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1.
PLoS Negl Trop Dis ; 14(7): e0008413, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32628667

RESUMO

Global Zika virus (ZIKV) outbreaks and their link to microcephaly have raised major public health concerns. However, the mechanism of maternal-fetal transmission remains largely unknown. In this study, we determined the role of yolk sac (YS) microglial progenitors in a mouse model of ZIKV vertical transmission. We found that embryonic (E) days 6.5-E8.5 were a critical window for ZIKV infection that resulted in fetal demise and microcephaly, and YS microglial progenitors were susceptible to ZIKV infection. Ablation of YS microglial progenitors significantly reduced the viral load in both the YS and the embryonic brain. Taken together, these results support the hypothesis that YS microglial progenitors serve as "Trojan horses," contributing to ZIKV fetal brain dissemination and congenital brain defects.


Assuntos
Feto/patologia , Microcefalia/patologia , Microglia/virologia , Complicações Infecciosas na Gravidez/patologia , Infecção por Zika virus/patologia , Zika virus/isolamento & purificação , Animais , Encéfalo/virologia , Modelos Animais de Doenças , Feminino , Feto/virologia , Humanos , Transmissão Vertical de Doença Infecciosa , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microcefalia/embriologia , Microcefalia/virologia , Microglia/metabolismo , Gravidez , Complicações Infecciosas na Gravidez/virologia , Carga Viral , Zika virus/fisiologia , Infecção por Zika virus/transmissão , Infecção por Zika virus/virologia
2.
PLoS One ; 15(7): e0235655, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32628740

RESUMO

Biallelic variants in RNU4ATAC, a non-coding gene transcribed into the minor spliceosome component U4atac snRNA, are responsible for three rare recessive developmental diseases, namely Taybi-Linder/MOPD1, Roifman and Lowry-Wood syndromes. Next-generation sequencing of clinically heterogeneous cohorts (children with either a suspected genetic disorder or a congenital microcephaly) recently identified mutations in this gene, illustrating how profoundly these technologies are modifying genetic testing and assessment. As RNU4ATAC has a single non-coding exon, the bioinformatic prediction algorithms assessing the effect of sequence variants on splicing or protein function are irrelevant, which makes variant interpretation challenging to molecular diagnostic laboratories. In order to facilitate and improve clinical diagnostic assessment and genetic counseling, we present i) an update of the previously reported RNU4ATAC mutations and an analysis of the genetic variations affecting this gene using the Genome Aggregation Database (gnomAD) resource; ii) the pathogenicity prediction performances of scores computed based on an RNA structure prediction tool and of those produced by the Combined Annotation Dependent Depletion tool for the 285 RNU4ATAC variants identified in patients or in large-scale sequencing projects; iii) a method, based on a cellular assay, that allows to measure the effect of RNU4ATAC variants on splicing efficiency of a minor (U12-type) reporter intron. Lastly, the concordance of bioinformatic predictions and cellular assay results was investigated.


Assuntos
RNA Nuclear Pequeno/metabolismo , Spliceossomos/metabolismo , Criança , Bases de Dados Genéticas , Nanismo/genética , Nanismo/patologia , Retardo do Crescimento Fetal/genética , Retardo do Crescimento Fetal/patologia , Fibroblastos/citologia , Fibroblastos/metabolismo , Variação Genética , Humanos , Microcefalia/genética , Microcefalia/patologia , Conformação de Ácido Nucleico , Osteocondrodisplasias/genética , Osteocondrodisplasias/patologia , Processamento de RNA , RNA Nuclear Pequeno/química , RNA Nuclear Pequeno/genética
3.
Nucleic Acids Res ; 48(12): 6672-6684, 2020 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-32504494

RESUMO

Hereditary mutations in polynucleotide kinase-phosphatase (PNKP) result in a spectrum of neurological pathologies ranging from neurodevelopmental dysfunction in microcephaly with early onset seizures (MCSZ) to neurodegeneration in ataxia oculomotor apraxia-4 (AOA4) and Charcot-Marie-Tooth disease (CMT2B2). Consistent with this, PNKP is implicated in the repair of both DNA single-strand breaks (SSBs) and DNA double-strand breaks (DSBs); lesions that can trigger neurodegeneration and neurodevelopmental dysfunction, respectively. Surprisingly, however, we did not detect a significant defect in DSB repair (DSBR) in primary fibroblasts from PNKP patients spanning the spectrum of PNKP-mutated pathologies. In contrast, the rate of SSB repair (SSBR) is markedly reduced. Moreover, we show that the restoration of SSBR in patient fibroblasts collectively requires both the DNA kinase and DNA phosphatase activities of PNKP, and the fork-head associated (FHA) domain that interacts with the SSBR protein, XRCC1. Notably, however, the two enzymatic activities of PNKP appear to affect different aspects of disease pathology, with reduced DNA phosphatase activity correlating with neurodevelopmental dysfunction and reduced DNA kinase activity correlating with neurodegeneration. In summary, these data implicate reduced rates of SSBR, not DSBR, as the source of both neurodevelopmental and neurodegenerative pathology in PNKP-mutated disease, and the extent and nature of this reduction as the primary determinant of disease severity.


Assuntos
Quebras de DNA de Cadeia Dupla , Quebras de DNA de Cadeia Simples , Enzimas Reparadoras do DNA/genética , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Proteína 1 Complementadora Cruzada de Reparo de Raio-X/genética , Apraxias/genética , Apraxias/patologia , Doença de Charcot-Marie-Tooth/genética , Doença de Charcot-Marie-Tooth/patologia , Reparo do DNA/genética , Fibroblastos/metabolismo , Fibroblastos/patologia , Humanos , Microcefalia/genética , Microcefalia/patologia , Mutação/genética , Convulsões/genética , Convulsões/patologia
4.
BMC Med Genet ; 21(1): 99, 2020 05 11.
Artigo em Inglês | MEDLINE | ID: mdl-32393352

RESUMO

BACKGROUND: To date, at least 746 genes have been identified to cause intellectual disability (ID). Among them, mutations in the Methyl CpG binding protein 2 (MECP2) gene are the leading cause of Rett syndrome and associated ID. METHODS: Considering the large number of ID-associated genes, we applied trio-based whole-exome sequencing (trio-WES) and in silico analysis for genetic diagnosis of 294 children with ID. RESULTS: Three de novo heterozygous mutations [NM_004992.3: c.502C > T, p.(Arg168*), c.916C > T, p.(Arg306Cys), and c.879C > G, p.(Ile293Met)] in MECP2 were identified in three unrelated girls. The first two mutations were detected in two patients who were diagnosed as typical Rett syndrome, X-linked ID and psychomotor retardation. The third mutation (c.879C > G), a previously unreported, was found in a 6-year-old girl with ID, microcephaly, severe underweight and psychomotor retardation. Particularly, this extremely rare de novo mutation (DNM) is located in the transcriptional repression domain (TRD) of MECP2, where at least 62 different causal mutations are identified. CONCLUSIONS: We identified three DNMs in MECP2 in a cohort of 294 individuals with ID. The novel c.879C > G mutation, as a likely pathogenic allele, may become a risk factor associated with X-linked ID, microcephaly and psychomotor retardation.


Assuntos
Predisposição Genética para Doença , Deficiência Intelectual/genética , Proteína 2 de Ligação a Metil-CpG/genética , Síndrome de Rett/genética , Criança , Pré-Escolar , Feminino , Heterozigoto , Humanos , Deficiência Intelectual/patologia , Microcefalia/genética , Microcefalia/patologia , Mutação , Linhagem , Fenótipo , Síndrome de Rett/patologia , Sequenciamento Completo do Exoma
5.
PLoS Pathog ; 16(5): e1008521, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32392268

RESUMO

Zika virus (ZIKV) infection may lead to congenital microcephaly and pregnancy loss in pregnant women. In the context of pregnancy, folic acid (FA) supplementation may reduce the risk of abnormal pregnancy outcomes. Intriguingly, FA may have a beneficial effect on the adverse pregnancy outcomes associated with ZIKV infection. Here, we show that FA inhibits ZIKV replication in human umbilical vein endothelial cells (HUVECs) and a cell culture model of blood-placental barrier (BPB). The inhibitory effect of FA against ZIKV infection is associated with FRα-AMPK signaling. Furthermore, treatment with FA reduces pathological features in the placenta, number of fetal resorptions, and stillbirths in two mouse models of in utero ZIKV transmission. Mice with FA treatment showed lower viral burden and better prognostic profiles in the placenta including reduced inflammatory response, and enhanced integrity of BPB. Overall, our findings suggest the preventive role of FA supplementation in ZIKV-associated abnormal pregnancy and warrant nutritional surveillance to evaluate maternal FA status in areas with active ZIKV transmission.


Assuntos
Ácido Fólico/farmacologia , Placenta , Complicações Infecciosas na Gravidez , Infecção por Zika virus/prevenção & controle , Zika virus/metabolismo , Animais , Modelos Animais de Doenças , Feminino , Células Endoteliais da Veia Umbilical Humana , Humanos , Camundongos , Microcefalia/metabolismo , Microcefalia/patologia , Microcefalia/prevenção & controle , Microcefalia/virologia , Placenta/metabolismo , Placenta/patologia , Placenta/virologia , Gravidez , Complicações Infecciosas na Gravidez/metabolismo , Complicações Infecciosas na Gravidez/patologia , Complicações Infecciosas na Gravidez/prevenção & controle , Infecção por Zika virus/metabolismo , Infecção por Zika virus/patologia
6.
Nat Commun ; 11(1): 1746, 2020 04 08.
Artigo em Inglês | MEDLINE | ID: mdl-32269212

RESUMO

In mammalian cell lines, the endosomal sorting complex required for transport (ESCRT)-III mediates abscission, the process that physically separates daughter cells and completes cell division. Cep55 protein is regarded as the master regulator of abscission, because it recruits ESCRT-III to the midbody (MB), the site of abscission. However, the importance of this mechanism in a mammalian organism has never been tested. Here we show that Cep55 is dispensable for mouse embryonic development and adult tissue homeostasis. Cep55-knockout offspring show microcephaly and primary neural progenitors require Cep55 and ESCRT for survival and abscission. However, Cep55 is dispensable for cell division in embryonic or adult tissues. In vitro, division of primary fibroblasts occurs without Cep55 and ESCRT-III at the midbody and is not affected by ESCRT depletion. Our work defines Cep55 as an abscission regulator only in specific tissue contexts and necessitates the re-evaluation of an alternative ESCRT-independent cell division mechanism.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Citocinese , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Animais , Animais Recém-Nascidos , Apoptose , Proteínas de Ciclo Celular/deficiência , Proteínas de Ciclo Celular/genética , Células Cultivadas , Córtex Cerebral/anormalidades , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Fibroblastos/metabolismo , Deleção de Genes , Genótipo , Rim/anormalidades , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microcefalia/patologia , Mitose
7.
PLoS Negl Trop Dis ; 14(3): e0008060, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32163415

RESUMO

The northeast (NE) region of Brazil commonly goes through drought periods, which favor cyanobacterial blooms, capable of producing neurotoxins with implications for human and animal health. The most severe dry spell in the history of Brazil occurred between 2012 and 2016. Coincidently, the highest incidence of microcephaly associated with the Zika virus (ZIKV) outbreak took place in the NE region of Brazil during the same years. In this work, we tested the hypothesis that saxitoxin (STX), a neurotoxin produced in South America by the freshwater cyanobacteria Raphidiopsis raciborskii, could have contributed to the most severe Congenital Zika Syndrome (CZS) profile described worldwide. Quality surveillance showed higher cyanobacteria amounts and STX occurrence in human drinking water supplies of NE compared to other regions of Brazil. Experimentally, we described that STX doubled the quantity of ZIKV-induced neural cell death in progenitor areas of human brain organoids, while the chronic ingestion of water contaminated with STX before and during gestation caused brain abnormalities in offspring of ZIKV-infected immunocompetent C57BL/6J mice. Our data indicate that saxitoxin-producing cyanobacteria is overspread in water reservoirs of the NE and might have acted as a co-insult to ZIKV infection in Brazil. These results raise a public health concern regarding the consequences of arbovirus outbreaks happening in areas with droughts and/or frequent freshwater cyanobacterial blooms.


Assuntos
Morte Celular/efeitos dos fármacos , Microcefalia/patologia , Envenenamento/complicações , Envenenamento/patologia , Saxitoxina/toxicidade , Infecção por Zika virus/complicações , Infecção por Zika virus/patologia , Animais , Toxinas Bacterianas/análise , Toxinas Bacterianas/toxicidade , Encéfalo/patologia , Brasil/epidemiologia , Células Cultivadas , Modelos Animais de Doenças , Surtos de Doenças , Feminino , Humanos , Incidência , Toxinas Marinhas/análise , Toxinas Marinhas/toxicidade , Camundongos Endogâmicos C57BL , Microcistinas/análise , Microcistinas/toxicidade , Modelos Teóricos , Neurotoxinas/análise , Neurotoxinas/toxicidade , Saxitoxina/análise , Água/química
8.
J Hum Genet ; 65(4): 387-396, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31965062

RESUMO

NGLY1 deficiency is the first and only autosomal recessive congenital disorder of N-linked deglycosylation (NGLY1-CDDG). To date, no patients with NGLY1 deficiency has been reported from mainland China or East Asia in English literature. Here, we present six patients with a diagnosis of NGLY1-CDDG on the basis of clinical phenotype, genetic testing, and functional studies. We retrospectively analyzed clinical phenotypes and NGLY1 genotypes of six cases from four families. Informed consent was obtained for diagnosis and treatment. In-silico tools and in vitro enzyme activity assays were used to determine pathogenicity of NGLY1 varaints. All patients had typical features of NGLY1-CDDG, including global developmental delay, microcephaly, hypotonia, hypertransaminasemia, alacrimia, and feeding difficulty. Dysmorphic features found in our patients include flat nasal bridge, loose and hollow cheeks, short stature, malnutrition, and ptosis. Pachylosis could be a novel cutaneous feature that may be explained by lack of sweat. We found three novel variants, including one missense (c.982C > G/p.Arg328Gly), one splice site (c.1003+3A > G), and one frame-shift (c.1637-1652delCATCTTTTGCTTATAT/p.Ser546PhefsTer) variant. All mutations were predicted to be disease causing with in-silico prediction tools, and affected at least one feature of gene splicing. Protein modeling showed missense variants may affect covalent bonding within the protein structure, or interrupt active/binding amino-acid residues. In vitro studies indicated that proteins carrying missense variants (p.Arg328Gly and p.Tyr342Cys) lost the enzyme activity. We expanded clinical phenotype and genetic mutation spectrum of NGLY1-CDDG by reporting six cases, three novel variants, and novel clinical features from mainland China.


Assuntos
Defeitos Congênitos da Glicosilação/genética , Deficiências do Desenvolvimento/genética , Oftalmopatias Hereditárias/genética , Transtornos da Alimentação e da Ingestão de Alimentos/genética , Doenças do Aparelho Lacrimal/genética , Microcefalia/genética , Hipotonia Muscular/genética , Mutação , Peptídeo-N4-(N-acetil-beta-glucosaminil) Asparagina Amidase/deficiência , Defeitos Congênitos da Glicosilação/patologia , Deficiências do Desenvolvimento/patologia , Oftalmopatias Hereditárias/patologia , Transtornos da Alimentação e da Ingestão de Alimentos/patologia , Feminino , Humanos , Lactente , Doenças do Aparelho Lacrimal/patologia , Masculino , Microcefalia/patologia , Hipotonia Muscular/patologia , Peptídeo-N4-(N-acetil-beta-glucosaminil) Asparagina Amidase/genética
9.
Elife ; 82019 12 03.
Artigo em Inglês | MEDLINE | ID: mdl-31794381

RESUMO

Occludin (OCLN) mutations cause human microcephaly and cortical malformation. A tight junction component thought absent in neuroepithelium after neural tube closure, OCLN isoform-specific expression extends into corticogenesis. Full-length and truncated isoforms localize to neuroprogenitor centrosomes, but full-length OCLN transiently localizes to plasma membranes while only truncated OCLN continues at centrosomes throughout neurogenesis. Mimicking human mutations, full-length OCLN depletion in mouse and in human CRISPR/Cas9-edited organoids produce early neuronal differentiation, reduced progenitor self-renewal and increased apoptosis. Human neural progenitors were more severely affected, especially outer radial glial cells, which mouse embryonic cortex lacks. Rodent and human mutant progenitors displayed reduced proliferation and prolonged M-phase. OCLN interacted with mitotic spindle regulators, NuMA and RAN, while full-length OCLN loss impaired spindle pole morphology, astral and mitotic microtubule integrity. Thus, early corticogenesis requires full-length OCLN to regulate centrosome organization and dynamics, revealing a novel role for this tight junction protein in early brain development.


Assuntos
Córtex Cerebral/crescimento & desenvolvimento , Córtex Cerebral/metabolismo , Ocludina/metabolismo , Junções Íntimas/metabolismo , Aneuploidia , Animais , Apoptose , Sistemas CRISPR-Cas , Diferenciação Celular , Proliferação de Células , Centrossomo/metabolismo , Córtex Cerebral/patologia , Modelos Animais de Doenças , Edição de Genes , Humanos , Camundongos , Camundongos Knockout , Microcefalia/genética , Microcefalia/patologia , Microtúbulos/metabolismo , Mutagênese , Mutação , Neurogênese/genética , Neurogênese/fisiologia , Ocludina/genética , Fuso Acromático/metabolismo , Junções Íntimas/genética
10.
Yi Chuan ; 41(10): 905-918, 2019 Oct 20.
Artigo em Chinês | MEDLINE | ID: mdl-31624053

RESUMO

Brain development diseases refer to a group of diseases that affect the development of the brain or the central nervous system. Autosomal recessive primary microcephaly (MCPH) is a typical neurodevelopmental disorder characterized by a decreased brain size, mental retardation and abnormal behaviors. To date, at least 25 genes have been discovered to cause MCPH when mutated. These genes were named MCPH1-25 according to the discovery order. MCPH proteins play important roles in regulating brain developmental signaling pathways. Here, we provide a timely review of the expression patterns, cellular localization, molecular functions, phenotypes, as well as animal models of these 25 MCPH proteins that will expedite our understanding of the pathogenesis of brain disorders at both molecular and cellular levels.


Assuntos
Proteínas de Ciclo Celular/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Microcefalia/genética , Proteínas do Tecido Nervoso/genética , Animais , Encéfalo , Humanos , Microcefalia/patologia , Mutação , Fenótipo
11.
Am J Pathol ; 189(12): 2440-2449, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31541646

RESUMO

Cells of the developing central nervous system are particularly susceptible to formation of double-stranded DNA breaks (DSBs) arising from physiological and/or environmental insults. Therefore, efficient repair of DSBs is especially vital for maintaining cellular health and proper functioning in the developing brain. Here, increased expression of DSB initiating and nonhomologous end joining repair machinery in newborn neurons in the developing brains of both mouse and human are demonstrated. In parallel, the first characterization is provided of the brain phenotype in the Lig4R278H/R278H (Lig4R/R) mouse model of DNA Ligase 4 (LIG4) syndrome, in which a hypomorphic Lig4 mutation, originally identified in patients, impedes nonhomologous end joining. It is shown that Lig4R/R mice develop nonprogressive microcephaly, resulting primarily from apoptotic death of newborn neurons that is both spatially and temporally specific during peak cortical neurogenesis. This apoptosis leads to a reduction in neurons throughout the postnatal cerebral cortex, but with a more prominent impact on those of the lower cortical layers. Together, these findings begin to uncover the pathogenesis of microcephaly in LIG4 syndrome and open avenues to more focused investigations on the critical roles of DSB formation and repair in vulnerable neuronal populations of the brain.


Assuntos
Apoptose , Córtex Cerebral/patologia , Anormalidades Craniofaciais/complicações , DNA Ligase Dependente de ATP/metabolismo , Modelos Animais de Doenças , Transtornos do Crescimento/complicações , Síndromes de Imunodeficiência/complicações , Microcefalia/etiologia , Neurônios/patologia , Animais , Córtex Cerebral/metabolismo , Quebras de DNA de Cadeia Dupla , DNA Ligase Dependente de ATP/genética , Feminino , Técnicas de Introdução de Genes , Masculino , Camundongos , Microcefalia/patologia , Mutação , Neurônios/metabolismo , Análise Espaço-Temporal
12.
Am J Hum Genet ; 105(4): 689-705, 2019 10 03.
Artigo em Inglês | MEDLINE | ID: mdl-31495489

RESUMO

Sphingomyelinases generate ceramide from sphingomyelin as a second messenger in intracellular signaling pathways involved in cell proliferation, differentiation, or apoptosis. Children from 12 unrelated families presented with microcephaly, simplified gyral pattern of the cortex, hypomyelination, cerebellar hypoplasia, congenital arthrogryposis, and early fetal/postnatal demise. Genomic analysis revealed bi-allelic loss-of-function variants in SMPD4, coding for the neutral sphingomyelinase-3 (nSMase-3/SMPD4). Overexpression of human Myc-tagged SMPD4 showed localization both to the outer nuclear envelope and the ER and additionally revealed interactions with several nuclear pore complex proteins by proteomics analysis. Fibroblasts from affected individuals showed ER cisternae abnormalities, suspected for increased autophagy, and were more susceptible to apoptosis under stress conditions, while treatment with siSMPD4 caused delayed cell cycle progression. Our data show that SMPD4 links homeostasis of membrane sphingolipids to cell fate by regulating the cross-talk between the ER and the outer nuclear envelope, while its loss reveals a pathogenic mechanism in microcephaly.


Assuntos
Artrogripose/genética , Microcefalia/genética , Transtornos do Neurodesenvolvimento/genética , Esfingomielina Fosfodiesterase/genética , Artrogripose/patologia , Linhagem da Célula , Criança , Retículo Endoplasmático/metabolismo , Feminino , Perfilação da Expressão Gênica , Células HEK293 , Humanos , Masculino , Microcefalia/patologia , Mitose , Transtornos do Neurodesenvolvimento/patologia , Linhagem , Processamento de RNA
13.
Rev Assoc Med Bras (1992) ; 65(6): 909-913, 2019 Jul 22.
Artigo em Inglês | MEDLINE | ID: mdl-31340324

RESUMO

INTRODUCTION: The Zika virus (ZIKV) is an arbovirus isolated for the first time in 1947 and transmitted to humans by the Aedes aegypti mosquito. In Brasil, it was first detected in May 2015. Since then, ZIKV has been identified as the etiological agent of acute exanthematous disease in Brasil, and Neuropediatricians of the Recife warned about an epidemic of microcephaly, and the Brazilian Ministry of Health confirmed the association between ZIKV and Congenital malformations and neurological syndromes. The eye, as an extension of the developing brain, has been examined in patients with microcephaly and maternal history of ZIKV infection. METHODS: Twenty newborn patients with microcephaly, whose mothers had presumed Zika virus during pregnancy, were analyzed through medical records. The nonparametric chi-square statistic was used to verify the association between head circumference and ocular alteration at a significance level of 0.0001. RESULTS: The significance of P = 0.000 in the value of non-parametric chi-square statistics was lower than the value of α = 0.0001, demonstrating that, at a level of 0.0001, there is an association between head circumference and ocular alteration. CONCLUSION: Although the knowledge of the natural evolution of the disease is still scarce, the current evidence is strong enough to establish a causal relationship between ZIKV infection during pregnancy and the increased incidence of the microcephaly and serious eye alterations that lead to the severe lower vision of these children.


Assuntos
Cefalometria , Oftalmopatias/virologia , Microcefalia/virologia , Infecção por Zika virus/complicações , Brasil , Oftalmopatias/patologia , Feminino , Cabeça/patologia , Humanos , Lactente , Masculino , Microcefalia/patologia , Valores de Referência
14.
Nucleic Acids Res ; 47(16): 8720-8733, 2019 09 19.
Artigo em Inglês | MEDLINE | ID: mdl-31276587

RESUMO

Expression of human mitochondrial DNA is indispensable for proper function of the oxidative phosphorylation machinery. The mitochondrial genome encodes 22 tRNAs, 2 rRNAs and 11 mRNAs and their post-transcriptional modification constitutes one of the key regulatory steps during mitochondrial gene expression. Cytosine-5 methylation (m5C) has been detected in mitochondrial transcriptome, however its biogenesis has not been investigated in details. Mammalian NOP2/Sun RNA Methyltransferase Family Member 2 (NSUN2) has been characterized as an RNA methyltransferase introducing m5C in nuclear-encoded tRNAs, mRNAs and microRNAs and associated with cell proliferation and differentiation, with pathogenic variants in NSUN2 being linked to neurodevelopmental disorders. Here we employ spatially restricted proximity labelling and immunodetection to demonstrate that NSUN2 is imported into the matrix of mammalian mitochondria. Using three genetic models for NSUN2 inactivation-knockout mice, patient-derived fibroblasts and CRISPR/Cas9 knockout in human cells-we show that NSUN2 is necessary for the generation of m5C at positions 48, 49 and 50 of several mammalian mitochondrial tRNAs. Finally, we show that inactivation of NSUN2 does not have a profound effect on mitochondrial tRNA stability and oxidative phosphorylation in differentiated cells. We discuss the importance of the newly discovered function of NSUN2 in the context of human disease.


Assuntos
5-Metilcitosina/metabolismo , Eczema/genética , Transtornos do Crescimento/genética , Deficiência Intelectual/genética , Metiltransferases/genética , Microcefalia/genética , Processamento Pós-Transcricional do RNA , RNA Mitocondrial/genética , RNA de Transferência/genética , Animais , Sistemas CRISPR-Cas , Eczema/metabolismo , Eczema/patologia , Facies , Fibroblastos/metabolismo , Fibroblastos/patologia , Edição de Genes , Técnicas de Inativação de Genes , Transtornos do Crescimento/metabolismo , Transtornos do Crescimento/patologia , Células HEK293 , Humanos , Deficiência Intelectual/metabolismo , Deficiência Intelectual/patologia , Metilação , Metiltransferases/deficiência , Camundongos , Camundongos Knockout , Microcefalia/metabolismo , Microcefalia/patologia , Mitocôndrias/genética , Mitocôndrias/metabolismo , Conformação de Ácido Nucleico , Fosforilação Oxidativa , Cultura Primária de Células , Transporte Proteico , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Mitocondrial/metabolismo , RNA de Transferência/metabolismo
15.
Int J Mol Sci ; 20(13)2019 Jul 02.
Artigo em Inglês | MEDLINE | ID: mdl-31269755

RESUMO

Hoyeraal-Hreidarsson syndrome (HHS), caused by several different germline mutations resulting in severe telomeropathy, presents with early-onset growth anomalies and neurologic/developmental disorders including characteristic cerebellar hypoplasia. Early mortalities may arise from immunodeficiency and bone marrow failure if not successfully salvaged by allogeneic hematopoietic stem cell transplantation (HSCT). Few reports have characterized the persistent somatic progression of HHS after successful HSCT. We present an HHS patient with an X-linked recessive DKC1 c.1058C > T; Ala353Val mutation who successfully underwent unrelated HSCT at 5 years of age. After months of early infections and organ toxicities immediately post-transplant, he had more than two years of excellent quality of life with correction of bone marrow failure and immunodeficiency. However, episodic massive variceal bleeding and progressive respiratory insufficiency, which were secondary to non-cirrhotic portal hypertension and pulmonary arteriovenous shunts, respectively, developed over 2 years after HSCT and resulted in his death from respiratory failure 4 years after HSCT. This outcome suggests that while HSCT can correct bone marrow failure and immunodeficiency, it may fail to prevent or even aggravate other fatal processes, such as portal hypertension and pulmonary arteriovenous shunting.


Assuntos
Proteínas de Ciclo Celular/genética , Disceratose Congênita/terapia , Retardo do Crescimento Fetal/terapia , Deficiência Intelectual/terapia , Microcefalia/terapia , Proteínas Nucleares/genética , Transplante de Células-Tronco de Sangue Periférico , Pré-Escolar , Disceratose Congênita/complicações , Disceratose Congênita/genética , Disceratose Congênita/patologia , Retardo do Crescimento Fetal/genética , Retardo do Crescimento Fetal/patologia , Humanos , Deficiência Intelectual/complicações , Deficiência Intelectual/genética , Deficiência Intelectual/patologia , Masculino , Microcefalia/complicações , Microcefalia/genética , Microcefalia/patologia , Transplante de Células-Tronco de Sangue Periférico/efeitos adversos , Transplante de Células-Tronco de Sangue Periférico/métodos , Mutação Puntual
16.
EMBO Mol Med ; 11(7): e10201, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31273937

RESUMO

PARN, poly(A)-specific ribonuclease, regulates the turnover of mRNAs and the maturation and stabilization of the hTR RNA component of telomerase. Biallelic PARN mutations were associated with Høyeraal-Hreidarsson (HH) syndrome, a rare telomere biology disorder that, because of its severity, is likely not exclusively due to hTR down-regulation. Whether PARN deficiency was affecting the expression of telomere-related genes was still unclear. Using cells from two unrelated HH individuals carrying novel PARN mutations and a human PARN knock-out (KO) cell line with inducible PARN complementation, we found that PARN deficiency affects both telomere length and stability and down-regulates the expression of TRF1, TRF2, TPP1, RAP1, and POT1 shelterin transcripts. Down-regulation of dyskerin-encoding DKC1 mRNA was also observed and found to result from p53 activation in PARN-deficient cells. We further showed that PARN deficiency compromises ribosomal RNA biogenesis in patients' fibroblasts and cells from heterozygous Parn KO mice. Homozygous Parn KO however resulted in early embryonic lethality that was not overcome by p53 KO. Our results refine our knowledge on the pleiotropic cellular consequences of PARN deficiency.


Assuntos
Disceratose Congênita/metabolismo , Exorribonucleases/deficiência , Retardo do Crescimento Fetal/metabolismo , Deficiência Intelectual/metabolismo , Microcefalia/metabolismo , RNA Ribossômico/biossíntese , Homeostase do Telômero , Telômero/metabolismo , Animais , Pré-Escolar , Modelos Animais de Doenças , Disceratose Congênita/genética , Disceratose Congênita/patologia , Exorribonucleases/metabolismo , Feminino , Retardo do Crescimento Fetal/genética , Retardo do Crescimento Fetal/patologia , Humanos , Deficiência Intelectual/genética , Deficiência Intelectual/patologia , Masculino , Camundongos , Camundongos Knockout , Microcefalia/genética , Microcefalia/patologia , RNA Ribossômico/genética , Telômero/genética , Telômero/patologia
18.
J Cell Biol ; 218(7): 2185-2197, 2019 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-31197030

RESUMO

In mammals, centrioles participate in brain development, and human mutations affecting centriole duplication cause microcephaly. Here, we identify a role for the mammalian homologue of yeast SFI1, involved in the duplication of the yeast spindle pole body, as a critical regulator of centriole duplication in mammalian cells. Mammalian SFI1 interacts with USP9X, a deubiquitylase associated with human syndromic mental retardation. SFI1 localizes USP9X to the centrosome during S phase to deubiquitylate STIL, a critical regulator of centriole duplication. USP9X-mediated deubiquitylation protects STIL from degradation. Consistent with a role for USP9X in stabilizing STIL, cells from patients with USP9X loss-of-function mutations have reduced STIL levels. Together, these results demonstrate that SFI1 is a centrosomal protein that localizes USP9X to the centrosome to stabilize STIL and promote centriole duplication. We propose that the USP9X protection of STIL to facilitate centriole duplication underlies roles of both proteins in human neurodevelopment.


Assuntos
Proteínas de Ciclo Celular/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Microcefalia/genética , Ubiquitina Tiolesterase/genética , Ciclo Celular/genética , Centríolos/genética , Centríolos/ultraestrutura , Centrossomo/ultraestrutura , Feminino , Fibroblastos/metabolismo , Células HEK293 , Células HeLa , Humanos , Microcefalia/patologia , Microscopia Eletrônica , Mutação , Transtornos do Neurodesenvolvimento/genética , Proteólise
19.
Nat Commun ; 10(1): 2612, 2019 06 13.
Artigo em Inglês | MEDLINE | ID: mdl-31197141

RESUMO

Primary microcephaly is caused by mutations in genes encoding centrosomal proteins including WDR62 and KIF2A. However, mechanisms underlying human microcephaly remain elusive. By creating mutant mice and human cerebral organoids, here we found that WDR62 deletion resulted in a reduction in the size of mouse brains and organoids due to the disruption of neural progenitor cells (NPCs), including outer radial glia (oRG). WDR62 ablation led to retarded cilium disassembly, long cilium, and delayed cell cycle progression leading to decreased proliferation and premature differentiation of NPCs. Mechanistically, WDR62 interacts with and promotes CEP170's localization to the basal body of primary cilium, where CEP170 recruits microtubule-depolymerizing factor KIF2A to disassemble cilium. WDR62 depletion reduced KIF2A's basal body localization, and enhanced KIF2A expression partially rescued deficits in cilium length and NPC proliferation. Thus, modeling microcephaly with cerebral organoids and mice reveals a WDR62-CEP170-KIF2A pathway promoting cilium disassembly, disruption of which contributes to microcephaly.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Cinesina/metabolismo , Microcefalia/patologia , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Fosfoproteínas/metabolismo , Proteínas Repressoras/metabolismo , Animais , Técnicas de Cultura de Células , Proteínas de Ciclo Celular/genética , Diferenciação Celular , Linhagem Celular , Proliferação de Células , Cílios/metabolismo , Modelos Animais de Doenças , Feminino , Técnicas de Inativação de Genes , Humanos , Masculino , Camundongos , Camundongos Knockout , Microcefalia/genética , Proteínas Associadas aos Microtúbulos/genética , Proteínas do Tecido Nervoso/genética , Células-Tronco Neurais/citologia , Células-Tronco Neurais/patologia , Neuroglia/citologia , Neuroglia/patologia , Organoides/patologia , Fosfoproteínas/genética , RNA Interferente Pequeno/metabolismo
20.
Int J Mol Sci ; 20(9)2019 Apr 28.
Artigo em Inglês | MEDLINE | ID: mdl-31035417

RESUMO

Glioblastoma multiforme and medulloblastoma are the most frequent high-grade brain tumors in adults and children, respectively. Standard therapies for these cancers are mainly based on surgical resection, radiotherapy, and chemotherapy. However, intrinsic or acquired resistance to treatment occurs almost invariably in the first case, and side effects are unacceptable in the second. Therefore, the development of new, effective drugs is a very important unmet medical need. A critical requirement for developing such agents is to identify druggable targets required for the proliferation or survival of tumor cells, but not of other cell types. Under this perspective, genes mutated in congenital microcephaly represent interesting candidates. Congenital microcephaly comprises a heterogeneous group of disorders in which brain volume is reduced, in the absence or presence of variable syndromic features. Genetic studies have clarified that most microcephaly genes encode ubiquitous proteins involved in mitosis and in maintenance of genomic stability, but the effects of their inactivation are particularly strong in neural progenitors. It is therefore conceivable that the inhibition of the function of these genes may specifically affect the proliferation and survival of brain tumor cells. Microcephaly genes encode for a few kinases, including CITK, PLK4, AKT3, DYRK1A, and TRIO. In this review, we summarize the evidence indicating that the inhibition of these molecules could exert beneficial effects on different aspects of brain cancer treatment.


Assuntos
Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Microcefalia/metabolismo , Microcefalia/patologia , Animais , Glioblastoma/metabolismo , Glioblastoma/patologia , Humanos , Meduloblastoma/metabolismo , Meduloblastoma/patologia , Proteínas Serina-Treonina Quinases/metabolismo
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