A concerted neuron-astrocyte program declines in ageing and schizophrenia.

Human brains vary across people and over time; such variation is not yet understood in cellular terms. Here we describe a relationship between people's cortical neurons and cortical astrocytes. We used single-nucleus RNA sequencing to analyse the prefrontal cortex of 191 human donors aged 22-97 years, including healthy individuals and people with schizophrenia. Latent-factor analysis of these data revealed that, in people whose cortical neurons more strongly expressed genes encoding synaptic components, cortical astrocytes more strongly expressed distinct genes with synaptic functions and genes for synthesizing cholesterol, an astrocyte-supplied component of synaptic membranes. We call this relationship the synaptic neuron and astrocyte program (SNAP). In schizophrenia and ageing-two conditions that involve declines in cognitive flexibility and plasticity1,2-cells divested from SNAP: astrocytes, glutamatergic (excitatory) neurons and GABAergic (inhibitory) neurons all showed reduced SNAP expression to corresponding degrees. The distinct astrocytic and neuronal components of SNAP both involved genes in which genetic risk factors for schizophrenia were strongly concentrated. SNAP, which varies quantitatively even among healthy people of similar age, may underlie many aspects of normal human interindividual differences and may be an important point of convergence for multiple kinds of pathophysiology.

Role of Post-Transcriptional Regulation in Learning and Memory in Mammals.

After many decades, during which most molecular studies on the regulation of gene expression focused on transcriptional events, it was realized that post-transcriptional control was equally important in order to determine where and when specific proteins were to be synthesized. Translational regulation is of the most importance in the brain, where all the steps of mRNA maturation, transport to different regions of the cells and actual expression, in response to specific signals, constitute the molecular basis for neuronal plasticity and, as a consequence, for structural stabilization/modification of synapses; notably, these latter events are fundamental for the highest brain functions, such as learning and memory, and are characterized by long-term potentiation (LTP) of specific synapses. Here, we will discuss the molecular bases of these fundamental events by considering both the role of RNA-binding proteins (RBPs) and the effects of non-coding RNAs involved in controlling splicing, editing, stability and translation of mRNAs. Importantly, it has also been found that dysregulation of mRNA metabolism/localization is involved in many pathological conditions, arising either during brain development or in the adult nervous system.

Functional labeling of individualized postsynaptic neurons using optogenetics and trans-Tango in Drosophila (FLIPSOT).

A population of neurons interconnected by synapses constitutes a neural circuit, which performs specific functions upon activation. It is essential to identify both anatomical and functional entities of neural circuits to comprehend the components and processes necessary for healthy brain function and the changes that characterize brain disorders. To date, few methods are available to study these two aspects of a neural circuit simultaneously. In this study, we developed FLIPSOT, or functional labeling of individualized postsynaptic neurons using optogenetics and trans-Tango. FLIPSOT uses (1) trans-Tango to access postsynaptic neurons genetically, (2) optogenetic approaches to activate (FLIPSOTa) or inhibit (FLIPSOTi) postsynaptic neurons in a random and sparse manner, and (3) fluorescence markers tagged with optogenetic genes to visualize these neurons. Therefore, FLIPSOT allows using a presynaptic driver to identify the behavioral function of individual postsynaptic neurons. It is readily applied to identify functions of individual postsynaptic neurons and has the potential to be adapted for use in mammalian circuits.

Electrical synapse structure requires distinct isoforms of a postsynaptic scaffold.

Electrical synapses are neuronal gap junction (GJ) channels associated with a macromolecular complex called the electrical synapse density (ESD), which regulates development and dynamically modifies electrical transmission. However, the proteomic makeup and molecular mechanisms utilized by the ESD that direct electrical synapse formation are not well understood. Using the Mauthner cell of zebrafish as a model, we previously found that the intracellular scaffolding protein ZO1b is a member of the ESD, localizing postsynaptically, where it is required for GJ channel localization, electrical communication, neural network function, and behavior. Here, we show that the complexity of the ESD is further diversified by the genomic structure of the ZO1b gene locus. The ZO1b gene is alternatively initiated at three transcriptional start sites resulting in isoforms with unique N-termini that we call ZO1b-Alpha, -Beta, and -Gamma. We demonstrate that ZO1b-Beta and ZO1b-Gamma are broadly expressed throughout the nervous system and localize to electrical synapses. By contrast, ZO1b-Alpha is expressed mainly non-neuronally and is not found at synapses. We generate mutants in all individual isoforms, as well as double mutant combinations in cis on individual chromosomes, and find that ZO1b-Beta is necessary and sufficient for robust GJ channel localization. ZO1b-Gamma, despite its localization to the synapse, plays an auxiliary role in channel localization. This study expands the notion of molecular complexity at the ESD, revealing that an individual genomic locus can contribute distinct isoforms to the macromolecular complex at electrical synapses. Further, independent scaffold isoforms have differential contributions to developmental assembly of the interneuronal GJ channels. We propose that ESD molecular complexity arises both from the diversity of unique genes and from distinct isoforms encoded by single genes. Overall, ESD proteomic diversity is expected to have critical impacts on the development, structure, function, and plasticity of electrical transmission.

Presynaptic Ube3a E3 ligase promotes synapse elimination through down-regulation of BMP signaling.

Inactivation of the ubiquitin ligase Ube3a causes the developmental disorder Angelman syndrome, whereas increased Ube3a dosage is associated with autism spectrum disorders. Despite the enriched localization of Ube3a in the axon terminals including presynapses, little is known about the presynaptic function of Ube3a and mechanisms underlying its presynaptic localization. We show that developmental synapse elimination requires presynaptic Ube3a activity in Drosophila neurons. We further identified the domain of Ube3a that is required for its interaction with the kinesin motor. Angelman syndrome-associated missense mutations in the interaction domain attenuate presynaptic targeting of Ube3a and prevent synapse elimination. Conversely, increased Ube3a activity in presynapses leads to precocious synapse elimination and impairs synaptic transmission. Our findings reveal the physiological role of Ube3a and suggest potential pathogenic mechanisms associated with Ube3a dysregulation.

SynLight: a bicistronic strategy for simultaneous active zone and cell labeling in the Drosophila nervous system.

At synapses, chemical neurotransmission mediates the exchange of information between neurons, leading to complex movement, behaviors, and stimulus processing. The immense number and variety of neurons within the nervous system make discerning individual neuron populations difficult, necessitating the development of advanced neuronal labeling techniques. In Drosophila, Bruchpilot-Short and mCD8-GFP, which label presynaptic active zones and neuronal membranes, respectively, have been widely used to study synapse development and organization. This labeling is often achieved via the expression of 2 independent constructs by a single binary expression system, but expression can weaken when multiple transgenes are expressed by a single driver. Recent work has sought to circumvent these drawbacks by developing methods that encode multiple proteins from a single transcript. Self-cleaving peptides, specifically 2A peptides, have emerged as effective sequences for accomplishing this task. We leveraged 2A ribosomal skipping peptides to engineer a construct that produces both Bruchpilot-Short-mStraw and mCD8-GFP from the same mRNA, which we named SynLight. Using SynLight, we visualized the putative synaptic active zones and membranes of multiple classes of olfactory, visual, and motor neurons and observed the correct separation of signal, confirming that both proteins are being generated separately. Furthermore, we demonstrate proof of principle by quantifying synaptic puncta number and neurite volume in olfactory neurons and finding no difference between the synapse densities of neurons expressing SynLight or neurons expressing both transgenes separately. At the neuromuscular junction, we determined that the synaptic puncta number labeled by SynLight was comparable to the endogenous puncta labeled by antibody staining. Overall, SynLight is a versatile tool for examining synapse density in any nervous system region of interest and allows new questions to be answered about synaptic development and organization.

Droplet-based transcriptome profiling of individual synapses.

Synapses are crucial structures that mediate signal transmission between neurons in complex neural circuits and display considerable morphological and electrophysiological heterogeneity. So far we still lack a high-throughput method to profile the molecular heterogeneity among individual synapses. In the present study, we develop a droplet-based single-cell (sc) total-RNA-sequencing platform, called Multiple-Annealing-and-Tailing-based Quantitative scRNA-seq in Droplets, for transcriptome profiling of individual neurites, primarily composed of synaptosomes. In the synaptosome transcriptome, or 'synaptome', profiling of both mouse and human brain samples, we detect subclusters among synaptosomes that are associated with neuronal subtypes and characterize the landscape of transcript splicing that occurs within synapses. We extend synaptome profiling to synaptopathy in an Alzheimer's disease (AD) mouse model and discover AD-associated synaptic gene expression changes that cannot be detected by single-nucleus transcriptome profiling. Overall, our results show that this platform provides a high-throughput, single-synaptosome transcriptome profiling tool that will facilitate future discoveries in neuroscience.

Synaptogenesis: unmasking molecular mechanisms using Caenorhabditis elegans.

The nematode Caenorhabditis elegans is a research model organism particularly suited to the mechanistic understanding of synapse genesis in the nervous system. Armed with powerful genetics, knowledge of complete connectomics, and modern genomics, studies using C. elegans have unveiled multiple key regulators in the formation of a functional synapse. Importantly, many signaling networks display remarkable conservation throughout animals, underscoring the contributions of C. elegans research to advance the understanding of our brain. In this chapter, we will review up-to-date information of the contribution of C. elegans to the understanding of chemical synapses, from structure to molecules and to synaptic remodeling.

Involvement of miR-135a-5p Downregulation in Acute and Chronic Stress Response in the Prefrontal Cortex of Rats.

Stress is a key risk factor in the onset of neuropsychiatric disorders. The study of the mechanisms underlying stress response is important to understand the etiopathogenetic mechanisms and identify new putative therapeutic targets. In this context, microRNAs (miRNAs) have emerged as key regulators of the complex patterns of gene/protein expression changes in the brain, where they have a crucial role in the regulation of neuroplasticity, neurogenesis, and neuronal differentiation. Among them, miR-135a-5p has been associated with stress response, synaptic plasticity, and the antidepressant effect in different brain areas. Here, we used acute unavoidable foot-shock stress (FS) and chronic mild stress (CMS) on male rats to study whether miR-135a-5p was involved in stress-induced changes in the prefrontal cortex (PFC). Both acute and chronic stress decreased miR-135a-5p levels in the PFC, although after CMS the reduction was induced only in animals vulnerable to CMS, according to a sucrose preference test. MiR-135a-5p downregulation in the primary neurons reduced dendritic spine density, while its overexpression exerted the opposite effect. Two bioinformatically predicted target genes, Kif5c and Cplx1/2, were increased in FS rats 24 h after stress. Altogether, we found that miR-135a-5p might play a role in stress response in PFC involving synaptic mechanisms.

Rabphilin-3A undergoes phase separation to regulate GluN2A mobility and surface clustering.

N-methyl-D-aspartate receptors (NMDARs) are essential for excitatory neurotransmission and synaptic plasticity. GluN2A and GluN2B, two predominant Glu2N subunits of NMDARs in the hippocampus and the cortex, display distinct clustered distribution patterns and mobility at synaptic and extrasynaptic sites. However, how GluN2A clusters are specifically organized and stabilized remains poorly understood. Here, we found that the previously reported GluN2A-specific binding partner Rabphilin-3A (Rph3A) has the ability to undergo phase separation, which relies on arginine residues in its N-terminal domain. Rph3A phase separation promotes GluN2A clustering by binding GluN2A's C-terminal domain. A complex formed by Rph3A, GluN2A, and the scaffolding protein PSD95 promoted Rph3A phase separation. Disrupting Rph3A's phase separation suppressed the synaptic and extrasynaptic surface clustering, synaptic localization, stability, and synaptic response of GluN2A in hippocampal neurons. Together, our results reveal the critical role of Rph3A phase separation in determining the organization and stability of GluN2A in the neuronal surface.

The role of genetic risk factors of Alzheimer's disease in synaptic dysfunction.

Alzheimer's disease (AD) is a neurodegenerative disease characterized by the progressive deterioration of cognitive functions. Due to the extended global life expectancy, the prevalence of AD is increasing among aging populations worldwide. While AD is a multifactorial disease, synaptic dysfunction is one of the major neuropathological changes that occur early in AD, before clinical symptoms appear, and is associated with the progression of cognitive deterioration. However, the underlying pathological mechanisms leading to this synaptic dysfunction remains unclear. Recent large-scale genomic analyses have identified more than 40 genetic risk factors that are associated with AD. In this review, we discuss the functional roles of these genes in synaptogenesis and synaptic functions under physiological conditions, and how their functions are dysregulated in AD. This will provide insights into the contributions of these encoded proteins to synaptic dysfunction during AD pathogenesis.

Far from home: the role of glial mRNA localization in synaptic plasticity.

Neurons and glia are highly polarized cells, whose distal cytoplasmic functional subdomains require specific proteins. Neurons have axonal and dendritic cytoplasmic extensions containing synapses whose plasticity is regulated efficiently by mRNA transport and localized translation. The principles behind these mechanisms are equally attractive for explaining rapid local regulation of distal glial cytoplasmic projections, independent of their cell nucleus. However, in contrast to neurons, mRNA localization has received little experimental attention in glia. Nevertheless, there are many functionally diverse glial subtypes containing extensive networks of long cytoplasmic projections with likely localized regulation that influence neurons and their synapses. Moreover, glia have many other neuron-like properties, including electrical activity, secretion of gliotransmitters and calcium signaling, influencing, for example, synaptic transmission, plasticity and axon pruning. Here, we review previous studies concerning glial transcripts with important roles in influencing synaptic plasticity, focusing on a few cases involving localized translation. We discuss a variety of important questions about mRNA transport and localized translation in glia that remain to be addressed, using cutting-edge tools already available for neurons.

Loss of Pex1 in Inner Ear Hair Cells Contributes to Cochlear Synaptopathy and Hearing Loss.

Peroxisome Biogenesis Disorders (PBD) and Zellweger syndrome spectrum disorders (ZSD) are rare genetic multisystem disorders that include hearing impairment and are associated with defects in peroxisome assembly, function, or both. Mutations in 13 peroxin (PEX) genes have been found to cause PBD-ZSD with ~70% of patients harboring mutations in PEX1. Limited research has focused on the impact of peroxisomal disorders on auditory function. As sensory hair cells are particularly vulnerable to metabolic changes, we hypothesize that mutations in PEX1 lead to oxidative stress affecting hair cells of the inner ear, subsequently resulting in hair cell degeneration and hearing loss. Global deletion of the Pex1 gene is neonatal lethal in mice, impairing any postnatal studies. To overcome this limitation, we created conditional knockout mice (cKO) using Gfi1Creor VGlut3Cre expressing mice crossed to floxed Pex1 mice to allow for selective deletion of Pex1 in the hair cells of the inner ear. We find that Pex1 excision in inner hair cells (IHCs) leads to progressive hearing loss associated with significant decrease in auditory brainstem responses (ABR), specifically ABR wave I amplitude, indicative of synaptic defects. Analysis of IHC synapses in cKO mice reveals a decrease in ribbon synapse volume and functional alterations in exocytosis. Concomitantly, we observe a decrease in peroxisomal number, indicative of oxidative stress imbalance. Taken together, these results suggest a critical function of Pex1 in development and maturation of IHC-spiral ganglion synapses and auditory function.

Diverse and Composite Roles of miRNA in Non-Neuronal Cells and Neuronal Synapses in Alzheimer's Disease.

Neurons interact with astrocytes, microglia, and vascular cells. These interactions become unbalanced in disease states, resulting in damage to neurons and synapses, and contributing to cognitive impairment. Importantly, synaptic loss and synaptic dysfunction have been considered for years as a main pathological factor of cognitive impairment in Alzheimer's disease (AD). Recently, miRNAs have emerged as essential regulators of physiological and pathological processes in the brain. Focusing on the role of miRNAs in regulating synaptic functions, as well as different cell types in the brain, offers opportunities for the early prevention, diagnosis, and potential treatment of AD-related cognitive impairment. Here, we review the recent research conducted on miRNAs regulating astrocytes, microglia, cerebrovasculature, and synaptic functions in the context of AD-related cognitive impairment. We also review potential miRNA-related biomarkers and therapeutics, as well as emerging imaging technologies relevant for AD research.

NMDA Receptor C-Terminal Domain Signalling in Development, Maturity, and Disease.

The NMDA receptor is a Ca2+-permeant glutamate receptor which plays key roles in health and disease. Canonical NMDARs contain two GluN2 subunits, of which 2A and 2B are predominant in the forebrain. Moreover, the relative contribution of 2A vs. 2B is controlled both developmentally and in an activity-dependent manner. The GluN2 subtype influences the biophysical properties of the receptor through difference in their N-terminal extracellular domain and transmembrane regions, but they also have large cytoplasmic Carboxyl (C)-terminal domains (CTDs) which have diverged substantially during evolution. While the CTD identity does not influence NMDAR subunit specific channel properties, it determines the nature of CTD-associated signalling molecules and has been implicated in mediating the control of subunit composition (2A vs. 2B) at the synapse. Historically, much of the research into the differential function of GluN2 CTDs has been conducted in vitro by over-expressing mutant subunits, but more recently, the generation of knock-in (KI) mouse models have allowed CTD function to be probed in vivo and in ex vivo systems without heterologous expression of GluN2 mutants. In some instances, findings involving KI mice have been in disagreement with models that were proposed based on earlier approaches. This review will examine the current research with the aim of addressing these controversies and how methodology may contribute to differences between studies. We will also discuss the outstanding questions regarding the role of GluN2 CTD sequences in regulating NMDAR subunit composition, as well as their relevance to neurodegenerative disease and neurodevelopmental disorders.

Mechanisms Underlying Circuit Dysfunction in Neurodevelopmental Disorders.

Recent advances in genomics have revealed a wide spectrum of genetic variants associated with neurodevelopmental disorders at an unprecedented scale. An increasing number of studies have consistently identified mutations-both inherited and de novo-impacting the function of specific brain circuits. This suggests that, during brain development, alterations in distinct neural circuits, cell types, or broad regulatory pathways ultimately shaping synapses might be a dysfunctional process underlying these disorders. Here, we review findings from human studies and animal model research to provide a comprehensive description of synaptic and circuit mechanisms implicated in neurodevelopmental disorders. We discuss how specific synaptic connections might be commonly disrupted in different disorders and the alterations in cognition and behaviors emerging from imbalances in neuronal circuits. Moreover, we review new approaches that have been shown to restore or mitigate dysfunctional processes during specific critical windows of brain development. Considering the heterogeneity of neurodevelopmental disorders, we also highlight the recent progress in developing improved clinical biomarkers and strategies that will help to identify novel therapeutic compounds and opportunities for early intervention.

Prediction of Synaptically Localized RNAs in Human Neurons Using Developmental Brain Gene Expression Data.

In the nervous system, synapses are special and pervasive structures between axonal and dendritic terminals, which facilitate electrical and chemical communications among neurons. Extensive studies have been conducted in mice and rats to explore the RNA pool at synapses and investigate RNA transport, local protein synthesis, and synaptic plasticity. However, owing to the experimental difficulties of studying human synaptic transcriptomes, the full pool of human synaptic RNAs remains largely unclear. We developed a new machine learning method, called PredSynRNA, to predict the synaptic localization of human RNAs. Training instances of dendritically localized RNAs were compiled from previous rodent studies, overcoming the shortage of empirical instances of human synaptic RNAs. Using RNA sequence and gene expression data as features, various models with different learning algorithms were constructed and evaluated. Strikingly, the models using the developmental brain gene expression features achieved superior performance for predicting synaptically localized RNAs. We examined the relevant expression features learned by PredSynRNA and used an independent test dataset to further validate the model performance. PredSynRNA models were then applied to the prediction and prioritization of candidate RNAs localized to human synapses, providing valuable targets for experimental investigations into neuronal mechanisms and brain disorders.

IRSp53 promotes postsynaptic density formation and actin filament bundling.

IRSp53 (aka BAIAP2) is a scaffold protein that couples membranes with the cytoskeleton in actin-filled protrusions such as filopodia and lamellipodia. The protein is abundantly expressed in excitatory synapses and is essential for synapse development and synaptic plasticity, although with poorly understood mechanisms. Here we show that specific multivalent interactions between IRSp53 and its binding partners PSD-95 or Shank3 drive phase separation of the complexes in solution. IRSp53 can be enriched to the reconstituted excitatory PSD (ePSD) condensates via bridging to the core and deeper layers of ePSD. Overexpression of a mutant defective in the IRSp53/PSD-95 interaction perturbs synaptic enrichment of IRSp53 in mouse cortical neurons. The reconstituted PSD condensates promote bundled actin filament formation both in solution and on membranes, via IRSp53-mediated actin binding and bundling. Overexpression of mutants that perturb IRSp53-actin interaction leads to defects in synaptic maturation of cortical neurons. Together, our studies provide potential mechanistic insights into the physiological roles of IRSp53 in synapse formation and function.

Complex regulation of Gephyrin splicing is a determinant of inhibitory postsynaptic diversity.

Gephyrin (GPHN) regulates the clustering of postsynaptic components at inhibitory synapses and is involved in pathophysiology of neuropsychiatric disorders. Here, we uncover an extensive diversity of GPHN transcripts that are tightly controlled by splicing during mouse and human brain development. Proteomic analysis reveals at least a hundred isoforms of GPHN incorporated at inhibitory Glycine and gamma-aminobutyric acid A receptors containing synapses. They exhibit different localization and postsynaptic clustering properties, and altering the expression level of one isoform is sufficient to affect the number, size, and density of inhibitory synapses in cerebellar Purkinje cells. Furthermore, we discovered that splicing defects reported in neuropsychiatric disorders are carried by multiple alternative GPHN transcripts, demonstrating the need for a thorough analysis of the GPHN transcriptome in patients. Overall, we show that alternative splicing of GPHN is an important genetic variation to consider in neurological diseases and a determinant of the diversity of postsynaptic inhibitory synapses.