cascades that result in the varied cytopathology of AD [23]. AC rods are independently induced in cell culture by the inhibition of mitochondrial ATP generation, thus potentially linking the formation and co-localization of AC rods and MAP inclusions to mitochondrial dysfunction [20]. Moreover, interaction of MAPs and actin in the organization of the cytoskeletal network is well- documented [28–35] and emerging evidence suggests that these interactions may be central to the processes involved in the initiation and development of early AD pathology [20,22,36,37]. Since AD neuropathology involves hyperphosphorylation and accumulation of numerous phosphoepitopes of tau and not just those in the MTBD recognized by 12E8, the aim of this study was to investigate the phosphorylation pattern and re-distribution of several other key phospho-tau epitopes in the primary neuronal culture model. We utilized the chick model since chick neurons express five forms of tau that are homologous to human tau isoforms and exhibit conserved phosphoepitopes recognized by antibodies against human tau [38]. Using a series of commercially available phospho-tau antibodies, we measured time-dependent effects of mitochondrial dysfunction on the levels and distribution patterns of these phosphoepitopes. We found that cellular ATP reduction gave rise to rapid overall dephosphorylation at most epitopes with the exception of the 12E8 epitope that exhibited persistent phosphorylation over the 120 min treatment time. Further, 12E8 was the only epitope predominantly recruited to AC rods generated under these conditions within the time frame investigated. These results highlight the MTBD of MAP/tau as a potential early player in the initiation of neuropil threads in neurons of the AD brain. Methods Ethics Statement Donated frontal cortex and hippocampal brain tissue from normal adult and confirmed Alzheimer’s disease patients were obtained from the Alzheimer Disease Research Center, University of California, San Diego, USA and approved for use by the Institutional Review Board (formerly the Human Research Committee), Colorado State University, USA (Approval ID: JR Bamburg 2001). Experiments involving the use of animals (cell cultures derived from chicken embryos) were approved by the Animal Ethics Committee, University of Sydney, Australia (Approval ID: CS Goldsbury K00/5-2008/3/4786). Antibodies and reagents Mouse monoclonal antibodies against phosphorylated tau epitopes were as follows: AT8 (which recognizes S202/T205 in the longest isoform of human tau and S193/T196 in the longest isoform of chick tau; Pierce USA), AT100 (T212/S214 in human, T203/S205 in chick; Sigma-Aldrich), AT180 (T231/S235 in human, T222/S226 in chick; Sigma-Aldrich), AT270 (T181 in human, T155 in chick; Sigma- Aldrich), 12E8 (‘KXGS’ motifs in MTBDs of tau and other MAPs. For tau, these motifs correspond to S262/S356 in human, S253/S378 in chick; Elan USA) [14]. Other mouse monoclonal antibodies included GAPDH and b-actin (Sigma-Aldrich). Rabbit polyclonal antibodies against phosphorylated tau epitopes were S214 (S205 in chick; GenScript USA), S396 (S418 in chick; Biosource), S404 (S426 in chick; Biosource), S422 (S444 in chick; Sigma-Aldrich), total tau (Dako, USA) and against chick ADF and mammalian ADF and cofilin (1439) [39]. Anti-mouse and anti-rabbit secondary antibodies included Alexa Fluor-conjugated 488, 555, 594 and 647 (Invitrogen) for immunoflu- orescence and horseradish peroxidase-conjugated (Amersham) for chemiluminescence detection of immunoblots. Changes in Phospho-MAP/Tau during Neuronal Stress Cell culture and treatments Primary chick tectal neurons were prepared from freshly dissected chicken embryos (E7), as previously described and cultured for 7 days in vitro (d.i.v) on poly-D-lysine-coated 30 mm culture dishes or glass coverslips [40]. For reduction of ATP, cells were treated with 1–2 mM antimycin (AM; a mitochondrial complex III inhibitor; Sigma-Aldrich) in warm phosphate buffered saline (PBS). The actin depolymerizing drug Latrunculin B (Calbiochem 428020, CA) and actin stabilizing drug Jasplakinolide (Calbiochem 420127, CA) were used at 1 mg/ml in warm PBS. Cells left without a medium change served as controls. After treatments, cells were immediately fixed for immunostaining (cells on coverslips) or lysed, harvested and prepared for Western blot analysis (cells on tissue culture dishes). Immunoblotting Primary neurons were treated in duplicate and prepared for SDS-PAGE. For equal gel loading, protein concentrations were determined by the Lowry assay (BioRad). Proteins transferred to nitrocellulose membranes were detected with an ECL Western Blotting Detection System (Amersham) on a ChemiDoc XRS (BioRad). For analysis, band densities were measured using Image J (v1.38x, National Institutes of Health freeware; http://rsb.info. nih.gov/ij), background intensity was subtracted for each individ- ual lane and normalized to GAPDH or b-actin loading controls. For quantification, blots within the linear range of detection were selected by plotting the band density versus exposure time. An average for each treatment condition was determined with error bars representing min/max intensities (where n = 2) or SEM (where n = 4). Results are presented as a percentage of the mean control band intensities. Microscopy Cells were fixed with 4% paraformaldehyde at 37uC for 35 minutes, permeabilized for 90 sec with 0.05% Triton X-100 in PBS or ice-cold 80% methanol/20% PBS (16), blocked in 5% goat serum and immunostained using primary antibodies and fluorescent secondary antibodies described above. Epifluorescence images were obtained on a Zeiss Axioplan 2 microscope, captured with a CCD camera driven by AxioVision software. Single labeled cells/sections were used to check for bleed-through in all double- label immunofluorescence studies. Confocal images were obtained on a Zeiss LSM 510 Meta driven by LSM 510 software. All captured images were converted to Tagged Image Files for subsequent analysis and presentation. Nuclear accumulation analysis For quantification of cells containing nuclear accumulation of AT100 label in control, AM- and Lat B-treated conditions, a number of randomly selected fields on each coverslip were selected and cells containing nuclear tau were counted and expressed as a percentage of the total number of counted cells. Inclusion criteria for nuclear accumulation were (i) general neuron morphology appeared normal (including intact nucleus) (ii) neuron exhibited obvious co-localization of AT100 label with DAPI nuclear label and (iii) nucleus exhibited AT100 fluorescence intensity .1.56the intensity of nuclei of surrounding, unaffected cells, as measured using Image J. Over 200 cells were counted for each condition in duplicate experiments using independently prepared cell cultures. Human tissue preparation Post-mortem time to fixation in formalin (for immunofluores- cence) was 3 h. For immunofluorescence, paraffin sections (6– PLoS ONE | www.plosone.org 2 June 2011 | Volume 6 | Issue 6 | e20878