Role of HDAC6 Inhibitor in Mitochondrial Transport Figure 3. Regulation of mitochondrial transport by Ab and TBA. (A) Representative kymographs of mitochondrial movement. 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. After being pretreated with Ab (2 mM) for 24 hrs, cells were treated with TBA (5 mM) for 3 hrs. Images were acquired every 1 sec for 2 min at microgrooves. X axis of kymograph is axonal length (152.7 mm). Proximal to distal indicates the soma to axon terminal direction. Mitochondria which move from proximal to distal region show anterograde movement. Y axis is the time that mitochondria have moved (2 min). (B) Pictures of motile mitochondria for each group were shown every 5 sec (Scale bars = 10 mm). Arrows indicate motile mitochondria. (C) Average velocity of motile mitochondria. Anterograde and retrograde velocity were analyzed separately. (D) Motility of mitochondria. Motility stands for percentage of motile mitochondria over total mitochondria. Anterograde and retrograde motility were analyzed separately. Data were acquired from 4 independent experiments (Veh n = 41, TBA n = 44, Ab n = 42, Ab+TBA n = 43, *P,0.05, **P,0.01, ***P,0.001). doi:10.1371/journal.pone.0042983.g003 Ab25–35 oligomer significantly reduced the motility of anterograde mitochondrial transport but not the velocity in mitochondrial transport in both directions. Moreover, Wang and colleagues [36] have shown that the motility of mitochondria was decreased in both directions. In the current study, we showed that the treatment of neurons with 2 mM Ab significantly reduced the motility and velocity in mitochondrial transport in both directions. The difference in the level and direction of mitochondrial motility and velocity between this and other studies may be relevant to the form or concentration of Ab. Our AFM assay showed that Ab Figure 4. Alteration of mitochondrial length by Ab and TBA. (A) Average length of total mitochondria, including both motile (anterograde, retrograde transported) and stationary mitochondria. (B) Average length of motile and stationary mitochondria. ***P,0.001 significance of stationary mitochondria vs. motile mitochondria; ++P,0.01, +++P,0.001 among motile mitochondria; ###P,0.001 among stationary mitochondria. (C) The average number of mitochondria per 100 mm of axon. Data were obtained from 4 independent experiments (Veh n = 35, TBA n = 42, Ab n = 38, Ab & TBA n = 38, *P,0.05, **P,0.01, ***P,0.001). doi:10.1371/journal.pone.0042983.g004 PLOS ONE | www.plosone.org 4 August 2012 | Volume 7 | Issue 8 | e42983