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1.
Proc Natl Acad Sci U S A ; 119(43): e2123476119, 2022 10 25.
Artigo em Inglês | MEDLINE | ID: mdl-36251998

RESUMO

Microglia, the resident immune cells of the central nervous system (CNS), are derived from yolk-sac macrophages that populate the developing CNS during early embryonic development. Once established, the microglia population is self-maintained throughout life by local proliferation. As a scalable source of microglia-like cells (MGLs), we here present a forward programming protocol for their generation from human pluripotent stem cells (hPSCs). The transient overexpression of PU.1 and C/EBPß in hPSCs led to a homogenous population of mature microglia within 16 d. MGLs met microglia characteristics on a morphological, transcriptional, and functional level. MGLs facilitated the investigation of a human tauopathy model in cortical neuron-microglia cocultures, revealing a secondary dystrophic microglia phenotype. Single-cell RNA sequencing of microglia integrated into hPSC-derived cortical brain organoids demonstrated a shift of microglia signatures toward a more-developmental in vivo-like phenotype, inducing intercellular interactions promoting neurogenesis and arborization. Taken together, our microglia forward programming platform represents a tool for both reductionist studies in monocultures and complex coculture systems, including 3D brain organoids for the study of cellular interactions in healthy or diseased environments.


Assuntos
Microglia , Células-Tronco Pluripotentes , Diferenciação Celular/genética , Sistema Nervoso Central , Humanos , Macrófagos , Neurônios
2.
Stem Cells ; 34(8): 2115-29, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-27068685

RESUMO

Adult neural stem cells with the ability to generate neurons and glia cells are active throughout life in both the dentate gyrus (DG) and the subventricular zone (SVZ). Differentiation of adult neural stem cells is induced by cell fate determinants like the transcription factor Prox1. Evidence has been provided for a function of Prox1 as an inducer of neuronal differentiation within the DG. We now show that within the SVZ Prox1 induces differentiation into oligodendrocytes. Moreover, we find that loss of Prox1 expression in vivo reduces cell migration into the corpus callosum, where the few Prox1 deficient SVZ-derived remaining cells fail to differentiate into oligodendrocytes. Thus, our work uncovers a novel function of Prox1 as a fate determinant for oligodendrocytes in the adult mammalian brain. These data indicate that the neurogenic and oligodendrogliogenic lineages in the two adult neurogenic niches exhibit a distinct requirement for Prox1, being important for neurogenesis in the DG but being indispensable for oligodendrogliogenesis in the SVZ. Stem Cells 2016;34:2115-2129.


Assuntos
Células-Tronco Adultas/citologia , Células-Tronco Adultas/metabolismo , Proteínas de Homeodomínio/metabolismo , Ventrículos Laterais/citologia , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Oligodendroglia/citologia , Proteínas Supressoras de Tumor/metabolismo , Animais , Padronização Corporal/genética , Diferenciação Celular/genética , Linhagem da Célula/genética , Movimento Celular/genética , Células Cultivadas , Elementos Facilitadores Genéticos/genética , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Camundongos , Neurogênese/genética , Bulbo Olfatório/citologia , Bulbo Olfatório/metabolismo , Fator de Transcrição 2 de Oligodendrócitos/genética , Fator de Transcrição 2 de Oligodendrócitos/metabolismo , Oligodendroglia/metabolismo , Regiões Promotoras Genéticas/genética , Ligação Proteica , Receptores Notch/genética , Receptores Notch/metabolismo
3.
Nucleic Acids Res ; 43(5): 2638-54, 2015 Mar 11.
Artigo em Inglês | MEDLINE | ID: mdl-25722370

RESUMO

In neural stem cells (NSCs), the balance between stem cell maintenance and neuronal differentiation depends on cell-fate determinants such as TRIM32. Previously, we have shown that TRIM32 associates with the RNA-induced silencing complex and increases the activity of microRNAs such as Let-7a. However, the exact mechanism of microRNA regulation by TRIM32 during neuronal differentiation has yet to be elucidated. Here, we used a mass spectrometry approach to identify novel protein-protein interaction partners of TRIM32 during neuronal differentiation. We found that TRIM32 associates with proteins involved in neurogenesis and RNA-related processes, such as the RNA helicase DDX6, which has been implicated in microRNA regulation. We demonstrate, that DDX6 colocalizes with TRIM32 in NSCs and neurons and that it increases the activity of Let-7a. Furthermore, we provide evidence that DDX6 is necessary and sufficient for neuronal differentiation and that it functions in cooperation with TRIM32.


Assuntos
Diferenciação Celular/genética , RNA Helicases DEAD-box/genética , MicroRNAs/genética , Células-Tronco Neurais/metabolismo , Proteínas Proto-Oncogênicas/genética , Ubiquitina-Proteína Ligases/genética , Animais , Proteínas Argonautas/genética , Proteínas Argonautas/metabolismo , Linhagem Celular Tumoral , Células Cultivadas , RNA Helicases DEAD-box/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Humanos , Immunoblotting , Camundongos , Camundongos Knockout , MicroRNAs/metabolismo , Microscopia de Fluorescência , Células NIH 3T3 , Neurogênese/genética , Ligação Proteica , Mapas de Interação de Proteínas/genética , Proteínas Proto-Oncogênicas/metabolismo , Interferência de RNA , Ubiquitina-Proteína Ligases/metabolismo
4.
Cell Mol Life Sci ; 72(4): 773-97, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25403878

RESUMO

Parkinson's disease (PD) is the second most common neurodegenerative disorder, leading to a variety of motor and non-motor symptoms. Interestingly, non-motor symptoms often appear a decade or more before the first signs of motor symptoms. Some of these non-motor symptoms are remarkably similar to those observed in cases of impaired neurogenesis and several PD-related genes have been shown to play a role in embryonic or adult neurogenesis. Indeed, animal models deficient in Nurr1, Pitx3, SNCA and PINK1 display deregulated embryonic neurogenesis and LRRK2 and VPS35 have been implicated in neuronal development-related processes such as Wnt/ß-catenin signaling and neurite outgrowth. Moreover, adult neurogenesis is affected in both PD patients and PD animal models and is regulated by dopamine and dopaminergic (DA) receptors, by chronic neuroinflammation, such as that observed in PD, and by differential expression of wild-type or mutant forms of PD-related genes. Indeed, an increasing number of in vivo studies demonstrate a role for SNCA and LRRK2 in adult neurogenesis and in the generation and maintenance of DA neurons. Finally, the roles of PD-related genes, SNCA, LRRK2, VPS35, Parkin, PINK1 and DJ-1 have been studied in NSCs, progenitor cells and induced pluripotent stem cells, demonstrating a role for some of these genes in stem/progenitor cell proliferation and maintenance. Together, these studies strongly suggest a link between deregulated neurogenesis and the onset and progression of PD and present strong evidence that, in addition to a neurodegenerative disorder, PD can also be regarded as a developmental disorder.


Assuntos
Células-Tronco Neurais/metabolismo , Doença de Parkinson/patologia , Animais , Modelos Animais de Doenças , Dopamina/farmacologia , Dopamina/uso terapêutico , Humanos , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina , MicroRNAs/metabolismo , Células-Tronco Neurais/citologia , Neurogênese/efeitos dos fármacos , Doença de Parkinson/tratamento farmacológico , Doença de Parkinson/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismo
5.
Biosens Bioelectron ; 228: 115223, 2023 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-36931193

RESUMO

Organoids are emerging in vitro models of human physiology. Neural models require the evaluation of functional activity of single cells and networks, which is commonly measured by microelectrode arrays. The characteristics of organoids clash with existing in vitro or in vivo microelectrode arrays. With inspiration from implantable mesh electronics and growth of organoids on polymer scaffolds, we fabricated suspended hammock-like mesh microelectrode arrays for neural organoids. We have demonstrated the growth of organoids enveloping these meshes and the culture of organoids on meshes for up to one year. Furthermore, we present proof-of-principle recordings of spontaneous electrical activity across the volume of an organoid. Our concept enables a new class of microelectrode arrays for in vitro models of three-dimensional electrically active tissue.


Assuntos
Técnicas Biossensoriais , Telas Cirúrgicas , Humanos , Microeletrodos , Organoides , Eletrofisiologia/métodos
6.
Cell Rep ; 37(3): 109864, 2021 10 19.
Artigo em Inglês | MEDLINE | ID: mdl-34686322

RESUMO

Increasing evidence suggests that neurodevelopmental alterations might contribute to increase the susceptibility to develop neurodegenerative diseases. We investigate the occurrence of developmental abnormalities in dopaminergic neurons in a model of Parkinson's disease (PD). We monitor the differentiation of human patient-specific neuroepithelial stem cells (NESCs) into dopaminergic neurons. Using high-throughput image analyses and single-cell RNA sequencing, we observe that the PD-associated LRRK2-G2019S mutation alters the initial phase of neuronal differentiation by accelerating cell-cycle exit with a concomitant increase in cell death. We identify the NESC-specific core regulatory circuit and a molecular mechanism underlying the observed phenotypes. The expression of NR2F1, a key transcription factor involved in neurogenesis, decreases in LRRK2-G2019S NESCs, neurons, and midbrain organoids compared to controls. We also observe accelerated dopaminergic differentiation in vivo in NR2F1-deficient mouse embryos. This suggests a pathogenic mechanism involving the LRRK2-G2019S mutation, where the dynamics of dopaminergic differentiation are modified via NR2F1.


Assuntos
Encéfalo/enzimologia , Fator I de Transcrição COUP/metabolismo , Neurônios Dopaminérgicos/enzimologia , Células-Tronco Pluripotentes Induzidas/enzimologia , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/metabolismo , Células-Tronco Neurais/enzimologia , Neurogênese , Doença de Parkinson/enzimologia , Animais , Encéfalo/patologia , Fator I de Transcrição COUP/genética , Ciclo Celular , Linhagem Celular , Proliferação de Células , Sobrevivência Celular , Neurônios Dopaminérgicos/patologia , Feminino , Humanos , Células-Tronco Pluripotentes Induzidas/patologia , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/genética , Masculino , Camundongos da Linhagem 129 , Camundongos Knockout , Mutação , Células-Tronco Neurais/patologia , Doença de Parkinson/genética , Doença de Parkinson/patologia , Fenótipo , RNA-Seq , Transdução de Sinais , Análise de Célula Única , Fatores de Tempo
7.
Stem Cell Reports ; 12(5): 878-889, 2019 05 14.
Artigo em Inglês | MEDLINE | ID: mdl-30982740

RESUMO

Emerging evidence suggests that Parkinson's disease (PD), besides being an age-associated disorder, might also have a neurodevelopment component. Disruption of mitochondrial homeostasis has been highlighted as a crucial cofactor in its etiology. Here, we show that PD patient-specific human neuroepithelial stem cells (NESCs), carrying the LRRK2-G2019S mutation, recapitulate key mitochondrial defects previously described only in differentiated dopaminergic neurons. By combining high-content imaging approaches, 3D image analysis, and functional mitochondrial readouts we show that LRRK2-G2019S mutation causes aberrations in mitochondrial morphology and functionality compared with isogenic controls. LRRK2-G2019S NESCs display an increased number of mitochondria compared with isogenic control lines. However, these mitochondria are more fragmented and exhibit decreased membrane potential. Functional alterations in LRRK2-G2019S cultures are also accompanied by a reduced mitophagic clearance via lysosomes. These findings support the hypothesis that preceding mitochondrial developmental defects contribute to the manifestation of the PD pathology later in life.


Assuntos
Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/genética , Mitocôndrias/genética , Mutação , Células-Tronco Neurais/metabolismo , Doença de Parkinson/genética , Idoso de 80 Anos ou mais , Diferenciação Celular/genética , Neurônios Dopaminérgicos/metabolismo , Feminino , Humanos , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/metabolismo , Masculino , Pessoa de Meia-Idade , Mitocôndrias/metabolismo , Doença de Parkinson/metabolismo , Doença de Parkinson/patologia
8.
Mol Neurobiol ; 55(4): 3490-3498, 2018 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-28508149

RESUMO

Parkinson's disease (PD) is the second most common neurodegenerative disorder. Accumulating evidences suggest that PD might have a strong neurodevelopmental component. Among the genetic cases, mutations in the leucine-rich repeat kinase 2 (LRRK2) are well known to be disease causing. Although the molecular mechanism of the pathogenic LRRK2 function is not fully clear, inhibition of microRNA (miRNA) activity has been suggested to be among the pathogenic LRRK2 targets. Here, we demonstrate that the miRNA activity inhibition function of pathogenic LRRK2 is directly antagonized by the neuronal cell fate determinant TRIM32. These findings suggest that TRIM32 might be a modifier for PD and could be a novel therapeutic target.


Assuntos
Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/genética , MicroRNAs/metabolismo , Mutação/genética , Doença de Parkinson/genética , Ubiquitina-Proteína Ligases/metabolismo , Animais , Proteínas Argonautas/metabolismo , Diferenciação Celular , Células HEK293 , Humanos , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/metabolismo , Camundongos Endogâmicos C57BL , MicroRNAs/genética , Neurônios/metabolismo , Neurônios/patologia , Ligação Proteica , Complexo de Inativação Induzido por RNA/metabolismo
9.
Sci Rep ; 5: 13456, 2015 Aug 26.
Artigo em Inglês | MEDLINE | ID: mdl-26307407

RESUMO

Induced pluripotent stem cells (iPSCs) have revolutionized the world of regenerative medicine; nevertheless, the exact molecular mechanisms underlying their generation and differentiation remain elusive. Here, we investigated the role of the cell fate determinant TRIM32 in modulating such processes. TRIM32 is essential for the induction of neuronal differentiation of neural stem cells by poly-ubiquitinating cMyc to target it for degradation resulting in inhibition of cell proliferation. To elucidate the role of TRIM32 in regulating somatic cell reprogramming we analysed the capacity of TRIM32-knock-out mouse embryonic fibroblasts (MEFs) in generating iPSC colonies. TRIM32 knock-out MEFs produced a higher number of iPSC colonies indicating a role for TRIM32 in inhibiting this cellular transition. Further characterization of the generated iPSCs indicated that the TRIM32 knock-out iPSCs show perturbed differentiation kinetics. Additionally, mathematical modelling of global gene expression data revealed that during differentiation an Oct4 centred network in the wild-type cells is replaced by an E2F1 centred network in the TRIM32 deficient cells. We show here that this might be caused by a TRIM32-dependent downregulation of Oct4. In summary, the data presented here reveal that TRIM32 directly regulates at least two of the four Yamanaka Factors (cMyc and Oct4), to modulate cell fate transitions.


Assuntos
Fibroblastos/citologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Fator 3 de Transcrição de Octâmero/metabolismo , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/fisiologia , Ubiquitina-Proteína Ligases/metabolismo , Animais , Diferenciação Celular/fisiologia , Células Cultivadas , Fibroblastos/fisiologia , Camundongos , Camundongos Endogâmicos C57BL
10.
J Biol Chem ; 284(32): 21139-56, 2009 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-19509292

RESUMO

The TP73 gene gives rise to transactivation domain-p73 isoforms (TAp73) as well as DeltaNp73 variants with a truncated N terminus. Although TAp73alpha and -beta proteins are capable of inducing cell cycle arrest, apoptosis, and differentiation, DeltaNp73 acts in many cell types as a dominant-negative repressor of p53 and TAp73. It has been proposed that p73 is involved in myeloid differentiation, and its altered expression is involved in leukemic degeneration. However, there is little evidence as to which p73 variants (TA or DeltaN) are expressed during differentiation and whether specific p73 isoforms have the capacity to induce, or hinder, this differentiation in leukemia cells. In this study we identify GATA1 as a direct transcriptional target of TAp73alpha. Furthermore, TAp73alpha induces GATA1 activity, and it is required for erythroid differentiation. Additionally, we describe a functional cooperation between TAp73 and DeltaNp73 in the context of erythroid differentiation in human myeloid cells, K562 and UT-7. Moreover, the impaired expression of GATA1 and other erythroid genes in the liver of p73KO embryos, together with the moderated anemia observed in p73KO young mice, suggests a physiological role for TP73 in erythropoiesis.


Assuntos
Proteínas de Ligação a DNA/fisiologia , Eritrócitos/metabolismo , Fator de Transcrição GATA1/fisiologia , Regulação Neoplásica da Expressão Gênica , Proteínas Nucleares/fisiologia , Proteínas Supressoras de Tumor/fisiologia , Animais , Apoptose , Diferenciação Celular , Proteínas de Ligação a DNA/biossíntese , Eritropoese , Fator de Transcrição GATA1/biossíntese , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Células K562 , Fígado/embriologia , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Nucleares/biossíntese , Proteína Tumoral p73 , Proteínas Supressoras de Tumor/biossíntese
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