7 mm) were deparaffinized for 20 min in Hemo-De (Fisher Scientific), rehydrated to distilled water through decreasing concentrations of ethanol, microwaved in water for 8 min, blocked with 5% goat serum for one hour and immunostained. Transmission Electron Microscopy Primary chick neurons were grown on glass coverslips, treated, washed with warm PBS and fixed for 30 min with 2.5% glutaraldehyde (Electron Microscopy Sciences, USA) in 0.1 M sodium cacodylate buffer (Sigma-Aldrich). Cells were rinsed with buffer and post-fixed for 30 min in cold 1% osmium tetroxide (Sigma-Aldrich). Cells were rinsed again and stained for 2 h with 1% uranyl acetate (Sigma-Aldrich), filtered through 0.22 mm Biofil Syringe filter in 0.05 M sodium acetate (Sigma-Aldrich). To prepare TEM grids, cells were dehydrated through increasing concentrations of ethanol (v/v) (50%, 70%, 95% and ultra-dry 100%), then infiltrated with Spurr’s resin in increasing ratios to 100% ethanol (1:1, 2:1 and 3:1). Cells on coverslips were embedded in 100% Spurr’s resin and cured at 60uC overnight. To remove coverslips, resin blocks were placed in liquid nitrogen for ,15 sec, transferred to warm water and carefully removed. Resin blocks were section using a diamond knife (70 nm) and placed on 200-mesh copper TEM grids that were subsequently post-stained for 10 min with uranyl acetate and lead citrate. Images were acquired with a Phillips CM 120 Biofilter TEM at 120 kV. Results Mitochondrial inhibition induces rapid dephosphorylation at all MAP/tau phosphoepitopes except for 12E8 In AD neurons, tau is hyperphosphorylated and abnormally redistributed to NFTs and neuropil threads in cell bodies and neurites respectively. The mechanisms by which these processes occur are not fully understood. The aim of this study was to test the hypothesis that inhibiting mitochondrial function influences the phosphorylation state and localization of tau protein. Primary neuronal cultures (prepared from E7 chick tectum) were treated for 10, 30, 60 or 120 min with the mitochondrial complex III inhibitor AM which has previously been shown to reduce the level of ATP in these neurons by 60% within 10 min [20]. Cells were then lysed or fixed for immunostaining and probed with a battery of phospho-tau antibodies - AT270, AT8, AT100, S214, AT180, 12E8, S396, S404 or S422 - that have been raised against different phosphoepitopes on human tau and have analogous correspond- ing epitopes in chicken tau (Fig. 1A) The 12E8 antibody recognizes phosphorylated KXGS motifs in the conserved MTBDs of both tau and other MAPs [14]. Western blot analysis of lysates revealed that compared to untreated (control) cells, ATP reduction leads to dephosphorylation of epitopes S396, AT8, AT270, AT180, S404 and S422 over the 120 min treatment time when normalized for loading to GAPDH (Fig. 1B, C). Total tau was observed to decrease transiently before returning to control levels at 120 min. By contrast, the blots revealed a strong and sustained signal for the epitopes recognized by the 12E8 antibody (Fig. 1D, E). Consistent with this, the ratio of 12E8 to total tau band intensities increased .2-fold by 60 min whereas the AT8 to total tau ratio declined over the same time frame to near zero (Fig. 1D). This trend of persistence of the 12E8 epitope signal was reproducible in a second independent experiment and combined (n = 4), these data reveal a peak increase to 232669% at 60 min and sustained phosphorylation at 120 min (15564%, p,0.05) (mean6SEM) (Fig. 1E). Changes in Phospho-MAP/Tau during Neuronal Stress Redistribution of MAP/tau phosphoepitopes to rod-like or spheroid inclusions in neurites Next, we conducted immunofluorescence imaging of the same phosphoepitopes in AM-treated (15 min.) neurons to investigate distribution patterns of these antibody labels throughout neurons following mitochondrial inhibition. While some phosphoepitope labeling remained relatively smoothly distributed throughout neurites of AM-treated cells, other labels were aggregated into spheroid inclusions in affected processes (Fig. 2A–J). Cell body staining intensity, indicative of phosphorylation state, of phos- phoepitopes AT8, S214 and AT180 was somewhat reduced (AT8 = ,30% intensity of control cells after 15 min AM treatment), but otherwise comparable to control cells with regards to its smooth distribution. By contrast, AT270, AT100, S396, S404, S422 and total tau labeling aggregated into spheroids that were rarely seen in untreated control cells. Overall cell body staining intensity for some of these epitopes was also found to be reduced (AT270 ,40% intensity compared to control cells). Although the 12E8 antibody labeled some spheroids, it predom- inantly labeled rod-shaped aggregates throughout neurites (Fig. 2F) which were previously shown to be AC rods [20]. The insets in Fig. 2 illustrate the appearance of spheroids and rods respectively. Rod-shaped structures were observed less often with the S404, S422 and AT270 phosphoepitope labels (Fig. 2A and G, insets). As shown for AT270 below, these structures only rarely co-localized with AC which was in contrast to 12E8 label which largely colocalized with AC rods. In AM-treated cells, in addition to labeling neuritic inclusions that co-localized with total tau immunolabeling, AT100 revealed a redistribution into cell nuclei in a subset of neurons (,36%), as indicated by colocalization with the nuclear stain DAPI (Fig. 2D, asterisks), which was never observed for any other tau epitope (Supplementary Fig. S1). However, further analysis demonstrated that this nuclear epitope does not represent tau protein as AT100 also labeled nuclei of mouse tau knockout neurons (LSM, ITW and JRB unpublished observations). Moreover, neither S214 (which shares the serine-214 recognition site of AT100) nor total tau labels were ever seen to accumulate in nuclei of AM-treated cells further suggesting this AT100 accumulation to be non-tau related. Interestingly however, the same nuclear localization of the AT100 label was seen in post mortem AD tissue (Supplementary Fig. S1F). Co-labeling studies of spheroids in AM-treated cells revealed co- localization of phospho-tau epitopes with total tau (Fig. 3A and results not shown), suggesting that these observed aggregates in ATP-depleted cells are comprised of tau phosphorylated at many sites. By contrast, rod-like structures immunolabeled for the 12E8 epitope did not co-label with other phosphoepitopes (Fig. 3B). We had previously demonstrated 12E8 label in AC rods [20], which we confirm here in both Triton X and methanol permeabilized cells (Fig. 4A and data not shown). We found that the 12E8 labeling of AC rods in AM treated cells was best preserved under Triton X permeabilization (Fig. 3B) whereas methanol permea- bilization resulted mostly in spheroid labeling (Fig. 3C). Further, 12E8-labeled AC rods did not co-localize with S396 label (Fig. 3B) which by contrast only localized to spheroid structures in the same cells (arrows Fig. 3B). This further documents the distinct and separate nature of AC rods versus spheroids. 12E8 but not other phosphoepitopes redistributes to AC rods during mitochondrial inhibition To further investigate whether other tau epitopes are present in AC rods, we carried out double-labeling for ADF and a variety of PLoS ONE | www.plosone.org 3 June 2011 | Volume 6 | Issue 6 | e20878