Activator of KAT3 histone acetyltransferase family ameliorates a neurodevelopmental disorder phenotype in the syntaxin 1A ablated mouse model.

Syntaxin-1A (stx1a) repression causes a neurodevelopmental disorder phenotype, low latent inhibition (LI) behavior, by disrupting 5-hydroxytryptaminergic (5-HTergic) systems. Herein, we discovered that lysine acetyltransferase (KAT) 3B increases stx1a neuronal transcription and TTK21, a KAT3 activator, induces stx1a transcription and 5-HT release in vitro. Furthermore, glucose-derived CSP-TTK21 could restore decreased stx1a expression, 5-HTergic systems in the brain, and low LI in stx1a (+/-) mice by crossing the blood-brain barrier, whereas the KAT3 inhibitor suppresses stx1a expression, 5-HTergic systems, and LI behaviors in wild-type mice. Finally, in wild-type and stx1a (-/-) mice treated with IKK inhibitors and CSP-TTK21, respectively, we show that KAT3 activator-induced LI improvement is a direct consequence of KAT3B-stx1a pathway, not a side effect. In conclusion, KAT3B can positively regulate stx1a transcription in neurons, and increasing neuronal stx1a expression and 5-HTergic systems by a KAT3 activator consequently improves the low LI behavior in the stx1a ablation mouse model.

Golgi stress induces upregulation of the ER-Golgi SNARE Syntaxin-5, altered ßAPP processing, and Caspase-3-dependent apoptosis in NG108-15 cells.

The involvement of secretory pathways and Golgi dysfunction in neuronal cells during Alzheimer's disease progression is poorly understood. Our previous overexpression and knockdown studies revealed that the intracellular protein level of Syntaxin-5, an endoplasmic reticulum-Golgi soluble N-ethylmaleimide-sensitive factor-attachment protein receptor (SNARE), modulates beta-amyloid precursor protein processing in neuronal cells. We recently showed that changes in endogenous Syntaxin-5 protein expression occur under stress induction. Syntaxin-5 was upregulated by endoplasmic reticulum stress but was degraded by Caspase-3 during apoptosis in neuronal cells. In addition, we showed that sustained endoplasmic reticulum stress promotes Caspase-3-dependent apoptosis during the later phase of the endoplasmic reticulum stress response in NG108-15 cells. In this study, to elucidate the consequences of secretory pathway dysfunction in beta-amyloid precursor protein processing that lead to neuronal cell death, we examined the effect of various stresses on endoplasmic reticulum-Golgi SNARE expression and beta-amyloid precursor protein processing. By using compounds to disrupt Golgi function, we show that Golgi stress promotes upregulation of the endoplasmic reticulum-Golgi SNARE Syntaxin-5, and prolonged stress causes Caspase-3-dependent apoptosis. Golgi stress induced intracellular beta-amyloid precursor protein accumulation and a concomitant decrease in total amyloid-beta production. We also examined the protective effect of the chemical chaperone 4-phenylbutylate on changes in amyloid-beta production and the activation of Caspase-3 induced by endoplasmic reticulum and Golgi stress. The compound alleviated the increase in the amyloid-beta 1-42/amyloid-beta 1-40 ratio induced by endoplasmic reticulum and Golgi stress. Furthermore, 4-phenylbutylate could rescue Caspase-3-dependent apoptosis induced by prolonged organelle stress. These results suggest that organelle stress originating from the endoplasmic reticulum and Golgi has a substantial impact on the amyloidogenic processing of beta-amyloid precursor protein and Caspase-3-dependent apoptosis, leading to neuronal cell death.

Disturbance of the reciprocal-interaction between the OXTergic and DAergic systems in the CNS causes atypical social behavior in syntaxin 1A knockout mice.

Several studies have shown that oxytocin (OXT) modulates social behavior. Similarly, monoamines such as dopamine (DA) play a role in regulating social behavior. Previous studies have demonstrated that the soluble N-ethylmaleimide-sensitive fusion attachment protein receptor (SNARE) protein syntaxin 1A (STX1A) regulates the secretion of OXT and monoamines, and that STX1A gene knockout (STX1A KO) mice exhibit atypical social behavior, such as deficient social recognition, due to reduced OXT release. In this study, we analyzed the neural mechanism regulating social behavior by OXT and/or DA using STX1A KO mice as a model animal. We found that OXT directly induced DA release from cultured DA neurons through OXT and V1a receptors. In STX1A KO mice, the atypical social behavior was partially improved by OXT administration, which was inhibited by D1 receptor blockade. In addition, the atypical social behavior in STX1A KO mice was partially improved by facilitation of DAergic signaling with the DA reuptake inhibitor GBR12909. Moreover, the amelioration by GBR12909 was inhibited by OXTR blockade. These results suggest that the reciprocal interaction between the DAergic and OXTergic neuronal systems in the CNS may be important in regulating social behavior.

Syntaxin 1A Gene Is Negatively Regulated in a Cell/Tissue Specific Manner by YY1 Transcription Factor, Which Binds to the -183 to -137 Promoter Region Together with Gene Silencing Factors Including Histone Deacetylase.

The HPC-1/syntaxin 1A (Stx1a) gene, which is involved in synaptic transmission and neurodevelopmental disorders, is a TATA-less gene with several transcription start sites. It is activated by the binding of Sp1 and acetylated histone H3 to the -204 to +2 core promoter region (CPR) in neuronal cell/tissue. Furthermore, it is depressed by the association of class 1 histone deacetylases (HDACs) to Stx1a-CPR in non-neuronal cell/tissue. To further clarify the factors characterizing Stx1a gene silencing in non-neuronal cell/tissue not expressing Stx1a, we attempted to identify the promoter region forming DNA-protein complex only in non-neuronal cells. Electrophoresis mobility shift assays (EMSA) demonstrated that the -183 to -137 OL2 promoter region forms DNA-protein complex only in non-neuronal fetal rat skin keratinocyte (FRSK) cells which do not express Stx1a. Furthermore, the Yin-Yang 1 (YY1) transcription factor binds to the -183 to -137 promoter region of Stx1a in FRSK cells, as shown by competitive EMSA and supershift assay. Chromatin immunoprecipitation assay revealed that YY1 in vivo associates to Stx1a-CPR in cell/tissue not expressing Stx1a and that trichostatin A treatment in FRSK cells decreases the high-level association of YY1 to Stx1a-CPR in default. Reporter assay indicated that YY1 negatively regulates Stx1a transcription. Finally, mass spectrometry analysis showed that gene silencing factors, including HDAC1, associate onto the -183 to -137 promoter region together with YY1. The current study is the first to report that Stx1a transcription is negatively regulated in a cell/tissue-specific manner by YY1 transcription factor, which binds to the -183 to -137 promoter region together with gene silencing factors, including HDAC.

Syntaxin 1B regulates synaptic GABA release and extracellular GABA concentration, and is associated with temperature-dependent seizures.

De novo heterozygous mutations in the STX1B gene, encoding syntaxin 1B, cause a familial, fever-associated epilepsy syndrome. Syntaxin 1B is an essential component of the pre-synaptic neurotransmitter release machinery as a soluble N-ethylmaleimide-sensitive factor attachment protein receptor protein that regulates the exocytosis of synaptic vesicles. It is also involved in regulating the functions of the SLC6 family of neurotransmitter transporters that reuptake neurotransmitters, including inhibitory neurotransmitters, such as γ-aminobutyric acid (GABA) and glycine. The purpose of the present study was to elucidate the molecular mechanisms underlying the development of febrile seizures by examining the effects of syntaxin 1B haploinsufficiency on inhibitory synaptic transmission during hyperthermia in a mouse model. Stx1b gene heterozygous knockout (Stx1b+/- ) mice showed increased susceptibility to febrile seizures and drug-induced seizures. In cultured hippocampal neurons, we examined the temperature-dependent properties of neurotransmitter release and reuptake by GABA transporter-1 (GAT-1) at GABAergic neurons using whole-cell patch-clamp recordings. The rate of spontaneous quantal GABA release was reduced in Stx1b+/- mice. The hyperthermic temperature increased the tonic GABAA current in wild-type (WT) synapses, but not in Stx1b+/- synapses. In WT neurons, recurrent bursting activities were reduced in a GABA-dependent manner at hyperthermic temperature; however, this was abolished in Stx1b+/- neurons. The blockade of GAT-1 increased the tonic GABAA current and suppressed recurrent bursting activities in Stx1b+/- neurons at the hyperthermic temperature. These data suggest that functional abnormalities associated with GABA release and reuptake in the pre-synaptic terminals of GABAergic neurons may increase the excitability of the neural circuit with hyperthermia.

Syntaxin 1B contributes to regulation of the dopaminergic system through GABA transmission in the CNS.

In neuronal plasma membrane, two syntaxin isoforms, HPC-1/syntaxin 1A (STX1A) and syntaxin 1B (STX1B), are predominantly expressed as soluble N-ethylmaleimide-sensitive fusion attachment protein receptors, also known as t-SNAREs. We previously reported that glutamatergic and GABAergic synaptic transmissions are impaired in Stx1b null mutant (Stx1b-/- ) mice but are almost normal in Stx1a null mutant (Stx1a-/- ) mice. These observations suggested that STX1A and STX1B have distinct functions in fast synaptic transmission in the central nervous system (CNS). Interestingly, recent studies indicated that Stx1a-/- or Stx1a+/- mice exhibit disruption in the monoaminergic system in the CNS, causing unusual behaviour that is similar to neuropsychological alterations observed in psychiatric patients. Here, we studied whether STX1B contributes to the regulation of monoaminergic system and if STX1B is related to neuropsychological properties in human neuropsychological disorders similar to STX1A. We found that monoamine release in vitro was normal in Stx1b+/- mice unlike Stx1a-/- or Stx1a+/- mice, but the basal extracellular dopamine (DA) concentration in the ventral striatum was increased. DA secretion in the ventral striatum is regulated by GABAergic neurons, and Stx1b+/- mice exhibited reduced GABA release both in vitro and in vivo, disrupting the DAergic system in the CNS of these mice. We also found that Stx1b+/- mice exhibited reduced pre-pulse inhibition (PPI), which is believed to represent one of the prominent schizotypal behavioural profiles of human psychiatric patients. The reduction in PPI was rescued by DA receptor antagonists. These observations indicated that STX1B contributes to excess activity of the DAergic system through regulation of GABAergic transmission.

Transcription regulation mechanism of the syntaxin 1A gene via protein kinase A.

Syntaxin 1A (Stx1a) is primarily involved in the docking of synaptic vesicles at active zones in neurons. Its gene is a TATA-less gene, with several transcription initiation sites, which is activated by the binding of Sp1 and acetylated histone H3 (H3) in the core promoter region (CPR) through the derepression of class I histone deacetylase (HDAC). In the present study, to clarify the factor characterizing Stx1a gene expression via the protein kinase A (PKA) pathway inducing the Stx1a mRNA, we investigated whether the epigenetic process is involved in the Stx1a gene transcription induced by PKA signaling. We found that the PKA activator forskolin induced Stx1a expression in non-neuronal cells, FRSK and 3Y1, which do not endogenously express Stx1a, unlike PC12. HDAC8 inhibition by shRNA knockdown and specific inhibitors induced Stx1a expression in FRSK. The PKA inhibitor H89 suppressed HDAC8-Ser39 phosphorylation, H3 acetylation and Stx1a induction by forskolin in FRSK cells. Finally, we also found that forskolin led to the dissociation of HDAC8-CPR interaction and the association of Sp1 and Ac-H3 to CPR in FRSK. The results of the current study suggest that forskolin phosphorylates HDAC8-Ser39 via the PKA pathway and increases histone H3 acetylation in cells expressing HDAC8, resulting in the induction of the Stx1a gene.

A part of patients with autism spectrum disorder has haploidy of HPC-1/syntaxin1A gene that possibly causes behavioral disturbance as in experimentally gene ablated mice.

Autism spectrum disorder (ASD) is highly heritable and encompasses a various set of neuropsychiatric disorders with a wide-ranging presentation. HPC-1/syntaxin1A (STX1A) encodes a neuronal plasma membrane protein that regulates the secretion of neurotransmitters and neuromodulators. STX1A gene ablated mice (null and heterozygote mutant) exhibit abnormal behavioral profiles similar to human autistic symptoms, accompanied by reduction of monoamine secretion. To determine whether copy number variation of STX1A gene and the change of its expression correlate with ASD as in STX1A gene ablated mice, we performed copy number assay and real-time quantitative RT-PCR using blood or saliva samples from ASD patients. We found that some ASD patients were haploid for the STX1A gene similar to STX1A heterozygote mutant mice. However, copy number of STX1A gene was normal in the parents and siblings of ASD patients with STX1A gene haploidy. In ASD patients with gene haploidy, STX1A mRNA expression was reduced to about half of their parents. Thus, a part of ASD patients had haploidy of STX1A gene and lower STX1A gene expression.

Unusual social behavior in HPC-1/syntaxin1A knockout mice is caused by disruption of the oxytocinergic neural system.

HPC-1/syntaxin1A (STX1A), a neuronal soluble N-ethylmaleimide-sensitive fusion attachment protein receptor, contributes to neural function in the CNS by regulating transmitter release. Recent studies reported that STX1A is associated with human neuropsychological disorders, such as autism spectrum disorder and attention deficit hyperactivity disorder. Previously, we showed that STX1A null mutant mice (STX1A KO) exhibit neuropsychological abnormalities, such as fear memory deficits, attenuation of latent inhibition, and unusual social behavior. These observations suggested that STX1A may be involved in the neuropsychological basis of these abnormalities. Here, to study the neural basis of social behavior, we analyzed the profile of unusual social behavior in STX1A KO with a social novelty preference test, which is a useful method for quantification of social behavior. Interestingly, the unusual social behavior in STX1A KO was partially rescued by intracerebroventricular administration of oxytocin (OXT). In vivo microdialysis studies revealed that the extracellular OXT concentration in the CNS of STX1A KO was significantly lower compared with wild-type mice. Furthermore, dopamine-induced OXT release was reduced in STX1A KO. These results suggested that STX1A plays an important role in social behavior through regulation of the OXTergic neural system. Dopamine (DA) release is reduced in CNS of syntaxin1A null mutant mice (STX1A KO). Unusual social behavior was observed in STX1A KO. We found that oxytocin (OXT) release, which was stimulated by DA, was reduced and was rescued the unusual social behavior in STX1A KO was rescued by OXT. These results indicated that STX1A plays an important role in promoting social behavior through regulation of DA-induced OXT release in amygdala.

Time lapse imaging analysis of the effect of ER stress modulators on apoptotic cell assessed by caspase3/7 activation in NG108-15 cells.

This paper reports the data from the long term time lapse imaging of neuronal cell line NG108-15 that were treated with apoptosis inducer or various ER stress inducers. Use of the fluorescent reporter for activated caspase3/7 in combination with the conventional light microscope allowed us to investigate the time course of apoptosis induction at the single cell level. Quantitative as well as qualitative data are presented here to show the effect of two different ER stress modulating chemical compounds on caspase3/7-dependent apoptosis in neuronal cell line NG108-15 cells. Additional results and interpretation of our data concerning ER stress and apoptosis in NG108-15 cells can be found in Suga et al. (2015) [1] and in Suga et al. (2015) [2].

The cell- and tissue-specific transcription mechanism of the TATA-less syntaxin 1A gene.

Syntaxin 1A (Stx1a) plays an important role in regulation of neuronal synaptic function. To clarify the mechanism of basic transcriptional regulation and neuron-specific transcription of Stx1a we cloned the Stx1a gene from rat, in which knowledge of the expression profile was accumulated, and elucidated that Stx1a consisting of 10 exons, possesses multiple transcription initiation sites and a 204-bp core promoter region (CPR) essential for transcription in PC12 cells. The TATA-less, conserved, GC-rich CPR has 2 specific protein (SP) sites that bind SP1 and are responsible for 65% of promoter activity. The endogenous CPR, including 23 CpG sites, is not methylated in PC12 cells, which express Stx1a and fetal rat skin keratinocyte (FRSK) cells, which do not, although an exogenous methylated CPR suppresses reporter activity in both lines. Trichostatin A (TSA) and class I histone deacetylase (HDAC) inhibitors, but not 5-azacytidine, induce Stx1a in FRSK cells. Acetylated histone H3 only associates to the CPR in FRSK cells after TSA addition, whereas the high acetylated histone H3-CPR association in PC12 cells was unchanged following treatment. HDAC inhibitor induction of Stx1a was negated by mithramycin A and deletion/mutation of 2 SP sites. HDAC1, HDAC2, and HDAC8 detach from the CPR when treated with TSA in FRSK cells and are associated with the CPR in lungs, and acetylated histone H3 associates to this region in the brain. In the first study characterizing a syntaxin promoter, we show that association of SP1 and acetylated histone H3 to CPR is important for Stx1a transcription and that HDAC1, HDAC2, and HDAC8 decide cell/tissue specificity in a suppressive manner.

Data supporting ER stress response in NG108-15 cells involves upregulation of syntaxin 5 expression and reduced amyloid ß peptide secretion.

This data contains insights into the upregulation of the ER-Golgi-soluble N-ethylmaleimide-sensitive factor-attachment protein receptors (ER-Golgi SNAREs) syntaxin 5 (Syx5) by ER stress and the downregulation of Syx5 by apoptosis induction. Use of the protein synthesis inhibitor verified the de novo synthesis of Syx5 under ER stress in NG108-15 cells. We also provide validation data for the increase of Syx5 expression caused by ER stress using different chemical compound and overexpression analysis. Interpretation of our data and further extensive insights into the role of Syx5 in ßAPP processing under ER stress can be found in Suga et al. (2015)[1].

Data for the effects of ER and Golgi stresses on the ER-Golgi SNARE Syntaxin5 expression and on the ßAPP processing in cultured hippocampal neurons.

This paper reports the data for the effects of organelle stresses on the ER-Golgi-soluble N-ethylmaleimide-sensitive factor-attachment protein receptors (ER-Golgi SNAREs) syntaxin 5 (Syx5) in neuronal cells. Quantitative as well as qualitative data are presented here to verify the upregulation of Syntaxin 5 (Syx5) under ER and Golgi stresses in hippocampal neurons. Changes in the processing of ß-amyloid precursor protein (ßAPP) under ER stress were analyzed by immunological assays. In addition, our data shows the specific increase of Syx5 expression under ER and Golgi stresses. Interpretation of our data and further extensive insights into the role of Syx5 in ßAPP processing under organelle stress can be found in "ER and Golgi stresses increase ER-Golgi SNARE Syntaxin5: Implications for organelle stress and ßAPP processing" [1].

ER and Golgi stresses increase ER-Golgi SNARE Syntaxin5: Implications for organelle stress and ßAPP processing.

Unresolved endoplasmic reticulum (ER) stress causes neuronal death and has been implicated in neurodegenerative conditions such as Alzheimer's disease (AD). However, the mechanisms by which stress signals propagate from the ER through the Golgi apparatus and their effects on the transport and processing of AD-related proteins, such as ß-amyloid precursor protein (ßAPP), are unknown. We recently found that in the NG108-15 cell line, ER stress upregulates ER-Golgi-soluble N-ethylmaleimide-sensitive factor-attachment protein receptors (ER-Golgi SNAREs) Syx5 and Bet1. In the present study, we examined the effects of apoptosis and ER stress inducers on the expression of ER-Golgi SNARE proteins and cell viability in a primary culture of rat hippocampal neurons. An apoptosis inducer significantly downregulated the expression of ER-Golgi SNARE Syx5. ER-stress inducers upregulated the expression of Syx5 isoforms and Bet1 proteins via de novo synthesis of their mRNA transcripts. Knockdown of Syx5 during apoptosis or ER stress induction enhanced vulnerability of neurons. Additionally, we examined the effects of Golgi stress on Syx5 expression and ßAPP processing. Golgi stress also induced upregulation of ER-Golgi SNARE Syx5, and concomitantly, suppressed amyloid-ß peptide secretion. These findings suggest that Syx5 is a potential stress responsive factor that participates in ßAPP processing and the survival pathways of neuronal cells.

ER stress response in NG108-15 cells involves upregulation of syntaxin 5 expression and reduced amyloid ß peptide secretion.

Endoplasmic reticulum (ER) stress plays a role in the pathogenesis of neurodegenerative diseases such as Alzheimer׳s disease (AD). We previously showed that manipulation of the ER-Golgi-soluble N-ethylmaleimide-sensitive factor-attachment protein receptors (ER-Golgi SNARE) syntaxin 5 (Syx5) causes changes in Golgi morphology and the processing of AD-related proteins. To understand the pathophysiologic significance of these phenomena, we examined whether the expression of Syx5 is altered by ER stress. De novo synthesis of ER-Golgi SNARE Syx5 and Bet1 was induced by various ER stressors. Elevated expression of Syx5 and Bet1 was associated with increased levels of these proteins in vesicular components, including ER-Golgi-intermediate-compartment/vesicular tubular clusters. In addition, ER stress diminished amyloid ß (Aß) peptide secretion. Knockdown of Syx5 expression enhanced the secretion of Aß peptides under condition without ER stress. Moreover, diminished Aß peptide secretion resulting from ER stress was significantly reversed by Syx5 knockdown. These findings suggest that Syx5 plays important roles in ß-amyloid precursor protein processing and in the ER stress response that precedes apoptotic cell death and may be involved in the crosstalk between these two pathways.

Syntaxin 1B, but not syntaxin 1A, is necessary for the regulation of synaptic vesicle exocytosis and of the readily releasable pool at central synapses.

Two syntaxin 1 (STX1) isoforms, HPC-1/STX1A and STX1B, are coexpressed in neurons and function as neuronal target membrane (t)-SNAREs. However, little is known about their functional differences in synaptic transmission. STX1A null mutant mice develop normally and do not show abnormalities in fast synaptic transmission, but monoaminergic transmissions are impaired. In the present study, we found that STX1B null mutant mice died within 2 weeks of birth. To examine functional differences between STX1A and 1B, we analyzed the presynaptic properties of glutamatergic and GABAergic synapses in STX1B null mutant and STX1A/1B double null mutant mice. We found that the frequency of spontaneous quantal release was lower and the paired-pulse ratio of evoked postsynaptic currents was significantly greater in glutamatergic and GABAergic synapses of STX1B null neurons. Deletion of STX1B also accelerated synaptic vesicle turnover in glutamatergic synapses and decreased the size of the readily releasable pool in glutamatergic and GABAergic synapses. Moreover, STX1A/1B double null neurons showed reduced and asynchronous evoked synaptic vesicle release in glutamatergic and GABAergic synapses. Our results suggest that although STX1A and 1B share a basic function as neuronal t-SNAREs, STX1B but not STX1A is necessary for the regulation of spontaneous and evoked synaptic vesicle exocytosis in fast transmission.

Enzyme-treated Asparagus officinalis extract shows neuroprotective effects and attenuates cognitive impairment in senescence-accelerated mice.

Increases in the number of patients with dementia involving Alzheimer's disease (AD) are seen as a grave public health problem. In neurodegenerative disorders involving AD, biological stresses, such as oxidative and inflammatory stress, induce neural cell damage. Asparagus (Asparagus officinalis) is a popular vegetable, and an extract prepared from this reportedly possesses various beneficial biological activities. In the present study, we investigated the effects of enzyme-treated asparagus extract (ETAS) on neuronal cells and early cognitive impairment of senescence-accelerated mouse prone 8 (SAMP8) mice. The expression of mRNAs for factors that exert cytoprotective and anti-apoptotic functions, such as heat-shock protein 70 and heme oxygenase-1, was upregulated in NG108-15 neuronal cells by treatment with ETAS. Moreover, when release of lactate dehydrogenase from damaged NG108-15 cells was increased for cells cultured in medium containing either the nitric oxide donor sodium nitroprusside or the hypoxia mimic reagent cobalt chloride, ETAS significantly attenuated this cell damage. Also, when contextual fear memory, which is considered to be a hippocampus-dependent memory, was significantly impaired in SAMP8 mice, ETAS attenuated the cognitive impairment. These results suggest that ETAS produces cytoprotective effects in neuronal cells and attenuates the effects on the cognitive impairment of SAMP8 mice.

HPC-1/syntaxin 1A and syntaxin 1B play distinct roles in neuronal survival.

Two types of syntaxin 1 isoforms, HPC-1/syntaxin 1A (STX1A) and syntaxin 1B (STX1B), are thought to have similar functions in exocytosis of synaptic vesicles. STX1A(-/-) mice which we generated previously develop normally, possibly because of compensation by STX1B. We produced STX1B(-/-) mice using targeted gene disruption and investigated their phenotypes. STX1B(-/-) mice were born alive, but died before postnatal day 14, unlike STX1A(-/-) mice. Morphologically, brain development in STX1B(-/-) mice was impaired. In hippocampal neuronal culture, the cell viability of STX1B(-/-) neurons was lower than that of WT or STX1A(-/-) neurons after 9 days. Interestingly, STX1B(-/-) neurons survived on WT or STX1A(-/-) glial feeder layers as well as WT neurons. However, STX1B(-/-) glial feeder layers were less effective at promoting survival of STX1B(-/-) neurons. Conditioned medium from WT or STX1A(-/-) glial cells had a similar effect on survival, but that from STX1B(-/-) did not promote survival. Furthermore, brain-derived neurotrophic factor (BDNF) or neurotrophin-3 supported survival of STX1B(-/-) neurons. BDNF localization in STX1B(-/-) glial cells was disrupted, and BDNF secretion from STX1B(-/-) glial cells was impaired. These results suggest that STX1A and STX1B may play distinct roles in supporting neuronal survival by glia. Syntaxin 1A (STX1A) and syntaxin 1B (STX1B) are thought to have similar functions as SNARE proteins. However, we found that STX1A and STX1B play distinct roles in neuronal survival using STX1A(-/-) mice and STX1B(-/-) mice. STX1B was important for neuronal survival, possibly by regulating the secretion of neurotrophic factors, such as BDNF, from glial cells.

Presynaptic inhibitory actions of pregabalin on excitatory transmission in superficial dorsal horn of mouse spinal cord: further characterization of presynaptic mechanisms.

Pregabalin is widely used as an analgesic for the treatment of neuropathic pain. In the present experiments using mouse spinal slices, we recorded electrically evoked glutamatergic excitatory postsynaptic currents (eEPSCs) from superficial dorsal horn neurons. Pregabalin reduced the amplitude of eEPSCs, and increased the paired pulse ratio. Pregabalin also inhibited the frequency of spontaneously occurring miniature EPSCs without affecting their amplitude. Partial ligation of the sciatic nerve increased the expression of the calcium channel α2δ-1 subunit, and increased the presynaptic inhibitory action of pregabalin. Intrathecal injection of antisense oligodeoxynucleotide against the α2δ-1 subunit, decreased the expression of α2δ-1 mRNA in the spinal dorsal horn, and decreased pregabalin's action. These results provide further evidence that pregabalin exerts its presynaptic inhibitory action via binding with the α2δ subunit in a state-dependent manner. Furthermore, presynaptic actions of pregabalin were attenuated in knockout mice lacking the protein syntaxin 1A, a component of the synaptic vesicle release machinery, indicating that syntaxin 1A is required for pregabalin to exert its full presynaptic inhibitory action. These observations might suggest that direct and/or indirect interactions with the presynaptic proteins composing the release machinery underlie at least some part of pregabalin's presynaptic actions.

Serotonin regulates glucose-stimulated insulin secretion from pancreatic ß cells during pregnancy.

In preparation for the metabolic demands of pregnancy, ß cells in the maternal pancreatic islets increase both in number and in glucose-stimulated insulin secretion (GSIS) per cell. Mechanisms have been proposed for the increased ß cell mass, but not for the increased GSIS. Because serotonin production increases dramatically during pregnancy, we tested whether flux through the ionotropic 5-HT3 receptor (Htr3) affects GSIS during pregnancy. Pregnant Htr3a(-/-) mice exhibited impaired glucose tolerance despite normally increased ß cell mass, and their islets lacked the increase in GSIS seen in islets from pregnant wild-type mice. Electrophysiological studies showed that activation of Htr3 decreased the resting membrane potential in ß cells, which increased Ca(2+) uptake and insulin exocytosis in response to glucose. Thus, our data indicate that serotonin, acting in a paracrine/autocrine manner through Htr3, lowers the ß cell threshold for glucose and plays an essential role in the increased GSIS of pregnancy.