Proteomic analysis of synaptosomal protein expression reveals that cerebral ischemia alters lysosomal Psap processing.
Cerebral ischemia (CI) induces dramatic changes in synaptic structure and function that precedes delayed post-ischemic neuronal death. Here, a proteomic analysis was used to identify the effects of focal CI on synaptosomal protein levels. Contralateral and ipsilateral synaptosomes, prepared from adult mice subjected to 60 min middle cerebral artery occlusion, were isolated following 3, 6 and 20 h of reperfusion. Synaptosomal protein samples (n=3) were labeled using the cleavable ICAT system 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 62 differentially expressed proteins were identified and Gene Ontology classification (cellular component) indicated that the majority of the proteins were located in the mitochondria and other components consistent with synaptic localization. The observed alterations in synaptic protein levels poorly correlated with gene expression, indicating the involvement of post-transcriptional regulatory mechanisms in determining post-ischemic synaptic protein content. Additionally, immunohistochemistry analysis of prosaposin (Psap) and saposin C (SapC) indicates that CI disrupts Psap processing and glycosphingolipid metabolism. These results demonstrate that the synapse is adversely affected by CI and may play a role in mediating post-ischemic neuronal viability.
Costain
Willard J
WJ
Glycosyltransferases and Neuroglycomics, Institute for Biological Sciences, National Research Council, Ottawa, ON, Canada. Will.Costain@nrc-cnrc.gc.ca
Haqqani
Arsalan S
AS
Rasquinha
Ingrid
I
Giguere
Marie-Soleil
MS
Slinn
Jacqueline
J
Zurakowski
Bogdan
B
Stanimirovic
Danica B
DB
D000818:Animals; D002545:Brain Ischemia / Q000235:genetics / Q000378:metabolism*; D005786:Gene Expression Regulation; D008247:Lysosomes / Q000737:chemistry* / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D040901:Proteomics; D054789:Ribosome Inactivating Proteins, Type 1 / Q000378:metabolism*; D000076985:Saporins; D013574:Synaptosomes / Q000737:chemistry* / Q000378:metabolism
D054789:Ribosome Inactivating Proteins, Type 1; D000076985:Saporins