Newly Discovered Mechanism Controls Levels and Efficacy of a Marijuana-Like Substance in the Brain

A newly discovered molecular mechanism helps control the amount and effectiveness of a substance that mimics an active ingredient in marijuana, but that is produced by the body's own nerve cells.

The results were reported in the latest Nature Neuroscience. The lead author on the study is William R. Marrs of the Neurobiology and Behavior program at the University of Washington (UW). The senior author is Dr. Nephi Stella, UW professor of pharmacology and psychiatry.

In previous papers, Stella and other scientists have noted that the body manufactures several cell signals that mimic the actions of marijuana-derived chemicals These signals are called endocannabinoids, a Latin-derived name for marijuana-like (cannabis) constituents created by the body's own cells (endo).

Marrs, Stella and their research team study endocannabinoids, their receptors on cells, and the cell functions controlled by these signals.

They hope their future work encourages the design of therapies to modulate these molecular communications. Specifically targeted treatments, for example, might give cancer and AIDS patients the same medicinal benefits as marijuana without its mind-altering properties.

Because cannabinoid signaling systems are common throughout the body and affect a variety of functions, therapies aimed at these systems might be more wide-ranging than simply a better substitute for medicinal marijuana. Stella is especially interest in the potential for helping people with conditions for which even symptomatic treatment is limited or non-existent, such as multiple sclerosis, brain tumors, Huntington's disease and other autoimmune or neurological disorders.

Earlier Stella's group discovered a key endocannabinoid, called 2-AG, that carries a type of messaging between brain cells. 2-AG is also implicated in brain cell migration and brain tissue inflammation. It does this by activating one type of cannabinoid receptor on neurons, and another type of cannabinoid receptor on microglia, the tiny cells that clean up debris, like damaged nerve cells and plaque, in the brain and spinal cord. As the brain's first line of defense against infection, microglia are attune to the most subtle clues suggesting an attack.

Stella's team further investigated 2-AG nerve cell signaling in the study just published in Nature Neuroscience. They looked at an enzyme called ABHD6, newly identified by other scientists using advanced protein profiling technology, also known as proteomics. ABHD6 is present in nerve cells in the brain.

Stella's team observed that this enzyme degrades the 2-AG nerve signaling substance by splitting it with water. This happens near the cell receptor for the 2-AG signal.

Breaking apart 2-AG reduced its accumulation and decreased its ability to prod other cells to action. In this case, the broken down 2-AG was less effective in stimulating the microglia -- the brain defenders -- to get moving.

The results provided by their study, the authors said, suggest that the enzyme ABDH6 "is a bona fide member of the endocannabinoid signaling system."

"The enzymatic steps that control the production and inactivation of endocannabinoids constitute promising molecular targets for indirectly modulating the activity of cannabinoid receptors," the authors noted. Designing treatments that manage the production and inactivation of important enzymes like ABHD6 could thereby control such conditions as brain inflammation or overactive brain signals. Other enzymes are involved in controlling the accumulation of different endocannabinoids.

Each of these enzymes, the researchers pointed out, provides a unique therapeutic opportunity. Inhibiting distinct enzymes would allow for the fine-tuned direction of endocannabinoid signaling. For example, blocking a specific enzyme to heighten a certain signal might ameliorate pain and also act as anti-anxiety and antidepressant therapy, the authors explained. Drugs that reduce the activity of the ABDH6 enzyme might prevent brain damage from an overactive response to a virus.

The study was supported by grants from the National Institute on Drug Abuse and the National Institute of General Medical Sciences, both part of the National Institutes of Health.

In addition to Marrs and Stella, other researchers on the study are Jacqueline L. Blankman, Jessica P. Alexander, Jonathan Z. Long, Weiwei Li, and Benjamin F. Cravatt, all of Scripps Research Institute; Eric A. Horne, Yi Hsing Lin, Jonathan Coy, and Cong Xu, all of the UW Department of Pharmacology; Aurore Thomazeau, Mathieu LaFourcade and Olivier J. Manzoni, all of INSERM, Bordeaux, France; Agnes L. Bodor of the UW Department of Otolaryngology; Giulio G. Muccioli of the Louvain Drug Research Institute, Bruxelles, Belgium; Sherry Shu-Jung Hu and Ken Mackie, of Indiana University; Grace Woodruff of the UW Neurobiology Undergraduate Program; Susan Fung of the UW Neurobiology and Behavior Graduate Program, and Thomas Moller of the UW Department of Neurology.

Timely Technology Sees Tiny Transitions

This computer simulation shows the electric field of a bipyramid. Molecules moving through the field shift the quality of light, which can be read via spectroscopy. (Credit: Nordlander lab)

Scientists can detect the movements of single molecules by using fluorescent tags or by pulling them in delicate force measurements, but only for a few minutes. A new technique by Rice University researchers will allow them to track single molecules without modifying them -- and it works over longer timescales.

In the current issue of Nanotechnology, a team led by Jason Hafner, an associate professor of physics and astronomy and of chemistry, has shown that the plasmonic properties of nanoparticles can "light up" molecular interactions at the single-molecule limit in ways that will be useful to scientists.

Hafner's method takes advantage of the ability of metal nanoparticles to focus light down to biomolecular scales through an effect called localized surface plasmon resonance (LSPR). The gold nanoparticles ultimately used in the experiment scatter light in visible wavelengths, which can be detected and spectrally analyzed in a microscope.

"The exact peak wavelength of the resonance is highly sensitive to small perturbations in the nearby dielectric environment," said graduate student Kathryn Mayer, the lead student on the experiment. "By tracking the peak with a spectrometer, we can detect molecular interactions near the surface of the nanoparticles."

Hafner first discussed their progress at a 2006 conference after a presentation on gold nanostars his lab had developed. "We had extremely preliminary data, and I said, 'Maybe we've got it.' I thought we were close," he recalled.

What took time was finding the right particle. "We started with nanorods, which don't scatter light well, at least not the small nanorods we produce in my lab. Then we tried nanostars and found they were very bright and sensitive, but each was a different shape and had a different peak wavelength."

The team settled on bipyramids, 140-nanometer-long, 10-sided gold particles that focus light at their sharp tips, creating a halo-like "sensing volume," the dielectric environment in which changes can be read by a spectrometer.

Hafner and his colleagues borrowed bioconjugate chemistry techniques, coating the bipyramids with antibodies and then adding antigens that strongly bind to them. Then the antigens were rinsed off. Whenever one was released from its bond to the bipyramid antibody, the researchers detected a slight shift toward the blue in the red light naturally scattered by gold bipyramids.

The process is "label-free," meaning the molecule itself is being detected, rather than a fluorescent tag that requires modification of the molecule, Hafner said. Also, the dielectric property being detected is permanent, so molecules could be tracked for more than 10 hours, as compared with only minutes with current methods.

"The ability to measure over long time scales opens the possibility to study systems with strong affinity at the single-molecule limit, such as lectin-carbohydrate interactions responsible for cell recognition and adhesion," Hafner said. "Other single-molecule methods based on fluorescence are limited by photo bleaching, and those based on force measurements are limited by radiation damage and mechanical instabilities."

Work needs to be done before LSPR becomes an ideal biological sensor, he said. The team plans to tweak the bipyramids and will test other particles.

"With this bipyramid, we went a little too red," he said. "It's a compromise. Make them long and they're really sensitive, but so red that we don't get much signal. Make them shorter, they're somewhat less sensitive but you have more signal.

"If we can get the signal-to-noise ratio up by a factor of two or three, we think it will be a powerful method for biological research."

In addition to Mayer, Hafner's co-authors included Peter Nordlander, a Rice professor of physics and astronomy and of electrical and computer engineering, former Rice graduate student Feng Hao, now a postdoctoral fellow at Sandia National Laboratories, and Rice graduate student Seunghyun Lee.

Funding for the project came from the National Science Foundation's Integrative Graduate Research and Educational Training program and Nanoscale Science and Engineering Initiative, the U.S. Army Research Office and the Welch Foundation.

Mimicking the Moon's Surface in the Basement

Ion Beam Materials Lab. (Credit: Image courtesy of DOE/Los Alamos National Laboratory)

A team of scientists used an ion beam in a basement room at Los Alamos National Laboratory to simulate solar winds on the surface of the Moon. The table-top simulation helped confirm that the Moon is inherently dry.

In research published in Science Express, Zachary Sharp of the University of New Mexico and a team of scientists from California, Texas and New Mexico -- including Yongqiang Wang, leader of Los Alamos' Ion Beam Materials Lab -- present an analysis of chlorine isotopic ratios in lunar rock samples that seem to indicate that the Moon never had water of its own.

Many scientists believe that the Moon formed when a large object collided with Earth early in its formative stages, leaving behind a blob of material that became trapped in orbit around the nascent Earth. Because most of the water on Earth likely came from water liberated from molten basalts as they cooled, researchers have often wondered whether the Moon's geology contains similar concentrations of trapped water.

Sharp and his team examined ratios of stable chlorine isotopes -- chlorine-35 and chlorine-37 -- in terrestrial and lunar rock samples. Chlorine readily interacts with hydrogen and is highly volatile. Consequently, the ratio and concentrations of these isotopes can provide a "fingerprint" of water content of volcanic rocks.

If the Moon were formed via cataclysmic collision of a foreign body with a fledgling Earth, it's reasonable to assume that lunar basalts would share a similarly soggy history as their earthen brethren. However, an analysis of the chlorine isotopic ratios of rocks from the Earth and Moon provided vastly different fingerprints. Sharp and his team came up with three possible explanations for the differences: 1) the moon-forming collision homogenized molten material from Earth and the colliding body into a material with a unique composition, 2) hydrogen-rich solar winds buffeting the moon preferentially stripped away one isotopic chlorine species from rocks, or 3) lunar basalts were inherently anhydrous.

The researchers dismissed the homogenization scenario after comparing observed chlorine isotope concentrations with other volatile elements in the basalts. The other volatile chemicals did not behave consistently with what would have been expected for the homogenization scenario.

To assess the effects of solar winds, Los Alamos researcher Wang took a thin film of sodium chloride -- the same chemical as ordinary table salt -- and bombarded it with a stream of protons (hydrogen ions) at Los Alamos' Ion Beam Materials Lab. If the rocks were to be affected by the solar winds, the lighter chlorine isotope, chlorine-35, would preferentially react with the protons and be carried away as hydrogen chloride (HCl) gas. If this scenario were true, researchers would then find slightly higher ratios of the heavier isotope in the rocks. After subjecting the sample to eons of "solar-wind" exposure, the research team found that the samples were essentially unaffected by the proton onslaught.

Furthermore, lunar rocks from the surface showed depleted values of chlorine-37 relative to the lighter chlorine-35 isotope, and subsurface lunar rock samples shielded from solar winds had higher, not lower, concentrations of chlorine-37. These findings helped dismiss the second scenario.

The research team found that the third scenario -- that the moon was inherently without water -- was supported by the lunar rock samples because the residual chlorine isotopes found in the rocks seem to have originated from metal chlorides such as sodium chloride, zinc chloride and iron chloride, which have been seen as surface coatings on lunar volcanic rocks.

With regard to scientific findings of water-ice in lunar surface samples, the likely source is from comets, not the Moon itself.

Other researchers have published papers contradicting the team's findings. Sharp says the reason behind the discrepancies in his team's research and previous research is not well understood yet, and will require further analysis.

Even though his laboratory helped simulate the moon, Los Alamos researcher Wang remains down to Earth.

"It was very gratifying to play a role in the research and to be able to exclude one argument more definitively than before," Wang said.

The research team included Sharp and Chip Shearer of the University of New Mexico; Kevin McKeegan of the University of California at Los Angeles; Jamie Barnes of the University of Texas; and Wang of Los Alamos National Laboratory.

Los Alamos National Laboratory's Ion Beam Materials Laboratory was supported by the U.S. Department of Energy's Office of Science's Office of Basic Energy Science. Further support for Los Alamos' efforts come from Los Alamos' Laboratory-Directed Research and Development program.