Hydrogen sulfide and polysulfides induce GABA/glutamate/D-serine release, facilitate hippocampal LTP, and regulate behavioral hyperactivity.

Hydrogen sulfide (H2S) and polysulfides (H2Sn, n ≥ 2) are signaling molecules produced by 3-mercaptopyruvate sulfurtransferase (3MST) that play various physiological roles, including the induction of hippocampal long-term potentiation (LTP), a synaptic model of memory formation, by enhancing N-methyl-D-aspartate (NMDA) receptor activity. However, the presynaptic action of H2S/H2Sn on neurotransmitter release, regulation of LTP induction, and animal behavior are poorly understood. Here, we showed that H2S/H2S2 applied to the rat hippocampus by in vivo microdialysis induces the release of GABA, glutamate, and D-serine, a co-agonist of NMDA receptors. Animals with genetically knocked-out 3MST and the target of H2S2, transient receptor potential ankyrin 1 (TRPA1) channels, revealed that H2S/H2S2, 3MST, and TRPA1 activation play a critical role in LTP induction, and the lack of 3MST causes behavioral hypersensitivity to NMDA receptor antagonism, as in schizophrenia. H2S/H2Sn, 3MST, and TRPA1 channels have therapeutic potential for psychiatric diseases and cognitive deficits.

Mature human induced pluripotent stem cell-derived cardiomyocytes promote angiogenesis through alpha-B crystallin.

BACKGROUND: Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) can be used to treat heart diseases; however, the optimal maturity of hiPSC-CMs for effective regenerative medicine remains unclear. We aimed to investigate the benefits of long-term cultured mature hiPSC-CMs in injured rat hearts. METHODS: Cardiomyocytes were differentiated from hiPSCs via monolayer culturing, and the cells were harvested on day 28 or 56 (D28-CMs or D56-CMs, respectively) after differentiation. We transplanted D28-CMs or D56-CMs into the hearts of rat myocardial infarction models and examined cell retention and engraftment via in vivo bioluminescence imaging and histological analysis. We performed transcriptomic sequencing analysis to elucidate the genetic profiles before and after hiPSC-CM transplantation. RESULTS: Upregulated expression of mature sarcomere genes in vitro was observed in D56-CMs compared with D28-CMs. In vivo bioluminescence imaging studies revealed increased bioluminescence intensity of D56-CMs at 8 and 12 weeks post-transplantation. Histological and immunohistochemical analyses showed that D56-CMs promoted engraftment and maturation in the graft area at 12 weeks post-transplantation. Notably, D56-CMs consistently promoted microvessel formation in the graft area from 1 to 12 weeks post-transplantation. Transcriptomic sequencing analysis revealed that compared with the engrafted D28-CMs, the engrafted D56-CMs enriched genes related to blood vessel regulation at 12 weeks post-transplantation. As shown by transcriptomic and western blot analyses, the expression of a small heat shock protein, alpha-B crystallin (CRYAB), was significantly upregulated in D56-CMs compared with D28-CMs. Endothelial cell migration was inhibited by small interfering RNA-mediated knockdown of CRYAB when co-cultured with D56-CMs in vitro. Furthermore, CRYAB overexpression enhanced angiogenesis in the D28-CM grafts at 4 weeks post-transplantation. CONCLUSIONS: Long-term cultured mature hiPSC-CMs promoted engraftment, maturation and angiogenesis post-transplantation in infarcted rat hearts. CRYAB, which was highly expressed in D56-CMs, was identified as an angiogenic factor from mature hiPSC-CMs. This study revealed the benefits of long-term culture, which may enhance the therapeutic potential of hiPSC-CMs.

Effects of riluzole on psychiatric disorders with anxiety or fear as primary symptoms: A systematic review.

AIM: Previous behavioral pharmacology studies involving rodents suggested riluzole had potential to be an ideal psychotropic drug for psychiatric disorders with anxiety or fear as primary symptoms. Several clinical studies have recently been conducted. The purpose of this study was to gather information about the efficacy and tolerability of riluzole for patients with those symptoms. METHODS: We searched PubMed, PsycINFO, CINAHL, EMBASE, and the Cochrane database from inception until April 2021, and performed manual searches for additional relevant articles. This review included: (1) studies involving participants that were patients with generalized anxiety disorder (GAD), social anxiety disorder, panic disorder, obsessive-compulsive disorder (OCD), posttraumatic stress disorder (PTSD), acute stress disorder, or phobias; and (2) randomized controlled trials (RCTs) or intervention studies (e.g., single arm trials) examining the effects and safety of riluzole. RESULTS: Of the 795 identified articles, four RCTs, one RCT subgroup-analysis, and three open-label trials without control groups met the inclusion criteria. Most trials evaluated the efficacy of riluzole as an augmentation therapy with selective serotonin reuptake inhibitors and other antidepressants for PTSD, OCD, or GAD. However, there was insufficient evidence to confirm the effects of riluzole for patients with these psychiatric disorders. Most trials demonstrated adequate study quality. CONCLUSIONS: This review found insufficient evidence to confirm the effects of riluzole for psychiatric disorders with anxiety or fear as primary symptoms. It would be worthwhile to conduct studies that incorporate novel perspectives, such as examining the efficacy of riluzole as a concomitant medication for psychotherapy.

[An emotional stress model using witnessing social defeat scenes in mice].

Chronic exposure to stress can lead to a variety of mental disorders such as depression. There are many treatment-resistant patients who do not respond adequately to the standard pharmacological treatments. Therefore, the development of novel therapeutic agents is highly expected. In rodents, socially defeated animals that were exposed to repeated physical aggression from other individuals are widely used in this field of research. The social defeat is considered as a stress that mimics human social stress. On the other hand, emotional stress, but not physical stress, is likely to contribute to the pathogenesis and etiology of depression in human. Therefore, there is a gap between the process of pathogenesis and the animal models, and this is one of the reasons why the development of new psychotropic drugs to treat depression has been difficult. Recently, a novel stress model has been introduced, in which mice are subjected to emotional stress without physical distress by witnessing social defeat scenes of their conspecifics. We have investigated the mechanisms by which emotional stress is transmitted by witnessing social defeat in mice, focusing on the insular cortex. In this article, we summarize and discuss the recent advancements in the neural basis of behavioral changes induced by emotional stress.

[Subregion-dependent synaptic changes in the orbitofrontal-amygdala pathways by adolescent social isolation in mice].

Because the early-life period is a critical window for the development and reorganization of neural circuits, the early life environment has a great impact on cognitive and emotional functions. It has been reported that a history of early life adversity such as child maltreatment and neglect increases the risk for psychiatric disorders with social and emotional problems. To develop treatments for these psychiatric disorders, it is important to understand the neural mechanisms of how early-life adversity causes social and emotional dysfunctions. In this article, we introduce our research that has revealed adolescent social isolation impairs social and stress-coping behaviors through subregion-dependent synaptic disruption in the orbitofrontal-amygdala circuit in mice.

Characterization of a WD-repeat family protein WDR3 in the brain of WDR3 hetero knockout mice.

The nuclear protein WDR3 is a member of the WD-repeat family and is a component of the 18S pre-rRNA processing complex. However, the expression and function of WDR3 in the brain remains unknown. To characterize WDR3 in the adult mouse brain, we developed Wdr3 heterozygous knockout (WDR3-HKO) mice. Notably, no homozygous Wdr3 knockout mice were born, suggesting that complete absence of WDR3 causes lethal abnormalities during embryogenesis. Brain Wdr3 mRNA expression was significantly reduced to 60% in the WDR3-HKO mice compared to wild type (WT) mice, while the expression of 18S rRNA did not decline. Using immunohistochemistry and X-gal staining, we demonstrated that WDR3 is widely expressed in the mouse brain, especially in the hippocampus, habenular nucleus, and cerebellum. We observed no differences in body weight during adulthood or developmental weight gain between the WDR3-HKO and WT mice. Interestingly, WDR3-HKO mice exhibited a slight but significant increase in spontaneous locomotor activity compared to WT littermates. In conclusion, the WDR3-HKO mice showed no significant phenotypic changes. Further studies are required to explore the behavioral characteristics of WDR3-HKO mice.

A reconstituted depolarization-induced Ca2+ release platform for validation of skeletal muscle disease mutations and drug discovery.

In skeletal muscle excitation-contraction (E-C) coupling, depolarization of the plasma membrane triggers Ca2+ release from the sarcoplasmic reticulum (SR), referred to as depolarization-induced Ca2+ release (DICR). DICR occurs through the type 1 ryanodine receptor (RyR1), which physically interacts with the dihydropyridine receptor Cav1.1 subunit in specific machinery formed with additional essential components including ß1a, Stac3 adaptor protein, and junctophilins. Exome sequencing has accelerated the discovery of many novel mutations in genes encoding DICR machinery in various skeletal muscle diseases. However, functional validation is time-consuming because it must be performed in a skeletal muscle environment. In this study, we established a platform of the reconstituted DICR in HEK293 cells. The essential components were effectively transduced into HEK293 cells expressing RyR1 using baculovirus vectors, and Ca2+ release was quantitatively measured with R-CEPIA1er, a fluorescent ER Ca2+ indicator, without contaminant of extracellular Ca2+ influx. In these cells, [K+]-dependent Ca2+ release was triggered by chemical depolarization with the aid of inward rectifying potassium channel, indicating a successful reconstitution of DICR. Using the platform, we evaluated several Cav1.1 mutations that are implicated in malignant hyperthermia and myopathy. We also tested several RyR1 inhibitors; whereas dantrolene and Cpd1 inhibited DICR, procaine had no effect. Furthermore, twitch potentiators such as perchlorate and thiocyanate shifted the voltage dependence of DICR to more negative potentials without affecting Ca2+-induced Ca2+ release. These results well reproduced the findings with the muscle fibers and the cultured myotubes. Since the procedure is simple and reproducible, the reconstituted DICR platform will be highly useful for the validation of mutations and drug discovery for skeletal muscle diseases.

Adolescent social isolation induces distinct changes in the medial and lateral OFC-BLA synapse and social and emotional alterations in adult mice.

Early-life social isolation is associated with social and emotional problems in adulthood. However, neural mechanisms underlying how social deprivation impairs social and emotional development are poorly understood. Recently, the orbitofrontal cortex (OFC) and basolateral amygdala (BLA) have been highlighted as key nodes for social and emotional functions. Hence, we hypothesize that early social deprivation disrupts the information processing in the OFC-BLA pathway and leads to social and emotional dysfunction. Here, we examined the effects of adolescent social isolation on the OFC-BLA synaptic transmission by optogenetic and whole-cell patch-clamp methods in adult mice. Adolescent social isolation decreased social preference and increased passive stress-coping behaviour in adulthood. Then, we examined excitatory synaptic transmissions to BLA from medial or lateral subregions of the OFC (mOFC or lOFC). Notably, adolescent social isolation decreased the AMPA/NMDA ratio in the mOFC-BLA synapse in adulthood, while the ratio was increased in the lOFC-BLA synapse. Furthermore, we optogenetically manipulated the mOFC-BLA or lOFC-BLA transmission in behaving mice and examined the effects on social and stress-coping behaviours. Optogenetic manipulation of the mOFC-BLA transmission altered social behaviour without affecting passive stress-coping behaviour, while optogenetic manipulation of the lOFC-BLA transmission altered passive stress-coping behaviour without affecting social behaviour. Our results suggest that adolescent social isolation induces distinct postsynaptic changes in the mOFC-BLA and lOFC-BLA synapses, and these changes may separately contribute to abnormalities in social and emotional development.

Brain Dp140 alters glutamatergic transmission and social behaviour in the mdx52 mouse model of Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is a muscle disorder caused by DMD mutations and is characterized by neurobehavioural comorbidities due to dystrophin deficiency in the brain. The lack of Dp140, a dystrophin short isoform, is clinically associated with intellectual disability and autism spectrum disorders (ASDs), but its postnatal functional role is not well understood. To investigate synaptic function in the presence or absence of brain Dp140, we utilized two DMD mouse models, mdx23 and mdx52 mice, in which Dp140 is preserved or lacking, respectively. ASD-like behaviours were observed in pups and 8-week-old mdx52 mice lacking Dp140. Paired-pulse ratio of excitatory postsynaptic currents, glutamatergic vesicle number in basolateral amygdala neurons, and glutamatergic transmission in medial prefrontal cortex-basolateral amygdala projections were significantly reduced in mdx52 mice compared to those in wild-type and mdx23 mice. ASD-like behaviour and electrophysiological findings in mdx52 mice were ameliorated by restoration of Dp140 following intra-cerebroventricular injection of antisense oligonucleotide drug-induced exon 53 skipping or intra-basolateral amygdala administration of Dp140 mRNA-based drug. Our results implicate Dp140 in ASD-like behaviour via altered glutamatergic transmission in the basolateral amygdala of mdx52 mice.

Cytokine receptor gp130 promotes postnatal proliferation of cardiomyocytes required for the normal functional development of the heart.

Mammalian ventricular cardiomyocytes are premature at birth and exhibit substantial phenotypic changes before weaning. Mouse ventricular myocytes undergo cell division several times after birth; however, the regulatory mechanisms and roles of cardiomyocyte division in postnatal heart development remain unclear. Here, we investigated the physiological role of glycoprotein 130 (gp130), the main subunit of multifunctional receptors for the IL-6 family of cytokines, in postnatal cardiomyocyte proliferation. Pharmacological inhibition of gp130 within the first month after birth induced significant systolic dysfunction of the left ventricle in mice. Consistently, mice with postnatal cardiomyocyte-specific gp130 depletion exhibited impaired left ventricular contractility compared with control mice. In these mice, cardiomyocytes exhibited a moderately decreased size and dramatically inhibited proliferation in the left ventricle but not in the right ventricle. Stereological analysis revealed that this change significantly decreased the number of cardiomyocytes in the left ventricle. Furthermore, IL-6 was mainly responsible for promoting ventricular cardiomyocyte proliferation by activating the JAK/STAT3 pathway. Taken together, the IL-6/gp130/JAK/STAT3 axis plays a crucial role in the physiological postnatal proliferation and hypertrophy of left ventricular cardiomyocytes to ensure normal cardiac functional development.NEW & NOTEWORTHY Although cardiomyocytes undergo proliferation in the early postnatal period, the regulatory mechanisms and physiological importance of this process have not been clarified. We found that the pharmacological and genetic depletion of gp130 in preweaning mice resulted in significant impairment of cardiomyocyte proliferation, thinning of the myocardium, and systolic dysfunction of the left but not right ventricle by perturbing JAK/STAT3 signaling. Thus, the IL-6/gp130/JAK/STAT3 axis is crucial for the postnatal functional development of the left ventricle.

[Regulation of localization and function of L-type calcium channels by junctophilins].

Striated muscle L-type calcium channels (LTCC) are localized specifically to the junctional membrane (JM) where the sarcolemma is closely apposed to the sarcoplasmic reticulum. Although this allocation of LTCC is critical for efficient excitation-contraction coupling in striated muscles, its underlying molecular mechanism has not been clarified. Junctophilins (JPs) stabilize the structure of JM by bridging the sarcolemmal and SR membranes. In addition, immunoprecipitation and pull-down assay revealed that the proximal C-terminus of CaV1.1 subunits directly binds to both JP1 and JP2, indicating that JPs might also directly recruit and hold LTCC in JM. Indeed, expression of a JP1 mutant lacking its C-terminus including the transmembrane domain in mouse skeletal muscles exerted a dominant-negative effect on endogenous JPs by impairing LTCC-RyR coupling at triads and reducing contractile force. To investigate a role of cardiac JP2 in a similar strategy, we injected adeno-associated virus vector expressing a C-terminus lacking JP2 mutant (JP2Δ427) driven by a cardiac troponin T promoter into C57BL/6 mice. Echocardiography recorded 4 weeks after the viral injection showed that the fractional shortening in JP2Δ427 group was significantly decreased compared to that of the control group. Calcium transient of isolated ventricular myocytes was significantly decreased by JP2Δ427 expression. Immunocytochemistry showed that JP2Δ427 recruited LTCC to the surface sarcolemma from T-tubules. Taken together, expression of C-terminus lacking JP mutants down-regulated contractile force by impairing ECC of skeletal and cardiac myocytes. Thus, the physical binding between LTCC and JP is essential for contraction of striated muscles.

Targeting Neurons with Functional Oxytocin Receptors: A Novel Set of Simple Knock-In Mouse Lines for Oxytocin Receptor Visualization and Manipulation.

The neuropeptide oxytocin (Oxt) plays important roles in modulating social behaviors. Oxt receptor (Oxtr) is abundantly expressed in the brain and its relationship to socio-behavioral controls has been extensively studied using mouse brains. Several genetic tools to visualize and/or manipulate Oxtr-expressing cells, such as fluorescent reporters and Cre recombinase drivers, have been generated by ES-cell based gene targeting or bacterial artificial chromosome (BAC) transgenesis. However, these mouse lines displayed some differences in their Oxtr expression profiles probably because of the complex context and integrity of their genomic configurations in each line. Here, we apply our sophisticated genome-editing techniques to the Oxtr locus, systematically generating a series of knock-in mouse lines, in which its endogenous transcriptional regulations are intactly preserved and evaluate their expression profiles to ensure the reliability of our new tools. We employ the epitope tagging strategy, with which C-terminally fused tags can be detected by highly specific antibodies, to successfully visualize the Oxtr protein distribution on the neural membrane with super-resolution imaging for the first time. By using T2A self-cleaving peptide sequences, we also induce proper expressions of tdTomato reporter, codon-improved Cre recombinase (iCre), and spatiotemporally inducible Cre-ERT2 in Oxtr-expressing neurons. Electrophysiological recordings from tdTomato-positive cells in the reporter mice support the validity of our tool design. Retro-orbital injections of AAV-PHP.eB vector into the Cre line further enabled visualization of recombinase activities in the appropriate brain regions. Moreover, the first-time Cre-ERT2 line drives Cre-mediated recombination in a spatiotemporally controlled manner on tamoxifen (TMX) administration. These tools thus provide an excellent resource for future functional studies in Oxt-responsive neurons and should prove of broad interest in the field.

NRSF-GNAO1 Pathway Contributes to the Regulation of Cardiac Ca2+ Homeostasis.

BACKGROUND: During the development of heart failure, a fetal cardiac gene program is reactivated and accelerates pathological cardiac remodeling. We previously reported that a transcriptional repressor, NRSF (neuron restrictive silencer factor), suppresses the fetal cardiac gene program, thereby maintaining cardiac integrity. The underlying molecular mechanisms remain to be determined, however. METHODS: We aim to elucidate molecular mechanisms by which NRSF maintains normal cardiac function. We generated cardiac-specific NRSF knockout mice and analyzed cardiac gene expression profiles in those mice and mice cardiac-specifically expressing a dominant-negative NRSF mutant. RESULTS: We found that cardiac expression of Gαo, an inhibitory G protein encoded in humans by GNAO1, is transcriptionally regulated by NRSF and is increased in the ventricles of several mouse models of heart failure. Genetic knockdown of Gnao1 ameliorated the cardiac dysfunction and prolonged survival rates in these mouse heart failure models. Conversely, cardiac-specific overexpression of GNAO1 in mice was sufficient to induce cardiac dysfunction. Mechanistically, we observed that increasing Gαo expression increased surface sarcolemmal L-type Ca2+ channel activity, activated CaMKII (calcium/calmodulin-dependent kinase-II) signaling, and impaired Ca2+ handling in ventricular myocytes, which led to cardiac dysfunction. CONCLUSIONS: These findings shed light on a novel function of Gαo in the regulation of cardiac Ca2+ homeostasis and systolic function and suggest Gαo may be an effective therapeutic target for the treatment of heart failure.

GAD67-mediated GABA Synthesis and Signaling Impinges on Directing Basket Cell Axonal Projections Toward Purkinje Cells in the Cerebellum.

Gamma-aminobutyric acid (GABA) is a major inhibitory neurotransmitter in the central nervous system, synthesized by two isoforms of glutamate decarboxylase (GAD): GAD65 and GAD67. GABA may act as a trophic factor during brain development, but its contribution to the development and maturation of cerebellar neural circuits is not known. To understand the roles of GABA in cerebellar organization and associated functions in motor coordination and balance, we examined GAD65 conventional knock out (KO) mice and mice in which GAD67 was eliminated in parvalbumin-expressing neurons (PV-Cre; GAD67flox/flox mice). We found aberrant subcellular localization of the Shaker-type K channel Kv1.1 in basket cell collaterals of PV-Cre; GAD67 flox/flox mice and abnormal projections from basket cells to Purkinje cells in both mouse strains. We also found that altered synaptic properties of basket cell terminals to Purkinje cells in PV-Cre; GAD67flox/flox mice. Furthermore, PV-Cre; GAD67 flox/flox mice exhibited abnormal motor coordination in the rotarod test. These results indicate that GABA signaling in the cerebellum is critical for establishing appropriate connections between basket cells and Purkinje cells and is associated with motor coordination in mice.

Neonatal blockade of NR2A-containing but not NR2B-containing NMDA receptor induces spatial working memory deficits in adult rats.

The immature brain is highly sensitive to disturbances in the functioning of N-methyl-d-aspartate (NMDA) receptor in rodents, and blockade of the receptor during postnatal brain development period causes schizophrenia-like behavior in adulthood. During the postnatal period, NR2A- and NR2B-containing NMDA receptors are highly expressed, and these two subunits show different expression patterns in the brain. However, the functions of these two NMDA receptors are unknown. In this study, we treated rats with an NR2A-preferring NMDA receptor antagonist (PEAQX, 10 mg/kg), an NR2B-selective NMDA receptor antagonist (ifenprodil, 7.5 mg/kg), or a nonselective blocker of the NMDA receptor (MK-801, 0.4 mg/kg) during the neonatal period. Rats neonatally treated with MK-801 or PEAQX showed spatial working memory deficits in the Y-maze test. PEAQX-treated rats also showed greater reactivity to acoustic stimuli and hypersensitivity to acute MK-801 challenge. However, ifenprodil treatment did not cause any detectable behavioral changes. These results suggest that the NR2A-containing NMDA receptor is indispensable for proper brain development in rats, and functional disturbances in this subunit impair hippocampus-dependent spatial working memory in adulthood.

[A novel way of modification of AT1 angiotensin receptors to alleviate neonatal and infantile heart failure].

Heart failure is an important cause of death of children. Especially, overt one within the preweaning period is fulminant and severe. However, there are no drugs with evidence for it. We recently found that angiotensin II (AngII) activates L-type Ca2+ channels through AT1 receptors (AT1R) and ß-arrestin 2 in murine cardiac myocytes only in the preweaning period, indicating that AT1R/ß-arrestin 2 pathway mediates positive inotropic effects before weaning. Indeed, ß-arrestin-bias AT1R agonist (BBA), TRV027 caused significant long-lasting positive inotropic effects in preweaning mice without increasing serum aldosterone concentrations or inducing tachycardia, arrhythmias, increased cardiac oxygen consumption, and reactive oxygen species generation. TRV027 increased the peak amplitude of twitch Ca2+ transients not only in preweaning mouse cardiac myocytes but in human iPS cell-derived cardiac myocytes exhibiting the fetal to neonatal phenotype. Moreover, TRV027 also increased contraction of the compromised heart of the model knock-in mice mimicking human congenital dilated cardiomyopathy. Although ~80% of these mice died before weaning, TRV027 significantly increased their survival rate. TRV027 did not cause any obvious adverse effects on their preweaning wildtype littermates. Thus, we reason in this review that BBA can be important therapeutics for preweaning heart failure.

[The contribution of orbitofrontal cortex to stress-related psychiatric disorders: stress induced synaptic change in the orbitofrontal-basolateral amygdala pathway].

Exposure to stress induces alterations in synaptic functions, and increases the risk of stress-related psychiatric disorders, such as major depression and PTSD. To develop new treatments for stress-related psychiatric disorders, it is important to understand the effect of stress on the emotional circuits, such as prefrontal cortex (PFC), amygdala and other limbic regions. The orbitofrontal cortex (OFC, ventral subregion of the PFC) has important roles for processing of negative emotion and has recently been highlighted as a critical region in stress-related psychiatric disorders. However, mechanisms how stress affect OFC circuit and induce psychiatric symptoms were less understood. OFC sends dense projection to the amygdala, which is one of the key nodes for processing of negative emotion. Taken together, there is a possibility that stress affects the information processing in the OFC-amygdala pathway, and it underlies stress-induced emotional alteration. In this article, we introduce our research that examined effects of stress on the excitatory synaptic transmission from OFC to the basolateral nucleus of the amygdala (BLA) using optogenetic and whole-cell patch-clamp methods in mice.