Molecular Mechanism Contributing to Neuronal Circuit Formation Found

ScienceDaily (Mar. 7, 2011) — Scientists at Helmholtz Zentrum München have discovered how sensory and motor fibers* interact during development of neuronal circuits in the limbs: Both types of nerve fibers can guide this process. With this finding, the researchers have made an important contribution to understanding how neural networks are formed during embryonic development and have found a new approach to explaining neurological disorders.

Researchers observed that motor and sensory axons were both able to guide and lead the formation of the spinal nerves of the arms and legs. 

During embryonic development, sensory and motor fibers interact to form nerves in the limbs. The research team led by Dr. Andrea Huber Brösamle of the Institute of Developmental Genetics of Helmholtz Zentrum München has now elucidated how this interaction functions at the molecular level: The cell surface receptor neuropilin-1 is present in both sensory and motor nerve fibers and controls their interaction in order to correctly regulate growth.

"We observed that motor and sensory axons were both able to guide and lead the formation of the spinal nerves of the arms and legs," said Rosa-Eva Hüttl and Heidi Söllner, lead authors of the study and doctoral students in Dr. Andrea Huber Brösamle's research group. This finding surprised the authors because it had previously been assumed that the motor axons were always responsible for establishing the correct trajectories. In the same study, the researchers created a model to better elucidate structural changes in human neurodegenerative disorders and following trauma : "Our next goal," said Dr. Huber Brösamle, "is to find out to what extent neuropilin-1 also controls the formation of fiber tracts in the brain."

Note:

*Nerves are bundles of sensory and motor fibers. The sensory fibers transmit physical sensation signs (e.g. pain), while the motor fibers control the contraction and movement of muscles.

Laminin's Role in Cancer Formation Illuminated

ScienceDaily (Mar. 7, 2011) — Laminin, long thought to be only a structural support protein in the microenvironment of breast and other epithelial tissue, is "famous" for its cross-like shape. However, laminin is far more than just a support player with a "pretty face." Two studies led by one of the world's foremost breast cancer scientists have shown how laminin plays a central role in the development of breast cancer, the second most leading cause of cancer death among women in the United States. In one study it was shown how laminin influences the genetic information inside a cell's nucleus. In the other study it was shown how destruction of laminin can play a detrimental role in the early stages of tumor development.

A 3D cell culture assay developed by Mina Bissell and her research group enables breast cells to reproduce actual structural units, an advantage that was essential for understanding the role of laminin in breast cancer development.

Mina Bissell is famous for having discovered the critical role in breast cancer development played by the extracellular matrix (ECM), the network of fibrous and globular proteins surrounding a breast cell. Her "dynamic reciprocity" theory holds that the fate of cells -- whether they stay healthy or become cancerous -- hinges on the chemical signals exchanged between the ECM and a cell's nucleus. In these latest studies, Bissell and her collaborators focused on laminin and its connections with two other proteins -- actin, a cytoplasmic protein that has been linked to nuclear activities; and MMP9, an enzyme that is secreted outside the cells and is known to break down ECM constituents.

Laminin and Cell Quiescence

"Quiescence" is the process by which a biological cell stops growing or dividing. This is the opposite of a cancerous state, in which cell growth and division is often unchecked. Signals from laminin-111, an ECM protein that helps the cell and its ECM stick together, have been linked to cell quiescence but the mechanism was unknown. Bissell and postdoctoral fellow, Virginia Spencer, in Berkeley Lab's Life Sciences Division, have now shown that the addition of laminin-111 leads to quiescence in breast epithelial cells through changes in nuclear actin.

"We found that high levels of laminin-111 depleted nuclear actin and this in turn induced cell quiescence," Bissell says. "Furthermore, this process can be prevented if a form of actin that can not exit the nucleus is introduced. Under these conditions the cells do not stop growing even in the presence of laminin."

In their study, Bissell and Spencer and their colleagues used a unique three-dimensional cell culture assay developed by Bissell's research group, and worked with mouse and human mammary epithelial cells. Through the addition of laminin-111, they were able to decrease nuclear actin levels in the cultured cells, which reduced DNA synthesis and transcription. When nuclear actin levels were deliberately over-expressed, the effects were reversed and cells were prevented from becoming quiescent even in the presence of laminin-111. Furthermore, the high levels of nuclear actin were concentrated in regions of the breast cells where there was little or no laminin-111. Taken together, the results implicate laminin-111 as the regulator of nuclear actin and nuclear actin as a key mediator of epithelialcell quiescence.

"In collaboration with Ole Petersen's laboratory, we had found previously that the ECM surrounding tissues from breast cancers has a dramatic reduction in laminin-111 in comparison to the ECM surrounding a normal breast cell, which is rich in laminin-111," Bissell says. "However, just giving laminin back to cancer cells was not enough to make them normal so other factors are clearly also involved and one such factor we now know is how laminin-111 and nuclear actin talk to each other!"

Says Spencer, "Ours is the first study to actually identify laminin-111 as the physiological regulator of nuclear actin and to implicate the loss of nuclear actin as a key step in reaching quiescence and homeostasis in the mammary gland in vivoand in culture."

Spencer believes that the interaction between laminin-111 and nuclear actin could provide a new target for diagnostic therapeutic efforts, but this will require further study.

"While it remains to be determined whether dysregulation of the levels or organization of nuclear actin is responsible for the inability of malignant cells to respond to growth-inhibitory signals from laminin-111, our preliminary results point in this direction," she says. "In addition, the findings that laminin-111 expression is lost in myoepithelial cells isolated from human tumors should place the interaction of laminin-111 and breast tumor cells at the forefront of future investigations."

A paper detailing the results of this study appears in theJournal of Cell Science. The paper is titled "Depletion of nuclear actin is a key mediator of quiescence in epithelial cells." Co-authoring the paper with Bissell and Spencer were Sylvain Costes, Jamie Inman, Ren Xu, James Chen and Michael Hendzel.

Laminin, MMP9 and Tumor Growth

In the second study, which was related to the role of laminin-111 in cell quiescence, Bissell and another group of collaborators examined laminin-111 in the context of matrix metalloproteinase-9 (MMP9), a zinc-dependent enzyme that plays a huge role in tissue function by virtue of its ability to cleave or degrade many of the ECM constituent proteins, including laminin-111.

"Organization into polarized three-dimensional tissue structures defines whether epithelial cells are normal or malignant," Bissell says. "We have shown that when MMP9 degrades laminin-111 in the ECM, the tissue architecture of breast cells becomes lost and cell proliferation is initiated. This is the first demonstration of how the degradation of laminin-111 by MMP9 in a physiological context contributes to tumor progression."

A paper detailing the results of this study has appeared in the journal Genes and Development. The paper is titled "Raf-induced MMP9 Disrupts Tissue Architecture of Human Breast Cells in Three-Dimensional Culture and is Necessary for Tumor Growth in vivo." Co-authoring the paper with Bissell were Alain Beliveau, Joni Mott, Alvin Lo, Emily Chen, Antonius Koller, Paul Yaswen and John Muschler.

Using a model of human breast cancer where breast epithelial cells were grown in three-dimensional cultures of basement membrane, a thin layer of ECM material that envelops breast and other glandular tissue, Bissell and her co-authors found that not only did excessive MMP9 activity disrupt tissue architecture, but that silencing MMP9 restored tissue architecture and decreased the ability of human beast cancer cells to form tumors in mice.

"We found that in all conditions where tumor cells could be reverted to a normal phenotype in our 3D assays, a novel signaling loop through a pathway of Raf/MEK/ERK proteins was responsible for MMP9 activity in the breast tumor cells," says co-author Joni Mott, a researcher with Bissell's group in Berkeley Lab's Life Sciences Division. "Once MMP9 was activated, the proteinase targeted the destruction of laminin-111 within the basement membrane."

Laminin-111 in the basement membrane, Mott explains, allows mammary epithelial cells to establish a normal polarized structural unit called an "acinus," which is responsible for storing milk and making it available for babies when they suckle.

In their Genes and Development paper, Bissell, Mott and their co-authors reported that when the integrity of the tissue architecture was compromised by laminin proteolysis, the basement membrane no longer provided the appropriate signals to restrain epithelial cell proliferation. The result was a sustained signaling of the Raf/MEK/ERK pathway that leads to continued MMP9 production and further disruption of tissue architecture and loss of cell growth control.

"This work is particularly poignant because it provides potential new therapeutic targets for controlling breast cancer and revitalizes the possibility of targeting MMPs in cancer therapy," Bissell says. "New information on how MMP9 and other MMPs truly function may provide highly targeted and effective therapeutic strategies to control MMP activity in cancer, and may soon lead to the development of novel cancer treatments."

Both studies were funded in part by grants from the U.S. Department of Energy's Office of Science, the National Cancer Institute, and the U.S. Department of Defense.

Enzyme Enhances, Erases Long-Term Memories in Rats; Can Restore Even Old, Fading Memories, Say Scientists

ScienceDaily (Mar. 7, 2011) — Even long after it is formed, a memory in rats can be enhanced or erased by increasing or decreasing the activity of a brain enzyme, say researchers supported, in part, by the National Institutes of Health.

A neuron in a rat brain's cortex over-expresses PKMzeta (blue).

"Our study is the first to demonstrate that, in the context of a functioning brain in a behaving animal, a single molecule, PKMzeta, is both necessary and sufficient for maintaining long-term memory," explained Todd Sacktor, of the SUNY Downstate Medical Center, New York City, a grantee of the NIH's National Institute of Mental Health.

Sacktor, Yadin Dudai, Ph.D., of the Weizmann Institute of Science, Rehovot, Israel, and colleagues, report of their discovery March 4, 2011 in the journal Science.

Unlike other recently discovered approaches to memory enhancement, the PKMzeta mechanism appears to work any time. It is not dependent on exploiting time-limited windows when a memory becomes temporarily fragile and changeable -- just after learning and upon retrieval -- which may expire as a memory grows older, says Sacktor.

"This pivotal mechanism could become a target for treatments to help manage debilitating emotional memories in anxiety disorders and for enhancing faltering memories in disorders of aging," said NIMH Director Thomas R. Insel, M.D.

In their earlier studies, Sacktor's team showed that even weeks after rats learned to associate a nauseating sensation with saccharin and shunned the sweet taste, their sweet tooth returned within a couple of hours after rats received a chemical that blocked the enzyme PKMzeta in the brain's outer mantle, or neocortex, where long-term memories are stored.

In the new study, they paired genetic engineering with the same aversive learning model to both confirm the earlier studies and to demonstrate, by increasing PKMzeta, the opposite effect. They harnessed a virus to infect the neocortex with the PKMzeta gene, resulting in overexpression of the enzyme and memory enhancement. Conversely, introducing a mutant inactive form of the enzyme, that replaced the naturally occurring one, erased the memory -- much as the chemical blocker did.

These effects applied generally to multiple memories stored in the targeted brain area -- raising questions about how specific memories might be targeted in any future therapeutic application.

The researchers turned up a clue that may hold the beginning of an answer.

"One explanation of the memory enhancement is that PKMzeta might go to some synapses, or connections between brain cells, and not others," said Sacktor. "Overexpressed PKMzeta may be selectively captured by molecular tags that mark just those brain connections where it's needed -- likely synapses that were holding the memory from the training."

Stretchable Balloon Electronics Get to the Heart of Cardiac Medicine

ScienceDaily (Mar. 7, 2011) — Cardiologists may soon be able to place sensitive electronics inside their patients' hearts with minimal invasiveness, enabling more sophisticated and efficient diagnosis and treatment of arrhythmias.

Fully inflated multifunctional balloon instrumented with temperature, tactile and EKG sensors on islands interconnected by non-coplanar serpentine wires.

A team of materials scientists, mechanical and electrical engineers, and physicians has successfully integrated stretchable electronics technology with standard endocardial balloon catheters. Led by John A. Rogers, the Lee J. Flory-Founder Chair in Engineering at Illinois, the team published its work in the March 6 online edition of Nature Materials.

The team previously demonstrated a sensor-laden sheet that could laminate to the surface of the heart in 2010. Now they have expanded their technology to endocardial balloon catheters, one of the most common, least-invasive devices for cardiac procedures.

Catheters are long, flexible tubes that can be threaded through a vein or artery to reach the inside of the heart. Catheters with balloons at the end are commonly used for angioplasty, stent placement and other procedures as passive mechanical instruments. When in place, the balloon inflates and gently presses against the surrounding tissue to open blood vessels or valves.

Invasive cardiologists specializing in heart rhythm disorders use catheters with electrodes at the end for detecting and mapping arrhythmias and for ablation, or selectively killing small patches of cells that beat off-rhythm. Current invasive arrhythmia procedures involve two separate, rigid catheter devices: one that maps the heart point-by-point as a cardiologist maneuvers the tube in search of irregularities, and one with an electrode at the end that ablates spots identified as aberrant, one at a time.

The balloon device Rogers' team developed can perform both functions over large areas of the heart simultaneously, using integrated arrays of multifunctional sensors and ablation electrodes.

"It's all in one, so it maps and zaps," said Rogers, a professor of materials science and engineering who also is affiliated with the Beckman Institute for Advanced Science and Technology at Illinois. "The idea here is instead of this single-point mapping and separate single-point zapping catheter, have a balloon that offers all that functionality, in a mode that can do spatial mapping in a single step. You just inflate it right into the cavity and softly push all of that electronics and functionality against the tissue."

The researchers created a meshwork of tiny sensor nodes that could mount directly onto a conventional catheter balloon. The device holds an array of sensors to measure electrical activity of the cardiac muscle, temperature, blood flow, and pressure as the balloon presses against the tissue, along with electrodes for ablation. The entire system is designed to operate reliably as the balloon inflates and deflates.

"It demands all the features and capabilities that we've developed in stretchable electronics over the years in a pretty aggressive way," Rogers says. "It also really exercises the technology in an extreme, and useful, manner -- we put everything on the soft surface of a rubber balloon and blow it up without any of the devices failing."

The Illinois team collaborated with cardiologists at the University of Arizona and Massachusetts General Hospital (MGH) to determine what types of features would be most useful for patient care.

For example, the researchers added temperature sensors and mapped temperature distribution on actual tissue as areas were ablated. From this data they developed a model to predict temperature distribution so cardiologists know how deep into the tissue they are ablating.

"Adding a feature such as temperature detection and distribution gives us greater insight as to what we are actually doing to the tissue," said co-author Dr. Marvin J. Slepian, a practicing cardiologist and a professor of medicine at the Sarver Heart Center of the University of Arizona. "This will enhance the safety and effectiveness of ablation catheters, providing a new level of precision that we have not had to date, while simultaneously shortening the length of procedure times, which is an overall 'win' for patients, physicians and hospitals."

Rogers' team also worked closely with mc10, a company he co-founded that is commercializing the underlying technology for both medical and non-medical applications. Several researchers at mc10 are co-authors of the paper. The company has tested the devices in live animal experiments with medical collaborators at Arizona and MGH.

The biggest challenge for the researchers was ensuring full functionality of the electronics at all levels of balloon inflation. Since the center of the balloon stretches more than the ends, they had to figure out the range of strain the sensors would encounter and how to accommodate it so that sensors at the most strained points would function the same as those at areas of lower strain.

Through a collaboration with researchers at Northwestern University, led by Younggang Huang, the team solved this problem by mounting the sensors and electrodes on tiny rigid islands so they wouldn't be affected by the balloon stretching. They also used spring-like interconnects between the sensors to handle the 100 percent distance increase between the islands when the balloon inflates.

The fabrication techniques the engineers used in developing the balloon device could be exploited for integrating many classes of advanced semiconductor devices on a variety of surgical instruments. For example, the team also demonstrated surgical gloves with sensor arrays mounted on the fingertips to show that the electronics could be applied to other biomedical platforms.

Next, Rogers would like to further increase the density of sensors on the balloon, up to thousands of tiny, multiplexed devices on the surface. This design would enable the integration of sophisticated electronic systems with the capability for even greater resolution for mapping and the ability to ablate the minimal amount of tissue. He also plans to continue exploring medical device applications for stretchable, flexible electronic arrays in other surgical tools.

"Being able to embed these kinds of advanced semiconductor devices into tissue-like formats creates all kinds of new ways to do minimally invasive procedures," Rogers said. "I'm hopeful that this will be the first of many devices that collectively can have a major impact on the way human health care is done."

This work was supported by the National Science Foundation and the Department of Energy. The authors will present related findings at the Heart Rhythm Society Meeting in San Francisco in May.

Multiple Sclerosis Blocked in Mouse Model: Barring Immune Cells from Brain Prevents Symptoms

ScienceDaily (Mar. 7, 2011) — Scientists have blocked harmful immune cells from entering the brain in mice with a condition similar to multiple sclerosis (MS).

New insights into how the brain and spine regulate immune cell entry have allowed scientists at Washington University School of Medicine to block the development of multiple sclerosis-like symptoms using a mouse model of the disease. When scientists gave mice a drug that suppressed the activity of a key molecule, immune cells (stained green in these images) lined up at the boundaries of the spine (right) instead of going in (left).

According to researchers from Washington University School of Medicine in St. Louis, this is important because MS is believed to be caused by misdirected immune cells that enter the brain and damage myelin, an insulating material on the branches of neurons that conduct nerve impulses.

New insights into how the brain regulates immune cell entry made the accomplishment possible. Washington University scientists had borrowed an anti-cancer drug in development by the company ChemoCentryx simply to test their theories.

"The results were so dramatic that we ended up producing early evidence that this compound might be helpful as a drug for MS," says Robyn Klein, MD, PhD, associate professor of pathology and immunology, of medicine and of neurobiology. "The harmful immune cells were unable to gain access to the brain tissue, and the mice that received the highest dosage were protected from disease."

ChemoCentryx is now testing the drug in Phase I safety trials. The study is published in The Journal of Experimental Medicine.

Klein and her colleagues discovered a chemical stairway that immune cells have to climb down to enter the brain. Immune cells that exit the blood remain along the vessels on the tissue side, climbing down from the meninges into the brain where they can then cross additional barriers and attack myelin on the branches of neurons.

"The effect of immune cell entry into the brain depends on context," Klein says. "In the case of viral infection, immune cell entry is required to clear the virus. But in autoimmune diseases like multiple sclerosis, their entry is associated with damage so we need to find ways to keep them out."

The stairway is located on the tissue side of the microvasculature, tiny vessels that carry blood into the central nervous system. The steps are made of a molecule called CXCL12 that localizes immune cells, acting like stairs that slow them down so that they can be evaluated to determine if they are allowed to enter the brain. Klein's lab previously discovered that the blood vessel cells of the microvasculature display copies of this molecule on their surfaces.

Klein also found that MS causes CXCL12 to be pulled inside blood vessel cells in humans and mice, removing the stairway's steps and the checkpoints they provide. In the new paper, she showed that blocking the internalization of the molecule prevented immune cells from getting into the brain and doing harm.

Work by another lab called Klein's attention to CXCR7, a receptor that binds to CXCL12. She showed that the receptor is made by the same cells in the microvasculature that display CXCL12. They watched the receptor take copies of CXCL12 and dump them in the cells' lysosomes, pockets for breakdown and recycling of molecules the cell no longer needs.

"After it dumps its cargo in the lysosome, the receptor can go right back to the cell surface to pull in another copy of CXCL12," Klein says. "There likely exists an equilibrium between expression and disposal of CXCL12. Some of the proteins expressed by the immune cells in MS patients affect CXCR7 expression and activity, disrupting the equilibrium and stripping the steps from this immune cell stairway we're studying."

Klein contacted researchers at ChemoCentryx, who were developing a blocker of the CXCR7 receptor as a cancer treatment. When they gave it to the mouse model of MS, immune cells stopped at the meninges.

Klein also found that immune factors could cause microvasculature cells to make more or less of CXCR7, ramping up or down the number of steps on the chemical stairway. She is currently investigating additional immune factors that impact on CXCR7 activity within the blood vessel cell. Whether a given factor promotes or suppresses the receptor may also differ depending upon what part of the brain is being considered.

"One of the biggest questions in MS has been why the location, severity and progression of disease varies so much from patient to patient," Klein says. "Getting a better understanding of how these factors regulate immune cell entry will be an important part of answering that question."

'Nano-Velcro' Technology Used to Improve Capture of Circulating Cancer Cells

ScienceDaily (Mar. 7, 2011) — Circulating tumor cells, which play a crucial role in cancer metastasis, have been known to science for more than 100 years, and researchers have long endeavored to track and capture them. Now, a UCLA research team has developed an innovative device based on Velcro-like nanoscale technology to efficiently identify and "grab" these circulating tumor cells, or CTCs, in the blood.

Nano-Velcro 2 'Nano-Velcro' microfluidic chip. An integrated chip for detecting circulating tumor cells in blood collected from prostate cancer patients. 

Metastasis is the most common cause of cancer-related death in patients with solid tumors and occurs when these marauding tumor cells leave the primary tumor site and travel through the blood stream to set up colonies in other parts of the body.

The current gold standard for determining the disease status of tumors involves the invasive biopsy of tumor samples, but in the early stages of metastasis, it is often difficult to identify a biopsy site. By capturing CTCs in blood samples, doctors can essentially perform a "liquid" biopsy, allowing for early detection and diagnosis, as well as improved monitoring of cancer progression and treatment responses.

In a study published this month and featured on the cover of the journal Angewandte Chemie, the UCLA researchers announce the successful demonstration of this "nano-Velcro" technology, which they engineered into a 2.5-by-5-centimeter microfluidic chip. This second-generation CTC-capture technology was shown to be capable of highly efficient enrichment of rare CTCs captured in blood samples collected from prostate cancer patients.

The new approach could be even faster and cheaper than existing methods, and it captures a greater number of CTCs, the researchers said.

The prostate cancer patients were recruited with the help of a clinical team led by physicians Dr. Matthew Rettig, of the UCLA Department of Urology, and Dr. Jiaoti Huang, of the UCLA Department of Pathology and Laboratory Medicine.

The new CTC enrichment technology is based on the research team's earlier development of 'fly-paper' technology, outlined in a 2009 paper in Angewandte Chemie. The technology involves a nanopillar-covered silicon chip whose "stickiness" resulted from the interaction between the nanopillars and nanostructures on CTCs known as microvilli, creating an effect much like the top and bottom of Velcro.

The new, second-generation device adds an overlaid microfluidic channel to create a fluid flow path that increases mixing. In addition to the Velcro-like effect from the nanopillars, the mixing produced by the microfluidic channel's architecture causes the CTCs to have greater contact with the nanopillar-covered floor, further enhancing the device's efficiency.

"The device features high flow of the blood samples, which travel at increased (lightning) speed," said senior study author Dr. Hsian-Rong Tseng, an associate professor of molecular and medical pharmacology at the UCLA Crump Institute for Molecular Imaging and the California NanoSystems Institute at UCLA.

"The cells bounce up and down inside the channel and get slammed against the surface and get caught," explained Dr. Clifton Shen, another study author.

The advantages of the new device are significant. The CTC-capture rate is much higher, and the device is easier to handle than its first-generation counterpart. It also features a more user-friendly, semi-automated interface that improves upon the earlier device's purely manual operation.

"This new CTC technology has the potential to be a powerful new tool for cancer researchers, allowing them to study cancer evolution by comparing CTCs with the primary tumor and the distant metastases that are most often lethal," said Dr. Kumaran Duraiswamy, a graduate of UCLA Anderson School of Management who became involved in the project while in school. "When it reaches the clinic in the future, this CTC-analysis technology could help bring truly personalized cancer treatment and management."