RESUMEN
Inherited retinal dystrophies (IRDs) are major causes of visual impairment and irreversible blindness worldwide, while the precise molecular and genetic mechanisms are still elusive. N6-methyladenosine (m6A) modification is the most prevalent internal modification in eukaryotic mRNA. YTH domain containing 2 (YTHDC2), an m6A reader protein, has recently been identified as a key player in germline development and human cancer. However, its contribution to retinal function remains unknown. Here, we explore the role of YTHDC2 in the visual function of retinal rod photoreceptors by generating rod-specific Ythdc2 knockout mice. Results show that Ythdc2 deficiency in rods causes diminished scotopic ERG responses and progressive retinal degeneration. Multi-omics analysis further identifies Ppef2 and Pde6b as the potential targets of YTHDC2 in the retina. Specifically, via its YTH domain, YTHDC2 recognizes and binds m6A-modified Ppef2 mRNA at the coding sequence and Pde6b mRNA at the 5'-UTR, resulting in enhanced translation efficiency without affecting mRNA levels. Compromised translation efficiency of Ppef2 and Pde6b after YTHDC2 depletion ultimately leads to decreased protein levels in the retina, impaired retinal function, and progressive rod death. Collectively, our finding highlights the importance of YTHDC2 in visual function and photoreceptor survival, which provides an unreported elucidation of IRD pathogenesis via epitranscriptomics.
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Células Fotorreceptoras de Vertebrados , Degeneración Retiniana , Animales , Humanos , Ratones , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 6/genética , Células Fotorreceptoras de Vertebrados/metabolismo , Células Fotorreceptoras de Vertebrados/patología , Degeneración Retiniana/genética , Degeneración Retiniana/metabolismo , Degeneración Retiniana/patología , ARN Helicasas/genética , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
The endoplasmic reticulum membrane protein complex (EMC) plays a critical role in the synthesis of multipass membrane proteins. Genetic studies indicated that mutations in EMC1 gene were associated with retinal degeneration diseases; however, the role of EMC1 in photoreceptor has not been confirmed. Here, we show that Emc1 ablation in the photoreceptor cells of mice recapitulated the retinitis pigmentosa phenotypes, including an attenuated scotopic electroretinogram response and the progressive degeneration of rod cells and cone cells. Histopathological examination of tissues from rod-specific Emc1 knockout mice revealed mislocalized rhodopsin and irregularly arranged cone cells at the age of 2 months. Further immunoblotting analysis revealed decreased levels of membrane proteins and endoplasmic reticulum chaperones in 1-month-old rod-specific Emc1 knockout mice retinae, and this led us to speculate that the loss of membrane proteins is the main cause of the degeneration of photoreceptors. EMC1 most likely regulated the membrane protein levels at an earlier step in the biosynthetic process before the proteins translocated into the endoplasmic reticulum. The present study demonstrates the essential roles of Emc1 in photoreceptor cells, and reveals the mechanism through which EMC1 mutations are linked to retinitis pigmentosa.
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Degeneración Retiniana , Retinitis Pigmentosa , Animales , Ratones , Modelos Animales de Enfermedad , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones Noqueados , Células Fotorreceptoras/metabolismo , Células Fotorreceptoras/patología , Degeneración Retiniana/genética , Degeneración Retiniana/metabolismo , Retinitis Pigmentosa/genética , Retinitis Pigmentosa/metabolismo , Retinitis Pigmentosa/patología , Retículo Endoplásmico/genética , Retículo Endoplásmico/metabolismoRESUMEN
BACKGROUND: As the most abundant epigenetic modification of eukaryotic mRNA, N6-methyladenosine (m6A) modification has been shown to play a role in mammalian nervous system development and function by regulating mRNA synthesis and degeneration. However, the role of m6A modification in retinal photoreceptors remains unknown. RESULTS: We generated the first retina-specific Mettl14-knockout mouse models using the Rho-Cre and HRGP-Cre lines and investigated the functions of Mettl14 in retinal rod and cone photoreceptors. Our data showed that loss of Mettl14 in rod cells causes a weakened scotopic photoresponse and rod degeneration. Further study revealed the ectopic accumulation of multiple outer segment (OS) proteins in the inner segment (IS). Deficiency of Mettl14 in cone cells led to the mislocalization of cone opsin proteins and the progressive death of cone cells. Moreover, Mettl14 depletion resulted in drastic decreases in METTL3/WTAP levels and reduced m6A methylation levels. Mechanistically, transcriptomic analyses in combination with MeRIP-seq illustrated that m6A depletion via inactivation of Mettl14 resulted in reduced expression levels of multiple phototransduction- and cilium-associated genes, which subsequently led to compromised ciliogenesis and impaired synthesis and transport of OS-residing proteins in rod cells. CONCLUSIONS: Our data demonstrate that Mettl14 plays an important role in regulating phototransduction and ciliogenesis events and is essential for photoreceptor function and survival, highlighting the importance of m6A modification in visual function.
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Metiltransferasas/metabolismo , Células Fotorreceptoras de Vertebrados , Retina , Animales , Mamíferos/genética , Metilación , Metiltransferasas/genética , Ratones , Células Fotorreceptoras de Vertebrados/metabolismo , Proteínas/metabolismo , ARN Mensajero/metabolismo , Retina/metabolismo , Células Fotorreceptoras Retinianas ConosRESUMEN
Yes-associated protein (YAP) is a major component of the Hippo pathway involved in development, growth, repair and homeostasis. Nonsense YAP1 mutations in humans result in autosomal dominant coloboma. Here, we generated a conditional knockout mouse model in which Yap1 was specifically deleted in embryonic retinal progenitor cells (RPCs) and in mature Müller cells using a Chx10-Cre driver. Our data demonstrated that the conditional ablation of Yap1 in embryonic RPCs does not prevent normal retinal development and caused no gross changes in retinal structure during embryonic and early postnatal life. Nevertheless, Yap1 deficient in retinal Müller cells in adult mice leads to impaired visual responses and extensive late-onset retinal degeneration, characterized by reduced cell number in all retinal layers. Immunofluorescence data further revealed the degeneration and death of rod and cone photoreceptors, bipolar cells, horizontal cells, amacrine cells and ganglion cells to varying degrees in aged knockout mice. Moreover, alteration of glial homeostasis and reactive gliosis were also observed. Finally, cell proliferation and TUNEL assay revealed that the broad retinal degeneration is mainly caused by enhanced apoptosis in late period. Together, this work uncovers that YAP is essential for the normal vision and retinal maintenance, highlighting the crucial role of YAP in retinal function and homeostasis.
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Degeneración Retiniana , Proteínas Señalizadoras YAP/metabolismo , Animales , Células Ependimogliales/metabolismo , Ratones , Ratones Noqueados , Neuroglía/metabolismo , Retina/metabolismo , Degeneración Retiniana/genéticaRESUMEN
N6-methyladenosine (m6A) modification, which is achieved by the METTL3/METTL14/WTAP methyltransferase complex, is the most abundant internal mRNA modification. Although recent evidence indicates that m6A can regulate neurodevelopment as well as synaptic function, the roles of m6A modification in the cerebellum and related synaptic connections are not well established. Here, we report that Purkinje cell (PC)-specific WTAP knockout mice display early-onset ataxia concomitant with cerebellar atrophy due to extensive PC degeneration and apoptotic cell death. Loss of Wtap also causes the aberrant degradation of multiple PC synapses. WTAP depletion leads to decreased expression levels of METTL3/14 and reduced m6A methylation in PCs. Moreover, the expression of GFAP and NF-L in the degenerating cerebellum is increased, suggesting severe neuronal injuries. In conclusion, this study demonstrates the critical role of WTAP-mediated m6A modification in cerebellar PCs, thus providing unique insights related to neurodegenerative disorders.
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Ataxia Cerebelosa , Células de Purkinje , Animales , Ataxia Cerebelosa/genética , Metilación , Metiltransferasas/genética , Ratones , Ratones Noqueados , Células de Purkinje/metabolismo , ARN Mensajero/genéticaRESUMEN
Type 2 diabetes mellitus (DM2) is associated closely with non-alcoholic fatty liver disease (NAFLD) by affecting lipid metabolism, which may lead to non-alcoholic steatohepatitis (NASH), fibrosis, and hepatocellular carcinoma (HCC). N6-methyladenosine (m6A) RNA methylation is an important epigenetic regulation for gene expression and is related to HCC development. We developed a new NAFLD model oriented from DM2 mouse, which spontaneously progressed to histological features of NASH, fibrosis, and HCC with high incidence. By RNA sequencing, protein expression and methylated RNA immunoprecipitation (MeRIP)-qPCR analysis, we found that enhanced expression of ACLY and SCD1 in this NAFLD model and human HCC samples was due to excessive m6A modification, but not elevation of mature SREBP1. Moreover, targeting METTL3/14 in vitro increases protein level of ACLY and SCD1 as well as triglyceride and cholesterol production and accumulation of lipid droplets. m6A sequencing analysis revealed that overexpressed METTL14 binds to mRNA of ACLY and SCD1 and alters their expression pattern. Our findings demonstrate a new NAFLD mouse model that provides a study platform for DM2-related NAFLD and reveals a unique epitranscriptional regulating mechanism for lipid metabolism via m6A-modified protein expression of ACLY and SCD1.
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Carcinoma Hepatocelular , Diabetes Mellitus Tipo 2 , Neoplasias Hepáticas , Enfermedad del Hígado Graso no Alcohólico , Animales , Carcinoma Hepatocelular/patología , Diabetes Mellitus Tipo 2/genética , Epigénesis Genética , Fibrosis , Lipogénesis/genética , Neoplasias Hepáticas/metabolismo , Ratones , Enfermedad del Hígado Graso no Alcohólico/metabolismo , ARN Mensajero/genéticaRESUMEN
Precise Norrin and ß-catenin (Norrin/ß-catenin; encoded by NDP and CTNNB1, respectively) signaling is critical for proper angiogenesis. Dysregulation of this signaling leads to various diseases, of which retinal exudative vitreoretinopathy is the most prevalent. Here, we used a global knockout mouse model to show that limb development membrane protein 1 like (LMBR1L), a transmembrane protein of unknown function in angiogenesis, is essential for retinal vascular development. In vitro experiments revealed that LMBR1L depletion results in aberrant activation of the Norrin/ß-catenin signaling pathway via decreased ubiquitylation of FZD4 and increased Norrin co-receptor LRP5 and p-GSK3ß-Ser9 expression levels, which cause accumulation of ß-catenin. Moreover, inhibition of LMBR1L in human retinal microvascular endothelial cells (HRECs) caused increased proliferation ability and defective cell migration, which might have occurred as a result of upregulated expression levels of the apical junction components. Treatment with p-GSK3ß-Ser9 inhibitor AR-A014418 restored the phenotypes in LMBR1L-null HRECs, which further demonstrated the important regulatory role of LMBR1L in the Norrin/ß-catenin signaling pathway. Taken together, our data reveal an essential role for LMBR1L in angiogenesis. This article has an associated First Person interview with the first author of the paper.
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Células Endoteliales , Receptores de Superficie Celular/metabolismo , beta Catenina , Animales , Proliferación Celular , Células Endoteliales/metabolismo , Proteínas del Ojo/genética , Proteínas del Ojo/metabolismo , Receptores Frizzled/metabolismo , Glucógeno Sintasa Quinasa 3 beta/metabolismo , Humanos , Ratones , Neovascularización Patológica/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Transducción de Señal/genética , beta Catenina/metabolismoAsunto(s)
Vitreorretinopatías Exudativas Familiares , Proteína-6 Relacionada a Receptor de Lipoproteína de Baja Densidad , Vía de Señalización Wnt , Vitreorretinopatías Exudativas Familiares/genética , Humanos , Proteína-6 Relacionada a Receptor de Lipoproteína de Baja Densidad/genética , Vía de Señalización Wnt/genéticaRESUMEN
Retinal angiogenesis is a critical process for normal retinal function. However, uncontrolled angiogenesis can lead to pathological neovascularization (NV), which is closely related to most irreversible blindness-causing retinal diseases. Understanding the molecular basis behind pathological NV is important for the treatment of related diseases. Twist-related protein 1 (TWIST1) is a well-known transcription factor and principal inducer of epithelial-mesenchymal transition (EMT) in many human cancers. Our previous study showed that Twist1 expression is elevated in pathological retinal NV. To date, however, the role of TWIST1 in retinal pathological angiogenesis remains to be elucidated. To study the role of TWIST1 in pathological retinal NV and identify specific molecular targets for antagonizing pathological NV, we generated an inducible vascular endothelial cell (EC)-specific Twist1 transgenic mouse model ( Tg-Twist1 iEC+ ). Whole-mount retinas from Tg-Twist1 iEC+ mice showed retarded vascular progression and increased vascular density in the front end of the growing retinal vasculature, as well as aneurysm-like pathological retinal NV. Furthermore, overexpression of Twist1 in the ECs promoted cell proliferation but disturbed cell polarity, thus leading to uncontrolled retinal angiogenesis. TWIST1 promoted pathological NV by activating the Wnt/ß-catenin signaling pathway and inducing the expression of NV formation-related genes, thereby acting as a 'valve' in the regulation of pathological angiogenesis. This study identified the critical role of TWIST1 in retinal pathological NV, thus providing a potential therapeutic target for pathological NV.
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Neovascularización Patológica , Neovascularización Retiniana , Enfermedades de los Roedores , Animales , Células Endoteliales , Ratones , Ratones Transgénicos , Neovascularización Patológica/genética , Neovascularización Patológica/veterinaria , Neovascularización Retiniana/genética , Neovascularización Retiniana/veterinaria , Proteína 1 Relacionada con Twist/genéticaRESUMEN
Glaucoma is a leading cause of blindness, affecting 70 million people worldwide. Owing to the similarity in anatomy and physiology between human and mouse eyes and the ability to genetically manipulate mice, mouse models are an invaluable resource for studying mechanisms underlying disease phenotypes and for developing therapeutic strategies. Here, we report the discovery of a new mouse model of early-onset glaucoma that bears a transversion substitution c. G344T, which results in a missense mutation, p. R115L in PITX2. The mutation causes an elevation in intraocular pressure (IOP) and progressive death of retinal ganglion cells (RGC). These ocular phenotypes recapitulate features of pathologies observed in human glaucoma. Increased oxidative stress was evident in the inner retina. We demonstrate that the mutant PITX2 protein was not capable of binding to Nuclear factor-like 2 (NRF2), which regulates Pitx2 expression and nuclear localization, and to YAP1, which is necessary for co-initiation of transcription of downstream targets. PITX2-mediated transcription of several antioxidant genes were also impaired. Treatment with N-Acetyl-L-cysteine exerted a profound neuroprotective effect on glaucoma-associated neuropathies, presumably through inhibition of oxidative stress. Our study demonstrates that a disruption of PITX2 leads to glaucoma optic pathogenesis and provides a novel early-onset glaucoma model that will enable elucidation of mechanisms underlying the disease as well as to serve as a resource to test new therapeutic strategies.
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Glaucoma/genética , Glaucoma/metabolismo , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Mutación Missense , Factor 2 Relacionado con NF-E2/metabolismo , Transducción de Señal/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Proteínas Señalizadoras YAP/metabolismo , Animales , Apoptosis/genética , Modelos Animales de Enfermedad , Femenino , Células HEK293 , Humanos , Presión Intraocular , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Fenotipo , Células Ganglionares de la Retina/metabolismo , Transfección , Proteína del Homeodomínio PITX2RESUMEN
Phosphatidylserine (PS) is distributed asymmetrically in the plasma membrane of eukaryotic cells. Phosphatidylserine flippase (P4-ATPase) transports PS from the outer leaflet of the lipid bilayer to the inner leaflet of the membrane to maintain PS asymmetry. The ß subunit TMEM30A is indispensable for transport and proper function of P4-ATPase. Previous studies have shown that the ATP11A and TMEM30A complex is the molecular switch for myotube formation. However, the role of Tmem30a in skeletal muscle regeneration remains elusive. In the current study, Tmem30a was highly expressed in the tibialis anterior (TA) muscles of dystrophin-null ( mdx) mice and BaCl 2-induced muscle injury model mice. We generated a satellite cell (SC)-specific Tmem30a conditional knockout (cKO) mouse model to investigate the role of Tmem30a in skeletal muscle regeneration. The regenerative ability of cKO mice was evaluated by analyzing the number and diameter of regenerated SCs after the TA muscles were injured by BaCl 2-injection. Compared to the control mice, the cKO mice showed decreased Pax7 + and MYH3 + SCs, indicating diminished SC proliferation, and decreased expression of muscular regulatory factors (MYOD and MYOG), suggesting impaired myoblast proliferation in skeletal muscle regeneration. Taken together, these results demonstrate the essential role of Tmem30a in skeletal muscle regeneration.
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Proteínas de la Membrana/metabolismo , Músculo Esquelético/fisiología , Regeneración/fisiología , Células Satélite del Músculo Esquelético/metabolismo , Animales , Proliferación Celular , Distrofina/genética , Distrofina/metabolismo , Antagonistas de Estrógenos/toxicidad , Regulación de la Expresión Génica/fisiología , Genotipo , Proteínas de la Membrana/genética , Ratones , Ratones Endogámicos mdx , Ratones Noqueados , Músculo Esquelético/efectos de los fármacos , Proteína MioD/genética , Proteína MioD/metabolismo , Miogenina/genética , Miogenina/metabolismo , Cadenas Pesadas de Miosina/genética , Cadenas Pesadas de Miosina/metabolismo , Factor de Transcripción PAX7/genética , Factor de Transcripción PAX7/metabolismo , Regeneración/genética , Tamoxifeno/toxicidadRESUMEN
The asymmetric distribution of phosphatidylserine (PS) in the cytoplasmic leaflet of eukaryotic cell plasma membranes is regulated by a group of P4-ATPases (named PS flippases) and the ß-subunit TMEM30A. Podocytes in the glomerulus form a filtration barrier to prevent the traversing of large cellular elements and macromolecules from the blood into the urinary space. Damage to podocytes can disrupt the filtration barrier and lead to proteinuria and podocytopathy. We observed reduced TMEM30A expression in patients with minimal change disease and membranous nephropathy, indicating potential roles of TMEM30A in podocytopathy. To investigate the role of Tmem30a in the kidney, we generated a podocyte-specific Tmem30a knockout (KO) mouse model using the NPHS2-Cre line. Tmem30a KO mice displayed albuminuria, podocyte degeneration, mesangial cell proliferation with prominent extracellular matrix accumulation and eventual progression to focal segmental glomerulosclerosis. Our data demonstrate a critical role of Tmem30a in maintaining podocyte survival and glomerular filtration barrier integrity. Understanding the dynamic regulation of the PS distribution in the glomerulus provides a unique perspective to pinpointing the mechanism of podocyte damage and potential therapeutic targets.
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Albuminuria/genética , Glomeruloesclerosis Focal y Segmentaria/genética , Proteínas de la Membrana/genética , Fosfatidilserinas/metabolismo , Podocitos/metabolismo , Animales , Línea Celular , Humanos , Ratones , Ratones NoqueadosRESUMEN
The endoplasmic reticulum (ER) membrane protein complex (EMC) regulates the synthesis and quality control of membrane proteins with multiple transmembrane domains. One of the membrane spanning subunits, EMC3, is a core member of the EMC complex that provides essential hydrophilic vestibule for substrate insertion. Here, we show that the EMC subunit Emc3 plays critical roles in the retinal vascular angiogenesis by regulating Norrin/Wnt signaling. Postnatal endothelial cell (EC)-specific deletion of Emc3 led to retarded retinal vascular development with a hyperpruned vascular network, the appearance of blunt-ended, aneurysm-like tip endothelial cells (ECs) with reduced numbers of filopodia and leakage of erythrocytes at the vascular front. Diminished tube formation and cell proliferation were also observed in EMC3 depleted human retinal endothelial cells (HRECs). We then discovered a critical role for EMC3 in expression of FZD4 receptor of ß-catenin signaling using RNA sequencing, real-time quantitative PCR (RT-qPCR) and luciferase reporter assay. Moreover, augmentation of Wnt activity via lithium chloride (LiCl) treatment remarkably enhanced ß-catenin signaling and cell proliferation of HRECs. Additionally, LiCl partially reversed the angiogenesis defects in Emc3-cKO mice. Our data reveal that Emc3 plays essential roles in angiogenesis through direct control of FZD4 expression and Norrin/ß-catenin signaling.
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Retículo Endoplásmico/metabolismo , Receptores Frizzled/metabolismo , Proteínas de la Membrana/metabolismo , Neovascularización Patológica/metabolismo , Retina/metabolismo , Vía de Señalización Wnt , Animales , Proliferación Celular , Células Cultivadas , Humanos , Cloruro de Litio/farmacología , Ratones , beta Catenina/metabolismoRESUMEN
The processing, maturation, and secretion of insulin are under precise regulation, and dysregulation causes profound defects in glucose handling, leading to diabetes. Tmem30a is the ß subunit of the phosphatidylserine (PS) flippase, which maintains the membrane asymmetric distribution of PS. Tmem30a regulates cell survival and the localization of subcellular structures and is thus critical to the normal function of multiple physiological systems. Here, we show that conditional knockout of Tmem30a specifically in pancreatic islet ß cells leads to obesity, hyperglycemia, glucose intolerance, hyperinsulinemia, and insulin resistance in mice, due to insufficient insulin release. Moreover, we reveal that Tmem30a plays an essential role in clathrin-mediated vesicle transport between the trans Golgi network (TGN) and the plasma membrane (PM), which comprises immature secretory granule (ISG) budding at the TGN. We also find that Tmem30a deficiency impairs clathrin-mediated vesicle budding and thus blocks both insulin maturation in ISGs and the transport of glucose-sensing Glut2 to the PM. Collectively, these disruptions compromise both insulin secretion and glucose sensitivity, thus contributing to impairments in glucose-stimulated insulin secretion. Taken together, our data demonstrate an important role of Tmem30a in insulin maturation and glucose metabolic homeostasis and suggest the importance of membrane phospholipid distribution in metabolic disorders.
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Intolerancia a la Glucosa/genética , Transportador de Glucosa de Tipo 2/metabolismo , Hiperglucemia/genética , Hiperinsulinismo/genética , Resistencia a la Insulina/genética , Insulina/metabolismo , Proteínas de la Membrana/genética , Obesidad/genética , Animales , Membrana Celular/metabolismo , Modelos Animales de Enfermedad , Técnicas de Inactivación de Genes , Glucosa/efectos adversos , Intolerancia a la Glucosa/metabolismo , Hiperglucemia/metabolismo , Hiperinsulinismo/metabolismo , Secreción de Insulina , Células Secretoras de Insulina/citología , Células Secretoras de Insulina/efectos de los fármacos , Células Secretoras de Insulina/metabolismo , Ratones , Obesidad/metabolismo , Fosfatidilserinas/metabolismo , Red trans-Golgi/metabolismoRESUMEN
The asparaginase and isoaspartyl peptidase 1 (ASRGL1) is an L-asparaginase and beta-aspartyl peptidase enzyme that may be involved in the formation of L-aspartate, a neurotransmitter that can operate as an excitatory neurotransmitter in some brain regions. Although variants in ASRGL1 have been reported in retinitis pigmentosa (RP) patients, the in vivo functions and mechanisms of ASRGL in RP remains unknown due to the lack of suitable disease models. To explore the role of ASRGL in RP, we generated an Asrgl1 knockout mouse model (Asrgl1 KO) using the CRISPR/Cas9 technique. Asrgl1 ablation in mice led to an attenuated electroretinogram (ERG) response around 8 months. The thickness of the outer nuclei layer (ONL) started to decrease around 9 months in Asrgl1 KO mice and gradually intensified at 12 and 15 months. Immunostaining revealed thinner inner segment (IS) and thinner outer segment (OS) as well as the progressive degeneration of rod and cone cells in Asrgl1 KO mice. One hundred forty-nine transcriptional differentially expressed genes (DEGs) were found by RNA-seq in Asrgl1 KO retina. These DEGs were linked to a number of biological processes that were considerably enriched, including gastrointestinal disease and organismal injury and abnormalities. By analysis of canonical pathways, glucocorticoid receptor signaling was the most significant canonical pathway altered in Asrgl1 KO retina. Several molecules, including NFE2L2, IL-4, Foxp3, and Fos, were in the central nodes of the interaction network in Asrgl1 KO retina. In summary, our study provided a knockout mouse model for a better understanding of the molecular mechanism for ASRGL1-related RP.
RESUMEN
Purpose: Retinitis pigmentosa (RP) is an inherited and progressive degenerative retinal disease that often results in severe vision loss and blindness. However, mutations in known RP disease genes account for only 60% of RP cases, indicating that there are additional pathogenic mutations are yet to be identified. We aimed to identify the causative mutations in the eyes shut homolog (EYS) gene in a cohort of Chinese RP and rod-cone dystrophy families. Materials and Methods: Targeted next-generation sequencing was applied to identify novel mutations in these patients. Candidate variants were evaluated using bioinformatics tools. Mutations were confirmed by Sanger sequencing. Results: We identified eight heterozygous mutations in the EYS gene in the four probands, including a novel frameshift deletion mutation, c.8242_8243del (p.L2748fs); a novel insertion mutation, c.5802_5803insT (p.I1935YfsX6); a novel splicing mutation, c.1300-1G>A; two heterozygous stop-gain mutations, c.1750G>T (p.E584X) and c.8805C>A (p.Y2935X); and three novel missense mutations, c.8269G>A (p.V2757I), c.2545C>T (p.R849C) and c.7506C>A (p.S2502R). Only c.8805C>A had been reported previously in RP patients. None of these mutations were present in 1000 control individuals. Conclusions: We identified seven novel mutations in the EYS gene, expanding the mutational specra of EYS in Chinese patients with RP and rod-cone dystrophy.
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Proteínas del Ojo/genética , Retinitis Pigmentosa/genética , Adulto , Pueblo Asiatico/genética , China , Biología Computacional/métodos , Análisis Mutacional de ADN/métodos , Proteínas del Ojo/metabolismo , Femenino , Mutación del Sistema de Lectura/genética , Genes Recesivos/genética , Heterocigoto , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Humanos , Masculino , Persona de Mediana Edad , Mutación/genética , Mutación Missense/genética , LinajeRESUMEN
Phosphatidylserine flippase (P4-ATPase) transports PS from the outer to the inner leaflet of the lipid bilayer in the membrane to maintain PS asymmetry, which is important for biological activities of the cell. ATP11A is expressed in multiple tissues and plays a role in myotube formation. However, the detailed cellular function of ATP11A remains elusive. Mutation analysis revealed that I91, L308, and E897 residues in ATP8A2 are important for flippase activity. In order to investigate the roles of these corresponding amino acid residues in ATP11A protein, we assessed the expression and cellular localization of the respective ATP11A mutant proteins. ATP11A mainly localizes to the Golgi and plasma membrane when coexpressed with the ß-subunit of the complex TMEM30A. Y300F mutation causes reduced ATP11A expression, and Y300F and D913K mutations affect correct localization of the Golgi and plasma membrane. In addition, Y300F and D913K mutations also affect PS flippase activity. Our data provides insight into important residues of ATP11A.
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Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/metabolismo , Mutación/genética , Animales , Células COS , Membrana Celular/metabolismo , Chlorocebus aethiops , Aparato de Golgi/genética , Aparato de Golgi/metabolismo , Células HEK293 , Humanos , Mutagénesis Sitio-Dirigida , Mutación/fisiología , Fosfolípidos/metabolismoRESUMEN
Retinitis pigmentosa (RP) is a group of inheritable blindness retinal diseases characterized by the death of photoreceptor cells and a gradual loss of peripheral vision. Mutations in Usher syndrome type 2 (USH2A) have been reported in RP with or without hearing loss. The present study aimed to identify causative mutations in a cohort of families with RP from China. A cohort of 62 nonsyndromic families with RP and 30 sporadic cases were enrolled in this study. All affected members underwent a complete ophthalmic examination, including fundus photography, visualfield test and optical coherence tomography examination. Nextgeneration sequencingtargeted sequencing of 163 genes involved in inheritable retinal disorders was performed on the probands. Stringent bioinformatics data analysis was applied to identify potential candidate variants. In total, 6 novel mutations and 2 known mutations of USH2A were identified in 4 families with RP. A stopgain mutation (c.C1731A) and a missense mutation (c.G8254A) were identified in RP family RP2148. In another RP family, RP2150, a known mutation (c.G802A) and a novel frameshift insertion mutation (c.12086dupA) were discovered. A novel stopgain mutation (c.G11754A) and a missense mutation (c.G13465A) were identified in family rpz05. A novel missense mutation (c.C9328G) and a known missense mutation (c.G8232C) were also identified. These mutations were subsequently confirmed by Sanger sequencing. All 6 novel mutations affected highly conserved amino acid residues, and were absent in 1,000 ethnically matched controls. Taken together, the present study has reported on 6 novel USH2A mutations in 4 families with RP, and has expanded the mutation spectrum of USH2A in autosomal recessive RP in the Chinese population, thus providing important information for the molecular diagnosis and screening of RP.
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Proteínas de la Matriz Extracelular/genética , Mutación , Retinitis Pigmentosa/genética , Adolescente , Adulto , Anciano , Análisis Mutacional de ADN , Femenino , Mutación del Sistema de Lectura , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Masculino , Persona de Mediana Edad , Mutación Missense , Linaje , Adulto JovenRESUMEN
Variants in interphotoreceptor matrix proteoglycans (IMPG2) have been reported in retinitis pigmentosa (RP) and vitelliform macular dystrophy (VMD) patients. However, the underlying molecular mechanisms remain elusive due to a lack of suitable disease models. We developed two independent Impg2 knockout (KO) mouse models using the CRISPR/Cas9 technique to assess the in vivo functions of Impg2 in the retina. Impg2 ablation in mice recapitulated the RP phenotypes of patients, including an attenuated electroretinogram (ERG) response and the progressive degeneration of photoreceptors. The histopathological examination of Impg2-KO mice revealed irregularly arranged rod cells and mislocalized rhodopsin protein in the inner segment at 6 months of age. In addition to the pathological changes in rod cells, cone cells were also affected in KO retinas. KO retinas exhibited progressive cone cell death and impaired cone cell elongation. Further immunoblotting analysis revealed increased levels of endoplasmic reticulum (ER) stress-related proteins, including C/EBP homologous protein (CHOP), immunoglobulin heavy-chain-binding protein (BIP) and protein disulfide isomerase (PDI), in Impg2-KO mouse retinas. Increased gliosis and apoptotic cell death were also observed in the KO retinas. As autophagy is closely associated with ER stress, we then checked whether autophagy was disturbed in Impg2-KO mouse retinas. The results showed that autophagy was impaired in KO retinas, as revealed by the increased accumulation of SQSTM1 and other proteins involved in autophagy. Our results demonstrate the essential roles of Impg2 in the retina, and this study provides novel models for mechanistic investigations and development of therapies for RP caused by IMPG2 mutations.
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Estrés del Retículo Endoplásmico/genética , Proteoglicanos/genética , Retina/metabolismo , Degeneración Retiniana/genética , Rodopsina/genética , Animales , Autofagia/genética , Sistemas CRISPR-Cas/genética , Muerte Celular/genética , Chaperón BiP del Retículo Endoplásmico , Proteínas de Choque Térmico/genética , Humanos , Ratones , Ratones Noqueados , Proteína Disulfuro Isomerasas/genética , Retina/patología , Células Fotorreceptoras Retinianas Conos/metabolismo , Células Fotorreceptoras Retinianas Conos/patología , Degeneración Retiniana/patología , Células Fotorreceptoras Retinianas Bastones/metabolismo , Células Fotorreceptoras Retinianas Bastones/patología , Factor de Transcripción CHOP/genéticaRESUMEN
Background: Familial exudative vitreoretinopathy (FEVR) is an inheritable retinal vascular disease, which often leads to severe vision loss and blindness in children. However, reported mutations can only account for 50-60% of patients with FEVR. The purpose of this study was to identify novel frizzled class receptor 4 (FZD4) and Norrin cystine knot growth factor NDP (NDP) mutations in a cohort of Indian patients with FEVR by whole-exome sequencing. Methods: We performed data filtering and bioinformatic analyses. Results: Two novel heterozygous mutations in FZD4 gene were identified, each in two different families: c.1499_1500del [p.500_500del] and c.G296C [p.C99S]. One novel mutation in NDP in another family was identified: c.A256G [p.K86E]. All FZD4 mutations affected conserved amino acid residues and were absent in 1000 control individuals. To assess the effect of these FZD4 mutations on the biological activity of the protein, we introduced each FZD4 mutation into FZD4 cDNA by the site-directed mutagenesis techniques. A Norrin/beta-catenin pathway-based luciferase reporter assay revealed that the c.1499_1500del failed to activate the luciferase reporter; in contrast, compared with the wild-type FZD4 protein, the, c.G296C [p.C99S] mutation exhibited increased luciferase reporter activity. Conclusion: Our study found two novel FZD4 mutations, with opposite effects regarding functional expression levels in Indian patients with FEVR and expands on the mutational spectrum of FZD4 in Indian FEVR patients.