Microglial SIRT1 activation attenuates synapse loss in retinal inner plexiform layer via mTORC1 inhibition.

BACKGROUND: Optic nerve injury (ONI) is a key cause of irreversible blindness and triggers retinal ganglion cells (RGCs) change and synapse loss. Microglia is the resistant immune cell in brain and retina and has been demonstrated to be highly related with neuron and synapse injury. However, the function of Sirtuin 1 (SIRT1), a neuroprotective molecule, in mediating microglial activation, retinal synapse loss and subsequent retinal ganglion cells death in optic nerve injury model as well as the regulatory mechanism remain unclear. METHOD: To this end, optic nerve crush (ONC) model was conducted to mimic optic nerve injury. Resveratrol and EX527, highly specific activator and inhibitor of SIRT1, respectively, were used to explore the function of SIRT1 in vivo and vitro. Cx3Cr1-CreERT2/RaptorF/F mice were used to delete Raptor for inhibiting mammalian target of rapamycin complex 1 (mTORC1) activity in microglia. HEK293 and BV2 cells were transfected with plasmids to explore the regulatory mechanism of SIRT1. RESULTS: We discovered that microglial activation and synapse loss in retinal inner plexiform layer (IPL) occurred after optic nerve crush, with later-development retinal ganglion cells death. SIRT1 activation induced by resveratrol inhibited microglial activation and attenuated synapse loss and retinal ganglion cells injury. After injury, microglial phagocytosed synapse and SIRT1 inhibited this process to protect synapse and retinal ganglion cells. Moreover, SIRT1 exhibited neuron protective effects via activating tuberous sclerosis complex 2 (TSC2) through deacetylation, and enhancing the inhibition effect of tuberous sclerosis complex 2 on mammalian target of rapamycin complex 1 activity. CONCLUSION: Our research provides novel insights into microglial SIRT1 in optic nerve injury and suggests a potential strategy for neuroprotective treatment of optic nerve injury disease.

TBK1 Knockdown Alleviates Axonal Transport Deficits in Retinal Ganglion Cells Via mTORC1 Activation in a Retinal Damage Mouse Model.

Purpose: Glaucoma is the leading cause of irreversible blindness worldwide and is characterized by progressive retinal ganglion cell (RGC) death and optic nerve degeneration. Axonal transport deficits are the earliest crucial pathophysiological changes in glaucoma. Genetic variation in the TANK-binding kinase 1 gene (TBK1) plays a role in the pathogenesis of glaucoma. This study was designed to investigate intrinsic factors underlying RGCs' damage and to explore the molecular mechanism of TBK1 involvement in glaucomatous pathogenesis. Methods: We established a mouse model of acute ocular hypertension and used TBK1 conditional knockdown mice to investigate the role of TBK1 in glaucoma. CTB-Alexa 555 was utilized to evaluate axonal transport in mice. To observe the efficiency of gene knockdown, we performed immunofluorescence staining. Immunoblotting and immunoprecipitation assays were performed to examine protein‒protein colocalization. RT‒qPCR was performed to measure the mRNA levels of Tbk1. Results: In this study, we found that conditional TBK1 knockdown in RGCs resulted in increased axonal transport and protection against axonal degeneration. Through mechanistic studies, we found that TBK1 inhibited mTORC1 pathway activation by phosphorylating RAPTOR at Ser1189. Phosphorylation of RAPTOR at Ser1189 abrogated the interaction of RAPTOR with the deubiquitinase USP9X, leading to an increase in RAPTOR ubiquitination and a subsequent decline in protein stabilization. Conclusions: Our study identified a novel mechanism involving an interaction between the glaucoma risk gene TBK1 and the pivotal mTORC1 pathway, which may provide new therapeutic targets in glaucoma and other neurodegenerative diseases.

Oxidative stress and epigenetics in ocular vascular aging: an updated review.

Vascular aging is an inevitable process with advancing age, which plays a crucial role in the pathogenesis of cardiovascular and microvascular diseases. Diabetic retinopathy (DR) and age-related macular degeneration (AMD), characterized by microvascular dysfunction, are the common causes of irreversible blindness worldwide, however there is still a lack of effective therapeutic strategies for rescuing the visual function. In order to develop novel treatments, it is essential to illuminate the pathological mechanisms underlying the vascular aging during DR and AMD progression. In this review, we have summarized the recent discoveries of the effects of oxidative stress and epigenetics on microvascular degeneration, which could provide potential therapeutic targets for DR and AMD.

Development and validation of immunogenic cell death-related signature for predicting the prognosis and immune landscape of uveal melanoma.

Introduction: Uveal melanoma (UM) is the most common primary intraocular malignant tumor in adults, and the main treatment for UM is currently surgery and plaque brachytherapy. UM is highly susceptible to metastasis, which eventually occurs in nearly half of all patients; once metastasis occurs, patients have a poor prognosis and the condition is difficult to treat. Therefore, the identification of new and effective UM biomarkers is vital for the application of therapeutic strategies. Immunogenic cell death (ICD) is a type of regulatory cell death that activates adaptive immune responses and generates long-term immunological memory. ICD can promote antitumor immunity, which may be a potential immunotherapeutic strategy for UM. Methods: The data of UM from the Cancer Genome Atlas (TCGA) was used as a training set and the data from Gene Expression Omnibus (GEO) was used as a validation set. To determine the expression pattern of ICD-related genes in UM, survival analysis and difference analysis was conducted. The ICD-related risk signature was constructed by employing the least absolute shrinkage and selection operator (LASSO) Cox regression. Subsequently, immune profile and somatic mutation analysis were performed. In addition, cell experiments were performed to verify the role of immunogenic cell death-related genes in UM. Results: In this study, we analyzed the relationship between ICD-related gene expression and UM patient prognosis, somatic mutations, and the tumor immune microenvironment. Importantly, we constructed a 5-gene ICD-related risk signature and confirmed it as a novel prognostic biomarker in UM patients. We found that the high-risk group had more immune cell infiltration and a worse prognosis than the low-risk group. In cellular experiments, we confirmed the high expression of FOXP3 inMUM2B andOCM-1A cell lines and that knockdown of FOXP3 markedly inhibited the proliferation of UM tumor cells. Discussion: ICD-related genes play a critical role in the tumor immune microenvironment. Our results may contribute to the development of effective immunotherapies.

Endothelial CYP2J2 overexpression restores the BRB via METTL3-mediated ANXA1 upregulation.

Blood-retinal barrier (BRB) breakdown is responsible for multiple ocular diseases, such as diabetic retinopathy, age-related macular degeneration, and retinal vascular occlusive diseases. Increased vascular permeability contributes to vasogenic edema and tissue damage, with consequent adverse effects on vision. Herein, we found that endothelial CYP2J2 overexpression maintained BRB integrity after ischemia-reperfusion injury and consequently protected against retinal ganglion cell loss. Oxidative stress repressed endothelial ANXA1 expression in vivo and in vitro. CYP2J2 upregulated methyltransferase-like 3 (METTL3) expression and hence promoted ANXA1 translation via ANXA1 m6 A modification in endothelium under oxidative stress. CYP2J2 maintained the distribution of endothelial tight junctions and adherens junctions in an ANXA1-dependent manner. Endothelial ANXA1 plays an indispensable role in vascular homeostasis and stabilization during development. Endothelial ANXA1 deletion disrupted retinal vascular perfusion as well as BRB integrity. CYP2J2 metabolites restored BRB integrity in the presence of ANXA1. Our findings identified the CYP2J2-METTL3-ANXA1 pathway as a potential therapeutic target for relieving BRB impairments.

CD82 protects against glaucomatous axonal transport deficits via mTORC1 activation in mice.

Glaucoma is a leading cause of irreversible blindness worldwide and is characterized by progressive optic nerve degeneration and retinal ganglion cell loss. Axonal transport deficits have been demonstrated to be the earliest crucial pathophysiological changes underlying axonal degeneration in glaucoma. Here, we explored the role of the tetraspanin superfamily member CD82 in an acute ocular hypertension model. We found a transient downregulation of CD82 after acute IOP elevation, with parallel emergence of axonal transport deficits. The overexpression of CD82 with an AAV2/9 vector in the mouse retina improved optic nerve axonal transport and ameliorated subsequent axon degeneration. Moreover, the CD82 overexpression stimulated optic nerve regeneration and restored vision in a mouse optic nerve crush model. CD82 exerted a protective effect through the upregulation of TRAF2, which is an E3 ubiquitin ligase, and activated mTORC1 through K63-linked ubiquitylation and intracellular repositioning of Raptor. Therefore, our study offers deeper insight into the tetraspanin superfamily and demonstrates a potential neuroprotective strategy in glaucoma treatment.

Modulation of Sirt1-mTORC1 Pathway in Microglia Attenuates Retinal Ganglion Cell Loss After Optic Nerve Injury.

PURPOSE: Optic nerve injury (ONI) causes neuroinflammation and neurodegeneration leading to visual deficits. The response of microglia has emerged as an impactful component of etiology in neurodegeneration. This study aimed to investigate the effect of SIRT1-mTORC1 signaling pathway in microglia regulation after ONI. METHODS: Cx3Cr1-CreERT2/Raptor F/F and Cx3Cr1-CreERT2/Sirt1 F/F mice were used to delete Raptor and Sirt1 in microglia, respectively. Optic nerve crush (ONC) model was established to mimic ONI. PLX5622, a highly specific inhibitor of the colony-stimulating factor 1 receptor (CSF1R), is used to eliminate microglia in optic nerve. Ionized calcium binding adaptor molecule 1 (Iba1) immunostaining was used to detect microglial activation. Retinal ganglion cells (RGCs) were quantified by Nissl staining and retinal whole-mount immunostaining with RNA-binding protein with multiple splicing (RBPMS). Axonal damage was valued by transmission electron microscopy (TEM). RESULTS: Microglial activation emerged on day 3 post ONC and was earlier than RGCs loss which occurred at day 5 after injury. Depleting microglia with PLX5622 could attenuate the loss of RGCs and axon damage after ONC. Gain- and loss-of-function studies revealed that SIRT1 determined the activation of microglia in optic nerve. In addition, microglia-specific deletion of Raptor resulted in decreased microglial activation. Interestingly, activating mTORC1 with CCT007093 could reverse the function of SIRT1 in regulating the process of microglial activation mediated RGCs loss. CONCLUSION: Our study reveals a potential novel mechanism of SIRT1-mTORC1 pathway in microglia regulation, and indicates a therapeutic potential for the protection of RGCs in ONI.

VIP Stabilizes the Cytoskeleton of Schlemm's Canal Endothelia via Reducing Caspase-3 Mediated ZO-1 Endolysosomal Degradation.

OBJECTIVES: In glaucomatous eyes, the main aqueous humor (AH) outflow pathway is damaged by accumulated oxidative stress arising from the microenvironment, vascular dysregulation, and aging, which results in increased outflow resistance and ocular hypertension. Schlemm's canal (SC) serves as the final filtration barrier of the main AH outflow pathway. The present study is aimed at investigating the possible regulation of vasoactive intestinal peptide (VIP) on the cytoskeleton by stabilizing ZO-1 in SC. METHODS: Model of chronic ocular hypertension (COH) induced by episcleral venous cauterization was treated with topical VIP. The ultrastructure of junctions, ZO-1 levels, and permeability of the SC inner wall to FITC-dextran (70 kDa) were detected in the COH models. The F-actin distribution, F/G-actin ratio, and ZO-1 degradation pathway in human umbilical vein endothelial cells (HUVECs) and HEK 293 cells were investigated. RESULTS: ZO-1 in the outer wall of the SC was less than that in the inner wall. COH elicited junction disruption, ZO-1 reduction, and increased permeability of the SC inner wall to FITC-dextran in rats. ZO-1 plays an essential role in maintaining the F/G-actin ratio and F-actin distribution. VIP treatment attenuated the downregulation of ZO-1 associated with COH or H2O2-induced oxidative damage. In H2O2-stimulated HUVECs, the caspase-3 inhibitor prevents ZO-1 disruption. Caspase-3 activation promoted endolysosomal degradation of ZO-1. Furthermore, a decrease in caspase-3 activation and cytoskeleton redistribution was demonstrated in VIP + H2O2-treated cells. The knockdown of ZO-1 or the overexpression of caspase-3 blocked the effect of VIP on the cytoskeleton. CONCLUSION: This study provides insights into the role of VIP in stabilizing the interaction between the actin cytoskeleton and cell junctions and may provide a promising targeted strategy for glaucoma treatment.