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Synaptic activity imposes large energy demands that are met by local adenosine triphosphate (ATP) synthesis through glycolysis and mitochondrial oxidative phosphorylation. ATP drives action potentials, supports synapse assembly and remodelling, and fuels synaptic vesicle filling and recycling, thus sustaining synaptic transmission. Given their polarized morphological features — including long axons and extensive branching in their terminal regions — neurons face exceptional challenges in maintaining presynaptic energy homeostasis, particularly during intensive synaptic activity. Recent studies have started to uncover the mechanisms and signalling pathways involved in activity-dependent and energy-sensitive regulation of presynaptic energetics, or ‘synaptoenergetics’. These conceptual advances have established the energetic regulation of synaptic efficacy and plasticity as an exciting research field that is relevant to a range of neurological disorders associated with bioenergetic failure and synaptic dysfunction. Numerous energy-demanding cellular processes contribute to synaptic activity and function. Li and Sheng describe the mechanisms that regulate presynaptic energy supply to ensure that neurons can meet these demands and maintain their functions during periods of intensive synaptic activity.

To gain insight into the potential protective mechanisms of low phenylalanine diet (LPD) in phenylketonuria (PKU), gene expression profiles were studied in the cerebral cortex and hippocampus of a PKU mouse model (BTBR-Pahenu2). PKU mice were fed with low Phe diet (LPD-PKU group) and normal diet (PKU group). Wild-type mice were treated with normal diet (WT group) as control. After 12 weeks, we detected gene expression in the cerebral cortex and hippocampus of the three groups by RNA-sequencing, and then screened the differentially-expressed genes (DEGs) among the groups by bioinformatics analyses. We found that the transcriptional profiles of both cerebral cortex and hippocampus changed markedly between PKU and WT mice. Furthermore, LPD changed the transcriptional profiles of the cerebral cortex and the hippocampus of PKU mice significantly, especially in the cerebral cortex, with overlaps of genes that changed with the disease and altered by LPD treatment. In the cerebral cortex, hundreds of DEGs enriched in a wide spectrum of biological processes, molecular function, and cellular component, including nervous system development, axon development and guidance, calcium ion binding, modulation of chemical synaptic transmission, and regulation of protein kinase activity. In the hippocampus, the overlapping genes were enriched in positive regulation of long term synaptic, negative regulation of excitatory postsynaptic potential, positive regulation of synapse assembly. Our results showed that genes impaired in PKU and then rescued by LPD might indicate the potential protective capability of LPD in the PKU brain.

The precise control of the nervous system function under the vitality of synapses is extremely critical. Efforts have been taken to explore the underlying cellular and molecular mechanisms for synapse formation. Cell adhesion molecules have been found important for synapse assembly in the brain. Many trans-adhesion complexes have been identified to modulate excitatory synapse formation. However, little is known about the synaptogenic mechanisms for inhibitory synapses. ErbB4 is a receptor tyrosine kinase enriched in interneurons. Here, we showed that overexpressing ErbB4 in HEK293T cells induced gephyrin or GABAAR α1 puncta in co-cultured primary hippocampal neurons. This induction of ErbB4 was independent of its kinase activity. K751M, a kinase-dead mutant of ErbB4, can also induce gephyrin or GABAAR α1 puncta in the co-culture system. We further constructed K751M knock-in mice and found that the homozygous were viable at birth and fertile without changes in gross brain structure. The number of interneurons and inhibitory synapses onto pyramidal neurons (PyNs) were comparable between K751M and wild-type mice but decreased in ErbB4-Null mice. Moreover, ErbB4 can interact in trans with Slitrk3, a transmembrane postsynaptic protein at inhibitory synapses, through the extracellular RLD domain of ErbB4. The deletion of RLD diminished the induction of gephyrin or GABAAR α1 puncta by ErbB4. Finally, disruption of ErbB4–Slitrk3 interaction through neutralization of Slitrk3 by secretable RLD decreased inhibitory synapses onto PyNs and impaired GABAergic transmission. These results identify that ErbB4, as a cell adhesion molecule, promotes inhibitory synapse formation onto PyNs by interacting with Slitrk3 and in a kinase-independent manner, providing an unexpected mechanism of ErbB4 in inhibitory synapse formation.

The site-specific recombinase Tn3 resolvase initiates DNA strand exchange when two res recombination sites and six resolvase dimers interact to form a synapse. The detailed architecture of this intricate recombination machine remains unclear. We have clarified which of the potential dimer-dimer interactions are required for synapsis and recombination, using a novel complementation strategy that exploits a previously uncharacterized resolvase from Bartonella bacilliformis ("Bart"). Tn3 and Bart resolvases recognize different DNA motifs, via diverged C-terminal domains (CTDs). They also differ substantially at N-terminal domain (NTD) surfaces involved in dimerization and synapse assembly. We designed NTD-CTD hybrid proteins, and hybrid res sites containing both Tn3 and Bart dimer binding sites. Using these components in in vivo assays, we demonstrate that productive synapsis requires a specific "R" interface involving resolvase NTDs at all three dimer-binding sites in res. Synapses containing mixtures of wild-type Tn3 and Bart resolvase NTD dimers are recombination-defective, but activity can be restored by replacing patches of Tn3 resolvase R interface residues with Bart residues, or vice versa. We conclude that the Tn3/Bart family synapse is assembled exclusively by R interactions between resolvase dimers, except for the one special dimer-dimer interaction required for catalysis.

Children with repeated inhalational anesthesia may develop cognitive disorders. This study aimed to investigate the transcriptome-wide response of hippocampus in young mice that had been exposed to multiple sevoflurane in the neonatal period. Mice received 3% sevoflurane for 2 h on postnatal day (PND) 6, 8, and 10, followed by arterial blood gas test on PND 10, behavioral experiments on PND 31-36, and RNA sequencing (RNA-seq) of hippocampus on PND 37. Functional annotation and protein-protein interaction analyses of differentially expressed genes (DEGs) and quantitative reverse transcription polymerase chain reaction (qPCR) were performed. Neonatal sevoflurane exposures induced cognitive and social behavior disorders in young mice. RNA-seq identified a total of 314 DEGs. Several enriched biological processes (ion channels, brain development, learning, and memory) and signaling pathways (oxytocin signaling pathway and glutamatergic, cholinergic, and GABAergic synapses) were highlighted. As hub-proteins, Pten was involved in nervous system development, synapse assembly, learning, memory, and behaviors, Nos3 and Pik3cd in oxytocin signaling pathway, and Cdk16 in exocytosis and phosphorylation. Some top DEGs were validated by qPCR. This study revealed a transcriptome-wide profile in mice hippocampus after multiple neonatal exposures to sevoflurane, promoting better understanding of underlying mechanisms and investigation of preventive strategies.

PSD-95 is a scaffolding protein that regulates the synaptic localization of many receptors, channels, and signaling proteins. The NLGN gene family encodes single-pass transmembrane postsynaptic cell adhesion molecules that are important for synapse assembly and function. At excitatory synapses, NLGN1 mediates transsynaptic binding with neurexin, a presynaptic cell adhesion molecule, and also binds to PSD-95, although the relevance of the PSD-95 interaction is not clear. We now show that disruption of the NLGN1 and PSD-95 interaction decreases surface expression of NLGN1 in cultured neurons. Furthermore, PKA phosphorylates NLGN1 on S839, near the PDZ ligand, and dynamically regulates PSD-95 binding. A phosphomimetic mutation of NLGN1 S839 significantly reduced PSD-95 binding. Impaired NLGN1/PSD-95 binding diminished synaptic NLGN1 expression and NLGN1-mediated synaptic enhancement. Our results establish a phosphorylation-dependent molecular mechanism that regulates NLGN1 and PSD-95 binding and provides insights into excitatory synaptic development and function.

Early exposure to anesthetics may interfere with synaptic development and lead to cognitive deficits. We previously demonstrated a decrease in vesicles docked at and within 100 nm from the presynaptic membrane in hippocampal nerve terminals of neonatal rats after anesthesia. Hence, we designed this study to assess the effects of neonatal anesthesia on Synapsin 1 (Syn 1) and Synaptotagmin 1 (Syt 1), two key regulators of vesicle docking and fusion. To test the link between changes in Syn 1 and Syt 1 and behavioral deficits observed after neonatal anesthesia, we also assessed retention memory and fear conditioning in adolescent rats after neonatal anesthesia. Pups received a combination of clinical anesthetics, then Syn 1 and Syt 1 mRNA and protein expression were determined at the peak (postnatal day 8, P8), part-way through (P12) and end of synaptogenesis (P24) in the CA1-subiculum by qPCR and Western Blotting. Anesthesia decreased Syn1 and Syt1 mRNA expression at P8 (p

Components of the intraflagellar transport (IFT) system that regulates the assembly of the primary cilium are co-opted by the non-ciliated T cell to orchestrate polarized endosome recycling and to sustain signaling during immune synapse formation. Here, we investigated the potential role of Bardet–Biedl syndrome 1 protein (BBS1), an essential core component of the BBS complex that cooperates with the IFT system in ciliary protein trafficking, in the assembly of the T cell synapse. We demonstrated that BBS1 allows for centrosome polarization towards the immune synapse. This function is achieved through the clearance of centrosomal F-actin and its positive regulator WASH1 (also known as WASHC1), a process that we demonstrated to be dependent on the proteasome. We show that BBS1 regulates this process by coupling the 19S proteasome regulatory subunit to the microtubule motor dynein for its transport to the centrosome. Our data identify the ciliopathy-related protein BBS1 as a new player in T cell synapse assembly that functions upstream of the IFT system to set the stage for polarized vesicular trafficking and sustained signaling. This article has an associated First Person interview with the first author of the paper.

Purpose Serine protease inhibitors (SERPINs) family has been discovered in many disorders with proteolysis mechanisms. Our study determined the SERPINBs protein expression via public‐based GEO databases and further validated by peri‐implant crevicular fluid (PICF) of peri‐implantitis patients and healthy recruiters. Methods This study is a retrospective analysis. A total of 123 participants of Fujian Medical University Fujian Stomatological Hospital, consisting of 58 cases of peri‐implantitis and 65 samples of healthy control were retrospectively analyzed by ELISA assays and explored the gene enrichment pathways and clinical significance of SERPINBs expression accompanied by two different cytokines (IL‐6 and TNF‐α). Moreover, the clinical significance of SERPINBs was evaluated in peri‐implantitis patients with PICF samples by the receiver operating curve (ROC) using the area under the curve (AUC). Results KEGG database showed that Starch and sucrose metabolism, Retrograde endocannabinoid signaling, Prion diseases, Pentose phosphate pathways, and Olfactory pathways are up‐regulated; GO database showed that synapse organization, synapse assembly, sequestering of triglyceride, sensory perception of smell, and regulation of synapse organization pathways are up‐regulated. SERPINBs were overexpressed in peri‐implant tissues and peri‐implantitis patients with PICF. SERPINBs was positively correlated to IL‐6 and TNF‐α in peri‐implantitis patients with PICF. The ROC‐AUCs of SERPINBs achieved a significantly higher range from 0.895 to 0.939 in peri‐implantitis patients with PICF. Therefore, certain SERPINBs expressions were not only perceived through PICF and peri‐implant tissues but also showed potential significance in peri‐implantitis. Conclusion SERPINBs play an influential role in the pathogenesis of peri‐implantitis via binding with other inflammatory cytokines., Figure 4. Comparison of the four SERPINBs in PICF of peri‐implantitis and healthy controls. (A) SERPINB1, (B) SERPINB3, (C) SERPINB4, (D) SERPINB5 were analyzed by scatter plots.

Thomas Südhof discusses the cell-biological principles underlying the assembly of specific synaptic connections with defined properties that control neural circuits., In a neural circuit, synapses transfer information rapidly between neurons and transform this information during transfer. The diverse computational properties of synapses are shaped by the interactions between pre- and postsynaptic neurons. How synapses are assembled to form a neural circuit, and how the specificity of synaptic connections is achieved, is largely unknown. Here, I posit that synaptic adhesion molecules (SAMs) organize synapse formation. Diverse SAMs collaborate to achieve the astounding specificity and plasticity of synapses, with each SAM contributing different facets. In orchestrating synapse assembly, SAMs likely act as signal transduction devices. Although many candidate SAMs are known, only a few SAMs appear to have a major impact on synapse formation. Thus, a limited set of collaborating SAMs likely suffices to account for synapse formation. Strikingly, several SAMs are genetically linked to neuropsychiatric disorders, suggesting that impairments in synapse assembly are instrumental in the pathogenesis of neuropsychiatric disorders.

The T cell Immunological Synapse (IS) is a pivotal hub for the regulation of adaptive immunity by endowing the exchange of information between cells engaged in physical contacts. Beyond the integration of antigen (signal one), co-stimulation (signal two), and cytokines (signal three), the IS facilitates the delivery of T-cell effector assemblies including supramolecular attack particles (SMAPs) and extracellular vesicles (EVs). How these particulate outputs differ among T -cell subsets and how subcellular compartments and signals exchanged at the synapse contribute to their composition is not fully understood. Here we harnessed bead-supported lipid bilayers (BSLBs) as a tailorable and versatile technology for the study of synaptic particle biogenesis and composition in different T-cell subsets, including CART. These synthetic antigen-presenting cells (APCs) facilitated the characterisation of trans-synaptic vesicles (tSV) as a heterogeneous population of EVs comprising among others PM-derived synaptic ectosomes and CD63+ exosomes. We harnessed BSLB to unveil the factors influencing the vesicular release of CD40L, as a model effector, identifying CD40 trans presentation, T-cell activation, ESCRT upregulation/recruitment, antigen density/potency, co-repression by PD-1 ligands, and its processing by ADAM10 as major determinants. Further, BSLB made possible the comparison of microRNA (miR) species associated with tSV and steadily released EVs. Altogether, our data provide evidence for a higher specialisation of tSV which are enriched not only in effector immune receptors but also in miR and RNA-binding proteins. Considering the molecular uniqueness and functional complexity of the tSV output, which is also accompanied by SMAPs, we propose their classification as signal four.Graphical abstractHighlightsBead Supported Lipid Bilayers (BSLB) reconstituting antigen-presenting cells support synapse assembly by T cells and the release of effector particles.BSLB facilitate the dissection of the cellular machineries and synapse composition shaping the released tSV.tSV and their steadily released counterparts have a different composition. TSV show a higher enrichment of effectors including immune receptors, miR, RNA- and other nucleic acid-binding proteins, than EVs.

The post-glacial colonization of Gander Lake in Newfoundland, Canada, by Arctic Charr (Salvelinus alpinus) provides the opportunity to study the genomic basis of adaptation to extreme deep-water environments. Colonization of deep-water (>50 m) habitats often requires extensive adaptation to cope with novel environmental challenges from high hydrostatic pressure, low temperature, and low light, but the genomic mechanisms underlying evolution in these environments are rarely known. Here, we compare genomic divergence between a deep-water morph adapted to depths of up to 288 m and a larger, piscivorous pelagic morph occupying shallower depths. Using both a SNP array and resequencing of whole nuclear and mitochondrial genomes, we find clear genetic divergence (FST = 0.11-0.15) between deep and shallow water morphs, despite an absence of morph divergence across the mitochondrial genome. Outlier analyses identified many diverged genomic regions containing genes enriched for processes such as gene expression and DNA repair, cardiac function, and membrane transport. Detection of putative copy number variants (CNVs) uncovered 385 genes with CNVs distinct to piscivorous morphs, and 275 genes with CNVs distinct to deep-water morphs, enriched for processes associated with synapse assembly. Demographic analyses identified evidence for recent and local morph divergence, and ongoing reductions in diversity consistent with postglacial colonization. Together, these results show that Arctic Charr morph divergence has occurred through genome-wide differentiation and elevated divergence of genes underlying multiple cellular and physiological processes, providing insight into the genomic basis of adaptation in a deep-water habitat following postglacial recolonization.

Matrix stiffness and its effects on tensional homeostasis act as major regulators of cellular states in health and disease. Stiffness-sensing studies are typically performed using cells that have acquired "mechanical memory" through prolonged propagation in rigid mechanical environments, e.g. tissue culture plastic (TCP). This may potentially mask the full extent of the stiffness-driven mechanosensing programs. To address this, we developed a biomaterial system composed of two-dimensional mechano-variant silicone substrates that is permissive to large-scale cell culture expansion processes. We broadly mapped the stiffness-mediated mechano-responses by performing RNA sequencing on human tendon-derived stromal cells. We find that matrix rigidities approximating tendon microscale stiffness range (E. ~35 kPa) distinctly favor programs related to chromatin remodeling and Hippo signaling; whereas more compliant stiffnesses (E. 2 kPa) were enriched in responses related to pluripotency, synapse assembly and angiogenesis. We also find that tendon stromal cells undergo dramatic phenotypic drift on conventional TCP, with near-complete suppression of tendon-related genes and emergence of expression signatures skewed towards fibro-inflammatory and metabolic activation. Strikingly, mechano-variant substrates abrogate fibroblasts activation, with tenogenic stiffnesses inducing a transcriptional program that strongly correlate with established tendon tissue-specific signatures. Computational inference predicted that AKT1 and ERK1/2 are major signaling hubs mediating stiffness-sensing in tendon cells. Together, our findings highlight how the underlying biophysical cues may dictate the transcriptional identity of resident cells, and how matrix mechano-reciprocity regulates diverse sets of previously underappreciated mechanosensitive processes in tendon stromal fibroblasts.

It has long been proposed that leukocyte common antigen-related receptor protein tyrosine phosphatases (LAR-RPTPs) are cell-adhesion proteins that control synapse assembly. Their synaptic nanoscale localization, however, is not established, and synapse fine structure after knockout of the three vertebrate LAR-RPTPs (PTPδ, PTPσ, and LAR) has not been tested. Here, superresolution microscopy reveals that PTPδ localizes to the synaptic cleft precisely apposed to postsynaptic scaffolds of excitatory and inhibitory synapses. We next assessed synapse structure in newly generated triple-conditional-knockout mice for PTPδ, PTPσ, and LAR, complementing a recent independent study of synapse function after LAR-RPTP ablation (Sclip and Südhof, 2020). While mild effects on synaptic vesicle clustering and active zone architecture were detected, synapse numbers and their overall structure were unaffected, membrane anchoring of the active zone persisted, and vesicle docking and release were normal. Hence, despite their localization at synaptic appositions, LAR-RPTPs are dispensable for presynapse structure and function.

Neural circuit assembly in the brain requires precise establishment of synaptic connections, but the mechanisms of synapse assembly remain incompletely understood. Latrophilins are postsynaptic adhesion-GPCRs that engage in trans-synaptic complexes with presynaptic teneurins and FLRTs. In CA1-region neurons, Latrophilin-2 and Latrophilin-3 are essential for formation of entorhinal-cortex-derived and Schaffer-collateral-derived synapses, respectively. However, it is unknown whether latrophilins function as GPCRs in synapse formation. Here, we show that Latrophilin-2 and Latrophilin-3 exhibit constitutive GPCR activity that increases cAMP levels, which was blocked by a mutation interfering with G-protein and arrestin interactions of GPCRs. The same mutation impaired the ability of Latrophilin-2 and Latrophilin-3 to rescue the synapse-loss phenotype in Latrophilin-2 and Latrophilin-3 knockout neurons in vivo. Our results suggest that Latrophilin-2 and Latrophilin-3 require GPCR signaling in synapse formation, indicating that latrophilins promote synapse formation in the hippocampus by activating a classical GPCR-signaling pathway.

The size and structure of the dendritic arbor play important roles in determining how synaptic inputs of neurons are converted to action potential output. The regulatory mechanisms governing the development of dendrites, however, are insufficiently understood. The evolutionary conserved Ste20/Hippo kinase pathway has been proposed to play an important role in regulating the formation and maintenance of dendritic architecture. A key element of this pathway, Ste20-like kinase (SLK), regulates cytoskeletal dynamics in non-neuronal cells and is strongly expressed throughout neuronal development. However, its function in neurons is unknown. We show that, during development of mouse cortical neurons, SLK has a surprisingly specific role for proper elaboration of higher, ≥ third-order dendrites both in male and in female mice. Moreover, we demonstrate that SLK is required to maintain excitation-inhibition balance. Specifically, SLK knockdown caused a selective loss of inhibitory synapses and functional inhibition after postnatal day 15, whereas excitatory neurotransmission was unaffected. Finally, we show that this mechanism may be relevant for human disease, as dysmorphic neurons within human cortical malformations revealed significant loss of SLK expression. Overall, the present data identify SLK as a key regulator of both dendritic complexity during development and inhibitory synapse maintenance. SIGNIFICANCE STATEMENT We show that dysmorphic neurons of human epileptogenic brain lesions have decreased levels of the Ste20-like kinase (SLK). Decreasing SLK expression in mouse neurons revealed that SLK has essential functions in forming the neuronal dendritic tree and in maintaining inhibitory connections with neighboring neurons.

Drosophila provides a powerful genetic system and an excellent model to study the development and function of the nervous system. The fly's small brain and complex behavior has been instrumental in mapping neuronal circuits and elucidating the neural basis of behavior. The fast pace of fly development and the wealth of genetic tools has enabled systematic studies on cell differentiation and fate specification, and has uncovered strategies for axon guidance and targeting. The accessibility of neuronal structures and the ability to edit and manipulate gene expression in selective cells and/or synaptic compartments has revealed mechanisms for synapse assembly and neuronal connectivity. Recent advances in single-cell RNA sequencing (scRNA-seq) have further enhanced our appreciation and understanding of neuronal diversity in a fly brain. However, due to the small size of the fly brain and its constituent cells, scRNA-seq methodologies require a few adaptations. Here, we describe a set of protocols optimized for scRNA-seq analysis of the Drosophila larval ventral nerve cord, starting from tissue dissection and cell dissociation to cDNA library preparation, sequencing, and data analysis. We apply this workflow to three separate samples and detail the technical challenges associated with successful application of scRNA-seq to studies on neuronal diversity. An accompanying article (Vicidomini, Nguyen, Choudhury, Brody, & Serpe, 2021) presents a custom multistage analysis pipeline that integrates modules contained in different R packages to ensure high-flexibility, high-quality RNA-seq data analysis. These protocols are developed for Drosophila larval ventral nerve cord, but could easily be adapted to other tissues and model organisms. © 2021 U.S. Government. Basic Protocol 1: Dissection of larval ventral nerve cords and preparation of single-cell suspensions Basic Protocol 2: Preparation and sequencing of single-cell transcriptome libraries Basic Protocol 3: Alignment of raw sequencing data to indexed genome and generation of count matrices.

Oligodendrocytes form myelin for central nervous system axons and release factors which signal to neurons during myelination. Here, we ask how oligodendroglial factors influence hippocampal GABAergic neuron physiology. In mixed hippocampal cultures GABAergic neurons fired action potentials of short duration and received high frequencies of excitatory synaptic events. In purified neuronal cultures without glial cells, GABAergic neuron excitability increased and the frequency of synaptic events decreased. These effects were largely reversed by adding oligodendrocyte conditioned medium. We compared the transcriptomic signature with the electrophysiological phenotype of single neurons in these three culture conditions. Genes expressed by single pyramidal or GABAergic neurons largely conformed to expected cell-type specific patterns. Multiple genes of GABAergic neurons were significantly downregulated by the transition from mixed cultures containing glial cells to purified neuronal cultures. Levels of these genes were restored by the addition of oligodendrocyte conditioned medium to purified cultures. Clustering genes with similar changes in expression between different culture conditions revealed processes affected by oligodendroglial factors. Enriched genes are linked to roles in synapse assembly, action potential generation and transmembrane ion transport, including of zinc. These results provide new insight into the molecular targets by which oligodendrocytes influence neuron excitability and synaptic function.

Components of the intraflagellar transport (IFT) system that regulates the assembly of the primary cilium are exploited by the non-ciliated T cell to orchestrate polarized endosome recycling to sustain signaling during immune synapse formation. Here we have investigated the potential role of BBS1, an essential core component of the Bardet-Biedl syndrome complex that cooperates with the IFT system in ciliary protein trafficking, in the assembly of the T cell synapse. We show that BBS1 allows for centrosome polarization towards the immune synapse by promoting its untethering from the nuclear envelope. This function is achieved through the clearance of centrosomal F-actin and its positive regulator WASH, a process that we demonstrate to be dependent on the proteasome. We show that BBS1 regulates this process by coupling the 19S proteasome regulatory subunit to the microtubule motor dynein for its transport to the centrosome. Our data identify the ciliopathy-related protein BBS1 as a new player in T cell synapse assembly that acts upstream of the IFT system to set the stage for polarized vesicular trafficking and sustained signaling.

Genome sequencing has identified a large number of putative autism spectrum disorder (ASD) risk genes, revealing possible disrupted biological pathways, however, the genetic and environmental underpinnings of ASD remain mostly unanswered. The presented methodology aimed to identify genetically related clusters of ASD individuals. By using the VariCarta dataset, which contains data retrieved from 13,069 people with ASD, we compared patients pairwise to build &ldquo, patient similarity matrices&rdquo, Hierarchical-agglomerative-clustering and heatmapping were performed, followed by enrichment analysis (EA). We analyzed whole-genome sequencing retrieved from 2062 individuals, and isolated 11,609 genetic variants shared by at least two people. The analysis yielded three clusters, composed, respectively, by 574 (27.8%), 507 (24.6%), and 650 (31.5%) individuals. Overall, 4187 variants (36.1%) were common to the three clusters. The EA revealed that the biological processes related to the shared genetic variants were mainly involved in neuron projection guidance and morphogenesis, cell junctions, synapse assembly, and in observational, imitative, and vocal learning. The study highlighted genetic networks, which were more frequent in a sample of people with ASD, compared to the overall population. We suggest that itemizing not only single variants, but also gene networks, might support ASD etiopathology research. Future work on larger databases will have to ascertain the reproducibility of this methodology.

Most research on neurodegenerative diseases has focused on neurons, yet glia help form and maintain the synapses whose loss is so prominent in these conditions. To investigate the contributions of glia to Huntington’s disease (HD), we studied transcriptomic changes in HD human, HD mice, andDrosophilaexpressing human mutantHuntingtin(mHTT) in either glia, neurons or both. A large portion of conserved genes are concordantly dysregulated across the three species; we tested these genes in a high-throughput behavioral assay and found that downregulation of genes involved in synapse assembly mitigated pathogenesis and behavioral deficits. To our surprise, mitigating glial pathogenesis by dNRXN3knockdown was sufficient to improve the phenotype of flies expressing mHTTin neurons, suggesting that mHTT’s toxic effects in glia ramify throughout the brain. This supports a model in which dampening synaptic function is protective because it attenuates the excitotoxicity that characterizes HD.

Sorting nexin 4 (SNX4) is an evolutionary conserved protein that mediates recycling from endosomes back to the plasma membrane in yeast and mammalian cells. SNX4 is expressed in the brain. Altered protein levels are associated with Alzheimer’s disease, but the neuronal localization and function of SNX4 have not been addressed. Using a new antibody, endogenous neuronal SNX4 co-localized with both early and recycling endosome markers, similar to the reported localization of SNX4 in non-neuronal cells. Neuronal SNX4 accumulated specifically in synaptic areas, with a predominant localization to presynaptic terminals. Acute depletion of neuronal SNX4 using independent short hairpin RNAs did not affect the levels of the transferrin receptor, a canonical SNX4 cargo. Quantitative mass spectrometry revealed that upon SNX4 knockdown the class of proteins involved in neurotransmission was the most dysregulated. This included integral membrane proteins at both the presynaptic and postsynaptic side of the synapse that participate in diverse synaptic processes such as synapse assembly, neurotransmission and the synaptic vesicle cycle. These data suggest that SNX4 is implicated in a variety of synaptic processes.

Presynaptic Ca(v)2 channels are essential for Ca2+-triggered exocytosis. In addition, there are two competing models for their roles in synapse structure. First, Ca2+ channels or Ca2+ entry may control synapse assembly. Second, active zone proteins may scaffold Ca(v)2s to presynaptic release sites, and synapse structure is Ca(v)2 independent. Here, we ablated all three Ca(v)2s using conditional knockout in cultured hippocampal neurons or at the calyx of Held, which abolished evoked exocytosis. Compellingly, synapse and active zone structure, vesicle docking, and transsynaptic nano-organization were unimpaired. Similarly, long-term blockade of action potentials and Ca2+ entry did not disrupt active zone assembly. Although Ca(v)2 knockout impaired the localization of beta subunits, alpha 2 delta-1 localized normally. Rescue with Ca(v)2 restored exocytosis, and Ca(v)2 active zone targeting depended on the intracellular C-terminus. We conclude that synapse assembly is independent of Ca(v)2s or Ca2+ entry through them. Instead, active zone proteins recruit and anchor Ca(v)2s via Ca(v)2 C-termini.

Purpose. The competing endogenous RNA (ceRNA) network regulatory has been investigated in the occurrence and development of many diseases. This research aimed at identifying the key RNAs of ceRNA network in pterygium and exploring the underlying molecular mechanism. Methods. Differentially expressed long noncoding RNAs (lncRNAs), microRNAs (miRNAs), and mRNAs were obtained from the Gene Expression Omnibus (GEO) database and analyzed with the R programming language. LncRNA and miRNA expressions were extracted and pooled by the GEO database and compared with those in published literature. The lncRNA-miRNA-mRNA network was constructed of selected lncRNAs, miRNAs, and mRNAs. Metascape was used to perform Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses on mRNAs of the ceRNA network and to perform Protein-Protein Interaction (PPI) Network analysis on the String website to find candidate hub genes. The Comparative Toxicogenomic Database (CTD) was used to find hub genes closely related to pterygium. The differential expressions of hub genes were verified using the reverse transcription-real-time fluorescent quantitative PCR (RT-qPCR). Result. There were 8 lncRNAs, 12 miRNAs, and 94 mRNAs filtered to construct the primary ceRNA network. A key lncRNA LIN00472 ranking the top 1 node degree was selected to reconstruct the LIN00472 network. The GO and KEGG pathway enrichment showed the mRNAs in ceRNA networks mainly involved in homophilic cell adhesion via plasma membrane adhesion molecules, developmental growth, regulation of neuron projection development, cell maturation, synapse assembly, central nervous system neuron differentiation, and PID FOXM1 PATHWAY. According to the Protein-Protein Interaction Network (PPI) analysis on mRNAs in LINC00472 network, 10 candidate hub genes were identified according to node degree ranking. Using the CTD database, we identified 8 hub genes closely related to pterygium; RT-qPCR verified 6 of them were highly expressed in pterygium. Conclusion. Our research found LINC00472 might regulate 8 hub miRNAs (miR-29b-3p, miR-183-5p, miR-138-5p, miR-211-5p, miR-221-3p, miR-218-5p, miR-642a-5p, miR-5000-3p) and 6 hub genes (CDH2, MYC, CCNB1, RELN, ERBB4, RB1) in the ceRNA network through mainly PID FOXM1 PATHWAY and play an important role in the development of pterygium.

A time course experiment is a widely used design in the study of cellular processes such as differentiation or response to stimuli. In this paper, we propose time course regulatory analysis (TimeReg) as a method for the analysis of gene regulatory networks based on paired gene expression and chromatin accessibility data from a time course. TimeReg can be used to prioritize regulatory elements, to extract core regulatory modules at each time point, to identify key regulators driving changes of the cellular state, and to causally connect the modules across different time points. We applied the method to analyze paired chromatin accessibility and gene expression data from a retinoic acid (RA)–induced mouse embryonic stem cells (mESCs) differentiation experiment. The analysis identified 57,048 novel regulatory elements regulating cerebellar development, synapse assembly, and hindbrain morphogenesis, which substantially extended our knowledge of cis-regulatory elements during differentiation. Using single-cell RNA-seq data, we showed that the core regulatory modules can reflect the properties of different subpopulations of cells. Finally, the driver regulators are shown to be important in clarifying the relations between modules across adjacent time points. As a second example, our method on Ascl1-induced direct reprogramming from fibroblast to neuron time course data identified Id1/2 as driver regulators of early stage of reprogramming.

Background 3,4-methylenedioxymethamphetamine (MDMA, “ecstasy”) is a widely used entactogenic drug known to impair cognitive functions on the long-run. Both hippocampal and frontal cortical regions have well established roles in behavior, memory formation and other cognitive tasks and damage of these regions is associated with altered behavior and cognitive functions frequently described in otherwise healthy MDMA users. Meanwhile, in post-traumatic stress disorder (PTSD) patients seem to benefit from therapeutic application of the drug, where damage in hippocampal cue extinction may play a role. The aim of this study was to examine the hippocampus, frontal cortex and dorsal raphe of Dark Agouti rats with gene expression arrays (Illumina RatRef bead arrays) looking for possible mechanisms and new candidates contributing to the consequences of a single dose of MDMA (15 mg/kg) 3 weeks earlier. Results The number of differentially expressed genes in the hippocampus, frontal cortex and the dorsal raphe were 481, 155, and 15, respectively. Gene set enrichment analysis of the microarray data revealed reduced expression of ‘memory’ and ‘cognition’, ‘dendrite development’ and ‘regulation of synaptic plasticity’ gene sets in the hippocampus, parallel to the downregulation of CaMK II subunits, glutamate-, CB1 cannabinoid- and EphA4, EphA5, EphA6 receptors. Downregulated gene sets in the frontal cortex were related to protein synthesis, chromatin organization, transmembrane transport processes, while ‘dendrite development’, ‘regulation of synaptic plasticity’ and ‘positive regulation of synapse assembly’ gene sets were upregulated besides elevated levels of a CaMK II subunit and NMDA2B glutamate receptor. Changes in the dorsal raphe region were mild and in most cases not significant. Conclusion The present data raise the possibility of new synapse formation / synaptic reorganization in the frontal cortex 3 weeks after a single neurotoxic dose of MDMA. In contrast, a prolonged depression of new neurite formation in the hippocampus is proposed by downregulations of members in long-term potentiation pathway and synaptic plasticity emphasizing the particular vulnerability of this brain region and proposing a mechanism responsible for cognitive problems in healthy individuals. At the same time, these results underpin benefits of MDMA in PTSD, where the drug may help memory extinction. Electronic supplementary material The online version of this article (10.1186/s12864-018-4929-x) contains supplementary material, which is available to authorized users.

Wnt signaling plays an important role in the adult brain function and its dysregulation has been implicated in some neurodegenerative pathways. Despite the functional role of the Wnt signaling in adult neural circuits, there is currently no evidence regarding the relationships between exogenously Wnt signaling activation or inhibition and hippocampal structural changes in vivo. Thus, we analyzed the effect of the chronic infusion of Wnt agonists, Wnt7a and Wnt5a, and antagonist, Dkk-1, on different markers of plasticity such as neuronal MAP-2, Tau, synapse number and morphology, and behavioral changes. We observed that Wnt7a and Wnt5a increased the number of perforated synapses and the content of pre-and postsynaptic proteins associated with synapse assembly compared to control and Dkk-1 infusion. These two Wnt agonists also reduced anxiety-like behavior. Conversely, the canonical antagonist, Dkk-1, increased anxiety and inhibited spatial memory recall. Therefore, the present study elucidates the potential participation of Wnt signaling in the remodeling of hippocampal circuits underlying plasticity events in vivo, and provides evidence of the potential benefits of Wnt agonist infusion for the treatment of some neurodegenerative conditions.

The Psychiatric Genomics Consortium (PGC) has recently identified 10 potential functional coding variants for schizophrenia. However, how these coding variants confer schizophrenia risk remains largely unknown. Here, we investigate the associations between eight potential functional coding variants identified by PGC and schizophrenia in a large Han Chinese sample (n = 4022 cases and 9270 controls). Among the eight tested single nucelotide polymorphisms (SNPs), rs3617 (a missense variant, p.K315Q in the ITIH3 gene) showed genome-wide significant association with schizophrenia in the Han Chinese population (P = 8.36 × 10−16), with the same risk allele as in PGC. Interestingly, rs3617 is located in a genomic region that is highly evolutionarily conserved, and its schizophrenia risk allele (C allele) was associated with lower ITIH3 mRNA and protein expression. Intriguingly, mouse neural stem cells stably overexpressing ITIH3 with different alleles of rs3617 exhibited significant differences in proliferation, migration, and differentiation, suggesting the impact of rs3617 on neurodevelopment. Subsequent transcriptome analysis found that the differentially expressed genes in neural stem cells stably overexpressing different alleles of rs3617 were significantly enriched in schizophrenia-related pathways, including cell adhesion, synapse assembly, MAPK and PI3K-AKT pathways. Our study provides convergent lines of evidence suggesting that rs3617 in ITIH3 likely affects protein function and neurodevelopment and thereby confers risk of schizophrenia.

Neurexins have been established as a major coordinator of synapse assembly, functioning through interactions with postsynaptic cell adhesion molecules. Kurshan et al. (2018) now show that a C. elegans "dwarf neurexin" lacking its extracellular interaction domains can conduct synapse formation independent of postsynaptic partners.

Gliomas are the most common primary brain cancers. In recent years, IDH mutation and 1p/19q codeletion have been suggested as biomarkers for the diagnosis, treatment and prognosis of gliomas. However, these biomarkers are only effective for a part of glioma patients and thus more biomarkers are still emergently needed. Recently, an electrochemical communication between normal neurons and glioma cells by neuro-glioma synapse has been reported. Moreover, it was discovered that breast-to-brain metastasis tumor cells have pseudo synapses with neurons and these synapses were indicated to promote tumor progression and metastasis. Based on the above observations, we first curated a panel of 66 SA genes and then proposed a metric, SA score, to quantify the synapseness for each sample of 12 glioma gene expression datasets from TCGA, CGGA, and GEO. Strikingly, SA score showed excellent predictive ability for the prognosis, diagnosis, and grading of gliomas. Moreover, being compared with the two established biomarkers, IDH mutation and 1p/19q codeletion, SA score was demonstrated independent and better predictive performance. In conclusion, this study revealed that SA genes contribute to glioma formation and development, and proposed a quantitative method, SA score, as an efficient biomarker for monitoring gliomas.

Copepods are the most numerous animals and play an essential role in the marine trophic web and biogeochemical cycles. The genus Oithona is described as having the highest numerical density, as the most cosmopolite copepod and iteroparous. The Oithona male paradox obliges it to alternate feeding (immobile) and mating (mobile) phases. As the molecular basis of this trade-off is unknown, we investigated this sexual dimorphism at the molecular level by integrating genomic, transcriptomic and protein-protein interaction analyses.While a ZW sex-determination system was predicted in O. nana, a fifteen-year time-series in the Toulon Little Bay showed a biased sex ratio toward females (male / female ratio < 0.15±0.11) highlighting a higher mortality in male. Here, the transcriptomic analysis of the five different developmental stages showed enrichment of Lin12-Notch Repeat (LNR) domains-containing proteins coding genes (LDPGs) in male transcripts. The male also showed enrichment in transcripts involved in proteolysis, nervous system development, synapse assembly and functioning and also amino acid conversion to glutamate. Moreover, several male down-regulated genes were involved in the increase of food uptake and digestion. The formation of LDP complexes was detected by yeast two-hybrid, with interactions involving proteases, extracellular matrix proteins and neurogenesis related proteins.Together, these results suggest that the O. nana male hypermotility is sustained by LDP-modulated proteolysis allowing the releases and conversions of amino acid into the excitatory neurotransmitter glutamate. This process could permit new axons and dendrites formation suggesting a sexual nervous system dimorphism. This could support the hypothesis of a sacrificial behaviour in males at the metabolic level.

SummaryGlutamate receptor auxiliary proteins control receptor distribution and function, ultimately controlling synapse assembly, maturation and plasticity. At the Drosophila neuromuscular junction (NMJ), a synapse with both pre- and post-synaptic kainate-type glutamate receptors (KARs), we show that the auxiliary protein Neto evolved functionally distinct isoforms to modulate synapse development and homeostasis. Using genetics, cell biology and electrophysiology we demonstrate that Neto-α functions on both sides of the NMJ. In muscle, Neto-α limits the size of the postsynaptic receptors field. In motor neurons, Neto-α controls neurotransmitter release in a KAR-dependent manner. Furthermore, Neto-α is both required and sufficient for the presynaptic increase in neurotransmitter release in response to reduced postsynaptic sensitivity. This KAR-independent function of Neto-α is involved in activity-induced cytomatrix remodeling. We propose that Drosophila ensured NMJ functionality by acquiring two Neto isoforms with differential expression patterns and activities.

Neurons differentiated in two-dimensional culture from human pluripotent stem-cell-derived neural progenitor cells (NPCs) represent a powerful model system to explore disease mechanisms and carry out high content screening (HCS) to interrogate compound libraries or identify gene mutation phenotypes. However, with human cells the transition from NPC to functional, mature neuron requires several weeks. Synapses typically start to form after 3 weeks of differentiation in monolayer culture, and several neuron-specific proteins, for example the later expressing pan-neuronal marker NeuN, or the layer 5/6 cerebral cortical neuron marker CTIP2, begin to express around 4-5 weeks post-differentiation. This lengthy differentiation time can be incompatible with optimal culture conditions used for small volume, multi-well HCS platforms. Among the many challenges are the need for well-adhered, uniformly distributed cells with minimal cell clustering, and culture procedures that foster long-term viability and functional synapse maturation. One approach is to differentiate neurons in a large volume format, then replate them at a later time point in HCS-compatible multi-wells. Some main challenges when using this replating approach concern reproducibility and cell viability, due to the stressful disruption of the dendritic and axonal network. Here we demonstrate a detailed and reliable procedure for enzymatically resuspending human induced pluripotent stem cell (hiPSC)-derived neurons after their differentiation for 4-8 weeks in a large-volume format, transferring them to 384-well microtiter plates, and culturing them for a further 1-3 weeks with excellent cell survival. This replating of human neurons not only allows the study of synapse assembly and maturation within two weeks from replating, but also enables studies of neurite regeneration and growth cone characteristics. We provide examples of scalable assays for neuritogenesis and synaptogenesis using a 384-well platform.

SummaryFollowing axon pathfinding, growth cones transition from stochastic filopodial exploration to the formation of a limited number of synapses. How the interplay of filopodia and synapse assembly ensures robust connectivity in the brain has remained a challenging problem. Here, we developed a new 4D analysis method for filopodial dynamics and a data-driven computational model of synapse formation for R7 photoreceptor axons in developing Drosophila brains. Our live data support a ‘serial synapse formation’ model, where at any time point only a single ‘synaptogenic’ filopodium suppresses the synaptic competence of other filopodia through competition for synaptic seeding factors. Loss of the synaptic seeding factors Syd-1 and Liprin-α leads to a loss of this suppression, filopodial destabilization and reduced synapse formation, which is sufficient to cause the destabilization of entire axon terminals. Our model provides a filopodial ‘winner-takes-all’ mechanism that ensures the formation of an appropriate number of synapses.

Three neuronal pentraxins are expressed in brain, the membrane-bound “neuronal pentraxin receptor” (NPR) and the secreted proteins NP1 and NARP (i.e., NP2). Neuronal pentraxins bind to AMPARs at excitatory synapses and play important, well-documented roles in the activity-dependent regulation of neural circuits via this binding activity. However, it is unknown whether neuronal pentraxins perform roles in synapses beyond modulating postsynaptic AMPAR-dependent plasticity, and whether they may even act in inhibitory synapses. Here, we show that NPR expressed in non-neuronal cells potently induces formation of both excitatory and inhibitory postsynaptic specializations in cocultured hippocampal neurons. Knockdown of NPR in hippocampal neurons, conversely, dramatically decreased assembly and function of both excitatory and inhibitory postsynaptic specializations. Overexpression of NPR rescued the NPR knockdown phenotype but did not in itself change synapse numbers or properties. However, the NPR knockdown decreased the levels of NARP, whereas NPR overexpression produced a dramatic increase in the levels of NP1 and NARP, suggesting that NPR recruits and stabilizes NP1 and NARP on the presynaptic plasma membrane. Mechanistically, NPR acted in excitatory synapse assembly by binding to the N-terminal domain of AMPARs; antagonists of AMPA and GABA receptors selectively inhibited NPR-induced heterologous excitatory and inhibitory synapse assembly, respectively, but did not affect neurexin-1β-induced synapse assembly as a control. Our data suggest that neuronal pentraxins act as signaling complexes that function as general trans-synaptic organizers of both excitatory and inhibitory synapses by a mechanism that depends, at least in part, on the activity of the neurotransmitter receptors at these synapses.SIGNIFICANCE STATEMENTNeuronal pentraxins comprise three neuronal proteins, neuronal pentraxin receptor (NPR) which is a type-II transmembrane protein on the neuronal surface, and secreted neuronal pentraxin-1 and NARP. The general functions of neuronal pentraxins at synapses have not been explored, except for their basic AMPAR binding properties. Here, we examined the functional role of NPR at synapses because it is the only neuronal pentraxin that is anchored to the neuronal cell-surface membrane. We find that NPR is a potent inducer of both excitatory and inhibitory heterologous synapses, and that knockdown of NPR in cultured neurons decreases the density of both excitatory and inhibitory synapses. Our data suggest that NPR performs a general, previously unrecognized function as a universal organizer of synapses.

BACKGROUND The molecular mechanism of recurrent spontaneous abortion is unclear. It has been suggested that dysregulated genes participate in the pathogenesis of recurrent spontaneous abortion. The aim of this study was to identify the differentially expressed genes (DEGs) and pathways in recurrent spontaneous abortion. MATERIAL AND METHODS Gene expression data series of GSE22490 and GSE26787 were obtained from the GEO database to identify the differentially expressed genes between patients with recurrent miscarriage (Case group) and patients with uncomplicated pregnancies matched for gestational age (Control group). Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEEG) were applied to enrich the biological functions and pathways of the identified differentially expressed genes. A protein-protein interaction (PPI) network was constructed thorough the STRING database. Thirty-one cases of recurrent spontaneous abortion (Case group) and 30 cases of artificial abortion (Control group) were included in the study. The protein expression of hub genes in the villi and decidua tissue of the 2 groups was detected by immunohistochemical assay. RESULTS Forty-six DEGs were identified with the enriched biological function mainly in the aspects of glutamate secretion and positive regulation of synapse assembly. KEGG pathway analysis indicated the dysregulated genes were only enriched in the glutamatergic synapse pathway. In the PPI network, 83 nodes and 273 edges with the average node degreed of 6.58 were enriched. The hub gene (ATP6V1G3) of the included 46 genes was identified using Cytohubba software. In the Case group, the high expression of ATP6V1G3 protein was detected in 13 (43.3%) and 10 (33.3%) for placental villus and decidual tissue, respectively. However, the high expression rate in the Control group was 23.3% and 16.7% for placental villus and decidual tissue, respectively. The ATP6V1G3 protein high expression rate was not significantly different between the Case and Control groups (P>0.05). CONCLUSIONS We found differential gene expression profiles in villous and decidual tissues between patients with recurrent miscarriage vs. those with uncomplicated pregnancies. Upregulation of the ATP6V1G3 gene may play an important role in the development of recurrent miscarriage.

Small vessel diseases, such as ischemic retinopathy and cerebral small vessel disease (CSVD), are increasingly recognized in patients with diabetes, dementia and cerebrovascular disease. The mechanisms of small vessel diseases are poorly understood, but the latest studies suggest a role for semaphorins. Initially identified as axon guidance cues, semaphorins are mainly studied in neuronal morphogenesis, neural circuit assembly, and synapse assembly and refinement. In recent years, semaphorins have been found to play important roles in regulating vascular growth and development and in many pathophysiological processes, including atherosclerosis, angiogenesis after stroke and retinopathy. Growing evidence indicates that semaphorins affect the occurrence, perfusion and regression of both the macrovasculature and microvasculature by regulating the proliferation, apoptosis, migration, barrier function and inflammatory response of endothelial cells, vascular smooth muscle cells (VSMCs) and pericytes. In this review, we concentrate on the regulatory effects of semaphorins on the cell components of the vessel wall and their potential roles in microvascular diseases, especially in the retina and cerebral small vessel. Finally, we discuss potential molecular approaches in targeting semaphorins as therapies for microvascular disorders in the eye and brain.

SUMMARY Glutamate receptor auxiliary proteins control receptor distribution and function, ultimately controlling synapse assembly, maturation, and plasticity. At the Drosophila neuromuscular junction (NMJ), a synapse with both pre- and postsynaptic kainate-type glutamate receptors (KARs), we show that the auxiliary protein Neto evolved functionally distinct isoforms to modulate synapse development and homeostasis. Using genetics, cell biology, and electrophysiology, we demonstrate that Neto-α functions on both sides of the NMJ. In muscle, Neto-α limits the size of the postsynaptic receptor field. In motor neurons (MNs), Neto-α controls neurotransmitter release in a KAR-dependent manner. In addition, Neto-α is both required and sufficient for the presynaptic increase in neurotransmitter release in response to reduced postsynaptic sensitivity. This KAR-independent function of Neto-α is involved in activity-induced cytomatrix remodeling. We propose that Drosophila ensures NMJ functionality by acquiring two Neto isoforms with differential expression patterns and activities., In Brief Han et al. report that Neto-α functions on both pre- and postsynaptic sides of individual synapses at the Drosophila NMJ. Postsynaptic Neto-α regulates the organization of glutamate receptor fields, whereas presynaptic Neto-αis required for normal physiology and homeostatic plasticity., Graphical Abstract

Introduction Patients with neuropathic pain have altered proteomic and neuropeptide constituents in cerebrospinal fluid (CSF) compared to controls. Tonic spinal cord stimulation (SCS) has demonstrated differential expression of neuropeptides in CSF before and after treatment suggesting potential mechanisms of action. Burst-SCS is an evidence-based paraesthesia free waveform utilised for neuropathic pain with a potentially different mechanistic action to tonic SCS. This study examines the dynamic biological changes of CSF at a cellular and proteome level after Burst-SCS. Methods Patients with neuropathic pain selected for SCS had CSF sampled prior to implant of SCS and following 8 weeks of continuous Burst-SCS. Baseline and 8-week pain scores with demographics were recorded. T cell frequencies were analysed by flow cytometry, proteome analysis was performed using mass spectrometry and secreted cytokines, chemokines and neurotrophins were measured by enzyme-linked immunosorbent assay (ELISA). Results 4 patients (2 females, 2 males) with a mean age of 51 years (+/-SEM 2.74, SD 5.48) achieved a reduction in pain of >50% following 8 weeks of Burst-SCS. Analysis of the CSF proteome indicated a significant alteration in protein expression most related to synapse assembly and immune regulators. There was significantly lower expression of the proteins: growth hormone A1 (PRL), somatostatin (SST), nucleobindin-2 (NUCB2), Calbindin (CALB1), acyl-CoA binding protein (DBI), proSAAS (PCSK1N), endothelin-3 (END3) and cholecystokinin (CCK) after Burst-SCS. The concentrations of secreted chemokines and cytokines and the frequencies of T cells were not significantly changed following Burst-SCS. Conclusion This study characterised the alteration in the CSF proteome in response to burst SCS in vivo. Functional analysis indicated that the alterations in the CSF proteome is predominately linked to synapse assembly and immune effectors. Individual protein analysis also suggests potential supraspinal mechanisms.

The presynaptic active zone, composed of evolutionarily conserved protein complexes, is a specialized area that serves to orchestrate precise and efficient neurotransmitter release by organizing various presynaptic proteins involved in mediating docking and priming of synaptic vesicles, recruiting voltage-gated calcium channels, and modulating presynaptic nerve terminals with aligned postsynaptic structures. Among membrane proteins localized to active zone, presynaptic neurexins and LAR-RPTPs (leukocyte common antigen-related receptor tyrosine phosphatase) have emerged as hubs that orchestrate both shared and distinct extracellular synaptic adhesion pathways. In this chapter, we discuss intracellular signaling cascades involved in recruiting various intracellular proteins at both excitatory and inhibitory synaptic sites. In particular, we highlight recent studies on key active zone proteins that physically and functionally link these cascades with neurexins and LAR-RPTPs in both vertebrate and invertebrate model systems. These studies allow us to build a general, universal view of how presynaptic active zones operate together with postsynaptic structures in neural circuits.