Apoptosis Genes in Photoreceptor Cell Death of the RD Mouse Determined through Gene Expression Profiling.

Abstract

Abstract: : Purpose: In the rd mouse, photoreceptor degeneration is due to a mutation of the rod–specific enzyme cGMP phosphodiesterase, resulting in permanently opened cGMP–gated cation channels in the rod outer segment membrane, allowing Na⁺ and Ca²⁺ ions to enter the cell, resulting in toxic levels of Ca²⁺. To identify pathways involved in the Ca²⁺–induced cell death of the rd rods, we evaluated gene expression (GE) in the rd and wild type (wt) mouse retina over the known time course of degeneration, and assessed simultaneous morphological changes with in vivo imaging. Methods: Total RNA was used to generate ds–cDNA, which served as a template to transcribe biotinylated cRNA. The labeled, fragmented probes were hybridized to U74A oligonucleatide arrays (Affymetrix). The raw data (PM–values) was normalized according to an algorithm developed to integrate binding interactions of probe sequences on microarrays (Zhang et al., 2003). Tools for hierarchical, functional and gene ontology (GO) term clustering were implemented in Matlab Statistical Toolbox functions. Selected genes were verified at the mRNA level using the QuantiTect Syber Green PCR Kit, and at the protein level using immunohistochemistry. Morphological in vivo data were obtained using a scanning laser ophthalmoscope (Heidelberg Engineering HRA). Results: (A) 182 genes passed the selection criteria (low standard deviation and high correlation between samples), falling into six clusters. For any given pair of genes, an expression profile correlation distance and a semantic distance (one for each class of GO terms) was established. (B) GE in rd started to deviate from wt by P10, with a reduction in photoreceptor– and an increase in apoptosis– and neuroinflammation–specific genes. (C) Quantitative RT–PCR and immunohistochemistry confirmed the increase in neuroinflammatory processes. (D) Persistent vitreal vessels and reduced retinal capillaries beyond P14 indicated a developmental delay. Between P14 and P21, an increase in retinal and vascular damage was observed. Conclusions: Our results suggest that photoreceptor degeneration in the rd mouse is a process starting with the primary insult of Ca²⁺–toxicity followed by a secondary insult involving multi–destructive pathways such as apoptosis and neuroinflammation, presumably boosting morphological changes. All of these components need to be addressed if rods are to be successfully protected.

Keywords: apoptosis/cell death • gene microarray • imaging/image analysis: non–clinical