Reperfusion rather than ischemia drives the formation of ubiquitin aggregates after middle cerebral artery occlusion.
OBJECTIVE
Cerebral ischemia leads to accumulation of ubiquitinated protein aggregates. However, the factors triggering ubiquitination and their impact on the outcome of cerebral ischemia remain poorly understood. Here we investigate the relationship between ubiquitin aggregation and duration of ischemia/reperfusion, infarct volume, and proteasomal activity in a mouse model of focal ischemia.


METHODS
Free ubiquitin and ubiquitin aggregate levels were examined by Western blotting in the mouse neocortex and striatum after different periods of ischemia/reperfusion and permanent ischemia induced by middle cerebral artery occlusion. Infarct volumes were measured in thionin-stained brain sections. Proteasome activity was studied by fluorometric peptidase activity assay.


RESULTS
Following transient ischemia, ubiquitin aggregates were detected in the ipsilateral neocortex and, to a lesser extent, striatum only after induction of reperfusion. In permanent ischemia, no ubiquitin aggregates were found. Shorter ischemic periods producing no or minimal tissue damage (10-15 minutes) resulted in ubiquitin aggregate levels similar to those produced by ischemia resulting in substantial infarction (30 minutes). Proteasomal impairment was greatest in ischemia without reperfusion, in which no ubiquitin aggregates were detected.


CONCLUSIONS
The data demonstrate that reperfusion rather than ischemia leads to the appearance of ubiquitinated aggregates, which form in the absence of major tissue damage and are not correlated with decreased proteasomal peptidase activity. Ubiquitin aggregates may form in potentially viable brain tissue, which may be later recruited into infarction by factors independent of ubiquitination.
Division of Neurobiology, Department of Neurology and Neuroscience, Weill Cornell Medical College, New York, NY 10065, USA.
Karin
K
Hochrainer
Katherine
K
Jackman
Josef
J
Anrather
Costantino
C
Iadecola
D000818:Animals; D015153:Blotting, Western; D001921:Brain / Q000473:pathology; D002545:Brain Ischemia / Q000378:metabolism / Q000473:pathology*; D003342:Corpus Striatum / Q000473:pathology; D005470:Fluorometry; D020244:Infarction, Middle Cerebral Artery / Q000378:metabolism / Q000473:pathology*; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D019579:Neocortex / Q000473:pathology; D010447:Peptide Hydrolases / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D015427:Reperfusion Injury / Q000378:metabolism / Q000473:pathology*; D013347:Subcellular Fractions / Q000378:metabolism / Q000473:pathology; D025801:Ubiquitin / Q000378:metabolism*
D025801:Ubiquitin; D010447:Peptide Hydrolases; D046988:Proteasome Endopeptidase Complex