Shigeyuki Namiki

Adiponectin and AdipoR1 regulate PGC-1α and mitochondria by Ca2+ and AMPK/SIRT1

Adiponectin is an anti-diabetic adipokine. Its receptors possess a seven-transmembrane topology with the amino terminus located intracellularly, which is the opposite of G-protein-coupled receptors. Here we provide evidence that adiponectin induces extracellular Ca2+ influx by adiponectin receptor 1 (AdipoR1), which was necessary for subsequent activation of Ca2+/calmodulin-dependent protein kinase kinase β (CaMKKβ), AMPK and SIRT1, increased expression and decreased acetylation of peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α), and increased mitochondria in myocytes. Moreover, muscle-specific disruption of AdipoR1 suppressed the adiponectin-mediated increase in intracellular Ca2+ concentration, and decreased the activation of CaMKK, AMPK and SIRT1 by adiponectin. Suppression of AdipoR1 also resulted in decreased PGC-1α expression and deacetylation, decreased mitochondrial content and enzymes, decreased oxidative type I myofibres, and decreased oxidative stress-detoxifying enzymes in skeletal muscle, which were associated with insulin resistance and decreased exercise endurance. Decreased levels of adiponectin and AdipoR1 in obesity may have causal roles in mitochondrial dysfunction and insulin resistance seen in diabetes.

Enzymatic production of RNAi libraries from cDNAs

RNA interference (RNAi) induced by small interfering (siRNA) or short hairpin RNA (shRNA) is an important research approach in mammalian genetics. Here we describe a technology called enzymatic production of RNAi library (EPRIL) by which cDNAs are converted by a sequence of enzymatic treatments into an RNAi library consisting of a vast array of different shRNA expression constructs. We applied EPRIL to a single cDNA source and prepared an RNAi library consisting of shRNA constructs with various RNAi efficiencies. High-throughput screening allowed us to rapidly identify the best shRNA constructs from the library. We also describe a new selection scheme using the thymidine kinase gene for obtaining efficient shRNA constructs. Furthermore, we show that EPRIL can be applied to constructing an RNAi library from a cDNA library, providing a basis for future whole-genome phenotypic screening of genes.

NO signalling decodes frequency of neuronal activity and generates synapse-specific plasticity in mouse cerebellum

Nitric oxide (NO) is an intercellular messenger regulating neuronal functions. To visualize NO signalling in the brain, we generated a novel fluorescent NO indicator, which consists of the heme‐binding region (HBR) of soluble guanylyl cyclase and the green fluorescent protein. The indicator (HBR–GFP) was expressed in the Purkinje cells of the mouse cerebellum and we imaged NO signals in acute cerebellar slices upon parallel fibre (PF) activation with a train of burst stimulations (BS, each BS consisting of five pulses at 50 Hz). Our results showed that the intensity of synaptic NO signal decays steeply with the distance from the synaptic input near PF–Purkinje cell synapses and generates synapse‐specific long‐term potentiation (LTP). Furthermore, the NO release level has a bell‐shaped dependence on the frequency of PF activity. At an optimal frequency (1 Hz), but not at a low frequency (0.25 Hz) of a train of 60 BS, NO release as well as LTP was induced. However, both NO release and LTP were significantly reduced at higher frequencies (2–4 Hz) of BS train due to cannabinoid receptor‐mediated retrograde inhibition of NO generation at the PF terminals. These results suggest that synaptic NO signalling decodes the frequency of neuronal activity to mediate synaptic plasticity at the PF–Purkinje cell synapse.

Cell Surface-Anchored Fluorescent Aptamer Sensor Enables Imaging of Chemical Transmitter Dynamics

A fluorescent aptamer sensor was applied to the analysis of extracellular chemical transmitter dynamics. We utilized a tocopherol-labeled aptamer, which allowed the direct anchoring of the fluorescent aptamer on the cell surface while retaining its performance as a fluorescent sensor. The fast-responsive fluorescent DNA aptamer sensor, which targets adenine compounds, was anchored on the surface of brain astrocytes. Fluorescence imaging of the aptamer-anchored astrocytes enabled the real-time monitoring of release of adenine compounds as a gliotransmitter, which was synchronized with calcium wave propagation in neighboring cells.

Optical glutamate sensor for spatiotemporal analysis of synaptic transmission.

Imaging neurotransmission is expected to greatly improve our understanding of the mechanisms and regulations of synaptic transmission. Aiming at imaging glutamate, a major excitatory neurotransmitter in the CNS, we developed a novel optical glutamate probe, which consists of a ligand‐binding domain of α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid (AMPA) receptor glutamate receptor GluR2 subunit and a small molecule fluorescent dye. We expected that such fluorescent conjugates might report the microenvironmental changes upon protein conformational changes elicited by glutamate binding. After more than 100 conjugates were tested, we finally obtained a conjugate named E (glutamate) optical sensor (EOS), which showed maximally 37% change in fluorescence intensity upon binding of glutamate with a dissociation constant of 148 nm. By immobilizing EOS on the cell surface of hippocampal neuronal culture preparations, we pursued in situ spatial mapping of synaptically released glutamate following presynaptic firing. Results showed that a single firing was sufficient to obtain high‐resolution images of glutamate release, indicating the remarkable sensitivity of this technique. Furthermore, we monitored the time course of changes in presynaptic activity induced by phorbol ester and found heterogeneity in presynaptic modulation. These results indicate that EOS can be generally applicable to evaluation of presynaptic modulation and plasticity. This EOS‐based glutamate imaging method is useful to address numerous fundamental issues about glutamatergic neurotransmission in the CNS.

Acidic‐pH‐Activatable Fluorescence Probes for Visualizing Exocytosis Dynamics

Live imaging of exocytosis dynamics is crucial for a precise spatiotemporal understanding of secretion phenomena, but current approaches have serious limitations. We designed and synthesized small-molecular fluorescent probes that were chemically optimized for sensing acidic intravesicular pH values, and established that they can be used to sensitively and reliably visualize vesicular dynamics following stimulation. This straightforward technique for the visualization of exocytosis as well as endocytosis/reacidification processes with high spatiotemporal precision is expected to be a powerful tool for investigating dynamic cellular phenomena involving changes in the pH value.

Glutamatergic neurotransmission in the procerebrum (Olfactory Center) of a terrestrial mollusk

The terrestrial slug Limax has the ability to learn odor associations. This ability depends on the function of the procerebrum, the secondary olfactory center in the brain. Among the various neurotransmitters that are thought to be involved in the function of the procerebrum, glutamate is one of the most important molecules. However, the existence and function of glutamate in this system have been proposed solely on the basis of a few lines of indirect evidence from pharmacological experiments. In the present study, we demonstrated the existence and release of glutamate as a neurotransmitter in the procerebrum of Limax, by using three different techniques: 1) immunohistochemistry of glutamate, 2) in situ hybridization to mRNA of the vesicular glutamate transporter, and 3) real‐time imaging of glutamate release within the procerebrum using the glutamate optical sensor EOS2. The release of glutamate within the cell mass layer of the procerebrum was synchronized with oscillation of the local field potential and had the same physiological properties as this oscillation; both were blocked by a serotonin antagonist and were propagated in an apical to basal direction in the procerebrum. Our observations suggest strongly that the oscillation of the local field potential is driven by the glutamate released by bursting neurons in the procerebrum.

Bis-N-nitroso-caged nitric oxides: photochemistry and biological performance test by rat aorta vasorelaxation

Three new caged nitric oxides (NOs)-BNN3, BNN5Na, and BNN5M were tested for biological use. BNNs have a strong ultraviolet (UV) absorption band (lambda(max): 300 nm, epsilon: 13.5 mM(-1) cm (-1)) extended to 420 nm and produce NO upon irradiation with 300-360 nm light in quantum yields about 2. A photoexcited BNN molecule yields two NOs with time constants of less than 10 ns for phase 1 and less than 20 micros for phase 2 at 37 degrees C, suggesting usefulness of BNNs for measuring in vivo and in vitro fast NO reactions. Upon irradiating with UV light, caged nitric oxides-loaded rat aortic strips maintained in a state of active tonic contraction effectively relaxed ( < 3 microM BNN5M loading solution concentration). BNN3 is incorporated in the lipid membrane. BNN5Na, insoluble in organic solvents but water soluble, localizes in the water phase. BNN5M, is muscle-cell-permeable and hydrolysed to BNN5Na to remain in cytosol. BNNs were thermally stable and demonstrated no observable toxicity.

Intracellular delivery of glutathione S-transferase into mammalian cells

Protein transduction domains (PTDs) derived from human immunodeficiency virus Tat protein and herpes simplex virus VP22 protein are useful for the delivery of non-membrane-permeating polar or large molecules into living cells. In the course of our study aiming at evaluating PTD, we unexpectedly found that the fluorescent-dye-labeled glutathione S-transferase (GST) from Schistosoma japonicum without known PTDs was delivered into COS7 cells. The intracellular transduction of GST was also observed in HeLa, NIH3T3, and PC12 cells, as well as in hippocampal primary neurons, indicating that a wide range of cell types is permissive for GST transduction. Furthermore, we showed that the immunosuppressive peptide VIVIT fused with GST successfully inhibits NFAT activation. These results suggest that GST is a novel PTD which may be useful in the intracellular delivery of biologically active molecules, such as small-molecule drugs, bioactive peptides, or proteins.

Synaptic weight set by Munc13-1 supramolecular assemblies

The weight of synaptic connections, which is controlled not only postsynaptically but also presynaptically, is a key determinant in neuronal network dynamics. The mechanisms controlling synaptic weight, especially on the presynaptic side, remain elusive. Using single-synapse imaging of the neurotransmitter glutamate combined with super-resolution imaging of presynaptic proteins, we identify a presynaptic mechanism for setting weight in central glutamatergic synapses. In the presynaptic terminal, Munc13-1 molecules form multiple and discrete supramolecular self-assemblies that serve as independent vesicular release sites by recruiting syntaxin-1, a soluble N-ethylmaleimide-sensitive-factor attachment receptor (SNARE) protein essential for synaptic vesicle exocytosis. The multiplicity of these Munc13-1 assemblies affords multiple stable states conferring presynaptic weight, potentially encoding several bits of information at individual synapses. Supramolecular assembling enables a stable synaptic weight, which confers robustness of synaptic computation on neuronal circuits and may be a general mechanism by which biological processes operate despite the presence of molecular noise.The authors show that Munc13-1 molecules form multiple supramolecular self-assemblies that serve as vesicular release sites. Having multiple Munc13-1 assemblies affords a stable synaptic weight, which confers robustness of synaptic computation.