Synaptic Ras GTPase-activating Protein Research Articles

Genetic variants that modify neuroendocrine gene expression and foraging behavior of C. elegans.

The molecular mechanisms underlying diversity in animal behavior are not well understood. A major experimental challenge is determining the contribution of genetic variants that affect neuronal gene expression to differences in behavioral traits. In Caenorhabditis elegans, the neuroendocrine transforming growth factor-β ligand, DAF-7, regulates diverse behavioral responses to bacterial food and pathogens. The dynamic neuron-specific expression of daf-7 is modulated by environmental and endogenous bacteria-derived cues. Here, we investigated natural variation in the expression of daf-7 from the ASJ pair of chemosensory neurons. We identified common genetic variants in gap-2, encoding a Ras guanosine triphosphatase (GTPase)-activating protein homologous to mammalian synaptic Ras GTPase-activating protein, which modify daf-7 expression cell nonautonomously and promote exploratory foraging behavior in a partially DAF-7-dependent manner. Our data connect natural variation in neuron-specific gene expression to differences in behavior and suggest that genetic variation in neuroendocrine signaling pathways mediating host-microbe interactions may give rise to diversity in animal behavior.

Impairments of Spatial Memory and N-methyl-d-aspartate Receptors and Their Postsynaptic Signaling Molecules in the Hippocampus of Developing Rats Induced by As, Pb, and Mn Mixture Exposure.

Exposure to metal mixtures is recognized as a real-life scenario, needing novel studies that can assess their complex effects on brain development. There is still a significant public health concern associated with chronic low levels of metal exposure. In contrast to other metals, these three metals (As, Pb, and Mn) are commonly found in various environmental and industrial contexts. In addition to additive or synergistic interactions, concurrent exposure to this metal mixture may also have neurotoxic effects that differ from those caused by exposure to single components. The NMDA receptor and several important signaling proteins are involved in learning, memory, and synaptic plasticity in the hippocampus, including CaMKII, postsynaptic density protein-95 (PSD-95), synaptic Ras GTPase activating protein (SynGAP), a negative regulator of Ras-MAPK activity, and CREB. We hypothesized that alterations in the above molecular players may contribute to metal mixture developmental neurotoxicity. Thus, the aim of this study was to investigate the effect of these metals and their mixture at low doses (As 4 mg, Pb 4 mg, and Mn 10 mg/kg bw/p.o) on NMDA receptors and their postsynaptic signaling proteins during developing periods (GD6 to PD59) of the rat brain. Rats exposed to As, Pb, and Mn individually or at the same doses in a triple-metal mixture (MM) showed impairments in learning and memory functions in comparison to the control group rats. Declined protein expressions of NR2A, PSD-95, p- CaMKII, and pCREB were observed in the metal mix-exposed rats, while the expression of SynGAP was found to be enhanced in the hippocampus as compared to the controls on PD60. Thereby, our data suggest that alterations in the NMDA receptor complex and postsynaptic signaling proteins could explain the cognitive dysfunctions caused by metal-mixture-induced developmental neurotoxicity in rats. These outcomes indicate that incessant metal mixture exposure may have detrimental consequences on brain development.

Generation of a transgene-free iPS cell line (SDQLCHi053-A) from a young girl carrying a heterozygous mutation (c.427C > T) in SYNGAP1 gene

The pathogenic mutations of Synaptic Ras GTPase-activating protein 1 (SYNGAP1) gene (OMIM #603384) have been tightly associated with a neurodevelopmental disease, also called autosomal dominant mental retardation type 5 (MRD5, OMIM #612621). We generated a human iPS cell line from a 34-month-old young girl bearing a recurrent heterozygous mutation (c.427C > T) of SYNGAP1. This cell line has great performance in pluripotency and shows differentiation potential towards three germ layers in vitro.

Identifying the building blocks for learning and memory in the carotid body

Glutamate is a major excitatory neurotransmitter with well-established roles for inducing neural plasticity, learning and memory in the central nervous system. In the carotid body, an organ derived in part from the neural crest, evidence suggests glutamatergic signalling is involved in its sensing of hypoxia. We hypothesized that altered glutamatergic signalling may underpin enhanced chemoreflex responses in carotid body in disease states. Therefore, we have initiated an investigation to assess the presence and location of glutamate receptors expressed in the carotid body of Wistar rats. We mined two independent RNA-sequencing (RNAseq) datasets to identify glutamate signalling components. Digital droplet PCR (ddPCR) was performed to validate the expression of selected glutamatergic target genes. Protein localisation was detailed using immunofluorescence and confocal microscopy in 12–16-week-old normotensive male Wistar rats. Neuronal phenotypes were identified using tyrosine hydroxylase (type I or glomus cells) and glial fibrillary acidic protein antibodies (type II cells). In carotid body extracts, RNAseq data revealed the presence of novel glutamatergic signalling components, not previously described. ddPCR analysis confirmed the expression of AMPA, NMDA and excitatory amino acid transporters. Glutamate ionotropic receptor AMPA type subunit 1 ( Gria1) showed co-localisation with the membrane of type I glomus cells. Glutamate ionotropic receptor NMDA type 1 ( Grin1) showed co-localisation with the nuclei membrane [JP1] of glomus cells. NMDA receptor type subunit 2C ( GRIN2C) showed co-localisation with the membranes of both glomus cells and type II cells. Expression of these receptors was not found on all glomus cells within a cluster suggesting functional heterogeneity within the carotid body. Synaptic Ras GTPase-activating protein 1 ( Syngap), a protein that interacts with glutamate synapses involved in learning, was expressed in both plasma and nuclear membranes of glomus cells. The excitatory amino acid transporter 5 ( EAAT5) showed co-localisation with both type I and type II cells. Additionally, we have observed that a local arterial injection of glutamate (via the internal carotid artery) evoked excitatory activity, followed by an inhibitory period as recorded from the carotid sinus nerve. Our results identified expression and location of glutamatergic AMPA and NMDA receptors in the carotid body[JP2]. We also found the presence of excitatory amino acid transporters, and proteins involved in chaperoning glutamate receptors for learning and memory, All told, we have identified the substrate for glutamate as a transmitter in the carotid body. Whether it plays a role in the learning and memory of carotid body function and/or sensitisation in disease remains to be elucidated. Funding: Health Research Council of New Zealand. Nothing to disclose. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Synaptoproteomic Analysis of the Prefrontal Cortex Reveals Spatio-Temporal Changes in SYNGAP1 Following Cannabinoid Exposure in Rat Adolescence

The regular use of cannabis during adolescence has been associated with a number of negative life outcomes, including psychopathology and cognitive impairments. However, the exact molecular mechanisms that underlie these outcomes are just beginning to be understood. Moreover, very little is known about the spatio-temporal molecular changes that occur following cannabinoid exposure in adolescence. To understand these changes, we exposed mid-adolescent male rats to a synthetic cannabinoid (WIN 55,212-2 mesylate; WIN) and, following drug abstinence through late adolescence, we subjected the synaptosomal fractions of the prefrontal cortex (PFC) to proteomic analyses. A total of N = 487 differentially expressed proteins were found in WIN-exposed animals compared to controls. Gene ontology analyses revealed enrichment of terms related to the gamma-aminobutyric acid (GABA)-ergic neurotransmitter system. Among the top differentially expressed proteins was the synaptic Ras GTPase-activating protein 1 (SYNGAP1). Using Western blotting experiments, we found that the WIN-induced upregulation of SYNGAP1 was spatio-temporal in nature, arising only in the synaptosomal fractions (not in the cytosol) and only following prolonged drug abstinence (not on abstinence day 1). Moreover, the SYNGAP1 changes were found to be specific to WIN-exposure in adolescence and not adulthood. Adolescent animals exposed to a natural cannabinoid (Δ9-tetrahydrocannabinol; THC) were also found to have increased levels of SYNGAP1 in the PFC. THC exposure also led to a pronounced upregulation of SYNGAP1 in the amygdala, but without any changes in the dorsal striatum, hippocampus, or nucleus accumbens. To our knowledge, this is the first study to uncover a link between cannabinoid exposure and changes in SYNGAP1 that are spatio-temporal and developmental in nature. Future studies are needed to investigate the putative role of SYNGAP1 in the negative behavioral consequences of cannabis use in adolescence.

Rho-Kinase/ROCK Phosphorylates PSD-93 Downstream of NMDARs to Orchestrate Synaptic Plasticity.

The N-methyl-D-aspartate receptor (NMDAR)-mediated structural plasticity of dendritic spines plays an important role in synaptic transmission in the brain during learning and memory formation. The Rho family of small GTPase RhoA and its downstream effector Rho-kinase/ROCK are considered as one of the major regulators of synaptic plasticity and dendritic spine formation, including long-term potentiation (LTP). However, the mechanism by which Rho-kinase regulates synaptic plasticity is not yet fully understood. Here, we found that Rho-kinase directly phosphorylated discs large MAGUK scaffold protein 2 (DLG2/PSD-93), a major postsynaptic scaffold protein that connects postsynaptic proteins with NMDARs; an ionotropic glutamate receptor, which plays a critical role in synaptic plasticity. Stimulation of striatal slices with an NMDAR agonist induced Rho-kinase-mediated phosphorylation of PSD-93 at Thr612. We also identified PSD-93-interacting proteins, including DLG4 (PSD-95), NMDARs, synaptic Ras GTPase-activating protein 1 (SynGAP1), ADAM metallopeptidase domain 22 (ADAM22), and leucine-rich glioma-inactivated 1 (LGI1), by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Among them, Rho-kinase increased the binding of PSD-93 to PSD-95 and NMDARs. Furthermore, we found that chemical-LTP induced by glycine, which activates NMDARs, increased PSD-93 phosphorylation at Thr612, spine size, and PSD-93 colocalization with PSD-95, while these events were blocked by pretreatment with a Rho-kinase inhibitor. These results indicate that Rho-kinase phosphorylates PSD-93 downstream of NMDARs, and suggest that Rho-kinase mediated phosphorylation of PSD-93 increases the association with PSD-95 and NMDARs to regulate structural synaptic plasticity.

RASAL3 predicts overall survival and CD8+ T lymphocyte infiltration in lung adenocarcinoma.

RAS-activating protein-like 3 (RASAL3) is a synaptic Ras GTPase-activating protein (SynGAP) and a potential novel biomarker of CD8+ T cell infiltration in lung adenocarcinoma (LUAD). This study explored RASAL3 expression in LUAD, the prognostic impact of RASAL3 and the relationship with immune cell infiltration. RASAL3 expression in LUAD tissues was considerably low, with high RASAL3 expression associated with better overall survival, whereas the low expression was linked to advanced T, N, M classifications, TNM stage and lower grade. Furthermore, RASAL3 expression positively correlated with CD8+ T lymphocyte infiltration. In conclusion, RASAL3 expression is a potential prognostic and immunological biomarker of LUAD.

Prenatal disruption of D1R-SynGAP complex causes cognitive deficits in adulthood

γ-aminobutyric acid (GABA)-ergic interneurons are essential for the physiological function of the mammalian central nervous system. Dysregulated GABAergic interneuron function has been implicated in the pathophysiology of a number of neurodevelopmental disorders including schizophrenia and autism spectrum disorder. Tangential migration is an important process to ensure the proper localization of GABAergic interneurons. Previously we found that disrupting the interaction between dopamine D1 receptor (D1R) and synaptic Ras GTPase- activating protein (SynGAP) using an interfering peptide (TAT-D1Rpep) during embryonic development impaired tangential migration. Here, we assessed the effects of prenatal disruption of D1R-SynGAP complex with the TAT-D1Rpep on the expression of several behaviours during adulthood. Mice with prenatal D1R-SynGAP disruption exhibited transiently reduced locomotor activity, abnormal sensorimotor gating, impaired sociability and deficits in visual discrimination associative learning compared to their control counterparts. Our findings reinforce the importance of GABAergic interneuron migration in the manifestation of normal motor, sensory, and cognitive behaviours of animals during adulthood.

SynGAP isoforms differentially regulate synaptic plasticity and dendritic development.

SynGAP is a synaptic Ras GTPase-activating protein (GAP) with four C-terminal splice variants: α1, α2, β, and γ. Although studies have implicated SYNGAP1 in several cognitive disorders, it is not clear which SynGAP isoforms contribute to disease. Here, we demonstrate that SynGAP isoforms exhibit unique spatiotemporal expression patterns and play distinct roles in neuronal and synaptic development in mouse neurons. SynGAP-α1, which undergoes liquid-liquid phase separation with PSD-95, is highly enriched in synapses and is required for LTP. In contrast, SynGAP-β, which does not bind PSD-95 PDZ domains, is less synaptically targeted and promotes dendritic arborization. A mutation in SynGAP-α1 that disrupts phase separation and synaptic targeting abolishes its ability to regulate plasticity and instead causes it to drive dendritic development like SynGAP-β. These results demonstrate that distinct intrinsic biochemical properties of SynGAP isoforms determine their function, and individual isoforms may differentially contribute to the pathogenesis of SYNGAP1-related cognitive disorders.

Importance of Functional Loss of FUS in FTLD/ALS.

Fused in sarcoma (FUS) is an RNA binding protein that regulates RNA metabolism including alternative splicing, transcription, and RNA transportation. FUS is genetically and pathologically involved in frontotemporal lobar degeneration (FTLD)/amyotrophic lateral sclerosis (ALS). Multiple lines of evidence across diverse models suggest that functional loss of FUS can lead to neuronal dysfunction and/or neuronal cell death. Loss of FUS in the nucleus can impair alternative splicing and/or transcription, whereas dysfunction of FUS in the cytoplasm, especially in the dendritic spines of neurons, can cause mRNA destabilization. Alternative splicing of the MAPT gene at exon 10, which generates 4-repeat Tau (4R-Tau) and 3-repeat Tau (3R-Tau), is one of the most impactful targets regulated by FUS. Additionally, loss of FUS function can affect dendritic spine maturations by destabilizing mRNAs such as Glutamate receptor 1 (GluA1), a major AMPA receptor, and Synaptic Ras GTPase-activating protein 1 (SynGAP1). Moreover, FUS is involved in axonal transport and morphological maintenance of neurons. These findings indicate that a biological link between loss of FUS function, Tau isoform alteration, aberrant post-synaptic function, and phenotypic expression might lead to the sequential cascade culminating in FTLD. Thus, to facilitate development of early disease markers and/or therapeutic targets of FTLD/ALS it is critical that the functions of FUS and its downstream pathways are unraveled.

Developmental stage-dependent regulation of spine formation by calcium-calmodulin-dependent protein kinase II\u03b1 and Rap1

The roles of calcium-calmodulin-dependent protein kinase II-alpha (CaMKIIα) in the expression of long-term synaptic plasticity in the adult brain have been extensively studied. However, how increased CaMKIIα activity controls the maturation of neuronal circuits remains incompletely understood. Herein, we show that pyramidal neurons without CaMKIIα activity upregulate the rate of spine addition, resulting in elevated spine density. Genetic elimination of CaMKIIα activity specifically eliminated the observed maturation-dependent suppression of spine formation. Enhanced spine formation was associated with the stabilization of actin in the spine and could be reversed by increasing the activity of the small GTPase Rap1. CaMKIIα activity was critical in the phosphorylation of synaptic Ras GTPase-activating protein (synGAP), the dispersion of synGAP from postsynaptic sites, and the activation of postsynaptic Rap1. CaMKIIα is already known to be essential in learning and memory, but our findings suggest that CaMKIIα plays an important activity-dependent role in restricting spine density during postnatal development.

Arsenite exposure altered the expression of NMDA receptor and postsynaptic signaling proteins in rat hippocampus

Chronic arsenic exposure has an adverse effect on neurobehavioral function. Our previous study demonstrated an elevated arsenic level, ultra-structure changes and reduced NR2A gene expression in hippocampus, and impaired spatial learning in arsenite-exposed rats. The NMDA receptor and the postsynaptic signaling proteins CaMKII, postsynaptic density protein 95 (PSD-95), synaptic Ras GTPase-activating protein (SynGAP) and nuclear activated extracellular-signal regulated kinase (ERK1/2) play important roles in synaptic plasticity, learning and memory. We hypothesized that the above molecular expression changes may contribute to arsenic neurotoxicity. In present study, the expression of NMDA receptor and postsynaptic signaling proteins in hippocampus were evaluated in rats exposed to 0, 2.72, 13.6 and 68mg/L sodium arsenite for 3 months. Decreased protein expression of NR2A, PSD-95 and p-CaMKII α in the hippocampus of arsenite-exposed rats was observed, while the expression of SynGAP, a negative regulator of Ras-MAPK activity, was increased when compared with the controls. Additionally, decreased p-ERK1/2 activity was found in the hippocampus of arsenite-exposed rats. These data suggest that altered expression of NMDA receptor complex and postsynaptic signaling proteins may explain arsenic-induced neurotoxicity.

Differential expression of two NMDA receptor interacting proteins, PSD‐95 and SynGAP during mouse development

Patterns of neural activity mediated by N-methyl-D-aspartate (NMDA) receptors are known to play important roles in development of the central nervous system. However, the signalling pathways downstream from NMDA receptors that are critical for normal neuronal development are not yet clearly understood. NMDA receptors interact with various signalling proteins via scaffolding proteins, which are important in adult neuronal and behavioural plasticity. For example, the NR2B subunits of the NMDA receptor interact with postsynaptic density 95 (PSD-95), which in turn binds to synaptic ras GTPase-activating protein (SynGAP). Interestingly, the developmental phenotype of mice carrying null mutations in these genes differ. NR2B and SynGAP homozygote mice die within the first week of birth whereas PSD-95 homozygote mice survive to adulthood. We therefore examined the expression patterns of PSD-95 and SynGAP genes from embryonic stages to adult using lacZ (beta-galactosidase) marker gene knock-in mice. Dramatic changes of expression were observed throughout development in brain and other tissues. Although SynGAP binds PSD-95, both genes had distinct, as well as overlapping expression. SynGAP expression peaked at times of synaptogenesis and developmental plasticity in contrast to PSD-95, which was expressed throughout the brain from early embryonic stages. Furthermore, SynGAP showed a more spatially restricted pattern as illustrated by its restriction to forebrain in contrast to PSD-95, which was also found in mid- and hindbrain. These data support the model that synaptic signalling complexes are heterogeneous and individual components show temporal and spatial specificity during development.

Impaired SynGAP expression and long-term spatial learning and memory in hippocampal CA1 area from rats previously exposed to perinatal hypoxia-induced insults: beneficial effects of A68930

Hypoxic encephalopathy is a common cause of neonatal seizures and long-term neurological cognitive deficits. In rats at postnatal days 10–12 (P10–P12), global hypoxia induced spontaneous seizures and chronic brain injury, mimicking clinical aspects of neonatal hypoxia. Synaptic Ras-GTPase activating protein (SynGAP) has important roles in RAS/MAPK-dependent synaptic plasticity and mammalian learning. We investigated possible alterations of SynGAP expression occurring in memory-impaired animals previously exposed to perinatal hypoxia insults. We also evaluated the therapeutic efficacy of A68930, a selective agonist of dopamine D1/D5 receptors, on perinatal hypoxia insults. In the hippocampal CA1 region, perinatal hypoxia insults (P10) led to a reduction in SynGAP expression associated with impairment in long-term spatial learning and memory performance at P45. The use of A68930 (at a dose of 1, 2, 3 mg/kg, P17–P23) effectively attenuated the deleterious effects as described above. Our results may indicate the involvement of SynGAP in certain forms of brain injury, leading to long-term learning and memory deficits. A68930 may have clinical potential as a therapeutic agent for alleviation of long-term cognitive deficits in rats and other animal models.

PSD-95 promotes CaMKII-catalyzed serine phosphorylation of the synaptic RAS-GTPase activating protein SynGAP after transient brain ischemia in rat hippocampus

Recent studies have indicated that cerebral ischemia induces rapid serine phosphorylation of synaptic RAS-GTPase activating protein (SynGAP) by calcium/Camodulin-dependent protein kinase II (CaMKII) in rat hippocampus. To further illustrate the mechanisms underlying these processes, we examined the effects of transient (15 min) brain ischemia followed by reperfusion (0, 30 min, 6 h, 1, 3 days) on serine phosphorylation of SynGAP and interactions involving SynGAP, postsynaptic density protein 95 (PSD95) and CaMKII in rat hippocampus. Transient brain ischemia was induced by the method of four-vessel occlusion in Sprague–Dawley rats. Serine phosphorylation of SynGAP increased immediately after brain ischemia and peaked at 30-min reperfusion, and the increase was maintained for 3 days. The association among SynGAP, PSD95 and CaMKII had a similar trend as serine phosphorylation of SynGAP. Intracrebroventricular infusion of PSD95 antisense oligodeoxynucleotide not only markedly decreased the protein levels of PSD95 but also attenuated the elevated serine phosphorylation of SynGAP and the associations among SynGAP, PSD95 and CaMKII induced by 30-min reperfusion following 15-min brain ischemia. The results suggest that the serine phosphorylation of SynGAP catalyzed by CaMKII is immediately increased and that PSD95 is critical for promoting SynGAP serine phosphorylation after transient brain ischemia.

Genomic organization of the HSET locus and the possible association of HLA-linked genes with immotile cilia syndrome (ICS).

The kinesin-related protein (HSET) gene belongs to the kinesin superfamily, the members of which are involved in cellular transport processes. The HSET gene product was previously characterized by partial cDNA sequencing. The gene is located on the short arm of human Chromosome 6 (6p21.3), at the centromeric end of the major histocompatibility complex. Here, we report the genomic structure of the complete HSET gene together with its flanking loci. Sequence analysis of the 40 kilobase (kb) cosmid clone containing the HSET gene also revealed the presence of several new genes not related to the kinesin superfamily. These include a 60S ribosomal protein L35A-like pseudogene (rPL35A-like) on the telomeric side and a polycomb-like gene (PHF1), a copper tolerance-like gene (CUTA1) and the 5' part of the synaptic ras-GTPase-activating protein (SynGAP) gene centromeric of HSET. In addition, a complete 60S ribosomal protein L12-like (rPL12L) gene in intron 3 of the HSET gene was identified which appears to have an open reading frame. The possible involvement of the HSET gene and a beta-tubulin gene (TUBB) in the pathogenesis of immotile cilia syndrome (ICS) was studied by screening two unrelated ICS families with microtubular defects and suspected HLA linkage for mutations within the HSET gene and the TUBB gene. Four single base substitutions were detected in the HSET gene, and none in the TUBB gene. On the basis of these data, a role of the HSET and TUBB products in the pathogenesis of ICS in the two families is unlikely.

Citron Binds to PSD-95 at Glutamatergic Synapses on Inhibitory Neurons in the Hippocampus

Synaptic NMDA-type glutamate receptors are anchored to the second of three PDZ (PSD-95/Discs large/ZO-1) domains in the postsynaptic density (PSD) protein PSD-95. Here, we report that citron, a protein target for the activated form of the small GTP-binding protein Rho, preferentially binds the third PDZ domain of PSD-95. In GABAergic neurons from the hippocampus, citron forms a complex with PSD-95 and is concentrated at the postsynaptic side of glutamatergic synapses. Citron is expressed only at low levels in glutamatergic neurons in the hippocampus and is not detectable at synapses onto these neurons. In contrast to citron, p135 SynGAP, an abundant synaptic Ras GTPase-activating protein that can bind to all three PDZ domains of PSD-95, and Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) are concentrated postsynaptically at glutamatergic synapses on glutamatergic neurons. CaM kinase II is not expressed and p135 SynGAP is expressed in less than half of hippocampal GABAergic neurons. Segregation of citron into inhibitory neurons does not occur in other brain regions. For example, citron is expressed at high levels in most thalamic neurons, which are primarily glutamatergic and contain CaM kinase II. In several other brain regions, citron is present in a subset of neurons that can be either GABAergic or glutamatergic and can sometimes express CaM kinase II. Thus, in the hippocampus, signal transduction complexes associated with postsynaptic NMDA receptors are different in glutamatergic and GABAergic neurons and are specialized in a way that is specific to the hippocampus.