Huntington’s Disease Model System Derived from Patient-Specific Induced Pluripotent Stem Cells


Huntington’s Chorea or Huntington’s disease is an inherited condition that results from the progressive and relentless degeneration of nerve cells in the central nervous system.  Huntington’s disease (HD) broadly affects the patient’s functional abilities and decreases his or her ability to move, think or behave properly.  Most of the time, patients develop the signs or symptoms of HD when they are 40 or 50 years old or slightly older.  In the case of Juvenile HD, symptoms begin before the age of 20.

Mutations in the HTT gene, which encodes the Huntingtin protein cause HD, and typically, the mutations in the gene that are associated with HD are so-called “triplet expansions.”  To understand triplet expansions, we must understand how genes encode proteins.  Genes are stretches of a DNA molecule that are transcribed into RNA copies.  The enzyme that synthesizes the RNA copy is called RNA polymerase, and a gene has a set of sequences that tell the RNA polymerase where to start making RNA copies and where to stop.  Once the RNA copy of the stretch of DNA is made, the RNA either has a function of its own, or the RNA is translated into protein.  Translation is the process by with RNA-protein complexes called “ribosomes” bind to the front of the RNA and use the nucleotide sequence to synthesize a protein that has a specific sequence of amino acids.  Amino acids are encoded in genes by a three-nucleotide sequence or codon, and ribosomes read the RNA molecules three nucleotides at a time.

The nucleotide sequence CAG (cytosine, adenine, guanine) encodes the amino acid glutamine.  The HTT gene has a stretch of these nucleotides, and they code for the amino acid glutamine.  Normal copies of the HTT gene will have anyways from nine to thirty-five glutamines in these stretches.  However, these CAG stretches have a tendency to expand because the enzymes that replicate DNA (DNA polymerases) have a tendency to slip when they get to CAG stretches, and this causes the CAG stretches to increase in size, or, occasionally, decrease in size.  The glutamine stretches can reach large numbers, and if the number of glutamines in the glutamine stretch exceeds 35, people will usually start showing symptoms.  The larger the number of glutamines in the glutamine stretches, the earlier the symptoms will appear (Juvenile HD usually occurs in patients with 60 or more glutamines in the glutamine stretch), and the more aggressive the disease.

How does the abnormal Huntingtin protein kill nerve cells?  This is unclear, but it is clear that Huntingtin proteins with abnormally large numbers of glutamines in their glutamine stretches are poisonous to cells, and the nerve cells that die tend to dump their neurotransmitters, which kills other nearby cells, which then cause them to dump their neurotransmitters, and the cascade of cell death begins.

Cell transplantation experiments in animals have produced a variety of positive results, but these results are probably not representative of the situation in human patients.  HD in animals, you see, is induced by the injection of chemicals into the brains of laboratory animals, and these chemicals kill off particular groups of nerve cells that cause the symptoms of the disease.  The rest of the brain is essentially normal.  Human patients have a brain that has been transformed into a toxic waste dump, and transplanted cells do not survive well in them.  I have other blogs on this site that speak about this here, here, and here.

To address this problem, a South Korean group has developed a model system for HD based on induced pluripotent stem cells made from an actual HD patient.  This paper was published in the journal Stem Cells on May 24, 2012 (doi: 10.1002/stem.1135), and is entitled:  Neuronal Properties, In Vivo Effects and Pathology of a Huntington’s Disease Patient-Derived Induced Pluripotent Stem Cells.  The lead author is I. Jeon from the CHA Stem Cell Institute, CHA University, Seoul, Korea.

In this paper, Jeon and colleagues took skin cells from HD patients and used them to make induced pluripotent stem cells (iPSCs).  By carefully manipulating the cells in culture, the South Korean scientists were able to convert the iPSCs into nerve cells.  The particular patient whose IPSCs were used in this experiment had a HTT gene that encoded a Huntington protein with 72 glutamines and the patient had a juvenile form of HD.

The specific nerve cells that degenerate in the brains of HD patients are neuron that produce the neurotransmitter GABA (gamma-amino butyric acid).  Therefore, Jeon and his coworkers had GABA-specific neurons from the iPSCs.  WHile the initial induction rate for nerve cell production from the iPSCs from the HD patient was low, they were able to produce a respectable quantity of GABA-neurons from the HD iPSCs.

Nest, they took rats and generated the types of lesions necessary to cause HD, but they transplanted the GABA-neurons that were made from the HD-iPSCs into the brains of the lesioned rats.  Interestingly, the rats recovered from the lesions and their behavior returned to normal.  At 12 weeks after the transplantations, the brains of the rats still looked normal.

However, once the rats were treated with a chemical that prevents cells from getting rid of excessive amounts of junk proteins, now the rats started to show the symptoms of HD and their brains showed pathologies that greatly resembled those found in HD patients.  Also, if the GABA neurons made from the HD iPSCs were implanted into the brains of neonatal rats, which grow very quickly, they produce HD-like pathology 33 weeks after transplantation.

What does this mean?  Even though these rats carried GABA neurons that contained a severe version of the HTT gene, the neurons still were able to work and give rise to normal neurons inside the body of the animals.  However, those animals were extremely susceptible to any sort of perturbations that caused junk proteins to build up.  If the levels of junk protein built up, they eventually killed the cells.  What are those triggers in human patients that cause cells to clog up with junk proteins?  Clearly this HD model will help neuroscience researchers answer some very vital questions about the cause and pathology of HD.  Answers that might lead to efficacious treatments that will reduce the extreme suffering of some patients.

Whitehead Scientists Discover Critical Role Played By One Enzyme In Embryonic Stem Cell Differentiation


Cells use gene expression programs to respond to external stimuli and maintain their present form and identity. Genes are stretches of DNA that encode a protein or RNA. Gene expression requires DNA sequences directly attached to the gene, and these sequences are called the “promoter” of the gene. The promoter is the binding site for the enzyme that executes the first step of gene expression. That enzyme is called “RNA polymerase.” RNA polymerase binds to the promoter and initiates the synthesis of an RNA copy of the gene, and process by which an RNA copy of the gene is synthesized rom the DNA template is called “Transcription.”

If the gene encodes a protein, the RNA is processed and sent from the nucleus of the cell to the cytoplasm, where protein/RNA complexes called “ribosomes” use the sequence in the RNA to make proteins that have amino acid sequences. Some genes, however, encode RNA molecules that are not used to direct the synthesis of protein, but are used for some other purpose.

Whatever the case, cells have a great deal of DNA in their nuclei. Almost every human cell, for example, contains so much DNA that if the DNA in one human cell was laid out end-to-end, it would stretch to a length of at least 1 meter. To pack all that DNA into the nucleus of a cell, the DNA is wound into a tight complex of DNA and proteins that is collectively called “chromatin.” Chromatin consists of DNA wound around proteins called histones, in ways that resemble the way thread is wound around a spool. These little histone spools are then wound into spirals that are then wound into a rosette of fibers. It is exceedingly for RNA polymerase to transcribe genes when they are wound into chromatin. How then are genes expressed? It turns out that particular proteins modify chromatin and cause it to loosen up so that RNA polymerase can access it.

Histone modifying proteins include those that encourage the formation of chromatin and tend to shut gene expression off (histone deacetylase, Polycomb-group proteins), and those that loosen chromatin and encourage gene expression (histone acetyl-transferases, histone methyltransferases). Therefore, we might expect to see such enzymes playing an important role in stem cell differentiation.

Therefore, we should not be surprised that stem cells researchers at the Whitehead Institute have discovered that a specific chromatin enzyme called lysine-specific demethylase 1 (LSD1) plays as embryonic stem cells differentiate into other cell types. Cell differentiation requires two key steps: 1) the genes active in the initial cell type must be deactivated; and 2) those genes important for the establishment of the new cell type must be activated. If the switch is not flawless, a transitioning cell may die or be driven to divide uncontrollably. Interestingly, LSD1 was known to be critical to development, but little was known about the key role it plays during differentiation, when cell-specific gene expression systems are switched on or off.

Paper author, Steve Bilodeau, who is also a postdoctoral research fellow in the laboratory of Whitehead Member, Richard Young, said; “We knew that cells express a new set of genes when the operating switch occurs. But this study shows it is also essential to shut off genes that were active in the prior cell state. If you don’t, the new cell is corrupted.”

Bilodeau and Warren Whyte, a Young lab graduate student and co-author, redefined LSD1’s role and described a previously unknown mechanism for silencing genes. They examined embryonic stem cell gene expression during differentiation and concentrated their efforts on those genes that must be shut off during differentiation. Whyte and Bilodeau found LSD1 was located on the promoters of those genes that had to be repressed in order for differentiation to occur. LSD1 was also found near DNA sequences called “enhancers,” which are associated with promoters and increase the ability of the promoters to activate gene expression.

What is LDS doing at the promoter and enhancer? When LSD1 receives the signal that the stem cell is going to differentiate, it transitions into an active conformation and silences those genes. Specifically, LSD1 hamstrings the ability of the enhancers of those genes to activate gene expression. With their enhancers rendered nonfunctional, transcription of these genes is silenced. While this occurs, other mechanisms switch on those genes necessary for the adoption of the new cell type.

Whyte added: “This reveals the critical function of LSD1 in cell differentiation. The enzyme decommissions the stem cell enhancers, thus allowing the new cell to function entirely within the parameters of the new operating system.”

Although this work focuses on one enzyme’s job in normal cells, Young sees broader implications, since LSD1 is a member of a class of molecules that regulate both gene activity and chromosome structure. Therefore, these findings about LSD1 could provide insights into how related regulators function. Similarly, understanding how a mechanism operates in normal cells provides a solid foundation for teasing apart what is going wrong in abnormal cells.

Young summed it up this way: This new knowledge brings us one important step closer to understanding defective operating systems in diseases such as cancer. And this may give us a new angle on drug development for these diseases.”

This work was published in “Enhancer decommissioning by LSD1 during embryonic stem cell differentiation;” Warren A. Whyte, Steve Bilodeau et al.; Nature, 2012; DOI: 10.1038/nature10805.