A physical model of axonal damage due to oxidative stress

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.

Fig. 1.

Schematic diagram of the experiment. MTs are encapsulated in lipid vesicles fluorescently labeled with 6-AS, a UV-excitable probe. Upon photoexcitation (t = 0), a free-radical cascade is initiated, causing breakdown of the MTs and relaxation of the membrane (t = τ).

Fig. 2.

Time progression of a representative experiment. In micrograph A, MT polymerization inside the vesicle causes deformation of the membrane into a long axon-like extension. Micrograph B shows a 50-msec excitation of the 6-AS membrane-associated probe. Micrographs C–E show the progressive changes in length of the MT-supported membrane extension. The change in length of the membrane extension is plotted as a function of time-elapsed post-photoexcitation.

Fig. 3.

Increasing 6-AS concentration increases the rate of MT degradation. Plot of MT degradation rate (or change in membrane extension length) as a function of the 6-AS concentration used to prepare the MT-encapsulating lipid vesicles. Error bars represent standard errors of the mean for measurements of four, six, and four vesicles (left to right; note that the error bars are partially occluded for the leftmost point because they are nearly the same size as the diamond). The linear fit has R² = 0.9926.

Fig. 4.

The position of the free-radical source within the membrane affects the degradation rate. Degradation rates observed for incorporation of different fluorescent lipid probes in vesicles having lipid compositions of 5:2 DLPC:DOPS and 1800:1 lipid:probe. The anthracene moiety is closer to the solvent–membrane interface in the 2-AS probe and farthest (most buried) using 16-AP. The degradation rates showed a striking decrease with distance of the anthracene moiety from the interface; the fastest rate was observed for 2-AS, and degradation was substantially slower when 16-AP was used. The standard error of the mean was used for 6-AS (calculated from measurements of six vesicles). For 2-AS and 16-AP, the standard error of the slope from the measurement of the degradation rate of one vesicle was used to calculate the error bar for each (note that the error bars for 16-AP are partially occluded because they are nearly the same size as the diamond).

Fig. 5.

Incorporating free-radical scavengers inhibits MT degradation. Typical plots of MT-extension length as a function of time after UV excitation for vesicles containing various antioxidants are shown. All data were recorded for vesicles having lipid compositions of 5:2 DLPC:DOPS and 1,800:1 lipid:6-AS. Vitamins E and K, which are membrane-soluble, were studied at concentrations of 11,000:1 lipid:vitamin (data represented by purple squares and blue triangles, respectively); water-soluble vitamin C was located in the buffer at a concentration of 5.7 × 10⁻³ M (data represented by green circles). Data for the control (without antioxidant present) are represented by dark-blue diamonds. For each antioxidant, data from more than one degradation curve are superimposed to provide a representation of the range of degradation rates observed; each data set is distinguished by a different symbol grayscale. Low concentrations of antioxidants greatly reduced the degradation rate.

Fig. 6.

Increasing lipid saturation decreases the rate of MT degradation. Typical plots of MT-extension length as a function of time for various degrees of lipid saturation are shown. The vesicle compositions studied were 5:2 DLPC:DLPG (fully saturated), 5:2 DLPC:DOPS (∼40% unsaturated lipids), and brain polar lipid extract (∼60% unsaturated lipids). All lipid compositions shown were studied by using 14,000:1 lipid:AS concentrations. As in Fig. 5, data from two degradation curves are superimposed for the control composition. Low levels of unsaturated lipid hindered degradation. Free radicals are known to attack unsaturated lipids in biological membranes, which then propagate the radical generation throughout the membrane.