RESUMO
Understanding the mechanisms that drive the differentiation of dopaminergic (DA) neurons is crucial for successful development of novel therapies for Parkinson's disease, in which DA neurons progressively degenerate. However, the mechanisms underlying the differentiation-promoting effects of Wnt5a on DA precursors are poorly understood. Here, we present the molecular and functional characterization of a signaling pathway downstream of Wnt5a, the Wnt/Dvl/Rac1 pathway. First, we characterize the interaction between Rac1 and Dvl and identify the N-terminal part of Dvl3 as necessary for Rac1 binding. Next, we show that Tiam1, a Rac1 guanosine exchange factor (GEF), is expressed in the ventral midbrain, interacts with Dvl, facilitates Dvl-Rac1 interaction, and is required for Dvl- or Wnt5a-induced activation of Rac1. Moreover, we show that Wnt5a promotes whereas casein kinase 1 (CK1), a negative regulator of the Wnt/Dvl/Rac1 pathway, abolishes the interactions between Dvl and Tiam1. Finally, using ventral midbrain neurosphere cultures, we demonstrate that the generation of DA neurons in culture is impaired after Tiam1 knockdown, indicating that Tiam1 is required for midbrain DA differentiation. In summary, our data identify Tiam1 as a novel regulator of DA neuron development and as a Dvl-associated and Rac1-specific GEF acting in the Wnt/Dvl/Rac1 pathway.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Neurônios Dopaminérgicos/citologia , Neurônios Dopaminérgicos/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Neuropeptídeos/metabolismo , Fosfoproteínas/metabolismo , Proteínas Wnt/metabolismo , Proteínas rac de Ligação ao GTP/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Caseína Quinase I/metabolismo , Diferenciação Celular , Proteínas Desgrenhadas , Fibroblastos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/imunologia , Humanos , Imunoprecipitação , Mesencéfalo/citologia , Mesencéfalo/embriologia , Mesencéfalo/metabolismo , Camundongos , Neuropeptídeos/genética , Fosfoproteínas/genética , Transdução de Sinais , Proteína 1 Indutora de Invasão e Metástase de Linfoma de Células T , Proteínas Wnt/genética , Via de Sinalização Wnt , Proteína Wnt-5a , Proteínas rac de Ligação ao GTP/genética , Proteínas rac1 de Ligação ao GTPRESUMO
Blood vessels and adjacent cells form perivascular stem cell niches in adult tissues. In this perivascular niche, a stem cell with mesenchymal characteristics was recently identified in some adult somatic tissues. These cells are pericytes that line the microvasculature, express mesenchymal markers and differentiate into mesodermal lineages but might even have the capacity to generate tissue-specific cell types. Here, we isolated, purified and characterized a previously unrecognized progenitor population from two different regions in the adult human brain, the ventricular wall and the neocortex. We show that these cells co-express markers for mesenchymal stem cells and pericytes in vivo and in vitro, but do not express glial, neuronal progenitor, hematopoietic, endothelial or microglial markers in their native state. Furthermore, we demonstrate at a clonal level that these progenitors have true multilineage potential towards both, the mesodermal and neuroectodermal phenotype. They can be epigenetically induced in vitro into adipocytes, chondroblasts and osteoblasts but also into glial cells and immature neurons. This progenitor population exhibits long-term proliferation, karyotype stability and retention of phenotype and multipotency following extensive propagation. Thus, we provide evidence that the vascular niche in the adult human brain harbors a novel progenitor with multilineage capacity that appears to represent mesenchymal stem cells and is different from any previously described human neural stem cell. Future studies will elucidate whether these cells may play a role for disease or may represent a reservoir that can be exploited in efforts to repair the diseased human brain.
Assuntos
Células-Tronco Adultas/citologia , Células-Tronco Mesenquimais/citologia , Células-Tronco Multipotentes/citologia , Actinas/biossíntese , Adulto , Diferenciação Celular , Linhagem da Célula , Ventrículos Cerebrais/citologia , Células Clonais , Humanos , Neocórtex/citologia , Pericitos/citologia , Receptor beta de Fator de Crescimento Derivado de Plaquetas/biossínteseRESUMO
BACKGROUND: Viral gene therapy of malignant brain tumors has been restricted by the limited vector distribution within the tumors. Multipotent mesenchymal stromal cells (MSCs) and other precursor cells have shown tropism for gliomas, and these cells are currently being explored as potential vehicles for gene delivery in glioma gene therapy. OBJECTIVE: To investigate MSC migration in detail after intratumoral and extratumoral implantation through syngeneic and orthotopic glioma models. METHODS: Adult rat bone marrow-derived MSCs were transduced to express enhanced green fluorescent protein and implanted either directly into or at a distance from rat gliomas. RESULTS: We found no evidence of long-distance MSC migration through the intact striatum toward syngeneic D74(RG2), N32, and N29 gliomas in the ipsilateral hemisphere or across the corpus callosum to gliomas located in the contralateral hemisphere. After intratumoral injection, MSCs migrated extensively, specifically within N32 gliomas. The MSCs did not proliferate within tumors, suggesting a low risk of malignant transformation of in vivo grafted cell vectors. Using a model for surgical glioma resection, we found that intratumorally grafted MSCs migrate efficiently within glioma remnants after partial surgical resection. CONCLUSION: The findings point to limitations for the use of MSCs as vectors in glioma gene therapy, although intratumoral MSC implantation provides a dense and tumor-specific vector distribution.
