RESUMO
Accumulation of unfolded proteins in the endoplasmic reticulum (ER) causes ER stress and activates a signaling network known as the unfolded protein response (UPR). Here we characterize how ER stress and the UPR inhibit insulin signaling. We find that ER stress inhibits insulin signaling by depleting the cell surface population of the insulin receptor. ER stress inhibits proteolytic maturation of insulin proreceptors by interfering with transport of newly synthesized insulin proreceptors from the ER to the plasma membrane. Activation of AKT, a major target of the insulin signaling pathway, by a cytosolic, membrane-bound chimera between the AP20187-inducible FV2E dimerization domain and the cytosolic protein tyrosine kinase domain of the insulin receptor was not affected by ER stress. Hence, signaling events in the UPR, such as activation of the JNK mitogen-activated protein (MAP) kinases or the pseudokinase TRB3 by the ER stress sensors IRE1α and PERK, do not contribute to inhibition of signal transduction in the insulin signaling pathway. Indeed, pharmacologic inhibition and genetic ablation of JNKs, as well as silencing of expression of TRB3, did not restore insulin sensitivity or rescue processing of newly synthesized insulin receptors in ER-stressed cells. [Media: see text].
Assuntos
Estresse do Retículo Endoplasmático/fisiologia , Resistência à Insulina/fisiologia , Precursores de Proteínas/metabolismo , Receptor de Insulina/metabolismo , Fator 6 Ativador da Transcrição/metabolismo , Animais , Antígenos CD/metabolismo , Antígenos CD/fisiologia , Técnicas de Cultura de Células , Membrana Celular/metabolismo , Retículo Endoplasmático/metabolismo , Fibroblastos , Células HEK293 , Humanos , Insulina/metabolismo , Resistência à Insulina/genética , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Camundongos , Fosforilação , Precursores de Proteínas/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Transporte Proteico , Receptor de Insulina/fisiologia , Transdução de Sinais , Resposta a Proteínas não Dobradas/fisiologia , eIF-2 Quinase/metabolismoRESUMO
Cardiac malformations (CVMs) are a leading cause of infant morbidity and mortality. CVMs are particularly prevalent when the developing fetus is exposed to high levels of phenylalanine in-utero in mothers with Phenylketonuria. Yet, elucidating the underlying molecular mechanism leading to CVMs has proven difficult. In this study we used RNA-Seq to investigate an avian model of MPKU and establish differential gene expression (DEG) characteristics of the early developmental stages HH10, 12, and 14. In total, we identified 633 significantly differentially expressed genes across stages HH10, 12, and 14. As expected, functional annotation of significant DEGs identified associations seen in clinical phenotypes of MPKU including CVMs, congenital heart defects, craniofacial anomalies, central nervous system defects, and growth anomalies. Additionally, there was an overrepresentation of genes involved in cardiac muscle contraction, adrenergic signaling in cardiomyocytes, migration, proliferation, metabolism, and cell survival. Strikingly, we identified significant changes in expression with multiple genes involved in Retinoic Acid (RA) metabolism and downstream targets. Using qRTPCR, we validated these findings and identified a total of 42 genes within the RA pathway that are differentially expressed. Here, we report the first elucidation of the molecular mechanisms of cardiovascular malformations in MPKU conducted at early developmental timepoints. We provide evidence suggesting a link between PHE exposure and the alteration of RA pathway. These results are promising and offer novel findings associated with congenital heart defects in MPKU.
Assuntos
Galinhas/genética , Modelos Animais de Doenças , Fenilcetonúria Materna/genética , Análise de Sequência de RNA , Animais , Feminino , Cardiopatias Congênitas , Redes e Vias Metabólicas/genética , Fenótipo , Fenilalanina/genética , Gravidez , Transcriptoma , Tretinoína/metabolismo , ZigotoRESUMO
The retinal disease gene peripherin 2 (PRPH2) is essential for the formation of photoreceptor outer segments (OSs), where it functions in oligomers with and without its homologue ROM1. However, the precise role of these proteins in OS morphogenesis is not understood. By utilizing a knock-in mouse expressing a chimeric protein comprised of the body of Rom1 and the C-terminus of Prph2 (termed RRCT), we find that the Prph2 C-terminus is necessary and sufficient for the initiation of OSs, while OS maturation requires the body of Prph2 and associated large oligomers. Importantly, dominant-negative physiological and biochemical defects in RRCT heterozygous rods are rescued by removing Rom1, suggesting Rom1 is a regulator for OS formation. Our experiments evaluating Prph2 trafficking show that Rom1 is a key determinant of whether Prph2 complexes utilize conventional versus unconventional (Golgi bypass) secretory pathways to reach the OS. These findings significantly advance our understanding of the molecular underpinnings of OS morphogenesis and particularly the role of Rom1.
Assuntos
Proteínas do Olho/fisiologia , Proteínas de Membrana/fisiologia , Periferinas/fisiologia , Células Fotorreceptoras de Vertebrados/fisiologia , Animais , Proteínas do Olho/genética , Proteínas do Olho/metabolismo , Feminino , Técnicas de Introdução de Genes/métodos , Heterozigoto , Masculino , Glicoproteínas de Membrana/genética , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Morfogênese , Mutação , Proteínas do Tecido Nervoso/genética , Periferinas/genética , Periferinas/metabolismo , Fenótipo , Degeneração Retiniana/genética , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Células Fotorreceptoras Retinianas Bastonetes/fisiologia , TetraspaninasRESUMO
Considerable progress has been made in the design and delivery of non-viral gene therapy vectors, but, like their viral counterparts, therapeutic levels of transgenes have not met the requirements for successful clinical applications so far. The biggest advantage of polymer-based nanoparticle vectors is the ease with which they can be modified to increase their ability to penetrate the cell membrane and target specific cells by simply changing the formulation of the nanoparticle compaction. We took advantage of this characteristic to improve transfection rates of our particles to meet the transgene levels which will be needed for future treatment of patients. For this study, we successfully investigated the possibility of our established pegylated polylysine particles to be administered via intravitreal rather than subretinal route to ease the damage during injection. We also demonstrated that our particles are flexible enough to sustain changes in the formulation to accommodate additional targeting sequences without losing their efficiency in transfecting neuronal cells in the retina. Together, these results give us the opportunity to even further improve our particles.
Assuntos
Técnicas de Transferência de Genes , Vetores Genéticos/administração & dosagem , Injeções Intraoculares/métodos , Nanopartículas/administração & dosagem , Polilisina/administração & dosagem , Epitélio Pigmentado da Retina/metabolismo , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Animais , DNA Recombinante/administração & dosagem , Injeções Intravítreas , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Mutantes , Tamanho da Partícula , Polietilenoglicóis/administração & dosagem , Epitélio Pigmentado da Retina/citologia , TransgenesRESUMO
INTRODUCTION: DNA nanoparticles (NPs) comprising polylysine conjugated to polyethylene glycol efficiently target murine photoreceptors and the retinal pigment epithelium (RPE) and lead to long-term phenotypic improvement in models of retinal degeneration. Advancing this technology requires testing in a large animal model, particularly with regard to safety. So, herein we evaluate NPs in non-human primates (baboon). METHODS AND RESULTS: NPs with plasmids carrying GFP and a ubiquitous, RPE-specific, or photoreceptor-specific promoter were delivered by either subretinal or intravitreal injection. We detected GFP message and protein in the retina/RPE from eyes dosed with NPs carrying ubiquitously expressed and RPE-specific vectors, and GFP message in eyes injected with NPs carrying photoreceptor-specific vectors. Importantly, we observed NP DNA in the retina/RPE following intravitreal injection, indicating the inner limiting membrane does not prevent NP diffusion into the outer retina. We did not observe any adverse events in any baboon, and there were no NP-associated changes in retinal function. Furthermore, no systemic or local inflammatory reaction to the vectors/injections was observed, and no NP DNA was found outside the eye. CONCLUSION: Taken together with the well-established rodent safety and efficacy data, these findings suggest that DNA NPs may be a safe and potentially clinically viable nonviral ocular therapy platform for retinal diseases.
Assuntos
DNA/química , Olho/efeitos dos fármacos , Nanopartículas/toxicidade , Animais , Encéfalo/metabolismo , Citocinas/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Mediadores da Inflamação/metabolismo , Injeções Intravítreas , Camundongos , Nanopartículas/química , Plasmídeos/metabolismo , Primatas , Epitélio Pigmentado da Retina/efeitos dos fármacos , Epitélio Pigmentado da Retina/metabolismoRESUMO
Mutations in peripherin 2 (PRPH2), also known as retinal degeneration slow/RDS, lead to various retinal degenerations including retinitis pigmentosa (RP) and macular/pattern dystrophy (MD/PD). PRPH2-associated disease is often characterized by a phenotypic variability even within families carrying the same mutation, raising interest in potential modifiers. PRPH2 oligomerizes with its homologue rod outer segment (OS) membrane protein 1 (ROM1), and non-pathogenic PRPH2/ROM1 mutations, when present together, lead to digenic RP. We asked whether ROM1 could modify the phenotype of a PRPH2 mutation associated with a high degree of intrafamilial phenotypic heterogeneity: Y141C. In vitro, Y141C-Prph2 showed signs of retention in the endoplasmic reticulum (ER), however co-expression with Rom1 rescued this phenotype. In the heterozygous Y141C knockin mouse model (Prph2Y/+), Y141C-Prph2 and Rom1 formed abnormal complexes but were present at normal levels. Abnormal complexes were eliminated in the absence of Rom1 (Prph2Y/+/Rom1-/-) and total Prph2 levels were reduced to those found in the haploinsufficient Prph2+/- RP model. The biochemical changes had functional and structural consequences; while Prph2Y/+ animals exhibited a cone-rod electroretinogram defect, Prph2Y/+/Rom1-/- animals displayed a rod-dominant phenotype and OSs similar to those seen in the Prph2+/-. These data show that ablation of Rom1 results in the conversion of an MD/PD phenotype characterized by cone functional defects and the formation of abnormal Prph2/Rom1 complexes to an RP phenotype characterized by rod-dominant functional defects and reductions in total Prph2 protein. Thus one method by which ROM1 may act as a disease modifier is by contributing to the large variability in PRPH2-associated disease phenotypes.
Assuntos
Periferinas/genética , Degeneração Retiniana/genética , Retinose Pigmentar/genética , Tetraspaninas/genética , Animais , Retículo Endoplasmático/genética , Retículo Endoplasmático/patologia , Proteínas do Olho , Regulação da Expressão Gênica , Técnicas de Introdução de Genes , Humanos , Degeneração Macular/genética , Degeneração Macular/patologia , Camundongos , Complexos Multiproteicos/biossíntese , Complexos Multiproteicos/química , Complexos Multiproteicos/genética , Mutação , Linhagem , Periferinas/biossíntese , Periferinas/química , Fenótipo , Células Fotorreceptoras de Vertebrados/química , Células Fotorreceptoras de Vertebrados/metabolismo , Multimerização Proteica , Degeneração Retiniana/patologia , Retinose Pigmentar/patologia , Tetraspaninas/biossíntese , Tetraspaninas/químicaRESUMO
Accumulation of unfolded proteins in the endoplasmic reticulum (ER) activates the unfolded protein response (UPR). In mammalian cells, UPR signals generated by several ER-membrane-resident proteins, including the bifunctional protein kinase endoribonuclease IRE1α, control cell survival and the decision to execute apoptosis. Processing of XBP1 mRNA by the RNase domain of IRE1α promotes survival of ER stress, whereas activation of the mitogen-activated protein kinase JNK family by IRE1α late in the ER stress response promotes apoptosis. Here, we show that activation of JNK in the ER stress response precedes activation of XBP1. This activation of JNK is dependent on IRE1α and TRAF2 and coincides with JNK-dependent induction of expression of several antiapoptotic genes, including cIap1 (also known as Birc2), cIap2 (also known as Birc3), Xiap and Birc6 ER-stressed Jnk1(-/-) Jnk2(-/-) (Mapk8(-/-) Mapk9(-/-)) mouse embryonic fibroblasts (MEFs) display more pronounced mitochondrial permeability transition and increased caspase 3/7 activity compared to wild-type MEFs. Caspase 3/7 activity is also elevated in ER-stressed cIap1(-/-) cIap2(-/-) and Xiap(-/-) MEFs. These observations suggest that JNK-dependent transcriptional induction of several inhibitors of apoptosis contributes to inhibiting apoptosis early in the ER stress response.