Reactive Oxygen Species in Mitochondrial Optic Neuropathies: Comment

Letters to the Editor Robert A. Egan, MD Oregon Neurology, Tualatin, Oregon The author reports no conflicts of interest. REFERENCES 1. International Stroke Trial Collaborative Group. The International Stroke Trial (IST): a randomised trial of aspirin, subcutaneous Reactive Oxygen Species in Mitochondrial Optic Neuropathies: Comment W e 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 (3,4) 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 Letters to the Editor: J Neuro-Ophthalmol 2015; 35: 444-446 heparin, both, or neither among 19 435 patients with acute ischaemic stroke. Lancet. 1997;349:1569-1581. 2. Sandercock PAG, Counsell C, Kamal AK. Anticoagulants for acute ischaemic stroke. Cochrane Database Syst Rev. 2008: CD000024. 3. Camerlingo M, Salvi P, Belloni G, Gamba T, Cesana BM, Mamoli A. Intravenous heparin started within the first 3 hours after onset of symptoms as a treatment for acute nonlacunar hemispheric cerebral infarctions. Stroke. 2005;36:2415- 2420. 4. The National Institute of Neurological Disorders and Stroke rtPA Stroke Study Group. Tissue plasminogen activator for acute ischemic stroke. N Engl J Med. 1995;333:1581-1587. 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 445 Copyright © North American Neuro-Ophthalmology Society. Unauthorized reproduction of this article is prohibited. Letters to the Editor 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. Alfredo A. Sadun, MD, PhD Rustum Karanjia, MD, PhD Department of Ophthalmology, David Geffen School of Medicine, Doheny Eye Centers, UCLA, Los Angeles, California Billy X. Pan, MD Fred N. Ross-Cisneros, BSc Doheny Eye Institute, Los Angeles, California Valerio Carelli, MD, PhD Department of Neurological Sciences, University of Bologna, Bologna, Italy The authors report no conflicts of interest. Reactive Oxygen Species in Mitochondrial Optic Neuropathies: Response I am grateful for the kind words of Dr. Sadun and colleagues agreeing with the suggestions made in my article with respect to Leber hereditary optic neuropathy (LHON). They make a seminal point with respect to the spread of injury in LHON, and the critical need to understand how it could occur. We are currently attempting to model this mathematically and try to explain why the injury in some optic nerve diseases spreads differently than in others. We look forward to further work in this area. With respect to the role of ATP deficiency vs superoxide signaling, the confusion probably arose because in their 2012 article (1), they explain the preferential involvement of the papillomacular bundle in LHON using the nerve fiber layer stress index, an energy-related measure. Specifically, they wrote: "The NFL-SI equation described by Sadun et al condenses down to the ratio of demand vs supply, . . .whereby the numerator reflects all the factors that require high-energy supply by the axon and the denominator, the source of that energy." It was 1 year later, in their excellent 2013 review (2) (not their reference 3) that they include the role of reactive 446 REFERENCES 1. Levin LA. Superoxide generation explains common features of optic neuropathies associated with cecocentral scotomas. J Neuroophthalmol. 2015;35:152-160. 2. Pan BX, Ross-Cisneros FN, Carelli V, Rue KS, Salomao SR, MoraesFilho MN, Moraes MN, Berezovsky A, Belfort R, Sadun AA. Mathematically modeling the involvement of axons in Leber's hereditary optic neuropathy. Invest Ophthalmol Vis Sci. 2012;53:7608-7617. 3. Sadun AA, La Morgia C, Carelli V. Mitochondrial optic neuropathies: additional facts and concepts-response. Clin Ophthalmol Exp. 2014;42:207-208. 4. Sadun AA, Win PH, Ross-Cisneros FN, Walker SO, Carelli V. Leber's hereditary optic neuropathy differentially affects smaller axons in the optic nerve. Trans Am Ophthalmol Soc. 2000;98:223-232; discussion 32-5. 5. Cortopassi G, Wang E. Modelling the effects of age-related mtDNA mutation accumulation; complex I deficiency, superoxide and cell death. Biochim Biophys Acta. 1995;1271:171-176. 6. Miller NR. Developing a human clinical trial from a scientific hypothesis. J Neuroophthalmol. 2015;35:160-161. 7. Lin CS, Sharpley MS, Fan W, Waymire KG, Sadun AA, Carelli V, Ross-Cisneros FN, Bacin P, Sung E, McManes MJ, Bx Pan, Gil DW, Macgregor GR, Wallace DC. Mouse mtDNA mutant model of Leber hereditary optic neuropathy. Proc Natl Acad Sci U S A. 2012;109:20065-20070. oxygen species signaling. I obviously agree with their more recent article, which matches our hypothesis first stated in 2007 (3). That said, the overwhelming impact of Dr. Sadun and colleagues' contributions in LHON far outweighs any small historical differences, and the most important issue is that we are converging on an exciting mechanism for LHON and possibly other optic neuropathies. Leonard A. Levin, MD, PhD McGill University, Montreal, Canada University of Wisconsin, Madison, WI The author reports no conflicts of interest. REFERENCES 1. Pan BX, Ross-Cisneros FN, Carelli V, Rue KS, Salomao SR, Moraes-Filho MN, Moraes MN, Berezovsky A, Belfort R, Sadun AA. Mathematically modeling the involvement of axons in Leber's hereditary optic neuropathy. Invest Ophthalmol Vis Sci. 2012;53:7608-7617. 2. Sadun AA, La Morgia C, Carelli V. Mitochondrial optic neuropathies: our travels from bench to bedside and back again. Clin Exp Ophthalmol. 2013;41:702-712. 3. Levin LA. Mechanisms of retinal ganglion specific-cell death in Leber hereditary optic neuropathy. Trans Am Ophthalmol Soc. 2007;105:379-391. Letters to the Editor: J Neuro-Ophthalmol 2015; 35: 444-446 Copyright © North American Neuro-Ophthalmology Society. Unauthorized reproduction of this article is prohibited.