Associate director, Proteomic Fractionation Core
University of Massachusetts Medical School
At A Glance
Name: Douglas Hinerfeld
Position: Associate director, Proteomic Fractionation Core, University of Massachusetts Medical School, since 2005. Research assistant professor.
Background: Scientist, Charles River Proteomic Services, 2003-2004.
Visiting scientist, Proteome Systems, as part of collaboration with the Buck Institute, 2002.
Postdoc, Simon Melov's laboratory, The Buck Institute, 2001-2003.
PhD, Gordon Churchward's laboratory, Emory University, 1996-2001.
The US National Institute of Allergy and Infectious Disease recently awarded $20 million to a consortium of institutions to further develop and test some catalytic scavenger compounds as a treatment for radiation exposure caused by terrorist attacks or industrial accidents (see story). ProteoMonitor talked with Douglas Hinerfeld, who was involved in testing the scavenger compounds on knockout mice, to find out more about the history of the scavenger compounds, and his background in antioxidant research.
How did you get interested in studying the mitochondrial proteome and antioxidants?
I actually got interested in science working as an undergraduate in the laboratory of Tom Johnson at the University of Colorado. He was fundamental in the genetics of aging and determining that genes could be associated with life span. I worked with a postdoc there part of the time named Simon Melov, and when I graduated from undergraduate, Simon had gone to Atlanta, where I'm from, to work with Doug Wallace, who's really a pioneer in mitochondrial genetics who has done work in relation to aging and oxidative stress. So I went back to Atlanta and worked with Simon for about a year and a half. That's when I first got involved with looking at free radical scavengers, particularly in the manganese superoxide dismutase knockout mouse. I worked on that for some time. I also worked on aging in mice, looking at mitochondrial DNA and deletions.
Then I started graduate school there at Emory University. I switched areas to the laboratory of Gordon Churchward, where I worked on DNA recombination in relation to transposition. That came largely from an interest that I had generated from working on mitochondrial DNA mutations and DNA metabolism. So I did my PhD there in Gordon's lab. Then I decided to go back to working on the SOD2 mice in relation to antioxidants, so I went and did a postdoc with Simon Melov. He was then a faculty at the Buck Institute in California. This sort of gets into the story of working with Eukarion, which is the company that had originally produced these [catalytic scavenger] compounds, and as it turns out, Proteome Systems.
I was interested in looking at neurodegeneration in the manganese SOD knockout mouse model. With these mice, a very small percentage of them survive out to a couple of weeks of age, at which point they succumb to a neurological phenotype. We noticed this, and we also realized that these mice had a dose-dependent response to catalytic antioxidants — the Eukarion compounds. So at a low dose, the life-span of the mouse is greatly extended, but they still get this neurological phenotype. And at a high dose of the drug, you can actually partially rescue this neurological phenotype. You can completely rescue the associated neuropathology. So the mice have neuropathology, you give them the drug, and you can rescue that neuropathology, and largely rescue the neurological phenotype.
What is the phenotype and pathology that the mice have?
The phenotype is largely [that] they get tremors — this is at around three weeks of age or so — and also they have ataxia, seizures. And the neuropathology was primarily a spongiform encephalopathy in the frontal cortex, and some in the brain stem. So that was the main pathology that I was interested in looking at. And I was interested in if the elevated levels of superoxide in the mitochondria which results from the loss of that particular gene. So manganese superoxide dismutase is the gene and the protein, and it scavenges superoxide. And the loss of that [gene and protein] presumably causes an excess of mitochondrial superoxide, which leads to this phenotype. If you give them antioxidants and you sop up those free radicals — and evidence points to these compounds doing that — the you can prevent mitochondrial damage.
Were you involved at all in developing the antioxidant compounds?
Not directly in developing the compounds. More in understanding their biological activity.
Is that mouse specifically engineered for radiation studies?
This mouse is specifically engineered to look at the effects of mitochondrial oxidative stress. That's its purpose. That's why it was made originally. It's not directly linked to radiation necessarily — it's just related to oxidative stress. And because the animals are succumbing to this oxidative stress, the idea was if we give them catalytic antioxidants, that we can suppress the level of oxidative stress, and hopefully cure the mice.
Oxidative stress is associated with many neurological diseases, including Alzheimer's and ALS and Friedrich's ataxia. So it could be a very good model for determining how antioxidants can prevent damage resulting from oxidative stress in the brain.
One thing important that this mouse study showed was that these compounds could cross the blood-brain barrier. That's strongly suggested by the data, because it was rescuing a neurological phenotype. That could be important in terms of the application of the drug to neurological diseases, which is an area that I know they have a lot of interest in.
How are these compounds administrated?
Through intraperatoneal injection. You couldn't feed these compounds to the mice, or put it in their drinking water. They had to be injected. They weren't bioavailable.
At the time, we worked with a couple of different compounds that were quite similar. One was Euk-8, and another was Euk-189. I think Euk-189 is the one that they're continuing to work on at Proteome Systems. I know that one of their goals is to make the compound bioavailable so that you can take it in pill form, but at that time, that technology wasn't available.
When was it that you did that work?
In 2001 was when I really got involved with it. At that point, all of what I've told you so far — that it rescues the neurological phenotype and neurodegeneration — had been known. That was done under Simon Melov. Then when I became involved, the goal was to understand first of all, what's going on with the mitochondria? Why are the animals so sick? And how is it that these antioxidants are rescuing or helping the mitochondria so that you don't get the neurological phenotype?
So that's how I came to Proteome Systems. Simon had had a good working relationship with a woman named Mary Lopez who was formerly at Proteome Systems, who is now at PerkinElmer. At that point I had been looking at the mitochondria and looking at enzyme assays — trying to understand which enzymes in the mitochondria were affected by oxidative stress, and how their levels of activity were altered by the antioxidants. But it became pretty clear that it's easier to let nature tell you what the problems are. So rather than picking this enzyme and this enzyme and this enzyme to study, why don't you just take an unbiased proteomic approach? So take all the mitochondria out of the brain, and do proteomic analysis to look at proteins that change as a result of the knockout. And then how do those proteins that change as the result of the knock out — how are they altered by the treatment of antioxidants? So that was the whole gist of bringing together the drug and using proteomics to understand the biology.
We published a paper in 2004 in the Journal of Neurochemistry on that work.
So you compared normal mice with knockout mice?
We did. We also compared the drug-treated mice to the normal mice and the knockout mice as well. I think the term is that a 'pharmacoproteomic approach' was used.
What proteins did you find that were affected?
Primarily components of the TCA cycle and the electron transport chain. So protein complexes such as Complex I, II, and III of the electron transport chain, as well as succinate dehydrogenase — a protein that's associated with both the TCA and the electron transport chain. That protein was the most profoundly affected protein — it is about 90 percent reduced in the knockout mouse. And we were able to significantly increase the activity of that with the drug. And that is true as well for other enzyme complexes. One of the other proteins affected was the alphaketoglutarate dehydrogenase complex in the TCA cycle. That protein was dramatically reduced in the knockout mouse as well, and could be slightly increased with the drug.
Once you have these proteins, what's the next step?
One of the major steps is trying to understand which of these protein complexes is limiting in activity. So some of these protein complexes have small reduction. Some of them have a very large reduction in activity. Which one of them tips the balance to the point where you start getting cell death? So the next step is really to ferret out which of these complexes that are affected are the most important in pathology? So is a 90 percent reduction in succinate dehydrogenase activity — if you can just increase that alone, is that going to stop the pathology? How do you target further drug development?
Is that something that's being worked on?
I think so, yes. But that's something that's being carried out at the Buck Institute, not at Proteome Systems. I think Proteome Systems' primary goal is to follow up on the application of these drugs.
What this study showed is that you can see real biological activity of these drugs in a mammal. Not in cell culture, but in vivo, and that they can 100 percent prevent neurodegeneration. So that was a pretty important first step to showing that these drugs have a great deal of potential for neurological diseases.
What are you working on at the present?
I've continued to work in the area of neurodegeneration and neurological disease. So I came to U. Mass Medical School about 10 months ago with my colleague Sunny Tam. We have a proteomics technology core here, and the main focus of our core is one: to help both the internal investigator and external for-profit companies and non-for-profit groups to do proteomics research. My main other focus is my own research, which is related to identifying antecedent biomarkers for Alzheimer's disease. So I'm identifying proteins in the CSF that could potentially tell an individual whether they're likely to get Alzheimer's disease. It's not something right now that you'd want to know about, but as therapies come along, the earlier you detect it, the better.
The subjects that I have samples from and that I'm funded to do research on have mutations that will certainly give them Alzheimer's disease at a young age. So they have familial Alzheimer's disease, but they have no sign of dementia yet. They're up to a decade away from having any dementia, and we already see protein changes happening in their cerebrospinal fluid related to Alzheimer's disease, up to a decade before they have any cognitive defects. So that's pretty important, because if you can translate those proteins into the general population, then what you might be able to do is predict up to a decade before a person has any cognitive defects that a person is likely to have Alzheimer's disease.
The other important application of this is if you want to study the effect of drugs on the development of Alzheimer's disease, it's very useful to have markers that you can follow. So for instance, if you treat these subjects that have mutations that are going to cause Alzheimer's disease — if you start treating them with a drug that you think is going to prevent Alzheimer's disease — you may not know for a decade if they've had an effect. Right now the only way to know is if they have cognitive defects. But if you can track a protein which is a good marker for Alzheimer's disease — if that protein is elevated in a person that has Alzheimer's disease, and if you can reduce it to a normal level, then that's a very good sign that your drug is having a beneficial effect on that patient. So these markers are very important for drug development.
How far along is drug development now for Alzheimer's disease?
There's nothing out there now that is conclusively able to have an effect. There have been some epidemiological studies that suggest that statins have some effect, and there are some NIH-funded studies looking at statin treatment in relation to Alzheimer's. There have been some antibody attempts, but they have resulted in inflammation, and in some cases, I believe, death. That was an early study where they thought if they could clear the plaques then you could prevent the onset of the disease, but that had some pretty nasty side-effects so it was discontinued. It was wonderful in mice, but you know, you've got to get to human studies.
Is there anything else that you're working on?
There are a lot of projects that I'm working on, but my main focus has been this particular project. I'm also working with a couple of investigators in developing proteomic strategies in zebrafish, which are a good model for studying and visualizing arterial development.