Irisin mediates exercise-induced glymphatic α-synuclein clearance by upregulating astrocytic REV-ERBα in Parkinson’s disease

Irisin mediates exercise-induced glymphatic α-synuclein clearance by upregulating astrocytic REV-ERBα in Parkinson’s disease | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Irisin mediates exercise-induced glymphatic α-synuclein clearance by upregulating astrocytic REV-ERBα in Parkinson’s disease Lingjing Jin, Ruoyu Li, Jinbao Zhang, Xining Ren, Bingyu Li, Xuerui Xiang, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9467573/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract Physical exercise alleviates motor deficits and neuropathology in Parkinson’s disease (PD), yet the underlying mechanisms remain elusive. Using a mouse model induced by α-synuclein (αSyn) preformed fibrils, we found that glymphatic transport was markedly impaired in PD mice, accompanied by disrupted perivascular aquaporin-4 (AQP4) polarization and αSyn accumulation. Chronic treadmill exercise restored glymphatic function, restored AQP4 polarity, and alleviated motor deficits and neuropathology; these effects were abrogated by AQP4 inhibition. We identified the exercise-induced myokine irisin as a critical systemic mediator linking peripheral activity to central clearance. Systemic irisin administration recapitulated the glymphatic benefits of exercise, whereas Fndc5 deficiency abolished them. Mechanistically, irisin acted on astrocytes to suppress STAT3 activation and upregulate the circadian gene Nr1d1 (encoding REV-ERBα). Astrocyte-specific knockdown of Nr1d1 disrupted AQP4 polarization and eliminated the glymphatic and neuroprotective effects of both exercise and irisin. Collectively, these findings establish an exercise-irisin-astrocyte axis that enhances glymphatic clearance of αSyn aggregates through REV-ERBα-dependent regulation of astrocytic function, revealing a previously unrecognized mechanism linking peripheral exercise to brain proteostasis. Health sciences/Neurology/Neurological disorders/Parkinson's disease Biological sciences/Neuroscience/Glial biology/Astrocyte Parkinson’s disease Exercise Glymphatic system α-Synuclein Irisin REV-ERBα Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Introduction Parkinson’s disease (PD) is a progressive neurodegenerative disorder characterized by the degeneration of dopaminergic neurons in the substantia nigra and the accumulation of misfolded α-synuclein (αSyn) 1 . Current therapies offer only symptomatic relief, underscoring the urgent need for interventions that target underlying pathogenic mechanisms 2 . In this context, physical exercise has emerged as a powerful non-pharmacological intervention, with epidemiological and preclinical studies consistently demonstrating its capacity to ameliorate motor impairments, mitigate dopaminergic degeneration, and lower αSyn burden 3-7 . However, the molecular pathways linking exercise to central pathology remain largely undefined, representing a critical barrier to the development of mechanism-based disease-modifying strategies 7,8 . Emerging evidence points to the glymphatic system as a critical player in brain proteostasis and a potential mediator of exercise’s benefits 9-11 . The glymphatic system is a glial-dependent perivascular network that facilitates the exchange of cerebrospinal fluid and interstitial fluid, promoting the clearance of metabolic waste and aggregation-prone proteins, including αSyn 12-14 . Glymphatic dysfunction, often characterized by the loss of perivascular aquaporin-4 (AQP4) polarization on astrocytic endfeet, has been implicated in PD pathogenesis, contributing to αSyn aggregation and neurodegeneration 15-19 . Notably, studies have linked exercise-induced improvements in meningeal lymphatic vessel function to enhanced perivascular clearance and have highlighted its capacity to reverse sleep-dependent glymphatic dysfunction 11,20,21 . Other studies have shown that exercise ameliorates glymphatic impairment and reduces amyloid-β pathology in Alzheimer's disease models 10,22,23 . Despite mounting evidences, the specific exercise-induced factors that link physical activity to central glymphatic clearance in PD remain elusive. The emerging concept of the “muscle–brain axis” has established skeletal muscle as a dynamic endocrine organ that communicates with the central nervous system through exercise-induced myokines 24,25 . Irisin, a cleavage product of the membrane protein FNDC5, is released from muscle into circulation during physical activity and has emerged as a mediator of exercise-induced neuroprotection. Importantly, irisin can cross the blood–brain barrier 26,27 , enabling it to directly influence central nervous system function. In our previous study, we observed that circulating irisin levels were significantly elevated in PD patients after 12 weeks of regular exercise, and the extent of this rise correlated strongly with improvements in balance performance 28 . However, whether irisin recapitulates the beneficial effects of exercise on αSyn pathology, as well as the underlying mechanisms, remains unexplored. In this study, we demonstrated that chronic treadmill exercise restored impaired glymphatic transport and reduced αSyn accumulation in a preformed fibrils (PFFs)-based PD model. Furthermore, we identified exercise-induced irisin as a key systemic mediator that acted on astrocytes to suppress STAT3 activation and increase the expression of circadian nuclear receptor REV-ERBα, thereby curbing inflammatory activation, restoring AQP4 polarization, and enhancing glymphatic clearance of αSyn. Importantly, astrocyte-specific knockdown of Nr1d1 (encoding REV-ERBα) abolished the glymphatic benefits of both exercise and irisin. Collectively, our findings established a muscle–brain signaling axis in which exercise-derived irisin promotes glymphatic function through upregulating REV-ERBα, providing a mechanistic framework linking physical activity to brain proteostasis. Results Exercise improves motor function and reduces nigrostriatal pathology in PD mice To investigate the effects of physical exercise on PD pathology, we established PD mouse model by unilateral striatal injection of PFFs, followed by a 3-month treadmill exercise intervention (Fig. 1a). PFFs-injected mice exhibited progressive motor impairment, as indicated by decreased locomotor activity in the open field test, increased descent time in the pole test and reduced latency to fall in the rotarod test. Exercise markedly improved motor performance across all behavioral assays, with significant recovery observed over time compared to sedentary PFFs mice (Fig. 1b–d). These findings indicate that sustained exercise alleviates motor deficits in this model. We next examined dopaminergic neurodegeneration and αSyn pathology in the nigrostriatal pathway. PFFs injection induced marked dopaminergic neurodegeneration, as evidenced by a substantial loss of tyrosine hydroxylase (TH)-positive neurons in the substantia nigra and reduced TH-positive fiber density in the striatum. This was accompanied by a pronounced accumulation of phosphorylated αSyn (pSer129) in the striatum, substantia nigra, and cortex. Notably, exercise significantly preserved TH-positive neurons and fiber density while concurrently reducing p-αSyn burden across these regions (Fig. 1e–k), indicating a coordinated attenuation of neurodegeneration and pathological protein accumulation. Together, these results demonstrate that long-term exercise mitigates motor deficits, preserves nigrostriatal integrity, and reduces p-αSyn burden in the PFFs model. Exercise enhances glymphatic transport and accelerates parenchymal clearance in PD mice Impaired clearance of pathological proteins is closely linked to PD progression 16,18 . We next asked whether exercise improves glymphatic function using dynamic contrast-enhanced MRI (DCE-MRI) in the PFFs model. Following intracisternal administration of Gd-DTPA as the contrast agent, control mice exhibited robust tracer influx and widespread distribution across brain regions, whereas PFFs mice showed markedly reduced signal intensity and impaired tracer propagation (Fig. 2a). Exercise significantly restored tracer influx and distribution in multiple regions, including the striatum, substantia nigra, cortex and hippocampus (Fig. 2b–e), indicating improved glymphatic transport. Time–signal intensity analysis further revealed that PFFs mice displayed delayed signal responses, consistent with impaired cerebrospinal–interstitial fluid exchange. In contrast, exercise enhanced both the magnitude and dynamics of tracer transport, suggesting a recovery of glymphatic flow. To more directly assess the effect of exercise on glymphatic clearance in the brain parenchyma, we examined parenchymal solute transport and clearance after intrastriatal co-injection of TR-d10 tracer and AF488-labeled αSyn PFFs (PFFs-488). In PFFs mice, both tracers were retained more strongly within the brain parenchyma, indicating impaired clearance (Fig. 2f–j). Exercise reduced the retention of both TR-d10 and PFFs-488 across coronal sections and enhanced their dispersion away from the injection site (Fig. 2f–j). Importantly, similar changes were observed for both tracers despite their different properties, indicating that exercise broadly improves parenchymal solute transport and clearance rather than affecting a single substrate. To further confirm that tracer clearance occurred via the glymphatic system, we also analyzed the amount of TR-d10 and PFFs-488 in the deep cervical lymph nodes (dCLNs), which are reported to drain lymph fluid from the brain. We found a significant reduction of both TR-d10 and PFFs-488 in the dCLNs of PFFs mice compared with the PBS group (Fig. 2k–m). However, this reduction was markedly alleviated by exercise (Fig. 2k–m). Together, these results demonstrate that exercise restores impaired glymphatic transport and promotes efficient brain clearance in PD mice. Exercise restores AQP4-dependent glymphatic function in PD mice We next examined whether AQP4 polarity underlies the exercise-induced improvement in glymphatic transport and parenchymal clearance 29 . Under control conditions, AQP4 was predominantly confined to perivascular endfeet, whereas administration of PFFs resulted in a marked disruption of this polarized distribution (Fig. 3a–c). Exercise restored AQP4 localization toward blood vessels, as reflected by a significant recovery of AQP4 polarity (Fig. 3a–c). Importantly, AQP4 polarity was negatively correlated with p-αSyn accumulation, suggesting a link between astrocytic structural integrity and pathological protein burden (Fig. 3d). To further determine whether AQP4-dependent mechanisms are required for the benefits of exercise, we pharmacologically inhibited AQP4 using TGN-020 during the intervention period (Fig. 3e). We found that the reduction in p-αSyn burden observed with exercise was lost after TGN-020 treatment, with p-αSyn levels remaining elevated and comparable to those in sedentary PFFs mice (Fig. 3f–h). Meanwhile, AQP4 inhibition largely abolished the exercise-induced improvements in motor performance, including performance in the open field test, pole test and rotarod test (Fig. 3i–k). These results revealed that enhanced AQP4 polarity is a key mechanism by which exercise promotes glymphatic clearance of αSyn aggregates. Irisin is the circulating mediator of exercise-induced glymphatic rescue We next asked whether exercise-responsive molecules could link peripheral activity to glymphatic regulation. A compelling candidate that may link exercise to neuroprotection is irisin, a cleavage product of the membrane protein FNDC5 that is released from skeletal muscle in response to exercise and can cross the blood–brain barrier 26,27,30 . We previously found that exercise elevated irisin levels in a PD mouse model, and that systemic administration of irisin sufficed to recapitulate the motor-improving effects of exercise 28 . We therefore sought to determine whether irisin acts by directly modulating AQP4 polarization and recapitulating the effect of exercise on glymphatic function in PFFs-injected mice. Immunofluorescence staining revealed that PFFs injection markedly disrupted the perivascular distribution of AQP4, whereas irisin treatment significantly restored AQP4 polarization around CD31-positive vessels (Fig. 4a–b). We then performed intrastriatal co-injection of TR-d10 and PFFs-488 in mice to determine whether irisin directly regulates parenchymal solute transport and clearance. Representative images across multiple rostrocaudal levels showed that irisin-treated mice exhibited a broader spatial distribution of both tracers compared to controls, indicating enhanced interstitial transport within the brain parenchyma (Fig. 4c). Quantitative analysis revealed a significant reduction in total TR-d10 signal in the injected hemisphere (Fig. 4d–e), consistent with accelerated clearance. Similarly, the parenchymal retention of PFFs-488 was markedly decreased following irisin administration (Fig. 4f–g), reflecting enhanced removal of protein tracers. Moreover, fluorescence intensity of both tracers in the deep cervical lymph nodes was significantly increased in irisin-treated mice (Fig. 4h–j), supporting enhanced brain-to-periphery efflux. To further assess whether endogenous irisin is necessary for exercise-induced enhancement of parenchymal clearance, we applied the same tracer clearance paradigm in wild-type and FNDC5-KO mice. In wild-type mice, exercise increased the spatial distribution of both TR-d10 and PFFs-488 and reduced their retention within the parenchyma, indicating enhanced clearance (Fig. 5a–e). In contrast, FNDC5-KO mice failed to exhibit these exercise-induced changes, with tracer distribution remaining restricted and no significant reduction in TR-d10 or PFFs-488 signal observed following exercise (Fig. 5a–e). Collectively, these findings demonstrate that irisin is required for exercise-induced enhancement of parenchymal solute transport and clearance. To determine whether elevating irisin levels is sufficient to recapitulate the protective effects of exercise at the organismal level, we overexpressed FNDC5 via systemic AAV delivery according to previous study 31 (Supplementary Fig. 2a). ELISA revealed decreased serum irisin levels in PD mice, whereas AAV treatment restored the levels to those of control mice (Supplementary Fig. 2b). Behavioral assessments revealed that FNDC5 overexpression markedly improved motor function, as evidenced by increased locomotor activity in the open field test, reduced descent time in the pole test and increased latency to fall in the rotarod test, compared to PFFs mice (Supplementary Fig. 2c–e). Histological analysis further showed that FNDC5 overexpression attenuated dopaminergic neurodegeneration. Specifically, TH immunostaining demonstrated preservation of dopaminergic neurons in the substantia nigra and restoration of TH-positive fiber density in the striatum (Supplementary Fig. 2f–h). In parallel, p-αSyn accumulation was significantly reduced in FNDC5-overexpressing mice (Supplementary Fig. 2i–j). Together, these results demonstrate that systemic elevation of irisin is sufficient to recapitulate the neuroprotective effects of exercise. Irisin restores a homeostatic astrocyte state and upregulates REV-ERBα The inflammatory status of astrocytes is closely linked to perivascular AQP4 polarization, wherein pro-inflammatory (A1-like) astrocytes typically exhibit disrupted AQP4 localization and impaired glymphatic function, whereas anti-inflammatory or homeostatic (A2-like) phenotypes support AQP4 polarity 10 . We therefore examined whether the effects of exercise on AQP4 polarity were associated with the functional state of astrocytes. Immunostaining for the astrocytic marker GFAP, the A1-associated marker C3d, and the A2-associated marker S100A10 revealed that PFFs injection induced a reactive astrocytic phenotype, whereas exercise reversed this shift (Fig. 6a–c). Specifically, PFFs-injected mice exhibited increased C3d expression and decreased S100A10 expression; in contrast, exercise significantly reduced C3d while upregulating S100A10, consistent with restoration of a more homeostatic astrocyte state (Fig. 6d–e). Sholl analysis confirmed this remodeling, showing increased summarized intersections and altered branching profiles after PFFs injection, both of which were attenuated by exercise (Fig. 6f–g). These findings demonstrate that exercise restores astrocytic AQP4 polarity and promotes a more homeostatic astrocyte state. To determine whether irisin could recapitulate the effects of exercise on astrocytic phenotype, we cultured primary astrocytes and treated them with with PFFs and/or irisin. Western blotting analysis revealed that C3d was significantly reduced in the PFFs+Irisin group, while S100A10 was significantly increased (Fig. 7a–c), suggesting that irisin shifts astrocytes from neurotoxic A1 phenotypes to neuroprotective A2 phenotypes, thereby counteracting PFF-induced reactive activation. Meanwhile, western blotting results also revealed significantly reduced p-STAT3 levels in the PFFs+Irisin group compared with PFFs group (Fig. 7d–e), indicating that irisin suppresses STAT3 activation. Previous studies have shown that p-STAT3 can directly repress transcription of the circadian gene Nr1d1 (encoding REV-ERBα) 32,33 , which in turn influences the functional state of astrocytes 34 . We therefore examined whether irisin modulates REV-ERBα expression. Notably, irisin treatment also upregulated the circadian regulator REV-ERBα, which was downregulated upon PFFs stimulation (Fig. 7d,7f). These findings suggest that irisin can suppress PFFs-induced astrocytic inflammatory activation and promotes a shift toward a neuroprotective phenotype via modulation of the STAT3/REV-ERBα signaling. REV-ERBα is indispensable for exercise- and irisin-induced glymphatic improvement To investigate the role of REV-ERBα in exercise-induced glymphatic improvement, we performed astrocyte-specific knockdown of Nr1d1 using AAV-shRNA and assessed its impact on astrocyte polarization. AQP4 polarization was significantly reduced in Nr1d1 -shRNA treated mice, as evidenced by immunostaining for AQP4 and CD31, demonstrating AQP4 depolarization in the astrocytes (Fig. 8a–b). Additionally, Nr1d1 -shRNA treatment in the striatum resulted in significant astrocyte activation, as indicated by Sholl analysis of astrocyte branching, showing an increase in summarized intersections and complexity (Fig. 8c–e). These findings indicate that REV-ERBα is required for maintaining both perivascular AQP4 polarity and proper astrocytic morphology. To further investigate the role of REV-ERBα in exercise- and irisin-induced glymphatic improvement, we assessed the effects of exercise and irisin on glymphatic function in Nr1d1 -shRNA mice. OVA-647 was used instead of TR-d10 to avoid spectral overlap with mCherry expressed from the AAV-shRNA vector. Representative images of OVA-647 and PFFs-488 showed that Nr1d1 knockdown markedly impaired tracer clearance (Fig. 9a). Quantitative analysis revealed that Nr1d1 deficiency significantly increased the retention of OVA-647 and PFFs-488, indicating impaired clearance. Importantly, neither exercise nor irisin administration was able to restore tracer distribution or clearance in Nr1d1 -deficient mice, as no significant differences were observed between Nr1d1 -shRNA, Nr1d1 -shRNA+Ex, and Nr1d1 -shRNA+Irisin groups (Fig. 9b–e). The results demonstrate that Nr1d1 is essential for the exercise- and irisin-mediated restoration of glymphatic function, as Nr1d1 knockdown prevents the exercise-induced enhancement of glymphatic clearance and AQP4 polarization in astrocytes. Discussion In this study, we identified a previously unrecognized muscle–brain–glymphatic axis through which physical exercise enhances the clearance of αSyn aggregates in a mouse model of PD. We demonstrated that chronic treadmill exercise restores impaired glymphatic transport and reduces αSyn pathology in the PFFs model, an effect not previously reported for exercise in the context of synucleinopathy. We further identified the exercise-induced myokine irisin as a critical systemic mediator that is both sufficient and necessary for the glymphatic benefits, revealing a novel role for irisin in promoting central protein clearance. Mechanistically, we showed that irisin acts on astrocytes to upregulate the circadian nuclear receptor REV-ERBα, which in turn restores perivascular AQP4 polarity and shifts astrocytes toward a homeostatic phenotype. Notably, the requirement of astrocytic REV-ERBα for exercise- and irisin-induced glymphatic enhancement represents a previously unidentified link between circadian control and astrocyte-dependent proteostasis in PD. Glymphatic dysfunction is increasingly recognized as a pathogenic feature across neurodegenerative conditions, including Alzheimer’s disease and PD 16,17,35-37 . Previous studies in Alzheimer’s disease and aged animals have shown that exercise promotes cerebrospinal fluid influx and restores AQP4 polarization at astrocytic endfeet, thereby facilitating amyloid-β clearance 9,10,38 . However, whether such mechanisms operate in PD and contribute to the clearance of αSyn aggregates remained unexplored. Our findings extend this knowledge by demonstrating that exercise improves glymphatic transport and reduces αSyn pathology in the PFFs model, positioning glymphatic restoration as a key mechanism underlying the neuroprotective effects of exercise in PD. Moreover, while earlier work has highlighted the importance of AQP4 for glymphatic function 10,38,39 , we provide direct evidence that AQP4 is required for the therapeutic benefits of exercise, as pharmacological inhibition of AQP4 abrogated exercise-induced reductions in αSyn pathology and motor recovery. These findings establish astrocyte-dependent fluid transport as a critical effector downstream of exercise and upstream of enhanced brain clearance in synucleinopathy. The systemic signals that link peripheral exercise to central glymphatic regulation remain poorly characterized. As a prototypical exercise-induced myokine which can cross the blood–brain barrier, irisin has emerged with neuroprotective properties in various neurological disorders 26,30,40,41 . Recent studies have shown that irisin can protect dopaminergic neurons, modulate glial cell activity, and reduce neuroinflammation in PD models 28,31 . However, the role of irisin in regulating global brain waste clearance had not been reported. Our study fills this gap by demonstrating that irisin is sufficient to enhance parenchymal solute transport and accelerate the clearance of αSyn aggregates, and that endogenous irisin is required for the glymphatic benefits of exercise. Moreover, we observed that exercise and irisin shift astrocytes from a pro-inflammatory A1-like state toward a more supportive A2-like phenotype, a transition tightly linked to the recovery of AQP4 polarity. This phenotypic remodeling might represent a common mechanism through which diverse interventions, including exercise, enhance brain clearance across neurodegenerative conditions. These findings expand the functional repertoire of irisin beyond direct neuronal modulation, implicating it as a systemic orchestrator of astrocyte-mediated proteostasis. Mechanistically, our findings established REV-ERBα as a critical transcriptional node linking exercise-induced peripheral signals to astrocyte-dependent glymphatic function, positioning circadian control at the center of brain proteostasis in PD. This was further supported by our observation that astrocyte-specific REV-ERBα disruption abolished the beneficial effects of both exercise and irisin on glymphatic clearance, underscoring the non-redundant role of this circadian factor in mediating central responses to peripheral physiological stimuli. Importantly, these findings suggested that the efficacy of exercise might depend not only on its intensity or duration but also on its temporal alignment with endogenous circadian rhythms. Given that glymphatic activity peaks during sleep and that REV-ERBα expression follows a daily oscillation 39,42 , it is plausible that exercise timing could synergize with rhythmic perivascular dynamics to optimize solute clearance 43,44 . Moreover, because PD is characterized by blunted circadian amplitude and fragmented rest–activity patterns 45,46 , exercise-induced restoration of REV-ERBα expression may help re-establish circadian homeostasis, thereby reinforcing the temporal architecture of glymphatic function 34,45 . This perspective raises the possibility that exercise interventions could be optimized as “chronotherapies,” tailored to align with individual circadian profiles to maximize neuroprotection. More broadly, our findings support a model in which peripheral signals such as irisin do not merely activate static molecular pathways but instead engage dynamic. This conceptual shift highlights the importance of integrating circadian biology into mechanistic studies of exercise and neurodegeneration, paving the way for chrono-exercise strategies in the management of PD and related disorders. Several limitations should be acknowledged. First, glymphatic transport is influenced by multiple physiological parameters, including sleep–wake state, vascular pulsatility, and circadian timing, which were not systematically controlled in the current study. Second, while our findings in the PFFs model provide mechanistic insight, the generalizability of this axis to other αSyn-based models or to aging remains to be established. Third, although we identified REV-ERBα as a critical mediator of exercise-induced glymphatic enhancement, our study did not manipulate the circadian timing of exercise. Given the known diurnal oscillations of REV-ERBα and glymphatic activity, it remains unknown whether exercise at specific zeitgeber times would produce differential effects. Future studies incorporating time-of-day interventions are warranted to explore this possibility. In summary, we describe a previously unrecognized pathway through which physical exercise engages a peripheral myokine to regulate astrocyte-dependent glymphatic clearance in PD. As illustrated in our working model (Fig. 10), physical exercise stimulates skeletal muscle to release irisin, which acts on astrocytes to restore REV-ERBα–dependent transcription, suppress inflammatory activation, and promote AQP4 polarization at perivascular endfeet. This astrocytic remodeling enhances glymphatic clearance of αSyn aggregates and contributes to dopaminergic neuroprotection. Together, these results establish a mechanistic framework linking systemic physical activity to astrocyte-mediated brain proteostasis and highlight the exercise/irisin/REV-ERBα axis as a promising therapeutic target for neurodegenerative diseases. Methods Animals Eight-week-old male C57BL/6J mice were obtained from Shanghai GemPharmatech Co., Ltd. FNDC5 knockout (FNDC5-KO) mice (Strain No. T014328, Cas9-mediated knockout; C57BL/6JGpt background) were also purchased from Shanghai GemPharmatech Co., Ltd. Mice were housed in groups of five per cage under SPF conditions in a ventilated animal facility. Animals were maintained on a 12-hour light/dark cycle with controlled temperature (20–23°C) and humidity (50–60%) and had ad libitum access to standard chow and water. All experimental procedures were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Ethics Committee of Tongji University (approval number: TJBC00322103). Preparation of αSyn PFFs Recombinant monomeric mouse αSyn protein was purchased from AnaSpec (AS-56082-100). The protein was dissolved in 100 mM NaCl to a final concentration of 5 mg/mL. For fibril formation, αSyn solution was incubated in a shaking dry bath (dryBATH-HS, WIX) at 37°C with continuous agitation at 1,000 rpm for 7 days. Prior to use, the formed fibrils were sonicated for 30 s at 15% amplitude with a 0.5 s on/off pulse cycle using a probe sonicator (JY92-IIN, SCIENTZ) to generate αSyn PFFs. For generation of PFFs-488, the sonicated PFFs were labeled using an Alexa Fluor™ 488 protein labeling kit (A30006, Thermo Fisher Scientific) according to the manufacturer’s instructions. Labeled fibrils were used immediately or stored under appropriate conditions until further use. Stereotaxic Injection of αSyn PFFs Two-month-old wild-type mice were anesthetized with 2.5% isoflurane for induction and maintained at 1% isoflurane during surgery via continuous inhalation. Animals were secured in a stereotaxic frame and Sodium hyaluronate ophthalmic solution were applied to prevent corneal drying during anesthesia. After exposing the skull, a small burr hole was drilled at the injection site targeting the right striatum (AP: 0.5 mm; ML: 2.0 mm; DV: –3.2 mm from bregma). Sonicated αSyn PFFs (5 μg in 2 μL) were injected using a 34-gauge Hamilton syringe at a rate of 0.2 μL/min. Upon completion of infusion, the needle was left in place for an additional 5 minutes to minimize reflux and then slowly withdrawn. Following surgery, mice received subcutaneous meloxicam (2.5 mg/kg) for postoperative analgesia and were placed on a heating pad until fully recovered from anesthesia. Pharmacology To modulate AQP4 function in mice, we used TGN-020 (MedChemExpress, HY-W008574), a selective AQP4 inhibitor. TGN-020 was prepared as a suspension in saline containing 0.5% methyl cellulose and 0.5% Tween-80 at a concentration of 5 mg/mL using sonication-assisted dissolution. Mice received intraperitoneal injections of TGN-020 at a dose of 100 mg/kg prior to each exercise session. To mimic exercise-induced circulating factors, we used recombinant irisin (R&D Systems, 8880-IR). Irisin was dissolved in sterile saline and administered at a dose of 200 μg/kg by intraperitoneal injection. Control mice received vehicle solution with the same composition. To assess the direct role of irisin in regulating astrocyte responses, recombinant irisin was applied to primary astrocyte cultures at a final concentration of 200 ng/mL. Viral vector delivery To manipulate systemic irisin levels, we used AAV-FNDC5, which was delivered by tail-vein injection on the same day as PFFs administration. To achieve astrocyte-specific knockdown of Nr1d1 , AAV- Nr1d1 -shRNA was stereotaxically injected into the striatum. Mice were anesthetized and fixed in a stereotaxic frame, and the virus was unilaterally injected at the following coordinates relative to bregma: AP, +0.5 mm; ML, +2.0 mm; DV, −3.2 mm. The injection was performed using a microsyringe at a constant rate, and the needle was left in place for 5 min before slow withdrawal to minimize backflow. Cisterna magna cannulation Cisterna magna cannulation was performed to enable intrathecal delivery of contrast agents during magnetic resonance imaging. Mice were anesthetized with isoflurane delivered via inhalation and positioned in a stereotaxic frame. The head was positioned at an angle of approximately 120–150 degrees relative to the body to optimize surgical exposure. Hair overlying the posterior skull and upper cervical region was removed, and a midline skin incision of approximately 5 mm was made. The bilateral trapezius muscles were separated along the midline, and deeper musculature was bluntly dissected using toothed forceps to expose the cisterna magna under direct visualization. A custom copper cannula (outer diameter 0.35 mm, inner diameter 0.28 mm) was implanted for intrathecal access. Copper was selected because of its diamagnetic properties, which minimize magnetic susceptibility artifacts during magnetic resonance imaging. The cannula was connected to PE10 tubing prefilled with artificial cerebrospinal fluid (ACSF; PH1851, PHYGENE) to prevent air bubble formation. After insertion into the cisterna magna, the cannula was secured using a mixture of ethyl cyanoacrylate adhesive and dental cement. An accelerator solution was applied to promote rapid polymerization. The distal end of the PE10 tubing was sealed using a thermal cauterizer. Following surgery, mice received subcutaneous meloxicam for postoperative analgesia and were placed on a heating pad until fully recovered from anesthesia. Parenchymal clearance assay To evaluate interstitial solute clearance from the brain, fluorescent tracers were stereotaxically injected into the unilateral striatum. PFFs-488 were mixed with either Texas Red–dextran (10 kDa; TR-d10, ThermoFisher Scientific, D1863) or Alexa Fluor™ 647–conjugated ovalbumin (OVA-647; Invitrogen, O34784). All tracers were adjusted to a final concentration of 2.5 mg/mL prior to injection. Mice were anesthetized with isoflurane and secured in a stereotaxic frame. After exposing the skull, a small burr hole was drilled at the striatal coordinates (AP: 0.5 mm; ML: 2.0 mm; DV: –3.2 mm). A total volume of 2 μL tracer mixture was injected at a rate of 0.2 μL/min using a micro-pump (KDS Legato 130, RWD Life Science). Following infusion, the needle was left in place for 5 minutes and then slowly withdrawn to minimize reflux. Two hours after injection, mice were sacrificed and brains were harvested and fixed in 4% PFA overnight at 4 °C. Brains were coronally sectioned at 100 μm thickness using a vibratome (Leica VT1200 S). Fluorescence images were acquired using an automated digital slide scanning system (Axioscan 7, Carl Zeiss) under identical exposure settings across all groups. For quantitative analysis, five coronal sections per animal, spaced at 400 μm intervals, were analyzed. Using ImageJ, the entire brain slice was manually outlined as the region of interest. Mean fluorescence intensity within this region was measured for each section after background subtraction. The average fluorescence intensity from the five sections was calculated to obtain a single value per animal for statistical analysis. DCE-MRI measurements All MRI experiments were performed on a horizontal bore 9.4 Tesla / 30 cm scanner (uMR 9.4T, United Imaging Life Science Instrument, Wuhan, China). An 86-mm inner diameter volume coil was used for radio frequency transmission, and a mouse brain surface coil was used for signal reception. Dynamic contrast-enhanced magnetic resonance imaging was conducted in adult C57BL/6 mice using a three-dimensional T1-weighted gradient-echo sequence with an isotropic spatial resolution of 0.15 × 0.15 × 0.15 mm³. A total of 80 dynamic frames were acquired over 78 seconds. Mice were surgically implanted with a cisterna magna cannula prior to imaging to enable controlled intrathecal delivery of contrast agent. The first dynamic frame was acquired as a pre-contrast baseline. Immediately after baseline acquisition, 10 μL of Gd-DTPA was infused through the pre-implanted cannula at a rate of 1 μL per minute using a microinfusion pump while dynamic acquisition continued. Raw DICOM images were converted to NIfTI format using dcm2niix. Motion correction of the four-dimensional dynamic series was performed using rigid-body registration implemented in Advanced Normalization Tools (version 2.6.2), with each frame aligned to a reference volume. A motion-corrected mean image was generated for region-of-interest delineation. Regions of interest were manually defined in subject space using ITK-SNAP (version 3.8.0). Mean signal intensity within each region was extracted across time to generate dynamic curves. Quantitative parameters, including area under the curve, peak signal intensity, and time-to-peak, were calculated for downstream analyses. Image processing and quantitative analyses were performed using Advanced Normalization Tools, ITK-SNAP, nibabel (version 5.2.1), and custom Python scripts executed in Jupyter Notebook. All procedures were applied uniformly across animals. Open field test To assess general locomotor activity and anxiety-like behavior, mice were tested in an open field arena (40 × 40 × 40 cm). Prior to testing, animals were transferred to the behavioral testing room at least 3 hours in advance to allow acclimatization to the experimental environment and minimize stress-related confounding effects. During the test session, each mouse was placed in the center of the arena and allowed to freely explore for 10 minutes. Locomotor activity was recorded using an automated video tracking system (Shanghai Xinruan Technology, China). For quantitative analysis, total distance traveled during the first 5 minutes was used to evaluate spontaneous locomotor activity. Time spent in the center area of the arena was recorded as an index of anxiety-like behavior. To eliminate olfactory cues between subjects, the arena was thoroughly cleaned with 75% ethanol after each trial. The next mouse was tested only after complete evaporation of the ethanol to avoid residual odor interference. Rotarod test Motor coordination and balance were assessed using a rotarod apparatus (Shanghai Xinruan Technology, China). Mice underwent a habituation phase prior to formal testing. Animals were trained for three consecutive days, with one session per day. During habituation, mice were placed on the rotating rod for 5 minutes each day. The rotation speed was maintained at 4 revolutions per minute on the first and second training days and increased from 4 to 10 revolutions per minute on the third day to facilitate adaptation to accelerating conditions. For the test session, mice were placed on the rod, which accelerated from 4 to 40 revolutions per minute over a period of 5 minutes. The latency to fall was recorded for each trial. Each mouse completed three trials with appropriate rest intervals between trials, and the average latency to fall was used for statistical analysis. To minimize olfactory cues and avoid confounding effects from residual scent, the rotarod apparatus and testing compartments were cleaned with 75% ethanol between trials. Pole test The pole test was performed to evaluate motor coordination as well as the ability of mice to turn and descend. The apparatus consisted of a vertical pole (50 cm in height, 1 cm in diameter) with a rough surface to facilitate grip. Prior to formal testing, mice underwent a habituation phase for two consecutive days. During habituation, each mouse performed three descent trials per day to acclimate to the task. For the test session, mice were placed head-upward on the top of the vertical pole. The time required to turn downward and descend to the base of the pole was recorded. Each mouse completed three trials, and the average descent time was used for statistical analysis. Mice were anesthetized with isoflurane prior to tissue collection. For both fresh tissue collection and perfusion-fixed tissue preparation, blood was first obtained via cardiac puncture and collected into 1.5 mL microcentrifuge tubes for serum isolation. Tissue collection Mice were anesthetized with isoflurane prior to tissue collection. Blood was first obtained via cardiac puncture and collected into 1.5 mL microcentrifuge tubes for serum isolation. For biochemical analyses, mice were transcardially perfused with pre-cooled phosphate-buffered saline to remove circulating blood. Brains were rapidly dissected and transferred onto sterile filter paper. Under a stereomicroscope, ipsilateral and contralateral brain regions, including the cortex, striatum, ventral midbrain, and hippocampus, were carefully separated. Dissected tissues were immediately frozen and stored at −80℃ for subsequent Western blotting and enzyme-linked immunosorbent assay analyses. For immunofluorescence and immunohistochemistry, mice were transcardially perfused with phosphate-buffered saline followed by 4% paraformaldehyde. Brains were dissected and post-fixed in 4% paraformaldehyde at 4℃for 24 h. Tissues were then washed with phosphate-buffered saline and cryoprotected in graded sucrose solutions (20%, 30%, and 30%) over three days. Subsequently, brains were embedded in Tissue-Tek OCT compound (SAKURA, USA) and stored at −80℃. Coronal sections (30μm) were prepared using a cryostat (Leica CM1950) and transferred to cryoprotectant solution containing 30% sucrose, 20% ethylene glycol, and 1% PVP-40 in 1× PBS for storage at −20℃ until further use. Immunohistochemistry and Immunofluorescence Frozen brain sections were retrieved from cryoprotectant solution and washed with phosphate-buffered saline (PBS) prior to staining. For immunohistochemical staining, sections were subjected to antigen retrieval and endogenous peroxidase blocking, followed by blocking at room temperature for 1 h in 1× PBS containing 0.3% Triton X-100 (Sigma-Aldrich, X100) and 10% horse serum (Gibco, 16050122). Sections were then incubated overnight at 4℃ with anti–tyrosine hydroxylase antibody. After washing, sections were incubated with biotinylated horse anti-mouse secondary antibody (Vector Laboratories, BA-2000), followed by signal amplification using the VECTASTAIN Elite ABC kit (Vector Laboratories, PK-6100). Immunoreactivity was visualized using DAB (Vector Laboratories, SK-4100). Sections were dehydrated, mounted, and imaged for quantitative analysis. For immunofluorescence staining, sections were subjected to antigen retrieval and then blocked for 1 h at room temperature in 1× PBS containing 0.3% Triton X-100 and 5% bovine serum albumin (Biosharp, BS114). Sections were incubated overnight at 4℃ with primary antibodies. After washing, sections were incubated for 2 h at room temperature with species-appropriate secondary antibodies, including goat anti-chicken Alexa Fluor 488 (Abcam, ab150169), donkey anti-goat Alexa Fluor 488 (Abcam, ab150129), donkey anti-rabbit Alexa Fluor 568 (Abcam, ab175470), donkey anti-mouse Alexa Fluor 488 (Abcam, ab150105), goat anti-rabbit Alexa Fluor 488 (Invitrogen, A-11008), and goat anti-rabbit Alexa Fluor 568 (Invitrogen, A-11011). Nuclear staining was performed using DAPI (Beyotime, C1002). Sections were mounted using antifade mounting medium (SouthernBiotech, 0100-01). For details of the primary antibodies, see Supplementary Table 1. Brightfield and fluorescence images were acquired using a Slide Scanner (VS200, Olympus), an automated digital slide scanning system (Axioscan 7, Carl Zeiss), and a confocal laser scanning microscope (TI2-E+A1, Nikon). Image acquisition was controlled using OLYMPUS OlyVIA (version 4.1), NIS-Elements Viewer (version 5.21), and ZEN lite (version 3.13). Image processing and quantitative analyses were performed using Fiji software (version 1.54p). All imaging parameters were kept constant across experimental groups for quantitative comparisons. AQP4 polarity calculation To quantify the polarization of AQP4 and evaluate its pathological mislocalization, we performed a radial fluorescence intensity analysis on AQP4 and CD31 co-stained images using the Line-plot tool in ImageJ. For each analyzed blood vessel, an 80-μm linear segment was drawn perpendicular to the vessel longitudinal axis, centered on the CD31 positive vascular lumen to ensure anatomical consistency. The Perivascular Intensity (Iperi) was defined as the mean fluorescence intensity within a 10-μm radius from the vessel center, representing AQP4 localization at the astrocytic endfeet. The Parenchymal Baseline (Ipara) was calculated as the mean intensity in the region 30 μm away from the vessel, reflecting the non-polarized AQP4 pool in the astrocytic soma and processes. The AQP4 Polarity Index was then calculated as the ratio of perivascular enrichment to the parenchymal background (Iperi/ Ipara), a metric specifically designed to capture the shift of AQP4 from the vascular interface to the brain parenchyma. To minimize inter-slice variability and ensure comparability across experimental groups, all polarity indices were normalized to the mean value of the control group. Primary astrocyte cultures Primary astrocytes were prepared from neonatal C57 mice at postnatal day 1–2. Under a stereomicroscope, the cerebral cortices were isolated and all meninges were carefully removed. Tissues were collected and processed at 4 °C, minced into small pieces using fine dissection scissors, and transferred into conical tubes containing Hibernate-E medium (A1247601, Gibco). After the tissue fragments had settled to the bottom of the tube, the supernatant was carefully removed, leaving only enough medium to immerse the tissue. Samples were then enzymatically digested in 0.25% trypsin (15050065, Gibco) containing DNase I (10 mg/mL, 18047019, Invitrogen) at 30 °C for 30 min. Enzymatic digestion was terminated by adding culture medium consisting of high-glucose Dulbecco’s modified Eagle medium (DMEM, 11965092, Gibco) supplemented with 10% fetal bovine serum (A5256701, Gibco), 10 mL/L penicillin–streptomycin, pH 7.4. The cell suspension was centrifuged at 150 × g for 5 min, and the pellet was resuspended in culture medium. Cells were seeded into poly-D-lysine-coated (0.1 mg/mL, A3890401, Gibco) T75 flasks at a density of 1 × 10 5 cells/cm 2 and maintained at 37 °C in a humidified 5% CO2 incubator until reaching confluence. Culture medium was replaced every 2–3 days. To enrich for astrocytes, microglia were removed from the mixed glial monolayer by shaking the flasks at 200 rpm for 2 h at 37 °C, followed by medium replacement to remove cells remaining in suspension. For stimulation experiments, astrocyte cultures were treated with PFFs and irisin as indicated, and cells were collected after 15 days of co-incubation for subsequent analyses. Western blotting analysis Brain tissues were homogenized in RIPA lysis buffer (Beyotime, P0013B) supplemented with protease and phosphatase inhibitor mixture (Beyotime, P1048). Lysates were centrifuged to remove debris, and protein concentrations were determined using a BCA protein assay kit (Epizyme Biotech, ZJ101). Equal amounts of protein were separated by SDS–PAGE and transferred onto PVDF membranes. Membranes were blocked for 1 h at room temperature in 5% BSA dissolved in TBST and incubated overnight at 4 °C with primary antibodies. After washing with 1×TBST, membranes were incubated with horseradish peroxidase–conjugated secondary antibodies for 1 h at room temperature. Protein bands were visualized using an enhanced chemiluminescence (Biosharp, BL520) substrate and imaged with a chemiluminescence detection system. Band intensities were quantified using ImageJ software and normalized to the corresponding loading controls for statistical analysis. For details of the primary antibodies, see Supplementary Table 1. ELISA For Irisin detection, blood samples were allowed to clot on ice for 30 min and then centrifuged at 12,000 rpm for 15 min to obtain serum. The supernatant was carefully collected and stored at –80 °C until analysis. Serum irisin levels were quantified using a Mouse Irisin ELISA Kit (Elabscience Biotechnology, E-EL-M2743) according to the manufacturer’s instructions. Absorbance was measured using a microplate reader, and concentrations were calculated from a standard curve generated using serial dilutions of the provided standards. Statistical analysis Animals from different cages were randomly allocated to experimental groups to minimize potential cage effects. Investigators were blinded to group allocation during data collection and analysis. Statistical analyses were performed using GraphPad Prism. For comparisons involving more than two groups, one - way or two - way analysis of variance (ANOVA) was applied as appropriate. When multiple comparisons were required, Dunnett’s post hoc test was used to compare each experimental group with the disease model group, thereby controlling for type I error associated with multiple testing. For comparisons between two groups only, a two - tailed unpaired Student’s t test was used. A P value < 0.05 was considered statistically significant. Data are presented as mean ± SEM. Declarations Data availability The data used in this study are available from the corresponding author upon request. Author Contributions L.J. and R.L. designed the study. R.L., J.Z., X.R., B.L., X.X. and Y.L. performed the experiments and collected data. R.L., J.Z., and X.R. analyzed the data. R.L., J.Z., X.R., B.L. and Y.Z. prepared the figures and drafted the original manuscript. Y.S. and L.J. revised the manuscript. All authors read and approved the final manuscript. Acknowledgements The authors would like to thank prof. Cong Liu (Institute of Organic Chemistry, Chinese Academy of Sciences, Shanghai, China) for the assistance with protein purification and PFFs labeling. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9467573","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":631776631,"identity":"d7192a06-1e90-4ba4-8807-8a09e14ddd90","order_by":0,"name":"Lingjing Jin","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA/ElEQVRIiWNgGAWjYBACPmYgIWHAwMDYzMD4ACQCZDAw8ODRwoakhdngAFFakNkSB2BMvFrYeQ+/sCi4Y9fcznus+kMFiAF04ds2BnlznA7jS7OQMHiW3NjMl3bjwBkQg4HZcG4bg+HOBlxaeMwMJAwOJzM285jdONgGYjCwSfO2MSQYHCBCSwFUC/tvAlqMHwC12IG0MAC12IFsYSZkCzCQDycAtRhLnDkDYjA2S845J2G4AYcWfv4zxp8l/hy2N+w/Y/ihogLEOHzww5syG3lctoAskpZgYEjc2ADhARmMIKYETvVAwPzxAwODvTyUB2eMglEwCkbBKIABAAMgVQjW1xOLAAAAAElFTkSuQmCC","orcid":"","institution":"School of Medicine, Tongji University","correspondingAuthor":true,"prefix":"","firstName":"Lingjing","middleName":"","lastName":"Jin","suffix":""},{"id":631776632,"identity":"d5b86e47-933f-48ac-a634-b51018f9c0a1","order_by":1,"name":"Ruoyu Li","email":"","orcid":"","institution":"Yangzhi Rehabilitation Hospital, School of Medicine, Tongji University","correspondingAuthor":false,"prefix":"","firstName":"Ruoyu","middleName":"","lastName":"Li","suffix":""},{"id":631776633,"identity":"495baac4-54b7-4b07-96c8-904a08afc142","order_by":2,"name":"Jinbao Zhang","email":"","orcid":"https://orcid.org/0000-0002-5688-5261","institution":"Yangzhi Rehabilitation Hospital (Shanghai Sunshine Rehabilitation Center), School of Medicine, Tongji University","correspondingAuthor":false,"prefix":"","firstName":"Jinbao","middleName":"","lastName":"Zhang","suffix":""},{"id":631776634,"identity":"d573f84c-f64e-49b6-9360-f8b9320902d2","order_by":3,"name":"Xining Ren","email":"","orcid":"","institution":"Yangzhi Rehabilitation Hospital, School of Medicine, Tongji University","correspondingAuthor":false,"prefix":"","firstName":"Xining","middleName":"","lastName":"Ren","suffix":""},{"id":631776635,"identity":"b06c3ac3-1a5d-412c-8dd1-c54a2a2cc21b","order_by":4,"name":"Bingyu Li","email":"","orcid":"","institution":"Yangzhi Rehabilitation Hospital (Shanghai Sunshine Rehabilitation Center), School of Medicine, Tongji University","correspondingAuthor":false,"prefix":"","firstName":"Bingyu","middleName":"","lastName":"Li","suffix":""},{"id":631776636,"identity":"b8cbcbb5-f2db-4220-8626-c62e65b73a10","order_by":5,"name":"Xuerui Xiang","email":"","orcid":"","institution":"Yangzhi Rehabilitation Hospital, School of Medicine, Tongji University","correspondingAuthor":false,"prefix":"","firstName":"Xuerui","middleName":"","lastName":"Xiang","suffix":""},{"id":631776637,"identity":"0de41228-0725-4979-94d3-7430b214705a","order_by":6,"name":"Yunxi Liu","email":"","orcid":"","institution":"Yangzhi Rehabilitation Hospital (Shanghai Sunshine Rehabilitation Center), School of Medicine, Tongji University","correspondingAuthor":false,"prefix":"","firstName":"Yunxi","middleName":"","lastName":"Liu","suffix":""},{"id":631776638,"identity":"e982b415-b29b-448b-ac8a-3fd0e7704243","order_by":7,"name":"Yunjiao Zhou","email":"","orcid":"","institution":"Tongji University","correspondingAuthor":false,"prefix":"","firstName":"Yunjiao","middleName":"","lastName":"Zhou","suffix":""},{"id":631776639,"identity":"966d5f50-10df-415e-84e9-65e4b3e4cf79","order_by":8,"name":"Yunping Song","email":"","orcid":"","institution":"Louisiana State University Health Sciences Center New Orleans","correspondingAuthor":false,"prefix":"","firstName":"Yunping","middleName":"","lastName":"Song","suffix":""}],"badges":[],"createdAt":"2026-04-20 06:20:21","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9467573/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9467573/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":108182718,"identity":"a37fd4d8-f12e-4091-ae91-8aa959bdf55d","added_by":"auto","created_at":"2026-04-30 08:59:31","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":4712034,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExercise alleviates dopaminergic degeneration and αSyn pathology in PD mice.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea,\u003c/strong\u003e Experimental schematic of treatments of PD mice. Two-month-old WT mice were injected with PBS or αSyn PFFs (5 μg) into the striatum. One month after PFFs injection, mice underwent treadmill exercise training (1 h/day, 5 days/week) for 3 months. Behavioral tests were performed during monthly the intervention period, and mice were sacrificed at month 4 for pathological and glymphatic analyses. \u003cstrong\u003eb–d, \u003c/strong\u003eBehavioral assessments. Motor performance was evaluated by open field test (b), pole test (c) and rotarod test (d). Data are presented as mean ± SEM, Two-way ANOVA followed by Dunnett’s post hoc test (n=12–14). \u003csup\u003e*\u003c/sup\u003e\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.05, \u003csup\u003e***\u003c/sup\u003e\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.001 for PFFs vs CTR; \u003csup\u003e##\u003c/sup\u003e\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.01, \u003csup\u003e###\u003c/sup\u003e\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.001 for PFFs+EX vs PFFs. \u003cstrong\u003ee, \u003c/strong\u003eRepresentative immunohistochemical staining of tyrosine hydroxylase (TH) in the striatum (CPu) (Scale bars, 1 mm) and substantia nigra pars compacta (SNc) (Scale bars, 200 μm). \u003cstrong\u003ef, \u003c/strong\u003eRepresentative immunofluorescence images showing TH (green), phosphorylated αSyn (p-αSyn, red), and DAPI (blue) in the CPu, SNc, and M1 cortex. Scale bars, 50 μm. \u003cstrong\u003eg,\u003c/strong\u003e Quantification of TH-positive fiber density in the lesioned striatum. \u003cstrong\u003eh,\u003c/strong\u003e Quantification of TH-positive dopaminergic neurons in the lesioned SNc. \u003cstrong\u003ei–k, \u003c/strong\u003eQuantification of p-αSyn immunoreactivity in the lesioned CPu (i), SNc (j), and M1 cortex (k). Data are presented as mean ± SEM. One-way ANOVA followed by Dunnett’s post hoc test for g–k (n=8 for g–h, n=12 for i–k). \u003csup\u003e**\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, \u003csup\u003e***\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-9467573/v1/b58939a79396160a9a6dfacb.png"},{"id":108164416,"identity":"13b5474c-a704-49e7-b22a-fb0bbd3f77bd","added_by":"auto","created_at":"2026-04-30 05:21:34","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":3309082,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExercise restores impaired glymphatic transport and clearance in PD mice.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea,\u003c/strong\u003e Representative sagittal MRI images showing the spatial distribution of the contrast signal at different time points following cisterna magna injection. Color maps represent changes in signal intensity relative to baseline. Scale bars, 5 mm. \u003cstrong\u003eb–e,\u003c/strong\u003e Quantification of MRI signal intensity changes over time in different brain regions, including the CPu (b), SN (c), cortex (d), and hippocampus (e) (n=5). \u003cstrong\u003ef,\u003c/strong\u003e Representative coronal brain sections showing the spatial distribution of TR-d10 (red) and PFFs-488 (green) across different bregma levels 2 h post injection. Scale bars, 2 mm. \u003cstrong\u003eg–h,\u003c/strong\u003e Quantification of TR-d10 fluorescence intensity (n=4). \u003cstrong\u003ei–j,\u003c/strong\u003e Quantification of PFFs-488 fluorescence intensity (n=4). \u003cstrong\u003ek, \u003c/strong\u003eRepresentative images of TR-d10 tracer (red), PFFs-488 (Green) and DAPI (blue) staining in the deep cervical lymph nodes (dCLNs). Scale bar, 250 μm. \u003cstrong\u003el, \u003c/strong\u003eQuantification of fluorescence intensity of the TR-d10 tracer in dCLNs (n=5). \u003cstrong\u003em,\u003c/strong\u003e Quantification of fluorescence intensity of the PFFs-488 in dCLNs (n=5). Data are presented as mean ± SEM. Statistics were analyzed using one-way ANOVA followed by Dunnett’s post hoc test. \u003csup\u003e*\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, \u003csup\u003e**\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, \u003csup\u003e***\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-9467573/v1/b855f4ff67f360ee5e57c0be.png"},{"id":108164418,"identity":"b8f09f8f-4826-4368-8674-27d9576c92e1","added_by":"auto","created_at":"2026-04-30 05:21:34","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":3057868,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePharmacological inhibition of AQP4 abolishes the protective effects of exercise in PD mice.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea,\u003c/strong\u003e Representative immunofluorescence images of astrocytic AQP4 (Red) and vascular marker CD31 (Green) in the CPu. Scale bars, 100 μm. \u003cstrong\u003eb,\u003c/strong\u003e Radial fluorescence intensity profiles of AQP4 signal relative to CD31-positive vasculature. \u003cstrong\u003ec,\u003c/strong\u003e Quantification of AQP4 polarity normalized to control levels (n=12). \u003cstrong\u003ed,\u003c/strong\u003e Correlation analysis between AQP4 polarity and p-αSyn density in the striatum.\u003cstrong\u003e e,\u003c/strong\u003e Experimental schematic of treatments of PD mice. One month after PFFs injection, mice underwent treadmill exercise training for 3 months. The AQP4 inhibitor TGN-020 (100 mg/kg) was administered intraperitoneally prior to each exercise session. Behavioral tests were performed monthly during the intervention period, and mice were sacrificed at month 4 for subsequent analyses. \u003cstrong\u003ef,\u003c/strong\u003e Representative immunofluorescence images showing TH (green), p-αSyn (red), and DAPI (blue) in the CPu and SNc across groups. Scale bars, 50 μm. \u003cstrong\u003eg–h,\u003c/strong\u003e Quantification of relative p-αSyn intensity in the lesioned CPu (g) and SNc (h) (n=5). \u003cstrong\u003ei–k,\u003c/strong\u003e Behavioral assessments. Open field test (i), pole test (j) and rotarod test (k) were performed to evaluate motor coordination and locomotor activity across experimental groups (n=9–11 mice for CTR, PFFs and PFFs+Ex; n=5 mice for PFFs+Ex+TGN-020). Data are presented as mean ± SEM. Statistics were analyzed using one-way ANOVA followed by Dunnett’s post hoc test. \u003csup\u003e*\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, \u003csup\u003e**\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, \u003csup\u003e***\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-9467573/v1/eaada5c6d43993a5db76f361.png"},{"id":108182861,"identity":"bc270b43-dc0b-40de-aee0-95b3f1fea464","added_by":"auto","created_at":"2026-04-30 08:59:38","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2197185,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIrisin modulates AQP4 polarity and enhances glymphatic tracer transport and clearance.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea, \u003c/strong\u003eRepresentative immunofluorescence images of astrocytic AQP4 (Red) and vascular marker CD31 (Green) in the CPu. Scale bars, 100 μm. \u003cstrong\u003eb,\u003c/strong\u003e Quantification of AQP4 polarity normalized to control levels (n=6). Statistics were analyzed using one-way ANOVA followed by Dunnett’s post hoc test. \u003csup\u003e*\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, \u003csup\u003e***\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001. \u003cstrong\u003ec,\u003c/strong\u003e Representative coronal sections showing the spatial distribution of TR-d10 (red) and PFFs-488 (green) across different bregma levels in CTR and CTR+Irisin mice. Scale bars, 2 mm. \u003cstrong\u003ed–e, \u003c/strong\u003eQuantification of TR-d10 fluorescence intensity (n=5).\u003cstrong\u003e f–g,\u003c/strong\u003e Quantification of PFFs-488 fluorescence intensity (n=5). \u003cstrong\u003eh,\u003c/strong\u003e Representative images of TR-d10 tracer (red), PFFs-488 (Green) and DAPI (blue) staining in the deep cervical lymph nodes (dCLNs). Scale bar, 250 μm. \u003cstrong\u003ei,\u003c/strong\u003e Quantification of fluorescence intensity of the TR-d10 tracer in dCLNs (n=5). \u003cstrong\u003ej,\u003c/strong\u003e Quantification of fluorescence intensity of the PFFs-488 in dCLNs (n=5). Data are presented as mean ± SEM. Statistics were analyzed using Student's t-test for e,g,i,j. \u003csup\u003e*\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, \u003csup\u003e**\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"Fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-9467573/v1/2d1cffaf0e8578de7a2e536d.png"},{"id":108183171,"identity":"44ed8206-f658-4f21-812a-f6ebd46841e9","added_by":"auto","created_at":"2026-04-30 08:59:50","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1726230,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIrisin deficiency abolishes exercise-induced glymphatic enhancement.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea, \u003c/strong\u003eRepresentative coronal brain sections showing the spatial distribution of TR-d10 (red) and PFFs-488 (green) across different bregma levels in CTR, CTR+Ex, FNDC5-KO, and KO+Ex mice. \u003cstrong\u003eb–c,\u003c/strong\u003e Quantification of TR-d10 fluorescence intensity. \u003cstrong\u003ed–e,\u003c/strong\u003e Quantification of PFFs-488 fluorescence intensity. Data are presented as mean ± SEM. Statistics were analyzed using one-way ANOVA followed by Dunnett’s post hoc test (n=5). \u003csup\u003e*\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, \u003csup\u003e**\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"Fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-9467573/v1/5ef9d3dbbb8ed90d1ca1c21d.png"},{"id":108183028,"identity":"5ffcf164-9839-4b83-b809-675825535e26","added_by":"auto","created_at":"2026-04-30 08:59:45","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":2216567,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExercise restores astrocyte remodeling in PD mice.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea,\u003c/strong\u003e Representative immunofluorescence images showing C3d (green), GFAP (red), and DAPI (blue) in the SNc across experimental groups. Scale bars, 20 μm. \u003cstrong\u003eb,\u003c/strong\u003e Representative immunofluorescence images showing S100A10 (green), GFAP (red), and DAPI (blue) in the SNc across experimental groups. Scale bars, 20 μm. \u003cstrong\u003ec,\u003c/strong\u003e Representative immunofluorescence images and reconstructions of GFAP staining in the SNc illustrating astrocytic morphological changes across groups. Scale bars, 20 μm. \u003cstrong\u003ed–e,\u003c/strong\u003e Quantification of the A1 astrocyte marker C3d (d) or A2 astrocyte marker S100A10 (e) integrated fluorescence intensity in the SNc (n=12). \u003cstrong\u003ef–g,\u003c/strong\u003e Quantification of astrocyte morphological complexity using Sholl analysis (n=12). Data are presented as mean ± SEM. Statistics were analyzed using one-way ANOVA followed by Dunnett’s post hoc test. \u003csup\u003e*\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, \u003csup\u003e***\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Fig6.png","url":"https://assets-eu.researchsquare.com/files/rs-9467573/v1/0245d64d75efe0f5179224b6.png"},{"id":108164422,"identity":"e3ebd86d-409b-4e66-9ba1-c478022029e4","added_by":"auto","created_at":"2026-04-30 05:21:34","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":224101,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIrisin reverses astrocytic phenotype and upregulates REV-ERBα.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea,\u003c/strong\u003e Representative Western blotting analysis of astrocyte phenotype markers following PFFs and irisin treatment, including the A1 astrocyte marker C3d and the A2 astrocyte marker S100A10. \u003cstrong\u003eb–c,\u003c/strong\u003e Quantification of C3d (b) and S100A10 (c) protein levels, normalized to GAPDH.\u003cstrong\u003e d,\u003c/strong\u003e Representative Western blotting analysis of p-STAT3, STAT3, and REV-ERBα in primary astrocytes treated with PFFs and irisin. \u003cstrong\u003ee,\u003c/strong\u003e Quantification of p-STAT3/STAT3 ratio. \u003cstrong\u003ef,\u003c/strong\u003e Quantification of REV-ERBα protein expression, normalized to GAPDH. Data are presented as mean ± SEM. Statistics were analyzed using one-way ANOVA followed by Dunnett’s post hoc test (n=4). \u003csup\u003e*\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, \u003csup\u003e**\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, \u003csup\u003e***\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Fig7.png","url":"https://assets-eu.researchsquare.com/files/rs-9467573/v1/7981a47bfcca2fc1df295b98.png"},{"id":108182927,"identity":"d8e08c50-0b1a-4fc1-b470-612459a3274b","added_by":"auto","created_at":"2026-04-30 08:59:41","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":846539,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAstrocytic REV-ERBα regulates astrocyte morphology and AQP4 polarization.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ed,\u003c/strong\u003e Representative immunofluorescence images of astrocytic AQP4 (Red) and vascular marker CD31 (Green) in the CPu. Scale bars, 100 μm. \u003cstrong\u003ee,\u003c/strong\u003e Quantification of AQP4 polarity normalized to control levels. \u003cstrong\u003ef,\u003c/strong\u003e Representative immunofluorescence images and reconstructions of GFAP staining in the CPu. Scale bars, 20 μm. \u003cstrong\u003eg–h,\u003c/strong\u003e Quantification of astrocyte morphological complexity using Sholl analysis. Data are presented as mean ± SEM. Student's t-test for b,e (n=6). \u0026nbsp;\u003csup\u003e***\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Fig8.png","url":"https://assets-eu.researchsquare.com/files/rs-9467573/v1/09fff9e60f5d2fc74701cf1d.png"},{"id":108164424,"identity":"9a0a15b1-052e-40fa-bc65-235d55fb69ba","added_by":"auto","created_at":"2026-04-30 05:21:34","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":1087776,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAstrocytic REV-ERBα is required for exercise- and irisin-induced glymphatic enhancement.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea,\u003c/strong\u003e Representative coronal brain sections showing the distribution of OVA-647 (red) and PFFs-488 (green) across different bregma levels in CTR, \u003cem\u003eNr1d1\u003c/em\u003e-shRNA, \u003cem\u003eNr1d1\u003c/em\u003e-shRNA+Ex, and \u003cem\u003eNr1d1\u003c/em\u003e-shRNA+Irisin groups. \u003cstrong\u003eb–c,\u003c/strong\u003e Quantification of OVA-647 fluorescence intensity. \u003cstrong\u003ed–e,\u003c/strong\u003e Quantification of PFFs-488 fluorescence intensity. Data are presented as mean ± SEM. Statistics were analyzed using one-way ANOVA followed by Dunnett’s post hoc test (n=3–4). \u003csup\u003e*\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e","description":"","filename":"Fig9.png","url":"https://assets-eu.researchsquare.com/files/rs-9467573/v1/852fc69acbc214c534429a96.png"},{"id":108164425,"identity":"9cdf3a7a-8172-4848-aec9-72d57bfb90e9","added_by":"auto","created_at":"2026-04-30 05:21:34","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":5754019,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExercise restores astrocytic AQP4 polarity and glymphatic αSyn clearance via irisin/REV-ERBα axis in PD mice.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePhysical exercise stimulates skeletal muscle to secrete irisin into the circulation. Irisin acts on astrocytes in the brain, where it suppresses STAT3 activation and upregulates the circadian transcriptional repressor REV-ERBα. This signaling cascade promotes an anti-inflammatory (A2-like) astrocytic phenotype and restores the perivascular polarization of AQP4 at astrocytic endfeet. Enhanced AQP4 polarity facilitates glymphatic transport and accelerates the clearance of αSyn aggregates, thereby mitigating nigrostriatal neurodegeneration.\u003c/p\u003e","description":"","filename":"Fig10.png","url":"https://assets-eu.researchsquare.com/files/rs-9467573/v1/c0b10008de5cfa679d19e38e.png"},{"id":108164415,"identity":"9614d19f-845d-4249-a91e-ce22cda31645","added_by":"auto","created_at":"2026-04-30 05:21:34","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":4996759,"visible":true,"origin":"","legend":"Supplementary information","description":"","filename":"Supplementaryinformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-9467573/v1/e2e9c629f5a2099371f1c037.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Irisin mediates exercise-induced glymphatic α-synuclein clearance by upregulating astrocytic REV-ERBα in Parkinson’s disease","fulltext":[{"header":"Introduction","content":"\u003cp\u003eParkinson\u0026rsquo;s disease (PD) is a progressive neurodegenerative disorder characterized by the degeneration of dopaminergic neurons in the substantia nigra and the accumulation of misfolded \u0026alpha;-synuclein (\u0026alpha;Syn)\u003csup\u003e1\u003c/sup\u003e. Current therapies offer only symptomatic relief, underscoring the urgent need for interventions that target underlying pathogenic mechanisms\u003csup\u003e2\u003c/sup\u003e. In this context, physical exercise has emerged as a powerful non-pharmacological intervention, with epidemiological and preclinical studies consistently demonstrating its capacity to ameliorate motor impairments, mitigate dopaminergic degeneration, and lower \u0026alpha;Syn burden\u003csup\u003e3-7\u003c/sup\u003e. However, the molecular pathways linking exercise to central pathology remain largely undefined, representing a critical barrier to the development of mechanism-based disease-modifying strategies\u003csup\u003e7,8\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eEmerging evidence points to the glymphatic system as a critical player in brain proteostasis and a potential mediator of exercise\u0026rsquo;s benefits\u003csup\u003e9-11\u003c/sup\u003e. The glymphatic system is a glial-dependent perivascular network that facilitates the exchange of cerebrospinal fluid and interstitial fluid, promoting the clearance of metabolic waste and aggregation-prone proteins, including \u0026alpha;Syn\u003csup\u003e12-14\u003c/sup\u003e. Glymphatic dysfunction, often characterized by the loss of perivascular aquaporin-4 (AQP4) polarization on astrocytic endfeet, has been implicated in PD pathogenesis, contributing to \u0026alpha;Syn aggregation and neurodegeneration\u003csup\u003e15-19\u003c/sup\u003e. Notably, studies have linked exercise-induced improvements in meningeal lymphatic vessel function to enhanced perivascular clearance and have highlighted its capacity to reverse sleep-dependent glymphatic dysfunction\u003csup\u003e11,20,21\u003c/sup\u003e. Other studies have shown that exercise ameliorates glymphatic impairment and reduces amyloid-\u0026beta; pathology in Alzheimer\u0026apos;s disease models\u003csup\u003e10,22,23\u003c/sup\u003e. Despite mounting evidences, the specific exercise-induced factors that link physical activity to central glymphatic clearance in PD remain elusive.\u003c/p\u003e\n\u003cp\u003eThe emerging concept of the \u0026ldquo;muscle\u0026ndash;brain axis\u0026rdquo; has established skeletal muscle as a dynamic endocrine organ that communicates with the central nervous system through exercise-induced myokines\u003csup\u003e24,25\u003c/sup\u003e. Irisin, a cleavage product of the membrane protein FNDC5, is released from muscle into circulation during physical activity and has emerged as a mediator of exercise-induced neuroprotection. Importantly, irisin can cross the blood\u0026ndash;brain barrier\u003csup\u003e26,27\u003c/sup\u003e, enabling it to directly influence central nervous system function. In our previous study, we observed that circulating irisin levels were significantly elevated in PD patients after 12 weeks of regular exercise, and the extent of this rise correlated strongly with improvements in balance performance\u003csup\u003e28\u003c/sup\u003e. However, whether irisin recapitulates the beneficial effects of exercise on \u0026alpha;Syn pathology, as well as the underlying mechanisms, remains unexplored.\u003c/p\u003e\n\u003cp\u003eIn this study, we demonstrated that chronic treadmill exercise restored impaired glymphatic transport and reduced \u0026alpha;Syn accumulation in a preformed fibrils (PFFs)-based PD model. Furthermore, we identified exercise-induced irisin as a key systemic mediator that acted on astrocytes to suppress STAT3 activation and increase the expression of circadian nuclear receptor REV-ERB\u0026alpha;, thereby curbing inflammatory activation, restoring AQP4 polarization, and enhancing glymphatic clearance of \u0026alpha;Syn. Importantly, astrocyte-specific knockdown of \u003cem\u003eNr1d1\u003c/em\u003e (encoding REV-ERB\u0026alpha;) abolished the glymphatic benefits of both exercise and irisin. Collectively, our findings established a muscle\u0026ndash;brain signaling axis in which exercise-derived irisin promotes glymphatic function through upregulating REV-ERB\u0026alpha;, providing a mechanistic framework linking physical activity to brain proteostasis.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eExercise improves motor function and reduces nigrostriatal pathology in PD mice\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo investigate the effects of physical exercise on PD pathology, we established PD mouse model by unilateral striatal injection of PFFs, followed by a 3-month treadmill exercise intervention (Fig. 1a). PFFs-injected mice exhibited progressive motor impairment, as indicated by decreased locomotor activity in the open field test, increased descent time in the pole test and reduced latency to fall in the rotarod test. Exercise markedly improved motor performance across all behavioral assays, with significant recovery observed over time compared to sedentary PFFs mice (Fig. 1b\u0026ndash;d). These findings indicate that sustained exercise alleviates motor deficits in this model. We next examined dopaminergic neurodegeneration and \u0026alpha;Syn pathology in the nigrostriatal pathway. PFFs injection induced marked dopaminergic neurodegeneration, as evidenced by a substantial loss of tyrosine hydroxylase (TH)-positive neurons in the substantia nigra and reduced TH-positive fiber density in the striatum. This was accompanied by a pronounced accumulation of phosphorylated \u0026alpha;Syn (pSer129) in the striatum, substantia nigra, and cortex. Notably, exercise significantly preserved TH-positive neurons and fiber density while concurrently reducing p-\u0026alpha;Syn burden across these regions (Fig. 1e\u0026ndash;k), indicating a coordinated attenuation of neurodegeneration and pathological protein accumulation. Together, these results demonstrate that long-term exercise mitigates motor deficits, preserves nigrostriatal integrity, and reduces p-\u0026alpha;Syn burden in the PFFs model.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExercise enhances glymphatic transport and accelerates parenchymal clearance in PD mice\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eImpaired clearance of pathological proteins is closely linked to PD progression\u003csup\u003e16,18\u003c/sup\u003e. We next asked whether exercise improves glymphatic function using dynamic contrast-enhanced MRI (DCE-MRI) in the PFFs model. Following intracisternal administration of Gd-DTPA as the contrast agent, control mice exhibited robust tracer influx and widespread distribution across brain regions, whereas PFFs mice showed markedly reduced signal intensity and impaired tracer propagation (Fig. 2a). Exercise significantly restored tracer influx and distribution in multiple regions, including the striatum, substantia nigra, cortex and hippocampus (Fig. 2b\u0026ndash;e), indicating improved glymphatic transport. Time\u0026ndash;signal intensity analysis further revealed that PFFs mice displayed delayed signal responses, consistent with impaired cerebrospinal\u0026ndash;interstitial fluid exchange. In contrast, exercise enhanced both the magnitude and dynamics of tracer transport, suggesting a recovery of glymphatic flow.\u003c/p\u003e\n\u003cp\u003eTo more directly assess the effect of exercise on glymphatic clearance in the brain parenchyma, we examined parenchymal solute transport and clearance after intrastriatal co-injection of TR-d10 tracer and AF488-labeled \u0026alpha;Syn PFFs (PFFs-488). In PFFs mice, both tracers were retained more strongly within the brain parenchyma, indicating impaired clearance (Fig. 2f\u0026ndash;j). Exercise reduced the retention of both TR-d10 and PFFs-488 across coronal sections and enhanced their dispersion away from the injection site (Fig. 2f\u0026ndash;j). Importantly, similar changes were observed for both tracers despite their different properties, indicating that exercise broadly improves parenchymal solute transport and clearance rather than affecting a single substrate. To further confirm that tracer clearance occurred via the glymphatic system, we also analyzed the amount of TR-d10 and PFFs-488 in the deep cervical lymph nodes (dCLNs), which are reported to drain lymph fluid from the brain. We found a significant reduction of both TR-d10 and PFFs-488 in the dCLNs of PFFs mice compared with the PBS group (Fig. 2k\u0026ndash;m). However, this reduction was markedly alleviated by exercise (Fig. 2k\u0026ndash;m). Together, these results demonstrate that exercise restores impaired glymphatic transport and promotes efficient brain clearance in PD mice.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExercise restores AQP4-dependent glymphatic function in PD mice\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe next examined whether AQP4 polarity underlies the exercise-induced improvement in glymphatic transport and parenchymal clearance\u003csup\u003e29\u003c/sup\u003e. Under control conditions, AQP4 was predominantly confined to perivascular endfeet, whereas administration of PFFs resulted in a marked disruption of this polarized distribution (Fig. 3a\u0026ndash;c). Exercise restored AQP4 localization toward blood vessels, as reflected by a significant recovery of AQP4 polarity (Fig. 3a\u0026ndash;c). Importantly, AQP4 polarity was negatively correlated with p-\u0026alpha;Syn accumulation, suggesting a link between astrocytic structural integrity and pathological protein burden (Fig. 3d). To further determine whether AQP4-dependent mechanisms are required for the benefits of exercise, we pharmacologically inhibited AQP4 using TGN-020 during the intervention period (Fig. 3e). We found that the reduction in p-\u0026alpha;Syn burden observed with exercise was lost after TGN-020 treatment, with p-\u0026alpha;Syn levels remaining elevated and comparable to those in sedentary PFFs mice (Fig. 3f\u0026ndash;h). Meanwhile, AQP4 inhibition largely abolished the exercise-induced improvements in motor performance, including performance in the open field test, pole test and rotarod test (Fig. 3i\u0026ndash;k). These results revealed that enhanced AQP4 polarity is a key mechanism by which exercise promotes glymphatic clearance of \u0026alpha;Syn aggregates.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIrisin is the circulating mediator of exercise-induced glymphatic rescue\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe next asked whether exercise-responsive molecules could link peripheral activity to glymphatic regulation.\u0026nbsp;A compelling candidate that may link exercise to neuroprotection is irisin, a cleavage product of the membrane protein FNDC5 that is released from skeletal muscle in response to exercise and can cross the blood\u0026ndash;brain barrier\u003csup\u003e26,27,30\u003c/sup\u003e. We previously found that exercise elevated irisin levels in a PD mouse model, and that systemic administration of irisin sufficed to recapitulate the motor-improving effects of exercise\u003csup\u003e28\u003c/sup\u003e. We therefore sought to determine whether irisin acts by directly modulating AQP4 polarization and recapitulating the effect of exercise on glymphatic function in PFFs-injected mice. Immunofluorescence staining revealed that PFFs injection markedly disrupted the perivascular distribution of AQP4, whereas irisin treatment significantly restored AQP4 polarization around CD31-positive vessels (Fig. 4a\u0026ndash;b). We then performed intrastriatal co-injection of TR-d10 and PFFs-488 in mice to determine whether irisin directly regulates parenchymal solute transport and clearance. Representative images across multiple rostrocaudal levels showed that irisin-treated mice exhibited a broader spatial distribution of both tracers compared to controls, indicating enhanced interstitial transport within the brain parenchyma (Fig. 4c). Quantitative analysis revealed a significant reduction in total TR-d10 signal in the injected hemisphere (Fig. 4d\u0026ndash;e), consistent with accelerated clearance. Similarly, the parenchymal retention of PFFs-488 was markedly decreased following irisin administration (Fig. 4f\u0026ndash;g), reflecting enhanced removal of protein tracers. Moreover, fluorescence intensity of both tracers in the deep cervical lymph nodes was significantly increased in irisin-treated mice (Fig. 4h\u0026ndash;j), supporting enhanced brain-to-periphery efflux.\u003c/p\u003e\n\u003cp\u003eTo further assess whether endogenous irisin is necessary for exercise-induced enhancement of parenchymal clearance, we applied the same tracer clearance paradigm in wild-type and FNDC5-KO mice. In wild-type mice, exercise increased the spatial distribution of both TR-d10 and PFFs-488 and reduced their retention within the parenchyma, indicating enhanced clearance (Fig. 5a\u0026ndash;e). In contrast, FNDC5-KO mice failed to exhibit these exercise-induced changes, with tracer distribution remaining restricted and no significant reduction in TR-d10 or PFFs-488 signal observed following exercise (Fig. 5a\u0026ndash;e). Collectively, these findings demonstrate that irisin is required for exercise-induced enhancement of parenchymal solute transport and clearance.\u003c/p\u003e\n\u003cp\u003eTo determine whether elevating irisin levels is sufficient to recapitulate the protective effects of exercise at the organismal level, we overexpressed FNDC5 via systemic AAV delivery according to previous study\u003csup\u003e31\u003c/sup\u003e (Supplementary Fig. 2a). ELISA revealed decreased serum irisin levels in PD mice, whereas AAV treatment restored the levels to those of control mice (Supplementary Fig. 2b). Behavioral assessments revealed that FNDC5 overexpression markedly improved motor function, as evidenced by increased locomotor activity in the open field test, reduced descent time in the pole test and increased latency to fall in the rotarod test, compared to PFFs mice (Supplementary Fig. 2c\u0026ndash;e). Histological analysis further showed that FNDC5 overexpression attenuated dopaminergic neurodegeneration. Specifically, TH immunostaining demonstrated preservation of dopaminergic neurons in the substantia nigra and restoration of TH-positive fiber density in the striatum (Supplementary Fig. 2f\u0026ndash;h). In parallel, p-\u0026alpha;Syn accumulation was significantly reduced in FNDC5-overexpressing mice (Supplementary Fig. 2i\u0026ndash;j). Together, these results demonstrate that systemic elevation of irisin is sufficient to recapitulate the neuroprotective effects of exercise.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIrisin restores a homeostatic astrocyte state and upregulates REV-ERB\u0026alpha;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe inflammatory status of astrocytes is closely linked to perivascular AQP4 polarization, wherein pro-inflammatory (A1-like) astrocytes typically exhibit disrupted AQP4 localization and impaired glymphatic function, whereas anti-inflammatory or homeostatic (A2-like) phenotypes support AQP4 polarity\u003csup\u003e10\u003c/sup\u003e. We therefore examined whether the effects of exercise on AQP4 polarity were associated with the functional state of astrocytes. Immunostaining for the astrocytic marker GFAP, the A1-associated marker C3d, and the A2-associated marker S100A10 revealed that PFFs injection induced a reactive astrocytic phenotype, whereas exercise reversed this shift (Fig. 6a\u0026ndash;c). Specifically, PFFs-injected mice exhibited increased C3d expression and decreased S100A10 expression; in contrast, exercise significantly reduced C3d while upregulating S100A10, consistent with restoration of a more homeostatic astrocyte state (Fig. 6d\u0026ndash;e). Sholl analysis confirmed this remodeling, showing increased summarized intersections and altered branching profiles after PFFs injection, both of which were attenuated by exercise (Fig. 6f\u0026ndash;g). These findings demonstrate that exercise restores astrocytic AQP4 polarity and promotes a more homeostatic astrocyte state.\u003c/p\u003e\n\u003cp\u003eTo determine whether irisin could recapitulate the effects of exercise on astrocytic phenotype, we cultured primary astrocytes and treated them with with PFFs and/or irisin. Western blotting analysis revealed that C3d was significantly reduced in the PFFs+Irisin group, while S100A10 was significantly increased (Fig. 7a\u0026ndash;c), suggesting that irisin shifts astrocytes from neurotoxic A1 phenotypes to neuroprotective A2 phenotypes, thereby counteracting PFF-induced reactive activation. Meanwhile, western blotting results also revealed significantly reduced p-STAT3 levels in the PFFs+Irisin group compared with PFFs group (Fig. 7d\u0026ndash;e), indicating that irisin suppresses STAT3 activation. Previous studies have shown that p-STAT3 can directly repress transcription of the circadian gene \u003cem\u003eNr1d1\u003c/em\u003e (encoding REV-ERB\u0026alpha;)\u003csup\u003e32,33\u003c/sup\u003e, which in turn influences the functional state of astrocytes\u003csup\u003e34\u003c/sup\u003e. We therefore examined whether irisin modulates REV-ERB\u0026alpha; expression. Notably, irisin treatment also upregulated the circadian regulator REV-ERB\u0026alpha;, which was downregulated upon PFFs stimulation (Fig. 7d,7f). These findings suggest that irisin can suppress PFFs-induced astrocytic inflammatory activation and promotes a shift toward a neuroprotective phenotype via modulation of the STAT3/REV-ERB\u0026alpha; signaling.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eREV-ERB\u0026alpha; is indispensable for exercise- and irisin-induced glymphatic improvement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo investigate the role of REV-ERB\u0026alpha; in exercise-induced glymphatic improvement, we performed astrocyte-specific knockdown of \u003cem\u003eNr1d1\u003c/em\u003e using AAV-shRNA and assessed its impact on astrocyte polarization. AQP4 polarization was significantly reduced in \u003cem\u003eNr1d1\u003c/em\u003e-shRNA treated mice, as evidenced by immunostaining for AQP4 and CD31, demonstrating AQP4 depolarization in the astrocytes (Fig. 8a\u0026ndash;b). Additionally, \u003cem\u003eNr1d1\u003c/em\u003e-shRNA treatment in the striatum resulted in significant astrocyte activation, as indicated by Sholl analysis of astrocyte branching, showing an increase in summarized intersections and complexity (Fig. 8c\u0026ndash;e). These findings indicate that REV-ERB\u0026alpha; is required for maintaining both perivascular AQP4 polarity and proper astrocytic morphology.\u003c/p\u003e\n\u003cp\u003eTo further investigate the role of REV-ERB\u0026alpha; in exercise- and irisin-induced glymphatic improvement, we assessed the effects of exercise and irisin on glymphatic function in \u003cem\u003eNr1d1\u003c/em\u003e-shRNA mice. OVA-647 was used instead of TR-d10 to avoid spectral overlap with mCherry expressed from the AAV-shRNA vector. Representative images of OVA-647 and PFFs-488 showed that \u003cem\u003eNr1d1\u003c/em\u003e knockdown markedly impaired tracer clearance (Fig. 9a). Quantitative analysis revealed that \u003cem\u003eNr1d1\u003c/em\u003e deficiency significantly increased the retention of OVA-647 and PFFs-488, indicating impaired clearance. \u0026nbsp;Importantly, neither exercise nor irisin administration was able to restore tracer distribution or clearance in \u003cem\u003eNr1d1\u003c/em\u003e-deficient mice, as no significant differences were observed between \u003cem\u003eNr1d1\u003c/em\u003e-shRNA, \u003cem\u003eNr1d1\u003c/em\u003e-shRNA+Ex, and \u003cem\u003eNr1d1\u003c/em\u003e-shRNA+Irisin groups (Fig. 9b\u0026ndash;e). The results demonstrate that \u003cem\u003eNr1d1\u003c/em\u003e is essential for the exercise- and irisin-mediated restoration of glymphatic function, as \u003cem\u003eNr1d1\u003c/em\u003e knockdown prevents the exercise-induced enhancement of glymphatic clearance and AQP4 polarization in astrocytes.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we identified a previously unrecognized muscle\u0026ndash;brain\u0026ndash;glymphatic axis through which physical exercise enhances the clearance of \u0026alpha;Syn aggregates in a mouse model of PD. We demonstrated that chronic treadmill exercise restores impaired glymphatic transport and reduces \u0026alpha;Syn pathology in the PFFs model, an effect not previously reported for exercise in the context of synucleinopathy. We further identified the exercise-induced myokine irisin as a critical systemic mediator that is both sufficient and necessary for the glymphatic benefits, revealing a novel role for irisin in promoting central protein clearance. Mechanistically, we showed that irisin acts on astrocytes to upregulate the circadian nuclear receptor REV-ERB\u0026alpha;, which in turn restores perivascular AQP4 polarity and shifts astrocytes toward a homeostatic phenotype. Notably, the requirement of astrocytic REV-ERB\u0026alpha; for exercise- and irisin-induced glymphatic enhancement represents a previously unidentified link between circadian control and astrocyte-dependent proteostasis in PD.\u003c/p\u003e\n\u003cp\u003eGlymphatic dysfunction is increasingly recognized as a pathogenic feature across neurodegenerative conditions, including Alzheimer\u0026rsquo;s disease and PD\u003csup\u003e16,17,35-37\u003c/sup\u003e. Previous studies in Alzheimer\u0026rsquo;s disease and aged animals have shown that exercise promotes cerebrospinal fluid influx and restores AQP4 polarization at astrocytic endfeet, thereby facilitating amyloid-\u0026beta; clearance\u003csup\u003e9,10,38\u003c/sup\u003e. However, whether such mechanisms operate in PD and contribute to the clearance of \u0026alpha;Syn aggregates remained unexplored. Our findings extend this knowledge by demonstrating that exercise improves glymphatic transport and reduces \u0026alpha;Syn pathology in the PFFs model, positioning glymphatic restoration as a key mechanism underlying the neuroprotective effects of exercise in PD. Moreover, while earlier work has highlighted the importance of AQP4 for glymphatic function\u003csup\u003e10,38,39\u003c/sup\u003e, we provide direct evidence that AQP4 is required for the therapeutic benefits of exercise, as pharmacological inhibition of AQP4 abrogated exercise-induced reductions in \u0026alpha;Syn pathology and motor recovery. These findings establish astrocyte-dependent fluid transport as a critical effector downstream of exercise and upstream of enhanced brain clearance in synucleinopathy.\u003c/p\u003e\n\u003cp\u003eThe systemic signals that link peripheral exercise to central glymphatic regulation remain poorly characterized. As a prototypical exercise-induced myokine which can cross the blood\u0026ndash;brain barrier, irisin has emerged with neuroprotective properties in various neurological disorders\u003csup\u003e26,30,40,41\u003c/sup\u003e. Recent studies have shown that irisin can protect dopaminergic neurons, modulate glial cell activity, and reduce neuroinflammation in PD models\u003csup\u003e28,31\u003c/sup\u003e. However, the role of irisin in regulating global brain waste clearance had not been reported. Our study fills this gap by demonstrating that irisin is sufficient to enhance parenchymal solute transport and accelerate the clearance of \u0026alpha;Syn aggregates, and that endogenous irisin is required for the glymphatic benefits of exercise. Moreover, we observed that exercise and irisin shift astrocytes from a pro-inflammatory A1-like state toward a more supportive A2-like phenotype, a transition tightly linked to the recovery of AQP4 polarity. This phenotypic remodeling might represent a common mechanism through which diverse interventions, including exercise, enhance brain clearance across neurodegenerative conditions. These findings expand the functional repertoire of irisin beyond direct neuronal modulation, implicating it as a systemic orchestrator of astrocyte-mediated proteostasis.\u003c/p\u003e\n\u003cp\u003eMechanistically, our findings established REV-ERB\u0026alpha; as a critical transcriptional node linking exercise-induced peripheral signals to astrocyte-dependent glymphatic function, positioning circadian control at the center of brain proteostasis in PD. This was further supported by our observation that astrocyte-specific REV-ERB\u0026alpha; disruption abolished the beneficial effects of both exercise and irisin on glymphatic clearance, underscoring the non-redundant role of this circadian factor in mediating central responses to peripheral physiological stimuli. Importantly, these findings suggested that the efficacy of exercise might depend not only on its intensity or duration but also on its temporal alignment with endogenous circadian rhythms. Given that glymphatic activity peaks during sleep and that REV-ERB\u0026alpha; expression follows a daily oscillation\u003csup\u003e39,42\u003c/sup\u003e, it is plausible that exercise timing could synergize with rhythmic perivascular dynamics to optimize solute clearance\u003csup\u003e43,44\u003c/sup\u003e. Moreover, because PD is characterized by blunted circadian amplitude and fragmented rest\u0026ndash;activity patterns\u003csup\u003e45,46\u003c/sup\u003e, exercise-induced restoration of REV-ERB\u0026alpha; expression may help re-establish circadian homeostasis, thereby reinforcing the temporal architecture of glymphatic function\u003csup\u003e34,45\u003c/sup\u003e. This perspective raises the possibility that exercise interventions could be optimized as \u0026ldquo;chronotherapies,\u0026rdquo; tailored to align with individual circadian profiles to maximize neuroprotection. More broadly, our findings support a model in which peripheral signals such as irisin do not merely activate static molecular pathways but instead engage dynamic. This conceptual shift highlights the importance of integrating circadian biology into mechanistic studies of exercise and neurodegeneration, paving the way for chrono-exercise strategies in the management of PD and related disorders.\u003c/p\u003e\n\u003cp\u003eSeveral limitations should be acknowledged. First, glymphatic transport is influenced by multiple physiological parameters, including sleep\u0026ndash;wake state, vascular pulsatility, and circadian timing, which were not systematically controlled in the current study. Second, while our findings in the PFFs model provide mechanistic insight, the generalizability of this axis to other \u0026alpha;Syn-based models or to aging remains to be established. Third, although we identified REV-ERB\u0026alpha; as a critical mediator of exercise-induced glymphatic enhancement, our study did not manipulate the circadian timing of exercise. Given the known diurnal oscillations of REV-ERB\u0026alpha; and glymphatic activity, it remains unknown whether exercise at specific zeitgeber times would produce differential effects. Future studies incorporating time-of-day interventions are warranted to explore this possibility.\u003c/p\u003e\n\u003cp\u003eIn summary, we describe a previously unrecognized pathway through which physical exercise engages a peripheral myokine to regulate astrocyte-dependent glymphatic clearance in PD. As illustrated in our working model (Fig. 10), physical exercise stimulates skeletal muscle to release irisin, which acts on astrocytes to restore REV-ERB\u0026alpha;\u0026ndash;dependent transcription, suppress inflammatory activation, and promote AQP4 polarization at perivascular endfeet. This astrocytic remodeling enhances glymphatic clearance of \u0026alpha;Syn aggregates and contributes to dopaminergic neuroprotection. Together, these results establish a mechanistic framework linking systemic physical activity to astrocyte-mediated brain proteostasis and highlight the exercise/irisin/REV-ERB\u0026alpha; axis as a promising therapeutic target for neurodegenerative diseases.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003eAnimals\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEight-week-old male C57BL/6J mice were obtained from Shanghai GemPharmatech Co., Ltd. FNDC5 knockout (FNDC5-KO) mice (Strain No. T014328, Cas9-mediated knockout; C57BL/6JGpt background) were also purchased from Shanghai GemPharmatech Co., Ltd. Mice were housed in groups of five per cage under SPF conditions in a ventilated animal facility. Animals were maintained on a 12-hour light/dark cycle with controlled temperature (20\u0026ndash;23\u0026deg;C) and humidity (50\u0026ndash;60%) and had ad libitum access to standard chow and water. All experimental procedures were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Ethics Committee of Tongji University (approval number: TJBC00322103).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePreparation of \u0026alpha;Syn PFFs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRecombinant monomeric mouse \u0026alpha;Syn protein was purchased from AnaSpec (AS-56082-100). The protein was dissolved in 100 mM NaCl to a final concentration of 5 mg/mL. For fibril formation, \u0026alpha;Syn solution was incubated in a shaking dry bath (dryBATH-HS, WIX) at 37\u0026deg;C with continuous agitation at 1,000 rpm for 7 days. Prior to use, the formed fibrils were sonicated for 30 s at 15% amplitude with a 0.5 s on/off pulse cycle using a probe sonicator (JY92-IIN, SCIENTZ) to generate \u0026alpha;Syn PFFs. For generation of PFFs-488, the sonicated PFFs were labeled using an Alexa Fluor\u0026trade; 488 protein labeling kit (A30006, Thermo Fisher Scientific) according to the manufacturer\u0026rsquo;s instructions. Labeled fibrils were used immediately or stored under appropriate conditions until further use.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStereotaxic Injection of \u0026alpha;Syn PFFs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTwo-month-old wild-type mice were anesthetized with 2.5% isoflurane for induction and maintained at 1% isoflurane during surgery via continuous inhalation. Animals were secured in a stereotaxic frame and Sodium hyaluronate ophthalmic solution were applied to prevent corneal drying during anesthesia. After exposing the skull, a small burr hole was drilled at the injection site targeting the right striatum (AP: 0.5 mm; ML: 2.0 mm; DV: \u0026ndash;3.2 mm from bregma). Sonicated \u0026alpha;Syn PFFs (5 \u0026mu;g in 2 \u0026mu;L) were injected using a 34-gauge Hamilton syringe at a rate of 0.2 \u0026mu;L/min. Upon completion of infusion, the needle was left in place for an additional 5 minutes to minimize reflux and then slowly withdrawn. Following surgery, mice received subcutaneous meloxicam (2.5 mg/kg) for postoperative analgesia and were placed on a heating pad until fully recovered from anesthesia.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePharmacology\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo modulate AQP4 function in mice, we used TGN-020 (MedChemExpress, HY-W008574), a selective AQP4 inhibitor. TGN-020 was prepared as a suspension in saline containing 0.5% methyl cellulose and 0.5% Tween-80 at a concentration of 5 mg/mL using sonication-assisted dissolution. Mice received intraperitoneal injections of TGN-020 at a dose of 100 mg/kg prior to each exercise session. To mimic exercise-induced circulating factors, we used recombinant irisin (R\u0026amp;D Systems, 8880-IR). Irisin was dissolved in sterile saline and administered at a dose of 200 \u0026mu;g/kg by intraperitoneal injection. Control mice received vehicle solution with the same composition. To assess the direct role of irisin in regulating astrocyte responses, recombinant irisin was applied to primary astrocyte cultures at a final concentration of 200 ng/mL.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eViral vector delivery\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo manipulate systemic irisin levels, we used AAV-FNDC5, which was delivered by tail-vein injection on the same day as PFFs administration. To achieve astrocyte-specific knockdown of \u003cem\u003eNr1d1\u003c/em\u003e, AAV-\u003cem\u003eNr1d1\u003c/em\u003e-shRNA was stereotaxically injected into the striatum. Mice were anesthetized and fixed in a stereotaxic frame, and the virus was unilaterally injected at the following coordinates relative to bregma: AP, +0.5 mm; ML, +2.0 mm; DV, \u0026minus;3.2 mm. The injection was performed using a microsyringe at a constant rate, and the needle was left in place for 5 min before slow withdrawal to minimize backflow.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCisterna magna cannulation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCisterna magna cannulation was performed to enable intrathecal delivery of contrast agents during magnetic resonance imaging. Mice were anesthetized with isoflurane delivered via inhalation and positioned in a stereotaxic frame. The head was positioned at an angle of approximately 120\u0026ndash;150 degrees relative to the body to optimize surgical exposure. Hair overlying the posterior skull and upper cervical region was removed, and a midline skin incision of approximately 5 mm was made. The bilateral trapezius muscles were separated along the midline, and deeper musculature was bluntly dissected using toothed forceps to expose the cisterna magna under direct visualization.\u003c/p\u003e\n\u003cp\u003eA custom copper cannula (outer diameter 0.35 mm, inner diameter 0.28 mm) was implanted for intrathecal access. Copper was selected because of its diamagnetic properties, which minimize magnetic susceptibility artifacts during magnetic resonance imaging. The cannula was connected to PE10 tubing prefilled with artificial cerebrospinal fluid (ACSF; PH1851, PHYGENE) to prevent air bubble formation. After insertion into the cisterna magna, the cannula was secured using a mixture of ethyl cyanoacrylate adhesive and dental cement. An accelerator solution was applied to promote rapid polymerization. The distal end of the PE10 tubing was sealed using a thermal cauterizer. Following surgery, mice received subcutaneous meloxicam for postoperative analgesia and were placed on a heating pad until fully recovered from anesthesia.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eParenchymal clearance assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo evaluate interstitial solute clearance from the brain, fluorescent tracers were stereotaxically injected into the unilateral striatum. PFFs-488 were mixed with either Texas Red\u0026ndash;dextran (10 kDa; TR-d10, ThermoFisher Scientific, D1863) or Alexa Fluor\u0026trade; 647\u0026ndash;conjugated ovalbumin (OVA-647; Invitrogen, O34784). All tracers were adjusted to a final concentration of 2.5 mg/mL prior to injection.\u003c/p\u003e\n\u003cp\u003eMice were anesthetized with isoflurane and secured in a stereotaxic frame. After exposing the skull, a small burr hole was drilled at the striatal coordinates (AP: 0.5 mm; ML: 2.0 mm; DV: \u0026ndash;3.2 mm). A total volume of 2 \u0026mu;L tracer mixture was injected at a rate of 0.2 \u0026mu;L/min using a micro-pump (KDS Legato 130, RWD Life Science). Following infusion, the needle was left in place for 5 minutes and then slowly withdrawn to minimize reflux.\u003c/p\u003e\n\u003cp\u003eTwo hours after injection, mice were sacrificed and brains were harvested and fixed in 4% PFA overnight at 4 \u0026deg;C. Brains were coronally sectioned at 100 \u0026mu;m thickness using a vibratome (Leica VT1200 S). Fluorescence images were acquired using an automated digital slide scanning system (Axioscan 7, Carl Zeiss) under identical exposure settings across all groups. For quantitative analysis, five coronal sections per animal, spaced at 400 \u0026mu;m intervals, were analyzed. Using ImageJ, the entire brain slice was manually outlined as the region of interest. Mean fluorescence intensity within this region was measured for each section after background subtraction. The average fluorescence intensity from the five sections was calculated to obtain a single value per animal for statistical analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDCE-MRI measurements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll MRI experiments were performed on a horizontal bore 9.4 Tesla / 30 cm scanner (uMR 9.4T, United Imaging Life Science Instrument, Wuhan, China). An 86-mm inner diameter volume coil was used for radio frequency transmission, and a mouse brain surface coil was used for signal reception.\u003c/p\u003e\n\u003cp\u003eDynamic contrast-enhanced magnetic resonance imaging was conducted in adult C57BL/6 mice using a three-dimensional T1-weighted gradient-echo sequence with an isotropic spatial resolution of 0.15 \u0026times; 0.15 \u0026times; 0.15 mm\u0026sup3;. A total of 80 dynamic frames were acquired over 78 seconds. Mice were surgically implanted with a cisterna magna cannula prior to imaging to enable controlled intrathecal delivery of contrast agent. The first dynamic frame was acquired as a pre-contrast baseline. Immediately after baseline acquisition, 10 \u0026mu;L of Gd-DTPA was infused through the pre-implanted cannula at a rate of 1 \u0026mu;L per minute using a microinfusion pump while dynamic acquisition continued.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eRaw DICOM images were converted to NIfTI format using dcm2niix. Motion correction of the four-dimensional dynamic series was performed using rigid-body registration implemented in Advanced Normalization Tools (version 2.6.2), with each frame aligned to a reference volume. A motion-corrected mean image was generated for region-of-interest delineation. Regions of interest were manually defined in subject space using ITK-SNAP (version 3.8.0). Mean signal intensity within each region was extracted across time to generate dynamic curves. Quantitative parameters, including area under the curve, peak signal intensity, and time-to-peak, were calculated for downstream analyses. Image processing and quantitative analyses were performed using Advanced Normalization Tools, ITK-SNAP, nibabel (version 5.2.1), and custom Python scripts executed in Jupyter Notebook. All procedures were applied uniformly across animals.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eOpen field test\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo assess general locomotor activity and anxiety-like behavior, mice were tested in an open field arena (40 \u0026times; 40 \u0026times; 40 cm). Prior to testing, animals were transferred to the behavioral testing room at least 3 hours in advance to allow acclimatization to the experimental environment and minimize stress-related confounding effects.\u003c/p\u003e\n\u003cp\u003eDuring the test session, each mouse was placed in the center of the arena and allowed to freely explore for 10 minutes. Locomotor activity was recorded using an automated video tracking system (Shanghai Xinruan Technology, China). For quantitative analysis, total distance traveled during the first 5 minutes was used to evaluate spontaneous locomotor activity. Time spent in the center area of the arena was recorded as an index of anxiety-like behavior.\u003c/p\u003e\n\u003cp\u003eTo eliminate olfactory cues between subjects, the arena was thoroughly cleaned with 75% ethanol after each trial. The next mouse was tested only after complete evaporation of the ethanol to avoid residual odor interference.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRotarod test\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMotor coordination and balance were assessed using a rotarod apparatus (Shanghai Xinruan Technology, China). Mice underwent a habituation phase prior to formal testing. Animals were trained for three consecutive days, with one session per day. During habituation, mice were placed on the rotating rod for 5 minutes each day. The rotation speed was maintained at 4 revolutions per minute on the first and second training days and increased from 4 to 10 revolutions per minute on the third day to facilitate adaptation to accelerating conditions.\u003c/p\u003e\n\u003cp\u003eFor the test session, mice were placed on the rod, which accelerated from 4 to 40 revolutions per minute over a period of 5 minutes. The latency to fall was recorded for each trial. Each mouse completed three trials with appropriate rest intervals between trials, and the average latency to fall was used for statistical analysis. To minimize olfactory cues and avoid confounding effects from residual scent, the rotarod apparatus and testing compartments were cleaned with 75% ethanol between trials.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePole test\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe pole test was performed to evaluate motor coordination as well as the ability of mice to turn and descend. The apparatus consisted of a vertical pole (50 cm in height, 1 cm in diameter) with a rough surface to facilitate grip. Prior to formal testing, mice underwent a habituation phase for two consecutive days. During habituation, each mouse performed three descent trials per day to acclimate to the task. For the test session, mice were placed head-upward on the top of the vertical pole. The time required to turn downward and descend to the base of the pole was recorded. Each mouse completed three trials, and the average descent time was used for statistical analysis. Mice were anesthetized with isoflurane prior to tissue collection. For both fresh tissue collection and perfusion-fixed tissue preparation, blood was first obtained via cardiac puncture and collected into 1.5 mL microcentrifuge tubes for serum isolation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTissue collection\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMice were anesthetized with isoflurane prior to tissue collection. Blood was first obtained via cardiac puncture and collected into 1.5 mL microcentrifuge tubes for serum isolation.\u003c/p\u003e\n\u003cp\u003eFor biochemical analyses, mice were transcardially perfused with pre-cooled phosphate-buffered saline to remove circulating blood. Brains were rapidly dissected and transferred onto sterile filter paper. Under a stereomicroscope, ipsilateral and contralateral brain regions, including the cortex, striatum, ventral midbrain, and hippocampus, were carefully separated. Dissected tissues were immediately frozen and stored at \u0026minus;80℃ for subsequent Western blotting and enzyme-linked immunosorbent assay analyses.\u003c/p\u003e\n\u003cp\u003eFor immunofluorescence and immunohistochemistry, mice were transcardially perfused with phosphate-buffered saline followed by 4% paraformaldehyde. Brains were dissected and post-fixed in 4% paraformaldehyde at 4℃for 24\u0026thinsp;h. Tissues were then washed with phosphate-buffered saline and cryoprotected in graded sucrose solutions (20%, 30%, and 30%) over three days. Subsequently, brains were embedded in Tissue-Tek OCT compound (SAKURA, USA) and stored at \u0026minus;80℃. Coronal sections (30\u0026mu;m) were prepared using a cryostat (Leica CM1950) and transferred to cryoprotectant solution containing 30% sucrose, 20% ethylene glycol, and 1% PVP-40 in 1\u0026times; PBS for storage at \u0026minus;20℃ until further use.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmunohistochemistry and Immunofluorescence\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFrozen brain sections were retrieved from cryoprotectant solution and washed with phosphate-buffered saline (PBS) prior to staining. For immunohistochemical staining, sections were subjected to antigen retrieval and endogenous peroxidase blocking, followed by blocking at room temperature for 1 h in 1\u0026times; PBS containing 0.3% Triton X-100 (Sigma-Aldrich, X100) and 10% horse serum (Gibco, 16050122). Sections were then incubated overnight at 4℃ with anti\u0026ndash;tyrosine hydroxylase antibody. After washing, sections were incubated with biotinylated horse anti-mouse secondary antibody (Vector Laboratories, BA-2000), followed by signal amplification using the VECTASTAIN Elite ABC kit (Vector Laboratories, PK-6100). Immunoreactivity was visualized using DAB (Vector Laboratories, SK-4100). Sections were dehydrated, mounted, and imaged for quantitative analysis. For immunofluorescence staining, sections were subjected to antigen retrieval and then blocked for 1 h at room temperature in 1\u0026times; PBS containing 0.3% Triton X-100 and 5% bovine serum albumin (Biosharp, BS114). Sections were incubated overnight at 4℃ with primary antibodies. After washing, sections were incubated for 2 h at room temperature with species-appropriate secondary antibodies, including goat anti-chicken Alexa Fluor 488 (Abcam, ab150169), donkey anti-goat Alexa Fluor 488 (Abcam, ab150129), donkey anti-rabbit Alexa Fluor 568 (Abcam, ab175470), donkey anti-mouse Alexa Fluor 488 (Abcam, ab150105), goat anti-rabbit Alexa Fluor 488 (Invitrogen, A-11008), and goat anti-rabbit Alexa Fluor 568 (Invitrogen, A-11011). Nuclear staining was performed using DAPI (Beyotime, C1002). Sections were mounted using antifade mounting medium (SouthernBiotech, 0100-01). For details of the primary antibodies, see Supplementary Table 1.\u003c/p\u003e\n\u003cp\u003eBrightfield and fluorescence images were acquired using a Slide Scanner (VS200, Olympus), an automated digital slide scanning system (Axioscan 7, Carl Zeiss), and a confocal laser scanning microscope (TI2-E+A1, Nikon). Image acquisition was controlled using OLYMPUS OlyVIA (version 4.1), NIS-Elements Viewer (version 5.21), and ZEN lite (version 3.13). Image processing and quantitative analyses were performed using Fiji software (version 1.54p). All imaging parameters were kept constant across experimental groups for quantitative comparisons.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAQP4 polarity calculation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo quantify the polarization of AQP4 and evaluate its pathological mislocalization, we performed a radial fluorescence intensity analysis on AQP4 and CD31 co-stained images using the Line-plot tool in ImageJ. For each analyzed blood vessel, an 80-\u0026mu;m linear segment was drawn perpendicular to the vessel longitudinal axis, centered on the CD31 positive vascular lumen to ensure anatomical consistency. The Perivascular Intensity (Iperi) was defined as the mean fluorescence intensity within a 10-\u0026mu;m radius from the vessel center, representing AQP4 localization at the astrocytic endfeet. The Parenchymal Baseline (Ipara) was calculated as the mean intensity in the region 30 \u0026mu;m away from the vessel, reflecting the non-polarized AQP4 pool in the astrocytic soma and processes. The AQP4 Polarity Index was then calculated as the ratio of perivascular enrichment to the parenchymal background (Iperi/ Ipara), a metric specifically designed to capture the shift of AQP4 from the vascular interface to the brain parenchyma. To minimize inter-slice variability and ensure comparability across experimental groups, all polarity indices were normalized to the mean value of the control group.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePrimary astrocyte cultures\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePrimary astrocytes were prepared from neonatal C57 mice at postnatal day 1\u0026ndash;2. Under a stereomicroscope, the cerebral cortices were isolated and all meninges were carefully removed. Tissues were collected and processed at 4\u0026thinsp;\u0026deg;C, minced into small pieces using fine dissection scissors, and transferred into conical tubes containing Hibernate-E medium (A1247601, Gibco). After the tissue fragments had settled to the bottom of the tube, the supernatant was carefully removed, leaving only enough medium to immerse the tissue. Samples were then enzymatically digested in 0.25% trypsin (15050065, Gibco) containing DNase I (10\u0026thinsp;mg/mL, 18047019, Invitrogen) at 30\u0026thinsp;\u0026deg;C for 30\u0026thinsp;min.\u003c/p\u003e\n\u003cp\u003eEnzymatic digestion was terminated by adding culture medium consisting of high-glucose Dulbecco\u0026rsquo;s modified Eagle medium (DMEM, 11965092, Gibco) supplemented with 10% fetal bovine serum (A5256701, Gibco), 10\u0026thinsp;mL/L penicillin\u0026ndash;streptomycin, pH 7.4. The cell suspension was centrifuged at 150 \u0026times; g for 5\u0026thinsp;min, and the pellet was resuspended in culture medium. Cells were seeded into poly-D-lysine-coated (0.1\u0026thinsp;mg/mL, A3890401, Gibco) T75 flasks at a density of 1 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells/cm\u003csup\u003e2\u003c/sup\u003e and maintained at 37\u0026thinsp;\u0026deg;C in a humidified 5% CO2 incubator until reaching confluence. Culture medium was replaced every 2\u0026ndash;3 days.\u003c/p\u003e\n\u003cp\u003eTo enrich for astrocytes, microglia were removed from the mixed glial monolayer by shaking the flasks at 200\u0026thinsp;rpm for 2\u0026thinsp;h at 37\u0026thinsp;\u0026deg;C, followed by medium replacement to remove cells remaining in suspension. For stimulation experiments, astrocyte cultures were treated with PFFs and irisin as indicated, and cells were collected after 15 days of co-incubation for subsequent analyses.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWestern blotting analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBrain tissues were homogenized in RIPA lysis buffer (Beyotime, P0013B) supplemented with protease and phosphatase inhibitor mixture (Beyotime, P1048). Lysates were centrifuged to remove debris, and protein concentrations were determined using a BCA protein assay kit (Epizyme Biotech, ZJ101). Equal amounts of protein were separated by SDS\u0026ndash;PAGE and transferred onto PVDF membranes. Membranes were blocked for 1 h at room temperature in 5% BSA dissolved in TBST and incubated overnight at 4 \u0026deg;C with primary antibodies. After washing with 1\u0026times;TBST, membranes were incubated with horseradish peroxidase\u0026ndash;conjugated secondary antibodies for 1 h at room temperature. Protein bands were visualized using an enhanced chemiluminescence (Biosharp, BL520) substrate and imaged with a chemiluminescence detection system. Band intensities were quantified using ImageJ software and normalized to the corresponding loading controls for statistical analysis. For details of the primary antibodies, see Supplementary Table 1.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eELISA\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor Irisin detection, blood samples were allowed to clot on ice for 30 min and then centrifuged at 12,000 rpm for 15 min to obtain serum. The supernatant was carefully collected and stored at \u0026ndash;80 \u0026deg;C until analysis. Serum irisin levels were quantified using a Mouse Irisin ELISA Kit (Elabscience Biotechnology, E-EL-M2743) according to the manufacturer\u0026rsquo;s instructions. Absorbance was measured using a microplate reader, and concentrations were calculated from a standard curve generated using serial dilutions of the provided standards.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAnimals from different cages were randomly allocated to experimental groups to minimize potential cage effects. Investigators were blinded to group allocation during data collection and analysis. Statistical analyses were performed using GraphPad Prism. For comparisons involving more than two groups, one - way or two - way analysis of variance (ANOVA) was applied as appropriate. When multiple comparisons were required, Dunnett\u0026rsquo;s post hoc test was used to compare each experimental group with the disease model group, thereby controlling for type I error associated with multiple testing. For comparisons between two groups only, a two - tailed unpaired Student\u0026rsquo;s t test was used. A P value \u0026lt; 0.05 was considered statistically significant. Data are presented as mean \u0026plusmn; SEM.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data used in this study are available from the corresponding author upon request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eL.J. and R.L. designed the study. R.L., J.Z., X.R., B.L., X.X. and Y.L. performed the experiments and collected data. R.L., J.Z., and X.R. analyzed the data. R.L., J.Z., X.R., B.L. and Y.Z. prepared the figures and drafted the original manuscript. Y.S. and L.J. revised the manuscript. All authors read and approved the final manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors would like to thank prof. Cong Liu (Institute of Organic Chemistry, Chinese Academy of Sciences, Shanghai, China) for the assistance with protein purification and PFFs labeling. This work was supported by grants from the National Natural Science Foundation of China (82230084), the National Key Clinical Specialty Discipline Construction Program of China (Z155080000004), Shanghai Rehabilitation Medical Research Center (Top Priority Research Center of Shanghai) (2023ZZ02027), Shanghai Clinical Research Ward (SHDC2023CRW018B) and the Postdoctoral Fellowship Program of CPSF (GZC20251541).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors declare no financial or non-financial competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBloem, B. R., Okun, M. S. \u0026amp; Klein, C. Parkinson\u0026apos;s disease. \u003cem\u003eLancet\u003c/em\u003e \u003cstrong\u003e397\u003c/strong\u003e, 2284-2303 (2021). https://doi.org:10.1016/S0140-6736(21)00218-X\u003c/li\u003e\n\u003cli\u003eStocchi, F., Bravi, D., Emmi, A. \u0026amp; Antonini, A. 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Circadian clock dysfunction in Parkinson\u0026apos;s disease: mechanisms, consequences, and therapeutic strategy. \u003cem\u003eNPJ Parkinsons Dis\u003c/em\u003e \u003cstrong\u003e11\u003c/strong\u003e, 213 (2025). https://doi.org:10.1038/s41531-025-01009-9\u003c/li\u003e\n\u003cli\u003eShen, Y.\u003cem\u003e et al.\u003c/em\u003e Circadian disruption and sleep disorders in neurodegeneration. \u003cem\u003eTransl Neurodegener\u003c/em\u003e \u003cstrong\u003e12\u003c/strong\u003e, 8 (2023). https://doi.org:10.1186/s40035-023-00340-6\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"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":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Parkinson’s disease, Exercise, Glymphatic system, α-Synuclein, Irisin, REV-ERBα","lastPublishedDoi":"10.21203/rs.3.rs-9467573/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9467573/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Physical exercise alleviates motor deficits and neuropathology in Parkinson’s disease (PD), yet the underlying mechanisms remain elusive. Using a mouse model induced by α-synuclein (αSyn) preformed fibrils, we found that glymphatic transport was markedly impaired in PD mice, accompanied by disrupted perivascular aquaporin-4 (AQP4) polarization and αSyn accumulation. Chronic treadmill exercise restored glymphatic function, restored AQP4 polarity, and alleviated motor deficits and neuropathology; these effects were abrogated by AQP4 inhibition. We identified the exercise-induced myokine irisin as a critical systemic mediator linking peripheral activity to central clearance. Systemic irisin administration recapitulated the glymphatic benefits of exercise, whereas Fndc5 deficiency abolished them. Mechanistically, irisin acted on astrocytes to suppress STAT3 activation and upregulate the circadian gene Nr1d1 (encoding REV-ERBα). Astrocyte-specific knockdown of Nr1d1 disrupted AQP4 polarization and eliminated the glymphatic and neuroprotective effects of both exercise and irisin. Collectively, these findings establish an exercise-irisin-astrocyte axis that enhances glymphatic clearance of αSyn aggregates through REV-ERBα-dependent regulation of astrocytic function, revealing a previously unrecognized mechanism linking peripheral exercise to brain proteostasis.","manuscriptTitle":"Irisin mediates exercise-induced glymphatic α-synuclein clearance by upregulating astrocytic REV-ERBα in Parkinson’s disease","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-30 05:21:28","doi":"10.21203/rs.3.rs-9467573/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"32a4e419-0289-4e9d-9fe4-b937b64bb646","owner":[],"postedDate":"April 30th, 2026","published":true,"recentEditorialEvents":[{"type":"editorInvitedReview","content":"This content is not available.","date":"2026-05-10T13:19:15+00:00","index":2,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2026-05-01T21:28:36+00:00","index":3,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2026-04-30T00:09:14+00:00","index":2,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2026-04-29T21:51:18+00:00","index":1,"fulltext":"This content is not available."}],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":67246543,"name":"Health sciences/Neurology/Neurological disorders/Parkinson's disease"},{"id":67246544,"name":"Biological sciences/Neuroscience/Glial biology/Astrocyte"}],"tags":[],"updatedAt":"2026-04-30T05:21:28+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-30 05:21:28","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9467573","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9467573","identity":"rs-9467573","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}