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New physical model reveals information on causes of cellular damage

Jun 2006
Research may lead to therapies for neurodegenerative disorders

Gary Boas

A variety of neurodegenerative disorders — including Alzheimer’s, Parkinson’s and Huntington’s diseases — have been linked to oxidative stress, in which free radicals chemically attack cells and parts of cells. Investigations of damage to the microtubule cytoskeletal proteins could reveal the mechanisms by which this occurs. But in vivo studies of these proteins are problematic at best because the many biomolecules necessary to cell survival tend to obscure these mechanisms.

Several groups have probed microtubule dynamics in vitro, shedding light, for example, on the roles of molecular motors, which use these protein polymers as “tracks” on which to walk. But the models used in these studies have been limited. To replicate the lipid bilayers that typically house the microtubules, some researchers have used rigid, microfabricated wells. This, however, overlooks the interactions between microtubules and bilayers that could affect the structural integrity of the proteins.

This physical model can mimic the interactions between microtubules — cytoskeletal proteins that may be involved in neurodegenerative disorders — and the lipid membranes surrounding the microtubules. Excitation of the lipid membrane (t = 0) — labeled with the ultraviolet-excitable probe 6-(9-anthroyloxy) stearic acid (6-AS) — initiated a free-radical cascade, which led to microtubule degradation and subsequent relaxation of the membrane (t = τ).

Investigators with Pennsylvania State University in University Park have employed a physical system that mimics these interactions. They described the system in the April 4 issue of PNAS and reported on a study in which they used it to explore microtubule degradation through differential interference contrast and epifluorescence imaging.

The study came about almost by accident. Paul S. Weiss and colleagues at the university had been looking for a means to control the shapes of membranes. They found that one of the simplest ways is to control the curvature of the membranes by including microtubules inside. “We thought that putting microtubules inside vesicles, which others have done, would give us a nice model system with very high and very low curvature in one package,” Weiss explained. “But we discovered that we were collapsing the microtubules, and, in fact, that the morphology we saw mimicked the morphology of neurodegeneration.”

Anne Milasincic Andrews, a colleague in the department of veterinary and biomedical science at the university, then suggested a series of experiments exploring how this relates to oxidative stress, and, furthermore, how they might use this knowledge to delay or prevent damage to the microtubules — suggesting a potential therapy for neurodegenerative disorders.

Good and bad actors

The first step was to develop a means to test the effects of different components on the system — “so we can step through the different lipids to see which are the good and bad actors in the brain,” Weiss said. Studies have shown that it is possible to induce morphological changes in liposomes by incorporating actin and tubulin into them, leading to protrusions at one or both ends of the lipid membrane surrounding the microtubule. By controlling the chemical composition of the membrane, the scientists could explore the ways in which lipids — as well as free radicals and free-radical scavengers — contribute to, prevent or suppress degradation of microtubules.

Specifically, they used an ultraviolet-excitable 6-(9-anthroyloxy) stearic acid probe to induce free-radical formation, leading to a free-radical cascade that causes microtubule degradation. They employed a 50-ms exposure from a mercury bulb and a long-pass filter cube made by Omega Optical Inc. of Brattleboro, Vt., to excite the probes. They determined the extent of degradation by measuring changes in the lengths of protrusions on the membrane surface with a series of differential interference contrast micrographs. For this they used a Nikon inverted microscope, outfitted for both differential interference contrast and epifluorescence imaging, and a Hamamatsu Photonics camera for detection.

This process was tracked through a series of differential interference contrast micrographs. Initially, the membrane appears as a long axonlike extension (A). Following excitation (B), this extension becomes progressively shorter (C-E).

They sought to determine the extent to which microtubule degradation is influenced by the lipid saturation level, different concentrations of free radicals and free radical scavengers. With respect to the former, they found that vesicles composed of approximately 60 percent unsaturated brain polar lipid extract exhibited the highest amount of degradation. This is an important first step toward developing therapies targeting neurodegenerative disorders. “If we can find the specific lipids that are really the ‘problem children,’ we can try to come up with strategies to address them,” Weiss said.

They also explored the effects of different concentrations of free radicals, finding that the rate of degradation grows as the concentration increases. In separate experiments, they examined the inhibiting effects of free-radical scavengers on microtubule degradation and noted that degradation was substantially slower in vesicles incorporating these scavengers, with respect to control experiments.

Thus, the researchers have begun to characterize microtubules’ response to a variety of factors that might affect their degradation via oxidative stress. The next step, Weiss said, is to explore the roles of other lipids and proteins that might be involved, by reconstituting the system with these. They hope to achieve this by incrementally increasing the complexity of the model, incorporating cellular components that will improve the extent to which they can mimic the neurodegenerative processes.

Ultimately, this could contribute to the development of new therapies for neurodegenerative disorders.

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