It is a great honor and pleasure and very humbling to deliver this lecture. I stand in awe of William Hoyt and the august list of past awardees. Most of my training in neuro-ophthalmology was with Simons Lessell and my debt to him can't be overstated. One of the many kindnesses he did for me was to send me to San Francisco to spend the last 6 weeks of my fellowship with Dr. Hoyt (see Supplemental Digital Content, Figure E1, http://links.lww.com/WNO/A78).
I am also humbled by the prospect of discussing the topic I may have foolishly chosen for myself. Are we there yet? Is neuro-ophthalmology at the cusp of a paradigm shift? This comment is in reference to the classical work of Thomas Kuhn. In 1962, Kuhn, a physicist, a philosopher, and a professor at Massachusetts Institute of Technology published “The Structure of Scientific Revolutions” (1). In this book, he described what became popularly known as the “paradigm shift.” Kuhn pointed out that scientists accumulate evidence that supports their model of the real world. However, such evidence eventually becomes messy and even self-contradictory until at such time as the revolution, or paradigm shift, re-establishes a fundamentally different model. Kuhn described 3 stages for each science as: the central paradigm, the anomalous elements, and the reconfiguration of the new paradigm.
What has all this got to do with neuro-ophthalmology? Well, the central paradigm for neuro-ophthalmology was that a single physical lesion produced a classical set of signs and symptoms. However, with the arrival of neuroimaging, it appeared that computed tomography and magnetic resonance imaging might do the job better than a neuro-ophthalmologist. In fact, when I was with Dr. Hoyt, I recall him bemoaning the possible end of neuro-ophthalmology with the success of neuroimaging. However, there are diseases that do not conform to the localizing lesion paradigm and are not detectable with neuroimaging techniques.
MITOCHONDRIAL OPTIC NEUROPATHIES
The metabolic injury that characterizes mitochondrial optic neuropathies (MON) might be characterized as a biochemical paradigm. MON can be genetic, as in Leber hereditary optic neuropathy (LHON) or dominant optic atrophy, secondary to nutritional deficiencies, such as with vitamin B12 or folic acid, or due to a variety of toxic agents, many of which are antibiotics that interfere with both bacterial and mitochondrial ribosomes (2,3).
The prototypic MON, LHON, was first described in 1858 by Von Graefe. In 1871, Leber reported 4 families and proposed that this was a hereditary and probably X-linked disease (4–7). However, it wasn't until 1988 when Wallace et al (8) described the 11778 mitochondrial DNA (mtDNA) mutation that we began to get a handle on both the genetics and the pathophysiology of this MON.
Neuro-ophthalmologists are well aware of the typical presentation of LHON. But you might find it interesting to know that what we often see in the United States is a little different in Brazil. In the United States, I might see a young man in good health without a family history of blindness. He goes off to college and adopts a lifestyle, which includes smoking and heavy drinking. A few months later he suffers subacute loss of vision in one eye and is seen at the student health center and diagnosed with optic neuritis. The work-up for multiple sclerosis is entirely negative and within a month or two he loses vision in the fellow eye. It's about then that he gets referred to neuro-ophthalmology and the diagnosis becomes clear. I’ll tell you later how this plays out a little bit differently in Brazil.
FOUR FUNDAMENTAL QUESTIONS
There are many mysteries in LHON. Let me point out 4:
1. Why are some individuals with the mutation affected and others not? (The problem of penetrance.)
2. Why are men much more likely to lose vision than women? (The problem of gender bias.)
3. Why is it almost always just the optic nerve that succumbs? (The problem of tissue specificity.)
4. Why does the catastrophic loss of vision occur during young adulthood? (The problem of mass tipping point.)
Mitochondria are remarkable organelles. We tend to describe them as individual little beans that float freely in the cytoplasm but of course that isn't really accurate. Mitochondria usually fuse into a syncytium, which follows the cytoskeleton and then sometimes they undergo fission, perhaps for special functions such as transport. The mitochondria coordinate with the nucleus in a variety of functions, including their own reproduction, which occurs in the soma of the retinal ganglion cell (RGC) in proximity to the nucleus. They may then hitch a ride on kinesin and dynein motors, to travel down the axons and provide adenosine triphosphate (ATP) to distal elements. In producing ATP, mitochondria utilize oxidative phosphorylation and, as a consequence, produce great amounts of reactive oxygen species (ROS) (9,10).
LHON PEDIGREE IN BRAZIL
I was extremely fortunate to lead a team of international investigators, numbering up to 40 at a time, in yearly field investigations to rural Brazil. We utilized an ophthalmology clinic in Colatina, Brazil, and received support from many volunteers organized through the Federal University of Sao Paulo (11). The first such visit in 2001 allowed us to define a large pedigree of 11778 LHON. In subsequent years, we worked out the psychophysics, came to understand that there was subclinical disease in carriers of LHON, monitored the natural history of conversions, and established a number of psychophysical, clinical, subclinical, and serum biomarkers (12–18). We characterized a pedigree with 362 members from 8 generations that all began in 1861 in Verona, Italy (Fig. 1). From there, a 16-year-old girl immigrated to Brazil to become the founder of this family. Almost all her progeny have remained in the same area in rural Brazil (11).
All of this came to our attention through a series of e-mails that originated from the mother of a 14-year-old boy who had just lost vision in one eye. This mother recalled that 30 years before, her 2 older brothers had experienced similar profound visual loss. By way of the Internet, she resolved to better understand her family's disease and then to contact experts in the field whom she hoped could provide a means of keeping her child from going blind in the second eye. Through the International Foundation for Optic Nerve Diseases, she found me and, despite the complications of language and culture, we concluded that the diagnosis that she proposed, LHON, was correct. She told of a 100 family members who might be carriers of LHON with several having already lost vision, and she was anxious that we evaluate her son. We resolved to assemble a large team to conduct a field investigation in Brazil. We arranged for her, with her son and 2 brothers, to be evaluated at the Federal University in Sao Paulo. Measurements of visual acuity, visual fields, and fundus photographs were sent to us along with blood for genetic testing. Unfortunately, by the time that they arrived in Sao Paulo, her 14-year-old son had already lost vision in the second eye. He had visual acuity of 20/200, right eye, and 20/800, left eye, and his fundus appearance is shown in Figure 2.
The logistics were challenging to bring expertise and sophisticated equipment from around the world into a remote area of Brazil. We managed these for 11 consecutive years and made a number of interesting findings. For example, although we found that the average age of onset for the entire family was 29 years, subgroup analysis showed this age to be 23 years for men and 33 years for women. Women had a bimodal distribution, with some losing vision at about the same age as the men but others losing vision as they approached menopause. The visual acuity of those who became affected and lost vision averaged 5/400. Remarkably, the penetrance rate was much higher if the mother herself had lost vision. In other words, if a mother lost vision, it was almost certain that her sons would be affected and her daughters were at high risk. But if a mother was an unaffected carrier, her daughters were very unlikely to lose vision and even her sons had good odds of preserving their sight. Similar to reports from other pedigrees (19–24), we found a male to female ratio of about 4:1 in this 11778 family. It was intriguing that the most common month for vision loss in Brazil was April. We will later revisit this seasonal phenomenon in the light of the likely 6-week lag period between the triggering insult and the loss of vision in LHON. We found that the likelihood of conversion from carrier status to affected was much higher in those exposed to pesticides, cigarette smoking, and alcohol (11,12). Sixty-five percent of our affected LHON patients smoked compared with 26.1% of the off-pedigree controls and 13.5% of our LHON carriers. With regards to the consumption of at least 2 drinks of alcohol per day, LHON affected (60%) were much more likely to consume compared with controls (38.2%) or carriers (33.8%). Thus, there was a statistically significant increased rate of smoking and alcohol use in LHON affected compared with either carriers or controls.
After examining close to 300 members of the LHON pedigree every year for 11 years, what did we learn? Our conclusions can be summarized as follows: Although there is an apoplectic loss of vision that occurs over a period of weeks to months, there is also clinical evidence of further progression that continues long term, year after year. This progression is hard to monitor because visual acuity does not continue to change but there is progressive visual field loss well beyond the central 30° angle. One lesson here is that you should keep seeing these patients, keep getting visual fields, and try to use visual field strategies that extend beyond 30° angle. This late progression is probably life-long. We obtained histopathological tissue from a 76-year-old patient who had lost her vision about 5 decades earlier. Her vision had been estimated as counting fingers for decades before she died of cardiovascular complications. In examination of her optic nerves, there was still active degeneration of some remaining fibers found in the superonasal quadrant, shown by light and electron microscopy (EM) (Fig. 3). The appearance on EM reflects degeneration that occurred in the last 4–6 days of her life or 50 years after she first noted visual loss!
What about the asymptomatic carriers? These patients may be asymptomatic but they often harbor subclinical disease. We found zones of mild sectoral disc edema, sometimes with telangiectatic vessels, in more than one-third of the carrier cases (Fig. 4) (15). Using more sophisticated psychophysical techniques (Cambridge color system), we were able to demonstrate some dyschromatopsia in about half of the carriers (16). This dyschromatopsia may be too subtle to show up on standard Ishihara color test plates. Finally, optical coherence tomography (OCT) turned out to be an excellent means of discerning structural changes in asymptomatic carriers. Several female carriers and almost 90% of male carriers demonstrated retinal nerve fiber layer (RNFL) thickening in the inferotemporal quadrant (25–27).
In this extensive longitudinal study, OCT was the most reliable method to demonstrate structural changes, which preceded and then reached a crescendo at the time of visual loss. This began with swelling of the peripapillary RNFL in the inferotemporal quadrant and was followed by swelling superiorly and temporally. Eventually, each quadrant took turns in first swelling and then undergoing atrophy. Barboni et al (26,27) demonstrated this RNFL thickening in carriers and the orderly wave of thickening followed by atrophy that occurs during conversion to affected status. However, not all LHON carriers with OCT changes or pseudo-disc edema lost vision. Some waxed and waned for many years and some even improved without any significant visual loss.
REACTIVE OXYGEN SPECIES
Many LHON patients in Brazil experienced vision loss each year in April. In our discussions, it became evident that the visual loss was preceded, in February, by attendance at Carnival, a week-long festivity in Brazil characterized by a great deal of binge drinking. In most cases, the carrier was a teenager who, for the first time, was allowed to accompany a family member to Carnival where they both participated in heavy consumption of alcohol.
The Brazil longitudinal studies take into consideration a number of epidemiological findings that suggest both genetic and environmental risk factors for conversion. Gene linkage analysis points to a “hot spot” on the X chromosome, which may harbor a nuclear modifying factor. There are many genes in this area. One candidate gene is for manganese superoxide dismutase (MnSOD) or SOD2, a specific form of superoxide dismutase used by mitochondria to help neutralize ROS. Of course, ROS may also come from the environment introduced by activities such as heavy drinking and exposure to smoke. We reported several members of the same LHON family who lost vision while being exposed to smoke from a tire fire (28).
This vulnerability to ROS has also been modeled in cybrid experiments. Cybrids are cell cultures that take advantage of an immortalized cell line, such as osteo-sarcoma, in which the original mitochondria have been destroyed and replaced with mitochondria obtained from the white blood cells taken from LHON patients. These cultures can be grown indefinitely and harbor only the mitochondria of interest. With these experiments, it has been shown that the 3 primary LHON mutations only produce a modest (approximately 20%) decrease in oxygen consumption and ATP production but proportionately a much greater increase in ROS production (29–32).
To look further at the role of ROS, we looked for the presence of nitrotyrosine in optic nerves from LHON patients. Nitrotyrosine is a product of accumulated ROS damage. As demonstrated in our optic nerve sections, nitrotyrosine immunostaining is minimal in control optic nerves and extreme in nerves from patients with LHON (Fig. 5). This suggests a life-long exposure to high levels of ROS in LHON.
ROS overproduction makes sense regarding the pathophysiology that leads to RGC death in LHON, for we know that the mtDNA mutation leads to insufficiency at complex I. This produces a small decrease in ATP but a very large increase in ROS (29,32). Smoke or drinking of alcohol can add significant amounts of ROS, which may then exceed a certain threshold, causing apoptotic cell death (33,34). Furthermore, RGCs may be at particular risk because of their long unmyelinated intraretinal segment.
Mitochondria are likely the lynch pin of this pathological process that ends with RGC apoptosis. To gain greater insight into the nature of mitochondria, let us consider their origin. The well-known theory of Margulis brings us back billions of years ago to when the world was populated by aerobic bacteria, anaerobic eukaryotes, and blue green algae. The blue green algae slowly brought the oxygen levels in the atmosphere from as low as 2% to about 22% (35). This was catastrophic for the anaerobic eukaryotes, which could not easily survive these high oxygen levels. Fortuitously, as Margulis et al (36) suggested, a small miracle of life occurred, which she termed “endosymbiosis.” That is to say, a eukaryote consumed but did not destroy a bacterium that it had ingested, and this surviving bacterium went on to become a proto mitochondrion. This unified cell prospered with the mitochondria providing relief from the oxygen and an efficient source of ATP for the new amalgam eukaryote. Mitochondria represent vestige bacteria within the modern eukaryote. The eukaryote, with many sophisticated subsystems and organelles, brought a lot to the table as well. Specifically, the modern eukaryote provided a safe haven in the form of a nucleus that was sheltered away from the high ROS levels naturally generated by mitochondria. With this cooperative system, some eukaryotes eventually became the multicellular organisms, including humans, that now populate the earth.
This leads us to ask a few more questions about LHON. Why does blocking oxidative phosphorylation in mitochondria lead to apoptosis? Why the tissue specificity (optic nerve) and why the predilection for the fibers of the papillomacular bundle (PMB)? Once again we have to think of the Margulis paradigm and understand that for a billion years preceding this symbiosis, bacteria were constantly destroying and being destroyed by eukaryotes (35). Consequently, each type of cell developed an arsenal of biochemical weapons as part of an arms race with the other. After the symbiosis, the DNA library for this arsenal sat as an unused time bomb. But this time bomb could be set off by unleashing the machinery of the protomitochondria. Generally, there is not much selection advantage for a single cell to commit suicide. However, once multicellularity occurred, this system became an extremely useful tool as part of what we now term apoptosis; it permitted a variety of key processes such as ontogeny, which allowed for the development of organs and appendages, and immunosurveillance for the consequent destruction of cancer cells. Developmental neuronal specificity can occur by the selective pruning of neurons based on their connections, which, if appropriate, provide chemical signals that modulate apoptosis. RGCs use this method as they go from 5 million to about 1 million during fetal development (37).
How does the mitochondrion control one aspect of apoptosis? The inner membrane of the mitochondrion has an electrical potential. This electrical potential is altered by a variety of signals. ROS lower the electrical potential of the membrane, which, at a key voltage gate, can lead to opening of the mitochondrial permeability transition pore (MPTP) (33,34). Once the MPTP opens, it releases cytochrome c, which in turn permits pro-caspase 9 to become caspase 9, and the entire cascade of apoptosis can be triggered (Fig. 6).
WHAT MAKES THE RGCs VULNERABLE?
The brain weighs approximately 2% of the body weight and yet consumes 20% of its oxygen (38). This reflects the extremely high energy needs required to maintain neurons and, specifically, axons, which bear the high-energy costs of membrane repolarization following every action potential. This is minimized by saltatory conduction made possible by myelination, which not only allows for faster conduction velocity but also a greater economy of energy expenditure and hence less strain on the greatest source of ATP—the mitochondria. However, this is problematic for RGCs whose axons, that must be transparent, run an unmyelinated course in the retina. We know from the usual clinical presentation of MON (poor visual acuity, dyschromatopsia, and central scotomas) that the PMB is selectively and initially affected (7). In LHON, not only is there the tissue specificity of the optic nerve but more selective still, early involvement of the PMB. We hypothesized that this is due to the small size of the fibers that constitute the PMB. We calculated that the energy required for repolarization of an axon potential should be the number of nodes (of Ranvier) times the average length of each node times the axonal circumference at the node (Fig. 7). This represents the numerator of an equation reflecting the mitochondrial axonal stress, where the denominator would be the number of mitochondria that can provide these quantities of ATP. This latter number is limited by the volume of the axon cylinder, which is calculated as the length times πr2, where r is the radius of the axon (small in the PMB). Further reduction of this equation leads to an index that goes as 2/r. The question then is, can we predict the areas of optic nerve involvement based solely on the distribution of axon fiber size?
To test this hypothesis, approximately 100,000 axons from normal human optic nerves were measured in 32 regions. The fibers in each region and then the regions were ranked according to size (producing a series of spectra). This was painstaking work by Pan et al (39) and included extensive computations. The histograms of the nerve fiber spectra showed a narrow range of small fibers coming from the PMB that were found in the inferotemporal regions (Fig. 8A) and a broader range of larger fibers coming from the superonasal regions (Fig. 8B). Seen another way, a gray scale representation of a normal human optic nerve shows that the smallest fibers are inferotemporal and central, and larger fibers are peripheral, and especially superonasal; this matches exactly the distribution as seen in the patterns of degeneration in LHON optic nerves (Fig. 7). In Figure 9, we have displayed a schematic superimposing on an optic nerve from a LHON patient with only mild pathology, the rank order distribution of fibers by size. The smallest fibers are found in the areas numbered 1 and the largest in the areas numbered 5. These numbers 1–5 also represent the order of progression of LHON degeneration and as predicted by the mitochondrial stress index equation.
So, are we there yet? Well, research in LHON and other MONs has moved in several important fronts leading to the consideration of new treatment options such as quinones that might potentially redirect the electron that spills off complex I back into complex III and reduce some ROS production. Recent publications have described the use of a second-generation quinone, idebenone, with some success (40–43). A third-generation quinone, EPI-743, has shown that, at least in some cases, there may be significant amelioration of vision and visual field loss in cases of LHON (44,45).
But are we there yet? I would have to say not quite, insofar as we lack testing on an appropriate animal model. But we have good reason to think that we are close. We and other groups are conducting such research on recently developed faithful animal models of LHON (46,47).
To paraphrase Enrico Fermi, who was describing the great advances in physics in the 20th century, “theory stimulates experimentation which in turn stimulates new theory.” And so yesterday's discovery becomes today's tool. Although neuro-ophthalmology is not physics, and our paradigm shift might not be as dramatic, we do well in avoiding polemics. We will watch with delight as the next generation of investigators take what we've done to a whole new level.
Much of this work has been in close collaborations with the author's colleagues. The author thanks Fred Ross-Cisneros, lab supervisor, who has patiently pushed himself to the limit in generating critical data and the perfect photomicrographs. The author thanks Michelle Wang for meeting every deadline and Billy Pan who spent countless hours measuring hundreds of thousands of axons. The author particularly thanks scientific siblings, Valerio Carelli and Chiara La Morgia, and the core group of field investigators of the Brazil-LHON team, Solange Salomao, Adriana Berezovsky, Rubens Belfort, Milton Moraes (father and son), Dora Ventura, Peter Quiros, Piero Barboni, Filipe Chicani, Federico Sadun, and Anna Maria DeNegri. The author also thanks the others who participated in the field investigation team. Finally, the author thanks the hundreds of members of the Brazilian family who are descendants of that intrepid immigrant, Maria Franchi.
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