RESUMO
X chromosome inactivation (XCI) in female placental mammals is a vital mechanism for dosage compensation between X-linked and autosomal genes. XCI starts with activation of Xist and silencing of the negative regulator Tsix, followed by cis spreading of Xist RNA over the future inactive X chromosome (Xi). Here, we show that XCI does not require physical contact between the two X chromosomes (X-pairing) but is regulated by trans-acting diffusible factors. We found that the X-encoded trans-acting and dose-dependent XCI-activator RNF12 acts in concert with the cis-regulatory region containing Jpx, Ftx, and Xpr to activate Xist and to overcome repression by Tsix. RNF12 acts at two subsequent steps; two active copies of Rnf12 drive initiation of XCI, and one copy needs to remain active to maintain XCI toward establishment of the Xi. This two-step mechanism ensures that XCI is very robust and fine-tuned, preventing XCI of both X chromosomes.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , RNA Longo não Codificante/genética , Ubiquitina-Proteína Ligases/genética , Inativação do Cromossomo X , Cromossomo X , Animais , Transporte Biológico , Linhagem Celular , Pareamento Cromossômico , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Feminino , Humanos , Camundongos Knockout , RNA Longo não Codificante/metabolismo , Transdução de Sinais , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Developmental and/or epileptic encephalopathies (DEEs) are a group of devastating genetic disorders, resulting in early-onset, therapy-resistant seizures and developmental delay. Here we report on 22 individuals from 15 families presenting with a severe form of intractable epilepsy, severe developmental delay, progressive microcephaly, visual disturbance and similar minor dysmorphisms. Whole exome sequencing identified a recurrent, homozygous variant (chr2:64083454A > G) in the essential UDP-glucose pyrophosphorylase (UGP2) gene in all probands. This rare variant results in a tolerable Met12Val missense change of the longer UGP2 protein isoform but causes a disruption of the start codon of the shorter isoform, which is predominant in brain. We show that the absence of the shorter isoform leads to a reduction of functional UGP2 enzyme in neural stem cells, leading to altered glycogen metabolism, upregulated unfolded protein response and premature neuronal differentiation, as modeled during pluripotent stem cell differentiation in vitro. In contrast, the complete lack of all UGP2 isoforms leads to differentiation defects in multiple lineages in human cells. Reduced expression of Ugp2a/Ugp2b in vivo in zebrafish mimics visual disturbance and mutant animals show a behavioral phenotype. Our study identifies a recurrent start codon mutation in UGP2 as a cause of a novel autosomal recessive DEE syndrome. Importantly, it also shows that isoform-specific start-loss mutations causing expression loss of a tissue-relevant isoform of an essential protein can cause a genetic disease, even when an organism-wide protein absence is incompatible with life. We provide additional examples where a similar disease mechanism applies.
Assuntos
Encefalopatias/genética , Síndromes Epilépticas/genética , Genes Essenciais/genética , UTP-Glucose-1-Fosfato Uridililtransferase/genética , Animais , Pré-Escolar , Feminino , Humanos , Lactente , Masculino , Mutação , Linhagem , Peixe-ZebraRESUMO
Schizophrenia is highly heritable, yet its underlying pathophysiology remains largely unknown. Among the most well-replicated findings in neurobiological studies of schizophrenia are deficits in myelination and white matter integrity; however, direct etiological genetic and cellular evidence has thus far been lacking. Here, we implement a family-based approach for genetic discovery in schizophrenia combined with functional analysis using induced pluripotent stem cells (iPSCs). We observed familial segregation of two rare missense mutations in Chondroitin Sulfate Proteoglycan 4 (CSPG4) (c.391G > A [p.A131T], MAF 7.79 × 10-5 and c.2702T > G [p.V901G], MAF 2.51 × 10-3). The CSPG4A131T mutation was absent from the Swedish Schizophrenia Exome Sequencing Study (2536 cases, 2543 controls), while the CSPG4V901G mutation was nominally enriched in cases (11 cases vs. 3 controls, P = 0.026, OR 3.77, 95% CI 1.05-13.52). CSPG4/NG2 is a hallmark protein of oligodendrocyte progenitor cells (OPCs). iPSC-derived OPCs from CSPG4A131T mutation carriers exhibited abnormal post-translational processing (P = 0.029), subcellular localization of mutant NG2 (P = 0.007), as well as aberrant cellular morphology (P = 3.0 × 10-8), viability (P = 8.9 × 10-7), and myelination potential (P = 0.038). Moreover, transfection of healthy non-carrier sibling OPCs confirmed a pathogenic effect on cell survival of both the CSPG4A131T (P = 0.006) and CSPG4V901G (P = 3.4 × 10-4) mutations. Finally, in vivo diffusion tensor imaging of CSPG4A131T mutation carriers demonstrated a reduction of brain white matter integrity compared to unaffected sibling and matched general population controls (P = 2.2 × 10-5). Together, our findings provide a convergence of genetic and functional evidence to implicate OPC dysfunction as a candidate pathophysiological mechanism of familial schizophrenia.
Assuntos
Proteoglicanas de Sulfatos de Condroitina/genética , Proteínas de Membrana/genética , Células Precursoras de Oligodendrócitos/metabolismo , Esquizofrenia/genética , Adulto , Antígenos/genética , Diferenciação Celular/fisiologia , Proteoglicanas de Sulfatos de Condroitina/metabolismo , Imagem de Tensor de Difusão , Família , Feminino , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Masculino , Proteínas de Membrana/metabolismo , Mutação/genética , Células Precursoras de Oligodendrócitos/fisiologia , Oligodendroglia/metabolismo , Linhagem , Proteoglicanas/genética , Esquizofrenia/metabolismo , Substância Branca/metabolismoRESUMO
The rare recessive developmental disorder Trichothiodystrophy (TTD) is characterized by brittle hair and nails. Patients also present a variable set of poorly explained additional clinical features, including ichthyosis, impaired intelligence, developmental delay and anemia. About half of TTD patients are photosensitive due to inherited defects in the DNA repair and transcription factor II H (TFIIH). The pathophysiological contributions of unrepaired DNA lesions and impaired transcription have not been dissected yet. Here, we functionally characterize the consequence of a homozygous missense mutation in the general transcription factor II E, subunit 2 (GTF2E2/TFIIEß) of two unrelated non-photosensitive TTD (NPS-TTD) families. We demonstrate that mutant TFIIEß strongly reduces the total amount of the entire TFIIE complex, with a remarkable temperature-sensitive transcription defect, which strikingly correlates with the phenotypic aggravation of key clinical symptoms after episodes of high fever. We performed induced pluripotent stem (iPS) cell reprogramming of patient fibroblasts followed by in vitro erythroid differentiation to translate the intriguing molecular defect to phenotypic expression in relevant tissue, to disclose the molecular basis for some specific TTD features. We observed a clear hematopoietic defect during late-stage differentiation associated with hemoglobin subunit imbalance. These new findings of a DNA repair-independent transcription defect and tissue-specific malfunctioning provide novel mechanistic insight into the etiology of TTD.
Assuntos
Fatores de Transcrição TFII/genética , Síndromes de Tricotiodistrofia/genética , Diferenciação Celular/genética , Reprogramação Celular/genética , DNA Helicases/genética , Reparo do DNA , Feminino , Humanos , Células-Tronco Pluripotentes Induzidas/patologia , Masculino , Mutação , Mutação de Sentido Incorreto , Especificidade de Órgãos , Linhagem , Fatores de Transcrição TFII/metabolismo , Transcrição Gênica , Síndromes de Tricotiodistrofia/metabolismo , Síndromes de Tricotiodistrofia/patologiaRESUMO
In somatic cells of female placental mammals, one of the two X chromosomes is transcriptionally silenced to accomplish an equal dose of X-encoded gene products in males and females. Initiation of random X chromosome inactivation (XCI) is thought to be regulated by X-encoded activators and autosomally encoded suppressors controlling Xist. Spreading of Xist RNA leads to silencing of the X chromosome in cis. Here, we demonstrate that the dose dependent X-encoded XCI activator RNF12/RLIM acts in trans and activates Xist. We did not find evidence for RNF12-mediated regulation of XCI through Tsix or the Xist intron 1 region, which are both known to be involved in inhibition of Xist. In addition, we found that Xist intron 1, which contains a pluripotency factor binding site, is not required for suppression of Xist in undifferentiated ES cells. Analysis of female Rnf12â»/â» knockout ES cells showed that RNF12 is essential for initiation of XCI and is mainly involved in the regulation of Xist. We conclude that RNF12 is an indispensable factor in up-regulation of Xist transcription, thereby leading to initiation of random XCI.
Assuntos
Inativação Gênica , RNA não Traduzido/genética , Proteínas Repressoras/fisiologia , Inativação do Cromossomo X/genética , Animais , Células-Tronco Embrionárias/metabolismo , Feminino , Imunofluorescência , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Vetores Genéticos , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Hibridização in Situ Fluorescente , Íntrons/genética , Masculino , Camundongos , Proteína Homeobox Nanog , RNA Longo não Codificante , Proteínas Repressoras/genética , Ubiquitina-Proteína LigasesRESUMO
Induced pluripotent stem cells (iPSCs) are a powerful tool for biomedical research, but their production presents challenges and safety concerns. Yamanaka and Takahashi revolutionised the field by demonstrating that somatic cells could be reprogrammed into pluripotent cells by overexpressing four key factors for a sufficient time. iPSCs are typically generated using viruses or virus-based methods, which have drawbacks such as vector persistence, risk of insertional mutagenesis, and oncogenesis. The application of less harmful nonviral vectors is limited as conventional plasmids cannot deliver the levels or duration of the factors necessary from a single transfection. Hence, plasmids that are most often used for reprogramming employ the potentially oncogenic Epstein-Barr nuclear antigen 1 (EBNA-1) system to ensure adequate levels and persistence of expression. In this study, we explored the use of nonviral SMAR DNA vectors to reprogram human fibroblasts into iPSCs. We show for the first time that iPSCs can be generated using nonviral plasmids without the use of EBNA-1 and that these DNA vectors can provide sufficient expression to induce pluripotency. We describe an optimised reprogramming protocol using these vectors that can produce high-quality iPSCs with comparable pluripotency and cellular function to those generated with viruses or EBNA-1 vectors.
Assuntos
Reprogramação Celular , Fibroblastos , Vetores Genéticos , Células-Tronco Pluripotentes Induzidas , Plasmídeos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Humanos , Vetores Genéticos/genética , Reprogramação Celular/genética , Fibroblastos/citologia , Fibroblastos/metabolismo , Plasmídeos/genética , Antígenos Nucleares do Vírus Epstein-Barr/genética , Células Cultivadas , Transfecção/métodosRESUMO
The etiopathology of Parkinson's disease has been associated with mitochondrial defects at genetic, laboratory, epidemiological, and clinical levels. These converging lines of evidence suggest that mitochondrial defects are systemic and causative factors in the pathophysiology of PD, rather than being mere correlates. Understanding mitochondrial biology in PD at a granular level is therefore crucial from both basic science and translational perspectives. In a recent study, we investigated mitochondrial alterations in fibroblasts obtained from PD patients assessing mitochondrial function in relation to clinical measures. Our findings demonstrated that the magnitude of mitochondrial alterations parallels disease severity. In this study, we extend these investigations to blood cells and dopamine neurons derived from induced pluripotent stem cells reprogrammed from PD patients. To overcome the inherent metabolic heterogeneity of blood cells, we focused our analyses on metabolically homogeneous, accessible, and expandable erythroblasts. Our results confirm the presence of mitochondrial anomalies in erythroblasts and induced dopamine neurons. Consistent with our previous findings in fibroblasts, we observed that mitochondrial alterations are reversible, as evidenced by enhanced mitochondrial respiration when PD erythroblasts were cultured in a galactose medium that restricts glycolysis. This observation indicates that suppression of mitochondrial respiration may constitute a protective, adaptive response in PD pathogenesis. Notably, this effect was not observed in induced dopamine neurons, suggesting their distinct bioenergetic behavior. In summary, we provide additional evidence for the involvement of mitochondria in the disease process by demonstrating mitochondrial abnormalities in additional cell types relevant to PD. These findings contribute to our understanding of PD pathophysiology and may have implications for the development of novel biomarkers and therapeutic strategies.
Assuntos
Doenças Mitocondriais , Doença de Parkinson , Humanos , Doença de Parkinson/metabolismo , Mitocôndrias/metabolismo , Metabolismo Energético/fisiologia , Fibroblastos/metabolismo , Doenças Mitocondriais/metabolismoRESUMO
The POU domain transcription factor Pou3f1 (Oct6/Scip/Tst1) initiates the transition from ensheathing, promyelinating Schwann cells to myelinating cells. Axonal and other extracellular signals regulate Oct6 expression through the Oct6 Schwann cell enhancer (SCE), which is both required and sufficient to drive all aspects of Oct6 expression in Schwann cells. Thus, the Oct6 SCE is pivotal in the gene regulatory network that governs the onset of myelin formation in Schwann cells and provides a link between myelin promoting signaling and activation of a myelin-related transcriptional network. In this study, we define the relevant cis-acting elements within the SCE and identify the transcription factors that mediate Oct6 regulation. On the basis of phylogenetic comparisons and functional in vivo assays, we identify a number of highly conserved core elements within the mouse SCE. We show that core element 1 is absolutely required for full enhancer function and that it contains closely spaced inverted binding sites for Sox proteins. For the first time in vivo, the dimeric Sox10 binding to this element is shown to be essential for enhancer activity, whereas monomeric Sox10 binding is nonfunctional. As Oct6 and Sox10 synergize to activate the expression of the major myelin-related transcription factor Krox20, we propose that Sox10-dependent activation of Oct6 defines a feedforward regulatory module that serves to time and amplify the onset of myelination in the peripheral nervous system.
Assuntos
Bainha de Mielina/metabolismo , Fator 6 de Transcrição de Octâmero/metabolismo , Fatores de Transcrição SOXE/metabolismo , Células de Schwann/metabolismo , Animais , Células Cultivadas , Imunoprecipitação da Cromatina , Ensaio de Desvio de Mobilidade Eletroforética , Regulação da Expressão Gênica no Desenvolvimento , Células HEK293 , Humanos , Ligação Proteica , Ratos , Células de Schwann/citologiaRESUMO
Mutations in ELANE cause severe congenital neutropenia (SCN), but how they affect neutrophil production and contribute to leukemia predisposition is unknown. Neutropenia is alleviated by CSF3 (granulocyte colony-stimulating factor) therapy in most cases, but dose requirements vary between patients. Here, we show that CD34+CD45+ hematopoietic progenitor cells (HPCs) derived from induced pluripotent stem cell lines from patients with SCN that have mutations in ELANE (n = 2) or HAX1 (n = 1) display elevated levels of reactive oxygen species (ROS) relative to normal iPSC-derived HPCs. In patients with ELANE mutations causing misfolding of the neutrophil elastase (NE) protein, HPCs contained elevated numbers of promyelocyte leukemia protein nuclear bodies, a hallmark of acute oxidative stress. This was confirmed in primary bone marrow cells from 3 additional patients with ELANE-mutant SCN. Apart from responding to elevated ROS levels, PML controlled the metabolic state of these ELANE-mutant HPCs as well as the expression of ELANE, suggestive of a feed-forward mechanism of disease development. Both PML deletion and correction of the ELANE mutation restored CSF3 responses of these ELANE-mutant HPCs. These findings suggest that PML plays a crucial role in the disease course of ELANE-SCN characterized by NE misfolding, with potential implications for CSF3 therapy.
Assuntos
Elastase de Leucócito/genética , Neutropenia , Proteínas Adaptadoras de Transdução de Sinal , Síndrome Congênita de Insuficiência da Medula Óssea , Fator Estimulador de Colônias de Granulócitos , Humanos , Mutação , Neutropenia/congênito , Neutropenia/genéticaRESUMO
Engineering brain organoids from human induced pluripotent stem cells (hiPSCs) is a powerful tool for modeling brain development and neurological disorders. Rett syndrome (RTT), a rare neurodevelopmental disorder, can greatly benefit from this technology, since it affects multiple neuronal subtypes in forebrain sub-regions. We have established dorsal and ventral forebrain organoids from control and RTT patient-specific hiPSCs recapitulating 3D organization and functional network complexity. Our data revealed a premature development of the deep-cortical layer, associated to the formation of TBR1 and CTIP2 neurons, and a lower expression of neural progenitor/proliferative cells in female RTT dorsal organoids. Moreover, calcium imaging and electrophysiology analysis demonstrated functional defects of RTT neurons. Additionally, assembly of RTT dorsal and ventral organoids revealed impairments of interneuron's migration. Overall, our models provide a better understanding of RTT during early stages of neural development, demonstrating a great potential for personalized diagnosis and drug screening.
RESUMO
Severe congenital neutropenia (SCN) patients treated with CSF3/G-CSF to alleviate neutropenia frequently develop acute myeloid leukemia (AML). A common pattern of leukemic transformation involves the appearance of hematopoietic clones with CSF3 receptor (CSF3R) mutations in the neutropenic phase, followed by mutations in RUNX1 before AML becomes overt. To investigate how the combination of CSF3 therapy and CSF3R and RUNX1 mutations contributes to AML development, we make use of mouse models, SCN-derived induced pluripotent stem cells (iPSCs), and SCN and SCN-AML patient samples. CSF3 provokes a hyper-proliferative state in CSF3R/RUNX1 mutant hematopoietic progenitors but does not cause overt AML. Intriguingly, an additional acquired driver mutation in Cxxc4 causes elevated CXXC4 and reduced TET2 protein levels in murine AML samples. Expression of multiple pro-inflammatory pathways is elevated in mouse AML and human SCN-AML, suggesting that inflammation driven by downregulation of TET2 activity is a critical step in the malignant transformation of SCN.
Assuntos
Transformação Celular Neoplásica/genética , Síndrome Congênita de Insuficiência da Medula Óssea/genética , Síndrome Congênita de Insuficiência da Medula Óssea/patologia , Proteínas de Ligação a DNA/genética , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Mutação/genética , Neutropenia/congênito , Fatores de Transcrição/genética , Animais , Linhagem Celular , Linhagem Celular Tumoral , Transformação Celular Neoplásica/patologia , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Células HEK293 , Humanos , Inflamação/genética , Inflamação/patologia , Células K562 , Camundongos , Neutropenia/genética , Neutropenia/patologia , Transdução de Sinais/genéticaRESUMO
Autosomal-dominant polycystic kidney disease (ADPKD) is the most common inherited kidney disease, leading to kidney failure in most patients. In approximately 85% of cases, the disease is caused by mutations in PKD1. How dysregulation of PKD1 leads to cyst formation on a molecular level is unknown. Induced pluripotent stem cells (iPSCs) are a powerful tool for in vitro modeling of genetic disorders. Here, we established ADPKD patient-specific iPSCs to study the function of PKD1 in kidney development and cyst formation in vitro. Somatic mutations are proposed to be the initiating event of cyst formation, and therefore, iPSCs were derived from cystic renal epithelial cells rather than fibroblasts. Mutation analysis of the ADPKD iPSCs revealed germline mutations in PKD1 but no additional somatic mutations in PKD1/PKD2. Although several somatic mutations in other genes implicated in ADPKD were identified in cystic renal epithelial cells, only few of these mutations were present in iPSCs, indicating a heterogeneous mutational landscape, and possibly in vitro cell selection before and during the reprogramming process. Whole-genome DNA methylation analysis indicated that iPSCs derived from renal epithelial cells maintain a kidney-specific DNA methylation memory. In addition, comparison of PKD1+/- and control iPSCs revealed differences in DNA methylation associated with the disease history. In conclusion, we generated and characterized iPSCs derived from cystic and healthy control renal epithelial cells, which can be used for in vitro modeling of kidney development in general and cystogenesis in particular.
Assuntos
Células Epiteliais/patologia , Células-Tronco Pluripotentes Induzidas/patologia , Rim/patologia , Rim Policístico Autossômico Dominante/patologia , Linhagem Celular , Reprogramação Celular , Metilação de DNA/genética , Análise Mutacional de DNA , Epigênese Genética , Humanos , Túbulos Renais/patologia , Mutação/genética , Rim Policístico Autossômico Dominante/genética , Canais de Cátion TRPP/genética , Canais de Cátion TRPP/metabolismoRESUMO
Homologous recombination is a versatile DNA damage repair pathway requiring Rad51 and Rad54. Here we show that a mammalian Rad54 paralog, Rad54B, displays physical and functional interactions with Rad51 and DNA that are similar to those of Rad54. While ablation of Rad54 in mouse embryonic stem (ES) cells leads to a mild reduction in homologous recombination efficiency, the absence of Rad54B has little effect. However, the absence of both Rad54 and Rad54B dramatically reduces homologous recombination efficiency. Furthermore, we show that Rad54B protects ES cells from ionizing radiation and the interstrand DNA cross-linking agent mitomycin C. Interestingly, at the ES cell level the paralogs do not display an additive or synergic interaction with respect to mitomycin C sensitivity, yet animals lacking both Rad54 and Rad54B are dramatically sensitized to mitomycin C compared to either single mutant. This suggests that the paralogs possibly function in a tissue-specific manner. Finally, we show that Rad54, but not Rad54B, is needed for a normal distribution of Rad51 on meiotic chromosomes. Thus, even though the paralogs have similar biochemical properties, genetic analysis in mice uncovered their nonoverlapping roles.
Assuntos
Dano ao DNA , DNA Helicases/fisiologia , Reparo do DNA , Proteínas Nucleares/fisiologia , Recombinação Genética , Animais , Antibióticos Antineoplásicos/farmacologia , Aberrações Cromossômicas , Cromossomos/química , DNA Helicases/genética , Proteínas de Ligação a DNA , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Humanos , Meiose , Camundongos , Camundongos Mutantes , Mitomicina/farmacologia , Proteínas Nucleares/deficiência , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Rad51 Recombinase/análise , Rad51 Recombinase/metabolismo , Tolerância a Radiação/genética , Células-Tronco/efeitos dos fármacos , Células-Tronco/enzimologia , Células-Tronco/efeitos da radiaçãoRESUMO
BACKGROUND: Most patients with Parkinson's disease, Parkinson's disease dementia, and dementia with Lewy bodies do not carry mutations in known disease-causing genes. The aim of this study was to identify a novel gene implicated in the development of these disorders. METHODS: Our study was done in three stages. First, we did genome-wide linkage analysis of an Italian family with dominantly inherited Parkinson's disease to identify the disease locus. Second, we sequenced the candidate gene in an international multicentre series of unrelated probands who were diagnosed either clinically or pathologically with Parkinson's disease, Parkinson's disease dementia, or dementia with Lewy bodies. As a control, we used gene sequencing data from individuals with abdominal aortic aneurysms (who were not examined neurologically). Third, we enrolled an independent series of patients diagnosed clinically with Parkinson's disease and controls with no signs or family history of Parkinson's disease, Parkinson's disease dementia, or dementia with Lewy bodies from centres in Portugal, Sardinia, and Taiwan, and screened them for specific variants. We also did mRNA and brain pathology studies in three patients from the international multicentre series carrying disease-associated variants, and we did functional protein studies in in-vitro models, including neurons from induced pluripotent stem-like cells. FINDINGS: Molecular studies were done between Jan 1, 2008, and Dec 31, 2017. In the initial kindred of ten affected Italian individuals (mean age of disease onset 59·8 years [SD 8·7]), we detected significant linkage of Parkinson's disease to chromosome 14 and nominated LRP10 as the disease-causing gene. Among the international series of 660 probands, we identified eight individuals (four with Parkinson's disease, two with Parkinson's disease dementia, and two with dementia with Lewy bodies) who carried different, rare, potentially pathogenic LRP10 variants; one carrier was found among 645 controls with abdominal aortic aneurysms. In the independent series, two of these eight variants were detected in three additional Parkinson's disease probands (two from Sardinia and one from Taiwan) but in none of the controls. Of the 11 probands from the international and independent cohorts with LRP10 variants, ten had a positive family history of disease and DNA was available from ten affected relatives (in seven of these families). The LRP10 variants were present in nine of these ten relatives, providing independent-albeit limited-evidence of co-segregation with disease. Post-mortem studies in three patients carrying distinct LRP10 variants showed severe Lewy body pathology. Of nine variants identified in total (one in the initial family and eight in stage 2), three severely affected LRP10 expression and mRNA stability (1424+5delG, 1424+5GâA, and Ala212Serfs*17, shown by cDNA analysis), four affected protein stability (Tyr307Asn, Gly603Arg, Arg235Cys, and Pro699Ser, shown by cycloheximide-chase experiments), and two affected protein localisation (Asn517del and Arg533Leu; shown by immunocytochemistry), pointing to loss of LRP10 function as a common pathogenic mechanism. INTERPRETATION: Our findings implicate LRP10 gene defects in the development of inherited forms of α-synucleinopathies. Future elucidation of the function of the LRP10 protein and pathways could offer novel insights into mechanisms, biomarkers, and therapeutic targets. FUNDING: Stichting ParkinsonFonds, Dorpmans-Wigmans Stichting, Erasmus Medical Center, ZonMw-Memorabel programme, EU Joint Programme Neurodegenerative Disease Research (JPND), Parkinson's UK, Avtal om Läkarutbildning och Forskning (ALF) and Parkinsonfonden (Sweden), Lijf and Leven foundation, and cross-border grant of Alzheimer Netherlands-Ligue Européene Contre la Maladie d'Alzheimer (LECMA).
Assuntos
Proteínas Relacionadas a Receptor de LDL/genética , Doença por Corpos de Lewy/genética , Doença de Parkinson/genética , Encéfalo/patologia , Cromossomos Humanos Par 14/genética , Demência/epidemiologia , Demência/etiologia , Demência/genética , Família , Feminino , Ligação Genética , Estudo de Associação Genômica Ampla , Heterozigoto , Humanos , Itália , Doença por Corpos de Lewy/epidemiologia , Masculino , Pessoa de Meia-Idade , Doença de Parkinson/complicações , Doença de Parkinson/epidemiologia , Linhagem , Células-Tronco Pluripotentes/metabolismo , RNA Mensageiro/química , RNA Mensageiro/genéticaRESUMO
In placental mammals, balanced expression of X-linked genes is accomplished by X chromosome inactivation (XCI) in female cells. In humans, random XCI is initiated early during embryonic development. To investigate whether reprogramming of female human fibroblasts into induced pluripotent stem cells (iPSCs) leads to reactivation of the inactive X chromosome (Xi), we have generated iPSC lines from fibroblasts heterozygous for large X-chromosomal deletions. These fibroblasts show completely skewed XCI of the mutated X chromosome, enabling monitoring of X chromosome reactivation (XCR) and XCI using allele-specific single-cell expression analysis. This approach revealed that XCR is robust under standard culture conditions, but does not prevent reinitiation of XCI, resulting in a mixed population of cells with either two active X chromosomes (Xas) or one Xa and one Xi. This mixed population of XaXa and XaXi cells is stabilized in naive human stem cell medium, allowing expansion of clones with two Xas.
Assuntos
Cromossomos Humanos X , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Ativação Transcricional , Linhagem Celular , Células Cultivadas , Mapeamento Cromossômico , Feminino , Fibroblastos/metabolismo , Expressão Gênica , Ordem dos Genes , Genes Ligados ao Cromossomo X , Loci Gênicos , Vetores Genéticos/genética , Humanos , Cariótipo , Transgenes , Inativação do Cromossomo XRESUMO
Standardization of culture methods for human pluripotent stem cell (PSC) neural differentiation can greatly contribute to the development of novel clinical advancements through the comprehension of neurodevelopmental diseases. Here, we report an approach that reproduces neural commitment from human induced pluripotent stem cells using dual-SMAD inhibition under defined conditions in a vitronectin-based monolayer system. By employing this method it was possible to obtain neurons derived from both control and Rett syndrome patients' pluripotent cells. During differentiation mutated cells displayed alterations in the number of neuronal projections, and production of Tuj1 and MAP2-positive neurons. Although investigation of a broader number of patients would be required, these observations are in accordance with previous studies showing impaired differentiation of these cells. Consequently, our experimental methodology was proved useful not only for the generation of neural cells, but also made possible to compare neural differentiation behavior of different cell lines under defined culture conditions. This study thus expects to contribute with an optimized approach to study the neural commitment of human PSCs, and to produce patient-specific neural cells that can be used to gain a better understanding of disease mechanisms.
Assuntos
Técnicas de Cultura de Células/métodos , Diferenciação Celular/genética , Células-Tronco Pluripotentes Induzidas/citologia , Neurogênese , Síndrome de Rett/genética , Linhagem Celular , Proliferação de Células/genética , Meios de Cultura , Células-Tronco Embrionárias/citologia , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Proteína 2 de Ligação a Metil-CpG/biossíntese , Proteína 2 de Ligação a Metil-CpG/genética , Células-Tronco Neurais/citologia , Neurônios/citologia , Síndrome de Rett/patologia , Síndrome de Rett/terapia , Proteínas Smad Inibidoras/genéticaRESUMO
Silencing of the FMR1 gene leads to fragile X syndrome, the most common cause of inherited intellectual disability. To study the epigenetic modifications of the FMR1 gene during silencing in time, we used fibroblasts and induced pluripotent stem cells (iPSCs) of an unmethylated full mutation (uFM) individual with normal intelligence. The uFM fibroblast line carried an unmethylated FMR1 promoter region and expressed normal to slightly increased FMR1 mRNA levels. The FMR1 expression in the uFM line corresponds with the increased H3 acetylation and H3K4 methylation in combination with a reduced H3K9 methylation. After reprogramming, the FMR1 promoter region was methylated in all uFM iPSC clones. Two clones were analyzed further and showed a lack of FMR1 expression, whereas the presence of specific histone modifications also indicated a repressed FMR1 promoter. In conclusion, these findings demonstrate that the standard reprogramming procedure leads to epigenetic silencing of the fully mutated FMR1 gene.
Assuntos
Metilação de DNA , Fibroblastos/metabolismo , Proteína do X Frágil da Deficiência Intelectual/genética , Inativação Gênica , Células-Tronco Pluripotentes Induzidas/metabolismo , Mutação , Adolescente , Animais , Estudos de Casos e Controles , Linhagem Celular , Reprogramação Celular , Criança , Pré-Escolar , Feminino , Fibroblastos/citologia , Proteína do X Frágil da Deficiência Intelectual/metabolismo , Histonas/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Masculino , Camundongos , Regiões Promotoras Genéticas , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
Constitutive activation of the Wnt pathway leads to adenoma formation, an obligatory step towards intestinal cancer. In view of the established role of Wnt in regulating stemness, we attempted the isolation of cancer stem cells (CSCs) from Apc- and Apc/KRAS-mutant intestinal tumours. Whereas CSCs are present in Apc/KRAS tumours, they appear to be very rare (<10(-6)) in the Apc-mutant adenomas. In contrast, the Lin(-)CD24(hi)CD29(+) subpopulation of adenocarcinoma cells appear to be enriched in CSCs with increased levels of active ß-catenin. Expression profiling analysis of the CSC-enriched subpopulation confirmed their enhanced Wnt activity and revealed additional differential expression of other signalling pathways, growth factor binding proteins, and extracellular matrix components. As expected, genes characteristic of the Paneth cell lineage (e.g. defensins) are co-expressed together with stem cell genes (e.g. Lgr5) within the CSC-enriched subpopulation. This is of interest as it may indicate a cancer stem cell niche role for tumor-derived Paneth-like cells, similar to their role in supporting Lgr5(+) stem cells in the normal intestinal crypt. Overall, our results indicate that oncogenic KRAS activation in Apc-driven tumours results in the expansion of the CSCs compartment by increasing ®-catenin intracellular stabilization.
Assuntos
Transformação Celular Neoplásica/genética , Genes APC , Genes ras , Neoplasias Intestinais/genética , Células-Tronco Neoplásicas/metabolismo , Animais , Antígeno CD24/metabolismo , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Análise por Conglomerados , Modelos Animais de Doenças , Regulação Neoplásica da Expressão Gênica , Imunofenotipagem , Integrina beta1/metabolismo , Neoplasias Intestinais/metabolismo , Espaço Intracelular/metabolismo , Camundongos , Camundongos Transgênicos , Mutação , Transdução de Sinais , Transcriptoma , beta Catenina/metabolismoRESUMO
Canonical Wnt signaling has been implicated in the regulation of hematopoiesis. By employing a Wnt-reporter mouse, we observed that Wnt signaling is differentially activated during hematopoiesis, suggesting an important regulatory role for specific Wnt signaling levels. To investigate whether canonical Wnt signaling regulates hematopoiesis in a dosage-dependent fashion, we analyzed the effect of different mutations in the Adenomatous polyposis coli gene (Apc), a negative modulator of the canonical Wnt pathway. By combining different targeted hypomorphic alleles and a conditional deletion allele of Apc, a gradient of five different Wnt signaling levels was obtained in vivo. We here show that different, lineage-specific Wnt dosages regulate hematopoietic stem cells (HSCs), myeloid precursors, and T lymphoid precursors during hematopoiesis. Differential, lineage-specific optimal Wnt dosages provide a unifying concept that explains the differences reported among inducible gain-of-function approaches, leading to either HSC expansion or depletion of the HSC pool.
Assuntos
Dosagem de Genes , Hematopoese , Via de Sinalização Wnt , Animais , Diferenciação Celular , Células Clonais , Citometria de Fluxo , Marcação de Genes , Sistema Hematopoético/metabolismo , Camundongos , Modelos Biológicos , Mutação/genética , Células Mieloides/citologia , Células Mieloides/metabolismo , Linfócitos T/citologiaRESUMO
The genetic hierarchy that controls myelination of peripheral nerves by Schwann cells includes the POU domain Oct-6/Scip/Tst-1and the zinc-finger Krox-20/Egr2 transcription factors. These pivotal transcription factors act to control the onset of myelination during development and tissue regeneration in adults following damage. In this report we demonstrate the involvement of a third transcription factor, the POU domain factor Brn-2. We show that Schwann cells express Brn-2 in a developmental profile similar to that of Oct-6 and that Brn-2 gene activation does not depend on Oct-6. Overexpression of Brn-2 in Oct-6-deficient Schwann cells, under control of the Oct-6 Schwann cell enhancer (SCE), results in partial rescue of the developmental delay phenotype, whereas compound disruption of both Brn-2 and Oct-6 results in a much more severe phenotype. Together these data strongly indicate that Brn-2 function largely overlaps with that of Oct-6 in driving the transition from promyelinating to myelinating Schwann cells.