Phase Transition in Postsynaptic Densities Underlies Formation of Synaptic Complexes and Synaptic Plasticity.

Postsynaptic densities (PSDs) are membrane semi-enclosed, submicron protein-enriched cellular compartments beneath postsynaptic membranes, which constantly exchange their components with bulk aqueous cytoplasm in synaptic spines. Formation and activity-dependent modulation of PSDs is considered as one of the most basic molecular events governing synaptic plasticity in the nervous system. In this study, we discover that SynGAP, one of the most abundant PSD proteins and a Ras/Rap GTPase activator, forms a homo-trimer and binds to multiple copies of PSD-95. Binding of SynGAP to PSD-95 induces phase separation of the complex, forming highly concentrated liquid-like droplets reminiscent of the PSD. The multivalent nature of the SynGAP/PSD-95 complex is critical for the phase separation to occur and for proper activity-dependent SynGAP dispersions from the PSD. In addition to revealing a dynamic anchoring mechanism of SynGAP at the PSD, our results also suggest a model for phase-transition-mediated formation of PSD.

Cross-modality supervised image restoration enables nanoscale tracking of synaptic plasticity in living mice.

Learning is thought to involve changes in glutamate receptors at synapses, submicron structures that mediate communication between neurons in the central nervous system. Due to their small size and high density, synapses are difficult to resolve in vivo, limiting our ability to directly relate receptor dynamics to animal behavior. Here we developed a combination of computational and biological methods to overcome these challenges. First, we trained a deep-learning image-restoration algorithm that combines the advantages of ex vivo super-resolution and in vivo imaging modalities to overcome limitations specific to each optical system. When applied to in vivo images from transgenic mice expressing fluorescently labeled glutamate receptors, this restoration algorithm super-resolved synapses, enabling the tracking of behavior-associated synaptic plasticity with high spatial resolution. This method demonstrates the capabilities of image enhancement to learn from ex vivo data and imaging techniques to improve in vivo imaging resolution.

Arc/Arg3.1 regulates an endosomal pathway essential for activity-dependent ß-amyloid generation.

Assemblies of ß-amyloid (Aß) peptides are pathological mediators of Alzheimer's Disease (AD) and are produced by the sequential cleavages of amyloid precursor protein (APP) by ß-secretase (BACE1) and γ-secretase. The generation of Aß is coupled to neuronal activity, but the molecular basis is unknown. Here, we report that the immediate early gene Arc is required for activity-dependent generation of Aß. Arc is a postsynaptic protein that recruits endophilin2/3 and dynamin to early/recycling endosomes that traffic AMPA receptors to reduce synaptic strength in both hebbian and non-hebbian forms of plasticity. The Arc-endosome also traffics APP and BACE1, and Arc physically associates with presenilin1 (PS1) to regulate γ-secretase trafficking and confer activity dependence. Genetic deletion of Arc reduces Aß load in a transgenic mouse model of AD. In concert with the finding that patients with AD can express anomalously high levels of Arc, we hypothesize that Arc participates in the pathogenesis of AD.

Engineered adhesion molecules drive synapse organization.

In multicellular organisms, cell-adhesion molecules connect cells into tissues and mediate intercellular signaling between these cells. In vertebrate brains, synaptic cell-adhesion molecules (SAMs) guide the formation, specification, and plasticity of synapses. Some SAMs, when overexpressed in cultured neurons or in heterologous cells co-cultured with neurons, drive formation of synaptic specializations onto the overexpressing cells. However, genetic deletion of the same SAMs from neurons often has no effect on synapse numbers, but frequently severely impairs synaptic transmission, suggesting that most SAMs control the function and plasticity of synapses (i.e., organize synapses) instead of driving their initial establishment (i.e., make synapses). Since few SAMs were identified that mediate initial synapse formation, it is difficult to develop methods that enable experimental control of synaptic connections by targeted expression of these SAMs. To overcome this difficulty, we engineered novel SAMs from bacterial proteins with no eukaryotic homologues that drive synapse formation. We named these engineered adhesion proteins "Barnoligin" and "Starexin" because they were assembled from parts of Barnase and Neuroligin-1 or of Barstar and Neurexin3ß, respectively. Barnoligin and Starexin robustly induce the formation of synaptic specializations in a specific and directional manner in cultured neurons. Synapse formation by Barnoligin and Starexin requires both their extracellular Barnase- and Barstar-derived interaction domains and their Neuroligin- and Neurexin-derived intracellular signaling domains. Our findings support a model of synapse formation whereby trans-synaptic interactions by SAMs drive synapse organization via adhesive interactions that activate signaling cascades.

Mouse models of SYNGAP1-related intellectual disability.

SYNGAP1 is a Ras-GTPase-activating protein highly enriched at excitatory synapses in the brain. De novo loss-of-function mutations in SYNGAP1 are a major cause of genetically defined neurodevelopmental disorders (NDDs). These mutations are highly penetrant and cause SYNGAP1-related intellectual disability (SRID), an NDD characterized by cognitive impairment, social deficits, early-onset seizures, and sleep disturbances. Studies in rodent neurons have shown that Syngap1 regulates developing excitatory synapse structure and function, and heterozygous Syngap1 knockout mice have deficits in synaptic plasticity, learning, and memory and have seizures. However, how specific SYNGAP1 mutations found in humans lead to disease has not been investigated in vivo. To explore this, we utilized the CRISPR-Cas9 system to generate knock-in mouse models with two distinct known causal variants of SRID: one with a frameshift mutation leading to a premature stop codon, SYNGAP1; L813RfsX22, and a second with a single-nucleotide mutation in an intron that creates a cryptic splice acceptor site leading to premature stop codon, SYNGAP1; c.3583-9G>A. While reduction in Syngap1 mRNA varies from 30 to 50% depending on the specific mutation, both models show ~50% reduction in Syngap1 protein, have deficits in synaptic plasticity, and recapitulate key features of SRID including hyperactivity and impaired working memory. These data suggest that half the amount of SYNGAP1 protein is key to the pathogenesis of SRID. These results provide a resource to study SRID and establish a framework for the development of therapeutic strategies for this disorder.

Experience-Induced Remodeling of the Hippocampal Post-synaptic Proteome and Phosphoproteome.

The postsynaptic density (PSD) of excitatory synapses contains a highly organized protein network with thousands of proteins and is a key node in the regulation of synaptic plasticity. To gain new mechanistic insight into experience-induced changes in the PSD, we examined the global dynamics of the hippocampal PSD proteome and phosphoproteome in mice following four different types of experience. Mice were trained using an inhibitory avoidance (IA) task and hippocampal PSD fractions were isolated from individual mice to investigate molecular mechanisms underlying experience-dependent remodeling of synapses. We developed a new strategy to identify and quantify the relatively low level of site-specific phosphorylation of PSD proteome from the hippocampus, by using a modified iTRAQ-based TiSH protocol. In the PSD, we identified 3938 proteins and 2761 phosphoproteins in the sequential strategy covering a total of 4968 unique protein groups (at least two peptides including a unique peptide). On the phosphoproteins, we identified a total of 6188 unambiguous phosphosites (75%

BRaf signaling principles unveiled by large-scale human mutation analysis with a rapid lentivirus-based gene replacement method.

Rapid advances in genetics are linking mutations on genes to diseases at an exponential rate, yet characterizing the gene-mutation-cell-behavior relationships essential for precision medicine remains a daunting task. More than 350 mutations on small GTPase BRaf are associated with various tumors, and ∼40 mutations are associated with the neurodevelopmental disorder cardio-facio-cutaneous syndrome (CFC). We developed a fast cost-effective lentivirus-based rapid gene replacement method to interrogate the physiopathology of BRaf and ∼50 disease-linked BRaf mutants, including all CFC-linked mutants. Analysis of simultaneous multiple patch-clamp recordings from 6068 pairs of rat neurons with validation in additional mouse and human neurons and multiple learning tests from 1486 rats identified BRaf as the key missing signaling effector in the common synaptic NMDA-R-CaMKII-SynGap-Ras-BRaf-MEK-ERK transduction cascade. Moreover, the analysis creates the original big data unveiling three general features of BRaf signaling. This study establishes the first efficient procedure that permits large-scale functional analysis of human disease-linked mutations essential for precision medicine.

All-or-none disconnection of pyramidal inputs onto parvalbumin-positive interneurons gates ocular dominance plasticity.

Disinhibition is an obligatory initial step in the remodeling of cortical circuits by sensory experience. Our investigation on disinhibitory mechanisms in the classical model of ocular dominance plasticity uncovered an unexpected form of experience-dependent circuit plasticity. In the layer 2/3 of mouse visual cortex, monocular deprivation triggers a complete, "all-or-none," elimination of connections from pyramidal cells onto nearby parvalbumin-positive interneurons (Pyr→PV). This binary form of circuit plasticity is unique, as it is transient, local, and discrete. It lasts only 1 d, and it does not manifest as widespread changes in synaptic strength; rather, only about half of local connections are lost, and the remaining ones are not affected in strength. Mechanistically, the deprivation-induced loss of Pyr→PV is contingent on a reduction of the protein neuropentraxin2. Functionally, the loss of Pyr→PV is absolutely necessary for ocular dominance plasticity, a canonical model of deprivation-induced model of cortical remodeling. We surmise, therefore, that this all-or-none loss of local Pyr→PV circuitry gates experience-dependent cortical plasticity.

Glutamate synapses in human cognitive disorders.

Accumulating data, including those from large genetic association studies, indicate that alterations in glutamatergic synapse structure and function represent a common underlying pathology in many symptomatically distinct cognitive disorders. In this review, we discuss evidence from human genetic studies and data from animal models supporting a role for aberrant glutamatergic synapse function in the etiology of intellectual disability (ID), autism spectrum disorder (ASD), and schizophrenia (SCZ), neurodevelopmental disorders that comprise a significant proportion of human cognitive disease and exact a substantial financial and social burden. The varied manifestations of impaired perceptual processing, executive function, social interaction, communication, and/or intellectual ability in ID, ASD, and SCZ appear to emerge from altered neural microstructure, function, and/or wiring rather than gross changes in neuron number or morphology. Here, we review evidence that these disorders may share a common underlying neuropathy: altered excitatory synapse function. We focus on the most promising candidate genes affecting glutamatergic synapse function, highlighting the likely disease-relevant functional consequences of each. We first present a brief overview of glutamatergic synapses and then explore the genetic and phenotypic evidence for altered glutamate signaling in ID, ASD, and SCZ.

An ultrasensitive biosensor for high-resolution kinase activity imaging in awake mice.

Protein kinases control nearly every facet of cellular function. These key signaling nodes integrate diverse pathway inputs to regulate complex physiological processes, and aberrant kinase signaling is linked to numerous pathologies. While fluorescent protein-based biosensors have revolutionized the study of kinase signaling by allowing direct, spatiotemporally precise kinase activity measurements in living cells, powerful new molecular tools capable of robustly tracking kinase activity dynamics across diverse experimental contexts are needed to fully dissect the role of kinase signaling in physiology and disease. Here, we report the development of an ultrasensitive, second-generation excitation-ratiometric protein kinase A (PKA) activity reporter (ExRai-AKAR2), obtained via high-throughput linker library screening, that enables sensitive and rapid monitoring of live-cell PKA activity across multiple fluorescence detection modalities, including plate reading, cell sorting and one- or two-photon imaging. Notably, in vivo visual cortex imaging in awake mice reveals highly dynamic neuronal PKA activity rapidly recruited by forced locomotion.

GRIP1 regulates synaptic plasticity and learning and memory.

Hebbian plasticity is a key mechanism for higher brain functions, such as learning and memory. This form of synaptic plasticity primarily involves the regulation of synaptic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) abundance and properties, whereby AMPARs are inserted into synapses during long-term potentiation (LTP) or removed during long-term depression (LTD). The molecular mechanisms underlying AMPAR trafficking remain elusive, however. Here we show that glutamate receptor interacting protein 1 (GRIP1), an AMPAR-binding protein shown to regulate the trafficking and synaptic targeting of AMPARs, is required for LTP and learning and memory. GRIP1 is recruited into synapses during LTP, and deletion of Grip1 in neurons blocks synaptic AMPAR accumulation induced by glycine-mediated depolarization. In addition, Grip1 knockout mice exhibit impaired hippocampal LTP, as well as deficits in learning and memory. Mechanistically, we find that phosphorylation of serine-880 of the GluA2 AMPAR subunit (GluA2-S880) is decreased while phosphorylation of tyrosine-876 on GluA2 (GluA2-Y876) is elevated during chemically induced LTP. This enhances the strength of the GRIP1-AMPAR association and, subsequently, the insertion of AMPARs into the postsynaptic membrane. Together, these results demonstrate an essential role of GRIP1 in regulating AMPAR trafficking during synaptic plasticity and learning and memory.

Tyrosine phosphorylation of the AMPA receptor subunit GluA2 gates homeostatic synaptic plasticity.

Hebbian plasticity, comprised of long-term potentiation (LTP) and depression (LTD), allows neurons to encode and respond to specific stimuli; while homeostatic synaptic scaling is a counterbalancing mechanism that enables the maintenance of stable neural circuits. Both types of synaptic plasticity involve the control of postsynaptic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor (AMPAR) abundance, which is modulated by AMPAR phosphorylation. To address the necessity of GluA2 phospho-Y876 in synaptic plasticity, we generated phospho-deficient GluA2 Y876F knock-in mice. We show that, while GluA2 phospho-Y876 is not necessary for Hebbian plasticity, it is essential for both in vivo and in vitro homeostatic upscaling. Bidirectional changes in GluA2 phospho-Y876 were observed during homeostatic scaling, with a decrease during downscaling and an increase during upscaling. GluA2 phospho-Y876 is necessary for synaptic accumulation of glutamate receptor interacting protein 1 (GRIP1), a crucial scaffold protein that delivers AMPARs to synapses, during upscaling. Furthermore, increased phosphorylation at GluA2 Y876 increases GluA2 binding to GRIP1. These results demonstrate that AMPAR trafficking during homeostatic upscaling can be gated by a single phosphorylation site on the GluA2 subunit.

The Immediate Early Gene Arc Is Not Required for Hippocampal Long-Term Potentiation.

Memory consolidation is thought to occur through protein synthesis-dependent synaptic plasticity mechanisms such as long-term potentiation (LTP). Dynamic changes in gene expression and epigenetic modifications underlie the maintenance of LTP. Similar mechanisms may mediate the storage of memory. Key plasticity genes, such as the immediate early gene Arc, are induced by learning and by LTP induction. Mice that lack Arc have severe deficits in memory consolidation, and Arc has been implicated in numerous other forms of synaptic plasticity, including long-term depression and cell-to-cell signaling. Here, we take a comprehensive approach to determine if Arc is necessary for hippocampal LTP in male and female mice. Using a variety of Arc knock-out (KO) lines, we found that germline Arc KO mice show no deficits in CA1 LTP induced by high-frequency stimulation and enhanced LTP induced by theta-burst stimulation. Temporally restricting the removal of Arc to adult animals and spatially restricting it to the CA1 using Arc conditional KO mice did not have an effect on any form of LTP. Similarly, acute application of Arc antisense oligodeoxynucleotides had no effect on hippocampal CA1 LTP. Finally, the maintenance of in vivo LTP in the dentate gyrus of Arc KO mice was normal. We conclude that Arc is not necessary for hippocampal LTP and may mediate memory consolidation through alternative mechanisms.SIGNIFICANCE STATEMENT The immediate early gene Arc is critical for maintenance of long-term memory. How Arc mediates this process remains unclear, but it has been proposed to sustain Hebbian synaptic potentiation, which is a key component of memory encoding. This form of plasticity is modeled experimentally by induction of LTP, which increases Arc mRNA and protein expression. However, mechanistic data implicates Arc in the endocytosis of AMPA-type glutamate receptors and the weakening of synapses. Here, we took a comprehensive approach to determine if Arc is necessary for hippocampal LTP. We find that Arc is not required for LTP maintenance and may regulate memory storage through alternative mechanisms.

Differential expression patterns of phospholipase D isoforms 1 and 2 in the mammalian brain and retina.

Phosphatidic acid is a key signaling molecule heavily implicated in exocytosis due to its protein-binding partners and propensity to induce negative membrane curvature. One phosphatidic acid-producing enzyme, phospholipase D (PLD), has also been implicated in neurotransmission. Unfortunately, due to the unreliability of reagents, there has been confusion in the literature regarding the expression of PLD isoforms in the mammalian brain which has hampered our understanding of their functional roles in neurons. To address this, we generated epitope-tagged PLD1 and PLD2 knockin mice using CRISPR/Cas9. Using these mice, we show that PLD1 and PLD2 are both localized at synapses by adulthood, with PLD2 expression being considerably higher in glial cells and PLD1 expression predominating in neurons. Interestingly, we observed that only PLD1 is expressed in the mouse retina, where it is found in the synaptic plexiform layers. These data provide critical information regarding the localization and potential role of PLDs in the central nervous system.

Twenty Years of SynGAP Research: From Synapses to Cognition.

SynGAP is a potent regulator of biochemical signaling in neurons and plays critical roles in neuronal function. It was first identified in 1998, and has since been extensively characterized as a mediator of synaptic plasticity. Because of its involvement in synaptic plasticity, SynGAP has emerged as a critical protein for normal cognitive function. In recent years, mutations in the SYNGAP1 gene have been shown to cause intellectual disability in humans and have been linked to other neurodevelopmental disorders, such as autism spectrum disorders and schizophrenia. While the structure and biochemical function of SynGAP have been well characterized, a unified understanding of the various roles of SynGAP at the synapse and its contributions to neuronal function remains to be achieved. In this review, we summarize and discuss the current understanding of the multifactorial role of SynGAP in regulating neuronal function gathered over the last two decades.

The C9orf72 repeat expansion disrupts nucleocytoplasmic transport.

The hexanucleotide repeat expansion (HRE) GGGGCC (G4C2) in C9orf72 is the most common cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Recent studies support an HRE RNA gain-of-function mechanism of neurotoxicity, and we previously identified protein interactors for the G4C2 RNA including RanGAP1. A candidate-based genetic screen in Drosophila expressing 30 G4C2 repeats identified RanGAP (Drosophila orthologue of human RanGAP1), a key regulator of nucleocytoplasmic transport, as a potent suppressor of neurodegeneration. Enhancing nuclear import or suppressing nuclear export of proteins also suppresses neurodegeneration. RanGAP physically interacts with HRE RNA and is mislocalized in HRE-expressing flies, neurons from C9orf72 ALS patient-derived induced pluripotent stem cells (iPSC-derived neurons), and in C9orf72 ALS patient brain tissue. Nuclear import is impaired as a result of HRE expression in the fly model and in C9orf72 iPSC-derived neurons, and these deficits are rescued by small molecules and antisense oligonucleotides targeting the HRE G-quadruplexes. Nucleocytoplasmic transport defects may be a fundamental pathway for ALS and FTD that is amenable to pharmacotherapeutic intervention.

Identification of long-lived synaptic proteins by proteomic analysis of synaptosome protein turnover.

Memory formation is believed to result from changes in synapse strength and structure. While memories may persist for the lifetime of an organism, the proteins and lipids that make up synapses undergo constant turnover with lifetimes from minutes to days. The molecular basis for memory maintenance may rely on a subset of long-lived proteins (LLPs). While it is known that LLPs exist, whether such proteins are present at synapses is unknown. We performed an unbiased screen using metabolic pulse-chase labeling in vivo in mice and in vitro in cultured neurons combined with quantitative proteomics. We identified synaptic LLPs with half-lives of several months or longer. Proteins in synaptic fractions generally exhibited longer lifetimes than proteins in cytosolic fractions. Protein turnover was sensitive to pharmacological manipulations of activity in neuronal cultures or in mice exposed to an enriched environment. We show that synapses contain LLPs that may underlie stabile long-lasting changes in synaptic structure and function.

SynGAP splice variants display heterogeneous spatio-temporal expression and subcellular distribution in the developing mammalian brain.

The SynGAP protein is a major regulator of synapse biology and neural circuit function. Genetic variants linked to epilepsy and intellectual disability disrupt synaptic function and neural excitability. SynGAP has been involved in multiple signaling pathways and can regulate small GTPases with very different roles. Yet, the molecular bases behind this pleiotropy are poorly understood. We hypothesize that different SynGAP isoforms will mediate different sets of functions and that deciphering their spatio-temporal expression and subcellular localization will accelerate understanding their multiple functions. Using isoform-specific antibodies recognizing SynGAP in mouse and human samples we found distinctive developmental expression patterns for all SynGAP isoforms in five mouse brain areas. Particularly noticeable was the delayed expression of SynGAP-α1 isoforms, which directly bind to postsynaptic density-95, in cortex and hippocampus during the first 2 weeks of postnatal development. Suggesting that during this period other isoforms would have a more prominent role. Furthermore, we observed subcellular localization differences between isoforms, particularly throughout postnatal development. Consistent with previous reports, SynGAP was enriched in the postsynaptic density in the mature forebrain. However, SynGAP was predominantly found in non-synaptic locations in a period of early postnatal development highly sensitive to SynGAP levels. While, α1 isoforms were always found enriched in the postsynaptic density, α2 isoforms changed from a non-synaptic to a mostly postsynaptic density localization with age and ß isoforms were always found enriched in non-synaptic locations. The differential expression and subcellular distribution of SynGAP isoforms may contribute to isoform-specific regulation of small GTPases, explaining SynGAP pleiotropy.

Subunit-Specific Augmentation of AMPA Receptor Ubiquitination by Phorbol Ester.

Excitatory neurotransmission relies on the precise targeting of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptors to the neuronal plasma membrane. Activity-dependent ubiquitination of AMPA receptor (AMPAR) subunits sorts internalised receptors to late endosomes for degradation, which ultimately determines the number of AMPARs on neuronal membrane. Our recent study has demonstrated a functional cross-talk between the phosphorylation and ubiquitination of the GluA1 subunit in mammalian central neurons. However, the existence of such a cross modulation for the GluA2 subunit remains unknown. Here, we have shown that bicuculline induced GluA2 ubiquitination on the same lysine residues (Lys-870 and Lys-882) in the C-terminal as those elicited by the AMPA treatment. Interestingly, bicuculline-induced ubiquitination was markedly enhanced by the phospho-mimetic GluA2 S880E mutant. Pharmacological activation of protein kinase C (PKC) by phorbol ester, which mediates the phosphorylation of GluA2 at Ser-880, augmented bicuculline-induced ubiquitination of GluA2 in cultured neurons. This effect was specific for the GluA2 subunit because phorbol ester did not alter the level of GluA1 ubiquitination. However, phorbol ester-induced enhancement of GluA2 ubiquitination did not require Ser-880 phosphorylation. This suggests that pseudo-phosphorylation of Ser-880 is sufficient but is not necessary for the augmentation of bicuculline-induced GluA2 ubiquitination. Collectively, these data provide the first demonstration of subunit-specific modulation of AMPAR ubiquitination by the PKC-dependent signalling pathway in mammalian central neurons.

O-GlcNAc transferase regulates excitatory synapse maturity.

Experience-driven synaptic plasticity is believed to underlie adaptive behavior by rearranging the way neuronal circuits process information. We have previously discovered that O-GlcNAc transferase (OGT), an enzyme that modifies protein function by attaching ß-N-acetylglucosamine (GlcNAc) to serine and threonine residues of intracellular proteins (O-GlcNAc), regulates food intake by modulating excitatory synaptic function in neurons in the hypothalamus. However, how OGT regulates excitatory synapse function is largely unknown. Here we demonstrate that OGT is enriched in the postsynaptic density of excitatory synapses. In the postsynaptic density, O-GlcNAcylation on multiple proteins increased upon neuronal stimulation. Knockout of the OGT gene decreased the synaptic expression of the AMPA receptor GluA2 and GluA3 subunits, but not the GluA1 subunit. The number of opposed excitatory presynaptic terminals was sharply reduced upon postsynaptic knockout of OGT. There were also fewer and less mature dendritic spines on OGT knockout neurons. These data identify OGT as a molecular mechanism that regulates synapse maturity.