Constitutive and AP20187-induced Ret activation in photoreceptors does not protect from light-induced damage

Abstract

Purpose: Delivery of glial cell-derived neurotrophic factor (GDNF), either as a recombinant protein or by retinal gene transfer results in photoreceptor (PR) neuroprotection in genetic models of retinitis pigmentosa (RP). The mechanism of GDNF action and its direct targets in the retina remain unknown. The goal of the present study was to test the neuroprotective effect of GDNF from light-induced damage, a commonly used stimulus of PR degeneration, and to determine whether protection occurs directly on PRs.

Methods: Adeno-associated viral vectors (AAV) were developed that expressed either GDNF or a constitutively (RetMen2A) or pharmacologically activated chimeric GDNF receptor (Fv2Ret). Fv2Ret homodimerization and activation are induced by the administration of the small dimerizer drug AP20187. AAV2/2 vectors and the cytomegalovirus (CMV) promoter were used to transduce GDNF in the retina, whereas RetMen2A and Fv2Ret were transduced by AAV2/5 vectors and their expression restricted to PRs by the rhodopsin promoter. In vivo GDNF levels were measured by ELISA, RetMen2A and Fv2Ret expression and activation in vitro and/or in vivo were assessed by Western blot and immunofluorescence analyses. ERG measurements and histologic analyses were performed to assess morphologic and functional rescue, respectively.

Results: GDNF gene transfer resulted in sustained protein expression in the eye. In addition, the results confirmed in vivo that PR-restricted activation of Ret signaling occurred after either AAV-mediated expression of RetMen2A or AP20187-dependent Fv2Ret activation. However, this or AAV-mediated GDNF retinal gene transfer did not result in functional or morphologic PR protection from light-induced damage.

Conclusions: The results suggest that the apoptotic pathways responsible for light-induced PR degeneration are not inhibited by GDNF. However, GDNF signaling was shown to be regulated in time and levels in the retina by the AP20187/Fv2Ret system which is therefore available to be tested as gene-based therapeutic strategy in models of PR degeneration responsive to GDNF.