Volume 3, Issue 24 December 2006/January 2007
The Molecular Cascade of Stress
Daniela Kaufer studies the effects of stress on the brain. Credit: Alon Friedman
Under stress, our bodies follow an ancient and arcane code of instructions. At the first sense of danger, the adrenal glands unleash a flood of hormones known as glucocorticoids. These so-called stress hormones ready the body for intense physical action. They speed up the heart, deepen breathing, muscles to tremble with unaccustomed tension. But glucocorticoids also set far more subtle changes into motion. Study after study has linked stress to immune system suppression, memory impairment, hypertension, and disrupted digestion, among other ailments.
UC Berkeley biologist Daniela Kaufer investigates how the body transforms the psychological signal of stress into physiological changes to the brain. Her research demonstrates that stress affects every level of functioning, from how genes are transcribed to what proteins are translated. These tiny but profound shifts ultimately alter how entire body systems perform.
Kaufer's interest in stress began as a graduate student at the Hebrew University in Jerusalem, Israel, in the Soreq lab. One of her collaborators, neurosurgeon Alon Friedman of Ben-Gurion University, was investigating the origins of Gulf War syndrome. The symptoms appeared consistent with neurological damage, though no nerve gas was used during the war.
Kaufer and Friedman found that stress opens temporary leaks in the blood-brain barrier, the membrane separating the circulatory system from the central nervous system. "It's a very dynamic structure; it's not at all a barrier in the sense you think of it," Kaufer says. "The fact that the barrier is open brings into the brain a lot of things that shouldn't be there."
During short-term stress, opening the membrane's junctions can be advantageous. "The organism can focus as much as possible on shuttling energy to the places that need it most, which means sending lots of glucose to the muscles and brain," she says. But among Gulf War soldiers, battle stress may have allowed anti-nerve gas agents or other toxic substances such as pesticides to contact and injure the brain.
Kaufer's research is providing a molecular explanation for why exposure to stress affects the growth and behavior of brain neurons. Here, a molecule of corticosterone, a principal stress hormone, is shown against a background of cultured rat neurons. Credit: Eyal Soreq
Many types of brain trauma, including stroke and infection, also open the blood-brain barrier. Friedman and Kaufer realized that this might explain a longstanding puzzle in neurology—why patients with brain trauma often go on to develop epilepsy. Their research, published in the journal Brain this November, establishes a robust molecular connection between brain trauma, protein production, and epilepsy.
An epileptic seizure is an electrical storm in the brain. Instead of firing in a smooth and coordinated manner, the neurons generate bursts of erratic electrical impulses. The scientists found they could trigger these neural storms by exposing slices of rat brain to a common blood protein called serum albumin.
The scientists figured the albumin was binding directly to neurons. But further experiments proved them wrong. Albumin labeled with fluorescent markers wound up not in neurons but in brain cells called astrocytes.
Astrocytes are the brain's chemical custodians. They buffer the concentration of ions around neurons, which affects how easily neurons will fire. Kaufer and colleagues noticed that potassium levels were abnormally high in rat brains exposed to albumin. They also found that astrocytes exposed to albumin had abnormally few potassium channels. The implication: albumin disrupts potassium channel production in astrocytes.
Further experiments revealed that astrocytes use a specific receptor to take up albumin. Blocking that receptor, the researchers found, protects rat brains exposed to albumin from becoming epileptic. Kaufer is now figuring out which genes turn on and off during the development of epilepsy. At the same time, she is seeking small molecules to block this cascade and prevent trauma-induced epilepsy altogether.
Kaufer is pursuing two additional avenues of research into the molecular underpinnings of stress. One is how stress prevents new neuron production in the brain. The answers could explain why people often draw a blank when recalling extremely traumatic events. With gene therapy, she can create cells that no longer react to glucocorticoids, and therefore can't tell the body is experiencing stress. "I can put these genes in stem cells or newborn neurons or mature neurons and ask, does this change memory ability in mice?" Kaufer says.
Kaufer is also discovering that cells fine-tune their machinery to produce proteins better adapted to stressful conditions. Instead of transcribing entirely different suites of genes, she's found, cells splice existing protein templates in new ways. "The groups of proteins that are changing dictate where the cutting and ligating occurs during splicing," Kaufer says
Kaufer hopes her work will lead to ways of inoculating the body against the most damaging forms of stress. "If I'm right, and cells have good means of dealing with stressful situations, then by uncovering these mechanisms we can tap into that resource, and design therapeutic tools that use the body's own plastic capabilities. Eventually maybe before you send somebody into a stressful situation like war, you might be able to better prepare their brains to deal with stress so that they will be less likely to develop post-traumatic stress disorder at the end of it."
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Catching Gravitational Waves
Don Backer and graduate student Paul Demorest work on optimizing detection methods for gravitational waves. (Photo by Andrew West)
Don Backer goes about his days attuned to some of the faintest rhythms in the cosmos. As a UC Berkeley professor of astronomy, he's not interested in the proverbial music of the spheres. Rather, he aims to detect surges rippling the very fabric of spacetime: gravitational waves.
Gravitational waves were first predicted by Einstein in 1916. According to his General Theory of Relativity, extremely massive objects deform three-dimensional coordinates of space as well as time. Imagine dropping a bowling ball onto a trampoline; the resulting dimple is how a hefty object such as our Sun warps spacetime. Two stars rotating around one another, also known as a binary system, will not only distort the surrounding space, but also send out waves of distortions through this fabric. The effect is much like a rotating lawn sprinkler spewing out spiraling streams of water. Those space ripples are known as gravitational waves.
Princeton University astronomers Joe Taylor and Russell Hulse discovered a stellar binary made up of two hyperdense "neutron stars" in 1973. This binary led to the proof of the existence of gravitational waves. Measurements by Taylor and colleagues over the following decade showed that the orbit of the two stars about each other was slowly shrinking. The rate at which their orbit decays exactly matches the predictions of Einstein's theory. The discovery won Hulse and Taylor the 1993 Nobel Prize in Physics.
The Arecibo telescope in Puerto Rico is used for the pulsar timing array experiment. The average power from a pulsar collected by this telescope is a billion-billion times fainter than a household lightbulb. (Courtesy NAIC - Arecibo Observatory, a facility of the NSF)
Yet to this day, no one has managed to detect gravitational waves directly affecting detectors on Earth. Don Backer seeks to change this.
Backer is working to detect the relatively strong gravitational waves generated by massive black hole binaries. These objects, which are found at the center of virtually all galaxies, tip the scales at billions of times the mass of the Sun.
Invisible, endless, and trillions of times fainter than the radiation emitted by the average toaster oven, gravitational radiation can't be "seen" directly. However, its effects can. So Backer tracks how gravitational waves alter the precise, metronomic signals received from objects known as millisecond pulsars. Backer and colleagues discovered the first of these highly magnetized neutron stars in 1982. Spinning like tops at nearly 1,000 times per second, millisecond pulsars act like cosmic lighthouses, sending out beacons of electromagnetic radiation from each pole. The signals are so regular that millisecond pulsars are considered among the universe's most precise clocks.
"When a pulsar signal is sent through space distorted by a gravitational wave, there is an effect," Backer says the blips will arrive sooner than expected, then later, in a repeating pattern. For that reason, he says, "we have to be exquisitely precise about any perturbations of that clock." With observations from the world's most powerful radio telescopes, he can time the arrival of each few-minute batch of pulses with microsecond precision .
Black holes orbiting one another produce ripples in space-time called gravitational waves. These ripples cause the signals traveling from two pulsars (P1, P2) to the telescope (T) to arrive faster or slower than they expected if they were traveling through flat space. (Courtesy JPL/NASA)
Black hole coalescence events emit broad, low-frequency gravitational waves that require up to ten years to cycle from beginning to end. Backer has observed one pulsar in particular for more than 17 years, with detector technology he has made increasingly sensitive over the past two decades. At the same time, he and colleague Andrew Jaffe have been fine-tuning theoretical models of what gravitational waves from coalescing black holes across the Universe will look like, enabling him to further perfect his detectors.
Idiosyncrasies in any one pulsar's timing are rare, but possible. To rule out such anomalies, Backer and colleagues are watching more than a dozen millisecond pulsars scattered across the sky as part of his Pulsar Timing Array experiment. "We're looking for a predictable pattern of perturbations amongst the different objects, which are spinning totally independent of each other, that we can't explain otherwise."
The signal itself, says Backer, "will be a cacophony of radiation coming in from all directions." Chaotic though it sounds, the data will open a window into a mysterious property of the unseen universe. "It will inform us about the overall rate of black hole coalescence events. We can't see particular sources. And it doesn't tell us whether there are more little ones than big ones. But it does reveal how much spacetime is being perturbed by the aggregate effects of waves from these distant events flowing through the solar system."
The Allen Telescope Array under construction at Hat Creek Radio Observatory near Mount Lassen in Northern California. These antennas are among the first 42 currently being outfitted and commissioned. The project goal is an array of 350. (Photo by J. R. Forster)
His discovery would also further the next generation of gravitational wave analysis. NASA and the European Space Agency are developing the Laser Interferometer Space Antenna (LISA), which should begin operating in space by 2015. The instrument is designed to detect the brilliant bursts of gravitational waves released by individual black hole mergers. Backer's gravitational wave data would tell astronomers how often these merging events are likely to occur.
Meanwhile, Backer continues to hone his pulsar detecting equipment and techniques. "We just have to make our measurements more precise with new equipment and new telescopes such as the Allen Telescope Array being built jointly by Berkeley's Radio Astronomy Laboratory and the SETI Institute at the Hat Creek Radio Observatory; find new pulsars to track; and dig deeper in the sensitivity."
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The 3D Language of Cells
Jay Groves is revealing how the information embedded in cell membrane patterns and other complex molecular environments affect cell behavior and organization. (courtesy A. Demond)
Beneath your skin, out of your sight, your cells are locked in heated conversation. Like teens glued to a telephone, they have much to say to one another. Who are you? What have you sensed? What do you need? Do you belong?
Though blind, deaf, and mute, cells face no communication quandary. They send out signaling molecules, tiny versions of a castaway's message in a bottle. They stud themselves with receptors, to snag other signaling molecules floating by. Each also plasters itself with molecular identity tags proclaiming citizenship in the body.
Now Jay Groves, a UC Berkeley professor of chemistry, is deciphering yet another mode of cellular discourse: spatial patterning. An expert in molecular self-organization, Groves' current research focuses on immune system components called T cells. Like customs agents, T cells use receptors on their surfaces to examine the identification molecules of every cell they encounter. When a T cell encounters another cell carrying foreign ID, two of its membrane proteins bond to other proteins on the alien cell. Then an astonishing thing occurs: those bonded proteins arrange themselves into a distinctive bull's-eye pattern. The T cell then releases a flood of intracellular calcium to sound the invasion alarm.
Groves has invented a half-live, half silicon cell junction by floating cell membrane molecules atop a silicon chip. Tiny walls (black) built atop the chip (white) restrict the movement of cell membrane molecules (red and yellow) floating in a sea of lipid molecules (blue). (Dr. Raghu Parthasarathy, former Groves postdoc)
Groves suspected the pattern conveyed additional information to the cell. But to find out for sure, he had to manipulate the physical locations of the membrane proteins themselves. "The goal was to see how forcing these clusters into different patterns might change how the T cells react," Groves says.
So Groves built a totally new experimental platform—a half-living, half artificial cell. He patterned bars of chromium in various patterns atop silicon wafers using the same techniques used to make computer chips. Fusing lipid molecules and cell membrane proteins to the surface of the chip results in what Groves calls a hybrid live cell-supported membrane junction.
T cells placed on top of the hybrid membrane junction can't tell the difference between the hybrid cell and a natural cell. "The T cell never sees the metal or the semiconductor. The only thing it interacts with is the protein," Groves says. "But with these structures we've snuck in underneath, we control the way the proteins move."
The patterned chips radically altered the T cells' behavior. Instead of forming one large, central bull's-eye pattern, the proteins clustered into many miniature bull's-eyes, each constrained within its own chromium-walled room. "It's like putting little barricades in there; the proteins can't get over them, so they stop the receptor clusters from moving around. The cell tries to drag them around, but they get stuck or redirected."
T cells arrange their receptors into a distinctive bull's-eye pattern (A) when they bind to foreign cells. Groves uses patterned silicon chips to disrupt this pattern (B-D) and determine its importance in cell signaling. (courtesy Jay Groves)
Disrupting the pattern clearly impacted the T cells' behavior. Those unable to pull their receptors into a central area released a much stronger alarm signal than normal. "The cell is using the bull's-eye pattern to control its sensitivity. It's moving its receptors out of a region of very high sensitivity to turn down its signal," Groves says.
This mechanism helps the body modulate its immune response. "If you have an extensive viral infection, the T cell in that case will initiate a response immediately because it will detect a large number of viral protein antigens. If it sees a smaller signal, it doesn't react that way, because you don't want to initiate a huge immune response to a small thing. That would take a huge amount of energy and you'd constantly feel sick."
Now Groves has developed a means to alter the reactive properties of molecules anywhere in a cell. The method takes advantage of the special properties of a compound known as NVOC. NVOC readily attaches to other molecules with a bond that dissolves in 320 nm UV light.
In the November issue of the Journal of the American Chemical Society, Groves reports using NVOC to cage the identity tag of a particular foreign protein. He then added the protein to one of his hybrid cell membrane junctions. T cells crawling atop the membrane never paused. But when Groves hit the membrane with UV light, the T cells stopped to form bull's-eye patterns. The beam of light had successfully uncaged the foreign protein sequences, allowing them to be recognized by the T cells.
Groves is now moving beyond manipulating surface proteins. "We can pick a spot inside of a cell, and uncage a protein or drug molecule only at that point," Groves says. By focusing an intense beam of longer wavelength light in a particular spot, he can uncage only proteins at the focal point, leaving all other cell molecules unaffected.
Groves' goal is to understand the forces of molecular organization that bring a cell to life. "It's like examining the sound wave of a symphony. It's fantastically complicated, but if you look at a larger scale, there are some patterns—a periodic beat there, and repeated phrases. And you realize there might be levels higher up in the complexity that might be simple, that organize the entire piece. There's something big out there that's regulating the chemistry of life, and that's where we're headed."