Antibodies used in this study were purchased as follows: anti-actin, anti-phospho-Drp1 Ser637 (Cell Signaling, Danvers, MA), anti-amyloid precursor protein, anti-glucose-regulated protein of 94 kDA (GRP94), anti-α tubulin, anti-receptor-interacting protein kinase (RIPK) (EMD-Millipore, Billerica, MA), anti-immunoglobulin-binding protein/glucose-regulated protein of 78 kDA (BiP/GRP78) (BD Biosciences, Franklin Lakes, NJ), anti-BiP/GRP78, anti-Drp1 (abcam, Cambridge, UK), anti-CD68 (Dako/Agilent, Markham, ON), anti-CCAAT/enhancer-binding protein (C/EBP) homologous protein (CHOP), anti-GRP75 (Pierce/Thermo, Waltham, MA), anti-CHOP (Enzo, Farmingdale, NY), anti-phosphofurin acidic cluster sorting protein 2 (PACS-2) (Protein Tech, Chicago, IL), anti-Rab32 (Sigma/Aldrich, St. Louis, MO), and anti-FLAG (Rockland, Limerick, PA). The antibody against calnexin has been described previously .
Isolation and maintenance of primary neuronal cultures
Cultures of human fetal neurons (HFN) were generated from 15–19-week fetal brains (obtained with consent from the University of Alberta Ethics Committee) as described .
Human frozen brain tissues and EAE mice tissue
For immunohistochemistry, snap-frozen blocks of post-mortem normal control (NC) or MS cerebral sub-ventricular deep white matter samples were obtained from the NeuroResource Tissue Bank, UCL Institute of Neurology, London, and UK MS Biobank, with next-of-kin informed consent for tissue donation and ethical approval from Central London REC1 (I.D.08/H0718/62) and approval for the study from the Local Research Ethics Committee (I.D.04/Q2102/111), UK MS brain bank charity number 1139257. A total of 12 MS patients (9 females, 3 males) who had been affected from secondary progressive (10), primary progressive (1) or relapsing progressive MS were used for this part of the study. Control tissue from individuals who had not been affected by disease (7) and 2 individuals who had been affected by Parkinson’s disease was also examined. Control patients died of non-inflammatory diseases (cardiac failure, lung cancer, bladder cancer, prostate cancer, tongue cancer, myelodysplastic syndrome; for two control cases, the cause of death was not known). Further information is contained in Additional file 1.
For Western blot and immunofluorescence analysis, tissues of two frozen MS brains (patient 1: secondary progressive MS, aged 54, male; patient 2: relapsing-remitting MS, aged 45, male) were obtained from the MS Tissue Bank at the University of Alberta. Post-mortem brain tissues were collected and processed as described . Frozen brain and spinal cord tissues of triplicate experimental autoimmune encephalomyelitis (EAE) mice, an animal model of MS, were generated with proper approvals as described . Control samples showed no signs of nervous disease. Disease peak samples were from clinical grade 1, whereas post-peak samples were from clinical grade 4 (hind limb paralysis at time of dissection).
Lysate preparation and analysis from tissues and cell lines
Tissue lysates were prepared from the human frozen brain as well as from the spinal cords of EAE mice in 1× sodium dodecyl sulphate (SDS) extraction buffer (0.125 M Tris-HCK pH 6.8, 2% SDS, 10% glycerol, 5% β-ME), followed by sonication on a 550 Sonic Dismembrator (Fisher Scientific, Ottawa, ON). Supernatants were collected, and protein concentrations were measured by NanoDrop Spectrophotometer ND1000 (Thermo/Life Technologies) at an absorbance of 280 nm. Cellular lysates from SH-SY5Y cells were prepared as described .
SH-SY5Y cells were cultured in mild hypoxia (4% O2, grown in the presence of 4% O2, 5% CO2 balanced N2, as is typical for brain tissue) in the presence of thapsigargin. After 24 h in culture, total RNA was extracted. The primers used for RT-PCR were as follows: Rab32 forward AGCAGGACTCTGGTGCGCCTG (position 211-231); Rab32 reverse CGGGCAGCTTCCTCTATGTTTATGTTATC (position 557-529). The result was normalized against the ribosomal 18S.
Sample sections were stained with hematoxylin and eosin (H&E) and luxol fast blue (LFB) as described . Lesions were classified into acute (referring to tissue phenotype, see below), sub-acute, and chronic on the basis of the number and distribution of oil red-O-positive macrophages, the extent of demyelination, cellularity in the borders and parenchyma of lesions, and perivascular cuffing as described in our previous work . Briefly, acute lesions were identified via demyelination, invading macrophages, hypercellularity at the lesion border, and cuffing around the blood vessels. Sub-acute lesions showed a demyelinated plaque with fewer macrophages, mostly at the lesion border, and less perivascular cuffing. A chronic lesion consisted of a hypocellular demyelinated plaque completely lacking of oil red-O-stained macrophages. Examination of MS brain tissue for Rab32 expression in specific cell types was performed employing enzyme immunohistochemistry using a Vectastain ABC system® (Vector Laboratories, Peterborough, UK), as described .
Transfection of constructs and shRNA, immunofluorescence, and quantification of apoptosis
mCherry-labeled Rab32 shRNA psi-mH1 plasmids (HSH001118) as well as scrambled control (CSHCTR001) were purchased from Genecopoeia (Rockland MD). FLAG-tagged Rab32 constructs were expressed from pcDNA3 as published  (wt, wild type; Q85L, dominant-active; T39N, dominant-negative) or transferred into the bi-cistronic pIRES2-EGFP plasmid (Clontech-Takara, Mountain View, CA) that allows for the expression of any protein, in parallel with nuclear EGFP. To do so, the described constructs contained in pcDNA3 were PCR-amplified using the SP6 and TS484 (ATATGCTAGCACCATGGACTACAAGGACGACGATGACAAG) oligos following cuts with the 5’ Nhe1 and 3’ Xho1 sites. Primary neurons or SH-SY5Y neuronal cell lines were transfected by nucleofection (Lonza, Mississauga, ON). Immunofluorescence was performed as described . To assay neurotoxicity, nuclear EGFP was used to identify transfected HFNs and SH-SY5Y. Apoptosis was then detected by Cy5-annexin V binding (BD Biosciences). Assays were repeated in the presence of bafilomycin (100 nM, Sigma-Aldrich), necrostatin-1 (Nec-1, 50 μM, Cayman Chemical), carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]- fluoromethylketone (zVAD-fmk) (10 μM, Enzo Life Sciences, Farmingdale, NY), or with a combination of nec-1 and zVAD-fmk.
Cells were rinsed in PBS and fixed in 3% paraformaldehyde and 0.05% glutaraldehyde (GA) containing 2% sucrose. Next, free aldehyde groups were quenched with ammonium chloride (50 mM), and samples were permeabilized with saponin (0.1%). The samples were blocked (PBS + 1% BSA + 0.05% FSG + Saponin 0.05%) for an hour and then were incubated with mouse anti-FLAG in the blocking buffer overnight in a wet chamber. Following washes (0.2%BSA + 0.05%FSG + 0.05% saponin), the samples were incubated with the secondary antibody (Fluoronanogold Anti-mouse Fab’Alexa Fluor 488, cat. 7202; Nanoprobes, NY) for 3 h at RT and washed with PBS three times. The samples then were fixed (2% GA in PBS + 2% sucrose) for an hour, followed by three rinses in water. Following a 1-min incubation with GoldEnhance EM Plus (Cat. 2114; Nanoprobes, NY), the samples were rinsed in water, scraped in 100 mM sodium cacodylate and pelleted. The pellet was incubated for 1 h with osmium tetroxide (1%), followed by overnight staining with uranyl acetate. After dehydration in increasing concentrations of ethanol and then propylene oxide treatments, the pellets were transferred to resin (Embed 812 kit, cat. 14120; Electron Microscopy Sciences, Hatfield, PA) and incubated at 60 °C for 48 h. Blocks were sectioned (70 nm) using Ultracut E (Reichert Jung) and imaged with a Philips 310 electron microscope, equipped with a digital camera (Mega View III Soft Imaging System, Emsis Gmbh, Muenster, Germany).