RESUMO
Isogenic pluripotent stem cells are critical tools for studying human neurological diseases by allowing one to study the effects of a mutation in a fixed genetic background. Of particular interest are the spectrum of autism disorders, some of which are monogenic such as Timothy syndrome (TS); others are multigenic such as the microdeletion and microduplication syndromes of the 16p11.2 chromosomal locus. Here, we report engineered human embryonic stem cell (hESC) lines for modeling these two disorders using locus-specific endonucleases to increase the efficiency of homology-directed repair (HDR). We developed a system to: (1) computationally identify unique transcription activator-like effector nuclease (TALEN) binding sites in the genome using a new software program, TALENSeek, (2) assemble the TALEN genes by combining golden gate cloning with modified constructs from the FLASH protocol, and (3) test the TALEN pairs in an amplification-based HDR assay that is more sensitive than the typical non-homologous end joining assay. We applied these methods to identify, construct, and test TALENs that were used with HDR donors in hESCs to generate an isogenic TS cell line in a scarless manner and to model the 16p11.2 copy number disorder without modifying genomic loci with high sequence similarity.
Assuntos
Engenharia Celular , Transtornos Globais do Desenvolvimento Infantil/genética , Células-Tronco Embrionárias , Modelos Genéticos , Transtorno Autístico , Sítios de Ligação , Linhagem Celular , Desoxirribonucleases/metabolismo , Marcação de Genes , Genoma Humano , Humanos , Síndrome do QT Longo/genética , Reparo de DNA por Recombinação , Software , Sindactilia/genéticaRESUMO
The mammalian cortex is comprised of cells with different morphological, physiological, and molecular properties that can be classified according to shared properties into cell types. Defining the contribution of each cell type to the computational and cognitive processes that are guided by the cortex is essential for understanding its function in health and disease. We use transcriptomic and epigenomic cortical cell type taxonomies from mice and humans to define marker genes and enhancers, and to build genetic tools for cortical cell types. Here, we present a large toolkit for selective targeting of cortical populations, including mouse transgenic lines and recombinant adeno-associated virus (AAV) vectors containing genomic enhancers. We report evaluation of fifteen new transgenic driver lines and over 680 different enhancer AAVs covering all major subclasses of cortical cells, with many achieving a high degree of specificity, comparable with existing transgenic lines. We find that the transgenic lines based on marker genes can provide exceptional specificity and completeness of cell type labeling, but frequently require generation of a triple-transgenic cross for best usability/specificity. On the other hand, enhancer AAVs are easy to screen and use, and can be easily modified to express diverse cargo, such as recombinases. However, their use depends on many factors, such as viral titer and route of administration. The tools reported here as well as the scaled process of tool creation provide an unprecedented resource that should enable diverse experimental strategies towards understanding mammalian cortex and brain function.
RESUMO
Experimental access to cell types within the mammalian spinal cord is severely limited by the availability of genetic tools. To enable access to lower motor neurons (LMNs) and LMN subtypes, which function to integrate information from the brain and control movement through direct innervation of effector muscles, we generated single cell multiome datasets from mouse and macaque spinal cords and discovered putative enhancers for each neuronal population. We cloned these enhancers into adeno-associated viral vectors (AAVs) driving a reporter fluorophore and functionally screened them in mouse. The most promising candidate enhancers were then extensively characterized using imaging and molecular techniques and further tested in rat and macaque to show conservation of LMN labeling. Additionally, we combined enhancer elements into a single vector to achieve simultaneous labeling of upper motor neurons (UMNs) and LMNs. This unprecedented LMN toolkit will enable future investigations of cell type function across species and potential therapeutic interventions for human neurodegenerative diseases.
RESUMO
The SORL1 gene (SORLA) is strongly associated with risk of developing Alzheimer's disease (AD). SORLA is a regulator of endosomal trafficking in neurons and interacts with retromer, a complex that is a "master conductor" of endosomal trafficking. Small molecules can increase retromer expression in vitro, enhancing its function. We treated hiPSC-derived cortical neurons that are either fully deficient, haploinsufficient, or that harbor one copy of SORL1 variants linked to AD with TPT-260, a retromer-enhancing molecule. We show significant increases in retromer subunit VPS26B expression. We tested whether endosomal, amyloid, and TAU pathologies were corrected. We observed that the degree of rescue by TPT-260 treatment depended on the number of copies of functional SORL1 and which SORL1 variant was expressed. Using a disease-relevant preclinical model, our work illuminates how the SORL1-retromer pathway can be therapeutically harnessed.
Assuntos
Doença de Alzheimer , Proteínas Relacionadas a Receptor de LDL , Proteínas de Membrana Transportadoras , Humanos , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Endossomos/metabolismo , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/metabolismo , Proteínas Relacionadas a Receptor de LDL/genética , Proteínas Relacionadas a Receptor de LDL/metabolismo , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Neurônios/metabolismoRESUMO
Dravet syndrome (DS) is a devastating developmental epileptic encephalopathy marked by treatment-resistant seizures, developmental delay, intellectual disability, motor deficits, and a 10-20% rate of premature death. Most DS patients harbor loss-of-function mutations in one copy of SCN1A , which has been associated with inhibitory neuron dysfunction. Here we developed an interneuron-targeting AAV human SCN1A gene replacement therapy using cell class-specific enhancers. We generated a split-intein fusion form of SCN1A to circumvent AAV packaging limitations and deliver SCN1A via a dual vector approach using cell class-specific enhancers. These constructs produced full-length Na V 1.1 protein and functional sodium channels in HEK293 cells and in brain cells in vivo . After packaging these vectors into enhancer-AAVs and administering to mice, immunohistochemical analyses showed telencephalic GABAergic interneuron-specific and dose-dependent transgene biodistribution. These vectors conferred strong dose-dependent protection against postnatal mortality and seizures in two DS mouse models carrying independent loss-of-function alleles of Scn1a, at two independent research sites, supporting the robustness of this approach. No mortality or toxicity was observed in wild-type mice injected with single vectors expressing either the N-terminal or C-terminal halves of SCN1A , or the dual vector system targeting interneurons. In contrast, nonselective neuronal targeting of SCN1A conferred less rescue against mortality and presented substantial preweaning lethality. These findings demonstrate proof-of-concept that interneuron-specific AAV-mediated SCN1A gene replacement is sufficient for significant rescue in DS mouse models and suggest it could be an effective therapeutic approach for patients with DS.
RESUMO
Proper brain function requires the assembly and function of diverse populations of neurons and glia. Single cell gene expression studies have mostly focused on characterization of neuronal cell diversity; however, recent studies have revealed substantial diversity of glial cells, particularly astrocytes. To better understand glial cell types and their roles in neurobiology, we built a new suite of adeno-associated viral (AAV)-based genetic tools to enable genetic access to astrocytes and oligodendrocytes. These oligodendrocyte and astrocyte enhancer-AAVs are highly specific (usually > 95% cell type specificity) with variable expression levels, and our astrocyte enhancer-AAVs show multiple distinct expression patterns reflecting the spatial distribution of astrocyte cell types. To provide the best glial-specific functional tools, several enhancer-AAVs were: optimized for higher expression levels, shown to be functional and specific in rat and macaque, shown to maintain specific activity in epilepsy where traditional promoters changed activity, and used to drive functional transgenes in astrocytes including Cre recombinase and acetylcholine-responsive sensor iAChSnFR. The astrocyte-specific iAChSnFR revealed a clear reward-dependent acetylcholine response in astrocytes of the nucleus accumbens during reinforcement learning. Together, this collection of glial enhancer-AAVs will enable characterization of astrocyte and oligodendrocyte populations and their roles across species, disease states, and behavioral epochs.
RESUMO
BACKGROUND: Mutations that alter splicing of X-linked ATP6AP2 cause a spectrum of neurodevelopmental and neurodegenerative pathologies including parkinsonism in affected males. All previously reported splicing mutations increase the level of a minor isoform with skipped exon 4 (Δe4) that encodes a functionally deficient protein. OBJECTIVES: We investigated the pathogenic mechanism of a novel c.168+6T>A variant reported in a family with X-linked intellectual disability, epilepsy, and parkinsonism. We also analyzed ATP6AP2 splicing defects in brains of carriers of a c.345C>T variant associated with X-linked spasticity and parkinsonism. METHODS: We generated induced pluripotent stem cells from patients with c.168+6T>A, reprogrammed them to neural progenitor cells and analyzed them by RNA-Seq and qRT-PCR. We also quantified ATP6AP2 isoforms in the brains of c.345C>T carriers by Nanostring nCounter. RESULTS: The c.168+6T>A increased skipping of ATP6AP2 exon 2 and usage of cryptic intronic donor splice sites. This results in out-of-frame splicing products and a reciprocal 50% reduction in functional full-length ATP6AP2 transcripts. Neural progenitors of patients with c.168+6T>A exhibited downregulated neural development gene networks. Analysis of blood transcriptomes of c.168+6T>A carriers identified potential biomarkers of ATP6AP2 deficiency in non-neural tissues. The c.345C>T variant increased exon 4 skipping with concomitant decrease of full length ATP6AP2 in brains of carriers. CONCLUSION: A common pathogenic consequence of splicing mutations affecting inclusion of different ATP6AP2 exons is reduction of the functional full-length transcript. The exacerbated ATP6AP2 splicing defect in brains of c.345C>T carriers is consistent with their CNS-restricted clinical presentations.
Assuntos
Transtornos Parkinsonianos , Receptores de Superfície Celular , ATPases Vacuolares Próton-Translocadoras , Éxons , Dosagem de Genes , Humanos , Masculino , Mutação , Transtornos Parkinsonianos/genética , Isoformas de Proteínas/genética , Sítios de Splice de RNA , Receptores de Superfície Celular/genética , ATPases Vacuolares Próton-Translocadoras/genéticaRESUMO
Viral genetic tools that target specific brain cell types could transform basic neuroscience and targeted gene therapy. Here, we use comparative open chromatin analysis to identify thousands of human-neocortical-subclass-specific putative enhancers from across the genome to control gene expression in adeno-associated virus (AAV) vectors. The cellular specificity of reporter expression from enhancer-AAVs is established by molecular profiling after systemic AAV delivery in mouse. Over 30% of enhancer-AAVs produce specific expression in the targeted subclass, including both excitatory and inhibitory subclasses. We present a collection of Parvalbumin (PVALB) enhancer-AAVs that show highly enriched expression not only in cortical PVALB cells but also in some subcortical PVALB populations. Five vectors maintain PVALB-enriched expression in primate neocortex. These results demonstrate how genome-wide open chromatin data mining and cross-species AAV validation can be used to create the next generation of non-species-restricted viral genetic tools.
Assuntos
Elementos Facilitadores Genéticos , Regulação da Expressão Gênica , Neocórtex/metabolismo , Animais , Cromatina/genética , Cromatina/metabolismo , Bases de Dados Genéticas , Dependovirus/genética , Doença/genética , Epigênese Genética , Vetores Genéticos/metabolismo , Genoma , Humanos , Camundongos , Neurônios/metabolismo , Parvalbuminas/metabolismo , Primatas , Especificidade da EspécieRESUMO
Spinal and bulbar muscular atrophy (SBMA) is an inherited neuromuscular disorder caused by a polyglutamine (polyQ) repeat expansion in the androgen receptor (AR). PolyQ-AR neurotoxicity may involve generation of an N-terminal truncation fragment, as such peptides occur in SBMA patients and mouse models. To elucidate the basis of SBMA, we expressed N-terminal truncated AR in motor neuron-derived cells and primary cortical neurons. Accumulation of polyQ-AR truncation fragments in the cytosol resulted in neurodegeneration and apoptotic, caspase-dependent cell death. Using primary neurons from mice transgenic or deficient for apoptosis-related genes, we determined that polyQ-AR apoptotic activation is fully dependent on Bax. Jun N-terminal kinase (JNK) was required for apoptotic pathway activation through phosphorylation of c-Jun. Expression of polyQ-AR in DP5/Hrk null neurons yielded significant protection against apoptotic activation, but absence of Bim did not provide protection, apparently due to compensatory upregulation of DP5/Hrk or other BH3-only proteins. Misfolded AR protein in the cytosol thus initiates a cascade of events beginning with JNK and culminating in Bax-dependent, intrinsic pathway activation, mediated in part by DP5/Hrk. As apoptotic mediators are candidates for toxic fragment generation and other cellular processes linked to neuron dysfunction, delineation of the apoptotic activation pathway induced by polyQ-expanded AR may shed light on the pathogenic cascade in SBMA and other motor neuron diseases.
Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Apoptose/genética , Neuropeptídeos/metabolismo , Peptídeos/metabolismo , Receptores Androgênicos/metabolismo , Expansão das Repetições de Trinucleotídeos/genética , Proteína X Associada a bcl-2/metabolismo , Animais , Proteínas Reguladoras de Apoptose/genética , Linhagem Celular , Células Cultivadas , Sistema Nervoso Central/metabolismo , Sistema Nervoso Central/patologia , Sistema Nervoso Central/fisiopatologia , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Camundongos , Camundongos Transgênicos , Atrofia Muscular Espinal/genética , Atrofia Muscular Espinal/metabolismo , Atrofia Muscular Espinal/fisiopatologia , Neurônios/metabolismo , Neurônios/patologia , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Dobramento de Proteína , Receptores Androgênicos/química , Receptores Androgênicos/genética , Transdução de Sinais/genéticaRESUMO
Single-cell transcriptomic approaches have found molecular heterogeneities within populations of pluripotent stem cells (PSCs). A tool that tracks single-cell lineages and their phenotypes longitudinally would reveal whether heterogeneity extends beyond molecular identity. Hence, we generated a stable Cre-inducible rainbow reporter human PSC line that provides up to 18 unique membrane-targeted fluorescent barcodes. These barcodes enable repeated assessments of single cells as they clonally expand, change morphology, and migrate. Owing to the cellular resolution of this reporter, we identified subsets of PSCs with enhanced clonal expansion, synchronized cell divisions, and persistent localization to colony edges. Reporter expression was stably maintained throughout directed differentiation into cardiac myocytes, cortical neurons, and hepatoblasts. Repeated examination of neural differentiation revealed self-assembled cortical tissues derive from clonally dominant progenitors. Collectively, these findings demonstrate the broad utility and easy implementation of this reporter line for tracking single-cell behavior.
Assuntos
Diferenciação Celular , Rastreamento de Células , Genes Reporter , Células-Tronco Pluripotentes/citologia , Engenharia Celular , Linhagem Celular , Proliferação de Células , Células Clonais , Camadas Germinativas/citologia , Humanos , Cinética , Modelos Biológicos , MorfogêneseRESUMO
SORL1/SORLA is a sorting receptor involved in retromer-related endosomal traffic and an Alzheimer's disease (AD) risk gene. Using CRISPR-Cas9, we deplete SORL1 in hiPSCs to ask if loss of SORL1 contributes to AD pathogenesis by endosome dysfunction. SORL1-deficient hiPSC neurons show early endosome enlargement, a hallmark cytopathology of AD. There is no effect of SORL1 depletion on endosome size in hiPSC microglia, suggesting a selective effect on neuronal endosomal trafficking. We validate defects in neuronal endosomal traffic by showing altered localization of amyloid precursor protein (APP) in early endosomes, a site of APP cleavage by the ß-secretase (BACE). Inhibition of BACE does not rescue endosome enlargement in SORL1-deficient neurons, suggesting that this phenotype is independent of amyloidogenic APP processing. Our data, together with recent findings, underscore how sporadic AD pathways regulating endosomal trafficking and autosomal-dominant AD pathways regulating APP cleavage independently converge on the defining cytopathology of AD.
Assuntos
Precursor de Proteína beta-Amiloide/metabolismo , Endossomos/metabolismo , Proteínas Relacionadas a Receptor de LDL/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Secretases da Proteína Precursora do Amiloide/metabolismo , Peptídeos beta-Amiloides/metabolismo , Sistemas CRISPR-Cas/genética , Diferenciação Celular , Linhagem Celular , Edição de Genes , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Proteínas Relacionadas a Receptor de LDL/antagonistas & inibidores , Proteínas Relacionadas a Receptor de LDL/genética , Proteínas de Membrana Transportadoras/genética , Neurônios/citologia , Neurônios/metabolismo , Transporte Proteico , Interferência de RNA , RNA Guia de Cinetoplastídeos/metabolismo , RNA Interferente Pequeno/metabolismoRESUMO
The Purkinje cell degeneration (pcd) mouse undergoes retinal photoreceptor degeneration and Purkinje cell loss. Nna1 is postulated to be the causal gene for pcd. We show that a BAC containing the Nna1 gene rescues retinal photoreceptor loss and Purkinje cell degeneration, confirming that Nna1 loss-of-function is responsible for these phenotypes. Mutation of the zinc-binding domain within the transgene destroyed its ability to rescue neuronal loss in pcd(5J) homozygous mice. In conclusion, Nna1 is required for survival of retinal photoreceptors and other neuron populations that degenerate in pcd mice. A functional zinc-binding domain is crucial for Nna1 to support neuron survival.
Assuntos
Ataxia Cerebelar/fisiopatologia , Proteínas de Ligação ao GTP/fisiologia , Células de Purkinje/patologia , Degeneração Retiniana/fisiopatologia , D-Ala-D-Ala Carboxipeptidase Tipo Serina/fisiologia , Zinco/metabolismo , Sequência de Aminoácidos , Animais , Ataxia Cerebelar/genética , Ataxia Cerebelar/patologia , Proteínas de Ligação ao GTP/genética , Proteínas de Ligação ao GTP/metabolismo , Genótipo , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase/métodos , Degeneração Retiniana/genética , Degeneração Retiniana/metabolismo , Degeneração Retiniana/patologia , D-Ala-D-Ala Carboxipeptidase Tipo Serina/genética , D-Ala-D-Ala Carboxipeptidase Tipo Serina/metabolismoRESUMO
Patient-specific stem cell technology from skin and other biopsy sources has transformed in vitro models of neurodegenerative disease, permitting interrogation of the effects of complex human genetics on neurotoxicity. However, the neuropathologic changes that underlie cognitive and behavioral phenotypes can only be determined at autopsy. To better correlate the biology of derived neurons with age-related and neurodegenerative changes, we generated leptomeningeal cell lines from well-characterized research subjects that have undergone comprehensive postmortem neuropathologic examinations. In a series of proof of principle experiments, we reprogrammed autopsy leptomeningeal cell lines to human-induced pluripotent stem cells (hiPSCs) and differentiated these into neurons. We show that leptomeningeal-derived hiPSC lines can be generated from fresh and frozen leptomeninges, are pluripotent, and retain the karyotype of the starting cell population. Additionally, neurons differentiated from these hiPSCs are functional and produce measurable Alzheimer disease-relevant analytes (Aß and Tau). Finally, we used direct conversion protocols to transdifferentiate leptomeningeal cells to neurons. These resources allow the generation of in vitro models to test mechanistic hypotheses as well as diagnostic and therapeutic strategies in association with neuropathology, clinical and cognitive data, and biomarker studies, aiding in the study of late-onset Alzheimer disease and other age-related neurodegenerative diseases.
Assuntos
Autopsia , Células-Tronco Pluripotentes Induzidas/fisiologia , Meninges/citologia , Doenças do Sistema Nervoso/patologia , Neurônios/fisiologia , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Diferenciação Celular , Linhagem Celular , Corpos Embrioides , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Cariótipo , Neurônios/metabolismo , Reação em Cadeia da Polimerase , Proteínas tau/metabolismoRESUMO
The complexity of gene regulatory networks that lead multipotent cells to acquire different cell fates makes a quantitative understanding of differentiation challenging. Using a statistical framework to analyze single-cell transcriptomics data, we infer the gene expression dynamics of early mouse embryonic stem (mES) cell differentiation, uncovering discrete transitions across nine cell states. We validate the predicted transitions across discrete states using flow cytometry. Moreover, using live-cell microscopy, we show that individual cells undergo abrupt transitions from a naïve to primed pluripotent state. Using the inferred discrete cell states to build a probabilistic model for the underlying gene regulatory network, we further predict and experimentally verify that these states have unique response to perturbations, thus defining them functionally. Our study provides a framework to infer the dynamics of differentiation from single cell transcriptomics data and to build predictive models of the gene regulatory networks that drive the sequence of cell fate decisions during development.
Assuntos
Diferenciação Celular , Células-Tronco Embrionárias/fisiologia , Animais , Citometria de Fluxo , Perfilação da Expressão Gênica , Camundongos , Análise de Célula ÚnicaRESUMO
During human brain development, multiple signaling pathways generate diverse cell types with varied regional identities. Here, we integrate single-cell RNA sequencing and clonal analyses to reveal lineage trees and molecular signals underlying early forebrain and mid/hindbrain cell differentiation from human embryonic stem cells (hESCs). Clustering single-cell transcriptomic data identified 41 distinct populations of progenitor, neuronal, and non-neural cells across our differentiation time course. Comparisons with primary mouse and human gene expression data demonstrated rostral and caudal progenitor and neuronal identities from early brain development. Bayesian analyses inferred a unified cell-type lineage tree that bifurcates between cortical and mid/hindbrain cell types. Two methods of clonal analyses confirmed these findings and further revealed the importance of Wnt/ß-catenin signaling in controlling this lineage decision. Together, these findings provide a rich transcriptome-based lineage map for studying human brain development and modeling developmental disorders.
Assuntos
Encéfalo/embriologia , Linhagem da Célula , Desenvolvimento Embrionário , Células-Tronco Embrionárias Humanas/citologia , Análise de Célula Única/métodos , Animais , Encéfalo/metabolismo , Linhagem Celular , Linhagem da Célula/genética , Células Clonais , Desenvolvimento Embrionário/genética , Humanos , Camundongos , Modelos Biológicos , Neurônios/citologia , Neurônios/metabolismo , Reprodutibilidade dos Testes , Análise de Sequência de RNA , Fatores de Transcrição/metabolismo , Transcriptoma/genética , Via de Sinalização Wnt/genéticaRESUMO
Spinocerebellar ataxia (SCA) type 7 is an inherited neurodegenerative disorder caused by expansion of a polyglutamine tract within the ataxin-7 protein. To determine the molecular basis of polyglutamine neurotoxicity in this and other related disorders, we produced SCA7 transgenic mice that express ataxin-7 with 24 or 92 glutamines in all neurons of the CNS, except for Purkinje cells. Transgenic mice expressing ataxin-7 with 92 glutamines (92Q) developed a dramatic neurological phenotype presenting as a gait ataxia and culminating in premature death. Despite the absence of expression of polyglutamine-expanded ataxin-7 in Purkinje cells, we documented severe Purkinje cell degeneration in 92Q SCA7 transgenic mice. We also detected an N-terminal truncation fragment of ataxin-7 in transgenic mice and in SCA7 patient material with both anti-ataxin-7 and anti-polyglutamine specific antibodies. The appearance of truncated ataxin-7 in nuclear aggregates correlates with the onset of a disease phenotype in the SCA7 mice, suggesting that nuclear localization and proteolytic cleavage may be important features of SCA7 pathogenesis. The non-cell-autonomous nature of the Purkinje cell degeneration in our SCA7 mouse model indicates that polyglutamine-induced dysfunction in adjacent or connecting cell types contributes to the neurodegeneration.
Assuntos
Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Peptídeos/genética , Células de Purkinje/patologia , Degenerações Espinocerebelares/etiologia , Animais , Ataxina-7 , Núcleo Celular/patologia , Marcha Atáxica/etiologia , Marcha Atáxica/metabolismo , Marcha Atáxica/patologia , Corpos de Inclusão/patologia , Cinética , Camundongos , Camundongos Transgênicos , Mutação , Proteínas do Tecido Nervoso/fisiologia , Degenerações Espinocerebelares/metabolismo , Degenerações Espinocerebelares/patologiaRESUMO
Many neurological and psychiatric disorders affect the cerebral cortex, and a clearer understanding of the molecular processes underlying human corticogenesis will provide greater insight into such pathologies. To date, knowledge of gene expression changes accompanying corticogenesis is largely based on murine data. Here we present a searchable, comprehensive, temporal gene expression data set encompassing cerebral cortical development from human embryonic stem cells (hESCs). Using a modified differentiation protocol that yields neurons suggestive of prefrontal cortex, we identified sets of genes and long noncoding RNAs that significantly change during corticogenesis and those enriched for disease-associations. Numerous alternatively spliced genes with varying temporal patterns of expression are revealed, including TGIF1, involved in holoprosencephaly, and MARK1, involved in autism. We have created a database (http://cortecon.neuralsci.org/) that provides online, query-based access to changes in RNA expression and alternatively spliced transcripts during human cortical development.
Assuntos
Córtex Cerebral/citologia , Córtex Cerebral/embriologia , Bases de Dados Genéticas , Células-Tronco Embrionárias/fisiologia , Perfilação da Expressão Gênica/métodos , Redes Reguladoras de Genes/genética , Animais , Diferenciação Celular/genética , Células Cultivadas , Bases de Dados Genéticas/tendências , Perfilação da Expressão Gênica/tendências , Humanos , Camundongos , Organogênese/fisiologia , Fatores de TempoRESUMO
Although autophagy maintains normal neural function by degrading misfolded proteins, little is known about how neurons activate this integral response. Furthermore, classical methods of autophagy induction used with nonneural cells, such as starvation, simply result in neuron death. To study neuronal autophagy, we cultured primary cortical neurons from transgenic mice that ubiquitously express green fluorescent protein-tagged LC3 and monitored LC3-I to LC3-II conversion by immunohistochemistry and immunoblotting. Evaluation of different culture media led us to discover that culturing primary neurons in Dulbecco's modified Eagle's medium without B27 supplementation robustly activates autophagy. We validated this nutrient-limited media approach for inducing autophagy by showing that 3-methyl-adenine treatment and Atg5 RNA interference knockdown each inhibits LC3-I to LC3-II conversion. Evaluation of B27 supplement components yielded insulin as the factor whose absence induced autophagy in primary neurons, and this activation was mammalian target of rapamycin-dependent. When we tested if nutrient-limited media could protect neurons expressing polyglutamine-expanded proteins against cell death, we observed a strong protective effect, probably due to autophagy activation. Our results indicate that nutrient deprivation can be used to understand the regulatory basis of neuronal autophagy and implicate diminished insulin signaling in the activation of neuronal autophagy.
Assuntos
Autofagia/efeitos dos fármacos , Insulina/farmacologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Transdução de Sinais/efeitos dos fármacos , Adenina/análogos & derivados , Adenina/farmacologia , Animais , Proteínas de Transporte/metabolismo , Células Cultivadas , Meios de Cultivo Condicionados , Camundongos , Neurônios/citologia , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Ácido Poliglutâmico/farmacologia , Receptores Androgênicos/metabolismo , Estresse Fisiológico/efeitos dos fármacos , Serina-Treonina Quinases TOR , Técnicas de Cultura de TecidosRESUMO
In the mouse, Purkinje cell degeneration (pcd) is a recessive mutation characterized by degeneration of cerebellar Purkinje cells, retinal photoreceptors, olfactory bulb mitral neurons, and certain thalamic neurons, and is accompanied by defective spermatogenesis. Previous studies of pcd have led to the identification of Nna1 as the causal gene; however, how loss of Nna1 function results in neurodegeneration remains unresolved. One useful approach for establishing which functional domains of a protein underlie a recessive phenotype has been to determine the genetic basis of the various alleles at the locus of interest. Because none of the pcd alleles analyzed at the time of the identification of Nna1 provided insight into the molecular basis of Nna1 loss-of-function, we obtained a recent pcd remutation--pcd5J, and after determining that its phenotype is comparable to existing pcd severe alleles, we sought its genetic basis by sequencing Nna1. In this article we report that pcd5J results from the insertion of a single GAC triplet encoding an aspartic acid residue at position 775 of Nna1. Although this insertion does not affect Nna1 expression at the RNA level, Nna1pcd-5J protein expression is markedly decreased. Pulse-chase experiments reveal that the aspartic acid insertion dramatically destabilizes Nna1pcd-5J protein, accounting for the observation that pcd5J is a severe allele. The presence of a readily detectable genetic mutation in pcd5J confirms that Nna1 loss-of-function alone underlies the broad pcd phenotype and will facilitate further studies of how Nna1 loss-of-function produces neurodegeneration and defective spermatogenesis in pcd mice.
Assuntos
Ácido Aspártico/genética , Proteínas de Ligação ao GTP/genética , Proteínas de Ligação ao GTP/metabolismo , Degeneração Neural/patologia , Células de Purkinje/fisiologia , D-Ala-D-Ala Carboxipeptidase Tipo Serina/genética , D-Ala-D-Ala Carboxipeptidase Tipo Serina/metabolismo , Alelos , Sequência de Aminoácidos , Animais , Ácido Aspártico/metabolismo , Linhagem Celular , Cerebelo/metabolismo , Cerebelo/patologia , Proteínas de Ligação ao GTP/biossíntese , Humanos , Masculino , Camundongos , Dados de Sequência Molecular , Mutação , Degeneração Neural/genética , Degeneração Neural/metabolismo , RNA Mensageiro/biossíntese , Retina/metabolismo , Retina/patologia , D-Ala-D-Ala Carboxipeptidase Tipo Serina/biossínteseRESUMO
Spinocerebellar ataxia type 7 (SCA7) is an autosomal dominant cerebellar ataxia caused by a CAG repeat expansion in the ataxin-7 gene. In humans, SCA7 is characterized by marked anticipation due to intergenerational repeat instability with a bias toward expansion, and is thus regarded as the most unstable of the polyglutamine diseases. To study the molecular basis of CAG/CTG repeat instability and its pathological significance, we generated lines of transgenic mice carrying either a SCA7 cDNA construct or a 13.5 kb SCA7 genomic fragment with 92 CAG repeats. While the cDNA transgenic mice showed little intergenerational repeat instability, the genomic fragment transgenic mice displayed marked intergenerational instability with an obvious expansion bias. We then went on to generate additional lines of genomic fragment transgenic mice, and observed that deletion of the 3' genomic region significantly stabilized intergenerational transmission of the SCA7 CAG92 repeat. These results suggest that cis-information present on the genomic fragment is driving the instability process. As the SCA7 genomic fragment contains a large number of replication-associated motifs, the presence of such sequence elements may make the SCA7 CAG repeat region more susceptible to instability. Small-pool and standard PCR analysis of tissues from genomic fragment mice revealed large repeat expansions in their brains and livers, but no such changes were found in any tissues from cDNA transgenic mice that have been shown to undergo neurodegeneration. As large somatic repeat expansions are absent from the brains of SCA7 cDNA mice, our results indicate that neurodegeneration can occur without marked somatic mosaicism, at least in these mice.