ABSTRACT
BACKGROUND: Moderate-to-severe hemophilia B is treated with lifelong, continuous coagulation factor IX replacement to prevent bleeding. Gene therapy for hemophilia B aims to establish sustained factor IX activity, thereby protecting against bleeding without burdensome factor IX replacement. METHODS: In this open-label, phase 3 study, after a lead-in period (≥6 months) of factor IX prophylaxis, we administered one infusion of adeno-associated virus 5 (AAV5) vector expressing the Padua factor IX variant (etranacogene dezaparvovec; 2×1013 genome copies per kilogram of body weight) to 54 men with hemophilia B (factor IX activity ≤2% of the normal value) regardless of preexisting AAV5 neutralizing antibodies. The primary end point was the annualized bleeding rate, evaluated in a noninferiority analysis comparing the rate during months 7 through 18 after etranacogene dezaparvovec treatment with the rate during the lead-in period. Noninferiority of etranacogene dezaparvovec was defined as an upper limit of the two-sided 95% Wald confidence interval of the annualized bleeding rate ratio that was less than the noninferiority margin of 1.8. Superiority, additional efficacy measures, and safety were also assessed. RESULTS: The annualized bleeding rate decreased from 4.19 (95% confidence interval [CI], 3.22 to 5.45) during the lead-in period to 1.51 (95% CI, 0.81 to 2.82) during months 7 through 18 after treatment, for a rate ratio of 0.36 (95% Wald CI, 0.20 to 0.64; P<0.001), demonstrating noninferiority and superiority of etranacogene dezaparvovec as compared with factor IX prophylaxis. Factor IX activity had increased from baseline by a least-squares mean of 36.2 percentage points (95% CI, 31.4 to 41.0) at 6 months and 34.3 percentage points (95% CI, 29.5 to 39.1) at 18 months after treatment, and usage of factor IX concentrate decreased by a mean of 248,825 IU per year per participant in the post-treatment period (P<0.001 for all three comparisons). Benefits and safety were observed in participants with predose AAV5 neutralizing antibody titers of less than 700. No treatment-related serious adverse events occurred. CONCLUSIONS: Etranacogene dezaparvovec gene therapy was superior to prophylactic factor IX with respect to the annualized bleeding rate, and it had a favorable safety profile. (Funded by uniQure and CSL Behring; HOPE-B ClinicalTrials.gov number, NCT03569891.).
Subject(s)
Factor IX , Genetic Therapy , Hemophilia B , Humans , Male , Factor IX/genetics , Factor IX/therapeutic use , Genetic Therapy/methods , Hemophilia B/complications , Hemophilia B/genetics , Hemophilia B/therapy , Hemorrhage/etiology , Hemorrhage/therapy , Genetic Vectors/administration & dosageABSTRACT
Store-operated Ca(2+) channels activated by the depletion of Ca(2+) from the endoplasmic reticulum (ER) are a major Ca(2+) entry pathway in nonexcitable cells and are essential for T cell activation and adaptive immunity. After store depletion, the ER Ca(2+) sensor STIM1 and the CRAC channel protein Orai1 redistribute to ER-plasma membrane (PM) junctions, but the fundamental issue of how STIM1 activates the CRAC channel at these sites is unresolved. Here, we identify a minimal, highly conserved 107-aa CRAC activation domain (CAD) of STIM1 that binds directly to the N and C termini of Orai1 to open the CRAC channel. Purified CAD forms a tetramer that clusters CRAC channels, but analysis of STIM1 mutants reveals that channel clustering is not sufficient for channel activation. These studies establish a molecular mechanism for store-operated Ca(2+) entry in which the direct binding of STIM1 to Orai1 drives the accumulation and the activation of CRAC channels at ER-PM junctions.
Subject(s)
Calcium Channels/metabolism , Calcium Signaling , Membrane Proteins/metabolism , Neoplasm Proteins/metabolism , Calcium Channels/chemistry , Cell Line , Cell Membrane/metabolism , Endoplasmic Reticulum/metabolism , Humans , ORAI1 Protein , Protein Structure, Tertiary , Stromal Interaction Molecule 1ABSTRACT
Recent advances in cell reprogramming enable investigators to generate pluripotent stem cells from somatic cells. These induced pluripotent cells can subsequently be differentiated into any cell type, making it possible for the first time to obtain functional human neurons in the lab from control subjects and patients with psychiatric disorders. In this review, we survey the progress made in generating various neuronal subtypes in vitro, with special emphasis on the characterization of these neurons and the identification of unique features of human brain development in a dish. We also discuss efforts to uncover neuronal phenotypes from patients with psychiatric disease and prospects for the use of this platform for drug development.
Subject(s)
Cell Culture Techniques/methods , Induced Pluripotent Stem Cells/cytology , Mental Disorders/physiopathology , Nervous System Diseases/physiopathology , Neurogenesis/physiology , Neurons/cytology , Animals , Humans , Induced Pluripotent Stem Cells/pathology , Mental Disorders/pathology , Nervous System Diseases/pathology , Neurons/pathologyABSTRACT
Phelan-McDermid syndrome (PMDS) is a complex neurodevelopmental disorder characterized by global developmental delay, severely impaired speech, intellectual disability, and an increased risk of autism spectrum disorders (ASDs). PMDS is caused by heterozygous deletions of chromosome 22q13.3. Among the genes in the deleted region is SHANK3, which encodes a protein in the postsynaptic density (PSD). Rare mutations in SHANK3 have been associated with idiopathic ASDs, non-syndromic intellectual disability, and schizophrenia. Although SHANK3 is considered to be the most likely candidate gene for the neurological abnormalities in PMDS patients, the cellular and molecular phenotypes associated with this syndrome in human neurons are unknown. We generated induced pluripotent stem (iPS) cells from individuals with PMDS and autism and used them to produce functional neurons. We show that PMDS neurons have reduced SHANK3 expression and major defects in excitatory, but not inhibitory, synaptic transmission. Excitatory synaptic transmission in PMDS neurons can be corrected by restoring SHANK3 expression or by treating neurons with insulin-like growth factor 1 (IGF1). IGF1 treatment promotes formation of mature excitatory synapses that lack SHANK3 but contain PSD95 and N-methyl-D-aspartate (NMDA) receptors with fast deactivation kinetics. Our findings provide direct evidence for a disruption in the ratio of cellular excitation and inhibition in PMDS neurons, and point to a molecular pathway that can be recruited to restore it.
Subject(s)
Chromosome Disorders/physiopathology , Insulin-Like Growth Factor I/pharmacology , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/metabolism , Neurons/physiology , Synapses/drug effects , Synapses/physiology , Cell Line , Child , Chromosome Deletion , Chromosome Disorders/genetics , Chromosomes, Human, Pair 22/genetics , Female , GABA Agents/pharmacology , Gene Expression Regulation/drug effects , Humans , Lentivirus/genetics , Male , Neurons/cytology , Neurons/drug effects , Pluripotent Stem Cells/cytology , Receptors, Glutamate/genetics , Sequence Deletion , Synapses/genetics , Synaptic Transmission/drug effects , Synaptic Transmission/geneticsABSTRACT
Individuals with congenital or acquired prolongation of the QT interval, or long QT syndrome (LQTS), are at risk of life-threatening ventricular arrhythmia. LQTS is commonly genetic in origin but can also be caused or exacerbated by environmental factors. A missense mutation in the L-type calcium channel Ca(V)1.2 leads to LQTS in patients with Timothy syndrome. To explore the effect of the Timothy syndrome mutation on the electrical activity and contraction of human cardiomyocytes, we reprogrammed human skin cells from Timothy syndrome patients to generate induced pluripotent stem cells, and differentiated these cells into cardiomyocytes. Electrophysiological recording and calcium (Ca(2+)) imaging studies of these cells revealed irregular contraction, excess Ca(2+) influx, prolonged action potentials, irregular electrical activity and abnormal calcium transients in ventricular-like cells. We found that roscovitine, a compound that increases the voltage-dependent inactivation of Ca(V)1.2 (refs 6-8), restored the electrical and Ca(2+) signalling properties of cardiomyocytes from Timothy syndrome patients. This study provides new opportunities for studying the molecular and cellular mechanisms of cardiac arrhythmias in humans, and provides a robust assay for developing new drugs to treat these diseases.
Subject(s)
Drug Evaluation, Preclinical/methods , Induced Pluripotent Stem Cells/pathology , Myocytes, Cardiac/drug effects , Myocytes, Cardiac/pathology , Action Potentials/drug effects , Autistic Disorder , Calcium Channels, L-Type/genetics , Calcium Channels, L-Type/metabolism , Calcium Signaling/drug effects , Cell Transdifferentiation , Cellular Reprogramming/genetics , Fibroblasts/cytology , HEK293 Cells , Humans , Long QT Syndrome/drug therapy , Long QT Syndrome/genetics , Long QT Syndrome/metabolism , Long QT Syndrome/pathology , Mutation, Missense/genetics , Myocytes, Cardiac/metabolism , Patch-Clamp Techniques , Phenotype , Purines/pharmacology , Roscovitine , Single-Cell Analysis , Syndactyly/drug therapy , Syndactyly/genetics , Syndactyly/metabolism , Syndactyly/pathologyABSTRACT
Neurogenic transcription factors and evolutionarily conserved signalling pathways have been found to be instrumental in the formation of neurons. However, the instructive role of microRNAs (miRNAs) in neurogenesis remains unexplored. We recently discovered that miR-9* and miR-124 instruct compositional changes of SWI/SNF-like BAF chromatin-remodelling complexes, a process important for neuronal differentiation and function. Nearing mitotic exit of neural progenitors, miR-9* and miR-124 repress the BAF53a subunit of the neural-progenitor (np)BAF chromatin-remodelling complex. After mitotic exit, BAF53a is replaced by BAF53b, and BAF45a by BAF45b and BAF45c, which are then incorporated into neuron-specific (n)BAF complexes essential for post-mitotic functions. Because miR-9/9* and miR-124 also control multiple genes regulating neuronal differentiation and function, we proposed that these miRNAs might contribute to neuronal fates. Here we show that expression of miR-9/9* and miR-124 (miR-9/9*-124) in human fibroblasts induces their conversion into neurons, a process facilitated by NEUROD2. Further addition of neurogenic transcription factors ASCL1 and MYT1L enhances the rate of conversion and the maturation of the converted neurons, whereas expression of these transcription factors alone without miR-9/9*-124 was ineffective. These studies indicate that the genetic circuitry involving miR-9/9*-124 can have an instructive role in neural fate determination.
Subject(s)
Cell Differentiation/genetics , Fibroblasts/cytology , Fibroblasts/metabolism , MicroRNAs/genetics , Neurons/cytology , Neurons/metabolism , Adult , Basic Helix-Loop-Helix Transcription Factors/genetics , Basic Helix-Loop-Helix Transcription Factors/metabolism , Biomarkers/analysis , Biomarkers/metabolism , Cell Line , Cell Lineage/genetics , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Excitatory Postsynaptic Potentials/physiology , Humans , Infant, Newborn , MicroRNAs/metabolism , Microtubule-Associated Proteins/analysis , Microtubule-Associated Proteins/metabolism , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/metabolism , Neuropeptides/genetics , Neuropeptides/metabolism , Transcription Factors/genetics , Transcription Factors/metabolism , Tubulin/analysis , Tubulin/metabolismABSTRACT
Copy number variants (CNVs) that delete or duplicate 30 genes within the 16p11.2 genomic region give rise to a range of neurodevelopmental phenotypes with high penetrance in humans. Despite the identification of this small region, the mechanisms by which 16p11.2 CNVs lead to disease are unclear. Relevant models, such as human cortical organoids (hCOs), are needed to understand the human-specific mechanisms of neurodevelopmental disease. We generated hCOs from 17 patients and controls, profiling 167,958 cells with single-cell RNA-sequencing analysis, which revealed neuronal-specific differential expression of genes outside the 16p11.2 region that are related to cell-cell adhesion, neuronal projection growth, and neurodevelopmental disorders. Furthermore, 16p11.2 deletion syndrome organoids exhibited reduced mRNA and protein levels of RBFOX1, a gene that can also harbor CNVs linked to neurodevelopmental phenotypes. We found that the genes previously shown to be regulated by RBFOX1 are also perturbed in organoids from patients with the 16p11.2 deletion syndrome and thus identified a novel link between independent CNVs associated with neuronal development and autism. Overall, this work suggests convergent signaling, which indicates the possibility of a common therapeutic mechanism across multiple rare neuronal diseases.
Subject(s)
Chromosome Deletion , DNA Copy Number Variations , Humans , DNA Copy Number Variations/genetics , Brain , Phenotype , Organoids , RNA Splicing Factors/geneticsABSTRACT
Ca(2+)-dependent inactivation (CDI) is a key regulator and hallmark of the Ca(2+) release-activated Ca(2+) (CRAC) channel, a prototypic store-operated Ca(2+) channel. Although the roles of the endoplasmic reticulum Ca(2+) sensor STIM1 and the channel subunit Orai1 in CRAC channel activation are becoming well understood, the molecular basis of CDI remains unclear. Recently, we defined a minimal CRAC activation domain (CAD; residues 342-448) that binds directly to Orai1 to activate the channel. Surprisingly, CAD-induced CRAC currents lack fast inactivation, revealing a critical role for STIM1 in this gating process. Through truncations of full-length STIM1, we identified a short domain (residues 470-491) C-terminal to CAD that is required for CDI. This domain contains a cluster of 7 acidic amino acids between residues 475 and 483. Neutralization of aspartate or glutamate pairs in this region either reduced or enhanced CDI, whereas the combined neutralization of six acidic residues eliminated inactivation entirely. Based on bioinformatics predictions of a calmodulin (CaM) binding site on Orai1, we also investigated a role for CaM in CDI. We identified a membrane-proximal N-terminal domain of Orai1 (residues 68-91) that binds CaM in a Ca(2+)-dependent manner and mutations that eliminate CaM binding abrogate CDI. These studies identify novel structural elements of STIM1 and Orai1 that are required for CDI and support a model in which CaM acts in concert with STIM1 and the N terminus of Orai1 to evoke rapid CRAC channel inactivation.
Subject(s)
Calcium Channels/metabolism , Calmodulin/metabolism , Ion Channel Gating/physiology , Membrane Proteins/metabolism , Neoplasm Proteins/metabolism , Cell Line , Computational Biology , DNA Primers/genetics , DNA, Complementary/genetics , Electrophysiology , Humans , Immunoblotting , Immunoprecipitation , Models, Biological , Mutagenesis , ORAI1 Protein , Plasmids/genetics , Protein Binding , Protein Structure, Tertiary/genetics , Stromal Interaction Molecule 1 , TransfectionABSTRACT
Background and Objectives: Huntington disease (HD) is a rare, inherited, and highly complex neurodegenerative disorder with no currently approved disease-modifying treatments. We investigated the effect of HD on health-related quality of life and other patient-reported outcomes in the Huntington's Disease Burden of Illness (HDBOI) study. Methods: The HDBOI study is a retrospective, cross-sectional study conducted between September 2020 and May 2021 in France, Germany, Italy, Spain, the United Kingdom, and the United States. People with symptomatic onset HD (PwHD) were recruited by their HD-treating physicians and categorized as early (ES), mid (MS), or advanced stage (AS) HD. Physicians provided sociodemographic and clinical information from the participant's medical records in electronic case report forms (eCRF); participants or their proxies completed online Patient Public Involvement Engagement questionnaires (PPIE-P). Patient-reported outcomes included the 5-level EQ-5D version (EQ-5D-5L), Short-Form-(SF)-36 v2 (and SF-6-Dimension [SF-6D] utility), Huntington Quality of Life Instrument (H-QoL-I), and the Work Productivity and Activity Impairment Specific Health Problem. All outcomes were summarized using descriptive statistics, and differences between disease stages were assessed by Kruskal-Wallis tests. Results: A total of 2,094 PwHD were enrolled with completed eCRFs (100%) and PPIE-P forms (n = 482, 23%). Participants' mean age was 47.3 years; they were generally evenly distributed across countries, with the majority being ES (40%) followed by MS (33%) and LS (26%). The mean EQ-5D-5L (n = 336) utility score was 0.59 (SD, 0.27), with the highest mean utility scores [SD] in ES (0.72 [0.22]) followed by MS (0.62 [0.18]) and AS (0.37 [0.30]), p < 0.001. The mean SF-6D score (n = 482) was 0.57 (SD, 0.10), with mean values decreasing with advanced disease (ES, 0.61; MS, 0.56; AS, 0.50, p < 0.001). H-QoL-I mean scores (n = 482) also worsened with more advanced disease, from 0.58 for ES to 0.49 for MS and 0.37 for AS, p < 0.001. Impairment in daily activities and in work productivity also increased with more advanced disease. Overall proxy respondents reported on average worse outcomes than PwHD (self-reported) across all outcomes and disease stages suggesting a possible unawareness of deficits by PwHD. Discussion: The HDBOI study provides new insights into the characteristics and humanistic burden of PwHD and offers a meaningful contribution to this underserved research area.
ABSTRACT
Voltage-gated calcium channels (VGCCs) convert electrical activity into calcium (Ca2+) signals that regulate cellular excitability, differentiation, and connectivity. The magnitude and kinetics of Ca2+ signals depend on the number of VGCCs at the plasma membrane, but little is known about the regulation of VGCC surface expression. We report that electrical activity causes internalization of the L-type Ca2+ channel (LTC) CaV1.2 and that this is mediated by binding to the tumor suppressor eIF3e/Int6 (eukaryotic initiation factor 3 subunit e). Using total internal reflection microscopy, we identify a population of CaV1.2 containing endosomes whose rapid trafficking is strongly regulated by Ca2+. We define a domain in the II-III loop of CaV1.2 that binds eIF3e and is essential for the activity dependence of both channel internalization and endosomal trafficking. These findings provide a mechanism for activity-dependent internalization and trafficking of CaV1.2 and provide a tantalizing link between Ca2+ homeostasis and a mammalian oncogene.
Subject(s)
Calcium Channels, N-Type/metabolism , Calcium/metabolism , Eukaryotic Initiation Factor-3/metabolism , Animals , Calcium Channel Blockers/pharmacology , Calcium Channels, N-Type/genetics , Cells, Cultured , Dose-Response Relationship, Drug , Dose-Response Relationship, Radiation , Dynamin I/genetics , Dynamin I/metabolism , Electric Stimulation/methods , Embryo, Mammalian , Hippocampus/cytology , Humans , Membrane Potentials/drug effects , Membrane Potentials/physiology , Membrane Potentials/radiation effects , Neurons/drug effects , Neurons/physiology , Neurons/radiation effects , Potassium Chloride/pharmacology , Protein Structure, Tertiary , Protein Transport/drug effects , Protein Transport/physiology , Rats , Rats, Sprague-Dawley , Transfection/methods , omega-Conotoxin GVIA/pharmacologyABSTRACT
Neurodevelopmental neuropsychiatric disorders, such as autism spectrum disorder and schizophrenia, have strong genetic risk components, but the underlying mechanisms have proven difficult to decipher. Rare, high-risk variants may offer an opportunity to delineate the biological mechanisms responsible more clearly for more common idiopathic diseases. Indeed, different rare variants can cause the same behavioral phenotype, demonstrating genetic heterogeneity, while the same rare variant can cause different behavioral phenotypes, demonstrating variable expressivity. These observations suggest convergent underlying biological and neurological mechanisms; identification of these mechanisms may ultimately reveal new therapeutic targets. At the 2021 Keystone eSymposium "Neuropsychiatric and Neurodevelopmental Disorders: Harnessing Rare Variants" a panel of experts in the field described significant progress in genomic discovery and human phenotyping and raised several consistent issues, including the need for detailed natural history studies of rare disorders, the challenges in cohort recruitment, and the importance of viewing phenotypes as quantitative traits that are impacted by rare variants.
Subject(s)
Congresses as Topic/trends , Genetic Variation/genetics , Mental Disorders/genetics , Neurodevelopmental Disorders/genetics , Penetrance , Research Report , Humans , Mental Disorders/diagnosis , Mental Disorders/psychology , Neurodevelopmental Disorders/diagnosis , Neurodevelopmental Disorders/psychologyABSTRACT
Microdeletions and microduplications of the 16p11.2 chromosomal locus are associated with syndromic neurodevelopmental disorders and reciprocal physiological conditions such as macro/microcephaly and high/low body mass index. To facilitate cellular and molecular investigations into these phenotypes, 65 clones of human induced pluripotent stem cells (hiPSCs) were generated from 13 individuals with 16p11.2 copy number variations (CNVs). To ensure these cell lines were suitable for downstream mechanistic investigations, a customizable bioinformatic strategy for the detection of random integration and expression of reprogramming vectors was developed and leveraged towards identifying a subset of 'footprint'-free hiPSC clones. Transcriptomic profiling of cortical neural progenitor cells derived from these hiPSCs identified alterations in gene expression patterns which precede morphological abnormalities reported at later neurodevelopmental stages. Interpreting clinical information-available with the cell lines by request from the Simons Foundation Autism Research Initiative-with this transcriptional data revealed disruptions in gene programs related to both nervous system function and cellular metabolism. As demonstrated by these analyses, this publicly available resource has the potential to serve as a powerful medium for probing the etiology of developmental disorders associated with 16p11.2 CNVs.
Subject(s)
Gene Deletion , Induced Pluripotent Stem Cells/physiology , Autism Spectrum Disorder/genetics , Autistic Disorder , Chromosome Deletion , Chromosome Disorders , Chromosomes, Human, Pair 16 , DNA Copy Number Variations , Gene Expression Regulation, Developmental/drug effects , Gene Expression Regulation, Developmental/genetics , Humans , Intellectual Disability , Neurodevelopmental Disorders , Neurons/physiology , TranscobalaminsABSTRACT
22q11.2 deletion syndrome (22q11DS) is a highly penetrant and common genetic cause of neuropsychiatric disease. Here we generated induced pluripotent stem cells from 15 individuals with 22q11DS and 15 control individuals and differentiated them into three-dimensional (3D) cerebral cortical organoids. Transcriptional profiling across 100 days showed high reliability of differentiation and revealed changes in neuronal excitability-related genes. Using electrophysiology and live imaging, we identified defects in spontaneous neuronal activity and calcium signaling in both organoid- and 2D-derived cortical neurons. The calcium deficit was related to resting membrane potential changes that led to abnormal inactivation of voltage-gated calcium channels. Heterozygous loss of DGCR8 recapitulated the excitability and calcium phenotypes and its overexpression rescued these defects. Moreover, the 22q11DS calcium abnormality could also be restored by application of antipsychotics. Taken together, our study illustrates how stem cell derived models can be used to uncover and rescue cellular phenotypes associated with genetic forms of neuropsychiatric disease.
Subject(s)
Calcium Signaling/genetics , Cerebral Cortex/ultrastructure , DiGeorge Syndrome/diagnosis , Neurons/ultrastructure , Adult , Cell Differentiation/genetics , Cerebral Cortex/pathology , DiGeorge Syndrome/pathology , Female , Humans , Induced Pluripotent Stem Cells/metabolism , Induced Pluripotent Stem Cells/ultrastructure , Male , Neurons/pathology , Organoids/pathology , Organoids/ultrastructure , Young AdultABSTRACT
Autism spectrum disorders (ASDs) are a group of developmental disorders characterized by social and emotional deficits, language impairments and stereotyped behaviors that manifest in early postnatal life. The molecular mechanisms that underlie ASDs are not known, but several recent developments suggest that some forms of autism are caused by failures in activity-dependent regulation of neural development. Mutations of several voltage-gated and ligand-gated ion channels that regulate neuronal excitability and Ca2+ signaling have been associated with ASDs. In addition, Ca2+-regulated signaling proteins involved in synapse formation and dendritic growth have been implicated in ASDs. These recent advances suggest a set of signaling pathways that might have a role in generating these increasingly prevalent disorders.
Subject(s)
Autistic Disorder/etiology , Autistic Disorder/physiopathology , Brain/growth & development , Brain/physiopathology , Calcium Signaling/genetics , Synaptic Transmission/genetics , Brain/metabolism , Cell Differentiation/genetics , Environment , Humans , Ion Channels/genetics , Ion Channels/metabolism , Receptors, Neurotransmitter/metabolism , Synapses/metabolismABSTRACT
The syndromic autism spectrum disorder (ASD) Timothy syndrome (TS) is caused by a point mutation in the alternatively spliced exon 8A of the calcium channel Cav1.2. Using mouse brain and human induced pluripotent stem cells (iPSCs), we provide evidence that the TS mutation prevents a normal developmental switch in Cav1.2 exon utilization, resulting in persistent expression of gain-of-function mutant channels during neuronal differentiation. In iPSC models, the TS mutation reduces the abundance of SATB2-expressing cortical projection neurons, leading to excess CTIP2+ neurons. We show that expression of TS-Cav1.2 channels in the embryonic mouse cortex recapitulates these differentiation defects in a calcium-dependent manner and that in utero Cav1.2 gain-and-loss of function reciprocally regulates the abundance of these neuronal populations. Our findings support the idea that disruption of developmentally regulated calcium channel splicing patterns instructively alters differentiation in the developing cortex, providing important in vivo insights into the pathophysiology of a syndromic ASD.
Subject(s)
Alternative Splicing/physiology , Autism Spectrum Disorder/metabolism , Calcium Channels/metabolism , Cell Differentiation/physiology , Animals , Autism Spectrum Disorder/genetics , Autism Spectrum Disorder/pathology , Autistic Disorder , Brain/embryology , Brain/growth & development , Calcium , Calcium Channels/genetics , Calcium Channels, L-Type/genetics , Calcium Channels, L-Type/metabolism , Cerebral Cortex/embryology , Cerebral Cortex/metabolism , Exons , Gene Expression Regulation, Developmental , Humans , Induced Pluripotent Stem Cells/metabolism , Long QT Syndrome , Matrix Attachment Region Binding Proteins/metabolism , Mice , Models, Animal , Mutation , Neurogenesis , Neurons/cytology , Neurons/metabolism , RNA Splicing , Repressor Proteins/genetics , Repressor Proteins/metabolism , Syndactyly , Transcription Factors/metabolism , Tumor Suppressor Proteins/genetics , Tumor Suppressor Proteins/metabolismABSTRACT
The transcription factor CREB mediates diverse responses in the nervous system. It is not known how CREB induces specific patterns of gene expression in response to different extracellular stimuli. We find that Ca(2+) influx into neurons induces CREB phosphorylation at Ser133 and two additional sites, Ser142 and Ser143. While CREB Ser133 phosphorylation is induced by many stimuli, phosphorylation at Ser142 and Ser143 is selectively activated by Ca(2+) influx. The triple phosphorylation of CREB is required for effective Ca(2+) stimulation of CREB-dependent transcription, but the phosphorylation of Ser142 and Ser143, in addition to Ser133, disrupts the interaction of CREB with its cofactor CBP. These results suggest that Ca(2+) influx triggers a specific program of gene expression in neurons by selectively regulating CREB phosphorylation.
Subject(s)
Calcium/metabolism , Cyclic AMP Response Element-Binding Protein/metabolism , Neurons/metabolism , Amino Acid Sequence/genetics , CREB-Binding Protein , Cells, Cultured , Cyclic AMP Response Element-Binding Protein/genetics , Electrophysiology , Immunohistochemistry , Neurons/physiology , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Nuclear Proteins/physiology , Phosphorylation , Trans-Activators/genetics , Trans-Activators/metabolism , Trans-Activators/physiology , Transcription, Genetic/physiologyABSTRACT
Dravet syndrome is an infantile epileptic encephalopathy primarily caused by loss-of-function variants of the gene SCN1A Standard treatment regimens have very limited efficacy to combat the life-threatening seizures in Dravet syndrome or the behavioral-cognitive comorbidities of the disease. Recently there has been encouraging progress in developing new treatments for this disorder. One of the clinical advances is cannabidiol (CBD), a compound naturally found in cannabis and shown to further reduce convulsive seizures in patients when used together with existing drug regimens. Like many other natural products, the exact therapeutic mechanism of CBD remains undefined. Previously we have established a human cellular model of Dravet syndrome by differentiating patient-derived induced pluripotent stem cells (iPSCs) into telencephalic inhibitory and excitatory neurons. Here we have applied this model to investigate the antiepileptic mechanism(s) of CBD at the cellular level. We first determined the effect of escalating the concentrations of CBD on neuronal excitability, using primary culture of rat cortical neurons. We found modulatory effects on excitability at submicromolar concentrations and toxic effects at high concentrations (15 µM). We then tested CBD at 50 nM, a concentration that corresponds to the estimated human clinical exposure, in telencephalic neurons derived from a patient iPSC line and control cell line H9. This 50 nM of CBD increased the excitability of inhibitory neurons but decreased the excitability of excitatory neurons, without changing the amplitude of sodium currents in either cell type. Our findings suggest a cell type-dependent mechanism for the therapeutic action of CBD in Dravet syndrome that is independent of sodium channel activity.
ABSTRACT
Compared with other medical fields, psychiatry is particularly challenging for rational drug discovery. The therapeutic endpoints are abstract measures of cognitive and behavioral performance, for which we have a very limited understanding of the underlying biological mechanisms. Existing preclinical disease models are also limited in their translational fidelity. Recently, there have been active discussions on the use of human induced pluripotent stem cells (iPSCs) as a catalyzing research tool in psychiatry, but very few review articles in the field have given specific considerations to their use at the interface between psychiatric research and drug discovery. Here, we discuss recent perspectives emerging from this interface. For physicians and researchers on the clinical side, we explain how iPSC-based experimental approaches are placed at the crossroads with psychiatric genetics and how representative studies in the field are addressing biological mechanisms underlying psychiatric disorders. For researchers who directly work with iPSCs and aspire to develop new research techniques, we direct their attention to the utility of this approach for unmet needs in drug discovery workflows.
Subject(s)
Drug Discovery , Induced Pluripotent Stem Cells , Mental Disorders/drug therapy , Psychiatry , Biomedical Research , Humans , Mental Disorders/genetics , Models, Biological , Molecular Targeted TherapyABSTRACT
SH3 and multiple ankyrin repeat domains 3 (SHANK3) haploinsufficiency is causative for the neurological features of Phelan-McDermid syndrome (PMDS), including a high risk of autism spectrum disorder (ASD). We used unbiased, quantitative proteomics to identify changes in the phosphoproteome of Shank3-deficient neurons. Down-regulation of protein kinase B (PKB/Akt)-mammalian target of rapamycin complex 1 (mTORC1) signaling resulted from enhanced phosphorylation and activation of serine/threonine protein phosphatase 2A (PP2A) regulatory subunit, B56ß, due to increased steady-state levels of its kinase, Cdc2-like kinase 2 (CLK2). Pharmacological and genetic activation of Akt or inhibition of CLK2 relieved synaptic deficits in Shank3-deficient and PMDS patient-derived neurons. CLK2 inhibition also restored normal sociability in a Shank3-deficient mouse model. Our study thereby provides a novel mechanistic and potentially therapeutic understanding of deregulated signaling downstream of Shank3 deficiency.
Subject(s)
Autism Spectrum Disorder/drug therapy , Nerve Tissue Proteins/genetics , Protein Serine-Threonine Kinases/antagonists & inhibitors , Protein-Tyrosine Kinases/antagonists & inhibitors , Amino Acid Sequence , Animals , Autism Spectrum Disorder/enzymology , Autism Spectrum Disorder/genetics , Chromosome Deletion , Chromosome Disorders/genetics , Chromosomes, Human, Pair 22/genetics , Disease Models, Animal , Down-Regulation , Gene Knockdown Techniques , Humans , Insulin-Like Growth Factor I/metabolism , Mechanistic Target of Rapamycin Complex 1 , Mice , Microfilament Proteins , Molecular Sequence Data , Multiprotein Complexes/metabolism , Neurons/enzymology , Phosphorylation , Protein Phosphatase 2/metabolism , Protein Serine-Threonine Kinases/metabolism , Protein-Tyrosine Kinases/metabolism , Proteomics , Proto-Oncogene Proteins c-akt/genetics , Proto-Oncogene Proteins c-akt/metabolism , Rats , Signal Transduction , TOR Serine-Threonine Kinases/metabolismABSTRACT
Dravet Syndrome is an intractable form of childhood epilepsy associated with deleterious mutations in SCN1A, the gene encoding neuronal sodium channel Nav1.1. Earlier studies using human induced pluripotent stem cells (iPSCs) have produced mixed results regarding the importance of Nav1.1 in human inhibitory versus excitatory neurons. We studied a Nav1.1 mutation (p.S1328P) identified in a pair of twins with Dravet Syndrome and generated iPSC-derived neurons from these patients. Characterization of the mutant channel revealed a decrease in current amplitude and hypersensitivity to steady-state inactivation. We then differentiated Dravet-Syndrome and control iPSCs into telencephalic excitatory neurons or medial ganglionic eminence (MGE)-like inhibitory neurons. Dravet inhibitory neurons showed deficits in sodium currents and action potential firing, which were rescued by a Nav1.1 transgene, whereas Dravet excitatory neurons were normal. Our study identifies biophysical impairments underlying a deleterious Nav1.1 mutation and supports the hypothesis that Dravet Syndrome arises from defective inhibitory neurons.