implicated in conferring tau toxicity in Drosophila models [15,56,57]. Consistent with this, activation of MARK correlates with elevated phosphorylation and dendritic accumulation of tau in stressed primary hippocampal neurons [47]. Extensive studies have demonstrated a role for tau and other MAPs in cross-linking and bundling of actin filaments [28–37]. Some studies have additionally demonstrated that MAP-actin association is regulated by the reversible phosphorylation of MAPs and that furthermore, tau and MAP2 interactions with actin occur via KXGS-containing motifs in the repeat regions of their MTBD domains [31,32,58]. That 12E8 labeled epitopes co-localized with AC rods in our study further supports a central role for KXGS motifs in the rapid association with actin. Moreover, EM analysis of AC rods following 15 min AM treatment in primary neurons demonstrate the presence of filamentous bundles (Fig. 5), and we speculate that AC-actin bundles form initially and serve as nucleation sites for subsequent recruitment of 12E8 epitopes. A recent study of human AD brains provides some support for this notion, which reported a correlation between increased cofilin expression and tau pathology in specific regions of the brain, including the temporal cortex [59–61]. Expression of the microRNA miR-107 has been shown to decrease in AD brain [60], which is associated with increased amyloid precursor protein and cofilin protein levels and formation of cofilin rod-like structures [61]. In regions of AD brains with confirmed reduction of miR-107 expression, a significant increase in NFTs was observed when compared with adjacent tissue expressing normal levels of miR-107 [59]. Further investigations are required to delineate the mechanisms facilitating the interaction between MAP and AC-actin rods and whether it is direct or indirect. Important questions that remain to be answered are whether the KXGS phosphorylation of tau or other MAPs is causative or correlative with its association with actin or AC rods and what mechanism facilitates the interaction between 12E8 epitopes and AC rods. Recent characterization of isolated AC rods demonstrated the presence of the scaffolding protein 14-3-3f [42] which was also shown to be associated with 12E8 within AC rods (unpublished observations LSM and JRB), suggesting that 14-3-3f may have a role either in rod structure or in a longer time-dependent maturation of MAP/tau phosphory- lation, as has previously been suggested in other studies [62–64]. Answering these questions is the subject of ongoing studies. In summary, we have shown that mitochondrial inhibition is associated with specific and sustained detection of 12E8 epitopes indicative of MAP phosphorylation in MTBD KXGS motifs, but a concomitant dephosphorylation of other phospho-tau epitopes. The combined effects of MT destabilization and accumulation of MAP/tau and cofilin-actin rods in the neurites is proposed to inhibit axonal transport, ultimately leading to synaptic pruning and neuronal death [47,65–68]. The present findings therefore suggest that targeting disrupted energy metabolism, mitochondrial dysfunction and downstream actin reorganization in aging cells References 1. Augustinack J, Schneider A, Mandelkow E, Hyman B (2002) Specific tau phosphorylation sites correlate with severity of neuronal cytopathology in Alzheimer’s disease. Acta Neuropathologica 103: 26–35. 2. Braak H, Alafuzoff I, Arzberger T, Kretzschmar H, Tredici K (2006) Staging of Alzheimer disease-associated neurofibrillary pathology using paraffin sections and immunocytochemistry. Acta Neuropathologica 112: 389–404. 3. Giannakopoulos P, von Gunten A, Kovari E, Gold G, Herrmann FR, et al. (2007) Stereological analysis of neuropil threads in the hippocampal formation: relationships with Alzheimer’s disease neuronal pathology and cognition. Neuropathology and Applied Neurobiology 33: 334–343. Changes in Phospho-MAP/Tau during Neuronal Stress may help prevent neurodegeneration associated with aberrant phosphorylation, redistribution and self-assembly of tau. Supporting Information Figure S1 AT100 labels a non-tau component in the nucleus during mitochondrial inhibition, actin depolymerization and in postmortem AD brain. Primary chick neurons were treated with 1 mM AM or 1 mg/ml Lat B for 15 min, fixed, immunostained and analyzed via confocal laser scanning. ATP reduction with AM induced nuclear accumulation of the AT100 label in a proportion of neurons (3662%; mean6SEM), as indicated by co-localization with DAPI (A, B). Co-stainings revealed no obvious translocation of total tau into the nuclear compartment in cells exhibiting AT100 nuclear accumulation (B, arrows), suggesting the AT100 antibody is labeling a protein other than tau. AT100 labeling in untreated cells (C) remained contained in the neurites. Similarly, actin depolymerization with Lat B gave rise to nuclear accumulation of AT100 label in 2262% cells (mean6SEM) (D) and again, lack of total tau and other epitopes such as S396 (D) in the nucleus. Interestingly, pre-treating cultures for 2 min with the F-actin stabilizing drug jasplakinolide prevented nuclear accumu- lation of AT100 in cultures subsequently treated with either AM or Lat-B (data not shown). Formalin-fixed 7 mm brain sections from the hippocampal region of confirmed AD cases were immuno- stained for phospho-tau epitopes. S396 label (E) and other tau epitopes (data not shown) often accumulated in flame-shaped tangles, though remained largely absent from the nucleus (E, arrows). By contrast, double-labeling studies showed AT100 label frequently appearing in the nuclear compartment as evidenced by its co-localization with DAPI (F, arrowhead). In the same neurons, S396 was exclusively contained within the somal compartment (F, arrow). Although characterization and identification of this AT100-positive protein is beyond the scope of the present study, further investigation of this stress-induced response would be valuable as the appearance of the epitope in the nucleus specifically occurs in stressed cells (ATP depleted) or AD brain and not control cells. Images in (A–D): confocal microscope; (E, F): optical microscope. Scale bars = 10 mm (A, F); 20 mm (B–E). (TIF) Acknowledgments We thank Prof. Peter Seubert, Elan Pharmaceuticals for providing the 12E8 antibody. We also thank Denise Nergenau for help with the TEM and Dr O’Neil Wiggan for valuable discussions and suggestions in the development of this manuscript. Author Contributions Conceived and designed the experiments: CG ITW LSM JRB. Performed the experiments: ITW DG LSM. Analyzed the data: ITW CG JRB LSM. Wrote the paper: ITW CG JRB. 4. Velasco ME, Smith MA, Siedlak SL, Nunomura A, Perry G (1998) Striation is the characteristic neuritic abnormality in Alzheimer disease. Brain Research 813: 329–333. 5. Mitchell TW, Nissanov J, Han L-Y, Mufson EJ, Schneider JA, et al. (2000) Novel Method to Quantify Neuropil Threads in Brains from Elders With or Without Cognitive Impairment. Journal of Histochemistry and Cytochemistry 48: 1627–1638. 6. Schmidt M, Murray J, Trojanowski J (1993) Continuity of neuropil threads with tangle-bearing and tangle-free neurons in Alzheimer disease cortex. Molecular and Chemical Neuropathology 18: 299–312. PLoS ONE | www.plosone.org 10 June 2011 | Volume 6 | Issue 6 | e20878