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Intraneuronal inclusions of misfolded α-synuclein (α-syn) and prion-like spread of the pathologic α-syn contribute to progressive neuronal death in Parkinson's disease (PD). Despite the pathologic significance, no efficient therapeutic intervention targeting α-synucleinopathy has been developed. In this study, we provide evidence that astrocytes, especially those cultured from the ventral midbrain (VM), show therapeutic potential to alleviate α-syn pathology in multiple in vitro and in vivo α-synucleinopathic models. Regulation of neuronal α-syn proteostasis underlies the therapeutic function of astrocytes. Specifically, VM-derived astrocytes inhibited neuronal α-syn aggregation and transmission in a paracrine manner by correcting not only intraneuronal oxidative and mitochondrial stresses but also extracellular inflammatory environments, in which α-syn proteins are prone to pathologic misfolding. The astrocyte-derived paracrine factors also promoted disassembly of extracellular α-syn aggregates. In addition to the aggregated form of α-syn, VM astrocytes reduced total α-syn protein loads both by actively scavenging extracellular α-syn fibrils and by a paracrine stimulation of neuronal autophagic clearance of α-syn. Transplantation of VM astrocytes into the midbrain of PD model mice alleviated α-syn pathology and protected the midbrain dopamine neurons from neurodegeneration. We further showed that cografting of VM astrocytes could be exploited in stem cell-based therapy for PD, in which host-to-graft transmission of α-syn pathology remains a critical concern for long-term cell therapeutic effects.
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Astrócitos , Transplante de Tecido Encefálico , Doença de Parkinson , Proteostase , alfa-Sinucleína , Animais , Astrócitos/transplante , Modelos Animais de Doenças , Neurônios Dopaminérgicos/metabolismo , Mesencéfalo/patologia , Mesencéfalo/cirurgia , Camundongos , Doença de Parkinson/patologia , Doença de Parkinson/terapia , alfa-Sinucleína/metabolismoRESUMO
Parkinson's disease (PD) is neurodegenerative movement disorder characterized by degeneration of midbrain-type dopamine (mDA) neurons in the substantia nigra (SN). The RNA-binding protein Lin28 plays a role in neuronal stem cell development and neuronal differentiation. In this study, we reveal that Lin28 conditional knockout (cKO) mice show degeneration of mDA neurons in the SN, as well as PD-related behavioral deficits. We identify a loss-of-function variant of LIN28A (R192G substitution) in two early-onset PD patients. Using an isogenic human embryonic stem cell (hESC)/human induced pluripotent stem cell (hiPSC)-based disease model, we find that the Lin28 R192G variant leads to developmental defects and PD-related phenotypes in mDA neuronal cells that can be rescued by expression of wild-type Lin28A. Cell transplantation experiments in PD model rats show that correction of the LIN28A variant in the donor patient (pt)-hiPSCs leads to improved behavioral phenotypes. Our data link LIN28A to PD pathogenesis and suggest future personalized medicine targeting this variant in patients.
Assuntos
Doença de Parkinson/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/fisiologia , Substância Negra/metabolismo , Animais , Comportamento Animal , Transplante de Células , Modelos Animais de Doenças , Dopamina/metabolismo , Neurônios Dopaminérgicos/fisiologia , Células-Tronco Embrionárias/fisiologia , Edição de Genes , Predisposição Genética para Doença , Humanos , Células-Tronco Pluripotentes Induzidas/fisiologia , Células-Tronco Pluripotentes Induzidas/transplante , Camundongos , Camundongos Knockout , Mutação , Células-Tronco Neurais/fisiologia , Células-Tronco Neurais/transplante , Doença de Parkinson/genética , Ratos , Transplante de Células-TroncoRESUMO
There is a compelling need to develop disease-modifying therapies for Alzheimer's disease (AD), the most common neuro-degenerative disorder. Together with recent progress in vector development for efficiently targeting the central nervous system, gene therapy has been suggested as a potential therapeutic modality to overcome the limited delivery of conventional types of drugs to and within the damaged brain. In addition, given increasing evidence of the strong link between glia and AD pathophysiology, therapeutic targets have been moving toward those addressing glial cell pathology. Nurr1 and Foxa2 are transcription/epigenetic regulators that have been reported to cooperatively regulate inflammatory and neurotrophic response in glial cells. In this study, we tested the therapeutic potential of Nurr1 and Foxa2 gene delivery to treat AD symptoms and pathologies. A series of functional, histologic, and transcriptome analyses revealed that the combined expression of Nurr1 and Foxa2 substantially ameliorated AD-associated amyloid ß and Tau proteinopathy, cell senescence, synaptic loss, and neuro-inflammation in multiple in vitro and in vivo AD models. Intra-cranial delivery of Nurr1 and Foxa2 genes using adeno-associated virus (AAV) serotype 9 improved the memory and cognitive function of AD model mice. The therapeutic benefits of gene delivery were attained mainly by correcting pathologic glial function. These findings collectively indicate that AAV9-mediated Nurr1 and Foxa2 gene transfer could be an effective disease-modifying therapy for AD.
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Brain organoids are three-dimensional (3D) structures derived from human pluripotent stem cells (hPSCs) that reflect early brain organization. These organoids contain different cell types, including neurons and glia, similar to those found in the human brain. Human brain organoids provide unique opportunities to model features of human brain development that are not well-reflected in animal models. Compared with traditional cell cultures and animal models, brain organoids offer a more accurate representation of human brain development and function, rendering them suitable models for neurodevelopmental diseases. In particular, brain organoids derived from patients' cells have enabled researchers to study diseases at different stages and gain a better understanding of disease mechanisms. Multi-brain regional assembloids allow for the investigation of interactions between distinct brain regions while achieving a higher level of consistency in molecular and functional characterization. Although organoids possess promising features, their usefulness is limited by several unresolved constraints, including cellular stress, hypoxia, necrosis, a lack of high-fidelity cell types, limited maturation, and circuit formation. In this review, we discuss studies to overcome the natural limitations of brain organoids, emphasizing the importance of combinations of all neural cell types, such as glia (astrocyte, oligodendrocytes, and microglia) and vascular cells. Additionally, considering the similarity of organoids to the developing brain, regionally patterned brain organoid-derived neural stem cells (NSCs) could serve as a scalable source for cell replacement therapy. We highlight the potential application of brain organoid-derived cells in disease cell therapy within this field.
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In this study, we found that undifferentiated human pluripotent stem cells (hPSCs; up to 30% of total cells) present in the cultures of neural stem or precursor cells (NPCs) completely disappeared within several days when cultured under neural differentiation culture conditions. Intriguingly, the disappearance of undifferentiated cells was not due to cell death but was instead mediated by neural conversion of hPSCs. Based on these findings, we propose pre-conditioning of donor NPC cultures under terminal differentiation culture conditions as a simple but efficient method of eliminating undifferentiated cells to treat neurologic disorders. In addition, we could establish a new neural differentiation protocol, in which undifferentiated hPSCs co-cultured with NPCs become differentiated neurons or NPCs in an extremely efficient, fast, and reproducible manner across the hESC and human-induced pluripotent stem cell (hiPSC) lines.
Assuntos
Técnicas de Cultura de Células/métodos , Diferenciação Celular , Células-Tronco Embrionárias/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Neurais/metabolismo , Linhagem Celular Tumoral , Técnicas de Cocultura , Fator 2 de Crescimento de Fibroblastos/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Humanos , Doenças do Sistema Nervoso/terapia , Fator 3 de Transcrição de Octâmero/metabolismo , Fenótipo , Transplante de Células-TroncoRESUMO
The original properties of tissue-specific stem cells, regardless of their tissue origins, are inevitably altered during in vitro culturing, lessening the clinical and research utility of stem cell cultures. Specifically, neural stem cells derived from the ventral midbrain lose their dopamine neurogenic potential, ventral midbrain-specific phenotypes, and repair capacity during in vitro cell expansion, all of which are critical concerns in using the cultured neural stem cells in therapeutic approaches for Parkinson's disease. In this study, we observed that the culture-dependent changes of neural stem cells derived from the ventral midbrain coincided with loss of RNA-binding protein LIN28A expression. When LIN28A expression was forced and sustained during neural stem cell expansion using an inducible expression-vector system, loss of dopamine neurogenic potential and midbrain phenotypes after long-term culturing was blocked. Furthermore, dopamine neurons that differentiated from neural stem cells exhibited remarkable survival and resistance against toxic insults. The observed effects were not due to a direct action of LIN28A on the differentiated dopamine neurons, but rather its action on precursor neural stem cells as exogene expression was switched off in the differentiating/differentiated cultures. Remarkable and reproducible behavioural recovery was shown in all Parkinson's disease rats grafted with neural stem cells expanded with LIN28A expression, along with extensive engraftment of dopamine neurons expressing mature neuronal and midbrain-specific markers. These findings suggest that LIN28A expression during stem cell expansion could be used to prepare therapeutically competent donor cells.
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Recent groundbreaking work has demonstrated that combined expression of the transcription factors Brn2, Ascl1, and Myt1L (BAM; also known as Wernig factors) convert mouse fibroblasts into postmitotic neuronal cells. However, questions remain regarding whether trans-conversion is achieved directly or involves an intermediary precursor stage. Trans-conversion toward expandable neural precursor cells (NPCs) is more useful than direct one-step neuron formation with respect to yielding a sufficient number of cells and the feasibility of manipulating NPC differentiation toward certain neuron subtypes. Here, we show that co-expression of Wernig factors and Bcl-xL induces fibroblast conversion into NPCs (induced NPCs (iNPCs)) that are highly expandable for >100 passages. Gene expression analyses showed that the iNPCs exhibited high expression of common NPC genes but not genes specific to defined embryonic brain regions. This finding indicated that a regional identity of iNPCs was not established. Upon induction, iNPCs predominantly differentiated into astrocytes. However, the differentiation potential was not fixed and could be efficiently manipulated into general or specific subtypes of neurons by expression of additional genes. Specifically, overexpression of Nurr1 and Foxa2, transcription factors specific for midbrain dopamine neuron development, drove iNPCs to yield mature midbrain dopamine neurons equipped with presynaptic DA neuronal functions. We further assessed the therapeutic potential of iNPCs in Parkinson disease model rats.
Assuntos
Transdiferenciação Celular , Dopamina/metabolismo , Neurônios Dopaminérgicos/citologia , Neurônios Dopaminérgicos/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Reprogramação Celular , Expressão Gênica , Fator 3-beta Nuclear de Hepatócito/genética , Mesencéfalo/citologia , Mesencéfalo/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos ICR , Camundongos Transgênicos , Proteínas do Tecido Nervoso/genética , Membro 2 do Grupo A da Subfamília 4 de Receptores Nucleares/genética , Fatores do Domínio POU/genética , Transtornos Parkinsonianos/metabolismo , Transtornos Parkinsonianos/patologia , Transtornos Parkinsonianos/terapia , Ratos , Ratos Endogâmicos Lew , Ratos Wistar , Fatores de Transcrição/genéticaRESUMO
Self-renewal and pluripotency are hallmark properties of pluripotent stem cells, including embryonic stem cells (ESCs) and iPS cells. Previous studies revealed the ESC-specific core transcription circuitry and showed that these core factors (e.g., Oct3/4, Sox2, and Nanog) regulate not only self-renewal but also pluripotent differentiation. However, it remains elusive how these two cell states are regulated and balanced during in vitro replication and differentiation. Here, we report that the transcription elongation factor Tcea3 is highly enriched in mouse ESCs (mESCs) and plays important roles in regulating the differentiation. Strikingly, altering Tcea3 expression in mESCs did not affect self-renewal under nondifferentiating condition; however, upon exposure to differentiating cues, its overexpression impaired in vitro differentiation capacity, and its knockdown biased differentiation toward mesodermal and endodermal fates. Furthermore, we identified Lefty1 as a downstream target of Tcea3 and showed that the Tcea3-Lefty1-Nodal-Smad2 pathway is an innate program critically regulating cell fate choices between self-replication and differentiation commitment. Together, we propose that Tcea3 critically regulates pluripotent differentiation of mESCs as a molecular rheostat of Nodal-Smad2/3 signaling.
Assuntos
Células-Tronco Embrionárias/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Células-Tronco Pluripotentes/metabolismo , Transdução de Sinais/genética , Fatores de Elongação da Transcrição/genética , Animais , Diferenciação Celular , Proliferação de Células , Células-Tronco Embrionárias/citologia , Endoderma/citologia , Endoderma/crescimento & desenvolvimento , Endoderma/metabolismo , Perfilação da Expressão Gênica , Fatores de Determinação Direita-Esquerda/genética , Fatores de Determinação Direita-Esquerda/metabolismo , Mesoderma/citologia , Mesoderma/crescimento & desenvolvimento , Mesoderma/metabolismo , Camundongos , Proteína Nodal/genética , Proteína Nodal/metabolismo , Células-Tronco Pluripotentes/citologia , Proteína Smad2/genética , Proteína Smad2/metabolismo , Fatores de Elongação da Transcrição/metabolismoRESUMO
Human pluripotent stem cells (hPSCs) such as human embryonic stem cells (hESCs), induced pluripotent stem cells, and somatic cell nuclear transfer (SCNT)-hESCs can permanently self-renew while maintaining their capacity to differentiate into any type of somatic cells, thereby serving as an important cell source for cell therapy. However, there are persistent challenges in the application of hPSCs in clinical trials, where one of the most significant is graft rejection by the patient immune system in response to human leukocyte antigen (HLA) mismatch when transplants are obtained from an allogeneic (non-self) cell source. Homozygous SCNT-hESCs (homo-SCNT-hESCs) were used to simplify the clinical application and to reduce HLA mismatch. Here, we present a xeno-free protocol that confirms the efficient generation of neural precursor cells in hPSCs and also the differentiation of dopaminergic neurons. Additionally, there was no difference when comparing the HLA expression patterns of hESC, homo-SCNT-hESCs and hetero-SCNT-hESCs. We propose that there are no differences in the differentiation capacity and HLA expression among hPSCs that can be cultured in vitro. Thus, it is expected that homo-SCNT-hESCs will possess a wider range of applications when transplanted with neural precursor cells in the context of clinical trials.
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Mitochondria are essential organelles for cell survival that manage the cellular energy supply by producing ATP. Mitochondrial dysfunction is associated with various human diseases, including metabolic syndromes, aging, and neurodegenerative diseases. Among the diseases related to mitochondrial dysfunction, Parkinson's disease (PD) is the second most common neurodegenerative disease and is characterized by dopaminergic neuronal loss and neuroinflammation. Recently, it was reported that mitochondrial transfer between cells occurred naturally and that exogenous mitochondrial transplantation was beneficial for treating mitochondrial dysfunction. The current study aimed to investigate the therapeutic effect of mitochondrial transfer on PD in vitro and in vivo. The results showed that PN-101 mitochondria isolated from human mesenchymal stem cells exhibited a neuroprotective effect against 1-methyl-4-phenylpyridinium, 6-hydroxydopamine and rotenone in dopaminergic cells and ameliorated dopaminergic neuronal loss in the brains of C57BL/6J mice injected 30 âmg/kg of methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) intraperitoneally. In addition, PN-101 exhibited anti-inflammatory effects by reducing the expression of pro-inflammatory cytokines in microglial cells and suppressing microglial activation in the striatum. Furthermore, intravenous mitochondrial treatment was associated with behavioral improvements during the pole test and rotarod test in the MPTP-induced PD mice. These dual effects of neuroprotection and anti-neuroinflammation support the potential for mitochondrial transplantation as a novel therapeutic strategy for PD.
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INTRODUCTION: Astrocytes are glial-type cells that protect neurons from toxic insults and support neuronal functions and metabolism in a healthy brain. Leveraging these physiological functions, transplantation of astrocytes or their derivatives has emerged as a potential therapeutic approach for neurodegenerative disorders. METHODS: To substantiate the clinical application of astrocyte-based therapy, we aimed to prepare human astrocytes with potent therapeutic capacities from human pluripotent stem cells (hPSCs). To that end, we used ventral midbrain patterning during the differentiation of hPSCs into astrocytes, based on the roles of midbrain-specific factors in potentiating glial neurotrophic/anti-inflammatory activity. To assess the therapeutic effects of human midbrain-type astrocytes, we transplanted them into mouse models of Parkinson's disease (PD) and Alzheimer's disease (AD). RESULTS: Through a comprehensive series of in-vitro and in-vivo experiments, we were able to establish that the midbrain-type astrocytes exhibited the abilities to effectively combat oxidative stress, counter excitotoxic glutamate, and manage pathological protein aggregates. Our strategy for preparing midbrain-type astrocytes yielded promising results, demonstrating the strong therapeutic potential of these cells in various neurotoxic contexts. Particularly noteworthy is their efficacy in PD and AD-specific proteopathic conditions, in which the midbrain-type astrocytes outperformed forebrain-type astrocytes derived by the same organoid-based method. CONCLUSION: The enhanced functions of the midbrain-type astrocytes extended to their ability to release signaling molecules that inhibited neuronal deterioration and senescence while steering microglial cells away from a pro-inflammatory state. This success was evident in both in-vitro studies using human cells and in-vivo experiments conducted in mouse models of PD and AD. In the end, our human midbrain-type astrocytes demonstrated remarkable effectiveness in alleviating neurodegeneration, neuroinflammation, and the pathologies associated with the accumulation of α-synuclein and Amyloid ß proteins.
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X-linked adrenoleukodystrophy (ALD) caused by the ABCD1 mutation, is the most common inherited peroxisomal disease. Previously, we generated an ALD patient-derived SCHi001-A iPSC model. In this study, we have performed the first genome editing of ALD patient-derived SCHi001-A iPSCs using homology-directed repair (HDR). The mutation site, c.1534G > A [GenBank: NM_000033.4], was corrected by introducing ssODN and the CRISPR/Cas9 system. The cell line exhibited normal iPSC plulipotency marker expression following genome editing. Mutation-corrected iPSCs from SCHi001-A iPSC line can be used in research into the pathophysiology of and therapeutics for ALD.
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Developing methods to improve the regenerative capacity of somatic stem cells (SSCs) is a major challenge in regenerative medicine. Here, we propose the forced expression of LIN28A as a method to modulate cellular metabolism, which in turn enhances self-renewal, differentiation capacities, and engraftment after transplantation of various human SSCs. Mechanistically, in undifferentiated/proliferating SSCs, LIN28A induced metabolic reprogramming from oxidative phosphorylation (OxPhos) to glycolysis by activating PDK1-mediated glycolysis-TCA/OxPhos uncoupling. Mitochondria were also reprogrammed into healthy/fused mitochondria with improved functional capacity. The reprogramming allows SSCs to undergo cell proliferation more extensively with low levels of oxidative and mitochondrial stress. When the PDK1-mediated uncoupling was untethered upon differentiation, LIN28A-SSCs differentiated more efficiently with an increase of OxPhos by utilizing the reprogrammed mitochondria. This study provides mechanistic and practical approaches of utilizing LIN28A and metabolic reprogramming in order to improve SSCs utility in regenerative medicine.
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Células-Tronco Adultas , Mitocôndrias , Células-Tronco Adultas/metabolismo , Diferenciação Celular , Reprogramação Celular , Glicólise , Humanos , Mitocôndrias/metabolismo , Fosforilação OxidativaRESUMO
Loss-of-function mutations of DNAJC6, encoding HSP40 auxilin, have recently been identified in patients with early-onset Parkinson's disease (PD). To study the roles of DNAJC6 in PD pathogenesis, we used human embryonic stem cells with CRISPR-Cas9-mediated gene editing. Here, we show that DNAJC6 mutations cause key PD pathologic features, i.e., midbrain-type dopamine (mDA) neuron degeneration, pathologic α-synuclein aggregation, increase of intrinsic neuronal firing frequency, and mitochondrial and lysosomal dysfunctions in human midbrain-like organoids (hMLOs). In addition, neurodevelopmental defects were also manifested in hMLOs carrying the mutations. Transcriptomic analyses followed by experimental validation revealed that defects in DNAJC6-mediated endocytosis impair the WNT-LMX1A signal during the mDA neuron development. Furthermore, reduced LMX1A expression during development caused the generation of vulnerable mDA neurons with the pathologic manifestations. These results suggest that the human model of DNAJC6-PD recapitulates disease phenotypes and reveals mechanisms underlying disease pathology, providing a platform for assessing therapeutic interventions.
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Doença de Parkinson , Proteínas de Choque Térmico HSP40/genética , Proteínas de Choque Térmico HSP40/metabolismo , Humanos , Mesencéfalo , Mutação , Organoides/metabolismo , Doença de Parkinson/metabolismo , FenótipoRESUMO
Astrocytes and microglia are brain-resident glia that can establish harmful inflammatory environments in disease contexts and thereby contribute to the progression of neuronal loss in neurodegenerative disorders. Correcting the diseased properties of glia is therefore an appealing strategy for treating brain diseases. Previous studies have shown that serum/ glucocorticoid related kinase 1 (SGK1) is upregulated in the brains of patients with various neurodegenerative disorders, suggesting its involvement in the pathogenesis of those diseases. In this study, we show that inhibiting glial SGK1 corrects the pro-inflammatory properties of glia by suppressing the intracellular NFκB-, NLRP3-inflammasome-, and CGAS-STING-mediated inflammatory pathways. Furthermore, SGK1 inhibition potentiated glial activity to scavenge glutamate toxicity and prevented glial cell senescence and mitochondrial damage, which have recently been reported as critical pathologic features of and therapeutic targets in Parkinson disease (PD) and Alzheimer disease (AD). Along with those anti-inflammatory/neurotrophic functions, silencing and pharmacological inhibition of SGK1 protected midbrain dopamine neurons from degeneration and cured pathologic synuclein alpha (SNCA) aggregation and PD-associated behavioral deficits in multiple in vitro and in vivo PD models. Collectively, these findings suggest that SGK1 inhibition could be a useful strategy for treating PD and other neurodegenerative disorders that share the common pathology of glia-mediated neuroinflammation.
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Glucocorticoides , Doença de Parkinson , Animais , Modelos Animais de Doenças , Neurônios Dopaminérgicos , Humanos , Modelos Animais , Neuroglia , Doença de Parkinson/tratamento farmacológicoRESUMO
Successful clinical translation of stem cell-based therapy largely relies on the scalable and reproducible preparation of donor cells with potent therapeutic capacities. In this study, midbrain organoids were yielded from human pluripotent stem cells (hPSCs) to prepare cells for Parkinson's disease (PD) therapy. Neural stem/precursor cells (NSCs) isolated from midbrain organoids (Og-NSCs) expanded stably and differentiated into midbrain-type dopamine(mDA) neurons, and an unprecedentedly high proportion expressed midbrain-specific factors, with relatively low cell line and batch-to-batch variations. Single cell transcriptome analysis followed by in vitro assays indicated that the majority of cells in the Og-NSC cultures are ventral midbrain (VM)-patterned with low levels of cellular senescence/aging and mitochondrial stress, compared to those derived from 2D-culture environments. Notably, in contrast to current methods yielding mDA neurons without astrocyte differentiation, mDA neurons that differentiated from Og-NSCs were interspersed with astrocytes as in the physiologic brain environment. Thus, the Og-NSC-derived mDA neurons exhibited improved synaptic maturity, functionality, resistance to toxic insults, and faithful expressions of the midbrain-specific factors, in vitro and in vivo long after transplantation. Consequently, Og-NSC transplantation yielded potent therapeutic outcomes that are reproducible in PD model animals. Collectively, our observations demonstrate that the organoid-based method may satisfy the demands needed in the clinical setting of PD cell therapy.
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Células-Tronco Neurais , Doença de Parkinson , Animais , Diferenciação Celular , Neurônios Dopaminérgicos , Humanos , Mesencéfalo , Organoides , Doença de Parkinson/terapiaRESUMO
In the developing mouse brain, the highest Bcl-X(L) expression is seen at the peak of neurogenesis, whereas the peak of Bax expression coincides with the astrogenic period. While such observations suggest an active role of the Bcl-2 family proteins in the generation of neurons and astrocytes, no definitive demonstration has been provided to date. Using combinations of gain- and loss-of-function assays in vivo and in vitro, we provide evidence for instructive roles of these proteins in neuronal and astrocytic fate specification. Specifically, in Bax knockout mice, astrocyte formation was decreased in the developing cortices. Overexpression of Bcl-X(L) and Bax in embryonic cortical precursors induced neural and astrocytic differentiation, respectively, while inhibitory RNAs led to the opposite results. Importantly, inhibition of caspase activity, dimerization, or mitochondrial localization of Bcl-X(L)/Bax proteins indicated that the differentiation effects of Bcl-X(L)/Bax are separable from their roles in cell survival and apoptosis. Lastly, we describe activation of intracellular signaling pathways and expression of basic helix-loop-helix transcriptional factors specific for the Bcl-2 protein-mediated differentiation.
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Córtex Cerebral/citologia , Córtex Cerebral/embriologia , Regulação da Expressão Gênica no Desenvolvimento , Proteína X Associada a bcl-2/metabolismo , Proteína bcl-X/metabolismo , Animais , Astrócitos/citologia , Astrócitos/fisiologia , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Inibidores de Caspase , Diferenciação Celular , Células Cultivadas , Materiais Revestidos Biocompatíveis/metabolismo , Cruzamentos Genéticos , Dimerização , Ativação Enzimática , Fibronectinas/metabolismo , Homozigoto , Camundongos , Camundongos Knockout , Mitocôndrias/metabolismo , Mutagênese Sítio-Dirigida , Neurônios/citologia , Neurônios/fisiologia , RNA Interferente Pequeno/genética , Retroviridae/genética , Transdução de Sinais/fisiologia , Transdução Genética , Proteína X Associada a bcl-2/química , Proteína X Associada a bcl-2/genética , Proteína bcl-X/química , Proteína bcl-X/genéticaRESUMO
Staufen1 (STAU1) and Lin28B are RNA-binding proteins that are involved in neuronal differentiation as a function of post-transcriptional regulation. STAU1 triggers post-transcriptional regulation, including mRNA export, mRNA relocation, translation and mRNA decay. Lin28B also has multiple functions in miRNA biogenesis and the regulation of translation. Here, we examined the connection between STAU1 and Lin28B and found that Lin28B regulates the abundance of STAU1 mRNA via miRNA maturation. Decreases in the expression of both STAU1 and Lin28B were observed during neuronal differentiation. Depletion of STAU1 or Lin28B inhibited neuronal differentiation, and overexpression of STAU1 or Lin28B enhanced neuronal differentiation. Interestingly, the stability of STAU1 mRNA was modulated by miR-142-3p, whose maturation was regulated by Lin28B. Thus, miR-142-3p expression increased as Lin28B expression decreased during differentiation, leading to the reduction of STAU1 expression. The transcriptome from Staufen-mediated mRNA decay (SMD) targets during differentiation was analyzed, confirming that STAU1 was a key factor in neuronal differentiation. In support of this finding, regulation of STAU1 expression in mouse neural precursor cells had the same effects on neuronal differentiation as it did in human neuroblastoma cells. These results revealed the collaboration of two RNA-binding proteins, STAU1 and Lin28B, as a regulatory mechanism in neuronal differentiation.