Abstract
Purpose: :
Nrl-/- is an enhanced S-cone photoreceptor model created by genetically ablating the key Nrl transcription factor for rod development. To elucidate cone phototransduction protein functions, including G-protein coupled receptor kinase 1 (Grk1), which is essential for light-activated cone opsin phosphorylation in phototransduction shutoff, a Nrl-/-Grk1-/- was created.1,2 We observed an age related light-independent cone degeneration in Nrl-/-Grk1-/- when compared to Nrl-/- mice.3 Our hypothesis is Grk1 has an essential role in photoreceptor homeostasis and survival.
Methods: :
AffymetrixTM mouse genome 430 2.0 microarrays were used to analyze mRNAs from adult Nrl-/- & Nrl-/-Grk1-/- retinas. Transcripts with statistically significant differences were categorized according to function using GeneCards® and Gene Ontology databases. These results were verified with quantitative RT-PCR and Northern blots to pinpoint differentially expressed mRNAs as putative markers for cone dystrophy. Isoelectric focus (IEF), immunoblot (IB) and immunohistochemistry analysis further characterized these candidates.
Results: :
Microarray analysis performed in triplicates on retinas from PN60 Nrl-/- & Nrl-/-Grk1-/- mice demonstrate statistically significant (p<0.05) changes in transcript levels of >400 genes. Of these, 66 had greater than 4 fold increases, 9 with known elements associated with the immune response linked to age-related macular degeneration (AMD). Kallikrein (Klk) and Pituitary Tumor Transforming Gene-1 (Pttg1) increased 90 and 40 fold, respectively, and both are linked to defects in cell homeostasis. Up-regulation of Pttg1 protein in Nrl-/-Grk1-/- retina was confirmed by IB and IEF experiments. A 4 fold decrease in transcript abundance was observed in 123 genes, including Crumbs homolog 1 (Crb1). Defects in Crb1 are linked to retinitis pigmentosa and Leber congenital amaurosis.
Conclusions: :
The Nrl-/-Grk1-/- mouse is an excellent model to study cone dystrophy due to an observed light independent age-related cone degeneration. We have utilized comprehensive gene expression analyses and biochemical approaches to elucidate the genetic and proteomic profiles leading to cone dystrophy.The knowledge of cellular and molecular mechanisms associated with cone apoptosis may lead to potential targets for therapeutic treatments of AMD.1 Zhu et al., Jour. Neurosci. 2007; 23:6152-6160.2 Nikonov et al., J. Gen. Physiol. 2006; 127; 359-374.3 Yetemian RM, et al. Invest. Ophthalmol. Vis. Sci. 2007; 48: ARVO E-Abstract 4655
Keywords: photoreceptors • apoptosis/cell death • gene microarray