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1.
bioRxiv ; 2023 May 26.
Article in English | MEDLINE | ID: mdl-37720016

ABSTRACT

Neurodevelopmental disorders are frequently linked to mutations in synaptic organizing molecules. MAM domain containing glycosylphosphatidylinositol anchor 1 and 2 (MDGA1 and MDGA2) are a family of synaptic organizers suggested to play an unusual role as synaptic repressors, but studies offer conflicting evidence for their localization. Using epitope-tagged MDGA1 and MDGA2 knock-in mice, we found that native MDGAs are expressed throughout the brain, peaking early in postnatal development. Surprisingly, endogenous MDGA1 was enriched at excitatory, but not inhibitory, synapses. Both shRNA knockdown and CRISPR/Cas9 knockout of MDGA1 resulted in cell-autonomous, specific impairment of AMPA receptor-mediated synaptic transmission, without affecting GABAergic transmission. Conversely, MDGA2 knockdown/knockout selectively depressed NMDA receptor-mediated transmission but enhanced inhibitory transmission. Our results establish that MDGA2 acts as a synaptic repressor, but only at inhibitory synapses, whereas both MDGAs are required for excitatory transmission. This nonoverlapping division of labor between two highly conserved synaptic proteins is unprecedented.

2.
Neuron ; 106(5): 759-768.e7, 2020 06 03.
Article in English | MEDLINE | ID: mdl-32243781

ABSTRACT

Autism spectrum disorder (ASD) is more prevalent in males; however, the etiology for this sex bias is not well understood. Many mutations on X-linked cell adhesion molecule NLGN4X result in ASD or intellectual disability. NLGN4X is part of an X-Y pair, with NLGN4Y sharing ∼97% sequence homology. Using biochemistry, electrophysiology, and imaging, we show that NLGN4Y displays severe deficits in maturation, surface expression, and synaptogenesis regulated by one amino acid difference with NLGN4X. Furthermore, we identify a cluster of ASD-associated mutations surrounding the critical amino acid in NLGN4X, and these mutations phenocopy NLGN4Y. We show that NLGN4Y cannot compensate for the functional deficits observed in ASD-associated NLGN4X mutations. Altogether, our data reveal a potential pathogenic mechanism for male bias in NLGN4X-associated ASD.


Subject(s)
Cell Adhesion Molecules, Neuronal/genetics , Chromosomes, Human, X/genetics , Chromosomes, Human, Y/genetics , Neurons/metabolism , Autism Spectrum Disorder/genetics , Cell Adhesion Molecules, Neuronal/metabolism , Female , Genetic Predisposition to Disease , HEK293 Cells , Humans , Induced Pluripotent Stem Cells , Intellectual Disability/genetics , Male , Mutation , Protein Transport/genetics
3.
Cell Rep ; 29(10): 2944-2952.e5, 2019 12 03.
Article in English | MEDLINE | ID: mdl-31801062

ABSTRACT

The RhoGEFs Kalirin-7 and Trio are regulators of synaptic plasticity, and their dysregulation is associated with a range of neurodevelopmental and neurodegenerative disorders. Although studies have implicated both Kalirin and Trio in certain diseases, such as tauopathies, they remarkably differ in their association with other disorders. Using unbiased proteomics, we identified interactomes of Kalirin-7 and Trio to ascertain distinct protein association networks associated with their respective function and revealed groups of proteins that preferentially interact with a particular RhoGEF. In comparison, we find Trio interacts with a range of axon guidance and presynaptic complexes, whereas Kalirin-7 associates with several synaptic adhesion molecules. Specifically, we show Kalirin-7 is an interactor of the cell adhesion molecule neuroligin-1 (NLGN1), and NLGN1-dependent synaptic function is mediated through Kalirin-7 in an interaction-dependent manner. Our data reveal not only the interactomes of two important disease-related proteins, but also provide an intracellular effector of NLGN1 function.


Subject(s)
Cell Adhesion Molecules, Neuronal/metabolism , Guanine Nucleotide Exchange Factors/metabolism , Protein Serine-Threonine Kinases/metabolism , Animals , Cell Line , Female , HEK293 Cells , Humans , Male , Mice , Mice, Inbred C57BL , Rats, Sprague-Dawley , Rho Guanine Nucleotide Exchange Factors/metabolism , Synapses/metabolism
4.
Mol Psychiatry ; 24(10): 1451-1460, 2019 10.
Article in English | MEDLINE | ID: mdl-30824864

ABSTRACT

Ionotropic glutamate delta receptors do not bind glutamate and do not generate ionic current, resulting in difficulty in studying the function and trafficking of these receptors. Here, we utilize chimeric constructs, in which the ligand-binding domain of GluD1 is replaced by that of GluK1, to examine its synaptic trafficking and plasticity. GluD1 trafficked to the synapse, but was incapable of expressing long-term potentiation (LTP). The C-terminal domain (CT) of GluD1 has a classic PDZ-binding motif, which is critical for the synaptic trafficking of other glutamate receptors, but we found that its binding to PSD-95 was very weak, and deleting the PDZ-binding motif failed to alter synaptic trafficking. However, deletion of the entire CT abolished synaptic trafficking, but not surface expression. We found that mutation of threonine (T) T923 to an alanine disrupted synaptic trafficking. Therefore, GluD1 receptors have strikingly different trafficking mechanisms compared with AMPARs. These results highlight the diversity of ionotropic glutamate receptor trafficking rules at a single type of synapse. Since this receptor is genetically associated with schizophrenia, our findings may provide an important clue to understand schizophrenia.


Subject(s)
Glutamate Dehydrogenase/metabolism , Receptors, Glutamate/metabolism , Animals , Carrier Proteins/genetics , Glutamate Dehydrogenase/physiology , Glutamic Acid/metabolism , Hippocampus/metabolism , Long-Term Potentiation , Membrane Proteins/metabolism , Mice , Neuronal Plasticity/physiology , Neurons/metabolism , Patch-Clamp Techniques , Protein Binding , Protein Transport/physiology , Receptors, AMPA/metabolism , Receptors, Glutamate/genetics , Receptors, Opioid, delta/metabolism , Synapses/metabolism
5.
Mol Psychiatry ; 24(1): 145-160, 2019 01.
Article in English | MEDLINE | ID: mdl-30242227

ABSTRACT

The assembly and maintenance of synapses are dynamic processes that require bidirectional contacts between the pre- and postsynaptic structures. A network of adhesion molecules mediate this physical interaction between neurons. How synapses are disassembled and if there are distinct mechanisms that govern the removal of specific adhesion molecules remain unclear. Here, we report isoform-specific proteolytic cleavage of neuroligin-3 in response to synaptic activity and protein kinase C signaling resulting in reduced synapse strength. Although neuroligin-1 and neuroligin-2 are not directly cleaved by this pathway, when heterodimerized with neuroligin-3, they too undergo proteolytic cleavage. Thus protein kinase C-dependent cleavage is mediated through neuroligin-3. Recent studies on glioma implicate the neuroligin-3 ectodomain as a mitogen. Here we demonstrate: (1) there are mechanisms governing specific adhesion molecule remodeling; (2) neuroligin-3 is a key regulator of neuroligin cleavage events; and (3) there are two cleavage pathways; basal and activity-dependent that produce the mitogenic form of neuroligin-3.


Subject(s)
Cell Adhesion Molecules, Neuronal/metabolism , Membrane Proteins/metabolism , Nerve Tissue Proteins/metabolism , Synapses/physiology , Animals , Cell Adhesion/physiology , Cells, Cultured , Female , HEK293 Cells , HeLa Cells , Hippocampus/metabolism , Humans , Male , Mice , Mice, Knockout , Nerve Growth Factors/metabolism , Neuregulin-1/metabolism , Neurons/metabolism , Protein Isoforms , Protein Kinase C/metabolism , Rats , Rats, Sprague-Dawley
6.
Proc Natl Acad Sci U S A ; 115(15): 3948-3953, 2018 04 10.
Article in English | MEDLINE | ID: mdl-29581259

ABSTRACT

Long-term potentiation (LTP) is a persistent strengthening of synaptic transmission in the brain and is arguably the most compelling cellular and molecular model for learning and memory. Previous work found that both AMPA receptors and exogenously expressed kainate receptors are equally capable of expressing LTP, despite their limited homology and their association with distinct auxiliary subunits, indicating that LTP is far more promiscuous than previously thought. What might these two subtypes of glutamate receptor have in common? Using a single-cell molecular replacement strategy, we demonstrate that the AMPA receptor auxiliary subunit TARP γ-8, via its PDZ-binding motif, is indispensable for both basal synaptic transmission and LTP. Remarkably, kainate receptors and their auxiliary subunits Neto proteins share the same requirement of PDZ-binding domains for synaptic trafficking and LTP. Together, these results suggest that a minimal postsynaptic requirement for LTP is the PDZ binding of glutamate receptors/auxiliary subunits to PSD scaffolding proteins.


Subject(s)
Calcium Channels/metabolism , Long-Term Potentiation , Receptors, AMPA/metabolism , Synapses/metabolism , Animals , Calcium Channels/chemistry , Calcium Channels/genetics , Humans , PDZ Domains , Protein Binding , Receptors, AMPA/chemistry , Receptors, AMPA/genetics , Receptors, Kainic Acid/genetics , Receptors, Kainic Acid/metabolism , Synapses/chemistry , Synapses/genetics
7.
Proc Natl Acad Sci U S A ; 114(50): 13266-13271, 2017 12 12.
Article in English | MEDLINE | ID: mdl-29180434

ABSTRACT

Bidirectional scaling of synaptic transmission, expressed as a compensatory change in quantal size following chronic activity perturbation, is a critical effector mechanism underlying homeostatic plasticity in the brain. An emerging model posits that the GluA2 AMPA receptor (AMPAR) subunit may be important for the bidirectional scaling of excitatory transmission; however, whether this subunit plays an obligatory role in synaptic scaling, and the identity of the precise domain(s) involved, remain controversial. We set out to determine the specific AMPAR subunit required for scaling up in CA1 hippocampal pyramidal neurons, and found that the GluA2 subunit is both necessary and sufficient. In addition, our results point to a critical role for a single amino acid within the membrane-proximal region of the GluA2 cytoplasmic tail, and suggest a distinct model for the regulation of AMPAR trafficking in synaptic homeostasis.


Subject(s)
CA1 Region, Hippocampal/metabolism , Receptors, AMPA/metabolism , Synaptic Potentials , Animals , CA1 Region, Hippocampal/cytology , CA1 Region, Hippocampal/physiology , Homeostasis , Mice , Protein Domains , Protein Transport , Pyramidal Cells/metabolism , Pyramidal Cells/physiology , Receptors, AMPA/chemistry , Synapses/metabolism , Synapses/physiology
8.
Trends Neurosci ; 38(8): 496-505, 2015 Aug.
Article in English | MEDLINE | ID: mdl-26209464

ABSTRACT

A fundamental physical interaction exists across the synapse. It is mediated by synaptic adhesion molecules, and is among the earliest and most indispensable of molecular events occurring during synaptogenesis. The regulation of adhesion molecules and their interactions with other synaptic proteins likely affect not only on synapse formation but also on ongoing synaptic function. We review research on one major family of postsynaptic adhesion molecules, neuroligins, which bind to their presynaptic partner neurexin across the synaptic cleft. We move from a structural overview to the broad cellular and synaptic context of neuroligins, intermolecular interactions, and molecular modifications that occur within a synapse. Finally, we examine evidence concerning the physiological functions of neuroligin in a cell and highlight areas requiring further investigation.


Subject(s)
Cell Adhesion Molecules, Neuronal/physiology , Neuronal Plasticity/physiology , Synapses/physiology , Amino Acid Sequence , Animals , Cell Adhesion Molecules, Neuronal/metabolism , Humans , Ligands , Models, Neurological , Molecular Sequence Data , Protein Interaction Domains and Motifs , Protein Multimerization , Synapses/metabolism
9.
Proc Natl Acad Sci U S A ; 112(8): 2551-6, 2015 Feb 24.
Article in English | MEDLINE | ID: mdl-25675530

ABSTRACT

Autism spectrum disorders (ASDs) comprise a highly heritable, multifarious group of neurodevelopmental disorders, which are characterized by repetitive behaviors and impairments in social interactions. Point mutations have been identified in X-linked Neuroligin (NLGN) 3 and 4X genes in patients with ASDs and all of these reside in their extracellular domains except for a single point mutation in the cytoplasmic domain of NLGN4X in which an arginine is mutated to a cysteine (R704C). Here we show that endogenous NLGN4X is robustly phosphorylated by protein kinase C (PKC) at T707, and R704C completely eliminates T707 phosphorylation. Endogenous NLGN4X is intensely phosphorylated on T707 upon PKC stimulation in human neurons. Furthermore, a phospho-mimetic mutation at T707 has a profound effect on NLGN4X-mediated excitatory potentiation. Our results now establish an important interplay between a genetic mutation, a key posttranslational modification, and robust synaptic changes, which can provide insights into the synaptic dysfunction of ASDs.


Subject(s)
Autistic Disorder/genetics , Cell Adhesion Molecules, Neuronal/genetics , Excitatory Postsynaptic Potentials , Mutation/genetics , Protein Kinase C/metabolism , Synapses/metabolism , Amino Acid Sequence , Animals , Cell Adhesion Molecules, Neuronal/chemistry , HEK293 Cells , Humans , Molecular Sequence Data , Mutant Proteins/metabolism , Neurons/metabolism , Phosphorylation , Phosphothreonine/metabolism , Rats, Sprague-Dawley
10.
J Neurosci ; 34(7): 2632-44, 2014 Feb 12.
Article in English | MEDLINE | ID: mdl-24523552

ABSTRACT

The regenerative failure of mammalian optic axons is partly mediated by Socs3-dependent inhibition of Jak/Stat signaling (Smith et al., 2009, 2011). Whether Jak/Stat signaling is part of the normal regenerative response observed in animals that exhibit an intrinsic capacity for optic nerve regeneration, such as zebrafish, remains unknown. Nor is it known whether the repression of regenerative inhibitors, such as Socs3, contributes to the robust regenerative response of zebrafish to optic nerve damage. Here we report that Jak/Stat signaling stimulates optic nerve regeneration in zebrafish. We found that IL-6 family cytokines, acting via Gp130-coupled receptors, stimulate Jak/Stat3 signaling in retinal ganglion cells after optic nerve injury. Among these cytokines, we found that CNTF, IL-11, and Clcf1/Crlf1a can stimulate optic axon regrowth. Surprisingly, optic nerve injury stimulated the expression of Socs3 and Sfpq (splicing factor, proline/glutamine rich) that attenuate optic nerve regeneration. These proteins were induced in a Jak/Stat-dependent manner, stimulated each other's expression and suppressed the expression of regeneration-associated genes. In vivo, the injury-dependent induction of Socs3 and Sfpq inhibits optic nerve regeneration but does not block it. We identified a robust induction of multiple cytokine genes in zebrafish retinal ganglion cells that may contribute to their ability to overcome these inhibitory factors. These studies not only identified mechanisms underlying optic nerve regeneration in fish but also suggest new molecular targets for enhancing optic nerve regeneration in mammals.


Subject(s)
Nerve Regeneration/physiology , Optic Nerve/metabolism , RNA-Binding Proteins/metabolism , STAT Transcription Factors/physiology , Signal Transduction/physiology , Suppressor of Cytokine Signaling Proteins/metabolism , Zebrafish Proteins/metabolism , Zebrafish/metabolism , Animals , Fluorescent Antibody Technique , In Situ Hybridization , Janus Kinases/physiology , PTB-Associated Splicing Factor , Polymerase Chain Reaction , Retinal Ganglion Cells/metabolism , Suppressor of Cytokine Signaling 3 Protein
11.
Nat Neurosci ; 17(1): 56-64, 2014 Jan.
Article in English | MEDLINE | ID: mdl-24336150

ABSTRACT

Neuroligins are postsynaptic cell adhesion molecules that are important for synaptic function through their trans-synaptic interaction with neurexins (NRXNs). The localization and synaptic effects of neuroligin-1 (NL-1, also called NLGN1) are specific to excitatory synapses with the capacity to enhance excitatory synapses dependent on synaptic activity or Ca(2+)/calmodulin kinase II (CaMKII). Here we report that CaMKII robustly phosphorylates the intracellular domain of NL-1. We show that T739 is the dominant CaMKII site on NL-1 and is phosphorylated in response to synaptic activity in cultured rodent neurons and sensory experience in vivo. Furthermore, a phosphodeficient mutant (NL-1 T739A) reduces the basal and activity-driven surface expression of NL-1, leading to a reduction in neuroligin-mediated excitatory synaptic potentiation. To the best of our knowledge, our results are the first to demonstrate a direct functional interaction between CaMKII and NL-1, two primary components of excitatory synapses.


Subject(s)
Calcium-Calmodulin-Dependent Protein Kinase Type 2/metabolism , Cell Adhesion Molecules, Neuronal/metabolism , Excitatory Postsynaptic Potentials/physiology , Neurons/physiology , Synapses/physiology , Animals , Animals, Newborn , Benzylamines/pharmacology , Bicuculline/pharmacology , Calcium-Calmodulin-Dependent Protein Kinase Type 2/genetics , Cell Adhesion Molecules, Neuronal/genetics , Cells, Cultured , Disks Large Homolog 4 Protein , Dose-Response Relationship, Drug , Electric Stimulation , Excitatory Amino Acid Antagonists/pharmacology , Excitatory Postsynaptic Potentials/genetics , Female , GABA Antagonists/pharmacology , Gene Expression Regulation/genetics , Guanylate Kinases/metabolism , Hippocampus/cytology , Humans , Immunoprecipitation , In Vitro Techniques , Luminescent Proteins/genetics , Male , Mass Spectrometry , Membrane Proteins/metabolism , Mice , Mice, Inbred C57BL , MicroRNAs/pharmacology , Mutation/genetics , Neurons/drug effects , Patch-Clamp Techniques , Phosphorylation/genetics , Protein Kinase Inhibitors/pharmacology , Receptors, AMPA/genetics , Sensory Deprivation/physiology , Sequence Analysis, Protein , Statistics, Nonparametric , Sulfonamides/pharmacology , Transfection , Vesicular Glutamate Transport Protein 1/metabolism , Visual Cortex/metabolism
12.
Mol Cell Neurosci ; 43(4): 370-83, 2010 Apr.
Article in English | MEDLINE | ID: mdl-20123021

ABSTRACT

We report that knockdown of the alpha1 tubulin isoform Tuba1a, but not the highly related Tuba1b, dramatically impedes nervous system formation during development and RGC axon regeneration following optic nerve injury in adults. Within the tuba1a promoter, a G/C-rich element was identified that is necessary for tuba1a induction during RGC differentiation and optic axon regeneration. KLF6a and 7a, which we previously reported are essential for optic axon regeneration (Veldman et al., 2007), bind this G/C-rich element and transactivate the tuba1a promoter. In vivo knockdown of KLF6a and 7a attenuate regeneration-dependent activation of the endogenous tuba1a and p27 genes. These results suggest tuba1a expression is necessary for CNS development and regeneration and that KLF6a and 7a mediate their effects, at least in part, via transcriptional control of tuba1a promoter activity.


Subject(s)
Nerve Regeneration/physiology , Nerve Tissue Proteins/metabolism , Optic Nerve Injuries/metabolism , Retina/metabolism , Tubulin/genetics , Zebrafish Proteins/metabolism , Animals , Animals, Genetically Modified , Electrophoretic Mobility Shift Assay , Gene Expression Regulation , Immunohistochemistry , In Situ Hybridization , Nerve Tissue Proteins/genetics , Promoter Regions, Genetic/genetics , RNA, Messenger/genetics , RNA, Messenger/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Tubulin/metabolism , Zebrafish , Zebrafish Proteins/genetics
13.
Dev Biol ; 312(2): 596-612, 2007 Dec 15.
Article in English | MEDLINE | ID: mdl-17949705

ABSTRACT

Unlike mammals, teleost fish are able to mount an efficient and robust regenerative response following optic nerve injury. Although it is clear that changes in gene expression accompany axonal regeneration, the extent of this genomic response is not known. To identify genes involved in successful nerve regeneration, we analyzed gene expression in zebrafish retinal ganglion cells (RGCs) regenerating their axons following optic nerve injury. Microarray analysis of RNA isolated by laser capture microdissection from uninjured and 3-day post-optic nerve injured RGCs identified 347 up-regulated and 29 down-regulated genes. Quantitative RT-PCR and in situ hybridization were used to verify the change in expression of 19 genes in this set. Gene ontological analysis of the data set suggests regenerating neurons up-regulate genes associated with RGC development. However, not all regeneration-associated genes are expressed in differentiating RGCs indicating the regeneration is not simply a recapitulation of development. Knockdown of six highly induced regeneration-associated genes identified two, KLF6a and KLF7a, that together were necessary for robust RGC axon re-growth. These results implicate KLF6a and KLF7a as important mediators of optic nerve regeneration and suggest that not all induced genes are essential to mount a regenerative response.


Subject(s)
Axons/physiology , Gene Expression Regulation , Nerve Regeneration/genetics , Nerve Tissue Proteins/physiology , Optic Nerve/physiology , Retinal Ganglion Cells/metabolism , Zebrafish Proteins/physiology , Animals , High Mobility Group Proteins/genetics , High Mobility Group Proteins/metabolism , Nerve Regeneration/physiology , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/metabolism , Optic Nerve Injuries/genetics , Optic Nerve Injuries/metabolism , SOX Transcription Factors , SOXC Transcription Factors , Zebrafish/genetics , Zebrafish/physiology , Zebrafish Proteins/genetics , Zebrafish Proteins/metabolism
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