RESUMEN
The sperm acrosome is a lysosome-related organelle that develops using membrane trafficking from the Golgi apparatus as well as the endolysosomal compartment. How vesicular trafficking is regulated in spermatids to form the acrosome remains to be elucidated. VPS13B, a RAB6-interactor, was recently shown involved in endomembrane trafficking. Here, we report the generation of the first Vps13b-knockout mouse model and show that male mutant mice are infertile due to oligoasthenoteratozoospermia. This phenotype was explained by a failure of Vps13b deficient spermatids to form an acrosome. In wild-type spermatids, immunostaining of Vps13b and Rab6 revealed that they transiently locate to the acrosomal inner membrane. Spermatids lacking Vps13b did not present with the Golgi structure that characterizes wild-type spermatids and showed abnormal targeting of PNA- and Rab6-positive Golgi-derived vesicles to Eea1- and Lamp2-positive structures. Altogether, our results uncover a function of Vps13b in the regulation of the vesicular transport between Golgi apparatus, acrosome, and endolysosome.
Asunto(s)
Acrosoma/metabolismo , Transporte Biológico/fisiología , Aparato de Golgi/metabolismo , Espermatogénesis/fisiología , Proteínas de Transporte Vesicular/metabolismo , Animales , Lisosomas/metabolismo , Masculino , Ratones , Ratones Noqueados , Transporte de Proteínas/fisiología , Espermátides/metabolismo , Espermatozoides/metabolismoRESUMEN
PIK3CA-related overgrowth spectrum is caused by mosaicism mutations in the PIK3CA gene. These mutations, which are also observed in various types of cancer, lead to a constitutive activation of the PI3K/AKT/mTOR pathway, increasing cell proliferation. Heat shock transcription factor 1 (HSF1) is the major stress-responsive transcription factor. Recent findings indicate that AKT phosphorylates and activates HSF1 independently of heat-shock in breast cancer cells. Here, we aimed to investigate the role of HSF1 in PIK3CA-related overgrowth spectrum. We observed a higher rate of proliferation and increased phosphorylation of AKT and p70S6K in mutant fibroblasts than in control cells. We also found elevated phosphorylation and activation of HSF1, which is directly correlated to AKT activation. Specific AKT inhibitors inhibit HSF1 phosphorylation as well as HSF1-dependent gene transcription. Finally, we demonstrated that targeting HSF1 with specific inhibitors reduced the proliferation of mutant cells. As there is currently no curative treatment for PIK3CA-related overgrowth spectrum, our results identify HSF1 as a new potential therapeutic target.
Asunto(s)
Proliferación Celular/efectos de los fármacos , Fosfatidilinositol 3-Quinasa Clase I/metabolismo , Descubrimiento de Drogas , Factores de Transcripción del Choque Térmico/antagonistas & inhibidores , Lipoma/metabolismo , Anomalías Musculoesqueléticas/metabolismo , Nevo/metabolismo , Malformaciones Vasculares/metabolismo , Células Cultivadas , Fosfatidilinositol 3-Quinasa Clase I/genética , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Fibroblastos/patología , Factores de Transcripción del Choque Térmico/metabolismo , Humanos , Lipoma/tratamiento farmacológico , Lipoma/genética , Lipoma/patología , Terapia Molecular Dirigida , Anomalías Musculoesqueléticas/tratamiento farmacológico , Anomalías Musculoesqueléticas/genética , Anomalías Musculoesqueléticas/patología , Mutación , Nevo/tratamiento farmacológico , Nevo/genética , Nevo/patología , Fosforilación/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal/efectos de los fármacos , Malformaciones Vasculares/tratamiento farmacológico , Malformaciones Vasculares/genética , Malformaciones Vasculares/patologíaRESUMEN
Retinitis pigmentosa (RP) is a disease that primarily affects the peripheral retina and ultimately causes visual impairment. X-chromosomal forms of RP are frequently caused by mutations in the retinitis pigmentosa GTPase regulator (RPGR) gene. We show that the novel splice donor site (SDS) mutation c.1245+3A>T in intron 10 of RPGR cosegregates with RP in a five-generation Caucasian family. The mutation causes in-frame skipping of exon 10 from RPGR transcripts in patient-derived primary fibroblasts. To correct the splice defect, we developed a gene therapeutic approach using mutation-adapted U1 small nuclear RNA (U1). U1 is required for SDS recognition of pre-mRNAs and initiates the splice process. The mutation described herein interferes with the recognition of the SDS by U1. To overcome the deleterious effects of the mutation, we generated four U1 isoforms with increasing complementarity to the SDS. Lentiviral particles were used to transduce patient-derived fibroblasts with these U1 variants. Full complementarity of U1 corrects the splice defect partially and increases recognition of the mutant SDS. The therapeutic effect is U1-concentration dependent as we show for endogenously expressed RPGR transcripts in patient-derived cells. U1-based gene therapeutic approaches constitute promising technologies to treat SDS mutations in inherited diseases including X-linked RP.
Asunto(s)
Proteínas del Ojo/genética , Mutación , Empalme del ARN , ARN Nuclear Pequeño/genética , Retinitis Pigmentosa/terapia , Células Cultivadas , Proteínas del Ojo/metabolismo , Terapia Genética , Células HEK293 , Humanos , Sitios de Empalme de ARN/genética , Retinitis Pigmentosa/genética , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Cohen syndrome (CS) is a rare syndromic form of rod-cone dystrophy. Recent case reports have suggested that cystoid maculopathy (CM) could affect CS patients with an early onset and high prevalence. Our study aims at improving our understanding and management of CM in CS patients through a retrospective case series of ten CS patients with identified pathogenic variants in VPS13B. Longitudinal optical coherence tomography (OCT) imaging was performed and treatment with carbonic anhydrase inhibitors (CAI) was provided to reduce the volume of cystoid spaces. CM affected eight out of ten patients in our cohort. The youngest patient showed a strong progression of macular cysts from the age of 4.5 to 5 years despite oral CAI medication. Other teenage and young adult patients showed stable macular cysts with and without treatment. One patient showed a moderate decrease of cystoid spaces in the absence of treatment at 22 years of age. Through a correlative analysis we found that the volume of cystoid spaces was positively correlated to the thickness of peripheral and macular photoreceptor-related layers. This study suggests that CAI treatments may not suffice to improve CM in CS patients, and that CM may resolve spontaneously during adulthood as photoreceptor dystrophy progresses.
Asunto(s)
Dedos/anomalías , Discapacidad Intelectual/patología , Degeneración Macular/patología , Edema Macular/patología , Microcefalia/patología , Hipotonía Muscular/patología , Miopía/patología , Obesidad/patología , Degeneración Retiniana/patología , Adolescente , Adulto , Niño , Preescolar , Discapacidades del Desarrollo/patología , Femenino , Dedos/patología , Humanos , Masculino , Estudios Retrospectivos , Tomografía de Coherencia Óptica/métodos , Adulto JovenRESUMEN
Purpose: Cohen syndrome (CS) is a rare genetic disorder caused by variants of the VPS13B gene. CS patients are affected with a severe form of retinal dystrophy, and in several cases cataracts also develop. The purpose of this study was to investigate the mechanisms and risk factors for cataract in CS, as well as to report on cataract surgeries in CS patients. Methods: To understand how VPS13B is associated with visual impairments in CS, we generated the Vps13b∆Ex3/∆Ex3 mouse model. Mice from 1 to 3 months of age were followed by ophthalmoscopy and slit-lamp examinations. Phenotypes were investigated by histology, immunohistochemistry, and western blot. Literature analysis was performed to determine specific characteristic features of cataract in CS and to identify potential genotype-phenotype correlations. Results: Cataracts rapidly developed in 2-month-old knockout mice and were present in almost all lenses at 3 months. Eye fundi appeared normal until cataract development. Lens immunostaining revealed that cataract formation was associated with the appearance of large vacuoles in the cortical area, epithelial-mesenchymal transition, and fibrosis. In later stages, cataracts became hypermature, leading to profound retinal remodeling due to inflammatory events. Literature analysis showed that CS-related cataracts display specific features compared to other forms of retinitis pigmentosa-related cataracts, and their onset is modified by additional genetic factors. Corroboratively, we were able to isolate a subline of the Vps13b∆Ex3/∆Ex3 model with delayed cataract onset. Conclusions: VPS13B participates in lens homeostasis, and the CS-related cataract development dynamic is linked to additional genetic factors.
Asunto(s)
Catarata/genética , Dedos/anomalías , Regulación de la Expresión Génica , Homeostasis/genética , Discapacidad Intelectual/complicaciones , Cristalino/metabolismo , Microcefalia/complicaciones , Hipotonía Muscular/complicaciones , Miopía/complicaciones , Obesidad/complicaciones , ARN/genética , Degeneración Retiniana/complicaciones , Proteínas de Transporte Vesicular/genética , Animales , Western Blotting , Catarata/etiología , Catarata/metabolismo , Discapacidades del Desarrollo/complicaciones , Discapacidades del Desarrollo/genética , Discapacidades del Desarrollo/metabolismo , Modelos Animales de Enfermedad , Discapacidad Intelectual/genética , Discapacidad Intelectual/metabolismo , Cristalino/patología , Ratones Endogámicos C57BL , Ratones Noqueados , Microcefalia/genética , Microcefalia/metabolismo , Hipotonía Muscular/genética , Hipotonía Muscular/metabolismo , Miopía/genética , Miopía/metabolismo , Obesidad/genética , Obesidad/metabolismo , Degeneración Retiniana/genética , Degeneración Retiniana/metabolismo , Proteínas de Transporte Vesicular/biosíntesisRESUMEN
Cohen syndrome (CS) is a rare genetic disorder due to mutations in VPS13B gene. Among various clinical and biological features, CS patients suffer from inconsistent neutropenia, which is associated with recurrent but minor infections. We demonstrate here that this neutropenia results from an exaggerate rate of neutrophil apoptosis. Besides this increased cell death, which occurs in the absence of any endoplasmic reticulum stress or defect in neutrophil elastase (ELANE) expression or localization, all neutrophil functions appeared to be normal. We showed a disorganization of the Golgi apparatus in CS neutrophils precursors, that correlates with an altered glycosylation of ICAM-1 in these cells, as evidenced by a migration shift of the protein. Furthermore, a striking decrease in the expression of SERPINB1 gene, which encodes a critical component of neutrophil survival, was detected in CS neutrophils. These abnormalities may account for the excessive apoptosis of neutrophils leading to neutropenia in CS. KEY MESSAGES: Cohen syndrome patients' neutrophils display normal morphology and functions. Cohen syndrome patients' neutrophils have an increased rate of spontaneous apoptosis compared to healthy donors' neutrophils. No ER stress or defective ELA2 expression or glycosylation was observed in Cohen syndrome patients' neutrophils. SerpinB1 expression is significantly decreased in Cohen syndrome neutrophils as well as in VPS13B-deficient cells.
Asunto(s)
Apoptosis , Dedos/anomalías , Discapacidad Intelectual/genética , Microcefalia/genética , Hipotonía Muscular/genética , Miopía/genética , Neutropenia/genética , Neutrófilos/patología , Obesidad/genética , Degeneración Retiniana/genética , Serpinas/genética , Adolescente , Adulto , Niño , Preescolar , Discapacidades del Desarrollo/complicaciones , Discapacidades del Desarrollo/genética , Discapacidades del Desarrollo/patología , Regulación hacia Abajo , Femenino , Dedos/patología , Humanos , Discapacidad Intelectual/complicaciones , Discapacidad Intelectual/patología , Masculino , Microcefalia/complicaciones , Microcefalia/patología , Persona de Mediana Edad , Hipotonía Muscular/complicaciones , Hipotonía Muscular/patología , Mutación , Miopía/complicaciones , Miopía/patología , Neutropenia/etiología , Neutropenia/patología , Neutrófilos/metabolismo , Obesidad/complicaciones , Obesidad/patología , Degeneración Retiniana/complicaciones , Degeneración Retiniana/patología , Adulto JovenRESUMEN
PURPOSE: Gene therapies to treat eye disorders have been extensively studied in the past 20 years. Frequently, adeno-associated viruses were applied to the subretinal or intravitreal space of the eye to transduce retinal cells with nucleotide sequences of therapeutic potential. In this study we describe a novel intravitreal injection procedure that leads to a reproducible adeno-associated virus (AAV)2/8-mediated transduction of more than 70% of the retina. METHODS: Prior to a single intravitreal injection of a enhanced green fluorescent protien (GFP)-expressing viral suspension, we performed an aspiration of vitreous tissue from wild-type C57Bl/6J mice. One and one-half microliters of AAV2/8 suspension was injected. Funduscopy, optical coherence tomography (OCT), laser scanning microscopy of retinal flat mounts, cryosections of eye cups, and ERG recordings verified the efficacy and safety of the method. RESULTS: The combination of vitreous aspiration and intravitreal injection resulted in an almost complete transduction of the retina in approximately 60% of the eyes and showed transduced cells in all retinal layers. Photoreceptors and RPE cells were predominantly transduced. Eyes presented with well-preserved retinal morphology. Electroretinographic recordings suggested that the new combination of techniques did not cause significant alterations of the retinal physiology. CONCLUSIONS: We show a novel application technique of AAV2/8 to the vitreous of mice that leads to widespread transduction of the retina. The results of this study have implications for virus-based gene therapies and basic science; for example, they might provide an approach to apply gene replacement strategies or clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 in vivo. It may further help to develop similar techniques for larger animal models or humans.
Asunto(s)
Dependovirus/genética , Terapia Genética/métodos , Vectores Genéticos/administración & dosificación , Enfermedades de la Retina/terapia , Succión/métodos , Transducción Genética/métodos , Animales , Dependovirus/patogenicidad , Modelos Animales de Enfermedad , Electrorretinografía , Inyecciones Intravítreas , Ratones , Ratones Endogámicos C57BL , Microscopía Confocal , Epitelio Pigmentado Ocular , Retina/patología , Retina/fisiopatología , Enfermedades de la Retina/diagnóstico , Tomografía de Coherencia Óptica , Transgenes , Cuerpo VítreoRESUMEN
The diagnoses of retinitis pigmentosa (RP) and stationary night blindness (CSNB) are two distinct clinical entities belonging to a group of clinically and genetically heterogeneous retinal diseases. The current study focused on the identification of causative mutations in the RP-affected index patient and in several members of the same family that reported a phenotype resembling CSNB. Ophthalmological examinations of the index patient confirmed a typical form of RP. In contrast, clinical characterizations and ERGs of another affected family member showed the Riggs-type CSNB lacking signs of RP. Applying whole exome sequencing we detected the non-synonymous substitution c.337G > A, p.E113 K in the rhodopsin (RHO) gene. The mutation co-segregated with the diseases. The identification of the pathogenic variant p.E113 K is the first description of a naturally-occurring mutation in the Schiff base counterion of RHO in human patients. The heterozygous mutation c.337G > A in exon 1 was confirmed in the index patient as well as in five CSNB-affected relatives. This pathogenic sequence change was excluded in a healthy family member and in 199 ethnically matched controls. Our findings suggest that a mutation in the biochemically well-characterized counterion p.E113 in RHO can be associated with RP or Riggs-type CSNB, even within the same family.