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SUMMARY Myelin is essential in vertebrates for the rapid propagation of action potentials, but the molecular mechanisms driving its formation remain largely unknown. Here we show that the initial stage of process extension and axon ensheathment by oligodendrocytes requires dynamic actin filament assembly by the Arp2/3 complex. Unexpectedly, subsequent myelin wrapping coincides with the upregulation of actin disassembly proteins, rapid disassembly of the oligodendrocyte actin cytoskeleton, and does not require Arp2/3. Inducing loss of actin filaments drives oligodendrocyte membrane spreading and myelin wrapping in vivo, and the actin disassembly factor gelsolin is required for normal wrapping. We show that myelin basic protein, a protein essential for CNS myelin wrapping whose role has been unclear, is required for actin disassembly, and its loss phenocopies loss of actin disassembly proteins. Together these findings provide insight into the molecular mechanism of myelin wrapping and identify it as an actin-independent form of mammalian cell motility.

Inhibitory synapses dampen neuronal activity through postsynaptic hyperpolarization. The composition of the inhibitory postsynapse and the mechanistic basis of its regulation, however, remains poorly understood. We used an in vivo chemico-genetic proximity-labeling approach to discover inhibitory postsynaptic proteins. Quantitative mass spectrometry not only recapitulated known inhibitory postsynaptic proteins, but also revealed a large network of new proteins, many of which are either implicated in neurodevelopmental disorders or are of unknown function. CRISPR-depletion of one of these previously uncharacterized proteins, InSyn1, led to decreased postsynaptic inhibitory sites, reduced frequency of miniature inhibitory currents, and increased excitability in the hippocampus. Our findings uncover a rich and functionally diverse assemblage of previously unknown proteins that regulate postsynaptic inhibition and might contribute to developmental brain disorders.

SUMMARY Proper establishment of synapses is critical for constructing functional circuits. Interactions between presynaptic neurexins and postsynaptic neuroligins coordinate the formation of synaptic adhesions. An isoform code determines the direct interactions of neurexins and neuroligins across the synapse. However, whether extracellular linker proteins can expand such a code is unknown. Using a combination of in vitro and in vivo approaches, we found that hevin, an astrocyte-secreted synaptogenic protein, assembles glutamatergic synapses by bridging neurexin-1α and neuroligin-1B, two isoforms that do not interact with each other. Bridging of neurexin-1α and neuroligin-1B via hevin is critical for the formation and plasticity of thalamocortical connections in the developing visual cortex. These results show that astrocytes promote the formation of synapses by modulating neurexin/neuroligin adhesions through hevin secretion. Our findings also provide an important mechanistic insight into how mutations in these genes may lead to circuit dysfunction in diseases such as autism.

We report here the cloning, expression, and characterization of a dual-substrate, cAMP and cGMP, cyclic nucleotide phosphodiesterase (PDE) from mouse. This PDE contains the consensus sequence for a PDE catalytic domain, but shares <50% sequence identity with the catalytic domains of all other known PDEs and, therefore, represents a new PDE gene family, designated PDE10A. The cDNA for PDE10A is 3,370 nt in length. It includes a full ORF, contains three in-frame stop codons upstream of the first methionine, and is predicted to encode a 779-aa enzyme. At the N terminus PDE10A has two GAF domains homologous to many signaling molecules, including PDE2, PDE5, and PDE6, which likely constitute a low-affinity binding site for cGMP. PDE10A hydrolyzes cAMP with a K m of 0.05 M and cGMP with a K m of 3 M. Although PDE10A has a lower K m for cAMP, the V max ratio (cGMP͞cAMP) is 4.7. RNA distribution studies indicate that PDE10A is expressed at highest levels in testis and brain.

A BSTR ACTCyclic nucleotide phosphodiesterases (PDEs) regulate intracellular levels of cAMP and cGMP by hydrolyzing them to their corresponding 5 monophosphates. We report here the cloning and characterization of a novel cAMP-specific PDE from mouse testis. This unique phosphodiesterase contains a catalytic domain that overall shares <40% sequence identity to the catalytic domain of all other known PDEs. Based on this limited homology, this new PDE clearly represents a previously unknown PDE gene family designated as PDE8. The cDNA for PDE8 is 3,678 nucleotides in length and is predicted to encode an 823 amino acid enzyme. The cDNA includes a full ORF as it contains an in-frame stop codon before the start methionine. PDE8 is specific for the hydrolysis of cAMP and has a K m of 0.15 M. Most common PDE inhibitors are ineffective antagonists of PDE8, including the nonspecific PDE inhibitor 3-isobutyl-1-methylxanthine. Dipyridamole, however, an inhibitor that is generally considered to be relatively specific for the cGMP selective PDEs, does inhibit PDE8 with an IC 50 of 4.5 M. Tissue distribution studies of 22 different mouse tissues indicates that PDE8 has highest expression in testis, followed by eye, liver, skeletal muscle, heart, 7-day embryo, kidney, ovary, and brain in decreasing order. In situ hybridizations in testis, the tissue of highest expression, shows that PDE8 is expressed in the seminiferous epithelium in a stage-specific manner. Highest levels of expression are seen in stages 7-12, with little or no expression in stages 1-6.

We report the cloning, expression, and characterization of a new family of cyclic nucleotide phosphodiesterase (PDE) that has unique kinetic and inhibitor specificities. A clone corresponding to the C terminus of this PDE was initially identified by a bioinformatic approach and used to isolate a cDNA that is likely full-length. This novel PDE, designated as MMPDE9A1, shows highest mRNA expression in kidney with lower levels in liver, lung, and brain The cyclic nucleotides cAMP and cGMP serve as second messengers for a wide variety of extracellular signals such as neurotransmitters, hormones, light, and odorants. The diverse cellular and behavioral responses to these second messengers are mediated by the action of cAMP and cGMP on their intracelluar targets, which include kinases, ion channels, transcriptional activators, and several isoforms of phosphodiesterases (PDEs). 1 Accordingly, these responses are regulated by the rates of synthesis of cyclic nucleotides by cyclases and their degradation by PDEs to biologically inactive 5Ј monophosphate nucleosides.Seven 2 different gene families of PDEs previously have been isolated based on their distinct kinetic and substrate characteristics, inhibitor profiles, allosteric activators and inhibitors, and amino acid sequence (1). Family 1 is activated by Ca 2ϩ / calmodulin and hydrolyzes both cAMP and cGMP; family 2 is stimulated by cGMP, and both cAMP and cGMP serve as substrate; family 3 is distinguished by cAMP hydrolysis that is inhibited by cGMP; family 4 is cAMP-specific; family 5 binds cGMP at a noncatalytic site and specifically hydrolyzes cGMP; family 6 is the retinal PDE that is inhibited by a ␥ subunit in the absence of activated transducin and hydrolyzes cGMP; and family 7 is a very low K m cAMP-specific PDE. Not only does each family of PDE have specialized substrate and regulatory features, but each PDE family and even members within a family also exhibit tissue-, cell-, and subcell-specific expression patterns and therefore participate in distinct signal transduction pathways. The precise cellular and subcellular profile of PDE expression then will determine the cyclic nucleotide phenotype of a cell and how it responds to first messengers. Identifying and characterizing these functionally distinct PDEs is therefore crucial for our understanding of the mechanisms by which cyclic nucleotides moderate their biologic effects.We report here the cloning, expression, and characterization of a previously unknown PDE designated as MMPDE9A1 (2). This PDE represents a new gene family because it shares less than 50% amino acid identity in the conserved catalytic domain with the other seven PDE families. A search of GenBank reveals that PDE9A1 has slightly higher sequence homology to a recently described Dictyostelium discoideum PDE referred to as RegA (3) rather than other mammalian PDEs. Additionally, expression of PDE9A1 in COS cells results in functional PDE activity that is unique kinetically from the other seven families in that it is cGMP-specific and has the l...

The scaffolding protein WAVE-1 (Wiskott-Aldrich syndrome protein family member 1) directs signals from the GTPase Rac through the Arp2/3 complex to facilitate neuronal actin remodeling. The WAVE-associated GTPase activating protein called WRP is implicated in human mental retardation, and WAVE-1 knock-out mice have altered behavior. Neuronal time-lapse imaging, behavioral analyses, and electrophysiological recordings from genetically modified mice were used to show that WAVE-1 signaling complexes control aspects of neuronal morphogenesis and synaptic plasticity. Gene targeting experiments in mice demonstrate that WRP anchoring to WAVE-1 is a homeostatic mechanism that contributes to neuronal development and the fidelity of synaptic connectivity. This implies that signaling through WAVE-1 complexes is essential for neural plasticity and cognitive behavior.