Compensatory role of neuregulin-1 in diabetic cardiomyopathy.

Diabetes mellitus is a prevalent risk factor for congestive heart failure. Diabetic cardiomyopathy patients present with left ventricular (LV) diastolic dysfunction at an early stage, then systolic dysfunction as the disease progresses. The mechanism underlying the development of diabetic cardiomyopathy has not yet been fully understood. This study aimed to elucidate the mechanisms by which diastolic dysfunction precedes systolic dysfunction at the early stage of diabetic cardiomyopathy. We hypothesized that the downregulation of cardioprotective factors is involved in the pathogenesis of diabetic cardiomyopathy. LV diastolic dysfunction, but not systolic dysfunction, was observed in type-1 diabetes mellitus model mice 4 weeks after STZ administration (STZ-4W), mimicking the early stage of diabetic cardiomyopathy. Counter to expectations, neuregulin-1 (NRG1) was markedly upregulated in the vascular endothelial cell in the ventricles of STZ-4W mice. To clarify the functional significance of the upregulated NRG1, we blocked its receptor ErbB2 with trastuzumab (TRZ). In STZ-4W mice, TRZ significantly reduced the systolic function without affecting diastolic function and caused a more prominent reduction in Akt phosphorylation levels. These results indicate that the compensatory upregulated NRG1 contributes to maintaining the LV systolic function, which explains why diastolic dysfunction precedes systolic dysfunction at the early stage of diabetic cardiomyopathy.

Pathogenic Mechanism of Dry Eye-Induced Chronic Ocular Pain and a Mechanism-Based Therapeutic Approach.

Purpose: Dry eye-induced chronic ocular pain is also called ocular neuropathic pain. However, details of the pathogenic mechanism remain unknown. The purpose of this study was to elucidate the pathogenic mechanism of dry eye-induced chronic pain in the anterior eye area and develop a pathophysiology-based therapeutic strategy. Methods: We used a rat dry eye model with lacrimal gland excision (LGE) to elucidate the pathogenic mechanism of ocular neuropathic pain. Corneal epithelial damage, hypersensitivity, and hyperalgesia were evaluated on the LGE side and compared with the sham surgery side. We analyzed neuronal activity, microglial and astrocytic activity, α2δ-1 subunit expression, and inhibitory interneurons in the trigeminal nucleus. We also evaluated the therapeutic effects of ophthalmic treatment and chronic pregabalin administration on dry eye-induced ocular neuropathic pain. Results: Dry eye caused hypersensitivity and hyperalgesia on the LGE side. In the trigeminal nucleus of the LGE side, neuronal hyperactivation, transient activation of microglia, persistent activation of astrocytes, α2δ-1 subunit upregulation, and reduced numbers of inhibitory interneurons were observed. Ophthalmic treatment alone did not improve hyperalgesia. In contrast, continuous treatment with pregabalin effectively ameliorated hypersensitivity and hyperalgesia and normalized neural activity, α2δ-1 subunit upregulation, and astrocyte activation. Conclusions: These results suggest that dry eye-induced hypersensitivity and hyperalgesia are caused by central sensitization in the trigeminal nucleus with upregulation of the α2δ-1 subunit. Here, we showed that pregabalin is effective for treating dry eye-induced ocular neuropathic pain even after chronic pain has been established.

[Pathophysiological functions of gas mediators in neurodegeneration].

Since the discovery of nitric oxide (NO) as gaseous signaling molecule, two other gaseous mediators, carbon monoxide (CO) and hydrogen sulfide (H2S) have been found to be also involved in many physiological and pathophysiological functions. This review will briefly summarize our recent progress in the pathophysiology of NO and H2S. In the photoreceptor cells, the level of intracellular Ca2+ is kept relatively low by H2S. Intraperitoneal injection of H2S donor to mice protected photoreceptor cells from light-induced retinal degeneration caused by oxidative stress and elevation of intracellular Ca2+. Another gaseous mediator NO induces Ca2+ release from the endoplasmic reticulum via S-nitrosylated type 1 ryanodine receptor (RyR1) Ca2+ release channel. NO-induced Ca2+ release (NICR) was abolished in primary cultured neurons from the knock-in mice, in which the S-nitrosylation site Cys-3636 of RyR1 was replaced by Ala (Ryr1C3636A). The neurons in hippocampal CA3 region of Ryr1C3636A mice were protected against seizure-induced neuronal cell death. The result indicates that NICR is critical for status epilepticus-induced neurodegeneration. The developments in the pathophysiology of gaseous mediators in the central nervous system will provide a better pharmacological advances for the treatment of neurodegenerative diseases.

Role of Endoplasmic Reticulum-Mediated Ca2+ Signaling in Neuronal Cell Death.

SIGNIFICANCE: Properly controlled intracellular Ca2+ dynamics is crucial for regulation of neuronal function and survival in the central nervous system. The endoplasmic reticulum (ER), a major intracellular Ca2+ store, plays a critical role as a source and sink for neuronal Ca2+. Recent Advances: Accumulating evidence indicates that disrupted ER Ca2+ signaling is involved in neuronal cell death under various pathological conditions, providing novel insight into neurodegenerative disease mechanisms. CRITICAL ISSUES: We summarize current knowledge concerning the relationship between abnormal ER Ca2+ dynamics and neuronal cell death. We also introduce recent technical advances for probing ER intraluminal Ca2+ dynamics with unprecedented spatiotemporal resolution. FUTURE DIRECTIONS: Further studies on ER Ca2+ signaling are expected to provide progress for unmet medical needs in neurodegenerative disease. Antioxid. Redox Signal. 29, 1147-1157.

Essential Roles of Natural Products and Gaseous Mediators on Neuronal Cell Death or Survival.

Although precise cellular and molecular mechanisms underlying neurodegeneration still remain enigmatic, key factors associated with degenerative disorders, such as glutamate toxicity and oxidative stress, have been recently identified. Accordingly, there has been growing interest in examining the effects of exogenous and endogenous molecules on neuroprotection and neurodegeneration. In this paper, we review recent studies on neuroprotective and/or neurodegenerative effects of natural products, such as caffeic acid and chlorogenic acid, and gaseous mediators, including hydrogen sulfide and nitric oxide. Furthermore, possible molecular mechanisms of these molecules in relation to glutamate signals are discussed. Insight into the pathophysiological role of these molecules will make progress in our understanding of molecular mechanisms underlying neurodegenerative diseases, and is expected to lead to potential therapeutic approaches.

Nitric Oxide-induced Activation of the Type 1 Ryanodine Receptor Is Critical for Epileptic Seizure-induced Neuronal Cell Death.

Status epilepticus (SE) is a life-threatening emergency that can cause neurodegeneration with debilitating neurological disorders. However, the mechanism by which convulsive SE results in neurodegeneration is not fully understood. It has been shown that epileptic seizures produce markedly increased levels of nitric oxide (NO) in the brain, and that NO induces Ca2+ release from the endoplasmic reticulum via the type 1 ryanodine receptor (RyR1), which occurs through S-nitrosylation of the intracellular Ca2+ release channel. Here, we show that through genetic silencing of NO-induced activation of the RyR1 intracellular Ca2+ release channel, neurons were rescued from seizure-dependent cell death. Furthermore, dantrolene, an inhibitor of RyR1, was protective against neurodegeneration caused by SE. These results demonstrate that NO-induced Ca2+ release via RyR is involved in SE-induced neurodegeneration, and provide a rationale for the use of RyR1 inhibitors for the prevention of brain damage following SE.

Whisker experience-dependent mGluR signaling maintains synaptic strength in the mouse adolescent cortex.

Sensory experience-dependent plasticity in the somatosensory cortex is a fundamental mechanism of adaptation to the changing environment not only early in the development but also in adolescence and adulthood. Although the mechanisms underlying experience-dependent plasticity during early development have been well documented, the corresponding understanding in the mature cortex is less complete. Here, we investigated the mechanism underlying whisker deprivation-induced synaptic plasticity in the barrel cortex in adolescent mice. Layer 4 (L4) to L2/3 excitatory synapses play a crucial role for whisker experience-dependent plasticity in rodent barrel cortex and whisker deprivation is known to depress synaptic strength at L4-L2/3 synapses in adolescent and adult animals. We found that whisker deprivation for 5 days or longer decreased the presynaptic glutamate release probability at L4-L2/3 synapses in the barrel cortex in adolescent mice. This whisker deprivation-induced depression was restored by daily administration of a positive allosteric modulator of the type 5 metabotropic glutamate receptor (mGluR5). On the other hand, the administration of mGluR5 antagonists reproduced the effect of whisker deprivation in whisker-intact mice. Furthermore, chronic and selective suppression of inositol 1,4,5-trisphosphate (IP3 ) signaling in postsynaptic L2/3 neurons decreased the presynaptic release probability at L4-L2/3 synapses. These findings represent a previously unidentified mechanism of cortical plasticity, namely that whisker experience-dependent mGluR5-IP3 signaling in the postsynaptic neurons maintains presynaptic function in the adolescent barrel cortex.

Chlorogenic acid, a polyphenol in coffee, protects neurons against glutamate neurotoxicity.

AIMS: The present study has been designed to explore the molecular mechanism of chlorogenic acid (CGA) in the protective effect against glutamate-induced neuronal cell death. MAIN METHODS: Cortical neurons in primary culture were exposed to 300 µM l-glutamic acid or vehicle, with or without 10 µM CGA or 10 µM MK-801. After 16 h, primary cultures were stained with propidium iodide (PI)/Hoechst or calcein. Double-staining with PI and Hoechst was performed to confirm whether cell death induced by glutamate was apoptotic. In addition, intracellular concentrations of Ca(2+) were observed using Ca(2+) indicator fura-2. KEY FINDINGS: We investigated the protective effects of CGA on glutamate-induced neuronal cell death using primary cultures of mouse cerebral cortex because the release of glutamate during brain ischemia triggers death of neurons. Glutamate-induced neuronal cell death was inhibited by treatment with CGA. In addition, CGA prevented the increase in intracellular concentrations of Ca(2+) caused by the addition of glutamate to cultured neurons. On the other hand, there was little effect of CGA on cell death induced by nitric oxide, which is downstream of the ischemic neuronal cell death. Our results suggested that the polyphenol CGA in coffee protects neurons from glutamate neurotoxicity by regulating Ca(2+) entry into neurons. SIGNIFICANCE: CGA in coffee may have clinical benefits for neurodegenerative diseases such as ischemic stroke.

Polysulfides are possible H2S-derived signaling molecules in rat brain.

Accumulating evidence shows that hydrogen sulfide (H2S) has a variety of physiological functions. H2S is produced from cysteine by 3 sulfurtransferases. H2S, in turn, generates polysulfides, the functions of which are not well understood. H2S induces Ca(2+) influx in astrocytes, a type of glia. However, the receptor that mediates the response has not been identified. Here, we have shown that polysulfides induce Ca(2+) influx by activating transient receptor potential (TRP)A1 channels in rat astrocytes (EC50 91 nM, Hill coefficient value 1.77±0.26) and that the maximum response was induced at 0.5 µM, which is 1/320 of the concentration of H2S required to achieve a response of similar magnitude (160 µM, EC50 116 µM). TRPA1-selective agonists, allyl isothiocyanate and cinnamaldehyde, induced Ca(2+) influx, and responses to polysulfides were suppressed by TRPA1-selective inhibitors, HC-030031 and AP-18, as well as by siRNAs selective to TRPA1. The present study suggests that polysulfides are possible H2S-derived signaling molecules that stimulate TRP channels in the brain.

Hydrogen sulfide is produced by cystathionine γ-lyase at the steady-state low intracellular Ca(2+) concentrations.

Hydrogen sulfide (H(2)S) is recognized as a physiologic mediator produced in a variety of tissues. It is produced by three enzymes, cystathionine ß-synthase (CBS), cystathionine γ-lyase (CSE) and 3-mercaptopyruvate sulfurtransferase (3MST). However, the regulation of H(2)S production by CSE has not well been understood. Here we show that H(2)S producing activity of CSE is regulated by intracellular Ca(2+) concentrations. In the presence of pyridoxal 5'-phosphate (PLP) CSE efficiently produces H(2)S at the steady-state low Ca(2+) concentrations but is suppressed at high Ca(2+) concentrations. In the absence of PLP H(2)S production maintains the suppressed levels at high Ca(2+) concentrations and decreased further at low Ca(2+) concentrations. These observations suggest that CSE produces H(2)S at the steady-state in cells and that the production is suppressed when the intracellular Ca(2+) concentrations are increased.

A mechanism of retinal protection from light-induced degeneration by hydrogen sulfide.

Since our initial demonstrations that hydrogen sulfide (H(2)S) may function as a neuromodulator in the brain and a smooth muscle relaxant in the vascular system, accumulating evidence shows that H(2)S may function as a signaling molecule. We and others also found that H(2)S has a cytoprotective effect. Because H(2)S is well-known toxic gas, a cytoprotective role has been overlooked. H(2)S protects neurons from oxidative stress. It also protects cardiac muscle from ischemia-reperfusion injury. The finding led to the application of H(2)S to the bypass surgery patients in Phase II clinical trial. Cystathionine ß-synthase (CBS) and cystathionine γ-lyase (CSE) are well known as H(2)S-producing enzymes. We recently demonstrated that the other H(2)S-producing enzyme, 3-mercaptopyruvate sulfurtransferase (3MST) along with cysteine aminotransferase (CAT) is localized to neurons in the brain and to the vascular endothelium. However, the regulation of H(2)S production by 3MST/CAT pathway had not been well understood. The present study shows that H(2)S production by 3MST/CAT pathway is regulated by Ca(2+) and that H(2)S protects retinal photoreceptor cells from light induced degeneration by suppressing excessive Ca(2+) influx caused by intense light.

Development of a highly selective fluorescence probe for hydrogen sulfide.

Hydrogen sulfide (H(2)S) has recently been identified as a biological response modifier. Here, we report the design and synthesis of a novel fluorescence probe for H(2)S, HSip-1, utilizing azamacrocyclic copper(II) ion complex chemistry to control the fluorescence. HSip-1 showed high selectivity and high sensitivity for H(2)S, and its potential for biological applications was confirmed by employing it for fluorescence imaging of H(2)S in live cells.

Hydrogen sulfide protects the retina from light-induced degeneration by the modulation of Ca2+ influx.

Hydrogen sulfide (H(2)S) has recently been recognized as a signaling molecule as well as a cytoprotectant. Cystathionine ß-synthase (CBS) and cystathionine γ-lyase (CSE) are well-known as H(2)S-producing enzymes. We recently demonstrated that 3-mercaptopyruvate sulfurtransferase (3MST) along with cysteine aminotransferase (CAT) produces H(2)S in the brain and in vascular endothelium. However, the cellular distribution and regulation of these enzymes are not well understood. Here we show that 3MST and CAT are localized to retinal neurons and that the production of H(2)S is regulated by Ca(2+); H(2)S, in turn, regulates Ca(2+) influx into photoreceptor cells by activating vacuolar type H(+)-ATPase (V-ATPase). We also show that H(2)S protects retinal neurons from light-induced degeneration. The excessive levels of light exposure deteriorated photoreceptor cells and increased the number of TUNEL- and 8-hydroxy-2'-deoxyguanosine (8-OHdG)-positive cells. Degeneration was greatly suppressed in the retina of mice administered with NaHS, a donor of H(2)S. The present study provides a new insight into the regulation of H(2)S production and the modulation of the retinal transmission by H(2)S. It also shows a cytoprotective effect of H(2)S on retinal neurons and provides a basis for the therapeutic target for retinal degeneration.

Protein tyrosine phosphatase σ regulates the synapse number of zebrafish olfactory sensory neurons.

The formation and refinement of synaptic connections are key steps of neural development to establish elaborate brain networks. To investigate the functional role of protein tyrosine phosphatase (PTP) σ, we employed an olfactory sensory neuron (OSN)-specific gene manipulation system in combination with in vivo imaging of transparent zebrafish embryos. Knockdown of PTPσ enhanced the accumulation of synaptic vesicles in the axon terminals of OSNs. The exaggerated accumulation of synaptic vesicles was restored to the normal level by the OSN-specific expression of PTPσ, indicating that presynaptic PTPσ is responsible for the regulation of synaptic vesicle accumulation. Consistently, transient expression of a dominant-negative form of PTPσ in OSNs enhanced the accumulation of synaptic vesicles. The exaggerated accumulation of synaptic vesicles was reproduced in transgenic zebrafish lines carrying an OSN-specific expression vector of the dominant-negative PTPσ. By electron microscopic analysis of the transgenic line, we found the significant increase of the number of OSN-mitral cell synapses in the central zone of the olfactory bulb. The density of docked vesicles at the active zone was also increased significantly. Our results suggest that presynaptic PTPσ controls the number of OSN-mitral cell synapses by suppressing their excessive increase.

Thioredoxin and dihydrolipoic acid are required for 3-mercaptopyruvate sulfurtransferase to produce hydrogen sulfide.

H2S (hydrogen sulfide) has recently been recognized as a signalling molecule as well as a cytoprotectant. We recently demonstrated that 3MST (3-mercaptopyruvate sulfurtransferase) produces H2S from 3MP (3-mercaptopyruvate). Although a reducing substance is required for an intermediate persulfide at the active site of 3MST to release H2S, the substance has not been identified. In the present study we show that Trx (thioredoxin) and DHLA (dihydrolipoic acid) associate with 3MST to release H2S. Other reducing substances, such as NADPH, NADH, GSH, cysteine and CoA, did not have any effect on the reaction. We also show that 3MST produces H2S from thiosulfate. The present study provides a new insight into a mechanism for the production of H2S by 3MST.

Identification and characterization of a novel zebrafish semaphorin.

The semaphorin gene family contains numerous secreted and transmembrane proteins. Some of them function as the repulsive and attractive axon guidance molecules during development. Herein, we report the cloning and characterization of a novel member of zebrafish semaphorin gene, semaphorin 6E (sema6E). Sema6E is expressed predominantly in the nervous system during embryogenesis. Results also show that Sema6E binds Plexin-A1, but not other Plexins. Sema6E chemorepels not only dorsal root ganglion axons but also sympathetic axons. Therefore, Sema6E might utilize Plexin-A1 as a receptor to repel axons of the specific types during development.

Vascular endothelium expresses 3-mercaptopyruvate sulfurtransferase and produces hydrogen sulfide.

Hydrogen sulfide (H(2)S) has been recognized as a smooth muscle relaxant. Cystathionine gamma-lyase, which is localized to smooth muscle, is thought to be the major H(2)S-producing enzyme in the thoracic aorta. Here we show that 3-mercaptopyruvate sulfurtransferase (3MST) and cysteine aminotransferase (CAT) are localized to vascular endothelium in the thoracic aorta and produce H(2)S. Both 3MST and CAT were localized to endothelium. Lysates of vascular endothelial cells produced H(2)S from cysteine and alpha-ketoglutarate. The present study provides a new insight into the production and release of H(2)S as a smooth muscle relaxant from vascular endothelium.

Identification and characterization of zebrafish semaphorin 6D.

The semaphorin gene family contains a large number of secreted and transmembrane proteins; some function as repulsive and attractive cues of axon guidance during development. Here, we report cloning and characterization of zebrafish transmembrane semaphorin gene, semaphorin 6D (sema6D). Sema6D is expressed predominantly in the nervous system during embryogenesis, as determined by in situ hybridization. We also found that Sema6D binds Plexin-A1 in vitro, but not other Plexins. It induces the repulsion of dorsal root ganglion axons, but not sympathetic axons. Consequently, Sema6D might use Plexin-A1 as a receptor to repel specific types of axons during development.

Identification of amino acid residues in the Ah receptor involved in ligand binding.

The Ah receptor (AhR) is a ligand-activated transcription factor. Five amino acids as candidate amino acids necessary for ligand binding within or near the ligand-binding domain were selected based on their evolutional conservation and their aromatic nature that could interact with xenobiotic ligands. These amino acids were changed to Ala, and the mutated AhRs were subjected to a test of their transactivation activity in HeLa cells. Mutation of Phe318 completely lost its activity whereas other mutations only weakly impaired activity. The Leu-substituted mutant, AhR(Phe318Leu), activated the luciferase activity to the level comparable to wild type in the cells treated with 3-methylcholanthrene (MC) but not at all with beta-naphthoflavone (beta-NF). Ligand-binding activity of mutants was examined with [3H]MC in vitro. AhR(Phe318Ala) could not bind to [3H]MC. [3H]MC bound by AhR(Phe318Leu) was competed with unlabeled MC but not with beta-NF. A structural model of the ligand-binding domain was constructed.

Expression of zebrafish glutamate receptor delta2 in neurons with cerebellum-like wiring.

Mammalian glutamate receptor (GluR) delta2 is selectively expressed in cerebellar Purkinje cells and plays key roles in cerebellar plasticity, motor learning, and neural wiring. Here, we isolated cDNA encoding the zebrafish ortholog of mammalian GluRdelta2. We found that in adult zebrafish brain, glurdelta2 mRNA was expressed not only in cerebellar Purkinje cells, but also in the crest cells of the medial octavolateral nucleus (MON) and the type I neurons of the optic tectum. Immunohistochemical analysis revealed that zebrafish GluRdelta2 proteins were selectively localized in the apical dendrites of these neurons. Interestingly, the crest cells of the MON and the type I neurons of the optic tectum receive large numbers of parallel fiber inputs at the apical dendrites and sensory inputs at the proximal or basal dendrites. These results suggest that the expression of zebrafish GluRdelta2 is selective for cerebellum-like neural wiring with large numbers of parallel fiber inputs.