The visual process begins in highly specialized photoreceptors (PRs), which are neurons with a complex structure and a unique ability to convert light photons into electrochemical messages. After the initial quantal light absorption by the rhodopsin visual pigments, a signal is generated in the PR and subsequently transmitted through two different synaptic pathways in the outer and inner plexiform layers. The information is then conveyed to higher visual centers by the ganglion cell axons. To subserve this role, a large number of genes are involved in PR specification, differentiation, and maintenance ; and mutations in many of these genes impair PR function and viability. Indeed, of the 246 loci that are associated with retinal degeneration in humans, in 206 the disease-causative genes have been identified . Several genes have also been associated with retinal degeneration in animals , and at least 24 mutations in 18 genes related to canine retinal degenerations have been identified .
Although the number of identified genetic mutations underlying different forms of retinal degeneration is systematically growing, the molecular events and key components that link specific mutations to PR degeneration remain poorly characterized. Multiple pathways, both pro-apoptotic and pro-survival, are associated with PR degeneration [5–9]. Furthermore, epigenetic mechanisms, including miRNA regulation, also play an essential role in the control of the complex visual processes during eye development and disease . miRNAs are small (~20-25 bp), endogenous, non-coding single-stranded regulatory RNA molecules that regulate various cellular functions, including differentiation, proliferation, and cell death/survival (reviewed by ). They are expressed in all living organisms in tissue- and developmental stage-specific manner, and are responsible for individual phenotypical variations. miRNAs silence gene expression via cleavage, degradation, and/or translational inhibition of their downstream target mRNAs (reviewed for mammalian miRNAs by ). Each miRNA has the potential to regulate multiple different mRNA targets simultaneously, while a given mRNA target might similarly be targeted by multiple miRNAs (reviewed by ). The number of known mature miRNAs is currently 30,424 in 193 species and approximately 2,580 have been discovered in humans . Of these, 349 have been linked to 163 different diseases .
Recently, the use of miRNA-microarray analysis in several tissues has enabled the identification of altered miRNA transcriptomes during development/aging and disease, including profiles of pathologically altered miRNAs in the eye and retina (reviewed by ). Also, it has been shown that miRNA pathways control important steps during the developmental timing of retinogenesis , and appear to regulate neuronal differentiation . The use of other technologies (i.e. deep sequencing) has also provided very comprehensive profiles of miRNAs and revealed a complex expression pattern of small RNA in the mouse retina and RPE/choroid . Of particular interest are “apoptomirs” , miRNAs that have been shown in many studies to be relevant mediators of cell death signaling [21–23]. Assessment of disease-related miRNAs in human retinal diseases obviously is limited by the availability of appropriately staged tissues from patients having the same disease and causative gene mutation. Notably however, the dog has been widely recognized as an ideal model for a variety of human retinal disease studies, as canine inherited retinopathies result from mutations in disease gene homologues and exhibit comparable phenotypic features, including age of onset and progression . Some models have the advantage of an early and predictable disease course, making the time window for experimentation very short and easily comparable. As such, they are an ideal system in which to determine if miRNAs are associated with PR death and if their involvement is dependent on the specific mutation driving disease.
To identify potential miRNAs associated with PR degeneration, we used the four following canine models: X-linked progressive retinal atrophy 2 (xlpra2), rod cone dysplasia 1 (rcd1), early retina degeneration (erd), and progressive rod-cone degeneration (prcd) that have mutations in RPGR, PDE6B, STK38L, and PRCD, respectively. The progression and histopathology in xlpra2, rcd1, and erd are comparable, and characterized by a fast degeneration of the PR cell layer and decreased number of PRs [24–27]. The first two models have mutations in genes that cause human inherited blindness, while no equivalent human disease for erd has been reported yet. In contrast, prcd is a post-developmental, slowly progressive disease where human patients and animal models show disease variation in the presence of the same mutation [28, 29].
For the initial microarray analysis, retinas of dogs affected with xlpra2 were compared to normal samples. To expand the microarray results, we then undertook a qRT-PCR analysis of the expression of selected apoptomirs in the three additional models, rcd1, erd, and prcd.
Our results show that although different mutations trigger the retinal diseases studied, there are commonalities in the miRNA expression pattern that appear to be associated with the PR cell death kinetics.