Clearing Stress

Stress is one of the major players in modern health problems. Those interested in the molecular mechanisms of the retina also know that oxidative stress is a major contributor to photoreceptor cell death in most retinal degenerative diseases. Of course, therapeutics to combat retinal damage are important to saving vision and the ideal targets are degenerative diseases such as retinitis pigmentosa (RP). Genes that protect against oxidative stress are useful targets for gene therapies – potentially allowing the activation of multiple antioxidative pathways in one fell swoop. This is the angle we are taking at the University of Massachusetts Medical School to treat RP by targeting the oxidation resistance 1 (OXR1) gene (1).

We found that elevating OXR1 protein levels increases cellular resistance to oxidative stress regardless of intracellular OXR1 expression levels. More importantly, we showed via electroretinography that subretinal OXR1 injections (with the human gene packaged in a viral vector for delivery) can delay retinal degeneration and photoreceptor loss twice as long as those injected with saline. Histological examination of the retina indicated that rod and cone photoreceptors were still present in transduced regions, but essentially absent elsewhere. The mouse model we used (rd1) has a very aggressive form of retinal degeneration, which suggests that this treatment will be even more effective in other, slower-degenerating mouse models. If slower degeneration correlates with lower levels of oxidative stress in the retina, then it is possible that our therapy will be more effective – with the same positive benefit of OXR1 elevation and a lower level of oxidative stress. We are currently in the process of investigating this further.

Strong as an OXR1
 

Why examine OXR1? Two studies of retinal degeneration showed that OXR1 levels declined shortly before the onset of degeneration (2,3), raising the possibility that the decline was related to the onset of degeneration. We decided that, if this hypothesis was correct, increasing OXR1 levels should prevent or delay neurodegeneration. The other feature that makes OXR1 an interesting candidate for gene therapy is that, even in cells that are not impaired in OXR1 expression, increasing OXR1 levels appears to be beneficial.

In our initial experiments, we tested the consequences of overexpressing OXR1 in a photoreceptor-like cell line that is not impaired in OXR1 expression. We made stable cell lines that incorporated an extra copy of OXR1 expressed from a strong promoter. These cells contain about seven times the normal amount of OXR1. This increased their resistance to peroxide-induced oxidative stress resistance such that it took twice the concentration of peroxide to kill 50 percent of the cells as needed to kill 50 percent of vector control cells. These cells also had lower levels of intracellular peroxide and reactive oxygen species than wild type and contained less cleaved caspase at all concentrations of peroxide, indicating lower levels of apoptosis. Combining these results shows that elevating OXR1 protein levels can increase oxidative stress resistance not only when OXR1 expression is impaired, but also when its expression is normal. This suggested that increasing OXR1 levels can be beneficial to cells regardless of their endogenous OXR1 status.

Widespread effects
 

Oxidative stress also contributes to neuronal cell death in many neurodegenerative diseases (4), and the retinal degeneration studies we performed are essentially a test for the general applicability of OXR1 to neurodegenerative diseases. Interestingly, in the last two years, research has revealed that OXR1 levels also decline shortly before neurodegeneration in several neurological diseases (5, 6). Human patients lacking OXR1 expression have also been identified; they are severely developmentally delayed, have severe neurological defects and, like the OXR1 deletion mutant mouse, develop ataxia.

Similar approach
 

OXR1 has a similar role to Nrf2, a transcription factor that binds to the antioxidant response element for an antioxidative effect. We have not compared our targeting directly to the Nrf2 gene (NFE2L2), but we are in the process of doing so. There are several features that differentiate OXR1 from Nrf2. Antioxidant genes controlled by Nrf2, including GPX2 and the heme oxygenase genes, also require OXR1 for expression. This suggests some regulatory overlap between these two genes. Because OXR1 functions upstream of multiple transcription factors, several of which are involved in oxidative stress resistance, Nrf2 may be another OXR1-controlled transcription factor. This idea is supported by the observation of Rolland and coworkers that OXR1 binds to Keap1, thereby regulating Nrf2 function (7). The relationship was later shown to be more complicated, with a number of direct and indirect interactions between OXR1 and Keap1 appearing to regulate the gene induction pathway of Nrf2 (8). OXR1 also controls other pathways that contribute to oxidative stress resistance. For example, OXR1 has been shown to control the expression of genes involved in DNA repair, caspase expression, caspase activation pathways, peroxide detoxification, and cell cycle control in response to oxidative stress (9). The recent results showing that OXR1 levels decline shortly before onset of degeneration in multiple examples of neurodegenerative diseases and that mice lacking OXR1 undergo rapid oxidative stress-mediated neurodegeneration, suggests that OXR1 expression levels and neurodegeneration may be closely linked.

Next steps
 

The immediate next steps are to determine whether the reduction in degeneration seen in the rd1 mouse model of neurodegeneration is also seen in other mouse models of retinal degenerative diseases and whether more slowly progressing retinal degeneration models exhibit better outcomes. The long-range goals are to see whether OXR1 gene therapy can be applied to other neurodegenerative diseases.