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Background: Brain calcium-calmodulin-dependent protein kinase II (CaMKII) is exclusively expressed in neurons, where it localizes to the post-synaptic density (PSD) to link calcium signals to changes in synaptic activity. Independent studies suggest that deregulation of CaMKII contributes to neuronal death associated with cerebral ischemia and could be a target for stroke therapy. However, the molecular events underlying CaMKII activity modification after ischemia are not understood. Cerebral ischemia induces ubiquitination of PSD proteins, raising the possibility that post-ischemic CaMKII function is modulated by ubiquitination. Here we investigate a novel link between CaMKIIα subunit ubiquitination and post-ischemic CaMKII regulation.Methods: Cerebral ischemia was induced in male C57BL6 mice by middle cerebral artery occlusion (MCAO) for 35 min. Whole tissue extracts as well as cytosolic, membrane and PSD fractions were prepared at different times after ischemia to assess CaMKIIα ubiquitination, protein levels and activity. Ubiquitination was reverted with recombinant USP2cc de-ubiquitinase.Results: MCAO induces CaMKIIα ubiquitination specifically in the neocortical PSD fraction peaking at 1-hour reperfusion. The ubiquitination is accompanied by translocation of CaMKIIα from cytosol/membrane to PSD (cytosol/membrane: 61±4%; PSD: 190±6% of sham, P<0.001, n=9/group), while total CaMKIIα protein levels are not affected (98±1% of sham, P=0.767, n=3/group). In the cytosol CaMKII activity normalized to protein content is not changed post-stroke (91±19% of sham; P<0.001, n=9/group), however at the PSD it is severely suppressed (31±6% of sham, P<0.001, n=9/group). De-ubiquitination leads to restoration of PSD-associated CaMKII activity (347±59% of untreated, P<0.001, n=6/group), identifying ubiquitination as reversible repressor of CaMKII activity at the PSD.Conclusions: CaMKIIα ubiquitination induced by cerebral ischemia-reperfusion constitutes a previously unappreciated mechanism of post-ischemic CaMKII regulation that may be important for modulating the ischemic outcome. Better understanding of how post-ischemic CaMKIIα ubiquitination is controlled may help to identify a new therapeutic stroke target.