‘‘Velocity (mm/sec) = Tangent (Angleu+90u) * 0.111276’’. Because the angles measured by ImageJ were all between 0u,2180u, the values of ‘‘Tangent (Angleu)’’ were all under 0. Therefore, we added 90u to the measured angles to distinguish the anterograde and retrograde mitochondrial movements as positive and negative values, respectively. In our imaging system, 0.111276 represents the length of one pixel (mm). Mitochondrial motility was tracked by hand using kymographs and the cut-off value of 0.0019 mm/sec (mean velocity) was used to distinguish motile from stationary mitochondria. Quantitative analysis of mitochondrial length was also based on manual tracking using ImageJ on the first picture of the live cell images. Western blot analysis To extract brain tissues, mice were anesthetized and perfused transcardially with phosphate-buffered saline (PBS). Cultured primary hippocampal neurons in 6 well plates were harvested as cell pellets. Brain tissues and harvested cell pellets were resuspended in RIPA buffer (150 mM NaCl, 1% Nonidet P-40, 0.5% deoxycholic acid, 0.1% SDS, and 50 mM Tris-HCl, pH 7.4) containing protease inhibitors (Sigma, St. Louis, MO), Phenyl- methylsulfonyl fluoride (PMSF; Sigma, St. Louis, MO), phospha- tase inhibitors (A.G. Scientific, Inc., San Diego, CA) and TBA and then incubated on ice for 20 min and sonicated for 5 sec. After centrifugation at 18,000 g for 10 min, the supernatant was collected to remove membrane lipids. Protein concentrations were determined by BCA assay (Sigma), and equal amounts of protein were loaded on 10% glycine gels. The separated samples were transferred to a PVDF membrane and incubated with antibodies against the target proteins. Protein bands were visualized by enhanced chemilumin-escence (ECL; Amersham Pharmacia Biotech, Buckinghamshire, England) using a bioimaging analyzer (LAS-3000; Fuji, Tokyo, Japan). This process has been also described in a previous report [44]. Immunocytochemistry analysis Primary hippocampal neurons were plated on 18-mm round coverslips coated with poly-D-lysine which were in a 12-well plate. After Ab and/or TBA treated, cells were washed with chilled PBS, and fixed with 4% paraformaldehyde (BIOSESANG, Inc., Gyeonggi-do, Korea) for 20 min at room temperature (RT). Cells were permeabilized and blocked with 1% Triton X-100 (Sigma- Aldrich, St. Louis, MO) and 1% goat serum (Vector Laboratories, Inc., Burlingame, CA) diluted in PBS for 10 min at RT. To detect acetylated a-tubulin, cells were incubated with primary antibodies References 1. Janke C, Kneussel M (2010) Tubulin post-translational modifications: encoding functions on the neuronal microtubule cytoskeleton. Trends Neurosci 33: 362– 372. 2. Fukushima N, Furuta D, Hidaka Y, Moriyama R, Tsujiuchi T (2009) Post- translational modifications of tubulin in the nervous system. J Neurochem 109: 683–693. 3. Hammond JW, Cai D, Verhey KJ (2008) Tubulin modifications and their cellular functions. Curr Opin Cell Biol 20: 71–76. 4. Dent EW, Gertler FB (2003) Cytoskeletal dynamics and transport in growth cone motility and axon guidance. Neuron 40: 209–227. 5. Reed NA, Cai D, Blasius TL, Jih GT, Meyhofer E, et al. (2006) Microtubule acetylation promotes kinesin-1 binding and transport. Curr Biol 16: 2166–2172. 6. Bulinski JC (2007) Microtubule modification: acetylation speeds anterograde traffic flow. Curr Biol 17: R18–20. 7. Zhang Y, Gilquin B, Khochbin S, Matthias P (2006) Two catalytic domains are required for protein deacetylation. J Biol Chem 281: 2401–2404. 8. Stokin GB, Lillo C, Falzone TL, Brusch RG, Rockenstein E, et al. (2005) Axonopathy and transport deficits early in the pathogenesis of Alzheimer’s disease. Science 307: 1282–1288. Role of HDAC6 Inhibitor in Mitochondrial Transport (Sigma-Aldrich, St. Louis, MO) overnight at 4uC and then with secondary antibodies for 1 hr at RT. Primary and secondary antibodies diluted in permeabilizing and blocking solution. DAPI (0.1 mg/ml; Sigma-Aldrich, St. Louis, MO) staining was per- formed for 10 min at room temperature. Finally, coverslips were mounted on slides using mounting reagent (Biomeda corp., Foster city, CA). Cells were washed by PBS twice every steps between incubations. Images were acquired using confocal microscope (Olympus FV10i; Olympus, Tokyo, Japan). Statistical analysis Significant differences between groups were examined for statistical analysis using t-test or one-way analysis of variance (ANOVA) and Bonferroni post-tests. All data were expressed as means 6 SEM (*P,0.05, **P,0.01, ***P,0.001). Supporting Information Mitochondrial movement of control neuron. Mitochondrial movement of TBA treated Mitochondrial movement of Ab treated neu- Movie S1 (MP4) Movie S2 neuron. (MP4) Movie S3 ron. (MP4) File S1 (DOC) Movie S4 Mitohcondrial movement of Ab and TBA treated neuron. Hippocampal neurons from rat (E18) were plated with densities of 66104 cells in the somal side of the microfluidic chamber. Cells were transfected with pDsRed2-Mito after 7 days of culture. Cells were treated with Ab (2 mM) for 24 hrs, and then treated with TBA (5 mM) for 3 hrs. Images were acquired every 1 sec for 2 min at microgrooves. (MP4) Supporting Materials and Methods. Author Contributions Conceived and designed the experiments: CK IMJ. Performed the experiments: CK HC WL. Analyzed the data: HC WL. Contributed reagents/materials/analysis tools: ESJ SO NLJ. Wrote the paper: CK HC IMJ. 9. Hempen B, Brion JP (1996) Reduction of acetylated alpha-tubulin immunore- activity in neurofibrillary tangle-bearing neurons in Alzheimer’s disease. J Neuropathol Exp Neurol 55: 964–972. 10. Henriques AG, Vieira SI, da Cruz ESEF, da Cruz ESOA (2010) Abeta promotes Alzheimer’s disease-like cytoskeleton abnormalities with consequences to APP processing in neurons. J Neurochem 113: 761–771. 11. 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PLOS ONE | www.plosone.org 6 August 2012 | Volume 7 | Issue 8 | e42983