Sex as a biological variable. Sex was not considered as a biological variable. Both male and female mice were used in this study.

Antibodies. The following antibodies were used for Western blotting: Nedd4 (07-049, EMD Millipore), Nedd4 (21698-1-AP, Proteintech), GFP (G1544, Sigma), GFP (632381, TaKaRa), GFP (GTX-113617, Genetex), T7-Tag (#6885, Cell Signaling Technology), huntingtin (MAB5490, MAB5492, MAB2166, MAB5374, MAB1574, MilliporeSigma), huntingtin (BML-PW0595A, Enzo), Huntingtin (ab109115, Abcam), HA- Tag (#3724, Cell Signaling Technology), HA-Tag (MMS-101P, Covance), HA-tag (66006-2-lg, Proteintech), α-tubulin (T5168, Sigma), p62 (18420-1-AP, Proteintech), p62 (P0067, Sigma), p62 (ab56416, Abcam), Ub (P4D1) (sc-8017, Santa Cruz), GAPDH (MAB374, MilliporeSigma), GAPDH (60004-1-lg, Proteintech), β-actin (ab6276, Abcam), Beclin-1 (#3738, Cell Signaling Technology), phospho-S6 (Ser235/236, #2211, Cell Signaling Technology), S6 (#2217, Cell Signaling Technology), Mdm2 (sc-965, Santa Cruz), Mdm2 (27883-1-AP, Proteintech), p53 (21891-1-AP, Proteintech), Vinculin (MAB3574, Millipore), and HRP-conjugated goat anti-rabbit and goat anti-mouse IgG (H+L) secondary antibodies (111-035-144 and 115-035-146, Jackson ImmunoResearch Laboratories). The following antibodies were used for IP: GFP (AFP5002, Qbiogene), GFP (G1544, Sigma), and huntingtin (MAB5490, MilliporeSigma). The following antibodies were used for immunocytochemistry: huntingtin (MAB5492, MilliporeSigma), huntingtin (5656S, Cell Signaling Technology), Nedd4 (21698-1-AP, Proteintech), NeuN (ABN90, Millipore), GFAP (sc-33673, Santa Cruz), MAP2 (ab11267, Abcam), Alexa Fluor 488 Phalloidin (A12379, Thermo Fisher), and Alexa Fluor 488 donkey anti-mouse IgG(H+L) secondary antibody (A-21202, Thermo Fisher).

Reagents. The following reagents were purchased as indicated: DMEM (11995065, Thermo Fisher), L-Glutamine (25030081, Thermo Fisher), Penicillin-Streptomycin (15140122, Thermo Fisher), Neurobasal media (21103049, Thermo Fisher), B-27 Supplement (17504044, Thermo Fisher), N-ethylmaleimde (23030, Thermo Fisher), PMSF (36978, Thermo Fisher), ZeptoMetrix RETROtek HIV-1 p24 Antigen ELISA (22-156-700, Fisher), FBS (100-106, Gemini Bio), CHX (14126, Cayman Chemical), bafilomycin A1 (11038, Cayman Chemical), bortezomib (#2204, Cell Signaling Technology), Ponseau S (P7170, Sigma), rapamycin (R8781, Sigma), cOmplete mini EDTA-free protease inhibitor cocktail (11836170001, Sigma), Protein A-Sepharose (P3391, Sigma), Protein G-Agarose (P4691, Sigma), Vectashield mounting medium with DAPI (H-1200, Vector Laboratories), Lenti-X concentrator (631232, TaKaRa), RNase-free DNase (79254, Qiagen), and Ub aldehyde (U-201-050, R&D Systems).

Animals. WT C57BL/6J mice were obtained from Guangdong Medical Laboratory Animal Center (Guangzhou, China). The HD140Q knock-in mice were described previously. All animals were housed under standard conditions (12:12 light-dark cycle) in the specific pathogen-free facility in the Division of Animal Resources at Jinan University, with all experimental protocols approved by the IACUC (application no. 106747, approval no. 20250630-04).

Genomic DNA was isolated from tail biopsies using a commercial extraction kit (CWBIO, CW2094S). PCR genotyping was performed using the following primer sets: WT-forward 5′-GCG GCT GAG GGG GTT GA-3′, HD-forward 5′-ACT GCT AAG TGG CGC CGC GTA G-3′, and common reverse 5′-GAG GCA GCA GCG GCT GTG CCT G-3′. Biological sex was not factored into the research design or data analysis for this study.

cDNAs, shRNAs and lentiviruses. HA-WT-Ub (#17608), HA-K0-Ub (#17603), scramble shRNA (#1864), GFP-p53 (#12091), and psPAX2 (#12260) were purchased from Addgene. pLP/VSV-G was obtained from Invitrogen. shNedd4-34 (TRCN0000092434) and shNedd4-35 (TRCN0000092435) were purchased from Open Biosystems. pEGFP was purchased from Clontech. Htt-Exon1-25Q-GFP and Htt-Exon1-46Q-GFP were provided by Aleksey Kazantsev (NIH). Htt-480-68Q and Htt-571-72Q were described previously (5). T7-tagged WT Nedd4 and catalytically inactive Nedd4 (C853S) were provided by Daniela Rotin (Hospital for Sick Children, Toronto, Canada). Human Mdm2 was provided by Karen Vousden (The Beatson Institute for Cancer Research). Nedd4 and Nedd4 CS inserts were amplified by PCR from pRc/CMV-Nedd4 and -Nedd4 CS (provided by Daniela Rotin) and were subcloned into pcDNA3 at EcoRI/NotI sites, which were used in some experiments. EGFP insert was amplified by PCR from pEGFP-C1 (Clontech) and was subcloned into the lentiviral expression vector pER4 at NheI/NotI sites. PRK-NEDD4, PRK-MDM2, and PRK-GFP vectors were custom made by IGEbio. The following lentiviral vectors were custom-made and packaged by IGEbio. pLKO.1-EF1α-HTTex1-72Q-P2A-mCherry, pLKO.1-EF1α-HTTEx1-25Q–P2A-mCherry, and pLKO.1-U6-shRNA-NEDD4-EF1α-copGFP (targeting sequence: 5′-GCG CAA ACA TTC TGG AGG ATT-3′).

Cell culture, transfection, and primary neuronal culture. HEK-293FT (Invitrogen R70007) and murine neuroblastoma cell line N2A (ATCC CCL-131) were cultured in DMEM with 10% FBS. For the experiments using HEK293 cell lines stably expressing full-length Htt with either 23 or 120 glutamine repeats (23Q/120Q), HEK293 cell lines were cultured in medium containing 500 μg/mL hygromycin B (Invivogen, ant-hg-5). Primary cortical neurons were isolated either from E16 embryos of C57BL/6 mice or from E18 embryos of Sprague Dawley rats as described previously (55). Neurons were plated in 6-well plates (400,000 cells per well), 12-well plates (160,000 cells per well), or 24-well plates with 12 mm coverslips in them (80,000 cells per well). Neurons were cultured in Neurobasal media with glutamine, penicillin/streptomycin, and 2% B-27 supplement.

Generation of lentiviruses and transductions. Lentiviruses expressing Htt-Exon1-25Q, Htt-Exon1-72Q, GFP, scramble shRNA, sh Nedd4-34, and shNedd4-35 were generated in 293FT cells (Thermo Fisher) by cotransfecting lentiviral vectors with helper plasmids (psPAX2 and pLP3) using X-tremeGene HP DNA Transfection Reagent (MilliporeSigma).

Media were replaced with fresh media 12 hours after transfection. Virus-containing culture supernatants were collected 48 hours after transfection and concentrated using Lenti-X concentrator. Virus titers were determined using the RETROtek HIV-1 p24 Antigen ELISA kit (ZeptoMetrix). In total, 1 ng p24 was considered equivalent to 5,000 infectious particles. Primary neurons were transduced with lentiviruses at the multiplicity of infection (moi) of 4–5 for expression vectors and 1.5 for knockdown, respectively, on DIV 9–13. Transduced neurons were harvested or fixed at 3–8 dpi (days post-infections) as indicated in the figures unless otherwise indicated.

Transfections. All cells were transfected with Lipofectamine 2000 for cDNA expression and shRNA knockdown according to the manufacturer’s instructions. Media were completely replaced with fresh media 4 h after transfection unless otherwise indicated.

Preparation of cell lysates and tissue homogenates. Total cell lysates were prepared by lysing cells in 2X LaemmLi sample buffer containing 2-mercaptoethanol, then boiling for 5 min. For time-course experiments, cells were harvested at the indicated time by removing media and freezing the sealed tissue culture plates in –20°C. The samples were lysed at the same time after harvesting all the time-course samples. For tissue homogenate preparation, WT and R6/2 cerebrum samples (110 day-old female brains, frozen) were homogenized in RIPA buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 1X protease inhibitor cocktail, 1 mM PMSF) using a tissuemiser homogenizer (Thermo Fisher) and then sonicated using a Q700 sonicator (Qsonica). Tissue homogenates were cleared by centrifugation at 21,000g for 15 minutes at 4°C. Protein concentrations were determined by BCA protein assay. In total, 30 μg of proteins were resolved by SDS-PAGE and analyzed by Western blotting. For the detection of Nedd4 in HD140Q KI mice, cortical tissues from HD140Q KI mice (16-month-old) were homogenized using a Dounce Tissue Grinder (Thermo Fisher, K8853000002) with 20–30 strokes. Tissue lysates were prepared in RIPA buffer (50 mM Tris, pH 8.0, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 0.1% SDS, 0.5% sodium deoxycholate, 1% Triton X-100) supplemented with protease inhibitors (Mei5bio, MF182-plus-10), followed by 6 cycles of sonication. Proteins were separated on SurePAGE Bis-Tris gels (GenScript, M00652) and transferred to nitrocellulose membranes. After blocking with 5% nonfat milk for 1 hour at room temperature, membranes were probed with primary antibodies diluted in 3% BSA overnight at 4°C. The following day, membranes were washed 3 times with PBS and incubated with HRP-conjugated secondary antibodies in 5% nonfat milk for 1 hour at room temperature. Following additional PBS washes, protein signals were detected using ECL substrate (Millipore, WBKLS0500) and visualized with a Clinx ChemiScope 6300 imaging system.

Co-IP assay. For co-IP assay of Htt-Exon1-GFP and Nedd4, 293FT cells grown on 6-well plates were transfected with plasmids. Forty-eight hours after transfection, cells were washed in PBS, and lysed on ice for 30 minutes in co-IP lysis buffer (40 mM HEPES, pH 7.4, 120 mM NaCl, 1 mM EDTA, 10 mM pyrophosphate, 10 mM glycerophosphate, 50 mM NaF, 0.3% CHAPS, 1 mM PMSF, 1X EDTA-free protease inhibitor cocktail). Cell lysates were cleared by centrifugation at 21,000g for 15 minutes. Protein concentration was determined by BCA assay. Equal amounts of proteins were precleared with 80 μL of protein G beads (20% slurry) for 2 hours to remove proteins that associate with protein G beads nonspecifically.

IP was performed with 1 μg of AFP antibody, 80 μL of protein G beads (20% slurry) and precleared lysates at 4°C overnight with rotation. IP samples were washed 3 times with co-IP lysis buffer. Inputs and IP samples were resolved by SDS-PAGE and analyzed by Western blotting. For co-IP assay of Ntt480-68Q and Nedd4, N2a cells grown on 6 cm dishes were transfected with plasmids. Cells were lysed on ice for 30 minutes in co-IP lysis buffer 26 hours after transfection. IP followed by Western blot analysis was performed. For the detection of interaction between Htt and Nedd4 in mouse brain lysates (Figure 1C), cortical tissue from 5-month-old WT C57BL/6J mice and cells were lysed in the buffer (40 mM HEPES, pH 7.4, 120 mM NaCl, 1 mM EDTA, 10 mM glycerophosphate, 50 mM NaF, 1 mM PMSF and protease inhibitors). The lysates (500 μg) were precleared with Protein G beads (30 μL, Thermo Fisher 10004D) and were then incubated with primary antibodies overnight at 4°C. Antibody complexes were captured with Protein G beads (50 μL, 1 hour at 4°C), washed 3× with 1% lysis buffer, magnetically isolated, and eluted in SDS loading buffer (95°C, 10 minutes).

Ub assay. For assessment of Htt571-72Q ubiquitination by Nedd4, N2a cells grown on 6 cm dishes were transfected with 0.5 μg HA-Ub (HA-Ub), 2 μg Htt571-72Q, and 1.5 μg vector (pRc/CMV), Nedd4, or Nedd4 CS. Cells were washed in PBS and collected 50 hours after transfection. Cells were lysed in 390 μL of lysis buffer (25 mM Tris, pH 7.5, 137 mM NaCl, 5 mM KCl, 1.5 mM Na2HPO4, 1 mM CaCl2·2H2O, 0.5 mM MgCl2·6H2O) by pipetting up and down. In total, 10 μL of 20% SDS was then added to the lysates and boiled at 70°C for 10 minutes to denature proteins. After lysates were cooled on ice, 1.6 mL of Ub dilution buffer (50 mM HEPES, pH 7.4, 150 mM NaCl, 10% glycerol, 1% Triton X-100, protease inhibitor cocktail [Roche], 2mM N-ethylmaleimide, 1mM PMSF) was added to the lysates. In total, 10 μL of RNase-free DNase was added and lysates were mixed by flipping the tubes. Cell lysates were incubated at room temperature until the viscosity went away. Lysates were cleared by centrifugation at 21,000g for 20 minutes, and protein concentration was determined by BCA assay. Cleared lysates were precleared with 80 μL of protein G beads (20% slurry) for 2 hours with rotation at 4°C. IP was performed by incubating precleared lysates, 80 μL of protein G beads (20% slurry), and 1.2 μL of anti-Htt antibody (MAB5490) at 4°C overnight. IP samples were washed 3 times with 1mL RIPA buffer (50mM Tris, pH7.4, 150mM NaCl, 1mM EDTA, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS). Inputs and IP samples were resolved by SDS-PAGE and analyzed by Western blotting. For determination of Htt480-68Q monoubiquitination, N2a cells grown on 6-well plates were transfected with 1.5 μg of GFP-Htt480-68Q and 0.5 μg of either WT HA-Ub or HA-K0-Ub. Twenty-five hours after transfection, cells were washed in PBS, lysed in 100 μL of lysis buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 1 mM EDTA, 0.5% Triton X-100, 1% SDS, 500 nM TSA, 5 mM nicotinamide, 1X protease inhibitor cocktail, 1 mM PMSF, 118 nM Ub aldehyde, 5 mM NEM, 1 mM DTT), boiled for 5 minutes, and cooled on ice. In total, 1 mL of Triton X-100 buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 1% Triton X-100, 5 mM NaF, 1 mM EDTA) was added to the lysates and incubated on ice for 30 minutes. Lysates were cleared by centrifugation at 21,000g for 15 minutes. Cleared lysates were precleared with 80 μL of protein G beads (20% slurry). IP was performed by rotating precleared lysates, 1 μg of anti- AFP antibody (AFP5002, Q-biogene) and 80 μL of protein G beads (20% slurry) at 4°C overnight. IP samples were washed 4 times with 1 mL RIPA buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS). Inputs and IP samples were resolved by SDS- PAGE and analyzed by Western blotting. For determination of GFP polyubiquitination, N2a cells grown on 6-well plates were transfected with 3 μg of GFP and 1 μg of HA-Ub or HA-K0-Ub. Cells were harvested 28 hours after transfection and immunoprecipitated as for the Htt480-68Q ubiquitination assay. For assessment of p53 ubiquitination by Nedd4, N2a cells grown on 6 cm dishes were transfected with p53-GFP, HA-Ub, and either Nedd4 or Nedd4 CS. Cells were harvested 28 hours after transfection, and immunoprecipitated as for the Htt480-68Q ubiquitination assay except the antibody used for IP was anti-GFP antibody from Sigma (G1544), and protein A beads were used in place of protein G beads.

CHX chase assay. For CHX chase of Htt with overexpression of Nedd4 or Nedd4 CS, N2a cells grown on 6 cm dishes were transfected with 4 μg of Htt571-72Q and 4 μg of vector (pcDNA 3.1+), Nedd4 or Nedd4 CS. Ten hours after transfection, cells in 6 cm dishes were trypsinized, collected, resuspended, and replated in 12-well plates. Twenty-four hours after transfection, t=0 h samples were harvested. At the same time, CHX was treated to t=6 h samples. Thirty hours after transfection, t=6 h samples were harvested. For CHX chase of Htt by knockdown of Nedd4, N2a cells grown on 6cm dishes were transfected with 4 μg of scramble shRNA or sh Nedd4-35. Twenty-four hours after transfection, cells were trypsinized and replated in 12-well plates. Forty-eight hours after transfection, shRNA-transfected cells were transfected with 0.8 μg of Htt571-72Q. Twenty-four hours after Htt transfection, t=0 h samples were harvested, and CHX was treated to the rest of the samples.

Samples were harvested 6, 12, and 24 hours after CHX treatment. For CHX chase with or without proteasome or lysosome inhibition, N2a cells grown on 6 cm dishes were transfected with 4 mg of Htt571-72Q and 4 μg of vector (pcDNA3.1+), Nedd4, or Nedd4 CS. Ten hours after transfection, cells in 6 cm dishes were trypsinized, collected, resuspended, and replated in 12-well plates. Twenty-four hours after transfection, t=0 h samples were harvested. At the same time, CHX was treated to t=6 h samples together with either vehicle (DMSO), bortezomib, or bafilomycin A1. Thirty hours after transfection, t=6 h samples were harvested. Samples were lysed, boiled for 5 minutes, resolved by SDS-PAGE, and analyzed by Western blotting.

Immunofluorescence staining of primary neurons. Primary neurons were fixed with 4% formaldehyde in 1X PBS for 15 minutes at room temperature. Coverslips were washed 2 times with PBS, blocked, and permeabilzed at the same time by incubating in 10% normal goat serum, 0.1% BSA, and 0.1% Triton X-100 in 1X PBS for 1 hour, incubated in anti-Htt antibody (MAB5492, 1:500) in 3% BSA in PBS at 4°C overnight. Coverslips were washed 3 times with PBS and incubated in secondary antibody (donkey anti-mouse IgG-Alexa Fluor 488, 1:600, Invitrogen, catalog A21202) for 1 hour at room temperature in the dark. Coverslips were washed 3 times with PBS, mounted using Vectashield mounting medium with DAPI, imaged using Leica DMI4000B confocal microscope and analyzed using ImageJ software (NIH).

Immunofluorescence staining of mouse brain section. Five-month-old WT C57BL/6J mice were anesthetized with isoflurane and transcardially perfused with warm saline (0.9%) followed by ice-cold 4% PFA in 0.1M PB. Brains were postfixed in 4% PFA overnight, then in 30% sucrose for 48 hours. After embedding in OCT compound (Sakura, 4583), 30 μm sections were obtained using a cryostat (Thermo Fisher). Sections and primary neurons were permeabilized with 0.5% Triton X-100/PBS (30 minutes) and blocked for 1 hour at room temperature. Primary antibody incubation was performed overnight at 4°C. After PBS washes, samples were stained with fluorescent secondary antibodies (1 hour) and DAPI (10 minutes), then imaged using a Zeiss AX10 or Olympus FV3000 microscope.

Ommatidium staining and imaging. Phalloidin staining for ommatidium was performed as described previously (34). Briefly, flies expressing Nedd4 RNAi with GMR-GAL4 (Nedd4 TRiP) or GMR-GAL4 only in the genetic background control for Nedd4 RNAi (TRiP Ctrl) were raised under 12:12 light/dark cycle, 25°C. Flies were collected within 1 day after eclosion and aged for 12 or 15 days prior to dissection. Adult eyes were dissected in PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4 and 1.8 mM KH2PO4); then, retinas were fixed in 4% formaldehyde solution for 1 hour. Retinas were washed 5 times with PBS containing 0.3% Triton X-100 and were then kept in 400 μM phalloidin in PBS containing 0.3% Triton X-100 and 5% normal goat serum at 4°C overnight on a shaker. Retinas were washed 5 times with PBS containing 0.5% Triton X-100 and mounted in 80% glycerol. Images were taken with Nikon C2 confocal microscope. The number of rhabdomeres in each ommatidium was quantified in a blinded manner.

Fly stock. GMR-GAL4, Nedd4 RNAi (BL34741, y[1] sc[*] v[1] sev[21]; PattP2) and its genetic control (BL36303, y[1] v[1]; PattP2) were obtained from Bloomington Stock Center. UAS-HttQ25 and UAS-HttQ103 with C-terminal GFP fusion were provided by Norbert Perrimon (Harvard Medical School, Cambridge, MA, USA).

qPCR. Flies expressing Nedd4 RNAi with elav-GAL4 (Nedd4 TRiP) or elav-GAL4 only in the genetic background control for Nedd4 RNAi (TRiP Ctrl) were raised under 12:12 light/dark cycle, 25°C. Flies were collected within 5 days after eclosion. Heads from ~45 flies were separated for RNA purification. RNA was extracted using Trizol reagent by according to the manufacturer’s protocol and treated with DNase (Promega) and cDNA synthesized using SuperScript III cDNA system. qPCR was done using Bio-Rad Sybr Green. CT values were normalized to Rp49 as the internal control for calculation of relative RNA abundance.

Statistics. All values in figures and text refer to mean ± SEM unless otherwise stated. Statistics and graphing were performed using GraphPad Prism software. Data were analyzed using unpaired 2-tailed Student t test (for 2 datasets) or 1-way ANOVA with Tukey’s post hoc test (for multiple data sets) unless otherwise indicated. The difference between the numbers of rhabdomere in each ommatidium was tested with Mann-Whitney U test. A P value less than 0.05 was considered significant.

Study approval. All animal experiments were conducted in accordance with institutional guidelines and were approved by the Institutional Animal Care and Use Committee (IACUC) of Jinan University (application no. 106747; approval no. 20250630-04).

Data availability. Values for all data points found in graphs are in the Supporting Data Values file. All data needed to evaluate the conclusions in the paper are present in the paper and/or the supplemental materials.