PLSCR1 promotes apoptosis and clearance of retinal ganglion cells in glaucoma pathogenesis.

Glaucoma is the leading cause of irreversible blindness worldwide. In the pathogenesis of glaucoma, activated microglia can lead to retinal ganglion cells (RGCs) apoptosis and death, however, the molecular mechanisms remain largely unknown. We demonstrate that phospholipid scramblase 1 (PLSCR1) is a key regulator promoting RGCs apoptosis and their clearance by microglia. As evidenced in retinal progenitor cells and RGCs of the acute ocular hypertension (AOH) mouse model, overexpressed PLSCR1 induced its translocation from the nucleus to the cytoplasm and cytomembrane, as well as elevated phosphatidylserine exposure and reactive oxygen species generation with subsequent RGCs apoptosis and death. These damages were effectively attenuated by PLSCR1 inhibition. In the AOH model, PLSCR1 led to an increase in M1 type microglia activation and retinal neuroinflammation. Upregulation of PLSCR1 resulted in strongly elevated phagocytosis of apoptotic RGCs by activated microglia. Taken together, our study provides important insights linking activated microglia to RGCs death in the glaucoma pathogenesis and other RGC-related neurodegenerative diseases.

Inhibiting HIF-1 signaling alleviates HTRA1-induced RPE senescence in retinal degeneration.

BACKGROUND: Age-related macular degeneration (AMD), characterized by the degeneration of retinal pigment epithelium (RPE) and photoreceptors, is the leading cause of irreversible vision impairment among the elderly. RPE senescence is an important contributor to AMD and has become a potential target for AMD therapy. HTRA1 is one of the most significant susceptibility genes in AMD, however, the correlation between HTRA1 and RPE senescence hasn't been investigated in the pathogenesis of AMD. METHODS: Western blotting and immunohistochemistry were used to detect HTRA1 expression in WT and transgenic mice overexpressing human HTRA1 (hHTRA1-Tg mice). RT-qPCR was used to detect the SASP in hHTRA1-Tg mice and ARPE-19 cells infected with HTRA1. TEM, SA-ß-gal was used to detect the mitochondria and senescence in RPE. Retinal degeneration of mice was investigated by fundus photography, FFA, SD-OCT and ERG. The RNA-Seq dataset of ARPE-19 cells treated with adv-HTRA1 versus adv-NC were analyzed. Mitochondrial respiration and glycolytic capacity in ARPE-19 cells were measured using OCR and ECAR. Hypoxia of ARPE-19 cells was detected using EF5 Hypoxia Detection Kit. KC7F2 was used to reduce the HIF1α expression both in vitro and in vivo. RESULTS: In our study, we found that RPE senescence was facilitated in hHTRA1-Tg mice. And hHTRA1-Tg mice became more susceptible to NaIO3 in the development of oxidative stress-induced retinal degeneration. Similarly, overexpression of HTRA1 in ARPE-19 cells accelerated cellular senescence. Our RNA-seq revealed an overlap between HTRA1-induced differentially expressed genes associated with aging and those involved in mitochondrial function and hypoxia response in ARPE-19 cells. HTRA1 overexpression in ARPE-19 cells impaired mitochondrial function and augmented glycolytic capacity. Importantly, upregulation of HTRA1 remarkably activated HIF-1 signaling, shown as promoting HIF1α expression which mainly located in the nucleus. HIF1α translation inhibitor KC7F2 significantly prevented HTRA1-induced cellular senescence in ARPE-19 cells, as well as improved the visual function in hHTRA1-Tg mice treated with NaIO3. CONCLUSIONS: Our study showed elevated HTRA1 contributes to the pathogenesis of AMD by promoting cellular senescence in RPE through damaging mitochondrial function and activating HIF-1 signaling. It also pointed out that inhibition of HIF-1 signaling might serve as a potential therapeutic strategy for AMD. Video Abstract.

Lipocalin 2 induces visual impairment by promoting ferroptosis in retinal ischemia-reperfusion injury.

Background: Retinal ischemia-reperfusion (RIR) is a common pathological condition that can lead to retinal ganglion cell (RGC) death and visual impairment. However, the pathogenesis of RGC loss and visual impairment caused by retinal ischemia remains unclear. Methods: A mouse model of elevated intraocular pressure (IOP)-induced RIR injury was used. Flash visual evoked potentials (FVEPs) and electroretinography (ERG) recordings were performed to assess visual function. The structural integrity of the retina and the number of RGC were assessed using hematoxylin and eosin (HE) staining and retinal flat mounts. Ferroptosis was evaluated by testing the levels of glutathione (GSH), malondialdehyde (MDA), glutathione peroxidase (GPX4), and ferritin light chains (FTL) in the retina of wild-type (WT) and lipocalin-2 transgenic (LCN2-TG) mice after RIR injury. Results: We found that LCN2 was mainly expressed in the RGC layer in the retina of wild-type mice and remarkably upregulated after RIR injury. Compared with wild-type mice, aggravated RGC death and visual impairment were exhibited in LCN2-TG mice with RIR injury. Moreover, LCN2 overexpression activated glial cells and upregulated proinflammatory factors. More importantly, we found that LCN2 strongly promoted ferroptosis signaling in RGC death and visual impairment. Liproxstatin-1, an inhibitor of ferroptosis, could significantly ameliorate RGC death and visual impairment. Furthermore, we found significantly alleviated RGC death and retinal damage in LCN2 heterozygous knockout mice. Conclusions: Our study provides important insights linking upregulated LCN2-mediated promotion of ferroptosis to RGC death and visual function impairment in the pathogenesis of ischemic retinopathy.

Identification of Nitric Oxide-Donating Ripasudil Derivatives with Intraocular Pressure Lowering and Retinal Ganglion Cell Protection Activities.

Based on the synergistic therapeutic effect of nitric oxide (NO) and Rho-associated protein kinase (ROCK) inhibitors on glaucoma, a new group of NO-donating ripasudil derivatives RNO-1-RNO-6 was designed, synthesized, and biologically evaluated. The results demonstrated that the most active compound RNO-6 maintained potent ROCK inhibitory and NO releasing abilities, reversibly depolymerized F-actin, and suppressed mitochondrial respiration in human trabecular meshwork (HTM) cells. Topical administration of RNO-6 (0.26%) in chronic ocular hypertension glaucoma mice exhibited significant IOP lowering and visual function and retinal ganglion cell (RGC) protection activities, superior to an equal molar dose of ripasudil. RNO-6 could be a promising agent for glaucoma or ocular hypertension, warranting further investigation.

TCF7L2 promotes ER stress signaling in diabetic retinopathy.

Diabetic retinopathy (DR) is one of the most common blindness in working-age adults. Transcription factor 7 like 2 (TCF7L2) is a susceptibility gene of DR, however, its roles in the pathogenesis of DR are still largely unknown. In this study, we found that TCF7L2 was mainly located in the cell nucleus of retinal ganglion cell layer (GCL) and inner nuclear layer (INL), while it was not expressed in the cell nucleus of retinal outer nuclear layer (ONL). Expression of TCF7L2 was significantly elevated in the retinas of db/db diabetic mice and oxygen-induced retinopathy (OIR) mice. Also, in Ad-hTCF7L2 treated hiPSCs-derived retinal progenitor cells (RPCs), activating transcription factor 6 (ATF6)-related endoplasmic reticulum (ER) stress signaling was remarkably activated. Moreover, knockdown of TCF7L2 significantly inhibited ATF6-related ER stress signaling. Furthermore, the data of endothelial permeability assay showed that RPCs pretreated with Ad-hTCF7L2 lead to enhanced monolayer permeability of human umbilical vein endothelial cells (HUVECs), and knockdown of TCF7L2 or ATF6 in RPCs could alleviate the monolayer permeability of HUVECs. Thus, our results showed that TCF7L2 could trigger ATF6-related ER stress signaling and promote vein endothelial cell permeability, which will provide important insight into the role of TCF7L2 in the pathogenesis of DR and contribute to designing potential therapies.

Defect of LSS Disrupts Lens Development in Cataractogenesis.

Congenital cataract is one of the leading causes of blindness in children worldwide. About one-third of congenital cataracts are caused by genetic defects. LSS, which encodes lanosterol synthase, is a causal gene for congenital cataracts. LSS is critical in preventing abnormal protein aggregation of various cataract-causing mutant crystallins; however, its roles in lens development remain largely unknown. In our study, we generated a mouse model harboring Lss G589S mutation, which is homologous to cataract-causing G588S mutation in human LSS. LssG589S/G589S mice exhibited neonatal lethality at postal day 0 (P0), whereas these mice showed severe opacity in eye lens. Also, we found that cataract was formed at E17.5 after we examined the opacity of embryonic lens from E13.5 to E18.5. Moreover, disrupted lens differentiation occurred at E14.5 prior to formation of the opacity of eye lens, shown as delayed differentiation of lens secondary fiber and disordered lens fiber organization. In addition, RNA-seq analysis indicated that cholesterol synthesis signaling pathways were significantly downregulated. Overall, our findings provide clear evidence that a mouse model harboring a homozygous Lss G589S mutation can recapitulate human congenital cataract. Our study points out that LSS functions as a critical determinant of lens development, which will contribute to better understanding LSS defects in cataractogenesis and developing therapies for cataracts.

Prohibitin 2 deficiency impairs cardiac fatty acid oxidation and causes heart failure.

Fatty acids are the most major substrate source for adult cardiac energy generation. Prohibitin 2 (PHB2), a highly conserved protein located in mitochondrial inner membrane, plays key roles in cellular energy metabolic homeostasis. However, its functions in regulating cardiac fatty acid metabolism have remained largely unknown. Our study demonstrates that cardiac-specific knockout of Phb2 leads to accumulation of lipid droplets and causes heart failure. Mechanistically, ablation of PHB2 impairs cardiac fatty acid oxidation (FAO) through downregulating carnitine palmitoyltransferase1b (CPT1b), a rate-limiting enzyme of cardiac mitochondrial FAO. Moreover, overexpression of CPT1b alleviates impaired FAO in PHB2-deficient cardiomyocytes. Thus, our study provides direct evidence for the link between PHB2 and cardiac fatty acid metabolism. Our study points out that PHB2 is a potential FAO regulator in cardiac mitochondrial inner membrane, as well as the connection between PHB2 and CPT1b and their relationships to cardiac pathology especially to cardiac fatty acid metabolic disorder.

In vivo detecting mouse persistent hyperplastic primary vitreous by Spectralis Optical Coherence Tomography.

To identify imaging characteristics of mouse persistent hyperplastic primary vitreous (PHPV) by Spectralis Optical Coherence Tomography (OCT), as well as to assess and compare the sensitivity and precision of OCT with color photography (CP) and Fundus Fluorescein Angiography (FFA) imaging in detecting mouse PHPV. Notch4-/- C57BL/6J mice (224 eyes) aged from 3 months to 7 months were examined in this study. CP, FFA and OCT imaging were utilized to examine vitreous cavity and retina of mouse eyes. Horizontal and radial OCT scan volume was centered on the optic nerve head. Hematoxylin and eosin (H&E) staining was performed to validate PHPV. For color photography and FFA imaging, retrolental irregular fibrovascular membrane-like tissues were found in 33 eyes with/without blood vessels in vitreous cavity. Among them, 31 eyes were visualized with lateral and oblique linear hyperreflective opacities in vitreous cavity using Spectralis OCT. Position of PHPV in posterior segment of eyes was also measured via OCT. Mouse PHPV was validated by H&E staining. Typical hyperreflective opacities in vitreous cavity were detected in PHPV mouse using Spectralis OCT. Spectralis OCT imaging can effectively detect mouse PHPV as color photography and FFA.

PRC2 specifies ectoderm lineages and maintains pluripotency in primed but not naïve ESCs.

Polycomb repressive complex 2 and the epigenetic mark that it deposits, H3K27me3, are evolutionarily conserved and play critical roles in development and cancer. However, their roles in cell fate decisions in early embryonic development remain poorly understood. Here we report that knockout of polycomb repressive complex 2 genes in human embryonic stem cells causes pluripotency loss and spontaneous differentiation toward a meso-endoderm fate, owing to de-repression of BMP signalling. Moreover, human embryonic stem cells with deletion of EZH1 or EZH2 fail to differentiate into ectoderm lineages. We further show that polycomb repressive complex 2-deficient mouse embryonic stem cells also release Bmp4 but retain their pluripotency. However, when converted into a primed state, they undergo spontaneous differentiation similar to that of hESCs. In contrast, polycomb repressive complex 2 is dispensable for pluripotency when human embryonic stem cells are converted into the naive state. Our studies reveal both lineage- and pluripotent state-specific roles of polycomb repressive complex 2 in cell fate decisions.Polycomb repressive complex 2 (PRC2) plays an essential role in development by modifying chromatin but what this means at a cellular level is unclear. Here, the authors show that ablation of PRC2 genes in human embryonic stem cells and in mice results in changes in pluripotency and the primed state of cells.