RESUMEN
Glaucoma is characterized by the progressive degeneration of retinal ganglion cells (RGCs) and their axons, and its risk increases with aging. Yet comprehensive insights into the complex mechanisms are largely unknown. Here, we found that anti-aging molecule Sirt6 was highly expressed in RGCs. Deleting Sirt6 globally or specifically in RGCs led to progressive RGC loss and optic nerve degeneration during aging, despite normal intraocular pressure (IOP), resembling a phenotype of normal-tension glaucoma. These detrimental effects were potentially mediated by accelerated RGC senescence through Caveolin-1 upregulation and by the induction of mitochondrial dysfunction. In mouse models of high-tension glaucoma, Sirt6 level was decreased after IOP elevation. Genetic overexpression of Sirt6 globally or specifically in RGCs significantly attenuated high tension-induced degeneration of RGCs and their axons, whereas partial or RGC-specific Sirt6 deletion accelerated RGC loss. Importantly, therapeutically targeting Sirt6 with pharmacological activator or AAV2-mediated gene delivery ameliorated high IOP-induced RGC degeneration. Together, our studies reveal a critical role of Sirt6 in preventing RGC and optic nerve degeneration during aging and glaucoma, setting the stage for further exploration of Sirt6 activation as a potential therapy for glaucoma.
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Envejecimiento , Modelos Animales de Enfermedad , Glaucoma , Nervio Óptico , Células Ganglionares de la Retina , Sirtuinas , Animales , Células Ganglionares de la Retina/metabolismo , Células Ganglionares de la Retina/patología , Ratones , Sirtuinas/metabolismo , Sirtuinas/genética , Glaucoma/metabolismo , Glaucoma/genética , Glaucoma/patología , Glaucoma/etiología , Nervio Óptico/metabolismo , Nervio Óptico/patología , Envejecimiento/metabolismo , Envejecimiento/genética , Presión Intraocular , Humanos , Axones/metabolismo , Axones/patología , Ratones Noqueados , Degeneración Nerviosa/metabolismoRESUMEN
Zika virus (ZIKV) infects pregnant women and causes devastating congenital zika syndrome (CZS). How the virus is vertically transmitted to the fetus and induces neuronal loss remains unclear. We previously reported that Pellino (Peli)1, an E3 ubiquitin ligase, promotes p38MAPK activation in microglia and induction of lethal encephalitis by facilitating the replication of West Nile virus (WNV), a closely related flavivirus. Here, we found that Peli1 expression was induced on ZIKV-infected human monocytic cells, peripheral blood mononuclear cells, human first-trimester placental trophoblasts, and neural stem cell (hNSC)s. Peli1 mediates ZIKV cell attachment, entry and viral translation and its expression is confined to the endoplasmic reticulum. Moreover, Peli1 mediated inflammatory cytokine and chemokine responses and induced cell death in placental trophoblasts and hNSCs. ZIKV-infected pregnant mice lacking Peli1 signaling had reduced placental inflammation and tissue damage, which resulted in attenuated congenital abnormalities. Smaducin-6, a membrane-tethered Smad6-derived peptide, blocked Peli1-mediated NF-κB activation but did not have direct effects on ZIKV infection. Smaducin-6 reduced inflammatory responses and cell death in placental trophoblasts and hNSCs, and diminished placental inflammation and damage, leading to attenuated congenital malformations in mice. Collectively, our results reveal a novel role of Peli1 in flavivirus pathogenesis and suggest that Peli1 promotes ZIKV vertical transmission and neuronal loss by mediating inflammatory cytokine responses and induction of cell death. Our results also identify Smaducin-6 as a potential therapeutic candidate for treatment of CZS.
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Síndrome de Guillain-Barré , Proteínas Nucleares/antagonistas & inhibidores , Péptidos/farmacología , Transducción de Señal/efectos de los fármacos , Ubiquitina-Proteína Ligasas/antagonistas & inhibidores , Infección por el Virus Zika , Virus Zika/metabolismo , Animales , Línea Celular , Femenino , Síndrome de Guillain-Barré/tratamiento farmacológico , Síndrome de Guillain-Barré/genética , Síndrome de Guillain-Barré/metabolismo , Síndrome de Guillain-Barré/patología , Humanos , Masculino , Ratones , Ratones Noqueados , FN-kappa B/genética , FN-kappa B/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Transducción de Señal/genética , Trofoblastos/metabolismo , Trofoblastos/patología , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , Virus Zika/genética , Infección por el Virus Zika/tratamiento farmacológico , Infección por el Virus Zika/genética , Infección por el Virus Zika/metabolismo , Infección por el Virus Zika/patologíaRESUMEN
The cornea is transparent and innervated by a dense collection of sensory nerves originating from the ocular branch of the trigeminal nerve. This study was designed to comprehensively analyze alterations of corneal sub-basal nerve plexus in a mouse model of tauopathy (P301L transgenic mice) to test the possibility of using corneal nerves as a biomarker for tauopathy. Corneal sensitivity, thickness and epithelial wound healing were measured non-invasively by aeshesiometer, optical coherence tomography and fluorescein staining, respectively. Tau, corneal nerves and immune cells were examined by immunohistochemistry or Western blot. At the early stage of tauopathy, although corneal sensitivity, thickness and nerve fiber density were not greatly altered, corneal nerve abnormalities were observed in the peripheral region of young P301L mice. With aging, the density of abnormal nerves increased, while corneal sensitivity, epithelial thickness, nerve fiber density and length decreased in middle-aged P301L mice compared with WT mice. After corneal epithelial injury in young mice, no difference in reepithelialization was observed between two groups of mice, however, the regeneration of corneal nerves in P301L mice lagged behind WT mice, which was reflected by delayed recovery of corneal sensitivity, decreased corneal nerve density and length and density of CD45+ dendriform cells in P301L mice. In conclusion, our data provide compelling evidence that corneal nerves were changed in a mouse model of tauopathy in an age-dependent manner. Moreover, tau overexpression impairs corneal nerve regeneration. These results suggest that cornea may serve as a promising ocular site for the early diagnosis of tauopathy.
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Enfermedades de la Córnea , Lesiones de la Cornea , Tauopatías , Animales , Córnea/inervación , Modelos Animales de Enfermedad , Ratones , Regeneración Nerviosa/fisiología , Nervio Trigémino/fisiologíaRESUMEN
Tauopathies are a family of neurodegenerative diseases which predominately afflict the rapidly growing aging population suffering from various brain disorders including Alzheimer's disease, frontotemporal dementia with parkinsonism-17 and Pick disease. As the only visually accessible region of the central nervous system, in recent years, the retina has attracted extensive attention for its potential as a target for visualizing and quantifying emerging biomarkers of neurodegenerative diseases. Our previous study has found that retinal vascular inflammation and leakage occur at the very early stage of tauopathic mouse model. Here, we aimed to non-invasively visualize age-dependent alterations of retinal vasculature assessing the potential for using changes in retinal vasculature as the biomarker for the early diagnosis of tauopathy. Optical coherence tomography angiography (OCTA), a non-invasive depth-resolved high-resolution imaging technique was used to visualize and quantify tauopathy-induced alterations of retinal vasculature in P301S transgenic mice overexpressing the P301S mutant form of human tau and age-matched wild type littermate mice at 3, 6 and 10 months of age. We observed significant alterations of vascular features in the intermediate capillary plexus (ICP) and deep capillary plexus (DCP) but not in the superficial vascular complex (SVC) of P301S mice at early stages of tauopathy. With aging, alterations of vascular features in P301S mice became more prominent in all three vascular plexuses. Staining of retinal vasculature in flatmounts and trypsin digests of P301S mice at 10 months of age revealed decreased vessel density and increased acellular capillary formation, indicating that vascular degeneration also occurs during tauopathy. Overall, our results demonstrate that the changes in retinal vascular features accelerate during the progression of tauopathy. Vessels in the ICP and DCP may be more susceptible to tauopathy than vessels in the SVC. Since changes in retinal vasculature often precede tau pathology in the brain, non-invasive identification of retinal vascular alterations with OCTA may be a useful biomarker for the early diagnosis of tauopathy and monitoring its progression.
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Tauopatías , Tomografía de Coherencia Óptica , Animales , Humanos , Ratones , Angiografía , Biomarcadores , Modelos Animales de Enfermedad , Angiografía con Fluoresceína/métodos , Ratones Transgénicos , Vasos Retinianos/diagnóstico por imagen , Vasos Retinianos/patología , Tauopatías/diagnóstico por imagen , Tauopatías/patología , Tomografía de Coherencia Óptica/métodosRESUMEN
Corneal neovascularization can cause devastating consequences including vision impairment and even blindness. Corneal inflammation is a crucial factor for the induction of corneal neovascularization. Current anti-inflammatory approaches are of limited value with poor therapeutic effects. Therefore, there is an urgent need to develop new therapies that specifically modulate inflammatory pathways and inhibit neovascularization in the cornea. The interaction of chemokines and their receptors plays a key role in regulating leukocyte migration during inflammatory response. CXCR3 is essential for mediating the recruitment of activated T cells and microglia/macrophages, but the role of CXCR3 in the initiation and promotion of corneal neovascularization remains unclear. Here, we showed that the expression of CXCL10 and CXCR3 was significantly increased in the cornea after alkali burn. Compared with WT mice, CXCR3-/- mice exhibited significantly increased corneal hemangiogenesis and lymphangiogenesis after alkali burn. In addition, exaggerated leukocyte infiltration and leukostasis, and elevated expression of inflammatory cytokines and angiogenic factor were also found in the corneas of CXCR3-/- mice subjected to alkali burn. With bone marrow (BM) transplantation, we further demonstrated that the deletion of CXCR3 in BM-derived leukocytes plays a key role in the acceleration of alkali burn-induced corneal neovascularization. Taken together, our results suggest that upregulation of CXCR3 does not exhibit its conventional action as a proinflammatory cytokine but instead serves as a self-protective mechanism for the modulation of inflammation and maintenance of corneal avascularity after corneal alkali burn.
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Quemaduras Químicas , Lesiones de la Cornea , Neovascularización de la Córnea , Quemaduras Oculares , Ratones , Animales , Neovascularización de la Córnea/tratamiento farmacológico , Quemaduras Químicas/tratamiento farmacológico , Álcalis/toxicidad , Quemaduras Oculares/tratamiento farmacológico , Lesiones de la Cornea/metabolismo , Córnea/metabolismo , Inflamación/metabolismo , Citocinas/metabolismo , Modelos Animales de EnfermedadRESUMEN
Sirt6 activation has emerged as a promising drug target for the treatment of various human diseases, while only limited Sirt6 activators have been reported. Herein, a series of novel pyrrolo[1,2-a]quinoxaline-based derivatives have been identified as potent and selective Sirt6 activators with low cytotoxicity. Sirt6-knockdown findings have validated the on-target effects of this class of Sirt6 activators. Docking studies indicate the protonated nitrogen on the side chain of 38 forms π-cation interactions with Trp188, further stabilizing it into this extended binding pocket. New compounds 35, 36, 38, 46, 47, and 50 strongly repressed LPS-induced proinflammatory cytokine/chemokine production, while 38 also significantly suppressed SARS-CoV-2 infection with an EC50 value of 9.3 µM. Moreover, compound 36 significantly inhibited the colony formation of cancer cells. These new molecules may serve as useful pharmacological tools or potential therapeutics against cancer, inflammation, and infectious diseases.
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COVID-19 , Sirtuinas , Humanos , Quinoxalinas/farmacología , Quinoxalinas/química , SARS-CoV-2/metabolismo , Sirtuinas/metabolismoRESUMEN
Optic neuritis, a characteristic feature of multiple sclerosis (MS), involves the inflammation of the optic nerve and the degeneration of retinal ganglion cells (RGCs). Although previous studies suggest that retinal blood flow alterations occur during optic neuritis, the precise location, the degree of impairment, and the underlying mechanisms remain unclear. In this study, we utilized two emerging non-invasive imaging techniques, laser speckle flowgraphy (LSFG) and optical coherence tomography angiography (OCTA), to investigate retinal vascular changes in a mouse model of MS, known as experimental autoimmune encephalomyelitis (EAE). We associated these changes with leukostasis, RGC injury, and the overall progression of EAE. LSFG imaging revealed a progressive reduction in retinal blood flow velocity and increased vascular resistance near the optic nerve head in the EAE model, indicating impaired ocular blood flow. OCTA imaging demonstrated significant decreases in vessel density, number of junctions, and total vessel length in the intermediate and deep capillary plexus of the EAE mice. Furthermore, our analysis of leukostasis revealed a significant increase in adherent leukocytes in the retinal vasculature of the EAE mice, suggesting the occurrence of vascular inflammation in the early development of EAE pathology. The abovechanges preceded or were accompanied by the characteristic hallmarks of optic neuritis, such as RGC loss and reduced visual acuity. Overall, our study sheds light on the intricate relationship between retinal vascular alterations and the progression of optic neuritis as well as MS clinical score. It also highlights the potential for the development of image-based biomarkers for the diagnosis and monitoring of optic neuritis as well as MS, particularly in response to emerging treatments.
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Encefalomielitis Autoinmune Experimental , Leucostasis , Esclerosis Múltiple , Neuritis Óptica , Ratones , Animales , Tomografía de Coherencia Óptica/métodos , Neuritis Óptica/diagnóstico por imagen , Neuritis Óptica/patología , Encefalomielitis Autoinmune Experimental/diagnóstico por imagen , Encefalomielitis Autoinmune Experimental/patología , Inflamación/patología , Modelos Animales de Enfermedad , AngiografíaRESUMEN
Purpose: Retinal ischemia is a common cause of a variety of eye diseases, such as retinopathy of prematurity, diabetic retinopathy, and vein occlusion. Protein kinase RNA-activated-like endoplasmic reticulum (ER) kinase (PERK), one of the main ER stress sensor proteins, has been involved in many diseases. In this study, we investigated the role of PERK in ischemia-induced retinopathy using a mouse model of oxygen-induced retinopathy (OIR). Methods: OIR was induced by subjecting neonatal pups to 70% oxygen at postnatal day 7 (P7) followed by returning to room air at P12. GSK2606414, a selective PERK inhibitor, was orally administrated to pups right after they were returned to room air once daily until 1 day before sample collection. Western blot, immunostaining, and quantitative PCR were used to assess PERK phosphorylation, retinal changes, and signaling pathways in relation to PERK inhibition. Results: PERK phosphorylation was prominently increased in OIR retinas, which was inhibited by GSK2606414. Concomitantly, PERK inhibition significantly reduced retinal neovascularization (NV) and retinal ganglion cell (RGC) loss, restored astrocyte network, and promoted revascularization. Furthermore, PERK inhibition downregulated the recruitment/proliferation of mononuclear phagocytes but did not affect OIR-upregulated canonical angiogenic pathways. Conclusions: Our results demonstrate that PERK is involved in ischemia-induced retinopathy and its inhibition using GSK2606414 could offer an effective therapeutic intervention aimed at alleviating retinal NV while preventing neuron loss during retinal ischemia.
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Enfermedades de la Retina , Neovascularización Retiniana , Retinopatía de la Prematuridad , eIF-2 Quinasa , Animales , Ratones , Animales Recién Nacidos , Modelos Animales de Enfermedad , Isquemia/metabolismo , Ratones Endogámicos C57BL , Neovascularización Patológica/metabolismo , Oxígeno/metabolismo , Retina , Enfermedades de la Retina/etiología , Enfermedades de la Retina/prevención & control , Células Ganglionares de la Retina/metabolismo , Neovascularización Retiniana/prevención & control , Neovascularización Retiniana/metabolismo , Retinopatía de la Prematuridad/metabolismo , eIF-2 Quinasa/metabolismoRESUMEN
Purpose: The pathogenic mechanisms behind the development of ischemic retinopathy are complex and poorly understood. This study investigates the involvement of exchange protein directly activated by cAMP (Epac)1 signaling in pericyte injury during ischemic retinopathy, including diabetic retinopathy, a disease that threatens vision. Methods: Mouse models of retinal ischemia-reperfusion injury and type 1 diabetes induced by streptozotocin were used to investigate the pathogenesis of these diseases. The roles of Epac1 signaling in the pathogenesis of ischemic retinopathy were determined by an Epac1 knockout mouse model. The cellular and molecular mechanisms of Epac1-mediated pericyte dysfunction in response to high glucose were investigated by specific modulation of Epac1 activity in primary human retinal pericytes using Epac1-specific RNA interference and a pharmacological inhibitor. Results: Ischemic injury or diabetes-induced retinal capillary degeneration were associated with an increased expression of Epac1 in the mouse retinal vasculature, including both endothelial cells and pericytes. Genetic deletion of Epac1 protected ischemic injury-induced pericyte loss and capillary degeneration in the mouse retina. Furthermore, high glucose-induced Epac1 expression in retinal pericytes was accompanied by increased Drp1 phosphorylation, mitochondrial fission, reactive oxygen species production, and caspase 3 activation. Inhibition of Epac1 via RNA interference or pharmacological approaches blocked high glucose-mediated mitochondrial dysfunction and caspase 3 activation. Conclusions: Our study reveals an important role of Epac1 signaling in mitochondrial dynamics, reactive oxygen species production, and apoptosis in retinal pericytes and identifies Epac1 as a therapeutic target for treating ischemic retinopathy.
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Factores de Intercambio de Guanina Nucleótido , Pericitos , Degeneración Retiniana , Animales , Humanos , Ratones , Apoptosis , Caspasa 3 , Células Endoteliales , Glucosa/farmacología , Dinámicas Mitocondriales , Especies Reactivas de Oxígeno , Regulación hacia ArribaRESUMEN
Purpose: Optic neuritis occurring in multiple sclerosis (MS) is a disease characterized by chronic inflammation and demyelination in the optic nerve. Although it has been well appreciated that leukocyte infiltration into the optic nerve is an early event during the course of the disease, there has been no study on visualizing and quantifying leukocyte trafficking in the retina during the progression of MS. Methods: In this study, we generated green fluorescent protein (GFP)+ bone marrow chimeric mice, in which GFP-labeled leukocytes facilitate the visualization of their trafficking in the retina. This reporter was then integrated with a well-established rodent model for MS-experimental autoimmune encephalomyelitis (EAE), allowing high resolution in vivo scanning laser ophthalmoscopy (SLO) to track leukocyte movement in the retina in real time. Quantification of leukocyte trafficking was accomplished through Imaris software. Results: Through SLO, we were able to localize the GFP signal, allowing us to clearly identify leukocytes within the vascular space. We observed more intense leukocyte migration in the retina of EAE mice, exhibiting three distinct movement behaviors: flowing, rolling/crawling and adherent. There was a marked increase in leukocyte rolling and adhesion in retinal vasculature, particularly in the veins and capillaries after induction of EAE. The velocity of rolling leukocytes ranged from 12.0 to 1065.0 µm/sec in the veins as compared to 14.1 to 942.0 in the capillaries. Furthermore, focal areas of recurrent leukocyte adhesion to endothelial surfaces were observed in EAE retinas. Conclusion: We generated a novel model that makes it possible to non-invasively track leukocyte trafficking in the retina of EAE mice. Our study demonstrates that leukocyte migration in an MS model is distinctly different from the control, suggesting that leukocytes may play a key role in the development of retinal vascular inflammation and optic neuritis during MS, warranting further investigation of the pathological roles of leukocytes in the disease onset and progression.
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Purpose: Optic nerve degeneration is a feature of neurodegenerative eye diseases and causes irreversible vision loss. Therefore, understanding the degenerating patterns of the optic nerve is critical to find the potential therapeutic target for optic neuropathy. However, the traditional method of optic nerve degeneration has the limitations of losing spatiotemporal tissue information. Light sheet fluorescence microscopy (LSFM) is a fluorescence microscopy technique that allows capturing 3D images rapidly with a high spatial optical resolution. In this study, we evaluated the availability of LSFM on the optic nerve with NMDA injected Thy1-CFP mice. Methods: NMDA injected to both eyes of Thy1-CFP mice. After 7 days from the injection, the retina and optic nerve were collected and immunostained with anti-Iba1 antibody. NMDA excitotoxicity induced RGC, and its axon loss and microglial activation in the retina were observed using confocal microscopy. The immunostained optic nerve was completed the optical clearing process with TDE and mounted for LSFM imaging. Results: We found that retinal flatmounts confirmed significant loss of CFP-expressing RGC and axon degradation and loss in Thy1-CFP mice at 7 days after NMDA injection. Together with these data verifying that NMDA induces RGC and its axon loss, we confirmed that NMDA excitotoxicity induced microglia activation and leukostasis, such as increased microglia number, transform its morphology to ameboid or round, and increase in attached leukocytes in vessels. Using LSFM, we observed that CFP expressing nerve fiber was well organized and arranged parallel in vehicle treated optic nerve, whileas NMDA injected optic nerve showed axon swelling and fragmentation and loss of axon density from the anterior to the posterior regions. Furthermore, LSFM enabled the observation of microglia phenotype transformation in the entire optic nerve. Unlike microglia in vehicle injected optic nerve, microglia in NMDA injected optic nerve displayed larger soma and short process with high Iba1 expression through the entire optic nerve from the anterior to posterior. Conclusions: In summary, we examined the applicability of the modified optic clearing protocol for the optic nerve and verified it enabled to acquiring of the 3D images of the optic nerve successfully revealing the complex spatial relationships between the axons, microglia and vasculature throughout the entire organ with single acquisitions. With these optimized techniques, we successfully obtained the high-resolution 3D images of NMDA-induced optic neuropathy, including the clues for optic nerve degeneration such as axon swelling, axonal fragmentation, and microglia activation. Overall, we believe that our current study could help understand the pathology of the optic nerve in neurodegenerative diseases, and it will be the basis for translational research.
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The retina, as the only visually accessible tissue in the central nervous system, has attracted significant attention for evaluating it as a biomarker for neurodegenerative diseases. Yet, most of studies focus on characterizing the loss of retinal ganglion cells (RGCs) and degeneration of their axons. There is no integrated analysis addressing temporal alterations of different retinal cells in the neurovascular unit (NVU) in particular retinal vessels. Here we assessed NVU changes in two mouse models of tauopathy, P301S and P301L transgenic mice overexpressing the human tau mutated gene, and evaluated the therapeutic effects of a tau oligomer monoclonal antibody (TOMA). We found that retinal edema and breakdown of blood-retina barrier were observed at the very early stage of tauopathy. Leukocyte adhesion/infiltration, and microglial recruitment/activation were constantly increased in the retinal ganglion cell layer of tau transgenic mice at different ages, while Müller cell gliosis was only detected in relatively older tau mice. Concomitantly, the number and function of RGCs progressively decreased during aging although they were not considerably altered in the very early stage of tauopathy. Moreover, intrinsically photosensitive RGCs appeared more sensitive to tauopathy. Remarkably, TOMA treatment in young tau transgenic mice significantly attenuated vascular leakage, inflammation and RGC loss. Our data provide compelling evidence that abnormal tau accumulation can lead to pathology in the retinal NVU, and vascular alterations occur more manifest and earlier than neurodegeneration in the retina. Oligomeric tau-targeted immunotherapy has the potential to treat tau-induced retinopathies. These data suggest that retinal NVU may serve as a potential biomarker for diagnosis and staging of tauopathy as well as a platform to study the molecular mechanisms of neurodegeneration.
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Retina/patología , Vasos Retinianos/patología , Tauopatías/patología , Animales , Anticuerpos Monoclonales/farmacología , Modelos Animales de Enfermedad , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Retina/efectos de los fármacos , Vasos Retinianos/efectos de los fármacos , Proteínas tau/antagonistas & inhibidores , Proteínas tau/genéticaRESUMEN
Zika virus (ZIKV), a mosquito-borne flavivirus, can cause severe eye disease and even blindness in newborns. However, ZIKV-induced retinal lesions have not been studied in a comprehensive way, mechanisms of ZIKV-induced retinal abnormalities are unknown, and no therapeutic intervention is available to treat or minimize the degree of vision loss in patients. Here, we developed a novel mouse model of ZIKV infection to evaluate its impact on retinal structure. ZIKV (20 plaque-forming units) was inoculated into neonatal wild type C57BL/6J mice at postnatal day (P) 0 subcutaneously. Retinas of infected mice and age-matched controls were collected at various ages, and retinal structural alterations were analyzed. We found that ZIKV induced progressive neuronal and vascular damage and retinal inflammation starting from P8. ZIKV-infected retina exhibited dramatically decreased thickness with loss of neurons, initial neovascular tufts followed by vessel dilation and degeneration, increased microglia and leukocyte recruitment and activation, degeneration of astrocyte network and gliosis. The above changes may involve inflammation and endoplasmic reticulum stress-mediated cell apoptosis and necroptosis. Moreover, we evaluated the efficacy of preclinical drugs and the safety of ZIKV vaccine candidate in this mouse model. We found that ZIKV-induced retinal abnormalities could be blocked by a selective flavivirus inhibitor NITD008 and a live-attenuated ZIKV vaccine candidate could potentially induce retinal abnormalities. Overall, we established a novel mouse model and provide a direct causative link between ZIKV and retinal lesion in vivo, which warrants further investigation of the underlying mechanisms of ZIKV-induced retinopathy and the development of effective therapeutics.
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Retina/crecimiento & desarrollo , Retina/virología , Degeneración Retiniana/patología , Degeneración Retiniana/virología , Infección por el Virus Zika/patología , Virus Zika , Animales , Animales Recién Nacidos , Ratones , Ratones Endogámicos C57BL , Vasculitis Retiniana/patología , Vasculitis Retiniana/virología , Vasos Retinianos/patología , Vasos Retinianos/virología , Virus Zika/aislamiento & purificaciónRESUMEN
Progressive loss of retinal ganglion cells (RGCs) leads to irreversible visual deficits in glaucoma. Here, we found that the level of cyclic AMP and the activity and expression of its mediator Epac1 were increased in retinas of two mouse models of ocular hypertension. Genetic depletion of Epac1 significantly attenuated ocular hypertension-induced detrimental effects in the retina, including vascular inflammation, neuronal apoptosis and necroptosis, thinning of ganglion cell complex layer, RGC loss, and retinal neuronal dysfunction. With bone marrow transplantation and various Epac1 conditional knockout mice, we further demonstrated that Epac1 in retinal neuronal cells (especially RGCs) was responsible for their death. Consistently, pharmacologic inhibition of Epac activity prevented RGC loss. Moreover, in vitro study on primary RGCs showed that Epac1 activation was sufficient to induce RGC death, which was mechanistically mediated by CaMKII activation. Taken together, these findings indicate that neuronal Epac1 plays a critical role in retinal neurodegeneration and suggest that Epac1 could be considered a target for neuroprotection in glaucoma.
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Glaucoma/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Células Ganglionares de la Retina/metabolismo , Animales , Apoptosis/genética , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , AMP Cíclico/metabolismo , Modelos Animales de Enfermedad , Femenino , Técnicas de Inactivación de Genes , Factores de Intercambio de Guanina Nucleótido/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Necroptosis/genética , Transducción de Señal/genéticaRESUMEN
Activated macrophages play an important role in many inflammatory diseases including septic shock and atherosclerosis. TRIM59 has been showed to participate in many pathological processes, such as inflammation, cytotoxicity and tumorigenesis. However, the molecular mechanisms controlling its expression in activated macrophages are not fully understood. Here we report that TRIM59 expression is regulated by Sp1 and Nrf1 in LPS-activated macrophages. TRIM59 is highly expressed in macrophages, and markedly decreased by LPS stimuli in vivo and in vitro. TRIM59 promoter activity is also significantly suppressed by LPS and further analysis demonstrated that Sp1 and Nrf1 directly bound to the proximal promoter of TRIM59 gene. LPS treatment significantly decreased Sp1 expression, nuclear translocation and reduced its binding to the promoter, whereas increased Nrf1 expression, nuclear translocation and enhanced its binding to the promoter. Moreover, LPS-decreased TRIM59 expression was reversed by JNK inhibitor. Finally, TRIM59 level is significantly decreased during atherosclerosis progression. Taken together, our results demonstrated that TRIM59 expression was precisely regulated by Sp1 and Nrf1 in LPS-activated macrophages, which may be dependent on the activation of JNK signaling pathway and TRIM59 may be a potential therapeutic target for inflammatory diseases such as atherosclerosis.
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Péptidos y Proteínas de Señalización Intracelular/metabolismo , Lipopolisacáridos/farmacología , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Activación de Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Factor 1 Relacionado con NF-E2/metabolismo , Factor de Transcripción Sp1/metabolismo , Proteínas de Motivos Tripartitos/metabolismo , Animales , Aterosclerosis/patología , Secuencia de Bases , Progresión de la Enfermedad , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Macrófagos/efectos de los fármacos , Macrófagos/enzimología , Masculino , Ratones , Ratones Endogámicos C57BL , Modelos Biológicos , Regiones Promotoras Genéticas , Transporte de Proteínas/efectos de los fármacos , Células RAW 264.7 , Transcripción Genética/efectos de los fármacos , Proteínas de Motivos Tripartitos/genéticaRESUMEN
Insulin is a key hormone for maintaining glucose homeostasis in organisms. In general, deficiency of insulin synthesis and secretion results in type I diabetes, whereas insulin resistance leads to type 2 diabetes. Cell division cycle 42 (CDC42), a member of Rho GTPases family, has been shown as an essential regulator in the second phase of glucose-induced insulin secretion in pancreatic islets ß cells in vitro. However, the effect of CDC42 on insulin expression has not been explored. Here we reported that the glucose-induced insulin expression and secretion were significantly inhibited in mice lacking CDC42 gene in pancreatic ß cells (Rip-CDC42cKO) in vivo and in vitro. Deletion of CDC42 gene in pancreatic ß cells did not affect survival or reproduction in mice. However, the Rip-CDC42cKO mice showed the systemic glucose intolerance and the decrease of glucose-induced insulin secretion without apparent alterations of peripheral tissues insulin sensitivity and the morphology of islets. Furthermore, we demonstrated that deletion of CDC42 gene in pancreatic ß cells significantly attenuated the insulin expression through inhibiting the ERK1/2-NeuroD1 signaling pathway. Taken together, our study presents novel evidence that CDC42 is an important modulator in glucose-induced insulin expression as well as insulin secretion in pancreatic ß cells.
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Glucosa/farmacología , Secreción de Insulina , Células Secretoras de Insulina/metabolismo , Insulina/genética , Proteína de Unión al GTP cdc42/genética , Animales , Células Cultivadas , Eliminación de Gen , Expresión Génica/efectos de los fármacos , Técnicas de Silenciamiento del Gen , Insulina/metabolismo , Resistencia a la Insulina/genética , Secreción de Insulina/efectos de los fármacos , Secreción de Insulina/genética , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Especificidad de Órganos/genética , Ratas , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genéticaRESUMEN
Previous studies showed that intratumoral 27-Hydroxycholesterol (27-HC), a metabolite of cholesterol, promotes growth, invasion and migration of breast cancer cells and that tumor-associated macrophages (TAMs) in breast cancers are closely related to tumor growth and metastatic progression. However, the relationship between 27-HC and TAMs in breast cancer remains unclear. In the present study, we observed that CYP27A1, the 27-HC synthesizing enzyme, was expressed in a much higher level in THP1 monocytes and THP1-derived macrophages than in breast cancer cells, and the promoter of CYP7B1, the degrading enzyme for 27-HC, was highly methylated in breast tumor cells. In addition, THP-1 monocytes and murine bone marrow cells were differentiated toward M2 type macrophages after being co-cultured with breast cancer cells or being exposed to exosomes derived from breast cancer cells. M2 type macrophages produced higher amounts of 27-HC than M0 and M1 type macrophages. 27-HC not only stimulated ER+ cancer cell proliferation as reported, but also promoted the recruitment of CCR2- and CCR5-expressing monocytes by inducing macrophages to express multiple chemokines including CCL2, CCL3 and CCL4. Taken together, our data demonstrate that the hypermethylation of CYP7B1 and recruitment of monocytes likely contribute to the accumulation of 27-Hydroxycholesterol in breast cancer and that the interaction of 27-HC with macrophages further promote the development of breast cancer.
RESUMEN
Glutathione (GSH), the most abundant biothiol in cells, not only plays a pivotal role in protective and detoxifying functions of the cell, but also serves as a very important mediator in many cellular functions. Especially, the difference of GSH level between cancer cells and normal cells is regarded as one of most important physiological parameters for cancer diagnosis. It is thereby extremely necessary to develop a simple, sensitive, and reliable analytical method for detection of GSH in cells. On the basis of the inhibition effect of GSH on the peroxidase-like activity of GSH stabilized gold nanoclusters, here a novel and facile strategy for colorimetric detection of cellular GSH level was well established. In this sensing system, GSH can effectively inhibit the oxidation of peroxidase substrate 3,3',5,5'-tetramethylbenzidine (TMB) to produce a blue colored product. Under the optimized conditions, the absorbance at 652 nm against GSH concentration shows a linear relationship within a range from 2 to 25 µM with detection limit of 420 nM. This excellent property allows our approach to be used to accurately evaluate the cellular GSH levels, and it is revealed that the overall GSH level in cancer cells was much higher than that in normal cells. The presented assay will enable a powerful tool for identifying cancer cells in a simple manner for biomedical diagnosis associated with GSH.
Asunto(s)
Colorimetría , Glutatión/análisis , Oro , Nanopartículas del Metal/química , Línea Celular Tumoral , Humanos , Límite de Detección , Neoplasias/diagnóstico , Oxidación-Reducción , Peroxidasa , Células THP-1RESUMEN
Activated macrophages play an important role in many inflammatory diseases including septic shock and atherosclerosis. However, the molecular mechanisms limiting macrophage activation are not completely understood. Members of the tripartite motif (TRIM) family have recently emerged as important players in innate immunity and antivirus. Here, we systematically analyzed mRNA expressions of representative TRIM molecules in human THP1-derived macrophages activated by different toll-like receptor (TLR) ligands. Twenty-nine TRIM members were highly induced (>3 fold) by one or more TLR ligands, among which 19 of them belong to TRIM C-IV subgroup. Besides TRIM21, TRIM22 and TRIM38 were shown to be upregulated by TLR3 and TLR4 ligands as previous reported, we identified a novel group of TRIM genes (TRIM14, 15, 31, 34, 43, 48, 49, 51 and 61) that were significantly up-regulated by TLR3 and TLR4 ligands. In contrast, the expression of TRIM59 was down-regulated by TLR3 and TLR4 ligands in both human and mouse macrophages. The alternations of the TRIM proteins were confirmed by Western blot. Finally, overexpression of TRIM59 significantly suppressed LPS-induced macrophage activation, whereas siRNA-mediated knockdown of TRIM59 enhanced LPS-induced macrophage activation. Taken together, the study provided an insight into the TLR ligands-induced expressions of TRIM family in macrophages.