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.
Table of Contents
- Becoming involved in HD research
- Getting involved in the HD community
- The “top-down” approach
- Current research projects
- Autophagy, or “self-eating”
- Autophagy and HD
- Autophagy research in the Kopito Lab
- More on Dr. Riley
- Life as a post-doctoral student
- Dr. Kopito, HD research, and the public
- For further reading
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.
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.
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.