To test the AP20187-dependent Fv2Ret and constitutive RetMen2A phosphorylation in vivo in PR cells, we generated recombinant AAV2/5.RHO.Fv2Ret, -.RetMen2A, and -.EGFP vectors. Four-week-old BALB/c mice received subretinal injections of AAV2/5.RHO.Fv2Ret or -.RetMen2A vectors in the right eye and of AAV2/5.RHO.EGFP vector in the left eye as control. To confirm that the combination of AAV5 capsid and rhodopsin promoter results in transgene expression restricted to PRs, 28 days after vector administration we analyzed some retinas injected with AAV2/5.RHO.Fv2Ret and .EGFP by immunofluorescence with anti-Ha antibody and by direct fluorescence, respectively.
Figure 3Ashows that EGFP expression was restricted to PR cell bodies and outer segments (left); Fv2Ret was localized to PR outer segments similarly to endogenous Ret
23 (right). Twenty-eight days after vector administration (a time point selected to allow sustained gene expression from AAV2/5 in the retina), the animals injected with AAV2/5.RHO.Fv2Ret were divided into three groups (
n = 3) as described in the Materials and Methods section. One group received IP administration of AP20187 (10 mg/kg) for 5 days, one group received combined AP20187 IP and intravitreal (IV, 4 μg) administration on days 1 and 5 of treatment, and the last group did not receive AP20187. The IV injections localize AP20187 directly to the vitreous, whereas IP injections require the drug to cross the blood–retina barrier. All retinas were harvested 5 hours after the last AP20187 injection (32 days after vector administration) and analyzed by Western blot with anti-Py and anti-Ha antibodies. The bands obtained by Western blot with anti-Py antibodies were quantified and the results are shown in
Fig. 3Bas the increase in Fv2Ret phosphorylation on AP20187 administration compared with AP20187-untreated, Fv2Ret-transduced control retinas. Fv2Ret tyrosine phosphorylation occurred in the absence of AP20187 increases of 3.7 ± 1.0- and 6.7 ± 2.2-fold after IP and combined IP and IV AP20187 injections, respectively
(Fig. 3B) . There was no increase in the Fv2Ret phosphorylation levels when higher doses (40 μg) of AP20187 were injected IV (data not shown). Our data indicate that AP20187 given systemically can cross the blood–retina barrier, although the levels of AP20187-dependent Fv2Ret phosphorylation are higher when the drug is given IV. Twenty-eight days after the vector was administered, the animals with AAV2/5.RHO.RetMen2A subretinally administered in one eye and AAV2/5.RHO.EGFP in the contralateral eye were killed and their retinas harvested and analyzed by Western blot
(Fig. 3C) . We detected a phosphorylated band with a molecular weight that corresponded to RetMen2A in the retinas injected with AAV2/5.RHO.RetMen2A, but not in those injected with AAV2/5.RHO.EGFP
(Fig. 3C) . In addition, all Shc isoforms are strongly phosphorylated on tyrosine residues in the retinas injected with AAV2/5.RHO.RetMen2A but not with AAV2/5.RHO.EGFP (data not shown). After subretinal administration of AAV2/5 vectors, recombinant Ret expression and tyrosine phosphorylation in PRs were constitutive and robust in the case of RetMen2A and dependent on AP20187 administration in the case of Fv2Ret. We therefore tested whether AAV-mediated overexpression of GDNF, Fv2Ret, or RetMen2A protects PRs from light-induced damage, a model widely used to test neurotrophic molecules.