RESUMEN
Fuchs endothelial corneal dystrophy (FECD) is an age-related cause of vision loss, and the most common repeat expansion-mediated disease in humans characterised to date. Up to 80% of European FECD cases have been attributed to expansion of a non-coding CTG repeat element (termed CTG18.1) located within the ubiquitously expressed transcription factor encoding gene, TCF4. The non-coding nature of the repeat and the transcriptomic complexity of TCF4 have made it extremely challenging to experimentally decipher the molecular mechanisms underlying this disease. Here we comprehensively describe CTG18.1 expansion-driven molecular components of disease within primary patient-derived corneal endothelial cells (CECs), generated from a large cohort of individuals with CTG18.1-expanded (Exp+) and CTG 18.1-independent (Exp-) FECD. We employ long-read, short-read, and spatial transcriptomic techniques to interrogate expansion-specific transcriptomic biomarkers. Interrogation of long-read sequencing and alternative splicing analysis of short-read transcriptomic data together reveals the global extent of altered splicing occurring within Exp+ FECD, and unique transcripts associated with CTG18.1-expansions. Similarly, differential gene expression analysis highlights the total transcriptomic consequences of Exp+ FECD within CECs. Furthermore, differential exon usage, pathway enrichment and spatial transcriptomics reveal TCF4 isoform ratio skewing solely in Exp+ FECD with potential downstream functional consequences. Lastly, exome data from 134 Exp- FECD cases identified rare (minor allele frequency <0.005) and potentially deleterious (CADD>15) TCF4 variants in 7/134 FECD Exp- cases, suggesting that TCF4 variants independent of CTG18.1 may increase FECD risk. In summary, our study supports the hypothesis that at least two distinct pathogenic mechanisms, RNA toxicity and TCF4 isoform-specific dysregulation, both underpin the pathophysiology of FECD. We anticipate these data will inform and guide the development of translational interventions for this common triplet-repeat mediated disease.
Asunto(s)
Distrofia Endotelial de Fuchs , Factor de Transcripción 4 , Expansión de Repetición de Trinucleótido , Humanos , Factor de Transcripción 4/genética , Factor de Transcripción 4/metabolismo , Expansión de Repetición de Trinucleótido/genética , Distrofia Endotelial de Fuchs/genética , Empalme Alternativo/genética , Transcriptoma/genética , Células Endoteliales/metabolismo , Endotelio Corneal/metabolismo , Endotelio Corneal/patología , MasculinoRESUMEN
ABCA4-related retinopathy is the most common inherited Mendelian eye disorder worldwide, caused by biallelic variants in the ATP-binding cassette transporter ABCA4. To date, over 2200 ABCA4 variants have been identified, including missense, nonsense, indels, splice site and deep intronic defects. Notably, more than 60% are missense variants that can lead to protein misfolding, mistrafficking and degradation. Currently no approved therapies target ABCA4. In this study, we demonstrate that ABCA4 misfolding variants are temperature-sensitive and reduced temperature growth (30 °C) improves their traffic to the plasma membrane, suggesting the folding of these variants could be rescuable. Consequently, an in vitro platform was developed for the rapid and robust detection of ABCA4 traffic to the plasma membrane in transiently transfected cells. The system was used to assess selected candidate small molecules that were reported to improve the folding or traffic of other ABC transporters. Two candidates, 4-PBA and AICAR, were identified and validated for their ability to enhance both wild-type ABCA4 and variant trafficking to the cell surface in cell culture. We envision that this platform could serve as a primary screen for more sophisticated in vitro testing, enabling the discovery of breakthrough agents to rescue ABCA4 protein defects and mitigate ABCA4-related retinopathy.
Asunto(s)
Transportadoras de Casetes de Unión a ATP , Pliegue de Proteína , Transporte de Proteínas , Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/metabolismo , Humanos , Pliegue de Proteína/efectos de los fármacos , Células HEK293 , Membrana Celular/metabolismo , Bibliotecas de Moléculas Pequeñas/farmacologíaRESUMEN
Fuchs endothelial corneal dystrophy (FECD) is a common disease for which corneal transplantation is the only treatment option in advanced stages, and alternative treatment strategies are urgently required. Expansion (≥50 copies) of a non-coding trinucleotide repeat in TCF4 confers >76-fold risk for FECD in our large cohort of affected individuals. An FECD subject-derived corneal endothelial cell (CEC) model was developed to probe disease mechanism and investigate therapeutic approaches. The CEC model demonstrated that the repeat expansion leads to nuclear RNA foci, with the sequestration of splicing factor proteins (MBNL1 and MBNL2) to the foci and altered mRNA processing. Antisense oligonucleotide (ASO) treatment led to a significant reduction in the incidence of nuclear foci, MBNL1 recruitment to the foci, and downstream aberrant splicing events, suggesting functional rescue. This proof-of-concept study highlights the potential of a targeted ASO therapy to treat the accessible and tractable corneal tissue affected by this repeat expansion-mediated disease.
Asunto(s)
Distrofia Endotelial de Fuchs/genética , Predisposición Genética a la Enfermedad , Oligonucleótidos Antisentido/farmacología , Factor de Transcripción 4/genética , Expansión de Repetición de Trinucleótido/genética , Anciano , Animales , Núcleo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Estudios de Cohortes , Células Endoteliales/metabolismo , Endotelio Corneal/patología , Femenino , Distrofia Endotelial de Fuchs/patología , Humanos , Masculino , Ratones Endogámicos C57BL , Especificidad de Órganos , Precursores del ARN/genética , Procesamiento Postranscripcional del ARN , Factores de Empalme de ARN/metabolismo , ARN Mensajero/metabolismo , Factores de RiesgoRESUMEN
PURPOSE: To demonstrate the utility of an amplification-free long-read sequencing method to characterize the Fuchs endothelial corneal dystrophy (FECD)-associated intronic TCF4 triplet repeat (CTG18.1). METHODS: We applied an amplification-free method, utilizing the CRISPR/Cas9 system, in combination with PacBio single-molecule real-time (SMRT) long-read sequencing, to study CTG18.1. FECD patient samples displaying a diverse range of CTG18.1 allele lengths and zygosity status (n = 11) were analyzed. A robust data analysis pipeline was developed to effectively filter, align, and interrogate CTG18.1-specific reads. All results were compared with conventional polymerase chain reaction (PCR)-based fragment analysis. RESULTS: CRISPR-guided SMRT sequencing of CTG18.1 provided accurate genotyping information for all samples and phasing was possible for 18/22 alleles sequenced. Repeat length instability was observed for all expanded (≥50 repeats) phased CTG18.1 alleles analyzed. Furthermore, higher levels of repeat instability were associated with increased CTG18.1 allele length (mode length ≥91 repeats) indicating that expanded alleles behave dynamically. CONCLUSION: CRISPR-guided SMRT sequencing of CTG18.1 has revealed novel insights into CTG18.1 length instability. Furthermore, this study provides a framework to improve the molecular diagnostic accuracy for CTG18.1-mediated FECD, which we anticipate will become increasingly important as gene-directed therapies are developed for this common age-related and sight threatening disease.
Asunto(s)
Distrofia Endotelial de Fuchs/genética , Predisposición Genética a la Enfermedad , Factor de Transcripción 4/genética , Expansión de Repetición de Trinucleótido/genética , Adulto , Anciano , Anciano de 80 o más Años , Alelos , Sistemas CRISPR-Cas/genética , Femenino , Distrofia Endotelial de Fuchs/patología , Genotipo , Humanos , Intrones/genética , Masculino , Persona de Mediana Edad , Análisis de Secuencia de ADN , Imagen Individual de Molécula , Repeticiones de Trinucleótidos/genéticaRESUMEN
PURPOSE: To characterise the phenotype and genotype of concurrent keratoconus and Fuchs endothelial corneal dystrophy (KC + FECD). METHODS: We recruited 20 patients with concurrent KC + FECD for a retrospective observational case series from the United Kingdom and the Czech Republic. We compared eight parameters of corneal shape (Pentacam, Oculus) with two groups of age-matched controls who had either isolated keratoconus (KC) or isolated FECD. We genotyped probands for an intronic triplet TCF4 repeat expansion (CTG18.1) and the ZEB1 variant c.1920G >T p.(Gln640His). RESULTS: The median age at diagnosis of patients with KC + FECD was 54 (interquartile range 46 to 66) years, with no evidence of KC progression (median follow-up 84 months, range 12 to 120 months). The mean (standard deviation (SD)) of the minimum corneal thickness, 493 (62.7) µm, was greater than eyes with KC, 458 (51.1) µm, but less than eyes with FECD, 590 (55.6) µm. Seven other parameters of corneal shape were more like KC than FECD. Seven (35%) probands with KC + FECD had a TCF4 repeat expansion of ≥50 compared to five controls with isolated FECD. The average of the largest TCF4 expansion in cases with KC + FECD (46 repeats, SD 36 repeats) was similar to the age-matched controls with isolated FECD (36 repeats, SD 28 repeats; p = 0.299). No patient with KC + FECD harboured the ZEB1 variant. CONCLUSIONS: The KC + FECD phenotype is consistent with KC but with superimposed stromal swelling from endothelial disease. The proportion of cases with a TCF4 expansion is similar in concurrent KC + FECD and age-matched controls with isolated FECD.
Asunto(s)
Distrofia Endotelial de Fuchs , Queratocono , Humanos , Distrofia Endotelial de Fuchs/complicaciones , Distrofia Endotelial de Fuchs/diagnóstico , Distrofia Endotelial de Fuchs/genética , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/genética , Factor de Transcripción 4/genética , Estudios Retrospectivos , Queratocono/complicaciones , Queratocono/diagnóstico , Queratocono/genética , Factores de Transcripción/genética , Genotipo , FenotipoRESUMEN
Corticotropin Releasing Factor (CRF) neuropeptides coordinate the stress response via two distinct membrane receptors (CRF-Rs). We have previously shown expression of both CRF-Rs in human breast cancer tissues. In the present study, we examined in vitro using the MCF-7 cell line model, the regulation of CRF-Rs expression and their signaling in hormone-dependent breast cancer growth. Our findings show that similarly to breast cancer biopsies, the predominant receptor type expressed in the cell line is CRF-R2α. The transcription of CRF-R1 and CRF-R2 is up and down-regulated respectively by exposure to estradiol (E2); however this effect seems not to be exerted at the level of promoter gene methylation, although in human breast cancer specimens, CRF-R1 methylation was found to be positively associated with the presence of steroid hormone receptors. Finally, we showed that specific activation of CRF-R2 increased the migration of MCF-7 cells and potentiated an estrogen-inducing effect. Our data support an involvement of CRF-R signaling in breast cancer pathophysiology via a regulatory steroid-hormone interplay.
Asunto(s)
Neoplasias de la Mama/metabolismo , Receptores de Hormona Liberadora de Corticotropina/metabolismo , Adulto , Anciano , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Línea Celular Tumoral , Movimiento Celular/genética , Movimiento Celular/fisiología , Proliferación Celular/genética , Proliferación Celular/fisiología , Técnica del Anticuerpo Fluorescente , Humanos , Células MCF-7 , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
Maintenance of protein homeostasis is vitally important in post-mitotic cells, particularly neurons. Neurodegenerative diseases such as polyglutamine expansion disorders-like Huntington's disease or spinocerebellar ataxia (SCA), Alzheimer's disease, fronto-temporal dementia (FTD), amyotrophic lateral sclerosis (ALS) and Parkinson's disease-are often characterized by the presence of inclusions of aggregated protein. Neurons contain complex protein networks dedicated to protein quality control and maintaining protein homeostasis, or proteostasis. Molecular chaperones are a class of proteins with prominent roles in maintaining proteostasis, which act to bind and shield hydrophobic regions of nascent or misfolded proteins while allowing correct folding, conformational changes and enabling quality control. There are many different families of molecular chaperones with multiple functions in proteostasis. The DNAJ family of molecular chaperones is the largest chaperone family and is defined by the J-domain, which regulates the function of HSP70 chaperones. DNAJ proteins can also have multiple other protein domains such as ubiquitin-interacting motifs or clathrin-binding domains leading to diverse and specific roles in the cell, including targeting client proteins for degradation via the proteasome, chaperone-mediated autophagy and uncoating clathrin-coated vesicles. DNAJ proteins can also contain ER-signal peptides or mitochondrial leader sequences, targeting them to specific organelles in the cell. In this review, we discuss the multiple roles of DNAJ proteins and in particular focus on the role of DNAJ proteins in protecting against neurodegenerative diseases caused by misfolded proteins. We also discuss the role of DNAJ proteins as direct causes of inherited neurodegeneration via mutations in DNAJ family genes.This article is part of the theme issue 'Heat shock proteins as modulators and therapeutic targets of chronic disease: an integrated perspective'.
Asunto(s)
Proteínas Fetales/genética , Proteínas del Choque Térmico HSP40/genética , Chaperonas Moleculares/genética , Enfermedades Neurodegenerativas/genética , Animales , Proteínas Fetales/metabolismo , Proteínas del Choque Térmico HSP40/metabolismo , Humanos , Ratones , Chaperonas Moleculares/metabolismo , Pliegue de Proteína , RatasRESUMEN
OBJECTIVE: The corticotropin-releasing factor (CRF) family consists of the neuropeptides CRF, Ucn I, II and III and the binding sites CRFR1, CRFR2 and CRF-BP. It regulates stress response and the homeostasis of an organism. In this study, we examined the presence of the CRF system in the human hearts of normal and pathological fetuses. DESIGN: Heart tissues from 40 archival human fetuses were divided into Group A (without pathology, 'normal'), Group B (with chromosomal abnormalities) and Group C (with congenital disorders). Immunohistochemistry was used to localize the CRF system. Results correlated to gestational trimester and pathology. RESULTS: Immunoreactivity for all antigens was found in cardiac myocytes of all groups, in almost all samples, except Ucn III which was present in almost half of the fetuses of Groups B and C and was not detected at all in Group A. Ucn III was more often present during the earlier stage of development (<21 weeks) and in fetuses with congenital disorders. In a fetus diagnosed with heart pathology, all but Ucn III antigens were also present. CONCLUSIONS: We localized a complete CRF system in the human fetal heart and correlated the presence of Ucn III to development and pathology. More studies are needed to verify and clarify the exact role of the CRF system in the human fetal heart.
RESUMEN
OBJECTIVE: The corticotropin-releasing factor (CRF) family consists of the neuropeptides CRF, Ucn I, II and III and the binding sites CRFR1, CRFR2 and CRF-BP. It regulates stress response and the homeostasis of an organism. In this study, we examined the presence of the CRF system in the human hearts of normal and pathological fetuses. DESIGN: Heart tissues from 40 archival human fetuses were divided into Group A (without pathology, 'normal'), Group B (with chromosomal abnormalities) and Group C (with congenital disorders). Immunohistochemistry was used to localize the CRF system. Results correlated to gestational trimester and pathology. RESULTS: Immunoreactivity for all antigens was found in cardiac myocytes of all groups, in almost all samples, except Ucn III which was present in almost half of the fetuses of Groups B and C and was not detected at all in Group A. Ucn III was more often present during the earlier stage of development (<21weeks) and in fetuses with congenital disorders. In a fetus diagnosed with heart pathology, all but Ucn III antigens were also present. CONCLUSIONS: We localized a complete CRF system in the human fetal heart and correlated the presence of Ucn III to development and pathology. More studies are needed to verify and clarify the exact role of the CRF system in the human fetal heart.