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1.
J Pharmacol Sci ; 154(2): 52-60, 2024 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-38246728

RESUMEN

Many glaucoma treatments focus on lowering intraocular pressure (IOP), with novel drugs continuing to be developed. One widely used model involves raising IOP by applying a laser to the trabecular iris angle (TIA) of cynomolgus monkeys to damage the trabecular meshwork. This model, however, presents challenges such as varying IOP values, potential trabecular meshwork damage, and risk of animal distress. This study investigated whether animals with naturally high IOP (>25 mmHg) could be used to effectively evaluate IOP-lowering drugs, thereby possibly replacing laser-induced models. Relationships between TIA size, IOP, and pupil diameter were also examined. Three representative IOP-lowering drugs (latanoprost, timolol, ripasudil) were administered, followed by multiple IOP measurements and assessment of corneal thickness, TIA, and pupil diameter via anterior segment optical coherence tomography (AS-OCT). There was a positive correlation was noted between IOP and corneal thickness before instillation, and a negative correlation between IOP and TIA before instillation. Our findings suggest animals with naturally high IOP could be beneficial for glaucoma research and development as a viable replacement for the laser-induced model and that measuring TIA using AS-OCT along with IOP yields a more detailed evaluation.


Asunto(s)
Glaucoma , Presión Intraocular , Animales , Macaca fascicularis , Timolol/farmacología , Malla Trabecular
2.
FASEB J ; 36(6): e22323, 2022 06.
Artículo en Inglés | MEDLINE | ID: mdl-35485981

RESUMEN

Neovascular glaucoma (NVG) is caused by the formation of new blood vessels in the angle, iris, and cornea in retinal ischemic disease, such as proliferative diabetic retinopathy (PDR) and retinal vein occlusion (RVO), which can reduce the visual acuity. However, the pathophysiological symptoms of NVG are still not well understood because there is no model for the formation of NVG in the angle, iris, and cornea. The aim of this study was to investigate the involvement of NVG during ischemic disease, in a murine model of retinal ischemia. We evaluated the changes of the intraocular pressure (IOP) and pathological symptoms in the anterior eye segment and retina in this model, and the changes in the RNA or protein expression of vascular endothelial growth factor (VEGF) and fibrosis-related factors were analyzed in the retina and cornea by quantitative real-time polymerase chain reaction or western blot, respectively. Furthermore, we examined the changes in IOP after intravitreal injection of an anti-VEGF antibody. First, NVG formed in the retinal ischemic murine model, and the IOP was elevated in mice with NVG formation. Interestingly, VEGF expression was decreased in the retina but increased in the cornea in the murine model of NVG. On the other hand, fibrosis-related factors were increased in the retina and also significantly increased in the cornea in NVG. Moreover, the administration of anti-VEGF antibody immediately after vessel occlusion suppressed the increase in IOP, but administration at 7 days after vessel occlusion accelerated the increase in IOP. These findings suggest that the formation of NVG may be correlated with the pathological symptoms of retinal ischemic disease, via changes in VEGF and fibrosis-related factor expression.


Asunto(s)
Glaucoma Neovascular , Enfermedades de la Retina , Animales , Segmento Anterior del Ojo/irrigación sanguínea , Modelos Animales de Enfermedad , Fibrosis , Glaucoma Neovascular/diagnóstico , Glaucoma Neovascular/etiología , Ratones , Retina , Factor A de Crecimiento Endotelial Vascular/genética
3.
J Pharmacol Sci ; 150(4): 279-288, 2022 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-36344051

RESUMEN

The corneal epithelium is located at the outermost layer of the ocular surface and continuously exposed to environmental factors, such as ultraviolet (UV) radiation from sunlight. UV irradiation causes excessive production of reactive oxygen species (ROS) in cells, which results in oxidative damage to membrane-bound organelles such as mitochondria, eventually leading to cell death. Crocetin, a natural carotenoid found in plants, has various biological properties including antioxidant activity. In this study, we investigated the effects of crocetin on UV-A-induced cell injury in the corneal epithelium. Using an in vitro system with the human corneal epithelial cell-transformed (HCE-T) cell line, pretreatment with 10 µM crocetin suppressed the reduction of cell viability induced by UV-A exposure. Crocetin ameliorated the decrease in oxygen consumption rates and the mitochondrial fragmentation that occurred following UV-A irradiation. Crocetin inhibited both ROS production and the activation of the apoptosis pathway; it also preserved the defects of epithelial cell polarity and barrier function in UV-A-irradiated HCE-T cells. The reduction in apical Mucin-16 expression was partially recovered in the presence of crocetin. Taking these findings together, we conclude that crocetin has a protective effect against UV-A irradiation-induced mitochondrial injury in corneal epithelial cells.


Asunto(s)
Células Epiteliales , Rayos Ultravioleta , Humanos , Especies Reactivas de Oxígeno/metabolismo , Rayos Ultravioleta/efectos adversos , Células Epiteliales/metabolismo , Estrés Oxidativo , Linfocitos T/metabolismo
4.
Int J Mol Sci ; 23(3)2022 Feb 04.
Artículo en Inglés | MEDLINE | ID: mdl-35163712

RESUMEN

Non-exudative age-related macular degeneration (AMD) is an irreversibly progressive retinal degenerative disease characterized by dysfunction and loss of retinal pigment epithelium (RPE). It has been suggested that impaired phagocytosis of the RPE is involved in the progression of non-exudative AMD, but the mechanism is not fully clear. In this study, we investigated the effect of lipid droplet accumulation on RPE function. Compared to young mice, the expression of lipid droplet-associated proteins increased in the RPE-choroidal complex, and lipid droplet in the RPE was observed in aged pigmented mice (12-month-old). Repeated treatment of the photoreceptor outer segment against ARPE-19 resulted in lipid droplets in ARPE-19 cells in vitro. Oleic acid treatment for ARPE-19 cells to form intracellular lipid droplet reduced the POS uptake into the ARPE-19 cells without causing a decrease in cell viability. The suppression of the POS uptake by lipid droplet formation improved by inhibiting lipid droplet formation using triacsin C. Moreover, the amount of intracellular reactive oxygen species was suppressed by the triacsin C treatment. These results indicate that lipid droplet is involved in the RPE dysfunction, and inhibiting lipid droplet formation may be a target for preventing and treating non-exudative AMD.


Asunto(s)
Gotas Lipídicas , Epitelio Pigmentado de la Retina , Animales , Transporte Biológico , Coroides/metabolismo , Gotas Lipídicas/metabolismo , Ratones , Especies Reactivas de Oxígeno/metabolismo , Epitelio Pigmentado de la Retina/metabolismo
5.
J Biol Chem ; 295(23): 8048-8063, 2020 06 05.
Artículo en Inglés | MEDLINE | ID: mdl-32358067

RESUMEN

TSPO2 (translocator protein 2) is a transmembrane protein specifically expressed in late erythroblasts and has been postulated to mediate intracellular redistribution of cholesterol. We identified TSPO2 as the causative gene for the HK (high-K+) trait with immature red cell phenotypes in dogs and investigated the effects of the TSPO2 defects on erythropoiesis in HK dogs with the TSPO2 mutation and Tspo2 knockout (Tspo2-/-) mouse models. Bone marrow-derived erythroblasts from HK dogs showed increased binucleated and apoptotic cells at various stages of maturation and shed large nuclei with incomplete condensation when cultured in the presence of erythropoietin, indicating impaired maturation and cytokinesis. The canine TSPO2 induces cholesterol accumulation in the endoplasmic reticulum and could thereby regulate cholesterol availability by changing intracellular cholesterol distribution in erythroblasts. Tspo2-/- mice consistently showed impaired cytokinesis with increased binucleated erythroblasts, resulting in compensated anemia, and their red cell membranes had increased Na,K-ATPase, resembling the HK phenotype in dogs. Tspo2-deficient mouse embryonic stem cell-derived erythroid progenitor (MEDEP) cells exhibited similar morphological defects associated with a cell-cycle arrest at the G2/M phase, resulting in decreased cell proliferation and had a depletion in intracellular unesterified and esterified cholesterol. When the terminal maturation was induced, Tspo2-/- MEDEP cells showed delays in hemoglobinization; maturation-associated phenotypic changes in CD44, CD71, and TER119 expression; and cell-cycle progression. Taken together, these findings imply that TSPO2 is essential for coordination of maturation and proliferation of erythroblasts during normal erythropoiesis.


Asunto(s)
Eritroblastos/citología , Eritroblastos/metabolismo , Eritropoyesis , Receptores Citoplasmáticos y Nucleares/metabolismo , Animales , Proliferación Celular , Células Cultivadas , Perros , Humanos , Células K562 , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Receptores Citoplasmáticos y Nucleares/deficiencia
6.
J Neuroinflammation ; 18(1): 164, 2021 Jul 25.
Artículo en Inglés | MEDLINE | ID: mdl-34304733

RESUMEN

BACKGROUND: Age-related macular degeneration (AMD) is the principal cause of permanent blindness among elderly individuals worldwide. Chronic inflammation in the subretinal space is associated with a progression of exudative AMD. Progranulin (PGRN) is a growth factor secreted from myeloid cells and plays an important role in controlling the lysosomal function. A deficiency in PGRN leads to inflammation of the neurons in the central nervous system. The purpose of this study was to investigate the role played by PGRN in the size of the choroidal neovascularization (CNV) in laser-induced CNV mice. METHODS: CNVs were induced in C57BL/6J mice by laser photocoagulation of the retina. The expression of PGRN and the accumulation of Iba-1+ cells around the sites of the CNVs were determined. Grn-/-, Grn+/-, and Grn+/+ mice with laser-induced CNVs were also studied. To evaluate the effect of macrophages on the inflammation, we used a macrophage cell line (RAW264.7) in which the expression of PGRN was knocked down by RNA interference and peritoneal macrophages derived from Grn-/- and Grn+/+ mice. These cells were incubated under hypoxic conditions (1% O2). RESULTS: Iba-1+ myeloid cells migrated and accumulated in the photocoagulation-induced CNV areas, and the CNV lesions secreted high levels of PGRN in Grn+/+ mice. The size of the CNVs was larger in Grn-/- mice than in Grn+/- and Grn+/+ mice. In Grn-/- mice, the number of ocular-infiltrating Iba-1+ cells around the CNV was higher, and these cells produced more VEGF-A than the cells in the Grn+/+ mice. PGRN-silencing of RAW264.7 cells led to abnormal activation of the cells. In addition, hypoxic conditions promoted the production of proangiogenic and proinflammatory cytokines from PGRN-deficient macrophages. Interestingly, the expression level of lysosome-associated proteins and the number of activated lysosomes increased in PGRN-deficient macrophages. CONCLUSIONS: These findings indicate that PGRN deficiency in Iba-1+ cells activates the lysosomal function that then leads to abnormal inflammation. The aberrant activation of Iba-1+ myeloid cells might contribute to the progression of the CNV and the regulation of these cells might be a novel therapeutic target for exudative AMD.


Asunto(s)
Proteínas de Unión al Calcio , Neovascularización Coroidal/metabolismo , Neovascularización Coroidal/patología , Inflamación/metabolismo , Lisosomas/patología , Proteínas de Microfilamentos , Células Mieloides/metabolismo , Progranulinas/metabolismo , Animales , Células Cultivadas , Citocinas/metabolismo , Modelos Animales de Enfermedad , Inflamación/patología , Lisosomas/metabolismo , Macrófagos/metabolismo , Degeneración Macular , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neuronas/metabolismo
7.
Exp Eye Res ; 213: 108800, 2021 12.
Artículo en Inglés | MEDLINE | ID: mdl-34688622

RESUMEN

Aging is a predominant risk factor for various eye diseases. Age-related macular degeneration (AMD) is the leading cause of blindness in the elderly, and its etiology remains unclear. Fragmented and dysfunctional mitochondria are associated with age-related diseases. The retinal pigment epithelium (RPE), a polarized cell layer that functions in visual pigment recycling and degeneration, is suspected as the primary region site of AMD. In the present study, we investigated the relationship between mitochondrial dysfunction and RPE aging. Compared to young mice, aged pigmented mice (C57BL/6J, 12-month-old) exhibit decreased visual function without retinal thinning. Consistently, the rhodopsin expression level decreased in the outer segment of aged mice. Moreover, the cell volume of the RPE increased in aged animals. Interestingly, the expression of mitochondria dynamics-related proteins, including Drp1, was altered in the RPE-choroid complex but not in the neural retina after aging. Electron microscopy revealed that mitochondrial size decreased and cristae width increased in aged RPE. The photoreceptor outer segment (POS) treatment of ARPE-19 cells causes Drp1 activation. Furthermore, pharmacological suppression of mitochondrial fission improved the phagocytosis of the POS. These findings indicate that mitochondrial dysfunction and fission in RPE impede phagocytosis and cause retardation of the visual cycle, which can be one of the age-related defects in the retina that may contribute to the onset of AMD.


Asunto(s)
Envejecimiento/fisiología , Mitocondrias/metabolismo , Fagocitosis/fisiología , Epitelio Pigmentado de la Retina/metabolismo , Animales , Western Blotting , Tamaño de la Célula , Células Cultivadas , Coroides/metabolismo , Dinaminas/metabolismo , Electrorretinografía , Etiquetado Corte-Fin in Situ , Masculino , Ratones , Ratones Endogámicos C57BL , Microscopía Electrónica de Transmisión , Segmento Externo de las Células Fotorreceptoras Retinianas/metabolismo , Rodopsina/metabolismo , Esclerótica/metabolismo , Porcinos , Tomografía de Coherencia Óptica
8.
Exp Eye Res ; 204: 108441, 2021 03.
Artículo en Inglés | MEDLINE | ID: mdl-33453278

RESUMEN

Retinal vein occlusion (RVO) is a vascular disease that represents characteristic retinal hemorrhage and dilated retinal veins. Despite its clinical importance, its pathogenesis remains largely unknown because of limited opportunities to acquire human retinal samples. Therefore, an animal model that reproduces the clinical features of RVO patients is required for further investigation. In this study, we established a pigmented murine RVO model that reproduced characteristic fundus appearances similar to human RVO findings. Retinal edema in this model was observed in both optical coherence tomography and histological analysis, which is a clinically important outcome. With quantitative real-time PCR analysis on retinal samples, we revealed that the mRNA level of vascular endothelial growth factor (VEGF) increased in the retina induced RVO. Moreover, this retinal edema was reduced by intravitreal injection of anti-VEGF antibody. These results were consistent with human clinical knowledge and suggested that this model could be a useful tool for research into new therapeutic approaches.


Asunto(s)
Modelos Animales de Enfermedad , Oclusión de la Vena Retiniana/patología , Animales , Anticuerpos Antiidiotipos/uso terapéutico , Angiografía con Fluoresceína , Humanos , Inmunoglobulina G/inmunología , Edema Macular/diagnóstico , Edema Macular/tratamiento farmacológico , Edema Macular/patología , Masculino , Ratones , Ratones Endogámicos C57BL , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Oclusión de la Vena Retiniana/diagnóstico por imagen , Oclusión de la Vena Retiniana/tratamiento farmacológico , Tomografía de Coherencia Óptica , Resultado del Tratamiento , Factor A de Crecimiento Endotelial Vascular/genética , Factor A de Crecimiento Endotelial Vascular/inmunología
9.
Biol Pharm Bull ; 44(7): 937-946, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-34193689

RESUMEN

The corneal epithelium is continuously exposed to oxygen, light, and environmental substances. Excessive exposure to those stresses is thought to be a risk factor for eye diseases. Photokeratitis is damage to the corneal epithelium resulting in a painful eye condition caused by unprotected exposure to UV rays, usually from sunlight, and is often found in people who spend a long time outdoors. In modern life, human eyes are exposed to artificial light from light-emitting diode (LED) displays of computers and smartphones, and it has been shown that short-wavelength (blue) LED light can damage eyes, especially photoreceptors. However, the effect of blue LED light on the cornea is less understood. In addition, it is important to develop new treatments for preserving human eyesight and eye health from light stress. Here, we used human corneal epithelial cells-transformed (HCE-T) cells as an in-vitro model to investigate the protective effect of NSP-116, an imidazolyl aniline derivative, against the oxidative stress induced by light in the corneal epithelium. Treatment with 10 µM NSP-116 significantly increased the cell viability and reduced the death ratio following UV or blue LED light exposure. Furthermore, NSP-116 treatment decreased light-induced reactive oxygen species production and preserved the mitochondrial membrane potential. Immunoblotting data showed that NSP-116 suppressed the stress response pathway. Finally, NSP-116 treatment prevented corneal epithelial apoptosis induced by blue LED light in an in-vivo mouse model. In conclusion, NSP-116 has a protective effect against oxidative stress and corneal cell death from both UV and blue LED light exposure.


Asunto(s)
Compuestos de Anilina/uso terapéutico , Lesiones de la Cornea/tratamiento farmacológico , Epitelio Corneal/efectos de los fármacos , Epitelio Corneal/efectos de la radiación , Depuradores de Radicales Libres/uso terapéutico , Imidazoles/uso terapéutico , Luz/efectos adversos , Traumatismos Experimentales por Radiación/tratamiento farmacológico , Protectores contra Radiación/uso terapéutico , Compuestos de Anilina/farmacología , Animales , Muerte Celular/efectos de los fármacos , Línea Celular , Supervivencia Celular/efectos de los fármacos , Lesiones de la Cornea/patología , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Células Epiteliales/fisiología , Epitelio Corneal/patología , Depuradores de Radicales Libres/farmacología , Humanos , Imidazoles/farmacología , Masculino , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Ratones , Traumatismos Experimentales por Radiación/patología , Protectores contra Radiación/farmacología , Especies Reactivas de Oxígeno/metabolismo
10.
J Neurosci ; 39(18): 3376-3393, 2019 05 01.
Artículo en Inglés | MEDLINE | ID: mdl-30819798

RESUMEN

Peripherin 2 (PRPH2) is a tetraspanin protein concentrated in the light-sensing cilium (called the outer segment) of the vertebrate photoreceptor. The mechanism underlying the ciliary targeting of PRPH2 and the etiology of cone dystrophy caused by PRPH2 mutations remain elusive. Here we show that the late endosome (LE) is the main waystation that critically sorts newly synthesized PRPH2 to the cilium. PRPH2 is expressed in the luminal membrane of the LE. We delineate multiple C-terminal motifs of PRPH2 that distinctively regulate its LE and ciliary targeting through ubiquitination and binding to ESCRT (Endosomal Sorting Complexes Required for Transport) component Hrs. Using the newly developed TetOn-inducible system in transfected male and female mouse cones in vivo, we show that the entry of nascent PRPH2 into the cone outer segment can be blocked by either cone dystrophy-causing C-terminal mutations of PRPH2, or by short-term perturbation of the LE or recycling endosomal traffic. These findings open new avenues of research to explore the biological role of the LE in the biosynthetic pathway and the etiology of cone dystrophy caused by PRPH2 mutations and/or malfunctions of the LE.SIGNIFICANCE STATEMENT Peripherin 2 (PRPH2) is a tetraspanin protein abundantly expressed in the light-sensing cilium, the outer segment, of the vertebrate photoreceptor. The mechanism underlying the ciliary transport of PRPH2 is unclear. The present study reveals a novel ciliary targeting pathway, in which the newly synthesized PRPH2 is first targeted to the lumen of the late endosome (LE) en route to the cilia. We deciphered the protein motifs and the machinery that regulates the LE trafficking of PRPH2. Using a novel TetOn-inducible system in transfected mouse cones, we showed that the LE pathway of PRPH2 is critical for its outer segment expression. A cone dystrophy-causing mutation impairs the LE and ciliary targeting of PRPH2, implicating the relevance of LE to cone/macular degenerative diseases.


Asunto(s)
Cilios/metabolismo , Endosomas/metabolismo , Periferinas/metabolismo , Células Fotorreceptoras Retinianas Conos/metabolismo , Células Fotorreceptoras Retinianas Bastones/metabolismo , Animales , Femenino , Células HEK293 , Humanos , Masculino , Ratones Endogámicos C57BL , Ratones Transgénicos
11.
Biochem Biophys Res Commun ; 526(2): 479-484, 2020 05 28.
Artículo en Inglés | MEDLINE | ID: mdl-32234235

RESUMEN

Exposure to blue light from light-emitting diodes (LEDs) is a source of damage for human eyes in today's modern life. Although it is well known that blue light can cause cellular damage and death, the molecular mechanism underlying this is still not fully understood. Here, we demonstrated that exposure to blue LED light increased lysosome levels and perinuclear cluster formation in 661W murine photoreceptor-derived cells. Irradiation with blue LED light promoted the nuclear transport of transcription factor EB (TFEB) and a subsequent increase in lysosomal-related gene expression. Moreover, blue LED light induced morphological changes in lysosomal structure and lysosomal membrane permeabilization (LMP). These effects were suppressed by an antioxidant, N-acetylcysteine (NAC). Finally, a calcium ion chelator, BAPTA-AM, attenuated blue LED light-induced lysosomal biogenesis and cell death. Taken together, these findings suggest that oxidative stress under blue LED light increases lysosome levels via the TFEB pathway in a calcium-dependent manner, resulting in the accumulation of damaged lysosomes and subsequently lysosomal cell death. Our results imply that lysosomal homeostasis plays a key role in the maintenance of eye function and the progression of retinal diseases.


Asunto(s)
Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/metabolismo , Muerte Celular/efectos de la radiación , Luz/efectos adversos , Lisosomas/efectos de la radiación , Células Fotorreceptoras de Vertebrados/efectos de la radiación , Transporte Activo de Núcleo Celular/efectos de la radiación , Animales , Línea Celular , Lisosomas/metabolismo , Ratones , Estrés Oxidativo/efectos de la radiación , Células Fotorreceptoras de Vertebrados/metabolismo
12.
EMBO Rep ; 18(8): 1460-1472, 2017 08.
Artículo en Inglés | MEDLINE | ID: mdl-28607034

RESUMEN

The primary cilium is a plasma membrane-protruding sensory organelle that undergoes regulated assembly and resorption. While the assembly process has been studied extensively, the cellular machinery that governs ciliary resorption is less well understood. Previous studies showed that the ciliary pocket membrane is an actin-rich, endocytosis-active periciliary subdomain. Furthermore, Tctex-1, originally identified as a cytoplasmic dynein light chain, has a dynein-independent role in ciliary resorption upon phosphorylation at Thr94. Here, we show that the remodeling and endocytosis of the ciliary pocket membrane are accelerated during ciliary resorption. This process depends on phospho(T94)Tctex-1, actin, and dynamin. Mechanistically, Tctex-1 physically and functionally interacts with the actin dynamics regulators annexin A2, Arp2/3 complex, and Cdc42. Phospho(T94)Tctex-1 is required for Cdc42 activation before the onset of ciliary resorption. Moreover, inhibiting clathrin-dependent endocytosis or suppressing Rab5GTPase on early endosomes effectively abrogates ciliary resorption. Taken together with the epistasis functional assays, our results support a model in which phospho(T94)Tctex-1-regulated actin polymerization and periciliary endocytosis play an active role in orchestrating the initial phase of ciliary resorption.


Asunto(s)
Actinas/fisiología , Cilios/fisiología , Dineínas/metabolismo , Línea Celular , Clatrina/fisiología , Dinaminas , Dineínas/genética , Endocitosis , Células Epiteliales , Humanos , Fosforilación , Multimerización de Proteína , Retina/citología
13.
J Biol Chem ; 288(25): 18521-32, 2013 Jun 21.
Artículo en Inglés | MEDLINE | ID: mdl-23658022

RESUMEN

Protein export from the endoplasmic reticulum (ER) depends on the interaction between a signal motif on the cargo and a cargo recognition site on the coatomer protein complex II. A hydrophobic sequence in the N terminus of the bovine anion exchanger 1 (AE1) anion exchanger facilitated the ER export of human AE1Δ11, an ER-retained AE1 mutant, through interaction with a specific Sec24 isoform. The cell surface expression and N-glycan processing of various substitution mutants or chimeras of human and bovine AE1 proteins and their Δ11 mutants in HEK293 cells were examined. The N-terminal sequence (V/L/F)X(I/L)X(M/L), (26)VSIPM(30) in bovine AE1, which is comparable with ΦXΦXΦ, acted as the ER export signal for AE1 and AE1Δ11 (Φ is a hydrophobic amino acid, and X is any amino acid). The AE1-Ly49E chimeric protein possessing the ΦXΦXΦ motif exhibited effective cell surface expression and N-glycan maturation via the coatomer protein complex II pathway, whereas a chimera lacking this motif was retained in the ER. A synthetic polypeptide containing the N terminus of bovine AE1 bound the Sec23A-Sec24C complex through a selective interaction with Sec24C. Co-transfection of Sec24C-AAA, in which the residues (895)LIL(897) (the binding site for another ER export signal motif IXM on Sec24C and Sec24D) were mutated to (895)AAA(897), specifically increased ER retention of the AE1-Ly49E chimera. These findings demonstrate that the ΦXΦXΦ sequence functions as a novel signal motif for the ER export of cargo proteins through an exclusive interaction with Sec24C.


Asunto(s)
Retículo Endoplásmico/metabolismo , Proteínas de la Membrana/metabolismo , Señales de Clasificación de Proteína , Proteínas de Transporte Vesicular/metabolismo , Secuencias de Aminoácidos/genética , Secuencia de Aminoácidos , Animales , Proteína 1 de Intercambio de Anión de Eritrocito/genética , Proteína 1 de Intercambio de Anión de Eritrocito/metabolismo , Sitios de Unión/genética , Unión Competitiva , Bovinos , Células HEK293 , Humanos , Immunoblotting , Proteínas de la Membrana/genética , Microscopía Confocal , Mutación , Péptidos/metabolismo , Unión Proteica , Transporte de Proteínas , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas de Transporte Vesicular/genética
14.
Nihon Yakurigaku Zasshi ; 159(4): 203-208, 2024 Jul 01.
Artículo en Japonés | MEDLINE | ID: mdl-38684400

RESUMEN

A photoreceptor is a specialized neuron that is responsible for the conversion of light into an electrical signal. Photoreceptors are classified into rods and cones, and both photoreceptors possess light-sensing ciliary organelles called outer segments (OSs), anchored in the cells by a microtubule-based axoneme. The OS consists of a stack of disc membranes, which are abundant for the retinal phototransduction proteins such as rhodopsin. Recently, modern protein synchronization techniques using in vivo transfection in rodents revealed that rhodopsin transits through Rab11-positive recycling endosomes, preferentially entering the OS in the dark. Moreover, Peripherin-2 (PRPH2, also called retinal degeneration slow, RDS), a photoreceptor-specific tetraspanin protein essential for the morphogenesis of disc membranes, is delivered to the OS following complementary to that of rhodopsin. Various PRPH2 disease-causing mutations have been found in humans, and most of the mutations in the cytosolic C-terminus of PRPH2 are linked to cone-dominant macular dystrophies. It has been shown that the late endosome is the waystation that sorts newly synthesized PRPH2 into the cilium. The multiple C-terminal motifs of PRPH2 regulate its late endosome and ciliary targeting through ubiquitination and binding to an Endosomal Sorting Complexes Required for Transport (ESCRT) component, Hrs. These findings suggest that the late endosomes play an important role in the biosynthetic pathway of ciliary proteins and can be a new therapeutic target for the diseases caused by ciliary defects.


Asunto(s)
Cilios , Endosomas , Endosomas/metabolismo , Animales , Cilios/metabolismo , Humanos , Periferinas/metabolismo , Membrana Celular/metabolismo , Transporte de Proteínas
15.
BMC Complement Med Ther ; 24(1): 3, 2024 Jan 02.
Artículo en Inglés | MEDLINE | ID: mdl-38167061

RESUMEN

BACKGROUND: Blue light exposure is known to induce reactive oxygen species (ROS) production and increased endoplasmic reticulum stress, leading to apoptosis of photoreceptors. Maqui berry (Aristotelia chilensis) is a fruit enriched in anthocyanins, known for beneficial biological activities such as antioxidation. In this study, we investigated the effects of Maqui berry extract (MBE) and its constituents on the subcellular damage induced by blue light irradiation in mouse retina-derived 661W cells. METHODS: We evaluated the effects of MBE and its main delphinidins, delphinidin 3-O-sambubioside-5-O-glucoside (D3S5G) and delphinidin 3,5-O-diglucoside (D3G5G), on blue light-induced damage on retinal cell line 661W cells. We investigated cell death, the production of ROS, and changes in organelle morphology using fluorescence microscopy. The signaling pathway linked to stress response was evaluated by immunoblotting in the whole cell lysates or nuclear fractions. We also examined the effects of MBE and delphinidins against rotenone-induced mitochondrial dysfunction. RESULTS: Blue light-induced cell death, increased intracellular ROS generation and mitochondrial fragmentation, decreased ATP-production coupled respiration, caused lysosomal membrane permeabilization, and increased ATF4 protein level. Treatment with MBE and its main constituents, delphinidin 3-O-sambubioside-5-O-glucoside and delphinidin 3,5-O-diglucoside, prevented these defects. Furthermore, MBE and delphinidins also protected 661W cells from rotenone-induced cell death. CONCLUSIONS: Maqui berry may be a useful protective agent for photoreceptors against the oxidative damage induced by exposure to blue light.


Asunto(s)
Antocianinas , Elaeocarpaceae , Animales , Ratones , Antocianinas/farmacología , Especies Reactivas de Oxígeno/metabolismo , Frutas , Luz Azul , Rotenona , Elaeocarpaceae/metabolismo , Glucósidos , Orgánulos/metabolismo
16.
Nat Commun ; 15(1): 5970, 2024 Jul 23.
Artículo en Inglés | MEDLINE | ID: mdl-39043666

RESUMEN

Vacuolar protein sorting 35 (VPS35), the core component of the retromer complex which regulates endosomal trafficking, is genetically linked with Parkinson's disease (PD). Impaired vision is a common non-motor manifestation of PD. Here, we show mouse retinas with VPS35-deficient rods exhibit synapse loss and visual deficit, followed by progressive degeneration concomitant with the emergence of Lewy body-like inclusions and phospho-α-synuclein (P-αSyn) aggregation. Ultrastructural analyses reveal VPS35-deficient rods accumulate aggregates in late endosomes, deposited as lipofuscins bound to P-αSyn. Mechanistically, we uncover a protein network of VPS35 and its interaction with HSC70. VPS35 deficiency promotes sequestration of HSC70 and P-αSyn aggregation in late endosomes. Microglia which engulf lipofuscins and P-αSyn aggregates are activated, displaying autofluorescence, observed as bright dots in fundus imaging of live animals, coinciding with pathology onset and progression. The Rod∆Vps35 mouse line is a valuable tool for further mechanistic investigation of αSyn lesions and retinal degenerative diseases.


Asunto(s)
Degeneración Retiniana , Proteínas de Transporte Vesicular , alfa-Sinucleína , Animales , alfa-Sinucleína/metabolismo , alfa-Sinucleína/genética , Proteínas de Transporte Vesicular/metabolismo , Proteínas de Transporte Vesicular/genética , Ratones , Degeneración Retiniana/genética , Degeneración Retiniana/metabolismo , Degeneración Retiniana/patología , Células Fotorreceptoras Retinianas Bastones/metabolismo , Células Fotorreceptoras Retinianas Bastones/patología , Endosomas/metabolismo , Microglía/metabolismo , Microglía/patología , Enfermedad de Parkinson/genética , Enfermedad de Parkinson/metabolismo , Enfermedad de Parkinson/patología , Retina/metabolismo , Retina/patología , Ratones Noqueados , Modelos Animales de Enfermedad , Humanos , Sinapsis/metabolismo , Sinapsis/patología , Masculino
17.
Biochem Biophys Res Commun ; 430(2): 839-45, 2013 Jan 11.
Artículo en Inglés | MEDLINE | ID: mdl-23200831

RESUMEN

The PDZ (PSD-95/Drosophila discs-large protein/zonula occludens protein) domain-containing proteins Na(+)/H(+) exchanger regulatory factor 1 (NHERF1) and NHERF2 interact with the glutamate transporter GLAST. To characterize the roles of these NHERF proteins in the plasma membrane targeting of GLAST, we examined the interaction of green fluorescent protein (EGFP)-tagged GLAST with epitope-tagged NHERF proteins in human embryonic kidney (HEK) 293T cells. Co-expression of either NHERF protein increased the cell surface expression of EGFP-GLAST. Deletion of the C-terminal PDZ domain-binding motif caused an increase in EGFP-GLAST with immature endoglycosidase H-sensitive N-linked oligosaccharides, suggesting impaired exit of EGFP-GLAST from the endoplasmic reticulum (ER). Immunoprecipitation experiments revealed that NHERF1 predominantly bound EGFP-GLAST containing immature N-glycans, whereas NHERF2 co-precipitated EGFP-GLAST with mature N-glycans. Expression of a dominant-negative mutant of the GTPase Sar1 increased the interaction of EGFP-GLAST with NHERF1 in the ER. By contrast, immunofluorescence microscopy showed that NHERF2 co-localized with EGFP-GLAST in ER-Golgi intermediate compartments (ERGICs), at the plasma membrane and in early endosomes, but not in the ER. These results suggest that NHERF1 interacts with GLAST during ER export, while NHERF2 interacts with GLAST in the secretory pathway from the ERGIC to the plasma membrane, thereby modulating the cell surface expression of GLAST.


Asunto(s)
Membrana Celular/metabolismo , Transportador 1 de Aminoácidos Excitadores/metabolismo , Fosfoproteínas/metabolismo , Intercambiadores de Sodio-Hidrógeno/metabolismo , Animales , Perros , Retículo Endoplásmico/metabolismo , Transportador 1 de Aminoácidos Excitadores/genética , Aparato de Golgi/metabolismo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , Humanos , Proteínas de Unión al GTP Monoméricas/genética , Proteínas de Unión al GTP Monoméricas/metabolismo , Dominios PDZ/genética , Fosfoproteínas/genética , Intercambiadores de Sodio-Hidrógeno/genética
18.
Front Mol Biosci ; 10: 1232188, 2023.
Artículo en Inglés | MEDLINE | ID: mdl-37780208

RESUMEN

The primary cilium is a single immotile microtubule-based organelle that protrudes into the extracellular space. Malformations and dysfunctions of the cilia have been associated with various forms of syndromic and non-syndromic diseases, termed ciliopathies. The primary cilium is therefore gaining attention due to its potential as a therapeutic target. In this review, we examine ciliary receptors, ciliogenesis, and ciliary trafficking as possible therapeutic targets. We first discuss the mechanisms of selective distribution, signal transduction, and physiological roles of ciliary receptors. Next, pathways that regulate ciliogenesis, specifically the Aurora A kinase, mammalian target of rapamycin, and ubiquitin-proteasome pathways are examined as therapeutic targets to regulate ciliogenesis. Then, in the photoreceptors, the mechanism of ciliary trafficking which takes place at the transition zone involving the ciliary membrane proteins is reviewed. Finally, some of the current therapeutic advancements highlighting the role of large animal models of photoreceptor ciliopathy are discussed.

19.
Nat Commun ; 13(1): 374, 2022 01 18.
Artículo en Inglés | MEDLINE | ID: mdl-35042858

RESUMEN

Age-related macular degeneration (AMD) is the leading cause of blindness among the elderly. Dry AMD has unclear etiology and no treatment. Lipid-rich drusen are the hallmark of dry AMD. An AMD mouse model and insights into drusenogenesis are keys to better understanding of this disease. Chloride intracellular channel 4 (CLIC4) is a pleomorphic protein regulating diverse biological functions. Here we show that retinal pigment epithelium (RPE)-specific Clic4 knockout mice exhibit a full spectrum of functional and pathological hallmarks of dry AMD. Multidisciplinary longitudinal studies of disease progression in these mice support a mechanistic model that links RPE cell-autonomous aberrant lipid metabolism and transport to drusen formation.


Asunto(s)
Canales de Cloruro/genética , Degeneración Macular/genética , Proteínas Mitocondriales/genética , Mutación/genética , Epitelio Pigmentado de la Retina/metabolismo , Animales , Muerte Celular , Canales de Cloruro/deficiencia , Modelos Animales de Enfermedad , Fondo de Ojo , Homeostasis , Metabolismo de los Lípidos , Degeneración Macular/diagnóstico por imagen , Degeneración Macular/fisiopatología , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas Mitocondriales/deficiencia , Especificidad de Órganos/genética , Drusas Retinianas/complicaciones , Drusas Retinianas/diagnóstico por imagen , Drusas Retinianas/patología , Epitelio Pigmentado de la Retina/diagnóstico por imagen , Epitelio Pigmentado de la Retina/fisiopatología , Epitelio Pigmentado de la Retina/ultraestructura , Factores de Riesgo , Transcripción Genética , Visión Ocular/fisiología
20.
Sci Rep ; 11(1): 18555, 2021 09 17.
Artículo en Inglés | MEDLINE | ID: mdl-34535730

RESUMEN

The cornea is directly exposed to cigarette smoke, and smoking is a risk factor for several corneal diseases including dry eye syndrome. Currently, heated tobacco products (HTPs) are widely used as substitutes for cigarette smoking around the world. In the present study, we investigated the molecular mechanism(s) leading to cellular injury induced by cigarette smoke extract (CSE) or HTPs. Exposure to CSE perturbed the formation of tight junctions, leading to an increase in cell volume, a decrease in transepithelial electrical resistance (TER) in the human corneal epithelial cell-transformed (HCE-T) cell line. Moreover, CSE exposure induced both lipid peroxidation and ferrous [Fe(II)] ion accumulation in autolysosomal compartments. Interestingly, a cleaved form of ferritin appeared when HCE-T cells were incubated with CSE. This aberrant ferritin processing was suppressed by treatment with autophagy inhibitors. Furthermore, the CSE-induced cell death was suppressed by either ferrostatin-1 or deferoxamine (DFO). CSE exposure also promoted the expression of cytokines whereas DFO treatment inhibited the CSE-induced expression of these cytokines. Exposure to HTPs also induced both HCE-T cell death and cleaved ferritin accumulation in a concentration- and time-dependent manner. These results indicated that CSE or HTPs activated the ferroptosis signaling pathway, which contributed to corneal epithelial cell injury.


Asunto(s)
Apoferritinas/metabolismo , Fumar Cigarrillos/metabolismo , Córnea/metabolismo , Ferritinas/metabolismo , Hierro/metabolismo , Oxidorreductasas/metabolismo , Línea Celular , Fumar Cigarrillos/efectos adversos , Córnea/citología , Células Epiteliales/citología , Células Epiteliales/metabolismo , Humanos
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