Mitochondrial DNA damage: an important marker for oxidative damage
Mitochondrial DNA (mtDNA) is more susceptible to oxidative damage than nuclear DNA (nDNA) because the former is in direct contact with mitochondrial produced reactive oxygen species; mtDNA are not protected by histones or other DNA-associated proteins [19–21]. In addition, the DNA repair machinery inherent in mitochondria is less efficient than nDNA [22, 23]. Therefore, in tissues rich in mitochondria, like the retina, the damaged or altered mtDNA serves as a reliable biomarker of oxidative stress.
Using a novel long quantitative polymerase chain reaction (QPCR technique), our laboratory demonstrated that mtDNA is damaged early in EAU (day 4 postimmunization). We also showed that in EAU, mitochondrial oxidative stress occurs before macrophage or other inflammatory cellular infiltration [7]. These findings support previous studies from our laboratory that determined that peroxynitrite-mediated nitration of photoreceptor mitochondrial proteins, as well as the presence of reactive oxidants and peroxynitrite, occurs in mitochondrial photoreceptors during early EAU [17, 18]. Oxidative stress appears to target the mitochondria as the original site of inflammation and thus damaged or altered mtDNA is a major target for oxidative damage.
iNOS-mediated oxidative stress results in the nitration of cytochrome c, a photoreceptor mitochondrial protein, which is then released into the cytosol; such cytochrome c release is known to cause apoptosis [6, 18]. Oxidative damage of mtDNA, if left unchecked, can lead to mitochondria dysregulation and cell death [24]. However, despite the presence of mtDNA damage, in our study apoptosis was not detected during early EAU (apoptosis occurred on day 12) [7]. Some of the reasons postulated by our laboratory for the lack of early cell death were that there may have been insufficient amounts of mtDNA damage to cause apoptotic cascades until day 12 or that protective mechanisms like heat shock proteins and crystallins, which are upregulated by oxidative stress, prevents photoreceptor apoptosis during early EAU by inhibiting components of the apoptotic cascade and functioning as protective and repair mechanisms [3, 7, 25].
Differential expression of mitochondrial proteins and mitochondrial dysfunction
During early EAU, retinal DNA damage is restricted to the mitochondria; this suggests that mitochondrial oxidative stress plays a role in retinal damage [7]. Mitochondria are a source of reactive oxygen species and are the main target for stress-mediated damage. Oxidative stress can alter mitochondrial protein levels and consequently alter the functions of mitochondria in EAU. ATP synthases maintain mitochondrial membrane potentials and morphology [26]. Using 2D-DIGE, mass spectrometry by MALDI-TOF MS analysis, and reconfirming findings with Western bolt analysis, our laboratory found that during early EAU, there was a significant decrease in ATP synthase protein levels in retinal mitochondria; this suggests that decreased levels of cellular ATP and loss of ATP synthase activity corresponds to mitochondrial oxidative damage [3]. Moreover, the expression of aconitase, a sensitive marker of oxidative stress in mitochondria, was upregulated suggesting that mitochondrial oxidative stress affects its expression during early EAU [3].
Other proteins with altered expression due to early EAU were calretinin, mitochondrial aspartate aminotransferase, and malate dehydrogenase; we found decreased levels of calretinin, increased levels of mitochondrial aspartate aminotransferase, and decreased levels of malate dehydrogenase when compared to controls. These findings suggest mitochondrial dysfunction during early EAU. As we reported in an earlier study, damaged or altered mtDNA is a primary target for oxidative damage [7]. mtDNA damage causes amplification of oxidative stress by decreasing the expression of essential proteins for electron transport like malate dehydrogenase, leading to reactive oxygen species and mitochondrial dysregulation which will eventually cause apoptosis.
The role of innate immunity in mediating photoreceptor mitochondrial oxidative stress
In early EAU, the mechanism that induces oxidative stress is still uncertain. Traditionally, it was believed that macrophages caused oxidative damage, but this seems unlikely since there is no histologic or immunohistochemical evidence of macrophage infiltration in the retina or uvea until days 11 to 12 whereas oxidative stress appears much earlier on days 5 to 7 [6, 15]. In addition to macrophages, retinal microglia also exhibit phagocytic and proinflammatory pathogenic functions, however, they too do not appear to migrate towards the outer retina during early EAU. Our laboratory has previously reported that there is some evidence of adaptive immunity playing a role in the induction of oxidative stress; the presence of a few CD3+ cells in the retina on day 5 postimmunization and real-time QPCR data showing an increase in CD28 transcripts in the retina, implicates the presence of activated T cells [18]. There is also a marked upregulation of inflammatory cytokines associated with the induction of oxidative stress, such as TNF-α, iNOS, IFNγ, and IL1α on day 5 postimmunization [18]. Such cytokines could be generated by the innate immune response in the retina; the number of activated T cell infiltration was minimal during early EAU. The presence of these cytokines coincides with the presence of mtDNA damage. TNF-α causes the upegulation of iNOS, which subsequently causes the production of nitric oxide and other oxidative factors that contribute to mitochondrial oxidative stress [27, 28]. The early upregulation of TNF-α, before the migration of retinal microglia and infiltration of macrophages, suggests that innate immunity could cause oxidative stress during early EAU.
The innate immune response may contribute to oxidative stress before the T cells have a chance to migrate into the retina. In a recent study, our laboratory used real-time PCR analysis to show that during early EAU, before leukocyte infiltration of the retina, there is increased expression of TNF-α and iNOS in nude mice compared with nonimmunized controls [4]. Since nude mice are deficient in T cells [29], the presence of these inflammatory cytokines confirms that innate immunity plays a role in the upregulation of cytokines.
Toll-like receptors (TLRs) are a group of transmembrane proteins that play an essential role in the innate immune response and in the upregulation of TNF-α [30, 31]. In vitro studies suggest that activation of TLR4 can cause mitochondrial oxidative stress in the central nervous system and in the liver [32]. Similar mitochondrial oxidative stress is seen in photoreceptors in early EAU [5]. Our recent study confirmed the importance of TLR4 in the generation of proinflammatory cytokines crucial to the induction of mitochondrial oxidative stress; using real-time PCR and Western blot analysis, we found that TNF-α and iNOS were markedly downregulated in TLR4-deficient mice when compared with wild-type mice with EAU [4]. TLR4 deficiency was shown to attenuate iNOS gene expression and expression of this cytokine is known to cause mitochondrial oxidative stress [18]. We confirmed our finding using immunohistochemistry, which revealed that in addition to TNF-α and iNOS, photoreceptor mitochondrial oxidative stress was abrogated in the absence of TLR4, as observed with TLR4 knockout mice with EAU [4].
Our laboratory also revealed that during early EAU, the presence of TLR4 is crucial for the initiation of DNA oxidative damage. As stated in a previous study, damaged or altered mtDNA is a reliable biomarker of oxidative stress in tissues rich in mitochondria like the photoreceptor inner segments of the retina [7]. 8-hydroxy-2′-deoxyguanosine (8-OHdG), an oxidized form of deoxyguanosine, is a reliable indicator of DNA oxidative damage; during oxidative stress, 8-OHdG levels increase preferentially in mitochondria as a result of the single-stranded nature of mtDNA, which makes it more susceptible to damage [33]. Our recent study showed that while there is substantial 8-OHdG staining and thus mitochondrial DNA damage in wild-type mice with EAU, this is substantially reduced in TLR4 knockout mice (Fig. 3) [4]. The intense 8-OHdG staining we found in the photoreceptor inner segments of WT mice with EAU reinforces previous findings from our laboratory which indicate that in EAU retinas, photoreceptor cells are the primary site of oxidative damage [12, 18]. Our recent studies also support previous reports that apoptosis is not a feature of early EAU; based on terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining, apoptotic cells were nor detected in WT or TLR4-deficient mice with EAU, suggesting the presence of a protective mechanism, such as αA crystallin upregulation, in the photoreceptors that prevents photoreceptor apoptosis in early EAU [4, 7, 25].
Although the presence of TLR4 is necessary for the upregulation of significant quantities of TNF-α, iNOS, and 8-OHdG, other TLRs may also play a minor role in the induction of these gene products in early EAU since mild upregulation of these cytokines and mild oxidative DNA damage still occurs when TLR4 is not present. However, based on our findings, TLR4 has a functional significance in EAU and further study of TLR4 and innate immunity will enhance our understanding of the EAU pathway.
Photoreceptor oxidative damage in sympathetic ophthalmia
A recent study from our laboratory reveals that like EAU, sympathetic ophthalmia (SO) also results in photoreceptor mitochondrial oxidative stress and damage. SO is a diffuse bilateral granulomatous uveitis that is a potential complication of penetrating ocular injury to one eye and has the potential to cause blindness to both eyes; in SO, leukocytic infiltration is seen in the uvea, but is absent in the choriocapillaries and retina [34, 35]. Using immunofluorescent techniques, we found increased levels of TNF-α in the photoreceptor layer, which suggests that TNF-α plays a role in the induction of iNOS in SO [36]. At the inner segments of the photoreceptors, iNOS was colocalized with cytochrome c, indicating its photoreceptor mitochondrial position. Thus, iNOS and nitrotyrosine were found in the mitochondria of the photoreceptors which clearly indicates the presence of photoreceptor mitochondrial oxidative stress in SO [11, 17, 18, 36]. The upregulation of both iNOS and nitrotyrosine occurred in the absence of CD3-positive T or CD45-positive leukocyes in SO retinas. Our study showed apoptosis of few photoreceptors, but the sections of the eye also revealed the absence of extensive apoptosis in the photoreceptor cell layer, despite the presence of diffuse photoreceptor mitochondrial oxidative stress (Fig. 4) [36]. This observation suggests that like EAU, SO also has a protective mechanisms in place, like crystallins, to prevent apoptotis.
Like EAU, the mechanism behind SO photoreceptor oxidative stress remains unclear; since SO and EAU appear so similar, perhaps the same approach should be taken in studying both diseases. In SO photoreceptor damage and eventual blindness occurs in the absence of T cells and leukocyte infiltration in the retina [34, 36], suggesting that perhaps the role of innate immunity in SO needs to be looked at more closely.