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1.
Brain Commun ; 6(3): fcae202, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38911266

RESUMO

While voltage-gated potassium channels have critical roles in controlling neuronal excitability, they also have non-ion-conducting functions. Kv8.1, encoded by the KCNV1 gene, is a 'silent' ion channel subunit whose biological role is complex since Kv8.1 subunits do not form functional homotetramers but assemble with Kv2 to modify its ion channel properties. We profiled changes in ion channel expression in amyotrophic lateral sclerosis patient-derived motor neurons carrying a superoxide dismutase 1(A4V) mutation to identify what drives their hyperexcitability. A major change identified was a substantial reduction of KCNV1/Kv8.1 expression, which was also observed in patient-derived neurons with C9orf72 expansion. We then studied the effect of reducing KCNV1/Kv8.1 expression in healthy motor neurons and found it did not change neuronal firing but increased vulnerability to cell death. A transcriptomic analysis revealed dysregulated metabolism and lipid/protein transport pathways in KCNV1/Kv8.1-deficient motor neurons. The increased neuronal vulnerability produced by the loss of KCNV1/Kv8.1 was rescued by knocking down Kv2.2, suggesting a potential Kv2.2-dependent downstream mechanism in cell death. Our study reveals, therefore, unsuspected and distinct roles of Kv8.1 and Kv2.2 in amyotrophic lateral sclerosis-related neurodegeneration.

2.
iScience ; 26(5): 106701, 2023 May 19.
Artigo em Inglês | MEDLINE | ID: mdl-37207277

RESUMO

Genetics have nominated many schizophrenia risk genes and identified convergent signals between schizophrenia and neurodevelopmental disorders. However, functional interpretation of the nominated genes in the relevant brain cell types is often lacking. We executed interaction proteomics for six schizophrenia risk genes that have also been implicated in neurodevelopment in human induced cortical neurons. The resulting protein network is enriched for common variant risk of schizophrenia in Europeans and East Asians, is down-regulated in layer 5/6 cortical neurons of individuals affected by schizophrenia, and can complement fine-mapping and eQTL data to prioritize additional genes in GWAS loci. A sub-network centered on HCN1 is enriched for common variant risk and contains proteins (HCN4 and AKAP11) enriched for rare protein-truncating mutations in individuals with schizophrenia and bipolar disorder. Our findings showcase brain cell-type-specific interactomes as an organizing framework to facilitate interpretation of genetic and transcriptomic data in schizophrenia and its related disorders.

3.
Cell Genom ; 3(3): 100250, 2023 Mar 08.
Artigo em Inglês | MEDLINE | ID: mdl-36950384

RESUMO

Autism spectrum disorders (ASDs) have been linked to genes with enriched expression in the brain, but it is unclear how these genes converge into cell-type-specific networks. We built a protein-protein interaction network for 13 ASD-associated genes in human excitatory neurons derived from induced pluripotent stem cells (iPSCs). The network contains newly reported interactions and is enriched for genetic and transcriptional perturbations observed in individuals with ASDs. We leveraged the network data to show that the ASD-linked brain-specific isoform of ANK2 is important for its interactions with synaptic proteins and to characterize a PTEN-AKAP8L interaction that influences neuronal growth. The IGF2BP1-3 complex emerged as a convergent point in the network that may regulate a transcriptional circuit of ASD-associated genes. Our findings showcase cell-type-specific interactomes as a framework to complement genetic and transcriptomic data and illustrate how both individual and convergent interactions can lead to biological insights into ASDs.

4.
Mol Ther ; 30(8): 2646-2663, 2022 08 03.
Artigo em Inglês | MEDLINE | ID: mdl-35690906

RESUMO

On August 18, 2021, the American Society of Gene and Cell Therapy (ASGCT) hosted a virtual roundtable on adeno-associated virus (AAV) integration, featuring leading experts in preclinical and clinical AAV gene therapy, to further contextualize and understand this phenomenon. Recombinant AAV (rAAV) vectors are used to develop therapies for many conditions given their ability to transduce multiple cell types, resulting in long-term expression of transgenes. Although most rAAV DNA typically remains episomal, some rAAV DNA becomes integrated into genomic DNA at a low frequency, and rAAV insertional mutagenesis has been shown to lead to tumorigenesis in neonatal mice. Currently, the risk of rAAV-mediated oncogenesis in humans is theoretical because no confirmed genotoxic events have been reported to date. However, because insertional mutagenesis has been reported in a small number of murine studies, there is a need to characterize this genotoxicity to inform research, regulatory needs, and patient care. The purpose of this white paper is to review the evidence of rAAV-related host genome integration in animal models and possible risks of insertional mutagenesis in patients. In addition, technical considerations, regulatory guidance, and bioethics are discussed.


Assuntos
Dependovirus , Vetores Genéticos , Animais , Dependovirus/genética , Vetores Genéticos/genética , Humanos , Camundongos , Mutagênese Insercional , Plasmídeos , Transgenes , Integração Viral
5.
Nat Commun ; 12(1): 2580, 2021 05 10.
Artigo em Inglês | MEDLINE | ID: mdl-33972534

RESUMO

Combining genetic and cell-type-specific proteomic datasets can generate biological insights and therapeutic hypotheses, but a technical and statistical framework for such analyses is lacking. Here, we present an open-source computational tool called Genoppi (lagelab.org/genoppi) that enables robust, standardized, and intuitive integration of quantitative proteomic results with genetic data. We use Genoppi to analyze 16 cell-type-specific protein interaction datasets of four proteins (BCL2, TDP-43, MDM2, PTEN) involved in cancer and neurological disease. Through systematic quality control of the data and integration with published protein interactions, we show a general pattern of both cell-type-independent and cell-type-specific interactions across three cancer cell types and one human iPSC-derived neuronal cell type. Furthermore, through the integration of proteomic and genetic datasets in Genoppi, our results suggest that the neuron-specific interactions of these proteins are mediating their genetic involvement in neurodegenerative diseases. Importantly, our analyses suggest that human iPSC-derived neurons are a relevant model system for studying the involvement of BCL2 and TDP-43 in amyotrophic lateral sclerosis.


Assuntos
Biologia Computacional/métodos , Estudo de Associação Genômica Ampla/métodos , Células-Tronco Pluripotentes Induzidas/metabolismo , Neoplasias/genética , Neoplasias/metabolismo , Neurônios/metabolismo , Software , Linhagem Celular Tumoral , Células Cultivadas , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Genômica , Humanos , Mutação , Polimorfismo de Nucleotídeo Único , Ligação Proteica , Proteômica , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteínas Proto-Oncogênicas c-mdm2/genética , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Espectrometria de Massas em Tandem
6.
Mol Autism ; 12(1): 10, 2021 02 08.
Artigo em Inglês | MEDLINE | ID: mdl-33557935

RESUMO

Autism spectrum disorder (ASD) comprises a group of neurodevelopmental disorders characterized by impaired social interactions as well as the presentation of restrictive and repetitive behaviors. ASD is highly heritable but genetically heterogenous with both common and rare genetic variants collaborating to predispose individuals to the disorder. In this review, we synthesize recent efforts to develop human induced pluripotent stem cell (iPSC)-derived models of ASD-related phenotypes. We firstly address concerns regarding the relevance and validity of available neuronal iPSC-derived models. We then critically evaluate the robustness of various differentiation and cell culture protocols used for producing cell types of relevance to ASD. By exploring iPSC models of ASD reported thus far, we examine to what extent cellular and neuronal phenotypes with potential relevance to ASD can be linked to genetic variants found to underlie it. Lastly, we outline promising strategies by which iPSC technology can both enhance the power of genetic studies to identify ASD risk factors and nominate pathways that are disrupted across groups of ASD patients that might serve as common points for therapeutic intervention.


Assuntos
Estudos de Associação Genética , Predisposição Genética para Doença , Células-Tronco Pluripotentes Induzidas/metabolismo , Transtorno do Espectro Autista/diagnóstico , Transtorno do Espectro Autista/genética , Transtorno do Espectro Autista/terapia , Biomarcadores , Técnicas de Cultura de Células , Estudos de Associação Genética/métodos , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Terapia de Alvo Molecular , Herança Multifatorial , Fenótipo
7.
Bio Protoc ; 10(17): e3748, 2020 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-33659408

RESUMO

The efficiency of cleavage of individual CRISPR/Cas9-sgRNAs remains difficult to predict based on the CRISPR target sequence alone. Different intracellular environments (dependent on cell type or cell cycle state for example) may affect sgRNA efficiency by altering accessibility of genomic DNA through DNA modifications such as epigenetic marks and DNA-binding proteins (e.g., histones) as well as alteration of the chromatin state of genomic DNA within the nucleus. We recently reported a multi-step screening method for the identification of efficient sgRNAs targeting the Herpes simplex virus (HSV-1) genome and reported a differential mechanism for viral inhibition by CRISPR-Cas9 in the latent versus lytic phase. The screening platform detailed in this protocol allows step-by-step testing of the efficiency of cleavage in a cell-free system and in the context of viral target cells such as human foreskin fibroblasts followed by functional testing of the effects of CRISPR/sgRNA on viral protein expression, replication, and reactivation. This strategy could be readily applied to other target cells such as pluripotent stem cell-derived human sensory neurons or other human DNA viruses.

8.
Elife ; 82019 12 02.
Artigo em Inglês | MEDLINE | ID: mdl-31789594

RESUMO

Herpes simplex virus (HSV) establishes lifelong latent infection and can cause serious human disease, but current antiviral therapies target lytic but not latent infection. We screened for sgRNAs that cleave HSV-1 DNA sequences efficiently in vitro and used these sgRNAs to observe the first editing of quiescent HSV-1 DNA. The sgRNAs targeted lytic replicating viral DNA genomes more efficiently than quiescent genomes, consistent with the open structure of lytic chromatin. Editing of latent genomes caused short indels while editing of replicating genomes produced indels, linear molecules, and large genomic sequence loss around the gRNA target site. The HSV ICP0 protein and viral DNA replication increased the loss of DNA sequences around the gRNA target site. We conclude that HSV, by promoting open chromatin needed for viral gene expression and by inhibiting the DNA damage response, makes the genome vulnerable to a novel form of editing by CRISPR-Cas9 during lytic replication.


Assuntos
Proteína 9 Associada à CRISPR/metabolismo , Sistemas CRISPR-Cas/genética , Edição de Genes , Genes Virais , Herpesviridae/genética , Animais , Sequência de Bases , Linhagem Celular , Reparo do DNA/genética , DNA Viral/genética , Regulação Viral da Expressão Gênica , Herpesvirus Humano 1/genética , Humanos , Modelos Genéticos , Mutagênese/genética , RNA Guia de Cinetoplastídeos/genética , Replicação Viral/genética
9.
Nat Neurosci ; 22(2): 229-242, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30664768

RESUMO

We generated cortical interneurons (cINs) from induced pluripotent stem cells derived from 14 healthy controls and 14 subjects with schizophrenia. Both healthy control cINs and schizophrenia cINs were authentic, fired spontaneously, received functional excitatory inputs from host neurons, and induced GABA-mediated inhibition in host neurons in vivo. However, schizophrenia cINs had dysregulated expression of protocadherin genes, which lie within documented schizophrenia loci. Mice lacking protocadherin-α showed defective arborization and synaptic density of prefrontal cortex cINs and behavioral abnormalities. Schizophrenia cINs similarly showed defects in synaptic density and arborization that were reversed by inhibitors of protein kinase C, a downstream kinase in the protocadherin pathway. These findings reveal an intrinsic abnormality in schizophrenia cINs in the absence of any circuit-driven pathology. They also demonstrate the utility of homogenous and functional populations of a relevant neuronal subtype for probing pathogenesis mechanisms during development.


Assuntos
Caderinas/metabolismo , Interneurônios/metabolismo , Córtex Pré-Frontal/metabolismo , Esquizofrenia/metabolismo , Transdução de Sinais/fisiologia , Animais , Caderinas/genética , Feminino , Humanos , Células-Tronco Pluripotentes Induzidas , Interneurônios/patologia , Masculino , Camundongos , Camundongos Knockout , Córtex Pré-Frontal/patologia , Protocaderinas , Esquizofrenia/patologia , Sinapses/genética , Sinapses/metabolismo
10.
J Assist Reprod Genet ; 35(10): 1763-1771, 2018 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-30120633

RESUMO

PURPOSE: To provide a commentary on our understanding of the role that the Hippo signaling pathway may play in patients with polycystic ovarian syndrome (PCOS) and how this understanding may impact the diagnosis of PCOS. METHODS: We assessed publications discussing the role of the Hippo signaling pathway in the ovary. In particular, we discuss how Hippo signaling disruption after ovarian fragmentation, combined with treating ovarian fragments with phosphatase and tensin homolog (PTEN) inhibitors and phosphoinositide-3-kinase stimulators to augment AKT signaling, has been used in treatment of patients with primary ovarian insufficiency. Furthermore, we discuss our own data on variations in Hippo signaling pathway gene expression in cumulus cells isolated from women undergoing IVF with a previous diagnosis of PCOS. RESULTS AND CONCLUSIONS: Aberrant Hippo signaling in PCOS patients is likely a contributing mechanism to the multifactorial etiology of the disease. Given the challenge of discerning the underlying etiology of oligo-ovulation in some patients, especially those with normal body mass indices, and the need for customized stimulation protocols for PCOS patients who have an increased risk of over-response and higher percentage of immature oocyte yield, it is important to identify these patients prior to treatment. Hippo gene expression fingerprints could potentially be used to more accurately define patients with PCOS. Additionally, targeting this pathway with pharmacologic agents could lead to non-surgical therapeutic options for PCOS.


Assuntos
Fertilização in vitro , Ovário/metabolismo , Síndrome do Ovário Policístico/genética , Proteínas Serina-Treonina Quinases/genética , Feminino , Via de Sinalização Hippo , Humanos , Infertilidade Feminina/genética , Infertilidade Feminina/patologia , Oócitos/crescimento & desenvolvimento , Oócitos/metabolismo , Síndrome do Ovário Policístico/metabolismo , Síndrome do Ovário Policístico/patologia , Transdução de Sinais
11.
Mol Biol Cell ; 25(17): 2571-8, 2014 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-25009283

RESUMO

Mutations in the RNA-binding protein FUS have been shown to cause the neurodegenerative disease amyotrophic lateral sclerosis (ALS). We investigate whether mutant FUS protein in ALS patient-derived fibroblasts affects normal FUS functions in the nucleus. We investigated fibroblasts from two ALS patients possessing different FUS mutations and a normal control. Fibroblasts from these patients have their nuclear FUS protein trapped in SDS-resistant aggregates. Genome-wide analysis reveals an inappropriate accumulation of Ser-2 phosphorylation on RNA polymerase II (RNA Pol II) near the transcription start sites of 625 genes for ALS patient cells and after small interfering RNA (siRNA) knockdown of FUS in normal fibroblasts. Furthermore, both the presence of mutant FUS protein and siRNA knockdown of wild-type FUS correlate with altered distribution of RNA Pol II within fibroblast nuclei. A loss of FUS function in orchestrating Ser-2 phosphorylation of the CTD of RNA Pol II is detectable in ALS patient-derived fibroblasts expressing mutant FUS protein, even when the FUS protein remains largely nuclear. A likely explanation for this loss of function is the aggregation of FUS protein in nuclei. Thus our results suggest a specific mechanism by which mutant FUS can have biological consequences other than by the formation of cytoplasmic aggregates.


Assuntos
Esclerose Lateral Amiotrófica/metabolismo , Núcleo Celular/metabolismo , Proteína FUS de Ligação a RNA/metabolismo , Esclerose Lateral Amiotrófica/genética , Células Cultivadas , Fibroblastos/metabolismo , Humanos , Fosforilação , Agregados Proteicos , Interferência de RNA , RNA Polimerase II/metabolismo , Proteína FUS de Ligação a RNA/antagonistas & inibidores , Proteína FUS de Ligação a RNA/genética , Sítio de Iniciação de Transcrição
12.
Neuron ; 81(3): 536-543, 2014 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-24507191

RESUMO

The RNA-binding protein TDP-43 regulates RNA metabolism at multiple levels, including transcription, RNA splicing, and mRNA stability. TDP-43 is a major component of the cytoplasmic inclusions characteristic of amyotrophic lateral sclerosis and some types of frontotemporal lobar degeneration. The importance of TDP-43 in disease is underscored by the fact that dominant missense mutations are sufficient to cause disease, although the role of TDP-43 in pathogenesis is unknown. Here we show that TDP-43 forms cytoplasmic mRNP granules that undergo bidirectional, microtubule-dependent transport in neurons in vitro and in vivo and facilitate delivery of target mRNA to distal neuronal compartments. TDP-43 mutations impair this mRNA transport function in vivo and in vitro, including in stem cell-derived motor neurons from ALS patients bearing any one of three different TDP-43 ALS-causing mutations. Thus, TDP-43 mutations that cause ALS lead to partial loss of a novel cytoplasmic function of TDP-43.


Assuntos
Esclerose Lateral Amiotrófica/patologia , Transporte Axonal/genética , Proteínas de Ligação a DNA/genética , Neurônios Motores/metabolismo , Mutação/genética , RNA Mensageiro/metabolismo , Esclerose Lateral Amiotrófica/genética , Animais , Animais Geneticamente Modificados , Células Cultivadas , Córtex Cerebral/citologia , Drosophila , Proteínas de Drosophila/genética , Humanos , Fator 4 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Proteínas Luminescentes/genética , Camundongos , Mitocôndrias/metabolismo , Neurônios Motores/ultraestrutura , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Proteínas de Ligação a RNA/metabolismo , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo
13.
Nature ; 495(7442): 474-80, 2013 Mar 28.
Artigo em Inglês | MEDLINE | ID: mdl-23474986

RESUMO

CLP1 was the first mammalian RNA kinase to be identified. However, determining its in vivo function has been elusive. Here we generated kinase-dead Clp1 (Clp1(K/K)) mice that show a progressive loss of spinal motor neurons associated with axonal degeneration in the peripheral nerves and denervation of neuromuscular junctions, resulting in impaired motor function, muscle weakness, paralysis and fatal respiratory failure. Transgenic rescue experiments show that CLP1 functions in motor neurons. Mechanistically, loss of CLP1 activity results in accumulation of a novel set of small RNA fragments, derived from aberrant processing of tyrosine pre-transfer RNA. These tRNA fragments sensitize cells to oxidative-stress-induced p53 (also known as TRP53) activation and p53-dependent cell death. Genetic inactivation of p53 rescues Clp1(K/K) mice from the motor neuron loss, muscle denervation and respiratory failure. Our experiments uncover a mechanistic link between tRNA processing, formation of a new RNA species and progressive loss of lower motor neurons regulated by p53.


Assuntos
Neurônios Motores/metabolismo , Neurônios Motores/patologia , RNA de Transferência de Tirosina/metabolismo , Fatores de Transcrição/metabolismo , Esclerose Lateral Amiotrófica , Animais , Animais Recém-Nascidos , Axônios/metabolismo , Axônios/patologia , Morte Celular , Diafragma/inervação , Perda do Embrião , Embrião de Mamíferos/metabolismo , Embrião de Mamíferos/patologia , Éxons/genética , Feminino , Fibroblastos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Atrofia Muscular Espinal , Doenças Neuromusculares/metabolismo , Doenças Neuromusculares/patologia , Estresse Oxidativo , Processamento Pós-Transcricional do RNA , RNA de Transferência de Tirosina/genética , Proteínas de Ligação a RNA , Respiração , Nervos Espinhais/citologia , Fatores de Transcrição/deficiência , Proteína Supressora de Tumor p53/metabolismo , Tirosina/genética , Tirosina/metabolismo
15.
Neuron ; 70(4): 626-44, 2011 May 26.
Artigo em Inglês | MEDLINE | ID: mdl-21609821

RESUMO

Among the disciplines of medicine, the study of neurological disorders is particularly challenging. The fundamental inaccessibility of the human neural types affected by disease prevents their isolation for in vitro studies of degenerative mechanisms or for drug screening efforts. However, the ability to reprogram readily accessible tissue from patients into pluripotent stem (iPS) cells may now provide a general solution to this shortage of human neurons. Gradually improving methods for directing the differentiation of patient-specific stem cells has enabled the production of several neural cell types affected by disease. Furthermore, initial studies with stem cell lines derived from individuals with pediatric, monogenic disorders have validated the stem cell approach to disease modeling, allowing relevant neural phenotypes to be observed and studied. Whether iPS cell-derived neurons will always faithfully recapitulate the same degenerative processes observed in patients and serve as platforms for drug discovery relevant to common late-onset diseases remains to be determined.


Assuntos
Modelos Teóricos , Doenças do Sistema Nervoso/cirurgia , Transplante de Células-Tronco/métodos , Células-Tronco , Animais , Diferenciação Celular/fisiologia , Humanos , Doenças do Sistema Nervoso/genética , Doenças do Sistema Nervoso/patologia , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/transplante , Células-Tronco/citologia , Células-Tronco/fisiologia
16.
Cell Stem Cell ; 3(6): 637-48, 2008 Dec 04.
Artigo em Inglês | MEDLINE | ID: mdl-19041780

RESUMO

It has been proposed that human embryonic stem cells could be used to provide an inexhaustible supply of differentiated cell types for the study of disease processes. Although methods for differentiating embryonic stem cells into specific cell types have become increasingly sophisticated, the utility of the resulting cells for modeling disease has not been determined. We have asked whether specific neuronal subtypes produced from human embryonic stem cells can be used to investigate the mechanisms leading to neural degeneration in amyotrophic lateral sclerosis (ALS). We show that human spinal motor neurons, but not interneurons, are selectively sensitive to the toxic effect of glial cells carrying an ALS-causing mutation in the SOD1 gene. Our findings demonstrate the relevance of these non-cell-autonomous effects to human motor neurons and more broadly demonstrate the utility of human embryonic stem cells for studying disease and identifying potential therapeutics.


Assuntos
Esclerose Lateral Amiotrófica/metabolismo , Células-Tronco Embrionárias/metabolismo , Biologia Molecular/métodos , Neurônios Motores/metabolismo , Degeneração Neural/metabolismo , Neuroglia/metabolismo , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/fisiopatologia , Animais , Animais Recém-Nascidos , Comunicação Celular/genética , Técnicas de Cultura de Células/métodos , Diferenciação Celular/genética , Linhagem Celular , Sobrevivência Celular/genética , Células Cultivadas , Técnicas de Cocultura , Células-Tronco Embrionárias/citologia , Humanos , Camundongos , Camundongos Transgênicos , Modelos Biológicos , Neurônios Motores/citologia , Mutação/genética , Degeneração Neural/genética , Degeneração Neural/fisiopatologia , Neurotoxinas/metabolismo , Receptores Imunológicos/genética , Receptores de Prostaglandina/genética , Superóxido Dismutase/genética , Superóxido Dismutase-1
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