Neuroprotective effects of hepatoma-derived growth factor in models of Huntington’s disease

Plasmids

The following plasmids were used for transfection: pcDNA3.1 mCherry, HTT-exon1-Q25-mCherry-Myc-His, and HTT-exon1-Q97-mCherry-Myc-His (Hipp et al, 2012); pcDNA3.1 HTT-exon1-Q25-His, and HTT-exon1-Q72-His (cloned from pPGK HTT-exon1-Q25, HTT-exon1-Q72) (Jeong et al, 2011); pCI-neo EGFP-HA (cloned from pCI-neo Fluc-EGFP) (Gupta et al, 2011); pCMV6-Entry HDGF-Myc-Flag (RC204148; OriGene); and cytHDGF-Myc-Flag. To generate cytHDGF, 12 point mutations were introduced into the two nuclear localization signals of HDGF by gene synthesis (Kishima et al, 2002), and a nuclear export sequence (SELQNKLEELDLDSYK) (Goedhart et al, 2012) was added C-terminally. For lentiviral expression, EYFP, EYFP-P2A-Flag-HDGF, and EYFP-P2A-Flag-cytHDGF were synthesized and cloned into pFhSynW2 (kindly provided by Dieter Edbauer) (May et al, 2014). psPAX2 and pcDNA3.1 VSV-G (kindly provided by Dieter Edbauer) were used for lentiviral packaging. Adenoviral plasmids were generated at the Viral Vector Production Unit, Universitat Autònoma de Barcelona: pAAV-CAG EYFP, EYFP-P2A-Flag-HDGF, and EYFP-P2A-Flag-cytHDGF. For recombinant protein production, HDGF and cytHDGF with a C-terminal Flag-TEV-His sequence were cloned into pET-17b (kindly provided by Kathrin Lang).

Mouse lines

All animal experiments were approved by the Government of Upper Bavaria, Germany (permit numbers 55.2-1-54-2532-168-14, 55.2-1-54-2532-19-2015, ROB-55.2-2532.Vet_02-20-05, and ROB-55.2-2532.Vet_02-19-083) and conducted in accordance with the relevant guidelines and regulations. Mice were housed with ad libitum access to food and water in the animal facilities of the Max Planck Institute for Biological Intelligence. Transgenic R6/2 mice (Mangiarini et al, 1996) (#002810; JAX stock) were maintained by breeding hemizygous R6/2 males with the female F1 progeny of a cross between CBA (Janvier Labs) and C57BL/6 (Janvier Labs) mice. zQ175DN (Menalled et al, 2012; Southwell et al, 2016) (#029928; JAX stock), Hdgf^−/− (Gallitzendoerfer et al, 2008) (kindly provided by Sørge Kelm), GAD2-Cre (Taniguchi et al, 2011) (#010802; JAX stock), and Ai9 tdTomato (Madisen et al, 2010) (#007909; JAX stock) mice were kept on C57BL/6 background. To generate HDGF-deficient R6/2 mice, hemizygous R6/2 males were crossed to homozygous Hdgf^−/− females, followed by mating the F1 offspring R6/2 transgenic males with heterozygous Hdgf^+/− females. For genetic labeling of cortical interneurons, heterozygous GAD2-Cre mice were crossed to homozygous tdTomato reporter mice. Immunostainings shown in Figs 2A and D and S2C and D, Western blots shown in Fig S5B and C, and survival analysis of striatal injected mice shown in Fig S5F were performed on female mice. In all other experiments, groups of mixed sex were used. To reduce animal numbers, parts of the behavioral experiments in Figs S6E–G and S9E–G were done in parallel with N = 9–10 WT, EYFP, and N = 6 R6/2; EYFP mice serving as controls for both wtHDGF- and cyt-HDGF-injected mice. CAG repeat length was determined by Laragen, Inc., and amounted to 207 ± 12 (SEQ CAG No., mean ± SD) for R6/2 mice.

Recombinant protein production

BDNF was purchased from R&D Systems (248-BD/CF). Recombinant HDGF protein was produced at the Protein Production Core Facility, Max Planck Institute of Biochemistry. In brief, pET-17b HDGF-Flag-TEV-His and cytHDGF-Flag-TEV-His were transformed into Rosetta (DE3) cells and expressed via autoinduction (Studier, 2005) at 20°C overnight. After pelleting, the cells were resuspended in His Binding Buffer (50 mM sodium phosphate pH 8, 500 mM NaCl, 10 mM imidazole, 1 mM TCEP, and 10% glycerol) supplemented with protease inhibitor mix (prepared in-house; 1 mM AEBSF HCl, 2 μg/ml aprotinin, 1 μg/ml leupeptin, 1 μg/ml pepstatin), 2.4 U/ml benzonase (produced in-house), and 2 mM MgCl₂. Cell disruption was conducted using an Emulsiflex C5 homogenizer (Avestin). Lysates were centrifuged for 30 min at 50,833g, 4°C. Per 50 ml supernatant, 2.5 ml of 5% polyethyleneimine solution was added for nucleic acid removal, followed by stirring for 10 min, and centrifugation for 30 min at 50,833g, 4°C. Proteins were precipitated by stirring for 1 h with 21.8g ammonium sulfate per 50 ml supernatant. After centrifugation for 30 min at 50,833g, 4°C, the pellet was resuspended in 50 ml His Binding Buffer and applied to a 1-ml BabyBio Ni-NTA column (45 655 103; Bio-Works) equilibrated in His Binding Buffer. The column was washed with 4% His Elution Buffer (50 mM sodium phosphate pH 8, 500 mM NaCl, 250 mM imidazole, 1 mM TCEP, and 10% glycerol), followed by protein elution in a straight gradient from 4% to 100% His Elution Buffer. The purest fractions were identified via SDS–PAGE and pooled accordingly. His-tag cleavage was performed by adding His-tagged TEV protease (produced in-house) and dialysis in PBS (137 mM NaCl, 2.7 mM KCl, 8.1 mM Na₂HPO₄, 1.5 mM KH₂PO₄, pH 7.4) with 1 mM TCEP overnight. The remaining tag and protease were removed by reverse Ni-NTA purification in batch mode (Ni-Sepharose High Performance; 17-5268-02; GE Healthcare). Final polishing of the target proteins was done through size exclusion on a HiLoad 26/600 Superdex 75 prep grade column (GE Healthcare) eluting in PBS with 1 mM DTT. Proteins were concentrated to 1 mg/ml using an Amicon Ultra 15 column (Amicon). The quality of recombinant proteins was checked via SDS–PAGE and DLS analysis. Identity was confirmed through LC-MS (micro-ToF). Protein aliquots were snap frozen in liquid nitrogen and stored at −80°C.

PC12 cell culture and LDH assay

Stable PC12 cell lines with inducible expression of EGFP-fused HTT-exon1-Q23 or HTT-exon1-Q74 were a gift from David Rubinsztein and cultured as described (Wyttenbach et al, 2001). For viability studies, cells were seeded on coverslips in 24-well plates and transfected 12 h later using Lipofectamine LTX with Plus Reagent (15338100; Thermo Fisher Scientific) following the manufacturer’s instructions. Induction of HTTQ23-EGFP or HTTQ74-EGFP was carried out 5 h after transfection by adding doxycycline at 1 μg/ml. After induction with doxycycline, cells were kept at 1% horse serum to maintain them in a quiescent-like state. LDH assay was performed with 50 μl of the medium taken at 60 h post-transfection according to the manufacturer's instructions (Pierce LDH Cytotoxicity Assay Kit; Thermo Fisher Scientific).

Primary neuronal cultures

Cultureware was coated with 1 mg/ml poly-D-lysine (P7886; Sigma-Aldrich) in borate buffer (50 mM boric acid and 12.5 mM sodium tetraborate, pH 8.5) for 4 h to overnight at 37°C, 5% CO₂. After washing three times with Dulbecco’s Phosphate Buffered Saline with calcium and magnesium (DPBS_Ca2+Mg2+; D8662; Sigma-Aldrich), 5 μg/ml laminin (23017-015; Gibco) in DPBS_Ca2+Mg2+ was applied for 2–4 h at 37°C, 5% CO₂. Meanwhile, a pregnant CD-1 female was euthanized by cervical dislocation at embryonic day 15.5. The uterus was dissected and washed in ice-cold DPBS_Ca2+Mg2+. Embryos were harvested and decapitated in ice-cold dissection medium containing 1x penicillin/streptomycin, 10 mM HEPES pH 7.4, and 10 mM MgSO₂ in HBSS (24020-091; Gibco). Brains were extracted and meninges were removed before dissection of neocortices. Digestion of collected neocortices was conducted at 37°C for 15 min in pre-warmed trypsin-EDTA solution (T4299; Sigma-Aldrich) with 7.5 μg/ml DNase I (10104159001; Roche). Trypsin activity was blocked by washing with pre-warmed neurobasal medium (21103-049; Gibco) supplemented with 5% heat-inactivated FBS (S0115; Biochrom). After washing with pre-warmed culture medium containing 1x B-27 (17504-044; Gibco), 1x penicillin/streptomycin and 2 mM L-Glutamine (25030-024; Gibco) in neurobasal medium, the cells were dissociated in pre-warmed culture medium by triturating and pelleted by centrifugation at 130g for 5 min. Meanwhile, coated plates were washed twice with DPBS_Ca2+Mg2+. Cells were resuspended and plated in 100-mm culture dishes (Western blot), 24-well plates with cover glasses (immunocytochemistry) or 96-well plates (MTT assay) in pre-warmed culture medium at a density of 60,000/cm². Neuronal cultures were maintained at 37°C, 5% CO₂.

Transfection of primary neurons

Neurons were transfected at day in vitro (DIV) 7 using CalPhos Mammalian Transfection Kit (631312; Takara Bio). Transfection solution was prepared by adding 1.5 μg DNA per construct (3 μg in total) in 200 mM CaCl₂ dropwise to 2x HEPES-Buffered Saline at a ratio of 1:1, followed by incubation for 30 min. Cells on cover glasses were equilibrated in a fresh pre-warmed culture medium and incubated with 30 μl transfection solution for 3 h at 37°C, 5% CO₂. Fresh culture medium was acidified for at least 30 min at 37°C, 10% CO₂ before transfer of transfected cells and incubation for 30 min at 37°C, 5% CO₂. Cells on cover glasses were transferred back to the original medium and incubated at 37°C, 5% CO₂ for protein expression.

Lentivirus preparation and transduction of primary neurons

Lenti-X 293T cells (632180; Takara Bio) were expanded until 80% confluency in a three-layer 525 cm² culture flask (353143; Corning) at 37°C, 5% CO₂ in DMEM (41965-039; Gibco) supplemented with 10% heat-inactivated FBS, 1% geneticin (10131-019; Gibco), 1% nonessential amino acids solution (11140-050; Gibco), and 10 mM HEPES pH 7.4. For virus production, cells were seeded in growth medium, containing 10% FBS, 1% nonessential amino acids, and 10 mM HEPES pH 7.4 in DMEM, at a split ratio of 1:2 in a new three-layer 525 cm² culture flask. The next day, transfection mix was prepared by combining 59.5 μg of the respective pFhSynW2 expression plasmid, 35.2 μg psPAX2, and 20.5 μg pcDNA3.1 VSV-G in 4.9 ml DMEM with 345 μl TransIT-Lenti Transfection Reagent (MIR 6600; Mirus Bio) added to 4.8 ml DMEM, followed by incubation for 20 min. Growth medium was exchanged and transfection mix was added to cells. After overnight incubation, the medium was replaced by fresh pre-warmed growth medium, collected 48–52 h later, and centrifuged at 1,200g for 10 min. Lentiviral supernatant was filtered through a 0.45-μm filter and concentrated using Lenti X Concentrator (631232; Takara Bio) according to the manufacturer’s instructions. Virus was suspended in 600 μl buffer containing 50 mM Tris-HCl pH 7.8, 130 mM NaCl, 10 mM KCl, and 5 mM MgCl₂. Single-use aliquots were stored at −80°C. Viral titers were estimated using Lenti-X GoStix Plus (631280; Takara Bio) following the manufacturer’s instructions using a reference value of 6.014 (TU/ml)/GV. Calculated titers were in the range of 1.35 × 10⁶–2.77 × 10⁶ TU/ml.

For transduction of primary neurons, 50 μl of the culture medium was removed and lentiviral vectors were added with 100 μl fresh pre-warmed culture medium at DIV 7. The amount of virus was adjusted according to the titer ranging from 0.25 to 0.50 μl.

Immunocytochemistry on primary neurons

Cells were fixed with 4% PFA in PBS (sc-281692; ChemCruz) for 20 min. After washing twice with PBS, 50 mM NH₄Cl in PBS was applied for 10 min. Cells were rinsed with PBS and permeabilized with 0.3% Triton X-100 in PBS for 5 min, followed by washing 5 min with PBS. To prevent unspecific antibody binding, cells were incubated in a blocking solution containing 0.2% BSA (8076; Carl Roth), 5% normal donkey serum (017-000-121; Jackson ImmunoResearch Laboratories), 0.2% L-lysine hydrochloride, 0.2% glycine, and 0.02% NaN₃ in PBS for 30 min. Primary antibodies were applied for 1 h in primary antibody solution (0.3% Triton X-100, 2% BSA, and 0.02% NaN₃ in PBS). The following primary antibodies were used: goat anti-mCherry (AB0040-200, 1:500; Origene), mouse anti-His (DIA-900-100, 1:1,000; Dianova), chicken anti-EGFP (A10262, 1:1,000; Invitrogen), mouse anti-Flag (TA50011, 1:1,000; Origene), goat anti-Flag (ab1257, 1:2,000; Abcam), and rabbit anti-cleaved Caspase-3 (9661, 1:500; Cell Signaling Technology). Cells were washed for 5 min in PBS and incubated for 30 min in the dark with Alexa Fluor 488, Cyanine Cy3, and/or Alexa Fluor 647-conjugated secondary antibodies derived from donkey (Jackson ImmunoResearch Laboratories) at 1:250 dilution and 0.5 μg/ml DAPI in a secondary antibody solution containing 0.3% Triton X-100, 3% normal donkey serum, and 0.02% NaN₃ in PBS. After three 5-min washes with PBS, coverslips were dipped in Milli-Q water and mounted with ProLong Glass Antifade Mountant (P36984; Invitrogen). Images were acquired with a Leica TCS SP8 confocal microscope. For viability analysis, cells negative for active caspase-3 with intact nuclear morphology were categorized as viable.

MTT assay

Medium was exchanged with a 5:1 mixture of pre-warmed culture medium and 5 mg/ml MTT (M5655; Sigma-Aldrich) in PBS for a total volume of 120 μl. After incubation for 3–4 h at 37°C and 5% CO₂, 100 μl solubilizer containing 10% SDS and 45% dimethylformamide, adjusted to pH 4.5 with acetic acid, was added. Crystals were dissolved at 37°C, 5% CO₂ for 4 h to overnight, and absorbance was measured at 570 nm.

iPSC culture and differentiation to NPCs

Control iPSCs (Q33, ND36997) were obtained from NINDS Human Cell and Data Repository through the Coriell Institute. The HD Q71-iPSC line was a gift from George Q. Daley and is established and fully characterized for pluripotency (Park et al, 2008). iPSCs were cultured on Geltrex (Thermo Fisher Scientific) using mTeSR1 media (Stem Cell Technologies). Undifferentiated iPSCs were passaged using Accutase (1 U/ml; Invitrogen). iPSC lines were tested for mycoplasma contamination at least once every 2 wk confirming the absence of mycoplasma. NPCs were generated by inducing neural differentiation of iPSCs with STEMdiff Neural Induction Medium (Stem Cell Technologies) after the monolayer culture method (Chambers et al, 2009). Briefly, iPSCs were rinsed once with PBS, followed by the addition of 1 ml Gentle Dissociation Reagent (Stem Cell Technologies). After incubation for 10 min, the cells were gently collected, 2 ml of DMEM/F12 (Thermo Fisher Scientific) with 10 μM ROCK inhibitor (Abcam) was added, and cells were centrifuged at 300g for 10 min. After centrifugation, the cells were resuspended on STEMdiff Neural Induction Medium with 10 μM ROCK inhibitor and plated on poly-ornithine (15 μg/ml)/laminin (10 μg/ml)-coated plates at a density of 200,000 cells/cm² for neural differentiation.

Exogenous HDGF treatment and proteasome inhibition in NPCs

60% confluent NPCs were treated with 250 ng/ml recombinant HDGF protein or PBS as control for 24 h. The next day, cells were treated with 250 ng/ml fresh recombinant HDGF protein or PBS for 8 h in the presence of 5 μM MG-132 for proteasome inhibition or DMSO as control treatment.

Immunocytochemistry on NPCs

Cells were fixed with 4% PFA in PBS for 20 min and permeabilized with 0.2% Triton X-100 in PBS for 10 min. After permeabilization, the cells were blocked with 3% BSA in 0.2% Triton X-100 in PBS for 10 min. The cells were incubated with mouse anti-polyQ (MAB1574, 1:50; Millipore) primary antibody for 2 h. After washing with 0.2% Triton-X in PBS, the cells were incubated with goat anti-mouse Alexa Fluor 488 (A-11029, 1:500; Thermo Fisher Scientific) secondary antibody and 2 μg/ml Hoechst 33342 (1656104; Life Technologies) for 1 h. The cells were washed with 0.2% Triton-X in PBS and with distilled water. The coverslips were mounted with FluorSave reagent (Merck Millipore).

Filter trap assay

Cells were lysed in a non-denaturing lysis buffer supplemented with EDTA-free protease inhibitor cocktail (Roche) on ice. Cell lysates were homogenized through a 27G syringe needle. The protein concentration was determined from the whole protein lysate with a standard BCA protein assay. The equilibrated whole lysates were centrifuged at 8,000g for 5 min at 4°C. The pellets were resuspended with 2% SDS and loaded onto a cellulose acetate membrane assembled in a slot blot apparatus (Bio-Rad). The membrane was washed with 0.2% SDS and retained SDS-insoluble mHTT aggregates were detected with mouse anti-polyQ antibody (MAB1574, 1:5,000; Millipore).

Stereotactic viral injections

Viral AAV8 vectors were produced by the Viral Vector Production Unit, Universitat Autònoma de Barcelona. For striatal injections, R6/2 mice and their littermates underwent stereotactic surgery at 4 wk of age. Mice were injected intraperitoneally with 15 ml/kg body weight (BW) 20% mannitol in 0.9% NaCl. Metamizol (200 mg/kg BW) was orally administered. Anesthesia was induced with 4% isoflurane and maintained at 1.5–2% isoflurane using an O₂ flow rate of 1 liter/min. Body temperature was maintained with a heating pad. Carprofen (5 mg/kg BW) was given subcutaneously. Per injection site, mice were injected with 10⁹ gc AAV8 in 0.2 μl with a final concentration of 6% mannitol in 0.9% NaCl at (X, Y, Z) = (±1.7, 1.0, −3.0) and (X, Y, Z) = (±2.1, 0.3, −3.0) mm from bregma. For each of the four injections, the glass capillary was left in position for 3 min. Skull holes were covered with bone wax and the incision was closed with sutures. Intracerebroventricular viral injections at postnatal day 0 (P0) were performed similarly as previously described (Kim et al, 2014). In brief, pregnant females were monitored for birth at least every 12 h starting 17 d after the plug date to ensure surgery of newborn pups within 24 h after birth. Anesthesia was induced with 5% isoflurane and maintained at 2% isoflurane using an O₂ flow rate of 1 liter/min. Xylocaine 2% jelly (6077215.00.00; Aspen) was applied on the prospective injection sites for local anesthetic blockade. Per hemisphere, 10¹⁰ gc AAV8 in 1–2 μl was stereotactically injected with 15 nl/sec using a microinjection system (Nanoliter 2010, WPI) at (X, Y, Z) = (±0.8, 1.5, −1.5) mm from lambda. Xylocaine 2% jelly was again applied on injection sites to seal wounds. During surgery and for recovery, neonates were kept on a warming pad. To ensure success of fostering with CD-1 females, fecal pellets from the foster mother were solved in water and rubbed on the back of the injected pups. Neonates were placed in the foster mother’s cage as soon as they recovered and were moving normally. Mice were transferred to an inverted light cycle after surgery.

Behavior and life span analysis

All behavioral assessments were conducted during the dark phase of the diurnal cycle. For the open field test, mice were video recorded while exploring a custom-made squared box (40 × 40 × 40 cm) with black walls and white floor for 10 min with lights on. EthoVision XT 14 software (Noldus Information Technology) was used for automated tracking to quantify the distance traveled. Rotarod analysis was conducted on a RotaRod NG (Ugo Basile). Mice were trained twice on two consecutive days at 5 rpm for 5 min. On the third day, the latency to fall was measured with accelerating speed from 5 to 40 rpm over a 5 min period, and averaged over three trials. Forepaw grip strength was determined using the BIO-GS3 Grip Strength Test (Bioseb) with the BIO-GRIPBS bar for mice (Bioseb) as grasping tool. Measurements were averaged from three consecutive trials. For survival analysis, endpoint measures were severe burden according to behavior, appearance, and body weight or loss of righting reflex.

Immunostaining and fluorescence in situ hybridization on mouse tissue

Mice were transcardially perfused with PBS for 4 min, followed by 4% PFA in PBS for 6 min at 3–3.5 ml/min under ketamine/xylazine anesthesia. Brains and, if indicated, spinal cords were extracted and postfixed overnight in 4% PFA in PBS at 4°C.

For immunostaining of free-floating sections, fixed tissue was embedded in 4% agarose in PBS and serial 70-μm-thick sections were cut in PBS with a vibratome. Sections were permeabilized with 0.5% Triton X-100 in PBS for 15 min. If indicated, antigen retrieval was performed in 10 mM trisodium citrate pH 6 with 0.05% Tween 20 at 80°C for 15 min at 300 rpm in an Eppendorf ThermoMixer. To prevent unspecific antibody binding, sections were incubated in the blocking solution for 1 h on a shaker. Primary antibodies were applied in the primary antibody solution overnight at 4°C on a shaker. The following primary antibodies were used: rabbit anti-HDGF (ab128921, 1:500; Abcam), mouse anti-HTT (EM48, MAB5374, 1:500; Chemicon), mouse anti-Flag (TA50011, 1:1,000; Origene), goat anti-ChAT (AB144, 1:500; Chemicon), goat anti-DARPP32 (LS-C150127, 1:300; LifeSpan Biosciences), mouse anti-NGRN (MAB7947, 1:60; R&D Systems), chicken anti-GFAP (AP31806PU-N, 1:2,000, with antigen retrieval; Origene), mouse anti-APC (OP80, 1:20, with antigen retrieval; Calbiochem), and goat anti-IBA1 (ab107159, 1:1,000, with antigen retrieval; Abcam). After three 10-min washes in PBS, sections were incubated for 1 h in the dark with Alexa Fluor 488, Cyanine Cy3, and/or Alexa Fluor 647-conjugated secondary antibodies derived from donkey at 1:250 dilution in the secondary antibody solution with gentle shaking. NeuroTrace 640/660 (N21483; Invitrogen) was added at 1:500 dilution to the secondary antibody solution if indicated. Sections were washed three times for 10 min in PBS with DAPI added in the middle washing step at a concentration of 0.5 μg/ml. ProLong Glass Antifade Mountant was used for mounting.

For fluorescence in situ hybridization, fixed brains were immersed in 15% sucrose in Dulbecco’s Phosphate Buffered Saline (DPBS; D8537; Sigma-Aldrich), followed by 30% sucrose in DPBS, until the tissue sunk. Cryopreserved brains were frozen in Tissue-Tek O.C.T. Compound (4583; Sakura Finetek) and coronally sectioned at 10 μm thickness on a cryostat. RNAscope Fluorescent Multiplex Assay (320850, 322000, and 322340; ACD) was conducted according to the manufacturer’s instructions (320293-USM and 320535-TN; ACD) with RNAscope Probe against Hdgf (524601; ACD). Immunostaining against HDGF protein was performed before counterstaining with DAPI similar as immunostaining of free-floating sections. Briefly, brain sections were incubated in the blocking solution for 2 h, followed by overnight incubation with rabbit anti-HDGF antibody (ab128921, 1:500; Abcam) in the primary antibody solution at 4°C. After washing four times for 5 min with RNAscope 1x Wash Buffer, Alexa Fluor 488-conjugated secondary antibody raised in donkey was applied at 1:250 dilution in the secondary antibody solution for 2 h. Sections were washed four times for 5 min with RNAscope 1x Wash Buffer, counterstained with RNAscope DAPI, and mounted with ProLong Glass Antifade Mountant. Images were acquired with a Leica TCS SP8 confocal microscope.

Western blotting

Primary neurons were lysed in 100 μl ice-cold lysis buffer containing 50 mM Tris–HCl pH 7.5, 150 mM NaCl, 1% Triton-X 100, 2 mM EDTA, protease inhibitor cocktail (04693159001; Roche), and phosphatase inhibitor (04906837001; Roche). Lysates were centrifuged for 10 min at 4,000g, 4°C, and supernatants were collected for Western blotting.

To obtain mouse brain tissue lysates, mice were euthanized by cervical dislocation. Brains were extracted and cerebellum, hippocampus, cortex, and striatum (per hemisphere) were dissected on ice. Dissected brain regions were homogenized in ice-cold lysis buffer, incubated on ice for at least 30 min, and centrifuged at 15,000g for 15 min at 4°C. Supernatants were collected and protein concentrations were determined with DC Protein Assay Kit (5000112; Bio-Rad) following the manufacturer’s instructions.

Samples were boiled at 95°C for 5 min with Laemmli sample buffer (1610747; Bio-Rad) containing 2-mercaptoethanol. Per lane, 25 μl cell lysate and 50 μg protein for tissue lysates was loaded on 4–15% (5678084; Bio-Rad) and 10% TGX Stain-Free Protein Gels (4568034 and 5678034; Bio-Rad), respectively. Proteins were separated along with 5 μl Precision Plus Protein All Blue Prestained Protein Standards (1610373; Bio-Rad) at 80–120 V in SDS–PAGE-running buffer containing 25 mM Tris, 192 mM glycine, and 0.1% SDS. After electrophoretic separation, stain-free gels were activated using the ChemiDoc MP Imaging System (17001402; Bio-Rad). Transfer onto low-fluorescence polyvinylidene fluoride membranes (1620264; Bio-Rad) was conducted at 2.5 A, up to 25 V for 10 min with the Trans-Blot Turbo Transfer System (1704150; Bio-Rad) according to the manufacturer’s instructions for using traditional semi-dry consumables. Once the protein transfer was complete, the stain-free blot image was acquired using the ChemiDoc MP imager. Membranes were then blocked for 1 h with 3% BSA and 5% powdered milk (T145; Carl Roth) in TBS (20 mM Tris-HCl pH 8, 150 mM NaCl) with 0.1% Tween 20 (TBS-T). After rinsing twice and washing for 5 min in TBS-T, primary antibodies were applied overnight at 1:1,000 dilution in 3% BSA and 0.01% NaN₃ in TBS-T at 4°C. The following primary antibodies were used: rabbit anti-HDGF (244498; Abcam), mouse anti-AKT (2920; Cell Signaling Technology), rabbit anti-p-AKT (4060; Cell Signaling Technology), rabbit anti-ERK (9102; Cell Signaling Technology), and mouse anti-p-ERK (9106; Cell Signaling Technology). The next day, membranes were rinsed twice and washed three times for 10 min in TBS-T. Rhodamine-conjugated anti-tubulin antibody (12004166; Bio-Rad) and StarBright Blue 520/700 secondary antibodies (Bio-Rad) were applied at 1:2,500 dilution in TBS for 1 h, followed by rinsing twice and washing three times for 10 min in TBS-T. Blot image was acquired using the ChemiDoc MP imager and quantified with Image Lab version 6.0.1, build 34 software (Bio-Rad). HDGF protein quantity was normalized to total protein per sample. For quantification of exogenous HDGF, the area under the curve was quantified at the band heights of EYFP-P2A-Flag-HDGF/cyt-HDGF and Flag-HDGF/cyt-HDGF irrespective of the presence or absence of a protein band. The sum was calculated and normalized to total protein per sample.

Immunohistochemistry on human brain sections

Formalin-fixed paraffin-embedded tissue sections of 5 μm thickness from the primary motor cortex of three HD autopsy cases and three age-matched controls were provided by the Neurobiobank Munich, Ludwig-Maximilians-Universität München. Informed consent was available for all cases. The experiments were approved by the ethics committee of the Max Planck Society and performed in accordance with the relevant guidelines and regulations. All HD cases were symptomatic; demographic information is described in Burgold et al (2019).

Immunohistochemistry was performed on a VENTATA BenchMark ULTRA (Roche). After standard pretreatment in CC1 buffer (Roche), sections were incubated with rabbit anti-HDGF antibody (ab244498; Abcam) at a dilution of 1:100 for 32 min. The UltraView Universal DAB Detection Kit (Roche) was used for detection and counterstaining was performed with hematoxylin for 4 min. Images were acquired with a Leica THUNDER imager. Control stainings without the primary antibody were imaged using an Olympus BX45 microscope with Olympus DP32 camera.

Data analysis and statistics

Images were analyzed and/or processed with the open-source image analysis software Fiji (Schindelin et al, 2012). GraphPad Prism 9.2.0 (GraphPad Software) was used for graphical representation and statistical analysis. Statistics are detailed in the figure legends. Differences were considered statistically significant with P < 0.05. In bar plots, columns with error bars represent mean ± SD. In violin plots, the thick black line and colored area indicate the median and interquartile range, respectively.