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SCN2A encodes NaV1.2, an excitatory neuron voltage-gated sodium channel and a major monogenic cause of neurodevelopmental disorders, including developmental and epileptic encephalopathies (DEE) and autism. Clinical presentation and pharmocosensitivity vary with the nature of SCN2A variant dysfunction and can be divided into gain-of-function (GoF) cases with pre- or peri-natal seizures and loss-of-function (LoF) patients typically having infantile spasms after 6 months of age. We established and assessed patient induced pluripotent stem cell (iPSC) - derived neuronal models for two recurrent SCN2A DEE variants with GoF R1882Q and LoF R853Q associated with early- and late-onset DEE, respectively. Two male patient-derived iPSC isogenic pairs were differentiated using Neurogenin-2 overexpression yielding populations of cortical-like glutamatergic neurons. Functional properties were assessed using patch clamp and multielectrode array recordings and transcriptomic profiles obtained with total mRNA sequencing after 2-4 weeks in culture. At 3 weeks of differentiation, increased neuronal activity at cellular and network levels was observed for R1882Q iPSC-derived neurons. In contrast, R853Q neurons showed only subtle changes in excitability after 4 weeks and an overall reduced network activity after 7 weeks in vitro. Consistent with the reported efficacy in some GoF SCN2A patients, phenytoin (sodium channel blocker) reduced the excitability of neurons to the control levels in R1882Q neuronal cultures. Transcriptomic alterations in neurons were detected for each variant and convergent pathways suggested potential shared mechanisms underlying SCN2A DEE. In summary, patient iPSC-derived neuronal models of SCN2A GoF and LoF pathogenic variants causing DEE show specific functional and transcriptomic in vitro phenotypes.
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Células-Tronco Pluripotentes Induzidas , Espasmos Infantis , Humanos , Masculino , Células-Tronco Pluripotentes Induzidas/metabolismo , Convulsões/genética , Espasmos Infantis/genética , Espasmos Infantis/metabolismo , Fenótipo , Neurônios/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.2/genéticaRESUMO
In vitro differentiation of stem cells into various cell lineages is valuable in developmental studies and an important source of cells for modelling physiology and pathology, particularly for complex tissues such as the brain. Conventional protocols for in vitro neuronal differentiation often suffer from complicated procedures, high variability and low reproducibility. Over the last decade, the identification of cell fate-determining transcription factors has provided new tools for cellular studies in neuroscience and enabled rapid differentiation driven by ectopic transcription factor expression. As a proneural transcription factor, Neurogenin 2 (Ngn2) expression alone is sufficient to trigger rapid and robust neurogenesis from pluripotent cells. Here, we established a stable cell line, by piggyBac (PB) transposition, that conditionally expresses Ngn2 for generation of excitatory neurons from mouse embryonic stem cells (ESCs) using an all-in-one PB construct. Our results indicate that Ngn2-induced excitatory neurons have mature and functional characteristics consistent with previous studies using conventional differentiation methods. This approach provides an all-in-one PB construct for rapid and high copy number gene delivery of dox-inducible transcription factors to induce differentiation. This approach is a valuable in vitro cell model for disease modeling, drug screening and cell therapy.
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Fatores de Transcrição Hélice-Alça-Hélice Básicos , Células-Tronco Embrionárias Murinas , Animais , Camundongos , Células-Tronco Embrionárias Murinas/metabolismo , Reprodutibilidade dos Testes , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Diferenciação Celular/genética , Neurônios/metabolismo , Linhagem Celular , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
CDKL5 Deficiency Disorder (CDD) is a debilitating epileptic encephalopathy disorder affecting young children with no effective treatments. CDD is caused by pathogenic variants in Cyclin-Dependent Kinase-Like 5 (CDKL5), a protein kinase that regulates key phosphorylation events in neurons. For therapeutic intervention, it is essential to understand molecular pathways and phosphorylation targets of CDKL5. Using an unbiased phosphoproteomic approach we identified novel targets of CDKL5, including GTF2I, PPP1R35, GATAD2A and ZNF219 in human iPSC-derived neuronal cells. The phosphoserine residue in the target proteins lies in the CDKL5 consensus motif. We validated direct phosphorylation of GTF2I and PPP1R35 by CDKL5 using complementary approaches. GTF2I controls axon guidance, cell cycle and neurodevelopment by regulating expression of neuronal genes. PPP1R35 is critical for centriole elongation and cilia morphology, processes that are impaired in CDD. PPP1R35 interacts with CEP131, a known CDKL5 phospho-target. GATAD2A and ZNF219 belong to the Nucleosome Remodelling Deacetylase (NuRD) complex, which regulates neuronal activity-dependent genes and synaptic connectivity. In-depth knowledge of molecular pathways regulated by CDKL5 will allow a better understanding of druggable disease pathways to fast-track therapeutic development.
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Síndromes Epilépticas , Células-Tronco Pluripotentes Induzidas , Neurônios , Proteínas Serina-Treonina Quinases , Espasmos Infantis , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/citologia , Neurônios/metabolismo , Neurônios/citologia , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Fosforilação , Síndromes Epilépticas/metabolismo , Síndromes Epilépticas/genética , Síndromes Epilépticas/patologia , Espasmos Infantis/metabolismo , Espasmos Infantis/genética , Espasmos Infantis/patologiaRESUMO
Brain pH is a critical factor for determining neuronal activity, with alkalosis increasing and acidosis reducing excitability. Acid shifts in brain pH through the breathing of carbogen (5% CO2/95% O2) reduces seizure susceptibility in animal models and patients. The molecular mechanisms underlying this seizure protection remain to be fully elucidated. Here, we demonstrate that male and female mice exposed to carbogen are fully protected from thermogenic-triggered seizures. Whole-cell patch-clamp recordings revealed that acid shifts in extracellular pH (pHo) significantly reduce action potential firing in CA1 pyramidal neurons but did not alter firing in hippocampal inhibitory interneurons. In real-time dynamic clamp experiments, acidification reduced simulated action potential firing generated in hybrid model neurons expressing the excitatory neuron predominant NaV1.2 channel. Conversely, acidification had no effect on action potential firing in hybrid model neurons expressing the interneuron predominant NaV1.1 channel. Furthermore, knockdown of Scn2a mRNA in vivo using antisense oligonucleotides reduced the protective effects of carbogen on seizure susceptibility. Both carbogen-mediated seizure protection and the reduction in CA1 pyramidal neuron action potential firing by low pHo were maintained in an Asic1a knock-out mouse ruling out this acid-sensing channel as the underlying molecular target. These data indicate that the acid-mediated reduction in excitatory neuron firing is mediated, at least in part, through the inhibition of NaV1.2 channels, whereas inhibitory neuron firing is unaffected. This reduction in pyramidal neuron excitability is the likely basis of seizure suppression caused by carbogen-mediated acidification.SIGNIFICANCE STATEMENT Brain pH has long been known to modulate neuronal excitability. Here, we confirm that brain acidification reduces seizure susceptibility in a mouse model of thermogenic seizures. Extracellular acidification reduced excitatory pyramidal neuron firing while having no effect on interneuron firing. Acidification also reduced dynamic clamp firing in cells expressing the NaV1.2 channel but not in cells expressing NaV1.1 channels. In vivo knockdown of Scn2a mRNA reduced seizure protection of acidification. In contrast, acid-mediated seizure protection was maintained in the Asic1a knock-out mouse. These data suggest NaV1.2 channel as an important target for acid-mediated seizure protection. Our results have implications on how natural variations in pH can modulate neuronal excitability and highlight potential antiseizure drug development strategies based on the NaV1.2 channel.
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Acidose Respiratória , Segmento Inicial do Axônio , Camundongos , Masculino , Animais , Feminino , Dióxido de Carbono , Convulsões/induzido quimicamente , Convulsões/genética , Células Piramidais , Potenciais de Ação , Camundongos Knockout , RNA MensageiroRESUMO
BACKGROUND: Ischemia-reperfusion injury (IRI) is one of the major risk factors implicated in morbidity and mortality associated with cardiovascular disease. During cardiac ischemia, the buildup of acidic metabolites results in decreased intracellular and extracellular pH, which can reach as low as 6.0 to 6.5. The resulting tissue acidosis exacerbates ischemic injury and significantly affects cardiac function. METHODS: We used genetic and pharmacologic methods to investigate the role of acid-sensing ion channel 1a (ASIC1a) in cardiac IRI at the cellular and whole-organ level. Human induced pluripotent stem cell-derived cardiomyocytes as well as ex vivo and in vivo models of IRI were used to test the efficacy of ASIC1a inhibitors as pre- and postconditioning therapeutic agents. RESULTS: Analysis of human complex trait genetics indicates that variants in the ASIC1 genetic locus are significantly associated with cardiac and cerebrovascular ischemic injuries. Using human induced pluripotent stem cell-derived cardiomyocytes in vitro and murine ex vivo heart models, we demonstrate that genetic ablation of ASIC1a improves cardiomyocyte viability after acute IRI. Therapeutic blockade of ASIC1a using specific and potent pharmacologic inhibitors recapitulates this cardioprotective effect. We used an in vivo model of myocardial infarction and 2 models of ex vivo donor heart procurement and storage as clinical models to show that ASIC1a inhibition improves post-IRI cardiac viability. Use of ASIC1a inhibitors as preconditioning or postconditioning agents provided equivalent cardioprotection to benchmark drugs, including the sodium-hydrogen exchange inhibitor zoniporide. At the cellular and whole organ level, we show that acute exposure to ASIC1a inhibitors has no effect on cardiac ion channels regulating baseline electromechanical coupling and physiologic performance. CONCLUSIONS: Our data provide compelling evidence for a novel pharmacologic strategy involving ASIC1a blockade as a cardioprotective therapy to improve the viability of hearts subjected to IRI.
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Canais Iônicos Sensíveis a Ácido/biossíntese , Canais Iônicos Sensíveis a Ácido/genética , Isquemia Miocárdica/genética , Isquemia Miocárdica/metabolismo , Traumatismo por Reperfusão Miocárdica/genética , Traumatismo por Reperfusão Miocárdica/metabolismo , Animais , Células Cultivadas , Feminino , Humanos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/metabolismo , Preparação de Coração Isolado/métodos , Masculino , Camundongos , Camundongos Knockout , Isquemia Miocárdica/terapia , Traumatismo por Reperfusão Miocárdica/terapia , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Polimorfismo de Nucleotídeo Único/fisiologia , Recuperação de Função Fisiológica/efeitos dos fármacos , Recuperação de Função Fisiológica/fisiologia , Venenos de Aranha/farmacologiaRESUMO
CDKL5 deficiency disorder (CDD) is an X-linked brain disorder of young children and is caused by pathogenic variants in the cyclin-dependent kinase-like 5 (CDKL5) gene. Individuals with CDD suffer infantile onset, drug-resistant seizures, severe neurodevelopmental impairment and profound lifelong disability. The CDKL5 protein is a kinase that regulates key phosphorylation events vital to the development of the complex neuronal network of the brain. Pathogenic variants identified in patients may either result in loss of CDKL5 catalytic activity or are hypomorphic leading to partial loss of function. Whilst the progressive nature of CDD provides an excellent opportunity for disease intervention, we cannot develop effective therapeutics without in-depth knowledge of CDKL5 function in human neurons. In this mini review, we summarize new findings on the function of CDKL5. These include CDKL5 phosphorylation targets and the consequence of disruptions on signaling pathways in the human brain. This new knowledge of CDKL5 biology may be leveraged to advance targeted drug discovery and rapid development of treatments for CDD. Continued development of effective humanized models will further propel our understanding of CDD biology and may permit the development and testing of therapies that will significantly alter CDD disease trajectory in young children.
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Síndromes Epilépticas , Espasmos Infantis , Criança , Pré-Escolar , Síndromes Epilépticas/tratamento farmacológico , Síndromes Epilépticas/terapia , Humanos , Neurônios/metabolismo , Proteínas Serina-Treonina Quinases/genética , Espasmos Infantis/tratamento farmacológico , Espasmos Infantis/genética , VirulênciaRESUMO
Epilepsy has a strong genetic component, with an ever-increasing number of disease-causing genes being discovered. Most epilepsy-causing mutations are germ line and thus present from conception. These mutations are therefore well positioned to have a deleterious impact during early development. Here we review studies that investigate the role of genetic lesions within the early developmental window, specifically focusing on genetic generalized epilepsy (GGE). Literature on the potential pathogenic role of sub-mesoscopic structural changes in GGE is also reviewed. Evidence from rodent models of genetic epilepsy support the idea that functional and structural changes can occur in early development, leading to altered seizure susceptibility into adulthood. Both animal and human studies suggest that sub-mesoscopic structural changes occur in GGE. The existence of sub-mesoscopic structural changes prior to seizure onset may act as biomarkers of excitability in genetic epilepsies. We also propose that presymptomatic treatment may be essential for limiting the long-term consequences of disease-causing mutations in genetic epilepsies.
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Desenvolvimento Embrionário/genética , Epilepsia Generalizada/genética , Animais , Encéfalo/embriologia , Humanos , MutaçãoRESUMO
The caudal neural plate is a distinct region of the embryo that gives rise to major progenitor lineages of the developing central and peripheral nervous system, including neural crest and floor plate cells. We show that dual inhibition of the glycogen synthase kinase 3ß and activin/nodal pathways by small molecules differentiate human pluripotent stem cells (hPSCs) directly into a preneuroepithelial progenitor population we named "caudal neural progenitors" (CNPs). CNPs coexpress caudal neural plate and mesoderm markers, and, share high similarities to embryonic caudal neural plate cells in their lineage differentiation potential. Exposure of CNPs to BMP2/4, sonic hedgehog, or FGF2 signaling efficiently directs their fate to neural crest/roof plate cells, floor plate cells, and caudally specified neuroepithelial cells, respectively. Neural crest derived from CNPs differentiated to neural crest derivatives and demonstrated extensive migratory properties in vivo. Importantly, we also determined the key extrinsic factors specifying CNPs from human embryonic stem cell include FGF8, canonical WNT, and IGF1. Our studies are the first to identify a multipotent neural progenitor derived from hPSCs, that is the precursor for major neural lineages of the embryonic caudal neural tube.
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Linhagem da Célula , Sistema Nervoso Central/citologia , Crista Neural/citologia , Células-Tronco Neurais/citologia , Tubo Neural/citologia , Sistema Nervoso Periférico/citologia , Células-Tronco Pluripotentes/citologia , Animais , Diferenciação Celular , Mesoderma/citologia , Camundongos Endogâmicos C57BL , Placa Neural/citologia , Células Neuroepiteliais/citologia , Ratos Sprague-DawleyRESUMO
Repressor element-1 silencing transcription factor (REST) is a transcriptional repressor involved in neurodevelopment and neuroprotection. REST forms a complex with the REST corepressors, CoREST1, CoREST2, or CoREST3 (encoded by RCOR1, RCOR2, and RCOR3, respectively). Emerging evidence suggests that the CoREST family can target unique genes independently of REST, in various neural and glial cell types during different developmental stages. However, there is limited knowledge regarding the expression and function of the CoREST family in human neurodevelopment. To address this gap, we employed 2D and 3D human pluripotent stem cell (hPSC) models to investigate REST and RCOR gene expression levels. Our study revealed a significant increase in RCOR3 expression in glutamatergic cortical and GABAergic ventral forebrain neurons, as well as mature functional NGN2-induced neurons. Additionally, a simplified astrocyte transdifferentiation protocol resulted in a significant decrease in RCOR2 expression following differentiation. REST expression was notably reduced in mature neurons and cerebral organoids. In summary, our findings provide the first insights into the cell-type-specific expression patterns of RCOR genes in human neuronal and glial differentiation. Specifically, RCOR3 expression increases in neurons, while RCOR2 levels decrease in astrocytes. The dynamic expression patterns of REST and RCOR genes during hPSC neuronal and glial differentiation underscore the potential distinct roles played by REST and CoREST proteins in regulating the development of these cell types in humans.
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INTRODUCTION: One-third of people with epilepsy continue to experience seizures despite treatment with existing anti-seizure medications (ASMs). The failure of modern ASMs to substantially improve epilepsy prognosis has been partly attributed to overreliance on acute rodent models in preclinical drug development as they do not adequately recapitulate the mechanisms of human epilepsy, are labor-intensive and unsuitable for high-throughput screening (HTS). There is an urgent need to find human-relevant HTS models in preclinical drug development to identify novel anti-seizure compounds. OBJECTIVES: This paper developed high-throughput preclinical screening models to identify new ASMs. METHODS: 14 natural compounds (α-asarone, curcumin, vinpocetine, magnolol, ligustrazine, osthole, tanshinone IIA, piperine, gastrodin, quercetin, berberine, chrysin, schizandrin A and resveratrol) were assessed for their ability to suppress epileptiform activity as measured by multi-electrode arrays (MEA) in neural cultures derived from human induced pluripotent stem cells (iPSCs). In parallel, they were tested for anti-seizure effects in zebrafish and mouse models, which have been widely used in development of modern ASMs. The effects of the compounds in these models were compared. Two approved ASMs were used as positive controls. RESULTS: Epileptiform activity could be induced in iPSCs-derived neurons following treatment with 4-aminopyridine (4-AP) and inhibited by standard ASMs, carbamazepine, and phenytoin. Eight of the 14 natural compounds significantly inhibited the epileptiform activity in iPSCs-derived neurons. Among them, piperine, magnolol, α-asarone, and osthole showed significant anti-seizure effects both in zebrafish and mice. Comparative analysis showed that compounds ineffective in the iPSCs-derived neural model also showed no anti-seizure effects in the zebrafish or mouse models. CONCLUSION: Our findings support the use of iPSCs-derived human neurons for first-line high-throughput screening to identify compounds with anti-seizure properties and exclude ineffective compounds. Effective compounds may then be selected for animal evaluation before clinical testing. This integrated approach may improve the efficiency of developing novel ASMs.
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Epilepsy is one of the most serious and common chronic neurological conditions, characterised by recurrent hypersynchronous electrical activity in the brain that lead to seizures. Despite over 50 million people being affected worldwide, only ~70% of people with epilepsy have their seizures successfully controlled with current pharmacotherapy, and many experience significant psychiatric and physical comorbidities. Adenosine, a ubiquitous purine metabolite, is a potent endogenous anti-epileptic substance that can abolish seizure activity via the adenosine A1 G protein-coupled receptor. Activation of A1 receptors decreases seizure activity in animal models, including models of drug-resistant epilepsy. Recent advances have increased our understanding of epilepsy comorbidities, highlighting the potential for adenosine receptors to modulate epilepsy-associated comorbidities, including cardiovascular dysfunction, sleep and cognition. This review provides an accessible resource of the current advances in understanding the adenosine system as a therapeutic target for epilepsy and epilepsy-associated comorbidities.
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Squamous cell carcinoma antigen recognized by T cells 3 (SART3) is an RNA-binding protein with numerous biological functions including recycling small nuclear RNAs to the spliceosome. Here, we identify recessive variants in SART3 in nine individuals presenting with intellectual disability, global developmental delay and a subset of brain anomalies, together with gonadal dysgenesis in 46,XY individuals. Knockdown of the Drosophila orthologue of SART3 reveals a conserved role in testicular and neuronal development. Human induced pluripotent stem cells carrying patient variants in SART3 show disruption to multiple signalling pathways, upregulation of spliceosome components and demonstrate aberrant gonadal and neuronal differentiation in vitro. Collectively, these findings suggest that bi-allelic SART3 variants underlie a spliceosomopathy which we tentatively propose be termed INDYGON syndrome (Intellectual disability, Neurodevelopmental defects and Developmental delay with 46,XY GONadal dysgenesis). Our findings will enable additional diagnoses and improved outcomes for individuals born with this condition.
Assuntos
Disgenesia Gonadal , Células-Tronco Pluripotentes Induzidas , Deficiência Intelectual , Masculino , Humanos , Testículo/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Antígenos de NeoplasiasRESUMO
CRISPR/Cas9 system is a powerful genome-editing technology for studying genetics and cell biology. Safe harbor sites are ideal genomic locations for transgene integration with minimal interference in cellular functions. Gene targeting of the AAVS1 locus enables stable transgene expression without phenotypic effects in host cells. Here, we describe the strategy for targeting the AAVS1 site with an inducible Neurogenin-2 (Ngn2) donor template by CRISPR/Cas9 in hiPSCs, which facilitates generation of an inducible cell line that can rapidly and homogenously differentiate into excitatory neurons.
Assuntos
Células-Tronco Pluripotentes Induzidas , Células-Tronco Pluripotentes , Sistemas CRISPR-Cas/genética , Edição de Genes , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , TransgenesRESUMO
Amyotrophic lateral sclerosis (ALS) is characterized by transactive response DNA-binding protein 43 (TDP-43) pathology, progressive loss of motor neurons and muscle dysfunction. Symptom onset can be insidious and diagnosis challenging. Conventional neuroimaging is used to exclude ALS mimics, however more advanced neuroimaging techniques may facilitate an earlier diagnosis. Here, we investigate the potential for neurite orientation dispersion and density imaging and diffusion tensor imaging (DTI) to detect microstructural changes in an experimental model of ALS with neuronal doxycycline (Dox)-suppressible overexpression of human TDP-43 (hTDP-43). In vivo diffusion-weighted imaging (DWI) was acquired 1- and 3- weeks following the initiation of hTDP-43 expression (post-Dox) to investigate whether neurite density imaging (NDI) and orientation dispersion imaging (ODI) are affected early in this preclinical model of ALS and if so, how these metrics compare to those derived from the diffusion tensor. Tract-based spatial statistics at 1-week post-Dox, i.e. very early in the disease stage, demonstrated increased NDI in TDP-43 mice but no change in ODI or DTI metrics. At 3-weeks post-Dox, a reduced pattern of increased NDI was observed along with widespread increases in ODI, and decreased fractional anisotropy (FA), apparent diffusion coefficient (ADC) and axial diffusivity (AD). A hypothesis driven analysis of the bilateral corticospinal tracts demonstrated that at 1-week post-Dox, ODI was significantly increased caudally but decreased in the motor cortex of TDP-43 mice. Decreased cortical ODI had normalized by 3-weeks post-Dox and only significant increases were observed. A similar, but inverse pattern in FA was also observed. Together, these results suggest a non-monotonic relationship between DWI metrics and pathophysiological progression with TDP-43 mice exhibiting significantly altered diffusion metrics consistent with early inflammation followed by progressive axonal degeneration. Importantly, significant group-wise changes were observed in the earliest stages of disease when subtle pathology may be more elusive to traditional structural imaging techniques.
Assuntos
Esclerose Lateral Amiotrófica , Imagem de Tensor de Difusão , Esclerose Lateral Amiotrófica/diagnóstico por imagem , Esclerose Lateral Amiotrófica/patologia , Animais , Proteínas de Ligação a DNA , Imagem de Difusão por Ressonância Magnética , Imagem de Tensor de Difusão/métodos , Humanos , Camundongos , Neuritos/patologiaRESUMO
BACKGROUND: Multiple lines of evidence suggest possible impairment of the glymphatic system in amyotrophic lateral sclerosis (ALS). To investigate this, we used in vivo magnetic resonance imaging (MRI) to assess glymphatic function early in the course of disease in a transgenic mouse with doxycycline (Dox)-controlled expression of cytoplasmic human TDP-43 (hTDP-43ΔNLS), mimicking the key pathology implicated in ALS. METHODS: Adult TDP-43 transgenic and littermate monogenic control mice underwent longitudinal multimodal MRI one and three weeks after the cessation of Dox feed, together with weekly rotarod assessments of motor performance. Glymphatic function was assessed using dynamic contrast-enhanced MRI to track the clearance of an MR contrast agent injected into the cisterna magna. RESULTS: Compared to their littermate controls, TDP-43 mice exhibited progressive neurodegeneration including that within the primary motor cortex, primary somatosensory cortex and corticospinal tract, significant weight loss including gastrocnemius atrophy, and shortened telomere length. Furthermore, in the presence of this ALS-like phenotype, these mice have significantly disrupted glymphatic function. CONCLUSIONS: Although the relationship between glymphatic clearance and ALS disease progression remains to be elucidated, these changes occurred very early in the disease course. This provides initial evidence to suggest that the glymphatic system might be a potential therapeutic target in the treatment of ALS.
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Esclerose Lateral Amiotrófica , Esclerose Lateral Amiotrófica/diagnóstico por imagem , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/metabolismo , Animais , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Modelos Animais de Doenças , Progressão da Doença , Camundongos , Camundongos Transgênicos , Telômero/metabolismo , Telômero/patologia , Encurtamento do TelômeroRESUMO
Integrating neurons into digital systems may enable performance infeasible with silicon alone. Here, we develop DishBrain, a system that harnesses the inherent adaptive computation of neurons in a structured environment. In vitro neural networks from human or rodent origins are integrated with in silico computing via a high-density multielectrode array. Through electrophysiological stimulation and recording, cultures are embedded in a simulated game-world, mimicking the arcade game "Pong." Applying implications from the theory of active inference via the free energy principle, we find apparent learning within five minutes of real-time gameplay not observed in control conditions. Further experiments demonstrate the importance of closed-loop structured feedback in eliciting learning over time. Cultures display the ability to self-organize activity in a goal-directed manner in response to sparse sensory information about the consequences of their actions, which we term synthetic biological intelligence. Future applications may provide further insights into the cellular correlates of intelligence.
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Developmental and epileptic encephalopathies (DEEs) are characterized by pharmaco-resistant seizures with concomitant intellectual disability. Epilepsy of infancy with migrating focal seizures (EIMFS) is one of the most severe of these syndromes. De novo variants in ion channels, including gain-of-function variants in KCNT1, which encodes for sodium activated potassium channel protein KNa1.1, have been found to play a major role in the etiology of EIMFS. Here, we test a potential precision therapeutic approach in KCNT1-associated DEE using a gene-silencing antisense oligonucleotide (ASO) approach. We generated a mouse model carrying the KCNT1 p.P924L pathogenic variant; only the homozygous animals presented with the frequent, debilitating seizures and developmental compromise that are seen in patients. After a single intracerebroventricular bolus injection of a Kcnt1 gapmer ASO in symptomatic mice at postnatal day 40, seizure frequency was significantly reduced, behavioral abnormalities improved, and overall survival was extended compared with mice treated with a control ASO (nonhybridizing sequence). ASO administration at neonatal age was also well tolerated and effective in controlling seizures and extending the life span of treated animals. The data presented here provide proof of concept for ASO-based gene silencing as a promising therapeutic approach in KCNT1-associated epilepsies.
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Encefalopatias , Camundongos , Animais , Convulsões/genética , Convulsões/terapiaRESUMO
De novo variation in SCN2A can give rise to severe childhood disorders. Biophysical gain of function in SCN2A is seen in some patients with early seizure onset developmental and epileptic encephalopathy (DEE). In these cases, targeted reduction in SCN2A expression could substantially improve clinical outcomes. We tested this theory by central administration of a gapmer antisense oligonucleotide (ASO) targeting Scn2a mRNA in a mouse model of Scn2a early seizure onset DEE (Q/+ mice). Untreated Q/+ mice presented with spontaneous seizures at P1 and did not survive beyond P30. Administration of the ASO to Q/+ mice reduced spontaneous seizures and significantly extended life span. Across a range of behavioral tests, Scn2a ASO-treated Q/+ mice were largely indistinguishable from WT mice, suggesting treatment is well tolerated. A human SCN2A gapmer ASO could likewise impact the lives of patients with SCN2A gain-of-function DEE.