Down Syndrome Neurons Grown from Stem Cells Show Signature Problems

Down Syndrome Neurons Grown from Stem Cells Show Signature Problems

Tuesday, 28 May 2013

Down syndrome, the most common genetic form of intellectual disability, results from an extra copy of one chromosome. Although people with Down syndrome experience intellectual difficulties and other problems, scientists have had trouble identifying why that extra chromosome causes such widespread effects.

In new research published this week, Anita Bhattacharyya, a neuroscientist at the Waisman Center at the University of Wisconsin-Madison, reports on brain cells that were grown from skin cells of individuals with Down syndrome.

"Even though Down syndrome is very common, it's surprising how little we know about what goes wrong in the brain," says Bhattacharyya. "These new cells provide a way to look at early brain development."

The study began when those skin cells were transformed into induced pluripotent stem cells, which can be grown into any type of specialized cell. Bhattacharyya's lab, working with Su-Chun Zhang and Jason Weick, then grew those stem cells into brain cells that could be studied in the lab.

One significant finding was a reduction in connections among the neurons, Bhattacharyya says.

"They communicate less, are quieter. This is new, but it fits with what little we know about the Down syndrome brain."

Brain cells communicate through connections called synapses, and the Down neurons had only about 60 percent of the usual number of synapses and synaptic activity.

"This is enough to make a difference," says Bhattacharyya.

"Even if they recovered these synapses later on, you have missed this critical window of time during early development."

The researchers looked at genes that were affected in the Down syndrome stem cells and neurons, and found that genes on the extra chromosome were increased 150 percent, consistent with the contribution of the extra chromosome.

However, the output of about 1,500 genes elsewhere in the genome was strongly affected.

"It's not surprising to see changes, but the genes that changed were surprising," says Bhattacharyya. The predominant increase was seen in genes that respond to oxidative stress, which occurs when molecular fragments called free radicals damage a wide variety of tissues.

"We definitely found a high level of oxidative stress in the Down syndrome neurons," says Bhattacharyya.

"This has been suggested before from other studies, but we were pleased to find more evidence for that. We now have a system we can manipulate to study the effects of oxidative stress and possibly prevent them."

Down syndrome includes a range of symptoms that could result from oxidative stress, Bhattacharyya says, including accelerated aging.

"In their 40s, Down syndrome individuals age very quickly. They suddenly get gray hair; their skin wrinkles, there is rapid aging in many organs, and a quick appearance of Alzheimer's disease. Many of these processes may be due to increased oxidative stress, but it remains to be directly tested."

Oxidative stress could be especially significant, because it appears right from the start in the stem cells.

"This suggests that these cells go through their whole life with oxidative stress," Bhattacharyya adds, "and that might contribute to the death of neurons later on, or increase susceptibility to Alzheimer's."

Other researchers have created neurons with Down syndrome from induced pluripotent stem cells, Bhattacharyya notes.

"However, we are the first to report this synaptic deficit, and to report the effects on genes on other chromosomes in neurons. We are also the first to use stem cells from the same person that either had or lacked the extra chromosome. This allowed us to look at the difference just caused by extra chromosome, not due to the genetic difference among people."

The research, published the week of May 27 in the Proceedings of the National Academy of Sciences, was a basic exploration of the roots of Down syndrome. Bhattacharyya says that while she did not intend to explore treatments in the short term, "we could potentially use these cells to test or intelligently design drugs to target symptoms of Down syndrome."

Source: University of Wisconsin-Madison 

Contact: Anita Bhattacharyya

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ZenMaster

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miRNA and Down Syndrome

New theory of Down Syndrome cause may lead to new therapies
Tuesday, 23 March 2010

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 over-expressed – meaning that more of it is present than needed in a cell – the protein level goes down, or is under-expressed. A total of at least five of these RNA segments are naturally over-expressed in persons with Down syndrome because the segments are housed on chromosome 21 – the chromosome that causes the disorder.

Terry Elton.

“We’re talking about a paradigm-shifting idea that maybe we should look for under-expressed proteins and not over-expressed 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.

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 bioinformatics database and found that five microRNAs sit on chromosome 21, and he and colleagues demonstrated in previous research that all five of them are over-expressed in the tissues, brains and hearts of Down syndrome patients.

“That means that whatever proteins these microRNAs work with are under-expressed,” 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 under-expressed. 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 under-expressed, the protein levels went up.

Next, the researchers examined adult and foetal human brain tissue from healthy and Down syndrome samples obtained from a national tissue bank.

“In both adult and foetal 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 under-expressed. 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.”

The study is published in a recent issue of the Journal of Biological Chemistry.
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Changes to Embryonic Stem Cells Caused by Down Syndrome

Changes to Embryonic Stem Cells Caused by Down Syndrome Thursday, 04 September 2008 Scientists investigating the mechanisms of Down Syndrome (DS) have revealed the earliest developmental changes in embryonic stem cells caused by an extra copy of human chromosome 21 – the aberrant inheritance of which results in the condition. Their study is published online today (Thursday 4 September) in the American Journal of Human Genetics. Lead by Dean Nizetic, Professor of Cellular and Molecular Biology at Barts and The London School of Medicine and Dentistry, the team utilised embryonic stem cells from a previously genetically engineered species of mice carrying a copy of human chromosome 21. They discovered that extra chromosome 21 - a genetic state known as trisomy 21 - disturbs a key regulating gene called NRSF or REST, which in turn disturbs the cascade of other genes that control normal development at the embryonic stem cell stage. Furthermore, they identified one gene (DYRK1A) on human chromosome 21, whose overdose in trisomy (DS) is responsible for the observed effects. Down Syndrome belongs to the group of conditions called 'aneuploidies', defined by an abnormal loss or gain of genetic material, i.e. fragments of chromosomes or whole chromosomes. Aneuploidies cause congenital anomalies that are a prime cause of infant death in Europe and the USA, and are currently on the increase with advancing maternal age in European countries. The number of people with DS in Europe exceeds half a million. The condition is more common than muscular dystrophy and cystic fibrosis. However, the development of new therapeutic concepts is hindered by the fact that unlike muscular dystrophy and cystic fibrosis, where a single mutated gene causing the disease is known, the entire human chromosome 21 (containing around 300 genes) still has to be dissected into individual gene-dose contributions to the DS symptoms. Professor Nizetic, calling for further research into the components of the disturbed cascade he and his team have revealed said: "We hope that further research might lead to clues for the design of new therapeutic approaches tackling developmental delay, mental retardation, ageing and regeneration of brain cells, and Alzheimer's disease. In other words, we hope our work will open new routes to tackle the genetics of these health disorders, approaching them from the "back entrance", as dominant component-symptoms of Down Syndrome." Reference: DYRK1A-Dosage Imbalance Perturbs NRSF/REST Levels, Deregulating Pluripotency and Embryonic Stem Cell Fate in Down Syndrome Claudia Canzonetta, Claire Mulligan, Samuel Deutsch, Sandra Ruf, Aideen O'Doherty, Robert Lyle, Christelle Borel, Nathalie Lin-Marq, Frederic Delom, Jürgen Groet, Felix Schnappauf, Serena De Vita, Sharon Averill, John V. Priestley, Joanne E. Martin, Janet Shipley, Gareth Denyer, Charles J. Epstein, Cristina Fillat, Xavier Estivill, Victor L.J. Tybulewicz, Elizabeth M.C. Fisher, Stylianos E. Antonarakis, Dean Nizetic The American Journal of Human Genetics, 04 September 2008, 10.1016/j.ajhg.2008.08.012 ......... ZenMaster

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