MDGAs perform activity-dependent synapse type-specific suppression via distinct extracellular mechanisms.

MDGA (MAM domain containing glycosylphosphatidylinositol anchor) family proteins were previously identified as synaptic suppressive factors. However, various genetic manipulations have yielded often irreconcilable results, precluding precise evaluation of MDGA functions. Here, we found that, in cultured hippocampal neurons, conditional deletion of MDGA1 and MDGA2 causes specific alterations in synapse numbers, basal synaptic transmission, and synaptic strength at GABAergic and glutamatergic synapses, respectively. Moreover, MDGA2 deletion enhanced both N-methyl-D-aspartate (NMDA) receptor- and α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor-mediated postsynaptic responses. Strikingly, ablation of both MDGA1 and MDGA2 abolished the effect of deleting individual MDGAs that is abrogated by chronic blockade of synaptic activity. Molecular replacement experiments further showed that MDGA1 requires the meprin/A5 protein/PTPmu (MAM) domain, whereas MDGA2 acts via neuroligin-dependent and/or MAM domain-dependent pathways to regulate distinct postsynaptic properties. Together, our data demonstrate that MDGA paralogs act as unique negative regulators of activity-dependent postsynaptic organization at distinct synapse types, and cooperatively contribute to adjustment of excitation-inhibition balance.

MDGA1 negatively regulates amyloid precursor protein-mediated synapse inhibition in the hippocampus.

Balanced synaptic inhibition, controlled by multiple synaptic adhesion proteins, is critical for proper brain function. MDGA1 (meprin, A-5 protein, and receptor protein-tyrosine phosphatase mu [MAM] domain-containing glycosylphosphatidylinositol anchor protein 1) suppresses synaptic inhibition in mammalian neurons, yet the molecular mechanisms underlying MDGA1-mediated negative regulation of GABAergic synapses remain unresolved. Here, we show that the MDGA1 MAM domain directly interacts with the extension domain of amyloid precursor protein (APP). Strikingly, MDGA1-mediated synaptic disinhibition requires the MDGA1 MAM domain and is prominent at distal dendrites of hippocampal CA1 pyramidal neurons. Down-regulation of APP in presynaptic GABAergic interneurons specifically suppressed GABAergic, but not glutamatergic, synaptic transmission strength and inputs onto both the somatic and dendritic compartments of hippocampal CA1 pyramidal neurons. Moreover, APP deletion manifested differential effects in somatostatin- and parvalbumin-positive interneurons in the hippocampal CA1, resulting in distinct alterations in inhibitory synapse numbers, transmission, and excitability. The infusion of MDGA1 MAM protein mimicked postsynaptic MDGA1 gain-of-function phenotypes that involve the presence of presynaptic APP. The overexpression of MDGA1 wild type or MAM, but not MAM-deleted MDGA1, in the hippocampal CA1 impaired novel object-recognition memory in mice. Thus, our results establish unique roles of APP-MDGA1 complexes in hippocampal neural circuits, providing unprecedented insight into trans-synaptic mechanisms underlying differential tuning of neuronal compartment-specific synaptic inhibition.

LRRTM3 regulates activity-dependent synchronization of synapse properties in topographically connected hippocampal neural circuits.

Synaptic cell-adhesion molecules (CAMs) organize the architecture and properties of neural circuits. However, whether synaptic CAMs are involved in activity-dependent remodeling of specific neural circuits is incompletely understood. Leucine-rich repeat transmembrane protein 3 (LRRTM3) is required for the excitatory synapse development of hippocampal dentate gyrus (DG) granule neurons. Here, we report that Lrrtm3-deficient mice exhibit selective reductions in excitatory synapse density and synaptic strength in projections involving the medial entorhinal cortex (MEC) and DG granule neurons, accompanied by increased neurotransmitter release and decreased excitability of granule neurons. LRRTM3 deletion significantly reduced excitatory synaptic innervation of hippocampal mossy fibers (Mf) of DG granule neurons onto thorny excrescences in hippocampal CA3 neurons. Moreover, LRRTM3 loss in DG neurons significantly decreased mossy fiber long-term potentiation (Mf-LTP). Remarkably, silencing MEC-DG circuits protected against the decrease in the excitatory synaptic inputs onto DG and CA3 neurons, excitability of DG granule neurons, and Mf-LTP in Lrrtm3-deficient mice. These results suggest that LRRTM3 may be a critical factor in activity-dependent synchronization of the topography of MEC-DG-CA3 excitatory synaptic connections. Collectively, our data propose that LRRTM3 shapes the target-specific structural and functional properties of specific hippocampal circuits.

LAR-RPTPs Directly Interact with Neurexins to Coordinate Bidirectional Assembly of Molecular Machineries.

Neurexins (Nrxns) and LAR-RPTPs (leukocyte common antigen-related protein tyrosine phosphatases) are presynaptic adhesion proteins responsible for organizing presynaptic machineries through interactions with nonoverlapping extracellular ligands. Here, we report that two members of the LAR-RPTP family, PTPσ and PTPδ, are required for the presynaptogenic activity of Nrxns. Intriguingly, Nrxn1 and PTPσ require distinct sets of intracellular proteins for the assembly of specific presynaptic terminals. In addition, Nrxn1α showed robust heparan sulfate (HS)-dependent, high-affinity interactions with Ig domains of PTPσ that were regulated by the splicing status of PTPσ. Furthermore, Nrxn1α WT, but not a Nrxn1α mutant lacking HS moieties (Nrxn1α ΔHS), inhibited postsynapse-inducing activity of PTPσ at excitatory, but not inhibitory, synapses. Similarly, cis expression of Nrxn1α WT, but not Nrxn1α ΔHS, suppressed the PTPσ-mediated maintenance of excitatory postsynaptic specializations in mouse cultured hippocampal neurons. Lastly, genetics analyses using male or female Drosophila Dlar and Dnrx mutant larvae identified epistatic interactions that control synapse formation and synaptic transmission at neuromuscular junctions. Our results suggest a novel synaptogenesis model whereby different presynaptic adhesion molecules combine with distinct regulatory codes to orchestrate specific synaptic adhesion pathways.SIGNIFICANCE STATEMENT We provide evidence supporting the physical interactions of neurexins with leukocyte common-antigen related receptor tyrosine phosphatases (LAR-RPTPs). The availability of heparan sulfates and alternative splicing of LAR-RPTPs regulate the binding affinity of these interactions. A set of intracellular presynaptic proteins is involved in common for Nrxn- and LAR-RPTP-mediated presynaptic assembly. PTPσ triggers glutamatergic and GABAergic postsynaptic differentiation in an alternative splicing-dependent manner, whereas Nrxn1α induces GABAergic postsynaptic differentiation in an alternative splicing-independent manner. Strikingly, Nrxn1α inhibits the glutamatergic postsynapse-inducing activity of PTPσ, suggesting that PTPσ and Nrxn1α might control recruitment of a different pool of postsynaptic machinery. Drosophila orthologs of Nrxns and LAR-RPTPs mediate epistatic interactions in controlling synapse structure and strength at neuromuscular junctions, underscoring the physiological significance in vivo.

Calsyntenin-3 interacts with both α- and ß-neurexins in the regulation of excitatory synaptic innervation in specific Schaffer collateral pathways.

Calsyntenin-3 (Clstn3) is a postsynaptic adhesion molecule that induces presynaptic differentiation via presynaptic neurexins (Nrxns), but whether Nrxns directly bind to Clstn3 has been a matter of debate. Here, using LC-MS/MS-based protein analysis, confocal microscopy, RNAscope assays, and electrophysiological recordings, we show that ß-Nrxns directly interact via their LNS domain with Clstn3 and Clstn3 cadherin domains. Expression of splice site 4 (SS4) insert-positive ß-Nrxn variants, but not insert-negative variants, reversed the impaired Clstn3 synaptogenic activity observed in Nrxn-deficient neurons. Consistently, Clstn3 selectively formed complexes with SS4-positive Nrxns in vivo Neuron-specific Clstn3 deletion caused significant reductions in number of excitatory synaptic inputs. Moreover, expression of Clstn3 cadherin domains in CA1 neurons of Clstn3 conditional knockout mice rescued structural deficits in excitatory synapses, especially within the stratum radiatum layer. Collectively, our results suggest that Clstn3 links to SS4-positive Nrxns to induce presynaptic differentiation and orchestrate excitatory synapse development in specific hippocampal neural circuits, including Schaffer collateral afferents.

Seizure progression triggered by IQSEC3 loss is mitigated by reducing activated microglia in mice.

IQSEC3, a guanine nucleotide exchange factor for ADP-ribosylation factors (ARF-GEFs) is specifically expressed at GABAergic synapses, and its loss increases seizure susceptibility in mice. However, the contribution of microglia to initiation and/or progression of seizures in IQSEC3-deficient mice has not been investigated. In the current study, we show that mice with hippocampal dentate gyrus (DG)-specific IQSEC3 knockdown (KD) exhibit microglial activation and death of DG granule cell. Furthermore, treatment of IQSEC3-KD mice with minocycline, an inhibitor of microglial activation, blocks DG granule neuron cell death and the occurrence of spontaneous seizures without affecting GABAergic synapse deficits or loss of somatostatin. Our results suggest that microglial activation is involved in a subset of IQSEC3-KD-induced epileptogenesis stages, and that its regulation could be an alternative strategy for managing epilepsy.

PTPσ Drives Excitatory Presynaptic Assembly via Various Extracellular and Intracellular Mechanisms.

Leukocyte common antigen-receptor protein tyrosine phosphatases (LAR-RPTPs) are hub proteins that organize excitatory and inhibitory synapse development through binding to various extracellular ligands. Here, we report that knockdown (KD) of the LAR-RPTP family member PTPσ reduced excitatory synapse number and transmission in cultured rat hippocampal neurons, whereas KD of PTPδ produced comparable decreases at inhibitory synapses, in both cases without altering expression levels of interacting proteins. An extensive series of rescue experiments revealed that extracellular interactions of PTPσ with Slitrks are important for excitatory synapse development. These experiments further showed that the intracellular D2 domain of PTPσ is required for induction of heterologous synapse formation by Slitrk1 or TrkC, suggesting that interaction of LAR-RPTPs with distinct intracellular presynaptic proteins, drives presynaptic machinery assembly. Consistent with this, double-KD of liprin-α2 and -α3 or KD of PTPσ substrates (N-cadherin and p250RhoGAP) in neurons inhibited Slitrk6-induced, PTPσ-mediated heterologous synapse formation activity. We propose a synaptogenesis model in presynaptic neurons involving LAR-RPTP-organized retrograde signaling cascades, in which both extracellular and intracellular mechanisms are critical in orchestrating distinct synapse types.SIGNIFICANCE STATEMENT In this study, we sought to test the unproven hypothesis that PTPσ and PTPδ are required for excitatory and inhibitory synapse formation/transmission, respectively, in cultured hippocampal neurons, using knockdown-based loss-of-function analyses. We further performed extensive structure-function analyses, focusing on PTPσ-mediated actions, to address the mechanisms of presynaptic assembly at excitatory synaptic sites. Using interdisciplinary approaches, we systematically applied a varied set of PTPσ deletion variants, point mutants, and splice variants to demonstrate that both extracellular and intracellular mechanisms are involved in organizing presynaptic assembly. Strikingly, extracellular interactions of PTPσ with heparan sulfates and Slitrks, intracellular interactions of PTPσ with liprin-α and its associated proteins through the D2 domain, as well as distinct substrates are all critical.

PTPσ functions as a presynaptic receptor for the glypican-4/LRRTM4 complex and is essential for excitatory synaptic transmission.

Leukocyte common antigen-related receptor protein tyrosine phosphatases--comprising LAR, PTPδ, and PTPσ--are synaptic adhesion molecules that organize synapse development. Here, we identify glypican 4 (GPC-4) as a ligand for PTPσ. GPC-4 showed strong (nanomolar) affinity and heparan sulfate (HS)-dependent interaction with the Ig domains of PTPσ. PTPσ bound only to proteolytically cleaved GPC-4 and formed additional complex with leucine-rich repeat transmembrane protein 4 (LRRTM4) in rat brains. Moreover, single knockdown (KD) of PTPσ, but not LAR, in cultured neurons significantly reduced the synaptogenic activity of LRRTM4, a postsynaptic ligand of GPC-4, in heterologous synapse-formation assays. Finally, PTPσ KD dramatically decreased both the frequency and amplitude of excitatory synaptic transmission. This effect was reversed by wild-type PTPσ, but not by a HS-binding-defective PTPσ mutant. Our results collectively suggest that presynaptic PTPσ, together with GPC-4, acts in a HS-dependent manner to maintain excitatory synapse development and function.

Neurotrophin-3 Regulates Synapse Development by Modulating TrkC-PTPσ Synaptic Adhesion and Intracellular Signaling Pathways.

UNLABELLED: Neurotrophin-3 (NT-3) is a secreted neurotrophic factor that binds neurotrophin receptor tyrosine kinase C (TrkC), which in turn binds to presynaptic protein tyrosine phosphatase σ (PTPσ) to govern excitatory synapse development. However, whether and how NT-3 cooperates with the TrkC-PTPσ synaptic adhesion pathway and TrkC-mediated intracellular signaling pathways in rat cultured neurons has remained unclear. Here, we report that NT-3 enhances TrkC binding affinity for PTPσ. Strikingly, NT-3 treatment bidirectionally regulates the synaptogenic activity of TrkC: at concentrations of 10-25 ng/ml, NT-3 further enhanced the increase in synapse density induced by TrkC overexpression, whereas at higher concentrations, NT-3 abrogated TrkC-induced increases in synapse density. Semiquantitative immunoblotting and optogenetics-based imaging showed that 25 ng/ml NT-3 or light stimulation at a power that produced a comparable level of NT-3 (6.25 µW) activated only extracellular signal-regulated kinase (ERK) and Akt, whereas 100 ng/ml NT-3 (light intensity, 25 µW) further triggered the activation of phospholipase C-γ1 and CREB independently of PTPσ. Notably, disruption of TrkC intracellular signaling pathways, extracellular ligand binding, or kinase activity by point mutations compromised TrkC-induced increases in synapse density. Furthermore, only sparse, but not global, TrkC knock-down in cultured rat neurons significantly decreased synapse density, suggesting that intercellular differences in TrkC expression level are critical for its synapse-promoting action. Together, our data demonstrate that NT-3 is a key factor in excitatory synapse development that may direct higher-order assembly of the TrkC/PTPσ complex and activate distinct intracellular signaling cascades in a concentration-dependent manner to promote competition-based synapse development processes. SIGNIFICANCE STATEMENT: In this study, we present several lines of experimental evidences to support the conclusion that neurotrophin-3 (NT-3) modulates the synaptic adhesion pathway involving neurotrophin receptor tyrosine kinase C (TrkC) and presynaptic protein tyrosine phosphatase σ (PTPσ) in a bidirectional manner at excitatory synapses. NT-3 acts in concentration-independent manner to facilitate TrkC-mediated presynaptic differentiation, whereas it acts in a concentration-dependent manner to exert differential effects on TrkC-mediated organization of postsynaptic development. We further investigated TrkC extracellular ligand binding, intracellular signaling pathways, and kinase activity in NT-3-induced synapse development. Last, we found that interneuronal differences in TrkC levels regulate the synapse number. Overall, these results suggest that NT-3 functions as a positive modulator of synaptogenesis involving TrkC and PTPσ.

IQ Motif and SEC7 Domain-containing Protein 3 (IQSEC3) Interacts with Gephyrin to Promote Inhibitory Synapse Formation.

Gephyrin is a central scaffold protein that mediates development, function, and plasticity of mammalian inhibitory synapses by interacting with various inhibitory synaptic proteins. Here, we show that IQSEC3, a guanine nucleotide exchange factor for ARF6, directly interacts with gephyrin, an interaction that is critical for the inhibitory synapse localization of IQSEC3. Overexpression of IQSEC3 increases inhibitory, but not excitatory, synapse density in a guanine nucleotide exchange factor activity-dependent manner. Conversely, knockdown of IQSEC3 decreases size of gephyrin cluster without altering gephyrin puncta density. Collectively, these data reveal that IQSEC3 acts together with gephyrin to regulate inhibitory synapse development.

Metabotropic glutamate receptor 5 couples cellular prion protein to intracellular signalling in Alzheimer's disease.

Alzheimer's disease-related phenotypes in mice can be rescued by blockade of either cellular prion protein or metabotropic glutamate receptor 5. We sought genetic and biochemical evidence that these proteins function cooperatively as an obligate complex in the brain. We show that cellular prion protein associates via transmembrane metabotropic glutamate receptor 5 with the intracellular protein mediators Homer1b/c, calcium/calmodulin-dependent protein kinase II, and the Alzheimer's disease risk gene product protein tyrosine kinase 2 beta. Coupling of cellular prion protein to these intracellular proteins is modified by soluble amyloid-ß oligomers, by mouse brain Alzheimer's disease transgenes or by human Alzheimer's disease pathology. Amyloid-ß oligomer-triggered phosphorylation of intracellular protein mediators and impairment of synaptic plasticity in vitro requires Prnp-Grm5 genetic interaction, being absent in transheterozygous loss-of-function, but present in either single heterozygote. Importantly, genetic coupling between Prnp and Grm5 is also responsible for signalling, for survival and for synapse loss in Alzheimer's disease transgenic model mice. Thus, the interaction between metabotropic glutamate receptor 5 and cellular prion protein has a central role in Alzheimer's disease pathogenesis, and the complex is a potential target for disease-modifying intervention.

Multifarious astrocyte-neuron dialog in shaping neural circuit architecture.

Astrocytes are multifaceted glial cell types that perform structural, functional, metabolic, and homeostatic roles in the brain. Recent studies have revealed mechanisms underlying the diversity of bidirectional communication modes between astrocytes and neurons - the fundamental organizing principle shaping synaptic properties at tripartite synapses. These astrocyte-neuron interactions are critical for the proper functioning of synapses and neural circuits. This review focuses on molecular mechanisms that direct these interactions, highlighting the versatile roles of multiple adhesion-based paths that likely modulate them, often in a context-dependent manner. It also describes how astrocyte-mediated processes go awry in certain brain disorders and provides a timely insight on the pivotal roles of astrocyte-neuron interactions in synaptic integrity and their relevance to understanding and treating neurological disorders.

Specification of neural circuit architecture shaped by context-dependent patterned LAR-RPTP microexons.

LAR-RPTPs are evolutionarily conserved presynaptic cell-adhesion molecules that orchestrate multifarious synaptic adhesion pathways. Extensive alternative splicing of LAR-RPTP mRNAs may produce innumerable LAR-RPTP isoforms that act as regulatory "codes" for determining the identity and strength of specific synapse signaling. However, no direct evidence for this hypothesis exists. Here, using targeted RNA sequencing, we detected LAR-RPTP mRNAs in diverse cell types across adult male mouse brain areas. We found pronounced cell-type-specific patterns of two microexons, meA and meB, in Ptprd mRNAs. Moreover, diverse neural circuits targeting the same neuronal populations were dictated by the expression of different Ptprd variants with distinct inclusion patterns of microexons. Furthermore, conditional ablation of Ptprd meA+ variants at presynaptic loci of distinct hippocampal circuits impaired distinct modes of synaptic transmission and objection-location memory. Activity-triggered alterations of the presynaptic Ptprd meA code in subicular neurons mediates NMDA receptor-mediated postsynaptic responses in CA1 neurons and objection-location memory. Our data provide the evidence of cell-type- and/or circuit-specific expression patterns in vivo and physiological functions of LAR-RPTP microexons that are dynamically regulated.

Intracellular signaling mechanisms that shape postsynaptic GABAergic synapses.

Postsynaptic GABAergic receptors interact with various membrane and intracellular proteins to mediate inhibitory synaptic transmission. They form structural and/or signaling synaptic protein complexes that perform a variety of postsynaptic functions. In particular, the key GABAergic synaptic scaffold, gephyrin, and its interacting partners govern downstream signaling pathways that are essential for GABAergic synapse development, transmission, and plasticity. In this review, we discuss recent researches on GABAergic synaptic signaling pathways. We also outline the main outstanding issues that need to be addressed in this field and highlight the association of dysregulated GABAergic synaptic signaling with the onset of various brain disorders.

LPS induces microglial activation and GABAergic synaptic deficits in the hippocampus accompanied by prolonged cognitive impairment.

Neuroinflammation impacts the brain and cognitive behavior through microglial activation. In this study, we determined the temporal sequence from microglial activation to synaptic dysfunction and cognitive behavior induced by neuroinflammation in mice. We found that LPS injection activated microglia within a short period, followed by impairments in GABAergic synapses, and that these events led to long-term cognitive impairment. We demonstrated that, 3 days after LPS injection, microglia in the hippocampus were significantly activated due to the LPS-induced inflammation in association with alterations in cellular morphology, microglial density, and expression of phagocytic markers. GABAergic synaptic impairments were detected at 4-6 days after LPS treatment, a time when microglia activity had returned to normal. Consequently, memory impairment persisted for 6 days after injection of LPS. Our results suggest that neuroinflammation induces microglia activation, GABAergic synaptic deficits and prolonged memory impairment over a defined temporal sequence. Our observations provide insight into the temporal sequence of neuroinflammation-associated brain pathologies. Moreover, the specific loss of inhibitory synapses accompanying the impaired inhibitory synaptic transmission provides mechanistic insight that may explain the prolonged cognitive deficit observed in patients with neuroinflammation. Thus, this study provides essential clues regarding early intervention strategies against brain pathologies accompanying neuroinflammation.

Neddylation positively regulates the ubiquitin E3 ligase activity of parkin.

Mutations in the parkin gene underlie a familial form of Parkinson's disease known as autosomal recessive juvenile Parkinsonism (AR-JP). Dysfunction of parkin, a ubiquitin E3 ligase, has been implicated in the accumulation of ubiquitin proteasome system-destined substrates and eventually leads to cell death. However, regulation of parkin enzymatic activity is incompletely understood. Here we investigated whether the ubiquitin E3 ligase activity of parkin could be regulated by neddylation. We found that parkin could be a target of covalent modification with NEDD8, a ubiquitin-like posttranslational modifier. In addition, NEDD8 attachment caused an increase of parkin activity through the increased binding affinity for ubiquitin-conjugating E2 enzyme as well as the enhanced formation of the complex containing parkin and substrates. These findings point to the functional importance of NEDD8 and suggest that neddylation is one to the diverse modes of parkin regulation, potentially linking it to the pathogenesis of AR-JP.

Reassessing synaptic adhesion pathways.

Multiple synaptic adhesion proteins are thought to collectively define the properties of specific synapses and thereby shape the architectures of neural circuits. Growing evidence supports a molecular model in which a set of central hub proteins interacts with a vast number of other proteins to organize multifarious synaptic adhesion pathways. However, several fundamental open questions remain, partly owing to drawbacks in current approaches and interpretations. In this opinion, we provide an overview of synaptic adhesion pathways, underscoring open questions to be addressed in future work, and highlighting approaches for advancing understanding of synaptic adhesion processes.

Differential Regional Vulnerability of the Brain to Mild Neuroinflammation Induced by Systemic LPS Treatment in Mice.

Background: Peripheral inflammation-triggered mild neuroinflammation impacts the brain and behavior through microglial activation. In this study, we performed an unbiased analysis of the vulnerability of different brain areas to neuroinflammation induced by systemic inflammation. Methods: We injected mice with a single low dose of LPS to induce mild inflammation and then analyzed microglial activation in 34 brain regions by immunohistochemical methods and whole-brain imaging using multi-slide scanning microscopy. We also conducted quantitative RT-PCR to measure the levels of inflammatory cytokines in selected brain regions of interest. Results: We found that microglia in different brain regions are differentially activated by mild, LPS-induced inflammation relative to the increase in microglia numbers or increased CD68 expression. The increased number of microglia induced by mild inflammation was not attributable to infiltration of peripheral immune cells. In addition, microglia residing in brain regions, in which a single low-dose injection of LPS produced microglial changes, preferentially generated pro-inflammatory cytokines. Conclusion: Our results suggest that mild neuroinflammation induces regionally different microglia activation, producing pro-inflammatory cytokines. Our observations provide insight into induction of possible region-specific neuroinflammation-associated brain pathologies through microglial activation.

IQSEC3 Deletion Impairs Fear Memory Through Upregulation of Ribosomal S6K1 Signaling in the Hippocampus.

BACKGROUND: IQSEC3, a gephyrin-binding GABAergic (gamma-aminobutyric acidergic) synapse-specific guanine nucleotide exchange factor, was recently reported to regulate activity-dependent GABAergic synapse maturation, but the underlying signaling mechanisms remain incompletely understood. METHODS: We generated mice with conditional knockout (cKO) of Iqsec3 to examine whether altered synaptic inhibition influences hippocampus-dependent fear memory formation. In addition, electrophysiological recordings, immunohistochemistry, and behavioral assays were used to address our question. RESULTS: We found that Iqsec3-cKO induces a specific reduction in GABAergic synapse density, GABAergic synaptic transmission, and maintenance of long-term potentiation in the hippocampal CA1 region. In addition, Iqsec3-cKO mice exhibited impaired fear memory formation. Strikingly, Iqsec3-cKO caused abnormally enhanced activation of ribosomal P70-S6K1-mediated signaling in the hippocampus but not in the cortex. Furthermore, inhibiting upregulated S6K1 signaling by expressing dominant-negative S6K1 in the hippocampal CA1 of Iqsec3-cKO mice completely rescued impaired fear learning and inhibitory synapse density but not deficits in long-term potentiation maintenance. Finally, upregulated S6K1 signaling was rescued by IQSEC3 wild-type, but not by an ARF-GEF (adenosine diphosphate ribosylation factor-guanine nucleotide exchange factor) inactive IQSEC3 mutant. CONCLUSIONS: Our results suggest that IQSEC3-mediated balanced synaptic inhibition in hippocampal CA1 is critical for the proper formation of hippocampus-dependent fear memory.