At A Glance
Name: Markus Stoffel
Position: Professor, Rockefeller University
Background: Assistant professor, Rockefeller University — 1995-1999; Research associate/assistant professor, University of Chicago — 1993-1995; Postdoc, University of Chicago — 1991-1993; Resident, University Medical Center, Hamburg — 1989-1991; PhD, molecular virology, Bonn University — 1991; MD, Bonn University — 1991; MA, Cambridge University — 1990
At Rockefeller University, Markus Stoffel focuses on metabolic diseases and the processes that regulate them, such as gene expression. It is no wonder, then, that his research has taken him into the field of microRNAs.
Recently, he co-authored a paper in Nature detailing the role of a new microRNA in controlling insulin secretion, garnering attention from both academics and the industry (Stoffel currently serves as a scientific advisor for Alnylam Pharmaceuticals).
Stoffel took time this week to speak with RNAi News about his research.
Could you give an overview of your lab and its focus?
We are interested in the molecular processes that regulate glucose and fat metabolism in humans using the mouse as a model system. We also study associated disease such as lipid disorders and diabetes.
One focus is the pancreatic beta cell. We identified a number of genes that are transcription factors — factors that regulate the expression of genes — [with] a set of them being responsible for inherited forms of diabetes. These forms are called MODYs, which stands for maturity-onset diabetes of the young, and that's a genetic subtype of type II diabetes. These patients develop diabetes early and can now be genetically classified.
Out of this work arose a program in which we tried to understand these transcription factors — what are the genes they regulate, and how do they influence insulin secretion? So we've been very interested over many years in studying the regulation of gene expression, mainly on the transcriptional level.
When microRNAs were discovered, we were really excited because here was a completely new class of genes that are believed to play an important role also in controlling gene expression on a post-transcriptional level. We were immediately interested in [the microRNA field] because of our broad interest in gene regulation.
About two years ago, we began asking, "Are there microRNAs in pancreatic beta cells, and if there are, what are they and what do they do?" We took an unbiased approach and we cloned every single microRNA in a pancreatic beta cell line initially. We found a number of microRNAs — some had been described previously, and others had neither been cloned nor predicted using bioinformatic approaches. In particular, there was one that was very specific, [and] just like the insulin message itself, you only find it in pancreatic islets.
We then asked the question, "What might these be doing?" and we simply increased the expression of the endogenous microRNA, which we call microRNA-375.
What we found was when we over-express it, we get less insulin secretion, and when we inhibit the endogenous microRNA, insulin secretion in response to glucose is increased.
When we found that out, we made adeno-viruses so that we could also study primary cells, not just cell lines. Then the next big question we wanted to address was: How does the microRNA regulate insulin secretion? That was, of course, at a time when we didn't have a clue about any targets of microRNAs. We collaborated with Nikolaus Rajewsky, a biostatistician at New York University, who wrote an algorithm predicting targets. However, these lists are very long, and it's very laborious to validate these targets experimentally.
We therefore set out to physiologically characterize the defect caused by microRNA-375 overexpression and to narrow down the spectrum of genes that we may want to validate from the bioinformatic target list. The way we did this was by systemically testing the pathway in which glucose stimulates insulin secretion. By studying the different steps of the pathway, we could narrow the defect to the very distal event, which is the fusion of the secretory granules with the plasma membrane.
When we found that microRNA-375 impairs exocytosis, we referred back to the target list and found two genes that we could possibly implicate in this process. When we checked these two putative targets, myotraphin and Vti-1a, we could show that over-expression of microRNA-375 inhibited the expression of these two targets. Furthermore, when we inhibited the endogenous microRNA, we increased the expression of these target genes. This, then, was really the first demonstration of a target regulated by a microRNA in mammalian systems.
Finally, what we had to do to close the loop was to find out if these two target genes play a role in the inhibition of insulin secretion. So we inhibited these by siRNAs and found out that reducing the expression of one of the targets, myotrophin, it leads to a defect in insulin secretion. That closed the loop showing that the target-gene regulation of microRNA-375 was at least partly responsible for the defect in insulin secretion.
This is the work that is published [in Nature]. Since then, we have expanded these studies to many other microRNAs that we found in pancreatic beta cells.
That leads to my next question. How is that effort proceeding? Do you have any good leads or is it still too early?
No, we have good leads, though not all of them have an apparent effect when we over-express or inhibit them. [MicroRNA]-375 [showed] the most marked effect, but since then we've found others that affect insulin secretion.
The way we are pursuing this [effort] is manifold. One, we're taking a genetic approach and knocking [the microRNAs] out. This is very important to do in order to understand the function in vivo, and also to understand the function in development. We are interested in seeing where they are expressed in development. We already know that they are expressed at high levels in early precursor cells for the pancreas, and therefore we hypothesized that they play a role in differentiation. We have knock-ins in which we express fluorescent proteins and find out in which cell type they are expressed, and also what happens when mice lack these microRNAs.
To take a more general approach, [we are also asking] the question: What happens when you don't have any microRNAs? — we are [investigating] the collective role of microRNAs by using mutant mice that don't have the Dicer protein, which is important for the maturation of microRNAs.
Lastly, we're looking at diabetic models to ask the question: Do [the microRNAs] play a role in disease, and is there any dysregulation of microRNAs in models of type II diabetes? These are in rat and mouse models. We're trying to link [our microRNA work] to pathophysiology.
We also work on trying to develop technologies that allow us to introduce these microRNAs, or inhibitory RNAs, into cells to use them as a pharmaceutical agent.
Since you brought that up, what is your take on [microRNAs as pharmaceuticals], specifically in terms of microRNA-375?
I think if we had a technology that could deliver a stable antisense RNA molecule capable of inhibiting the endogenous microRNA-375, then — based on all the in vitro data — this would lead to more insulin secretion. It could also have other effects that we don't know about. We find it exciting, but the technology is not there yet to deliver, with high efficiency, these molecules into cells.
What about a small molecule targeting microRNAs?
That may be feasible. The wonderful thing about the microRNA itself is that you can get specificity. One would have to see if a small molecule would have the same specificity.
Another approach is to attach a small molecule to a microRNA, and let the microRNA guide that molecule to a certain site. There are many ways in which one can think about using this pharmaceutically.
We are pursuing a few leads of trying to get microRNAs into cells. That's not just beta cells, but other cell types.
Have you gotten any interest from industry in your work?
There is interest. There are companies that are licensing microRNAs, and there are companies that are very interested in developing technologies that allow for the delivery into cells.
In the same journal that our journal was in, there was a nice paper by Alnylam (see RNAi News, 11/12/2004).
What about collaborations? You're at Rockefeller with Tom Tuschl
We very closely collaborate with Tom's group. We have a project that was just funded by the NIH.
We also closely work with Nikolaus Rajewsky, and we developed an improved algorithm that uses many more sequences in its predictions. It basically asks the question: Can we come up with a better algorithm if you propose there are multiple, highly expressed microRNAs targeting the same messenger RNA. It turns out this is the case, and we'll hopefully soon have a paper out that describes this algorithm and describes how powerful it is.
It turns out that many targets are not targeted by a single microRNA, but multiple microRNAs.
What about this NIH-funded collaboration with Tom [Tuschl]?
It has many aims. It involves Tom's group, my group, and [Chris] Sander's group [at Memorial Sloan-Kettering Cancer Center].
It aims to identify all the microRNAs, all the targets, work out the biology of how they work, and even look at genetics. A question we will answer is: Are there genetic variants in the microRNA or the microRNA target site that can account for disease.
We can do this because we have a huge population-based genetic study with many, many clinically characterized patients, and much laboratory data.