Purification of Neurofilaments and proteomics
Briefly, 10 spinal cords from 6 months-old Sacs+/+ or Sacs−/− male mice were pulled to purify endogenous NF proteins: NFH, NFM and NFL [23]. The spinal cords were homogenized in 5ml of buffer A (0.1 M NaCl, 1 mM EDTA, 10 mM NaH2PO4, pH:6.8) and centrifuged (30 min, 10,000g, 4°C). The supernatant was discarded and the pellet was re-suspended in ice-cold buffer A-TPS (0.1 M NaCl, 1 mM EDTA, 10 mM NaH2PO4, 1% (v/v) Triton X-100, 0.85 M sucrose, protease inhibitors cocktail (Sigma Aldrich), pH: 6.8) and then centrifuged (1 hr, 10,000g, 4°C). Crude NFs were solubilised in buffer 1 (8M urea, deionized and filtered, 10mM sodium phosphate buffer, 0.1% (v/v) 2-mercaptoethanol, protease inhibitors cocktail (Sigma Aldrich), pH 7.4), centrifuged 30 min at 10,000g, 4°C. An affinity chromatography column of Bio-Gel-HTP rehydrated with 10mM NaH₂PO₄, pH 7.4 was prepared and then equilibrated with buffer1. Crude NF proteins were then applied to the affinity chromatography at room temperature and washed with buffer1. Non-NFs proteins were eluted with buffer2 (8M deionized and filtered urea, 100mM sodium phosphate buffer, 0.1% (v/v) 2-mercaptoethanol, protease inhibitors cocktail (Sigma Aldrich), pH 7.0). Then purified NF proteins were eluted with buffer4 (8M deionized and filtered urea, 300mM sodium phosphate buffer, 0.1% (v/v) 2-mercaptoethanol protease inhibitors cocktail (Sigma Aldrich). Eluted purified NFs protein were then analysed using SDS-PAGE and stained with Coomassie blue. In gel digestion with trypsin of NFL, NFM and NFH bands and proteomics analysis using mass spectrometry LC-MS was carried out by the Proteomics and Molecular Analysis Platform of the MUHC (McGill University) and results were analysed using scaffold software 4 to assess the purity of NFL, NFM and NFH. Only peptides carrying post-translational modifications identified with a ≥ 95% probability were considered.
Cell Culture
Sacs −/− mice were described previously[13] and breeding and animal procedures were carried out with the approval of the McGill Animal Care Committee in accordance with the Canadian Council on Animal Care guidelines. Immortalized Human skin fibroblasts control MCH74 and from ARSACS patient expressing the homozygous ARSACS-causing p.2801delQ variant (ARSACS7373) were previously described [30, 31]. The study was approved by McGill University Ethics Research Board. Immortalized fibroblasts were cultured in DMEM with 5–10% fetal bovine serum. Primary cultures of dissociated spinal cord-dorsal root ganglia were prepared from C57Bl6 E13 Sacs−/− mice and wild-type (Sacs+/+). Cells were maintained in EMEM as previously described [32]. Cultures were used 6 weeks following plating to allow neuronal maturation and appearance of NF bundles in more than 80% of Sacs−/− motor neurons.
Biochemical and Cellular analysis
Protein samples were analyzed with SDS-PAGE, and electrotransfered to nitrocellulose membranes (Merck Millipore). The following primary antibodies were used for the immunoblotting; monoclonal anti-NFL antibody (clone NR4), monoclonal anti-NFH antibody (clone N52), monoclonal anti-NFM (clone NN18), GAPDH (MediMabs, MM-0163-P), anti-P62 (Proteintech, 18420-1-AP), anti-LC3 (Proteintech, 14600-1-AP) and anti-acetyl-lysine (cell signaling 9441L). Horse radish peroxidase conjugated secondary antibodies were from Jackson ImmunoResearch and Images were the Intas Imaging System (Intas GmbH) and densitometry of bands was measured using NIH ImageJ software.
Indirect immunofluorescence was carried out after fixation with 4% Paraformaldehyde using anti- LAMP1(Proteintech cat# 67300-1-Ig), anti-LC3 (Proteintech, 14600-1-AP). Cy2 and Cy3 conjugated secondary antibodies were from Jackson ImmunoResearch. Images were captured using an Zeiss Observer.Z1 microscope with the ZenBlue 2.6 Software (Carl Zeiss Canada Ltd.).
Quantitation of the percentage of motor neurons carrying NF bundling was done as described in Gentil et al [31]. NF bundles are defined by a continuous filament labeled by indirect immunofluorescence with anti-NFL and crossing the cell body from dendrite to dendrite[31]. After image capture using ZenBlue2.6 software, colocalization measurements were performed, and the Pearson Correlation Coefficient, a reliable and simple measure for evaluating fluorescent probe overlap and enabling further statistical analysis, was obtained through the colocalization tool.
Statistics
If not otherwise specified, all experiments were replicated independently three times form different cell batch when cell culture is involved. Data are given as average values ± SD. Statistical significance was calculated using one-way ANOVA with HSD test as a posthoc analysis or Two-tailed Student’s t-test as indicated in legend (* p < 0.05, ** p < 0.01).