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1.
Cell ; 173(3): 677-692.e20, 2018 04 19.
Artigo em Inglês | MEDLINE | ID: mdl-29677512

RESUMO

RNA-binding proteins (RBPs) with prion-like domains (PrLDs) phase transition to functional liquids, which can mature into aberrant hydrogels composed of pathological fibrils that underpin fatal neurodegenerative disorders. Several nuclear RBPs with PrLDs, including TDP-43, FUS, hnRNPA1, and hnRNPA2, mislocalize to cytoplasmic inclusions in neurodegenerative disorders, and mutations in their PrLDs can accelerate fibrillization and cause disease. Here, we establish that nuclear-import receptors (NIRs) specifically chaperone and potently disaggregate wild-type and disease-linked RBPs bearing a NLS. Karyopherin-ß2 (also called Transportin-1) engages PY-NLSs to inhibit and reverse FUS, TAF15, EWSR1, hnRNPA1, and hnRNPA2 fibrillization, whereas Importin-α plus Karyopherin-ß1 prevent and reverse TDP-43 fibrillization. Remarkably, Karyopherin-ß2 dissolves phase-separated liquids and aberrant fibrillar hydrogels formed by FUS and hnRNPA1. In vivo, Karyopherin-ß2 prevents RBPs with PY-NLSs accumulating in stress granules, restores nuclear RBP localization and function, and rescues degeneration caused by disease-linked FUS and hnRNPA2. Thus, NIRs therapeutically restore RBP homeostasis and mitigate neurodegeneration.


Assuntos
Transporte Ativo do Núcleo Celular , Príons/química , Proteínas de Ligação a RNA/química , Receptores Citoplasmáticos e Nucleares/química , Adulto , Idoso , Animais , Citoplasma/química , Proteínas de Ligação a DNA/química , Drosophila melanogaster , Feminino , Proteínas de Fluorescência Verde/química , Células HEK293 , Células HeLa , Homeostase , Humanos , Carioferinas/química , Masculino , Pessoa de Meia-Idade , Chaperonas Moleculares/química , Mutação , Doenças Neurodegenerativas/patologia , Domínios Proteicos , Proteína EWS de Ligação a RNA/química , Fatores Associados à Proteína de Ligação a TATA/química , beta Carioferinas/química
2.
Nature ; 568(7753): 561-565, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30944467

RESUMO

Current programmable nuclease-based methods (for example, CRISPR-Cas9) for the precise correction of a disease-causing genetic mutation harness the homology-directed repair pathway. However, this repair process requires the co-delivery of an exogenous DNA donor to recode the sequence and can be inefficient in many cell types. Here we show that disease-causing frameshift mutations that result from microduplications can be efficiently reverted to the wild-type sequence simply by generating a DNA double-stranded break near the centre of the duplication. We demonstrate this in patient-derived cell lines for two diseases: limb-girdle muscular dystrophy type 2G (LGMD2G)1 and Hermansky-Pudlak syndrome type 1 (HPS1)2. Clonal analysis of inducible pluripotent stem (iPS) cells from the LGMD2G cell line, which contains a mutation in TCAP, treated with the Streptococcus pyogenes Cas9 (SpCas9) nuclease revealed that about 80% contained at least one wild-type TCAP allele; this correction also restored TCAP expression in LGMD2G iPS cell-derived myotubes. SpCas9 also efficiently corrected the genotype of an HPS1 patient-derived B-lymphoblastoid cell line. Inhibition of polyADP-ribose polymerase 1 (PARP-1) suppressed the nuclease-mediated collapse of the microduplication to the wild-type sequence, confirming that precise correction is mediated by the microhomology-mediated end joining (MMEJ) pathway. Analysis of editing by SpCas9 and Lachnospiraceae bacterium ND2006 Cas12a (LbCas12a) at non-pathogenic 4-36-base-pair microduplications within the genome indicates that the correction strategy is broadly applicable to a wide range of microduplication lengths and can be initiated by a variety of nucleases. The simplicity, reliability and efficacy of this MMEJ-based therapeutic strategy should permit the development of nuclease-based gene correction therapies for a variety of diseases that are associated with microduplications.


Assuntos
Proteínas Associadas a CRISPR/metabolismo , Conectina/genética , Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades/genética , Síndrome de Hermanski-Pudlak/genética , Síndrome de Hermanski-Pudlak/terapia , Distrofia Muscular do Cíngulo dos Membros/genética , Distrofia Muscular do Cíngulo dos Membros/terapia , Alelos , Proteína 9 Associada à CRISPR/metabolismo , Células Cultivadas , Mutação da Fase de Leitura/genética , Humanos , Mioblastos/citologia , Mioblastos/metabolismo , Poli(ADP-Ribose) Polimerase-1/antagonistas & inibidores , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Sequências Repetitivas de Ácido Nucleico/genética
3.
J Cell Sci ; 132(3)2019 02 11.
Artigo em Inglês | MEDLINE | ID: mdl-30659111

RESUMO

Intraflagellar transport (IFT), which is essential for the formation and function of cilia in most organisms, is the trafficking of IFT trains (i.e. assemblies of IFT particles) that carry cargo within the cilium. Defects in IFT cause several human diseases. IFT trains contain the complexes IFT-A and IFT-B. To dissect the functions of these complexes, we studied a Chlamydomonas mutant that is null for the IFT-A protein IFT140. The mutation had no effect on IFT-B but destabilized IFT-A, preventing flagella assembly. Therefore, IFT-A assembly requires IFT140. Truncated IFT140, which lacks the N-terminal WD repeats of the protein, partially rescued IFT and supported formation of half-length flagella that contained normal levels of IFT-B but greatly reduced amounts of IFT-A. The axonemes of these flagella had normal ultrastructure and, as investigated by SDS-PAGE, normal composition. However, composition of the flagellar 'membrane+matrix' was abnormal. Analysis of the latter fraction by mass spectrometry revealed decreases in small GTPases, lipid-anchored proteins and cell signaling proteins. Thus, IFT-A is specialized for the import of membrane-associated proteins. Abnormal levels of the latter are likely to account for the multiple phenotypes of patients with defects in IFT140.This article has an associated First Person interview with the first author of the paper.


Assuntos
Proteínas de Algas/genética , Membrana Celular/metabolismo , Chlamydomonas reinhardtii/genética , Cílios/metabolismo , Flagelos/metabolismo , Proteínas Ligadas a Lipídeos/genética , Proteínas de Algas/química , Proteínas de Algas/metabolismo , Axonema/metabolismo , Axonema/ultraestrutura , Proteínas de Transporte/química , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Membrana Celular/ultraestrutura , Ataxia Cerebelar/genética , Ataxia Cerebelar/metabolismo , Ataxia Cerebelar/patologia , Chlamydomonas reinhardtii/metabolismo , Chlamydomonas reinhardtii/ultraestrutura , Cílios/ultraestrutura , Síndrome de Ellis-Van Creveld/genética , Síndrome de Ellis-Van Creveld/metabolismo , Síndrome de Ellis-Van Creveld/patologia , Flagelos/ultraestrutura , Expressão Gênica , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Proteínas Ligadas a Lipídeos/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Proteínas Monoméricas de Ligação ao GTP/genética , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Mutação , Organismos Geneticamente Modificados , Transporte Proteico , Retinose Pigmentar/genética , Retinose Pigmentar/metabolismo , Retinose Pigmentar/patologia , Transdução de Sinais , Proteína Vermelha Fluorescente
4.
Metabolomics ; 16(9): 98, 2020 09 11.
Artigo em Inglês | MEDLINE | ID: mdl-32915320

RESUMO

INTRODUCTION: Salivary metabolite profiles are altered in adults with HIV compared to their uninfected counterparts. Less is known about youth with HIV and how oral disorders that commonly accompany HIV infection impact salivary metabolite levels. OBJECTIVE: As part of the Adolescent Master Protocol multi-site cohort study of the Pediatric HIV/AIDS Cohort Study (PHACS) network we compared the salivary metabolome of youth with perinatally-acquired HIV (PHIV) and youth HIV-exposed, but uninfected (PHEU) and determined whether metabolites differ in PHIV versus PHEU. METHODS: We used three complementary targeted and discovery-based liquid chromatography-tandem mass spectrometry (LC-MS/MS) workflows to characterize salivary metabolite levels in 20 PHIV and 20 PHEU youth with and without moderate periodontitis. We examined main effects associated with PHIV and periodontal disease, and the interaction between them. RESULTS: We did not identify differences in salivary metabolite profiles that remained significant under stringent control for both multiple between-group comparisons and multiple metabolites. Levels of cadaverine, a known periodontitis-associated metabolite, were more abundant in individuals with periodontal disease with the difference being more pronounced in PHEU than PHIV. In the discovery-based dataset, we identified a total of 564 endogenous peptides in the metabolite extracts, showing that proteolytic processing and amino acid metabolism are important to consider in the context of HIV infection. CONCLUSION: The salivary metabolite profiles of PHIV and PHEU youth were overall very similar. Individuals with periodontitis particularly among the PHEU youth had higher levels of cadaverine, suggesting that HIV infection, or its treatment, may influence the metabolism of oral bacteria.


Assuntos
Infecções por HIV/complicações , Doenças Periodontais/metabolismo , Saliva/metabolismo , Adolescente , Bactérias , Criança , Cromatografia Líquida , Estudos de Coortes , Estudos Transversais , Feminino , Humanos , Masculino , Metabolômica , Saúde Bucal , Espectrometria de Massas em Tandem , Adulto Jovem
5.
Nature ; 495(7442): 467-73, 2013 Mar 28.
Artigo em Inglês | MEDLINE | ID: mdl-23455423

RESUMO

Algorithms designed to identify canonical yeast prions predict that around 250 human proteins, including several RNA-binding proteins associated with neurodegenerative disease, harbour a distinctive prion-like domain (PrLD) enriched in uncharged polar amino acids and glycine. PrLDs in RNA-binding proteins are essential for the assembly of ribonucleoprotein granules. However, the interplay between human PrLD function and disease is not understood. Here we define pathogenic mutations in PrLDs of heterogeneous nuclear ribonucleoproteins (hnRNPs) A2B1 and A1 in families with inherited degeneration affecting muscle, brain, motor neuron and bone, and in one case of familial amyotrophic lateral sclerosis. Wild-type hnRNPA2 (the most abundant isoform of hnRNPA2B1) and hnRNPA1 show an intrinsic tendency to assemble into self-seeding fibrils, which is exacerbated by the disease mutations. Indeed, the pathogenic mutations strengthen a 'steric zipper' motif in the PrLD, which accelerates the formation of self-seeding fibrils that cross-seed polymerization of wild-type hnRNP. Notably, the disease mutations promote excess incorporation of hnRNPA2 and hnRNPA1 into stress granules and drive the formation of cytoplasmic inclusions in animal models that recapitulate the human pathology. Thus, dysregulated polymerization caused by a potent mutant steric zipper motif in a PrLD can initiate degenerative disease. Related proteins with PrLDs should therefore be considered candidates for initiating and perhaps propagating proteinopathies of muscle, brain, motor neuron and bone.


Assuntos
Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/patologia , Demência Frontotemporal/genética , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/química , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/metabolismo , Distrofia Muscular do Cíngulo dos Membros/genética , Proteínas Mutantes/genética , Mutação/genética , Miosite de Corpos de Inclusão/genética , Osteíte Deformante/genética , Príons/química , Sequência de Aminoácidos , Esclerose Lateral Amiotrófica/metabolismo , Animais , Drosophila melanogaster/citologia , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Feminino , Demência Frontotemporal/metabolismo , Demência Frontotemporal/patologia , Células HeLa , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/genética , Humanos , Corpos de Inclusão/genética , Corpos de Inclusão/metabolismo , Corpos de Inclusão/patologia , Masculino , Camundongos , Dados de Sequência Molecular , Distrofia Muscular do Cíngulo dos Membros/metabolismo , Distrofia Muscular do Cíngulo dos Membros/patologia , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Miosite de Corpos de Inclusão/metabolismo , Miosite de Corpos de Inclusão/patologia , Osteíte Deformante/metabolismo , Osteíte Deformante/patologia , Fatores de Terminação de Peptídeos/química , Fatores de Terminação de Peptídeos/genética , Fatores de Terminação de Peptídeos/metabolismo , Príons/genética , Príons/metabolismo , Estrutura Terciária de Proteína/genética , RNA/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
6.
Mol Cell ; 43(1): 72-84, 2011 Jul 08.
Artigo em Inglês | MEDLINE | ID: mdl-21726811

RESUMO

Sequences rich in glutamine (Q) and asparagine (N) residues often fail to fold at the monomer level. This, coupled to their unusual hydrogen-bonding abilities, provides the driving force to switch between disordered monomers and amyloids. Such transitions govern processes as diverse as human protein-folding diseases, bacterial biofilm assembly, and the inheritance of yeast prions (protein-based genetic elements). A systematic survey of prion-forming domains suggested that Q and N residues have distinct effects on amyloid formation. Here, we use cell biological, biochemical, and computational techniques to compare Q/N-rich protein variants, replacing Ns with Qs and Qs with Ns. We find that the two residues have strong and opposing effects: N richness promotes assembly of benign self-templating amyloids; Q richness promotes formation of toxic nonamyloid conformers. Molecular simulations focusing on intrinsic folding differences between Qs and Ns suggest that their different behaviors are due to the enhanced turn-forming propensity of Ns over Qs.


Assuntos
Asparagina/química , Glutamina/química , Fatores de Terminação de Peptídeos/química , Príons/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/metabolismo , Sequência de Aminoácidos , Amiloide/química , Amiloide/metabolismo , Asparagina/metabolismo , Asparagina/fisiologia , Glutamina/metabolismo , Glutamina/fisiologia , Dados de Sequência Molecular , Fatores de Terminação de Peptídeos/metabolismo , Fatores de Terminação de Peptídeos/fisiologia , Príons/metabolismo , Príons/fisiologia , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/fisiologia , Análise de Sequência de Proteína
7.
Mol Ther ; 24(8): 1405-11, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-27378237

RESUMO

Derepression of DUX4 in skeletal muscle has emerged as a likely cause of pathology in facioscapulohumeral muscular dystrophy (FSHD). Here we report on the use of antisense phosphorodiamidate morpholino oligonucleotides to suppress DUX4 expression and function in FSHD myotubes and xenografts. The most effective was phosphorodiamidate morpholino oligonucleotide FM10, which targets the polyadenylation signal of DUX4. FM10 had no significant cell toxicity, and RNA-seq analyses of FSHD and control myotubes revealed that FM10 down-regulated many transcriptional targets of DUX4, without overt off-target effects. Electroporation of FM10 into FSHD patient muscle xenografts in mice also down-regulated DUX4 and DUX4 targets. These findings demonstrate the potential of antisense phosphorodiamidate morpholino oligonucleotides as an FSHD therapeutic option.


Assuntos
Inativação Gênica , Terapia Genética , Proteínas de Homeodomínio/genética , Morfolinos/genética , Distrofia Muscular Facioescapuloumeral/genética , Animais , Modelos Animais de Doenças , Perfilação da Expressão Gênica , Técnicas de Silenciamento de Genes , Marcação de Genes , Xenoenxertos , Sequenciamento de Nucleotídeos em Larga Escala , Proteínas de Homeodomínio/metabolismo , Humanos , Camundongos , Morfolinos/administração & dosagem , Fibras Musculares Esqueléticas , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Distrofia Muscular Facioescapuloumeral/metabolismo , Distrofia Muscular Facioescapuloumeral/patologia , Distrofia Muscular Facioescapuloumeral/terapia , Transcriptoma
8.
Hum Mol Genet ; 23(12): 3180-8, 2014 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-24452336

RESUMO

Development of novel therapeutics requires good animal models of disease. Disorders for which good animal models do not exist have very few drugs in development or clinical trial. Even where there are accepted, albeit imperfect models, the leap from promising preclinical drug results to positive clinical trials commonly fails, including in disorders of skeletal muscle. The main alternative model for early drug development, tissue culture, lacks both the architecture and, usually, the metabolic fidelity of the normal tissue in vivo. Herein, we demonstrate the feasibility and validity of human to mouse xenografts as a preclinical model of myopathy. Human skeletal muscle biopsies transplanted into the anterior tibial compartment of the hindlimbs of NOD-Rag1(null) IL2rγ(null) immunodeficient host mice regenerate new vascularized and innervated myofibers from human myogenic precursor cells. The grafts exhibit contractile and calcium release behavior, characteristic of functional muscle tissue. The validity of the human graft as a model of facioscapulohumeral muscular dystrophy is demonstrated in disease biomarker studies, showing that gene expression profiles of xenografts mirror those of the fresh donor biopsies. These findings illustrate the value of a new experimental model of muscle disease, the human muscle xenograft in mice, as a feasible and valid preclinical tool to better investigate the pathogenesis of human genetic myopathies and to more accurately predict their response to novel therapeutics.


Assuntos
Marcadores Genéticos , Xenoenxertos/fisiologia , Músculo Esquelético/transplante , Distrofia Muscular Facioescapuloumeral/cirurgia , Animais , Modelos Animais de Doenças , Feminino , Perfilação da Expressão Gênica , Humanos , Camundongos , Camundongos Endogâmicos NOD , Músculo Esquelético/patologia , Distrofia Muscular Facioescapuloumeral/patologia
9.
Proc Natl Acad Sci U S A ; 110(36): 14759-64, 2013 Sep 03.
Artigo em Inglês | MEDLINE | ID: mdl-23959875

RESUMO

In man, mutations in different regions of the prion protein (PrP) are associated with infectious neurodegenerative diseases that have remarkably different clinical signs and neuropathological lesions. To explore the roots of this phenomenon, we created a knock-in mouse model carrying the mutation associated with one of these diseases [Creutzfeldt-Jakob disease (CJD)] that was exactly analogous to a previous knock-in model of a different prion disease [fatal familial insomnia (FFI)]. Together with the WT parent, this created an allelic series of three lines, each expressing the same protein with a single amino acid difference, and with all native regulatory elements intact. The previously described FFI mice develop neuronal loss and intense reactive gliosis in the thalamus, as seen in humans with FFI. In contrast, CJD mice had the hallmark features of CJD, spongiosis and proteinase K-resistant PrP aggregates, initially developing in the hippocampus and cerebellum but absent from the thalamus. A molecular transmission barrier protected the mice from any infectious prion agents that might have been present in our mouse facility and allowed us to conclude that the diseases occurred spontaneously. Importantly, both models created agents that caused a transmissible neurodegenerative disease in WT mice. We conclude that single codon differences in a single gene in an otherwise normal genome can cause remarkably different neurodegenerative diseases and are sufficient to create distinct protein-based infectious elements.


Assuntos
Códon/genética , Modelos Animais de Doenças , Mutação , Doenças Priônicas/genética , Príons/genética , Animais , Western Blotting , Encéfalo/metabolismo , Encéfalo/patologia , Síndrome de Creutzfeldt-Jakob/genética , Síndrome de Creutzfeldt-Jakob/metabolismo , Síndrome de Creutzfeldt-Jakob/transmissão , Feminino , Humanos , Imuno-Histoquímica , Insônia Familiar Fatal/genética , Insônia Familiar Fatal/metabolismo , Estimativa de Kaplan-Meier , Masculino , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Modelos Genéticos , Fenótipo , Doenças Priônicas/metabolismo , Doenças Priônicas/transmissão , Príons/metabolismo , Antígeno Nuclear de Célula em Proliferação/metabolismo
10.
Bioinformatics ; 30(17): 2501-2, 2014 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-24825614

RESUMO

UNLABELLED: Prions are self-templating protein aggregates that stably perpetuate distinct biological states and are of keen interest to researchers in both evolutionary and biomedical science. The best understood prions are from yeast and have a prion-forming domain with strongly biased amino acid composition, most notably enriched for Q or N. PLAAC is a web application that scans protein sequences for domains with P: rion- L: ike A: mino A: cid C: omposition. Users can upload sequence files, or paste sequences directly into a textbox. PLAAC ranks the input sequences by several summary scores and allows scores along sequences to be visualized. Text output files can be downloaded for further analyses, and visualizations saved in PDF and PNG formats. AVAILABILITY AND IMPLEMENTATION: http://plaac.wi.mit.edu/. The Ruby-based web framework and the command-line software (implemented in Java, with visualization routines in R) are available at http://github.com/whitehead/plaac under the MIT license. All software can be run under OS X, Windows and Unix.


Assuntos
Aminoácidos/química , Príons/química , Software , Algoritmos , Internet , Proteínas de Saccharomyces cerevisiae/química , Análise de Sequência de Proteína
11.
Proc Natl Acad Sci U S A ; 109(40): 16234-9, 2012 Oct 02.
Artigo em Inglês | MEDLINE | ID: mdl-22988124

RESUMO

Facioscapulohumeral muscular dystrophy (FSHD) is a progressive neuromuscular disorder caused by contractions of repetitive elements within the macrosatellite D4Z4 on chromosome 4q35. The pathophysiology of FSHD is unknown and, as a result, there is currently no effective treatment available for this disease. To better understand the pathophysiology of FSHD and develop mRNA-based biomarkers of affected muscles, we compared global analysis of gene expression in two distinct muscles obtained from a large number of FSHD subjects and their unaffected first-degree relatives. Gene expression in two muscle types was analyzed using GeneChip Gene 1.0 ST arrays: biceps, which typically shows an early and severe disease involvement; and deltoid, which is relatively uninvolved. For both muscle types, the expression differences were mild: using relaxed cutoffs for differential expression (fold change ≥1.2; nominal P value <0.01), we identified 191 and 110 genes differentially expressed between affected and control samples of biceps and deltoid muscle tissues, respectively, with 29 genes in common. Controlling for a false-discovery rate of <0.25 reduced the number of differentially expressed genes in biceps to 188 and in deltoid to 7. Expression levels of 15 genes altered in this study were used as a "molecular signature" in a validation study of an additional 26 subjects and predicted them as FSHD or control with 90% accuracy based on biceps and 80% accuracy based on deltoids.


Assuntos
Biomarcadores/metabolismo , Perfilação da Expressão Gênica , Músculo Esquelético/metabolismo , Distrofia Muscular Facioescapuloumeral/genética , Distrofia Muscular Facioescapuloumeral/metabolismo , RNA Mensageiro/metabolismo , Humanos , Modelos Logísticos , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase em Tempo Real
12.
Hum Mol Genet ; 21(20): 4419-30, 2012 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-22798623

RESUMO

Facioscapulohumeral muscular dystrophy (FSHD), the most prevalent myopathy afflicting both children and adults, is predominantly associated with contractions in the 4q35-localized macrosatellite D4Z4 repeat array. Recent studies have proposed that FSHD pathology is caused by the misexpression of the DUX4 (double homeobox 4) gene resulting in production of a pathogenic protein, DUX4-FL, which has been detected in FSHD, but not in unaffected control myogenic cells and muscle tissue. Here, we report the analysis of DUX4 mRNA and protein expression in a much larger collection of myogenic cells and muscle biopsies derived from biceps and deltoid muscles of FSHD affected subjects and their unaffected first-degree relatives. We confirmed that stable DUX4-fl mRNA and protein were expressed in myogenic cells and muscle tissues derived from FSHD affected subjects, including several genetically diagnosed adult FSHD subjects yet to show clinical manifestations of the disease in the assayed muscles. In addition, we report DUX4-fl mRNA and protein expression in muscle biopsies and myogenic cells from genetically unaffected relatives of the FSHD subjects, although at a significantly lower frequency. These results establish that DUX4-fl expression per se is not sufficient for FSHD muscle pathology and indicate that quantitative modifiers of DUX4-fl expression and/or function and family genetic background are determinants of FSHD muscle disease progression.


Assuntos
Proteínas de Homeodomínio/genética , Distrofia Muscular Facioescapuloumeral/genética , Distrofia Muscular Facioescapuloumeral/patologia , Adulto , Idoso , Estudos de Coortes , Progressão da Doença , Proteínas de Homeodomínio/metabolismo , Humanos , Imuno-Histoquímica , Pessoa de Meia-Idade , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Distrofia Muscular Facioescapuloumeral/metabolismo , RNA Mensageiro/metabolismo
13.
Hum Mol Genet ; 21(13): 2899-911, 2012 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-22454397

RESUMO

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease affecting motor neurons. Mutations in related RNA-binding proteins TDP-43, FUS/TLS and TAF15 have been connected to ALS. These three proteins share several features, including the presence of a bioinformatics-predicted prion domain, aggregation-prone nature in vitro and in vivo and toxic effects when expressed in multiple model systems. Given these commonalities, we hypothesized that a related protein, EWSR1 (Ewing sarcoma breakpoint region 1), might also exhibit similar properties and therefore could contribute to disease. Here, we report an analysis of EWSR1 in multiple functional assays, including mutational screening in ALS patients and controls. We identified three missense variants in EWSR1 in ALS patients, which were absent in a large number of healthy control individuals. We show that disease-specific variants affect EWSR1 localization in motor neurons. We also provide multiple independent lines of in vitro and in vivo evidence that EWSR1 has similar properties as TDP-43, FUS and TAF15, including aggregation-prone behavior in vitro and ability to confer neurodegeneration in Drosophila. Postmortem analysis of sporadic ALS cases also revealed cytoplasmic mislocalization of EWSR1. Together, our studies highlight a potential role for EWSR1 in ALS, provide a collection of functional assays to be used to assess roles of additional RNA-binding proteins in disease and support an emerging concept that a class of aggregation-prone RNA-binding proteins might contribute broadly to ALS and related neurodegenerative diseases.


Assuntos
Esclerose Lateral Amiotrófica/genética , Proteínas de Ligação a Calmodulina/genética , Neurônios Motores/patologia , Proteínas de Ligação a RNA/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Esclerose Lateral Amiotrófica/metabolismo , Esclerose Lateral Amiotrófica/patologia , Animais , Animais Geneticamente Modificados , Proteínas de Ligação a Calmodulina/metabolismo , Células Cultivadas , Criança , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Drosophila/genética , Feminino , Genes Reguladores , Variação Genética , Genótipo , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Neurônios Motores/metabolismo , Mutação de Sentido Incorreto , Proteína EWS de Ligação a RNA , Proteína FUS de Ligação a RNA/genética , Proteína FUS de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/metabolismo , Alinhamento de Sequência , Fatores Associados à Proteína de Ligação a TATA/genética , Fatores Associados à Proteína de Ligação a TATA/metabolismo , Adulto Jovem
14.
Nat Methods ; 8(2): 159-64, 2011 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-21217751

RESUMO

Phenotypes that might otherwise reveal a gene's function can be obscured by genes with overlapping function. This phenomenon is best known within gene families, in which an important shared function may only be revealed by mutating all family members. Here we describe the 'green monster' technology that enables precise deletion of many genes. In this method, a population of deletion strains with each deletion marked by an inducible green fluorescent protein reporter gene, is subjected to repeated rounds of mating, meiosis and flow-cytometric enrichment. This results in the aggregation of multiple deletion loci in single cells. The green monster strategy is potentially applicable to assembling other engineered alterations in any species with sex or alternative means of allelic assortment. To test the technology, we generated a single broadly drug-sensitive strain of Saccharomyces cerevisiae bearing precise deletions of all 16 ATP-binding cassette transporters within clades associated with multidrug resistance.


Assuntos
Deleção de Genes , Técnicas de Inativação de Genes/métodos , Proteínas de Fluorescência Verde/análise , Família Multigênica , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Proteínas de Fluorescência Verde/genética , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismo
15.
Proc Natl Acad Sci U S A ; 108(52): 20881-90, 2011 Dec 27.
Artigo em Inglês | MEDLINE | ID: mdl-22065782

RESUMO

Amyotrophic lateral sclerosis (ALS) is a devastating and universally fatal neurodegenerative disease. Mutations in two related RNA-binding proteins, TDP-43 and FUS, that harbor prion-like domains, cause some forms of ALS. There are at least 213 human proteins harboring RNA recognition motifs, including FUS and TDP-43, raising the possibility that additional RNA-binding proteins might contribute to ALS pathogenesis. We performed a systematic survey of these proteins to find additional candidates similar to TDP-43 and FUS, followed by bioinformatics to predict prion-like domains in a subset of them. We sequenced one of these genes, TAF15, in patients with ALS and identified missense variants, which were absent in a large number of healthy controls. These disease-associated variants of TAF15 caused formation of cytoplasmic foci when expressed in primary cultures of spinal cord neurons. Very similar to TDP-43 and FUS, TAF15 aggregated in vitro and conferred neurodegeneration in Drosophila, with the ALS-linked variants having a more severe effect than wild type. Immunohistochemistry of postmortem spinal cord tissue revealed mislocalization of TAF15 in motor neurons of patients with ALS. We propose that aggregation-prone RNA-binding proteins might contribute very broadly to ALS pathogenesis and the genes identified in our yeast functional screen, coupled with prion-like domain prediction analysis, now provide a powerful resource to facilitate ALS disease gene discovery.


Assuntos
Esclerose Lateral Amiotrófica/genética , Neurônios Motores/metabolismo , Estrutura Terciária de Proteína , Proteínas de Ligação a RNA/genética , Medula Espinal/citologia , Fatores Associados à Proteína de Ligação a TATA/genética , Animais , Células Cultivadas , Biologia Computacional , Drosophila melanogaster/genética , Estudos de Associação Genética/métodos , Humanos , Imuno-Histoquímica , Mutação de Sentido Incorreto/genética , Saccharomyces cerevisiae/genética , Fatores Associados à Proteína de Ligação a TATA/metabolismo
16.
Mol Ther Methods Clin Dev ; 32(3): 101295, 2024 Sep 12.
Artigo em Inglês | MEDLINE | ID: mdl-39139628

RESUMO

Adeno-associated viral vectors (AAVs) are a leading delivery system for gene therapy in animal models and humans. With several Food and Drug Administration-approved AAV gene therapies on the market, issues related to vector manufacturing have become increasingly important. In this study, we focused on potentially toxic DNA contaminants that can arise from AAV proviral plasmids, the raw materials required for manufacturing recombinant AAV in eukaryotic cells. Typical AAV proviral plasmids are circular DNAs containing a therapeutic gene cassette flanked by natural AAV inverted terminal repeat (ITR) sequences, and a plasmid backbone carrying prokaryotic sequences required for plasmid replication and selection in bacteria. While the majority of AAV particles package the intended therapeutic payload, some capsids instead package the bacterial sequences located on the proviral plasmid backbone. Since ITR sequences also have promoter activity, potentially toxic bacterial open reading frames can be produced in vivo, thereby representing a safety risk. In this study, we describe a new AAV proviral plasmid for vector manufacturing that (1) significantly decreases cross-packaged bacterial sequences, (2) increases correctly packaged AAV payloads, and (3) blunts ITR-driven transcription of cross-packaged material to avoid expressing potentially toxic bacterial sequences. This system may help improve the safety of AAV vector products.

17.
J Neuromuscul Dis ; 10(6): 1031-1040, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37899061

RESUMO

BACKGROUND: Facioscapulohumeral muscular dystrophy (FSHD) is a progressive myopathy caused by misexpression of the double homeobox 4 (DUX4) embryonic transcription factor in skeletal muscle. Identifying quantitative and minimally invasive FSHD biomarkers to report on DUX4 activity will significantly accelerate therapeutic development. OBJECTIVE: The goal of this study was to analyze secreted proteins known to be induced by DUX4 using the commercially available Olink Proteomics platform in order to identify potential blood-based molecular FSHD biomarkers. METHODS: We used high-throughput, multiplex immunoassays from Olink Proteomics to measure the levels of several known DUX4-induced genes in a cellular myoblast model of FSHD, in FSHD patient-derived myotube cell cultures, and in serum from individuals with FSHD. Levels of other proteins on the Olink Proteomics panels containing these DUX4 targets were also examined in secondary exploratory analysis. RESULTS: Placental alkaline phosphatase (ALPP) levels correlated with DUX4 expression in both cell-based FSHD systems but did not distinguish FSHD patient serum from unaffected controls. CONCLUSIONS: ALPP, as measured with the Olink Proteomics platform, is not a promising FSHD serum biomarker candidate but could be utilized to evaluate DUX4 activity in discovery research efforts.


Assuntos
Proteínas de Homeodomínio , Distrofia Muscular Facioescapuloumeral , Feminino , Humanos , Gravidez , Biomarcadores , Genes Homeobox , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Distrofia Muscular Facioescapuloumeral/diagnóstico , Distrofia Muscular Facioescapuloumeral/tratamento farmacológico , Placenta/metabolismo , Proteômica
18.
J Cell Sci ; 123(Pt 8): 1191-201, 2010 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-20356930

RESUMO

Prions are proteins that access self-templating amyloid forms, which confer phenotypic changes that can spread from individual to individual within or between species. These infectious phenotypes can be beneficial, as with yeast prions, or deleterious, as with mammalian prions that transmit spongiform encephalopathies. However, the ability to form self-templating amyloid is not unique to prion proteins. Diverse polypeptides that tend to populate intrinsically unfolded states also form self-templating amyloid conformers that are associated with devastating neurodegenerative disorders. Moreover, two RNA-binding proteins, FUS and TDP-43, which form cytoplasmic aggregates in amyotrophic lateral sclerosis, harbor a 'prion domain' similar to those found in several yeast prion proteins. Can these proteins and the neurodegenerative diseases to which they are linked become 'infectious' too? Here, we highlight advances that define the transmissibility of amyloid forms connected with Alzheimer's disease, Parkinson's disease and Huntington's disease. Collectively, these findings suggest that amyloid conformers can spread from cell to cell within the brains of afflicted individuals, thereby spreading the specific neurodegenerative phenotypes distinctive to the protein being converted to amyloid. Importantly, this transmissibility mandates a re-evaluation of emerging neuronal graft and stem-cell therapies. In this Commentary, we suggest how these treatments might be optimized to overcome the transmissible conformers that confer neurodegeneration.


Assuntos
Doenças Priônicas/transmissão , Príons/patogenicidade , Peptídeos beta-Amiloides/metabolismo , Animais , Humanos , Neurônios/transplante , Peptídeos/metabolismo , Príons/metabolismo , Transplante de Células-Tronco , alfa-Sinucleína/metabolismo , Proteínas tau/metabolismo
19.
Front Neurosci ; 16: 972201, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36817096

RESUMO

This study examines cortical organoids generated from a panel of isogenic trisomic and disomic iPSC lines (subclones) as a model of early fetal brain development in Down syndrome (DS). An initial experiment comparing organoids from one trisomic and one disomic line showed many genome-wide transcriptomic differences and modest differences in cell-type proportions, suggesting there may be a neurodevelopmental phenotype that is due to trisomy of chr21. To better control for multiple sources of variation, we undertook a highly robust study of ∼1,200 organoids using an expanded panel of six all-isogenic lines, three disomic, and three trisomic. The power of this experimental design was indicated by strong detection of the ∼1.5-fold difference in chr21 genes. However, the numerous expression differences in non-chr21 genes seen in the smaller experiment fell away, and the differences in cell-type representation between lines did not correlate with trisomy 21. Results suggest that the initial smaller experiment picked up differences between small organoid samples and individual isogenic lines, which "averaged out" in the larger panel of isogenic lines. Our results indicate that even when organoid and batch variability are better controlled for, variation between isogenic cell lines (even subclones) may obscure, or be conflated with, subtle neurodevelopmental phenotypes that may be present in ∼2nd trimester DS brain development. Interestingly, despite this variability between organoid batches and lines, and the "fetal stage" of these organoids, an increase in secreted Aß40 peptide levels-an Alzheimer-related cellular phenotype-was more strongly associated with trisomy 21 status than were neurodevelopmental shifts in cell-type composition.

20.
Elife ; 112022 01 25.
Artigo em Inglês | MEDLINE | ID: mdl-35076017

RESUMO

Skeletal muscle myoblasts (iMyoblasts) were generated from human induced pluripotent stem cells (iPSCs) using an efficient and reliable transgene-free induction and stem cell selection protocol. Immunofluorescence, flow cytometry, qPCR, digital RNA expression profiling, and scRNA-Seq studies identify iMyoblasts as a PAX3+/MYOD1+ skeletal myogenic lineage with a fetal-like transcriptome signature, distinct from adult muscle biopsy myoblasts (bMyoblasts) and iPSC-induced muscle progenitors. iMyoblasts can be stably propagated for >12 passages or 30 population doublings while retaining their dual commitment for myotube differentiation and regeneration of reserve cells. iMyoblasts also efficiently xenoengrafted into irradiated and injured mouse muscle where they undergo differentiation and fetal-adult MYH isoform switching, demonstrating their regulatory plasticity for adult muscle maturation in response to signals in the host muscle. Xenograft muscle retains PAX3+ muscle progenitors and can regenerate human muscle in response to secondary injury. As models of disease, iMyoblasts from individuals with Facioscapulohumeral Muscular Dystrophy revealed a previously unknown epigenetic regulatory mechanism controlling developmental expression of the pathological DUX4 gene. iMyoblasts from Limb-Girdle Muscular Dystrophy R7 and R9 and Walker Warburg Syndrome patients modeled their molecular disease pathologies and were responsive to small molecule and gene editing therapeutics. These findings establish the utility of iMyoblasts for ex vivo and in vivo investigations of human myogenesis and disease pathogenesis and for the development of muscle stem cell therapeutics.


Muscular dystrophies are a group of inherited genetic diseases characterised by progressive muscle weakness. They lead to disability or even death, and no cure exists against these conditions. Advances in genome sequencing have identified many mutations that underly muscular dystrophies, opening the door to new therapies that could repair incorrect genes or rebuild damaged muscles. However, testing these ideas requires better ways to recreate human muscular dystrophy in the laboratory. One strategy for modelling muscular dystrophy involves coaxing skin or other cells from an individual into becoming 'induced pluripotent stem cells'; these can then mature to form almost any adult cell in the body, including muscles. However, this approach does not usually create myoblasts, the 'precursor' cells that specifically mature into muscle during development. This limits investigations into how disease-causing mutations impact muscle formation early on. As a response, Guo et al. developed a two-step protocol of muscle maturation followed by stem cell growth selection to isolate and grow 'induced myoblasts' from induced pluripotent stem cells taken from healthy volunteers and muscular dystrophy patients. These induced myoblasts can both make more of themselves and become muscle, allowing Guo et al. to model three different types of muscular dystrophy. These myoblasts also behave as stem cells when grafted inside adult mouse muscles: some formed human muscle tissue while others remained as precursor cells, which could then respond to muscle injury and start repair. The induced myoblasts developed by Guo et al. will enable scientists to investigate the impacts of different mutations on muscle tissue and to better test treatments. They could also be used as part of regenerative medicine therapies, to restore muscle cells in patients.


Assuntos
Células-Tronco Pluripotentes Induzidas/transplante , Distrofia Muscular Facioescapuloumeral/terapia , Mioblastos/transplante , Animais , Diferenciação Celular , Linhagem Celular , Linhagem da Célula , Células Cultivadas , Modelos Animais de Doenças , Proteínas de Homeodomínio/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Camundongos , Desenvolvimento Muscular , Distrofia Muscular Facioescapuloumeral/patologia , Fator de Transcrição PAX3/metabolismo , Recuperação de Função Fisiológica , Regeneração
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