Mammalian oocytes store proteins for the early embryo on cytoplasmic lattices.

Mammalian oocytes are filled with poorly understood structures called cytoplasmic lattices. First discovered in the 1960s and speculated to correspond to mammalian yolk, ribosomal arrays, or intermediate filaments, their function has remained enigmatic to date. Here, we show that cytoplasmic lattices are sites where oocytes store essential proteins for early embryonic development. Using super-resolution light microscopy and cryoelectron tomography, we show that cytoplasmic lattices are composed of filaments with a high surface area, which contain PADI6 and subcortical maternal complex proteins. The lattices associate with many proteins critical for embryonic development, including proteins that control epigenetic reprogramming of the preimplantation embryo. Loss of cytoplasmic lattices by knocking out PADI6 or the subcortical maternal complex prevents the accumulation of these proteins and results in early embryonic arrest. Our work suggests that cytoplasmic lattices enrich maternally provided proteins to prevent their premature degradation and cellular activity, thereby enabling early mammalian development.

The RacGAP ß2-Chimaerin selectively mediates axonal pruning in the hippocampus.

Axon pruning and synapse elimination promote neural connectivity and synaptic plasticity. Stereotyped pruning of axons that originate in the hippocampal dentate gyrus (DG) and extend along the infrapyramidal tract (IPT) occurs during postnatal murine development by neurite retraction and resembles axon repulsion. The chemorepellent Sema3F is required for IPT axon pruning, dendritic spine remodeling, and repulsion of DG axons. The signaling events that regulate IPT axon pruning are not known. We find that inhibition of the small G protein Rac1 by the Rac GTPase-activating protein (GAP) ß2-Chimaerin (ß2Chn) mediates Sema3F-dependent pruning. The Sema3F receptor neuropilin-2 selectively binds ß2Chn, and ligand engagement activates this GAP to ultimately restrain Rac1-dependent effects on cytoskeletal reorganization. ß2Chn is necessary for axon pruning both in vitro and in vivo, but it is dispensable for axon repulsion and spine remodeling. Therefore, a Npn2/ß2Chn/Rac1 signaling axis distinguishes DG axon pruning from the effects of Sema3F on repulsion and dendritic spine remodeling.

Complexin has a dual synaptic function as checkpoint protein in vesicle priming and as a promoter of vesicle fusion.

The presynaptic SNARE-complex regulator complexin (Cplx) enhances the fusogenicity of primed synaptic vesicles (SVs). Consequently, Cplx deletion impairs action potential-evoked transmitter release. Conversely, though, Cplx loss enhances spontaneous and delayed asynchronous release at certain synapse types. Using electrophysiology and kinetic modeling, we show that such seemingly contradictory transmitter release phenotypes seen upon Cplx deletion can be explained by an additional of Cplx in the control of SV priming, where its ablation facilitates the generation of a "faulty" SV fusion apparatus. Supporting this notion, a sequential two-step priming scheme, featuring reduced vesicle fusogenicity and increased transition rates into the faulty primed state, reproduces all aberrations of transmitter release modes and short-term synaptic plasticity seen upon Cplx loss. Accordingly, we propose a dual presynaptic function for the SNARE-complex interactor Cplx, one as a "checkpoint" protein that guarantees the proper assembly of the fusion machinery during vesicle priming, and one in boosting vesicle fusogenicity.

GABAB receptors induce phasic release from medial habenula terminals through activity-dependent recruitment of release-ready vesicles.

GABAB receptor (GBR) activation inhibits neurotransmitter release in axon terminals in the brain, except in medial habenula (MHb) terminals, which show robust potentiation. However, mechanisms underlying this enigmatic potentiation remain elusive. Here, we report that GBR activation on MHb terminals induces an activity-dependent transition from a facilitating, tonic to a depressing, phasic neurotransmitter release mode. This transition is accompanied by a 4.1-fold increase in readily releasable vesicle pool (RRP) size and a 3.5-fold increase of docked synaptic vesicles (SVs) at the presynaptic active zone (AZ). Strikingly, the depressing phasic release exhibits looser coupling distance than the tonic release. Furthermore, the tonic and phasic release are selectively affected by deletion of synaptoporin (SPO) and Ca2+-dependent activator protein for secretion 2 (CAPS2), respectively. SPO modulates augmentation, the short-term plasticity associated with tonic release, and CAPS2 retains the increased RRP for initial responses in phasic response trains. The cytosolic protein CAPS2 showed a SV-associated distribution similar to the vesicular transmembrane protein SPO, and they were colocalized in the same terminals. We developed the "Flash and Freeze-fracture" method, and revealed the release of SPO-associated vesicles in both tonic and phasic modes and activity-dependent recruitment of CAPS2 to the AZ during phasic release, which lasted several minutes. Overall, these results indicate that GBR activation translocates CAPS2 to the AZ along with the fusion of CAPS2-associated SVs, contributing to persistency of the RRP increase. Thus, we identified structural and molecular mechanisms underlying tonic and phasic neurotransmitter release and their transition by GBR activation in MHb terminals.

A fluorescent nanosensor paint detects dopamine release at axonal varicosities with high spatiotemporal resolution.

The neurotransmitter dopamine (DA) controls multiple behaviors and is perturbed in several major brain diseases. DA is released from large populations of specialized structures called axon varicosities. Determining the DA release mechanisms at such varicosities is essential for a detailed understanding of DA biology and pathobiology but has been limited by the low spatial resolution of DA detection methods. We used a near-infrared fluorescent DA nanosensor paint, adsorbed nanosensors detecting release of dopamine (AndromeDA), to detect DA secretion from cultured murine dopaminergic neurons with high spatial and temporal resolution. We found that AndromeDA detects discrete DA release events and extracellular DA diffusion and observed that DA release varies across varicosities. To systematically detect DA release hotspots, we developed a machine learning­based analysis tool. AndromeDA permitted the simultaneous visualization of DA release for up to 100 dopaminergic varicosities, showing that DA release hotspots are heterogeneous and occur at only ∼17% of all varicosities, indicating that many varicosities are functionally silent. Using AndromeDA, we determined that DA release requires Munc13-type vesicle priming proteins, validating the utility of AndromeDA as a tool to study the molecular and cellular mechanism of DA secretion.

Flowers for synaptic endocytosis.

Exocytosis and endocytosis of synaptic vesicles are tightly coordinated to maintain a steady supply of new vesicles during periods of extended neuronal stimulation. Yao et al. (2009) now report that a synaptic vesicle membrane protein named Flower promotes endocytosis at neuromuscular junctions in the fruit fly Drosophila.

Synaptic PRG-1 modulates excitatory transmission via lipid phosphate-mediated signaling.

Plasticity related gene-1 (PRG-1) is a brain-specific membrane protein related to lipid phosphate phosphatases, which acts in the hippocampus specifically at the excitatory synapse terminating on glutamatergic neurons. Deletion of prg-1 in mice leads to epileptic seizures and augmentation of EPSCs, but not IPSCs. In utero electroporation of PRG-1 into deficient animals revealed that PRG-1 modulates excitation at the synaptic junction. Mutation of the extracellular domain of PRG-1 crucial for its interaction with lysophosphatidic acid (LPA) abolished the ability to prevent hyperexcitability. As LPA application in vitro induced hyperexcitability in wild-type but not in LPA(2) receptor-deficient animals, and uptake of phospholipids is reduced in PRG-1-deficient neurons, we assessed PRG-1/LPA(2) receptor-deficient animals, and found that the pathophysiology observed in the PRG-1-deficient mice was fully reverted. Thus, we propose PRG-1 as an important player in the modulatory control of hippocampal excitability dependent on presynaptic LPA(2) receptor signaling.

De Novo Missense Variants in SLC32A1 Cause a Developmental and Epileptic Encephalopathy Due to Impaired GABAergic Neurotransmission.

OBJECTIVE: Rare inherited missense variants in SLC32A1, the gene that encodes the vesicular gamma-aminobutyric acid (GABA) transporter, have recently been shown to cause genetic epilepsy with febrile seizures plus. We aimed to clarify if de novo missense variants in SLC32A1 can also cause epilepsy with impaired neurodevelopment. METHODS: Using exome sequencing, we identified four individuals with a developmental and epileptic encephalopathy and de novo missense variants in SLC32A1. To assess causality, we performed functional evaluation of the identified variants in a murine neuronal cell culture model. RESULTS: The main phenotype comprises moderate-to-severe intellectual disability, infantile-onset epilepsy within the first 18 months of life, and a choreiform, dystonic, or dyskinetic movement disorder. In silico modeling and functional analyses reveal that three of these variants, which are located in helices that line the putative GABA transport pathway, result in reduced quantal size, consistent with impaired filling of synaptic vesicles with GABA. The fourth variant, located in the vesicular gamma-aminobutyric acid N-terminus, does not affect quantal size, but increases presynaptic release probability, leading to more severe synaptic depression during high-frequency stimulation. Thus, variants in vesicular gamma-aminobutyric acid can impair GABAergic neurotransmission through at least two mechanisms, by affecting synaptic vesicle filling and by altering synaptic short-term plasticity. INTERPRETATION: This work establishes de novo missense variants in SLC32A1 as a novel cause of a developmental and epileptic encephalopathy. SUMMARY FOR SOCIAL MEDIA IF PUBLISHED: @platzer_k @lemke_johannes @RamiJamra @Nirgalito @GeneDx The SLC family 32 Member 1 (SLC32A1) is the only protein identified to date, that loads gamma-aminobutyric acid (GABA) and glycine into synaptic vesicles, and is therefore also known as the vesicular GABA transporter (VGAT) or vesicular inhibitory amino acid transporter (VIAAT). Rare inherited missense variants in SLC32A1, the gene that encodes VGAT/vesicular inhibitory amino acid transporter, have recently been shown to cause genetic epilepsy with febrile seizures plus. We aimed to clarify if de novo missense variants in SLC32A1 can also cause epilepsy with impaired neurodevelopment. We report on four individuals with de novo missense variants in SLC32A1 and a developmental and epileptic encephalopathy with infantile onset epilepsy. We establish causality of the variants via in silico modeling and their functional evaluation in a murine neuronal cell culture model. SLC32A1 variants represent a novel genetic etiology in neurodevelopmental disorders with epilepsy and a new GABA-related disease mechanism. ANN NEUROL 2022;92:958-973.

The α3 subunit of GABAA receptors promotes formation of inhibitory synapses in the absence of collybistin.

Signaling at nerve cell synapses is a key determinant of proper brain function, and synaptic defects-or synaptopathies-are at the basis of many neurological and psychiatric disorders. Collybistin (CB), a brain-specific guanine nucleotide exchange factor, is essential for the formation of γ-aminobutyric acidergic (GABAergic) postsynapses in defined regions of the mammalian forebrain, including the hippocampus and basolateral amygdala. This process depends on a direct interaction of CB with the scaffolding protein gephyrin, which leads to the redistribution of gephyrin into submembranous clusters at nascent inhibitory synapses. Strikingly, synaptic clustering of gephyrin and GABAA type A receptors (GABAARs) in several brain regions, including the cerebral cortex and certain thalamic areas, is unperturbed in CB-deficient mice, indicating that the formation of a substantial subset of inhibitory postsynapses must be controlled by gephyrin-interacting proteins other than CB. Previous studies indicated that the α3 subunit of GABAARs (GABAAR-α3) binds directly and with high affinity to gephyrin. Here, we provide evidence (i) that a homooligomeric GABAAR-α3A343W mutant induces the formation of submembranous gephyrin clusters independently of CB in COS-7 cells, (ii) that gephyrin clustering is unaltered in the neuronal subpopulations endogenously expressing the GABAAR-α3 in CB-deficient brains, and (iii) that exogenous expression of GABAAR-α3 partially rescues impaired gephyrin clustering in CB-deficient hippocampal neurons. Our results identify an important role of GABAAR-α3 in promoting gephyrin-mediated and CB-independent formation of inhibitory postsynapses.

Two Functional Epithelial Sodium Channel Isoforms Are Present in Rodents despite Pronounced Evolutionary Pseudogenization and Exon Fusion.

The epithelial sodium channel (ENaC) plays a key role in salt and water homeostasis in tetrapod vertebrates. There are four ENaC subunits (α, ß, γ, δ), forming heterotrimeric αßγ- or δßγ-ENaCs. Although the physiology of αßγ-ENaC is well understood, for decades the field has stalled with respect to δßγ-ENaC due to the lack of mammalian model organisms. The SCNN1D gene coding for δ-ENaC was previously believed to be absent in rodents, hindering studies using standard laboratory animals. We analyzed all currently available rodent genomes and discovered that SCNN1D is present in rodents but was independently lost in five rodent lineages, including the Muridae (mice and rats). The independent loss of SCNN1D in rodent lineages may be constrained by phylogeny and taxon-specific adaptation to dry habitats, however habitat aridity does not provide a selection pressure for maintenance of SCNN1D across Rodentia. A fusion of two exons coding for a structurally flexible region in the extracellular domain of δ-ENaC appeared in the Hystricognathi (a group that includes guinea pigs). This conserved pattern evolved at least 41 Ma and represents a new autapomorphic feature for this clade. Exon fusion does not impair functionality of guinea pig (Cavia porcellus) δßγ-ENaC expressed in Xenopus oocytes. Electrophysiological characterization at the whole-cell and single-channel level revealed conserved biophysical features and mechanisms controlling guinea pig αßγ- and δßγ-ENaC function as compared with human orthologs. Guinea pigs therefore represent commercially available mammalian model animals that will help shed light on the physiological function of δ-ENaC.

Neuroligin-2 dependent conformational activation of collybistin reconstituted in supported hybrid membranes.

The assembly of the postsynaptic transmitter sensing machinery at inhibitory nerve cell synapses requires the intimate interplay between cell adhesion proteins, scaffold and adaptor proteins, and γ-aminobutyric acid (GABA) or glycine receptors. We developed an in vitro membrane system to reconstitute this process, to identify the essential protein components, and to define their mechanism of action, with a specific focus on the mechanism by which the cytosolic C terminus of the synaptic cell adhesion protein Neuroligin-2 alters the conformation of the adaptor protein Collybistin-2 and thereby controls Collybistin-2-interactions with phosphoinositides (PtdInsPs) in the plasma membrane. Supported hybrid membranes doped with different PtdInsPs and 1,2-dioleoyl-sn-glycero-3-{[N-(5-amino-1-carboxypentyl)iminodiacetic acid]succinyl} nickel salt (DGS-NTA(Ni)) to allow for the specific adsorption of the His6-tagged intracellular domain of Neuroligin-2 (His-cytNL2) were prepared on hydrophobically functionalized silicon dioxide substrates via vesicle spreading. Two different collybistin variants, the WT protein (CB2SH3) and a mutant that adopts an intrinsically 'open' and activated conformation (CB2SH3/W24A-E262A), were bound to supported membranes in the absence or presence of His-cytNL2. The corresponding binding data, obtained by reflectometric interference spectroscopy, show that the interaction of the C terminus of Neuroligin-2 with Collybistin-2 induces a conformational change in Collybistin-2 that promotes its interaction with distinct membrane PtdInsPs.

Evolution of the Autism-Associated Neuroligin-4 Gene Reveals Broad Erosion of Pseudoautosomal Regions in Rodents.

Variants in genes encoding synaptic adhesion proteins of the neuroligin family, most notably neuroligin-4, are a significant cause of autism spectrum disorders in humans. Although human neuroligin-4 is encoded by two genes, NLGN4X and NLGN4Y, that are localized on the X-specific and male-specific regions of the two sex chromosomes, the chromosomal localization and full genomic sequence of the mouse Nlgn4 gene remain elusive. Here, we analyzed the neuroligin-4 genes of numerous rodent species by direct sequencing and bioinformatics, generated complete drafts of multiple rodent neuroligin-4 genes, and examined their evolution. Surprisingly, we find that the murine Nlgn4 gene is localized to the pseudoautosomal region (PAR) of the sex chromosomes, different from its human orthologs. We show that the sequence differences between various neuroligin-4 proteins are restricted to hotspots in which rodent neuroligin-4 proteins contain short repetitive sequence insertions compared with neuroligin-4 proteins from other species, whereas all other protein sequences are highly conserved. Evolutionarily, these sequence insertions initiate in the clade eumuroidea of the infraorder myomorpha and are additionally associated with dramatic changes in noncoding sequences and gene size. Importantly, these changes are not exclusively restricted to neuroligin-4 genes but reflect major evolutionary changes that substantially altered or even deleted genes from the PARs of both sex chromosomes. Our results show that despite the fact that the PAR in rodents and the neuroligin-4 genes within the rodent PAR underwent massive evolutionary changes, neuroligin-4 proteins maintained a highly conserved core structure, consistent with a substantial evolutionary pressure preserving its physiological function.

Deletion of LRRTM1 and LRRTM2 in adult mice impairs basal AMPA receptor transmission and LTP in hippocampal CA1 pyramidal neurons.

Leucine-rich repeat transmembrane (LRRTM) proteins are synaptic cell adhesion molecules that influence synapse formation and function. They are genetically associated with neuropsychiatric disorders, and via their synaptic actions likely regulate the establishment and function of neural circuits in the mammalian brain. Here, we take advantage of the generation of a LRRTM1 and LRRTM2 double conditional knockout mouse (LRRTM1,2 cKO) to examine the role of LRRTM1,2 at mature excitatory synapses in hippocampal CA1 pyramidal neurons. Genetic deletion of LRRTM1,2 in vivo in CA1 neurons using Cre recombinase-expressing lentiviruses dramatically impaired long-term potentiation (LTP), an impairment that was rescued by simultaneous expression of LRRTM2, but not LRRTM4. Mutation or deletion of the intracellular tail of LRRTM2 did not affect its ability to rescue LTP, while point mutations designed to impair its binding to presynaptic neurexins prevented rescue of LTP. In contrast to previous work using shRNA-mediated knockdown of LRRTM1,2, KO of these proteins at mature synapses also caused a decrease in AMPA receptor-mediated, but not NMDA receptor-mediated, synaptic transmission and had no detectable effect on presynaptic function. Imaging of recombinant photoactivatable AMPA receptor subunit GluA1 in the dendritic spines of cultured neurons revealed that it was less stable in the absence of LRRTM1,2. These results illustrate the advantages of conditional genetic deletion experiments for elucidating the function of endogenous synaptic proteins and suggest that LRRTM1,2 proteins help stabilize synaptic AMPA receptors at mature spines during basal synaptic transmission and LTP.

SUMOylation controls the neurodevelopmental function of the transcription factor Zbtb20.

SUMOylation is a dynamic post-translational protein modification that primarily takes place in cell nuclei, where it plays a key role in multiple DNA-related processes. In neurons, the SUMOylation-dependent control of a subset of neuronal transcription factors is known to regulate various aspects of nerve cell differentiation, development, and function. In an unbiased screen for endogenous SUMOylation targets in the developing mouse brain, based on a His6 -HA-SUMO1 knock-in mouse line, we previously identified the transcription factor Zinc finger and BTB domain-containing 20 (Zbtb20) as a new SUMO1-conjugate. We show here that the three key SUMO paralogues SUMO1, SUMO2, and SUMO3 can all be conjugated to Zbtb20 in vitro in HEK293FT cells, and we confirm the SUMOylation of Zbtb20 in vivo in mouse brain. Using primary hippocampal neurons from wild-type and Zbtb20 knock-out (KO) mice as a model system, we then demonstrate that the expression of Zbtb20 is required for proper nerve cell development and neurite growth and branching. Furthermore, we show that the SUMOylation of Zbtb20 is essential for its function in this context, and provide evidence indicating that SUMOylation affects the Zbtb20-dependent transcriptional profile of neurons. Our data highlight the role of SUMOylation in the regulation of neuronal transcription factors that determine nerve cell development, and they demonstrate that key functions of the transcription factor Zbtb20 in neuronal development and neurite growth are under obligatory SUMOylation control.

Hair cell synaptic dysfunction, auditory fatigue and thermal sensitivity in otoferlin Ile515Thr mutants.

The multi-C2 domain protein otoferlin is required for hearing and mutated in human deafness. Some OTOF mutations cause a mild elevation of auditory thresholds but strong impairment of speech perception. At elevated body temperature, hearing is lost. Mice homozygous for one of these mutations, OtofI515T/I515T, exhibit a moderate hearing impairment involving enhanced adaptation to continuous or repetitive sound stimulation. In OtofI515T/I515T inner hair cells (IHCs), otoferlin levels are diminished by 65%, and synaptic vesicles are enlarged. Exocytosis during prolonged stimulation is strongly reduced. This indicates that otoferlin is critical for the reformation of properly sized and fusion-competent synaptic vesicles. Moreover, we found sustained exocytosis and sound encoding to scale with the amount of otoferlin at the plasma membrane. We identified a 20 amino acid motif including an RXR motif, presumably present in human but not in mouse otoferlin, which reduces the plasma membrane abundance of Ile515Thr-otoferlin. Together, this likely explains the auditory synaptopathy at normal temperature and the temperature-sensitive deafness in humans carrying the Ile515Thr mutation.

Autism Related Neuroligin-4 Knockout Impairs Intracortical Processing but not Sensory Inputs in Mouse Barrel Cortex.

Neuroligin-4 (Nlgn4) is a cell adhesion protein that regulates synapse organization and function. Mutations in human NLGN4 are among the causes of autism spectrum disorders. In mouse, Nlgn4 knockout (KO) perturbs GABAergic synaptic transmission and oscillatory activity in hippocampus, and causes social interaction deficits. The complex profile of cellular and circuit changes that are caused by Nlgn4-KO is still only partly understood. Using Nlgn4-KO mice, we found that Nlgn4-KO increases the power in the alpha frequency band of spontaneous network activity in the barrel cortex under urethane anesthesia in vivo. Nlgn4-KO did not affect single-whisker-induced local field potentials, but suppressed the late evoked multiunit activity in vivo. Although Nlgn4-KO did not affect evoked EPSCs in layer 4 (L4) spiny stellate cells in acute thalamocortical slices elicited by electrical stimulation of thalamocortical inputs, it caused a lower frequency of both miniature (m) IPSCs and mEPSCs, and a decrease in the number of readily releasable vesicles at GABAergic and glutamatergic connections, weakening both excitatory and inhibitory transmission. However, Nlgn4 deficit strongly suppresses glutamatergic activity, shifting the excitation-inhibition balance to inhibition. We conclude that Nlgn4-KO does not influence the incoming whisker-mediated sensory information to the barrel cortex, but modifies intracortical information processing.

Endosomal Phosphatidylinositol 3-Phosphate Promotes Gephyrin Clustering and GABAergic Neurotransmission at Inhibitory Postsynapses.

The formation of neuronal synapses and the dynamic regulation of their efficacy depend on the proper assembly of the postsynaptic neurotransmitter receptor apparatus. Receptor recruitment to inhibitory GABAergic postsynapses requires the scaffold protein gephyrin and the guanine nucleotide exchange factor collybistin (Cb). In vitro, the pleckstrin homology domain of Cb binds phosphoinositides, specifically phosphatidylinositol 3-phosphate (PI3P). However, whether PI3P is required for inhibitory postsynapse formation is currently unknown. Here, we investigated the role of PI3P at developing GABAergic postsynapses by using a membrane-permeant PI3P derivative, time-lapse confocal imaging, electrophysiology, as well as knockdown and overexpression of PI3P-metabolizing enzymes. Our results provide the first in cellula evidence that PI3P located at early/sorting endosomes regulates the postsynaptic clustering of gephyrin and GABAA receptors and the strength of inhibitory, but not excitatory, postsynapses in cultured hippocampal neurons. In human embryonic kidney 293 cells, stimulation of gephyrin cluster formation by PI3P depends on Cb. We therefore conclude that the endosomal pool of PI3P, generated by the class III phosphatidylinositol 3-kinase, is important for the Cb-mediated recruitment of gephyrin and GABAA receptors to developing inhibitory postsynapses and thus the formation of postsynaptic membrane specializations.

A conformational switch in collybistin determines the differentiation of inhibitory postsynapses.

The formation of neuronal synapses and the dynamic regulation of their efficacy depend on the assembly of the postsynaptic neurotransmitter receptor apparatus. Receptor recruitment to inhibitory GABAergic and glycinergic synapses is controlled by the scaffold protein gephyrin and the adaptor protein collybistin. We derived new insights into the structure of collybistin and used these to design biochemical, cell biological, and genetic analyses of collybistin function. Our data define a collybistin-based protein interaction network that controls the gephyrin content of inhibitory postsynapses. Within this network, collybistin can adopt open/active and closed/inactive conformations to act as a switchable adaptor that links gephyrin to plasma membrane phosphoinositides. This function of collybistin is regulated by binding of the adhesion protein neuroligin-2, which stabilizes the open/active conformation of collybistin at the postsynaptic plasma membrane by competing with an intramolecular interaction in collybistin that favors the closed/inactive conformation. By linking trans-synaptic neuroligin-dependent adhesion and phosphoinositide signaling with gephyrin recruitment, the collybistin-based regulatory switch mechanism represents an integrating regulatory node in the formation and function of inhibitory postsynapses.

Proteomic screening of glutamatergic mouse brain synaptosomes isolated by fluorescence activated sorting.

For decades, neuroscientists have used enriched preparations of synaptic particles called synaptosomes to study synapse function. However, the interpretation of corresponding data is problematic as synaptosome preparations contain multiple types of synapses and non-synaptic neuronal and glial contaminants. We established a novel Fluorescence Activated Synaptosome Sorting (FASS) method that substantially improves conventional synaptosome enrichment protocols and enables high-resolution biochemical analyses of specific synapse subpopulations. Employing knock-in mice with fluorescent glutamatergic synapses, we show that FASS isolates intact ultrapure synaptosomes composed of a resealed presynaptic terminal and a postsynaptic density as assessed by light and electron microscopy. FASS synaptosomes contain bona fide glutamatergic synapse proteins but are almost devoid of other synapse types and extrasynaptic or glial contaminants. We identified 163 enriched proteins in FASS samples, of which FXYD6 and Tpd52 were validated as new synaptic proteins. FASS purification thus enables high-resolution biochemical analyses of specific synapse subpopulations in health and disease.