Speaker: Willard Costain Glycosyltranseferases and Neuroglycomics, National Research Council – Institute for Biological Sciences Proteomic Analysis Of The Temporal Effects Of Cerebral Ischemia On Synaptic Protein Expression Cerebral ischemia induces dramatic and rapidly reversible structural changes in dendritic spine morphology that precede delayed post-ischemic neuronal death. This suggests that synaptic stress may initiate signals that propagate toward the cell body and instigate delayed cell death through a process described as synaptic apoptosis. To elucidate the effects of cerebral ischemia on synaptic function, we performed proteomic analysis of post-ischemic synaptosomes. Synaptosomes were prepared from contralateral and ipsilateral hemispheres from adult male C57 mice subjected to a transient (60 min) middle cerebral artery occlusion (MCAO) followed by 3 - 20 hrs of reperfusion. Synaptosomal protein samples (n=3) were labeled using the cleavable ICAT™ system (Applied Biosystems) prior to analysis with nanoLC-MS/MS. Each sample was analyzed by LC-MS to identify differential expressions using InDEPT software and differentially expressed peptides were identified by targeted LC-MS/MS. A total of 55 proteins were identified and Gene Ontology classification (cellular component) of these proteins indicated that the majority of the proteins were located in the mitochondria, synapse, extracellular space and plasma membrane. Importantly, three glycoproteins (Psap, Negr1 & Gpm6a), known to regulate neurite outgrowth and spine formation, were identified. The observed alterations in synaptic protein levels poorly correlated with gene expression, indicating post-transcriptional regulation of synaptic protein content. IHC experiments, conducted on MCAO treated mouse brain, indicated that Negr1 and Gpm6a exhibited marked alterations in their distribution in the ischemic hippocampus. These results demonstrate that synaptic glycoproteins are affected by cerebral ischemia and may play a role in mediating post-ischemic events.