mTOR-dependent dysregulation of autophagy contributes to the retinal ganglion cell loss in streptozotocin-induced diabetic retinopathy.

BACKGROUND: Neurodegeneration, an early event in the pathogenesis of diabetic retinopathy (DR), precedes clinically detectable microvascular damage. Autophagy dysregulation is considered a potential cause of neuronal cell loss, however underlying mechanisms remain unclear. The mechanistic target of rapamycin (mTOR) integrates diverse environmental signals to coordinate biological processes, including autophagy. Here, we investigated the role of mTOR signaling in neuronal cell death in DR. METHODS: Diabetes was induced by a single intraperitoneal injection of streptozotocin and tissue samples were harvested at 1, 2, 3, 4, and 6 months of diabetes. Early-stage of DR was investigated in 1-month-diabetic mice treated with phlorizin (two daily subcutaneous injections at a dose of 200 mg/kg of body weight during the last 7 full days of the experiment and the morning of the 8th day, 3 h before sacrifice) or rapamycin (daily intraperitoneal injections, at a dose of 3 mg/kg for the same period as for phlorizin treatment). The effect of autophagy modulation on retinal ganglion cells was investigated in 3-months-diabetic mice treated with phlorizin (two daily subcutaneous injections during the last 10 full days of the experiment and the morning of the 11th day, 3 h before sacrifice) or MHY1485 (daily i.p. injections, at a dose of 10 mg/kg for the same period as for phlorizin treatment). Tissue samples obtained from treated/untreated diabetic mice and age-matched controls were used for Western blot and histologic analysis. RESULTS: mTOR-related proteins and glucose transporter 1 (GLUT1) was upregulated at 1 month and downregulated in the following period up to 6 months. Diabetes-induced neurodegeneration was characterized by an increase of apoptotic marker-cleaved caspase 3, a decrease of the total number of cells, and NeuN immunoreactivity in the ganglion cell layer, as well as an increase of autophagic protein. Insulin-independent glycemic control restored the mTOR pathway activity and GLUT1 expression, along with a decrease of autophagic and apoptotic proteins in 3-months-diabetic mice neuroretina. However, blockade of autophagy using MHY1485 resulted in a more protective effect on ganglion cells compared with phlorizin treatment. CONCLUSION: Collectively, our study describes the mechanisms of neurodegeneration through the hyperglycemia/ mTOR/ autophagy/ apoptosis pathway. Video Abstract.

mTORC1 and mTORC2 are differentially engaged in the development of laser-induced CNV.

BACKGROUND: The mechanistic target of rapamycin (mTOR) pathway is a potential target to inhibit pathologic processes in choroidal neovascularization. However, the exact role of mTOR signaling in the development of CNV remains obscure. In this study, we assessed the role of mTORC1 and mTORC2 as well as the effect of rapamycin (sirolimus) on choroidal neovascularization (CNV) in a laser-induced mouse model. METHODS: In experiment A, we observed the natural course of CNV development and the dynamics of mTOR-related proteins during the 12 days after the laser injury. The expression of mTOR-related proteins was evaluated using Western blot (WB). Cryosections of CNV-induced mice were immunostained for the visualization of the vascular and extravascular components of the CNV. Experiment B was performed to confirm the critical period of mTOR signaling in the development of laser-induced CNV, we administered rapamycin before and/or during the active period of mTOR complexes. WB and immunofluorescence staining was performed to evaluate the mode of action and the effect of mTOR inhibition on CNV development. RESULTS: In experiment A, we detected high levels of p-mTOR S2448 and p-mTOR S2481 from the 5th to 12th day of laser injury. Immunofluorescence imaging of cryosections of mice sacrificed on day 7 revealed greater co-immunoreactivity of p-mTOR S2448 positive cells with CD11b and F4/80, while p-mTOR S2481 positive cells showed colocalization with CD31, α-SMA, and cytokeratin. In experiment B, rapamycin injection during the active period of mTOR signaling demonstrated near-complete inhibition of CNV lesion as well as significant induction of autophagy. CONCLUSION: Our study suggests the mTOR as a critical player during CNV development in laser-induced mouse model through differentially acting with the mTORC1 and mTORC2. mTORC1 activity was high predominantly in inflammatory cells in CNV lesion, while mTORC2 activity was higher in vascular components and the RPE.

Essential Role of mTOR Signaling in Human Retinal Pigment Epithelial Cell Regeneration After Laser Photocoagulation.

This study assessed the role of mechanistic target of rapamycin (mTOR) pathway in the human adult retinal pigment epithelial (ARPE) cell response after laser photocoagulation (LP). The effect of mTOR inhibition on ARPE-19 cell was investigated by rapamycin treatment after LP. Cell viability and proliferation were explored using MTT and EdU assays, respectively. The expression of mTOR-related proteins and epithelial-mesenchymal transition (EMT) markers was verified by Western blot. Rapamycin retarded the LP area recovery in a dose-dependent manner by the 120 h, while LP+DMSO vehicle-treated cells completely restored the lesion zone (P ≤ 0.01). ARPE-19 cell viability is significantly lower in LP + rapamycin 80 and 160 ng/ml treated cultures compared to LP control at 120 h (P ≤ 0.001). LP control group demonstrated significantly more proliferative cells compared to untreated cells at the 72 and 120 h, whereas EdU-positive cell numbers in cultures treated with rapamycin at concentrations of 80 and 160 ng/ml were similar to baseline values (P ≤ 0.01). mTOR pathway activation is essential for regulation of the RPE cell migration and proliferation after LP. mTOR inhibition with rapamycin effectively blocks the migration and proliferation of the RPE cells. Our results demonstrate that mTOR has an important role in ARPE-19 cell as a regulator of cell behavior under stress conditions, suggesting that mTOR could be a promising therapeutic target for numerous retinal diseases.

The retinal pigment epithelial response after retinal laser photocoagulation in diabetic mice.

To investigate the characteristics of regenerated retinal pigment epithelial (RPE) cells after retinal laser photocoagulation in diabetic mice. C57BL/6J mice were used to induce diabetes using intraperitoneal injection of streptozotocin. The proliferation of RPE cells after laser photocoagulation was determined using the 5-ethynyl-2'-deoxyuridine (EdU) assay in both diabetic and wild-type mice. The morphological changes of RPE cells were evaluated by using Voronoi diagram from immunostaining for ß-catenin. Characteristics of regenerated cells were evaluated by quantifying the mRNA and protein levels of RPE and epithelial-mesenchymal transition (EMT) markers. There were significantly less EdU-positive cells in laser-treated areas in diabetic mice than wild-type mice. Hexagonality was extensively lost in diabetic mice. Many EdU-positive cells were co-localized with Otx2-positive cells in the center of the laser-treated areas in wild-type mice, but only EdU-positive cells were widely distributed in diabetic mice. Quantitative analysis of mRNA and protein levels showed that the expression levels of RPE markers, Pax6, Mitf, and Otx2, were significantly decreased in RPE of diabetic mice compared with that of wild-type mice, whereas the expression levels of EMT markers, vimentin and fibronectin, were significantly increased. The proliferation and hexagonality of regenerating RPE cells were impaired after laser photocoagulation, and the regenerated RPE cells lost their original properties in diabetic mice. Further clinical research is needed to elucidate the RPE response after laser photocoagulation in diabetic patients.

The role of Wnt/ß-catenin signaling in the restoration of induced pluripotent stem cell-derived retinal pigment epithelium after laser photocoagulation.

To investigate the role of Wnt/ß-catenin signaling pathway in the restoration of induced pluripotent stem cell-derived retinal pigment epithelium (hiPSC-RPE) after laser photocoagulation. After differentiation of RPE cells from hiPSCs, laser photocoagulation was performed. Activation of Wnt/ß-catenin signaling at days 1 and 5 after laser photocoagulation was evaluated by expression of ß-catenin. Cell proliferation and alteration in cell-to-cell contact at day 5 after laser photocoagulation with or without Dickkopf-1 (Dkk-1) treatment were studied using ethynyl-2'-deoxyuridine (EdU) assay and zonula occludens-1 (ZO-1) expression analysis, respectively. The mRNA levels of Wnt genes at day 5 after laser photocoagulation were evaluated by quantitative real-time polymerase chain reaction (qRT-PCR). Activation of Wnt/ß-catenin signaling at days 1 and 5 after laser photocoagulation was confirmed by ß-catenin accumulation in the cytoplasm and nucleus of hiPSC-RPE. Many EdU-positive cells also expressed ß-catenin, and the number of EdU-positive cells was decreased at day 5 after laser photocoagulation after Dkk-1 treatment, indicating that Wnt/ß-catenin signaling mediated hiPSC-RPE proliferation. ZO-1 expression was not decreased with Dkk-1 treatment at day 5 after laser photocoagulation, indicating that Wnt/ß-catenin signaling mediated hiPSC-RPE restoration. At day 5, after laser photocoagulation, mRNA levels of Wnt2b, Wnt3, Wnt5a, Wnt7a, and Wnt10b were increased. Wnt/ß-catenin signaling has a crucial role in restoration of hiPSC-RPE proliferation after laser photocoagulation. Manipulation of Wnt/ß-catenin signaling while elucidating the underlying mechanisms of RPE restoration might have a therapeutic potential in retinal degenerative diseases.

Role of mTORC1 activity during early retinal development and lamination in human-induced pluripotent stem cell-derived retinal organoids.

Retinal organoids derived from human-induced pluripotent stem cells (hiPSC) are powerful tools for studying retinal development as they model spatial and temporal differentiation of retinal cell types. Vertebrate retinal development involves a delicate and coordinated process of retinal progenitor cell (RPC) differentiation, and the mammalian target of rapamycin complex 1 (mTORC1) has been reported to play a significant role in this complex process. Herein, using hiPSC-derived retinal organoids, we identify the time-dependent role of mTORC1 in retinal development, specifically in retinal ganglion cell (RGC) differentiation and the retinal lamination process, during the early stages of retinal organoid (RO) development. mTORC1 activity in ROs was the highest at 40 days of differentiation. MHY1485-induced hyperactivation of mTORC1 during this period resulted in a significant increase in the overall size of ROs compared to the untreated controls and rapamycin-treated Ros; there was also a marked increase in proliferative activity within the inner and outer layers of ROs. Moreover, the MHY1485-treated ROs showed a significant increase in the number of ectopic RGCs in the outer layers (indicating disruption of retinal laminar structure), with robust expression of HuC/D-binding proteins in the inner layers. These results demonstrate that mTORC1 plays a critical role in the development of hiPSC-derived ROs, especially during the early stages of differentiation.

Peripapillary Intravitreal Injection Improves AAV-Mediated Retinal Transduction.

The intravitreal (IVT) injection method is a choice when targeting the inner retina for gene therapy. However, the transduction efficiency of adeno-associated virus (AAV) vectors administered by the IVT route is usually low and may be affected by several factors. To improve the transduction efficiency, we developed a novel illuminated long-needle attached injection system and injected AAV2-CMV (cytomegalovirus)-EGFP in front of the retina in rabbit eyes. Ophthalmological examinations were performed and the levels of pro-inflammatory cytokines in the aqueous humor were assessed at the baseline and 1 month, and the results were compared with those of the conventional injection method. Retinal tissues were used for immunohistochemistry. In the ophthalmological examinations, no significant inflammatory signs were detected in both groups, except for transient, mild hyperemia. In the tissues of the rabbits in the peripapillary injection group, significantly increased GFP expression was detected at the ganglion cell and the inner nuclear layers (p < 0.01). There were no differences between groups in glial activation and expressions of interleukin (IL)-6 and IL-8. These results suggest that peripapillary IVT injection in front of the retina would be safe and efficiently transduce viral vectors into the retina of large animals and is considered as a potential method for use in clinical trials.