Solving Rett Syndrome

Intro

Anna Estep and her daughter, Dominique, who has Rett syndrome.

Is there anybody in there?” That was her recurring thought when Aleksandra “Sasha” Djukic, M.D., Ph.D., started seeing children with Rett syndrome, a rare genetic disorder that severely compromises muscle control early in life.
Since Rett girls (affected boys rarely survive infancy) are effectively “locked in”—unable to talk, gesture or communicate in any meaningful way—neurologists long thought they had little cognitive ability.

Rett girls are effectively “locked in”—unable to talk, gesture or communicate in any meaningful way.

“But their eyes told a different story,” says Dr. Djukic, a professor of clinical neurology in the Saul R. Korey Department of Neurology and an associate professor of clinical pediatrics at Einstein. “These children had such a piercing gaze.” Many Rett parents agreed, insisting their children could follow conversations and even communicate using subtle eye movements. In truth, no one really knew what—if anything—these kids were thinking. Dr. Djukic was determined to find out.

Rare diseases such as Rett syndrome are defined as disorders or syndromes affecting fewer than 200,000 Americans. Most have no known treatments. At Einstein and Montefiore, researchers are collaborating on investigations into Rett syndrome and several other rare diseases, including Niemann-Pick C and 22q11.2 deletion syndrome.

Dr. Djukic set out to devise techniques to assess the Rett girls’ cognitive abilities and perhaps find ways to help them communicate. Rett syndrome impairs speech and hand control, rendering most neuropsychological testing useless. So Dr. Djukic focused on the girls’ eyes. If there was a touch of poetry to her approach—the eyes being “windows to the soul”—there was also a healthy dose of science.

For several years, scientists had been studying human perception and cognition using computerized eye-tracking technology (which employs reflected infrared light to measure precisely where a person is looking). A few studies had tried this approach with Rett girls, but the results were inconclusive. Then, in 2011, Dr. Djukic, director of the Tri-State Rett Syndrome Center at the Children’s Hospital at Montefiore (CHAM), worked with neuropsychology colleagues at Einstein to design a study of Rett patients that combined eye-tracking technology with visual paired-comparison testing.

In visual paired-comparison testing, a patient is repeatedly shown two identical images (of a person’s face, for example) so that the patient becomes familiar with them. Next, one of the familiar images is paired with a novel one, and eye-tracking assesses where the patient gazes and for how long. Since our brains are hardwired to favor novelty, a test subject with normal attention and memory will tend to favor the new stimulus when it’s paired with a familiar one.

Tests were conducted on 27 girls with Rett syndrome and 30 age- and sex-matched controls. Results showed that Rett patients favored the novel stimuli at a rate greater than chance. Their performance was significantly poorer than that of the typically developing controls—not surprising given the nature of the disease. But more important, as Dr. Djukic had suspected, the study showed that there is somebody inside. “It’s a human tragedy,” she says. “Communication is a basic human need, and these girls have been robbed of that ability.”

A Rett Specialist Is Born

A team found that in an animal model of Rett (mice with inactivated MECP2), most symptoms could be reversed, raising hopes for new therapies or even a cure.

Dr. Djukic, who joined the Einstein faculty in 2006, never intended to become a Rett syndrome specialist. Her clinical practice at CHAM initially focused on children with autism, epilepsy and other common neurologic disorders. A girl with Rett would come in for treatment every so often, but Dr. Djukic and her colleagues could do little except offer supportive care.

Progress against Rett syndrome has come in fits and starts. The syndrome was first described in 1966 by Austrian physician Andreas Rett. In 1999, after a 16-year search for a cause, Huda Zoghbi, M.D., of Baylor College of Medicine found that nearly all cases of Rett syndrome arise from mutations in a single gene known as methyl CpG binding protein 2, or MECP2. The next major development came in 2007, when a University of Edinburgh team found that in an animal model of Rett (mice with inactivated MECP2), most symptoms could be reversed by reactivating the MECP2 gene. This stunning turnabout was achieved by genetic manipulations that would be impossible in humans, but it raised hopes for new therapies and perhaps even a cure.

The next major development came in 2007, when a University of Edinburgh team found that in an animal model of Rett (mice with inactivated MECP2), most symptoms could be reversed by reactivating the MECP2 gene. This stunning turnabout was achieved by genetic manipulations that would be impossible in humans, but it raised hopes for new therapies and perhaps even a cure.

In 2008, Dr. Djukic established the Tri-State Rett Syndrome Center at CHAM, just the third such center in the country. “Now that there was proof of principle that these children could get better, I felt an obligation to promote research and provide better care,” she says. In just six years, the center has evolved into the nation’s largest clinical site for people with Rett syndrome, serving about 350 patients, and has spurred a variety of research projects, ranging from basic studies to clinical trials.

About Rett Syndrome

globe1 in 10,000
female births worldwide

Rett syndrome occurs in about one in every 10,000 female births worldwide. Most cases are caused by mutations to an X chromosome gene called MECP2, which synthesizes a protein that regulates genes involved in neuronal development. At about 6 to 18 months of age, girls who have been developing normally start to experience a host of symptoms that characterize Rett syndrome, including loss of speech; loss of motor abilities affecting the hands, arms and legs; seizures; and difficulties with learning, heart function, breathing, chewing, swallowing and digestion. The severity of the disabilities varies widely, depending on the underlying genetic mutations.

Rett syndrome is often misdiagnosed as autism, cerebral palsy or nonspecific developmental delay. Treatment is largely supportive, including medications for improving motor difficulties and anticonvulsants for controlling seizures. Occupational therapy can help patients develop skills for performing activities of daily living, while physical therapy and adaptive equipment can enhance mobility. Many Rett patients live into their 40s, although little is known about the potential longevity of people who have the syndrome.

Damage to a Key Protein

The MECP2 gene produces the protein MECP2, responsible for the normal functioning of many types of cells, including brain cells. “MECP2 may well be the most important protein for guiding normal development of the human brain,” says Steven U. Walkley, D.V.M., Ph.D., a professor in the Dominick P. Purpura Department of Neuroscience, in the Saul R. Korey Department of Neurology and in the department of pathology and director of Einstein’s Rose F. Kennedy Intellectual and Developmental Disabilities Research Center (IDDRC).

John J. Foxe, Ph.D., (left) and Steven U. Walkley, D.V.M., Ph.D.
John J. Foxe, Ph.D., left, and Steven U. Walkley, D.V.M., Ph.D., discuss using mice as model organisms in Rett syndrome research.

The MECP2 protein functions as a transcription factor, meaning it controls the expression of many genes. In the brain, this protein regulates genes important in forming neurons—silencing some genes and increasing the activity of others. MECP2 mutations result in structurally abnormal forms of the MECP2 protein that presumably can’t properly orchestrate gene expression in neurons.

Scientists don’t yet know how “normal” MECP2 protein does its job—“It affects the expression of so many genes in ways we don’t yet understand,” notes Dr. Walkley—nor why defective forms of the protein cause the intellectual disability and other problems associated with Rett syndrome. Much of the Rett research at Einstein and Montefiore is aimed at answering those two questions.

Dr. Galanopoulou’s lab is now using a mouse model of Rett syndrome to determine just how MECP2 mutations interfere with GABA signaling.

Mutations in the MECP2 gene seem to have little impact until Rett girls reach 6 to 18 months of age, when affected neurons lose the ability make new dendrites, the all-important branches essential for neuron-to-neuron communication. Patients then begin to regress, losing varying degrees of speech and movement, depending on the specific MECP2 mutation. But what happens at the molecular level to cause this clinical tragedy?

Aristea S. Galanopoulou, M.D., Ph.D., a professor of neurology and of neuroscience at Einstein and an attending physician in neurology, neurophysiology and epilepsy at Montefiore, suspects that MECP2 mutations severely disrupt signaling pathways that are controlled by gamma-aminobutyric acid (GABA)—the most important inhibitory neurotransmitter in the adult central nervous system. GABA molecules activate so-called inhibitory neurons that help keep overactive neurons in check.

Aristea S. Galanopoulou, M.D., Ph.D., and Michael D. Brenowitz, Ph.D.
Aristea S. Galanopoulou, M.D., Ph.D., and Michael D. Brenowitz, Ph.D., study whether shifts in ion level affect the function of defective MECP2 protein during nerve development.

Dr. Galanopoulou first got interested in GABA signaling because of its involvement in her primary research area: epilepsy, a condition in which clusters of neurons fire abnormally. Epileptic seizures are thought to reflect the nervous system’s failure to maintain balance between neuronal excitation and inhibition. Since epilepsy is a common characteristic of Rett syndrome, she wondered whether GABA irregularities might contribute to Rett as well.

In her epilepsy research, Dr. Galanopoulou had shown that seizures very early in life can change the way neurons respond to GABA; neurons that were inhibited by GABA may instead become excited. She had also found that seizures may deprive very young neurons of a key GABA function: helping neurons develop and mature normally. A similar sort of “GABA deprivation” may contribute to Rett syndrome.

Recent findings by Dr. Galanopoulou and other researchers suggest that MECP2 mutations may cause the problems associated with Rett syndrome by interfering with neurons’ ability to produce GABA. GABA’s absence appears to have devastating consequences for neurons.

“During brain development, GABA activates a cascade of signals within nerve cells that are critical for normal neuronal differentiation and synapse formation,” says Dr. Galanopoulou, research director of the Rett Syndrome Center and an attending physician at Montefiore’s Comprehensive Epilepsy Center. “We’ve found that if you stop this process in mice, the brain doesn’t fully develop, and you get something like Rett syndrome.”

Dr. Galanopoulou’s lab is now using a mouse model of Rett syndrome to determine just how MECP2 mutations interfere with GABA signaling. She is focusing on the substantia nigra, a brain region involved in motor control, with the goal of restoring normal signaling and reversing symptoms such as repetitive motor movements and seizures.

Recent findings suggest that Rett syndrome begins well before the first signs and symptoms appear. So Dr. Galanopoulou’s team is also seeking biomarkers that might reveal the syndrome’s presence at the earliest possible stage. “The earlier you can intervene,” she says, “the better the chance that therapies might have some benefit.”

Dr. Galanopoulou does not believe that altered GABA signaling fully explains Rett syndrome. “It’s more likely that a combination of abnormalities leads to the final clinical presentation,” she says. “A further complication is that each patient will probably have a different set of abnormalities, depending on the underlying genetic mutations, and thus each will require a personalized therapy. Nonetheless, I’m optimistic. Rett is one of very few genetic diseases that can be reversed after disease onset, at least in an animal model. It means that we should never lose hope.”

Ions and Dimers

Michael D. Brenowitz, Ph.D., a professor of biochemistry, is probing aspects of the MECP2 gene that are even more fundamental. A biophysicist by training, Dr. Brenowitz studies the behaviors and interactions of large molecules engaged in phenomena such as protein folding and protein binding—the everyday stuff of life at the cellular level.

Three years ago, at the request of colleagues in Einstein’s Center for Epigenomics (where researchers study gene regulation), he tackled a question that had vexed molecular biologists: How does the MECP2 protein (the product of the MECP2 gene) recognize and bind to the DNA of the genes it regulates? This question is critically important in Rett syndrome. A number of the MECP2 mutations that cause Rett do so by altering the MECP2 protein’s binding domain—the “key” that fits into a particular gene’s “lock.”

The odd thing about the MECP2 protein was how little specificity it showed for its targets. It was more like a skeleton key, able to fit into all sorts of DNA locks.

a portion of the MECP2 protein that binds to DNA
Above: a portion of the MECP2 protein that binds to DNA. Colors indicate where MECP2 changes shape in response to shifts in ion levels within the cell. This image resulted from a nuclear magnetic resonance study conducted by Dr. Brenowitz and Mark E. Girvin, Ph.D., a professor of biochemistry and scientific director of Einstein’s Structural NMR Facility.

“This lack of specificity struck us as very unusual,” says Dr. Brenowitz. “Proteins that control gene transcription typically bind to their target DNA with up to a million-times greater specificity than to nontarget DNA. We finally figured out that both the type and the concentration of ions, such as sodium chloride, seem to govern MECP2’s specificity. The protein’s binding domain shows little preference for its target gene at low concentrations, but it has high specificity at high ion concentrations.”

If we can understand the nature of MECP2-DNA binding, we may be able to identify drugs that stabilize this interaction and help patients
with Rett.

This finding dovetails nicely with Dr. Galanopoulou’s observation that ion types and levels change within neurons as animals develop and that Rett syndrome may be associated with these altered levels in developing neurons. The two researchers will soon team up to investigate whether these shifts in ion types and levels affect the ability of defective versions of the MECP2 protein to bind to genes during neuronal development.

Dr. Brenowitz may also have solved another puzzle associated with MECP2. Previous research had indicated that MECP2’s DNA binding domain was in the form of a monomer, or single macromolecule. But his latest structural analyses suggest that the portion of MECP2 that binds to DNA is actually in the form of a dimer (two macromolecules). This finding suggests that some MECP2 mutations alter MECP2’s ability to form this dimer, thus impeding the expression of genes associated with the protein.

“The take-home message,” he says, “is that disruption of any facet of MECP2 activity can interfere with normal neuronal development and cause disease. We can potentially fix what we understand, so if we can understand the nature of MECP2-DNA binding, we may be able to identify drugs that stabilize this interaction and, we hope, help patients with Rett.

“It’s amusing that I got into neuroscience at this point in my career,” adds Dr. Brenowitz, a bench scientist for three decades. “I’ve never done anything remotely related to neuronal function, much less a neurologic disease.

Today, however, he’s well versed in the intricacies of neurons as well as the clinical impact of MECP2 mutations. Every month or so, he and seven other scientists and clinicians meet to compare notes and chart new avenues of research. They belong to Einstein’s Rett Syndrome Interest Group, which Dr. Walkley started in early 2013.

“As director of the IDDRC, one of my roles is to get clinicians and basic scientists together to work on issues related to intellectual disability,” says Dr. Walkley. “When I came here, I was surprised to learn we had a fair number of people at Einstein and Montefiore who were working on Rett, as well as a world-class Rett clinic. But they weren’t necessarily working together. I’m encouraged to see new collaborations emerging and plan to extend this model to other diseases.”

Dr. Djukic (left) discusses new data on brain function in Rett syndrome with Sophie Molholm, Ph.D.
Dr. Djukic, left, discusses new data on brain function in Rett syndrome with Sophie Molholm, Ph.D., associate professor of pediatrics and of neuroscience, the Muriel and Harold Block Faculty Scholar in Mental Illness and the newly appointed director of the Sheryl and Daniel R. Tishman Cognitive Neurophysiology Laboratory.

Beeps and Boops

One such collaboration involves Dr. Djukic and John J. Foxe, Ph.D. ’99, formely a professor of pediatrics and of neuroscience and director of research at the Children’s Evaluation and Rehabilitation Center, and now a visiting professor. Dr. Foxe studies the neural underpinnings of vision, hearing and cognition and how these processes are compromised in autism, schizophrenia and other diseases. Soon after joining the Einstein faculty in 2010, Dr. Foxe met with Dr. Djukic to learn more about Rett syndrome, which shares some characteristics with autism.

Dr. Djukic’s Rett Syndrome Center launched a communication clinic offering Rett families a variety of services.

“Frankly, when I first heard Sasha talking about these girls and how she could see something in their eyes, I was deeply skeptical,” he says. “But seeing the girls myself, I thought, ‘Wait a minute—there may be something here.’ There was at least the possibility of a lot of cognitive function, but we needed some objective biomarker to tell us that information is indeed going in and being understood.”

As it happened, Dr. Foxe and his colleagues were developing new EEG (electroencephalograph) recording techniques to assess the brain’s electrophysiological response to spoken language. Compared with conventional EEGs, the new techniques yield a higher signal-to-noise ratio and, therefore, more-useful data.

“We could use this as our biomarker for showing whether an individual is comprehending speech,” Dr. Foxe says. “It doesn’t involve asking the child to do anything at all. We’re just ‘asking’ the brain whether it can discern a difference in a set of auditory signals. For instance, if we present a series of beeps and then a boop, every time that boop happens, because it is rare, the brain kicks off a little EEG signal that says, ‘I heard a change.’ It’s called a mismatch negativity.”

Montefiore speech-language pathologist Elaine Williams, M.A., C.C.C.-S.L.P., helps Rett patients and family members communicate.
Montefiore speech-language pathologist Elaine Williams, M.A., C.C.C.-S.L.P., helps Rett patients and family members communicate.

In a study funded by a National Institutes of Health “exploratory” research grant, Drs. Foxe and Djukic demonstrated that Rett girls have relatively normal auditory capabilities, at least at the most basic level.

Ongoing studies are assessing their higher-order speech recognition. “For example,” Dr. Foxe explains, “we might present a Rett syndrome patient with the sentence, ‘I woke up this morning and poured myself a bowl of socks.’ Because that last word is out of place, the brain of a normal individual will produce a measurable electrical response, what we call the semantic incongruence response.”

Dr. Foxe’s EEG measurements could also be used to indicate early on whether
experimental therapies are having any effect. At present, assessing whether a given intervention improves clinical outcomes can take years.

“It would speed things up to have a sensitive measure of a therapy’s impact at the neuronal level,” he says. “If, say, you get a 2 or 3 percent change in neurons over the course of the first few weeks of treatment, that therapy is highly unlikely to change the clinical picture in any measurable way. But that small percentage change says that you’re on the right track and making fundamental changes in brain activity.”

Einstein Hosts Rett Symposium

From left, Aleksandra Djukic, M.D., Ph.D.; Steven U. Walkley, D.V.M., Ph.D.; Huda Zoghbi, M.D.; and Monica Coenraads.

Rett syndrome was the focus of Einstein’s third annual Isabelle Rapin Conference on Communication Disorders, held last December. Attendees packed LeFrak Auditorium to hear experts from Einstein and other medical institutions present the latest information about the devastating condition.

One of the speakers was Huda Zoghbi, M.D., of Baylor College of Medicine, who in 1999 discovered the genetic defect responsible for nearly all cases of Rett syndrome (see page 28). Many Einstein and Montefiore researchers mentioned in these pages also spoke, including Drs. Aleksandra Djukic, John Foxe, Michael Brenowitz and Aristea Galanopoulou. The overall message: there is slow but steady progress toward better treatments and, ideally, a cure.

Monica Coenraads, co-founder and executive director of the Rett Syndrome Research Trust (RSRT), opened the program with a video about her daughter, Chelsea.

“The Rett research landscape was dismal when Chelsea was diagnosed in 1998,” recalled Ms. Coenraads. “My conviction that treatments and a cure were possible came from intuition and a mother’s love. Today that conviction is based on science.” The RSRT and an earlier organization Ms. Coenraads co-founded have raised $36 million to support Rett syndrome research at Einstein, Montefiore and other academic research centers.

People with Rett syndrome can’t speak, so researchers have developed systems that allow them to communicate. Dr. Djukic taught Chelsea to “talk” with her eyes, and she can now express her thoughts, needs and feelings.

Treating Rett

A physician as well as a scientist, Dr. Djukic has quickly translated her research findings into clinical practice. After her initial eye-gaze studies, for example, her Rett Syndrome Center launched a communication clinic offering Rett families a variety of services. They include training in use of assistive devices such as My Tobii, a commercially available product that tracks eye movements and the direction of gaze, allowing people with limited mobility to communicate via computer.

For some kids with Rett syndrome, the results are remarkable. “Through Tobii, the girls can tell us which music they like, what people they want to see, what parts of their body hurt,” says Dr. Djukic. “They even make jokes, for instance, saying dinner is ‘yucky.’ They don’t elaborate, they don’t make metaphors, but they’re able to communicate their needs and immediate thoughts.”

One high-functioning Rett girl, Gaby Valner, uses the device to write e-mails and make blog posts.

“Think of yourself in a cage in a room full of people, and the cage is soundproof,” she wrote in one heart-wrenching post. “You feel uncomfortable, maybe thirsty, or hungry, in need of some assistance or wanting to ask a question. Think about how frustrated you are when nobody hears. No matter how loudly you shout your requests inside your cage, none of the people in the room with you can hear. Now imagine instead of a cage you are trapped in, it is your own body. That is my life—an intelligent mind imprisoned in my body.”

More interventions may be coming. Dr. Djukic and her team recently completed the first clinical trial of a drug for treating Rett syndrome. A second drug will soon be evaluated in a clinical trial.

The first trial, which is supported by the Rett Syndrome Research Trust, evaluated Copaxone—an injectable drug already approved for treating multiple sclerosis. It works by increasing levels of brain-derived neurotropic factor (BDNF), which contributes to neuronal development. BDNF levels are typically low in Rett patients, presumably due to MECP2 mutations. Animal studies have shown that raising BDNF levels can ameliorate Rett symptoms—and researchers hope that the same will prove true in humans.

Dr. Djukic tested Copaxone on 20 girls ages 10 and older who have at least some ability to walk. The yearlong study looked primarily for improvements in gait but is also assessing changes in respiratory function, visual attention, memory and overall quality of life. “We’re hoping for a 20 percent improvement,” says Dr. Djukic. It’s too early to release results, but a few girls have reportedly shown remarkable responses.

The second trial, also funded by the Rett Syndrome Research Trust, will test the effects of Mevacor, a so-called statin drug that lowers serum cholesterol levels. A few years ago, researchers found that cholesterol metabolism is disrupted in mouse models of Rett syndrome. Symptoms improved when the mice were treated with a statin.

A later analysis of more than 100 Rett patients at CHAM found that 54 percent had abnormally high lipid levels, unrelated to body mass index levels. Since too much cholesterol can contribute to neurologic problems, it seemed logical that Rett patients might benefit from statin treatment. In a trial beginning this spring, Mevacor will be tested on 20 Rett girls ages 19 and younger.

Blue Sky Girls Event

Aiming for Blue Skies

On October 15, 2011, Dr. Djukic stood atop the steps of Tweed Courthouse in lower Manhattan to inaugurate the first annual Blue Sky Girls day, an international event she created to raise awareness of Rett syndrome.

“The sky is blue and that is why we are here, to reach the sky without clouds,” she said to the assembled crowd. Rett girls, she continued, “ask for very little: for recognition of the burden of isolation … for their silence not to be mistaken for their lack of understanding, for strategies that can help them communicate not to remain underutilized. Our girls have thoughts, emotions and dreams … [but are] trapped in their bodies. Today, I salute these brave girls, and I promise the day will come when this microphone will be theirs.”

Above: Constance and Christopher Steinon and daughter Ysolde climb the steps of Tweed Courthouse in New York City.