McKnight Awards Go to Two from Caltech

PASADENA, Calif.--Richard Andersen, Boswell Professor of Neuroscience, and Kai Zinn, professor of biology, both of the California Institute of Technology, have each received a 2005 McKnight Neuroscience of Brain Disorder Award.

Andersen's work focuses on severely paralyzed human patients. These patients can think about making movements, but due to brain lesions from trauma, stroke, or peripheral neuropathies, can no longer make movements. The McKnight funding will allow Andersen's group to further their research in creating brain-implant technology that will interface between a patient's thoughts for movement and artificial limbs, computers, and other devices that will "read out" the patient's desires.

Zinn's work on prions, which are commonly known to the public as the cause of mad cow disease, addresses the mechanisms involved in the accumulation of these proteins. Aggregates composed of prion proteins are known to cause fatal human brain diseases. Through his study of prion propagation, in Drosophila and yeast, Zinn hopes to uncover how prions are formed and whether prions might have functions in the normal brain.

The Research Projects Award from the McKnight Foundation, established in 1977, and its Endowment Fund for Neuroscience, established in 1986, will provide $300,000 each to the Caltech professors over three years to further their work in neuroscience.

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Toward a Longer, Healthier Life

PASADENA, Calif. - The Spanish explorer Ponce de Leon spent a fair amount of his time in 1513 looking for the fountain of youth. The upside was that he discovered Florida. The downside was that the fountain was a myth. Now in two separate awards from the Ellison Medical Foundation, two scientists from the California Institute of Technology are taking a much more scholarly approach to the ravages of aging. Harry Gray, a chemist, has been awarded $970,000 to reveal the structure of a protein and a peptide that underlie two age-related diseases, Alzheimer's and Parkinson's, while biologist Alexander Varshavsky has been awarded $972,000 to conduct a systematic investigation of the genetics and biochemistry of aging.

Gray, the Arnold O. Beckman Professor of Chemistry, notes that approximately one million Americans suffer from Parkinson's, while 4.5 million have Alzheimer's. In order to design a drug to combat these two diseases, a key step is to understand the critical structural differences between normal proteins and the malignant proteins that comprise these diseases.

Both Alzheimer's and Parkinson's are associated with the accumulation in the brain of aggregates of proteins known as fibrils. In Parkinson's, the fibrils are composed of the protein alpha-synuclein, while in Alzheimer's, the fibrils or plaques are composed of the AB amyloid peptide. Alpha-synuclein and AB amyloid peptide are known as "disordered biopolymers," meaning that they do not have well-defined structures. Because of this lack of structure, the traditional tools used by chemists, such as x-ray crystallography and nuclear magnetic resonance spectroscopy, are virtually useless. They are only effective if the peptides and proteins being studied have well-defined structures in crystals or solutions.

Instead, Gray and his colleagues plan to use laser spectroscopic methods developed in Caltech's Beckman Institute to gain new insights into the structures, dynamics, and misfolding of malignant proteins and peptides. One of the most powerful methods they will use will employ an ultrafast camera to obtain distances between atoms in disordered structures that are constantly changing.

"We're very excited about the possibility of applying our laser methods to study proteins and peptides that are involved in disease in older people," says Gray. "We have a chance to identify toxic species that lead to these diseases, and point the way to successful interventions."

For Alexander Varshavsky, the Howard and Gwen Laurie Smits Professor of Cell Biology, it is the causes and alterations of the aging process that interest him. Every cell contains within it a molecular machine to eventually destroy its own proteins, he notes. The mechanisms and functions of this so-called regulated protein degradation became (mostly) understood over the last 25 years, in large part through discoveries in Varshavsky's lab. When a protein called ubiquitin is linked to another protein in a cell, that protein is marked for destruction. The molecular machines inside a cell that link ubiquitin to other proteins, and the intracellular machinery that "recognizes" ubiquitin-linked proteins and destroys them, are elaborate and complex. "Detailed understanding of these protein-destruction pathways will have a profound impact on the practice of medicine," says Varshavsky, "because all kinds of things that go wrong with us, from cancer and infectious diseases, to neurodegenerative syndromes and even normal aging, have a lot to do with either inherent imperfections of the ubiquitin system, or with an overt damage to it in a specific disease." Many clinical drugs of the future, he notes, will be designed to suppress, enhance, or otherwise modify various aspects of the ubiquitin system.

In this research Varshavsky will overexpress, selectively and in a controlled manner, specific components of the mouse ubiquitin system in intact mice, in order to determine the effects of such alterations on the rate of aging. He also plans to use analogous approaches with a much simpler organism, S. cerevisiae, common baker's yeast. His aim is to discover the molecular circuits that contribute to normal aging, and also to see whether some of the alterations that he plans to introduce could slow down the aging process.

The Ellison Medical Foundation is a nonprofit corporation that was established by a gift from Mr. Lawrence J. Ellison to support basic biomedical research to understand aging processes and age-related diseases and disabilities. Through various award mechanisms, including the Senior Scholar and New Scholar award programs, the foundation fosters research by means of grants-in-aid to investigators at universities and laboratories within the United States.

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Scientists Discover What You Are Thinking

PASADENA, Calif. - By decoding signals coming from neurons, scientists at the California Institute of Technology have confirmed that an area of the brain known as the ventrolateral prefrontal cortex (vPF) is involved in the planning stages of movement, that instantaneous flicker of time when we contemplate moving a hand or other limb. The work has implications for the development of a neural prosthesis, a brain-machine interface that will give paralyzed people the ability to move and communicate simply by thinking.

By piggybacking on therapeutic work being conducted on epileptic patients, Daniel Rizzuto, a postdoctoral scholar in the lab of Richard Andersen, the Boswell Professor of Neuroscience, was able to predict where a target the patient was looking at was located, and also where the patient was going to move his hand. The work currently appears in the online version of Nature Neuroscience.

Most research in this field involves tapping into the areas of the brain that directly control motor actions, hoping that this will give patients the rudimentary ability to move a cursor, say, or a robotic arm with just their thoughts. Andersen, though, is taking a different tack. Instead of the primary motor areas, he taps into the planning stages of the brain, the posterior parietal and premotor areas.

Rizzuto looked at another area of the brain to see if planning could take place there as well. Until this work, the idea that spatial processing or movement planning took place in the ventrolateral prefrontal cortex has been a highly contested one. "Just the fact that these spatial signals are there is important," he says. "Based upon previous work in monkeys, people were saying this was not the case." Rizzuto's work is the first to show these spatial signals exist in humans.

Rizzuto took advantage of clinical work being performed by Adam Mamelak, a neurosurgeon at Huntington Memorial Hospital in Pasadena. Mamelak was treating three patients who suffered from severe epilepsy, trying to identify the brain areas where the seizures occurred and then surgically removing that area of the brain. Mamelak implanted electrodes into the vPF as part of this process.

"So for a couple of weeks these patients are lying there, bored, waiting for a seizure," says Rizzuto, "and I was able to get their permission to do my study, taking advantage of the electrodes that were already there." The patients watched a computer screen for a flashing target, remembered the target location through a short delay, then reached to that location. "Obviously a very basic task," he says.

"We were looking for the brain regions that may be contributing to planned movements. And what I was able to show is that a part of the brain called the ventrolateral prefrontal cortex is indeed involved in planning these movements." Just by analyzing the brain activity from the implanted electrodes using software algorithms that he wrote, Rizzuto was able to tell with very high accuracy where the target was located while it was on the screen, and also what direction the patient was going to reach to when the target wasn't even there.

Unlike most labs doing this type of research, Andersen's lab is looking at the planning areas of the brain rather than the primary motor area of the brain, because they believe the planning areas are less susceptible to damage. "In the case of a spinal cord injury," says Rizzuto, "communication to and from the primary motor cortex is cut off." But the brain still performs the computations associated with planning to move. "So if we can tap into the planning computations and decode where a person is thinking of moving," he says, then it just becomes an engineering problem--the person can be hooked up to a computer where he can move a cursor by thinking, or can even be attached to a robotic arm.

Andersen notes, "Dan's results are remarkable in showing that the human ventral prefrontal cortex, an area previously implicated in processing information about objects, also processes the intentions of subjects to make movements. This research adds ventral prefrontal cortex to the list of candidate brain areas for extracting signals for neural prosthetics applications."

In Andersen's lab, Rizzuto's goal is to take the technology they've perfected in animal studies to human clinical trials. "I've already met with our first paralyzed patient, and graduate student Hilary Glidden and I are now doing noninvasive studies to see how the brain reorganizes after paralysis," he says. If it does reorganize, he notes, all the technology that has been developed in non-paralyzed humans may not work. "This is why we think our approach may be better, because we already know that the primary motor area shows pathological reorganization and degeneration after paralysis. We think our area of the brain is going to reorganize less, if at all. After this we hope to implant paralyzed patients with electrodes so that they may better communicate with others and control their environment."

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Potential New Approach to Fighting Cancer

PASADENA, Calif. - The immune system is a remarkable defense mechanism, able to defend the body against a lifetime's worth of pathogens and pathogen attractors--bacteria and viruses, toxins and parasites, splinters and cuts--everything, that is, except cancer. Although the immune system handles most of these disease-causing organisms and insults well, it does a poor job of suppressing the growth of tumors.

A major goal of cancer immunotherapy has been to bolster the immune system by generating large numbers of white blood cells (T cells) that could specifically seek and destroy cancer cells. Now, two scientists from the California Institute of Technology have come up with a novel and promising approach to antitumor immunotherapy. Reporting in the online edition of the Proceedings of the National Academy of Sciences (http://www.pnas.org/papbyrecent.shtml), Lili Yang, a postdoctoral scholar, and David Baltimore, professor of biology, Caltech president, and Nobel Prize recipient, have developed a new methodology they are calling "instructive immunotherapy" that someday may fight human cancer.

In mice and humans, hematopoietic stem cells (HSC) form both red blood cells and immune system cells. In mice, Yang and Baltimore succeeded in altering some HSCs so that they would generate specific kinds of T cells that aggressively attack and destroy specific cancer cells. Once the mouse immune system received this enhancement, it became able to generate its own cancer-specific T cells on a long-term basis. When helped by dendritic cells (another type of immune system cell) carrying a piece of the tumor's marker protein, the methodology achieved the complete elimination of large, established tumors. While the work is preliminary and was done with mice, says Baltimore, instructive immunotherapy could eventually be used for controlling the growth of tumors in humans.

"We've achieved something that could one day prove important," says Baltimore, who was awarded the 1975 Nobel Prize in Physiology or Medicine, "but the first caveat is that this is all with mice, and mice are often not predictive of behavior in humans." Still, he notes, "everything we have done is in principle possible to do in humans, so we plan to try to develop a system for optimizing the ability to program human stem cells."

Yang, a former graduate student of Dr. Baltimore, says current cancer strategies fall into two categories: developing a cancer vaccine, or developing a drug that can be given when cancer is diagnosed. "Our strategy is threefold," she says, "a combination of gene therapy, stem cell therapy, and immunotherapy. When these three methodologies work together, it is possible to provide life-long immunity."

In addition to making billions of new blood cells each day, HSCs are responsible for providing immune protection of every cell type in the body. In fact, HSC transplants are routinely used to treat patients with cancers. In their case, Yang and Baltimore chose to manipulate HSCs for three reasons--because HSCs normally make T cells, they make them by the billions, and they exist in humans through their lifetime.

The first step was to design a retrovirus vector that could deliver genes for both chains of the T cell receptor to HSCs. This was actually the key to the whole study. For this work, two vectors delivering two sets of genes were developed. The HSCs then gave rise to both of the major types of T cells known as CD4 helper cells and CD8 killer cells. Together, these two cell types can recognize the foreign nature of the test cancer cells used in the study and can kill them. The researchers were successful in programming up to a quarter of the mouse's T cells to react to the model tumor. Even better, once modified, the mouse's immune system continued to produce these antigen-specific T cells on its own. However, with this method alone, Yang and Baltimore found that mice were only partially resistant to the tumor cells.

To achieve complete protection required boosting the animal's immune system with dendritic cells carrying a fragment of the tumor cell's marker protein. These dendritic cells are thought to use their long tentacle-like branches (called dendrites) to stimulate the T cells and make them more active. With this combination, Yang and Baltimore were able to achieve the complete shrinkage and suppression of even large, well-established tumors.

Dr. Yang recalled her reaction to the first positive results: "It was a great surprise that the method worked so well. This level of efficacy makes us believe that the method may have real therapeutic potential."

The next step, says Yang, will be to repeat the experiment, this time using conditions that more closely approximate human tumors. After that, if things hold up, the next step will be to start thinking about human trials.

"Producing a state of antitumor immunity has been a dream of immunologists for years, but has been unrealized in humans," says Baltimore. "Here we've developed a methodology that provides a new opportunity to realize this goal. We certainly hope that it will prove to be effective in humans."

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Caltech computer scientists embed computation in a DNA crystal to create microscopic patterns

PASADENA, Calif.--In a demonstration that holds promise for future advances in nanotechnology, California Institute of Technology computer scientists have succeeded in building a DNA crystal that computes as it grows. As the computation proceeds, it creates a triangular fractal pattern in the DNA crystal.

This is the first time that a computation has been embedded in the growth of any crystal, and the first time that computation has been used to create a complex microscopic pattern. And, the researchers say, it is one step in the dream of nanoscientists to master construction techniques at the molecular level.

Reporting in the December issue of the journal Public Library of Science (PLoS) Biology, Caltech assistant professor Erik Winfree and his colleagues show that DNA "tiles" can be programmed to assemble themselves into a crystal bearing a pattern of progressively smaller "triangles within triangles," known as a Sierpinski triangle. This fractal pattern is more complex than patterns found in natural crystals because it never repeats. Natural crystals, by contrast, all bear repeating patterns like those commonly found in the tiling of a bathroom floor. And, because each DNA tile is a tiny knot of DNA with just 150 base pairs (an entire human genome has some 3 billion), the resulting Sierpinski triangles are microscopic. The Winfree team reports growing micron-size DNA crystals (about a hundredth the width of a human hair) that contain numerous Sierpinski triangles.

A key feature of the Caltech team's approach is that the DNA tiles assemble into a crystal spontaneously. Comprising a knot of four DNA strands, each DNA tile has four loose ends known as "sticky ends." These sticky ends are what binds one DNA tile to another. A sticky end with a particular DNA sequence can be thought of as a special type of glue, one that only binds to a sticky end with a complementary DNA sequence, a special "anti-glue''. For their experiments, the authors just mixed the DNA tiles into salt water and let the sticky ends do the work, self-assembling the tiles into a Sierpinski triangle. In nanotechnology this "hands off" approach to manufacturing is a desirable property, and a common theme.

The novel aspect of the research is the translation of an algorithm--the basic method underlying a computer program--into the process of crystal growth. A well-known algorithm for drawing a Sierpinski triangle starts with a sequence of 0s and 1s. It redraws the sequence over and over again, filling up successive rows on a piece of paper, each time performing binary addition on adjacent digits.

The result is a Sierpinski triangle built out of 0s and 1s. To embed this algorithm in crystal growth, the scientists represented written rows of binary "0s" and "1s" as rows of DNA tiles in the crystal--some tiles stood for 0, and others for 1. To emulate addition, the sticky ends were designed to ensure that whenever a free tile stuck to tiles already in the crystal, it represented the sum of the tiles it was sticking to.

The process was not without error, however. Sometimes DNA tiles stuck in the wrong place, computing the wrong sum, and destroying the pattern. The largest perfect Sierpinski triangle that grew contained only about 200 DNA tiles. But it is the first time any such thing has been done and the researchers believe they can reduce errors in the future.

In fact the work is the first experimental demonstration of a theoretical concept that Winfree has been developing since 1995--his proposal that any algorithm can be embedded in the growth of a crystal. This concept, according to Winfree's coauthor and Caltech research fellow Paul W. K. Rothemund, has inspired an entirely new research field, "algorithmic self-assembly," in which scientists study the implications of embedding computation into crystal growth.

"A growing group of researchers has proposed a series of ever more complicated computations and patterns for these crystals, but until now it was unclear that even the most basic of computations and patterns could be achieved experimentally," Rothemund says.

Whether larger, more complicated computations and patterns can be created depends on whether Winfree's team can reduce the errors. Whether the crystals will be useful in nanotechnology may depend on whether the patterns can be turned into electronic devices and circuits, a possibility being explored at other universities including Duke and Purdue.

Nanotechnology applications aside, the authors contend that the most important implication of their work may be a better understanding of how computation shapes the physical world around us. "If algorithmic concepts can be successfully adapted to the molecular context," the authors write, "the algorithm would join energy and entropy as essential concepts for understanding how physical processes create order."

Winfree is an assistant professor of computation and neural systems and computer science; Rothemund is a senior research fellow in computer science and computation and neural systems. The third author is Nick Papadakis, a former staff member in computer science.

 

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A "Smoking Gun" For Nicotine Addiction

Embargoed for Release at 11 a.m. PST, Thursday, November 4, 2004

PASADENA, Calif. - Nicotine is responsible for more than four million smoking-related deaths each year. Yet people still smoke. Why? One reason is the stranglehold of addiction, started when nicotine enhances the release of a neurotransmitter called dopamine, a chemical messenger that induces a feeling of pleasure. That's what smoking, presumably, is all about.

Knowing specifically which receptor molecules are activated by nicotine in the dopamine-releasing cells would be a promising first step in developing a therapeutic drug to help people kick the habit. But that's a challenging goal, since there are many cell receptor proteins, each in turn comprising a set of "subunit proteins" that may respond to nicotine, or instead, to a completely different chemical signal. Reporting in the November 5 issue of the journal Science, California Institute of Technology postdoctoral scholar Andrew Tapper, Professor Allan Collins of the University of Colorado, seven other colleagues, and Henry A. Lester, the Bren Professor of Biology at Caltech, have determined that when receptors with a specific subunit known as alpha4 are activated by nicotine, it's sufficient for some addiction-related events, such as pleasure response, sensitization, and tolerance to repeated doses of nicotine. This research suggests that alpha4, and the molecules that are triggered in turn by alpha4, may prove to be useful targets for addiction therapies.

When cells communicate in the brain, nerve impulses jump chemically across a gap between two nerve cells called the synapse, using a neurotransmitter such as acetylcholine. Acetylcholine activates specific receptors on the post-synaptic nerve cell. This starts the firing of electrical impulses and, in cells that manufacture dopamine, that pleasure-inducing messenger is released as well. Having completed its task, acetylcholine is then rapidly broken down by an enzyme called acetylcholinesterase. It's a clever and wondrous biological machine, says Lester. But, he says, "nicotine is clever too, because it mimics acetylcholine." Worse, nicotine is not broken down by acetylcholinesterase. "So it persists at the synapse for minutes rather than milliseconds, and excites the post-synaptic neurons to fire rapidly for long periods, releasing large amounts of dopamine. Most scientists believe that's a key reason why nicotine is so addictive."

Previous work in several laboratories in the 1990s had suggested that, of the many so-called nicotinic acetylcholine receptors, one consisting of subunits called alpha4 and beta2 was important for nicotine addiction. This had been determined by the use of so-called "knockout" mice. In this method, scientists identify a gene that may affect a behavior, then interrupt or knock it out in a specially bred strain of mice. In this case, the effects of nicotine on the knockout mice and normal mice were compared, and the mice without the beta2 subunit lacked some responses to nicotine.

In work to identify receptor subunits that were sufficient to cause nicotine dependence, Lester's group examined the "partner" subunit, alpha4. But instead of experimenting with a knockout mouse, they developed "hypersensitive knock-in" mice. Lester's group replaced a naturally occurring bit of DNA with a mutation that changed a single amino acid in a single gene among the mouse's 30,000 genes. That change was enough to make the alpha4 subunit highly sensitive to nicotine.

Lester's group first selected the proper mutation with tests in frog eggs and cultures, then bred the strain of mice. As they hypothesized, the mice with the re-engineered alpha4 receptor proved to be highly sensitive even to very small doses of nicotine that didn't activate other nicotinic receptor types. This finding shows the alpha4 subunit is a prime candidate to be studied at the molecular and cellular level, says Lester, "and it may be a possible target for developing a medication that would reduce the release of dopamine caused by nicotine, and hopefully reduce nicotine's addictive grip."

Knockout mice leave nothing to measure, explains Lester. Knock-in mice have the advantage, he says, of isolating and amplifying the "downstream" chain of molecular signals within nerve cells that occur after nicotine activates its receptors. These signals, when repeatedly stimulated by nicotine, eventually cause the-as-yet unknown, long-term changes in nerve cells that are presumably the biological basis of addiction. Lester and his colleagues are now tracking down those signals, molecules, and newly activated genes, using their hypersensitive mice. The eventual hope: one of those signals might provide a molecular target that can be specifically blocked, providing a therapy against addiction. This strategy would resemble modern cancer drugs, such as Gleevec, which block only specific signaling molecules needed by proliferating cancer cells. Hypersensitive knock-in mice may also prove useful in gaining further insight into diseases such as epilepsy and Parkinson's disease.

Lester is optimistic about ultimately defeating nicotine's addictive power. "It's a complicated pathway that still must be broken down into individual steps before we can understand it fully," he says, "but I personally believe that nicotine addiction will be among the first addictions to be solved, because we already have so many tools to study it."

This research was supported by the California Tobacco-Related Disease Research Program, the W. M. Keck Foundation, the Plum Foundation, the National Institute of Mental Health, the National Institute of Neurological Disorders and Stroke, and the National Institute on Drug Abuse.

MEDIA CONTACT: Mark Wheeler (626) 395-8733 wheel@caltech.edu

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Caltech Biologists Pursue Promising New Approach in Treatment of HIV/AIDS and Cancer

PASADENA, Calif.—In response to the arduously slow progress in finding cures for AIDS and cancer, Caltech researchers are now investigating a promising new approach in the treatment of these diseases.

With a $1.5 million matching grant from the Skirball Foundation in New York, Caltech biologists have established the Engineering Immunity project, designed to create a novel immunological approach to treating–and even some day preventing–HIV infection and some cancers like melanoma.

The immune system provides humans with a powerful defense against infectious diseases–but sometimes, it fails. Utilizing an innovative, integrated approach, the Engineering Immunity project will combine gene therapy, stem cell biology, and immunotherapy to arm the immune system; this integrative methodology offers groundbreaking potential for treatment of these diseases and others for which the immune system currently fails to provide defense.

Caltech President David Baltimore, who won the Nobel Prize in 1975 for his work in virology and cancer research, stated, "The Engineering Immunity project advocates a new approach to therapy for AIDS and cancer with revolutionary implications for the treatment of these and many other diseases. It is an innovative research project that holds special significance for the future of biomedical sciences."

In the fight against HIV, the virus that causes AIDS, T-cell immunity and T-cell-focused therapies and vaccines have been methods widely investigated and pursued. However, antibodies often provide the best protection against viruses, and virtually all vaccines for other viral diseases are designed to elicit antibody-based immunity. Antibodies against HIV do appear during HIV infections, but heretofore, they had not been able to provide therapeutic advantage to most patients. Rare neutralizing antibodies have been identified, but have not proven valuable because a general way to elicit their production in all patients has not been found. Moreover, most of them are effective only at very high concentrations that are hard to maintain in a person by conventional means. Thus, early attempts to elicit antibody-based immunity against HIV have largely failed.

The Engineering Immunity integrated methodology involves utilizing retroviruses, which are natural carriers of genes. Retrovirus vectors will be produced that encode antibodies found to be effective against HIV. Utilizing retroviruses, the Baltimore Laboratory at Caltech, in collaboration with Caltech structural biologist Pamela Bjorkman, will introduce specific genes into stem cells. These genes will encode specificity molecules on the immune cells, thereby arming the immune cells to kill selected agents or cells, i.e., the cells that are growing HIV or particular cancer cells.

The Engineering Immunity initiative will provide a new route to the production of antibodies with therapeutic, and even protective, ability for a potential cure of AIDS, melanoma, and other diseases ultimately.

The Skirball Foundation, an independent foundation created in 1950 by Jack H. Skirball, is dedicated primarily to medical research and care, educational and social needs of disadvantaged children, and advancing the highest values of the Jewish heritage. Among the many institutions that the Foundation has supported are the Skirball Cultural Center, the Salk Institute, the Venice Family Clinic, the Jewish Childcare Association in New York City, and the Skirball Institute of Biomolecular Medicine at New York University.

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Contact: Deborah Williams-Hedges (626) 395-3227 debwms@caltech.edu

Visit the Caltech Media Relations Web site at: http://pr.caltech.edu/media

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Memory Lane in the Brain

PASADENA, Calif.- Biologist Erin Schuman is interested in how memories are formed--or forgotten. The landscape the professor of biology at the California Institute of Technology explores is the hippocampus, the part of the brain known to be crucial for memory in humans and other animals.

In 2002, Schuman and Miguel Remondes, her graduate student, published a paper in the journal Nature that suggested a possible role for a well-known but poorly understood part of the brain known as the temporoammonic (TA) pathway. Using rat hippocampal slices, they suggested two possible roles for the TA pathway that were not previously known: to serve as a memory gatekeeper that can either enhance or diminish memories, and to provide information to help animals know where they are in their environments.

The researchers' next step was to prove their theories by looking at a possible role for the TA in memory at a behavioral level. That is, says Remondes, now a postdoctoral fellow at MIT, "to do the real test."

To understand how memories are formed, many scientists have focused on the "trisynaptic circuit," which involves three areas of the hippocampus: input from the senses is first sent from the cortex to the dentate gyrus, where this signal is processed by two sets of synapses, then sent back to the cortex. That's the circuit. An often overlooked separate input to the hippocampus, though, is the TA pathway. It makes direct contact with the neurons that are at the last station in the trisynaptic circuit, thus short-circuiting the traditional trisynaptic pathway.

Reporting in the October 7 issue of the journal Nature, Remondes and Schuman, also an associate investigator for the Howard Hughes Medical Institute, now show they were correct in their belief that the TA Pathway is important in spatial or location memory. The scientists used rats as their experimental animal, and the Morris Water Maze, a standard test for location memory in rodents. The animals swim in a pool of opaque water until they find a hidden goal--a platform which allows them to escape the water. To find the platform, the animals rely on the geometrical relationships of cues away from the pool (e.g., on the walls of the maze). In other words, says Remondes, "they have to navigate and remember where the platform is in order to escape the water."

The researchers tested both short-term (24 hours) and long-term memory (four weeks). The TA pathway was lesioned (disabled) in one set of rats; another set was used as a control. Having learned the location of the platform, both sets of rats still remembered where it was 24 hours later. But when tested four weeks later, only the control rats remembered where it was. The lesioned rats forgot, which showed that the TA pathway played some role in the retention of long-term memories. But what was the role?

"It led to a second question," says Schuman. "Because long-term memories require something called consolidation, an exchange of information between the cortex and hippocampus, we wanted to know if the TA pathway was working in the acquisition phase of memory or in its consolidation."

Using two other groups of rats, the pair conducted a second set of tests. After confirming the rat's memory of the platform after 24 hours, one group was immediately lesioned. These animals lost their long-term memory when tested 4 weeks later, indicating to Schuman and Remondes that ongoing TA pathway activity was required on days after learning to stabilize or consolidate the long-term memory.

The second group of rats was also lesioned, but not until three weeks later. The researchers found that this group remembered the platform's location, showing their memory had already been adequately consolidated after three weeks. This proved the TA pathway is required to consolidate long-term location memory.

"These data indicate there must be a dialogue between the hippocampus and the cortex during long-term memory consolidation," says Schuman. "Clearly, the TA pathway plays an important role in this discussion." Further, she notes, "understanding the mechanisms of memory formation and retention may shed light on diseases like Alzheimers, where memory is impaired. "

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New Target for Future Therapeutic Drugs

PASADENA, Calif. - "Sometimes letting nature tell you what's important is the better way to go," says Raymond Deshaies, an associate professor of biology at the California Institute of Technology. Deshaies is referring to new work to come out of his lab and the lab of Randall King at Harvard that defies conventional thinking--they've discovered a chemical that stops a key cell function, but, more importantly, suggests a new possible target within a cell, once thought to be untenable, for future therapeutic drugs.

In a report in this week's issue of the journal Science, lead author Rati Verma, a Howard Hughes Medical Institute (HHMI) Research Specialist in the Deshaies lab, Deshaies, also an assistant investigator for the HHMI, and nine other authors report that a small molecule called ubistatin blocked an important step in the so-called cell cycle, a process fundamental to life where a cell makes duplicate copies of its own DNA for distribution to two daughter cells. Knowing how to stop cell duplication is critical in preventing diseases like cancer, when mutated cells go out of control and proliferate madly. Further, ubistatin blocked the cell cycle by preventing two proteins from interacting together. Prior to this, it was thought unlikely that a compound with low molecular weight like ubistatin--or any future drug--would have much impact on the interaction of proteins with each other.

While ubistatin has other properties that preclude it from being a drug candidate, its stoppage of the cell cycle provides an important clue for future drug development, says Deshaies. "We've found a chemical Achilles' heel in this cell pathway, at least from the viewpoint of these small molecules that comprise most therapeutic drugs."

Because the cell cycle is maddeningly complex, researchers usually pick a single pathway (a pathway is a series of chemical events within a cell that perform some task), then try to make a chemical to block it. They may find such a chemical, but often find it difficult to discover where in the pathway--the target--their drug hit. Finding the target is like finding the proverbial needle in a haystack.

Deshaies's colleague Rati Verma found the needle. Instead of using the typical "top down" approach of starting with a specific target, then looking for a drug to block it, the researchers took a "bottom up" approach of starting with a drug and then searching for the target it blocks. They decided to test a large number of molecules to see if any of them might block any step in one particular pathway called the ubiquitin-proteasome pathway (UP pathway): within the cell cycle, when a protein's job is done, another chain of proteins called ubiquitin attaches to it. That serves as a signal to yet another protein called proteasome. The proteasome, says Deshaies, is the biological equivalent of a Cuisinart. "It attaches to these ubiquitin-marked proteins, then ingests them and chews them up."

The researchers examined an entire cell, specifically that of a frog's egg. The King group decided to screen 110,000 molecules to see if any had an impact on the cell. First, they weeded out those molecules that had no effect on cellular function in the UP pathway. King attached a molecule of luciferase ("the stuff that makes a firefly light up," says Deshaies) to certain proteins that are normally destroyed during cell division. Next, he added this newly created protein (now a readily detectable biological "flashlight") to droplets of cellular material extracted from the frog's egg that had been placed in individual chambers. As the egg extract conducted its normal cell division, the luciferase flashlight was destroyed and the chambers went dark. That meant those proteins had been destroyed as part of the normal progress of the cell cycle. He then separately added the 110,000 small molecules to see if any of them would prevent the loss of the luciferase--essentially looking for a lit-up reaction chamber in a field of darkness.

Using this approach, the researchers eventually narrowed the molecules they were testing down to a few that were operating in a specific part of the pathway--downstream from where ubiquitin attaches to the soon-to-be doomed protein, but before the proteasome ingested and chewed it up. But given that numerous proteins are involved in this process, the question remained--where specifically was the molecule they were testing working? In short, where was the target?

To find out, Deshaies turned to work they had done over the last five years with ubiquitin, which examined how it interacted with various other proteins, including proteasome. Through a process of elimination, says Deshaies, "we figured out that these small molecules called ubistatins were blocking the recognition of the ubiquitin chain by the proteasome." Graphic evidence for how this occurs was provided by a 'picture' taken by David Fushman at the University of Maryland with a nuclear magnetic resonance spectrometer.

This step blocked by ubistatin involves a protein-protein interaction, a surprise to Deshaies. "One interesting thing about our discovery is that it is further evidence that you can affect a protein-protein interaction with a small molecule. The conventional thinking was that if you look at a footprint of a drug binding a protein, the drugs are small, but the footprint that corresponds to one protein binding to another is big. So most people thought that the idea of trying to block the huge footprint of protein-protein interaction with a tiny drug was extremely unlikely. So if I were asked to predict what we would find, I would never have proposed that a drug could prevent the ubiquitin chain from binding to the proteasome, because I was also influenced by this conventional wisdom."

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MW
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Caltech Bioinformatics Experts DevelopNew Literature Search Engine for Biologists

PASADENA, Calif.—When it comes to finding a used book on the Internet, one merely needs to Google the title, and a few suitable items for sale will soon be just a click away. But for the biologist or medical researcher looking for information on how two nematode genes interrelate in hopes of better understanding human disease, there is a clear need for a more focused search engine.

Bioinformatics experts from the California Institute of Technology are formally announcing today the Textpresso search engine, which they hope will revolutionize the way that genetic information is retrieved by researchers worldwide. The Textpresso search engine is specifically built to serve researchers who work on the small worm known as C. elegans, but the basic design should lead to the creation of new search engines for researchers who specialize in other living organisms that are intensively studied.

In the current issue of the journal PLOS, published by the Public Library of Science, Caltech biology professor Paul Sternberg and his colleagues--research associate Hans-Michael Muller and bioinformatics specialist Eimear Kenny--write that the new "text-mining system for scientific literature" will be invaluable to specialists trying to cope with the vast amount of information now available on C. elegans. This information has vastly increased in recent years due to the large-scale gene-sequencing initiative as well as the more traditional small-scale projects by individual researchers. As a result, the need for a way to scan the vast literature has become much more important.

"Textpresso gives me, as a researcher, more time to actually read papers because I don't have to skim papers anymore," says Sternberg, who is leader of the federally funded WormBase project that has already put online the entire genome sequence of C. elegans and the closely related organism C. briggsae, as well as genes for some 20 other nematode species.

The four-year-old WormBase project, a linchpin in the worldwide effort to better understand how genes interrelate, also makes a host of other information freely available in addition to the 100.2 million base-pairs that make up the millimeter-long worm's genome. There are now 28,000 gene-disruption experiments in WormBase, along with 2 million DNA expression ("chip") microarray observations, as well as detailed information on the expression of more than 1,700 of the worm's 20,000 genes. The Textpresso search engine is a logical product for the WormBase team to develop in the ongoing quest to put genetic information to work in curing and preventing human disease.

Lest anyone assume that the genes of a millimeter-long nematode have little to do with humans, it should be pointed out that the two organisms are similar in about 40 percent of their genes. A very realistic motivation for funding the genome sequencing of the fruit fly, the small mustardlike plant known as Arabidopsis, the chimp, and various other species, has been the expectation of finding underlying common mechanisms.

Thus, a cancer researcher who discovers that a certain gene is expressed in cancer cells can use the WormBase to see if the gene exists in nematodes, and if so, what is known about the gene's function. And now that Textpresso is available, the researcher can do so much more efficiently.

"The idea is distilling down the information so it can be extracted easier," says Muller, the lead author of the paper and codeveloper of Textpresso with Kenny. The idea for the name of the search engine, in fact, comes from its resemblance to "espresso," which is a process used to get the caffeine and flavor out of coffee in a minimal volume.

According to Kenny, the search engine is designed with a special kind of search in mind, which establishes categories of terms and organizes them as an ontology--that is, a catalog of types of objects and concepts and their relationships. For example, if the researcher wants to find out whether any other researcher has worked on the relationship between the nematode gene called "lin-12" and the anchor cell, then typing the two terms into a conventional search engine like Google results in more than 400 hits. And if the researcher wants to know which genes are important in the anchor cell, the task is even more arduous. But Textpresso is designed to get the information in a much simpler, more efficient, more straightforward way.

Textpresso is a text-processing system that splits research papers into sentences, and sentences into words or phrases. All words and phrases are labeled so that they are searchable, and the labels are then condensed into 33 ontological categories. So far, the database includes 4,420 scientific papers on C. elegans, as well as bibliographic information from WormBase, information on various scientific meetings, the "Worm Breeder's Gazette," and various other links and WormBase information. Therefore, the engine already searches through millions of sentences to allow researchers to find a paper of interest or information of interest with great efficiency.

Finally, the Textpresso search engine should be a useful prototype for search engines to serve other biological databases--some of which have even larger piles of data for the specialist to cope with. "Yeast currently has 25,000 papers," Kenny says.

Textpresso can be accessed at www.textpresso.org or via WormBase at www.wormbase.org.

Writer: 
Robert Tindol
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