In vitro autoradiography aims to visualize the anatomical distribution of a protein of interest in tissue from experimental animals as well as humans. The method is based on the specific binding of a radioligand to its biological target. Therefore, frozen tissue sections are incubated with radioligand solution, and the binding to the target is subsequently localized by the detection of radioactive decay, for example, by using photosensitive film or phosphor imaging plates. Resulting digital autoradiograms display remarkable spatial resolution, which enables quantification and localization of radioligand binding in distinct anatomical structures. Moreover, quantification allows for the pharmacological characterization of ligand affinity by means of dissociation constants (K_d), inhibition constants (K_i) as well as the density of binding sites (B_max) in selected tissues. Thus, the method provides information about both target localization and ligand selectivity. Here, the technique is exemplified with autoradiographic characterization of the high-affinity γ-hydroxybutyric acid (GHB) binding sites in mammalian brain tissue, with special emphasis on methodological considerations regarding the binding assay parameters, the choice of the radioligand and the detection method.

Excitotoxicity, a neuronal death process in neurological disorders such as stroke, is initiated by the overstimulation of ionotropic glutamate receptors. Although dysregulation of proteolytic signaling networks is critical for excitotoxicity, the identity of affected proteins and mechanisms by which they induce neuronal cell death remain unclear. To address this, we used quantitative N-terminomics to identify proteins modified by proteolysis in neurons undergoing excitotoxic cell death. We found that most proteolytically processed proteins in excitotoxic neurons are likely substrates of calpains, including key synaptic regulatory proteins such as CRMP2, doublecortin-like kinase I, Src tyrosine kinase and calmodulin-dependent protein kinase IIβ (CaMKIIβ). Critically, calpain-catalyzed proteolytic processing of these proteins generates stable truncated fragments with altered activities that potentially contribute to neuronal death by perturbing synaptic organization and function. Blocking calpain-mediated proteolysis of one of these proteins, Src, protected against neuronal loss in a rat model of neurotoxicity. Extrapolation of our N-terminomic results led to the discovery that CaMKIIα, an isoform of CaMKIIβ, undergoes differential processing in mouse brains under physiological conditions and during ischemic stroke. In summary, by identifying the neuronal proteins undergoing proteolysis during excitotoxicity, our findings offer new insights into excitotoxic neuronal death mechanisms and reveal potential neuroprotective targets for neurological disorders.

Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) is a major mediator of Ca²⁺-dependent signaling pathways in various cell types throughout the body. Its neuronal isoform CaMKIIα (alpha) centrally integrates physiological but also pathological glutamate signals directly downstream of glutamate receptors and has thus emerged as a target for ischemic stroke. Previous studies provided evidence for the involvement of CaMKII activity in ischemic cell death by showing that CaMKII inhibition affords substantial neuroprotection. However, broad inhibition of this central kinase is challenging because various essential physiological processes like synaptic plasticity rely on intact CaMKII regulation. Thus, specific strategies for targeting CaMKII after ischemia are warranted which would ideally only interfere with pathological activity of CaMKII. This review highlights recent advances in the understanding of how ischemia affects CaMKII and how pathospecific pharmacological targeting of CaMKII signaling could be achieved. Specifically, we discuss direct targeting of CaMKII kinase activity with peptide inhibitors versus indirect targeting of the association (hub) domain of CaMKIIα with analogues of γ-hydroxybutyrate (GHB) as a potential way to achieve more specific pharmacological modulation of CaMKII activity after ischemia.

The α_4/6βδ-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, ¹¹C-labeling, and subsequent evaluation of [¹¹C]DS2OMe in a domestic pig as a PET radioligand for visualization of the δ-containing GABA_A receptors.

γ‐Hydroxybutyric acid (GHB) analogs are small molecules that bind competitively to a specific cavity in the oligomeric CaMKIIα hub domain. Binding affects conformation and stability of the hub domain, which may explain the neuroprotective action of some of these compounds. Here, we describe molecular details of interaction of the larger‐type GHB analog 2‐(6‐(4‐chlorophenyl)imidazo[1,2‐b]pyridazine‐2‐yl)acetic acid (PIPA). Like smaller‐type analogs, PIPA binding to the CaMKIIα hub domain promoted thermal stability. PIPA additionally modulated CaMKIIα activity under sub‐maximal CaM concentrations and ultimately led to reduced substrate phosphorylation. A high‐resolution X‐ray crystal structure of a stabilized CaMKIIα (6x mutant) hub construct revealed details of the binding mode of PIPA, which involved outward placement of tryptophan 403 (Trp403), a central residue in a flexible loop close to the upper hub cavity. Small‐angle X‐ray scattering (SAXS) solution structures and mass photometry of the CaMKIIα wild‐type hub domain in the presence of PIPA revealed a high degree of ordered self‐association (stacks of CaMKIIα hub domains). This stacking neither occurred with the smaller compound 3‐hydroxycyclopent‐1‐enecarboxylic acid (HOCPCA), nor when Trp403 was replaced with leucine (W403L). Additionally, CaMKIIα W403L hub was stabilized to a larger extent by PIPA compared to CaMKIIα hub wild type, indicating that loop flexibility is important for holoenzyme stability. Thus, we propose that ligand‐induced outward placement of Trp403 by PIPA, which promotes an unforeseen mechanism of hub domain stacking, may be involved in the observed reduction in CaMKIIα kinase activity. Altogether, this sheds new light on allosteric regulation of CaMKIIα activity via the hub domain.