ABSTRACT
Ca2+/calmodulin-dependent protein kinase II alpha (CaMKIIα) is a major contributor to physiological and pathological glutamate-mediated Ca2+ signals, and its involvement in various critical cellular pathways demands specific pharmacological strategies. We recently presented γ-hydroxybutyrate (GHB) ligands as the first small molecules selectively targeting and stabilizing the CaMKIIα hub domain. Here, we report that the cyclic GHB analogue 3-hydroxycyclopent-1-enecarboxylic acid (HOCPCA), improves sensorimotor function after experimental stroke in mice when administered at a clinically relevant time and in combination with alteplase. Further, we observed improved hippocampal neuronal activity and working memory after stroke. On the biochemical level, we observed that hub modulation by HOCPCA results in differential effects on distinct CaMKII pools, ultimately alleviating aberrant CaMKII signalling after cerebral ischemia. As such, HOCPCA normalised cytosolic Thr286 autophosphorylation after ischemia in mice and downregulated ischemia-specific expression of a constitutively active CaMKII kinase proteolytic fragment. Previous studies suggest holoenzyme stabilisation as a potential mechanism, yet a causal link to in vivo findings requires further studies. Similarly, HOCPCA's effects on dampening inflammatory changes require further investigation as an underlying protective mechanism. HOCPCA's selectivity and absence of effects on physiological CaMKII signalling highlight pharmacological modulation of the CaMKIIα hub domain as an attractive neuroprotective strategy.
Subject(s)
Sodium Oxybate , Stroke , Mice , Animals , Sodium Oxybate/metabolism , Calcium-Calmodulin-Dependent Protein Kinase Type 2/metabolism , CognitionABSTRACT
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 IIß (CaMKIIß). 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 CaMKIIß, 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.
Subject(s)
Cell Death , Neurons , Synapses , Animals , Male , Mice , Rats , Calpain/metabolism , Cells, Cultured , Cysteine Proteinase Inhibitors/pharmacology , Intercellular Signaling Peptides and Proteins/metabolism , Mice, Inbred C57BL , Nerve Tissue Proteins/metabolism , Neurons/pathology , Neurons/physiology , Neuroprotection , Proteome/analysis , Rats, Wistar , Stroke/pathology , Synapses/pathology , Synapses/physiologyABSTRACT
Ca2+ /calmodulin-dependent protein kinase II alpha (CaMKIIα) is a key regulator of neuronal signaling and synaptic plasticity. Synaptic activity and neurotransmitter homeostasis are closely coupled to the energy metabolism of both neurons and astrocytes. However, whether CaMKIIα function is implicated in brain energy and neurotransmitter metabolism remains unclear. Here, we explored the metabolic consequences of CaMKIIα deletion in the cerebral cortex using a genetic CaMKIIα knockout (KO) mouse. Energy and neurotransmitter metabolism was functionally investigated in acutely isolated cerebral cortical slices using stable 13 C isotope tracing, whereas the metabolic function of synaptosomes was assessed by the rates of glycolytic activity and mitochondrial respiration. The oxidative metabolism of [U-13 C]glucose was extensively reduced in cerebral cortical slices of the CaMKIIα KO mice. In contrast, metabolism of [1,2-13 C]acetate, primarily reflecting astrocyte metabolism, was unaffected. Cellular uptake, and subsequent metabolism, of [U-13 C]glutamate was decreased in cerebral cortical slices of CaMKIIα KO mice, whereas uptake and metabolism of [U-13 C]GABA were unaffected, suggesting selective metabolic impairments of the excitatory system. Synaptic metabolic function was maintained during resting conditions in isolated synaptosomes from CaMKIIα KO mice, but both the glycolytic and mitochondrial capacities became insufficient when the synaptosomes were metabolically challenged. Collectively, this study shows that global deletion of CaMKIIα significantly impairs cellular energy and neurotransmitter metabolism, particularly of neurons, suggesting a metabolic role of CaMKIIα signaling in the brain.
ABSTRACT
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.
ABSTRACT
Ca2+/calmodulin-dependent protein kinase II alpha (CaMKIIα) is a potential target for acute neuroprotection due to its key role in physiological and pathological glutamate signaling. The hub domain organizes the CaMKII holoenzyme into large oligomers, and additional functional effects on holoenzyme activation have lately emerged. We recently reported that compounds related to the proposed neuromodulator γ-hydroxybutyrate (GHB) selectively bind to the CaMKIIα hub domain and increase hub thermal stabilization, which is believed to have functional consequences and to mediate neuroprotection. However, the detailed molecular mechanism is unknown. In this study, we functionally characterize the novel and brain permeable GHB analog (E)-2-(5-hydroxy-2-phenyl-5,7,8,9-tetrahydro-6H-benzo[7]annulen-6-ylidene)acetic acid (Ph-HTBA). Administration of a single dose of Ph-HTBA at a clinically relevant time point (3-6 h after photothrombotic stroke) promotes neuroprotection with a superior effect at low doses compared to the smaller GHB analog 3-hydroxycyclopent-1-enecarboxylic acid (HOCPCA). In contrast to HOCPCA, Ph-HTBA reduces Ca2+-stimulated CaMKIIα Thr286 autophosphorylation in primary cortical neurons and substrate phosphorylation of recombinant CaMKIIα, potentially contributing to its neuroprotective effect. Supported by previous in silico docking studies, we suggest that Ph-HTBA makes distinct molecular interactions with the hub cavity, which may contribute to its differential functional profile and superior neuroprotective effect compared to HOCPCA. Together, this highlights Ph-HTBA as a promising tool to study hub functionality, but also as a good candidate for clinical development.
Subject(s)
Ischemic Stroke , Neuroprotective Agents , Sodium Oxybate , Humans , Ligands , Sodium Oxybate/metabolism , Neuroprotection , Neuroprotective Agents/pharmacology , Calcium-Calmodulin-Dependent Protein Kinase Type 2/metabolism , HoloenzymesABSTRACT
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.
Subject(s)
Calcium-Calmodulin-Dependent Protein Kinase Type 2/metabolism , Sodium Oxybate/metabolism , Binding Sites , Calcium-Calmodulin-Dependent Protein Kinase Type 2/genetics , Carboxylic Acids/pharmacology , Crystallography, X-Ray , Cyclopentanes/pharmacology , Gene Expression Regulation, Enzymologic/drug effects , HEK293 Cells , Humans , Neuroprotection , Protein Binding , Protein Domains , Signal TransductionABSTRACT
γ-Aminobutyric acid (GABA) acting through heteropentameric GABAA receptors plays a pivotal role in the sleep-promoting circuitry. Whereas the role of the different GABAA receptor α-subunits in sleep regulation and in mediating the effect of benzodiazepines for treatment of insomnia is well-described, the ß-subunits are less studied. Here we report the first study characterizing sleep in mice lacking the GABAA receptor ß1 -subunit (ß1-/- mice). We show that ß1-/- mice have a distinct and abnormal sleep phenotype characterized by increased delta power in non-rapid eye movement (NREM) sleep and decreased theta activity in rapid eye movement (REM) sleep compared to ß1+/+ mice, without any change in the overall sleep-wake architecture. From GABAA receptor-specific autoradiography, it is further demonstrated that functional ß1 -subunit-containing GABAA receptors display the highest binding levels in the hippocampus and frontal cortex. In conclusion, this study suggests that the GABAA receptor ß1 -subunit does not play an important role in sleep initiation or maintenance but instead regulates the power spectrum and especially the expression of theta rhythm. This provides new knowledge on the complex role of GABAA receptor subunits in sleep regulation. In addition, ß1-/- mice could provide a useful mouse model for future studies of the physiological role of delta and theta rhythms during sleep.
Subject(s)
Receptors, GABA-A , Sleep, REM , Animals , Electroencephalography , Mice , Mice, Knockout , Receptors, GABA-A/genetics , Sleep , gamma-Aminobutyric AcidABSTRACT
The small-molecule ligand (E)-2-(5-hydroxy-5,7,8,9-tetrahydro-6H-benzo[7]annulen-6-ylidene)acetic acid (NCS-382) is an analogue of γ-hydroxybutyric acid (GHB) and is widely used for probing the brain-specific GHB high-affinity binding sites. To reach these, brain uptake is imperative, and it is therefore important to understand the molecular mechanisms of NCS-382 transport in order to direct in vivo studies. In this study, we hypothesized that NCS-382 is a substrate for the monocarboxylate transporter subtype 1 (MCT1) which is known to mediate blood-brain barrier (BBB) permeation of GHB. For this purpose, we investigated NCS-382 uptake by MCT subtypes endogenously expressed in tsA201 and MDA-MB-231 cell lines in assays of radioligand-based competition and fluorescence-based intracellular pH measurements. To further verify the results, we measured NCS-382 uptake by means of mass spectrometry in Xenopus laevis oocytes heterologously expressing MCT subtypes. As expected, we found that NCS-382 is a substrate for MCT1 with half-maximal effective concentrations in the low millimolar range. Surprisingly, NCS-382 also showed substrate activity at MCT4 as well as uptake in water-injected oocytes, suggesting a component of passive diffusion. In conclusion, transport of NCS-382 across membranes differs from GHB as it also involves MCT4 and/or passive diffusion. This should be taken into consideration when designing pharmacological studies with this compound and its closely related analogues. The combination of MCT assays used here exemplifies a setup that may be suitable for a reliable characterization of MCT ligands in general.
Subject(s)
Benzocycloheptenes/pharmacology , Monocarboxylic Acid Transporters/metabolism , Muscle Proteins/metabolism , Symporters/metabolism , Animals , Cell Line , Humans , Hydroxybutyrates , Ligands , Monocarboxylic Acid Transporters/genetics , Muscle Proteins/genetics , Oocytes/metabolism , Symporters/genetics , Xenopus laevisABSTRACT
The α4/6ßδ-containing GABAA 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 GABAA receptors.
ABSTRACT
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 (Kd), inhibition constants (Ki) as well as the density of binding sites (Bmax) 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.