Down Syndrome – A Possible Treatment in Sight?

William Mobley, the newly appointed chairman of the Department of Neurosciences at UCSD published a paper today in Science Translational Medicine showing that boosting levels of the neurotransmitter, neuropinephrine, in a particular part of the brain, the locus coeruleus, reversed some of the cognitive deficits in a mouse model of Down Syndrome.  This work adds to the fascinating data in recent years showing that neurodevelopmental disorders such as Down, Rett Syndrome, Fragile X, and others may be treatable.

UCSD Press Release
The Scientist – A Fix For Down Syndrome Brains?

Rett Syndrome Awareness Video

RETT SYNDROME RESEARCH TRUST WEBSITE

McCann Erickson graciously created this 90-second awareness video.  We are indebted to Steve Levit and Kenny Gilbreath for the pro bono effort.  Steve, who is chief creative officer of McCann Erickson recently joined the RSRT Professional Advisory Council (PAC).

The awareness video was launched at the recent Hope for Hannah event which was held in the home of FOX CEO, Jim Gianopulos and his lovely wife Ann, both members of the PAC.

[To share this video on Facebook, etc use this link: http://blip.tv/file/2756182]

Where and How Does Rett Research Happen?

RETT SYNDROME RESEARCH TRUST WEBSITE

A video presentation by Monica Coenraads

On September 9, 2009 the Rett Syndrome Center at The Children’s Hospital at Montefiore in the Bronx hosted its second Parent Gathering. The Director of the Center, Dr. Aleksandra Djukic, gave a presentation entitled Rett Syndrome: What Went Right in the Brain?   Dr. Chhavi Agarwal, the pediatric endocrinologist of the Rett Center, gave a talk entitled Osteopenia in Rett Syndrome.

In this presentation, I address some basic questions regarding Rett research. The focus of the presentation is not the actual scientific data but rather the logistics.  What are the fields of expertise who are involved in the current research?  How does the data get communicated? Where do scientists find funding? How do NIH, pharma and biotech fit into the picture?

As always, I welcome your questions and comments. My email is monica@rsrt.org.

Podcast: Mt. Sinai researcher interviews Monica Coenraads

RETT SYNDROME RESEARCH TRUST WEBSITE

Monica Coenraads, Executive Director of RSRT, was interviewed on October 1st by John Fossella, PhD, MBA, a neuroscientist at Mount Sinai in New York City.

Click here to visit his blog and access the podcast (15 min)

The Upside of Genetic Mutations

RETT SYNDROME RESEARCH TRUST WEBSITE

by Monica Coenraads

Last week I attended a scientific meeting held in Stresa, Italy and organized by a parent group, Pro Rett Ricerca. One of the most well received talks of the speaking program was presented by Monica Justice, PhD of Baylor College of Medicine, who discussed data collected from her RSRT-funded project. What follows is an excerpt from a recent conversation between the two Monicas following the Italian meeting.

MC: Dr. Justice, it was great to see you in Italy. I thought it might be helpful to give our readers some insight into your project. But first I’d like to start with you, the soul behind this impressive undertaking. How did you end up in science?

MJ: My grandfather was a vet and I had an uncle who was a physician. I have always had a deep love for animals so as a child and young adult I wanted to be a vet.  My father thought that was not an appropriate career for a woman and he and my uncle encouraged me towards the medical field. Early on, however, I realized that my true passion was in basic research.  I went to graduate school thinking I would focus on immunology and microbiology but my very first class would change my career path forever. My professor was switching into the mouse genetics field and invited me to join his lab.  I loved mouse genetics from the very start and knew immediately this was exactly where I wanted to be.

MC: Most lay readers of this blog will not have realized that mouse genetics as a field exists.  Can you elaborate on this specialty?

MJ: When I entered the field most mouse genetics was being carried out in a few labs, The Jackson Laboratories being the primary one in the US, and research centered around a few mouse mutations that primarily altered mouse coat color.  I think the perception from the science community at large was that we weren’t doing particularly important work. That perception changed with the introduction of very powerful research tools.   One such tool was the ability to alter genes in mouse embryonic stem cells to engineer DNA mutations at will. The second was the ability to use a strong mutagen, called N-ethyl-N-nitrosurea or ENU, to do forward genetics – more on that later. This was also the time when molecular biology was exploding.  Rather quickly the mouse became THE model organism of choice. I’ve ridden that wave since my graduate student days. Today nearly every institution that is doing cutting edge research has a mouse genetics core.  I suspect there are now about 2000 true mouse geneticists worldwide.  Nearly every person who works on human disease now works with mouse models.

MC: Please tell our readers the basics behind your Rett project.

MJ: Our Rett project is based on two discoveries:  1) that you could make the symptoms of Mecp2 mutant mice better if you  introduced brain derived neurotrophic factor (BDNF) and 2) Adrian Bird’s  finding that you could actually reverse very severe symptoms in the mice by reintroducing the gene. Because of those two findings I believe that Rett symptoms can be altered by other genetic mutations. I felt strongly that the genetic approach that I was familiar with would be an ideal strategy to try and identify suppressors of the symptoms of Mecp2 knockout (ko) mice.

So let me tell you a bit about the screen. I use a powerful mutagen, ENU, that induces mutations in mouse sperm at a very high rate. We give the mutagen to wildtype (normal) male mice and then mate them to female Mecp2 knockout mice. A certain percentage of their offspring will have no Mecp2 and a sporadic mutation somewhere in their genome.  We then analyze the mice very closely and look for any that appear healthier than your typical Mecp2 ko mouse.  For example, a Mecp2 male ko mouse is dead by 6-14 weeks. If a mouse in our screen lives much longer than that, we hypothesize that there is a mutation in another gene that suppresses the ill effects of having no Mecp2. We currently have mice that are over a year old and still do not show signs of Rett.

MC: How many mice has your project generated?

MJ: We have used about 10,000 mice at this point and envision needing 5,000 more to find and understand the current genes of interest. To reach saturation for our screen, meaning that we are confident that the mutagen has generated mutations in every gene that could potentially be a suppressor, we would need to screen through five times the number of males that we have done thus far. Statistically, I estimate that there are 25-50 suppressor genes that we would expect to find were we to hit saturation.

MC: What kind of precedents are there for success using ENU modifier screens?

MJ: The first successful modifier screens were done in bacteria and yeast. The technique gained momentum in the late 1980’s early 1990’s when Gerry Rubin carried out a modifier screen in the fruit fly for genes that would interfere with a particular pathway. Dr. Rubin is a famous scientist who now is the Director of the Howard Hughes Medical Institute Janelia Farm Research Campus.   Historically, people thought that fruit flies were the only organism that you could do this with. It’s clear now that the mouse is an equally powerful organism.  My graduate mentor, Vernon Bode, did a screen in mice for PKU modifiers, which was finished by Bill Dove at the University of Wisconsin Madison.  And an Australian group that works on diseases of the blood did an ENU mouse screen looking for genes that influence platelet counts.   Each of these screens was very successful.

MC: Is there data to show that modifier genes are the rule or the exception in disease?

MJ: That’s a very interesting question. I work in the Department of Human and Molecular Genetics at Baylor. What I see from many of my colleagues’ work is that genetic modification of disease is the rule and not the exception.

MC: Can you envision a situation where you find modifiers in the Mecp2 ko mice but those genes are not implicated in the human disorder?

MJ: I do not think that we will find modifiers that are mouse specific only. I believe that because the mouse model for Rett Syndrome is amazingly similar to the human disease.  Also, DNA methylation (which is critical to MECP2) and some of the possible functions of the MECP2 gene are highly conserved between species. So it’s very likely that the MECP2 gene in people and in mice is doing the same thing.

MC: What do you foresee as the best possible outcome?

MJ: I foresee finding a molecule that would help forge neuronal connections, and help these connections be maintained and molded.
Furthermore, whatever molecules we find that suppress Rett symptoms may also give us important biochemical information on other genes that may interact with Mecp2.

MC: Did being at Baylor, a Rett hotspot, impact your decision of taking on this Rett project?

MJ: I have been very much aware of Rett since I moved to Baylor in 1998, a year before Huda Zoghbi identified the gene. I have been on the student committee of some of Dr. Zoghbi’s students, which kept me up- to- date on the ongoing work. MECP2 is a transcription factor and I’ve always been interested in transcriptional regulation. What really brought me into the project was when you called me up with a proposition.

MC: When I was the Director of Research at the Rett Syndrome Research Foundation (several years before the merger with IRSA to form IRSF) I piggybacked an early morning think tank during the RSRF Rett Syndrome Symposium in Chicago. About two dozen creative thinkers were kind enough to drag themselves out of bed and brainstorm with me about potential key experiments that could significantly move the field forward.  At the top of that list was an ENU mouse modifier screen. The group also gave me a list of potential people who could undertake such a laborious and intense project…it was a small list and your name was on it.  As you well know, RSRF then organized a workshop at the Mouse Genetics meeting, which took place in Charleston, SC in 2006. Those discussions led to the funding of your project.

MJ: I was so thrilled when you called. There was a time when I would have turned down this project.  But the timing of your call was perfect. The project appealed to me very strongly as a geneticist but also as a compassionate person who wants to make a difference in the life of others.

Your readers should also know that this is a project that the NIH would never have funded.  It was too risky and too “out there”.  I knew this was a viable technique and I was confident with the expertise of my lab with regards to mouse breeding, husbandry and handling that would move this project along quickly so I am very grateful to have had the funding to pursue this.   People should be aware of how much private foundations such as RSRT move the field forward by funding high risk, but high impact projects.

MC: Speaking of compassionate person, at the end of your talk in Italy you got a little emotional.  I was quite touched by that. Can you tell our readers what was going through your mind?

MJ: Yes, I got a little “verklempt” and I got teased quite a bit at dinner for that. I take this project very seriously because I feel that what we are doing could have an impact on people’s lives – an impact that perhaps wouldn’t happen without the screen – I guess as I was up there in front of scientists and the organizers of this meeting who have children suffering from Rett – the importance of our efforts hit me hard.

MC: Are you able to give our readers a hint at the data that your project has thus far yielded?

MJ: The project is at an exciting point.  We are close to identifying our first suppressor gene and we have a few more potential genes that we are pursuing as well.  Once they are identified we will begin experiments to confirm that they are indeed interacting with Mecp2m, first in the mice and then in people.  I also think the modifiers we have so far are just the tip of the iceberg.  So we have a lot more screening ahead of us.

I love this project, it’s fun, it’s exciting, and each new piece of data that we identify brings us closer to our goal.

MC: On behalf of families everywhere who love a child with Rett Syndrome we wish you Godspeed. We look forward to hearing about future progress.  Thank you also to Pro Rett Ricerca and especially Rita Negri and Laura Rassetti for their tireless work to organize this meeting.

MJ: It was my pleasure and honor to attend the meeting in this most beautiful area of Italy, near where you were born.

Drug Discovery for Rett Syndrome: A Fresh Angle

RETT SYNDROME RESEARCH TRUST WEBSITE

Stavros Lomvardas, a young investigator at the University of California San Francisco, has just been awarded funding from RSRT for an innovative project which has the potential to not only help to clarify the function of the MeCP2 protein but also discover drugs to reverse the deficits incurred when the protein is mutated. Below are excerpts from a recent conversation with Dr. Stavros.

MC: I am always fascinated by how scientists end up in their chosen career. Was it a relatively late decision for you or did you know as a boy that you were destined for science?

SL: I guess in my heart I always knew. Even when I was a very little kid I was always designing little experiments. I grew up in Athens, Greece.  My father was dentist who had a deep interest in biology.  I found his love for biology very inspiring. He would always bring me books and talk to me about experiments and help me to design my own. So from a very young age I knew I wanted to be a scientist.

MC: It’s my impression that most scientists go into this field for the love of basic science and not with the goal of understanding or treating disease. Would that be a fair statement with regards to you?

SL: Yes, it would be fair. To be quite frank until 5 years ago I was completely dedicated to basic science and did not have any real interest in applying my knowledge to treat disease. I thought it was utopic to expect that basic science could be used, at least in the near future, to treat diseases.  But the last 5 years have changed me dramatically. Maybe it has to do with having my own children. Or maybe dealing with health issues that have cropped up in my extended family and realizing that there are people out there who are really suffering. I realize now that scientists cannot sit at our lab benches preoccupied only with the joy of doing pure science. We have an obligation to also help people. The idea of doing experiments that might actually have medical applications is very appealing to me right now. Maybe I’ve just matured but I find the concept very fulfilling.

MC: For a young investigator you have a very impressive training and publication record.  In fact, you have worked with not one but two Nobel Laureates.

SL: I came to the U.S. as a graduate student in 1998 and trained in the lab of Dimitri Thanos at Columbia University. The Thanos lab and the lab of Nobel Laureate, Eric Kandel, also of Columbia had an extensive collaboration that lasted a number of years. It was a very interesting experience for me personally. After I received my PhD in 2002 I transferred to the lab of Richard Axel for my post-doc training.
Dr. Axel, together with Linda Buck, shared the Nobel Prize in 2004 during my second year in the lab for solving the question of how we recognize and remember thousands of different smells. They discovered a large gene family comprised of about 1500 different genes that correlate to an equal number of olfactory receptors, each of which detect a small number of smells.  These receptors are located on the olfactory receptor cells which in turn are located in the upper part of the nasal epithelium.
Interestingly, each olfactory receptor cell has only one type of odorant receptor, and each receptor can detect a limited number of odorant substances.

MC: Did the Nobel Prize make working in the Axel lab any different?

SL: Dr. Axel was an extremely admired scientist among his peers. I don’t think receiving the Nobel Prize changed how his peers viewed him. While the prize brought lots of attention from the outside community it did not change his already existing celebrity status in the scientific world.  What did change is the demands on his time – for a number of years, and to some extent still now, he was insanely busy.

I will always remember the call I received from him while he was in Sweden receiving his Nobel. He was eager to know how an important experiment that I was undertaking was proceeding. The call is an example of Dr. Axel’s philosophy – science first. For me the experience was quite surreal.

Overall I was very fortunate to have trained in labs where the bar of what is considered good science and ground-breaking research was set very high.

MC: How did your research come to include MeCP2?

SL: Toward the end of my post-doc I had started working on DNA methylation and other epigenetic modifications of olfactory receptors. I was a fellow of the Helen Hay Whitney Foundation and at a Foundation meeting I saw a friend, Joe Zhou, who worked on MeCP2 in Michael Greenberg’s lab at Harvard. I had certainly heard of MeCP2 and knew of its involvement in Rett Syndrome but had never considered working on this protein myself. Joe gave an interesting talk which enticed me to do some preliminary experiments. I asked Joe for some antibodies against MeCP2 and I quickly noticed a peculiar expression pattern in the olfactory epithelium. Also the Ronnet lab in John’s Hopkins had published some very interesting results regarding MeCP2 function in the development of the olfactory epithelium, so I got really interested. We then ordered the knockout mice and started working seriously.  So my entry into the Rett field was, like many things in science, quite serendipitous.

MC: Please give our readers some insight into your RSRT funded experiment.

SL: My experiment leverages the discovery that MeCP2 deficient olfactory receptor neurons (ORN) have a very robust readout. They co-express molecules that are never expressed in the same neuron in wild type mice. Part of our experiment will capitalize on this finding to screen for drugs that can reverse the deficit – in other words find drugs, using high throughput screens (HTS) that can turn off one of ectopically expressed molecular markers.

MC: The reviewers of your proposal as well as our advisors had a common observation. They commented that the leap to the HTS was exceptionally clever and unconventional.

SL: The idea to undertake a HTS came to me last December during at a scientific meeting organized by RSRT.  As you know RSRT brought together about two dozen scientists, including the top minds in epigenetics and Rett Syndrome, to brainstorm about MeCP2 function. During the stimulating discussions it hit me that as far as I could tell there were no existing straightforward assays for MeCP2 deficiency.  It occurred to me that our finding could be leveraged to develop an assay for drug screening. At first it seemed like a crazy idea. But the more I spoke to people the more they liked the idea.   I am quite hopeful that we can find some molecules that can turn off one of the two markers and be effective also in vivo.

MC: This is a prime example of translational research – in other words a logical next step of taking a basic science discovery and designing an experiment with clinical applications – that most individuals outside the science arena believes happens naturally – but actually needs to be pushed, facilitated and funded – often by research organizations such as RSRT.

SL: Yes, absolutely. For me the first key turning point was reading Adrian Bird’s reversal paper. It was a beautiful paper; the experiments were so elegant and the results so dramatic. The second key turning point was attending the RSRT meeting which led to the idea of the HTS.

MC: You’ve been studying MeCP2 for about 3 years now. Has anything surprised you?

SL: Before I started working on MeCP2 I could not understand why people were struggling so much to understand the function of this protein. After all, there is a knockout, with a severe set of symptoms. So why don’t we have a clear list of target genes that are regulated by MeCP2. Now that my lab is focused on this protein I am realizing that we are not dealing with a traditional molecule. MeCP2 is not a traditional repressor or activator in that when you remove it you do not see huge effects on other genes.  It’s a very intriguing molecule indeed.  My personal hypothesis is that MeCP2 is a modulator involved in the fine tuning of nuclear processes. This is where my lab as an edge in choosing the olfactory epithelium because we can work on a very pure population of neurons.

MC: Parents reading this who have children with Rett Syndrome are probably wondering if their child has problems with smell.

SL: I don’t know. We are doing experiments now to delete the protein in the olfactory epithelium and will test whether there are olfaction deficits in the mice. I would expect that there may be subtle problems of discriminating between smells.

MC: This is a pretty difficult time economically for young scientists. Are you also feeling the impact?

SL: Yes I am certainly feeling the impact, especially being in CA.  My first two years of having my own lab have been very rough.  I had serious problems attracting funding from anywhere. It was a very painful experience because I was establishing a lab, recruiting and training people and trying to get enough funds to stay afloat. Fortunately my department and the UCSF supported me during this very difficult phase. Things have improved slightly but the situation remains challenging. Funding from RSRT is greatly appreciated.

MC: Over the past decade I have administered peer review for almost 1000 Rett grants.  I have seldom seen the kind of unanimous enthusiasm that your application generated. I wish you success and look forward to updating our readers about your progress.

Rett Gene Discovery: Ten-Year Anniversary

RETT SYNDROME RESEARCH TRUST WEBSITE

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 and research assistant

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.

Curing Rett Syndrome: How Do We Get There?

RETT SYNDROME RESEARCH TRUST WEBSITE

A video presentation by Monica Coenraads

On June 28, 2009 the Rett Syndrome Center at The Children’s Hospital at Montefiore in the Bronx hosted a Parent Gathering. The Director of the Center, Dr. Aleksandra Djukic, warmly welcomed the audience and introduced the first of what will be quarterly Gatherings. Dr. Djukic introduced R.E.T.T. (Rethink Education, Therapy & Technology) an engaged group of parents who have designed a survey to gather information to better assess what programs, techniques and settings are most effective for educating individuals with Rett Syndrome. Darcy Minsky followed with some IEP tips to get next year’s educational year off to a good start. The next Gathering will take place on September 27th.

I spoke about an issue that is dear to the heart of anyone who loves a child with Rett Syndrome: How do we get to a cure? The presentation, which is about an hour in length, highlights the key research discoveries of the last decade and lays out the current thinking on treatment/cure approaches in easy to understand language. The presentation is divided into four sections:

• Genetics of Rett Syndrome
• Functions of MECP2
• Reversal
• Treatments and Cures

If you are the parent, relative or friend of a child with Rett Syndrome, I hope this video will give you a glimpse of the excitement that the scientific community feels about the possibilities that lie ahead for our children.

I welcome your thoughts and questions. I can be reached at monica@rsrt.org.

New uses for existing drugs

RETT SYNDROME RESEARCH TRUST WEBSITE

A recently published article in the NYT lends support for RSRT’s rationale behind testing FDA approved drugs and compounds in an animal model of Rett Syndrome.

RSRT Project
Sponsor a Drug

With Aid of Drug Library, New Remedies From Old

Some of the more than 3,000 drugs at Johns Hopkins.

By Kate Murphy
Published: April 27, 2009

REPRINTED FROM THE NEW YORK TIMES

Housed in a row of white freezers in a nondescript laboratory at the Johns Hopkins University School of Medicine in Baltimore are more than 3,000 of the estimated 10,000 drugs known to medicine. There is no sign on the door to indicate that this is perhaps the largest public drug library available to researchers interested in finding new uses for old and often forgotten drugs.

Already, researchers have used the library to discover that itraconazole, a drug used for decades to treat toenail fungus, may also inhibit the growth of some kinds of tumors and may forestall macular degeneration. Another drug, clofazimine, used more than a century ago to treat leprosy, may be effective against autoimmune disorders like multiple sclerosis and psoriasis.

“It takes 15 years and costs close to a billion dollars to develop a new drug,” said Jun O. Liu, professor of pharmacology and director of the Johns Hopkins Drug Library. “Why not start with compounds that already have proven safety and efficacy?”

He and his colleagues have been building the collection since 2002 and hope to have it complete by 2011. They acquire the drugs through donations, purchases and sometimes lab synthesis. And they will send researchers a complete set — minuscule amounts of every drug in the library — for $5,000, which covers the cost of shipping and replenishment.

Since the toenail and leprosy drugs are approved for use in the United States and are no longer under patent protection, clinical trials to test their new uses are either under way or close to regulatory approval, Dr. Liu said.

Drugs still under patent protection are more complicated; patent holders seldom allow independent research on alternative uses. “The drug companies haven’t been too keen on helping us,” Dr. Liu said.

There are other drug libraries, both commercial and noncommercial. Commercial suppliers offer considerably fewer drugs than Johns Hopkins (though they may have medicines it does not), and they charge much more. Noncommercial drug libraries include those at the National Institutes of Health; the University of California, San Francisco; and McMaster University in Hamilton, Ontario. But they will usually not send drugs to unaffiliated researchers. And like the commercial libraries, their holdings are smaller and composed largely of compounds from Hopkins.

Regardless of the source, researchers typically order copies of entire collections rather than individual drugs they think may work in their experiments.

“We’ve found drugs that are active in ways no one would have ever hypothesized,” said Marc G. Caron, a professor of cell biology at Duke who is using the Johns Hopkins library to find drugs that might quell the cravings of substance abusers.

Testing of these compounds has become much easier in recent years as a result of an automated technology called H.T.S., for high-throughput screening. The drugs are dissolved in a solution and stored in rectangular, compartmented plates reminiscent of ice trays; they can then be delivered to researchers for testing of their efficacy against various diseases, or disease mechanisms like inflammation.

Computerized droppers, plate agitators and microscope image readers can now accomplish in days what it once took bench scientists years to do.

Although H.T.S. has been around for at least a decade, it is just within the last five years that the technology has been widely available. Previously, only big pharmaceutical companies could afford to screen thousands of compounds; now more public and academic institutions are doing so, and their emphasis tends to be on rediscovering or tweaking the chemical structure of old drugs rather than developing new ones.

“The instrumentation to do sophisticated, large-scale screening of drugs has gotten significantly better and cheaper,” said Michelle Arkin, associate director of the Small Molecule Discovery Center at U.C. San Francisco.

Some institutions, like McMaster in Ontario and Rockefeller University in New York City, allow outside researchers to use their H.T.S. facilities for $10,000 to $20,000, depending on the complexity of the project.

Access to such facilities has increased demand for compounds, particularly already approved and off-patent drugs, to analyze. Johns Hopkins and commercial suppliers report a surge in orders over the last two years — because there are more H.T.S. laboratories, they said, and because of efforts to find cheaper therapies against third world scourges like malaria and tuberculosis.

“Old drugs are the low hanging fruit in terms of finding safe and inexpensive treatments for these diseases,” said Carl Nathan, chairman of microbiology at Weill Cornell Medical College in New York. Dr. Nathan receives plates of drugs from Johns Hopkins as well as commercial suppliers and does high-throughput screening at Rockefeller, which has a partnership with Weill.

“I’m addicted to it,” he said.

And then there were six

RETT SYNDROME RESEARCH TRUST WEBSITE
ROCKEFELLER PRESS RELEASE
HEINTZ LAB

Isolated nuclei from cerebellum. Blue color represents DNA. Green spot is the nucleolus of a Purkinje cell. (photo from Heintz lab)

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.

Nat Heintz, Ph.D.

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)?

Skirmantas Kriaucionis, Ph.D.

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.