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A paper authored by Huda Zoghbi and Jianrong Tang at Baylor College of Medicine and published in Nature describes improvement in learning and memory paradigms in mice models of Rett after deep brain stimulation (DBS). This research was funded, in part, by RSRT.
DBS is a surgical procedure that involves implanting electrodes in specific areas of the brain. The electrodes are attached to a pacemaker-like device placed under the skin in your upper chest that generated electrical impulses.
The disorders most commonly treated with DBS include Parkinson’s disease, essential tremor and dystonia. The procedure is also being studied as a treatment for epilepsy, cluster headaches, Tourette syndrome, chronic pain and depression.
While the procedure looks daunting neurosurgeons view it as rather routine.
Here is a remarkable video showing DBS surgery for a violinist who was having difficulty playing due to tremors.
The experiments conducted at Baylor targeted a brain region called the fornix. While improvements were seen in learning and memory no changes were observed in other symptoms such as anxiety, motor coordination, social behavior, body weight. It will now be important to see whether targeting other brain regions via DBS will result in improvements in these symptoms.
Below is a podcast between Dr. Zoghbi, Dr. Tang and the RSRT Executive Director, Monica Coenraads. The scientists describe the highlights of their experiments and key next steps.
by Kelly Rae Chi
Rett Syndrome is caused by a variety of mutations in the MeCP2 protein, but in some instances, MeCP2’s end is missing. A graduate student in Developmental Biology at the Baylor College of Medicine in Houston, Steven Baker, who is also in the medical scientist training program, was sifting through the clinical literature on boys with such mutations when he noticed that a tiny difference in how much of the protein’s tail is shortened—by just three amino acids—seemed to make the difference between decades of life (albeit with Rett-like deficits) and death in infancy.
Baker asked his adviser, Huda Zoghbi, whether she thought those extra few amino acids could so dramatically change the clinical progression of Rett.
“I don’t know,” Zoghbi, Howard Hughes Medical Institute Investigator and director of the Jan and Dan Duncan Neurological Research Institute at Texas Children’s Hospital, recalls telling him, because other genetic factors could be contributing the dramatic difference in the progression of Rett. “The only way to know is to make mice that have the two different mutations.”
So they did. One mouse had the end of its MeCP2 cut off at the 270 mark (‘R270X’ mice) while another’s protein was shortened at the 273 mark (‘G273X’ mice). The stories of these mice were reminiscent of the boys Baker had noticed. The R270X mice died prematurely, around the same time as mice with no MeCP2. In contrast, the G273X mice, with their extra three amino acids, survived longer and showed symptoms later, although these features became more severe and the mice died before healthy mice did.
What do those amino acids do? In trying to find out, the scientists have refined their understanding of how MeCP2 works. Their results are published this week in Cell.
A first look at DNA binding:
Researchers know that MeCP2 binds throughout the genome, coating the DNA in neurons more heavily than in other cells, but what exactly it does after that is less clear. It’s thought to turn genes on or off, or alter the overall structure of DNA.
Zoghbi’s team thought that the two truncated forms of MeCP2 might bind to DNA differently — in a way that would help explain the different clinical progressions of the boys — but when they initially looked at several spots within the genome they saw that both forms bound to those spots similarly.
In fact, looking more broadly across the genome the group found that overall binding of the MeCP2 was pretty much the same, and it looked normal. (The latter wasn’t too surprising, though, because the front end of MeCP2 was already known to bind to DNA.) Both mutations also interfered with the normal ability of MeCP2 to repress genes.
Looking more closely at gene expression at various time points in brain development, however, the group found a key difference in the two mutants: at 4 weeks of age, a small group of genes was improperly expressed in the R270X mutant but not in the G273X mutant. Interestingly, most of those genes eventually became misregulated in the G273X group by 9 weeks.
The hook from the animal kingdom:
Meanwhile, the scientists asked evolutionary biologist, Olivier Lichtarge, a Professor at Baylor College of Medicine who uses computational tools to study the evolution of protein sequences, to help them compare the sequences of the protein’s tail across different species (the idea being that any regions of the protein that were highly similar across species might be important for the protein’s function). “We worked with Angela Wilkins in the Lichtarge lab and asked, ‘Is there something in this domain that’s really unique?” says Zoghbi, who is also a Professor at Baylor College of Medicine.
They found that three clusters of MeCP2’s tail were highly conserved across fish, frog, rat, mouse, and human. Shortening MeCP2 at the 273 mark removed the third of those conserved clusters, whereas cutting the tail at the 270 mark deleted the second and third clusters.
What are these clusters in MeCP2’s tail and what do they do? It turns out that they’re called AT-hooks. In 2005, a study by Adrian Bird’s group described the first of those three AT-hooks (which is the one still present in both of Zoghbi’s new models), though its function was unclear.
AT-hooks are regions of a protein that are already well known to bind DNA, however, so the team went back to the idea that the two truncated proteins might differ from one another in how they attached to the genome, even though their initial results had shown that binding was similar. Using a different assay, they found that missing the second AT-hook domain impaired the ability of the R270X to steadily interact with certain sequences in the genome.
The interaction between MeCP2 and DNA:
Our genomes are wound tightly around spool-like proteins called histones; the DNA and histones (together, called chromatin, which looks like beads on a string) are then packed in even more so that it can all fit inside cells. Using an experimental model of compaction in vitro, the team found results suggesting that R270X mice (which, remember, are missing an two AT-hooks instead of one) don’t pack up chromatin as well as 273X mice do.
The initial finding that both mutated forms of MeCP2 bind to DNA are still important though, Zoghbi says. “It tells us that the major binding to DNA happens. That’s the first step.” The researchers think that once the front end of MeCP2 sits on DNA, the AT-hook clusters on its tail come in manipulate the DNA further, likely bending or altering the structure to help it pack further into the cell.
“There were hints previously that MeCP2 might cause a change in the overall conformation of DNA. The new study is probably the most direct evidence,” says Howard Hughes Medical Institute Investigator Gail Mandel, at the Oregon Health and Science University in Portland, who was not involved with the study.
The protein partner ATRX:
When the MeCP2 sits on DNA, and likely alters the way it packs into a cell, there are other molecular partners that come and join it. One of those is the protein ATRX—whose mutations have been linked to Alpha-thalassemia mental retardation syndrome—and Adrian Bird’s lab has previously shown that its interaction with DNA is disrupted in mice missing MeCP2. Zoghbi’s team decided to look at this protein in their new mutant mice.
Compared with the healthy mice and the G273X mice, ATRX goes missing from the tightly packed DNA of neurons earlier in life for the R270X mouse, and this loss mirrors the quicker onset of Rett symptoms. “To us, that was really interesting,” Zoghbi says, “because this change is not because the neurons are sick, it’s because you don’t have MeCP2 functioning properly.”
Studying female mutant mice that are missing a copy of MeCP2, the researchers found those brain cells with no MeCP2 also had less ATRX bound to tightly packed DNA compared to controls. In female mutants with too much MeCP2, an excess of ATRX latched on to DNA. MeCP2’s absence from liver and non-brain organs didn’t affect ATRX binding in those organs, suggesting that MeCP2 has a mechanism that’s specific for the brain.
Taken together, these results suggest that in the brain, the AT-hook clusters on MeCP2’s tail are manipulating DNA in a way that’s crucial for the other protein partners to bind and do their jobs, Zoghbi says.
“This new paper is beginning to shed light on the complexity of this interaction between MeCP2 and ATRX,” says Mandel. In addition, “we don’t know all the other proteins that bind to MeCP2, but the guess would be that there are likely more partners affecting whether genes are on or off.”
Zoghbi’s team hopes to understand how shortening MeCP2’s tail changed chromatin structure without dramatically changing gene expression — as well as the mutation’s affect on brain activity. They also plan to do biochemical and molecular experiments to figure out where ATRX is going and what it’s doing when its distribution is altered in the brain cells of the MeCP2 mutants.
For Zoghbi, the new findings underscore the importance of going back to patients to look for clues about MeCP2’s function. In 1999, Zoghbi first showed that various mutations in MeCP2 caused Rett. “Here we are 14 years later, some of these human mutations are teaching us lessons,” she says. “The variety you get and the breadth of human features you can dissect and go back and study in the mouse are really very humbling.”
Kelly Rae Chi
[links to podcasts are below]
That mutations in the MECP2 gene cause Rett Syndrome has been known for over a decade. But what exactly the protein does is not yet clear.
In the early 90s, Adrian Bird’s group purified MeCP2—which stands for methyl-CpG binding protein 2—and named the protein for its ability to bind parts of the DNA with a chemical tag called a methyl. Methyls tend to dampen the expression of genes, suggesting that MeCP2’s function is to silence genes.
Studies published since then suggest MeCP2 activates or represses the expression of many genes. Other results suggest that the protein binds throughout the genome, influencing the way DNA packs into a cell.
New evidence, published today (December 21) in Cell, shows that MeCP2 binds to spots throughout the genome that are tagged with the chemical, 5-hydroxymethylcytosine (5hmC) in mice, and that this interaction may be important for understanding Rett Syndrome.
“[The study is] a very interesting new development in studying the functional significance of MeCP2, which we have to understand if we’re going to understand Rett Syndrome,” says Bird, professor of genetics at the University of Edinburgh in Scotland who was not involved with work.
Abundant in the DNA of certain brain cells, 5hmC seems to be a signpost of sorts for active spots within the genome — that is, the regions that are churning out new protein — the study found. MeCP2’s latching onto these sites supports its potential role as a gene activator, though it’s clear that the case of what MeCP2 does is far from closed.
“Whether or not [MeCP2 is] directly involved in activation is still a matter of further investigation. But we know that it can localize to a region that contains 5hmC and active genes,” says Skirmantas Kriaucionis, group head in the University of Oxford Nuffield Department of Medicine, who co-led the study with Nathaniel Heintz of the Rockefeller University in New York.
Podcast with Skirmantas Kriaucionis
Figuring out 5hmC
As a postdoctoral researcher in Heintz’s lab, Kriaucionis found 5hmC in 2009 by accident when he was looking at how a closely related chemical, 5-methylcytosine (5mC), influences genome structure in brain cells. Anjana Rao’s group, then at Harvard Medical School, working independently from Heintz’s lab, confirmed the existence of 5hmC in the same issue of the journal.
Researchers consider 5mC a fifth base, and 5hmC a sixth base, of DNA, which is traditionally thought of as a string of four different chemicals called nucleotides. 5mC and 5hmC resemble the traditional base cytosine, but with a methyl group added on, making 5mC, and a hydroxy group added to the methyl, creating 5hmC. The new study confirmed that patterns of these chemical modifications to the genome are different in each cell and influence which genes are turned on or off and when.
The discovery of 5hmC opened up a new area of work—and hundreds of new papers—focused on where the nucleotide is in the genomes of different cell types, and what it’s doing.
In the new study, using an explorative molecular assay to fish for a binding partner for 5hmC, the group identified the molecule as MeCP2, and nothing else. “It was really a surprise,” Kriaucionis says.
“This paper is the second paper to suggest a candidate binding protein for 5hmC,” says Rao, now a professor of signaling and gene expression research at the La Jolla Institute for Allergy and Immunology, who was not involved with the new study. Other findings have proposed a different molecule, MBD3, as a candidate, but those and the new results need further investigation, she adds.
“So far, both candidates — MBD3 and MeCP2—also bind 5mC, so an exclusive binding protein for 5hmC has not yet emerged,” Rao says.
Indeed, contradicting the new evidence that MeCP2 binds 5hmC and 5mC equally, some previous studies show that MeCP2 much prefers binding to 5mC over 5hmC. For example, a study published earlier this year shows that says MeCP2 is nearly 20 times more likely to bind 5mC than 5hmC.
Relating to Rett
In the new study, Kriaucionis and his colleagues observed that a certain Rett-causing mutation, called R133C—which is responsible for a relatively milder form of the disorder—disrupts MeCP2’s binding to 5hmC.
“[The R133C mutation] is really interesting because it allows us to speculate that MeCP2 binding to 5hmC is important as a part of the function which causes Rett Syndrome,” Kriaucionis says.
The evidence now “strongly suggests” the potential involvement of 5hmC in Rett, says Peng Jin, an associate professor of human genetics who was not involved with the new study. A study his group published last year in Nature Neuroscience found that patterns of 5hmC are altered in mouse models of Rett.
Interestingly, the R133C mutation only slightly dampens MeCP2’s interaction with 5mC, suggesting that MeCP2’s binding with 5mC serves a different purpose than that of MeCP2 and 5hmC.
Other Rett-causing mutations in MeCP2 examined by the group don’t seem to affect binding to 5hmC, meaning 5hmC binding does not fully explain the symptoms of Rett. “It will be important to test further the contribution of 5hmC to Rett Syndrome,” notes Jin, adding that there are mouse models available to do so. Those mutant mice lack the enzymes needed to convert 5mC to 5hmC.
In the new study, researchers studied only a few types of neurons, but there are hundreds of cell types in the brain. Kriaucionis thinks that MeCP2 binds to 5hmC in other cells.
The 5hmC patterns themselves are cell-specific, however, perhaps further complicating the story of MeCP2.
“We really need to get more data to understand whether or not, how different 5hmC and MeCP2 localization would be in different cell types,” Kriaucionis says. “It’s an important component to understand MeCP2 function,” and how scientists might think about future treatment.
Cell podcast with Nat Heintz (click on Paperclick on right)
by Kelly Rae Chi
In September of 2011, RSRT met with the National Institute of Neurological Disorders (NINDS) and other public and private organizations that fund Rett Syndrome research to discuss crucial knowledge gaps in the field. The main findings of the workshop were published recently in Disease Models & Mechanisms.
In particular, the meeting focused on how the research community can improve its chances of success in clinical trials. Preclinical studies require a huge investment of time and effort studying disease in rodent models. Even then, for a variety of reasons, drugs that show promise in preclinical studies will often fail in the clinic.
Here are a few big hurdles in preclinical animal studies — some that are specific to Rett research — and how experts are meeting those challenges.
1. Studying female mouse models of Rett.
“It’s important that when we do a drug trial, that we really impact features that are clinically meaningful, features that are going to impact patients,” Huda Zoghbi of the Baylor College of Medicine in Houston, Texas, told RSRT in a recent interview.
Like girls with Rett, however, female mouse models of the disorder vary in the type and severity of their symptoms, which makes them harder to study than males.
That’s because the gene missing or mutated in Rett, MECP2, is located on the X chromosome. Female mice — which, like girls, have two X chromosomes, only one of which is active — will have either mutated protein or normal protein levels, depending on which copy is expressed in the cell. Rarely are they missing all of their MeCP2 protein.
In contrast, male mouse models missing the Rett gene have no protein at all. Although these mice have paved the way in understanding the protein’s role in the brain, when it comes to treating Rett, results from studies of male mouse models may not be the ideal model to work with.
More researchers are turning to female mouse models. Zoghbi and Rodney Samaco, also at Baylor, for example, published a study in October in Human Molecular Genetics, describing two different female mouse models of Rett in detail. Detailed characterization of these mice will help lay the groundwork for preclinical studies.
2. Unknowns about how an animal’s environment affects therapeutic efficacy.
No two research labs are alike. The ways in which they differ, including animals’ access to food, housing, lighting or other environmental factors, might well influence an animal’s response to a drug.
What’s more, an individual mouse’s genetic environment — meaning the genetic background on which Rett mutations are made — affects some of its symptoms, such as obesity and abnormalities in social behavior. These genetic differences may also affect how animals respond to treatment.
Variability in genetic and environmental conditions plague scientists studying many conditions, not just Rett syndrome. One way to help address this obstacle, according to Rett researchers in the Disease Models & Mechanisms workshop summary, is to study symptoms and potential therapies across a variety of models and in many lab settings. Those mouse models that show consistent results across different environments will be most useful for translational studies.
3. Recapitulating speech problems in mice.
Of the many symptoms seen in Rett, loss of speech is among the most challenging to study in a mouse model. Some groups have shown that Rett mouse pups produce unusual vocalizations when they’re separated from their mothers in early postnatal life. These sounds are either more or less frequent than in healthy controls, depending on the mouse model studied. Future work will need to sort out these conflicting results and identify a mouse model that best captures this hallmark symptom of Rett, researchers say.
4. Avoiding bias, which can prevent preclinical errors.
Unintended biases can creep into animal studies. This can lead researchers to conclude a treatment is effective when it isn’t, or it can cause overestimations of a drug’s efficacy.
In recent years, researchers across numerous fields have stepped up efforts to improve study rigor. In June of this year, NINDS convened a panel of scientists, funders and journal editors to talk about how researchers can do a better job reporting methods in preclinical animal studies; both in grant applications and journal publications. At the very least, the panel concluded in a perspective published October in Nature, researchers should report on the following practices:
- Randomization, where animals are randomly assigned to receive either treatment or placebo;
- Blinding, where researchers doing the experiments or analyzing the data are unaware of whether of which animals are receiving treatment or placebo;
- Sample-size estimation, a calculation of an appropriate sample size at the study’s outset;
- And how data is handled, for example, deciding on study’s primary endpoints, or how to handle missing data points or outliers, before starting the study.
Not reporting such details has, in the past, been linked to overestimations of therapeutic efficacy, according to the NINDS report.
Now Rett researchers have added their voices to the mix in the Disease Models & Mechanisms report, voicing their support of NINDS’s recommendations and emphasizing the need for rigorous experimental design.
Ever wondered why most labs use male Rett mice for their experiments even though the females are the better model? What human symptoms are replicated in the Rett mice? What are some of the surprises these mice have in store for us? What are the complexities of doing well-designed and executed trials in mice? What are some of the pitfalls that the Rett field needs to avoid? What is the potential for the newly unveiled Rett rat? Listen and find out….
This past week, more than 30,000 neuroscientists convened in New Orleans for the annual Society for Neuroscience meeting. Here are some of their interesting (unpublished) findings on Rett Syndrome.
Recent progress in genetic engineering has made it possible to model Rett Syndrome in rats – whose behavior is easier to study than mice. Researchers led by Richard Paylor from the Baylor College of Medicine in Houston, Texas, designed a set of behavioral tests to capture the animals’ social interest, anxiety, vocalizations and sensory and motor abilities. The team found that male rats whose Mecp2 (the gene that is missing or mutated in girls with Rett Syndrome) is disrupted were less active, showed impairments in a memory task, and behaved in a way that suggested they have disrupted connections between sensory and motor brain areas.
IGF-1 on trial
Researchers administered a full-length version of insulin-like growth factor 1 (IGF-1) — which is now under clinical investigation for treating Rett Syndrome — to mice lacking Mecp2. This particular form of IGF-1 boosted neuron-to-neuron signaling and the ability of neural connections to change in strength, compared with untreated mutant mice. According to a report by SFARI.org, however, treatment did not improve mutant mice’s performance on a task of motor coordination and learning.
In a separate study of mutant mice, led by Jeffrey Neul’s team at Baylor, scientists administered a slow-release form of IGF-1, or PEG-IGF1, finding that it only slightly lengthened lifespan but did not improve heart rate, body temperature, breathing, motor function, or behavior. And a higher dose of PEG-IGF1 cut the lifespan of mice, the group found.
Neul is planning a clinical trial in adult women with Rett with a shorter, tripeptide form of IGF-1, which in 2009 scientists found delayed the onset of several symptoms of the disorder in a mouse model.
In 2010, Huda Zoghbi and her colleagues at Baylor showed that neurons that dampen brain signals through their production of the inhibitory chemical GABA (gamma-aminobutyric acid) play an important role in the development of Rett.
For a preliminary study presented this week, Zoghbi’s group found that reactivating Mecp2 expression exclusively in GABA neurons well into adulthood (6 and 9 weeks) improved two symptoms of Rett — obesity and ataxia — in male mice missing Mecp2. Her group is also measuring cognitive and breathing symptoms after selectively reactivating the gene in GABA neurons.
Reprogrammed Rett cells
Alysson Muotri at the Scripps Research Institute in La Jolla and his collaborators took cells from human males with Rett and converted them into induced pluripotent stem (iPS) cells, which have the ability to form any other cell in the body.
The group found that several molecular signaling pathways differ between the iPS cells of healthy people and individuals with Rett as their cells begin to form neurons. These early changes may underlie neuronal features of Rett. The scientists are working to validate the biochemistry, but report that the new findings suggest that iPS cells derived from people with Rett may help identify new drug targets.
Progress on point mutations
Two years ago, researchers from the Barrow Neurological Institute in Phoenix, Arizona, described a new Mecp2 mouse — the A140V model —that reproduces a point mutation (meaning a single “letter” of the DNA code is replaced).
Male mice with the mutation survive, though they have X-linked mental retardation and show some brain abnormalities — such as less intricate neuronal branching and more tightly packed cells – compared with healthy mice. Unlike other mutants, however, the A140V has a normal lifespan and weight gain and no seizures or trouble breathing. The same group presented a detailed protocol to characterize the shape and size of brain cells of female mice that carry the mutation.
by Monica Coenraads
It would be difficult to overestimate the importance of what we have learned from the mouse models of Rett Syndrome. After all, without them we would not know that Rett is reversible.
It may come as a surprise that there is no single mouse model of Rett but rather a variety of genetic models, from “KO” or “knock-out” mice, which have no MeCP2 at all, to those in which the precise MeCP2 mutations that are seen in humans suffering from Rett Syndrome have been duplicated.
Jackson Laboratories in Bar Harbor, Maine currently distributes almost a dozen mouse models of Rett. Jackson (or Jax, as most scientists refer to it) is a non-profit organization that specializes in this work to advance the understanding of human disease. Although no animal model perfectly capitulates human symptoms or responses, 95% of our genomic information exists also in rodents. Maintaining an extraordinary level of care and attention to detail in this sensitive field, Jax conducts its own research as well as breeding and managing colonies of thousands of models.
To learn more about Jax please read an earlier blog post, Of Mice and Men…Or in the Case of Rett…Of Mice and Women.
Having access to the various models of Rett Syndrome is crucial to the advancement of research and this is an area that RSRT and its predecessor, RSRF, have been actively involved in since the first animal models were published in 2001 by RSRT Trustee and advisor, Adrian Bird and by Rudolf Jaenisch.
From the Jax website: “Partners in the fight against Rett syndrome,” a story about how Monica Coenraads, the mother of a daughter with Rett syndrome and co-founder of two organizations focused on treating it, is working with Dr. Cathy Lutz to develop and distribute new mouse models of Rett syndrome.
Sharing mouse models is not always the norm as this article about a mouse model for Angelman Syndrome illustrates. We are extremely fortunate that researchers in the Rett field have been stellar about quickly sharing their models. Adrian Bird, Rudolf Jaenisch and Huda Zoghbi have set an exemplary high bar when it comes to making their own mouse models available as soon as they publish.
In some cases sharing the mouse has even preceded publication. Last year Nature reported on a situation regarding a Rett mouse that had been developed by Novartis. The model had been engineered so the Rett protein glowed so it could be tracked visually. Many researchers were eager to access the model but could not due to legal issues. (Nature article – Licence rules hinder work on rare disease. Animal model off-limits to Rett syndrome researchers.) I knew that Adrian Bird after being denied access had created his own model and I asked him whether he would be willing to share it through Jax. He agreed immediately even though he had not published yet. The mouse is now available to any researcher worldwide that needs it.
Besides teaching us about the molecular underpinnings of disease, mouse models may be effective to test treatments. We have all heard many stories about how drugs tested in mice with success are found to be ineffective in humans. The next few years will be extremely interesting as we begin to explore how predictive the Rett mice models really are.
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
Last month brought me to Houston, Texas to attend a fascinating meeting organized by Huda Zoghbi and Morgan Sheng and co-sponsored by RSRT. Entitled Disorders of Synaptic Dysfunction, the event was the inaugural symposium of the recently established Jan and Dan Duncan Neurological Research Institute, directed by Dr. Zoghbi.
The two-day meeting brought together a heterogeneous group of scientists from academia (senior and junior faculty as well as post-docs and graduate students), industry, NIH and other funding agencies.
The focus was not on a single disease but rather on a group of disorders (Rett, Angelman, Fragile X, autism, Tuberous Sclerosis) that share a common cellular phenotype: abnormal synapse activity.
It’s no surprise that some of the talks that generated the most buzz came from labs that are doing very clinically relevant research. These include the labs of Mark Bear at MIT, working on Fragile X, and Ben Philpot at UNC whose lab works on Angelman Syndrome.
Like Rett Syndrome, Fragile X is a single gene disorder, caused by mutations in a gene called Fmr1. When Fmr1 is mutated, protein synthesis fails to shut down, leading to excess. Some years ago Dr. Bear proposed that compounds which can block a certain type of receptor, mGluR5 (which triggers the burst of synaptic protein synthesis) might counteract over-expression of protein and thereby cancel out the damaging effect of Fmr1 deficiency. His theory has proved correct, and clinical trials of mGluR5 antagonists are currently ongoing at multiple pharmaceutical companies.
I first met Dr. Bear almost a decade ago, when he was just beginning to formulate what is now commonly known as the mGluR5 theory of Fragile X. His lab is currently funded by RSRT to explore protein synthesis in the Rett mouse models. Dr. Bear hypothesizes that Rett may be due to under-expression of proteins. If his hypothesis holds up, pharmacological manipulations of mGluR signaling will be pursued.
Ben Philpot’s talk also generated excitement. He discussed a high-throughput screen that has yielded a compound which can activate the silenced Angelman Syndrome gene, UBE3A. Dr. Philpot is currently funded by RSRT to pursue a similar approach for the silent MECP2 gene on the inactive X chromosome.
Mike Greenberg spoke about MECP2 and shared unpublished data that has come about from his collaboration with Adrian Bird via the RSRT funded MECP2 Consortium. (More on that in the months to come.)
Jackie Crawley of the NIH gave a brilliant talk on how “autistic mice” are being characterized to yield a plethora of new information. For me the highlight of her talk was hearing recordings of mouse “speech”. She shared a variety recordings and I was taken aback by the complexity and richness of the sounds, which left me yearning for an analysis of Rett mouse vocalizations.
After a lively cocktail hour it was back to work with dinner plates in hand. Drs. Zoghbi and Sheng divided the attendees into three working groups: 1) dysfunction of proteins of the synapse 2) dysfunction of nuclear/cytoplasmic proteins 3) young investigators and junior faculty. Masquerading as a 30-something I happily joined the third group. I was struck by the fearlessness and boldness of these young scientists. There were not shy about criticizing the status quo and what could be done differently to enhance the research progress. I came away feeling buoyed and reassured that science is in good hands with this new generation.
The following several hours of discussion, led by Rodney Samaco and Mingshan Xue and facilitated by NIMH Director, Tom Insel, were intellectually stimulating and entertaining. Below is a visual output of our intense discussion.
A few personal reflections on the symposium
- Over and over again throughout the meeting I heard comments from autism researchers such as: “Where would we be without the syndromic autism animal models like Rett and Fragile X? We’ve learned so much from them”. More than once I found myself thinking that as horrible as Rett is at least the genetics of the disorder are clear-cut – Rett’s silver lining.
- The meeting provided an opportunity to meet some scientists with whom I had communicated by email and/or phone, but never met in person. People like Pat Levitt, Freda Miller and Michael Palfreyman. It was a reminder of how many people over the years have taken the time to discuss their work and possible synergies to Rett Syndrome.
- Drs. Zoghbi and Sheng kept everyone busy from the moment the meeting started to the moment we left, including an intense working dinner. I tend to do the same thing at meetings that I organize, but always feel like I’m being a bit of a slave driver. Never again, however, will I feel guilty. If Dr. Zoghbi thinks it’s acceptable, then so do I!
Kudos to Drs. Zoghbi and Sheng for a stimulating meeting and thank you both for inviting me.
Science Translational Medicine, which co-organized the meeting, will be publishing a white paper on the proceedings.
RSRT will let you know when the paper is available.
by Monica Coenraads
Many of you know that my involvement in Rett Syndrome is personal. I have a daughter who suffers greatly from every Rett symptom in the book. She is now 15 years old and every year brings new challenges. In the last six months she has developed severe Parkinsonian symptoms: violent tremors, increased rigidity, difficulty initiating movement.
Despite the increased hardships I cannot help but be optimistic. The news from the scientific community continues to be encouraging and I have not heard one shred of data to dampen my optimism. As I reflect on the state of the current research I am particularly struck by one thing: the number of potential treatment approaches that we are pursuing in parallel. From gene therapy and exploration of modifier genes to repurposing of drugs, there is certainly no lack of ideas about how to reverse Rett Syndrome or modulate symptoms. That simple fact lifts me up even on those dark “Rett days.”
Today RSRT is pleased to announce that we are adding to our portfolio of potential treatment options with $515,054 of new funding for Huda Zoghbi and her lab. Dr. Zoghbi needs no introduction to anyone familiar with Rett Syndrome. She identified MECP2 mutations as the cause of Rett Syndrome in 1999 and has consistently added to our body of knowledge about the disorder, the animal models and the protein in the years since then. Simply put, the field of Rett would look very different without Dr. Zoghbi.
This latest award, entitled “Investigating Novel Therapeutic Approaches for Rett Syndrome” includes three separate objectives, each of which has potential clinical relevance.
The first objective tests a pharmacological intervention while the other two are aimed at altering the activity of the neural network.
1) Test drugs on Rett mouse models to enhance the cholinergic pathway.
This neurotransmitter pathway is critical for learning, memory and regulation of the autonomic nervous system. Drugs exist that can be used alone or in combination. If we find the data from mouse models encouraging, then the findings could be immediately transitioned into clinical trials.
2) Explore deep brain stimulation (DBS) as novel treatment strategy.
DBS has revolutionized the treatment of Parkinson’s and is now also used for depression, OCD, Alzheimer’s and more recently in pediatric disorders such as dystonia and Tourette. The availability of Rett mouse models allows us the opportunity to explore potential benefits of this procedure for Rett. Again, encouraging data can be quickly moved to the clinic.
3) Boosting Mecp2 levels in normal cells.
Girls with Rett have approximately 50% normal cells and 50% cells which lack the MeCP2 protein. Dr. Zoghbi will explore whether boosting MeCP2 levels in the cells that already have normal amount could enhance the overall neural network activity even though the other 50% have no protein. If boosting levels in normal cells rescues some of the symptoms this would set the stage for a large scale effort to identify targets that can modulate MeCP2 levels.
Please join me in congratulating Dr. Zoghbi on this award and wishing her the very best as she pursues these new lines of inquiry. I’d also like to take this opportunity to congratulate her once again on being awarded the prestigious 2011 Gruber Neuroscience Prize which was presented during last month’s annual meeting of the Society for Neuroscience.
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.
Anyone who keeps up with Rett research knows that the different mouse models of the disease have given us a rich knowledge base. But have you ever stopped to think of how scientists get access to these crucial models? Today we share with you a conversation between Cathleen Lutz of The Jackson Laboratory in Bar Harbor, Maine, and Monica Coenraads, Executive Director of the Rett Syndrome Research Trust. Jackson is the gold standard for the colonization and distribution of mouse models of disease.
MC: Thank you, Dr. Lutz, for spending some time with us. Tell us a bit about the background behind Jackson Laboratories.
CL: Jackson Laboratories was established by Clarence Cooks Little and Roscoe B. Jackson in 1929 as a genetics institute. Financial support came from Detroit industrialists such as Edsel Ford and Roscoe Jackson, president of the Hudson Motorcar Company, with land donated by family friend George B. Dorr. Of course, Bar Harbor has a long history of philanthropic summer residents who supported the Laboratory, for example the Rockefellers had settled on Bar Harbor.
Off the coast of Maine may seem like a strange place to have a genetics facility. The advantage to the location is that at the time there wasn’t any air conditioning, so the ocean breezes really kept the animal facilities cool. In the early years we didn’t have the ability to do genetic engineering, so essentially we relied on spontaneous mutations that resulted in interesting things to study.
MC: I’ve recently learned of veterinary schools setting up facilities to diagnose animals with spontaneous genetic mutations. For example, it’s possible that a dog with a mutation in MECP2 would be taken to vet and a bright geneticist might be able to diagnose the animal. This would allow different species to be studied without having to do all the expensive and time consuming genetic engineering involved with making models.
CL: In fact I just attended a seminar on this. Recently a naturally occurring form of ALS was identified in dogs. What is particularly interesting is that the canine form of ALS progresses slowly, unlike the human ALS where patients usually die within 5 years of diagnosis. The key question is what is genetically protecting these dogs?
MC: The hope is that genetic modifiers are protecting these dogs from their mutations in SOD1, an ALS gene. And if you can identify these modifiers it may open up avenues for intervention. We have the same situation in Rett. Currently RSRT is funding a project in the lab of Monica Justice at Baylor to look for genetic modifiers in the Rett mice models.
How many disease models would you estimate that Jackson has?
CL: We have over 5000 different strains here at the Jackson Laboratory.
MC: How many new strains are imported every year?
CL: We’re importing about 600 new strains every year.
MC: Is Jackson struggling to keep up given such large numbers?
CL: We have over 1300 strains live on the shelf and over the years have worked to meticulously manage the supply and demand of the strains so investigators can get a jump start on their experiments. We also scale up our colony sizes for individual investigators who need a larger supply of animals than we currently may have. For strains that have low demand, those mice are available from our cryopreserved stocks. Cryopreservation involves either freezing embryos or sperm. Dr. Robert Taft at Jackson has been on the cutting edge of that technology and recently published his technique that helps recover sperm much more easily. Animals can then be recovered from cryopreserved stocks as needed.
So instead of having to super ovulate 50 or 60 females, fertilize, and bring embryos to the two cell stage for cryopreservation, all we have to do is take two males and freeze down the sperm and that particular model is completely archived. We cut down on shelf space and cost.
MC: When a laboratory needs a particular strain which is cryopreserved, that means you don’t have a live colony; what do you actually send them?
CL: It depends on where the requesting laboratory is physically located and the level of their expertise. Cryopreservation is still a rather novel technology so some labs are not equipped to handle the technique of thawing sperm and doing in vitro fertilization (IVF). In those cases we can take the sperm, thaw it and do an IVF to donor females and then we’ll send them live mice. Alternatively we can send frozen viable embryos. This works well especially if the lab is an international customer because we have all kinds of handcuffs regarding transportation of live animals and tissues outside the country.
MC: How many scientists do you estimate have purchased from Jackson?
CL: Last year over 19,000 investigators from 50 countries purchased 2.7 million mice.
MC: That is unbelievable! How is Jackson funded?
CL: We are a not for profit organization with three prongs. We are a research organization; a resource organization, that’s the mouse distribution portion of our institution; and we run courses and conferences where we teach people the latest technologies.
Most of the research and courses are funded mainly through NIH grants. A large portion of our Mouse Repository is also funded through NIH program grants. The rest of the funding required for running the Repository comes from the fees we charge for the mice we distribute. The proceeds go right back into the operation to acquire more mice and outfit new facilities to expand the program. It’s very expensive to distribute mice because we have to maintain high health standards so that any institutions can receive mice knowing that they are free of viruses and pathogens that could contaminate their facility. We also have philanthropic donations.
MC: When I was the Director of Research at the Rett Syndrome Research Foundation we financially supported the importation and colonization of several Rett animal models at Jackson. That was money very well spent as those mice have now been distributed to hundreds of labs and have formed the foundation of much of what we have learned about Rett Syndrome.
You shared that in 2009, 95 different labs ordered Rett mice. The first Rett mouse model made by Adrian Bird was published in 2001, so Jackson had it ready for purchase in 2002. So eight years later almost 100 researchers bought this mouse.
CL: Yes, there is still a lot of demand for that animal, partly because it’s one of the better models of neurological disease. But it’s always going to take more than one model to really dissect what it is that you’re looking for. So if you want to ask specific questions it’s very helpful to be able to utilize more than one type of mouse model. So one model may have a point mutation, another may have a complete exon deleted, yet another may be a conditional mutation so you can just make that mouse gene defective in certain tissues and not others. When you put the collection all together it makes for a really good research resource…your toolbox, so to speak.
MC: I want to acknowledge the scientists who have developed the Rett mouse models: Adrian Bird, Rudolf Jaenisch and Huda Zoghbi. All of them quickly deposited their mice with either Jackson or the Mutant Mouse Regional Resource Center, thereby giving the research community at large access to the mice. This type of sharing does not always happen and I’m so grateful that they set a high standard for our community in terms of accessibility to these models. I hope that it’s a standard that others will follow.
MC: The recent ability to manipulate rat embryonic cells now makes it possible to create rat models of genetic disease. Does Jackson plan to expand into rat models?
CL: We’ve really talked about it a lot as genetic engineering in rats has come a long way in the last few years. One problem is that sperm cryopreservation in rats is still not as efficient as it is in mice. And the housing of rats is so much more expensive because they are so much bigger than mice.
So we have to realize analyze what the advantages of working in rats versus mice are.
MC: Rats are considered smarter than mice.
CL: Yes, they are. They are probably a better model for studying behavior, as well as learning and memory, which will be important in many neurological diseases. But the advantages of studying diabetes in a rat versus mice, for example, is less clear. There is a rat repository in Missouri run by John Critser. I think that Jackson will basically rely on the Missouri repository, working with them when and if needed.. But certainly we’d like to see the cryopreservation and the sperm recovery be just as easy and cost effective and efficient for rats as it is for mice so that we could we could cut the cost and make the process feasible.
MC: I wonder then how many labs would purchase rats. It would be a big learning curve to switch and the costs would be so much higher.
CL: Yes. That’s absolutely true. So there again I think researchers will really need to ask themselves what the advantage to using rats is for their particular research.
MC: Jackson also does its own research and has some high profile scientists on staff.
CL: We have 35 staff scientists on site working right now in a variety of areas. We have cancer biologists, neuroscientists, bioinformaticians. We have investigators who specialize in metabolic diseases like diabetes and obesity. We try to be as diverse as we possibly can in that respect.
MC: And why do you think the scientists would choose to work at Jackson and not at an academic institution?
CL: There are many factors but I think one of the attractions is the availability on site of all of the different mouse models. Also the sheer size of our operation means we can offer economies of scale. The per diem costs of mouse experiments are much lower than they would be at other institutions. That is a very attractive feature for scientists. If researchers need large numbers for their studies then this is the place to do it.
MC: Is there anything you would like to say to families of children with Rett Syndrome?
CL: I’d Iike to let people know that our mission at the Jackson Laboratories is really for the families, for the patients, and for biomedical research. We have, as I described, the repository and the disease model resources. It is quite an undertaking and we really feel that it is within our scientific mission to be collecting these animals and to be making them as readily available to the scientific community as we possibly can. That’s why we’re here and we feel that over the years we’ve really developed the expertise to do that and to manage the sheer numbers of strains that we have live on the shelf.
MC: Jackson truly provides an important resource for the scientific community. Thank you, Dr. Lutz, for sharing some of your knowledge with us today.
As many parents may already know, the Diagnostic and Statistical Manual of Mental Disorders, known as the DSM, is in the process of reevaluating criteria for the new edition to be published in 2013, the DSM V. There is discussion among members of the Rett community and the Asperger’s community about the decisions to drop both diagnoses from the manual. How this change might impact services, particularly intensive educational intervention for Rett children, is unknown and will probably vary from state to state. People who would like to express their opinions to the DSM committee may do so until April 20, 2010.
RSRT scientific advisory board member and Rett Syndrome researcher Huda Zoghbi , M.D. discusses the DSM reclassification with Monica Coenraads.
Huda Zoghbi will be appearing on the Charlie Rose Show on Tuesday, February 23. The episode, entitled “The Developing Brain” is part of the “Charlie Rose Brain Series” hosted jointly with Nobel Laureate, Eric Kandel, Ph.D. of Columbia University.
MC: What do you think was the impetus behind removing Rett Syndrome from the DSM?
HZ: My understanding is Rett was originally included in the DSM because it was a disorder with autistic features of an unknown cause. Now that the genetic cause has been identified, the rationale for removing Rett is that it is more its own distinct entity. Another reason pertains to the transient nature of autism features in Rett patients but this is not exactly the case. Rett patients do not have language skills and continue to manifest stereotyped behaviors for decades. Although some might acquire some social interaction skills through eye-pointing this is not true for all cases.
MC: Yet, if knowing the genetic cause of a disorder is the rationale for exclusion, in time, as more genetic underpinnings of disease are identified there will be fewer and fewer left for categorization by the DSM.
HZ: Correct. That is why I actually do not agree with this approach. I think the approach should be to see what clinically fulfills criteria for autism. I would be in favor of a more precise categorization and dividing DSM V into two types: DSM V A and B. One would be used for syndromic autism and one would be non-syndromic autism. There would be genetic etiologies for both syndromic and non-syndromic. Currently most of the known genetic causes are for syndromic autism but in time, as we do more sophisticated sequencing and we study patients with simplex autism (one case in a family, with no features other than classic autism) we will find etiologies for non-syndromic as well. In my view this would be a much more useful distinction. Bottom-line: having a known genetic cause should not eliminate a disorder from DSM V.
MC: The decision to remove disorders identified with a genetic cause seems very black and white to me. While knowing the root cause of a disorder is hugely important it often also brings many unanswered questions. Let’s look at Rett Syndrome itself – a certain percentage of girls/women with a clinical diagnosis of Rett do not have an identified mutation. And then we have individuals with MECP2 mutations who do not have Rett Syndrome symptoms. So, using Rett as an example of a genetic disorder, the situation is certainly not black and white.
HZ: Absolutely. In fact, the girls who have MECP2 mutations who fit the clinical criteria for autism and do not have Rett symptoms make a very compelling case against the current draft of DSM V. They represent a troubling scenario for an important patient population – what diagnosis do we give them? Where do they belong? So now with the proposed DSM criteria we have a category of patients that are left unattended to in this manual.
MC: It seems to me that the Rett clinical community, in general, was in favor of removing Rett. Do you have any insight into their reasoning?
HZ: The medical community appropriately focuses on clinical management: what treatments can be delivered to the patient, what code is used in the medical records for billing purposes, etc. These issues have probably driven the support of the clinical community for removal. On the other hand, if you are about solving the puzzle of these brain disorders and understanding the pathogenesis of the autism phenotype in Rett and beyond, then removal doesn’t make much sense to me. If you believe that the DSM manual is a tool to help us better understand brain diseases and to highlight the commonalities and differences between them, then I don’t feel taking out Rett serves the cause of disease-oriented research.
MC: Once DSM V is finalized will we be able to call Rett an autism spectrum disorder or will that be a misnomer?
HZ: I don’t think it will be a misnomer because clinically Rett is an autism spectrum disorder. Just because it’s taken out of a manual does not change the phenotype of the disorder. Imagine, for a moment, a girl comes to see me in the clinic. She used to speak but has experienced a loss of language, she has no social interactions, she has stereotypic behaviors. We evaluate her using ADOS (The Autism Diagnostic Observation Schedule – a standardized protocol for assessing social and communicative behavior) and the ADIR (Autism Diagnostic Interview-Revised) and the child fulfills all the criteria for an autism diagnosis. Yet she has a MECP2 mutation. What do you put in her chart? It can’t say MECP2 mutation because that is not a clinical diagnosis, it’s a genetic one.
MC: Of course we are already facing these complicated issues. And I think the proposed changes in the DSM may further complicate things. As you can imagine, I’ve received a plethora of emails and phone calls from parents who are wondering what this may mean for their child in terms of losing services. I think worries about losing medical services are probably not warranted. Worries about educational services, however, I’m much more concerned about. For example, it may become more difficult to obtain intensive ABA (applied behavioral analysis) programs and other educational supports where autism has blazed a trail.
HZ: Yes, I would agree with that prediction. It is really important to remember that autism spectrum disorders do not only overlap clinically but that some of their features respond to similar therapies in spite of different molecular causes. Therefore keeping an eye on the clinical similarities in face of genetic heterogeneity is one path to gain insight about the mechanisms underlying their common features and to develop therapies that might benefit more than one disease. I do hope the committee will take these far-reaching ramifications into account as they contemplate disease classifications.
MC: Thank you so much for sharing these thoughts with our readers. Parents and other advocates for those with Rett Syndrome, such as therapists, teachers or personal physicians, are encouraged to weigh in on this matter. Remember, the cut-off date for submitting comments to the committee is April 20.
Tenacity, talent and pure luck coincided ten years ago this week in a crucial experiment that forever changed the landscape of Rett Syndrome research.
by Monica Coenraads
Dr. Zoghbi examined her first patient with Rett Syndrome in the mid 1980’s and was so emotionally and intellectually hooked that she decided to put her nascent neurology clinical practice on hold and move instead into basic science. Her ambitious goal to locate the gene mutations responsible for this puzzling disorder was successfully realized sixteen years later.
Because Rett Syndrome is a sporadic disorder “gene hunters” could not employ traditional strategies to identify the culprit gene. Fortunately significant clues came courtesy of several families with multiple affected members and the location was narrowed to a specific section of the X chromosome – Xq28. What followed was a painstaking candidate gene approach analyzing each of the hundreds of genes located on Xq28. Visit an earlier blog post to read in Dr. Zoghbi’s own words the details of the gene discovery.
During the summer of 1999 my daughter, then three years old, had been diagnosed for less than a year. As any parent of a newly diagnosed child will testify the year had been marked by a rollercoaster of emotions. With the shock and the grief came also the urgent desire to understand the lay of the land in current Rett research and how I might help to speed things along. I spent my days juggling Chelsea’s therapy visits, caring for my 5-month-old son and speaking to as many scientists as I could.
Late one night in early September I received an instant message from a fellow mom who had taken her disabled child to see a well-known autism spectrum disorder neurologist in the Boston area earlier that day. The doctor mentioned that the “Rett gene” had finally been found. I had heard similar claims in the past year that turned out to be unsubstantiated rumors, so I spent the next few days doing detective work. To my surprise and delight, this time it was true. A few days later I spoke to Dr. Zoghbi and she confirmed the wonderful news. A few excruciating weeks followed during which the discovery had to be kept under wraps until the embargo was lifted, and the paper was published in Nature Genetics on October 1, 1999.
I spent hours on PubMed learning about this gene/protein with the strange name, methyl CpG binding protein 2. Eager to identify the leading labs, I poured through every publication on the subject. Two names flew out at me: Adrian Bird and Alan Wolffe. That same week I called them both and a few months later had an opportunity to meet them at Rett Syndrome meeting in Washington DC. Both quickly became cherished mentors. I was devastated to learn in May of 2001 that a traffic accident in Rio de Janeiro had claimed the life of Dr. Wolffe at the age of 41, leaving behind two young children and a devoted wife.
It is hard to convey to parents and relatives whose children were diagnosed after the gene discovery the excitement felt by the Rett community. For me it was the realization that the limited world of Rett research was about to burst wide open and that we would soon welcome scientists from the fields of epigenetics, DNA methylation, X inactivation, gene therapy and more. It was exhilarating to think that Rett might be able to leverage decades of research already underway in these many laboratories.
It was this excitement and promise that prompted me and five other parents to start the Rett Syndrome Research Foundation in the fall of 1999. During the next eight years RSRF’s funding contributed to nearly every major publication in the field culminating in Adrian Bird’s reversal experiments of 2007. I left shortly thereafter to establish RSRT.
Scientists and their institutions and funding agencies often trumpet any progress as a breakthrough. In reality true breakthroughs are few and far between. They are always unpredictable and they indelibly change the course of research. The Zoghbi Lab’s discovery on that hot, humid Houston day in mid-August certainly fits the bill.
The Rett community owes a tremendous debt of gratitude to Dr. Zoghbi, not only for her fortitude during the difficult 16-year search for the gene, but also for the plethora of key scientific papers she has written since.
I often hear Dr. Zoghbi described as one of the most accomplished female neuroscientists of our time. Her impressive body of work and the respect she commands on the international scientific world stage have played an enormous part in making Rett Syndrome a high-profile disorder.
Over the ensuing years I have been fortunate to count Dr. Zoghbi as an advisor and a friend. I ask the Rett community to join me in congratulating her and her colleagues, in particular Ruthie Amir, on the 10-year anniversary of their momentous discovery.
May we all have much to celebrate before another decade has passed.
The readers of this blog will have noted the frequent mention of epigenetics – a young but hot area of research which holds promise for novel therapeutic interventions for a myriad of diseases. The term epigenetic means over and above the genome. It refers to changes in gene expression caused by mechanisms other than changes in the underlying DNA sequence. It helps to think of epigenetics in terms of the charm bracelet analogy – the DNA is the charm bracelet and epigenetic modifications are the charms that can be added to or removed from the bracelet.
Every cell has about 30,000 genes. What distinguishes a heart cell from a liver cell is the pattern of gene expression (what genes are on and what genes are off). Considering the vast number of cell types in the human body it’s easy to appreciate how the instructions for turning genes on and off must be finely tuned and exquisitely calibrated. When missteps occur – disease often follows.
Epigenetic modifications catalyze gene expression changes. The best known epigenetic modification is methylation. Adding methyl “charms” to DNA leads to gene silencing. MeCP2, the protein that is mutated in Rett syndrome as well as a variety of other disorders, binds to DNA that has already been methylated and plays a role in regulating downstream genes.
The excitement surrounding epigenetics is that it’s dynamic. “Charms” can be added and they can be removed. The ability to catalyze these modifications through drugs has already led to some treatments for cancers. The hope is that these treatments are the mere tip of the iceberg.
The latest breakthrough in epigenetics comes to us from the laboratory of RSRT advisor Nat Heintz of Rockefeller University. The connection to Rett Syndrome, however, does not end there. The post-doc in the Heintz lab who made this discovery is Skirmantas Kriaucionis. As a graduate student, he spent five years in the lab of RSRT trustee Professor Adrian Bird who in 2007 announced the dramatic Rett reversal experiments. The Heintz and Kriaucionis paper was published online, April 16th, in the high profile journal Science.
We all remember the four nucleotide bases (A-adenine, T-thymine, G-guanine, C-cystosine) from high school biology. Together these four bases make up DNA. If we were taking high school biology today we would also learn about a fifth nucleotide, methylcytosine (mC) that replaces cytosine depending on whether a gene needs to be turned on or off. Kriaucionis has now added a sixth, hydroxymethylcytosine (hmC), a nucleotide base previously observed only in the simplest of organisms, bacterial viruses.
Drs. Heintz and Kriaucionis discuss their findings with Monica Coenraads.
MC: It’s not every day that a new nucleotide is discovered. Congratulations! Were you surprised by this finding?
NH: Yes, I was. I like to think that in most cases I know where my research is heading. But, in this particular case, the finding was completely unexpected. Broadly speaking, I think these experiments validate that it’s worthwhile spending time analyzing detailed properties of cells as they exist and operate in vivo, rather than studying their properties after they have been adapted for growth in culture.
Ironically, as a graduate student in David Shub’s lab I worked on bacterial viruses carrying modified nucleotides in their genomes. Now, many years later, that research has come full circle.
MC: As is often the case in science, the discovery was rather accidental. Dr. Kriaucionis, can you tell us what kind of research you were undertaking and how you stumbled on hydroxymethylcytosine (hmC)?
SK: The discovery of hmC can be traced back to several key reasons. One, we were interested in investigating chromatin structure (DNA/protein material which makes up the chromosomes) in different brain cell types. A finding which attracted our attention is that certain cell types have distinct looking chromatin. A dramatic example is Purkinje cells which have a very open chromatin configuration, in contrast to cerebellar granule cells which have very condensed looking chromatin (see above picture) We wanted to investigate this further. Second, was the availability of technological tools in the Heintz lab, which not only gave us access to these specific cells but allowed us to isolate very pure material to investigate. Thirdly, was the rather outdated technique that I was using to quantify the absolute levels of the different nucleotides. Scientists rarely use this technique now because it does not tell you where in the genome the nucleotides are. Since location gives insight into biological function this technique is rarely employed. But the decision to use this method turned out to play a large part of how I was able to identify the hmC.
At first I didn’t believe my results, and it took several months to reproduce the results many times using different experimental conditions to give us the necessary confidence that indeed, we were seeing a novel nucleotide and not an artifact of the experiment.
MC: So hmC is an example of a further modification to a nucleotide base, cytosine, that has already been modified by a methyl group. Has your discovery added more complexity to the process by which genes are regulated?
SK: Yes, indeed it does add a layer of complexity to what sort of biological message is being encoded by these modifications. The next important step will be to figure out what outcome is expected when DNA sequences have unmethylated cytosine, methylated cytosine or hydroxymethylated cystosine.
It’s interesting to note that while histone proteins have many epigenetic modifications mammalian DNA had, until now, only one – methylation. This finding is totally unexpected. Although RNA has plenty of modifications, its functional repertoire is complex as well including structural, enzymatic and coding roles.
MC: Do you have any hypothesis about what hmC might be doing?
SK: hmC is very abundant in brain so this gives us confidence that it’s doing something biologically significant. We currently have two hypotheses regarding its function. One hypothesis is that the hmC might be an intermediate step to get methylated cytosine back to its original unmethylated state.
MC: So in essence hmC might transform a silent gene (methylated) to an active gene (unmethylated).
SK: Correct. Another possibility is that hmC is a stable final modification. It could still activate a silent gene without demethylation. We see different amounts of hmC in different cell types so it may be that in some cell types hmC is an intermediate while in other cell types it’s a stable modification. And obviously we are keeping open mind as there may be an unpredicted function of hmC.
MC: There was a second paper in the same issue of Science as your paper that identified the enzyme that catalyzes the conversion from mC to hmC. Can you elaborate on the synergies between these two papers?
NH: The second paper is from the laboratory of Anjana Rao at Harvard. We were unaware of her ongoing research until very recently. Our papers complement and actually help each other quite a bit. The Rao lab identified an enzyme which catalyzes the conversion from mC to hmC. This is a very significant finding. A criticism that our paper was receiving was that hydroxylation of methyl groups could be a spontaneous reaction that was happening due to the oxidation of DNA. We addressed this question in our study, but the accompanying paper completely puts this criticism to rest since the group found the enzyme catalyzing the conversion of mC to hmC. So in that respect their paper was helpful to us. Our data strengthened their paper because we showed that hmC was highly enriched in brain, providing a link to the biology of neurons and, perhaps, neurological disease. Taken together, the two papers establish the importance of hmC in the mammalian genome, and suggest that this new epigenetic mark will provide an entry into a previously unanticipated and important field of biology.
MC: Dr. Kriaucionis, previous to your post-doc position in the Heintz lab you were in Adrian Bird’s lab for five years. Prof Bird’s lab focuses on DNA methylation and discovered MECP2, the “Rett gene” in the early 1990s. You co-authored 5 papers on MECP2 and Rett Syndrome with Prof. Bird. Do you think there is any possible connection between MECP2 and hmC?
SK: I think there is indeed a connection. As your readers know, MECP2 binds to methylated DNA. There is data showing that MECP2 resists binding to DNA that contains hmC. If these findings hold up, then hydroxylating mC could release MECP2 from the chromatin and influence for example nearby gene expression. Finding genomic MeCP2, mC and hmC distribution in neuronal cell types will provide us with valuable insights into the biological function of MECP2 and the role of DNA modifications.
NH: So Skirmantas’ hypothesis implies that hmC can modulate the distribution of where MeCP2 binds in the nucleus. There is an alternative scenario in which a different and, as of yet, unidentified protein binds to hmC. My theory is that this protein is likely not a repressor, like MeCP2, but perhaps is acting as an activator. The beauty of science is that we can test these hypotheses. I suspect we’ll have our answer in a relatively short amount of time.
MC: What are the next steps that the lab is pursuing?
SK: Of utmost importance is to answer the following question: where is hmC and what is it doing? To answer that question we are developing a new set of tools which will allow us to adequately map where hmC is in the genome. I hope that having a clear understanding of location will provide clues as to its role.
MC: Dr. Heintz, you are known in the scientific world as a “big idea man” who identifies and then develops the necessary tools to go after key neurobiological questions. Can you elaborate on the development of the novel tools to which Dr Kriaucionis just alluded?
NH: The fact that as currently applied bisulfate sequencing techniques,which are used to detect sites of methylation in the genome, do not distinguish between a cytosine that is methylated or hydroxymethylated is a major problem. We hope to develop new methodology that will allow us to map the precise sites of genomic hmC and mC separately.
Our strength as a laboratory fits in nicely with the task ahead. We have at our disposal gene expression data that is specific to a large variety of specific cell types. This information will be very useful as we begin to map where in the genome hmC is found. These two separate but very complementary sides of our lab will help elucidate not only the function of hmC but perhaps also the function of the proteins that bind to it.
MC: Dr. Heintz, your lab does not have a history of working on epigenetics and yet you made a remarkable discovery. Given this finding do you envision changing the focus of your lab in any way?
NH: Yes, I think it will change our focus somewhat. We feel this discovery is a critically important finding and the role of hmC in neurons will be of high interest both fundamentally to understand brain function and also for investigation of epigenetic influences on disease states. So we will be focusing a lot of our attention on what the biological role of hmC is in the healthy and diseased brain .
MC: You are not a “Rett researcher” yet you have kept up with the literature, attended a number of Rett scientific meetings that I have organized over the years and have been a highly respected advisor to the field. Do you have any insight on recent progress and where you think the field is heading?
NH: I’m very enthusiastic about the developments of the last few years. Adrian Bird’s reversal experiments are stunning. It has also become very clear, largely as a consequence of Huda Zoghbi’s work, that the impact of MeCP2 in different brain cell types is distinct. This means that strategies aimed at treating particular symptoms of the disease can be devised in the nearer term while approaches to reversing the entire phenotype, a much taller order, are explored.
MC: On behalf of our readers I thank you both for your time and wish you the very best for your ongoing research. I look forward to staying in touch and hearing how this work unravels. Congratulations again on your paper.
To continue our two-part interview with Huda Zoghbi, MD, we have just added these topics to our interview page:
- Jan and Dan Duncan Neurological Research Institute (NRI)
- Rett Syndrome and autism
Dr. Zoghbi has recently been appointed as Director of NRI which upon completion in 2010 will be the world’s first comprehensive children’s neurological center.
Our inaugural post includes interviews with Huda Zoghbi, M.D. and Adrian Bird, Ph.D. — two people whose names have become almost synonymous with Rett Syndrome. It was due to Dr. Zoghbi’s tenacity and commitment that the Rett gene, MECP2, was identified in her lab in 1999. After seeing her first Rett patient in 1983 she became determined to find the disorder’s genetic cause. It took 16 years of hard work and determination. Dr. Zoghbi’s efforts ushered in the appearance of Professor Adrian Bird. He had discovered the MeCP2 protein earlier that same decade, years before anyone knew it was related to a human disease. During this decade they have both made many key contributions to the Rett field. Rett Syndrome is high-profile disorder in the neuroscience community, in large part, due to their efforts. They play a key role at RSRT as trustee and advisors.
Huda Zoghbi, M.D. is an internationally renowned physician-scientist at Baylor College of Medicine and an investigator of the Howard Hughes Medical Institute. Click here for the first part of a two-part interview.
Adrian Bird, Ph.D. is the world’s leading expert in the gene MECP2. He is the Buchanan Professor of Genetics at the University of Edinburgh and the Deputy Chair of the Wellcome Trust. Click here to read his interview.