AAV-mediated MANF (mesencephalic astrocyte-derived neurotrophic factor) expression protects retinal ganglion cells in a retinal ischemia/reperfusion mouse model

Abstract

Purpose : Mesencephalic astrocyte-derived neurotrophic factor (MANF) is a member of a novel evolutionarily conserved protein family with neurotrophic capabilities. Previously we reported that recombinant human MANF protects photoreceptors in a retinal degeneration rat model. The present work investigates potential neuroprotective effects of MANF on retinal ganglion cells (RGC) using a mouse retinal ischemia–reperfusion (IR) model.

Methods : A viral vector AAV-MANF was used to deliver the MANF gene to retinal cells. The expression of MANF was driven by a CMV promoter. A control viral vector, AAV-GFP, was constructed by replacing the ORF of MANF with the ORF of GFP. Adult C57B/6J mice (3 months old) were used in all experiments. Animals were divided into 3 groups: IR only (n=5), IR+AAV-GFP (n=3), and IR+AAV-MANF (n=5). Three microliters of AAV-GFP (2x10¹³ vg/ml) or AAV-MANF (2x10¹³ vg/ml) were injected intravitreally into each eye 3 weeks prior to IR. The expression levels of MANF were assessed by Western blot analysis. Ischemia was achieved by increasing the intraocular pressure (IOP) to 120 mmHg for 45 min in the left eye. A 33-gauge needle connected to a reservoir of saline was inserted into the anterior chamber to get desired IOP by rasising the reservoir. The right eye was untreated as a control. Retinas were harvested 7 days after IR, stained with NeuN, and flat-mounted. NeuN positive cells in the retinas from both eyes were counted, and cell survival rate in the IR retinas was calculated as a percentile of the mean cell density of the control fellow eye.

Results : Seven days after IR, the remaining of RGCs in the control ischemic retinas without injection was 25±1%, indicating about 75% cell loss. A similar survival rate (21±3%) was found in retinas injected with AAV-GFP. In AAV-MANF treated retinas, however, the survival rate was 78±8%, significantly higher than the two control groups (P<0.001 compared with each control group). Analysis also showed the levels of MANF protein in the retina and vitreous in the AAF-MANF treated eyes were significantly higher than those in the two control groups.

Conclusions : Our results demonstrated that AAV-mediated MANF expression significantly protects against RGC loss. MANF thus could be considered as a clinical neuroprotective agent for retinal degenerative disorders.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.