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.”

Turning Adult Cells into Early Stage Neurons and Bypassing the Pluripotent Cell Stage

Researchers at the University of Wisconsin, Madison have converted skin cells from monkeys and humans into early neural stem cells that can form a wide variety of nervous system-specific cells. This reprogramming did not require converting adult cells into induced pluripotent stem cells or iPSCs. Su-Chun Zhang, professor of neuroscience and neurology at the University of Wisconsin, Madison, served as the senior author of this research. Bypassing the ultraflexible iPSC stage was the key advantage in this research, accord to Zhang.

Zhang added, “IPSC cells [sic] can generate any cell type , which could be a problem for cell-based therapy to repair damage due to disease in the nervous system.” In particular, the absence of iPSCs greatly reduces the risk of tumor formation in the recipient of the stem cell therapy.

There is a second advantage to this procedure. Namely that iPSC generation usually requires the recombinant viruses that deliver genes to the adult cells. These viruses, retroviruses, insert their genes directly into the genomes of the host cell. While there are ways are using such viruses, the use of retroviruses is definitely the most popular strategy for converting adult cells into iPSCs.

Retroviral life cycle
Retroviral life cycle

However, the procedure used in Zhang’s laboratory, utilized recombinant Sendai viruses that do not integrate their genes into the genome of the host cell, but expressed them transiently, after which, the exogenous genes are degraded.

Sendai virus
Sendai virus

Jaingfeng Lu, a postdoctoral researcher in Zhang’s lab, removed skin cells from monkeys and people, and exposed them to recombinant Sendai viruses that contained the four genes normally used to make iPSCs for 24 hours. Then Lu heated the cells to thirty-nine degrees to kill the viruses and prevent the cells from becoming iPSCs. However, 13 days later, Lu found that the cells had become induced neural progenitors or iNPs. When implanted into newborn mice, the iNPs grew normally and differentiated into neural cell types without forming any tumors.

While other researchers have managed to convert adult cells directly into neurons, Zhang admitted that he had a different goal. “our idea was to turn skin cells into neural progenitors, cells that can produce cells relating to the neural tissue. These progenitors can be propagated in large numbers.”

the research overcomes limitations of previous efforts, according to Zhang. The Sendai, which produces little more than a cold, is not a severe pathogen, does not integrate its genes into the genome of the host cell, does not cause tumors, and is considered safe, since it can be killed by heat within 24 hours. This illustrates how fevers in our bodies can kill off cold viruses. Secondly, the iNPs have a greater ability to grow in culture. Third, iNPs are far enough along in their differentiation so that they can only form nervous system-specific cell types. They cannot form muscle or live. However, the iNPs can form many more specialized cells.

Interestingly, the neurons produced from the iNPs had the characteristics of neurons normally formed in the back part of the brain, something that is potentially helpful. As Zhang noted, “For therapeutic use, it is essential to use specific types of neural progenitors. We need region-specific and function-specific neuronal types for specific neurological diseases.”

Progenitor cells grown from the skin of ALS or spinal muscular atrophy patients can be used to make a whole host of neural cells in order to model each disease and allow rapid drug screening. Such cells could also be used to treat patients with neurological disease too.

“These transplantation experiments confirmed that the reprogrammed cells indeed belong to ells of the intended brain regions and the progenitors produced the three major classes of neural cells: neurons, astrocytes, and oligodendrocytes. This proof-of-principle study highlights the possibility to generate [sic] many specialized neural progenitors for specific neurological disorders.”

Neural progenitors
Neural progenitors

Lu, Jianfeng, Liu, Huisheng, Huang, Cindy Tzu-Ling, Chen, Hong, Du, Zhongwei, Liu, Yan, Sherafat, Mohammad Amin, Zhang, Su-Chun.  Generation of Integration-free and Region-Specific Neural Progenitors from Primate Fibroblasts.  2013/05/02. Cell Reports 2211-1247.