Complexin Mutants Reveal Partial Segregation between Recycling Pathways That Drive Evoked and Spontaneous Neurotransmission.

Synaptic vesicles fuse at morphological specializations in the presynaptic terminal termed active zones (AZs). Vesicle fusion can occur spontaneously or in response to an action potential. Following fusion, vesicles are retrieved and recycled within nerve terminals. It is still unclear whether vesicles that fuse spontaneously or following evoked release share similar recycling mechanisms. Genetic deletion of the SNARE-binding protein complexin dramatically increases spontaneous fusion, with the protein serving as the synaptic vesicle fusion clamp at Drosophila synapses. We examined synaptic vesicle recycling pathways at complexin null neuromuscular junctions, where spontaneous release is dramatically enhanced. We combined loading of the lipophilic dye FM1-43 with photoconversion, electron microscopy, and electrophysiology to monitor evoked and spontaneous recycling vesicle pools. We found that the total number of recycling vesicles was equal to those retrieved through spontaneous and evoked pools, suggesting that retrieval following fusion is partially segregated for spontaneous and evoked release. In addition, the kinetics of FM1-43 destaining and synaptic depression measured in the presence of the vesicle-refilling blocker bafilomycin indicated that spontaneous and evoked recycling pools partially intermix during the release process. Finally, FM1-43 photoconversion combined with electron microscopy analysis indicated that spontaneous recycling preferentially involves synaptic vesicles in the vicinity of AZs, whereas vesicles recycled following evoked release involve a larger intraterminal pool. Together, these results suggest that spontaneous and evoked vesicles use separable recycling pathways and then partially intermix during subsequent rounds of fusion. SIGNIFICANCE STATEMENT: Neurotransmitter release involves fusion of synaptic vesicles with the plasma membrane in response to an action potential, or spontaneously in the absence of stimulation. Upon fusion, vesicles are retrieved and recycled, and it is unclear whether recycling pathways for evoked and spontaneous vesicles are segregated after fusion. We addressed this question by taking advantage of preparations lacking the synaptic protein complexin, which have elevated spontaneous release that enables reliable tracking of the spontaneous recycling pool. Our results suggest that spontaneous and evoked recycling pathways are segregated during the retrieval process but can partially intermix during stimulation.

A Drosophila model of Huntington disease-like 2 exhibits nuclear toxicity and distinct pathogenic mechanisms from Huntington disease.

Huntington disease-like 2 (HDL2) and Huntington disease (HD) are adult-onset neurodegenerative diseases characterized by movement disorders, psychiatric disturbances and cognitive decline. Brain tissue from HD and HDL2 patients shows degeneration of the striatum and ubiquitinated inclusions immunoreactive for polyglutamine (polyQ) antibodies. Despite these similarities, the diseases result from different genetic mutations. HD is caused by a CAG repeat expansion in the huntingtin (HTT) gene, while HDL2 results from an expansion at the junctophilin 3 (JPH3) locus. Recent evidence indicates that the HDL2 expansion may give rise to a toxic polyQ protein translated from an antisense mRNA derived from the JPH3 locus. To investigate this hypothesis, we generated and characterized a Drosophila HDL2 model and compared it with a previously established HD model. We find that neuronal expression of HDL2-Q15 is not toxic, while the expression of an expanded HDL2-Q138 protein is lethal. HDL2-Q138 forms large nuclear aggregates, with only smaller puncta observed in the cytoplasm. This is in contrast to what is observed in a Drosophila model of HD, where polyQ aggregates localize exclusively to the cytoplasm. Altering localization of HLD2 with the addition of a nuclear localization or nuclear export sequence demonstrates that nuclear accumulation is required for toxicity in the Drosophila HDL2 model. Directing HDL2-Q138 to the nucleus exacerbates toxicity in multiple tissue types, while confining HDL2-Q138 to the cytoplasm restores viability to control levels. We conclude that while HD and HDL2 have similar clinical profiles, distinct pathogenic mechanisms are likely to drive toxicity in Drosophila models of these disorders.

Shank Modulates Postsynaptic Wnt Signaling to Regulate Synaptic Development.

UNLABELLED: Prosap/Shank scaffolding proteins regulate the formation, organization, and plasticity of excitatory synapses. Mutations in SHANK family genes are implicated in autism spectrum disorder and other neuropsychiatric conditions. However, the molecular mechanisms underlying Shank function are not fully understood, and no study to date has examined the consequences of complete loss of all Shank proteins in vivo Here we characterize the single Drosophila Prosap/Shank family homolog. Shank is enriched at the postsynaptic membrane of glutamatergic neuromuscular junctions and controls multiple parameters of synapse biology in a dose-dependent manner. Both loss and overexpression of Shank result in defects in synaptic bouton number and maturation. We find that Shank regulates a noncanonical Wnt signaling pathway in the postsynaptic cell by modulating the internalization of the Wnt receptor Fz2. This study identifies Shank as a key component of synaptic Wnt signaling, defining a novel mechanism for how Shank contributes to synapse maturation during neuronal development. SIGNIFICANCE STATEMENT: Haploinsufficiency for SHANK3 is one of the most prevalent monogenic causes of autism spectrum disorder, making it imperative to understand how the Shank family regulates neurodevelopment and synapse function. We created the first animal model lacking all Shank proteins and used the Drosophila neuromuscular junction, a model glutamatergic synapse, to characterize the role of Shank at synapses. We identified a novel function of Shank in synapse maturation via regulation of Wnt signaling in the postsynaptic cell.

Genetic analysis of the Complexin trans-clamping model for cross-linking SNARE complexes in vivo.

Complexin (Cpx) is a SNARE-binding protein that regulates neurotransmission by clamping spontaneous synaptic vesicle fusion in the absence of Ca(2+) influx while promoting evoked release in response to an action potential. Previous studies indicated Cpx may cross-link multiple SNARE complexes via a trans interaction to function as a fusion clamp. During Ca(2+) influx, Cpx is predicted to undergo a conformational switch and collapse onto a single SNARE complex in a cis-binding mode to activate vesicle release. To test this model in vivo, we performed structure-function studies of the Cpx protein in Drosophila. Using genetic rescue approaches with cpx mutants that disrupt SNARE cross-linking, we find that manipulations that are predicted to block formation of the trans SNARE array disrupt the clamping function of Cpx. Unexpectedly, these same mutants rescue action potential-triggered release, indicating trans-SNARE cross-linking by Cpx is not a prerequisite for triggering evoked fusion. In contrast, mutations that impair Cpx-mediated cis-SNARE interactions that are necessary for transition from an open to closed conformation fail to rescue evoked release defects in cpx mutants, although they clamp spontaneous release normally. Our in vivo genetic manipulations support several predictions made by the Cpx cross-linking model, but unexpected results suggest additional mechanisms are likely to exist that regulate Cpx's effects on SNARE-mediated fusion. Our findings also indicate that the inhibitory and activating functions of Cpx are genetically separable, and can be mapped to distinct molecular mechanisms that differentially regulate the SNARE fusion machinery.

Postsynaptic actin regulates active zone spacing and glutamate receptor apposition at the Drosophila neuromuscular junction.

Synaptic communication requires precise alignment of presynaptic active zones with postsynaptic receptors to enable rapid and efficient neurotransmitter release. How transsynaptic signaling between connected partners organizes this synaptic apparatus is poorly understood. To further define the mechanisms that mediate synapse assembly, we carried out a chemical mutagenesis screen in Drosophila to identify mutants defective in the alignment of active zones with postsynaptic glutamate receptor fields at the larval neuromuscular junction. From this screen we identified a mutation in Actin 57B that disrupted synaptic morphology and presynaptic active zone organization. Actin 57B, one of six actin genes in Drosophila, is expressed within the postsynaptic bodywall musculature. The isolated allele, act(E84K), harbors a point mutation in a highly conserved glutamate residue in subdomain 1 that binds members of the Calponin Homology protein family, including spectrin. Homozygous act(E84K) mutants show impaired alignment and spacing of presynaptic active zones, as well as defects in apposition of active zones to postsynaptic glutamate receptor fields. act(E84K) mutants have disrupted postsynaptic actin networks surrounding presynaptic boutons, with the formation of aberrant actin swirls previously observed following disruption of postsynaptic spectrin. Consistent with a disruption of the postsynaptic actin cytoskeleton, spectrin, adducin and the PSD-95 homolog Discs-Large are all mislocalized in act(E84K) mutants. Genetic interactions between act(E84K) and neurexin mutants suggest that the postsynaptic actin cytoskeleton may function together with the Neurexin-Neuroligin transsynaptic signaling complex to mediate normal synapse development and presynaptic active zone organization.

Dan forms condensates in neuroblasts and regulates nuclear architecture and progenitor competence in vivo.

Genome organization is thought to underlie cell type specific gene expression, yet how it is regulated in progenitors to produce cellular diversity is unknown. In Drosophila, a developmentally-timed genome reorganization in neural progenitors terminates competence to produce early-born neurons. These events require downregulation of Distal antenna (Dan), part of the conserved pipsqueak DNA-binding superfamily. Here we find that Dan forms liquid-like condensates with high protein mobility, and whose size and subnuclear distribution are balanced with its DNA-binding. Further, we identify a LARKS domain, a structural motif associated with condensate-forming proteins. Deleting just 13 amino acids from LARKS abrogates Dan's ability to retain the early-born neural fate gene, hunchback, in the neuroblast nuclear interior and maintain competence in vivo. Conversely, domain-swapping with LARKS from known phase-separating proteins rescues Dan's effects on competence. Together, we provide in vivo evidence for condensate formation and the regulation of progenitor nuclear architecture underlying neuronal diversification.

Complexin controls spontaneous and evoked neurotransmitter release by regulating the timing and properties of synaptotagmin activity.

Neurotransmitter release following synaptic vesicle (SV) fusion is the fundamental mechanism for neuronal communication. Synaptic exocytosis is a specialized form of intercellular communication that shares a common SNARE-mediated fusion mechanism with other membrane trafficking pathways. The regulation of synaptic vesicle fusion kinetics and short-term plasticity is critical for rapid encoding and transmission of signals across synapses. Several families of SNARE-binding proteins have evolved to regulate synaptic exocytosis, including Synaptotagmin (SYT) and Complexin (CPX). Here, we demonstrate that Drosophila CPX controls evoked fusion occurring via the synchronous and asynchronous pathways. cpx(-/-) mutants show increased asynchronous release, while CPX overexpression largely eliminates the asynchronous component of fusion. We also find that SYT and CPX coregulate the kinetics and Ca(2+) co-operativity of neurotransmitter release. CPX functions as a positive regulator of release in part by coupling the Ca(2+) sensor SYT to the fusion machinery and synchronizing its activity to speed fusion. In contrast, syt(-/-); cpx(-/-) double mutants completely abolish the enhanced spontaneous release observe in cpx(-/-) mutants alone, indicating CPX acts as a fusion clamp to block premature exocytosis in part by preventing inappropriate activation of the SNARE machinery by SYT. CPX levels also control the size of synaptic vesicle pools, including the immediate releasable pool and the ready releasable pool-key elements of short-term plasticity that define the ability of synapses to sustain responses during burst firing. These observations indicate CPX regulates both spontaneous and evoked fusion by modulating the timing and properties of SYT activation during the synaptic vesicle cycle.

Interaction of the N-terminal domain of the AMPA receptor GluR4 subunit with the neuronal pentraxin NP1 mediates GluR4 synaptic recruitment.

Synaptogenesis requires recruitment of neurotransmitter receptors to developing postsynaptic specializations. We developed a coculture system reconstituting artificial synapses between neurons and nonneuronal cells to investigate the molecular components required for AMPA-receptor recruitment to synapses. With this system, we find that excitatory axons specifically express factors that recruit the AMPA receptor GluR4 subunit to sites of contact between axons and GluR4-transfected nonneuronal cells. Furthermore, the N-terminal domain (NTD) of GluR4 is necessary and sufficient for its recruitment to these artificial synapses and also for GluR4 recruitment to native synapses. Moreover, we show that axonally derived neuronal pentraxins NP1 and NPR are required for GluR4 recruitment to artificial and native synapses. RNAi knockdown and knockout of the neuronal pentraxins significantly decreases GluR4 targeting to synapses. Our results indicate that NP1 and NPR secreted from presynaptic neurons bind to the GluR4 NTD and are critical trans-synaptic factors for GluR4 recruitment to synapses.

Comparative analysis of Drosophila and mammalian complexins as fusion clamps and facilitators of neurotransmitter release.

The SNARE-binding protein complexin (Cpx) has been demonstrated to regulate synaptic vesicle fusion. Previous studies are consistent with Cpx functioning either as a synaptic vesicle fusion clamp to prevent premature exocytosis, or as a facilitator to directly stimulate release. Here we examined conserved roles of invertebrate and mammalian Cpx isoforms in the regulation of neurotransmitter release using the Drosophila neuromuscular junction as a model synapse. We find that SNARE binding by Cpx is required for its role as a fusion clamp. All four mammalian Cpx proteins (mCpx), which have been demonstrated to facilitate release, also function as fusion clamps when expressed in Drosophila cpx null mutants, though their clamping abilities vary between isoforms. Moreover, expression of mCpx I, II or III isoforms dramatically enhance evoked release compared to mCpx IV or Drosophila Cpx. Differences in the clamping and facilitating properties of complexin isoforms can be partially attributed to differences in the C-terminal membrane tethering domain. Our findings indicate that the function of complexins as fusion clamps and facilitators of fusion are conserved across evolution, and that these roles are genetically separable within an isoform and across different isoforms.

"Empty" sac in pregnant women with bleeding: are measurements answering the right question?

PURPOSE: To assess the percentage of first-trimester pregnancies with bleeding that demonstrate a visible sac but lack an identifiable embryo and have a mean sac diameter (MSD) in the controversial range of 16-20 mm. METHODS: Retrospective study of all first-trimester sonograms among women with vaginal bleeding during a 4-year interval. RESULTS: The study cohort consisted of 546 first- trimester sonograms. An embryo was not seen in 132 cases (24%). Of these, the MSD in 69 cases (52%) was <16 mm, between 16 and 19 mm in 20 cases (15%), or >or=20 mm in 39 cases (30%). The percentage of women who were threatening to abort who demonstrated a visible sac but lacked an identifiable embryo and had a MSD in the controversial range of 16-20 mm was 3.7% (20/546). CONCLUSION: We found that of 546 sonograms undertaken in pregnant women with vaginal bleeding in the first trimester, only 20 patients (3.7%) fell in the MSD range of 16-20 mm. Therefore, even among those diagnosticians who adopt the most stringent criterion (MSD = 20 mm), an additional examination would be requested in fewer than 1 in 25 patients.

Narp and NP1 form heterocomplexes that function in developmental and activity-dependent synaptic plasticity.

Narp is a neuronal immediate early gene that plays a role in excitatory synaptogenesis. Here, we report that native Narp in brain is part of a pentraxin complex that includes NP1. These proteins are covalently linked by disulfide bonds into highly organized complexes, and their relative ratio in the complex is dynamically dependent upon the neuron's activity history and developmental stage. Complex formation is dependent on their distinct N-terminal coiled-coil domains, while their closely homologous C-terminal pentraxin domains mediate association with AMPA-type glutamate receptors. Narp is substantially more effective in assays of cell surface cluster formation, coclustering of AMPA receptors, and excitatory synaptogenesis, yet their combined expression results in supraadditive effects. These studies support a model in which Narp can regulate the latent synaptogenic activity of NP1 by forming mixed pentraxin assemblies. This mechanism appears to contribute to both activity-independent and activity-dependent excitatory synaptogenesis.

Five-millimeter and smaller embryos without embryonic cardiac activity: outcomes in women with vaginal bleeding.

OBJECTIVE: The purpose of this study was to assess outcomes in embryos with a crown-rump length (CRL) of 5 mm or less without embryonic cardiac activity (ECA) among pregnant women with vaginal bleeding in the first trimester. METHODS: A retrospective study of all first-trimester sonograms in women with vaginal bleeding from 1999 to 2002 was conducted. RESULTS: Thirty-seven embryos without detectable ECA that had a CRL of 5 mm or less were identified. All resulted in pregnancy failure. The breakdown of these embryos by CRL was as follows: 13 were 5 mm; 10 ranged from 4 to 4.9 mm; 11 ranged from 3 to 3.9 mm; and 3 ranged from 2 to 2.9 mm. CONCLUSIONS: In pregnant women with vaginal bleeding, embryos of 5 mm and smaller without a heartbeat all resulted in pregnancy failure.

Neuronal pentraxins mediate synaptic refinement in the developing visual system.

Neuronal pentraxins (NPs) define a family of proteins that are homologous to C-reactive and acute-phase proteins in the immune system and have been hypothesized to be involved in activity-dependent synaptic plasticity. To investigate the role of NPs in vivo, we generated mice that lack one, two, or all three NPs. NP1/2 knock-out mice exhibited defects in the segregation of eye-specific retinal ganglion cell (RGC) projections to the dorsal lateral geniculate nucleus, a process that involves activity-dependent synapse formation and elimination. Retinas from mice lacking NP1 and NP2 had cholinergically driven waves of activity that occurred at a frequency similar to that of wild-type mice, but several other parameters of retinal activity were altered. RGCs cultured from these mice exhibited a significant delay in functional maturation of glutamatergic synapses. Other developmental processes, such as pathfinding of RGCs at the optic chiasm and hippocampal long-term potentiation and long-term depression, appeared normal in NP-deficient mice. These data indicate that NPs are necessary for early synaptic refinements in the mammalian retina and dorsal lateral geniculate nucleus. We speculate that NPs exert their effects through mechanisms that parallel the known role of short pentraxins outside the CNS.

Phosphorylation of Complexin by PKA Regulates Activity-Dependent Spontaneous Neurotransmitter Release and Structural Synaptic Plasticity.

Synaptic plasticity is a fundamental feature of the nervous system that allows adaptation to changing behavioral environments. Most studies of synaptic plasticity have examined the regulated trafficking of postsynaptic glutamate receptors that generates alterations in synaptic transmission. Whether and how changes in the presynaptic release machinery contribute to neuronal plasticity is less clear. The SNARE complex mediates neurotransmitter release in response to presynaptic Ca(2+) entry. Here we show that the SNARE fusion clamp Complexin undergoes activity-dependent phosphorylation that alters the basic properties of neurotransmission in Drosophila. Retrograde signaling following stimulation activates PKA-dependent phosphorylation of the Complexin C terminus that selectively and transiently enhances spontaneous release. Enhanced spontaneous release is required for activity-dependent synaptic growth. These data indicate that SNARE-dependent fusion mechanisms can be regulated in an activity-dependent manner and highlight the key role of spontaneous neurotransmitter release as a mediator of functional and structural plasticity.

Association of housing first implementation and key outcomes among homeless persons with problematic substance use.

OBJECTIVES: Housing First is a supportive housing model for persons with histories of chronic homelessness that emphasizes client-centered services, provides immediate housing, and does not require treatment for mental illness or substance abuse as a condition of participation. Previous studies of Housing First have found reduced governmental costs and improved personal well-being among participants. However, variations in real-world program implementation require better understanding of the relationship between implementation and outcomes. This study investigated the effects of Housing First implementation on housing and substance use outcomes. METHODS: Study participants were 358 individuals with histories of chronic homelessness and problematic substance use. Clients were housed in nine scatter-site Housing First programs in New York City. Program fidelity was judged across a set of core Housing First components. Client interviews at baseline and 12 months were used to assess substance use. RESULTS: Clients in programs with greater fidelity to consumer participation components of Housing First were more likely to be retained in housing and were less likely to report using stimulants or opiates at follow-up. CONCLUSIONS: Consistently implemented Housing First principles related to consumer participation were associated with superior housing and substance use outcomes among chronically homeless individuals with a history of substance use problems. The study findings suggest that program implementation is central to understanding the potential of Housing First to help clients achieve positive housing and substance use outcomes.

Second trimester prenatal ultrasound for the detection of pregnancies at increased risk of Trisomy 18 based on serum screening.

OBJECTIVE: To describe ultrasound findings in fetuses with Trisomy 18. METHODS: Prospective population-based cohort study of second trimester ultrasound among Californian women who were at increased risk of chromosome abnormality based on serum screening between November 1999 and April 2001. Structural anomalies plus the following soft markers were assessed: nuchal fold thickening, choroid plexus cyst (CPC), echogenic intracardiac focus, echogenic bowel, renal pyelectasis, clenched hands; clinodactyly; short femur, short humerus and a single umbilical artery (SUA). RESULTS: Overall, 8763 women underwent ultrasound evaluation, including 56 whose fetuses had Trisomy 18. Ultrasound anomalies were seen in 89% of Trisomy 18 fetuses, as compared with 14% of normal fetuses. If the genetic sonogram was normal (no structural anomaly and no soft marker), the risk was reduced by approximately 90%. The ultrasound soft markers were typically seen in conjunction with structural anomalies in affected fetuses and in the absence of a structural anomaly, most isolated ultrasound soft markers were not associated with Trisomy 18. The only exception was an isolated CPC, seen as the only finding in 11% of fetuses with Trisomy 18. CONCLUSIONS: If the genetic sonogram is used as a sequential test following serum biochemistry, a normal ultrasound study reduces the likelihood of Trisomy 18 substantially even if a woman has abnormal serum biochemistry. The presence of an isolated CPC raises the risk, but not high enough to prompt invasive testing.

mGluR1/5-dependent long-term depression requires the regulated ectodomain cleavage of neuronal pentraxin NPR by TACE.

Matrix metalloproteases (MMPs) play a role in remodeling the extracellular matrix during brain development and have been implicated in synaptic plasticity. Here, we report that a member of the neuronal pentraxin (NP) family, neuronal pentraxin receptor (NPR), undergoes regulated cleavage by the MMP tumor necrosis factor-alpha converting enzyme (TACE). NPR is enriched at excitatory synapses where it associates with AMPA-type glutamate receptors (AMPAR) and enhances synaptogenesis. However, in response to activation of group 1 mGluRs (mGluR1/5), TACE cleaves NPR and releases the pentraxin domain from its N-terminal transmembrane domain. Cleaved NPR rapidly accumulates in endosomes where it colocalizes with AMPAR. This process is necessary for mGluR1/5-dependent LTD in hippocampal and cerebellar synapses. These observations suggest that cleaved NPR functions to "capture" AMPAR for endocytosis and reveal a bifunctional role of NPs in both synapse strengthening and weakening.