Ca2+/calmodulin-dependent protein kinase II alpha subunit (CaMKIIα) is a key neuronal signaling protein and an emerging drug target. The central hub domain regulates the activity of CaMKIIα by organizing the holoenzyme complex into functional oligomers, yet pharmacological modulation of the hub domain has never been demonstrated. Here, using a combination of photoaffinity labeling and chemical proteomics, we show that compounds related to the natural substance γ-hydroxybutyrate (GHB) bind selectively to CaMKIIα. By means of a 2.2-Å x-ray crystal structure of ligand-bound CaMKIIα hub, we reveal the molecular details of the binding site deep within the hub. Furthermore, we show that binding of GHB and related analogs to this site promotes concentration-dependent increases in hub thermal stability believed to alter holoenzyme functionality. Selectively under states of pathological CaMKIIα activation, hub ligands provide a significant and sustained neuroprotection, which is both time and dose dependent. This is demonstrated in neurons exposed to excitotoxicity and in a mouse model of cerebral ischemia with the selective GHB analog, HOCPCA (3-hydroxycyclopent-1-enecarboxylic acid). Together, our results indicate a hitherto unknown mechanism for neuroprotection by a highly specific and unforeseen interaction between the CaMKIIα hub domain and small molecule brain-penetrant GHB analogs. This establishes GHB analogs as powerful tools for investigating CaMKII neuropharmacology in general and as potential therapeutic compounds for cerebral ischemia in particular.

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The α 4/6 βδ-containing GABA A receptors are involved in a number of brain diseases. Despite the potential of a δ-selective imaging agent, no PET radioligand is currently available for in vivo imaging. Here, we report the characterization of DS2OMe ( 1 ) as a candidate radiotracer, 11 C-labeling, and subsequent evaluation of [ 11 C]DS2OMe in a domestic pig as a PET radioligand for visualization of the δ-containing GABA A receptors.

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.

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