Gain and Loss in Optimistic Versus Pessimistic Brains

PASADENA, Calif.—Our belief as to whether we will likely succeed or fail at a given task—and the consequences of winning or losing—directly affects the levels of neural effort put forth in movement-planning circuits in the human cortex, according to a new brain-imaging study by neuroscientists at the California Institute of Technology (Caltech). 

A paper about the research—led by Richard A. Andersen, the James G. Boswell Professor of Neuroscience at Caltech—appears in the August issue of PLoS Biology.

Research in Andersen's laboratory includes work to understand the neural mechanisms of action planning and decision-making. The lab is working toward the development of implanted neural prosthetic devices that would serve as an interface between severely paralyzed individuals' brain signals and artificial limbs—allowing their planned actions to control the limbs' movements.

 

In particular, Andersen's group focuses on a high-level area of the brain called the posterior parietal cortex (PPC), where sensory stimuli are transformed into movement plans.

In the current study, Andersen and his colleagues used a functional magnetic resonance imaging scanner to monitor activity in the PPC and other brain areas in subjects who were asked to perform a complex task. Using a trackball, they had to move a cursor to a number of memorized locations on a computer screen, in a predetermined order. 

"The subjects were given 1 second to memorize the sequence, 15 seconds to plan their movements in advance, and then only 10 seconds to finish the task," says Igor Kagan, a senior research fellow in biology in the Andersen lab, and a coauthor of the PLoS Biology paper. "We intentionally made the task hard—I couldn't do it myself," he says. 

The subjects received monetary compensation for participating in the experiment, with their earnings tied to their performance. The amount of money that would be gained (or lost) varied from trial to trial. In one trial, for example, success might net the participant $5, while failure would cause him to lose $1. In another trial, completing the task correctly would earn $1, while failure would cost $5. Alternatively, success and failure might produce an equivalent gain or loss (say, +$5 versus -$5). The subjects were told the stakes in advance of each trial.

Prior to receiving their earnings, the subjects reported—in a post-test questionnaire—how they perceived their performance. Interestingly, those perceptions did not correlate with their actual performance; individuals in the group who believed they had performed well were just as likely to have performed poorly, and vice versa for individuals in the group who believed they had done badly.

Furthermore, the researchers found that the pattern of brain activity in the PPC was linked to how well the subjects believed they had done on the tasks—that is, their subjective perception of their performance, rather than their actual performance—as well as by the monetary gain or loss they expected from success or failure. 

How hard an individual subject's brain "worked" at the task was dependent upon their personal approach. For example, Andersen says, "subjects who are 'optimists' and believe they are doing well will put out the most effort—and exhibit an increase in activity in their PPC—when they expect to earn a larger reward for being successful." Conversely, those individuals who believe they are doing poorly—the pessimists—show the most brain activity when there is a higher price for failure.

"They're trying harder to avoid losses and seem to care less about potential gains," Kagan adds.

"This study demonstrates that the process of planning and action is influenced by our subjective, but often incorrect, idea of how well we are doing, as well as by the potential gain or loss," Andersen says. The results suggest that the cortical areas involved in planning actions are also likely to be involved in decision-making, and take into account higher-order cognitive as well as subjective factors when deciding among potential actions.  

The paper, "Motor Preparatory Activity in Posterior Parietal Cortex is Modulated by Subjective Absolute Value," was also coauthored by former Caltech graduate student Asha Iyer, the first author of the study, now a resident at Mount Sinai Medical School, and former Caltech postdoctoral fellow Axel Lindner, now a group leader at the University of Tübingen. The research was funded by the Gordon and Betty Moore Foundation, the James G. Boswell Foundation, and the National Eye Institute.

 

Writer: 
Kathy Svitil
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Of Bugs & Brains: Caltech Researchers Discover that Gut Bacteria Affect Multiple Sclerosis

PASADENA, Calif.—Biologists at the California Institute of Technology (Caltech) have demonstrated a connection between multiple sclerosis (MS)—an autoimmune disorder that affects the brain and spinal cord-and gut bacteria.

The work—led by Sarkis K. Mazmanian, an assistant professor of biology at Caltech, and postdoctoral scholar Yun Kyung Lee—was published July 26 in the early online edition of the Proceedings of the National Academy of Sciences.

Multiple sclerosis results from the progressive deterioration of the protective fatty myelin sheath surrounding nerve cells. The loss of myelin hinders nerve cells from communicating with one another, leading to a host of neurological symptoms including loss of sensation, muscle spasms and weakness, fatigue, and pain. Multiple sclerosis is estimated to affect about half a million people in the United States alone, with rates of diagnosis rapidly increasing. There is currently no cure for MS.

Although the cause of MS is unknown, microorganisms seem to play some sort of role. "In the literature from clinical studies, there are papers showing that microbes affect MS," Mazmanian says. "For example, the disease gets worse after viral infections, and bacterial infections cause an increase in MS symptoms."

On the other hand, he concedes, "it seems counterintuitive that a microbe would be involved in a disease of the central nervous system, because these are sterile tissues."

And yet, as Mazmanian found when he began examining the multiple sclerosis literature, the suggestion of a link between bacteria and the disease is more than anecdotal. Notably, back in 1993, Caltech biochemist Leroy Hood—who was then at the University of Washington—published a paper describing a genetically engineered strain of mouse that developed a lab-induced form of multiple sclerosis known as experimental autoimmune encephalomyelitis, or EAE.

When Hood's animals were housed at Caltech, they developed the disease. But, oddly, when the mice were shipped to a cleaner biotech facility—where their resident gut bacterial populations were reduced—they didn't get sick. The question was, why? At the time, Mazmanian says, "the authors speculated that some environmental component was modulating MS in these animals." Just what that environmental component was, however, remained a mystery for almost two decades.

But Mazmanian—whose laboratory examines the relationships between gut microbes, both harmful and helpful, and the immune systems of their mammalian hosts—had a hunch that intestinal bacteria were the key. "As we gained an appreciation for how profoundly the gut microbiota can affect the immune system, we decided to ask if symbiotic bacteria are the missing variable in these mice with MS," he says.

To find out, Mazmanian and his colleagues tried to induce MS in animals that were completely devoid of the microbes that normally inhabit the digestive system. "Lo and behold, these sterile animals did not get sick," he says.

Then the researchers decided to see what would happen if bacteria were reintroduced to the germ-free mice. But not just any bacteria. They inoculated mice with one specific organism, an unculturable bug from a group known as segmented filamentous bacteria. In prior studies, these bacteria had been shown to lead to intestinal inflammation and, more intriguingly, to induce in the gut the appearance of a particular immune-system cell known as Th17. Th17 cells are a type of T helper cell—cells that help activate and direct other immune system cells. Furthermore, Th17 cells induce the inflammatory cascade that leads to multiple sclerosis in animals.

"The question was, if this organism is inducing Th17 cells in the gut, will it be able to do so in the brain and central nervous system?" Mazmanian says. "Furthermore, with that one organism, can we restore to sterile animals the entire inflammatory response normally seen in animals with hundreds of species of gut bacteria?"

The answer? Yes on all counts. Giving the formerly germ-free mice a dose of one species of segmented filamentous bacteria induced Th17 not only in the gut but in the central nervous system and brain—and caused the formerly healthy mice to become ill with MS-like symptoms.

"It definitely shows that gut microbes have a strong role in MS, because the genetics of the animals were the same. In fact, everything was the same except for the presence of those otherwise benign bacteria, which are clearly playing a role in shaping the immune system," Mazmanian says. "This study shows for the first time that specific intestinal bacteria have a significant role in affecting the nervous system during MS—and they do so from the gut, an anatomical location very, very far from the brain."

Mazmanian and his colleagues don't, however, suggest that gut bacteria are the direct cause of multiple sclerosis, which is known to be genetically linked. Rather, the bacteria may be helping to shape the immune system's inflammatory response, thus creating conditions that could allow the disease to develop. Indeed, multiple sclerosis also has a strong environmental component; identical twins, who possess the same genome and share all of their genes, only have a 25 percent chance of sharing the disease. "We would like to suggest that gut bacteria may be the missing environmental component," he says.

For their part, Th17 cells are needed for the immune system to properly combat infection. Problems only arise when the cells are activated in the absence of infection—just as disease can arise, Mazmanian and others suspect, when the species composition of gut bacteria become imbalanced, say, by changes in diet, because of improved hygiene (which kills off the beneficial bacteria as well as the dangerous ones), or because of stress or antibiotic use. One impact of the dysregulation of normal gut bacterial populations—a phenomenon dubbed "dysbiosis"—may be the rising rate of multiple sclerosis seen in recent years in more hygienic societies.

"As we live cleaner, we're not just changing our exposure to infectious agents, but we're changing our relationship with the entire microbial world, both around and inside us, and we may be altering the balance between pro- and anti-inflammatory bacteria," leading to diseases like MS, Mazmanian says. "Perhaps treatments for diseases such as multiple sclerosis may someday include probiotic bacteria that can restore normal immune function in the gut... and the brain."

The paper, "Pro-inflammatory T-cell responses to gut microbiota promote experimental autoimmune encephalomyelitis," was coauthored by former Caltech postdoctoral scholar Juscilene Menezes, now at the Scripps Research Institute, and Yoshinori Umesaki of the Yakult Central Institute for Microbiological Research in Japan; Lee and Menezes contributed equally to the work. The work was supported by funding from the California Institute of Technology, the Weston Havens Foundation, and the Edward Mallinckrodt, Jr. Foundation.

Writer: 
Kathy Svitil
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Caltech Biologists Discover How T Cells Make a Commitment

PASADENA, Calif.—When does a cell decide its particular identity? According to biologists at the California Institute of Technology (Caltech), in the case of T cells—immune system cells that help destroy invading pathogens—the answer is when the cells begin expressing a particular gene called Bcl11b.

The activation of Bcl11b is a "clean, nearly perfect indicator of when cells have decided to go on the T-cell pathway," says Ellen Rothenberg, the Albert Billings Ruddock Professor of Biology at Caltech and senior author of a paper about the discovery that appears in the July 2 issue of the journal Science. The paper, coauthored by Caltech postdoctoral scholar Long Li, is one of three in the issue to examine this powerful gene. 

The Bcl11b gene produces what is known as a transcription factor—a protein that controls the activity of other genes. Specifically, the gene is a repressor, which means it shuts off other genes. This is crucial for T cells, because T cells are derived from multipotent hematopoietic stem cells—stem cells that express a wide variety of genes and have the capacity to differentiate into a host of other blood cell types, including the various cells of the immune system. 

"Stem cells and their multipotent descendents follow one set of growth rules, and T cells another," says Rothenberg, "so if T-cell precursors don't give up certain stem-cell functions, bad things happen." Like stem cells, T cells have a remarkable ability to grow—but as part of their T-cell-ness, she says, they do so "under incredibly strict regulation. Their growth is restricted unless certain conditions are met." The cells need to shift their growth-control rules during development; after development, because they still need to grow, the cells and their daughters need an active mechanism to make the change irreversible. Bcl11b is a long-sought part of that mechanism. 

"For cells that never divide again, maintaining identity is trivial. What they are at that moment is what they are forever," Rothenberg says. Once T cells mature, their abilities to keep dividing and migrating around the body also give them the opportunity to have their daughters adopt different roles in the immune system as they encounter and interact with other types of cells. "Even so, their central T-cell nature remains unchanged, which means that they must have a strong sense of identity," she adds. 

The conversion from T-cell precursors to actual T cells takes place in the thymus, a specialized organ located near the heart. "When the future T cells move into the thymus," Rothenberg explains, "they are expressing a variety of genes that give them the option to become other cells," such as mast cells (which are involved in allergic reactions), killer cells (which kill cells infected by viruses), and antigen-presenting cells (which help T cells recognize targeted foreign cells). 

As they enter the thymus, the organ sends molecular signals to the cells, directing them down the T-cell pathway. At this point, the Rothenberg lab found, the Bcl11b gene gets turned on. Li, the lead author on the Science paper, found that this confirms the T cells' identity by blocking other pathways. The Bcl11b protein is also needed for the cells to make the break from their stem-cell heritage. "It is like a switch that allows the cells to shut off stem-cell genes and other regulatory genes," Rothenberg says. "It keeps them clean—and may be necessary to 'guard' the T cell from becoming some other type of cell." 

Although it is thought that many genes are involved in the process of creating and maintaining T cells, "Bcl11b is the only regulatory gene in the whole genome to be turned on at this stage," she adds, "and it is probably always active in all T cells. It is the most T-cell specific of all of the regulatory factors discovered so far." Among blood cells, this gene is only expressed in T cells, she says. "The gene is used in other cells in completely different types of tissue, such as brain and skin and mammary tissue, but that’s how the body works. There's no confusion, because something like brain tissue and mammary tissue will never be a T cell." 

When Bcl11b is not present—as in mice genetically altered to lack the gene—T cells "don't turn out right," Rothenberg says. Indeed, T cells in individuals with T-cell leukemia have been found to lack the gene. "It may make them more susceptible to the effects of radiation, because the cells don’t know when to stop growing," she says. "We think that the loss of one of the two copies of the gene is enough to prevent cells from growing appropriately."

The work in the paper, "An Early T Cell Lineage Commitment Checkpoint Dependent on the Transcription Factor Bcl11b" —coauthored by Rothenberg, Li, and Mark Leid of Oregon State University—was supported by a California Institute for Regenerative Medicine fellowship to Li, and by the National Institutes of Health, the Caltech–City of Hope Biomedical Research Initiative, the Louis A. Garfinkle Memorial Laboratory Fund, the Al Sherman Foundation, and the Albert Billings Ruddock Professorship.

Writer: 
Kathy Svitil
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Caltech Researchers Show How Active Immune Tolerance Makes Pregnancy Possible

Understanding of mouse immune-system response to specific fetal antigens also may provide insight into issues that arise during human pregnancies

PASADENA, Calif.—The concept of pregnancy makes no sense—at least not from an immunological point of view. After all, a fetus, carrying half of its father's genome, is biologically distinct from its mother. The fetus is thus made of cells and tissues that are very much not "self"—and not-self is precisely what the immune system is meant to search out and destroy.

Women's bodies manage to ignore this contradiction in the vast majority of cases, making pregnancy possible. Similarly, scientists have generally paid little attention to this phenomenon—called "pregnancy tolerance"—and its biological details.

Now, a pair of scientists from the California Institute of Technology (Caltech) have shown that females actively produce a particular type of immune cell in response to specific fetal antigens—immune-stimulating proteins—and that this response allows pregnancy to continue without the fetus being rejected by the mother's body.

Their findings were detailed in a recent issue of the Proceedings of the National Academy of Sciences (PNAS).

“Our finding that specific T regulatory cells protect the mother is a step to learning how the mother avoids rejection of her fetus. This central biological mechanism is important for the health of both the fetus and the mother,” says David Baltimore, Caltech's Robert Andrews Millikan Professor of Biology, recipient of the 1975 Nobel Prize in Physiology or Medicine, and the principal investigator on the research.

Scientists had long been "hinting around at the idea that the mother's immune system makes tolerance possible," notes paper coauthor Daniel Kahn, a visiting associate in biology at Caltech, and an assistant professor of maternal–fetal medicine at the University of California, Los Angeles (UCLA). What they didn't have were the details of this tolerance—or proof that it was immune-related.

Now they do. To pin down those details, the two scientists began looking at the immune system's T regulatory cells (Tregs) in a strain of inbred mice that are all genetically identical—except for one seemingly tiny detail. Male mice—including male fetuses—carry on their cells' surfaces a protein known as a "minor transplantation antigen." Female mice lack this antigen.

Under normal circumstances, this antigen's existence isn’t a problem for the male fetuses because the pregnancy tolerance phenomenon kicks in and protects them from any maternal immune repercussions.

To demonstrate the role of Tregs, Baltimore and Kahn used a drug to selectively target and destroy the cells. If the Tregs were indeed the source of pregnancy tolerance, they reasoned, their destruction would give the immune system free rein to go after the antigen-laden fetuses.

"In this case," says Kahn, "we knew the only possible immune response would be against the males—that the males would be at risk."

Indeed they were. When Baltimore and Kahn looked at the offspring of mice who'd been treated with the toxin, they found that fewer of the male fetuses survived to birth; those males that did survive were of significantly lower birthweight, presumably because of the inflammation caused by the mother's immune response to that single antigen.

"These T cells are functioning in an antigen-specific manner," Kahn notes. "In other words, their function requires the presence of the specific fetal antigens."

In their studies of these animals, the scientists also found that pregnancy tolerance "develops actively as a consequence of pregnancy," says Kahn. "The mice are not born with it." Indeed, virgin mice showed no signs of these pregnancy-specific Treg cells. Conversely, the cells were found in larger numbers in those individual mice that had given birth to more male babies, with the level of Treg cells increasing with the number of male births.

The next step, Kahn adds, is to look at Tregs and their role in pregnancy tolerance in humans—a line of research that may lead to new insights into such pregnancy-related conditions as preeclampsia, in which high blood pressure and other symptoms develop in the second half of pregnancy. Preeclampsia is a major cause of maternal mortality around the world.

"There's a lot to be learned," he says. "Pregnancy is often ignored in research because it's usually successful, and because—from an immunologic standpoint—it has such complexity. Until now, it's been difficult to grab a handle on how the immunology of pregnancy really works."

The work described in the PNAS article, "Pregnancy induces a fetal antigen-specific maternal T regulatory cell response that contributes to tolerance," was supported in part by a research grant from the Skirball Foundation. Kahn is supported by the National Institutes of Health's Building Interdisciplinary Research Careers in Women's Health Center at UCLA.

Writer: 
Lori Oliwenstein
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Caltech Biologists Provide Molecular Explanation for the Evolution of Tamiflu Resistance

PASADENA, Calif.—Biologists at the California Institute of Technology (Caltech) have pinpointed molecular changes that helped allow the global spread of resistance to the antiviral medication Tamiflu (oseltamivir) among strains of the seasonal H1N1 flu virus. 

The study—led by David Baltimore, Caltech's Robert Andrews Millikan Professor of Biology and recipient of the 1975 Nobel Prize in Physiology or Medicine, and postdoctoral scholar Jesse D. Bloom—appears in the June 4 issue of the journal Science

Tamiflu and other antiviral drugs directly target viruses, unlike vaccines, which instead stimulate our body's immune system to respond to the pathogens after an infection is established. 

In a flu infection, viruses bind to sialic acid on the surface of a host cell using a protein called hemagglutinin (the "H" in H1N1). The viruses then enter the cell and replicate. When the newly minted viruses exit the cell, they too bind to sialic acid. The viruses then use a protein called neuraminidase (the "N" in H1N1) to cut the sialic acid, freeing themselves to infect new cells. 

This process, however, is blocked by Tamiflu, which prevents neuraminidase from cleaving the sialic acid. "It does this by binding in the 'active site' of the neuraminidase molecule, where neuraminidase normally cleaves sialic acid," Bloom says.

In general, for a virus to become resistant to Tamiflu, the neuraminidase protein has to be able to tell the difference between sialic acid (the thing it cleaves) and Tamiflu (the drug "decoy").

Such recognition is possible in viruses that have a mutation, known as H274Y, in the neuraminidase protein. The mutation swaps out one amino acid for another at a particular location on the neuraminidase protein, producing a slight conformational change in a crucial region of the protein’s three-dimensional structure. "Once that happens," Bloom says, "the neuraminidase no longer strongly binds to Tamiflu, and it is still able to cleave sialic acid.” 

"People have known about this H274Y mutation for over a decade," he adds, "but the mutation seemed to interfere with the virus’s ability to replicate and be transmitted. The molecular basis for that interference was not clear, but it seemed that the H274Y viruses weren’t of great clinical significance."

However, during the 2007-2008 flu season, resistant H1N1 viruses with the H274Y mutation began cropping up all over the world. By the following year, essentially all seasonal H1N1 flu viruses suddenly were resistant to Tamiflu because of the mutation.

The only difference: They now were growing just as well as regular viruses.

"We thought it was an interesting evolutionary mystery," Bloom says. "Something happened to make the Tamiflu-resistant virus also capable of replicating and spreading like wild-type flu viruses." The question was, what?

The first step in finding out was to determine why the H274Y mutation usually hampers the growth and spread of a virus.

"Our hypothesis," Bloom says, "was that the resistance mutation was—as an incidental effect—preventing neuraminidase from reaching the cell membrane." This decreased availability of neuraminidase—the protein, remember, that cleaves newly formed viruses from their sialic-acid mooring on the host cell, allowing them to spread to infect other cells—decreased the rate of viral replication. The researchers confirmed this in cell cultures.

"Now, if you've got a second mutation that fixes this problem in H274Y mutants," Bloom says, "you'll have a virus that grows very well and is resistant to Tamiflu. And that's bad—for us, not the virus."

The researchers discovered just such a secondary mutation—two of them, in fact—in the neuraminidase gene of Tamiflu-resistant seasonal flu strains dating from the 2007-2008 flu season.

Interestingly, an examination of flu sequences showed that the two secondary mutations had cropped up before the H274Y mutation had begun to spread. The existence of these "pre-adaptive mutations," say the researchers, permitted the survival and spread of subsequent occurrences of the H274Y mutation.

Genetic changes that set the stage for later adaptations may represent a fairly common event in evolution.

"This study shows how combining an understanding of molecular mechanisms underlying evolution with the extensive sequencing data on historical isolates of influenza virus can bring about a deeper understanding of the challenge that this virus presents to the human population," says Baltimore. "Only by marshaling a wide range of available information was it possible to understand why the virus could suddenly tolerate mutations that were previously deleterious. It shows that mutations are not necessarily 'good' or 'bad,' but that their effects may depend on the context in which they appear." 

So far, the H274Y mutation has not become widespread in either the avian H5N1 influenza or the recent swine-origin influenza pandemic, although it has cropped up in isolated cases. "We hope that understanding the basis of the evolution of Tamiflu resistance in seasonal H1N1 might help in understanding what might be needed for H274Y to spread widely in these other strains as well," Bloom says.

The paper, "Permissive Secondary Mutations Enable the Evolution of Influenza Oseltamivir Resistance," was coauthored by Duke University undergraduate student Lizhi Ian Gong, who worked on the study at Caltech as part of a Summer Undergraduate Research Fellowship. The research was supported by a Beckman Institute Postdoctoral Fellowship and the Irvington Institute Fellowship Program of the Cancer Research Institute.

Writer: 
Kathy Svitil
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Caltech Researchers Identify Genes and Brain Centers That Regulate Meal Size in Flies

PASADENA, Calif.—Biologists from the California Institute of Technology (Caltech) and Yale University have identified two genes, the leucokinin neuropeptide and the leucokinin receptor, that appear to regulate meal sizes and frequency in fruit flies. Both genes have mammalian counterparts that seem to play a similar role in food intake, indicating that the steps that control meal size and meal frequency are not just behaviorally similar but are controlled by the same genes throughout the animal kingdom.

A paper describing the work will appear in the June 8 issue of the journal Current Biology.

In animals, food intake is regulated to keep body weight constant over a long period of time. Most animals consume food in discrete bouts—that is, in meals. "Identifying the genes and molecules that regulate meal-related parameters is essential for understanding the relationships between body weight and caloric intake," says Bader Al-Anzi, a research scientist at Caltech and the lead author of the Current Biology study.

In hungry animals, meal size and frequency are regulated in three phases. In the first phase, the smell and taste of food initiates feeding. Once a meal has begun, other factors assure that the feeding bout will continue for given period of time (representing the second phase). In the final phase, feeding is terminated—usually when the amount of stomach distension passes a given threshold. The three phases of feeding behavior have been observed in animals ranging from mammals to insects. However, what was unknown was whether similarities in behavior actually reflected an evolutionarily conserved process that employed similar genes and molecules across animal species.

To help answer this question, Al-Anzi and his colleagues developed an assay to examine feeding behavior in the common fruit fly, Drosophila melanogaster. In this assay, genetically normal flies were starved for one day and then transferred into a vial containing sugar meal mixed with red food dye. Invariably, the flies became satiated during their exposure to red food, and their small abdomens turned red.

Next, the researchers performed the same experiment using mutant fly strains. "Our hope," says Al-Anzi, "was that if flies contained mutations in genes involved in meal regulation, those flies would eat excessive amounts of red food, making them visibly bloated with red abdomens."

Two mutant fly strains produced notable results. One strain contained a mutation in the gene encoding the leucokinin neuropeptide (a peptide initially identified for its ability to induce insect gut contraction), and the second strain contained mutated versions of the receptor that binds to leucokinin. In the assay, both types of fly mutants ate to such excess that they became visibly bloated, with their crops—food storage organs—stretched to the limit with red-dyed food.

Surprisingly, Al-Anzi says, "although in the short term these flies tend to overeat, in the long run they consume a similar amount of food as normal flies. This was largely due to the fact that they are compensating for the large increase in meal size by reducing the number of times they eat." Whereas mutant flies consumed four or five large meals in a single day, normal flies ate seven or eight small meals.

In additional experiments, Al-Anzi and his colleagues found that although the leucokinin neuropeptide is found exclusively in the brain, the leucokinin receptor is found in neurons located in both the brain and the foregut—an area of the gut that contains stretch receptors known to be responsible for monitoring meal size in other insects. The researchers also found that introducing a normal copy of the leucokinin neuropeptide or of the leucokinin receptor gene to these neurons in their corresponding mutant flies fully restored normal feeding behavior.

Furthermore, when these same neurons were destroyed in normal, nonmutant flies, the flies began to consume abnormally large meals, just like mutants. "This proves that we identified the right genes responsible for the flies' bingeing as well as the fly brain center that regulates meal size and frequency," Al-Anzi says.

These results suggest that in normal flies, the stretch receptors signal to the brain that it is time to stop eating when the gut becomes full. In flies in which the leucokinin neuropeptide or leucokinin receptors are not functioning properly as result of mutations or the destruction of the brain centers that express the genes, the "time to stop" signal isn't properly relayed, and the flies—unaware that their bellies are full—continue to eat.

Both leucokinin and its receptor are homologous to tachykinins—vertebrate pathway genes known to cause a reduction in food intake when injected into the brain. Indeed, some tachykinin pathway genes are expressed within or close to mammalian brain centers that regulate body weight and food intake, including a region known as the arcuate nucleus. The observation that a fly tachykinin plays a similar role in food-intake regulation indicates an evolutionarily conserved role for this signaling system in controlling food intake.

"Despite our disparate body forms, the functions of many genes are conserved across the animal kingdom-including in the lowly fruit fly," Al-Anzi says. Because of this, he says, "if we know what a given gene does in a fly, it is likely that its counterpart in humans would play a similar role. However," he adds, "I was still surprised that this conservation extends even to behavioral phenomena like meal-size regulation. The fruit fly is a powerful model organism for studying the genetic basis of many biological phenomena, and the evolutionarily conserved role of the leucokinin pathway in meal-size regulation indicates that, when it comes to food intake, we can now further exploit the genetic powers of Drosophila to understand the molecular basis of food intake regulation in humans."

The paper, "The Leukokinin Pathway and Its Neurons Regulate Meal Size in Drosophila," is the result of research originally led by Caltech biologist Seymour Benzer, a pioneer in the study of genes and behavior. Kai Zinn, professor of biology at Caltech, continued this research with Al-Anzi after Benzer's death in late 2007. In addition to Al-Anzi, Zinn, and Benzer, the other authors on the Current Biology paper are Caltech laboratory assistant Elena Armand and research technician Viveca Sapin; Christopher Waters and Paul Nagami, formerly of Caltech; and Margaret Olszewski and Robert J. Wyman of Yale University. The work was supported by the National Institutes of Health and a Life Sciences Resources Foundation grant from Bristol-Myers Squibb.

Writer: 
Kathy Svitil
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Flower Organ's Cells Make Random Decisions that Determine Size

Caltech-led team provides evidence of key roles for cell-cycle length and chromosome duplication without division

PASADENA, Calif.—The sepals of the plant Arabidopsis thaliana—commonly known as the mouse-eared cress—are characterized by an outer layer of cells that vary widely in their sizes, and are distributed in equally varied patterns and proportions.

Scientists have long wondered how the plant regulates cell division to create these patterns—in other words, how it decides which and how many cells will be large, which slightly smaller, and which very small.

Melding time-lapse imaging and computer modeling, a team of scientists led by biologists from the California Institute of Technology (Caltech) has provided a somewhat unexpected answer to this question.

"We conclude that probabilistic decisions of individual cells—rather than organ-wide mechanisms—can produce a characteristic and robust cell-size pattern in development," says Elliot Meyerowitz, the George W. Beadle Professor of Biology and chair of the Division of Biology at Caltech.

These findings were published on May 11 in the online journal PLoS Biology.

A plant's sepals are the small green leaf-like organs that cup the petals of a flower, enclosing and protecting the flower before it blooms. The outer layer, or epidermis, of the Arabidopsis sepal consists of cells of widely varying sizes. These cells range in size from very small to very large; the largest cells are a type found only in the sepals and are dubbed, appropriately, "giant cells."

Each of the four sepals that cup an Arabidopsis flower has a unique pattern of cell sizes. What the Caltech-led team wanted to find out was what determines this pattern.

To gain insight into the process, Meyerowitz and Caltech postdoctoral scholar Adrienne Roeder imaged sepals during their early development. They tracked each round of cell divisions to determine how the different cell sizes were created, and what influences their distribution pattern.

They then worked with senior postdoctoral scholar Vijay Chickarmane, who had designed a computer model to test the team's hypotheses about how the observed size-related patterning in the sepals comes to pass.

"We started using the live imaging of the sepals to gather data to make a hypothesis about the patterning," Roeder explains. "Then we ran that hypothesis as a model in the computer, to see whether it would give us the patterns we were seeing in the imaging."

At first, the computer model was unable to produce the patterns found in the actual sepals. So the team tweaked the model until it independently produced the range of cell sizes the team had seen in the living organ.

They found the sepal generates its epidermal cell-size pattern based not on an organ-wide control mechanism, but on when or whether each individual cell decides to divide and on the length of its cell cycle. This sort of random, probabilistic development process results in sepal patterns that not only differ from flower to flower, but from sepal to sepal within an individual flower.

"This is so contrary to our normal way of thinking," says Roeder, "in which we assume that there's always something dictating exactly what each cell is going to do."

Scanning electron micrographs of an Arabidopsis sepal shows that the outer surface contains cells in a wide range of sizes from the highly elongated giant cells (falsely colored in red) to a variety of smaller cells.
Credit: Caltech/Adrienne Roeder

Cells in the sepal can undergo one of two growth cycles. The first is normal cell division, in which the cell duplicates its chromosomes and then splits into two smaller cells. The other is a specialized type of cell cycle called endoreduplication, in which the cell duplicates its chromosomes but does not split in two; instead, it simply continues to grow ever larger.

The team's original hypothesis was that "the earlier a cell decides to endoreduplicate, the longer it will have to grow," says Roeder. "And the more endocycles it goes through, the bigger it will get."

For a cell to become a giant cell, she explains, it will generally need to endoreduplicate during its first cell cycle. If it waits a cycle or two to stop dividing, it will have less time to grow, and thus will be a slightly smaller cell. Cells that never endoreduplicate—i.e., cells that continue to divide with each cell cycle—will be among the smallest cells in the sepal.

"Each cell starts out with a chance to become a giant cell," Roeder says. "It's a probabilistic thing; each cell has a certain probability of making that decision. Once it makes the decision, however, its fate is determined."

But endoreduplication isn't the only thing that decides the ultimate size of a sepal cell, the research team found. A cell that endoreduplicates early can grow to be an even larger giant if its cell cycles are longer than average, giving it plenty of growing time.

To prove their point, the team performed a series of experiments in which they altered the levels of cell-cycle inhibitors in the sepal cells. When they decreased the inhibitor—increasing the frequency with which the cell divides, and thus reducing the length of the cell cycle—the sepal cells were unable to grow into giant cells.

"These findings back up our hypothesis," says Roeder. "And when you change the parameters in the computer model, as if you were reducing the level of a cell-cycle inhibitor, the model shows the same pattern."

Understanding exactly how sepal cells decide whether to grow big or small could some day lead to practical applications, Roeder notes. For instance, the utility of various crops as biofuels depends on how much cellulose they contain. A sepal with a large number of giant cells has much less cell-wall surface area than a sepal with lots of smaller cells; since the cell wall is where cellulose is found, giant-cell-laden sepals would be less useful as biofuel.

"This work gives us ideas about how growth happens in these plants," says Roeder. "And once we better understand plant growth and cell division, we can better manipulate them."

In addition to Meyerowitz, Roeder, and Chickarmane, the other authors on the PLoS Biology paper, "Variability in the Control of Cell Division Underlies Sepal Epidermal Patterning in Arabidopsis thaliana," were Caltech computational scientist Alexandre Cunha; Boguslaw Obara, formerly at the University of California, Santa Barbara (UCSB), and now at the University of Oxford in the United Kingdom; and B.S. Manjunath from UCSB.

Their work was funded by a Helen Hay Whitney Foundation fellowship; the Gordon and Betty Moore Foundation Cell Center at Caltech; a grant from the Division of Chemical Sciences, Geosciences, and Biosciences in the Department of Energy's Office of Basic Energy Sciences; and the National Science Foundation.

Writer: 
Lori Oliwenstein
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Caltech-led Team Uncovers New Functions of Mitochondrial Fusion

Finds cells without mitochondrial fusion have less mtDNA, more mutations in mtDNA, less ability to tolerate mutations

PASADENA, Calif.— A typical human cell contains hundreds of mitochondria—energy-producing organelles—that continually fuse and divide. Relatively little is known, however, about why mitochondria undergo this behavior.

In a paper published in the April 16 issue of the journal Cell, a team of researchers—led by scientists at the California Institute of Technology (Caltech)—have taken steps toward a fuller understanding of this process by revealing just what happens to the organelle, its DNA (mtDNA), and its energy-producing ability when mitochondrial fusion fails.

In the process, the researchers show that fusion (the merging of two mitochondria) is "highly protective, allowing the mitochondria to tolerate very high loads of mitochondrial DNA mutations," says David Chan, associate professor of biology at Caltech and a Howard Hughes Medical Institute (HHMI) investigator.

These findings, Chan adds, help to shed light on the pathogenesis behind human mitochondrial encephalomyopathies—a class of neuromuscular diseases caused by mutations in mtDNA. In these diseases, muscle weakness occurs due to the loss of energy production by mitochondria.

When first discovered, mitochondrial fusion was thought simply to control the shape of mitochondria. And indeed, Chan says, that is at least partially the case. "If you don’t have fusion to balance division, the mitochondria get smaller and smaller as they divide," he explains.

But what hadn't been appreciated in the past, he says—and what the research described in the Cell paper makes clear—is that these smaller mitochondria undergo much more than a cosmetic change. "We've showed that in mammalian cells, there are physiological consequences if there's no mitochondrial fusion," says Chan.

To show just what happens, the team created mice with defects in two proteins known as mitofusins—mfn1 and mfn2—which are located on the surface of the mitochondria and are essential to the process of fusion. "We were able to specifically delete these mitofusins in skeletal muscle," Chan explains.

As it turns out, when fusion is blocked, not only are the mitochondria smaller, but the mtDNA levels in the mitochondria drop precipitously. As for the mice themselves? While they are born looking relatively normal, over the next couple of months they show signs that something is going wrong. Their growth is severely stunted and they die by 7–8 weeks of age, just at the onset of adulthood.

The mtDNA that remains in these unfused mitochondria "has a higher accumulation of point mutations and deletions," says Chan. In other words, without fusion, the mtDNA contains more mistakes, suggesting that fusion is "necessary for mtDNA stability."

This work may be important to our understanding of how and why human mitochondrial encephalomyopathies come to pass. Scientists have noted that most cells have a remarkably high tolerance for the mtDNA mutations that cause these conditions; in fact, somewhere between 60 and 90 percent of mtDNA has to carry the mutation before symptoms will begin to appear in a person with the mtDNA mutation. "Cells can tolerate a very high load of mtDNA mutations," Chan notes.

Why? Possibly because each cell carries so many copies of mtDNA that the "normal" versions are able to make up for the miscues of the mutated versions—but only if the mitochondria are able to fuse and combine their contents from time to time.

Chan and colleagues showed this to be the case in another set of experiments, in which they looked at a mouse model known to carry a high number of mtDNA mutations. Due to these mtDNA mutations, Chan explains, the mouse line has a lifespan less than half that of a normal mouse.

Still, it could be much worse—as Chan and colleagues showed when they tweaked the mouse model so that its mitochondria could no longer fuse. "When we added the mfn1 mutation into this model, we found that the mice died at birth instead of surviving to one year of age," he says. These results suggest that mitochondrial fusion is highly protective in cells carrying mtDNA mutations, as would be the case in encephalomyopathies.

Now that they've identified the problems that lack of fusion cause, the team plans to address the mechanisms by which these issues arise. "Why is there less mtDNA?" asks Chan. "Why is there less fidelity in the mtDNA genome? That's what we're going to study now."

In addition to Chan, the other authors on the Cell paper, "Mitochondrial Fusion is Required for mtDNA Stability in Skeletal Muscle and Tolerance of mtDNA Mutations," are Caltech senior research scientist Hsiuchen Chen; Marc Vermulst, formerly a postdoctoral scholar at Caltech now at the University of North Carolina, Chapel Hill; Caltech graduate student Yun Elisabeth Wang; Anne Chomyn, an HHMI research specialist and senior research associate emerita at Caltech; Tomas Prolla from the University of Wisconsin, Madison; and J. Michael McCaffery from the Johns Hopkins University.

Their work was funded by an RO1 grant from the National Institutes of Health (NIH), an Ellison Medical Foundation Senior Scholar Award, and a grant from the NIH's National Center for Research Resources.

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Lori Oliwenstein
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Caltech Scientists Uncover Structure of Key Protein in Common HIV Subgroup

Finding looks at clade C gp120; also reveals unusual autoreactivity between 21c antibody and CD4 receptor

PASADENA, Calif.—Scientists from the California Institute of Technology (Caltech) have provided the first-ever glimpse of the structure of a key protein—gp120—found on the surface of a specific subgroup of the human immunodeficiency virus, HIV-1. In addition, they demonstrated that a particular antibody to gp120 makes contact not only with the protein, but with the CD4 receptor that gp120 uses to gain entrance into the body's T cells.

This three-dimensional understanding of how gp120 is built is more than just a basic scientific advance. "There's a tremendous continuing effort to develop a vaccine for HIV," says Caltech postdoctoral scholar Ron Diskin, "and most of those efforts use gp120. Having more structural information will facilitate better vaccine design."

The findings are detailed in a paper published in the advance online edition of the journal Nature Structural & Molecular Biology.

The team looked specifically at gp120 from what is known as clade C HIV-1. To explain what that means, here's a brief HIV family history: Most people who get HIV and proceed to AIDS are infected with a member of the HIV-1 family of viruses. HIV-1 is divided into groups; most AIDS-related strains of the virus come from group M. The groups are further subdivided into what are known as clades.

Clade B is the form of group M HIV-1 most often found in the United States and western Europe, and the one that is probably best-studied to date. Clade C, the clade studied by the Caltech team, is "the one that is devastating Africa and Asia," says Diskin. "It's the one that probably causes the largest number of infections worldwide."

Previous studies had looked at the structure of clade B gp120, and it had been assumed—but not proven—that clade C's version would look much the same.

In order to uncover the structure of clade C gp120—and determine if the hypothesis about its similarities was indeed true—the Caltech team needed to crystallize the protein. That was no easy task. Turns out, says Diskin, the protein itself is not stiff enough for crystallization. And so the researchers created a complex of molecules consisting of a gp120 monomer, a CD4 receptor, and an anti-HIV antibody known as 21c.

This configuration facilitated crystallization, and allowed the scientists to look not only at gp120—which, indeed, "looks pretty much the same in clade C as in clade B," says Diskin—but to visualize the entire binding site and to see how the various components in the complex interact with one another.

That was when they noticed something unusual: Antibody 21c was not only reacting to—and thus making contact with—the gp120 protein sticking out from HIV's envelope, but also was reacting to the CD4 receptors on the body's own T cells. It is the first time this sort of polyreactivity—a response to more than one antigen—had been visualized in the 3-D structure of an HIV-targeting antibody.

"The most interesting aspect of our structure is the unexpected contact between the antibody and CD4," says Pamela Bjorkman, the Max Delbruck Professor of Biology at Caltech, a Howard Hughes Medical Institute investigator, and the Caltech team's leader. "The binding to CD4 suggests that this class of anti-HIV antibodies has autoreactive properties, which raises many interesting questions about how anti-HIV immune responses affect an HIV-infected individual."

Does this autoreactivity mean that 21c is too dangerous to work with, because clinicians might be courting a potential autoimmune response with a vaccine that elicits 21c-like antibodies?

Not necessarily, says Diskin.

"Other data out there show that some of the best neutralizing antibodies are also autoreactive," he explains.

What it does mean, however, is that there would be additional hurdles to overcome in eliciting such antibody responses, Diskin says. The body tends to eliminate autoreactive antibodies, in an attempt to keep autoimmune diseases at bay. "In order to create a good vaccine to produce 21c-like antibodies, researchers will have to overcome this elimination mechanism."

The next step for the Caltech team is to try to improve on the relatively low-resolution structure worked out in the current paper. In addition, Diskin says, the team would like to try to resolve the structure of a gp120 trimer—a more complex, three-pronged version of the protein.

In addition to Bjorkman and Diskin, the other coauthor on the paper, "Structure of a clade C HIV gp120 bound to CD4 and CD4-induced antibody reveals anti-CD4 polyreactivity," is Paola Marcovecchio, a Caltech research technician. Their work was made possible by a fellowship from the European Molecular Biology Organization, a Collaboration for AIDS Vaccine Discovery grant (supported by the Bill & Melinda Gates Foundation), and support from Caltech's Molecular Observatory (funded by the Gordon and Betty Moore Foundation).

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Lori Oliwenstein
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Caltech-led Team Provides Proof in Humans of RNA Interference Using Targeted Nanoparticles

Researchers unveil scientific results from siRNA Phase I clinical trial in cancer patients

PASADENA, Calif.—A California Institute of Technology (Caltech)-led team of researchers and clinicians has published the first proof that a targeted nanoparticle—used as an experimental therapeutic and injected directly into a patient's bloodstream—can traffic into tumors, deliver double-stranded small interfering RNAs (siRNAs), and turn off an important cancer gene using a mechanism known as RNA interference (RNAi). Moreover, the team provided the first demonstration that this new type of therapy, infused into the bloodstream, can make its way to human tumors in a dose-dependent fashion—i.e., a higher number of nanoparticles sent into the body leads to a higher number of nanoparticles in the tumor cells.

These results, published in the March 21 advance online edition of the journal Nature, demonstrate the feasibility of using both nanoparticles and RNAi-based therapeutics in patients, and open the door for future "game-changing" therapeutics that attack cancer and other diseases at the genetic level, says Mark Davis, the Warren and Katharine Schlinger Professor of Chemical Engineering at Caltech, and the research team's leader.

The discovery of RNA interference, the mechanism by which double strands of RNA silence genes, won researchers Andrew Fire and Craig Mello the 2006 Nobel Prize in Physiology or Medicine. The scientists first reported finding this novel mechanism in worms in a 1998 Nature paper. Since then, the potential for this type of gene inhibition to lead to new therapies for diseases like cancer has been highly touted.

"RNAi is a new way to stop the production of proteins," says Davis. What makes it such a potentially powerful tool, he adds, is the fact that its target is not a protein. The vulnerable areas of a protein may be hidden within its three-dimensional folds, making it difficult for many therapeutics to reach them. In contrast, RNA interference targets the messenger RNA (mRNA) that encodes the information needed to make a protein in the first place.

"In principle," says Davis, "that means every protein now is druggable because its inhibition is accomplished by destroying the mRNA. And we can go after mRNAs in a very designed way given all the genomic data that are and will become available."

Still, there have been numerous potential roadblocks to the application of RNAi technology as therapy in humans. One of the most problematic has been finding a way to ferry the therapeutics, which are made up of fragile siRNAs, into tumor cells after direct injection into the bloodstream. Davis, however, had a solution.

The targeted nanoparticle used in the study and shown in this schematic is made of a unique polymer and can make its way to human tumor cells in a dose-dependent fashion.
Credit: Caltech/Derek Bartlett

Even before the discovery of RNAi, he and his team had begun working on ways to deliver nucleic acids into cells via systemic administration. They eventually created a four-component system—featuring a unique polymer—that can self-assemble into a targeted, siRNA-containing nanoparticle. The siRNA delivery system is under clinical development by Calando Pharmaceuticals, Inc., a Pasadena-based nanobiotech company.

"These nanoparticles are able to take the siRNAs to the targeted site within the body," says Davis. Once they reach their target—in this case, the cancer cells within tumors—the nanoparticles enter the cells and release the siRNAs.

The scientific results described in the Nature paper are from a Phase I clinical trial of these nanoparticles that began treating patients in May 2008. Phase I trials are, by definition, safety trials; the idea is to see if and at what level the drug or other therapy turns harmful or toxic. These trials can also provide an in-human scientific proof of concept—which is exactly what is being reported in the Nature paper.

Using a new technique developed at Caltech, the team was able to detect and image nanoparticles inside cells biopsied from the tumors of several of the trial's participants. In addition, Davis and his colleagues were able to show that the higher the nanoparticle dose administered to the patient, the higher the number of particles found inside the tumor cells—the first example of this kind of dose-dependent response using targeted nanoparticles.

Even better, Davis says, the evidence showed the siRNAs had done their job. In the tumor cells analyzed by the researchers, the mRNA encoding the cell-growth protein ribonucleotide reductase had been degraded. This degradation, in turn, led to a loss of the protein.

More to the point, the mRNA fragments found were exactly the length and sequence they should be if they'd been cleaved in the spot targeted by the siRNA, notes Davis. "It's the first time anyone has found an RNA fragment from a patient's cells showing the mRNA was cut at exactly the right base via the RNAi mechanism," he says. "It proves that the RNAi mechanism can happen using siRNA in a human."

"There are many cancer targets that can be efficiently blocked in the laboratory using siRNA, but blocking them in the clinic has been elusive," says Antoni Ribas, associate professor of medicine and surgery at UCLA's Jonsson Comprehensive Cancer Center. "This is because many of these targets are not amenable to be blocked by traditionally designed anti-cancer drugs. This research provides the first evidence that what works in the lab could help patients in the future by the specific delivery of siRNA using targeted nanoparticles. We can start thinking about targeting the untargetable."

"Although these data are very early and more research is needed, this is a promising study of a novel cancer agent, and we are proud of our contribution to the initial clinical development of siRNA for the treatment of cancer," says Anthony Tolcher, director of clinical research at South Texas Accelerated Research Therapeutics (START).

"Promising data from the clinical trials validates our years of research at City of Hope into ribonucleotide reductase as a target for novel gene-based therapies for cancer," adds coauthor Yun Yen, associate director for translational research at City of Hope. "We are seeing for the first time the utility of siRNA as a cancer therapy and how nanotechnology can target cancer cells specifically."

The Phase I trial—sponsored by Calando Pharmaceuticals—is proceeding at START and UCLA's Jonsson Comprehensive Cancer Center, and the clinical results of the trial will be presented at a later time. "At the very least, we've proven that the RNAi mechanism can be used in humans for therapy and that the targeted delivery of siRNA allows for systemic administration," Davis says. "It is a very exciting time."

In addition to Davis, Ribas, Tolcher, and Yen, the coauthors on the Nature paper, "Evidence of RNAi in humans from systemically administered siRNA via targeted nanoparticles," are Caltech graduate students Jonathan Zuckerman (an MD/PhD student doing his MD work at UCLA) and Chung Hang Choi; former Caltech graduate student Christopher Alabi, now a postdoctoral scholar at the Massachusetts Institute of Technology; David Seligson, director of the UCLA Tissue Array Core Facility at the David Geffen School of Medicine; and Jeremy Heidel, who is currently a consultant for Calando Pharmaceuticals.

The work described in the paper was supported in part by the National Cancer Institute and the Daljit S. and Elaine Sarkaria Biomarker Laboratories. Caltech, Davis, and Heidel have a financial interest in Calando Pharmaceuticals.

Writer: 
Jon Weiner
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