Micro-RNA29 mitigates neurotoxic pathways induced by 12/15-lipoxygenase metabolites in Müller cells

Abstract

Purpose : Diabetes induces neurotoxic pathways in Müller glial cells(MC) by impairing glutamate transport causing glutamate excitotoxicity. Our goal was to investigate the role of 12/15-lipoxygenase(12/15-LO) metabolites 12- and 15-HETEs in activating MC and neurodegeneration in diabetic retinopathy(DR) and the neuroprotective function of mirR29.

Methods : We tested the effect of intravitreal injection of 12-HETE(0.1 µM) on mouse MC by immunostaining of retinal sections with the glial cell marker GFAP. We generated diabetic mice that lack 12/15-LO (Ins2^Akita/12/15-LO^-/-). We compared retinal thickness using H&E-stained retinal sections and retinal function using ERG in 6 month-diabetic 12/15-LO^-/- mice to normal wild type, normal 12/5-LO^-/- and diabetic (Ins2^Akita) groups(N=5). We performed microRNA array of retina samples from the same groups. Retinal levels of 12- and 15-HETEs receptors GPR31 and GPR39 respectively were studied by western blot (WB)(N=4). In vitro, we used rat MC (rMC1) treated with 12-HETE (0.1 mM) in the presence or absence of miR29a mimic (30 nM). Levels of glutamate, TNFa and reactive oxygen species (ROS) in rMc1 were studied by ELISA and CellROX® Green Reagent(N=6). We also assessed the levels of Nrf2 and the neurotrophic factor PEDF by immunofluorescence and WB(N=4). Expression levels of GPR31 and GPR39 mouse MC and rMC1 were assessed by immunofluorescence and Flow Cytometer.

Results : 12-HETE induced a remarkable increase in retinal expression of MC stress marker GFAP(gliosis) in mouse retina. Diabetes significantly increased expression of GPR31 and GPR39 in mouse retina compared to the control (P<0.05). Immunofluorescence and Flow Cytometer showed notable expression levels of GPR31 and GPR39 in mouse and rMC. Deletion of 12/15-LO preserved normal retinal thickness and function as well as normal levels of miR29 family in diabetic mice(P<0.05). Treatment of MC with 12-HETE significantly increased glutamate production compared to the control. Also treatment of MC with glutamate or 12-HETE caused upregulation of TNFa and ROS, while suppressed PEDF expression and nuclear levels of Nrf2 compared to the control(P<0.05). These effect of 12-HETE were reversed in the presence of miR29a mimic.

Conclusions : 12/15-LO contribute to diabetes-induced retinal neurodegeneration. GPR31, GPR39 and miR29a are potential targets to mitigate the neurodegenerative effects of 12/15-LO-metabolites in DR.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.