Breeding pairs of mice lacking exon 3 of the X chromosome-linked Mecp2 gene (B6.Cg-Mecp2^tm1.1Jae, “Jaenisch” strain in a pure C57BL/6 background) (Chen et al., 2001) were purchased from the Mutant Mouse Regional Resource Center at the University of California, Davis. A colony was established at The University of Alabama at Birmingham (UAB) by mating wildtype males with heterozygous Mecp2^tm1.1Jae mutant females, as recommended by the supplier. Genotyping was performed by PCR of DNA sample from tail clips. Hemizygous Mecp2^tm1.1Jae mutant males are healthy until 5–6 weeks of age, when they exhibit RTT-like motor symptoms, such as hypoactivity, hind limb clasping, and reflex impairments (Chen et al., 2001). Animals were handled and housed according to the Committee on Laboratory Animal Resources of the National Institutes of Health. All experimental protocols were annually reviewed and approved by the Institutional Animals Care and Use Committee of UAB.

Both hippocampi were dissected from anesthetized postnatal day 0 or 1 (P0-1) male Mecp2 knockout mice and wildtype littermates, and dissociated in papain (20 U/ml) plus DNAse I (Worthington, Lakewood, NJ) for 20-30 min at 37°C, as described (Amaral and Pozzo-Miller, 2007). The tissue was then triturated to obtain a single-cell suspension, and the cells were plated at a density of 50,000 cells/cm² on 12 mm poly-L-lysine/laminin coated glass coverslips, and immersed in Neurobasal medium supplemented with 2% B27 and 0.5 mM glutamine (Life Technologies, Carlsbad, CA). Neurons were grown in 37°C, 5% CO₂, 90% relative humidity incubators (Thermo-Forma), with half of the fresh medium changed every 3-4 days. After 11 days in vitro (DIV), neurons were transfected with cDNA encoding BDNF-YFP (a gift from M. Kojima) using Lipofectamine 2000 (Life Technologies) (0.8 μg DNA) according to the manufacturer's protocol.

All experiments were performed at 12-14 DIV. For localization of endogenous native BDNF, neurons were fixed with 4% (wt/vol) paraformaldehyde/sucrose in phosphate buffer (PB; 23 mM NaH₂PO4, 2 mM Na₂HPO4 pH 7.4) for 10 min, and incubated in 0.25% (vol/vol) Triton X-100 for 10 min, then washed with PB saline (PBS). After blocking with 10% (vol/vol) goat serum in PBS, cells were incubated with anti-BDNF antibody (2μ g/ml, Santa Cruz Biotechnology #SC-546) overnight at 4°C, rinsed in PBS, and incubated with Alexa Fluor-488 secondary antibody (Life Technologies) for 1 h; coverslips were then mounted with Vectashield (Vector Laboratories). Images were acquired in a laser-scanning confocal microscope using a solid-state 488 nm laser for excitation, and a 60 X 1.4 NA oil immersion lens, and standard FITC dichroic and emission filters (FluoView-300, Olympus; Center Valley, PA). For dual immunocytochemistry, BDNF-YFP-expressing neurons were fixed with 3% formaldehyde in PB at 0°C for 20 min, permeabilized for 10 min at room temperature with 3% formaldehyde containing 0.25% Triton X-100, and blocked with 10% bovine serum albumin (BSA) for 1 h at 37°C. Primary goat anti-chromogranin B (1:100; Santa Cruz) and rabbit anti-BDNF (1:100, Santa Cruz) antibodies, as well as fluorescently-conjugated anti-goat and anti-rabbit secondary antibodies (Molecular Probes) were diluted in 1X PBS and 3% horse serum. Coverslips were mounted with Vectashield (Vector Laboratories), and fluorescence images were acquired with a cooled CCD camera (CoolSnap HQ2, Photometics) on a wide-field fluorescence microscope (Eclipse TE2000-U, Nikon Instruments); BDNF-YFP was imaged with a standard FITC filter, and fluorescently-conjugated secondary antibodies with standard FITC and TRITC filters. Images were deconvolved using Metamorph (Molecular Devices).

Time-lapse imaging was performed 24–48 h after BDNF-YFP transfection. For mitochondria trafficking, neurons were incubated for 15 min with MitoTracker Red (200 nM; Life Technologies) prior to live imaging. Individual coverslips with cultured neurons were transferred to a recording chamber mounted on a fixed-stage upright microscope (Leica DM-LFS with either a 63 × 0.9 NA or a Zeiss 63 × 1.0 NA water-immersion objective), and continuously perfused (1 ml/min) with HEPES buffered artificial CSF (aCSF) at 32–34°C, containing (mM): 119 NaCl, 5 KCl, 2 CaCl₂, 1.3 MgCl₂, 10 glucose, 10 HEPES (pH 7.4). YFP was excited with 490 ± 12 nm light using a galvanometric monochromator (Polychrome-II, TILL Photonics; Germany), and its emission (>510 nm, FITC-LP cube) was filtered and detected with an electron-multiplying cooled CCD camera operating in frame-transfer mode (QuantEM: 512SC, Photometrics, Tucson AZ). MitoTracker Red was excited with 560 ± 12 nm and imaged through a standard TRITC cube. Digital images were acquired every 5 s (50–100 ms exposures for ~100 × 200 pixel sub-arrays, 1 × 1 binning) for a total time of 10 min. The position of fluorescent puncta was tracked as a function of time using the Particle Tracking module of Imaris (Bitplane).

BDNF-YFP-transfected neurons were treated with the HDAC6 inhibitor Tubastatin A (1 μM, prepared in 0.01% DMSO vehicle) for 48 h; 0.01% DMSO was used as control. Cell viability was assessed by trypan blue exclusion. Cells were rinsed with HBSS, and incubated with 0.4% trypan blue solution (Gibco) for 2 min at room temperature. After washing with HBSS, dead and live cells were counted in ten random fields per coverslip.

The procedure for BDNF-YFP immunostaining on the surface of cultured neurons was as described (Sadakata et al., 2012). Twenty-four hours after BDNF-YFP transfection, cultured neurons were stimulated with 50 mM KCl for 10 min in the absence or presence of the voltage-gated Ca²⁺ channel blocker nifedipine (50 μ M), the ionotropic glutamate receptor antagonists CNQX (10 μ M) and D-APV (50 μ M), the GABA_AR antagonist bicuculline (10 μ M), or the voltage-gated Na⁺ channel blocker TTX (0.5 μ M), fixed with 4% (wt/vol) paraformaldehyde/sucrose for 5 min, and then washed with PBS. After blocking with 10% (vol/vol) goat serum in PBS, cells were incubated with anti-GFP antibody (which also recognized YFP; Abcam) overnight at 4°C, rinsed in PBS, and incubated with anti-rabbit secondary antibody conjugated to Cy3 (Millipore) for 1 h; coverslips were then mounted with Vectashield medium (Vector Laboratories). Images were acquired in a laser-scanning confocal microscope using a solid-state 488 nm laser for YFP excitation, a 543 nm HeNe green laser for Cy3 excitation, a 60 × 1.4 NA oil immersion lens, and standard FITC and TRITC dichroic and emission filters (FluoView-300, Olympus). Quantification of co-localized pixels was performed using Colocalization module in Imaris. The ratio of surface-bound BDNF-YFP to total BDNF-YFP was estimated as volumetric percentage of co-localized signals over the threshold.

For biochemical measurements of the levels of acetylated and total α-tubulin, total cell lysates were obtained by rinsing 4 × 10⁵ treated hippocampal neurons with cold PBS followed by lysis in NP-40 lysis buffer (20 mM Tris at pH 8.0, 137 mM NaCl, 10% glycerol, 1% Nonidet P-40, 2 mM EDTA) containing protease inhibitor. The cell lysates were maintained with constant agitation for 30 min at 4°C and centrifuged at 12,000 g for 20 min. The supernatants were aspirated and protein concentrations were quantified by the Lowry method. Fifteen micrograms of total lysate were subjected to SDS-PAGE (Bio-Rad) and Western blot analysis with primary antibodies against acetylated α-tubulin (1:1000; Sigma) and total α-tubulin (1:2000; Life Technologies). Immunodetection was performed using Odyssey infrared imaging system (Li-Cor Bioscience).

All the experiments were performed at least 3 different times, from at least 3 different neuronal culture preparations. Data are presented as mean ± standard error of the mean (SEM), and were compared using unpaired Student's t-test for two groups or One Way ANOVA with Tukey post test for more than three groups; percentages were compared using Chi-square test. All analyses were performed using Prism software (GraphPad Software, San Diego, CA). P < 0.05 was considered significant.

The cellular localization, processing and secretion of exogenously expressed BDNF-xFP have been reported to be identical to those of endogenous native BDNF, including its co-localization to secretory granule cargoes like chromogranin-B and SGA2 (Haubensak et al., 1998; Kohara et al., 2001; Lessmann and Brigadski, 2009; Hartmann et al., 2012) (Supplemental Figure 1). To evaluate whether the dynamics of dendritic BDNF trafficking is altered by Mecp2 deletion, time-lapse fluorescence imaging of BDNF-YFP was performed in primary cultures of hippocampal neurons (12–14 days in vitro, DIV) from male Mecp2 knockouts and wildtype littermates. BDNF-YFP puncta are widely distributed and move bi-directionally in live neurons (Figures 1A,B), as previously described (Park et al., 2008; Matsuda et al., 2009). The average velocity of BDNF-YFP puncta was significantly slower in Mecp2 knockout neurons (0.10 ± 0.01 μm/s, n = 261 puncta from 8 cells) compared to wildtype cells (WT 0.25 ± 0.01 μm/s, n = 225 puncta from 8 cells; p < 0.001). Analyses of the distributions of the velocity of BDNF-YFP puncta revealed that the number of fast moving puncta (velocity >0.4 μm/s) is significantly smaller in Mecp2 knockout neurons (Mecp2 1.30 % vs. WT 16.77 %; p < 0.001; Figures 1C,D). To quantify BDNF transport efficiency, we compared the persistence of each BDNF-YFP puncta, estimated as the ratio of total distance traveled during an image sequence over the minimum displacement between image frames (Gauthier et al., 2004). Consistent with impaired and slower BDNF trafficking, the persistence of BDNF-YFP puncta was higher in Mecp2 knockout neurons (Mecp2 7.14 ± 0.98 vs. WT 3.91 ± 0.43; p < 0.05; Figure 1E). Then, we tested whether the transport of other organelles such as mitochondria is also affected by Mecp2 deletion. Intriguingly, time-lapse imaging of MitoTracker-Red puncta revealed a lower percentage of fast moving mitochondria in Mecp2 knockout neurons (Mecp2 6.08 % vs. WT 12.39 %; p < 0.01; Figure 1F), but no significant differences in their average velocity or persistence between Mecp2 knockout (n = 410 puncta from 13 cells) and wildtype neurons (n = 359 puncta from 14 cells; p > 0.05; Figures 1G,H).

Anterograde BDNF transport from somata to dendrites and axon terminals is important for activity-dependent BDNF release to participate in synaptic plasticity, while retrograde BDNF transport back to the soma is critical for neurotrophin recycling and nuclear signaling (Egan et al., 2003; Park et al., 2008). When BDNF-YFP puncta were separated by their trafficking direction, Mecp2 knockout neurons showed significantly fewer BDNF puncta moving in both anterograde and retrograde directions than wildtype neurons, with a significantly shorter total distance traveled in either direction in Mecp2 knockout cells (p < 0.001; Figures 2A,B). Consistently, the average velocity of BDNF-YFP puncta moving in both anterograde and retrograde directions was significantly slower (p < 0.001; Figure 2C), and their persistence higher (p < 0.05 in retrograde direction; Figure 2D), in Mecp2 knockout neurons.

HDAC6 removes acetyl groups from α-tubulin (Hubbert et al., 2002; Matsuyama et al., 2002; Zhang et al., 2003), which makes microtubules less stable. Since BDNF is transported along microtubules (Gauthier et al., 2004), inhibiting HDAC6 is expected to improve BDNF trafficking by stabilizing microtubules (Dompierre et al., 2007). We tested whether treatment with the selective HDAC6 inhibitor enhances BDNF-YFP trafficking in cultured hippocampal neurons. Treatment with Tubastatin A for 48 h (TBA, 1 μM, prepared in 0.01% DMSO) was not neurotoxic, as determined by trypan blue exclusion (p > 0.05; Supplemental Figure 2A). Then, we confirmed that TBA affected the acetylation state of α-tubulin under our experimental conditions. Western immunoblots of cultured hippocampal neurons (DIV12) treated with TBA showed that the levels of acetylated tubulin were significantly increased in wildtype neurons (p < 0.01; Supplemental Figure 2B), and elevated by two times in Mecp2 knockout neurons (p < 0.001; Supplemental Figure 2B).

Next, we tested the effect of HDAC6 inhibition on BDNF-YFP trafficking. Compared to vehicle-treated neurons (162 puncta from 7 cells), TBA increased the proportion of retrogradely moving BDNF puncta (159 puncta from 7 cells; p < 0.01; Figure 3A), as well as the average velocity of puncta moving anterogradely and retrogradely in Mecp2 knockout neurons (p < 0.001; Figure 3B). However, TBA did not affect the persistence of BDNF puncta in Mecp2 knockout neurons (p > 0.05; Figure 3C). On the other hand, TBA had no effect in any of these BDNF-YFP trafficking parameters in wildtype neurons (DMSO 106 puncta from 5 cells; TBA 149 puncta from 7 cells; p > 0.05; Figures 3A–C).

Improving BDNF transport to release sites could in principle also enhance its activity-dependent release. To estimate activity-dependent BDNF secretion, BDNF-YFP-expressing neurons were depolarized with 50 mM KCl, followed by immunostaining with anti-YFP antibody before cell permeabilization. The degree of co-colocalization between immunopositive puncta (labeled with red-conjugated anti-YFP secondary antibody), and native YFP fluorescent puncta after fixation is directly proportional to BDNF-YFP secreted during neuronal depolarization (Figure 4A), as described (Sadakata et al., 2012). KCl-induced postsynaptic secretion of BDNF depends on Ca²⁺ influx (Hartmann et al., 2001; Kolarow et al., 2007), mainly through L-type voltage-gated Ca²⁺ channels (Wang et al., 2002). Here, we confirmed that elevated KCl-induced BDNF secretion was absent in a Ca²⁺ free solution (n = 18 cells), or in the presence of the L-type Ca²⁺ channel blocker nifedipine (50 μM; n = 16; p < 0.05; Figure 4B). Ca²⁺ channels can also be activated by depolarization from GABA_A receptor activity in immature neurons with high intracellular Cl⁻ concentration (Fiorentino et al., 2009; Porcher et al., 2011). Indeed, the GABA_A receptor antagonist bicuculline also prevented KCl-induced BDNF secretion (10 μM; n = 15; p < 0.05; Figure 4B). In addition, KCl-induced BDNF secretion required functional ionotropic glutamate receptor activity (10 μM CNQX, 50 μM D-APV; n = 17; p < 0.01), and voltage-sensitive Na⁺ channels (0.5 μM TTX; n = 19; p < 0.05; Figure 4B), as previously described (Lessmann et al., 2003). Figure 5 illustrates the different mechanisms leading to Ca²⁺-dependent BDNF release during KCl-induced depolarization.

Consistent with reports of impaired activity-dependent BDNF release using different assays (Li et al., 2012), the proportion of BDNF secreted during KCl-induced depolarization was significantly smaller in Mecp2 knockout neurons (n = 21) than in wildtype neurons (n = 25; p < 0.01; Figure 4B). In addition, there was no evidence of BDNF secretion in Mecp2 knockout neurons under any of the conditions of activity blockade described above (Ca²⁺ free n = 15; nifedipine n = 13; CNQX/D-APV n = 16; bicuculline n = 15; TTX n = 13; p > 0.05; Figure 4B). Intriguingly, TBA significantly improved the proportion of BDNF secreted during KCl-induced depolarization in Mecp2 knockout neurons (n = 14; p < 0.001; Figure 4B), but not in wildtype neurons (n = 22; p > 0.05; Figure 4B). These results suggest that improved microtubule-dependent trafficking allowed a larger pool of BDNF to be transported to release sites and be available for activity-dependent release, underscoring the potential therapeutic benefit of this approach to restore BDNF signaling in RTT.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.