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UTMB s Helen Hellmich on RNAi and Brain Trauma

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At A Glance

Name: Helen Hellmich

Position: Assistant professor, University of Texas Medical Branch

Background: PhD, zoology, University of Massachusetts, Amherst — 1989; BA, biology, University of Pennsylvania — 1981


After completing postdoctoral stints at the Marine Biological Laboratory in Woods Hole, Mass., and at the National Institutes of Health, Helen Hellmich made her way to the University of Texas Medical Branch where she works in the department of anesthesiology. Focusing on brain injury, Hellmich recently began using RNAi to examine genes that may be associated with the neurodegenerative effects of this kind of trauma.

She was recently awarded a two-year grant, worth roughly $275,000, to use siRNAs to examine the use of RNAi in rat hippocampal neurons following brain injury. Hellmach recently spoke with RNAi News about the work.

Could you give an overview of what your lab focuses on?

I am a neurobiologist by training, and I used to work at the NIH studying neurotrophic factors that are involved in brain development. But this is a brain injury lab; we study brain trauma. My first project was doing gene-expression analysis of the effects of brain injury in a rat model.

We actually focus directly on the hippocampus because one of the primary symptoms of brain injury patients — and there's almost 2 million people who suffer brain injury every year in the US alone, and about 50,000 of them die eventually — is some mental deficient.

The most common deficient is memory problems. The reason for that is the hippocampus, the area of the brain responsible for learning and memory, is very suceptible to brain injury. It's deep inside the brain, but when the brain is injured, the hippocampus is one of the most vulnerable areas. So, people who have any sort of brain injury whatsoever tend to have problems with their memory, and that is one of the most visible aspects of [the condition.] The problem is that when the cells in the hippocampus are injured, a lot of them die [initially], but for the next several years they [continue] to die at a higher rate [than other brain cells.] Nobody knows why. It's the long-term, progressive neurodegeneration that apparently causes the problems with memory — if you start losing cells in the hippocampus, you're going to have problems with memory because those cells are involved in memory and learning.

Our interest is, 'Why are these cells so vulnerable, and what can we do to prevent the degeneration?' My first few projects involved doing microarray analysis. First, I started with the whole brain, then I focused on the hippocampus. I use a technique called laser capture microdissection to specifically pick up groups of individual cells from the rat hippocampus after injury. Then we can make RNA from those cells, and I use a technique called linear amplification to amplify the RNA from those cells, which can be used for real-time PCR, conventional PCR, for microarray analysis, for ribonuclease protection analysis, and so forth.

The last few projects in my lab have had to do with profiling the differences between injured and uninjured neurons, and we use a specific dye called Fluoro Jade, [which] seems to be specific for dying degenerated, injured neurons. So at different times after injury, we take out the rat brain, we section it, and we stain with with Fluoro Jade, and the injured neurons light up and the uninjured neurons do not. So we pick up two different populations of neurons, then we make RNA, amplify it, and we do gene-expression analysis. From this I found out that the uninjured cells seem to stay uninjured because they seem to have much higher levels of protective gene expression. But the other thing is the injured cells have higher levels of genes that are technically bad.

When we started doing these RNA interference experiments, like everybody else we started out in culture. I buy my siRNA from Ambion. I use a lot of Ambion products … and since they offer a service where they design the oligos and you can try them out, and they worked, I stay with Ambion. There's no other reason then that I started out with them and I was happy with their products. I'm not saying anything bad about Dharmacon or any other company.

The particular molecule we're working with now … is nitric oxide synthase. We know that neuronal nitric oxide synthase is an important molecule. It has physiological functions that are very well-known and characterized. The problem is that, after brain injury, in the hippocampus massive amounts of neuronal NOS are released and apparently that trauma-induced release of nNOS seems to increase the intracellular concentration on free radicals, and there's a lot of free radical damage. We hypothesize that some of that nNOS leads to high levels of nitric oxide, which leads to high levels of peroxynitrate, and it causes cellular damage via the generation of free radicals. Actually, it's very simplistic.

People have used RNA interference in culture a lot, and a few people have tried it in the intact brain in mice and rats to knock down certain diseases like Huntington's [disease], but as far as I know, nobody has ever tried it in the traumatized brain. I'm going to try to see if knocking down neuronal NOS may alleviate some of the initial brain damage. After brain injury, we can take out the brain 24 hours later and [see] a lot of injured cells as assessed by Fluoro Jade staining.

This experiment itself is very simple. We've already made three different [siRNA-expressing] adenoviral constructs, and this week we started injecting the first construct … stereotactically into the hippocampus directly. We're waiting three days … and then we do the brain injury. A day later … we can assess the neuronal damage — that's today, actually.

What about the viral construct?

I did not have time to work out expression systems, so again I'm using an Ambion system — it's the pSilencer adenoviral system. It's relatively straightforward. We first tested three different siRNA oligos in culture in a rat hippocampal cell line. We assessed whether any of these would work in silencing gene expression, and they actually all worked to some extent … but truthfully, you cannot assess how something is going to work in vivo until you do it. Everything may work beautifully in a cultured cell line, but it may not work at all in vivo. So originally I was going to determine which [oligo] worked best in culture, and only make a virus for that. Then I thought about if it didn't work very well, so I decided to [experiment with] all three siRNAs.

I [went with an adenovirus construct] because I didn't want to use other viruses, [which] integrate into the host genome, and adenoviruses are episomally expressed. It's basically a transient infection, and I wanted to do a transient infection because usually something that is transiently expressed then cleared would be better for situations like injury or trauma. For long-term diseases, ones that are genetic, of course you want something that is permanent. But for something like brain trauma you just want to knock down something temporarily that is bad for you in the first few days, and then it hopefully it goes away. You cannot knock down a gene like neuronal NOS and expect the animal to be healthy, because it's essential.

The purpose of this [research] is not necessarily to knock down any one particular thing, but is to show that you can actually do an in vivo gene-silencing experiment using adenoviruses … in a brain traumatized animal, then to show that it has a functional effect. If we can use this technology to stop a physiologically deleterious process, at least temporarily to prevent further neurodegeneration, that's important.

 

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