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).
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