Distinct Signature of Altered Homeostasis in Aging Rod Photoreceptors: Implications for Retinal Diseases

Advanced age contributes to clinical manifestations of many retinopathies and represents a major risk factor for age-related macular degeneration, a leading cause of visual impairment and blindness in the elderly. Rod photoreceptors are especially vulnerable to genetic defects and changes in microenvironment, and are among the first neurons to die in normal aging and in many retinal degenerative diseases. The molecular mechanisms underlying rod photoreceptor vulnerability and potential biomarkers of the aging process in this highly specialized cell type are unknown.To discover aging-associated adaptations that may influence rod function, we have generated gene expression profiles of purified rod photoreceptors from mouse retina at young adult to early stages of aging (1.5, 5, and 12 month old mice). We identified 375 genes that showed differential expression in rods from 5 and 12 month old mouse retina compared to that of 1.5 month old retina. Quantitative RT-PCR experiments validated expression change for a majority of the 25 genes that were examined. Macroanalysis of differentially expressed genes using gene class testing and protein interaction networks revealed overrepresentation of cellular pathways that are potentially photoreceptor-specific (angiogenesis and lipid/retinoid metabolism), in addition to age-related pathways previously described in several tissue types (oxidative phosphorylation, stress and immune response).Our study suggests a progressive shift in cellular homeostasis that may underlie aging-associated functional decline in rod photoreceptors and contribute to a more permissive state for pathological processes involved in retinal diseases

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Purpose: To evaluate and optimize methods of target labeling and microarray hybridization using eye gene microarrays. Standardized protocols that consistently produce low background and high intensity hybridization with small amounts of starting RNA are needed to extract differentially expressed genes from a pool of thousands of unaltered genes. Methods: Two identical aliquots of RNA from P19 cell line were labeled with Cy3 or Cy5 dyes using four different methods and self-against-self hybridization was performed on mouse eye gene arrays. The validity and reproducibility of these protocols were further examined using target RNAs isolated from wild-type or neural retinal leucine zipper (Nrl) knockout mouse retinas. Hybridizations were also carried out on human gene array slides with different amounts of starting RNA from human retina. Results: Using self-against-self hybridization, we optimized the protocols for direct labeling (R-square = 0.93), aminoallyl indirect labeling (R-square = 0.97), Genisphere 3DNA labeling (R-square = 0.96), and for microarray hybridization and washing. Although small amounts of initial RNA can be used in TSA method, inconsistent labeling was encountered under our experimental conditions. When retinal RNA targets from Nrl +/+ and Nrl -/-mice were tested by direct and aminoallyl indirect labeling protocols, both produced varying hybridization results with low intensity spots and non-uniform backgrounds. However, the Genisphere 3DNA labeling procedure consistently yielded strong hybridization and R-square values of 0.92 or higher. Furthermore, expression profiles were compatible with prior knowledge of this mouse model. Serial analysis of hybridizations with various starting amounts of RNA showed that the Genisphere 3DNA protocol could produce reliable signal intensity with 3 µg of total RNA. Conclusions: We have systematically evaluated and optimized methods for target labeling, microarray hybridization and washing. These procedures have been used for expression profiling with 3 µg of starting RNA. Our studies should encourage further use of microarray technology for gene profiling during eye development and in retinal diseases

Strong Association of the Y402H Variant in Complement Factor H at 1q32 with Susceptibility to Age-Related Macular Degeneration

Using a large sample of cases and controls from a single center, we show that a T→C substitution in exon 9 (Y402H) of the complement factor H gene is strongly associated with susceptibility to age-related macular degeneration, the most common cause of blindness in the elderly. Frequency of the C allele was 0.61 in cases, versus 0.34 in age-matched controls (P<1×10(-24)). Genotype frequencies also differ markedly between cases and controls (χ(2)=112.68 [2 degrees of freedom]; P<1×10(-24)). A multiplicative model fits the data well, and we estimate the population frequency of the high-risk C allele to be 0.39 (95% confidence interval 0.36–0.42) and the genotype relative risk to be 2.44 (95% confidence interval 2.08–2.83) for TC heterozygotes and 5.93 (95% confidence interval 4.33–8.02) for CC homozygotes

CFH haplotypes without the Y402H coding variant show strong association with susceptibility to age-relatedmacular degeneration,”Nature

In developed countries, age-related macular degeneration is a common cause of blindness in the elderly. A common polymorphism, encoding the sequence variation Y402H in complement factor H (CFH), has been strongly associated with disease susceptibility. Here, we examined 84 polymorphisms in and around CFH in 726 affected individuals (including 544 unrelated individuals) and 268 unrelated controls. In this sample, 20 of these polymorphisms showed stronger association with disease susceptibility than the Y402H variant. Further, no single polymorphism could account for the contribution of the CFH locus to disease susceptibility. Instead, multiple polymorphisms defined a set of four common haplotypes (of which two were associated with disease susceptibility and two seemed to be protective) and multiple rare haplotypes (associated with increased susceptibility in aggregate). Our results suggest that there are multiple disease susceptibility alleles in the region and that noncoding CFH variants play a role in disease susceptibility. Age-related macular degeneration (AMD; OMIM 603075) is a complex degenerative disorder that primarily affects the elderly. Disease susceptibility is influenced by multiple genetic 1-5 and environmental factors After quality assessment of genotype data (see Methods), we tested each SNP for association in 544 unrelated affected individuals and 268 unrelated control