Abstract
Purpose :
Understanding gene expression changes in bipolar cells following the loss of photoreceptors will enhance the development of cell and gene therapy based therapies aimed at restoring sight to patients with photoreceptor loss. We tested the hypothesis that genes are differentially expressed in bipolar cells of retinal degenerate vs. non-degenerate retina. Gene expression data was validated with protein studies, using a murine model of inherited retinal degeneration (rd1).
Methods :
Bipolar cells were isolated by fluorescence activated cell sorting (FACS) of dissociated retinae from 90 day old C57BL/C3H mice selectively expressing EYFP in rod bipolar cells and cone(types 2 & 6) bipolar cells by crossing L7-Cre and floxed EYFP strains. 6 mice (3M 3F) additionally homozygous for the rd1 mutation were compared to 6 (3M 3F) age matched controls lacking the rd1 mutation “non-rd1.”
RNA was extracted and assessed by Illumina mouseWG-6 microarray with appropriate controls. Quintile normalisation and false discovery rate testing was performed, probes with an adjusted significance level P<0.05 were considered to be different between the groups.
A literature review of these potential genes of interest (GOI) was performed: four in particular were identified for further investigation. Immunohistochemistry (IHC) with antibodies for these four GOI proteins was performed on retinal sections from non-rd and rd1 mice at postnatal day 30, 80, 120 & >150.
Results :
Expression of 65 genes was significantly different between rd1 & non-rd1 groups, with four of these highlighted by the literature review for further investigation with IHC:
Sulfatase 2 (log fold Δ +0.31, p=0.034 on gene array) showed Increased expression in rd1 at P30, reducing over IHC time course
Contactin 1 (log fold Δ -0.51, p=0.049) showed decreased expression in rd1 at P30, reducing over the on IHC time course
Shroom 2 (log fold Δ +0.52, p=0.042) showed increased expression in rd1 with stable expression over the IHC time course
Annexin a7 (log fold Δ +0.39, p=0.024) showed decreased expression rd1 with stable expression over the IHC time course
Conclusions :
We identified 65 genes differentially expressed in bipolar cells of mice carrying the rd1 mutation. Two of these (Sulf2 and Cntn1) appear to alter protein expression over the course of degeneration.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.