All posts in Protein aggregation

Prion-like Behavior in the Huntingtin Protein

Prion-Like Behavior in the Huntingtin Protein:

Protein aggregates are a hallmark feature of Huntington’s disease (HD)[1], as well as a number of other neurodegenerative diseases. These protein aggregates, composed of misfolded proteins that clump together, are traditionally thought to develop in vulnerable neurons individually. However, recent research suggests that these misfolded proteins may be transmitted from neuron to neuron.


Transmission of disease-causing proteins between cells is not new in the scientific literature. The idea of an infectious agent composed only of proteins, called prions, was first proposed in 1967[2], and is now known to cause a number of neurodegenerative diseases in animals and humans. Prions cause other proteins to fold into the wrong shape. Some research suggests that proteins demonstrating prion-like behavior may play a role in other neurodegenerative diseases, including Parkinson’s, Alzheimer’s, and Amyotrophic Lateral Sclerosis (ALS)[3]. This destructive process mainly appears in functioning, connected neural networks. Unlike prions, proteins involved in these neurodegenerative diseases are not infectious between individuals or species [4]. A study published in Nature in August 2014 by Pecho-Vrieseling et al. [5] suggests that the protein that creates protein aggregates in HD patients, mutant huntingtin (mHTT), may spread from cell to cell. This study provides valuable insight into what is currently understood about the role of protein aggregates and HD.

The study examines whether mHTT can spread among and propagate in vulnerable neurons using R6/2 HD mouse models and normal human stem cells. The researchers used genetically modified mice that express human mHTT fragments (only small portion of mHTT containing the polyglutamine stretch) and have accelerated HD-like pathophysiology. They began by implanting human neuronal progenitor cells without mHTT (derived from normal human stem cells), into HD mouse brain slices. The researchers determined that the human cells were successfully integrated in the brain slice as functional neurons. Then, they demonstrated that the healthy human cells were able to acquire aggregates of mouse mHTT protein and underwent similar changes as the sick mouse cells, including fewer projections from neurons and loss in medium spiny neurons.

Finally, in order to investigate if mHTT was transmitted from cell to cell via synapses, the researchers treated co-cultures of human neurons and HD mouse brain slices with a neurotoxin, botulinum toxin, which blocks vesicle fusion with the plasma membrane and prevents the neurons from releasing neurotransmitters. In the presence of this neurotoxin, the HD mouse neurons contained mHTT aggregates but the human cells did not. This evidence suggests that mHTT is transmitted along the cortical striatal pathway and is transmitted across neurons via synapses.
If mHTT is passed from neuron to neuron, it could have important implications on therapeutic interventions because the propagation can be experimentally blocked. In the past, neural transplants have been tested as a therapeutic for HD patients. If mHTT is indeed able to escape between cells, this could lead to a failure of neural transplants in HD patients. The biological mechanisms by which misfolding proteins are transmitted to functioning neural networks in this study are still unclear. The authors of the paper speculate that mHTT transfer depends on synaptic activity, and suggest that mHTT is transmitted at the synapse. However, much more research is still needed to determine whether this prion-like process actually affects human HD onset and/or progression.

This recent study adds to the understanding of the development of protein aggregates in HD by demonstrating that in certain lab conditions, mHTT can escape one cell and enter another. It is possible that cell-to cell propagation of mHTT may be another factor in the development of protein aggregates in HD. However, although this work is very well done and novel, it is unclear whether this process has any relevance to disease development. The role of protein aggregates in HD development is still widely debated.

[1]Arrasate, Montserrat and Steven Finkbeiner, “S. Protein aggregates in Huntington’s disease. Exp. Neurol. 2012, 238,:11-11.
[2] Griffith, J.S. “Nature of the Scrapie Agent: Self-replication and the Scrapie.” Nature 2 Sept. 1967: 1043-1044.
[3]Guo, Jing L. and Virginia M Y Lee. “Cell-to-cell transmission of pathogenic proteins in neurodegenerative diseases.” Nature Medicine (2014) 20: 130-138.
[4]Aguzzi et al. “Cell Biology: Beyond the prion principle.” Nature, 18 Jun. 2009, 459: 924-925.
[5]Pecho-Vrieseling et al. “Transneuronal propagation of mutant huntingtin
contributes to non-cell autonomous pathology in neurons.” Nature Neuroscience Aug. 2014; 1(8): 1064-1072.


TRiC and Huntingtin Protein Aggregation

In Huntington’s disease (HD), an abnormal increase in the number of CAG repeats in the mutant Huntington gene corresponds to a long tract of glutamine amino acids in the huntingtin protein (Htt). This excessively long glutamine tract is sticky and leads to the formation of protein aggregates in brain cells. Whether these aggregates are toxic only upon formation or are formed as a cellular defense mechanism against free toxic mutant huntingtin protein (mHtt) is still hotly debated (more information about huntingtin protein aggregation can be found here); however, what is certain is that these protein aggregates are directly linked to neuron dysfunction and death. Moreover, a larger number of CAG repeats is clearly correlated with earlier disease onset and increased severity.

Although these findings suggest that HD is solely a result of the CAG expansion and subsequent protein aggregation, several observations indicate that other factors are also at play. For example, patients with the same number of CAG repeats may exhibit different disease patterns. In addition, there are genes other than the Huntington gene that also contain a CAG repeat region. Some of these genes may have a long region of upwards of 50 CAG repeats, but still produce functional non-harmful proteins (recall that HD can occur in patients with as few as 36 CAG repeats in the Huntington gene).  Evidently, there are other cellular mechanisms, though not well understood, that govern how the expanded number of CAG repeats translates into the toxic behavior of mHtt and aggregates seen in the specific case of HD.

Recently, researchers have found increasing evidence that the cellular mechanisms contributing to neurodegenerative diseases resulting from protein aggregates involve disruptions to the quality control machinery in the cell responsible for identifying and eliminating these toxic proteins. In particular, a protein known as TRiC (TCP-1 ring complex) has been found to be involved in controlling whether proteins with a long glutamine tract like Htt will fold and aggregate.  This article will first review how certain classes of proteins can control protein folding, then go over the case of TRiC and its role in Htt folding, and finally discuss how this knowledge might be helpful for researching HD therapeutics.

Protein Folding and Molecular Chaperones^

To understand how TRiC is involved in huntingtin (Htt) protein aggregation, it is useful to first review the players that make proteins in the cell. First, mRNA instructions are made based on the DNA blueprint in the nucleus, or the control center, of the cell. These instructions are then sent out into the cytoplasm of the cell and are read by structures known as ribosomes, the actual molecular machines that make proteins. Proteins are synthesized as long chains of amino acid building blocks, which are assembled one by one as the ribosome reads the mRNA instructions. For a more detailed breakdown of the protein synthesis process, go here.

However, proteins are not functional as a linear chain; they need to take on a certain shape or conformation in order to exert their proper functions. To assist in this process known as protein folding, proteins known as molecular chaperones bind to chains of amino acids that are newly made by ribosomes. Molecular chaperones, as their name suggests, are present during the protein-making process to ensure that newly made proteins behave normally and appropriately for their intended function. They do this by binding to the amino acid chain, thereby stabilizing it and facilitating proper folding. Another function that chaperones perform is to prevent folding errors and inappropriate aggregation, by recognizing incorrectly folded proteins and targeting them for repair or elimination. More information about molecular chaperones and proper protein folding can be found here.

Proteins folding incorrectly can lead to protein aggregation and disease. Moreover, there is a natural decline in a cell’s ability to continually make and fold protein correctly as one ages. As a result, in the study of HD, many scientists have focused their attention to the study of molecular chaperones and their role in responding to protein aggregation. TRiC is a molecular chaperone that has recently been found to be directly involved in regulating aggregation seen in proteins containing a long tract of glutamine amino acids, such as Htt.

What is TRiC and how is it involved in protein folding?^

TCP-1 ring complex, or TRiC, is a type of molecular chaperone that is composed of 8 different protein subunits, called CCT1, CCT2, all the way to CCT8 (CCT = chaperonin containing TCP-1). Together, these CCT subunits form a structure resembling two donuts stacked on top of each other.  Like other molecular chaperones, TRiC’s primary role in the cell is to act as a protein-folding machine: they bind to a specific set of newly made proteins and fold them into shapes that allow them to function correctly. This process occurs first with the unfolded protein entering and binding into the center of one of the rings in TRiC. TRiC then closes up the ring with the protein inside. During this time when the protein is enveloped in TRiC, protein folding occurs. After the protein is folded, TRiC can open up its ring again and eject the folded protein. In addition to huntingtin (Htt), some other proteins that are known to be regulated by TRiC include actin and tubulin, which are very important proteins that maintain the structural framework of the cell.

In order to identify molecular chaperones that are involved in proper folding of proteins with glutamine repeats, researchers looked at a roundworm often used in lab research. This 2006 study examined which proteins are important for minimizing aggregation of proteins with a polyglutamine expansion (“poly” means “many”, so “polyglutamine” means many glutamines). The researchers achieved this by using a roundworm that was genetically engineered to produce yellow fluorescent protein containing a polyglutamine expansion of 40 glutamine repeats. Then, they suppressed each gene in the roundworm one by one and looked for changes in the polyglutamine expanded fluorescent protein. The logic behind this experiment is that if a gene is important for preventing proteins with polyglutamine expansions, like Htt, from aggregating, then blocking that gene would increase the amount of protein aggregation. As you might have expected, one of the genes that the scientists identified through this experiment is the one that encodes the molecular chaperone TRiC.

Although roundworm models are easy to manipulate in the lab, it is much more relevant to study Htt aggregation in human cells. Consistent with the previous result, it has also been found that blocking TRiC in human cells will result in increased aggregation of proteins with an expanded glutamine repeat region.

How does TRiC actually suppress huntingtin aggregation?^

So far, we’ve discussed how TRiC is involved in folding newly formed proteins, but how does TRiC prevent already formed mutant huntingtin (mHtt) from aggregating?  Studies in this area of research first found that TRiC is able to directly bind to the Htt protein. This result aligns with what we previously knew about TRiC – that it acts as a molecular chaperone by assisting in protein folding. In particular, scientists have found, through several different experiments, that CCT1, one of the eight subunits forming TRiC, is especially important for binding to Htt. First, an in vivo study of the brain tissue of a transgenic mouse expressing mHtt revealed that TRiC and, specifically, CCT1 directly interacts with the Htt protein. This is very strong evidence indicating that TRiC regulates Htt activity in the brain in some way. Furthermore, by suppressing the CCT1 subunit alone, researchers still observe the same increase in Htt aggregation as when TRiC is suppressed. Even more significantly, expressing more CCT1 in cells can actually decrease the amount of Htt aggregation and toxicity in human cell culture models. All of these experiments suggest that TRiC can somehow control Htt aggregation through the CCT1 subunit.

Recent studies conducted between 2009 and 2013 from Judith Frydman’s lab at Stanford have shed light on how TRiC might control Htt aggregation through the CCT1 subunit.  First, researchers from the lab found that CCT1 binds to the Htt protein on a region of 17 amino acids, called N17, right next to the tract of glutamine repeats. This N17 region is significant because deleting this region in an Htt protein with 51 glutamine repeats appears to slow down the toxic protein aggregation process normally observed. In other words, the N17 region right next to the glutamine tract plays a role in speeding up the process of Htt protein aggregation.  Scientists also found that the N17 region of one Htt protein can bind to the N17 region and the glutamine tract of other Htt proteins.  Based on this evidence, they hypothesize that perhaps the N17 region of the Htt protein acts as a sticky end that other Htt proteins can attach onto and gradually form aggregates.

TRiC is another protein that can bind to the N17 region of Htt through its CCT1 subunit. Using advanced imaging techniques, the Stanford researchers found that when Htt and TRiC were put together, TRiC can either encapsulate single Htt proteins or small Htt aggregates in its ring chamber. It can also attach onto the tips of larger Htt aggregate fibers. From this experiment, the researchers concluded that TRiC can inhibit Htt aggregation perhaps by blocking the N17 region of Htt protein from endlessly sticking to other Htt proteins to form large, fibrous protein aggregates associated with neuron death. In the presence of TRiC, Htt will be maintained as a small single protein or small protein aggregates that are not toxic to the cell.

How can these findings about TRiC translate to an HD therapeutic?^

Research about TRiC is still in its preliminary stages, since much of the mechanisms behind TRiC’s ability to suppress huntingtin (Htt) aggregation remains to be understood. However, the fact that we do know TRiC and specifically its CCT1 subunit are potent inhibitors of aggregation is useful for researchers who are thinking of ways to stop the formation of dangerous aggregates that are toxic to brain cells.

A study published in 2012 by Sontag et al. took this knowledge and asked the following question: because cells that express more of the CCT1 subunit had less Htt protein aggregation, can this CCT1 subunit be applied as a drug and be directly delivered to brain cells? In order to answer this question, the researchers used cell models of HD and first showed that a modified fragment of CCT1 can penetrate the cell membrane and enter the cell to exert its effects. With regards to therapeutic benefits, the researchers observed that when higher doses of CCT1 were applied to rat cells engineered to express mHtt, there was a corresponding decrease in the amount of visible Htt aggregates formed. The scientists followed up with another experiment where they applied CCT1 to cells of HD mice that were removed from the striatum, the brain area most affected in HD.  They obtained perhaps an even more encouraging result: the cells receiving CCT1 treatment exhibited a higher survival rate compared to cells without treatment.  This suggests that CCT1 may exert therapeutic benefits for cells expressing mHtt beyond just suppressing protein aggregation.


The above research is simply a small-scale experiment that demonstrates the potential that this line of research can have for HD therapeutics.  Evidently, there is a still a lot that we don’t know about how molecular chaperones like TRiC can suppress huntingtin (Htt) aggregation.  The experiments up to this point have all been performed with cells in laboratory settings, and these results will have to be verified in subsequent animal studies before any conclusions can be made about their actual therapeutic benefit.  However, studying TRiC has emphasized the importance of looking beyond the infamous glutamine repeats in Htt and looking at regions such as N17 to understand the mechanism of Htt aggregation and toxicity. Furthermore, the fact that direct protein delivery to the brain is already being done in mice for Parkinson’s disease research could lead the way for testing CCT1 in HD mouse models as well.

Further Reading^

Meyer AS, Gillespie JR, Walther D, Millet IS, Doniach S, Frydman J. (2003) Closing the Folding Chamber of the Eukaryotic Chaperonin Requires the Transition State of ATP Hydrolysis. Cell. 113(3): 369-381.

Behrands C, Langer CA, Boteva R, Böttcher UM, Stemp MJ, Schaffer G, Rao BR, Giese A, Kretzschmar H, Siegers K, Hartl FU. (2006). Chaperonin TRiC Promotes the Assembly of polyQ Expansion Proteins into Nontoxic Oligomers. Mol Cell. 23(6): 887-897.

Tam S, Geller R, Spiess C, Frydman J. (2006). The chaperonin TRiC controls polyglutamine aggregation and toxicity through subunit-specific interactions. Nat. Cell Biol. 8(10): 1155-1162.

Tam S, Spiess C, Auyeung W, Joachimiak L, Chen B, Poirier MA, Frydman J. (2009) The Chaperonin TRiC Blocks a Huntingtin Sequence Element that promotes the Conformational Switch to Aggregation. Nat. Struct. Mol. Biol. 16(12): 1279-1285.

Shen K, Frydman J. (2013). The interplay between the chaperonin TRiC and N-terminal region of Huntingtin mediates Huntington’s Disease aggregation and pathogenesis. Protein Quality Control in Neurodegenerative Diseases. Eds. Morimoto  RI, Christen Y. 121-132.

Shahmoradian SH, Galaz-Montoya JG, Schmid MF, Cong Y, Ma B, Spiess C, Frydman J, Ludtke SJ, Chiu W. (2013). TRiC’s tricks inhibit huntingtin aggregation. eLife. 2:e00710.

Sontag EM, Joachimiak LA, Tan Z, Tomlinson A, Housman DE, Glabes CG, Potkin SG, Frydman J, Thompson LM. (2012). Exogenous delivery of chaperonin subunit fragment ApiCCT1 modulates mutant Huntingtin cellular phenotypes. Proc. Natl. Acad. Sci. USA. 110(8): 3077-3082.

– J. Choi, 02-03-14


Genome Editing

Although the pathology of Huntington’s disease (HD) is still not completely understood, we know that HD is a genetic disorder where the root cause of every HD case is a longer-than-normal series of three repeated DNA base pairs, CAG, in the HD gene. A DNA sequence provides the instructions for the cell to make mRNA (messenger RNA), which in turn contains the instructions for making a protein – the building blocks and machines of cells. The process of using the information contained in genetic material (DNA and RNA) to form protein is called gene expression. Genetic changes in the HD gene sequence are thus propagated into the mRNA sequence and result in production of a mutated version of the huntingtin protein that ultimately results in degeneration of brain cells.

In order to develop a therapy that prevents the production of the mutant huntingtin protein, many scientists are currently using a technology known as gene silencing. This approach creates molecules that directly target and bind to the mRNA copies that contain the instructions for producing huntingtin protein. These molecules then use the cell’s own molecular machinery to destroy the problematic mRNA instructions (for more information about gene silencing, click here).

Gene silencing is a very promising approach, and many scientists are focusing their efforts on conducting studies and clinical trials to assess the feasibility and efficacy of gene silencing treatments for HD and other diseases. However, it has recently become possible to go one step further in manipulating gene expression. Instead of targeting the intermediate mRNA copies as in gene silencing, some scientists are pursuing strategies that will directly modify the DNA blueprint in cells and ultimately living organisms. This article will discuss this nascent technology known as genome editing and its potential as an HD therapeutic.

What is Genome Editing?^

Just as editing text involves adding, removing, or replacing words, genome editing is an approach in which the genome sequence is directly changed by adding, replacing, or removing DNA bases. However, the genome is relatively resistant to change. DNA in the body is not only responsible for encoding all of the necessary functions within a cell, but it is also crucial to determining differences between individuals. If DNA could be easily altered, many essential cell functions would be disrupted in undesirable ways. To deter any changes from being inadvertently made to DNA, cells have inherent mechanisms to proofread and repair their genetic code. These repair mechanisms and the overall stability of DNA is what makes genome editing such a novel approach and so difficult to achieve.


Remarkably, researchers have been able to take advantage of the cell’s DNA repair mechanisms to achieve genome editing. To accomplish this, scientists can use artificially engineered enzymes called nucleases to cleave DNA strands. In effect, these nucleases act as molecular scissors that form a break in the DNA double-stranded helix. Once a break is introduced in the DNA, the cell will detect a problem in its genetic code and quickly activate its repair machinery.

There are two major methods by which a cell can repair a break in its DNA. First, the cell can employ various enzymes to directly join the two ends of the DNA break back together. This process, known as nonhomologous end-joining, is very error-prone and often results in mutations – such as small insertions or deletions of nucleotides – in the resulting DNA strand. These small mutations can be neutral, but they can also render the entire gene in that location nonfunctional, achieving the disruption or knockout of the gene.

Second, the cell can also repair a DNA break by using another DNA sequence as a template. In genome editing applications, a DNA sequence can be designed to be inserted along with a nuclease, such that when a cut is made in the DNA, the cell’s own repair mechanisms can use the DNA sequence supplied to replace an existing DNA sequence as it repairs the break. This method allows scientists to directly change genetic information in cells by introducing a correct version of a DNA sequence to replace an unwanted mutation.

How can a specific gene be edited?^

By using a cell’s own repair mechanisms, scientists can disrupt or correct a mutation by genome editing, and both approaches could prove useful in the context of HD treatment. However, there must be a way to direct a nuclease to the desired location where a DNA break is to be introduced. To address this issue, many different types of nucleases have been developed. All nucleases consist of 2 components – the nuclease itself that is responsible for DNA cleavage and a secondary component responsible for recognizing a specific DNA sequence. There are three main classes of nucleases engineered for genomic editing purposes:

Zinc finger nucleases (ZFNs)^

ZFNs consist of a nuclease component linked to a DNA-binding component derived from an array of zinc finger proteins. Each zinc finger protein can bind three nucleotides, so combinations of zinc fingers linked together can be designed to recognize specific genomic sequences.

Transcription activator-like effector nucleases (TALENs)^

TALENs are very similar to ZFNs in that they also have a nuclease domain linked to a DNA recognition domain. The difference lies in the fact that in TALENs, the DNA recognition domain is a series of amino acid repeats. Each repeat corresponds to a single nucleotide base (A, G, C, or T), and TALENs can be designed to have different combinations of repeats to recognize specific genomic sequences.

CRISPR/Cas System^

The CRISPR/Cas system employs a nuclease called Cas9 to introduce a DNA double strand break. Unlike ZFNs or TALENs, this approach does not use a protein-based DNA recognition domain. In order to guide the Cas9 nuclease to a specific DNA binding site, an RNA sequence is designed to precisely bind to a complementary DNA sequence, allowing for the Cas9 nuclease to make a cut.


Unlike ZFNs or TALENs, each meganuclease has a long recognition sequence that allows them to make DNA breaks at specific sites. However, these long recognition sequences are naturally defined and cannot be engineered, thus meganucleases can only be used for some target genetic sequences.

Can genome editing be used therapeutically?^

Just like gene silencing, genome editing is already being used by scientists as one of their many tools to develop cell and animal models for studying different diseases. Is there a possibility that genome editing can be used in humans to cure genetic diseases like HD? Preliminary research suggests that genome editing may be a promising therapeutic approach, but more work is needed prior to clinical testing in humans.

Research has shown that simply delivering engineered zinc finger proteins (ZFPs) that do not have any nuclease activity is able to reduce the levels of mutant huntingtin. A study published in 2012 by a research group in Spain found that ZFPs can be designed to bind longer CAG repeats more strongly than shorter repeats, which means that these ZFPs could specifically recognize the mutant huntingtin gene with the CAG expansion. They further demonstrated that ZFPs reduced the levels of mutant huntingtin by 95% without affecting the levels of the wild-type huntingtin protein in an in vitro model of HD using mouse cells expressing a human version of the mutant HD gene. Moreover, they were able to demonstrate similar results in an HD mouse model, where ZFP treatment reduced the level of mutant huntingtin up to 60% and motor performance as measured on a rotarod was significantly improved. This study not only demonstrated that ZFPs can specifically bind to the mutant huntingtin gene, but also suggested that ZFPs can accomplish gene silencing by simply binding to the DNA and preventing the gene from being transcribed. These findings support the use of zinc finger nucleases (ZFNs), which could add to the repressive effect of ZFPs by actually disrupting or correcting the mutant gene.

ZFNs have been tested as a therapeutic approach in other diseases. For example, Sangamo Biosciences, a biopharmaceutical company, has explored the potential of ZFNs in treating hemophilia, a genetic disorder in which the ability of the blood to clot is impaired. The researchers used ZFNs to replace the mutated gene responsible for causing hemophilia with a correct gene that allows for normal function in a mouse model of hemophilia, and found that clotting times of the mice returned to normal after treatment. This study suggests that ZFNs are a viable strategy for correcting the genome in genetic diseases such as HD.


Many factors will still need to be considered before genome editing can be used as a viable therapeutic option for HD. Designing nucleases to be specific for one genetic sequence is a difficult and often expensive process, as it requires linking together the right combination of zinc fingers in ZFNs or the right combination of amino acid repeats in TALENs. Moreover, just like in gene silencing, DNA recognition by ZFNs or TALENs is not perfect and can result in off-target effects (binding to the wrong genetic sequence). Genome editing is still in its early stages, and it will be awhile before we will know if it can be used as a gene therapy for HD patients. But, many scientists are pursuing this new avenue of research with promising results.

Further Reading^

Jasin, Maria. (1996) Genetic manipulation of genomes with rare-cutting endonucleases. Trends in Genetics. 12(6): 224-228.

de Souza, Natalie. (2012) Primer: genome editing with engineered nucleases. Nature Methods. 9: 27.

Hsu PD, Scott DA, Weinstein JA, Ran FA, Konermann S, Agarwala V, Li Y, Fine EJ, Wu X, Shalem O, Cradick TJ, Marraffini LA, Bao G, Zhang F. (2013) DNA targeting specificity of RNA-guided Cas9 nucleases. Nature Biotech. 31: 827-832.

Carroll, Jeff. (2011) Cut-and-paste DNA: fixing mutations with ‘genome editing’. HDBuzz.

Sangamo Biosciences. ZFP nucleases: Huntington’s disease:

Garriga-Canut¬¬¬ M, Agustin-Pavón C, Hermann F, Sánchez A, Dierssen M, Fillat C, Isalan M. (2012) Synthetic zinc finger repressors reduce mutant huntingtin expression in the brain of R6/2 mice. Proc Natl Acad Sci USA. 109(45): E3136-E3145.

-J. Choi, 10-24-13


Gene Silencing

Huntington’s disease (HD) is a genetic disease due to the abnormal CAG expansion of the mutated huntingtin gene.  The mutated gene instructs cells in the body to produce a version of the huntingtin protein that ultimately leads to neuronal damage in the brain and creates the symptoms of HD.  Currently, available HD therapies can only provide relief of symptoms, but what if there were a way to eliminate the root of the problem by correcting the mutated gene or preventing the production of the “bad” huntingtin protein?  We are still years away from being able to directly edit the genome in human patients, but much progress has been made in the field of gene silencing, a scientific technique which can hinder or stop the production of a protein.  This article discusses the mechanism of gene silencing, its potential as an HD therapeutic, and challenges this possible therapy faces on the path to clinical implementation.


What is Gene Silencing?^

As the name implies, gene silencing is a technique that aims to reduce or eliminate the production of a protein from its corresponding gene.  Genes are sections of DNA that contain the instructions for making proteins.  Proteins are essential molecules that perform an array of functions including signaling between cells, speeding up biochemical reactions, and providing structural support for the cell.  Each gene is responsible for producing a corresponding protein in a two-step process.  First, a copy of the information encoded in a gene is made in the form of messenger RNA (mRNA), a process known as transcription.  This occurs in the nucleus of the cell, the cellular structure where all of the cell’s genetic material is contained.  The mRNA subsequently travels out of the nucleus, and the genetic information it carries is used to produce a specific protein, a process known as translation.  (For more information about proteins and how they are made, click here.)

Instead of directly editing DNA or inhibiting the transcription process, the key idea behind gene silencing is intervening in gene expression prior to translation.  By designing a molecule that can specifically identify and breakdown the mRNA carrying instructions for making a certain protein, scientists have been able to effectively decrease levels of that protein.  Imagine the gene silencing molecule as a censor and mRNA as messages from genes that are broadcast into proteins:  the molecule will censor out a specified mRNA message, preventing the corresponding protein from being broadcast into the cell, and thus silencing the gene that is providing these instructions.  The ability to significantly lower the levels of a specific protein opens up many possibilities in scientific research and drug development, since proteins are critically involved in the proper function and structure of cells.


Types of Gene Silencing Techniques^

There are various gene silencing methods currently employed in research and being developed as potential disease therapeutics.  Nearly all of them involve disabling the function of mRNA by preventing it from being translated into a protein.  However, they differ in the design of the molecule used to disrupt mRNA and the manner of mRNA breakdown.  As a result, different silencing methods have specific advantages and drawbacks.  Two of the leading and most understood methods of gene silencing are RNA interference (RNAi) and antisense oligonucleotides (ASOs).

RNA Interference

In RNAi, the molecules that identify the target mRNA are called small-interfering RNAs (siRNAs). Unlike normal single-stranded RNA found in cells – such as mRNA – siRNAs are short, synthetically made double-stranded RNA molecules designed to pair with a specific mRNA strand.  This association of the siRNAs with a particular target mRNA causes the breakdown of the target mRNA by recruiting other proteins that degrade the mRNA target.  Because siRNAs are double-stranded, they are more stable and less susceptible to degradation than ASOs, allowing them to continue to perform their silencing function for a longer period of time in the cell.  For a more detailed description of how RNAi works, click here.

Antisense Oligonucleotides

Similar to siRNAs, ASOs are engineered by scientists to associate with a target mRNA strand.  The binding of the ASO to mRNA directs a protein to breakdown the mRNA.  However, unlike siRNAs, ASOs are smaller, single-stranded RNA molecules.  As mentioned above, single-stranded RNAs are not as stable as double-stranded ones; thus, ASOs are often chemically modified to increase their durability in a biological environment.  However, their smaller size and chemical structure allow ASOs to be transported in cells and living tissues much more effectively than siRNAs.  For a more detailed description of how ASOs work, click here.

Is one gene silencing method better than the other?

In terms of developing a drug therapy based on gene silencing, how do RNAi and ASOs compare to each other in effectiveness?  In cell culture experiments, gene silencing is often used to intentionally decrease levels of a certain protein for research purposes.  In such applications, siRNAs have sometimes been shown to produce stronger and longer lasting gene silencing than ASOs.  However, when developing silencing therapeutics, the strength and duration of gene silencing needed for treatment may vary; sometimes a shorter-acting or less complete gene silencing may be required.  Furthermore, when considering the efficacy of each method in live animal models, the results are not as clear-cut.  For example, as mentioned earlier, ASOs can often be distributed more easily than siRNAs throughout the target tissue because of their size and structure.  This observation would be expected to simplify delivery and lower costs of a therapeutic application.  The fact that there is no definitive answer to which gene silencing method is more effective has resulted in continued active research and development of both areas.

Gene Silencing and HD^

HD is characterized by a mutation causing excess CAG repeats in the Huntington gene and the consequent production of the mutant huntingtin protein results in disease. As such, silencing of the mutant version of the huntingtin gene is a potential therapeutic strategy for HD treatment.  Indeed, HD and other related neurodegenerative diseases involving mutant CAG repeats, such as spinobulbar muscular atrophy and some types of spinocerebellar ataxias, have been at the frontier of the therapeutic development of gene silencing (for more information about trinucleotide repeat disorders, click here).

Approaches to Huntingtin Gene Silencing

Recall that everyone’s DNA is composed of two copies of a gene, called alleles, one from each parent.  In the majority of individuals with HD, one copy of the gene is mutated with excess CAG repeats, while the other copy is an allele with a number of CAG repeats within the normal range.  As a result, the body not only produces the mutant version of the huntingtin protein, but also makes the normal protein.  When considering gene silencing as a therapeutic approach to HD, it is crucial to think about the difference between silencing huntingtin mRNA in general and selectively disrupting mRNA that encodes for the mutant, and not the normal, huntingtin protein.

The huntingtin protein has many roles in proper development. Studies in mouse models have shown that completely eliminating huntingtin protein results in mice that do not survive past the embryonic stages of development, while mice that were induced to lose huntingtin after birth experienced severe neuronal degeneration (for more information about the function of wild-type huntingtin protein, click here). Thus, it is important for scientists to develop gene silencing drugs that specifically target mutant huntingtin mRNA.  This type of approach is known as allele-specific gene silencing.

While it may seem straightforward to target the excess CAG repeats to specifically decrease the levels of mutant huntingtin, it is important to remember that the molecules used for silencing are short RNA sequences, about 25 nucleotides in length.  Hence, they cannot effectively distinguish between the size of the normal and the expanded CAG repeats of the huntingtin gene. This is particularly true when trying to differentiate between 30 CAGs and 40 CAGs, CAG repeat ranges that are near normal.  To get around this obstacle, scientists are developing an approach that identifies single nucleotide polymorphisms (SNPs) – changes in a single nucleotide in the DNA sequence –closely linked with the mutant gene and not the normal allele.  SNPs are mutations that differ by a single nucleotide (e.g. ‘A’ à ‘C’) and result in the genetic variation between individuals.  What researchers have found is that many HD patients have common SNPs that are associated with the mutated huntingtin allele. Using silencing molecules to identify these SNPs provides a potential approach to allele-specific silencing of the mutant gene.

A recent study explored allele-specific silencing of the mutant huntingtin protein by targeting associated SNPs.  Since not all HD patients have the same SNPs, they sought to develop a panel of ASOs to maximize the coverage of the HD population.  They found that 85% of HD patients can be covered by targeting as few as three SNPs.  Moreover, injecting ASOs targeting an HD-associated SNP into HD mice showed a greater than 50% decrease in mutant huntingtin protein, while normal mice receiving the same treatment showed only a 3% drop in huntingtin levels.  These results indicate a relatively strong and selective silencing effect on mutant huntingtin.  Although further work must be done to expand coverage of the HD population by this approach and to assess its therapeutic efficacy outside of animal models, this study represents an initial step forward toward using allele-specific gene silencing as an HD therapeutic.

Although the above results are encouraging, some researchers have suggested using nonallele-specific silencing of huntingtin protein because  of the lack of a single SNP that will specifically target mutant huntingtin in all HD patients.  In this approach, instead of trying to decrease levels of the mutant huntingtin only, both the normal and mutated versions of the huntingtin protein are targeted and decreased.  This method is also advantageous because instead of employing different silencing molecules for different SNPs in different individuals, a single therapy can be developed for all HD patients, thus minimizing costs.  Although huntingtin has important functions in the body, interestingly, a study of nonallele-specific silencing using siRNA in HD mouse models found that when both mutant huntingtin and normal huntingtin levels were decreased by 75%, the treated mice demonstrated improved motor control and increased survival compared to controls.  This result suggests that nonallele-specific silencing may be a beneficial therapeutic for HD.  An important caveat to consider is that many therapies that show an effect in HD mouse models may not directly translate to humans.  For example, the mouse brain may be better able to tolerate a decrease in normal huntingtin than the human brain.  In any case, since both allele-specific and nonallele-specific silencing methods have their pros and cons, both continue to be under investigation as potential therapeutic options.

Challenges to Gene Silencing Therapeutics^

Even though gene silencing is a promising strategy for treating HD, there are still many hurdles to overcome before it can be applied in the clinic.  First and foremost, gene-silencing molecules have to be effectively delivered to the relevant parts of the body, which, in the case of HD, are the afflicted areas of the brain.  The blood-brain barrier prevents passage into the brain of most molecules that are injected or absorbed into the blood, making drug delivery difficult.  Some methods that scientists have used in animal models include direct injection of the silencing drug or implanting pumps that infuse the molecules into the brain.

Once past the blood-brain barrier, silencing molecules have to locate neurons and other affected cells and enter these cells to silence huntingtin expression.  As mentioned earlier, due to their structure, ASOs distribute and enter cells more effectively than siRNAs.  To effectively deliver siRNAs into cells, scientists currently use viral-based delivery systems, which essentially take advantage of the machinery viruses use to infect our cells.  One of the drawbacks of using a viral delivery mechanism is the potential for an immune response against the molecules.  As an alternative to this method, Dr. Jan Nolta’s group at the University of California Davis has begun studying the possibility of using mesenchymal stem cells (MSCs) as a delivery system for siRNA (for more information about MSCs, click here).  A possible advantage of using a viral or stem cell delivery system is that they might be able to become a production facility for siRNA molecules, allowing long-term therapeutic treatment of HD, a great benefit for a chronic illness. Ongoing research is currently investigating whether this theoretical possibility could become a reality.

There are other concerns associated with gene silencing therapeutics.  For example, researchers have observed that high dosages of silencing molecules could have a toxic effect, highlighting the importance of finding an optimal dosage that is safe and effective.  In addition, there is the possibility for ASOs and siRNAs to accidentally bind to an undesired mRNA (mRNA coding for a protein that is not huntingtin).  To deal with this so-called off-target gene silencing phenomenon, researchers are studying the selectivity of how siRNAs and ASOs bind to the huntingtin mRNA, in order to better develop specific, effective, and safe HD gene silencing therapeutics.


Gene silencing as a therapeutic strategy is a highly active area of research and may one day yield an effective treatment for HD, since it acts by directly reducing the production of the mutant huntingtin protein.  However, the technology is still in preclinical stages, and there remain many issues to address and resolve before it can be approved for clinical trials.  Delivery methods, dosages, and selectivity of gene silencing drugs must be optimized to ensure safety and efficacy of treatment.  Clinical trials have begun using gene silencing for therapeutic applications in other diseases. These studies will help to inform current efforts to develop gene silencing for HD treatment.

Further Reading^



1.  Bennett CF, Swayze EE.  RNA Targeting Therapeutics: Molecular Mechanisms of Antisense Oligonucleotides as a Therapeutic Platform.  Annu. Rev. Pharmacol. Toxicol. 2010. 50:259–93.

This is a technical article published by Isis Pharmaceuticals that gives an in-depth review of the mechanisms and pharmacology involved in developing RNA-targeting gene silencing therapeutics.

2.  Boudreau RL, McBride JL, Martins I, Shen S, Xing Y, Carter BJ, Davidson BL.  Nonallele-specific silencing of mutant and wild-type Huntingtin demonstrates therapeutic efficacy in Huntington’s disease mice. Molecular Therapy (2009) 17 6, 1053–1063.

A technical article that explains the potential benefit of nonallele-specific silencing in HD mouse models.

3.  Boudreau RL, Rodriguez-Lebrón E, Davidson, BL.  RNAi medicine for the brain: progresses and challenges.  Hum Mol Genet. 2011 Apr 15;20(R1):R21-7.

A medium-difficulty article that discusses the development of RNAi as a therapeutic, current preclinical data, and the key challenges that remain for its clinical implementation.

4.  Carroll JB, Warby SC, Southwell AL, Doty CN, Greenlee S, Skotte N, Hung G, Bennett CF, Freier SM, Hayden MR.  Potent and selective antisense oligonucleotides targeting single-nucleotide polymorphisms in the Huntington disease gene/ allele-specific silencing of mutant huntingtin.  Mol Ther. 2011 Dec;19(12):2178-85.

A technical article that explains the results of a study to develop a panel of ASOs for allele-specific gene silencing of mutant huntingtin in mouse models.

5.  Dessy A, Gorman JM.  The emerging therapeutic role of RNA interference in disorders of the central nervous system.  Clinical Pharmacology & Therapeutics (2011) 89 3, 450–454.

A medium-difficulty article that gives a broad overview of the current status of RNAi as a developing therapy for neurodegenerative diseases.

6.  Scholefield J, Wood MJ.  Therapeutic gene silencing strategies for polyglutamine disorders.  Trends Genet. 2010 Jan;26(1):29-38.

A technical article that reviews the mechanism of gene silencing and discusses therapeutic studies that have been done and challenges that remain to be addressed for allele-specific silencing of polyglutamine disorders.

7.  Sah DWY, Aronin N.  Oligonucleotide therapeutic approaches for Huntington disease.  J Clin Invest. 2011;121(2):500–507.

Another technical article that explains and compares the various approaches to gene silencing therapeutics for HD.

8.  Sass, Meghan; Aronin, Neil. “RNA- and DNA- Based Therapies for Huntington’s Disease.” Neurobiology of Huntington’s Disease: Applications to Drug Discovery. Ed. Donald C. Lo and Robert E. Hughes. Boca Raton: CRC Press, 2010.

A technical article that broadly covers the mechanisms, current studies, and challenges of both RNAi and ASO therapeutics for HD.

J. Choi 04.04.12


About Protein Aggregation

The altered huntingtin protein responsible for HD has been shown to contain many more molecules of the amino acid glutamine than regular huntingtin. This abundance of glutamine is due to repetitive copies of the CAG codon in the Huntington gene. The extended glutamine tracts of these proteins have affinity for one another, and tend to “stick together,” leading to the formation of “clumps” or aggregations of the protein in the cell’s nucleus. These protein aggregations are often referred to as neuronal inclusions (NIs).

It is not absolutely clear whether NIs are the cause or the result of HD, or whether they might even be a defense mechanism against it. Scientists are not certain whether the NIs themselves are toxic, or whether the intermediates or building blocks in the aggregation process are the toxic agents. These questions aside, there is mounting evidence supporting NIs as a primary mediator of cellular toxicity in Huntington’s disease.

There are two major lines of thought regarding the toxic mechanisms of polyglutamine protein aggregation. One is that other normal molecules with glutamine tracts get “trapped” within the NIs, and are therefore prevented from performing their normal functions. Another is that the NIs “clog” the Ubiquitin-Proteasome System (UPS), a kind of disposal system that is essential for normal cell function. For more information on the role of protein aggregation in the progression of HD, click here.

Scientists have shown that reducing the amount of protein aggregation in the cell may be beneficial for patients with HD. The drugs listed under the “Protein aggregation” navigation menu potentially reduce the amount of NIs in the cell, and are therefore being researched as possible treatments for HD.

-E. Tan, 9-21-01


Antisense Gene Therapy

Antisense gene therapy is a gene silencing technique similar to RNA interference, but uses a slightly different mechanism. The therapy is called a gene silencing technique because, instead of repairing the gene, it aims to “silence” the gene’s effect. Recall that people with Huntington’s disease (HD) have two copies, or alleles, of the Huntington gene, one of which codes for the normal huntingtin protein and one of which codes for the “bad” huntingtin protein that is associated with the symptoms of the disease. Like all proteins, both good and bad huntingtin proteins are formed using the information present in the genetic code. Proteins are produced in two steps. First, the genetic code of DNA is transcribed into mRNA, which then travels to ribosomes. Next, ribosomes use mRNA as instructions to build the correct protein in a step known as translation. The step is called translation because the information is translated from the language of genetic code into the language of amino acids, which are the building blocks of proteins. To learn more about the relationship between DNA and proteins, click here. Antisense gene therapy seeks to “silence” a gene by inhibiting the translation step and preventing the bad protein from being formed.

How does antisense gene therapy work?^

In antisense gene therapy, short single-stranded pieces of chemically modified nucleotides, known as oligonucleotides are inserted into cells. These short strands are sometimes abbreviated as “oligos” and are chemically engineered to be complementary to specific mRNA in the cell. For example, in treating HD, these strands would be complimentary to the mRNA that codes for the harmful huntingtin protein. After being inserted into the cell, these oligos bind to the target mRNA and inhibit the protein from being produced in one of two ways: physically blocking translation or recruiting an enzyme known as RNase H to degrade the mRNA. This way, the information in the DNA that codes for the bad huntingtin protein never makes it to the ribosome and the mutated form of the protein is never formed.

How is antisense gene therapy different from RNA interference?^

The intended effect of both RNA interference (RNAi) and antisense gene therapy is the same, but their mechanisms are slightly different. Like antisense therapy, RNAi is a gene silencing technique that inhibits the actions of genes by interfering with the translation of proteins. However, antisense technology destroys target mRNA by recruiting the enzyme RNase H, while RNAi recruits a different RNase enzyme known as dicer. In addition, RNAi molecules are twice as large as antisense oligonucleotides (because they are double stranded rather than single stranded). Their larger size makes it more difficult for them to get into the body’s cells where they can have a beneficial effect. For more information on RNAi, please click here.

Challenges to antisense technology^

One major challenge to antisense technology (and RNAi) is the difficulty of getting it into the body. Delivery of the treatment to the brain, for use in diseases like HD, is especially challenging because it must cross the blood-brain barrier. Brain entry is very difficult and cannot be accomplished through a simple injection or pill that contains the antisense oligos. Isis Pharmaceuticals, the leading company in antisense technology, plans to solve this problem by inserting a pump into the chest that can carry the drug to the brain through a catheter, or tube. Another possible solution is to use an inactivated virus to “infect” cells with the drug.

The second major challenge to antisense technology is its inevitable toxic effects. Although antisense technology is engineered to be very specific, it can still cause unintended damage because it would regulate both the mutant and normal Huntington alleles. The challenge is to determine the right dosage and composition of an antisense molecule to strike a balance between reducing the symptoms of HD and avoiding side effects caused by altering the “good” Huntington gene. For this reason, it is likely that antisense therapy will only be able to go so far in mitigating the symptoms of HD. It is also possible that antisense technology may be most effective if engineered specifically for each patient’s needs, but this type of personalized medicine is not now of primary concern.

Current development of antisense gene therapy^

Isis Pharmaceuticals has taken the lead in the arena of antisense gene therapy technology. Though they have not yet engineered an antisense drug for HD, they have already been successful in getting one antisense drug, Vitravene©, on the market. This drug uses antisense technology to treat an eye disease associated with AIDS. In addition, Isis has made a number of promising advances in scientific trials of antisense technology to treat other conditions including high cholesterol and Parkinson’s disease. Perhaps most exciting is that in 2007, CHDI, a Los Angeles non-profit dedicated to finding a cure for HD, granted Isis 9.9 million to work on developing an antisense drug for HD. So far, Isis has successfully used antisense technology to inhibit the action of the Huntington gene in mouse cells, mouse brains and human cells. Isis is currently testing the technology on transgenic mice (mice that are genetically modified to have the mutated HD allele). If that goes well, the next step will be to try it in monkeys, and then eventually humans.

For further reading^

– C. Garnett, 10-19-09




Drug summary: Rapamycin, also known as sirolimus, is an FDA-approved antibiotic and immunosuppressant. It is already being used in organ transplant patients and is currently being tested in phase II and III clinical trials in cancer patients for its antitumor activity. Rapamycin inhibits the activity of a protein called mTOR which, among its other functions, inhibits a process called autophagy. Autophagy is the process by which a cell breaks down its own molecules and other components that are no longer needed. Since mTOR functions to inhibit autophagy, by inhibiting mTOR, rapamycin promotes autophagy, allowing for the breakdown of unnecessary components of the cell. Researchers have shown in fly and mouse models of HD that by inducing autophagy, rapamycin helps nerve cells break down huntingtin aggregates.

Whether these protein aggregates are a cause or result of the HD disease process is not yet known. However, nerve cells that build up huntingtin aggregates in the brains of people with HD often die. (To read more about huntingtin protein aggregation and its role in HD, click here.) Thus, rapamycin may help prevent cell death by helping nerve cells clear out huntingtin aggregates. Rapamycin could be an especially promising treatment if started before or shortly after the onset of symptoms in people with HD, when the levels of huntingtin aggregates in the nerve cells are still manageable.

What is autophagy, the cellular mechanism affected by rapamycin?^

Rapamycin prevents the protein mTOR from performing its normal functions in the cell. mTOR is a member of a whole family of “TOR” (“target of rapamycin”) proteins. While mTOR is involved in many different cell functions, it mainly helps regulate when the cell makes and breaks down proteins. The decision to make or break down proteins depends on what proteins are needed by the cell at specific times and on the conditions around the cell. If the cell has enough available amino acids, which are the building blocks of proteins, mTOR is free to signal to other molecules that will tell the cell to build new proteins. On the other hand, if the cell is running low on nutrients, it has to break down already existing proteins and other cell components to free the building blocks so that they can be reused.

Fig H-3: The Process of Autophagy

The process by which the cell breaks down its own components is called autophagy, which basically means “eating of the self.” The part of the cell that is to be degraded is first engulfed by a double membrane to separate it from the rest of the cell; the resulting membrane-enclosed bubble of cytosol (and the proteins it contains) becomes what is called the autophagosome. The autophagosome eventually fuses with a cellular organelle called a lysosome, a much larger membrane-enclosed bubble that contains a variety of enzymes that can break down all sorts of cellular components (which is why lysosomes are sometimes referred to as the “garbage disposals” of the cell). In order to protect the rest of the cell from being degraded, these enzymes only work in a very acidic environment, so the pH inside lysosomes is much lower than the neutral pH in the rest of the cell. This pH barrier protects the rest of the cell from being degraded should the enzymes somehow leak out. Once the contents of the autophagosome are delivered to the lysosome, the lysosomal enzymes break down the new contents, which can then be recycled for new use within the cell.

mTOR comes into this picture because it inhibits the process of autophagy; since mTOR signaling means that the cell has plenty of nutrients to build with, autophagy is not necessary to break down already existing molecules. The discovery that rapamycin inhibits mTOR prompted researchers to see if its ability to stimulate autophagy could also help nerve cells get rid of huntingtin aggregates.

How could rapamycin be used to help prevent build-up of huntingtin aggregates?^

Until a couple of years ago, it was believed that the main mechanism by which the cell got rid of huntingtin aggregates involved what is called the ubiquitin-proteasome system, which is responsible for tagging and degrading improperly formed proteins. However, recent research shows that proteins with abnormally expanded stretches of the amino acid glutamine, like the altered huntingtin protein (which causes HD), are also disposed of through a particular kind of autophagy. In this process, the proteins are gathered up and transported to the lysosome, where they are broken down and their component amino acids recycled. Studies of nerve cells have shown that huntingtin can often be found in autophagosomes, the membrane-bound sacs that carry cell parts to the lysosome for degradation.

Fig H-4: Rapamycin's Effects on mTOR & Autophagy

Rapamycin could potentially be used to treat HD by taking advantage of the autophagy process. The drug has been shown to induce autophagy and to help prevent toxicity caused by huntingtin aggregates in both cell and animal models of HD. The basic process by which this occurs can be summarized as follows: Rapamycin inhibits the protein mTOR -> mTOR can no longer inhibit autophagy -> autophagy is activated -> huntingtin aggregates are broken down in the lysosome.

Unfortunately, the mechanism by which rapamycin could help people with HD is more complicated than the process outlined above. Researchers that tested rapamycin’s ability to reduce huntingtin aggregates in cell cultures and animal models found that the drug only works in cells that have been expressing the altered (HD-causing) huntingtin protein for a short time. In cells and animals that have already had time to build up huntingtin aggregates, rapamycin fails to stimulate autophagy enough to clear out the aggregates. The current explanation for this finding is that mTOR is actually sequestered, or trapped, by the huntingtin aggregates themselves. (For more information about huntingtin aggregates, click here.) This reasoning could help explain the typical late onset of Huntington’s disease: early in life, the huntingtin aggregates sequester mTOR and in doing so induce autophagy, which initially helps get rid of the aggregates. However, as more and more huntingtin aggregates form, the autophagy that is set off by inactivation of mTOR can no longer keep up the pace as aggregates begin to form faster than they can be degraded – and the symptoms of HD begin to appear.

When rapamycin is administered to cells that already contain a lot of huntingtin aggregates, there is no visible improvement because the aggregates in these cells have already inactivated mTOR. Further inactivation of mTOR by rapamycin cannot clear out aggregates which have already become too numerous to be totally cleared out by the resulting autophagy. However, rapamycin does have protective effects in cells that don’t yet have much aggregate build-up. Researchers found that the drug decreases death in cell cultures, fruit flies, and in a mouse model of HD that mimics the late onset of the disease in humans. The severity of symptoms can also be decreased in mice treated with rapamycin. This finding offers hope that rapamycin could be used early in patients that have tested to be at risk for developing HD in order to delay the onset of symptoms even further. (For more information on genetic testing, click here.)

How promising is the use of rapamycin to treat HD?^

A slightly modified form of rapamycin, called CCI-779, has better properties as a drug and has been shown to have only mild and treatable side effects in humans. In a clinical study of CCI-779 in cancer patients, the most common side effects were usually treatable acne-like rashes or lesions, and no significant suppression of the immune system was seen even at the highest dose tested. There is also evidence that mTOR is highly involved in learning and memory, but so far researchers have not seen any harmful effects of rapamycin on these processes. However, neither form of rapamycin has yet been tested for efficacy in people with HD. More testing needs to be done to determine whether rapamycin would be safe for the kind of long-term use necessary should the drug be used to delay symptoms starting from the early stages of the disease.

Research on rapamycin and HD^

Ravikumar, et al. (2002) investigated whether proteins with expanded sections of the amino acid glutamine (like the altered huntingtin protein) and the amino acid alanine (which causes other diseases) could be degraded by cells using the process of autophagy. They compared autophagy with the ubiquitin-proteasome process, which was originally thought to be the only process by which these harmful proteins are degraded. The researchers used cells that expressed these proteins and tagged them with green fluorescent protein (GFP) in order to visualize their fate within the cells. The use of GFP allows researchers to see the amount of a specific protein present in the cell because it fluoresces, or glows, when viewed under a special microscope. To study how huntingtin aggregates are broken down by the cell, they used cells that expressed the part of the HD allele that contained either 55 or 74 CAG repeats, and thus produced proteins with stretches of 55 or 74 glutamines.

To determine whether autophagy is indeed a key process in the clearance of huntingtin aggregates, the researchers first used two different compounds to inhibit autophagy at different points of the process and observed the effect on aggregate formation. The first compound they used inhibits autophagy by preventing a membrane from surrounding the cell contents that are about to be degraded; if the autophagosome can’t form, the contents cannot be delivered to the lysosome to be broken down. The second compound they used prevents the autophagosome from fusing with the lysosome and releasing its contents, which also prevents autophagy from occurring. Treatment with these compounds resulted in visibly higher levels of huntingtin aggregates in cell cultures, which showed that autophagy does play a role in the breakdown of aggregates. Along with the increase in aggregates, the researchers also saw increased cell death when the cells were treated with autophagy-inhibiting compounds.

The researchers then tested the effects of rapamycin on aggregate formation in the cells. It had no effect on the degree of aggregation in cells that had been producing the altered huntingtin protein for 48 hours. They repeated the experiment with cells that had only been producing the protein for 24 hours, and in this case they found that rapamycin did reduce aggregate formation and cell death. This finding showed that rapamycin may only be effective when the degree of huntingtin aggregation is still low. They also noted that rapamycin promotes autophagy by inhibiting mTOR, but that the exact nature of this interaction is unknown.

Finally, the researchers tested the role of the ubiquitin-proteasome system in reducing protein aggregation in the same cell cultures. Most previous experiments have used a certain compound to inhibit the proteasome that can apparently inhibit the function of the lysosome as well. Because they wanted to test the role of the proteasome only, the researchers used a different compound that inhibits the proteasome and has no effect on lysosomes. They found that inhibiting the proteasome increased aggregate formation in one cell line but not in another. While these results are somewhat inconclusive, they may suggest that the ubiquitin-proteasome process is not the main mechanism by which cells get rid of the altered huntingtin protein. More research needs to be done about the role of autophagy in degrading mutant huntingtin.

Ravikumar, et al. (2004) took these studies further by testing the effects of rapamycin in fly and mouse models of HD. Before testing the drug in animals, the researchers set out to show how mTOR interacts with huntingtin protein aggregates. After showing that mTOR is indeed sequestered by huntingtin aggregates in cell cultures, they went on to show that mTOR does not function properly in cells that have huntingtin aggregates. To set off different cellular processes, mTOR signals to other molecules in the cell by working as a kinase, which is a molecule that adds a phosphate group onto another molecule (or “phosphorylates” it) in order to turn that molecule on or off. The researchers showed that certain molecules phosphorylated by mTOR, were phosphorylated less often in cells that contained huntingtin aggregates. This finding indicates that the interaction between mTOR and the aggregates prevents mTOR from performing its usual functions. By phosphorylating these molecules, mTOR is supposed to stimulate the synthesis of certain proteins. The experiment also showed that in cells with huntingtin aggregates, these proteins were produced at lower levels, probably because mTOR was inactivated. The researchers also found that increasing mTOR activity, which would prevent autophagy, increased aggregate levels and cell death.

The first model the researchers used were flies that expressed the altered huntingtin protein in their photoreceptors, which are specialized cells that receive light in the eyes. The researchers found that treatment with rapamycin decreased degeneration of these cells. The next experiment tested CCI-779, a more water-soluble form of rapamycin, in a mouse model of HD. The researchers used a mouse model that mimics the late onset of disease symptoms that occurs in humans so that they would have time to administer rapamycin treatment before severe symptoms appeared. Throughout the study, the mice treated with CCI-779 performed better on four different motor tasks than did mice treated with a placebo. Afterwards, the researchers found that there were also fewer aggregates in the brains of mice treated with CCI-779 than in the brains of control mice. These findings show that rapamycin plays a role in helping nerve cells get rid of huntingtin aggregates and that it may have promise as a therapeutic agent for HD. However, more research needs to be done on the safety and efficacy of rapamycin humans.

For further reading^

  1. Raught, et al. “The target of rapamycin (TOR) proteins.” Proceedings of the National Academy of Sciences of the United States of America. 2001 Jun 19;98(13):7037-44.
    This is a very technical article that describes the functions of the TOR family of proteins. It does not specifically address HD.
  2. Ravikumar, et al. “Aggregate-prone proteins with polyglutamine and polyalanine expansions are degraded by autophagy.” Human Molecular Genetics. 2002 May 1;11(9):1107-17.
    This is a highly technical article describing the study that showed the role of autophagy in breaking down HD-causing huntingtin.
  3. Ravikumar, et al. “Inhibition of mTOR induces autophagy and reduces toxicity of polyglutamine expansions in fly and mouse models of Huntington disease.” Nature Genetics 2004 Jun;36(6):585-95.
    This is a highly technical article that describes the most recent study of rapamycin treatment in fly and mouse models of HD.
  4. Raymond, et al. “Safety and pharmacokinetics of escalated doses of weekly intravenous infusion of CCI-779, a novel mTOR inhibitor, in patients with cancer.” Journal of Clinical Oncology. 2004 Jun 15;22(12):2336-47.
    This is an article of medium difficulty that recounts a clinical safety study of CCI-779 in cancer patients. It lists all of the side effects that were encountered during the study.
  5. Thoreen, et al. “Huntingtin aggregates ask to be eaten.” Nature Genetics. 2002 Jun;36(6):553-4.
    This is a comment on the 2004 Ravikumar study that appeared in the same issue of Nature Genetics. It is shorter and less technical than the full article and provides a good summary of the findings.

-A. Milczarek, 12/29/04




Drug summary: Cystamine is a drug that is thought to inhibit transglutaminase (TGase), an enzyme involved in the formation of huntingtin protein aggregates. Studies of cystamine in mouse models of HD has shown that cystamine improves physical symptoms, and decreases nerve cell death. Raptor Pharmaceuticals is currently conducting phase II clinical trials to determine whether cysteamine – a form of cystamine – can benefit people with HD.

Problem: Protein Aggregation^

Research suggests that one problem in HD is protein aggregation: copies of the mutant huntingtin protein stick to eachother and form clumps. These clumps, called protein aggregates or neuronal inclusions (NIs), are thought to cause some of the problems of HD by interfering with the many important processes that occur in neurons, as described in more detail here. While there is some debate over the exact role of protein aggregates – with some scientists speculating that aggregates are helpful, as they isolate mutant huntingtin protein and prevent it from doing further harm – some studies have shown that decreasing the number of protein aggregates might improve HD in animal models.

These protein aggregates are formed in part through the action of the enzyme transglutaminase (TGase), which links mutant huntingtin together. While scientists are still unsure whether protein aggregates are harmful or helpful, evidence about TGase is more clear: TGase seems to make HD worse. TGase levels are higher in the brains of HD patients. Scientists have genetically engineered some HD mice so that they lack the TGase gene, and therefore don’t have TGase. These mice live longer, lose less weight, have healthier brains, and have a later onset of motor symptoms (Bailey and Johnson 2006). Therefore, scientists are studying the effects of cystamine, a molecule that reduces TGase activity.

How can cystamine help treat HD?^

Cystamine is a competitive inhibitor: it blocks the region of TGase that allows the enzyme to hold onto copies of the mutant huntingtin protein and link them together. So in theory, cystamine should prevent TGase from forming protein aggregates.

Cystamine may also have other properties that could help treat HD. It may also help prevent early cell death by interacting with another type of enzyme, called a caspase. There are many different types of caspases, but they all contribute to early cell death in HD by playing a role in the cascade leading to apoptosis, or programmed cell death. (For more information on caspases and apoptosis, click here.). Cystamine inhibits caspase 3 in cells, and therefore might reduce apoptosis.

Cystamine may also promote brain health through other paths. Cystamine is thought to be an antioxidant, relieving the harmful effects caused by oxidative stress. (For more information on antioxidant treatments, click here.). It also increases levels of brain-derived neurotrophic factor (BDNF), a chemical in the brain that promotes neuron health (Borrell-Pages et al., 2006).


So in these ways – and possibly others – cystamine might reverse some of the problems caused by HD.

Research on cystamine^

Karpuj, et al. (2002) investigated how treating mouse models of HD with cystamine injections affected their physical symptoms and nerve cells. The researchers began treating the mice after seven weeks of age, when symptoms of HD had already begun to appear. Treatment was evaluated by measuring the amount of TGase activity, looking for abnormal mouse movement, charting weight loss, and counting the number of protein aggregates in the nerve cells in the brain.

Following treatment, the mice showed signs of improvement: the tremors and abnormal movement became less prominent, survival was extended by 20 percent, and weight loss was not as severe. TGase is normally very active in the mouse model of HD, but it was greatly reduced by treatment with cystamine.

To the researchers’ surprise, however, cystamine treatment did not influence the appearance or number of NIs. Instead, the researchers found increased production of the protein products of certain genes. In the fruit fly, these genes are known to exert protective effects in nerve cells against toxicity that results from polyglutamine diseases similar to HD. (For more information on polyglutamine diseases, click here.) One of the protein products of these genes, known as HDJ1 in humans and Hsp40 in mice, was found in elevated concentrations after treatment with cystamine. The researchers hypothesized that elevated HDJ1/Hsp40 in HD brains might indicate that the level of this protein was increased in a failed attempt at recovery. Releasing high levels of HDJ1/Hsp40 is probably a response to the HD disease process that was initiated by the altered huntingtin protein.

Dedeoglu, et al. (2002) examined the effects of cystamine in a mouse model of HD on TGase activity, brain and body weight, and survival. The mice received the drug in two ways: 1) through injections; and, 2) orally, in their drinking water. Treatment began when the mice were in the womb – by injecting pregnant mothers or putting cystamine in their water – and continued after birth. About 80 out of 180 mice in the first group and 26 out of 56 mice in the second group were not treated but were used for comparison to mice receiving cystamine.

The effect on survival largely depended on the dose of cystamine. In the group where mice were injected with the drug, those given the lower doses lived longer. However, all of the mice given the highest dose died, probably because of drug toxicity. The mice that were given the drug in their water survived longer than the mice that were not given cystamine at all. Injection and oral administration of cystamine were found to help survival equally at the appropriate dosage.

Both treatments also improved body weight gain compared to untreated mice. (HD often causes weight loss, so improved weight gain is sometimes thought to be a beneficial result because it combats a symptom of HD.) Also, mice treated with cystamine lost much less brain weight compared to mice not receiving cystamine treatment, which implies that cystamine protects nerve cells from degenerating in HD mice.

In the mouse model of HD, TGase activity is usually much higher than in normal mice (because, as mentioned above, TGase helps form huntingtin protein aggregates). However, when the HD mice in this study were treated with cystamine, their TGase activity fell within the normal range of non-HD mice. This finding may explain why treated mice were found to have fewer huntingtin protein aggregates. This study, along with that conducted by Karpuj, et al. (who started cystamine therapy after HD symptoms were already present), suggests that cystamine may be able to prevent protein aggregation in people with HD if given before the onset of symptoms.

Clinical Trials^

These promising results in animal studies have led researchers to investigate whether cystamine can be used to treat HD.

Dubinsky and Gray (2006) conducted a phase I clinical trial on 9 patients with HD, and found that 20 mg/kg of cysteamine – another form of cystamine – is safe and tolerable in HD patients. This study set the stage for future, larger studies.

Raptor Pharmaceuticals is currently conducting a phase II clinical trial, which began in October 2010. Eight clinical centers in France are recruiting 96 patients, who will be treated with either cystamine or a placebo for 18 months. After that, there will be an “open-label” phase in which all patients will be treated with cystamine for another 18 months.

The Food and Drug Administration (FDA), which regulates drugs in the US, gave Raptor an orphan drug designation for its formula for cysteamine in 2008. The orphan drug designation is a status sometimes given to drugs intended to treat rare medical conditions, and is meant to promote research in those areas by making it easier for that drug to pass through the approval process.

For further reading^

  1. Bailey CD, Johnson GV. The protective effects of cystamine in the R6/2 Huntington’s disease mouse involve mechanisms other than the inhibition of tissue transglutaminase. Neurobiol Aging. 2006 Jun;27(6):871-9. PubMed PMID: 15896882. This technical article discusses some ways that cystamine might fight HD.
  2. Prolonged survival and decreased abnormal movements in transgenic model of Huntington disease, with administration of the transglutaminase inhibitor cystamine. 2002. Nature Medicine 8: 143-149. Online.
    This article reports that cystamine treatment increased survival rates and decreased abnormal movements in a mouse model of HD. It is a scientific article of medium difficulty with several technical parts.
  3. Therapeutic effects of cystamine in a murine model of Huntington’s disease. 2002. The Journal of Neuroscience 22(20): 8942-8950. Online.
    This article presents the research findings of a study examining cystamine in a mouse model of HD. It is a scientific article of high difficulty.
  4. Cystamine inhibits caspase activity: implications for the treatment of polyglutamine disorders. 2003. Journal of Biological Chemistry 278(6): 3825-3830. Online.
    This is a highly technical article that presents the research findings of a study examining the effects of cystamine on caspases in HD cells.
  5. Dubinsky R, Gray C. CYTE-I-HD: phase I dose finding and tolerability study of cysteamine (Cystagon) in Huntington’s disease. Mov Disord. 2006 Apr;21(4):530-3. This is paper about the phase I clinical trial of cysteamine for the treatment of HD.

-K. Taub, 11/21/04, updated by M. Hedlin 8.10.11




This chapter discusses a small molecule called trehalose that may help prevent protein aggregation. Everyone has a certain copy, or allele, of the Huntington gene, but people with Huntington’s disease (HD) have one copy that is longer than normal. The longer section of this HD allele consists of a repeated sequence, CAG, which codes for glutamine, an amino acid. (For more information on CAG repeats and HD, click here.) Since the Huntington gene codes for the huntingtin protein, the HD allele, with its extra CAGs, codes for a huntingtin protein with too many glutamines. The extra glutamines cause the protein to have an abnormal shape, which prevents it from functioning as it should. Instead, many of these altered huntingtin proteins clump together and trap other useful and important molecules. These “clumps” are called protein aggregates, and they may prevent the normal functioning of nerve cells. (For more information on protein aggregation, click here.) Scientists are not sure if the formation of protein aggregates is a cause or only a symptom of HD, but many agree that it would be beneficial to prevent them from forming in the first place.

What is trehalose ?^

Trehalose is a disaccharide (two sugar) molecule composed of two smaller glucose molecules linked together. It is naturally produced by the body and can also be found in common foods. The U.S. Food and Drug Administration lists trehalose as a compound under the category of “generally regarded as safe.” Since trehalose is a sugar, it is used as a sweetener in products such as chewing gum. It also has a very important property that helps it to stabilize proteins and can thus be used as a biological preservative. It is this very feature that may useful for treating Huntington’s disease.

How can trehalose be used to treat HD?^

A protein is made up of a string of amino acids. As the amino acids are strung together, the protein begins to fold up on itself until it gets to its final three-dimensional (3D) shape. Normal, stable proteins have no problem maintaining their shapes and functions in the cell. However, the huntingtin proteins formed from the HD allele are not very stable on their own, so they form into clumps known as protein aggregates.

Scientists think that if these proteins can be stabilized before they are fully folded, the protein aggregations will not form. A recent study in a mouse model of HD has shown the efficacy of trehalose in reducing traditional physiological, motor, and cognitive HD symptoms. The researchers added trehalose, as well as multiple other non-toxic disaccharides, to the water that the mice drank. Trehalose was shown to be the most effective of the disaccharides in inhibiting protein aggregation of mutant huntingtin, in both the brain and the liver. The researchers hypothesized that this improvement was a result of trehalose’s binding to the multiple polyglutamines that arise from the CAG repeats characteristic of HD and thereby stabilizing the mutant huntingtin protein,arresting it in a partially unfolded state. Although it is still under debate whether aggregate formation in HD is a driver of disease symptoms or the body’s attempt to collectand remove mutant huntingtin from actively harming the body, the results of trehalose on HD symptoms persuasively showed the benefit of keeping mutant huntingtin in this partially unfolded, non-aggregate configuration.

Mutant huntingtin is non-soluble, which makes it difficult to directly screen for potential molecules that can inhibit aggregation in vitro. In this study researchers used mutant sperm whale myoglobin protein that contained the same expanded polyglutamine sequence that is the hallmark of HD to create a cellular model of the disease. With this mutant protein the researchers could screen for inhibitors of protein-aggregation driven by the polyglutamine repeats. They focused exclusively on inhibitors that were non-toxic and had the potential for oral administration as an HD treatment. They found that many disaccharides, but most effectively trehalose, reduced protein aggregate in a dependable and repeatable manner.

The researchers then tested the effects of trehalose in a mouse model of HD, and found that administration of trehalose reduced mutant huntingtin aggregation in a dose-dependent manner without showing any toxic effects when given to the mice as part of their diet. The researchers showed that this reduced aggregation was not the result of production of heat-shock proteins, but rather trehalose’s own abilities to stabilize mutant proteins. Administration of trehalose increased cell survival by more than fifty percent in a cellular model of HD, and in the mouse model was added to the drinking water of the mice in various doses. Trehalose-treated mice had reduced weight loss, and it was shown that 2% trehalose had the most drastic effect on this HD symptom. These mice also had reduced neurodegeneration as seen by comparing atrophy in their striatums to that of control mice. Like in the cellular studies performed previously, trehalose reduced mutant huntingtin aggregates in the brains of these HD mice, as well as in their livers. The mice were then analyzed for motor symptoms. Trehalose improved the rotarod abilities of HD model mice but not non-HD mice, and the mice were better able to pace their steps while walking, as measured by the average distance and width of walking strides. Finally, trehalose extended the lifetime of HD mice in a small but statistically significant manner.

Interestingly, the researchers also tested glucose for effects on HD mice, as trehalose is metabolized into glucose in the body. Glucose did not reduce mutant huntingtin aggregation or extend the lifespan of HD mice, showing that it is trehalose itself that seems to have beneficial effects on HD. By showing that trehalose did not induce the production of heat-shock proteins, the researchers showed that trehalose does not induce responses to cell stress like other small molecules that affect mutant huntingtin aggregation, such as geldanamycin, and the fact that trehalose affected both mutant huntingtin and the myoglobin with polyglutamine repeats suggests that trehalose may bind directly to protein regions of polyglutamine repeats. Because trehalose seems to bind to these regions, it may be that trehalose binding prevents the mutant huntingtin proteins from folding completely, and from misfolding. A non-folded protein is usually not functional, but in this case by preventing protein folding trehalose is preventing the formation of toxic, misfolded proteins that form harmful aggregates. It is also likely that trehalose performs its normal function as a protein stabilizer by keeping the normal copies of the huntingtin protein folded correctly so they can function properly. . In addition, the researchers proposed that trehalose may prevent huntingtin aggregates from entering the nucleus, a process previously shown to be essential to HD progression. It is thought that the stability promoted by trehalose would make the proteins more resistant to being broken down by caspases, which is required for transport of cleaved protein fragments into the nucleus.

From this study the researchers suggest that trehalose reduces mutant huntingtin aggregation not by breaking up aggregates but by preventing their formation in the first place by keeping mutant huntingtin from folding into a shape that allows for aggregate formation. Trehalose is not quickly metabolized into glucose, and so would be available even if administered in low concentrations to perform these beneficial effects in HD patients. Trehalose has great potential to be used therapeutically as it is non-toxic and highly soluble, and shown to be effective when administered orally. The fact that trehalose already is in our diet means that it would not have to undergo a lengthy and arduous process of proving its non-toxicity in clinical trials.

Trehalose may be a promising treatment of HD symptoms that can soon become available because of its demonstrated safety, if further studies show its usefulness in treating HD.

For further reading:^

  1. Alper, Joe. Delivering Huntington disease its coup de GRAS. 2004. Preclinica 2(3): 162-163. Online.
    This short article discusses the commercial and therapeutic uses of trehalose and debates the extension of it to humans. It is of medium difficulty and an important caveat to other highly enthusiastic reactions to trehalose.
  2. Katsuno et al. Sweet relief for Huntington disease. 2004. Nature Medicine 10: 123-124. Online.
    This article comments on HD and other polyglutamine diseases and the recent study done by Tanaka et al. It is fairly scientific and of medium to high difficulty.
  3. Pilcher, Helen R. Simple sugar eases Huntington’s disease in mice. 2004. Online.
    This is an easy to understand explanation of the original study of trehalose and HD.
  4. Tanaka, et al. Trehalose alleviates polyglutamine-mediated pathology in a mouse model of Huntington disease. 2004. Nature Medicine 10: 148-154. Online.
    This is the report written by the researchers who conducted the original study on trehalose and HD. It is highly technical and recommended only for a scientific audience
  5. Trehalose description and manufacture. Online.
    This is a short webpage of medium difficulty that explains what trehalose is and how it is made. It has a diagram of the structural formula.

Updated by A. Lanctot, 11-08-13


RNA Interference (RNAi)

People with Huntington’s disease have two different copies, or alleles, of the Huntington gene. As we discussed here, genes are sections of DNA that provide the information for making proteins. The non-HD allele produces a normally functioning protein, but the HD allele produces a protein that is either the cause or result of many problems in nerve cells. The proteins produced by the HD allele form clumps, or protein aggregates that prevent normal functioning of nerve cells. (For more information on protein aggregation, click here). Although the topic is still up for debate, some researchers suggest that these aggregations may somehow contribute to the progression of the disease. The next step, therefore, is to figure out how to remove the huntingtin protein aggregations or, better still, prevent them from ever forming in the first place. This chapter discusses RNA interference, a gene therapy technique that may do just that.

What is RNAi? How does it work?^

RNA interference (RNAi) is a way to “silence” genes by preventing the formation of the proteins that they code for. A type of gene therapy, it takes advantage of an intermediate step between DNA and protein. DNA acts as a blueprint for the final protein but it uses a kind of “middleman,” called messenger RNA (mRNA), in order to get there. Going from gene to protein is a two step process. The first step in protein synthesis, transcription, takes place in a cell’s nucleus, where the DNA template is used to make a single strand of mRNA. The mRNA then exits the nucleus and enters the cytoplasm, where now it serves as the template for making the protein. With the help of several different molecules, a string of amino acids forms according to the order of the mRNA bases, which are very similar to DNA bases. This process is called translation because the mRNA code is translated into the language of amino acids, the building blocks of proteins.

RNAi comes into play between the steps of transcription and translation. RNA is introduced into the cell and binds to and destroys its mRNA target. Scientists can tailor make pieces of RNA that are complementary (matched up) to a specific strand of mRNA. In some organisms, the whole strand of complementary RNA can be introduced and an enzyme called dicer cuts it up into small fragments once it is inside the cell. Experiments have shown that introducing large strands of RNA into mammals does not work, so scientists were able to overcome this problem by making small interfering RNA (siRNA), also called short interference RNA. This is basically creating smaller chunks of double-stranded RNA (RNA is usually single stranded, except in some viruses) before injecting it into the cell. When these pieces of siRNA match up with the mRNA, they initiate a process that cuts up the mRNA into small fragments. The cell recognizes these fragments as waste and degrades them, and the proteins never form. (See figure below for a representation of the RNAi mechanism.)

Fig H-2: RNA Interference - Simplified Mechanism

How can RNAi be used to treat HD?^

DNA serves as the template for mRNA. This means that the mRNA from the HD allele will be different from the mRNA from the non-HD allele in the same way that their DNA differs. Since the non-HD allele makes a functional protein, it is important that we only silence the disruptive mRNA from the HD allele (mouse studies have shown that shutting down both Huntington genes could be fatal). In order to do this we must first find a difference between the HD and non-HD mRNA. Unfortunately, targeting the most obvious difference between these two molecules, the extra CAG repeats, has proven ineffective (For more information on CAG repeats and HD, click here.) However, there are other differences within the HD allele that are present in most of the people who have it. These differences can be as small as one base substitution (remember how the DNA “alphabet” consists of only four letters, A, C, G, and T? This would be like substituting an “A” for a “C” somewhere in the middle of the chain.). These small differences are called single nucleotide polymorphisms (SNPs). Scientists can create pieces of RNA that are only complementary to the HD mRNA containing a SNP so that only the disruptive HD protein is prevented from being formed. This would allow the non-HD allele to continue to make the normal protein and prevent the harmful protein aggregations that form from the HD protein.

What are some of the challenges of RNAi?^

RNAi is a very promising new tool for treating many kinds of genetic disorders, but much more research and testing need to be done before it can be put to use. One of the main challenges right now is finding a vector, or delivery system, to bring the therapeutic RNA into the nerve cells in the brain. Some researchers have successfully used certain modified viruses for this purpose. This is done by first removing the virus’ own genetic material, thus removing its potential to cause harm, and then replacing it with the therapeutic RNA. One of the first successful RNAi tests was done on mice with a disease similar to HD called spinocerebellar ataxia type 1. (For more information on spinocerebellar ataxia type 1, click here). After injecting the mice with a modified virus, their condition improved. Mice receiving RNAi treatment stopped producing the mutant protein. With the disruptive protein out of the way, the mice no longer experience physical symptoms and are able to move around more easily.

While these are very promising results, we must remember that the testing was done on mice with a similar but different disease than human HD. Much work still needs to be done before the lessons learned from mouse experiments can be safely adapted for use in humans with HD. For instance, further testing has shown that the virus used in the mouse experiment will not work on humans, so another virus must be used. So far, researchers have had success with treating human cells in the laboratory using a virus similar to HIV that normally infects cats, not humans. It is also very important that the siRNA only silence the mRNA from the HD allele. As mentioned before, this requires finding a difference between the two alleles other than the extra CAG repeats. One problem with this in developing an effective treatment is to find a difference that is present in most or all people with HD. In the testing done on mice, the difference was a SNP that was present in 70% of mice with spinocerebellar ataxia. This begs the question, what about the other 30%? Scientists will need to find SNPs present in all people with HD so that they can eventually treat 100% of the population.

Many other important questions about RNAi have yet to be answered. At what age should people start to receive treatment? Would this be before or after they start showing symptoms of HD? Researchers must also run trials to see how much and how often patients should receive treatment. Right now we have no idea if RNAi therapy is long-term or only temporary. In addition, researchers need to continue their work on finding a vector that is both safe and effective. RNAi holds great promise for future treatment of HD but several more years of research and clinical trials need to be done before it can be widely available to the HD community.

For further reading:^

  1. Ambion, Inc. The RNA interference resource. Online
    This is a website with great articles of medium difficulty explaining RNAi and links to new research. Recommended are the articles listed under “New to RNAi? Start Here!”
  2. Check, Erika. Cure hoped for Huntington’s sufferers. 2004. Nature. Online
    This is an easy to read article explaining the initial successful research using RNAi.
  3. Crittenden, Jill. RNA modalities in Huntington’s disease therapy (Workshop). 2002. Online
    This is a discussion of what went on at a workshop of gene therapy researchers. It is of medium difficulty and touches on several key issues that must be worked on before RNAi can be put to use.
  4. Davidson, B.L. and Paulson, H.L. Molecular medicine for the brain: silencing of disease genes with RNA interference. 2004. The Lancet Neurology 3(3): 145-149.
  5. Dykxhoorn, et al. Killing the messenger: short RNAs that silence gene expression. 2003. Nature Reviews Molecular Cell Biology 4: 457-467. Online.
    This is a technical article that explains how RNAi is used to silence genes.
  6. Miller, Marsha. A major breakthrough in gene therapy. 2004. HDAC News Commentary. Online.
    This is a short commentary on the news that RNAi may eventually be able to treat Huntington’s disease.
  7. Miller, et al. Allele-specific silencing of dominant disease genes. 2003. Proceedings of the National Academy of Sciences. Online.
    This is a technical scientific article that explains research using siRNA technology to only silence the dominant allele in diseases similar to HD.
  8. Wexler, Nancy. RNA to our rescue. 2003. Hereditary Disease Foundation 10: 2, 7-9. Online.
    This is a very non-technical explanation of how RNAi may be applied to treat HD and some of the problems with achieving this goal.

-K. Taub, 11/14/04



Drug Summary: Geldanamycin (GA) is a naturally-occurring drug produced by microorganisms to protect themselves from disease-causing substances. GA binds to a special kind of protein called a heat shock protein. All cells produce a common set of heat shock proteins (Hsp) in response to a variety of stresses, including heat, exposure to toxic compounds, or other conditions that cells normally do not experience. Experiments with bacteria, yeast, fruit flies, and mice have shown that increased production of heat shock proteins can protect an organism against stress-induced damage. There are many different kinds of heat shock proteins – each one of them performs a variety of functions that help the cell in both stressful and non-stressful conditions. Most, but not all, heat shock proteins play the role of “molecular chaperones.” Molecular chaperones are substances inside the cell that bind and stabilize proteins at intermediate stages of folding, assembly, movement across membranes, and degradation.

How do Heat Shock Proteins work?^

Fig H-1: How Heat Shock Proteins Work

How does Geldanamycin work in preventing huntingtin aggregation?^

First of all, it is helpful to understand how GA works as an anti-tumor drug because its mode of action against cancer and huntingtin aggregation is similar. Scientists have shown that GA binds to the heat shock protein Hsp 90, which acts as a molecular chaperone. Some of the proteins that Hsp 90 chaperones are proteins involved in the progression of cancer. Once GA binds to Hsp 90, Hsp 90 loses its ability to act as a chaperone. Hsp 90 is then unable to help the cancer-causing proteins fold properly, leaving them malformed. Cells in the human body continually degrade improperly folded proteins, so the loss of function of Hsp 90 causes the degradation of proteins involved in the progression of cancer.

How does this mechanism help in reducing the huntingtin aggregations in HD cells? In addition to its role in directing the folding of proteins involved in cancer, Hsp 90 also associates with another protein called HSF-1 (Heat Shock Factor 1). When GA is absent in cells, Hsp 90 and HSF 1 commonly bind to each other and perform various functions as a unit. When GA is added to cells, it binds to Hsp 90, interfering with Hsp 90’s ATP-binding site and making Hsp 90 unable to associate with HSF 1. The free HSF 1 is then able to enter the cell nucleus where it initiates the production of other heat shock proteins, specifically Hsp 70 and Hsp 40. Once Hsp 70 and Hsp 40 are produced, they associate with the misfolded huntingtin protein and prevent its aggregation. Earlier studies in animals affected with another polyglutamine condition, called Machado-Joseph Disease (For more on Machado-Joseph Disease, click here), have also demonstrated that the overproduction of Hsp 70 and Hsp 40 suppressed protein aggregation and subsequent nerve cell death.

In summary, recent research suggests that GA works against huntingtin aggregations by triggering the following chain of events: (1) GA binds to the heat stress protein Hsp 90, creating free HSF 1 within HD neurons; (2) the free HSF 1 triggers increased production of Hsp 70 and Hsp 40 within the cells; and (3) high levels of Hsp 70 and Hsp 40 then prevent the aggregation of mutant huntingtin.

Other studies have also revealed that GA has the potential to reduce nerve cell damage caused by HD and other polyglutamine diseases. However, GA is also known to be toxic for many cells, and this fact limits its usefulness for patient treatment over long periods of time.

A recent study has proposed the use of GA derivatives that prevent mutant protein aggregation like GA but are not as toxic and so may be viable as therapies for neurodegenerative diseases like HD. These chemicals aid the cell’s defense mechanisms against stress by producing a heat shock response, allowing molecular chaperones to prevent protein aggregation by properly targeting misfolded proteins and aggregates for degradation.

GA is unsafe for use in medical therapy as it is not soluble in water and so not stable in the water-based biological fluids of the body. For this reason researchers created two GA derivatives called 17-DMAG and 17-AAG, which are sufficiently non-toxic to be used in medical therapies. 17-AAG is currently in phase II clinical trials for various cancers, and 17-DMAG is in phase I clinical trials for cancers as well. It was shown that 17-DMAG upregulated Hsp40, Hsp70, and Hsp105 in mammalian cells, three heat shock proteins which are known to inhibit huntingtin aggregation, with greater efficiency than non-modified GA.  The quantity of heat shock proteins increased proportionally with the concentration of 17-DMAG administered. Levels of mutant huntingtin aggregates were also directly tested and were demonstrated to decrease with the increasing doses of 17-DMAG and 17-AAG, and with the number of cells including aggregations decreasing as well. 17-AAG was further shown to improve motor abilities in a mouse model of spinal and bulbar muscular atrophy (SBMA), a disease caused by a nucleotide expansion and mutant protein aggregation like HD.

While it is controversial whether large, insoluble aggregates are in fact the most toxic elements of HD, with some evidence suggesting soluble mutant proteins may in fact contribute more to disease progression, Hsp40, HSp70, and Hsp105 interfere with the early stages of protein misfolding. This means GA and its analogs effectively treat these non-aggregated mutant proteins as well. The concentrations of GA derivatives sufficient to inhibit aggregation are low enough to be safe for medical use. While 17-AAG is similar to GA in its poor solubility, making it a difficult drug to administer orally, 17-DMAG is water-soluble and so conceivably could be taken as an orally-administered drug. With its safety and efficacy already being tested in clinical trials for other diseases, specifically cancer and other nucleotide repeat disorders, 17-DMAG could be expedited through the drug pipeline as a therapy for HD if further studies show its benefit in HD. These initial studies demonstrate that non-toxic derivatives of GA are still able to produce heat shock proteins and induce a stress response reducing mutant huntingtin misfolding and aggregation, thus paving the way for further studies of GA and the heat shock response, and how it can be harnessed by engineered non-toxic drugs to combat HD progression.

Research on GA and heat shock proteins^

Sittler, et al. (2001) demonstrated that GA is capable of suppressing huntingtin aggregation in cells. To test what happens when GA is added to neurons from mice whose gene had been modified so that they express symptoms similar to Huntington’s Disease, the researchers attached a chemical marker to the huntingtin protein and watched what happened to it inside a collection of HD nerve cells. They found out that as more GA was added, the cells produced increased amounts of several heat shock proteins, including Hsp 90, Hsp 70, and Hsp 40. When a large amount of GA was added, they also found that huntingtin aggregates were reduced by as much as 80%. The researchers then asked whether the overproduction of Hsp 90 caused the reduction of aggregates or whether it was increased production of Hsp 70 and Hsp 40 that had reduced the aggregates. They found that simply increasing the amounts of Hsp 70 and Hsp 40 without increasing the amounts of Hsp 90 is enough to reduce the aggregates. Increased production of Hsp 90 had no discernable effect on huntingtin aggregation.

For further reading:^

  1. Sittler, A., et al. “Geldanamycin activates a heat shock response and inhibits huntingtin aggregation in a cell culture model of Huntington’s disease.” Human Molecular Genetics. 2001 Jun 1; 10(12): 1307-15.
    This article reports that Geldanamycin supplementation resulted in decreased huntingtin aggregation in HD nerve cells.

-E. Tan, 9-21-01; A. Lanctot, updated 11-6-13