InfoStat software version 2009 (InfoStat Group, FCA, Cordoba National University, Argentina) was used for the analysis of variance and followed by a post hoc LSD Fisher's test. Data are expressed as mean +/- S.E.M. *P < 0.05;**P < 0.01;***P < 0.001.
Constructs and siRNA
The plasmid encoding BDNF-eGFP was previously described [5, 56]. BDNF-eGFP shows cellular localization, processing, and secretion properties indistinguishable from those of endogenous BDNF. The plasmid encoding for the huntingtin-associated protein 1 (HAP1) tagged with GFP was a gift of XJ Li (Emory University, Atlanta, USA). The siRNA targeting human huntingtin (siHtt-hu585, Eurogentec, Seraing, Belgium) corresponds to the coding region 279-298 of human htt mRNA (NCBI ref. seq. NM_002111). The control RNA (scRNA, ATCGAGCTACCACGAACGCTT, Eurogentec) has a unique sequence which does not match to any sequence in the genome of interest.
Construction of pARIS-htt
pARIS-htt was engineered based on the cDNA of full-lengh human htt using OptGene (Ocimum Biosolutions, Hyderabad, India) gene optimizing tool. Gene synthesis was performed by assembly of oligonucleotides using proprietary in-house protocols of BaseClear BV (Leiden, Netherlands). The original sequence was designed with a polyglutamine stretch of 23 glutamines. Glutamine repeats were encoded by alternate CAG/CAA codons to provide more genetic stability. The first base on the start translation codon is considered position number 1.
The pARIS-htt sequence has been rendered insensitive to different siRNAs commonly used in our laboratory: siHtt-1.1 AAGAACTTTCAGCTACCAA (human specific, position 275-293); siHtt-hu585 AACTTTCAGCTACCAAGAAAG (human specific, position 279-298); siHtt-6: AAGCTTTGATGGATTCTAA (human specific, position 474-492); siHtt-13: GCAGCTTGTCCAGGTTTAT (human, rat and mouse specific, position 1062-1080). siHtt-hu585 was used in this study because it is particularly effective to knock-down endogenous htt expression in cells of human origin (referred as siRNA-htt throughout the text).
The full-length engineered pARIS-htt construct was entirely sequenced and inserted into HindIII/BamHI sites of a pUC19 variant (Baseclear BV) for amplification. The pARIS-htt sequence contains flanking attL1 and attL2 sites to allow recombination into pDON201 donor vector (BP clonase reaction, Invitrogen, Carlsbad, USA). A second recombination with pcDNA3.2-DEST (Invitrogen) using LR clonase was necessary to generate a pcDNA3-based destination vector. Recombinations were done in a 10 μl final volume following instructions provided by the manufacturer. Amplification of the constructs was done in TOP10 or DH5α E. coli strains (Invitrogen). A DNA fragment of htt containing a polyQ stretch of 100 glutamines was synthesized using alternative protocols by Geneart AG (Regensburg, Germany) and inserted into NotI/SacI sites of pARIS-htt to replace the 23Q stretch. Vector maps are available in additional file 1.
We use the following nomenclature to describe the first step constructs in the Entry vector: pARIS-htt-N[His-mCherry]Q23-C[HA-TC] and pARIS-htt-N[His-mCherry]Q100-C[HA-TC]. These constructs were transposed to pcDNA3.2 to generate pARIS-httpcDNA3.2-N[His-mCherry]Q23-C[HA-TC] (pARIS-mCherry-httQ23) and pARIS-httpcDNA3.2-N[His-mCherry]Q100-C[HA-TC] (pARIS-mCherry-httQ100).
To generate the htt construct deleted for the dynein-interacting domain, the deletion was first generated within the F3 fragment (pUC19-F3Δ633-672), transposed to Entry-based pARIS-htt by insertion of SacII/KpnI (fragment 3) generating pARIS-htt-N[His-mCherry]Q23-Δ633-672-C[HA-TC] and next transposed to pcDNA3.2 to generate pARIS-httpcDNA3.2-N[His-mCherry]Q23-Δ633-672-C[HA-TC] hereafter denoted pARIS-mCherry-httQ23-Δdyn. To generate the htt construct deleted for the HAP1 binding domain, the deletion was first generated within the F2 fragment (pUC19-F2Δ170-268), transposed to Entry-based pARIS-htt by insertion of SacI/SacII (fragment 2) generating pARIS-htt-N[His-mCherry]Q23-Δ170-268-C[HA-TC] and next transposed to pcDNA3.2 to generate pARIS-httpcDNA3.2-N[His-mCherry]Q23-Δ170-268-C[HA-TC] hereafter denoted pARIS-mCherry-httQ23-ΔHAP1. Requests for constructs may be sent to the following e-mail address: email@example.com.
HEK and Cos7 cells were grown at 37°C in 5% CO2 in Dulbeco's modified Eagle's medium (DMEM) supplemented with 10% bovine calf serum, 1% L-glutamine and antibiotics (50 units/ml penicillin and 50 μg/ml streptomycin). HeLa cells stably expressing GFP-mannosidase II (gift of F. Perez, Institut Curie, Paris, France), were grown at 37°C in 5% CO2 and cultured in DMEM supplemented with 10% bovine calf serum, 1% L-glutamine and 400 μg/ml geneticin (Gibco, Carlsbad, USA). Mouse neuronal cells, STHdh+/+ cells derived from immortalized striatal progenitor cells were grown as previously described .
For pARIS-htt expression analysis, HEK cells were transfected with pARIS-mCherry-httQ23, pARIS-mCherry-httQ100 or equivalent amount of empty vector, using the calcium phosphate method . Western blot analysis was performed after 24-48 h.
For immunofluorescence experiments, Cos7 cells seeded in 12-well plates with 18 mm coverslips were transfected with pARIS-mCherry-httQ23, pARIS-mCherry-httQ100 or equivalent amount of empty vector using FuGENE reagent (Roche, Mannheim, Germany) according to the manufacturer's instructions. Immunostaining was done after 48 h.
For gene replacement experiments, HeLa cells stably expressing GFP-mannosidase II were seeded in 12-well plates with 18 mm coverslips. Sequential transfection was performed as following: attached cells were first transfected using Lipofectamine 2000 (Invitrogen) with siRNA-htt or scRNA. After 24 h, cells were transfected again with pARIS-mCherry-httQ23, pARIS-mCherry-httQ100, pARIS-mCherry-httQ23-Δdyn or pARIS-mCherry-httQ23-ΔHAP1. Cells were processed for western blotting or immunostaining 24 h after. DNA, siRNA and Lipofectamine 2000 quantities were used according to the manufacturer's instructions.
To perform co-immunoprecipitation experiments, HEK cells were transfected using Lipofectamine 2000 with siRNA-htt as described above. After 24 h the cells were transfected with pARIS-mCherry-Htt constructs and/or HAP1-GFP using the calcium phosphate method. Immunoprecipitation assays were performed after 24 h.
For videomicroscopy experiments mouse neuronal cells were electroporated with Kit L Nucleofector according to the supplier's manual (Amaxa, Köln, Germany). BDNF-eGFP and pARIS-htt DNA or equivalent amount of empty vector were added to the electroporation mix. After electroporation, cells were seeded in 12 well plates with 18 mm coverslips.
Transfected HeLa GFP-mannosidase II cells were treated with 4 μM nocodazole for 30 min at 4°C and 90 min at 37°C to allow a complete depolymerization of microtubules. Cells were washed twice with DMEM prior to methanol fixation (2 min at -20°C).
Anti-huntingtin antibodies used in this study htt-4C8, htt-2C1 and 1C2 were previously described [29, 31], α-tubulin was from Sigma (St Louis, USA), high affinity anti-HA and anti-GFP were from Roche, anti-dynein intermediate chain (DIC) was from Chemicon (Billerica, USA), secondary IgG-HRP antibodies were from Jackson ImmunoResearch (WestGrove, USA), the mouse monoclonal antibody against the cis/medial Golgi marker CTR433 was previously described . Alexa Fluor secondary antibodies used in immunofluorescence experiments were from Invitrogen.
Transfected cells were harvested and lyzed in 50 mM Tris-HCl, pH 7.5, containing 0.1% Triton X-100, 2 mM EDTA, 2 mM EGTA, 50 mM NaF, 10 mM β-glycerophosphate, 5 mM sodium pyrophosphate, 1 mM sodium orthovanadate, 0.1% (v/v) β-mercaptoethanol, 250 μM PMSF, 10 mg/ml aprotinin and leupeptin. Cell lysates were centrifuged at 20,000 g for 10 min at 4°C. Equal amounts of protein were subjected to SDS-PAGE on 6% polyacrylamide gels and transferred to nitrocellulose membranes (Whatman, Dassel, Germany). Blocked membranes (5% milk in TBS-0.1% Tween-20) were incubated with mouse anti-huntingtin antibodies (htt-4C8, htt-2C1), mouse anti-polyQ expansion (1C2), rat anti-HA, mouse anti-GFP, mouse anti-DIC or mouse anti-α-tubulin antibodies and washed three times with TBS-0.1% Tween-20 for 10 min. Membranes were then labelled with secondary IgG-HRP antibodies raised against each corresponding primary antibody. After three washes, the membranes were incubated with SuperSignal West Pico Chemiluminescent Substrate (Pierce, Erembodegem, Belgium) according to the instructions of the supplier. Membranes were exposed to Amersham Hyperfilm™ MP (GE Healthcare, Buckinghamshire, UK) films and developed.
Immunoprecipitations were performed as described  with minor modifications. Cell lysis and wash of the immunocomplexes were done in 50 mM of Tris 1 M (pH 8), 150 mM NaCl and 1% of NP40 containing protease and phosphatase inhibitors. Briefly, transfected cells were harvested and lyzed on ice. Lysates were centrifuged at 16,000 g (15 min at 4°C) and precleared (30 min at 4°C) using protein A-Sepharose beads (Sigma). Cleared lysates were incubated for 2-3 h at 4°C with protein A-Sepharose beads conjugated to mouse htt-4C8, mouse DIC or rat HA antibodies. Immunoprecipitates were washed three times and analyzed by Western blot as described.
After methanol fixation cells were blocked for 1 h at RT with PBS-BSA 3% and incubated with primary antibodies for 1 h prior staining with Alexa Fluor secondary antibodies. Nuclei were stained with DAPI (Roche). The mounting medium was 0.1 g/ml Mowiol 4-88 (Calbiochem, Darmstadt, Germany) in 20% glycerol.
Images on fixed samples were acquired at RT with a Leica SP5 laser scanning confocal microscope equipped with a 63 × oil-immersion objective or with a Leica DM RXA microscope with a PL APO oil 63 × NA of 1.4 objective coupled to a piezzo and a Micromax RTE/CCD-1300-Y/HS camera controlled by Metamorph software (Molecular Devices, Sunnyvale, CA). Z-stack step was of 0.2 μm. All stacks were treated by automatic batch deconvolution using the PSF of the optical system, Meinel algorithm with parameters set at 7 iterations, 0.7 sigma and 4 frequencies.
Computer morphometric analysis of the Golgi apparatus
Images of fixed cells were acquired as described (see above, image aquisition). Only HeLa cells stably expressing GFP-mannosidase II and transfected with our pARIS-htt constructs were analyzed. Once deconvolved, images were analyzed with ImageJ software using 3D object counter plugin (; available at http://imagejdocu.tudor.lu/doku.php?id=plugin:analysis:3d_object_counter:start). The quantification was achieved tagging each identified object within the z-stacks (around 30 z-stacks per image), treating each Golgi particle as an individual object. Statistics about each object were calculated, volume as: number of voxels of the object × x calibration × y calibration × z calibration. The overall measurements were obtained from 3 independent experiments and analyzed to determine the mean volume per particle for each condition.
Mouse neuronal transfected cells were grown on glass coverslips and mounted in a Ludin's chamber. The microscope and the chamber were kept at 33°C. Live videomicroscopy was carried out using a Leica DM IRBE microscope and a PL APO oil 100 × objective with a numerical aperture of 1.40-0.70, coupled to a piezo device (PI) and recorded with Photometrics CoolSNAP HQ2 camera (Roper Scientific, Trenton, NJ) controlled by Metamorph software. Images were collected in stream set at 2 × 2 binning with an exposure time of 50-150 ms (frequency of 2 s) with a Z-step of 0.3 μm. Deconvolution was performed as described for fixed samples. All dynamic parameters of intracellular transport were obtained from three independent experiments with a total of about 1500-5000 measures from 18-39 independent cells. Dynamics were characterized by tracking positions of eGFP vesicles as a function of time with an especially developed plugin (available at http://rsb.info.nih.gov/ij/plugins/track/track.html) for Image J.