RESUMO
Mammalian SWI/SNF/BAF chromatin remodeling complexes influence cell lineage determination. While the contribution of these complexes to neural progenitor cell (NPC) proliferation and differentiation has been reported, little is known about the transcriptional profiles that determine neurogenesis or gliogenesis. Here, we report that BCL7A is a modulator of the SWI/SNF/BAF complex that stimulates the genome-wide occupancy of the ATPase subunit BRG1. We demonstrate that BCL7A is dispensable for SWI/SNF/BAF complex integrity, whereas it is essential to regulate Notch/Wnt pathway signaling and mitochondrial bioenergetics in differentiating NPCs. Pharmacological stimulation of Wnt signaling restores mitochondrial respiration and attenuates the defective neurogenic patterns observed in NPCs lacking BCL7A. Consistently, treatment with an enhancer of mitochondrial biogenesis, pioglitazone, partially restores mitochondrial respiration and stimulates neuronal differentiation of BCL7A-deficient NPCs. Using conditional BCL7A knockout mice, we reveal that BCL7A expression in NPCs and postmitotic neurons is required for neuronal plasticity and supports behavioral and cognitive performance. Together, our findings define the specific contribution of BCL7A-containing SWI/SNF/BAF complexes to mitochondria-driven NPC commitment, thereby providing a better understanding of the cell-intrinsic transcriptional processes that connect metabolism, neuronal morphogenesis, and cognitive flexibility.
Assuntos
Diferenciação Celular , Proteínas dos Microfilamentos , Células-Tronco Neurais , Animais , Camundongos , Adenosina Trifosfatases/metabolismo , Montagem e Desmontagem da Cromatina , Metabolismo Energético , Mitocôndrias/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteínas dos Microfilamentos/metabolismo , Células-Tronco Neurais/citologiaRESUMO
Charcot-Marie-Tooth (CMT) disease 4A is an autosomal-recessive polyneuropathy caused by mutations of ganglioside-induced differentiation-associated protein 1 (GDAP1), a putative glutathione transferase, which affects mitochondrial shape and alters cellular Ca2+ homeostasis. Here, we identify the underlying mechanism. We found that patient-derived motoneurons and GDAP1 knockdown SH-SY5Y cells display two phenotypes: more tubular mitochondria and a metabolism characterized by glutamine dependence and fewer cytosolic lipid droplets. GDAP1 interacts with the actin-depolymerizing protein Cofilin-1 and beta-tubulin in a redox-dependent manner, suggesting a role for actin signaling. Consistently, GDAP1 loss causes less F-actin close to mitochondria, which restricts mitochondrial localization of the fission factor dynamin-related protein 1, instigating tubularity. GDAP1 silencing also disrupts mitochondria-ER contact sites. These changes result in lower mitochondrial Ca2+ levels and inhibition of the pyruvate dehydrogenase complex, explaining the metabolic changes upon GDAP1 loss of function. Together, our findings reconcile GDAP1-associated phenotypes and implicate disrupted actin signaling in CMT4A pathophysiology.
Assuntos
Actinas , Proteínas do Tecido Nervoso/metabolismo , Neuroblastoma , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Humanos , Mitocôndrias/metabolismo , Neuroblastoma/metabolismo , Complexo Piruvato Desidrogenase/metabolismoRESUMO
Mitochondria sustain the energy demand of the cell. The composition and functional state of the mitochondrial oxidative phosphorylation system are informative indicators of organelle bioenergetic capacity. Here, we describe a highly sensitive and reproducible method for a single-cell quantification of mitochondrial CI- and CIV-containing respiratory supercomplexes (CI∗CIV-SCs) as an alternative means of assessing mitochondrial respiratory chain integrity. We apply a proximity ligation assay (PLA) and stain CI∗CIV-SCs in fixed human and mouse brains, tumorigenic cells, induced pluripotent stem cells (iPSCs) and iPSC-derived neural precursor cells (NPCs), and neurons. Spatial visualization of CI∗CIV-SCs enables the detection of mitochondrial lesions in various experimental models, including complex tissues undergoing degenerative processes. We report that comparative assessments of CI∗CIV-SCs facilitate the quantitative profiling of even subtle mitochondrial variations by overcoming the confounding effects that mixed cell populations have on other measurements. Together, our PLA-based analysis of CI∗CIV-SCs is a sensitive and complementary technique for detecting cell-type-specific mitochondrial perturbations in fixed materials.
Assuntos
Complexo IV da Cadeia de Transporte de Elétrons , Células-Tronco Neurais , Camundongos , Animais , Humanos , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Células-Tronco Neurais/metabolismo , Mitocôndrias/metabolismo , Membranas Mitocondriais/metabolismo , Fosforilação OxidativaRESUMO
The incidence of Alzheimer's disease is increasing with the aging population, and it has become one of the main health concerns of modern society. The dissection of the underlying pathogenic mechanisms and the development of effective therapies remain extremely challenging, also because available animal and cell culture models do not fully recapitulate the whole spectrum of pathological changes. The advent of human pluripotent stem cells and cell reprogramming has provided new prospects for tackling these challenges in a human and even patient-specific setting. Yet, experimental modeling of non-cell autonomous and extracellular disease-related alterations has remained largely inaccessible. These limitations are about to be overcome by advances in the development of 3D cell culture systems including organoids, neurospheroids and matrix-embedded 3D cultures, which have been shown to recapitulate extracellular pathologies such as plaque formation in vitro. Recent xenograft studies have even taken human stem cell-based disease modeling to an in vivo scenario where grafted neurons are probed in a disease background in the context of a rodent brain. Here, we review the latest developments in this emerging field along with their advantages, challenges, and future prospects.
Assuntos
Doença de Alzheimer/metabolismo , Medicina de Precisão/métodos , Cultura Primária de Células/métodos , Doença de Alzheimer/etiologia , Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Animais , Edição de Genes/métodos , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/patologia , Transplante Heterólogo/métodosRESUMO
The controlled differentiation of pluripotent stem cells (PSCs) into neurons and glia offers a unique opportunity to study early stages of human central nervous system development under controlled conditions in vitro. With the advent of cell reprogramming and the possibility to generate induced pluripotent stem cells (iPSCs) from any individual in a scalable manner, these studies can be extended to a disease- and patient-specific level. Autism spectrum disorder (ASD) is considered a neurodevelopmental disorder, with substantial evidence pointing to early alterations in neurogenesis and network formation as key pathogenic drivers. For that reason, ASD represents an ideal candidate for stem cell-based disease modeling. Here, we provide a concise review on recent advances in the field of human iPSC-based modeling of syndromic and non-syndromic forms of ASD, with a particular focus on studies addressing neuronal dysfunction and altered connectivity. We further discuss recent efforts to translate stem cell-based disease modeling to 3D via brain organoid and cell transplantation approaches, which enable the investigation of disease mechanisms in a tissue-like context. Finally, we describe advanced tools facilitating the assessment of altered neuronal function, comment on the relevance of iPSC-based models for the assessment of pharmaceutical therapies and outline potential future routes in stem cell-based ASD research.
Assuntos
Transtorno do Espectro Autista/patologia , Transtorno do Espectro Autista/fisiopatologia , Células-Tronco Pluripotentes Induzidas/patologia , Modelos Biológicos , Neurônios/patologia , Animais , Reprogramação Celular/genética , Humanos , Organoides/patologiaRESUMO
Nuclear clearance of TDP-43 into cytoplasmic aggregates is a key driver of neurodegeneration in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD), but the mechanisms are unclear. Here, we show that TDP-43 knockdown specifically reduces the number and motility of RAB11-positive recycling endosomes in dendrites, while TDP-43 overexpression has the opposite effect. This is associated with delayed transferrin recycling in TDP-43-knockdown neurons and decreased ß2-transferrin levels in patient CSF Whole proteome quantification identified the upregulation of the ESCRT component VPS4B upon TDP-43 knockdown in neurons. Luciferase reporter assays and chromatin immunoprecipitation suggest that TDP-43 represses VPS4B transcription. Preventing VPS4B upregulation or expression of its functional antagonist ALIX restores trafficking of recycling endosomes. Proteomic analysis revealed the broad reduction in surface expression of key receptors upon TDP-43 knockdown, including ErbB4, the neuregulin 1 receptor. TDP-43 knockdown delays the surface delivery of ErbB4. ErbB4 overexpression, but not neuregulin 1 stimulation, prevents dendrite loss upon TDP-43 knockdown. Thus, impaired recycling of ErbB4 and other receptors to the cell surface may contribute to TDP-43-induced neurodegeneration by blocking trophic signaling.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Endossomos/metabolismo , Neurônios/metabolismo , Receptor ErbB-4/metabolismo , ATPases Associadas a Diversas Atividades Celulares , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/metabolismo , Animais , Células Cultivadas , Proteínas de Ligação a DNA/genética , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Degeneração Lobar Frontotemporal/genética , Degeneração Lobar Frontotemporal/metabolismo , Técnicas de Silenciamento de Genes , Hipocampo/citologia , Humanos , Transporte Proteico , Ratos , Receptor ErbB-4/genética , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/metabolismo , Transdução de SinaisRESUMO
Spinal muscular atrophy (SMA) is a devastating motoneuron (MN) disorder caused by homozygous loss of SMN1. Rarely, SMN1-deleted individuals are fully asymptomatic despite carrying identical SMN2 copies as their SMA III-affected siblings suggesting protection by genetic modifiers other than SMN2. High plastin 3 (PLS3) expression has previously been found in lymphoblastoid cells but not in fibroblasts of asymptomatic compared to symptomatic siblings. To find out whether PLS3 is also upregulated in MNs of asymptomatic individuals and thus a convincing SMA protective modifier, we generated induced pluripotent stem cells (iPSCs) from fibroblasts of three asymptomatic and three SMA III-affected siblings from two families and compared these to iPSCs from a SMA I patient and control individuals. MNs were differentiated from iPSC-derived small molecule neural precursor cells (smNPCs). All four genotype classes showed similar capacity to differentiate into MNs at day 8. However, SMA I-derived MN survival was significantly decreased while SMA III- and asymptomatic-derived MN survival was moderately reduced compared to controls at day 27. SMN expression levels and concomitant gem numbers broadly matched SMN2 copy number distribution; SMA I presented the lowest levels, whereas SMA III and asymptomatic showed similar levels. In contrast, PLS3 was significantly upregulated in mixed MN cultures from asymptomatic individuals pinpointing a tissue-specific regulation. Evidence for strong PLS3 accumulation in shaft and rim of growth cones in MN cultures from asymptomatic individuals implies an important role in neuromuscular synapse formation and maintenance. These findings provide strong evidence that PLS3 is a genuine SMA protective modifier.
Assuntos
Glicoproteínas de Membrana/metabolismo , Proteínas dos Microfilamentos/metabolismo , Neurônios Motores/patologia , Atrofia Muscular Espinal/genética , Células-Tronco Neurais/citologia , Proteína 1 de Sobrevivência do Neurônio Motor/genética , Regulação para Cima , Biópsia , Diferenciação Celular , Feminino , Fibroblastos/citologia , Fibroblastos/metabolismo , Deleção de Genes , Inativação Gênica , Vetores Genéticos , Genótipo , Humanos , Imuno-Histoquímica , Células-Tronco Pluripotentes Induzidas/citologia , Cariotipagem , Linfócitos/citologia , Masculino , Microscopia Confocal , Mutação , Linhagem , Fenótipo , RNA Interferente Pequeno/metabolismo , Pele/patologiaRESUMO
Neural precursor cells (NPCs) derived from human pluripotent stem cells (hPSCs) represent an attractive tool for the in vitro generation of various neural cell types. However, the developmentally early NPCs emerging during hPSC differentiation typically show a strong propensity for neuronal differentiation, with more limited potential for generating astrocytes and, in particular, for generating oligodendrocytes. This phenomenon corresponds well to the consecutive and protracted generation of neurons and GLIA during normal human development. To obtain a more gliogenic NPC type, we combined growth factor-mediated expansion with pre-exposure to the differentiation-inducing agent retinoic acid and subsequent immunoisolation of CD133-positive cells. This protocol yields an adherent and self-renewing population of hindbrain/spinal cord radial glia (RG)-like neural precursor cells (RGL-NPCs) expressing typical neural stem cell markers such as nestin, ASCL1, SOX2, and PAX6 as well as RG markers BLBP, GLAST, vimentin, and GFAP. While RGL-NPCs maintain the ability for tripotential differentiation into neurons, astrocytes, and oligodendrocytes, they exhibit greatly enhanced propensity for oligodendrocyte generation. Under defined differentiation conditions promoting the expression of the major oligodendrocyte fate-determinants OLIG1/2, NKX6.2, NKX2.2, and SOX10, RGL-NPCs efficiently convert into NG2-positive oligodendroglial progenitor cells (OPCs) and are subsequently capable of in vivo myelination. Representing a stable intermediate between PSCs and OPCs, RGL-NPCs expedite the generation of PSC-derived oligodendrocytes with O4-, 4860-, and myelin basic protein (MBP)-positive cells that already appear within 7 weeks following growth factor withdrawal-induced differentiation. Thus, RGL-NPCs may serve as robust tool for time-efficient generation of human oligodendrocytes from embryonic and induced pluripotent stem cells.
Assuntos
Técnicas de Cultura de Células/métodos , Células Ependimogliais/fisiologia , Oligodendroglia/fisiologia , Células-Tronco Pluripotentes/fisiologia , Antígeno AC133 , Animais , Antígenos CD/metabolismo , Diferenciação Celular/fisiologia , Linhagem Celular , Transplante de Células , Células-Tronco Embrionárias/fisiologia , Fatores de Crescimento de Fibroblastos/metabolismo , Perfilação da Expressão Gênica , Glicoproteínas/metabolismo , Proteína Homeobox Nkx-2.2 , Proteínas de Homeodomínio , Humanos , Imuno-Histoquímica , Camundongos Knockout , Camundongos Mutantes , Proteínas Nucleares , Peptídeos/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição , Tretinoína/metabolismoRESUMO
Human induced pluripotent stem cells (iPS cells) resemble embryonic stem cells and can differentiate into cell derivatives of all three germ layers. However, frequently the differentiation efficiency of iPS cells into some lineages is rather poor. Here, we found that fusion of iPS cells with human hematopoietic stem cells (HSCs) enhances iPS cell differentiation. Such iPS hybrids showed a prominent differentiation bias toward hematopoietic lineages but also toward other mesendodermal lineages. Additionally, during differentiation of iPS hybrids, expression of early mesendodermal markers-Brachyury (T), MIX1 Homeobox-Like Protein 1 (MIXL1), and Goosecoid (GSC)-appeared with faster kinetics than in parental iPS cells. Following iPS hybrid differentiation there was a prominent induction of NODAL and inhibition of NODAL signaling blunted mesendodermal differentiation. This indicates that NODAL signaling is critically involved in mesendodermal bias of iPS hybrid differentiation. In summary, we demonstrate that iPS cell fusion with HSCs prominently enhances iPS cell differentiation.
Assuntos
Diferenciação Celular , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Mesoderma/metabolismo , Antígenos de Diferenciação/biossíntese , Fusão Celular , Células-Tronco Hematopoéticas/citologia , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Mesoderma/citologiaRESUMO
A comprehensive understanding of the functional network of transcription factors establishing and maintaining pluripotency is key for the development of biomedical applications of stem cells. Nanog plays an important role in early development and is essential to induce natural pluripotency in embryonic stem cells (ESCs). Inducible gain-of-function systems allowing a precise control over time and dosage of Nanog activity would be highly desirable to study its vital role in the establishment and maintenance of pluripotency at molecular level. Here we engineered a recombinant cell permeable version of Nanog by fusing it with the cell penetrating peptide TAT. Nanog-TAT can be readily expressed in and purified from E. coli and binds to a consensus Nanog DNA sequence. At cellular level it enhances proliferation and self-renewal of ESCs in the absence of leukemia inhibitory factor (LIF). Nanog-TAT together with LIF acts synergistically as judged by enhanced clonogenicity and activation of an Oct4-promoter-driven GFP reporter gene. Furthermore Nanog-TAT, in the absence of LIF, promotes pluripotency by inhibiting endodermal specification in a Stat3-independent manner. Our results demonstrate that Nanog protein transduction is an attractive tool allowing control over dose and time of addition to the cells for studying the molecular control of pluripotency without genetic manipulation.
Assuntos
Células-Tronco Embrionárias/metabolismo , Endoderma/citologia , Proteínas de Homeodomínio/metabolismo , Células-Tronco Pluripotentes/metabolismo , Animais , Diferenciação Celular , Peptídeos Penetradores de Células/genética , Peptídeos Penetradores de Células/metabolismo , Células-Tronco Embrionárias/citologia , Endoderma/metabolismo , Proteínas de Homeodomínio/genética , Fator Inibidor de Leucemia/metabolismo , Camundongos , Proteína Homeobox Nanog , Células-Tronco Pluripotentes/citologia , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Human pluripotent stem cells (hPSCs) are a promising source of cells for clinical applications, such as transplantation of clinically engineered tissues and organs, and drug discovery programs due to their ability to self-renew and to be differentiated into cells from the three embryonic germ layers. In this study, the differentiation of two hPSC-lines into neural precursors (NPs) was accomplished with more than 80% efficiency, by means of the dual-SMAD inhibition protocol, based on the use of two small molecules (SB431542 and LDN193189) to generate Pax6 and Nestin-positive neural entities. One of the major hurdles related to the in vitro generation of PSC-derived populations is the tumorigenic potential of cells that remain undifferentiated. These remaining hPSCs have the potential to generate teratomas after being transplanted, and may interfere with the outcome of in vitro differentiation protocols. One strategy to tackle this problem is to deplete these "contaminating" cells during the differentiation process. Magnetic activated cell sorting (MACS) was used for the first time for purification of hPSC-derived NPs after the neural commitment stage using anti-Tra-1-60 micro beads for negative selection of the unwanted hPSCs. The depletion had an average efficiency of 80.4 ± 5% and less than 1.5% of Tra-1-60 positive cells were present in the purified populations. After re-plating, the purified neural precursors maintained their phenotype, and the success of the preparative purification with MACS was further confirmed with a decrease of 94.3% in the number of Oct4-positive proliferating hPSC colonies. Thus, the integration of the MACS depletion step with the neural commitment protocol paves the way towards the establishment of a novel bioprocess for production of purified populations of hPSC-derived neural cells for different applications.
Assuntos
Células-Tronco Pluripotentes Induzidas/fisiologia , Células-Tronco Neurais/fisiologia , Técnicas de Cultura de Células , Diferenciação Celular , Linhagem Celular , Proliferação de Células , Separação Celular , Citometria de Fluxo , HumanosRESUMO
Transcription factors such as Scl/Tal1, Lmo2, and Runx1 are essential for the development of hematopoietic stem cells (HSCs). However, the precise mechanisms by which these factors interact to form transcriptional networks, as well as the identity of the genes downstream of these regulatory cascades, remain largely unknown. To this end, we generated an Scl(-/-) yolk sac cell line to identify candidate Scl target genes by global expression profiling after reintroduction of a TAT-Scl fusion protein. Bioinformatics analysis resulted in the identification of 9 candidate Scl target transcription factor genes, including Runx1 and Runx3. Chromatin immunoprecipitation confirmed that both Runx genes are direct targets of Scl in the fetal liver and that Runx1 is also occupied by Scl in the yolk sac. Furthermore, binding of an Scl-Lmo2-Gata2 complex was demonstrated to occur on the regions flanking the conserved E-boxes of the Runx1 loci and was shown to transactivate the Runx1 element. Together, our data provide a key component of the transcriptional network of early hematopoiesis by identifying downstream targets of Scl that can explain key aspects of the early Scl(-/-) phenotype.