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Q&A: Large-scale Study Explores Protein Changes that Persist in Cocaine Use, Even After Long Periods of Abstinence

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This story was originally published on May 4.

Willard.JPGName: Willard Freeman
Position: director, Functional Genomics Facility, Penn State College of Medicine, 2004 to present; assistant professor of pharmacology, Penn State College of Medicine, 2004 to present
Background: Postdoc fellow, Yerkes National Primate Research Center, Emory University School of Medicine, 2003 to 2004; postdoc fellow, Vollum Institute, department of behavioral neuroscience, Oregon Health & Science University, 2001 to 2003

Lull pic.JPGName: Melinda Lull
Position: PhD candidate in pharmacology, Penn State College of Medicine, 2004 to present
Background: BA in anthropology, St. John Fisher College, 2000 to 2004



In a study published March 17 in the online edition of Proteomics-Clinical Applications, a team of researchers looked at the protein changes in rats that had been exposed to cocaine.

Specifically, the researchers were interested in looking at changes after the rats had stopped using cocaine. Previous work by the same team had shown that rats who had been previously exposed to cocaine but who were then starved of cocaine for prolonged periods more actively sought out the drug than rats who either were not forced to abstain from drug consumption or those that abstained for shorter periods.

In the current work, "large scale discovery proteomic techniques were used to gain a better understanding of the proteomic changes occurring with cocaine administration that persist with abstinence, and those occurring specifically during abstinence, in the medial prefrontal cortex," they wrote in the study.

They looked at changes in rat brains at one day of abstinence and 100 days of abstinence and compared them to the brains of rats that had never been exposed to cocaine: In the rats that had been exposed to cocaine, the researchers found that some proteins that had changed at the 1-day mark returned to normal levels at 100 days. Others stayed changed even after 100 days of abstinence, and some proteins were unchanged at the 1-day mark, but changed at 100 days.

"These protein expression changes that do not reset to pre-cocaine exposure levels may contribute to the persistent relapse potential that occurs in response to cocaine abstinence," the researchers wrote.

ProteoMonitor spoke with Melinda Lull, the first author of the Proteomics-Clinical Applications study, and Willard Freeman, the corresponding author on the study, recently about the work. Below is an edited version of the conversation.

Describe the research.

Willard Freeman: Our drug abuse efforts are specifically focused on persistent changes in the brain even after drug abuse has stopped. We're very interested in understanding that and the nerve biology of relapse.

It's well known [that with] all sorts of abuse drugs, from nicotine to cocaine to methamphetamine and heroin [to] alcohol, [that] one of the biggest problems is we can get people clean, they can go to a hospital, especially an in-patient facility and then be in-patient for two weeks or six weeks … and they'll be clean when they leave the hospital, but the problem is keeping folks clean.

And it's well known across a lot of clinical studies that there's this heightened potential for relapse. Specifically, relapse is often caused by stress, exposure to the environment, or people that you previously abused drugs with.

The majority of folks relapse. That's the major problem — getting people clean and keeping them clean. Despite their best efforts, these are folks who are generally motivated to stop, but this is addiction.

We're interested in understanding the neurobiology of that. Naturally, we use animal models that we can intervene and manipulate in different ways. And we're interested in understanding that after drug self-administration … in long periods of abstinence [whether] there are things that remain changed. Does the brain ever go back to normal, or is there a new normal that leaves a susceptibility to relapse? And are there additional changes that occur during abstinence?

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So we're interested in finding what changes with drug abuse and never goes back to normal, even with abstinence: Are there things that change during abstinence after a period of drug abuse, and how would these combine … as a neurobiological basis for relapse?

If we can understand what is more or less permanently changed about the brain in addiction, that's what we can go after as therapeutic targets to help people stay clean.

These persistent changes that you observed in the rats, did they occur in all of them or only in some but not others?

WF: They were pretty consistent across all the rats in that 100 days of abstinence. They're subtle changes. That's one of the biggest findings that we have from this study. This is a tough nut to crack.

There [are] subtle, but we believe important, changes that persist, unlike the field of cancer where you have major demodeling of the cell from a normal cell to a [diseased] cell.

We're talking about subtle but important differences that are going to alter neural transmission in the brain. This is some tough stuff to do because we're never going to see these huge and widespread changes. It's always going to be a small number of relatively modest changes.

In terms of the number of proteins, or even within a single protein, the changes are going to be subtle?

WF: I would say both: the total number of proteins that change and then the magnitude of change for a specific protein.

Did you look at the level of cocaine use and what effect that had on the rats?

Melinda Lull: There was no significant difference between the amount of cocaine that the animals took while they were administering cocaine.

WF: It's a … very specific aspect of the behavior in this model system that we used that was developed by Dave Roberts at Wake Forest University.

The animals have free access with some limitations [to cocaine]. They're pressing on a bar to give themselves cocaine. They have an in-dwelling line into a jugular vein. They press on a bar to receive cocaine injections and they have … pretty much open access to cocaine.

We have to provide some limitation. Otherwise, they'll overdose and kill themselves, to be brutally honest. …And pretty much all the rats do the maximum that we allow them to take.

The paper says that proteins associated with cell structure were overrepresented with these changes. Were these changes that were persistent? Or changes that occurred only during abstinence? Or did they occur only during the period when cocaine was being ingested by the rats?

ML: It was a mixture of both. Some of the changes that we saw, [such as those in] neurofilament or alpha-internexin, were short-term changes that returned to normal. Some were between one day of abstinence and 100 days of abstinence. Some of the other proteins that we saw, like dynamine-1, were persistently altered up to the level of 100 days.

What do you draw from this observation that cell structure proteins were overrepresented?

WF: We were very interested to find that because there are several reports in the drug-abuse literature that have shown [that] changes in synapse structure and formation, and even possibly numbers of synapses … [may be] how the brain encode[s] information that lasts for a long time.

There's still a huge black box there, and whoever figures out how memory works will get their Nobel Prize and we'll know all their names.

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But we were interested that [our findings] fit well with what had been seen previously in the anatomical work that other groups had done. We're seeing perhaps that these are individual proteins that can contribute to those morphological changes in neurons that other people have seen.

You also have found that there are proteins that don't change during cocaine use but do during abstinence. Have you drawn any conclusions about why this is happening and its significance?

WF: There's a growing understanding both in clinical research and in animal research that you have a period of drug use and then you have a period of abstinence. Cravings can actually increase with abstinence.

So there's some sort of additive effect. The period of drug use and then the period of abstinence may combine [to] heighten the potential for relapse. When we saw the changes were occurring when the drug was no longer being taken, that fits into what has been seen behaviorally — there's some kind of combination of these two time periods that results in a new state of relapse reliability.

So we expected it. It makes sense in that it has a parallel to the behavioral work.

You looked at one day of abstinence and 100 days of abstinence. Did you look at time periods between these two and what happens?

WF: That's what we're doing currently. Another technical finding that's perhaps of interest to the proteomics field … is we were very interested and excited about the findings that we have from this study, but it also points to the fact that for tissue proteomics, some sort of fractionation has to be done.

We saw some very interesting changes, but we want to see more of the proteins that are related to synapses and neural transmission, so what [Lull] is working on is synaptosomal preparations where we pull out the pre-synaptic and post-synaptic elements and examine proteomic changes on that specific part of the neuronal architecture.

We're also adding more time points. We really want to see how these obvious changes occur with smaller time frames.

We've done all the proteomics experiments [and] we're analyzing the data. We haven't quite gotten to the point of saying, 'Here's the final answer in relation to drug abuse.'

But something we can definitively say is that when we do these synaptosomal preparations or fractionations, we see much more of the proteins that we would like to see from a standpoint of the synapse. I think everyone who's doing tissue proteomics is getting to a point where they're saying, 'We really can't just look at whole tissue homogeneously. We have to look at some sort of fractionation that simplifies the proteome around those things that we're interested in.'

Are you looking at post-translational modifications that may occur?

WF: Yes we are. One problem with looking at PTMs has always been the ability — especially of these phosphorylation events as you prepare your samples — [to] go away, or you can add spurious new modifications.

We just got a new instrument, which we think will be a critical, enabling technology. It's a Denator Stabilizer. This was developed by a group in Sweden in collaboration with the Karolinska Institute.

What this does is through a heat and pressure process, very rapidly in a matter of maybe 30 seconds for a piece of tissue, stop protease, phosphatase activity, things like these, so that you're not losing your post-translational modifications or getting protein degradation in intact pieces of tissue.

We've done some initial pilot work, enough that we wanted to get the instrument and we've been very happy with what we've found for keeping these PTMs stable.

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How are you going to use it and what exactly are you trying to address with this instrument?

WF: There are a couple of different routes: one is just for our studies … when we do our animal dissections, we'll stabilize the tissue at dissection. Then we can use it … for just focused work where we have developed targets out of broad screens whether by DIGE or iTRAQ, and as we do immunoblotting or MRM, we can specifically look at phosphorylation, which is a critical part of some of these structural proteins and for synaptic proteins.

Are you going to look at other types of PTMs or [are you] concentrating solely [on] phosphorylations?

WF: Initially it's phosphorylation. There's also a great deal of interest in glycosylation. We just haven't gotten to it yet — how it can regulate insertion or retraction of proteins from the membrane or from complexes.

But phosphorylation is the first place to start because the most is known about what these mean functionally.

You said that you're currently looking at time points between one day of abstinence and 100 days of abstinence. Are you also looking at what may happen beyond 100 days of abstinence?

WF: So far, the 100 days is as far out as we're going in this animal model. There doesn't seem to be much in the way of changes in the behavioral phenotype [beyond 100 days], so it doesn't give us a lot more to tie to the behavior. The 100 days in a rat is a pretty long time.

Is there any reason, though, to think that there may be protein changes that are observed at 100 days that may be mitigated later, at 150 days, 200 days, et cetera?

WF: It certainly is possible. What we don’t know terribly well in humans or in animals is the very long-term phenotype. From the clinical standpoint, if we have a drug abuser, and let's say they reach five years of drug-free time, it's not totally clear [they're unlikely to relapse].

There's not a lot of data to say that if you can get to five years, you're going to be good. We would sort of say that's likely to be the case, but there's not a lot of data [to support that].

Your paper mentions some of the limitations of the proteomics technology. Were there specific limitations that you encountered in your work with the technology?

WF: I don't think we have much that's specific to drug abuse or behavior or neural proteomics. I think we have a lot of the same issues that anyone else working with tissue has. We're talking about synapses, ways to fractionate proteins so that we can get more of the proteins we want to see and less of the highly abundant proteins that are not as pertinent.

We want to get to smaller and smaller and more discrete pieces of tissue ensuring that we have enough material for proteomics studies. We still believe that this technology still has a great deal to offer because the number of samples that we can do … and the quantitation is quite accurate.

When we confirm proteins by immunoblotting or something like that, the quantitation is really quite accurate.

As we continue to move into our different proteomics studies, our standard operating procedure has come to be, if possible, [to] perform DIGE. We perform iTRAQ; we also, in certain studies, perform a direct analysis through Luminex [technology] and then we combine all of these datasets because we see only a limited overlap in proteome coverage between the different methods.

So to get the broadest proteome coverage possible, what we're doing these days is multiple different technologies.

Has there been new technology since you did your research that you believe would be able to elucidate some things you couldn't explore with the technology you were using?

WF: We've done, I'd say, three things: The first is, having a background in transcriptomic work for a decade, we've been well aware of the [limits] regarding statistical power …When we're trying to do studies like this … we're going to see small magnitude changes.

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With 8-plex iTRAQ coming out — it came out after this initial DIGE study — that finally gets us to a point where we're using iTRAQ more routinely because we can analyze the number of samples that we need to analyze.

We never adopted ICAT because it didn’t give us the statistical power that we needed.

But with the 4-plex iTRAQ, we've looked at the technology, but it still wasn't high enough throughput in terms of samples. But with 8-plex iTRAQ, that's now a standard workflow, as well. We can do two runs of iTRAQ and analyze 15 samples. At that point, you can have a well-powered study.

How are you going to do validation work moving forward? You mention in your paper that that is going to be a challenge.

WF: One [approach] has been a sort of scaling up of our traditional immunoblotting approaches to where we can do more antibodies and more samples rapidly. As much as possible, we're now trying to do fluorescent immunoblotting because that speeds up that process.

And the other [thing we're doing] is we're just starting now on our first set of developments for MRM assays.

Have you been able to draw anything that may be of use for your own research from other researchers doing proteomics in the drug abuse arena?

WF: Drug abuse [research] in proteomics is still pretty small. One thing that we do that we've been trying to make better is how we can pull datasets that are generated in those studies and do dataset-to-dataset comparisons.

Currently we're using Ingenuity Pathway Analysis software for that. A big challenge has always been, let's say a colleague is working on a nicotine-generated set of data and he has protein identifier ascension numbers, but there's a lot of redundancy. There [are] a lot of different ascension numbers for the same protein.

Making your dataset match up with their dataset has been a real challenge in the past. What we're doing with the Ingenuity software is rather than having a protein ascension number … [because ] protein ascension numbers are all underneath one gene ID … if we translate everything into a gene ID, that allows us to line datasets up and make comparisons.

It sounds like your work so far has really been on the discovery level and then will progress to the validation level. Have you started any work looking into protein interactions?

WF: We're not doing much in that regard currently. One of the next likely goals is with our behavioral collaborators at Wake Forest — they're working a great deal on viral vectors to introduce antisense or hairpin RNAs, things like this to where we can intervene in a specific brain region to alter the expression of one of the proteins we find and see the behavioral outcome of that intervention.

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