COLUMBUS, Ohio – Conventional wisdom among scientists for years has suggested that because individuals with Down syndrome have an extra chromosome, the disorder most likely results from the presence of too many genes or proteins contained in that additional structure.
But a recent study reveals that just the opposite could be true – that a deficiency of a protein in the brain of Down syndrome patients could contribute to the cognitive impairment and congenital heart defects that characterize the syndrome.
Scientists have shown in a series of experiments that there are lower levels of this protein in the brains of humans and mice with Down syndrome than are present in humans and mice without the disorder.
The researchers also showed that manually manipulating pieces of RNA that regulate the protein could increase protein levels in both human cell lines and mouse brains. In fact, an experimental drug that acts on those RNA segments returned this protein to normal levels in mice that model the syndrome.
When this RNA segment is overexpressed – meaning that more of it is present than needed in a cell – the protein level goes down, or is underexpressed. A total of at least five of these RNA segments are naturally overexpressed in persons with Down syndrome because the segments are housed on chromosome 21 – the chromosome that causes the disorder.
"We're talking about a paradigm-shifting idea that maybe we should look for underexpressed proteins and not overexpressed proteins in Down syndrome," said Terry Elton, senior author of the study and a professor of pharmacology at Ohio State University.
"What this offers to the Down syndrome community is the potential for at least five new therapeutic targets to pursue."
The Centers for Disease Control and Prevention estimates that about 13 of every 10,000 babies born in the United States each year have Down syndrome, characterized primarily by a mild-to-moderate range of intellectual disabilities, possible delayed language development and difficulties with physical coordination.
The study is published in a recent issue of the Journal of Biological Chemistry.
Elton, also interim director of Ohio State's Davis Heart and Lung Research Institute, stumbled upon this theory about Down syndrome while working on a different protein associated with cardiovascular disease. It turns out the protein he has studied for 25 years was regulated by one of these microRNAs that is known to be housed on chromosome 21.
A key role of RNA in a cell is to make protein, and proteins are the building blocks of all life. But the process has many steps. MicroRNAs are small pieces of RNA that bind to messenger RNA, which contains the actual set of instructions for building proteins. When that connection is made, however, the microRNA inhibits the building of the protein. Why that occurs is not completely understood, but increasingly microRNAs are considered tiny molecules that have a big impact in a number of physiological processes.
For his cardiovascular disease research, Elton found that a genetic trait in some people caused one specific microRNA to be bad at its job, leading to high protein levels that contribute to cardiovascular disease. This malfunctioning molecule is called microRNA-155, or miR-155.
"So we became interested in miR-155, and it is on chromosome 21. That's how we jumped to Down syndrome," Elton said.
There is also a strong link between the heart and Down syndrome. About half of those with the syndrome are born with congenital heart defects – problems with the heart's anatomy, not coronary arteries. But they do not experience cardiovascular disease or high blood pressure.
The advent of biomedical informatics has allowed scientists to use supercomputers to explore the human genome in a search for genes and their various relationships in the context of human disease. Elton consulted a bioinformatic database and found that five microRNAs sit on chromosome 21, and he and colleagues demonstrated in previous research that all five of them are overexpressed in the tissues, brains and hearts of Down syndrome patients.
"That means that whatever proteins these microRNAs work with are underexpressed," Elton said.
Further database exploration suggested that these five microRNAs target 1,695 proteins, all of which could cause problems in Down syndrome because they are underexpressed. To narrow that to a more manageable number, Elton's group had to make an educated guess based on a variety of data, including which proteins that are connected to these microRNAs are made by cells in the brain and heart – two areas most commonly affected by Down syndrome.
A protein surfaced as an attractive target to study: methyl-CpG-binding protein 2, known as MeCP2. Among the reasons it seemed important: A mutation in this protein is already known to lead to Rett syndrome, a cognitive disorder.
"So we thought that it was more than a coincidence that this protein plays a role in normal brain development, and if the protein doesn't function right, you're going to have cognitive impairment. Maybe this is the connection," Elton said. "We still don't know if this is the most important protein related to Down syndrome. But we were able to go on and prove scientifically that MeCP2 is a target of these microRNAs on chromosome 21."
The researchers used just two of the five microRNAs on chromosome 21 for the experiments in this study, miR-155 and miR-802, to match the only microRNAs available in the genetically engineered mouse model of Down syndrome.
First, the researchers made copies of the relevant microRNAs. In human brain cell lines, they manipulated levels of those two molecules to show the inverse relationship with the protein. If the microRNAs were more active, the level of the MeCP2 protein went down. When the microRNAs were underexpressed, the protein levels went up.
Next, the researchers examined adult and fetal human brain tissue from healthy and Down syndrome samples obtained from a national tissue bank.
"In both adult and fetal Down syndrome brain samples, it didn't matter which area of the brain we were looking at, the MeCP2 proteins were down. These are just observations with no manipulation on our part, and the MeCP2 is almost non-existent in the Down syndrome brain," Elton said. "We marked the protein with a fluorescent molecule, and by comparison, we could visualize and appreciate how much MeCP2 was being made by neurons in the control samples."
MeCP2 is a transcription factor, meaning it turns genes on and off. If its levels are too low in the brain, this suggests that genes influenced by its presence should be malfunctioning, too. Based on previous research by another group, Elton and colleagues focused on two genes affected by the MeCP2 protein for their next set of experiments.
Looking again at the human brain tissue samples, they found that the genes were indeed affected by the lowered protein level in Down syndrome brains – one gene that MeCP2 normally silences was in abundance, and the gene that should have been activated was underexpressed. Because the two genes examined have known roles in neural development, Elton said the results suggested even more strongly that the lowered protein's effects on the genes likely contribute to cognitive problems associated with Down syndrome.
Finally, the researchers tested an experimental drug called an antagomir on mice that serve as models for Down syndrome research. Antagomirs are relatively new agents that render microRNAs inactive. The scientists injected an antagomir into the brains of these mice to silence the miR-155 with the intent to increase levels of the MeCP2 protein. Seven days after the injection, the level of the protein in the treated mouse brains resembled levels in normal mouse brains.
"We showed that we can fix the protein abnormality in mice that model Down syndrome. But we can't undo the pathology that has already occurred," Elton said. "It's a starting point, but it appears that we have new therapeutic targets to consider."
###This work was supported by grants from the National Institutes of Health and the Foundation Jerome Lejeune.
Co-authors of the study were Donald Kuhn, Gerard Nuovo, Mickey Martin, Geraldine Malana, Sarah Sansom, Adam Pleister and David Feldman of Ohio State's Davis Heart and Lung Research Institute; Alvin Terry Jr. and Wayne Beck of Medical College of Georgia's Department of Pharmacology and Toxicology; and Elizabeth Head of the Institute for Brain Aging and Dementia, Department of Neurology, University of California, Irvine.
Contact: Terry Elton, (614) 292-1400; terry.elton@osumc.edu
Written by Emily Caldwell, (614) 292-8310; caldwell.151@osu.edu
Wednesday, March 24, 2010
Sunday, March 14, 2010
MeCP2 Goes Global: Redefining the Function of the Rett Syndrome Protein
FEBRUARY 25, 2010
A paper published online today in Molecular Cell proposes that Methyl CpG binding protein 2 (MeCP2) impacts the entire genome in neurons, rather than acting as a regulator of specific genes. Mutations in MeCP2 cause the autism spectrum disorder Rett Syndrome as well as some cases of neuropsychiatric problems including autism, schizophrenia and learning disabilities.
The discovery of MeCP2’s global reach was made in the laboratory of Adrian Bird, Ph.D. of the University of Edinburgh. Bird’s seminal contributions in the Rett Syndrome field include cloning the MeCP2 protein in the early 1990’s and the dramatic reversal of severe symptoms in fully mature mice models of the disease published in Science in 2007. He is a Trustee and Scientific Advisor of the Rett Syndrome Research Trust, a nonprofit organization intensively focused on the development of treatments and cures for Rett Syndrome and related MECP2 disorders.
Rett Syndrome strikes little girls almost exclusively, with first symptoms usually appearing before the age of 18 months. These children lose speech, motor control and functional hand use, and many suffer from seizures, orthopedic and severe digestive problems, breathing and other autonomic impairments. Most live into adulthood, and require total, round-the-clock care.
Historically, MeCP2 has been viewed as a classic transcription factor, but Bird’s data establishes MeCP2 as one of the most abundant neuronal nuclear proteins, with levels 100 to 1,000 times higher than typical transcription factors. In fact, there are nearly as many molecules of MeCP2 in the nucleus as there are nucleosomes, the fundamental repeating structural units of chromatin which in turn make up chromosomes. To put this in perspective, there is enough MeCP2 to cover nearly the entire genome.
Peter Skene, a post-doctoral fellow in the Bird lab and first author of the paper confirmed via chromatin immunoprecipitation and high throughput sequencing that this huge abundance of MeCP2 meticulously tracks the DNA methylation pattern of the cell. As a result, Skene observed that most regions of the genome bind to MeCP2, calling into question the previously assigned role of this protein as a target-specific transcription factor. This may explain why few clear gene targets for MeCP2 have been identified in the last decade.
“The brain contains many types of neurons with different functions, but interestingly it appears that the pattern of MeCP2 binding to chromosomes is broadly similar in all of them. This raises the possibility that the neuronal defect brought about by mutations in this gene affect all neurons in a similar way. If there really is a generic defect shared by many neurons, then the causes of Rett Syndrome may be less complicated than we feared. This idea now needs to be tested by further work,” said Professor Bird.
In line with its genome-wide distribution, the scientists found that MeCP2 globally impacts the packaging of the DNA in the cell. Histones are proteins which act as spools around which DNA is wound. This winding, or compaction, allows the 1.8 meters of DNA material to fit inside each of our cells. There are two classes of histones – core histones and linker histones. Core histones form the spool around which DNA winds - resembling beads on a string. The linker histones, such as histone H1, seal the DNA onto the spool formed by the core histones. In this way linker histones act as a padlock to hold the DNA in this structure and stop inappropriate access to the DNA outside of genes. In the absence of MeCP2, the amount of linker histone H1 doubles, suggesting an attempt to compensate for the lack of MeCP2.
The Bird lab also found an increase in histone acetylation in MeCP2-deficient neurons, but not in glia. These chemical modifications lead to an unwinding of the chromatin spools and potentially leave the DNA open for inappropriate expression. This suggests that the role of MeCP2 is to globally suppress the genome.
“Consistent with MeCP2 coating the entire genome, we observed global changes in the chromatin composition and activity. In the absence of MeCP2, we discovered an increase in the spurious transcription of the so-called ‘junk DNA’ which lies between genes. This suggests to us that rather than targeting specific genes, MeCP2 functions on a genome-wide level and may act as the watchdog of the neuronal genome,” said Skene.
“RSRT is pursuing two parallel approaches to interventions for Rett Syndrome. One is to find assays for MeCP2 function and then screen for anything that fixes the defect. The other is to understand as much as possible about what MeCP2 does in the brain and then design rational treatments. Understanding that MeCP2 acts in a global manner rather than as a gene-specific regulator gives us a new perspective on the molecular basis of Rett Syndrome that will aid in guiding drug development and other treatment modalities,” comments Monica Coenraads, Executive Director of RSRT and parent of a child with the disorder.
For an in-depth interview with Adrian Bird please visit the RSRT Blog, http://rettsyndrome.wordpress.com/
Source: Rett Syndrome Research Trust
A paper published online today in Molecular Cell proposes that Methyl CpG binding protein 2 (MeCP2) impacts the entire genome in neurons, rather than acting as a regulator of specific genes. Mutations in MeCP2 cause the autism spectrum disorder Rett Syndrome as well as some cases of neuropsychiatric problems including autism, schizophrenia and learning disabilities.
The discovery of MeCP2’s global reach was made in the laboratory of Adrian Bird, Ph.D. of the University of Edinburgh. Bird’s seminal contributions in the Rett Syndrome field include cloning the MeCP2 protein in the early 1990’s and the dramatic reversal of severe symptoms in fully mature mice models of the disease published in Science in 2007. He is a Trustee and Scientific Advisor of the Rett Syndrome Research Trust, a nonprofit organization intensively focused on the development of treatments and cures for Rett Syndrome and related MECP2 disorders.
Rett Syndrome strikes little girls almost exclusively, with first symptoms usually appearing before the age of 18 months. These children lose speech, motor control and functional hand use, and many suffer from seizures, orthopedic and severe digestive problems, breathing and other autonomic impairments. Most live into adulthood, and require total, round-the-clock care.
Historically, MeCP2 has been viewed as a classic transcription factor, but Bird’s data establishes MeCP2 as one of the most abundant neuronal nuclear proteins, with levels 100 to 1,000 times higher than typical transcription factors. In fact, there are nearly as many molecules of MeCP2 in the nucleus as there are nucleosomes, the fundamental repeating structural units of chromatin which in turn make up chromosomes. To put this in perspective, there is enough MeCP2 to cover nearly the entire genome.
Peter Skene, a post-doctoral fellow in the Bird lab and first author of the paper confirmed via chromatin immunoprecipitation and high throughput sequencing that this huge abundance of MeCP2 meticulously tracks the DNA methylation pattern of the cell. As a result, Skene observed that most regions of the genome bind to MeCP2, calling into question the previously assigned role of this protein as a target-specific transcription factor. This may explain why few clear gene targets for MeCP2 have been identified in the last decade.
“The brain contains many types of neurons with different functions, but interestingly it appears that the pattern of MeCP2 binding to chromosomes is broadly similar in all of them. This raises the possibility that the neuronal defect brought about by mutations in this gene affect all neurons in a similar way. If there really is a generic defect shared by many neurons, then the causes of Rett Syndrome may be less complicated than we feared. This idea now needs to be tested by further work,” said Professor Bird.
In line with its genome-wide distribution, the scientists found that MeCP2 globally impacts the packaging of the DNA in the cell. Histones are proteins which act as spools around which DNA is wound. This winding, or compaction, allows the 1.8 meters of DNA material to fit inside each of our cells. There are two classes of histones – core histones and linker histones. Core histones form the spool around which DNA winds - resembling beads on a string. The linker histones, such as histone H1, seal the DNA onto the spool formed by the core histones. In this way linker histones act as a padlock to hold the DNA in this structure and stop inappropriate access to the DNA outside of genes. In the absence of MeCP2, the amount of linker histone H1 doubles, suggesting an attempt to compensate for the lack of MeCP2.
The Bird lab also found an increase in histone acetylation in MeCP2-deficient neurons, but not in glia. These chemical modifications lead to an unwinding of the chromatin spools and potentially leave the DNA open for inappropriate expression. This suggests that the role of MeCP2 is to globally suppress the genome.
“Consistent with MeCP2 coating the entire genome, we observed global changes in the chromatin composition and activity. In the absence of MeCP2, we discovered an increase in the spurious transcription of the so-called ‘junk DNA’ which lies between genes. This suggests to us that rather than targeting specific genes, MeCP2 functions on a genome-wide level and may act as the watchdog of the neuronal genome,” said Skene.
“RSRT is pursuing two parallel approaches to interventions for Rett Syndrome. One is to find assays for MeCP2 function and then screen for anything that fixes the defect. The other is to understand as much as possible about what MeCP2 does in the brain and then design rational treatments. Understanding that MeCP2 acts in a global manner rather than as a gene-specific regulator gives us a new perspective on the molecular basis of Rett Syndrome that will aid in guiding drug development and other treatment modalities,” comments Monica Coenraads, Executive Director of RSRT and parent of a child with the disorder.
For an in-depth interview with Adrian Bird please visit the RSRT Blog, http://rettsyndrome.wordpress.com/
Source: Rett Syndrome Research Trust
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