Mitochondria are the powerhouses of the cell. Most people have heard this basic fact so many times, they could recite it in their sleep. But beyond earning middle school students A⁺ grades on their science tests, this fundamental principle of cell biology could hold the key to a new therapy for a severe eye disease.

Fuchs endothelial corneal dystrophy is characterized by aberrant death of endothelial cells that make up the inner lining of the cornea. “There's a natural aging process of the corneal endothelium; we tend to lose those cells as we get older and older. In Fuchs dystrophy, basically, this process is accelerated,” said Stephan Ong Tone, a clinician-scientist specializing in cornea and external eye disease at the University of Toronto. “When the corneal endothelial cells die off, there’s a very limited regenerative response.” If the density of cells drops too low, the endothelium’s function of pumping fluid out of the cornea is compromised. As a result, the cornea swells, leading to vision decline and painful blistering on the surface of the eye. Corneal endothelial cell death is accompanied by the formation of guttae, an extracellular matrix component that contributes to visual impairment and appears to drive further cell death. 

If we can incorporate new, healthy mitochondria in the cells, maybe we can delay their death or revive them or make them functional for at least a period of time. 
- Patrick Rochette, Laval University 

The disease, which affects approximately four percent of the United States population, is triggered by a complex combination of genetic, epigenetic, and environmental factors and manifests in gradual deterioration of the corneal endothelium throughout a patient’s lifetime. Corneal swelling can be managed with a high salt solution treatment, but the gold standard for correcting the root cause of the disease is surgical transplantation of donor corneal tissue. While these procedures generally yield positive outcomes, they carry the risk of donor cornea shortages, surgical complications, and graft rejection.

Now, researchers at Laval University and the University of Montreal are exploring an alternative approach to treating Fuchs endothelial corneal dystrophy that harnesses the power of the cell’s powerhouse. They found that by internalizing healthy mitochondria, Fuchs corneal endothelial cells are rescued from a positive feedback loop of cell death. The team is adapting their proof-of-concept study to a clinically translatable therapy, aiming to provide an early intervention that could postpone or prevent the need for a cornea transplant. 

Breaking a vicious cycle 

A growing body of research has revealed a critical role for dysfunction in mitochondria, which produce the cell’s energy currency in the form of adenosine triphosphate (ATP), in cell death in Fuchs endothelial corneal dystrophy (1-3). Researchers at Laval University and the University of Montreal observed that Fuchs corneal endothelial cells show significant variability in their mitochondrial mass. “The cells are not all at the same stage of the pathology, and mitochondrial mass is a marker to look at what stage each cell is in in the progression of the pathology,” said Patrick Rochette, an ocular photobiologist at Laval University. 

To piece together the sequence of events as cells move from one stage to the next, the team used cells from a Fuchs endothelial corneal dystrophy explant removed during a corneal transplant (4). They grouped cells based on their mitochondrial content and evaluated various measures of cellular and mitochondrial function. They observed that cells initially bulk up their mitochondrial mass and strain to boost ATP production, which increases the generation of reactive oxygen species. Eventually, mitochondrial mass and all signs of mitochondrial activity plummet, and the cells undergo apoptosis.  

Their findings support the role of “mitochondrial burnout” as a driving force for Fuchs endothelial corneal dystrophy. As corneal endothelial cells die, mitochondria in the remaining cells must compensate in order to meet the high energy demand needed to pump fluid out of the cornea. But like a star student who is the powerhouse of a group project, these mitochondria can become exhausted from picking up the slack. Overworked and further damaged by oxidative stress from the reactive oxygen species, dysfunctional mitochondria are removed as waste through the process of mitophagy until the cell doesn’t have enough left to survive. “And then, the other [cells] have even more pressure, so it’s kind of a vicious cycle,” Rochette said. 

The researchers wondered if they could break this loop by restoring mitochondrial health. “If we can incorporate new, healthy mitochondria in the cells, maybe we can delay their death or revive them or make them functional for at least a period of time,” Rochette said. Scientists are currently investigating exogenous mitochondria delivery as a therapy for other disorders associated with mitochondrial dysfunction, including Parkinson’s disease and ischemia–reperfusion injury (5,6).

To explore this approach as a treatment for Fuchs endothelial corneal dystrophy, the team extracted healthy mitochondria from cultured immortalized human embryonic kidney cells and stained them with a fluorescent dye (7). They then incubated the mitochondria with explant tissue and observed that the cells engulfed the mitochondria, most likely through macropinocytosis, a nonspecific internalization process for large quantities of extracellular material. “The corneal endothelial cells eat all the mitochondria they can. You can see that the incorporation is impressive,” Rochette said. “Then, they make a clean up; they eliminate by mitophagy the bad mitochondria to just give the best ones. The cells really are brilliant.”

The researchers found that cells with either the lowest or highest amounts of native mitochondria incorporated the most exogenous organelle. While they aren’t yet sure of the reason for this trend, mitochondria uptake in healthy, mitochondria-rich cells could prevent these cells from progressing to subsequent stages of burnout, and internalization in stressed, mitochondria-poor cells could rescue these cells from death. Although apoptosis was long believed to be a one-way ticket to the cellular grave, recent studies have demonstrated that apoptosis is reversible even at late stages of the process under certain conditions, including sustained energy production by healthy mitochondria (8). And unlike cultured cells, which are all too prone to dying after a weekend without fresh media, cells in the body can take up to years to undergo apoptosis, providing an ample window of opportunity to abort the process. 

The team observed that explant tissue incubated with healthy mitochondria showed a higher mitochondrial membrane potential, the electrical charge gradient across the membrane that signals efficient ATP production, than an untreated portion. The incubated tissue also displayed lower levels of oxidative stress and mitophagy. “It tells us that the pool of mitochondria is in better shape than before incorporation,” Rochette said. 

The researchers found that the abundance of a protein signature of apoptosis decreased from approximately 60 percent in the untreated tissue to approximately 10 percent in the incubated explant, indicating that internalization of healthy mitochondria successfully saves cells that are in the process of dying. “Understanding the pathology is one thing, but using this understanding to reverse or delay the pathology is something really exciting,” Rochette said. 

This demonstration of the restorative power of exogenous mitochondria could pave the way for a novel intervention for Fuchs endothelial corneal dystrophy. “This, to me, seems to be quite an innovative approach, and, at least ex vivo in these specimens, seems to be quite promising,” Ong Tone said. 

From explants to eyes

The team is now trying to translate this strategy into a therapy that works in the real environment of the eye. They envision that mitochondria could be injected into the anterior chamber, the space between the iris and the corneal endothelium. When performing this procedure in rabbits, they observed that most of the mitochondria are internalized in the endothelial cells, but some are sidetracked into the iris and the trabecular meshwork (a cluster of tiny channels that drain fluid from the eye). “It's not surprising because if it's a nonspecific mechanism of how these mitochondria are uptaken by cells, it's probably not a [mechanism specific] to the corneal endothelium,” Ong Tone said. “I'm not sure what the sequela is of having more mitochondria in those cell types.” The team plans to conduct animal studies to identify any side effects from off-target mitochondria incorporation and monitor other safety parameters, such as intraocular pressure. 

Another important step is identifying a source of healthy mitochondria that is sustainable and unlikely to face major regulatory hurdles. The researchers are collaborating with blood supplier Héma-Québec to obtain mitochondria from a patient’s platelets, a major reserve of mitochondria in circulating blood. They have also developed a protocol to extract mitochondria from a blood sample in less than an hour, allowing the patient to have their blood drawn and mitochondria injected in the same visit. This approach “offers a lot of advantages from a therapeutic point of view by trying to take advantage of using some sort of autologous transplantation,” Ong Tone said. “The question becomes, ‘are all mitochondria created equal?’ Is a mitochondrion from a peripheral blood cell the same as a mitochondrion from a corneal endothelium? ...It's one of the things that they'll have to show in their preclinical studies.”

In vivo preclinical studies are also necessary to understand the long-term effects of healthy mitochondria internalization. The researchers acquired their measurements within a period of 48 hours due to limitations in explant culture but plan to investigate the duration of the benefits of the treatment to guide the dosing regimen. “The next question is, ‘how often would the therapy be required?’ Is it just one injection, and then it lasts for years? Or is it something that has to be repeated?” said Stéphanie Proulx, an ophthalmology and tissue engineering researcher at Laval University and collaborator on the project. 

Patients who are diagnosed with Fuchs endothelial corneal dystrophy early based on genetic testing or an eye exam that looks for the first signs of the guttae disease hallmark would make ideal candidates for the mitochondrial internalization therapy. “The idea would be to treat them early on before the disease is too spread out in order to see if it would progress more slowly and perhaps prevent the graft transplantation altogether,” Proulx said. “If we could slow it down, then perhaps the patient will be able to keep their vision for 100 years instead of 60 years.” 

It may also be more effective to intervene at an early stage of the disease because a dense mass of vision-impairing guttae has not yet formed, said Ula Jurkunas, an ophthalmologist and Fuchs endothelial corneal dystrophy researcher at Harvard University. The team at Laval University and the University of Montreal observed that the levels of markers of apoptosis and mitochondrial burnout correlate with guttae content, suggesting that internalization of healthy mitochondria might prevent or reduce additional guttae formation (9). Since corneal explants are collected from patients with end-stage Fuchs endothelial corneal dystrophy, the researchers will need to use other methods to demonstrate the potential of the treatment in early-stage disease, Jurkunas added.  

Toward this end, Proulx and her colleagues discovered that they could isolate a subset of proliferative endothelial cells from the basement membrane of the corneal endothelium of Fuchs endothelial corneal dystrophy patients. The researchers cultured the cells in vitro, allowing them to develop a 3D tissue model of the disease (10). “It tells another part of the story: the early events and what happens first,” Proulx said. The team could use this platform to investigate the effect of the mitochondria therapy on cell death and guttae formation at this point in the pathological process.

With further evaluation of safety and efficacy, Fuchs endothelial corneal dystrophy researchers are hopeful that internalization of healthy mitochondria could provide a minimally invasive treatment option for a poorly publicized but prominent disease. “Any sort of development in this condition has tremendous potential to impact patients with this disease and hopefully address a significant cause of vision loss worldwide,” Ong Tone said. 

References

1. Jurkunas, U.V., Bitar, M. S., Funaki, T. & Azizi, B. Evidence of oxidative stress in the pathogenesis of Fuchs endothelial corneal dystrophy. Am J Pathol  177, 2278–2289 (2010). https://ajp.amjpathol.org/article/S0002-9440(10)60281-7/fulltext
2. Czarny, P. et al. Mutagenesis of mitochondrial DNA in Fuchs endothelial corneal dystrophy. Mutat Res  760, 42–47 (2014). https://www.sciencedirect.com/science/article/abs/pii/S002751071300198X
3. Jurkunas, U.V. Fuchs endothelial corneal dystrophy through the prism of oxidative stress. Cornea  37, S50–S54 (2018). https://journals.lww.com/corneajrnl/abstract/2018/11001/fuchs_endothelial_corneal_dystrophy_through_the.4.aspx
4. Méthot, S.J., Proulx, S., Brunette, I. & Rochette, P.J. Chronology of cellular events related to mitochondrial burnout leading to cell death in Fuchs endothelial corneal dystrophy. Sci Rep 10, 5811 (2020). 
5. Chang, J.C. et al. Allogeneic/xenogeneic transplantation of peptide-labeled mitochondria in Parkinson’s disease: Restoration of mitochondria functions and attenuation of 6-hydroxydopamine-induced neurotoxicity. Transl Res  170, 40–56 (2016). 
6. Masuzawa, A. et al. Transplantation of autologously derived mitochondria protects the heart from ischemia-reperfusion injury. Am Physiol Heart Circ Physiol 304, 966–982 (2013). 
7. Méthot, S.J., Proulx, S., Brunette, I. & Rochette, P.J. Rescuing cellular function
   in Fuchs endothelial corneal dystrophy by healthy exogenous mitochondrial internalization. Sci Rep 13, 3380 (2023). 
8. Tang, H.M. & Tang, H. L. Anastasis: Recovery from the brink of cell death. R Soc Open Sci  5, 180442 (2018). https://royalsocietypublishing.org/doi/10.1098/rsos.180442
9. Méthot, S.J., Proulx, S., Brunette, I. & Rochette, P.J. The presence of guttae in Fuchs endothelial corneal dystrophy explants correlates with cellular markers of disease progression. IOVS  64, 13 (2023). 
10. Zaniolo, K. et al. Culture of human corneal endothelial cells isolated from corneas with Fuchs endothelial corneal dystrophy. Exp Eye Res  94, 22-31 (2012). https://www.sciencedirect.com/science/article/abs/pii/S0014483511003344?via%3Dihub