Biology subjects

Huganir, R. L.

Publications and source records attributed to Huganir, R. L..

36 records · Page 2Linked to original sources

bioRxiv2023

Context-dependent hyperactivity in syngap1a and syngap1b zebrafish autism models.

Background and AimsSYNGAP1 disorder is a prevalent genetic form of Autism Spectrum Disorder and Intellectual Disability (ASD/ID) and is caused by de novo or inherited mutations in one copy of the SYNGAP1 gene. In addition to ASD/ID, SYNGAP1 disorder is associated with comorbid symptoms including treatment-resistant-epilepsy, sleep disturbances, and gastrointestinal distress. Mechanistic links between these diverse symptoms and SYNGAP1 variants remain obscure, therefore, our goal was to generate a zebrafish model in which this range of symptoms can be studied. MethodsWe used CRISPR/Cas9 to introduce frameshift mutations in the syngap1a and syngap1b zebrafish duplicates (syngap1ab) and validated these stable models for Syngap1 loss-of-function. Because SYNGAP1 is extensively spliced, we mapped splice variants to the two zebrafish syngap1a and b genes and identified mammalian-like isoforms. We then quantified locomotory behaviors in zebrafish syngap1ab larvae under three conditions that normally evoke different arousal states in wild type larvae: aversive, high-arousal acoustic, medium-arousal dark, and low-arousal light stimuli. ResultsWe show that CRISPR/Cas9 indels in zebrafish syngap1a and syngap1b produced loss-of-function alleles at RNA and protein levels. Our analyses of zebrafish Syngap1 isoforms showed that, as in mammals, zebrafish Syngap1 N- and C-termini are extensively spliced. We identified a zebrafish syngap1 1-like variant that maps exclusively to the syngap1b gene. Quantifying locomotor behaviors showed that syngap1ab larvae are hyperactive compared to wild type but to differing degrees depending on the stimulus. Hyperactivity was most pronounced in low arousal settings, with overall movement increasing with the number of mutant syngap1 alleles. ConclusionsOur data support mutations in zebrafish syngap1ab as causal for hyperactivity associated with elevated arousal that is especially pronounced in low-arousal environments.

neuroscience↗

bioRxiv2023

SynGAP regulates synaptic plasticity and cognition independent of its catalytic activity

SynGAP is an abundant synaptic GTPase-activating protein (GAP) critical for synaptic plasticity, learning, memory, and cognition. Mutations in SYNGAP1 in humans result in intellectual disability, autistic-like behaviors, and epilepsy. Heterozygous Syngap1 knockout mice display deficits in synaptic plasticity, learning, and memory, and exhibit seizures. It is unclear whether SynGAP imparts structural properties at synapses independent of its GAP activity. Here, we report that inactivating mutations within the SynGAP GAP domain do not inhibit synaptic plasticity or cause behavioral deficits. Instead, SynGAP modulates synaptic strength by physically competing with the AMPA- receptor-TARP complex, the major excitatory receptor complex in the brain, in the formation of molecular condensates with synaptic scaffolding proteins. These results have significant implications for the development of therapeutic treatments for SYNGAP1- related neurodevelopmental disorders. One-Sentence SummarySynGAP regulates synaptic plasticity and cognition due to its phase separation properties instead of its catalytic activity.

neuroscience↗

bioRxiv2023

Calcium-permeable AMPA receptors govern PV neuron feature selectivity

The brain helps us survive by forming internal representations of the external world1,2. Excitatory cortical neurons are often precisely tuned to specific external stimuli3,4. However, inhibitory neurons, such as parvalbumin-positive (PV) interneurons, are generally less selective5. PV interneurons differ from excitatory cells in their neurotransmitter receptor subtypes, including AMPA receptors6,7. While excitatory neurons express calcium-impermeable AMPA receptors containing the GluA2 subunit, PV interneurons express receptors that lack the GluA2 subunit and are calcium-permeable (CP-AMPARs). Here we demonstrate a causal relationship between CP-AMPAR expression and the low feature selectivity of PV interneurons. We find a low expression stoichiometry of GluA2 mRNA relative to other subunits in PV interneurons which is conserved across ferrets, rodents, marmosets, and humans, causing abundant CP-AMPAR expression. Replacing CP-AMPARs in PV interneurons with calcium-impermeable AMPARs increased their orientation selectivity in the visual cortex. Sparse CP-AMPAR manipulations demonstrated that this increase was cell-autonomous and could occur well beyond development. Interestingly, excitatory-PV interneuron connectivity rates and unitary synaptic strength were unaltered by CP-AMPAR removal, suggesting that the selectivity of PV interneurons can be altered without drastically changing connectivity. In GluA2 knockout mice, where all AMPARs are calcium-permeable, excitatory neurons showed significantly reduced orientation selectivity, suggesting that CP-AMPARs are sufficient to drive lower selectivity regardless of cell type. Remarkably, hippocampal PV interneurons, which usually exhibit low spatial tuning, became more spatially selective after removing CP-AMPARs, indicating that CP-AMPARs suppress the feature selectivity of PV interneurons independent of modality. These results reveal a novel role of CP-AMPARs in maintaining a low-selectivity sensory representation in PV interneurons and suggest a conserved molecular mechanism that distinguishes the unique synaptic computations of inhibitory and excitatory neurons.

neuroscience↗

bioRxiv2023

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 (NDD). These mutations are highly penetrant and cause SYNGAP1-related intellectual disability (SRID), a NDD characterized by cognitive impairment, social deficits, early-onset seizures, and sleep disturbances (1-5). Studies in rodent neurons have shown that Syngap1 regulates developing excitatory synapse structure and function (6-11), and heterozygous Syngap1 knockout mice have deficits in synaptic plasticity, learning and memory, and have seizures (9, 12-14). 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 30-50% 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. Significance StatementSYNGAP1 is a protein enriched at excitatory synapses in the brain that is an important regulator of synapse structure and function. SYNGAP1 mutations cause SYNGAP1-related intellectual disability (SRID), a neurodevelopmental disorder with cognitive impairment, social deficits, seizures, and sleep disturbances. To explore how SYNGAP1 mutations found in humans lead to disease, we generated the first knock-in mouse models with causal SRID variants: one with a frameshift mutation and a second with an intronic mutation that creates a cryptic splice acceptor site. Both models show decreased Syngap1 mRNA and Syngap1 protein and recapitulate key features of SRID including hyperactivity and impaired working memory. These results provide a resource to study SRID and establish a framework for the development of therapeutic strategies. HighlightsO_LITwo mouse models with SYNGAP1-related intellectual disability (SRID) mutations found in humans were generated: one with a frameshift mutation that results in a premature stop codon and the other with an intronic mutation resulting in a cryptic splice acceptor site and premature stop codon. C_LIO_LIBoth SRID mouse models show 35[~]50% reduction in mRNA and [~]50% reduction in Syngap1 protein. C_LIO_LIBoth SRID mouse models display deficits in synaptic plasticity and behavioral phenotypes found in people. C_LIO_LIRNA-seq confirmed cryptic splice acceptor activity in one SRID mouse model and revealed broad transcriptional changes also identified in Syngap1+/- mice. C_LIO_LINovel SRID mouse models generated here provide a resource and establish a framework for development of future therapeutic intervention. C_LI

neuroscience↗

bioRxiv2022

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 Neurexins, 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 Bastar-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.

neuroscience↗

bioRxiv2022

Cell-specific regulation of gene expression using splicing-dependent frameshifting.

Precise and reliable cell-specific gene delivery remains technically challenging. Here we report a splicing-based approach for controlling gene expression whereby separate translational reading frames are coupled to the inclusion or exclusion of cell-specific alternative exons. Candidate exons are identified by analyzing thousands of publicly available RNA sequencing datasets and filtering by cell specificity, sequence conservation, and local intron length. This method, which we denote splicing-linked expression design (SLED), can be combined in a Boolean manner with existing techniques such as minipromoters and viral capsids. SLED vectors can leverage the strong expression of constitutive promoters, without sacrificing precision, by decoupling the tradeoff between promoter strength and selectivity. We generated SLED vectors to selectively target all neurons, photoreceptors, or excitatory neurons, and demonstrated that specificity was retained in vivo when delivered using AAVs. We further demonstrated the utility of SLED by creating what would otherwise be unobtainable research tools, specifically a GluA2 flip/flop reporter and a dual excitatory/inhibitory neuronal calcium indicator. Finally, we show the translational potential of SLED by rescuing photoreceptor degeneration in Prph2rds/rds mice and by developing an oncolytic vector that can selectively induce apoptosis in SF3B1 mutant cancer cells. The flexibility of SLED technology enables new avenues for basic and translational research.

molecular biology↗

bioRxiv2022

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

Synaptic plasticity encodes learning as changes in the strength of synapses, sub-micron structures that mediate communication between brain cells. Due to their small size and high density, synapses are extremely difficult to image in vivo, limiting our ability to directly relate synaptic plasticity with 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. Applied to in vivo images from transgenic mice expressing fluorescently labeled synaptic proteins, this restoration algorithm super-resolved diffraction-limited synapses, enabling identification and logitudinal tracking of synaptic plasticity underlying behavior with unprecedented spatial resolution. More generally, our method demonstrates the capabilities of image enhancement to learn from ex vivo data and imaging techniques to improve in vivo imaging resolution.

neuroscience↗

bioRxiv2021

Btbd11 is an inhibitory interneuron specific synaptic scaffolding protein that supports excitatory synapse structure and function

Synapses in the brain exhibit cell-type-specific differences in basal synaptic transmission and plasticity. Here, we evaluated cell-type-specific differences in the composition of glutamatergic synapses, identifying Btbd11, as an inhibitory interneuron-specific synapse-enriched protein. Btbd11 is highly conserved across species and binds to core postsynaptic proteins including Psd-95. Intriguingly, we show that Btbd11 can undergo liquid-liquid phase separation when expressed with Psd-95, supporting the idea that the glutamatergic post synaptic density in synapses in inhibitory and excitatory neurons exist in a phase separated state. Knockout of Btbd11 from inhibitory interneurons decreased glutamatergic signaling onto parvalbumin-positive interneurons. Further, both in vitro and in vivo, we find that Btbd11 knockout disrupts network activity. At the behavioral level, Btbd11 knockout from interneurons sensitizes mice to pharmacologically induced hyperactivity following NMDA receptor antagonist challenge. Our findings identify a cell-type-specific protein that supports glutamatergic synapse function in inhibitory interneurons--with implication for circuit function and animal behavior.

neuroscience↗

bioRxiv2021

Experience-induced remodeling of the hippocampal post-synaptic proteome and phosphoproteome

The post synaptic density (PSD) of excitatory synapses contains a highly organized protein network with thousands of proteins and is 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 PSD proteome and phosphoproteome in mice following various treatments. Mice were trained using an inhibitory avoidance (IA) task and hippocampal PSD fractions were isolated for quantitative proteomic and phosphoproteomics analysis. We used a sequential enrichment strategy to explore the concurrent events of protein expression and phosphorylation in the hippocampal PSD following IA training (IA) or immediate shock (Shock). We identified more than 6,200 proteins and 3,000 phosphoproteins in the sequential strategy covering a total of 7,429 proteins. On the phosphoproteins we identified a total of 9,589 phosphosites. Strikingly, of the significantly IA-regulated proteins and phosphoproteins, a large fraction of the proteins displayed an overall decrease in phosphorylation level. Bioinformatic analysis of proteins and phosphoproteins that were regulated by IA were annotated for an involvement in regulation of glutamate receptor functionality, calcium signaling, and synaptic plasticity. We also identified synaptic kinases, phosphatases and their respective phosphosites regulated by IA training or immediate shock. Furthermore, we found that AMPA receptor surface expression was regulated by protein phosphatase, Mg2+/Mn2+ dependent 1H (Ppm1h). Together, these results unravel the dynamic remodeling of the PSD upon IA learning or immediate shock and serve as a resource for elucidating the synaptic proteome dynamics induced by experience-dependent plasticity. HighlightsO_LIThe proteome and phosphoproteome of mouse hippocampal PSD fractions were examined using quantitative phosphoproteomics and bioinformatics following inhibitory avoidance training or non-associative immediate shock. C_LIO_LIApproximately 6,200 proteins and 3,000 phosphoproteins were identified and quantified in the hippocampal PSD fractions. C_LIO_LIIA mediates widespread decreases in the abundance and phosphorylation of proteins in the hippocampal PSD fraction. C_LIO_LIKinases, phosphatases and their phosphorylation status were dynamically and significantly regulated by IA and immediate shock. C_LIO_LIFunctional validation shows that the protein phosphatase Ppm1h is linked to the regulation of synaptic plasticity in vitro and in vivo. C_LI In BriefQuantitative proteomics and phosphoproteomics combined with subcellular protein fractionation and bioinformatic analysis identifies a highly dynamic regulation of synaptic protein phosphorylation at the postsynaptic density following IA training and immediate shock.

neuroscience↗

bioRxiv2021

KIBRA-PKCγ signaling pathway modulates memory performance in mice and humans

Human memory is a polygenic cognitive trait that is fundamental to individual competence. Genome-wide association studies (GWAS) have identified KIBRA as a novel gene associated with human memory performance. KIBRA interacts with AMPA receptors (AMPARs) and proteins essential for synaptic plasticity. The deletion of Kibra in mice impairs synaptic plasticity and learning and memory. However, the molecular basis through which KIBRA regulates dynamic AMPAR trafficking underlying synaptic plasticity is still unknown. Here we report that KIBRA interacts with the neuronal specific kinase PKC{gamma} to modulate AMPAR trafficking upon learning, and KIBRA-PKC{gamma} signaling pathway also associates with human memory performance. We find PKC{gamma} is an essential kinase that phosphorylates AMPARs upon learning, and the loss of KIBRA in mouse brain impedes PKC{gamma}-AMPAR interaction. Activation of PKC{gamma} enables KIBRA to recruit phosphorylated AMPARs to the synapse to promote LTP and learning. We further performed transcriptomic and genetic analyses in human postmortem brain samples, and behavioral and fMRI evaluations in living human subjects, to demonstrate the genetic interactions between KIBRA and PRKCG on memory performance and memory associated physiological engagement of the hippocampal memory system. Overall, our results support that the KIBRA-PKC{gamma} signaling pathway is crucial for modulating memory performance in mice and humans.

neuroscience↗

bioRxiv2021

Tagging active neurons by soma-targeted Cal-Light

Verifying causal effects of neural circuits is essential for proving direct a circuit-behavior relationship. However, techniques for tagging only active neurons with high spatiotemporal precision remain at the beginning stages. Here we developed the soma-targeted Cal-Light (ST-Cal-Light) which selectively converts somatic calcium rise triggered by action potentials into gene expression. Such modification simultaneously increases the signal-to-noise ratio (SNR) of reporter gene expression and reduces the light requirement for successful labeling. Because of the enhanced efficacy, the ST-Cal-Light enables the tagging of functionally engaged neurons in various forms of behaviors, including context-dependent fear conditioning, leverpressing choice behavior, and social interaction behaviors. We also targeted kainic acid-sensitive neuronal populations in the hippocampus which subsequently suppressed seizure symptoms, suggesting ST-Cal-Lights applicability in controlling disease-related neurons. Furthermore, the generation of a conditional ST-Cal-Light knock-in (KI) mouse provides an opportunity to tag active neurons in a region- or cell-type specific manner via crossing with other Cre-driver lines. Thus, the versatile ST-Cal-Light system links somatic action potentials to behaviors with high temporal precision, and ultimately allows functional circuit dissection at a single cell resolution.

neuroscience↗

bioRxiv2020

An Optimized CRISPR/Cas9 Approach for Precise Genome Editing in Neurons

The efficient knock-in of large DNA fragments to label endogenous proteins remains especially challenging in non-dividing cells such as neurons. We developed Targeted Knock-In with Two-guides (TKIT) as a novel CRISPR/Cas9 based approach for efficient, and precise, genomic knock-in. Through targeting non-coding regions TKIT is resistant to INDEL mutations. We demonstrate TKIT labelling of endogenous synaptic proteins with various tags, with efficiencies up to 42% in mouse primary cultured neurons. Utilizing in utero electroporation or viral injections in mice TKIT can label AMPAR subunits with Super Ecliptic pHluorin, enabling visualization of endogenous AMPARs in vivo using two-photon microscopy. We further use TKIT to assess the mobility of endogenous AMPARs using fluorescence recovery after photobleaching. Finally, we show that TKIT can be used to tag AMPARs in rat neurons, demonstrating precise genome editing in another model organism and highlighting the broad potential of TKIT as a method to visualize endogenous proteins.

neuroscience↗

bioRxiv2020

Visualizing synaptic plasticity in vivo by large-scale imaging of endogenous AMPA receptors

Elucidating how synaptic molecules such as AMPA receptors mediate neuronal communication and tracking their dynamic expression during behavior is crucial to understand cognition and disease, but current technological barriers preclude large-scale exploration of molecular dynamics in vivo. We have developed a suite of innovative methodologies that break through these barriers: a new knockin mouse line with fluorescently tagged endogenous AMPA receptors, two-photon imaging of hundreds of thousands of labeled synapses in behaving mice, and computer-vision-based automatic synapse detection. Using these tools, we can longitudinally track how the strength of synapses changes during behavior. We used this approach to generate an unprecedentedly detailed spatiotemporal map of synaptic plasticity underlying sensory experience. More generally, these tools can be used as an optical probe capable of measuring functional synapse strength across entire brain areas during any behavioral paradigm, describing complex system-wide changes with molecular precision.

neuroscience↗

bioRxiv2020

SynGAP splice isoforms differentially regulate synaptic plasticity and dendritic development

SynGAP is a synaptic Ras GTPase-activating protein (GAP) with four C-terminal splice variants: 1, 2, {beta}, and {gamma}. Although recent studies have implicated SYNGAP1 haploinsufficiency in ID/ASD pathogenesis, the degree to which each SynGAP isoform contributes to disease pathogenesis remains elusive. Here we demonstrate that individual SynGAP isoforms exhibit unique spatiotemporal expression and have distinct roles in neuronal and synaptic development. The SynGAP-1 isoform, which undergoes robust liquid-liquid phase-separation with PSD-95 and is highly-enriched in synapses, is expressed late in development and disperses from synaptic spines in response to LTP-inducing synaptic activity to allow for AMPA receptor insertion and spine enlargement. In contrast, the SynGAP-{beta} isoform, which undergoes less liquid-liquid phase-separation with PSD95 and is less synaptically targeted, is expressed early in development and promotes dendritic arborization. Interestingly, a SynGAP-1 mutation that disrupts phase separation and synaptic targeting abolishes its function in plasticity and instead drives dendritic arbor development like the {beta} isoform. These results demonstrate that distinct phase separation and synaptic targeting properties of SynGAP isoforms determine their function. HighlightsO_LISynGAP-1, 2, {beta}, {gamma} isoforms have distinct spatiotemporal expression and function in the brain. C_LIO_LISynGAP-1 is required for plasticity, while {beta} is required for dendritic development. C_LIO_LILiquid-liquid phase separation of SynGAP-1 is required for its role in plasticity. C_LIO_LISynGAP isoforms may differentially contribute to SYNGAP1 related human NDDs. C_LI

neuroscience↗

bioRxiv2020

The immediate early gene Arc is not required for hippocampal long-term potentiation.

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 modelled experimentally by induction of long-term potentiation (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 must regulate memory storage through alternative mechanisms.

neuroscience↗

bioRxiv2019

Perampanel rescues cortical gamma dysregulation associated with parvalbumin interneuron GluA2 upregulation in epileptic Syngap1+/- mice

Loss-of-function SYNGAP1 mutations cause a neurodevelopmental disorder characterized by intellectual disability and epilepsy. SYNGAP1 is a Ras-GTPase-activating protein that underlies the formation and experience-dependent regulation of postsynaptic densities. The mechanisms that contribute to this proposed monogenic cause of intellectual disability and epilepsy remain unresolved. Here, we establish the phenotype of the epileptogenesis in a Syngap1+/- mouse model using 24h video electroencephalogram/electromyogram (vEEG/EMG) recordings at advancing ages. A progressive worsening of clinically-similar seizure phenotypes, interictal spike frequency, sleep dysfunction, and hyperactivity was identified in Syngap1+/- mice. Interictal spikes emerged predominantly during NREM in 24h vEEG of Syngap1+/- mice. Myoclonic seizures occurred at behavioral-state transitions both in Syngap1+/- mice and during an overnight EEG from a child with SYNGAP1 haploinsufficiency. In Syngap1+/- mice, EEG spectral power analyses identified a significant loss of cortical gamma homeostasis during behavioral-state transitions from NREM to Wake and NREM to REM. The loss of gamma homeostasis was associated with a region- and location-specific significant increase of GluA2 AMPA receptor subunit expression in the somas of parvalbumin-positive (PV+) interneurons. Acute dosing with Perampanel, an FDA approved AMPA antagonist significantly rescued cortical gamma homeostasis, identifying a novel mechanism implicating Ca2+ impermeable AMPARs on PV+ interneurons underlying circuit dysfunction in SYNGAP1 haploinsufficiency.

neuroscience↗

bioRxiv2019

Syngap Splice Variants Display Heterogeneous Spatio-Temporal Expression And Subcellular Distribution In The Developing Mammalian Brain

The Syngap1 gene 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. The SynGAP protein has been involved in multiple signaling pathways and can regulate small GTPases with very different functions. 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 our understanding of the multiple functions performed by SynGAP. Using antibodies that detect all isoforms of SynGAP, we found that its subcellular localization changed throughout postnatal development. Consistent with previous reports, SynGAP was enriched in the postsynaptic density in the mature forebrain. However, this was age-dependent and SynGAP was predominantly found in non-synaptic locations in a period of postnatal development highly sensitive to SynGAP levels. Furthermore, we identified different expression patterns in the spatial and temporal axes for different SynGAP isoforms. Particularly noticeable was the delayed expression of SynGAP 1 isoforms, which bind to PSD-95 at the postsynaptic density, in cortex and hippocampus during the first two weeks of postnatal development. The subcellular localization of SynGAP was also isoform-dependent. While, 1 isoforms were highly enriched in the postsynaptic density, other C-terminal isoforms were less enriched or even more abundant in non-synaptic locations, particularly during the postnatal period. Thus, the regulation of expression and subcellular distribution of SynGAP isoforms may contribute to isoform-specific regulation of small GTPases, explaining SynGAP pleiotropy.

neuroscience↗

bioRxiv2019

Neuronal histone methyltransferase EZH2 regulates neuronal morphogenesis, synaptic plasticity, and cognitive behavior of mice

Recent studies showed that in the nervous system histone methyltransferase EZH2-mediated trimethylation of histone H3 lysine 27 (H3K27me3) acts to regulate neural stem cell proliferation and fate specificity through silencing different gene sets. Here we explored the function of EZH2 in early post-mitotic excitatory neurons by generating a neuronal specific Ezh2 conditional knockout mouse line. The results showed that lack of neuronal EZH2 led to delayed neuronal migration, more complex dendritic arborization, and significantly increased dendritic spine density. RNA-sequencing (RNA-seq) experiments comparing control and Ezh2 knockout neurons revealed that neuronal EZH2 regulated genes related to neuronal morphogenesis. In particular, Pak3 was identified as a target gene suppressed by EZH2 and H3K27me3, and expression of dominant negative PAK3 reversed Ezh2 knockout-induced higher dendritic spine density. Lastly, lack of neuronal EZH2 resulted in impaired memory behaviors in adult mice. Our results demonstrated that neuronal EZH2 played important roles in controlling multiple steps of neuronal morphogenesis during development, which had long-lasting effects on cognitive function in adult mice.

neuroscience↗