Cell culture

The iPSC lines from CMT2A patients and healthy subjects were derived from fibroblast samples collected by our institutional biobank with appropriate consent and local ethical committee approval and were already available in our laboratory [33]. The iPSCs were maintained in culture on Matrigel-coated dishes with Essential 8 medium (Thermo Fisher Scientific). Lentiviral transductions, selection and expansion of stable iPSC lines were performed in mTeSR1 (Stem Cell Technologies). HeLa cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM)/F12 supplemented with 15% fetal bovine serum, 1% penicillin/streptomycin (Pen-Strep), and 1% amphotericin B (all from Thermo Fisher Scientific). All cell cultures were maintained at 37 °C in 5% CO2. All cell lines were tested for mycoplasma contamination once a month.

Plasmids

For transient knockdown, pSUPuro-MFN2-shRNAtg1 and pSUPuro-MFN2-shRNAtg2 were cloned by inserting double-stranded oligos into pSUPERpuro between the BglII and HindIII sites as described previously [35, 36]. The shRNAs expressed from pSUPuro-MFN2-shRNAtg1 and pSUPuro-MFN2-shRNAtg2 target nucleotides 1564–1582 (5′-CCTCAAGGTTTATAAGAAT-3′) and 2381–2399 (5-GCAAAGCTGCTCAGGAATA-3′) of MFN2 mRNA (numbering according to NM_014874.4). pSUPuro-scrambled (5′-ATTCTCCGAACGTGTCACG-3) is described elsewhere [37]. C-terminally Myc-DDK-tagged MFN2 was from Origene (pCMV6-MFN2-Myc-DDK; #RC202218; Myc-tag: EQKLISEEDL  ~ 1202 Daltons; FLAG-tag: DYKDDDDK  ~ 1012 Da).

To render the MFN2 cDNA RNAi-resistant, silent mutations were introduced into the shRNA target sites in the MFN2 cDNA using the Quikchange Lightning Multi-Site Kit (Agilent) and primers QC-MFN-t1 (5′-gacttccacccttctccagtagtgctgaaagtctacaaaaacgagctgcaccgccacatagagga-3′) and QC-MFN-t2 (5′-cttgactcacttcagagcaaagctaaactcctgagaaacaaagccggttggttggacagtga-3′) according to the manufacturer’s protocol to generate pCMV6-MFN2Rtg1-Myk-DDK and pCMV6-MFN2Rtg2-Myk-DDK, respectively. For stable knockdown, the shRNAtg2 sequence was inserted into pEco-Lenti-H1-shRNA-Blasticidin (Gentarget). To increase the expression of blasticidin resistance in iPSCs, the RSV promoter between the MluI and KpnI sites of pEco-Lenti-H1-MFN2-shRNAtg2-shRNA-Blasticidin was replaced by a gene-synthesized fragment (GeneArt Gene Synthesis) containing a CMV promoter with a chimeric intron to generate pEco-Lenti-H1-MFN2-shRNAtg2-CMV-BSD. To generate the lentiviral MFN2 cDNA expression construct (pLVX-EF1a-MFN2Rtg2-Myk-DDK-IRES-Puro), MFN2 cDNA was excised from pCMV6-MFN2Rtg2-Myk-DDK using EcoRI and PmeI (New England Biolabs) and cloned into the EcoRI-BamHI (Blunt) sites of PLVX-EF1a-IRES-Puro (Clontech). All constructs were verified by Sanger sequencing.

Transient transfection, lentiviral vector production, and transduction

HeLa cells were transfected with 7.5 µg shRNAtg1 or shRNAtg2 using Lipofectamine LTX reagent (ThermoFisher Scientific) or Dreamfect (OZ Biosciences). Twenty-four hours after transfection, cells were selected in DMEM/F12 medium supplemented with 1.5 µg/mL puromycin (Sigma Aldrich). Co-transfection experiments with 7.5 µg shRNAtg1/MFN2Rtg1 or shRNAtg2/MFN2Rtg2 were performed in parallel in a 1:1 (3.75 µg shRNA to 3.75 µg pCMV6) ratio. After 24 h, cells were cultured in the presence of 500 µg/mL G418 (Sigma Aldrich). Cells were transfected with pSUPuro-SCR under the same conditions as controls. Virus production for protein expression was essentially performed as follows. HEK 293 T cells were transfected with pEco-Lenti-H1-shRNAt2-CMV-BSD supplemented with Lentiviral Packaging plasmid (Gentarget; #HT-Pack) or pLVX-EF1a-MFN2Rtg2-Myc-DDK-IRES-Puro supplemented with Lenti-X HTX packaging mix (Clontech: #631248) according to the manufacturer’s protocol and established methods [36]. Lentiviral supernatants were collected 48 and 72 h post-transfection, filtered through a 0.45-μM polyethersulfone sterile filter (Millipore), followed by concentration using Lenti-X-Concentrator (Clontech: #631232). CMT2A iPSCs were transduced three times with pEco-Lenti-H1-shRNAt2-CMV-BSD supernatant to increase the number of transduced cells without diluting the essential growth factors in the iPSC medium. The cells were incubated twice with lentiviral supernatant overnight. An initial assessment indicated an insufficient MFN2 silencing, so the cells were re-transduced again one month later. Eight hours after the final transduction, cells were expanded under blasticidin selection at a final concentration of 2.5–5 μg/mL, followed by a final 5-day selection with 40 µg/mL blasticidin. To rescue MFN2 expression, pLVX-EF1a-MFN2Rtg2-Myc-DDK-IRES-Puro viral supernatant was added twice to pEco-Lenti-H1-shRNAt2-CMV-BSD transduced cells. Forty-eight hours after the final transduction, cells were expanded with puromycin to a final concentration of 0.5 μg/mL.

Differentiation of iPSCs into MNs

iPSCs were differentiated into MNs using a multistep protocol modified from Maury et al. [38]. To induce embryoid body (EB) formation from iPSCs, on day 0, iPSCs were dissociated with accutase and resuspended in differentiation N2B27 medium (1:1 DMEM/F12-Neurobasal media, supplemented with N2, B27, 2 mM L-glutamine, 1% Pen-Strep, 0.1 mM β-ME; all from ThermoFisher Scientific), with 10 μM Y-27632 (Cell Signaling Technology), 0.1 μM LDN 193189 (MiltenyiBiotec), 20 μM SB431542, and 3 μM CHIR-99021 (both from Sigma Aldrich). The media was replaced every 2 days, adding small molecules as follows: on day 2, 1 μM LDN 193189 (MiltenyiBiotec), 20 μM SB431542, 3 μM CHIR-99021, and 100 nM retinoic acid (RA, all three from Sigma Aldrich); on day 4: 0.1 μM LDN 193189, 20 μM SB431542, 3 μM CHIR-99021, 100 nM RA, and 500 nM Smoothened Agonist (SAG, Sigma Aldrich); on day 7: 100 nM RA and 500 nM SAG; finally, on day 9: 100 nM RA, 500 nM SAG, and 10 μM DAPT (Stem Cell Technologies). BDNF (20 ng/mL) and GDNF (10 ng/mL; both from Peprotech) were added to the differentiation medium on day 10. On day 11 or 14, EBs were dissociated and the cells seeded on poly-L-ornithine (20 µg/mL) and laminin (20 µg/mL; both from Sigma Aldrich) coated plates. Three or four days after seeding, cells were stained for live imaging analyses, and 5–8 days after seeding, they were fixed for immunocytochemistry and harvested for mtDNA and Western blot analysis.

Immunocytochemistry of iPSCs and MNs

Cells were fixed in 4% paraformaldehyde for 20 min at 37 °C, permeabilized with 0.25% Triton X-100, and then blocked with 10% bovine serum albumin in phosphate-buffered saline (PBS) containing 0.25% Triton X-100 (all from Sigma Aldrich) for 1 h at room temperature. We incubated the cells with primary antibodies (Table S1) overnight at 4 °C, and then with secondary antibodies (Table S2) for 1 h at room temperature. The nuclei were stained with 0.5 µg/mL DAPI (Sigma Aldrich). Images were acquired using a laser-scanning Leica TCS SP5 confocal microscope (Leica Microsystems, Wetzlar, Germany) or a LED-based Nikon ECLIPSE Ti/CREST microscope (Nikon).

Mitochondrial area analysis

Accurate analysis of the area occupied by mitochondrial structures requires sharp, high contrast images with minimal background noise. Single maximum intensity projection images of TOM20 immunolabeled MNs obtained by laser-scanning confocal microscopy (Leica TCS SP5, Leica Microsystems) were pre-processed in Fiji software using an unsharp mask filter and then binarized by thresholding (Otsu threshold 20560–65535). To perform single cell analysis of the mitochondrial area, each cell was subdivided into seven adjacent regions of interest (ROIs), the first spanning from the center of the cell to the axon hillock (7 × 10 µm) and the other six covering the axon (3 × 10 µm), for a total length of 70 µm. The mean fluorescence intensity (Iaverage) was measured.

Quantification of the mitochondrial area was performed according to the specifications of Valente et al. [39]. The mitochondrial area represents the total area in the image covered by signal after being separated from the background and consists of the number of pixels in the binary image containing the signal multiplied by the area of a pixel. I represents the pixel intensity in the binarized image, x represents the width of the image in pixels, y represents the height of the image in pixels, and s represents the calibrated length of one pixel:

$$}\frac}}} }}}}} }} \cdot x \cdot y \cdot s^$$

Data were graphed and two groups of data were compared by multiple Student’s unpaired t-tests using GraphPad Prism 8 (GraphPad Software, San Diego, California, USA). Data were considered to be significantly different if p < 0.05.

Cell imaging of lysosomes and mitochondria

For mitochondrial and lysosome cell imaging, MNs plated in optical 4- or 8-well µ-Slides (Ibidi GmbH) pre-coated with poli-L-ornithine and laminin (both from Sigma Aldrich) were transduced with CellLight™ Mitochondria-RFP BacMam 2.0 and/or CellLight™ lysosome-GFP BacMam 2.0 reagents (both from ThermoFisher Scientific) (30 particles per cell) and incubated for 48 h at 37 °C. Imaging was performed with a Crest Optics Spinning Disk module (Crest-Crisel Instruments) mounted on a fully automated inverted Nikon ECLIPSE Ti microscope (Nikon) and acquisitions achieved with an Andor DU-888 EM-CCD camera (Andor) for fast recordings and NIS-Elements v.5 software (Nikon).

mtDNA analysis

Total DNA was extracted from MNs using a standard protocol (Flexigene, Qiagen). The mtDNA was quantified by quantitative real-time PCR using the ΔΔCt method on a 7500 Real Time PCR system (Software 2.01, Applied Biosystems, ThermoFisher Scientific) and the Taqman assay with probes for human mitochondrial genes CYTB (VIC-CAC CAG ACG CCT CAA CCG CCT T-TAMRA) and ND4 (FAM-CCG ACA TCA TTA CCG GGT TTT CCT CTT G-MGB), normalizing for nuclear APP (FAM-CCC TGA ACT GCA GAT CAC CAA TGT GGT AC-TAMRA) and RNAseP (TaqMan™ Copy Number Reference Assay, human, RNase P; 4,403,326) genes, respectively. All quantifications were carried out in triplicate using 25 ng of total DNA as the template. The mtDNA levels were normalized to nuclear DNA and expressed as relative values using the amount of mtDNA in the cells of healthy controls as a reference (relative quantification = 1).

AAV vectors

AAV9 vectors were produced by Virovek Laboratories (Hayward, CA). Self-complementary AAV9-KD (AAV9-KD) contained an shRNAtg2 sequence (5′-GCAAAGCTGCTCAGGAATA-3′) under the control of the U6 promoter to silence MFN2 (numbering according to NM_014874.4) and incorporated a GFP tag under the control of the CMV promoter. Single-stranded (ss)AAV9-KD-rMFN2 (AAV9-KD-rMFN2) contained an shRNAtg2 sequence (5′-GCAAAGCTGCTCAGGAATA-3′) under the control of the U6 promoter and Myc-DDK-tagged MFN2 cDNA mutated to be resistant to the shRNA (as previously described) under the control of the CMV promoter. As a control, an AAV9-null vector was produced with a non-coding sequence under the control of the CMV promoter.

Animal procedures

The MitoCharc1 transgenic mice (B6;D2-Tg(Eno2-MFN2*R94Q)L51Ugfm/J) have the neuron-specific rat enolase (Eno2) promoter directing the expression of human R94Q (arginine to glutamine) MNF2 mainly in neurons, mimicking the most common mutation found in CMT2A patients [34]. Hemizygous mutant mice (MFN2) express two copies of mouse WT murine mfn2 and one copy of human mutant MFN2; they are viable, fertile, normal in size. MFN2 mice and WT littermates were used for the experiments and analyses. The genotypes of the mice were confirmed using a PCR-based assay as described previously [34]. All transgenic animals were purchased from the Jackson Laboratory (stock #012812), and they were maintained according to standard conditions, including ad libitum access to food and water and 12-h dark/light cycle. All animal experiments were approved by the Italian Ministry of Health review boards in compliance with U.S. National Institutes of Health Guide for the Care and Use of Laboratory Animals. AAV9-KD or AAV9-KD-rMFN2 (1.8 × 1013 vg/kg) were injected into MitoCharc1 pups (P1, n = 3) via intracerebroventricular injection [40, 41]. AAV9::null was used as a control vector (n = 3). To evaluate MFN2 expression, brains were collected after 2 weeks.

Protein analysis

Western blotting was performed as described previously [33, 42]. Protein lysate was separated by 4–12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) in MES SDS Running Buffer 20x (B0002; life Technologies) for 30 min at 200 V. To separate endoMFN2 and exoMFN2, the gel was ran in MOPSs SDS Running Buffer 20× (B0001; Life Technologies) for 90 min at 130 V and then 30 min at 90 V. Proteins were transferred to a nitrocellulose membrane (GE Healthcare) and incubated with primary antibodies (Table S1) overnight at 4 °C. The membranes were then incubated in secondary antibodies (Table S2) and the immune complexes revealed using the Odyssey® Fc Imaging System (LI-COR Biosciences). Anti-actin antibody was used as a loading control. Semi-quantitative analysis was performed using Image Studio™ Lite software (LI-COR Biosciences).

Statistical analysis

Statistical analysis was carried out utilizing StatsDirect for Windows (version 2.6.4) or GraphPad Prism 8 software. Multiple comparisons on a single data set were performed with one-way analysis of variance (ANOVA) and, when several variables were considered, two-way ANOVA was used, followed by appropriate post hoc analysis. Two-tailed, unpaired Student’s t-test was employed to compare two groups. All experiments were carried out at least in triplicate. The experimental results are shown as mean + SEM or mean + SD as needed. The null hypothesis was rejected at the 0.05 level of significance.