Cells and reagents
Dermal fibroblasts derived from two healthy individuals (control fibroblasts) and from three CMT2B patients belonging to the same Italian family carrying the RAB7V162M mutation were obtained as previously described . After informed consent for pathological diagnosis, samples were anonymously encoded to protect patient confidentiality and used under protocols approved by the Azienda Ospedaliera Universitaria "Federico II" Ethics Committee (Ethical Committee Approval Protocol # 107/05). Fibroblasts were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 15% fetal bovine serum (FBS), 2 mM l-glutamine, 100 U/ml penicillin and 10 mg/ml streptomycin in a 5% CO2 incubator at 37 °C. Chemicals and tissue culture reagents were from Sigma-Aldrich (St-Louis, MO, USA). HeLa cells were maintained in DMEM supplemented with 10% FBS, 2 mM l-glutamine, 100 U/ml penicillin and 10 mg/ml streptomycin in a 5% CO2 incubator at 37 °C.
Primary antibodies used in this study were: mouse monoclonal anti-RAB7 (1:500, sc-376362), anti-RAB9 (1:200, sc-74482), anti-β-actin (1:1000, sc-47778), anti-RACGAP1 (1:200, sc-271110), anti-OCT3/4 (1:200; sc-5279) and anti EAP30 (1:200, sc-100892), rabbit polyclonal anti-RAB4 (1:200, sc-312), anti-RAB5 (1:200, sc-309), anti-ERK1 (1:200, sc-93), anti-HA (1:100, sc-805) and anti-pAKT (1:200, sc-7985-R), goat polyclonal anti-cathepsin D (1:500, sc-6486) from Santa Cruz Biotechnology, (Dallas, TX, USA); mouse monoclonal anti-tubulin (1:10,000, T5168) and anti-Smooth Muscle Actin (1:200; A2547) from Sigma-Aldrich (St. Louis, MO, USA); mouse monoclonal anti-cytokeratin CK8/18 (1:400; NCL-L-5D3) from Leica (Wetzlar, Germany); mouse monoclonal anti-LAMP2 (1:5000, H4B4), developed by J.T. August and J.E.K. Hildreth and obtained from the Developmental Studies Hybridoma Bank, (Iowa City, IA 52242); rabbit polyclonal anti-CI-MPR (1:2000, ab32815), anti-TSG101 (1:500, ab30871) anti-LAMP1 (1:4000, for immunoblot analysis and 1:1000 for immunofluorescence analysis, ab24170) and goat polyclonal anti-CGRP (1:200; ab36001) from Abcam (Cambridge, UK); rabbit polyclonal anti-peripherin (1:100, AB1530) and anti-Brn3a (1:100; AB5945) from Merck Millipore (Burlington, MS, USA); sheep polyclonal anti-TGN46 (1:500, AHP500) from Bio-Rad (Hercules, CA, USA); sheep polyclonal anti EGFR (1:1000, 20-ES04) from Fitzgerald (North Acton, MS, USA); rabbit monoclonal anti-AKT (1:1000, 4691) and phospho-p44/42 (Erk 1/2) (1:2000, 4370) from Cell Signaling Technology (Leiden, The Netherlands); mouse monoclonal anti-RAC1 (1:600, ARC03) from Cytoskeleton (Denver, CO, USA); rabbit polyclonal anti-Nanog (1:100; RCAB0002P-F) from ReproCELL (Glasgow, UK); rabbit polyclonal anti-Pax-6 (1:100; PRB-278P) from Covance (Princeton, NJ, USA); chicken polyclonal anti-Neurofilament M (1:1000; 822701) from BioLegend (San Diego, CA, USA); rabbit polyclonal anti-ARHGEF6 (1:1000; A302-558A) from Bethyl Laboratories (Montgomery, TX, USA). Secondary antibodies conjugated to fluorochromes (used at 1:600 dilution) or HRP (used at 1:5000 dilution) were from Invitrogen (Carlsbad, CA, USA), Fitzgerald, SouthernBiotech (Birmingham, AL, USA) or Jackson ImmunoResearch (Cambridgeshire, UK).
Plasmids encoding HA-tagged RAB7WT and the CMT2B-causing RAB7V162M mutant protein have been previously described . The pcDNA3-HA plasmid was constructed by inserting a DNA sequence coding for a 2xHA-tag into the KpnI restriction site of the pCDNA3.1 (Invitrogen, V79020) and it was used as empty vector in control transfections.
Induced pluripotent stem cells generation
iPSCs were generated reprogramming fibroblasts from healthy controls and CMT2B patients carrying the RAB7V162M mutation by Sendai viral transduction of the transcription factors OCT4, SOX2, KLF4, and c-MYC (CytoTune2.0-iPS Sendai Reprogramming Kit, Thermo Fisher, Waltham, MS, USA). iPSCs were then tested for Sendai virus clearance (CytoTune2.0-iPS Sendai Reprogramming Kit, Thermo Fisher) and pluripotency (Trilineage differentiation Kit, Stem Cell Technologies, Vancouver, Canada). Cells were regularly screened and confirmed negative for mycoplasma during both maintenance and differentiation (MycoAlert kit, Lonza, Basel, Switzerland).
Induced pluripotent stem cells maintenance and differentiation in dorsal root ganglia (DRG) sensory neurons
iPSCs were maintained in feeder-free conditions using mTeSR1 medium (Stem Cell Technologies) on hES qualified Matrigel (Corning, Corning, NY, USA) coated plates. At 80–90% of confluency, iPSCs were passaged using 0.5 mM EDTA in PBS (Sigma-Aldrich) or ReLeSR (Stem Cell Technologies), in the presence of ROCK inhibitor (Stem Cell Technologies). Prior to differentiation, iPSCs were single cell dissociated with Accumax (Sigma-Aldrich) and plated onto hES qualified Matrigel coated six-well plates in the presence of ROCK inhibitor. Cells were grown in mTeSR1 medium for 24 h, then shifted to MEF-CM (Mouse embryo fibroblasts-conditioned medium) produced according to Lee et al. , supplemented with 10 ng/ml bFGF (Thermo Fisher). At approximately 40–50% of confluency (usually 24/48 h after the shift to CM medium), differentiation was started [45, 46]. The medium was replaced with KSR medium high glucose DMEM (Thermo Fisher) supplemented with 15% KSR (KO serum replacement, Thermo Fisher), 1 × l-glutamine (Thermo Fisher), 1 × Pen/Strep (Thermo Fisher), 1 × sodium pyruvate (Thermo Fisher), non-essential amino acids (Thermo Fisher), and 100 µM 2-mercaptoethanol (Thermo Fisher). Smad inhibitors 10 µM SB431542 (Tocris, Bristol, UK) + 0.1 µM LDN193189 (Stemgent, Cambridge, MS, USA) were added to the medium from day #0 to day #5 (see Supplemental Table 1). Three more small molecules 3 µM CHIR99021 (Tocris), 10 µM DAPT 10 µM (Tocris), and 10 µM SU5402 (Tocris) were added from day #2 to day #12. If overconfluent, cultures were split on day #2/day #3 to guarantee optimal differentiation . From day #5 to day #12, the KSR medium was gradually transitioned to N2B27 medium (Neurobasal, Thermo Fisher) and added with 1% N2 supplement (Thermo Fisher), 1% B27 supplement (Thermo Fisher), 1 × glutamine (Thermo Fisher), and 1 × pen strep (Thermo Fisher). On day #11, young neurons were detached with TrypLE Express (Thermo Fisher) and replated on GFR (growth factor reduced) Matrigel (Corning)-coated coverslips. On day #13, the cultures were shifted to neural differentiation medium (Neurobasal, Thermo Fisher), supplemented with 1% N2 supplement (Thermo Fisher), 1% B27 (Thermo Fisher), 1% Pen-Strep (Thermo Fisher), 1% l-glutamine (Thermo Fisher), 25 ng/ml GDNF (Peprotech, London, UK), 25 ng/ml BDNF (Peprotech), 25 ng/ml NGF (Peprotech), 25 ng/ml NT3 (Peprotech), and 1 µM laminin (Sigma-Aldrich). CHIR99021 was added to neural differentiation medium for 3 days (from day #13 to day #15). 1 µM AraC (Sigma-Aldrich) was included in differentiation medium for 1 week (from day #13 to day #20) to kill off dividing cells. The treatment with AraC was repeated, if necessary, every other week (up to 3 cycles). Differentiation was usually extended from 2 to 4 weeks.
Transfection and RNA interference
Transfection of HeLa cells was performed using Metafectene Pro from Biontex (Martinsried, Germany), as indicated by the manufacturer. After 20 h of transfection, cells were processed for DQ-BSA dequenching assay. Transfection of cells with siRNA was performed using Metafectene SI from Biontex (Martinsried, Germany) as indicated by the manufacturer. Cells were analyzed after 5 days of transfection. Small interfering RNAs (siRNAs) were purchased from MWG-Biotech (Ebersberg, Germany).
Rab7a siRNA efficiency in silencing was reported previously : sense sequence 5′-GGAUGACCUCUAGGAAGAATT-3′ and antisense sequence 5′-UUCUUCCUAGAGGUCAUCCTT-3′. Control RNA was used as a negative control: sense sequence 5′-ACUUCGAGCGUGCAUGGCUTT-3′ and antisense sequence 5′-AGCCAUGCACGCUCGAAGUTT-3′.
EGF internalization and EGFR degradation assays
For the EGF internalization assay, control and CMT2B skin fibroblasts were incubated overnight in starvation medium (0.5% BSA, 20 mm HEPES, pH 7.3, in DMEM). Cells were subsequently incubated for 1 h at 4 °C with 0.8 mg/ml rhodamine-labeled EGF (Thermo Fisher, E3481) in starvation medium and then washed several times with starvation medium. After incubation at 37 °C in complete DMEM medium for different time points (30 min, 1 h and 2 h), cells were fixed, mounted on slides, and processed for confocal microscopy. Zen 2011 software (Carl Zeiss, Oberkochen, Germany) was used for image capture and to calculate weighted colocalization coefficient of EGF and LAMP1. For EGFR degradation assay, control and CMT2B skin fibroblasts were incubated for 1 h at 37 °C with serum- and antibiotic-free DMEM and 10 μg/ml cycloheximide, then stimulated with EGF (50 ng/ml) for different times (15, 60, 180, 360 min) and lysed with RIPA buffer (50 mM Tris–HCl, pH 8.0, with 150 mM sodium chloride, 1.0% Igepal CA-630 (NP-40), 0.5% sodium deoxycholate, and 0.1% sodium dodecyl sulfate) plus protease inhibitor cocktail (Roche, Mannheim, Germany). The levels of degraded EGFR were determined by western blotting. When indicated, 100 μM chloroquine was added to the cells 3 h before harvesting.
Analysis of cathepsin D synthesis
Cells were treated with 10 μg/ml cycloheximide (to inhibit protein synthesis) for 50 h, lysed with 2 × Laemmli buffer supplemented with DTT (dithiothreitol) and subjected to western blot analysis. Bands were quantified by densitometry using ImageJ software (National Institutes of Health).
Cathepsin activity assays
Cathepsin D Activity Fluorometric Assay (K143-100, BioVision, Milpitas, CA, USA) utilizes the cathepsin-D substrate sequence GKPILFFRLK(Dnp)-D-R-NH2 labeled with fluorescent MCA (7-methoxycoumarin-4-acetic acid). 2 × 104 control and CMT2B cells were collected and read in a fluorometer equipped with a 328-nm excitation filter and 460-nm emission filter. Cathepsin D activity was expressed by relative fluorescence units (RFU) per million cells and by RFU fold increase of CMT2B fibroblasts against control fibroblasts. Cathepsin B and Cathepsin L Activity Assay Kits (BioVision, K140-100 e K142-100) utilize, respectively, the preferred cathepsin-B RR and cathepsin-L FR substrate sequence labeled with fluorescent AFC (7-amino-4-trifluoromethylcoumarin). 2 × 104 control and CMT2B fibroblasts were collected and read in a fluorometer equipped with a 400-nm excitation filter and 505-nm emission filter. Fold-increase in cathepsin-B or cathepsin-L activity was determined by comparing the relative fluorescence units (RFU) measured in CMT2B and control fibroblasts.
DQ-BSA dequenching assay
Cells were grown on glass coverslips and treated with DQ-BSA. Fibroblasts were treated with DQ Red BSA (10 μg/ml, Thermo Fisher, D12051) for 48 h, neurons with DQ Green BSA (50 μg/ml, Thermo Fisher, D12050) for 24 h and HeLa cells with DQ Green BSA (10 μg/ml) for 6 h. Fluorescence was quantified with Zeiss LSM 700 confocal microscope.
Control and CMT2B fibroblasts and neurons were lysed with Laemmli buffer [100 mM Tris–HCl, pH 6.8, 4% (w/v) SDS, 0.2% (w/v) bromophenol blue, 20% glycerol and 200 mM DTT (dithiothreitol)]. Lysates were loaded on SDS-PAGE and separated proteins were transferred onto PVDF membrane (Merck Millipore). The filter was blocked in 5% milk in PBS for 30 min at room temperature, incubated with the appropriate primary antibody and then with a secondary antibody conjugated with HRP (diluted 1:5000). When phosphorylation was monitored, cells were lysed in the presence of phosphatase inhibitors (PhosSTOP, Roche). Bands were visualized using Western blot Luminol Reagent (Santa Cruz) or Western Bright ECL kit (Advansta, Menlo Park, CA, USA) or ClarityMax (Bio-Rad). The signal was captured on a film, avoiding saturation, to ideally compare samples in the linear range of detection. To prevent saturation of the signal, we previously estimated proper loading amounts. To reduce variability, we did not quantify signals that decayed too quickly and we avoided comparing very weak bands that may be outside the linear range. Films were then scanned at 600 dpi resolution and quantified using ImageJ (National Institutes of Health). Each band was quantified selecting rectangular areas and subtracting the film background. Bands relative to the protein of interest and to the related tubulin loading control were quantified. Measurement of each protein was then normalized on the related tubulin loading control and CMT2B normalized measurements were compared to control normalized measurements obtained from the same gel.
Confocal immunofluorescence microscopy
Fibroblasts grown on coverslips were permeabilized, fixed and incubated with primary and secondary antibodies as described previously  and viewed with Zeiss LSM 700 confocal microscope. HeLa cells were fixed for 20 min in 3% paraformaldehyde, permeabilized with 0.1% TX100 in PBS and then incubated with primary and secondary antibodies diluted in 0.1% saponin in PBS. Then, washing, cells were stained with DAPI and coverslips were mounted and viewed with Zeiss LSM 700 confocal microscope.
iPS cells or peripheral neurons were fixed for 20 min in 4% paraformaldehyde, blocked and permeabilized for 20 min with 10% NGS, 1% BSA, and 0.1% TX100. Alternatively, cells were fixed and permeabilized for 10 min in ice‐cold methanol at − 20 °C. After incubation with primary antibody overnight at 4 °C, the coverslips were washed, incubated with the secondary antibody for 30 min, washed, and mounted with DAPI (H-1200, Vector Laboratories, Burlingame, CA, USA) and viewed with Leica SP5 confocal microscope.
Cells were fixed in 2% glutaraldehyde in 0.2 M Hepes, pH 7.4, at room temperature for 2 h. During fixation, the cells were scraped off the culture dish and pelleted. After postfixation in 1% osmium tetroxide, cells were dehydrated in ethanol and embedded in epoxy resin. Sections were cut with a diamond knife and contrasted with uranyl acetate and lead citrate. Microscopy was performed with a Jeol JEM 1400 Plus transmission electron microscope.
For RT-PCR on fibroblasts, RNA was isolated from cells using an RNeasy Micro kit according to the manufacturer’s instructions (Qiagen, Hilden, Germany). For RNA retrotranscription protocol, we used SuperScript II Reverse Transcriptase (Invitrogen) according to the manufacturer’s instructions. Briefly, a mixture (12 µl) containing 4 µg of RNA, 10 mM of deoxynucleotides and 40 ng of random primers (Promega, Madison, WI, USA) was heated at 65 °C for 5 min. First-strand cDNA synthesis was carried out with SuperScript II Reverse Transcriptase in the presence of dithiothreitol (0.01 M) and ribonuclease inhibitor (40 U, RNaseOUT, Invitrogen) at 42 °C for 50 min. Reactions were stopped by heat inactivation at 70 °C for 15 min.
Quantitative real-time PCR was carried out with Power SYBR Green (Applied Biosystems, Foster City, USA) using Applied Biosystems 7900HT Fast Real-time PCR System.
The primers used were:
GAPDH: forward: 5′-GGTGGTCTCCTCTGACTTCAACA-3′,
Cathepsin B: forward: 5′-CTGTCGGATGAGCTGGTCAAC-3′,
Cathepsin D: forward: 5′-CAGAAGCTGGTGGACCAGAAC-3′,
Cathepsin L: forward: 5′-GCTAATGACACCGGCTTTGT-3′,
LAMP1: forward: 5′-ACGTTACAGCGTCCAGCTCAT-3′,
LAMP2: forward: 5′-TGCTGGCTACCATGGGGCTG-3′,
These were purchased from Eurofins Genomics (Ebersberg, Germany).
The thermal profile used for Real-time PCR was as follows: 1 cycle of 2 min at 50 °C; 1 cycle of 10 min at 95 °C; 40 cycles of 15 s at 95 °C, 1 min at 55 °C; 1 cycle of 15 s at 95 °C and 15 s at 60 °C. The specificity of PCR products was checked by performing a melting-curve test. The relative expression level was calculated using the comparative CT method and expressed as a “fold change”. The quantitative values were obtained from the mean minimal cycle threshold (CT) calculated from triplicate reactions. The fold change, measured as the amount of target gene normalized to the endogenous reference gene GAPDH, was given by 2−∆∆CT, where ∆C = CTtarget − CTGAPDH and ∆∆CT = ∆CTsample − ∆Ccontrol. Negative fold change was calculated using the formula − 1/2−∆∆CT. The relative quantification was considered significant when there was a minimum of twofold change.
For RT-PCR on iPS or iPS-derived cells, in brief, iPS cell clones and differentiated iPSCs tested for pluripotency (Trilineage differentiation Kit, Stem Cell Technologies) were homogenized with TRIzol reagent (15596026; Invitrogen) and total RNA was extracted with chloroform and precipitated with isopropanol. A portion (500 ng) of total RNA was reverse transcribed using High-Capacity cDNA Reverse Transcription Kits (4368814, Applied Biosystems), according to the manufacturer’s instructions. Semiquantitative RT-PCR analyses were performed with GoTaq G2 Flexi DNA Polymerase (M7805, Promega). Primer sequences are shown in Supplemental Table 2, while primers used to test iPSCs for Sendai virus clearance derived from CytoTune2.0‐iPS Sendai Reprogramming Kit (Thermo Fisher).
Confluent monolayer of fibroblasts was wounded with a pipette tip. The cell debris was washed out with PBS. Cells were imaged at the moment of the scratch (T0) and after 15 h using a 10 × objective of EVOS digital microscope. Accumulated distance was measured using photoshop software as the total distance that cells traveled in a certain amount of time.
The conditioned medium was collected from cells cultured for 24 h in serum-free medium, the sample was concentrated using Amicon® Ultra (Merck Millipore) and quantified by Bradford assay. Each sample was adjusted to 15 µg/ml, and 10 µl was loaded in non-reduced protein sample buffer on 7.5% SDS-PAGE gel containing 1 mg/ml gelatin (Biorad). The gel was washed in a specific buffer (2.5% Triton X-100, 50 mM Tris–HCl pH 7.5, 5 mM CaCl2, 1 µM ZnCl2) to remove SDS and then three times in distilled water. After washing, the gel was incubated at 37 °C for 24 h in a buffer containing 1% Triton X-100, 50 mM Tris–HCl pH 7.5, 5 mM CaCl2, 1 µM ZnCl2 and then was stained with 0.5% Coomassie Blue R-250 (Sigma-Aldrich) and destained in 40% methanol and 10% acetic acid.
Rac1 activity assay
Rac1 activity was tested following the manufacturer’s protocol with Rac1 Pull-down Activation Assay Biochem Kit (BK035, Cytoskeleton). Briefly, lysates were mixed with GST-Pak-PBD beads for 1 h at 4 °C. After washing, the pelleted beads were resuspended in Laemmli sample buffer and subjected to SDS–polyacrylamide gel electrophoresis. GTP-bound Rac1 was detected by western blot analysis using anti-Rac1 antibody. The total amount of Rac1 was detected by immunoblotting of the whole cell lysates.
iPSCs were preincubated with mTeSR1 (Stem Cell Technologies), added with ROCK inhibitor (Stem Cell Technologies) for 1 h, harvested by Accumax (Sigma-Aldrich) treatment, collected into tubes, and centrifuged, and the pellets were suspended in PBS 1 × (Sigma-Aldrich) with ROCK inhibitor. One-third of the cells from a confluent 35 mm dish was injected in the testis of 8 weeks old Fox Chase SCID Beige mice (Strain Code: 250, Charles River). Six weeks after injection, tumors were dissected and fixed with formaldehyde solution. Paraffin-embedded tissue was sliced and stained with hematoxylin and eosin. All animal experiments were approved and performed in compliance with the guidelines of San Raffaele Institutional Animal Care and Use Committee.
DNA sequence analysis of RAB7
Genomic DNA was extracted from pelleted neurons using GenElute Mammalian Genomic DNA Miniprep Kit (Sigma-Aldrich) according to the manufacturer’s protocol. 100 ng of genomic DNA was amplified by PCR using the following primers for exon 4: 5′ CTGTGTCCTCACCTGTACTACC 3′ and 5′ GAAAAGAGTGGGTTAGGGAAGAAG 3′. PCR conditions for this reaction are: 94 °C for 7 min, (94 °C for 1 min, 55 °C for 30 s, 72 °C for 45 s) × 30 cycles followed by a final step at 72 °C for 7 min. PCR products were loaded on agarose gel and extracted using QIAEX II Gel Extraction Kit (Qiagen, Hilden, Germany). Samples were shipped to Eurofins Genomics (Ebersberg, Germany) for sequencing.
Neurite length analysis
Neurite length was measured by IncuCyte® S3 Live-Cell Analysis System (Essen BioSCience). Briefly, young neurons were plated in 24-well plate (20,000 cells/well). Bright field (contrast) real-time automated acquisition and measurements for live cells were performed every 4 h for the first six consecutively days after neuron plating. Two wells per clones were plated and sixteen field per well were analyzed (objective 20×). Neurite length was measured as detected by the IncuCyte Zoom software as the total combined length of all neurites (mm) detected per mm2. All data are expressed relative to the respective day 0 of maturation. Each group is represented by mean ± SEM (n = 32).
Data were statistically analyzed using Student's t test and Mann–Whitney U- test (GraphPad Prism4 software) (*p < 0.05, **p < 0.01 and p < 0.001). Error bars represent SEM. Experiments were performed at least in triplicate.