Changes in Phospho-MAP/Tau during Neuronal Stress Figure 4. Recruitment to AC rods during ATP reduction is specific for ‘KXGS’ phosphorylated species. (A) Confirmation of 12E8-positive MAP/tau in AC rods during ATP reduction [20]. These inclusions often form in tandem arrays along neurites (arrows). Inset shown at higher magnification in lower panel of (A). Additional double-labeling was performed with AT270 (B) or AT100 (C) and ADF, to see if there was any relationship between these tau epitopes and AC rods. AC rods only sometimes overlapped with AT270 (B, arrows) and were never observed to co- localize with AT100, under either Triton-X or methanol permeabilization (C and D, arrows). These results suggest AC rods are distinct from MAP/tau- containing neuritic spheroids and recruit only the 12E8-phosphorylated MAP/tau isoform into distinct rod structures during ATP reduction. (A, B and D) = Triton X permeabilized. Scale bar = 20 mm; 5 mm (inset in (A)). doi:10.1371/journal.pone.0020878.g004 AD marker’ antibody raised against ‘KXGS’ motifs in the MTBD of human tau [1,14] (Fig. 1). Notably, we previously demonstrated that this epitope is incorporated into AC rods, which are also generated under ATP depleting conditions [20] (confirmed here in Fig. 4A). The MAP protein family, including the vertebrate proteins tau, MAP2, doublecortin and MAP4 (the latter of which is non-neuronal) contain homologous conserved ‘KXGS’ repeat motifs in MTBDs [8,48,49]. In light of this, although tau is probably the most predominant protein recognized by 12E8, we cannot completely discount that the 12E8-immunoreactivity we are observing in AC rods might be due to the presence of other MAPs in addition to tau. In animal models of ischemia-reperfusion, rapid dephosphory- lation of tau occurs during the initial ischemia and we have confirmed in our primary neuronal cell culture model that tau dephosphorylation not hyperphosphorylation is the immediate PLoS ONE | www.plosone.org 7 June 2011 | Volume 6 | Issue 6 | e20878