Faulty Stem Cell Regulation Contributes to Down Syndrome Deficits

People who have three copies of chromosome 21 have a genetic condition known as Down Syndrome (DS). In particular, patients who have an extra copy of a small portion of chromosome 21 (q22.13–q22.2) known as the Down Syndrome Critical Region or DSCR have the symptoms of DS. The DSCR contains at least 30 genes or so and some of them tightly correlate to the pathology of DS. For example, the APP (amyloid protein precursor) gene accounts for the accumulation of amyloid protein in the brains of DS patients. DS patients develop Alzheimer disease-like pathology by the fourth decade of life, and the APP protein is overexpressed in the adult Down syndrome brain. Another gene found in the DSCR called DYRK1A (dual-specificity tyrosine phosphorylation-regulated kinase 1A) encodes a member of the dual-specificity tyrosine phosphorylation-regulated kinase family and this protein participates in various cellular processes. Overproduction of DYRK1A seems to cause the abnormal brain development observed in DS babies.

Another gene found in the DSCR is called USP16 and this gene encodes a protein that removes small peptides called ubiquitin from other proteins. Ubiquitin attachment marks a protein for degradation, but it can also mark a protein to do a specific job. USP16 removes ubiquitin an either stops the protein from acting or prevents the proteins from being degraded. Overexpression of UPS16 occurs in DS patients, and too much UPS16 protein affects stem cell function.

Michael Clarke, professor of cancer biology at the Stanford University School of Medicine, said, “There appear to be defects in the stem cells in all the tissues we tested, including the brain.” Clarke continued, “We believe USP16 overexpression is a major contributor to the neurological deficits seen in Down Syndrome.” Clarke’s laboratory conducted their experiments in mouse and human cells.

Additional work by Clarke and his colleagues showed that downregulation of USP16 partially rescues the stem cell proliferation defects found in DS patients.

Clarke’s study suggests that drugs that reduce the activity of USP16 could reduce the some of the most profound deficits in DS patients.

This paper also details some of the pathological mechanisms of DS. DS patients age faster and exhibit early Alzheimer’s disease. The reason for this seems to rely on the overexpression of UPS16, which accelerates the rate at which stem cells are used during early development. This accelerated rate of stem cell use burns out and exhausts the stem cell reserves and, consequently, the brains age faster and are susceptible to the early onset of neurodegenerative diseases.

After examining laboratory mice that had a rodent form of DS, Clarke and his coworkers turned their attention to USP16 overexpression in human cells. Clarke collaborated with a Stanford University neurosurgeon named Samuel Cheshier and their study showed that skin cells from normal volunteers grew much more slowly when the Usp16 gene was overexpressed. Furthermore, neural stem cells, which normally clump into little balls of cells called neurospheres, no longer formed these structures when Usp16 was overexpressed in them.

a, Proliferation analysis, as well as SA-βgal and p16Ink4a staining, of three control and four Down’s syndrome (DS) human fibroblast cultures show growth impairment and senescence of Down’s syndrome cells. b, c, Lentiviral-induced overexpression of USP16 decreases the proliferation of two different control fibroblast lines (b), whereas downregulation of USP16 in Down’s syndrome fibroblasts promotes proliferation (c). d, Overexpression of USP16 reduces the formation of neurospheres derived from human adult SVZ cells. The right panel quantifies the number of spheres in the first and second passages. P < 0.0001. All the experiments were replicated at least twice. Luc, luciferase.
a, Proliferation analysis, as well as SA-βgal and p16Ink4a staining, of three control and four Down’s syndrome (DS) human fibroblast cultures show growth impairment and senescence of Down’s syndrome cells. b, c, Lentiviral-induced overexpression of USP16 decreases the proliferation of two different control fibroblast lines (b), whereas downregulation of USP16 in Down’s syndrome fibroblasts promotes proliferation (c). d, Overexpression of USP16 reduces the formation of neurospheres derived from human adult SVZ cells. The right panel quantifies the number of spheres in the first and second passages. P < 0.0001. All the experiments were replicated at least twice. Luc, luciferase.

Conversely, when cultured cells from DS patients had their USP16 activity levels knocked down, their proliferation defects disappeared. In Clarke’s words, “This gene is clearly regulating processes that are central to aging in mice and humans, and stem cells are severely compromised. Reducing Usp16 expression gives an unambiguous rescue at the stem cell level. The fact that it’s also involved in this human disorder highlights how critical stem cells are to our well-being.”

Drug Corrects Brain Abnormalities in Mice With Down Syndrome

Down syndrome (DS) results when human babies have three copies of chromosome 21 rather than the normal two copies. However, three copies of pieces of chromosome 21 can also cause DS, and the region of chromosome 21 called the “Down Syndrome Critical Region” can also cause the symptoms of DS. The Down Syndrome Critical Region is located 21q21–21q22.3. Within this region are several genes, that, when present in three copies, seem to be responsible for the symptoms of DS. These genes are APP or amyloid beta4 precursor protein, SOD1 or Superoxide dismutase, DYRK or Tyrosine Phosphorylation-Regulated Kinase 1A, IFNAR or Interferon, Alpha, Beta, and Omega, Receptor, DSCR1 or the Down Syndrome Critical Region Gene 1 (some sort of signaling protein), COL6A1 or Collagen, type I, alpha 1, ETS2 or Avian Erythroblastosis Virus E26 Oncogene Homolog 2, and CRYAz or alpha crystalline (a protein that makes the lens of the eye).

All of these genes have been studied in laboratory animals, and the overproduction of each one of them can produce some of the symptoms of DS. For example, APP overproduction in mice leads to the death of neurons in the brain and inadequate transport of growth factors in the brain (see A.Salehi et al., Neuron, July 6, 2006; and S.G. Dorsey et al., Neuron, July 6, 2006). Also, the overexpression of CRYA1 seems to cause the increased propensity of DS patients to suffer from cataracts. Likewise, overexpression of ETS2 leads to the head and facial abnormalities in mice that are normally seen in human DS patients (Sumarsono SH, et al. (1996). Nature 379 (6565): 534–537).

People can also have only portions of the DS Critical Region triplicated and this leads to graded types of DS that only have some but not all of the symptoms of DS.

Why all this introduction to DS? It is among the most frequent genetic causes of intellectual disability. Therefore, finding a way to improve the cognitive abilities of DS patients is a major goal. T

There is a mouse strain called Ts65Dn mice that recapitulates some major brain structural and behavioral symptoms of DS and these include reduced size and cellularity of the cerebellum and learning deficits associated with the hippocampus.

Roger Reeves at Johns Hopkins University has used a drug that activates the hedgehog signaling pathway to reverse the brain deficits of Ts65Dn mice. Yes you read that right.

A single treatment given the newborn mice of the Sonic hedgehog pathway agonist SAG 1.1 (SAG) results in normal cerebellar morphology in adults.


But wait, there’s more. SAG treatment at birth also improved the hippocampal structure and function. The hippocampus is involved in learning and memory.


SAG treatment resulted in behavioral improvements and normalized performance in a test called the “Morris water maze task for learning and memory. The Morris water maze test essentially takes a mouse from a platform in shallow water and then moves the mouse through the maze and then leave it there. The mouse has to remember how they got there and retrace their steps to get back to the platform before they get too tired from all that swimming. Normally Ts65Dn mice do very poorly at this test. However, after treating newborn Ts65Dn mice with SAG, they improved their ability to find their way back.

SAG treatment also produced other effects in the brain. For example, the ratios of different types of receptors in the brain associated with memory are skewed in Ts65Dn mice, but after treatment with SAG, these ratios became far more normal. Also, the physiology of learning and memory was also more normal in the brains of SAG-treated Ts65Dn mice.

These results are extremely exciting. They confirm an important role for the hedgehog pathway in cerebellar development. Also, they suggest that the development of the cerebellum (a small lobe at the back of the brain involved in coordination and fine motor skills, direct influences the development of the hippocampus. These results also suggest that it might be possible to provide a viable therapeutic intervention to improve cognitive function for DS patients.

This excitement must be tempered. This is an animal model and not a perfect animal model. Also, it is unclear if such a compound will work in humans. Much more work must be done, but this is a fascinating start.

Neurons Made from the Skin Cells of Down Syndrome Patients Show Reduced Connectivity

The most common form of intellectual disability in the United States is caused by Down syndrome (DS). DS results when babies are born with an extra copy of an extra piece of chromosome 21. Individuals with DS show various types of intellectual deficits and other health problems as well, such as heart problems, poor muscle tone, an under-active thyroid, respiratory infections, hearing problems, celiac disease, eye conditions, depression or behavior problems associated with attention-deficit hyperactivity disorder or autism.

Even though Down syndrome patients have symptoms and health problems that are well described, how the extra chromosome causes such widespread effects is still largely mysterious.

In recently published research, Anita Bhattacharyya, who is a neuroscientist at the Waisman Center at the University of Wisconsin-Madison, reported that brain cells that were grown from skin cells taken from 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 skin cells taken from DS patients were grown in culture and genetically engineered to so that a fraction of them were transformed into induced pluripotent stem cells (iPSCs). Since iPSCs can be differentiated into any adult cell type, Bhattacharyya’s lab, working with collaboration with Su-Chun Zhang and Jason Weick, grew those iPSCs in culture and differentiated them into dorsal forebrain neurons, which they could test in the laboratory.

Neurophysiological tests of the DS neurons revealed that these neurons formed a reduced number of connections between them each other. 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.”

Bhattacharyya and colleagues also examined the genes that were affected in the Down syndrome stem cells and neurons. They discovered that those genes on the extra chromosome were increased 150 percent, which is 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 molecules with unpaired electrons 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.”

DS includes a range of symptoms that might 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 DS 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 this work, she did note that “we could potentially use these cells to test or intelligently design drugs to target symptoms of Down syndrome.”

Scientists Remove Extra Chromosome 21 from the Cells of Down Syndrome Patient

University of Washington researchers have done something seemingly impossible: they have removed the extra copy of chromosome 21 in cells taken from a patient with Down syndrome. This gene therapy technique targets only the extra genetic material in the cell, and scientists were able to successfully remove the extra chromosome 21 without damaging the integrity of the rest of the chromosomes present in the nucleus.

The first reaction to this news is to shout, “there’s a cure for Down Syndrome!” Unfortunately that is not the case. However, it might be a way to treat Down Syndrome patients who have blood cancers. Down syndrome patients are at increased risk for leukemia, and this technique, pioneered by Dr. David Russell and his colleagues is meant to fix the errant bone marrow cells in culture and then reintroduce the fixed cells back into the patient.

Dr. Russell explained: “We are certainly not proposing that the method we describe would lead to a treatment for Down syndrome. What we are looking at is the possibility that medical scientists could create cell therapies for some of the blood-forming disorders that accompany Down syndrome.” Dr. Russell is from the University of Washington’s Department of Medicine.

This technique works on cultured cells grown in a laboratory. The cells are infected with an engineered virus that inserts into the extra chromosome. Then the cells are grown under conditions that kill all cells with the viral DNA. Only those cells that spontaneously lose the extra copy of chromosome 21 survive the culture conditions.

This protocol could potentially treat Down syndrome patients with leukemia with genetically-modified stem cells that are derived from their own cells, but lack the extra chromosome. Stem cells could be taken from the bone marrow of the patients, the doctors could remove the extra chromosome, and then the healthy cells could then be grown and transplanted back into the bone marrow of the patient. This same technique could also be used for leukemia patients whose bone marrow cells have an extra chromosome, but do not have Down syndrome.

This is great news for those with Down syndrome and for all those who live with any kind of trisomy. Also, since gene therapy can introduce new defects into the patient’s DNA, this technique could potentially remove unwanted extra bits of DNA without adversely affecting other chromosomes. This is certainly a major achievement.