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

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

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

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

Fig H-3: The Process of Autophagy

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

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

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

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

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

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

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

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

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

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

Research on rapamycin and HD^

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

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

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

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

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

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

For further reading^

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

-A. Milczarek, 12/29/04