Piccolino is required for ribbon architecture at cochlear inner hair cell synapses and for hearing.

Cochlear inner hair cells (IHCs) form specialized ribbon synapses with spiral ganglion neurons that tirelessly transmit sound information at high rates over long time periods with extreme temporal precision. This functional specialization is essential for sound encoding and is attributed to a distinct molecular machinery with unique players or splice variants compared to conventional neuronal synapses. Among these is the active zone (AZ) scaffold protein piccolo/aczonin, which is represented by its short splice variant piccolino at cochlear and retinal ribbon synapses. While the function of piccolo at synapses of the central nervous system has been intensively investigated, the role of piccolino at IHC synapses remains unclear. In this study, we characterize the structure and function of IHC synapses in piccolo gene-trap mutant rats (Pclogt/gt ). We find a mild hearing deficit with elevated thresholds and reduced amplitudes of auditory brainstem responses. Ca2+ channel distribution and ribbon morphology are altered in apical IHCs, while their presynaptic function seems to be unchanged. We conclude that piccolino contributes to the AZ organization in IHCs and is essential for normal hearing.

Microglia actively remove NR1 autoantibody-bound NMDA receptors and associated post-synaptic proteins in neuron microglia co-cultures.

Autoantibodies against the NR1 subunit of NMDA receptors (NMDARs) have been shown to promote crosslinking and internalization of bound receptors in NMDAR encephalitis (NMDARE). This internalization-mediated loss of NMDARs is thought to be the major mechanism leading to pathogenic outcomes in patients. However, the role of bound autoantibody in engaging the resident immune cells, microglia, remains poorly understood. Here, using a patient-derived monoclonal NR1 autoantibody (hNR1-mAb) and a co-culture system of microglia and neurons, we could show that hNR1-mAb bound to hippocampal neurons led to microglia-mediated removal of hNR1-mAb bound NMDARs. These complexes were found to accumulate inside endo-lysosomal compartments of microglia. Utilizing another patient isolated monoclonal autoantibody, against the α1-subunit of GABAA receptors (α1-GABAA -mAb), such removal of receptors was found to be specific to the antibody-bound receptor targets. Interestingly, along with receptor removal, we also observed a reduction in synapse number, more specifically in the numbers of post-synaptic proteins like PSD95 and Homer 1, when microglia were present in the culture. Importantly, mutations in the Fc region of hNR1-mAb, blocking its Fcγ receptor (FcγR) and complement binding, attenuated hNR1-mAb driven loss of NMDARs and synapses, indicating that microglia engagement by bound hNR1-mAb is critical for receptor and synapse loss. Our data argues for an active involvement of microglia in removal of NMDARs and other receptors in individuals with autoimmune encephalitis, thereby contributing to the etiology of these diseases.

Synaptic logistics: The presynaptic scaffold protein Piccolo a nodal point tuning synaptic vesicle recycling, maintenance and integrity.

Properly working synapses are one important guarantor for a functional and healthy brain. They are small, densely packed structures, where information is transmitted through the release of neurotransmitters from synaptic vesicles (SVs). The latter cycle within the presynaptic terminal as they first fuse with the plasma membrane to deliver their neurotransmitter, and afterwards become recycled and prepared for a new release event. The synapse is an autonomous structure functioning mostly independent of the neuronal soma. Dysfunction in synaptic processes associated with local insults or genetic abnormalities can directly compromise synapse function and integrity and subsequently lead to the onset of neurodegenerative diseases. Therefore, measures need to be in place counteracting these threats for instance through the continuous replacement of old and damaged SV proteins. Interestingly recent studies show that the presynaptic scaffolding protein Piccolo contributes to health, function and integrity of synapses, as it mediates the delivery of synaptic proteins from the trans-Golgi network (TGN) towards synapses, as well as the local recycling and turnover of SV proteins within synaptic terminals. It can fulfill these various tasks through its multi-domain structure and ability to interact with numerous binding partners. In addition, Piccolo has recently been linked with the early onset neurodegenerative disease Pontocerebellar Hypoplasia Type 3 (PCH3) further underlying its importance for neuronal health. In this review, we will focus on Piccolo's contributions to synapse function, health and integrity and make a connection how those may contribute to the disease pattern of PCH3.

Differential Modes of Action of α1- and α1γ2-Autoantibodies Derived from Patients with GABAAR Encephalitis.

Autoantibodies against central nervous system proteins are increasingly being recognized in association with neurologic disorders. Although a growing number of neural autoantibodies have been identified, a causal link between specific autoantibodies and disease symptoms remains unclear, as most studies use patient-derived CSF-containing mixtures of autoantibodies. This raises questions concerning mechanism of action and which autoantibodies truly contribute to disease progression. To address this issue, monoclonal autoantibodies were isolated from a young girl with a range of neurologic symptoms, some of which reacted with specific GABAA receptor (GABAAR) subunits, α1-subunit and α1γ2-subunit, which in this study we have characterized in detail using a combination of cellular imaging and electrophysiological techniques. These studies in neurons from wild-type mice (C57BL/6J; RRID:IMSR_JAX:000664) of mixed-sex revealed that the α1 and α1γ2 subunit-specific antibodies have differential effects on the GABAA receptor. Namely, the α1-antibody was found to directly affect GABAA receptor function on a short time scale that diminished GABA currents, leading to increased network excitability. On longer time scales those antibodies also triggered a redistribution of the GABAA receptor away from synapses. In contrast, the α1γ2-antibody had no direct effect on GABAA receptor function and could possibly mediate its effect through other actors of the immune system. Taken together, these data highlight the complexity underlying autoimmune disorders and show that antibodies can exert their effect through many mechanisms within the same disease.

Organization of Presynaptic Autophagy-Related Processes.

Brain synapses pose special challenges on the quality control of their protein machineries as they are far away from the neuronal soma, display a high potential for plastic adaptation and have a high energy demand to fulfill their physiological tasks. This applies in particular to the presynaptic part where neurotransmitter is released from synaptic vesicles, which in turn have to be recycled and refilled in a complex membrane trafficking cycle. Pathways to remove outdated and damaged proteins include the ubiquitin-proteasome system acting in the cytoplasm as well as membrane-associated endolysosomal and the autophagy systems. Here we focus on the latter systems and review what is known about the spatial organization of autophagy and endolysomal processes within the presynapse. We provide an inventory of which components of these degradative systems were found to be present in presynaptic boutons and where they might be anchored to the presynaptic apparatus. We identify three presynaptic structures reported to interact with known constituents of membrane-based protein-degradation pathways and therefore may serve as docking stations. These are (i) scaffolding proteins of the cytomatrix at the active zone, such as Bassoon or Clarinet, (ii) the endocytic machinery localized mainly at the peri-active zone, and (iii) synaptic vesicles. Finally, we sketch scenarios, how presynaptic autophagic cargos are tagged and recruited and which cellular mechanisms may govern membrane-associated protein turnover in the presynapse.

Patient-Derived Anti-NMDAR Antibody Disinhibits Cortical Neuronal Networks through Dysfunction of Inhibitory Neuron Output.

Anti-NMDA receptor (NMDAR) encephalitis is a severe neuropsychiatric disorder associated with autoantibodies against NMDARs, which cause a variety of symptoms from prominent psychiatric and cognitive manifestations to seizures and autonomic instability. Previous studies mainly focused on hippocampal effects of these autoantibodies, helping to explain mechanistic causes for cognitive impairment. However, antibodies' effects on higher cortical network function, where they could contribute to psychosis and/or seizures, have not been explored in detail until now. Here, we employed a patient-derived monoclonal antibody targeting the NR1 subunit of NMDAR and tested its effects on in vitro cultures of rodent cortical neurons, using imaging and electrophysiological techniques. We report that this hNR1 antibody drives cortical networks to a hyperexcitable state and disrupts mechanisms stabilizing network activity such as Npas4 signaling. Network hyperactivity is in part a result of a reduced synaptic output of inhibitory neurons, as indicated by a decreased inhibitory drive and levels of presynaptic inhibitory proteins, specifically in inhibitory-to-excitatory neuron synapses. Importantly, on a single-cell level hNR1 antibody selectively impairs NMDAR-mediated currents and synaptic transmission of cortical inhibitory neurons, yet has no effect on excitatory neurons, which contrasts with its effects on hippocampal neurons. Together, these findings provide a novel, cortex-specific mechanism of antibody-induced neuronal hyperexcitability, highlighting regional specificity underlying the pathology of autoimmune encephalitis.SIGNIFICANCE STATEMENT It is increasingly appreciated that the inadvertent activation of the immune system within CNS can underlie pathogenesis of neuropsychiatric disorders. Although the exact mechanisms remain elusive, autoantibodies derived from patients with autoimmune encephalitis pose a unique tool to study pathogenesis of neuropsychiatric states. Our analysis reveals that autoantibody against the NMDA receptor (NMDAR) has a distinct mechanism of action in the cortex, where it impairs function of inhibitory neurons leading to increased cortical network excitability, in contrast to previously described hippocampal synaptic mechanisms of information encoding, highlighting brain regional specificity. Notably, similar mechanism of NMDAR-mediated inhibitory hypofunction leading to cortical disinhibition has been suggested to underlie pathology of schizophrenia, hence our data provide new evidence for common mechanisms underlying neuropsychiatric disorders.

Encephalitis patient-derived monoclonal GABAA receptor antibodies cause epileptic seizures.

Autoantibodies targeting the GABAA receptor (GABAAR) hallmark an autoimmune encephalitis presenting with frequent seizures and psychomotor abnormalities. Their pathogenic role is still not well-defined, given the common overlap with further autoantibodies and the lack of patient-derived mAbs. Five GABAAR mAbs from cerebrospinal fluid cells bound to various epitopes involving the α1 and γ2 receptor subunits, with variable binding strength and partial competition. mAbs selectively reduced GABAergic currents in neuronal cultures without causing receptor internalization. Cerebroventricular infusion of GABAAR mAbs and Fab fragments into rodents induced a severe phenotype with seizures and increased mortality, reminiscent of encephalitis patients' symptoms. Our results demonstrate direct pathogenicity of autoantibodies on GABAARs independent of Fc-mediated effector functions and provide an animal model for GABAAR encephalitis. They further provide the scientific rationale for clinical treatments using antibody depletion and can serve as tools for the development of antibody-selective immunotherapies.

In vitro zinc supplementation alters synaptic deficits caused by autism spectrum disorder-associated Shank2 point mutations in hippocampal neurons.

Autism Spectrum Disorders (ASDs) are neurodevelopmental disorders characterised by deficits in social interactions and repetitive behaviours. ASDs have a strong genetic basis with mutations involved in the development and function of neural circuitry. Shank proteins act as master regulators of excitatory glutamatergic synapses, and Shank mutations have been identified in people with ASD. Here, we have investigated the impact of ASD-associated Shank2 single nucleotide variants (SNVs) at the synaptic level, and the potential of in vitro zinc supplementation to prevent synaptic deficits. Dissociated rat hippocampal cultures expressing enhanced green fluorescent protein (EGFP) tagged Shank2-Wildtype (WT), and ASD-associated Shank2 single nucleotide variants (SNVs: S557N, V717F, and L1722P), were cultured in the absence or presence of 10 µM zinc. In comparison to Shank2-WT, ASD-associated Shank2 SNVs induced significant decreases in synaptic density and reduced the frequency of miniature excitatory postsynaptic currents. These structural and functional ASD-associated synaptic deficits were prevented by chronic zinc supplementation and further support zinc supplementation as a therapeutic target in ASD.

BSN (bassoon) and PRKN/parkin in concert control presynaptic vesicle autophagy.

Maintaining the integrity and function of the presynaptic neurotransmitter release apparatus is a demanding process for a post-mitotic neuron; the mechanisms behind it are still unclear. BSN (bassoon), an active zone scaffolding protein, has been implicated in the control of presynaptic macroautophagy/autophagy, a process we recently showed depends on poly-ubiquitination of synaptic proteins. Moreover, loss of BSN was found to lead to smaller synaptic vesicle (SV) pools and younger pools of the SV protein SV2. Of note, the E3 ligase PRKN/parkin appears to be involved in BSN deficiency-related changes in autophagy levels, as shRNA-mediated knockdown of PRKN counteracts BSN-deficiency and rescues decreased SV protein levels as well as impaired SV recycling in primary cultured neurons. These data imply that BSN and PRKN act in concert to control presynaptic autophagy and maintain presynaptic proteostasis and SV turnover at the physiologically required levels.

Serine-Arginine Protein Kinase SRPK2 Modulates the Assembly of the Active Zone Scaffolding Protein CAST1/ERC2.

Neurons release neurotransmitters at a specialized region of the presynaptic membrane, the active zone (AZ), where a complex meshwork of proteins organizes the release apparatus. The formation of this proteinaceous cytomatrix at the AZ (CAZ) depends on precise homo- and hetero-oligomerizations of distinct CAZ proteins. The CAZ protein CAST1/ERC2 contains four coiled-coil (CC) domains that interact with other CAZ proteins, but also promote self-assembly, which is an essential step for its integration during AZ formation. The self-assembly and synaptic recruitment of the Drosophila protein Bruchpilot (BRP), a partial homolog of CAST1/ERC2, is modulated by the serine-arginine protein kinase (SRPK79D). Here, we demonstrate that overexpression of the vertebrate SRPK2 regulates the self-assembly of CAST1/ERC2 in HEK293T, SH-SY5Y and HT-22 cells and the CC1 and CC4 domains are involved in this process. Moreover, the isoform SRPK2 forms a complex with CAST1/ERC2 when co-expressed in HEK293T and SH-SY5Y cells. More importantly, SRPK2 is present in brain synaptic fractions and synapses, suggesting that this protein kinase might control the level of self-aggregation of CAST1/ERC2 in synapses, and thereby modulate presynaptic assembly.

Autism-associated Shank3 mutations alter mGluR expression and mGluR-dependent but not NMDA receptor-dependent long-term depression.

SHANK3 is a postsynaptic structural protein localized at excitatory glutamatergic synapses in which deletions and mutations have been implicated in patients with autism spectrum disorders (ASD). The expression of Shank3 ASD mutations causes impairments in ionotropic glutamate receptor-mediated synaptic responses in neurons, which is thought to underlie ASD-related behaviors, thereby indicating glutamatergic synaptopathy as one of the major pathogenic mechanisms. However, little is known about the functional consequences of ASD-associated mutations in Shank3 on another important set of glutamate receptors, group I metabotropic glutamate receptors (mGluRs). Here, we further assessed how Shank3 mutations identified in patients with ASD (one de novo InsG mutation and two inherited point mutations, R87C and R375C) disrupt group I mGluR (mGluR1 and mGluR5) expression and function. To identify potential isoform-specific deficits induced by ASD-associated Shank3 mutations on group I mGluRs, we surface immunolabeled mGluR1 and mGluR5 independently. We also induced mGluR-dependent synaptic plasticity (R,S-3,5-dihydroxyphenylglycine [DHPG]-induced long-term depression [LTD]) as well as N-methyl-D-aspartate receptor (NMDAR)-dependent LTD. ASD-associated mutations in Shank3 differentially interfered with the ability of cultured hippocampal neurons to express mGluR5 and mGluR1 at synapses. Intriguingly, all ASD Shank3 mutations impaired mGluR-dependent LTD without altering NMDAR-dependent LTD. Our data show that the specific perturbation in mGluR-dependent synaptic plasticity occurs in neurons expressing ASD-associated Shank3 mutations, which may underpin synaptic dysfunction and subsequent behavioral deficits in ASD.

N-methyl-D-aspartate receptor dysfunction by unmutated human antibodies against the NR1 subunit.

Anti-N-methyl-D-aspartate receptor (NMDAR) encephalitis is the most common autoimmune encephalitis related to autoantibody-mediated synaptic dysfunction. Cerebrospinal fluid-derived human monoclonal NR1 autoantibodies showed low numbers of somatic hypermutations or were unmutated. These unexpected germline-configured antibodies showed weaker binding to the NMDAR than matured antibodies from the same patient. In primary hippocampal neurons, germline NR1 autoantibodies strongly and specifically reduced total and synaptic NMDAR currents in a dose- and time-dependent manner. The findings suggest that functional NMDAR antibodies are part of the human naïve B cell repertoire. Given their effects on synaptic function, they might contribute to a broad spectrum of neuropsychiatric symptoms. Ann Neurol 2019;85:771-776.

A Multiple Piccolino-RIBEYE Interaction Supports Plate-Shaped Synaptic Ribbons in Retinal Neurons.

Active zones at chemical synapses are highly specialized sites for the regulated release of neurotransmitters. Despite a high degree of active zone protein conservation in vertebrates, every type of chemical synapse expresses a given set of protein isoforms and splice variants adapted to the demands on neurotransmitter release. So far, we know little about how specific active zone proteins contribute to the structural and functional diversity of active zones. In this study, we explored the nanodomain organization of ribbon-type active zones by addressing the significance of Piccolino, the ribbon synapse-specific splice variant of Piccolo, for shaping the ribbon structure. We followed up on previous results, which indicated that rod photoreceptor synaptic ribbons lose their structural integrity in a knockdown of Piccolino. Here, we demonstrate an interaction between Piccolino and the major ribbon component RIBEYE that supports plate-shaped synaptic ribbons in retinal neurons. In a detailed ultrastructural analysis of three different types of retinal ribbon synapses in Piccolo/Piccolino-deficient male and female rats, we show that the absence of Piccolino destabilizes the superstructure of plate-shaped synaptic ribbons, although with variable manifestation in the cell types examined. Our analysis illustrates how the expression of a specific active zone protein splice variant (e.g., Piccolino) contributes to structural diversity of vertebrate active zones.SIGNIFICANCE STATEMENT Retinal ribbon synapses are a specialized type of chemical synapse adapted for the regulated fast and tonic release of neurotransmitter. The hallmark of retinal ribbon synapses is the plate-shaped synaptic ribbon, which extends from the release site into the terminals' cytoplasm and tethers hundreds of synaptic vesicles. Here, we show that Piccolino, the synaptic ribbon specific splice variant of Piccolo, interacts with RIBEYE, the main component of synaptic ribbons. This interaction occurs via several PxDLS-like motifs located at the C terminus of Piccolino, which can connect multiple RIBEYE molecules. Loss of Piccolino disrupts the characteristic plate-shaped structure of synaptic ribbons, indicating a role of Piccolino in synaptic ribbon assembly.

Cognitive impairment and autistic-like behaviour in SAPAP4-deficient mice.

In humans, genetic variants of DLGAP1-4 have been linked with neuropsychiatric conditions, including autism spectrum disorder (ASD). While these findings implicate the encoded postsynaptic proteins, SAPAP1-4, in the etiology of neuropsychiatric conditions, underlying neurobiological mechanisms are unknown. To assess the contribution of SAPAP4 to these disorders, we characterized SAPAP4-deficient mice. Our study reveals that the loss of SAPAP4 triggers profound behavioural abnormalities, including cognitive deficits combined with impaired vocal communication and social interaction, phenotypes reminiscent of ASD in humans. These behavioural alterations of SAPAP4-deficient mice are associated with dramatic changes in synapse morphology, function and plasticity, indicating that SAPAP4 is critical for the development of functional neuronal networks and that mutations in the corresponding human gene, DLGAP4, may cause deficits in social and cognitive functioning relevant to ASD-like neurodevelopmental disorders.

Light-Activated ROS Production Induces Synaptic Autophagy.

The regulated turnover of synaptic vesicle (SV) proteins is thought to involve the ubiquitin-dependent tagging and degradation through endo-lysosomal and autophagy pathways. Yet, it remains unclear which of these pathways are used, when they become activated, and whether SVs are cleared en masse together with SV proteins or whether both are degraded selectively. Equally puzzling is how quickly these systems can be activated and whether they function in real-time to support synaptic health. To address these questions, we have developed an imaging-based system that simultaneously tags presynaptic proteins while monitoring autophagy. Moreover, by tagging SV proteins with a light-activated ROS generator, Supernova, it was possible to temporally control the damage to specific SV proteins and assess their consequence to autophagy-mediated clearance mechanisms and synaptic function. Our results show that, in mouse hippocampal neurons of either sex, presynaptic autophagy can be induced in as little as 5-10 min and eliminates primarily the damaged protein rather than the SV en masse. Importantly, we also find that autophagy is essential for synaptic function, as light-activated damage to, for example, Synaptophysin only compromises synaptic function when autophagy is simultaneously blocked. These data support the concept that presynaptic boutons have a robust highly regulated clearance system to maintain not only synapse integrity, but also synaptic function.SIGNIFICANCE STATEMENT The real-time surveillance and clearance of synaptic proteins are thought to be vital to the health, functionality, and integrity of vertebrate synapses and are compromised in neurodegenerative disorders, yet the fundamental mechanisms regulating these systems remain enigmatic. Our analysis reveals that presynaptic autophagy is a critical part of a real-time clearance system at synapses capable of responding to local damage of synaptic vesicle proteins within minutes and to be critical for the ongoing functionality of these synapses. These data indicate that synapse autophagy is not only locally regulated but also crucial for the health and functionality of vertebrate presynaptic boutons.

The Exocyst Component Exo70 Modulates Dendrite Arbor Formation, Synapse Density, and Spine Maturation in Primary Hippocampal Neurons.

Neurons are highly polarized cells displaying an elaborate architectural morphology. The design of their dendritic arborization and the distribution of their synapses contribute importantly to information processing in the brain. The growth and complexity of dendritic arbors are driven by the formation of synapses along their lengths. Synaptogenesis is augmented by the secretion of factors, like BDNF, Reelin, BMPs, or Wnts. Exo70 is a component of the exocyst complex, a protein complex that guides membrane addition and polarized exocytosis. While it has been linked to cytokinesis and the establishment of cell polarity, its role in synaptogenesis is poorly understood. In this report, we show that Exo70 plays a role in the arborization of dendrites and the development of synaptic connections between cultured hippocampal neurons. Specifically, while the overexpression of Exo70 increases dendritic arborization, synapse number, and spine density, the inhibition of Exo70 expression reduces secondary and tertiary dendrite formation and lowers synapse density. Moreover, increasing Exo70 expression augmented synaptic vesicle recycling as evaluated by FM4-64 dye uptake and the inverse was observed with downregulation of endogenous Exo70. Monitoring the formation of dendritic spines by super-resolution microscopy, we also observed that mRFP-Exo70 accumulates at the tip of EGFP-ß-actin-positive filopodia. Together, these results suggest that Exo70 is essentially involved in the formation of synapses and neuronal dendritic morphology.

Shank and Zinc Mediate an AMPA Receptor Subunit Switch in Developing Neurons.

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.

Dietary Zinc Supplementation Prevents Autism Related Behaviors and Striatal Synaptic Dysfunction in Shank3 Exon 13-16 Mutant Mice.

The SHANK family of synaptic proteins (SHANK1-3) are master regulators of the organizational structure of excitatory synapses in the brain. Mutations in SHANK1-3 are prevalent in patients with autism spectrum disorders (ASD), and loss of one copy of SHANK3 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 Shank3 -/- mouse model of ASD (Shank3 ex13-1616-/-), which displays ASD-related behaviors and structural and functional deficits at striatal synapses. We observed that 6 weeks of dietary zinc supplementation in Shank3 ex13-16-/- 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 N-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.

LuTHy: a double-readout bioluminescence-based two-hybrid technology for quantitative mapping of protein-protein interactions in mammalian cells.

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.