Reactive Oxygen Species in Mitochondrial Optic Neuropathies (2015)

We read with great interest the recent article by Leonard Levin (1), who emphasized the role of superoxide generation in several metabolic optic neuropathies, all characterized by cecocentral scotomas. He suggests that electrons spilled from the electron transport chain (e.g., in Leber hereditary optic neuropathy [LHON]), leads, in the presence of oxygen, to superoxide formation and that this can specifically signal retinal ganglion cell (RGC) death. Citing a variety of experiments, Levin makes a compelling argument that superoxide is not a consequence of cell death but a causative factor. We agree. Dr. Levin correctly interprets our mathematical model (2), which uses a nerve fiber layer stress index to predict the order of fiber loss (small to large and hence papillomacular bundle first). But Levin suggested that we believe this to be due to problems of bioenergetics. So a clarification is in order. We did not intend to suggest that lack of adenosine triphosphate (ATP) directly leads to RGC death. On the contrary, in our other article pertaining to this mathematical model, we emphasized “…that the mitochondria could use cell signaling systems to help initiate and promote apoptosis.” and further that, “Reactive oxygen species (ROS) are not only the toxic by-products of oxygenation reactions but also a signaling system for mitochondria.” (3) In LHON, these electrons probably spill from Complex I to produce ROS. As Dr. Levin points out, superoxide is the predominant ROS. Levin cited our work (3,43,4) in which we show that the smallest fibers of the optic nerve, beginning in the papillomacular bundle, are affected first, and then, in more severe cases, this proceeds like a wave to involve larger and larger fibers. Indeed true. But what is fascinating and remains unexplained is that the histological measurements show the affected sectors of LHON optic nerves (corresponding to areas that in normals contain smaller than average caliber axons) “were completely devoid of axons, with losses not limited to just the smallest…” (2). In other words, some sort of signal or effect must spread from the small and susceptible axons to their larger neighbors. This suggests a mass action effect such that a cascading event includes adjacent fibers regardless of their size. This cannot be explained by just bioenergetics. That the RGC injury arises in the unmyelinated retinal nerve fiber layer is no surprise, given that repolarization only occurs where there is no myelin. But again, this does not necessarily mean that the problem is a lack of ATP, as a bioenergetic problem may lead to compensatory changes that produce an abundance of ROS in the context of dysfunctional mitochondria (3). It should also be noted that although ROS may mediate apoptosis, ROS are also important and useful as intracellular signals. For example, LHON cybrid studies have shown that mitochondrial biogenesis is likely signaled by high ROS levels (5). Furthermore, although superoxide is a prominent member of the ROS family, this chemical is ephemeral. It is very reactive and thus changes quickly in time and space. Superoxide, therefore, is hard to pin down and be reliably measured in any experimental system. In the same issue of Journal of Neuro-Ophthalmology, Neil Miller (6) provides us with his thoughts on what a human clinical trial would need to test these hypotheses. Dr. Miller would like to see a clinical trial with an agent that not only ameliorates the optic neuropathy of LHON but also works in B-12 deficiency and ethambutol optic neuropathy. All other considerations aside, such a trial would be a challenge insofar as the treatment of B-12 deficiency and ethambutol toxicity is to supplement the former and discontinue the latter. And if an agent is added to that management, seeing the small additional therapeutic effect will not be easy. Rather, I would like to see any purported agents tried in a faithful animal model. Fortunately, this model now exists (7). Wallace's team introduced the human optic atrophy mtDNA ND6 P25L mutation into the mouse thus creating a genetically identical animal model of LHON. These mice went blind as demonstrated by a number of tests including electroretinograms and their small caliber axons were selectively lost in the optic nerves. Perhaps most importantly, in vitro analysis of the mice brains demonstrated “…decreased Complex I activity and increased ROS but no diminution of ATP production. Thus, LHON pathophysiology may result from oxidative stress.” (7) It seems that in this, we all agree. And we are pleased that Dr. Levin stresses ROS-induced apoptosis over bioenergetics. But let us remember that ROS are necessary, and most likely, it is only when the ROS are out of balance within the signaling systems and cross a critical threshold, that they induce apoptosis of the RGC.