Subunit Of Voltage-gated Calcium Channels Research Articles

Inhibitory effects on L- and N-type calcium channels by a novel CaV\u03b21 variant identified in a patient with autism spectrum disorder

Voltage-gated calcium channel (VGCC) subunits have been genetically associated with autism spectrum disorders (ASD). The properties of the pore-forming VGCC subunit are modulated by auxiliary β-subunits, which exist in four isoforms (CaVβ1-4). Our previous findings suggested that activation of L-type VGCCs is a common feature of CaVβ2 subunit mutations found in ASD patients. In the current study, we functionally characterized a novel CaVβ1b variant (p.R296C) identified in an ASD patient. We used whole-cell and single-channel patch clamp to study the effect of CaVβ1b_R296C on the function of L- and N-type VGCCs. Furthermore, we used co-immunoprecipitation followed by Western blot to evaluate the interaction of the CaVβ1b-subunits with the RGK-protein Gem. Our data obtained at both, whole-cell and single-channel levels, show that compared to a wild-type CaVβ1b, the CaVβ1b_R296C variant inhibits L- and N-type VGCCs. Interaction with and modulation by the RGK-protein Gem seems to be intact. Our findings indicate functional effects of the CaVβ1b_R296C variant differing from that attributed to CaVβ2 variants found in ASD patients. Further studies have to detail the effects on different VGCC subtypes and on VGCC expression.

P083 An integrative analysis of DNA methylation and RNA-Seq data in human adipose-stem cells of Crohn’s Disease patients with different clinical activity

Abstract Background Crohn’s disease (CD) is characterized by the expansion of mesenteric fat attached to the inflamed segments of the intestine, also known as “creeping fat” which seems to be directly related to disease activity. Our group revealed that adipose-stem cells (hASCs) isolated from the creeping fat of CD subjects are dysfunctional (showing a high inflammatory profile, high invasive and phagocytic capacities, and worse immunosuppressive properties); and this dysfunction is maintained even in hASCs isolated from CD subjects in remission of the disease.1 Methods 1) Culture and characterization of hASCs isolated from adipose tissue biopsies of active CD, inactive CD, and healthy subjects (n=25). Visceral origin: creeping fat in active CD subjects and mesenteric fat in CD subjects in remission and healthy subjects. 2) Comparative DNA methylome analysis in hASCs isolated from the included population (Infinium Human-Methylation EPIC (850K) BeadChip; Illumina, Inc). 3) Transcriptomic study in hASCs isolated from the included population (Illumina HiSeq 2500 system). 4) Bioinformatic analysis & data integration. Results Methylation and transcriptomic studies revealed a multi-omic profile of hASCs isolated from CD subjects compared to control individuals (Heatmaps Fig 1A). Indeed, the integration of genome-wide methylation changes and gene expression differences revealed the main candidate’s genes (Figure 1B). The most significant upregulated gene in CD was Mab-21 like 2 (MAB21L2) (Fig 1B-C). This gene encodes a downstream target of transforming growth factor-beta signaling. MAB21L2 was validated in an independent cohort of hASC isolated from control, active, and inactive CD subjects (n=7/each group) using qPCR. In agreement, MAB21L2 gene expression was significantly higher in hASCs isolated from the active and inactive group than in the control group (Fig 1D). Another interesting candidate gene found was tyrosine kinase 2 (TYK2) which is significantly upregulated in active CD subjects. This gene promulgates cytokine signals by phosphorylating receptor subunits. It is also a component of both the type I and III interferon signaling pathways.2 Finally, another gene upregulated in remission of the disease is the calcium voltage-gated channel subunit alpha 1H (CACNA1H), which has been involved in low-grade inflammation and has been proposed as an important player in IBD during the remission phase.3 Conclusion Integration of data revealed genes involved in the dysregulation of hASCs associated with CD and genes related to disease remission. Reference 1. Serena C Stem Cell Rep 2017 9(4):1109–23; 2De Vries LCS JCC 2021 617-30; 3Picard E Br J Pharmacol 2019;176:950–63

Enhanced long-term potentiation and impaired learning in mice lacking alternative exon 33 of CaV1.2 calcium channel

The CACNA1C (calcium voltage-gated channel subunit alpha 1 C) gene that encodes the CaV1.2 channel is a prominent risk gene for neuropsychiatric and neurodegenerative disorders with cognitive and social impairments like schizophrenia, bipolar disorders, depression and autistic spectrum disorders (ASD). We have shown previously that mice with exon 33 deleted from CaV1.2 channel (CaV1.2-exon 33−/−) displayed increased CaV1.2 current density and single channel open probability in cardiomyocytes, and were prone to develop arrhythmia. As Ca2+ entry through CaV1.2 channels activates gene transcription in response to synaptic activity, we were intrigued to explore the possible role of Cav1.2Δ33 channels in synaptic plasticity and behaviour. Homozygous deletion of alternative exon 33 resulted in enhanced long-term potentiation (LTP), and lack of long- term depression (LTD), which did not correlate with enhanced learning. Exon 33 deletion also led to a decrease in social dominance, sociability and social novelty. Our findings shed light on the effect of gain-of-function of CaV1.2Δ33 signalling on synaptic plasticity and behaviour and provides evidence for a link between CaV1.2 and distinct cognitive and social behaviours associated with phenotypic features of psychiatric disorders like schizophrenia, bipolar disorder and ASD.

Long read sequencing reveals novel isoforms and insights into splicing regulation during cell state changes

BackgroundAlternative splicing is a key mechanism underlying cellular differentiation and a driver of complexity in mammalian neuronal tissues. However, understanding of which isoforms are differentially used or expressed and how this affects cellular differentiation remains unclear. Long read sequencing allows full-length transcript recovery and quantification, enabling transcript-level analysis of alternative splicing processes and how these change with cell state. Here, we utilise Oxford Nanopore Technologies sequencing to produce a custom annotation of a well-studied human neuroblastoma cell line SH-SY5Y, and to characterise isoform expression and usage across differentiation.ResultsWe identify many previously unannotated features, including a novel transcript of the voltage-gated calcium channel subunit gene, CACNA2D2. We show differential expression and usage of transcripts during differentiation identifying candidates for future research into state change regulation.ConclusionsOur work highlights the potential of long read sequencing to uncover previously unknown transcript diversity and mechanisms influencing alternative splicing.

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.

Hydroxychloroquine in a COVID-19 patient on chronic Pregabalin therapy- Is it safe?

Dear Editor, A 58-year-old male patient, weighing 90 kg presented with fever, shortness of breath, and cough for one week. The RT-PCR was tested positive for COVID-19. Patient had hypertension for past 15 years and controlled on amlodipine 5-mg OD and pregabalin 75-mg and clonazepam 0.5-mg OD for cervical spondylosis for the last 4 years. Pulse rate was 106/min, blood pressure 118/74 mm Hg, and respiratory rate was 28 breaths/min. His condition deteriorated and was shifted to ICU where his oxygen saturation improved to 98% on partial rebreathing face mask from 83% on room air. On auscultation, crepitations were present in bilateral lower lung fields. All routine investigations were within normal limit except for high WBC count (15000/µL) and electrocardiogram (ECG) demonstrating a heart rate of 150/min and QTc interval of 480ms(Bazett’sformula). Chest X-ray revealed bilateral lower zone infiltrates. He received tazobactam-piperacllin, teicoplanin, ivermectin, enoxaparin, dexamethasone, salmeterol MDI, vitamin C, zinc and had already received azithromycin. Considering the ECG changes, hydroxychloroquine (HCQ) was not administered. It was revealed that he was on both pregabalin and clonazepam till a day before and was immediately stopped. The ECG changes resolved on next day i.e. within 36 h. Following which, HCQ was administered in a dose of 400-mg OD for 5 days; ECG on all days was normal. Patient improved clinically and discharged thereafter. Cardiac events with pregabalin are rarely reported and is related to its binding to a2d2 subunits of voltage-gated calcium channel causing potassium-evoked accentuation of calcium influx. Recently, a population-based database concluded the relation between new exposure to pregabalin and initiating AF treatment with the incidence of 8.6 per 1000 and RR 2.79 (95% CI 1.05–7.40) when compared to the patients started on opiates.[1] Also, in an animal model, increase in HR and QTc prolongation was observed with pregabalin.[2] The role of HCQ has been controversial in the therapeutic management of COVID19. However, it is shown to significantly reduce the viral load in COVID-19 patients further reinforced by coadministration of azithromycin.[3] This particular evidence led to the exponential increase in its use for therapeutic management in the initial few months of this pandemic and was the time the present case was managed. Recently, a cohort study observed significant QTc prolongation in patients on HCQ and azithromycin combination therapy; however, did not report any arrhythmogenic deaths.[4] In this case, we attribute QTc prolongation to chronic pregabalin and/or clonazepam therapy. The oral bioavailability of pregabalin is approximately 90% and is not plasma protein bound with elimination half-life of 6.3 h. The steady-state plasma concentration is achieved within 24–48 h.[2] This explains the normalization of ECG approximately within 36 h of the last pregabalin dose. Similarly, literature search reveals only a single case report implicating clonazepam, a long-acting benzodiazepine to be a potential cause of QTc prolongation in a paediatric patient.[5] To conclude, the judicious and cautious use of proarrhythmic drugs like hydroxychloroquine and/or azithromycin in patients with COVID-19 on chronic pregabalin and/or clonazepam therapy as it may potentiate the risk of arrhythmia. Declaration of patient consent The authors certify that they have obtained all appropriate patient consent forms. In the form, the patient has given his consent for his images and other clinical information to be reported in the journal. The patient understands that name and initials will not be published and due efforts will be made to conceal identity, but anonymity cannot be guaranteed. Financial support and sponsorship Nil. Conflicts of interest There are no conflicts of interest.

Mirogabalin as a Novel Gabapentinoid for the Treatment of Chronic Pain Conditions: An Analysis of Current Evidence.

Neuropathic pain is a challenge for physicians to treat and often requires a multimodal approach with both pharmacologic and lifestyle interventions. Mirogabalin, a potent, selective ligand of the α2δ-1 and α2δ-2 subunits of voltage-gated calcium channels (VGCCs), provides analgesia by inhibiting neurotransmitter release at the presynaptic end of the neuron. Mirogabalin offers more sustained analgesia than its gabapentinoid counterparts in addition to a wider safety margin for adverse events. Recent clinical trials of mirogabalin have demonstrated both efficacy and tolerability of the drug for the treatment of diabetic peripheral neuropathic pain and postherpetic neuralgia, leading to its approval in Japan. While still not yet FDA approved, mirogabalin is still in its infancy and offers potential into the treatment of neuropathic pain and its associated comorbidities.

Effects of clozapine and risperidone antipsychotic drugs on the expression of CACNA1C and behavioral changes in rat ‘Ketamine model of schizophrenia

Calcium Voltage-Gated Channel Subunit Alpha1 C (CACNA1C) is one of the most important genes associated with schizophrenia. In this study, 45 male Wistar rats were divided into 5 groups of saline, control, ketamine, clozapine, and risperidone. Animals in ketamine, risperidone, and clozapine groups received ketamine (30 mg/kg-i.p.) for 10 days. After the last injection of ketamine, we started injecting clozapine (7.5 mg/kg-i.p.), risperidone (1 mg/kg-i.p.), up to 28 days. Twenty-four hours after the last injection, open field, social interaction, and elevated plus-maze tests and gene expression in hippocampus were performed. The results of the social interaction test revealed a significant decrease in cumulative time with ketamine, compared with the saline group, and an increase with clozapine and risperidone compared with the ketamine group. Moreover, results from the elevated plus-maze test demonstrated a critical decrease in open arm time and increase in close arm time with ketamine compared with saline, as well as increased in open arm time with risperidone compared with ketamine. Further results revealed a significant increase in rearing and grooming with ketamine compared to saline, as well as a decrease with risperidone and clozapine compared to ketamine. There were no significant differences in CACNA1C gene expression between groups in the rat hippocampus. In brief, the results of this study indicated that clozapine and risperidone could partially improve cognitive impairments in the rat. However, our findings demonstrated that this treatment is not related to CACNA1C gene expression.

Three-Week-Old Rabbit Ventricular Cardiomyocytes as a Novel System to Study Cardiac Excitation and EC Coupling.

Cardiac arrhythmias significantly contribute to cardiovascular morbidity and mortality. The rabbit heart serves as an accepted model system for studying cardiac cell excitation and arrhythmogenicity. Accordingly, primary cultures of adult rabbit ventricular cardiomyocytes serve as a preferable model to study molecular mechanisms of human cardiac excitation. However, the use of adult rabbit cardiomyocytes is often regarded as excessively costly. Therefore, we developed and characterized a novel low-cost rabbit cardiomyocyte model, namely, 3-week-old ventricular cardiomyocytes (3wRbCMs). Ventricular myocytes were isolated from whole ventricles of 3-week-old New Zealand White rabbits of both sexes by standard enzymatic techniques. Using wheat germ agglutinin, we found a clear T-tubule structure in acutely isolated 3wRbCMs. Cells were adenovirally infected (multiplicity of infection of 10) to express Green Fluorescent Protein (GFP) and cultured for 48 h. The cells showed action potential duration (APD90 = 253 ± 24 ms) and calcium transients similar to adult rabbit cardiomyocytes. Freshly isolated and 48-h-old-cultured cells expressed critical ion channel proteins: calcium voltage-gated channel subunit alpha1 C (Cavα1c), sodium voltage-gated channel alpha subunit 5 (Nav1.5), potassium voltage-gated channel subfamily D member 3 (Kv4.3), and subfamily A member 4 (Kv1.4), and also subfamily H member 2 (RERG. Kv11.1), KvLQT1 (K7.1) protein and inward-rectifier potassium channel (Kir2.1). The cells displayed an appropriate electrophysiological phenotype, including fast sodium current (INa), transient outward potassium current (Ito), L-type calcium channel peak current (ICa,L), rapid and slow components of the delayed rectifier potassium current (IKr and IKs), and inward rectifier (IK1). Although expression of the channel proteins and some currents decreased during the 48 h of culturing, we conclude that 3wRbCMs are a new, low-cost alternative to the adult-rabbit-cardiomyocytes system, which allows the investigation of molecular mechanisms of cardiac excitation on morphological, biochemical, genetic, physiological, and biophysical levels.

De novo variants in CACNA1E found in patients with intellectual disability, developmental regression and social cognition deficit but no seizures

BackgroundDe novo variants in the voltage-gated calcium channel subunit α1 E gene (CACNA1E) have been described as causative of epileptic encephalopathy with contractures, macrocephaly and dyskinesias.MethodsFollowing the observation of an index patient with developmental delay and autism spectrum disorder (ASD) without seizures who had a de novo deleterious CACNA1E variant, we screened GeneMatcher for other individuals with CACNA1E variants and neurodevelopmental phenotypes without epilepsy. The spectrum of pathogenic CACNA1E variants was compared to the mutational landscape of variants in the gnomAD control population database.ResultsWe identified seven unrelated individuals with intellectual disability, developmental regression and ASD-like behavioral profile, and notably without epilepsy, who had de novo heterozygous putatively pathogenic variants in CACNA1E. Age of onset of clinical manifestation, presence or absence of regression and degree of severity were variable, and no clear-cut genotype–phenotype association could be recognized. The analysis of disease-associated variants and their comparison to benign variants from the control population allowed for the identification of regions in the CACNA1E protein that seem to be intolerant to substitutions and thus more likely to harbor pathogenic variants. As in a few reported cases with CACNA1E variants and epilepsy, one patient showed a positive clinical behavioral response to topiramate, a specific calcium channel modulator.LimitationsThe significance of our study is limited by the absence of functional experiments of the effect of identified variants, the small sample size and the lack of systematic ASD assessment in all participants. Moreover, topiramate was given to one patient only and for a short period of time.ConclusionsOur results indicate that CACNA1E variants may result in neurodevelopmental disorders without epilepsy and expand the mutational and phenotypic spectrum of this gene. CACNA1E deserves to be included in gene panels for non-specific developmental disorders, including ASD, and not limited to patients with seizures, to improve diagnostic recognition and explore the possible efficacy of topiramate.

Piperazinyl Bicyclic Derivatives as Selective Ligands of the α2δ-1 Subunit of Voltage-Gated Calcium Channels.

The synthesis and pharmacological activities of a new series of piperazinyl quinazolin-4-(3H)-one derivatives acting toward the α2δ-1 subunit of voltage-gated calcium channels (Cavα2δ-1) are reported. Different positions of a micromolar HTS hit were explored, and best activities were obtained for compounds containing a small alkyl group in position 3 of the quinazolin-4-(3H)-one scaffold and a 3-methyl-piperazin-1-yl- or 3,5-dimethyl-piperazin-1-yl-butyl group in position 2. The activity was shown to reside in the R enantiomer of the chain in position 2, and several eutomers reached single digit nanomolar affinities. Final modification of the central scaffold to reduce lipophilicity provided the pyrido[4,3-d]pyrimidin-4(3H)-one 16RR, which showed high selectivity for Cavα2δ-1 versus Cavα2δ-2, probably linked to its improved analgesic efficacy-safety ratio in mice over pregabalin.

Editors' Note: What Is the Mechanism of Therapeutic and Adverse Effects of Gabapentinoids?

Dr. Benarroch reviewed the mechanisms underlying the therapeutic and adverse effects of gabapentinoids such as gabapentin and pregabalin. The author noted that the efficacy of these drugs for neuropathic pain, restless legs syndrome, and focal seizures is primarily attributed to their inhibition of the α2δ subunit of presynaptic voltage-gated calcium channels, which decreases neurotransmitter release. He discussed the relevance of this inhibitory action in the potential induction of cerebellar ataxia by gabapentinoids. In response, Dr. Gazulla et al. argue that GABAergic effects of these medications are also important, noting that gabapentin has been shown to increase the expression of δGABAA receptors, inhibitory tone in the cerebellum, and brain GABA concentration in patients, whereas pregabalin has been shown to increase neuronal calcium influx, which facilitates neurotransmission. They argue that gabapentinoids could actually help treat ataxia in diseases involving GABA deficiency. Responding to these comments, Dr. Benarroch notes that he did cite work finding increased expression of GABAA receptors with gabapentin use but did not cite a pilot study of pregabalin for ataxia because the review was focused on mechanisms relating to the current clinical use of gabapentinoids. He agrees that increased GABAergic transmission by these drugs may benefit disorders affecting Purkinje cells and other inhibitory neurons such as periodic alternating nystagmus but cautions that the mechanisms of ataxia are complex and that further studies are needed to confirm potential beneficial effects of gabapentinoids in cerebellar ataxia. This exchange highlights uncertainties regarding the potential therapeutic vs adverse effects of gabapentinoids in ataxia and other cerebellar disorders. Dr. Benarroch reviewed the mechanisms underlying the therapeutic and adverse effects of gabapentinoids such as gabapentin and pregabalin. The author noted that the efficacy of these drugs for neuropathic pain, restless legs syndrome, and focal seizures is primarily attributed to their inhibition of the α2δ subunit of presynaptic voltage-gated calcium channels, which decreases neurotransmitter release. He discussed the relevance of this inhibitory action in the potential induction of cerebellar ataxia by gabapentinoids. In response, Dr. Gazulla et al. argue that GABAergic effects of these medications are also important, noting that gabapentin has been shown to increase the expression of δGABAA receptors, inhibitory tone in the cerebellum, and brain GABA concentration in patients, whereas pregabalin has been shown to increase neuronal calcium influx, which facilitates neurotransmission. They argue that gabapentinoids could actually help treat ataxia in diseases involving GABA deficiency. Responding to these comments, Dr. Benarroch notes that he did cite work finding increased expression of GABAA receptors with gabapentin use but did not cite a pilot study of pregabalin for ataxia because the review was focused on mechanisms relating to the current clinical use of gabapentinoids. He agrees that increased GABAergic transmission by these drugs may benefit disorders affecting Purkinje cells and other inhibitory neurons such as periodic alternating nystagmus but cautions that the mechanisms of ataxia are complex and that further studies are needed to confirm potential beneficial effects of gabapentinoids in cerebellar ataxia. This exchange highlights uncertainties regarding the potential therapeutic vs adverse effects of gabapentinoids in ataxia and other cerebellar disorders.

Reader Response: What Is the Mechanism of Therapeutic and Adverse Effects of Gabapentinoids?

Dr. Benarroch's article about gabapentinoids describes that the α2δ subunit of voltage-gated calcium channels (VGCC) allows for neuronal calcium influx. A2δ is expressed in Purkinje cells, which use gamma aminobutyric acid (GABA) as a neurotransmitter. The author states that gabapentinoids inhibit the α2δ subunit and reduce neurotransmitter release, resulting in the induction of cerebellar ataxia.1,2

Advances in Imaging Genetics of Suicidal Behavior

Suicide,a major public health problem,is the death caused by injuring oneself with the intent to die.In this paper,we reviewed the genes encoding serotonin system,calcium voltage-gated channel subunit alpha1 C,γ-aminobutyric acid,and spindle and kinetochore associated complex subunit 2,as well as their related brain regions,from the perspective of imaging genetics,aiming to provide new ideas for the research and intervention on suicidal behavior.

Differences of DNA methylation patterns in the placenta of large for gestational age infant

To investigate the molecular mechanisms of later metabolic health changes in large for gestational age (LGA) newborns by analyzing deoxyribonucleic acid (DNA) methylation patterns in the placenta of LGA and appropriate for gestational age (AGA) newborns.A total of 6 placentas of LGA and 6 placentas of AGA newborns were enrolled as LGA group and AGA group. DNA methylation was analyzed using the Illumina Infinium Human MethylationEPIC BeadChip microarrays and verified via pyrosequencing and reverse transcription-quantitative real-time polymerase chain reaction. Functional enrichment analysis were constructed by gene ontology and Kyoto Encyclopedia of Genes and Genomes pathway analysis based on the differentially methylated regions between LGA and AGA groups.Clinical investigation showed that LGA newborns had significantly lower hemoglobin and blood glucose compared to AGA newborns. Birth weight was negatively correlated to hemoglobin and blood glucose. Genome-wide DNA methylation analysis identified 17 244 methylation variable positions achieving genome-wide significance (adjusted P < .05). 34% methylation variable positions were located in the gene promoter region. A total of 117 differentially methylated regions were revealed by bump hunting analysis, which mapped to 107 genes. Function analysis showed 13 genes enriched in adhesion and infection process, endocrine and other factor-regulated calcium reabsorption, calcium signaling pathway and transmembrane transport. Four genes linked to type II diabetes mellitus. Among the 13 genes, we selected GNAS and calcium voltage-gated channel subunit alpha1 G for independent verification of pyrosequencing, and the messenger ribonucleic acid levels of guanine nucleotide binding protein, calcium voltage-gated channel subunit alpha1 G, DECR1, and FK506 binding protein 11 were verified by reverse transcription-quantitative real-time polymerase chain reaction.DNA methylation variation and gene expression differences in placental samples were associated with LGA newborns, which linking the effect of intrauterine environment to regulation of the offspring's gene expression. Furthermore, pathway analysis suggested that intrauterine environment affecting fetal growth might had a functional impact on multiple signaling pathways involved in fetal growth, metabolism, and inflammation. Further studies were required to understand the differences of methylation patterns.

Adaptive Mechanisms of Somatostatin-Positive Interneurons after Traumatic Brain Injury through a Switch of α Subunits in L-Type Voltage-Gated Calcium Channels.

Unilateral traumatic brain injury (TBI) causes cortical dysfunctions spreading to the primarily undamaged hemisphere. This phenomenon, called transhemispheric diaschisis, is mediated by an imbalance of glutamatergic versus GABAergic neurotransmission. This study investigated the role of GABAergic, somatostatin-positive (SST) interneurons in the contralateral hemisphere 72h after unilateral TBI. The brain injury was induced to the primary motor/somatosensory cortex of glutamate decarboxylase 67-green fluorescent protein (GAD67-GFP) knock-in mice at postnatal days 19-21 under anesthesia in vivo. Single GFP+ interneurons of the undamaged, contralateral cortex were isolated by fluorescence-activated cell sorting and analyzed by mass spectrometry. TBI caused a switch of 2 α subunits of pore-forming L-type voltage-gated calcium channels (VGCC) in GABAergic interneurons, an increased expression of CaV1.3, and simultaneous ablation of CaV1.2. This switch was associated with 1) increased excitability of single SST interneurons in patch-clamp recordings and (2) a recovery from early network hyperactivity in the contralateral hemisphere in microelectrode array recordings of acute slices. The electrophysiological changes were sensitive to pharmacological blockade of CaV1.3 (isradipine, 100nM). These data identify a switch of 2 α subunits of VGCCs in SST interneurons early after TBI as a mechanism to counterbalance post-traumatic hyperexcitability.

Cerebrospinal fluid proteomics targeted for central nervous system processes in bipolar disorder.

The etiopathology of bipolar disorder is largely unknown. We collected cerebrospinal fluid (CSF) samples from two independent case-control cohorts (total n = 351) to identify proteins associated with bipolar disorder. A panel of 92 proteins targeted towards central nervous system processes identified two proteins that replicated across the cohorts: the CSF concentrations of testican-1 were lower, and the CSF concentrations of C-type lectin domain family 1 member B (CLEC1B) were higher, in cases than controls. In a restricted subgroup analysis, we compared only bipolar type 1 with controls and identified two additional proteins that replicated in both cohorts: draxin and tumor necrosis factor receptor superfamily member 21 (TNFRSF21), both lower in cases than controls. This analysis additionally revealed several proteins significantly associated with bipolar type 1 in one cohort, falling just short of replicated statistical significance in the other (tenascin-R, disintegrin and metalloproteinase domain-containing protein 23, cell adhesion molecule 3, RGM domain family member B, plexin-B1, and brorin). Next, we conducted genome-wide association analyses of the case-control-associated proteins. In these analyses, we found associations with the voltage-gated calcium channel subunit CACNG4, and the lipid-droplet-associated gene PLIN5 with CSF concentrations of TNFRSF21 and CLEC1B, respectively. The reported proteins are involved in neuronal cell-cell and cell-matrix interactions, particularly in the developing brain, and in pathways of importance for lithium's mechanism of action. In summary, we report four novel CSF protein associations with bipolar disorder that replicated in two independent case-control cohorts, shedding new light on the central nervous system processes implicated in bipolar disorder.

Dietary Supplementation of Octacosanol Improves Exercise-Induced Fatigue and Its Molecular Mechanism.

Several publications report that octacosanol (OCT) has different biological functions. This study was designed to evaluate the antifatigue effect and molecular mechanism of octacosanol (200 mg/(kg day)) in forced exercise-induced fatigue models of trained male C57BL/6 mice. Results showed that octacosanol ameliorated the mice's autonomic activities, forelimb grip strength, and swimming endurance, and the levels of liver glycogen (LG), muscle glycogen (MG), blood lactic acid (BLA), lactate dehydrogenase (LDH), superoxide dismutase (SOD), and glutathione peroxidase (GSH-Px) were also regulated. Gene analysis results showed that treatment with OCT upregulated 29 genes, while 38 genes were downregulated in gastrocnemius tissue. Gene ontology (GO) analyses indicated that these genes enriched functions in relation to myofibril, contractile fiber, and calcium-dependent adenosinetriphosphatase (ATPase) activity. Octacosanol supplementation significantly adjusted the messenger RNA (mRNA) and protein expression levels related to fatigue performance. Octacosanol has an observably mitigating effect in exercise-induced fatigue models, and its molecular mechanism may be related to the regulation of tripartite motif-containing 63 (Trim63), periaxin (Prx), calcium voltage-gated channel subunit α1 H (Cacna1h), and myosin-binding protein C (Mybpc3) expression.

MiR-208b Reduces the Expression of Kcnj5 in a Cardiomyocyte Cell Line.

MicroRNAs (miRs) contribute to different aspects of cardiovascular pathology, among them cardiac hypertrophy and atrial fibrillation. Cardiac miR expression was analyzed in a mouse model with structural and electrical remodeling. Next-generation sequencing revealed that miR-208b-3p was ~25-fold upregulated. Therefore, the aim of our study was to evaluate the impact of miR-208b on cardiac protein expression. First, an undirected approach comparing whole RNA sequencing data to miR-walk 2.0 miR-208b 3′-UTR targets revealed 58 potential targets of miR-208b being regulated. We were able to show that miR-208b mimics bind to the 3′ untranslated region (UTR) of voltage-gated calcium channel subunit alpha1 C and Kcnj5, two predicted targets of miR-208b. Additionally, we demonstrated that miR-208b mimics reduce GIRK1/4 channel-dependent thallium ion flux in HL-1 cells. In a second undirected approach we performed mass spectrometry to identify the potential targets of miR-208b. We identified 40 potential targets by comparison to miR-walk 2.0 3′-UTR, 5′-UTR and CDS targets. Among those targets, Rock2 and Ran were upregulated in Western blots of HL-1 cells by miR-208b mimics. In summary, miR-208b targets the mRNAs of proteins involved in the generation of cardiac excitation and propagation, as well as of proteins involved in RNA translocation (Ran) and cardiac hypertrophic response (Rock2).

Basolateral Na+/Ca2+ exchanger 1 and Na+/K+-ATPase, which display light-enhanced gene and protein expression levels, could be involved in the absorption of exogenous Ca2+ through the ctenidium of the giant clam, Tridacna squamosa

Giant clams perform light-enhanced shell formation (calcification) and therefore need to increase the uptake of exogenous Ca2+ during illumination. The ctenidium of the fluted giant clam, Tridacna squamosa, is involved in light-enhanced Ca2+ uptake. It expresses the pore-forming voltage-gated calcium channel (VGCC) subunit alpha 1 (CACNA1) in the apical membrane of the epithelial cells, and the protein expression level of CACNA1 is upregulated in the ctenidium during illumination. This study aimed to elucidate the mechanism involved in the transport of the absorbed Ca2+ across the basolateral membrane of the ctenidial epithelial cells into the hemolymph. We obtained a homolog of Na+/Ca2+exchanger 1 (NCX1-like) from the ctenidium of T. squamosa, which comprised 2418 bp, encoding a protein of 806 amino acids (88.9 kDa). NCX1-like had a basolateral localization in the epithelial cells of the ctenidial filaments and tertiary water channels. Illumination resulted in significant increases in the transcript and protein levels of NCX1-like/NCX1-like in the ctenidium. Hence, NCX1-like could operate in conjunction with VGCC to increase the transport of Ca2+ from the ambient seawater into the hemolymph during illumination. Illumination also resulted in the upregulation of the gene and protein expression levels of Na+/K+-ATPase (NKA) α-subunit (NKAα/NKAα) in the ctenidium of T. squamosa. As light-enhanced extrusion of Ca2+ into the hemolymph through NCX1-like would lead to a greater influx of extracellular Na+, the increased expression of the basolateral NKA was required to augment the capacity of intracellular Na+ homeostasis.