Role of HDAC6 Inhibitor in Mitochondrial Transport Figure 2. Modulation of acetylated a-tubulin by Ab and TBA. (A) Western blot of acetylated a-tubulin in the rat hippocampal neurons. After being pretreated with Ab (2 mM) for 24 hrs, cells were treated with TBA (5 mM) for 3 hrs and lysed with RIPA buffer. Actin is a loading control. (B) Quantitation of the acetylated a-tubulin normalized by total a-tubulin is shown as means 6 SEM. Data were acquired from 4 independent experiments (*P,0.05, ***P,0.001). (C) Immunocytochemistry of acetylated a-tubulin in hippocampal neurons. Anti-acetylated a-tubulin antibody detects a-tubulin only when acetylated at Lys 40 (Scale bar = 100 mm). doi:10.1371/journal.pone.0042983.g002 measured mitochondrial length. In parallel, to compare the length and motility of mitochondria, we separately determined the length of stationary and motile mitochondria. Overall, mitochondrial length was decreased by Ab and increased in the group co-treated with Ab and TBA (Fig. 4A; Veh: 1.16860.02, Ab: 0.90460.02, TBA: 1.22860.02, Ab & TBA: 1.15960.02, ***p,0.001). Although both motile and stationary mitochondria showed a similar pattern as shown in Fig. 4B, the length of stationary mitochondria was shorter than that of motile mitochondria (Fig. 4B, **p,0.01, ***p,0.001), suggesting that longer mito- chondria have higher motility than shorter ones. This demon- strates that mitochondrial length is related to mitochondrial transport and that TBA treatment affects both mitochondrial length and motility in neurons. However, there was no significant difference among the four groups in the number of mitochondria per 100 mm of axon (Fig. 4C). These findings suggest that Ab and HDAC6 are important in mitochondrial length and axonal transport. Discussion Impaired axonal transport and mitochondrial dysfunctions occur in the early stages of AD [8,15]. These changes are induced by Ab which forms amyloid plaques, one of the major hallmarks of AD [26,27,28,29]. Increased GSK3b activity induces hyperpho- sphorylation of tau, resulting in the formation of neurofibrillary tangles (NFT) and/or an increase in Ab generation[30]. HDAC6 activity, as a substrate of GSK3b, is increased by GSK3b activation [31], suggesting a decrease in both the level of a- tubulin acetylation and axonal transport. To modulate HDAC6 activity, we used TBA as a potent HDAC6 inhibitor, which is more selective than other HDAC6 inhibitors, such as TSA and tubacin [21,32]. The specificity of TBA to HDAC6 was confirmed in previous studies by homology modeling and enzyme inhibition experiments using 11 HDAC isozymes [21,32]. TBA affects the acetylation of cytosolic proteins like a-tubulin but not histones [21]. The knockdown of HDAC6 by siRNA resulted in an increased level of acetylated a-tubulin, consistent with the results with the HDAC6 inhibitor [33]. It has been reported that TBA treatment increases not only axonal transport and the number of moving mitochondria but also the level of acetylated a-tubulin in neuropathies of a Charcot-Marie-Tooth model that have been induced by mutant heat-shock protein gene (HSPB1) [32]. Given the results from the current and previous studies, it is increasingly clear that TBA specifically inhibits HDAC6 and, thus, affects the acetylation of a-tubulin and axonal transport of cellular cargoes, such as mitochondria. Motor proteins, such as kinesin for anterograde transport and dynein for retrograde transport, are involved in mitochondrial transport [34]. Adaptor proteins, such as milton, syntabulin, miro (for kinesin), and dynactin (for dynein), are also required [17,18]. Axonal transport is regulated by the interaction between adaptor and motor proteins as well as their stability is important in axonal transport [17,18,34]. It has been also known that a-tubulin acetylation at Lys 40 increases the interaction between kinesin and the microtubule, resulting in improved transport of cargo proteins [3]. Dompierre et al. have reported that MT acetylation increases both anterograde and retrograde transport, which was attributed to the increased interaction of both kinesin family motor protein 5 (KIF5) and dynein with MTs [35]. However, the mechanism by which acetylation of a-tubulin regulates mitochondrial transport under pathological conditions, such as the accumulation of Ab, is unclear. Du et al. [15] have reported that mitochondrial motility was significantly reduced in both directions (anterograde and retrograde) in neurons treated with 200 nM Ab oligomer. By contrast, Calkins et al. [12] found that the treatment with 20 mM PLOS ONE | www.plosone.org 3 August 2012 | Volume 7 | Issue 8 | e42983