Voltage-dependent Calcium Channel Subunit Research Articles

Landscape of extrachromosomal circular DNAs, transcriptome, and proteome analysis reveals insights into alcoholic liver cirrhosis

Alcoholic liver cirrhosis (ALC) is a result of excessive and chronic alcohol consumption. Because alchol can cause DNA damage, extrachromosomal circular DNA (eccDNA) was investigated in ALC liver due to it can be a result of DNA damage. Considering eccDNA has ability to lead to genomic instability as an enhancer of gene transcription, we utilized Circle-Seq to identify differences in eccDNA profiles and gene expression patterns in liver samples obtained from ALC patients (n = 3) and healthy controls (n = 3) to investigate the role of eccDNA in the development of ALC. The abundance of eccDNA in ALC (mean = 13,349) were higher than the healthy control (mean = 11,557) without significant difference (pvalue = 0.6530). We observed 1,032 eccDNA containing genes showed higher expression in ALC patients compared to healthy controls (p < 0.05, log2FC > 1). Notably, we discovered seven genes that exhibited a significant positive correlation between eccDNA abundance and gene expression levels. These genes include A disintegrin and metalloproteinase with thrombospondin motifs 2 (ADAMTS2), Voltage-dependent L-type calcium channel subunit alpha-1C (CACNA1C), Protein TANC1 (TANC1), Integrin alpha-2 (ITGA2), EH domain-containing protein 4 (EHD4), Phosphofurin acidic cluster sorting protein 1 (PACS1), and Neuron navigator 2 (NAV2). Through mass spectrometry proteomics, ITGA2 were found to have significantly higher abbudance in ALC. Integrins are a family of proteins plays key roles in the fibrosis development of liver. Thus, our study opens a new perspective for liver fibrosis development.

Proteomic analysis of blood serum in bipolar disorder

IntroductionBipolar disorder (BD) often has symptoms similar to other mental disorders (BD), and there are no paraclinical criteria for differential diagnosis. (Geoffroy et al. Bip Dis 2017; 5 7). Published work on MD proteomics is scarce and focused on schizophrenia. (Dmitrieva et al. PeerJ. 2022; 10 e13907). Therefore, it is important to study potential biomarkers of BD using easily accessible material—blood serum (Rhee et al. Transl Psy 2023; 13 44). Identification of proteins involved in the pathogenesis of BD will help in the study of the pathogenetic mechanisms of BD, the development of differential diagnostic methods and pathogenetically based drugs.ObjectivesCarrying out a comparative proteomic analysis of blood serum from patients with BD and healthy individuals to identify potential biomarkersMethodsWe analyzed the protein spectrum of the blood serum of 14 patients with BD who were admitted during a depressive episode at the age of 32 [21;52] years with a disease duration of 8[5;11] years. The control group consisted of 10 mentally and somatically healthy individuals corresponding to the gender and age of the BD group. Blood serum was purified from 14 major proteins using affinity chromatography and separated by electrophoresis using the Laemmli method. After trypsinolysis, proteins were identified using HPLC/mass spectrometry on an Orbitrap instrument. Mass spectrometric analysis was performed on the Advanced Mass Spectrometry Core Facility of Skolkovo Institute of Science and Technology. Protein identification was carried out using the UniProtKB database using the Mascot search engine. The results were tested for significance using the nonparametric Fisher exact test with Yates correction.ResultsIn patients with BD, qualitative mass spectrometry revealed differential expression of 21 neurospecific proteins. Among them: Protein dispatched homolog 3, Ceroid-lipofuscinosis neuronal protein 6, SWI/SNF complex subunit SMARCC1, Neurogenic differentiation factor 4, Protein furry homolog-like, REST corepressor 1 – are involved in the proliferation, development and differentiation of neurons; Hemicentin-2, Dystrophin, Voltage-dependent L-type calcium channel subunit alpha-1D, Syntaxin-binding protein 5, Small conductance calcium-activated potassium channel protein 1– participate in synaptic transmission of ion transport and form receptors.ConclusionsStudying the role of these proteins in BD and their quantitative content in a larger number of patients is promising. This will help in the development of new diagnostic criteria and targets for drug therapy for BD.Support by the Russian Science Foundation grant No. 23-75-00023.Disclosure of InterestNone Declared

Potential application of bisoprolol derivative compounds as antihypertensive drugs: synthesis and in silico study.

Two bisoprolol derivatives, N-acetyl bisoprolol and N-formyl bisoprolol, belonging to the beta-blocker class of antihypertensive drugs, were synthesized using acetylation and formylation reactions. The yields of the reactions were determined to be 32.40% for N-acetyl bisoprolol and 20.20% for N-formyl bisoprolol. In silico methods such as molecular docking, molecular dynamics simulation and SwissADME prediction were employed to evaluate the potential of these bisoprolol derivatives as antihypertensive drugs. These methods were used to assess the interaction between the bisoprolol derivatives and various receptors associated with hypertension, including human angiotensin I-converting enzyme (PDB ID: 1O8A), renin (PDB ID: 2V0Z), beta-1 adrenergic receptors (PDB ID: 4BVN, 7BVQ), voltage-dependent L-type calcium channel subunit alpha-1S (PDB ID: 6JP5) and mineralocorticoid receptor (PDB ID: 6L88). Our results demonstrated the highest binding energy when bisoprolol and its derivatives bound to 4BVN, with binding energy values of 6.74 kcal mol-1, 7.03 kcal mol-1 and 7.63 kcal mol-1 for bisoprolol, N-acetyl bisoprolol and N-formyl bisoprolol, respectively. The stability of these complexes was confirmed by molecular dynamics simulations, with a root-mean-square deviation value of approximately 2. Furthermore, the SwissADME results indicated that both derivatives exhibited similar properties to the reference drug bisoprolol.

Evaluation of trans- and cis-4-[18F]Fluorogabapentin for Brain PET Imaging.

Gabapentin, a selective ligand for the α2δ subunit of voltage-dependent calcium channels, is an anticonvulsant medication used in the treatment of neuropathic pain, epilepsy, and other neurological conditions. We recently described two radiofluorinated derivatives of gabapentin (trans-4-[18F]fluorogabapentin, [18F]tGBP4F, and cis-4-[18F]fluorogabapentin, [18F]cGBP4F) and showed that these compounds accumulate in the injured nerves in a rodent model of neuropathic pain. Given the use of gabapentin in brain diseases, here we investigate whether these radiofluorinated derivatives of gabapentin can be used for imaging α2δ receptors in the brain. Specifically, we developed automated radiosynthesis methods for [18F]tGBP4F and [18F]cGBP4F and conducted dynamic PET imaging in adult rhesus macaques with and without preadministration of pharmacological doses of gabapentin. Both radiotracers showed very high metabolic stability, negligible plasma protein binding, and slow accumulation in the brain. [18F]tGBP4F, the isomer with higher binding affinity, showed low brain uptake and could not be displaced, whereas [18F]cGBP4F showed moderate brain uptake and could be partially displaced. Kinetic modeling of brain regional time-activity curves using a metabolite-corrected arterial input function shows that a one-tissue compartment model accurately fits the data. Graphical analysis using Logan or multilinear analysis 1 produced similar results as compartmental modeling, indicating robust quantification. This study advances our understanding of how gabapentinoids work and provides an important advancement toward imaging α2δ receptors in the brain.

Mechanism of Lingbao Huxin Dan in the treatment of bradyarrhythmia complicated with coronary heart disease: a network pharmacology analysis.

To investigate the mechanism of action of the Lingbao Huxin Dan in treating bradycardia arrhythmia with coronary heart disease (BA-CHD) by network pharmacology. The active ingredients of the Lingbao Huxin Dan were screened on the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform and Bioinformatics tools designed for the analysis of molecular mechanisms of Chinese medicine platform; target prediction was conducted with the SwissTargetPrediction database, and Cytoscape 3.8 was used to construct a drug ingredient-target network. The Genecards, Online Mendelian Inheritance in Man, and DrugBank databases were searched for disease targets. Venn plots were used to display the common targets of BA-CHD and active ingredients. The STRING platform was used to construct a protein-protein interaction network. The Metascape data platform was used for Gene Ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis to construct a signaling pathway network of the active ingredients of the Lingbao Huxin Dan. There were 121 active ingredients, 899 related targets, 39 targets important in BA-CHD and 14 targets which intersected between the active ingredients and BA-CHD. There were 27 core therapeutic ingredients, 153 biological processes, 18 cell ingredients and 20 molecular functions obtained by GO enrichment analysis. The KEGG pathway analysis yielded 19 signaling pathways. RBA-CHD may treat BA-CHD by regulating adrenergic receptor beta-1, alpha 1-α adrenergic receptor, calcium voltage-gated channel subunit alpha1 C, alpha-1β-adrenergic receptor, nitric oxide synthase 2, beta-2 adrenergic receptor, voltage-dependent calcium channel subunit alpha-2/delta-1, an- giotensin-converting enzyme, Raf-1 proto-oncogene serine/threonine-protein kinase, and other targets, potentially by affecting adrenergic receptor binding and calcium channel opening, to regulate the activity of cardiomyocytes.

Effect of semaglutide and empagliflozin on cognitive function and hippocampal phosphoproteomic in obese mice.

Objective: Based on the 4D label-free phosphoproteomic technique, we examined the differences in cognitive function and hippocampal phosphorylated protein expression in high-fat diet-induced obese mice after the intervention of semaglutide and empagliflozin, as well as the effects of both on protein activity and function in obese mice's hippocampal tissues and the signaling pathways involved. Methods: Thirty-two C57BL/6JC male mice were assigned to two groups randomly: A control group (group C, 10% of energy is from fat, n = 8) and a high-fat diet group (group H, 60% of energy is from fat, n = 24). The high-fat diet-induced obese mice were screened after 12weeks of feeding based on the criterion that the bodyweight of mice in fat rich diet group was greater than or equal to 20% of the average body weight of the mice in the blank control group. Group H separate into group H (n = 8), group Semaglutide (group S, n = 8), and group empagliflozin (group E, n = 8). For a total of 12weeks, group S received 30nmol/kg/d bodyweight of semaglutide intraperitoneally, group E received 10mg/kg/d bodyweight of empagliflozin via gavage, and groups C and H received equal amounts of saline by intraperitoneal injection and gavage. At the end of treatment, the mice were appraised for cognitive function employing the Morris water maze (MWM), and serum fasting glucose, lipids, and inflammatory parameters were measured. The 4D label-free phosphoproteomics method was employed to screen the differential phosphoproteins and loci in hippocampal tissues of mice in different treatment groups, and bioinformatics was used to analyze the biological processes, signaling pathways, and related protein-protein interaction (PPI) network analysis of these differentially phosphorylated proteins. Results: In comparison to normal controls, The escape latency of obese mice induced by high-fat diet was prolonged, the percentage of swimming time in the target quadrant was reduced, and the number of times of crossing the platform was reduced, whereas semaglutide and empagliflozin treatment reduced escape latency, increase the percentage of swim time in the target quadrant and increase the frequency of passing through the platform area, although there is little difference in the effect of the two drugs. The phosphoproteomic results showed 20,493 unique phosphorylated peptides, representing 21,239 phosphorylation sites and 4,290 phosphorylated proteins. Further analysis revealed that the proteins corresponding to these differentially phosphorylated sites are jointly distributed in signaling pathways such as dopaminergic synapses and axon guidance, and are involved in biological processes such as neuronal projection development, synaptic plasticity, and axonogenesis. Notably, the key factors voltage-dependent L-type calcium channel subunit alpha-1D (CACNA1D), voltage-dependent P/Q-type calcium channel subunit alpha-1A (CACNA1A), and voltage-dependent N-type calcium channel subunit alpha-1B (CACNA1B) were all found to be involved in the dopaminergic synapse pathway, and their expression was upregulated by semaglutide and empagliflozin. Conclusion: We found for the first time that a high-fat diet decreased CACNA1D, CACNA1A, and CACNA1B protein serine phosphorylation, which may affect neuronal development, synaptic plasticity, and cognitive function in mice. Notably, semaglutide and empagliflozin increased the phosphorylation of these proteins.

Lack of panmixia of Bothnian Bay vendace - Implications for fisheries management

Overexploitation of fisheries is recognized as a major environmental and socioeconomic problem that threats biodiversity and ecosystem functioning. Inappropriate management policies of fish stocks have been applied as a consequence of inadequate characterization of subtle genetic structure in many fish species. In this study, we aim to assess the extent of genetic differentiation and structure of vendace (Coregonus albula) collected from eight locations in the Bothnian Bay, the northernmost part of the Baltic Sea. Specifically, we test if this species forms a single panmictic population or is divided into several genetically distinct units. We used restriction site-associated DNA sequencing (RAD-seq) to identify 21,792 SNPs based on 266 individuals. We identified a clear pattern of genetic differentiation between River Kalix and the other sampling locations, and a weak structuring between samples from Swedish and Finnish coast. Outlier analysis detected 41 SNPs putatively under divergent selection, mainly reflecting the divergence between River Kalix and the other samples. The outlier SNPs were located within or near 25 genes, including voltage-dependent calcium channel subunit alpha-2 (CACNA2D2), cadherin 26 (CDH26) and carbonic anhydrase 4-like (CA4) that have earlier been associated with salt-tolerance and salinity stress. Our study provides the first genome-wide perspective on genetic structuring of Baltic Sea vendace and rejects the hypothesis of panmixia in the Bothnian Bay. As such, our work demonstrates the power of RAD-sequencing to detect low but significant genetic structuring relevant for fisheries management.

Development of a PET radioligand for α2δ-1 subunit of calcium channels for imaging neuropathic pain

Neuropathic pain affects 7–10% of the adult population. Being able to accurately monitor biological changes underlying neuropathic pain will improve our understanding of neuropathic pain mechanisms and facilitate the development of novel therapeutics. Positron emission tomography (PET) is a noninvasive molecular imaging technique that can provide quantitative information of biochemical changes at the whole-body level by using radiolabeled ligands. One important biological change underlying the development of neuropathic pain is the overexpression of α2δ-1 subunit of voltage-dependent calcium channels (the target of gabapentin). Thus, we hypothesized that a radiolabeled form of gabapentin may allow imaging changes in α2δ-1 for monitoring the underlying pathophysiology of neuropathic pain. Here, we report the development of two 18F-labeled derivatives of gabapentin (trans-4-[18F]fluorogabapentin and cis-4-[18F]fluorogabapentin) and their evaluation in healthy rats and a rat model of neuropathic pain (spinal nerve ligation model). Both isomers were found to selectively bind to the α2δ-1 receptor with trans-4-[18F]fluorogabapentin having higher affinity. Both tracers displayed around 1.5- to 2-fold increased uptake in injured nerves over the contralateral uninjured nerves when measured by gamma counting ex vivo. Although the small size of the nerves and the signal from surrounding muscle prevented visualizing these changes using PET, this work demonstrates that fluorinated derivatives of gabapentin retain binding to α2δ-1 and that their radiolabeled forms can be used to detect pathological changes in vitro and ex vivo. Furthermore, this work confirms that α2δ-1 is a promising target for imaging specific features of neuropathic pain.

Abnormal levels of mitochondrial Ca2+ channel proteins in plasma neuron-derived extracellular vesicles of early schizophrenia.

Structural alterations or quantitative abnormalities of some mitochondrial ion channels and exchangers are associated with altered neuronal functions and increased susceptibility to mental illness. Here we have assessed levels of functionally prominent mitochondrial calcium ion channel proteins in plasma neuron-derived extracellular vesicles (NDEVs) of living patients with first episodes of psychosis (FP) and matched controls (Cs). NDEVs were enriched with an established method of precipitation and immunoabsorption by anti-human CD171 neural adhesion protein (L1CAM) antibody and extracted proteins quantified with ELISAs. CD81 exosome marker-normalized NDEV levels of leucine zipper EF-hand containing transmembrane 1 protein (LETM1), transient receptor potential cation channel subfamily M, member 4 (TRPM4), and solute carrier family 8 member B1 (SLC24A6) or mitochondrial Na+ /Ca2+ exchanger (NCLX) were significantly lower for FP patients (n=10) than Cs (n=10), whereas NDEV levels of voltage-dependent L-type calcium channel subunit α-1C (CACNA-1C) were significantly higher for FP patients than Cs. Abnormal structures or mitochondrial levels of LETM1, NCLX, and CACNA-1C have been linked through analyses of individual proteins, genome-wide association studies, and whole exome protein-coding sequence studies to neurodevelopmental disorders, mental retardation, schizophrenia, and major depressive diseases. A greater understanding of the altered calcium homeostasis in schizophrenia, that is attributable to underlying mitochondrial calcium channel abnormalities, will lead to improved diagnosis and treatment.

175P Mutations and clinical significance of calcium voltage-gated channel subunit alpha 1E (CACNA1E) in non-small cell lung cancer

CACNA1E is a gene encoding the ion-conducting α1 subunit of R-type voltage-dependent calcium channels. We previously showed that CACNA1E was significantly mutated in patients with non-small cell lung cancer (NSCLC) who were long-term exposed to household air pollution, with a mutation rate of 19% (15 of 79 cases). However, the roles for CACNA1E to play in lung tumorigenesis remain largely unknown.

Time-related accurate investigation of multicomponent drug interventions in heart failure

BackgroundChronic heart failure (CHF) is one of the most common disabling, painful, and deadly syndromes. As a syndrome, heart failure (HF) has been divided into more specific processes, and these processes may occur at the same time or have a precedence. In addition, drug intervention in the process of HF has the most appropriate time point, which means that it is not always the best time to intervene with this drug during the entire course of HF. Traditional Chinese medicine (TCM) Dengzhan Shengmai capsule (DZSMC), a TCM formula, is known as a multitarget complementary medicine that is widely used clinically to treat HF in Asian countries. However, because the mechanism by which DZSMCs treat HF has not been demonstrated comprehensively, the uncertainty of the occasion of drug use makes the precise intervention of DZSMCs a challenge. MethodsIn this article, for precise research on the time point of DZSMC intervention during the process of HF, we applied the time-related proteomics data we measured combined with proteomics data reported and online database to perform a precise prediction to learn DZSMC intervention in specific progression and time points of HF. Then the precise prediction was validation in rats after ligation of left anterior descending coronary artery. ResultsWe demonstrate that DZSMC can improve the cardiac function of myocardial infarction (MI) rats by raising the cardiac ejection fraction (EF) and slowing down the thinning of the anterior wall of the left ventricle. In addition, we found that the appropriate time point for DZSMC intervention is when the serum level of voltage-dependent calcium channel subunit alpha-2/delta-2 (Cacna2d2) increases and that DZSMCs play their role in delaying HF mainly by reducing the myocardial load through decreasing Cacna2d2. ConclusionIn this study, we used a multidimensional data integration strategy by combining the proteomics data and bioinformatic tools to explore the suitable time points for DZSMC intervention in HF induced by MI on a rat model. We verified the efficiency of DZSMC in treating HF and found that DZSMC can improve HF by increasing the cardiac EF, FS and retarding the thinning of the anterior wall of the left ventricle by echocardiography. Futhermore, the upregulated Cacna2d2 in 3 weeks post MI could be a potential signal for DZSMC intervention and DZSMC can downregulate the abnormal decrease of Cacna2d2 in 4weeks post MI. This study provides a practical and feasible method for determining a suitable time point for TCM intervention, and this is the first report of DZSMCs decreasing the serum level of Cacna2d2 to improve cardiac function.

Mutations and clinical significance of calcium voltage-gated channel subunit alpha 1E (CACNA1E) in non-small cell lung cancer

CACNA1E is a gene encoding the ion-conducting α1 subunit of R-type voltage-dependent calcium channels, whose roles in tumorigenesis remain to be determined. We previously showed that CACNA1E was significantly mutated in patients with non-small cell lung cancer (NSCLC) who were long-term exposed to household air pollution, with a mutation rate of 19% (15 of 79 cases). Here we showed that CACNA1E was also mutated in 207 (12.8%) of the 1616 patients with NSCLC in The Cancer Genome Atlas (TCGA) datasets. At mRNA and protein levels, CACNA1E was elevated in tumor tissues compared to counterpart non-tumoral lung tissues in NSCLCs of the public datasets and our settings, and its expression level was inversely associated with clinical outcome of the patients. Overexpression of wild type (WT) or A275S or R249G mutant CACNA1E transcripts promoted NSCLC cell proliferation with activation of epidermal growth factor receptor (EGFR) signaling pathway, whereas knockdown of this gene exerted inhibitory effects on NSCLC cells in vitro and in vivo. CACNA1E increased current density and Ca2+ entrance, whereas calcium channel blockers inhibited NSCLC cell proliferation. These data indicate that CACNA1E is required for NSCLC cell proliferation, and blockade of this oncoprotein may have therapeutic potentials for this deadly disease.

Input-dependent synaptic suppression by pregabalin in the central amygdala in male mice with inflammatory pain

Pregabalin (PGB) is a synthetic amino acid compound most widely prescribed for chronic peripheral and central neuropathic pain. PGB is a ligand for the α2δ1 subunit of voltage-dependent calcium channels, and its binding reduces neurotransmitter release and thus inhibits synaptic transmission. The central nucleus of the amygdala (CeA) is a kernel site for the enhanced nociception-emotion link in chronic pain. The nociceptive information is conveyed to the CeA via the following two pathways: 1) the pathway arising from the basolateral amygdala (BLA), which carries nociceptive information mediated by the thalamocortical system, and 2) that arising from the external part of the pontine lateral parabrachial nucleus (LPB), that forms the final route of the spino-parabrachio-amygdaloid pathway that conveys nociceptive information directly from the superficial layer of the spinal dorsal horn. We compared the effects of PGB on the excitatory postsynaptic currents of neurons in the right CeA in response to electrical stimulation of BLA and LPB pathways using the whole-cell patch-clamp technique. Inflammatory pain was induced by intraplantar injection of formalin solution at the left hind paw.At eight hours post-formalin, PGB reduced EPSCs amplitude of the BLA-to-CeA synaptic transmission, accompanied by a significant increase in the PPR, suggesting a decreased release probability from the presynaptic terminals. In addition, these effects of PGB were only seen in inflammatory conditions. PGB did not affect the synaptic transmission at the LPB-to-CeA pathway, even in formalin-treated mice. These results suggest PGB improves not simply the aberrantly enhanced nociception but also various pain-associated cognitive and affective consequences in patients with chronic nociplastic pain.

Rapid Repurposing of Novel Combination Drugs for the Treatment of Heart Failure via a Computationally Guided Network Screening Approach.

Combination drugs, characterized by high efficacy and few side effects, have received extensive attention from pharmaceutical companies and researchers for the treatment of complex diseases such as heart failure (HF). Traditional combination drug discovery depends on large-scale high-throughput experimental approaches that are time-consuming and costly. Herein we developed a novel, rapid, and potentially universal computer-guided combination drug-network-screening approach based on a set of databases and web services that are easy for individuals to obtain and operate, and we discovered for the first time that the menthol-allethrin combination screened by this approach exhibited a significant synergistic cardioprotective effect in vitro. Further mechanistic studies indicated that allethrin and menthol could synergistically block calcium channels. Allethrin bound to the central cavity of the voltage-dependent L-type calcium channel subunit alpha-1S (CACNA1S) lead to a conformational change in an allosteric site of CACNA1S, thereby enhancing the binding of menthol to this allosteric site. In summary, we reported a potentially universal computational approach to combination drug screening that has been used to discover a new combination of menthol-allethrin against HF in vitro, providing a new synergistic mechanism and prospective agent for HF treatment.

Zika E Glycan Loop Region and Guillain-Barré Syndrome-Related Proteins: A Possible Molecular Mimicry to Be Taken in Account for Vaccine Development.

The neurological complications of infection by the mosquito-borne Zika virus (ZIKV) include Guillain–Barré syndrome (GBS), an acute inflammatory demyelinating polyneuritis. GBS was first associated with recent ZIKV epidemics caused by the emergence of the ZIKV Asian lineage in South Pacific. Here, we hypothesize that ZIKV-associated GBS relates to a molecular mimicry between viral envelope E (E) protein and neural proteins involved in GBS. The analysis of the ZIKV epidemic strains showed that the glycan loop (GL) region of the E protein includes an IVNDT motif which is conserved in voltage-dependent L-type calcium channel subunit alpha-1C (Cav1.2) and Heat Shock 70 kDa protein 12A (HSP70 12A). Both VSCC-alpha 1C and HSP70 12A belong to protein families which have been associated with neurological autoimmune diseases in central nervous system. The purpose of our in silico analysis is to point out that IVNDT motif of ZIKV E-GL region should be taken in consideration for the development of safe and effective anti-Zika vaccines by precluding the possibility of adverse neurologic events including autoimmune diseases such as GBS through a potent mimicry with Heat Shock 70 kDa protein 12A (HSP70 12A).

Mitochondrial-Protective Effects of R-Phenibut after Experimental Traumatic Brain Injury.

Altered neuronal Ca2+ homeostasis and mitochondrial dysfunction play a central role in the pathogenesis of traumatic brain injury (TBI). R-Phenibut ((3R)-phenyl-4-aminobutyric acid) is an antagonist of the α2δ subunit of voltage-dependent calcium channels (VDCC) and an agonist of gamma-aminobutyric acid B (GABA-B) receptors. The aim of this study was to evaluate the potential therapeutic effects of R-phenibut following the lateral fluid percussion injury (latFPI) model of TBI in mice and the impact of R- and S-phenibut on mitochondrial functionality in vitro. By determining the bioavailability of R-phenibut in the mouse brain tissue and plasma, we found that R-phenibut (50 mg/kg) reached the brain tissue 15 min after intraperitoneal (i.p.) and peroral (p.o.) injections. The maximal concentration of R-phenibut in the brain tissues was 0.6 μg/g and 0.2 μg/g tissue after i.p. and p.o. administration, respectively. Male Swiss-Webster mice received i.p. injections of R-phenibut at doses of 10 or 50 mg/kg 2 h after TBI and then once daily for 7 days. R-Phenibut treatment at the dose of 50 mg/kg significantly ameliorated functional deficits after TBI on postinjury days 1, 4, and 7. Seven days after TBI, the number of Nissl-stained dark neurons (N-DNs) and interleukin-1beta (IL-1β) expression in the cerebral neocortex in the area of cortical impact were reduced. Moreover, the addition of R- and S-phenibut at a concentration of 0.5 μg/ml inhibited calcium-induced mitochondrial swelling in the brain homogenate and prevented anoxia-reoxygenation-induced increases in mitochondrial H2O2 production and the H2O2/O ratio. Taken together, these results suggest that R-phenibut could serve as a neuroprotective agent and promising drug candidate for treating TBI.

Gene expression profile of lipopolysaccharide‑induced apoptosis of nucleus pulposus cells reversed by syringic acid.

Apoptosis of nucleus pulposus (NP) cells has an important role in the process of intervertebral disc degeneration (IDD), and the search for novel compounds to prevent apoptosis from occurring is urgently required. In the present study, syringic acid (SyrA) was found to exhibit no cytotoxicity on NP cells, and was able to reverse the cytotoxicity, as well as the abnormal expression of Bcl-2 and caspase-3, that were induced by lipopolysaccharide (LPS). The transcriptomes of each group were then analyzed using RNA-Seq. A total of 65 differentially expressed genes (DEGs) were identified in LPS-stimulated groups (LPS group vs. control group), 819 DEGs were identified in the SyrA-reversed groups (SyrA plus LPS group vs. LPS group), and a further 25 DEGs were identified in the SyrA plus LPS group compared with the control group. Reverse transcription-quantitative PCR validation indicated that the alterations in expression of uroplakin 3B-like 1 (UPK3BL1), voltage-dependent calcium channel subunit α-2/δ-1 (CACNA2D1) and polo-like kinase 4 (PLK4) were consistent with the corresponding results of RNA-Seq, and that these genes were involved in both LPS-stimulation and SyrA-reversion processes. Kyoto Encyclopedia of Genes and Genomes analyses indicated that the DEGs in SyrA-reversed groups were involved in, amongst other pathways, ‘Autophagy-other’ and ‘Apoptosis-multiple species’. In conclusion, the addition of SyrA to the NP cells co-incubated with LPS appeared to help prevent the abnormal expression of mRNAs and apoptosis that had been identified in NP cells incubated with LPS alone. The potential mechanism underlying the reversion of SyrA might be attributed to the regulation of CACNA2D1 and PLK4.

Functional α6β4 acetylcholine receptor expression enables pharmacological testing of nicotinic agonists with analgesic properties.

The α6β4 nicotinic acetylcholine receptor (nAChR) is enriched in dorsal root ganglia neurons and is an attractive non-opioid therapeutic target for pain. However, difficulty expressing human α6β4 receptors in recombinant systems has precluded drug discovery. Here, genome-wide screening identified accessory proteins that enable reconstitution of human α6β4 nAChRs. BARP, an auxiliary subunit of voltage-dependent calcium channels, promoted α6β4 surface expression while IRE1α, an unfolded protein response sensor, enhanced α6β4 receptor assembly. Effects on α6β4 involve BARP's N-terminal region and IRE1α's splicing of XBP1 mRNA. Furthermore, clinical efficacy of nicotinic agents in relieving neuropathic pain best correlated with their activity on α6β4. Finally, BARP-knockout, but not NACHO-knockout mice lacked nicotine-induced antiallodynia, highlighting the functional importance of α6β4 in pain. These results identify roles for IRE1α and BARP in neurotransmitter receptor assembly and unlock drug discovery for the previously elusive α6β4 receptor.

Retinal teratogenicity of pregabalin in chick embryo model

Background Pregabalin is a gamma-aminobutyric acid analog that binds to the α2-δ subunits of the pre-synaptic voltage-dependent calcium channels of nerves with a high affinity and selectivity. In this study, the retinal teratogenic potential of pregabalin was investigated in a chick embryo model. Materials and methods Fertilised chicken eggs were divided into groups for administration with different doses of pregabalin. All eggs were opened on the 10th day of incubation. The embryos were dissected and the effects of pregabalin on the retina were investigated histopathologically, morphometrically, and immunohistochemically (Caspase-3). Results There was no statistically significant difference between the low dose pregabalin, control, or vehicle control groups in terms of the number of retina layers and retinal thickness. Medium and high dose pregabalin caused a statistically significant decrease in the number of retina layers, as well as sensory retinal and pigment epithelium layer thicknesses. The outer nuclear and outer plexiform layer did not form in the group administered a medium dose. Similarly, the outer nuclear, outer plexiform, inner nuclear, and inner plexiform layer did not form in the high-dose group. No statistically significant difference was observed between the groups in terms of cellular damage and Caspase-3 expression. Conclusion The use of pregabalin during pregnancy compromises retinal development in a dose-dependent manner. The use of pregabalin in pregnancy causes the aforementioned defects in this system and it may have developmental effects that needs to be further evaluated.

Effect and mechanism of the CACNA2D1-CGRP pathway in osteoarthritis-induced ongoing pain

This study built an OA model in rats by monosodium iodoacetate (MIA) injection to determine the effects and mechanism of the voltage-dependent calcium channel subunit alpha-2/delta-1 (CACNA2D1)-calcitonin gene-related protein (CGRP) pathway in osteoarthritis (OA)-induced ongoing pain. CACNA2D1 expression was measured by qPCR assay, western blotting assay, and immunofluorescence. Pain behaviors in rats were assessed with the measurement of thermal paw withdrawal latency (PWL) and mechanical paw withdrawal threshold (PWT). The expression of CACNA2D1, neuropeptide Y (NPY), activating transcription factor 3 (ATF3), CGRP, protein kinase A (PKA), phosphorylated (p)-PKA, adenylyl cyclase (AC), protein kinase C (PKC), p-PKC, phospholipase C (PLC), and mitogen-activated protein kinase (MAPK) signaling pathway proteins were measured, OA rats had higher CACNA2D1 expression than normal rats. Knockdown of CACNA2D1 led to the elevation of the pain threshold of OA rats, and CACNA2D1 over-expression decreased the pain threshold of normal rats. Moreover, CACNA2D1 over-expression inhibited the expression of CGRP, up-regulated the expressions of NPY, ATF3, p-PKA, AC, p-PKC, PLC, p-Jun N-terminal kinase (JNK), and p-p38, and had no significant effect on phosphorylated extracellular signal-regulated kinase (p-ERK) expression in vivo and in vitro. Using this model of MIA-induced OA, we demonstrated that CACNA2D1 might be involved in the process of pain by modulating the CGRP and AC-PKA/PKC/MAPK signaling pathways in the dorsal root ganglion.