Irgm1 Promotes Microglial Clearance of α-Synuclein via the TFEB-Dependent Autophagy- Lysosome Pathway in Parkinson’s Disease

Irgm1 Promotes Microglial Clearance of α-Synuclein via the TFEB-Dependent Autophagy- Lysosome Pathway 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 Article Irgm1 Promotes Microglial Clearance of α-Synuclein via the TFEB-Dependent Autophagy- Lysosome Pathway in Parkinson’s Disease Sijia Wen, Xiaojing Li, Junling Yang, Jiawen Chen, Chenxi Xue, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9004768/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 Parkinson’s disease (PD) is a common neurodegenerative disorder characterized by the progressive loss of nigral dopaminergic neurons and abnormal α-synuclein (α-syn) aggregation. Growing evidence indicates that impaired autophagy–lysosome pathway (ALP) activity in microglia exacerbates the pathological process; however, the precise regulatory mechanisms involved remain elusive. In this study, we found that Irgm1, a key autophagy regulator, was markedly upregulated in the midbrains of PD mice. Irgm1-deficient mice exhibited accelerated PD progression, more severe motor deficits, greater TH⁺ neuronal loss, increased α-syn deposition and aggravated ALP damage, indicating a neuroprotective role for Irgm1. Moreover, Irgm1 was selectively enriched in the microglia of PD mice, and Irgm1 knockdown amplifiedα-syn-preformed fibril (PFF)-induced ALP impairment: the LC3-II and cathepsin D levels decreased, p62 and α-syn aggregates accumulated, the colocalization of α-syn with Lamp1 declined, lysosomal acidification decreased, and TFEB nuclear translocation was blocked. The TFEB-activating autophagy agonist rapamycin restored TFEB nuclear translocation, reactivated the ALP, accelerated α-syn clearance, and abolished the increased toxicity of Irgm1-deficient microglia toward cocultured SH-SY5Y neurons after PFFs preexposure. Thus, Irgm1 promotes microglial α-syn clearance via the TFEB-ALP axis, and targeting this pathway could be a potential therapeutic strategy for PD. Biological sciences/Cell biology Biological sciences/Drug discovery Health sciences/Neurology Biological sciences/Neuroscience Parkinson’s disease Irgm1 Autophagy-lysosomal pathway TFEB Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Parkinson’s disease (PD) is the second most common neurodegenerative disorder after Alzheimer’s disease [ 1 , 2 ]. Clinically, it presents with motor symptoms of resting tremor, muscular rigidity and bradykinesia [ 3 ], as well as nonmotor symptoms, including hyposmia, constipation and sleep disturbances [ 4 ]. Current pharmacological and surgical interventions only ameliorate motor deficits; they do not halt the progressive loss of dopaminergic neurons. Therefore, clarifying the core mechanism of disease progression and identifying potential intervention targets have become urgent priorities. Pathologically, massive degeneration of dopaminergic neurons in the substantia nigra pars compacta and the formation of Lewy bodies composed of misfolded α-synuclein (α-syn) are hallmarks of PD [ 5 – 7 ]. These prion-like aggregates can propagate between neurons and glial cells, triggering chronic neuroinflammation and creating a vicious cycle of protein deposition, microglial hyperactivation, and ever-amplifying inflammation [ 8 – 11 ]. Thus, increasing the capacity of microglia to clear α-syn aggregates is considered a promising therapeutic approach. The autophagy–lysosome pathway (ALP) is the principal route for disposing of damaged organelles and abnormal proteins: cargo is sequestered by autophagosomes that subsequently fuse with lysosomes for degradation [ 12 ]. α-syn aggregates are cleared mainly through this route [ 13 ], yet the pathway is markedly impaired in the brains of Parkinson’s disease patients. Transcription factor EB (TFEB) acts as the master switch of the ALP, accelerating waste disposal and preserving intracellular homeostasis by coordinately upregulating autophagy and lysosomal genes [ 14 , 15 ]. Although ALP function in neurons has been intensively studied over the past two decades, microglial autophagy in Parkinson’s disease remains in its infancy. How microglial ALP is regulated and how extracellular α-syn influences this regulation remain poorly understood. The immune-related GTPase M1 (Irgm1) is a key autophagy-regulating protein that enhances autophagy by activating the ULK1/Beclin-1 pathway, promotes LC3II conversion and P62 degradation, and suppresses NLRP3 inflammasome assembly as well as the release of IL-1β and TNF-α [ 16 , 17 ]. However, direct evidence that Irgm1 mediates microglial clearance of α-syn is still lacking in Parkinson’s disease, a disorder characterized by impaired ALP and prominent neuroinflammation. In this study, through the establishment of an α-syn-overexpressing PD mouse model, we demonstrated that Irgm1 is significantly upregulated in the midbrain and mice lacking Irgm1 showed accelerated progression of PD. Using microglia stimulated with α-syn-preformed fibrils (PFFs), we further showed that Irgm1 deficiency impedes TFEB nuclear translocation, exacerbates ALP dysfunction, promotes intracellular α-syn accumulation, and amplifies the toxic spread of α-syn to dopaminergic neurons. Pharmacological activation of TFEB with rapamycin reversed this pathological cascade. Collectively, our findings identify the Irgm1–TFEB–ALP axis as a critical regulatory node for microglial α-syn clearance and provide a potential therapeutic target for arresting PD progression. Materials and methods Animals C57BL/6 WT mice were purchased from Changsheng Bio-Technology Co., Ltd. Systemic stable Irgm1 knockout mice (Irgm1⁻ / ⁻) were generated by crossing Irgm1-flox mice with Dppa3-Cre mice, both of which were purchased from the Shanghai Model Organisms Center. Mice were housed on a 12 h light/dark cycle at 23 ± 1°C with free access to food and water. The animal experimental plan was approved by the Ethics Committee of Harbin Medical University. Animal studies were in accordance with the National Guidelines for the Care and Use of Laboratory Animals of China. Mouse model of PD To induce α-syn overexpression in vivo, 8-week-old male C57BL/6 WT and Irgm1 -/- mice received a unilateral injection of AAV5-hα-syn (AAV-SNCA, 1.2E+13 vg), under the control of the human synapsin promoter (Genechem, China) into the right substantia nigra (SN). Control mice received AAV-GFP or saline into the right SN. During surgery, mice were anesthetized with 2% isoflurane and stereotaxically injected with 2 μl of AAV-SNCA, AAV-GFP, or saline at a rate of 0.3 μl /min, using the following coordinates: AP −3.0 mm, ML −1.25 mm, and DV +4.5 mm relative to bregma. The needle was left in place for 5 min before retraction. Behavioral test Behavioral tests were performed on mice three months after construction of the α-syn-overexpression PD model. All tests were performed between 10 am and 3 pm by experimenters who were blind to subgrouping. Before the test, mice were allowed to acclimate undisturbed in a dimly lit behavioral room for at least 1 h. Equipment for open field test and rotarod test was purchased from SansBio (Jiangsu, China). Open field test Locomotor activity was measured in an open field box (40 cm × 40 cm × 40 cm). The box was cleaned with 75% alcohol before each trial to remove odors, with at least 10 min between testing different mice. After placing the mouse in the center, locomotor activity was recorded for 15 min, and total distance traveled was used for statistical analysis. Rotarod test Prior to the formal test, mice were trained on the rotarod for 5 min per day over 5 consecutive days. During the test, the rod accelerated from 4 rpm to 30 rpm over 300 seconds. The trial ended when the mouse fell from the rod. Fall latency was recorded for each of 5 trials per mouse, with at least 30 min between trials. The average latency to fall was used for statistical analysis. Cell culture The mouse microglia cell line BV-2 and human neuroblastoma cell line SH-SY5Y were purchased from Pricella Biotechnology (Wuhan, China) and were tested for mycoplasma contamination. Cells were cultured using Dulbecco’s modified Eagle’s medium (DMEM; Gibco, USA) supplemented with 10% fetal bovine serum (FBS; Sigma, USA) and 1% penicillin-streptomycin (Gibco, USA) and incubated in a humidified incubator at 37 °C with 5% CO₂. PFFs were a gift from Prof. Zhentao Zhang (Department of Neurology, Renmin Hospital of Wuhan University, China). Transfection siRNA transfection was performed using Lipofectamine 3000 (L3000015, Invitrogen, USA). Cells were transfected at 70–80% confluence in serum-free Opti-MEM (11058021, Gibco, USA) according to the manufacturer’s instructions. Cells were harvested 48–72 h later for the subsequent experiments. Irgm1 siRNA and control siRNA (si-NC) were purchased from OBiO Technology (Shanghai, China). The sequences are as follows: for si-Irgm1: P1:5′- GGUCAGUAGGAGCACCGAATT -3′, P2:5′- UUCGGUGCUCCUACUGACCTT -3′; si-NC: P1:5′- UUCUCCGAACGUGUCACGUTT -3′, P2:5′- ACGUGACACGUUCGGAGAATT -3′. Flow cytometric analysis of apoptosis Flow cytometry was performed to evaluate the apoptosis of SH-SY5Y cells cocultured with BV2 cells pretreated with PFFs, and the Annexin V-FITC/PI Cell Apoptosis Detection Kit (TransGen, China) was used according to the manufacturer’s protocol. Unstained controls and single-staining controls were set up, and each experiment was performed at least three times. The apoptosis rate of SH-SY5Y cells was evaluated via flow cytometry (BD, USA) within 1 h. Quantitative real-time PCR Total RNA was extracted from the midbrain of mice in each group using RNAisoPlus (9109, TaKaRa) according to the manufacturer’s instructions. cDNA was synthesized from individual RNA samples with the PrimeScript FAST RT Reagent Kit (RR092A, TaKaRa). The Irgm1 primers were 5′-CGCGATCAGACCTCCTCTTG-3′ and 5′-CAAGAGAGGAGGTCTGATCGCG-3′. The GAPDH primers were 5′-CAGAAGACTGTGGATGGCCG-3′ and 5′-CGGCCATCCACAGTCTTCTG-3′. Real-time qPCR was performed in a 25-μL reaction system containing cDNA, forward and reverse primers, and TB Green Premix (CN830A, TaKaRa). Relative Irgm1 mRNA expression was calculated using the 2^(-ΔΔCt) method, and each experiment was repeated at least three times. Immunofluorescence staining After cardiac perfusion and isolation, brain tissues were fixed overnight in 4% PFA at 4 °C and dehydrated in 30% sucrose for 48 h. Frozen brain tissue sections (5 μm) near the midbrain, as well as BV2 and SH-SY5Y cells, were fixed with 4% PFA for 15 min, permeabilized with 0.1% Triton X-100 for 15 min, and blocked with QuickBlock™Blocking Buffer (P0260, Beyotime) for 15 min. Sections and cells were then incubated with primary antibodies against TH (1:100, ab112, Abcam), Iba-1 (1:100, ab178846, Abcam), Irgm1 (1:100, IC11, AbMart), α-syn (1:100, ab138501, Abcam), Lamp1 (1:100, ab25245, Abcam), or cleaved caspase-3 (1:100, 9664S, CST) at 4 °C overnight. After three washes with PBS, corresponding Alexa Fluor-conjugated secondary antibodies were applied for 2 h at room temperature. Following three additional PBS washes, samples were counterstained with DAPI for 5 min. Images were acquired using a fluorescence microscope (Nikon, Japan and Zeiss, Germany), and fluorescence intensity was quantified using ImageJ software. All experiments and analyses were performed by an investigator blinded to group allocation. Each experiment was repeated at least three times. Lysosome staining Cells grown on PDL-coated coverslips were stained with the live-cell dyes LysoTracker Green DND-26 (50 nM, 3 min) and LysoSensor Green DND-189 (1 μM, 30 min) following the manufacturer’s protocol. After staining at 37 °C in the dark, cells were washed twice with PBS and imaged with a fluorescence microscope (Nikon, Japan). Fluorescence intensity was quantified using ImageJ. All procedures were performed by an investigator blinded to group allocation, and each experiment was repeated at least three times. Western blotting Total proteins from cells or mice brain tissues were lysed in RIPA buffer containing protease inhibitor and PMSF. Nuclear proteins and cytoplasm proteins were extracted from cells via Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime, China) according to the manufacturer’s protocol. After ultrasonic crushing and high-speed centrifugation at 4 °C, protein concentrations were measured with a bicinchoninic acid (BCA) protein assay kit (Beyotime, China). 20 μg of proteins were separated via SDS-PAGE and transferred onto Nitrocellulose membrane (Cytiva, USA) with a 0.22 μm pore size. After being blocked with 5% skimmed milk in tris-buffered saline with 0.1% Triton X-100 (TBST) for 2 h, the membranes were incubated overnight at 4 °C with the following primary antibodies: Irgm1 (1:1000, 14979S, CST), α-syn (1:1000, ab138501, Abcam), TH (1:1000, ab112, Abcam), p62 (1:1000, P0067, Sigma), LC3 (1:1000, 2775S, CST), Lamp1 (1:1000, ab25245, Abcam), Ctsd (1:1000, 21327-1-AP, Proteintech), TFEB (1:1000, 13372-1-AP, Proteintech), Bak (), Bcl2(), Histone-H3 (1:5000, 17168-1-AP, Proteintech), β-actin (1:20000, A3854, Sigma) and GAPDH (1:40,000, 10494-1-AP, Proteintech). After washing 3 times for 15 min with TBST, the membrane was incubated in the HRP-conjugated secondary antibody for 2 h at room temperature. After washes, the signals were detected via super sensitive ECL luminescence reagent (MeilunBio, China) and a chemiluminescence system (Tanon, China). Images were analyzed via ImageJ software. Each experiment was repeated at least three times. Statistical analysis The statistical significance of the data was analyzed with GraphPad Prism version 9.5. Shapiro–Wilk test and F test were used to examine the normality of the date and equality of variance, respectively. Data were analyzed by unpaired t test for two independent groups and one-way ANOVA with Tukey's multiple comparisons test for multiple independent groups that were normally distributed and had the same variance. Data were analyzed by Welch's t test for two independent groups that were normally distributed and different variances. Assuming sphericity depending on the situation, two-way ANOVA with Tukey's multiple comparisons test was used for comparisons between multiple groups and regions. Data in figures were represented as mean ± SEM. P-values <0.05 were considered statistically significant (* P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001). Results Irgm1 expression was elevated in the midbrain of PD mice, and its deficiency further accelerated disease progression. To investigate whether Irgm1, an immune-related GTPase with autophagy-regulating functions, is involved in PD pathogenesis, we observed the expression of Irgm1 in the AAV-SNCA PD mouse model in which wild-type human α-syn is selectively overexpressed in dopaminergic neurons. Compared with mice receiving AAV-GFP or saline, those injected with AAV-SNCA into the SNpc presented consistent upregulation of Irgm1 at both the protein and mRNA levels in midbrain tissue (Fig. 1 a-b). To explore the effects of Irgm1 on PD pathogenesis, we compared motor performance and neuronal degeneration between WT and Irgm1 -/- mice after stereotaxic injection of AAV-SNCA. At 12weeks after virus transduction, Irgm1 -/- mice exhibited significantly shorter total travel distances in the open-field test and reduced latency to fall in the rotarod test, compared with WT mice, indicating aggravated motor deficits (Fig. 1 c-e). Immunofluorescence and western blotting revealed robust loss of midbrain TH + neurons in the PD model, validating the paradigm, and Irgm1 deficiency further exacerbated this neuronal loss (Fig. 1 f-i). Notably, Irgm1 deficiency alone (in control mice) neither altered impaired motor behavior nor TH + neuronal survival. Collectively, these findings suggest that Irgm1 likely plays a neuroprotective role in the context of α-syn-driven Parkinson’s disease. Irgm1 deficiency exacerbated autophagy‒lysosome dysfunction Given that Irgm1 is a known regulator of autophagy and its expression was upregulated in our PD model (Fig. 1 ), we next investigated whether Irgm1 deficiency could modulate the critical nexus between α-syn aggregation and ALP dysfunction in the PD midbrain. Western blotting revealed that, within the AAV-SNCA group, α-syn aggregation was significantly greater in Irgm1 -/- mice than in WT mice (Fig. 2 a, b). Moreover, there was a significant reduction in LC3Ⅱ, Ctsd, and Lamp1 but an increase in p62 in PD mice, indicating autophagy–lysosome dysfunction, and this impairment was markedly amplified by Irgm1 knockout (Fig. 2 a, b). Next, we investigated where and how Irgm1 affects α-syn aggregation and the ALP in PD. The fluorescence results revealed that Irgm1 was significantly colocalized with microglia in the midbrains of PD mice (Fig. 2 c). Studies have shown that impaired microglial ALP fails to clear α-syn and damaged mitochondria, promoting chronic neuroinflammation that accelerates dopaminergic neuron loss and Parkinson’s disease progression [ 18 – 20 ]. Dysregulation of microglial autophagy is emerging as a core regulator of brain development and diseases [ 21 , 22 ]. To determine whether Irgm1 contributes to Parkinson’s disease pathogenesis by modulating microglial ALP, we exposed BV2 microglia to α-syn-preformed fibrils (PFFs). After 24 h of exposure to PFFs (1 µg/ml), western blotting revealed that Irgm1 expression was upregulated, accompanied by increased p62 levels and decreased levels of LC3-II and Ctsd (Fig. 2 d, e), indicating suppressed ALP activity, which is consistent with the findings of a recent study [ 19 ]. Compared with the control conditions, Irgm1 knockdown exacerbated these impairments and elevated the intracellular α-syn level (Fig. 2 d, e). Immunofluorescence further revealed that, compared with si-NC-treated BV2 cells, si-Irgm1-treated BV2 cells accumulated more α-syn and showed a marked reduction in the colocalization of α-syn and Lamp1 (Fig. 2 f, g). Moreover, the PFF-induced reduction in Lamp1 intensity was significantly exacerbated (Fig. 2 f, g). These results demonstrate that the loss of Irgm1 aggravates PFF-induced ALP dysfunction and α-syn accumulation. We subsequently examined the lysosomal activity of BV2 cells. Prior to PFFs exposure, no differences in LysoTracker or LysoSensor fluorescence intensities were detected between si-NC and si-Irgm1 BV2 cells. After 24 h of PFFs exposure, both LysoTracker and LysoSensor fluorescence intensities were significantly reduced, and this reduction was further exacerbated by Irgm1 knockdown (Fig. 2 h, i). These findings indicate that Irgm1 deficiency amplifies α-syn-induced lysosomal dysfunction, further impairing autophagy–lysosome pathway activity and thereby hindering PFFs degradation. Irgm1 deficiency exacerbates ALP dysfunction by inhibiting TFEB nuclear translocation Transcription factor EB (TFEB) is a master regulator of autophagy and lysosomal gene expression. Its nuclear translocation enhances ALP activity and promotes the clearance of cellular waste [ 15 ]. We therefore asked whether Irgm1 deficiency compromises the ALP by interfering with TFEB subcellular localization, thereby impairing α-syn degradation. Western blotting revealed that after 24 h of exposure to PFFs (1 µg/ml), the nuclear TFEB level decreased, whereas the cytoplasmic TFEB level increased, and this effect was exaggerated in si-Irgm1 BV2 cells (Fig. 3 a, b). Treatment with rapamycin, a TFEB-activating autophagy agonist, significantly reversed the alterations caused by Irgm1 knockdown, restoring TFEB nuclear expression and reducing its cytoplasmic accumulation (Fig. 3 a, b). Immunofluorescence further confirmed these findings. Compared with si-NC BV2 cells, si-Irgm1 BV2 cells presented a greater reduction in TFEB nuclear translocation following PFF exposure, which was abolished by rapamycin treatment (Fig. 3 c, d). These results suggest that Irgm1 may play a role in regulating the subcellular localization of TFEB. Next, we investigated whether rapamycin could restore α-syn clearance and lysosomal function in si-Irgm1 BV2 cells exposed to PFFs. Immunofluorescence revealed that rapamycin treatment markedly increased the colocalization of α-syn with Lamp1 and increased the fluorescence intensity of Lamp1, accompanied by a clear reduction in the α-syn aggregates (Fig. 3 e, f). Concomitantly, both LysoTracker and LysoSensor fluorescence intensities were significantly increased (Fig. 3 g, h), indicating that lysosomal acidification and functional activity were restored. Collectively, these results demonstrate that rapamycin reactivates ALP and accelerates α-syn degradation in Irgm1-deficient microglia, an effect associated with restored TFEB nuclear translocation. PFF-exposed Irgm1-deficient microglia exacerbate neurotoxic effects To examine the impact of Irgm1-deficient microglia on neurons, we established a contact coculture assay in which SH-SY5Y cells were exposed to BV2 cells that had been pretreated with PFFs. After a further 24 h of coculture, western blotting revealed that SH-SY5Y cells exposed to PFF-primed BV2 cells presented increased levels of α-syn and elevated Bak/Bcl-2 ratios (Fig. 4 a, b), indicating increased apoptotic priming. Knocking down Irgm1 in BV2 cells further increased α-syn levels and increased the Bak/Bcl-2 ratio in SH-SY5Y cells, both of which were reversed by pretreatment with rapamycin (Fig. 4 a, b). Immunofluorescence analysis revealed a greater increase in cleaved caspase-3 intensity in SH-SY5Y cells cocultured with PFF-primed si-Irgm1 BV2 cells compared with those cocultured with si-NC BV2 cells (Fig. 4 c, d), while this increase was substantially attenuated by rapamycin treatment. Flow cytometric apoptosis analysis revealed that, compared with the control conditions, PFF-primed BV2 cells significantly increased the percentage of apoptotic SH-SY5Y cells (Fig. 4 e, f). Irgm1 knockdown in BV2 cells further intensified this proapoptotic effect, whereas rapamycin treatment effectively abolished neuronal cell death (Fig. 4 e, f). Collectively, these results indicate that Irgm1 deficiency impairs the capacity of microglia to clear internalized PFFs, leading to greater α-syn accumulation in cocultured SH-SY5Y cells and a corresponding increase in neuronal apoptosis. Rapamycin ameliorates this neurotoxicity, most likely by reactivating the ALP in microglia and thereby reducing the extracellular α-syn burden on neurons. Discussion After misfolded and aggregated α-syn accumulates inside neurons, it spreads into the extracellular space in a “prion-like” manner and is rapidly taken up by microglia. Under normal physiological conditions, microglia clear most internalized α-syn aggregates via the efficient ALP. When the ALP is impaired, undegraded α-syn accumulates within the cells, activating the NLRP3 inflammasome and inducing the massive release of IL-1β, TNF-α, and ROS, thereby establishing a vicious cycle characterized by 'failed clearance leading to amplified inflammation, which in turn leads to neuronal damage’. This cascade has been repeatedly validated in various models of AD, HD, and PD [23, 24]. IRGM/Irgm1 is a molecular link between autophagy and inflammation. Previous studies have shown that IRGM directly binds ATG16L1, Beclin-1 and ULK1, thereby promoting autophagosome formation. It also accelerates lysosome–autophagosome fusion by activating AMPK and inhibiting mTOR [16]. More importantly, IRGM targets NLRP3 and PYCARD/ASC for autophagic degradation, limiting the maturation and release of IL-1β and thus maintaining immune homeostasis [17]. Our group has shown that Irgm1 is neuroprotective in the acute phase of ischemic stroke and traumatic brain injury by increasing neuronal autophagy [25, 26]. In CCCP-treated SH-SY5Y cells, IRGM enhances PINK1/Parkin-mediated mitophagy by facilitating the autophagic degradation of mitofilin, which stabilizes PINK1 and recruits Parkin, thereby exerting a neuroprotective effect [27]. However, how Irgm1 regulates microglial autophagy and whether it modulates the disease progression of PD remain unclear. In this study, we found that Irgm1 expression was elevated in an AAV-SNCA-PD mouse model and that Irgm1-knockout mice exhibited more severe neurodegeneration. After confirming that Irgm1 colocalized with microglia, we treated BV2 cells with PFFs to model microglial clearance of α-syn pathology. Western blotting revealed the following ALP impairments: decreased levels of LC3-II and Ctsd, and elevated levels of p62, which is consistent with the experimental results of Choi I [19]. Moreover, Irgm1 knockdown not only aggravated ALP dysfunction but also reduced the colocalization of α-syn with lysosomes, accompanied by a decrease in lysosomal activity. Mechanistically, we focused on TFEB, the master regulator of the ALP. Irgm1 promotes TFEB dephosphorylation and nuclear translocation via mTOR inhibition, thereby driving lysosomal biogenesis and autophagy-related gene transcription; this Irgm1-mTOR-TFEB axis has been shown to be involved in infection and atherosclerosis [28, 29]. Here, Irgm1 knockdown markedly suppressed TFEB nuclear translocation after PFFs exposure in BV2 cells. In a coculture system, PFF-treated Irgm1-deficient microglia inefficiently degraded α-syn, leading to elevated Bax/Bcl-2 ratio, increased cleaved caspase-3 intensity, and enhanced apoptosis in SH-SY5Y neurons. Treating BV2 cells with rapamycin restored TFEB nuclear translocation, enhanced α-syn degradation and lysosomal function, and alleviated the neurotoxic phenotype of Irgm1-deficient microglia. However, this study has the following limitations. First, our in vivo experiments employed whole-body Irgm1-knockout mice instead of microglia-specific mutants, so we cannot exclude the possibility that Irgm1 expression changes in other brain cell types may influence PD progression. Future studies employing microglia-specific Irgm1 knockout mice will be crucial to confirm the cell-autonomous role of microglial Irgm1 in PD. Second, our in vitro work was performed in Irgm1-knockdown BV2 cells, so Irgm1-overexpressing models and additional interventions targeting the TFEB-ALP axis remain to be tested. Moreover, investigating whether Irgm1 modulates microglial inflammatory responses, phagocytic capacity and energy metabolism beyond its ALP-related role is worthwhile as such insights could open new therapeutic avenues for PD. In conclusion, we identified the “Irgm1-TFEB-ALP” axis as a critical mechanism by which microglia regulate α-syn clearance. Irgm1 deficiency impaired TFEB nuclear translocation, exacerbated α-syn-induced ALP dysfunction in microglia, intensified dopaminergic neuronal injury and accelerated PD progression. Our study delineates a previously unrecognized Irgm1-TFEB-ALP axis specifically in microglia, which is crucial for α-syn clearance and neuroprotection in PD, extending its known functions beyond inflammasome regulation and neuronal autophagy. Statements & Declarations Funding This work was funded by the National Natural Science Foundation of China (U23A20428, 32370962), the Joint Funds for the Innovation of Science and Technology of Fujian Province (2024Y9178), the National Natural Science Foundation of China (31870894) and the Natural Science Foundation of Heilongjiang Province of China (LH2022H005). Competing Interests The authors declare no competing interests. Author Contributions S.W. and C.P. designed the study, drafted the manuscript, and provided overall supervision for the study. S.W., X.L., J.Y., J.C., and C.X. conducted the animal-related experiments. S.W. and X.L. performed the cell-related experiments. S.W. and C.P. revised the manuscript. B.L., H.X., and R.L. participated in the discussion and review of the experimental results. H.X., R.L. and C.P. supported the project. Data Availability The datasets generated, used, and analyzed during the current study, as well as the materials used in this manuscript, are available from the corresponding author upon reasonable request. Ethics approval This study was performed under a protocol approved by the Ethics Committee of Harbin Medical University (approval number: [HMUIRB2026008]). Consent to Participate Not applicable. Consent to publication Not applicable. Acknowledgements The authors would like to thank the research group of Professor Zhentao Zhang for kindly providing α-synuclein preformed fibrils (α-syn PFFs). References Corti, O., Lesage, S. & Brice, A. What genetics tells us about the causes and mechanisms of Parkinson's disease. Physiol. Rev. 91 (4), 1161–1218 (2011). Mason, A. R., Ziemann, A. & Finkbeiner, S. Targeting the low-hanging fruit of neurodegeneration. Neurology 83 (16), 1470–1473 (2014). Goedert, M., Spillantini, M. G., Del Tredici, K. & Braak, H. 100 years of Lewy pathology, Nature reviews. Neurology 9 (1), 13–24 (2013). Schapira, A. H. V., Chaudhuri, K. R. & Jenner, P. Non-motor features of Parkinson disease, Nature reviews. Neuroscience 18 (7), 435–450 (2017). Spillantini, M. G. et al. Alpha-synuclein in Lewy bodies. Nature 388 (6645), 839–840 (1997). Braak, H. et al. Staging of brain pathology related to sporadic Parkinson's disease. Neurobiol. Aging . 24 (2), 197–211 (2003). Balestrino, R. & Schapira, A. H. V. Parkinson disease. Eur. J. Neurol. 27 (1), 27–42 (2020). Brück, D., Wenning, G. K., Stefanova, N. & Fellner, L. Glia and alpha-synuclein in neurodegeneration: A complex interaction. Neurobiol. Dis. 85 , 262–274 (2016). Guo, M. et al. Microglial exosomes facilitate α-synuclein transmission in Parkinson's disease. Brain: J. Neurol. 143 (5), 1476–1497 (2020). Luk, K. C. et al. Intracerebral inoculation of pathological α-synuclein initiates a rapidly progressive neurodegenerative α-synucleinopathy in mice. J. Exp. Med. 209 (5), 975–986 (2012). Peng, C., Trojanowski, J. Q. & Lee, V. M. Protein transmission in neurodegenerative disease. Nat. reviews Neurol. 16 (4), 199–212 (2020). Mizushima, N. & Komatsu, M. Autophagy: renovation of cells and tissues. Cell 147 (4), 728–741 (2011). Ebrahimi-Fakhari, D. et al. Distinct roles in vivo for the ubiquitin-proteasome system and the autophagy-lysosomal pathway in the degradation of α-synuclein. J. neuroscience: official J. Soc. Neurosci. 31 (41), 14508–14520 (2011). Sardiello, M. et al. A gene network regulating lysosomal biogenesis and function, Science (New York, N.Y.) 325(5939) 473-7. (2009). Settembre, C. et al. TFEB links autophagy to lysosomal biogenesis, Science (New York, N.Y.) 332(6036) 1429-33. (2011). Chauhan, S., Mandell, M. A. & Deretic, V. IRGM governs the core autophagy machinery to conduct antimicrobial defense. Mol. Cell . 58 (3), 507–521 (2015). Mehto, S. et al. The Crohn's Disease Risk Factor IRGM Limits NLRP3 Inflammasome Activation by Impeding Its Assembly and by Mediating Its Selective Autophagy. Mol. Cell . 73 (3), 429–445e7 (2019). Cheng, J. et al. Microglial autophagy defect causes parkinson disease-like symptoms by accelerating inflammasome activation in mice. Autophagy 16 (12), 2193–2205 (2020). Choi, I. et al. Microglia clear neuron-released α-synuclein via selective autophagy and prevent neurodegeneration. Nat. Commun. 11 (1), 1386 (2020). Jeon, Y. M. et al. The Role of Glial Mitochondria in α-Synuclein Toxicity. Front. cell. Dev. biology . 8 , 548283 (2020). Kim, H. J. et al. Deficient autophagy in microglia impairs synaptic pruning and causes social behavioral defects. Mol. Psychiatry . 22 (11), 1576–1584 (2017). Larabi, A., Barnich, N. & Nguyen, H. T. T. New insights into the interplay between autophagy, gut microbiota and inflammatory responses in IBD. Autophagy 16 (1), 38–51 (2020). Guan, Y. & Han, F. Key Mechanisms and Potential Targets of the NLRP3 Inflammasome in Neurodegenerative Diseases. Front. Integr. Nuerosci. 14 , 37 (2020). Holbrook, J. A. et al. Neurodegenerative Disease and the NLRP3 Inflammasome. Front. Pharmacol. 12 , 643254 (2021). He, S. et al. Immune-related GTPase M (IRGM1) regulates neuronal autophagy in a mouse model of stroke. Autophagy 8 (11), 1621–1627 (2012). Pei, C. et al. Neuron-specific deficiency of autophagy increases neuronal loss in traumatic brain injury. PNAS nexus . 3 (10), pgae457 (2024). Guo, X. et al. IRGM promotes the PINK1-mediated mitophagy through the degradation of Mitofilin in SH-SY5Y cells, FASEB journal: official publication of the Federation of American Societies for. Experimental Biology . 34 (11), 14768–14779 (2020). Cai, H. et al. A potential early-atheroprotective target: Irgm1 mediates lymphangiogenesis through LEC autophagy by Tfeb translocation, Biochimica et biophysica acta. Mol. basis disease . 1870 (6), 167238 (2024). Kumar, S. et al. Mammalian Atg8 proteins and the autophagy factor IRGM control mTOR and TFEB at a regulatory node critical for responses to pathogens. Nat. Cell Biol. 22 (8), 973–985 (2020). Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9004768","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":603763972,"identity":"6cc9201f-fe7f-4a25-be96-d38bae2c78a9","order_by":0,"name":"Sijia Wen","email":"","orcid":"","institution":"Harbin Medical University","correspondingAuthor":false,"prefix":"","firstName":"Sijia","middleName":"","lastName":"Wen","suffix":""},{"id":603763975,"identity":"306f3c93-9684-4afe-aa17-a500274298de","order_by":1,"name":"Xiaojing Li","email":"","orcid":"","institution":"Harbin Medical University","correspondingAuthor":false,"prefix":"","firstName":"Xiaojing","middleName":"","lastName":"Li","suffix":""},{"id":603763976,"identity":"79ce84c2-fcfc-4ed2-8395-fc9423ebb4fc","order_by":2,"name":"Junling Yang","email":"","orcid":"","institution":"Harbin Medical University","correspondingAuthor":false,"prefix":"","firstName":"Junling","middleName":"","lastName":"Yang","suffix":""},{"id":603763977,"identity":"e12acc01-70da-44ba-af4e-aec98e3288a3","order_by":3,"name":"Jiawen Chen","email":"","orcid":"","institution":"Harbin Medical University","correspondingAuthor":false,"prefix":"","firstName":"Jiawen","middleName":"","lastName":"Chen","suffix":""},{"id":603763978,"identity":"ea8b4824-1d17-471f-9563-28f2df4644c0","order_by":4,"name":"Chenxi Xue","email":"","orcid":"","institution":"Harbin Medical University","correspondingAuthor":false,"prefix":"","firstName":"Chenxi","middleName":"","lastName":"Xue","suffix":""},{"id":603763980,"identity":"b8f487d0-c8b0-4193-bc3f-c6994222f8c0","order_by":5,"name":"Bo Li","email":"","orcid":"","institution":"Harbin Medical University","correspondingAuthor":false,"prefix":"","firstName":"Bo","middleName":"","lastName":"Li","suffix":""},{"id":603763983,"identity":"0d8f935e-b87c-44ed-9b50-63ea22a8799d","order_by":6,"name":"Hongwei Xu","email":"","orcid":"","institution":"Harbin Medical University","correspondingAuthor":false,"prefix":"","firstName":"Hongwei","middleName":"","lastName":"Xu","suffix":""},{"id":603763984,"identity":"23cdfad5-75a2-4871-b1b3-1eecfff5ed22","order_by":7,"name":"Rui Li","email":"","orcid":"","institution":"Fujian Medical University","correspondingAuthor":false,"prefix":"","firstName":"Rui","middleName":"","lastName":"Li","suffix":""},{"id":603763985,"identity":"af857c9f-2051-4aaf-95b6-388f4f875dc5","order_by":8,"name":"Chunying Pei","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA9UlEQVRIiWNgGAWjYFACxgZmEMUGZD1mYDgAFpMgVguzMZFagEqhNJs0UVrM2ZsbPxe23Unsk26/Vl245060wQHmg7d5GOzycGmx7DnYLD2z7Vlim8yZstsznj3L3XCALdmahyG5GJcWgxuJbcy8bYcT2yRy0m7zHDgM1MJjJs3DcCCxAZeW+w8RWoohWvi/4ddygxGmJf0YM9QWNrxaLHsSm6V5zh02BtrCLM1z4FnuzMNsxpZzDJJxajFnP/7wM0/ZYdn5M9KBjAN3cvuONz+88abCDrfDoLRjAwMPlM2MLI5Hiz0DA/sD3MpGwSgYBaNgRAMADuNd2Aymo4AAAAAASUVORK5CYII=","orcid":"","institution":"Harbin Medical University","correspondingAuthor":true,"prefix":"","firstName":"Chunying","middleName":"","lastName":"Pei","suffix":""}],"badges":[],"createdAt":"2026-03-02 02:23:17","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9004768/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9004768/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":104380796,"identity":"b6cc5058-679d-421d-882a-a50aee20d9ee","added_by":"auto","created_at":"2026-03-11 07:35:59","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":348971,"visible":true,"origin":"","legend":"\u003cp\u003eIrgm1 expression was elevated in the midbrain of PD mice, and its deficiency further accelerated disease progression. (\u003cstrong\u003ea\u003c/strong\u003e) Representative western blotting images and quantification of Irgm1 protein relative expression (n=3). (\u003cstrong\u003eb\u003c/strong\u003e) Quantification of Irgm1 mRNA relative expression (n=3). (\u003cstrong\u003ec\u003c/strong\u003e) Representative image showing the trajectory of spontaneous activity obtained from each group by open field test. (\u003cstrong\u003ed\u003c/strong\u003e) Quantification of total distance of movement by open field test (n=6). (\u003cstrong\u003ee\u003c/strong\u003e) Quantification of latency to fall by rotarod test (n=6). (\u003cstrong\u003ef\u003c/strong\u003e) Representative immunofluorescence images of TH (red) in the midbrain in each group. Scale bar, 500 μm. (\u003cstrong\u003eg\u003c/strong\u003e) Quantification of mean fluorescence intensity of TH (n=3). (\u003cstrong\u003eh\u003c/strong\u003e) Representative western blotting images of TH expression in each group. (\u003cstrong\u003ei\u003c/strong\u003e) Quantification of TH protein relative expression (n=3). Data are presented as means ± SEM. **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001, ****\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001; ns, non-significant. Statistics were calculated by one-way ANOVA with Tukey’s multiple comparisons test and two-way ANOVA with Tukey’s multiple comparisons test.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-9004768/v1/41430c2d44267d62c2dfc5a5.png"},{"id":104405703,"identity":"5c0dd22a-226a-420f-ad0f-5bc71a9614ad","added_by":"auto","created_at":"2026-03-11 12:23:38","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":378513,"visible":true,"origin":"","legend":"\u003cp\u003eIrgm1 deficiency exacerbated autophagy‒lysosome dysfunction. (\u003cstrong\u003ea\u003c/strong\u003e) Representative western blotting images of Irgm1, p62, LC3, Lamp1, Ctsd, and α-syn expression in each group. (\u003cstrong\u003eb\u003c/strong\u003e) Quantification of Irgm1, p62, LC3Ⅱ, Lamp1, Ctsd, and α-syn protein relative expression (n=3). (\u003cstrong\u003ec\u003c/strong\u003e) Representative immunofluorescence images of Iba-1 (red) and Irgm1 (green) in the midbrain in each group. Scale bar, 25 μm. (\u003cstrong\u003ed\u003c/strong\u003e) Representative western blotting images of Irgm1, p62, LC3, Ctsd, and α-syn expression in each group. (\u003cstrong\u003ee\u003c/strong\u003e) Quantification of Irgm1, p62, LC3Ⅱ, Ctsd, and α-syn protein relative expression (n=3). (\u003cstrong\u003ef\u003c/strong\u003e) Representative immunofluorescence images of α-syn (red) and Lamp1 (green) in each group. Scale bar, 25 μm. (\u003cstrong\u003eg\u003c/strong\u003e) Quantification of MCC of α-syn and Lamp1, and mean fluorescence intensity of α-syn and Lamp1. The fluorescence intensity of each cell was estimated by examining 50 random cells. (\u003cstrong\u003eh\u003c/strong\u003e) Representative images of LysoTracker (green) and LysoSensor (green) in each group. Scale bar, 25 μm. (\u003cstrong\u003ei\u003c/strong\u003e) Quantification of integrated density of LysoTracker and LysoSensor. The integrated density of each cell was estimated by examining 50 random cells. Data are presented as means ± SEM. *\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; ns, non-significant. Statistics were calculated by unpaired t-test or one-way ANOVA with Tukey’s multiple comparisons test. MCC, Manders’ Colocalization Coefficients; a.u., arbitrary unit.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-9004768/v1/1ee6f8c33c87b93733b37f68.png"},{"id":104380798,"identity":"3e32b598-8aed-45a9-8101-07cf40b1487d","added_by":"auto","created_at":"2026-03-11 07:35:59","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":428552,"visible":true,"origin":"","legend":"\u003cp\u003eIrgm1 deficiency exacerbates ALP dysfunction by inhibiting TFEB nuclear translocation. (\u003cstrong\u003ea\u003c/strong\u003e) Representative western blotting images of nuclear TFEB and cytoplasmic TFEB expression in each group. (\u003cstrong\u003eb\u003c/strong\u003e) Quantification of nuclear TFEB and cytoplasmic TFEB protein relative expression (n=3). (\u003cstrong\u003ec\u003c/strong\u003e) Representative immunofluorescence images of TFEB (red) in each group. Scale bar, 25 μm. (\u003cstrong\u003ed\u003c/strong\u003e) Quantification of the nuclear-cytoplasmic TFEB fluorescence ratio. The fluorescence intensity of each cell was estimated by examining 50 random cells. (\u003cstrong\u003ee\u003c/strong\u003e) Representative immunofluorescence images of α-syn (red) and Lamp1 (green) in each group. Scale bar, 25 μm. (\u003cstrong\u003ef\u003c/strong\u003e) Quantification of MCC of α-syn and Lamp1, and mean fluorescence intensity of α-syn and Lamp1. The fluorescence intensity of each cell was estimated by examining 50 random cells. (\u003cstrong\u003eg\u003c/strong\u003e) Representative images of LysoTracker (green) and LysoSensor (green) in each group. Scale bar, 25 μm. (\u003cstrong\u003eh\u003c/strong\u003e) Quantification of integrated density of LysoTracker and LysoSensor. The integrated density of each cell was estimated by examining 50 random cells. Data are presented as means ± SEM. *\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. Statistics were calculated by one-way ANOVA with Tukey’s multiple comparisons test. MCC, Manders’ Colocalization Coefficients; a.u., arbitrary unit.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-9004768/v1/b4988bca80c5ec379e05f437.png"},{"id":104380799,"identity":"b6b9bebe-f5a2-41b2-be2f-800dc27cadae","added_by":"auto","created_at":"2026-03-11 07:35:59","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":268440,"visible":true,"origin":"","legend":"\u003cp\u003ePFF-exposed Irgm1-deficient microglia exacerbate neurotoxic effects. (\u003cstrong\u003ea\u003c/strong\u003e) Representative western blotting images of Bak, Bcl2, and α-syn expression in SH-SY5Y cells in each group. (\u003cstrong\u003eb\u003c/strong\u003e) Quantification of the Bak/Bcl2 ratio and α-syn protein relative expression (n=3). (\u003cstrong\u003ec\u003c/strong\u003e) Representative immunofluorescence images of cleaved caspase-3 (green) in SH-SY5Y cells in each group. Scale bar, 25 μm. (\u003cstrong\u003ed\u003c/strong\u003e) Quantification of mean fluorescence intensity of cleaved caspase-3. The fluorescence intensity of each cell was estimated by examining 50 random cells. (\u003cstrong\u003ee\u003c/strong\u003e) Representative flow cytometry images of SH-SY5Y cells in each group. (\u003cstrong\u003ef\u003c/strong\u003e) Quantification of apoptosis of SH-SY5Y cells (n=3). Data are presented as means ± SEM. *\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. Statistics were calculated by one-way ANOVA with Tukey’s multiple comparisons test. a.u., arbitrary unit.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-9004768/v1/b4a2e14cde720246eb1a11cc.png"},{"id":109176350,"identity":"5ce93691-13ab-40a5-98f2-dc7a691c19c7","added_by":"auto","created_at":"2026-05-13 09:31:12","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1604308,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9004768/v1/439a483a-ecd0-425b-a082-2b2665fe6d18.pdf"},{"id":104380800,"identity":"4308199a-b890-4483-a31b-6d72519e4efb","added_by":"auto","created_at":"2026-03-11 07:35:59","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":1317214,"visible":true,"origin":"","legend":"","description":"","filename":"WenSupplementaryFigures.docx","url":"https://assets-eu.researchsquare.com/files/rs-9004768/v1/63bacbfd0d392cda10c81f3c.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Irgm1 Promotes Microglial Clearance of α-Synuclein via the TFEB-Dependent Autophagy- Lysosome Pathway in Parkinson’s Disease","fulltext":[{"header":"Introduction","content":"\u003cp\u003eParkinson\u0026rsquo;s disease (PD) is the second most common neurodegenerative disorder after Alzheimer\u0026rsquo;s disease [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Clinically, it presents with motor symptoms of resting tremor, muscular rigidity and bradykinesia [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e], as well as nonmotor symptoms, including hyposmia, constipation and sleep disturbances [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Current pharmacological and surgical interventions only ameliorate motor deficits; they do not halt the progressive loss of dopaminergic neurons. Therefore, clarifying the core mechanism of disease progression and identifying potential intervention targets have become urgent priorities.\u003c/p\u003e \u003cp\u003ePathologically, massive degeneration of dopaminergic neurons in the substantia nigra pars compacta and the formation of Lewy bodies composed of misfolded α-synuclein (α-syn) are hallmarks of PD [\u003cspan additionalcitationids=\"CR6\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. These prion-like aggregates can propagate between neurons and glial cells, triggering chronic neuroinflammation and creating a vicious cycle of protein deposition, microglial hyperactivation, and ever-amplifying inflammation [\u003cspan additionalcitationids=\"CR9 CR10\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Thus, increasing the capacity of microglia to clear α-syn aggregates is considered a promising therapeutic approach.\u003c/p\u003e \u003cp\u003eThe autophagy\u0026ndash;lysosome pathway (ALP) is the principal route for disposing of damaged organelles and abnormal proteins: cargo is sequestered by autophagosomes that subsequently fuse with lysosomes for degradation [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. α-syn aggregates are cleared mainly through this route [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], yet the pathway is markedly impaired in the brains of Parkinson\u0026rsquo;s disease patients. Transcription factor EB (TFEB) acts as the master switch of the ALP, accelerating waste disposal and preserving intracellular homeostasis by coordinately upregulating autophagy and lysosomal genes [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Although ALP function in neurons has been intensively studied over the past two decades, microglial autophagy in Parkinson\u0026rsquo;s disease remains in its infancy. How microglial ALP is regulated and how extracellular α-syn influences this regulation remain poorly understood.\u003c/p\u003e \u003cp\u003eThe immune-related GTPase M1 (Irgm1) is a key autophagy-regulating protein that enhances autophagy by activating the ULK1/Beclin-1 pathway, promotes LC3II conversion and P62 degradation, and suppresses NLRP3 inflammasome assembly as well as the release of IL-1β and TNF-α [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. However, direct evidence that Irgm1 mediates microglial clearance of α-syn is still lacking in Parkinson\u0026rsquo;s disease, a disorder characterized by impaired ALP and prominent neuroinflammation.\u003c/p\u003e \u003cp\u003eIn this study, through the establishment of an α-syn-overexpressing PD mouse model, we demonstrated that Irgm1 is significantly upregulated in the midbrain and mice lacking Irgm1 showed accelerated progression of PD. Using microglia stimulated with α-syn-preformed fibrils (PFFs), we further showed that Irgm1 deficiency impedes TFEB nuclear translocation, exacerbates ALP dysfunction, promotes intracellular α-syn accumulation, and amplifies the toxic spread of α-syn to dopaminergic neurons. Pharmacological activation of TFEB with rapamycin reversed this pathological cascade. Collectively, our findings identify the Irgm1\u0026ndash;TFEB\u0026ndash;ALP axis as a critical regulatory node for microglial α-syn clearance and provide a potential therapeutic target for arresting PD progression.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003eAnimals\u003c/p\u003e\n\u003cp\u003eC57BL/6 WT mice were purchased from Changsheng Bio-Technology Co., Ltd. Systemic stable Irgm1 knockout mice (Irgm1⁻\u003csup\u003e/\u003c/sup\u003e⁻) were generated by crossing Irgm1-flox mice with Dppa3-Cre mice, both of which were purchased from the Shanghai Model Organisms Center.\u003c/p\u003e\n\u003cp\u003eMice were housed on a 12 h light/dark cycle at 23 ± 1°C with free access to food and water. The animal experimental plan was approved by the Ethics Committee of Harbin Medical University. Animal studies were in accordance with the National Guidelines for the Care and Use of Laboratory Animals of China.\u003c/p\u003e\n\u003cp\u003eMouse model of PD\u003c/p\u003e\n\u003cp\u003eTo induce α-syn overexpression in vivo, 8-week-old male C57BL/6 WT and Irgm1\u003csup\u003e-/-\u003c/sup\u003emice received a unilateral injection of AAV5-hα-syn (AAV-SNCA, 1.2E+13 vg),\u0026nbsp;under the control of the human synapsin\u0026nbsp;promoter (Genechem, China) into the right substantia nigra (SN). Control mice received AAV-GFP or saline into the right SN. During surgery, mice were anesthetized with 2% isoflurane and stereotaxically\u0026nbsp;injected with 2 μl\u0026nbsp;of AAV-SNCA, AAV-GFP, or saline at a rate of 0.3 μl\u0026nbsp;/min, using the following coordinates: AP −3.0 mm, ML −1.25 mm, and DV +4.5 mm relative to bregma. The needle was left in place for 5 min before retraction.\u003c/p\u003e\n\u003cp\u003eBehavioral test\u003c/p\u003e\n\u003cp\u003eBehavioral tests were performed on mice three months after construction of the α-syn-overexpression PD model. All tests were performed between 10 am and 3 pm by experimenters who were blind to subgrouping. Before the test, mice were allowed to acclimate undisturbed in a dimly lit behavioral room for at least 1 h. Equipment for open field test and rotarod test was purchased from SansBio\u0026nbsp;(Jiangsu, China).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eOpen field test\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLocomotor activity was measured in an open field box (40 cm × 40 cm × 40 cm). The box was cleaned with 75% alcohol before each trial to remove odors, with at least 10 min between testing different mice. After placing the mouse in the center, locomotor activity was recorded for 15 min, and total distance traveled was used for statistical analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRotarod test\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePrior to the formal test, mice were trained on the rotarod for 5 min per day over 5 consecutive days. During the test, the rod accelerated from 4 rpm to 30 rpm over 300 seconds. The trial ended when the mouse fell from the rod. Fall latency was recorded for each of 5 trials per mouse, with at least 30 min between trials. The average latency to fall was used for statistical analysis.\u003c/p\u003e\n\u003cp\u003eCell culture\u003c/p\u003e\n\u003cp\u003eThe mouse microglia cell line BV-2 and human neuroblastoma cell line SH-SY5Y were purchased from Pricella Biotechnology (Wuhan, China) and were tested for mycoplasma contamination. Cells were cultured using Dulbecco’s modified Eagle’s medium (DMEM; Gibco, USA) supplemented with 10% fetal bovine serum (FBS; Sigma, USA) and 1% penicillin-streptomycin (Gibco, USA) and incubated in a humidified incubator at 37 °C with 5% CO₂. PFFs were a gift from Prof. Zhentao\u0026nbsp;Zhang (Department of Neurology, Renmin Hospital of Wuhan University, China).\u003c/p\u003e\n\u003cp\u003eTransfection\u003c/p\u003e\n\u003cp\u003esiRNA transfection was performed using Lipofectamine 3000 (L3000015, Invitrogen, USA). Cells were transfected at 70–80% confluence in serum-free Opti-MEM (11058021, Gibco, USA) according to the manufacturer’s instructions. Cells were harvested 48–72 h later for the subsequent experiments. Irgm1 siRNA and control siRNA (si-NC) were purchased from OBiO\u0026nbsp;Technology (Shanghai, China). The sequences are as follows: for si-Irgm1: P1:5′- GGUCAGUAGGAGCACCGAATT -3′, P2:5′- UUCGGUGCUCCUACUGACCTT -3′; si-NC: P1:5′- UUCUCCGAACGUGUCACGUTT -3′, P2:5′- ACGUGACACGUUCGGAGAATT -3′.\u003c/p\u003e\n\u003cp\u003eFlow cytometric analysis of apoptosis\u003c/p\u003e\n\u003cp\u003eFlow cytometry was performed to evaluate the apoptosis of SH-SY5Y cells cocultured with BV2 cells pretreated with PFFs, and the Annexin V-FITC/PI Cell Apoptosis Detection Kit (TransGen, China) was used according to the manufacturer’s protocol. Unstained controls and single-staining controls were set up, and each experiment was performed at least three times. The apoptosis rate of SH-SY5Y cells was evaluated via flow cytometry (BD, USA) within 1 h.\u003c/p\u003e\n\u003cp\u003eQuantitative real-time PCR\u003c/p\u003e\n\u003cp\u003eTotal RNA was extracted from the midbrain of mice in each group using RNAisoPlus (9109, TaKaRa) according to the manufacturer’s instructions. cDNA was synthesized from individual RNA samples with the PrimeScript\u0026nbsp;FAST RT Reagent Kit (RR092A, TaKaRa). The Irgm1 primers were 5′-CGCGATCAGACCTCCTCTTG-3′ and 5′-CAAGAGAGGAGGTCTGATCGCG-3′. The GAPDH primers were 5′-CAGAAGACTGTGGATGGCCG-3′ and 5′-CGGCCATCCACAGTCTTCTG-3′. Real-time qPCR was performed in a 25-μL reaction system containing cDNA, forward and reverse primers, and TB Green Premix (CN830A, TaKaRa). Relative Irgm1 mRNA expression was calculated using the 2^(-ΔΔCt) method, and each experiment was repeated at least three times.\u003c/p\u003e\n\u003cp\u003eImmunofluorescence staining\u003c/p\u003e\n\u003cp\u003eAfter cardiac perfusion and isolation, brain tissues were fixed overnight in 4% PFA at 4 °C and dehydrated in 30% sucrose for 48 h. Frozen brain tissue sections (5 μm) near the midbrain, as well as BV2 and SH-SY5Y cells, were fixed with 4% PFA for 15 min, permeabilized with 0.1% Triton X-100 for 15 min, and blocked with QuickBlock™Blocking Buffer (P0260, Beyotime) for 15 min. Sections and cells were then incubated with primary antibodies against TH (1:100, ab112, Abcam), Iba-1 (1:100, ab178846, Abcam), Irgm1 (1:100, IC11, AbMart), α-syn (1:100, ab138501, Abcam), Lamp1 (1:100, ab25245, Abcam), or cleaved caspase-3 (1:100, 9664S, CST) at 4 °C overnight. After three washes with PBS, corresponding Alexa Fluor-conjugated secondary antibodies were applied for 2 h at room temperature. Following three additional PBS washes, samples were counterstained with DAPI for 5 min. Images were acquired using a fluorescence microscope\u0026nbsp;(Nikon, Japan and Zeiss, Germany), and fluorescence intensity was quantified using ImageJ software. All experiments and analyses were performed by an investigator blinded to group allocation. Each experiment was repeated at least three times.\u003c/p\u003e\n\u003cp\u003eLysosome staining\u003c/p\u003e\n\u003cp\u003eCells grown on PDL-coated coverslips were stained with the live-cell dyes LysoTracker\u0026nbsp;Green DND-26 (50 nM, 3 min) and LysoSensor\u0026nbsp;Green DND-189 (1 μM, 30 min) following the manufacturer’s protocol. After staining at 37 °C in the dark, cells were washed twice with PBS and imaged with a fluorescence microscope (Nikon, Japan). Fluorescence intensity was quantified using ImageJ. All procedures were performed by an investigator blinded to group allocation, and each experiment was repeated at least three times.\u003c/p\u003e\n\u003cp\u003eWestern blotting\u003c/p\u003e\n\u003cp\u003eTotal proteins from cells or mice brain tissues were lysed in RIPA buffer containing protease inhibitor and PMSF. Nuclear proteins and cytoplasm proteins were extracted from cells via Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime, China) according to the manufacturer’s protocol. After ultrasonic crushing and high-speed centrifugation at 4 °C, protein concentrations were measured with a bicinchoninic acid (BCA) protein assay kit (Beyotime, China). 20 μg\u0026nbsp;of proteins were separated via SDS-PAGE and transferred onto Nitrocellulose membrane (Cytiva, USA) with a 0.22 μm\u0026nbsp;pore size. After being blocked with 5% skimmed milk in tris-buffered saline with 0.1% Triton X-100 (TBST) for 2 h, the membranes were incubated overnight at 4 °C with the following primary antibodies: Irgm1 (1:1000, 14979S, CST), α-syn (1:1000, ab138501, Abcam), TH (1:1000, ab112, Abcam), p62\u0026nbsp;(1:1000, P0067, Sigma), LC3 (1:1000, 2775S, CST), Lamp1 (1:1000, ab25245, Abcam), Ctsd\u0026nbsp;(1:1000, 21327-1-AP, Proteintech), TFEB (1:1000, 13372-1-AP, Proteintech), Bak (), Bcl2(), Histone-H3 (1:5000, 17168-1-AP, Proteintech), β-actin (1:20000, A3854, Sigma) and GAPDH (1:40,000, 10494-1-AP, Proteintech). After washing 3 times for 15 min with TBST, the membrane was incubated in the HRP-conjugated secondary antibody for 2 h at room temperature.\u0026nbsp;After\u0026nbsp;washes, the signals were detected via super sensitive ECL luminescence reagent (MeilunBio, China) and a chemiluminescence system (Tanon, China). Images were analyzed via ImageJ software. Each experiment was repeated at least three times.\u003c/p\u003e\n\u003cp\u003eStatistical analysis\u003c/p\u003e\n\u003cp\u003eThe statistical significance of the data was analyzed with GraphPad Prism version 9.5. Shapiro–Wilk test and F test were used to examine the normality of the date and equality of variance, respectively. Data were analyzed by unpaired t test for two independent groups and one-way ANOVA with Tukey's multiple comparisons test for multiple independent groups that were normally distributed and had the same variance. Data were analyzed by Welch's t test for two independent groups that were normally distributed and different variances. Assuming sphericity depending on the situation, two-way ANOVA with Tukey's multiple comparisons test was used for comparisons between multiple groups and regions. Data in figures were represented as mean ± SEM. P-values \u0026lt;0.05 were considered statistically significant (*\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).\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eIrgm1 expression was elevated in the midbrain of PD mice, and its deficiency further accelerated disease progression.\u003c/p\u003e \u003cp\u003eTo investigate whether Irgm1, an immune-related GTPase with autophagy-regulating functions, is involved in PD pathogenesis, we observed the expression of Irgm1 in the AAV-SNCA PD mouse model in which wild-type human α-syn is selectively overexpressed in dopaminergic neurons. Compared with mice receiving AAV-GFP or saline, those injected with AAV-SNCA into the SNpc presented consistent upregulation of Irgm1 at both the protein and mRNA levels in midbrain tissue (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea-b).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo explore the effects of Irgm1 on PD pathogenesis, we compared motor performance and neuronal degeneration between WT and Irgm1\u003csup\u003e-/-\u003c/sup\u003emice after stereotaxic injection of AAV-SNCA. At 12weeks after virus transduction, Irgm1\u003csup\u003e-/-\u003c/sup\u003e mice exhibited significantly shorter total travel distances in the open-field test and reduced latency to fall in the rotarod test, compared with WT mice, indicating aggravated motor deficits (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec-e). Immunofluorescence and western blotting revealed robust loss of midbrain TH\u003csup\u003e+\u003c/sup\u003e neurons in the PD model, validating the paradigm, and Irgm1 deficiency further exacerbated this neuronal loss (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ef-i). Notably, Irgm1 deficiency alone (in control mice) neither altered impaired motor behavior nor TH\u003csup\u003e+\u003c/sup\u003e neuronal survival. Collectively, these findings suggest that Irgm1 likely plays a neuroprotective role in the context of α-syn-driven Parkinson\u0026rsquo;s disease.\u003c/p\u003e \u003cp\u003e \u003cdiv description=\"\" class=\"Drawing\" id=\"1458668822\" name=\"图片 1\"\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eIrgm1 deficiency exacerbated autophagy‒lysosome dysfunction\u003c/p\u003e \u003cp\u003eGiven that Irgm1 is a known regulator of autophagy and its expression was upregulated in our PD model (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), we next investigated whether Irgm1 deficiency could modulate the critical nexus between α-syn aggregation and ALP dysfunction in the PD midbrain. Western blotting revealed that, within the AAV-SNCA group, α-syn aggregation was significantly greater in Irgm1\u003csup\u003e-/-\u003c/sup\u003emice than in WT mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea, b). Moreover, there was a significant reduction in LC3Ⅱ, Ctsd, and Lamp1 but an increase in p62 in PD mice, indicating autophagy\u0026ndash;lysosome dysfunction, and this impairment was markedly amplified by Irgm1 knockout (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea, b).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eNext, we investigated where and how Irgm1 affects α-syn aggregation and the ALP in PD. The fluorescence results revealed that Irgm1 was significantly colocalized with microglia in the midbrains of PD mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec). Studies have shown that impaired microglial ALP fails to clear α-syn and damaged mitochondria, promoting chronic neuroinflammation that accelerates dopaminergic neuron loss and Parkinson\u0026rsquo;s disease progression [\u003cspan additionalcitationids=\"CR19\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Dysregulation of microglial autophagy is emerging as a core regulator of brain development and diseases [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. To determine whether Irgm1 contributes to Parkinson\u0026rsquo;s disease pathogenesis by modulating microglial ALP, we exposed BV2 microglia to α-syn-preformed fibrils (PFFs). After 24 h of exposure to PFFs (1 \u0026micro;g/ml), western blotting revealed that Irgm1 expression was upregulated, accompanied by increased p62 levels and decreased levels of LC3-II and Ctsd (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed, e), indicating suppressed ALP activity, which is consistent with the findings of a recent study [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Compared with the control conditions, Irgm1 knockdown exacerbated these impairments and elevated the intracellular α-syn level (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed, e). Immunofluorescence further revealed that, compared with si-NC-treated BV2 cells, si-Irgm1-treated BV2 cells accumulated more α-syn and showed a marked reduction in the colocalization of α-syn and Lamp1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ef, g). Moreover, the PFF-induced reduction in Lamp1 intensity was significantly exacerbated (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ef, g). These results demonstrate that the loss of Irgm1 aggravates PFF-induced ALP dysfunction and α-syn accumulation. We subsequently examined the lysosomal activity of BV2 cells. Prior to PFFs exposure, no differences in LysoTracker or LysoSensor fluorescence intensities were detected between si-NC and si-Irgm1 BV2 cells. After 24 h of PFFs exposure, both LysoTracker and LysoSensor fluorescence intensities were significantly reduced, and this reduction was further exacerbated by Irgm1 knockdown (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eh, i). These findings indicate that Irgm1 deficiency amplifies α-syn-induced lysosomal dysfunction, further impairing autophagy\u0026ndash;lysosome pathway activity and thereby hindering PFFs degradation.\u003c/p\u003e \u003cp\u003e \u003cdiv description=\"\" class=\"Drawing\" id=\"1538161810\" name=\"图片 2\"\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv description=\"\" class=\"Drawing\" id=\"1921661509\" name=\"图片 3\"\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eIrgm1 deficiency exacerbates ALP dysfunction by inhibiting TFEB nuclear translocation\u003c/p\u003e \u003cp\u003eTranscription factor EB (TFEB) is a master regulator of autophagy and lysosomal gene expression. Its nuclear translocation enhances ALP activity and promotes the clearance of cellular waste [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. We therefore asked whether Irgm1 deficiency compromises the ALP by interfering with TFEB subcellular localization, thereby impairing α-syn degradation. Western blotting revealed that after 24 h of exposure to PFFs (1 \u0026micro;g/ml), the nuclear TFEB level decreased, whereas the cytoplasmic TFEB level increased, and this effect was exaggerated in si-Irgm1 BV2 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea, b). Treatment with rapamycin, a TFEB-activating autophagy agonist, significantly reversed the alterations caused by Irgm1 knockdown, restoring TFEB nuclear expression and reducing its cytoplasmic accumulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea, b). Immunofluorescence further confirmed these findings. Compared with si-NC BV2 cells, si-Irgm1 BV2 cells presented a greater reduction in TFEB nuclear translocation following PFF exposure, which was abolished by rapamycin treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec, d). These results suggest that Irgm1 may play a role in regulating the subcellular localization of TFEB.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eNext, we investigated whether rapamycin could restore α-syn clearance and lysosomal function in si-Irgm1 BV2 cells exposed to PFFs. Immunofluorescence revealed that rapamycin treatment markedly increased the colocalization of α-syn with Lamp1 and increased the fluorescence intensity of Lamp1, accompanied by a clear reduction in the α-syn aggregates (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee, f). Concomitantly, both LysoTracker and LysoSensor fluorescence intensities were significantly increased (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eg, h), indicating that lysosomal acidification and functional activity were restored. Collectively, these results demonstrate that rapamycin reactivates ALP and accelerates α-syn degradation in Irgm1-deficient microglia, an effect associated with restored TFEB nuclear translocation.\u003c/p\u003e \u003cp\u003e \u003cdiv description=\"\" class=\"Drawing\" id=\"992918345\" name=\"图片 4\"\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv description=\"\" class=\"Drawing\" id=\"1055136809\" name=\"图片 6\"\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003ePFF-exposed Irgm1-deficient microglia exacerbate neurotoxic effects\u003c/p\u003e \u003cp\u003eTo examine the impact of Irgm1-deficient microglia on neurons, we established a contact coculture assay in which SH-SY5Y cells were exposed to BV2 cells that had been pretreated with PFFs. After a further 24 h of coculture, western blotting revealed that SH-SY5Y cells exposed to PFF-primed BV2 cells presented increased levels of α-syn and elevated Bak/Bcl-2 ratios (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea, b), indicating increased apoptotic priming. Knocking down Irgm1 in BV2 cells further increased α-syn levels and increased the Bak/Bcl-2 ratio in SH-SY5Y cells, both of which were reversed by pretreatment with rapamycin (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea, b). Immunofluorescence analysis revealed a greater increase in cleaved caspase-3 intensity in SH-SY5Y cells cocultured with PFF-primed si-Irgm1 BV2 cells compared with those cocultured with si-NC BV2 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec, d), while this increase was substantially attenuated by rapamycin treatment.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFlow cytometric apoptosis analysis revealed that, compared with the control conditions, PFF-primed BV2 cells significantly increased the percentage of apoptotic SH-SY5Y cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee, f). Irgm1 knockdown in BV2 cells further intensified this proapoptotic effect, whereas rapamycin treatment effectively abolished neuronal cell death (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee, f). Collectively, these results indicate that Irgm1 deficiency impairs the capacity of microglia to clear internalized PFFs, leading to greater α-syn accumulation in cocultured SH-SY5Y cells and a corresponding increase in neuronal apoptosis. Rapamycin ameliorates this neurotoxicity, most likely by reactivating the ALP in microglia and thereby reducing the extracellular α-syn burden on neurons.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eAfter misfolded and aggregated α-syn accumulates inside neurons, it spreads into the extracellular space in a “prion-like” manner and is rapidly taken up by microglia. Under normal physiological conditions, microglia clear most internalized α-syn aggregates via the efficient\u0026nbsp;ALP.\u0026nbsp;When the ALP is impaired, undegraded α-syn accumulates within the cells, activating the NLRP3 inflammasome and inducing the massive release of IL-1β, TNF-α, and ROS, thereby establishing a vicious cycle characterized by 'failed clearance leading to amplified inflammation, which in turn leads to neuronal damage’. This cascade has been repeatedly validated in various models of AD, HD, and PD\u0026nbsp;[23, 24].\u003c/p\u003e\n\u003cp\u003eIRGM/Irgm1 is a molecular link between autophagy and inflammation. Previous studies have shown that IRGM directly binds ATG16L1, Beclin-1 and ULK1, thereby promoting autophagosome formation. It also accelerates lysosome–autophagosome fusion by activating AMPK and inhibiting mTOR\u0026nbsp;[16]. More importantly, IRGM targets NLRP3 and PYCARD/ASC for autophagic degradation, limiting the maturation and release of IL-1β and thus maintaining immune homeostasis\u0026nbsp;[17]. Our group has shown that Irgm1 is neuroprotective in the acute phase of ischemic stroke and traumatic brain injury by increasing neuronal autophagy\u0026nbsp;[25, 26]. In CCCP-treated SH-SY5Y cells, IRGM enhances PINK1/Parkin-mediated mitophagy by facilitating the autophagic degradation of mitofilin, which stabilizes PINK1 and recruits Parkin, thereby exerting a neuroprotective effect\u0026nbsp;[27]. However, how Irgm1 regulates microglial autophagy and whether it modulates the disease progression of PD remain unclear.\u003c/p\u003e\n\u003cp\u003eIn this study, we found that Irgm1 expression was elevated in an AAV-SNCA-PD mouse model and that Irgm1-knockout mice exhibited more severe neurodegeneration. After confirming that Irgm1 colocalized with microglia, we treated BV2 cells with PFFs to model microglial clearance of α-syn pathology. Western blotting revealed the following ALP impairments: decreased levels of LC3-II and Ctsd, and elevated levels of p62, which is consistent with the experimental results of Choi I\u0026nbsp;[19].\u0026nbsp;Moreover, Irgm1 knockdown not only aggravated ALP dysfunction but also reduced the colocalization of α-syn with lysosomes, accompanied by a decrease in lysosomal activity.\u003c/p\u003e\n\u003cp\u003eMechanistically, we focused on TFEB, the master regulator of the ALP. Irgm1 promotes TFEB dephosphorylation and nuclear translocation via mTOR inhibition, thereby driving lysosomal biogenesis and autophagy-related gene transcription; this Irgm1-mTOR-TFEB axis has been shown to be involved in infection and atherosclerosis\u0026nbsp;[28, 29]. Here, Irgm1 knockdown markedly suppressed TFEB nuclear translocation after PFFs exposure in BV2 cells. In a coculture system, PFF-treated Irgm1-deficient microglia inefficiently degraded α-syn, leading to elevated Bax/Bcl-2 ratio, increased cleaved caspase-3 intensity, and enhanced apoptosis in SH-SY5Y neurons. Treating BV2 cells with rapamycin restored TFEB nuclear translocation, enhanced α-syn degradation and lysosomal function, and alleviated the neurotoxic phenotype of Irgm1-deficient microglia.\u003c/p\u003e\n\u003cp\u003eHowever, this study has the following limitations. First, our in vivo experiments employed whole-body Irgm1-knockout mice instead of microglia-specific mutants, so we cannot exclude the possibility that Irgm1 expression changes in other brain cell types may influence PD progression.\u0026nbsp;Future\u0026nbsp;studies employing microglia-specific Irgm1 knockout mice will be crucial to confirm the cell-autonomous role of microglial Irgm1 in PD. Second, our in vitro work was performed in Irgm1-knockdown BV2 cells, so Irgm1-overexpressing models and additional interventions targeting the TFEB-ALP axis remain to be tested. Moreover, investigating whether Irgm1 modulates microglial inflammatory responses, phagocytic capacity and energy metabolism beyond its ALP-related role is worthwhile as such insights could open new therapeutic avenues for PD.\u003c/p\u003e\n\u003cp\u003eIn conclusion, we identified the “Irgm1-TFEB-ALP” axis as a critical mechanism by which microglia regulate α-syn clearance. Irgm1 deficiency impaired TFEB nuclear translocation, exacerbated α-syn-induced ALP dysfunction in microglia, intensified dopaminergic neuronal injury and accelerated PD progression. Our study delineates a previously unrecognized Irgm1-TFEB-ALP axis specifically in microglia, which is crucial for α-syn clearance and neuroprotection in PD, extending its known functions beyond inflammasome regulation and neuronal autophagy.\u003c/p\u003e"},{"header":"Statements \u0026 Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was funded by the National Natural Science Foundation of China (U23A20428, 32370962), the Joint Funds for the Innovation of Science and Technology of Fujian Province (2024Y9178), the National Natural Science Foundation of China (31870894) and the Natural Science Foundation of Heilongjiang Province of China (LH2022H005).\u003c/p\u003e\n\u003cp\u003eCompeting Interests\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003eAuthor Contributions\u003c/p\u003e\n\u003cp\u003eS.W. and C.P. designed the study, drafted the manuscript, and provided overall supervision for the study. S.W., X.L., J.Y., J.C., and C.X. conducted the animal-related experiments. S.W. and X.L. performed the cell-related experiments. S.W. and C.P. revised the manuscript. B.L., H.X., and R.L. participated in the discussion and review of the experimental results. H.X.,\u0026nbsp;R.L. and C.P. supported the project.\u003c/p\u003e\n\u003cp\u003eData Availability\u003c/p\u003e\n\u003cp\u003eThe datasets generated, used, and analyzed during the current study, as well as the materials used in this manuscript, are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003eEthics approval\u003c/p\u003e\n\u003cp\u003eThis study was performed under a protocol approved by the Ethics Committee of Harbin Medical University\u0026nbsp;(approval number: [HMUIRB2026008]).\u003c/p\u003e\n\u003cp\u003eConsent to Participate\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003eConsent to publication\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003eAcknowledgements\u003c/p\u003e\n\u003cp\u003eThe authors would like to thank the research group of Professor Zhentao Zhang for kindly providing α-synuclein preformed fibrils (α-syn PFFs).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eCorti, O., Lesage, S. \u0026amp; Brice, A. What genetics tells us about the causes and mechanisms of Parkinson's disease. \u003cem\u003ePhysiol. Rev.\u003c/em\u003e \u003cb\u003e91\u003c/b\u003e (4), 1161\u0026ndash;1218 (2011).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMason, A. R., Ziemann, A. \u0026amp; Finkbeiner, S. Targeting the low-hanging fruit of neurodegeneration. \u003cem\u003eNeurology\u003c/em\u003e \u003cb\u003e83\u003c/b\u003e (16), 1470\u0026ndash;1473 (2014).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGoedert, M., Spillantini, M. G., Del Tredici, K. \u0026amp; Braak, H. 100 years of Lewy pathology, Nature reviews. \u003cem\u003eNeurology\u003c/em\u003e \u003cb\u003e9\u003c/b\u003e (1), 13\u0026ndash;24 (2013).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSchapira, A. H. V., Chaudhuri, K. R. \u0026amp; Jenner, P. Non-motor features of Parkinson disease, Nature reviews. \u003cem\u003eNeuroscience\u003c/em\u003e \u003cb\u003e18\u003c/b\u003e (7), 435\u0026ndash;450 (2017).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSpillantini, M. G. et al. Alpha-synuclein in Lewy bodies. \u003cem\u003eNature\u003c/em\u003e \u003cb\u003e388\u003c/b\u003e (6645), 839\u0026ndash;840 (1997).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBraak, H. et al. Staging of brain pathology related to sporadic Parkinson's disease. \u003cem\u003eNeurobiol. Aging\u003c/em\u003e. \u003cb\u003e24\u003c/b\u003e (2), 197\u0026ndash;211 (2003).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBalestrino, R. \u0026amp; Schapira, A. H. V. Parkinson disease. \u003cem\u003eEur. J. Neurol.\u003c/em\u003e \u003cb\u003e27\u003c/b\u003e (1), 27\u0026ndash;42 (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBr\u0026uuml;ck, D., Wenning, G. K., Stefanova, N. \u0026amp; Fellner, L. Glia and alpha-synuclein in neurodegeneration: A complex interaction. \u003cem\u003eNeurobiol. Dis.\u003c/em\u003e \u003cb\u003e85\u003c/b\u003e, 262\u0026ndash;274 (2016).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGuo, M. et al. Microglial exosomes facilitate α-synuclein transmission in Parkinson's disease. \u003cem\u003eBrain: J. Neurol.\u003c/em\u003e \u003cb\u003e143\u003c/b\u003e (5), 1476\u0026ndash;1497 (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLuk, K. C. et al. Intracerebral inoculation of pathological α-synuclein initiates a rapidly progressive neurodegenerative α-synucleinopathy in mice. \u003cem\u003eJ. Exp. Med.\u003c/em\u003e \u003cb\u003e209\u003c/b\u003e (5), 975\u0026ndash;986 (2012).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePeng, C., Trojanowski, J. Q. \u0026amp; Lee, V. M. Protein transmission in neurodegenerative disease. \u003cem\u003eNat. reviews Neurol.\u003c/em\u003e \u003cb\u003e16\u003c/b\u003e (4), 199\u0026ndash;212 (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMizushima, N. \u0026amp; Komatsu, M. Autophagy: renovation of cells and tissues. \u003cem\u003eCell\u003c/em\u003e \u003cb\u003e147\u003c/b\u003e (4), 728\u0026ndash;741 (2011).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEbrahimi-Fakhari, D. et al. Distinct roles in vivo for the ubiquitin-proteasome system and the autophagy-lysosomal pathway in the degradation of α-synuclein. \u003cem\u003eJ. neuroscience: official J. Soc. Neurosci.\u003c/em\u003e \u003cb\u003e31\u003c/b\u003e (41), 14508\u0026ndash;14520 (2011).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSardiello, M. et al. A gene network regulating lysosomal biogenesis and function, Science (New York, N.Y.) 325(5939) 473-7. (2009).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSettembre, C. et al. TFEB links autophagy to lysosomal biogenesis, Science (New York, N.Y.) 332(6036) 1429-33. (2011).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChauhan, S., Mandell, M. A. \u0026amp; Deretic, V. IRGM governs the core autophagy machinery to conduct antimicrobial defense. \u003cem\u003eMol. Cell\u003c/em\u003e. \u003cb\u003e58\u003c/b\u003e (3), 507\u0026ndash;521 (2015).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMehto, S. et al. The Crohn's Disease Risk Factor IRGM Limits NLRP3 Inflammasome Activation by Impeding Its Assembly and by Mediating Its Selective Autophagy. \u003cem\u003eMol. Cell\u003c/em\u003e. \u003cb\u003e73\u003c/b\u003e (3), 429\u0026ndash;445e7 (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCheng, J. et al. Microglial autophagy defect causes parkinson disease-like symptoms by accelerating inflammasome activation in mice. \u003cem\u003eAutophagy\u003c/em\u003e \u003cb\u003e16\u003c/b\u003e (12), 2193\u0026ndash;2205 (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChoi, I. et al. Microglia clear neuron-released α-synuclein via selective autophagy and prevent neurodegeneration. \u003cem\u003eNat. Commun.\u003c/em\u003e \u003cb\u003e11\u003c/b\u003e (1), 1386 (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJeon, Y. M. et al. The Role of Glial Mitochondria in α-Synuclein Toxicity. \u003cem\u003eFront. cell. Dev. biology\u003c/em\u003e. \u003cb\u003e8\u003c/b\u003e, 548283 (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKim, H. J. et al. Deficient autophagy in microglia impairs synaptic pruning and causes social behavioral defects. \u003cem\u003eMol. Psychiatry\u003c/em\u003e. \u003cb\u003e22\u003c/b\u003e (11), 1576\u0026ndash;1584 (2017).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLarabi, A., Barnich, N. \u0026amp; Nguyen, H. T. T. New insights into the interplay between autophagy, gut microbiota and inflammatory responses in IBD. \u003cem\u003eAutophagy\u003c/em\u003e \u003cb\u003e16\u003c/b\u003e (1), 38\u0026ndash;51 (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGuan, Y. \u0026amp; Han, F. Key Mechanisms and Potential Targets of the NLRP3 Inflammasome in Neurodegenerative Diseases. \u003cem\u003eFront. Integr. Nuerosci.\u003c/em\u003e \u003cb\u003e14\u003c/b\u003e, 37 (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHolbrook, J. A. et al. Neurodegenerative Disease and the NLRP3 Inflammasome. \u003cem\u003eFront. Pharmacol.\u003c/em\u003e \u003cb\u003e12\u003c/b\u003e, 643254 (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHe, S. et al. Immune-related GTPase M (IRGM1) regulates neuronal autophagy in a mouse model of stroke. \u003cem\u003eAutophagy\u003c/em\u003e \u003cb\u003e8\u003c/b\u003e (11), 1621\u0026ndash;1627 (2012).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePei, C. et al. Neuron-specific deficiency of autophagy increases neuronal loss in traumatic brain injury. \u003cem\u003ePNAS nexus\u003c/em\u003e. \u003cb\u003e3\u003c/b\u003e (10), pgae457 (2024).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGuo, X. et al. IRGM promotes the PINK1-mediated mitophagy through the degradation of Mitofilin in SH-SY5Y cells, FASEB journal: official publication of the Federation of American Societies for. \u003cem\u003eExperimental Biology\u003c/em\u003e. \u003cb\u003e34\u003c/b\u003e (11), 14768\u0026ndash;14779 (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCai, H. et al. A potential early-atheroprotective target: Irgm1 mediates lymphangiogenesis through LEC autophagy by Tfeb translocation, Biochimica et biophysica acta. \u003cem\u003eMol. basis disease\u003c/em\u003e. \u003cb\u003e1870\u003c/b\u003e (6), 167238 (2024).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKumar, S. et al. Mammalian Atg8 proteins and the autophagy factor IRGM control mTOR and TFEB at a regulatory node critical for responses to pathogens. \u003cem\u003eNat. Cell Biol.\u003c/em\u003e \u003cb\u003e22\u003c/b\u003e (8), 973\u0026ndash;985 (2020).\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, Irgm1, Autophagy-lysosomal pathway, TFEB","lastPublishedDoi":"10.21203/rs.3.rs-9004768/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9004768/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eParkinson\u0026rsquo;s disease (PD) is a common neurodegenerative disorder characterized by the progressive loss of nigral dopaminergic neurons and abnormal α-synuclein (α-syn) aggregation. Growing evidence indicates that impaired autophagy\u0026ndash;lysosome pathway (ALP) activity in microglia exacerbates the pathological process; however, the precise regulatory mechanisms involved remain elusive. In this study, we found that Irgm1, a key autophagy regulator, was markedly upregulated in the midbrains of PD mice. Irgm1-deficient mice exhibited accelerated PD progression, more severe motor deficits, greater TH⁺ neuronal loss, increased α-syn deposition and aggravated ALP damage, indicating a neuroprotective role for Irgm1. Moreover, Irgm1 was selectively enriched in the microglia of PD mice, and Irgm1 knockdown amplifiedα-syn-preformed fibril (PFF)-induced ALP impairment: the LC3-II and cathepsin D levels decreased, p62 and α-syn aggregates accumulated, the colocalization of α-syn with Lamp1 declined, lysosomal acidification decreased, and TFEB nuclear translocation was blocked. The TFEB-activating autophagy agonist rapamycin restored TFEB nuclear translocation, reactivated the ALP, accelerated α-syn clearance, and abolished the increased toxicity of Irgm1-deficient microglia toward cocultured SH-SY5Y neurons after PFFs preexposure. Thus, Irgm1 promotes microglial α-syn clearance via the TFEB-ALP axis, and targeting this pathway could be a potential therapeutic strategy for PD.\u003c/p\u003e","manuscriptTitle":"Irgm1 Promotes Microglial Clearance of α-Synuclein via the TFEB-Dependent Autophagy- Lysosome Pathway in Parkinson’s Disease","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-11 07:35:54","doi":"10.21203/rs.3.rs-9004768/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":"e8708a80-3cf4-43b0-ad42-0ec8f990f5b3","owner":[],"postedDate":"March 11th, 2026","published":true,"recentEditorialEvents":[{"type":"decision","content":"Withdrawn","date":"2026-05-13T09:07:08+00:00","index":"","fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":64245570,"name":"Biological sciences/Cell biology"},{"id":64245571,"name":"Biological sciences/Drug discovery"},{"id":64245572,"name":"Health sciences/Neurology"},{"id":64245573,"name":"Biological sciences/Neuroscience"}],"tags":[],"updatedAt":"2026-05-13T09:25:30+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-11 07:35:54","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9004768","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9004768","identity":"rs-9004768","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}