Cell culture

Mouse ESC lines (Rex1GFP-d2 and E14IVc¹³⁷) were cultured in feeder free conditions [plastic coated with 0.2% gelatine (Sigma, cat. G1890)] and replated every 3–4 days at a split ratio of 1:10 following dissociation with Accutase (GE Healthcare, cat. L11-007) or 0.25% Trypsin (Life Technologies). Cells were cultured in serum-free N2B27-based medium [DMEM/F12 and Neurobasal in 1:1 ratio, 0.1 mM β-mercaptoethanol, 2 mM L-glutamine, 1:200 N2 and 1:100 B27 (all reagents from Life Technologies)] or serum-containing KSR medium [GMEM (Sigma, cat. G5154) supplemented with 10% KSR (Life Technologies), 2% FBS (Sigma, cat. F7524), 100 mM β-mercaptoethanol (Sigma, cat. M7522), 1× MEM non-essential amino acids (Invitrogen, cat. 1140-036), 2 mM L-glutamine, 1 mM sodium pyruvate (both from Invitrogen)], supplemented with two small-molecule inhibitors (2i) PD (PD0325901, 1 μM), CH (CHIR99021, 3 μM) from Axon (cat. 1386 and 1408) and LIF (100 units/mL purchased from Qkine – Cambridge UK).

Human iPSCs CS09iHD-109n5 (here referred as iPSC_Q109) and CS14iCTR-21n3 (here referred as iPSC_Q21) purchased from Cedars-Sinai Biomanufacturing Center (Los Angeles, California), were maintained on pre-coated 0.5% Matrigel (CORNING, cat. 356231) plates in E8 medium made in house (according to Chen et al., 2011¹³⁸) or in mTeSR (StemCell Technologies, cat. 05850) at 37 °C, 5% O², 5% CO². Human iPSCs were dissociated in clumps with 0.5 mM EDTA (Gibco, cat. AM99260G) and replated at 1:6 dilution every 3-4 days with 10 μM ROCK inhibitor (Y27632-dihydrochloride Axon Medchem, cat. 1683) for 24 hours. Medium was changed every day. All cell lines were mycoplasma-negative.

Genotyping of iPSC_Q21 and iPSC_Q109

Genomic DNA from iPSC_Q21 and iPSC_Q109 was isolated using the DNeasy Blood and Tissue kit (Qiagen, cat. 69504) following manufacturer’s instructions and quantified by Nanodrop ND-1000. PCR was performed using primers listed in Supplementary Table 1 and described in Mangiarini et al.²⁹, designed to amplify polyQ tract in HTT exon 1. For a 25 μL reaction we used 200 ng of genomic DNA, 0.25 μL of Taq Phusion High fidelity (ThermoFisher, cat. F-530L), 2.5 μL of Buffer GC 5x + MgCl² (7.5 mM), 2 μL of dNTPs (10 mM) and 1.25 μL of Dimethyl sulfoxide (DMSO, 100%). After 3 minutes at 94 °C, we performed 35 cycles of: 94 °C for 1 minute, 63 °C for 45 seconds, 72 °C for 1 minute, followed by a final elongation at 70 °C for 7 minutes. Gel electrophoresis was performed on agarose gel at 2.5%, loading 20 μL of PCR products with Purple Loading Dye 6x (NEB, cat. B7024S). β-Actin was used as loading control. Results were digitally acquired by VWR Imager CHEMI Premium.

Generation of HTT-expressing ESC lines

Q15 and Q128 cells were generated by DNA transfection of vectors containing N-terminal of human huntingtin gene, with 128 or 15 CAG repeats respectively (courtesy of Professor Elena Cattaneo). Overnight linearization of plasmid DNA was performed with the restriction enzyme PvuI. For DNA transfection, we used Lipofectamine 2000 (Life Technologies, cat. 11668-019) and performed reverse transfection. For one well of a 6-well plate, we used 6 μl of transfection reagent, 2 μg of plasmid DNA and 300,000 cells in 2 mL of medium. The medium was changed after overnight incubation. Antibiotic selection (Puromycin 1 μg/mL) started 24 hours after transfection.

Generation of mouse ESCs stably expressing genes of interest

Stable transgenic mouse ESCs expressing candidates were generated by transfecting HTT-expressing cells with PB transposon plasmids (1 μg of CAG-Mtf1, CAG-Kdm2b, CAG-Kdm5b and CAG-Fbxo34), purchased from VectorBuilder (VectorBuilder Inc, Chicago, IL, USA), with PB transposase expression vector pBase (1 μg). We used Lipofectamine 2000 and performed reverse transfection as described for HD lines generation. Antibiotic selection (Hygromycin B, 150 μg/mL; Invitrogen, cat. 10687010) started 24 hours after transfection.

NPCs differentiation

iPSC_Q21 and iPSC_Q109 were differentiated into NPCs according to Li et al., 2011⁹² protocol. iPSCs at 80% confluency were dissociated in single cells with Accutase (Gibco, cat. A1110-501) and plated 45,000 cells/cm² in E8 medium with 10 μM ROCK inhibitor. After 1 day, E8 medium was substituted with N2B27 induction medium composed of Advanced DMEM/F12 (Gibco, cat. 12634-010): Neurobasal (Gibco, cat. 21103-049) (1:1 ratio), BSA 50 mg/mL (Gibco, cat. 15260-037), Glutamax 1% (Gibco, cat. 35050-038), Penicillin/Streptomycin 1% (Gibco, cat. 15140122), N2 Supplement 1:200 (Gibco, cat. 17502-048), B27 Supplement 1:100 (Gibco, cat. 17504-044), supplemented with small molecules human LIF 10 ng/mL (Qkine, cat. Qk036), SB431542 2 μM (Axon Medchem, cat. 1661), CHIR99021 3 μM (Axon Medchem, cat. 1386), Compound E 0.1 μM (Sigma-Aldrich, cat. 209986-17-4). N2B27 induction medium was changed every day, for 7 days. On day 7, NPCs were splitted at 1:6 dilution in N2B27 maintenance medium which is composed of Advanced DMEM/F12 (Gibco, cat. 12634-010): Neurobasal (Gibco, cat. 21103-049) (1:1 ratio), BSA 50 mg/mL (Gibco, cat. 15260-037), Glutamax 1% (Gibco, cat. 35050-038), Penicillin/Streptomycin 1% (Gibco, cat. 15140122), N2 Supplement 1:200 (Gibco, cat. 17502-048), B27 Supplement 1:100 (Gibco, cat. 17504-044), supplemented with small molecules human LIF 10 ng/ml (Qkine, cat. Qk036), SB431542 2 μM (Axon Medchem, cat. 1661), CHIR99021 3 μM (Axon Medchem, cat. 1386), supplemented with EGF 20 ng/mL (R&D, cat. 236-EG) and FGF2 20 ng/mL (Qkine, cat. Qk002, recombinant zebrafish FGF2). NPCs were maintained for 6 passages. h-iPSCs and NPCs morphology data were digitally collected with microscope Zeiss Axio Vert A1 FL-LED.

Generation of NPCs transiently expressing genes of interest

For DNA transfection, 250,000 NPCs were dissociated as single cells with Accutase (Gibco, cat. A1110-501) and were transfected with PB constructs (1 μg) using FuGENE HD Transfection (Promega, cat. E2311), following the protocol for reverse transfection. For one well of a 12-well plate, we used 3.9 μL of transfection reagent, 1 μg of plasmid DNA and 250,000 cells in 1 mL of N2B27 maintenance medium medium with 10 µM Y27632 [ROCKi, Rho-associated kinase (ROCK) inhibitor, Axon Medchem cat. 1683]. The medium was changed after overnight incubation. After 48 hours post transfection, cells were treated with Rotenone 30 µM for 24 hours and then analysed as indicated in Fig. 8f.

Proliferation assay

Mouse ESCs were conditioned for one passage in KSR + 2iL medium in presence of Puromycin 6 μg/mL. Proliferation of ESCs was assessed by plating 15,000 cells in a 24-well plate (7,500 cells/cm²) in KSR + 2iL medium in presence of Puromycin 6 μg/mL. Cells were dissociated with 0.25% Trypsin (Life Technologies) and counted every 24 hours for 4 days.

Proliferation of iPSCs was measured by plating 40,000 single cells on pre-coated 0.5% Matrigel (CORNING, cat. 356231) 12-well plates (11,428 cells/cm²) in E8 medium with 10 µM ROCK inhibitor (Y27632-dihydrochloride, Axon Medchem, cat. 1683) for 24 hours. Medium was changed every day. Cells were dissociated with Accutase (GE Healthcare, cat. L11-007) and counted every 24 hours for 4 days.

Proliferation of NPCs was measured by plating 100,000 cells on pre-coated 0.5% Matrigel (CORNING, cat. 356231) 12-well plates (28,571 cells/cm²) in N2B27 maintenance medium with 10 µM ROCK inhibitor (Y27632-dihydrochloride Axon Medchem, cat. 1683) for 24 hours. Cells were dissociated with Accutase (GE Healthcare, cat. L11-007) and counted every 24 hours for 4 days.

Stressors treatment and Crystal violet (CV) staining

For experiments in Figs. 2b, 5,000 mouse ESCs were plated in a 24-well plate (2,500 cells/cm²) in KSR + 2iL medium in the presence of the inhibitors (and Puromycin 6 μg/ml) for 48 hours and scored by quantification of the number of surviving cells by CV staining [CV solution: 0.05% w/v Crystal Violet (Sigma), 1% of formaldehyde solution 37% (Sigma), 1% methanol, 10% PBS] and quantification of mean intensity was performed with Fiji software (v2.0.0).

For PB-mutagenesis followed by stressor treatments, cells were plated at density 2,500 cells/cm² in Puromycin 6 μg/mL and selected for 5 days in the presence of MG132 (Sigma-Aldrich, cat. C2211) or Tamoxifen (Sigma-Aldrich, cat. T2859).

For experiments in Fig. 4g, 5,000 cells were plated in a 24-well plate in KSR + 2iL medium with Puromycin 3 μg/mL. Stressors (MG132 12.5 nM or Tamoxifen 13.4 µM) were added after 12 hours. Scoring of surviving cells was performed as described above.

For experiments in Supplementary Fig. 4d, 2,500 cells were plated in a 48-well plate in KSR + 2iL medium. Stressors [Rotenone (Sigma-Aldrich, cat. R8875), Cumene (Sigma-Aldrich, cat. 247502), 5-Azacytidine (Sigma-Aldrich, cat. A1287), MG132 (Sigma-Aldrich, cat. C2211), Bafilomycin (Sigma-Aldrich, cat. B1793), Staurosporine (Sigma-Aldrich, cat. S6942), Tamoxifen (Sigma-Aldrich, cat. T2859)] were added after 12 hours at the indicated concentrations. After 48 hours, surviving cells were stained with CV solution, scoring of surviving cells was performed as described above.

Electroporation of the PB system in ESCs

PB-mediated mutagenesis by electroporation was performed for genome-wide screening. PB vectors integrate stably in the genome after random insertion in TTAA sites. The PB pGG134 vector used (shown in Fig. 2a) was optimised for gain-of-function screens⁵⁴: it consists of the MSCV enhancer/promoter followed by a splice donor site from exon 1 of Foxf2 gene, which allows the over-activation of nearby genes. The PB 5’-ITR has also weak directional promoter activity, i.e. this construct can activate genes in either orientation. The vector contains also a second cassette, including a constitutive promoter followed by DsRed and Hygromycin resistance genes, which was used to identify cells with stable vector integration.

We optimised the conditions in order to achieve a low number of integration events, by adjusting the ratio of PB vector vs transposase pBase. For the screening procedure, mutagenesis was performed using the optimised amount of 0.5 μg pGG134 and 20 μg pBase.

For a single electroporation, 10⁷ cells and 20.5 μg DNA were mixed and placed into an electroporation cuvette (Biorad Gene Pulser Cuvette, cat. 165-2088). Cells were electroporated by placing the cuvette in the electroporation holder of the Biorad GenePulser (cat. 165-2076). Settings used: 250 V, 500 μF, time constant should be between 5.6 and 7.5. Electroporated cells were gently recovered from the cuvettes and plated. Antibiotic selection started 24 hours after electroporation.

Genomic DNA extraction and Splinkerette-PCR

Cells were harvested and incubated overnight at 56 °C with lysis buffer [10 mM Tris-HCl, pH 7.5; 10 mM EDTA; 10 mM NaCl; 0.5% w/v Sarcosyl, supplemented with proteinase K (Sigma, cat. P2308) to a final concentration of 1 mg/mL]. In order to obtain DNA precipitates, the next day 2 mL of a mixture of NaCl and ethanol (30 µL of 5 M NaCl mixed with 20 mL of cold absolute ethanol) was added. Cellular extracts were centrifuged for 45 minutes at 4 °C to remove soluble fraction. Precipitated gDNA was rinsed three times by dripping 2 mL of 70% ethanol and finally resuspended in 70 °C milliQ water.

Splinkerette-PCR procedure for PB-integration mapping was adapted from Potter and Luo ¹³⁹ and consisted of the following steps: a) 2 μg of genomic DNA were digested with 10 U BstYI (10,000 U/mL, NEB) in a volume of 30 μL. Reaction was incubated at 60 °C overnight, the following day the enzyme was inactivated at 80 °C for 20 minutes. Adapters for Splinkerette-PCR were generated by annealing of 150 pmol of AdapterA and B primers (Supplementary Table 1) in a final volume of 100 μL (10x NEB Buffer 2). Oligos were denatured at 65 °C for 5 min, then cooled; b) Ligation was performed in a total volume of 6 μl including a 2x Ligation mix (Takara), 2.5 μL of digested gDNA and 0.5 μL of annealed adapters for Splinkerette-PCR. Ligation reaction was incubated at 16 °C overnight, the next day 65 °C for 10 minutes for enzyme inactivation. A purification step was included before step C, using QIaquick PCR Purification Kit, following manufacturer’s instructions. For PCR amplifications we used Phusion HF DNA Pol (NEB) in 5x Phusion GC Buffer recommended in case GC-rich templates or those with secondary structures. PCR mix included 5x GC Buffer, 10 mM dNTPs, DMSO and Phusion Pol; c) First round PCR was amplified with 15 μL of ligated DNA (or 50% of ligation product for each reaction for PB5’ and PB3’ transposon/host junctions), 0.5 μM for each primer (Adaptor-PCR1 and PB5’ or PB3’-ITR PCR1), 6.5 μL PCR mix, final volume of 25 μL. Splinkerette-PCR1 program: 95 °C for 2 minutes; two cycles of 95 °C for 20 seconds, 65 °C for 30 seconds, 68 °C for 2 minutes; then 30 cycles of 95 °C for 30 seconds, 60 °C for 30 seconds, 68 °C for 2 minutes; then 68 °C for 10 minutes; d) For second round PCR, we used 5 μL of 1:500 dilution of PCR1 product, 0.5 μM for each primer (Adapter-PCR2 and PB5’ or PB3’-ITR PCR2), 6.5 μL PCR mix, final volume of 25 μL. Splinkerette-PCR2 program: 95 °C for 2 minutes; two cycles of 95 °C for 20 seconds, 65 °C for 30 seconds, 68 °C for 2 minutes; then 5 cycles of 95 °C for 30 seconds, 60 °C for 30 seconds, 68 °C for 2 minutes; then 25 cycles of 95 °C for 30 seconds, 58 °C for 30 seconds, 68 °C for 2 minutes; then 68 °C for 10 minutes; e) PCR2 products were treated with Antarctic Phosphatase and Exonuclease I (both from NEB) and sequenced using PB5’- or PB3’-ITR PCR2 primers. Primers and adaptor sequences are listed in Supplementary Table 1.

Next Generation Sequencing analysis of genomic integration sites

Genomic DNA from entire populations of mutants was extracted using a Gentra Puregene Cell Kit. Library preparation and sequencing was performed as previously described¹⁴⁰. A bespoke bioinformatics pipeline allowed to map each single read to a genomic locus and to associate each site of integration to a gene within 20 kb of distance. Data was then organised into the network of HD interacting genes by means of Cytoscape software (v3.8.2)¹⁴¹.

Propidium iodide (PI) staining

PI staining was performed on live single mouse ESCs according to the manufacturer’s instructions (Ebioscience, cat. 88-8007-72). After washing in PBS, 10⁵ live cells were resuspended in 200 μL of 1x Binding Buffer and 5 μL of PI Staining Solution (cat. 00-6990) were added. Flow cytometry analysis was performed using a BD FACSCanto^TM II cytometer within 1 hour, storing samples at 2-8 °C in the dark. Data were analysed with BD FACSDiva^TM (v. 9.0) and FlowJo (10.8.1) software. Representative gating strategy is available in Supplementary Fig. 10a.

ROS measurement assay

ROS production was detected by staining single live mouse ESCs and human NPCs cells with 2′,7′-dichlorodihydrofluorescein diacetate (H₂DCFDA; Life Technologies, cat. D399), performing the following steps: a) ROS indicator was freshly reconstituted in order to make a concentrated stock solution (10 mM); b) 3-5×10⁵ cells were harvested, c) washed once with 500 μL of PBS and d) resuspend in 300 μL PBS containing the probe to provide a final working concentration of 0.5 μM dye; e) cells were incubated at 37 °C for 10 minutes in the dark; f) after removal of the staining solution, samples were g) washed twice in PBS. Samples were collected by flow cytometry using a BD FACSCanto^TM II cytometer or BIO-RAD S3e Cell Sorter and analysis was performed with BD FACSDiva^TM (v. 9.0), ProSort^TM (v. 1.6) and FlowJo (10.8.1) software. Representative gating strategy is available in Supplementary Fig. 10b-c.

Annexin V staining

Live NPCs, transiently transfected with the gene of interest and treated with Rotenone 30 μM for 24 hours, were stained with Annexin V according to the manufacturer’s instructions (Ebioscience, cat. 88-8007-72). Cells were washed once in PBS, then once in 1x Binding Buffer (cat. 00-0055). 5×10⁵ cells were resuspended in 200 μL of 1x Binding Buffer and incubated with 5 μL of fluorochrome-conjugated Annexin V (cat. 17-8007) for 10 minutes at room temperature. Cells were then washed in 500 μL of 1x Binding Buffer. Finally, cells were resuspended in 200 μL of 1x Binding Buffer. Flow cytometry analysis was performed using the BIO-RAD S3e Cell Sorter within 1 hour, storing samples at 2-8 °C in the dark. Data were analysed with ProSort^TM (v. 1.6) and FlowJo (10.8.1) software. Representative gating strategy is available in Supplementary Fig. 10c.

Western Blotting

Cells were washed in cold PBS and harvested in lysis buffer (50 mM Hepes pH 7.8, 200 mM NaCl, 5 mM EDTA, 1% NP40, 5% glycerol), freshly supplemented with 1 mM DTT, protease inhibitor (Roche, cat. 39802300) and phosphatase inhibitor (Sigma-Aldrich, cat. P5726). Samples were exposed to ultrasound in a sonicator (Diagenode Bioruptor) and centrifuged at 15,871 rcf for 10 minutes to prepare supernatant. Protein concentration was determined by Bradford quantification. For experiments in Figs. 1b, 2g, 4d and Supplementary Fig. 1d, total protein (10 µg) was fractionated on 4-12% Nupage MOPS acrylamide gel (Life Technologies, cat. BG04125BOX/BG00105BOX) and electrophoretically transferred on a PVDF membrane (Millipore, cat. IPFL00010) in a Transfer solution (50 mM Tris-HCl, 40 mM glycine, 20% methanol, 0.04% SDS). Membranes were then saturated with 5% Non-Fat Dry Milk powder (BioRad; 170-6405-MSDS) in TBST (8 g NaCl, 2.4 g Tris-HCl, 0.1% Tween20/litres, pH 7.5) for 1 hour at room temperature and incubated overnight at 4 °C with anti-HTT (clone 1HU-4C8) or anti-GAPDH (clone 6C5) primary antibody (Supplementary Table 2). Membranes were then incubated with secondary antibodies conjugated with a peroxidase, diluted in 1% milk in TBST. Pico SuperSignal West chemiluminescent reagent (Thermo Scientific, cat. 34078) was used to incubate membranes and images were digitally acquired by ImageQuant LAS 4000 (GE Healthcare). Uncropped gels and numerical values are provided in the Source data file.

Mice were sacrificed by cervical dislocation and tissues were homogenised in lysis buffer containing 20 mM Tris-HCl, pH 7.4, 1% Nonidet P-40, 1 mM EDTA, 20 mM NaF, 2 mM Na₃VO₄ and 1:1000 protease inhibitor mixture (Sigma-Aldrich) and sonicated. For experiment in Supplementary Fig. 1b, 50 µg of total protein lysate of WT mouse, R6/2 mouse, Q15 cells and Q128 cells were loaded in a handcast 5-11% acrylamide stacking-gel and incubated with the following antibodies: anti-HTT (clone EM48) and anti-GAPDH (Supplementary Table 2). For experiments in Supplementary Fig. 8a, d, e and g, 40 µg of total protein lysate were immunoblotted with the following antibodies: anti-GFP, anti-ACTIN (clone 8H10D10), anti-HTT (clone EM48), anti-TUBULIN (clone B-5-1-2) (Supplementary Table 2). Membranes were processed as described above. Images were digitally acquired by VWR Imager CHEMI Premium or ChemiDoc XRS+ (model n°: Universal Hood II) with Image Lab Software, BioRad (v. 6.1). Quantification was performed using Fiji 2.9.0 with background subtraction and normalising on the loading control (GAPDH, ACTIN or TUBULIN). Uncropped gels are provided in the Source data files.

Immunofluorescence

Immunofluorescence for MTF1 detection in mouse ESCs was performed on cells plated on fibronectin (Merck, cat. FC010)-coated glass coverslips. Cells were fixed in 4% formaldehyde (Sigma-Aldrich, cat. F8775) in PBS for 10 minutes at room temperature, washed in PBS, permeabilised for 10 minutes in PBS + 0.1% Triton X-100 (PBST) at room temperature and blocked in PBST + 3% of horse serum (HS; Gibco, cat. 16050-122) for 45 minutes at room temperature. Cells were incubated overnight at 4 °C with primary antibodies (for primary and secondary antibodies details see Supplementary Table 2) in PBST + 3% of HS. After washing with PBST, cells were incubated with secondary antibodies (Alexa, Life Technologies) for 45 minutes at room temperature. Nuclei were stained with DAPI (4′,6-diamidino-2-phenylindole; Sigma-Aldrich, cat. F6057). Images were acquired with Leica SP5 confocal microscopes. Image analysis was performed using Fiji 2.9.0. Quantification of nuclear and cytoplasmic intensity was performed with CellProfiler software (v4.1.3). Briefly, nuclei were detected by DAPI staining. Cytoplasm was arbitrarily defined by radially expanding nuclei by 10 pixels. Ratio of the integrated nuclear intensity of MTF1 signal over the cellular integrated intensity of MTF1 signal (nucleus + cytoplasm) was calculated and plotted.

Immunofluorescence analysis on human iPSCs and NPCs was performed on 1% Matrigel-coated glass coverslips in wells. Cells were fixed in 4% formaldehyde (Sigma-Aldrich, cat. F8775) in PBS for 10 minutes at room temperature, washed in PBS, permeabilised for 1 hour in PBST at room temperature and blocked in PBST + 5% of HS (Gibco, cat. 16050-122) for 5 hours at room temperature. Cells were incubated overnight at 4 °C with primary antibodies (for primary and secondary antibodies details see Supplementary Table 2) in PBST + 3% of HS. After washing with PBS, cells were incubated with secondary antibodies (Alexa, Life Technologies) for 45 minutes at room temperature. Nuclei were stained with DAPI (4′,6-diamidino-2-phenylindole; Sigma-Aldrich, cat. F6057). Images were acquired with Zeiss LSM900 Airyscan 2 confocal microscopes. Image analysis was performed using Fiji 2.9.0.

RNA isolation, reverse transcription and quantitative PCR

For cellular lysate, RNA was isolated using Total RNA Purification Kit (Norgen Biotek, cat. 37500) and complementary DNA (cDNA) was made from 500 ng using M-MLV reverse transcriptase (Invitrogen, cat. 28025-013), RNaseOUT (40 units/µL), random primers (200 nM), dNTPs (10 mM), First-Strand Buffer 5x, DTT (0.1 M). For zebrafish larvae, total RNA was isolated taking advantage of the phenol-chloroform extraction. Total RNA was isolated from pools of 10 animals by using TRIzol Reagent (Life Technologies, cat. 15596026), following manufacturer’s instructions for standard trizol-chloroform-ethanol extraction procedure. RNase-free glycogen was used as suggested by the protocol, to increase the yield of the RNA precipitation step. 2 μg of total RNA were reverse transcribed into cDNA by using Superscript III Reverse Transcriptase (Invitrogen, cat. 18080044) and a mixture of oligo(dT)18 primers (500 μg/mL); dNTP mix (10 mM); DTT (0.1 M); 5x First-Strand Buffer; RNaseOUT (40 units/μL).

For real-time PCR, SYBR Green Master mix (Bioline, cat. BIO-94020) was used. Primers are detailed in Supplementary Table 3. Technical replicates were carried out for all quantitative PCR. For mouse ESCs, human iPSCs and NPCs, Gapdh and GAPDH were used as endogenous control to normalise expression. The Ct mean of zebrafish gapdh, eef1a1l1, tuba1b and b2m was used as an endogenous housekeeping control for normalisation, due to the variability shown looking at the expression levels of those genes. qPCR data were acquired with QuantStudio™ 6&7 Flex Software 1.0 and 1.3 version.

RNA sequencing: Library Preparation

Total RNA was quantified using the Qubit 4.0 fluorimetric Assay (Thermo Fisher Scientific). Libraries were prepared from 125 ng of total RNA using the NEGEDIA Digital mRNA-seq research grade sequencing service (Negedia srl)¹⁴⁰ which included library preparation, quality assessment and sequencing on a NovaSeq 6000 sequencing system using a single-end, 100 cycles strategy (Illumina Inc.).

Bioinformatics workflow

The raw data were analysed by Next Generation Diagnostics srl proprietary NEGEDIA Digital mRNA-seq pipeline, which involves a cleaning step by quality filtering and trimming, alignment to the reference genome and counting by gene^142,143. Genes were sorted removing those that had a total number of counts below 5 in at least 3 samples out of 30. After applying this filter, we identified 11,851 expressed genes that were considered for further analyses.

Differential expression analysis was carried out in the R environment (v. 4.1.0) with Bioconductor (v. 3.14) exploiting the DESeq2 R package. (v. 1.34.0)¹⁴⁴ and edgeR (v. 3.36.0)¹⁴⁵. DESeq2 performs the estimation of size factors, the estimation of dispersion for each gene and fits a negative binomial generalised linear model with two-tailed Wald statistics. Biological significance of DEGs between Q128 and Q15 (Fig. 1g), of DEGs between Q128_Mtf1 and Q128 (Supplementary Fig. 5b) and of genes rescued by Mtf1 (Fig. 5c) was explored by Gene Ontology (GO) term enrichment analysis using Enrichr software (v. 3.0) and including the categories of Biological Processes (BP) (2021) and Molecular Function (MF) (2021).

To perform the enrichment of cell population proliferation (GO:0008283 and¹⁴⁶) and apoptosis (R-HSA-109581 and WP1351) gene signatures in Q128 and Q15 cell lines overexpressing Mtf1, we used Gene Set Enrichment Analysis (GSEA) software (v 4.3.2)¹⁴⁷. Pre-ranked gene set lists were generated based on log₂ fold-change (FC) values as obtained by the differential expression analysis between Q128_Mtf1 vs Q128 and Q15_Mtf1 vs Q15.

Motif enrichment analysis was performed using the Motif Analysis tool from the Regulatory Genomics Toolbox suite (https://reg-gen.readthedocs.io). Mtf1 MRE was obtained from the Jaspar database (9th version, http://jaspar.genereg.net/)¹⁴⁸. The command “rgt-motifanalysis matching” was used to search for binding sites on the promoter region of all the genes of interest; then, “rgt-motifanalysis enrichment” was used to perform a Fisher’s exact test to evaluate if the proportion of binding sites in the gene set of interest is higher than expected by chance.

One representative biological replicate of RNA sequencing data and Mtf1 MRE were visualised as tracks in the Integrated Genomics Viewer (IGV v. 2.16.0) and shown in Fig. 5h.

Volcano plots and scatter plots were produced with log₂ FC and -log₁₀ p-value exploiting the ggscatter function from ggpubr R package (v. 0.4.0.5). Heatmaps were made using CPM values with the pheatmap function from pheatmap R package (v. 1.0.12).

Alignment of Mtf1 orthologues

The Mtf1 sequence alignment was performed using the Clustal Omega software (v1.2.4, https://www.ebi.ac.uk/Tools/msa/clustalo/)¹⁴⁹. The identity between the sequences was calculated using the Sequence Manipulation Suite program (https://www.bioinformatics.org/sms2/ident_sim.html).

Metal analysis

After a 48 hours conditioning treatment, 10⁷ Q15 and Q128 cells were collected, centrifuged in Eppendorf tubes, the supernatant was removed and the cell pellets were stored at −80 °C. Immediately before the analysis, cells were thawed and resuspended in concentrated HNO₃ (68%), 1 mL added to each sample. After complete dissolution, we added 2 mL of ultrapure water and fully mineralised the sample by means of microwave heating, using a high efficiency high pressure microwave reactor (Ultrawave, Milestone, Bergamo, Italy). The same procedure was applied to the culture medium, before and after the conditioning treatment. The calibration curves for the metals quantitation were obtained by preparing six standard solutions at different concentrations (0.005-0.010-0.025-0.050-0.100-0.200 ppm), making use of certified multi-element and single-element standards (Agilent). All the samples were then filtered through a 0.20 µm syringe filter. The metal analysis, with the atomisation and ionisation of the samples obtained in an Argon plasma, was performed by Inductively Coupled Plasma – Optical Emission Spectrometry (ICP-OES, mod 5110, Agilent).

Generation of Zebrafish HD model

HD zebrafish were generated by microinjection in one-cell stage embryos of mRNA encoding the first exon of human HTT including 74Q or 16Q, fused in-frame to eGFP coding sequence. Q74eGFP and Q16eGFP were cloned into pCS2+ plasmid in order to allow for the in vitro transcription. pCS2_Mtf1 and pCS2_mCherry plasmids were also generated to obtain Mtf1 and mCherry mRNAs used for injections in HD zebrafish embryos.

For RNA in vitro transcription, 2.5 μg of pCS2_Q74eGFP, pCS2_Q16eGFP, pCS2_Mtf1, and pCS2_mCherry were linearised by overnight digestion at 37 °C with HF-Not I (New England Biolabs, cat. R3189S). The digestion volume was then concentrated by the DNA Clean & Concentrator kit (Zymo Research, cat. D4003) and used for the capped transcription reaction (mMESSAGE mMACHINETM SP6 Transcription Kit, Thermo Fisher Scientific, cat. AM1340) by SP6 RNA polymerase. After removing the DNA template by DNase treatment (Thermo Fisher Scientific, cat. AM2238) for 15 minutes at 37 °C, RNA was purified by Phenol-Chloroform extraction (as discussed in ‘RNA isolation, reverse transcription and quantitative PCR’ paragraph). RNA was quantified by Nanodrop ND-1000 and then diluted according to the need in a mix of 10% Danieau buffer [8 mM NaCl, 0.7 mM KCl, 0.4 mM MgSO₄, 0.6 mM Ca(NO₃)₂, 2.5 mM HEPES, pH 7.6], 10% Phenol Red (Merck, cat. 1072410025) and RNase-free water.

In order to select the injection dose that caused the highest rate of malformations with the lowest level of death, we injected increasing doses of Q74eGFP mRNA ranging from 150 to 1000 pg/embryo and phenotypically scored 24 hpf embryos. Once established the dose of 250 pg/embryo, under a light microscope, embryos were injected with in vitro transcribed mRNAs. Microinjected embryos were then transferred to fish water and incubated at 28 °C. Unfertilised eggs were recognised and discarded 4 hours post-microinjection. 24 hpf tadpoles were dechorionated using dedicated needles under a light microscope.

Whole-mount stainings

Injected embryos were anaesthetised with tricaine and immobilised in 1.5% Methylcellulose or 2% low melting agarose and analysed using a Leica M165FC fluorescence microscope. Confocal zebrafish images were acquired with a Nikon C2 H600L confocal microscope.

For Acridine Orange hemi (zinc chloride) salt in vivo staining (Merck, cat. A6014), 24 hpf embryos were dechorionated, transferred into a 6-well plate and incubated in about 2 mL of Acridine Orange (20 μg/mL) per well in fish water for 15 minutes at 28 °C. The Acridine solution was then removed and embryos were washed three times with 1 mL of fish water. Before being observed on a glass slide by a fluorescence microscope, tadpoles were anaesthetised by Tricaine.

For the TUNEL assay, ApopTag Fluorescein In Situ Apoptosis Detection Kit (Merck, cat. S7110) and collagenase (Merck, cat. C9891) were used. 7 embryos-30 hours post-microinjection per condition were placed in an Eppendorf, anaesthetised with Tricaine and fixed in 4% paraformaldehyde (PFA) at 4 °C overnight. Then, PFA was removed and samples were washed with PBS (3 times, 10 minutes each), while shaking. Embryos were dehydrated through a series of methanol solutions ranging from 10% to 100% and frozen at −20 °C overnight. Then, embryos were rehydrated with a series of 70-50-30% methanol solutions and washed with PBS with Tween-20 for 10 minutes, while shaking. After that, collagenase was applied for 8 minutes while shaking and the excess was washed away by PBS with Tween-20 washing steps (3 times, 5 minutes each). Samples were incubated for 1 hour in the equilibration buffer while shaking, then for 2 hours at 37 °C in working strength TdT. The reaction was stopped by washing twice the samples in the working strength Stop/Wash buffer. Next, there was a blocking step of 1 hour with PBS with Tween-20 while shaking, and then embryos were incubated overnight in a Working Strength anti-digoxigenin conjugate solution at 4 °C in the dark. The morning after, the antibody solution was removed, samples were washed with PBS (4 times, 10 minutes each) and analysed by a confocal microscope. Zebrafish larvae anterior structures were scanned in 70 stacks of 3.475 μm each, spanning their entire depth. We quantified the fractions of the fluorescent positive area over the total area (excluding the yolk region). For quantification analyses, all images were acquired with the same exposure parameters and processed using Fiji software (v2.9.0). Statistical analyses were carried out with Past (v.4.03) and Prism (v.9.5.0).

AAV-PHP.eB vector injection, mouse phenotyping and tissue collection

AAV-PHP.eB viral particles were produced and titrated in Broccoli’s lab as described previously⁷⁵. This viral vector was modified to express under the control of the Ef-1ɑ promoter the candidate gene Mtf1 or either eGFP as a control. Vascular injection was performed in a restrainer that positioned the tail in a heated groove. The tail was swabbed with alcohol and then injected intravenously. WT and R6/2 mice were randomised in groups and injected in the tail vein at 4.2 weeks of age. Following injection, all mice were weighed twice a week. Phenotyping was carried out, blind to genotype and treatment, twice a week. The balance and the motor coordination were assessed by the Rotarod test and Horizontal Ladder Task. Total DNA was isolated from animal tissues (cortex and striatum) using the Qiagen DNeasy Blood and Tissue Kits (QIAGEN, cat. 9504).

Animal husbandry

All Zebrafish experiments were carried out at the Fish Facility in the Department of Biology of the University of Padova. Zebrafish larvae were kept at most three days in Petri dishes with fish water (60 mg of Instant Ocean, cat. SS15-10, per litre of distilled water) at neutral pH at 28 °C, according to standard procedures (http://ZFIN.org).

Mouse colonies were established at IRCCS Neuromed. Breeding pairs of the R6/2 line of transgenic female mice [strain name: B6CBA-tgN (HDexon1) 62Gpb/1 J] with ∼160  ±  10 CAG-repeat expansions were purchased from the Jackson Laboratories. Mice were housed under standard conditions (22 ± 1 °C, 60% relative humidity, 12 h light/dark schedule, 3–4 mice/cage, with free access to food and water). Male R6/2 mice (5-6 weeks of age) were crossed with female B6CBA WT mice (5-6 weeks of age) for colony maintenance; the resultant WT and R6/2 mice were used for all the experiments performed in this study. A complete list of mice used in this study, indicating age, sex, treatments and measurements, is reported in Supplementary Table 4. All experimental procedures were approved by the IRCCS Neuromed Animal Care Review Board ethics committee and by Istituto Superiore di Sanità (ISS permit number: 548/2022-PR) and were conducted according to the 2010/63/EU directive for animal experiments.

Motor behaviour tests

All behaviour tests were carried out during the light phase of the light/dark cycle. Mice were tested before and after treatment at the indicated time points. Before training and testing, mice underwent a period of habituation to the testing room and equipment. All mice received training for two consecutive days on each instrument and task before performing motor behaviour measurements. Mice were tested at fixed speed (0.1 rcf) on a rotarod apparatus for 1 min. Each mouse was tested in three consecutive trials of 1 min each, with 1 min rest between trials. The time spent on the rotarod in each of the three trials was averaged to give the overall time for each mouse. In the horizontal ladder task, the mice spontaneously walked along a horizontal ladder with variable and irregular spacing between rungs. In each test session, the mouse performance was evaluated using an established footfall scoring system¹⁵⁰, which allows for qualitative and quantitative evaluation of forelimb and hindlimb placement on the ladder rungs. All motor tests were conducted by the same experimenter who was blinded to mouse genotype and experimental group throughout the entire course of the analysis.

Clasping analysis

The clasping score is determined over 30 seconds. In particular, mice were suspended by their tails from a height of 50 cm and a limb-clasping response was defined as the withdrawal of any limb to the torso for more than 2 seconds. The following scores were used: 0 (absence of clasping), 0.5 (withdrawal of any single limb), 1 (withdrawal of any two limbs), 1.5 (withdrawal of any three limbs), 2 (withdrawal of all four limbs).

Dihydroethidium (DHE)

WT and R6/2 mice were sacrificed by cervical dislocation. Brains were removed and trimmed by removing the olfactory bulbs and spinal cord. The remaining brain was processed and embedded in paraffin wax, 10 µm coronal sections were cut on an RM 2245 microtome (Leica Microsystems) and floated in a 40 °C water bath containing distilled water. Sections were transferred onto glass slides suitable for immunohistochemistry and let dry overnight at room temperature. Samples were deparaffinized in xylene for 30 minutes, transferred to 100% alcohol for 10 minutes and then once through 95%, 70% and 50% alcohol respectively for 10 minutes each, washed in PBS twice. In situ superoxide generation production was detected by fluorescence with DHE (Sigma-Aldrich, cat. D7008). Samples were incubated with DHE (2 µM) in a light-protected humidified chamber at 37 °C for 30 minutes. Slides were rinsed with PBS twice and observed at the microscope. For each staining, four mice per experimental group were used and three coronal sections for each animal were acquired with the Nikon ECLIPSE Ni microscope and analysed by NIS-Elements Image Software (v. 4.40, Nikon) and Fiji software 2.9.0.

mHTT aggregates immunostaining

WT and R6/2 mice were sacrificed by cervical dislocation. Brains were removed and trimmed by removing the olfactory bulbs and spinal cord. The remaining brain was processed and embedded in paraffin wax, 10 µm coronal sections were cut on an RM 2245 microtome (Leica Microsystems) and floated in a 40 °C water bath containing distilled water. Sections were transferred onto glass slides suitable for immunohistochemistry and let dry overnight at room temperature. Samples were deparaffinized in xylene for 30 minutes, transferred to 100% alcohol for 10 minutes and then once through 95%, 70% and 50% alcohol respectively for 10 minutes each, washed in PBS twice. To unmask the antigenic epitope, antigen retrieval was performed using citrate buffer method (incubate with citrate buffer 10 mM, pH 6.0 at 95-100 °C for 15 minutes then allow slides to cool for 15 minutes). Slides were washed twice with PBS, permeabilized in TBS-Triton 0.1% for 10 minutes, then incubated in a humidified chamber at room temperature with blocking buffer (Horse serum 10% in PBS) for 1 h. Blocking buffer was removed and slides were incubated in a humidified chamber at 4 °C overnight using a mouse anti-HTT antibody (clone EM48, for details see Supplementary Table 2). After washing three times with PBS, cells were incubated with secondary antibodies for 1 h in a humidified chamber at room temperature, protected from light. Nuclei were stained with DAPI (4′,6-diamidino-2-phenylindole; Sigma-Aldrich, cat. F6057). Four mice per experimental group were used and three coronal sections for each animal were acquired with the Nikon ECLIPSE Ni microscope and analysed by NIS-Elements Image Software (v. 4.40, Nikon) and Fiji software 2.9.0.

Statistics and reproducibility

No statistical method was used to predetermine sample size, but our sample sizes are similar to those commonly used in our field of research. No data were excluded from the analyses. Data distribution was assumed to be normal but this was not formally tested. P-values for experiments involving repeated measures (Fig. 1c, Fig. 4f, Fig. 7b (left), 7c (left) and 7g, Fig. 8d,f, Supplementary Fig. 1e, Supplementary Fig. 4a,c, Supplementary Fig. 8c, Supplementary Fig. 9c) were calculated with Two-way Repeated Measure ANOVA with Bonferroni’s correction. For experiments with cell lines, we randomly allocated a fraction of each cell population to different biological replicates. For the analysis of immunostaining and flow cytometry data, we analysed random fields or random fraction of cells. Other kinds of experiments were not randomised. Data collection and analysis were not performed blind to the conditions of the experiments, but data analyses have been performed with identical parameters and software. Analysis of mouse motor behaviours was performed in blind. Data representation and statistical analyses were performed using R software (v. 4.0.0 and v. 4.1.0) and PAST (v4.03), unless stated otherwise. All bars, error bars and box plots are defined in figure legends. The number of biological replicates and independent experiments, both >2, is indicated in figures legends. The statistical tests used are indicated in figure legends. All qPCR experiments were performed with three technical replicates. Key experimental results have been obtained by 2 independent operators.

Reporting summary

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

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• Source: https://www.nature.com/articles/s41467-023-39552-9