A physical model of axonal damage due to oxidative stress

doi:10.1073/pnas.0504134103

A physical model of axonal damage due to oxidative stress

*Departments of Chemistry and Physics, and
^§Department of Veterinary and Biomedical Science and Huck Institutes of the Life Sciences, Pennsylvania State University, University Park, PA 16802-6300

Edited by Harry B. Gray, California Institute of Technology, Pasadena, CA, and approved January 17, 2006 (received for review May 18, 2005)

Abstract

Oxidative damage is implicated in the pathogenesis of neurodegenerative disorders, including Alzheimer's, Parkinson's, and Huntington's diseases, and in normal aging. Here, we model oxidative stress in neurons using photogenerated radicals in a simplified membrane-encapsulated microtubule system. Using fluorescence and differential interference contrast microscopies, we monitor photochemically induced microtubule breakdown on the supported region of membrane in encapsulating synthetic liposomes as a function of lipid composition and environment. Degradation of vesicle-encapsulated microtubules is caused by attack from free radicals formed upon UV excitation of the lipid-soluble fluorescent probe, 6-(9-anthroyloxy)stearic acid. Probe concentration was typically limited to a regime in which microtubule degradation was slow, and microtubule degradation was monitored by changes in the observed protrusion of the membrane surface. The kinetics of microtubule degradation are influenced by lipid saturation level, fluorescent probe concentration, and the presence of free-radical scavengers. This system is sufficient to reproduce some degenerative morphologies found in vivo.

Footnotes

^¶To whom correspondence should be addressed at:
104 Davey Laboratory, Departments of Chemistry and Physics, Pennsylvania State University, University Park, PA 16802-6300.
E-mail: stm{at}psu.edu
↵ ^†Present address: Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520.
↵ ^‡Present address: U.S. Army Edgewood Chemical Biological Center, Aberdeen Proving Ground, MD 21010.
Author contributions: A.E.C., T.G.D., A.M.A., and P.S.W. designed research; A.E.C. and T.G.D. performed research; A.E.C., T.G.D., A.M.A., and P.S.W. analyzed data; and A.E.C., T.G.D., A.M.A., and P.S.W. wrote the paper.
↵ ‖ When we extrapolated down to the concentration of vitamin K actually used in degradation experiments, we found the absorption to be 2.98 × 10⁻³ at 362 nm, the peak for 6-AS, yielding an extinction coefficient of ≈1,000. This is lower than the extinction coefficient of 6-AS. Additionally, 6-AS was present in the membrane at 10-fold higher concentrations than vitamin K. In our vesicle preparations, the absorption of vitamin K was lower than that of 6-AS by a factor of 27. Thus, spectral overlap should not have significantly influenced the results obtained for vesicles doped with vitamin K.
Conflict of interest statement: No conflicts declared.
This paper was submitted directly (Track II) to the PNAS office.
Abbreviations:

Abbreviations:

2-AS,

2-(9-anthroyloxy)stearic acid;

6-AS,

6-(9-anthroyloxy)stearic acid;

16-AP,

16-(9-anthroyloxy)palmitic acid;

DLPC,

1,2-dilauroyl-sn-glycero-3-phosphocholine;

DOPS,

1,2-dioleoyl-sn-glycero-3-[phospho-l-serine];

MT,

microtubule