PROTEIN DAMAGE & AGGREGATION AFTER BRAIN INJURIES

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 人类的缺血性中风通常会导致急性、迟发性的神经细胞丢失和广泛的慢性行为和认知缺陷。已经建立了几种动物模型来了解缺血后的神经细胞丢失和神经功能障碍，包括全脑缺血和局灶性缺血。大鼠短暂性全脑和局灶性脑缺血在缺血后24-72小时开始，选择性地在部分神经元亚群中产生延迟性神经元细胞死亡，并在缺血后阶段导致神经功能障碍。脑缺血后的突触功能障碍和细胞丢失是有文献记载的。归根结底，中风后神经功能的恢复需要通过突触进行精确的细胞间沟通，这是中风患者的关键问题。因此，对突触的形态、分子和功能变化的研究对于了解缺血性神经元丢失的机制和神经预后具有重要意义。我们最近建立了一系列实验技术来研究脑缺血后突触的变化，并记录了突触的戏剧性超结构、分子和生化改变。在我们的团队和NCMIR的科学家们非常成功和富有成效的合作中，我们开发了一系列基于EPTA细胞化学染色方法的定量和三维(3D)电子显微镜技术，用于研究缺血病理条件下突触的超微结构变化。与显示突触结构变化不大的常规Os-铀-铅染色方法不同，在脑缺血后的脑切片EPTA染色中观察到突触的超微结构发生了强烈的变化。 在使用EPTA染色分析突触结构时，我们注意到，虽然EPTA染色仅限于对照组脑中的突触复合体和核异染色质，但在缺血后的脑中，它显示出广泛的染色遍及细胞质，通常与膜结构有关。染色图案的分布类似于早期电子显微镜研究中对嗜奥米暗物质的描述，但要广泛得多。在一系列研究中，我们确定EPTA染色的材料是高度泛素的，表明这些材料是由聚集的蛋白质组成的。异常蛋白质聚集体已在多种慢性神经退行性疾病中被描述(Soto，2003)。然而，我们的结果首次表明异常蛋白聚集体可能参与了脑缺血后的病理状态。我们最近获得了R01奖金，与NCMIR合作，继续我们对缺血中蛋白质聚集的研究。这项建议包含合作研究，使用EPTA染色和电子断层扫描来调查和量化蛋白质聚集体与蛋白质运输途径的组件，如高尔基体的关联。我们正在继续使用已经建立的EPTA和断层扫描方法进行这些研究，并已经开始获取海马体锥体细胞核周区域的断层数据集，并将我们的分析扩展到其他神经退行性模型，例如作为肌嗜性侧索硬化症模型的超氧化物歧化酶基因敲除小鼠。此外，我们还围绕NCMIR正在开发的新染色技术设计了一组新的实验，以跟踪不同分辨率水平的基因标记蛋白质。特别是，我们将使用基于四半胱氨酸的方法来研究膜蛋白运输的关键限速因子N-乙基马来酰亚胺敏感因子(NSF)在延迟性缺血细胞死亡中的作用。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Ischemic stroke in humans often results in acute, delayed neuronal cell loss and a wide range of chronic behavioral and cognitive deficits. Several animal models have been established to understand neuronal cell loss and neurological deficit after ischemia, including both global and focal ischemia. Transient global and focal ischemia in rat produces delayed neuronal cell death selectively in some populations of neurons, in subpopulations of neurons, beginning 24-72 hours after the ischemic event, and leads to neurological deficit during the post-ischemic phase. Synaptic dysfunction and cell loss after brain ischemia are well documented. Ultimately, recovery of neurological function after stroke requires precise cell-to-cell communication through synapses and is a key issue for stroke patients. Thus, studies of morphological, molecular and functional alterations of synapses are relevant in the understanding of the mechanisms of ischemic neuronal loss and neurological outcome. We have recently established a series of experimental techniques to study synaptic changes after brain ischemia and documented dramatic ultra-structural, molecular and biochemical modifications of synapses. In a highly successful and productive collaboration between our groups and scientists at NCMIR, we developed a series of quantitative and three-dimensional (3D) electron microscopic techniques based on the EPTA cytochemical staining method for the study of synaptic ultrastructural changes in ischemic pathological conditions. In contrast to the conventional osmium-uranium-lead staining method that reveals little change in synaptic structure, robust ultrastructural alterations of the synapse were observed in the EPTA stainings of brain sections after ischemia . When analyzing synaptic structures using EPTA staining, we noted that while EPTA staining was restricted to synaptic complexes and nuclear heterochromatin in control brains, in post-ischemic brains it revealed extensive staining throughout the cytoplasm, often associated with membranous structures. The distribution of the staining pattern was similar to that described for osmiophilic dark materials in early electron microscopic studies , but was much more widespread. In a series of studies, we determined that the EPTA stained materials were highly ubiquinated, suggesting that these materials were composed of aggregated proteins. Abnormal protein aggregates have been described in multiple chronic neurodegenerative disorders (Soto, 2003). However, our results were the first to indicate that abnormal protein aggregates may be involved in the pathological conditions seen after cerebral ischemia. We recently were awarded an R01 grant to continue our investigations of protein aggregation in ischemia in collaboration with NCMIR. This proposal contains collaborative studies to use EPTA staining and electron tomography to investigate and quantify the association of protein aggregates with components of the protein trafficking pathway such as the Golgi apparatus. We are continuing these studies using the EPTA and tomography methods already established and have begun to acquire tomographic data sets of the perinuclear region of hippocampal pyramidal cells and also extend our analysis to additional neurodegenerative models, such as a super oxide dismutase knock out mouse as a model of amyotropic lateral sclerosis. In addition, we have designed a new set of experiments around new staining techniques being developed at NCMIR to follow genetically tagged proteins at different resolution levels. In particular, we will employ tetracysteine-based methods to investigate the role in delayed ischemia cell death of a key rate-limiting factor for membranous protein trafficking, N-ethyl-maleimide-sensitive factor (NSF).