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ECU s Alexander Murashov on RNAi and the Nervous System


At A Glance

Name: Alexander Murashov

Position: Associate professor of physiology, East Carolina University School of Medicine

Education: PhD, physiology, Institute of Physiology, Russian Academy of Medical Sciences — 1987

MD, Moscow Medical School — 1983

Though trained as a physician, Alexander Murashov always had a strong interest in life science research. As such, the day after receiving his medical degree, he enrolled in a PhD program that has led him to conduct postdoctoral work in Australia, at the University of Melbourne, and New York City, at Columbia Presbyterian Medical School.

Now, he runs his own lab at East Carolina University School of Medicine and studies cell signaling in the nervous system during neuronal injury and peripheral nerve regeneration. Recently, he spoke with RNAi News about how RNAi has become a part of his work.

Could you give us an overview of how you went from being an MD to getting involved in RNAi?

My initial interest was sparked by my discovery of antisense transcription from one of the known genes, HSP70.2. This gene was initially described as a spermatogenesis-specific gene that is expressed mostly during meiosis. When I started to work with this gene, I found its expression in the brain and other reproductive tissues. I became interested, of course, in the expression in the brain because I was initially trained as a neuroscientist, and I was interested in the nervous system.

When I probed my Northern [blots] with a sense probe, I found that this probe also recognizes some band. Initially, it started because I did in situ hybridizations and I used sense probes as a negative control, so I always got some signal that looks very specific to me. Finally, I decided to check the sense probe on the Northern blot, and I found that it recognizes a specific band, especially in the nervous system. So I started to look at the signal, and I found … expression in the brain, and actually there was a specific pattern of expression during brain development in the mouse. I also tried to clone this gene, but it was very difficult because there was a complete match to the sense part of the gene. Basically, the sense and antisense transcripts completely overlapped with each other. It was almost impossible to get it from the cDNA libraries. So I used PCR and RACE-PCR techniques to get this gene. But basically I couldn't get the 5' and 3' ends — that was the most difficult thing.

Eventually I left this project because I moved to Greenville [in North Carolina] to accept a faculty position here. My projects shifted into slightly different directions, but I was always interested in antisense. At that point it was 1996, and I found around 20 different publications describing antisense transcription from known genes. Again, I was not able to follow up on this HSP70.2 story, but I still have it in mind to get the full sequence.

Later on, this interest was renewed when I was already here in Greenville and one of the faculty members here, Dr. Michael Van Scott, started to collaborate with Alnylam Pharmaceuticals. They wanted us to test the distribution of siRNAs in the lung. We did some work with them — it was almost three years ago. When we started to work with siRNAs, I became curious if we could see what the distribution of siRNAs in the nervous system may be, and if peripheral nerves pick up siRNA molecules, and whether siRNAs can inhibit, for example, local protein synthesis. We ordered some siRNAs from Dharmacon against anti-tubulin, and we did some labeling also with fluorescent probes. What we found was very interesting. We transected the sciatic nerve, and then we applied a little pouch on the proximal stump that had fluorescently labeled siRNAs in it. We found that peripheral nerves can pick up siRNAs, and those siRNAs can be transported by retrograde axonal transport to motor neurons in the ventral horn of the lumbar spinal cord. When we did sections of the spinal cord and looked under a confocal microscope, we saw granules of siRNAs in motor neurons. That was a very interesting observation because it was pure siRNA — there were duplexes without any transfection reagent, viral vector, or anything. It was just siRNA.

We also did similar experiments where we used siRNAs against beta-tubulin, and we looked at the expression of beta-tubulin on the level of proteins by Western [blot] and also the level of mRNA by real-time RT-PCR. We just cut out the proximal and distal parts of the sciatic nerve, extracted proteins or RNA correspondingly, and used it for analysis. We also did an immunohistochemical analysis.

What we found was that if we applied anti-beta-tubulin siRNA to the sciatic nerve, we saw a decrease in expression, and we confirmed that at the level of immunohistochemistry, in situ hybridization, PCR, and Western blot. We saw a decrease in the expression of tubulin.

The interesting about peripheral nerves is that they are very long, so there is some local synthesis of proteins that takes place. There was a recent paper by Twiss' group that showed peripheral nerves can actually synthesize a lot of different proteins. The nerves extend over long distances away from neuronal cell bodies, which are allocated for example in the spinal cord. In humans it may be two or three feet. How is the function of the nerve supported, if it's so distant from the cell body? Of course, we have retrograde transport and antegrade transport, but we cannot explain really how the axons survive. For example, you can transect a nerve and put it in cell culture, and it will survive for several days without a neuronal cell body. We thought, and several groups have proposed, that there is local protein synthesis that takes place in the axon as it supports its axonal function. This has been shown before for dendrites and is accepted. For axons, it's kind of accepted by some groups, but it is still debated by others.

We thought that RNAi may be a mechanism for the regulation of local protein synthesis in axons. So what we did next was look at the expression of different proteins that are part of RISC. If we see that when we apply siRNAs to the peripheral nerve, we see inhibition of local protein synthesis, we should then see components of the RISC. We found that there is actually activation of the RNA interference machinery. We detected Argonaute 2 nuclease, Fragile X mental retardation protein, p100 nuclease, and Gemin 3 helicase — they all are components, as you know, of the RNA-induced silencing complex.

We found that these factors exist in peripheral nerves and moreover, if you apply siRNAs you see up-regulation of these proteins. Then we were able to precipitate these protein complexes after application of siRNA. This paper is, right now, prepared for submission and it was also submitted as an abstract to the Society for Neuroscience's annual meeting. It's already completed work.

That's where you are right now, then.

Yes. But we do not stop here. We have a collaboration with Scott Hammond at UNC Chapel Hill. He's studying microRNAs, and he recently published a paper in Nature wherein he characterized clusters of microRNAs that are specifically expressed in different cancers. What we did [with him] was get isolated RNA from a crushed or injured peripheral nerve, and Scott did a microarray analysis for microRNAs. [Again,] we found that there are specific clusters of microRNAs that are up-regulated in peripheral nerves after crush injury. There are [also] some clusters that are down-regulated by the crush. These are data that are preliminary. We now want to repeat this microRNA analysis, then think about publishing. It's very interesting because it's a little bit like what Scott saw in cancers.

But in peripheral nerve crush, there are also specific groups of microRNAs that are up-regulated during injury and during regeneration. We found that there are certain microRNAs that are expressed at day 7 [after injury] or at day 4, or at both. Again, it proves the existence of the RNA interference machinery in peripheral nerves and shows that it regulates local protein synthesis, and that it's especially important during injury and regeneration as nerves need to synthesize a lot of new proteins to rebuild themselves.

Jumping back, is the work with Alnylam still ongoing?

Yes. We're still collaborating with Alnylam. We have some siRNAs from them and we're looking at the distribution in the nervous system. We're testing some siRNAs for their distribution in the spinal cord.

That's right. Alnylam is looking into RNAi for spinal cord injury with Merck.

Right. We're looking to see if siRNAs can be taken up by neurons, or if we need certain reagents to facilitate [delivery]. We can use adenoviral vectors, but with viral vectors there is always some danger involved. It will be nice to develop a method of delivery that doesn't involve viruses and can basically work like a drug to penetrate the cell and do its job. So we're working on non-viral delivery systems.

Can you give any insight into what approaches you're looking at?

It's too early.

What about collaborations with other companies? Are you working with anybody else?

We're having some conversations with Dharmacon, and we hope that we will begin collaborating with them.

They're not a drug company, so what kind of collaboration would you have with them?

They are not a drug company, but they're open-minded. They seem interested in some research applications. They're also developing microarrays for microRNAs. There are different things we've been discussing. One of the ideas is to maybe try siRNAs against diseases such as amyotrophic lateral sclerosis. There were several papers that showed an RNAi strategy may be successful in treating ALS, so one of the ideas was to try to treat ALS with siRNAs.

This was something you were discussing with Dharmacon?

Yes, but that is very preliminary.

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