Exploring the Pathogenesis of Rett Syndrome

QUEBEC CITY —New research in Rett syndrome is illuminating the anatomic pathways responsible for specific features of the disorder and is laying the groundwork for development of new therapies, reported Huda Zoghbi, MD, at the 36th Annual Meeting of the Child Neurology Society.

Since the discovery of the Rett syndrome gene methyl-CpG binding protein 2 (MECP2) in 1999, Dr. Zoghbi and others have learned more about its role in the disease. MECP2 encodes a methylcytosine-binding protein. During development of Rett syndrome, many genes become silenced when methyl groups are added to their bases, including cytosine. MECP2 helps maintain that silence by binding to the methylated cytosines, preventing other proteins from demethylating the gene and reactivating it.

The mutation causes a loss of MECP2 function, which begins to manifest clinically after a period of normal development. Affected females lose early milestones, eventually becoming autistic or mute, with stereotyped movements, seizures, and autonomic dysfunction. Rett syndrome is not degenerative, however, and with good care, patients may live a normal life span; the oldest known patients with Rett syndrome are now in their early 70s, said Dr. Zoghbi, Professor of Molecular and Human Genetics, Pediatrics, Neurology, and Neuroscience at the Baylor College of Medicine in Houston.

The MECP2 gene is carried on the X chromosome. Males, therefore, have only one copy, and in most instances, lack of a normal MECP2 gene is incompatible with life. Females, on the other hand, have two copies. Random inactivation leads to variable levels of normal protein among the body’s tissues. In the brains of a few patients with Rett syndrome that have been studied, about half the tissue expressed the normal gene, and the other half expressed the mutated one.

Partial phenotypes exist as well, according to Dr. Zoghbi, in which a milder clinical disease is associated with expression of the normal gene in close to 85% of tissue (the figure is based on peripheral tissue, not brain tissue, which has not been studied in these individuals). In healthy mothers of girls with Rett syndrome, the figure is closer to 99%. Boys with milder mutations, in which there is some residual protein activity, develop a range of phenotypes, including seizures, balance disorder, tremor, mental retardation, and neuropsychiatric conditions, and they die before the fifth decade.

THE MOLECULAR BASIS OF ANXIETY

Several animal models have been developed to allow researchers to explore the pathogenesis of the disorder. “Our experience really shows how you can shuttle back and forth between basic and clinical research,” Dr. Zoghbi said. One of the first observations made with the use of the mouse model was that the mice trembled when they were handled.

Wondering if this was a display of anxiety, Dr. Zoghbi tested the mice in several settings and found that they quickly fled open areas and preferred the dark to the light. “This suggested they are indeed anxious,” she stated. “So we went back to the parents [of children with Rett syndrome], who indicated that anxiety is one of the most troubling features they encounter.”

Further work with the animal models led the researchers to determine that although the Rett mice have similar levels of corticosterone at baseline, the levels increase abnormally when the mice are stressed. The reason is that expression of corticotropin-releasing hormone was elevated. “This was quite an important discovery for us, because MECP2 normally regulates the expression of this gene,” Dr. Zoghbi said.
In this gene, explained Dr. Zoghbi, MECP2’s normal action was not to completely repress expression but to modulate it.

“It’s not an on-off light switch but more like a dimmer,” she said, in which expression is altered to match the physiologic needs of the animal. “It also told us that MECP2 regulates specific genes in specific types of neurons, rather than acting throughout the brain.”

Clinically, the finding suggested that corticotropin-releasing hormone receptor antagonists might be effective in Rett syndrome, a concept that is approaching the clinical trial stage.

TREATMENT STRATEGIES

Gene therapy is a potential therapeutic strategy. However, when the technique was tried in mice, Dr. Zoghbi’s group made a surprising finding. Doubling the amount of normal protein caused a progressive neurologic syndrome that included seizures. “This was worrisome,” she said, “because it told us that gene therapy is probably not an option for this gene, because its levels are too tightly regulated. But it raised the possibility that there might be humans with a duplication who have a neurologic phenotype.”

Such a condition was found recently, with MECP2 duplication leading to profound mental retardation, autism, and death, usually from infection, in teenagers, according to Van Esch et al. The condition may be a common cause of severe mental retardation. “So the question for us was ‘How do both the loss and the doubling of this protein produce a neurologic phenotype?’”

Dr. Zoghbi’s group explored this question in individual neurons and showed that loss of MECP2 reduced the number of glutamatergic synapses formed by hippocampal neurons, while excess MECP2 increased the number. The same result was found in hippocampal slices. “This shows us that this protein is a key rate-limiting factor in regulating glutamatergic synapse formation,” said Dr. Zoghbi.

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