Modulation of GABA A receptor trafficking by WWC2 reveals class-specific mechanisms of synapse regulation by WWC family proteins.

WWC2 (WW and C2 domain-containing protein) is implicated in several neurological disorders, however its function in the brain has yet to be determined. Here, we demonstrate that WWC2 interacts with inhibitory but not excitatory postsynaptic scaffolds, consistent with prior proteomic identification of WWC2 as a putative component of the inhibitory postsynaptic density. Using mice lacking WWC2 expression in excitatory forebrain neurons, we show that WWC2 suppresses GABA A R incorporation into the plasma membrane and regulates HAP1 and GRIP1, which form a complex promoting GABA A R recycling to the membrane. Inhibitory synaptic transmission is dysregulated in CA1 pyramidal cells lacking WWC2. Furthermore, unlike the WWC2 homolog KIBRA (WWC1), a key regulator of AMPA receptor trafficking at excitatory synapses, deletion of WWC2 does not affect synaptic AMPAR expression. In contrast, loss of KIBRA does not affect GABA A R membrane expression. These data reveal unique, synapse class-selective functions for WWC proteins as regulators of ionotropic neurotransmitter receptors and provide insight into mechanisms regulating GABA A R membrane expression.

SynGAP regulates synaptic plasticity and cognition independently 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 independently of its GAP activity. Here, we report that inactivating mutations within the GAP domain do not inhibit synaptic plasticity or cause behavioral deficits. Instead, SynGAP modulates synaptic strength by physically competing with the AMPA-receptor-TARP excitatory receptor complex in the formation of molecular condensates with synaptic scaffolding proteins. These results have major implications for developing therapeutic treatments for SYNGAP1-related neurodevelopmental disorders.

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.

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 Statement: SYNGAP1 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. Highlights: Two 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. Both SRID mouse models show 35∼50% reduction in mRNA and ∼50% reduction in Syngap1 protein.Both SRID mouse models display deficits in synaptic plasticity and behavioral phenotypes found in people. RNA-seq confirmed cryptic splice acceptor activity in one SRID mouse model and revealed broad transcriptional changes also identified in Syngap1 +/- mice. Novel SRID mouse models generated here provide a resource and establish a framework for development of future therapeutic intervention.

Btbd11 supports cell-type-specific synaptic function.

Synapses in the brain exhibit cell-type-specific differences in basal synaptic transmission and plasticity. Here, we evaluated cell-type-specific specializations 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 postsynaptic density in synapses in inhibitory interneurons exists in a phase-separated state. Knockout of Btbd11 decreased glutamatergic signaling onto parvalbumin-positive interneurons. Further, both in vitro and in vivo, Btbd11 knockout disrupts network activity. At the behavioral level, Btbd11 knockout from interneurons alters exploratory behavior, measures of anxiety, and sensitizes mice to pharmacologically induced hyperactivity following NMDA receptor antagonist challenge. Our findings identify a cell-type-specific mechanism that supports glutamatergic synapse function in inhibitory interneurons-with implications for circuit function and animal behavior.

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.

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 populations of synapses changes during behavior. We used this approach to generate an unprecedentedly detailed spatiotemporal map of synapses undergoing changes in strength following 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.

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 (TKIT) guides 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 labeling 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.

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.

Strong patterns of intraspecific variation and local adaptation in Great Basin plants revealed through a review of 75 years of experiments.

Variation in natural selection across heterogeneous landscapes often produces (a) among-population differences in phenotypic traits, (b) trait-by-environment associations, and (c) higher fitness of local populations. Using a broad literature review of common garden studies published between 1941 and 2017, we documented the commonness of these three signatures in plants native to North America's Great Basin, an area of extensive restoration and revegetation efforts, and asked which traits and environmental variables were involved. We also asked, independent of geographic distance, whether populations from more similar environments had more similar traits. From 327 experiments testing 121 taxa in 170 studies, we found 95.1% of 305 experiments reported among-population differences, and 81.4% of 161 experiments reported trait-by-environment associations. Locals showed greater survival in 67% of 24 reciprocal experiments that reported survival, and higher fitness in 90% of 10 reciprocal experiments that reported reproductive output. A meta-analysis on a subset of studies found that variation in eight commonly measured traits was associated with mean annual precipitation and mean annual temperature at the source location, with notably strong relationships for flowering phenology, leaf size, and survival, among others. Although the Great Basin is sometimes perceived as a region of homogeneous ecosystems, our results demonstrate widespread habitat-related population differentiation and local adaptation. Locally sourced plants likely harbor adaptations at rates and magnitudes that are immediately relevant to restoration success, and our results suggest that certain key traits and environmental variables should be prioritized in future assessments of plants in this region.

Activity-Dependent Ubiquitination of GluA1 and GluA2 Regulates AMPA Receptor Intracellular Sorting and Degradation.

AMPA receptors (AMPARs) have recently been shown to undergo post-translational ubiquitination in mammalian neurons. However, the underlying molecular mechanisms are poorly understood and remain controversial. Here, we report that all four AMPAR subunits (GluA1-4) are rapidly ubiquitinated upon brief application of AMPA or bicuculline in cultured neurons. This process is Ca2+ dependent and requires the activity of L-type voltage-gated Ca2+ channels and Ca2+/calmodulin-dependent kinase II. The ubiquitination of all subunits occurs exclusively on AMPARs located on the plasma membrane post-endocytosis. The sites of ubiquitination were mapped to Lys-868 in GluA1 and Lys-870/Lys-882 in GluA2 C-terminals. Mutation of these lysines did not affect basal surface expression or AMPA-induced internalization of GluA1 and GluA2 subunits. Instead, it reduced the intracellular trafficking of AMPARs to the late endosomes and thus protein degradation. These data indicate that ubiquitination is an important regulatory signal for controlling AMPAR function, which may be crucial for synaptic plasticity.

Relating adaptive genetic traits to climate for Sandberg bluegrass from the intermountain western United States.

Genetic variation for potentially adaptive traits of the key restoration species Sandberg bluegrass (Poa secunda J. Presl) was assessed over the intermountain western United States in relation to source population climate. Common gardens were established at two intermountain west sites with progeny from two maternal parents from each of 130 wild populations. Data were collected over 2 years at each site on fifteen plant traits associated with production, phenology, and morphology. Analyses of variance revealed strong population differences for all plant traits (P < 0.0001), indicating genetic variation. Both the canonical correlation and linear correlation established associations between source populations and climate variability. Populations from warmer, more arid climates had generally lower dry weight, earlier phenology, and smaller, narrower leaves than those from cooler, moister climates. The first three canonical variates were regressed with climate variables resulting in significant models (P < 0.0001) used to map 12 seed zones. Of the 700 981 km(2) mapped, four seed zones represented 92% of the area in typically semi-arid and arid regions. The association of genetic variation with source climates in the intermountain west suggested climate driven natural selection and evolution. We recommend seed transfer zones and population movement guidelines to enhance adaptation and diversity for large-scale restoration projects.

Genetic variation in adaptive traits and seed transfer zones for Pseudoroegneria spicata (bluebunch wheatgrass) in the northwestern United States.

A genecological approach was used to explore genetic variation in adaptive traits in Pseudoroegneria spicata, a key restoration grass, in the intermountain western United States. Common garden experiments were established at three contrasting sites with seedlings from two maternal parents from each of 114 populations along with five commercial releases commonly used in restoration. Traits associated with size, flowering phenology, and leaf width varied considerably among populations and were moderately correlated with the climates of the seed sources. Pseudoroegneria spicata populations from warm, arid source environments were smaller with earlier phenology and had relatively narrow leaves than those from mild climates with cool summers, warm winters, low seasonal temperature differentials, high precipitation, and low aridity. Later phenology was generally associated with populations from colder climates. Releases were larger and more fecund than most of the native ecotypes, but were similar to native populations near their source of origin. Differences among native populations associated with source climates that are logical for survival, growth, and reproduction indicate that genetic variation across the landscape is adaptive and should be considered during restoration. Results were used to delineate seed transfer zones and population movement guidelines to ensure adapted plant materials for restoration activities.

Regulation of AMPA receptor function by the human memory-associated gene KIBRA.

KIBRA has recently been identified as a gene associated with human memory performance. Despite the elucidation of the role of KIBRA in several diverse processes in nonneuronal cells, the molecular function of KIBRA in neurons is unknown. We found that KIBRA directly binds to the protein interacting with C-kinase 1 (PICK1) and forms a complex with α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate receptors (AMPARs), the major excitatory neurotransmitter receptors in the brain. KIBRA knockdown accelerates the rate of AMPAR recycling following N-methyl-D-aspartate receptor-induced internalization. Genetic deletion of KIBRA in mice impairs both long-term depression and long-term potentiation at hippocampal Schaffer collateral-CA1 synapses. Moreover, KIBRA knockout mice have severe deficits in contextual fear learning and memory. These results indicate that KIBRA regulates higher brain function by regulating AMPAR trafficking and synaptic plasticity.

Enhanced synaptic plasticity in mice with phosphomimetic mutation of the GluA1 AMPA receptor.

Phosphorylation of the GluA1 subunit of AMPA receptors has been proposed to regulate receptor trafficking and synaptic transmission and plasticity. However, it remains unclear whether GluA1 phosphorylation is permissive or sufficient for enacting these functional changes. Here we investigate the role of GluA1 phosphorylation at S831 and S845 residues in the hippocampus through the analyses of GluA1 S831D/S845D phosphomimetic knock-in mice. S831D/S845D mice showed normal total and surface expression and subcellular localization of GluA1 as well as intact basal synaptic transmission. In addition, theta-burst stimulation, a protocol that was sufficient to induce robust long-term potentiation (LTP) in WT mice, resulted in LTP of similar magnitude in S831D/S845D mice. However, S831D/S845D mice showed LTP induced with 10-Hz stimulation, a protocol that is weaker than theta-burst stimulation and was not sufficient to induce LTP in WT mice. Moreover, S831D/S845D mice exhibited LTP induced with spike-timing-dependent plasticity (STDP) protocol at a long pre-post interval that was subthreshold for WT mice, although a suprathreshold STDP protocol at a short pre-post interval resulted in similarly robust LTP for WT and S831D/S845D mice. These results indicate that phosphorylation of GluA1 at S831 and S845 is sufficient to lower the threshold for LTP induction, increasing the probability of synaptic plasticity.

Mechanism of Ca2+/calmodulin-dependent kinase II regulation of AMPA receptor gating.

The function, trafficking and synaptic signaling of AMPA receptors are tightly regulated by phosphorylation. Ca(2+)/calmodulin-dependent kinase II (CaMKII) phosphorylates the GluA1 AMPA receptor subunit at Ser831 to increase single-channel conductance. We show that CaMKII increases the conductance of native heteromeric AMPA receptors in mouse hippocampal neurons through phosphorylation of Ser831. In addition, co-expression of transmembrane AMPA receptor regulatory proteins (TARPs) with recombinant receptors is required for phospho-Ser831 to increase conductance of heteromeric GluA1-GluA2 receptors. Finally, phosphorylation of Ser831 increases the efficiency with which each subunit can activate, independent of agonist efficacy, thereby increasing the likelihood that more receptor subunits will be simultaneously activated during gating. This underlies the observation that phospho-Ser831 increases the frequency of openings to larger conductances rather than altering unitary conductance. Together, these findings suggest that CaMKII phosphorylation of GluA1-Ser831 decreases the activation energy for an intrasubunit conformational change that regulates the conductance of the receptor when the channel pore opens.

Mutational activation of ErbB2 reveals a new protein kinase autoinhibition mechanism.

Autoinhibition plays a key role in the control of protein kinase activity. ErbB2 is a unique receptor-tyrosine kinase that does not bind ligand but possesses an extracellular domain poised to engage other ErbBs. Little is known about the molecular mechanism for ErbB2 catalytic regulation. Here we show that ErbB2 kinase is strongly autoinhibited, and a loop connecting the alphaC helix and beta4 sheet within the kinase domain plays a major role in the control of kinase activity. Mutations of two Gly residues at positions 776 and 778 in this loop dramatically increase ErbB2 catalytic activity. Kinetic analysis demonstrates that mutational activation is due to approximately 10- and approximately 7-fold increases in ATP binding affinity and turnover number, respectively. Expression of the activated ErbB2 mutants in cells resulted in elevated ligand-independent ErbB2 autophosphorylation, ErbB3 phosphorylation, and stimulation of mitogen-activated protein kinase. Molecular modeling suggests that the ErbB2 kinase domain is stabilized in an inactive state via a hydrophobic interaction between the alphaC-beta4 and activation loops. Importantly, many ErbB2 human cancer mutations have been identified in the alphaC-beta4 loop, including the activating G776S mutation studied here. Our findings reveal a new kinase regulatory mechanism in which the alphaC-beta4 loop functions as an intramolecular switch that controls ErbB2 activity and suggests that loss of alphaC-beta4 loop-mediated autoinhibition is involved in oncogenic activation of ErbB2.

Synaptic incorporation of AMPA receptors during LTP is controlled by a PKC phosphorylation site on GluR1.

Incorporation of GluR1-containing AMPA receptors into synapses is essential to several forms of neural plasticity, including long-term potentiation (LTP). Numerous signaling pathways that trigger this process have been identified, but the direct modifications of GluR1 that control its incorporation into synapses are unclear. Here, we show that phosphorylation of GluR1 by PKC at a highly conserved serine 818 residue is increased during LTP and critical for LTP expression. GluR1 is phosphorylated by PKC at this site in vitro and in vivo. In addition, acute phosphorylation at GluR1 S818 by PKC, as well as a phosphomimetic mutation, promotes GluR1 synaptic incorporation. Conversely, preventing GluR1 S818 phosphorylation reduces LTP and blocks PKC-driven synaptic incorporation of GluR1. We conclude that the phosphorylation of GluR1 S818 by PKC is a critical event in the plasticity-driven synaptic incorporation of AMPA receptors.