PROTEIN DAMAGE & AGGREGATION AFTER BRAIN INJURIES

该子项目是利用NIH/NCRR资助的中心赠款提供的资源的许多研究子项目之一。子项目和研究者（PI）可能从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。所列机构为中心，不一定是研究者所在机构。人类缺血性中风通常会导致急性、迟发性神经元细胞丢失以及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元细胞损失和神经功能缺损，包括全局和局灶性缺血。大鼠的短暂性全脑和局灶性缺血在缺血事件后24-72小时开始在某些神经元群体、神经元亚群中选择性地产生延迟的神经元细胞死亡，并导致缺血后阶段的神经功能缺损。脑缺血后的突触功能障碍和细胞丢失是有据可查的。最终，中风后神经功能的恢复需要通过突触进行精确的细胞间通信，这对中风患者来说是一个关键问题。因此，突触的形态学、分子和功能改变的研究对于理解缺血性神经元损失和神经学结果的机制是相关的。我们最近建立了一系列的实验技术来研究脑缺血后突触的变化，并记录了戏剧性的突触超微结构，分子和生化修饰。在我们的小组和科学家之间的一个非常成功和富有成效的合作在NCMIR，我们开发了一系列的定量和三维（3D）电子显微镜技术的基础上EPTA细胞化学染色方法的突触超微结构的变化在缺血性病理条件下的研究。 与传统的锇-铀-铅染色方法相比，该方法揭示突触结构的变化很小，在缺血后脑切片的EPTA染色中观察到突触的强烈超微结构改变。当使用EPTA染色分析突触结构时，我们注意到，虽然EPTA染色仅限于对照脑中的突触复合体和核异染色质，但在缺血后脑中，它显示了整个细胞质的广泛染色，通常与膜结构相关。染色模式的分布与早期电子显微镜研究中描述的嗜锇性深色物质相似，但更广泛。在一系列的研究中，我们确定EPTA染色的材料是高度泛素化的，这表明这些材料是由聚集的蛋白质组成的。已在多种慢性神经退行性疾病中描述了异常蛋白质聚集体（Soto，2003）。然而，我们的研究结果首次表明，异常蛋白质聚集体可能参与脑缺血后的病理状态。我们最近获得了R 01资助，继续与NCMIR合作研究缺血中的蛋白质聚集。该提案包含使用EPTA染色和电子断层扫描的合作研究，以调查和量化的蛋白质聚集体与蛋白质运输途径的组成部分，如高尔基体的协会。我们正在继续这些研究，使用EPTA和断层扫描方法已经建立，并已开始获取海马锥体细胞核周区的断层扫描数据集，并将我们的分析扩展到其他神经退行性模型，如超氧化物歧化酶敲除小鼠作为肌萎缩侧索硬化症的模型。 此外，我们设计了一套新的实验，围绕新的染色技术正在开发的NCMIR在不同的分辨率水平以下的基因标记的蛋白质。特别是，我们将采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).