海马和皮层文化，即使是最大的VGCC激活不促进显着的细胞死亡，因为它诱导低得多的钙升高比毒性NMDAR激活。然而，在一小部分神经元中，去极化引起更强的钙升高，接近毒性NMDA诱导的钙升高。这些神经元的特征在于VGCC的表达升高，这导致Ca 2+负荷增强、线粒体功能障碍和细胞死亡。结果表明，神经元的脆弱性跟踪的程度Ca 2+负载，从而加强了这样的想法，即毒性并不明确依赖于Ca 2+进入的路线。 目的#2：阐明双相情感障碍中钙稳态丧失的细胞基础。 双相情感障碍的特征是细胞内钙稳态改变。这种异常背后的分子机制已经被各种各样地提出涉及内质网（ER）和/或线粒体钙转运功能障碍，可能由Bcl-2介导，Bcl-2是一种与这些细胞器相互作用以调节Ca 2+信号的关键蛋白。本研究探讨了Bcl-2基因单核苷酸多态性（SNP）rs 956572对双相情感障碍患者细胞内Ca 2+动力学的影响。我们观察到增加的基础细胞质Ca 2+水平的风险变异AA和一般的反相关性与Bcl-2水平在所有的SNP变异。腔内ER Ca 2+浓度和释放速率的测量表明，Bcl-2通过InsP 3受体直接靶向ER Ca 2+释放而在变体AA细胞中发挥其作用。结果揭示了一个关键机制，通过该机制，BPD相关SNP变体中的异常Bcl-2基因表达导致钙动力学功能障碍。 目标#3：确定锌在谷氨酸兴奋性毒性和氧-葡萄糖剥夺中的作用。 短暂性脑缺血后细胞内锌（Zn 2+）浓度升高可导致神经元损伤，但Zn 2+毒性机制尚不清楚。最近的实验表明，Zn 2+可以通过VGCC进入神经元，并且Ca 2+和Zn 2+都在线粒体中积累并导致线粒体肿胀。线粒体内的锌与钙共沉淀，以及锌诱导的线粒体肿胀中的Ca 2 +-无介质，表明线粒体功能障碍的可能性，可能是一个早期步骤的锌毒性机制。我们计划进一步评估Zn 2+和Ca 2+在谷氨酸诱导的毒性过程中对线粒体功能障碍的相对贡献，近期目标是了解这两种阳离子之间的相互作用。 目标4：应用一种新的成像技术，在单个细胞器水平上定量绘制细胞内钙-阐明线粒体内钙沉淀形成在Ca 2+信号转导中的作用。 最近，我们合作开发了一种能量过滤透射电子显微镜（EFTEM）技术，能够获得定量的，百万像素的细胞内钙的亚细胞分辨率的图像。这种方法被用来研究交感神经元的线粒体内富含钙的沉淀物，在去极化诱导的线粒体Ca 2+的积累和持续复极后许多分钟的功能意义。这个问题是重要的，因为持续的刺激后胞浆Ca 2+升高与复极化诱导的线粒体Ca 2+释放同时严重调节许多细胞功能，如基因表达，但控制Ca 2+升高的时间过程的因素是有争议的。EFTEM钙图现在表明，复极化后线粒体Ca 2+释放的持续时间比标准计算模型预测的要长得多。我们发现，这种延长可以解释考虑上述线粒体钙沉淀在一个扩展的模型。在模型的当前迭代中，沉淀物的放电以适当地解释Ca 2+恢复的时间过程的速率维持线粒体Ca 2+释放。