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.

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.

RNA editing regulates glutamatergic synapses in the frontal cortex of a molecular subtype of Amyotrophic Lateral Sclerosis.

BACKGROUND: Amyotrophic Lateral Sclerosis (ALS) is a highly heterogenous neurodegenerative disorder that primarily affects upper and lower motor neurons, affecting additional cell types and brain regions. Underlying molecular mechanisms are still elusive, in part due to disease heterogeneity. Molecular disease subtyping through integrative analyses including RNA editing profiling is a novel approach for identification of molecular networks involved in pathogenesis. METHODS: We aimed to highlight the role of RNA editing in ALS, focusing on the frontal cortex and the prevalent molecular disease subtype (ALS-Ox), previously determined by transcriptomic profile stratification. We established global RNA editing (editome) and gene expression (transcriptome) profiles in control and ALS-Ox cases, utilizing publicly available RNA-seq data (GSE153960) and an in-house analysis pipeline. Functional annotation and pathway analyses identified molecular processes affected by RNA editing alterations. Pearson correlation analyses assessed RNA editing effects on expression. Similar analyses on additional ALS-Ox and control samples (GSE124439) were performed for verification. Targeted re-sequencing and qRT-PCR analysis targeting CACNA1C, were performed using frontal cortex tissue from ALS and control samples (n = 3 samples/group). RESULTS: We identified reduced global RNA editing in the frontal cortex of ALS-Ox cases. Differentially edited transcripts are enriched in synapses, particularly in the glutamatergic synapse pathway. Bioinformatic analyses on additional ALS-Ox and control RNA-seq data verified these findings. We identified increased recoding at the Q621R site in the GRIK2 transcript and determined positive correlations between RNA editing and gene expression alterations in ionotropic receptor subunits GRIA2, GRIA3 and the CACNA1C transcript, which encodes the pore forming subunit of a post-synaptic L-type calcium channel. Experimental data verified RNA editing alterations and editing-expression correlation in CACNA1C, highlighting CACNA1C as a target for further study. CONCLUSIONS: We provide evidence on the involvement of RNA editing in the frontal cortex of an ALS molecular subtype, highlighting a modulatory role mediated though recoding and gene expression regulation on glutamatergic synapse related transcripts. We report RNA editing effects in disease-related transcripts and validated editing alterations in CACNA1C. Our study provides targets for further functional studies that could shed light in underlying disease mechanisms enabling novel therapeutic approaches.

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.

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.

Rbm24 modulates neuronal RNA splicing to restrict cognitive dysfunction.

Synaptic dysfunction is associated with early neurodegenerative changes and cognitive deficits. Neuronal cell-specific alternative splicing (AS) programs exclusively encode unique neuron- and synapse-specific proteins. However, it remains unclear whether splicing disturbances in neurons influence the pathogenesis of cognitive impairment. Here, we observed that RNA-binding motif protein 24 (RBM24) expression was decreased in Alzheimer's disease (AD) patients. Using conditional RBM24 knockout mice, we demonstrated that deletion of RBM24 in the brain resulted in learning and memory impairment. Electrophysiological recordings from hippocampal slices from mice lacking RBM24 revealed multiple defects in excitatory synaptic function and plasticity. Furthermore, RNA sequencing and splicing analysis showed that RBM24 regulates a network of genes related to cognitive function. Deletion of RBM24 disrupted the AS of synapse-associated genes, including GluR2 and Prrt1, the major disease genes involved in cognitive impairment and memory loss, leading to cognitive dysfunction. Together, our results suggest that the regulation of mRNA splicing by RBM24 is a key process involved in maintaining normal synaptic function and provide novel mechanistic insights into the pathogenesis of AD.

CRISPR-Cas9 editing of synaptic genes in human embryonic stem cells for functional analysis in induced human neurons.

Generating stable human embryonic stem cells (hESCs) with targeted genetic mutations allows for the interrogation of protein function in numerous cellular contexts while maintaining a relatively high degree of isogenicity. We describe a step-by-step protocol for generating knockout hESC lines with mutations in genes involved in synaptic transmission using CRISPR-Cas9. We describe steps for gRNA design, cloning, stem cell transfection, and clone isolation. We then detail procedures for gene knockout validation and differentiation of stem cells into functional induced neurons.

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.

Rare heterozygous genetic variants of NRXN and NLGN gene families involved in synaptic function and their association with neurodevelopmental disorders.

The interaction of neurexins (NRXNs) in the presynaptic membrane with postsynaptic cell adhesion molecules called neuroligins (NLGNs) is critical for this synaptic function. Impaired synaptic functions are emphasized in neurodevelopmental disorders to uncover etiological factors. We evaluated variants in NRXN and NLGN genes encoding molecules located directly at the synapse in patients with neuropsychiatric disorders using clinical exome sequencing and chromosomal microarray. We presented detailed clinical findings of cases carrying heterozygous NRXN1 (c.190C > T, c.1679C > T and two copy number variations [CNVs]), NRXN2 (c.808dup, c.1901G > T), NRXN3 (c.3889C > T), and NLGN1 (c.269C > G, c.473T > A) gene variants. In addition, three novel variants were identified in the NRXN1 (c.1679C > T), NRXN3 [c.3889C > T (p.Pro1297Ser)], and NLGN1 [c.473T > A (p.Ile158Lys)] genes. We emphasize the clinical findings of CNVs of the NRXN1 gene causing a more severe clinical presentation than single nucleotide variants of the NRXN1 gene in this study. We detected an NRXN2 gene variant (c.808dup) with low allelic frequency in two unrelated cases with the same diagnosis. We emphasize the importance of this variant for future studies. We suggest that NRXN2, NRXN3, and NLGN1 genes, which are less frequently reported than NRXN1 gene variants, may also be associated with neurodevelopmental disorders.

Network analysis of S-nitrosylated synaptic proteins demonstrates unique roles in health and disease.

Nitric oxide can covalently modify cysteine thiols on target proteins to alter that protein's function in a process called S-nitrosylation (SNO). S-nitrosylation of synaptic proteins plays an integral part in neurotransmission. Here we review the function of the SNO-proteome at the synapse and whether clusters of SNO-modification may predict synaptic dysfunction associated with disease. We used a systematic search strategy to concatenate SNO-proteomic datasets from normal human or murine brain samples. Identified SNO-modified proteins were then filtered against proteins reported in the Synaptome Database, which provides a detailed and experimentally verified annotation of all known synaptic proteins. Subsequently, we performed an unbiased network analysis of all known SNO-synaptic proteins to identify clusters of SNO proteins commonly involved in biological processes or with known disease associations. The resulting SNO networks were significantly enriched in biological processes related to metabolism, whereas significant gene-disease associations were related to Schizophrenia, Alzheimer's, Parkinson's and Huntington's disease. Guided by an unbiased network analysis, the current review presents a thorough discussion of how clustered changes to the SNO-proteome influence health and disease.

ZCCHC17 knockdown phenocopies Alzheimer's disease-related loss of synaptic proteins and hyperexcitability.

ZCCHC17 is a master regulator of synaptic gene expression and has recently been shown to play a role in splicing of neuronal mRNA. We previously showed that ZCCHC17 protein declines in Alzheimer's disease (AD) brain tissue before there is significant gliosis and neuronal loss, that ZCCHC17 loss partially replicates observed splicing abnormalities in AD brain tissue, and that maintenance of ZCCHC17 levels is predicted to support cognitive resilience in AD. Here, we assessed the functional consequences of reduced ZCCHC17 expression in primary cortical neuronal cultures using siRNA knockdown. Consistent with its previously identified role in synaptic gene expression, loss of ZCCHC17 led to loss of synaptic protein expression. Patch recording of neurons shows that ZCCHC17 loss significantly disrupted the excitation/inhibition balance of neurotransmission, and favored excitatory-dominant synaptic activity as measured by an increase in spontaneous excitatory post synaptic currents and action potential firing rate, and a decrease in spontaneous inhibitory post synaptic currents. These findings are consistent with the hyperexcitable phenotype seen in AD animal models and in patients. We are the first to assess the functional consequences of ZCCHC17 knockdown in neurons and conclude that ZCCHC17 loss partially phenocopies AD-related loss of synaptic proteins and hyperexcitability.

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.

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.

AFM Imaging Reveals MicroRNA-132 to be a Positive Regulator of Synaptic Functions.

The modification of synaptic and neural connections in adults, including the formation and removal of synapses, depends on activity-dependent synaptic and structural plasticity. MicroRNAs (miRNAs) play crucial roles in regulating these changes by targeting specific genes and regulating their expression. The fact that somatic and dendritic activity in neurons often occurs asynchronously highlights the need for spatial and dynamic regulation of protein synthesis in specific milieu and cellular loci. MicroRNAs, which can show distinct patterns of enrichment, help to establish the localized distribution of plasticity-related proteins. The recent study using atomic force microscopy (AFM)-based nanoscale imaging reveals that the abundance of miRNA(miR)-134 is inversely correlated with the functional activity of dendritic spine structures. However, the miRNAs that are selectively upregulated in potentiated synapses, and which can thereby support prospective changes in synaptic efficacy, remain largely unknown. Using AFM force imaging, significant increases in miR-132 in the dendritic regions abutting functionally-active spines is discovered. This study provides evidence for miR-132 as a novel positive miRNA regulator residing in dendritic shafts, and also suggests that activity-dependent miRNAs localized in distinct sub-compartments of neurons play bi-directional roles in controlling synaptic transmission and synaptic plasticity.

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.

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.

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.

GRID1/GluD1 homozygous variants linked to intellectual disability and spastic paraplegia impair mGlu1/5 receptor signaling and excitatory synapses.

The ionotropic glutamate delta receptor GluD1, encoded by the GRID1 gene, is involved in synapse formation, function, and plasticity. GluD1 does not bind glutamate, but instead cerebellin and D-serine, which allow the formation of trans-synaptic bridges, and trigger transmembrane signaling. Despite wide expression in the nervous system, pathogenic GRID1 variants have not been characterized in humans so far. We report homozygous missense GRID1 variants in five individuals from two unrelated consanguineous families presenting with intellectual disability and spastic paraplegia, without (p.Thr752Met) or with (p.Arg161His) diagnosis of glaucoma, a threefold phenotypic association whose genetic bases had not been elucidated previously. Molecular modeling and electrophysiological recordings indicated that Arg161His and Thr752Met mutations alter the hinge between GluD1 cerebellin and D-serine binding domains and the function of this latter domain, respectively. Expression, trafficking, physical interaction with metabotropic glutamate receptor mGlu1, and cerebellin binding of GluD1 mutants were not conspicuously altered. Conversely, upon expression in neurons of dissociated or organotypic slice cultures, we found that both GluD1 mutants hampered metabotropic glutamate receptor mGlu1/5 signaling via Ca2+ and the ERK pathway and impaired dendrite morphology and excitatory synapse density. These results show that the clinical phenotypes are distinct entities segregating in the families as an autosomal recessive trait, and caused by pathophysiological effects of GluD1 mutants involving metabotropic glutamate receptor signaling and neuronal connectivity. Our findings unravel the importance of GluD1 receptor signaling in sensory, cognitive and motor functions of the human nervous system.

Neurodevelopmental and synaptic defects in DNAJC6 parkinsonism, amenable to gene therapy.

DNAJC6 encodes auxilin, a co-chaperone protein involved in clathrin-mediated endocytosis (CME) at the presynaptic terminal. Biallelic mutations in DNAJC6 cause a complex, early-onset neurodegenerative disorder characterized by rapidly progressive parkinsonism-dystonia in childhood. The disease is commonly associated with additional neurodevelopmental, neurological and neuropsychiatric features. Currently, there are no disease-modifying treatments for this condition, resulting in significant morbidity and risk of premature mortality. To investigate the underlying disease mechanisms in childhood-onset DNAJC6 parkinsonism, we generated induced pluripotent stem cells (iPSC) from three patients harbouring pathogenic loss-of-function DNAJC6 mutations and subsequently developed a midbrain dopaminergic neuronal model of disease. When compared to age-matched and CRISPR-corrected isogenic controls, the neuronal cell model revealed disease-specific auxilin deficiency as well as disturbance of synaptic vesicle recycling and homeostasis. We also observed neurodevelopmental dysregulation affecting ventral midbrain patterning and neuronal maturation. To explore the feasibility of a viral vector-mediated gene therapy approach, iPSC-derived neuronal cultures were treated with lentiviral DNAJC6 gene transfer, which restored auxilin expression and rescued CME. Our patient-derived neuronal model provides deeper insights into the molecular mechanisms of auxilin deficiency as well as a robust platform for the development of targeted precision therapy approaches.