Calmodulin-dependent Kinase II Research Articles

Enhanced NMDA receptor pathway and glutamate transmission in the hippocampal dentate gyrus mediate the spatial learning and memory impairment of obese rats.

Obesity has been linked with the impairment of spatial memory and synaptic plasticity but the molecular mechanisms remained unidentified. Since glutamatergic transmission and NMDA receptor neural pathways in hippocampal dentate gyrus (DG) are essential in the learning and memory, we aimed to investigate glutamate (Glu) and NMDA receptor signaling of DG in spatial learning and memory in diet-induced obesity (DIO) rats. Spatial learning and memory were assessed via Morris water maze (MWM) test on control (Ctr) and DIO rats. Extracellular concentration of Glu in the DG was determined using in vivo microdialysis and HPLC. The protein expressions of NMDA receptor subunit 2B (NR2B), brain-derived neurotrophic factor (BDNF), the activation of calcium/calmodulin-dependent kinase II (CaMKII) and cAMP-response-element-binding protein (CREB) in the DG were observed by western blot. Spatial learning and memory were impaired in DIO rats compared to those of Ctr. NR2B expression was increased, while BDNF expression and CaMKII and CREB activation were decreased in DG of DIO rats. Extracellular concentration of Glu was increased in Ctr on the 3rd and 4th days of the MWM test, but significant further increment was observed in DIO rats. Microinjection of an NMDA antagonist (MK-801) into the DG reversed spatial learning and memory impairment. Such effects were accompanied by greater BDNF expression and CaMKII/CREB activation in the DG of DIO rats. In conclusion, the enhancement of Glu-NMDA receptor transmission in the hippocampal DG contributes to the impairment of spatial learning and memory in DIO rats, maybe via the modulation of CaMKII-CREB-BDNF signaling pathway.

Temporal-Specific Sex and Injury-Dependent Changes on Neurogranin-Associated Synaptic Signaling After Controlled Cortical Impact in Rats.

Extensive effort has been made to study the role of synaptic deficits in cognitive impairment after traumatic brain injury (TBI). Neurogranin (Ng) is a calcium-sensitive calmodulin (CaM)-binding protein essential for Ca2+/CaM-dependent kinase II (CaMKII) autophosphorylation which subsequently modulates synaptic plasticity. Given the loss of Ng expression after injury, additional research is warranted to discern changes in hippocampal post-synaptic signaling after TBI. Under isoflurane anesthesia, adult, male and female Sprague-Dawley rats received a sham/control or controlled cortical impact (CCI) injury. Ipsilateral hippocampal synaptosomes were isolated at 24h and 1, 2, and 4weeks post-injury, and western blot was used to evaluate protein expression of Ng-associated signaling proteins. Non-parametric Mann-Whitney tests were used to determine significance of injury for each sex at each time point. There were significant changes in the hippocampal synaptic expression of Ng and associated synaptic proteins such as phosphorylated Ng, CaMKII, and CaM up to 4weeks post-CCI, demonstrating TBI alters hippocampal post-synaptic signaling. This study furthers our understanding of mechanisms of cognitive dysfunction within the synapse sub-acutely after TBI.

Abstract C101: Calcium signaling rewiring activates epithelial-to-mesenchymal transition

Abstract Purpose: Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer-related deaths in the United States and is often not diagnosed until it has already metastasized. Calcium and downstream signaling cascades contribute to epithelial-to-mesenchymal transition (EMT) and facilitate metastasis formation in PDAC. Ca2+/calmodulin-dependent kinase-II (CaMK2), a downstream effector kinase activated by Ca2+, has previously been shown in vitro to be an important mediator of a partial/hybrid EMT program that gives tumor cells high metastatic proclivity. We hypothesize that CaMK2 is a critical node in the partial EMT state and thus deleted Camk2b in vivo to study the effect of its loss on tumor progression and metastasis. Methods: We generated a genetically engineered mouse model where Camk2b was knocked out in the KPCY autochthonous PDAC mouse model (herein KPCY-Camk2bcKO). Isolated cell lines were treated with the EMT-inducing ligand TGF-b to study the effect of Camk2b on tumor cell plasticity. Results: Prior in vitro work indicated that CAMK2 is required for hybrid EMT and thus we expected KPCY-Camk2bcKO mice to have fewer metastases and live longer. Surprisingly, the genetic deletion of Camk2b accelerated tumor formation and reduced overall survival relative to KPCY controls. Despite dying sooner, there was no difference in overall tumor burden in KPCY-Camk2bcKO mice. Pathological and immunofluorescence analysis of KPCY-Camk2bcKO tumors showed an increased number of poorly differentiated regions and EMT as indicated by loss of the epithelial cell marker ECADHERIN (E-CAD) in YFP lineage-labeled tumor cells. In stark contrast to prior work, the loss of Camk2b increased tumor cell plasticity, potentially highlighting a rewiring of calcium signaling circuits in the Camk2b-null state. To this end, KPCY-Camk2bcKO tumor cell lines displayed a baseline mesenchymal morphology in vitro and had lower expression of Cdh1 (the gene encoding E-CAD) mRNA. Using a flow cytometry-based approach, we also found baseline E-CAD protein levels to be diminished relative to KPCY lines. Additionally, KPCY-Camk2bcKO tumor cells were more responsive to the EMT-inducing ligand TGF-b and downregulated their E-CAD levels more than KPCY cells. Notably, treatment with low dose TGF-b also dramatically reduced E-CAD expression in KPCY-Camk2bcKO but not KPCY cells, indicating that the knockout cells are easily pushed towards a mesenchymal state. Conclusions: While CAMK2 has been studied previously as a facilitator of the EMT program, this work paradoxically demonstrates that Camk2b loss enhances cellular plasticity within PDAC. In this context, we speculate that loss of Camk2b rewires calcium signaling to drive EMT. In future studies, we will investigate the effect of Camk2b loss on the metastatic cascade and uncover mechanisms through which Camk2b loss makes PDAC tumor cells more plastic. We will also begin to characterize the non-cell autonomous roles of CAMK2B in crosstalk with the tumor microenvironment. Citation Format: Jessica Peura, Yamini Ogoti, Jason Pitarresi. Calcium signaling rewiring activates epithelial-to-mesenchymal transition [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr C101.

Secretoneurin levels are higher in dilated cardiomyopathy than in ischaemic cardiomyopathy: preliminary results.

Secretoneurin (SN) is a neuropeptide with potential utility as a biomarker of cardiovascular episodes. The main effect of SN is mediated through its inhibition of calmodulin-dependent kinase II (CaMKII), which influences calcium handling. We aimed to associate the levels of SN in plasma with different causes of heart failure. We prospectively enrolled consecutive patients with ischaemic (ICM) and dilated (DCM) cardiomyopathy from the outpatient heart failure clinic and healthy individuals. SN was analysed from venous blood by use of the ELISA method. SN plasma levels were compared in DCM, ICM and healthy individuals with non-parametric tests. A total of 53 patients (81.1% male, 18.9% female; mean age 67.9 ± 12.6 years) and 34 healthy individuals (38% male, 62% female) were included in the analysis. Plasma SN levels were significantly higher in the dilated cardiomyopathy (38.8 ± 27 pmol/L) as compared with the ischaemic cardiomyopathy (19.7 ± 22.6 pmol/L) group (P = 0.006). There was no significant difference between females vs. males (27.1 ± 23 vs. 25.5 ± 26.2 pmol/L, P = NS). Plasma SN levels allowed DCM and ICM to be differentiated with 88% sensitivity and 61% specificity (P = 0.007), the cut of value is 13.3 pmol/L. Plasma SN levels differed significantly between healthy volunteers and both ICM (P < 0.0001) and DCM (P = 0.049). Plasma SN levels did not differ according to age and were not associated with comorbidities, left ventricular ejection fraction, heart failure medication, troponin, creatinine, or natriuretic peptide plasma levels. Plasma secretoneurin levels differed significantly in DCM vs. ICM, being higher in the former. Based on plasma SN levels, discrimination between DCM and ICM might be possible. Healthy individuals produce higher SN plasma levels than stable HFrEF patients.

Development of early prediction and discriminating techniques for Lewy body diseases

The advent of a super-aged society poses urgent challenges in overcoming age-related neurological disorders and extending a healthy lifespan. Neurodegenerative diseases such as Alzheimer's disease, dementia with Lewy bodies, and Parkinson's disease are characterized by the accumulation of pathogenic proteins in the brain, leading to the formation of intracellular aggregates known as pathological hallmarks. In the early stages of protein accumulation, before the onset of clinical symptoms such as cognitive impairment or motor dysfunction, brain inflammation begins to occur. Subsequently, neuronal death progresses, and clinical symptoms manifest as dementia or Parkinson's disease. Therefore, there is a need for early prediction of neurodegeneration and the development of disease-modifying drugs for pre-symptomatic prevention. To address this issue, we have focused on enhancing the degradation of amyloid-β protein by targeting Ca2+/calmodulin-dependent kinase II (CaMKII)/proteasome system and on suppressing the propagation and uptake mechanisms of α-synuclein by targeting fatty acid-binding proteins (FABPs) coupled with the long isoform of dopamine D2 (D2L) receptor. Additionally, our analysis of FABP knockout mice has revealed an increased expression of FABPs in the neurodegenerative process, suggesting their involvement in mitochondrial dysfunction and neuronal death. Based on these findings, this article highlights the physiological significance of FABP family proteins in neurodegeneration and discusses the analysis of plasma biomarkers for predicting neurodegenerative disorders and the discriminatory methods for distinguishing between Alzheimer's disease, dementia with Lewy bodies, and Parkinson's disease. Furthermore, we explore the potential of ultra-early prediction of neurodegenerative disorders.

Increased Risk for Atrial Alternans in Rabbit Heart Failure: The Role of Ca2+/Calmodulin-Dependent Kinase II and Inositol-1,4,5-trisphosphate Signaling.

Heart failure (HF) increases the probability of cardiac arrhythmias, including atrial fibrillation (AF), but the mechanisms linking HF to AF are poorly understood. We investigated disturbances in Ca2+ signaling and electrophysiology in rabbit atrial myocytes from normal and failing hearts and identified mechanisms that contribute to the higher risk of atrial arrhythmias in HF. Ca2+ transient (CaT) alternans-beat-to-beat alternations in CaT amplitude-served as indicator of increased arrhythmogenicity. We demonstrate that HF atrial myocytes were more prone to alternans despite no change in action potentials duration and only moderate decrease of L-type Ca2+ current. Ca2+/calmodulin-dependent kinase II (CaMKII) inhibition suppressed CaT alternans. Activation of IP3 signaling by endothelin-1 (ET-1) and angiotensin II (Ang II) resulted in acute, but transient reduction of CaT amplitude and sarcoplasmic reticulum (SR) Ca2+ load, and lowered the alternans risk. However, prolonged exposure to ET-1 and Ang II enhanced SR Ca2+ release and increased the degree of alternans. Inhibition of IP3 receptors prevented the transient ET-1 and Ang II effects and by itself increased the degree of CaT alternans. Our data suggest that activation of CaMKII and IP3 signaling contribute to atrial arrhythmogenesis in HF.

Beta Blockade Prevents Cardiac Morphological and Molecular Remodelling in Experimental Uremia.

Heart failure and chronic kidney disease (CKD) share several mediators of cardiac pathological remodelling. Akin to heart failure, this remodelling sets in motion a vicious cycle of progressive pathological hypertrophy and myocardial dysfunction in CKD. Several decades of heart failure research have shown that beta blockade is a powerful tool in preventing cardiac remodelling and breaking this vicious cycle. This phenomenon remains hitherto untested in CKD. Therefore, we set out to test the hypothesis that beta blockade prevents cardiac pathological remodelling in experimental uremia. Wistar rats had subtotal nephrectomy or sham surgery and were followed up for 10 weeks. The animals were randomly allocated to the beta blocker metoprolol (10 mg/kg/day) or vehicle. In vivo and in vitro cardiac assessments were performed. Cardiac tissue was extracted, and protein expression was quantified using immunoblotting. Histological analyses were performed to quantify myocardial fibrosis. Beta blockade attenuated cardiac pathological remodelling in nephrectomised animals. The echocardiographic left ventricular mass and the heart weight to tibial length ratio were significantly lower in nephrectomised animals treated with metoprolol. Furthermore, beta blockade attenuated myocardial fibrosis associated with subtotal nephrectomy. In addition, the Ca++- calmodulin-dependent kinase II (CAMKII) pathway was shown to be activated in uremia and attenuated by beta blockade, offering a potential mechanism of action. In conclusion, beta blockade attenuated hypertrophic signalling pathways and ameliorated cardiac pathological remodelling in experimental uremia. The study provides a strong scientific rationale for repurposing beta blockers, a tried and tested treatment in heart failure, for the benefit of patients with CKD.

Megalencephalic leukoencephalopathy with subcortical cysts protein-1: A new calcium-sensitive protein functionally activated by endoplasmic reticulum calcium release and calmodulin binding in astrocytes

BackgroundMLC1 is a membrane protein highly expressed in brain perivascular astrocytes and whose mutations account for the rare leukodystrophy (LD) megalencephalic leukoencephalopathy with subcortical cysts disease (MLC). MLC is characterized by macrocephaly, brain edema and cysts, myelin vacuolation and astrocyte swelling which cause cognitive and motor dysfunctions and epilepsy. In cultured astrocytes, lack of functional MLC1 disturbs cell volume regulation by affecting anion channel (VRAC) currents and the consequent regulatory volume decrease (RVD) occurring in response to osmotic changes. Moreover, MLC1 represses intracellular signaling molecules (EGFR, ERK1/2, NF-kB) inducing astrocyte activation and swelling following brain insults. Nevertheless, to date, MLC1 proper function and MLC molecular pathogenesis are still elusive. We recently reported that in astrocytes MLC1 phosphorylation by the Ca2+/Calmodulin-dependent kinase II (CaMKII) in response to intracellular Ca2+ release potentiates MLC1 activation of VRAC. These results highlighted the importance of Ca2+ signaling in the regulation of MLC1 functions, prompting us to further investigate the relationships between intracellular Ca2+ and MLC1 properties. MethodsWe used U251 astrocytoma cells stably expressing wild-type (WT) or mutated MLC1, primary mouse astrocytes and mouse brain tissue, and applied biochemistry, molecular biology, video imaging and electrophysiology techniques. ResultsWe revealed that WT but not mutant MLC1 oligomerization and trafficking to the astrocyte plasma membrane is favored by Ca2+ release from endoplasmic reticulum (ER) but not by capacitive Ca2+ entry in response to ER depletion. We also clarified the molecular events underlining MLC1 response to cytoplasmic Ca2+ increase, demonstrating that, following Ca2+ release, MLC1 binds the Ca2+ effector protein calmodulin (CaM) at the carboxyl terminal where a CaM binding sequence was identified. Using a CaM inhibitor and generating U251 cells expressing MLC1 with CaM binding site mutations, we found that CaM regulates MLC1 assembly, trafficking and function, being RVD and MLC-linked signaling molecules abnormally regulated in these latter cells. ConclusionOverall, we qualified MLC1 as a Ca2+ sensitive protein involved in the control of volume changes in response to ER Ca2+ release and astrocyte activation. These findings provide new insights for the comprehension of the molecular mechanisms responsible for the myelin degeneration occurring in MLC and other LD where astrocytes have a primary role in the pathological process.

Mitochondrial protein prohibitin promotes learning memory recovery in mice following intracerebral hemorrhage via CAMKII/CRMP signaling pathway

Prohibitin (PHB) is a mitochondrial inner membrane protein with neuroprotective, antioxidant, and apoptosis-reducing effects. This study aimed to explore the role of PHB in pathological symptoms, behavioral deficits, and cognitive impairment in a collagenase-IV-induced intracerebral hemorrhage (ICH) murine model. In this study, mice that received collagenase IV injection were pretreated with PHB or saline 21 days prior to modeling. The role of PHB in memory and learning ability was monitored using the Morris water maze, Y-maze, and rotarod, social, startle, and nest-building tests. The effect of PHB on depression-like symptoms was examined using the forced swimming, tail suspension, and sucrose preference tests. Subsequently, mouse samples were analyzed using immunohistochemistry, western blotting, Perls staining, Nissl staining, and gene sequencing. Results showed that collagenase IV significantly induced behavioral deficits, brain edema, cognitive impairment, and depressive symptoms. PHB overexpression effectively alleviated memory, learning, and motor deficits in mice with ICH. PHB markedly inhibited the number of terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling-positive cells and protein levels of ionized calcium-binding adapter molecule 1, glial fibrillary acidic protein, and interleukin-1β in the perihematomal region of ICH mice. PHB overexpression also remarkably promoted production of neurologin1 (NLGL1), and upregulated levels of Ca2+-calmodulin-dependent kinase II (CaMKII) and collapsin response mediator protein-1 (CRMP1) proteins. In conclusion, PHB overexpression can effectively alleviate the neurological deficits and neurodegeneration around the hematoma region. This may play a protective role by upregulating the expression of NLGL1 and promoting expression of CaMKII and CRMP1.

Direct regulation of the cardiac ryanodine receptor (RyR2) by O-GlcNAcylation

BackgroundO-GlcNAcylation is the enzymatic addition of a sugar, O-linked β-N-Acetylglucosamine, to the serine and threonine residues of proteins, and is abundant in diabetic conditions. We have previously shown that O-GlcNAcylation can trigger arrhythmias by indirectly increasing pathological Ca2+ leak through the cardiac ryanodine receptor (RyR2) via Ca2+/calmodulin-dependent kinase II (CaMKII). However, RyR2 is well known to be directly regulated by other forms of serine and threonine modification, therefore, this study aimed to determine whether RyR2 is directly modified by O-GlcNAcylation and if this also alters the function of RyR2 and Ca2+ leak.MethodsO-GlcNAcylation of RyR2 in diabetic human and animal hearts was determined using western blotting. O-GlcNAcylation of RyR2 was pharmacologically controlled and the propensity for Ca2+ leak was determined using single cell imaging. The site of O-GlcNAcylation within RyR2 was determined using site-directed mutagenesis of RyR2.ResultsWe found that RyR2 is modified by O-GlcNAcylation in human, animal and HEK293 cell models. Under hyperglycaemic conditions O-GlcNAcylation was associated with an increase in Ca2+ leak through RyR2 which persisted after CaMKII inhibition. Conversion of serine-2808 to alanine prevented an O-GlcNAcylation induced increase in Ca2+ leak.ConclusionsThese data suggest that the function of RyR2 can be directly regulated by O-GlcNAcylation and requires the presence of serine-2808.

Dysfunction of inhibitory interneurons contributes to synaptic plasticity via GABABR-pNR2B signaling in a chronic migraine rat model.

According to our previous study, the loss of inhibitory interneuron function contributes to central sensitization in chronic migraine (CM). Synaptic plasticity is a vital basis for the occurrence of central sensitization. However, whether the decline in interneuron-mediated inhibition promotes central sensitization by regulating synaptic plasticity in CM remains unclear. Therefore, this study aims to explore the role of interneuron-mediated inhibition in the development of synaptic plasticity in CM. A CM model was established in rats by repeated dural infusion of inflammatory soup (IS) for 7 days, and the function of inhibitory interneurons was then evaluated. After intraventricular injection of baclofen [a gamma-aminobutyric acid type B receptor (GABABR) agonist] or H89 [a protein kinase A (PKA) inhibitor), behavioral tests were performed. The changes in synaptic plasticity were investigated by determining the levels of the synapse-associated proteins postsynaptic density protein 95 (PSD95), synaptophysin (Syp) and synaptophysin-1(Syt-1)]; evaluating the synaptic ultrastructure by transmission electron microscopy (TEM); and determining the density of synaptic spines via Golgi-Cox staining. Central sensitization was evaluated by measuring calcitonin gene-related peptide (CGRP), brain-derived neurotrophic factor (BDNF), c-Fos and substance P (SP) levels. Finally, the PKA/Fyn kinase (Fyn)/tyrosine-phosphorylated NR2B (pNR2B) pathway and downstream calcium-calmodulin-dependent kinase II (CaMKII)/c-AMP-responsive element binding protein (pCREB) signaling were assessed. We observed dysfunction of inhibitory interneurons, and found that activation of GABABR ameliorated CM-induced hyperalgesia, repressed the CM-evoked elevation of synapse-associated protein levels and enhancement of synaptic transmission, alleviated the CM-triggered increases in the levels of central sensitization-related proteins, and inhibited CaMKII/pCREB signaling via the PKA/Fyn/pNR2B pathway. The inhibition of PKA suppressed the CM-induced activation of Fyn/pNR2B signaling. These data reveal that the dysfunction of inhibitory interneurons contributes to central sensitization by regulating synaptic plasticity through the GABABR/PKA/Fyn/pNR2B pathway in the periaqueductal gray (PAG) of CM rats. Blockade of GABABR-pNR2B signaling might have a positive influence on the effects of CM therapy by modulating synaptic plasticity in central sensitization.

Cardiac Protein Kinase D1 ablation alters the myocytes β-adrenergic response

β-adrenergic (β-AR) signaling is essential for the adaptation of the heart to exercise and stress. Chronic stress leads to the activation of Ca2+/calmodulin-dependent kinase II (CaMKII) and protein kinase D (PKD). Unlike CaMKII, the effects of PKD on excitation-contraction coupling (ECC) remain unclear. To elucidate the mechanisms of PKD-dependent ECC regulation, we used hearts from cardiac-specific PKD1 knockout (PKD1 cKO) mice and wild-type (WT) littermates. We measured calcium transients (CaT), Ca2+ sparks, contraction and L-type Ca2+ current in paced cardiomyocytes under acute β-AR stimulation with isoproterenol (ISO; 100 nM). Sarcoplasmic reticulum (SR) Ca2+ load was assessed by rapid caffeine (10 mM) induced Ca2+ release. Expression and phosphorylation of ECC proteins phospholambam (PLB), troponin I (TnI), ryanodine receptor (RyR), sarcoendoplasmic reticulum Ca2+ ATPase (SERCA) were evaluated by western blotting. At baseline, CaT amplitude and decay tau, Ca2+ spark frequency, SR Ca2+ load, L-type Ca2+ current, contractility, and expression and phosphorylation of ECC protein were all similar in PKD1 cKO vs. WT. However, PKD1 cKO cardiomyocytes presented a diminished ISO response vs. WT with less increase in CaT amplitude, slower [Ca2+]i decline, lower Ca2+ spark rate and lower RyR phosphorylation, but with similar SR Ca2+ load, L-type Ca2+ current, contraction and phosphorylation of PLB and TnI. We infer that the presence of PKD1 allows full cardiomyocyte β-adrenergic responsiveness by allowing optimal enhancement in SR Ca2+ uptake and RyR sensitivity, but not altering L-type Ca2+ current, TnI phosphorylation or contractile response. Further studies are necessary to elucidate the specific mechanisms by which PKD1 is regulating RyR sensitivity. We conclude that the presence of basal PKD1 activity in cardiac ventricular myocytes contributes to normal β-adrenergic responses in Ca2+ handling.

Mangiferin depresses vesicular glutamate release in synaptosomes from the rat cerebral cortex by decreasing synapsin I phosphorylation

Mangiferin is a glucosyl xanthone that has been shown to be a neuroprotective agent against brain disorders involving excess glutamate. However, the effect of mangiferin on the function of the glutamatergic system has not been investigated. In this study, we used synaptosomes from the rat cerebral cortex to investigate the effect of mangiferin on glutamate release and identify the possible underlying mechanism. We observed that mangiferin produced a concentration-dependent reduction in the release of glutamate elicited by 4-aminopyridine with an IC50 value of 25 μM. Inhibition of glutamate release was blocked by removing extracellular calcium and by treatment with the vacuolar-type H+-ATPase inhibitor bafilomycin A1, which prevents the uptake and storage of glutamate in vesicles. Moreover, we showed that mangiferin decreased the 4-aminopyridine-elicited FM1-43 release and synaptotagmin 1 luminal domain antibody (syt1-L ab) uptake from synaptosomes, which correlated with decreased synaptic vesicle exocytosis. Transmission electron microscopy in synaptosomes also showed that mangiferin attenuated the 4-aminopyridine-elicited decrease in the number of synaptic vesicles. In addition, antagonism of Ca2+/calmodulin-dependent kinase II (CaMKII) and protein kinase A (PKA) counteracted mangiferin's effect on glutamate release. Mangiferin also decreased the phosphorylation of CaMKII, PKA, and synapsin I elicited by 4-aminopyridine treatment. Our data suggest that mangiferin reduces PKA and CaMKII activation and synapsin I phosphorylation, which could decrease synaptic vesicle availability and lead to a subsequent reduction in vesicular glutamate release from synaptosomes.

Reduced O-GlcNAcylation diminishes cardiomyocyte Ca2+ dependent facilitation and frequency dependent acceleration of relaxation

Ca2+ dependent facilitation (CDF) and frequency dependent acceleration of relaxation (FDAR) are regulatory mechanisms that potentiate cardiomyocyte Ca2+ channel function and increase the rate of Ca2+ sequestration following a Ca2+-release event, respectively, when depolarization frequency increases. CDF and FDAR likely evolved to maintain EC coupling at increased heart rates. Ca2+/calmodulin-dependent kinase II (CaMKII) was shown to be indispensable to both; however, the mechanisms remain to be completely elucidated. CaMKII activity can be modulated by post-translational modifications but if and how these modifications impact CDF and FDAR is unknown. Intracellular O-linked glycosylation (O-GlcNAcylation) is a post-translational modification that acts as a signaling molecule and metabolic sensor. In hyperglycemic conditions, CaMKII was shown to be O-GlcNAcylated resulting in pathologic activity. Here we sought to investigate whether O-GlcNAcylation impacts CDF and FDAR through modulation of CaMKII activity in a pseudo-physiologic setting. Using voltage-clamp and Ca2+ photometry we show that cardiomyocyte CDF and FDAR are significantly diminished in conditions of reduced O-GlcNAcylation. Immunoblot showed that CaMKIIδ and calmodulin expression are increased but the autophosphorylation of CaMKIIδ and the muscle cell-specific CaMKIIβ isoform are reduced by 75% or more when O-GlcNAcylation is inhibited. We also show that the enzyme responsible for O-GlcNAcylation (OGT) can likely be localized in the dyad space and/or at the cardiac sarcoplasmic reticulum and is precipitated by calmodulin in a Ca2+ dependent manner. These findings will have important implications for our understanding of how CaMKII and OGT interact to impact cardiomyocyte EC coupling in normal physiologic settings as well as in disease states where CaMKII and OGT may be aberrantly regulated.

Activation of CB1R alleviates central sensitization by regulating HCN2-pNR2B signaling in a chronic migraine rat model

BackgroundCentral sensitization has been widely accepted as an underlying pathophysiological mechanism of chronic migraine (CM), activation of cannabinoid type-1 receptor (CB1R) exerts antinociceptive effects by relieving central sensitization in many pain models. However, the role of CB1R in the central sensitization of CM is still unclear.MethodsA CM model was established by infusing inflammatory soup (IS) into the dura of male Wistar rats for 7 days, and hyperalgesia was assessed by the mechanical and thermal thresholds. In the periaqueductal gray (PAG), the mRNA and protein levels of CB1R and hyperpolarization-activated cyclic nucleotide-gated cation channel 2 (HCN2) were measured by qRT–PCR and western blotting. After intraventricular injection of Noladin ether (NE) (a CB1R agonist), ZD 7288 (an HCN2 blocker), and AM 251 (a CB1R antagonist), the expression of tyrosine phosphorylation of N-methyl-D-aspartate receptor subtype 2B (pNR2B), calcium-calmodulin-dependent kinase II (CaMKII), and phosphorylated cAMP-responsive element binding protein (pCREB) was detected, and central sensitization was evaluated by the expression of calcitonin gene-related peptide (CGRP), c-Fos, and substance P (SP). Synaptic-associated protein (postsynaptic density protein 95 (PSD95) and synaptophysin (Syp)) and synaptic ultrastructure were detected to explore synaptic plasticity in central sensitization.ResultsWe observed that the mRNA and protein levels of CB1R and HCN2 were both significantly increased in the PAG of CM rats. The application of NE or ZD 7288 ameliorated IS-induced hyperalgesia; repressed the pNR2B/CaMKII/pCREB pathway; reduced CGRP, c-Fos, SP, PSD95, and Syp expression; and inhibited synaptic transmission. Strikingly, the application of ZD 7288 relieved AM 251-evoked elevation of pNR2B, CGRP, and c-Fos expression.ConclusionsThese data reveal that activation of CB1R alleviates central sensitization by regulating HCN2-pNR2B signaling in CM rats. The activation of CB1R might have a positive influence on the prevention of CM by mitigating central sensitization.

Autophosphorylation of αCaMKII regulates alcohol consumption by controlling sedative effects of alcohol and alcohol-induced loss of excitatory synapses.

Calcium/calmodulin-dependent kinase II (CaMKII) is a key enzyme at the glutamatergic synapses. CAMK2A gene variants have been linked with alcohol use disorder (AUD) by an unknown mechanism. Here, we looked for the link between αCaMKII autophosphorylation and the AUD aetiology. Autophosphorylation-deficient heterozygous αCaMKII mutant mice (T286A+/- ) were trained in the IntelliCages to test the role of αCaMKII activity in AUD-related behaviours. The glutamatergic synapses morphology in CeA was studied in the animals drinking alcohol using 3D electron microscopy. We found that T286A+/- mutants consumed less alcohol and were more sensitive to sedating effects of alcohol, as compared to wild-type littermates (WT). After voluntary alcohol drinking, T286A+/- mice had less excitatory synapses in the CeA, as compared to alcohol-naive animals. This change correlated with alcohol consumption was not reversed after alcohol withdrawal and not observed in WT mice. Our study suggests that αCaMKII autophosphorylation affects alcohol consumption by controlling sedative effects of alcohol and preventing synaptic loss in the individuals drinking alcohol. This finding advances our understanding of the molecular processes that regulate alcohol dependence.

Experiment and Simulation Reveal Residue Details for How Target Binding Tunes Calmodulin's Calcium-Binding Properties.

We aim to elucidate the molecular mechanism of the reciprocal relation of calmodulin's (CaM) target binding and its affinity for calcium ions (Ca2+), which is central to decoding CaM-dependent Ca2+ signaling in a cell. We employed stopped-flow experiments and coarse-grained molecular simulations that learn the coordination chemistry of Ca2+ in CaM from first-principle calculations. The associative memories as part of the coarse-grained force fields built on known protein structures further influence CaM's selection of its polymorphic target peptides in the simulations. We modeled the peptides from the Ca2+/CaM-binding domain of Ca2+/CaM-dependent kinase II (CaMKII), CaMKIIp (293-310) and selected distinctive mutations at the N-terminus. Our stopped-flow experiments have shown that the CaM's affinity for Ca2+ in the bound complex of Ca2+/CaM/CaMKIIp decreased significantly when Ca2+/CaM bound to the mutant peptide (296-AAA-298) compared to that bound to the wild-type peptide (296-RRK-298). The coarse-grained molecular simulations revealed that the 296-AAA-298 mutant peptide destabilized the structures of Ca2+-binding loops at the C-domain of CaM (c-CaM) due to both loss of electrostatic interactions and differences in polymorphic structures. We have leveraged a powerful coarse-grained approach to advance a residue-level understanding of the reciprocal relation in CaM, that could not be possibly achieved by other computational approaches.

Differential changes in cyclic adenosine 3'-5' monophosphate (cAMP) effectors and major Ca2+ handling proteins during diabetic cardiomyopathy.

Diabetic cardiomyopathy (DCM) is associated with differential and time-specific regulation of β-adrenergic receptors and cardiac cyclic nucleotide phosphodiesterases with consequences for total cyclic adenosine 3'-5' monophosphate (cAMP) levels. We aimed to investigate whether these changes are associated with downstream impairments in cAMP and Ca2+ signalling in a type 1 diabetes (T1D)-induced DCM model. T1D was induced in adult male rats by streptozotocin (65 mg/kg) injection. DCM was assessed by cardiac structural and molecular remodelling. We delineated sequential changes affecting the exchange protein (Epac1/2), cAMP-dependent protein kinase A (PKA) and Ca2+ /Calmodulin-dependent kinase II (CaMKII) at 4, 8 and 12 weeks following diabetes, by real-time quantitative PCR and western blot. Expression of Ca2+ ATPase pump (SERCA2a), phospholamban (PLB) and Troponin I (TnI) was also examined. Early upregulation of Epac1 transcripts was noted in diabetic hearts at Week 4, followed by increases in Epac2 mRNA, but not protein levels, at Week 12. Expression of PKA subunits (RI, RIIα and Cα) remained unchanged regardless of the disease stage, whereas CaMKII increased at Week 12 in DCM. Moreover, PLB transcripts were upregulated in diabetic hearts, whereas SERCA2a and TnI gene expression was unchanged irrespective of the disease evolution. PLB phosphorylation at threonine-17 was increased in DCM, whereas phosphorylation of both PLB at serine-16 and TnI at serine-23/24 was unchanged. We show for the first time differential and time-specific regulations in cardiac cAMP effectors and Ca2+ handling proteins, data that may prove useful in proposing new therapeutic approaches in T1D-induced DCM.

Differential roles of CaMKII isoforms in phase separation with NMDA receptors and in synaptic plasticity

Calcium calmodulin-dependent kinase II (CaMKII) is critical for synaptic transmission and plasticity. Two major isoforms of CaMKII, CaMKIIα and CaMKIIβ, play distinct roles in synaptic transmission and long-term potentiation (LTP) with unknown mechanisms. Here, we show that the length of the unstructured linker between the kinase domain and the oligomerizing hub determines the ability of CaMKII to rescue the basal synaptic transmission and LTP defects caused by removal of both CaMKIIα and CaMKIIβ (double knockout [DKO]). Remarkably, although CaMKIIβ binds to GluN2B with a comparable affinity as CaMKIIα does, only CaMKIIα with the short linker forms robust dense clusters with GluN2B via phase separation. Lengthening the linker of CaMKIIα with unstructured "Gly-Gly-Ser" repeats impairs its phase separation with GluN2B, and the mutant enzyme cannot rescue the basal synaptic transmission and LTP defects of DKO mice. Our results suggest that the phase separation capacity of CaMKII with GluN2B is critical for its cellular functions in the brain.

Neuronal circuits within the homing pigeon hippocampal formation.

The current study aimed to reveal in detail patterns of intrahippocampal connectivity in homing pigeons (Columba livia). In light of recent physiological evidence suggesting differences between dorsomedial and ventrolateral hippocampal regions and a hitherto unknown laminar organization along the transverse axis, we also aimed to gain a higher-resolution understanding of the proposed pathway segregation. Both in vivo and high-resolution in vitro tracing techniques were employed and revealed a complex connectivity pattern along the subdivisions of the avian hippocampus. We uncovered connectivity pathways along the transverse axis that started in the dorsolateral hippocampus and continued to the dorsomedial subdivision, from where information was relayed to the triangular region either directly or indirectly via the V-shaped layers. The often-reciprocal connectivity along these subdivisions displayed an intriguing topographical arrangement such that two parallel pathways could be discerned along the ventrolateral (deep) and dorsomedial (superficial) aspects of the avian hippocampus. The segregation along the transverse axis was further supported by expression patterns of the glial fibrillary acidic protein and calbindin. Moreover, we found strong expression of Ca2+ /calmodulin-dependent kinase IIα and doublecortin in the lateral but not medial V-shape layer, indicating a difference between the two V-shaped layers. Overall, our findings provide an unprecedented, detailed description of avian intrahippocampal pathway connectivity, and confirm the recently proposed segregation of the avian hippocampus along the transverse axis. We also provide further support for the hypothesized homology of the lateral V-shape layer and the dorsomedial hippocampus with the dentate gyrus and Ammon's horn of mammals, respectively.