Ubiquitin Specific Peptidase 11 Knockdown Slows Huntington's Disease Progression via Regulating Mitochondrial Dysfunction and Neuronal Apoptosis Depending on PTEN- mediated AKT Pathway

Ubiquitin Specific Peptidase 11 Knockdown Slows Huntington's Disease Progression via Regulating Mitochondrial Dysfunction and Neuronal Apoptosis Depending on PTEN- mediated AKT Pathway | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Ubiquitin Specific Peptidase 11 Knockdown Slows Huntington's Disease Progression via Regulating Mitochondrial Dysfunction and Neuronal Apoptosis Depending on PTEN- mediated AKT Pathway Bai Gao, Yuchen Jing, Xi Li, Shuyan Cong This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4923322/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 08 Jan, 2025 Read the published version in Molecular Medicine → Version 1 posted 10 You are reading this latest preprint version Abstract Background Mitochondrial dysfunction and neuronal apoptosis are major symptoms of cytopathology in Huntington's disease (HD), a neurodegenerative disease. Ubiquitin Specific Peptidase 11 (USP11) is a deubiquitinating enzyme involved in various physiological processes through regulating protein degradation. However, its specific role in HD is unclear. Methods To interfere with USP11 expression, adeno-associated viruses 2 containing USP11-specific shRNA were injected into the bilateral striatum of 12-week-old R6/1 and WT mice. In vitro , the inducible PC12 cell model of HD was used in which the expression of an N-terminal truncation of huntingtin, with either wild type (Q23) or expanded polyglutamine (Q74) can be induced by the doxycycline. USP11 was knocked down to study its role in HD. The protein expression patterns in Q74 cells were quantified by label-free proteomics to further explore the target protein of USP11. Detecting the association between USP11 and Phosphatase and Tensin Homolog (PTEN) through Co-IP. Results Herein, USP11 was found to be upregulated in the striatum of R6/1 mice (an HD model with gradual development of symptoms) in an age-dependent manner. The spontaneous HD was alleviated by silencing USP11, as evidenced by improved locomotor activity and spatial memory, attenuated striatal atrophy in R6/1 mice, reduced accumulation of mutant huntingtin protein and neuronal apoptosis, and restored mitochondrial function in vitro and in vivo . The results of label-free proteomics revealed a significant change in the protein expression profile. Through functional enrichment, we focused on PTEN, known as a negative regulator of the AKT pathway. We demonstrated that USP11 downregulation promoted ubiquitination modification of PTEN and activated the AKT pathway, and PTEN overexpression reversed the effects of USP11 knockdown. Conclusions Collectively, USP11 knockdown protects R6/1 mouse neurons from apoptosis, thereby preventing the HD progression. This is achieved by inhibiting PTEN expression, which in turn activates the AKT pathway. This study suggests that USP11-PTEN-AKT signaling pathway may be a new attractive therapeutic target for HD. Huntington's Disease (HD) Ubiquitin Specific Peptidase 11 (USP11) Phosphatase and Tensin Homolog (PTEN) Neuronal Apoptosis Mitochondria Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Introduction Huntington's disease (HD) is a prevalent autosomal dominant neurodegenerative disorder caused by a repetition of the cytosine-adenine-guanine (CAG) sequence in the HD gene coding region [ 1 ]. As the number of CAG repetitions increases, the possibility of polyglutamine expansion in the N-terminal region of the protein also increases. This results in the misfolding of huntingtin (HTT) and the formation of insoluble polymers. If this repetition occurs more than 36 times, it is considered a pathogenic mutation. The repetitive sequence of CAG encodes the mutant huntingtin (mHtt) [ 2 ]. Current treatment of HD focuses largely on managing symptoms. However, as of now, the Food and Drug Administration (FDA) has not approved any disease-modifying treatments for HD [ 3 ]. Therefore, revealing the molecular mechanisms of HD and identifying potential therapeutic targets are promising strategies for treating patients with HD. Ubiquitin-specific processing proteases were reported to be involved in the regulation of neuroinflammation [ 4 , 5 ]. Ubiquitin Specific Peptidase (USP) 11 is a deubiquitinating enzyme that belongs to the ubiquitin-specific processing protease family [ 6 – 8 ]. The upregulation of USP11 exacerbated traumatic brain injury in rats by inducing neurological impairment and neuronal apoptosis [ 9 ]. After suppressing the expression of USP11, the neural damage in rats with cerebral hemorrhage was alleviated, accompanied by a reduction in neuronal apoptosis, microglial polarization, and inflammation. [ 8 , 10 ]. This evidence suggests a potential connection between USP11 and neurological disease, but our understanding of the relationship between USP11 and HD is limited. Translational modifications enhanced protein versatility by increasing phosphorylation, ubiquitination, methylation, and other processes [ 11 ]. As a deubiquitinating enzyme, USP11 was reported to be involved in regulating neural function by mediating the deubiquitination modification of downstream proteins to increase their stability, such as Beclin 1 [ 7 ]. Based on this, we analyzed the protein expression patterns in PC12 Q74 cells using label-free proteomics. PTEN, a phosphatase that removes phosphate groups from its bound substrates, was screened out by functional enrichment for mechanistic research. The AKT Serine/Threonine Kinase (AKT) is a crucial protein regulated by PTEN, an important tumor suppressor that promotes apoptosis by dephosphorylating AKT [ 12 , 13 ]. Knockdown of PTEN protected hippocampal neurons from oxidative stress damage [ 14 ]. Promoting AKT phosphorylation inhibited striatal neuronal damage caused by misfolded HTT [ 15 ]. In this study, we reported the establishment of a mouse model with HD by crossbreeding male R6/1 mice with female C57BL/6J mice. We investigated the regulatory effect of USP11 on HD by knocking down USP11 in the striatum of WT and HD mice. The effect of USP11 on protein expression was evaluated using label-free proteomics in doxycycline (Dox)-induced PC12 cells employing the Tet-on system in vitro . Through the enrichment of genes associated with phosphorylation and cell growth, we concentrated on PTEN, which is recognized as a negative regulator of the AKT pathway. Furthermore, the regulatory mechanism of USP11 and PTEN on mitochondrial dysfunction and neuronal apoptosis was investigated in vitro . Our study revealed the mechanism through which USP11 and PTEN safeguard neurons in HD. Materials and Methods Antibodies The primary antibodies used for immunofluorescence in myocardial tissues included mHtt antibody (Sigma-Aldrich, St. Louis, MO, USA), USP11 antibody (Santa Cruz, Dallas, TX, USA), Neuronal Nuclei (NeuN) antibody (Abcam, Boston, MA, USA), PTEN antibody (Proteintech, Rosemont, IL, USA), p-AKT antibody (Affinity, Cincinnati, OH, USA), and AKT antibody (Affinity). FITC-conjugated secondary antibody (goat anti-rabbit IgG) and Cy3-conjugated secondary antibody (goat anti-mouse IgG) were from Proteintech. For the Western blot analysis, the primary antibodies used included the USP11 antibody purchased from Santa Cruz, the mHtt antibody purchased from Sigma-Aldrich, the Ubi antibody obtained from Wanleibo (Shenyang, China), the cytochrome c antibody, the PTEN antibody, the COX IV antibody, and the β-actin antibody obtained from Proteintech. The HRP-conjugated secondary antibodies used were goat anti-rabbit IgG and goat anti-mouse IgG (Proteintech). Adeno-associated Virus (AAV) To downregulate USP11 mRNA expression, oligonucleotides targeting the mouse USP11 (shUSP11: 5’-GGTGGAAGTGTACCCACTAGA-3’) or the sequence without any predicted target gene were cloned into the pAAV2-CMV-U6 Track vector. Then, the pAAV2-CMV-U6 Track vector was co-transfected with the pHelper plasmid and pRC2-mi342 plasmid into AAV-293 cells (iCell Bioscience Inc, Shanghai, China) using Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) to generate infectious AAV viral particles containing a plasmid with USP11 shRNA and NC shRNA. Animal experiments Male R6/1 mice (C57BL/6J background), expressing exon1 of the human HTT gene, were crossbred with female C57BL/6J mice. Genotypes of the F1 individuals were determined using PCR analysis. Part 1 A total of 36 littermates of F1, including 18 R6/1 mice and 18 WT mice, were used in this part. The WT mice and R6/1 mice were kept for 8, 16, and 20 weeks, respectively. Subsequently, the mice were euthanized, and the striatum was collected. Part 2 Thirty-six WT mice and 36 R6/1 mice were divided into two groups (shNC and shUSP11, n = 18), respectively. Three mice were required to complete all the analyses. The mice were injected with 2 µl of AAV-2 containing USP11-specific shRNA (shUSP11) and NC shRNA (shNC) at a concentration of 1.53×10 9 genomic copies. The injections were performed in the striatum using coordinates relative to bregma as previously described [ 16 ]: (1) anteroposterior (AP), + 0.8; mediolateral (ML), + 1.8; and dorsoventral (DV), 2.9 mm, and (2) AP, + 0.3; ML, + 2; and DV, 3 mm below the dural surface, with the incisor bar positioned 3 mm above the interaural line. After 4 weeks of injections, the motor ability and spatial recognition memory of each group were evaluated using the open field test and Y-maze test, as previously reported [ 17 , 18 ]. Subsequently, the mice were euthanized, and the striatum was collected. All animal experiments have been approved by the Medical Ethics Committee of Shengjing Hospital of China Medical University and followed the Guide for the Care and Use of Laboratory Animals (Eighth Edition). Cell Culture Inducible rat PC12 cells expressing an exon 1 fragment of HTT with 23 (Q23) or 74 (Q74) glutamine repeats fused to the Green Fluorescent Protein (GFP) were maintained in DMEM culture medium (Servicebio, Wuhan, China) supplemented with 10% fetal calf serum (Tianhang Biotech, Huzhou, China) and cultured at 37℃ with 10% CO 2 . To induce the expression of HTT, the cells were treated with Dox (1 µg/ml) (Macklin, Shanghai, China) and after 0, 1, 3, and 6 days. The induced cells were exposed to cycloheximide (CHX) (Aladdin, Shanghai, China) for 0, 0.5, 1, and 2 h, respectively, to inhibit protein synthesis. Cell Transfection The sequence of shUSP11 or shNC was cloned into a plasmid vector. The cells were transfected with the plasmids using Lipofectamine 3000. The cell viability was detected using a CCK-8 cell proliferation detection kit (KeyGEN, Nanjing, China). MitoSOX staining was performed to assess the level of reactive oxygen species (ROS) in cells. Real-time PCR The samples were homogenized in TRIpure lysis buffer (BioTeke Bio., Beijing, China) to extract total RNA. The RNA concentration was analyzed using a UV spectrophotometer (NANO 2000, ThermoFisher Scientific, Pittsburgh, PA, USA). The cDNA synthesis was performed using BeyoRT II M-MLV reverse transcriptase (BeyotimeBiotech, Shanghai, China). The mRNA expression levels were evaluated using real-time PCR with a fluorescent quantitative PCR instrument (Exicycler 96, Bioneer, Daejeon, Korea). The results were calculated using the 2 -△△CT method. The primer sequences are as follows: Rat USP11, 5’-GGCAGCCTATGTCTTGT-3’ (F), 3’-GATGTCAGAGTTGGGTGTA-5’ (R). Mus USP11: 5’-GATGTACCGACTTTCACG-3’ (F), 3’-GCTGTTGTCTAAGAGGGAT-5’ (R). Protein Extraction and Western Blot For the total protein analysis, the sample was lysed for 30 min on ice in RIPA lysis buffer (Proteintech) containing 1% protease inhibitor (Proteintech). After centrifugation, the lysate was separated, and the supernatant was collected. The mitochondrial protein was extracted using a mitochondrial isolation and protein extraction kit (Proteintech), following the provided instructions. The BCA protein concentration determination kit (Proteintech) was used to measure the protein concentration. The protein sample was separated using SDS-PAGE and then transferred from the gels to polyvinylidene difluoride (PVDF) membranes (Thermo Fisher Scientific, Pittsburgh, PA, USA). The membranes were then blocked in 5% skim milk (Proteintech). The blocked membranes were incubated with the primary antibodies (USP11 antibody, 1:300; cytochrome c antibody, 1:2000; mHtt antibody, 1:500; PTEN antibody, 1:5000; Ubi antibody, 1:1000; COX IV, 1:5000; β-actin, 1:20000) overnight at 4°C. Incubation with antibodies against β-actin or COX IV was performed to obtain loading controls. After the primary antibody incubation, the membranes were washed and then incubated for 40 min at 37°C with the secondary antibodies (1:10000). Finally, the reaction was visualized using the hypersensitive ECL chemiluminescence test kit (Proteintech). Co-Immunoprecipitation (Co-IP) The cells were lysed in a lysis solution for 30 min. AminoLink® coupling resin cross-linked with the antibody was washed using lysis buffer, and the liquid flowed through the resin was discarded. Next, the lysate was added to the resin and incubated overnight 4℃. Subsequently, then the resin was with a with lysis buffer. Elution buffer was added to release the precipitate from binding to the resin. The eluate was then was using the western blot procedure. Immunofluorescence The tissues were embedded in paraffin and then cut into 5-µm sections, which were dewaxed and boiled in an antigen retrieval solution for 10 min. The cells grown on coverslips were fixed with 4% paraformaldehyde and permeabilized using 0.1% Triton X-100 (BeyotimeBiotech). The slices were blocked in 1% BSA (Sangon, Shanghai, China) for 15 min. Then, the blocked slices were incubated with the primary antibodies (1:50) overnight at 4°C. The slices were incubated with secondary antibodies (1:200) for 1 h at 37°C. Finally, the nuclei were counterstained using DAPI (Aladdin Reagents Co. Ltd., Shanghai, China). Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) Assay The cells grown on coverslips and the tissue sections were permeabilized using 0.1% Triton X-100. The TUNEL assay was conducted using the In Situ Cell Death Detection Kit (Roche, Basel, Switzerland), according to the manufacturer's instructions. The nuclei were counterstained with DAPI. For the TUNEL assay combined with immunofluorescence for NeuN, the TUNEL assay was performed first. The slides were then rinsed before performing immunofluorescence for NeuN using the previously described methods. Caspase-3 Activity Assay The protein was extracted using the lysis buffer, and its concentration was determined using the Bradford protein concentration assay kit (BeyotimeBiotech). The Caspase-3 activity was detected using the Caspase-3 activity detection kit (BeyotimeBiotech) according to the manufacturer's instructions. Optical density was measured using a microplate reader (ELX-800, BioTek, Winooski, VT, USA). Hematoxylin Eosin (H&E) Staining The dewaxed sections were stained with hematoxylin (Solarbio, Beijing, China) for 5 min and then cleared in distilled water. Afterward, the sections were washed and counterstained with Eosin (Sangon) for 3 min, then washed and photographed for later analysis of the lateral ventricle area. Transmission Electron Microscope (TEM) The sample was immersed in acetone and an embedding agent (SPI, West Chester, PA, USA), and then polymerized at 60°C for 48 h. Subsequently, the sample was cut into 60–80 nm sections. The slices were stained with a 2% uranium acetate solution in saturated alcohol for 8 min in the dark. After washing, the sections were stained with a 2.6% lead citrate solution in carbon dioxide for 8 min. The stained sections were observed using a TEM (H-7650, Hitachi, Tokyo, Japan). Label-Free Proteomics Protein samples were incubated with dithiothreitol (DTT) for 1 h at 37°C to break down disulfide bonds. Then, iodoacetamide was added and incubated for 45 min at room temperature in the dark. Samples were diluted with NH 4 HCO 3 and incubated overnight with trypsin at 37℃ for enzymatic digestion. Formic acid was added to stop the reaction. The sample was desalted using a C18 column. The peptide mixture was separated using the RIGOL L-3000 high-performance liquid chromatography system (Beijing RIGOL Technology Co., Ltd., Beijing, China) and analyzed using the ORBITRAP ECLIPSE mass spectrometer (Thermo Fisher Scientific). The gene ontology (GO) annotation and enrichment analysis were conducted on the differentially expressed proteins. Data Analysis All data were presented as mean ± standard deviation (SD). Data analysis was performed using GraphPad Prism 9.5 (GraphPad Software Inc., La Jolla, CA, USA). The t-test or ANOVA was used to compare the differences in means. Differences were considered statistically significant at p < 0.05. Result USP11 was highly expressed in mice with HD We have established an HD mouse model by crossing male R6/1 with female C57 mice, as indicated in Fig. 1 a. The R6/1 transgenic mice and the WT mice were obtained from the F1. We detected the expression of mHtt in the striatum of R6/1 transgenic mice using immunofluorescence. As shown in Fig. 1 b, no obvious red fluorescence was observed in the 8-week R6/1 mice. The red fluorescence intensity increased with aging, indicating that the expression of mHtt increased as the age of R6/1 mice increased, and reached its peak in the 20-week R6/1 mice. The levels of USP11 in the striatum were analyzed. In the WT mice, the level of USP11 did not show a remarkable change among all groups, but it increased in R6/1 mice as they aged. The level of USP11 was highest in the 20-week R6/1 mice (Fig. 1 c-d). Knocking down USP11 improved the locomotor abilities and spatial memory of mice with HD Based on the observed changes in USP11 in the striatum, we injected AAV-2 containing USP11-specific shRNA into the striatum of WT and R6/1 mice to downregulate USP11 mRNA expression (Fig. 2 a). After 16 weeks, the results of the open field test showed that the distance covered by R6/1 mice decreased compared to the WT mice (Fig. 2 b). The low expression of USP11 did not have a significant effect on the WT mice. However, the R6/1 mice with USP11 knockdown showed an increase in the distance they covered. The results of the Y-maze test indicated that the residence time in the familiar arm of R6/1 mice increased, while in the novel arm it decreased compared to that of WT mice (Fig. 2 c). The silencing of USP11 resulted in R6/1 mice spending less time in the familiar arm and more time in the new arm. It can be observed from the H&E staining results (Fig. 2 d) that there was no obvious difference in the morphology of the striatum between the WT groups. The lateral ventricular area enlargement and striatum atrophy can be observed in R6/1 mice. However, the R6/1 mice with silenced USP11 showed a reduction in the lateral ventricular area, as indicated by the yellow arrows, and the atrophy of the striatum has been alleviated. The level of USP11 in the striatum of WT mice was lower than that in R6/1 mice (Fig. 2 e), indicating that the AAV-2 infection successfully reduced the expression of USP11. The immunofluorescence staining also showed a similar result (Fig. 2 f). Downregulation of USP11 reduced mHtt polymer formation and prevented mitochondria-related apoptosis in neuron cells As shown in Fig. 3 a, a lot of mHtt polymer was observed in the R6/1 mice, however, the USP11 knockdown cleared most of the mHtt polymer. The western blot results indicated the same outcome (Fig. 3 b). As shown in Fig. 3 c, the WT groups exhibited weak green fluorescence and strong red fluorescence, indicating that the neuron cells in WT mice had minimal apoptosis. However, the R6/1 groups showed the opposite effect, with the neuron cells largely undergoing apoptosis. The silencing of USP11 inhibited neuron cell apoptosis in the R6/1 mice. The Caspase-3 activity in the WT groups did not show a distinct difference and was lower than in R6/1 mice. The reduced expression of USP11 led to a decrease in Caspase-3 activity in the R6/1 mice (Fig. 3 d). Bax expression was increased, and Bcl-2 expression was decreased in the R6/1 mice. USP11 silence inhibited Bax expression, and promoted Bcl-2 expression (Fig. 3 e). Mitochondria in the striatum were observed using TEM (Fig. 3 f). The striatum in the WT mice exhibited intact mitochondrial ultrastructure, which was altered in the R6/1 mice. The absence of mitochondrial cristae can be observed in the R6/1 mice. The knockdown of USP11 prevented the alteration of mitochondrial ultrastructure. The yellow arrows indicate the mitochondrial cristae. We detected the level of cytochrome c in the mitochondria (Fig. 3 g). The expression of cytochrome c was higher in the WT groups compared to the R6/1 groups. The level of cytochrome c increased in the R6/1 mice with a low level of USP11. Downregulation of USP11 reduced apoptosis in Q74 cells To confirm these results in vitro , we established an inducible HD cell model. The GFP tag was fused to the N-terminal of HTT exon 1, which contained 23 CAG repeats and 74 CAG repeats in PC-12 cells, respectively (Fig. 4 a). The GFP tag was expressed after induction by culture in the presence of Dox, based on the Tet-on system. The HTT expression was determined by the fluorescence intensity. Fluorescence microscopy observations revealed that the fluorescence intensity increased as the induction time extended, reaching its peak after 6 days of induction (Fig. 4 b). In Q23 cells, HTT showed a diffuse staining, while in Q74 cells, large aggregates are visible. We examined the expression of GFP. It can be observed that the bands from Q74 cells exhibited a higher molecular weight, indicating the fusion expression of mHtt and GFP (Fig. 4 c). The expression of USP11 in the Q23 cells did not exhibit a significant change during culture (Fig. 4 d-e). In the Q74 cell, the expression of USP11 increased as the induction time was extended. The 6-day cells were selected for further experiments. The expression of USP11 in the Q74 cell was interfered with a plasmid containing USP11 shRNA (Fig. 4 f-g). We examined the effect of downregulation of USP11 on mHtt expression in the cells. A large amount of mHtt polymer appeared in the Q74 cells (Fig. 5 a-b). Downregulation of USP11 reduced the expression of mHtt polymer. Compared to the Q23 cell, the viability of the Q74 cell decreased, but it was improved by USP11 silencing (Fig. 5 c). The results of TUNEL staining and flow cytometry revealed that the apoptosis rate of the Q74 cell was higher than that of the Q23 cell. The apoptosis in the Q74 cell was inhibited by the low expression of USP11 (Fig. 5 d-e), and it was also demonstrated by the expression of Bax and Bcl-2 (Fig. 5 f). Downregulation of USP11 prevented mitochondrial dysfunction in Q74 cells The Q23 cells exhibited intact mitochondrial ultrastructure (Fig. 6 a). The absence of mitochondrial cristae can be observed in the Q74 cells. The silencing of USP11 protected the mitochondrial cristae. We detected the level of ROS in the mitochondria using MitoSox staining (Fig. 6 b). The level of ROS in the Q74 cells was higher than in the Q23 cells, but it decreased when USP11 was silenced. The level of cytochrome c in the mitochondria was also detected (Fig. 6 c). The expression of cytochrome c was lower in the Q74 cells than in the Q23 cells, but it increased in USP11-silenced Q74 cells. Mitochondrial damage in Q47 cells was also reflected in the decrease of mitochondrial membrane potential, and the downregulation of USP11 expression increased the mitochondrial membrane potential (Fig. 6 d). Knocking down USP11 affected the protein expression in Q74 cells Figure 7 a shows a decrease in the level of USP11 in the Q74-shUSP11 cell. The proteomic analysis results are shown in Fig. 7 b. Silencing USP11 altered protein expression in Q74 cells. GO enrichment results (Fig. 7 c) indicated that the knockdown of USP11 altered the biological processes related to the regulation of protein aggregation, neuronal development, and phosphorylation. The molecular functions that regulate protein phosphorylation are primarily changed. Various differential proteins were localized in neurons, mitochondria, or the endoplasmic reticulum. Based on the above enrichment results, we screened genes involved in both phosphorylation and cell growth in the HD cell model to further explore the role of the phosphorylation-related target gene regulated by USP11 in HD (Fig. 8 a). The PTEN gene, which was common to all GO terms (GO:0045936, GO:0048638, GO:0042326, GO:0001558, GO:0016311, GO:0052745, GO:0042578, GO:0016791, GO:0002020, GO:0004438, GO:0099524, GO:0044309, GO:0043197, GO:0099522), was found (Fig. 8 b). Knockdown of USP11 promoted degradation of PTEN Compared to the R6/1 mice, the expression of USP11 and PTEN was lower in the WT mice. In the WT mice, USP11 had no significant effect on the expression of PTEN. In the R6/1 mice, USP11 positively regulates the expression of PTEN (Fig. 9 a). The expression of PTEN in the Q74 cells was higher than in the Q23 cells, however, it was also inhibited by USP11 silencing (Fig. 9 b). In the striatum of WT and R6/1 mice, the colocalization of USP11 and PTEN was observed (Fig. 9 c). We then investigated the relationship between USP11 and PTEN in the Q74 cells. The results of molecular simulation docking show that USP11 and PTEN can spontaneously bind (Fig. 9 d). The existence of multiple potential ubiquitination modification sites on the amino acid sequence of PTEN was predicted by MusiteDeep software (Fig. 9 e). The CoIP results revealed that USP11 and PTEN interact with each other at the protein level (Fig. 9 f). The production of proteins in the Q74 cells was restrained after treatment with CHX, and the PTEN level decreased as the treatment time extended (Fig. 9 g). Additionally, the degradation rate of PTEN protein was faster in the USP11-silenced cells than in the control cells. The detection of ubiquitination modification of PTEN revealed that USP11 silencing increased the ubiquitination modification level of PTEN, thereby promoting the degradation of PTEN (Fig. 9 h). Knockdown of USP11 prevented mitochondrial dysfunction by inhibiting PTEN expression To explore the mechanism of PTEN on neuronal apoptosis, we examined the activation of AKT pathway. The AKT level did not differ among all cells, but the p-AKT level showed a change. The level of p-AKT expression in the Q74 cells was lower than that in the Q23 cells. The downregulation of USP11 led to increased phosphorylation of AKT (Fig. 10 a). The expression of PTEN decreased, and the expression of p-AKT increased in the cells with silenced USP11. However, these effects were abolished by overexpression of PTEN (Fig. 10 b). The silencing of USP11 inhibited apoptosis, which was rescued by the high expression of PTEN (Fig. 10 c). The ROS produced by mitochondria were affected in the same manner (Fig. 10 d). Discussion HD is a devastating disease, and existing treatments mainly center on delivering symptomatic drugs. However, there are currently no available treatments that can improve or prevent HD. As more information was revealed about the pathogenesis of HD, targeted RNA therapy may offer hope for patients with HD. In this study, a transgenic mouse model of HD was constructed, and it was observed that the expression of USP11 was elevated in the HD mice. USP11 may play a crucial role in HD. We then conducted additional research to explore the role of USP11 and its downstream protein PTEN in regulating mitochondrial function and neuronal growth during HD in vivo and in vitro . The mechanism of action may involve the PTEN-AKT signaling pathway. These findings revealed the intricate interactions among USP11, PTEN, mitochondrial dysfunction, and neuronal apoptosis in the context of HD. This study provided new insights into targeted RNA therapy for HD. The main cause of HD is the toxic function of the mHtt protein, which is typically located in the neuronal nuclei of the brain's striatum [ 19 ]. High levels of mHtt polymer expression were detected in the striatum of 16-week-old R6/1 mice, indicating that these mice develop HD by 16 weeks of age. Meanwhile, high levels of USP11 expression were detected in the striatum of 16-week-old R6/1 mice. HD is characterized by progressive cognitive impairment and abnormal motor symptoms. Clinical manifestations include slow movement, impaired fine motor skills, cognitive decline, and behavioral or mental changes [ 20 ]. In this study, the motor ability and spatial recognition of HD mice were assessed using the open field test and Y-maze. Mice with HD had significantly weaker locomotor ability and engaged in fewer walking behaviors compared to WT mice. This was typical of HD onset and is consistent with a previously reported [ 17 , 21 ]. The HD mice with silenced USP11 showed increased walking behavior and improved locomotor activity. Mice with HD typically exhibited significant deficits of spatial recognition, likely due to cognitive impairment, which was consistent with previous findings [ 18 ]. The USP11 silencing also improved the situation. In histopathology, the enlargement of the lateral ventricles and severe atrophy of the striatum are neuropathological features of HD [ 22 ]. In this study, H&E staining of the striatum of HD mice revealed a severely atrophied striatum and lateral ventricular enlargement, consistent with previous reports [ 23 ]. Downregulation of USP11 alleviated striatum degeneration. Based on the above phenomena, we further explored the molecular mechanism of USP11 in HD. The striatum lesions were always caused by the loss of striatal neurons [ 24 ]. The insolubility and toxicity of aggregated protein mHtt were major causes of neuronal apoptosis [ 25 ]. At the same time, the loss of striatal neurons was also considered to be the most significant morphological correlate of clinical symptoms of HD patients, such as cognitive decline [ 26 ]. The high level of mHtt polymer in the striatum was a marker of HD. TUNEL combined with NeuN staining was used to detect neuronal loss in the mouse striatum. There was minimal neuronal loss in the striatum of WT mice, whereas a significant number of neurons underwent apoptosis in the striatum of HD mice. Meanwhile, the activity of Caspase-3 increased in the striatum of HD mice, further explaining the apoptosis of neurons. The neuronal apoptosis was inhibited by silencing USP11 in mice with HD. Neurons are high-energy-demanding cells, and their normal function is closely related to mitochondrial function. Mitochondria play key roles in supplying ATP, producing ROS, and providing resistance to oxidative stress in neurons [ 27 ]. In HD, the accumulation of mHtt may lead to mitochondrial dysfunction, ultimately resulting in neuronal apoptosis [ 28 ]. ROS was the primary factor in oxidative stress, a condition that is universally present in neurodegenerative diseases. Damage to mitochondrial function increased levels of ROS, leading to neuronal apoptosis [ 29 ]. We observed morphological abnormalities in the mitochondria of the striatum in HD mice using TEM. These abnormalities were mainly characterized by the disappearance of mitochondrial cristae, which is consistent with previous reports [ 30 ]. Cytochrome c is released from the mitochondria. Downregulation of USP11 in HD mice reduced mHtt polymers in the striatum, suppressed the release of cytochrome c, and improved mitochondrial morphology. The same results were obtained in vitro . Elevated levels of ROS were observed in cells with impaired mitochondrial morphology. Downregulation of USP11 inhibited the production of ROS and preserved mitochondrial function. Label-free proteomic analysis revealed that silencing USP11 significantly altered protein expression in Q74 cells. The levels of proteins associated with neuron growth, protein polymerization, and protein phosphorylation were significantly altered. Protein phosphorylation is closely related to HD pathogenesis [ 31 ]. It has been reported that there was a widespread disorder of protein phosphorylation in the cerebral cortex in mice before the onset of HD [ 32 ]. So, we focused on the protein regulated by USP11 that was related to phosphorylation. PTEN, which was involved in both the phosphorylation pathway and cell growth, was highly expressed in the striatum of HD mice and Q74 cells. Its expression was downregulated after the silencing of USP11. It was reported that USP11 mediated PTEN deubiquitination, thereby enhancing PTEN stability [ 33 ]. Studies demonstrated that USP11 suppressed PI3K/AKT activity by reversing polyubiquitination and increasing PTEN expression in vitro and in vivo [ 34 ]. We revealed that USP11 knockdown promoted the ubiquitination degradation of PTNE in Q74 cells, which once again demonstrated the deubiquitination effect of USP11 on PTEN. This finding led us to hypothesize that USP11 regulates HD progression by interacting with PTEN. PTEN negatively regulated AKT phosphorylation and mediated spinal developmental disorders and neuroinflammation in mice [ 35 , 36 ]. The increase of AKT phosphorylation level was conducive to the decrease of mHtt formation [ 15 ]. To delve deeper into the regulatory mechanism of PTEN on HD, we examined the expression of PTEN downstream target protein AKT and its phosphorylated form, p-AKT, in vitro . The results showed that the downregulation of USP11 promoted AKT phosphorylation. Overexpression of PTEN reversed this regulatory effect. The USP11-PTEN-AKT axis may be involved in HD progress. PTEN played a critical role in mitochondria-dependent apoptosis in neurodegenerative diseases. After PTEN was knocked out in hippocampal cells, the increase in ROS levels, release of cytochrome c, and activity of Caspase-3 were inhibited [ 14 ]. The knockout of PTEN restrained the apoptosis by preserving mitochondrial function in an ischemic stroke-like model [ 37 ]. In addition, the AKT signaling pathway plays a key role in neuronal survival and growth, thereby influencing brain development and function, and impacting a variety of neurological diseases [ 38 ]. Activation of AKT signal reduced neuronal apoptosis after cerebral hemorrhage in mice [ 39 ]. Elevated AKT phosphorylation levels mediated protection of cardiac mitochondrial function [ 40 ]. In this study, when USP11 was inhibited and PTEN was simultaneously highly expressed in Q74 cells, the regulation of apoptosis and mitochondrial ROS by USP11 was abolished. This suggested that USP11 silencing prevented apoptosis and mitochondrial damage by decreasing PTEN expression, which was mediated by the negative regulation of AKT activation by PTEN. Conclusion The present paper provides important evidence that USP11, a key target in HD treatment, regulated mitochondrial function and nerve cell apoptosis through the PTEN-mediated AKT pathway. The downregulation of USP11 inhibits the expression of PTEN and promotes the phosphorylation of AKT, thereby restoring mitochondrial function and reducing nerve cell apoptosis. This study demonstrates that the USP11-PTEN-AKT signaling pathway may represent a promising new therapeutic target for HD. Declarations Ethics approval and consent to participate All animal experiments have been approved by the Medical Ethics Committee of Shengjing Hospital of China Medical University and followed the Guide for the Care and Use of Laboratory Animals (Eighth Edition). Consent for publication Not applicable. Availability of data and materials The datasets generated and/or analyzed during the current study are available from the corresponding author on reasonable request. Competing interest There are no competing financial conflicts to declare. Funding This research was supported by the National Natural Science Foundation of China (General Program, grant number 81371271) and the Basic Research Project of the Educational Department of Liaoning Province (Youth Project, grant number LJKQZ20222350). Authors’ contributions BG: methodology, formal Analysis, and writing-original draft; YCJ: resources, methodology, and writing-original draft; XL: validation and writing-review editing; SYC: funding acquisition and supervision. Acknowledgements Not applicable. References MacDonald ME, Ambrose CM, Duyao MP, Myers RH, Lin C, Srinidhi L, et al. A novel gene containing a trinucleotide repeat that is expanded and unstable on Huntington's disease chromosomes. Cell. 1993;72(6):971-83. doi: 10.1016/0092-8674(93)90585-E Semaka A, Kay C, Doty C, Collins JA, Bijlsma EK, Richards F, et al. CAG size-specific risk estimates for intermediate allele repeat instability in Huntington disease. J Med Genet. 2013;50(10):696-703. doi: 10.1136/jmedgenet-2013-101796 Pan L, Feigin A. Huntington's Disease: New Frontiers in Therapeutics. Curr Neurol Neurosci Rep. 2021;21(3):10. doi: 10.1007/s11910-021-01093-3 Jiang X, Yu M, Ou Y, Cao Y, Yao Y, Cai P, et al. Downregulation of USP4 Promotes Activation of Microglia and Subsequent Neuronal Inflammation in Rat Spinal Cord After Injury. Neurochem Res. 2017;42(11):3245-53. doi: 10.1007/s11064-017-2361-2 Zhu L, Bi W, Lu D, Zhang C, Shu X, Wang H, et al. Regulation of ubiquitin-specific processing protease 8 suppresses neuroinflammation. Mol Cell Neurosci. 2015;64:74-83. doi: 10.1016/j.mcn.2014.05.004 Jacko AM, Nan L, Li S, Tan J, Zhao J, Kass DJ, et al. De-ubiquitinating enzyme, USP11, promotes transforming growth factor β-1 signaling through stabilization of transforming growth factor β receptor II. Cell Death Dis. 2016;7(11):e2474. doi: 10.1038/cddis.2016.371 Rong Y, Fan J, Ji C, Wang Z, Ge X, Wang J, et al. USP11 regulates autophagy-dependent ferroptosis after spinal cord ischemia-reperfusion injury by deubiquitinating Beclin 1. Cell Death Differ. 2022;29(6):1164-75. doi: 10.1038/s41418-021-00907-8 Zhang X, Liu T, Xu S, Gao P, Dong W, Liu W, et al. A pro-inflammatory mediator USP11 enhances the stability of p53 and inhibits KLF2 in intracerebral hemorrhage. Mol Ther Methods Clin Dev. 2021;21:681-92. doi: 10.1016/j.omtm.2021.01.015 Fang Y, Zhao T, Ni H, Li Y, Zhu Y, Gao R, et al. USP11 exacerbates neuronal apoptosis after traumatic brain injury via PKM2-mediated PI3K/AKT signaling pathway. Brain Res. 2023;1807:148321. doi: 10.1016/j.brainres.2023.148321 Xu Z, Li X, Chen J, Zhao J, Wang J, Ji Y, et al. USP11, Deubiquitinating Enzyme, Associated with Neuronal Apoptosis Following Intracerebral Hemorrhage. J Mol Neurosci. 2016;58(1):16-27. doi: 10.1007/s12031-015-0644-0 Basak S, Lu C, Basak A. Post-Translational Protein Modifications of Rare and Unconventional Types: Implications in Functions and Diseases. Curr Med Chem. 2016;23(7):714-45. doi: 10.2174/0929867323666160118095620 Piguet AC, Dufour JF. PI(3)K/PTEN/AKT pathway. J Hepatol. 2011;54(6):1317-9. doi: 10.1016/j.jhep.2010.12.013 Deng L, Meng T, Chen L, Wei W, Wang P. The role of ubiquitination in tumorigenesis and targeted drug discovery. Signal Transduct Target Ther. 2020;5(1):11. doi: 10.1038/s41392-020-0107-0 Zhu Y, Hoell P, Ahlemeyer B, Krieglstein J. PTEN: a crucial mediator of mitochondria-dependent apoptosis. Apoptosis. 2006;11(2):197-207. doi: 10.1007/s10495-006-3714-5 Humbert S, Bryson EA, Cordelières FP, Connors NC, Datta SR, Finkbeiner S, et al. The IGF-1/Akt pathway is neuroprotective in Huntington's disease and involves Huntingtin phosphorylation by Akt. Dev Cell. 2002;2(6):831-7. doi: 10.1016/s1534-5807(02)00188-0 Creus-Muncunill J, Rué L, Alcalá-Vida R, Badillos-Rodríguez R, Romaní-Aumedes J, Marco S, et al. Increased Levels of Rictor Prevent Mutant Huntingtin-Induced Neuronal Degeneration. Mol Neurobiol. 2018;55(10):7728-42. doi: 10.1007/s12035-018-0956-5 Angeles-López QD, García-Lara L, Aguirre-Pineda N, Castañeda-Arellano R, Elizondo-Azuela G, Pérez-Severiano F, et al. The absence of the aryl hydrocarbon receptor in the R6/1 transgenic mouse model of Huntington's disease improves the neurological phenotype. Behav Brain Res. 2021;408:113230. doi: 10.1016/j.bbr.2021.113230 Ragot A, Pietropaolo S, Vincent J, Delage P, Zhang H, Allinquant B, et al. Genetic deletion of the Histone Deacetylase 6 exacerbates selected behavioral deficits in the R6/1 mouse model for Huntington's disease. Brain Behav. 2015;5(9):e00361. doi: 10.1002/brb3.361 DiFiglia M, Sapp E, Chase KO, Davies SW, Bates GP, Vonsattel JP, et al. Aggregation of huntingtin in neuronal intranuclear inclusions and dystrophic neurites in brain. Science. 1997;277(5334):1990-3. doi: 10.1126/science.277.5334.1990 Rüb U, Seidel K, Heinsen H, Vonsattel JP, den Dunnen WF, Korf HW. Huntington's disease (HD): the neuropathology of a multisystem neurodegenerative disorder of the human brain. Brain Pathol. 2016;26(6):726-40. doi: 10.1111/bpa.12426 Ransome MI, Hannan AJ. Behavioural state differentially engages septohippocampal cholinergic and GABAergic neurons in R6/1 Huntington's disease mice. Neurobiol Learn Mem. 2012;97(2):261-70. doi: 10.1016/j.nlm.2012.01.004 Paulsen JS, Magnotta VA, Mikos AE, Paulson HL, Penziner E, Andreasen NC, et al. Brain structure in preclinical Huntington's disease. Biol Psychiatry. 2006;59(1):57-63. doi: 10.1016/j.biopsych.2005.06.003 Ramírez-Jarquín UN, Sharma M, Zhou W, Shahani N, Subramaniam S. Deletion of SUMO1 attenuates behavioral and anatomical deficits by regulating autophagic activities in Huntington disease. Proc Natl Acad Sci U S A. 2022;119(5). doi: 10.1073/pnas.2107187119 Bunner KD, Rebec GV. Corticostriatal Dysfunction in Huntington's Disease: The Basics. Front Hum Neurosci. 2016;10:317. doi: 10.3389/fnhum.2016.00317 Soto C. Unfolding the role of protein misfolding in neurodegenerative diseases. Nat Rev Neurosci. 2003;4(1):49-60. doi: 10.1038/nrn1007 Shepherd GM. Corticostriatal connectivity and its role in disease. Nat Rev Neurosci. 2013;14(4):278-91. doi: 10.1038/nrn3469 López-Doménech G, Kittler JT. Mitochondrial regulation of local supply of energy in neurons. Curr Opin Neurobiol. 2023;81:102747. doi: 10.1016/j.conb.2023.102747 Jodeiri Farshbaf M, Ghaedi K. Huntington's Disease and Mitochondria. Neurotox Res. 2017;32(3):518-29. doi: 10.1007/s12640-017-9766-1 Sharma A, Behl T, Sharma L, Aelya L, Bungau S. Mitochondrial Dysfunction in Huntington's Disease: Pathogenesis and Therapeutic Opportunities. Curr Drug Targets. 2021;22(14):1637-67. doi: 10.2174/1389450122666210224105945 Hyeon SJ, Park J, Yoo J, Kim SH, Hwang YJ, Kim SC, et al. Dysfunction of X-linked inhibitor of apoptosis protein (XIAP) triggers neuropathological processes via altered p53 activity in Huntington's disease. Prog Neurobiol. 2021;204:102110. doi: 10.1016/j.pneurobio.2021.102110 White A, McGlone A, Gomez-Pastor R. Protein Kinase CK2 and Its Potential Role as a Therapeutic Target in Huntington's Disease. Biomedicines. 2022;10(8). doi: 10.3390/biomedicines10081979 Mees I, Tran H, Roberts A, Lago L, Li S, Roberts BR, et al. Quantitative Phosphoproteomics Reveals Extensive Protein Phosphorylation Dysregulation in the Cerebral Cortex of Huntington's Disease Mice Prior to Onset of Symptoms. Mol Neurobiol. 2022;59(4):2456-71. doi: 10.1007/s12035-021-02698-y Christine A, Park MK, Song SJ, Song MS. The equilibrium of tumor suppression: DUBs as active regulators of PTEN. Exp Mol Med. 2022;54(11):1814-21. doi: 10.1038/s12276-022-00887-w Park MK, Yao Y, Xia W, Setijono SR, Kim JH, Vila IK, et al. PTEN self-regulates through USP11 via the PI3K-FOXO pathway to stabilize tumor suppression. Nat Commun. 2019;10(1):636. doi: 10.1038/s41467-019-08481-x Wu C, Liu H, Zhong D, Yang X, Liao Z, Chen Y, et al. Mapk7 deletion in chondrocytes causes vertebral defects by reducing MEF2C/PTEN/AKT signaling. Genes Dis. 2024;11(2):964-77. doi: 10.1016/j.gendis.2023.02.012 Fan Q, Liu Y, Sheng L, Lv S, Yang L, Zhang Z, et al. Chaihu-Shugan-San inhibits neuroinflammation in the treatment of post-stroke depression through the JAK/STAT3-GSK3β/PTEN/Akt pathway. Biomed Pharmacother. 2023;160:114385. doi: 10.1016/j.biopha.2023.114385 Farajdokht F, Mohaddes G, Karimi-Sales E, Kafshdooz T, Mahmoudi J, Aberoumandi SM, et al. Inhibition of PTEN protects PC12 cells against oxygen-glucose deprivation induced cell death through mitoprotection. Brain Res. 2018;1692:100-9. doi: 10.1016/j.brainres.2018.05.026 Manning BD, Toker A. AKT/PKB Signaling: Navigating the Network. Cell. 2017;169(3):381-405. doi: 10.1016/j.cell.2017.04.001 Chen S, Peng J, Sherchan P, Ma Y, Xiang S, Yan F, et al. TREM2 activation attenuates neuroinflammation and neuronal apoptosis via PI3K/Akt pathway after intracerebral hemorrhage in mice. J Neuroinflammation. 2020;17(1):168. doi: 10.1186/s12974-020-01853-x Li X, Luo W, Tang Y, Wu J, Zhang J, Chen S, et al. Semaglutide attenuates doxorubicin-induced cardiotoxicity by ameliorating BNIP3-Mediated mitochondrial dysfunction. Redox Biol. 2024;72:103129. doi: 10.1016/j.redox.2024.103129 Additional Declarations No competing interests reported. Supplementary Files SupplementaryfileuncroppedBlotsimages.zip Cite Share Download PDF Status: Published Journal Publication published 08 Jan, 2025 Read the published version in Molecular Medicine → Version 1 posted Editorial decision: Revision requested 29 Nov, 2024 Reviews received at journal 28 Nov, 2024 Reviewers agreed at journal 20 Nov, 2024 Reviews received at journal 09 Oct, 2024 Reviewers agreed at journal 28 Aug, 2024 Reviewers agreed at journal 27 Aug, 2024 Reviewers invited by journal 27 Aug, 2024 Editor assigned by journal 16 Aug, 2024 Submission checks completed at journal 16 Aug, 2024 First submitted to journal 16 Aug, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4923322","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":349846279,"identity":"18b92fa9-6088-4610-9fa4-4d53f27a3232","order_by":0,"name":"Bai Gao","email":"","orcid":"","institution":"Shengjing Hospital of China Medical University","correspondingAuthor":false,"prefix":"","firstName":"Bai","middleName":"","lastName":"Gao","suffix":""},{"id":349846281,"identity":"dc8288c8-ae2d-4532-b89f-4d7fae850915","order_by":1,"name":"Yuchen Jing","email":"","orcid":"","institution":"The First Hospital of China Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yuchen","middleName":"","lastName":"Jing","suffix":""},{"id":349846285,"identity":"b05aed1d-cc78-47bb-b47c-5b614ece01f5","order_by":2,"name":"Xi Li","email":"","orcid":"","institution":"The First Hospital of China Medical University","correspondingAuthor":false,"prefix":"","firstName":"Xi","middleName":"","lastName":"Li","suffix":""},{"id":349846286,"identity":"0ff7be7e-9112-48a1-a458-5a43058ac309","order_by":3,"name":"Shuyan Cong","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA3UlEQVRIie2QsQrCMBCGT4TaIZJNCiK+womLQ0F8E7N0qtBH8AGErCm+RMEHMHprwbWggyA4Z+zgYALONqNgvuH+5f+4SwACgR+FWkxt9LU2rZ/RIygym5E4lTs/pU9gzjbZnOLIo89ljVSgFpLXhoDBlI/0dyVpciSFN1GqTUXFAmblfv1dwYShZvgUVTOsSDFY47VL4bVTSBwv9Z1Y5KOAPcwpFeTgpyRNVpwUZnPVZGg/Oel+C5d0MOaVTqSkhzFtOuXjDgUgRjdX28/WrrpjcHdz6VMNBAKBP+UNpxBO79vv4PYAAAAASUVORK5CYII=","orcid":"","institution":"Shengjing Hospital of China Medical University","correspondingAuthor":true,"prefix":"","firstName":"Shuyan","middleName":"","lastName":"Cong","suffix":""}],"badges":[],"createdAt":"2024-08-16 07:44:53","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4923322/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4923322/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s10020-024-01061-w","type":"published","date":"2025-01-08T15:57:08+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":64404106,"identity":"9da45d55-f60e-4a97-84e5-83fac9280b8f","added_by":"auto","created_at":"2024-09-12 16:40:01","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2222180,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e(a)\u003c/strong\u003e The male R6/1 mice were crossbred with female C57BL/6J mice to obtain the F1 (WT, R6/1). \u003cstrong\u003e(b)\u003c/strong\u003e The expression of mHtt was detected using immunofluorescence staining. \u003cstrong\u003e(c)\u003c/strong\u003e The USP11 mRNA expression was analyzed using real-time PCR. \u003cstrong\u003e(d)\u003c/strong\u003e The USP11 protein level was analyzed using western blotting. ns, p\u0026gt;0.05, \u003csup\u003e###\u003c/sup\u003ep\u0026lt;0.001. mHtt, mutant huntingtin. USP11, Ubiquitin Specific Peptidase 11.\u003c/p\u003e","description":"","filename":"Fig.1.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4923322/v1/464e3651f742bb091fc2b7b3.jpg"},{"id":64404108,"identity":"c1199f51-a397-496a-9849-dcc4e5156689","added_by":"auto","created_at":"2024-09-12 16:40:02","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":5878227,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e(a)\u003c/strong\u003e The adeno-associated virus carrying USP11-specific shRNA was injected into the striatum. \u003cstrong\u003e(b)\u003c/strong\u003e The results of the open field test. \u003cstrong\u003e(c)\u003c/strong\u003e The results of the Y-maze test. \u003cstrong\u003e(d)\u003c/strong\u003e The results of H\u0026amp;E staining and quantitative measurement of the lateral ventricular area in the striatum tissue (the yellow arrows in the images represented the location of the lateral ventricle). \u003cstrong\u003e(e)\u003c/strong\u003e The USP11 protein level was analyzed using western blotting. \u003cstrong\u003e(f)\u003c/strong\u003e The expression of USP11 in the striatum tissue was detected using immunofluorescence staining. ns, p\u0026gt;0.05,\u003csup\u003e #\u003c/sup\u003ep\u0026lt;0.05,\u003csup\u003e ##\u003c/sup\u003ep\u0026lt;0.01,\u003csup\u003e ###\u003c/sup\u003ep\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"Fig.2.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4923322/v1/6739e13b926c4ec85e6a242e.jpg"},{"id":64404110,"identity":"e2bd5904-a133-4023-91ec-30fd0b5cf641","added_by":"auto","created_at":"2024-09-12 16:40:02","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":5065386,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e(a)\u003c/strong\u003e The expression of mHtt polymer was detected using immunofluorescence staining. \u003cstrong\u003e(b)\u003c/strong\u003e The mHtt protein level was analyzed using western blotting. \u003cstrong\u003e(c)\u003c/strong\u003e The neuronal apoptosis was analyzed using TUNEL and immunofluorescence staining. \u003cstrong\u003e(d)\u003c/strong\u003e The caspase 3 activity was detected using the kit. \u003cstrong\u003e(e)\u003c/strong\u003e The protein level of Bax and Bcl-2 was analyzed using western blotting. \u003cstrong\u003e(f)\u003c/strong\u003e The mitochondria were observed using the transmission electron microscope (the yellow arrows in the images represented the mitochondrial cristae). \u003cstrong\u003e(g)\u003c/strong\u003e The cytochrome c protein level in mitochondria was analyzed using western blotting. ns, p\u0026gt;0.05, \u003csup\u003e###\u003c/sup\u003ep\u0026lt;0.001. mHtt, mutant huntingtin, Bax, BCL2 Associated X, Apoptosis Regulator, Bcl-2, BCL2 Apoptosis Regulator.\u003c/p\u003e","description":"","filename":"Fig.3.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4923322/v1/e5d6b8f8b870eefe36a53760.jpg"},{"id":64404495,"identity":"e16c37a3-3b88-4d78-b2d7-3738d8aaebbe","added_by":"auto","created_at":"2024-09-12 16:48:02","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":3399239,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e(a)\u003c/strong\u003e The HD cell model was established by inducing PC-12 cells based on the Tet-on system. \u003cstrong\u003e(b)\u003c/strong\u003e The induced cells were observed using a fluorescence microscope. \u003cstrong\u003e(c)\u003c/strong\u003e The GFP protein expression was analyzed using western blotting. \u003cstrong\u003e(d)\u003c/strong\u003e The USP11 mRNA expression was analyzed using real-time PCR. \u003cstrong\u003e(e)\u003c/strong\u003e The USP11 protein level was analyzed using western blotting. \u003cstrong\u003e(f)\u003c/strong\u003e The USP11 mRNA expression was analyzed using real-time PCR after transfection. \u003cstrong\u003e(g)\u003c/strong\u003e The USP11 protein level was analyzed using western blotting after transfection. ns, p\u0026gt;0.05, \u003csup\u003e##\u003c/sup\u003ep\u0026lt;0.01, \u003csup\u003e###\u003c/sup\u003ep\u0026lt;0.001. HD, Huntington's Disease, USP11, Ubiquitin Specific Peptidase 11, GFP, Green Fluorescent Proteins.\u003c/p\u003e","description":"","filename":"Fig.4.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4923322/v1/9a11d7fe2045ec9ea68b0f67.jpg"},{"id":64404115,"identity":"8afe3497-ca4e-4573-a2e4-bd18a9fdc854","added_by":"auto","created_at":"2024-09-12 16:40:02","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":6117523,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e(a) \u003c/strong\u003eThe expression of mHtt polymer was observed using a fluorescence microscope. \u003cstrong\u003e(b) \u003c/strong\u003eThe GFP protein expression was analyzed using western blotting. \u003cstrong\u003e(c)\u003c/strong\u003e The cell viability was detected using the CCK-8 assay. The apoptosis was analyzed using a TUNEL assay \u003cstrong\u003e(d)\u003c/strong\u003e and flow cytometry \u003cstrong\u003e(e)\u003c/strong\u003e. \u003cstrong\u003e(f)\u003c/strong\u003e The protein level of Bax and Bcl-2 was analyzed using western blotting. ns, p\u0026gt;0.05, \u003csup\u003e#\u003c/sup\u003ep\u0026lt;0.05, \u003csup\u003e###\u003c/sup\u003ep\u0026lt;0.001. mHtt, mutant huntingtin, GFP, Green Fluorescent Proteins, Bax, BCL2 Associated X, Apoptosis Regulator, Bcl-2, BCL2 Apoptosis Regulator.\u003c/p\u003e","description":"","filename":"Fig.5.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4923322/v1/b46cbd6f931bb006a3cd3414.jpg"},{"id":64404496,"identity":"8f99812e-8c22-46bd-982e-3ef23540b5a7","added_by":"auto","created_at":"2024-09-12 16:48:02","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1099204,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e(a)\u003c/strong\u003e The mitochondria were observed using the transmission electron microscope. \u003cstrong\u003e(b)\u003c/strong\u003e The level of ROS was detected using MitSox staining.\u003cstrong\u003e (c)\u003c/strong\u003e The cytochrome c protein level in mitochondria was analyzed using western blotting. \u003cstrong\u003e(d)\u003c/strong\u003e The mitochondrial membrane potential was analyzed by JC-1 staining. \u003csup\u003e#\u003c/sup\u003ep\u0026lt;0.05,\u003csup\u003e ###\u003c/sup\u003ep\u0026lt;0.001. ROS, Reactive Oxygen Species.\u003c/p\u003e","description":"","filename":"Fig.6.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4923322/v1/8d7d47ba92b9e5a108576df7.jpg"},{"id":64404112,"identity":"6457fb56-71e2-4545-a98b-25951eaa0c05","added_by":"auto","created_at":"2024-09-12 16:40:02","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":6776363,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e(a)\u003c/strong\u003eThe USP11 protein level was analyzed using western blotting. \u003cstrong\u003e(b)\u003c/strong\u003e The heatmap of differential proteins. \u003cstrong\u003e(c)\u003c/strong\u003e The differential proteins were analyzed using GO annotation and enrichment. USP11, Ubiquitin Specific Peptidase 11, GO, gene ontology.\u003c/p\u003e","description":"","filename":"Fig.7.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4923322/v1/5b90c6584dd1e5bcfb7e1dba.jpg"},{"id":64404114,"identity":"0da6956f-4b22-41a7-bd4b-f920ea696ea0","added_by":"auto","created_at":"2024-09-12 16:40:02","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":5143240,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e(a)\u003c/strong\u003eThe genes involved in phosphorylation and cell growth in the HD cell model were analyzed using GO annotation and enrichment. \u003cstrong\u003e(b)\u003c/strong\u003e The common genes involved in both phosphorylation and cell growth were presented via the Venn diagram. HD, Huntington's Disease, GO, gene ontology.\u003c/p\u003e","description":"","filename":"Fig.8.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4923322/v1/2ab0c866abb0fe8561ec1ce5.jpg"},{"id":64404107,"identity":"1986803e-46fd-4b33-af5a-3c63e44b0c56","added_by":"auto","created_at":"2024-09-12 16:40:02","extension":"jpg","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":2308417,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e(a-b)\u003c/strong\u003eThe PTEN protein level was analyzed using western blotting. \u003cstrong\u003e(c)\u003c/strong\u003e The location of USP11 and PTEN in the striatum tissue was detected using immunofluorescence staining. \u003cstrong\u003e(d)\u003c/strong\u003e Molecular simulation docking of USP11 and PTEN. \u003cstrong\u003e(e)\u003c/strong\u003ePrediction of ubiquitination sites of PTEN. \u003cstrong\u003e(f)\u003c/strong\u003e Interaction of USP11 and PTEN was analyzed using co-immunoprecipitation. \u003cstrong\u003e(g)\u003c/strong\u003e The PTEN protein level was analyzed using western blotting. \u003cstrong\u003e(h)\u003c/strong\u003e Ubiquitination levels of PTEN was analyzed using co-immunoprecipitation. USP11, Ubiquitin Specific Peptidase 11, PTEN, Phosphatase and Tensin Homolog.\u003c/p\u003e","description":"","filename":"Fig.9.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4923322/v1/31cff12b8a56d6b459e82c76.jpg"},{"id":64404497,"identity":"360c8e06-9516-4fe4-8535-0940216ce57b","added_by":"auto","created_at":"2024-09-12 16:48:02","extension":"jpg","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":3426946,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e(a)\u003c/strong\u003eThe protein level of p-AKT and AKT was analyzed using western blotting. \u003cstrong\u003e(b)\u003c/strong\u003eThe protein level of PTEN, p-AKT, and AKT was analyzed using western blotting.\u003cstrong\u003e(c)\u003c/strong\u003e The cell apoptosis was analyzed using a TUNEL assay. \u003cstrong\u003e(d)\u003c/strong\u003e The level of ROS was detected using MitSox staining. PTEN, Phosphatase and Tensin Homolog. AKT, AKT Serine/Threonine Kinase, p-AKT, Phospho-AKT Serine/Threonine Kinase.\u003c/p\u003e","description":"","filename":"Fig.10.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4923322/v1/12499b6e7e5eade748d19f9e.jpg"},{"id":73693831,"identity":"cbfdf05a-0572-4be5-ad72-79cfb357daf2","added_by":"auto","created_at":"2025-01-13 16:08:11","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":42558474,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4923322/v1/a87ed1c0-ff97-458a-8fd3-c1470a4065e3.pdf"},{"id":64404498,"identity":"5ce4fdac-209a-4d4b-b829-71580c61f807","added_by":"auto","created_at":"2024-09-12 16:48:02","extension":"zip","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":5428562,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryfileuncroppedBlotsimages.zip","url":"https://assets-eu.researchsquare.com/files/rs-4923322/v1/a6ced63b169d9c8279f4a435.zip"}],"financialInterests":"No competing interests reported.","formattedTitle":"Ubiquitin Specific Peptidase 11 Knockdown Slows Huntington's Disease Progression via Regulating Mitochondrial Dysfunction and Neuronal Apoptosis Depending on PTEN- mediated AKT Pathway","fulltext":[{"header":"Introduction","content":"\u003cp\u003eHuntington's disease (HD) is a prevalent autosomal dominant neurodegenerative disorder caused by a repetition of the cytosine-adenine-guanine (CAG) sequence in the HD gene coding region [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. As the number of CAG repetitions increases, the possibility of polyglutamine expansion in the N-terminal region of the protein also increases. This results in the misfolding of huntingtin (HTT) and the formation of insoluble polymers. If this repetition occurs more than 36 times, it is considered a pathogenic mutation. The repetitive sequence of CAG encodes the mutant huntingtin (mHtt) [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Current treatment of HD focuses largely on managing symptoms. However, as of now, the Food and Drug Administration (FDA) has not approved any disease-modifying treatments for HD [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Therefore, revealing the molecular mechanisms of HD and identifying potential therapeutic targets are promising strategies for treating patients with HD.\u003c/p\u003e \u003cp\u003eUbiquitin-specific processing proteases were reported to be involved in the regulation of neuroinflammation [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Ubiquitin Specific Peptidase (USP) 11 is a deubiquitinating enzyme that belongs to the ubiquitin-specific processing protease family [\u003cspan additionalcitationids=\"CR7\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. The upregulation of USP11 exacerbated traumatic brain injury in rats by inducing neurological impairment and neuronal apoptosis [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. After suppressing the expression of USP11, the neural damage in rats with cerebral hemorrhage was alleviated, accompanied by a reduction in neuronal apoptosis, microglial polarization, and inflammation. [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. This evidence suggests a potential connection between USP11 and neurological disease, but our understanding of the relationship between USP11 and HD is limited.\u003c/p\u003e \u003cp\u003eTranslational modifications enhanced protein versatility by increasing phosphorylation, ubiquitination, methylation, and other processes [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. As a deubiquitinating enzyme, USP11 was reported to be involved in regulating neural function by mediating the deubiquitination modification of downstream proteins to increase their stability, such as Beclin 1 [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Based on this, we analyzed the protein expression patterns in PC12 Q74 cells using label-free proteomics. PTEN, a phosphatase that removes phosphate groups from its bound substrates, was screened out by functional enrichment for mechanistic research. The AKT Serine/Threonine Kinase (AKT) is a crucial protein regulated by PTEN, an important tumor suppressor that promotes apoptosis by dephosphorylating AKT [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Knockdown of PTEN protected hippocampal neurons from oxidative stress damage [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Promoting AKT phosphorylation inhibited striatal neuronal damage caused by misfolded HTT [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn this study, we reported the establishment of a mouse model with HD by crossbreeding male R6/1 mice with female C57BL/6J mice. We investigated the regulatory effect of USP11 on HD by knocking down USP11 in the striatum of WT and HD mice. The effect of USP11 on protein expression was evaluated using label-free proteomics in doxycycline (Dox)-induced PC12 cells employing the Tet-on system \u003cem\u003ein vitro\u003c/em\u003e. Through the enrichment of genes associated with phosphorylation and cell growth, we concentrated on PTEN, which is recognized as a negative regulator of the AKT pathway. Furthermore, the regulatory mechanism of USP11 and PTEN on mitochondrial dysfunction and neuronal apoptosis was investigated \u003cem\u003ein vitro\u003c/em\u003e. Our study revealed the mechanism through which USP11 and PTEN safeguard neurons in HD.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eAntibodies\u003c/h2\u003e \u003cp\u003eThe primary antibodies used for immunofluorescence in myocardial tissues included mHtt antibody (Sigma-Aldrich, St. Louis, MO, USA), USP11 antibody (Santa Cruz, Dallas, TX, USA), Neuronal Nuclei (NeuN) antibody (Abcam, Boston, MA, USA), PTEN antibody (Proteintech, Rosemont, IL, USA), p-AKT antibody (Affinity, Cincinnati, OH, USA), and AKT antibody (Affinity). FITC-conjugated secondary antibody (goat anti-rabbit IgG) and Cy3-conjugated secondary antibody (goat anti-mouse IgG) were from Proteintech. For the Western blot analysis, the primary antibodies used included the USP11 antibody purchased from Santa Cruz, the mHtt antibody purchased from Sigma-Aldrich, the Ubi antibody obtained from Wanleibo (Shenyang, China), the cytochrome c antibody, the PTEN antibody, the COX IV antibody, and the β-actin antibody obtained from Proteintech. The HRP-conjugated secondary antibodies used were goat anti-rabbit IgG and goat anti-mouse IgG (Proteintech).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eAdeno-associated Virus (AAV)\u003c/h2\u003e \u003cp\u003eTo downregulate USP11 mRNA expression, oligonucleotides targeting the mouse USP11 (shUSP11: 5\u0026rsquo;-GGTGGAAGTGTACCCACTAGA-3\u0026rsquo;) or the sequence without any predicted target gene were cloned into the pAAV2-CMV-U6 Track vector. Then, the pAAV2-CMV-U6 Track vector was co-transfected with the pHelper plasmid and pRC2-mi342 plasmid into AAV-293 cells (iCell Bioscience Inc, Shanghai, China) using Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) to generate infectious AAV viral particles containing a plasmid with USP11 shRNA and NC shRNA.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eAnimal experiments\u003c/h2\u003e \u003cp\u003eMale R6/1 mice (C57BL/6J background), expressing exon1 of the human HTT gene, were crossbred with female C57BL/6J mice. Genotypes of the F1 individuals were determined using PCR analysis.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003ePart 1\u003c/h3\u003e\n\u003cp\u003eA total of 36 littermates of F1, including 18 R6/1 mice and 18 WT mice, were used in this part. The WT mice and R6/1 mice were kept for 8, 16, and 20 weeks, respectively. Subsequently, the mice were euthanized, and the striatum was collected.\u003c/p\u003e\n\u003ch3\u003ePart 2\u003c/h3\u003e\n\u003cp\u003eThirty-six WT mice and 36 R6/1 mice were divided into two groups (shNC and shUSP11, n = 18), respectively. Three mice were required to complete all the analyses. The mice were injected with 2 µl of AAV-2 containing USP11-specific shRNA (shUSP11) and NC shRNA (shNC) at a concentration of 1.53×10\u003csup\u003e9\u003c/sup\u003e genomic copies. The injections were performed in the striatum using coordinates relative to bregma as previously described [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]: (1) anteroposterior (AP), + 0.8; mediolateral (ML), + 1.8; and dorsoventral (DV), 2.9 mm, and (2) AP, + 0.3; ML, + 2; and DV, 3 mm below the dural surface, with the incisor bar positioned 3 mm above the interaural line. After 4 weeks of injections, the motor ability and spatial recognition memory of each group were evaluated using the open field test and Y-maze test, as previously reported [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Subsequently, the mice were euthanized, and the striatum was collected. All animal experiments have been approved by the Medical Ethics Committee of Shengjing Hospital of China Medical University and followed the Guide for the Care and Use of Laboratory Animals (Eighth Edition).\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eCell Culture\u003c/h2\u003e \u003cp\u003eInducible rat PC12 cells expressing an exon 1 fragment of HTT with 23 (Q23) or 74 (Q74) glutamine repeats fused to the Green Fluorescent Protein (GFP) were maintained in DMEM culture medium (Servicebio, Wuhan, China) supplemented with 10% fetal calf serum (Tianhang Biotech, Huzhou, China) and cultured at 37℃ with 10% CO\u003csub\u003e2\u003c/sub\u003e. To induce the expression of HTT, the cells were treated with Dox (1 µg/ml) (Macklin, Shanghai, China) and after 0, 1, 3, and 6 days. The induced cells were exposed to cycloheximide (CHX) (Aladdin, Shanghai, China) for 0, 0.5, 1, and 2 h, respectively, to inhibit protein synthesis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eCell Transfection\u003c/h2\u003e \u003cp\u003eThe sequence of shUSP11 or shNC was cloned into a plasmid vector. The cells were transfected with the plasmids using Lipofectamine 3000. The cell viability was detected using a CCK-8 cell proliferation detection kit (KeyGEN, Nanjing, China). MitoSOX staining was performed to assess the level of reactive oxygen species (ROS) in cells.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eReal-time PCR\u003c/h2\u003e \u003cp\u003eThe samples were homogenized in TRIpure lysis buffer (BioTeke Bio., Beijing, China) to extract total RNA. The RNA concentration was analyzed using a UV spectrophotometer (NANO 2000, ThermoFisher Scientific, Pittsburgh, PA, USA). The cDNA synthesis was performed using BeyoRT II M-MLV reverse transcriptase (BeyotimeBiotech, Shanghai, China). The mRNA expression levels were evaluated using real-time PCR with a fluorescent quantitative PCR instrument (Exicycler 96, Bioneer, Daejeon, Korea). The results were calculated using the 2\u003csup\u003e-△△CT\u003c/sup\u003e method. The primer sequences are as follows: Rat USP11, 5’-GGCAGCCTATGTCTTGT-3’ (F), 3’-GATGTCAGAGTTGGGTGTA-5’ (R). Mus USP11: 5’-GATGTACCGACTTTCACG-3’ (F), 3’-GCTGTTGTCTAAGAGGGAT-5’ (R).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eProtein Extraction and Western Blot\u003c/h2\u003e \u003cp\u003eFor the total protein analysis, the sample was lysed for 30 min on ice in RIPA lysis buffer (Proteintech) containing 1% protease inhibitor (Proteintech). After centrifugation, the lysate was separated, and the supernatant was collected. The mitochondrial protein was extracted using a mitochondrial isolation and protein extraction kit (Proteintech), following the provided instructions. The BCA protein concentration determination kit (Proteintech) was used to measure the protein concentration. The protein sample was separated using SDS-PAGE and then transferred from the gels to polyvinylidene difluoride (PVDF) membranes (Thermo Fisher Scientific, Pittsburgh, PA, USA). The membranes were then blocked in 5% skim milk (Proteintech). The blocked membranes were incubated with the primary antibodies (USP11 antibody, 1:300; cytochrome c antibody, 1:2000; mHtt antibody, 1:500; PTEN antibody, 1:5000; Ubi antibody, 1:1000; COX IV, 1:5000; β-actin, 1:20000) overnight at 4°C. Incubation with antibodies against β-actin or COX IV was performed to obtain loading controls. After the primary antibody incubation, the membranes were washed and then incubated for 40 min at 37°C with the secondary antibodies (1:10000). Finally, the reaction was visualized using the hypersensitive ECL chemiluminescence test kit (Proteintech).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eCo-Immunoprecipitation (Co-IP)\u003c/h2\u003e \u003cp\u003eThe cells were lysed in a lysis solution for 30 min. AminoLink® coupling resin cross-linked with the antibody was washed using lysis buffer, and the liquid flowed through the resin was discarded. Next, the lysate was added to the resin and incubated overnight 4℃. Subsequently, then the resin was with a with lysis buffer. Elution buffer was added to release the precipitate from binding to the resin. The eluate was then was using the western blot procedure.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eImmunofluorescence\u003c/h2\u003e \u003cp\u003eThe tissues were embedded in paraffin and then cut into 5-µm sections, which were dewaxed and boiled in an antigen retrieval solution for 10 min. The cells grown on coverslips were fixed with 4% paraformaldehyde and permeabilized using 0.1% Triton X-100 (BeyotimeBiotech). The slices were blocked in 1% BSA (Sangon, Shanghai, China) for 15 min. Then, the blocked slices were incubated with the primary antibodies (1:50) overnight at 4°C. The slices were incubated with secondary antibodies (1:200) for 1 h at 37°C. Finally, the nuclei were counterstained using DAPI (Aladdin Reagents Co. Ltd., Shanghai, China).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eTerminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) Assay\u003c/h2\u003e \u003cp\u003eThe cells grown on coverslips and the tissue sections were permeabilized using 0.1% Triton X-100. The TUNEL assay was conducted using the In Situ Cell Death Detection Kit (Roche, Basel, Switzerland), according to the manufacturer's instructions. The nuclei were counterstained with DAPI. For the TUNEL assay combined with immunofluorescence for NeuN, the TUNEL assay was performed first. The slides were then rinsed before performing immunofluorescence for NeuN using the previously described methods.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eCaspase-3 Activity Assay\u003c/h2\u003e \u003cp\u003eThe protein was extracted using the lysis buffer, and its concentration was determined using the Bradford protein concentration assay kit (BeyotimeBiotech). The Caspase-3 activity was detected using the Caspase-3 activity detection kit (BeyotimeBiotech) according to the manufacturer's instructions. Optical density was measured using a microplate reader (ELX-800, BioTek, Winooski, VT, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eHematoxylin Eosin (H\u0026amp;E) Staining\u003c/h2\u003e \u003cp\u003eThe dewaxed sections were stained with hematoxylin (Solarbio, Beijing, China) for 5 min and then cleared in distilled water. Afterward, the sections were washed and counterstained with Eosin (Sangon) for 3 min, then washed and photographed for later analysis of the lateral ventricle area.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eTransmission Electron Microscope (TEM)\u003c/h2\u003e \u003cp\u003eThe sample was immersed in acetone and an embedding agent (SPI, West Chester, PA, USA), and then polymerized at 60°C for 48 h. Subsequently, the sample was cut into 60–80 nm sections. The slices were stained with a 2% uranium acetate solution in saturated alcohol for 8 min in the dark. After washing, the sections were stained with a 2.6% lead citrate solution in carbon dioxide for 8 min. The stained sections were observed using a TEM (H-7650, Hitachi, Tokyo, Japan).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eLabel-Free Proteomics\u003c/h2\u003e \u003cp\u003eProtein samples were incubated with dithiothreitol (DTT) for 1 h at 37°C to break down disulfide bonds. Then, iodoacetamide was added and incubated for 45 min at room temperature in the dark. Samples were diluted with NH\u003csub\u003e4\u003c/sub\u003eHCO\u003csub\u003e3\u003c/sub\u003e and incubated overnight with trypsin at 37℃ for enzymatic digestion. Formic acid was added to stop the reaction. The sample was desalted using a C18 column. The peptide mixture was separated using the RIGOL L-3000 high-performance liquid chromatography system (Beijing RIGOL Technology Co., Ltd., Beijing, China) and analyzed using the ORBITRAP ECLIPSE mass spectrometer (Thermo Fisher Scientific). The gene ontology (GO) annotation and enrichment analysis were conducted on the differentially expressed proteins.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eData Analysis\u003c/h2\u003e \u003cp\u003eAll data were presented as mean ± standard deviation (SD). Data analysis was performed using GraphPad Prism 9.5 (GraphPad Software Inc., La Jolla, CA, USA). The t-test or ANOVA was used to compare the differences in means. Differences were considered statistically significant at p \u0026lt; 0.05.\u003c/p\u003e \u003c/div\u003e "},{"header":"Result","content":"\u003ch2\u003eUSP11 was highly expressed in mice with HD\u003c/h2\u003e\u003cp\u003eWe have established an HD mouse model by crossing male R6/1 with female C57 mice, as indicated in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea. The R6/1 transgenic mice and the WT mice were obtained from the F1. We detected the expression of mHtt in the striatum of R6/1 transgenic mice using immunofluorescence. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb, no obvious red fluorescence was observed in the 8-week R6/1 mice. The red fluorescence intensity increased with aging, indicating that the expression of mHtt increased as the age of R6/1 mice increased, and reached its peak in the 20-week R6/1 mice. The levels of USP11 in the striatum were analyzed. In the WT mice, the level of USP11 did not show a remarkable change among all groups, but it increased in R6/1 mice as they aged. The level of USP11 was highest in the 20-week R6/1 mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec-d).\u003c/p\u003e\u003ch2\u003eKnocking down USP11 improved the locomotor abilities and spatial memory of mice with HD\u003c/h2\u003e\u003cp\u003eBased on the observed changes in USP11 in the striatum, we injected AAV-2 containing USP11-specific shRNA into the striatum of WT and R6/1 mice to downregulate USP11 mRNA expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). After 16 weeks, the results of the open field test showed that the distance covered by R6/1 mice decreased compared to the WT mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). The low expression of USP11 did not have a significant effect on the WT mice. However, the R6/1 mice with USP11 knockdown showed an increase in the distance they covered. The results of the Y-maze test indicated that the residence time in the familiar arm of R6/1 mice increased, while in the novel arm it decreased compared to that of WT mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec). The silencing of USP11 resulted in R6/1 mice spending less time in the familiar arm and more time in the new arm. It can be observed from the H\u0026amp;E staining results (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed) that there was no obvious difference in the morphology of the striatum between the WT groups. The lateral ventricular area enlargement and striatum atrophy can be observed in R6/1 mice. However, the R6/1 mice with silenced USP11 showed a reduction in the lateral ventricular area, as indicated by the yellow arrows, and the atrophy of the striatum has been alleviated. The level of USP11 in the striatum of WT mice was lower than that in R6/1 mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee), indicating that the AAV-2 infection successfully reduced the expression of USP11. The immunofluorescence staining also showed a similar result (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ef).\u003c/p\u003e\u003ch2\u003eDownregulation of USP11 reduced mHtt polymer formation and prevented mitochondria-related apoptosis in neuron cells\u003c/h2\u003e\u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea, a lot of mHtt polymer was observed in the R6/1 mice, however, the USP11 knockdown cleared most of the mHtt polymer. The western blot results indicated the same outcome (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec, the WT groups exhibited weak green fluorescence and strong red fluorescence, indicating that the neuron cells in WT mice had minimal apoptosis. However, the R6/1 groups showed the opposite effect, with the neuron cells largely undergoing apoptosis. The silencing of USP11 inhibited neuron cell apoptosis in the R6/1 mice. The Caspase-3 activity in the WT groups did not show a distinct difference and was lower than in R6/1 mice. The reduced expression of USP11 led to a decrease in Caspase-3 activity in the R6/1 mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed). Bax expression was increased, and Bcl-2 expression was decreased in the R6/1 mice. USP11 silence inhibited Bax expression, and promoted Bcl-2 expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee). Mitochondria in the striatum were observed using TEM (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ef). The striatum in the WT mice exhibited intact mitochondrial ultrastructure, which was altered in the R6/1 mice. The absence of mitochondrial cristae can be observed in the R6/1 mice. The knockdown of USP11 prevented the alteration of mitochondrial ultrastructure. The yellow arrows indicate the mitochondrial cristae. We detected the level of cytochrome c in the mitochondria (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eg). The expression of cytochrome c was higher in the WT groups compared to the R6/1 groups. The level of cytochrome c increased in the R6/1 mice with a low level of USP11.\u003c/p\u003e\u003ch2\u003eDownregulation of USP11 reduced apoptosis in Q74 cells\u003c/h2\u003e\u003cp\u003eTo confirm these results \u003cem\u003ein vitro\u003c/em\u003e, we established an inducible HD cell model. The GFP tag was fused to the N-terminal of HTT exon 1, which contained 23 CAG repeats and 74 CAG repeats in PC-12 cells, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). The GFP tag was expressed after induction by culture in the presence of Dox, based on the Tet-on system. The HTT expression was determined by the fluorescence intensity. Fluorescence microscopy observations revealed that the fluorescence intensity increased as the induction time extended, reaching its peak after 6 days of induction (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). In Q23 cells, HTT showed a diffuse staining, while in Q74 cells, large aggregates are visible. We examined the expression of GFP. It can be observed that the bands from Q74 cells exhibited a higher molecular weight, indicating the fusion expression of mHtt and GFP (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec). The expression of USP11 in the Q23 cells did not exhibit a significant change during culture (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed-e). In the Q74 cell, the expression of USP11 increased as the induction time was extended. The 6-day cells were selected for further experiments. The expression of USP11 in the Q74 cell was interfered with a plasmid containing USP11 shRNA (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ef-g). We examined the effect of downregulation of USP11 on mHtt expression in the cells. A large amount of mHtt polymer appeared in the Q74 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea-b). Downregulation of USP11 reduced the expression of mHtt polymer. Compared to the Q23 cell, the viability of the Q74 cell decreased, but it was improved by USP11 silencing (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec). The results of TUNEL staining and flow cytometry revealed that the apoptosis rate of the Q74 cell was higher than that of the Q23 cell. The apoptosis in the Q74 cell was inhibited by the low expression of USP11 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed-e), and it was also demonstrated by the expression of Bax and Bcl-2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ef).\u003c/p\u003e\u003ch2\u003eDownregulation of USP11 prevented mitochondrial dysfunction in Q74 cells\u003c/h2\u003e\u003cp\u003eThe Q23 cells exhibited intact mitochondrial ultrastructure (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea). The absence of mitochondrial cristae can be observed in the Q74 cells. The silencing of USP11 protected the mitochondrial cristae. We detected the level of ROS in the mitochondria using MitoSox staining (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb). The level of ROS in the Q74 cells was higher than in the Q23 cells, but it decreased when USP11 was silenced. The level of cytochrome c in the mitochondria was also detected (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec). The expression of cytochrome c was lower in the Q74 cells than in the Q23 cells, but it increased in USP11-silenced Q74 cells. Mitochondrial damage in Q47 cells was also reflected in the decrease of mitochondrial membrane potential, and the downregulation of USP11 expression increased the mitochondrial membrane potential (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ed).\u003c/p\u003e\u003ch2\u003eKnocking down USP11 affected the protein expression in Q74 cells\u003c/h2\u003e\u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea shows a decrease in the level of USP11 in the Q74-shUSP11 cell. The proteomic analysis results are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eb. Silencing USP11 altered protein expression in Q74 cells. GO enrichment results (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ec) indicated that the knockdown of USP11 altered the biological processes related to the regulation of protein aggregation, neuronal development, and phosphorylation. The molecular functions that regulate protein phosphorylation are primarily changed. Various differential proteins were localized in neurons, mitochondria, or the endoplasmic reticulum. Based on the above enrichment results, we screened genes involved in both phosphorylation and cell growth in the HD cell model to further explore the role of the phosphorylation-related target gene regulated by USP11 in HD (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ea). The PTEN gene, which was common to all GO terms (GO:0045936, GO:0048638, GO:0042326, GO:0001558, GO:0016311, GO:0052745, GO:0042578, GO:0016791, GO:0002020, GO:0004438, GO:0099524, GO:0044309, GO:0043197, GO:0099522), was found (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eb).\u003c/p\u003e\u003ch2\u003eKnockdown of USP11 promoted degradation of PTEN\u003c/h2\u003e\u003cp\u003eCompared to the R6/1 mice, the expression of USP11 and PTEN was lower in the WT mice. In the WT mice, USP11 had no significant effect on the expression of PTEN. In the R6/1 mice, USP11 positively regulates the expression of PTEN (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003ea). The expression of PTEN in the Q74 cells was higher than in the Q23 cells, however, it was also inhibited by USP11 silencing (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eb). In the striatum of WT and R6/1 mice, the colocalization of USP11 and PTEN was observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003ec). We then investigated the relationship between USP11 and PTEN in the Q74 cells. The results of molecular simulation docking show that USP11 and PTEN can spontaneously bind (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003ed). The existence of multiple potential ubiquitination modification sites on the amino acid sequence of PTEN was predicted by MusiteDeep software (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003ee). The CoIP results revealed that USP11 and PTEN interact with each other at the protein level (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003ef). The production of proteins in the Q74 cells was restrained after treatment with CHX, and the PTEN level decreased as the treatment time extended (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eg). Additionally, the degradation rate of PTEN protein was faster in the USP11-silenced cells than in the control cells. The detection of ubiquitination modification of PTEN revealed that USP11 silencing increased the ubiquitination modification level of PTEN, thereby promoting the degradation of PTEN (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eh).\u003c/p\u003e\u003ch2\u003eKnockdown of USP11 prevented mitochondrial dysfunction by inhibiting PTEN expression\u003c/h2\u003e\u003cp\u003eTo explore the mechanism of PTEN on neuronal apoptosis, we examined the activation of AKT pathway. The AKT level did not differ among all cells, but the p-AKT level showed a change. The level of p-AKT expression in the Q74 cells was lower than that in the Q23 cells. The downregulation of USP11 led to increased phosphorylation of AKT (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003ea). The expression of PTEN decreased, and the expression of p-AKT increased in the cells with silenced USP11. However, these effects were abolished by overexpression of PTEN (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003eb). The silencing of USP11 inhibited apoptosis, which was rescued by the high expression of PTEN (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003ec). The ROS produced by mitochondria were affected in the same manner (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003ed).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eHD is a devastating disease, and existing treatments mainly center on delivering symptomatic drugs. However, there are currently no available treatments that can improve or prevent HD. As more information was revealed about the pathogenesis of HD, targeted RNA therapy may offer hope for patients with HD. In this study, a transgenic mouse model of HD was constructed, and it was observed that the expression of USP11 was elevated in the HD mice. USP11 may play a crucial role in HD. We then conducted additional research to explore the role of USP11 and its downstream protein PTEN in regulating mitochondrial function and neuronal growth during HD \u003cem\u003ein vivo\u003c/em\u003e and \u003cem\u003ein vitro\u003c/em\u003e. The mechanism of action may involve the PTEN-AKT signaling pathway. These findings revealed the intricate interactions among USP11, PTEN, mitochondrial dysfunction, and neuronal apoptosis in the context of HD. This study provided new insights into targeted RNA therapy for HD.\u003c/p\u003e \u003cp\u003eThe main cause of HD is the toxic function of the mHtt protein, which is typically located in the neuronal nuclei of the brain's striatum [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. High levels of mHtt polymer expression were detected in the striatum of 16-week-old R6/1 mice, indicating that these mice develop HD by 16 weeks of age. Meanwhile, high levels of USP11 expression were detected in the striatum of 16-week-old R6/1 mice. HD is characterized by progressive cognitive impairment and abnormal motor symptoms. Clinical manifestations include slow movement, impaired fine motor skills, cognitive decline, and behavioral or mental changes [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. In this study, the motor ability and spatial recognition of HD mice were assessed using the open field test and Y-maze. Mice with HD had significantly weaker locomotor ability and engaged in fewer walking behaviors compared to WT mice. This was typical of HD onset and is consistent with a previously reported [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. The HD mice with silenced USP11 showed increased walking behavior and improved locomotor activity. Mice with HD typically exhibited significant deficits of spatial recognition, likely due to cognitive impairment, which was consistent with previous findings [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. The USP11 silencing also improved the situation. In histopathology, the enlargement of the lateral ventricles and severe atrophy of the striatum are neuropathological features of HD [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. In this study, H\u0026amp;E staining of the striatum of HD mice revealed a severely atrophied striatum and lateral ventricular enlargement, consistent with previous reports [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Downregulation of USP11 alleviated striatum degeneration.\u003c/p\u003e \u003cp\u003eBased on the above phenomena, we further explored the molecular mechanism of USP11 in HD. The striatum lesions were always caused by the loss of striatal neurons [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. The insolubility and toxicity of aggregated protein mHtt were major causes of neuronal apoptosis [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. At the same time, the loss of striatal neurons was also considered to be the most significant morphological correlate of clinical symptoms of HD patients, such as cognitive decline [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. The high level of mHtt polymer in the striatum was a marker of HD. TUNEL combined with NeuN staining was used to detect neuronal loss in the mouse striatum. There was minimal neuronal loss in the striatum of WT mice, whereas a significant number of neurons underwent apoptosis in the striatum of HD mice. Meanwhile, the activity of Caspase-3 increased in the striatum of HD mice, further explaining the apoptosis of neurons. The neuronal apoptosis was inhibited by silencing USP11 in mice with HD. Neurons are high-energy-demanding cells, and their normal function is closely related to mitochondrial function. Mitochondria play key roles in supplying ATP, producing ROS, and providing resistance to oxidative stress in neurons [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. In HD, the accumulation of mHtt may lead to mitochondrial dysfunction, ultimately resulting in neuronal apoptosis [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. ROS was the primary factor in oxidative stress, a condition that is universally present in neurodegenerative diseases. Damage to mitochondrial function increased levels of ROS, leading to neuronal apoptosis [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. We observed morphological abnormalities in the mitochondria of the striatum in HD mice using TEM. These abnormalities were mainly characterized by the disappearance of mitochondrial cristae, which is consistent with previous reports [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Cytochrome c is released from the mitochondria. Downregulation of USP11 in HD mice reduced mHtt polymers in the striatum, suppressed the release of cytochrome c, and improved mitochondrial morphology. The same results were obtained \u003cem\u003ein vitro\u003c/em\u003e. Elevated levels of ROS were observed in cells with impaired mitochondrial morphology. Downregulation of USP11 inhibited the production of ROS and preserved mitochondrial function.\u003c/p\u003e \u003cp\u003eLabel-free proteomic analysis revealed that silencing USP11 significantly altered protein expression in Q74 cells. The levels of proteins associated with neuron growth, protein polymerization, and protein phosphorylation were significantly altered. Protein phosphorylation is closely related to HD pathogenesis [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. It has been reported that there was a widespread disorder of protein phosphorylation in the cerebral cortex in mice before the onset of HD [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. So, we focused on the protein regulated by USP11 that was related to phosphorylation. PTEN, which was involved in both the phosphorylation pathway and cell growth, was highly expressed in the striatum of HD mice and Q74 cells. Its expression was downregulated after the silencing of USP11.\u003c/p\u003e \u003cp\u003eIt was reported that USP11 mediated PTEN deubiquitination, thereby enhancing PTEN stability [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Studies demonstrated that USP11 suppressed PI3K/AKT activity by reversing polyubiquitination and increasing PTEN expression \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. We revealed that USP11 knockdown promoted the ubiquitination degradation of PTNE in Q74 cells, which once again demonstrated the deubiquitination effect of USP11 on PTEN. This finding led us to hypothesize that USP11 regulates HD progression by interacting with PTEN. PTEN negatively regulated AKT phosphorylation and mediated spinal developmental disorders and neuroinflammation in mice [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. The increase of AKT phosphorylation level was conducive to the decrease of mHtt formation [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. To delve deeper into the regulatory mechanism of PTEN on HD, we examined the expression of PTEN downstream target protein AKT and its phosphorylated form, p-AKT, \u003cem\u003ein vitro\u003c/em\u003e. The results showed that the downregulation of USP11 promoted AKT phosphorylation. Overexpression of PTEN reversed this regulatory effect. The USP11-PTEN-AKT axis may be involved in HD progress. PTEN played a critical role in mitochondria-dependent apoptosis in neurodegenerative diseases. After PTEN was knocked out in hippocampal cells, the increase in ROS levels, release of cytochrome c, and activity of Caspase-3 were inhibited [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. The knockout of PTEN restrained the apoptosis by preserving mitochondrial function in an ischemic stroke-like model [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. In addition, the AKT signaling pathway plays a key role in neuronal survival and growth, thereby influencing brain development and function, and impacting a variety of neurological diseases [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Activation of AKT signal reduced neuronal apoptosis after cerebral hemorrhage in mice [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Elevated AKT phosphorylation levels mediated protection of cardiac mitochondrial function [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. In this study, when USP11 was inhibited and PTEN was simultaneously highly expressed in Q74 cells, the regulation of apoptosis and mitochondrial ROS by USP11 was abolished. This suggested that USP11 silencing prevented apoptosis and mitochondrial damage by decreasing PTEN expression, which was mediated by the negative regulation of AKT activation by PTEN.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThe present paper provides important evidence that USP11, a key target in HD treatment, regulated mitochondrial function and nerve cell apoptosis through the PTEN-mediated AKT pathway. The downregulation of USP11 inhibits the expression of PTEN and promotes the phosphorylation of AKT, thereby restoring mitochondrial function and reducing nerve cell apoptosis. This study demonstrates that the USP11-PTEN-AKT signaling pathway may represent a promising new therapeutic target for HD.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll animal experiments have been approved by the Medical Ethics Committee of Shengjing Hospital of China Medical University and followed the Guide for the Care and Use of Laboratory Animals (Eighth Edition).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated and/or analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThere are no competing financial conflicts to declare.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was supported by the National Natural Science Foundation of China (General Program, grant number 81371271) and the Basic Research Project of the Educational Department of Liaoning Province (Youth Project, grant number LJKQZ20222350).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBG: methodology, formal Analysis, and writing-original draft; YCJ: resources, methodology, and writing-original draft; XL: validation and writing-review editing; SYC: funding acquisition and supervision.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eMacDonald ME, Ambrose CM, Duyao MP, Myers RH, Lin C, Srinidhi L, et al. A novel gene containing a trinucleotide repeat that is expanded and unstable on Huntington\u0026apos;s disease chromosomes. Cell. 1993;72(6):971-83. doi: 10.1016/0092-8674(93)90585-E\u003c/li\u003e\n\u003cli\u003eSemaka A, Kay C, Doty C, Collins JA, Bijlsma EK, Richards F, et al. CAG size-specific risk estimates for intermediate allele repeat instability in Huntington disease. J Med Genet. 2013;50(10):696-703. doi: 10.1136/jmedgenet-2013-101796\u003c/li\u003e\n\u003cli\u003ePan L, Feigin A. Huntington\u0026apos;s Disease: New Frontiers in Therapeutics. Curr Neurol Neurosci Rep. 2021;21(3):10. doi: 10.1007/s11910-021-01093-3\u003c/li\u003e\n\u003cli\u003eJiang X, Yu M, Ou Y, Cao Y, Yao Y, Cai P, et al. Downregulation of USP4 Promotes Activation of Microglia and Subsequent Neuronal Inflammation in Rat Spinal Cord After Injury. Neurochem Res. 2017;42(11):3245-53. doi: 10.1007/s11064-017-2361-2\u003c/li\u003e\n\u003cli\u003eZhu L, Bi W, Lu D, Zhang C, Shu X, Wang H, et al. Regulation of ubiquitin-specific processing protease 8 suppresses neuroinflammation. Mol Cell Neurosci. 2015;64:74-83. doi: 10.1016/j.mcn.2014.05.004\u003c/li\u003e\n\u003cli\u003eJacko AM, Nan L, Li S, Tan J, Zhao J, Kass DJ, et al. De-ubiquitinating enzyme, USP11, promotes transforming growth factor \u0026beta;-1 signaling through stabilization of transforming growth factor \u0026beta; receptor II. Cell Death Dis. 2016;7(11):e2474. doi: 10.1038/cddis.2016.371\u003c/li\u003e\n\u003cli\u003eRong Y, Fan J, Ji C, Wang Z, Ge X, Wang J, et al. USP11 regulates autophagy-dependent ferroptosis after spinal cord ischemia-reperfusion injury by deubiquitinating Beclin 1. Cell Death Differ. 2022;29(6):1164-75. doi: 10.1038/s41418-021-00907-8\u003c/li\u003e\n\u003cli\u003eZhang X, Liu T, Xu S, Gao P, Dong W, Liu W, et al. A pro-inflammatory mediator USP11 enhances the stability of p53 and inhibits KLF2 in intracerebral hemorrhage. Mol Ther Methods Clin Dev. 2021;21:681-92. doi: 10.1016/j.omtm.2021.01.015\u003c/li\u003e\n\u003cli\u003eFang Y, Zhao T, Ni H, Li Y, Zhu Y, Gao R, et al. USP11 exacerbates neuronal apoptosis after traumatic brain injury via PKM2-mediated PI3K/AKT signaling pathway. Brain Res. 2023;1807:148321. doi: 10.1016/j.brainres.2023.148321\u003c/li\u003e\n\u003cli\u003eXu Z, Li X, Chen J, Zhao J, Wang J, Ji Y, et al. USP11, Deubiquitinating Enzyme, Associated with Neuronal Apoptosis Following Intracerebral Hemorrhage. J Mol Neurosci. 2016;58(1):16-27. doi: 10.1007/s12031-015-0644-0\u003c/li\u003e\n\u003cli\u003eBasak S, Lu C, Basak A. Post-Translational Protein Modifications of Rare and Unconventional Types: Implications in Functions and Diseases. Curr Med Chem. 2016;23(7):714-45. doi: 10.2174/0929867323666160118095620\u003c/li\u003e\n\u003cli\u003ePiguet AC, Dufour JF. PI(3)K/PTEN/AKT pathway. J Hepatol. 2011;54(6):1317-9. doi: 10.1016/j.jhep.2010.12.013\u003c/li\u003e\n\u003cli\u003eDeng L, Meng T, Chen L, Wei W, Wang P. The role of ubiquitination in tumorigenesis and targeted drug discovery. Signal Transduct Target Ther. 2020;5(1):11. doi: 10.1038/s41392-020-0107-0\u003c/li\u003e\n\u003cli\u003eZhu Y, Hoell P, Ahlemeyer B, Krieglstein J. PTEN: a crucial mediator of mitochondria-dependent apoptosis. Apoptosis. 2006;11(2):197-207. doi: 10.1007/s10495-006-3714-5\u003c/li\u003e\n\u003cli\u003eHumbert S, Bryson EA, Cordeli\u0026egrave;res FP, Connors NC, Datta SR, Finkbeiner S, et al. The IGF-1/Akt pathway is neuroprotective in Huntington\u0026apos;s disease and involves Huntingtin phosphorylation by Akt. Dev Cell. 2002;2(6):831-7. doi: 10.1016/s1534-5807(02)00188-0\u003c/li\u003e\n\u003cli\u003eCreus-Muncunill J, Ru\u0026eacute; L, Alcal\u0026aacute;-Vida R, Badillos-Rodr\u0026iacute;guez R, Roman\u0026iacute;-Aumedes J, Marco S, et al. Increased Levels of Rictor Prevent Mutant Huntingtin-Induced Neuronal Degeneration. Mol Neurobiol. 2018;55(10):7728-42. doi: 10.1007/s12035-018-0956-5\u003c/li\u003e\n\u003cli\u003eAngeles-L\u0026oacute;pez QD, Garc\u0026iacute;a-Lara L, Aguirre-Pineda N, Casta\u0026ntilde;eda-Arellano R, Elizondo-Azuela G, P\u0026eacute;rez-Severiano F, et al. The absence of the aryl hydrocarbon receptor in the R6/1 transgenic mouse model of Huntington\u0026apos;s disease improves the neurological phenotype. Behav Brain Res. 2021;408:113230. doi: 10.1016/j.bbr.2021.113230\u003c/li\u003e\n\u003cli\u003eRagot A, Pietropaolo S, Vincent J, Delage P, Zhang H, Allinquant B, et al. Genetic deletion of the Histone Deacetylase 6 exacerbates selected behavioral deficits in the R6/1 mouse model for Huntington\u0026apos;s disease. Brain Behav. 2015;5(9):e00361. doi: 10.1002/brb3.361\u003c/li\u003e\n\u003cli\u003eDiFiglia M, Sapp E, Chase KO, Davies SW, Bates GP, Vonsattel JP, et al. Aggregation of huntingtin in neuronal intranuclear inclusions and dystrophic neurites in brain. Science. 1997;277(5334):1990-3. doi: 10.1126/science.277.5334.1990\u003c/li\u003e\n\u003cli\u003eR\u0026uuml;b U, Seidel K, Heinsen H, Vonsattel JP, den Dunnen WF, Korf HW. Huntington\u0026apos;s disease (HD): the neuropathology of a multisystem neurodegenerative disorder of the human brain. Brain Pathol. 2016;26(6):726-40. doi: 10.1111/bpa.12426\u003c/li\u003e\n\u003cli\u003eRansome MI, Hannan AJ. Behavioural state differentially engages septohippocampal cholinergic and GABAergic neurons in R6/1 Huntington\u0026apos;s disease mice. Neurobiol Learn Mem. 2012;97(2):261-70. doi: 10.1016/j.nlm.2012.01.004\u003c/li\u003e\n\u003cli\u003ePaulsen JS, Magnotta VA, Mikos AE, Paulson HL, Penziner E, Andreasen NC, et al. Brain structure in preclinical Huntington\u0026apos;s disease. Biol Psychiatry. 2006;59(1):57-63. doi: 10.1016/j.biopsych.2005.06.003\u003c/li\u003e\n\u003cli\u003eRam\u0026iacute;rez-Jarqu\u0026iacute;n UN, Sharma M, Zhou W, Shahani N, Subramaniam S. Deletion of SUMO1 attenuates behavioral and anatomical deficits by regulating autophagic activities in Huntington disease. Proc Natl Acad Sci U S A. 2022;119(5). doi: 10.1073/pnas.2107187119\u003c/li\u003e\n\u003cli\u003eBunner KD, Rebec GV. Corticostriatal Dysfunction in Huntington\u0026apos;s Disease: The Basics. Front Hum Neurosci. 2016;10:317. doi: 10.3389/fnhum.2016.00317\u003c/li\u003e\n\u003cli\u003eSoto C. Unfolding the role of protein misfolding in neurodegenerative diseases. Nat Rev Neurosci. 2003;4(1):49-60. doi: 10.1038/nrn1007\u003c/li\u003e\n\u003cli\u003eShepherd GM. Corticostriatal connectivity and its role in disease. Nat Rev Neurosci. 2013;14(4):278-91. doi: 10.1038/nrn3469\u003c/li\u003e\n\u003cli\u003eL\u0026oacute;pez-Dom\u0026eacute;nech G, Kittler JT. Mitochondrial regulation of local supply of energy in neurons. Curr Opin Neurobiol. 2023;81:102747. doi: 10.1016/j.conb.2023.102747\u003c/li\u003e\n\u003cli\u003eJodeiri Farshbaf M, Ghaedi K. Huntington\u0026apos;s Disease and Mitochondria. Neurotox Res. 2017;32(3):518-29. doi: 10.1007/s12640-017-9766-1\u003c/li\u003e\n\u003cli\u003eSharma A, Behl T, Sharma L, Aelya L, Bungau S. Mitochondrial Dysfunction in Huntington\u0026apos;s Disease: Pathogenesis and Therapeutic Opportunities. Curr Drug Targets. 2021;22(14):1637-67. doi: 10.2174/1389450122666210224105945\u003c/li\u003e\n\u003cli\u003eHyeon SJ, Park J, Yoo J, Kim SH, Hwang YJ, Kim SC, et al. Dysfunction of X-linked inhibitor of apoptosis protein (XIAP) triggers neuropathological processes via altered p53 activity in Huntington\u0026apos;s disease. Prog Neurobiol. 2021;204:102110. doi: 10.1016/j.pneurobio.2021.102110\u003c/li\u003e\n\u003cli\u003eWhite A, McGlone A, Gomez-Pastor R. Protein Kinase CK2 and Its Potential Role as a Therapeutic Target in Huntington\u0026apos;s Disease. Biomedicines. 2022;10(8). doi: 10.3390/biomedicines10081979\u003c/li\u003e\n\u003cli\u003eMees I, Tran H, Roberts A, Lago L, Li S, Roberts BR, et al. Quantitative Phosphoproteomics Reveals Extensive Protein Phosphorylation Dysregulation in the Cerebral Cortex of Huntington\u0026apos;s Disease Mice Prior to Onset of Symptoms. Mol Neurobiol. 2022;59(4):2456-71. doi: 10.1007/s12035-021-02698-y\u003c/li\u003e\n\u003cli\u003eChristine A, Park MK, Song SJ, Song MS. The equilibrium of tumor suppression: DUBs as active regulators of PTEN. Exp Mol Med. 2022;54(11):1814-21. doi: 10.1038/s12276-022-00887-w\u003c/li\u003e\n\u003cli\u003ePark MK, Yao Y, Xia W, Setijono SR, Kim JH, Vila IK, et al. PTEN self-regulates through USP11 via the PI3K-FOXO pathway to stabilize tumor suppression. Nat Commun. 2019;10(1):636. doi: 10.1038/s41467-019-08481-x\u003c/li\u003e\n\u003cli\u003eWu C, Liu H, Zhong D, Yang X, Liao Z, Chen Y, et al. Mapk7 deletion in chondrocytes causes vertebral defects by reducing MEF2C/PTEN/AKT signaling. Genes Dis. 2024;11(2):964-77. doi: 10.1016/j.gendis.2023.02.012\u003c/li\u003e\n\u003cli\u003eFan Q, Liu Y, Sheng L, Lv S, Yang L, Zhang Z, et al. Chaihu-Shugan-San inhibits neuroinflammation in the treatment of post-stroke depression through the JAK/STAT3-GSK3\u0026beta;/PTEN/Akt pathway. Biomed Pharmacother. 2023;160:114385. doi: 10.1016/j.biopha.2023.114385\u003c/li\u003e\n\u003cli\u003eFarajdokht F, Mohaddes G, Karimi-Sales E, Kafshdooz T, Mahmoudi J, Aberoumandi SM, et al. Inhibition of PTEN protects PC12 cells against oxygen-glucose deprivation induced cell death through mitoprotection. Brain Res. 2018;1692:100-9. doi: 10.1016/j.brainres.2018.05.026\u003c/li\u003e\n\u003cli\u003eManning BD, Toker A. AKT/PKB Signaling: Navigating the Network. Cell. 2017;169(3):381-405. doi: 10.1016/j.cell.2017.04.001\u003c/li\u003e\n\u003cli\u003eChen S, Peng J, Sherchan P, Ma Y, Xiang S, Yan F, et al. TREM2 activation attenuates neuroinflammation and neuronal apoptosis via PI3K/Akt pathway after intracerebral hemorrhage in mice. J Neuroinflammation. 2020;17(1):168. doi: 10.1186/s12974-020-01853-x\u003c/li\u003e\n\u003cli\u003eLi X, Luo W, Tang Y, Wu J, Zhang J, Chen S, et al. Semaglutide attenuates doxorubicin-induced cardiotoxicity by ameliorating BNIP3-Mediated mitochondrial dysfunction. Redox Biol. 2024;72:103129. doi: 10.1016/j.redox.2024.103129\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"molecular-medicine","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mome","sideBox":"Learn more about [Molecular Medicine](https://molmed.biomedcentral.com)","snPcode":"10020","submissionUrl":"https://submission.springernature.com/new-submission/10020/3","title":"Molecular Medicine","twitterHandle":"@MolecularMedic1","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Huntington's Disease (HD), Ubiquitin Specific Peptidase 11 (USP11), Phosphatase and Tensin Homolog (PTEN), Neuronal Apoptosis, Mitochondria","lastPublishedDoi":"10.21203/rs.3.rs-4923322/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4923322/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eMitochondrial dysfunction and neuronal apoptosis are major symptoms of cytopathology in Huntington's disease (HD), a neurodegenerative disease. Ubiquitin Specific Peptidase 11 (USP11) is a deubiquitinating enzyme involved in various physiological processes through regulating protein degradation. However, its specific role in HD is unclear.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eTo interfere with USP11 expression, adeno-associated viruses 2 containing USP11-specific shRNA were injected into the bilateral striatum of 12-week-old R6/1 and WT mice. \u003cem\u003eIn vitro\u003c/em\u003e, the inducible PC12 cell model of HD was used in which the expression of an N-terminal truncation of huntingtin, with either wild type (Q23) or expanded polyglutamine (Q74) can be induced by the doxycycline. USP11 was knocked down to study its role in HD. The protein expression patterns in Q74 cells were quantified by label-free proteomics to further explore the target protein of USP11. Detecting the association between USP11 and Phosphatase and Tensin Homolog (PTEN) through Co-IP.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eHerein, USP11 was found to be upregulated in the striatum of R6/1 mice (an HD model with gradual development of symptoms) in an age-dependent manner. The spontaneous HD was alleviated by silencing USP11, as evidenced by improved locomotor activity and spatial memory, attenuated striatal atrophy in R6/1 mice, reduced accumulation of mutant huntingtin protein and neuronal apoptosis, and restored mitochondrial function \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e. The results of label-free proteomics revealed a significant change in the protein expression profile. Through functional enrichment, we focused on PTEN, known as a negative regulator of the AKT pathway. We demonstrated that USP11 downregulation promoted ubiquitination modification of PTEN and activated the AKT pathway, and PTEN overexpression reversed the effects of USP11 knockdown.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eCollectively, USP11 knockdown protects R6/1 mouse neurons from apoptosis, thereby preventing the HD progression. This is achieved by inhibiting PTEN expression, which in turn activates the AKT pathway. This study suggests that USP11-PTEN-AKT signaling pathway may be a new attractive therapeutic target for HD.\u003c/p\u003e","manuscriptTitle":"Ubiquitin Specific Peptidase 11 Knockdown Slows Huntington's Disease Progression via Regulating Mitochondrial Dysfunction and Neuronal Apoptosis Depending on PTEN- mediated AKT Pathway","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-09-12 16:39:57","doi":"10.21203/rs.3.rs-4923322/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-11-29T16:28:51+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-11-28T17:30:45+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"123202549699212960889248217065219802570","date":"2024-11-20T12:55:19+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-10-09T06:50:17+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"252121290050854105274146185816993674469","date":"2024-08-28T07:09:49+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"200969523418091503716458622569334357046","date":"2024-08-27T17:54:42+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-08-27T17:12:22+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-08-16T10:22:47+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-08-16T10:21:27+00:00","index":"","fulltext":""},{"type":"submitted","content":"Molecular Medicine","date":"2024-08-16T07:43:22+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"molecular-medicine","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mome","sideBox":"Learn more about [Molecular Medicine](https://molmed.biomedcentral.com)","snPcode":"10020","submissionUrl":"https://submission.springernature.com/new-submission/10020/3","title":"Molecular Medicine","twitterHandle":"@MolecularMedic1","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"bb383607-cbaf-4f03-bef8-d18759475783","owner":[],"postedDate":"September 12th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-01-13T15:59:58+00:00","versionOfRecord":{"articleIdentity":"rs-4923322","link":"https://doi.org/10.1186/s10020-024-01061-w","journal":{"identity":"molecular-medicine","isVorOnly":false,"title":"Molecular Medicine"},"publishedOn":"2025-01-08 15:57:08","publishedOnDateReadable":"January 8th, 2025"},"versionCreatedAt":"2024-09-12 16:39:57","video":"","vorDoi":"10.1186/s10020-024-01061-w","vorDoiUrl":"https://doi.org/10.1186/s10020-024-01061-w","workflowStages":[]},"version":"v1","identity":"rs-4923322","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4923322","identity":"rs-4923322","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}