Hyperactivity with Disrupted Attention by Activation of an Astrocyte Synaptogenic Cue.

Hyperactivity and disturbances of attention are common behavioral disorders whose underlying cellular and neural circuit causes are not understood. We report the discovery that striatal astrocytes drive such phenotypes through a hitherto unknown synaptic mechanism. We found that striatal medium spiny neurons (MSNs) triggered astrocyte signaling via γ-aminobutyric acid B (GABAB) receptors. Selective chemogenetic activation of this pathway in striatal astrocytes in vivo resulted in acute behavioral hyperactivity and disrupted attention. Such responses also resulted in upregulation of the synaptogenic cue thrombospondin-1 (TSP1) in astrocytes, increased excitatory synapses, enhanced corticostriatal synaptic transmission, and increased MSN action potential firing in vivo. All of these changes were reversed by blocking TSP1 effects. Our data identify a form of bidirectional neuron-astrocyte communication and demonstrate that acute reactivation of a single latent astrocyte synaptogenic cue alters striatal circuits controlling behavior, revealing astrocytes and the TSP1 pathway as therapeutic targets in hyperactivity, attention deficit, and related psychiatric disorders.

Regulation of Neuronal Differentiation, Function, and Plasticity by Alternative Splicing.

Posttranscriptional mechanisms provide powerful means to expand the coding power of genomes. In nervous systems, alternative splicing has emerged as a fundamental mechanism not only for the diversification of protein isoforms but also for the spatiotemporal control of transcripts. Thus, alternative splicing programs play instructive roles in the development of neuronal cell type-specific properties, neuronal growth, self-recognition, synapse specification, and neuronal network function. Here we discuss the most recent genome-wide efforts on mapping RNA codes and RNA-binding proteins for neuronal alternative splicing regulation. We illustrate how alternative splicing shapes key steps of neuronal development, neuronal maturation, and synaptic properties. Finally, we highlight efforts to dissect the spatiotemporal dynamics of alternative splicing and their potential contribution to neuronal plasticity and the mature nervous system.

Histone variant H2BE enhances chromatin accessibility in neurons to promote synaptic gene expression and long-term memory.

Histone proteins affect gene expression through multiple mechanisms, including through exchange with histone variants. Recent findings link histone variants to neurological disorders, yet few are well studied in the brain. Most notably, widely expressed variants of H2B remain elusive. We applied recently developed antibodies, biochemical assays, and sequencing approaches to reveal broad expression of the H2B variant H2BE and defined its role in regulating chromatin structure, neuronal transcription, and mouse behavior. We find that H2BE is enriched at promoters, and a single unique amino acid allows it to dramatically enhance chromatin accessibility. Further, we show that H2BE is critical for synaptic gene expression and long-term memory. Together, these data reveal a mechanism linking histone variants to chromatin accessibility, transcriptional regulation, neuronal function, and memory. This work further identifies a widely expressed H2B variant and uncovers a single histone amino acid with profound effects on genomic structure.

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.

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.

Key genes and convergent pathogenic mechanisms in Parkinson disease.

Parkinson disease (PD) is a neurodegenerative disorder marked by the preferential dysfunction and death of dopaminergic neurons in the substantia nigra. The onset and progression of PD is influenced by a diversity of genetic variants, many of which lack functional characterization. To identify the most high-yield targets for therapeutic intervention, it is important to consider the core cellular compartments and functional pathways upon which the varied forms of pathogenic dysfunction may converge. Here, we review several key PD-linked proteins and pathways, focusing on the mechanisms of their potential convergence in disease pathogenesis. These dysfunctions primarily localize to a subset of subcellular compartments, including mitochondria, lysosomes and synapses. We discuss how these pathogenic mechanisms that originate in different cellular compartments may coordinately lead to cellular dysfunction and neurodegeneration in PD.

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.

Genetic mechanisms for impaired synaptic plasticity in schizophrenia revealed by computational modeling.

Schizophrenia phenotypes are suggestive of impaired cortical plasticity in the disease, but the mechanisms of these deficits are unknown. Genomic association studies have implicated a large number of genes that regulate neuromodulation and plasticity, indicating that the plasticity deficits have a genetic origin. Here, we used biochemically detailed computational modeling of postsynaptic plasticity to investigate how schizophrenia-associated genes regulate long-term potentiation (LTP) and depression (LTD). We combined our model with data from postmortem RNA expression studies (CommonMind gene-expression datasets) to assess the consequences of altered expression of plasticity-regulating genes for the amplitude of LTP and LTD. Our results show that the expression alterations observed post mortem, especially those in the anterior cingulate cortex, lead to impaired protein kinase A (PKA)-pathway-mediated LTP in synapses containing GluR1 receptors. We validated these findings using a genotyped electroencephalogram (EEG) dataset where polygenic risk scores for synaptic and ion channel-encoding genes as well as modulation of visual evoked potentials were determined for 286 healthy controls. Our results provide a possible genetic mechanism for plasticity impairments in schizophrenia, which can lead to improved understanding and, ultimately, treatment of the disorder.

Tissue-specific knockout in the Drosophila neuromuscular system reveals ESCRT's role in formation of synapse-derived extracellular vesicles.

Tissue-specific gene knockout by CRISPR/Cas9 is a powerful approach for characterizing gene functions during development. However, this approach has not been successfully applied to most Drosophila tissues, including the Drosophila neuromuscular junction (NMJ). To expand tissue-specific CRISPR to this powerful model system, here we present a CRISPR-mediated tissue-restricted mutagenesis (CRISPR-TRiM) toolkit for knocking out genes in motoneurons, muscles, and glial cells. We validated the efficacy of CRISPR-TRiM by knocking out multiple genes in each tissue, demonstrated its orthogonal use with the Gal4/UAS binary expression system, and showed simultaneous knockout of multiple redundant genes. We used CRISPR-TRiM to discover an essential role for SNARE components in NMJ maintenance. Furthermore, we demonstrate that the canonical ESCRT pathway suppresses NMJ bouton growth by downregulating retrograde Gbb signaling. Lastly, we found that axon termini of motoneurons rely on ESCRT-mediated intra-axonal membrane trafficking to release extracellular vesicles at the NMJ. Thus, we have successfully developed an NMJ CRISPR mutagenesis approach which we used to reveal genes important for NMJ structural plasticity.

mRNA and circRNA mislocalization to synapses are key features of Alzheimer's disease.

Proper transport of RNAs to synapses is essential for localized translation of proteins in response to synaptic signals and synaptic plasticity. Alzheimer's disease (AD) is a neurodegenerative disease characterized by accumulation of amyloid aggregates and hyperphosphorylated tau neurofibrillary tangles followed by widespread synapse loss. To understand whether RNA synaptic localization is impacted in AD, we performed RNA sequencing on synaptosomes and brain homogenates from AD patients and cognitively healthy controls. This resulted in the discovery of hundreds of mislocalized mRNAs in AD among frontal and temporal brain regions. Similar observations were found in an APPswe/PSEN1dE9 mouse model. Furthermore, major differences were observed among circular RNAs (circRNAs) localized to synapses in AD including two overlapping isoforms of circGSK3ß, one upregulated, and one downregulated. Expression of these distinct isoforms affected tau phosphorylation in neuronal cells substantiating the importance of circRNAs in the brain and pointing to a new class of therapeutic targets.

Reduced synaptic depression in human neurons carrying homozygous disease-causing STXBP1 variant L446F.

MUNC18-1 is an essential protein of the regulated secretion machinery. De novo, heterozygous mutations in STXBP1, the human gene encoding this protein, lead to a severe neurodevelopmental disorder. Here, we describe the electrophysiological characteristics of a unique case of STXBP1-related disorder caused by a homozygous mutation (L446F). We engineered this mutation in induced pluripotent stem cells from a healthy donor (STXBP1LF/LF) to establish isogenic cell models. We performed morphological and electrophysiological analyses on single neurons grown on glial micro-islands. Human STXBP1LF/LF neurons displayed normal morphology and normal basal synaptic transmission but increased paired-pulse ratios and charge released, and reduced synaptic depression compared to control neurons. Immunostainings revealed normal expression levels but impaired recognition by a mutation-specific MUNC18-1 antibody. The electrophysiological gain-of-function phenotype is in line with earlier overexpression studies in Stxbp1 null mouse neurons, with some potentially human-specific features. Therefore, the present study highlights important differences between mouse and human neurons critical for the translatability of pre-clinical studies.

Alzheimer's disease risk gene CD2AP is a dose-sensitive determinant of synaptic structure and plasticity.

CD2-Associated protein (CD2AP) is a candidate susceptibility gene for Alzheimer's disease, but its role in the mammalian central nervous system remains largely unknown. We show that CD2AP protein is broadly expressed in the adult mouse brain, including within cortical and hippocampal neurons, where it is detected at pre-synaptic terminals. Deletion of Cd2ap altered dendritic branching and spine density, and impaired ubiquitin-proteasome system activity. Moreover, in mice harboring either one or two copies of a germline Cd2ap null allele, we noted increased paired-pulse facilitation at hippocampal Schaffer-collateral synapses, consistent with a haploinsufficient requirement for pre-synaptic release. Whereas conditional Cd2ap knockout in the brain revealed no gross behavioral deficits in either 3.5- or 12-month-old mice, Cd2ap heterozygous mice demonstrated subtle impairments in discrimination learning using a touchscreen task. Based on unbiased proteomics, partial or complete loss of Cd2ap triggered perturbation of proteins with roles in protein folding, lipid metabolism, proteostasis, and synaptic function. Overall, our results reveal conserved, dose-sensitive requirements for CD2AP in the maintenance of neuronal structure and function, including synaptic homeostasis and plasticity, and inform our understanding of possible cell-type specific mechanisms in Alzheimer's Disease.

Immunoglobulin-Like Receptors and Their Impact on Wiring of Brain Synapses.

Synapse formation is mediated by a surprisingly large number and wide variety of genes encoding many different protein classes. One of the families increasingly implicated in synapse wiring is the immunoglobulin superfamily (IgSF). IgSF molecules are by definition any protein containing at least one Ig-like domain, making this family one of the most common protein classes encoded by the genome. Here, we review the emerging roles for IgSF molecules in synapse formation specifically in the vertebrate brain, focusing on examples from three classes of IgSF members: ( a) cell adhesion molecules, ( b) signaling molecules, and ( c) immune molecules expressed in the brain. The critical roles for IgSF members in regulating synapse formation may explain their extensive involvement in neuropsychiatric and neurodevelopmental disorders. Solving the IgSF code for synapse formation may reveal multiple new targets for rescuing IgSF-mediated deficits in synapse formation and, eventually, new treatments for psychiatric disorders caused by altered IgSF-induced synapse wiring.

Neurexins: molecular codes for shaping neuronal synapses.

The function of neuronal circuits relies on the properties of individual neuronal cells and their synapses. We propose that a substantial degree of synapse formation and function is instructed by molecular codes resulting from transcriptional programmes. Recent studies on the Neurexin protein family and its ligands provide fundamental insight into how synapses are assembled and remodelled, how synaptic properties are specified and how single gene mutations associated with neurodevelopmental and psychiatric disorders might modify the operation of neuronal circuits and behaviour. In this Review, we first summarize insights into Neurexin function obtained from various model organisms. We then discuss the mechanisms and logic of the cell type-specific regulation of Neurexin isoforms, in particular at the level of alternative mRNA splicing. Finally, we propose a conceptual framework for how combinations of synaptic protein isoforms act as 'senders' and 'readers' to instruct synapse formation and the acquisition of cell type-specific and synapse-specific functional properties.

Plasticity mechanisms of genetically distinct Purkinje cells.

Despite its uniform appearance, the cerebellar cortex is highly heterogeneous in terms of structure, genetics and physiology. Purkinje cells (PCs), the principal and sole output neurons of the cerebellar cortex, can be categorized into multiple populations that differentially express molecular markers and display distinctive physiological features. Such features include action potential rate, but also their propensity for synaptic and intrinsic plasticity. However, the precise molecular and genetic factors that correlate with the differential physiological properties of PCs remain elusive. In this article, we provide a detailed overview of the cellular mechanisms that regulate PC activity and plasticity. We further perform a pathway analysis to highlight how molecular characteristics of specific PC populations may influence their physiology and plasticity mechanisms.

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.

Small Differences and Big Changes: The Many Variables of MicroRNA Expression and Function in the Brain.

MicroRNAs are emerging as crucial regulators within the complex, dynamic environment of the synapse, and they offer a promising new avenue for the treatment of neurological disease. These small noncoding RNAs modify gene expression in several ways, including posttranscriptional modulation via binding to complementary and semicomplementary sites on target mRNAs. This rapid, finely tuned regulation of gene expression is essential to meet the dynamic demands of the synapse. Here, we provide a detailed review of the multifaceted world of synaptic microRNA regulation. We discuss the many mechanisms by which microRNAs regulate gene expression at the synapse, particularly in the context of neuronal plasticity. We also describe the various factors, such as age, sex, and neurological disease, that can influence microRNA expression and activity in neurons. In summary, microRNAs play a crucial role in the intricate and quickly changing functional requirements of the synapse, and context is essential in the study of microRNAs and their potential therapeutic applications.

ALS-Linked VapB P56S Mutation Alters Neuronal Mitochondrial Turnover at the Synapse.

Mitochondrial population maintenance in neurons is essential for neuron function and survival. Contact sites between mitochondria and the endoplasmic reticulum (ER) are poised to regulate mitochondrial homeostasis in neurons. These contact sites can facilitate transfer of calcium and lipids between the organelles and have been shown to regulate aspects of mitochondrial dynamics. Vesicle-associated membrane protein-associated protein B (VapB) is an ER membrane protein present at a subset of ER-mitochondrial contact sites. A proline-to-serine mutation in VapB at amino acid 56 (P56S) correlates with susceptibility to amyotrophic lateral sclerosis (ALS) type 8. Given the relationship between failed mitochondrial health and neurodegenerative disease, we investigated the function of VapB in mitochondrial population maintenance. We demonstrated that transgenic expression of VapBP56S in zebrafish larvae (sex undetermined) increased mitochondrial biogenesis, causing increased mitochondrial population size in the axon terminal. Expression of wild-type VapB did not alter biogenesis but, instead, increased mitophagy in the axon terminal. Using genetic manipulations to independently increase mitochondrial biogenesis, we show that biogenesis is normally balanced by mitophagy to maintain a constant mitochondrial population size. VapBP56S transgenics fail to increase mitophagy to compensate for the increase in mitochondrial biogenesis, suggesting an impaired mitophagic response. Finally, using a synthetic ER-mitochondrial tether, we show that VapB's function in mitochondrial turnover is likely independent of ER-mitochondrial tethering by contact sites. Our findings demonstrate that VapB can control mitochondrial turnover in the axon terminal, and this function is altered by the P56S ALS-linked mutation.

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.

Phospholipid-flippase chaperone CDC50A is required for synapse maintenance by regulating phosphatidylserine exposure.

Synaptic refinement is a critical physiological process that removes excess synapses to establish and maintain functional neuronal circuits. Recent studies have shown that focal exposure of phosphatidylserine (PS) on synapses acts as an "eat me" signal to mediate synaptic pruning. However, the molecular mechanism underlying PS externalization at synapses remains elusive. Here, we find that murine CDC50A, a chaperone of phospholipid flippases, localizes to synapses, and that its expression depends on neuronal activity. Cdc50a knockdown leads to phosphatidylserine exposure at synapses and subsequent erroneous synapse removal by microglia partly via the GPR56 pathway. Taken together, our data support that CDC50A safeguards synapse maintenance by regulating focal phosphatidylserine exposure at synapses.