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Docking experiments

The docking analysis was performed with AutoDock4.2 software73 building the coordinates of the VPS29-VPS35 heterodimer74 (pdb-id 2r17). Each ligand was drawn with the program “Marvin Sketch v20.13.0” (Chemaxon packages; https://chemaxon.com/), converted in a 3D structure and then transformed in the pdbqt format with the addition of partial charges and rotatable bonds (http://mgltools.scripps.edu). A cubic docking grid centered around the side chain of Gln530 of VPS35 with 15 Å side length was used for explorations. We performed 40 independent, global-local Lamarkian genetic-algorithm runs for each tested compound, extracting the conformation, the binding energy and the calculated Kd of the best pose together with the number of poses clustered around such best conformation (Python Molecular Viewer http://mgltools.scripps.edu). Additionally, we utilized a sigmoidal rectification function shown in Supplementary Fig. 4a to weight the calculated Kd with recurrence of best docking poses and generate a normalized Kd, as explained in the main text.

Druggability parameters

pKa and clogP values were calculated using the “Calculator Plugins” module of the ChemAxon’s Marvin suite (https://chemaxon.com/marvin-archive). We used the default parameters without any correction.

Synthesis

General procedures: 1H-NMR spectra were recorded on a Bruker Avance 400 MHz instrument in CDCl3, CD3OD, or D2O as solvent at 400 MHz. 13C-NMR spectra were recorded in CDCl3, CD3OD, or D2O as solvent at 101 MHz. Coupling constants are expressed in Hertz and are rounded to the nearest 0.1 Hz. LC-MS data were collected with a Waters AcquityTM Ultra performance LC equipped with an Acquity UPLCTM HSS T3 column (2.1 × 50 mm, 1.8 µm) and a SQD detector. Purifications were carried out either by flash chromatography on silica gel (particle size 60 μm, 230–400 mesh), on Kieselgel, or by BiotageTM flash chromatography [Biotage columns Si-25-M (150 × 25 mm; silica gel (40–63 μm), flow rate 25 mL/min)], or by BiotageTM C18 reverse phase chromatography [Biotage column C18HS (150 × 25 mm; KP-C18-HS (35–70 μm), flow rate 25 mL/min)]. Some final compounds were purified by C18 reverse phase semi-preparative HPLC using a Waters X-Bridge column (19 mm × 15.0 cm, 5 μm). Melting points were determined with a Stuart Scientific SMP3 melting point apparatus. Solvents were distilled and dried according to standard procedures, and reactions requiring anhydrous conditions were performed under nitrogen or argon atmosphere.

Synthesis of bis-1,3-phenyl guanylhydrazone 2a

Compound 2a (280 mg, 0.88 mmoles, 88% yield, ≥95% purity, m.p. 200–202 °C, white solid) was prepared in a single step from commercially available isophthalic aldehyde and aminoguanidine dihydrochloride. Diode Array LC trace, TIC trace and ES scan for compound 2a are available in Supplementary Fig. 4e; 1H-NMR and 13C-NMR are provided in the Supplementary Fig. 4f and g, respectively.

Animals and treatments

Mice were maintained under pathogen-free conditions at San Raffaele Hospital mouse facility (Milan, Italy). All efforts were made to minimize animal suffering and to reduce the number of mice used in accordance with the European Communities Council Directive of 24 November 1986 (86/609/EEC). The Ethics Review Committee approved experimental protocols according guidelines from the Italian Ministry of Health and from the Institutional Animal Care and Use Committee of the San Raffaele Scientific Institute (protocol number 704/2015PR). Transgenic mutant SOD1 mice carrying the SOD1G93A allele (strain B6SJL-TgN[SOD1G93A]1GUR)75 were purchased from Jackson laboratories, while C57BL/6J mice, used for breeding and experimental purposes, were purchased from Charles River (Italy). Both transgenic and WT mice had food and water freely available in the home-cage. The holding room was on a 12-h light–dark cycle, the temperature of the room was 22 ± 0.2 °C. Starting from day 30 we intraperitoneally injected compound 2a (10 mg/Kg) or vehicle in G93A mice as well as in C57BL/6J mice for 70 days. Additional experiments were performed injecting mice from day 83 to day 103. An operator blind to the treatment performed injections and manipulation of mice. At the day of the sacrifice, mice received an overdose of anesthetic drugs and they were perfused via vascular system with saline followed by 70–80 ml 4% paraformaldehyde (PF) in PBS, pH 7.2 (Sigma–Aldrich). Spinal cords were coronally sliced at ~6-mm thickness and postfixed in 4% PF in PBS, pH 7.2 for 12 h at +4 °C. Tissues were cryoprotected in PBS 1X/30% Sucrose (Sigma–Aldrich), embedded in OCT inclusion media and stored at −80 °C before processing. Lumbar spinal cords were 12-µm sectioned, labelled and digital images were acquired every 330 µm in a region encompassing 2.4 mm of the lumbar spinal cord. We determined numbers of mice by preliminary results or literature precedent76.

Motor function

Motor activity of mice receiving compound 2a or vehicle was assessed on 50-, 60-, 70-, 80-, 90-, and 100-days-old animals. Briefly, mice were trained 1 min on a static rotor and 1 min at constant speed (4 rpm) for two times and then we ran two trials performed over two consecutive days (one per day). Each trial consisted of 6-test sessions with 15 min interval between sessions. For each session, we placed mice on an accelerating rotor (4–40 rpm) and the latency to fall was recorded, with a maximum limit for individual animal set at 600 seconds.

Mass spectrometry quantitative analysis of compound 2a

Quantification of compound 2a in CNS extracts of C57BL/6J mice was performed at ProMeFa facility (Proteomics and Metabolomics Facility) of San Raffaele Scientific Institute. Wild-type C57BL/6J males mice (P60) were intraperitoneally injected with compound 2a (10 mg/kg) or vehicle, daily. We sacrificed mice at the following time points: 1, 7, 15, and 30 days. Before brains collection, mice received saline perfusion. Brains were weighted (~350 mg each) and rapidly frozen at −80 °C. The extraction was performed with methanol and each biological sample was resuspended in 60 µl of LC/MS grade water and 5 µL were analyzed by LC-MS/MS using the UPLC 1290 (Agilent Technologies) coupled to the TripleTOF 5600+ mass spectrometer (SCIEX). The mass spectrometry analysis was carried out in positive mode, in the range of 50-500 m/z for both the TOF-MS scan and the product ion scan, with a selected product ion of 247.1. A Waters ACQUITY UPLC BEH HILIC column (2.1 × 10mm, 1.7 µm) was used for the chromatographic separation through a gradient of solvent A (acetonitrile + 0.1% formic acid) and solvent B (water + 0.1% formic acid) from 2% up to 29% B in 5 minutes with a flow rate of 600 µl/min. Calibration curve in brain was constructed by plotting the peak area versus corresponding quantities of compound 2a (0; 0.1; 0.26; 1 nmol, run in quadruplicates, R2 = 0.9443), using MultiQuant 2.1 software (SCIEX).

NMR spectroscopy

NMR spectra were acquired at 298K on a Bruker Avance NEO 700 MHz spectrometer equipped with a Z-gradient cryoprobe. The spectra were processed with the Bruker TOPSPIN 4.0.5 software packages. For NMR studies of compound 2a with VPS29-VPS35, 5 μl from a DMSO-d6 stock 20 mM solution were dissolved in 175 μl of buffer (50 mM KH2PO4, 50 mM Na2HPO4, pH 8.0, 150 mM NaCl, 2 mM DTT) and 20 μL of 2H2O. Saturation Transfer Difference spectra (1H spectral window = 16 ppm; relaxation delay = 3.0 s; number of points = 32K) were acquired with 1024 scans with on-resonance irradiation at −1.0 ppm for selective saturation of protein resonances, and off-resonance irradiation at 40 ppm for reference spectra. A train of 40 Gaussian shaped pulses of 50 ms with 1 ms delay between pulses were used, for a total saturation time of 2 s. 1D 1H spectra were recorded using a 1 s presaturation pulse with a B1 power of 200 Hz. WaterLOGSY experiments (WL) were acquired using the ePHOGSY sequence33. WL experiments (1H spectral window = 16 ppm; relaxation delay = 3.0 s; number of points = 16K, scan = 1024) employed a 20 ms selective Gaussian 180° pulse at the water signal frequency and a NOE mixing time of 1 s. Both for STD and WaterLOGSY, FIDs were multiplied by an exponential weighting (lb = 5 Hz) before Fourier transformation.

Cell lines, culture conditions, and treatments

Neuro2a cells (ATCC® CCL-131™) were seeded in Dulbecco’s modified Eagle’s media (DMEM, Gibco) supplemented with FBS 10% (Gibco), L-glutamine (Gibco) 2 mM and penicillin/streptomycin 1% (Gibco). Depending on the experimental setting, we used 6-well, 12-well or 96-well dishes. Neuro2a were grow at 37 °C in a humidified 5% CO2 atmosphere. All experiments were carried out at least in triplicate. Compounds and transfections were done on cells showing 60–70% of confluence. Compounds were usually kept on cells for 48 h; Bortezomib (BTZ) was used at 10 nM for 6 h to inhibit proteasome. Transient transfections were obtained using lipofectamin LTX reagent (Thermofisher) with the following plasmids: pCMV-LacZ; pcDNA3.1(+)SOD1G93A;77 pcCDNA3.1(+)VPS35; pCAAG-GFP according the manufacturer’s instructions.

VPS35 short interference: we used Lipofectamine LTX (Thermofisher) to transfect Neuro2a cells with the following plasmids: plKO short interference scramble; TRCN0000111556 (Sh56); TRCN0000111558 (Sh58); TRCN0000111559 (Sh59), (Mission, Sigma–Aldrich). Protein and total mRNA extracts were obtained according the experimental design.

Cell survival experiments were run on 96-well plates using Cell Counting Kit-8 (CCK8) survival kit (96992-Sigma–Aldrich) according the manufacturer’s recommendations. Compound 2a cytotoxicity was tested on Neuro2a cells receiving increasing amounts of compound 2a (1, 10, 50, 100, 200, 500, and 1000 µM) for 48 h. Data were acquired using an Epoch spectrophotometer equipped with the GEN5 (2.03.1) software (Agilent).

LDH cytotoxicity was run on 96-well plates using LDH-Glo™ Cytotoxicity Assay (Promega). Briefly, Neuro2a cells were transfected with plasmids encoding pCMV-LacZ; pcDNA3.1(+)SOD1G93A77 with or without VPS35 short interfering plasmids (Sh56) or compound 2a (10 µM). After 48 h, 5 µl of supernatants were 100-fold diluted in LDH storage buffer (Tris-HCl 200mM, 10% Glycerol, 1% BSA, Sigma–Aldrich) and used to determine LDH, according to manufacturer’s recommendations. Maximum LDH Release Control was obtained lysing untreated Neuro2a cells with 2 μL of 10% Triton X-100 (Sigma–Aldrich). In each experiment we calculated(% ,{mathrm{cytotoxicity}} = frac{{{mathrm{Exp}}.{mathrm{LDH}},{mathrm{release}} – {mathrm{medium}},{mathrm{background}}}}{{{mathrm{Max}},{mathrm{LDH}},{mathrm{release}},{mathrm{control}} – {mathrm{medium}},{mathrm{background}}}}), and we run parallel positive controls treating cells with 200 µM H2O2. Data were acquired using a Victor3 spectrophotometer equipped with Wallac 1420 software (PerkinElmer)

Cycloheximide (CHX) assay was done on Neuro2a cells seeded in 6-well plates and incubated with compound 2a (10 µM) or vehicle for 24 h. We next added CHX (Sigma–Aldrich) at 10 µg/mL and cells were collected at 0, 2, 4, and 8 h for the time-course assay of VPS35 levels by WB.

Generation of human iPSC lines and derived MNs

Skin biopsies from ALS patients and healthy volunteers were performed under local anesthesia, after informed consent at San Raffaele Hospital (BANCA INSPE/8-10-19 and MND Genotipo Fenotipo/16-5-19, approved by the Ospedale San Raffaele Etic Committee). Primary fibroblasts were grown in Dulbecco’s modified Eagle’s medium with Glutamax I (GIBCO). Human iPS cell lines were generated using non integrating Sendai virus, maintained in feeder-free conditions in mTeSR-1 (Stem Cell Technologies) on embryonic stem cell (HESC) Matrigel (Corning). MNs were generated following the protocol published by Du et al.78 with minimal modification. Briefly, when hiPSCs reached confluence, they have been detached using EDTA and plated 1:4 on Matrigel ES coated dishes in mTeSR-1 supplemented with ROCK inhibitor (StemMACS, Miltenyi Biotec). On the following day the medium was replaced with a chemically defined neural medium: DMEM/F12, Neurobasal medium at 1:1 ratio, 1% B27, 0.5% N2 (all from Gibco, ThermoFisher Scientific), 1% P/S (Gibco), 1% Glutamax (Gibco) and 0.1 mM Ascorbic Acid (Sigma–Aldrich). CHIR99021 (3 μM, Tocris), DMH1 (2 μM, Tocris) and SB431542 (2 μM, Miltenyi Biotec) were added to the medium. The culture medium was changed every other day, until day 6. On day 7, cells have been dissociated with Dispase (1U/mL), split 1:4 and kept on Matrigel growth factor reduced (MaGR) for 6 days in the basal neural medium described above, supplemented with 1 μM CHIR99021, 2 μM DMH1, 2 μM SB431542, 0.1 μM Retinoic Acid (RA, Sigma–Aldrich) and 0.5 μM Smoothened Agonist, (SAG, Calbiochem). On day 13, cells have been dissociated with Dispase (1U/mL), split 1:4, and kept for 6 days in neural medium supplemented with 3 μM CHIR99021, 2 μM DMH1, 2 μM SB431542, 0.1 μM RA, 0.5 μM SAG, and 0.5 mM Valproic Acid (VPA, Tocris). To induce MN differentiation, cells were dissociated with Dispase (1U/mL) and cultured in suspension for 6 days in neural medium with 0.5 μM RA and 0.1 μM SAG. On day 25, cells were dissociated into single cell with Accumax (Sigma–Aldrich) and plated on Matrigel Growth Factor Reduced (Thermofisher) coated plates, in neural medium with 0.5 μM RA, 0.1 μM SAG, 0.1 μM Compound E (Calbiochem) until day 35. Half of the medium has usually been changed every other day. The karyotyping analysis was performed by ISENET Biobanking service unit in Milan, Italy (www.isenetbiobanking.com).

Immunoblotting

Lumbar SCs were dissected after mice received saline perfusion and were rapidly homogenized in the following lysis buffer: Tris-HCl 10 mM pH 8, EDTA pH 8 1 mM, NaCl 100 mM, NP40 1% and protease inhibitor cocktail (Sigma–Aldrich). Protein extracts were obtained using a tight-fitting glass Potter tissue grinder (1 mL; Wheaton) and then sonicated at a frequency of 20 kHz (10 times-1s). Protein lysates from cells cultures were collected from plates receiving PBS 1× washing and subsequently the lysis buffer followed by sonication. Protein concentrations were measured by BCA protein assay kit (Thermofisher) according to the manufacturer’s recommendations. Ten µg of protein extract was loaded on 10% Mini-PROTEAN TGX Stain-Free precast gels for PAGE (Bio-Rad). Gel electrophoresis was performed at 100 V for 2 h. Gels were transferred on Immobilon®-P PVDF membrane according to the manufacturer’s instructions (Millipore). Blots were blocked in 5% BSA in Tris buffered saline plus 0.1% Tween-20 (TBS-T) for 1 h before receiving primary antibodies: goat α-VPS35 1:800 (ab10099, Abcam); rabbit α-VPS26 1:1000 (ab23892 Abcam), rabbit α-VPS26b 1:1000 (15915-1-AP, ProteinTech); rabbit α-VPS29 1:1000 (ab236796 Abcam); mouse α-βActin 1:25000 (Sigma–Aldrich); mouse α-Golgin97 1:1000 (Invitrogen); mouse α-Ubiquitin 1:500 (Merk); rabbit α-CD14 1:1000 (Bioss Inc); mouse α-Tubulin 1:5000 (Immunological Sciences MAB-80143) overnight at 4 °C on shaker. Following several washes with TBS-T, blots were incubated in appropriate HRP-labeled secondary antibodies (Bio-Rad) for 1 h at room temperature. Visualization of protein bands was obtained using ClarityMax-ECL (Bio-Rad) according to the manufacturer’s instructions and images were acquired on ChemiDoc (Bio-Rad). Quantifications were done using the Image Lab 6.0.1 software (Bio-Rad).

Blue-Native gel electrophoresis

G93A mice and their WT litters were injected daily with vehicle or compound 2a (10 mg/kg, from day 83 to day 103). Lumbar spinal cords dissected from saline-perfused mice were lysed in Tris-HCl 10 mM pH 8, EDTA 1 mM pH 8, NaCl 100 mM, NP40 1% and protease inhibitor cocktail (Sigma–Aldrich). Extracts were obtained using a tight-fitting glass Potter tissue grinder (1 mL; Wheaton). We incubated 250 µL of each extract with 100 mM Iodoacetamide (Sigma–Aldrich). Samples were mixed with 4× Sample NativePAGE (Thermofisher) sample buffer and the G-250 additive (Thermofisher) and proteins were resolved on native 4–15% Bis-Tris gel (Bio-Rad) with Dark Blue Cathode buffer (Thermofisher) at 150 V for 1/3 of gel length. We replaced the running buffer with Light Blue Cathode Buffer to complete the run. At the end of the electrophoresis, gels were washed with 5 mM tris(2-carboxyethyl)phosphine (Sigma–Aldrich) for 15 min and then proteins were transferred to nitrocellulose and fixed on the membrane by 8% acetic acid for 15 min. Rabbit α-SOD1 (1:2000, Genetex) was incubated overnight as above described. Visualization of proteins was performed using ClarityMax-ECL (Bio-Rad) according to the manufacturer’s instructions and images were acquired on ChemiDoc (Bio-Rad).

Immunohistochemistry, immunofluorescence, and morphometric analyses

Paraffin embedded SCs were obtained from ALS patients and non-neurological controls from the Target ALS Human Postmortem Tissue Core. Sections were stored at the INSPE tissue Bank (San Raffaele Scientific Institute). Sections were incubated with a polyclonal VPS35 antibody (1:400 Abcam ab97545) and with the polyclonal VPS26 antibody (1:200, Abcam ab23892) after rehydration and antigen retrieval (slides were cooled at 97 °C in TRIS EDTA pH = 9 by water bath). Peroxidase-diaminobenzidine (DAB) reaction was used for detecting primary antibody, biotinylated anti-rabbit IgG (H+L) (1:500 Vector laboratories) and avidin-biotin complex (ABC 1:100 Vector laboratories). An operator that was blind to the disease status and demographic data subsequently evaluated these sections.

Sections from mice or glass covers containing cells were washed three times in PBS 1× (Lonza, 5 min each), and incubated in the following blocking mix: PBS 1×/FBS 10% (Invitrogen), BSA 1 mg/mL (Sigma–Aldrich), TritonX100 0.1% (Sigma Aldrich), for 1 h at room temperature. Antibodies were diluted in blocking mix and incubated at +4 °C overnight according manufacturer’s instructions. The following day, sections were rinsed in PBS and fluorescent secondary antibodies—i.e. never deriving from the species from which the primary antibodies are derived- (Alexafluor conjugated) diluted in blocking mix, were used according to the manufacturer’s instructions. Slides were incubated in Hoechst 33342 (Sigma–Aldrich) for nuclei counterstaining. When necessary, we performed antigens retrieval on tissue sections by boiling samples in 10 mM sodium citrate (pH 6) for 5 min. The following antibodies and working concentrations were used: rabbit α-Iba1 1:500 (Wako); rabbit α-VPS35 1:1000 (ab97545 Abcam); goat α-VPS35 1:800 (ab10099 Abcam); rabbit α-VPS26 1:1000 (ab23892 Abcam); rabbit α-Sortilin 1:1000 (ab16640 Abcam); mouse α-CI-MPR 1:100 (Novus bio. NB-300-514), mouse α-NeuN 1:800 (Millipore MAB377); mouse α-Ubiquitin 1:500 (Millipore MAB1510); mouse α-Golgin97 1:700 (Thermofisher A21270); rabbit α-ISLET1:1000 (ab20670 Abcam); goat α-ChAT 1:200 (Millipore ab144p); chicken α-GFP 1:1000 (Invitrogen); α-SOX2 1:100 (R&D, MAB2018); goat α-NANOG 1:200 (R&D, AF1997); mouse α-OCT3-4 1:500 (Santa Cruz, sc-5279); mouse α-SSEA4 1:400 (Millipore MAB4304); mouse α-TRA1-60 1:200 (Millpore MAB4360); chicken α-Neurofilament-medium 1:500 (Biolegend PCK-593P); rat α-MBP 1:100 (kindly provided by Dr. A. Bolino); mouse α-GM130 1:100 (BD biosciences, 610823); rat α-CTSD 1:100 (R&D system MAB1029). Tyramide Signal Amplification (TSA, PerkinElmer) was used, when appropriate, to improve fluorescent intensity in single and double immunofluorescence.

Quantification of nerves degeneration was done on sciatic nerves obtained from saline-perfused mice. Tissues were fixed in 2% buffered glutaraldehyde (Sigma–Aldrich), postfixed in 1% osmium tetroxide and cut to obtain 1 µm thick sections. We stained sections with toluidine blue and specimens were blindly examined by light microscopy using ×40 objective. For each nerve, we merged images to obtain the entire nerve cross section. Five consecutive nonoverlapping semi-thin sections from each sample were analyzed with NHI-Image J software (US National Institutes of Health). Degenerating fibers were counted in the entire nerve cross section.

Quantification of myelin was done on sagittal sciatic nerves by Luxol fast blue (LFB). Briefly, nerve sections were incubated the ethanol series before receiving 0.1% LFB overnight at 60 °C. Sections were then incubated 5 min in 0.05% lithium carbonate before receiving the ethanol series and xylene. Bright field microcopy of nerves at ×63 was used for quantification. Positive myelin staining is expressed as a percentage of the total area examined.

Imaging

We used Olympus, BX51 with the following objectives: ×20, ×40, and ×63 equipped with the following cameras: Leica CCD Microscope DFC3000 G and DMC2900 to obtain 16-bit light and fluorescence images (1296×966 pixels). Fluorescence images were merged using Photoshop (Adobe) CS4 or NHI-Image J software (US National Institutes of Health). We obtained confocal images using Leica SP8 equipped with ×40 and ×64 objectives and super-sensitive HyD detectors. We recorded each fluorescence signal as square 8-bit images (1024 × 1024 pixels). Images were postprocessed to generate maximal projections of Z-stacks (acquired with a 0.4–0.8 µm step) and cross-sectional profiles of the Z-stack (acquired with a 0.3 µm step) and pseudo-colored using Leica Application Suite X (2.0.0.14332).

Quantification of MFI in tissue MNs

Fluorescence intensity levels of VPS35, VPS26, CI-MPR, Sortilin, CTSD and Ubiquitin were calculated using NHI-Image J software (US National Institutes of Health) according to the following protocol: stacks of confocal images were used to generate the maximal projections using the Leica Application Suite X (2.0.0.14332) software and then postprocessed by NHI-Image J to crop single NeuN+ MNs from the ventral horn of the spinal cord. We next performed background (calculated on adjacent slices only receiving secondary antibodies) subtraction for each channel, before applying the NHI-Image J mean filter (with radius 2.0 pixels) to calculate mean NeuN fluorescence levels in individual MNs. Using this approach, we established a region of interest (ROI) to gate single MNs. On this ROI, we measured mean fluoresce levels (MFI) of target proteins.

Morphological assessment of Golgi apparatus in Neuro2a

Golgi morphology was assessed in control or in Neuro2a cells receiving G93A plasmids with or without compound 2a (10 µM) as well as in GFP-transfected cells as additional control. We used Golgin97 immunofluorescence to label trans-Golgi network membranes. We classified Golgi morphology as follow: normal (polarized network), intermediate (partially fragmented) or fragmented (severely fragmented with Golgi stacks dispersed in the cytoplasm throughout). In each experiment, cells were randomly sampled across three coverslips. Golgi subclasses were expressed as a percent of the total number of Golgi scored for each experimental condition.

Micro electrode array (MEA) recordings

Primary neuronal cultures were established from the cerebral cortex of E16.5 C57BL6J embryos. Upon dissection, cells were plated on MEA biochips (60 electrode MEA biochips with 200 µm electrode spacing and 30 µm electrodes diameter with an integrated reference electrode, Multichannel Systems GmbH) at the density of 3 × 105 cells/MEA and kept in Neurobasal medium supplemented with B27 for 14 days. At day 14 neuronal cultures were exposed to increasing concentration of compound 2a (1, 3, 10, 30, and 100 µM) in Krebs–Ringer–Hepes buffer (KRH, 130 mM NaCl, 5 mM KCl, 1.2 mM KH2PO4, 1.2 mM MgSO4, 2 mM CaCl2, 25 mM Hepes, 0.6% glucose, 1% glutamine, Sigma–Aldrich). Firing activity was recorded using a pre-amplifier stage (MEA-1060-Inv-BC-Standard, gain: 55, bandwidth: 300 Hz–8 kHz, MCS GmbH), an amplification and filtering stage (FA64S, gain 20, bandwidth: 10 Hz–8 kHz, MCS GmbH) and a data acquisition device (sampling frequency: 25 kHz). Then, off-line signal processing was performed, and raw data were analyzed by MC-Rack Software version 4.6.2 (MCS GmbH).

Real-time PCR

Lumbar spinal tracts dissected from saline-perfused mice or Neuro2A cells were lysed to extract total RNAs using the RNeasy Mini Kit (Qiagen) according to the manufacturer’s recommendations, including DNase (Promega) digestion. We synthetized the cDNA using ThermoScript RT-PCR System (Invitrogen) and Random Hexamer (Invitrogen), according to the manufacturer’s instructions. The LightCycler 480 System (Roche) and LightCycler 480 SYBR Green I Master Mix (Roche) were used for real-time PCR experiments. Normalization was obtained using the housekeeping gene Histone H3:

F: 5-GGTGAAGAAACCTCATCGTTACAGGCCTGGTAC-3

R: 5-CTGCAAAGCACCAATAGCTGCACTCTGGAAGC-3

The following specific primers were used for gene expression analysis: VPS35F: 5-GTGCCGTGGTGTGCAGCATCCG-3′VPS35R: 5-ATGCTGCATCCGCACCCAGAGC-3′VPS26A F: 5-GCTAAGTATGAAATAATGGATGGGGC-3′VPS26A R: 5-CTTGTTCACATCTCTCATCGTGGGG-3′VPS29F: 5-CCAGCACATCCTCTGCACCGGC-3′VPS29R: 5-CCACGGAATAACTTGGTGTCCGTG-3′.

Statistics

We express data as the mean value (±standard error of the mean (SEM) or ±standard deviation (SD)) of independent experiments. We assessed normality of data applying either Kolmogorov–Smirnov test (with Dallas–Wilkinson–Lille for P-value) or D’Agostino & Pearson omnibus normality test. Comparisons were made using the following tests: unpaired t-test, one-way or two-way analysis of variance (ANOVA) tests, followed by Tukey’s multiple Comparison test or by Bonferroni multiple comparison test. Statistical analyses were performed using PRISM5.01, PRISM8.4.2 (GraphPad Software, La Jolla, CA, USA) or BioVinci 1.1.5 (BioTuring Inc.). Values lower than 0.05 were considered statistically significant.

Reporting summary

Further information on research design is available in the Nature Research Reporting Summary linked to this article.



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