Professor, physiology/ophthalmology and visual sciences, University of Kentucky
• Assistant professor, ophthalmology & visual sciences, University of Kentucky — 2001-2004
• Instructor, ophthalmology, Harvard Medical School — 1999-2001
• Chief resident/instructor, ophthalmology, University of Rochester — 1997-1998
• MD, SUNY Health Science Center — 1994
Earlier this month, a team of researchers led by the University of Kentucky's Jayakrishna Ambati reported in Nature that depletion of the microRNA-processing enzyme Dicer1 resulted in toxic accumulations of Alu RNAs in human cells and the eyes of mouse models, triggering the degeneration of retinal pigmented epithelium cells — a hallmark of the severest form of dry age-related macular degeneration.
This week, Gene Silencing News spoke with Ambati about the paper and this ostensibly miRNA-independent role of Dicer.
Let's start with a bit about geographic atrophy.
AMD comes in two flavors: the so-called dry form and the so-called wet form. Until now, most of the research and therapeutic development has revolved around the wet form of the disease. But in terms of the numbers of people [that] AMD affects, the vast majority have the dry form 80 to 90 percent. Of this group, a substantial number go on to develop the late stage of dry AMD, called geographic atrophy, which is characterized by the actual death of cells in the retina over time. Right now, there is no treatment for that condition.
The paper focuses on Dicer, a component of microRNA processing, but in terms of its activity outside of that pathway. What brought you to that?
Actually, it was a circuitous path. The paper starts out talking about Dicer, but that was one of the last things we found. We were concentrating on double-stranded RNAs that were accumulating in the eyes of [GA patients], and found those to be Alu RNA transcripts. We then worked backwards to determine what might be causing the accumulation of these transcripts and looked at a whole panel of enzymes including Dicer, and found that it was unique [in being] down-regulated.
Can you give an overview of the experiments you conducted and described in the paper?
We took human donor eyes and looked at the area of the retina affected in geographic atrophy, and found that there are RNA duplexes in abundance. We did unbiased RNA sequencing to determine the identity of the RNA molecules and that revealed they were these Alu RNA transcripts. Then we looked at the levels of Dicer and a variety of other enzymes in these same eyes and found that Dicer was dramatically reduced.
We tried to connect those two [findings] together, knocking out Dicer in mouse models and in human cell culture models, and found that both of those led to increased levels of accumulating amounts of Alu RNA transcripts and to retinal cell death. We showed that retinal degeneration could be prevented by blocking Alu RNA or by increasing Dicer. These and other experiments tied together those two systems.
How exactly do those transcripts lead to GA?
We have some insight into the mechanism, but not a lot. We're actively investigating that now [but it appears that] these Alu transcripts cause reduction of cell viability by activating caspase-3 cleavage, so there is some element of apoptosis there. We have [had] more insight into it since the paper came out, but it's still a work in progress.
And what about the role of Dicer in the process?
There are more than a million Alu DNA elements in the human genome, and for a long time it was thought that these are transcribed in modest amounts. Even though there are [so many of them] in the genome, there are maybe a few dozen RNA transcripts in each cell. The thought was that they are somehow silenced, but it turns out that they are being actively transcribed all the time and that Dicer is one mechanism used by the cell to chop them up to prevent them from reaching toxic levels. It's just an ongoing balance.
And this is completely independent of any microRNA mechanism?
In biology, it's hard to say that anything is completely independent of anything, but … we knocked out a variety of other enzymes that are involved in microRNA biogenesis or their functioning, and found that that did not lead, in these cells, to cell death or retinal degeneration. We also showed that when we knocked down Dicer, as we expect, we see microRNA deficits on a global level. But when we block Alu, we don't get any recovery of the microRNA deficits, even though we get recovery of cell viability. That tells us that there is probably not a major role played by microRNAs in the phenomenon of retinal degeneration in the setting of Dicer depletion.
There may be other subtle modulating effects that still need to be teased out, but in the system we're looking at, the effects of Dicer depletion seem to be specific to Dicer and Alu and not due to microRNA disturbances.
Do you think this phenomenon might be at play in other kinds of diseases, ocular or otherwise?
I don't think it's unique to these cells. In other cell culture systems, when you knock down Dicer, these Alu transcripts accumulate. So I think it's entirely reasonable — and we have some data in some other organ systems, as well — that during development Dicer depletion has profound effects that are due to microRNA disturbances. In the adult context, however, I think it's worthwhile to explore the possibility that it's actually accumulation of Alu transcripts that might be explaining some of the phenomena people see.
There are lots and lots of papers showing that Dicer deletion in this system or in another leads to apoptosis. People generically attribute it to microRNA disturbances, and I'm sure that in many or even most of those cases that's probably true. In addition to our paper, we have some data suggesting that it's also important to determine whether Alu perturbations [are involved] in what we see when Dicer is depleted.
This work suggests possible new targets for GA. What form might therapeutic intervention take? Can these transcripts be approached using antisense? Small molecules?
In the paper, we showed that Alu antisense molecules can actually block the toxic effects of Dicer depletion, and we're actually exploring a variety of other platforms including siRNA technology. That's what we're principally focusing on: blocking Alu RNA molecules.