RESUMO
SignificanceStudies in multiple experimental systems have demonstrated that an increase in proteolytic capacity of post-mitotic cells improves cellular resistance to a variety of stressors, delays cellular aging and senescence. Therefore, approaches to increase the ability of cells to degrade misfolded proteins could potentially be applied to the treatment of a broad spectrum of human disorders. An example would be retinal degenerations, which cause irreversible loss of vision and are linked to impaired protein degradation. This study suggests that chronic activation of the mammalian target of rapamycin complex 1 (mTORC1) pathway in degenerating photoreceptor neurons could stimulate the degradation of ubiquitinated proteins and enhance proteasomal activity through phosphorylation.
Assuntos
Complexo de Endopeptidases do Proteassoma , Proteólise , Células Fotorreceptoras Retinianas Bastonetes , Retinose Pigmentar , Ubiquitina , Animais , Modelos Animais de Doenças , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Camundongos , Camundongos Knockout , Complexo de Endopeptidases do Proteassoma/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Células Fotorreceptoras Retinianas Bastonetes/patologia , Retinose Pigmentar/genética , Retinose Pigmentar/metabolismo , Retinose Pigmentar/patologia , Proteína 2 do Complexo Esclerose Tuberosa/genética , Proteína 2 do Complexo Esclerose Tuberosa/metabolismo , Ubiquitina/genética , Ubiquitina/metabolismo , Proteínas Ubiquitinadas/metabolismoRESUMO
Usher syndrome type 3 (USH3) is an autosomal recessively inherited disorder caused by mutations in the gene clarin-1 (CLRN1), leading to combined progressive hearing loss and retinal degeneration. The cellular distribution of CLRN1 in the retina remains uncertain, either because its expression levels are low or because its epitopes are masked. Indeed, in the adult mouse retina, Clrn1 mRNA is developmentally downregulated, detectable only by RT-PCR. In this study we used the highly sensitive RNAscope in situ hybridization assay and single-cell RNA-sequencing techniques to investigate the distribution of Clrn1 and CLRN1 in mouse and human retina, respectively. We found that Clrn1 transcripts in mouse tissue are localized to the inner retina during postnatal development and in adult stages. The pattern of Clrn1 mRNA cellular expression is similar in both mouse and human adult retina, with CLRN1 transcripts being localized in Müller glia, and not photoreceptors. We generated a novel knock-in mouse with a hemagglutinin (HA) epitope-tagged CLRN1 and showed that CLRN1 is expressed continuously at the protein level in the retina. Following enzymatic deglycosylation and immunoblotting analysis, we detected a single CLRN1-specific protein band in homogenates of mouse and human retina, consistent in size with the main CLRN1 isoform. Taken together, our results implicate Müller glia in USH3 pathology, placing this cell type to the center of future mechanistic and therapeutic studies to prevent vision loss in this disease. © 2019 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.
Assuntos
Células Ependimogliais/metabolismo , Proteínas de Membrana/biossíntese , Retina/metabolismo , Síndromes de Usher/metabolismo , Animais , Glicosilação , Humanos , Hibridização In Situ , Proteínas de Membrana/genética , Camundongos Endogâmicos C57BL , Neuroglia/metabolismo , RNA Mensageiro/genética , Síndromes de Usher/patologiaRESUMO
Strong experimental evidence from studies in human donor retinas and animal models supports the idea that the retinal pathology associated with age-related macular degeneration (AMD) involves mitochondrial dysfunction and consequent altered retinal metabolism. This chapter provides a brief overview of mitochondrial structure and function, summarizes evidence for mitochondrial defects in AMD, and highlights the potential ramifications of these defects on retinal health and function. Discussion of mitochondrial haplogroups and their association with AMD brings to light how mitochondrial genetics can influence disease outcome. As one of the most metabolically active tissues in the human body, there is strong evidence that disruption in key metabolic pathways contributes to AMD pathology. The section on retinal metabolism reviews cell-specific metabolic differences and how the metabolic interdependence of each retinal cell type creates a unique ecosystem that is disrupted in the diseased retina. The final discussion includes strategies for therapeutic interventions that target key mitochondrial pathways as a treatment for AMD.
Assuntos
DNA Mitocondrial , Degeneração Macular , Animais , DNA Mitocondrial/metabolismo , Ecossistema , Humanos , Degeneração Macular/genética , Degeneração Macular/metabolismo , Mitocôndrias/genética , Retina , Epitélio Pigmentado da Retina/metabolismoRESUMO
Retinal degenerative diseases are generally characterized by a permanent loss of light-sensitive retinal neurons known as photoreceptors, or their support cells, the retinal pigmented epithelium (RPE). Metabolic dysfunction has been implicated as a common mechanism of degeneration. In this study, we used the drug metformin in a gain-of-function approach to activate adenosine monophosphate-activated protein kinase (AMPK). We found that treatment protected photoreceptors and the RPE from acute injury and delayed inherited retinal degeneration. Protection was associated with decreased oxidative stress, decreased DNA damage, and increased mitochondrial energy production. To determine whether protection was a local or a systemic effect of metformin, we used AMPK retinal knockout mice and found that local expression of AMPK catalytic subunit α2 was required for metformin-induced protection. Our data demonstrate that increasing the activity of AMPK in retinal neurons or glia can delay or prevent degeneration of photoreceptors and the RPE from multiple types of cell-death triggers.
Assuntos
Proteínas Quinases Ativadas por AMP/fisiologia , Modelos Animais de Doenças , Metformina/farmacologia , Células Fotorreceptoras/efeitos dos fármacos , Degeneração Retiniana/prevenção & controle , Epitélio Pigmentado da Retina/efeitos dos fármacos , Animais , Feminino , Hipoglicemiantes/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Células Fotorreceptoras/metabolismo , Degeneração Retiniana/metabolismo , Degeneração Retiniana/patologia , Epitélio Pigmentado da Retina/metabolismoRESUMO
Retinal photoreceptor cells contain the highest concentration of docosahexaenoic acid (DHA) in our bodies, and it has been long assumed that this is critical for supporting normal vision. Indeed, early studies using DHA dietary restriction documented reduced light sensitivity by DHA-deprived retinas. Recently, it has been demonstrated that a major route of DHA entry in the retina is the delivery across the blood-retina barrier by the sodium-dependent lipid transporter, Mfsd2a. This discovery opened a unique opportunity to analyze photoreceptor health and function in DHA-deprived retinas using the Mfsd2a knock-out mouse as animal model. Our lipidome analyses of Mfsd2a-/- retinas and outer segment membranes corroborated the previously reported decrease in the fraction of DHA-containing phospholipids and a compensatory increase in phospholipids containing arachidonic acid. We also revealed an increase in the retinal content of monounsaturated fatty acids and a reduction in very long chain fatty acids. These changes could be explained by a combination of reduced DHA supply to the retina and a concomitant upregulation of several fatty acid desaturases controlled by sterol regulatory element-binding transcription factors, which are upregulated in Mfsd2a-/- retinas. Mfsd2a-/- retinas undergo slow progressive degeneration, with â¼30% of photoreceptor cells lost by the age of 6 months. Despite this pathology, the ultrastructure Mfsd2a-/- photoreceptors and their ability to produce light responses were essentially normal. These data demonstrate that, whereas maintaining the lysophosphatidylcholine route of DHA supply to the retina is essential for long-term photoreceptor survival, it is not important for supporting normal phototransduction.SIGNIFICANCE STATEMENT Phospholipids containing docosahexaenoic acid (DHA) are greatly enriched in the nervous system, with the highest concentration found in the light-sensitive membranes of photoreceptor cells. In this study, we analyzed the consequences of impaired DHA transport across the blood-retina barrier. We have found that, in addition to a predictable reduction in the DHA level, the affected retinas undergo a complex, transcriptionally-driven rebuilding of their membrane lipidome in a pattern preserving the overall saturation/desaturation balance of retinal phospholipids. Remarkably, these changes do not affect the ability of photoreceptors to produce responses to light but are detrimental for the long-term survival of these cells.
Assuntos
Barreira Hematorretiniana/metabolismo , Barreira Hematorretiniana/patologia , Lisofosfatidilcolinas/metabolismo , Células Fotorreceptoras de Vertebrados/patologia , Transdução de Sinais/fisiologia , Animais , Ácidos Docosa-Hexaenoicos/deficiência , Ácidos Docosa-Hexaenoicos/metabolismo , Ácidos Graxos não Esterificados/metabolismo , Feminino , Metabolismo dos Lipídeos/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Estimulação Luminosa , Células Fotorreceptoras de Vertebrados/metabolismo , Gravidez , Retina/metabolismo , Degeneração Retiniana/genética , Degeneração Retiniana/patologia , Segmento Externo da Célula Bastonete/metabolismo , Simportadores/genética , Simportadores/metabolismoRESUMO
Retinal degenerations are a large cluster of diseases characterized by the irreversible loss of light-sensitive photoreceptors that impairs the vision of 9.1 million people in the US. An attractive treatment option is to use gene therapy to deliver broad-spectrum neuroprotective factors. However, this approach has had limited clinical translation because of the inability to control transgene expression. To address this problem, we generated an adeno-associated virus vector named RPF2 that was engineered to express domains of leukemia inhibitory factor fused to the destabilization domain of bacterial dihydrofolate reductase. Fusion proteins containing the destabilization domain are degraded in mammalian cells but can be stabilized with the binding of the drug trimethoprim. Our data show that expression levels of RPF2 are tightly regulated by the dose of trimethoprim and can be reversed by trimethoprim withdrawal. We further show that stabilized RPF2 can protect photoreceptors and prevent blindness in treated mice.
Assuntos
Terapia Genética , Fator Inibidor de Leucemia/genética , Degeneração Retiniana/terapia , Animais , Dependovirus/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Fator Inibidor de Leucemia/administração & dosagem , Camundongos , Neuroproteção/genética , Células Fotorreceptoras/efeitos dos fármacos , Células Fotorreceptoras/patologia , Retina/efeitos dos fármacos , Retina/patologia , Degeneração Retiniana/genética , Degeneração Retiniana/patologia , Tetra-Hidrofolato Desidrogenase/genética , Transgenes/efeitos dos fármacos , Trimetoprima/administração & dosagemRESUMO
Evidence suggests that metabolic dysregulation plays an important role in disease etiology of retinal degenerations. Several studies suggest that preserving the retinal metabolic ecosystem may be protective against retinal degenerations. We investigated whether activation of 5' adenosine monophosphate protein kinase (AMPK) is protective to the retina in several preclinical mouse models of retinal degeneration and found that metformin-induced activation of AMPK was able to delay or prevent retinal degeneration in the rd10 model of retinitis pigmentosa, the NaIO3 model of RPE and retinal injury, and the light damage model of retinal degeneration. This protection was associated with increased mitochondrial DNA copy number, increased levels of ATP, and a reduction in oxidative stress and oxidative DNA damage. We propose that AMPK plays an important role in regulation of the retinal metabolic ecosystem and that activation of AMPK may promote metabolic processes to prevent retinal degeneration.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Retina/enzimologia , Degeneração Retiniana/prevenção & controle , Animais , Dano ao DNA , DNA Mitocondrial/genética , Modelos Animais de Doenças , Dosagem de Genes , Metformina/farmacologia , Camundongos , Estresse Oxidativo , Retinose Pigmentar/enzimologia , Retinose Pigmentar/prevenção & controleRESUMO
The title of the chapter is "Melatonin as the Possible Link Between Age-Related Retinal Degeneration and the Disrupted Circadian Rhythm in Elderly" but degeneration was incorrectly published as regeneration. Now this has been corrected to degeneration.
RESUMO
Müller cells provide support to photoreceptors under many conditions of stress and degeneration. Leukemia inhibitory factor is known to be expressed in Müller cells, which is necessary to promote photoreceptor survival in stress. We hypothesize that Müller cells that express LIF are undergoing other biological processes or functions which may benefit photoreceptors in disease. In this study, we analyze an existing single Müller cell microarray dataset to determine which processes are upregulated in Müller cells that express LIF, by correlating LIF expression to the expression of other genes using a robust correlation method. Some enriched processes include divalent inorganic cation homeostasis, negative regulation of stem cell proliferation, and gamma-glutamyl transferase activity.
Assuntos
Células Ependimogliais/metabolismo , Fator Inibidor de Leucemia/biossíntese , Regiões 3' não Traduzidas , Animais , Cálcio/metabolismo , Cátions/metabolismo , Autorrenovação Celular , Conjuntos de Dados como Assunto , Células Ependimogliais/citologia , Regulação da Expressão Gênica , Fator Inibidor de Leucemia/genética , Camundongos , Camundongos Knockout , Células Fotorreceptoras de Vertebrados/citologia , Células Fotorreceptoras de Vertebrados/metabolismo , Receptores de Superfície Celular/metabolismo , Degeneração Retiniana/metabolismo , Degeneração Retiniana/patologia , Rodopsina/deficiência , Rodopsina/genética , Análise de Célula Única , Análise Serial de Tecidos , Regulação para Cima , gama-Glutamiltransferase/metabolismoRESUMO
Age-related macular degeneration (AMD) is the leading cause of blindness in older adults in developed countries. The molecular mechanisms of disease pathogenesis remain poorly understood; however, evidence suggests that mitochondrial dysfunction may contribute to the progression of the disease. Studies have shown that mitochondrial DNA lesions are increased in the retinal pigment epithelium (RPE) of human patients with the disease and that the number of these lesions increases with disease severity. Additionally, microscopy of human RPE from patients with dry AMD shows severe disruptions in mitochondrial inner and outer membrane structure, mitochondrial size, and mitochondrial cellular organization. Thus, improving our understanding of mitochondrial dysfunction in dry AMD pathogenesis may lead to the development of targeted therapies. We propose that mitochondrial dysfunction in the RPE can lead to the chronic oxidative stress associated with the disease. Therefore, one protective strategy may involve the use of small molecule therapies that target the regulation of mitochondrial biogenesis and mitochondrial fission and mitophagy.
Assuntos
DNA Mitocondrial/metabolismo , Degeneração Macular/metabolismo , Mitocôndrias/patologia , Terapia de Alvo Molecular , Epitélio Pigmentado da Retina/patologia , Adenilato Quinase/fisiologia , Animais , DNA Mitocondrial/genética , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Atrofia Geográfica/patologia , Humanos , Iodatos/toxicidade , Degeneração Macular/tratamento farmacológico , Degeneração Macular/genética , Metformina/farmacologia , Camundongos , Mitocôndrias/efeitos dos fármacos , Dinâmica Mitocondrial/efeitos dos fármacos , Estresse Oxidativo , Espécies Reativas de Oxigênio/metabolismo , Epitélio Pigmentado da Retina/efeitos dos fármacos , Epitélio Pigmentado da Retina/metabolismoRESUMO
Damage to mitochondria is a common mechanism of cell death in inherited neurodegenerative disorders. Therefore, mitochondrial protection and mitochondrial repair are promising strategies to induce retinal neuroprotection. Peroxisome proliferator-activated receptor γ coactivator-α (PGC-1α) and ß (PGC-1ß) are transcriptional coactivators that are the main regulators of mitochondrial biogenesis. We propose that PGC-1α and PGC-1ß could play a role in regulating retina cell survival, and may be important therapeutic targets to prevent retinal degeneration.
Assuntos
Proteínas de Transporte/metabolismo , Mitocôndrias/metabolismo , Retina/metabolismo , Fatores de Transcrição/metabolismo , Sobrevivência Celular , Replicação do DNA , DNA Mitocondrial/genética , Humanos , Mitocôndrias/genética , Biogênese de Organelas , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo , Proteínas de Ligação a RNA , Espécies Reativas de Oxigênio/metabolismo , Retina/citologia , Degeneração Retiniana/genética , Degeneração Retiniana/metabolismo , Degeneração Retiniana/patologiaRESUMO
Adenosine monophosphate-activated kinase (AMPK) is a highly conserved protein kinase found in all eukaryotic genomes. It exists as heterotrimeric protein consisting of α, ß, and γ subunits. AMPK is activated by elevated levels of adenosine mono-phosphate (AMP), which is produced during conditions of low ATP production and perhaps mitochondrial dysfunction. Activation of AMPK has been shown to regulate a large number of downstream pathways. These will either increase energy production such as increase oxidation of fatty acids and glucose, or decrease energy utilization such as inhibiting synthesis of glycogen, fatty acid synthesis, and protein synthesis. In addition, being a key regulator of physiological energy dynamics, AMPK has been demonstrated to play roles in regulating various cellular processes such as mitochondrial biogenesis (Jager et al. Proc Natl Acad Sci U S A 104:12017-12022, 2007), autophagy (Hyttinen et al. Rejuven Res 14:651-660, 2011) and inflammation and immune responses (Giri et al. 2004). Retinal neurons have a high energy demand but have a poor energy storage capacity. Because of this, it is likely that the AMPK signaling pathway plays an important role in maintaining energy balance, and therefore may be a therapeutic target to prevent or delay retinal degeneration.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Metabolismo Energético/fisiologia , Neuroproteção/fisiologia , Transdução de Sinais/fisiologia , Animais , Fenômenos Fisiológicos Celulares/fisiologia , Humanos , Biogênese de Organelas , Degeneração Retiniana/metabolismo , Degeneração Retiniana/fisiopatologia , Neurônios Retinianos/metabolismoRESUMO
Caveolin-1 (Cav-1), the scaffolding protein of caveolae, is expressed in several retinal cell types and is associated with ocular pathologies. Cav-1 modulates neuroinflammatory/neuroprotective responses to central nervous system injury. We have shown that loss of Cav-1 results in a blunted cytokine response in retinas challenged with inflammatory stimuli. As neuroinflammatory and neuroprotective signaling overlap in their cytokine production and downstream signaling pathways, we hypothesized that loss of Cav-1 may also suppress neuroprotective signaling in the retina. To test this, we subjected mice in which Cav-1 was deleted specifically in the retina to a neurodegenerative insult induced by sodium iodate (NaIO3) and measured STAT3 activation, a measure of neuroprotective signaling. Our results show that Cav-1 ablation blunts STAT3 activation induced by NaIO3. STAT3 activation in response to intravitreal administration of the IL-6 family cytokine, leukemia inhibitory factor (LIF), was not affected by Cav-1 deletion indicating a competent gp130 receptor response. Thus, Cav-1 modulates neuroprotective signaling by regulating the endogenous production of neuroprotective factors.
Assuntos
Caveolina 1/genética , Neuroproteção/genética , Retina/metabolismo , Transdução de Sinais/genética , Animais , Western Blotting , Caveolina 1/deficiência , Feminino , Imuno-Histoquímica , Injeções Intraperitoneais , Iodatos/administração & dosagem , Iodatos/farmacologia , Fator Inibidor de Leucemia/administração & dosagem , Fator Inibidor de Leucemia/farmacologia , Masculino , Camundongos , Camundongos Knockout , Neuroproteção/efeitos dos fármacos , Retina/efeitos dos fármacos , Epitélio Pigmentado da Retina/efeitos dos fármacos , Epitélio Pigmentado da Retina/metabolismo , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
Müller cells are major macroglia and play many essential roles as a supporting cell in the retina. As Müller cells only constitute a small portion of retinal cells, investigating the role of Müller glia in retinal biology and diseases is particularly challenging. To overcome this problem, we first generated a Cre/lox-based conditional gene targeting system that permits the genetic manipulation and functional dissection of gene of interests in Müller cells. To investigate diabetes-induced alteration of Müller cells, we recently adopted methods to analyze Müller cells survival/death in vitro and in vivo. We also used normal and genetically altered primary cell cultures to reveal the mechanistic insights for Müller cells in biological and disease processes. In this article, we will discuss the applications and limitations of these methodologies, which may be useful for research in retinal Müller cell biology and pathophysiology.
Assuntos
Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/patologia , Retinopatia Diabética/genética , Retinopatia Diabética/patologia , Células Ependimogliais/patologia , Animais , Apoptose/fisiologia , Modelos Animais de Doenças , Humanos , Integrases/genética , Camundongos , Mutagênese , Cultura Primária de Células/métodosRESUMO
The gene encoding Elongation of Very Long Chain Fatty Acids-4 (ELOVL4) is mutated in patients with autosomal dominant Stargardt's Macular Dystrophy Type 3 (STDG3). ELOVL4 catalyzes the initial condensation step in the elongation of polyunsaturated fatty acids (PUFA) containing more than 26 carbons (26C) to very long chain PUFA (VLC-PUFA; C28 and greater). To investigate the role of VLC-PUFA in rod photoreceptors, we generated mice with rod-specific deletion of Elovl4 (RcKO). The mosaic deletion of rod-expressed ELOVL4 protein resulted in a 36 % lower amount of VLC-PUFA in the retinal phosphatidylcholine (PC) fraction compared to retinas from wild-type mice. However, this reduction was not sufficient to cause rod dysfunction at 7 months or photoreceptor degeneration at 9 or 15 months.
Assuntos
Proteínas do Olho/metabolismo , Ácidos Graxos Insaturados/metabolismo , Proteínas de Membrana/metabolismo , Fosfatidilcolinas/metabolismo , Degeneração Retiniana/metabolismo , Células Fotorreceptoras Retinianas Bastonetes/citologia , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Animais , Eletrorretinografia , Proteínas do Olho/genética , Proteínas de Membrana/genética , Camundongos , Camundongos Mutantes , Mosaicismo , Degeneração Retiniana/genéticaRESUMO
The retina is light-sensitive neuronal tissue in the back of the eye. The phospholipid composition of the retina is unique and highly enriched in polyunsaturated fatty acids, including docosahexaenoic fatty acid (DHA). While it is generally accepted that a high DHA content is important for vision, surprisingly little is known about the mechanisms of DHA enrichment in the retina. Furthermore, the biological processes controlled by DHA in the eye remain poorly defined as well. Here, we combined genetic manipulations with lipidomic analysis in mice to demonstrate that acyl-CoA synthetase 6 (Acsl6) serves as a regulator of the unique composition of retinal membranes. Inactivation of Acsl6 reduced the levels of DHA-containing phospholipids, led to progressive loss of light-sensitive rod photoreceptor neurons, attenuated the light responses of these cells, and evoked distinct transcriptional response in the retina involving the Srebf1/2 (sterol regulatory element binding transcription factors 1/2) pathway. This study identifies one of the major enzymes responsible for DHA enrichment in the retinal membranes and introduces a model allowing an evaluation of rod functioning and pathology caused by impaired DHA incorporation/retention in the retina.
Assuntos
Coenzima A Ligases , Fosfolipídeos , Células Fotorreceptoras Retinianas Bastonetes , Animais , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Camundongos , Fosfolipídeos/metabolismo , Coenzima A Ligases/metabolismo , Coenzima A Ligases/genética , Retina/metabolismo , Ácidos Docosa-Hexaenoicos/metabolismo , Camundongos Knockout , Camundongos Endogâmicos C57BLRESUMO
Oncostatin M (OSM) and leukemia inhibitory factor are pleiotropic cytokines that belong to the interleukin-6 (IL-6) family. These cytokines play a crucial role in diverse biological events like inflammation, neuroprotection, hematopoiesis, metabolism, and development. The family is grouped together based on structural similarities and their ability to activate the transmembrane receptor glycoprotein 130 (gp130). The common structure among these cytokines defines the spacing and the orientation of binding sites for cell surface receptors. OSM is unique in this family as it can signal using heterodimers of gp130 with either leukemia inhibitory factor receptor (LIFR) (type I) or oncostatin M receptor (OSMR) (type II). We have identified a unique helical loop on OSM between its B and C helices that is not found on other IL-6 family cytokines. This loop is located near the "FXXK" motif in active site III, which is essential for OSM's binding to both LIFR and OSMR. In this study, we show that the BC loop does not play a role in OSM's unique ability to bind OSMR. Shortening of the loop enhanced OSM's interaction with OSMR and LIFR as shown by kinetic and equilibrium binding analysis, suggesting the loop may hinder receptor interactions. As a consequence of improved binding, these structurally modified OSMs exhibited enhanced biological activity, including suppressed proliferation of A375 melanoma cells.
Assuntos
Subunidade alfa de Receptor de Fator Inibidor de Leucemia/química , Subunidade alfa de Receptor de Fator Inibidor de Leucemia/metabolismo , Subunidade beta de Receptor de Oncostatina M/química , Subunidade beta de Receptor de Oncostatina M/metabolismo , Oncostatina M/química , Oncostatina M/imunologia , Motivos de Aminoácidos , Linhagem Celular Tumoral , Humanos , Cinética , Subunidade alfa de Receptor de Fator Inibidor de Leucemia/genética , Oncostatina M/genética , Subunidade beta de Receptor de Oncostatina M/genética , Ligação Proteica , Estrutura Terciária de ProteínaRESUMO
Leukemia inhibitory factor (LIF), an interleukin-6 family neurocytokine, is up-regulated in response to different types of retinal stress and has neuroprotective activity through activation of the gp130 receptor/STAT3 pathway. We observed that LIF induces rapid, robust, and sustained activation of STAT3 in both the retina and retinal pigmented epithelium (RPE). Here, we tested whether LIF-induced STAT3 activation within the RPE can down-regulate RPE65, the central enzyme in the visual cycle that provides the 11-cis-retinal chromophore to photoreceptors in vivo. We generated conditional knock-out mice to specifically delete STAT3 or gp130 in RPE, retina, or both RPE and retina. After intravitreal injection of LIF, we analyzed the expression levels of visual cycle genes and proteins, isomerase activity of RPE65, levels of rhodopsin protein, and the rates of dark adaptation and rhodopsin regeneration. We found that RPE65 protein levels and isomerase activity were reduced and recovery of bleachable rhodopsin was delayed in LIF-injected eyes. In mice with functional gp130/STAT3 signaling in the retina, rhodopsin protein was also reduced by LIF. However, the LIF-induced down-regulation of RPE65 required a functional gp130/STAT3 cascade intrinsic to RPE. Our data demonstrate that a single cytokine, LIF, can simultaneously and independently affect both RPE and photoreceptors through the same signaling cascade to reduce the generation and utilization of 11-cis-retinal.
Assuntos
Fator Inibidor de Leucemia/farmacologia , Epitélio Pigmentado da Retina/metabolismo , Animais , Western Blotting , Receptor gp130 de Citocina/genética , Receptor gp130 de Citocina/metabolismo , Regulação para Baixo/efeitos dos fármacos , Eletrorretinografia , Feminino , Humanos , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Knockout , Reação em Cadeia da Polimerase em Tempo Real , Retina/efeitos dos fármacos , Retina/metabolismo , Epitélio Pigmentado da Retina/efeitos dos fármacos , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , cis-trans-Isomerases/genética , cis-trans-Isomerases/metabolismoRESUMO
Caveolin-1 (Cav-1), an integral component of caveolar membrane domains, is expressed in several retinal cell types, including photoreceptors, retinal vascular endothelial cells, Müller glia, and retinal pigment epithelium (RPE) cells. Recent evidence links Cav-1 to ocular diseases, including autoimmune uveitis, diabetic retinopathy, and primary open angle glaucoma, but its role in normal vision is largely undetermined. In this report, we show that ablation of Cav-1 results in reduced inner and outer retinal function as measured, in vivo, by electroretinography and manganese-enhanced MRI. Somewhat surprisingly, dark current and light sensitivity were normal in individual rods (recorded with suction electrode methods) from Cav-1 knock-out (KO) mice. Although photoreceptor function was largely normal, in vitro, the apparent K(+) affinity of the RPE-expressed α1-Na(+)/K(+)-ATPase was decreased in Cav-1 KO mice. Cav-1 KO retinas also displayed unusually tight adhesion with the RPE, which could be resolved by brief treatment with hyperosmotic medium, suggesting alterations in outer retinal fluid homeostasis. Collectively, these findings demonstrate that reduced retinal function resulting from Cav-1 ablation is not photoreceptor-intrinsic but rather involves impaired subretinal and/or RPE ion/fluid homeostasis.
Assuntos
Caveolina 1/metabolismo , Microambiente Celular/fisiologia , Homeostase/fisiologia , Células Fotorreceptoras de Vertebrados/metabolismo , Epitélio Pigmentado da Retina/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismo , Junções Íntimas/metabolismo , Animais , Caveolina 1/genética , Camundongos , Camundongos Knockout , Células Fotorreceptoras de Vertebrados/citologia , Potássio/metabolismo , Doenças Retinianas/genética , Doenças Retinianas/metabolismo , Epitélio Pigmentado da Retina/citologia , ATPase Trocadora de Sódio-Potássio/genética , Junções Íntimas/genéticaRESUMO
Proteasomes are the central proteolytic machines that are critical for breaking down most of the damaged and abnormal proteins in human cells. Although universally applicable drugs are not yet available, the stimulation of proteasomal activity is being analyzed as a proof-of-principle strategy to increase cellular resistance to a broad range of proteotoxic stressors. These approaches have included the stimulation of proteasomes through the overexpression of individual proteasome subunits, phosphorylation, or conformational changes induced by small molecules or peptides. In contrast to these approaches, we evaluated a transcription-driven increase in the total proteasome pool to enhance the proteolytic capacity of degenerating retinal neurons. We show that overexpression of nuclear factor erythroid-2-like 1 (Nfe2l1) transcription factor stimulated proteasome biogenesis and activity, improved the clearance of the ubiquitin-proteasomal reporter, and delayed photoreceptor neuron loss in a preclinical mouse model of human blindness caused by misfolded proteins. The findings highlight Nfe2l1 as an emerging therapeutic target to treat neurodegenerative diseases linked to protein misfolding.