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RETT SYNDROME RESEARCH TRUST WEBSITE

From today’s Press Release:

A paper published online today in Nature reveals that glia play a key role in preventing the progression of the most prominent Rett Syndrome symptoms displayed by mouse models of the disease: lethality, irregular breathing and apneas, hypoactivity and decreased dendritic complexity. The discovery, funded in part by the Rett Syndrome Research Trust (RSRT) was led by Gail Mandel, Ph.D., an investigator of the Howard Hughes Medical Institute at Oregon Health and Science University.

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INTERVIEW WITH GAIL MANDEL AND DAN LIOY

Brain cells can be divided into two broad categories: neurons and glia. The three types of glial cells are the star-shaped astrocytes, the oligodendrocytes, and microglia. Historically, neurons have received most of the attention, while glia were thought to play a secondary supporting role. During the past several years it  has become increasingly clear that glial cells contribute in very complex and dynamic ways to healthy brain function and are important players in Rett Syndrome.

Monica Coenraads speaks with Gail Mandel, PhD and Dan Lioy, a graduate student in her lab, to discuss their data published online today in Nature, concerning the influence of glial cells on the progression of Rett Syndrome symptoms.

Gail Mandel and Dan Lioy in the lab - Oregon Health and Sciences University

MC:  It’s good to speak with you both again, and congratulations on the new Nature publication.

GM:  Thanks, Monica.

MC:  Let’s go back in time a little bit to give this work some context for our readers. Soon after the discovery that mutations in MECP2 cause Rett Syndrome, a researcher with a longstanding interest in chromatin, Alan Wolffe, organized a scientific meeting in Washington DC. I had just co-founded the Rett Syndrome Research Foundation, which provided financial support for this meeting. It was the first scientific meeting I attended and it stands out very prominently in my mind. And it was there, over a decade ago, that I first met you. After the meeting we stayed in touch, and I remember trying to get you involved in Rett. At first you were somewhat resistant, but eventually you really jumped in with both feet.  What triggered that shift?

GM:  My lab was, and is still today, working on a gene called REST, which is a repressor. We had been doing biochemical experiments and we noticed that MECP2 was one of the proteins that were in the vicinity of the REST binding sites. We didn’t know much about MECP2 so we started reading about it.

This was in 2002.  Nurit Ballas was in my lab at the time, and she and I became interested in where MECP2 was in the nervous system.  And we were perplexed, because it was supposed to be a ubiquitous protein, but people were thinking it wasn’t in glial cells.  Basically, Nurit and I were skeptical, because we know a lot about repressors, and ubiquitous repressors in particular, and from a molecular biology standpoint, it just didn’t make a lot of sense to us that MECP2 would be excluded from glia.

So Nurit did her own experiments to search for MECP2, and she found it in purified glia, and using immunocytochemistry she showed it was in tissue—in glia in tissue. And that made us consider the possibility that MECP2 could be regulating something in glia.

Gail Mandel

MC:  And that finding is a perfect example of why it’s always good in a field to reach out to new people who will bring their own experience, curiosity and fresh ways of looking at a problem. You published your first paper on the subject in early 2009 in Nature Neuroscience. Dan, please give our readers the highlights of the new paper that just came out in Nature.

DL:  The key point of the paper is that in a mouse model that has no MECP2, putting MECP2 back just in astrocytes goes a long way toward correcting the Rett phenotype, especially the respiration problems. We also document that knocking out MECP2 only in astrocytes causes a phenotype, including a respiratory phenotype. But interestingly, and I’ll be the first to admit that I’m not yet clear on why this is—the phenotype isn’t complimentary to the extent of the rescue.  In a simple world one would do an experiment and find that if putting MECP2 back in one cell type corrects the phenotype then removing it from that cell type should, logically cause the phenotype with equal severity.  And so far, that doesn’t seem to be the case.

What I take away from our experiments is that neither MECP2-deficient neurons nor glia alone are sufficient to cause the full-blown Rett phenotype.  But conversely, putting MECP2 back in just neurons or glia can go an extremely long way in correcting the phenotype.  A scientifically interesting question is:   Why is that?

GM:  That was really the unexpected part.

Dan Lioy

DL:  One possibility that we’ve discussed at length is that both cell types, neurons and glia, must be mutated to get the full phenotype.  There is precedence from other diseases, like ALS, that says that neurons and glia contribute to the pathogenesis of the disease, but they do so differently.  And we wonder whether or not this may actually be a general model that’s also applicable to Rett.

MC:  Was this finding surprising?

GM:  Yes, it was. Thankfully we did both experiments pretty much at the same time, knocking it out and putting it back. Otherwise I’m not sure we would have pursued putting it back based on the knockout.  Since the knockout was not as dramatic as the null we might have concluded that glia are not very important and the disease is mostly neuronal. But, luckily, we did them both at the same time.

MC:  Another example of the serendipitous nature of science. Regarding the hypothesis that neurons initiate and astrocytes play a role in the progression of the disease, do you think that’s going to become more of a common theme across diseases?

GM:  That model was presented initially by Don Cleveland for ALS.  I do think it is going to become a more common theme. I think it also means that we need to understand more about how neurons and glia talk to each other.

MC:  Historically glia have not attracted the type of attention that neurons have, but that is changing. Gone are the days where glia were thought of simply as structural components and nursemaids to the neurons.

GM: Yes, that’s right.

MC:  RSRT is funding a collaborative gene therapy project between your lab and Brian Kaspar. You are using a vector called AAV9, which, depending on when you administer it, seems to target astrocytes. If indeed replacing MECP2 in astrocytes is beneficial then this vector might be quite interesting.

GM:  So this project is a big risk for my lab to take on in terms of manpower because I don’t know if it’s going to work.  But I think the project is so interesting and clinically relevant. In terms of targeting the underlying genetic problem in Rett, either we turn the silent MECP2 on from the inactive X, or we have to think about how to add the gene back. That’s why I think AAV9 may hold promise, because it has good expression and it crosses the blood-brain barrier.  There are a lot of labs working on gene therapy approaches so the field is competitive, but I think that is a good thing.

MC:  Well, we are risk-takers—we can’t afford not to be—so we’re delighted that your lab has become so engaged with Rett and opened up new research vistas. I agree with you about the value of competition, and will follow the unfolding of the next developments in your work with great interest. There is certainly no lack of complexities to explore. We hope the Rett community will continue to benefit from your scientific curiosity and perseverance. Thanks for your time with us today, and we look forward to the next updates.

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RETT SYNDROME RESEARCH TRUST WEBSITE
GERMAN TRANSLATION

Monica Justice, Ph.D. – Baylor College of Medicine

By Monica Coenraads

Last week the trustees of RSRT voted to award continued funding to Monica Justice of Baylor College of Medicine.  Dr. Justice is a mouse geneticist (yes…there really is such a thing) who is spearheading one of the most unique projects in the Rett research arena today. For background information please read the September 2009 blog interview with Dr. Justice.

This project is a mammoth undertaking – the kind that requires a certain sense of fearlessness on the part of the investigator (and the funding agency). The risks are high but are in line with the potential rewards.

I feel a certain sense of attachment to this particular project as I was present when the idea was first suggested. At the time I was the Director of Research at the Rett Syndrome Research Foundation (RSRF) and I organized the annual Rett Syndrome Symposiums that spanned three days at the end of every June. At the 2006 meeting I cajoled two dozen of the brightest and most creative scientists to join me for an early morning, pre-meeting gathering I called the “knowledge gap meeting”.  I posed the following challenge: develop a prioritized list of high-impact experiments that no one was currently undertaking and that was unlikely to be funded by traditional agencies. After a two-hour discussion the group delivered their top contender: a modifier screen to identify genes that suppress the effects of an MECP2 mutation.

My next task was to find the best possible person to undertake the screen and talk them into taking it on.  I organized a Rett workshop a few months later at a “Mouse Genetics Meeting” (yes…they have those) in Charleston, SC, which attracted a number of candidates.  Bottom line – I courted Dr. Justice and she enthusiastically agreed to pursue the project.

Fast-forward almost five years and I’m delighted that the project is thriving and yielding a wealth of information. I recently caught up with Dr. Justice to discuss how the project is faring.

MC:  Dr. Justice, thank you for taking time away from your work to bring our readers up to date on your fascinating project.  Please tell us how things are going.

MJ:  My pleasure, Monica. I want to start by saying that this project would never have been funded by the NIH [editor’s note: National Institutes of Health] nor would I have proposed this to your foundation, had you not approached me about it.  I knew little about Rett Syndrome when I started this project.

MC:  Tell us a bit about modifiers. Do you think they exist for every disease?

MJ:  I think that most diseases can be influenced by mutations but I don’t think every disease necessarily has modifiers.

MC:  Do you have a sense of how many screens like yours are ongoing right now?

MJ: I know of only a few in the mouse.

MC:  Do you know of any screens that have yielded modifiers that suggest a drug for the particular disease?

MJ:   That’s a great question, Monica. I do not. The goal of most of the modifier screens is not to identify drugs but rather to understand some developmental or biochemical pathway. I think with our screen we are learning a lot about the biology of the pathway, but our hope is to find a drug-targetable pathway.

MC:  Can you summarize the progress and the ups and downs of the project?

MJ:  At the inception of the project we encountered several problems. We really had no idea that the Mecp2 mutant females would be such poor breeders. We tried all sorts of tricks to improve their breeding capacity, because we needed a lot of them to do the screen, and nothing worked.  We kept at it, though, just with basic mouse breeding, and eventually got up to speed. Also, in the beginning we had some communication issues within the lab. I’m not sure we were expecting quite as many modifiers as we found. But we then developed a system, and that system worked very well; then things started moving along beautifully. I also realized early on that I needed to participate in a very hands-on way with this project.

As we isolated each modifier line, we realized that each one was different: that is, none suppressed the disease entirely, but each modifier line appeared to suppress a subset of Rett symptoms.  Even so, each line allowed the mice to live longer, function better and be healthier. However some of them developed other symptoms, such as inflammation and susceptibility to infection, which could also shorten their lifespan.  So, we worked with our veterinarians who helped us decide how to treat these mice. For example, we found that putting them on antibiotics worked extremely well.

And so, there were some unforeseen glitches in the beginning that are moving very smoothly now. We have isolated five modifier lines. We have put the screen on hold for now and are concentrating on understanding these five modifier lines. We have identified three suppressor genes so far and have candidates for the other two.  We feel that the screen and the gene identification have gone extremely well. Each of the modifiers has a different phenotype in that they rescue different symptoms, and we find that each modifier locus is affecting a different gene.  For example, in one line that we named “Romeo,” the suppressor delays the onset of Rett-like symptoms, and the mice have no inflammatory lesions, but eventually, the mice succumb with symptoms.  Therefore, the onset of neurological symptoms is later in their life, making their lifespan longer.  Another line, “Henry” develops almost no Rett-like symptoms, and lives nearly a full long life, with only a few mild inflammatory skin lesions late in life.  Another line, “Cletus”, rescues some of the neurological symptoms so the mice live longer, but the long-lived mice have severe inflammatory lesions, even early in life.  Remember that each of these lines carries the Mecp2 (Bird) null allele, along with a second site mutation that alleviates its symptoms.

MC:  It’s possible to figure out statistically how many mice you have to go through so that you’ve saturated the genome – in other words, ensure that you have a mutation in every gene. What percentage of the genome has your screen hit so far?

MJ:  I think we are between ten and twenty percent of the way through.

MC:  So there is a way to go.

MJ:  Based on our work thus far we could find another 25 modifiers.

MC:  The ideal situation is that the suppressors you find are in pathways that are somewhat known and, ideally, for which there are already drugs associated. But you also have to figure out just how the suppressor inhibits the ill effects of having an MeCP2 mutation.

MJ:  Yes, there are many steps that we have to take after we find a modifier gene, to understand what’s going on.

MC:  One of the surprising things that have already come out of your screen is the mere fact that MeCP2 has so many modifiers. Why do you think that is the case?

MJ:  My hypothesis is that the biological system in Rett is sort of poised on the edge, and it’s not so detrimental that it, for instance, causes death immediately. It’s very easy to tip the scale toward a little bit better—– or a little bit worse.  And my feeling after doing the screens is that MeCP2 creates that kind of situation in the cells, that they’re poised to go one direction or another pretty easily.

MC:  Your hypothesis suggests that there could be lots of things that improve Rett symptoms.

MJ:  Absolutely. And maybe combining them will provide an amazing improvement.

MC:  What is your time frame for restarting the screen?

MJ:  I’m usually an optimist about time; my hope would be that, within a year, we could start the screen again.

MC:  One of my favorite things about the screen is that it is completely unbiased. Preconceptions and favored hypothesis don’t play a role here. The animal tells you what is important and you simply go where the data leads you. I find that very reassuring.

MJ:  I completely agree.  Often, we have been totally surprised, then after generating more data, the mode of suppression makes complete sense.  We are learning a lot from these animals.

MC:  I will leave our readers with one last comment.  Over the past 12 years I’ve overseen peer-review for well over a thousand research applications for funding. So I’ve read my fair share of reviewer comments. Typically reviewers are a conservative bunch; even if they are enthusiastic about a project they tend to be cautious. Your recent proposal, however, generated comments like  “Wow”. That’s truly unusual and a real tribute to your creativity, risk-taking and perseverance. I am thrilled that RSRT can partner with you and hope that together we can deliver some much-needed treatments to kids and adults who are in such desperate need.

RETT SYNDROME RESEARCH TRUST WEBSITE
GERMAN TRANSLATION

Huda Zoghbi, M.D.

Huda Zoghbi’s watershed discovery of the genetic cause of Rett Syndrome in 1999 ushered in a new era of research.  The first mouse models for the disease came on the scene in 2001. The male mice are missing the Mecp2 protein completely and are called knockouts; the females, due to X chromosome inactivation, have approximately half of their cells lacking the protein.

These are what most people think of when discussing “Rett mice.” However, in the past few years more types of mouse models have been created, each of them developed to answer a specific question and to teach us something about the disorder. Differences between these various models help form the foundation for much of the current drug discovery efforts.

Data regarding the latest animal model was published today in the high-profile journal, Science.  The model was developed in the Zoghbi lab by MD/PhD student, Christopher McGraw.  Through genetic engineering techniques he created mice that were missing Mecp2 only as adults.


MONICA COENRAADS, RSRT EXECUTIVE DIRECTOR, INTERVIEWS HUDA ZOGHBI, M.D.

MC:  Dr. Zoghbi, please tell us about your decision to undertake this experiment and the results.

HZ:  There were two main reasons we wanted to perform this study.  We know that Rett symptoms start after birth and we wanted to understand whether there are any developmental components to the disease. In other words, did the Mecp2 protein have some function that is important during early development or childhood?  That was the first question we wanted to answer.

The 2007 experiments from Adrian Bird’s lab told us that if Mecp2 is restored in adult mice that had developed abnormally without the protein, their Rett-like symptoms are reversed. We were curious to see whether brain cells that had properly developed and matured with Mecp2 being present, and had gone through typical experiences of learning and memory and then had the protein removed as adults – would their phenotype be milder? And the answer was a resounding NO. The big surprise for us was how similar the knockout mice that had Mecp2 missing from conception were to the mice that had Mecp2 missing only as adults.  This told us that bypassing the critical period of development did not affect the severity of the symptoms.

The experiment tells us that you need Mecp2 all the time. It also tells us that you need Mecp2 not for development but rather to maintain normal brain function.

MC:   So the timing of the appearance of Rett symptoms has nothing to do with development and everything to do with what happens in the brain after you remove Mecp2.   Can we safely say now that Rett is not a neurodevelopmental disease?

HZ:  Our experiments were done on mice and not humans so we must always be cognizant of that caveat.  But I think you are right.  It’s how long the cells are without the protein that matters.

MC:  Your experiment certainly strengthens the idea that Rett is not neurodevelopmental. The reversal experiments of 2007 provided the first clue. You and I have been at meetings together where the issue has been debated. Some pointed out that the debate was not worth having because it was a matter of semantics.  But I disagree. This is not just semantics; there are clinical implications.

HZ:  You are right. It’s not semantics. I now call Rett a post-natal neurological disorder. Mecp2 is a factor that is critical for the normal function of brain cells. It’s a factor that is constantly needed for normal neurological function and this has implications for therapies.  Therapies will need to be maintained for the long term.

MC:  Your findings have implications for other diseases that are post-natal, like autism.  Can you elaborate?

HZ:  There are many disorders that show up after birth and we have assumed that, just like for Rett, the absence of a protein was affecting normal development.  I think this paper is telling us that maybe this is not the case.  In the case of Rett the protein affects transcription; in other cases the proteins are doing something different but the end effect is the same – some molecule is not being made in the right amount when it is needed in the brain cell.

So for disorders like Fragile X, Angelman Syndrome, Tuberous Sclerosis, if we take away their particular protein the cells are sensitive to the deficiency, but if we bring the protein back the chances for recovery are high.

MC:  The key is that Rett symptoms are not hard-wired since the same symptoms can be found also in the adult knockout. That is hopeful, encouraging news.

It’s quite fascinating to me that despite being a rare disorder and having relatively small number of investigators working on Rett, the field seems to be tearing away at some long- standing neuroscience dogma.

Your discovery in 1999 made Rett the first sporadic neurological disorder that had a gene associated with it.  Rett was the first childhood neurological disorder to be shown to be reversible, thereby teaching us about the plasticity of the brain.  We now know that Rett is not developmental and this fact calls into question the neurodevelopmental status for other disorders such as autism, Fragile X, Angelman Syndrome, Tuberous Sclerosis and others. It’s quite remarkable.

It’s been almost 12 years since you discovered the genetic cause of Rett.   Are we as far along as you would have expected in our search for treatments?

HZ:  In many ways things are going well, as we’ve learned so much about the disease. We know the anatomy of the brain is normal, we know the cells can recover if you bring back this protein.  Our challenge is that the protein is so essential for so many cells. Finding a pharmacological intervention that can hit a great majority of the cells will be key. I don’t underestimate the difficulties; it will take some very good pharmacology to bring the symptoms under control.

MC:  I know I speak for every Rett family around the world – we are tremendously grateful that you are working on behalf of our children. Thank you for your commitment, your determination and your hard work.