All posts by Stephanie Liou

Dr. Elena Cattaneo
Department of Pharmacological Sciences and Center of Excellence on Neurodegenerative Diseases, University of Milan
Milan, Italy

In September of 2004, HOPES team members Devon McGee and Agnieszka Milczarek visited the Laboratory of Stem Cell Biology and Pharmacology of Neurodegenerative Diseases, headed by Dr. Elena Cattaneo, at the University of Milan in Italy. HOPES would like to thank Dr. Cattaneo and the rest of the members of the lab for taking time out of their busy schedules to meet with us and share with us the philosophy behind their research. The following is the lab´s mission statement:

The ingenuity of mankind, often anonymous, has taught humanity to always make better use of water which runs ceaselessly… The mill is the processor of the river directing simplicity and chaos into a powerful and productive force. Our laboratory strives to remain a mill in constant operation.

Introduction

Dr. Elena Cattaneo did her graduate work at the Massachusetts Institute of Technology in the lab of Professor R.D.G. McKay, where she became interested in neural stem cells and their potential therapeutic applications to neurodegenerative diseases. She decided to focus her studies on neural stem cells from a human brain structure called the striatum because she realized that “the striatum is at the center of many biophysiological events.” The striatum forms part of the basal ganglia, and is the part of the brain most affected in HD. (For more information on the basal ganglia and their role in HD, click here.) Remarkably, while at MIT, she was able to “immortalize” stem cells from the striatum and create a new cell line called ST14A. The cells exhibit quite accurately many of the properties of nerve cells from the striatum and can be engineered to express either normal or mutant huntingtin. (For more information about huntingtin, click here.) The cell line offers a great model of the Huntington´s disease and researchers around the world now use it in their labs.

Presently, Dr. Cattaneo works in the Department of Pharmacological Sciences and in the Center of Excellence on Neurodegenerative Diseases at the University of Milan, where she is a full professor of pharmaceutical biotechnology. Since 1995, she has collaborated on Coalition for the Cure, an international research group organized by the Huntington´s Disease Society of America. (For more information on the Huntington´s Disease Society of America, click here.) She has also collaborated on the Cure HD Initiative sponsored by the Hereditary Disease Foundation. Since 1988, she has been an investigator in the Italian Telethon Foundation. Dr. Cattaneo has received many awards and distinctions, including the “Le Scienze” Prize for Medicine and a medal from the president of Italy for her work on stem cells and HD. In addition, she coordinates a national program on stem cells and participates in Eurostemcell (www.eurostemcell.org), a group for research on stem cells funded by the European Union.

Dr. Cattaneo´s lab is comprised of senior scientists (similar to post doctoral fellows), junior scientists (similar to graduate students), and undergraduate students. Overall, there are about fifteen people working in the lab. Each member of the lab chooses and proposes his/her own research project to work on. Although the members of the lab have diverse research interests, the lab is unified in its quest to someday find a cure for HD. There are weekly lab meetings and each person is at least partially involved in all ongoing research projects.

Currently, the lab is focused on the mechanisms of neurodegeneration in Huntington´s disease, as well as neural stem cell biology and its potential application to HD and other neurodegenerative diseases. (For more information on stem cells and their potential to treat HD, click here.) The ultimate goals of the lab are to identify cellular pathways that might be suitable for therapeutic intervention and to find new methods for drug testing. While a majority of HD research is focused on the harmful effects of the mutant huntingtin protein, Dr. Cattaneo´s lab looks at the other side of the problem by examining the normal function of huntingtin protein. This is important because not much is known about how the huntingtin protein is supposed to work normally. By investigating the normal function, the researchers hope to find new ways of treating HD. Dr. Cattaneo´s research has revealed that normal huntingtin possesses a variety of anti-apoptotic capabilities, which means that it helps keep cells alive.

Thus, on the basis of Dr. Cattaneo´s work, the damaging effects of HD should be attributed not only to the toxic function of mutant huntingtin, but also to the loss of beneficial function of normal huntingtin. With this in mind, the lab hopes to completely make clear the physiological role of normal huntingtin and its mode of action in order to develop new ways to combat the disease. For example, once more is known about normal huntingtin, researchers can attempt to restore normal huntingtin function in people with HD, and this restoration of normal huntingtin function may significantly alleviate many HD symptoms.

Members of the research lab are also involved in the study of the biology of neural stem cells derived from the developing human brain. Neural stem cells seem promising because they are the developmental source of nerve cells. The number one problem in neurodegenerative diseases like HD is the death of nerve cells, which are usually unable to regenerate themselves. Neural stem cell transplantation may help restore nerve cells and may even prevent nerve cells from dying in the first place.

Neural stem cell therapy for neurodegenerative diseases is still a distant goal and many obstacles remain before it can be used as a treatment. One obstacle, for example, is that it is difficult to properly integrate neural stem cells into a recipient brain. Presently, Dr. Cattaneo´s lab is trying to identify the signals that instruct neural stem cells to undergo division and differentiate into nerve cells. Understanding the mechanisms that regulate nerve cell survival and differentiation will make it easier to develop ways to integrate neural stem cells into the brain. In addition to studying nerve cell division and differentiation, the lab is also producing cell and animal models of brain diseases for studies of cellular mechanisms and of drug efficacy.

HOPES members Devon McGee and Agnieszka Milczarek with Dr. Elena Cattaneo, center.

What´s special to Dr. Cattaneo about HD research?

HD research involves a great deal of obligation to the HD community and this obligation is what drives Dr. Cattaneo and her coworkers to produce promising results in the lab. The lab often receives not only phone calls but also visits from individuals and families affected by HD, who bring assortments of jams and cookies and cakes to show their gratitude for the researchers´ work. Dr. Cattaneo and the other members of the lab recognize that it is important for the patients and their families to know that someone is constantly working on uncovering the mysteries of HD. For this exact reason, Cattaneo and some of the other researchers occasionally present their scientific results, in non-technical terms at the meetings of Italian associations for people with HD. This constant interaction between the researchers and individuals affected by HD has such a positive effect on the lab that the researchers become not only “driven by curiosity, but also by the desire to help patients.” All the researchers in the lab confess that they feel increased pressure to produce and publish results because they know that they are working for real patients – and this “healthy pressure,” as Dr. Cattaneo calls it, is one of the factors that make HD research so special.

Dr. Cattaneo also believes that HD research is extraordinary because of the close-knit HD research community that continues to develop around the world. Funding and support from associations such as the Hereditary Disease Foundation and the Huntington´s Disease Society of America (HDSA) in the United States and others in the European Union help bring together researchers who share one goal – to find a cure for HD – so that they can work as a team rather than compete with each other for results. Since 1997, the HDSA has coordinated “Coalition for the Cure,” which organizes researchers from almost twenty different laboratories and organizes them into five teams that work on different aspects of HD. Cattaneo says that coalitions such as these not only promote but also require sharing of unpublished results among research labs, which helps the coalition as a whole make progress as researchers build on each other´s ideas. Furthermore, labs that are part of such alliances have the advantage of sharing research materials such as useful genes or experimental mouse strains (for more information on animal models in HD research, click here), which speeds up the research, keeps costs down, and makes possible experiments that might not otherwise be possible. This type of sharing helps research labs circumvent financial and legal obstacles that might prevent them from obtaining materials developed by other research institutions.

On the one hand, being part of a scientific community that emphasizes sharing goes a long way to reduce competitive barriers commonly seen between labs working on similar projects. On the other hand, Cattaneo says it also adds pressure to be constantly productive. Researchers are expected to present new results at each of the annual or biannual meetings they must attend for each coalition or organization. Cattaneo admits that such expectations can add stress to her life, but being part of a community of researchers who have placed complete trust in each other and have vowed to work together for families affected by HD makes it worthwhile. She emphasizes that the formation of such a close-knit community could only have been possible with a disease like HD, because there has been historically much less interest in HD research than in diseases such as Alzheimer´s or Parkinson´s. (For more information on these diseases and how they compare to HD, click here.) Through her work on HD, Dr. Cattaneo has forged strong relationships with researchers from across the globe, including Drs. James Gusella and Marcy MacDonald at Massachusetts General Hospital, whom two other HOPES members visited in the summer of 2004. (For more information about the latter, click here.) In the process, Cattaneo´s obligation to patients has been complemented by the obligation she now feels to produce valuable results that she can share with fellow researchers.

Approaching HD

According to Dr. Cattaneo, to be conceptualized correctly, HD must be seen as a cascade in which every single disease event leads to more events that cause further dysfunction and damage (see Figure AH-0.) In some way, HD researchers are lucky because they have a definite single starting point of the disease: a CAG expansion in the Huntington gene. Cattaneo agrees that the genetic nature of HD makes it easier to research than other neurodegenerative diseases, which can have varying genetic and environmental causes. However, the disease cascade has yet to be completely figured out, and Cattaneo stresses that it will be important not only to find a majority of the disease events, but also to arrange them in the correct temporal order; that is, to figure out when each dysfunction occurs, what causes it, and what other dysfunctions it causes itself. As is evident from Figure AH-0, this will not be an easy task.

Researchers in Dr. Cattaneo´s lab are already working on putting together the schematic cascade, but Cattaneo says that the work is difficult since “every result leads to more experimentation.” With every new mechanism she and the researchers discover, there is more they need to find out about its causes and effects. Cattaneo says that often, “your brain goes faster than your hands,” and while she or her colleagues may form hypotheses and ideas rapidly, the funding and time may not allow for immediate experimentation to follow up on those ideas. However, one researcher in the lab, Chiara Zuccato, was recently very successful in figuring out a very important aspect of the disease cascade. After learning that normal huntingtin protein increases the amounts of brain-derived neurotrophic factor (BDNF) in nerve cells, she set out to find the mechanism by which it does so. Zuccato found that normal huntingtin increases the production of BDNF by indirectly inhibiting a molecule called NRSE, which normally prevents the production of BDNF (this finding will be explained in more detail in a later section).

Because BDNF is a protective growth factor in the brain and is depleted in the nerve cells of people with HD, researchers in Dr. Cattaneo´s lab are now focusing their energies on the functions of normal huntingtin and what prevents it from performing these functions in people with HD. This approach is different from the classic research approach that has typically focused only on mutant huntingtin. However, mutant and normal huntingtin may be linked earlier on in the disease cascade and Cattaneo´s lab is now on a quest to figure out how mutant huntingtin´s toxicity could cause normal huntingtin´s loss of function in HD.

Unfortunately, BDNF production is only one of many cellular processes that are disturbed in HD, and Cattaneo says real progress will be made when researchers are finally able to link together all the dysfunctions in a cause-and-effect relationship. Transcriptional dysregulation is a major disease mechanism in HD that affects the production of proteins needed by nerve cells (one of these is BDNF, mentioned above). Mitochondrial dysfunction, which causes abnormalities in energy metabolism, is another major disease mechanism. (For more information on problems with energy metabolism in HD, click here.) As of now, researchers only understand bits and pieces of each of these major mechanisms, and Cattaneo believes that mapping out precisely what events lead to these dysfunctions will be crucial in understanding HD and developing a cure.

Because HD involves a complicated cascade of events, Dr. Cattaneo believes that an effective cure will actually involve a combination of treatments. She is very hopeful about the use of new techniques such as RNA interference, which attack the disease as close as possible to the beginning of the cascade by “silencing” the mutant Huntington gene. However, Cattaneo doesn´t think that such techniques will ever be able to fully block the production of the mutant huntingtin protein. (For more information on RNA interference, click here.) Cattaneo says that “a cure with only one strategy is not realistic; to battle HD you need a more global approach.” In the future, she believes that the most successful therapy, which could turn into a cure, will involve different types of treatments at different stages of the disease. According to Cattaneo, these treatments will ideally begin before the onset of symptoms and change accordingly as the disease progresses from the nerve cell dysfunction stage to the nerve cell death stage. Future treatment of HD may include:

• Stage I: Early treatment with RNA interference to prevent expression of mutant huntingtin; continues throughout the following stages.
• Stage II: Dysfunction of nerve cells combated by drugs that prevent toxic functions of mutant huntingtin and drugs that restore the functions of normal huntingtin.
• Stage III: Nerve cell death combated by cell replacement therapy (possibly with the use of stem cells) as well as protective strategies involving therapy with growth factors such as BDNF.

Dr. Cattaneo believes that for patients, “a cure is anything that gives them more time,” and she hopes that a treatment regimen like the one outlined above could postpone and decrease the symptoms of the disease to such an extent that it could in fact be considered an effective cure.

However, because potential drugs will be used to combat the disease at different time points in different individuals, the researchers will need biomarkers. Biomarkers are biological indicators that can be measured in patients to determine the severity of disease and to show whether drugs are effective in specific stages of the disease. Measuring the severity of symptoms is not sufficient because symptoms can vary so widely between patients and between stages of the disease. A possible biomarker that the Cattaneo lab is considering using is the level and activity of the A2A receptor, a receptor molecule that is expressed by nerve cells in the striatum and becomes more active in the presence of mutant huntingtin. Another possibility involves measuring levels of BDNF, which can also be used to track the progression and severity of the disease.

Dr. Cattaneo´s lab tries to simultaneously identify drug targets as they learn about new disease mechanisms. She stresses that successful drug development cannot happen without a deep understanding of the disease mechanism that the drug is meant to attack. Therefore, she believes that studies on compounds such as nutritional supplements and their effects in people with HD are not as promising at they may seem since most of the studies are conducted without an understanding of how the supplements exert their effects. An understanding of the disease mechanism is essential to picking drug targets – molecules that the drug will act on to alter some pathway – that will be both effective and safe. The drug must act early enough in the pathway to successfully stop the disease cascade, but it must also be specific enough so that it does not cause significant side effects.

Main Findings

Dr. Cattaneo´s lab has contributed to significant breakthroughs in HD research. Prior to the research done by the Cattaneo lab, it was assumed that since the HD allele gives rise to mutant huntingtin, mutant huntingtin must be toxic to nerve cells and it must be the only reason that nerve cells die. Indeed, numerous studies support this notion. However, Dr. Cattaneo´s lab showed that mutant huntingtin is not the only reason that nerve cells die. In fact, it may be only half the reason. Whereas earlier research had simply disregarded normal huntingtin´s role in the HD disease process, the Cattaneo lab found that normal huntingtin is actually crucial to nerve cell survival in the brain. They have found that individuals with the HD allele experience nerve cell death not only because their cells produce toxic mutant huntingtin, but also because the normal huntingtin they produce loses its normal functions.

Dr. Cattaneo and two other members of her lab, Dorotea Rigamonti and Chiara Zuccato, are leading experts in the field of normal huntingtin function. They have amassed a remarkable amount of evidence supporting the notion that normal huntingtin is, in fact, beneficial to nerve cells. They began their research in this area by first inserting extra copies of either normal huntingtin protein or mutant huntingtin protein into nerve cells grown in culture dishes in the lab. In 2000, they reported that nerve cells overproducing normal huntingtin can survive even when deprived of key growth factors or when subjected to other conditions that would normally cause them to die. Furthermore, they found that normal huntingtin appears to keep nerve cells alive by stopping the cascade of events that usually leads to apoptosis, or programmed cell death. They therefore concluded that normal huntingtin serves as a kind of protein “lifesaver” for nerve cells.

Other researchers, such as Scott Zeitlin of Columbia University in the USA, have confirmed these findings by creating knockout mice that express neither mutant huntingtin nor normal huntingtin. In such cases, the mice develop severe brain damage. In this instance, the brain damage can be explained not by mutant huntingtin (because it isn´t present), but by the absence of normal huntingtin (which also is not present). Interrupting huntingtin production at various points in the mice´s lives also leads to apoptosis. Remarkably, mice lacking normal huntingtin display very similar neurological symptoms to mice that express mutant huntingtin, suggesting that the absence of normal huntingtin and the presence of mutant huntingtin might be different sides of the same coin.

Lab members Evangelia Papadimou, Erika Reitano, and Alessia Tarditi, left to right.

The discovery of the importance of normal huntingtin function was a significant contribution to the field of HD research. However, it does not explain why nerve cells in the striatum are targeted preferentially in HD. The huntingtin protein is produced in many cells throughout the body. Since normal huntingtin does not function correctly in people with HD, and since it is present in all types of cells, scientists were puzzled by the fact that only nerve cells in the striatum were seriously affected by the disease. Intrigued by this question, Zuccato and her colleagues undertook an examination of brain-derived neurotrophic factor (BDNF). As discussed above, BDNF is a growth factor that is known to be crucial to the development and survival of nerve cells in the striatum. It is usually produced in the cell bodies of nerve cells in the cortex and then travels to the striatum along fibers that connect the two brain regions. With this is mind, the researchers hypothesized that there might be a connection between BDNF and huntingtin. Amazingly, they found that normal huntingtin stimulates the production of BDNF in nerve cells grown in lab culture dishes. Specifically, huntingtin seems to indirectly activate the “on” switch, or promoter, of the gene that encodes BDNF. When this gene is turned on, it prompts nerve cells to make more BDNF. Normal huntingtin enables the gene to be turned on by inhibiting a molecule called NRSE, which normally turns the gene off. Contrarily, mutant huntingtin does not inhibit NRSE, which means that NRSE stays around and keeps the gene turned off so that no BDNF can be produced. Zuccato and her colleagues confirmed this link between huntingtin and BDNF by carrying out experiments involving genetically engineered mice. They found that mice overproducing normal huntingtin have elevated amounts of BDNF in their brains, whereas mice with mutant huntingtin have lower than normal levels of BDNF in their brains.

The lab´s studies are both enlightening and intriguing because the findings do not necessarily conform to previous hypotheses about the disease. They reveal the true complexity of the disorder. In addition to generating mutant huntingtin which is thought to interfere with several crucial cellular proteins and systems, HD also somehow deprives the brain of normal huntingtin – which would otherwise turn on the gene for the growth factor BDNF and protect nerve cells from apoptosis. As mentioned previously, Cattaneo´s lab believes that mutant huntingtin´s toxicity and normal huntingtin´s loss of function may be related. In fact, another group of researchers has recently shown in genetically engineered mice that mutant huntingtin can destroy normal huntingtin. It is also clear that normal huntingtin interacts with the brain in complex ways and there are many aspects of huntingtin that need to be further explored. As Dr. Cattaneo noted, “Much additional research must be completed before these findings can help patients, and we want to be clear that this is not a cure. But we are optimistic that our work will help guide the development of new therapies, such as drugs to replace or boost the activity of normal huntingtin, or to increase levels of another brain protein.”

By more clearly exposing the complexities of HD, Dr. Cattaneo´s lab has opened the door for the production of better treatments for the disease. Many of the drugs currently available only alleviate some of the symptoms of HD and can have serious side effects. Furthermore, the drugs often treat one symptom only to make another symptom worse. For example, doctors often prescribe sedatives to control involuntary movements, but these drugs also decrease levels of the neurotransmitter dopamine in the brain, worsening the patient´s depressive symptoms.

As a result of the findings of Dr. Cattaneo´s lab, several more innovative treatments for HD are currently being tested, such as replacing the damaged nerve cells with transplants of fetal tissue or injecting neurotrophic factors such as BDNF into the striatum. The use of fetal tissues is very controversial and raises many ethical questions, but the preliminary results do look promising. Researchers at the School of Medicine in Creteil, France have transplanted fetal nerve cells into the striata of five people with HD. Three of the people improved significantly in terms of motor and intellectual function. Currently, clinical trials with a larger number of patients are being conducted.

Obstacles and Challenges

Although Dr. Cattaneo certainly enjoys the rewards of being a part of an HD research team, she emphasizes that she and her coworkers face numerous obstacles. The intense pressure and time constraints that accompany HD research force the researchers to live demanding and hectic lifestyles. Because the lab is part of an HD coalition that meets every six months, as well as other organizations that meet regularly, the Cattaneo lab is expected to maintain constant progress. The labs in the coalition are the best in the world and Dr. Cattaneo said she feels pressure to live up to high expectations. She confesses that she is usually unable to sleep the night before coalition meetings because the excitement and pressure are great.

In order to be able to report new results every six months, members of the lab cannot afford to waste any time. They generally arrive at the lab at 8:30 am and leave between 9:00 pm and 11:00 pm. If they leave at 6 pm, they consider it a half-day. Furthermore, they do not take weekends off. As researcher Chiara Zuccato put it, “There are twenty-four hours in a day – I need to sleep about seven hours and it takes over an hour to get to and from work. For pretty much the rest of the day, I´m in the lab.” Dr. Cattaneo has two children and it is very hard for her to spend so much time away from them. Luckily, it is an Italian way of life to have support from the family and her mother-in-law helps take care of the children.

Many people are not able to handle the pace of this lifestyle, and it is not uncommon for researchers to leave the lab after a year or two. Ironically, Dr. Cattaneo enjoys her work so much that it is hard for her to be away from the lab. The last time she took a vacation she got up very early every morning before her family woke up to check her e-mail and maintain correspondence with the lab.

A big obstacle in Italian academia is the lack of available jobs. Currently, the government is attempting to abolish assistant professorships. If this happens, even individuals with many qualifications will not be able to find positions within academic settings. An individual could be highly prolific for fifteen years and make significant research discoveries and still not be able to advance in the field. This situation in the US would be equivalent to post doctoral students never being able to become professors. Dr. Cattaneo sees this lack of job positions as an obstacle because it will be even harder to recruit new people into her lab.

Because the lab uses stem cells and there are numerous ethical issues surrounding stem cells, it has encountered a few obstacles in the form of religious and political interference. (For a concise discussion on stem cell ethics, click here.) However, members of the lab believe that this problem is a result of the public being misinformed. They believe that once the public better understands stem cell research this issue will no longer be an obstacle.

Another obstacle arises when the researchers must decide whether they should devote to making an old experiment more complete or whether they should devote time to starting a new experiment. On one hand, they want the experiment to be completely thorough. On the other hand, their minds are racing with new ideas and they want to start new experiments. This situation is worsened by the fact that it is difficult for the researchers to satisfy both their curiosities and their own expectations. In one instance, Zuccato had already written and submitted a paper about an experiment when she realized that there was something missing. Despite having a ton of other things to do, she immediately stopped submission of the paper and decided to redo the entire experiment with added conditions to make it more thorough. Thus, for researchers, it is a constant struggle to balance thoroughness with productivity.

Surprisingly, Dr. Cattaneo explains that money is not normally a major obstacle for her lab, because it is supported by many sources, including the University of Milan, the European community, and many American foundations. Happily, for at least the next four years, there is adequate funding. Dr. Cattaneo is very thankful for this: she points out that “research always begets new research,” and funding is crucial for exploring new possibilities.

Conclusion

Between their work on normal huntingtin function, mutant huntingtin function, and the development of stem cells into useful nerve cell models, the members of the Cattaneo lab will have their hands full for many years to come. Dr. Cattaneo knows that she and her fellow lab members will continue working their twelve hour days in hopes of finding new treatments for HD. As Cattaneo puts it, in Italy, the researcher´s reward “is not monetary; it is a dot on a gel” – that is, a single dot showing the successful results of an experiment. For Cattaneo, helping young enthusiastic researchers get started in the field is almost as rewarding. Dr. Cattaneo reminds us that “science has no guarantees but one: that researchers will keep working.” She encourages people affected by HD to learn about the research efforts going on around the world, and she hopes that the knowledge that “someone is thinking about this [disease] every minute of every day” can be comforting and reassuring.

For further reading

1. To find out more about the Cattaneo lab, please visit its website at http://users.unimi.it/DPS/struttura.php?id=10.
2. For more information about the Huntington´s Disease Society of America and their Coalition for the Cure, visit the HDSA website at www.hdsa.org.
3. Cattaneo E., Rigamonti D. Goffredo D. and Zuccato C. (2001) Loss of normal huntingtin function: new developments in Huntington´s disease research. Trends in Neurosciences, 24:3, 182-187.
   This article is fairly easy to read. It succinctly summarizes the background of HD as well as the various hypotheses about its molecular pathology.
4. Rossi F. and Cattaneo E. (2002) Neural stem cell therapy for neurological diseases: dreams and reality. Nature Reviews Neuroscience, 3, 401-409.
   This is a very in-depth article about the therapeutic potential of neural stem cell therapy. It also explains all of the obstacles that must be overcome before neural stem cell therapy can become a viable treatment.
5. Zuccato C. et al. (2001) Loss of huntingtin-mediated BDNF gene transcription in Huntington´s disease. Science, 293, 493-496.
   This is a technical article that describes how the beneficial activity of huntingtin is lost in people with HD and how this leads to decreased production of BDNF.
6. Zuccato C., Tartari T., Crotti C., Goffredo D., Valenza M., Conti L., Cataudella T., Leavitt B. R., Hayden M. R.,Timmusk T., Rigamonti D. and Cattaneo E. (2003) Huntingtin interacts with REST/NRSF to modulate the transcription of NRSE-controlled neuronal genes. Nature Genetics, 35: 76-83.
   This article is very technical. It describes in detail how normal huntingtin increases transcription of BDNF by silencing NRSE.

– A. Milczarek and D. McGee, 04/29/05

Dr. Steinman’s research has focused on the use of cystamine, a transglutaminase (TGase) inhibitor, as a potential treatment for Huntington’s Disease. Previous studies have shown that HD is associated with the formation of protein clumps, or aggregates, in the brain. Protein aggregation has been linked with some of the neurological symptoms of HD. (To learn more about protein aggregation, click here.) Earlier research has also shown that cystamine inactivates transglutaminase (TGase), an enzyme that helps produce these clumps of huntingtin protein. Therefore, Steinman and his former graduate student, Marcela Karpuj, PhD, reasoned that cystamine might control the disease by preventing the formation of huntingtin protein clumps.

Cystamine has been shown to inhibit the activity of TGase in two ways – one that flips the “off switch” and one that blocks huntingtin from binding. First, cystamine inactivates TGase through a disulfide-exchange reaction, a process by which proteins are activated or inactivated by the folding and unfolding of the amino acid chains that make the proteins. Second, cystamine competitively inhibits TGase. Since cystamine and huntingtin are both capable of binding to the same active site on the TGase enzyme, cystamine will block huntingtin from binding to TGase and thus limit TGase activity.

Steinman and Karpuj tested the effects of cystamine on mice that had a portion of the human HD allele of the Huntington gene (exon 1) inserted into their DNA. They knew that mice with exon 1 exhibited clinical pathologies very similar to the symptoms of human juvenile HD (including the presence of huntingtin protein aggregates and increased TGase activity in the brain), making them good test subjects for the study. (To learn more about juvenile HD, click here.)When cystamine was injected into the mice, TGase activity in the brain was reduced by up to 40% after only ten minutes. To test the effects of cystamine on the clinical symptoms of the HD mice, the drug was administered to mice that clearly demonstrated tremors and abnormal movement. Mice treated with cystamine generally exhibited fewer tremors, decreased abnormal movement, and a lower incidence and delayed onset of HD symptoms. They also lived 20% longer on average.

To the researchers’ surprise, however, cystamine did not reduce the number of huntingtin clumps in the brain. So although their hypothesized clinical outcomes were realized, their hypothetical mechanism was not. This unexpected finding led the Steinman lab to search for an explanation of the alleviated tremors and prolonged life. They started by looking for differences in gene expression between the mice that were treated with cystamine and the mice that were not. The researchers found two genes that were expressed much more in the cystamine-treated mice. A third, related gene showed increased activity in human HD patients, although not in the mice. All three genes code for proteins that are known to protect brain cells from damage. Therefore, cystamine seems to boost the levels of the brain’s natural protective proteins.

Although these findings suggest that cystamine could be a new treatment for HD, research for even more effective and specific drugs continues. Steinman suggests that a combination of cystamine and other drugs may have even greater benefits.

For more information on the research done by Lawrence Steinman, visit the following website: http://steinmanlab.stanford.edu/

For Further Reading:

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-J. Czaja 8-28-02

HOPES interview notes
Dr. Ron Kopito and Dr. Brigit Riley
April 5, 2006

In April 2006, Christina Chen and Justine Seidenfeld visited the Kopito Laboratory in the Department of Biological Sciences at Stanford University. HOPES would like to thank Dr. Ron Kopito and the members of his lab, particularly Dr. Brigit Riley, for taking time out of their busy schedules to meet with us, and share their perspectives on Huntington’s Disease research.

Introduction

In a cell, proteins are constantly being produced or destroyed in order to ensure that a healthy balance is sustained. Many neurodegenerative diseases are associated with an imbalance of proteins. They often involve mutated or misfolded proteins that are toxic to the nerve cell. The Kopito lab looks at one of the most well-known systems for clearing out misfolded proteins, the proteosome. The proteosome is a protein complex that digests unwanted proteins into short chains of amino acids. The proteosome has many roles in the cell and plays a part in many processes, such as the cell cycle, communication between proteins, and the production of proteins. Research about the proteosome is an increasingly important part of our understanding of disorders involving misfolded proteins like HD.

Becoming involved in HD research

When asked how he became interested in HD research, Dr. Kopito acknowledges that he has a different background than most scientists currently studying the disorder. He was originally interested in researching Cystic Fibrosis (CF)- a genetic disorder that, like HD, is associated with misfolded proteins. The most common symptom of CF is difficult breathing, which is caused by lung infections that can be treated (but not cured) by antibiotics. There are other symptoms, including sinus infections, poor growth, diarrhea, and infertility.

A study using a model of CF demonstrated that, by impairing the proteosome, mutant CFTR proteins were not broken down and recycled. Instead, they tended to form protein aggregates. This finding indicates that the proteosome plays a key role in delaying or preventing diseases involving misfolded proteins, like CF and HD. If something goes wrong with degradation, misfolded proteins accumulate in the cell and cause problems.

If the proteosome becomes impaired, misfolded proteins tend to form concentrated clusters of proteins aggregates known as inclusion bodies (IBs). IBs were initially discovered in experiments in the Kopito Lab. At first researchers did not know if IBs occurred naturally in a diseased cell. It was possible that they only appeared because of laboratory conditions used for the experiment. To make sure that IBs occurred in the normal course of a disease involving misfolded proteins, Dr. Kopito and his coworkers looked at other diseases that were similar to CF. He wanted to see if these disease also involve the formation of IBs from mutated, misfolded proteins. Kopito’s group discovered that in addition to CF, most neurodegenerative diseases involve proteins that misfold and form IBs. So, the Kopito lab concluded that IBs were a real part of diseases involving protein misfolding.

Next, they looked closer at the IBs to see what other kinds of proteins were found in addition to the misfolded disease-related protein (i.e. huntingtin or CFTR). They found that another protein called ubiquitin (Ub) was always present. Ub is one of the proteins in the proteosome protein complex. If Ub is found trapped in IBs, it means that Ub cannot work with the proteosome to degrade proteins. Therefore, the proteosome cannot function properly. In this case, proteins in the cell that need to be degraded and cleared (like altered huntingtin) will not be. These proteins can then interfere with other parts of the cell that would otherwise be functioning properly.

Getting involved in the HD community

For some time, the Kopito lab was very interested in studying IBs in general. They were not interested in how IBs were related to specific diseases (like HD), since they were found in so many different genetic disorders. Rather, the lab was using lots of different disease models to study IBs- from Cystic Fibrosis, to HD, and more. But in 1998, the Huntington’s Disease Society of America (HDSA) asked Dr. Kopito to give a talk at a conference on his work with IBs. He went to several of their meetings, met many researchers and patients who were part of the HD community, and found the HDSA to be a “cohesive, vibrant organization”. Dr. Kopito says that the HD community and the HDSA are remarkably well-organized and mobilized, much more so than many organizations involved in advocacy for other disorders.

At these HDSA meetings he saw that many people, scientists and non-scientists alike, attended and participated. The HDSA gives grants for research, and encourages scientists to become involved in the advocacy and politics of HD. When he saw how patients, their families, and advocates supported scientists involved with HD research, Dr. Kopito became very motivated; a few years later, he decided to concentrate on HD more seriously. He has been focusing his research on the role of IBs and the proteosome in HD ever since.

The “top-down” approach

On a basic level, it is known that HD is caused by a mutation in huntingtin that causes it to gain a new toxic function and harm the cell. But, as Dr. Kopito explains, there is not a single, direct route from the genetic mutation to the disease. Rather, the mutated huntingtin protein has different effects all over the cell. Dr. Kopito feels that when studying HD, it is hard to start by isolating each of the possible small problems that may be caused by the mutated huntingtin protein. Starting from the endpoint of the disease cascade will not provide much insight into understanding the disease. Rather, you have to study the disease as a whole to gain insight into each of the small steps, and how they interact with one another. This approach would be considered a top-down approach. So, Dr. Kopito likes to use animal models like mice to study the disease, rather than looking at HD only in tissue culture or in vitro. That way, he can look at the whole disease in the context of an animal, rather than just a fraction of it in the context of a test tube or Petri dish.

Current research projects

Dr. Kopito mentions two current projects in his lab that he finds particularly exciting at the moment. The first project involves looking at the timing and appearance of inclusion bodies in HD. Dr. Kopito says he believes that to understand how IBs play a role in HD, he first has to know at what time point they appear over the course of the disease. When Ub accumulates and is incorporated into IBs, it indicates that something is wrong with the proteosome. Dr. Kopito thinks that this happens because at some point over the course of the disease, the proteosome just stops working. All of the extra Ub that should have been degraded needs somewhere to go, and it gets incorporated into an IB. But for Dr. Kopito, the question is when does that occur? When does the proteosome stop working? For more on the proteosome, UB, and HD click here and here.

To answer this question, the Kopito lab is using a model system with mice that have HD. They take samples from the brain tissues of these mice over time and test how well the proteosome is functioning at each time point in order to find the time when the proteosome stops working. Dr. Kopito says that the data from this experiment is forthcoming, and so they should have results to further investigate. He is pleased that he will be able to tell if his pet hypothesis (that the proteosome shuts down early and is important in the onset of HD) is right or wrong. Either way, it will give him a great direction in which to move his research.

Dr. Kopito’s second project is in partnership with a local biotechnology company. Together, they are trying to find biomarkers for HD. A biomarker for HD would be a type of molecule that indicates when neurodegeneration has started. A good marker could be a molecule that has a recognizable change in concentration or structure when neurodegeneration begins. It would be nice to have a biomarker for HD so that you do not have to wait for the motor, behavioral, or cognitive symptoms to appear to know when neurodegeneration has begun. (For more on the symptoms of HD, click here). Biomarkers would allow doctors and scientists to intervene in the course of the disease early on, before it is too late. Ideally, if you could test a blood sample and find a molecule that indicated that neurodegeneration had begun, it would be an important step in being able to test drugs and therapies.

This second project is actually closely related to the first project. Dr. Kopito hopes that the proteosome, or a piece of the proteosome, might be a good biomarker molecule for HD. If the experiments described for the first project prove that the proteosome stops functioning early in course of the disease, it could serve to indicate when it is time to intervene before the motor, behavioral, or cognitive symptoms set in. However, right now, the only ways to test how well the proteosome functions is by doing a biopsy, which is a complicated and very invasive procedure. Dr. Kopito proposes that a cerebral spinal tap might be an alternative, simpler option to test how well the proteosome is functioning.

Autophagy, or “self-eating”

While Dr. Kopito’s lab focuses mainly on the role of the proteosome in HD, some members of his lab look at other mechanisms that the cell uses to break down and clear out unwanted proteins. Christina and Justine met with Dr. Brigit Riley, a post-doctoral student in the Kopito lab who studies a process known as autophagy, and how it is involved in HD. Autophagy literally means “self-eating”. In autophagy, the cell’s membrane encircles organelles, proteins, and parts of the cytoplasm into spheres called vesicles. These vesicles are sent to a part of the cell called the lysosome to release their contents. Then, proteins in the lysosome degrade the contents of the vesicles . See Figure 1 below). Both the proteosome and autophagy are methods the cell has to recycle proteins. But, there are some important differences between the two processes. While the proteosome can selectively target certain proteins that are supposed to live for a short period of time, autophagy is used to digest large organelles and long-lived proteins, and it cannot select for certain proteins.

Figure 1

Autophagy has many purposes in the cell. During development, it helps the cell take up amino acids. If the cell is starving and does not have enough nutrients or energy, autophagy is used to recycle unnecessary proteins to make more important proteins. Autophagy can also be used to defend the cell against invading bacteria or viruses. It is also thought to play a protective role against the progression of human diseases like cancer, Alzheimer’s disease, Parkinson’s disease, and HD. (For more on other neurodegenerative and related diseases, click here.

Autophagy and HD

There have been many scientific models recently developed to look at the role of autophagy in HD. Scientists think that autophagy is triggered to help protect the cell when the proteosome system is overwhelmed by too much aggregated protein. That is, the proteosome is the first line of defense, whereas autophagy is the second line of defense against altered huntingtin protein.

In this case, the proteosome can deal with small huntingtin aggregates by selectively targeting them for degradation. However, when there are too many of these misfolded proteins, the system gets overwhelmed. Then, the excess of misfolded proteins are shuttled along the microtubules (which are the tracks that allow protein to move across the cell) to form clumps of misfolded proteins called aggresomes (the name for IBs found in the cytoplasm as opposed to in the nucleus of the cell). Logically, the cell would form aggresomes as protection because it is much easier to autophage large clusters of altered huntingtin proteins than small proteins floating all over the cell.

However, there are still a lot of questions surrounding this process. We do not know if only the aggresomes are degraded by autophagy. It could be that any altered huntingtin proteins that are not incorporated into aggresomes are also autophaged. We also don’t know if the molecular signals that activate autophagy to degrade aggresomes also trigger autophagy for other purposes, like cell development or cell starvation.

Autophagy research in the Kopito Lab

Dr. Riley looks specifically at the role of the microtubules in triggering autophagy. Microtubules may assist the fusion of the vesicles and the lysosome to form the active autolysosome that carries out autophagy. Dr. Riley believes that this is the link between microtubules and autophagy. In fact, experiments have shown that mutating the microtubules to disrupt their structure interferes with autophagy.

Researchers do not know for sure that autophagy is completely protective for the cell. It is possible that aggresomes activate autophagy, but instead of only targeting the aggresomes, autophagy targets everything in the cell. In this case, autophagy would be blindly degrading essential proteins and organelles along with the aggresomes, thus harming the cell. Autophagy would be a good temporary solution, but bad for the cell in the long run.

Dr. Riley also suggests that when aggresomes get degraded during autophagy, the resulting fragments of altered huntingtin protein act as the toxic molecule that harms the cell. Another possibility is that the aggresomes clog the lysosome, preventing it from acting normally in other parts of the cell. As she says, there are a lot of options to explain how autophagy functions in HD. However, autophagy research is relatively new, so a lot remains to be done.

There are a lot of experimental models used in the lab, and Dr. Riley mentions a specific tissue culture cell line that comes from mouse nerve cells. These cells have the altered huntingtin protein, and it is under the control of a “conditional promoter”. By adding or taking away a certain chemical called tetracycline, researchers can turn the production of the altered huntingtin protein either on or off at will. This is a very powerful model system because it allows you to produce enough altered huntingtin protein for HD to begin. Then, you can turn off production of the protein, and look at how autophagy is clearing it out of the cell. Dr. Riley particularly likes working with cell lines like these. She thinks it is a good model system for preliminary studies before taking her findings and applying them to more complex models, such as mice. But she has a few concerns. She recognizes that mice nerve cells do not behave the same way as human nerve cells, so she wonders if findings from model systems like cell lines and mice will apply to humans well.

More on Dr. Riley

So how did Dr. Riley become interested in studying HD? As an undergraduate, she started as an organic chemist, working in a lab that studies nitric oxide synthase in Parkinson’s and Alzheimer’s disease. Both of these are neurodegenerative diseases that have many similarities to HD. At the time, she was also volunteering in a local nursing home, and began to interact with patients with the very same neurodegenerative disorders she was studying in the lab. As a graduate student, she decided to study biochemistry. She began looking at the role of the proteosome in spinocerebellar ataxia, which is a neurodegenerative disorder associated with expanded polyglutamine chains, like HD. Specifically, she was looking at a set of proteins that acted as an alternative to ubiquitin. This line of inquiry led to her interest in autophagy as an alternative method of recycling proteins compared to the well-studied proteosome system. Since then, Dr. Riley has switched model systems and is now studying HD, but she plans to always stay in the area of science devoted to studying neurodegenerative disorders.

Who does Dr. Riley find to be an inspirational scientist? She first identifies Dr. Yee Yamamoto, who created the first tetracycline-regulatable model system for an HD mouse. This cell line allows scientists to control when the altered huntingtin protein is produced in the mice, and when to turn off its production. Many other scientists applied this idea to other model systems, including the tissue culture cell line that Dr. Riley mentioned using earlier.

Life as a post-doctoral student

Dr. Riley came to the Kopito lab as a post-doctoral fellow. She entered the lab with a few other scientists who were interested in working on autophagy, so she had a new group of people to collaborate with on projects. She found that the Kopito lab is unique because it has a good environment and good energy. Everyone works together, especially within the group of graduate students and post-doctoral fellows working on autophagy. She also notes they are unusually dynamic, and they like to act on an idea quickly rather than wait around. She also comments that Dr. Kopito is great about helping his graduate students and post-docs to act quickly on a project idea by supplying them with necessary equipment, materials, and advice.

As a post-doctoral student, Dr. Riley finds that it is a challenge to balance bench work and experiments with meetings, writing papers, meeting people, and making contacts, all of which are important parts of a post-doctoral student’s work. She found that it was not as hard to balance responsibilities as a graduate student, but there are more tasks and more pressure now. She also wants to stay on top of current research about HD and autophagy, but finds it more difficult than she expected. Because there are so many different approaches to the subject, it is hard to know what is going on in the field. Furthermore, the lack of communication between other scientists in the field makes it hard to design experiments.

When asked if her fellow researchers at the Kopito lab have focused their goals on contributing to a cure for HD, or rather on the joy of scientific discovery, she says the Kopito lab is a combination of attitudes. Her old lab (where she did her graduate studies) focused on either curing or understanding the initial steps of HD. At the Kopito lab, there is more of a balance between both attitudes. Personally, when she thinks only about the joy of discovery or the intellectual pursuit of understanding HD, she becomes concerned that it isn’t really relevant to the people who are affected by HD. She thinks that scientists are really far removed from the fact that this is a fascinating scientific question, but also involves patients and their families.

For Dr. Riley, the excitement in studying autophagy is in the hopes of “find[ing] a drug that would promote it”. But it also prompts questions about the nature of HD treatment- do people want to take a drug treatment for their whole lives? Is it too inconvenient or difficult of a method? Optimistically, she says, maybe this drug approach will be used to treat symptoms starting in 5 to 10 years, but it would be nice if gene therapy was possible. If they were developed around the same time, it would give people options in how they want to be treated, which she believes is an important aspect of scientific research and medicine.

Dr. Kopito, HD research, and the public

When asked what the most rewarding parts of research are to him, Dr. Kopito stressed that he most enjoys the satisfaction of discovering and understanding something new. In his role as a professor and researcher, he is also a mentor to the graduate students and post-doctoral students in his lab. He emphasizes how much he enjoys teaching and nurturing other people’s sense of discovery. But Dr. Kopito further elaborates by saying that his research program is not intended to directly cure HD, but rather to further understand the background and the cause of the disease. As he explains, he finds HD to be a “compelling medical problem, an interesting scientific problem, and really, a human problem”. But when asked if he hopes he is making an impact on lives, he recognizes that his role is indirect and not concrete. While he feels he is not directly accountable to HD patients, he also recognizes that scientists have to be responsible to the HD community. Scientists have to spend any grant money on research that will get scientists or doctors closer to a place where they can find a treatment.

This line of questioning raises many ethical questions for Dr. Kopito. He wonders- what if it turns out that inclusion bodies are not the problem to target in HD? Can he still work on studying IBs in other diseases, because he finds it to be interesting and compelling research? Or would it be more ethical to switch the focus of his research, and look at other mechanisms that may be involved in causing HD? He thinks about these questions a lot, meeting with other people to talk about inspiring new ideas within the group of researchers working on HD.

Finally, Dr. Kopito emphasizes that the biggest myth about research is that it proceeds in a very dry, set manner from hypothesis to experiment to truth. The public does not often get to see how qualities like intuition, passion, and creativity are involved in conducting research. If scientists were to fit the cold, objective stereotype, there would be no important discoveries. Rather, when scientists get ideas, some people see it as crazy risks, but they have to have enough faith in their own ideas to work on it, and they have to convince other fellow scientists and students to take risks along with them. Scientists have to design an experiment with objectivity and creativity. An experiment must be designed to test, not prove, a hypothesis- scientists must be willing to acknowledge when they are wrong or their hypothesis is incorrect. But, that can be hard because the ego gets wrapped up in it. Throughout their careers, scientists have to utilize ego, passion, commitment, and risk to conduct experiments. For Dr. Kopito, research is a vital, dynamic profession.

For further reading

• Find the Kopito Lab at: http://www.stanford.edu/group/kopito/
• Debnath, J, et al. “Does Autophagy Contribute to Cell Death?” Autophagy 2005. 1 (2): 66-74.
  A good review of autophagy, not too technical language. Discusses roles of autophagy in both cell death and cell survival.
• Ross, CA, et al. “What is the role of protein aggregation in neurodegenation?” Nature Reviews Molecular Cell Biology 2005. (11):891-8.
  A more complex review of disease involving protein aggregation, inclusion bodies, and the roles of both in causing diseases or acting as a protective response.
• Yorimitsu T, et al. “Autophagy: molecular machinery for self-eating.” Cell Death Differ.2005. 12 (2):1542-52.
  A technical paper on autophagy, how it works, and the various roles it plays in the cell.
• Iwata, A, et al. “HDAC6 and microtubules are required for autophagic degradation of aggregated huntingtin.” J Biol Chem. 2005. 280(48):40282-92.
  A technical research paper on the role of microtubules on forming aggregates and how they participate in forming vesicles and activating the lysosome.
• Levine, B, et al. “Development by self-digestion: molecular mechanisms and biological functions of autophagy.” Dev Cell. 2004. 6(4):463-77.
  A good review of autophagy that goes into a bit more detail about the sets of genes involved in autophagy and different analogous genes in model organisms.
• Kopito RR. “Aggresomes, inclusion bodies and protein aggregation.” Trends Cell Biol. 2000. 10 (12):524-30.
  A review that clarifies the similarities, differences, and relationship between aggregates, IBs, and aggresomes.

Dr. Rick Morimoto
Department of Biochemistry, Molecular Biology and Cell Biology
Northwestern University
Evanston, IL

Introduction

In November 2005, HOPES team member Justine Seidenfeld visited the Morimoto Laboratory in the Department of Biochemistry, Molecular Biology and Cell Biology at Northwestern University’s graduate school in Evanston, IL. HOPES would like to thank Dr. Morimoto and the members of his lab for taking time to meet with us and share their outlooks on research and Huntington’s Disease.

The Morimoto Lab studies how cells protect themselves by sensing and responding to environmental and physiological stress. These kinds of stress often lead to damage and problems within the cell. Stress can come from a wide variety of sources, like oxidative damage, over heating (heat shock), bacterial infections, and misfolded proteins (For more on proteins and their folding structure, click here) The cell has many different ways of recognizing and responding to stress induced problems. Under normal circumstances the cell can maintain a balanced state known as homeostasis. When stress causes proteins to misfold, the cell responds through a mechanism called the heat shock response. This response increases the amount of molecular chaperones and proteases produced within the cell. Molecular chaperones and proteases correct misfolded proteins, repair damage, and bring the cell back to balance. Sometimes, however, there are so many misfolded proteins that the heat shock response cannot keep up. The misfolded proteins accumulate into protein aggregates. Aggregates are a hallmark of neurodegenerative diseases like HD.

Approaching HD

The Morimoto Lab studies the cell’s response to the stress-related buildup of misfolded proteins within the cell. These researchers are attempting to understand the mechanisms that lead to the appearance of misfolded proteins in the cell in the first place. They also study how these misfolded proteins affect the heat shock response, and the consequences for the cell’s overall health.

The Morimoto Lab approaches these questions from a variety of angles. They have pioneered the use of a roundworm, or C. elegans, model to study HD. They created a mutant of C. elegans which carries the gene for an expanded polyglutamine chain protein that is characteristic of HD. Furthermore, they have shown that the polyglutamine chain by itself is toxic, not only when it is part of an altered huntingtin protein. (For more on the structure of the altered huntingtin protein, click here) They have also conducted a genetic screen of the entire genome of these nematodes to determine what other proteins interact with the polyglutamine chain protein, and find out how to suppress polyglutamine protein aggregation. Among the 186 proteins identified, many are molecular chaperones involved in the heat shock response. So in order to understand exactly how altered huntingin protein aggregation occurs in the cell, the lab is studying the interaction between molecular chaperones like Hsp70, its co-chaperone Hsp40, and aggregates .

Research

Dr. Morimoto’s lab looks at HD from the very beginning of the disease cascade. He describes the disease cascade process like this: a protein with an expanded polyglutamine chain (which is toxic) impairs cells and their function over time, eventually leading to nerve cell death. His lab studies the beginning of the cascade, asking the question “What goes wrong in protein quality control?” Dr. Morimoto believes that protein quality control is one of the cell’s most ancient mechanisms. In the very first forms of life, RNA was the main hereditary genetic material in cells, instead of DNA as it is now. In this “RNA world,” cells were much less complex. In order to survive all of the sources of stress they were subjected to, they must have had very good protein quality control mechanisms. These mechanisms have been preserved in the “DNA world” of the present day. Additionally, the question of protein quality control is especially intriguing because protein misfolding is common among neurodegenerative diseases, HD included. Dr. Morimoto’s findings have the potential to be useful in the study of other neurodegenerative diseases. “The implications are broad,” he says of his research program.

Why does Dr. Morimoto focus on HD if is research is so broad? As he puts it, “HD is a leader as a genetic dominant.” As opposed to some other neurodegenerative diseases, HD is a disease that is easy to identify by using genetics. The presence of at least one allele for the altered Huntingtin gene always indicates a person will have HD. The genetic link is not so clear in other neurodegenerative diseases. For example, only 2% of cases of amyotrophic lateral sclerosis are actually related to an inherited genetic mutation. These cases are called Familial Amyotrophic Lateral Sclerosis, or FALS. In short, using HD to study protein quality control is valuable because it has straightforward genetic links, and it has broader implications for other neurodegenerative diseases.

The Morimoto Lab has also created C. elegans models related to tau pathologies, amyotrophic lateral sclerosis, and other polyglutamine diseases besides HD. All of these models help to understand the genetic basis of protein misfolding by comparing the different molecules associated with each of these diseases. In addition to C. elegans, the lab uses use in vitro studies and tissue culture (cells grown in a Petri dish), among others as model systems to answer these questions. Dr. Morimoto describes these multiple approaches as “the only way to run a lab.” These different models allow the lab to run multiple tests on hypotheses, so they can be much more confident about their results. For example, Dr. Morimoto says that there are certain advantages to doing experiments with tissue culture, but that they need to be verified in studies using whole organisms. In doing this, they can see if they get the same in an entire living animal, not just isolated tissue in a Petri dish, and the C. elegans model is a good way to test any hypothesis in his lab.

Dr. Morimoto also discusses the pharmacological studies done in his lab. In these studies, the researchers discover small molecules involved in protein folding. These molecules could serve as a basis for therapeutic drugs or therapy treatments. In a large collaborative effort with 26 other laboratories worldwide, the Morimoto lab helped identify a small molecule called celastrol, from a plant often used for its anti-inflammatory properties in Chinese herbal medicine. Celastrol was identified because it can activate important genes involved in the heat shock response and elevate the level of molecular chaperones in the cell. These molecular chaperones could help protect the cell from the damage caused by misfolded proteins. Members of the Morimoto lab are now working with Dr. Richard Silverman, a professor in the Department of Chemistry, to discover the specific structure of celastrol and how it works with other proteins to activate the heat shock response. Dr. Morimoto believes a detailed understanding of exactly how celastrol works is important before it is turned into a therapy drug and put into clinical trials. However, the potential of celastrol certainly seems promising.

HD Research and the Public

Dr. Morimoto discussed his views on the responsibilities of a scientist to the public. “It is our responsibility and obligation to talk to the public about research,” he says. Dr. Morimoto wants patients and their families to understand how research on neurodegenerative disorders works: how scientists pose questions; how drugs and therapies are created; and why animal models like flies, worms, and mice, are used instead of humans at certain stages of the research process.

Dr. Morimoto explains that using a model system like C. elegans is helpful because this organism has a lot of important similarities to humans, but also differences that make them easier to study. C. elegans has a much simpler nervous system than humans. C. elegans has only 302 nerve cells, as compared to the human nervous system which can have 10 to 100 billion nerve cells. Although the C. elegans nervous system is less complex than ours, each individual nerve cell (as opposed to the whole system) is just as complex as a human’s nerve cell. C. elegans is good in vivo model for the Morimoto Lab, because they are focusing on protein balance and health within the individual nerve cell rather than an entire nervous system.

Dr. Morimoto believes that he and other academic scientists are fortunate because they can choose to focus their research on whatever interests them. They can be well-funded, but that gives them an obligation to educate people on how their money is being spent. Yet not all scientists are trained to do this. Dr. Morimoto believes that more effort is needed to increase communication between laboratory researchers and the public.

Dr. Morimoto is one of the 17 Huntington’s Disease Society of America (HDSA) Coalition for the Cure Senior Researchers. He is committed to long-term HD research and collaborates with other scientists around the world who also study HD. Through the local chapter of the HDSA, HD patients and their families often contact him, and he will sometimes bring them to his lab to meet the postdoctoral fellows, graduate, and undergraduate students working to discover the mechanisms behind HD. Dr. Morimoto says that it helps him get a sense of clarity apart from his research, and he thinks that it is equally beneficial to the patients and their families. He also attends Illinois HDSA meetings to give talks. Since he knows that patients and families are always at these meetings, he tries to make his talks especially relevant for them. One of the great things about science, he says, is that all scientific information is accessible. And the local chapters of the HDSA get scientists to appreciate the public, to make sure that information reaches them in an appropriate way. To learn more about the HDSA, click here.

A unique feature of HD is that it is relatively rare compared to some other neurodegenerative disorders like Alzheimer’s and Parkinson’s (for more information on these diseases, click here). Because less people are affected by HD, there is less funding from the National Institutes of Health (NIH) devoted to HD. Dr. Morimoto says that the community of HD research advocates is amazingly strong and plays a big role is raising necessary money. As an example, he cites the Fiore family of Highwood, IL. The Fiores have a history of HD in their family, and have raised money for HD research by partnering with the HDSA and holding their own fundraising events like golf and poker tournaments (you can visit their site by clicking here). Their work has helped to raise money to open an HDSA Center of Excellence at Rush St. Luke Medical Center in Chicago. According to Dr. Morimoto, families like the Fiores have single-handedly used their personal adversity to “increase awareness of the disease for all.”

More on Dr. Morimoto

Dr. Morimoto received his Ph.D. in molecular biology from the University of Chicago in 1978. He says that he became interested in his line of research when, as a graduate student, he heard a talk by Dr. Matthew Meselson of Harvard University. Dr. Meselson spoke about the newly discovered heat-shock response system in fruit flies. Dr. Morimoto was intrigued by this talk, and did his postdoctoral fellowship at Harvard in Dr. Meselson’s lab. While he was there, he was the first to clone the human Heat-Shock Protein 70 (Hsp70) in 1985, This means that he was the first to discover the location and sequence of the gene that codes for the protein. Now it could be used in genetic studies related to the heat shock response. Hsp70 was identified as a key protein in protein quality control. Multiple studies showed that by making an unusually high amount of Hsp70 in cells prone to protein misfolding, it would suppress the toxic effects of these misfolded proteins. Dr. Morimoto came to Northwestern University in 1982 and continued his research on the heat shock response in neurodegenerative diseases. As he puts it, the 1970s and 80s were full of important discoveries about the regulation of protein folding related to the heat-shock response. The 90s brought increasing awareness that neurodegenerative diseases were related to the misfolding of protein aggregates. Seven years ago, his lab began to use C. elegans to study the heat shock response. They began to also study HD in C. elegans, and this is a major portion of his current research. Dr. Morimoto says that his interest in science stemmed from, and continues to be, a desire for pure knowledge, to understand the natural world.

Future of HD Research

What are some of the greatest obstacles facing the community of researchers studying on HD? Dr. Morimoto believes that one of the most pressing questions involves the use of model systems like C. elegans, mice, or tissue culture to study HD. We can certainty understand the fundamentals of protein folding, aggregation, toxicity, and nerve cell death by using these model systems. However, to what extent can we take these fundamental discoveries and apply them to humans- a vastly more complex creature than mice, worms, or yeast? Dr. Morimoto believes that discoveries in model systems will prove to be useful and important because the machinery behind protein quality control is an ancient and conserved process. He believes that most of the discoveries made in less complex model systems will probably also be found in humans. However, he is concerned that researchers will have to take into account the fact that humans have very complex systems (such as human metabolism) when developing therapies. Another obstacle will be that of developing effective drug therapies. He explains that there has been limited success in discovering which proteins to target with therapeutic drugs . One in one hundred options may work, but there are not yet one hundred likely targets. In order to discover new targets, Dr. Morimoto argues that there must be more consensus among different branches of the scientific community. He strongly believes that when research laboratories partner up, they are much more effective and successful than when they work on their own.

Dr. Morimoto emphasizes that, among the myths of research that he would like to dispel, research labs like his are wonderfully diverse in their scope of cultural backgrounds, scientific experience, and intellectual perspectives. He stresses that the lab environment can be a very cooperative and social one, as opposed to the stereotype of fierce competition. Diversity and collaboration are important characteristics for successful scientific research.

-J. Seidenfeld, 3/14/06

Dr. Diana Rosas
Massachusetts General Hospital
Charlestown, MA

Dr. Rosas is a clinician who treats people with HD. The complexity and mysterious nature of the illness inspired her to get involved with Dr. Nancy Wexler’s Venezuela project in the early 1980s—a genetic study of large families that were all descended from a single ancestor with HD. The goal of the project was to generate a precise map of genetic markers.

Since then, Rosas has dedicated her life to treating the symptoms experienced by people with HD, as well as to investigating the effects of HD on the brain. In contrast to researchers like Drs. James Gusella and Marcy MacDonald (please see Chapter 1, Part 1 of Research Frontiers who direct their efforts toward the root causes of HD and the beginning of the disease cascade, Rosas and other clinicians focus on the end of the cascade, in which motor, cognitive, and psychiatric functions begin to decline. Rosas defines the age of onset of HD as the age when motor symptoms (irregular eye movements, chorea, etc.) appear. Interestingly, some people are able to suppress their chorea during a doctor’s examination, and, as a result, their HD goes undiagnosed for a long period of time.

Rosas also discussed the unique onset of juvenile HD. Children with greater than 45 CAG codon repeats in the Huntington gene have an earlier and much more severe onset than people with traditional HD. While these children experience less chorea than their adult counterparts, they often have hallucinations and symptoms that resemble those of Parkinson’s disease.

Although she acknowledges that it is important for clinicians to concentrate on the striatum, where neurodegeneration starts, Rosas mostly takes a holistic approach to the brain. She emphasizes that the entire brain, not just the striatum, deteriorates as a result of HD.

“The motor cortex is affected across the board,” said Rosas. Located on the frontal lobe, the motor cortex carries out the initial processing of motor information in the brain and is involved in the control of movement.

The earliest changes in the brain occur in the posterior regions (the parietal, occipital, and temporal lobes). During the course of the disease, however, a person experiences large reductions in volume in almost all areas of the brain. For example:

To learn more about the different parts of the brain, please click here.

The treatment of HD requires an integrated, multidisciplinary approach that includes the medical management of symptoms; physical, occupational, and/or speech therapy; and genetic and psychological counseling. In addition to treating people who already have the disease, Rosas counsels people who are at risk for developing it. While the decision to undergo genetic testing is a very personal one, Rosas does recommend it for those who are planning to have children.

She encourages all others at risk not to undergo testing for several reasons. First, there is no cure for HD. Second, insurance issues and genetic discrimination in the workplace may result if an at-risk person finds out that he or she is likely to get the disease. Third, knowing that one is likely to get HD would cause unnecessary worry. He or she would constantly wonder, “When will I get sick?” Fourth, even negative test results may cause emotional distress, such as “survivor’s guilt” if one’s parents died from HD.

Finally, Rosas strongly advocates seeing a doctor for a complete neurological examination as soon as one notices the first physical symptoms of HD. She advises people not to wait. A doctor is the best judge of whether or not someone has the disease. Sometimes, though, people do experience sexual, mood, and marital problems even before the onset of motor symptoms. These problems are symptoms associated with the psychosocial part of the disease, and are most common in men. Many psychosocial symptoms can be treated by clinicians and psychologists.

For those who have been diagnosed with HD and are interested in participating in clinical trials, Rosas recommends visiting the website of the Huntington Study Group. Among its many resources, the website provides information about clinical trials in progress and new clinical trial initiatives.

-T. Altman, 5/5/06

Drs. James Gusella and Marcy MacDonald
Molecular Neurogenetics Unit, Massachusetts General Hospital
Charlestown, MA

Introduction

This summer two HOPES team members, Taylor Altman and Shawn Fu, spent several days with Drs. James Gusella and Marcy MacDonald at their laboratories in the Molecular Neurogenetics Unit of Massachusetts General Hospital (MGH) in the Boston area. HOPES would like to thank Gusella and MacDonald for taking the time to share many valuable insights with us.

The Molecular Neurogenetics Unit, under the direction of Gusella, is dedicated to using molecular genetic techniques in humans and mice to understand and treat neurological disorders. Gusella and his colleagues pioneered the use of DNA sequence polymorphisms as genetic markers and mapped the Huntington gene to chromosome 4 in 1983 (please click here for more information about this breakthrough). He and his fellow researchers have since applied this genetic mapping approach to numerous neurological diseases, including Alzheimer’s disease, amyotrophic lateral sclerosis (ALS), Batten disease, and familial dysautonomia (FD) in an effort to pinpoint the chromosomal locations of the disease genes and/or the nature of the genetic defect itself. In 1993, Gusella, MacDonald, and their colleagues, as part of an international collaboration, identified an expanded, unstable CAG trinucleotide repeat in a novel gene as the cause of Huntington’s disease.

Both Gusella and MacDonald are currently trying to understand the pathogenesis of HD and find ways to interfere with the disease cascade in order to halt its progression. According to the webpage of the MacDonald laboratory, she and her colleagues follow three interrelated steps in order to investigate the complex biology behind HD and other neurological disorders:

1. The use of polymorphic DNA markers in genetic family studies to isolate gene defects that cause inherited disorders of the nervous system and test loci that alter the disease phenotype in some way.
2. The creation of model systems (such as the mouse model of HD) in order to understand and clarify the functions of the products (essentially, proteins) of the abnormal gene at the whole animal, cellular, and biochemical levels.
3. The use of model systems to explore novel gene therapy based strategies, which aim to intervene in the disease process.

MacDonald uses knock-in mice that carry human mutations, such as the gene defects in HD and Batten disease, to identify early cellular and molecular events in the disease pathways. She is also in the process of identifying cellular partners (the parts of the cell that a chemical or molecule interacts with) and pathways for the HD and Batten disease proteins, huntingtin and battenin.

The MacDonald lab

What’s special about HD research?

Altman and Fu sat down with Gusella and MacDonald to discuss their passion for investigating HD. What’s special about HD research? Both stated that they have the opportunity to do a great service for those with HD and, at the same time, learn about the biology behind the disease.

“HD, in particular, gets scientists interested,” said MacDonald. “What makes [HD research] fun is the discovery part, being in the middle of nowhere and finding new biology.” Take, for example, the mutant huntingtin protein: “We have no clue what it does, but we can connect it with old things and see things that are brand-new.” When studying the biology behind non-genetic (traditional) disease, researchers learn from what they already know, which is “not great for discovery,” according to MacDonald. Conversely, when studying HD, researchers do not know what they are looking for, which sets the stage for some amazing new findings.

“What’s disturbing is that the more you know about how something works, the less interesting it is,” Gusella said, pointing out a negative aspect of scientific discovery. “But working on HD is different”, MacDonald asserted. Unlike other degenerative disorders of the nervous system like Alzheimer’s and Parkinson’s disease, whose “ etiologies are myriad” (meaning that there is a wide range of possible causes), everyone who develops HD has the same mutation. The hypothesis that things will be found that counteract the effects of the single mutation in the single gene that triggers the disease in everyone is exciting to work on because it still poses new and interesting questions and challenges for the scientists who work on it. (For a comparison of Alzheimer’s, Parkinson’s, and HD, please click here.)

Although MacDonald, Gusella, and their colleagues are interested in many aspects of the Huntington gene and related proteins, the overarching purpose of their work is finding a way to treat HD sufferers. “The ultimate goal of our research is a treatment, not understanding HD completely,” said Gusella. MacDonald added, “If you know how the disease starts, there will be a solution.” However, Gusella believes that an actual cure will not be possible until the HD allele vanishes from the population – something that is not likely anytime in the foreseeable future (he defines “cure” as the disappearance of the HD allele from the gene pool). (For more information on the distinction between a treatment and a cure, please click here.)

Conceptualizing HD

MacDonald drew a diagram of the disease (please see Figure AH-2: “HD pathogenesis and progression” below), its original CAG repeat mutation, and the age of onset. She and Gusella are currently looking for genes that interact with the original mutation. They conceptualize the disease in the human body as a series of steps in time from the HD CAG mutation to the onset of nerve cell death, to clinical symptoms and then the progression of the symptoms with factors that can effect each step (modifiers) determining the rapidity of each step and the age of onset of symptoms. Their labs are working to produce information about the genetic factors that determine the age of onset in the HD-MAPS (HD Modifiers in Age of Onset in Pairs of Siblings) study, coordinated by Dr. Richard H. Myers of the Boston University School of Medicine. By understanding the biological modifiers of onset, researchers may be able to develop methods to delay the onset of HD. Similarly, in the Huntington’s Disease PREDICT-HD (Neurobiological Predictors of Huntington’s Disease) study, conducted by Dr. Jane S. Paulsen of the University of Iowa, individuals who are known to have the gene expansion for HD are continually being recruited for a brief study examining changes in the DNA (genetic factors) that influence the age at onset of the disease.

Figure AH-2: A conceptual diagram of HD

Approaching HD

How do scientists go about studying HD? Gusella and MacDonald explained the approach they take to their research. They look at HD from a genetic angle, choosing to study the basics – in other words, the root cause(s) of the disease – not just the symptoms and pathology (the clinical aspect that interests doctors). If we envision the disease cascade as a timeline, Gusella and MacDonald work at the very beginning, understanding the various mechanisms that trigger the disease, including the early biochemical and metabolic changes that take place. Clinicians, on the other hand, work near the end of the cascade, looking not at the cause(s) of the disease, but rather at the consequences (the movement, cognitive, and psychiatric symptoms). Whereas clinicians generally try to intervene toward the end of the disease cascade with drugs or other therapies, Gusella and MacDonald attempt research that aims to intervene at the start of the cascade via experimental methods on the level of RNA, most notably RNA interference (RNAi), which will be discussed later. The researchers even anticipate that someday they may be able to change the slope of the cascade so that the age of onset is later and the symptoms are less severe. (As you may already know, the later the age of onset, the less severe the symptoms.)

Genetic disease is fundamentally different than non-genetic disease in that it has a definite starting point, even if symptoms are not yet apparent. In contrast, non-genetic disease usually has apparent symptoms that clinicians try to block, but not always an easily identifiable starting point. In the study of HD, researchers like Gusella and MacDonald have the broadest definition for the “disease” part of Huntington’s disease. They define it as the genetic mutation present at the time of conception all the way to the last stages of the cascade near the end of the person’s life. Neuropathologists (professionals who study diseases of the nervous system) define the disease as nerve cell death. Clinicians, who have the narrowest definition, consider it to be the visible symptoms in the last stages of the cascade.

MacDonald argues that huntingtin protein aggregation and eventual nerve cell death are not the causes of the disease, but rather the consequences. She and Gusella, therefore, strive to discover the things that happen before, without worrying about integrating the results of their experiments into the HD “story.” For a decade, the HD story has sounded the theme of huntingtin aggregation as a cause of HD. Here the term “story” describes a paradigm created by the scientific community – something of an umbrella idea under which many subsequent research findings huddle. While a story of this kind may one day be proven false, the scientific community has a hard time letting go or changing the paradigm. Similar to the HD story, the Alzheimer’s story emphasizes the buildup of amyloid plaques in the brain as the cause of the disease (the events that start the disease cascade). This story, too, may turn out to be inaccurate. Protein clusters may not cause either disease, but are considered to be the result of events that occur further upstream in the disease cascade. In other words, once the original trigger (as yet unidentified) sets off the disease cascade, it often takes a long time for protein clumps or tangles to form.

To hypothesize or not to hypothesize?

“The first step [of experimentation] is observation, not a hypothesis”, Gusella said. The scientific community has been so focused on having a hypothesis, he said, that getting funding without one was almost impossible because research projects that appeared purely descriptive (that is, projects that seemed to merely describe a phenomenon rather than answer a research question) were rarely awarded grants in the recent past. The scientific establishment’s penchant for storytelling may still prompt some researchers to formulate questions that they already know the answers to (“I can map and clone this gene”, for instance) simply to get funding for experimental research that may inhibit the careful observation necessary to formulate a meaningful hypothesis.

“Unfortunately”, said Gusella, “scientists must tailor their results to fit a hypothesis, which then must fit the overarching story that the research community is trying to tell.” This need for storytelling often results in unnecessary hype over a “rediscovered” discovery. For example, Alzheimer’s researchers have presented the same or similar findings about amyloid plaques over and over again, and the scientific community treats these findings as new discoveries. The way MacDonald views it, “It’s like mentioning Wilt Chamberlain to today’s great basketball players, and they say, ’Who?’”

Gusella and MacDonald think of science as a field in which researchers must continually build on the past, or else they run the risk of repeating the past (as in the Alzheimer’s example) or even losing it (as in the Wilt Chamberlain example). Gusella, though, cited an example of a scientific endeavor that did not have a hypothesis but was ultimately successful – the Human Genome Project, a thirteen-year effort coordinated by the U.S. Department of Energy and the National Institutes of Health to determine the complete sequence of the DNA in the human genome. The project gathered genetic information and developed a hypothesis later. Thankfully, said Gusella, the scientific community is beginning to come around; nowadays, more value is placed on descriptive research.

“Regardless of the type of research one is doing,” Gusella concluded, “the results can be interpreted in various ways, but one must select the correct interpretation.” Scientists must be willing to rule out certain hypotheses, as well as present failed hypotheses to their peers and to the public. Negative results are just as important as positive results because the data from a failed experiment may be useful in future research.

Accomplishments

Gusella, MacDonald, and their colleagues in the field of HD research have accomplished much in their careers. As mentioned earlier, Gusella mapped the Huntington gene to chromosome 4 in 1983. Dr. Gillian Bates of King’s College, London, cloned the tip of the chromosome, where the Huntington gene is located, and with Gusella and MacDonald, made a road map that pointed to the location of the gene. From there, researcher Christine Ambrose narrowed down the search for the gene to the two most promising segments of the tip, IT15 and IT16 (IT is lab lingo for “Interesting Transcript”). She discovered that these ITs were actually one gene, the Huntington gene. (Several neighboring ITs are now known to be inherited together with this gene.) Then, with her colleague Mabel Duyao, Ambrose made an assay that found that excess CAG repeats on the HD allele of the Huntington gene cause HD.

MacDonald got involved in the research effort in 1985, discovering new pieces of DNA on chromosome 4. In 1987, enough pieces of the chromosome were cloned to show various gene recombination events. Recombination events occur when genes lying farther apart on the same chromosome are often not inherited together due to the transfer of segments of DNA between homologous chromosomes during meiosis, the reproductive process by which sperm and egg cells are made. A haplotype study (a study describing the genetic makeup of an individual with respect to a specific pair of alleles or genes) was published in 1992. The objective of the study was to find out if the chromosomes on which the Huntington gene resides looked similar at a particular region in different people who had HD (in other words, the researchers wanted to know if the same mutation causes HD in everyone who has it). Through linkage studies (for more information, please click here), the researchers found that there was more than one mutation in ancestral populations (the predecessors of various groups of people who suffer from HD today). Interestingly, they also found the same mutation at the same spot in chromosome 4 in unrelated people with HD, so the same type of mutation in the same gene causes the disease in all people.

MacDonald’s Masterminds

Altman and Fu were fortunate enough to get a behind-the-scenes look at where HD breakthroughs are being made. They attended a lab meeting, where several of MacDonald’s postdoctoral (post-PhD) research fellows and interns gave presentations about their individual research projects. “Presenting research and sharing results is an important part of science,” said MacDonald.

Research fellow Julie Woda discussed her work on huntingtin normal function. Past research has shown that the protein is quite large and contains 36 HEAT-like repeats. The acronym HEAT comes from four proteins in which these repeated sequences have been found. These HEAT repeat sequences, which fold up into a spiral structure (alpha helix-loop-alpha helix), may serve as docking sites for other proteins. Most huntingtin partner proteins (“binding partners”) bind to one of its end segments. Based on its binding partners, huntingtin is involved in a variety of processes in the cell, including signal transduction, regulation of transcription (a big step in the process of turning DNA code into a protein), trafficking of molecules and other materials within the cell, maintaining the function of the cell’s cytoskeleton, and gene splicing.

Woda believes that the normal huntingtin protein is essential for the development of the human embryo. Studies conducted in the past have revealed that inactivating the Huntington gene (which produces the huntingtin protein) in a mouse results in abnormal embryo development. Supplying the mouse with either the altered (mutant) or wild-type (normal) huntingtin protein can rescue the non-huntingtin phenotype, meaning that the mouse can be brought back to a normal state. This phenomenon occurs because altered huntingtin acts like wild-type huntingtin, but gains some kind of new functions, such as a kind of “stickiness” that might make it more likely to interact inappropriately with another protein.

While touring the MacDonald lab, Altman and Fu spoke with some other postdoctoral research fellows about their research projects. Each trainee approaches HD from a different angle. For example, research fellow Elisa Fossale and intern Sony Mysore work together to prepare tissue cultures (a technique used to grow body tissue outside the body on a culture medium, a liquid or gel-like substance containing nutrients). The tissues kept in the culture medium are from the striatum of the brains of mice that carry the Huntington gene homolog (the mouse version of the gene). This brain region is known to deteriorate during the course of HD. The researchers are currently looking at differences in energy metabolism in mutant and wild-type striatal nerve cell clones. Preparing the cells for experimentation is a laborious process, generally taking about four to six hours. After this preparation, they can operationalize the changes in energy metabolism as changes in levels of the cell’s major energy carrier, ATP. These researchers have observed that ATP levels decrease in mutant cells, suggesting that metabolism is sluggish. Therefore, the cells are weakened through decreased efficiency in producing energy for their own survival. This information may be important to other researchers who use these types of cells for screening drugs that have the potential to combat decreases in energy metabolism.

Intern Sony Mysore learns how to perform a tissue culture.

HOPES team member Shawn Fu looks at striatal nerve cells under a microscope.

Other researchers and technicians work on a type of tissue culture called cell lines. Cell lines represent generations of a primary or original culture, such as a bacterial colony that arose from a single cell. Cell lines are “immortalized” biochemically so that they continue to reproduce themselves and can subsequently be used to develop tests called screening assays for potential drugs that are available from biotechnology companies and academic laboratories under contract. Once researchers have figured out the appropriate target for a drug (such as mitochondria that make ATP or nerve cell transportation machinery slowed by the effects of mutated huntingtin), they can test these chemical compounds to see which one of them really goes after the target.

Cell lines in cold storage in the MacDonald lab.

Alex Lloret, another research fellow in the lab, studies sections of the brains of transgenic mice with different genetic backgrounds to observe the formation of huntingtin in the nucleus and later formation of neuronal inclusions (NI) in the nuclei of nerve cells. Neuronal inclusions are clumps of mutated huntingtin protein fragments that result from having HD. There is generally one inclusion per nucleus in mutant mice (those with excess CAG repeats).

Lloret also looks for proteins that interact with the normal huntingtin protein. He has found that huntingtin’s function is similar to that of a scaffold, or facilitator, because it organizes groups of proteins that play various roles in signal transduction in nerve cells. He is currently in the process of mapping one end fragment of the huntingtin protein in order to understand how other protein complexes bind to it. He is also looking at HEAT repeat sequences in huntingtin and trying to determine how these sequences interact with many protein complexes.

Researcher Alex Lloret prepares sections of transgenic mouse brains for experimentation.

Sections of transgenic mouse brains are mounted on slides for viewing under a microscope.

Other methods of investigating HD include the genomic, RNA interference (RNAi), and biochemical approaches. The genomic approach entails screening various genes in a human or mouse’s body for genetic markers, measuring the transcription level (amount of transcription) of each gene, and measuring the amount of RNA produced. The RNAi approach consists, in part, of describing the normal function of the huntingtin protein. Working with HD cell models, researcher Songshan Jiang uses short interference RNA (siRNA) to stop the translation of the huntingtin protein. Nerve cells treated with siRNA do not contain huntingtin at all. Jiang then examines the phenotype of the cells, looking at the way in which they are affected by the lack of huntingtin. This kind of observation can tell him what huntingtin’s regular function is within the nerve cells.

The biochemical approach involves understanding and describing how the structural and physical properties of biological molecules (such as proteins) influence the functions of those molecules. To study these molecules, they must first be purified. One of the biggest breakthroughs in biochemistry was the introduction of chromatography, which made it possible to separate and isolate different kinds of molecules quickly and efficiently. In the MacDonald lab, a technique called gravitation chromatography is used to compare the mass of the mutant and wild-type huntingtin protein. Researcher Ihn-Sik Seong has found that mutant huntingtin is in large complexes that are even bigger (have more mass) than the complexes with normal huntingtin. They also regularly use high pressure liquid chromatography (HPLC) when studying changes in the biochemical molecules that are due the presence of mutant huntingtin in cells. Generally, protein molecules are separated according to their physical properties such as their size, shape, and affinity for other molecules. HPLC facilitates the separation of molecules under high pressure in a stainless steel column filled with a special chemical substance called a matrix. A computer controls both a pump and a means of collecting data. By using HPLC, researchers can describe various properties of mutated and wild-type huntingtin. Knowing more about the characteristics of wild-type huntingtin will give the researchers a basis of comparison for mutant huntingtin since they each have different properties.

High pressure liquid chromatography (HPLC) machines in the MacDonald lab.

Debunking the Myths

For those of us who are not familiar with the ins and outs of the scientific community, Gusella and MacDonald believe that there are many misconceptions about researcher work because then many different activities that are necessary to attack a disease like HD tend to be lumped together. In reality, these activities require quite different skills, levels of funding, and working environments.

• Myth #1: All labs are the same.Reality: There are important distinctions between academic labs (like the MacDonald and Gusella labs), industrial labs, pharmaceutical labs, clinical research labs, and clinical testing labs. All carry out different kinds of activities that are important to HD research. Generally, academic labs are responsible for discovering the biology behind a disease. Well-funded industrial and pharmaceutical labs are responsible for developing drugs that treat the disease and testing them on animals raised for that purpose. Clinical research labs are responsible for conducting clinical trials of treatments and drugs using consenting human subjects. Clinical testing labs do not do research, but instead conduct diagnostic tests for disease, in a government-approved manner, that are ordered by physicians.
• Myth #2: Researchers spend all their time researching.Reality: In fairly large academic labs like MacDonald’s, the principal researcher does little or no actual lab work (often referred to as “bench work” because the lab tables are called benches). Rather, MacDonald hires postdoctoral students and technicians to do the bench work, dividing up the different tasks and projects among them and spending time to direct their efforts. MacDonald herself spends much of her time interpreting results, strategizing for future projects, and doing administrative work, such as writing and reviewing grant proposals and sitting on scientific committees.
• Myth #3: Researchers are paid by the hospitals they work for.Reality: Researchers like MacDonald rent lab space from their institution, such as a hospital, and must do their own fundraising to obtain money for equipment and employees’ salaries. This fundraising is accomplished by applying for grants.
• Myth #4: Treatments that work on mice will usually work on humans.Reality: Most experimental drugs and treatments tested on mouse models of various diseases do not work on humans. Mice are often used to test the toxicity of the chemical compounds involved in various treatments before drugs are developed and approved for human consumption. The process of testing treatments on mice, developing drugs, getting government approval for the drugs, and putting them on the market is quite slow, has many steps, and involves making enough of a particular compound for large groups of people.Some scientists think that a larger animal model may be a promising alternative to a mouse model of HD in terms of shortening the clinical trial timeframe. A large animal model may be physiologically more similar to a human, which will give scientists a better sense of what kinds of treatments may work on humans. MacDonald mentioned that a researcher named Russell Snell at the University of Auckland in New Zealand is currently developing a sheep model of HD. Because researchers will see much more nerve cell death in sheep than in mice or rats, they might be able to get a more accurate picture of the progression of the disease in people. It will thus be easier to study the pathology of the disease in its final stages. Not only is the physiology of a sheep is closer to that of a human than a rodent’s, but a sheep model can provide other benefits in terms of HD experimentation. For example, living experiments (experiments conducted in the living animal), transplantation of cells and tissues, and protection of the nerve cells against deterioration (a process called neuroprotection) will be easier to perform.
• Myth #5: The nerve cell is healthy, and then it changes and gets sick after the onset of HD.Reality: The nerve cell in a presymptomatic person with HD is never normal. If a genetic mutation is present, then the cell is abnormal from the start. It is a common misconception that if a cell is not presently sick, it is normal. Yet, according to MacDonald, the cell merely gives the appearance of being normal by trying its best to remain healthy for as long as possible.
• Myth #6: There is one big, important change that occurs in the body to make a person sick with HD.Reality: There is certainly more than one big, important change; in fact, there is a whole series of changes! Visualizing HD in terms of a timeline allows us to think about the changes sequentially (i.e. what happens, when, and in what order during the disease cascade). MacDonald says that one of the earliest changes involves the RRS1 gene. RRS, which stands for regulator of ribosome synthesis, directs the production of this small organelle that, in turn, directs the synthesis of nuclear proteins (proteins found in the nucleus of the cell). At the beginning of the disease cascade, more RRS1 is made, perhaps because the cell needs to change its protein synthesis. Other subsequent changes take place, leading to “derangements;” for example, in energy metabolism, which lead to other changes, and so on.
• Myth #7: Scientists will find a “magic bullet” to cure HD.Reality: MacDonald thinks that it is highly unlikely that one chemical compound will work well in all people with HD. Yet she believes that HD researchers may, as a first step, be able to develop a drug to delay the onset of disease symptoms for about five years, which is the size of the effect in some individuals with a certain version of the GRIK2 (Glu6) gene. The GRIK2 gene is a genetic modifier in HD, meaning that its expression in different people can have an effect on age of onset. For instance, by manipulating this gene in a certain way, scientists can perhaps stall the onset of HD and thus shift the disease cascade in such a way that when onset actually does occur, the symptoms will be less severe.

Toward a Treatment: The future of HD research

What big questions remain in the field of HD research? Gusella said that the question, “How does mutant huntingtin trigger the disease cascade?” is, by far, the most important one because learning about the root cause of the disease will help researchers intervene early in the cascade before the onset of symptoms. Secondary questions include:

• What is the normal function of the huntingtin protein?
• Is there a point at which the disease pathway becomes independent of its trigger?
• What are the phenotypes in tissues other than the brain of a person with HD?
• Why are cancer rates lower in people with HD?
• What is the overall effect of HD on metabolism (not just in certain fat cells)?
• What is the origin of the chromosome that gives rise to new mutations?
• What produces the long length of the HD allele?
• Why don’t nematodes (a type of worm) have a Huntington gene? (Nematodes are multicellular – made up of many cells – just like humans and mice.)

MacDonald discussed what she believes will be an important future discovery: the development of a biomarker to measure the progression of HD in a patient. She thinks that a rating scale that measures the functional decline (the physical deterioration) of people with the disease may prove to be important. She would also like to see a quantitative test that reflects the state of a person’s disease – not just for HD, but for Parkinson’s and cancer, as well. Unlike MacDonald, Gusella thinks that a biomarker would not be particularly meaningful; rather, he would like to see researchers direct their efforts toward developing a marker that could indicate a reversible disease state.

What level of specificity within the body will be targeted by a breakthrough treatment – the cell, gene, protein, DNA, or RNA? After a drug, which would be their first choice, MacDonald and Gusella said that RNA, but not DNA, is a promising level for a treatment. RNA is promising in terms of delivery to nerve cells (siRNA is delivered to the cells via the RNAi technique). At the protein level, it is possible to deliver normal huntingtin to the nerve cells in a similar way. However, the only drawback is that the technology involved in these processes is relatively unknown and underdeveloped. On the bright side, scientists and doctors can target the mutant huntingtin with small-molecule pharmacological drugs that are already available. At the level of the nerve cell, it seems promising to target treatments to nerve cell-nerve cell interactions at a distance from the trigger of the disease cascade. Unfortunately, this may be difficult due to the fact that the trigger is still unknown. On the other hand, targeting drugs to genetic modifiers would be a faster route than targeting nerve cell-nerve cell interaction – if such a route were available.

Conclusion

Gusella, MacDonald, and their fellow researchers in the United States and around the world will continue to work diligently to find a treatment for HD. Much has already been accomplished by dedicated research teams, and many more breakthroughs are soon to come with advances in technology and the development of more accurate animal models of the disease. Once researchers identify the trigger, they can develop an effective treatment that stops the disease cascade early enough to prevent the onset of symptoms.

In the meantime, how can people with HD help the researchers in their quest for a breakthrough? MacDonald said that they can ask their doctors if they can participate in ongoing research, such as clinical trials, genetic studies, or observational studies. To learn more about current research, people with HD should read informative websites like the Huntington Study Group site. MacDonald added that newly diagnosed people should ask their doctors for something to read (a booklet or pamphlet) about managing the symptoms of HD, such as A Physician’s Guide to the Management of Huntington’s Disease, Second Edition by Drs. Adam Rosenblatt, Neal G. Ranen, Martha A. Nance, and Jane S. Paulsen.

“Research is a winding journey from the starting point to where you want to end up,” MacDonald said of the experience of working on HD (or any scientific phenomenon). It has been a long road, and she and Gusella still have miles to go, but they can see their destination – a much-anticipated treatment – on the horizon.

L-R: HOPES team member Shawn Fu, Dr. Marcy MacDonald, and HOPES team member Taylor Altman

-T. Altman, S. Fu, M. Stenerson, 12/07/04