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Ts65Dn mice, a model for Down syndrome, have excessive inhibition in the dentate gyrus, a condition that could compromise synaptic plasticity and mnemonic processing. We show that chronic systemic treatment of these mice with GABA A antagonists at non-epileptic doses causes a persistent post-drug recovery of cognition and long-term potentiation. These results suggest that over-inhibition contributes to intellectual disabilities associated with Down syndrome and that GABA A antagonists may be useful therapeutic agents for this disorder.

The SHANK family of synaptic proteins (SHANK1-3) are master regulators of the organizational structure of excitatory synapses in the brain. Mutations in are prevalent in patients with autism spectrum disorders (ASD), and loss of one copy of causes Phelan-McDermid Syndrome, a syndrome in which Autism occurs in >80% of cases. The synaptic stability of SHANK3 is highly regulated by zinc, driving the formation of postsynaptic protein complexes and increases in excitatory synaptic strength. As ASD-associated SHANK3 mutations retain responsiveness to zinc, here we investigated how increasing levels of dietary zinc could alter behavioral and synaptic deficits that occur with ASD. We performed behavioral testing together with cortico-striatal slice electrophysiology on a mouse model of ASD ( ), which displays ASD-related behaviors and structural and functional deficits at striatal synapses. We observed that 6 weeks of dietary zinc supplementation in mice prevented ASD-related repetitive and anxiety behaviors and deficits in social novelty recognition. Dietary zinc supplementation also increased the recruitment of zinc sensitive SHANK2 to synapses, reduced synaptic transmission specifically through -methyl-D-aspartate (NMDA)-type glutamate receptors, reversed the slowed decay tau of NMDA receptor (NMDAR)-mediated currents and occluded long term potentiation (LTP) at cortico-striatal synapses. These data suggest that alterations in NMDAR function underlie the lack of NMDAR-dependent cortico-striatal LTP and contribute to the reversal of ASD-related behaviors such as compulsive grooming. Our data reveal that dietary zinc alters neurological function from synapses to behavior, and identifies dietary zinc as a potential therapeutic agent in ASD.

The presynaptic active zone (AZ) is a specialized microdomain designed for the efficient and repetitive release of neurotransmitter. Bassoon and Piccolo are two high molecular weight components of the AZ, with hypothesized roles in its assembly and structural maintenance. However, glutamatergic synapses lacking either protein exhibit relatively minor defects, presumably due to their significant functional redundancy. In the present study, we have used interference RNAs to eliminate both proteins from glutamatergic synapses, and find that they are essential for maintaining synaptic integrity. Loss of Bassoon and Piccolo leads to the aberrant degradation of multiple presynaptic proteins, culminating in synapse degeneration. This phenotype is mediated in part by the E3 ubiquitin ligase Siah1, an interacting partner of Bassoon and Piccolo whose activity is negatively regulated by their conserved zinc finger domains. Our findings demonstrate a novel role for Bassoon and Piccolo as critical regulators of presynaptic ubiquitination and proteostasis. The presynaptic proteins Bassoon and Piccolo interact with and inhibit the activity of the E3 ubiquitin ligase Siah1 to regulate protein homeostasis and synaptic integrity.

The formation of synapses in the vertebrate central nervous system is a complex process that occurs over a protracted period of development. Recent work has begun to unravel the mysteries of synaptogenesis, demonstrating the existence of multiple molecules that influence not only when and where synapses form but also synaptic specificity and stability. Some of these molecules act at a distance, steering axons to their correct receptive fields and promoting neuronal differentiation and maturation, whereas others act at the time of contact, providing positional information about the appropriateness of targets and/or inductive signals that trigger the cascade of events leading to synapse formation. In addition, correlated synaptic activity provides critical information about the appropriateness of synaptic connections, thereby influencing synapse stability and elimination. Although synapse formation and elimination are hallmarks of early development, these processes are also fundamental to learning, memory, and cognition in the mature brain.

Down’s syndrome results from full or partial trisomy of chromosome 21. However, the consequences of the underlying gene–dosage imbalance on adult tissues remain poorly understood. Here we show that in Ts65Dn mice, which are trisomic for 132 genes homologous to genes on human chromosome 21, triplication of Usp16 reduces the self-renewal of haematopoietic stem cells and the expansion of mammary epithelial cells, neural progenitors and fibroblasts. In addition, Usp16 is associated with decreased ubiquitination of Cdkn2a and accelerated senescence in Ts65Dn fibroblasts. Usp16 can remove ubiquitin from histone H2A on lysine 119, a critical mark for the maintenance of multiple somatic tissues. Downregulation of Usp16, either by mutation of a single normal Usp16 allele or by short interfering RNAs, largely rescues all of these defects. Furthermore, in human tissues overexpression of USP16 reduces the expansion of normal fibroblasts and postnatal neural progenitors, whereas downregulation of USP16 partially rescues the proliferation defects of Down’s syndrome fibroblasts. Taken together, these results suggest that USP16 has an important role in antagonizing the self-renewal and/or senescence pathways in Down’s syndrome and could serve as an attractive target to ameliorate some of the associated pathologies. An analysis of somatic tissues derived from mouse models of Down’s syndrome shows reduced self-renewal capacities in various cell types, with these defects partially dependent on triplication of the Usp16 gene; overexpression and knockout studies in human cells shows that USP16 has a role in Down’s syndrome-related proliferation defects, making this gene an attractive option for further study. Excess Usp16 linked to Down's syndrome People with Down's syndrome have abnormalities in multiple tissues including mental retardation and early ageing. The disease is often the result of full or partial trisomy of chromosome 21, but the molecular mechanisms underlying the observed cellular defects remain largely unknown. An analysis of haematopoietic stem cells in the Down's syndrome mouse model Ts65Dn has revealed a reduced self-renewal associated with the proliferation of cells expressing three copies of the Usp16 gene, which encodes a deubiquitination enzyme involved in chromatin remodelling and cell cycle progression. In a second Down's syndrome mouse model, Ts1Cje, haematopoietic stem cells were not defective. Downregulation of USP16 rescued the functional defects of affected Ts65Dn cells. Overexpression of USP16 in normal human fibroblasts reduced their proliferative capacity and USP16 downregulation partially rescued human Down's syndrome fibroblast proliferation defects. The authors propose that USP16 is a potential target for therapeutics designed to ameliorate the pathologies associated with this syndrome.

Neuronal morphology and number of synapses is not static, but can change in response to a variety of factors, a process called synaptic plasticity. These structural and molecular changes are believed to represent the basis for learning and memory, thereby underling both the developmental and activity‐dependent remodelling of excitatory synapses. Here, we report that Zn 2+ ions, which are highly enriched within the postsynaptic density (PSD), are able to influence the recruitment of ProSAP/Shank proteins to PSDs in a family member‐specific manner during the course of synaptogenesis and synapse maturation. Through selectively overexpressing each family member at excitatory postsynapses and comparing this to shRNA‐mediated knockdown, we could demonstrate that only the overexpression of zinc‐sensitive ProSAP1/Shank2 or ProSAP2/Shank3 leads to increased synapse density, although all of them cause a decrease upon knockdown. Furthermore, depletion of synaptic Zn 2+ along with the knockdown of zinc‐insensitive Shank1 causes the rapid disintegration of PSDs and the loss of several postsynaptic molecules including Homer1, PSD‐95 and NMDA receptors. These findings lead to the model that the concerted action of ProSAP/Shank and Zn 2+ is essential for the structural integrity of PSDs and moreover that it is an important element of synapse formation, maturation and structural plasticity. ProSAP/Shank are scaffolding proteins that localize to the postsynaptic density (PSD). This study shows that Zn 2+ ions directly regulate the localization and recruitment of Shank/ProSAP1/2 to PSDs to facilitate synapse formation and maturation.

The dynamic assembly of filamentous (F) actin plays essential roles in the assembly of presynaptic boutons, the fusion, mobilization and recycling of synaptic vesicles (SVs), and presynaptic forms of plasticity. However, the molecular mechanisms that regulate the temporal and spatial assembly of presynaptic F-actin remain largely unknown. Similar to other F-actin rich membrane specializations, presynaptic boutons contain a set of molecules that respond to cellular cues and trans-synaptic signals to facilitate activity-dependent assembly of F-actin. The presynaptic active zone (AZ) protein Piccolo has recently been identified as a key regulator of neurotransmitter release during SV cycling. It does so by coordinating the activity-dependent assembly of F-Actin and the dynamics of key plasticity molecules including Synapsin1, Profilin and CaMKII. The multidomain structure of Piccolo, its exquisite association with the AZ, and its ability to interact with a number of actin-associated proteins suggest that Piccolo may function as a platform to coordinate the spatial assembly of F-actin. Here we have identified Daam1, a Formin that functions with Profilin to drive F-actin assembly, as a novel Piccolo binding partner. We also found that within cells Daam1 activation promotes Piccolo binding, an interaction that can spatially direct the polymerization of F-Actin. Moreover, similar to Piccolo and Profilin, Daam1 loss of function impairs presynaptic-F-actin assembly in neurons. These data suggest a model in which Piccolo directs the assembly of presynaptic F-Actin from the AZ by scaffolding key actin regulatory proteins including Daam1.

This study shows that, although AMPA-type glutamate receptors are trafficked to dendrites through the normal Golgi secretory pathway, NMDA receptors bypass the somatic Golgi apparatus and instead move from the endoplasmic reticulum in endoplasmic reticulum–like vesicles to Golgi 'outposts' located in the dendrites. Synaptic plasticity is dependent on the differential sorting, delivery and retention of neurotransmitter receptors, but the mechanisms underlying these processes are poorly understood. We found that differential sorting of glutamate receptor subtypes began in the endoplasmic reticulum of rat hippocampal neurons. As AMPA receptors (AMPARs) were trafficked to the plasma membrane via the conventional somatic Golgi network, NMDA receptors (NMDARs) were diverted from the somatic endoplasmic reticulum into a specialized endoplasmic reticulum subcompartment that bypasses somatic Golgi, merging instead with dendritic Golgi outposts. This endoplasmic reticulum subcompartment was composed of highly mobile vesicles containing the NMDAR subunits NR1 and NR2B, the microtubule-dependent motor protein KIF17, and the postsynaptic adaptor proteins CASK and SAP97. Our data demonstrate that the retention and trafficking of NMDARs in this endoplasmic reticulum subcompartment requires both CASK and SAP97. These findings indicate that NMDARs are sorted away from AMPARs via a non-conventional secretory pathway that utilizes dendritic Golgi outposts.

During development, pyramidal neurons undergo dynamic regulation of AMPA receptor (AMPAR) subunit composition and density to help drive synaptic plasticity and maturation. These normal developmental changes in AMPARs are particularly vulnerable to risk factors for Autism Spectrum Disorders (ASDs), which include loss or mutations of synaptic proteins and environmental insults, such as dietary zinc deficiency. Here, we show how Shank2 and Shank3 mediate a zinc-dependent regulation of AMPAR function and subunit switch from GluA2-lacking to GluA2-containing AMPARs. Over development, we found a concomitant increase in Shank2 and Shank3 with GluA2 at synapses, implicating these molecules as potential players in AMPAR maturation. Since Shank activation and function require zinc, we next studied whether neuronal activity regulated postsynaptic zinc at glutamatergic synapses. Zinc was found to increase transiently and reversibly with neuronal depolarization at synapses, which could affect Shank and AMPAR localization and activity. Elevated zinc induced multiple functional changes in AMPAR, indicative of a subunit switch. Specifically, zinc lengthened the decay time of AMPAR-mediated synaptic currents and reduced their inward rectification in young hippocampal neurons. Mechanistically, both Shank2 and Shank3 were necessary for the zinc-sensitive enhancement of AMPAR-mediated synaptic transmission and act in concert to promote removal of GluA1 while enhancing recruitment of GluA2 at pre-existing Shank puncta. These findings highlight a cooperative local dynamic regulation of AMPAR subunit switch controlled by zinc signaling through Shank2 and Shank3 to shape the biophysical properties of developing glutamatergic synapses. Given the zinc sensitivity of young neurons and its dependence on Shank2 and Shank3, genetic mutations and/or environmental insults during early development could impair synaptic maturation and circuit formation that underlie ASD etiology.

Information on protein–protein interactions (PPIs) is of critical importance for studying complex biological systems and developing therapeutic strategies. Here, we present a double‐readout bioluminescence‐based two‐hybrid technology, termed LuTHy, which provides two quantitative scores in one experimental procedure when testing binary interactions. PPIs are first monitored in cells by quantification of bioluminescence resonance energy transfer (BRET) and, following cell lysis, are again quantitatively assessed by luminescence‐based co‐precipitation (LuC). The double‐readout procedure detects interactions with higher sensitivity than traditional single‐readout methods and is broadly applicable, for example, for detecting the effects of small molecules or disease‐causing mutations on PPIs. Applying LuTHy in a focused screen, we identified 42 interactions for the presynaptic chaperone CSPα, causative to adult‐onset neuronal ceroid lipofuscinosis (ANCL), a progressive neurodegenerative disease. Nearly 50% of PPIs were found to be affected when studying the effect of the disease‐causing missense mutations L115R and ∆L116 in CSPα with LuTHy. Our study presents a robust, sensitive research tool with high utility for investigating the molecular mechanisms by which disease‐associated mutations impair protein activity in biological systems. Synopsis A new double‐readout bioluminescence‐based two‐hybrid method deepens the coverage of protein interaction maps. It provides two quantitative interaction scores, recovers transient associations and monitors the effects of missense mutations and small molecules on interactions. LuTHy is a double‐readout technology that provides two quantitative scores for protein‐protein interactions in mammalian cells. Low‐ and high‐affinity interactions can be detected with high sensitivity. Nearly 50% of CSPα interactions with synaptic proteins are affected by ANCL‐disease mutations. Graphical Abstract A new double‐readout bioluminescence‐based two‐hybrid method deepens the coverage of protein interaction maps. It provides two quantitative interaction scores, recovers transient associations and monitors the effects of missense mutations and small molecules on interactions.