Research matters

News from the Harvard Medical School research community.
Research matters
By Harvard Medical International
Mon 17 Sep 2007 04:00 AM

News from the Harvard Medical School research community.

Neurology

Blocker of longevity-associated protein cuts down Parkinson's in animal models.

Good things come in large packages. In the presence of the SIRT2 inhibitor AGK2, toxic alpha-synuclein is packaged into fewer, larger inclusions (white spots) compared to those found in control cells treated with the common solvent DMSO. Larger inclusions may protect nerve cells.

Blocking the activity of sirtuin 2 (SIRT2), one of a family of proteins that regulate longevity, may offer protection from Parkinson's disease, according to new findings by HMS researchers. Writing in the June 21 online Science, Aleksey Kazantsev, HMS assistant professor of neurology at Massachusetts General Hospital, and colleagues report that small molecule inhibitors of SIRT2 protect cell and animal models against the toxicity of mutated alpha-synuclein. The same mutations in humans cause degeneration of dopaminergic neurons in a specific region of the brain, the substantia nigra, and are responsible for certain inherited forms of Parkinson's disease.

About two out of three people with epilepsy do not achieve the goal of seizure freedom without side effects.

"This discovery suggests that we can develop neuroprotective compounds that block cells from dying and help them retain their functionality," said Kazantsev. Current therapies for Parkinson's supplement dopamine levels, but do not address the inherent degenerative nature of the disease.

Kazantsev and colleagues discovered the SIRT2 inhibitors by using high-throughput, phenotypic screens to identify molecules that promote protein aggregation. Last year, after screening around 37,000 small molecules, the researchers reported that a lead compound called B2 promotes the formation of inclusion bodies, intracellular aggregates of toxic proteins such as alpha-synuclein and mutant huntingtin, which causes Huntington's disease.

Inclusion bodies are believed to be protective because they sequester neurotoxic intermediates that form protein aggregates. The researchers then faced the daunting challenge of modifying that lead compound so it would work in animal models and eventually humans. "Lead development is a very tedious and difficult process, and it really requires that you have a molecular target," said Kazantsev.

Unfortunately, one of the problems with cell-based screens is that the molecular target is initially unknown, he explained.

To address this, the researchers tested B2 directly on a variety of proteins implicated in neurodegenerative diseases, including proteases, chaperones, and histone deacetylases like SIRT2. The compound weakly, though consistently, inhibited SIRT2.

"This was very exciting for us because this class of histone deacetylase has been implicated in the pathology of both Parkinson's and Huntington's diseases," said Kazantsev.

With this B2 target identified, the researchers next set about optimising the molecule. They generated about 200 structural analogs. One of these, AGK2, was about 10-fold more potent against SIRT2 and appeared quite specific, having very little inhibitory action on SIRT1 or SIRT3 (there are seven known human SIRT proteins). Tiago Fleming Outeiro, a postdoctoral fellow in collaborator Bradley Hyman's lab at MGH and first author on the Science paper, found that AGK2 inhibited SIRT2 in human cells and that the compound promoted the formation of large inclusion bodies in neuroglioma cells overexpressing alpha-synuclein. It also protected dopaminergic neurons against cell death caused by a Parkinson's disease-associated variant of the protein. When fed to fruit flies (in a collaborative experiment with Mel Feany's lab at Brigham and Women's Hospital), AGK2 also had a dramatic effect on alpha-synuclein-driven loss of dopaminergic neurons. At the highest doses tested, neuronal loss was only about 10% versus the 70% seen in untreated animals.

"Our next step will be to develop a safe SIRT2 inhibitor for use in human phase I clinical trials for Parkinson's disease, first testing various compounds in mouse models," said Kazantsev. Because inclusion bodies are also hallmarks of Huntington's and Alzheimer's diseases, Kazantsev plans to test SIRT2 inhibitors in animal models of those diseases as well.

If a painting's worth were measured by the money it fetched, van Gogh's famous rendering of his friend and physician Dr. Gachet would be among the most valuable in all of art. "Portrait of Dr. Gachet"-which depicts a languid man holding a purple foxglove, the plant from which the drug digitalis is derived-was sold in 1990 for an astounding 82 million dollars.

The great and famously tortured artist had his own reasons for valuing the portrait. He suffered from severe epilepsy and depended heavily on Gachet's prescription of digitalis to treat his debilitating seizures.

Public health

Obesity spreads through social networks.

Public health officials have been working hard to account for the dramatic rise in U.S. obesity rates. Many obvious factors, such as poor diet and a sedentary lifestyle, certainly contribute to the swelling statistics. However, these and other explanations tend to focus exclusively on how individuals' choices and behaviours affect their own weight.
Now, researchers from Harvard Medical School and the University of California, San Diego have found that obesity is hardly a private matter. Reported in the July 26 edition of the New England Journal of Medicine, the researchers found that obesity spreads through social ties. When an individual gains weight, it dramatically increases the chances that their friends, siblings, and spouses will likewise gain weight. The closer two people are in a social network, the stronger the effect. Interestingly, geographical distance between persons in a social network appears to have no effect.

"What we see here is that one person's obesity can influence numerous others to whom he or she is connected both directly and indirectly," says Nicholas Christakis, MD, PhD, a professor in Harvard Medical School's Department of Health Care Policy. "In other words, it's not that obese or non-obese people simply find other similar people to hang out with. Rather, there is a direct, causal relationship."

Heart disease is the number one cause of death in the Western world.

Over the last 25 years, the incidence of obesity among U.S. adults has more than doubled, shooting from 15 to 32%. In addition, roughly 66% of U.S. adults are considered overweight. Christakis and U.C. San Diego researcher James Fowler, PhD, decided to analyse data from the Framingham Heart Study (an ongoing cardiovascular study begun in 1948) to see if any social patterns might elucidate these alarming rates.

Christakis and Fowler derived information from archived, handwritten administrative tracking sheets dating back to 1971. All family changes for each study participant, such as birth, marriage, death, and divorce, were recorded. In addition, participants had also listed contact information for their closest friends. Coincidentally, many of these friends were also study participants. Focusing on 12,067 individuals, Christakis and Fowler observed a total of 38,611 social and family ties. As they analysed the data, the researchers also looked closely at the influence of gender, smoking, socioeconomic status, and geographic distance.

The study found that when an individual becomes obese, the chances that a friend of theirs will become obese increase by 57%. Their siblings have a 40% increased risk of obesity, and their spouse a 37% increased risk. However, that person's neighbour, if not a part of their social network, has no effect.

Gender played an important role in how these statistics broke down. In same-sex friendships, individuals experienced a 71% increased risk if a friend of theirs became obese. This pattern was also observed in siblings. Here, if a man's brother became obese, his chances of becoming obese increased by 44%. Among sisters, the risk was 67%. Friends and siblings of opposite genders showed no increased risk. While the researchers note that correlations among siblings provide evidence for a biological, and possibly even a genetic, component to obesity, patterns seen among friends indicate that there's more than biology at work.

Social connections seem to be key. Moreover, as Christakis notes, "The fact that neighbours don't affect each other and that geographic separation doesn't influence the risk among siblings or friends tells us that environmental factors are not essential here," says Christakis. "Most likely, the interpersonal, social network effects we observe arise not because friends and siblings adopt each other's lifestyles. It's more subtle that that. What appears to be happening is that a person becoming obese most likely causes a change of norms about what counts as an appropriate body size. People come to think that it is okay to be bigger since those around them are bigger, and this sensibility spreads."

Obesity, the authors conclude, needs to be seen not simply as a clinical issue but as a public health problem.

"We need to understand that a significant part of an individual's health is embedded in their network," says Fowler. "In fact, we really need to revisit our whole notion of cost-effectiveness. The fact that certain healthcare approaches won't just affect the individual but will also cascade through their social ties means that healthcare interventions are far more cost-effective than previously thought."

"The rising rate of obesity threatens to reverse the decline in disability in the older population, with major implications for the health care system," says Richard Suzman, Ph.D., director of the NIA's Behavioural and Social Research Program. "This seminal study breaks important new ground in showing how social networks may amplify other factors and help account for the dramatic increase in obesity across the population."

Oncology

Promising treatment target found in Hodgkin lymphoma.

Dana-Farber Cancer Institute scientists have identified a protein that prevents the body's immune system from recognizing and attacking Hodgkin lymphoma cells. Based on this finding, the researchers are now investigating targeted therapies to disable this molecular "bodyguard" and boost a patient's ability to fight the blood cancer.

If the strategy proves successful, patients might escape some of the long-term complications - like heart damage and the threat of a second cancer - caused by standard treatments that include radiation, said Margaret Shipp, MD, of Dana-Farber, who headed the study. A report was posted online by the Proceedings of the National Academy of Sciences on July 30 and will appear in an upcoming print issue of the journal.

"We're excited about this treatment lead," said Shipp, a medical oncologist. "We are currently generating antibodies that can neutralize the ‘bodyguard' protein, and we'd like to fast-track this experimental therapy into clinical trials."

Nearly 8200 people in the United States - the great majority of them young adults - will be diagnosed with Hodgkin lymphoma in 2007, according to the American Cancer Society, with an estimated 1070 deaths. The cancer begins in the lymph nodes and channels that distribute infection-fighting white blood cells around the body. Its symptoms can include swollen glands in the neck, night sweats and fatigue.

The biological trademark of Hodgkin lymphoma is a type of giant, mutant white blood cell called the Reed-Sternberg cell that is found in the lymph node tumours. While most solid cancers consist almost entirely of tumour cells, says Shipp, Hodgkin tumours, which can reach the size of a basketball, contain only about 5% cancerous Reed-Sternberg cells; the rest are different types of immune cells recruited to fight the tumour, but they are ineffective.
"You would expect with all these host immune cells attracted to the area of the tumour cells that they would mount a great anti-tumour response," Shipp says. "But that's not the case. There are a lot of immune cells, but they're the wrong kind."

The immune army includes different types of T cells, such as T helper 1 (Th1) cells designed to recognize and kill foreign infectious agents and sometimes tumours, T helper 2 (Th2) cells, which normally control allergic responses, and T regulatory (Treg) cells that suppress other T-cell types and shut down an immune response when the job is done. The Hodgkin tumours are overloaded with Th2 and Treg cells that act as bodyguards for the cancer by weakening the Th1 immune response against it.

Researchers found that obesity spreads through social ties.

Jing Ouyang and Przemyslaw Juszczynski are part of a team that identified a treatment lead for Hodgkin lymphoma.

Przemyslaw Juszczynski, MD, PhD, Jing Ouyang, PhD, and colleagues from the Shipp laboratory, together with collaborators from Brigham and Women's Hospital, the Broad Institute and the University of Buenos Aires, hunted for the source of the cancer cells' protection. Using gene microarray chips, the scientists looked for genes that were active in Reed-Sternberg cells but not in cells of another non-Hodgkin B-cell lymphoma. The comparison revealed that a gene called Gal1 was up to 30 times more active in the Reed-Sternberg cells, causing them to secrete large quantities of a protein - Gal1 or Galectin 1 - that turns down the Th1 immune response.

The Shipp team then defined the mechanism for Gal1 overexpression in Hodgkin lymphoma. Next, they demonstrated that Th1 immune cells underwent apoptosis, or cell death, when treated with Gal1, leaving increased numbers of Th2 cells and the suppressive Treg cells.

Using a gene-silencing technique, RNA interference or RNAi, they then turned off the Gal1 gene in Hodgkin Reed-Sternberg cells and showed that it blocked the death of infiltrating normal Th1 cells, making them an equal force to the Th2 cells.

"Likely what's happening here is that the tumour cells essentially hijack a normal regulatory program and use it to avoid being knocked off by the immune response," explains Shipp, who is also a professor of medicine at Harvard Medical School. "These observations provide an important explanation for why you have this ineffective immune response in Hodgkin lymphoma."

She adds that this bodyguard strategy may not be limited to Hodgkin lymphoma. One of the collaborating authors, Gabriel Rabinovich, PhD, of the University of Buenos Aires, has blocked Gal1 in mice with a form of the deadly skin cancer melanoma, and the animal's immune system succeeded in eliminating the cancer, Shipp says. "We think it's very possible that this strategy will be applicable to other types of cancer."

Cardiology

Study helps explain origins of cardiac fibrosis in patients with heart disease.

A report led by researchers at Beth Israel Deaconess Medical Center (BIDMC) helps explain the origins of cardiac fibrosis, a stiffening of the heart muscle that leads to a variety of cardiac diseases, most notably heart failure. The animal study, which appeared in the July issue of Advance Online Publication of Nature Medicine, also demonstrates that a bone morphogenic molecule known as rhBMP7 can reverse the cardiac fibrosis process, offering the possibility of a therapeutic target for this debilitating condition.

"Heart disease is the number one cause of death in the Western world," explains the study's lead author Elisabeth Zeisberg, MD, a scientist in the Division of Matrix Biology at BIDMC and an Instructor of Medicine at Harvard Medical School (HMS). "And most people who suffer from heart disease have developed scarring of the heart tissue, known as fibrosis."

Fibrosis develops when the body's natural wound-healing process goes awry. Under normal conditions, specialized cells known as fibroblasts deposit layers of collagen protein to form a scar and thereby enable wounds to heal. However, in abnormal circumstances - and for unknown reasons - excessive production of matrix proteins, such as collagen, results in pathological scarring, or fibrosis. In the heart, the buildup of matrix leaves the organ stiff and inflexible, unable to properly relax and function.

"Fibrosis can develop in any organ in the body," explains Zeisberg. "While it's known that fibroblast cells are responsible for cardiac fibrosis, the source of these fibroblasts has remained unknown until now."

Zeisberg and senior author Raghu Kalluri, PhD, Chief of the Division of Matrix Biology at BIDMC, speculated that a specialized form of epithelial-mesenchymal transition (EMT) known as endothelial-mesenchymal transition might be the mechanism behind this turn-of-events.

"Our laboratory has had a longstanding interest in the area of organ fibrosis and the origin of fibroblasts in this setting," explains Kalluri, who is also Associate Professor of Medicine at HMS. "We have previously demonstrated that in the kidney, liver and the lung, epithelial cells under stress can convert into fibroblasts via epithelial-mesenchymal transition."

So, using knockout mice in which endothelial cells had been marked genetically, the investigators confirmed that during cardiac fibrosis, these cells were indeed converting into activated fibroblasts, which were depositing scar material and impeding the proper function and electrical conduction of the heart.
In the second part of the study, the investigators turned to the rhBMP7 protein to determine if it could successfully reverse the EndMT process and thereby reduce the development of fibroblasts and lead to the improvement of heart function.

"The rhBMP-7 protein was quite impressive in its ability to recover the function of damaged hearts," says Kalluri. "These findings provide compelling proof that the process of fibrosis can be reversed in the heart and offers the possibility of new therapies for patients who have developed cardiac fibrosis as the result of myocardial infarction, hypertension, valvular diseases or heart transplantation."

Neurology

Trial turns over new leaf for traditional herb.

The ranks of epilepsy medications have expanded considerably in the past hundred years, due mostly to the addition of pharmaceutically derived compounds. Still, people with epilepsy, who account for up to 2% of the population, may continue to suffer, either from seizures or from secondary effects associated with their medicines. "About two out of three people with epilepsy do not achieve the goal of therapy, which is seizure freedom without side effects," said Steven Schachter, associate director of clinical research at the HMS Osher Institute and HMS professor of neurology and epileptologist at Beth Israel Deaconess Medical Center.

Part of the problem is that epilepsy is a brain disorder that has several underlying mechanisms. A drug that works in one patient may not work in another. Yet all epileptic seizures are characterized by uncontrolled electrical activity. One way to control them would be to block substances in the brain, such as glutamate, that cause neurons to fire. Pharmaceutical companies have been pressing to find glutamate-inhibiting compounds, with very limited success.

Schachter has hit upon a compound that does just that. And he has done so by drawing upon the same centuries-old botanical tradition that yielded the drug digitalis.

Applying modern methods of drug discovery, he and colleagues have identified a compound derived from the spiky-looking Chinese club moss that when tested in rodents, had the power to prevent seizures. The seizures are considered to be representative of the highly debilitating grand mal, or tonic-clonic, episodes that many patients with epilepsy experience, and which are often refractory to treatment. During this fall, he hopes to launch a small clinical trial of the compound, huperzine A.

It will not be the substance's first foray into the medical arena. For centuries, Chinese healers have been using extracts of huperzine A to quell inflammation and fever and, more recently, to treat schizophrenia. Clinical trials are under way in China and the United States to test huperzine A's power against Alzheimer's disease. Meanwhile, the compound is being widely marketed as an over-the-counter memory aid. But in all these years, it has not been used for the treatment of epilepsy.

Schachter, who has been seeking new methods for treating epilepsy for more than 20 years, was alerted to the promise of botanicals several years ago when he was invited by David Eisenberg to become associate director of clinical research at the Osher Institute.

Eisenberg, the Bernard Osher associate professor of medicine at HMS, had been pursuing the use of botanicals in the treatment of cancer and introduced Schachter to plant-minded colleagues in this country and East Asia. Schachter set out to find herb-derived compounds for the treatment of epilepsy "using the same methods that were being used to identify potentially efficacious pharmaceuticals," he said.

In Alzheimer's disease, huperzine A is thought to work by blocking the enzyme that degrades acetylcholine, a neurotransmitter associated with memory. But huperzine also blocks glutamate, which suggested to Schachter that it might have potential as an anti-epileptic agent.

Working with the National Institute of Neurological Disorders and Stroke and colleagues at the University of Utah, he set about testing huperzine's mettle in a well-known rodent model of seizures, the 6-Hz model.

The researchers administered the compound to the animals and then exposed them to three levels of seizure-inducing electrical currents. Huperzine A potently prevented seizures, and it did so at all three levels. More remarkably, the dose required at the highest and lowest currents was not much different. "All other drugs that are effective in this model have shown that it takes larger doses to stop seizures as the voltage goes up, and often the difference is huge," he said.

What makes the compound particularly ready for the prime time of clinical trials is that it is already in use, and apparently safely so. Extremely pure preparations of huperzine are available as supplements. Schachter, with funding from the Epilepsy Research Foundation, plans to give huperzine to about 20 patients who have not responded to available anti-epileptic drugs or who are experiencing side effects.

"We'll look to see how well they tolerate different dosages of huperzine, whether there's any indication that it's benefiting their seizures, and perhaps some early indication of any effects on their memory," he said. He and colleagues will also be looking at how the compound might interact with other seizure medications taken by the patients.

Huperzine is just the first of several promising botanicals that Schachter hopes to test in humans. "Over the last couple of years, we've been building a pipeline of herbal extracts and extract-derived compounds and moving them as far as we can with basic research," he said. In some cases, he will try to improve upon nature. For example, though huperzine worked impressively well in rodents, it was most effective when given an hour before the rats received the seizure-inducing current. "We'd like to have a drug that is effective 24 hours a day," he said, which could require tinkering with huperzine's pharmacokinetics.

"The overall goal of my work is to identify novel compounds that can then be developed as prescription drugs for people with epilepsy-especially those who have ongoing seizures despite available therapies or who are experiencing intolerable side effects," said Schachter.

Research Matters brings together selected research being conducted at Harvard Medical School's 18 affiliated institutions. For more information, visit the Harvard Medical School website at www.hms.harvard.edu.

This article is provided courtesy of Harvard Medical International.

© 2007 President and Fellows of Harvard College.

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