DNase2a downregulated by the cGAS-STING-IFN-MEF2C pathway prevents α-synuclein ubiquitination via NEDD4 in Parkinson's disease

DNase2a downregulated by the cGAS-STING-IFN-MEF2C pathway prevents α-synuclein ubiquitination via NEDD4 in Parkinson's disease | 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 DNase2a downregulated by the cGAS-STING-IFN-MEF2C pathway prevents α-synuclein ubiquitination via NEDD4 in Parkinson's disease Jie-wen Zhang, Hao-han Zhang, Lei Li, Fei Chen, Jie Zhu, Fang Cui, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7903266/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background DNase2a, a key enzyme responsible for clearing cytoplasmic double-stranded DNA, prevents cytosolic DNA accumulation. Accumulating evidence suggests that aberrant cytosolic DNA accumulation contributes to Parkinson’s disease (PD) pathogenesis, yet the role of DNase2a in PD remains unclear. Methods We examined the effects of neuronal DNase2a and cytosolic damaged DNA on α-synuclein (α-Syn) accumulation in cultured neurons and male A53T transgenic mice, and investigated the underlying mechanism by which α-Syn modulates DNase2a expression. Results The levels of DNase2a were markedly reduced in the brain of A53T α-Syn transgenic mice, accompanied by increased cytoplasmic DNA accumulation. Decreased neuronal DNase2a led to persistent cytosolic DNA accumulation and suppressed NEDD4-mediated α-Syn ubiquitination and degradation, exacerbating α-Syn accumulation and PD pathology in vitro and in vivo. Moreover, A53T α-Syn further aggravated cytosolic DNA accumulation and then repressed MEF2C-mediated DNase2a transcription via activating the cGAS-STING-IFN pathway, forming a deleterious loop between DNase2a and α-Syn. Consistently, neuronal DNase2a deficiency in WT mice drove α-Syn pathology and dopaminergic neuronal degeneration, leading to motor deficits characteristic of PD, while neuronal DNase2a overexpression in A53T transgenic mice significantly ameliorated motor deficits by reducing α-Syn accumulation and preserving dopaminergic neuron integrity. Conclusions Our findings reveal that DNase2a deficiency disrupts α-Syn degradation and accelerates PD pathogenesis, suggesting that DNase2a is a potential therapeutic target for PD. Parkinson's disease DNase2a DNA damage response Ubiquitination cGAS-STING-IFN pathway Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Parkinson’s disease (PD), an age-related neurodegenerative disorder marked by progressive motor deficits, is expected to affect more than 14 million people globally by 2040[ 1 , 2 ]. Pathologically, PD is characterized by the degeneration of dopaminergic neurons, and the presence of Lewy bodies and Lewy neurites composed of abnormally folded and aggregated α-synuclein (α-Syn)[ 3 ]. Therefore, understanding the mechanisms underlying the abnormal production and impaired clearance of α-Syn is crucial for PD treatment [ 4 – 6 ]. Emerging evidence suggests a link between DNA accumulation and α-Syn pathology [ 7 – 11 ]. However, the precise mechanisms related to this process remain largely unclear. Deoxyribonuclease 2α (DNase2a) is a lysosomal enzyme that degrades cytosolic double-strand DNA (dsDNA), a predominant form of damaged DNA. In the absence of DNase2a, undigested DNA activates cytosolic DNA-sensing pathways, triggering inflammation and autoimmunity[ 12 ]. Previous studies have indicated that the levels of dsDNA breaks were increased and DNase2a levels were reduced in aging and Alzheimer’s disease models [ 13 , 14 ]. In senescent cells, downregulation of DNase2a leads to the accumulation of cytosolic DNA, which promotes the senescence-associated secretory phenotype [ 15 ]. Additionally, DNase2a deficiency promotes tau phosphorylation in neurons and accelerates Alzheimer’s disease progression [ 14 ]. Notably, excessive dsDNA breaks and impaired DNA damage response (DDR) have been reported in various α-Syn pathology models[ 7 – 10 ] and in the brains of PD patients[ 16 – 18 ]. Recent studies found that excess cytosolic DNA can activate the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway, triggering neuroinflammation and cell death[ 8 , 11 , 19 – 21 ]. Alternatively, dsDNA acted as a template that interacted with wild-type α-Syn, thereby facilitating its pathological aggregation[ 22 ]. More importantly, overexpression of DNase2a reduced cytosolic DNA accumulation and dopaminergic neuronal loss, and ameliorated motor deficits in PD models[ 11 ]. Therefore, DNase2a and cytosolic DNA accumulation may play an important role in the development of PD neuropathology. In this study, we investigated the effect of neuronal DNase2a and cytosolic damaged DNA on the accumulation and generation of α-Syn in vitro and in vivo , and explored the effect of α-Syn on the expression of DNase2a and the underlying mechanism. Materials and methods Animals Ten-month-old C57BL/6 male mice were purchased from Si Pei Fu (Beijing) Biotechnology Co., Ltd. (Beijing, China). Eight-month-old A53T-Tg mice (Stock No. 004479, The Jackson Laboratory) and age-matched WT controls were purchased from Jiangsu Huangchuang Xinnuo Pharmaceutical Technology Co., Ltd. (Jiangsu, China). Only male mice were used in this study to minimize variability associated with the estrous cycle and to ensure consistency with previously established PD models. All mice were housed under specific pathogen-free conditions (temperature: 22 ± 2°C; humidity: 45% ± 10%; 12-hour light/dark cycle), with food and water provided ad libitum. All animal procedures adhered to the guidelines approved by the Institutional Animal Care and Use Committee of the Institute of Process Engineering, Chinese Academy of Sciences (Approval code: IPEAECA2024113). To evaluate the effects of DNase2a deficiency in WT mice, ten-month-old C57BL/6 mice were randomly assigned to two groups: (1) WT-CON, receiving injections of adeno-associated virus (AAV)-shCON; and (2) WT-KD, receiving injections of AAV-shDNase2a. To evaluate the effects of DNase2a overexpression on PD pathology, eight-month-old WT and A53T-Tg mice were each randomly assigned to two groups. WT mice received either AAV-CON (WT-CON) or AAV-DNase2a (WT-DNase2a), while A53T-Tg mice were administered either AAV-CON (PD-CON) or AAV-DNase2a (PD-DNase2a). Cells Mouse primary neurons Primary cortical neurons were obtained from the cortex of 14- to 15-day-old C57BL/6 mouse embryos. The cortex was isolated, dissociated, and cells were plated at a density of 5 × 10^5 cells per well in 12-well dishes, as previously described[ 23 ]. Cells were cultured in Neurobasal medium supplemented with B-27 (Gibco, #17504-044), GlutaMAX (Gibco, #35050061), and 0.5% penicillin-streptomycin (Gibco, #15070063) for 7–11 days in vitro (DIV7-11), with half of the medium replaced every 3 days. Neuronal maturation was confirmed by immunocytochemistry (ICC) using anti-MAP2 antibodies[ 23 – 25 ]. Neuro2a (N2a) cell line and HEK 293T cells N2a cells were purchased from China’s National Infrastructure of Cell Line Resource (NICR), and HEK 293T cells from ATCC (CRL-3216)[ 24 ]. Both cell lines were cultured in T25 or T75 flasks with DMEM medium (Gibco, #C11965500CP), supplemented with 10% fetal bovine serum (FBS, Gibco, #10099141) and 0.5% penicillin-streptomycin. Cultures were kept in a humidified incubator at 37°C with 5% CO₂. Transfection procedures are described below. Cells were passaged at 80–90% confluency. Plasmid construction and lentiviral production Lentiviruses carrying short hairpin RNA (shRNA) targeting DNase2a (sh-DNase2a) and NEDD4 (sh-NEDD4) were used to downregulate endogenous gene expression, respectively. The shRNA sequences were used as follows: sh-DNase2a, 5′-GGGTCTAGGGATACTCCAAAG-3′; sh-NEDD4, 5′-GCAAACATTCTGGAGGATTCT-3′. The negative control shRNA (sh-NC) sequence was 5′-TTCTCCGAACGTGTCACGT-3′. The pSicor packaging vector was utilized for knockdown experiments. Lentiviruses carrying DNase2a , NEDD4 , or A53T α -Syn genes were used to overexpress each gene, respectively. The mRNA sequences of DNase2a (mRNA: NM_010062.4; protein: P56542) and NEDD4 (mRNA: NM_010890.4; protein: P56935) were retrieved from NCBI (National Center for Biotechnology Information). A53T α-Syn expression plasmids were constructed as previously described [ 26 ]. For overexpression experiments, the pCDH packaging vector was used. All constructs were verified via Sanger sequencing. Lentiviruses were generated by co-transfecting HEK 293T cells with these recombinant plasmids or control vectors, along with the packaging plasmids pMD2.G and psPAX2, as previously described [ 14 , 24 ]. Briefly, the culture medium containing viral particles was harvested 48 hours post-transfection. The supernatant was then filtered and ultracentrifuged to concentrate viral particles, yielding high-titer viral stocks. The resulting pellet was resuspended in PBS and stored at -80°C until use. Lentivirus infection and cell treatments To investigate the impact of DNase2a on α-Syn expression and the underlying molecular mechanisms, primary cortical neurons (DIV7) were infected with lentivirus expressing shRNA targeting DNase2a (sh-DNase2a) or non-targeting control (sh-NC), as well as DNase2a overexpression construct (OE-DNase2a) or empty vector control (OE-Ctrl). After 48 hours of infection, the efficiency of transfection was evaluated by immunofluorescence analysis. Neurons were harvested 72 hours post-transduction (DIV10) for mRNA analysis and 96 hours post-transduction (DIV11) for protein analysis. To further examine the role of NEDD4 in DNase2a-mediated regulation of α-Syn, primary cortical neurons were infected with lentivirus carrying a NEDD4 overexpression construct (OE-NEDD4) or empty vector control (OE-Ctrl), as well as shRNA targeting NEDD4 (sh-NEDD4) or non-targeting control (sh-NC). The procedures for infection, transduction efficiency evaluation, and subsequent mRNA and protein analyses were performed as described above for DNase2a. To determine how A53T α-Syn regulates DNase2a level, primary cortical neurons (DIV7) were infected with lentivirus expressing A53T α-Syn (LV-A53T). Cells for analysis were harvested at DIV10-11. For stable cell line generation, N2a cells infected with LV-A53T were selected in fresh medium supplemented with 50 µg/mL puromycin starting 72 hours post-transduction. The efficiency of gene overexpression or knockdown was confirmed by quantitative reverse transcription PCR (qRT-PCR) and Western blot analyses. At 72 hours post-infection, primary cortical neurons (DIV10) were treated with the indicated compounds and subsequently harvested at the time points shown in the respective figure legends. For degradation experiments involving α-Syn, DNase2a, or MEF2C, neurons were treated with the following inhibitors: 100 µg/mL cycloheximide[ 24 ](CHX, MedChemExpress, Cat.#HY-12320) to block protein synthesis; 20 µM chloroquine[ 27 ](MedChemExpress, Cat.#HY-17589AR ) to inhibit lysosomal degradation; or 20 µM MG132[ 28 ](MedChemExpress, Cat.#HY-13259 ) to inhibit proteasomal degradation. To inhibit cGAS signaling, neurons were treated with 50 µM RU.521[ 29 ](MedChemExpress, Cat.#HY-114180) for 24 hours. Following treatment, cells were harvested on ice in radioimmunoprecipitation assay (RIPA) buffer (Beyotime, Shanghai, China, Cat. # P0013C) supplemented with protease and phosphatase inhibitors. Lysates were then centrifuged at 13,000 ×g for 15 minutes at 4°C, and the supernatants were collected for Western blot analysis. Co-immunoprecipitation (Co-IP) assay Co-IP assays were performed in primary cortical neurons to assess the interactions between α-Syn and NEDD4, α-Syn and ubiquitin, and MEF2C and p-STAT1, as previously described[ 24 ]. Neurons were subjected to the indicated treatments, as outlined in the respective figure legends, and then lysed on ice using a weak RIPA lysis buffer (Beyotime Biotechnology, Shanghai, China; Cat. #P0013D) with protease inhibitors. The lysates were pre-cleared with Protein A/G agarose beads (MedChemExpress; Cat. #HY-K0202) at 4°C for 1 hour to reduce nonspecific binding. Then, the clarified supernatants were incubated overnight at 4°C with the indicated primary antibodies or control IgG (Santa Cruz Biotechnology; Cat. #sc-2025) under gentle rotation. Immune complexes were captured using Protein A/G magnetic beads with continuous rotation at 4°C for 2 hours. Beads were then washed five times with 0.1% PBST (PBS containing 0.1% Tween-20) to remove nonspecific interactions. Bound proteins were eluted with 20 mM glycine buffer (pH 2.0) for 3 minutes, immediately neutralized, and analyzed by Western blotting. Dual-luciferase reporter assays To investigate whether the transcription factor MEF2C activates the DNase2a promoter, we conducted a luciferase reporter assay. Putative MEF2C binding sites within the DNase2a promoter were predicted using the PROMO ( http://alggen.lsi.upc.es/cgi-bin/promo_v3/promo/promoinit.cgi?dirDBnsTF_8.3 ) and TRAP ( http://trap.molgen.mpg.de/cgi-bin/home.cgi ) databases. Six high-scoring candidate binding sites (-1576/-1565, -1523/-1513, -1224/-1213, -1110/-1099, -582/-571, and − 560/-549; Fig. 3 l), along with their corresponding mutant sequences and full-length DNase2a, were individually cloned into the pGL3-basic luciferase reporter vector. The MEF2C coding sequence was subcloned into the pcDNA3.1 expression vector. HEK293T cells were seeded in 6-well plates and transfected at approximately 70% confluence with the firefly luciferase reporter construct, a Renilla luciferase control vector, and the MEF2C expression plasmid using the Y-20 transfection reagent (Yoshi, Wuhan, China; Cat. #A2011). Forty-eight hours post-transfection, cells were harvested. Luciferase activities were measured in cell lysates using the Dual-Luciferase Reporter Assay Kit (Beyotime, Shanghai, China; Cat. #RG029S) following the manufacturer's protocol. The ratio of firefly to Renilla luciferase activity was calculated to evaluate MEF2C-mediated transactivation of the DNase2a promoter[ 30 ]. Chromatin immunoprecipitation (ChIP) analysis. ChIP assays were conducted in primary cortical neurons to examine the interaction between MEF2C and the DNase2a promoter. ChIP analysis was performed using the ChIP Assay Kit (Beyotime, Shanghai, China, Cat. #P2078) under the manufacturer’s instructions. Briefly, chromatin was extracted from neurons (DIV7) and cross-linked with formaldehyde, followed by sonication to shear the genomic DNA into fragments ranging from 200 to 1000 bp. The chromatin lysates were incubated overnight at 4°C with an anti-MEF2C antibody (Abcam; Cat. #ab211493) or normal IgG (Santa Cruz; Cat. #sc-2025) as a negative control. Immune complexes were captured using Protein A/G agarose beads (MedChemExpress; Cat. #HY-K0202). After extensive washing, the DNA–protein crosslinks were reversed, and DNA was purified using the DNA Purification Kit (Beyotime, Shanghai, China; Cat. #D0033). Quantitative real-time PCR was then performed using primers flanking the predicted MEF2C-binding site (–1576 to − 1565 bp) within the DNase2a promoter region. The following primer sequences were used: Forward: 5′-CAGACTCAGCGTTGCCTTTT-3′; Reverse: 5′-CGAGGGTACAGACTCCTCCC-3′. Stereotaxic injection of AAV To manipulate DNase2a expression in vivo , adeno-associated viruses (AAVs) with a serotype of AAV2/9 were used. All recombinant AAVs were purchased from Obio Technology (Shanghai, China). Neuron-specific expression was achieved by using the human synapsin (hSyn) promoter in all constructs. For DNase2a knockdown, an shRNA targeting mouse DNase2a—identical in sequence to that used in in vitro experiments—was cloned into the pAAV-hSyn-EGFP-3Flag-WPRE vector (Fig. 4 a). For overexpression, the full-length coding sequence of mouse DNase2a was subcloned into the pAAV-hSyn-MCS-3Flag-EGFP-WPRE vector (Fig. 5 a). Mice were deeply anesthetized via intraperitoneal injection of ketamine (100 mg/kg) and xylazine (10 mg/kg). According to the grouping described above, 10-month-old male C57BL/6 mice ( n = 10 per group) were injected with AAV-shDNase2a (WT-KD) or AAV-shCON (WT-CON) for DNase2a knockdown, while 8-month-old male C57BL/6 and A53T-Tg mice ( n = 10 per group) received AAV-DNase2a or control vector (AAV-CON) for overexpression experiments. Stereotactic injections of AAV (titer: 8 × 10¹² vg/mL) were bilaterally administered into the substantia nigra (SN; A/P − 3.5 mm, M/L ± 1.25 mm, D/V − 4.5 mm) and striatum (A/P 0.5 mm, M/L ± 2 mm, D/V − 3.5 mm) at a rate of 0.5 µL/min, with 1 µL delivered at each injection site. To prevent reflux, the needle was held in place for 3 minutes following injection. The surgical site was cleaned with sterile saline, and the incision was sutured. After surgery, animals were monitored and given appropriate post-surgical care. One month after stereotaxic injection, the mice underwent training and testing for their motor coordination and anxiety-like behavior, after which they were sacrificed for biochemical and histological analyses (Fig. 4 b; Fig. 5 b). Behavioral tests These animals underwent a series of behavioral tests one month after AAV treatment. Motor coordination was evaluated using the rotarod test, pole test, and wire hang test[ 25 ]. Anxiety-like behavior was assessed via the open-field test[ 14 ]. Mice were acclimated to the testing environment before test initiation. Apparatuses were cleaned with 75% ethanol between trials to prevent cross-contamination. Rotarod Test : Mice were subjected to an accelerating rotarod system. Before testing, mice underwent training on the rotarod for 3 consecutive days: each training session involved placing mice on the rotarod at a constant speed of 20 rpm for 5 minutes. For the test, mice were placed on the rotating cylinder, which accelerated from 0 to 40 rpm within 10 minutes. The latency to fall off the cylinder was recorded for each mouse. Each mouse completed three trials, and results were expressed as the average latency across trials. Pole Test Mice were placed head-up at the top of a vertical pole (50 cm high, 1 cm in diameter). Before testing, mice underwent training for 3 consecutive days, with three trials per training session. On the test day, each mouse was tested in three trials, during which both turning latency and total descent time were recorded. A 60-second cutoff time was applied to terminate prolonged trials. Results were expressed as the average of the three trials. Wire hang test Mice were placed on an inverted wire cage lid, which was suspended 60 cm above a padded platform. The lid was then manually shaken at a constant frequency (4 times per second). The latency to fall off the wire grid was recorded for each mouse. Each mouse underwent three trials, with a 1-hour inter-trial interval. Results for each mouse were calculated as the mean of the three trials. Open field test Mice were individually placed in a square arena (27 × 27 × 20.3 cm). After a 5-minute acclimation period, locomotor activity was recorded for 10 minutes using a video-tracking system. Assessed parameters included total distance traveled, distance traveled in the center zone, duration in the center zone, number of entries into the center zone, and rearing frequency. Brain lysate preparation Following the completion of behavioral testing, mice were deeply anesthetized with sodium pentobarbital and transcardially perfused with ice-cold phosphate-buffered saline (PBS) containing heparin (10 U/mL). Brains were then rapidly removed and sagittally bisected. One hemisphere was fixed in 4% paraformaldehyde for subsequent immunohistochemical analysis, while the other hemisphere was immediately placed on ice for tissue dissection. The striatum and brainstem were carefully isolated, snap-frozen in liquid nitrogen, and stored at − 80°C for subsequent protein and RNA extraction. For protein extraction, tissue samples were homogenized in RIPA buffer (Beyotime, Shanghai, China, Cat. #P0013B) supplemented with protease and phosphatase inhibitors. Homogenization was performed mechanically on ice for 30 minutes. The lysates were centrifuged at 20,000 × g for 30 minutes at 4°C, and the supernatants were collected as the soluble protein fraction. The remaining pellets were resuspended and sonicated in 2% SDS lysis buffer, followed by centrifugation at 22,000 × g for 20 minutes. The resulting supernatants were collected as the insoluble protein fraction[ 25 , 31 ]. Western blot Protein concentrations of both cell lysates and mouse tissue samples were determined using a BCA protein assay kit (Beyotime, Shanghai, China, Cat. #P0009) according to the manufacturer’s instructions. Equal amounts of protein were separated by 4–20% gradient SDS-PAGE gels (Meilunbio, Dalian, China, Cat. #MA0287), run at 80 V for 20 minutes followed by 120 V for 90–120 minutes. Proteins were then transferred to nitrocellulose membranes (PALL, Cat. #P-N66485). Membranes were blocked with 5% skim milk (Solarbio, Beijing, China, Cat. #D8340) in TBST for 1 hour at room temperature, then incubated overnight at 4°C with primary antibodies. The following day, membranes were washed and incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (Zsbio, Beijing, China, Cat. #ZB2301/ZB2305) for 1 hour at room temperature. Protein bands were visualized using a chemiluminescent detection system (A1600, GE Healthcare, Chicago, IL, USA) following the manufacturer's instructions. Band intensities were quantified using ImageJ software (NIH). Unless otherwise specified, β-actin was used as the internal loading control for normalization. Primary antibodies used in this study were as follows: β-actin (Zsbio, Beijing, China, Cat. #TA-9; 1:1000), β-Tubulin (Zsbio, Beijing, China, Cat. #TA-9; 1:1000), DNase2a (Proteintech, Cat. #15934-1-AP; 1:1000), human α-Syn (Abcam, Cat. #ab138501), mouse α-Syn (BD Biosciences, Cat. #610787), ubiquitin (Beyotime, Shanghai, China, Cat. #AF1705; 1:1000), NEDD4 (Beyotime, Shanghai, China, Cat. #AF7554; 1:1000), cGAS (Proteintech, Cat. #29958-1-AP; 1:1000), p-STAT1 Tyr701 (Beyotime, Shanghai, China, Cat. #AF5935), STING (Beyotime, Shanghai, China, Cat. #AG5348; 1:1000), p-STING Ser366 (Affinity, Cat. #AF7416), TBK1 (Abcam, Cat. #ab40676; 1:1000), p-TBK1/NAK Ser172 (Cell Signaling Technology, Cat. #5483; 1:1000), MEF2C (Abcam, Cat. #ab211493; 1:1000), and TREX1 (Beyotime, Shanghai, China, Cat. #AF8232; 1:1000). RNA extraction and Quantitative real-time PCR (RT-qPCR) Total RNA was extracted from cultured cells using TRIzol reagent (Invitrogen, Carlsbad, CA, USA, 15596026CN) following the manufacturer’s instructions. For in vivo samples, RNA was isolated from snap-frozen striatum and brainstem tissues using the RNAeasy™ Mini Kit (Beyotime, Shanghai, China, Cat. #R0026) according to the manufacturer’s protocol. Complementary DNA (cDNA) was synthesized from purified RNA using the Evo M-MLV Reverse Transcription Kit (Accurate Biology, Hunan, China, Cat. #AG11728). Quantitative real-time PCR (RT-qPCR) was performed using the SYBR Green method on a PE ABI PRISM 7700 Sequence Detection System. Primer sequences used in this study are listed in Table S1 . Immunocytochemistry and Immunohistochemistry (IHC) For cultured cells, neurons were gently rinsed three times with PBS, fixed with 4% paraformaldehyde (PFA) for 20 minutes at room temperature, permeabilized with 0.3% Triton X-100 for 20 minutes, and then blocked with 10% donkey serum in PBS for 1 hour at room temperature. Cells were subsequently incubated with primary antibodies overnight at 4°C, followed by incubation with the corresponding fluorophore-conjugated secondary antibodies for 1 hour at room temperature in the dark. Fluorescence images were acquired using a Leica TCS SP8 confocal microscope. For tissue sections, one hemisphere of each brain was post-fixed in 4% PFA overnight at 4°C, dehydrated in a graded ethanol series, cleared with xylene, embedded in paraffin, and sectioned into 8 µm slices. Sections were deparaffinized, rehydrated through xylene/ethanol gradients, and subjected to antigen retrieval in 0.01 M sodium citrate buffer (pH 6.0, 0.05% Tween-20) at 95°C for 20 minutes. After permeabilization with 0.1% Triton X-100 for 15 minutes and blocking with 10% donkey serum in PBS for 1 hour at room temperature, sections were incubated with primary antibodies (diluted in blocking buffer) overnight at 4°C, followed by incubation with the appropriate fluorophore-conjugated secondary antibodies for 1 hour at room temperature in the dark. Confocal imaging was performed using a Leica TCS SP8 microscope. For 3′-diaminobenzidine (DAB) staining, sections were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies for 1 hour at room temperature, developed using a DAB substrate kit (Zsbio, Beijing, China, Cat. #ZLI-9018). For Nissl staining, tissue sections were stained with Nissl Stain Kit (Solarbio, #G1434) according to the manufacturer’s protocols. Images were then acquired with an Olympus IX73 inverted microscope equipped with a DP80 camera. Image acquisition settings were kept constant across all samples. Primary antibodies used for ICC and IHC included: MAP2 (Invitrogen, Cat. #PA1-16751; 1:200), Iba-1 (GeneTex, Cat. # GTX100042; 1:200), GFAP (Cell Signaling Technology, Cat. #3670S; 1:200), GFP (Abcam, Cat. #ab5450; 1:200), DNase2a (Proteintech, Cat. #15934-1-AP; 1:200), TH (R&D Systems, Cat. #MAB7566; 1:200), γ-H2AX Ser139 (Beyotime Biotechnology, Shanghai, China, Cat. # AF5836; 1:100), NEDD4 (Beyotime Biotechnology, Shanghai, China, Cat. # AF7554; 1:100), and p-α-Syn 129 (Cell Signaling Technology, Cat. #23706; 1:200). Secondary antibodies included: donkey anti-mouse IgG H&L (Alexa Fluor® 488; Abcam, Cat. #ab150105; 1:500), donkey anti-mouse IgG H&L (Alexa Fluor® 555; Abcam, Cat. #ab150110; 1:500), donkey anti-rabbit IgG H&L (Alexa Fluor® 488; Abcam, Cat. #ab150073; 1:500), donkey anti-rabbit IgG H&L (Alexa Fluor® 647; Abcam, Cat. #ab150063; 1:500), donkey anti-chicken IgY (H + L) (Alexa Fluor® 488; Yeasen Biotechnology, Shanghai, China, Cat. #34606ES60; 1:500), donkey anti-chicken IgY (H + L) (Alexa Fluor® 594; Yeasen Biotechnology, Shanghai, China, Cat. #34612ES60; 1:500), goat anti-mouse IgG (HRP; Zsbio, Beijing, China, Cat. #zb2305; 1:300), and goat anti-rabbit IgG (HRP; Zsbio, Beijing, China, Cat. #zb2301; 1:300). Statistical analysis Statistical analyses were performed using GraphPad Prism 10 (GraphPad Software, San Diego, CA), as described in the figure legends and Methods section. Data normality was evaluated using the Shapiro–Wilk test. For comparisons between two groups, normally distributed data were analyzed with a two-tailed unpaired t-test; non-normally distributed data were analyzed using the nonparametric Mann–Whitney U test. For comparisons among more than two groups, normally distributed data were assessed via one-way or two-way analysis of variance (ANOVA), followed by Tukey’s post hoc test for multiple comparisons; when normality was violated, the Kruskal–Wallis H test was used, with post hoc analyses performed using the two-stage step-up Benjamini-Krieger-Yekutieli test. Data are expressed as group mean ± SD, and p < 0.05 was considered statistically significant. Results The level of DNase2a is decreased in the brain of A53T transgenic mice Previous reports have demonstrated that cytosolic dsDNA accumulation induces cytotoxicity in animal models of PD, which can be rescued by the overexpression of DNase2a, a lysosomal DNase that degrades cytosolic dsDNA, indicating that DNase2a may be involved in the pathological development of PD[ 11 , 20 , 21 ]. To investigate the relationship between DNase2a and PD risk, we analyzed DNase2a expression in A53T α-Syn transgenic (A53T-Tg) mice. Compared to wild-type (WT) controls, DNase2a protein levels were significantly reduced in A53T-Tg mice both in the brainstem and striatum (Fig. 1 a, b). Moreover, immunofluorescence staining revealed that DNase2a levels were obviously decreased in the substantia nigra (SN) of A53T-Tg mice (Fig. 1 c, d), while the level of γH2AX, a well-established marker of dsDNA breaks, was significantly increased in the same region (Fig. 1 e, f). In contrast, the level of TREX1, another cytosolic DNase, showed no significant changes in either the brainstem or the striatum of A53T-Tg mice when compared to WT mice (Fig. 1 g, h). These findings indicated that DNase2a deficiency may contribute to the pathogenesis of PD by promoting cytosolic DNA accumulation. DNase2a affects α-Syn ubiquitination and degradation To investigate the potential role of DNase2a in PD in vitro , primary cortical neurons were cultured and infected with lentiviral vectors carrying either DNase2a-targeting shRNA (sh-DNase2a) or DNase2a gene construct (OE-DNase2a) to achieve DNase2a deficiency or DNase2a overexpression (Fig. 2 a, b; Fig. S1 a-c). As expected, DNase2a deficiency significantly induced the accumulation of cytosolic dsDNA, showing the increase in γH2AX levels, whereas DNase2a overexpression effectively eliminated the cytosolic dsDNA fragments in neurons (Fig. S1 d-f). Since α-Syn plays a central role in PD pathogenesis, we further explored the association of DNase2a with α-Syn. DNase2a knockdown significantly increased α-Syn level in neurons, whereas DNase2a overexpression markedly decreased it (Fig. 2 a, b). These changes in α-Syn protein levels were not attributed to transcriptional regulation, as SNCA mRNA levels remained unchanged in neurons infected with either sh-DNase2a or OE-DNase2a compared to their respective controls (Fig. S1 g, h). Consistently, RT-qPCR analysis showed that the levels of transcription factors of α-Syn, including CEBPδ, EMX2, NKX6-1[ 32 ], GATA2[ 33 ], PARP1[ 34 ], ZNF219, and ZSCAN21[ 35 ], remained unchanged following DNase2a knockdown (Fig. S1 i). Next, we investigated whether the DNase2a regulated α-Syn protein levels through the protein degradation pathway. Neurons with DNase2a knockdown or overexpression were treated with CHX, a protein synthesis inhibitor, and the results indicated that DNase2a knockdown significantly slowed the degradation rate of α-Syn in the neurons relative to control (Fig. 2 c, d), whereas overexpressing DNase2a accelerated this process (Fig. 2 e, f). These results suggested that DNase2a affected the degradation of α-Syn protein. The intracellular protein degradation pathways mainly include two types: the autophagy-lysosome pathway and the ubiquitin-proteasome pathway[ 4 , 6 ].To investigate how DNase2 affects the degradation of α-Syn, primary cortical neurons were pretreated with either OE-DNase2a or an empty vector (OE-Ctrl), followed by treatment with chloroquine (a lysosomal inhibitor), MG132 (a proteasomal inhibitor), or a combination of both for 24 hours. MG132, either alone or combined with chloroquine, significantly inhibited DNase2a-mediated α-Syn degradation, whereas chloroquine alone had no such effect (Fig. 2 g, h). To further investigate the role of DNase2a in the proteasomal degradation of α-Syn, neurons were treated with the proteasome inhibitor MG132, and the ubiquitination levels of α-Syn were measured. Immunoblotting showed that, upon proteasomal inhibition, α-Syn levels in DNase2a-overexpressing neurons and DNase2a-knockdown neurons became similar to those in the control group (Fig. 2 i). Moreover, IP with anti-α-Syn antibody showed elevated ubiquitin-conjugated α-Syn in OE-DNase2a neurons and reduced ubiquitin-conjugated α-Syn in sh-DNase2a neurons (Fig. 2 i). Overall, these findings indicated that DNase2a affected α-Syn degradation through the ubiquitin-proteasome pathway. DNase2a regulates α-Syn ubiquitination and degradation through NEDD4 Subsequently, we investigated which E3 ubiquitin ligase is involved in DNase2a-regulated α-Syn ubiquitination. Previous studies have demonstrated that neural precursor cell-expressed, developmentally downregulated protein 4 (NEDD4), an E3 ubiquitin ligase, was involved in both α-Syn degradation[ 36 ] and the DDR[ 37 ]. As a regulator of DNA damage sensing and the subsequent response, NEDD4 represses p53 signaling and facilitates DNA repair[ 37 , 38 ], while NEDD4 knockout results in elevated p53 activity and an amplified DDR[ 39 ]. To investigate whether DNase2a regulated α-Syn degradation by modulating NEDD4 expression, we first examined NEDD4 expression in neurons with either DNase2a overexpression or knockdown. NEDD4 protein levels were significantly decreased in sh-DNase2a neurons (Fig. 2 j-l) and elevated in OE-DNase2a neurons (Fig. 2 j, k, m). Next, we silenced NEDD4 in DNase2a-overexpressing neurons and assessed α-Syn abundance. NEDD4 knockdown attenuated DNase2a-induced α-Syn ubiquitination and degradation (Fig. 2 n). Conversely, overexpressing NEDD4 in DNase2a-deficient neurons restored α-Syn ubiquitination and rescued its degradation defect (Fig. 2 o). Moreover, DNase2a overexpression enhanced, whereas its knockdown impaired, both the interaction between α-Syn and NEDD4 and the levels of ubiquitin-conjugated α-Syn. (Fig. 2 p, q). Thus, DNase2a overexpression alleviates α-Syn accumulation by upregulating NEDD4, thereby enhancing α-Syn ubiquitination and degradation. While the activation of the cGAS-STING pathway has been documented in DNase2a-deficient neurons[ 40 ], it remains unclear whether this pathway directly regulates α-Syn accumulation. To examine this, we treated DNase2a-deficient primary cortical neurons with RU.521, a cGAS inhibitor, and measured cytosolic α-Syn levels. Strikingly, RU.521 treatment failed to alter α-Syn levels in sh-DNase2a neurons relative to vector controls (Fig. S1 j, k). These observations indicated that the cGAS-STING pathway did not participate in regulating the abundance of α-Syn mediated by DNase2a. A53T α-Syn suppresses the expression of DNase2a by reducing MEF2C levels To investigate why DNase2a levels were decreased in the brains of A53T-Tg mice, we examined DNase2a levels in primary neurons and N2a cells overexpressing α-Syn through LV-A53T infection. A significant reduction in DNase2a protein levels was observed in both primary neurons and N2a cells following A53T overexpression (OE-A53T) (Fig. 3 a, b; Fig. S2 a, b). This finding was further supported by ICC assay in primary neurons (Fig. 3 c, d). Consistently, the levels of γH2AX were significantly elevated after A53T α-Syn overexpression (Fig. S2 c, d). To determine the causes of DNase2a reduction, CHX was added to the primary cortical neurons to inhibit protein synthesis, and then DNase2a protein levels were analyzed. A53T α-Syn did not accelerate DNase2a degradation (Fig. S2 e, f), and treatment with either autophagy or proteasome inhibitors also failed to rescue DNase2a reduction (Fig. S2 g). Instead, A53T α-Syn markedly decreased DNase2a mRNA levels (Fig. 3 e; Fig. S2 h), demonstrating that A53T α-Syn reduced DNase2a expression by decreasing its transcription. To reveal the underlying mechanism by which A53T α-Syn mediated DNase2a transcription, we first identified the TF governing DNase2a transcription by promoter analysis tools, including GTRD, FIMO-JASPAR, and Animal TFEB. Myocyte enhancer factor 2C (MEF2C), a TF linked to PD pathogenesis and critical for nervous system development, was consistently predicted as the TF of DNase2a by all three datasets (Fig. 3 f). Therefore, we examined MEF2C levels in A53T α-Syn overexpression cells. Compared to controls, the mRNA levels and protein levels of MEF2C were significantly reduced in primary neurons (Fig. 3 g- 3 i) and N2a cells (Fig. S2 i-k) overexpressing A53T α-Syn, which was consistent with the reports demonstrating lower MEF2C levels in PD models [ 41 , 42 ]. This finding was further supported by ICC assay in primary neurons (Fig. 3 j, k). Luciferase reporter assays and ChIP assays with primers flanking the predicted binding region showed that the sequence spanning − 1576 to − 1565 nt had the highest promoter activity (Fig. 3 l–o), indicating MEF2C bound to and enriched at the DNase2a promoter. Consistently, analysis of healthy brain tissues in the GEPIA database ( http://gepia.cancer-pku.cn/ ) revealed a positive correlation between MEF2C and DNase2a expression (Pearson R = 0.2, p < 0.001; Fig. S2 l). These findings demonstrated that MEF2C was the TF of DNase2a and was involved in the regulation of the DNase2a expression via A53T α-Syn. A53T α-Syn reduces MEF2C levels by activating cGAS-STING-IFN pathway MEF2C, a transcription factor regulating immune and neuronal processes, is repressed by the cGAS-IFN pathway via mitochondrial DNA leakage in AD models[ 43 ]. We measured the protein levels related to the cGAS-IFN pathway, including cGAS, p-STING/STING, p-TBK1/TBK1, and p-STAT1/STAT1 in primary neurons (Fig. 3 p, q) and N2a cells (Fig. S2 m, n) overexpressing A53T α-Syn, and found that these protein levels were significantly upregulated, consistent with activation of the cGAS-STING-IFN pathway in PD models[ 11 , 20 , 44 ]. However, how the cGAS-STING-IFN axis downregulates MEF2C remains unclear. To explore this, we treated A53T-overexpressing neurons with CHX and analyzed MEF2C levels. A53T overexpression did not affect MEF2C degradation (Fig. S2 o, p). Consistently, blockade of autophagy or proteasomal activity failed to rescue MEF2C loss (Fig. S2 g), supporting that the reduction of MEF2C was driven by transcriptional downregulation rather than enhanced degradation. Next, we used promoter analysis tools to identify transcription factors that regulate MEF2C expression. We selected high-confidence candidates by cross-referencing predictions from six independent databases (KnockTF, hTFtarget, CHIP-Atlas, GTRD, FIMO-JASPAR, CHEA), including only those selected by four or more tools. Notably, we focused on factors involved in the cGAS-IFN pathway, such as STAT1, a key pathway mediator (Fig. 3 r) and YY1[ 44 ], a major p-STING-binding protein (Fig. S2 q). Immunoblotting revealed significant p-STAT1 upregulation in A53T neurons compared to controls (Fig. 3 p, q; Fig. S2 m, n), while YY1 levels remained unchanged (Fig. S2 r-t). These findings suggested that STAT1, rather than YY1, mediated MEF2C downregulation. Previous reports showed that p-STAT1 interacted with MEF2C and repressed MEF2C transcription[ 45 ]. Here, our CoIP assay results showed that p-STAT1 bound to MEF2C in neurons, and the binding was significantly increased in primary cortical neurons infected with LV-A53T (Fig. 3 s). We further treated A53T-overexpressing neurons with the cGAS inhibitor RU.521 to verify that MEF2C-mediated DNase2a regulation depends on the cGAS-STING pathway. RU.521 treatment suppressed the levels of p-STAT1/STAT1, and rescued the downregulation of DNase2a and MEF2C induced by A53T α-Syn (Fig. S2 u, v). These data supported that A53T α-Syn suppressed DNase2a expression via the cGAS-IFN-MEF2C pathway. Consistently, the levels of cGAS, p-STING/STING, and p-STAT1/STAT1 in A53T-Tg mice were significantly increased, while the levels of MEF2C and DNase2a were notably decreased in the brainstem and striatum of A53T-Tg mice relative to WT mice (Fig. S3a-c). Additionally, qPCR analysis also showed elevated cGAS levels as well as reduced MEF2C and DNase2a levels in the brainstems and striatum of A53T-Tg mice (Fig. S3d-f). Neuronal DNase2a deficiency induces PD-like phenotypes in WT mice To investigate the effect of neuronal DNase2a on PD progression, we downregulated DNase2a expression in neurons by injecting AAVs expressing shRNA against DNase2a (AAV-shDNase2a) or control shRNA (AAV-CON) with the neuron-specific hSyn promoter into the SN and striatum of 10-month-old C57BL/6 mice (Fig. 4 a, b). One month post-injection, robust GFP fluorescence produced by AAV vectors was observed throughout the SN and striatum (Fig. S4a, b), which co-located specifically with MAP2 + neurons but not with microglia or astrocytes (Fig. S4c, d), confirming neuron-specific expression. Knockdown efficiency was confirmed by quantitative immunoblotting of DNase2a in both SN and striatum (Fig. 4 f, g). Immunofluorescence staining further revealed a significant reduction of DNase2a in the SN of WT-KD mice relative to WT-CON controls (Fig. S5a, b), which was accompanied by increased dsDNA accumulation with increased γ-H2AX immunostaining (Fig. S5c, d). Next, we performed a series of behavioral tests to evaluate the impact of DNase2a knockdown on motor function and anxiety-like behaviors in the mice. In the pole test, WT-KD mice showed longer turn and descent times compared to WT-CON mice (Fig. 4 c). Similarly, DNase2a knockdown reduced the latency to maintain balance on the rod in the rotarod test (Fig. 4 d). WT-KD mice also exhibited weaker grip strength than WT-CON mice in the wire hang test (Fig. 4 e), suggesting that DNase2a deficiency exacerbated motor deficits. In the open field test, WT-KD mice traveled shorter total distances, explored the center less, spent less time in the center zone, and made fewer central entries, indicating that DNase2a knockdown increased anxiety-like behavior (Fig. S5e, f). These results aligned with the literature showing that cytosolic DNA accumulation induced PD-like behavioral symptoms in WT mice[ 20 ]. We next examined the impact of DNase2a knockdown on NEDD4 and α-Syn expression in vivo . Western blot analyses showed a reduction of NEDD4 level in the lysates of brainstem and striatum WT-KD mice (Fig. 4 f, g), which was further confirmed by decreased NEDD4 immunostaining in the SN of WT-KD mice (Fig. 4 h, i). Moreover, α-Syn monomer levels in the soluble fraction were significantly increased in both the brainstem and striatum of WT-KD mice (Fig. 4 f, g). Consistent with this, the insoluble fraction from WT-KD mice showed a pronounced accumulation of both monomeric α-Syn and higher-molecular-weight species compared with that in WT controls (Fig. 4 j, k). Since phosphorylation of α-Syn at serine 129 (p-α-Syn 129 ) is typically associated with the formation of aggregates[ 46 , 47 ], we next examined p-α-Syn 129 in WT-KD mice. p-α-Syn 129 was undetectable in both WT-CON and WT-KD mice (Fig. S5g), which was consistent with previous findings that undamaged mitochondrial DNA accumulation does not induce α-Syn phosphorylation[ 20 ]. In addition, we evaluated dopaminergic neuron integrity and found a marked reduction in TH protein levels in the SN and striatum of WT-KD mice (Fig. 4 l, m). IHC analysis further confirmed an exacerbated loss of TH-positive neurons (Fig. 4 n, o) and dopaminergic fibers (Fig. 4 n, p) with DNase2a knockdown. Moreover, Nissl staining of midbrain slices revealed that DNase2a knockdown induced more severe neuronal loss compared to controls (Fig. 4 q, r). These results demonstrated that neuronal DNase2a deficiency drove α-Syn pathology and dopaminergic neuronal degeneration, leading to motor deficits characteristic of PD. Neuronal DNase2a overexpression attenuates behavioral deficits in A53T-Tg mice To assess the impact of DNase2a overexpression on PD pathology, we stereotaxically injected AAV expressing the DNase2a gene (AAV-DNase2a) or control vector (AAV-CON) with neuron-specific hSyn promoter into the bilateral SN and striatum of 8-month-old A53T-Tg and WT mice to drive neuronal DNase2a expression (Fig. 5 a, b). IHC assay confirmed robust AAV expression by showing GFP-positive neurons widely distributed in both SN and striatum one month post-injection (Fig. S4a, b), and AAV expression was restricted to neurons and absent in microglia or astrocytes (Fig. S4e, f). As expected, AAV-DNase2a significantly elevated DNase2a expression in infected neurons (Fig. 5 c, d; Fig. 6 a, b) and alleviated damaged DNA levels (Fig. 5 e, f). We next examined the effect of DNase2a overexpression on the motor and anxiety-like behaviors in WT and A53T-Tg mice. In the pole test, A53T mice injected with DNase2a initiated movement more quickly and descended faster (Fig. 5 g, h), while they showed an extended latency to fall in the rotarod test (Fig. 5 i). Similarly, in the wire hang test, DNase2a overexpression resulted in longer suspension time for A53T mice (Fig. 5 j). Moreover, anxiety-like behavior was also alleviated in A53T-Tg overexpressing DNase2a, as shown by increased distance traveled in the center, increased center time, and more center entries in the open field test (Fig. 5 k, l). Collectively, these results demonstrated that DNase2a overexpression ameliorated PD-like behavioral symptoms of A53T-Tg mice. Neuronal DNase2a overexpression attenuates α-Syn pathology and neurotoxicity in A53T mice We then performed Western blot and IHC assays to assess the effect of neuronal DNase2a overexpression on the levels of NEDD4 and α-Syn in vivo . A53T-Tg mice injected with AAV-DNase2a showed increased NEDD4 levels (Fig. 6 a, b) compared with A53T-Tg mouse controls, which was further confirmed in the SN by IHC (Fig. 6 c, e). Neuronal DNase2a overexpression significantly reduced soluble α-Syn levels in both the brainstem and striatum (Fig. 6 a, b), as well as in the insoluble fractions of these regions (Fig. 6 d, f). ICC revealed markedly reduced p-α-Syn129 signals in the SN (Fig. 6 g, h) and striatum (Fig. 6 g, i) of A53T-Tg mice injected with AAV-DNase2a. Consistently, Western blot analysis confirmed significantly lower p-α-Syn129 levels in the brainstem and striatum (Fig. 6 j, k). Collectively, these data indicated that neuronal DNase2a overexpression elevated NEDD4 expression and reduced α-Syn load in A53T-Tg mice. Previous studies have shown that p-α-Syn pathology is toxic to TH-positive dopaminergic neurons, leading to motor deficits in PD[ 48 , 49 ]. We next investigated TH levels by Western blot and ICC assays. Western blot results showed that DNase2a overexpression markedly attenuated dopaminergic neuron loss in the brainstem and striatum of A53T-Tg mice (Fig. 6 l, m). TH immunostaining demonstrated preservation of TH-positive neurons in the SNpc (Fig. 6 n, o) and reduced dopaminergic fiber degeneration in the striatum of A53T-Tg mice with DNase2a overexpression (Fig. 6 n, p). Furthermore, Nissl staining of midbrain slices revealed a significant decrease in Nissl-positive cells in the SN of A53T-Tg mice, which was partially restored by DNase2a overexpression (Fig. 6 q, r). Together, these findings indicated that neuronal DNase2a mitigated both α-Syn pathology and α-Syn-induced neurotoxicity in vivo . Discussion DNase2a serves as a lysosomal nuclease that hydrolyzes dsDNA, and its downregulation results in cytosolic accumulation of DNA. Although previous studies have investigated the role of DNase2a in senescent cells[ 15 ] and Alzheimer’s disease[ 14 ], its effects on α-Syn and the pathology of PD remain elusive. In our study, we identified DNase2a as a key regulator, connecting cytosolic DNA clearance, the DDR, and α-Syn pathology in PD. DNase2a deficiency hampered cytosolic DNA removal, triggered aberrant DDR, and inhibited NEDD4-mediated α-Syn ubiquitination and degradation, inducing α-Syn accumulation and PD progression. Conversely, DNase2a overexpression restored NEDD4 levels and promoted α-Syn clearance, consistent with the reports that NEDD4 upregulation mitigated PD pathology in vitro and in vivo [ 36 , 50 ]. Importantly, neuronal DNase2a overexpression in A53T mice lowered α-Syn levels, protected dopaminergic neurons, and improved motor and cognitive functions, highlighting its therapeutic potential. DNA leakage from mitochondria or nuclei triggers cGAS-IFN activation in the models of Alzheimer’s disease[ 51 ] , [ 43 ], PD[ 11 , 20 , 52 ], and cellular senescence[ 19 , 53 , 54 ]. In our study, DNA leaked into the cytosol induced by A53T α-Syn activated the cGAS-IFN pathway and repressed MEF2C-dependent DNase2a transcription, thereby hindering the clearance of cytosolic dsDNA and promoting cytosolic DNA accumulation, which formed a self-amplifying pathogenic loop. Evidence from AD models has demonstrated reduced DNase2a expression[ 14 ], cGAS-IFN activation, and MEF2C downregulation[ 43 ], and comparable changes have been observed in aging models[ 15 , 55 ]. However, the interconnectedness of these events has not been systematically investigated. Based on our findings, we speculate that the cGAS-IFN-MEF2C-DNase2a cascade may act as a common pathway contributing to AD, aging, and PD pathology. Notably, this finding also expands our understanding of the mechanisms underlying cytosolic DNA accumulation: whereas previous studies have mostly focused on the "passive leakage" of mitochondrial DNA or nuclear DNA into the cytoplasm[ 11 , 20 , 52 ], our results suggest that impaired DNA clearance due to A53T α-Syn-induced DNase2a reduction prevents the effective removal of the leaked DNA, which is another important driver for sustained cytosolic DNA accumulation. This highlights DNA clearance as a potentially effective target to mitigate aberrant DNA accumulation-induced neurodegeneration. Whether DNase2a expression downregulation is a cause or consequence of PD pathology remains under debate. In our study, neuronal DNase2a knockdown in WT mice led to PD-like neuropathology and induced both motor deficits and anxiety-like behavior, indicating that neuronal DNase2a deficiency may act as an early trigger of PD. This is consistent with previous reports that mitochondrial DNA accumulation alone can trigger PD-like phenotypes[ 20 ]. A53T α-Syn can activate the cGAS-STING pathway by inducing mitochondrial or nuclear DNA leakage[ 56 ]. However, cGAS-STING activation can also occur independently of pathological α-Syn, and low-level cGAS activity is detectable even under physiological conditions[ 57 ]. Furthermore, age-associated cGAS-STING activation promotes neuroinflammation and neurodegeneration[ 15 ], indicating cGAS-STING signaling may be involved in early disease stages independent of α-Syn accumulation. Taken together, our findings support the following model: neuronal DNase2a deficiency and the accumulation of cytosolic DNA act as an early trigger that activates DDR and downregulates NEDD4, impairing α-Syn degradation and inducing PD pathology. Then, α-Syn aggregation exacerbates DNA leakage[ 10 , 52 ] and cGAS-IFN signaling, which further suppresses DNase2a transcription via decreased MEF2C, establishing a deleterious feedback loop. Overall, our study not only supplements the association between cytosolic DNA clearance defects and abnormal α-Syn degradation in PD but also offers a novel molecular perspective for understanding PD pathogenesis. In conclusion, our findings demonstrate that the DNase2a levels are decreased in the brains of A53T-Tg mice, leading to cytosolic DNA accumulation. Decreased DNase2a level impairs α-Syn ubiquitination and degradation by the DNase2a-DDR-NEDD4 axis in vitro and in vivo , resulting in α-Syn accumulation and PD pathogenesis. Moreover, A53T α-Syn promotes cytosolic DNA accumulation and suppresses MEF2C-mediated transcriptional expression of DNase2a via cGAS-STING-IFN pathway, forming a vicious circle. Our present findings reveal that DNase2a deficiency is an early event, rather than merely a secondary consequence in PD pathogenesis, highlighting DNase2a and cytosolic DNA as potential therapeutic targets for PD. Abbreviations AAVs adeno-associated viruses α-Syn α-synuclein ChIP Chromatin immunoprecipitation Co-IP Co-immunoprecipitation cGAS cyclic GMP–AMP synthase DAB Diaminobenzidine DDR DNA damage response DNase2a Deoxyribonuclease 2α dsDNA double-stranded DNA ICC immunocytochemistry NEDD4 Neural precursor cell expressed developmentally downregulated protein 4 (NEDD4) N2a Neuro2a PD Parkinson’s disease qRT-PCR quantitative reverse transcription PCR STING stimulator of interferon genes SN substantia nigra SNpc substantia nigra pars compacta TH tyrosine hydroxylase Declarations Ethics approval and consent to participate All animal experiments were performed in accordance with the China Public Health Service Guide for the Care and Use of Laboratory Animals. Experiments involving mice and protocols were approved by the Institutional Animal Care and Use Committee of the Institute of Process Engineering, Chinese Academy of Sciences (Approval code: IPEAECA2024113). Consent for publication All authors have reviewed the final manuscript and consent to publication. Data Availability All data generated and/or analyzed during this study are either included in this article or are available from the corresponding author on reasonable request. This study did not generate new unique reagents. Conflict of interest The authors have no conflicts of interest to declare that are relevant to the content of this article. Funding This study was supported by the National Natural Science Foundation of China (No.82401674 and No. 82171196). Authors’ contribution R.-t. L., J.-w. Z., H.-h. Z., Y.-r. H., and S.C. designed the experiments; L.L., Fei. C., and J. Z. performed behavioral experiments; H.-h. Z., Y.-r. H., and Fang. C. performed ICC and IHC; Fei. C. and H.-h. Z. conducted the biochemistry experiments; R.-h. S., K. M., and Z.-x. Z. analyzed the data; R.-t. L., J.-w. Z., H.-h. Z., and Y.-r. H. wrote the manuscript. 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08:24:04","extension":"html","order_by":32,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":222464,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7903266/v1/93bf8a6104cc4bbaac682b97.html"},{"id":95613304,"identity":"33c432cf-91f2-4805-ae8f-9f1f8ba26c6c","added_by":"auto","created_at":"2025-11-11 08:24:06","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":889917,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDNase2a levels are decreased in A53T transgenic mice.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea, b \u003c/strong\u003eRepresentative immunoblot images (a) and quantification (b) of DNase2a in the lysates of brainstem and striatum from 8-month-old WT and A53T-Tg mice. \u003cem\u003en\u003c/em\u003e = 5.\u003cstrong\u003e c, d \u003c/strong\u003eRepresentative confocal images (c) and MFI quantification (d) of DNase2a in the substantia nigra(SN) section of WT and A53T-Tg mice. Scale bar, 20 µm; \u003cem\u003en\u003c/em\u003e = 5.\u003cstrong\u003e e, f \u003c/strong\u003eRepresentative confocal images (e) and MFI quantification (f) of γ-H2AX in the SN of WT and A53T-Tg. Scale bar, 100 µm; \u003cem\u003en\u003c/em\u003e = 6. \u003cstrong\u003eg, h \u003c/strong\u003eRepresentative immunoblot images (g) and quantification (h) of TREX1 in the lysates of brainstem and striatum from WT and A53T-Tg mice. \u003cem\u003en\u003c/em\u003e = 5.\u003cstrong\u003e \u003c/strong\u003eFor all panels, n denotes the number of mice per group. Data are mean ± SD. \u003cem\u003eP\u003c/em\u003e-values were determined using two-tailed unpaired Student’s \u003cem\u003et\u003c/em\u003e-tests for comparisons between two groups. * \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05; **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01; ****\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001; \u003cem\u003ens\u003c/em\u003e, not significant.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7903266/v1/90a580b204576155532bd126.png"},{"id":95613297,"identity":"d4d2e97f-69bc-4e38-b7b5-31064e66a43d","added_by":"auto","created_at":"2025-11-11 08:24:05","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2050156,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDNase2a affects α-synuclein ubiquitination and degradation via NEDD4 in primary cortical neurons.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea, b\u003c/strong\u003e Immunoblots (a) and quantification (b) of DNase2a and α-synuclein (α-Syn) in primary cortical neurons (DIV11) infected with lentivirus carrying DNase2a shRNA (sh-DNase2a; upper) or DNase2a gene (OE-DNase2a; lower). β-Tubulin served as a loading control. \u003cem\u003en\u003c/em\u003e= 5.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ec\u003c/strong\u003e -\u003cstrong\u003ef\u003c/strong\u003e Analysis of α-Syn degradation in neurons infected with sh-DNase2a (c, d) or OE-DNase2a (e, f)after CHX treatment for the indicated time. Two-way ANOVA with Tukey’s test. \u003cem\u003en\u003c/em\u003e = 3.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eg, h \u003c/strong\u003eImmunoblots (g) and quantification (h) of α-Syn in neurons infected with sh-NC or sh-DNase2a, and treated with DMSO, chloroquine (CQ), MG132, or MG132+ CQ for 24 h. \u003cem\u003en\u003c/em\u003e = 4.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ei\u003c/strong\u003e Ubiquitination of α-Syn in neurons expressing sh-DNase2a (left) or OE-DNase2a (right) with ±MG132 for 24 h. Cell lysates were immunoprecipitated with α-Syn antibody and probed with anti-ubiquitin (Ub) antibody. \u003cstrong\u003ej, k \u003c/strong\u003eImmunoblots (j) and quantification (k) of NEDD4 in neurons infected with sh-DNase2a or OE-DNase2a. \u003cstrong\u003el, m \u003c/strong\u003eConfocal images and quantification of NEDD4 (purple) in neurons infected with sh-DNase2a (l) or OE-DNase2a (m). Scale bar, 20 μm; \u003cem\u003en\u003c/em\u003e = 5.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003en, o \u003c/strong\u003eUbiquitination of α-Syn in indicated cells. Neurons were co-infected with OE-Ctrl or OE-DNase2a along with sh-NC or sh-NEDD4 (n), or with sh-NC or sh-DNase2a along with OE-Ctrl or OE-NEDD4 (o). α-Syn was immunoprecipitated and blotted with anti-Ub antibody.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ep, q \u003c/strong\u003eCo-IP showing the interaction of α-Syn with NEDD4 and Ub in neurons expressing sh-DNase2a (p) or OE-DNase2a (q) after ±MG132 treatment for 24 h. α-Syn was immunoprecipitated and blotted with anti-NEDD4 and anti-Ub antibodies. \u003cem\u003en\u003c/em\u003e, biologically replicates. CHX, MG132, and CQ were used at 100 μg/mL, 20 μM, and 20 μM, respectively. Data are mean ± SD; two-tailed unpaired t-tests were used unless otherwise indicated. *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01; \u003cem\u003ens\u003c/em\u003e, not significant. See also Fig. S1.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7903266/v1/3f757a7f5b13b78d3ae97bf3.png"},{"id":95657786,"identity":"a758d79c-befb-4a3d-a779-63a65353a841","added_by":"auto","created_at":"2025-11-11 16:21:57","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1567455,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eA53T α-Syn represses DNase2a expression via the cGAS-STING-IFN-MEF2C pathway in primary neurons.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea, b\u003c/strong\u003e Immunoblots (a) and quantification (b) of DNase2a in neurons infected with LV-A53T (OE-A53T) or control vector (OE-Ctrl).\u003cstrong\u003e \u003c/strong\u003e\u003cem\u003en\u003c/em\u003e = 5.\u003cstrong\u003e c, d\u003c/strong\u003e Confocal images (c) and MFI quantification (d) of DNase2a (red) in OE-Ctrl and OE-A53T neurons. Scale bar, 20 μm. \u003cem\u003en\u003c/em\u003e = 6.\u003cstrong\u003e e\u003c/strong\u003e RT-qPCR of DNase2a mRNA in OE-Ctrl and OE-A53T neurons. \u003cem\u003en \u003c/em\u003e= 5.\u003cstrong\u003e f \u003c/strong\u003eVenn diagram showing the overlap of predicted DNase2a transcription factors from GTRD, FIMO-JASPAR, and Animal TFEB.\u003cstrong\u003eg\u003c/strong\u003e RT-qPCR of MEF2C mRNA in OE-Ctrl and OE-A53T neurons. \u003cem\u003en \u003c/em\u003e= 5.\u003cstrong\u003e h, i \u003c/strong\u003eImmunoblots (h) and quantification (i) of MEF2C in OE-Ctrl and OE-A53T neurons. \u003cem\u003en \u003c/em\u003e= 5.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ej, k\u003c/strong\u003e Confocal images (j) and quantification (k) of MEF2C in OE-Ctrl and OE-A53T neurons. Scale bar, 20μm; \u003cem\u003en \u003c/em\u003e= 5. \u003cstrong\u003el\u003c/strong\u003e Predicted MEF2C-binding sites on the DNase2a promoter identified by PROMO and TRAP, with corresponding sequences. \u003cstrong\u003em\u003c/strong\u003e Luciferase reporter assays validating MEF2C binding to the DNase2a promoter in 293T cells. \u003cem\u003en \u003c/em\u003e= 3. \u003cstrong\u003en, o \u003c/strong\u003eChIP-PCR (n) and ChIP-qPCR (o) showing MEF2C binding to the DNase2a promoter in neurons using anti-MEF2C or control IgG. \u003cem\u003en \u003c/em\u003e= 4.\u003cstrong\u003e p, q \u003c/strong\u003eImmunoblots (p) and quantification (q) of α-Syn, cGAS, STING, p-STING, TBK1, p-TBK1, STAT1, p-STAT1 in OE-Ctrl and OE-A53T neurons. \u003cem\u003en \u003c/em\u003e= 5.\u003cstrong\u003e r \u003c/strong\u003eVenn diagram showing the overlap of the predicted transcription factors of MEF2C among the hTFtarget, GTRD, CHIP-Atlas, and CHEA datasets.\u003cstrong\u003e s \u003c/strong\u003eThe interaction between MEF2C and p-STAT1 in OE-Ctrl or OE-A53T neurons was detected by CoIP assay. β-actin served as a loading control, and MEF2C served as a loading control for IP lysates.\u003cstrong\u003e \u003c/strong\u003e\u003cem\u003en\u003c/em\u003e, biologically replicates. Data are mean ± SD. Two-tailed unpaired t-tests were used unless otherwise indicated. *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ****\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001, \u003cem\u003ens\u003c/em\u003e, not significant. See also Fig. S2.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7903266/v1/1368feeff1d032461f8bb7a2.png"},{"id":95656070,"identity":"59b3910c-8f47-45ec-8171-310f383f4131","added_by":"auto","created_at":"2025-11-11 16:17:44","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1813904,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNeuronal DNase2a knockdown induces PD-like phenotypes in WT mice.\u003c/strong\u003e \u003cstrong\u003ea \u003c/strong\u003eSchematic of AAV expressing GFP and DNase2a shRNA.\u003cstrong\u003e b\u003c/strong\u003e Experimental design and pharmacological treatment timeline. \u003cstrong\u003ec \u003c/strong\u003eTime to turn and descend in the pole test. n = 7–8.\u003cstrong\u003ed\u003c/strong\u003e Latency to fall in the rotarod test. \u003cem\u003en\u003c/em\u003e = 7–8.\u003cstrong\u003e e\u003c/strong\u003e The latency of mice falling from the wire mesh in the wire hang test. \u003cem\u003en\u003c/em\u003e = 7–8.\u003cstrong\u003e f, g \u003c/strong\u003eImmunoblots (f) and quantification (g) of DNase2a, NEDD4, and α-Syn in both brainstem and striatal homogenates of mice. \u003cem\u003en\u003c/em\u003e = 5–6.\u003cstrong\u003e h, i\u003c/strong\u003e Confocal images (h) and MFI quantification (i) of NEDD4 in the SN of WT-CON and WT-KD mice. Scale bars, 50 μm; \u003cem\u003en\u003c/em\u003e = 5. \u003cstrong\u003ej, k \u003c/strong\u003eImmunoblots (j) and quantification (k) of insoluble α-Syn in both brainstem and striatal lysates of mice. \u003cem\u003en\u003c/em\u003e = 5.\u003cstrong\u003e l, m \u003c/strong\u003eImmunoblots (l) and quantification (m) of tyrosine hydroxylase (TH) in both brainstem and striatal lysates of mice. \u003cem\u003en\u003c/em\u003e = 6. \u003cstrong\u003en\u003c/strong\u003e Representative IHC images of TH in the substantia nigra pars compacta (SNpc; upper) and striatum (lower) sections of WT-CON and WT-KD mice; the right panel shows enlarged striatum. Scale bars, 400 μm (SNpc),800 μm (striatum), 300 μm (enlarged striatum). \u003cstrong\u003eo\u003c/strong\u003e Counting of TH\u003csup\u003e+\u003c/sup\u003e neurons in the SNpc of (o). \u003cem\u003en\u003c/em\u003e = 6. \u003cstrong\u003ep\u003c/strong\u003e Quantification of TH fiber density in the striatum of (p). \u003cem\u003en\u003c/em\u003e = 5. \u003cstrong\u003eq, r \u003c/strong\u003eRepresentative Nissl-stained images (q) and quantification of Nissl-positive cells (r) in the SN of mice. \u003cem\u003en\u003c/em\u003e = 6. Scale bars, 300 μm. For all panels, \u003cem\u003en\u003c/em\u003e denotes the number of mice per group. Data are mean ± SD. Two-tailed unpaired Student’s t-test was used for comparisons between two groups. *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, ****\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001. See also Fig. S4 and Fig. S5.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7903266/v1/3d153306753934f47aa3cf5a.png"},{"id":95613294,"identity":"d9496bfa-56e8-4d70-88f4-8d07f1276d45","added_by":"auto","created_at":"2025-11-11 08:24:05","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1668400,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNeuronal DNase2a overexpression mitigates motor deficits and anxiety-like behavior in A53T-Tg mice.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea\u003c/strong\u003e Schematic of AAV expressing GFP and DNase2a. \u003cstrong\u003eb\u003c/strong\u003e Schematic representation of the pharmacological treatment and experimental measurement. \u003cstrong\u003ec\u003c/strong\u003e-\u003cstrong\u003ef \u003c/strong\u003eRepresentative confocal images and MFI quantification of DNase2a (c, d) and γ-H2AX (e, f) in the SN of mice from the indicated group. Scale bars, 50 µm (c),100 μm (e);\u003cstrong\u003e \u003c/strong\u003e\u003cem\u003en\u003c/em\u003e = 5. \u003cstrong\u003eg, h \u003c/strong\u003eThe time for the mouse to initiate the turn on the pole and face downwards (g) and the time taken to descend from the top to the bottom of the pole (h) in the pole test.\u003cstrong\u003e \u003c/strong\u003e\u003cem\u003en\u003c/em\u003e = 8. \u003cstrong\u003ei\u003c/strong\u003e The fall latency of mice in the rotarod test. \u003cem\u003en\u003c/em\u003e = 8.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ej\u003c/strong\u003e The latency of mice falling from the wire mesh in the wire hang test. \u003cem\u003en\u003c/em\u003e = 8. \u003cstrong\u003ek\u003c/strong\u003e Representative movement tracks in the open-field test for the indicated groups. \u003cstrong\u003el \u003c/strong\u003eQuantification of distance traveled in the central area, time spent in the central area, and number of entries into the central area in mice of the indicated group. \u003cem\u003en\u003c/em\u003e = 8. For all panels, \u003cem\u003en\u003c/em\u003edenotes the number of mice per group. Data are mean ± SD. Statistical analysis was performed using one-way ANOVA followed by Tukey’s multiple comparison test. *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, ****\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001; \u003cem\u003ens\u003c/em\u003e, not significant. See also Fig. S4.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-7903266/v1/7b8e45628e3b67885218df51.png"},{"id":97664615,"identity":"08cb037a-5652-4bae-8d95-98975c2bd7bb","added_by":"auto","created_at":"2025-12-08 09:11:35","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":9425038,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7903266/v1/90da14ad-d453-4229-8a74-49ef3d9b10a2.pdf"},{"id":95613314,"identity":"770de5f7-d121-47fc-9cc5-28c7f73e17dc","added_by":"auto","created_at":"2025-11-11 08:24:06","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1742962,"visible":true,"origin":"","legend":"","description":"","filename":"supplementaryfile.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7903266/v1/dc24d3bb423b8a34464862f7.pdf"},{"id":95613316,"identity":"943f80f6-26b7-45ba-9366-f7adc30c02b3","added_by":"auto","created_at":"2025-11-11 08:24:06","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":26384194,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryInformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-7903266/v1/e61d56b091fb9cb8c6e08083.docx"},{"id":95656467,"identity":"b76ad020-0d78-4c3f-a912-5447a8c2a37b","added_by":"auto","created_at":"2025-11-11 16:18:45","extension":"jpg","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":120412,"visible":true,"origin":"","legend":"\u003cp\u003eGraphical abstract\u003c/p\u003e","description":"","filename":"Graphicalabstract.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7903266/v1/7d01a15652d3f4dab5db5d03.jpg"}],"financialInterests":"","formattedTitle":"DNase2a downregulated by the cGAS-STING-IFN-MEF2C pathway prevents α-synuclein ubiquitination via NEDD4 in Parkinson's disease","fulltext":[{"header":"Introduction","content":"\u003cp\u003eParkinson\u0026rsquo;s disease (PD), an age-related neurodegenerative disorder marked by progressive motor deficits, is expected to affect more than 14\u0026nbsp;million people globally by 2040[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Pathologically, PD is characterized by the degeneration of dopaminergic neurons, and the presence of Lewy bodies and Lewy neurites composed of abnormally folded and aggregated α-synuclein (α-Syn)[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Therefore, understanding the mechanisms underlying the abnormal production and impaired clearance of α-Syn is crucial for PD treatment [\u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Emerging evidence suggests a link between DNA accumulation and α-Syn pathology [\u003cspan additionalcitationids=\"CR8 CR9 CR10\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. However, the precise mechanisms related to this process remain largely unclear.\u003c/p\u003e\u003cp\u003eDeoxyribonuclease 2α (DNase2a) is a lysosomal enzyme that degrades cytosolic double-strand DNA (dsDNA), a predominant form of damaged DNA. In the absence of DNase2a, undigested DNA activates cytosolic DNA-sensing pathways, triggering inflammation and autoimmunity[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Previous studies have indicated that the levels of dsDNA breaks were increased and DNase2a levels were reduced in aging and Alzheimer\u0026rsquo;s disease models [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. In senescent cells, downregulation of DNase2a leads to the accumulation of cytosolic DNA, which promotes the senescence-associated secretory phenotype [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Additionally, DNase2a deficiency promotes tau phosphorylation in neurons and accelerates Alzheimer\u0026rsquo;s disease progression [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Notably, excessive dsDNA breaks and impaired DNA damage response (DDR) have been reported in various α-Syn pathology models[\u003cspan additionalcitationids=\"CR8 CR9\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e] and in the brains of PD patients[\u003cspan additionalcitationids=\"CR17\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Recent studies found that excess cytosolic DNA can activate the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway, triggering neuroinflammation and cell death[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan additionalcitationids=\"CR20\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Alternatively, dsDNA acted as a template that interacted with wild-type α-Syn, thereby facilitating its pathological aggregation[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. More importantly, overexpression of DNase2a reduced cytosolic DNA accumulation and dopaminergic neuronal loss, and ameliorated motor deficits in PD models[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Therefore, DNase2a and cytosolic DNA accumulation may play an important role in the development of PD neuropathology.\u003c/p\u003e\u003cp\u003eIn this study, we investigated the effect of neuronal DNase2a and cytosolic damaged DNA on the accumulation and generation of α-Syn \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e, and explored the effect of α-Syn on the expression of DNase2a and the underlying mechanism.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eAnimals\u003c/h2\u003e\u003cp\u003eTen-month-old C57BL/6 male mice were purchased from Si Pei Fu (Beijing) Biotechnology Co., Ltd. (Beijing, China). Eight-month-old A53T-Tg mice (Stock No. 004479, The Jackson Laboratory) and age-matched WT controls were purchased from Jiangsu Huangchuang Xinnuo Pharmaceutical Technology Co., Ltd. (Jiangsu, China). Only male mice were used in this study to minimize variability associated with the estrous cycle and to ensure consistency with previously established PD models. All mice were housed under specific pathogen-free conditions (temperature: 22\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C; humidity: 45% \u0026plusmn; 10%; 12-hour light/dark cycle), with food and water provided ad libitum. All animal procedures adhered to the guidelines approved by the Institutional Animal Care and Use Committee of the Institute of Process Engineering, Chinese Academy of Sciences (Approval code: IPEAECA2024113).\u003c/p\u003e\u003cp\u003eTo evaluate the effects of DNase2a deficiency in WT mice, ten-month-old C57BL/6 mice were randomly assigned to two groups: (1) WT-CON, receiving injections of adeno-associated virus (AAV)-shCON; and (2) WT-KD, receiving injections of AAV-shDNase2a. To evaluate the effects of DNase2a overexpression on PD pathology, eight-month-old WT and A53T-Tg mice were each randomly assigned to two groups. WT mice received either AAV-CON (WT-CON) or AAV-DNase2a (WT-DNase2a), while A53T-Tg mice were administered either AAV-CON (PD-CON) or AAV-DNase2a (PD-DNase2a).\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eCells\u003c/h3\u003e\n\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003eMouse primary neurons\u003c/h2\u003e\u003cp\u003ePrimary cortical neurons were obtained from the cortex of 14- to 15-day-old C57BL/6 mouse embryos. The cortex was isolated, dissociated, and cells were plated at a density of 5 \u0026times; 10^5 cells per well in 12-well dishes, as previously described[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Cells were cultured in Neurobasal medium supplemented with B-27 (Gibco, #17504-044), GlutaMAX (Gibco, #35050061), and 0.5% penicillin-streptomycin (Gibco, #15070063) for 7\u0026ndash;11 days \u003cem\u003ein vitro\u003c/em\u003e (DIV7-11), with half of the medium replaced every 3 days. Neuronal maturation was confirmed by immunocytochemistry (ICC) using anti-MAP2 antibodies[\u003cspan additionalcitationids=\"CR24\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eNeuro2a (N2a) cell line and HEK 293T cells\u003c/h3\u003e\n\u003cp\u003eN2a cells were purchased from China\u0026rsquo;s National Infrastructure of Cell Line Resource (NICR), and HEK 293T cells from ATCC (CRL-3216)[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Both cell lines were cultured in T25 or T75 flasks with DMEM medium (Gibco, #C11965500CP), supplemented with 10% fetal bovine serum (FBS, Gibco, #10099141) and 0.5% penicillin-streptomycin. Cultures were kept in a humidified incubator at 37\u0026deg;C with 5% CO₂. Transfection procedures are described below. Cells were passaged at 80\u0026ndash;90% confluency.\u003c/p\u003e\n\u003ch3\u003ePlasmid construction and lentiviral production\u003c/h3\u003e\n\u003cp\u003eLentiviruses carrying short hairpin RNA (shRNA) targeting \u003cem\u003eDNase2a\u003c/em\u003e (sh-DNase2a) and \u003cem\u003eNEDD4\u003c/em\u003e (sh-NEDD4) were used to downregulate endogenous gene expression, respectively. The shRNA sequences were used as follows: sh-DNase2a, 5\u0026prime;-GGGTCTAGGGATACTCCAAAG-3\u0026prime;; sh-NEDD4, 5\u0026prime;-GCAAACATTCTGGAGGATTCT-3\u0026prime;. The negative control shRNA (sh-NC) sequence was 5\u0026prime;-TTCTCCGAACGTGTCACGT-3\u0026prime;. The pSicor packaging vector was utilized for knockdown experiments.\u003c/p\u003e\u003cp\u003eLentiviruses carrying \u003cem\u003eDNase2a\u003c/em\u003e, \u003cem\u003eNEDD4\u003c/em\u003e, or \u003cem\u003eA53T\u003c/em\u003e α\u003cem\u003e-Syn\u003c/em\u003e genes were used to overexpress each gene, respectively. The mRNA sequences of \u003cem\u003eDNase2a\u003c/em\u003e (mRNA: NM_010062.4; protein: P56542) and \u003cem\u003eNEDD4\u003c/em\u003e (mRNA: NM_010890.4; protein: P56935) were retrieved from NCBI (National Center for Biotechnology Information). \u003cem\u003eA53T α-Syn\u003c/em\u003e expression plasmids were constructed as previously described [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. For overexpression experiments, the pCDH packaging vector was used. All constructs were verified via Sanger sequencing.\u003c/p\u003e\u003cp\u003eLentiviruses were generated by co-transfecting HEK 293T cells with these recombinant plasmids or control vectors, along with the packaging plasmids pMD2.G and psPAX2, as previously described [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Briefly, the culture medium containing viral particles was harvested 48 hours post-transfection. The supernatant was then filtered and ultracentrifuged to concentrate viral particles, yielding high-titer viral stocks. The resulting pellet was resuspended in PBS and stored at -80\u0026deg;C until use.\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eLentivirus infection and cell treatments\u003c/h2\u003e\u003cp\u003eTo investigate the impact of DNase2a on α-Syn expression and the underlying molecular mechanisms, primary cortical neurons (DIV7) were infected with lentivirus expressing shRNA targeting DNase2a (sh-DNase2a) or non-targeting control (sh-NC), as well as DNase2a overexpression construct (OE-DNase2a) or empty vector control (OE-Ctrl). After 48 hours of infection, the efficiency of transfection was evaluated by immunofluorescence analysis. Neurons were harvested 72 hours post-transduction (DIV10) for mRNA analysis and 96 hours post-transduction (DIV11) for protein analysis.\u003c/p\u003e\u003cp\u003eTo further examine the role of NEDD4 in DNase2a-mediated regulation of α-Syn, primary cortical neurons were infected with lentivirus carrying a NEDD4 overexpression construct (OE-NEDD4) or empty vector control (OE-Ctrl), as well as shRNA targeting NEDD4 (sh-NEDD4) or non-targeting control (sh-NC). The procedures for infection, transduction efficiency evaluation, and subsequent mRNA and protein analyses were performed as described above for DNase2a.\u003c/p\u003e\u003cp\u003eTo determine how A53T α-Syn regulates DNase2a level, primary cortical neurons (DIV7) were infected with lentivirus expressing A53T α-Syn (LV-A53T). Cells for analysis were harvested at DIV10-11. For stable cell line generation, N2a cells infected with LV-A53T were selected in fresh medium supplemented with 50 \u0026micro;g/mL puromycin starting 72 hours post-transduction. The efficiency of gene overexpression or knockdown was confirmed by quantitative reverse transcription PCR (qRT-PCR) and Western blot analyses.\u003c/p\u003e\u003cp\u003eAt 72 hours post-infection, primary cortical neurons (DIV10) were treated with the indicated compounds and subsequently harvested at the time points shown in the respective figure legends. For degradation experiments involving α-Syn, DNase2a, or MEF2C, neurons were treated with the following inhibitors: 100 \u0026micro;g/mL cycloheximide[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e](CHX, MedChemExpress, Cat.#HY-12320) to block protein synthesis; 20 \u0026micro;M chloroquine[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e](MedChemExpress, Cat.#HY-17589AR ) to inhibit lysosomal degradation; or 20 \u0026micro;M MG132[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e](MedChemExpress, Cat.#HY-13259 ) to inhibit proteasomal degradation. To inhibit cGAS signaling, neurons were treated with 50 \u0026micro;M RU.521[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e](MedChemExpress, Cat.#HY-114180) for 24 hours.\u003c/p\u003e\u003cp\u003eFollowing treatment, cells were harvested on ice in radioimmunoprecipitation assay (RIPA) buffer (Beyotime, Shanghai, China, Cat. # P0013C) supplemented with protease and phosphatase inhibitors. Lysates were then centrifuged at 13,000 \u0026times;g for 15 minutes at 4\u0026deg;C, and the supernatants were collected for Western blot analysis.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eCo-immunoprecipitation (Co-IP) assay\u003c/h3\u003e\n\u003cp\u003eCo-IP assays were performed in primary cortical neurons to assess the interactions between α-Syn and NEDD4, α-Syn and ubiquitin, and MEF2C and p-STAT1, as previously described[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Neurons were subjected to the indicated treatments, as outlined in the respective figure legends, and then lysed on ice using a weak RIPA lysis buffer (Beyotime Biotechnology, Shanghai, China; Cat. #P0013D) with protease inhibitors. The lysates were pre-cleared with Protein A/G agarose beads (MedChemExpress; Cat. #HY-K0202) at 4\u0026deg;C for 1 hour to reduce nonspecific binding. Then, the clarified supernatants were incubated overnight at 4\u0026deg;C with the indicated primary antibodies or control IgG (Santa Cruz Biotechnology; Cat. #sc-2025) under gentle rotation. Immune complexes were captured using Protein A/G magnetic beads with continuous rotation at 4\u0026deg;C for 2 hours. Beads were then washed five times with 0.1% PBST (PBS containing 0.1% Tween-20) to remove nonspecific interactions. Bound proteins were eluted with 20 mM glycine buffer (pH 2.0) for 3 minutes, immediately neutralized, and analyzed by Western blotting.\u003c/p\u003e\n\u003ch3\u003eDual-luciferase reporter assays\u003c/h3\u003e\n\u003cp\u003eTo investigate whether the transcription factor MEF2C activates the DNase2a promoter, we conducted a luciferase reporter assay. Putative MEF2C binding sites within the DNase2a promoter were predicted using the PROMO (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://alggen.lsi.upc.es/cgi-bin/promo_v3/promo/promoinit.cgi?dirDBnsTF_8.3\u003c/span\u003e\u003cspan address=\"http://alggen.lsi.upc.es/cgi-bin/promo_v3/promo/promoinit.cgi?dirDBnsTF_8.3\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and TRAP (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://trap.molgen.mpg.de/cgi-bin/home.cgi\u003c/span\u003e\u003cspan address=\"http://trap.molgen.mpg.de/cgi-bin/home.cgi\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) databases. Six high-scoring candidate binding sites (-1576/-1565, -1523/-1513, -1224/-1213, -1110/-1099, -582/-571, and \u0026minus;\u0026thinsp;560/-549; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003el), along with their corresponding mutant sequences and full-length DNase2a, were individually cloned into the pGL3-basic luciferase reporter vector. The MEF2C coding sequence was subcloned into the pcDNA3.1 expression vector.\u003c/p\u003e\u003cp\u003eHEK293T cells were seeded in 6-well plates and transfected at approximately 70% confluence with the firefly luciferase reporter construct, a Renilla luciferase control vector, and the MEF2C expression plasmid using the Y-20 transfection reagent (Yoshi, Wuhan, China; Cat. #A2011). Forty-eight hours post-transfection, cells were harvested. Luciferase activities were measured in cell lysates using the Dual-Luciferase Reporter Assay Kit (Beyotime, Shanghai, China; Cat. #RG029S) following the manufacturer's protocol. The ratio of firefly to Renilla luciferase activity was calculated to evaluate MEF2C-mediated transactivation of the DNase2a promoter[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003cb\u003eChromatin immunoprecipitation (ChIP) analysis.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eChIP assays were conducted in primary cortical neurons to examine the interaction between MEF2C and the DNase2a promoter. ChIP analysis was performed using the ChIP Assay Kit (Beyotime, Shanghai, China, Cat. #P2078) under the manufacturer\u0026rsquo;s instructions. Briefly, chromatin was extracted from neurons (DIV7) and cross-linked with formaldehyde, followed by sonication to shear the genomic DNA into fragments ranging from 200 to 1000 bp. The chromatin lysates were incubated overnight at 4\u0026deg;C with an anti-MEF2C antibody (Abcam; Cat. #ab211493) or normal IgG (Santa Cruz; Cat. #sc-2025) as a negative control. Immune complexes were captured using Protein A/G agarose beads (MedChemExpress; Cat. #HY-K0202). After extensive washing, the DNA\u0026ndash;protein crosslinks were reversed, and DNA was purified using the DNA Purification Kit (Beyotime, Shanghai, China; Cat. #D0033). Quantitative real-time PCR was then performed using primers flanking the predicted MEF2C-binding site (\u0026ndash;1576 to \u0026minus;\u0026thinsp;1565 bp) within the DNase2a promoter region. The following primer sequences were used: Forward: 5\u0026prime;-CAGACTCAGCGTTGCCTTTT-3\u0026prime;; Reverse: 5\u0026prime;-CGAGGGTACAGACTCCTCCC-3\u0026prime;.\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eStereotaxic injection of AAV\u003c/h2\u003e\u003cp\u003eTo manipulate DNase2a expression \u003cem\u003ein vivo\u003c/em\u003e, adeno-associated viruses (AAVs) with a serotype of AAV2/9 were used. All recombinant AAVs were purchased from Obio Technology (Shanghai, China). Neuron-specific expression was achieved by using the human synapsin (hSyn) promoter in all constructs. For DNase2a knockdown, an shRNA targeting mouse DNase2a\u0026mdash;identical in sequence to that used in \u003cem\u003ein vitro\u003c/em\u003e experiments\u0026mdash;was cloned into the pAAV-hSyn-EGFP-3Flag-WPRE vector (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). For overexpression, the full-length coding sequence of mouse DNase2a was subcloned into the pAAV-hSyn-MCS-3Flag-EGFP-WPRE vector (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea).\u003c/p\u003e\u003cp\u003eMice were deeply anesthetized via intraperitoneal injection of ketamine (100 mg/kg) and xylazine (10 mg/kg). According to the grouping described above, 10-month-old male C57BL/6 mice (\u003cem\u003en\u0026thinsp;=\u003c/em\u003e\u0026thinsp;10 per group) were injected with AAV-shDNase2a (WT-KD) or AAV-shCON (WT-CON) for DNase2a knockdown, while 8-month-old male C57BL/6 and A53T-Tg mice (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;10 per group) received AAV-DNase2a or control vector (AAV-CON) for overexpression experiments. Stereotactic injections of AAV (titer: 8 \u0026times; 10\u0026sup1;\u0026sup2; vg/mL) were bilaterally administered into the substantia nigra (SN; A/P \u0026minus;\u0026thinsp;3.5 mm, M/L\u0026thinsp;\u0026plusmn;\u0026thinsp;1.25 mm, D/V \u0026minus;\u0026thinsp;4.5 mm) and striatum (A/P 0.5 mm, M/L\u0026thinsp;\u0026plusmn;\u0026thinsp;2 mm, D/V \u0026minus;\u0026thinsp;3.5 mm) at a rate of 0.5 \u0026micro;L/min, with 1 \u0026micro;L delivered at each injection site. To prevent reflux, the needle was held in place for 3 minutes following injection. The surgical site was cleaned with sterile saline, and the incision was sutured. After surgery, animals were monitored and given appropriate post-surgical care. One month after stereotaxic injection, the mice underwent training and testing for their motor coordination and anxiety-like behavior, after which they were sacrificed for biochemical and histological analyses (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb; Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eBehavioral tests\u003c/h2\u003e\u003cp\u003eThese animals underwent a series of behavioral tests one month after AAV treatment. Motor coordination was evaluated using the rotarod test, pole test, and wire hang test[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Anxiety-like behavior was assessed via the open-field test[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Mice were acclimated to the testing environment before test initiation. Apparatuses were cleaned with 75% ethanol between trials to prevent cross-contamination.\u003c/p\u003e\u003cp\u003e\u003cb\u003eRotarod Test\u003c/b\u003e: Mice were subjected to an accelerating rotarod system. Before testing, mice underwent training on the rotarod for 3 consecutive days: each training session involved placing mice on the rotarod at a constant speed of 20 rpm for 5 minutes. For the test, mice were placed on the rotating cylinder, which accelerated from 0 to 40 rpm within 10 minutes. The latency to fall off the cylinder was recorded for each mouse. Each mouse completed three trials, and results were expressed as the average latency across trials.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003ePole Test\u003c/strong\u003e\u003cp\u003eMice were placed head-up at the top of a vertical pole (50 cm high, 1 cm in diameter). Before testing, mice underwent training for 3 consecutive days, with three trials per training session. On the test day, each mouse was tested in three trials, during which both turning latency and total descent time were recorded. A 60-second cutoff time was applied to terminate prolonged trials. Results were expressed as the average of the three trials.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eWire hang test\u003c/strong\u003e\u003cp\u003eMice were placed on an inverted wire cage lid, which was suspended 60 cm above a padded platform. The lid was then manually shaken at a constant frequency (4 times per second). The latency to fall off the wire grid was recorded for each mouse. Each mouse underwent three trials, with a 1-hour inter-trial interval. Results for each mouse were calculated as the mean of the three trials.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eOpen field test\u003c/strong\u003e\u003cp\u003eMice were individually placed in a square arena (27 \u0026times; 27 \u0026times; 20.3 cm). After a 5-minute acclimation period, locomotor activity was recorded for 10 minutes using a video-tracking system. Assessed parameters included total distance traveled, distance traveled in the center zone, duration in the center zone, number of entries into the center zone, and rearing frequency.\u003c/p\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eBrain lysate preparation\u003c/h2\u003e\u003cp\u003eFollowing the completion of behavioral testing, mice were deeply anesthetized with sodium pentobarbital and transcardially perfused with ice-cold phosphate-buffered saline (PBS) containing heparin (10 U/mL). Brains were then rapidly removed and sagittally bisected. One hemisphere was fixed in 4% paraformaldehyde for subsequent immunohistochemical analysis, while the other hemisphere was immediately placed on ice for tissue dissection. The striatum and brainstem were carefully isolated, snap-frozen in liquid nitrogen, and stored at \u0026minus;\u0026thinsp;80\u0026deg;C for subsequent protein and RNA extraction.\u003c/p\u003e\u003cp\u003eFor protein extraction, tissue samples were homogenized in RIPA buffer (Beyotime, Shanghai, China, Cat. #P0013B) supplemented with protease and phosphatase inhibitors. Homogenization was performed mechanically on ice for 30 minutes. The lysates were centrifuged at 20,000 \u0026times; g for 30 minutes at 4\u0026deg;C, and the supernatants were collected as the soluble protein fraction. The remaining pellets were resuspended and sonicated in 2% SDS lysis buffer, followed by centrifugation at 22,000 \u0026times; g for 20 minutes. The resulting supernatants were collected as the insoluble protein fraction[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eWestern blot\u003c/h2\u003e\u003cp\u003eProtein concentrations of both cell lysates and mouse tissue samples were determined using a BCA protein assay kit (Beyotime, Shanghai, China, Cat. #P0009) according to the manufacturer\u0026rsquo;s instructions. Equal amounts of protein were separated by 4\u0026ndash;20% gradient SDS-PAGE gels (Meilunbio, Dalian, China, Cat. #MA0287), run at 80 V for 20 minutes followed by 120 V for 90\u0026ndash;120 minutes. Proteins were then transferred to nitrocellulose membranes (PALL, Cat. #P-N66485). Membranes were blocked with 5% skim milk (Solarbio, Beijing, China, Cat. #D8340) in TBST for 1 hour at room temperature, then incubated overnight at 4\u0026deg;C with primary antibodies. The following day, membranes were washed and incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (Zsbio, Beijing, China, Cat. #ZB2301/ZB2305) for 1 hour at room temperature. Protein bands were visualized using a chemiluminescent detection system (A1600, GE Healthcare, Chicago, IL, USA) following the manufacturer's instructions. Band intensities were quantified using ImageJ software (NIH). Unless otherwise specified, β-actin was used as the internal loading control for normalization.\u003c/p\u003e\u003cp\u003ePrimary antibodies used in this study were as follows: β-actin (Zsbio, Beijing, China, Cat. #TA-9; 1:1000), β-Tubulin (Zsbio, Beijing, China, Cat. #TA-9; 1:1000), DNase2a (Proteintech, Cat. #15934-1-AP; 1:1000), human α-Syn (Abcam, Cat. #ab138501), mouse α-Syn (BD Biosciences, Cat. #610787), ubiquitin (Beyotime, Shanghai, China, Cat. #AF1705; 1:1000), NEDD4 (Beyotime, Shanghai, China, Cat. #AF7554; 1:1000), cGAS (Proteintech, Cat. #29958-1-AP; 1:1000), p-STAT1 Tyr701 (Beyotime, Shanghai, China, Cat. #AF5935), STING (Beyotime, Shanghai, China, Cat. #AG5348; 1:1000), p-STING Ser366 (Affinity, Cat. #AF7416), TBK1 (Abcam, Cat. #ab40676; 1:1000), p-TBK1/NAK Ser172 (Cell Signaling Technology, Cat. #5483; 1:1000), MEF2C (Abcam, Cat. #ab211493; 1:1000), and TREX1 (Beyotime, Shanghai, China, Cat. #AF8232; 1:1000).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003eRNA extraction and Quantitative real-time PCR (RT-qPCR)\u003c/h2\u003e\u003cp\u003eTotal RNA was extracted from cultured cells using TRIzol reagent (Invitrogen, Carlsbad, CA, USA, 15596026CN) following the manufacturer\u0026rsquo;s instructions. For \u003cem\u003ein vivo\u003c/em\u003e samples, RNA was isolated from snap-frozen striatum and brainstem tissues using the RNAeasy\u0026trade; Mini Kit (Beyotime, Shanghai, China, Cat. #R0026) according to the manufacturer\u0026rsquo;s protocol. Complementary DNA (cDNA) was synthesized from purified RNA using the Evo M-MLV Reverse Transcription Kit (Accurate Biology, Hunan, China, Cat. #AG11728). Quantitative real-time PCR (RT-qPCR) was performed using the SYBR Green method on a PE ABI PRISM 7700 Sequence Detection System. Primer sequences used in this study are listed in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003eImmunocytochemistry and Immunohistochemistry (IHC)\u003c/h2\u003e\u003cp\u003eFor cultured cells, neurons were gently rinsed three times with PBS, fixed with 4% paraformaldehyde (PFA) for 20 minutes at room temperature, permeabilized with 0.3% Triton X-100 for 20 minutes, and then blocked with 10% donkey serum in PBS for 1 hour at room temperature. Cells were subsequently incubated with primary antibodies overnight at 4\u0026deg;C, followed by incubation with the corresponding fluorophore-conjugated secondary antibodies for 1 hour at room temperature in the dark. Fluorescence images were acquired using a Leica TCS SP8 confocal microscope.\u003c/p\u003e\u003cp\u003eFor tissue sections, one hemisphere of each brain was post-fixed in 4% PFA overnight at 4\u0026deg;C, dehydrated in a graded ethanol series, cleared with xylene, embedded in paraffin, and sectioned into 8 \u0026micro;m slices. Sections were deparaffinized, rehydrated through xylene/ethanol gradients, and subjected to antigen retrieval in 0.01 M sodium citrate buffer (pH 6.0, 0.05% Tween-20) at 95\u0026deg;C for 20 minutes. After permeabilization with 0.1% Triton X-100 for 15 minutes and blocking with 10% donkey serum in PBS for 1 hour at room temperature, sections were incubated with primary antibodies (diluted in blocking buffer) overnight at 4\u0026deg;C, followed by incubation with the appropriate fluorophore-conjugated secondary antibodies for 1 hour at room temperature in the dark. Confocal imaging was performed using a Leica TCS SP8 microscope. For 3\u0026prime;-diaminobenzidine (DAB) staining, sections were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies for 1 hour at room temperature, developed using a DAB substrate kit (Zsbio, Beijing, China, Cat. #ZLI-9018). For Nissl staining, tissue sections were stained with Nissl Stain Kit (Solarbio, #G1434) according to the manufacturer\u0026rsquo;s protocols. Images were then acquired with an Olympus IX73 inverted microscope equipped with a DP80 camera. Image acquisition settings were kept constant across all samples.\u003c/p\u003e\u003cp\u003ePrimary antibodies used for ICC and IHC included: MAP2 (Invitrogen, Cat. #PA1-16751; 1:200), Iba-1 (GeneTex, Cat. # GTX100042; 1:200), GFAP (Cell Signaling Technology, Cat. #3670S; 1:200), GFP (Abcam, Cat. #ab5450; 1:200), DNase2a (Proteintech, Cat. #15934-1-AP; 1:200), TH (R\u0026amp;D Systems, Cat. #MAB7566; 1:200), γ-H2AX Ser139 (Beyotime Biotechnology, Shanghai, China, Cat. # AF5836; 1:100), NEDD4 (Beyotime Biotechnology, Shanghai, China, Cat. # AF7554; 1:100), and p-α-Syn\u003csup\u003e129\u003c/sup\u003e (Cell Signaling Technology, Cat. #23706; 1:200).\u003c/p\u003e\u003cp\u003eSecondary antibodies included: donkey anti-mouse IgG H\u0026amp;L (Alexa Fluor\u0026reg; 488; Abcam, Cat. #ab150105; 1:500), donkey anti-mouse IgG H\u0026amp;L (Alexa Fluor\u0026reg; 555; Abcam, Cat. #ab150110; 1:500), donkey anti-rabbit IgG H\u0026amp;L (Alexa Fluor\u0026reg; 488; Abcam, Cat. #ab150073; 1:500), donkey anti-rabbit IgG H\u0026amp;L (Alexa Fluor\u0026reg; 647; Abcam, Cat. #ab150063; 1:500), donkey anti-chicken IgY (H\u0026thinsp;+\u0026thinsp;L) (Alexa Fluor\u0026reg; 488; Yeasen Biotechnology, Shanghai, China, Cat. #34606ES60; 1:500), donkey anti-chicken IgY (H\u0026thinsp;+\u0026thinsp;L) (Alexa Fluor\u0026reg; 594; Yeasen Biotechnology, Shanghai, China, Cat. #34612ES60; 1:500), goat anti-mouse IgG (HRP; Zsbio, Beijing, China, Cat. #zb2305; 1:300), and goat anti-rabbit IgG (HRP; Zsbio, Beijing, China, Cat. #zb2301; 1:300).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eStatistical analyses were performed using GraphPad Prism 10 (GraphPad Software, San Diego, CA), as described in the figure legends and Methods section. Data normality was evaluated using the Shapiro\u0026ndash;Wilk test. For comparisons between two groups, normally distributed data were analyzed with a two-tailed unpaired t-test; non-normally distributed data were analyzed using the nonparametric Mann\u0026ndash;Whitney U test. For comparisons among more than two groups, normally distributed data were assessed via one-way or two-way analysis of variance (ANOVA), followed by Tukey\u0026rsquo;s post hoc test for multiple comparisons; when normality was violated, the Kruskal\u0026ndash;Wallis H test was used, with post hoc analyses performed using the two-stage step-up Benjamini-Krieger-Yekutieli test. Data are expressed as group mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD, and \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003eThe level of DNase2a is decreased in the brain of A53T transgenic mice\u003c/h2\u003e\u003cp\u003ePrevious reports have demonstrated that cytosolic dsDNA accumulation induces cytotoxicity in animal models of PD, which can be rescued by the overexpression of DNase2a, a lysosomal DNase that degrades cytosolic dsDNA, indicating that DNase2a may be involved in the pathological development of PD[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. To investigate the relationship between DNase2a and PD risk, we analyzed DNase2a expression in A53T α-Syn transgenic (A53T-Tg) mice. Compared to wild-type (WT) controls, DNase2a protein levels were significantly reduced in A53T-Tg mice both in the brainstem and striatum (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea, b). Moreover, immunofluorescence staining revealed that DNase2a levels were obviously decreased in the substantia nigra (SN) of A53T-Tg mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec, d), while the level of γH2AX, a well-established marker of dsDNA breaks, was significantly increased in the same region (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee, f). In contrast, the level of TREX1, another cytosolic DNase, showed no significant changes in either the brainstem or the striatum of A53T-Tg mice when compared to WT mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eg, h). These findings indicated that DNase2a deficiency may contribute to the pathogenesis of PD by promoting cytosolic DNA accumulation.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003eDNase2a affects α-Syn ubiquitination and degradation\u003c/h2\u003e\u003cp\u003eTo investigate the potential role of DNase2a in PD \u003cem\u003ein vitro\u003c/em\u003e, primary cortical neurons were cultured and infected with lentiviral vectors carrying either DNase2a-targeting shRNA (sh-DNase2a) or DNase2a gene construct (OE-DNase2a) to achieve DNase2a deficiency or DNase2a overexpression (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea, b; Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003ea-c). As expected, DNase2a deficiency significantly induced the accumulation of cytosolic dsDNA, showing the increase in γH2AX levels, whereas DNase2a overexpression effectively eliminated the cytosolic dsDNA fragments in neurons (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003ed-f). Since α-Syn plays a central role in PD pathogenesis, we further explored the association of DNase2a with α-Syn. DNase2a knockdown significantly increased α-Syn level in neurons, whereas DNase2a overexpression markedly decreased it (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea, b). These changes in α-Syn protein levels were not attributed to transcriptional regulation, as SNCA mRNA levels remained unchanged in neurons infected with either sh-DNase2a or OE-DNase2a compared to their respective controls (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eg, h). Consistently, RT-qPCR analysis showed that the levels of transcription factors of α-Syn, including CEBPδ, EMX2, NKX6-1[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e], GATA2[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e], PARP1[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e], ZNF219, and ZSCAN21[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e], remained unchanged following DNase2a knockdown (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003ei). Next, we investigated whether the DNase2a regulated α-Syn protein levels through the protein degradation pathway. Neurons with DNase2a knockdown or overexpression were treated with CHX, a protein synthesis inhibitor, and the results indicated that DNase2a knockdown significantly slowed the degradation rate of α-Syn in the neurons relative to control (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec, d), whereas overexpressing DNase2a accelerated this process (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee, f). These results suggested that DNase2a affected the degradation of α-Syn protein.\u003c/p\u003e\u003cp\u003eThe intracellular protein degradation pathways mainly include two types: the autophagy-lysosome pathway and the ubiquitin-proteasome pathway[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].To investigate how DNase2 affects the degradation of α-Syn, primary cortical neurons were pretreated with either OE-DNase2a or an empty vector (OE-Ctrl), followed by treatment with chloroquine (a lysosomal inhibitor), MG132 (a proteasomal inhibitor), or a combination of both for 24 hours. MG132, either alone or combined with chloroquine, significantly inhibited DNase2a-mediated α-Syn degradation, whereas chloroquine alone had no such effect (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eg, h). To further investigate the role of DNase2a in the proteasomal degradation of α-Syn, neurons were treated with the proteasome inhibitor MG132, and the ubiquitination levels of α-Syn were measured. Immunoblotting showed that, upon proteasomal inhibition, α-Syn levels in DNase2a-overexpressing neurons and DNase2a-knockdown neurons became similar to those in the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ei). Moreover, IP with anti-α-Syn antibody showed elevated ubiquitin-conjugated α-Syn in OE-DNase2a neurons and reduced ubiquitin-conjugated α-Syn in sh-DNase2a neurons (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ei). Overall, these findings indicated that DNase2a affected α-Syn degradation through the ubiquitin-proteasome pathway.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\u003ch2\u003eDNase2a regulates α-Syn ubiquitination and degradation through NEDD4\u003c/h2\u003e\u003cp\u003eSubsequently, we investigated which E3 ubiquitin ligase is involved in DNase2a-regulated α-Syn ubiquitination. Previous studies have demonstrated that neural precursor cell-expressed, developmentally downregulated protein 4 (NEDD4), an E3 ubiquitin ligase, was involved in both α-Syn degradation[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e] and the DDR[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. As a regulator of DNA damage sensing and the subsequent response, NEDD4 represses p53 signaling and facilitates DNA repair[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e], while NEDD4 knockout results in elevated p53 activity and an amplified DDR[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. To investigate whether DNase2a regulated α-Syn degradation by modulating NEDD4 expression, we first examined NEDD4 expression in neurons with either DNase2a overexpression or knockdown. NEDD4 protein levels were significantly decreased in sh-DNase2a neurons (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ej-l) and elevated in OE-DNase2a neurons (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ej, k, m). Next, we silenced NEDD4 in DNase2a-overexpressing neurons and assessed α-Syn abundance. NEDD4 knockdown attenuated DNase2a-induced α-Syn ubiquitination and degradation (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003en). Conversely, overexpressing NEDD4 in DNase2a-deficient neurons restored α-Syn ubiquitination and rescued its degradation defect (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eo). Moreover, DNase2a overexpression enhanced, whereas its knockdown impaired, both the interaction between α-Syn and NEDD4 and the levels of ubiquitin-conjugated α-Syn. (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ep, q). Thus, DNase2a overexpression alleviates α-Syn accumulation by upregulating NEDD4, thereby enhancing α-Syn ubiquitination and degradation.\u003c/p\u003e\u003cp\u003eWhile the activation of the cGAS-STING pathway has been documented in DNase2a-deficient neurons[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e], it remains unclear whether this pathway directly regulates α-Syn accumulation. To examine this, we treated DNase2a-deficient primary cortical neurons with RU.521, a cGAS inhibitor, and measured cytosolic α-Syn levels. Strikingly, RU.521 treatment failed to alter α-Syn levels in sh-DNase2a neurons relative to vector controls (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003ej, k). These observations indicated that the cGAS-STING pathway did not participate in regulating the abundance of α-Syn mediated by DNase2a.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\u003ch2\u003eA53T α-Syn suppresses the expression of DNase2a by reducing MEF2C levels\u003c/h2\u003e\u003cp\u003eTo investigate why DNase2a levels were decreased in the brains of A53T-Tg mice, we examined DNase2a levels in primary neurons and N2a cells overexpressing α-Syn through LV-A53T infection. A significant reduction in DNase2a protein levels was observed in both primary neurons and N2a cells following A53T overexpression (OE-A53T) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea, b; Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003ea, b). This finding was further supported by ICC assay in primary neurons (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec, d). Consistently, the levels of γH2AX were significantly elevated after A53T α-Syn overexpression (Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003ec, d). To determine the causes of DNase2a reduction, CHX was added to the primary cortical neurons to inhibit protein synthesis, and then DNase2a protein levels were analyzed. A53T α-Syn did not accelerate DNase2a degradation (Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003ee, f), and treatment with either autophagy or proteasome inhibitors also failed to rescue DNase2a reduction (Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eg). Instead, A53T α-Syn markedly decreased DNase2a mRNA levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee; Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eh), demonstrating that A53T α-Syn reduced DNase2a expression by decreasing its transcription.\u003c/p\u003e\u003cp\u003eTo reveal the underlying mechanism by which A53T α-Syn mediated DNase2a transcription, we first identified the TF governing DNase2a transcription by promoter analysis tools, including GTRD, FIMO-JASPAR, and Animal TFEB. Myocyte enhancer factor 2C (MEF2C), a TF linked to PD pathogenesis and critical for nervous system development, was consistently predicted as the TF of DNase2a by all three datasets (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ef). Therefore, we examined MEF2C levels in A53T α-Syn overexpression cells. Compared to controls, the mRNA levels and protein levels of MEF2C were significantly reduced in primary neurons (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eg-\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ei) and N2a cells (Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003ei-k) overexpressing A53T α-Syn, which was consistent with the reports demonstrating lower MEF2C levels in PD models [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. This finding was further supported by ICC assay in primary neurons (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ej, k). Luciferase reporter assays and ChIP assays with primers flanking the predicted binding region showed that the sequence spanning \u0026minus;\u0026thinsp;1576 to \u0026minus;\u0026thinsp;1565 nt had the highest promoter activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003el\u0026ndash;o), indicating MEF2C bound to and enriched at the DNase2a promoter. Consistently, analysis of healthy brain tissues in the GEPIA database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://gepia.cancer-pku.cn/\u003c/span\u003e\u003cspan address=\"http://gepia.cancer-pku.cn/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) revealed a positive correlation between MEF2C and DNase2a expression (Pearson \u003cem\u003eR\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.2, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003el). These findings demonstrated that MEF2C was the TF of DNase2a and was involved in the regulation of the DNase2a expression via A53T α-Syn.\u003c/p\u003e\u003cdiv id=\"Sec23\" class=\"Section3\"\u003e\u003ch2\u003eA53T α-Syn reduces MEF2C levels by activating cGAS-STING-IFN pathway\u003c/h2\u003e\u003cp\u003eMEF2C, a transcription factor regulating immune and neuronal processes, is repressed by the cGAS-IFN pathway via mitochondrial DNA leakage in AD models[\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. We measured the protein levels related to the cGAS-IFN pathway, including cGAS, p-STING/STING, p-TBK1/TBK1, and p-STAT1/STAT1 in primary neurons (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ep, q) and N2a cells (Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003em, n) overexpressing A53T α-Syn, and found that these protein levels were significantly upregulated, consistent with activation of the cGAS-STING-IFN pathway in PD models[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eHowever, how the cGAS-STING-IFN axis downregulates MEF2C remains unclear. To explore this, we treated A53T-overexpressing neurons with CHX and analyzed MEF2C levels. A53T overexpression did not affect MEF2C degradation (Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eo, p). Consistently, blockade of autophagy or proteasomal activity failed to rescue MEF2C loss (Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eg), supporting that the reduction of MEF2C was driven by transcriptional downregulation rather than enhanced degradation. Next, we used promoter analysis tools to identify transcription factors that regulate MEF2C expression. We selected high-confidence candidates by cross-referencing predictions from six independent databases (KnockTF, hTFtarget, CHIP-Atlas, GTRD, FIMO-JASPAR, CHEA), including only those selected by four or more tools. Notably, we focused on factors involved in the cGAS-IFN pathway, such as STAT1, a key pathway mediator (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003er) and YY1[\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e], a major p-STING-binding protein (Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eq). Immunoblotting revealed significant p-STAT1 upregulation in A53T neurons compared to controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ep, q; Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003em, n), while YY1 levels remained unchanged (Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003er-t). These findings suggested that STAT1, rather than YY1, mediated MEF2C downregulation. Previous reports showed that p-STAT1 interacted with MEF2C and repressed MEF2C transcription[\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. Here, our CoIP assay results showed that p-STAT1 bound to MEF2C in neurons, and the binding was significantly increased in primary cortical neurons infected with LV-A53T (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003es).\u003c/p\u003e\u003cp\u003eWe further treated A53T-overexpressing neurons with the cGAS inhibitor RU.521 to verify that MEF2C-mediated DNase2a regulation depends on the cGAS-STING pathway. RU.521 treatment suppressed the levels of p-STAT1/STAT1, and rescued the downregulation of DNase2a and MEF2C induced by A53T α-Syn (Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eu, v). These data supported that A53T α-Syn suppressed DNase2a expression via the cGAS-IFN-MEF2C pathway. Consistently, the levels of cGAS, p-STING/STING, and p-STAT1/STAT1 in A53T-Tg mice were significantly increased, while the levels of MEF2C and DNase2a were notably decreased in the brainstem and striatum of A53T-Tg mice relative to WT mice (Fig. S3a-c). Additionally, qPCR analysis also showed elevated cGAS levels as well as reduced MEF2C and DNase2a levels in the brainstems and striatum of A53T-Tg mice (Fig. S3d-f).\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec24\" class=\"Section2\"\u003e\u003ch2\u003eNeuronal DNase2a deficiency induces PD-like phenotypes in WT mice\u003c/h2\u003e\u003cp\u003eTo investigate the effect of neuronal DNase2a on PD progression, we downregulated DNase2a expression in neurons by injecting AAVs expressing shRNA against DNase2a (AAV-shDNase2a) or control shRNA (AAV-CON) with the neuron-specific hSyn promoter into the SN and striatum of 10-month-old C57BL/6 mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea, b). One month post-injection, robust GFP fluorescence produced by AAV vectors was observed throughout the SN and striatum (Fig. S4a, b), which co-located specifically with MAP2\u003csup\u003e+\u003c/sup\u003e neurons but not with microglia or astrocytes (Fig. S4c, d), confirming neuron-specific expression. Knockdown efficiency was confirmed by quantitative immunoblotting of DNase2a in both SN and striatum (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ef, g). Immunofluorescence staining further revealed a significant reduction of DNase2a in the SN of WT-KD mice relative to WT-CON controls (Fig. S5a, b), which was accompanied by increased dsDNA accumulation with increased γ-H2AX immunostaining (Fig. S5c, d).\u003c/p\u003e\u003cp\u003eNext, we performed a series of behavioral tests to evaluate the impact of DNase2a knockdown on motor function and anxiety-like behaviors in the mice. In the pole test, WT-KD mice showed longer turn and descent times compared to WT-CON mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec). Similarly, DNase2a knockdown reduced the latency to maintain balance on the rod in the rotarod test (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed). WT-KD mice also exhibited weaker grip strength than WT-CON mice in the wire hang test (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee), suggesting that DNase2a deficiency exacerbated motor deficits. In the open field test, WT-KD mice traveled shorter total distances, explored the center less, spent less time in the center zone, and made fewer central entries, indicating that DNase2a knockdown increased anxiety-like behavior (Fig. S5e, f). These results aligned with the literature showing that cytosolic DNA accumulation induced PD-like behavioral symptoms in WT mice[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eWe next examined the impact of DNase2a knockdown on NEDD4 and α-Syn expression \u003cem\u003ein vivo\u003c/em\u003e. Western blot analyses showed a reduction of NEDD4 level in the lysates of brainstem and striatum WT-KD mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ef, g), which was further confirmed by decreased NEDD4 immunostaining in the SN of WT-KD mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eh, i). Moreover, α-Syn monomer levels in the soluble fraction were significantly increased in both the brainstem and striatum of WT-KD mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ef, g). Consistent with this, the insoluble fraction from WT-KD mice showed a pronounced accumulation of both monomeric α-Syn and higher-molecular-weight species compared with that in WT controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ej, k). Since phosphorylation of α-Syn at serine 129 (p-α-Syn\u003csup\u003e129\u003c/sup\u003e) is typically associated with the formation of aggregates[\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e], we next examined p-α-Syn\u003csup\u003e129\u003c/sup\u003ein WT-KD mice. p-α-Syn\u003csup\u003e129\u003c/sup\u003e was undetectable in both WT-CON and WT-KD mice (Fig. S5g), which was consistent with previous findings that undamaged mitochondrial DNA accumulation does not induce α-Syn phosphorylation[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. In addition, we evaluated dopaminergic neuron integrity and found a marked reduction in TH protein levels in the SN and striatum of WT-KD mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003el, m). IHC analysis further confirmed an exacerbated loss of TH-positive neurons (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003en, o) and dopaminergic fibers (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003en, p) with DNase2a knockdown. Moreover, Nissl staining of midbrain slices revealed that DNase2a knockdown induced more severe neuronal loss compared to controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eq, r). These results demonstrated that neuronal DNase2a deficiency drove α-Syn pathology and dopaminergic neuronal degeneration, leading to motor deficits characteristic of PD.\u003c/p\u003e\u003cdiv id=\"Sec25\" class=\"Section3\"\u003e\u003ch2\u003eNeuronal DNase2a overexpression attenuates behavioral deficits in A53T-Tg mice\u003c/h2\u003e\u003cp\u003eTo assess the impact of DNase2a overexpression on PD pathology, we stereotaxically injected AAV expressing the DNase2a gene (AAV-DNase2a) or control vector (AAV-CON) with neuron-specific hSyn promoter into the bilateral SN and striatum of 8-month-old A53T-Tg and WT mice to drive neuronal DNase2a expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea, b). IHC assay confirmed robust AAV expression by showing GFP-positive neurons widely distributed in both SN and striatum one month post-injection (Fig. S4a, b), and AAV expression was restricted to neurons and absent in microglia or astrocytes (Fig. S4e, f). As expected, AAV-DNase2a significantly elevated DNase2a expression in infected neurons (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec, d; Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea, b) and alleviated damaged DNA levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ee, f).\u003c/p\u003e\u003cp\u003eWe next examined the effect of DNase2a overexpression on the motor and anxiety-like behaviors in WT and A53T-Tg mice. In the pole test, A53T mice injected with DNase2a initiated movement more quickly and descended faster (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eg, h), while they showed an extended latency to fall in the rotarod test (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ei). Similarly, in the wire hang test, DNase2a overexpression resulted in longer suspension time for A53T mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ej). Moreover, anxiety-like behavior was also alleviated in A53T-Tg overexpressing DNase2a, as shown by increased distance traveled in the center, increased center time, and more center entries in the open field test (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ek, l). Collectively, these results demonstrated that DNase2a overexpression ameliorated PD-like behavioral symptoms of A53T-Tg mice.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec26\" class=\"Section3\"\u003e\u003ch2\u003eNeuronal DNase2a overexpression attenuates α-Syn pathology and neurotoxicity in A53T mice\u003c/h2\u003e\u003cp\u003eWe then performed Western blot and IHC assays to assess the effect of neuronal DNase2a overexpression on the levels of NEDD4 and α-Syn \u003cem\u003ein vivo\u003c/em\u003e. A53T-Tg mice injected with AAV-DNase2a showed increased NEDD4 levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea, b) compared with A53T-Tg mouse controls, which was further confirmed in the SN by IHC (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec, e). Neuronal DNase2a overexpression significantly reduced soluble α-Syn levels in both the brainstem and striatum (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea, b), as well as in the insoluble fractions of these regions (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ed, f). ICC revealed markedly reduced p-α-Syn129 signals in the SN (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eg, h) and striatum (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eg, i) of A53T-Tg mice injected with AAV-DNase2a. Consistently, Western blot analysis confirmed significantly lower p-α-Syn129 levels in the brainstem and striatum (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ej, k). Collectively, these data indicated that neuronal DNase2a overexpression elevated NEDD4 expression and reduced α-Syn load in A53T-Tg mice.\u003c/p\u003e\u003cp\u003ePrevious studies have shown that p-α-Syn pathology is toxic to TH-positive dopaminergic neurons, leading to motor deficits in PD[\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. We next investigated TH levels by Western blot and ICC assays. Western blot results showed that DNase2a overexpression markedly attenuated dopaminergic neuron loss in the brainstem and striatum of A53T-Tg mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003el, m). TH immunostaining demonstrated preservation of TH-positive neurons in the SNpc (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003en, o) and reduced dopaminergic fiber degeneration in the striatum of A53T-Tg mice with DNase2a overexpression (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003en, p). Furthermore, Nissl staining of midbrain slices revealed a significant decrease in Nissl-positive cells in the SN of A53T-Tg mice, which was partially restored by DNase2a overexpression (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eq, r). Together, these findings indicated that neuronal DNase2a mitigated both α-Syn pathology and α-Syn-induced neurotoxicity \u003cem\u003ein vivo\u003c/em\u003e.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eDNase2a serves as a lysosomal nuclease that hydrolyzes dsDNA, and its downregulation results in cytosolic accumulation of DNA. Although previous studies have investigated the role of DNase2a in senescent cells[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e] and Alzheimer\u0026rsquo;s disease[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], its effects on α-Syn and the pathology of PD remain elusive. In our study, we identified DNase2a as a key regulator, connecting cytosolic DNA clearance, the DDR, and α-Syn pathology in PD. DNase2a deficiency hampered cytosolic DNA removal, triggered aberrant DDR, and inhibited NEDD4-mediated α-Syn ubiquitination and degradation, inducing α-Syn accumulation and PD progression. Conversely, DNase2a overexpression restored NEDD4 levels and promoted α-Syn clearance, consistent with the reports that NEDD4 upregulation mitigated PD pathology \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. Importantly, neuronal DNase2a overexpression in A53T mice lowered α-Syn levels, protected dopaminergic neurons, and improved motor and cognitive functions, highlighting its therapeutic potential.\u003c/p\u003e\u003cp\u003eDNA leakage from mitochondria or nuclei triggers cGAS-IFN activation in the models of Alzheimer\u0026rsquo;s disease[\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]\u003csup\u003e,\u003c/sup\u003e[\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e], PD[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e], and cellular senescence[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e, \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. In our study, DNA leaked into the cytosol induced by A53T α-Syn activated the cGAS-IFN pathway and repressed MEF2C-dependent DNase2a transcription, thereby hindering the clearance of cytosolic dsDNA and promoting cytosolic DNA accumulation, which formed a self-amplifying pathogenic loop. Evidence from AD models has demonstrated reduced DNase2a expression[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], cGAS-IFN activation, and MEF2C downregulation[\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e], and comparable changes have been observed in aging models[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. However, the interconnectedness of these events has not been systematically investigated. Based on our findings, we speculate that the cGAS-IFN-MEF2C-DNase2a cascade may act as a common pathway contributing to AD, aging, and PD pathology. Notably, this finding also expands our understanding of the mechanisms underlying cytosolic DNA accumulation: whereas previous studies have mostly focused on the \"passive leakage\" of mitochondrial DNA or nuclear DNA into the cytoplasm[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e], our results suggest that impaired DNA clearance due to A53T α-Syn-induced DNase2a reduction prevents the effective removal of the leaked DNA, which is another important driver for sustained cytosolic DNA accumulation. This highlights DNA clearance as a potentially effective target to mitigate aberrant DNA accumulation-induced neurodegeneration.\u003c/p\u003e\u003cp\u003eWhether DNase2a expression downregulation is a cause or consequence of PD pathology remains under debate. In our study, neuronal DNase2a knockdown in WT mice led to PD-like neuropathology and induced both motor deficits and anxiety-like behavior, indicating that neuronal DNase2a deficiency may act as an early trigger of PD. This is consistent with previous reports that mitochondrial DNA accumulation alone can trigger PD-like phenotypes[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. A53T α-Syn can activate the cGAS-STING pathway by inducing mitochondrial or nuclear DNA leakage[\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. However, cGAS-STING activation can also occur independently of pathological α-Syn, and low-level cGAS activity is detectable even under physiological conditions[\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. Furthermore, age-associated cGAS-STING activation promotes neuroinflammation and neurodegeneration[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], indicating cGAS-STING signaling may be involved in early disease stages independent of α-Syn accumulation. Taken together, our findings support the following model: neuronal DNase2a deficiency and the accumulation of cytosolic DNA act as an early trigger that activates DDR and downregulates NEDD4, impairing α-Syn degradation and inducing PD pathology. Then, α-Syn aggregation exacerbates DNA leakage[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e] and cGAS-IFN signaling, which further suppresses DNase2a transcription via decreased MEF2C, establishing a deleterious feedback loop. Overall, our study not only supplements the association between cytosolic DNA clearance defects and abnormal α-Syn degradation in PD but also offers a novel molecular perspective for understanding PD pathogenesis.\u003c/p\u003e\u003cp\u003eIn conclusion, our findings demonstrate that the DNase2a levels are decreased in the brains of A53T-Tg mice, leading to cytosolic DNA accumulation. Decreased DNase2a level impairs α-Syn ubiquitination and degradation by the DNase2a-DDR-NEDD4 axis \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e, resulting in α-Syn accumulation and PD pathogenesis. Moreover, A53T α-Syn promotes cytosolic DNA accumulation and suppresses MEF2C-mediated transcriptional expression of DNase2a via cGAS-STING-IFN pathway, forming a vicious circle. Our present findings reveal that DNase2a deficiency is an early event, rather than merely a secondary consequence in PD pathogenesis, highlighting DNase2a and cytosolic DNA as potential therapeutic targets for PD.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eAAVs\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eadeno-associated viruses\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eα-Syn\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eα-synuclein\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eChIP\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eChromatin immunoprecipitation\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eCo-IP\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eCo-immunoprecipitation\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003ecGAS\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003ecyclic GMP\u0026ndash;AMP synthase\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eDAB\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eDiaminobenzidine\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eDDR\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eDNA damage response\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eDNase2a\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eDeoxyribonuclease 2α\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003edsDNA\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003edouble-stranded DNA\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eICC\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eimmunocytochemistry\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eNEDD4\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eNeural precursor cell expressed developmentally downregulated protein 4 (NEDD4)\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eN2a\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eNeuro2a\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003ePD\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eParkinson\u0026rsquo;s disease\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eqRT-PCR\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003equantitative reverse transcription PCR\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eSTING\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003estimulator of interferon genes\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eSN\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003esubstantia nigra\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eSNpc\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003esubstantia nigra pars compacta\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eTH\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003etyrosine hydroxylase\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll animal experiments were performed in accordance with the China Public Health Service Guide for the Care and Use of Laboratory Animals. Experiments involving mice and protocols were approved by the Institutional Animal Care and Use Committee of the Institute of Process Engineering, Chinese Academy of Sciences (Approval code: IPEAECA2024113).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors have reviewed the final manuscript and consent to publication.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated and/or analyzed during this study are either included in this article or are available from the corresponding author on reasonable request. This study did not generate new unique reagents.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have no conflicts of interest to declare that are relevant to the content of this article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by the National Natural Science Foundation of China (No.82401674 and No. 82171196).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contribution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eR.-t. L., J.-w. Z., H.-h. Z., Y.-r. H., and S.C. designed the experiments; L.L., Fei. C., and J. Z. performed behavioral experiments; H.-h. Z., Y.-r. H., and Fang. C. performed ICC and IHC; Fei. C. and H.-h. Z. conducted the biochemistry experiments; R.-h. S., K. M., and Z.-x. Z. analyzed the data; R.-t. L., J.-w. Z., H.-h. Z., and Y.-r. H. wrote the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Dr. Ling-jie Li, Dr. Qi-xin Huang, and Ms. Fang-jing Lu for their technical support. We are also grateful to Dr. Chao Jiang, Dr. Shaoxun Li, and Fan Yang for assistance with manuscript editing.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor details\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e1\u003c/sup\u003eDepartment of Neurology, Zhengzhou University People\u0026apos;s Hospital, Henan Provincial People\u0026apos;s Hospital, Zhengzhou, Henan, 450003, China. \u003csup\u003e2\u003c/sup\u003eNational Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, China. \u003csup\u003e3\u003c/sup\u003ePeking Union Medical College Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100730, China. \u003csup\u003e4\u003c/sup\u003eSchool of Life Science, Ningxia University, Yinchuan, Ningxia, 750021, China.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eTabar V, Sarva H, Lozano AM, Fasano A, Kalia SK, Yu KKH, Brennan C, Ma Y, Peng S, Eidelberg D, et al. 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Nat Rev Immunol. 2025;25:425\u0026ndash;44.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Parkinson's disease, DNase2a, DNA damage response, Ubiquitination, cGAS-STING-IFN pathway","lastPublishedDoi":"10.21203/rs.3.rs-7903266/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7903266/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e\u003cp\u003eDNase2a, a key enzyme responsible for clearing cytoplasmic double-stranded DNA, prevents cytosolic DNA accumulation. Accumulating evidence suggests that aberrant cytosolic DNA accumulation contributes to Parkinson\u0026rsquo;s disease (PD) pathogenesis, yet the role of DNase2a in PD remains unclear.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e\u003cp\u003eWe examined the effects of neuronal DNase2a and cytosolic damaged DNA on α-synuclein (α-Syn) accumulation in cultured neurons and male A53T transgenic mice, and investigated the underlying mechanism by which α-Syn modulates DNase2a expression.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eThe levels of DNase2a were markedly reduced in the brain of A53T α-Syn transgenic mice, accompanied by increased cytoplasmic DNA accumulation. Decreased neuronal DNase2a led to persistent cytosolic DNA accumulation and suppressed NEDD4-mediated α-Syn ubiquitination and degradation, exacerbating α-Syn accumulation and PD pathology in vitro and in vivo. Moreover, A53T α-Syn further aggravated cytosolic DNA accumulation and then repressed MEF2C-mediated DNase2a transcription via activating the cGAS-STING-IFN pathway, forming a deleterious loop between DNase2a and α-Syn. Consistently, neuronal DNase2a deficiency in WT mice drove α-Syn pathology and dopaminergic neuronal degeneration, leading to motor deficits characteristic of PD, while neuronal DNase2a overexpression in A53T transgenic mice significantly ameliorated motor deficits by reducing α-Syn accumulation and preserving dopaminergic neuron integrity.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e\u003cp\u003eOur findings reveal that DNase2a deficiency disrupts α-Syn degradation and accelerates PD pathogenesis, suggesting that DNase2a is a potential therapeutic target for PD.\u003c/p\u003e","manuscriptTitle":"DNase2a downregulated by the cGAS-STING-IFN-MEF2C pathway prevents α-synuclein ubiquitination via NEDD4 in Parkinson's disease","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-11 08:23:38","doi":"10.21203/rs.3.rs-7903266/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"fcf6a1db-2ad5-40d7-a204-d4591e814e6e","owner":[],"postedDate":"November 11th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-12-03T07:34:46+00:00","versionOfRecord":[],"versionCreatedAt":"2025-11-11 08:23:38","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7903266","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7903266","identity":"rs-7903266","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}