Gephyrin promotes autonomous assembly and synaptic localization of GABAergic postsynaptic components without presynaptic GABA release.

Synapses containing γ-aminobutyric acid (GABA) constitute the primary centers for inhibitory neurotransmission in our nervous system. It is unclear how these synaptic structures form and align their postsynaptic machineries with presynaptic terminals. Here, we monitored the cellular distribution of several GABAergic postsynaptic proteins in a purely glutamatergic neuronal culture derived from human stem cells, which virtually lacks any vesicular GABA release. We found that several GABAA receptor (GABAAR) subunits, postsynaptic scaffolds, and major cell-adhesion molecules can reliably coaggregate and colocalize at even GABA-deficient subsynaptic domains, but remain physically segregated from glutamatergic counterparts. Genetic deletions of both Gephyrin and a Gephyrin-associated guanosine di- or triphosphate (GDP/GTP) exchange factor Collybistin severely disrupted the coassembly of these postsynaptic compositions and their proper apposition with presynaptic inputs. Gephyrin-GABAAR clusters, developed in the absence of GABA transmission, could be subsequently activated and even potentiated by delayed supply of vesicular GABA. Thus, molecular organization of GABAergic postsynapses can initiate via a GABA-independent but Gephyrin-dependent intrinsic mechanism.

Plexin-B1 and Plexin-B2 play non-redundant roles in GABAergic synapse formation.

Synapse formation in the mammalian brain is a complex and dynamic process requiring coordinated function of dozens of molecular families such as cell adhesion molecules (CAMs) and ligand-receptor pairs (Ephs/Ephrins, Neuroligins/Neurexins, Semaphorins/Plexins). Due to the large number of molecular players and possible functional redundancies within gene families, it is challenging to determine the precise synaptogenic roles of individual molecules, which is key to understanding the consequences of mutations in these genes for brain function. Furthermore, few molecules are known to exclusively regulate either GABAergic or glutamatergic synapses, and cell and molecular mechanisms underlying GABAergic synapse formation in particular are not thoroughly understood. We previously demonstrated that Semaphorin-4D (Sema4D) regulates GABAergic synapse development in the mammalian hippocampus while having no effect on glutamatergic synapse development, and this effect occurs through binding to its high affinity receptor, Plexin-B1. In addition, we demonstrated that RNAi-mediated Plexin-B2 knock-down decreases GABAergic synapse density suggesting that both receptors function in this process. Here, we perform a structure-function study of the Plexin-B1 and Plexin-B2 receptors to identify the protein domains in each receptor which are required for its synaptogenic function. Further, we examine whether Plexin-B2 is required in the presynaptic neuron, the postsynaptic neuron, or both to regulate GABAergic synapse formation. Our data reveal that Plexin-B1 and Plexin-B2 function non-redundantly to regulate GABAergic synapse formation and suggest that the transmembrane domain may underlie functional distinctions. We also provide evidence that Plexin-B2 expression in presynaptic GABAergic interneurons, as well as postsynaptic pyramidal cells, regulates GABAergic synapse formation in hippocampus. These findings lay the groundwork for future investigations into the precise signaling pathways required for synapse formation downstream of Plexin-B receptor signaling.

Shank3 Deficiency Results in a Reduction in GABAergic Postsynaptic Puncta in the Olfactory Brain Areas.

Dysfunctional sensory systems, including altered olfactory function, have recently been reported in patients with autism spectrum disorder (ASD). Disturbances in olfactory processing can potentially result from gamma-aminobutyric acid (GABA)ergic synaptic abnormalities. The specific molecular mechanism by which GABAergic transmission affects the olfactory system in ASD remains unclear. Therefore, the present study aimed to evaluate selected components of the GABAergic system in olfactory brain regions and primary olfactory neurons isolated from Shank3-deficient (-/-) mice, which are known for their autism-like behavioral phenotype. Shank3 deficiency led to a significant reduction in GEPHYRIN/GABAAR colocalization in the piriform cortex and in primary neurons isolated from the olfactory bulb, while no change of cell morphology was observed. Gene expression analysis revealed a significant reduction in the mRNA levels of GABA transporter 1 in the olfactory bulb and Collybistin in the frontal cortex of the Shank3-/- mice compared to WT mice. A similar trend of reduction was observed in the expression of Somatostatin in the frontal cortex of Shank3-/- mice. The analysis of the expression of other GABAergic neurotransmission markers did not yield statistically significant results. Overall, it appears that Shank3 deficiency leads to changes in GABAergic synapses in the brain regions that are important for olfactory information processing, which may represent basis for understanding functional impairments in autism.

GABAergic synapses suppress intestinal innate immunity via insulin signaling in Caenorhabditis elegans.

GABAergic neurotransmission constitutes a major inhibitory signaling mechanism that plays crucial roles in central nervous system physiology and immune cell immunomodulation. However, its roles in innate immunity remain unclear. Here, we report that deficiency in the GABAergic neuromuscular junctions (NMJs) of Caenorhabditis elegans results in enhanced resistance to pathogens, whereas pathogen infection enhances the strength of GABAergic transmission. GABAergic synapses control innate immunity in a manner dependent on the FOXO/DAF-16 but not the p38/PMK-1 pathway. Our data reveal that the insulin-like peptide INS-31 level was dramatically decreased in the GABAergic NMJ GABAAR-deficient unc-49 mutant compared with wild-type animals. C. elegans with ins-31 knockdown or loss of function exhibited enhanced resistance to Pseudomonas aeruginosa PA14 exposure. INS-31 may act downstream of GABAergic NMJs and in body wall muscle to control intestinal innate immunity in a cell-nonautonomous manner. Our results reveal a signaling axis of synapse-muscular insulin-intestinal innate immunity in vivo.

Recruitment of Plasma Membrane GABA-A Receptors by Submembranous Gephyrin/Collybistin Clusters.

It has been shown that subunit composition is the main determinant of the synaptic or extrasynaptic localization of GABAA receptors (GABAARs). Synaptic and extrasynaptic GABAARs are involved in phasic and tonic inhibition, respectively. It has been proposed that synaptic GABAARs bind to the postsynaptic gephyrin/collybistin (Geph/CB) lattice, but not the typically extrasynaptic GABAARs. Nevertheless, there are no studies of the direct binding of various types of GABAARs with the submembranous Geph/CB lattice in the absence of other synaptic proteins, some of which are known to interact with GABAARs. We have reconstituted GABAARs of various subunit compositions, together with the Geph/CB scaffold, in HEK293 cells, and have investigated the recruitment of surface GABAARs by submembranous Geph/CB clusters. Results show that the typically synaptic α1ß3γ2 GABAARs were trapped by submembranous Geph/CB clusters. The α5ß3γ2 GABAARs, which are both synaptic and extrasynaptic, were also trapped by Geph/CB clusters. Extrasynaptic α4ß3δ GABAARs consistently showed little or no trapping by the Geph/CB clusters. However, the extrasynaptic α6ß3δ, α1ß3, α6ß3 (and less α4ß3) GABAARs were highly trapped by the Geph/CB clusters. AMPA and NMDA glutamate receptors were not trapped. The results suggest: (I) in the absence of other synaptic molecules, the Geph/CB lattice has the capacity to trap not only synaptic but also several typically extrasynaptic GABAARs; (II) the Geph/CB lattice is important but does not play a decisive role in the synaptic localization of GABAARs; and (III) in neurons there must be mechanisms preventing the trapping of several typically extrasynaptic GABAARs by the postsynaptic Geph/CB lattice.

Collybistin SH3-protein isoforms are expressed in the rat brain promoting gephyrin and GABA-A receptor clustering at GABAergic synapses.

Collybistin (CB) is a guanine nucleotide exchange factor (GEF) selectively localized at GABAergic and glycinergic postsynapses. Analysis of mRNA shows that several isoforms of collybistin are expressed in the brain. Some of the isoforms have a SH3 domain (CBSH3+) and some have no SH3 domain (CBSH3-). The CBSH3+ mRNAs are predominantly expressed over CBSH3-. However, in an immunoblot study of mouse brain homogenates, only CBSH3+ protein isoforms were detected, proposing that CBSH3- protein might not be expressed in the brain. The expression or lack of expression of CBSH3- protein is an important issue because CBSH3- has a strong effect in promoting the postsynaptic clustering of gephyrin and GABA-A receptors (GABAA Rs). Moreover CBSH3- is constitutively active; therefore lower expression of CBSH3- protein might play a relatively stronger functional role than the more abundant but self-inhibited CBSH3+ isoforms, which need to be activated. We are now showing that: (a) CBSH3- protein is expressed in the brain; (b) parvalbumin positive (PV+) interneurons show higher expression of CBSH3- protein than other neurons; (c) CBSH3- is associated with GABAergic synapses in various regions of the brain and (d) knocking down CBSH3- in hippocampal neurons decreases the synaptic clustering of gephyrin and GABAA Rs. The results show that CBSH3- protein is expressed in the brain and that it plays a significant role in the size regulation of the GABAergic postsynapse.

Syntaxin 1B regulates synaptic GABA release and extracellular GABA concentration, and is associated with temperature-dependent seizures.

De novo heterozygous mutations in the STX1B gene, encoding syntaxin 1B, cause a familial, fever-associated epilepsy syndrome. Syntaxin 1B is an essential component of the pre-synaptic neurotransmitter release machinery as a soluble N-ethylmaleimide-sensitive factor attachment protein receptor protein that regulates the exocytosis of synaptic vesicles. It is also involved in regulating the functions of the SLC6 family of neurotransmitter transporters that reuptake neurotransmitters, including inhibitory neurotransmitters, such as γ-aminobutyric acid (GABA) and glycine. The purpose of the present study was to elucidate the molecular mechanisms underlying the development of febrile seizures by examining the effects of syntaxin 1B haploinsufficiency on inhibitory synaptic transmission during hyperthermia in a mouse model. Stx1b gene heterozygous knockout (Stx1b+/- ) mice showed increased susceptibility to febrile seizures and drug-induced seizures. In cultured hippocampal neurons, we examined the temperature-dependent properties of neurotransmitter release and reuptake by GABA transporter-1 (GAT-1) at GABAergic neurons using whole-cell patch-clamp recordings. The rate of spontaneous quantal GABA release was reduced in Stx1b+/- mice. The hyperthermic temperature increased the tonic GABAA current in wild-type (WT) synapses, but not in Stx1b+/- synapses. In WT neurons, recurrent bursting activities were reduced in a GABA-dependent manner at hyperthermic temperature; however, this was abolished in Stx1b+/- neurons. The blockade of GAT-1 increased the tonic GABAA current and suppressed recurrent bursting activities in Stx1b+/- neurons at the hyperthermic temperature. These data suggest that functional abnormalities associated with GABA release and reuptake in the pre-synaptic terminals of GABAergic neurons may increase the excitability of the neural circuit with hyperthermia.

HIV gp120-induced neuroinflammation potentiates NMDA receptors to overcome basal suppression of inhibitory synapses by p38 MAPK.

HIV-associated neurocognitive disorder affects about half of HIV-infected patients. HIV impairs neuronal function through indirect mechanisms mainly mediated by inflammatory cytokines and neurotoxic viral proteins, such as the envelope protein gp120. HIV gp120 elicits a neuroinflammatory response that potentiates NMDA receptor function and induces the loss of excitatory synapses. How gp120 influences neuronal inhibition remains unknown. In this study, we expressed a green fluorescent protein (GFP)-tagged recombinant antibody-like protein that binds to the post-synaptic scaffolding protein gephyrin to label inhibitory synapses in living neurons. Treatment with 600 pM gp120 for 24 h increased the number of labeled inhibitory synapses. HIV gp120 evoked the release of interleukin-1ß (IL-1ß) from microglia to activate IL-1 receptors on neurons. Subsequent activation of the tyrosine kinase Src and GluN2A-containing NMDA receptors increased the number of inhibitory synapses via a process that required protein synthesis. In naïve cultures, inhibition of neuronal p38 mitogen-activated protein kinase (p38 MAPK) increased the number of inhibitory synapses suggesting that p38 MAPK produces a basal suppression of inhibitory synapses that is overcome in the presence of gp120. Direct activation of a mutant form of p38 MAPK expressed in neurons mimicked basal suppression of inhibitory synapses. This study shows for the first time that gp120-induced neuroinflammation increases the number of inhibitory synapses and that this increase overcomes a basal suppression of synaptic inhibition. Increased inhibition may be an adaptive mechanism enabling neurons to counteract excess excitatory input in order to maintain network homeostasis. OPEN SCIENCE BADGES: This article has received a badge for *Open Materials* because it provided all relevant information to reproduce the study in the manuscript. The complete Open Science Disclosure form for this article can be found at the end of the article. More information about the Open Practices badges can be found at https://cos.io/our-services/open-science-badges/.

Diverse Actions of Astrocytes in GABAergic Signaling.

An imbalance of excitatory and inhibitory neurotransmission leading to over excitation plays a crucial role in generating seizures, while enhancing GABAergic mechanisms are critical in terminating seizures. In recent years, it has been reported in many studies that astrocytes are deeply involved in synaptic transmission. Astrocytes form a critical component of the "tripartite" synapses by wrapping around the pre- and post-synaptic elements. From this location, astrocytes are known to greatly influence the dynamics of ions and transmitters in the synaptic cleft. Despite recent extensive research on excitatory tripartite synapses, inhibitory tripartite synapses have received less attention, even though they influence inhibitory synaptic transmission by affecting chloride and GABA concentration dynamics. In this review, we will discuss the diverse actions of astrocytic chloride and GABA homeostasis at GABAergic tripartite synapses. We will then consider the pathophysiological impacts of disturbed GABA homeostasis at the tripartite synapse.

Activity-dependent inhibitory synapse remodeling through gephyrin phosphorylation.

Maintaining a proper balance between excitation and inhibition is essential for the functioning of neuronal networks. However, little is known about the mechanisms through which excitatory activity can affect inhibitory synapse plasticity. Here we used tagged gephyrin, one of the main scaffolding proteins of the postsynaptic density at GABAergic synapses, to monitor the activity-dependent adaptation of perisomatic inhibitory synapses over prolonged periods of time in hippocampal slice cultures. We find that learning-related activity patterns known to induce N-methyl-D-aspartate (NMDA) receptor-dependent long-term potentiation and transient optogenetic activation of single neurons induce within hours a robust increase in the formation and size of gephyrin-tagged clusters at inhibitory synapses identified by correlated confocal electron microscopy. This inhibitory morphological plasticity was associated with an increase in spontaneous inhibitory activity but did not require activation of GABAA receptors. Importantly, this activity-dependent inhibitory plasticity was prevented by pharmacological blockade of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII), it was associated with an increased phosphorylation of gephyrin on a site targeted by CaMKII, and could be prevented or mimicked by gephyrin phospho-mutants for this site. These results reveal a homeostatic mechanism through which activity regulates the dynamics and function of perisomatic inhibitory synapses, and they identify a CaMKII-dependent phosphorylation site on gephyrin as critically important for this process.

Phenotype-dependent Ca(2+) dynamics in single boutons of various anatomically identified GABAergic interneurons in the rat hippocampus.

Interneurons (INs) of the hippocampus exert versatile inhibition on pyramidal cells by silencing the network at different oscillation frequencies. Although IN discharge can phase-lock to various rhythms in the hippocampus, under high-frequency axon firing, the boutons may not be able to follow the fast activity. Here, we studied Ca(2+) responses to action potentials (APs) in single boutons using combined two-photon microscopy and patch clamp electrophysiology in three types of INs: non-fast-spiking (NFS) neurons showing cannabinoid 1 receptor labelling and dendrite targeting, fast-spiking partially parvalbumin-positive cells synapsing with dendrites (DFS), and parvalbumin-positive cells with perisomatic innervation (PFS). The increase in [Ca(2+) ]i from AP trains was substantially higher in NFS boutons than in DFS or PFS boutons. The decay of bouton Ca(2+) responses was markedly faster in DFS and PFS cells compared with NFS neurons. The bouton-to-bouton variability of AP-evoked Ca(2+) transients in the same axon was surprisingly low in each cell type. Importantly, local responses were saturated after shorter trains of APs in NFS cells than in PFS cells. This feature of fast-spiking neurons might allow them to follow higher-frequency gamma oscillations for a longer time than NFS cells. The function of NFS boutons may better support asynchronous GABA release. In conclusion, we demonstrate several neuron-specific Ca(2+) transients in boutons of NFS, PFS and DFS neurons, which may serve differential functions in hippocampal networks.

Synaptic Conductance Estimates of the Connection Between Local Inhibitor Interneurons and Pyramidal Neurons in Layer 2/3 of a Cortical Column.

Stimulation of a principal whisker yields sparse action potential (AP) spiking in layer 2/3 (L2/3) pyramidal neurons in a cortical column of rat barrel cortex. The low AP rates in pyramidal neurons could be explained by activation of interneurons in L2/3 providing inhibition onto L2/3 pyramidal neurons. L2/3 interneurons classified as local inhibitors based on their axonal projection in the same column were reported to receive strong excitatory input from spiny neurons in L4, which are also the main source of the excitatory input to L2/3 pyramidal neurons. Here, we investigated the remaining synaptic connection in this intracolumnar microcircuit. We found strong and reliable inhibitory synaptic transmission between intracolumnar L2/3 local-inhibitor-to-L2/3 pyramidal neuron pairs [inhibitory postsynaptic potential (IPSP) amplitude -0.88 ± 0.67 mV]. On average, 6.2 ± 2 synaptic contacts were made by L2/3 local inhibitors onto L2/3 pyramidal neurons at 107 ± 64 µm path distance from the pyramidal neuron soma, thus overlapping with the distribution of synaptic contacts from L4 spiny neurons onto L2/3 pyramidal neurons (67 ± 34 µm). Finally, using compartmental simulations, we determined the synaptic conductance per synaptic contact to be 0.77 ± 0.4 nS. We conclude that the synaptic circuit from L4 to L2/3 can provide efficient shunting inhibition that is temporally and spatially aligned with the excitatory input from L4 to L2/3.

Shank1 regulates excitatory synaptic transmission in mouse hippocampal parvalbumin-expressing inhibitory interneurons.

The Shank genes (SHANK1, 2, 3) encode scaffold proteins highly enriched in postsynaptic densities where they regulate synaptic structure in spiny neurons. Mutations in human Shank genes are linked to autism spectrum disorder and schizophrenia. Shank1 mutant mice exhibit intriguing cognitive phenotypes reminiscent of individuals with autism spectrum disorder. However, the molecular mechanisms leading to the human pathophysiological phenotypes and mouse behaviors have not been elucidated. In this study it is shown that Shank1 protein is highly localized in parvalbumin-expressing (PV+) fast-spiking inhibitory interneurons in the hippocampus. Importantly, a lack of Shank1 in hippocampal CA1 PV+ neurons reduced excitatory synaptic inputs and inhibitory synaptic outputs to pyramidal neurons. Furthermore, it is demonstrated that hippocampal CA1 pyramidal neurons in Shank1 mutant mice exhibit a shift in the excitatory and inhibitory balance (E-I balance), a pathophysiological hallmark of autism spectrum disorder. The mutant mice also exhibit lower expression of gephyrin (a scaffold component of inhibitory synapses), supporting the dysregulation of E-I balance in the hippocampus. These results suggest that Shank1 scaffold in PV+ interneurons regulates excitatory synaptic strength and participates in the maintenance of E-I balance in excitatory neurons.

Layer-specific endocannabinoid-mediated long-term depression of GABAergic neurotransmission onto principal neurons in mouse visual cortex.

Visually induced endocannabinoid-mediated long-term depression of GABAergic neurotransmission (iLTD) mediates the maturation of GABAergic release in layer 2/3 of visual cortex. Here we examined whether the maturation of GABAergic transmission in other layers of visual cortex also requires endocannabinoids. The developmental plasticity of GABAergic neurotransmission onto the principal neurons in different layers of mouse visual cortex was examined in cortical slices by whole-cell recordings of inhibitory postsynaptic currents evoked by presynaptic inhibitory inputs. Theta burst stimulation of GABAergic inputs induced an endocannabinoid-mediated long-term depression of GABAergic neurotransmission onto pyramidal cells in layer 2/3 from postnatal day (P)10 to 30 and in layer 5 from P10 to 40, whereas that of GABAergic inputs did not induce iLTD onto star pyramidal neurons in layer 4 at any time postnatally, indicating that this plasticity is laminar-specific. The developmental loss of iLTD paralleled the maturation of GABAergic inhibition in both layer 2/3 and layer 5. Visual deprivation delayed the developmental loss of iLTD in layers 3 and 5 during a critical period, while 2 days of light exposure eliminated iLTD in both layers. Furthermore, the GABAergic synapses in layers 2/3 and 5 did not normally mature in the type 1 cannabinoid receptor knock-out mice, whereas those in layer 4 did not require endocannabinoid receptor for maturation. These results suggest that visually induced endocannabinoid-dependent iLTD mediates the maturation of GABAergic release in extragranular layer rather than in granular layer of mouse visual cortex.

The role of Down syndrome cell adhesion molecule in Down syndrome.

Down syndrome (DS) is caused by the presence of an extra copy of the entire or a portion of human chromosome 21 (HSA21). This genomic alteration leads to elevated expression of numerous HSA21 genes, resulting in a variety of health issues in individuals with DS. Among the genes located in the DS "critical region" of HSA21, Down syndrome cell adhesion molecule (DSCAM) plays an important role in neuronal development. There is a growing body of evidence underscoring DSCAM's involvement in various DS-related disorders. This review aims to provide a concise overview of the established functions of DSCAM, with a particular focus on its implications in DS. We delve into the roles that DSCAM plays in DS-associated diseases. In the concluding section of this review, we explore prospective avenues for future research to further unravel DSCAM's role in DS and opportunities for therapeutic treatments.

Antagonistic interaction between caffeine and ketamine in zebrafish: Implications for aquatic toxicity.

The coexistence of caffeine (CF) and ketamine (KET) in surface waters across Asia has been widely reported. Previous studies have implied that CF and KET may share a mechanism of action. However, the combined toxicity of these two chemicals on aquatic organisms remains unclear at environmental levels, and the underlying mechanisms are not well understood. Here we demonstrate that KET antagonizes the adverse effects of CF on zebrafish larvae by modulating the gamma-aminobutyric acid (GABA)ergic synapse pathway. Specifically, KET (10-250 ng L-1) ameliorates the locomotor hyperactivity and impaired circadian rhythms in zebrafish larvae induced by 2 mg L-1 of CF, showing a dose-dependent relationship. Additionally, the developmental abnormalities in zebrafish larvae exposed to CF are mitigated by KET, with an incidence rate reduced from 26.7% to 6.7%. The competition between CF and KET for binding sites on the GABA-A receptor (in situ and in silico) elucidates the antagonistic interactions between the two chemicals. Following a seven-day recovery period, the adverse outcomes of CF exposure persist in the fish, whereas the changes observed in the CF + KET groups are significantly alleviated, especially with KET at 10 ng L-1. Based on these results, it is imperative to further assess the environmental risks associated with CF and KET co-pollution. This pilot study underscores the utility of systems toxicology approaches in estimating the combined toxicity of environmental chemicals on aquatic organisms. Moreover, the nighttime behavioral functions of fish could serve as a sensitive biomarker for evaluating the toxicity of psychoactive substances.

Human mutations in SLITRK3 implicated in GABAergic synapse development in mice.

This study reports on biallelic homozygous and monoallelic de novo variants in SLITRK3 in three unrelated families presenting with epileptic encephalopathy associated with a broad neurological involvement characterized by microcephaly, intellectual disability, seizures, and global developmental delay. SLITRK3 encodes for a transmembrane protein that is involved in controlling neurite outgrowth and inhibitory synapse development and that has an important role in brain function and neurological diseases. Using primary cultures of hippocampal neurons carrying patients' SLITRK3 variants and in combination with electrophysiology, we demonstrate that recessive variants are loss-of-function alleles. Immunostaining experiments in HEK-293 cells showed that human variants C566R and E606X change SLITRK3 protein expression patterns on the cell surface, resulting in highly accumulating defective proteins in the Golgi apparatus. By analyzing the development and phenotype of SLITRK3 KO (SLITRK3-/-) mice, the study shows evidence of enhanced susceptibility to pentylenetetrazole-induced seizure with the appearance of spontaneous epileptiform EEG as well as developmental deficits such as higher motor activities and reduced parvalbumin interneurons. Taken together, the results exhibit impaired development of the peripheral and central nervous system and support a conserved role of this transmembrane protein in neurological function. The study delineates an emerging spectrum of human core synaptopathies caused by variants in genes that encode SLITRK proteins and essential regulatory components of the synaptic machinery. The hallmark of these disorders is impaired postsynaptic neurotransmission at nerve terminals; an impaired neurotransmission resulting in a wide array of (often overlapping) clinical features, including neurodevelopmental impairment, weakness, seizures, and abnormal movements. The genetic synaptopathy caused by SLITRK3 mutations highlights the key roles of this gene in human brain development and function.

GABAA receptors and neuroligin 2 synergize to promote synaptic adhesion and inhibitory synaptogenesis.

GABAA receptors (γ-aminobutyric acid-gated receptors type A; GABAARs), the major structural and functional postsynaptic components of inhibitory synapses in the mammalian brain, belong to a family of GABA-gated Cl-/HCO3 - ion channels. They are assembled as heteropentamers from a family of subunits including: α (1-6), ß(1-3), γ(1-3), δ, ε, π, θ and ρ(1-3). GABAARs together with the postsynaptic adhesion protein Neuroligin 2 (NL2) and many other pre- and post-synaptic proteins guide the initiation and functional maturation of inhibitory GABAergic synapses. This study examined how GABAARs and NL2 interact with each other to initiate the formation of synapses. Two functionally distinct GABAAR subtypes, the synaptic type α2ß2γ2-GABAARs versus extrasynaptic type α4ß3δ-GABAARs were expressed in HEK293 cells alone or together with NL2 and co-cultured with striatal GABAergic medium spiny neurons to enable innervation of HEK293 cells by GABAergic axons. When expressed alone, only the synaptic α2ß2γ2-GABAARs induced innervation of HEK293 cells. However, when GABAARs were co-expressed with NL2, the effect on synapse formation exceeded the individual effects of these proteins indicating a synergistic interaction, with α2ß2γ2-GABAAR/NL2 showing a significantly greater synaptogenic activity than α4ß3δ-GABAAR/NL2 or NL2 alone. To investigate the molecular basis of this interaction, different combinations of GABAAR subunits and NL2 were co-expressed, and the degree of innervation and synaptic activity assessed, revealing a key role of the γ2 subunit. In biochemical assays, the interaction between NL2 and α2ß2γ2-GABAAR was established and mapped to the large intracellular domain of the γ2 subunit.

Structural and functional characterization of the IgSF21-neurexin2α complex and its related signaling pathways in the regulation of inhibitory synapse organization.

The prevailing model behind synapse development and specificity is that a multitude of adhesion molecules engage in transsynaptic interactions to induce pre- and postsynaptic assembly. How these extracellular interactions translate into intracellular signal transduction for synaptic assembly remains unclear. Here, we focus on a synapse organizing complex formed by immunoglobulin superfamily member 21 (IgSF21) and neurexin2α (Nrxn2α) that regulates GABAergic synapse development in the mouse brain. We reveal that the interaction between presynaptic Nrxn2α and postsynaptic IgSF21 is a high-affinity receptor-ligand interaction and identify a binding interface in the IgSF21-Nrxn2α complex. Despite being expressed in both dendritic and somatic regions, IgSF21 preferentially regulates dendritic GABAergic presynaptic differentiation whereas another canonical Nrxn ligand, neuroligin2 (Nlgn2), primarily regulates perisomatic presynaptic differentiation. To explore mechanisms that could underlie this compartment specificity, we targeted multiple signaling pathways pharmacologically while monitoring the synaptogenic activity of IgSF21 and Nlgn2. Interestingly, both IgSF21 and Nlgn2 require c-jun N-terminal kinase (JNK)-mediated signaling, whereas Nlgn2, but not IgSF21, additionally requires CaMKII and Src kinase activity. JNK inhibition diminished de novo presynaptic differentiation without affecting the maintenance of formed synapses. We further found that Nrxn2α knockout brains exhibit altered synaptic JNK activity in a sex-specific fashion, suggesting functional linkage between Nrxns and JNK. Thus, our study elucidates the structural and functional relationship of IgSF21 with Nrxn2α and distinct signaling pathways for IgSF21-Nrxn2α and Nlgn2-Nrxn synaptic organizing complexes in vitro. We therefore propose a revised hypothesis that Nrxns act as molecular hubs to specify synaptic properties not only through their multiple extracellular ligands but also through distinct intracellular signaling pathways of these ligands.

Delineation of biomarkers and molecular pathways of residual effects of fluoxetine treatment in juvenile rhesus monkeys by proteomic profiling.

Fluoxetine (Prozac™) is the only antidepressant approved by the US Food and Drug Administration (FDA) for the treatment of major depressive disorder (MDD) in children. Despite its considerable efficacy as a selective serotonin reuptake inhibitor, the possible long-term effects of fluoxetine on brain development in children are poorly understood. In the current study, we aimed to delineate molecular mechanisms and protein biomarkers in the brains of juvenile rhesus macaques (Macaca mulatta) one year after the discontinuation of fluoxetine treatment using proteomic and phosphoproteomic profiling. We identified several differences in protein expression and phosphorylation in the dorsolateral prefrontal cortex (DLPFC) and cingulate cortex (CC) that correlated with impulsivity in animals, suggesting that the GABAergic synapse pathway may be affected by fluoxetine treatment. Biomarkers in combination with the identified pathways contribute to a better understanding of the mechanisms underlying the chronic effects of fluoxetine after discontinuation in children.