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Proc Natl Acad Sci U S A. 2006 April 4; 103(14): 5262–5266. Published online 2006 March 28. doi: 10.1073/pnas.0504134103. | PMCID: PMC1459344 |
Copyright © 2006 by The National Academy of Sciences of the USA Chemistry, Biophysics A physical model of axonal damage due to oxidative stress Anne E. Counterman,*† Terrence G. D'Onofrio,*‡ Anne Milasincic Andrews,§ and Paul S. Weiss*¶ *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 Received May 18, 2005. 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. Keywords: membrane, neurodegeneration, Alzheimer's disease, Parkinson's disease, microtubule Oxidative modifications to proteins in neurons have been implicated in a number of degenerative disorders and have become a controversial topic in elucidating the progression of Parkinson's, Huntington's, and Alzheimer's diseases ( 1– 7). Microtubules (MTs) are key cytoskeletal proteins in nerve cell axons. These protein polymers are responsible for many disparate functions of cells, including structural support and imparting polarity, and they are involved in motility and transport of intracellular cargo ( 8). Fundamental studies of MTs in vivo are complicated by the presence of the full complement of biomolecules that are required for cell survival. Thus, a substantial effort has been devoted to characterizing MT dynamics in vitro, and these studies have led to increased understanding of issues such as dynamic instability ( 9) and the involvement of molecular motors in producing motility ( 10). To mimic the confinement of a bilayer, MT assemblies (asters) have previously been examined in rigid, microfabricated wells ( 11). The drawback of such studies is that interactions between MTs and components of the elastic lipid membrane are neglected. It is important to be able to isolate and to quantify specific factors that impact the structural integrity of the cytoskeleton, while maintaining a simplified cell-like environment. Toward this aim, we have developed a minimal physical system with which to investigate the effects of oxidative stress on the cytoskeleton. Previous studies have demonstrated that incorporating actin and tubulin into liposomes and inducing these proteins to polymerize results in a morphological change in the liposome; a protrusion of the membrane is observed at one or both ends of the protein polymer ( 12– 15). The asymmetry occurs frequently, when the MT wraps around within the encapsulating liposome ( 16). Here, we employ the membrane as a chemical reservoir to examine individual factors that lead to degradation or stabilization of MTs. In our model, the chemical composition of the membrane is controlled during liposome assembly and the elasticity of the bilayer is retained (and is tunable according to lipid composition). The roles of individual components (e.g., specific lipids, MT-associated proteins, or protective molecules that function as radical scavengers) can be elucidated by reconstituting the liposomes with varying concentrations of these species. We demonstrate that the rate of MT degradation is influenced by the lipid saturation level, fluorescent probe (radical source) concentration, fluorescent probe location, and presence of free-radical scavengers. Overview. An intrinsic property of MTs is their dynamic instability: the ability to alternate between periods of polymerization and rapid depolymerization, or collapse ( 9). We focus here on inducing catastrophic collapse and measuring the rate of that collapse. A general schematic of our experiment is shown in . We polymerize MTs within lipid vesicles prepared from known compositions of synthetic lipids and fluorescent probe(s). Upon heating vesicles containing tubulin and GTP to 37°C, tubulin polymerizes to form MTs. A mechanism that explains the formation of a single filament of MTs by alignment of individual filaments (and thus creating a single-membrane protrusion) has previously been outlined by Hotani and coworkers ( 17). Most of the experiments that we describe here incorporate the UV-excitable probe 6-(9-anthroyloxy)stearic acid (6-AS) into the lipid membrane. UV excitation of anthracene has been shown to promote free-radical formation ( 18); in our studies, we exploit anthracene-based fluorescent probes to photoinitiate a free-radical cascade that leads to MT degradation. We track the extent of MT degradation by measuring the perturbation of the membrane extension. illustrates this process for a single 50-msec exposure of a vesicle-encapsulated MT to UV light. Comparison of the micrographs recorded before and after photoexcitation of the 6-AS probe shows a progression of decreasing membrane extension, corresponding to degradation of the supporting MT filament. Note that the placement of the radical source in the membrane enables chemical coupling of the photogenerated radical to the adjacent (encapsulated) MT ( vide infra). In an initial experiment, after obtaining a fluorescence micrograph of the vesicle with the tubulin extension, we observed that the extended region of the membrane exhibited pearling and that the MT had depolymerized ( 19). Pearling was typically observed only at high 6-AS concentrations, and is consistent with the behavior of a membrane tether after the release of stress ( 20, 21). Importantly, pearling has been noted in axonal dystrophy of aging primates and in Alzheimer's disease patients, both of which have been attributed to axon degradation but were thought to require the MT-associated protein tau, which is not included in our physical model ( 22– 25). We have systematically examined the dynamics of this system to understand and to quantify factors that affect MT degradation. Confirming Free-Radical Involvement. Control experiments were carried out to confirm that free-radical formation from 6-AS was responsible for MT degradation. To generate free radicals in vitro, MTs were polymerized on a slide in the presence of low levels of iron, and then hydrogen peroxide was introduced. The mixture of iron and hydrogen peroxide is a well known system for free-radical generation. Within minutes, degradation of the in vitro MTs was observed. Next, to confirm that 6-AS was required for degradation of the membrane-encapsulated MTs, two sets of experiments were performed: UV irradiation of MT-containing liposomes that did not contain a fluorescent probe and substitution of other fluorescent probes (that were not expected to produce free radicals) in place of 6-AS. No MT degradation was observed for UV irradiation of non-fluorophore-containing liposomes or for vesicles containing 1,1′-didodecyl-3,3,3′,3′- tetramethylindocarbocyanine (DiI-C12) or 1,1′-dioctadecyl-3,3,3′,3′- tetramethylindodicarbocyanine (DiD) (excited at their respective peak absorption wavelengths). Thus, we conclude that (i) MT degradation can be induced by free-radical attack and (ii) the membrane-soluble free-radical source 6-AS is responsible for the photoinduced degradation that we observe in our experiments on MTs encapsulated in liposomes. In , the length of the stretched membrane region is plotted as a function of time elapsed after 50-msec photoinitiation. The degradation trend that we observed under these conditions was approximately linear over the first minute and appeared to plateau after 2 min. Periods of zero degradation (which appear as steps in the degradation plot, e.g., at 70–78 sec, 95–101 sec, and 117–128 sec) were typical. There did not appear to be a consistent pattern in the frequency or duration of these “pauses” in MT degradation. The variability we observed during MT catastrophic collapse may arise from factors such as lattice discontinuities in the MT assembly or variations in MT capping ( 26). Below, we demonstrate that this model system can be used to quantify effects associated with modulating components of the lipid membrane that influence MT degradation, by altering the free-radical source (concentration and location longitudinally within the lipid leaflet), the lipid bilayer (lipid unsaturation), and the presence of free-radical scavengers in the membrane or the vesicle-encapsulated fluid volume. We note that within a given sample of synthetic lipid vesicles, the lengths and widths of membrane protrusions observed varied widely. To ensure the reliability of quantitative comparisons for degradation rates measured from different samples, we examined numerous vesicles from each preparation and also compared different preparations having the same composition. Effect of Varying the Free-Radical Source: Concentration and Localization. To monitor the effect of changing the concentration of free radicals on MT degradation rate, we performed several experiments on vesicles synthesized by using different concentrations of 6-AS (spanning a range of two orders of magnitude) in a matrix of 5:2 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC):1,2-dioleoyl-sn-glycero-3-[phospho-l-serine] (DOPS) (). As expected, increasing the concentration of 6-AS increased the rate at which MTs were degraded. However, we note that the variability among vesicles was high (). A question that arises when considering the free-radical source is the exact location of the anthracene moieties with respect to the encapsulated MT. We cannot control the lateral position of the incorporated fluorophore within the lipid bilayer; in this case, because 6-AS partitions into fluid-phase lipids, it is found throughout the bilayer. However, the position of the anthracene group longitudinally with respect to the lipid leaflet (i.e., farther away or closer to the leaflet interior) can be controlled by using different fluorescent probes. We synthesized batches of 5:2 DLPC:DOPS vesicles using identical 1,800:1 lipid:probe concentrations of 2-(9-anthroyloxy)stearic acid (2-AS) (in which the anthracene group should be incorporated closest to the lipid headgroup and thus closest to the encapsulated MT in the inner leaflet), 6-AS, and 16-(9-anthroyloxy)palmitic acid (16-AP) (in which the anthracene group should reside near the interior of the lipid bilayer). Comparison of the MT-degradation rates for these batches of vesicles gives a relative ordering of 2-AS > 6-AS > 16-AP; MT-degradation rates measured for 2-AS-containing vesicles were >30 times higher than those measured for the 16-AP-containing vesicles (). Effect of Incorporating Free-Radical Scavengers. To inhibit free-radical-induced MT degradation, we incorporated free-radical scavengers into the vesicle-encapsulated system. Studies were performed by using vitamin C, which is water-soluble, and vitamins E and K, which are soluble in the lipid bilayer. Note that the results for vitamin C cannot be directly compared with those for vitamins E and K because of the difference in localization, solution vs. membrane; thus, the experiments described here are each referenced to a control (which contained no free-radical scavengers). shows that inclusion of these free-radical scavengers in the vesicle-encapsulated MT system substantially slowed MT degradation relative to control experiments. A dramatic effect was observed upon inclusion of vitamin C in the solution; on average, MT degradation was observed to be very slow or did not occur in these experiments. At similar concentrations, vitamin C has previously been shown to stop radical-induced damage in human-serum samples ( 27). The MT-degradation rates measured for systems including the two membrane-soluble vitamins (E and K) were identical within experimental uncertainty (−0.034 ± 0.003 μm/sec and −0.036 ± 0.006 μm/sec, respectively), and both slowed MT degradation relative to control by over an order of magnitude under the given lipid, probe, and scavenger concentrations. We also considered the possibility that the decreases in MT-degradation rates observed upon addition of the antioxidants were due to spectral overlap between the vitamins and the 6-AS fluorescent probe; however, no spectral overlap with 6-AS is observed for vitamins C or E, and the overlap for vitamin K appears to be small. ‖
Thus, we conclude that the observed reduction in MT degradation is due to free-radical scavenging. Varying Lipid Saturation. Another key parameter in our model system is the degree of lipid saturation. Unsaturated lipids, which are more susceptible to peroxidation than saturated lipids, produce 4-hydroxynonenal upon peroxidation ( 28), which may cause MT breakdown. We investigated the influence of lipid saturation by varying the composition of the synthetic vesicles; typical results are shown in . The observed MT degradation rate was highest for vesicles composed of ≈60% unsaturated brain polar lipid extract. At the concentration of 6-AS used (14,000:1 lipid:probe), collapse of membrane extensions for this lipid composition was very fast; collapse was typically complete in <10 sec (depending on the initial length of the membrane extension). The MT-degradation rate was more than an order of magnitude lower for liposomes composed solely of fully saturated lipids [DLPC and 1,2-dilauroyl- sn-glycero-3-phosphatidylglycerol (DLPG)]. The simple model system that we have demonstrated here provides a “bottom-up” approach to the study of damage to MTs in a cell-mimetic lipid container. This type of system, in which the cytoskeletal proteins and components of the lipid membrane and fluid volume can be well controlled, provides a means of quantitatively examining factors that influence MT degradation. We have characterized the response of MTs in this model system with respect to several factors. Specifically, decreasing the concentration of the free-radical source (6-AS), increasing the lipid-saturation level, or increasing the concentration of free-radical scavengers led to decreased rates of MT degradation. Positioning the free-radical source (in our studies, the anthracene moiety of the UV-excitable fluorophore) toward the center of the lipid bilayer also led to decreased degradation rates. A key aspect of the model system we have developed is the incorporation of the lipid membrane. In contrast with studies of MTs in solution, this allows us to examine effects arising from the interaction of cytoskeletal proteins with membrane-associated species. Increasing the complexity of the model in a stepwise fashion by adding cellular components will allow us to mimic processes involved in neuronal dystrophies more closely. Vesicle Preparation. Vesicles were prepared by using the gentle hydration method of Evans and Needham ( 29). Lipids were procured from Avanti Polar Lipids. Lipid purity was verified by using thin-layer chromatography on activated silica plates (Keystone Scientific, Bellefonte, PA), with a chloroform:methanol:water (64:25:4) (vol:vol:vol) solution or a chloroform:methanol:ammonium hydroxide (64:25:4) (vol:vol:vol) solution and developed with molybdenum blue solution (Sigma-Aldrich). Lipids and fluorescent probes dissolved in chloroform were mixed and deposited on a scored Teflon disk in a beaker. The chloroform was allowed to evaporate, and the resulting film was dried in a vacuum desiccator for 6 h. Upon removal from the desiccator, the film was hydrated with 10 ml of Pipes buffer (pH 6.9; containing 1 mM EGTA, 2 mM MgSO 4, and 100 mM Pipes) that had been warmed to at least 55°C. The solution was blanketed with argon, and the beaker was sealed with aluminum foil. The system was then incubated at 55°C for 4 h and allowed to cool slowly in an insulated box. Tubulin Purification. Tubulin was purified from bovine brain by using a protocol provided by W. O. Hancock (Department of Bioengineering, Pennsylvania State University). Briefly, we extracted the cerebrums from fresh bovine brains by dissection on ice. The cerebrums were processed in a chilled blender in the presence of protease inhibitors; the homogenate was clarified in a centrifuge at 54,000 × gand 4°C. All subsequent spins were performed at 300,000 × g(achieved by using a Beckman Ultra 50.2 Ti rotor). The supernatant was collected and warmed to 37°C to induce tubulin polymerization. This solution was clarified in a centrifuge, and the MT pellet was retained. The MTs were depolymerized on ice and clarified again by centrifugation at 4°C. This polymerization/depolymerization cycle was repeated. Finally, the resulting supernatant was passed through a phosphocellulose column to concentrate MTs and remove MT-associated proteins. Microscopy Methods. For the MT degradation experiments, 20 μl of vesicles was combined with 10 μl of 10 mM GTP (Sigma-Aldrich) solution and 10 μl of 100 mM tubulin dimer into a cryovial (66008-251; VWR Scientific). We encapsulated tubulin inside vesicles with a freeze–thaw cycle; the cryovial containing vesicles and tubulin was dipped into liquid nitrogen, and the mixture was thawed on ice. During inspection by using optical microscopy, samples were heated with an objective heater (Bioptechs, Butler, PA) set to 40°C to induce tubulin polymerization. Experiments were performed on a Nikon Eclipse TE300 inverted microscope that was equipped for differential interference contrast and epifluorescence. A mercury bulb and a long-pass filter cube (XF02; Omega Optical, Brattleboro, VT) were used for fluorescence excitation, and an ORCA 100 camera (Hamamatsu Photonics) was used for detection. A 50-msec exposure to UV light was used to initiate MT degradation; progress of the depolymerization was monitored by collecting differential interference contrast micrographs. We thank Profs. Will Hancock and Donald Schmechel for insightful discussions, Prof. Hancock for the use of his laboratory for purification of tubulin, Dr. Anat Hatzor for preliminary studies on liposome-encapsulated MTs in our laboratory, and Dr. Michael Elbaum for initial guidance in the encapsulation protocol. This work was supported by the National Science Foundation. A.E.C. was supported by a Ruth L. Kirschstein National Research Service Award from the National Institutes of Health. | | 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 |
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