attempted to elucidate the mechanism that links the acetylation of a-tubulin and Ab-induced impairment of mitochondrial transport in hippocampal neurons cultured in a microfluidic system. To increase a-tubulin acetylation, we used the Tubastatin A (TBA) as the HDAC6 inhibitor. Mitochondrial axonal transport was analyzed by measuring the velocity, motility and length of mitochondria. We found that pharmacological inhibition of HDAC6 significantly restored the compromised velocity and motility of the mitochondria of Ab hippocampal neurons to a normal level in both anterograde and retrograde axonal transports. The inhibition of HDAC6 also recovered the length of mitochondria that had been shortened by Ab. These results show that the inhibition of HDAC6 rescued neuronal cells from Ab-induced impairment of mitochondrial axonal transport as well as mitochondrial length, identifying HDAC6 as a potential therapeutic target to modulate AD pathogenesis. Results Decrease in the acetylation of a-tubulin in the brains of 5XFAD mice The reduced level of acetylated a-tubulin [9,10] correlates to the increased level of HDAC6 in the AD patients’ brains [11]. To determine the role of acetylation of a-tubulin in AD, we examined the level of acetylated a-tubulin in the brains of 5XFAD mice, an AD animal model. Western blot analysis showed that the level of acetylated a-tubulin in the 5XFAD mice was significantly lower than that of wild type mice (WT: 1.14560.059, 5XFAD: 0.9660.026, Fig. 1A and 1B, *p,0.05), whereas no difference was observed in the amount of total a-tubulin. We, therefore, conclude that acetylated a-tubulin level is decreased in the brains of 5XFAD mice. Regulation of the acetylation of a-tubulin by Ab and HDAC6 inhibitor It has been reported that Ab alters the level of acetylated a- tubulin in primary neuronal cultures and cell lines [10] whereas the HDAC6 inhibitor, TBA, promotes the acetylation of a-tubulin [19,20,21]. To examine the role of Ab in reducing acetylated a- tubulin, primary hippocampal neurons were characterized. Consistent with previous studies [10,22], the level of acetylated a-tubulin was significantly decreased by the Ab treatment (Fig. 2), as shown by Western blot analysis (Fig. 2A and 2B, *p,0.05, ***P,0.001) as well as immunocytochemistry (Fig. 2C). Western blot analysis (Fig. 2A and 2B, *p,0.05, ***P,0.001), together with immunocytochemistry (Fig. 2C), revealed that TBA treat- ment in the presence of Ab restored Ab-induced reduction in the level of acetylated a-tubulin level, compared to Ab treatment alone. This indicates that TBA increased a-tubulin acetylation even in the presence of Ab. The effect of Ab and TBA on the velocity and motility in mitochondrial axonal transport Because it has been reported that mitochondrial transport along the microtubule was disrupted in the presence of Ab [12], the effect of TBA in the presence of Ab on mitochondrial transport was examined with primary hippocampal neuron cultures in microfluidic system, which could isolate neuronal compartments between the soma and axon. To visualize mitochondrial move- ment, pDsRed2-Mito construct was transfected into the DIV 7 neurons, and live cell imaging was performed after the neurons were treated with Ab and/or TBA. 24 hrs after transfection, the neurons were pretreated with 2 mM Ab for 24 hrs and then Role of HDAC6 Inhibitor in Mitochondrial Transport Figure 1. Reduction of acetylated a-tubulin in 5XFAD. (A) Western blot of acetylated a-tubulin in the brains of both wild type (WT) and 5XFAD mice. Brain extracts were prepared from frontal cortex of 13-month-old mice. Actin is a loading control. (B) Quantitation of the acetylated a-tubulin normalized by total a-tubulin is shown as means 6 SEM (WT n = 4, 5XFAD n = 3, *P,0.05). doi:10.1371/journal.pone.0042983.g001 treated with 5 mM TBA for 3 hrs in both somal and axonal sides. Images were taken every sec for 2 mins. To analyze the direction of velocity and motility of mitochondria, we used z-projection and multiple Kymograph plugins functions of the ImageJ program. We found that Ab greatly reduced mitochondrial movement, and TBA recovered the reduced mitochondrial movement in the presence of Ab, as shown by the kymographs (Fig. 3A, B, and Movie S1, S2, S3, S4). In Ab treated neurons, we observed the decreased velocity in both anterograde and retrograde movements of mitochondria. TBA treatment with Ab induced a recovery of the reduced velocity of mitochondria by Ab (Fig. 3C, ***p,0.001). The results for motility were consistent with those for velocity (Fig. 3D, *p,0.05, **p,0.01, ***p,0.001). In this experiment, the ration of motile mitochondria over total mitochondria was calculated to present the motility of mitochondria. Consistent with the data for mitochondrial velocity (Fig. 3C), mitochondrial motility was also decreased by Ab and recovered by TBA treatment in both directions (Fig. 3D). In the group treated with both Ab with TBA, the defects caused by Ab were all rescued by TBA treatment (Fig. 3), suggesting that acetylated a-tubulin regulates mitochondrial transport even in the presence of Ab. The effect of Ab and TBA on mitochondrial length in axon It has been shown that the motor protein dynein is involved in mitochondrial morphology [23,24] and that pharmacological inhibition of HDAC affects mitochondrial elongation [25]. To test the hypothesis that the acetylation of a-tubulin affects mitochondrial length in hippocampal neurons, we determined the effect of HDAC6 inhibition on mitochondrial length by treating the cells with TBA in the absence or presence of Ab and PLOS ONE | www.plosone.org 2 August 2012 | Volume 7 | Issue 8 | e42983