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Molecular Autism Apr 2024Glutamatergic synapse dysfunction is believed to underlie the development of Autism Spectrum Disorder (ASD) and Intellectual Disability (ID) in many individuals....
BACKGROUND
Glutamatergic synapse dysfunction is believed to underlie the development of Autism Spectrum Disorder (ASD) and Intellectual Disability (ID) in many individuals. However, identification of genetic markers that contribute to synaptic dysfunction in these individuals is notoriously difficult. Based on genomic analysis, structural modeling, and functional data, we recently established the involvement of the TRIO-RAC1 pathway in ASD and ID. Furthermore, we identified a pathological de novo missense mutation hotspot in TRIO's GEF1 domain. ASD/ID-related missense mutations within this domain compromise glutamatergic synapse function and likely contribute to the development of ASD/ID. The number of ASD/ID cases with mutations identified within TRIO's GEF1 domain is increasing. However, tools for accurately predicting whether such mutations are detrimental to protein function are lacking.
METHODS
Here we deployed advanced protein structural modeling techniques to predict potential de novo pathogenic and benign mutations within TRIO's GEF1 domain. Mutant TRIO-9 constructs were generated and expressed in CA1 pyramidal neurons of organotypic cultured hippocampal slices. AMPA receptor-mediated postsynaptic currents were examined in these neurons using dual whole-cell patch clamp electrophysiology. We also validated these findings using orthogonal co-immunoprecipitation and fluorescence lifetime imaging (FLIM-FRET) experiments to assay TRIO mutant overexpression effects on TRIO-RAC1 binding and on RAC1 activity in HEK293/T cells.
RESULTS
Missense mutations in TRIO's GEF1 domain that were predicted to disrupt TRIO-RAC1 binding or stability were tested experimentally and found to greatly impair TRIO-9's influence on glutamatergic synapse function. In contrast, missense mutations in TRIO's GEF1 domain that were predicted to have minimal effect on TRIO-RAC1 binding or stability did not impair TRIO-9's influence on glutamatergic synapse function in our experimental assays. In orthogonal assays, we find most of the mutations predicted to disrupt binding display loss of function but mutants predicted to disrupt stability do not reflect our results from neuronal electrophysiological data.
LIMITATIONS
We present a method to predict missense mutations in TRIO's GEF1 domain that may compromise TRIO function and test for effects in a limited number of assays. Possible limitations arising from the model systems employed here can be addressed in future studies. Our method does not provide evidence for whether these mutations confer ASD/ID risk or the likelihood that such mutations will result in the development of ASD/ID.
CONCLUSIONS
Here we show that a combination of structure-based computational predictions and experimental validation can be employed to reliably predict whether missense mutations in the human TRIO gene impede TRIO protein function and compromise TRIO's role in glutamatergic synapse regulation. With the growing accessibility of genome sequencing, the use of such tools in the accurate identification of pathological mutations will be instrumental in diagnostics of ASD/ID.
Topics: Humans; Autism Spectrum Disorder; HEK293 Cells; Intellectual Disability; Mutation; Mutation, Missense; Neurons
PubMed: 38566250
DOI: 10.1186/s13229-024-00590-9 -
Molecular Brain Apr 2024Synaptopodin (SP), an actin-associated protein found in telencephalic neurons, affects activity-dependant synaptic plasticity and dynamic changes of dendritic spines....
Synaptopodin (SP), an actin-associated protein found in telencephalic neurons, affects activity-dependant synaptic plasticity and dynamic changes of dendritic spines. While being required for long-term depression (LTD) mediated by metabotropic glutamate receptor (mGluR-LTD), little is known about its role in other forms of LTD induced by low frequency stimulation (LFS-LTD) or spike-timing dependent plasticity (STDP). Using electrophysiology in ex vivo hippocampal slices from SP-deficient mice (SPKO), we show that absence of SP is associated with a deficit of LTD at Sc-CA1 synapses induced by LFS-LTD and STDP. As LTD is known to require AMPA- receptors internalization and IP3-receptors calcium signaling, we tested by western blotting and immunochemistry if there were changes in their expression which we found to be reduced. While we were not able to induce LTD, long-term potentiation (LTP), albeit diminished in SPKO, can be recovered by using a stronger stimulation protocol. In SPKO we found no differences in NMDAR, which are the primary site of calcium signalling to induce LTP. Our study shows, for the first time, the key role of the requirement of SP to allow induction of activity-dependant LTD at Sc-CA1 synapses.
Topics: Animals; Mice; Depression; Hippocampus; Long-Term Potentiation; Long-Term Synaptic Depression; Neuronal Plasticity; Schaffer Collaterals; Synapses
PubMed: 38566234
DOI: 10.1186/s13041-024-01089-3 -
BMC Psychiatry Apr 2024Glutamatergic function abnormalities have been implicated in the etiology of treatment-resistant schizophrenia (TRS), and the efficacy of clozapine may be attributed to...
BACKGROUND
Glutamatergic function abnormalities have been implicated in the etiology of treatment-resistant schizophrenia (TRS), and the efficacy of clozapine may be attributed to its impact on the glutamate system. Recently, evidence has emerged suggesting the involvement of immune processes and increased prevalence of antineuronal antibodies in TRS. This current study aimed to investigate the levels of multiple anti-glutamate receptor antibodies in TRS and explore the effects of clozapine on these antibody levels.
METHODS
Enzyme linked immunosorbent assay (ELISA) was used to measure and compare the levels of anti-glutamate receptor antibodies (NMDAR, AMPAR, mGlur3, mGluR5) in clozapine-treated TRS patients (TRS-C, n = 37), clozapine-naïve TRS patients (TRS-NC, n = 39), and non-TRS patients (nTRS, n = 35). Clinical symptom severity was assessed using the Positive and Negative Symptom Scale (PANSS), while cognitive function was evaluated using the MATRICS Consensus Cognitive Battery (MCCB).
RESULT
The levels of all four glutamate receptor antibodies in TRS-NC were significantly higher than those in nTRS (p < 0.001) and in TRS-C (p < 0.001), and the antibody levels in TRS-C were comparable to those in nTRS. However, no significant associations were observed between antibody levels and symptom severity or cognitive function across all three groups after FDR correction.
CONCLUSION
Our findings suggest that TRS may related to increased anti-glutamate receptor antibody levels and provide further evidence that glutamatergic dysfunction and immune processes may contribute to the pathogenesis of TRS. The impact of clozapine on anti-glutamate receptor antibody levels may be a pharmacological mechanism underlying its therapeutic effects.
Topics: Humans; Clozapine; Schizophrenia; Schizophrenia, Treatment-Resistant; Receptors, Glutamate; Glutamic Acid; Antipsychotic Agents
PubMed: 38566016
DOI: 10.1186/s12888-024-05689-0 -
Cell Reports Apr 2024Type I spiral ganglion neurons (SGNs) convey sound information to the central auditory pathway by forming synapses with inner hair cells (IHCs) in the mammalian cochlea....
Type I spiral ganglion neurons (SGNs) convey sound information to the central auditory pathway by forming synapses with inner hair cells (IHCs) in the mammalian cochlea. The molecular mechanisms regulating the formation of the post-synaptic density (PSD) in the SGN afferent terminals are still unclear. Here, we demonstrate that brain-specific angiogenesis inhibitor 1 (BAI1) is required for the clustering of AMPA receptors GluR2-4 (glutamate receptors 2-4) at the PSD. Adult Bai1-deficient mice have functional IHCs but fail to transmit information to the SGNs, leading to highly raised hearing thresholds. Despite the almost complete absence of AMPA receptor subunits, the SGN fibers innervating the IHCs do not degenerate. Furthermore, we show that AMPA receptors are still expressed in the cochlea of Bai1-deficient mice, highlighting a role for BAI1 in trafficking or anchoring GluR2-4 to the PSDs. These findings identify molecular and functional mechanisms required for sound encoding at cochlear ribbon synapses.
Topics: Animals; Receptors, AMPA; Mice; Spiral Ganglion; Hearing; Cochlea; Post-Synaptic Density; Mice, Knockout; Hair Cells, Auditory, Inner; Mice, Inbred C57BL; Synapses; Receptors, G-Protein-Coupled
PubMed: 38564333
DOI: 10.1016/j.celrep.2024.114025 -
BioRxiv : the Preprint Server For... Mar 2024WWC2 (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...
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 R incorporation into the plasma membrane and regulates HAP1 and GRIP1, which form a complex promoting GABA 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 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 R membrane expression.
PubMed: 38559047
DOI: 10.1101/2024.03.11.584487 -
BioRxiv : the Preprint Server For... Mar 2024The human memory-associated protein KIBRA regulates synaptic plasticity and trafficking of AMPA-type glutamate receptors, and is implicated in multiple neuropsychiatric...
The human memory-associated protein KIBRA regulates synaptic plasticity and trafficking of AMPA-type glutamate receptors, and is implicated in multiple neuropsychiatric and cognitive disorders. How KIBRA forms complexes with and regulates AMPA receptors remains unclear. Here, we show that KIBRA does not interact directly with the AMPA receptor subunit GluA2, but that PICK1, a key regulator of AMPA receptor trafficking, can serve as a bridge between KIBRA and GluA2. We identified structural determinants of KIBRA-PICK1-AMPAR complexes by investigating interactions and cellular expression patterns of different combinations of KIBRA and PICK1 domain mutants. We find that the PICK1 BAR domain, a coiled-coil structure, is sufficient for interaction with KIBRA, whereas mutation of the BAR domain disrupts KIBRA-PICK1-GluA2 complex formation. In addition, KIBRA recruits PICK1 into large supramolecular complexes, a process which requires KIBRA coiled-coil domains. These findings reveal molecular mechanisms by which KIBRA can organize key synaptic signaling complexes.
PubMed: 38558978
DOI: 10.1101/2024.03.12.584494 -
The Journal of Biological Chemistry May 2024Tauopathies are neurodegenerative disorders characterized by the deposition of aggregates of the microtubule-associated protein tau, a main component of neurofibrillary...
Tauopathies are neurodegenerative disorders characterized by the deposition of aggregates of the microtubule-associated protein tau, a main component of neurofibrillary tangles. Alzheimer's disease (AD) is the most common type of tauopathy and dementia, with amyloid-beta pathology as an additional hallmark feature of the disease. Besides its role in stabilizing microtubules, tau is localized at postsynaptic sites and can regulate synaptic plasticity. The activity-regulated cytoskeleton-associated protein (Arc) is an immediate early gene that plays a key role in synaptic plasticity, learning, and memory. Arc has been implicated in AD pathogenesis and regulates the release of amyloid-beta. We found that decreased Arc levels correlate with AD status and disease severity. Importantly, Arc protein was upregulated in the hippocampus of Tau KO mice and dendrites of Tau KO primary hippocampal neurons. Overexpression of tau decreased Arc stability in an activity-dependent manner, exclusively in neuronal dendrites, which was coupled to an increase in the expression of dendritic and somatic surface GluA1-containing α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors. The tau-dependent decrease in Arc was found to be proteasome-sensitive, yet independent of Arc ubiquitination and required the endophilin-binding domain of Arc. Importantly, these effects on Arc stability and GluA1 localization were not observed in the commonly studied tau mutant, P301L. These observations provide a potential molecular basis for synaptic dysfunction mediated through the accumulation of tau in dendrites. Our findings confirm that Arc is misregulated in AD and further show a physiological role for tau in regulating Arc stability and AMPA receptor targeting.
Topics: Animals; Humans; Mice; Alzheimer Disease; Cytoskeletal Proteins; Dendrites; Hippocampus; Mice, Knockout; Nerve Tissue Proteins; Neurons; Proteasome Endopeptidase Complex; Protein Stability; tau Proteins; Ubiquitin; Ubiquitination
PubMed: 38552740
DOI: 10.1016/j.jbc.2024.107237 -
Frontiers in Molecular Neuroscience 2024The nucleus accumbens (NAc) and the ventral tegmental area (VTA) are integral brain regions involved in reward processing and motivation, including responses to drugs of...
The nucleus accumbens (NAc) and the ventral tegmental area (VTA) are integral brain regions involved in reward processing and motivation, including responses to drugs of abuse. Previously, we have demonstrated that activation of NAc-VTA afferents during the acquisition of cocaine conditioned place preference (CPP) reduces the rewarding properties of cocaine and diminished the activity of VTA dopamine neurons. In the current study, we examined the impact of enhancing these inhibitory inputs on molecular changes and neurotransmission associated with cocaine exposure. Our results unveiled significant reductions in extracellular signal-regulated kinase (ERK) levels in the VTA and medial prefrontal cortex (mPFC) of both cocaine-treated groups compared with the saline control group. Furthermore, optic stimulation of NAc-VTA inputs during cocaine exposure decreased the expression of GluA1 subunit of AMPA receptor in the VTA and mPFC. Notably, in the NAc, cocaine exposure paired with optic stimulation increased ERK levels and reduced GluA1 phosphorylation at Ser845 as compared with all other groups. Additionally, both cocaine-treated groups exhibited decreased levels of GluA1 phosphorylation at Ser831 in the NAc compared with the saline control group. Moreover, cocaine exposure led to reduced ERK, GluA1, and GluA1 phosphorylation at Ser845 and Ser831 in the mPFC. Augmentation of GABAergic tone from the NAc during cocaine conditioning mitigated changes in GluA1 phosphorylation at Ser845 in the mPFC but reduced ERK, GluA1, and GluA1 phosphorylation at Ser831 compared with the saline control group. Interestingly, enhancing GABAergic tone during saline conditioning decreased GluA1 phosphorylation at Ser831 compared with the saline control group in the mPFC. Our findings highlight the influence of modulating inhibitory inputs from the NAc to the VTA on molecular signaling and glutamatergic neurotransmission in cocaine-exposed animals. Activation of these inhibitory inputs during cocaine conditioning induced alterations in key signaling molecules and AMPA receptor, providing valuable insights into the neurobiological mechanisms underlying cocaine reward and cocaine use disorder. Further exploration of these pathways may offer potential therapeutic targets for the treatment of substance use disorder.
PubMed: 38528956
DOI: 10.3389/fnmol.2024.1271654 -
ENeuro Apr 2024As the global elderly population grows, age-related cognitive decline is becoming an increasingly significant healthcare issue, often leading to various neuropsychiatric...
As the global elderly population grows, age-related cognitive decline is becoming an increasingly significant healthcare issue, often leading to various neuropsychiatric disorders. Among the many molecular players involved in memory, AMPA-type glutamate receptors are known to regulate learning and memory, but how their dynamics change with age and affect memory decline is not well understood. Here, we examined the in vivo properties of the AMPA-type glutamate receptor GLR-1 in the AVA interneuron of the nervous system during physiological aging. We found that both total and membrane-bound GLR-1 receptor levels decrease with age in wild-type worms, regardless of their location along the axon. Using fluorescence recovery after photobleaching, we also demonstrated that a reduction in GLR-1 abundance correlates with decreased local, synaptic GLR-1 receptor dynamics. Importantly, we found that reduced GLR-1 levels strongly correlate with the age-related decline in short-term associative memory. Genetic manipulation of GLR-1 stability, by either deleting or expressing a ubiquitination-defective GLR-1 (4KR) variant, prevented this age-related reduction in receptor abundance and improved the short-term memory performance in older animals, which reached performance levels similar to those of young animals. Overall, our data indicate that AMPA-type glutamate receptor abundance and dynamics are key factors in maintaining memory function and that changes in these parameters are linked to age-dependent short-term memory decline.
Topics: Animals; alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid; Caenorhabditis elegans; Caenorhabditis elegans Proteins; Memory, Short-Term; Mutation; Receptors, AMPA; Receptors, Glutamate
PubMed: 38519128
DOI: 10.1523/ENEURO.0420-23.2024 -
Communications Biology Mar 2024The scaffolding A-kinase anchoring protein 150 (AKAP150) is critically involved in kinase and phosphatase regulation of synaptic transmission/plasticity, and neuronal...
The scaffolding A-kinase anchoring protein 150 (AKAP150) is critically involved in kinase and phosphatase regulation of synaptic transmission/plasticity, and neuronal excitability. Emerging evidence also suggests that AKAP150 signaling may play a key role in brain's processing of rewarding/aversive experiences, however its role in the lateral habenula (LHb, as an important brain reward circuitry) is completely unknown. Using whole cell patch clamp recordings in LHb of male wildtype and ΔPKA knockin mice (with deficiency in AKAP-anchoring of PKA), here we show that the genetic disruption of PKA anchoring to AKAP150 significantly reduces AMPA receptor-mediated glutamatergic transmission and prevents the induction of presynaptic endocannabinoid-mediated long-term depression in LHb neurons. Moreover, ΔPKA mutation potentiates GABA receptor-mediated inhibitory transmission while increasing LHb intrinsic excitability through suppression of medium afterhyperpolarizations. ΔPKA mutation-induced suppression of medium afterhyperpolarizations also blunts the synaptic and neuroexcitatory actions of the stress neuromodulator, corticotropin releasing factor (CRF), in mouse LHb. Altogether, our data suggest that AKAP150 complex signaling plays a critical role in regulation of AMPA and GABA receptor synaptic strength, glutamatergic plasticity and CRF neuromodulation possibly through AMPA receptor and potassium channel trafficking and endocannabinoid signaling within the LHb.
Topics: Animals; Male; Mice; A Kinase Anchor Proteins; Corticotropin-Releasing Hormone; Endocannabinoids; Habenula; Neuronal Plasticity; Neurons; Receptors, AMPA; Receptors, GABA-A; Synaptic Transmission
PubMed: 38509283
DOI: 10.1038/s42003-024-06041-8