PrPC-Mediated Ca²⁺/Calcineurin/TFEB Signaling Enhances Autophagic-Lysosomal Function and Anti-inflammatory Astrocyte Transition to Alleviate Cerebral Ischemia-Reperfusion Injury

PrPC-Mediated Ca²⁺/Calcineurin/TFEB Signaling Enhances Autophagic-Lysosomal Function and Anti-inflammatory Astrocyte Transition to Alleviate Cerebral Ischemia-Reperfusion Injury | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article PrP C -Mediated Ca² ⁺ /Calcineurin/TFEB Signaling Enhances Autophagic-Lysosomal Function and Anti-inflammatory Astrocyte Transition to Alleviate Cerebral Ischemia-Reperfusion Injury Jie Shao, Xiang Yin, Yue Lang, Jie Yang, Menghan Jia, Tengfei Su, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7212124/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 An increasing number of studies have focused on understanding the role of macroautophagy/autophagy and the autophagy-lysosomal pathway (ALP) in cerebral ischemic injury. Transcription factor EB (TFEB) is a central regulator of genes involved in autophagy and plays a pivotal role in the regulation of the ALP; however, the mechanisms controlling TFEB activity remain incompletely understood. In this study, we investigated the role of cellular prion protein (PrPC)-targeted TFEB in mediating ALP dysfunction and inflammatory phenotypic changes in mouse cortical astrocytes after cerebral ischemia-reperfusion injury (CIRI). Our findings indicate that during the ultra-early phase of CIRI, intracellular Ca2+ levels are low, with inhibited PPP3/calcineurin activity and reactivation of mTOR, leading to TFEB phosphorylation and retention in the cytoplasm. As reperfusion time increases, elevated intracellular Ca2+ levels activate PPP3/calcineurin, resulting in TFEB dephosphorylation, nuclear translocation, and the subsequent induction of autophagy lysosome-associated gene transcription. This process promotes astrocyte survival and shifts the cellular phenotype toward an anti-inflammatory state. Furthermore, increased PrPC expression was observed to maintain intracellular Ca2+ homeostasis and sustain PPP3/calcineurin activation, facilitating continuous TFEB dephosphorylation and nuclear translocation. These results clarify the regulatory role of PrPC in astrocytic autophagy-lysosomal pathways following cerebral ischemia/reperfusion injury, providing new insights for more targeted interventions in ischemic stroke. Transcription factor EB Cellular prion protein Autophagy-lysosomal pathway calcineurin astrocyte Cerebral ischemia reperfusion injury Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 Figure 13 Figure 14 Figure 15 Introduction Stroke remains a leading cause of death and disability worldwide. Currently, recanalization strategies are the most effective treatment for ischemic stroke; however, reperfusion often exacerbates secondary injury. Numerous studies indicate that the astrocyte-mediated immune-inflammatory response plays a significant role in the pathophysiology of cerebral ischemia-reperfusion injury (CIRI) [1-2]. Autophagy, an essential intracellular catabolic pathway, helps maintain cellular homeostasis by degrading damaged proteins and organelles through lysosomal pathways [3-4] . Increasing evidence suggests that CIRI induces autophagy activation, and impaired autophagic function is implicated in its pathogenesis. Nonetheless, the outcomes of modulating autophagy in this context have been inconsistent. Some studies suggest that autophagy-lysosomal pathway (ALP) activation exacerbates CIRI. For instance, research involving a transient middle cerebral artery occlusion (tMCAO) model in mice demonstrated that administering autophagy inhibitors such as 3-Methyladenine (3-MA) or Bafilomycin A1 reduced neuronal damage in ischemic regions and improved neurological outcomes [5]. Similarly, an in vitro oxygen-glucose deprivation and re-oxygenation (OGD/R) study confirmed that 3-MA reduced astrocyte apoptosis by inhibiting ALP activity [6]. However, other studies have reported contrasting findings. Research by Gabryel [7] and Zhao [8] observed in OGD/R models that autophagy inhibitors like 3-MA or chloroquine (CQ) significantly decreased astrocyte viability. These conflicting data suggest that ALP modulation in CIRI may involve a dynamic process. Additionally, the timing of autophagy modulation is crucial. Carloni's study found that rapamycin-induced autophagy during ischemia and hypoxia mitigated neuronal damage, while 3-MA-induced autophagy inhibition produced the opposite effect [9]. Conversely, in another mouse tMCAO study, 3-MA administered 48 to 72 hours post-reperfusion significantly reduced infarct size and improved neurological function [10]. These findings indicate that moderate autophagy activation during ischemia and hypoxia supports cellular homeostasis, while excessive autophagy during reperfusion may overwhelm cellular adaptive mechanisms and lead to cell death [11]. Thus, a comprehensive understanding of ALP dynamics in astrocytes post-CIRI may serve as a foundation for its precise therapeutic modulation. Transcription factor EB (TFEB) has recently been recognized as a key regulator of the autophagy-lysosomal pathway (ALP), orchestrating the expression of autophagy and lysosomal genes essential for ALP regulation [12]. Dysregulation of TFEB has been linked to various pathological conditions. For instance, Zhang’s study on a manganese-induced Parkinson’s model revealed that manganese exposure significantly inhibited TFEB nuclear translocation in astrocytes within the mouse striatum, disrupting ALP function. In this model, TFEB overexpression alleviated manganese-induced mitochondrial dysfunction in astrocytes [13]. Similarly, Gu's study on cardiomyocyte ischemia-reperfusion injury showed that downregulation of LAPTM4B via the mTORC1/TFEB pathway led to ALP dysfunction, resulting in cardiomyocyte death. Conversely, TFEB upregulation reversed ischemia-reperfusion injury in cardiomyocytes following LAPTM4 knockdown [14]. Currently, most studies investigating TFEB in the context of CIRI have focused on its neuronal roles. Enhancing TFEB nuclear translocation in neurons has been shown to exert neuroprotective effects by restoring ALP function, thereby mitigating ischemic injury [15]. However, the specific changes in TFEB within astrocytes after CIRI, its upstream regulatory mechanisms, and its impact on ALP function in these cells remain unknown. Cellular prion protein (PrP C ) is a cell surface glycoprotein encoded by the Prnp gene, widely distributed throughout the central nervous system. When PrP C undergoes a conformational change, it is converted to the pathogenic and infectious scrapie isoform (PrP Sc) , which is implicated in prion diseases such as bovine spongiform encephalopathy in animals and Creutzfeldt-Jakob disease in humans [16]. PrP C exerts a notable neuroprotective effect in CIRI. Overexpression of PrP C in microglia promotes an anti-inflammatory phenotype, likely due to its ability to delay lysosomal depletion and sustain ALP functionality, although the precise molecular mechanisms remain unclear [17-18]. To investigate this, we assessed changes in ALP patterns following CIRI, with a focus on whether TFEB can modulate ALP function and thereby protect astrocytes from ischemic injury. Our findings provide the first evidence that CIRI induces dynamic alterations in ALP. In the early phase of CIRI, ALP partially blockade occurs alongside decreased TFEB expression and nuclear translocation, driven by mTOR reactivation and inhibition of PPP3/calcineurin activity. With extended reperfusion, PPP3/calcineurin activation restores TFEB nuclear translocation, alleviating ALP impairment, which ultimately supports astrocyte survival and promotes a shift to an anti-inflammatory phenotype. Notably, overexpression of PrP C maintains PPP3/calcineurin activation by preserving intracellular Ca 2+ homeostasis, which in turn sustains TFEB dephosphorylation and nuclear translocation. This study offers new insights for potential therapeutic interventions in ischemic stroke. Materials and Methods Animals One-day-old neonatal FVB/N wild-type (WT; obtained from the Vital River Laboratory Animal Technology Co., Ltd., Beijing, China), Prnp -/- , and Prnp -overexpressing mice (obtained from the Institute of Medical Laboratory Animals, Chinese Academy of Medical Sciences) were used in this study. All experimental protocols were approved by the Institutional Animal Care and Use Committee at the First Hospital of Jilin University. Mouse transient Middle Cerebral Artery Occlusion (tMCAO) Model Mice underwent isoflurane anesthesia. Following a ventral midline neck incision, the right common (CCA), external (ECA), and internal carotid (ICA) arteries were exposed. The proximal CCA and distal ECA were ligated. A paraffin-coated 4-0 nylon suture (diameter 0.26 mm) was advanced from the CCA into the ICA and positioned to occlude the origin of the right middle cerebral artery at the Circle of Willis. Sham-operated animals received identical procedures excluding the suture occlusion. Animals were euthanized at designated time points for subsequent analysis. Primary cortical astrocyte culture and PrP C protein assay Primary cortical astrocytes were cultured from one-day-old FVB/N mice as previously described [19]. Briefly, cortices from neonatal mouse brains were aseptically dissected, meninges were removed, and single-cell suspensions were prepared by gentle trituration. Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, USA, 8123234) supplemented with 10% fetal bovine serum (FBS; Gibco, USA, 10010023) and 1% penicillin/streptomycin (ThermoFisher, USA, 15240062). Mixed glial cultures were maintained in vitro until confluent, and the uppermost layer of microglia was removed by shaking on an orbital shaker at 200 rpm for 4 hours. The middle layer of astrocytes was isolated by incubation in 0.25% trypsin-EDTA (diluted 1:2 in DMEM; Gibco, USA, 25200072) for 15–25 minutes, leaving the lower layer of microglia adhered to the culture flask. The mid-layer cells were then collected and incubated until confluent, with the process repeated to obtain purified astrocytes. Astrocyte purity was verified by immunofluorescence staining with rat anti-glial fibrillary acidic protein (GFAP, an astrocyte-specific marker; 1:200, Abcam, China, ab27929) ( Figure S1A). Flow cytometry was also performed using rat anti-CD11b-FITC (a microglia-specific marker; ThermoFisher, USA, 11-0112-82) to exclude microglial contamination ( Figure S1B) . PrP C protein levels in WT, Prnp - /- , and Prnp -overexpressing astrocytes were assessed by immunofluorescence staining with rabbit anti-PrP antibody (1:600, Invitrogen, USA, MA5-32202) ( Figure S5) . OGD/R in primary cortical astrocytes For oxygen-glucose deprivation (OGD), astrocytes were washed three times with phosphate-buffered saline (PBS; Gibco, 10010023) and then incubated in glucose-free DMEM (Gibco, 11966-025). Cells were cultured in MIC-101 hypoxic chambers maintained at 37°C, with a gas mixture of 0.1% O₂, 94.9% N₂, and 5% CO₂ for 2 hours. Following OGD, glucose-containing medium was reintroduced, and cells were transferred to a normoxic incubator with 5% CO₂ and 95% air to initiate oxygen-glucose resupply (OGR). Astrocyte samples were collected at 3, 6, 12, 24, 48, and 72 hours post-OGD/R to analyze the dynamic response. At the onset of OGD/R, astrocytes were treated with various drugs, including the lysosome inhibitor chloroquine (CQ, 50 µM; Selleck, S6999), mechanistic target of rapamycin kinase inhibitor rapamycin (mTOR, 200 nM; Selleck, S1039), mTOR agonist MHY1485 (500 µM; Selleck, S7811), and calcineurin inhibitor cyclosporin A (CsA, 10 µM; Selleck, S2286). The maximum concentration of DMSO used in the experiments (0.1%) was confirmed to be non-toxic to the cells. Astrocyte Activity assay Astrocyte activity was assessed using the Cell Counting Kit-8 (CCK-8; Beyotime Biotech, C0041) according to the manufacturer’s instructions. Briefly, purified astrocytes were seeded into a 96-well plate, and 10 µL of CCK-8 solution was added 1 hour before the end of OGD/R. Absorbance was then measured at an excitation wavelength of 450 nm using a microplate reader (Thermo Fisher Scientific, USA). Immunoblotting Astrocytes were lysed using Cell lysis buffer for Western and IP (Beyotime Biotech, P0013J) supplemented with a protease and phosphatase inhibitor mixture (Solarbio, P1260). The cell lysates were then centrifuged at 12,000 × g for 15 minutes at 4°C. The resulting pellets (Triton X-100-insoluble fractions) were washed three times with lysis buffer and resuspended in SDS lysis buffer (Beyotime Biotech, P0013G), followed by another round of centrifugation at 12,000 × g for 15 minutes. Total protein concentration was measured using a BCA protein assay kit (Epizyme, China, 23225). Protein aliquots (10 µg) from each fraction were separated by SDS-PAGE (4-20%, GenScript, USA, M00657), and the gel-separated proteins were transferred onto 0.22 µm polyvinylidene fluoride membranes, which were blocked with 5% skimmed milk in TBST (0.1% Tween 20 in Tris-buffered saline). After blocking, the membranes were incubated overnight at 4°C with primary antibodies: rabbit anti- LC3B (1:1000, Abcam, USA, ab192890), SQSTM1 (, 1:1000, Abcam, USA, ab91526), CTSD (1:1000, Abcam, USA, ab65302), CTSB (1:2000, Abcam, USA, ab214428), ubiquitin (1:1000, ProteinTech, USA, 10201-2-AP), β-actin (1:5000, ProteinTech, USA, 81115-1-RR), TFEB (1:1000, ProteinTech, USA, 13372-1AP), PPP3 (1:1000, Cell Signaling Technology, USA, 2614), mTOR (1:1000, Cell Signaling Technology, USA, 2983), phospho-mTOR (1:1000, Cell Signaling Technology, USA, 2974), and rat anti- LAMP1 (1:2000, eBioscience, USA, 14-1071-82). After three washes, membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (1:10,000, Cohesion Biosciences, CSA2115 and CSA2133) for 2 hours at room temperature. Protein bands were visualized using Omni-ECL™ Chemiluminescent Substrate (Epizyme, China, SQ201) and a ChemiDoc MP imaging system (Bio-Rad Laboratories, CA, USA). The intensity of the protein bands was quantified using ImageJ software, with the intensity expressed as the relative value compared to the control. Immunofluorescence staining Astrocytes in vitro : Cells were washed twice with ice-cold PBS, followed by fixation and permeabilization with 4% (w:v) paraformaldehyde supplemented with 0.2% (v:v) Triton X-100 for 30 minutes at room temperature. After washing three times with PBS, the cells were blocked with 10% goat serum in PBS for 2 hours at room temperature. The cells were then incubated overnight at 4°C with primary antibodies: rabbit anti-LC3B (1:200, Abcam, USA, ab192890), CTSD (1:200, Abcam, USA, ab65302), TFEB (1:200, eBioscience, USA, 14-1071-82), and rat anti-LAMP1 (1:200, eBioscience, USA, 14-1071-82). Following primary antibody incubation, cells were washed three times with PBS and incubated with Alexa Fluor 488- and Alexa Fluor 594-conjugated secondary antibodies (1:200, Abcam, ab150165 and ab150080) for 2 hours at room temperature, in the dark, in PBS containing 2% goat serum. The cells were then washed three times and counterstained with DAPI (Solarbio, 0065) for nuclear labeling. After final washes with PBS, cells on glass coverslips were mounted onto glass slides using Anti-fluorescent Attenuation Sealer (Solarbio, 188105). Images were captured using a laser confocal microscope and analyzed using ImageJ software. Mouse brain tissue sections : Following deparaffinization and antigen retrieval, paraffin sections were subjected to serum blocking and then processed through three sequential cycles of immunostaining. Each cycle consisted of: overnight incubation (4°C) with a primary antibody, application of a species-matched HRP-conjugated secondary antibody, tyramide signal amplification (TSA) with the corresponding fluorophore, and thorough antibody stripping followed by serum reblocking prior to the subsequent cycle. After completing the third cycle, nuclei were counterstained with DAPI, and sections were mounted for imaging using laser scanning confocal microscopy. Cathepsin D activity assay Astrocyte lysates (n = 3 per group) from normal and OGD/R conditions were collected at the indicated time points using CTSD lysis buffer. The samples were centrifuged at 12,000 × g for 10 minutes at 4°C, and the supernatants were collected. Protein concentration was quantified using a BCA protein assay kit (Epizyme, ZJ102). CTSD activity was measured using a fluorometric CTSD activity assay kit (Abcam, ab65302), following the manufacturer's instructions. Cellular Bead Array Cytokine levels in astrocyte culture media (n = 3 per group) were measured using the mouse Th1/Th2/Th17 cytokine kit (BD Biosciences, Franklin Lakes, NJ, USA, 560485), following the manufacturer's instructions. Interleukin (IL)-6, IL-10, tumor necrosis factor-α (TNF-α), and interferon-gamma (IFN-γ) were selected as representative cytokines for assessing pro-inflammatory and anti-inflammatory states in astrocytes. Protein phosphatase assay Astrocyte lysates (n = 3 per group) from normal and OGD/R conditions were collected at the specified time points using lysis buffer. The samples were centrifuged at 100,000–200,000 × g for 45 minutes at 4°C, and the supernatants were collected. Protein concentrations were determined using the Bradford method. PPP3/calcineurin activity was assessed using a calcineurin phosphatase assay kit (ZNEO, BML-AK816-0001), following the manufacturer's instructions. Calcium imaging Astrocyte samples from normal and OGD/R groups were washed three times with calcium-free Hank's balanced salt solution (HBSS, Solarbio, H1040) to remove residual serum. The cells were then incubated with 5 µM Fluo-4 AM (Beyotime, S1060) in calcium-free HBSS for 30 minutes at 37°C. Afterward, the cells were washed three times with calcium-free HBSS and further incubated in the same solution for an additional 30 minutes to complete deesterification. Fluorescence measurements were taken at an excitation wavelength of 494 nm and emission wavelength of 516 nm using a multifunctional enzyme reader to determine the intracellular calcium ion concentration. Small interfering RNA (siRNA) is used to silence Tfeb expression in astrocytes We used siRNA to silence Tfeb in primary mouse astrocytes, with siRNA purchased from Integrated Biotech Solutions. The si- Tfeb primer sequences were as follows: Sense: 5'-GACGCAGGUUUCAACAUCAAUG-3' Antisense: 5'-UUGAUGUUGAACCUGCGUCUUU-3' The si-Control primer sequences were: Sense: 5'-UUCUCCGAACGUGUCACGUTT-3' Antisense: 5'-ACGUGACACGUUCGGAGAATT-3' The siRNA was transfected into primary astrocytes using the JiePRIME Transfection Kit (Polyplus, France, 101000046) according to the manufacturer’s instructions. Transfection efficiency was determined by immunofluorescence staining, and silencing efficiency of si-T feb was verified by both immunofluorescence staining and immunoblotting ( Figure S3 ). Plasmids is used to induce the overexpression of Tfeb in astrocytes We used plasmids to induce Tfeb overexpression in astrocytes, which were purchased from Integrated Biotech Solutions. The plasmids used were: Target plasmid: pcDNA3.1(+)-mouse Tfeb-3×FLAG Amp+ (1 µg/µL). Control plasmid: pcDNA3.1(+) Amp+ (1 µg/µL). Fluorescent control plasmid: Pegfp-N1 Amp+ (1 µg/µL). The plasmids were amplified and transfected into primary astrocytes using the Plasmid Extraction Kit (Tengen Biochemistry, Germany, DP120) and the Lipofectamine™3000 Transfection Kit (Thermo Fisher Scientific, USA, L3000001), according to the manufacturer's instructions. Transfection efficiency was assessed by immunofluorescence staining, and the efficiency of Tfeb overexpression in astrocytes was confirmed through immunoblotting ( Figure S4) . Statistical analysis All experimental data were derived from at least three independent replicate experiments. Data processing, analysis, and plotting were conducted using GraphPad Prism 8 and Excel software. Normally distributed data are presented as means ± SEM. Statistical analyses included one-way or two-way ANOVA followed by Tukey’s or Dunnett’s post hoc tests for multiple comparisons, and Student’s t-test for two-group comparisons. Statistical significance was set at P < 0.05. Results Transient cerebral ischemia results in a dynamic change of ALP The tMCAO mice and OGD/R astrocytes served as in vivo and in vitro models of cerebral ischemia-reperfusion, respectively. Elevated neurological deficit scores in tMCAO mice, alongside reduced astrocyte viability post-OGD/R (specifically: significant decline at 3-6 h, recovery at 12 h, and return to baseline at 48-72 h), confirmed the successful establishment of their respective models (Figure S2) . However, the extent to which these changes reflect alterations in ALP processing remains controversial and warrants further investigation. ALP is a complex process with three primary stages—autophagosome formation, autophagosome-lysosome fusion, and lysosomal degradation. Dysfunction in any of these stages can impair ALP [20-22]. Thus, we monitored dynamic changes in ALP function through markers of autophagic and lysosomal activity. Immunoblotting was performed to analyze the expression of autophagy-related proteins in mouse cortical astrocytes from 3 to 72 hours post-OGD/R. Results showed a significant increase in the autophagic marker MAP1LC3B-II/I from 3 to 6 hours ( Figure 1A1, A2) , as well as elevated levels of the autophagic substrate SQSTM1 in both Triton X-100-soluble and -insoluble fractions from 3 to 12 hours post-OGD/R (Figure 1A1, A3-A4). In contrast, no significant changes were observed in MAP1LC3-II/I or SQSTM1 levels between 48 and 72 hours post-OGD/R. However, levels of another autophagic substrate, ubiquitin, decreased significantly during this later period ( Figure 1A1, A5 ). These observations suggest an abnormal accumulation of autophagosomes and substrates during early OGD/R, which diminishes in the later phase. Lysosomes are critical organelles for degrading autophagosome contents [23-24]. We assessed lysosomal quantity and function in astrocytes from 3 to 72 hours following CIRI, with LAMP1 used as a marker of lysosomal quantity, and CTSD and CTSB as markers of lysosomal function. Results indicated that LAMP1 expression remained unchanged from 3 to 24 hours post-OGD/R ( Figure 1A8 ). However, there was a significant decrease in pro-CTSD and mCTSB expression ( Figure 1A6-A7 ), CTSD enzymatic activity (Figure 1B ), and the percentage of functional lysosomes (LAMP1 + CTSD + ) (Figure 1C ), suggesting lysosomal dysfunction despite an unchanged lysosome count in early OGD/R. In contrast, from 48 to 72 hours post-OGD/R, LAMP1 expression increased significantly, with pro-CTSD and mCTSB expression, CTSD activity, and functional lysosome levels (LAMP1 + CTSD + ) returning to baseline, indicating both replenishment of lysosome quantity and restoration of function in late OGD/R. Similarly, in astrocytes of MCAO/R mice, functional lysosome levels (LAMP1 + CTSD + ) decreased at 6 h and recovered to baseline levels at 48 h ( Figure 1D ). These results are consistent with in vitro data. To further investigate the relationship between autophagosome and lysosome dynamics, we used MAP1LC3B-positive particles as markers for autophagosomes, LAMP1-positive particles for lysosomes, and co-localized MAP1LC3B and LAMP1 particles for autolysosomes. Immunofluorescence staining analysis revealed that autophagosome numbers significantly increased from 3 to 24 hours post-OGD/R, while lysosome numbers remained stable, suggesting that lysosomal insufficiency contributed to autophagosome accumulation. Conversely, from 48 to 72 hours post-OGD/R, autophagosome and lysosome numbers were balanced, both showing increases ( Figure 2A ). Similarly, in astrocytes of MCAO/R mice, autophagosome numbers were significantly elevated at 6 h post-reperfusion, whereas lysosome counts remained unaltered. In contrast, both autophagosomes and lysosomes increased significantly by 48 h ( Figure 2B ). These results are consistent with in vitro data. These findings indicate that in early CIRI, lysosomal insufficiency and dysfunction lead to autophagosome and substrate accumulation. In late CIRI, lysosomal upregulation and restored function alleviate this accumulation of autophagic substrates. To further elucidate changes in autophagic flux, we assessed MAP1LC3B levels in OGD/R-treated astrocytes during early and late OGD/R phases, with and without CQ treatment, which inhibits lysosomal proteases or blocks autophagosome-lysosome fusion ( Figure 2C ). Immunoblotting revealed that CQ treatment significantly increased MAP1LC3-II/I level at both 6 and 48 hours post-OGD/R, as well as under normal conditions, indicating that OGD/R did not cause a complete ALP blockade ( Figure 2C1-C2 ). Notably, the degree of CQ-induced MAP1LC3B-II/I elevation was significantly reduced at 6 hours and returned to baseline at 48 hours post-OGD/R compared to normal conditions ( Figure 2C3 ), suggesting that while ALP was partially impaired in early OGD/R, its function improved during late OGD/R. Transient cerebral ischemia induces the activation of TFEB To explore the association between dynamic changes in ALP and TFEB nuclear translocation, we examined TFEB levels in total fractions via immunoblotting and TFEB's intracellular distribution using immunofluorescence. Immunoblotting analysis showed that total TFEB expression significantly decreased between 3 and 6 hours, began to increase at 12 hours, peaked at 24 hours, and returned to baseline levels by 72 hours post-OGD ( Figure 3A ). Immunofluorescence analysis showed diffuse cytoplasmic TFEB staining under normal conditions, with perinuclear accumulation observed starting at 3 hours post-OGD/R, prOGD/Ressing towards the nucleus at 12 hours, and ceasing by 72 hours. This shift was supported by an increase in mean optical density and a notable rise in the Mander’s overlap coefficient, indicating enhanced nuclear localization ( Figure 3B ). Similarly, in mouse astrocytes, TFEB exhibited cytoplasmic staining in the sham group and the MCAO/R(6 h) group, while in the MCAO/R( 6 h) group, TFEB exhibited significant nuclear staining ( Figure 3C ). These results are consistent with in vitro data. These findings suggest that transient cerebral ischemia induces TFEB nuclear translocation in astrocytes. TFEB nuclear translocation is partially regulated by phosphorylation via the mTOR and Ca 2+ /PPP3 pathways. To investigate the impact of the Ca 2+ /PPP3 pathway on TFEB nuclear translocation in WT primary astrocytes from 3 to 72 hours following OGD/R, we measured intracellular Ca 2+ levels and PPP3 activity. Ca 2+ levels decreased at 6 hours, began to increase at 12 hours, peaked at 24 hours, and returned to baseline at 72 hours post-OGD/R ( Figure 4A) . Although PPP3 protein expression remained stable, PPP3 enzyme activity significantly increased between 12 and 48 hours post-OGD/R ( Figure 4B-C ). When CsA, a PPP3 inhibitor, was applied, it markedly inhibited TFEB nuclear translocation from 12 to 48 hours post-OGD/R, as shown by immunofluorescence ( Figure 4D) . These findings indicate that the Ca 2+ /PPP3 pathway is crucial in regulating TFEB nuclear translocation in astrocytes following OGD/R. We also examined the role of the mTOR pathway in TFEB nuclear translocation in WT primary astrocytes from 3 to 72 hours following OGD/R. The p-mTOR/mTOR ratio remained unchanged between 3 and 6 hours but significantly increased from 12 to 72 hours post-OGD/R( Figure 4E ). Although mTOR theoretically inhibits TFEB nuclear translocation, this observation contrasts with the actual nuclear translocation of TFEB seen from 12 to 48 hours post-OGD/R. To further clarify, we treated astrocytes with Rapamycin to inhibit p-mTOR activity and MHY1485 to activate it. Results showed that Rapamycin significantly promoted TFEB nuclear translocation from 3 to 6 hours, while MHY1485 markedly inhibited it from 12 to 72 hours post-OGD/R, compared to the untreated group ( Figure 4F ). These findings suggest that the mTOR pathway primarily acts to inhibit TFEB nuclear translocation during OGD/R, likely to prevent excessive autophagy in astrocytes. Astrocyte-targeted TFEB enhances ALP function, boosts cellular activity, and promotes a shift toward an anti-inflammatory phenotype following OGD/R Based on the observations above, we propose two hypotheses: 1) Is TFEB involved in regulating the ALP and influencing phenotypic transformation of astrocytes during late OGD/R? 2) Can upregulation of TFEB expression alleviate ALP dysfunction in the early phase of OGD/R? To investigate whether TFEB regulates the ALP and influences astrocyte phenotypic transformation during late OGD/R, we used siRNA to silence TFEB expression in WT mouse primary astrocytes. Immunoblotting results revealed that TFEB expression in the si- Tfeb group at 48 h following OGD/R was significantly reduced compared to the si-Con group. Concurrently, the expression of MAP1LC3B-II/I and ubiquitin was significantly increased, while the expression of LAMP1, CTSD, and mCTSB was significantly decreased ( Figure 5A ). CCK8 assays showed a significant reduction in astrocyte viability in the si- Tfeb group at 48 h following OGD/R compared to the si-Con group ( Figure 5B ). CBA analysis demonstrated that the levels of the anti-inflammatory cytokine IL-10 were reduced, while pro-inflammatory cytokines TNF-α and IL-6 were significantly elevated in the culture supernatants of the OGD/R 48 h si- Tfeb group compared to the si-Con group ( Figure 5C ). These results suggest that TFEB plays a role in promoting the conversion of astrocytes to an anti-inflammatory phenotype by enhancing ALP function during late OGD/R. To investigate whether upregulation of TFEB expression during early OGD/R could alleviate ALP blockade, we overexpressed TFEB in astrocytes using a plasmid. The results showed that the expression of MAP1LC3B-II/I, LAMP1, CTSD, and mCTSB did not significantly change in the OGD/R 6 h Tfeb overexpression group compared to the vector group ( Figure 6A ). These findings suggest that upregulation of TFEB expression does not improve ALP blockade in astrocytes during early OGD/R. Overexpression of PrP C preserves ALP functionality following CIRI, enhances astrocyte viability, and promotes astrocyte phenotypic switching from pro-inflammatory to anti-inflammatory states. Previous studies have shown that PrP C has neuroprotective effects in cerebral ischemic injury [25]. To investigate whether these protective effects are related to ALP, we examined PrP C expression from 3 h to 72 h following OGD/R in WT mouse primary astrocytes. Immunoblotting results revealed a significant increase in PrP C expression from 24 h to 48 h following OGD/R ( Figure 7A ), suggesting that PrP C may play a role in improving ALP function in astrocytes during late OGD/R. Next, we cultured WT, Prnp -/- ,and Prnp- overexpressing mouse astrocytes i n vitro ( Figure S5 ) and subjected them to OGD/R treatment. CCK-8 assays indicated that astrocyte activity in WT mice decreased between 3 and 6 hours post-OGD/R showed an increasing trend by 12 hours, and returned to baseline levels by 48 hours ( Figure S2 ). In Prnp - /- astrocytes, activity remained reduced from 3 h to 72 h following OGD/R. In contrast, Prnp -overexpressing astrocytes showed decreased activity from 3 h to 6 h, but increased activity from 48 h to 72 h following OGD/R, compared to normal conditions. Furthermore, Prnp - /- astrocytes exhibited significantly lower activity from 12 h to 72 h compared with WT astrocytes, while Prnp -overexpressing astrocytes showed improved activity both from 3 h to 6 h and 48 h to 72 h ( Figure 7B ). These results suggest that overexpression of PrP C ameliorates impaired astrocyte activity induced by OGD/R. CBA assays revealed that IL-10 levels in the supernatants of Prnp - /- astrocytes were significantly lower at 72 h following OGD/R compared to WT astrocytes, while TNF and IFN-γ levels were significantly higher from 24 h to 48 h. In contrast, IL-10 levels in the supernatants of Prnp -overexpressing astrocytes were significantly elevated from 3 h to 72 h, whereas TNF and IFN-γ levels were significantly reduced from 3 h to 72 h, with IL-6 levels significantly decreased from 6 h to 12 h ( Figure 7C ). These findings suggest that overexpression of PrP C facilitates the transformation of astrocytes from a pro-inflammatory to an anti-inflammatory phenotype following OGD/R. Subsequently, we examined the dynamics of ALP in cortical astrocytes from Prnp - /- and Prnp- overexpressing mice. As demonstrated in the previous section, ALP was partially blocked in WT mouse primary astrocytes during early CIRI and alleviated during late CIRI. In Prnp- overexpressing astrocytes, we observed the following: 1) The expression of the autophagy marker MAP1LC3-II/I decreased or showed a decreasing trend from 6 h to 72 h following OGD/R ( Figure 8A1-A2 ), which suggests two possibilities: either autophagosome formation is inhibited or autophagosomes are rapidly degraded. 2) The expression levels of LAMP1, CTSD, and mCTSB, as well as CTSD enzyme activity, increased or showed an upward trend from 3 h to 72 h following OGD/R ( Figure 8A1, A6-A8, B ), indicating that Prnp overexpression contributes to the replenishment of lysosomes and the improvement of lysosomal dysfunction at various stages of OGD/R. 3) The proportion of functional lysosomes (LAMP + CTSD + ) significantly increased from 3 h to 72 h following OGD/R and MCAO/R ( Figure 8C-D ), suggesting that Prnp overexpression helps maintain the number of functional lysosomes throughout different periods of CIRI. 4) Fluorescence co-localization of MAP1LC3B with LAMP1 showed that the number of autophagosomes decreased significantly from 3 h to 72 h following OGD/R and MCAO/R, while the number of lysosomes increased ( Figure 9A-B ). This suggests that a sufficient number of lysosomes can rapidly degrade CIRI-induced accumulation of autophagosomes. 5) Assessment of ALP activity using the lysosomal enzyme inhibitor CQ revealed that, compared with the normal group, the degree of elevation of MAP1LC3B-II/I following chloroquine treatment did not change significantly at 6 h post-OGD/R but increased significantly at 48 h ( Figure 9C ). These findings suggest that Prnp overexpression contributes to the maintenance of ALP function during both the early and late stages of CIRI. In contrast, we observed in Prnp -/- astrocytes that: 1) the expression of autophagy markers MAP1LC3-II/I and the autophagy substrate insoluble SQSTM1 increased from 3 h to 72 h following OGD/R ( Figure 10A1-A3 ), indicating the accumulation of autophagosomes and substrates. 2) Although LAMP1 expression did not change significantly from 3 h to 72 h following OGD/R, the expression levels of CTSD and mCTSB, as well as CTSD enzyme activity, decreased significantly over this period ( Figure 10A1, A6-A8, B ), suggesting that the absence of PrP C impedes the restoration of lysosomal number and function in late OGD/R, exacerbating and prolonging autophagosome accumulation. 3) The proportion of functional lysosomes (LAMP + CTSD + ) decreased significantly from 3 h to 72 h following OGD/R and MCAO/R ( Figure 10C-D ), suggesting that Prnp -/- interferes with the recovery of lysosomal dysfunction in late CIRI. 4) Fluorescence co-localization of MAP1LC3B and LAMP1 showed a significant increase in the number of autophagosomes from 3 h to 72 h following OGD/R and MCAO/R ( Figure 11A-B) , with no significant change in lysosomes. This suggests that the relative insufficiency of lysosomes contributes to autophagosome accumulation. 5) Assessment of ALP function using the lysosomal inhibitor CQ revealed a significant decrease in the elevation of MAP1LC3B-II/I following chloroquine treatment at 6 h and 48 h after OGD/R ( Figure 11C ), These findings suggest that the absence of PrP C impairs the recovery of ALP function during late CIRI by inhibiting lysosomal upregulation and its associated functional improvements. These findings indicate that overexpression of PrP C ameliorates lysosomal dysfunction by upregulating lysosomal numbers at different stages of CIRI, thereby maintaining ALP patency. Overexpression of PrP C sustains ALP function during both early and late CIRI by promoting TFEB nuclear translocation, thereby facilitating the shift of astrocytes from a pro-inflammatory to an anti-inflammatory phenotype The above observations led us to hypothesize that PrP C modulation of ALP may be linked to TFEB. To investigate this, we examined total TFEB expression and its nuclear translocation in WT, P rnp - /- , and Prnp -overexpressing astrocytes following CIRI. Immunoblotting results revealed that total TFEB expression in Prnp - /- astrocytes decreased significantly from 3 h to 24 h following OGD/R compared to the normal condition. In contrast, total TFEB expression in Prnp -overexpressing astrocytes decreased from 3 h to 6 h but significantly increased at 12 h and remained elevated from 48 h to 72 h following OGD/R. Furthermore, compared to WT astrocytes, total TFEB expression in Prnp- overexpressing astrocytes was significantly higher at 12 h and from 48 h to 72 h following OGD/R, while total TFEB expression in Prnp - /- astrocytes decreased significantly at 12 h to 24 h after OGD/R ( Figure 12A ). Immunofluorescence staining showed that TFEB nuclear translocation was observed only in Prnp - /- astrocytes at 12 h following OGD/R, as evidenced by an increase in the mean optical density and a significant rise in the Mander’s overlap coefficient. In contrast, TFEB nuclear aggregation was detected in Prnp -overexpressing astrocytes from 12 h to 72 h following OGD/R and normal condition. Notably, TFEB nuclear aggregation was significantly higher in Prnp -overexpressing astrocytes at all time points (3 h to 72 h) compared to WT astrocytes, whereas TFEB nuclear aggregation in Prnp - /- astrocytes was significantly lower from 24 h to 48 h following OGD/R ( Figure 12B ). Similarly, the phenomena observed in mouse MCAO/R astrocytes were consistent with those in vitro ( Figure 12C ). These results suggest that overexpression of PrP C promotes TFEB nuclear translocation during CIRI and sustains it over an extended period. Subsequently, we used siRNA to inhibit TFEB expression in Prnp -overexpressing astrocytes. Immunoblotting results demonstrated that TFEB expression was significantly reduced in the si- Tfeb group compared to the si-Con group at 6 h following OGD/R. Concurrently, the expression of autophagosome markers MAP1LC3B-II/Ⅰand the autophagy substrate ubiquitin was significantly elevated, while pro-CTSD and mCTSB expression was significantly reduced. No significant changes were observed in LAMP1 and mCTSD expression. At 48 h following OGD/R, TFEB expression remained significantly decreased in the si- Tfeb group compared to the si-Con group, along with a notable increase in MAP1LC3B-II/Ⅰand ubiquitin expression, and a significant decrease in LAMP1, CTSD, and mCTSB expression ( Figure 13A ). These results suggest that PrP C overexpression maintains ALP patency during both early and late OGD/R by regulating TFEB nuclear translocation. CCK8 and CBA assays showed that, at both 6 h and 48 h following OGD/R, cellular activity was significantly reduced in the si- Tfeb group compared to the si-Con group. In addition, cytokine IL-10 levels were decreased, while TNF and IL-6 levels were significantly increased ( F igure 13B-C ). These findings suggest that PrP C overexpression enhances astrocyte activity and modulates the inflammatory phenotype during OGD/R through TFEB regulation. In conclusion, our results indicate that overexpression of PrP C preserves ALP patency in both early and late stages of CIRI and promotes the transition of astrocytes from a pro-inflammatory to an anti-inflammatory phenotype by facilitating TFEB nuclear translocation. Overexpression of PrP C promotes nuclear translocation of TFEB following OGD/R via the Ca 2+ /PPP3 pathway Finally, we aimed to explore whether PrP C regulates TFEB following OGD/R through the Ca 2+ /PPP3 pathway. The results showed that intracellular Ca 2+ concentration in Prnp- overexpressing astrocytes decreased at 6 h and subsequently increased from 12 h to 72 h following OGD/R. Although the expression of PPP3 did not change significantly, PPP3 activity increased notably from 3 h to 6 h and from 24 h to 48 h following OGD/R. In contrast, intracellular Ca 2+ concentration in Prnp -/ - astrocytes decreased at 6 h and then increased at 24 h following OGD/R, with no significant changes in PPP3 expression. However, PPP3 activity was significantly reduced from 3 h to 6 h and from 48 h to 72 h following OGD/R. Compared to WT mouse astrocytes, PPP3 ctivity in Prnp- overexpressing astrocytes was significantly higher from 3 h to 6 h and from 24 h to 48 h following OGD/R, while intracellular Ca 2+ concentration increased from 3 h to 12 h and from 48 h to 72 h, and decreased at 24 h following OGD/R. Conversely, in Prnp -/- astrocytes, PPP3 activity was significantly lower at 12 h and from 48 h to 72 h, and intracellular Ca 2+ concentration remained reduced from 3 h to 72 h expect 6 h following OGD/R. These findings suggest that PrP C overexpression helps maintain Ca 2+ homeostasis in astrocytes following OGD/R, which in turn sustains moderate PPP3 activity ( Figure 14A-C ). To further validate this pathway, we inhibited PPP3 in Prnp -overexpressing astrocytes using CsA. CsA significantly reduced TFEB nuclear translocation from 3 h to 72 h following OGD/R compared to the untreated group ( Figure 14D ). These results suggest that PrP C promotes TFEB nuclear translocation via the Ca 2+ /PPP3 pathway. Discussion The present study demonstrates that PrP C mitigates OGD/R-induced ALP dysfunction through the Ca 2+ /PPP3/TFEB pathway and promotes the conversion of astrocytes from a pro-inflammatory to an anti-inflammatory phenotype. Our observations revealed that ALP function was partially impaired during early OGD/R, accompanied by reduced TFEB expression and nuclear translocation, which was attributed to the combined effects of mTOR signaling and PPP3/calcineurin. In contrast, during late OGD/R, lysosomal activity was enhanced, autophagosome and substrate accumulation was alleviated, and cellular damage was reduced, accompanied by increased nuclear translocation of TFEB, primarily mediated by the Ca 2+ /PPP3 pathway. While upregulation of TFEB expression alone did not rescue ALP dysfunction in the early stages of OGD/R, promoting TFEB nuclear translocation via the Ca2 +/ PPP3 pathway was able to attenuate OGD/R injury by restoring ALP function. PrP C modulates TFEB nuclear translocation through the Ca 2+ /PPP3 pathway, improving ALP function and facilitating the shift of astrocytes from a pro-inflammatory to an anti-inflammatory phenotype, thereby alleviating OGD/R-induced damage. In this study, we investigated the dynamic changes in ALP function induced by CIRI. Our findings indicate that at the early stage of OGD/R, autophagosome marker LC3B and substrate SQSTM1 accumulated in WT mouse astrocytes, suggesting that the abnormal accumulation of autophagosomes and substrates begins early in CIRI. This observation aligns with findings reported by Xia Zhang and colleagues [23]. Furthermore, lysosomal function was impaired during early OGD/R, as evidenced by decreased expression of lysosomal markers CTSB and CTSD, reduced CTSD activity, and diminished co-localization of LAMP1 and CTSD. In the presence of CQ, LC3B-II/I levels decreased in the early stages of OGD/R but returned to baseline levels in the later stages. These results suggest that ALP dysfunction in early OGD/R leads to the accumulation of autophagosomes, whereas ALP function gradually improves in the later stages, facilitating the degradation of autophagosomes. This phenomenon raises three key questions: 1) What are the underlying molecular mechanisms responsible for early ALP blockade in OGD/R? 2) What molecular mechanisms contribute to the amelioration of ALP dysfunction in late OGD/R? 3) Is there any overlap in the molecular mechanisms of early ALP blockade and late recovery during OGD/R, and are they regulated by the same proteins? Investigating these mechanisms may help identify potential drug targets for ischemic stroke treatment. TFEB is a key regulator of ALP [12] and has been identified as a potential therapeutic target for rescuing myocardial ischemia-reperfusion injury [14] and permanent cerebral ischemia injury [15]. While much of the existing research focuses on neurons [26-27], we are specifically interested in how TFEB regulates astrocyte ALP following CIRI. In this study, we observed that total TFEB expression initially decreased from 3 to 6 hours and then gradually increased, peaking at 12 hours before returning to baseline levels at 48 hours following OGD/R. TFEB nuclear translocation increased from 12 to 48 hours after OGD/R. These findings suggest that TFEB function is minimal during the ultra-early stages of OGD/R, as indicated by its low accumulation in the nucleus. As the recovery period for oxygen and glucose was extended, TFEB function became gradually activated, translocating to the nucleus and accompanied by an increase in its total expression. This pattern of TFEB expression and nuclear translocation corresponds with changes in ALP function following OGD/R, with the increase in TFEB expression and nuclear translocation slightly preceding the improvement in ALP function. Further, we explored the critical role of TFEB in OGD/R-mediated ALP dysfunction. We found that TFEB function was inhibited during the ultra-early phase of OGD/R, as evidenced by the absence of nuclear translocation, which was regulated by both the mTOR and calcineurin/PPP3 pathways. As the recovery time for oxygen and glucose was prolonged, TFEB function was gradually activated, leading to an increase in nuclear TFEB accumulation, which alleviated ALP blockade and reduced cellular injury. Conversely, si- Tfeb- mediated silencing of TFEB expression reversed the improvement in ALP function and exacerbated astrocyte injury in the later stages of OGD/R. Interestingly, upregulation of TFEB expression did not improve ALP blockade in astrocytes during the early stages of OGD/R, which contrasts with findings from studies on permanent brain ischemia [15]. The discrepancy between our results and those from permanent brain ischemia may be attributed to different molecular mechanisms in CIRI versus permanent brain ischemia. It is well known that the phosphorylation of TFEB by mTORC1 is dependent on intracellular nutrient levels [28]. In the case of cerebral ischemia, mTORC1 is inactive, whereas after reperfusion, mTORC1 is rapidly activated and transformed into its active form, p-mTORC1, a phenomenon confirmed in both our and previous studies [28-29]. Intracellular TFEB exists as dimers [30-32]. In nutrient-rich environments, activated p-mTORC1 phosphorylates TFEB, forming a phosphorylated TFEB homodimer, which is inactive and predominates in the cytoplasm. After ischemic injury, dephosphorylation of TFEB leads to the formation of active homodimers of dephosphorylated TFEB, as well as inactive heterodimers made of phosphorylated and non-phosphorylated TFEB. In other words, phosphorylated TFEB interferes with its nuclear translocation by forming heterodimers with dephosphorylated TFEB [33]. Based on these insights, we hypothesize that the upregulated TFEB expression observed in our study was largely in its phosphorylated form due to activation of p-mTORC1. This phosphorylated TFEB form likely further reduced TFEB activity, and thus, although TFEB is a promising therapeutic target for CIRI, simply upregulating its expression was insufficient to improve outcomes in CIRI. As mentioned previously, TFEB is a member of the microphthalmia family. which also includes microphthalmia-associated transcription factor, TFEC, and TFE3 [32, 34]. TFEB plays a pivotal role in regulating ALP function, and this process is not significantly interfered with by the other members of the the microphthalmia family [35-36]. Consequently, this study focused specifically on the dynamic changes of TFEB following OGD/R. However, our study is not exhaustive, and future research will be necessary to investigate the changes in microphthalmia-associated transcription factor, TFEC, and TFE3 during CIRI. We also observed that the transfection reagent Lipofectamine™ 3000 inhibited ALP function after OGD/R. Upon reviewing the literature, we found that Lipofectamine™ 3000 uses lipid nanoparticle technology to facilitate the entry of exogenous DNA into cells, making it a classical non-viral vector for gene delivery. Recently, an increasing number of studies have highlighted the impact of this cationic liposome transfection reagent on the regulation of ALP [37-38]. Therefore, our choice of transfection reagent may be problematic, and this represents a limitation in our study. The transcriptional activity of TFEB is primarily regulated by its ability to translocate to the nucleus, which is influenced by phosphorylation modifications. As discussed previously, mTOR phosphorylates TFEB to retain it in the cytoplasm, while the Ca 2+ /PPP3 signaling cascade facilitates TFEB nuclear translocation after dephosphorylation [39-40]. To explore the signals upstream of CIRI-induced TFEB nuclear translocation, we monitored the dynamic changes of PPP3/calcineurin and mTOR after OGD/R, followed by pharmacological interventions to determine whether TFEB nuclear translocation depends on these two upstream pathways. These pathways may potentially be targeted to promote TFEB nuclear translocation, improve ALP function, and enhance prognosis. Our findings showed that the trend in intracellular PPP3 enzyme activity largely paralleled TFEB nuclear translocation, and that CsA reversed the OGD/R-induced TFEB nuclear translocation. This suggests that PPP3/calcineurin activation during OGD/R promotes TFEB nuclear translocation. Previous studies have demonstrated that PPP3/calcineurin activation is associated with ischemia-induced neuronal death [41] and that PPP3/calcineurin-dependent TFEB nuclear translocation plays a role in autophagy induction [42-44] . These findings confirm the potential of the PPP3/calcineurin pathway in regulating TFEB nuclear translocation after CIRI. Furthermore, a recent study indicated that the lysosomal calcium-dependent PPP3/calcineurin signaling pathway regulates autophagy through activation of a TFEB-mediated transcriptional prOGD/Ram, independent of mTOR [45]. It is plausible that intracellular Ca 2+ levels serve as an upstream regulatory signal for the PPP3/calcineurin-TFEB axis, as ischemia and hypoxia can induce elevated cytoplasmic Ca 2+ levels [15, 45]. In our study, we observed that intracellular Ca 2+ levels in astrocytes decreased, then increased, and finally returned to baseline levels after OGD/R, which differs from the pattern observed in OGD-treated neurons [15]. This discrepancy may be due to the different pathophysiological mechanisms underlying ischemic injury and ischemia-reperfusion injury. Additionally, previous evidence suggests that intracellular Ca 2+ may partially regulate TFEB dephosphorylation through the PPP3/calcineurin signaling cascade [46-47], which in turn modulates ALP [48]. These data highlight the intracellular Ca 2+ -dependent PPP3/calcineurin pathway as a critical upstream regulatory mechanism of TFEB function in the late stage of CIRI. In general, mTOR inhibits TFEB nuclear translocation by phosphorylating TFEB under nutrient-rich conditions [49-51]. In our study, we observed that mTOR was rephosphorylated into activated p-mTOR following OGD/R. Theoretically, this activated p-mTOR should further phosphorylate TFEB, inhibiting its nuclear translocation. However, our findings show that OGD/R-induced TFEB nuclear translocation occurred despite this rephosphorylation, which presents an apparent discrepancy. The mTOR activator MHY1485 reversed OGD/R-induced TFEB nuclear translocation, suggesting that p-mTOR-mediated inhibition of TFEB nuclear translocation might be a protective mechanism to avoid uncontrolled autophagy during the early phases of CIRI. This idea, however, warrants further investigation in future studies to better understand the role of mTOR in regulating TFEB under ischemic conditions. Additionally, we observed that Rapamycin, an inhibitor of p-mTOR activity, significantly enhanced TFEB nuclear translocation during the ultra-early phase of OGD/R. This enhancement is likely due to the low activity of the Ca 2+ /PPP3 pathway during this early period, which may influence the dynamics of TFEB nuclear translocation. This suggests that while mTOR can modulate TFEB function, the interplay between mTOR and the Ca 2+ /PPP3 pathway may be crucial for determining the timing and extent of TFEB nuclear translocation and autophagic regulation during ischemia-reperfusion injury. Furthermore, autophagic lysosomal remodeling (ALR) is a critical process for maintaining lysosomal homeostasis and represents an evolutionarily conserved cycle of lysosomal regeneration [52]. T Reactivation of mTOR is the initiating event for autophagic lysosomal remodeling (ALR). Following mTOR reactivation, autolysosomes typically undergo tubulization and an increase in vesicle formation, ultimately maturing into functional lysosomes [52]. In our study, we observed that p-mTOR rapidly returned to basal levels after OGD/R. However, the basal mTOR activity was insufficient to meet the elevated demand for lysosomal function following ischemic stroke. As reperfusion continued, mTOR activation levels gradually increased, which in turn alleviated lysosomal dysfunction. These findings suggest that strategies targeting mTOR-related pathways to promote lysosomal accumulation after ischemic stress may hold promise as a therapeutic approach for mitigating lysosomal dysfunction in the future. Previous studies have indeed demonstrated that PrP C has neuroprotective effects during CIRI [18, 53], which aligns with our findings that astrocyte activity in WT mice declines in early OGD/R but recovers to baseline levels in late OGD/R. In contrast, Prnp knockdown inhibited the recovery of cellular activity in late OGD/R and exacerbated cellular activity impairment in early OGD/R. On the other hand, Prnp overexpression significantly attenuated cell injury during OGD/R, highlighting its protective role. Furthermore, our investigation into the potential role of PrP C in regulating ALP function revealed that Prnp overexpression increased lysosome number, enhanced lysosomal enzyme activity, and reduced autophagosome accumulation in both early and late OGD/R. This improvement in ALP function was significant compared to WT astrocytes and was further supported by a reduction in CQ-induced autophagosome accumulation. Conversely, Prnp knockdown astrocytes showed significantly reduced lysosomal function, with exacerbated autophagosome accumulation in late OGD/R. These findings suggest that PrP C plays a crucial role in maintaining ALP function during OGD/R by enhancing lysosomal function and mitigating lysosomal dysfunction, which is consistent with previous reports of impaired ALP in Prnp knockout Purkinje cells [54]. However, while PrP C is a key regulator of ALP in CIRI, directly upregulating its expression is not a feasible therapeutic strategy due to the risk of prion diseases. Therefore, understanding the molecular mechanisms through which PrP C regulates ALP could help identify alternative drug targets to enhance ALP function without the risks associated with PrP C overexpression. Exploring this regulatory pathway will be critical for developing safer and more effective treatments for ischemic stroke. The role of PrP C in regulating immune-related phenotypes, particularly its impact on the inflammatory response in astrocytes after OGD/R, has gained considerable attention. In this study, we observed that Prnp knockdown in astrocytes significantly reduced the concentration of the anti-inflammatory cytokine IL-10, while increasing the levels of pro-inflammatory cytokines INF-γ and TNF after OGD/R. This suggests that PrP C is essential for promoting the anti-inflammatory response in astrocytes following ischemia-reperfusion injury. On the other hand, Prnp overexpression enhanced IL-10 production and suppressed INF-γ and TNF levels, further supporting PrP C 's role in mediating the switch from a pro-inflammatory to an anti-inflammatory phenotype. These findings align with previous studies demonstrating that Prnp overexpression in microglial cells promotes an anti-inflammatory phenotype, whereas Prnp knockdown in microglia leads to a pro-inflammatory phenotype [17]. Moreover, our results emphasize the importance of anti-inflammatory cytokines, such as IL-10, in the prOGD/Ression of neuronal repair after cerebral ischemic stroke. We found that Prnp knockdown suppressed IL-10 expression in late OGD/R, indicating that PrP C may contribute to regulating the expression of key cytokines involved in the resolution of inflammation during ischemic stroke recovery. However, we also noted a discrepancy with a previous study, where P rnp si lencing did not alter IL-10 expression in Mycoplasma bovis-infected microglia [55]. This difference may arise due to the distinct disease models used, suggesting that PrP C 's role in immune regulation might be context-dependent. Overall, our study adds new evidence to the growing body of literature on the potential therapeutic benefits of modulating PrP C expression in ischemic stroke, particularly through its effects on immune responses. Targeting PrP C could provide a promising avenue for enhancing recovery and reducing neuroinflammation following stroke. The study's findings regarding the dynamics of TFEB expression and nuclear translocation in different PrP C -expressing astrocytes provide further insight into the protective mechanisms of PrP C in ischemic conditions. In Prnp knockdown astrocytes, we observed a significant decrease in total TFEB expression from 3 h to 24 h after OGD/R, with perinuclear aggregation of TFEB evident at 12 h. This was reflected by an increase in the mean optical density and Mander's overlap coefficient, suggesting impaired TFEB function. On the other hand, Prnp -overexpressing astrocytes exhibited a more pronounced and sustained nuclear translocation of TFEB, with total TFEB expression decreasing initially from 3 h to 6 h, but then increasing at 12 h and from 48 h to 72 h, along with intranuclear and perinuclear aggregates. This pattern indicates enhanced activation of TFEB function in the later stages of OGD/R in Prnp-overexpressing cells. Interestingly, compared with WT mouse astrocytes, Prnp- overexpressing cells showed a consistent increase in TFEB nuclear translocation from 3 h to 72 h, whereas in Prnp knockout astrocytes, this nuclear translocation decreased from 24 h to 48 h. This suggests that PrP C plays a key role in enhancing TFEB function and promoting its nuclear translocation during OGD/R. Notably, despite the increase in TFEB nuclear translocation in Prnp- overexpressing astrocytes, total TFEB expression dropped back to baseline levels at 24 h post-OGD/R. This warrants further investigation to understand the underlying mechanisms behind this observation, as it could have significant implications for therapeutic strategies targeting PrP C . Further evidence supporting the role of PrP C in promoting TFEB nuclear translocation came from experiments where silencing TFEB in Prnp- overexpressing astrocytes with si- Tfeb significantly reversed the expression of ALP-related proteins. This suggests that PrP C maintains ALP function by promoting TFEB nuclear translocation during OGD/R. Given these findings, PrP C appears to be a promising therapeutic target for ischemic stroke, with its ability to regulate TFEB and maintain autophagic flux playing a key role in cellular protection during CIRI. When Prnp was knocked down, there was no significant change in the trend of intracellular Ca 2+ levels and PPP3 activity after OGD/R, but the rise time of both was shortened. This suggests that PrP C may play a role in fine-tuning the timing of these signaling events rather than directly altering their overall trajectory. Conversely, in Prnp -overexpressing astrocytes, intracellular Ca 2+ levels were better maintained, and PPP3 activity was sustained for a longer period. The addition of CsA, a calcineurin inhibitor, reversed the nuclear translocation of TFEB after OGD/R, supporting the hypothesis that PrP C regulates TFEB activity through the Ca 2+ /PPP3 pathway. This finding aligns with previous studies indicating PrP C 's role in maintaining Ca 2+ homeostasis in cells, though the specific molecular mechanisms behind this regulation remain to be fully elucidated. The findings from this study highlight the crucial role of PrP C in regulating TFEB nuclear translocation during OGD/R through the Ca 2+ /PPP3 pathway, which is tied to its ability to maintain intracellular Ca 2+ homeostasis. This regulation appears to be pivotal in controlling the dynamic process of TFEB activation during the ischemia-reperfusion injury. The model presented in Figure 15 outlines the dynamic changes in the upstream mechanisms of CIRI-induced TFEB nuclear translocation. In the ultra-early stages of CIRI, low intracellular Ca2+ levels and inhibited PPP3/calcineurin activity, along with reactivation of mTOR, result in TFEB phosphorylation and retention in the cytoplasm. As reperfusion time increases, intracellular Ca2+ levels increase, and PPP3/calcineurin is activated, leading to TFEB dephosphorylation, nuclear translocation, and the subsequent transcription of its target genes. This process enhances lysosomal function, alleviates autophagosome accumulation, and promotes astrocyte survival and an anti-inflammatory phenotype. PrPC overexpression sustains intracellular Ca2+ homeostasis and PPP3/calcineurin activity, ensuring the continuous dephosphorylation and nuclear translocation of TFEB. This results in the maintenance of ALP function throughout CIRI and ultimately promotes astrocyte survival and their shift to an anti-inflammatory phenotype, which is essential for neuronal repair after ischemic injury. These insights further emphasize the therapeutic potential of targeting PrPC in ischemic stroke to regulate autophagy and inflammatory responses for better outcomes. In conclusion, we demonstrate a novel mechanism of neuroprotection during CIRI triggered by PrP C . PrP C activates PPP3/calcineurin by maintaining intracellular Ca 2+ homeostasis, PPP3/calcineurin induces nuclear translocation of TFEB, and TFEB within the nucleus transcribe ALP-related genes to modulate their function, which contributes to the transformation of astrocytes from a pro-inflammatory phenotype to an anti-inflammatory phenotype. Declarations Funding Statement This work was supported by a grant from the National Natural Science Foundation of China (No. 82371371) and the Doctor of Excellence Program (DEP), The First Hospital of Jilin University (No. JDYY-DEP-2024039). Disclosure statement The authors declare no conflicts of interests. Data Availability The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. Ethics approval The experimental protocol was approved by the Ethics Committee for Laboratory Animal Research of the First Hospital of Jilin University. Authors' contributions JS, LC, and XY contributed to the conception of this review. JS, XY, YL, and LC participated in writing the manuscript. JS, JY, and TS prepared the figures and tables, while XY and LC critically revised the manuscript. All authors contributed to the article and approved the final version. Consent to Participate Not applicable. Consent for publication Not applicable. References Shi, K.; Tian, D. C.; Li, Z. G.; Ducruet, A. F.; Lawton, M. T.; Shi, F. D., Global brain inflammation in stroke. Lancet Neurol 2019, 18 (11), 1058-1066. Liu, M.; Xu, Z.; Wang, L.; Zhang, L.; Liu, Y.; Cao, J.; Fu, Q.; Liu, Y.; Li, H.; Lou, J.; Hou, W.; Mi, W.; Ma, Y., Cottonseed oil alleviates ischemic stroke injury by inhibiting the inflammatory activation of microglia and astrocyte. J Neuroinflammation 2020, 17 (1), 270. Mizushima, N.; Komatsu, M., Autophagy: renovation of cells and tissues. Cell 2011, 147 (4), 728-41. Liu, S.; Yao, S.; Yang, H.; Liu, S.; Wang, Y., Autophagy: Regulator of cell death. Cell Death Dis 2023, 14 (10), 648. Qin, A. P.; Liu, C. F.; Qin, Y. Y.; Hong, L. Z.; Xu, M.; Yang, L.; Liu, J. Q.; Qin, Z. H.; Zhang, H. L., Autophagy was activated in injured astrocytes and mildly decreased cell survival following glucose and oxygen deprivation and focal cerebral ischemia. Autophagy 2010, 6 (6), 738-753. Pan, R.; Timmins, G. S.; Liu, W.; Liu, K. J., Autophagy Mediates Astrocyte Death During Zinc-Potentiated Ischemia--Reperfusion Injury. Biol Trace Elem Res 2015, 166 (1), 89-95. Gabryel, B.; Kost, A.; Kasprowska, D.; Liber, S.; Machnik, G.; Wiaderkiewicz, R.; Labuzek, K., AMP-activated protein kinase is involved in induction of protective autophagy in astrocytes exposed to oxygen-glucose deprivation. Cell Biol Int 2014, 38 (10), 1086-1097. Zhao, F.; Qu, Y.; Wang, H.; Huang, L.; Zhu, J.; Li, S.; Tong, Y.; Zhang, L.; Li, J.; Mu, D., The effect of miR-30d on apoptosis and autophagy in cultured astrocytes under oxygen-glucose deprivation. Brain Res 2017, 1671 , 67-76. Carloni, S.; Buonocore, G.; Balduini, W., Protective role of autophagy in neonatal hypoxia-ischemia induced brain injury. Neurobiol Dis 2008, 32 (3), 329-39. Shi, R.; Weng, J.; Zhao, L.; Li, X. M.; Gao, T. M.; Kong, J., Excessive autophagy contributes to neuron death in cerebral ischemia. CNS Neurosci Ther 2012, 18 (3), 250-60. Wang, P.; Shao, B. Z.; Deng, Z.; Chen, S.; Yue, Z.; Miao, C. Y., Autophagy in ischemic stroke. Prog Neurobiol 2018, 163-164 , 98-117. Shao, J.; Lang, Y.; Ding, M. Q.; Yin, X.; Cui, L., Transcription Factor EB: A Promising Therapeutic Target for Ischemic Stroke. Current Neuropharmacology 2024, 22 (2), 170-190. Zhang, Z.; Yan, J.; Bowman, A. B.; Bryan, M. R.; Singh, R.; Aschner, M., Dysregulation of TFEB contributes to manganese-induced autophagic failure and mitochondrial dysfunction in astrocytes. Autophagy 2020, 16 (8), 1506-1523. Gu, S.; Tan, J.; Li, Q.; Liu, S.; Ma, J.; Zheng, Y.; Liu, J.; Bi, W.; Sha, P.; Li, X.; Wei, M.; Cao, N.; Yang, H. T., Downregulation of LAPTM4B Contributes to the Impairment of the Autophagic Flux via Unopposed Activation of mTORC1 Signaling During Myocardial Ischemia/Reperfusion Injury. Circ Res 2020, 127 (7), e148-e165. Liu, Y.; Xue, X.; Zhang, H.; Che, X.; Luo, J.; Wang, P.; Xu, J.; Xing, Z.; Yuan, L.; Liu, Y.; Fu, X.; Su, D.; Sun, S.; Zhang, H.; Wu, C.; Yang, J., Neuronal-targeted TFEB rescues dysfunction of the autophagy-lysosomal pathway and alleviates ischemic injury in permanent cerebral ischemia. Autophagy 2019, 15 (3), 493-509. Bolton, D. C.; McKinley, M. P.; Prusiner, S. B., Identification of a protein that purifies with the scrapie prion. Science 1982, 218 (4579), 1309-11. Shao, J.; Yin, X.; Lang, Y.; Ding, M.; Zhang, B.; Sun, Q.; Jiang, X.; Song, J.; Cui, L., Cellular Prion Protein Attenuates OGD/R-Induced Damage by Skewing Microglia toward an Anti-inflammatory State via Enhanced and Prolonged Activation of Autophagy. Mol Neurobiol 2023, 60 (3), 1297-1316. Zhang, B. Z.; Yin, X.; Lang, Y.; Han, X. O.; Shao, J.; Bai, R. R.; Cui, L., Role of cellular prion protein in splenic CD4 T cell differentiation in cerebral ischaemic/reperfusion. Ann Clin Transl Neur 2021, 8 (10), 2040-2051. Yin, X.; Feng, L. S.; Ma, D.; Yin, P.; Wang, X. Y.; Hou, S.; Hao, Y. L.; Zhang, J. D.; Xin, M. Y.; Feng, J. C., Roles of astrocytic connexin-43, hemichannels, and gap junctions in oxygen-glucose deprivation/reperfusion injury induced neuroinflammation and the possible regulatory mechanisms of salvianolic acid B and carbenoxolone. J Neuroinflamm 2018, 15 . Wu, M.; Lao, Y. Z.; Tan, H. S.; Lu, G.; Ren, Y.; Zheng, Z. Q.; Yi, J.; Fu, W. W.; Shen, H. M.; Xu, H. X., Oblongifolin C suppresses lysosomal function independently of TFEB nuclear translocation. Acta Pharmacol Sin 2019, 40 (7), 929-937. Boya, P.; Reggiori, F.; Codogno, P., Emerging regulation and functions of autophagy. Nat Cell Biol 2013, 15 (7), 713-20. Koerver, L.; Papadopoulos, C.; Liu, B.; Kravic, B.; Rota, G.; Brecht, L.; Veenendaal, T.; Polajnar, M.; Bluemke, A.; Ehrmann, M.; Klumperman, J.; Jaattela, M.; Behrends, C.; Meyer, H., The ubiquitin-conjugating enzyme UBE2QL1 coordinates lysophagy in response to endolysosomal damage. EMBO Rep 2019, 20 (10), e48014. Zhang, X.; Wei, M.; Fan, J.; Yan, W.; Zha, X.; Song, H.; Wan, R.; Yin, Y.; Wang, W., Ischemia-induced upregulation of autophagy preludes dysfunctional lysosomal storage and associated synaptic impairments in neurons. Autophagy 2021, 17 (6), 1519-1542. Hossain, M. I.; Marcus, J. M.; Lee, J. H.; Garcia, P. L.; Singh, V.; Shacka, J. J.; Zhang, J. H.; Gropend, T. I.; Falany, C. N.; Andrabi, S. A., Restoration of CTSD (cathepsin D) and lysosomal function in stroke is neuroprotective. Autophagy 2021, 17 (6), 1330-1348. Spudich, A.; Frigg, R.; Kilic, E.; Kilic, U.; Oesch, B.; Raeber, A.; Bassetti, C. L.; Hermann, D. M., Aggravation of ischemic brain injury by prion protein deficiency: role of ERK-1/-2 and STAT-1. Neurobiol Dis 2005, 20 (2), 442-9. Xiao, Q.; Yan, P.; Ma, X.; Liu, H.; Perez, R.; Zhu, A.; Gonzales, E.; Tripoli, D. L.; Czerniewski, L.; Ballabio, A.; Cirrito, J. R.; Diwan, A.; Lee, J. M., Neuronal-Targeted TFEB Accelerates Lysosomal Degradation of APP, Reducing Abeta Generation and Amyloid Plaque Pathogenesis. J Neurosci 2015, 35 (35), 12137-51. Polito, V. A.; Li, H.; Martini-Stoica, H.; Wang, B.; Yang, L.; Xu, Y.; Swartzlander, D. B.; Palmieri, M.; di Ronza, A.; Lee, V. M.; Sardiello, M.; Ballabio, A.; Zheng, H., Selective clearance of aberrant tau proteins and rescue of neurotoxicity by transcription factor EB. EMBO Mol Med 2014, 6 (9), 1142-60. Szwed, A.; Kim, E.; Jacinto, E., Regulation and metabolic functions of mTORC1 and mTORC2. Physiol Rev 2021, 101 (3), 1371-1426. Hwang, S. K.; Kim, H. H., The functions of mTOR in ischemic diseases. BMB Rep 2011, 44 (8), 506-11. Pogenberg, V.; Ballesteros-Alvarez, J.; Schober, R.; Sigvaldadottir, I.; Obarska-Kosinska, A.; Milewski, M.; Schindl, R.; Ogmundsdottir, M. H.; Steingrimsson, E.; Wilmanns, M., Mechanism of conditional partner selectivity in MITF/TFE family transcription factors with a conserved coiled coil stammer motif. Nucleic Acids Res 2020, 48 (2), 934-948. La Spina, M.; Contreras, P. S.; Rissone, A.; Meena, N. K.; Jeong, E.; Martina, J. A., MiT/TFE Family of Transcription Factors: An Evolutionary Perspective. Front Cell Dev Biol 2020, 8 , 609683. Puertollano, R.; Ferguson, S. M.; Brugarolas, J.; Ballabio, A., The complex relationship between TFEB transcription factor phosphorylation and subcellular localization. Embo Journal 2018, 37 (11). Sha, Y. B.; Rao, L.; Settembre, C.; Ballabio, A.; Eissa, N. T., STUB1 regulates TFEB-induced autophagy-lysosome pathway. Embo J 2017, 36 (17), 2544-2552. Cheli, Y.; Ohanna, M.; Ballotti, R.; Bertolotto, C., Fifteen-year quest for microphthalmia-associated transcription factor target genes. Pigment Cell Melanoma Res 2010, 23 (1), 27-40. Sardiello, M.; Palmieri, M.; di Ronza, A.; Medina, D. L.; Valenza, M.; Gennarino, V. A.; Di Malta, C.; Donaudy, F.; Embrione, V.; Polishchuk, R. S.; Banfi, S.; Parenti, G.; Cattaneo, E.; Ballabio, A., A gene network regulating lysosomal biogenesis and function. Science 2009, 325 (5939), 473-7. Settembre, C.; Di Malta, C.; Polito, V. A.; Garcia Arencibia, M.; Vetrini, F.; Erdin, S.; Erdin, S. U.; Huynh, T.; Medina, D.; Colella, P.; Sardiello, M.; Rubinsztein, D. C.; Ballabio, A., TFEB links autophagy to lysosomal biogenesis. Science 2011, 332 (6036), 1429-33. Bhagya, N.; Chandrashekar, K. R., Liposome encapsulated anticancer drugs on autophagy in cancer cells - Current and future perspective. Int J Pharm 2023, 642 , 123105. Zhou, F.; Li, X. J. Y.; Xue, X. Y.; Li, S.; Fan, G. F.; Cai, Y. J.; Chang, Z. H.; Qu, J. R.; Liu, R. P., A Novel Tri-Functional Liposome Re-Educates "Cold Tumor" and Abrogates Tumor Growth by Synergizing Autophagy Inhibition and PD-L1 Blockade. Adv Healthc Mater 2023, 12 (11). Pena-Llopis, S.; Vega-Rubin-de-Celis, S.; Schwartz, J. C.; Wolff, N. C.; Tran, T. A. T.; Zou, L. H.; Xie, X. J.; Corey, D. R.; Brugarolas, J., Regulation of TFEB and V-ATPases by mTORC1. Embo J 2011, 30 (16), 3242-3258. Pena-Llopis, S.; Brugarolas, J., TFEB, a novel mTORC1 effector implicated in lysosome biogenesis, endocytosis and autophagy. Cell Cycle 2011, 10 (23), 3987-3988. Chen, Y.; Holstein, D. M.; Aime, S.; Bollo, M.; Lechleiter, J. D., Calcineurin beta protects brain after injury by activating the unfolded protein response. Neurobiol Dis 2016, 94 , 139-56. Singh, N.; Kansal, P.; Ahmad, Z.; Baid, N.; Kushwaha, H.; Khatri, N.; Kumar, A., Antimycobacterial effect of IFNG (interferon gamma)-induced autophagy depends on HMOX1 (heme oxygenase 1)-mediated increase in intracellular calcium levels and modulation of PPP3/calcineurin-TFEB (transcription factor EB) axis. Autophagy 2018, 14 (6), 972-991. Zhang, X. L.; Cheng, X. P.; Yu, L.; Yang, J. S.; Calvo, R.; Patnaik, S.; Hu, X.; Gao, Q.; Yang, M. M.; Lawas, M.; Delling, M.; Marugan, J.; Ferrer, M.; Xu, H. X., MCOLN1 is a ROS sensor in lysosomes that regulates autophagy. Nature Communications 2016, 7 . Song, R. W.; Li, J.; Zhang, J.; Wang, L.; Tong, L.; Wang, P.; Yang, H.; Wei, Q.; Cai, H. B.; Luo, J., Peptides derived from transcription factor EB bind to calcineurin at a similar region as the NFAT-type motif. Biochimie 2017, 142 , 158-167. Medina, D. L.; Di Paola, S.; Peluso, I.; Armani, A.; De Stefani, D.; Venditti, R.; Montefusco, S.; Scotto-Rosato, A.; Prezioso, C.; Forrester, A.; Settembre, C.; Wang, W. Y.; Gao, Q.; Xu, H. X.; Sandri, M.; Rizzuto, R.; De Matteis, M. A.; Ballabio, A., Lysosomal calcium signalling regulates autophagy through calcineurin and TFEB. Nature Cell Biology 2015, 17 (3), 288-+. Jin, Y. P.; Bai, Y. Y.; Ni, H. Z.; Qiang, L.; Ye, L. C.; Shan, Y. F.; Zhou, M. T., Activation of autophagy through calcium-dependent AMPK/mTOR and PKCθ pathway causes activation of rat hepatic stellate cells under hypoxic stress. Febs Letters 2016, 590 (5), 672-682. Yang, J.; Yu, J.; Li, D. D.; Yu, S. J.; Ke, J. B.; Wang, L. Y.; Wang, Y. W.; Qiu, Y. Z.; Gao, X. B.; Zhang, J. H.; Huang, L., Store-operated calcium entry-activated autophagy protects EPC proliferation via the CAMKK2-MTOR pathway in ox-LDL exposure. Autophagy 2017, 13 (1), 82-98. Mizushima, N.; Yoshimori, T.; Levine, B., Methods in Mammalian Autophagy Research. Cell 2010, 140 (3), 313-326. Martina, J. A.; Chen, Y.; Gucek, M.; Puertollano, R., MTORC1 functions as a transcriptional regulator of autophagy by preventing nuclear transport of TFEB. Autophagy 2012, 8 (6), 903-914. Roczniak-Ferguson, A.; Petit, C. S.; Froehlich, F.; Qian, S.; Ky, J.; Angarola, B.; Walther, T. C.; Ferguson, S. M., The Transcription Factor TFEB Links mTORC1 Signaling to Transcriptional Control of Lysosome Homeostasis. Sci Signal 2012, 5 (228). Vega-Rubin-de-Celis, S.; Pena-Llopis, S.; Konda, M.; Brugarolas, J., Multistep regulation of TFEB by MTORC1. Autophagy 2017, 13 (3), 464-472. Yu L, McPhee CK, Zheng L, et al. Termination of autophagy and reformation of lysosomes regulated by mTOR. Nature. 2010 ;465(7300):942–946. Shyu, W. C.; Lin, S. Z.; Chiang, M. F.; Ding, D. C.; Li, K. W.; Chen, S. F.; Yang, H. I.; Li, H., Overexpression of PrPC by adenovirus-mediated gene targeting reduces ischemic injury in a stroke rat model. J Neurosci 2005, 25 (39), 8967-77. Shin, H. Y.; Oh, J. M.; Kim, Y. S., The Functional Role of Prion Protein (PrPC) on Autophagy. Pathogens 2013, 2 (3), 436-45. Ding, T.; Zhou, X.; Kouadir, M.; Shi, F.; Yang, Y.; Liu, J.; Wang, M.; Yin, X.; Yang, L.; Zhao, D., Cellular prion protein participates in the regulation of inflammatory response and apoptosis in BV2 microglia during infection with Mycobacterium bovis. J Mol Neurosci 2013, 51 (1), 118-26. Additional Declarations No competing interests reported. Supplementary Files shaojieAdditionalfile1.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-7212124","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":492844469,"identity":"b7d0f6e7-8711-44f4-a22a-46591c36a827","order_by":0,"name":"Jie Shao","email":"","orcid":"","institution":"The First Hospital of Jilin University, Jilin University","correspondingAuthor":false,"prefix":"","firstName":"Jie","middleName":"","lastName":"Shao","suffix":""},{"id":492844471,"identity":"b55d39b2-cd87-4948-986e-0811e1750e97","order_by":1,"name":"Xiang Yin","email":"","orcid":"","institution":"The First Hospital of Jilin University, Jilin University","correspondingAuthor":false,"prefix":"","firstName":"Xiang","middleName":"","lastName":"Yin","suffix":""},{"id":492844473,"identity":"5825104c-1b5b-4a37-9709-962ea8d28572","order_by":2,"name":"Yue Lang","email":"","orcid":"","institution":"The First Hospital of Jilin University, Jilin University","correspondingAuthor":false,"prefix":"","firstName":"Yue","middleName":"","lastName":"Lang","suffix":""},{"id":492844474,"identity":"0975c24c-16f7-47f9-b6f2-2fe1cb82e134","order_by":3,"name":"Jie Yang","email":"","orcid":"","institution":"The First Hospital of Jilin University, Jilin University","correspondingAuthor":false,"prefix":"","firstName":"Jie","middleName":"","lastName":"Yang","suffix":""},{"id":492844475,"identity":"49674e92-b144-481a-8d7f-d555504b817c","order_by":4,"name":"Menghan Jia","email":"","orcid":"","institution":"The First Hospital of Jilin University, Jilin University","correspondingAuthor":false,"prefix":"","firstName":"Menghan","middleName":"","lastName":"Jia","suffix":""},{"id":492844476,"identity":"0d77a5d1-310c-49ec-baee-140680de5ca1","order_by":5,"name":"Tengfei Su","email":"","orcid":"","institution":"The First Hospital of Jilin University, Jilin University","correspondingAuthor":false,"prefix":"","firstName":"Tengfei","middleName":"","lastName":"Su","suffix":""},{"id":492844477,"identity":"caa9cb16-6b2e-458f-89cd-753d452c0d4e","order_by":6,"name":"Li Cui","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAp0lEQVRIiWNgGAWjYBACPiA+8IHHAsQ2IE4LGxAfnMEjQaIWZh4GkrSwnzE8bCMjkdjA3rxNgqHmDhFaeHIMDufwALXwHCuTYDj2jAgtEjxQLRI5ZhKMDYeJ1GIB0iL/hhQtDGBbeIjVwpNWcLCHR8K4jSet2CLhGBFa+NkPb/7ws8dGtp/98MYbH2qI0AIGjD2QCGJIIFIDEPwgXukoGAWjYBSMQAAAZLwu4AssADcAAAAASUVORK5CYII=","orcid":"","institution":"The First Hospital of Jilin University, Jilin University","correspondingAuthor":true,"prefix":"","firstName":"Li","middleName":"","lastName":"Cui","suffix":""}],"badges":[],"createdAt":"2025-07-25 08:38:14","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7212124/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7212124/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":87979559,"identity":"1f4a3d56-9812-41a7-8a6f-448004671868","added_by":"auto","created_at":"2025-07-31 05:32:40","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":877000,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003etMCAO induces dynamic alterations in ALP function in astrocytes of WT mice. \u003c/strong\u003e(\u003cstrong\u003eA\u003c/strong\u003e) Time-dependent alterations in the expression of ALP-related proteins in primary astrocyte protein extracts from 3 h to 72 h following OGD/R. Quantification of immunoblotted proteins was performed using ImageJ. Statistical analysis was conducted using ANOVA followed by Tukey’s test or Dunnett’s test. Data are presented as mean ± SEM from more than 4 independent experiments. *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01 vs. normal condition group. (\u003cstrong\u003eB\u003c/strong\u003e) Histograms showing changes in CTSD activity in astrocytes from the normal condition group and OGD/R-treated groups. CTSD activity was assessed using a fluorimetric assay. Statistical analysis was performed using ANOVA followed by Dunnett’s test. Data are presented as mean ± SEM from 3 independent experiments. *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01 vs. normal condition group. (\u003cstrong\u003eC\u003c/strong\u003e) Immunofluorescence images show the colocalization of LAMP1-positive lysosomes (green) with the lysosomal enzyme CTSD (red) in the normal condition group and OGD/R groups. Nuclei are stained with DAPI (blue). Scale bar: 20 μm. Histogram shows changes in the percentage of LAMP1\u003csup\u003e+\u003c/sup\u003eCTSD\u003csup\u003e+\u003c/sup\u003e puncta and LAMP1\u003csup\u003e+\u003c/sup\u003e puncta in each astrocyte. Data are expressed as mean ± SEM from at least 3 independent experiments. *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01 vs. normal condition group. (\u003cstrong\u003eD\u003c/strong\u003e) Immunofluorescence images show colocalization of LAMP1-positive lysosomes (green) with the lysosomal enzyme CTSD (yellow) in astrocytes (red) of the cerebral cortex of sham-operated and MCAO/R mice. Nuclei are stained with DAPI (blue). Scale bar: 50 μm and 5 μm.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-7212124/v1/0b796389b14f013e7c1d187d.png"},{"id":87980074,"identity":"45672f1d-f33b-4e64-917f-406694638698","added_by":"auto","created_at":"2025-07-31 05:40:40","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":680993,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003etMCAO induces dynamic alterations in ALP function in astrocytes of WT mice\u003c/strong\u003e. (\u003cstrong\u003eA\u003c/strong\u003e) Immunofluorescence images showing the colocalization of LAMP1-positive lysosomes (green) with LC3B-positive autophagosomes (red) in the normal condition group and OGD/R groups. Nuclei are stained with DAPI (blue). Scale bar: 20 μm. Histogram shows changes in the number of LAMP1\u003csup\u003e+\u003c/sup\u003e puncta, LC3B\u003csup\u003e+\u003c/sup\u003e puncta, and LAMP1\u003csup\u003e+\u003c/sup\u003eLC3B\u003csup\u003e+\u003c/sup\u003e puncta. Data are presented as mean ± SEM from more than 3 independent experiments. *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, #\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ##\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, \u0026amp;\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 vs. normal condition group. (\u003cstrong\u003eB\u003c/strong\u003e) Immunofluorescence images show colocalization of LAMP1-positive lysosomes (green) with LC3B-positive autophagosomes (yellow) in cortical astrocytes (red) of the sham group and MCAO/R group. Cell nuclei were stained with DAPI (blue). Scale bar: 50 μm and 5 μm. (\u003cstrong\u003eC\u003c/strong\u003e) Expression of LC3B in the normal condition group and OGD/R-treated groups with or without CQ. Quantification of immunoblotted proteins was performed using ImageJ. Statistical analysis was performed using ANOVA. Data are presented as mean ± SEM from 4 independent experiments. (\u003cstrong\u003eC2\u003c/strong\u003e) **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 vs. non-dosed group. (\u003cstrong\u003eC3\u003c/strong\u003e) **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 vs. the normal condition group.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-7212124/v1/08e106e13d6d05ec0a1b9d25.png"},{"id":87980075,"identity":"ae6ff078-88be-45f8-85fc-7910db2cbd45","added_by":"auto","created_at":"2025-07-31 05:40:40","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":920013,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003etMCAO induces TFEB activation in cortical astrocytes of WT mice. \u003c/strong\u003e(\u003cstrong\u003eA\u003c/strong\u003e) Time-dependent changes in TFEB expression from 3 h to 72 h following OGD/R in astrocyte protein extracts. Quantification of immunoblotted proteins was performed with ImageJ. Statistical analysis was carried out using ANOVA followed by Dunnett’s test. Data are shown as mean ± SEM from 4 independent experiments. *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 vs. normal condition group. (\u003cstrong\u003eB\u003c/strong\u003e) Immunofluorescence images showing TFEB distribution in astrocytes under normal conditions and after OGD/R treatment, observed with laser confocal microscopy using antibodies against TFEB (red). Nuclei are stained with DAPI (blue). Scale bar: 50 μm. High-magnification images of the boxed areas are shown in the inserts. Columns represent Mander’s overlap coefficient. Data are presented as mean ± SEM from at least 5 independent experiments per group. **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 vs. normal condition group. (\u003cstrong\u003eC\u003c/strong\u003e) Immunofluorescence staining visualized TFEB distribution (green) in cortical astrocytes (red) across sham and MCAO/R groups. Nuclei were counterstained with DAPI (blue). Scale bars: 20 μm and 5 μm.​\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-7212124/v1/6d75ed2ebf825951ca7af073.png"},{"id":87979562,"identity":"bdd635ac-44f0-4617-b146-9136f5606188","added_by":"auto","created_at":"2025-07-31 05:32:40","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":893232,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003etMCAO induces TFEB activation in WT mice cortical astrocytes via the Ca\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e2+\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e/PPP3 signaling pathway.\u003c/strong\u003e (\u003cstrong\u003eA\u003c/strong\u003e) Quantification of Ca²⁺ concentration dynamics in WT mouse primary astrocytes. Statistical comparisons were conducted with ANOVA followed by Tukey’s test. Data are presented as mean ± SEM from 18 independent experiments. *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01 vs. normal condition group. (\u003cstrong\u003eB\u003c/strong\u003e) Immunoblot analysis showing dynamic changes in PPP3/calcineurin expression following OGD/R in WT mouse primary astrocytes. Quantification of immunoblotted proteins was performed with ImageJ. Statistical analysis was conducted using ANOVA followed by Dunnett’s test. Data are presented as mean ± SEM from 5 independent experiments. (\u003cstrong\u003eC\u003c/strong\u003e) Histogram displaying alterations in PPP3/calcineurin activity in normal and OGD/R-treated astrocytes. Each sample was analyzed in triplicate. Data are shown as mean ± SEM, with statistical comparisons using ANOVA followed by Dunnett’s test. *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 vs. normal condition group. (\u003cstrong\u003eD\u003c/strong\u003e) WT mouse primary astrocytes were treated with the PPP3/calcineurin inhibitor CsA from 3 h to 72 h following OGD/R. TFEB distribution was visualized by immunofluorescence using a TFEB antibody. Enlarged images of the boxed areas are shown in the inserts. Scale bar: 50 μm. Columns represent Mander’s overlap coefficient. Data are presented as mean ± SEM from at least 6 independent experiments per group. **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 vs. untreated group. (\u003cstrong\u003eE\u003c/strong\u003e) Time-dependent changes in mTOR and p-mTOR expression from 3 h to 72 h following OGD/R in astrocyte protein extracts. Quantification was performed with ImageJ. Statistical analysis was done using ANOVA followed by Dunnett’s test. Data are shown as mean ± SEM from 6 independent experiments. **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 vs. normal condition group. (\u003cstrong\u003eF\u003c/strong\u003e) WT mouse primary astrocytes were treated with mTOR inhibitors Rapamycin or activator MHY1485 from 3 h to 72 h following OGD/R. TFEB distribution was visualized by immunofluorescence using a TFEB antibody. Enlarged images of the boxed areas are shown in the inserts. Scale bar: 50 μm. Columns represent Mander’s overlap coefficient. Data are shown as mean ± SEM from at least 6 independent experiments per group. **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 vs. untreated group.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-7212124/v1/43e8078bf1d1bdb2a2c2d718.png"},{"id":87979563,"identity":"d0c8ea3e-3e06-442d-9056-b76cb3874f90","added_by":"auto","created_at":"2025-07-31 05:32:40","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":479529,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAstrocyte-targeted silencing of TFEB impairs ALP function, reduces cellular viability, and induces a pro-inflammatory phenotype during late OGD/R. \u003c/strong\u003e(\u003cstrong\u003eA\u003c/strong\u003e) Immunoblots showing the expression of ALP-related proteins in astrocytes under normal conditions and after OGD/R treatment, incubated with either si-Con or si-\u003cem\u003eTfeb\u003c/em\u003e at 48 h post-OGR. Quantitative analysis of immunoblotted proteins was performed using ImageJ. Statistical comparisons were carried out with ANOVA followed by Tukey’s test. Data are shown as mean ± SEMfrom at least 3 independent experiments. **\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.01, *\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.05 vs. si-Con group. (\u003cstrong\u003eB\u003c/strong\u003e) CCK-8 assay results showing cellular viability in astrocytes under normal conditions and after OGD/R treatment, incubated with si-Con or si-\u003cem\u003eTfeb\u003c/em\u003e at 48 h post-OGR. Statistical comparisons were carried out with ANOVA followed by Tukey’s test. Data are presented as mean ± SEM from 6 independent experiments. **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01 vs. si-Con group. (\u003cstrong\u003eC\u003c/strong\u003e) Flow cytometry analysis using CBAs showing concentrations of pro- and anti-inflammatory cytokines in astrocytes under normal conditions and after OGD/R treatment, incubated with si-Con or si-\u003cem\u003eTfeb\u003c/em\u003e at 48 h post-OGR. Statistical comparisons were performed with ANOVA followed by Tukey’s test. Data are shown as mean ± SEM from 3 independent experiments. *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01 vs. si-Con group.\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-7212124/v1/7993e2a37709bba776921930.png"},{"id":87979560,"identity":"74b33c70-4ebd-4f3e-8228-fee0412ec32c","added_by":"auto","created_at":"2025-07-31 05:32:40","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":269693,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAstrocyte-targeted overexpression of TFEB does not enhance ALP function during the early stage of OGD/R. \u003c/strong\u003e(\u003cstrong\u003eA\u003c/strong\u003e) Immunoblots showing the expression of ALP-related proteins in astrocytes under normal conditions or after OGD/R treatment, incubated with \u003cem\u003eTfeb\u003c/em\u003eoverexpression vector or control vector at 6 h post-OGR. Quantitative analysis of immunoblotted proteins was performed using ImageJ. Statistical comparisons were conducted with ANOVA followed by Tukey’s test. Data are presented as mean ± SEM from at least 3 independent experiments. **\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.01, *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 vs. Vector group.\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-7212124/v1/cc0def595405800049fb946b.png"},{"id":87979569,"identity":"43e61305-fff7-4c23-8640-2f5ac1f12439","added_by":"auto","created_at":"2025-07-31 05:32:40","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":310057,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eOverexpression of PrP\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003eC\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e enhances astrocyte activity and promotes a shift from a pro-inflammatory to an anti-inflammatory phenotype following OGD/R. \u003c/strong\u003e(\u003cstrong\u003eA\u003c/strong\u003e) Time-dependent changes in PrP\u003csup\u003eC\u003c/sup\u003e expression from 3 h to 72 h post-OGR in protein extracts from WT mouse primary astrocytes. Quantitative analysis of immunoblotted proteins was conducted using ImageJ. Statistical comparisons were performed with ANOVA followed by Dunnett’s test. Data are presented as mean ± SEM from 4 independent experiments. *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 vs. normal condition group. (\u003cstrong\u003eB\u003c/strong\u003e) Time-dependent changes in cellular viability, as assessed by CCK-8 assay, in WT, \u003cem\u003ePrnp\u003c/em\u003e\u003csup\u003e-/-\u003c/sup\u003e, and \u003cem\u003ePrnp\u003c/em\u003e-overexpressing mouse primary astrocytes following OGR. Statistical comparisons were performed with ANOVA followed by Dunnett’s test. Data are presented as mean ± SEM from 6 independent experiments. *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01 vs. normal condition group. (\u003cstrong\u003eC\u003c/strong\u003e) Time-dependent changes in concentrations of pro- and anti-inflammatory cytokines, detected by flow cytometry (CBA assay), in WT, \u003cem\u003ePrnp\u003c/em\u003e\u003csup\u003e-/-\u003c/sup\u003e, and\u003cem\u003e Prnp\u003c/em\u003e-overexpressing mouse primary astrocytes following OGR. Statistical comparisons were carried out with ANOVA followed by Tukey’s test. Data are presented as mean ± SEM from 3 independent experiments. *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01 vs. normal condition group.\u003c/p\u003e","description":"","filename":"image7.png","url":"https://assets-eu.researchsquare.com/files/rs-7212124/v1/f67784e3156b3c2120c8200a.png"},{"id":87979566,"identity":"690fa52a-b35d-4357-b496-731edffa06d6","added_by":"auto","created_at":"2025-07-31 05:32:40","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":963822,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003etMCAO induces dynamic alterations in ALP function in astrocytes of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ePrnp\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e-overexpressing mice. \u003c/strong\u003e(\u003cstrong\u003eA\u003c/strong\u003e) Time-dependent alterations in the expression of ALP-related proteins in primary astrocyte protein extracts from 3 h to 72 h following OGD/R. Quantification of immunoblotted proteins was performed using ImageJ. Statistical analysis was conducted using ANOVA followed by Tukey’s test or Dunnett’s test. Data are presented as mean ± SEM from more than 4 independent experiments. *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01 vs. normal condition group. (\u003cstrong\u003eB\u003c/strong\u003e) Histograms showing changes in CTSD activity in astrocytes from the normal condition group and OGD/R-treated groups. CTSD activity was assessed using a fluorimetric assay. Statistical analysis was performed using ANOVA followed by Dunnett’s test. Data are presented as mean ± SEM from 3 independent experiments. *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01 vs. normal condition group. (\u003cstrong\u003eC\u003c/strong\u003e) Immunofluorescence images show the colocalization of LAMP1-positive lysosomes (green) with the lysosomal enzyme CTSD (red) in the normal condition group and OGD/R groups. Nuclei are stained with DAPI (blue). Scale bar: 20 μm. Histogram shows changes in the percentage of LAMP1\u003csup\u003e+\u003c/sup\u003eCTSD\u003csup\u003e+\u003c/sup\u003e puncta and LAMP1\u003csup\u003e+\u003c/sup\u003e puncta in each astrocyte. Data are expressed as mean ± SEM from at least 3 independent experiments. *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01 vs. normal condition group. (\u003cstrong\u003eD\u003c/strong\u003e) Immunofluorescence images show colocalization of LAMP1-positive lysosomes (green) with the lysosomal enzyme CTSD (yellow) in astrocytes (red) of the cerebral cortex of sham-operated and MCAO/R mice. Nuclei are stained with DAPI (blue). Scale bar: 50 μm and 5 μm.\u003c/p\u003e","description":"","filename":"image8.png","url":"https://assets-eu.researchsquare.com/files/rs-7212124/v1/d4ab549019416d97cb601f25.png"},{"id":87979573,"identity":"68484746-7227-4547-9185-7194baec90d1","added_by":"auto","created_at":"2025-07-31 05:32:41","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":924423,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003etMCAO induces dynamic alterations in ALP function in astrocytes of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ePrnp\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e-overexpressing mice\u003c/strong\u003e. (\u003cstrong\u003eA\u003c/strong\u003e) Immunofluorescence images showing the colocalization of LAMP1-positive lysosomes (green) with LC3B-positive autophagosomes (red) in the normal condition group and OGD/R groups. Nuclei are stained with DAPI (blue). Scale bar: 20 μm. Histogram shows changes in the number of LAMP1\u003csup\u003e+\u003c/sup\u003e puncta, LC3B\u003csup\u003e+\u003c/sup\u003e puncta, and LAMP1\u003csup\u003e+\u003c/sup\u003eLC3B\u003csup\u003e+\u003c/sup\u003e puncta. Data are presented as mean ± SEM from more than 3 independent experiments. *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, #\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ##\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, \u0026amp;\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 vs. normal condition group. (\u003cstrong\u003eB\u003c/strong\u003e) Immunofluorescence images show colocalization of LAMP1-positive lysosomes (green) with LC3B-positive autophagosomes (yellow) in cortical astrocytes (red) of the sham group and MCAO/R group. Cell nuclei were stained with DAPI (blue). Scale bar: 50 μm and 5 μm. (\u003cstrong\u003eC\u003c/strong\u003e) Expression of LC3B in the normal condition group and OGD/R-treated groups with or without CQ. Quantification of immunoblotted proteins was performed using ImageJ. Statistical analysis was performed using ANOVA. Data are presented as mean ± SEM from 4 independent experiments. (\u003cstrong\u003eC2\u003c/strong\u003e) **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 vs. non-dosed group. (\u003cstrong\u003eC3\u003c/strong\u003e) **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 vs. the normal condition group.\u003c/p\u003e","description":"","filename":"image9.png","url":"https://assets-eu.researchsquare.com/files/rs-7212124/v1/d690973ac127956a32e13991.png"},{"id":87979568,"identity":"2071ace1-04e3-4dae-93fa-497ca5016d22","added_by":"auto","created_at":"2025-07-31 05:32:40","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":892682,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003etMCAO induces dynamic alterations in ALP function in astrocytes of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ePrnp\u003c/strong\u003e\u003c/em\u003e\u003csup\u003e\u003cstrong\u003e-/-\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e mice. \u003c/strong\u003e(\u003cstrong\u003eA\u003c/strong\u003e) Time-dependent alterations in the expression of ALP-related proteins in primary astrocyte protein extracts from 3 h to 72 h following OGD/R. Quantification of immunoblotted proteins was performed using ImageJ. Statistical analysis was conducted using ANOVA followed by Tukey’s test or Dunnett’s test. Data are presented as mean ± SEM from more than 4 independent experiments. *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01 vs. normal condition group. (\u003cstrong\u003eB\u003c/strong\u003e) Histograms showing changes in CTSD activity in astrocytes from the normal condition group and OGD/R-treated groups. CTSD activity was assessed using a fluorimetric assay. Statistical analysis was performed using ANOVA followed by Dunnett’s test. Data are presented as mean ± SEM from 3 independent experiments. *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01 vs. normal condition group. (\u003cstrong\u003eC\u003c/strong\u003e) Immunofluorescence images show the colocalization of LAMP1-positive lysosomes (green) with the lysosomal enzyme CTSD (red) in the normal condition group and OGD/R groups. Nuclei are stained with DAPI (blue). Scale bar: 20 μm. Histogram shows changes in the percentage of LAMP1\u003csup\u003e+\u003c/sup\u003eCTSD\u003csup\u003e+\u003c/sup\u003e puncta and LAMP1\u003csup\u003e+\u003c/sup\u003e puncta in each astrocyte. Data are expressed as mean ± SEM from at least 3 independent experiments. *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01 vs. normal condition group. (\u003cstrong\u003eD\u003c/strong\u003e) Immunofluorescence images show colocalization of LAMP1-positive lysosomes (green) with the lysosomal enzyme CTSD (yellow) in astrocytes (red) of the cerebral cortex of sham-operated and MCAO/R mice. Nuclei are stained with DAPI (blue). Scale bar: 50 μm and 5 μm.\u003c/p\u003e","description":"","filename":"image10.png","url":"https://assets-eu.researchsquare.com/files/rs-7212124/v1/78f6ea4a086d9e9933fbb813.png"},{"id":87980202,"identity":"9086d824-92b6-4f0f-95f5-2b4b041e42ec","added_by":"auto","created_at":"2025-07-31 05:48:40","extension":"png","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":994919,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003etMCAO induces dynamic alterations in ALP function in astrocytes of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ePrnp\u003c/strong\u003e\u003c/em\u003e\u003csup\u003e\u003cstrong\u003e-/-\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e mice\u003c/strong\u003e. (\u003cstrong\u003eA\u003c/strong\u003e) Immunofluorescence images showing the colocalization of LAMP1-positive lysosomes (green) with LC3B-positive autophagosomes (red) in the normal condition group and OGD/R groups. Nuclei are stained with DAPI (blue). Scale bar: 20 μm. Histogram shows changes in the number of LAMP1\u003csup\u003e+\u003c/sup\u003e puncta, LC3B\u003csup\u003e+\u003c/sup\u003e puncta, and LAMP1\u003csup\u003e+\u003c/sup\u003eLC3B\u003csup\u003e+\u003c/sup\u003e puncta. Data are presented as mean ± SEM from more than 3 independent experiments. *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, #\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ##\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, \u0026amp;\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 vs. normal condition group. (\u003cstrong\u003eB\u003c/strong\u003e) Immunofluorescence images show colocalization of LAMP1-positive lysosomes (green) with LC3B-positive autophagosomes (yellow) in cortical astrocytes (red) of the sham group and MCAO/R group. Cell nuclei were stained with DAPI (blue). Scale bar: 50 μm and 5 μm. (\u003cstrong\u003eC\u003c/strong\u003e) Expression of LC3B in the normal condition group and OGD/R-treated groups with or without CQ. Quantification of immunoblotted proteins was performed using ImageJ. Statistical analysis was performed using ANOVA. Data are presented as mean ± SEM from 4 independent experiments. (\u003cstrong\u003eC2\u003c/strong\u003e) **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 vs. non-dosed group. (\u003cstrong\u003eC3\u003c/strong\u003e) **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 vs. the normal condition group.\u003c/p\u003e","description":"","filename":"image11.png","url":"https://assets-eu.researchsquare.com/files/rs-7212124/v1/ff1a48e34918fc7b4c398317.png"},{"id":87979570,"identity":"3bb477ea-20bb-46ed-b4b8-abdfe85d990e","added_by":"auto","created_at":"2025-07-31 05:32:41","extension":"png","order_by":12,"title":"Figure 12","display":"","copyAsset":false,"role":"figure","size":949861,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eOverexpression of PrP\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003eC\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e promotes the nuclear translocation of TFEB following CIRI. \u003c/strong\u003e(\u003cstrong\u003eA\u003c/strong\u003e) Time-dependent changes in total TFEB expression from 3 h to 72 h following OGR in \u003cem\u003ePrnp\u003c/em\u003e\u003csup\u003e-/-\u003c/sup\u003e and \u003cem\u003ePrnp\u003c/em\u003e-overexpressing mouse primary astrocyte protein extracts. Quantitative analysis of immunoblotted proteins was performed using ImageJ. Statistical comparisons were carried out with ANOVA followed by Dunnett’s test. Data are presented as mean ± SEM from 4 independent experiments. *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01 vs. normal condition group. (\u003cstrong\u003eB\u003c/strong\u003e) Immunofluorescence images showing TFEB distribution in \u003cem\u003ePrnp\u003c/em\u003e\u003csup\u003e-/-\u003c/sup\u003e and \u003cem\u003ePrnp\u003c/em\u003e-overexpressing mouse primary astrocytes under normal and OGD/R-treated conditions, detected by laser confocal microscopy with TFEB antibodies (red). Nuclei are stained with DAPI (blue). Scale bar: 50 μm. Enlarged images of the boxed areas are shown in the inserts. Columns represent Mander’s overlap coefficient. Data are expressed as mean ± SEM from at least 5 independent experiments per group. **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 vs. normal condition group. (\u003cstrong\u003eC\u003c/strong\u003e) Immunofluorescence staining visualized TFEB distribution (green) in \u003cem\u003ePrnp\u003c/em\u003e\u003csup\u003e-/-\u003c/sup\u003e and \u003cem\u003ePrnp\u003c/em\u003e-overexpressing cortical astrocytes (red) across sham and MCAO/R groups. Nuclei were counterstained with DAPI (blue). Scale bars: 20 μm and 5 μm.\u003c/p\u003e","description":"","filename":"image12.png","url":"https://assets-eu.researchsquare.com/files/rs-7212124/v1/f2c2c384102a87fff22820e1.png"},{"id":87979571,"identity":"6e5dcdef-a452-4a7f-b631-416b4b915046","added_by":"auto","created_at":"2025-07-31 05:32:41","extension":"png","order_by":13,"title":"Figure 13","display":"","copyAsset":false,"role":"figure","size":583256,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAstrocyte-targeted TFEB silencing in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ePrnp\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e-overexpressing mice inhibits ALP function, impairs cellular activity, and induces a pro-inflammatory phenotype during both early and late OGD/R. \u003c/strong\u003e(\u003cstrong\u003eA\u003c/strong\u003e) Immunoblots showing ALP-related protein expression in normal or OGR-treated \u003cem\u003ePrnp\u003c/em\u003e-overexpressing astrocytes incubated with si-Con or si-\u003cem\u003eTfeb\u003c/em\u003e at 6 h and 48 h post-OGR. Quantitative analysis of immunoblotted proteins was performed using ImageJ. Statistical comparisons were carried out with ANOVA followed by Tukey’s test. Data are presented as mean ± SEM from at least 3 independent experiments. **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 vs. si-Con group. (\u003cstrong\u003eB\u003c/strong\u003e) CCK-8 assay results showing cellular viability in normal or OGD/R-treated \u003cem\u003ePrnp\u003c/em\u003e-overexpressing astrocytes incubated with si-Con or si-\u003cem\u003eTfeb\u003c/em\u003e at 6 h and 48 h post-OGR. Statistical comparisons were carried out with ANOVA followed by Tukey’s test. Data are presented as mean ± SEM from 12 independent experiments. **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 vs. si-Con group. (\u003cstrong\u003eC\u003c/strong\u003e) Flow cytometry results from CBAs showing pro- and anti-inflammatory cytokine concentrations in normal or OGD/R-treated \u003cem\u003ePrnp\u003c/em\u003e-overexpressing astrocytes incubated with si-Con or si-\u003cem\u003eTfeb\u003c/em\u003e at 6 h and 48 h post-OGR. Statistical comparisons were carried out with ANOVA followed by Tukey’s test. Data are presented as mean ± SEM from 3 independent experiments. *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01 vs. si-Con group.\u003c/p\u003e","description":"","filename":"image13.png","url":"https://assets-eu.researchsquare.com/files/rs-7212124/v1/ea47518233099f430133edbf.png"},{"id":87979574,"identity":"815112df-3981-4e31-a663-a5c3eb2cb74c","added_by":"auto","created_at":"2025-07-31 05:32:41","extension":"png","order_by":14,"title":"Figure 14","display":"","copyAsset":false,"role":"figure","size":612417,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eOverexpression of PrP\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003eC\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e promotes TFEB nuclear translocation following OGD/R via the Ca²⁺/PPP3 pathway \u003c/strong\u003e(\u003cstrong\u003eA\u003c/strong\u003e) Immunoblot analysis of dynamic PPP3/calcineurin expression changes following OGR in \u003cem\u003ePrnp\u003c/em\u003e\u003csup\u003e-/-\u003c/sup\u003e and \u003cem\u003ePrnp\u003c/em\u003e-overexpressing mouse primary astrocytes. Quantitative analysis of immunoblotted proteins was performed using ImageJ. Statistical comparisons were carried out with ANOVA followed by Dunnett’s test. Data are presented as mean ± SEM from 5 independent experiments. (\u003cstrong\u003eB\u003c/strong\u003e) Histograms showing PPP3/calcineurin activity changes in normal and OGD/R-treated \u003cem\u003ePrnp\u003c/em\u003e\u003csup\u003e-/-\u003c/sup\u003e and \u003cem\u003ePrnp\u003c/em\u003e-overexpressing mouse primary astrocytes. Each sample was analyzed in triplicate. Data are presented as mean ± SEM. Statistical comparisons were carried out with ANOVA followed by Dunnett’s test. **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01 vs. normal condition group. (\u003cstrong\u003eC\u003c/strong\u003e) Quantification of Ca\u003csup\u003e2+\u003c/sup\u003e concentration changes in \u003cem\u003ePrnp\u003c/em\u003e\u003csup\u003e-/-\u003c/sup\u003e and \u003cem\u003ePrnp\u003c/em\u003e-overexpressing mouse primary astrocytes. Statistical comparisons were carried out with ANOVA followed by Tukey’s test. Data are presented as mean ± SEM from 18 independent experiments. *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01 vs. normal condition group. (\u003cstrong\u003eD\u003c/strong\u003e) Immunofluorescence images showing TFEB distribution in \u003cem\u003ePrnp\u003c/em\u003e-overexpressing mouse primary astrocytes incubated with the PPP3/calcineurin inhibitor CsA from 3 h to 72 h following OGR. Enlarged images of the boxed areas are shown in the inserts. Scale bar: 50 μm. Columns represent Mander’s overlap coefficient. Data are presented as mean ± SEM from at least 6 independent experiments. **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 vs. untreated group.\u003c/p\u003e","description":"","filename":"image14.png","url":"https://assets-eu.researchsquare.com/files/rs-7212124/v1/c9c91991cfb70c58ea51de0a.png"},{"id":87979572,"identity":"769e5728-7883-4093-bd6b-34782f74ad71","added_by":"auto","created_at":"2025-07-31 05:32:41","extension":"png","order_by":15,"title":"Figure 15","display":"","copyAsset":false,"role":"figure","size":424818,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePrP\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003eC\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e-Mediated Ca²⁺/Calcineurin/TFEB Signaling Enhances Autophagic-Lysosomal Function and Anti-inflammatory Astrocyte Transition to Alleviate Cerebral Ischemia-Reperfusion Injury.\u003c/strong\u003e During early CIRI, low intracellular Ca\u003csup\u003e2+\u003c/sup\u003e levels inhibit PPP3/calcineurin activity. In late CIRI, elevated Ca\u003csup\u003e2+\u003c/sup\u003e levels activate PPP3/calcineurin, resulting in TFEB dephosphorylation and its nuclear translocation. Nuclear TFEB then drives the transcription of target genes, significantly enhancing lysosomal function and reducing autophagosome accumulation. Upregulated PrP\u003csup\u003eC\u003c/sup\u003e expression helps sustain intracellular Ca\u003csup\u003e2+\u003c/sup\u003e homeostasis, enabling continuous PPP3/calcineurin activation and subsequent TFEB nuclear translocation. This pathway ensures ALP function throughout CIRI, supporting astrocyte survival and anti-inflammatory transformation.\u003c/p\u003e","description":"","filename":"image15.png","url":"https://assets-eu.researchsquare.com/files/rs-7212124/v1/d7808f9dda2d472a9804951e.png"},{"id":91791285,"identity":"0abeb797-cbcb-43a0-a50d-fd91c91d67d7","added_by":"auto","created_at":"2025-09-21 12:31:39","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":12317687,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7212124/v1/602cd055-5e5a-49c8-930a-8435919c74ff.pdf"},{"id":87979564,"identity":"0cc79065-9af1-4d69-8758-104b6db75e21","added_by":"auto","created_at":"2025-07-31 05:32:40","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":2797726,"visible":true,"origin":"","legend":"","description":"","filename":"shaojieAdditionalfile1.docx","url":"https://assets-eu.researchsquare.com/files/rs-7212124/v1/893168fd2f48d8e26bb4f361.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003ePrP\u003csup\u003eC\u003c/sup\u003e-Mediated Ca²\u003csup\u003e⁺\u003c/sup\u003e/Calcineurin/TFEB Signaling Enhances Autophagic-Lysosomal Function and Anti-inflammatory Astrocyte Transition to Alleviate Cerebral Ischemia-Reperfusion Injury\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eStroke remains a leading cause of death and disability worldwide. Currently, recanalization strategies are the most effective treatment for ischemic stroke; however, reperfusion often exacerbates secondary injury. Numerous studies indicate that the astrocyte-mediated immune-inflammatory response plays a significant role in the pathophysiology of cerebral ischemia-reperfusion injury (CIRI) [1-2].\u003c/p\u003e\n\u003cp\u003eAutophagy, an essential intracellular catabolic pathway, helps maintain cellular homeostasis by degrading damaged proteins and organelles through lysosomal pathways [3-4] . Increasing evidence suggests that CIRI induces autophagy activation, and impaired autophagic function is implicated in its pathogenesis. Nonetheless, the outcomes of modulating autophagy in this context have been inconsistent.\u003c/p\u003e\n\u003cp\u003eSome studies suggest that autophagy-lysosomal pathway (ALP) activation exacerbates CIRI. For instance, research involving a transient middle cerebral artery occlusion (tMCAO) model in mice demonstrated that administering autophagy inhibitors such as 3-Methyladenine (3-MA) or Bafilomycin A1 reduced neuronal damage in ischemic regions and improved neurological outcomes [5]. Similarly, an \u003cem\u003ein vitro\u003c/em\u003e oxygen-glucose deprivation and re-oxygenation (OGD/R) study confirmed that 3-MA reduced astrocyte apoptosis by inhibiting ALP activity [6]. However, other studies have reported contrasting findings. Research by Gabryel [7] and Zhao [8] observed in OGD/R models that autophagy inhibitors like 3-MA or chloroquine (CQ) significantly decreased astrocyte viability.\u003c/p\u003e\n\u003cp\u003eThese conflicting data suggest that ALP modulation in CIRI may involve a dynamic process. Additionally, the timing of autophagy modulation is crucial. Carloni\u0026apos;s study found that rapamycin-induced autophagy during ischemia and hypoxia mitigated neuronal damage, while 3-MA-induced autophagy inhibition produced the opposite effect [9]. Conversely, in another mouse tMCAO study, 3-MA administered 48 to 72 hours post-reperfusion significantly reduced infarct size and improved neurological function [10]. These findings indicate that moderate autophagy activation during ischemia and hypoxia supports cellular homeostasis, while excessive autophagy during reperfusion may overwhelm cellular adaptive mechanisms and lead to cell death [11]. Thus, a comprehensive understanding of ALP dynamics in astrocytes post-CIRI may serve as a foundation for its precise therapeutic modulation.\u003c/p\u003e\n\u003cp\u003eTranscription factor EB (TFEB) has recently been recognized as a key regulator of the autophagy-lysosomal pathway (ALP), orchestrating the expression of autophagy and lysosomal genes essential for ALP regulation [12]. Dysregulation of TFEB has been linked to various pathological conditions. For instance, Zhang\u0026rsquo;s study on a manganese-induced Parkinson\u0026rsquo;s model revealed that manganese exposure significantly inhibited TFEB nuclear translocation in astrocytes within the mouse striatum, disrupting ALP function. In this model, TFEB overexpression alleviated manganese-induced mitochondrial dysfunction in astrocytes\u0026nbsp;[13]. Similarly, Gu\u0026apos;s study on cardiomyocyte ischemia-reperfusion injury showed that downregulation of LAPTM4B via the mTORC1/TFEB pathway led to ALP dysfunction, resulting in cardiomyocyte death. Conversely, TFEB upregulation reversed ischemia-reperfusion injury in cardiomyocytes following LAPTM4 knockdown\u0026nbsp;[14]. Currently, most studies investigating TFEB in the context of CIRI have focused on its neuronal roles. Enhancing TFEB nuclear translocation in neurons has been shown to exert neuroprotective effects by restoring ALP function, thereby mitigating ischemic injury\u0026nbsp;[15]. However, the specific changes in TFEB within astrocytes after CIRI, its upstream regulatory mechanisms, and its impact on ALP function in these cells remain unknown.\u003c/p\u003e\n\u003cp\u003eCellular prion protein (PrP\u003csup\u003eC\u003c/sup\u003e) is a cell surface glycoprotein encoded by the \u003cem\u003ePrnp\u0026nbsp;\u003c/em\u003egene, widely distributed throughout the central nervous system. When PrP\u003csup\u003eC\u003c/sup\u003e undergoes a conformational change, it is converted to the pathogenic and infectious scrapie isoform (PrP\u003csup\u003eSc)\u003c/sup\u003e, which is implicated in prion diseases such as bovine spongiform encephalopathy in animals and Creutzfeldt-Jakob disease in humans [16]. PrP\u003csup\u003eC\u003c/sup\u003e exerts a notable neuroprotective effect in CIRI. Overexpression of PrP\u003csup\u003eC\u003c/sup\u003e in microglia promotes an anti-inflammatory phenotype, likely due to its ability to delay lysosomal depletion and sustain ALP functionality, although the precise molecular mechanisms remain unclear [17-18].\u003c/p\u003e\n\u003cp\u003eTo investigate this, we assessed changes in ALP patterns following CIRI, with a focus on whether TFEB can modulate ALP function and thereby protect astrocytes from ischemic injury. Our findings provide the first evidence that CIRI induces dynamic alterations in ALP. In the early phase of CIRI, ALP partially blockade occurs alongside decreased TFEB expression and nuclear translocation, driven by mTOR reactivation and inhibition of PPP3/calcineurin activity. With extended reperfusion, PPP3/calcineurin activation restores TFEB nuclear translocation, alleviating ALP impairment, which ultimately supports astrocyte survival and promotes a shift to an anti-inflammatory phenotype. Notably, overexpression of PrP\u003csup\u003eC\u003c/sup\u003e maintains PPP3/calcineurin activation by preserving intracellular Ca\u003csup\u003e2+\u0026nbsp;\u003c/sup\u003ehomeostasis, which in turn sustains TFEB dephosphorylation and nuclear translocation. This study offers new insights for potential therapeutic interventions in ischemic stroke.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cstrong\u003eAnimals\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOne-day-old neonatal FVB/N wild-type (WT; obtained from the Vital River Laboratory Animal Technology Co., Ltd., Beijing, China), \u003cem\u003ePrnp\u003c/em\u003e\u003csup\u003e-/-\u003c/sup\u003e, and \u003cem\u003ePrnp\u003c/em\u003e-overexpressing mice (obtained from the Institute of Medical Laboratory Animals, Chinese Academy of Medical Sciences) were used in this study. All experimental protocols were approved by the Institutional Animal Care and Use Committee at the First Hospital of Jilin University.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMouse transient Middle Cerebral Artery Occlusion (tMCAO) Model\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMice underwent isoflurane anesthesia. Following a ventral midline neck incision, the right common (CCA), external (ECA), and internal carotid (ICA) arteries were exposed. The proximal CCA and distal ECA were ligated. A paraffin-coated 4-0 nylon suture (diameter 0.26 mm) was advanced from the CCA into the ICA and positioned to occlude the origin of the right middle cerebral artery at the Circle of Willis. Sham-operated animals received identical procedures excluding the suture occlusion. Animals were euthanized at designated time points for subsequent analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePrimary cortical astrocyte culture and PrP\u003csup\u003eC\u003c/sup\u003e protein assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePrimary cortical astrocytes were cultured from one-day-old FVB/N mice as previously described [19]. Briefly, cortices from neonatal mouse brains were aseptically dissected, meninges were removed, and single-cell suspensions were prepared by gentle trituration. Cells were cultured in Dulbecco\u0026rsquo;s modified Eagle\u0026rsquo;s medium (DMEM; Gibco, USA, 8123234) supplemented with 10% fetal bovine serum (FBS; Gibco, USA, 10010023) and 1% penicillin/streptomycin (ThermoFisher, USA, 15240062). Mixed glial cultures were maintained \u003cem\u003ein vitro\u003c/em\u003e until confluent, and the uppermost layer of microglia was removed by shaking on an orbital shaker at 200 rpm for 4 hours. The middle layer of astrocytes was isolated by incubation in 0.25% trypsin-EDTA (diluted 1:2 in DMEM; Gibco, USA, 25200072) for 15\u0026ndash;25 minutes, leaving the lower layer of microglia adhered to the culture flask. The mid-layer cells were then collected and incubated until confluent, with the process repeated to obtain purified astrocytes.\u003c/p\u003e\n\u003cp\u003eAstrocyte purity was verified by immunofluorescence staining with rat anti-glial fibrillary acidic protein (GFAP, an astrocyte-specific marker; 1:200, Abcam, China, ab27929) (\u003cstrong\u003eFigure S1A).\u003c/strong\u003e Flow cytometry was also performed using rat anti-CD11b-FITC (a microglia-specific marker; ThermoFisher, USA, 11-0112-82) to exclude microglial contamination (\u003cstrong\u003eFigure S1B)\u003c/strong\u003e. PrP\u003csup\u003eC\u0026nbsp;\u003c/sup\u003eprotein levels in WT, \u003cem\u003ePrnp\u003csup\u003e-\u003c/sup\u003e\u003c/em\u003e\u003csup\u003e/-\u003c/sup\u003e, and \u003cem\u003ePrnp\u003c/em\u003e-overexpressing astrocytes were assessed by immunofluorescence staining with rabbit anti-PrP antibody (1:600, Invitrogen, USA, MA5-32202) (\u003cstrong\u003eFigure S5)\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eOGD/R in primary cortical astrocytes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor oxygen-glucose deprivation (OGD), astrocytes were washed three times with phosphate-buffered saline (PBS; Gibco, 10010023) and then incubated in glucose-free DMEM (Gibco, 11966-025). Cells were cultured in MIC-101 hypoxic chambers maintained at 37\u0026deg;C, with a gas mixture of 0.1% O₂, 94.9% N₂, and 5% CO₂ for 2 hours. Following OGD, glucose-containing medium was reintroduced, and cells were transferred to a normoxic incubator with 5% CO₂ and 95% air to initiate oxygen-glucose resupply (OGR). Astrocyte samples were collected at 3, 6, 12, 24, 48, and 72 hours post-OGD/R to analyze the dynamic response. At the onset of OGD/R, astrocytes were treated with various drugs, including the lysosome inhibitor chloroquine (CQ, 50 \u0026micro;M; Selleck, S6999), mechanistic target of rapamycin kinase inhibitor rapamycin (mTOR, 200 nM; Selleck, S1039), mTOR agonist MHY1485 (500 \u0026micro;M; Selleck, S7811), and calcineurin inhibitor cyclosporin A (CsA, 10 \u0026micro;M; Selleck, S2286). The maximum concentration of DMSO used in the experiments (0.1%) was confirmed to be non-toxic to the cells.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAstrocyte Activity assay\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAstrocyte activity was assessed using the Cell Counting Kit-8 (CCK-8; Beyotime Biotech, C0041) according to the manufacturer\u0026rsquo;s instructions. Briefly, purified astrocytes were seeded into a 96-well plate, and 10 \u0026micro;L of CCK-8 solution was added 1 hour before the end of OGD/R. Absorbance was then measured at an excitation wavelength of 450 nm using a microplate reader (Thermo Fisher Scientific, USA).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmunoblotting\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAstrocytes were lysed using Cell lysis buffer for Western and IP (Beyotime Biotech, P0013J) supplemented with a protease and phosphatase inhibitor mixture (Solarbio, P1260). The cell lysates were then centrifuged at 12,000\u0026nbsp;\u0026times;\u0026nbsp;g for 15 minutes at 4\u0026deg;C. The resulting pellets (Triton X-100-insoluble fractions) were washed three times with lysis buffer and resuspended in SDS lysis buffer (Beyotime Biotech, P0013G), followed by another round of centrifugation at 12,000\u0026nbsp;\u0026times;\u0026nbsp;g for 15 minutes. Total protein concentration was measured using a BCA protein assay kit (Epizyme, China, 23225). Protein aliquots (10 \u0026micro;g) from each fraction were separated by SDS-PAGE (4-20%, GenScript, USA, M00657), and the gel-separated proteins were transferred onto 0.22 \u0026micro;m polyvinylidene fluoride membranes, which were blocked with 5% skimmed milk in TBST (0.1% Tween 20 in Tris-buffered saline). After blocking, the membranes were incubated overnight at 4\u0026deg;C with primary antibodies: rabbit anti-\u0026nbsp;LC3B (1:1000, Abcam, USA, ab192890), SQSTM1 (, 1:1000, Abcam, USA, ab91526), CTSD (1:1000, Abcam, USA, ab65302), CTSB (1:2000, Abcam, USA, ab214428), ubiquitin (1:1000, ProteinTech, USA, 10201-2-AP),\u0026nbsp;\u0026beta;-actin (1:5000, ProteinTech, USA, 81115-1-RR), TFEB (1:1000, ProteinTech, USA, 13372-1AP), PPP3 (1:1000, Cell Signaling Technology, USA, 2614), mTOR (1:1000, Cell Signaling Technology, USA, 2983), phospho-mTOR (1:1000, Cell Signaling Technology, USA, 2974), and rat anti-\u0026nbsp;LAMP1\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e(1:2000, eBioscience, USA, 14-1071-82). After three washes, membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (1:10,000, Cohesion Biosciences, CSA2115 and CSA2133) for 2 hours at room temperature. Protein bands were visualized using Omni-ECL\u0026trade;\u0026nbsp;Chemiluminescent Substrate (Epizyme, China, SQ201) and a ChemiDoc MP imaging system (Bio-Rad Laboratories, CA, USA). The intensity of the protein bands was quantified using ImageJ software, with the intensity expressed as the relative value compared to the control.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmunofluorescence staining\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAstrocytes \u003cem\u003ein vitro\u003c/em\u003e\u003c/strong\u003e: Cells were washed twice with ice-cold PBS, followed by fixation and permeabilization with 4% (w:v) paraformaldehyde supplemented with 0.2% (v:v) Triton X-100 for 30 minutes at room temperature. After washing three times with PBS, the cells were blocked with 10% goat serum in PBS for 2 hours at room temperature. The cells were then incubated overnight at 4\u0026deg;C with primary antibodies: rabbit anti-LC3B (1:200, Abcam, USA, ab192890), CTSD (1:200, Abcam, USA, ab65302), TFEB (1:200, eBioscience, USA, 14-1071-82), and rat anti-LAMP1 (1:200, eBioscience, USA, 14-1071-82). Following primary antibody incubation, cells were washed three times with PBS and incubated with Alexa Fluor 488- and Alexa Fluor 594-conjugated secondary antibodies (1:200, Abcam, ab150165 and ab150080) for 2 hours at room temperature, in the dark, in PBS containing 2% goat serum. The cells were then washed three times and counterstained with DAPI (Solarbio, 0065) for nuclear labeling. After final washes with PBS, cells on glass coverslips were mounted onto glass slides using Anti-fluorescent Attenuation Sealer (Solarbio, 188105). Images were captured using a laser confocal microscope and analyzed using ImageJ software.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMouse brain tissue sections\u003c/strong\u003e: Following deparaffinization and antigen retrieval, paraffin sections were subjected to serum blocking and then processed through three sequential cycles of immunostaining. Each cycle consisted of: overnight incubation (4\u0026deg;C) with a primary antibody, application of a species-matched HRP-conjugated secondary antibody, tyramide signal amplification (TSA) with the corresponding fluorophore, and thorough antibody stripping followed by serum reblocking prior to the subsequent cycle. After completing the third cycle, nuclei were counterstained with DAPI, and sections were mounted for imaging using laser scanning confocal microscopy.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCathepsin D activity assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAstrocyte lysates (n = 3 per group) from normal and OGD/R conditions were collected at the indicated time points using CTSD lysis buffer. The samples were centrifuged at 12,000\u0026nbsp;\u0026times;\u0026nbsp;g for 10 minutes at 4\u0026deg;C, and the supernatants were collected. Protein concentration was quantified using a BCA protein assay kit (Epizyme, ZJ102). CTSD activity was measured using a fluorometric CTSD activity assay kit (Abcam, ab65302), following the manufacturer\u0026apos;s instructions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCellular Bead Array\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCytokine levels in astrocyte culture media (n = 3 per group) were measured using the mouse Th1/Th2/Th17 cytokine kit (BD Biosciences, Franklin Lakes, NJ, USA, 560485), following the manufacturer\u0026apos;s instructions. Interleukin (IL)-6, IL-10, tumor necrosis factor-\u0026alpha;\u0026nbsp;(TNF-\u0026alpha;), and interferon-gamma (IFN-\u0026gamma;) were selected as representative cytokines for assessing pro-inflammatory and anti-inflammatory states in astrocytes.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eProtein phosphatase assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAstrocyte lysates (n = 3 per group) from normal and OGD/R conditions were collected at the specified time points using lysis buffer. The samples were centrifuged at 100,000\u0026ndash;200,000\u0026nbsp;\u0026times;\u0026nbsp;g for 45 minutes at 4\u0026deg;C, and the supernatants were collected. Protein concentrations were determined using the Bradford method. PPP3/calcineurin activity was assessed using a calcineurin phosphatase assay kit (ZNEO, BML-AK816-0001), following the manufacturer\u0026apos;s instructions.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCalcium imaging\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAstrocyte samples from normal and OGD/R groups were washed three times with calcium-free Hank\u0026apos;s balanced salt solution (HBSS, Solarbio, H1040) to remove residual serum. The cells were then incubated with 5 \u0026micro;M Fluo-4 AM (Beyotime, S1060) in calcium-free HBSS for 30 minutes at 37\u0026deg;C. Afterward, the cells were washed three times with calcium-free HBSS and further incubated in the same solution for an additional 30 minutes to complete deesterification. Fluorescence measurements were taken at an excitation wavelength of 494 nm and emission wavelength of 516 nm using a multifunctional enzyme reader to determine the intracellular calcium ion concentration.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSmall interfering RNA (siRNA) is used to silence \u003cem\u003eTfeb\u003c/em\u003e expression in astrocytes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe used siRNA to silence \u003cem\u003eTfeb\u003c/em\u003e in primary mouse astrocytes, with siRNA purchased from Integrated Biotech Solutions. The si-\u003cem\u003eTfeb\u003c/em\u003e primer sequences were as follows:\u003c/p\u003e\n\u003cp\u003eSense: 5\u0026apos;-GACGCAGGUUUCAACAUCAAUG-3\u0026apos;\u003c/p\u003e\n\u003cp\u003eAntisense: 5\u0026apos;-UUGAUGUUGAACCUGCGUCUUU-3\u0026apos;\u003c/p\u003e\n\u003cp\u003eThe si-Control primer sequences were:\u003c/p\u003e\n\u003cp\u003eSense: 5\u0026apos;-UUCUCCGAACGUGUCACGUTT-3\u0026apos;\u003c/p\u003e\n\u003cp\u003eAntisense: 5\u0026apos;-ACGUGACACGUUCGGAGAATT-3\u0026apos;\u003c/p\u003e\n\u003cp\u003eThe siRNA was transfected into primary astrocytes using the JiePRIME Transfection Kit (Polyplus, France, 101000046) according to the manufacturer\u0026rsquo;s instructions. Transfection efficiency was determined by immunofluorescence staining, and silencing efficiency of si-T\u003cem\u003efeb\u0026nbsp;\u003c/em\u003ewas verified by both immunofluorescence staining and immunoblotting (\u003cstrong\u003eFigure S3\u003c/strong\u003e).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePlasmids is used to\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;induce the overexpression of \u003cem\u003eTfeb\u003c/em\u003e in astrocytes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe used plasmids to induce \u003cem\u003eTfeb\u003c/em\u003e overexpression in astrocytes, which were purchased from Integrated Biotech Solutions. The plasmids used were: Target plasmid: pcDNA3.1(+)-mouse Tfeb-3\u0026times;FLAG Amp+ (1 \u0026micro;g/\u0026micro;L). Control plasmid: pcDNA3.1(+) Amp+ (1 \u0026micro;g/\u0026micro;L). Fluorescent control plasmid: Pegfp-N1 Amp+ (1 \u0026micro;g/\u0026micro;L). The plasmids were amplified and transfected into primary astrocytes using the Plasmid Extraction Kit (Tengen Biochemistry, Germany, DP120) and the Lipofectamine\u0026trade;3000 Transfection Kit (Thermo Fisher Scientific, USA, L3000001), according to the manufacturer\u0026apos;s instructions. Transfection efficiency was assessed by immunofluorescence staining, and the efficiency of \u003cem\u003eTfeb\u003c/em\u003e overexpression in astrocytes was confirmed through immunoblotting (\u003cstrong\u003eFigure S4)\u003c/strong\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll experimental data were derived from at least three independent replicate experiments. Data processing, analysis, and plotting were conducted using GraphPad Prism 8 and Excel software. Normally distributed data are presented as means\u0026nbsp;\u0026plusmn;\u0026nbsp;SEM. Statistical analyses included one-way or two-way ANOVA followed by Tukey\u0026rsquo;s or Dunnett\u0026rsquo;s post hoc tests for multiple comparisons, and Student\u0026rsquo;s t-test for two-group comparisons. Statistical significance was set at \u003cem\u003eP\u003c/em\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u0026lt; 0.05.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eTransient cerebral ischemia results in a dynamic change of ALP\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe tMCAO mice and OGD/R astrocytes served as \u003cem\u003ein vivo\u003c/em\u003e and \u003cem\u003ein vitro\u003c/em\u003e models of cerebral ischemia-reperfusion, respectively. Elevated neurological deficit scores in tMCAO mice, alongside reduced astrocyte viability post-OGD/R (specifically: significant decline at 3-6 h, recovery at 12 h, and return to baseline at 48-72 h), confirmed the successful establishment of their respective models\u003cstrong\u003e\u0026nbsp;(Figure S2)\u003c/strong\u003e. However, the extent to which these changes reflect alterations in ALP processing remains controversial and warrants further investigation. ALP is a complex process with three primary stages\u0026mdash;autophagosome formation, autophagosome-lysosome fusion, and lysosomal degradation. Dysfunction in any of these stages can impair ALP [20-22]. Thus, we monitored dynamic changes in ALP function through markers of autophagic and lysosomal activity.\u003c/p\u003e\n\u003cp\u003eImmunoblotting was performed to analyze the expression of autophagy-related proteins in mouse cortical astrocytes from 3 to 72 hours post-OGD/R. Results showed a significant increase in the autophagic marker MAP1LC3B-II/I from 3 to 6 hours (\u003cstrong\u003eFigure 1A1, A2)\u003c/strong\u003e, as well as elevated levels of the autophagic substrate SQSTM1 in both Triton X-100-soluble and -insoluble fractions from 3 to 12 hours post-OGD/R (Figure 1A1, A3-A4). In contrast, no significant changes were observed in MAP1LC3-II/I or SQSTM1 levels between 48 and 72 hours post-OGD/R. However, levels of another autophagic substrate, ubiquitin, decreased significantly during this later period (\u003cstrong\u003eFigure 1A1, A5\u003c/strong\u003e). These observations suggest an abnormal accumulation of autophagosomes and substrates during early OGD/R, which diminishes in the later phase.\u003c/p\u003e\n\u003cp\u003eLysosomes are critical organelles for degrading autophagosome contents\u0026nbsp;[23-24]. We assessed lysosomal quantity and function in astrocytes from 3 to 72 hours following CIRI, with LAMP1 used as a marker of lysosomal quantity, and CTSD and CTSB as markers of lysosomal function. Results indicated that LAMP1 expression remained unchanged from 3 to 24 hours post-OGD/R (\u003cstrong\u003eFigure 1A8\u003c/strong\u003e). However, there was a significant decrease in pro-CTSD and mCTSB expression (\u003cstrong\u003eFigure 1A6-A7\u003c/strong\u003e), CTSD enzymatic activity \u003cstrong\u003e(Figure 1B\u003c/strong\u003e), and the percentage of functional lysosomes (LAMP1\u003csup\u003e+\u003c/sup\u003eCTSD\u003csup\u003e+\u003c/sup\u003e) \u003cstrong\u003e(Figure 1C\u003c/strong\u003e), suggesting lysosomal dysfunction despite an unchanged lysosome count in early OGD/R. In contrast, from 48 to 72 hours post-OGD/R, LAMP1 expression increased significantly, with pro-CTSD and mCTSB expression, CTSD activity, and functional lysosome levels (LAMP1\u003csup\u003e+\u003c/sup\u003eCTSD\u003csup\u003e+\u003c/sup\u003e) returning to baseline, indicating both replenishment of lysosome quantity and restoration of function in late OGD/R. Similarly, in astrocytes of MCAO/R mice, functional lysosome levels (LAMP1\u003csup\u003e+\u003c/sup\u003eCTSD\u003csup\u003e+\u003c/sup\u003e) decreased at 6 h and recovered to baseline levels at 48 h (\u003cstrong\u003eFigure 1D\u003c/strong\u003e). These results are consistent with \u003cem\u003ein vitro\u003c/em\u003e data.\u003c/p\u003e\n\u003cp\u003eTo further investigate the relationship between autophagosome and lysosome dynamics, we used MAP1LC3B-positive particles as markers for autophagosomes, LAMP1-positive particles for lysosomes, and co-localized MAP1LC3B and LAMP1 particles for autolysosomes. Immunofluorescence staining analysis revealed that autophagosome numbers significantly increased from 3 to 24 hours post-OGD/R, while lysosome numbers remained stable, suggesting that lysosomal insufficiency contributed to autophagosome accumulation. Conversely, from 48 to 72 hours post-OGD/R, autophagosome and lysosome numbers were balanced, both showing increases (\u003cstrong\u003eFigure 2A\u003c/strong\u003e). Similarly, in astrocytes of MCAO/R mice, autophagosome numbers were significantly elevated at 6 h post-reperfusion, whereas lysosome counts remained unaltered. In contrast, both autophagosomes and lysosomes increased significantly by 48 h (\u003cstrong\u003eFigure 2B\u003c/strong\u003e). These results are consistent with \u003cem\u003ein vitro\u003c/em\u003e data.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThese findings indicate that in early CIRI, lysosomal insufficiency and dysfunction lead to autophagosome and substrate accumulation. In late CIRI, lysosomal upregulation and restored function alleviate this accumulation of autophagic substrates.\u003c/p\u003e\n\u003cp\u003eTo further elucidate changes in autophagic flux, we assessed MAP1LC3B levels in OGD/R-treated astrocytes during early and late OGD/R phases, with and without CQ treatment, which inhibits lysosomal proteases or blocks autophagosome-lysosome fusion (\u003cstrong\u003eFigure 2C\u003c/strong\u003e). Immunoblotting revealed that CQ treatment significantly increased MAP1LC3-II/I level at both 6 and 48 hours post-OGD/R, as well as under normal conditions, indicating that OGD/R did not cause a complete ALP blockade (\u003cstrong\u003eFigure 2C1-C2\u003c/strong\u003e). Notably, the degree of CQ-induced MAP1LC3B-II/I elevation was significantly reduced at 6 hours and returned to baseline at 48 hours post-OGD/R compared to normal conditions (\u003cstrong\u003eFigure 2C3\u003c/strong\u003e), suggesting that while ALP was partially impaired in early OGD/R, its function improved during late OGD/R.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTransient cerebral ischemia induces the activation of TFEB\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo explore the association between dynamic changes in ALP and TFEB nuclear translocation, we examined TFEB levels in total fractions via immunoblotting and TFEB\u0026apos;s intracellular distribution using immunofluorescence. Immunoblotting analysis showed that total TFEB expression significantly decreased between 3 and 6 hours, began to increase at 12 hours, peaked at 24 hours, and returned to baseline levels by 72 hours post-OGD (\u003cstrong\u003eFigure 3A\u003c/strong\u003e). Immunofluorescence analysis showed diffuse cytoplasmic TFEB staining under normal conditions, with perinuclear accumulation observed starting at 3 hours post-OGD/R, prOGD/Ressing towards the nucleus at 12 hours, and ceasing by 72 hours. This shift was supported by an increase in mean optical density and a notable rise in the Mander\u0026rsquo;s overlap coefficient, indicating enhanced nuclear localization (\u003cstrong\u003eFigure 3B\u003c/strong\u003e). Similarly, in mouse astrocytes, TFEB exhibited cytoplasmic staining in the sham group and the MCAO/R(6 h) group, while in the MCAO/R( 6 h) group, TFEB exhibited significant nuclear staining (\u003cstrong\u003eFigure 3C\u003c/strong\u003e). These results are consistent with \u003cem\u003ein vitro\u003c/em\u003e data. These findings suggest that transient cerebral ischemia induces TFEB nuclear translocation in astrocytes.\u003c/p\u003e\n\u003cp\u003eTFEB nuclear translocation is partially regulated by phosphorylation via the mTOR and Ca\u003csup\u003e2+\u003c/sup\u003e/PPP3 pathways. To investigate the impact of the Ca\u003csup\u003e2+\u003c/sup\u003e/PPP3 pathway on TFEB nuclear translocation in WT primary astrocytes from 3 to 72 hours following OGD/R, we measured intracellular Ca\u003csup\u003e2+\u003c/sup\u003e levels and PPP3 activity. Ca\u003csup\u003e2+\u003c/sup\u003e levels decreased at 6 hours, began to increase at 12 hours, peaked at 24 hours, and returned to baseline at 72 hours post-OGD/R (\u003cstrong\u003eFigure 4A)\u003c/strong\u003e. Although PPP3 protein expression remained stable, PPP3 enzyme activity significantly increased between 12 and 48 hours post-OGD/R (\u003cstrong\u003eFigure 4B-C\u003c/strong\u003e). When CsA, a PPP3 inhibitor, was applied, it markedly inhibited TFEB nuclear translocation from 12 to 48 hours post-OGD/R, as shown by immunofluorescence (\u003cstrong\u003eFigure 4D)\u003c/strong\u003e. These findings indicate that the Ca\u003csup\u003e2+\u003c/sup\u003e/PPP3 pathway is crucial in regulating TFEB nuclear translocation in astrocytes following OGD/R.\u003c/p\u003e\n\u003cp\u003eWe also examined the role of the mTOR pathway in TFEB nuclear translocation in WT primary astrocytes from 3 to 72 hours following OGD/R. The p-mTOR/mTOR ratio remained unchanged between 3 and 6 hours but significantly increased from 12 to 72 hours post-OGD/R(\u003cstrong\u003eFigure 4E\u003c/strong\u003e). Although mTOR theoretically inhibits TFEB nuclear translocation, this observation contrasts with the actual nuclear translocation of TFEB seen from 12 to 48 hours post-OGD/R. To further clarify, we treated astrocytes with Rapamycin to inhibit p-mTOR activity and MHY1485 to activate it. Results showed that Rapamycin significantly promoted TFEB nuclear translocation from 3 to 6 hours, while MHY1485 markedly inhibited it from 12 to 72 hours post-OGD/R, compared to the untreated group (\u003cstrong\u003eFigure 4F\u003c/strong\u003e). These findings suggest that the mTOR pathway primarily acts to inhibit TFEB nuclear translocation during OGD/R, likely to prevent excessive autophagy in astrocytes.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAstrocyte-targeted TFEB enhances ALP function, boosts cellular activity, and promotes a shift toward an anti-inflammatory phenotype following OGD/R\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBased on the observations above, we propose two hypotheses: 1) Is TFEB involved in regulating the ALP and influencing phenotypic transformation of astrocytes during late OGD/R? 2) Can upregulation of TFEB expression alleviate ALP dysfunction in the early phase of OGD/R?\u003c/p\u003e\n\u003cp\u003eTo investigate whether TFEB regulates the ALP and influences astrocyte phenotypic transformation during late OGD/R, we used siRNA to silence TFEB expression in WT mouse primary astrocytes. Immunoblotting results revealed that TFEB expression in the si-\u003cem\u003eTfeb\u003c/em\u003e group at 48 h following OGD/R was significantly reduced compared to the si-Con group. Concurrently, the expression of MAP1LC3B-II/I and ubiquitin was significantly increased, while the expression of LAMP1, CTSD, and mCTSB was significantly decreased (\u003cstrong\u003eFigure 5A\u003c/strong\u003e). CCK8 assays showed a significant reduction in astrocyte viability in the si-\u003cem\u003eTfeb\u003c/em\u003e group at 48 h following OGD/R compared to the si-Con group (\u003cstrong\u003eFigure 5B\u003c/strong\u003e). CBA analysis demonstrated that the levels of the anti-inflammatory cytokine IL-10 were reduced, while pro-inflammatory cytokines TNF-\u0026alpha;\u0026nbsp;and IL-6 were significantly elevated in the culture supernatants of the OGD/R 48 h si-\u003cem\u003eTfeb\u003c/em\u003e group compared to the si-Con group (\u003cstrong\u003eFigure 5C\u003c/strong\u003e). These results suggest that TFEB plays a role in promoting the conversion of astrocytes to an anti-inflammatory phenotype by enhancing ALP function during late OGD/R.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo investigate whether upregulation of TFEB expression during early OGD/R could alleviate ALP blockade, we overexpressed TFEB in astrocytes using a plasmid. The results showed that the expression of MAP1LC3B-II/I, LAMP1, CTSD, and mCTSB did not significantly change in the OGD/R 6 h \u003cem\u003eTfeb\u003c/em\u003e overexpression group compared to the vector group (\u003cstrong\u003eFigure 6A\u003c/strong\u003e). These findings suggest that upregulation of TFEB expression does not improve ALP blockade in astrocytes during early OGD/R.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eOverexpression of PrP\u003csup\u003eC\u003c/sup\u003e preserves ALP functionality following CIRI, enhances astrocyte viability, and promotes astrocyte phenotypic switching from pro-inflammatory to anti-inflammatory states.\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePrevious studies have shown that PrP\u003csup\u003eC\u0026nbsp;\u003c/sup\u003ehas neuroprotective effects in cerebral ischemic injury\u0026nbsp;[25]. To investigate whether these protective effects are related to ALP, we examined PrP\u003csup\u003eC\u0026nbsp;\u003c/sup\u003eexpression from 3 h to 72 h following OGD/R in WT mouse primary astrocytes. Immunoblotting results revealed a significant increase in PrP\u003csup\u003eC\u003c/sup\u003e expression from 24 h to 48 h following OGD/R (\u003cstrong\u003eFigure 7A\u003c/strong\u003e), suggesting that PrP\u003csup\u003eC\u0026nbsp;\u003c/sup\u003emay play a role in improving ALP function in astrocytes during late OGD/R.\u003c/p\u003e\n\u003cp\u003eNext, we cultured WT, \u003cem\u003ePrnp\u003c/em\u003e\u003csup\u003e-/-\u003c/sup\u003e,and \u003cem\u003ePrnp-\u003c/em\u003eoverexpressing mouse astrocytes i\u003cem\u003en vitro\u0026nbsp;\u003c/em\u003e(\u003cstrong\u003eFigure S5\u003c/strong\u003e) and subjected them to OGD/R treatment. CCK-8 assays indicated that astrocyte activity in WT mice decreased between 3 and 6 hours post-OGD/R showed an increasing trend by 12 hours, and returned to baseline levels by 48 hours (\u003cstrong\u003eFigure S2\u003c/strong\u003e). In \u003cem\u003ePrnp\u003csup\u003e-\u003c/sup\u003e\u003c/em\u003e\u003csup\u003e/-\u0026nbsp;\u003c/sup\u003eastrocytes, activity remained reduced from 3 h to 72 h following OGD/R. In contrast, \u003cem\u003ePrnp\u003c/em\u003e-overexpressing astrocytes showed decreased activity from 3 h to 6 h, but increased activity from 48 h to 72 h following OGD/R, compared to normal conditions. Furthermore, \u003cem\u003ePrnp\u003csup\u003e-\u003c/sup\u003e\u003c/em\u003e\u003csup\u003e/-\u0026nbsp;\u003c/sup\u003eastrocytes exhibited significantly lower activity from 12 h to 72 h compared with WT astrocytes, while \u003cem\u003ePrnp\u003c/em\u003e-overexpressing astrocytes showed improved activity both from 3 h to 6 h and 48 h to 72 h (\u003cstrong\u003eFigure 7B\u003c/strong\u003e). These results suggest that overexpression of PrP\u003csup\u003eC\u0026nbsp;\u003c/sup\u003eameliorates impaired astrocyte activity induced by OGD/R. CBA assays revealed that IL-10 levels in the supernatants of \u003cem\u003ePrnp\u003csup\u003e-\u003c/sup\u003e\u003c/em\u003e\u003csup\u003e/-\u0026nbsp;\u003c/sup\u003eastrocytes were significantly lower at 72 h following OGD/R compared to WT astrocytes, while TNF and IFN-\u0026gamma;\u0026nbsp;levels were significantly higher from 24 h to 48 h. In contrast, IL-10 levels in the supernatants of\u003cem\u003e\u0026nbsp;Prnp\u003c/em\u003e-overexpressing astrocytes were significantly elevated from 3 h to 72 h, whereas TNF and IFN-\u0026gamma;\u0026nbsp;levels were significantly reduced from 3 h to 72 h, with IL-6 levels significantly decreased from 6 h to 12 h (\u003cstrong\u003eFigure 7C\u003c/strong\u003e). These findings suggest that overexpression of PrP\u003csup\u003eC\u0026nbsp;\u003c/sup\u003efacilitates the transformation of astrocytes from a pro-inflammatory to an anti-inflammatory phenotype following OGD/R.\u003c/p\u003e\n\u003cp\u003eSubsequently, we examined the dynamics of ALP in cortical astrocytes from \u003cem\u003ePrnp\u003csup\u003e-\u003c/sup\u003e\u003c/em\u003e\u003csup\u003e/-\u0026nbsp;\u003c/sup\u003eand \u003cem\u003ePrnp-\u003c/em\u003eoverexpressing mice. As demonstrated in the previous section, ALP was partially blocked in WT mouse primary astrocytes during early CIRI and alleviated during late CIRI. In \u003cem\u003ePrnp-\u003c/em\u003eoverexpressing astrocytes, we observed the following: 1) The expression of the autophagy marker MAP1LC3-II/I decreased or showed a decreasing trend from 6 h to 72 h following OGD/R (\u003cstrong\u003eFigure 8A1-A2\u003c/strong\u003e), which suggests two possibilities: either autophagosome formation is inhibited or autophagosomes are rapidly degraded. 2) The expression levels of LAMP1, CTSD, and mCTSB, as well as CTSD enzyme activity, increased or showed an upward trend from 3 h to 72 h following OGD/R (\u003cstrong\u003eFigure 8A1, A6-A8, B\u003c/strong\u003e), indicating that \u003cem\u003ePrnp\u003c/em\u003e overexpression contributes to the replenishment of lysosomes and the improvement of lysosomal dysfunction at various stages of OGD/R. 3) The proportion of functional lysosomes (LAMP\u003csup\u003e+\u003c/sup\u003eCTSD\u003csup\u003e+\u003c/sup\u003e) significantly increased from 3 h to 72 h following OGD/R and MCAO/R (\u003cstrong\u003eFigure 8C-D\u003c/strong\u003e), suggesting that\u003cem\u003e\u0026nbsp;Prnp\u003c/em\u003e overexpression helps maintain the number of functional lysosomes throughout different periods of CIRI. 4) Fluorescence co-localization of MAP1LC3B with LAMP1 showed that the number of autophagosomes decreased significantly from 3 h to 72 h following OGD/R and MCAO/R, while the number of lysosomes increased (\u003cstrong\u003eFigure 9A-B\u003c/strong\u003e). This suggests that a sufficient number of lysosomes can rapidly degrade CIRI-induced accumulation of autophagosomes. 5) Assessment of ALP activity using the lysosomal enzyme inhibitor CQ revealed that, compared with the normal group, the degree of elevation of MAP1LC3B-II/I following chloroquine treatment did not change significantly at 6 h post-OGD/R but increased significantly at 48 h (\u003cstrong\u003eFigure 9C\u003c/strong\u003e). These findings suggest that \u003cem\u003ePrnp\u0026nbsp;\u003c/em\u003eoverexpression contributes to the maintenance of ALP function during both the early and late stages of CIRI.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn contrast, we observed in \u003cem\u003ePrnp\u003c/em\u003e\u003csup\u003e-/-\u003c/sup\u003e astrocytes that: 1) the expression of autophagy markers MAP1LC3-II/I and the autophagy substrate insoluble SQSTM1 increased from 3 h to 72 h following OGD/R (\u003cstrong\u003eFigure 10A1-A3\u003c/strong\u003e), indicating the accumulation of autophagosomes and substrates. 2) Although LAMP1 expression did not change significantly from 3 h to 72 h following OGD/R, the expression levels of CTSD and mCTSB, as well as CTSD enzyme activity, decreased significantly over this period (\u003cstrong\u003eFigure 10A1, A6-A8, B\u003c/strong\u003e), suggesting that the absence of PrP\u003csup\u003eC\u003c/sup\u003e impedes the restoration of lysosomal number and function in late OGD/R, exacerbating and prolonging autophagosome accumulation. 3) The proportion of functional lysosomes (LAMP\u003csup\u003e+\u003c/sup\u003eCTSD\u003csup\u003e+\u003c/sup\u003e) decreased significantly from 3 h to 72 h following OGD/R and MCAO/R (\u003cstrong\u003eFigure 10C-D\u003c/strong\u003e), suggesting that \u003cem\u003ePrnp\u003c/em\u003e\u003csup\u003e-/-\u003c/sup\u003e interferes with the recovery of lysosomal dysfunction in late CIRI. 4) Fluorescence co-localization of MAP1LC3B and LAMP1 showed a significant increase in the number of autophagosomes from 3 h to 72 h following OGD/R and MCAO/R (\u003cstrong\u003eFigure 11A-B)\u003c/strong\u003e, with no significant change in lysosomes. This suggests that the relative insufficiency of lysosomes contributes to autophagosome accumulation. 5) Assessment of ALP function using the lysosomal inhibitor CQ revealed a significant decrease in the elevation of MAP1LC3B-II/I following chloroquine treatment at 6 h and 48 h after OGD/R (\u003cstrong\u003eFigure 11C\u003c/strong\u003e), These findings suggest that the absence of PrP\u003csup\u003eC\u003c/sup\u003e impairs the recovery of ALP function during late CIRI by inhibiting lysosomal upregulation and its associated functional improvements.\u003c/p\u003e\n\u003cp\u003eThese findings indicate that overexpression of PrP\u003csup\u003eC\u003c/sup\u003e ameliorates lysosomal dysfunction by upregulating lysosomal numbers at different stages of CIRI, thereby maintaining ALP patency.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eOverexpression of PrP\u003csup\u003eC\u003c/sup\u003e sustains ALP function during both early and late CIRI by promoting TFEB nuclear translocation, thereby facilitating the shift of astrocytes from a pro-inflammatory to an anti-inflammatory phenotype\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe above observations led us to hypothesize that PrP\u003csup\u003eC\u003c/sup\u003e modulation of ALP may be linked to TFEB. To investigate this, we examined total TFEB expression and its nuclear translocation in WT, P\u003cem\u003ernp\u003csup\u003e-\u003c/sup\u003e\u003c/em\u003e\u003csup\u003e/-\u0026nbsp;\u003c/sup\u003e, and \u003cem\u003ePrnp\u003c/em\u003e-overexpressing astrocytes following CIRI. Immunoblotting results revealed that total TFEB expression in\u003cem\u003e\u0026nbsp;Prnp\u003csup\u003e-\u003c/sup\u003e\u003c/em\u003e\u003csup\u003e/-\u0026nbsp;\u003c/sup\u003eastrocytes decreased significantly from 3 h to 24 h following OGD/R compared to the normal condition. In contrast, total TFEB expression in \u003cem\u003ePrnp\u003c/em\u003e-overexpressing astrocytes decreased from 3 h to 6 h but significantly increased at 12 h and remained elevated from 48 h to 72 h following OGD/R. Furthermore, compared to WT astrocytes, total TFEB expression in \u003cem\u003ePrnp-\u003c/em\u003eoverexpressing astrocytes was significantly higher at 12 h and from 48 h to 72 h following OGD/R, while total TFEB expression in \u003cem\u003ePrnp\u003csup\u003e-\u003c/sup\u003e\u003c/em\u003e\u003csup\u003e/-\u0026nbsp;\u003c/sup\u003eastrocytes decreased significantly at 12 h to 24 h after OGD/R (\u003cstrong\u003eFigure 12A\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003eImmunofluorescence staining showed that TFEB nuclear translocation was observed only in \u003cem\u003ePrnp\u003csup\u003e-\u003c/sup\u003e\u003c/em\u003e\u003csup\u003e/-\u003c/sup\u003eastrocytes at 12 h following OGD/R, as evidenced by an increase in the mean optical density and a significant rise in the Mander\u0026rsquo;s overlap coefficient. In contrast, TFEB nuclear aggregation was detected in\u003cem\u003e\u0026nbsp;Prnp\u003c/em\u003e-overexpressing astrocytes from 12 h to 72 h following OGD/R and normal condition. Notably, TFEB nuclear aggregation was significantly higher in \u003cem\u003ePrnp\u003c/em\u003e-overexpressing astrocytes at all time points (3 h to 72 h) compared to WT astrocytes, whereas TFEB nuclear aggregation in \u003cem\u003ePrnp\u003csup\u003e-\u003c/sup\u003e\u003c/em\u003e\u003csup\u003e/-\u0026nbsp;\u003c/sup\u003eastrocytes was significantly lower from 24 h to 48 h following OGD/R (\u003cstrong\u003eFigure 12B\u003c/strong\u003e). Similarly, the phenomena observed in mouse MCAO/R astrocytes were consistent with those in vitro (\u003cstrong\u003eFigure 12C\u003c/strong\u003e). These results suggest that overexpression of PrP\u003csup\u003eC\u003c/sup\u003e promotes TFEB nuclear translocation during CIRI and sustains it over an extended period.\u003c/p\u003e\n\u003cp\u003eSubsequently, we used siRNA to inhibit TFEB expression in \u003cem\u003ePrnp\u003c/em\u003e-overexpressing astrocytes. Immunoblotting results demonstrated that TFEB expression was significantly reduced in the si-\u003cem\u003eTfeb\u0026nbsp;\u003c/em\u003egroup compared to the si-Con group at 6 h following OGD/R. Concurrently, the expression of autophagosome markers MAP1LC3B-II/Ⅰand the autophagy substrate ubiquitin was significantly elevated, while pro-CTSD and mCTSB expression was significantly reduced. No significant changes were observed in LAMP1 and mCTSD expression. At 48 h following OGD/R, TFEB expression remained significantly decreased in the si-\u003cem\u003eTfeb\u0026nbsp;\u003c/em\u003egroup compared to the si-Con group, along with a notable increase in MAP1LC3B-II/Ⅰand ubiquitin expression, and a significant decrease in LAMP1, CTSD, and mCTSB expression (\u003cstrong\u003eFigure 13A\u003c/strong\u003e). These results suggest that PrP\u003csup\u003eC\u0026nbsp;\u003c/sup\u003eoverexpression maintains ALP patency during both early and late OGD/R by regulating TFEB nuclear translocation.\u003c/p\u003e\n\u003cp\u003eCCK8 and CBA assays showed that, at both 6 h and 48 h following OGD/R, cellular activity was significantly reduced in the si-\u003cem\u003eTfeb\u0026nbsp;\u003c/em\u003egroup compared to the si-Con group. In addition, cytokine IL-10 levels were decreased, while TNF and IL-6 levels were significantly increased (\u003cstrong\u003eF\u003c/strong\u003e\u003cstrong\u003eigure 13B-C\u003c/strong\u003e). These findings suggest that PrP\u003csup\u003eC\u0026nbsp;\u003c/sup\u003eoverexpression enhances astrocyte activity and modulates the inflammatory phenotype during OGD/R through TFEB regulation.\u003c/p\u003e\n\u003cp\u003eIn conclusion, our results indicate that overexpression of PrP\u003csup\u003eC\u003c/sup\u003e preserves ALP patency in both early and late stages of CIRI and promotes the transition of astrocytes from a pro-inflammatory to an anti-inflammatory phenotype by facilitating TFEB nuclear translocation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eOverexpression of PrP\u003csup\u003eC\u0026nbsp;\u003c/sup\u003epromotes nuclear translocation of TFEB following OGD/R via the Ca\u003csup\u003e2+\u003c/sup\u003e/PPP3 pathway\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFinally, we aimed to explore whether PrP\u003csup\u003eC\u0026nbsp;\u003c/sup\u003eregulates TFEB following OGD/R through the Ca\u003csup\u003e2+\u003c/sup\u003e/PPP3 pathway. The results showed that intracellular Ca\u003csup\u003e2+\u003c/sup\u003e concentration in \u003cem\u003ePrnp-\u003c/em\u003eoverexpressing astrocytes decreased at 6 h and subsequently increased from 12 h to 72 h following OGD/R. Although the expression of PPP3 did not change significantly, PPP3 activity increased notably from 3 h to 6 h and from 24 h to 48 h following OGD/R. In contrast, intracellular Ca\u003csup\u003e2+\u003c/sup\u003e concentration in \u003cem\u003ePrnp\u003csup\u003e-/\u003c/sup\u003e\u003c/em\u003e\u003csup\u003e-\u0026nbsp;\u003c/sup\u003eastrocytes decreased at 6 h and then increased at 24 h following OGD/R, with no significant changes in PPP3 expression. However, PPP3 activity was significantly reduced from 3 h to 6 h and from 48 h to 72 h following OGD/R. Compared to WT mouse astrocytes, PPP3 ctivity in \u003cem\u003ePrnp-\u003c/em\u003eoverexpressing astrocytes was significantly higher from 3 h to 6 h and from 24 h to 48 h following OGD/R, while intracellular Ca\u003csup\u003e2+\u003c/sup\u003e concentration increased from 3 h to 12 h and from 48 h to 72 h, and decreased at 24 h following OGD/R. Conversely, in\u003cem\u003e\u0026nbsp;Prnp\u003c/em\u003e\u003csup\u003e-/-\u0026nbsp;\u003c/sup\u003eastrocytes, PPP3 activity was significantly lower at 12 h and from 48 h to 72 h, and intracellular Ca\u003csup\u003e2+\u003c/sup\u003e concentration remained reduced from 3 h to 72 h expect 6 h following OGD/R. These findings suggest that PrP\u003csup\u003eC\u0026nbsp;\u003c/sup\u003eoverexpression helps maintain Ca\u003csup\u003e2+\u003c/sup\u003e homeostasis in astrocytes following OGD/R, which in turn sustains moderate PPP3 activity (\u003cstrong\u003eFigure 14A-C\u003c/strong\u003e). To further validate this pathway, we inhibited PPP3 in \u003cem\u003ePrnp\u003c/em\u003e-overexpressing astrocytes using CsA. CsA significantly reduced TFEB nuclear translocation from 3 h to 72 h following OGD/R compared to the untreated group (\u003cstrong\u003eFigure 14D\u003c/strong\u003e). These results suggest that PrP\u003csup\u003eC\u003c/sup\u003e promotes TFEB nuclear translocation via the Ca\u003csup\u003e2+\u003c/sup\u003e/PPP3 pathway.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe present study demonstrates that PrP\u003csup\u003eC\u0026nbsp;\u003c/sup\u003emitigates OGD/R-induced ALP dysfunction through the Ca\u003csup\u003e2+\u003c/sup\u003e/PPP3/TFEB pathway and promotes the conversion of astrocytes from a pro-inflammatory to an anti-inflammatory phenotype. Our observations revealed that ALP function was partially impaired during early OGD/R, accompanied by reduced TFEB expression and nuclear translocation, which was attributed to the combined effects of mTOR signaling and PPP3/calcineurin. In contrast, during late OGD/R, lysosomal activity was enhanced, autophagosome and substrate accumulation was alleviated, and cellular damage was reduced, accompanied by increased nuclear translocation of TFEB, primarily mediated by the Ca\u003csup\u003e2+\u003c/sup\u003e/PPP3 pathway. While upregulation of TFEB expression alone did not rescue ALP dysfunction in the early stages of OGD/R, promoting TFEB nuclear translocation via the Ca2\u003csup\u003e+/\u003c/sup\u003ePPP3 pathway was able to attenuate OGD/R injury by restoring ALP function. PrP\u003csup\u003eC\u0026nbsp;\u003c/sup\u003emodulates TFEB nuclear translocation through the Ca\u003csup\u003e2+\u003c/sup\u003e/PPP3 pathway, improving ALP function and facilitating the shift of astrocytes from a pro-inflammatory to an anti-inflammatory phenotype, thereby alleviating OGD/R-induced damage.\u003c/p\u003e\n\u003cp\u003eIn this study, we investigated the dynamic changes in ALP function induced by CIRI. Our findings indicate that at the early stage of OGD/R, autophagosome marker LC3B and substrate SQSTM1 accumulated in WT mouse astrocytes, suggesting that the abnormal accumulation of autophagosomes and substrates begins early in CIRI. This observation aligns with findings reported by Xia Zhang and colleagues [23]. Furthermore, lysosomal function was impaired during early OGD/R, as evidenced by decreased expression of lysosomal markers CTSB and CTSD, reduced CTSD activity, and diminished co-localization of LAMP1 and CTSD. In the presence of CQ, LC3B-II/I levels decreased in the early stages of OGD/R but returned to baseline levels in the later stages. These results suggest that ALP dysfunction in early OGD/R leads to the accumulation of autophagosomes, whereas ALP function gradually improves in the later stages, facilitating the degradation of autophagosomes. This phenomenon raises three key questions: 1) What are the underlying molecular mechanisms responsible for early ALP blockade in OGD/R? 2) What molecular mechanisms contribute to the amelioration of ALP dysfunction in late OGD/R? 3) Is there any overlap in the molecular mechanisms of early ALP blockade and late recovery during OGD/R, and are they regulated by the same proteins? Investigating these mechanisms may help identify potential drug targets for ischemic stroke treatment.\u003c/p\u003e\n\u003cp\u003eTFEB is a key regulator of ALP [12] and has been identified as a potential therapeutic target for rescuing myocardial ischemia-reperfusion injury [14] and permanent cerebral ischemia injury [15]. While much of the existing research focuses on neurons [26-27], we are specifically interested in how TFEB regulates astrocyte ALP following CIRI. In this study, we observed that total TFEB expression initially decreased from 3 to 6 hours and then gradually increased, peaking at 12 hours before returning to baseline levels at 48 hours following OGD/R. TFEB nuclear translocation increased from 12 to 48 hours after OGD/R. These findings suggest that TFEB function is minimal during the ultra-early stages of OGD/R, as indicated by its low accumulation in the nucleus. As the recovery period for oxygen and glucose was extended, TFEB function became gradually activated, translocating to the nucleus and accompanied by an increase in its total expression. This pattern of TFEB expression and nuclear translocation corresponds with changes in ALP function following OGD/R, with the increase in TFEB expression and nuclear translocation slightly preceding the improvement in ALP function. Further, we explored the critical role of TFEB in OGD/R-mediated ALP dysfunction. We found that TFEB function was inhibited during the ultra-early phase of OGD/R, as evidenced by the absence of nuclear translocation, which was regulated by both the mTOR and calcineurin/PPP3 pathways. As the recovery time for oxygen and glucose was prolonged, TFEB function was gradually activated, leading to an increase in nuclear TFEB accumulation, which alleviated ALP blockade and reduced cellular injury. Conversely, si-\u003cem\u003eTfeb-\u003c/em\u003emediated silencing of TFEB expression reversed the improvement in ALP function and exacerbated astrocyte injury in the later stages of OGD/R. Interestingly, upregulation of TFEB expression did not improve ALP blockade in astrocytes during the early stages of OGD/R, which contrasts with findings from studies on permanent brain ischemia [15]. The discrepancy between our results and those from permanent brain ischemia may be attributed to different molecular mechanisms in CIRI versus permanent brain ischemia. It is well known that the phosphorylation of TFEB by mTORC1 is dependent on intracellular nutrient levels [28]. In the case of cerebral ischemia, mTORC1 is inactive, whereas after reperfusion, mTORC1 is rapidly activated and transformed into its active form, p-mTORC1, a phenomenon confirmed in both our and previous studies [28-29]. Intracellular TFEB exists as dimers [30-32]. In nutrient-rich environments, activated p-mTORC1 phosphorylates TFEB, forming a phosphorylated TFEB homodimer, which is inactive and predominates in the cytoplasm. After ischemic injury, dephosphorylation of TFEB leads to the formation of active homodimers of dephosphorylated TFEB, as well as inactive heterodimers made of phosphorylated and non-phosphorylated TFEB. In other words, phosphorylated TFEB interferes with its nuclear translocation by forming heterodimers with dephosphorylated TFEB [33]. Based on these insights, we hypothesize that the upregulated TFEB expression observed in our study was largely in its phosphorylated form due to activation of p-mTORC1. This phosphorylated TFEB form likely further reduced TFEB activity, and thus, although TFEB is a promising therapeutic target for CIRI, simply upregulating its expression was insufficient to improve outcomes in CIRI.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAs mentioned previously, TFEB is a member of the microphthalmia family. which also includes microphthalmia-associated transcription factor, TFEC, and TFE3 [32, 34]. TFEB plays a pivotal role in regulating ALP function, and this process is not significantly interfered with by the other members of the the microphthalmia family [35-36]. Consequently, this study focused specifically on the dynamic changes of TFEB following OGD/R. However, our study is not exhaustive, and future research will be necessary to investigate the changes in microphthalmia-associated transcription factor, TFEC, and TFE3 during CIRI.\u003c/p\u003e\n\u003cp\u003eWe also observed that the transfection reagent Lipofectamine\u0026trade;\u0026nbsp;3000 inhibited ALP function after OGD/R. Upon reviewing the literature, we found that Lipofectamine\u0026trade;\u0026nbsp;3000 uses lipid nanoparticle technology to facilitate the entry of exogenous DNA into cells, making it a classical non-viral vector for gene delivery. Recently, an increasing number of studies have highlighted the impact of this cationic liposome transfection reagent on the regulation of ALP [37-38]. Therefore, our choice of transfection reagent may be problematic, and this represents a limitation in our study.\u003c/p\u003e\n\u003cp\u003eThe transcriptional activity of TFEB is primarily regulated by its ability to translocate to the nucleus, which is influenced by phosphorylation modifications. As discussed previously, mTOR phosphorylates TFEB to retain it in the cytoplasm, while the Ca\u003csup\u003e2+\u003c/sup\u003e/PPP3 signaling cascade facilitates TFEB nuclear translocation after dephosphorylation [39-40]. To explore the signals upstream of CIRI-induced TFEB nuclear translocation, we monitored the dynamic changes of PPP3/calcineurin and mTOR after OGD/R, followed by pharmacological interventions to determine whether TFEB nuclear translocation depends on these two upstream pathways. These pathways may potentially be targeted to promote TFEB nuclear translocation, improve ALP function, and enhance prognosis. Our findings showed that the trend in intracellular PPP3 enzyme activity largely paralleled TFEB nuclear translocation, and that CsA reversed the OGD/R-induced TFEB nuclear translocation. This suggests that PPP3/calcineurin activation during OGD/R promotes TFEB nuclear translocation. Previous studies have demonstrated that PPP3/calcineurin activation is associated with ischemia-induced neuronal death [41] and that PPP3/calcineurin-dependent TFEB nuclear translocation plays a role in autophagy induction [42-44] . These findings confirm the potential of the PPP3/calcineurin pathway in regulating TFEB nuclear translocation after CIRI. Furthermore, a recent study indicated that the lysosomal calcium-dependent PPP3/calcineurin signaling pathway regulates autophagy through activation of a TFEB-mediated transcriptional prOGD/Ram, independent of mTOR [45]. It is plausible that intracellular Ca\u003csup\u003e2+\u0026nbsp;\u003c/sup\u003elevels serve as an upstream regulatory signal for the PPP3/calcineurin-TFEB axis, as ischemia and hypoxia can induce elevated cytoplasmic Ca\u003csup\u003e2+\u003c/sup\u003e levels [15, 45]. In our study, we observed that intracellular Ca\u003csup\u003e2+\u003c/sup\u003e levels in astrocytes decreased, then increased, and finally returned to baseline levels after OGD/R, which differs from the pattern observed in OGD-treated neurons [15]. This discrepancy may be due to the different pathophysiological mechanisms underlying ischemic injury and ischemia-reperfusion injury. Additionally, previous evidence suggests that intracellular Ca\u003csup\u003e2+\u003c/sup\u003e may partially regulate TFEB dephosphorylation through the PPP3/calcineurin signaling cascade [46-47], which in turn modulates ALP [48]. These data highlight the intracellular Ca\u003csup\u003e2+\u003c/sup\u003e-dependent PPP3/calcineurin pathway as a critical upstream regulatory mechanism of TFEB function in the late stage of CIRI.\u003c/p\u003e\n\u003cp\u003eIn general, mTOR inhibits TFEB nuclear translocation by phosphorylating TFEB under nutrient-rich conditions\u0026nbsp;[49-51]. In our study, we observed that mTOR was rephosphorylated into activated p-mTOR following OGD/R. Theoretically, this activated p-mTOR should further phosphorylate TFEB, inhibiting its nuclear translocation. However, our findings show that OGD/R-induced TFEB nuclear translocation occurred despite this rephosphorylation, which presents an apparent discrepancy. The mTOR activator MHY1485 reversed OGD/R-induced TFEB nuclear translocation, suggesting that p-mTOR-mediated inhibition of TFEB nuclear translocation might be a protective mechanism to avoid uncontrolled autophagy during the early phases of CIRI. This idea, however, warrants further investigation in future studies to better understand the role of mTOR in regulating TFEB under ischemic conditions. Additionally, we observed that Rapamycin, an inhibitor of p-mTOR activity, significantly enhanced TFEB nuclear translocation during the ultra-early phase of OGD/R. This enhancement is likely due to the low activity of the Ca\u003csup\u003e2+\u003c/sup\u003e/PPP3 pathway during this early period, which may influence the dynamics of TFEB nuclear translocation. This suggests that while mTOR can modulate TFEB function, the interplay between mTOR and the Ca\u003csup\u003e2+\u003c/sup\u003e/PPP3 pathway may be crucial for determining the timing and extent of TFEB nuclear translocation and autophagic regulation during ischemia-reperfusion injury. Furthermore, autophagic lysosomal remodeling (ALR) is a critical process for maintaining lysosomal homeostasis and represents an evolutionarily conserved cycle of lysosomal regeneration [52]. T Reactivation of mTOR is the initiating event for autophagic lysosomal remodeling (ALR). Following mTOR reactivation, autolysosomes typically undergo tubulization and an increase in vesicle formation, ultimately maturing into functional lysosomes [52]. In our study, we observed that p-mTOR rapidly returned to basal levels after OGD/R. However, the basal mTOR activity was insufficient to meet the elevated demand for lysosomal function following ischemic stroke. As reperfusion continued, mTOR activation levels gradually increased, which in turn alleviated lysosomal dysfunction. These findings suggest that strategies targeting mTOR-related pathways to promote lysosomal accumulation after ischemic stress may hold promise as a therapeutic approach for mitigating lysosomal dysfunction in the future.\u003c/p\u003e\n\u003cp\u003ePrevious studies have indeed demonstrated that PrP\u003csup\u003eC\u0026nbsp;\u003c/sup\u003ehas neuroprotective effects during CIRI [18, 53], which aligns with our findings that astrocyte activity in WT mice declines in early OGD/R but recovers to baseline levels in late OGD/R. In contrast, \u003cem\u003ePrnp\u0026nbsp;\u003c/em\u003eknockdown inhibited the recovery of cellular activity in late OGD/R and exacerbated cellular activity impairment in early OGD/R. On the other hand, \u003cem\u003ePrnp\u0026nbsp;\u003c/em\u003eoverexpression significantly attenuated cell injury during OGD/R, highlighting its protective role. Furthermore, our investigation into the potential role of PrP\u003csup\u003eC\u0026nbsp;\u003c/sup\u003ein regulating ALP function revealed that \u003cem\u003ePrnp\u003c/em\u003e overexpression increased lysosome number, enhanced lysosomal enzyme activity, and reduced autophagosome accumulation in both early and late OGD/R. This improvement in ALP function was significant compared to WT astrocytes and was further supported by a reduction in CQ-induced autophagosome accumulation. Conversely, \u003cem\u003ePrnp\u0026nbsp;\u003c/em\u003eknockdown astrocytes showed significantly reduced lysosomal function, with exacerbated autophagosome accumulation in late OGD/R. These findings suggest that PrP\u003csup\u003eC\u003c/sup\u003e plays a crucial role in maintaining ALP function during OGD/R by enhancing lysosomal function and mitigating lysosomal dysfunction, which is consistent with previous reports of impaired ALP in \u003cem\u003ePrnp\u0026nbsp;\u003c/em\u003eknockout Purkinje cells [54]. However, while PrP\u003csup\u003eC\u0026nbsp;\u003c/sup\u003eis a key regulator of ALP in CIRI, directly upregulating its expression is not a feasible therapeutic strategy due to the risk of prion diseases. Therefore, understanding the molecular mechanisms through which PrP\u003csup\u003eC\u0026nbsp;\u003c/sup\u003eregulates ALP could help identify alternative drug targets to enhance ALP function without the risks associated with PrP\u003csup\u003eC\u0026nbsp;\u003c/sup\u003eoverexpression. Exploring this regulatory pathway will be critical for developing safer and more effective treatments for ischemic stroke.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe role of PrP\u003csup\u003eC\u003c/sup\u003e in regulating immune-related phenotypes, particularly its impact on the inflammatory response in astrocytes after OGD/R, has gained considerable attention. In this study, we observed that \u003cem\u003ePrnp\u003c/em\u003e knockdown in astrocytes significantly reduced the concentration of the anti-inflammatory cytokine IL-10, while increasing the levels of pro-inflammatory cytokines INF-\u0026gamma;\u0026nbsp;and TNF after OGD/R. This suggests that PrP\u003csup\u003eC\u003c/sup\u003e is essential for promoting the anti-inflammatory response in astrocytes following ischemia-reperfusion injury. On the other hand, \u003cem\u003ePrnp\u0026nbsp;\u003c/em\u003eoverexpression enhanced IL-10 production and suppressed INF-\u0026gamma;\u0026nbsp;and TNF levels, further supporting PrP\u003csup\u003eC\u0026nbsp;\u003c/sup\u003e\u0026apos;s role in mediating the switch from a pro-inflammatory to an anti-inflammatory phenotype. These findings align with previous studies demonstrating that \u003cem\u003ePrnp\u0026nbsp;\u003c/em\u003eoverexpression in microglial cells promotes an anti-inflammatory phenotype, whereas \u003cem\u003ePrnp\u0026nbsp;\u003c/em\u003eknockdown in microglia leads to a pro-inflammatory phenotype [17]. Moreover, our results emphasize the importance of anti-inflammatory cytokines, such as IL-10, in the prOGD/Ression of neuronal repair after cerebral ischemic stroke. We found that \u003cem\u003ePrnp\u0026nbsp;\u003c/em\u003eknockdown suppressed IL-10 expression in late OGD/R, indicating that PrP\u003csup\u003eC\u0026nbsp;\u003c/sup\u003emay contribute to regulating the expression of key cytokines involved in the resolution of inflammation during ischemic stroke recovery. However, we also noted a discrepancy with a previous study, where P\u003cem\u003ernp si\u003c/em\u003elencing did not alter IL-10 expression in Mycoplasma bovis-infected microglia [55]. This difference may arise due to the distinct disease models used, suggesting that PrP\u003csup\u003eC\u0026nbsp;\u003c/sup\u003e\u0026apos;s role in immune regulation might be context-dependent. Overall, our study adds new evidence to the growing body of literature on the potential therapeutic benefits of modulating PrP\u003csup\u003eC\u003c/sup\u003e expression in ischemic stroke, particularly through its effects on immune responses. Targeting PrP\u003csup\u003eC\u003c/sup\u003e could provide a promising avenue for enhancing recovery and reducing neuroinflammation following stroke.\u003c/p\u003e\n\u003cp\u003eThe study\u0026apos;s findings regarding the dynamics of TFEB expression and nuclear translocation in different PrP\u003csup\u003eC\u003c/sup\u003e-expressing astrocytes provide further insight into the protective mechanisms of PrP\u003csup\u003eC\u003c/sup\u003e in ischemic conditions. In\u003cem\u003e\u0026nbsp;Prnp\u003c/em\u003e knockdown astrocytes, we observed a significant decrease in total TFEB expression from 3 h to 24 h after OGD/R, with perinuclear aggregation of TFEB evident at 12 h. This was reflected by an increase in the mean optical density and Mander\u0026apos;s overlap coefficient, suggesting impaired TFEB function. On the other hand, \u003cem\u003ePrnp\u003c/em\u003e-overexpressing astrocytes exhibited a more pronounced and sustained nuclear translocation of TFEB, with total TFEB expression decreasing initially from 3 h to 6 h, but then increasing at 12 h and from 48 h to 72 h, along with intranuclear and perinuclear aggregates. This pattern indicates enhanced activation of TFEB function in the later stages of OGD/R in Prnp-overexpressing cells. Interestingly, compared with WT mouse astrocytes, \u003cem\u003ePrnp-\u003c/em\u003eoverexpressing cells showed a consistent increase in TFEB nuclear translocation from 3 h to 72 h, whereas in\u003cem\u003e\u0026nbsp;Prnp\u003c/em\u003e knockout astrocytes, this nuclear translocation decreased from 24 h to 48 h. This suggests that PrP\u003csup\u003eC\u0026nbsp;\u003c/sup\u003eplays a key role in enhancing TFEB function and promoting its nuclear translocation during OGD/R. Notably, despite the increase in TFEB nuclear translocation in \u003cem\u003ePrnp-\u003c/em\u003eoverexpressing astrocytes, total TFEB expression dropped back to baseline levels at 24 h post-OGD/R. This warrants further investigation to understand the underlying mechanisms behind this observation, as it could have significant implications for therapeutic strategies targeting PrP\u003csup\u003eC\u003c/sup\u003e.\u003csup\u003e\u0026nbsp;\u003c/sup\u003eFurther evidence supporting the role of PrP\u003csup\u003eC\u003c/sup\u003e in promoting TFEB nuclear translocation came from experiments where silencing TFEB in \u003cem\u003ePrnp-\u003c/em\u003eoverexpressing astrocytes with si-\u003cem\u003eTfeb\u0026nbsp;\u003c/em\u003esignificantly reversed the expression of ALP-related proteins. This suggests that PrP\u003csup\u003eC\u003c/sup\u003e maintains ALP function by promoting TFEB nuclear translocation during OGD/R. Given these findings, PrP\u003csup\u003eC\u003c/sup\u003e appears to be a promising therapeutic target for ischemic stroke, with its ability to regulate TFEB and maintain autophagic flux playing a key role in cellular protection during CIRI.\u003c/p\u003e\n\u003cp\u003eWhen \u003cem\u003ePrnp\u0026nbsp;\u003c/em\u003ewas knocked down, there was no significant change in the trend of intracellular Ca\u003csup\u003e2+\u0026nbsp;\u003c/sup\u003elevels and PPP3 activity after OGD/R, but the rise time of both was shortened. This suggests that PrP\u003csup\u003eC\u0026nbsp;\u003c/sup\u003emay play a role in fine-tuning the timing of these signaling events rather than directly altering their overall trajectory. Conversely, in \u003cem\u003ePrnp\u003c/em\u003e-overexpressing astrocytes, intracellular Ca\u003csup\u003e2+\u003c/sup\u003e levels were better maintained, and PPP3 activity was sustained for a longer period. The addition of CsA, a calcineurin inhibitor, reversed the nuclear translocation of TFEB after OGD/R, supporting the hypothesis that PrP\u003csup\u003eC\u0026nbsp;\u003c/sup\u003eregulates TFEB activity through the Ca\u003csup\u003e2+\u003c/sup\u003e/PPP3 pathway. This finding aligns with previous studies indicating PrP\u003csup\u003eC\u003c/sup\u003e\u0026apos;s role in maintaining Ca\u003csup\u003e2+\u0026nbsp;\u003c/sup\u003ehomeostasis in cells, though the specific molecular mechanisms behind this regulation remain to be fully elucidated. The findings from this study highlight the crucial role of PrP\u003csup\u003eC\u0026nbsp;\u003c/sup\u003ein regulating TFEB nuclear translocation during OGD/R through the Ca\u003csup\u003e2+\u003c/sup\u003e/PPP3 pathway, which is tied to its ability to maintain intracellular Ca\u003csup\u003e2+\u003c/sup\u003e homeostasis. This regulation appears to be pivotal in controlling the dynamic process of TFEB activation during the ischemia-reperfusion injury.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe model presented in \u003cstrong\u003eFigure 15\u003c/strong\u003e outlines the dynamic changes in the upstream mechanisms of CIRI-induced TFEB nuclear translocation. In the ultra-early stages of CIRI, low intracellular Ca2+ levels and inhibited PPP3/calcineurin activity, along with reactivation of mTOR, result in TFEB phosphorylation and retention in the cytoplasm. As reperfusion time increases, intracellular Ca2+ levels increase, and PPP3/calcineurin is activated, leading to TFEB dephosphorylation, nuclear translocation, and the subsequent transcription of its target genes. This process enhances lysosomal function, alleviates autophagosome accumulation, and promotes astrocyte survival and an anti-inflammatory phenotype. PrPC overexpression sustains intracellular Ca2+ homeostasis and PPP3/calcineurin activity, ensuring the continuous dephosphorylation and nuclear translocation of TFEB. This results in the maintenance of ALP function throughout CIRI and ultimately promotes astrocyte survival and their shift to an anti-inflammatory phenotype, which is essential for neuronal repair after ischemic injury. These insights further emphasize the therapeutic potential of targeting PrPC in ischemic stroke to regulate autophagy and inflammatory responses for better outcomes.\u003c/p\u003e\n\u003cp\u003eIn conclusion, we demonstrate a novel mechanism of neuroprotection during CIRI triggered by PrP\u003csup\u003eC\u003c/sup\u003e. PrP\u003csup\u003eC\u003c/sup\u003e activates PPP3/calcineurin by maintaining intracellular Ca\u003csup\u003e2+\u003c/sup\u003e homeostasis, PPP3/calcineurin induces nuclear translocation of TFEB, and TFEB within the nucleus transcribe ALP-related genes to modulate their function, which contributes to the transformation of astrocytes from a pro-inflammatory phenotype to an anti-inflammatory phenotype.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eFunding Statement\u003c/p\u003e\n\u003cp\u003eThis work was supported by a grant from the National Natural Science Foundation of China (No. 82371371) and the Doctor of Excellence Program (DEP), The First Hospital of Jilin University (No. JDYY-DEP-2024039).\u003c/p\u003e\n\u003cp\u003eDisclosure statement\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflicts of interests.\u003c/p\u003e\n\u003cp\u003eData Availability\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003eEthics approval\u003c/p\u003e\n\u003cp\u003eThe experimental protocol was approved by the Ethics Committee for Laboratory Animal Research of the First Hospital of Jilin University.\u003c/p\u003e\n\u003cp\u003eAuthors\u0026apos; contributions\u003c/p\u003e\n\u003cp\u003eJS, LC, and XY contributed to the conception of this review. JS, XY, YL, and LC participated in writing the manuscript. JS, JY, and TS prepared the figures and tables, while XY and LC critically revised the manuscript. All authors contributed to the article and approved the final version.\u003c/p\u003e\n\u003cp\u003eConsent to Participate\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003eConsent for publication\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eShi, K.; Tian, D. C.; Li, Z. G.; Ducruet, A. F.; Lawton, M. T.; Shi, F. D., Global brain inflammation in stroke. \u003cem\u003eLancet Neurol \u003c/em\u003e\u003cstrong\u003e2019,\u003c/strong\u003e \u003cem\u003e18\u003c/em\u003e (11), 1058-1066.\u003c/li\u003e\n\u003cli\u003eLiu, M.; Xu, Z.; Wang, L.; Zhang, L.; Liu, Y.; Cao, J.; Fu, Q.; Liu, Y.; Li, H.; Lou, J.; Hou, W.; Mi, W.; Ma, Y., Cottonseed oil alleviates ischemic stroke injury by inhibiting the inflammatory activation of microglia and astrocyte. \u003cem\u003eJ Neuroinflammation \u003c/em\u003e\u003cstrong\u003e2020,\u003c/strong\u003e \u003cem\u003e17\u003c/em\u003e (1), 270.\u003c/li\u003e\n\u003cli\u003eMizushima, N.; Komatsu, M., Autophagy: renovation of cells and tissues. \u003cem\u003eCell \u003c/em\u003e\u003cstrong\u003e2011,\u003c/strong\u003e \u003cem\u003e147\u003c/em\u003e (4), 728-41.\u003c/li\u003e\n\u003cli\u003eLiu, S.; Yao, S.; Yang, H.; Liu, S.; Wang, Y., Autophagy: Regulator of cell death. \u003cem\u003eCell Death Dis \u003c/em\u003e\u003cstrong\u003e2023,\u003c/strong\u003e \u003cem\u003e14\u003c/em\u003e (10), 648.\u003c/li\u003e\n\u003cli\u003eQin, A. P.; Liu, C. F.; Qin, Y. Y.; Hong, L. Z.; Xu, M.; Yang, L.; Liu, J. Q.; Qin, Z. H.; Zhang, H. L., Autophagy was activated in injured astrocytes and mildly decreased cell survival following glucose and oxygen deprivation and focal cerebral ischemia. \u003cem\u003eAutophagy \u003c/em\u003e\u003cstrong\u003e2010,\u003c/strong\u003e \u003cem\u003e6\u003c/em\u003e (6), 738-753.\u003c/li\u003e\n\u003cli\u003ePan, R.; Timmins, G. S.; Liu, W.; Liu, K. J., Autophagy Mediates Astrocyte Death During Zinc-Potentiated Ischemia--Reperfusion Injury. \u003cem\u003eBiol Trace Elem Res \u003c/em\u003e\u003cstrong\u003e2015,\u003c/strong\u003e \u003cem\u003e166\u003c/em\u003e (1), 89-95.\u003c/li\u003e\n\u003cli\u003eGabryel, B.; Kost, A.; Kasprowska, D.; Liber, S.; Machnik, G.; Wiaderkiewicz, R.; Labuzek, K., AMP-activated protein kinase is involved in induction of protective autophagy in astrocytes exposed to oxygen-glucose deprivation. \u003cem\u003eCell Biol Int \u003c/em\u003e\u003cstrong\u003e2014,\u003c/strong\u003e \u003cem\u003e38\u003c/em\u003e (10), 1086-1097.\u003c/li\u003e\n\u003cli\u003eZhao, F.; Qu, Y.; Wang, H.; Huang, L.; Zhu, J.; Li, S.; Tong, Y.; Zhang, L.; Li, J.; Mu, D., The effect of miR-30d on apoptosis and autophagy in cultured astrocytes under oxygen-glucose deprivation. \u003cem\u003eBrain Res \u003c/em\u003e\u003cstrong\u003e2017,\u003c/strong\u003e \u003cem\u003e1671\u003c/em\u003e, 67-76.\u003c/li\u003e\n\u003cli\u003eCarloni, S.; Buonocore, G.; Balduini, W., Protective role of autophagy in neonatal hypoxia-ischemia induced brain injury. \u003cem\u003eNeurobiol Dis \u003c/em\u003e\u003cstrong\u003e2008,\u003c/strong\u003e \u003cem\u003e32\u003c/em\u003e (3), 329-39.\u003c/li\u003e\n\u003cli\u003eShi, R.; Weng, J.; Zhao, L.; Li, X. M.; Gao, T. M.; Kong, J., Excessive autophagy contributes to neuron death in cerebral ischemia. \u003cem\u003eCNS Neurosci Ther \u003c/em\u003e\u003cstrong\u003e2012,\u003c/strong\u003e \u003cem\u003e18\u003c/em\u003e (3), 250-60.\u003c/li\u003e\n\u003cli\u003eWang, P.; Shao, B. Z.; Deng, Z.; Chen, S.; Yue, Z.; Miao, C. Y., Autophagy in ischemic stroke. \u003cem\u003eProg Neurobiol \u003c/em\u003e\u003cstrong\u003e2018,\u003c/strong\u003e \u003cem\u003e163-164\u003c/em\u003e, 98-117.\u003c/li\u003e\n\u003cli\u003eShao, J.; Lang, Y.; Ding, M. Q.; Yin, X.; Cui, L., Transcription Factor EB: A Promising Therapeutic Target for Ischemic Stroke. \u003cem\u003eCurrent Neuropharmacology \u003c/em\u003e\u003cstrong\u003e2024,\u003c/strong\u003e \u003cem\u003e22\u003c/em\u003e (2), 170-190.\u003c/li\u003e\n\u003cli\u003eZhang, Z.; Yan, J.; Bowman, A. B.; Bryan, M. R.; Singh, R.; Aschner, M., Dysregulation of TFEB contributes to manganese-induced autophagic failure and mitochondrial dysfunction in astrocytes. \u003cem\u003eAutophagy \u003c/em\u003e\u003cstrong\u003e2020,\u003c/strong\u003e \u003cem\u003e16\u003c/em\u003e (8), 1506-1523.\u003c/li\u003e\n\u003cli\u003eGu, S.; Tan, J.; Li, Q.; Liu, S.; Ma, J.; Zheng, Y.; Liu, J.; Bi, W.; Sha, P.; Li, X.; Wei, M.; Cao, N.; Yang, H. T., Downregulation of LAPTM4B Contributes to the Impairment of the Autophagic Flux via Unopposed Activation of mTORC1 Signaling During Myocardial Ischemia/Reperfusion Injury. \u003cem\u003eCirc Res \u003c/em\u003e\u003cstrong\u003e2020,\u003c/strong\u003e \u003cem\u003e127\u003c/em\u003e (7), e148-e165.\u003c/li\u003e\n\u003cli\u003eLiu, Y.; Xue, X.; Zhang, H.; Che, X.; Luo, J.; Wang, P.; Xu, J.; Xing, Z.; Yuan, L.; Liu, Y.; Fu, X.; Su, D.; Sun, S.; Zhang, H.; Wu, C.; Yang, J., Neuronal-targeted TFEB rescues dysfunction of the autophagy-lysosomal pathway and alleviates ischemic injury in permanent cerebral ischemia. \u003cem\u003eAutophagy \u003c/em\u003e\u003cstrong\u003e2019,\u003c/strong\u003e \u003cem\u003e15\u003c/em\u003e (3), 493-509.\u003c/li\u003e\n\u003cli\u003eBolton, D. C.; McKinley, M. P.; Prusiner, S. B., Identification of a protein that purifies with the scrapie prion. \u003cem\u003eScience \u003c/em\u003e\u003cstrong\u003e1982,\u003c/strong\u003e \u003cem\u003e218\u003c/em\u003e (4579), 1309-11.\u003c/li\u003e\n\u003cli\u003eShao, J.; Yin, X.; Lang, Y.; Ding, M.; Zhang, B.; Sun, Q.; Jiang, X.; Song, J.; Cui, L., Cellular Prion Protein Attenuates OGD/R-Induced Damage by Skewing Microglia toward an Anti-inflammatory State via Enhanced and Prolonged Activation of Autophagy. \u003cem\u003eMol Neurobiol \u003c/em\u003e\u003cstrong\u003e2023,\u003c/strong\u003e \u003cem\u003e60\u003c/em\u003e (3), 1297-1316.\u003c/li\u003e\n\u003cli\u003eZhang, B. Z.; Yin, X.; Lang, Y.; Han, X. O.; Shao, J.; Bai, R. R.; Cui, L., Role of cellular prion protein in splenic CD4\u003c/li\u003e\n\u003cli\u003eT cell differentiation in cerebral ischaemic/reperfusion. \u003cem\u003eAnn Clin Transl Neur \u003c/em\u003e\u003cstrong\u003e2021,\u003c/strong\u003e \u003cem\u003e8\u003c/em\u003e (10), 2040-2051.\u003c/li\u003e\n\u003cli\u003eYin, X.; Feng, L. S.; Ma, D.; Yin, P.; Wang, X. Y.; Hou, S.; Hao, Y. L.; Zhang, J. D.; Xin, M. Y.; Feng, J. C., Roles of astrocytic connexin-43, hemichannels, and gap junctions in oxygen-glucose deprivation/reperfusion injury induced neuroinflammation and the possible regulatory mechanisms of salvianolic acid B and carbenoxolone. \u003cem\u003eJ Neuroinflamm \u003c/em\u003e\u003cstrong\u003e2018,\u003c/strong\u003e \u003cem\u003e15\u003c/em\u003e.\u003c/li\u003e\n\u003cli\u003eWu, M.; Lao, Y. Z.; Tan, H. S.; Lu, G.; Ren, Y.; Zheng, Z. Q.; Yi, J.; Fu, W. W.; Shen, H. M.; Xu, H. X., Oblongifolin C suppresses lysosomal function independently of TFEB nuclear translocation. \u003cem\u003eActa Pharmacol Sin \u003c/em\u003e\u003cstrong\u003e2019,\u003c/strong\u003e \u003cem\u003e40\u003c/em\u003e (7), 929-937.\u003c/li\u003e\n\u003cli\u003eBoya, P.; Reggiori, F.; Codogno, P., Emerging regulation and functions of autophagy. \u003cem\u003eNat Cell Biol \u003c/em\u003e\u003cstrong\u003e2013,\u003c/strong\u003e \u003cem\u003e15\u003c/em\u003e (7), 713-20.\u003c/li\u003e\n\u003cli\u003eKoerver, L.; Papadopoulos, C.; Liu, B.; Kravic, B.; Rota, G.; Brecht, L.; Veenendaal, T.; Polajnar, M.; Bluemke, A.; Ehrmann, M.; Klumperman, J.; Jaattela, M.; Behrends, C.; Meyer, H., The ubiquitin-conjugating enzyme UBE2QL1 coordinates lysophagy in response to endolysosomal damage. \u003cem\u003eEMBO Rep \u003c/em\u003e\u003cstrong\u003e2019,\u003c/strong\u003e \u003cem\u003e20\u003c/em\u003e (10), e48014.\u003c/li\u003e\n\u003cli\u003eZhang, X.; Wei, M.; Fan, J.; Yan, W.; Zha, X.; Song, H.; Wan, R.; Yin, Y.; Wang, W., Ischemia-induced upregulation of autophagy preludes dysfunctional lysosomal storage and associated synaptic impairments in neurons. \u003cem\u003eAutophagy \u003c/em\u003e\u003cstrong\u003e2021,\u003c/strong\u003e \u003cem\u003e17\u003c/em\u003e (6), 1519-1542.\u003c/li\u003e\n\u003cli\u003eHossain, M. I.; Marcus, J. M.; Lee, J. H.; Garcia, P. L.; Singh, V.; Shacka, J. J.; Zhang, J. H.; Gropend, T. I.; Falany, C. N.; Andrabi, S. A., Restoration of CTSD (cathepsin D) and lysosomal function in stroke is neuroprotective. \u003cem\u003eAutophagy \u003c/em\u003e\u003cstrong\u003e2021,\u003c/strong\u003e \u003cem\u003e17\u003c/em\u003e (6), 1330-1348.\u003c/li\u003e\n\u003cli\u003eSpudich, A.; Frigg, R.; Kilic, E.; Kilic, U.; Oesch, B.; Raeber, A.; Bassetti, C. L.; Hermann, D. M., Aggravation of ischemic brain injury by prion protein deficiency: role of ERK-1/-2 and STAT-1. \u003cem\u003eNeurobiol Dis \u003c/em\u003e\u003cstrong\u003e2005,\u003c/strong\u003e \u003cem\u003e20\u003c/em\u003e (2), 442-9.\u003c/li\u003e\n\u003cli\u003eXiao, Q.; Yan, P.; Ma, X.; Liu, H.; Perez, R.; Zhu, A.; Gonzales, E.; Tripoli, D. L.; Czerniewski, L.; Ballabio, A.; Cirrito, J. R.; Diwan, A.; Lee, J. M., Neuronal-Targeted TFEB Accelerates Lysosomal Degradation of APP, Reducing Abeta Generation and Amyloid Plaque Pathogenesis. \u003cem\u003eJ Neurosci \u003c/em\u003e\u003cstrong\u003e2015,\u003c/strong\u003e \u003cem\u003e35\u003c/em\u003e (35), 12137-51.\u003c/li\u003e\n\u003cli\u003ePolito, V. A.; Li, H.; Martini-Stoica, H.; Wang, B.; Yang, L.; Xu, Y.; Swartzlander, D. B.; Palmieri, M.; di Ronza, A.; Lee, V. M.; Sardiello, M.; Ballabio, A.; Zheng, H., Selective clearance of aberrant tau proteins and rescue of neurotoxicity by transcription factor EB. \u003cem\u003eEMBO Mol Med \u003c/em\u003e\u003cstrong\u003e2014,\u003c/strong\u003e \u003cem\u003e6\u003c/em\u003e (9), 1142-60.\u003c/li\u003e\n\u003cli\u003eSzwed, A.; Kim, E.; Jacinto, E., Regulation and metabolic functions of mTORC1 and mTORC2. \u003cem\u003ePhysiol Rev \u003c/em\u003e\u003cstrong\u003e2021,\u003c/strong\u003e \u003cem\u003e101\u003c/em\u003e (3), 1371-1426.\u003c/li\u003e\n\u003cli\u003eHwang, S. K.; Kim, H. H., The functions of mTOR in ischemic diseases. \u003cem\u003eBMB Rep \u003c/em\u003e\u003cstrong\u003e2011,\u003c/strong\u003e \u003cem\u003e44\u003c/em\u003e (8), 506-11.\u003c/li\u003e\n\u003cli\u003ePogenberg, V.; Ballesteros-Alvarez, J.; Schober, R.; Sigvaldadottir, I.; Obarska-Kosinska, A.; Milewski, M.; Schindl, R.; Ogmundsdottir, M. H.; Steingrimsson, E.; Wilmanns, M., Mechanism of conditional partner selectivity in MITF/TFE family transcription factors with a conserved coiled coil stammer motif. \u003cem\u003eNucleic Acids Res \u003c/em\u003e\u003cstrong\u003e2020,\u003c/strong\u003e \u003cem\u003e48\u003c/em\u003e (2), 934-948.\u003c/li\u003e\n\u003cli\u003eLa Spina, M.; Contreras, P. S.; Rissone, A.; Meena, N. K.; Jeong, E.; Martina, J. A., MiT/TFE Family of Transcription Factors: An Evolutionary Perspective. \u003cem\u003eFront Cell Dev Biol \u003c/em\u003e\u003cstrong\u003e2020,\u003c/strong\u003e \u003cem\u003e8\u003c/em\u003e, 609683.\u003c/li\u003e\n\u003cli\u003ePuertollano, R.; Ferguson, S. M.; Brugarolas, J.; Ballabio, A., The complex relationship between TFEB transcription factor phosphorylation and subcellular localization. \u003cem\u003eEmbo Journal \u003c/em\u003e\u003cstrong\u003e2018,\u003c/strong\u003e \u003cem\u003e37\u003c/em\u003e (11).\u003c/li\u003e\n\u003cli\u003eSha, Y. B.; Rao, L.; Settembre, C.; Ballabio, A.; Eissa, N. T., STUB1 regulates TFEB-induced autophagy-lysosome pathway. \u003cem\u003eEmbo J \u003c/em\u003e\u003cstrong\u003e2017,\u003c/strong\u003e \u003cem\u003e36\u003c/em\u003e (17), 2544-2552.\u003c/li\u003e\n\u003cli\u003eCheli, Y.; Ohanna, M.; Ballotti, R.; Bertolotto, C., Fifteen-year quest for microphthalmia-associated transcription factor target genes. \u003cem\u003ePigment Cell Melanoma Res \u003c/em\u003e\u003cstrong\u003e2010,\u003c/strong\u003e \u003cem\u003e23\u003c/em\u003e (1), 27-40.\u003c/li\u003e\n\u003cli\u003eSardiello, M.; Palmieri, M.; di Ronza, A.; Medina, D. L.; Valenza, M.; Gennarino, V. A.; Di Malta, C.; Donaudy, F.; Embrione, V.; Polishchuk, R. S.; Banfi, S.; Parenti, G.; Cattaneo, E.; Ballabio, A., A gene network regulating lysosomal biogenesis and function. \u003cem\u003eScience \u003c/em\u003e\u003cstrong\u003e2009,\u003c/strong\u003e \u003cem\u003e325\u003c/em\u003e (5939), 473-7.\u003c/li\u003e\n\u003cli\u003eSettembre, C.; Di Malta, C.; Polito, V. A.; Garcia Arencibia, M.; Vetrini, F.; Erdin, S.; Erdin, S. U.; Huynh, T.; Medina, D.; Colella, P.; Sardiello, M.; Rubinsztein, D. C.; Ballabio, A., TFEB links autophagy to lysosomal biogenesis. \u003cem\u003eScience \u003c/em\u003e\u003cstrong\u003e2011,\u003c/strong\u003e \u003cem\u003e332\u003c/em\u003e (6036), 1429-33.\u003c/li\u003e\n\u003cli\u003eBhagya, N.; Chandrashekar, K. R., Liposome encapsulated anticancer drugs on autophagy in cancer cells - Current and future perspective. \u003cem\u003eInt J Pharm \u003c/em\u003e\u003cstrong\u003e2023,\u003c/strong\u003e \u003cem\u003e642\u003c/em\u003e, 123105.\u003c/li\u003e\n\u003cli\u003eZhou, F.; Li, X. J. Y.; Xue, X. Y.; Li, S.; Fan, G. F.; Cai, Y. J.; Chang, Z. H.; Qu, J. R.; Liu, R. P., A Novel Tri-Functional Liposome Re-Educates \u0026quot;Cold Tumor\u0026quot; and Abrogates Tumor Growth by Synergizing Autophagy Inhibition and PD-L1 Blockade. \u003cem\u003eAdv Healthc Mater \u003c/em\u003e\u003cstrong\u003e2023,\u003c/strong\u003e \u003cem\u003e12\u003c/em\u003e (11).\u003c/li\u003e\n\u003cli\u003ePena-Llopis, S.; Vega-Rubin-de-Celis, S.; Schwartz, J. C.; Wolff, N. C.; Tran, T. A. T.; Zou, L. H.; Xie, X. J.; Corey, D. R.; Brugarolas, J., Regulation of TFEB and V-ATPases by mTORC1. \u003cem\u003eEmbo J \u003c/em\u003e\u003cstrong\u003e2011,\u003c/strong\u003e \u003cem\u003e30\u003c/em\u003e (16), 3242-3258.\u003c/li\u003e\n\u003cli\u003ePena-Llopis, S.; Brugarolas, J., TFEB, a novel mTORC1 effector implicated in lysosome biogenesis, endocytosis and autophagy. \u003cem\u003eCell Cycle \u003c/em\u003e\u003cstrong\u003e2011,\u003c/strong\u003e \u003cem\u003e10\u003c/em\u003e (23), 3987-3988.\u003c/li\u003e\n\u003cli\u003eChen, Y.; Holstein, D. M.; Aime, S.; Bollo, M.; Lechleiter, J. D., Calcineurin beta protects brain after injury by activating the unfolded protein response. \u003cem\u003eNeurobiol Dis \u003c/em\u003e\u003cstrong\u003e2016,\u003c/strong\u003e \u003cem\u003e94\u003c/em\u003e, 139-56.\u003c/li\u003e\n\u003cli\u003eSingh, N.; Kansal, P.; Ahmad, Z.; Baid, N.; Kushwaha, H.; Khatri, N.; Kumar, A., Antimycobacterial effect of IFNG (interferon gamma)-induced autophagy depends on HMOX1 (heme oxygenase 1)-mediated increase in intracellular calcium levels and modulation of PPP3/calcineurin-TFEB (transcription factor EB) axis. \u003cem\u003eAutophagy \u003c/em\u003e\u003cstrong\u003e2018,\u003c/strong\u003e \u003cem\u003e14\u003c/em\u003e (6), 972-991.\u003c/li\u003e\n\u003cli\u003eZhang, X. L.; Cheng, X. P.; Yu, L.; Yang, J. S.; Calvo, R.; Patnaik, S.; Hu, X.; Gao, Q.; Yang, M. M.; Lawas, M.; Delling, M.; Marugan, J.; Ferrer, M.; Xu, H. X., MCOLN1 is a ROS sensor in lysosomes that regulates autophagy. \u003cem\u003eNature Communications \u003c/em\u003e\u003cstrong\u003e2016,\u003c/strong\u003e \u003cem\u003e7\u003c/em\u003e.\u003c/li\u003e\n\u003cli\u003eSong, R. W.; Li, J.; Zhang, J.; Wang, L.; Tong, L.; Wang, P.; Yang, H.; Wei, Q.; Cai, H. B.; Luo, J., Peptides derived from transcription factor EB bind to calcineurin at a similar region as the NFAT-type motif. \u003cem\u003eBiochimie \u003c/em\u003e\u003cstrong\u003e2017,\u003c/strong\u003e \u003cem\u003e142\u003c/em\u003e, 158-167.\u003c/li\u003e\n\u003cli\u003eMedina, D. L.; Di Paola, S.; Peluso, I.; Armani, A.; De Stefani, D.; Venditti, R.; Montefusco, S.; Scotto-Rosato, A.; Prezioso, C.; Forrester, A.; Settembre, C.; Wang, W. Y.; Gao, Q.; Xu, H. X.; Sandri, M.; Rizzuto, R.; De Matteis, M. A.; Ballabio, A., Lysosomal calcium signalling regulates autophagy through calcineurin and TFEB. \u003cem\u003eNature Cell Biology \u003c/em\u003e\u003cstrong\u003e2015,\u003c/strong\u003e \u003cem\u003e17\u003c/em\u003e (3), 288-+.\u003c/li\u003e\n\u003cli\u003eJin, Y. P.; Bai, Y. Y.; Ni, H. Z.; Qiang, L.; Ye, L. C.; Shan, Y. F.; Zhou, M. T., Activation of autophagy through calcium-dependent AMPK/mTOR and PKC\u0026theta; pathway causes activation of rat hepatic stellate cells under hypoxic stress. \u003cem\u003eFebs Letters \u003c/em\u003e\u003cstrong\u003e2016,\u003c/strong\u003e \u003cem\u003e590\u003c/em\u003e (5), 672-682.\u003c/li\u003e\n\u003cli\u003eYang, J.; Yu, J.; Li, D. D.; Yu, S. J.; Ke, J. B.; Wang, L. Y.; Wang, Y. W.; Qiu, Y. Z.; Gao, X. B.; Zhang, J. H.; Huang, L., Store-operated calcium entry-activated autophagy protects EPC proliferation via the CAMKK2-MTOR pathway in ox-LDL exposure. \u003cem\u003eAutophagy \u003c/em\u003e\u003cstrong\u003e2017,\u003c/strong\u003e \u003cem\u003e13\u003c/em\u003e (1), 82-98.\u003c/li\u003e\n\u003cli\u003eMizushima, N.; Yoshimori, T.; Levine, B., Methods in Mammalian Autophagy Research. \u003cem\u003eCell \u003c/em\u003e\u003cstrong\u003e2010,\u003c/strong\u003e \u003cem\u003e140\u003c/em\u003e (3), 313-326.\u003c/li\u003e\n\u003cli\u003eMartina, J. A.; Chen, Y.; Gucek, M.; Puertollano, R., MTORC1 functions as a transcriptional regulator of autophagy by preventing nuclear transport of TFEB. \u003cem\u003eAutophagy \u003c/em\u003e\u003cstrong\u003e2012,\u003c/strong\u003e \u003cem\u003e8\u003c/em\u003e (6), 903-914.\u003c/li\u003e\n\u003cli\u003eRoczniak-Ferguson, A.; Petit, C. S.; Froehlich, F.; Qian, S.; Ky, J.; Angarola, B.; Walther, T. C.; Ferguson, S. M., The Transcription Factor TFEB Links mTORC1 Signaling to Transcriptional Control of Lysosome Homeostasis. \u003cem\u003eSci Signal \u003c/em\u003e\u003cstrong\u003e2012,\u003c/strong\u003e \u003cem\u003e5\u003c/em\u003e (228).\u003c/li\u003e\n\u003cli\u003eVega-Rubin-de-Celis, S.; Pena-Llopis, S.; Konda, M.; Brugarolas, J., Multistep regulation of TFEB by MTORC1. \u003cem\u003eAutophagy \u003c/em\u003e\u003cstrong\u003e2017,\u003c/strong\u003e \u003cem\u003e13\u003c/em\u003e (3), 464-472.\u003c/li\u003e\n\u003cli\u003eYu L, McPhee CK, Zheng L, et al. Termination of autophagy and reformation of lysosomes regulated by mTOR. Nature. \u003cstrong\u003e2010\u003c/strong\u003e;465(7300):942\u0026ndash;946.\u003c/li\u003e\n\u003cli\u003eShyu, W. C.; Lin, S. Z.; Chiang, M. F.; Ding, D. C.; Li, K. W.; Chen, S. F.; Yang, H. I.; Li, H., Overexpression of PrPC by adenovirus-mediated gene targeting reduces ischemic injury in a stroke rat model. \u003cem\u003eJ Neurosci \u003c/em\u003e\u003cstrong\u003e2005,\u003c/strong\u003e \u003cem\u003e25\u003c/em\u003e (39), 8967-77.\u003c/li\u003e\n\u003cli\u003eShin, H. Y.; Oh, J. M.; Kim, Y. S., The Functional Role of Prion Protein (PrPC) on Autophagy. \u003cem\u003ePathogens \u003c/em\u003e\u003cstrong\u003e2013,\u003c/strong\u003e \u003cem\u003e2\u003c/em\u003e (3), 436-45.\u003c/li\u003e\n\u003cli\u003eDing, T.; Zhou, X.; Kouadir, M.; Shi, F.; Yang, Y.; Liu, J.; Wang, M.; Yin, X.; Yang, L.; Zhao, D., Cellular prion protein participates in the regulation of inflammatory response and apoptosis in BV2 microglia during infection with Mycobacterium bovis. \u003cem\u003eJ Mol Neurosci \u003c/em\u003e\u003cstrong\u003e2013,\u003c/strong\u003e \u003cem\u003e51\u003c/em\u003e (1), 118-26.\u003c/li\u003e\n\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":"Transcription factor EB, Cellular prion protein, Autophagy-lysosomal pathway, calcineurin, astrocyte, Cerebral ischemia reperfusion injury","lastPublishedDoi":"10.21203/rs.3.rs-7212124/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7212124/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"An increasing number of studies have focused on understanding the role of macroautophagy/autophagy and the autophagy-lysosomal pathway (ALP) in cerebral ischemic injury. Transcription factor EB (TFEB) is a central regulator of genes involved in autophagy and plays a pivotal role in the regulation of the ALP; however, the mechanisms controlling TFEB activity remain incompletely understood. In this study, we investigated the role of cellular prion protein (PrPC)-targeted TFEB in mediating ALP dysfunction and inflammatory phenotypic changes in mouse cortical astrocytes after cerebral ischemia-reperfusion injury (CIRI). Our findings indicate that during the ultra-early phase of CIRI, intracellular Ca2+ levels are low, with inhibited PPP3/calcineurin activity and reactivation of mTOR, leading to TFEB phosphorylation and retention in the cytoplasm. As reperfusion time increases, elevated intracellular Ca2+ levels activate PPP3/calcineurin, resulting in TFEB dephosphorylation, nuclear translocation, and the subsequent induction of autophagy lysosome-associated gene transcription. This process promotes astrocyte survival and shifts the cellular phenotype toward an anti-inflammatory state. Furthermore, increased PrPC expression was observed to maintain intracellular Ca2+ homeostasis and sustain PPP3/calcineurin activation, facilitating continuous TFEB dephosphorylation and nuclear translocation. These results clarify the regulatory role of PrPC in astrocytic autophagy-lysosomal pathways following cerebral ischemia/reperfusion injury, providing new insights for more targeted interventions in ischemic stroke.","manuscriptTitle":"PrPC-Mediated Ca²⁺/Calcineurin/TFEB Signaling Enhances Autophagic-Lysosomal Function and Anti-inflammatory Astrocyte Transition to Alleviate Cerebral Ischemia-Reperfusion Injury","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-31 05:32:35","doi":"10.21203/rs.3.rs-7212124/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":"87e73dce-3102-435e-a170-bd34b91b75c9","owner":[],"postedDate":"July 31st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-09-21T12:23:18+00:00","versionOfRecord":[],"versionCreatedAt":"2025-07-31 05:32:35","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7212124","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7212124","identity":"rs-7212124","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}