טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentBilgoray-Brenner Liat
SubjectSaving Photoreceptors in a Rat Model of Rod Degenerative
Retinal Disease
DepartmentDepartment of Medicine
Supervisor Professor Emeritus Ido Perlman
Full Thesis textFull thesis text - English Version


Abstract

Retinitis Pigmentosa (RP) is a group of inherited retinal diseases. Many of the mutated genes underlying RP encode proteins that are specific for rod photoreceptors, yet cone photoreceptors degenerate too. Understanding the mechanisms underlying the spread of degeneration from affected rods to healthy cones may lead to development of novel strategies that will slow down or will prevent the spread of degeneration and may provide useful day vision. The most prevalent cause of autosomal dominant RP is the P23H mutation of opsin, the protein of the rod visual pigment. The misfolding of the P23H-opsin causes its accumulation in the Endoplasmic Reticulum (ER) stimulating the ER-stress that triggers the Unfolded-Protein-Response (UPR) and leading to apoptosis. Autophagy is a cellular process that acts to remove defective proteins and organelles and to maintain cell viability during starvation. The drug rapamycin induces autophagy, and thus promotes clearance of damaged proteins from cells.

The purpose of the first part was to create a rat model of RP mimicking the slow spread of photoreceptor degeneration. We used sub-retinal injection of Adeno-Associated Viruses (AAVs) that carried the P23H-opsin gene in order to produce a rat model that expressed the P23H-opsin in a limited retinal area. We found slow deterioration of the Electroretinogram (ERG) responses, reflecting progressive loss of photoreceptors. TUNEL assay, used to identify apoptotic cells, showed positive staining in the pAAV-P23H-IRES-hrGFP injected eyes. There was no positive TUNEL staining in the control injected eyes. Another indication for the spread of retinal degeneration is the wide spread Müller cell gliosis as evident by the expression of GFAP protein in regions remote from the site of injection of the pAAV-P23H-IRES-hrGFP. We found up-regulation in the expression levels of two ER stress-related genes in the pAAV-P23H-IRES-hrGFP injected retinas compared to the control ones. These findings indicate that we succeeded in creating a rat model for studying the spread of degeneration from affected photoreceptors to non-affected ones.

In the second part, we used rapamycin in order to induce autophagy in retinas of P23H transgenic rats. We found that rapamycin slowed down the rate of retinal degeneration. This was evident as slower rate of ERG deterioration and prolonged photoreceptors survival. Rapamycin treatment showed up-regulation in expression of autophagy-related genes and reduction in the activation of ER stress-related markers. In conclusion, rapamycin serves as a neuroprotective agent in the RP animal model. Therefore, Rapamycin hold promise as potential neuroprotective agent for RP.