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Monica Coenraads, Executive Director of the Rett Syndrome Research Trust, interviews David Katz, PhD about his Journal of Neuroscience paper published 10/3/2012 and entitled “Brain Activity Mapping in Mecp2 Mutant Mice Reveals Functional Deficits in Forebrain Circuits, Including Key Nodes in the Default Mode Network, that are Reversed with Ketamine Treatment.”
Adrian Bird and colleagues recently published their latest paper on MeCP2 in the journal Human Molecular Genetics. The series of experiments described in the paper were designed to explore what happens when the MeCP2 protein is removed from mice of various ages, including in a fully adult mouse. This work was funded in part by RSRT with generous support from RSRT UK, Rett Syndrome Research & Treatment Foundation (Israel) and other organizations who financially support our research effort.
Below are excerpts from a conversation with joint first authors Hélène Cheval and Jacky Guy.
MC Dr. Cheval, you trained as a neuroscientist. What attracted you to the Bird lab, which is very biochemistry-based, and where you are the sole neuroscientist?
HC My previous lab, run by Serge Laroche, was a pure neuroscience lab focused on learning and memory. However, I was actually doing biochemistry and I was very much interested in how to get from molecule to behavior, and I was also quite interested in chromatin. I had read the Bird lab reversal paper of 2007 and thought it was one of the most exciting papers I had ever seen. Upon receiving my PhD I applied for a post-doc position, convinced that it would be a great experience for me but also thinking that perhaps the lab would benefit from having someone with a neuroscience background. I joined the lab in 2009.
MC Dr. Guy, you co-authored your first paper on Rett Syndrome in 2001. That was the paper that described the MeCP2 knockout mouse model made in the lab, one that is now used in hundreds of labs around the world.
JG I joined the lab in 1997. My first project was to make the conditional mouse models of Mecp2, meaning mice where the protein can be removed at will. At that stage we didn’t yet know about the link between MECP2 and Rett Syndrome. That came about as I was working on the project. It was a very exciting time.
MC It’s unusual for people to stay in a lab so long. This gives you an amazing depth of uninterrupted knowledge about the field.
JG I took a rather unconventional path. I’m very happy to do bench work and being able to work in the same field has been wonderful.
MC Dr. Guy, perhaps you can start us off. What are the key questions you were trying to answer with this series of experiments?
JG This was actually an experiment we had been wanting to do for a long time. We have always been interested in defining when MeCP2 is important. Rett had been thought of as a neurodevelopmental disease. Since we were completely new to Rett, we thought maybe it’s not neurodevelopmental. So we set out to remove the protein at different ages and see what happens. Removing the protein is not quite as simple as reactivating the gene, which we had already done in the reversal experiment. When you reactivate the gene it makes protein right away. In this experiment, however, when you deactivate the gene you have to wait for the protein to decay away. We found it takes about two weeks for the amount of MeCP2 protein to fall by half.
HC Jacky’s reversal experiment suggested that MeCP2 is implicated in adulthood. But many papers were still describing Rett as a neurodevelopmental disease. We also wanted to confirm a hypothesis that we all shared in the lab that MeCP2 is required throughout life.
MC That is a hypothesis that was also put forth in Huda Zoghbi’s 2011 Science paper. She showed that removing Mecp2 in adult mice aged 9 weeks and older caused Rett symptoms. Do you think that her paper and your new data have definitively put to rest the notion that Rett is neurodevelopmental?
HC To my mind it’s clear that it’s not merely neurodevelopmental.
JG I think “merely” is the key word here. The phenotypes we analyze in mice are those that are quite easy to see; for example, lifespan, breathing, gait. There might be more subtle things that we are not observing, or that are not affected by knocking out the protein in adulthood. And we are not analyzing cognitive aspects. So we can’t completely rule out the possibility that there could be some things that are indeed of a neurodevelopmental origin that we are not seeing in these experiments.
JG Mecp2 can be deleted by treating the mouse with tamoxifen in the same way the protein was reactivated in the reversal paper. In this paper we picked three different time points to turn off the gene: three weeks (which is when mice are weaned and begin to live independently) eleven weeks and twenty weeks. In all three scenarios the tamoxifen was able to delete Mecp2 in about 80% of the cells.
What you might expect is that at whatever age you delete the gene, there will be a certain amount of time for the protein to disappear and then the effects of not having the protein will appear.
In fact, what we found is that the time it took for symptoms to appear varied with the age at which we inactivated the gene. It took longer for the symptoms to appear when we deactivated Mecp2 at 3 weeks. When we removed MeCP2 in older mice, the symptoms appeared more rapidly. So it seems that younger mice are able to live symptom-free without MeCP2 for a longer period of time. There is a certain period when the need for MeCP2 becomes more important in mice. This is the first critical time period that we talk about in the paper; it happens around eleven weeks.
As we followed the mice treated at all three time periods, eventually they all started to die at about the same age, approximately thirty-nine weeks, regardless of when MeCP2 was removed. We concluded that this time period centered around thirty-nine weeks represented a second critical period for MeCP2 requirement. This is a time in a mouse that roughly coincides with middle age in humans. We think that maybe MeCP2 is playing a role in maintaining the brain as it ages.
Interestingly, this time frame of thirty-nine weeks is when female mice that are MeCP2-deficient in about 50% of their cells from conception begin to show symptoms. The male mice which have zero MeCP2 can’t make it past the first critical time period of eleven weeks. When you delete MeCP2 in 80% of the cells, the male mice show symptoms at 11 weeks and die at 39 weeks. So having about 20% normally expressing cells allows you to survive the first critical period but not the second.
MC I’ve heard clinicians say that women with Rett in their 30s and 40s and beyond look older than they are. I wonder if this has anything to do with your hypothesis that MeCP2 may play a role in aging. Of course we don’t know if the premature aging is primary or secondary. It may have to do with the effects of dealing with a chronic illness for many years.
JC We are quite interested to learn about a potential late deterioration in women with Rett but there is very little published on the subject.
MC There are two potentially critically relevant points made in your paper. One is the fact that the half-life of the MeCP2 protein is two weeks. That could be relevant and encouraging for a protein replacement approach.
JG We certainly had this in mind when we were doing the experiment. The half-life of MeCP2 is longer than we expected. And could in fact bode well for protein replacement therapy. One caveat, ours was a bulk brain experiment. It could very well be that if you looked regionally in the brain or by cell type you might find varying results.
MC The other potentially clinically relevant information comes from comparing the severity of symptoms seen in the mice in this study versus the adult knockout done in the Zoghbi lab and correlating symptoms to amount of MeCP2 protein. Your experiments yielded 3% more protein and resulted in less severely affected animals. Can you elaborate?
HC That such a small difference in protein could have such a significant impact on survival is unexpected and indeed may be relevant for therapeutic interventions. We may not need to get the protein back to wildtype levels to have an effect. It may be possible that even small increases may be helpful.
MC Congratulations to you both on this publication. The Bird lab has made numerous seminal contributions to the Rett field. The Rett parent community doesn’t typically have a chance to glimpse the researchers behind the experiments, doing the day-to-day work, so I’m delighted to provide an opportunity for our readers to get to know you a bit. I look forward to the next publication. Best wishes for your ongoing work.
Photos courtesy of Kevin Coloton
Doris Tulcin – A Mother’s Love Raises the Bar For All Non-Profits
Half a century ago, a mother whose baby daughter was diagnosed with a life-threatening genetic disorder decided to fight it. Doris Tulcin is that mother, and Cystic Fibrosis is the disease against which she went to war. First identified in 1938, Cystic Fibrosis affects roughly 30,000 children and adults in the United States and is therefore, like Rett, classified as a rare disease. In 2010 the Cystic Fibrosis Foundation generated donations in excess of $300 million, $120 million of which was raised from the general public. Now, Vertex Pharmaceutical’s VX-770, the first drug that treats not just symptoms but the underlying genetic cause of Cystic Fibrosis, has been fast-tracked through FDA approval.
Doris Tulcin has been a valued advisor to RSRT since our inception three years ago. The following in an excerpt from a recent conversation between Mrs. Tulcin and RSRT Executive Director, Monica Coenraads.
MC: Congratulations! You must be thrilled with the early FDA approval—it’s been a long road and I know CF families are rejoicing today!
DT: It’s mind-blowing! VX-770 is for a small group of patients that have a rare mutation but will literally change the disease and these kids completely. And that’s what we are working on now for 90% of the patients with the major mutation. Things are moving really well.
MC: CFF provided substantial scientific, financial and clinical support for the development of VX-770, including the investment of $75 million. When I think about the vast amount of money CFF raises, with a patient population similar to Rett, I can’t help but ask myself, How have you done it? Are there specific decisions that you think have been pivotal?
DT: Well, you must remember that we have a long history; we’ve been in business since 1955. So it took over fifty years to get to where we are today. Going back in time, it is so hard to pinpoint any one thing. But we really focused on research, as you are doing.
MC: Of course, the scientific tools and knowledge that were available fifty years ago are so very different from research today. Everything has accelerated. Yet I know that getting to this point with Cystic Fibrosis has been a process with many components.
DT: From 1980 on is where we really took off with developing research programs with major universities, and by the 1990’s we became increasingly business-oriented and
focused on working with the biomedical community and pharmaceutical companies for drug development.
MC: As with Rett syndrome, the Cystic Fibrosis population is small. You know RSRT has a PSA that’s running in Times Square, the same screen that was used for the CFF. So 1.5 million people will see it every day for three months. How important do you feel it is to raise general public awareness, in terms of the ability to raise research funds?
DT: Name recognition is very important. The general public doesn’t have to understand the disease but they certainly have to recognize the name. You know, people will say “I’ve heard of Cystic Fibrosis”— well, what do you think it is? They can’t describe it. They haven’t the vaguest idea, but they’ve heard of it.
MC: And you think that correlates to the amount of money you raise?
DT: I do. Our chapters raise roughly $100 million a year. But keep in mind that it took decades to get where we are today.
MC: For RSRT, at the three-year mark, most of our revenue comes directly from affected families and their networks. Do you think the same applies to you, or do you have people just spontaneously donating to CFF without any personal connection?
DT: I think now we do, yes. But what we started with and built on was indeed that personal commitment and involvement to develop networks of family and friends. The activism of those who are personally affected is crucial.
MC: How would you compare the current philanthropic landscape to what you’ve seen in past decades? These are difficult economic times for many.
DT: Yes, we are still in a bad economy now and that reflects on contributions, there’s no doubt. It’s tough, and there’s always competition and distraction.
MC: On a personal note, how is your daughter doing?
DT: Oh, thank God, she’s doing well. She’s a grandmother! My grandchildren have introduced me to Facebook, so we communicate in that way, because they’re all grown up and don’t have time for other things. But we do that too with the foundation, we’re on everything you can think of— Facebook, Twitter, YouTube. Everything now is sent electronically. The website is being updated all the time, you can get every bit of information at any time off that website.
MC: Yes, the fact that electronic media is now ubiquitous worldwide has really changed the flow of information in important ways for families dealing with something like a rare disease. The ability to be in instant communication with others who really understand relieves some of the isolating strain of constant and intense caregiving, as well as keeping people informed of current developments. That families can so easily connect and learn from each other is vital and sustaining. The day-to-day struggles can be so overwhelming sometimes for Rett parents that there’s not always a lot of energy left for what I feel is truly going to change our children’s lives—the research. What are your thoughts?
DT: There is no doubt that you really have to find ways to stay focused, and I think that was one of our biggest successes. Care has been important but it is research that’s absolutely critical; it’s the only way to move forward.
MC: Research has always been my commitment, and I hope the work of RSRT will prosper as brilliantly as that of the CFF. Congratulations again on VX-770 and
thank you for your guidance and inspiration.
DT: Monica, I know how tremendously devoted you are to this fight. The Rett community is very blessed to have you.
MC: Thank you, Doris.
On a chilly day in early spring, an unlikely group gathered in a spacious office at Harvard Medical School – the office of Michael Greenberg, Chairman of the Department of Neurobiology, one of the most respected and prolific neurobiology departments in the world. Joining Dr. Greenberg was Adrian Bird of the University of Edinburgh and Gail Mandel, a Howard Hughes Medical Investigator from Oregon Health & Sciences University. These names are well known to anyone who is at all familiar with the Rett research literature, yet none of these distinguished scientists would describe themselves as a “Rett Syndrome researcher.” The questions that have kept them busy throughout their careers revolve around basic science phenomena such as DNA methylation, gene expression and brain plasticity.
Each of these scientists has been drawn to Rett Syndrome via a different route, and their combined interests will now create a powerful synergy to explore the most basic mystery of Rett: What is the precise function of MeCP2 in the brain?
RSRT Invests Record $1.8 million in Three-Way Collaborative Experiments To Speed Path to Drug Development
Dr. Greenberg called me one day last year and said “I’m coming to you with a far-out proposition.” He confessed that elucidating the role of MeCP2 was the most challenging problem he had ever worked on (a striking remark, coming from a scientist as accomplished as Dr. Greenberg) and that the chances of success would be greatly increased if he could put his head together with outstanding researchers with complementary expertise. He asked me to explore whether there might be any mutual interest on the part of Drs. Bird and Mandel. I did so, and the response was enthusiastically positive. Synchronicity was on our side. RSRT Trustee Tony Schoener and his wife, Kathy, were interested in funding a high-impact project: the MECP2 Consortium was born.
I recently caught up with the investigators to discuss this novel and non-traditional collaboration.
Coenraads: How would the three of you define the goal of the Consortium?
Bird: The goal of the Consortium is to bring about a step-change in our understanding of the function of MeCP2 in relation to Rett Syndrome, which we believe will be vital for designing rational treatment therapies. Unlike most other autism spectrum disorders, we know exactly the root cause of this disorder, but explaining in molecular terms just why absence of functional MeCP2 brings about Rett’s particular constellation of symptoms still eludes us.
We already have useful information about what MeCP2 might do in cells – we know it is a chromosome binding protein that targets DNA methylation; we know it becomes chemically altered when nerve cells are active; and we know that other types of cells in the brain apart from nerve cells also need MeCP2 for the brain to function normally – but there is no consensus among scientists about why MeCP2 is needed for the brain to work properly.
Our joint view is that solving this tricky problem calls for cooperation between laboratories with different expertise. Gail, Mike and I have rather different slants on biology due to our training and backgrounds, but we appear to complement each other nicely. Our view is that the next few years will see advances in our understanding of both MeCP2 and the brain. The timing feels right and it will be exciting to see what happens.
Mandel: The goal of the Consortium, from my point of view, is to put our heads together to generate new ideas, and to critically evaluate each other’s ideas and experiments, and to collaborate on experiments where the expertise is complimentary. I also view it as an opportunity to engage our young scientists in training in rigorous translational biology.
Coenraads: That is a good point Dr. Mandel. The Consortium goes well beyond the three of you. It requires the active participation of all of your lab members, who will be interacting with each other on a regular basis.
Greenberg: I propose that “speed” is a part of the equation as well. The goal of the Consortium is to gain rapid understanding of the molecular and cellular basis of Rett Syndrome through a collaborative effort.
Coenraads: During the 12 years that I’ve been working with the scientific community the concept of consortiums has been discussed from time to time. It strikes me that what differentiates a true collaboration from one that is superficial and in name only is that the desire to collaborate has to come from the scientists themselves. Collaborations cannot be imposed from above and made attractive with the bribe of money. Meaningful collaborations come from the bottom up and are nurtured by mutual respect and trust and a strong sense that the whole will be greater than the sum of its parts.
How is working with the Consortium different than how you’ve worked in the past? Has it required any kind of mental shift in your personal working style?
Mandel: Having had a long-term collaboration with my husband, who is also a scientist, I have first hand knowledge of the virtue of consortiums. My personal style has also, I think, been open to collaboration. Similarly, my lab members work very well as a team.
Bird: Science is normally a competitive activity. Discretion at least is required, if not complete secrecy, if one is to avoid the trauma of being beaten to your goal by other laboratories and scooped by their prior publication. This dog-eat-dog culture among many researchers has its advantages in that it can accelerate discovery, but is often at odds with the needs of a charity like RSRT, which may wish to have scientists putting their heads together to solve pressing, clinically relevant problems.
Our consortium intends to do the latter. We share unpublished data and resources. We speak regularly on the phone and meet several times a year to bring each other up to date on what’s new. The Consortium is still at the beginning, but already it is having an impact on the research going on in our laboratories. To be honest, I find it refreshing to be part of an endeavor that transcends our personal ambitions for a higher purpose.
Greenberg: I agree. I feel that although the Consortium research effort began just a few months ago we are already seeing a benefit. The pace of progress in understanding Rett Syndrome is already beginning to accelerate. My expectation is that through collaborative interactions with the Bird and Mandel laboratories we will be able to overcome current obstacles to understanding the molecular basis of the disorder. I think that we can expect to make key discoveries that will lead to new ideas for therapies for treating Rett Syndrome in the near future.
Coenraads: I think it’s also important to point out that the discoveries that the Consortium will likely yield will help not only Rett Syndrome but also the MECP2 Duplication Syndrome and all disorders caused by alterations in MECP2.
RSRT has committed $1.8 million to the MECP2 Consortium. The Schoeners have contributed $1 million to the endeavor. It’s an understatement to say that without them it’s unlikely we could have launched the Consortium so quickly. I thank them for their generosity, commitment and frankly, their belief in the scientific process.
To the three of you I wish you much success. I look forward to our monthly Consortium calls and in-person meetings and to keeping our readers apprised of your progress.
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.
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.
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.
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.
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.
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.
Mark Bear, Ph.D. of MIT is the most recent addition to RSRT’s portfolio of funded scientists. Prof. Bear studies synapses, the gaps between nerve cells where chemical or electrical signals are exchanged. The strengthening and weakening of synapses contributes to learning and memory but when impaired can lead to neurological disorders.
Much of the excitement in the Fragile X community comes courtesy of the Bear lab. His discoveries have spawned a series of clinical trials.
New York Times
Monica Coenraads, Executive Director of RSRT, recently caught up with Prof. Bear to discuss his Fragile X research and how it might extend to Rett Syndrome.
MC: Prof. Bear, thank you for taking time to discuss your research with us. Many of our readers will have heard of the ongoing Fragile X clinical trials and are eager to understand how your research might also impact Rett Syndrome. Please explain the so called “mGluR Theory of Fragile X” which was discovered in your lab.
MB: Sure. Synaptic function requires the synthesis of proteins in the synapses, so that supply can keep up with demand. Demand is registered, in part, by activating metabotropic glutamate receptors (mGluR). So the more active the synapses are, the more glutamate is released and the more protein is made. Like in many systems there are checks and balances, and one of those is the negative regulation of protein synthesis by FMRP, the protein made by the Fragile X gene, FMR1. Normal synaptic function requires a sense of balance between driving protein synthesis through mGluRs, and inhibiting protein synthesis through FMRP. In Fragile X the FMRP protein is missing so it’s like driving a car with no brakes – your foot is on the gas but there is no way to stop. So there’s excessive protein synthesis which leads to a myriad of deleterious consequences. The approach that holds a lot of promise is to inhibit mGluR which in essence takes your foot off the gas.
Now that theory has been pretty widely validated and at least in the animal models of Fragile X many features of the disorder can be corrected by inhibiting mGluR.
MC: You theorize that Rett Syndrome is at the other end of the spectrum, instead of too much protein synthesis, there’s too little protein synthesis. What’s behind this hypothesis for you?
MB: Once we had the success in Fragile X, we started thinking more broadly about other single gene disorders that are characterized by autism, seizures, and impaired learning. I was influenced by a paper that was published by Christian Rosenmund and Huda Zoghbi. They analyzed synaptic connectivity of hippocampal-cultured neurons that either were over or under expressing MeCP2, the Rett Syndrome protein. They found that reducing expression of MeCP2 reduced the connectivity, and over expressing it increased the connectivity.
We think about Fragile X as a hyper-connectivity disorder: too much protein synthesis, too many synapses, or too much synaptic turnover…and so, the Rosenmund/Zoghbi results made me think about Rett in terms of diminished protein synthesis. Also, in terms of morphology in Rett tissues we see signs of reduced connectivity –for example too few spines on dendrites.
MC: You were recently at a Fragile X meeting in Edinburgh where you spent some time discussing your theory with Adrian Bird. Tell us a bit about that.
MB: I was starting to mull this theory over then I ran into Adrian and had a great conversation with him. He was very encouraging – he didn’t think that this was a ridiculous idea. So that really got me charged up. We agreed that the most exciting thing is that we have drugs that can correct both excessive and diminished protein synthesis.
MC: Prof. Bird called me after you and he had this discussion – he was charged up too. I organized a conference call and the three of us rather quickly decided on a collaboration and a division of labor with regards to experiments. Please tell our readers a bit about the drugs that are in existence.
MB: There are two types of mGluR drugs that have been developed. One of them is the negative modulators that will inhibit mGluR. These would be used for Fragile X. The others are positive modulators that will promote mGluR activation – these might be helpful for Rett. The negative modulators were developed originally as a potential treatment for generalized anxiety disorder with the goal of creating the next generation of anxiolytics. That’s what motivated industry and they invested hundreds of millions of dollars into developing these compounds. We are really lucky in that there’s already a lot of great chemistry around our target. The positive modulators were developed for schizophrenia.
MC: Novartis recently released data on a phase 2 clinical trial for Fragile X. What did you think of the outcome of that trial?
MB: I think the best news is that they’ve decided to go forward into phase 3. Overall I think there is tremendous hope for disorders like Rett and Fragile X even for interventions in adults. So we are extremely optimistic and very energized to help people affected by Rett. And we thank RSRT for giving us funds to explore the disease and for facilitating a collaboration with Adrian.
MC: Talk to us about Seaside Therapeutics, the biotech that you started to develop drugs for neurodevelopmental disorders.
MB: When we first realized that mGluR inhibitors might be beneficial for individuals with Fragile X we reached out to big Pharma and we got a very cool reception. In those days, about ten years ago, big Pharma had very little interest in rare genetic disorders. As a consequence, I founded Seaside. So far we have been pretty successful in advancing a drug that shows great promise in both Fragile X and autism. Seaside is committed to tackling the single gene disorders. And although we do not currently have a Rett program, there easily could be if we get a promising lead, so we are eager to get to work.
MC: I remember sitting in your office at MIT 6 or 7 years ago talking to you about Rett Syndrome. It’s taken a bit of time but I’m so pleased that you are now working on Rett. Our readers and I wish you much luck. We hope to hear of your success soon.
The recently opened Jan and Dan Duncan Neurological Research Institute (NRI) in Houston, Texas is dedicated to scientific exploration of childhood neurological disorders. Director Huda Zoghbi, whose laboratory established that mutations in MECP2 cause Rett Syndrome, envisioned a center where researchers with diverse interests could work within an environment of ongoing, cross-disciplinary dialogue. The soaring new structure is located in the heart of the Texas Medical Center, close to the basic science campus of Baylor College of Medicine and Texas Children’s Hospital. At full capacity the NRI will provide laboratory facilities for 50 to 60 investigators. All NRI investigators are Baylor College of Medicine faculty.
Below are excerpts from a recent conversation between Dr. Zoghbi and Monica Coenraads, RSRT Executive Director.
MC: Dr. Zoghbi, it was wonderful to witness the recent opening of the NRI, the culmination of a lead fifty million dollar gift by the Duncans, an outpouring of support from the local community and years of work. The unique architecture reflects a specific functional goal: the creation of a powerful center for collaborative research on children’s neurological disorders. In shepherding this concept from an idea to a most impressive reality, I know you were involved in every aspect of its development. Congratulations are in order! And now that this beautiful facility is open for business, tell us how the next steps are progressing.
HZ: I think there are really two phases now that are moving in parallel. One is the recruitment of talented faculty to occupy the laboratories of the first five floors that have been completed. The second will be to continue our expansion, which is being built by stimulus money and will hopefully be ready for additional recruits in 2012.
MC: I know the physical layout of the building goes beyond its striking appearance. You had a particular vision in mind. In fact, you coined a new descriptive term: collaboratory.
HZ: Yes. The design is specifically intended to promote and enhance interaction between investigators within the building, and communication with adjoining faculties. We have tried to structure this within individual labs as well as the institution as a whole. In an age where most people will text or send an e-mail message rather than walk across the hallway to talk to someone, we have arranged work areas that are conducive to actual conversation, and social spaces that invite and encourage movement and exchange. Investigators with different areas of expertise will be able to access shared resources. The collaboratory is a beautiful, very open glass tower, modeled after the DNA double helix; the stairwell is very spacious and pleasant. People will be drawn here, moving from floor to floor to lunch, have a cappuccino, take a break, use the exercise machines, and in doing so will naturally be interacting with fellow scientists from labs on different stories.
So this collaboratory, this getting together people of different disciplines is still rather new, a kind of paradigm-changing shift from traditional science boundaries. As you recruit faculty, I imagine personality will have to play an equal role with intellectual excellence in considering a candidate.
HZ: Yes, it is really important that the scientists we recruit be generous and receptive. Generous means they are willing to help and to share their ideas and contribute to others’ projects if their skills would be useful in a particular area. Receptive scientists are open to hearing input about their work. These are very important qualities and are key for an interactive research environment; we dream of a generation of scientists who really cherish such a philosophy. We are also establishing programs to help scientists transition to independence as soon as they are ready. Toward this end we will be creating NRI fellowship positions, to give brilliant young PhD graduates (two per year) the opportunity to work within an unusually supportive and nurturing environment. If their projects are successful, they will then be well positioned for highly competitive faculty appointments.
MC: And this philosophy of the collaboratory is expanded even beyond the architecture of the new building, by the way the site was chosen. I know the location was very critical to you.
HZ: The NRI is a Texas Children’s Hospital building, but I wanted it in a location where scientists from very different disciplines would have access to it. I also wanted our own scientists to be only steps away from institutions where the focus and expertise are on scientific problems that are quite different from problems seen in childhood neurodevelopmental disorders. For example, a researcher at Mitchell research building at MD Anderson (attached to NRI) who studies cancer and the epigenetics of cancer might make a discovery that has relevance to epigenetics in the nervous system. You really don’t know where the breakthroughs will come from, and so this cross-cultivation of work and ideas from different institutions has great potential value.
MC: Tell us about the potential of this approach to accelerate and validate new work.
HZ: If you know one technique very well, or even have multiple skills in one discipline, this is still not enough when you are trying to understand something as intricate as brain development and brain function. Somebody might come here with expertise in basic synaptic biology and neurophysiology, but is very willing to engage and think about how they could maximize the impact of their work by collaborating with someone who might be studying a model of Rett Syndrome or Fragile X. You truly need a great variety of specializations, including those from the physical sciences, to begin to tackle complex problems. Even with all of the expertise you can begin to put together, these problems are still challenging.
MC: The readers of this blog are of course interested in Rett Syndrome. Can you speak about the kinds of resources that you envision being allocated for Rett research?
HZ: We’ve recruited eight faculty members so far, and one of our first recruits was somebody who works in Rett Syndrome, Jeff Neul. In addition, we’ve really strengthened the physiology core. Our colleagues in neuroscience are doing some work using two-photon imaging of cortical neurons in animal models of Rett, so the NRI has purchased equipment for these experiments. (Editor’s note: Two-photon imaging is a type of microscopy that allows researchers to look in depth at living tissue.) Our behavioral core is designed to address the needs of large scale preclinical trials in Rett mouse models so we can expand the number of trials we do and expand our behavioral assays. Some of our new recruits will be investigators who bring in a skill set to look at Rett from different angles. Since Rett encompasses so many symptoms, the more we learn about it, the more we’ll gain knowledge that may be applicable to a very large range of neurological and neuropsychiatric disorders.
MC: Along those lines, many children with neurological disorders suffer from seizures, chronic GI problems, and orthopedic issues. The approach thus far has been to try to ameliorate symptoms, but often standard treatments don’t work well and they really don’t address the underlying causes. Will existing faculty members or new recruits be focusing on looking more deeply into the mechanisms of these problems across different diagnoses?
HZ: Yes, absolutely. One of the ways information will be exchanged at the NRI will be through series of regularly scheduled seminars, and some of these will focus on a specific symptom. We bring together clinicians with basic scientists, presenting problems from both points of view. We will invite GI experts, bone experts. The very serious problem of uncontrolled epilepsy may be the first topic we explore in this way. A symposium on this topic is currently in the planning stage.
MC: And this leads into the situation of children who have symptoms but no diagnosis. There are girls who have a clinical diagnosis of Rett but no MECP2 mutations have been found for them. Will the NRI be a resource for these families?
HZ: Sequencing costs are coming down, so it’s feasible to look not only at the children but the parents as well. We are beginning an initiative between our NRI investigators and the genome center to do large-scale medical sequencing for these patients.
MC: On all fronts, then, the NRI is gearing up: Creative collaborative strategies, fresh angles of approach, in-depth examination of the symptoms that children suffer from in Rett and many other neurological disorders, and genomic investigation. You are really launching a powerful new interdisciplinary model for 21st century medical research. Thank you so much for your dedication to Rett research all these years, and for this interview. We hope to check in with you periodically for updates and anticipate great work from the Institute.
On November 11th the high-profile journal Cell published a paper by Alysson Muotri, Ph.D. entitled A Model for Neural Development and Treatment of Rett Syndrome Using Human Induced Pluripotent Stem Cells. The stem cell field has seen amazing progress in the last few years. Induced Pluripotent Stem Cells (iPS cells) is an especially hot area because of the clinical implications. Simply put, iPS cells allow you to study diseased cells up close and personal through their entire lifecycle. Importantly, any deficits that are identified in the cells can be used as read-outs in drug screening endeavors.
I’ve had the pleasure of knowing Dr. Muotri for a number of years, in fact since his introduction to Rett about six years ago. He became interested in the disorder while doing his post-doc in the lab of Fred (Rusty) Gage at the Salk Institute in La Jolla, CA. Thankfully his interest has continued now that he is an independent investigator at UCSD.
Dr. Muotri and colleagues discuss their new findings in a 4 minute video.
Below is an excerpt from a conversation Dr. Muotri and I had regarding his paper.
MC Dr. Muotri, congratulations on your Cell paper which has strong implications for drug development and therefore is of interest to anyone who loves a child with Rett Syndrome. I know this is a very hectic time so thank you for taking time out to speak with me.
I’m curious, what drew you to a science career?
AM I’ve always been interested in understanding how things work. I reasoned that science was the most obvious way to achieve that. You know that I’m from Brazil. I received my PhD in genetics from the University of São Paulo. I started off in the cancer biology field but quickly switched to neuroscience in 2002 when I moved to the Salk Institute. I was there for 6 years until I got my current position here at UCSD two years ago.
MC I’m assuming it was a significant switch moving into neuroscience from cancer biology.
AM Yes, it was a bit intimidating in the beginning because there was so much to learn. But I welcomed the challenge. And as it turned out my experiences from cancer were beneficial for my transition into neuroscience. For example, when I moved to Rusty’s lab one of the first observations we made was related to a phenomenon called transposons. I knew from my previous work that retrotransposons are very active in cancer cells and I remember discussing this with my neuroscience colleagues. Most of them were not very familiar with this phenomenon and assumed it was insignificant. My feeling was that if these transpositions were really happening in the brain it would be better to look at it closely because it could be involved in a new mechanism related to brain development.
MC Since retrotransposons are actually the topic of your next Rett paper coming out in Nature soon let me take a moment to give our readers a little background information.
Retrotransposons are sequences of DNA that move around and insert themselves into new positions within the genome. Barbara McClintock received the Nobel Prize in 1983 for her discovery of this phenomenon. Historically retrotransposons have been considered “junk DNA” because they occupy around 50% of the mammalian genome and do not have a clear function in the cell. It’s probably more likely that transposons have a biological function which remains, for the moment, unknown to us. Retrotransposons have been linked to disease.
Dr. Muotri, would you like to give us a glimpse into your upcoming paper that deals with retrotransposons in Rett Syndrome?
AM So the idea is that retrotransposons , which jump around inserting themselves into the genome, result in neurons, in the same individual, which are genetically different from each other. We observed that MECP2, the gene involved in Rett Syndrome, is a major repressor of this activity. Also, we determined that these jumping events are pretty much exclusive to the brain and MECP2 seems to be one of the gate keepers controlling the amount of the activity.
MC So in a brain that is deficient in the MeCP2 protein there would be increased jumping events?
AM That is right. It remains to be seen whether these extra events contribute to the symptoms of Rett or whether the brain simply compensates and manages to work around them. We are working on this question now.
MC Fascinating. I look forward to continuing our dialogue on this subject as your research progresses. Two high profile papers in one month – very impressive.
Now, getting back to iPS. This is a field that has seen amazing advances in a short period of time. Can you highlight for our readers the excitement surrounding these cells?
AM The dream of neuroscientists is to understand the early stages of a neurological disorder. Until recently we had two options to achieve this. One is to develop a mouse model that will hopefully recapitulate the symptoms seen in humans. Of course a limitation of a mouse model is that it’s a mouse and not a human – the brain of a human is so much more complex. The other option is post-mortem brain tissue. The problem is that at that stage the damage is already done and what you see is the end stage of a disease. To really study a disease it’s beneficial to have the most primitive cell line possible and then to coax these cells into a variety of different cell populations and to study what happens at various time points.
An important breakthrough happened a few years ago that has made this type of work very feasible. The Japanese group headed by Shinya Yamanaka surprised the world when they showed that you can reprogram cells that have already differentiated back to a more naïve state resembling a human embryonic stem cell. This allows us to capture the genome of a person, including any genetic mutations, and allows us to study the neurons and other cells of interest and see how a disease progresses and what changes happen at the molecular level.
MC In general the Rett field has relied on the mouse models as their standard assay. In terms of drug screening that’s a very expensive assay. Having iPS lines with MECP2 mutations gives scientists the ability to have a cellular assay to screen for therapeutics. Thousands, and in fact, hundreds of thousands of compounds can be efficiently and quickly screened in cell lines using either low or high throughput technologies.
Can you tell us about the phenotypes that you have identified in the cells. (a phenotype is an observable characteristic or trait)
AM One of the phenotypes was related to cell soma size (cell body) of a neuron. Just looking at neurons under the microscope we saw that Rett neurons are reduced in size by 10%. That might not seem like a big deal but when you consider the 3 dimensional structure of the neuron; a 10% reduction is very significant. So size was the simplest read-out that we found.
Another phenotype is related to the morphology of neurons. (Morphology is the study of the structure and form of an organism.) The idea to look at morphology was inspired by the reports over the last decade from post-mortem brain tissue in both people and animal models. We focused on the number of spine densities in neurons and we also saw a reduction. (A dendritic spine, or simply spine, is a small membranous protrusion from a neuron’s dendrite that typically receives input from a synapse.) We looked at neuronal networks and found deficiencies in their ability to communicate.
MC You made iPS cells with different MECP2 mutations. You found that the phenotypes were consistent among mutations. Can you elaborate?
AM Yes, the four different mutations we studied led to similar phenotypes. At least the phenotypes we looked at. We were convinced that this was a strong suggestion pointing to a loss of MeCP2 function. Thus, we knocked down MeCP2 expression from control neurons and obtained the same result. We then, restored the normal MeCP2 gene in Rett neurons, suppressing the phenotypes. In combination, these experiments suggest that MeCP2 is responsible for the alterations in Rett neurons. The fact that several MeCP2 mutants revealed a similar phenotype has clinical relevancy because it may indicate that a single drug may correct them all.
MC So the goal is to use these cells as a platform for drug screening.
AM Absolutely. As a proof of principle we added a growth factor, IGF1, to the cells. As you know a paper was published in PNAS in early 2009 showing that a compound similar to IGF1 improved some of the symptoms in mice so we decided to try it in our system. We found that IGF1 corrected the phenotype, in fact it over-corrected. The over-correction is something that needs to be considered in terms of the clinical trial, a proper dose tuning in each patient is desirable. Also, something to keep in mind is that while I put IGF1 directly into the cells in the clinical trial the IGF1 has to get into the brain and we know that that doesn’t happen as much as we would like.
The other drug we tried is gentamicin , an antibiotic that has the ability to “read through” premature stop codons (nonsense mutations that end in X, such as 255X, 168X) . We found that gentamicin restored levels of the MeCP2 and phenotypically rescued the cells.
MC That is pretty interesting especially in light of the fact that read through drugs act by substituting the stop codon with a random amino acid. So in effect they swap out one mutation for another.
AM We checked that and found that the protein level was normal but there was no way for us to see what mutation was inserted. Part of the new protein that is synthesized in the presence of gentamicin, is probably correct and we believe that is exactly what is reverting the phenotypes.
MC It’s important to note that gentamicin is highly toxic and doesn’t cross the blood brain barrier very well either so this is not a drug that can be used now for the treatment of Rett Syndrome. There are however other drugs with similar modes of action that being tested in animal models.
But the take home message from your data is that iPS cell lines are an in vitro model system for Rett Syndrome and can be utilized in a drug screening platform. What are next steps to utilize the iPS lines as a platform for drug screening?
AM The next step is to scale this up and that is not easy to do. Because the experiments are very sensitive to variables and there are many steps during the conversion of the iPS cells to neurons. Thus, we need to systematically validate all the variables and make the system as robust as possible. Finally, we need to choose the appropriate read-outs (the cellular phenotypes) we would like to use. It is important to design these experiments carefully so one doesn’t lose time with false positives. My lab was recently awarded a CIRM grant (California Institute for Regenerative Medicine) exactly to optimize these steps, so I would like to start this as soon as possible. Finally, I would like to test libraries of drugs that previously failed clinical trials for other diseases. Drug repositioning, as this concept is called, is attractive because repurposed drugs can bypass much of the early cost and time needed to bring a drug to market.
MC I found your paper very encouraging for a number of reasons. Firstly, your data continues to confirm and validate the concept that Rett is reversible. Secondly, you showed that the iPS platform can be used for drug screening. Thirdly, your data suggests that while there may be many mutations in MECP2, they may share common phenotypes. That may be an important issue in terms of treatment strategies.
Dr. Muotri, I’m sure I speak for every Rett family who reads this interview … we wish you great luck and god speed in your work.