Team of Proteins Works Together to Turn on T Cells

The fates of various cells in our bodies—whether they become skin or another type of tissue—are controlled by genetic switches. In a new study, Caltech scientists investigate the switch for T cells, which are immune cells produced in the thymus that destroy virus-infected cells and cancers. The researchers wanted to know how cells make the choice to become T cells.

"We already know which genetic switch directs cells to commit to becoming T cells, but we wanted to figure out what enables that switch to be turned on," says Hao Yuan Kueh, a postdoctoral scholar at Caltech and lead author of a Nature Immunology report about the work, published on July 4.

The study found that a group of four proteins, specifically DNA-binding proteins known as transcription factors, work in a multi-tiered fashion to control the T-cell genetic switch in a series of steps. This was a surprise because transcription factors are widely assumed to work in a simultaneous, all-at-once fashion when collaborating to regulate genes.

The results may ultimately allow doctors to boost a person's T-cell population. This has potential applications in fighting various diseases, including AIDS, which infects mature T cells.

"In the past, combinatorial gene regulation was thought to involve all the transcription factors being required at the same time," says Kueh, who works in the lab of  Ellen Rothenberg, Caltech's Albert Billings Ruddock Professor of Biology. "This was particularly true in the case of the genetic switch for T-cell commitment, where it was thought that a quorum of the factors working simultaneously was needed to ensure that the gene would only be expressed in the right cell type."

The authors report that a key to their finding was the ability to image live cells in real-time. They genetically engineered mouse cells so that a gene called Bcl11b—the key switch for T cells—would express a fluorescent protein in addition to its own Bcl11b protein. This caused the mouse cells to glow when the Bcl11b gene was turn on. By monitoring how different transcription factors, or proteins, affected the activation of this genetic switch in individual cells, the researchers were able to isolate the distinct roles of the proteins.

The results showed that four proteins work together in three distinct steps to flip the switch for T cells. Kueh says to think of the process as a team of people working together to get a light turned on. He says first two proteins in the chain (TCF1 and GATA3) open a door where the main light switch is housed, while the next protein (Notch) essentially switches the light on. A fourth protein (Runx1) controls the amplitude of the signal, like sliding a light dimmer.

"We identify the contributions of four regulators of Bcl11b, which are all needed for its activation but carry out surprisingly different functions in enabling the gene to be turned on," says Rothenberg. "It's interesting—the gene still needs the full quorum of transcription factors, but we now find that it also needs them to work in the right order. This makes the gene respond not only to the cell's current state, but also to the cell's recent developmental history."

Team member Kenneth Ng, a visiting student from California Polytechnic State University, says he was surprised by how much detail they could learn about gene regulation using live imaging of cells.

"I had read about this process in textbooks, but here in this study we could pinpoint what the proteins are really doing," he says.

The next step in the research is to get a closer look at precisely how the T cell genetic switch itself works. Kueh says he wants to "unscrew the panels" of the switch and understand what is physically going on in the chromosomal material around the Bcl11b gene.

The Nature Immunology paper, titled, "Asynchronous combinatorial action of four regulatory factors activates Bcl11b for T cell commitment," includes seven additional Caltech coauthors: Mary Yui, Shirley Pease, Jingli Zhang, Sagar Damle, George Freedman, Sharmayne Siu, and Michael Elowitz; as well as a collaborator at the Fred Hutchinson Cancer Research Center, Irwin Bernstein. The work at Caltech was funded by a CRI/Irvington Postdoctoral Fellowship, the National Institutes of Health, the California Institute for Regenerative Medicine, the Al Sherman Foundation, and the Louis A. Garfinkle Memorial Laboratory Fund.

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Scientists Transform Lower-Body Cells into Facial Cartilage

Caltech scientists have converted cells of the lower-body region into facial tissue that makes cartilage, in new experiments using bird embryos. The researchers discovered a "gene circuit," composed of just three genes, that can alter the fate of cells destined for the lower bodies of birds, turning them instead into cells that produce cartilage and bones in the head.

The results, published in the June 24 issue of the journal Science, could eventually lead to therapies for conditions where facial bone or cartilage is lost. For example, cartilage destroyed in the nose due to cancer is particularly hard to replace. Understanding the genetic pathways that lead to the development of facial cartilage may help in future stem-cell therapies, where a patient's own skin cells could be transformed and used to repair the nose.

"When facial cartilage and bone is lost, from cancer or an accident, it has been difficult to replace," says Marianne Bronner, the Albert Billings Ruddock Professor of Biology at Caltech, and senior author of the Science report. "Our long term hope is that uncovering this gene circuit may be useful in reprogramming a patient's own stem cells to make facial cartilage."

The bones below our necks, referred to by scientists as the "long" bones, originate from a different source of tissue than the bones in our head. As embryos, we are born with a type of early tissue called the neural crest that forms along the entire body, from the head to the end of the spinal cord. Those neural crest cells which originate in the head, called cranial neural crest, differentiate into the cartilage and bone of our faces, including the jaws and skull. In contrast, the so-called trunk neural crest cells, forming below the neck, do not make cartilage or bone but instead turn into nerve cells and pigment cells elsewhere in our bodies. Bronner and her colleagues want to understand what genes regulate the development of cranial neural crest cells and enable them to make cartilage and bones in the head.

To this end, they divided the trunk and cranial neural crest cells of bird embryos into separate groups, and looked for differences in gene activity. Fifteen genes were initially identified as being turned on in only the cranial cells. The researchers chose six of these genes for further study. All six code for transcription factors—molecules that bind to DNA to turn on and off the expression of other genes. After studying how these factors interact with each other, the scientists focused on three, called Sox8, Tfap2b and Ets, that are part of the cranial neural crest circuit.

These three genes were then inserted into the bodies of developing bird embryos, in particular the trunk neural crest, using a technique called electroporation. In this method, electric current is applied to cells to open up pores through which molecules such as DNA may pass. Next, the researchers transplanted the altered trunk cells to the cranial region of the embryos. Five days later, the trunk cells were doing something entirely new: producing cartilage.

"Normally, these trunk cells will not make cartilage," says Bronner. "Introducing just three genes into these cells reprogrammed them to acquire the ability to do so."

Bronner said that she hopes other researchers will use this information for experiments in cell culture. By adding the new-found gene circuit, perhaps with other known factors, to skin cells in a petri dish it may be possible to turn them into cartilage-producing cells—a key next step in creating future therapies for facial bone and cartilage loss.

The first author of the Science paper, titled, "Reprogramming of avian neural crest axial identity and cell fate," is Marcos Simoes-Costa of Caltech. The research is funded by the National Institutes of Health and the Pew Fellows Program in Biomedical Sciences.

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Creating Facial Cartilage in the Lab
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Dietary Fiber and Microbes Change the Gel That Lines Our Gut

In the ongoing hustle and bustle of our intestines, where bacteria and food regularly intermingle, there is another substance that, to the surprise of researchers, has been found to rapidly change: the gel that lines the gut. A new Caltech study is the first to show how the structure of this gut gel, or mucus, can change in the presence of certain substances, such as bacteria and polymers—a class of long-chained molecules that includes dietary fiber.

The work, to be published online the week of June 13 in the Proceedings of the National Academy of Sciences, could lead to the development of new drugs or diets for intestinal conditions such as irritable bowel disease.

Our intestinal tracts are lined with a mucus gel that acts as a protective barrier between the insides of our bodies and the outside world. The gel lets in nutrients and largely blocks out bacteria, preventing infections. It also regulates how some drugs are delivered elsewhere in our bodies.

Researchers had previously studied how the gel can be damaged, for instance when bacteria feed on the gut's lining. The Caltech study is the first to look at the structure of the gel and how it morphs in the presence of other substances naturally found in the gut.

Performing their experiments in mice, the team tested the effects of polymers, which include dietary fiber as well as therapeutics such as medicines for constipation. The researchers fed some mice a diet rich in polymers and others (the controls) a polymer-free diet. Using a technique called confocal reflectance microscopy they measured the thickness of the gut gel and the degree to which the gel was compressed as a result of the consumed polymers. Mice given a high-polymer diet, they found, had a more compressed gel layer.

"The gel is like a sponge with holes that let material through," says the paper's lead author, Sujit Datta, a postdoctoral scholar in the laboratory of Rustem Ismagilov, Ethel Wilson Bowles and Robert Bowles Professor of Chemistry and Chemical Engineering. "We are seeing that polymers, including dietary fiber, can compress the gel, potentially making the holes smaller, and we think that this might offer protective benefits," Datta adds.

In addition, the researchers applied different kinds of polymers—including dietary fibers like pectin, found in apples—directly to the gel lining to test its response. All of the polymers tested compressed the gel layer.

"It's too early to draw any conclusions, but it may be that eating an apple a day will affect the shape of the lining in your gut," says Asher Preska Steinberg, a Caltech graduate student and coauthor of the study.

The researchers also found that dietary fiber and gut bacteria—which are part of a community of microorganisms collectively known as gut microbiota—can work together to influence how the gut gel changes shape. They performed the same polymer/fiber experiments in germ-free mice, which are mice carefully raised to not have any bacteria in their gut. The results showed that the polymers compressed the gut gels of these germ-free mice to a greater degree. This implies that species of bacteria in our gut that are known to break down polymers can weaken the compressing effect.

"We previously thought of the gel as a static structure, so it was unexpected to find an interplay between diet and gut microbiota that rapidly and dynamically changes the biological structures that protect a host," says Ismagilov.

Both dietary fiber and certain gut microbes have been linked to good health. Fiber has been shown to lower cholesterol and regulate blood sugar levels—factors in heart disease and diabetes, respectively. Meanwhile, some bacteria, including the good "probiotics," can help treat digestive disorders and may even play a beneficial role in mental health. For instance, a separate Caltech-led study found that probiotics can alleviate autism-like behaviors in mice—a finding that could potentially lead to new therapies for the disorder in humans.

The entire collection of bacteria in our gut can include 1,000 different species or more and weigh a total of three pounds. Exactly how these microscopic organisms influence our health, for good and bad, is an area of active research with many unanswered questions. The White House recently announced the National Microbiome Initiative, with federal funding worth $121 million, to investigate the mysteries of microbes not only living in our bodies but all over the planet. In addition, more than 100 nonfederal agencies have pledged money and support toward researching microbial communities.

"Our study gives biologists and scientists studying diseases of the gut something else to think about," says Datta. "Now they can take the structure of the gut mucus, and how it responds to its environment, into account."

This research was funded by the Defense Advanced Research Projects Agency (DARPA) and National Science Foundation.

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Microbes & Dietary Fiber Change the Gut Lining
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The study is the first to look at the structure of the gut's mucus gel lining and how it morphs in the presence of other substances naturally found in the gut.

Newly Named Pew Scholar to Image Gut Bacteria with Sound Waves

Caltech's Mikhail Shapiro, assistant professor of chemical engineering, has been selected as a 2016 Pew scholar by the Pew Scholars Program in the Biomedical Sciences. As a Pew Scholar, Shapiro will receive $240,000 over the next four years in support of his research program to image the location and activities of microbes in the body using ultrasound.

Our guts, or intestines, are alive with colonies of bacteria. Some species are good for us but others are bad and can lead to medical conditions, such as food poisoning and irritable bowel disease. Observing these bacteria in action is difficult because they are hidden deep inside the body. Typically, researchers culture the microbes outside the body to learn more about them, but this does not reveal where the bacteria are in the gut, or how they interact.

Shapiro plans to solve this problem with bacteria genetically engineered to be visible to ultrasound. The same ultrasound imaging techniques used by doctors to take pictures of a developing baby could be used to visualize communities of bacteria in the gut.

"Imaging techniques that rely on photons, such as fluorescence or luminescence, don't penetrate very deeply into the body," says Shapiro. "We are developing proteins that cells can make that will allow them to interact with sound waves and magnetic fields, which can penetrate more deeply."

The key to the approach is a unique class of proteins normally employed by certain photosynthetic, single-celled organisms to control how much they float, a trait needed to regulate access to light and other nutrients. The proteins form gas-filled structures that, Shapiro's team discovered, can scatter sound waves in a manner that makes them detectable by ultrasound. The researchers plan to genetically engineer bacteria to produce the proteins, then image them in mice.

The technique could ultimately lead to better ways to diagnose conditions such as irritable bowel disease.

Shapiro came to Caltech from UC Berkeley in 2014. Before that, he was a postdoctoral fellow at the University of Chicago, and earned his PhD from the Massachusetts Institute of Technology.

The Pew Scholars Program in the Biomedical Sciences, according to their website, "provides funding to young investigators of outstanding promise in science relevant to the advancement of human health. The program makes grants to selected academic institutions to support the independent research of outstanding individuals who are in their first few years of their appointment at the assistant professor level."

In addition to engineering bacteria, Shapiro's lab works on other methods to image and control cells deep in our body—such as tumor cells, immune cells, and neurons—with ultrasound and magnetic resonance.

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Oka Receives McKnight Award

Yuki Oka, assistant professor of biology, has been named one of six recipients of the 2016 McKnight Scholars Award. The McKnight Endowment Fund for Neuroscience awards $75,000 per year for three years to support young scientists who are establishing their own laboratories and research centers. The award is only available to researchers in the first four years of a tenure-track faculty position.

 

"A McKnight Scholar Award is one of the most prestigious early-career honors that a young neuroscientist can receive," said Anthony Movshon, chair of the awards committee and professor at New York University, in a press release. "This year's Scholars are a superbly talented group, with as much promise as any selected in the past. … Their work will help us to understand the brain's function in health and in disease, and will shape the neuroscience of the future."

 

Oka studies the neural mechanisms controlling thirst. These mechanisms help the body maintain a healthy balance of water and salt. He is attempting to isolate exactly which circuits in the brain regulate thirst and to determine how those circuits are triggered by external signals. Understanding these key brain functions may lead to new treatments for appetite-related disorders.

 

The McKnight Foundation of Minneapolis, Minnesota has supported neuroscience research since 1977. It created the Endowment Fund in 1986 in honor of William L. McKnight, an early leader of the 3M Company who had a personal interest in neurological diseases and wanted his legacy to help find cures. Previous awardees from Caltech include Athanossios Siapas, professor of computation and neural systems, and Kai Zinn, professor of biology.

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A Feeling Touch

Using funding from the BRAIN Initiative, Caltech biologists are developing neuroprosthetics to bring tactile sensations to the users of robotic arms.

Caltech biologist Richard Andersen is working to incorporate a sense of touch into the neural prosthetics he has been helping develop for years—devices implanted in the brain that allow a paralyzed patient to manipulate a robotic arm.

Andersen and colleagues first reported success of their original implant in early 2015. The team, led by Andersen, placed their prosthesis in the posterior parietal cortex, an area that controls the intent to move rather than controlling movement directly as previous experiments had done. This allowed Erik Sorto, a 35-year-old man who has been paralyzed from the neck down for more than 10 years, to use a robotic arm placed next to his body to perform a fluid hand-shaking gesture, play rock-paper-scissors, and even grasp a bottle of beer and bring it to his mouth for a sip—something he had long dreamed of doing.

This research on how to make a robotic arm move resulted in a 2015 National Science Foundation grant to Andersen from President Obama's Brain Research through Advancing Innovative Neurotechnology—or BRAIN—Initiative, as well as seed money from the California Blueprint for Research to Advance Innovations in Neuroscience (Cal-BRAIN) program, the California complement to the federal initiative, which gave out its first-ever monetary awards last year to a group of researchers that included Andersen.

Andersen is now using those Cal-BRAIN funds—designed to bring together interdisciplinary teams of scientists and engineers from diverse fields for fundamental brain research—to take his team's work to the next level. His hope is to enable people using robotic arms to literally regain their sense of touch—their ability to feel an object in their "hands."

For more on Andersen's work, read A Feeling Touch on E&S+

 

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Caltech biologists are developing neuroprosthetics to bring tactile sensations to the users of robotic arms.

DNA Origami: Folded DNA as a Building Material for Molecular Devices

Living things use DNA to store the genetic information that makes each plant, bacterium, and human being unique. The reproduction of this information is made possible because DNA's nucleotides—A's and T's, G's and C's—fit together perfectly, like matching jigsaw puzzle pieces. Engineers can take advantage of the matching between long strands of DNA nucleotides to use DNA as a kind of molecular origami, folding it into everything from nanoscale smiley face artwork to serious drug-delivery devices.

On Wednesday, May 25, at 8 p.m. in Beckman Auditorium, Paul Rothemund (BS '94), the inventor of the DNA origami technique, will explain how his group and groups around the world are using DNA origami in applications ranging from potential cancer treatments to devices for computing. Rothemund is research professor of bioengineering, computing and mathematical sciences, and computation and neural systems in the Division of Engineering and Applied Science at Caltech. Admission is free.

What do you do?

I use DNA and RNA as building materials to create shapes and patterns with a resolution of just a few nanometers. The smallest features in the DNA structures we make are about 20,000 times smaller than the pixels in the fanciest computer displays, which are each about 80 microns across. A large part of our work over the last 20 years has been just figuring out how to get DNA or RNA strands to fold themselves into a desired computer-designed shape. As we've mastered the ability to make whatever shape or pattern we desire, we've moved on to using these shapes as "pegboards" for arranging other nano-sized objects, such as protein enzymes, carbon-nanotube transistors, and fluorescent molecules.

Why is this important?

Every task in your body, from digesting food to moving your muscles to sensing light, is powered by tiny nanometer-scale biological machines, all built from the "bottom up" via the self-folding of molecules such as proteins and RNAs. The billions of transistors that make up the chips in our cell phones and computers are tens of nanometers in size, but they are built in a "top down" fashion using fancy printing processes in billion-dollar factories. Our goal is to learn how to build complex artificial devices the way biology builds natural ones—that is, starting from self-folding molecules that assemble together into larger more complex structures. In addition to vastly cheaper devices, this will enable completely new applications, such as man-made molecular machines that can make complex therapeutic decisions and apply drugs only where needed.

How did you get into this line of work?

As an undergraduate at Caltech, I had great difficulty trying to decide how to combine my diverse interests in computer science, chemistry, and biology. Fortunately, the late Jan L. A. van de Snepscheut introduced his computer science class to the hypothetical idea of building a DNA Turing machine—a very simple machine which can nevertheless run every possible computer program. He challenged us, suggesting that someone who knew about both biochemistry and computer science could come up with a concrete way to build such a DNA computer. For a project class in information theory with Yaser Abu-Mostafa, a professor of electrical engineering and computer science, I came up with a pretty inefficient, yet possible, way to do this. At the time, I couldn't interest any Caltech professors in building my DNA computer, but shortly after, USC professor Len Adleman published a paper on a more practical DNA computer in Science. I joined Adleman's lab at USC as a graduate student, and I've been trying to use DNA to build computers or other complex devices ever since. I returned to Caltech as a postdoc in 2001 and became a research professor in 2008.

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A preview of Paul Rothemund's upcoming Watson Lecture.

When Beneficial Bacteria Knock But No One is Home

The community of beneficial bacteria that live in our intestines, known as the gut microbiome, are important for the development and function of the immune system. There has been growing evidence that certain probiotics—therapies that introduce beneficial bacteria into the gut—may help alleviate some of the symptoms of intestinal disorders such as Crohn's disease. By studying the interplay between genetic risk factors for Crohn's and the bacteria that populate the gut, researchers at Caltech have discovered a new potential cause for this disorder in some patients—information that may lead to advances in probiotic therapies and personalized medicine.

The results were published online in the May 5 edition of the journal Science.

Previously, scientists had found that patients with Crohn's disease often exhibit alterations in both their genome and their gut microbiome—the diverse collection of bacteria that reside in the intestine. More than 200 genes have been implicated as having a role in the susceptibility to Crohn's. For years, researchers in the field have believed that these are genes that normally function by sensing pathogenic bacteria and deploying an immune response to kill the unwanted microbes; when these genes are defective, the pathogenic bacteria survive, multiply in the gut, and lead to disease.

"While we believe that all of that is true, in this study we were curious to see if some of the genes that are important in sensing pathogenic bacteria may also be important in sensing beneficial bacteria to promote immune health," says the study's first author, Hiutung Chu, a postdoctoral scholar in biology and biological engineering at Caltech. "Typically, the signals from these beneficial commensal microbes promote anti-inflammatory responses that dampen inflammation in the gut. However, mutations in genes that sense and respond to pathogenic bacteria would also impair the response to the beneficial ones. So it's kind of a new spin on the existing dogma."

To figure this out, Chu and her colleagues in the laboratory of Sarkis Mazmanian, the Luis B. and Nelly Soux Professor of Microbiology, designed several experiments to study how genetic mutations might interrupt the immune-enhancing effects of a known beneficial bacterium, Bacteroides fragilis. The researchers tested their new theory by using B. fragilis to treat mice that had nonfunctional versions of two genes known to play a role in Crohn's disease risk, called ATG16L1 and NOD2.

The researchers found that if just one of these two genes was absent, the mice were unable to develop disease-protective immune cells called regulatory T cells in response to B. fragilis—and that even after treatment with B. fragilis, symptoms in an ATG16L1-deficient mouse model of intestinal disease remained unchanged.

Chu and Mazmanian then obtained blood samples from both healthy patients and patients with Crohn's disease at the Cedars-Sinai Medical Center in Los Angeles. "We could see that certain patients' immune cells responded to Bacteroides fragilis, while immune cells from other patients didn't respond at all," Chu says. "Because the cells from Cedars had already been genotyped, we were able to match up our results with the patients' genotypes: immune cells from individuals with the protective version of ATG16L1 responded to the treatment, but cells from patients who had the mutated version of the gene showed no anti-inflammatory response to B. fragilis."

Mazmanian says the results suggest that the faulty versions of these genes may cause Crohn's disease in two different ways: by being unable to assist in destroying pathogenic bacteria and by preventing the beneficial immune signals usually elicited by "good" bacteria. "What Hiutung has shown is that there are specific bacteria in the human microbiome that appear to utilize the pathways that are encoded by these genes—genes normally involved in killing bacteria—to send beneficial signals to the host," he says.

This work reveals the important relationship between the genome and the microbiome—and it may also one day be used to improve the use of probiotics in clinical trials, Mazmanian says. "For example, our previous work has suggested using B. fragilis as a probiotic treatment for certain disorders. What this new study suggests is that there are certain populations that wouldn't benefit from this treatment because they have this genetic predisposition," he notes. "Right now, clinical trials don't do a good job of identifying which patients might respond best to treatment, but our experiments in mouse models suggest that, conceptually, you could design clinical trials that are more effective."

The research described in the paper, "Gene-Microbiota Interactions Contribute to the Pathogenesis of Inflammatory Bowel Disease," was funded by the National Institutes of Health, the Cedars-Sinai F. Widjaja Foundation Inflammatory Bowel and Immunobiology Research Institute, the Lupus Research Institute, the European Union, the Crohn's and Colitis Foundation of America, the Leona M. and Harry B. Helmsley Charitable Trust, and the Heritage Medical Research Institute.

In addition to Chu and Mazmanian, other Caltech coauthors include former graduate students Arya Khosravi (PhD '14) and Yue Shen (PhD '12); research technician assistants Indah Kusumawardhani and Alice Kwon; and Wei-Li Wu, a postdoctoral scholar in biology and biological engineering. Coauthors from other institutions include: Anilton Vasconcelos and Peter Ernst from UC San Diego; Larissa Cunha and Douglas Green from St. Jude Children's Research Hospital in Memphis; Anne Mayer, Amal Kambal, and Herbert Virgin from the Washington University School of Medicine in St. Louis; Stephan Targan and Dermot McGovern from Cedars-Sinai Medical Center; and Ramnik Xavier from Harvard Medical School.

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Mapping Neurons to Improve the Treatment of Parkinson's

Because billions of neurons are packed into our brain, the neuronal circuits that are responsible for controlling our behaviors are by necessity highly intermingled. This tangled web makes it complicated for scientists to determine exactly which circuits do what. Now, using two laboratory techniques pioneered in part at Caltech, Caltech researchers have mapped out the pathways of a set of neurons responsible for the kinds of motor impairments—such as difficulty walking—found in patients with Parkinson's disease.

The work—from the laboratory of Viviana Gradinaru (BS '05), assistant professor of biology and biological engineering—was published on April 20 in the journal Neuron.

In patients with Parkinson's disease, gait disorders and difficulty with balance are often caused by the degeneration of a specific type of neuron—called cholinergic neurons—in a region of the brainstem called the pedunculopontine nucleus (PPN). Damage to this same population of neurons in the PPN is also linked to reward-based behaviors and disorders, such as addiction.

Previously, researchers had not been able to untangle the neural circuitry originating in the PPN to understand how both addictions and Parkinson's motor impairments are modulated within the same population of cells. Furthermore, this uncertainty created a barrier to treating those motor symptoms. After all, deep brain stimulation—in which a device is inserted into the brain to deliver electrical pulses to a targeted region—can be used to correct walking and balance difficulties in these patients, but without knowing exactly which part of the PPN to target, the procedure can lead to mixed results.

"The circuits responsible for controlling our behaviors are not nicely lined up, where this side does locomotion and this side does reward," Gradinaru says, and this disordered arrangement arises from the way neurons are structured. Much as a tree extends into the ground with long roots, neurons are made up of a cell body and a long string-like axon that can diverge and project elsewhere into different areas of the brain. Because of this shape, the researchers realized they could follow the neuron's "roots" to an area of the brain less crowded than the PPN. This would allow them to more easily look at the two very different behaviors and how they are implemented.

Cheng Xiao, a senior research scientist at Caltech and first author on the study, began by mapping the projections of the cholinergic neurons in the PPN of a rat using a technique developed by the Gradinaru lab called Passive CLARITY Technique, or PACT. In this technique, a solution of chemicals is applied to the brain; the chemicals dissolve the lipids in the tissue and render that region of the brain optically transparent—see-through, in other words—and able to take up fluorescent markers that can label different types of neurons. The researchers could then follow the path of the PPN neurons of interest, marked by a fluorescent protein, by simply looking through the rest of the brain.

Using this method, Gradinaru and Xiao were able to trace the axons of the PPN neurons as they extended into two regions of the midbrain: the ventral substantia nigra, a landmark area for Parkinson's disease that had been previously associated with locomotion; and the ventral tegmental area, a region of the brain that had been previously associated with reward.

Next, the researchers used an electrical recording technique to keep track of the signals sent by PPN neurons—confirming that these neurons do, in fact, communicate with their associated downstream structures in the midbrain. Then, the scientists went on to determine how this specific population of neurons affects behavior. To do this, they used a technique that Gradinaru helped develop called optogenetics, which allows researchers to manipulate neural activities—in this case, by either exciting or inhibiting the PPN neural projections in the midbrain—using different colors of light.

Using the optogenetic approach in rats, the researchers found that exciting the neuronal projections in the ventral substantia nigra would stimulate the animal to walk around its environment; by contrast, they could stop the animal's movement by inhibiting these same projections. Furthermore, they found that they could stimulate reward-seeking behavior by exciting the neuronal projections in the ventral tegmental area, but could cause aversive behavior by inhibiting these projections.

"Our results show that the cholinergic neurons from the PPN indeed have a role in controlling both behaviors," Gradinaru says. "Although the neurons are very densely packed and intermingled, these pathways are, to some extent, dedicated to very specialized behaviors." Determining which pathways are associated with which behaviors might also improve future treatments, she adds.

"In the past it's been difficult to target treatment to the PPN because the specific neurons associated with different behaviors are intermingled at the source—the PPN. Our results show that you could target the axonal projections in the substantia nigra for movement disorders and projections in the ventral tegmental area for reward disorders, as addiction is," Gradinaru says. In addition, she notes, these projections in the midbrain are much easier to access surgically than their source in the PPN.

Although this new information could inform clinical treatments for Parkinson's disease, the PPN is only one region of the brain and there are many more important examples of connectivity that need to be explored, Gradinaru says. "These results highlight the need for brain-wide functional and anatomical maps of these long-range neuronal projections; we've shown that tissue clearing and optogenetics are enabling technologies in the creation of these maps."

These results are published in a paper titled, "Cholinergic mesopontine signals govern locomotion and reward through dissociable midbrain pathways." In addition to Gradinaru and Xiao, other Caltech coauthors include Jounhong Ryan Cho, Chunyi Zhou, Jennifer Treweek, Ken Chan, Sheri McKinney, and Bin Yang. The work was supported by the National Institutes of Health, the Heritage Medical Research Institute, the Pew Charitable Trust, the Michael J. Fox Foundation, and the Sloan Foundation; the Beckman Institute supports the Resource Center on CLARITY, Optogenetics, and Vector Engineering (CLOVER) for technology development and broad dissemination.

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Mapping Neurons to Improve Parkinson's
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Caltech researchers have mapped out a circuit of neurons responsible for motor impairment in patients with Parkinson's disease.

Rerouting Cancer

Cancer is capable of rapidly developing resistance to therapeutic drugs, rendering those drugs harmless—often before they have a chance to work. Now, researchers at Caltech and their colleagues have identified how at least one brain cancer, called glioblastoma multiforme (GBM), adapts so fast—and they show that by formulating the right combination of drugs, doctors could potentially overcome this resistance and stop a tumor in its tracks.

The work appears in the April 12 issue of the journal Cancer Cell.

Some cancer drugs are designed to target a cell's chemical circuitry. This network of signaling pathways controls how a healthy cell functions, but in many cancers, the pathways are hyperactivated, directly leading to the aggressive nature of the disease. By blocking a key pathway, a drug can, in principle, stop the tumor from growing.

"The concept is that if you block a key node in the pathway, then the communication can't proceed and the cells can't get the signals to divide and multiply," explains Jim Heath, the Elizabeth W. Gilloon Professor of Chemistry and co-corresponding author on the paper.

In reality, however, tumors can become resistant to a drug even if the drug works exactly as designed. With GBM, such resistance develops in almost every patient. "In some patients, you can treat with a drug that does everything you could want it to do, but you would never know that the drug hit the target because the tumor adapts so quickly," Heath says.

Some scientists have suspected that the cancer becomes resistant through Darwinian-type evolution, in a process similar to how bacteria develop resistance to antibiotics. That is, the genetic differences of certain cancer cells may make those cells resistant to a drug. Nonresistant cells are killed by the drug and their death leaves room for the naturally resistant cells—and tumors—to grow and multiply.

However, this mechanism was not what Heath and his colleagues found in studies of tissue from glioblastoma patients. Instead, the researchers discovered that the cancer cells that developed resistance to a drug were the same cells that had responded to the drug. When the drug blocks a signaling pathway in a cancer cell, they realized, the cell simply finds a detour, like a GPS navigator that reroutes to avoid traffic.

"You can block a key part," Heath says, "and the cells will respond to route around that part you blocked."

This notion of shifting pathways is not new, but the work is the first to show that the process can happen in as little as two days. In particular, the researchers found that the changes occur with a specific drug (CC214‑2) that targets a central GBM signaling-protein called mTOR. When mTOR is inhibited, certain GBM signaling pathways are repressed, but others are activated.

To map the detours, the researchers separated individual GBM cells from patient tumors and measured the levels of several key proteins in the cells. These proteins—called phosphoproteins because they are activated by the addition of a phosphoryl group to a molecule—carry signals throughout the cell. Measurements of the abundance of the proteins showed that the drug was effective.

The story was different at the single-cell level, at which Heath and his colleagues not only measured the levels of proteins in individual cells, but also the signaling between those proteins. For example, if protein A signals protein B, then the levels of A, as measured across many single cells, will correlate with the levels of B.  By measuring the presence of several such proteins, the researchers could infer the structure of the protein signaling network.

They discovered that after the drug was introduced, the cell activated new pathways that previously had been dormant. This drug-induced pathway activation suggested several combination therapies that might halt the development of drug resistance, as well as drugging strategies that would have no effect.

In mice, Heath and his team tested seven therapies or therapy combinations that they predicted would—or would not—halt resistance development. The four that they predicted would not work were, indeed, ineffectual; the three they thought would work, did. The researchers then showed that they could see similar effects in GBM patient tissues, as well as in melanoma tumor models. This kind of rapid drug adaptation by tumors may occur in many cancer types, and helps explain how cancers can develop resistance to targeted drugs so quickly, Heath says.

The good news is that, by identifying the drug-activated signaling pathways, one may be able to find drug combinations that will suppress resistance, Heath says. Eventually, he says, clinicians may be able to analyze a patient's tumor at the single-cell level to determine the best therapy strategy.

These kinds of drug combinations would likely remain a secondary therapy against cancer—used when treatments like chemotherapy, radiation, and surgery fail. But, Heath says, they are essential for staving off the resistance that has severely limited the benefits that patients currently receive from targeted therapies.

The first authors of the Cancer Cell paper, titled "Single cell phosphoproteomics resolves adaptive signaling dynamics and informs targeted combination therapy in glioblastoma," are Wei Wei (PhD '14), a visitor in chemistry at Caltech and assistant professor at UCLA, and Young Shik Shin (MS '06, PhD '11), who now works at a biotech startup. Both are former graduate students of Heath's. A third key contributor to the work was Beatrice Gini, formerly a member of the UC San Diego (UCSD) laboratory of co-corresponding author Paul Mischel and now at UC San Francisco. Other Caltech authors include Min Xue and Kiwook Hwang (PhD '13) and graduate students Jungwoo Kim and Yapeng Su. Authors also include researchers from UCSD, the University of Verona in Italy, Northwestern University, and the Celgene Corporation. Heath is board member of and holds a financial interest in IsoPlexis, a company that is commercializing a microchip technology similar to what was used for single-cell analyses in the research described. 

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Rerouting Cancer
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A new strategy may help overcome cancer cells' drug resistance.

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