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1.
Brain ; 142(11): 3382-3397, 2019 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-31637422

RESUMO

CTP:phosphoethanolamine cytidylyltransferase (ET), encoded by PCYT2, is the rate-limiting enzyme for phosphatidylethanolamine synthesis via the CDP-ethanolamine pathway. Phosphatidylethanolamine is one of the most abundant membrane lipids and is particularly enriched in the brain. We identified five individuals with biallelic PCYT2 variants clinically characterized by global developmental delay with regression, spastic para- or tetraparesis, epilepsy and progressive cerebral and cerebellar atrophy. Using patient fibroblasts we demonstrated that these variants are hypomorphic, result in altered but residual ET protein levels and concomitant reduced enzyme activity without affecting mRNA levels. The significantly better survival of hypomorphic CRISPR-Cas9 generated pcyt2 zebrafish knockout compared to a complete knockout, in conjunction with previously described data on the Pcyt2 mouse model, indicates that complete loss of ET function may be incompatible with life in vertebrates. Lipidomic analysis revealed profound lipid abnormalities in patient fibroblasts impacting both neutral etherlipid and etherphospholipid metabolism. Plasma lipidomics studies also identified changes in etherlipids that have the potential to be used as biomarkers for ET deficiency. In conclusion, our data establish PCYT2 as a disease gene for a new complex hereditary spastic paraplegia and confirm that etherlipid homeostasis is important for the development and function of the brain.

2.
Am J Hum Genet ; 101(6): 1021-1033, 2017 Dec 07.
Artigo em Inglês | MEDLINE | ID: mdl-29220674

RESUMO

ACTB encodes ß-actin, an abundant cytoskeletal housekeeping protein. In humans, postulated gain-of-function missense mutations cause Baraitser-Winter syndrome (BRWS), characterized by intellectual disability, cortical malformations, coloboma, sensorineural deafness, and typical facial features. To date, the consequences of loss-of-function ACTB mutations have not been proven conclusively. We describe heterozygous ACTB deletions and nonsense and frameshift mutations in 33 individuals with developmental delay, apparent intellectual disability, increased frequency of internal organ malformations (including those of the heart and the renal tract), growth retardation, and a recognizable facial gestalt (interrupted wavy eyebrows, dense eyelashes, wide nose, wide mouth, and a prominent chin) that is distinct from characteristics of individuals with BRWS. Strikingly, this spectrum overlaps with that of several chromatin-remodeling developmental disorders. In wild-type mouse embryos, ß-actin expression was prominent in the kidney, heart, and brain. ACTB mRNA expression levels in lymphoblastic lines and fibroblasts derived from affected individuals were decreased in comparison to those in control cells. Fibroblasts derived from an affected individual and ACTB siRNA knockdown in wild-type fibroblasts showed altered cell shape and migration, consistent with known roles of cytoplasmic ß-actin. We also demonstrate that ACTB haploinsufficiency leads to reduced cell proliferation, altered expression of cell-cycle genes, and decreased amounts of nuclear, but not cytoplasmic, ß-actin. In conclusion, we show that heterozygous loss-of-function ACTB mutations cause a distinct pleiotropic malformation syndrome with intellectual disability. Our biological studies suggest that a critically reduced amount of this protein alters cell shape, migration, proliferation, and gene expression to the detriment of brain, heart, and kidney development.


Assuntos
Anormalidades Múltiplas/genética , Actinas/genética , Deficiências do Desenvolvimento/genética , Haploinsuficiência/genética , Actinas/biossíntese , Adolescente , Adulto , Idoso , Animais , Ciclo Celular/genética , Proliferação de Células/genética , Criança , Pré-Escolar , Códon sem Sentido/genética , Coloboma/genética , Facies , Feminino , Mutação da Fase de Leitura/genética , Deleção de Genes , Humanos , Lactente , Recém-Nascido , Deficiência Intelectual/genética , Masculino , Malformações do Desenvolvimento Cortical/genética , Camundongos , Interferência de RNA , RNA Interferente Pequeno/genética , Adulto Jovem
3.
Oncotarget ; 8(39): 64974-64983, 2017 Sep 12.
Artigo em Inglês | MEDLINE | ID: mdl-29029405

RESUMO

B cell precursor acute lymphoblastic leukemia (BCP-ALL) is the most frequent type of cancer in children. Despite progresses in curative treatment, intensive chemotherapy regimens still cause life threatening complications. A better understanding of the molecular mechanisms underlying the emergence and maintenance of BCP-ALL is fundamental for the development of novel therapies. Here, we establish that SOX7 is frequently and specifically expressed in BCP-ALL and that the expression of this transcription factor does not correlate with any specific cytogenetic abnormalities. Using human leukemia model systems, we establish that the down-regulation of SOX7 in BCP-ALL causes a significant decrease in proliferation and clonogenicity in vitro that correlates with a delay in leukemia initiation and burden in vivo. Overall, these results identify a novel and important functional role for the transcription factor SOX7 in promoting the maintenance of BCP-ALL.

4.
FEBS Lett ; 590(22): 4116-4125, 2016 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-27404333

RESUMO

Well into the second decade of the 21st century, the field of regenerative medicine is bursting with hopes and promises to heal young and old. The bespoken generation of cells is thought to offer unprecedented cures for a vast range of diseases. Haematological disorders have already benefited tremendously from stem cell therapy in the form of bone marrow transplantation. However, lack of compatible donors often means that patients remain on transplantation waiting lists for too long. The in vitro derivation of haematopoietic stem cells offers the possibility to generate tailor-made cells for the treatment of these patients. Promising approaches to generate in vitro-derived blood progenitors include the directed differentiation of pluripotent stem cells and the reprogramming of somatic cells.


Assuntos
Transplante de Medula Óssea , Diferenciação Celular/genética , Células-Tronco Hematopoéticas/citologia , Células-Tronco/citologia , Animais , Reprogramação Celular/genética , Humanos , Camundongos , Medicina Regenerativa
5.
Open Biol ; 6(7)2016 07.
Artigo em Inglês | MEDLINE | ID: mdl-27411892

RESUMO

During embryogenesis, the three SOXF transcription factors, SOX7, SOX17 and SOX18, regulate the specification of the cardiovascular system and are also involved in the development of haematopoiesis. The ectopic expression of SOX17 in both embryonic and adult blood cells enhances self-renewal. Likewise, the enforced expression of SOX7 during embryonic development promotes the proliferation of early blood progenitors and blocks lineage commitment. However, whether SOX7 expression can also affect the self-renewal of adult blood progenitors has never been explored. In this study, we demonstrate using an inducible transgenic mouse model that the enforced expression of Sox7 ex vivo in bone marrow/stroma cell co-culture promotes the proliferation of blood progenitors which retain multi-lineage short-term engrafting capacity. Furthermore, SOX7 expression induces a profound block in the generation of B lymphocytes. Correspondingly, the ectopic expression of SOX7 in vivo results in dramatic alterations of the haematopoietic system, inducing the proliferation of blood progenitors in the bone marrow while blocking B lymphopoiesis. In addition, SOX7 expression induces extra-medullary haematopoiesis in the spleen and liver. Together, these data demonstrate that the uncontrolled expression of the transcription factor SOX7 in adult haematopoietic cells has dramatic consequences on blood homeostasis.


Assuntos
Linfócitos B/citologia , Células da Medula Óssea/citologia , Células-Tronco Mesenquimais/citologia , Fatores de Transcrição SOXF/genética , Fatores de Transcrição SOXF/metabolismo , Animais , Linfócitos B/metabolismo , Células da Medula Óssea/metabolismo , Diferenciação Celular , Linhagem da Célula , Proliferação de Células , Autorrenovação Celular , Técnicas de Cocultura , Feminino , Hematopoese , Homeostase , Células-Tronco Mesenquimais/metabolismo , Camundongos , Camundongos Transgênicos , Gravidez
6.
Stem Cell Reports ; 4(3): 431-44, 2015 Mar 10.
Artigo em Inglês | MEDLINE | ID: mdl-25660408

RESUMO

The generation of in vivo repopulating hematopoietic cells from in vitro differentiating embryonic stem cells has remained a long-standing challenge. To date, hematopoietic engraftment has mostly been achieved through the enforced expression of ectopic transcription factors. Here, we describe serum-free culture conditions that allow the generation of in vivo repopulating hematopoietic cells in the absence of ectopically expressed factors. We show that repopulating activity arises immediately upon the commitment of mesodermal precursors to the blood program, within the first wave of hematopoietic specification. We establish that the formation of these progenitors is extremely transient and exquisitely sensitive to the cytokine milieu. Our findings define the precise differentiating stage at which hematopoietic repopulating activity first appears in vitro, and suggest that during embryonic stem cell differentiation, all hematopoietic programs are unraveled simultaneously from the mesoderm in the absence of cues that restrict the coordinated emergence of each lineage as is normally observed during embryogenesis.


Assuntos
Diferenciação Celular , Células-Tronco Embrionárias/citologia , Hematopoese , Células-Tronco Hematopoéticas/citologia , Ativinas/genética , Ativinas/metabolismo , Animais , Proteína Morfogenética Óssea 4/genética , Proteína Morfogenética Óssea 4/metabolismo , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Citocinas/farmacologia , Células-Tronco Embrionárias/efeitos dos fármacos , Células-Tronco Embrionárias/metabolismo , Fatores de Crescimento de Fibroblastos , Hematopoese/efeitos dos fármacos , Hematopoese/genética , Células-Tronco Hematopoéticas/metabolismo , Imunofenotipagem , Masculino , Camundongos , Camundongos Transgênicos , Fenótipo , Glicoproteína IIb da Membrana de Plaquetas/metabolismo , Proteínas Proto-Oncogênicas c-kit/metabolismo , Transplante de Células-Tronco , Fatores de Crescimento do Endotélio Vascular/genética , Fatores de Crescimento do Endotélio Vascular/metabolismo
7.
J Bone Miner Res ; 27(6): 1322-34, 2012 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-22367914

RESUMO

The MET oncogene is aberrantly overexpressed in human osteosarcomas. We have previously converted primary cultures of human bone-derived cells into osteosarcoma cells by overexpressing MET. To determine whether MET transforms mesenchymal stem cells or committed progenitor cells, here we characterize distinct MET overexpressing osteosarcoma (MET-OS) clones using genome-wide expression profiling, cytometric analysis, and functional assays. All the MET-OS clones consistently display mesenchymal and stemness markers, but not most of the mesenchymal­stem cell-specific markers. Conversely, the MET-OS clones express genes characteristic of early osteoblastic differentiation phases, but not those of late phases. Profiling of mesenchymal stem cells induced to differentiate along osteoblast, adipocyte, and chondrocyte lineages confirms that MET-OS cells are similar to cells at an initial phase of osteoblastic differentiation. Accordingly, MET-OS cells cannot differentiate into adipocytes or chondrocytes, but can partially differentiate into osteogenic-matrix-producing cells. Moreover, in vitro MET-OS cells form self-renewing spheres enriched in cells that can initiate tumors in vivo. MET kinase inhibition abrogates the self-renewal capacity of MET-OS cells and allows them to progress toward osteoblastic differentiation. These data show that MET initiates the transformation of a cell population that has features of osteo-progenitors and suggest that MET regulates self-renewal and lineage differentiation of osteosarcoma cells.


Assuntos
Linhagem da Célula , Transformação Celular Neoplásica/patologia , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/patologia , Osteoblastos/patologia , Osteossarcoma/patologia , Proteínas Proto-Oncogênicas c-met/metabolismo , Biomarcadores/metabolismo , Diferenciação Celular/genética , Linhagem Celular Tumoral , Linhagem da Célula/genética , Proliferação de Células , Transformação Celular Neoplásica/genética , Células Cultivadas , Células Clonais , Análise por Conglomerados , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Análise de Sequência com Séries de Oligonucleotídeos , Osteoblastos/metabolismo , Osteossarcoma/genética , Fenótipo
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