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.

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.

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.

Wnt5a is essential for hippocampal dendritic maintenance and spatial learning and memory in adult mice.

Stability of neuronal connectivity is critical for brain functions, and morphological perturbations are associated with neurodegenerative disorders. However, how neuronal morphology is maintained in the adult brain remains poorly understood. Here, we identify Wnt5a, a member of the Wnt family of secreted morphogens, as an essential factor in maintaining dendritic architecture in the adult hippocampus and for related cognitive functions in mice. Wnt5a expression in hippocampal neurons begins postnatally, and its deletion attenuated CaMKII and Rac1 activity, reduced GluN1 glutamate receptor expression, and impaired synaptic plasticity and spatial learning and memory in 3-mo-old mice. With increased age, Wnt5a loss caused progressive attrition of dendrite arbors and spines in Cornu Ammonis (CA)1 pyramidal neurons and exacerbated behavioral defects. Wnt5a functions cell-autonomously to maintain CA1 dendrites, and exogenous Wnt5a expression corrected structural anomalies even at late-adult stages. These findings reveal a maintenance factor in the adult brain, and highlight a trophic pathway that can be targeted to ameliorate dendrite loss in pathological conditions.

Purkinje cell-specific Grip1/2 knockout mice show increased repetitive self-grooming and enhanced mGluR5 signaling in cerebellum.

Cerebellar Purkinje cell (PC) loss is a consistent pathological finding in autism. However, neural mechanisms of PC-dysfunction in autism remain poorly characterized. Glutamate receptor interacting proteins 1/2 (Grip1/2) regulate AMPA receptor (AMPAR) trafficking and synaptic strength. To evaluate role of PC-AMPAR signaling in autism, we produced PC-specific Grip1/2 knockout mice by crossing Grip2 conventional and Grip1 conditional KO with L7-Cre driver mice. PCs in the mutant mice showed normal morphology and number, and a lack of Grip1/2 expression. Rodent behavioral testing identified normal ambulation, anxiety, social interaction, and an increase in repetitive self-grooming. Electrophysiology studies revealed normal mEPSCs but an impaired mGluR-LTD at the Parallel Fiber-PC synapses. Immunoblots showed increased expression of mGluR5 and Arc, and enhanced phosphorylation of P38 and AKT in cerebellum of PC-specific Grip1/2 knockout mice. Results indicate that loss of Grip1/2 in PCs contributes to increased repetitive self-grooming, a core autism behavior in mice. Results support a role of AMPAR trafficking defects in PCs and disturbances of mGluR5 signaling in cerebellum in the pathogenesis of repetitive behaviors.

Differential vesicular sorting of AMPA and GABAA receptors.

In mature neurons AMPA receptors cluster at excitatory synapses primarily on dendritic spines, whereas GABAA receptors cluster at inhibitory synapses mainly on the soma and dendritic shafts. The molecular mechanisms underlying the precise sorting of these receptors remain unclear. By directly studying the constitutive exocytic vesicles of AMPA and GABAA receptors in vitro and in vivo, we demonstrate that they are initially sorted into different vesicles in the Golgi apparatus and inserted into distinct domains of the plasma membrane. These insertions are dependent on distinct Rab GTPases and SNARE complexes. The insertion of AMPA receptors requires SNAP25-syntaxin1A/B-VAMP2 complexes, whereas insertion of GABAA receptors relies on SNAP23-syntaxin1A/B-VAMP2 complexes. These SNARE complexes affect surface targeting of AMPA or GABAA receptors and synaptic transmission. Our studies reveal vesicular sorting mechanisms controlling the constitutive exocytosis of AMPA and GABAA receptors, which are critical for the regulation of excitatory and inhibitory responses in neurons.

Enhancing social behavior in an autism spectrum disorder mouse model: investigating the underlying mechanisms of Lactiplantibacillus plantarum intervention.

Autism spectrum disorder (ASD) is a neurodevelopmental disorder affecting over 1% of the global population. Individuals with ASD often exhibit complex behavioral conditions, including significant social difficulties and repetitive behaviors. Moreover, ASD often co-occurs with several other conditions, including intellectual disabilities and anxiety disorders. The etiology of ASD remains largely unknown owing to its complex genetic variations and associated environmental risks. Ultimately, this poses a fundamental challenge for the development of effective ASD treatment strategies. Previously, we demonstrated that daily supplementation with the probiotic Lactiplantibacillus plantarum PS128 (PS128) alleviates ASD symptoms in children. However, the mechanism underlying this improvement in ASD-associated behaviors remains unclear. Here, we used a well-established ASD mouse model, induced by prenatal exposure to valproic acid (VPA), to study the physiological roles of PS128 in vivo. Overall, we showed that PS128 selectively ameliorates behavioral abnormalities in social and spatial memory in VPA-induced ASD mice. Morphological examination of dendritic architecture further revealed that PS128 facilitated the restoration of dendritic arborization and spine density in the hippocampus and prefrontal cortex of ASD mice. Notably, PS128 was crucial for restoring oxytocin levels in the paraventricular nucleus and oxytocin receptor signaling in the hippocampus. Moreover, PS128 alters the gut microbiota composition and increases the abundance of Bifidobacterium spp. and PS128-induced changes in Bifidobacterium abundance positively correlated with PS128-induced behavioral improvements. Together, our results show that PS128 treatment can effectively ameliorate ASD-associated behaviors and reinstate oxytocin levels in VPA-induced mice, thereby providing a promising strategy for the future development of ASD therapeutics.

ICA69 regulates activity-dependent synaptic strengthening and learning and memory.

Long-term potentiation (LTP) is one of the major cellular mechanisms for learning and memory. Activity-dependent increases in surface AMPA receptors (AMPARs) are important for enhanced synaptic efficacy during LTP. Here, we report a novel function of a secretory trafficking protein, ICA69, in AMPAR trafficking, synaptic plasticity, and animal cognition. ICA69 is first identified as a diabetes-associated protein well characterized for its function in the biogenesis of secretory vesicles and trafficking of insulin from ER, Golgi to post-Golgi in pancreatic beta cells. In the brain, ICA69 is found in the AMPAR protein complex through its interaction with PICK1, which binds directly to GluA2 or GluA3 AMPAR subunits. Here, we showed that ICA69 regulates PICK1's distribution in neurons and stability in the mouse hippocampus, which in turn can impact AMPAR function in the brain. Biochemical analysis of postsynaptic density (PSD) proteins from hippocampi of mice lacking ICA69 (Ica1 knockout) and their wild-type littermates revealed comparable AMPAR protein levels. Electrophysiological recording and morphological analysis of CA1 pyramidal neurons from Ica1 knockout also showed normal AMPAR-mediated currents and dendrite architecture, indicating that ICA69 does not regulate synaptic AMPAR function and neuron morphology at the basal state. However, genetic deletion of ICA69 in mice selectively impairs NMDA receptor (NMDAR)-dependent LTP but not LTD at Schaffer collateral to CA1 synapses, which correlates with behavioral deficits in tests of spatial and associative learning and memory. Together, we identified a critical and selective role of ICA69 in LTP, linking ICA69-mediated synaptic strengthening to hippocampus-dependent learning and memory.

radish encodes a phospholipase-A2 and defines a neural circuit involved in anesthesia-resistant memory.

BACKGROUND: In both vertebrate and invertebrate animals, anesthetic agents cause retrograde amnesia for recently experienced events. In contrast, older memories are resistant to the same treatments. In Drosophila, anesthesia-resistant memory (ARM) and long-term memory (LTM) are genetically distinct forms of long-lasting memory that exist in parallel for at least a day after training. ARM is disrupted in radish mutants but is normal in transgenic flies overexpressing a CREB repressor transgene. In contrast, LTM is normal in radish mutants but is disrupted in CREB repressor transgenic flies. To date, nothing is known about the molecular, genetic, or cell biological pathways underlying ARM. RESULTS: Here, we report the molecular identification of radish as a phospholipase-A2, providing the first clue about signaling pathways underlying ARM in any animal. An enhancer-trap allele of radish (C133) reveals expression in a novel anatomical pathway. Transgenic expression of PLA2 under control of C133 restores normal levels of ARM to radish mutants, whereas transient disruption of neural activity in C133 neurons inhibits memory retention. Notably, expression of C133 is not in mushroom bodies, the primary anatomical focus of olfactory memory research in Drosophila. CONCLUSIONS: Identification of radish as a phospholipase-A2 and the neural expression pattern of an enhancer-trap allele significantly broaden our understanding of the biochemistry and anatomy underlying olfactory memory in Drosophila.

Mice lacking GRIP1/2 show increased social interactions and enhanced phosphorylation at GluA2-S880.

Glutamate receptor interacting proteins 1 and 2 (GRIP1/2) play an important role in regulating synaptic trafficking of AMPA receptor 2/3 (GluA2/3) and synaptic strength. Gain-of-function GRIP1 mutations are implicated in social behavioral deficits in autism. To study mechanisms of Grip1/2-mediated AMPA signaling in the regulation of social behaviors, we performed social behavioral testing on neuron-specific Grip1/2-double knockout (DKO) and wild type (WT) mice that are matched for age, sex, and strain background. We determined the expression profile of key signaling proteins in AMPAR, mGluR, mTOR, and GABA pathways in frontal cortex, striatum, and cerebellum of DKO mice. Compared to WT mice, DKO mice show increased sociability in a modified three-chamber social behavioral test [mean±sem for interaction time in seconds; WT: 44.0±5.0; n=10; DKO: 81.0±9.0; n=9; two factor repeated measures ANOVA: F(1,37)=14.45; p<0.01 and planned t-test; p<0.01] and in a dyadic male-male social interaction test (mean±sem for total time in seconds: sniffing, WT-WT, 18.9±1.1; WT-DKO, 42.5±2.1; t-test: p<0.001; following, WT-WT, 7.7±0.72; WT-DKO,14.4±1.8; t-test: p<0.001). Immunoblot studies identified an increase in phosphorylation at GluA2-Serine 880 (GluA2-pS880) in frontal cortex (mean±sem; WT: 0.69±0.06, n=5; DKO: 0.96±0.06, n=6; t-test; p<0.05) and reduced GABAß3 expression in striatum (mean±sem; WT: 1.16±0.04, n=4; DKO: 0.95±0.06, n=4; t-test; p<0.05) in DKO mice. GluA2-S880 phosphorylation is known to regulate GluA2synaptic recycling, AMPA signaling strength and plasticity. GABAß3 has been implicated in the etiology and pathogenesis in autism. These data support an important role of Grip1/2-mediated AMPA signaling in regulating social behaviors and disturbance of glutamate- and GABA-signaling in specialized brain regions in autism-related social behavioral deficits.

GRASP1 Regulates Synaptic Plasticity and Learning through Endosomal Recycling of AMPA Receptors.

Learning depends on experience-dependent modification of synaptic efficacy and neuronal connectivity in the brain. We provide direct evidence for physiological roles of the recycling endosome protein GRASP1 in glutamatergic synapse function and animal behavior. Mice lacking GRASP1 showed abnormal excitatory synapse number, synaptic plasticity, and hippocampal-dependent learning and memory due to a failure in learning-induced synaptic AMPAR incorporation. We identified two GRASP1 point mutations from intellectual disability (ID) patients that showed convergent disruptive effects on AMPAR recycling and glutamate uncaging-induced structural and functional plasticity. Wild-type GRASP1, but not ID mutants, rescued spine loss in hippocampal CA1 neurons in Grasp1 knockout mice. Together, these results demonstrate a requirement for normal recycling endosome function in AMPAR-dependent synaptic function and neuronal connectivity in vivo, and suggest a potential role for GRASP1 in the pathophysiology of human cognitive disorders.

Neuropilin-2/PlexinA3 Receptors Associate with GluA1 and Mediate Sema3F-Dependent Homeostatic Scaling in Cortical Neurons.

Regulation of AMPA-type glutamate receptor (AMPAR) number at synapses is a major mechanism for controlling synaptic strength during homeostatic scaling in response to global changes in neural activity. We show that the secreted guidance cue semaphorin 3F (Sema3F) and its neuropilin-2 (Npn-2)/plexinA3 (PlexA3) holoreceptor mediate homeostatic plasticity in cortical neurons. Sema3F-Npn-2/PlexA3 signaling is essential for cell surface AMPAR homeostatic downscaling in response to an increase in neuronal activity, Npn-2 associates with AMPARs, and Sema3F regulates this interaction. Therefore, Sema3F-Npn-2/PlexA3 signaling controls both synapse development and synaptic plasticity.

Palmitoylation by DHHC5/8 targets GRIP1 to dendritic endosomes to regulate AMPA-R trafficking.

Palmitoylation, a key regulatory mechanism controlling protein targeting, is catalyzed by DHHC-family palmitoyl acyltransferases (PATs). Impaired PAT activity is linked to neurodevelopmental and neuropsychiatric disorders, suggesting critical roles for palmitoylation in neuronal function. However, few substrates for specific PATs are known, and functional consequences of palmitoylation events are frequently uncharacterized. Here, we identify the closely related PATs DHHC5 and DHHC8 as specific regulators of the PDZ domain protein GRIP1b. Binding, palmitoylation, and dendritic targeting of GRIP1b require a PDZ ligand unique to DHHC5/8. Palmitoylated GRIP1b is targeted to trafficking endosomes and may link endosomes to kinesin motors. Consistent with this trafficking role, GRIP1b's palmitoylation turnover rate approaches the highest of all reported proteins, and palmitoylation increases GRIP1b's ability to accelerate AMPA-R recycling. To our knowledge, these findings identify the first neuronal DHHC5/8 substrate, define novel mechanisms controlling palmitoylation specificity, and suggest further links between dysregulated palmitoylation and neuropathological conditions.

Insulin receptor signaling in the development of neuronal structure and function.

Sensory experience plays a crucial role in regulating neuronal shape and in developing synaptic contacts during brain formation. These features are required for a neuron to receive, integrate, and transmit signals within the neuronal network so that animals can adapt to the constant changing environment. Insulin receptor signaling, which has been extensively studied in peripheral organ systems such as liver, muscle and adipocyte, has recently been shown to play important roles in the central nervous system. Here we review the current understanding of the underlying mechanisms that regulate structural and functional aspects of circuit development, particularly with respect to the role of insulin receptor signaling in synaptic function and the development of dendritic arbor morphology. The potential link between insulin receptor signaling malfunction and neurological disorders will also be discussed.

Membrane targeted horseradish peroxidase as a marker for correlative fluorescence and electron microscopy studies.

Synaptic dynamics and reorganization are fundamental features of synaptic plasticity both during synaptic circuit development and in the mature CNS underlying learning, memory, and experience-dependent circuit rearrangements. Combining in vivo time-lapse fluorescence imaging and retrospective electron microscopic analysis provides a powerful technique to decipher the rules governing dynamics of neuronal structure and synaptic connections. Here we have generated a membrane-targeted horseradish peroxidase (mHRP) that allows identification of transfected cells without obscuring the intracellular ultrastructure or organelles and in particular allows identification of synaptic sites using electron microscopy. The expression of mHRP does not affect dendritic arbor growth or dynamics of transfected neurons. Co-expression of EGFP and mHRP was used to study neuronal morphology at both the light and electron microscopic levels. mHRP expression greatly facilitates 3D reconstruction based on serial EM sections. We expect this reagent will be valuable for studying the mechanisms that guide construction of neuronal networks.

Co-expression of Argonaute2 Enhances Short Hairpin RNA-induced RNA Interference in Xenopus CNS Neurons In Vivo.

RNA interference (RNAi) is an evolutionarily conserved mechanism for sequence-specific gene silencing. Recent advances in our understanding of RNAi machinery make it possible to reduce protein expression by introducing short hairpin RNA (shRNA) into cells of many systems, however, the efficacy of RNAi-mediated protein knockdown can be quite variable, especially in intact animals, and this limits its application. We built adaptable molecular tools, pSilencer (pSi) and pReporter (pRe) constructs, to evaluate the impact of different promoters, shRNA structures and overexpression of Ago2, the key enzyme in the RNA-induced silencing complex, on the efficiency of RNAi. The magnitude of RNAi knockdown was evaluated in cultured cells and intact animals by comparing fluorescence intensity levels of GFP, the RNAi target, relative to mCherry, which was not targeted. Co-expression of human Ago2 with shRNA significantly enhanced efficiency of GFP knockdown in cell lines and in neurons of intact Xenopus tadpoles. Human H1- and U6-promotors alone or the U6-promotor with an enhancer element were equally effective at driving GFP knockdown. shRNA derived from the microRNA-30 design (shRNA(mir30)) enhanced the efficiency of GFP knockdown. Expressing pSi containing Ago2 with shRNA increased knockdown efficiency of an endogenous neuronal protein, the GluR2 subunit of the AMPA receptor, functionally accessed by recording AMPA receptor-mediated spontaneous synaptic currents in Xenopus CNS neurons. Our data suggest that co-expression of Ago2 and shRNA is a simple method to enhance RNAi in intact animals. While morpholino antisense knockdown is effective in Xenopus and Zebrafish, a principle advantage of the RNAi method is the possibility of spatial and temporal control of protein knockdown by use of cell type specific and regulatable pol II promoters to drive shRNA and Ago2. This should extend the application of RNAi to study gene function of intact brain circuits.

Insulin receptor signaling regulates synapse number, dendritic plasticity, and circuit function in vivo.

Insulin receptor signaling has been postulated to play a role in synaptic plasticity; however, the function of the insulin receptor in CNS is not clear. To test whether insulin receptor signaling affects visual system function, we recorded light-evoked responses in optic tectal neurons in living Xenopus tadpoles. Tectal neurons transfected with dominant-negative insulin receptor (dnIR), which reduces insulin receptor phosphorylation, or morpholino against insulin receptor, which reduces total insulin receptor protein level, have significantly smaller light-evoked responses than controls. dnIR-expressing neurons have reduced synapse density as assessed by EM, decreased AMPA mEPSC frequency, and altered experience-dependent dendritic arbor structural plasticity, although synaptic vesicle release probability, assessed by paired-pulse responses, synapse maturation, assessed by AMPA/NMDA ratio and ultrastructural criteria, are unaffected by dnIR expression. These data indicate that insulin receptor signaling regulates circuit function and plasticity by controlling synapse density.

In vivo single-cell electroporation for transfer of DNA and macromolecules.

Single-cell electroporation allows transfection of plasmid DNA or macromolecules into individual living cells using modified patch electrodes and common electrophysiological equipment. This protocol is optimized for rapid in vivo electroporation of Xenopus laevis tadpole brains with DNA, dextrans, morpholinos and combinations thereof. Experienced users can electroporate roughly 40 tadpoles per hour. The technique can be adapted for use with other charged transfer materials and in other systems and tissues where cells can be targeted with a micropipette. Under visual guidance, an electrode filled with transfer material is placed in a cell body-rich area of the tadpole brain and a train of voltage pulses applied, which electroporates a nearby cell. We show examples of successfully electroporated single cells, instances of common problems and troubleshooting suggestions. Single-cell electroporation is an affordable method to fluorescently label and genetically manipulate individual cells. This powerful technique enables observation of single cells in an otherwise normal environment.

Insect NMDA receptors mediate juvenile hormone biosynthesis.

In vertebrates, the N-methyl-D-aspartate subtype of glutamate receptors (NMDAR) appears to play a role in neuronal development, synaptic plasticity, memory formation, and pituitary activity. However, functional NMDAR have not yet been characterized in insects. We have now demonstrated immunohistochemically glutamatergic nerve terminals in the corpora allata of an adult female cockroach, Diploptera punctata. Cockroach corpus allatum (CA) cells, exposed to NMDA in vitro, exhibited elevated cytosolic [Ca(2+)], but not in culture medium nominally free of calcium or containing NMDAR-specific channel blockers: MK-801 and Mg(2+). Sensitivity of cockroach corpora allata to NMDA changed cyclically during the ovarian cycle. Highly active glands of 4-day-old mated females, exposed to 3 microM NMDA, produced 70% more juvenile hormone (JH) in vitro, but the relatively inactive glands of 8-day-old mated females showed little response to the agonist. The stimulatory effect of NMDA was eliminated by augmenting the culture medium with MK-801, conantokin, or high Mg(2+). Having obtained substantive evidence of functioning NMDAR in insect corpora allata, we used reverse transcription PCR to demonstrate two mRNA transcripts, DNMDAR1 and DNMDAR2, in the ring gland and brain of last-instar Drosophila melanogaster. Immunohistochemical labeling, using mouse monoclonal antibody against rat NMDAR1, showed that only one of the three types of endocrine cells in the ring gland, CA cells, expressed rat NMDAR1-like immunoreactive protein. This antibody also labeled two brain neurons in the lateral protocerebrum, one neuron per brain hemisphere. Finally, we used the same primers for DNMDAR1 to demonstrate a fragment of putative NMDA receptor in the corpora allata of Diploptera punctata. Our results suggest that the NMDAR has a role in regulating JH synthesis and that ionotropic-subtype glutamate receptors became specialized early in animal evolution.