Long-term exercise enhances meningeal lymphatic vessel plasticity and drainage in a mouse model of Alzheimer's disease

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This makes it a promising therapeutic target for Alzheimer's disease (AD). Long-term exercise can reduce the risk of AD; however, the underlying mechanism is not fully understood. In this study, we investigated whether exercise alleviates AD-related pathological changes by improving meningeal lymphatic drainage and explored its potential mechanisms. Methods The morphological and functional features of meningeal lymphatic vessels, as well as Aβ and reactive gliosis in the brain, were compared between 6.5-month-old 5×FAD mice with or without 1 month of treadmill exercise. RNA sequencing analysis, protein interactions analysis, adeno-associated virus (AAV)-mediated gene knockdown, and lymphatic endothelial cell culture were conducted to investigate the mechanism underlying exercise-induced meningeal lymphatic vessel plasticity of 5×FAD mice. Results The structural integrity of meningeal lymphatic vessels was compromised in 5×FAD mice, compared with the wild-type mice. Treadmill exercise increased the diameter and drainage capacity of the meningeal lymphatic vessels, reduced Aβ deposition, reactive gliosis, and astrocyte senescence in the hippocampus and frontal cortex, and improved cognitive function in 5×FAD mice. Mechanistically, exercise reduced the up-regulation of thrombospondin-1 (TSP-1), a lymphangiogenesis inhibitor, in activated astrocytes of AD mice. TSP-1 exacerbated the inhibitory effect of Aβ on lymphatic vessel formation and plasticity through interactions with CD36 and CD47, respectively. Additionally, exercise decreased the expression of TSP-1 in reactive astrocytes of AD mice by downregulating eleven-nineteen lysine-rich leukemia-associated factor 2 (EAF2), which facilitates the transcription of the TSP-1 encoding gene Thbs-1 via its binding partner p53. Ultimately, we discovered that hippocampal astrocyte-specific knockdown of Thbs-1 or Eaf2 enhanced meningeal lymphatic drainage and alleviated AD-like pathology in the hippocampus of 5×FAD mice. Conclusions These findings collectively unveil a novel mechanism through which long-term exercise combats AD. It enhances the plasticity and drainage of meningeal lymphatic vessels by downregulating the EAF2-p53-TSP-1 pathway, which is associated with reactive astrocytes. Alzheimer's disease Lymphangiogenesis Meningeal lymphatics Treadmill exercise EAF2-p53-TSP-1 Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Alzheimer's disease (AD) is one of the most common age-dependent neurodegenerative diseases, posing a serious threat to the health and life of older adults. Although monoclonal antibodies such as Aducanumab and Lecanemab can specifically reduce the deposition of amyloid β (Aβ) plaques, their long-term efficacy and potential complications still present challenges [ 1 ]. It is known that the imbalance between the production and clearance of Aβ occurs prior to the onset of cognitive impairment, causing excessive aggregation of Aβ and a series of neuropathological cascades [ 2 ]. Notably, a large number of astrocytes are persistently activated and undergo senescence, losing their ability to maintain brain homeostasis and exhibiting the senescence-associated secretory phenotype, which in turn accelerates neurodegeneration [ 3 , 4 ]. Therefore, timely and effectively facilitating clearance of Aβ from the brain and preventing astrocyte senescence may be beneficial for delaying or even preventing the onset of AD [ 5 , 6 ]. Meningeal lymphatic vessels have recently been characterized in humans and rodents [ 7 ]. They mediate the drainage of macromolecular waste [ 8 , 9 ], cellular debris [ 10 ], neurotropic viruses [ 11 ], and brain tumor cells [ 12 ] from the brain. Meningeal lymphatic drainage progressively deteriorates during natural aging and in AD [ 13 – 15 ]. Blocking meningeal lymphatic vessels exacerbates Aβ load and memory deficits in transgenic mouse models of AD [ 8 , 14 ]. Therefore, enhancing meningeal lymphatic drainage could be a novel therapeutic target for AD. Notably, the specialized morphological features of meningeal lymphatic vessels are associated with their drainage functions [ 15 ]. A continuous, zipper-like vascular endothelial (VE)-Cadherin junction and a discontinuous, button-like junctional pattern in lymphatic endothelial cells (LECs) are associated with distinct modes of cerebrospinal fluid (CSF) macromolecule transport by meningeal lymphatic vessels. Zipper-like junctions form tight, continuous barriers that support directional fluid flow, while button-like junctions are more permissive and facilitate macromolecule uptake. The balance between these junction types is crucial for efficient CSF drainage and is disrupted in aged mice, potentially contributing to impaired brain waste clearance [ 15 ]. Vascular endothelial growth factor C (VEGFC) overexpression induces meningeal lymphangiogenesis [ 16 ], whereas inhibitors of lymphangiogenesis, such as pigment epithelium-derived factor, suppress peripheral nasopharyngeal lymphangiogenesis [ 17 ]. Nonetheless, the mechanisms that impair the integrity and plasticity of meningeal lymphatic vessels during the progression of AD remain unclear. Investigating this issue could lead to the discovery of therapeutic targets that enhance the draining function of dural lymphatic vessels, potentially alleviating or delaying Aβ-related neurodegeneration. Exercise intervention is one of the most effective nonpharmacologic therapeutic modalities, exerting beneficial effects on various organs, particularly the brain [ 18 , 19 ]. For example, long-term exercise not only enhances the production of brain-derived neurotrophic factor [ 20 ] and synaptic plasticity [ 21 ] but also diminishes brain oxidative stress [ 22 ] and age-related gliosis [ 23 ]. Additionally, recent evidence suggests that astrocytic aquaporin 4 (AQP4)-mediated glymphatic transport plays a role in the neuroprotective effects of voluntary exercise [ 24 – 26 ]. Nonetheless, it remains undetermined whether exercise can enhance meningeal lymphatic plasticity during the progression of AD. Here, we have screened and identified activated astrocyte-derived thrombospondin-1 (TSP-1) as a key inhibitor of meningeal lymphangiogenesis in 5×FAD transgenic mice. The hippocampal astrocyte-specific knockdown of Thbs-1 enhanced meningeal lymphatic drainage and alleviated AD-like pathology. Notably, long-term exercise decreased TSP-1 and its transcriptional regulator, eleven-nineteen lysine-rich leukemia-associated factor 2 (EAF2), promoting glymphatic-meningeal lymphatic transport of Aβ, thereby blocking the vicious cycle of parenchymal Aβ accumulation and astrocyte activation and senescence, ultimately improving the cognitive function of 5×FAD mice. These results not only uncover new mechanisms of dysfunctional meningeal lymphatic vessels in AD but also highlight new targets for long-term exercise in combating Aβ-related neurodegeneration. Methods Animal care and use B6.Cg-Tg (APPSwFlLon, PSEN1*M146L*L286V) 6799Vas/Mmjax (5×FAD, strain# 008730) mice were obtained from Jackson Laboratories. The mice, along with their age-matched wild-type (WT) littermates, were housed in controlled ambient temperatures and exposed to a 12-hour light/12-hour dark cycle. They had unlimited access to standard rodent chow and clean water. Each group of mice consisted of an equal number of males and females. All animal experiments were conducted under the approval of the Institutional Animal Care and Use Committee (IACUC-1812054). Treadmill exercise training 5×FAD mice aged 5.5 months were randomly assigned to either a treadmill exercise group or a sedentary group. The treadmill exercise (Exe) regimen consisted of twice-daily sessions (at zeitgeber time (ZT) 1 and ZT 12, with lights on at 08:00 am designated as ZT 0), six days a week. Mice were acclimated and trained on a 10° uphill treadmill, beginning with 30 minutes of running at 8 m/min, followed by 30 minutes at 10 m/min for the first two days as a warm-up. Starting on the third day, mice were subjected to increasing treadmill speeds, with increments of 1 m/min every 20 minutes, for a total of 90 minutes per session [ 27 ]. Sedentary (Sed) control mice were kept in identical conditions but remained in their natural state. After one month of repeated training, the animals underwent behavioral tests followed by pathological analyses. In vivo two-photon imaging After being anesthetized with an intraperitoneal injection of a mixed solution containing ketamine (80 mg/kg) and xylazine (8 mg/kg) in saline, 6-month-old wild-type (WT) mice and 5×FAD mice with 10 days of treadmill exercise training were secured in a stereotaxic device. The skin on their heads and necks was shaved and cleaned with iodine and 75% ethanol. Following a surgical skin incision in the parietal skull bone, the skull bone was thinned using an electrical micro drill. A total of 3 µL of A488-Lyve-1 (Invitrogen; Cat# 53-0443-82) was injected into the cisterna magna of the mice within 5 minutes via a 5 µL Hamilton syringe. The syringe was left in place for an additional 5 minutes before being slowly withdrawn. After suturing the exposed incision, the mice were returned to their home cage. Twenty-four hours later, the mice received an intrahippocampal injection (anteroposterior − 2.0 mm, mediolateral ± 1.8 mm, dorsoventral − 2.0 mm) of 1 µL of Fluor 555-labeled Aβ (at 1 mg/mL, AnaSpec; Cat# AS-60480-01), and one hour later, in vivo two-photon imaging was performed. The heads of the mice were fixed on a metal holder to minimize movement during live imaging. As previously described [ 11 ], a confocal scanning system (Zeiss ZEN) equipped with a two-photon laser scanning microscope and a 20× water-immersion lens installed on an upright microscope (Zeiss LSM880, Germany) was used for imaging the meninges. AAV-mediated astrocytes selective knockdown of Thbs-1 or Eaf2 The rAAV-GFaABC1D-mCherry-5′miR-30a-shRNA (Thbs1)-3′miR-30a-WPREs or rAAV-GFaABC1D-EGFP-5′miR-30a-shRNA (Eaf2)-3′miR-30a-WPREs, designed for specific knockdown of Thbs1 or Eaf2, were obtained from BrainVTA. An shRNA sequence verified to efficiently knockdown mouse TSP-1 or Eaf2 was utilized: siRNA Thbs1, 5′-GAUGACUACGCUGGCUUUGUU-3′, siRNA Eaf2, 5′-GGACUUCCAAUCUUGUACATT-3′. As described above, after anesthetization, 5-month-old WT and 5×FAD mice were secured in a stereotaxic apparatus. rAAV2/5-GFaABC1D-Thbs1-shRNA-mCherry, rAAV2/9-GFaABC1D-scramble-shRNA-mCherry, rAAV2/5-GFaABC1D-Eaf2-shRNA-EGFP, or rAAV2/5-GFaABC1D-scramble-shRNA-EGFP was injected into the bilateral hippocampal region (anteroposterior − 2.0 mm, mediolateral ± 1.8 mm, dorsoventral − 2.0 mm, 1 µL for each hemisphere). Four weeks post-virus injection, the behavior and pathology of Thbs1 or Eaf2 protein knockdown were evaluated. Y-maze test The Y-maze test (27 cm × 9 cm × 24 cm) consisted of a start arm, another arm, and a novel arm. During the training stage, mice were placed in the start arm to explore for 5 minutes with the novel arm blocked. Two hours later, the mice were allowed to freely explore the entire maze for 5 minutes. The percentage of time spent in the novel arm and the number of entries into the novel arm were recorded by video tracking software (TopScan, CleverSys, Inc.). Novel object recognition (NOR) test The NOR test was conducted in two phases: familiarization and testing [ 10 ]. During the familiarization phase, each animal was permitted to freely explore an open arena (40 cm × 40 cm × 30 cm) containing two identical objects placed in opposite diagonal corners for a 5-minute trial. In the testing phase, mice were reintroduced to the arena for another 5-minute trial, this time with one familiar object replaced by a novel object (differing in color and shape) after a two-hour interval. Exploration of both objects was defined as the mouse sniffing or interacting with an object from within 2 cm and was quantified using video tracking software (TopScan, CleverSys, Inc.). The discrimination index, a measure of recognition memory, was calculated by dividing the time spent exploring the novel object by the total time spent exploring both objects, then subtracting the time spent exploring the familiar object. Elevated plus maze (EPM) Test The EPM consisted of two open arms (35 cm × 6 cm) and two closed arms (35 cm × 6 cm × 15 cm), each elevated 75 cm above the floor. Mice were placed in the central hub and allowed to freely explore the maze for 5 minutes. Video tracking software (TopScan, CleverSys, Inc.) was utilized to quantify the time spent in the open arms and the frequency of entries into the open arms. Primary astrocytes and cell line cultures and treatment Primary astrocytes were isolated from neonatal mice as previously described [ 10 ]. In brief, after removing the meningeal vessels, the hippocampus and surrounding cortices were microdissected and subjected to trypsin digestion. Tissue homogenates were passed through a 70 µm mesh filter, resuspended in astrocyte growth media (DMEM (Gibco, Cat# 11960), 10% FBS, 100 U/mL penicillin/streptomycin (Gibco, Cat# 15-140-122)), and plated on 10 cm petri dishes. These were incubated at 37°C in 5% CO 2 . The medium was fully replaced every 3 to 4 days, and cells were passaged using 0.05% Trypsin (Gibco, Cat# 25-200-056) when they reached full growth. For experiments involving primary astrocytes treated with Aβ 1−42 oligomers, cultured 12–15 days old primary astrocytes were transferred to complete serum medium and then incubated in the presence or absence of oligomeric Aβ 1−42 (at concentrations of 0, 5, and 10 µM) for 48 hours. Some experiments continued with the transfection of siR-Eaf2, after which the pellet and supernatant were collected 72 hours later. The culture of the Mus musculus lymphoid endothelial cell line (SVEC4-10) (ATCC, Cat# CRL-2181), endothelial cell line (human aortic endothelial cells, HAECs) and human-derived lymphatic endothelial cell line (HLEC) were routinely maintained in DMEM medium supplemented with 10% FBS and 100 U/mL penicillin/streptomycin. The SVEC4-10 cells were planted on a 24-well plate and then treated with a gradient concentration of recombinant TSP-1 protein (R&D Systems, USA; Cat# 3074-TH-050) or oligomer Aβ 1−42 . To investigate the role of the TSP-1-CD47 pathway on junctional patterns in vitro , SVEC4-10 cells were pretreated with siR-CD47 before recombinant TSP-1 intervention. The sequences were as follows: siR-Eaf2, 5′-CAAAGGCUGCUCCAGCUCUdTT-3′; siR-Cd47 (Mus), 5′- CUUGCAUCGUCCGUAAUGUTT-3′; Negative control (NC), 5′-UUCUCCGAACGUGUCACGUTT-3′. CSF and tissue collection After anesthesia, CSF was collected from the cisterna magna using a borosilicate glass pipette with an internal filament. The sample was then centrifuged at 1000 g for 15 minutes. The supernatant was carefully transferred to a collection tube and stored at − 80°C. Subsequently, the deep cervical lymph nodes (dCLNs) preserved in 4% paraformaldehyde (PFA) were removed. The mice were then transcardially perfused with ice-cold phosphate-buffered saline (PBS). Following the removal of the skin and muscle from the head, the hippocampi were collected and stored at − 80°C until further processing. The brains and skullcaps were kept in 4% PFA for an additional 24 hours. The fixed meninges, including the dura mater and arachnoid, were carefully dissected from the skullcaps using a stereomicroscope. Fixed brains and dCLNs were subsequently subjected to gradient dehydration using 20% and 30% sucrose solutions and embedded in tissue-plus OCT compound (Sakura, USA; Cat# 4583). The brain segments encompassing the hippocampus and adjacent cerebral cortex were sliced into coronal sections at a thickness of 20 µm, while the frozen lymph nodes were sectioned into 10 µm slices using a cryostat (Leica, CM1950, Germany). The sections were then transferred to cryoprotectant (50% glycerol, 50% 0.1 M PBS, pH 7.4) and stored at − 20°C until needed. SVEC4-10 cells were fixed with 4% PFA for 15 minutes at room temperature, washed with PBS, and subsequently stored at 4°C until further staining was to be performed. Immunofluorescence Frozen brain sections, lymph node slices, meningeal whole mounts, SVEC4-10 cells, and primary astrocytes on glass coverslips were blocked and permeabilized for one hour at room temperature using a block/stain buffer (PBS with 0.3% Triton X-100 and 10% bovine serum). The sections were then incubated with primary antibodies (Supplementary Table 1) in block/stain buffer overnight at 4°C, washed three times in PBS, and incubated with secondary antibodies (at a 1:1000 dilution) for 2 hours at room temperature. After being washed in PBS and incubated with 4',6-diamidino-2-phenylindole dihydrochloride (DAPI) at a concentration of 1 µg/mL in PBS, the sections were coverslipped until images were acquired using either a wide-field microscope (DM4000B, Leica) or a confocal microscope (Zeiss LSM710, Germany). Thioflavin-S staining Frozen brain sections were stained with 1% Thioflavin-S (Sigma, St. Louis, USA; Cat# 1326-12-1) for 5 minutes. After rinsing with distilled water, they were differentiated with 70% alcohol for 1 minute. The sections were then washed with PBS and mounted with glass coverslips. Western blot Total protein was extracted from siRNA-transfected or oligomer Aβ 1− 42 -treated primary astrocytes, and mouse hippocampus using lysis buffer (25 mM Tris pH 7.4, 150 mM NaCl, 1 mM CaCl 2 , 1 mM MgCl 2 , 0.5% NP-40, and protease inhibitors). Briefly, after washing the cultured cells twice with ice-cold PBS, the cells or tissues were incubated on ice in lysis buffer for 10 minutes. The lysates were then centrifuged at 13,000 g at 4°C for 20 minutes, and the supernatant was collected. Protein concentration was estimated using the BCA protein assay (Beyotime Biotechnology) for all protein samples. Thirty micrograms of protein samples were mixed with 6× loading buffer (Thermo Scientific) and boiled at 95°C for 5 minutes. The samples were loaded onto 8–15% gradient gels and transferred to PVDF membranes, which were then blocked with 5% non-fat milk for 1 hour. The membranes were subsequently incubated with primary antibodies (Supplementary Table 1) overnight at 4°C. After washing the membranes three times with TBST, they were incubated with secondary antibodies for 1 hour at room temperature. Finally, all bands were washed three times with TBST and imaged using an imaging system (ImageQuant™ LAS 4000 mini, version 1.2). ELISA The samples of CSF, hippocampus, pellet and supernatant of primary astrocytes were diluted appropriately in a dilution buffer in the 96-well ELISA plate to analyze TSP-1 or EAF2 levels using ELISA commercial kits (Jingmei Co., Ltd. Cat# JM-04106H1, JM-13432M2) following the manufacturer’s instructions. RNA extraction and qRT-PCR Total RNA was extracted from brain or cell samples using Trizol reagent (TaKaRa, Japan), and cDNA was generated using a reverse transcriptase kit (Vazyme Biotech Co., Ltd., Cat# R323) following the manufacturer’s instructions. Relative qRT-PCR was performed on an ABI 7300 Fast Real-Time PCR System (Applied Biosystems, Foster City, CA, USA), with qRT-PCR SYBR master mix (Vazyme Biotech Co., Ltd. Cat# Q712). The 2 −ΔΔCt method was used to calculate mRNA levels as in previous reports [ 10 ]. GAPDH was used as an internal control. The primers used for analysis are listed in Supplementary Table 2. Tube formation assay A tube formation assay was performed to assess the effects of recombinant TSP-1 on the tube formation of LECs lines of murine and human species in vitro . A coating of 10 µL Matrigel (Coring, Cat# 354243) was applied to the inner walls of 15-well Ibidi µ-slides (Ibidi, Martinsried, Germany; Cat# 81506) and allowed to polymerize for one hour at 37°C to form a gel-like surface. The SVEC4-10 or HLEC in complete culture media (DMEM, Gibco) with 10% FBS, 100 U/mL penicillin/streptomycin, 1X endothelial cell growth supplement containing exogenous recombinant TSP-1 (R&D Systems, USA; Cat# 3074-TH-050), Aβ 1−42 (Nanjing Peptide Biotech Ltd. China; Cat# 107761-42-2) or CD36 blocking peptide (Fab Gennix, USA; Cat# P-CD36) were seeded, then plated (1×10 5 cells/well) into the duplicate and incubated for 4 hours at 37°C in 5% CO 2 . Images were captured and the numbers of nodes, sprouts and total tube length were measured and quantified using Image J (NIH, Bethesda, MD, USA). RNA sequencing data analysis Publicly available RNA sequencing (RNA-seq) data for meningeal lymphatic endothelial cells from 6-month-old 5×FAD male mice and WT mice were obtained from the Gene Expression Omnibus (GEO) database under the accession number GSE245658. Additional bulk RNA-seq data from three different time points (4, 8, and 18 months) in the hippocampus of 5×FAD mice and control mice were sourced from the database with the accession number GSE168137. The count data were utilized for the quantification of gene expression. Normalization and differential expression analysis were performed using the DESeq2 package (version 1.32.0) in R (version 4.3.1). Genes with an adjusted p-value less than 0.05 were considered differentially expressed. Principal component analysis (PCA) and hierarchical clustering were conducted to evaluate the overall variance and sample clustering. A specific list of genes, obtained from literature screening, was analyzed to ascertain differential expression changes across various groups. The expression levels of these genes were extracted and normalized. The gene expression levels in the histogram were normalized using the FPKM value, subtracted by the mean and divided by the standard deviation. Differential expression analysis was conducted for each gene in the list across different groups. Quantification and statistical analysis Quantification is detailed in the methods and figure legends. Experimenters were blinded to the identity of experimental groups from the time of euthanasia until the end of data collection. An unpaired Student's t-test was used to compare differences between the two groups. A one-way ANOVA with a Tukey post hoc test was employed to compare three independent groups. For the comparison of multiple factors (e.g., genotype versus treatment), a two-way ANOVA with a Tukey post hoc test was utilized. A repeated-measures two-way ANOVA with a Tukey post hoc test was applied for repeated observations of multiple factors. Statistical analysis (data are consistently presented as mean ± S.E.M.) was conducted using R (version 4.3.1) and Prism 8.0 (GraphPad Software, Inc.). Results Impaired meningeal lymphatic vessels in 5×FAD mice Recent studies have revealed that meningeal lymphatic vessels drain brain macromolecule metabolites into the peripheral system, which was impaired in both aged mice and AD mouse models [ 10 , 13 , 14 ]. Consistently, the present results showed significant decreases in the coverage of lymphatic vascular endothelial hyaluronan receptor 1 (LYVE-1) and prospero homeobox protein 1 (PROX1) positive lymphatic vessels along the transverse sinus (TS) of 6.5-month-old 5×FAD mice, compared with age-matched WT mice (Fig. 1 a-b, d-f). Additionally, under normal physiological conditions, the TS lymphatic vessels consist mostly of a zipper-like junctional pattern of LECs, and insufficient continuous zipper connections are related to impaired lymphatic flow [ 15 , 28 ]. The results indicated that in TS lymphatic vessels of 5×FAD mice, there was a decrease in tight zipper-like LEC junctions and an increase in button-like LEC junctions compared to those in WT mice (Fig. 1 c, g-h). Quantitative analyses also showed a reduction in the diameter and number of sprouts of TS lymphatic vessels in 5×FAD mice, further indicating impaired lymphangiogenesis (Fig. 1 i-j). Activated astrocytes with increased production of TSP-1 in 5×FAD mice We investigated the underlying mechanism of impaired meningeal lymphangiogenesis in 5×FAD mice. Initially, we conducted an RNA-seq analysis of LECs sorted from the meninges of 6-month-old WT and 5×FAD mice (GSE245658). Notably, the classical factors regulating lymphangiogenesis, such as Vegfc and Vegfd , were not significantly different in meningeal LECs between WT and 5×FAD mice (Fig. S1 a-c). This suggested that the soluble cytokines regulating meningeal lymphangiogenesis are not produced by LECs but may originate from brain cells. Consequently, we further evaluated published RNA-sequence data from the hippocampus of WT and 5×FAD mice at 4, 8, and 18 months of age (GSE168137) and screened the literature for factors that regulate lymphangiogenesis. We discovered that Thbs1 , the gene encoding the protein TSP-1, was age-dependently elevated in the 5×FAD group (Fig. 2 a-b, Fig. S2 ). TSP-1, an endogenous inhibitor of lymphangiogenesis, plays a crucial role in tumor metastasis and transplant outcome [ 29 ]. Notably, TSP-1 is a secreted protein primarily produced by astrocytes in the CNS, particularly up-regulated in activated astrocytes [ 30 , 31 ]. Consistently, double immunofluorescence revealed that astrocytes surrounding the plaques were activated with an upregulation of TSP-1 expression in the hippocampus of 6.5-month-old 5×FAD mice (Fig. 2 c and e, Fig. S3 ). High concentrations of TSP-1 were found in the hippocampus and CSF of 5×FAD mice, as shown by ELISA (Fig. 2 f and g). AD mice also exhibited high mRNA levels of TSP-1 receptors CD36 and CD74 in the meningeal lymphatic endothelial cells, compared with WT mice (Fig. 2 i). Furthermore, when treated with Aβ 1−42 in vitro , the expression of TSP-1 increased in primary astrocytes in a dose-dependent manner (Fig. 2 d and h). Collectively, these data suggest that under Aβ stimulation, astrocytes are activated and enhance the production of TSP-1. Astrocyte-specific knockdown of TSP-1 enhanced meningeal lymphatic vessel plasticity and drainage in 5×FAD mice To investigate the contribution of astrocyte-derived TSP-1 to impaired meningeal lymphatic drainage under AD-like pathology, we utilized an AAV-Thbs1-shRNA-mCherry construct (controlled by the GfaABC1D promoter) to selectively knock down TSP-1 expression in the hippocampal astrocytes of 5×FAD mice (Fig. 3 a). We confirmed reduced TSP-1 levels at the hippocampal injection site and in the CSF of 5×FAD mice, demonstrating the efficacy of the Thbs1 -shRNA virus (Fig. 3 b and Fig. S4 a, c). Astrocyte-specific knockdown of TSP-1 also resulted in an increased diameter of LYVE-1 + vessels and continuous zipper-like patterns of LECs in the meningeal tissue (Fig. 3 c-d and g-h), as well as a reduction in the accumulation of Aβ plaques and senescent astrocytes, along with decreased glial activation in the brain parenchyma of 5×FAD mice (Fig. 3 e-f, i-k, Fig. S4 b, d-e). Furthermore, immunofluorescence analyses employing monoclonal antibodies against glial fibrillary acidic protein (GFAP) and rabbit IgG antibodies specific for human Aβ revealed the presence of non-tissue self-produced antigens or non-specific markers in the dCLNs. This supports the notion that these macromolecules are cleared from the brain to the peripheral lymph system [ 32 ]. Elevated Aβ and GFAP signals were observed in the dCLNs of 5×FAD mice following hippocampal astrocyte-specific knockdown of TSP-1 (Fig. S5 a-c). As anticipated, the astrocyte-specific knockdown of TSP-1 in 5×FAD mice notably increased the number of entries into the novel arm during the Y-maze test, yet it did not influence behavioral performance in the NOR test (Fig. S6 a-d), suggesting a partial mitigation of cognitive impairment. These findings indicate that the astrocyte-specific knockdown of TSP-1 exerts a therapeutic effect on AD model mice by enhancing meningeal lymphatic plasticity and drainage. TSP-1 aggravated the inhibitory role of Aβ on lymphatic vessel formation and plasticity To confirm the involvement of TSP-1 in lymphatic vessel formation and plasticity, a tube formation assay was initially performed using SVEC4-10 cells, a cell line of LECs [ 33 ]. SVEC4-10 cells highly expressed lymphatic endothelial markers such as LYVE1, PROX1, and vascular endothelial growth factor receptor 3 (VEGFR3), while they exhibited low expression of vascular endothelial cell markers, including CD31, CD34, and FLI-1, compared with an endothelial cell line of HAECs (Fig. S7a-d). The results indicated that treatment with 200 ng/mL TSP-1 led to reductions in the number of nodes, sprouts, and total tube length in SVEC4-10 cells, demonstrating that TSP-1 inhibited lymphangiogenesis in vitro (Fig. S8a-b and e). Additionally, previous studies have suggested that meningeal Aβ deposition may impact LEC plasticity [ 13 ]. In the lymphangiogenesis assay using the tube formation assay of SVEC4-10 cells, no significant changes were observed at lower concentrations of Aβ 1−42 treatment (0, 0.5, and 2.5 µM). However, the number of nodes, sprouts, and total tube length was notably decreased after treatment with 5 µM Aβ 1−42 (Fig. S8c and f). We further investigated whether the combination of lower concentrations of Aβ 1−42 and TSP-1 had a synergistic inhibitory effect on lymphangiogenesis. The results demonstrated that treatment with 2 µM Aβ 1−42 and 100 ng/mL TSP-1 together significantly suppressed tubule formation of the SVEC4-10 cells (Fig. S8d and g). To further investigate how TSP-1 facilitates lymphatic vessel formation and plasticity, we conducted a tube formation assay and visualized lymphatic vessel junction patterns on SVEC4-10 cells. The results indicated that TSP-1 regulates lymphangiogenesis and the junctions of lymphatic vessels through interactions with its various receptors, such as CD36 and CD47. Pretreatment with a CD36-blocking peptide diminished the inhibitory effect of TSP-1 on the lymphangiogenesis of SVEC4-10 cells, as demonstrated by a tube formation assay (Fig. 4 a-e). On the other hand, TSP-1 dose-dependently suppressed VE-Cadherin-formed zipper-like junctions (Fig. S9a, b). In addition, the inhibitory effect of TSP-1 on tube-forming and VE-Cadherin-positive continuous junctions was also confirmed in a human-derived lymphatic endothelial cell line on HLEC (Fig. S9c-g). Furthermore, the inhibitory effect of TSP-1 at a concentration of 2 µg/mL on these zipper-like junctions was nullified after the knockdown of CD47 on SVEC4-10 cells (Fig. S9h-j and Fig. 4 f-i). In summary, these in vitro data suggest that TSP-1 inhibits lymphangiogenesis and junction plasticity through interactions with CD36 and CD47, respectively. Treadmill exercise reduced brain Aβ load and related pathophysiological changes Brain Aβ deposition is a typical hallmark in AD transgenic mice [ 34 ]. We assessed whether treadmill exercise could attenuate Aβ-related brain pathology and cognitive dysfunction in 5×FAD mice (Fig. 5 a). After one month of treadmill exercise, Thioflavin-S positive plaques, reactive gliosis, and accumulation of senescent astrocytes were significantly reduced in the hippocampus and adjacent cortical region of 6.5-month-old 5×FAD mice (Fig. S10a-g). Treadmill exercise training also had a beneficial effect on the short-term learning and memory of 5×FAD mice (Fig. S11a-d). However, exercise intervention did not ameliorate the anxiety-like behavior of 5×FAD mice in the EPM test (Fig. S11e-f). Treadmill exercise increased meningeal lymphatic plasticity and drainage in 5×FAD mice We determined whether long-term exercise reduced brain Aβ load is associated with enhanced meningeal lymphatic plasticity. As expected, 6.5-month-old 5×FAD mice that received treadmill exercise for one month showed a marked increase in the area fraction, diameter, and number of sprouts of meningeal lymphatic vessels, as well as continuous lymphatic junctions (Fig. 5 b-h). Furthermore, compared with the sedentary control group, more Aβ and GFAP-positive products were detected in the dCLNs of 5×FAD mice that underwent treadmill exercise training, indicating that exercise facilitated the drainage of brain Aβ and GFAP from the brain to the peripheral system (Fig. 5 i, k-l). These results together revealed that treadmill exercise improves meningeal lymphatic vessel plasticity and drainage under AD-like pathology. To further confirm this conclusion, in vivo two-photon imaging was used to monitor the dynamic distribution of fluorescent-labeled Aβ 1−42 at 24 hours post-intrahippocampal administration in the meningeal lymphatic vessels of 6-month-old WT and 5×FAD mice that had undergone 10 days of treadmill exercise training (Fig. S12a). As previously reported [ 35 ], Aβ 1−42 fluorescent signals were detected in the A488-Lyve1-labeled meningeal lymphatic vessels running along the TS. Notably, a comparison of Aβ 1−42 -555 drainage through the TS regions at consecutive time points revealed a delayed clearance of the Aβ tracer in 5×FAD mice. The treadmill exercise significantly enhanced the drainage of Aβ 1−42 -555 from the meningeal lymphatic vessels in WT mice, and there was a trend toward improvement in AD mice as well (Fig. S12b-c and Supplementary Movies 1–4). Consistently, there were increased Aβ 1−42 -555 signals in the dCLNs of 5×FAD mice after treadmill exercise (Fig. S12d-e). These results verified that exercise promoted the meningeal lymphatic drainage of Aβ from the brain to the peripheral system. Treadmill exercise down-regulated TSP-1 expression in astrocytes of 5×FAD mice We examined the expression level of TSP-1 in the hippocampus of 5×FAD mice, with or without exercise, and found that the upregulated expression of TSP-1 in GFAP-positive astrocytes was reversed by treadmill exercise (Fig. 5 j and m). ELISA analysis of the hippocampus confirmed this conclusion (Fig. 5 n). The ELISA results also indicated a significant decrease in TSP-1 levels in the CSF after treadmill exercise in 5×FAD mice (Fig. 5 o). Consistently, CD36, a receptor for TSP-1, was highly expressed in meningeal lymphatic vessels, which were partially normalized by exercise treatment in 5×FAD mice (Fig. S13a-b). Besides, the exchange of CSF and interstitial fluid (ISF) mediated by AQP4 is responsible for the clearance of harmful metabolites from the brain [ 36 ]. We observed that, compared with WT littermates, the perivascular localization of astrocytic AQP4 was impaired in the hippocampus of 5×FAD mice, a condition that was reversed by treadmill exercise (Fig. S14a-c). This suggests that treadmill exercise also facilitates AQP4-mediated glymphatic clearance of Aβ. However, the expression levels of APP and its secretases, as well as proteins involved in the transport or degradation of Aβ, were not significantly altered in the hippocampus of WT mice and 5×FAD mice following exercise training (Fig. S15a and b). Collectively, these data suggest that long-term exercise enhances the glymphatic-meningeal lymphatic transport of Aβ, which in turn inhibits the pathological cascades of parenchymal Aβ accumulation, astrocyte activation, TSP1 secretion, and meningeal lymphatic dysfunction, thereby improving the cognitive function of 5×FAD mice. EAF2 regulating TSP-1 expression in activated astrocytes exposed to Aβ To further explore how exercise reduces TSP-1 expression, we screened the upstream transcription factors (TFs) of TSP-1 in the hippocampus of WT mice and 5×FAD mice following exercise training, which brought p53 into our focus (Fig. 6 a and b). The regulation of TSP-1 by p53 varies across different tissues and cells [ 37 – 39 ]. For instance, the downregulation of TSP-1 has been shown to increase angiogenesis in the liver of EAF2 knockout mice. However, the transfection of EAF2 alone had minimal impact on the TSP-1 promoter [ 40 ]. Functional protein association networks analysis indicated that EAF2 has a direct regulatory relationship with p53, but not with TSP-1 (Fig. 6 c). Additional evidence was provided by primary astrocytes exposed to varying concentrations of Aβ 1−42 for 48 hours, which demonstrated that the levels of p53, EAF2, and TSP-1 were all elevated at a concentration of 10 µM (Fig. 6 d-g). Furthermore, increased TSP-1 levels in astrocyte cells and their culture medium upon exposure to Aβ 1−42 were reversed by Eaf2 knockdown (Fig. 6 h-l). Consequently, we hypothesized that EAF2 plays a role in meningeal lymphangiogenesis by modulating TSP-1 levels through its interaction with p53. As anticipated, there were elevated mRNA levels of p53 in the hippocampus of 5×FAD mice (Fig. 6 b). Additionally, we observed that EAF2 was heavily co-localized with activated astrocytes and that there was an increase in EAF2 expression in the hippocampus of 5×FAD mice compared to WT mice (Fig. 6 m-n). Collectively, these findings suggest that Aβ triggers the activation of the EAF2-p53-TSP-1 pathway in astrocytes. Astrocyte-specific knockdown of EAF2 improved meningeal lymphatic drainage and AD-like pathology in 5×FAD mice We next investigated whether the drainage of meningeal lymphatic vessels could be enhanced through selective knockdown of astrocyte EAF2. Five-month-old 5×FAD mice were administered an AAV-Eaf2-shRNA-EGFP construct (controlled by the GfaABC1D promoter) to selectively knock down EAF2 in hippocampal astrocytes (Fig. 7 a and Fig. S16a-b). Four weeks later, EAF2 levels also significantly decreased in the CSF of 5×FAD mice (Fig. 7 b). These 5×FAD mice, with selective knockdown of the Eaf2 gene specifically in astrocytes, exhibited improved behavioral performance in the NOR test and Y-maze test (Fig. S17a-d). Furthermore, the hippocampal level of TSP-1 decreased after EAF2 knockdown in astrocytes of 5×FAD mice (Fig. 7 c-e). Additionally, down-regulating EAF2 in astrocytes significantly increased the coverage and diameter of LYVE1 + lymphatic vessels and the continuity of the zipper-like patterns of meningeal LECs (Fig. 7 f-h and k). This transformation of LEC junction patterns may facilitate Aβ drainage, as evidenced by reductions in Aβ plaques, reactive gliosis, and astrocyte senescence in the hippocampus (Fig. 7 i-j, l-o). In line with these findings, Aβ and GFAP levels were observed to be higher in the dCLNs compared to control groups (Fig. S18a-c). Collectively, these outcomes suggest that EAF2, derived from activated astrocytes, plays an inhibitory role in meningeal lymphangiogenesis during AD-like pathology. Discussion Impaired meningeal lymphatic drainage was accompanied by a decrease in macromolecules draining into lymph nodes, as well as cognitive decline in aged or AD mice [ 13 , 15 ]. Considering the plasticity of meningeal lymphatic vessels, overexpression of VEGFC-induced meningeal lymphangiogenesis [ 16 ]. Consistently, our previous data also showed a significant increase in the drainage of GFAP into the dCLNs in aged mice after AAV1-CMV-mVEGFC treatment [ 10 ]. In the current study, we observed that classically regulated lymphatic vessel molecules such as VEGFC in the meninges are unchanged, but there is significant upregulation of TSP-1, a secreted protein associated with lymphangiogenesis, in the hippocampus of 6.5-month-old AD mice, compared with WT mice. The identification of clinical biomarkers for meningeal lymphatic injury is essential for early detection and monitoring of neurodegenerative diseases. Advanced neuroimaging techniques, such as dynamic contrast-enhanced magnetic resonance imaging (MRI) and positron emission tomography (PET), could provide valuable insights into changes in cerebrospinal fluid flow and meningeal lymphatic function [ 15 , 41 , 42 ]. Additionally, CSF biomarkers, encompassing proteins such as Aβ, tau, and GFAP, as well as lymphatic markers like podoplanin or LYVE-1, may indicate impaired lymphatic drainage [ 11 , 43 ]. Beyond these markers, neuroinflammatory cytokines and matrix metalloproteinases (MMPs), which play roles in blood-brain barrier disruption and lymphatic vessel remodeling, could offer additional evidence of lymphatic dysfunction [ 44 , 45 ]. Collectively, these biomarkers could improve our capacity to detect and monitor meningeal lymphatic injury in clinical settings. However, this hypothesis requires more evidence. Previous studies have shown that TSP-1 plays a role in peripheral tumor lymphatic metastasis and corneal lymphatic vessel remodeling [ 29 , 46 ]. However, the expression level of TSP-1 in CNS lymphangiogenesis has seldom been investigated. Buee et al. (1992) reported that the distribution of TSPs staining in the brains of AD patients was comparable to that in control subjects [ 47 ]. In contrast, Son et al. (2015) found evidence of downregulation of TSP-1 in cortical samples from individuals with AD [ 48 ]. Such inconsistent findings might be attributed to the heterogeneity of astrocyte distribution and the variable disease processes involved in AD. TSP-1 also acts as a promoter of aging and age-associated diseases. The accumulation of TSP-1 in the extracellular matrix is frequently observed in age-related diseases [ 49 ]. In this study, we found that treadmill exercise reduced the expression level of TSP-1 in the brain parenchyma of AD mice following treadmill exercise. Similarly, RNA sequencing analysis (refer to GSE164401) also observed that compared with control plasma-treated mice, hippocampal transcriptome levels of Thbs1 had a decreasing trend in recipient mice injected with the donor plasma from exercising mice. Altogether, these data highlighted TSP-1 as a potential target for exercise interventions that alleviate AD pathology by increasing meningeal lymphangiogenesis. CD36 acts as a receptor for TSP-1 and serves as a negative regulator of angiogenesis and lymphangiogenesis [ 50 ]. In our current study, we have further demonstrated that TSP-1 can effectively suppress the proliferation of meningeal lymphatic vessels in vitro by binding to the CD36 receptor on LECs. CD36 has been implicated in maintaining lymphatic vessel integrity, which is associated with obesity and type 2 diabetes models [ 51 ]. The deletion of CD36 has been shown to protect cerebral arteries from the harmful effects of Aβ 1−40 , thereby enhancing the cognitive performance of AD model mice [ 52 ]. CD47 is another receptor for TSP-1. The TSP-1-CD47 interaction has been reported to modulate apoptosis in meningeal lymphatic endothelial cells within a subarachnoid hemorrhage model [ 53 ]. Elevated levels of TSP-1 have been found to impede lymphangiogenesis by activating CD47 in aortic LECs of a mouse model of atherosclerosis [ 54 ]. Our findings further indicate that TSP-1-CD47 regulates the junctional pattern of meningeal lymphatic vessels. EAF2 is preferentially expressed in the CNS during mouse embryonic development [ 55 ]. Overexpression of EAF2 has been shown to induce apoptosis and inhibit cell growth in several peripheral studies [ 56 , 57 ]. However, its role in the CNS has been scarcely reported. Our data indicates that EAF2 expression is upregulated in activated astrocytes of 5×FAD mice. Exercise was found to down-regulate EAF2 and its binding partner p53 in 5×FAD mice, thereby promoting the inhibition of the EAF2-p53 complex on TSP-1. We also demonstrated that knocking down EAF2 in specific astrocytes reduced TSP-1 expression in the brain, which in turn improved meningeal lymphatic vessel function in AD mice. Collectively, these findings underscore that the EAF2-p53-TSP-1 pathway plays a crucial role in regulating meningeal lymphatic plasticity. Epidemiological studies have reported that treadmill exercise is a generally applicable form of physical therapy, playing a role in delaying the aging process and improving cognitive function in patients with AD or mild cognitive impairment [ 58 , 59 ]. However, there is also literature that challenges this view [ 60 , 61 ]. Several studies have indicated that treadmill exercise has the potential to reduce Aβ load in both AD patients and transgenic AD mice [ 62 , 63 ]. Here, we confirmed that treadmill exercise decreased the parenchymal Aβ deposition in 6.5-month-old 5×FAD mice. Accumulating evidence supports the notion that Aβ aggregation and deposition accelerate neuroinflammation and also trigger cellular senescence [ 3 ], particularly, astrocytes are susceptible to senescence within the CNS [ 4 ]. Utilizing pharmacological or genetic models, the elimination of senescent glial cells preserves cognitive function in mouse models of tau-dependent neurodegenerative disease [ 5 ]. This study demonstrated that the enhancement of learning and memory in AD mice through treadmill exercise was accompanied by an increase in the removal of senescent astrocytes from the hippocampus. Furthermore, it has been reported that targeting the clearance of senescent cells, such as cardiomyocytes, pancreatic β cells, and osteocytes, alleviates disease symptoms and exerts beneficial effects in slowing the aging process [ 64 – 66 ]. Our findings further suggest that targeting the elimination of Aβ and senescent astrocytes may offer a therapeutic approach to the benefits of treadmill exercise against AD pathology. Additionally, volunteers underwent assessment of brain waste clearance using noninvasive MRI following either a single session or a 12-week regimen of cycling exercise. The findings indicated that glymphatic influx in the putamen, as well as the size and flow of meningeal lymphatics, significantly increased after the long-term exercise regimen [ 67 ]. This suggests that sustained physical exercise promotes the flow of putative glymphatic and meningeal lymphatic vessels, thereby enhancing the clearance of brain metabolites in humans. This is consistent with the outcomes of our animal studies. Furthermore, by comparing various durations and intensities of exercise, it was determined that there was no significant difference in lymphatic and meningeal lymphatic vessel flow before and after a single exercise session. Studies involving animals have shown an increase in CSF influx in mice after 5 weeks of voluntary running-wheel exercise [ 25 ]. Similarly, human studies have demonstrated increased compliance of the middle cerebral artery in volunteers who reported engaging in moderate-to-vigorous recreational aerobic exercise [ 68 ]. Based on these findings, it is hypothesized that moderate to high-intensity exercise may also significantly contribute to cerebrospinal fluid flow and the clearance of metabolites from the brain. Growing evidence suggests that the glymphatic system acts as a functional pathway for the removal of metabolic waste from the brain's parenchyma [ 36 ]. Voluntary exercise in young, awake mice or aged mice has been shown to enhance glymphatic function, leading to pro-cognitive effects [ 24 , 25 ]. Furthermore, our previous study indicated that the improvement of cognitive deficits in APP/PS1 mice through voluntary exercise is dependent on the polarity of astrocytic AQP4 [ 26 ]. In the present study, we confirmed that treadmill exercise significantly reduced the activation of astrocytes and improved AQP4 polarity in the hippocampus of 5×FAD mice. These findings suggest that the glymphatic system, which is responsible for the clearance of Aβ, could be an important target for the preventive and therapeutic effects of treadmill exercise on AD. Furthermore, the meningeal lymphatic system serves as a crucial pathway for the elimination of metabolites and brain antigens from the CSF. Studies have demonstrated that improving meningeal lymphatic function can positively influence learning and memory performance in both aged and AD model mice [ 13 , 14 ]. Conversely, inhibiting lymphatic drainage in aged mice has been found to worsen the accumulation of perivascular senescent astrocytes within the brain parenchyma and exacerbate cognitive behavioral deficits [ 10 ]. Additionally, previous research has shown that cerebral blood flow and CSF flow dynamics significantly increase during exercise in both human and rodent studies [ 25 , 69 ]. In our study, the results indicated that treadmill exercise expanded the diameter and increased the number of sprouts and the continuity of VE-Cadherin junctions in the meningeal lymphatic vessels of AD mice, thereby enhancing their drainage function. This approach could potentially be a promising strategy to improve the clearance of Aβ, potentially slowing the progression of AD. It is important to note that we conducted treadmill exercise training specifically at two time points, day and night, on mice to avoid any argument regarding the effects of circadian rhythms on running. We did not ascertain which time point for treadmill exercise would yield the greatest benefit for the mice. Human studies have indicated that morning and evening exercise have distinct effects on the skeletal muscle molecular clock and nocturnal sleep [ 70 , 71 ]. The circadian rhythm impact of treadmill exercise on the enhancement of meningeal lymphatic vessels in humans requires further clarification. Investigating these matters will maximize the benefits of treadmill exercise, particularly for the elderly or individuals with AD. Conclusions The plasticity of meningeal lymphatics has been identified as a significant target for the drainage of metabolites from the brain. We demonstrated that activated astrocyte-derived TSP-1 is crucial for impaired meningeal lymphangiogenesis in 5×FAD transgenic mice. Astrocyte-specific knockdown of Thbs1 or Eaf2 promoted functional meningeal lymphatic vessel plasticity and mitigated AD-like pathology. Our results also suggested that exercise modulated the lymphangiogenesis and junctional patterns of meningeal lymphatic vessels by down-regulating the reactive astrocytes-related EAF2-p53-TSP-1 pathway (Fig. 8 ). This finding revealed a novel mechanism by which exercise improves meningeal lymphatic vessel plasticity, thereby alleviating AD-related pathology. Abbreviations AAV Adeno-associated virus AD Alzheimer’s disease AQP4 aquaporin 4 Aβ amyloid-β CNS central nervous system CSF cerebrospinal fluid DAPI 4',6-diamidino-2-phenylindole dihydrochloride dCLNs deep cervical lymph nodes EAF2 eleven nineteen lysine rich leukemia -associated factor 2 ELISA enzyme-linked immunosorbent assay EPM elevated plus maze GFAP glial fibrillary acidic protein Iba-1 ionized calcium-binding adaptor molecule 1 ISF interstitial fluid LECs lymphatic endothelial cells LYVE-1 lymphatic vascular endothelial hyaluronan receptor 1 NOR novel object recognition PROX1 prospero homeobox protein 1 TFs transcription factors TS transverse sinus TSP-1 thrombospondin-1 VEGFC vascular endothelial growth factor C WT wide type ZT zeitgeber time Declarations Acknowledgments Not applicable. Author contributions Q.L., Y.C., J. C., X. H., H.F., X. L. and W. Y., carried out and analyzed immunostaining and behavior tests. L. Y., Y.F., M. Y., Y. S., and Y.C. carried out biochemistry, in vitro experiments and data analysis. Y. J. performed cartoon diagrams. W. Z. and Y.C. carried out and analyzed in vivo two-photon imaging. M.X., Q.L., F.D., and Y.C. designed the experiments. M.X., Q.L., C. S., J. G., and Y.C. wrote and modified the manuscript. All authors read and approved the final manuscript. Funding This work was supported by grants from the National Natural Science Foundation of China (Grant No. 82204365, 82071199 and 81871117), the Natural Science Foundation of Jiangsu Province (Grant No. BK20230057), Shandong Postdoctoral Science Foundation (Grant No. SDCX-ZG-202400044), Shandong Postdoctoral Innovative Talents Program (Grant No. SDBX2023056) and the Natural Science Foundation of the Jiangsu Higher Education Institutions of China (Grant No. 23KJB310009). Data Availability All supporting information and data are available in the article and supplementary files. 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Circadian rhythms and exercise - re-setting the clock in metabolic disease. Nat Rev Endocrinol. 2019;15:197–206. Supplementary Files RevisedSupplementalinformation.docx RevisedSupplementaryfile.docx SupplementaryMovie1.mp4 SupplementaryMovie2.mp4 SupplementaryMovie3.mp4 Cite Share Download PDF Status: Published Journal Publication published 25 Jul, 2025 Read the published version in Translational Neurodegeneration → Version 1 posted Reviewers agreed at journal 28 Apr, 2025 Reviewers invited by journal 28 Apr, 2025 Editor assigned by journal 27 Apr, 2025 First submitted to journal 25 Apr, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5720097","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":449154427,"identity":"5b1ed59a-6881-4b8c-b8b7-bd9c3b87f7ec","order_by":0,"name":"Yan Chen","email":"","orcid":"","institution":"Nanjing Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yan","middleName":"","lastName":"Chen","suffix":""},{"id":449154428,"identity":"20aa408b-ec28-40e3-a698-2591153f4083","order_by":1,"name":"Jiachen Cai","email":"","orcid":"","institution":"Nanjing Medical 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08:26:09","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5720097/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5720097/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s40035-025-00497-2","type":"published","date":"2025-07-25T15:57:15+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":81731370,"identity":"224410c0-b12f-4ff9-9677-9c8837ae6478","added_by":"auto","created_at":"2025-04-30 19:15:01","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":808864,"visible":true,"origin":"","legend":"\u003cp\u003eImpaired meningeal lymphatic plasticity in 5×FAD mice. \u003cstrong\u003ea-c\u003c/strong\u003e Representative image of LYVE1\u003csup\u003e+ \u003c/sup\u003e(a), PROX1\u003csup\u003e+ \u003c/sup\u003e(b)\u003csup\u003e \u003c/sup\u003eand VE-Cadherin\u003csup\u003e+\u003c/sup\u003e LYVE1\u003csup\u003e+\u003c/sup\u003e (c) meningeal lymphatic vessels in meninges. Scale bar, 800 μm (left) and 50 μm (right) in (a). 50 μm in (b),\u003cstrong\u003e \u003c/strong\u003e100 μm (left) and 20 μm (right) in (c). Arrowheads indicate the dominant junctional pattern. Zipper-like junctions (green arrowheads) were defined as continuous junctions at cell-cell borders of LECs, while button-like junctions (blue arrowheads) were defined as dot-like, discontinuous junctions, roughly parallel linear segments of VE-Cadherin. \u003cstrong\u003ed-e\u003c/strong\u003e Quantification of LYVE1 coverage area on the TS (d) and superior sagittal sinus (SSS) (e) (n = 6 per group). \u003cstrong\u003ef\u003c/strong\u003e Quantification of PROX1\u003csup\u003e+ \u003c/sup\u003ecoverage area on the TS (n = 6 per group).\u003cstrong\u003e g-h\u003c/strong\u003e Cartoon diagram (g) and quantification of VE-Cadherin\u003csup\u003e+\u003c/sup\u003e lymphatic vessels junctions (h). VE-Cadherin immunostaining at least 3.5 μm in length was recognized as zipper junctions, while button-like junctions were defined as a length of 0.5 μm - 3.2 μm and a spacing of 2.9 ± 0.3 μm. \u003cstrong\u003ei-j\u003c/strong\u003e Quantification of the diameters (i) and sprout numbers (j) of lymphatic extensions in adjacent sections of meningeal lymphatics along TS (n = 6 per group; bilateral TS per mouse). The lymphatics on the left and right TS were divided into 10 segments (400 μm each), respectively. Data represent the mean ± SEM; significance was evaluated with unpaired Student’s t-test (\u003cstrong\u003ed-f\u003c/strong\u003e) or one-way ANOVA with Tukey post hoc test (\u003cstrong\u003eh\u003c/strong\u003e) or repeated-measures two-way ANOVA with Tukey post hoc test (\u003cstrong\u003ei-j\u003c/strong\u003e). *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-5720097/v1/30d938380cc088cda82474fc.png"},{"id":81731365,"identity":"f6fb9b17-a203-4455-92dc-ad30fae34eea","added_by":"auto","created_at":"2025-04-30 19:15:01","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":740327,"visible":true,"origin":"","legend":"\u003cp\u003eIncreased TSP-1 levels in the hippocampus of 5×FAD mice and cultured astrocytes exposed to Aβ\u003csub\u003e1-42\u003c/sub\u003e. \u003cstrong\u003ea\u003c/strong\u003e Heat map showing the relative expression level of genes associated with lymphangiogenesis in the hippocampus. \u003cstrong\u003eb\u003c/strong\u003e The line graph of the log2 fold change (5×FAD vs. WT) of \u003cem\u003eThbs1\u003c/em\u003e expression in the hippocampus at three different ages. \u003cstrong\u003ec\u003c/strong\u003e,\u003cstrong\u003e e\u003c/strong\u003e Representative images of GFAP, 6E10 and TSP-1 staining and quantification of the intensity of TSP-1 in the hippocampus (n = 6 per group). Scale bar, 20 μm (top) and 50 μm (bottom). \u003cstrong\u003ed\u003c/strong\u003e,\u003cstrong\u003e h\u003c/strong\u003e Representative images of astrocytes (labeled with GFAP and S100β) and TSP-1 staining and quantification of the fluorescence intensity of TSP-1 in primary astrocytes (n = 12 per group). Scale bar, 20 μm. \u003cstrong\u003ef-g\u003c/strong\u003e ELISA assay for TSP-1 levels from the hippocampus (f) (n = 6 per group) and CSF samples (g) (2 mouse CSF were pooled per sample, 6 samples per group). \u003cstrong\u003ei\u003c/strong\u003e Relative mRNA levels of \u003cem\u003eCD36\u003c/em\u003e and \u003cem\u003eCD47\u003c/em\u003e in meninges (n = 6 per group). Data represent the mean ± SEM; significance was evaluated with unpaired Student’s t-test (\u003cstrong\u003ee-g\u003c/strong\u003e,\u003cstrong\u003e i\u003c/strong\u003e) or one-way ANOVA with Tukey post hoc test (\u003cstrong\u003eh\u003c/strong\u003e). *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-5720097/v1/6a2a6adc4253fb5ca5d7d362.png"},{"id":81731366,"identity":"0ce76b16-ada6-4f7d-ace2-d4d54f1c3c10","added_by":"auto","created_at":"2025-04-30 19:15:01","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1152560,"visible":true,"origin":"","legend":"\u003cp\u003eAstrocyte-specific Thbs1 knockdown enhanced meningeal lymphatic vessel plasticity and alleviated accumulation of Aβ and senescent astrocytes in the hippocampus of 5×FAD mice. \u003cstrong\u003ea\u003c/strong\u003e Schematic of 5-month-old 5×FAD mice treatment with astrocyte-specific knockdown of Thbs1, including an image of the injection site of the hippocampus. Scale bar, 500 μm. \u003cstrong\u003eb\u003c/strong\u003e ELISA assay for TSP-1 levels from the CSF sample of mice (2 mouse CSF were pooled per sample, 4 samples per group). \u003cstrong\u003ec-d\u003c/strong\u003e,\u003cstrong\u003e g-h\u003c/strong\u003e Representative image of LYVE1 and VE-Cadherin staining (d) and quantification of VE-Cadherin\u003csup\u003e+\u003c/sup\u003e lymphatic vessel junctions (c), LYVE1\u003csup\u003e+\u003c/sup\u003e area and diameter of LYVE1\u003csup\u003e+\u003c/sup\u003e vessels (h) among TS region (g) (n = 6 per group). Scale bar, 40 μm (top) and 20 μm (bottom). Arrowheads indicate the dominant junctional pattern, zipper junctions (green arrowheads) and button junctions (blue arrowheads). \u003cstrong\u003ee\u003c/strong\u003e,\u003cstrong\u003e i-j\u003c/strong\u003e Representative images of GFAP\u003csup\u003e+\u003c/sup\u003e senescent astrocytes (white arrowheads) characterized by higher expressing p16 (e) and quantification of GFAP\u003csup\u003e+\u003c/sup\u003e area (i) and p16\u003csup\u003e+\u003c/sup\u003e GFAP\u003csup\u003e+\u003c/sup\u003e astrocytes in the hippocampal lacunosum moleculare layer (LMol) (j) (n = 6 per group). Scale bar, 30 μm. \u003cstrong\u003ef\u003c/strong\u003e,\u003cstrong\u003e k\u003c/strong\u003e Representative images of Thioflavin-S staining and quantification of the positive area of Aβ plaques in the hippocampus and its surrounding cortical area (n = 6 per group). Scale bar, 400 μm (top) and 100 μm (bottom). Data represent the mean ± SEM; significance was evaluated with two-way ANOVA with Tukey post hoc test (\u003cstrong\u003ec\u003c/strong\u003e, \u003cstrong\u003eg-k\u003c/strong\u003e, *p \u0026lt; 0.05, AAV-ctrl-shRNA vs AAV-Thbs1-shRNA, ##p \u0026lt; 0.01, ###p \u0026lt; 0.001, WT vs 5×FAD) or one-way ANOVA with Tukey post hoc test (\u003cstrong\u003eb\u003c/strong\u003e, *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001).\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-5720097/v1/5d87b73dfd3f4da0863d637d.png"},{"id":81731367,"identity":"1de04c95-c64b-4571-8644-4c3a3c680b89","added_by":"auto","created_at":"2025-04-30 19:15:01","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":988450,"visible":true,"origin":"","legend":"\u003cp\u003eTSP-1-CD36/CD47 signaling pathway modulated lymphangiogenesis and junction plasticity \u003cem\u003ein vitro\u003c/em\u003e, respectively. \u003cstrong\u003ea\u003c/strong\u003e Schematic for tube formation in the SVEC4-10 cells pre-treated exogenous recombinant TSP-1 together with CD36 blocking peptide or control peptide. \u003cstrong\u003eb\u003c/strong\u003e Representative images of tube formation assay in the SVEC4-10 cells pre-treated gradient concentrations of exogenous recombinant TSP-1 plus CD36 blocking peptide or control peptide. Scale bar, 100 μm. \u003cstrong\u003ec-e\u003c/strong\u003e Quantification of the numbers of nodes, \u003cu\u003esprouts \u003c/u\u003e\u003cdel\u003ebranch points \u003c/del\u003eand total tube length in random fields (3.1 × 5.6 mm\u003csup\u003e2\u003c/sup\u003e), n = 8 per group. \u003cstrong\u003ef\u003c/strong\u003e Schematic of SVEC4-10 cells pre-transfected with siRNA-CD47 for 36 h followed by treatment with 2 μg/mL recombinant TSP-1. \u003cstrong\u003eg\u003c/strong\u003e Representative images of VE-Cadherin and CD47 staining in the SVEC4-10 cells. Scale bar, 20 μm. \u003cstrong\u003eh-i\u003c/strong\u003e Quantification of the fluorescence intensity of CD47 and percentage of zipper-like junctions (n = 4 per group). Data represent the mean ± SEM; significance was evaluated with one-way ANOVA with Tukey post hoc test. *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-5720097/v1/265c336e803b705e077f158c.png"},{"id":81731601,"identity":"8afdb1da-68ee-47ec-928a-99eba7341ac5","added_by":"auto","created_at":"2025-04-30 19:23:01","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1132151,"visible":true,"origin":"","legend":"\u003cp\u003eTreadmill exercise increased the plasticity of meningeal lymphatic vessels and reduced TSP-1 levels in 5×FAD mice. \u003cstrong\u003ea\u003c/strong\u003e Schematic of 5.5-month-old WT and 5×FAD mice with treadmill training at a 10-degree incline with increasing running speeds (m/min) twice a day for 90 min for 30 days. \u003cstrong\u003eb-c\u003c/strong\u003e Representative images of PROX1\u003csup\u003e+ \u003c/sup\u003eLYVE1\u003csup\u003e+\u003c/sup\u003e (b) and VE-Cadherin\u003csup\u003e\u003cstrong\u003e+ \u003c/strong\u003e\u003c/sup\u003eLYVE1\u003csup\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/sup\u003e (c) meningeal lymphatic vessels in the TS. Scale bar, 800 μm (left) and 50 μm (right) in (b), 100 μm (top) and 20 μm (bottom) in (c). Arrowheads indicate the dominant junctional pattern, zipper-like junctions (green arrowheads) and button-like junctions (blue arrowheads). \u003cstrong\u003ed-f\u003c/strong\u003e Quantification of LYVE1\u003csup\u003e+ \u003c/sup\u003e(d), PROX1\u003csup\u003e+ \u003c/sup\u003e(e) coverage area and VE-Cadherin\u003csup\u003e+\u003c/sup\u003e lymphatic vessels junctions (f) on the TS (n = 6 per group). \u003cstrong\u003eg-h\u003c/strong\u003e Quantification of the diameters (g) and numbers (h) of lymphatic extensions in adjacent sections of meningeal lymphatics along TS (n = 6 per group; bilateral TS per mouse). \u003cstrong\u003ei\u003c/strong\u003e, \u003cstrong\u003ek-l\u003c/strong\u003e Representative images of Aβ and GFAP staining in the dCLNs (i) and quantification of Aβ (k) and GFAP positive area (l) in the dCLNs (n = 6 per group). Scale bar, 200 μm (left) and 50 μm (right). \u003cstrong\u003ej\u003c/strong\u003e,\u003cstrong\u003e m\u003c/strong\u003e Representative images of GFAP and TSP-1 staining (j) and quantification of the intensity of TSP-1 in the hippocampus (m) (n = 6 per group). Scale bar, 50 μm. \u003cstrong\u003en-o\u003c/strong\u003e ELISA assay for TSP-1 levels from the hippocampus (n) and CSF samples (o) (2 mouse CSF were pooled per sample, 6 samples per group). Data represent the mean ± SEM; significance was evaluated with unpaired Student’s t-test (\u003cstrong\u003ed\u003c/strong\u003e-\u003cstrong\u003ee\u003c/strong\u003e,\u003cstrong\u003e k-o\u003c/strong\u003e) or one-way ANOVA with Tukey post hoc test (\u003cstrong\u003ef\u003c/strong\u003e) or repeated-measures two-way ANOVA with Tukey post hoc test (\u003cstrong\u003eg-h\u003c/strong\u003e). *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-5720097/v1/3c70f3a4fb9b957bf479cf67.png"},{"id":81731371,"identity":"2c072827-e83a-4f72-b07e-98342c177e08","added_by":"auto","created_at":"2025-04-30 19:15:01","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":697644,"visible":true,"origin":"","legend":"\u003cp\u003eEAF2 decreased TSP-1 levels through binding to p53. \u003cstrong\u003ea\u003c/strong\u003e Lists show transcription factors (TFs) that regulate Thbs1. \u003cstrong\u003eb\u003c/strong\u003e Relative mRNA levels of upstream regulators factors of TSP-1 (\u003cem\u003ep53\u003c/em\u003e, \u003cem\u003eEgr1\u003c/em\u003e, \u003cem\u003eFosl1\u003c/em\u003e, \u003cem\u003eAtf1\u003c/em\u003e, \u003cem\u003eRunx2,\u003c/em\u003e and \u003cem\u003eId1\u003c/em\u003e) in the hippocampus from 6-month-old WT and 5×FAD mice (n = 5 per group). \u003cstrong\u003ec\u003c/strong\u003e Functional protein association networks among p53, EAF2 and TSP-1. \u003cstrong\u003ed-g\u003c/strong\u003e Representative Western blot bands (d) and densitometry analysis of p53 (e), EAF2 (f) and TSP-1 levels (g) in the pellet of primary astrocytes treated with gradient Aβ\u003csub\u003e1-42\u003c/sub\u003e for 48 h (n = 4 per group). \u003cstrong\u003eh\u003c/strong\u003e Schematic of primary astrocytes treated with gradient Aβ\u003csub\u003e1-42\u003c/sub\u003e for 48 h followed by knocking down of Eaf2. \u003cstrong\u003ei-j\u003c/strong\u003e Representative Western blot bands (i) and densitometry analysis (j) of EAF2 levels in the pellet of primary astrocytes treated with gradient Aβ\u003csub\u003e1-42\u003c/sub\u003e for 48 h followed by knocking down of Eaf2 (n = 4 per group). \u003cstrong\u003ek-l\u003c/strong\u003e ELISA assay for TSP-1 levels from the supernatant (k) and pellet (l) of primary astrocytes treated with gradient Aβ\u003csub\u003e1-42\u003c/sub\u003e for 48 h followed by knocking down of Eaf2 (n = 4 per group). \u003cstrong\u003em\u003c/strong\u003e Representative images of EAF2 and GFAP staining and quantification of the fluorescence intensity of EAF2 in the hippocampus. Scale bar, 20 μm, n = 6 per group. Data represent the mean ± SEM; significance was evaluated with one-way ANOVA with Tukey post hoc test. *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-5720097/v1/210261f40a8134a71503135d.png"},{"id":81732185,"identity":"22e22225-61fc-4536-8565-d88205b882f2","added_by":"auto","created_at":"2025-04-30 19:31:01","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":1167294,"visible":true,"origin":"","legend":"\u003cp\u003eAstrocytes-specific Eaf2 knockdown enhanced meningeal lymphatic vessel plasticity and alleviated accumulation Aβ and senescent astrocytes in the hippocampus of 5×FAD mice. \u003cstrong\u003ea\u003c/strong\u003e Schematic of 5-month-old 5×FAD mice treatment with astrocyte-specific knockdown of Eaf2, including an image of the injection site of the hippocampus. Scale bar, 500 μm..\u003cstrong\u003e b\u003c/strong\u003e ELISA assay for EAF2 levels from the CSF samples of mice (2 mouse CSF were pooled per sample, 4 samples per group). \u003cstrong\u003ec-e\u003c/strong\u003e Representative Western blot bands (d) and densitometry analysis of EAF2 (c) and TSP-1 (e) levels in the hippocampus (n = 6 per group). \u003cstrong\u003ef-h\u003c/strong\u003e,\u003cstrong\u003e k\u003c/strong\u003e Representative image of LYVE1 and VE-Cadherin staining (h) and quantification of VE-Cadherin\u003csup\u003e+\u003c/sup\u003e lymphatic vessels junctions (f), the percentage of LYVE1\u003csup\u003e+\u003c/sup\u003e area (j) and diameter of LYVE1\u003csup\u003e+\u003c/sup\u003e vessels (k) among TS region in meninges. n = 6 per group. Scale bar, 40 μm (top) and 20 μm (bottom). Arrowheads indicate the dominant junctional pattern, zipper junctions (green arrowheads) and button junctions (blue arrowheads). \u003cstrong\u003ei\u003c/strong\u003e Representative images of GFAP\u003csup\u003e+\u003c/sup\u003e senescent astrocytes (white arrowheads) characterized by higher expressing p16 in the hippocampal LMol. Scale bar, 30 μm. \u003cstrong\u003ej\u003c/strong\u003e Representative image of 6E10 and Iba-1 staining in the hippocampus. Scale bar, 30 μm. \u003cstrong\u003el-m\u003c/strong\u003e Quantification of GFAP\u003csup\u003e+\u003c/sup\u003e area and GFAP\u003csup\u003e+ \u003c/sup\u003ep16\u003csup\u003e+\u003c/sup\u003e astrocytes in the LMol (n = 6 per group). \u003cstrong\u003en-o\u003c/strong\u003e Quantification of the percentage of 6E10\u003csup\u003e+\u003c/sup\u003e and Iba-1\u003csup\u003e+\u003c/sup\u003e area in the hippocampus, respectively (n = 6 per group). Data represent the mean ± SEM; significance was evaluated with two-way ANOVA with Tukey post hoc test (\u003cstrong\u003ec\u003c/strong\u003e, \u003cstrong\u003ee\u003c/strong\u003e-\u003cstrong\u003ef\u003c/strong\u003e, \u003cstrong\u003ek-o\u003c/strong\u003e, *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001, AAV-ctrl-shRNA vs AAV-Thbs1-shRNA, ##p \u0026lt; 0.01, ###p \u0026lt; 0.001, WT vs 5×FAD) or one-way ANOVA with Tukey post hoc test (\u003cstrong\u003eb\u003c/strong\u003e, *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001).\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-5720097/v1/e98d4be89e1172e64022ed02.png"},{"id":81731381,"identity":"6f8f0c6a-c63c-4d00-85ab-886d5f417896","added_by":"auto","created_at":"2025-04-30 19:15:01","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":280464,"visible":true,"origin":"","legend":"\u003cp\u003eThe working model of impaired lymphangiogenesis in the AD mouse model was enhanced by long-term exercise. Exercise via the down-regulating EAF2-p53-TSP-1 pathway increases the zipper-like junctions and diameter of meningeal lymphatics, which in turn facilitates drainage of brain Aβ and reduces astrocyte activation and senescence.\u003c/p\u003e","description":"","filename":"floatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-5720097/v1/c6531f0e1f098abb91f52ff0.png"},{"id":88506619,"identity":"b1ad45ad-e4cf-4417-bb89-dfe596dc1dbf","added_by":"auto","created_at":"2025-08-07 07:33:05","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":8342886,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5720097/v1/92abc134-c20e-4ab4-bd36-2cd64b0ee733.pdf"},{"id":81731379,"identity":"7e440397-0f92-4c97-82cc-b11e6c568a1e","added_by":"auto","created_at":"2025-04-30 19:15:01","extension":"docx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":12720790,"visible":true,"origin":"","legend":"","description":"","filename":"RevisedSupplementalinformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-5720097/v1/0d14f881ede4c8b394b165e5.docx"},{"id":81732193,"identity":"cd183557-b8ea-4aba-a8a7-89d0c38de924","added_by":"auto","created_at":"2025-04-30 19:31:01","extension":"docx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":755209,"visible":true,"origin":"","legend":"","description":"","filename":"RevisedSupplementaryfile.docx","url":"https://assets-eu.researchsquare.com/files/rs-5720097/v1/8c3033ef25a245c948227ee4.docx"},{"id":81732187,"identity":"4989e388-1f1c-46a4-aee6-8c598c52af67","added_by":"auto","created_at":"2025-04-30 19:31:01","extension":"mp4","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":4856347,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryMovie1.mp4","url":"https://assets-eu.researchsquare.com/files/rs-5720097/v1/cee2b82767bc860b2ba48b5e.mp4"},{"id":81731391,"identity":"76467875-904a-4621-81d6-7d1d4348e13a","added_by":"auto","created_at":"2025-04-30 19:15:01","extension":"mp4","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":3662014,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryMovie2.mp4","url":"https://assets-eu.researchsquare.com/files/rs-5720097/v1/806e659600ecd1f1b43514e9.mp4"},{"id":81731396,"identity":"a87b83d8-6df6-4b56-b771-220b3d2742fc","added_by":"auto","created_at":"2025-04-30 19:15:01","extension":"mp4","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":4695104,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryMovie3.mp4","url":"https://assets-eu.researchsquare.com/files/rs-5720097/v1/2b4842ae0a469fb4adbe20a8.mp4"}],"financialInterests":"","formattedTitle":"Long-term exercise enhances meningeal lymphatic vessel plasticity and drainage in a mouse model of Alzheimer's disease","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAlzheimer's disease (AD) is one of the most common age-dependent neurodegenerative diseases, posing a serious threat to the health and life of older adults. Although monoclonal antibodies such as Aducanumab and Lecanemab can specifically reduce the deposition of amyloid β (Aβ) plaques, their long-term efficacy and potential complications still present challenges [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. It is known that the imbalance between the production and clearance of Aβ occurs prior to the onset of cognitive impairment, causing excessive aggregation of Aβ and a series of neuropathological cascades [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Notably, a large number of astrocytes are persistently activated and undergo senescence, losing their ability to maintain brain homeostasis and exhibiting the senescence-associated secretory phenotype, which in turn accelerates neurodegeneration [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Therefore, timely and effectively facilitating clearance of Aβ from the brain and preventing astrocyte senescence may be beneficial for delaying or even preventing the onset of AD [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eMeningeal lymphatic vessels have recently been characterized in humans and rodents [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. They mediate the drainage of macromolecular waste [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], cellular debris [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], neurotropic viruses [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], and brain tumor cells [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e] from the brain. Meningeal lymphatic drainage progressively deteriorates during natural aging and in AD [\u003cspan additionalcitationids=\"CR14\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Blocking meningeal lymphatic vessels exacerbates Aβ load and memory deficits in transgenic mouse models of AD [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Therefore, enhancing meningeal lymphatic drainage could be a novel therapeutic target for AD.\u003c/p\u003e \u003cp\u003eNotably, the specialized morphological features of meningeal lymphatic vessels are associated with their drainage functions [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. A continuous, zipper-like vascular endothelial (VE)-Cadherin junction and a discontinuous, button-like junctional pattern in lymphatic endothelial cells (LECs) are associated with distinct modes of cerebrospinal fluid (CSF) macromolecule transport by meningeal lymphatic vessels. Zipper-like junctions form tight, continuous barriers that support directional fluid flow, while button-like junctions are more permissive and facilitate macromolecule uptake. The balance between these junction types is crucial for efficient CSF drainage and is disrupted in aged mice, potentially contributing to impaired brain waste clearance [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Vascular endothelial growth factor C (VEGFC) overexpression induces meningeal lymphangiogenesis [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], whereas inhibitors of lymphangiogenesis, such as pigment epithelium-derived factor, suppress peripheral nasopharyngeal lymphangiogenesis [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Nonetheless, the mechanisms that impair the integrity and plasticity of meningeal lymphatic vessels during the progression of AD remain unclear. Investigating this issue could lead to the discovery of therapeutic targets that enhance the draining function of dural lymphatic vessels, potentially alleviating or delaying Aβ-related neurodegeneration.\u003c/p\u003e \u003cp\u003eExercise intervention is one of the most effective nonpharmacologic therapeutic modalities, exerting beneficial effects on various organs, particularly the brain [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. For example, long-term exercise not only enhances the production of brain-derived neurotrophic factor [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e] and synaptic plasticity [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e] but also diminishes brain oxidative stress [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e] and age-related gliosis [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Additionally, recent evidence suggests that astrocytic aquaporin 4 (AQP4)-mediated glymphatic transport plays a role in the neuroprotective effects of voluntary exercise [\u003cspan additionalcitationids=\"CR25\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Nonetheless, it remains undetermined whether exercise can enhance meningeal lymphatic plasticity during the progression of AD.\u003c/p\u003e \u003cp\u003eHere, we have screened and identified activated astrocyte-derived thrombospondin-1 (TSP-1) as a key inhibitor of meningeal lymphangiogenesis in 5\u0026times;FAD transgenic mice. The hippocampal astrocyte-specific knockdown of Thbs-1 enhanced meningeal lymphatic drainage and alleviated AD-like pathology. Notably, long-term exercise decreased TSP-1 and its transcriptional regulator, eleven-nineteen lysine-rich leukemia-associated factor 2 (EAF2), promoting glymphatic-meningeal lymphatic transport of Aβ, thereby blocking the vicious cycle of parenchymal Aβ accumulation and astrocyte activation and senescence, ultimately improving the cognitive function of 5\u0026times;FAD mice. These results not only uncover new mechanisms of dysfunctional meningeal lymphatic vessels in AD but also highlight new targets for long-term exercise in combating Aβ-related neurodegeneration.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eAnimal care and use\u003c/h2\u003e \u003cp\u003eB6.Cg-Tg (APPSwFlLon, PSEN1*M146L*L286V) 6799Vas/Mmjax (5\u0026times;FAD, strain# 008730) mice were obtained from Jackson Laboratories. The mice, along with their age-matched wild-type (WT) littermates, were housed in controlled ambient temperatures and exposed to a 12-hour light/12-hour dark cycle. They had unlimited access to standard rodent chow and clean water. Each group of mice consisted of an equal number of males and females. All animal experiments were conducted under the approval of the Institutional Animal Care and Use Committee (IACUC-1812054).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eTreadmill exercise training\u003c/h3\u003e\n\u003cp\u003e5\u0026times;FAD mice aged 5.5 months were randomly assigned to either a treadmill exercise group or a sedentary group. The treadmill exercise (Exe) regimen consisted of twice-daily sessions (at zeitgeber time (ZT) 1 and ZT 12, with lights on at 08:00 am designated as ZT 0), six days a week. Mice were acclimated and trained on a 10\u0026deg; uphill treadmill, beginning with 30 minutes of running at 8 m/min, followed by 30 minutes at 10 m/min for the first two days as a warm-up. Starting on the third day, mice were subjected to increasing treadmill speeds, with increments of 1 m/min every 20 minutes, for a total of 90 minutes per session [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Sedentary (Sed) control mice were kept in identical conditions but remained in their natural state. After one month of repeated training, the animals underwent behavioral tests followed by pathological analyses.\u003c/p\u003e \u003cp\u003e \u003cb\u003eIn vivo\u003c/b\u003e \u003cb\u003etwo-photon imaging\u003c/b\u003e\u003c/p\u003e \u003cp\u003eAfter being anesthetized with an intraperitoneal injection of a mixed solution containing ketamine (80 mg/kg) and xylazine (8 mg/kg) in saline, 6-month-old wild-type (WT) mice and 5\u0026times;FAD mice with 10 days of treadmill exercise training were secured in a stereotaxic device. The skin on their heads and necks was shaved and cleaned with iodine and 75% ethanol. Following a surgical skin incision in the parietal skull bone, the skull bone was thinned using an electrical micro drill. A total of 3 \u0026micro;L of A488-Lyve-1 (Invitrogen; Cat# 53-0443-82) was injected into the cisterna magna of the mice within 5 minutes via a 5 \u0026micro;L Hamilton syringe. The syringe was left in place for an additional 5 minutes before being slowly withdrawn. After suturing the exposed incision, the mice were returned to their home cage. Twenty-four hours later, the mice received an intrahippocampal injection (anteroposterior \u0026minus;\u0026thinsp;2.0 mm, mediolateral\u0026thinsp;\u0026plusmn;\u0026thinsp;1.8 mm, dorsoventral \u0026minus;\u0026thinsp;2.0 mm) of 1 \u0026micro;L of Fluor 555-labeled Aβ (at 1 mg/mL, AnaSpec; Cat# AS-60480-01), and one hour later, in vivo two-photon imaging was performed. The heads of the mice were fixed on a metal holder to minimize movement during live imaging. As previously described [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], a confocal scanning system (Zeiss ZEN) equipped with a two-photon laser scanning microscope and a 20\u0026times; water-immersion lens installed on an upright microscope (Zeiss LSM880, Germany) was used for imaging the meninges.\u003c/p\u003e\n\u003ch3\u003eAAV-mediated astrocytes selective knockdown of Thbs-1 or Eaf2\u003c/h3\u003e\n\u003cp\u003eThe rAAV-GFaABC1D-mCherry-5\u0026prime;miR-30a-shRNA (Thbs1)-3\u0026prime;miR-30a-WPREs or rAAV-GFaABC1D-EGFP-5\u0026prime;miR-30a-shRNA (Eaf2)-3\u0026prime;miR-30a-WPREs, designed for specific knockdown of Thbs1 or Eaf2, were obtained from BrainVTA. An shRNA sequence verified to efficiently knockdown mouse TSP-1 or Eaf2 was utilized: siRNA Thbs1, 5\u0026prime;-GAUGACUACGCUGGCUUUGUU-3\u0026prime;, siRNA Eaf2, 5\u0026prime;-GGACUUCCAAUCUUGUACATT-3\u0026prime;. As described above, after anesthetization, 5-month-old WT and 5\u0026times;FAD mice were secured in a stereotaxic apparatus. rAAV2/5-GFaABC1D-Thbs1-shRNA-mCherry, rAAV2/9-GFaABC1D-scramble-shRNA-mCherry, rAAV2/5-GFaABC1D-Eaf2-shRNA-EGFP, or rAAV2/5-GFaABC1D-scramble-shRNA-EGFP was injected into the bilateral hippocampal region (anteroposterior \u0026minus;\u0026thinsp;2.0 mm, mediolateral\u0026thinsp;\u0026plusmn;\u0026thinsp;1.8 mm, dorsoventral \u0026minus;\u0026thinsp;2.0 mm, 1 \u0026micro;L for each hemisphere). Four weeks post-virus injection, the behavior and pathology of Thbs1 or Eaf2 protein knockdown were evaluated.\u003c/p\u003e\n\u003ch3\u003eY-maze test\u003c/h3\u003e\n\u003cp\u003eThe Y-maze test (27 cm \u0026times; 9 cm \u0026times; 24 cm) consisted of a start arm, another arm, and a novel arm. During the training stage, mice were placed in the start arm to explore for 5 minutes with the novel arm blocked. Two hours later, the mice were allowed to freely explore the entire maze for 5 minutes. The percentage of time spent in the novel arm and the number of entries into the novel arm were recorded by video tracking software (TopScan, CleverSys, Inc.).\u003c/p\u003e\n\u003ch3\u003eNovel object recognition (NOR) test\u003c/h3\u003e\n\u003cp\u003eThe NOR test was conducted in two phases: familiarization and testing [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. During the familiarization phase, each animal was permitted to freely explore an open arena (40 cm \u0026times; 40 cm \u0026times; 30 cm) containing two identical objects placed in opposite diagonal corners for a 5-minute trial. In the testing phase, mice were reintroduced to the arena for another 5-minute trial, this time with one familiar object replaced by a novel object (differing in color and shape) after a two-hour interval. Exploration of both objects was defined as the mouse sniffing or interacting with an object from within 2 cm and was quantified using video tracking software (TopScan, CleverSys, Inc.). The discrimination index, a measure of recognition memory, was calculated by dividing the time spent exploring the novel object by the total time spent exploring both objects, then subtracting the time spent exploring the familiar object.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eElevated plus maze (EPM) Test\u003c/h2\u003e \u003cp\u003eThe EPM consisted of two open arms (35 cm \u0026times; 6 cm) and two closed arms (35 cm \u0026times; 6 cm \u0026times; 15 cm), each elevated 75 cm above the floor. Mice were placed in the central hub and allowed to freely explore the maze for 5 minutes. Video tracking software (TopScan, CleverSys, Inc.) was utilized to quantify the time spent in the open arms and the frequency of entries into the open arms.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003ePrimary astrocytes and cell line cultures and treatment\u003c/h3\u003e\n\u003cp\u003ePrimary astrocytes were isolated from neonatal mice as previously described [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. In brief, after removing the meningeal vessels, the hippocampus and surrounding cortices were microdissected and subjected to trypsin digestion. Tissue homogenates were passed through a 70 \u0026micro;m mesh filter, resuspended in astrocyte growth media (DMEM (Gibco, Cat# 11960), 10% FBS, 100 U/mL penicillin/streptomycin (Gibco, Cat# 15-140-122)), and plated on 10 cm petri dishes. These were incubated at 37\u0026deg;C in 5% CO\u003csub\u003e2\u003c/sub\u003e. The medium was fully replaced every 3 to 4 days, and cells were passaged using 0.05% Trypsin (Gibco, Cat# 25-200-056) when they reached full growth. For experiments involving primary astrocytes treated with Aβ\u003csub\u003e1\u0026minus;42\u003c/sub\u003e oligomers, cultured 12\u0026ndash;15 days old primary astrocytes were transferred to complete serum medium and then incubated in the presence or absence of oligomeric Aβ\u003csub\u003e1\u0026minus;42\u003c/sub\u003e (at concentrations of 0, 5, and 10 \u0026micro;M) for 48 hours. Some experiments continued with the transfection of siR-Eaf2, after which the pellet and supernatant were collected 72 hours later. The culture of the Mus musculus lymphoid endothelial cell line (SVEC4-10) (ATCC, Cat# CRL-2181), endothelial cell line (human aortic endothelial cells, HAECs) and human-derived lymphatic endothelial cell line (HLEC) were routinely maintained in DMEM medium supplemented with 10% FBS and 100 U/mL penicillin/streptomycin. The SVEC4-10 cells were planted on a 24-well plate and then treated with a gradient concentration of recombinant TSP-1 protein (R\u0026amp;D Systems, USA; Cat# 3074-TH-050) or oligomer Aβ\u003csub\u003e1\u0026minus;42\u003c/sub\u003e. To investigate the role of the TSP-1-CD47 pathway on junctional patterns \u003cem\u003ein vitro\u003c/em\u003e, SVEC4-10 cells were pretreated with siR-CD47 before recombinant TSP-1 intervention. The sequences were as follows:\u003c/p\u003e \u003cp\u003esiR-Eaf2, 5\u0026prime;-CAAAGGCUGCUCCAGCUCUdTT-3\u0026prime;;\u003c/p\u003e \u003cp\u003esiR-Cd47 (Mus), 5\u0026prime;- CUUGCAUCGUCCGUAAUGUTT-3\u0026prime;;\u003c/p\u003e \u003cp\u003eNegative control (NC), 5\u0026prime;-UUCUCCGAACGUGUCACGUTT-3\u0026prime;.\u003c/p\u003e\n\u003ch3\u003eCSF and tissue collection\u003c/h3\u003e\n\u003cp\u003eAfter anesthesia, CSF was collected from the cisterna magna using a borosilicate glass pipette with an internal filament. The sample was then centrifuged at 1000 g for 15 minutes. The supernatant was carefully transferred to a collection tube and stored at \u0026minus;\u0026thinsp;80\u0026deg;C. Subsequently, the deep cervical lymph nodes (dCLNs) preserved in 4% paraformaldehyde (PFA) were removed. The mice were then transcardially perfused with ice-cold phosphate-buffered saline (PBS). Following the removal of the skin and muscle from the head, the hippocampi were collected and stored at \u0026minus;\u0026thinsp;80\u0026deg;C until further processing. The brains and skullcaps were kept in 4% PFA for an additional 24 hours. The fixed meninges, including the dura mater and arachnoid, were carefully dissected from the skullcaps using a stereomicroscope. Fixed brains and dCLNs were subsequently subjected to gradient dehydration using 20% and 30% sucrose solutions and embedded in tissue-plus OCT compound (Sakura, USA; Cat# 4583). The brain segments encompassing the hippocampus and adjacent cerebral cortex were sliced into coronal sections at a thickness of 20 \u0026micro;m, while the frozen lymph nodes were sectioned into 10 \u0026micro;m slices using a cryostat (Leica, CM1950, Germany). The sections were then transferred to cryoprotectant (50% glycerol, 50% 0.1 M PBS, pH 7.4) and stored at \u0026minus;\u0026thinsp;20\u0026deg;C until needed. SVEC4-10 cells were fixed with 4% PFA for 15 minutes at room temperature, washed with PBS, and subsequently stored at 4\u0026deg;C until further staining was to be performed.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eImmunofluorescence\u003c/h2\u003e \u003cp\u003eFrozen brain sections, lymph node slices, meningeal whole mounts, SVEC4-10 cells, and primary astrocytes on glass coverslips were blocked and permeabilized for one hour at room temperature using a block/stain buffer (PBS with 0.3% Triton X-100 and 10% bovine serum). The sections were then incubated with primary antibodies (Supplementary Table\u0026nbsp;1) in block/stain buffer overnight at 4\u0026deg;C, washed three times in PBS, and incubated with secondary antibodies (at a 1:1000 dilution) for 2 hours at room temperature. After being washed in PBS and incubated with 4',6-diamidino-2-phenylindole dihydrochloride (DAPI) at a concentration of 1 \u0026micro;g/mL in PBS, the sections were coverslipped until images were acquired using either a wide-field microscope (DM4000B, Leica) or a confocal microscope (Zeiss LSM710, Germany).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eThioflavin-S staining\u003c/h2\u003e \u003cp\u003eFrozen brain sections were stained with 1% Thioflavin-S (Sigma, St. Louis, USA; Cat# 1326-12-1) for 5 minutes. After rinsing with distilled water, they were differentiated with 70% alcohol for 1 minute. The sections were then washed with PBS and mounted with glass coverslips.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eWestern blot\u003c/h2\u003e \u003cp\u003eTotal protein was extracted from siRNA-transfected or oligomer Aβ\u003csub\u003e1\u0026minus;\u0026thinsp;42\u003c/sub\u003e-treated primary astrocytes, and mouse hippocampus using lysis buffer (25 mM Tris pH 7.4, 150 mM NaCl, 1 mM CaCl\u003csub\u003e2\u003c/sub\u003e, 1 mM MgCl\u003csub\u003e2\u003c/sub\u003e, 0.5% NP-40, and protease inhibitors). Briefly, after washing the cultured cells twice with ice-cold PBS, the cells or tissues were incubated on ice in lysis buffer for 10 minutes. The lysates were then centrifuged at 13,000 g at 4\u0026deg;C for 20 minutes, and the supernatant was collected. Protein concentration was estimated using the BCA protein assay (Beyotime Biotechnology) for all protein samples. Thirty micrograms of protein samples were mixed with 6\u0026times; loading buffer (Thermo Scientific) and boiled at 95\u0026deg;C for 5 minutes. The samples were loaded onto 8\u0026ndash;15% gradient gels and transferred to PVDF membranes, which were then blocked with 5% non-fat milk for 1 hour. The membranes were subsequently incubated with primary antibodies (Supplementary Table\u0026nbsp;1) overnight at 4\u0026deg;C. After washing the membranes three times with TBST, they were incubated with secondary antibodies for 1 hour at room temperature. Finally, all bands were washed three times with TBST and imaged using an imaging system (ImageQuant\u0026trade; LAS 4000 mini, version 1.2).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eELISA\u003c/h2\u003e \u003cp\u003eThe samples of CSF, hippocampus, pellet and supernatant of primary astrocytes were diluted appropriately in a dilution buffer in the 96-well ELISA plate to analyze TSP-1 or EAF2 levels using ELISA commercial kits (Jingmei Co., Ltd. Cat# JM-04106H1, JM-13432M2) following the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eRNA extraction and qRT-PCR\u003c/h2\u003e \u003cp\u003eTotal RNA was extracted from brain or cell samples using Trizol reagent (TaKaRa, Japan), and cDNA was generated using a reverse transcriptase kit (Vazyme Biotech Co., Ltd., Cat# R323) following the manufacturer\u0026rsquo;s instructions. Relative qRT-PCR was performed on an ABI 7300 Fast Real-Time PCR System (Applied Biosystems, Foster City, CA, USA), with qRT-PCR SYBR master mix (Vazyme Biotech Co., Ltd. Cat# Q712). The 2\u003csup\u003e\u0026minus;ΔΔCt\u003c/sup\u003e method was used to calculate mRNA levels as in previous reports [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. GAPDH was used as an internal control. The primers used for analysis are listed in Supplementary Table\u0026nbsp;2.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eTube formation assay\u003c/h2\u003e \u003cp\u003eA tube formation assay was performed to assess the effects of recombinant TSP-1 on the tube formation of LECs lines of murine and human species \u003cem\u003ein vitro\u003c/em\u003e. A coating of 10 \u0026micro;L Matrigel (Coring, Cat# 354243) was applied to the inner walls of 15-well Ibidi \u0026micro;-slides (Ibidi, Martinsried, Germany; Cat# 81506) and allowed to polymerize for one hour at 37\u0026deg;C to form a gel-like surface. The SVEC4-10 or HLEC in complete culture media (DMEM, Gibco) with 10% FBS, 100 U/mL penicillin/streptomycin, 1X endothelial cell growth supplement containing exogenous recombinant TSP-1 (R\u0026amp;D Systems, USA; Cat# 3074-TH-050), Aβ\u003csub\u003e1\u0026minus;42\u003c/sub\u003e (Nanjing Peptide Biotech Ltd. China; Cat# 107761-42-2) or CD36 blocking peptide (Fab Gennix, USA; Cat# P-CD36) were seeded, then plated (1\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells/well) into the duplicate and incubated for 4 hours at 37\u0026deg;C in 5% CO\u003csub\u003e2\u003c/sub\u003e. Images were captured and the numbers of nodes, sprouts and total tube length were measured and quantified using Image J (NIH, Bethesda, MD, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eRNA sequencing data analysis\u003c/h2\u003e \u003cp\u003ePublicly available RNA sequencing (RNA-seq) data for meningeal lymphatic endothelial cells from 6-month-old 5\u0026times;FAD male mice and WT mice were obtained from the Gene Expression Omnibus (GEO) database under the accession number GSE245658. Additional bulk RNA-seq data from three different time points (4, 8, and 18 months) in the hippocampus of 5\u0026times;FAD mice and control mice were sourced from the database with the accession number GSE168137. The count data were utilized for the quantification of gene expression. Normalization and differential expression analysis were performed using the DESeq2 package (version 1.32.0) in R (version 4.3.1). Genes with an adjusted p-value less than 0.05 were considered differentially expressed. Principal component analysis (PCA) and hierarchical clustering were conducted to evaluate the overall variance and sample clustering. A specific list of genes, obtained from literature screening, was analyzed to ascertain differential expression changes across various groups. The expression levels of these genes were extracted and normalized. The gene expression levels in the histogram were normalized using the FPKM value, subtracted by the mean and divided by the standard deviation. Differential expression analysis was conducted for each gene in the list across different groups.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eQuantification and statistical analysis\u003c/h2\u003e \u003cp\u003eQuantification is detailed in the methods and figure legends. Experimenters were blinded to the identity of experimental groups from the time of euthanasia until the end of data collection. An unpaired Student's t-test was used to compare differences between the two groups. A one-way ANOVA with a Tukey post hoc test was employed to compare three independent groups. For the comparison of multiple factors (e.g., genotype versus treatment), a two-way ANOVA with a Tukey post hoc test was utilized. A repeated-measures two-way ANOVA with a Tukey post hoc test was applied for repeated observations of multiple factors. Statistical analysis (data are consistently presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;S.E.M.) was conducted using R (version 4.3.1) and Prism 8.0 (GraphPad Software, Inc.).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eImpaired meningeal lymphatic vessels in 5\u0026times;FAD mice\u003c/h2\u003e \u003cp\u003eRecent studies have revealed that meningeal lymphatic vessels drain brain macromolecule metabolites into the peripheral system, which was impaired in both aged mice and AD mouse models [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Consistently, the present results showed significant decreases in the coverage of lymphatic vascular endothelial hyaluronan receptor 1 (LYVE-1) and prospero homeobox protein 1 (PROX1) positive lymphatic vessels along the transverse sinus (TS) of 6.5-month-old 5\u0026times;FAD mice, compared with age-matched WT mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea-b, d-f). Additionally, under normal physiological conditions, the TS lymphatic vessels consist mostly of a zipper-like junctional pattern of LECs, and insufficient continuous zipper connections are related to impaired lymphatic flow [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. The results indicated that in TS lymphatic vessels of 5\u0026times;FAD mice, there was a decrease in tight zipper-like LEC junctions and an increase in button-like LEC junctions compared to those in WT mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec, g-h). Quantitative analyses also showed a reduction in the diameter and number of sprouts of TS lymphatic vessels in 5\u0026times;FAD mice, further indicating impaired lymphangiogenesis (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ei-j).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eActivated astrocytes with increased production of TSP-1 in 5\u0026times;FAD mice\u003c/h2\u003e \u003cp\u003eWe investigated the underlying mechanism of impaired meningeal lymphangiogenesis in 5\u0026times;FAD mice. Initially, we conducted an RNA-seq analysis of LECs sorted from the meninges of 6-month-old WT and 5\u0026times;FAD mice (GSE245658). Notably, the classical factors regulating lymphangiogenesis, such as \u003cem\u003eVegfc\u003c/em\u003e and \u003cem\u003eVegfd\u003c/em\u003e, were not significantly different in meningeal LECs between WT and 5\u0026times;FAD mice (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003ea-c). This suggested that the soluble cytokines regulating meningeal lymphangiogenesis are not produced by LECs but may originate from brain cells. Consequently, we further evaluated published RNA-sequence data from the hippocampus of WT and 5\u0026times;FAD mice at 4, 8, and 18 months of age (GSE168137) and screened the literature for factors that regulate lymphangiogenesis. We discovered that \u003cem\u003eThbs1\u003c/em\u003e, the gene encoding the protein TSP-1, was age-dependently elevated in the 5\u0026times;FAD group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea-b, Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTSP-1, an endogenous inhibitor of lymphangiogenesis, plays a crucial role in tumor metastasis and transplant outcome [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Notably, TSP-1 is a secreted protein primarily produced by astrocytes in the CNS, particularly up-regulated in activated astrocytes [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Consistently, double immunofluorescence revealed that astrocytes surrounding the plaques were activated with an upregulation of TSP-1 expression in the hippocampus of 6.5-month-old 5\u0026times;FAD mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec and e, Fig. \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e). High concentrations of TSP-1 were found in the hippocampus and CSF of 5\u0026times;FAD mice, as shown by ELISA (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ef and g). AD mice also exhibited high mRNA levels of TSP-1 receptors CD36 and CD74 in the meningeal lymphatic endothelial cells, compared with WT mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ei). Furthermore, when treated with Aβ\u003csub\u003e1\u0026minus;42\u003c/sub\u003e \u003cem\u003ein vitro\u003c/em\u003e, the expression of TSP-1 increased in primary astrocytes in a dose-dependent manner (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed and h). Collectively, these data suggest that under Aβ stimulation, astrocytes are activated and enhance the production of TSP-1.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eAstrocyte-specific knockdown of TSP-1 enhanced meningeal lymphatic vessel plasticity and drainage in 5\u0026times;FAD mice\u003c/h2\u003e \u003cp\u003eTo investigate the contribution of astrocyte-derived TSP-1 to impaired meningeal lymphatic drainage under AD-like pathology, we utilized an AAV-Thbs1-shRNA-mCherry construct (controlled by the GfaABC1D promoter) to selectively knock down TSP-1 expression in the hippocampal astrocytes of 5\u0026times;FAD mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). We confirmed reduced TSP-1 levels at the hippocampal injection site and in the CSF of 5\u0026times;FAD mice, demonstrating the efficacy of the \u003cem\u003eThbs1\u003c/em\u003e-shRNA virus (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb and Fig. \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003ea, c). Astrocyte-specific knockdown of TSP-1 also resulted in an increased diameter of LYVE-1\u003csup\u003e+\u003c/sup\u003e vessels and continuous zipper-like patterns of LECs in the meningeal tissue (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec-d and g-h), as well as a reduction in the accumulation of Aβ plaques and senescent astrocytes, along with decreased glial activation in the brain parenchyma of 5\u0026times;FAD mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee-f, i-k, Fig. \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003eb, d-e). Furthermore, immunofluorescence analyses employing monoclonal antibodies against glial fibrillary acidic protein (GFAP) and rabbit IgG antibodies specific for human Aβ revealed the presence of non-tissue self-produced antigens or non-specific markers in the dCLNs. This supports the notion that these macromolecules are cleared from the brain to the peripheral lymph system [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Elevated Aβ and GFAP signals were observed in the dCLNs of 5\u0026times;FAD mice following hippocampal astrocyte-specific knockdown of TSP-1 (Fig. \u003cspan refid=\"MOESM5\" class=\"InternalRef\"\u003eS5\u003c/span\u003ea-c). As anticipated, the astrocyte-specific knockdown of TSP-1 in 5\u0026times;FAD mice notably increased the number of entries into the novel arm during the Y-maze test, yet it did not influence behavioral performance in the NOR test (Fig. \u003cspan refid=\"MOESM6\" class=\"InternalRef\"\u003eS6\u003c/span\u003ea-d), suggesting a partial mitigation of cognitive impairment. These findings indicate that the astrocyte-specific knockdown of TSP-1 exerts a therapeutic effect on AD model mice by enhancing meningeal lymphatic plasticity and drainage.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003eTSP-1 aggravated the inhibitory role of Aβ on lymphatic vessel formation and plasticity\u003c/h2\u003e \u003cp\u003eTo confirm the involvement of TSP-1 in lymphatic vessel formation and plasticity, a tube formation assay was initially performed using SVEC4-10 cells, a cell line of LECs [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. SVEC4-10 cells highly expressed lymphatic endothelial markers such as LYVE1, PROX1, and vascular endothelial growth factor receptor 3 (VEGFR3), while they exhibited low expression of vascular endothelial cell markers, including CD31, CD34, and FLI-1, compared with an endothelial cell line of HAECs (Fig. S7a-d). The results indicated that treatment with 200 ng/mL TSP-1 led to reductions in the number of nodes, sprouts, and total tube length in SVEC4-10 cells, demonstrating that TSP-1 inhibited lymphangiogenesis in vitro (Fig. S8a-b and e). Additionally, previous studies have suggested that meningeal Aβ deposition may impact LEC plasticity [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. In the lymphangiogenesis assay using the tube formation assay of SVEC4-10 cells, no significant changes were observed at lower concentrations of Aβ\u003csub\u003e1\u0026minus;42\u003c/sub\u003e treatment (0, 0.5, and 2.5 \u0026micro;M). However, the number of nodes, sprouts, and total tube length was notably decreased after treatment with 5 \u0026micro;M Aβ\u003csub\u003e1\u0026minus;42\u003c/sub\u003e (Fig. S8c and f). We further investigated whether the combination of lower concentrations of Aβ\u003csub\u003e1\u0026minus;42\u003c/sub\u003e and TSP-1 had a synergistic inhibitory effect on lymphangiogenesis. The results demonstrated that treatment with 2 \u0026micro;M Aβ\u003csub\u003e1\u0026minus;42\u003c/sub\u003e and 100 ng/mL TSP-1 together significantly suppressed tubule formation of the SVEC4-10 cells (Fig. S8d and g).\u003c/p\u003e \u003cp\u003eTo further investigate how TSP-1 facilitates lymphatic vessel formation and plasticity, we conducted a tube formation assay and visualized lymphatic vessel junction patterns on SVEC4-10 cells. The results indicated that TSP-1 regulates lymphangiogenesis and the junctions of lymphatic vessels through interactions with its various receptors, such as CD36 and CD47. Pretreatment with a CD36-blocking peptide diminished the inhibitory effect of TSP-1 on the lymphangiogenesis of SVEC4-10 cells, as demonstrated by a tube formation assay (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea-e). On the other hand, TSP-1 dose-dependently suppressed VE-Cadherin-formed zipper-like junctions (Fig. S9a, b). In addition, the inhibitory effect of TSP-1 on tube-forming and VE-Cadherin-positive continuous junctions was also confirmed in a human-derived lymphatic endothelial cell line on HLEC (Fig. S9c-g). Furthermore, the inhibitory effect of TSP-1 at a concentration of 2 \u0026micro;g/mL on these zipper-like junctions was nullified after the knockdown of CD47 on SVEC4-10 cells (Fig. S9h-j and Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ef-i). In summary, these \u003cem\u003ein vitro\u003c/em\u003e data suggest that TSP-1 inhibits lymphangiogenesis and junction plasticity through interactions with CD36 and CD47, respectively.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003eTreadmill exercise reduced brain Aβ load and related pathophysiological changes\u003c/h2\u003e \u003cp\u003eBrain Aβ deposition is a typical hallmark in AD transgenic mice [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. We assessed whether treadmill exercise could attenuate Aβ-related brain pathology and cognitive dysfunction in 5\u0026times;FAD mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). After one month of treadmill exercise, Thioflavin-S positive plaques, reactive gliosis, and accumulation of senescent astrocytes were significantly reduced in the hippocampus and adjacent cortical region of 6.5-month-old 5\u0026times;FAD mice (Fig. S10a-g). Treadmill exercise training also had a beneficial effect on the short-term learning and memory of 5\u0026times;FAD mice (Fig. S11a-d). However, exercise intervention did not ameliorate the anxiety-like behavior of 5\u0026times;FAD mice in the EPM test (Fig. S11e-f).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec25\" class=\"Section3\"\u003e \u003ch2\u003eTreadmill exercise increased meningeal lymphatic plasticity and drainage in 5\u0026times;FAD mice\u003c/h2\u003e \u003cp\u003eWe determined whether long-term exercise reduced brain Aβ load is associated with enhanced meningeal lymphatic plasticity. As expected, 6.5-month-old 5\u0026times;FAD mice that received treadmill exercise for one month showed a marked increase in the area fraction, diameter, and number of sprouts of meningeal lymphatic vessels, as well as continuous lymphatic junctions (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb-h). Furthermore, compared with the sedentary control group, more Aβ and GFAP-positive products were detected in the dCLNs of 5\u0026times;FAD mice that underwent treadmill exercise training, indicating that exercise facilitated the drainage of brain Aβ and GFAP from the brain to the peripheral system (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ei, k-l). These results together revealed that treadmill exercise improves meningeal lymphatic vessel plasticity and drainage under AD-like pathology.\u003c/p\u003e \u003cp\u003eTo further confirm this conclusion, \u003cem\u003ein vivo\u003c/em\u003e two-photon imaging was used to monitor the dynamic distribution of fluorescent-labeled Aβ\u003csub\u003e1\u0026minus;42\u003c/sub\u003e at 24 hours post-intrahippocampal administration in the meningeal lymphatic vessels of 6-month-old WT and 5\u0026times;FAD mice that had undergone 10 days of treadmill exercise training (Fig. S12a). As previously reported [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e], Aβ\u003csub\u003e1\u0026minus;42\u003c/sub\u003e fluorescent signals were detected in the A488-Lyve1-labeled meningeal lymphatic vessels running along the TS. Notably, a comparison of Aβ\u003csub\u003e1\u0026minus;42\u003c/sub\u003e-555 drainage through the TS regions at consecutive time points revealed a delayed clearance of the Aβ tracer in 5\u0026times;FAD mice. The treadmill exercise significantly enhanced the drainage of Aβ\u003csub\u003e1\u0026minus;42\u003c/sub\u003e-555 from the meningeal lymphatic vessels in WT mice, and there was a trend toward improvement in AD mice as well (Fig. S12b-c and Supplementary Movies 1\u0026ndash;4). Consistently, there were increased Aβ\u003csub\u003e1\u0026minus;42\u003c/sub\u003e-555 signals in the dCLNs of 5\u0026times;FAD mice after treadmill exercise (Fig. S12d-e). These results verified that exercise promoted the meningeal lymphatic drainage of Aβ from the brain to the peripheral system.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section3\"\u003e \u003ch2\u003eTreadmill exercise down-regulated TSP-1 expression in astrocytes of 5\u0026times;FAD mice\u003c/h2\u003e \u003cp\u003eWe examined the expression level of TSP-1 in the hippocampus of 5\u0026times;FAD mice, with or without exercise, and found that the upregulated expression of TSP-1 in GFAP-positive astrocytes was reversed by treadmill exercise (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ej and m). ELISA analysis of the hippocampus confirmed this conclusion (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003en). The ELISA results also indicated a significant decrease in TSP-1 levels in the CSF after treadmill exercise in 5\u0026times;FAD mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eo). Consistently, CD36, a receptor for TSP-1, was highly expressed in meningeal lymphatic vessels, which were partially normalized by exercise treatment in 5\u0026times;FAD mice (Fig. S13a-b).\u003c/p\u003e \u003cp\u003eBesides, the exchange of CSF and interstitial fluid (ISF) mediated by AQP4 is responsible for the clearance of harmful metabolites from the brain [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. We observed that, compared with WT littermates, the perivascular localization of astrocytic AQP4 was impaired in the hippocampus of 5\u0026times;FAD mice, a condition that was reversed by treadmill exercise (Fig. S14a-c). This suggests that treadmill exercise also facilitates AQP4-mediated glymphatic clearance of Aβ. However, the expression levels of APP and its secretases, as well as proteins involved in the transport or degradation of Aβ, were not significantly altered in the hippocampus of WT mice and 5\u0026times;FAD mice following exercise training (Fig. S15a and b). Collectively, these data suggest that long-term exercise enhances the glymphatic-meningeal lymphatic transport of Aβ, which in turn inhibits the pathological cascades of parenchymal Aβ accumulation, astrocyte activation, TSP1 secretion, and meningeal lymphatic dysfunction, thereby improving the cognitive function of 5\u0026times;FAD mice.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec27\" class=\"Section3\"\u003e \u003ch2\u003eEAF2 regulating TSP-1 expression in activated astrocytes exposed to Aβ\u003c/h2\u003e \u003cp\u003eTo further explore how exercise reduces TSP-1 expression, we screened the upstream transcription factors (TFs) of TSP-1 in the hippocampus of WT mice and 5\u0026times;FAD mice following exercise training, which brought p53 into our focus (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea and b). The regulation of TSP-1 by p53 varies across different tissues and cells [\u003cspan additionalcitationids=\"CR38\" citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. For instance, the downregulation of TSP-1 has been shown to increase angiogenesis in the liver of EAF2 knockout mice. However, the transfection of EAF2 alone had minimal impact on the TSP-1 promoter [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Functional protein association networks analysis indicated that EAF2 has a direct regulatory relationship with p53, but not with TSP-1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec). Additional evidence was provided by primary astrocytes exposed to varying concentrations of Aβ\u003csub\u003e1\u0026minus;42\u003c/sub\u003e for 48 hours, which demonstrated that the levels of p53, EAF2, and TSP-1 were all elevated at a concentration of 10 \u0026micro;M (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ed-g). Furthermore, increased TSP-1 levels in astrocyte cells and their culture medium upon exposure to Aβ\u003csub\u003e1\u0026minus;42\u003c/sub\u003e were reversed by Eaf2 knockdown (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eh-l). Consequently, we hypothesized that EAF2 plays a role in meningeal lymphangiogenesis by modulating TSP-1 levels through its interaction with p53. As anticipated, there were elevated mRNA levels of p53 in the hippocampus of 5\u0026times;FAD mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb). Additionally, we observed that EAF2 was heavily co-localized with activated astrocytes and that there was an increase in EAF2 expression in the hippocampus of 5\u0026times;FAD mice compared to WT mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003em-n). Collectively, these findings suggest that Aβ triggers the activation of the EAF2-p53-TSP-1 pathway in astrocytes.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec28\" class=\"Section2\"\u003e \u003ch2\u003eAstrocyte-specific knockdown of EAF2 improved meningeal lymphatic drainage and AD-like pathology in 5\u0026times;FAD mice\u003c/h2\u003e \u003cp\u003eWe next investigated whether the drainage of meningeal lymphatic vessels could be enhanced through selective knockdown of astrocyte EAF2. Five-month-old 5\u0026times;FAD mice were administered an AAV-Eaf2-shRNA-EGFP construct (controlled by the GfaABC1D promoter) to selectively knock down EAF2 in hippocampal astrocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea and Fig. S16a-b). Four weeks later, EAF2 levels also significantly decreased in the CSF of 5\u0026times;FAD mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eb). These 5\u0026times;FAD mice, with selective knockdown of the Eaf2 gene specifically in astrocytes, exhibited improved behavioral performance in the NOR test and Y-maze test (Fig. S17a-d). Furthermore, the hippocampal level of TSP-1 decreased after EAF2 knockdown in astrocytes of 5\u0026times;FAD mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ec-e). Additionally, down-regulating EAF2 in astrocytes significantly increased the coverage and diameter of LYVE1\u003csup\u003e+\u003c/sup\u003e lymphatic vessels and the continuity of the zipper-like patterns of meningeal LECs (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ef-h and k). This transformation of LEC junction patterns may facilitate Aβ drainage, as evidenced by reductions in Aβ plaques, reactive gliosis, and astrocyte senescence in the hippocampus (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ei-j, l-o). In line with these findings, Aβ and GFAP levels were observed to be higher in the dCLNs compared to control groups (Fig. S18a-c). Collectively, these outcomes suggest that EAF2, derived from activated astrocytes, plays an inhibitory role in meningeal lymphangiogenesis during AD-like pathology.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eImpaired meningeal lymphatic drainage was accompanied by a decrease in macromolecules draining into lymph nodes, as well as cognitive decline in aged or AD mice [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Considering the plasticity of meningeal lymphatic vessels, overexpression of VEGFC-induced meningeal lymphangiogenesis [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Consistently, our previous data also showed a significant increase in the drainage of GFAP into the dCLNs in aged mice after AAV1-CMV-mVEGFC treatment [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. In the current study, we observed that classically regulated lymphatic vessel molecules such as VEGFC in the meninges are unchanged, but there is significant upregulation of TSP-1, a secreted protein associated with lymphangiogenesis, in the hippocampus of 6.5-month-old AD mice, compared with WT mice.\u003c/p\u003e \u003cp\u003eThe identification of clinical biomarkers for meningeal lymphatic injury is essential for early detection and monitoring of neurodegenerative diseases. Advanced neuroimaging techniques, such as dynamic contrast-enhanced magnetic resonance imaging (MRI) and positron emission tomography (PET), could provide valuable insights into changes in cerebrospinal fluid flow and meningeal lymphatic function [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Additionally, CSF biomarkers, encompassing proteins such as Aβ, tau, and GFAP, as well as lymphatic markers like podoplanin or LYVE-1, may indicate impaired lymphatic drainage [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Beyond these markers, neuroinflammatory cytokines and matrix metalloproteinases (MMPs), which play roles in blood-brain barrier disruption and lymphatic vessel remodeling, could offer additional evidence of lymphatic dysfunction [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. Collectively, these biomarkers could improve our capacity to detect and monitor meningeal lymphatic injury in clinical settings. However, this hypothesis requires more evidence. Previous studies have shown that TSP-1 plays a role in peripheral tumor lymphatic metastasis and corneal lymphatic vessel remodeling [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. However, the expression level of TSP-1 in CNS lymphangiogenesis has seldom been investigated. Buee et al. (1992) reported that the distribution of TSPs staining in the brains of AD patients was comparable to that in control subjects [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. In contrast, Son et al. (2015) found evidence of downregulation of TSP-1 in cortical samples from individuals with AD [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. Such inconsistent findings might be attributed to the heterogeneity of astrocyte distribution and the variable disease processes involved in AD.\u003c/p\u003e \u003cp\u003eTSP-1 also acts as a promoter of aging and age-associated diseases. The accumulation of TSP-1 in the extracellular matrix is frequently observed in age-related diseases [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. In this study, we found that treadmill exercise reduced the expression level of TSP-1 in the brain parenchyma of AD mice following treadmill exercise. Similarly, RNA sequencing analysis (refer to GSE164401) also observed that compared with control plasma-treated mice, hippocampal transcriptome levels of Thbs1 had a decreasing trend in recipient mice injected with the donor plasma from exercising mice. Altogether, these data highlighted TSP-1 as a potential target for exercise interventions that alleviate AD pathology by increasing meningeal lymphangiogenesis.\u003c/p\u003e \u003cp\u003eCD36 acts as a receptor for TSP-1 and serves as a negative regulator of angiogenesis and lymphangiogenesis [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. In our current study, we have further demonstrated that TSP-1 can effectively suppress the proliferation of meningeal lymphatic vessels \u003cem\u003ein vitro\u003c/em\u003e by binding to the CD36 receptor on LECs. CD36 has been implicated in maintaining lymphatic vessel integrity, which is associated with obesity and type 2 diabetes models [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. The deletion of CD36 has been shown to protect cerebral arteries from the harmful effects of Aβ\u003csub\u003e1\u0026minus;40\u003c/sub\u003e, thereby enhancing the cognitive performance of AD model mice [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. CD47 is another receptor for TSP-1. The TSP-1-CD47 interaction has been reported to modulate apoptosis in meningeal lymphatic endothelial cells within a subarachnoid hemorrhage model [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. Elevated levels of TSP-1 have been found to impede lymphangiogenesis by activating CD47 in aortic LECs of a mouse model of atherosclerosis [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. Our findings further indicate that TSP-1-CD47 regulates the junctional pattern of meningeal lymphatic vessels.\u003c/p\u003e \u003cp\u003eEAF2 is preferentially expressed in the CNS during mouse embryonic development [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. Overexpression of EAF2 has been shown to induce apoptosis and inhibit cell growth in several peripheral studies [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e, \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. However, its role in the CNS has been scarcely reported. Our data indicates that EAF2 expression is upregulated in activated astrocytes of 5\u0026times;FAD mice. Exercise was found to down-regulate EAF2 and its binding partner p53 in 5\u0026times;FAD mice, thereby promoting the inhibition of the EAF2-p53 complex on TSP-1. We also demonstrated that knocking down EAF2 in specific astrocytes reduced TSP-1 expression in the brain, which in turn improved meningeal lymphatic vessel function in AD mice. Collectively, these findings underscore that the EAF2-p53-TSP-1 pathway plays a crucial role in regulating meningeal lymphatic plasticity.\u003c/p\u003e \u003cp\u003eEpidemiological studies have reported that treadmill exercise is a generally applicable form of physical therapy, playing a role in delaying the aging process and improving cognitive function in patients with AD or mild cognitive impairment [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e, \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e]. However, there is also literature that challenges this view [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e, \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e]. Several studies have indicated that treadmill exercise has the potential to reduce Aβ load in both AD patients and transgenic AD mice [\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e, \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e]. Here, we confirmed that treadmill exercise decreased the parenchymal Aβ deposition in 6.5-month-old 5\u0026times;FAD mice. Accumulating evidence supports the notion that Aβ aggregation and deposition accelerate neuroinflammation and also trigger cellular senescence [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e], particularly, astrocytes are susceptible to senescence within the CNS [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Utilizing pharmacological or genetic models, the elimination of senescent glial cells preserves cognitive function in mouse models of tau-dependent neurodegenerative disease [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. This study demonstrated that the enhancement of learning and memory in AD mice through treadmill exercise was accompanied by an increase in the removal of senescent astrocytes from the hippocampus. Furthermore, it has been reported that targeting the clearance of senescent cells, such as cardiomyocytes, pancreatic β cells, and osteocytes, alleviates disease symptoms and exerts beneficial effects in slowing the aging process [\u003cspan additionalcitationids=\"CR65\" citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e]. Our findings further suggest that targeting the elimination of Aβ and senescent astrocytes may offer a therapeutic approach to the benefits of treadmill exercise against AD pathology.\u003c/p\u003e \u003cp\u003eAdditionally, volunteers underwent assessment of brain waste clearance using noninvasive MRI following either a single session or a 12-week regimen of cycling exercise. The findings indicated that glymphatic influx in the putamen, as well as the size and flow of meningeal lymphatics, significantly increased after the long-term exercise regimen [\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e]. This suggests that sustained physical exercise promotes the flow of putative glymphatic and meningeal lymphatic vessels, thereby enhancing the clearance of brain metabolites in humans. This is consistent with the outcomes of our animal studies. Furthermore, by comparing various durations and intensities of exercise, it was determined that there was no significant difference in lymphatic and meningeal lymphatic vessel flow before and after a single exercise session. Studies involving animals have shown an increase in CSF influx in mice after 5 weeks of voluntary running-wheel exercise [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Similarly, human studies have demonstrated increased compliance of the middle cerebral artery in volunteers who reported engaging in moderate-to-vigorous recreational aerobic exercise [\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e]. Based on these findings, it is hypothesized that moderate to high-intensity exercise may also significantly contribute to cerebrospinal fluid flow and the clearance of metabolites from the brain.\u003c/p\u003e \u003cp\u003eGrowing evidence suggests that the glymphatic system acts as a functional pathway for the removal of metabolic waste from the brain's parenchyma [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Voluntary exercise in young, awake mice or aged mice has been shown to enhance glymphatic function, leading to pro-cognitive effects [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Furthermore, our previous study indicated that the improvement of cognitive deficits in APP/PS1 mice through voluntary exercise is dependent on the polarity of astrocytic AQP4 [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. In the present study, we confirmed that treadmill exercise significantly reduced the activation of astrocytes and improved AQP4 polarity in the hippocampus of 5\u0026times;FAD mice. These findings suggest that the glymphatic system, which is responsible for the clearance of Aβ, could be an important target for the preventive and therapeutic effects of treadmill exercise on AD.\u003c/p\u003e \u003cp\u003eFurthermore, the meningeal lymphatic system serves as a crucial pathway for the elimination of metabolites and brain antigens from the CSF. Studies have demonstrated that improving meningeal lymphatic function can positively influence learning and memory performance in both aged and AD model mice [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Conversely, inhibiting lymphatic drainage in aged mice has been found to worsen the accumulation of perivascular senescent astrocytes within the brain parenchyma and exacerbate cognitive behavioral deficits [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Additionally, previous research has shown that cerebral blood flow and CSF flow dynamics significantly increase during exercise in both human and rodent studies [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e]. In our study, the results indicated that treadmill exercise expanded the diameter and increased the number of sprouts and the continuity of VE-Cadherin junctions in the meningeal lymphatic vessels of AD mice, thereby enhancing their drainage function. This approach could potentially be a promising strategy to improve the clearance of Aβ, potentially slowing the progression of AD.\u003c/p\u003e \u003cp\u003eIt is important to note that we conducted treadmill exercise training specifically at two time points, day and night, on mice to avoid any argument regarding the effects of circadian rhythms on running. We did not ascertain which time point for treadmill exercise would yield the greatest benefit for the mice. Human studies have indicated that morning and evening exercise have distinct effects on the skeletal muscle molecular clock and nocturnal sleep [\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e, \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e]. The circadian rhythm impact of treadmill exercise on the enhancement of meningeal lymphatic vessels in humans requires further clarification. Investigating these matters will maximize the benefits of treadmill exercise, particularly for the elderly or individuals with AD.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThe plasticity of meningeal lymphatics has been identified as a significant target for the drainage of metabolites from the brain. We demonstrated that activated astrocyte-derived TSP-1 is crucial for impaired meningeal lymphangiogenesis in 5\u0026times;FAD transgenic mice. Astrocyte-specific knockdown of Thbs1 or Eaf2 promoted functional meningeal lymphatic vessel plasticity and mitigated AD-like pathology. Our results also suggested that exercise modulated the lymphangiogenesis and junctional patterns of meningeal lymphatic vessels by down-regulating the reactive astrocytes-related EAF2-p53-TSP-1 pathway (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e). This finding revealed a novel mechanism by which exercise improves meningeal lymphatic vessel plasticity, thereby alleviating AD-related pathology.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eAAV Adeno-associated virus\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAD Alzheimer’s disease\u003c/p\u003e\n\u003cp\u003eAQP4 aquaporin 4\u003c/p\u003e\n\u003cp\u003eAβ amyloid-β\u003c/p\u003e\n\u003cp\u003eCNS central nervous system\u003c/p\u003e\n\u003cp\u003eCSF cerebrospinal fluid\u003c/p\u003e\n\u003cp\u003eDAPI 4',6-diamidino-2-phenylindole dihydrochloride\u003c/p\u003e\n\u003cp\u003edCLNs deep cervical lymph nodes\u003c/p\u003e\n\u003cp\u003eEAF2 eleven nineteen lysine rich leukemia -associated factor 2\u003c/p\u003e\n\u003cp\u003eELISA enzyme-linked immunosorbent assay\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eEPM elevated plus maze\u003c/p\u003e\n\u003cp\u003eGFAP glial fibrillary acidic protein\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIba-1 ionized calcium-binding adaptor molecule 1\u003c/p\u003e\n\u003cp\u003eISF interstitial fluid\u003c/p\u003e\n\u003cp\u003eLECs lymphatic endothelial cells\u003c/p\u003e\n\u003cp\u003eLYVE-1 lymphatic vascular endothelial hyaluronan receptor 1\u003c/p\u003e\n\u003cp\u003eNOR novel object recognition\u003c/p\u003e\n\u003cp\u003ePROX1 prospero homeobox protein 1\u003c/p\u003e\n\u003cp\u003eTFs transcription factors\u003c/p\u003e\n\u003cp\u003eTS transverse sinus\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTSP-1 thrombospondin-1\u003c/p\u003e\n\u003cp\u003eVEGFC vascular endothelial growth factor C\u003c/p\u003e\n\u003cp\u003eWT wide type\u003c/p\u003e\n\u003cp\u003eZT zeitgeber time\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eQ.L., Y.C., J. C., X. H., H.F., X. L. and W. Y., carried out and analyzed immunostaining and behavior tests. L. Y., Y.F., M. Y., Y. S., and Y.C. carried out biochemistry, in vitro experiments and data analysis. Y. J. performed cartoon diagrams. W. Z. and Y.C. carried out and analyzed \u003cem\u003ein vivo\u003c/em\u003e two-photon imaging. M.X., Q.L., F.D., and Y.C. designed the experiments. M.X., Q.L., C. S., J. G., and Y.C. wrote and modified the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by grants from the National Natural Science Foundation of China (Grant No. 82204365, 82071199 and 81871117), the Natural Science Foundation of Jiangsu Province (Grant No. BK20230057), Shandong Postdoctoral Science Foundation (Grant No. SDCX-ZG-202400044), Shandong Postdoctoral Innovative Talents Program (Grant No. SDBX2023056) and the Natural Science Foundation of the Jiangsu Higher Education Institutions of China (Grant No. 23KJB310009).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll supporting information and data are available in the article and supplementary files.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclarations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical Approval and Consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll animal studies were approved by the Care and Use of Laboratory Animals of Nanjing Medical University (IACUC-1812054).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors have consented to the publication of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eHardy J, Selkoe DJ. 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Nat Rev Endocrinol. 2019;15:197\u0026ndash;206.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"translational-neurodegeneration","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"tneu","sideBox":"Learn more about [Translational Neurodegeneration](http://translationalneurodegeneration.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/tneu/default.aspx","title":"Translational Neurodegeneration","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Alzheimer's disease, Lymphangiogenesis, Meningeal lymphatics, Treadmill exercise, EAF2-p53-TSP-1","lastPublishedDoi":"10.21203/rs.3.rs-5720097/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5720097/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eMeningeal lymphatic drainage is crucial for the clearance of amyloid β (Aβ), supporting the maintenance of brain homeostasis. This makes it a promising therapeutic target for Alzheimer's disease (AD). Long-term exercise can reduce the risk of AD; however, the underlying mechanism is not fully understood. In this study, we investigated whether exercise alleviates AD-related pathological changes by improving meningeal lymphatic drainage and explored its potential mechanisms.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eThe morphological and functional features of meningeal lymphatic vessels, as well as Aβ and reactive gliosis in the brain, were compared between 6.5-month-old 5\u0026times;FAD mice with or without 1 month of treadmill exercise. RNA sequencing analysis, protein interactions analysis, adeno-associated virus (AAV)-mediated gene knockdown, and lymphatic endothelial cell culture were conducted to investigate the mechanism underlying exercise-induced meningeal lymphatic vessel plasticity of 5\u0026times;FAD mice.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eThe structural integrity of meningeal lymphatic vessels was compromised in 5\u0026times;FAD mice, compared with the wild-type mice. Treadmill exercise increased the diameter and drainage capacity of the meningeal lymphatic vessels, reduced Aβ deposition, reactive gliosis, and astrocyte senescence in the hippocampus and frontal cortex, and improved cognitive function in 5\u0026times;FAD mice. Mechanistically, exercise reduced the up-regulation of thrombospondin-1 (TSP-1), a lymphangiogenesis inhibitor, in activated astrocytes of AD mice. TSP-1 exacerbated the inhibitory effect of Aβ on lymphatic vessel formation and plasticity through interactions with CD36 and CD47, respectively. Additionally, exercise decreased the expression of TSP-1 in reactive astrocytes of AD mice by downregulating eleven-nineteen lysine-rich leukemia-associated factor 2 (EAF2), which facilitates the transcription of the TSP-1 encoding gene Thbs-1 via its binding partner p53. Ultimately, we discovered that hippocampal astrocyte-specific knockdown of Thbs-1 or Eaf2 enhanced meningeal lymphatic drainage and alleviated AD-like pathology in the hippocampus of 5\u0026times;FAD mice.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eThese findings collectively unveil a novel mechanism through which long-term exercise combats AD. It enhances the plasticity and drainage of meningeal lymphatic vessels by downregulating the EAF2-p53-TSP-1 pathway, which is associated with reactive astrocytes.\u003c/p\u003e","manuscriptTitle":"Long-term exercise enhances meningeal lymphatic vessel plasticity and drainage in a mouse model of Alzheimer's disease","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-30 19:14:56","doi":"10.21203/rs.3.rs-5720097/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2025-04-28T13:59:57+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-04-28T13:52:55+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-04-28T00:44:49+00:00","index":"","fulltext":""},{"type":"submitted","content":"Translational Neurodegeneration","date":"2025-04-26T01:58:59+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"translational-neurodegeneration","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"tneu","sideBox":"Learn more about [Translational Neurodegeneration](http://translationalneurodegeneration.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/tneu/default.aspx","title":"Translational Neurodegeneration","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"0c22a970-1ea5-40ab-9d21-109aee523f8f","owner":[],"postedDate":"April 30th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-08-07T07:23:49+00:00","versionOfRecord":{"articleIdentity":"rs-5720097","link":"https://doi.org/10.1186/s40035-025-00497-2","journal":{"identity":"translational-neurodegeneration","isVorOnly":false,"title":"Translational Neurodegeneration"},"publishedOn":"2025-07-25 15:57:15","publishedOnDateReadable":"July 25th, 2025"},"versionCreatedAt":"2025-04-30 19:14:56","video":"","vorDoi":"10.1186/s40035-025-00497-2","vorDoiUrl":"https://doi.org/10.1186/s40035-025-00497-2","workflowStages":[]},"version":"v1","identity":"rs-5720097","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5720097","identity":"rs-5720097","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}