Peptide based targeting of PTEN and Kv2.1 Rescues RGCs Following Optic Nerve Transection

Abstract

Purpose: The hallmark of glaucoma is the degeneration of RGCs (retinal ganglion cells) by apoptosis. We studied the potential neuroprotective effects of antagonistic peptides corresponding to the c-termini of PTEN and Kv2.1, two proteins that have been shown to promote RGC apoptosis after injury.

Methods: Optic nerve transection was performed in adult rats and RGCs were retrogradely labeled with Fluorogold in order to assess survival. Peptides (conjugated to TAT- or R9- protein transduction sequences; 10mg/ml; n=4-6 per group) were administered by intraocular (IO) injection or direct application to the cut end of the optic nerve. Controls received injections of TAT-conjugated poly-alanine peptide, or vehicle. RGC survival was quantified at 14 days postaxotomy via fixed flat-mounted retinas and epifluorescence micrographs. To assess RGC regeneration, peptides were similarly administered after optic nerve crush. The number of regenerating axons distal to the crush site was quantified in frozen, GAP-43 immunostained, sections of the optic nerve at 21 days post-crush. Eye drop delivery of peptides was tested by applying drops once per day for the first 7 days after axotomy; RGC survival was quantified at 14 days post-axotomy. Data was analyzed via one-way ANOVA followed by Tukey’s post-hoc test in order to identify statistically significant differences between control and experimental groups.

Results: Intraocular injection of TAT-PTEN after axotomy increased survival by 3x, and nerve injection produced a 2x increase. Nerve application of TAT-Kv2.1 resulted in a ~5x increase in RGC survival and intraocular injection a 3x augmentation after intraocular delivery. Eye drop administration of R9-conjugated PTEN or Kv2.1 peptides increased survival 2-3x. Immunoprecipitation demonstrated that PTEN peptide reduces co-precipitation of MAGI-2 and Mast205, indicating that PTEN peptide blocks the recruitment of PTEN to protein complexes. Both TAT-PTEN and R9-PTEN significantly enhanced RGC regeneration after optic nerve crush.

Conclusions: Cell-permeable peptides corresponding to the c-termini of PTEN or Kv2.1 enhance RGC survival after optic nerve injury. These peptides can be delivered via several routes (intraocular injection, eye-drops) suggesting that these approaches may have therapeutic potential for visual disease.