Changes in Phospho-MAP/Tau during Neuronal Stress Figure 3. Distinct rod-shaped aggregates are specific to 12E8 and are best preserved with Triton-X permeabilization. ATP-depleted cells were permeabilized for 90 sec with 80% methanol or 0.05% Triton-X and co-labeled for epitopes of tau. (A) Under both methanol and Triton-X methods, aggregations containing AT270 epitopes co-labeled with total tau (shown here with methanol permeabilized cells) suggesting spheroid neuritic aggregates are comprised of tau phosphorylated at numerous epitopes. (B) By contrast, 12E8-labeling was predominantly in distinct rod- shaped aggregates that did not co-label with other phospho-tau epitopes (shown here for S396). 12E8-positive AC rods were best visualized following Triton-X permeabilization. (C) Using methanol permeabilization protocols, 12E8 by contrast more often labeled spheroid structures that as described above could be co-labeled with many other tau epitopes (shown here for S396). Scale bar = 20 mm. doi:10.1371/journal.pone.0020878.g003 43]. By contrast, unaffected neurites of treated cells contain individually spaced microtubules approximately 24 nm in diameter (Fig. 5C). Actin depolymerization mimics effects of mitochondrial inhibition on 12E8 epitope redistribution but does not affect the distribution of other phosphoepitopes Similar to mitochondrial inhibition, we previously demonstrated that pharmacologically-induced depolymerization of actin induces the formation of AC rods which subsequently recruit 12E8 epitopes [20]. In order to determine whether actin depolymeriza- tion induces redistribution of other phosphoepitopes, we treated primary neurons with Latrunculin B (Lat B) which induces depolymerization of actin filaments in the absence of ATP reduction. The formation and co-localization of AC rods with 12E8 label under these conditions was confirmed (not shown). In contrast to AM-treated cells, staining for other phosphoepitopes including AT100 (Fig. 6A) and S396 (Supplementary Fig. S1D) in Lat B treated cells revealed an even distribution along neurites. Western blot analysis of Lat B-treated neurons revealed a 2.9-fold increase in 12E8 immunoreactivity after 2 min treatment while other phosphoepitopes such as S396 remained largely unchanged (Fig. 6B, C). These results confirm that 12E8 is the primary epitope recruited to Lat B-induced AC rods during actin reorganization, as was also found to be the case for AC rods induced by ATP-reduction. Together these results provide evidence for an important role of KXGS phosphorylation in the initial recruitment of tau or other MAPs to AC rods during times of cellular stress. Discussion Disrupted energy metabolism, mitochondrial dysfunction and increases in oxidative stress in the ageing brain have been linked with the onset of AD pathologies, including aberrant distribution and accumulation of hyperphosphorylated tau and deposition of amyloid-b [24,44–47]. The aim of this study was to investigate whether induced mitochondrial dysfunction could elicit changes in tau phosphorylation and distribution in healthy neurons in cell culture, since mechanisms leading to hyperphosphorylation and redistribution of tau remain elusive, yet are central to understand- ing the pathogenesis of AD. Here, we treated primary neuronal cell cultures derived from chick tecta with the mitochondrial inhibitor AM that rapidly depletes cellular ATP [20] and subsequently analyzed changes to phosphorylation and sub-cellular distribution of tau using Western blotting and immunolabeling. Whereas dephosphorylation was observed for every other epitope over 120 min, we observed a substantial increase in phosphorylation at the 12E8 site, an ‘early PLoS ONE | www.plosone.org 6 June 2011 | Volume 6 | Issue 6 | e20878