Thermodynamic modulation of gephyrin condensation by inhibitory synapse components.

Phase separation drives compartmentalization of intracellular contents into various biomolecular condensates. Individual condensate components are thought to differentially contribute to the organization and function of condensates. However, how intermolecular interactions among constituent biomolecules modulate the phase behaviors of multicomponent condensates remains unclear. Here, we used core components of the inhibitory postsynaptic density (iPSD) as a model system to quantitatively probe how the network of intra- and intermolecular interactions defines the composition and cellular distribution of biomolecular condensates. We found that oligomerization-driven phase separation of gephyrin, an iPSD-specific scaffold, is critically modulated by an intrinsically disordered linker region exhibiting minimal homotypic attractions. Other iPSD components, such as neurotransmitter receptors, differentially promote gephyrin condensation through distinct binding modes and affinities. We further demonstrated that the local accumulation of scaffold-binding proteins at the cell membrane promotes the nucleation of gephyrin condensates in neurons. These results suggest that in multicomponent systems, the extent of scaffold condensation can be fine-tuned by scaffold-binding factors, a potential regulatory mechanism for self-organized compartmentalization in cells.

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.

Peer effects on organizational commitment: Evidence from military cadets.

The commitment of soldiers to the military is essential because it could lead to increased morale, motivation and retention. Despite the accumulation of knowledge about predictors of organizational commitment (OC), efforts to investigate environmental factors influencing OC are in their infancy. We note that individuals shape their attitudes toward the environment based on information obtained from their surroundings, and we investigate peer effects on OC using data from a natural experiment of randomly-assigned military academy roommates. A total of 400 cadets (Sex ratio: 93.5% male, Age: 21.13 ± 1.43 years) from 136 living quarters participated in this quantitative study. In both self- and roommate-reports, we found that the average affective commitment (AC), continuance commitment (CC), and normative commitment (NC) of roommates in a living quarter can still predict AC, CC, and NC of the remaining individual in that same living quarter, respectively, even after controlling for the personal predictors of that remaining individual. Additionally, in self-report, we discovered that when there is a high heterogeneity in AC among roommates within a living quarter, the AC of the remaining individual in that living quarter tends to be higher, even after controlling for the personal predictors of that remaining individual. These findings provide initial evidence that attempting to assign soldiers with low OC to the same living quarters as those with high OC may be worthwhile.

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.

Molecular Decrowding by Tissue Expansion Allows Precise Determination of the Spatial Distribution of Synaptic Proteins at a Nanometer Scale by exTEM.

To understand how the molecular machinery of synapses works, it is essential to determine an inventory of synaptic proteins at a subsynaptic resolution. Nevertheless, synaptic proteins are difficult to localize because of the low expression levels and limited access to immunostaining epitopes. Here, we report on the exTEM (epitope-exposed by expansion-transmission electron microscopy) method that enables the imaging of synaptic proteins in situ. This method combines TEM with nanoscale resolution and expandable tissue-hydrogel hybrids for enhanced immunolabeling with better epitope accessibility via molecular decrowding, allowing successful probing of the distribution of various synapse-organizing proteins. We propose that exTEM can be employed for studying the mechanisms underlying the regulation of synaptic architecture and function by providing nanoscale molecular distribution of synaptic proteins in situ. We also envision that exTEM is widely applicable for investigating protein nanostructures located in densely packed environments by immunostaining of commercially available antibodies at nanometer resolution.

Orchestration of synaptic functions by WAVE regulatory complex-mediated actin reorganization.

The WAVE regulatory complex (WRC), composed of five components-Cyfip1/Sra1, WAVE/Scar, Abi, Nap1/Nckap1, and Brk1/HSPC300-is essential for proper actin cytoskeletal dynamics and remodeling in eukaryotic cells, likely by matching various patterned signals to Arp2/3-mediated actin nucleation. Accumulating evidence from recent studies has revealed diverse functions of the WRC in neurons, demonstrating its crucial role in dictating the assembly of molecular complexes for the patterning of various trans-synaptic signals. In this review, we discuss recent exciting findings on the physiological role of the WRC in regulating synaptic properties and highlight the involvement of WRC dysfunction in various brain disorders.

Trans-synaptic mechanisms orchestrated by mammalian synaptic cell adhesion molecules.

Bidirectional trans-synaptic signaling is essential for the formation, maturation, and plasticity of synaptic connections. Synaptic cell adhesion molecules (CAMs) are prime drivers in shaping the identities of trans-synaptic signaling pathways. A series of recent studies provide evidence that diverse presynaptic cell adhesion proteins dictate the regulation of specific synaptic properties in postsynaptic neurons. Focusing on mammalian synaptic CAMs, this article outlines several exemplary cases supporting this notion and highlights how these trans-synaptic signaling pathways collectively contribute to the specificity and diversity of neural circuit architecture.

SLITRK2 variants associated with neurodevelopmental disorders impair excitatory synaptic function and cognition in mice.

SLITRK2 is a single-pass transmembrane protein expressed at postsynaptic neurons that regulates neurite outgrowth and excitatory synapse maintenance. In the present study, we report on rare variants (one nonsense and six missense variants) in SLITRK2 on the X chromosome identified by exome sequencing in individuals with neurodevelopmental disorders. Functional studies showed that some variants displayed impaired membrane transport and impaired excitatory synapse-promoting effects. Strikingly, these variations abolished the ability of SLITRK2 wild-type to reduce the levels of the receptor tyrosine kinase TrkB in neurons. Moreover, Slitrk2 conditional knockout mice exhibited impaired long-term memory and abnormal gait, recapitulating a subset of clinical features of patients with SLITRK2 variants. Furthermore, impaired excitatory synapse maintenance induced by hippocampal CA1-specific cKO of Slitrk2 caused abnormalities in spatial reference memory. Collectively, these data suggest that SLITRK2 is involved in X-linked neurodevelopmental disorders that are caused by perturbation of diverse facets of SLITRK2 function.

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.

Deletion of Calsyntenin-3, an atypical cadherin, suppresses inhibitory synapses but increases excitatory parallel-fiber synapses in cerebellum.

Cadherins contribute to the organization of nearly all tissues, but the functions of several evolutionarily conserved cadherins, including those of calsyntenins, remain enigmatic. Puzzlingly, two distinct, non-overlapping functions for calsyntenins were proposed: As postsynaptic neurexin ligands in synapse formation, or as presynaptic kinesin adaptors in vesicular transport. Here, we show that, surprisingly, acute CRISPR-mediated deletion of calsyntenin-3 in mouse cerebellum in vivo causes a large decrease in inhibitory synapse, but a robust increase in excitatory parallel-fiber synapses in Purkinje cells. As a result, inhibitory synaptic transmission was suppressed, whereas parallel-fiber synaptic transmission was enhanced in Purkinje cells by the calsyntenin-3 deletion. No changes in the dendritic architecture of Purkinje cells or in climbing-fiber synapses were detected. Sparse selective deletion of calsyntenin-3 only in Purkinje cells recapitulated the synaptic phenotype, indicating that calsyntenin-3 acts by a cell-autonomous postsynaptic mechanism in cerebellum. Thus, by inhibiting formation of excitatory parallel-fiber synapses and promoting formation of inhibitory synapses in the same neuron, calsyntenin-3 functions as a postsynaptic adhesion molecule that regulates the excitatory/inhibitory balance in Purkinje cells.

A chemical tool for blue light-inducible proximity photo-crosslinking in live cells.

We developed a proximity photo-crosslinking method (Spotlight) with a 4-azido-N-ethyl-1,8-naphthalimide (AzNP) moiety that can be converted to reactive aryl nitrene species using ambient blue light-emitting diode light. Using an AzNP-conjugated HaloTag ligand (VL1), blue light-induced photo-crosslinked products of various HaloTag-conjugated proteins of interest were detected in subcellular spaces in live cells. Chemical or heat stress-induced dynamic changes in the proteome were also detected, and photo-crosslinking in the mouse brain tissue was enabled. Using Spotlight, we further identified the host interactome of SARS-CoV-2 nucleocapsid (N) protein, which is essential for viral genome assembly. Mass analysis of the VL1-crosslinked product of N-HaloTag in HEK293T cells showed that RNA-binding proteins in stress granules were exclusively enriched in the cross-linked samples. These results tell that our method can reveal the interactome of protein of interest within a short distance in live cells.

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.

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.

SALM4 negatively regulates NMDA receptor function and fear memory consolidation.

Many synaptic adhesion molecules positively regulate synapse development and function, but relatively little is known about negative regulation. SALM4/Lrfn3 (synaptic adhesion-like molecule 4/leucine rich repeat and fibronectin type III domain containing 3) inhibits synapse development by suppressing other SALM family proteins, but whether SALM4 also inhibits synaptic function and specific behaviors remains unclear. Here we show that SALM4-knockout (Lrfn3-/-) male mice display enhanced contextual fear memory consolidation (7-day post-training) but not acquisition or 1-day retention, and exhibit normal cued fear, spatial, and object-recognition memory. The Lrfn3-/- hippocampus show increased currents of GluN2B-containing N-methyl-D-aspartate (NMDA) receptors (GluN2B-NMDARs), but not α-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptors (AMPARs), which requires the presynaptic receptor tyrosine phosphatase PTPσ. Chronic treatment of Lrfn3-/- mice with fluoxetine, a selective serotonin reuptake inhibitor used to treat excessive fear memory that directly inhibits GluN2B-NMDARs, normalizes NMDAR function and contextual fear memory consolidation in Lrfn3-/- mice, although the GluN2B-specific NMDAR antagonist ifenprodil was not sufficient to reverse the enhanced fear memory consolidation. These results suggest that SALM4 suppresses excessive GluN2B-NMDAR (not AMPAR) function and fear memory consolidation (not acquisition).

Npas4 regulates IQSEC3 expression in hippocampal somatostatin interneurons to mediate anxiety-like behavior.

Activity-dependent GABAergic synapse plasticity is important for normal brain functions, but the underlying molecular mechanisms remain incompletely understood. Here, we show that Npas4 (neuronal PAS-domain protein 4) transcriptionally regulates the expression of IQSEC3, a GABAergic synapse-specific guanine nucleotide-exchange factor for ADP-ribosylation factor (ARF-GEF) that directly interacts with gephyrin. Neuronal activation by an enriched environment induces Npas4-mediated upregulation of IQSEC3 protein specifically in CA1 stratum oriens layer somatostatin (SST)-expressing GABAergic interneurons. SST+ interneuron-specific knockout (KO) of Npas4 compromises synaptic transmission in these GABAergic interneurons, increases neuronal activity in CA1 pyramidal neurons, and reduces anxiety behavior, all of which are normalized by the expression of wild-type IQSEC3, but not a dominant-negative ARF-GEF-inactive mutant, in SST+ interneurons of Npas4-KO mice. Our results suggest that IQSEC3 is a key GABAergic synapse component that is directed by Npas4 and ARF activity, specifically in SST+ interneurons, to orchestrate excitation-to-inhibition balance and control anxiety-like behavior.

Impaired formation of high-order gephyrin oligomers underlies gephyrin dysfunction-associated pathologies.

Gephyrin is critical for the structure, function, and plasticity of inhibitory synapses. Gephyrin mutations have been linked to various neurological disorders; however, systematic analyses of the functional consequences of these mutations are lacking. Here, we performed molecular dynamics simulations of gephyrin to predict how six reported point mutations might change the structural stability and/or function of gephyrin. Additional in silico analyses revealed that the A91T and G375D mutations reduce the binding free energy of gephyrin oligomer formation. Gephyrin A91T and G375D displayed altered clustering patterns in COS-7 cells and nullified the inhibitory synapse-promoting effect of gephyrin in cultured neurons. However, only the G375D mutation reduced gephyrin interaction with GABAA receptors and neuroligin-2 in mouse brain; it also failed to normalize deficits in GABAergic synapse maintenance and neuronal hyperactivity observed in hippocampal dentate gyrus-specific gephyrin-deficient mice. Our results provide insights into biochemical, cell-biological, and network-activity effects of the pathogenic G375D mutation.