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.

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. Highthroughput 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.Taking advantage of the huge quantity of genome data currently available 1-5 , reverse genetics approaches that can determine the role of each gene by loss-of-function are important in determining gene-function relationships. Gene targeting 6 using homologous recombination is widely used, but its labor-intensive processes preclude its convenient and versatile application. Although antisense oligonucleotides can be more conveniently used, outcomes from this approach are often characterized by toxicity, instability and nonspecific effects 7 .RNAi, a gene suppression phenomenon triggered by doublestranded RNA 8 , is a good alternative 7,9 , because it is usually specific and can be achieved with unprecedented speed in a wide range of organisms. RNAi has been applied to genome-wide reverse genetics in Caenorhabditis elegans 10 . But the initial use of RNAi had been limited to invertebrates because long (>30 nucleotides, nt) double-stranded RNAs elicit interferon responses in higher vertebrates. This problem was overcome by the finding that a short (21-23 nt) double-stranded RNA (siRNA) directs RNAi in mammals without adverse effects 11 . The transient nature of siRNA effects has been overcome by the development of DNA-based vectors by which siRNA or shRNA is expressed intracellularly [12][13][14][15][16][17] .Despite this progress, there are still no general rules for designing siRNA or shRNA constructs with efficient gene-silencing activity; thus, it costs much time and money to identify suitable constructs. We developed a new technology called EPRIL to produce a library of shRNA expression constructs that can be systematically screened. We have also developed a technology for selecting the most efficient shRNA constructs from the library using the thymidine kinase gene. We furthermore showed that EPRIL can be used to produce an RNAi library from a cDNA library. RESULTS Production of an RNAi libraryEPRIL comprises several steps of enzymatic treatments to produce an shRNA expression vector library from cDNAs of interest (Fig. 1a). First, double-stranded DNAs are quasi-randomly fragmented with DNase I (ref. 18). The fragments are...

Astroglia are essential for brain development, homeostasis, and metabolic support. They also contribute actively to the formation and regulation of synaptic circuits, by successfully handling, integrating, and propagating physiological signals of neural networks. The latter occurs mainly by engaging a versatile mechanism of internal Ca 21 fluctuations and regenerative waves prompting targeted release of signaling molecules into the extracellular space. Astroglia also show substantial structural plasticity associated with age-and use-dependent changes in neural circuitry. However, the underlying cellular mechanisms are poorly understood, mainly because of the extraordinary complex morphology of astroglial compartments on the nanoscopic scale. This complexity largely prevents direct experimental access to astroglial processes, most of which are beyond the diffraction limit of optical microscopy. Here we employed super-resolution microscopy (direct stochastic optical reconstruction microscopy; dSTORM), to visualize astroglial organization on the nanoscale, in culture and in thin brain slices, as an initial step to understand the structural basis of astrocytic nano-physiology. We were able to follow nanoscopic morphology of GFAP-enriched astrocytes, which adapt a flattened shape in culture and a sponge-like structure in situ, with GFAP fibers of varied diameters. We also visualized nanoscopic astrocytic processes using the ubiquitous cytosolic astrocyte marker proteins S100b and glutamine synthetase. Finally, we overexpressed and imaged membrane-targeted pHluorin and lymphocytespecific protein tyrosine kinase (N-terminal domain) -green fluorescent protein (lck-GFP), to better understand the molecular cascades underlying some common astrogliatargeted fluorescence imaging techniques. The results provide novel, albeit initial, insights into the cellular organization of astroglia on the nanoscale, paving the way for functionspecific studies. V C 2017 Wiley Periodicals, Inc.

Genome-wide association studies have linked polymorphisms in the gene G72 to schizophrenia risk in several human populations. Although controversial, biochemical experiments have suggested that the mechanistic link of G72 to schizophrenia is due to the G72 protein product, pLG72, exerting a regulatory effect on human D-amino acid oxidase (hDAAO) activity. In an effort to identify hDAAO inhibitors of novel mechanism of action, we designed a pLG72-directed hDAAO activity assay suitable for high-throughput screening (HTS). During assay development, we confirmed that pLG72 was an inhibitor of hDAAO. Thus, our assay employed an IC 20 pLG72 concentration that was high enough to allow dynamic pLG72-hDAAO complexes to form but with sufficient remaining hDAAO activity to measure during an HTS. After conducting an approximately 150,000-compound HTS, we further characterized a class of compound hits that were less potent hDAAO inhibitors when pLG72 was present. Focusing primarily on compound, we demonstrated that these compounds inhibited hDAAO via an allosteric, covalent mechanism. Although there is significant interest in the therapeutic potential of compound 2 and its analogues, their sensitivity to reducing agents and their capacity to bind cysteines covalently would need to be addressed during therapeutic drug development.

Introduction: Gain-of-function mutations in the L-type Ca2+ channel Cav1.2 cause Timothy syndrome (TS), a multisystem disorder associated with neurologic symptoms, including autism spectrum disorder (ASD), seizures, and intellectual disability. Cav1.2 plays key roles in neural development, and its mutation can affect brain development and connectivity through Ca2+-dependent and -independent mechanisms. Recently, a gain-of-function mutation, I1166T, in Cav1.2 was identified in patients with TS-like disorder. Its channel properties have been analyzed in vitro but in vivo effects of this mutation on brain development remain unexplored.Methods:In utero electroporation was performed on ICR mice at embryonic day 15 to express GFP, wild-type, and mutant Cav1.2 channels into cortical layer 2/3 excitatory neurons in the primary somatosensory area. The brain was fixed at postnatal days 14–16, sliced, and scanned using confocal microscopy. Neuronal migration of electroporated neurons was examined in the cortex of the electroporated hemisphere, and callosal projection was examined in the white matter and contralateral hemisphere.Results: Expression of the I1166T mutant in layer 2/3 neurons caused migration deficits in approximately 20% of electroporated neurons and almost completely diminished axonal arborization in the contralateral hemisphere. Axonal projection in the white matter was not affected. We introduced second mutations onto Cav1.2 I1166T; L745P mutation blocks Ca2+ influx through Cav1.2 channels and inhibits the Ca2+-dependent pathway, and the W440A mutation blocks the interaction of the Cav1.2 α1 subunit to the β subunit. Both second mutations recovered migration and projection.Conclusion: This study demonstrated that the Cav1.2 I1166T mutation could affect two critical steps during cerebrocortical development, migration and axonal projection, in the mouse brain. This is mediated through Ca2+-dependent pathway downstream of Cav1.2 and β subunit-interaction.

RNA interference (RNAi) using small interfering (siRNA) or short hairpin RNA (shRNA) has become the first choice for gene silencing maneuver in mammalian cells. Because different siRNAs of the same gene have variable silencing efficacy and only limited siRNAs are functional, many candidates are necessary to identify optimal siRNAs. We have previously reported a method named enzymatic production of RNAi library (EPRIL), by which a great variety of shRNA expression constructs (RNAi library) can be produced simultaneously from cDNAs of interest. Recently, we have improved this method and developed a more efficient method. We describe in this chapter detailed protocols for the improved version of EPRIL and high-throughput selection of effective shRNA expression constructs from an RNAi library.