Shota Tanifuji

Licochalcones suppress degranulation by decreasing the intracellular Ca2+ level and tyrosine phosphorylation of ERK in RBL-2H3 cells

Mast cells play a key role in allergic inflammation by releasing various mediators, such as histamine, serotonin, leukotrienes and cytokines. A signaling cascade of events activated by stimulation with antigens contributes to the regulation of mast cell degranulation. While various anti-inflammatory and anti-allergic drugs have been developed that inhibit degranulation of mast cells, the inhibitory mechanism has been poorly understood. Licochalcone A (Lico A) is a retrochalcone isolated from the root of Xinjiang liquorice and has been reported to exhibit various biological activities such as anti-inflammatory activity. We examined the effects of Lico A and related chalcones on degranulation in a rat basophilic leukemia cell line, RBL-2H3. Whereas Lico A and licochalcone C (Lico C) exhibited inhibitory activity with cytotoxicity, licochalcone D (Lico D) significantly inhibited the degranulation in RBL-2H3 cells with low cytotoxicity. Moreover, Lico D significantly inhibited the Ca2+ influx and phosphorylation of extracellular signal regulated kinase (ERK) and MEK. These results suggest that Lico D inhibits mast cell degranulation via the inhibition of both extracellular Ca2+ influx and activation of the MEK-ERK pathway.

Dynamin isoforms decode action potential firing for synaptic vesicle recycling.

Background: The molecular mechanism linking variation in presynaptic neuronal activity to vesicle trafficking is unknown. Results: Three isoforms of dynamin, an essential endocytic protein, mediate vesicle reuse, having distinct rate and time constants with physiological action potential frequencies. Conclusion: Dynamin isoforms select appropriate vesicle reuse pathways associated with specific neuronal firing patterns. Significance: Individual dynamin isoforms regulate distinct synaptic vesicle reuse pathways that cover the full range of physiological action potential frequencies. Presynaptic nerve terminals must maintain stable neurotransmission via synaptic vesicle membrane recycling despite encountering wide fluctuations in the number and frequency of incoming action potentials (APs). However, the molecular mechanism linking variation in neuronal activity to vesicle trafficking is unknown. Here, we combined genetic knockdown and direct physiological measurements of synaptic transmission from paired neurons to show that three isoforms of dynamin, an essential endocytic protein, work individually to match vesicle reuse pathways, having distinct rate and time constants with physiological AP frequencies. Dynamin 3 resupplied the readily releasable pool with slow kinetics independently of the AP frequency but acted quickly, within 20 ms of the incoming AP. Under high-frequency firing, dynamin 1 regulated recycling to the readily releasable pool with fast kinetics in a slower time window of greater than 50 ms. Dynamin 2 displayed a hybrid response between the other isoforms. Collectively, our findings show how dynamin isoforms select appropriate vesicle reuse pathways associated with specific neuronal firing patterns.

Kinetic Organization of Ca2+ Signals That Regulate Synaptic Release Efficacy in Sympathetic Neurons

Calcium regulation of neurotransmitter release is essential for maintenance of synaptic transmission. However, the temporal and spatial organization of Ca2+ dynamics that regulate synaptic vesicle (SV) release efficacy in sympathetic neurons is poorly understood. Here, we investigate the N-type Ca2+ channel–mediated kinetic structure of Ca2+ regulation of cholinergic transmission of sympathetic neurons. We measured the effect of Ca2+ chelation with fast 1,2-bis(2-aminophenoxy) ethane-tetraacetic acid (BAPTA) and slow ethyleneglycol-tetraacetic acid (EGTA) buffers on exocytosis, synaptic depression, and recovery of the readily releasable vesicle pool (RRP), after both single action potential (AP) and repetitive APs. Surprisingly, postsynaptic potentials peaking at ∼12 milliseconds after the AP was inhibited by both rapid and slow Ca2+ buffers suggests that, in addition to the well known fast Ca2+ signals at the active zone (AZ), slow Ca2+ signals at the peak of Ca2+ entry also contribute to paired-pulse or repetitive AP responses. Following a single AP, discrete Ca2+ transient increase regulated synaptic depression in rapid (<30-millisecond) and slow (<120-millisecond) phases. In contrast, following prolonged AP trains, synaptic depression was reduced by a slow Ca2+ signal regulation lasting >200 milliseconds. Finally, after an AP burst, recovery of the RRP was mediated by an AP-dependent rapid Ca2+ signal, and the expansion of releasable SV number by an AP firing activity–dependent slow Ca2+ signal. These data indicate that local Ca2+ signals operating near Ca2+ sources in the AZ are organized into discrete fast and slow temporal phases that remodel exocytosis and short-term plasticity to ensure long-term stability in acetylcholine release efficacy.

Neural Activity Selects Myosin IIB and VI with a Specific Time Window in Distinct Dynamin Isoform-Mediated Synaptic Vesicle Reuse Pathways

Presynaptic nerve terminals must maintain stable neurotransmissions via synaptic vesicle (SV) resupply despite encountering wide fluctuations in the number and frequency of incoming action potentials (APs). However, the molecular mechanism linking variation in neural activity to SV resupply is unknown. Myosins II and VI are actin-based cytoskeletal motors that drive dendritic actin dynamics and membrane transport, respectively, at brain synapses. Here we combined genetic knockdown or molecular dysfunction and direct physiological measurement of fast synaptic transmission from paired rat superior cervical ganglion neurons in culture to show that myosins IIB and VI work individually in SV reuse pathways, having distinct dependency and time constants with physiological AP frequency. Myosin VI resupplied the readily releasable pool (RRP) with slow kinetics independently of firing rates but acted quickly within 50 ms after AP. Under high-frequency AP firing, myosin IIB resupplied the RRP with fast kinetics in a slower time window of 200 ms. Knockdown of both myosin and dynamin isoforms by mixed siRNA microinjection revealed that myosin IIB-mediated SV resupply follows amphiphysin/dynamin-1-mediated endocytosis, while myosin VI-mediated SV resupply follows dynamin-3-mediated endocytosis. Collectively, our findings show how distinct myosin isoforms work as vesicle motors in appropriate SV reuse pathways associated with specific firing patterns.

Synaptic vesicle glycoprotein 2A modulates vesicular release and calcium channel function at peripheral sympathetic synapses

Synaptic vesicle glycoprotein (SV)2A is a transmembrane protein found in secretory vesicles and is critical for Ca2+‐dependent exocytosis in central neurons, although its mechanism of action remains uncertain. Previous studies have proposed, variously, a role of SV2 in the maintenance and formation of the readily releasable pool (RRP) or in the regulation of Ca2+ responsiveness of primed vesicles. Such previous studies have typically used genetic approaches to ablate SV2 levels; here, we used a strategy involving small interference RNA (siRNA) injection to knockdown solely presynaptic SV2A levels in rat superior cervical ganglion (SCG) neuron synapses. Moreover, we investigated the effects of SV2A knockdown on voltage‐dependent Ca2+ channel (VDCC) function in SCG neurons. Thus, we extended the studies of SV2A mechanisms by investigating the effects on vesicular transmitter release and VDCC function in peripheral sympathetic neurons. We first demonstrated an siRNA‐mediated SV2A knockdown. We showed that this SV2A knockdown markedly affected presynaptic function, causing an attenuated RRP size, increased paired‐pulse depression and delayed RRP recovery after stimulus‐dependent depletion. We further demonstrated that the SV2A–siRNA‐mediated effects on vesicular release were accompanied by a reduction in VDCC current density in isolated SCG neurons. Together, our data showed that SV2A is required for correct transmitter release at sympathetic neurons. Mechanistically, we demonstrated that presynaptic SV2A: (i) acted to direct normal synaptic transmission by maintaining RRP size, (ii) had a facilitatory role in recovery from synaptic depression, and that (iii) SV2A deficits were associated with aberrant Ca2+ current density, which may contribute to the secretory phenotype in sympathetic peripheral neurons.

Dynamin Is a Key Molecule to Decode Action Potential Firing

The maintenance of neurotransmission relies on the replenishment of synaptic vesicles (SVs) adapted to wide variations in the number and frequency of incoming action potentials (APs). A candidate mechanism for SV recycling indexed to AP firing activity could involve a protein-initiating endocytosis. Dynamin is a GTPase, which mediates fission of SVs from the presynaptic terminal membrane. There are three dynamin isoforms, dynamin 1, 2 and 3, in mammalian neurons. Knockout of dynamin 1 and 3 in central neurons suggests a role of each dynamin isoform in neuronal activity. We have carefully accessed the SV replenishment into the release site (readily releasable pool, RRP) in relation to AP firing activity and dynamin 1, 2 and 3 mediation. The three isoforms in sympathetic superior cervical ganglion neurons, an ideal model for direct physiological measurement of synaptic transmission combined with genetic knockdown, mediate the RRP replenishment, having distinct rate and time constants. Individual isoforms regulate distinct SV recycling pathways that cover the full range of physiological AP frequency. Thus, dynamin 1, 2 and 3 decode AP firing for SV recycling in sympathetic neurons. In this chapter, we review dynamin, in mammalian central and peripheral neurons, that is a key molecule for the selection and regulation of distinct SV recycling pathways in sensing of presynaptic AP firing patterns.