Tatsuya Mishima

Analysis of Knock-Out Mice to Determine the Role of HPC-1/Syntaxin 1A in Expressing Synaptic Plasticity

The protein HPC-1/syntaxin 1A is abundantly expressed in neurons and localized in the neuronal plasma membrane. It forms a complex with SNAP-25 (25 kDa synaptosomal-associated protein) and VAMP-2 (vesicle-associated membrane protein)/synaptobrevin called SNARE (a soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor) complex, which is considered essential for synaptic vesicle exocytosis; thus, HPC-1/syntaxin 1A is considered crucial for synaptic transmission. To examine the physiological function of HPC-1/syntaxin 1A in vivo, we produced knock-out (KO) mice by targeted gene disruption. Although HPC-1/syntaxin 1A expression was completely depleted without any effect on the expression of other SNARE proteins, the KO mice were viable. They grew normally, were fertile, and displayed no difference in appearance compared with control littermate. In cultured hippocampal neurons derived from the KO mice, the basic synaptic transmission in vitro was normal. However, the mutant mice had impaired long-term potentiation in the hippocampal slice. Also, although KO mice exhibited normal spatial memory in the hidden platform test, consolidation of conditioned fear memory was impaired. Interestingly, the KO mice had impaired conditioned fear memory extinction. These observations suggest that HPC-1/syntaxin 1A may be closely related to synaptic plasticity.

Mitochondrial hyperpolarization after transient oxygen-glucose deprivation and subsequent apoptosis in cultured rat hippocampal neurons

Mitochondrial membrane potential (MMP) regulates the production of high-energy phosphate and apoptotic cascade, both occurring after ischemic impact. The timed profile of MMP differing from grading ischemic impact has to be determined. Primary rat hippocampal cultures were exposed to oxygen-glucose deprivation (OGD) for 30, 60, and 90 min and then were reoxygenated. MMP was expressed as a voltage-dependent dye, JC-1 fluorescence, under confocal microscopy. Cell viability was assessed by calcein AM and ethidium homodimer, each at 3 hours and 24 hours after 30, 60, and 90 min of OGD. The appearance of apoptosis was also evaluated by the TUNEL method at 24 hours. Hyperpolarization of MMP (2.31+/-0.94 normalized JC-1 fluorescence ratio between red and green) was observed during reoxygenation after 30 min OGD, while 60 min OGD induced depolarization (0.66+/-0.22, Valinomycin (potassium ionophore)-induced depolarization: 0.53+/-0.19). The fluorescence of mitochondria became weak after 90 min OGD. Most of the neurons were shrunken after 90 min and neurons were TUNEL-positive 24 hours after 30 min OGD, although most neurons were viable at 3 hours. A longer period of OGD induced necrosis, and most neurons remained viable after only 3 hours. Our data present that the short (30 min) OGD induced hyperpolarization of MMP during reoxygenation, while a longer OGD (60 or 90 min) induced depolarization and acute necrosis. Neurons were still viable even during hyperpolarization of mitochondria, but this hyperpolarization appears to be linked to subsequent apoptotic change.

Mitochondrial membrane potential and intracellular ATP content after transient experimental ischemia in the cultured hippocampal neuron.

Ischemia limits the delivery of oxygen and glucose to cells and disturbs the maintenance of mitochondrial membrane potential (MMP). MMP regulates the production of high-energy phosphate and apoptotic cascading. Thus, MMP is an important parameter determining the fate of neurons. Differences in the time course of MMP according to the grading of the ischemic impact have not been clarified. MMP and intracellular ATP contents were monitored before and after short-term oxygen-glucose deprivation. A primary hippocampal culture seeded in a 35 mm fenestrated dish for fluorescence microscopy was mounted in a sealed chamber for an anaerobic incubation. A continuous flow of 100% nitrogen into the chamber and a replacement of glucose-free medium allowed the condition of oxygen-glucose deprivation (OGD), thereby extrapolating ischemia. MMP was evaluated by the fluorescence of a voltage-dependent dye, JC-1, under fluorescence microscopy. The intracellular ATP content was evaluated in a hippocampal culture seeded in a 96-well plate by the luciferin-luciferase reaction after a designated period of OGD. During OGD, MMP decreased to 0.72+/-0.03 (normalized JC-1 fluorescence), then increased to the hyperpolarized level 1.99+/-0.12 during 60 min reoxygenation after 30 min OGD. MMP after 60 min OGD decreased and recovered occasionally during reoxygenation. After 90 min OGD and reoxygenation, MMP was reduced and never recovered. The intracellular ATP content was 8.1+/-6.6 and 3.2+/-1.9% after 30 min OGD and 30 min reoxygenation following 30 min OGD, respectively; 60 min OGD did not significantly change these levels (7.1+/-5.8, 2.6+/-0.5%). Hyperpolarization after OGD did not accompany ATP production. This observation suggests the inhibition of electron reentry into an inner membrane during reoxygenation and the disturbance of FoF1-ATP synthase. This pathological finding of an energy-producing system after OGD may provide a clue to explain post-ischemic energy failure.

Neuroprotective effect of propofol on necrosis and apoptosis following oxygen–glucose deprivation—Relationship between mitochondrial membrane potential and mode of death

Mitochondrial membrane potential (MMP) appears to play an important role in apoptotic cascade and has been proposed as an index for apoptosis or necrosis. We examined the neuroprotective effect of propofol on mode of death, focusing on MMP. Hippocampal cell culture was divided into three groups: control, oxygen-glucose deprivation for 30 min (30OGD), 90 min (90OGD). Propofol was added to each culture group at a concentration of 0 microM (Vehicle), 0.1 microM (Pro0.1) or 1.0 microM (Pro1.0). MMP was expressed as normalized JC-1 fluorescence. ATP content was assayed using the luciferin-luciferase reaction. Neuronal viability and appearance of apoptosis were also assessed. ATP content was decreased after OGD (0.276 +/- 0.115 microM/microg (control), 0.172 +/- 0.125 microM/microg (30OGD) and 0.096 +/- 0.092 microM/microg (90OGD)). Propofol did not alter ATP content. MMP was hyperpolarized after 30OGD (1.26 +/- 0.23 (vehicle), 1.29 +/- 0.13 (Pro0.1) and 1.18 +/- 0.06 (Pro1.0)) but was depolarized after 90OGD (0.77 +/- 0.04 (vehicle), 0.89 +/- 0.04 (Pro0.1), but Pro1.0 prevented depolarization (1.03 +/- 0.15 (P < 0.05)). Viability of cells significantly decreased to 50.3 +/- 5.7% (vehicle), 46.1 +/- 7.5% (Pro0.1), but Pro1.0 significantly salvaged neurons 65.1 +/- 6.2% (higher than vehicle and Pro0.1 value, P < 0.05) after 90OGD. At 24 h after OGD, TUNEL-positive cells were increased to 34.5 +/- 6.2% (vehicle), 26.7 +/- 7.9% (Pro0.1) and 30.4 +/- 7.1% (Pro1.0) in the 30OGD group. No pharmacological effect of propofol on the incidence of apoptosis was found. Propofol inhibited acute neuronal death accompanied with the maintenance of MMP but did not prevent subsequent apoptosis. Propofol induces a moratorium on neuronal death, during which pharmacological intervention might be able to prevent cell death.

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.

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.

Impairment of Catecholamine Systems during Induction of Long-Term Potentiation at Hippocampal CA1 Synapses in HPC-1/Syntaxin 1A Knock-out Mice

The membrane protein HPC-1/syntaxin 1A is believed to play a key role in synaptic vesicle exocytosis, and it was recently suggested to be required for synaptic plasticity. Despite evidence for the function of HPC-1/syntaxin 1A in synaptic plasticity, the underlying cellular mechanism is unclear. We found that although fast synaptic transmission and long-term depression were unaffected, HPC-1/syntaxin 1A knock-out (STX1A−/−) mice showed impaired long-term potentiation (LTP) in response to theta-burst stimulation in CA1 hippocampal slices. The impairment in LTP was rescued by the application of forskolin, an adenylyl cyclase activator, or more robust stimulation, suggesting that cAMP/protein kinase A signaling was suppressed in these mice. In addition, catecholamine release from the hippocampus was significantly reduced in STX1A−/− mice. Because HPC-1/syntaxin 1A regulates exocytosis of dense-core synaptic vesicles, which contain neuromodulatory transmitters such as noradrenaline, dopamine and 5-HT, we examined the effect of neuromodulatory transmitters on LTP induction. Noradrenaline and dopamine enhanced LTP induction in STX1A−/− mice, whereas catecholamine depletion reduced LTP induction in wild-type mice. Theses results suggest that HPC-1/syntaxin 1A regulates catecholaminergic systems via exocytosis of dense-core synaptic vesicles, and that deletion of HPC-1/syntaxin 1A causes impairment of LTP induction.

Reduction of neurotransmitter release by the exogenous H3 domain peptide of HPC-1/syntaxin 1A in cultured rat hippocampal neurons

The membrane protein HPC-1/syntaxin 1A plays a key role in synaptic vesicle exocytosis in the presynaptic terminal. In particular, the H3 domain of HPC-1/syntaxin 1A participates in several protein-protein interactions that regulate neurotransmitter release. To investigate H3 domain function in neurotransmitter release, we used paired whole-cell patch clamping to record the evoked inhibitory postsynaptic currents in cultured hippocampal neurons. Introducing H3 domain peptide into the presynaptic neuron with a patch electrode depressed neurotransmitter release in a stimulation-frequency-dependent manner. Recovery from synaptic vesicle depletion induced by tetanic stimulation was significantly slowed by exogenous H3 domain peptide. These results suggest that the H3 domain peptide reduces neurotransmitter release by retarding the refilling of readily releasable vesicles.

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

Unresolved endoplasmic reticulum (ER) stress causes neuronal death and has been implicated in neurodegenerative conditions such as Alzheimers 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 β-amyloid precursor protein (βAPP), 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 βAPP processing. Golgi stress also induced upregulation of ER-Golgi SNARE Syx5, and concomitantly, suppressed amyloid-β peptide secretion. These findings suggest that Syx5 is a potential stress responsive factor that participates in βAPP processing and the survival pathways of neuronal cells.

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.