Membrane fusion is mediated by complexes formed by SNAPreceptor (SNARE) and Secretory 1 (Sec1)/mammalian uncoordinated-18 (Munc18)-like (SM) proteins, but it is unclear when and how these complexes assemble. Here we describe an improved two-color fluorescence nanoscopy technique that can achieve effective resolutions of up to 7.5-nm full width at half maximum (3.2-nm localization precision), limited only by stochastic photon emission from single molecules. We use this technique to dissect the spatial relationships between the neuronal SM protein Munc18-1 and SNARE proteins syntaxin-1 and SNAP-25 (25 kDa synaptosomeassociated protein). Strikingly, we observed nanoscale clusters consisting of syntaxin-1 and SNAP-25 that contained associated Munc18-1. Rescue experiments with syntaxin-1 mutants revealed that Munc18-1 recruitment to the plasma membrane depends on the Munc18-1 binding to the N-terminal peptide of syntaxin-1. Our results suggest that in a primary neuron, SNARE/SM protein complexes containing syntaxin-1, SNAP-25, and Munc18-1 are preassembled in microdomains on the presynaptic plasma membrane. Our superresolution imaging method provides a framework for investigating interactions between the synaptic vesicle fusion machinery and other subcellular systems in situ.neurotransmission | exocytosis | single-molecule | colocalization | active stabilization I ntracellular trafficking as well as transmission of signals across cell membranes, such as release of neurotransmitters at neuronal synapses, is mediated by fusion of vesicles with target membranes. The energy required for membrane juxtaposition and fusion is provided by folding of cognate vesicular-and targetmembrane SNAP (soluble NSF attachment protein) receptors (SNAREs) into tight helical bundles that bring the two lipid bilayers into close apposition (1). However, in vitro and in vivo neuronal SNAREs do not efficiently overcome transition barriers to proceed to full fusion, requiring the action of Secretory 1 (Sec1)/mammalian uncoordinated-18 (Munc18)-like (SM) proteins (2), as deletion of SM protein genes results in severe fusion defects in yeast (3), flies (4), worms (5, 6), and mice (7). Moreover, at a synapse the SNARE/SM protein fusion machinery is regulated by the Ca 2+ sensor synaptotagmin-1 (8) in coordination with complexin (9) to efficiently trigger neurotransmitter release (10, 11). Despite their central role, the exact mechanisms by which SM and SNARE proteins mediate fusion in vivo have been the subject of considerable debate.At least four modes of interactions between SM proteins and SNAREs have been reported: (i) Many SM proteins bind to their cognate syntaxins via a conserved N-terminal peptide that is exposed in the SNARE complex, resulting in SM protein/ SNARE complex assemblies (12-14); note that this interaction plays an important role when syntaxin is in either binary or ternary SNARE complexes. (ii) In addition to engaging in the first mode, SM proteins involved in exocytosis (Munc18-1, -2, and -3) also bind to their cognate synta...

Most neuropsychiatric disease risk variants are in noncoding sequences and lack functional interpretation. Because regulatory sequences often reside in open chromatin, we reasoned that neuropsychiatric disease risk variants may affect chromatin accessibility during neurodevelopment. Using human induced pluripotent stem cell (iPSC)–derived neurons that model developing brains, we identified thousands of genetic variants exhibiting allele-specific open chromatin (ASoC). These neuronal ASoCs were partially driven by altered transcription factor binding, overrepresented in brain gene enhancers and expression quantitative trait loci, and frequently associated with distal genes through chromatin contacts. ASoCs were enriched for genetic variants associated with brain disorders, enabling identification of functional schizophrenia risk variants and their cis-target genes. This study highlights ASoC as a functional mechanism of noncoding neuropsychiatric risk variants, providing a powerful framework for identifying disease causal variants and genes.

SUMMARY Down syndrome (DS) is a common neurodevelopmental disorder, and cognitive defects in DS patients may arise from imbalances in excitatory and inhibitory neurotransmission. Understanding the mechanisms underlying such imbalances may provide opportunities for therapeutic intervention. Here, we show that human induced pluripotent stem cells (hiPSCs) derived from DS patients overproduce OLIG2+ ventral forebrain neural progenitors. As a result, DS hiPSC-derived cerebral organoids excessively produce specific subclasses of GABAergic interneurons and cause impaired recognition memory in neuronal chimeric mice. Increased OLIG2 expression in DS cells directly upregulates interneuron lineage-determining transcription factors. shRNA-mediated knockdown of OLIG2 largely reverses abnormal gene expression in early-stage DS neural progenitors, reduces interneuron production in DS organoids and chimeric mouse brains, and improves behavioral deficits in DS chimeric mice. Thus, altered OLIG2 expression may underlie neurodevelopmental abnormalities and cognitive defects in DS patients.

SUMMARY Glucagon Like Peptide 1 (GLP-1)-expressing neurons in the hindbrain send robust projections to the paraventricular nucleus of the hypothalamus (PVN), which is involved in the regulation of food intake. Here, we describe that stimulation of GLP-1 afferent fibers within the PVN is sufficient to suppress food intake independent of glutamate release. We also show that GLP-1 receptor (GLP-1R) activation augments excitatory synaptic strength in PVN corticotropin-releasing hormone (CRH) neurons, with GLP-1R activation promoting a protein kinase A (PKA) dependent signaling cascade leading to phosphorylation of serine S845 on GluA1 AMPA receptors and their trafficking to the plasma membrane. Finally, we show that postnatal depletion of GLP-1R in the PVN increases food intake and causes obesity. This study provides a comprehensive multi-level (circuit, synaptic, and molecular) explanation of how food intake behavior and body weight are regulated by endogenous central GLP-1.