N-Terminomic Changes of Neurons During Excitotoxicity Reveal Proteolytic Events Associated with Synaptic Dysfunctions and Inform Potential Targets for Neuroprotection

Abstract

Excitotoxicity is a neuronal death process initiated by over-stimulation of ionotropic glutamate receptors. Although dysregulation of proteolysis and protein phosphorylation signaling networks is critical for excitotoxicity, the identity of affected proteins and mechanisms by which they induce neuronal cell death remains unclear. To address this, we used quantitative N-terminomics to identify proteins modified by proteolysis in neurons undergoing excitotoxic cell death. Our investigation led to the discovery of proteins that, upon proteolysis by calpains, perturb synaptic organization and function. These included key synaptic regulatory proteins including CRMP2, doublecortin-like kinase I, Src tyrosine kinase and calmodulin-dependent protein kinase IIβ (CaMKIIβ), which we found to undergo calpain-catalyzed proteolytic processing to generate stable truncated fragments with altered activities. We further show that blockade of proteolysis of Src by calpains could protect against neuronal loss in a rat model of neurotoxicity, and that CaMKIIβ and its isoform CaMKIIα undergo differential processing by calpains in mouse brains under physiological conditions and during ischemic stroke. Our findings thus reveal new insights into excitotoxic neuronal death mechanisms and suggest potential therapeutic targets for neurological disorders.

CaMKIIα as a Promising Drug Target for Ischemic Grey Matter

“…Recently, another pathological regulatory mechanism of CaMKII was described, which could contribute to the hyperactivity of a subpopulation of CaMKII after ischemia. Specifically, CaMKII was found to be proteolytically processed by the protease calpain after ischemia, which generates a stable kinase fragment with uncontrolled kinase activity (∆CaMKII, 31 kDa) [ 34 , 113 ]. As the cleavage site was mapped to the beginning of the regulatory domain (i.e., M 281 ↓H 282 ), cleavage of CaMKII at this site generates a N-terminal fragment consisting of the kinase domain only ( Figure 4 ).…”

“…Although proteolytic processing of CaMKII by calpain could already be described in vitro in 1989 [ 116 , 117 , 118 ], its relevance in vivo was only recently addressed. Intriguingly, ∆CaMKII has been detected exclusively after ischemic stroke (photothrombotic stroke) [ 113 ]. Furthermore, in vitro experiments showed that proteolytic processing of CaMKII at this site requires prior Ca 2+ -stimulation and Thr286 autophosphorylation [ 113 ], resembling ischemic conditions of CaMKII [ 26 ].…”

“…Intriguingly, ∆CaMKII has been detected exclusively after ischemic stroke (photothrombotic stroke) [ 113 ]. Furthermore, in vitro experiments showed that proteolytic processing of CaMKII at this site requires prior Ca 2+ -stimulation and Thr286 autophosphorylation [ 113 ], resembling ischemic conditions of CaMKII [ 26 ]. Proteolytic processing of CaMKII might thus represent a pathological mechanism downstream of ischemia-activated and Thr286 phosphorylated CaMKII, and the uncontrolled kinase activity of ∆CaMKII might contribute to the neurotoxic role of sustained CaMKII activity after ischemia by phosphorylating specific neuronal proteins to direct cell death.…”

“…This peptide could successfully restore ischemia-mediated downregulation of GABA B receptors and increased neuronal survival after glutamate insult in vitro [ 158 ]. Other intriguing strategies could be to target the interaction of calpain with CaMKII with the goal to prevent ischemia-specific proteolytic processing of CaMKII [ 113 ].…”

“… CaMKII cleavage by calpain after ischemia [ 113 ]. Ischemic conditions lead to overactivation of calpains (red scissors) as well as increased Thr286/287 CaMKII autophosphorylation, facilitating cleavage of CaMKII by overactivated calpains at M 281 ↓H 282 .…”