used in this experiment is mostly in oligomer form even though other types of Ab are present in the mixture (see the figure in File S1). Although the exact mechanism underlying TBA-mediated enhancement of axonal transport is not fully studied, we suggest that TBA promotes the binding of motor proteins to MTs, contributing to the restoration of mitochondrial transport that has been impaired by Ab. The size and structure of mitochondria are dynamically regulated by mitochondrial fission and fusion [37,38]. Ab activates two mitochondrial fission proteins, Fis1 and Drp1, leading to excessive fragmentation of mitochondria that may disrupt mitochondrial transport [12]. Misko et al. (2010) also reported that mitofusin 2, an outer mitochondrial membrane protein involved in regulating mitochondrial dynamics, is directly involved in mitochondrial axonal transport [39]. Our results (Fig. 4B) show that motile mitochondria are longer than stationary ones. Thus, it is likely that the change in mitochondrial length is coupled with mitochondrial transport. Therefore, we tentatively anticipate that HADC6 is more likely to regulate the mitochon- drial dynamics. An outstanding issue that remains to be further investigated is how HDAC6 inhibitors affect the interaction between an adaptor protein and MTs and the functions of the mitochondria. In addition, it remains to be further examined whether mitochondrial elongation directly leads to increased mitochondrial transport. Since Ab has been shown to reduce ATP production in neurons [40], we examined whether TBA-treated neurons are recovered from Ab-induced impairment in ATP generation. When ATP production was measured in this system, Ab treatment reduced ATP production significantly, but co- treatment of Ab with TBA did not rescue Ab-induced reduction of ATP production (data not shown), suggesting that ATP production is not associated with axonal transport and morphology of the mitochondria in cells treated with TBA. Regarding the relation- ship of changes in membrane potential to mitochondria and axonal transport, Miller and Sheetz (2004) reported that mitochondria with high membrane potential moved toward the axon terminus, whereas mitochondria with low membrane potential moved toward the soma [41], suggesting that membrane potential is another factor that influences mitochondrial axonal transport. Overall, this study showed that Ab impaired bidirec- tional mitochondrial transport, which was subsequently rescued by the HDAC6 inhibitor. Furthermore, we observed that the changes in both acetylation of a-tubulin and mitochondrial elongation correlate to the alteration of mitochondrial transport in cells treated with Ab and/or an HDAC6 inhibitor. Therefore, these results suggest that an HDAC6 inhibitor may be a potential therapeutic target for the treatment of AD. Materials and Methods Animals Female Sprague-Dawley (SD) rats whose embryos (E18) were used for primary hippocampal neuronal culture were purchased from KOATECH (Gyeonggido, Korea). The brain tissues of 13- month-old 5XFAD mice (Tg6799; B6SJL-Tg (APPSwFlLon, PSEN* M146L*L286V) 6799Vas/J, stock number 006554, Jackson Labs, Bar Harbor, ME) overexpressed three mutations (Swedish, Florida, and London) of human APP 695 and two mutations (M146L and L286V) of human PS1 under transcrip- tional control of the murine Thy-1-promoter and B6SJL wild type (WT) mice were used for western blot analysis. Animals were treated and maintained as per the Helsinki Treaty, the Principles of Laboratory Animal Care (NIH publication No. 85-23, revised 1985), and the Animal Care and Use Guidelines of Seoul National University, Seoul, Korea. All procedures for animal experiments Role of HDAC6 Inhibitor in Mitochondrial Transport were approved by the Ethics Review Committee for Animal Experimentation in Seoul National University (approval number, SNU060519-5, IACUC). DNA construct and reagents pDsRed2-Mito (Clontech, Mountain View, CA) is a mamma- lian expression vector that encodes a red fluorescent protein and the mitochondrial targeting sequence, resulting in neuronal mitochondria appearing as a red fluorescence. Acetylated a- tubulin and b-actin antibodies were purchased from Sigma- Aldrich (St. Louis, MO) and a-tubulin antibodies were from abm (Applied Biological Materials Inc., Richmond, BC). Alexa Fluor 488 donkey anti-mouse IgG was used in immunofluorescence analysis. Tubastatin A (TBA; BioVision, Mountain View, Cali- fornia) and Ab1–42 peptides (American peptide, Sunnyvale, CA) were also used. Cell cultures and transfection Primary hippocampal neurons were obtained from the brain tissue of SD rat embryos (E18). Preparation of primary hippo- campal neurons was followed in previous reports with minor modifications [15,42]. Briefly, brain tissues were isolated in ice- cold Hank’s Balanced Salt Solution (HBSS; WelGENE, Daegu, Korea) and digested with 0.5% trypsin (using 2.5% trypsin; Sigma, St. Louis, MO). Cells were dissociated in Neurobasal/B27 medium (Invitrogen, Carlsbad, California) with 0.1 mg/ml penicilin/streptomycin (Sigma, St. Louis, MO) and plated onto poly-D-lysine (Sigma, St. Louis, MO) coated plates and micro- fluidic chambers. Half of the culture medium was replaced by fresh medium every 2 days for plates and every day for microfluidic chambers. For labeling mitochondria, pDsRed2-Mito was transfected into neurons at 7 days in vitro (DIV) using Lipofectamine 2000 (Invitrogen, Carlsbad, California). After being pretreated with Ab1–42 peptides (2 mM) at DIV 8 for 24 hrs, cells were treated with TBA (5 mM) for 3 hrs [21]. Microfluidic chamber system The microfluidic chambers for separating axons were fabricated as described in the reference [43]. After assembling the chambers, the chambers were coated by poly-D-lysine. Cells were loaded in only one compartment of the chamber, the somal compartment. As neurites were growing up, the other compartment was filled with axon which we called the axonal compartment. All chambers have a microgroove length of 900 mm and a width of 8 mm. Live cell imaging and image analysis Living cells were imaged using Olympus IX81 microscope (Tokyo, Japan) equipped with a Cool SNAP HQ2 CCD camera (Photometrics, Ltd., Tucson, AZ 85706), controlled by Meta- Morph Software (Universal Imaging, PA, USA). During imaging, cells were maintained in an incubating chamber at 37uC and supply atmosphere of 5% CO2/95% air (Live cell instrument, Seoul, Korea). Time-lapse image recordings were acquired at an exposure time of 500 ms, 1 sec interval and duration up to 2 min. Live cell images were obtained from before and after TBA treatment and focused on axons in microgroove. Movies were processed using MetaMorph. Quantitative analysis of mitochon- drial velocity and motility was performed manually using ImageJ (rsb.info.nih.gov, by W. Rasband) installed multiple kymograph plugins (by J. Rietdorf and A. Seitz) at each axon. In order to analyze mitochondrial velocity, we measured the angle of motile mitochondria on the kymographs using ImageJ and then calculated the velocity according to the following equation: PLOS ONE | www.plosone.org 5 August 2012 | Volume 7 | Issue 8 | e42983