In some patients, at advanced stages of age-related macular degeneration (AMD), regions of the retina waste away, resulting in a “geography of atrophy” that creates a gradually expanding blind spot in the visual field.
In a new study, scientists at the University of Virginia, School of Medicine show complementary DNA reverse transcribed from noncoding RNA called Alu retrotransposons—repetitive DNA elements that comprise over 10% of the human genome—pile up in the back of the eye under the retina, in the retinal pigmented epithelium (RPE). These Alu cDNAs trigger the activation of the inflammasome—a multiprotein complex of the innate immune system in the cell cytosol that triggers inflammation—causing the untreatable pathological condition called Geographic Atrophy.
In an earlier study, the researchers had shown Alu RNA induces inflammasome activation leading to cell death in the RPE. In a more recent study they showed Alu RNA is reverse transcribed in the cytosol. In the current study reported in Science Advances article, “Alu complementary DNA is enriched in atrophic macular degeneration and triggers retinal pigmented epithelium toxicity via cytosolic innate immunity,” the team shows Alu cDNA also activates the inflammasome to induce cell death in Geographic Atrophy.
The findings establish a new molecular pathway in the pathophysiology of Geographic Atrophy and provide a rationale for clinical testing of inflammasome-inhibiting drugs for this untreatable form of blindness.
Jayakrishna Ambati, MD, Professor of Ophthalmology at the University of Virginia and senior author on the study says, “Dozens of clinical trials for Geographic Atrophy have not succeeded. Nearly all these trials have targeted the complement pathway. However, Geographic Atrophy is a multifactorial disease where an excess of multiple toxic substances including Alu, amyloid-beta, complement, and iron are present. We believe a different approach—one that targets a final common pathway [inflammasome activation]—is more likely to be successful in Geographic Atrophy.”
Atrophic mechanisms
In healthy young individuals, an enzyme called DICER1 keeps the levels of Alu repeats in check, acting as a protective shield for the RPE. Unfortunately, DICER1 levels decline with age.
“In Geographic Atrophy, reduced levels of DICER1 lead to accumulation of Alu RNA. We show Alu RNA accumulation leads to Alu cDNA formation via L1 reverse transcriptase activity,” says Ambati.
In fact, Alu cDNA is more toxic to the RPE than Alu RNA. “This might reflect stoichiometric inefficiencies in reverse transcription of Alu RNA. In addition, it is conceivable that some fraction of the pool of Alu RNA does not encounter reverse transcriptase,” explains Ambati.
As part of the mechanism uncovered in the study, the researchers show Alu cDNA engages a DNA sensing enzyme called cGAS to trigger the escape of mitochondrial DNA into the cell’s cytosol. This, in turn, amplifies cGAS in a feedback loop and triggers the inflammasome, resulting in the degeneration of the RPE.
The mechanism that triggers the initial RPE lesion and the mechanism by which the atrophy of RPE cells spreads centrifugally, taking along with it the overlying retina and resulting in a visible “geography of atrophy” has remained a longstanding mystery.
Maria Grant, MD, from the Department of Ophthalmology and Visual Science at the University of Alabama at Birmingham, who is not involved in the current study, says “By meticulously studying human eye specimens and identifying endogenous Alu cDNA as enriched in the active leading edge of the growing lesion, this stunning work provides a potential answer to this question.”
“Although we don’t know for certain, we believe that the initial lesion is a combination of the systems failure that accompanies aging coupled with accumulation of various toxic molecules,” says Ambati. “The etiology of centrifugal spread is well studied but poorly understood. Our finding that toxic Alu cDNA is enriched at the leading edge of the growing lesion in human Geographic Atrophy eyes, provides a strong clue that this toxic substance is involved in the centrifugal expansion of the degeneration.”
Andrew Dick, FMedSci, professor of Ophthalmology, University of Bristol, U.K., who is not involved in the current study says, “The terminal activation of the inflammasome has always been an enigma as to whether this is pivotal to RPE degeneration in AMD or Geographic Atrophy. This work goes toward supporting this, at least experimentally, by demonstrating mechanisms of the role of Alu RNA and inflammasome activation. The implication for AMD is significant where demonstrating in human and rodent the enrichment of Alu cDNA and inflammasome activation via cGAS opens therapeutic avenues.”
Carl Schmid, PhD, professor emeritus, Department of Molecular and Cellular Biology, College of Biological Sciences at UC Davis, who also is not involved in the study says, “Remembering that Alu and L1 repeats are widely regarded as junk DNA, this deleterious activity might result from an otherwise normal function gone awry or from the simple risk that such retrotransposons pose. The authors show endogenous L1 RNA and Alu cDNA accumulate within the RPE of human Geographic Atrophy eyes, thereby, directly implicating them in macular degeneration and they further demonstrate a specific pathway by which Alu cDNA promotes RPE degeneration. Of special significance, their novel findings in molecular biology advance testable hypotheses for treating this eye disease.”
As part of the study, the authors develop a new method they call “equator blotting” to detect and size extrachromosomal Alu cDNA. “We coined this term because it has aspects of both northern and Southern blotting procedures, which are used to study RNA and genomic DNA, respectively. Equator blotting is like a Southern blot in that it probes for a target DNA sequence. However, it does not involve restriction digestion of the DNA because we want to determine the size of the DNA species, which is important in establishing that Alu cDNA is non-genomic and non-embedded. Rather, the DNA is separated without undergoing enzymatic digestion prior to hybridization, like northern blotting,” says Ambati.
Tracking Treatments
Toward developing a treatment for this intractable pathology, the researchers demonstrate that FDA-approved nucleoside reverse transcriptase inhibitors (NRTIs) and their alkylated derivatives (Kamuvudines) inhibit Alu cDNA-induced activation of the inflammasome, effectively blocking retinal degeneration.
Toxic NRTIs inhibit both the reverse transcription of Alu RNA into cytoplasmic Alu cDNA and inflammasome activation while the safer Kamuvudines, inhibit inflammasome activation but not the reverse transcription of Alu RNA.
Grant says, “This work shows that small-molecule inflammasome inhibitors called Kamuvudines blocked the toxic effects of Alu cDNA. It would be interesting to test these drugs in clinical trials for Geographic Atrophy because, at present, there is no treatment for this devastating disease.”
Ambati and his team chose to target the inflammasome rather than upstream points of the pathway resulting in Geographic Atrophy, such as the transport of Alu cDNA to cGAS or cGAS mediated release of mitochondrial DNA.
Justifying the rationale behind this approach, Ambati says, “Alu cDNA causes RPE degeneration via the DNA sensor cGAS. However, there are other toxic substances such as amyloid-beta, complement, and iron, which also play a role in Geographic Atrophy. Therefore, we believe that Kamuvudines which block inflammasome activation induced by these various toxic substances are a rational therapeutic approach to block this multipronged assault.”
“The enticement of data showing NRTIs or derivatives block Alu cDNA induced RPE death and terminal inflammasome activation, questions when during AMD or Geographic Atrophy do you start considering repurposing NRTI use if targeting this pathway. Given the slow progression of Geographic Atrophy, what protection is operative to prevent a more evident, acute, or subacute RPE loss during geographic atrophic spread remains unanswered. Their data nevertheless is compelling,” says Dick.
Ambati agrees that the progression of Geographic Atrophy is indeed slow, starting in the mid-periphery of the retina and radiating outward to engulf the center of the retina over a 2-to-2.5-year period. “We know from our earlier paper that inflammasome activation is evident in human eyes at this stage of Geographic Atrophy. Therefore, the planned clinical trial will enroll patients who have early stages of Geographic Atrophy, not involving the center of the retina. The goal of Kamuvudine treatment is to stop or reduce the rate of progression of the disease. This has been the typical strategy of most clinical trials to date with other drug candidates.”
James Rosenbaum, MD, professor of ophthalmology, Oregon Health & Science University who is not involved in the study says, “Limitations of this work include the reliance on animal and in vitro models and the extrapolation from Geographic Atrophy, a very severe form of macular degeneration, to macular degeneration in general. The Ambati lab has also previously implicated a form of amyloid as a cause of a similar result. Despite these limitations, a novel approach to dry macular degeneration would be a major clinical advance so this is a welcome addition to the understanding of this potentially blinding disease.”
Looking ahead
Inflammasome Therapeutics, a private company in Newton, Massachusetts founded by Ambati and Paul Ashton, PhD, has licensed Kamuvudines and is poised to start clinical trials next year.
Ambati says, “We will pursue every opportunity to accelerate the development of Kamuvudines. Phase III clinical trials for Geographic Atrophy typically run for 12–24 months. We will, of course, have to demonstrate safety and efficacy, but the great thing about Kamuvudines is that while the clinical trial process may not be faster we can be more confident about the outcome because of the health insurance database analyses showing that NRTI use is associated with protection against the disease in people.”
Grant says, “By discovering the existence of endogenous Alu cDNA in a human disease, this work raises the intriguing question of whether it might also be playing a role in other diseases where reverse transcriptase activity is enhanced.”
The list of inflammatory diseases is long and varied and includes diseases such as Alzheimer’s, Multiple Sclerosis, and Parkinson’s disease. Successful targeting of inflammation to check the progress of age-related macular degeneration may launch analogous therapeutic strategies for other diseases where inflammation is the terminal effector that drives disease progression.
