Phenotypic variability based on genetic background differences observed in mice (28
) can provide models for complex human conditions and explain the variable susceptibility to certain conditions among different individuals with the same allelic variant of disease. With no reliable markers to date for ARD before it becomes clinically evident, genetic studies have involved only affected individuals, making it difficult to identify factors that trigger this condition. Compared with BALB/c and B6 mice, A/J mice in this study displayed accelerated retinal degeneration stemming from differences in their natural genetic background. Declining visual function with age, associated with worsening RPE and photoreceptor cell pathology, was documented in these mice. Given that the retina is fully developed after 1 month in mice, we used a comprehensive set of approaches to identify genetic factors in 1-month-old mice that led to severe ARD at 8 months in A/J mice, but not BALB/c or B6 mice.
Identifying global gene variations associated with complex diseases of multigenic origin can be difficult. The present studies highlighted that CSS panels, although capable of revealing some complex disease associations (15
), lacked the breadth to identify distant interacting genetic factors characteristic of a complex disease like ARD. RNA-Seq allows for more complete documentation of genetic changes relevant to complex disease etiology (31
). This is especially true for age-related neurodegenerative conditions, as illustrated by the recent discovery of 2 novel genetic loci associated with Alzheimer disease by high-throughput sequencing methodology (32
). The challenge of RNA-Seq technology lies in the large-scale translation of genetic variations into knowledge about the molecular pathogenesis of complex diseases. By studying cellular pathways encoded by these genes as integrated systems, we can begin to understand their derived phenotypes (33
) and identify genes that critically contribute to complex disease traits. In this study, RNA-Seq of young mouse eye tissue across different genetic backgrounds was performed before substantial pathology was present. This analysis revealed global gene expression changes that identified factors apparently predisposing A/J mice to a severe ARD phenotype, as well as factors in BALB/c and B6 mice that may prevent this pathology. Complementing morphologic assessment of ARD with deep sequencing technology allowed us to identify early signatures of disease in inbred mice with different genetic backgrounds.
RPE cells are critical for the maintenance of the blood/retina barrier and retinal neurons and must possess mechanisms that protect the retina against oxidative stress generated by exposure to light and high oxygen tension. Compared with BALB/c and B6 mice, A/J mice displayed decreased expression of key gene families needed by RPE cells to mitigate such stress. Most notable were those families involved in glutathione-mediated oxidative stress protection. Abundant in the retina, the GPX family encodes a critical group of retinal detoxification enzymes. Only Gpx3
expression from this family was markedly decreased in A/J mice, and GPX3 largely localized to RPE cells. Inadequate activity of antioxidant enzymes like GPX in other organ systems has been implicated in the progression to chronic inflammatory pathology (34
), and polymorphisms of Gpx3
have been implicated in human phenotypes of ARD (35
). This finding extends to other protein families, such as the HSPs, of which both Hspa8
showed decreased expression in A/J mice. In addition to chaperone-mediated activity (37
), HSPs combat oxidative stress by increasing levels of glutathione and modulating the redox status of cells (38
). Decreasing levels of glutathione and associated proteins with age (39
), coupled with constitutively decreased expression of these important homeostatic gene families in young A/J mice compared with BALB/c and B6 mice, could explain the accelerated ARD phenotype found in A/J mice. Pathologic changes seen in A/J RPE, characterized by cell swelling and buildup of undigested disc elements, have also been reported in albino and pigmented rats deprived of selenium (41
), a critical cofactor for activation of antioxidant proteins such as GPX3. Inadequate protection against oxidative stress was also accompanied by accelerated loss of photoreceptors, especially in the central retina of these rats, which suggests that oxidative damage to RPE cells may initiate the detrimental process and that photoreceptor loss is a secondary effect. An inadequate oxidative stress response could thus produce wide-ranging adverse effects in the retina. One detrimental age-related effect — namely, cellular senescence — was evidenced by increased BMP4 expression in A/J mice as young as 1 month of age, and this could lead to secretion of various factors that fuel chronic inflammation (43
The RPE is a central site of immune regulation in the retina (44
). Moreover, constant stress and cellular senescence can exacerbate immune responses that lead to chronic inflammation, which results in disease progression because of changes in homeostatic set points (45
). Although A/J mice have natural deficiencies of complement (C5a
) and NOD (Naip5
) genes involved in susceptibility to bacterial/fungal infection, recombinant congenic strains (46
) have shown that these genes are controlled by additional factors and have little effect on the inflammatory-priming pathways highlighted in this study. In young A/J mice (and to a lesser degree BALB/c mice, but not B6 mice), RNA-Seq revealed an inflammatory-primed network characterized by increased expression of IFN gene products, such as Stat1
and its downstream effectors (23
). The inflammatory-primed state identified in A/J mice may not be limited to the retina, as it also likely contributes to the enhanced A/J inflammatory response in experimental allergic asthma (48
). Moreover, the graded retinal inflammatory changes we saw from A/J to BALB/c to B6 mice are consistent with studies of allergic airway inflammation in the lungs of these strains (49
). These prior studies indicate that A/J mice are most susceptible to inflammation in tissues of high oxygen tension (retina and lungs), further implicating an inadequate oxidative stress response as a driving force of late stages of inflammatory disease progression. In the present study, the inflammatory-primed state of the retina in A/J mice was compounded by markedly lower levels of immune-regulatory enzymes. Compared with BALB/c mice, A/J mice exhibited decreased expression of genes such as Tyro3
and its downstream effector Socs1
, which serve to control this inflammatory response (50
). Moreover, inadequate function of enzymes like GPX3 would lead to increased oxidative stress and hydrogen peroxide production, which reduce the expression and function of complement factor H (51
), a key complement-regulatory enzyme implicated in disease pathogenesis (52
). Thus, the inflammatory-primed state of the retina in young A/J mice reflects a parainflammatory state (53
) that, accentuated by increased oxidative stress, transitions to chronic inflammation and cellular dysfunction with increasing age (Figure ), just as observed in diseased patients (54
). Priming of RPE cells can translate to secondary effects on critical cellular processes such as phagocytosis and digestion of POS, which are increasingly affected in A/J mice with age. Another secondary effect is the production of anti-retinal antibodies to α-crystallin, GFAP, and α-enolase found in sera of ARD patients (55
). Preferential activation of Stat1
has been detected in several autoimmune/inflammatory conditions (23
), and this increased significantly and exclusively in A/J mice with age. We also noted increased GFAP expression with age, and α-enolase was elevated at the transcript level in A/J mice as early as 1 month of age. RPE/retina barrier function is most detrimentally affected when both oxidative stress and immune activation are induced (56
), which suggests that these additive effects of aging in A/J mice could potentially lead to autoimmune responses. Ocular immune privilege is normally responsible for the impaired tolerance to retinal antigens (57
), but exposure to such antigens upon age-related barrier compromise could further contribute to disease progression.
Inadequate protection by the RPE from stress drives the retina from an inflammatory-primed state to a chronic disease state.
Our preliminary RNA-Seq analysis of whole eye tissues from both nocturnal (Long-Evans rat) and diurnal (Nile rat) rodent species at a young age revealed gene signatures most closely related to B6 mice and clearly distinct from A/J mice (Supplemental Table 3). This observation emphasizes that our present findings may extend beyond model mouse species and provide an additional frameworks for understanding etiologic factors contributing to complex age-associated diseases in higher organisms. This is especially relevant to human blinding conditions associated with aging, such as age-related macular degeneration (AMD), the leading cause of blindness in the industrial world and now considered one of the major blinding diseases worldwide (58
). The complex etiology of AMD is reflected by the relative paucity of effective compounds for its early prevention and treatment, with the main risk factor being increasing age. Complex trait analysis of genome-wide association studies — having identified the vast majority of variations in noncoding regions (59
) and the recent identification of large intervening noncoding RNAs implicated in controlling a diverse set of biological processes (60
), including differentiation of the murine retina (62
), a discovery accelerated by application of next-generation sequencing technology (63
) — could serve as the next frontier to facilitate understanding of complex disease phenotypes.
In conclusion, we used 3 inbred background strains of mice with differing susceptibilities to ARD and used RNA-Seq to identify genes encoding components of cellular pathways that contribute to this blinding condition at a young age, before phenotypic changes are apparent. We identified relatively high expression of proinflammatory factors coupled with low expression of homeostatic protective factors, such as those involved in oxidative stress response, and localized them to the A/J mouse retina, most notably to the RPE. This combination made A/J mice especially vulnerable to rapidly progressive ARD compared with BALB/c and B6 mice. A/J mice reared in the dark still developed ARD, which indicates that the pathogenic mechanisms described here differ from those involving excessive accumulation of the all-trans
-retinal chromophore (64
). These results, in conjunction with imaging techniques that demonstrated elevated level of ROS and changes in phagocytotic processing in the RPE, allow a more comprehensive understanding of a complex neuronal degeneration. With advances in phenotypic and genotypic characterization technology, the methodologies outlined herein represent a powerful paradigm to unveil cellular changes that trigger and drive progression of complex neurodegenerative diseases extending beyond the eye.