What causes the onset of HD? Current research shows that there is an abundance of information to be learned regarding the genetic origins of HD. Let’s trace HD’s beginnings from the molecular level, exploring the relationships between a gene, a protein, aggregation “clumps,” neural cell death, and the disease itself.
An Explanation of Terms:
Please note that although “Huntington’s disease” is spelled with an “o,” the correct spelling of the protein involved is “huntingtin” with an “i.” The scientific literature on HD refers to the gene as both the “Huntington gene” and the “huntingtin gene.” For the purposes of this website, we will refer to the gene as the “Huntington gene” or the “HD gene.”
The Huntington Gene^
Recall that a gene is a section of DNA made up of four different nucleotide bases, abbreviated by the letters A, T, C, and G. The order of these bases determines the protein “product” of the gene. (To read more about DNA, click here.) Everyone has a gene that codes for huntingtin protein, a protein found in the cells of the body, which we will discuss later. Towards the beginning of this gene, the three-letter codon sequence C-A-G is repeated a few times. Each C-A-G sequence codes for the amino acid glutamine, a protein building block. People with HD simply have an increased number of these C-A-G repeats toward the beginning of their Huntington gene, coding for an excess number of glutamines in their huntingtin protein. This glutamine extension in altered huntingtin leads to further problems, as we will see.
What is the function of huntingtin protein?^
Although researchers are still investigating huntingtin protein’s exact purpose, it appears to play a critical role in nerve cell function. Huntingtin regularly interacts with proteins found only in the brain. Thus, altered huntingtin is most disruptive to nerve cells, even though it is found throughout the body. For more information, click here.
How can the structure of huntingtin be altered?^
The altered form of huntingtin protein has been found in the autopsied brains of patients who have died of HD. Normal, functioning huntingtin protein contains 10-35 glutamines. (Click here to read more about glutamine expansion numbers.) In contrast, altered HD huntingtin protein (called “Htt” by researchers), contains 40 or more glutamine repeats, resulting from the genetic mutation discussed above. The extended number of glutamine repeats in Htt characterizes HD as one of nine polyglutamine expansion disorders. (To read more about these disorders, click here.)
Because glutamine is a polar, or “charged” molecule, the overabundance of glutamine causes links to form within and between proteins. Htt molecules “stick” to one another, forming strands that are held together by hydrogen bonds. Rather than folding into functional proteins, they develop into tangled, rigid groupings known as protein aggregates. (See Figure P-1.) These fibrous protein aggregates accumulate and interfere with nerve cell function by entrapping key cell regulatory factors. Researchers are exploring whether protein aggregates are a cause or a consequence of neurodegenerative diseases such as HD. Are aggregates always harmful, causing nerve cell dysfunction and death, or are they simply part of a cellular defense mechanism? We will explore this question throughout this article.
Recently, researchers have more clearly elucidated how the glutamine repeats cause protein misfolding and aggregation. The translation of irregular Htt proteins, with expanded polyglutamine domains, signals to the cell that these proteins are abnormal, causing the cell to recruit proteases such as caspase-3 and caspase-6 in order to cut these proteins up into small fragments that can be recycled and digested by the cell. Unfortunately, some of these fragments, particularly those from the N-terminal domain of the Htt protein, are harmful to the cell and cause protein aggregation. These fragments can get into the nucleus of cells, forming aggregates there that disrupt the functions of the cell carried out by the nucleus, such as transcription.
The smallest, and most harmful, Htt protein fragments are caused not by it being cut up by proteases but rather by abnormal alternative splicing of the htt mRNA, leading to a very small protein fragment that can enter the nucleus. This aberrant splicing has been shown to occur in all HD mouse models and at a wide range of CAG repeat numbers, from fifty to a hundred. Aberrant splicing also occurs in human mutant Htt protein, as shown in fibroblasts and blood cells from HD patients. This splicing is mediated by the protein SRSF6, which has been shown to bind to the beginning of the mutant htt mRNA transcript and generate these small protein fragments. This study brings up the question as to whether RNA-targeted therapies, which target the larger fragment of the mutant Htt protein, will be effective, since they would not eliminate these smaller fragments, which may be causing the bulk of damage due to protein misfolding and aggregation.
Another study has added weight to the idea that the N-terminal fragments of mutant Htt play a role in disease progression by studying the role of this fragment in regulating the localization of the Htt protein. The authors found that the nuclear export sequence, a specific sequence of amino acids encoded in the first seventeen amino acids of the Htt protein allows the protein to move out of the nucleus and into the cytoplasm of the cell. When this sequence is mutated in HD, the protein will accumulate and aggregate in the nucleus.
This region is also significantly modified after translation, such as by phosphorylation, which influences the degree to which the fragment aggregates, accumulates in the nucleus, and harms the cell. Additionally, this region can promote association of the Htt protein with the endoplasmic reticulum or mitochondria, further preventing nuclear accumulation. Clearly, mutations in this region, which occur in HD, can have very detrimental effects on the cell. The authors found that the nuclear export sequence consisted of four amino acids in the first fourteen amino acids of the protein sequence, and loss of any of these amino acids caused nuclear accumulation. The first seventeen amino acids on their own, not with the rest of the Htt protein, also accumulates in the nucleus rather than the cytoplasm. Htt protein requires the protein exportin 1 to be transported out of the nucleus, and point mutations in these amino acids cause aggregation of the Htt protein in the nucleus, especially in neurons. How this region of the Htt protein regulates protein aggregation is not known. It could be that mutations in this region change the structure of the protein to one that accommodates aggregation, or disturbs protein stability, or it could specifically promote aggregation in neuronal cells. Regardless, it is clear that the Htt itself, and its sequence, play a role in protein aggregation and HD progression.
In a process similar to the formation of aggregates, the excess glutamines in Htt can also lead to a type of protein bundling known as neuronal inclusions (NI), or inclusion bodies. NIs initially form at the axons and dendrites of nerve cells in specific areas of the human brain, producing the damaged neurons characteristic of HD. Subsequently, an enzyme cuts Htt into smaller fragments which enter the nerve cell nuclei, forming more clumps at the centrosomes. (See Figure P-2.)
Neuronal inclusions cause problems for the cell. They can cause significant changes in cell structure, “trap” and interfere with the normal production of other proteins, and ultimately become toxic to the nerve cell. Thus, the formation of NIs and the neurodegenerative symptoms of HD are clearly linked.
Does mutant huntingtin protein itself kill nerve cells?^
Not directly. As we have seen, one way Htt indirectly leads to nerve cell damage and toxicity is through the formation of protein aggregates and neuronal inclusions. These structures can interfere with several crucial cellular proteins and systems. Researchers have explored a number of hypotheses regarding the mechanisms by which huntingtin aggregates cause cell dysfunction in HD.
Altered Huntingtin and CREB-binding Protein^
Recent studies have shown that altered huntingtin can “kidnap” smaller proteins from their usual locations, preventing them from functioning normally within nerve cells. A recent Johns Hopkins University study showed that Htt entangles and inhibits CREB-binding protein (also known as CBP), a smaller regulatory protein that is key for cell survival. CBP has its own tract of 18 glutamines, and these glutamines interact directly with the expanded Htt glutamine chain. Huntingtin aggregates pull CBP away from its normal position alongside the DNA in the nucleus. Live mouse models with altered huntingtin and autopsied brains of patients who have died of HD show very reduced amounts of CBP, suggesting that it has been pulled away from the DNA and sequestered by the altered huntingtin.
Once seized, CBP is out of service. It can no longer accomplish its normal function of activatingtranscription or “turning on” genes for survival pathways. Fewer proteins are produced, ultimately leading to nerve cell death. However, researchers were able to fully halt and reverse this degenerative process in the laboratory. This reversal was accomplished by inserting an engineered form of CBP that did not have glutamine repeats. Since Htt interacts directly with the CBP’s glutamines, the modified CBP was not recognized by Htt. The modified CBP was not sequestered in huntingtin inclusion bodies, and the nerve cells survived.
In addition, other compounds known as histone-deacetylase inhibitors (HDAC inhibitors) have been shown to compensate for the negative effects of CBP. It has been shown in fruit flies that HDAC inhibitors reduce the lethal effects of the altered huntingtin. These developments indicate potential targets for new drug treatments, but further research is still needed.
Altered Huntingtin and the Ubiquitin-Proteasome System^
Normal cells have a mechanism for quality control called the ubiquitin-proteasome system (UPS). The UPS is a protein-chaperone system which tags misfolded or damaged proteins for re-folding, or more commonly, for degradation. In contrast, protein aggregates resulting from the altered HD gene have a high molecular mass and are sequestered to the aggresome region of a cell. (See Figure P-3 above.)
Why is the altered form of huntingtin not broken down by the cell, as normally occurs with irregular proteins?^
A recent Stanford University study suggests that protein aggregates impair UPS function, another explanation for how these huntingtin bundles can lead to the death of neurons. Large clumps of defective protein accumulate and scar neurons, which then cannot survive and reproduce. This conclusion about the relationship between protein aggregation, UPS function, and neurodegenerative disease was drawn from an experiment involving mutant cystic fibrosis protein and mutant huntingtin protein. Both of these proteins contain a polyglutamine repeat sequence, giving them the tendency to aggregate. Within human embryonic kidney cells, proteins were tagged with a special form of green fluorescent protein (GFP) which glows only when proteins remain intact. Throughout the experiment, mutant, aggregation-prone proteins continued to glow, showing that they were resistant to degradation, and thus, that the UPS system was stalled. Fluorescence was higher in cells containing aggregates than in cells without, suggesting that protein aggregation led to the UPS disruption.
What is the process by which protein aggregates interfere with the UPS system?^
Well, one clue is that levels of the normal protein ubiquitin are not diminished, and it continues to tag mutant huntingtin for degradation. Thus, it has been suggested that the abnormal proteins clog the other component of the UPS system, the proteasomes. Proteasomes are “master controllers” which normally break down proteins that have been tagged by ubiquitin. Protein aggregates may occupy the proteasomes, inhibiting their normal activity.
Future research in this area may focus on how and why the proteasomes shut down in the presence of protein aggregates. Answers to these questions may lead to treatments that can dissolve aggregate proteins or prevent them from forming, if it is proven that they are indeed only harmful.
Are huntingtin aggregates a cause or consequence of HD?^
This remains a matter of some debate, as the precise role of aggregates in HD is still unclear. On one hand, large inclusions of aggregates seem to be correlated with the development of HD, which suggests a toxic role. There is evidence that huntingtin aggregates accumulate in the nuclei of cells, where they interfere with the process of transcription, and that they also affect other cellular processes such as axonal transport in neurons. A study by Chang et al showed that aggregates can block the movement of mitochondria in neurons, consequently impairing energy production and metabolism. In addition, the previously mentioned Stanford study concludes that aggregates themselves can contribute to cell toxicity by impairing the UPS system. In a vicious cycle, the aggregates inhibit the very pathway that destroys them. Hence, they continue to build up, resulting in further impairment of UPS function. This accumulation of mutant proteins is gradual, which may explain the late onset of HD.
However, there is also much evidence to support the idea that aggregates do not cause HD, but that they are rather an attempt by cells to isolate toxic cell fragments that result from HD, in order to prevent these fragments from causing too much harm. A 2005 study conducted by Slow et al. demonstrated that HD mice with frequent and widespread aggregates did not display any evidence of neuronal dysfunction or degeneration, indicating that aggregation may not be sufficient to cause neurodegeneration. Furthermore, another study conducted by Arrasate et al. demonstrated that aggregates are not directly correlated with neuronal death, and in fact neurons containing aggregates have been shown to survive significantly longer than neurons without aggregates. This suggests that aggregates may actually play a neuroprotective role in HD, acting as a cellular coping mechanism to temporarily store toxic fragments before they are degraded by the proteasome.
One way to reconcile these opposing theories is that aggregate formation does in fact decrease toxicity, but that the UPS system also needs to degrade these aggregates properly and clear toxic fragments from the cell. In other words, aggregate formation might be working together with the UPS system to reduce the levels of toxic mutant huntingtin. Additional research on how to enhance the ability of the UPS system to degrade aggregates may be important for identifying therapeutic targets.
Future Challenges for Huntingtin Research^
Clearly, we have much to learn from exploring the relationships between the Huntington gene, huntingtin protein, protein aggregation, and neural cell death that characterize HD. The effects of HD huntingtin protein on the CBP and UPS pathways are examples of molecular-level problems leading to neural cell death. Future research in this area will likely continue to explore the following questions:
- What is the process by which the proteins aggregate?
- How can the cell get rid of these aggregates?
- What other protein-protein interactions are involved in this disease?
Answers to these questions may point to further possibilities for treatment of HD.
- Arrasate M, Mitra S, Schweitzer ES, Segal MR, Finkbeiner S. “Inclusion body formation reduces levels of mutant huntingtin and the risk of neuronal death.” Nature 431: 805–810, 2004.
- Chang D, et al. “Mutant huntingtin aggregates impair mitochondrial movement and trafficking in cortical neurons” Neurobiol Dis. 2006 May;22(2):388-400.
- Slow EJ,Graham RK,Osmand AP,Devon RS,Lu G,Deng Y,Pearson J,Vaid K,Bissada N,Wetzel R,Leavitt BR,Hayden MR. “Absence of behavioral abnormalities and neurodegeneration in vivo despite widespread neuronal huntingtin inclusions.” Proc Natl Acad Sci USA 102: 11402–11407, 2005.
For further reading^
- Bence NF, Sampat RM, Kopito RR. “Impairment of the ubiquitin-proteasome system by protein aggregation.” Science 2001. 292: 1552-1555.
A fairly technical paper, reporting a study which shows that protein aggregation impairs the ubiquitin-proteasome system.
- Helmuth, Laura. “Protein clumps hijack cell’s clearance system.” Science 2001. 292: 1467-1468.
A less technical, more reader-friendly summary of the paper listed above.
- Kopito RR, Ron D. “Conformational disease.” Nature Cell Biology. 2: 207-209.
A fairly technical overview of diseases caused by protein aggregation pathways.
- Nucifora FC, Jr, et al. “Interference by huntingtin and atrophin-1 with CBP-medicated transcription leading to cellular toxicity.” Science 2001. 291(5512): 2423-2428.
A highly technical paper regarding the negative effect produced when the altered huntingtin protein “kidnaps” a smaller protein called CBP.
- Steffan JS, et al. “Histone deacetylase inhibitors arrest polyglutamine-dependent neurodegeneration in Drosophila.” Nature 2001. 413: 739-743.
A fairly technical paper that discusses the interaction between altered huntingtin protein and CBP, as well as the effects of HDAC inhibitors.
- Waelter S et al. “Accumulation of mutant huntingtin fragments in aggresome-like inclusion bodies as a result of insufficient protein degradation.” Molecular Biology of the Cell 2001. 12(5): 1393-1407.
A fairly technical paper regarding the relationship between inclusion bodies and the ubiquitin-proteasome system.
- Zuccato C, Valenza M, Cattaneo E. “Molecular Mechanisms and Potential Therapeutical Targets in Huntington’s Disease.” Physiol Rev July 1, 2010 90(3): 905-981.
A fairly technical review article summarizing research about mutant huntingtin and potential therapeutic targets for HD.
- Zheng Z, Li A, Holmes BB, Marasa JC, Diamond MI. “An N-terminal Nuclear Export Signal Regulates Trafficking and Aggregation of Htt exon 1.” J Biol Chem. 2013 288(9): 6063-6071. A technical research article showing the role of the nuclear export sequence of Htt in mediating intracellular trafficking.
C. Barnard, 1-28-02; update: J. Nguyen, 5-23-12; update: A. Lanctot, 11-06-13