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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.

After intracranial hemorrhage (ICH), iron is released from the hematoma and induces secondary brain injury. However, the detail effect of iron on blood-brain barrier (BBB) function is still unknown. We investigated whether hemoglobin (Hb), ferrous ammonium sulfate (FAS) or hemin which contains iron have the detrimental effect on both human brain microvascular endothelial cells and pericytes by cellular function analysis in vitro . We developed an iron (Fe 2+ )-detectable probe, Si-RhoNox-1, to investigate intracellular Fe 2+ accumulation (Fe 2+ intra ). After FAS treatment, there was the correlation between Fe 2+ intra and cell death. Moreover, Hb or hemin treatment induced cell death, increased reactive oxygen species and promoted Fe 2+ intra in both cells. These changes were inhibited by the Fe 2+ chelator, 2,2′-bipyridil (BP). Furthermore, hemin induced endothelial barrier dysfunction via disruption of junction integrity. Based on in vitro studies, we used a hemin-injection ICH mice model in vivo . Hemin injection (10 mM/10 µL, i.c.) induced deleterious effects including BBB hyper-permeability, neuronal deficits, neuronal damage, altered proteins expression, and Fe 2+ intra in BBB composed cells. Lastly, BP (40 mg/kg, i.p.) administration attenuated neuronal deficits at 3 days after surgery. Collectively, Hb or hemin damaged BBB composed cells via Fe 2+ intra . Therefore, the regulation of the Fe 2+ movement in BBB might be effective for treatment of ICH.

Sodium-glucose transporter 2 (SGLT2) inhibitors are antidiabetic drugs affecting SGLT2. Recent studies have shown various cancers expressing SGLT2, and SGLT2 inhibitors attenuating tumor proliferation. We evaluated the antitumor activities of canagliflozin, a SGLT2 inhibitor, on glioblastoma (GBM). Three GBM cell lines, U251MG (human), U87MG (human), and GL261 (murine), were used. We assessed the expression of SGLT2 of GBM through immunoblotting, specimen-use, cell viability assays, and glucose uptake assay with canagliflozin. Then, we assessed phosphorylation of AMP-activated protein kinase (AMPK), p70 S6 kinase, and S6 ribosomal protein by immunoblotting. Concentrations of 5, 10, 20, and 40 μM canagliflozin were used in these tests. We also evaluated cell viability and immunoblotting using U251MG with siRNA knockdown of SGLT2. Furthermore, we divided the mice into vehicle group and canagliflozin group. The canagliflozin group was administrated with 100 mg/kg of canagliflozin orally for 10 days starting from the third days post-GBM transplant. The brains were removed and the tumor volume was evaluated using sections. SGLT2 was expressed in GBM cell and GBM allograft mouse. Canagliflozin administration at 40 μM significantly inhibited cell proliferation and glucose uptake into the cell. Additionally, canagliflozin at 40 μM significantly increased the phosphorylation of AMPK and suppressed that of p70 S6 kinase and S6 ribosomal protein. Similar results of cell viability assays and immunoblotting were obtained using siRNA SGLT2. Furthermore, although less effective than in vitro, the canagliflozin group significantly suppressed tumor growth in GBM-transplanted mice. This suggests that canagliflozin can be used as a potential treatment for GBM.

The present study investigated the vitrification-induced deterioration of mitochondrial functions that may reduce the developmental ability of post-warming bovine embryos. In addition, the effect of supplementation of the culture medium with resveratrol on the mitochondrial functions and post-warming embryonic development was examined. Two days after in vitro fertilization, embryos with 8-12 cells (referred to hereafter as 8-cell embryos) were vitrified and warmed, followed by in vitro incubation for 5 days in a culture medium containing either the vehicle or 0.5 μM resveratrol. Vitrification reduced embryonic development until the blastocyst stage, reduced the ATP content of embryos, and impaired the mitochondrial genome integrity, as determined by real-time polymerase chain reaction. Although the total cell number and mitochondrial DNA copy number (Mt-number) of blastocysts were low in the vitrified embryos, the Mt-number per blastomere was similar among the blastocysts derived from fresh (non-vitrified) and vitrified-warmed embryos. Supplementation of the culture medium with resveratrol enhanced the post-warming embryonic development and reduced the Mt-number and reactive oxygen species level in blastocysts and blastomeres without affecting the ATP content. An increase in the content of cell-free mitochondrial DNA in the spent culture medium was observed following cultivation of embryos with resveratrol. These results suggested that vitrification induces mitochondrial damages and that resveratrol may enhance the development of post-warming embryos and activates the degeneration of damaged mitochondria, as indicated by the increase in the cell-free mitochondrial DNA content in the spent culture medium and the decrease in the Mt-number of blastocysts and blastomeres.

Spinal muscular atrophy (SMA) is a motor neuron disease, typically resulting from loss-of-function mutations in the survival motor neuron 1 ( SMN1 ) gene. Nusinersen/SPINRAZA, a splice-switching oligonucleotide that modulates SMN2 (a paralog of SMN1 ) splicing and consequently increases SMN protein levels, has a therapeutic effect for SMA. Previously reported small-molecule SMN2 splicing modulators such as risdiplam/EVRYSDI and its analog SMN-C3 modulate not only the splicing of SMN2 but also that of secondary splice targets, including forkhead box protein M1 ( FOXM1 ). Through screening SMA patient-derived fibroblasts, a novel small molecule, designated TEC-1, was identified that selectively modulates SMN2 splicing over three secondary splice targets. TEC-1 did not strongly affect the splicing of FOXM1 , and unlike risdiplam, did not induce micronucleus formation. In addition, TEC-1 showed higher selectively on galactosylceramidase and huntingtin gene expression compared to previously reported compounds (e.g., SMN-C3) due to off-target effects on cryptic exon inclusion and nonsense-mediated mRNA decay. Moreover, TEC-1 significantly ameliorated the disease phenotype in an SMA murine model in vivo. Thus, TEC-1 may have promising therapeutic potential for SMA, and our study demonstrates the feasibility of RNA-targeting small-molecule drug development with an improved tolerability profile.

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.

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% O 2 ). 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.

Leber’s hereditary optic neuropathy (LHON) is a mitochondrial disease caused by mitochondrial DNA mutations, leading to central vision loss and retinal ganglion cell (RGC) degeneration. Progress in understanding LHON and developing treatments has been limited by the lack of human-like models. In this study, we aimed to establish a human retinal model of LHON using retinal organoids (ROs) from LHON patient-derived induced pluripotent stem cells (LHON-iPSCs). We first confirmed LHON-iPSCs were successfully differentiated into ROs (LHON-ROs). LHON-RO showed a reduction in RGC numbers and the density of neural axons. Additionally, both mitochondrial membrane potential and ATP production were decreased in LHON-RO. Finally, treatment with idebenone, the only approved therapeutic agent for LHON, improved RGC numbers in LHON-RO. This model replicates key clinical features of LHON, including RGC and axonal loss, and demonstrates idebenone’s therapeutic potential. Furthermore, a comprehensive analysis of the LHON-RO model revealed impaired mitophagy, suggesting novel therapeutic targets for LHON. Thus, the LHON-RO model offers a valuable platform for studying LHON pathogenesis and evaluating treatments.

Neovascular age-related macular degeneration (nAMD) featuring choroidal neovascularization (CNV) is the principal cause of irreversible blindness in elderly people in the world. Integrated stress response (ISR) is one of the intracellular signals to be adapted to various stress conditions including endoplasmic reticulum (ER) stress. ISR signaling results in the upregulation of activating transcription factor 4 (ATF4), which is a mediator of ISR. Although recent studies have suggested ISR contributes to the progression of some age-related disorders, the effects of ATF4 on the development of CNV remain unclear. Here, we performed a murine model of laser-induced CNV and found that ATF4 was highly expressed in endothelial cells of the blood vessels of the CNV lesion site. Exposure to integrated stress inhibitor (ISRIB) reduced CNV formation, vascular leakage, and the upregulation of vascular endothelial growth factor (VEGF) in retinal pigment epithelium (RPE)-choroid-sclera complex. In human retinal microvascular endothelial cells (HRMECs), ISRIB reduced the level of ATF4 and VEGF induced by an ER stress inducer, thapsigargin, and recombinant human VEGF. Moreover, ISRIB decreased the VEGF-induced cell proliferation and migration of HRMECs. Collectively, our findings showed that pro-angiogenic effects of ATF4 in endothelial cells may be a potentially therapeutic target for patients with nAMD.

Homozygous loss-of-function progranulin gene (GRN) mutation carriers develop adult-onset neuronal ceroid lipofuscinosis (NCL), a lysosomal storage disease. Clinically, NCL patients display retinal degeneration and visual dysfunction. However, there is little information about the effects of progranulin dysfunction on lysosomal function of the retinal pigment epithelium (RPE). We performed RNA interference knock down of progranulin in primary human RPE (hRPE) cells and observed RPE function and lysosomal activity. Progranulin localized to the lysosome in RPE cells. Loss of progranulin did not affect the biogenesis of lysosomes in RPE cells, while it was necessary for the activation of lysosomal proteases. Furthermore, progranulin deficiency decreased cell viability and disrupted the cell-cell junctions. Our results demonstrate that progranulin insufficiency disturbs lysosomal activity and physiological functions in RPE cells.