Involvement of Multiple Pathways in the Retinitis Pigmentosa 1 (RP1) Disease

Abstract

Abstract: : Purposes: To identify molecular pathways involved in the RP1 disease using a mouse model of RP1 (Gao et al., 2002). Methods: Affymetrix U74Av2 microarrays (>12,000 cDNA/ESTs) were used to examine retinal gene expression profiles from P7 to P21 in Rp1+/+ and -/- mice. P7 is when the Rp1 protein is first expressed in photoreceptor connecting cilia in Rp1+/+ and when the first sign of outer segment abnormality occurs in Rp1-/-; P21 is when no significant photoreceptor cell loss is yet observed in Rp1-/- (Liu et al., 2002; Gao et al., 2002). Triplicates of RNA samples from Rp1+/+ and -/- neural retinas for hybridization were collected at each of the following time points: P7, 10, 14, 18 and 21. Each RNA sample included a pool of neural retinas from 3-4 mice. Retinas were all collected at 1-2 pm of the day. Two-sample t-test was used to identify genes whose expressions were significantly different between Rp1+/+ and -/- at each of the five time points. Two-factor ANOVA was used to identify genes whose expressions were significantly affected by the interaction between genotype and time point, showing the effect of the Rp1 disruption on development at a global level. Real-time PCR and in situ hybridization were performed to verify expression levels and cellular localization of selected genes in Rp1+/+ and -/- at the five time points. Results: 876/4,506 genes in the NEI retinal cDNA library and 223/264 genes in the photoreceptor SAGE library (Blackshaw et al., 2001) are present in U74Av2, suggesting that U74Av2 contains most of photoreceptor genes. ~100 genes are differentially expressed (t-test p<0.01) at each time point between Rp1+/+ and -/- with surprisingly small numbers (<5%) in common among the time points, suggesting that Rp1-/- induced molecular responses change dramatically from P7 to P21. 77 genes show significant effects of the Rp1 mutation on development (ANOVA interactions p<0.01), and they are further grouped into 33 clusters by hierarchical rankings. They implicate multiple pathways in metabolism, cytoskeletal structure, inflammatory responses, transcriptional regulation, and ion transport. In support of the hypothesized role of RP1 in transporting outer segment proteins, several genes are likely involved in vesicle transport in photoreceptors: a clathrin-associated protein, an adaptor-related protein, a myosin, a retinol binding protein, and peripherin2. Conclusions: Our data provided basis for analysis of molecular mechanisms associated with RP1 gene defects and normal retinal development.

Keywords: gene microarray • retinitis • retinal development