Neural Cell Adhesion Molecule Found to Have Alcohol Binding Site

HMS researchers have discovered a novel alcohol binding site on the neural cell adhesion molecule L1. The study, an early step to finding drugs to reduce ethanol toxicity on the nervous system, appears in the Jan. 8 Proceedings of the National Academy of Sciences.

Courtesy Keith Miller

Form and function. Based on this domain structure of the neural cell adhesion molecule L1, HMS researchers speculate that small alcohols break the hydrogen bond between two amino acids on immunoglobulins 1 and 4, causing the L1 molecule to lose its horseshoe shape and thus its adhesive properties. Mutations in nearby residues cause neurological disorders similar to those observed in children with fetal alcohol spectrum disorders (FASD).

L1 molecules on one nerve cell adhere to L1s on other nerve cells and are critical for normal brain development. Children with L1 gene mutations have mental retardation, thinning or absence of the corpus callosum, hydrocephalus, and malformation of the cerebellum. Michael Charness, co–senior author and an HMS professor of neurology at the VA Boston Healthcare System, began studying L1 when he noticed that children with fetal alcohol spectrum disorders (FASD) have brain lesions similar to those of children with L1 mutations.

Charness and colleagues have reported that alcohols with fewer than four carbons inhibit L1 adhesion, while certain alcohols containing five or more carbons block the action of small alcohols on L1 and prevent ethanol teratogenesis in mice.

Initially, Charness could not easily determine if L1 had an alcohol binding site because of alcohol’s low affinity for its targets. “Alcohol comes off the receptor almost instantly,” he said. “That’s why you need a high concentration of alcohol in your blood to have an effect.”

He joined forces with the lab of co–senior author Keith Miller, the Mallinckrodt professor of pharmacology in the Department of Anesthesia at Massachusetts General Hospital. The researchers used a photolabeling technique to pinpoint the binding site and identified the critical amino acids via mass spectrometry. The site was located where two domains of the L1 molecule interact, stabilizing the molecule’s horseshoe-shaped structure.

The researchers hypothesize that ethanol breaks the hydrogen bond holding two particular amino acids together. Once the bond is broken, the L1 molecule is more likely to unfold and lose its adhesive properties, the authors said.

Charness and Miller hope to crystallize the L1 molecule to obtain a three-dimensional image of the binding site so they can begin searching for drugs that could protect L1 from ethanol. “The ultimate goal of the research is to develop medications that will decrease alcohol toxicity in the nervous system,” said Charness. “If a woman who is eight weeks pregnant comes into an emergency room with severe alcohol intoxication, that might be an opportunity for using a drug that blocks some of the effects of alcohol on development.”

—Laura Geggel

Microchip Detects Rare Circulating Cancer Cells

For more than 200 years, doctors have known that tumor cells can circulate in the bloodstream. Yet circulating tumor cells (CTCs) are a problematic diagnostic marker: in eight milliliters of blood there are about 60 to 80 billion cells, but only a handful of CTCs.

Now, using a silicon microchip the size of a business card, HMS researchers have created a technology that can sift through two milliliters of blood per hour and catch roaming CTCs. The study appears in the Dec. 20 issue of Nature.

“It is our hope that the CTC chip will one day become part of the routine checkup for early detection and screening,” said Mehmet Toner, senior author and HMS professor of surgery at Massachusetts General Hospital.

The microchip is equipped with 78,000 posts that are each 50 microns in diameter and coated with antibodies that recognize epithelial tumor cells.

“Out of 60 billion cells, all of them end up touching the posts,” said Toner. He explained that 85 to 90 percent of all cancers originate in epithelial cells, which normally do not circulate in the blood. Once the fragile epithelial tumor cells bind to the posts, the researchers can enumerate them as well as analyze their nucleic acids.

Using the CTC chip, the researchers tested blood samples from 68 patients with five different types of tumor. Of the 116 samples collected, only one microchip did not identify CTC cells, giving the chip a 99 percent sensitivity reading. In the control group of samples from 20 cancer-free subjects, no CTC cells were found.
Toner and his colleagues set two essential parameters when designing the microchip. The first, flow velocity, influences the duration of cell–micropost contact. The second, shear force, ensures maximum cell–micropost attachment. Accordingly, the researchers designed the microchip so that peripheral blood would run through it at one tenth the speed it travels in humans.

The researchers hope that the microchip will help them understand the process of blood-borne metastasis, which is the path most cancers use in spreading to other parts of the body and the ultimate cause of most cancer deaths.

In addition, the microchip may help doctors tailor treatment to patients.
“It turns out that when a patient responds to a treatment, you can see a decline in the number of circulating tumor cells relatively quickly,” said co-author Daniel Haber, the Laurel Schwartz professor of medicine at HMS and MGH and director of the hospital’s cancer center. “The ability to follow these cells, test them for genetic abnormalities and for evidence that drugs are effectively suppressing their targets may revolutionize the way we test the effectiveness of new cancer treatments.”

—Laura Geggel

Receptor Plays Leading Role In Pulmonary Fibrosis

HMS researchers are one receptor closer to understanding the pathogenesis of idiopathic pulmonary fibrosis (IPF), a highly lethal disorder that scars and stiffens the lungs, impeding gas exchange.

Patients with IPF live only an average of three to five years following diagnosis and rely on lung transplants as the sole effective treatment. Many mediators have been implicated in the disorder, but the authors discovered that the bioactive lipid lysophosphatidic acid (LPA) and one of its receptors, LPA1, represent a critical pathway in the pathology. The crucial role of the LPA–LPA1 cascade was underscored by the authors’ findings that this mediator–receptor pair is largely responsible for both fibroblast recruitment and vascular leak induced by lung injury. The two processes are thought to go into overdrive when lung injury leads to fibrosis rather than repair of normal lung structures.

In the study, published in the January Nature Medicine, the researchers used bleomycin, an anti-cancer agent that can cause pulmonary fibrosis as an unwanted side effect in humans, to induce pulmonary fibrosis in wild-type and LPA1-knockout mice. Compared to wild-type mice with normal LPA1 receptors, mice lacking these receptors were protected from the deposition of collagen as well as from death produced by bleomycin lung injury, and both vascular leak and fibroblast recruitment induced by bleomycin were markedly attenuated.

Andrew Luster, senior author and the Persis, Cyrus and Marlow B. Harrison professor of medicine at HMS and Massachusetts General Hospital, and Andrew Tager, lead author and HMS assistant professor of medicine at MGH, also found elevated amounts of LPA in the bronchoalveolar lavage (BAL) fluid of patients with IPF. Further, of the five known LPA receptors, they found that only LPA1 was highly expressed on the fibroblasts found in the BAL of these patients.

The researchers reconfirmed results from earlier investigators that had shown that the BAL fluid of IPF patients contains chemoattractants that cause fibroblasts to migrate into the lung. The MGH researchers now postulated that LPA was the critical compound.

The next step featured a chemical LPA1 antagonist called Ki16425. “When we treated the responding fibroblasts with the inhibitor that targets the LPA1 receptor, it inhibited virtually all of the chemoattractant activity that was in the BAL from the IPF patients,” said Luster.

When the researchers tested the fibroblasts with another chemoattractant, the cells continued to migrate just fine. “It wasn’t that the inhibitor killed the fibroblasts or prevented them from moving to anything, it just prevented their LPA1 receptors from responding to LPA,” said Tager. The results suggest that LPA is the chemoattractant predominantly responsible for recruiting fibroblasts into the lungs of IPF patients and consequently is a new therapeutic target for the disease.

—Laura Geggel

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