RESUMO
Patients with mitochondrial disorders present with clinically diverse symptoms, largely driven by heterogeneous mutations in mitochondrial-encoded and nuclear-encoded mitochondrial genes. These mutations ultimately lead to complex biochemical disorders with a myriad of clinical manifestations, often accumulating during childhood on into adulthood, contributing to life-altering and sometimes fatal events. It is therefore important to diagnose and characterize the associated disorders for each mitochondrial mutation as early as possible since medical management might be able to improve the quality and longevity of life in mitochondrial disease patients. Here we identify a novel mitochondrial variant in a mitochondrial transfer RNA for histidine (mt-tRNA-his) [m.12148T>C], that is associated with the development of ocular, aural, neurological, renal, and muscular dysfunctions. We provide a detailed account of a family harboring this mutation, as well as the molecular underpinnings contributing to cellular and mitochondrial dysfunction. In conclusion, this investigation provides clinical, biochemical, and morphological evidence of the pathogenicity of m.12148T>C. We highlight the importance of multiple tissue testing and in vitro disease modeling in diagnosing mitochondrial disease.
RESUMO
Translational Relevance: Mitochondria are viable therapeutic targets for a broad spectrum of ocular diseases.
Assuntos
Oftalmopatias , Mitocôndrias , Oftalmopatias/tratamento farmacológico , HumanosRESUMO
The transplantation of human embryonic stem cell (hESC)-derived insulin-producing ß cells for the treatment of diabetes is finally approaching the clinical stage. However, even with state-of-the-art differentiation protocols, a significant percentage of undefined non-endocrine cell types are still generated. Most importantly, there is the potential for carry-over of non-differentiated cell types that may produce teratomas. We sought to modify hESCs so that their differentiated progeny could be selectively devoid of tumorigenic cells and enriched for cells of the desired phenotype (in this case, ß cells). Here we report the generation of a modified hESC line harboring two suicide gene cassettes, whose expression results in cell death in the presence of specific pro-drugs. We show the efficacy of this system at enriching for ß cells and eliminating tumorigenic ones both in vitro and in vivo. Our approach is innovative inasmuch as it allows for the preservation of the desired cells while eliminating those with the potential to develop teratomas.
Assuntos
Carcinogênese/patologia , Células-Tronco Embrionárias Humanas/patologia , Células Secretoras de Insulina/patologia , Animais , Carcinogênese/genética , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Linhagem Celular , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Teratoma/genética , Teratoma/patologiaRESUMO
Understanding the molecular mechanisms that underlie neurodegenerative disorders has been hampered by a lack of readily available model systems that replicate the complexity of the human disease. Recent advances in stem cell technology have facilitated the derivation of patient-specific stem cells from a variety of differentiated cell types. These induced pluripotent stem cells (iPSCs) are attractive disease models since they can be grown and differentiated to produce large numbers of disease-relevant cell types. However, most iPSC lines are derived in advance of, and without the benefit of, neuropathological confirmation of the donor - the gold standard for many disease classifications and measurement of disease severity. While others have reported the generation of autopsy-confirmed iPSC lines from patient explants, these methods require outgrowth of cadaver tissue, which require additional time and is often only successful â¼50% of the time. Here we report the rapid generation of autopsy-confirmed iPSC lines from peripheral blood mononuclear cells (PBMCs) drawn postmortem. Since this approach doesn't require the propagation of previously frozen cadaver tissue, iPSC can be rapidly and efficiently produced from patients with autopsy-confirmed pathology. These matched iPSC-derived patient-specific neurons and postmortem brain tissue will support studies of specific mechanisms that drive the pathogenesis of neurodegenerative diseases.
Assuntos
Diferenciação Celular/fisiologia , Células-Tronco Pluripotentes Induzidas/citologia , Leucócitos Mononucleares/citologia , Doenças Neurodegenerativas/patologia , Autopsia/métodos , Separação Celular , Fibroblastos/metabolismo , Humanos , Neurônios/patologiaRESUMO
Among several genetic mutations known to cause amyotrophic lateral sclerosis (ALS), a hexanucleotide repeat expansion in the C9orf72 gene is the most common. In approximately 30% of C9orf72-ALS cases, 5-methylcytosine (5mC) levels within the C9orf72 promoter are increased, resulting in a modestly attenuated phenotype. The developmental timing of C9orf72 promoter hypermethylation and the reason why it occurs in only a subset of patients remain unknown. In order to model the acquisition of C9orf72 hypermethylation and examine the potential role of 5-hydroxymethylcytosine (5hmC), we generated induced pluripotent stem cells (iPSCs) from an ALS patient with C9orf72 promoter hypermethylation. Our data show that 5mC levels are reduced by reprogramming and then re-acquired upon neuronal specification, while 5hmC levels increase following reprogramming and are highest in iPSCs and motor neurons. We confirmed the presence of 5hmC within the C9orf72 promoter in post-mortem brain tissues of hypermethylated patients. These findings show that iPSCs are a valuable model system for examining epigenetic perturbations caused by the C9orf72 mutation and reveal a potential role for cytosine demethylation.
Assuntos
Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/patologia , Células-Tronco Pluripotentes Induzidas/fisiologia , Mutação/genética , Regiões Promotoras Genéticas/fisiologia , Proteínas/genética , 5-Metilcitosina/metabolismo , Encéfalo/patologia , Proteína C9orf72 , Técnicas de Cocultura , Ilhas de CpG/fisiologia , Citosina/análogos & derivados , Metilação de DNA/genética , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Proteínas de Homeodomínio/metabolismo , Humanos , Linfócitos/fisiologia , Neurônios Motores/fisiologia , Proteína Homeobox Nanog , Nestina/metabolismo , RNA Mensageiro/metabolismo , Fatores de Transcrição SOXB1/metabolismo , Fatores de TempoRESUMO
BACKGROUND: GABAergic synaptic transmission is known to play a critical role in the assembly of neuronal circuits during development and is responsible for maintaining the balance between excitatory and inhibitory signaling in the brain during maturation into adulthood. Importantly, defects in GABAergic neuronal function and signaling have been linked to a number of neurological diseases, including autism spectrum disorders, schizophrenia, and epilepsy. With patient-specific induced pluripotent stem cell (iPSC)-based models of neurological disease, it is now possible to investigate the disease mechanisms that underlie deficits in GABAergic function in affected human neurons. To that end, tools that enable the labeling and purification of viable GABAergic neurons from human pluripotent stem cells would be of great value. RESULTS: To address the need for tools that facilitate the identification and isolation of viable GABAergic neurons from the in vitro differentiation of iPSC lines, a cell type-specific promoter-driven fluorescent reporter construct was developed that utilizes the human vesicular GABA transporter (hVGAT) promoter to drive the expression of mCherry specifically in VGAT-expressing neurons. The transduction of iPSC-derived forebrain neuronal cultures with the hVGAT promoter-mCherry lentiviral reporter construct specifically labeled GABAergic neurons. Immunocytochemical analysis of hVGAT-mCherry expression cells showed significant co-labeling with the GABAergic neuronal markers for endogenous VGAT, GABA, and GAD67. Expression of mCherry from the VGAT promoter showed expression in several cortical interneuron subtypes to similar levels. In addition, an effective and reproducible protocol was developed to facilitate the fluorescent activated cell sorting (FACS)-mediated purification of high yields of viable VGAT-positive cells. CONCLUSIONS: These studies demonstrate the utility of the hVGAT-mCherry reporter construct as an effective tool for studying GABAergic neurons differentiated in vitro from human pluripotent stem cells. This approach could provide a means of obtaining large quantities of viable GABAergic neurons derived from disease-specific hiPSCs that could be used for functional assays or high-throughput screening of small molecule libraries.
Assuntos
Neurônios GABAérgicos/metabolismo , Proteínas Luminescentes/metabolismo , Células-Tronco Pluripotentes/fisiologia , Proteínas Vesiculares de Transporte de Aminoácidos Inibidores/metabolismo , Calbindina 2/metabolismo , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/fisiologia , Sobrevivência Celular , Células Cultivadas , Citometria de Fluxo , Fator Neurotrófico Derivado de Linhagem de Célula Glial/farmacologia , Proteína Glial Fibrilar Ácida/metabolismo , Glutamato Descarboxilase/metabolismo , Humanos , Proteínas Luminescentes/genética , Mutação/genética , Fator de Crescimento Neural/farmacologia , Parvalbuminas/metabolismo , Células-Tronco Pluripotentes/efeitos dos fármacos , Prosencéfalo/citologia , Somatostatina/metabolismo , Sinapsinas/metabolismo , Transfecção , Proteínas Vesiculares de Transporte de Aminoácidos Inibidores/genética , Proteína Vermelha FluorescenteRESUMO
A hexanucleotide repeat expansion residing within the C9ORF72 gene represents the most common known cause of amyotrophic lateral sclerosis (ALS) and places the disease among a growing family of repeat expansion disorders. The presence of RNA foci, repeat-associated translation products, and sequestration of RNA binding proteins suggests that toxic RNA gain-of-function contributes to pathology while C9ORF72 haploinsufficiency may be an additional pathological factor. One viable therapeutic strategy for treating expansion diseases is the use of small molecule inhibitors of epigenetic modifier proteins to reactivate expanded genetic loci. Indeed, previous studies have established proof of this principle by increasing the drug-induced expression of expanded (and abnormally heterochromatinized) FMR1, FXN and C9ORF72 genes in respective patient cells. While epigenetic modifier proteins are increasingly recognized as druggable targets, there have been few screening strategies to address this avenue of drug discovery in the context of expansion diseases. Here we utilize a semi-high-throughput gene expression based screen to identify siRNAs and small molecule inhibitors of epigenetic modifier proteins that regulate C9ORF72 RNA in patient fibroblasts, lymphocytes and reprogrammed motor neurons. We found that several bromodomain small molecule inhibitors increase the expression of C9ORF72 mRNA and pre-mRNA without affecting repressive epigenetic signatures of expanded C9ORF72 alleles. These data suggest that bromodomain inhibition increases the expression of unexpanded C9ORF72 alleles and may therefore compensate for haploinsufficiency without increasing the production of toxic RNA and protein products, thereby conferring therapeutic value.