RESUMO
The human genome expresses thousands of natural antisense transcripts (NAT) that can regulate epigenetic state, transcription, RNA stability or translation of their overlapping genes1,2. Here we describe MAPT-AS1, a brain-enriched NAT that is conserved in primates and contains an embedded mammalian-wide interspersed repeat (MIR), which represses tau translation by competing for ribosomal RNA pairing with the MAPT mRNA internal ribosome entry site3. MAPT encodes tau, a neuronal intrinsically disordered protein (IDP) that stabilizes axonal microtubules. Hyperphosphorylated, aggregation-prone tau forms the hallmark inclusions of tauopathies4. Mutations in MAPT cause familial frontotemporal dementia, and common variations forming the MAPT H1 haplotype are a significant risk factor in many tauopathies5 and Parkinson's disease. Notably, expression of MAPT-AS1 or minimal essential sequences from MAPT-AS1 (including MIR) reduces-whereas silencing MAPT-AS1 expression increases-neuronal tau levels, and correlate with tau pathology in human brain. Moreover, we identified many additional NATs with embedded MIRs (MIR-NATs), which are overrepresented at coding genes linked to neurodegeneration and/or encoding IDPs, and confirmed MIR-NAT-mediated translational control of one such gene, PLCG1. These results demonstrate a key role for MAPT-AS1 in tauopathies and reveal a potentially broad contribution of MIR-NATs to the tightly controlled translation of IDPs6, with particular relevance for proteostasis in neurodegeneration.
Assuntos
Biossíntese de Proteínas/genética , Proteostase/genética , RNA Antissenso/genética , Tauopatias/genética , Tauopatias/metabolismo , Proteínas tau/genética , Proteínas tau/metabolismo , Idoso , Animais , Sítios de Ligação , Encéfalo/metabolismo , Encéfalo/patologia , Estudos de Casos e Controles , Diferenciação Celular , Progressão da Doença , Feminino , Humanos , Sítios Internos de Entrada Ribossomal/genética , Masculino , Camundongos , Camundongos Transgênicos , Pessoa de Meia-Idade , Neurônios/metabolismo , Neurônios/patologia , Ribossomos/metabolismo , Proteínas tau/biossínteseRESUMO
Even though hematopoietic stem cells (HSC) are characterized by their ability to self-renew and differentiate, they primarily reside in quiescence. Despite the immense importance of this quiescent state, its maintenance and regulation is still incompletely understood. Schlafen2 (Slfn2) is a cytoplasmic protein known to be involved in cell proliferation, differentiation, quiescence, interferon response, and regulation of the immune system. Interestingly, Slfn2 is highly expressed in primitive hematopoietic cells. In order to investigate the role of Slfn2 in the regulation of HSC we have studied HSC function in the elektra mouse model, where the elektra allele of the Slfn2 gene contains a point mutation causing loss of function of the Slfn2 protein. We found that homozygosity for the elektra allele caused a decrease of primitive hematopoietic compartments in murine bone marrow. We further found that transplantation of elektra bone marrow and purified HSC resulted in a significantly reduced regenerative capacity of HSC in competitive transplantation settings. Importantly, we found that a significantly higher fraction of elektra HSC (as compared to wild-type HSC) were actively cycling, suggesting that the mutation in Slfn2 increases HSC proliferation. This additionally caused an increased amount of apoptotic stem and progenitor cells. Taken together, our findings demonstrate that dysregulation of Slfn2 results in a functional deficiency of primitive hematopoietic cells, which is particularly reflected by a drastically impaired ability to reconstitute the hematopoietic system following transplantation and an increase in HSC proliferation. This study thus identifies Slfn2 as a novel and critical regulator of adult HSC and HSC quiescence.
Assuntos
Proteínas de Ciclo Celular , Hematopoese , Células-Tronco Hematopoéticas , Animais , Camundongos , Medula Óssea , Diferenciação Celular/genética , Proliferação de Células , Células-Tronco Hematopoéticas/metabolismo , Proteínas de Ciclo Celular/genéticaRESUMO
Diamond-Blackfan anemia is a congenital erythroid hypoplasia and is associated with physical malformations and a predisposition to cancer. Twenty-five percent of patients with Diamond-Blackfan anemia have mutations in a gene encoding ribosomal protein S19 (RPS19). Through overexpression of RPS19 using a lentiviral vector with the spleen focus-forming virus promoter, we demonstrated that the Diamond-Blackfan anemia phenotype can be successfully treated in Rps19-deficient mice. In our present study, we assessed the efficacy of a clinically relevant promoter, the human elongation factor 1α short promoter, with or without the locus control region of the ß-globin gene for treatment of RPS19-deficient Diamond-Blackfan anemia. The findings demonstrate that these vectors rescue the proliferation defect and improve erythroid development of transduced RPS19-deficient bone marrow cells. Remarkably, bone marrow failure and severe anemia in Rps19-deficient mice was cured with enforced expression of RPS19 driven by the elongation factor 1α short promoter. We also demonstrate that RPS19-deficient bone marrow cells can be transduced and these cells have the capacity to repopulate bone marrow in long-term reconstituted mice. Our results collectively demonstrate the feasibility to cure RPS19-deficient Diamond-Blackfan anemia using lentiviral vectors with cellular promoters that possess a reduced risk of insertional mutagenesis.
Assuntos
Anemia de Diamond-Blackfan/genética , Medula Óssea/metabolismo , Medula Óssea/patologia , Vetores Genéticos/genética , Lentivirus/genética , Regiões Promotoras Genéticas , Anemia de Diamond-Blackfan/diagnóstico , Anemia de Diamond-Blackfan/terapia , Animais , Células da Medula Óssea/citologia , Células da Medula Óssea/metabolismo , Transplante de Medula Óssea , Diferenciação Celular/genética , Proliferação de Células , Modelos Animais de Doenças , Expressão Gênica , Ordem dos Genes , Terapia Genética , Sobrevivência de Enxerto/genética , Hematopoese/genética , Humanos , Camundongos , Fenótipo , Interferência de RNA , RNA Interferente Pequeno/genética , Proteínas Ribossômicas/genética , Transdução Genética , Transgenes , Integração ViralRESUMO
Diamond-Blackfan anaemia (DBA) is a rare congenital disease causing severe anaemia and progressive bone marrow failure. The majority of patients carry mutations in ribosomal proteins, which leads to depletion of erythroid progenitors in the bone marrow. As many as 40% of all DBA patients receive glucocorticoids to alleviate their anaemia. However, despite their use in DBA treatment for more than half a century, the therapeutic mechanisms of glucocorticoids remain largely unknown. Therefore we sought to study disease specific effects of glucocorticoid treatment using a ribosomal protein s19 (Rps19) deficient mouse model of DBA. This study determines for the first time that a mouse model of DBA can respond to glucocorticoid treatment, similar to DBA patients. Our results demonstrate that glucocorticoid treatment reduces apoptosis, rescues erythroid progenitor depletion and premature differentiation of erythroid cells. Furthermore, glucocorticoids prevent Trp53 activation in Rps19-deficient cells- in a disease-specific manner. Dissecting the therapeutic mechanisms behind glucocorticoid treatment of DBA provides indispensible insight into DBA pathogenesis. Identifying mechanisms important for DBA treatment also enables development of more disease-specific treatments of DBA.
Assuntos
Anemia de Diamond-Blackfan/tratamento farmacológico , Eritropoese/efeitos dos fármacos , Prednisolona/uso terapêutico , Proteínas Ribossômicas/deficiência , Proteína Supressora de Tumor p53/fisiologia , Adolescente , Anemia de Diamond-Blackfan/sangue , Animais , Apoptose/efeitos dos fármacos , Células Cultivadas , Inibidor de Quinase Dependente de Ciclina p21/biossíntese , Inibidor de Quinase Dependente de Ciclina p21/genética , Dexametasona/farmacologia , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Células Precursoras Eritroides/efeitos dos fármacos , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Prednisolona/farmacologia , Quimera por Radiação , Proteínas Ribossômicas/genética , Fatores de Transcrição/biossíntese , Fatores de Transcrição/genética , Regulação para Cima/efeitos dos fármacos , Proteína X Associada a bcl-2/biossíntese , Proteína X Associada a bcl-2/genéticaRESUMO
Nucleic acid therapeutics have demonstrated an impressive acceleration in recent years. They work through multiple mechanisms of action, including the downregulation of gene expression and the modulation of RNA splicing. While several drugs based on the former mechanism have been approved, few target the latter, despite the promise of RNA splicing modulation. To improve our ability to discover novel RNA splicing-modulating therapies, we developed HCS-Splice, a robust cell-based High-Content Screening (HCS) assay. By implementing the use of a two-colour (GFP/RFP) fluorescent splicing reporter plasmid, we developed a versatile, effective, rapid, and robust high-throughput strategy for the identification of potent splicing-modulating molecules. The HCS-Splice strategy can also be used to functionally confirm splicing mutations in human genetic disorders or to screen drug candidates. As a proof-of-concept, we introduced a dementia-related splice-switching mutation in the Microtubule-Associated Protein Tau (MAPT) exon 10 splicing reporter. We applied HCS-Splice to the wild-type and mutant reporters and measured the functional change in exon 10 inclusion. To demonstrate the applicability of the method in cell-based drug discovery, HCS-Splice was used to evaluate the efficacy of an exon 10-targeting siRNA, which was able to restore the correct alternative splicing balance.
Assuntos
Processamento Alternativo , Splicing de RNA , Humanos , Splicing de RNA/genética , Processamento Alternativo/genética , Mutação/genéticaRESUMO
Diamond-Blackfan anemia (DBA) is a rare genetic disorder in which patients present a scarcity of erythroid precursors in an otherwise normocellular bone marrow. Most, but not all, patients carry mutations in ribosomal proteins such as RPS19, suggesting that compromised mRNA translation and ribosomal stress are pathogenic mechanisms causing depletion of erythroid precursors. To gain further insight to disease mechanisms in DBA, we performed a custom short hairpin RNA (shRNA) based screen against 750 genes hypothesized to affect DBA pathophysiology. Among the hits were two shRNAs against the erythroid specific heme-regulated eIF2α kinase (HRI), which is a negative regulator of mRNA translation. This study shows that shRNA-mediated HRI silencing or loss of one HRI allele improves expansion of Rps19-deficient erythroid precursors, as well as improves the anemic phenotype in Rps19-deficient animals. We found that Rps19-deficient erythroblasts have elevated levels of unbound intracellular heme, which is normalized by HRI heterozygosity. Additionally, targeting elevated heme levels by treating cells with the heme scavenger alpha-1-microglobulin (A1M), increased proliferation of Rps19-deficient erythroid precursors and decreased heme levels in a disease-specific manner. HRI heterozygosity, but not A1M treatment, also decreased the elevated p53 activity observed in Rps19-deficient cells, indicating that p53 activation is caused by ribosomal stress and aberrant mRNA translation and not heme overload in Rps19-deficiency. Together, these findings suggest that targeting elevated heme levels is a promising new treatment strategy for DBA.
Assuntos
alfa-Globulinas/uso terapêutico , Anemia de Diamond-Blackfan/terapia , Heme/análise , Anemia de Diamond-Blackfan/sangue , Anemia de Diamond-Blackfan/genética , Animais , Células Cultivadas , Modelos Animais de Doenças , Feminino , Deleção de Genes , Inativação Gênica , Terapia Genética , Heme/genética , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Serina-Treonina Quinases/genética , Proteínas Recombinantes/uso terapêutico , Proteínas Ribossômicas/genéticaRESUMO
Diamond-Blackfan anemia (DBA) is a congenital erythroid hypoplasia caused by a functional haploinsufficiency of genes coding for ribosomal proteins. Among these genes, the ribosomal protein S19 (RPS19) gene is the most frequently mutated. Previously, a mouse model deficient in RPS19 was developed by our laboratory, which recapitulates the hematopoietic disease phenotype by manifesting pathologic features and clinical symptoms of DBA. Characterization of this model revealed that chronic RPS19 deficiency leads to exhaustion of hematopoietic stem cells and subsequent bone marrow (BM) failure. In this study, we evaluated a nonmyeloablative conditioning protocol for BM transplants in RPS19-deficient mice by transplanting wild-type BM cells to RPS19-deficient recipients given no conditioning or sublethal doses of irradiation before transplant. We describe full correction of the hematopoietic phenotype in mice given sublethal doses of irradiation, as well as in animals completely devoid of any preceding irradiation. In comparison, wild-type animals receiving the same preconditioning regimen and number of transplanted cells exhibited significantly lower engraftment levels. Thus, robust engraftment and repopulation of transplanted cells can be achieved in reduced-intensity conditioned RPS19-deficient recipients. As gene therapy studies with autologous gene-corrected hematopoietic stem cells are emerging, we propose the results described here can guide determination of the level of conditioning for such a protocol in RPS19-deficient DBA. On the basis of our findings, a relatively mild conditioning strategy would plausibly be sufficient to achieve sufficient levels of engraftment and clinical success.
Assuntos
Anemia de Diamond-Blackfan/metabolismo , Transplante de Medula Óssea , Aloenxertos , Anemia de Diamond-Blackfan/genética , Anemia de Diamond-Blackfan/patologia , Animais , Modelos Animais de Doenças , Camundongos , Camundongos Knockout , Proteínas Ribossômicas/deficiência , Proteínas Ribossômicas/metabolismo , Condicionamento Pré-TransplanteRESUMO
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMO
The fate options of hematopoietic stem cells (HSCs) include self-renewal, differentiation, migration, and apoptosis. HSCs self-renewal divisions in stem cells are required for rapid regeneration during tissue damage and stress, but how precisely intracellular calcium signals are regulated to maintain fate options in normal hematopoiesis is unclear. S100A6 knockout (KO) HSCs have reduced total cell numbers in the HSC compartment, decreased myeloid output, and increased apoptotic HSC numbers in steady state. S100A6KO HSCs had impaired self-renewal and regenerative capacity, not responding to 5-Fluorouracil. Our transcriptomic and proteomic profiling suggested that S100A6 is a critical HSC regulator. Intriguingly, S100A6KO HSCs showed decreased levels of phosphorylated Akt (p-Akt) and Hsp90, with an impairment of mitochondrial respiratory capacity and a reduction of mitochondrial calcium levels. We showed that S100A6 regulates intracellular and mitochondria calcium buffering of HSC upon cytokine stimulation and have demonstrated that Akt activator SC79 reverts the levels of intracellular and mitochondrial calcium in HSC. Hematopoietic colony-forming activity and the Hsp90 activity of S100A6KO are restored through activation of the Akt pathway. We show that p-Akt is the prime downstream mechanism of S100A6 in the regulation of HSC self-renewal by specifically governing mitochondrial metabolic function and Hsp90 protein quality.
RESUMO
Diamond-Blackfan anemia (DBA) results from haploinsufficiency of ribosomal protein subunits in hematopoietic progenitors in the earliest stages of committed erythropoiesis. Nemo-like kinase (NLK) is chronically hyperactivated in committed erythroid progenitors and precursors in multiple human and murine models of DBA. Inhibition of NLK activity and suppression of NLK expression both improve erythroid expansion in these models. Metformin is a well-tolerated drug for type 2 diabetes with multiple cellular targets. Here we demonstrate that metformin improves erythropoiesis in human and zebrafish models of DBA. Our data indicate that the effects of metformin on erythroid proliferation and differentiation are mediated by suppression of NLK expression through induction of miR-26a, which recognizes a binding site within the NLK 3' untranslated region (3'UTR) to facilitate transcript degradation. We propose that induction of miR-26a is a potentially novel approach to treatment of DBA and could improve anemia in DBA patients without the potentially adverse side effects of metformin in a DBA patient population.
Assuntos
Anemia de Diamond-Blackfan/tratamento farmacológico , Eritropoese/efeitos dos fármacos , Hematínicos/uso terapêutico , Metformina/uso terapêutico , MicroRNAs/biossíntese , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Regiões 3' não Traduzidas/genética , Anemia de Diamond-Blackfan/genética , Animais , Células Cultivadas , Ensaio de Unidades Formadoras de Colônias , Modelos Animais de Doenças , Eritropoese/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Genes Reporter , Hematínicos/farmacologia , Humanos , Metformina/farmacologia , MicroRNAs/genética , Estabilidade de RNA , RNA Interferente Pequeno/farmacologia , Proteínas Recombinantes/metabolismo , Especificidade da Espécie , Regulação para Cima/efeitos dos fármacos , Peixe-ZebraRESUMO
Diamond-Blackfan anemia (DBA) is a rare hematopoietic disease characterized by a block in red cell differentiation. Most DBA cases are caused by mutations in ribosomal proteins and characterized by higher than normal activity of the tumor suppressor p53. Higher p53 activity is thought to contribute to DBA phenotypes by inducing apoptosis during red blood cell differentiation. Currently, there are few therapies available for patients with DBA. We performed a chemical screen using zebrafish ribosomal small subunit protein 29 (rps29) mutant embryos that have a p53-dependent anemia and identified calmodulin inhibitors that rescued the phenotype. Our studies demonstrated that calmodulin inhibitors attenuated p53 protein amount and activity. Treatment with calmodulin inhibitors led to decreased p53 translation and accumulation but does not affect p53 stability. A U.S. Food and Drug Administration-approved calmodulin inhibitor, trifluoperazine, rescued hematopoietic phenotypes of DBA models in vivo in zebrafish and mouse models. In addition, trifluoperazine rescued these phenotypes in human CD34+ hematopoietic stem and progenitor cells. Erythroid differentiation was also improved in CD34+ cells isolated from a patient with DBA. This work uncovers a potential avenue of therapeutic development for patients with DBA.
Assuntos
Anemia de Diamond-Blackfan , Anemia de Diamond-Blackfan/tratamento farmacológico , Animais , Apoptose , Calmodulina , Eritropoese , Humanos , Proteína Supressora de Tumor p53 , Peixe-ZebraRESUMO
Diamond-Blackfan anemia (DBA) is a bone marrow failure syndrome caused by mutations in ribosomal protein genes. Pathogenic mechanisms are poorly understood but involve severely reduced proliferation of erythroid precursors. Because current DBA therapies are ineffective and associated with severe side effects, disease-specific therapies are urgently needed. We hypothesized that druggable molecular pathways underlying the defect can be revealed through phenotypic small-molecule screens. Accordingly, a screening assay was developed using c-kit+ fetal liver erythroid progenitors from a doxycycline-inducible DBA mouse model. The addition of doxycycline to the culture medium induces the phenotype and reduces proliferation to <10% of normal, such that rescue of proliferation can be used as a simple readout for screening. Here, we describe the assay rationale and efforts toward validation of a microtiter plate-compatible assay and its application in a pilot screen of 3871 annotated compounds. Ten hits demonstrated concentration-dependent activity, and we report a brief follow-up of one of these compounds. In conclusion, we established a robust scalable assay for screening molecules that rescue erythropoiesis in DBA.
Assuntos
Anemia de Diamond-Blackfan/tratamento farmacológico , Fenótipo , Anemia de Diamond-Blackfan/patologia , Animais , Transplante de Medula Óssea , Proliferação de Células/efeitos dos fármacos , Modelos Animais de Doenças , Doxiciclina/farmacologia , Doxiciclina/uso terapêutico , Ensaios de Triagem em Larga Escala , Humanos , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Embryonic stem (ES) cells are an invaluable model for identifying subtle phenotypes as well as severe outcomes of perturbing gene function that may otherwise result in lethality. However,though ES cells of different origins are regarded as equally pluripotent,their in vitro differentiation potential varies, suggesting that their response to developmental signals is different. The R1 cell line is widely used for gene manipulation due to its good growth characteristics and highly efficient germline transmission. Hence, we analysed the expression of Notch, Wnt and Sonic Hedgehog (Shh) pathway genes during differentiation of R1 cells into early vascular lineages. Notch-, Wnt- and Shh-mediated signalling is important during embryonic development. Regulation of gene expression through these signalling molecules is a frequently used theme, resulting in context-dependent outcomes during development. Perturbing these pathways can result in severe and possibly lethal developmental phenotypes often due to primary cardiovascular defects. We report that during early spontaneous differentiation of R1 cells, Notch-1 and the Wnt target Brachyury are active whereas the Shh receptor is not detected. This expression pattern is similar to that seen in a mouse endothelial cell line. This temporal study of expression of genes representative of all three pathways in ES cell differentiation will aid in further analysis of cell signalling during vascular development.
Assuntos
Vasos Sanguíneos/metabolismo , Diferenciação Celular , Sequência Conservada/genética , Células-Tronco Embrionárias/citologia , Células Endoteliais/citologia , Regulação da Expressão Gênica , Transdução de Sinais/genética , Animais , Vasos Sanguíneos/citologia , Linhagem Celular , Células-Tronco Embrionárias/metabolismo , Células Endoteliais/metabolismo , Camundongos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptor Notch1/metabolismo , beta Catenina/metabolismoRESUMO
Pigment epithelium derived factor (PEDF), a ubiquitously expressed 50 kDa secreted glycoprotein, was recently discovered to regulate self-renewal of neural stem cells and have a supportive effect on human embryonic stem cell growth. Here, we analyzed expression of PEDF in the murine hematopoietic stem cell (HSC) compartments and found that PEDF is highly expressed in primary long-term HSCs. Therefore, we characterized the hematopoietic system in a knockout mouse model for PEDF and using this model we surprisingly found that PEDF is dispensable for HSC regulation. PEDF knockout mice exhibit normal hematopoiesis in steady state conditions and the absence of PEDF lead to normal regeneration capacity in a serial competitive transplantation setting. Additionally, PEDF-deficient cells exhibit unaltered lineage distribution upon serial transplantations. When human cord blood stem and progenitor cells were cultured in media supplemented with recombinant PEDF they did not show changes in growth potential. Taken together, we report that PEDF is not a critical regulatory factor for HSC function during regeneration in vivo or growth of human stem/progenitor cells in vitro.
Assuntos
Proteínas do Olho/metabolismo , Hematopoese , Células-Tronco Hematopoéticas/metabolismo , Fatores de Crescimento Neural/metabolismo , Serpinas/metabolismo , Animais , Células Cultivadas , Proteínas do Olho/genética , Células-Tronco Hematopoéticas/citologia , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Fatores de Crescimento Neural/genética , Serpinas/genéticaRESUMO
We describe a novel murine gene rudhira that is expressed at high levels in embryonic stem cells and is restricted to blood islands and the erythroid lineage during embryonic development. Rudhira is expressed in angiogenic precursors but is excluded from the differentiated endothelium. Rudhira-expressing cells are seen in close proximity to endothelial cells in angiogenic blood vessels. Rudhira encodes a predicted cytoplasmic WD40 protein that is 98% identical to human BCAS3. The gene encoding BCAS3 maps to a breakpoint of hematological neoplasms on human chromosome 17q23, but its expression and function remain to be determined. We demonstrate that mouse Rudhira is a novel marker for analysis of the erythroid lineage.
Assuntos
Eritropoese/genética , Proteínas/genética , Células-Tronco/metabolismo , Processamento Alternativo , Sequência de Aminoácidos , Animais , Sequência de Bases , Linhagem Celular , Linhagem Celular Tumoral , Desenvolvimento Embrionário/genética , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Camundongos , Dados de Sequência Molecular , Proteínas de Neoplasias/genética , Proteínas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Homologia de Sequência de Aminoácidos , Especificidade da EspécieRESUMO
During development, hematopoietic stem cells (HSCs) undergo a rapid expansion in the fetal liver (FL) before settling in the adult bone marrow. We recently reported that proliferating adult HSCs are vulnerable to ER stress caused by accumulation of mis-folded proteins. Here, we find that FL-HSCs, despite an increased protein synthesis rate and a requirement for protein folding, do not upregulate ER chaperones. Instead, bile acids (BAs), secreted from maternal and fetal liver, coordinate to serve as chemical chaperones. Taurocholic acid, the major BA in FL, supports growth of HSCs in vitro by inhibiting protein aggregation. In vivo, reducing BA levels leads to ER stress elevation and accumulation of aggregated proteins and significantly decreases the number of FL-HSCs. Taken together, these findings reveal that BA alleviation of ER stress is a mechanism required for HSC expansion during fetal hematopoiesis.
Assuntos
Ácidos e Sais Biliares/farmacologia , Retículo Endoplasmático/efeitos dos fármacos , Células-Tronco Hematopoéticas/efeitos dos fármacos , Fígado/efeitos dos fármacos , Prenhez , Resposta a Proteínas não Dobradas/efeitos dos fármacos , Animais , Retículo Endoplasmático/metabolismo , Feminino , Células-Tronco Hematopoéticas/metabolismo , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Estresse Oxidativo/efeitos dos fármacos , Gravidez , Agregados Proteicos/efeitos dos fármacosRESUMO
Erythroid cell commitment and differentiation proceed through activation of a lineage-restricted transcriptional network orchestrated by a group of well characterized genes. However, the minimal set of factors necessary for instructing red blood cell (RBC) development remains undefined. We employed a screen for transcription factors allowing direct lineage reprograming from fibroblasts to induced erythroid progenitors/precursors (iEPs). We show that Gata1, Tal1, Lmo2, and c-Myc (GTLM) can rapidly convert murine and human fibroblasts directly to iEPs. The transcriptional signature of murine iEPs resembled mainly that of primitive erythroid progenitors in the yolk sac, whereas addition of Klf1 or Myb to the GTLM cocktail resulted in iEPs with a more adult-type globin expression pattern. Our results demonstrate that direct lineage conversion is a suitable platform for defining and studying the core factors inducing the different waves of erythroid development.
Assuntos
Linhagem da Célula , Eritropoese , Fatores de Transcrição/metabolismo , Envelhecimento , Animais , Diferenciação Celular/genética , Linhagem da Célula/genética , Reprogramação Celular/genética , Ensaio de Unidades Formadoras de Colônias , Eritroblastos/citologia , Eritroblastos/metabolismo , Eritropoese/genética , Fibroblastos/citologia , Fibroblastos/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Globinas/genética , Globinas/metabolismo , Humanos , Camundongos Endogâmicos C57BLRESUMO
Frontotemporal dementia (FTD) is a neurodegenerative disorder characterized by degeneration of the fronto temporal lobes and abnormal protein inclusions. It exhibits a broad clinicopathological spectrum and has been linked to mutations in seven different genes. We will provide a picture, which connects the products of these genes, albeit diverse in nature and function, in a network. Despite the paucity of information available for some of these genes, we believe that RNA processing and post-transcriptional regulation of gene expression might constitute a common theme in the network. Recent studies have unraveled the role of mutations affecting the functions of RNA binding proteins and regulation of microRNAs. This review will combine all the recent findings on genes involved in the pathogenesis of FTD, highlighting the importance of a common network of interactions in order to study and decipher the heterogeneous clinical manifestations associated with FTD. This approach could be helpful for the research of potential therapeutic strategies.
RESUMO
Alternative splicing is an important regulator of the transcriptome. However, mutations may cause alteration of splicing patterns, which in turn leads to disease. During the past 10 years, exon skipping has been looked upon as a powerful tool for correction of missplicing in disease and progress has been made towards clinical trials. In this review, we discuss the use of antisense oligonucleotides to correct splicing defects through exon skipping, with a special focus on diseases affecting the nervous system, and the latest stage achieved in its progress.
Assuntos
Doenças Genéticas Inatas/genética , Doenças Genéticas Inatas/terapia , Doenças do Sistema Nervoso/genética , Doenças do Sistema Nervoso/terapia , Oligonucleotídeos Antissenso/genética , Oligonucleotídeos Antissenso/uso terapêutico , Reparo Gênico Alvo-Dirigido/métodos , Animais , Ataxia Telangiectasia/genética , Ataxia Telangiectasia/terapia , Barreira Hematoencefálica , Defeitos Congênitos da Glicosilação/genética , Defeitos Congênitos da Glicosilação/terapia , Cistos/genética , Cistos/terapia , Sistemas de Liberação de Medicamentos , Éxons , Demência Frontotemporal/genética , Demência Frontotemporal/terapia , Doenças Desmielinizantes Hereditárias do Sistema Nervoso Central/genética , Doenças Desmielinizantes Hereditárias do Sistema Nervoso Central/terapia , Humanos , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/terapia , Mutação , Neurofibromatoses/genética , Neurofibromatoses/terapia , Doença de Niemann-Pick Tipo C/genética , Doença de Niemann-Pick Tipo C/terapia , Oligonucleotídeos Antissenso/química , Doença de Pelizaeus-Merzbacher/genética , Doença de Pelizaeus-Merzbacher/terapia , Fosfotransferases (Fosfomutases)/deficiência , Fosfotransferases (Fosfomutases)/genética , Splicing de RNARESUMO
A wide variety of mammalian cell types is used in gene transfection studies. Establishing transfection methods that enable highly efficient DNA uptake has become increasingly important. PC12 is an established rat pheochromocytoma cell line, which responds to exposure to NGF with cessation of growth, expression of cytoplasmic processes, and differentiation into cells resembling sympathetic neurons. Although PC12 cells represent an important model system to study a variety of neuronal functions, they proved relatively difficult to transfect. We have compared the efficiency of three different chemical transfection reagents (Lipofectamine 2000, Lipofectamine LTX and TransIT-LT1) and of two electroporation systems (Neon and Gene Pulser Xcell) in transiently transfecting undifferentiated PC12 cells. By comparing efficiencies from replicate experiments we proved electroporation (in particular Neon) to be the method of choice. By optimizing different parameters (voltage, pulse width and number of pulses) we reached high efficiency of transfection (90 %) and viability (99 %). We also demonstrated that, upon electroporation, cells are not altered by the transfection and maintain their ability to differentiate.