Changes in Phospho-MAP/Tau during Neuronal Stress Figure 6. 12E8 but not other tau epitopes aggregates and co-localizes with AC rods during actin depolymerization. Primary chick neurons were treated with 1 mg/ml Latrunculin B (Lat B) for 15 min, fixed and immunostained. We first confirmed formation of both AC and 12E8- MAP/tau positive rods in Lat B treated cultures (data not shown), as has previously been observed [20] and then co-stained cultures for ADF with numerous tau epitopes. Whereas an abundance of AC rods were induced by Lat B (A, arrows), label for all tau epitopes, except 12E8, remained relatively smooth and evenly distributed throughout neurites, as shown here for AT100 (A) (other epitopes not shown). Western blots of lysates of Lat B-treated neurons were probed with the same battery of antibodies to tau phospho-epitopes. While most epitopes such as S396 (shown here) showed little change in phosphorylation during Lat B treatment, the 12E8 epitope was strongly phosphorylated within 2 min of treatment (B, C). Band intensities were quantified for each time-point for 12E8 and S396 immunoblots (C) with each lane normalized to individual b-actin loading controls and then calculated as a percentage of control band intensities. At 2 min, 12E8 phosphorylation was 2.960.1-fold higher than control means (mean 6min/max intensities). Scale bar = 10 mm. doi:10.1371/journal.pone.0020878.g006 and highly localized cytopathology could reflect a sub-optimal or failing local microvasculature [52]. In contrast to 12E8, other phosphoepitopes including AT270, S422, AT100, S396 and S404 mostly accumulated in small neuritic spheroid swellings in ATP depleted neurons. We found these phosphoepitopes only occasionally co-localized with ADF immunostaining in rod-shaped structures (Fig. 2 and 3), and this observation was rare compared to the high level of co-localization of ADF and 12E8 (Fig. 4). We can conclude that recruitment to AC rods during these conditions is primarily specific to KXGS- phosphorylated species in the initial stages. The occasional rod- shaped structure formed by S404 and AT270 label (Fig. 2) and rare co-localization of AT270 with ADF in AC rods, suggests that other phosphoepitopes of tau may appear in AC rods later, a subject of ongoing investigation. In primary neurons, Lat B- induced actin depolymerization also resulted in specific recruit- ment of 12E8 epitopes to AC rods, with a concomitant ,3-fold increase in the signal of the band recognized by 12E8 on immunoblots following 2 min Lat-B treatment (Fig. 6). By contrast, S396 was neither seen to redistribute into spheroid or rod-like aggregates nor deviate from control levels of phosphor- ylation. These data suggest that actin rearrangement in this cell model triggered either by changes in cellular ATP levels or directly by actin depolymerizing drugs may be an upstream effector of KXGS phosphorylation and redistribution of MAP into AC rods. Converging or independent signaling mechanisms triggered by mitochondrial dysfunction, excess glutamate, oxidative stress, calcium deregulation or other effectors leading to AC rods and MAP/tau redistribution in vivo are potentially multifold [22,43,47,53]. Oxidative stress and calcium have previously been implicated in the cytoskeletal pathology of AD and studies have shown both calcium-dependent and –independent pathways leading to AC rods in various model systems [22,43,53]. More work is needed to delineate the involvement of these pathways in AD. A predominant function of tau protein is to bind and stabilize axonal MTs – the tracks for cargo transport to synaptic terminals. Hyperphosphorylation of tau in the MTBD by microtubule affinity regulating kinase (MARK)/PAR1 or other kinases gives rise to a destabilized MT network and concomitant self-assembly of the increased concentration of unbound tau [13,15,16]. This self-assembled tau is purported to serve as a seed for further assembly of redistributed tau into PHFs along neurites (neuropil threads) and in the somatodendritic domain (neurofibrillary tangles) [1,2,54,55]. Albeit in a cell model and not demonstrated in situ in brain tissue, our results suggest that AC rods may contribute to the sequestration and accumulation of unbound tau via its phosphorylated MTBD in neurites leading to neuropil thread-like inclusions and potentially PHFs. We speculate that rapid depolymerization and rearrangement of the actin cytoskel- eton during rod assembly may induce activation of MARK/Par-1 or other kinases, the former of which has previously been PLoS ONE | www.plosone.org 9 June 2011 | Volume 6 | Issue 6 | e20878