Beneficial effects of voluntary exercise therapy on an Alzheimer’s mouse model are dependent on aquaporin 4-mediated glymphatic transport

Beneficial effects of voluntary exercise therapy on an Alzheimer’s mouse model are dependent on aquaporin 4-mediated glymphatic transport | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Beneficial effects of voluntary exercise therapy on an Alzheimer’s mouse model are dependent on aquaporin 4-mediated glymphatic transport Yun Liu, Weixi Feng, Panpan Hu, Shuang Zhai, Rui Zhang, Qian Li, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-108136/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background: Epidemiological and clinical evidence suggest there is an effective time window for the ability of exercise to slow the progression of Alzheimer's disease (AD). Astrocyte aquaporin-4 (AQP4) dependent glymphatic transport is necessary for clearance of extracellular amyloid-β (Aβ) from the brain. The purpose of this study is to explore whether this Aβ clearance mechanism is involved in the time-dependent benefit of exercise on Aβ related pathology. Method: 3- and 7-month-old male wild type (WT) mice, APP695 swe /PS1-dE9 (APP/PS1) mice and AQP4 knockout (AQP4 −/− )/APP/PS1 mice received voluntary wheel exercise intervention for 2 months, followed by behavioral and pathological analyses. Results: 9-month-old APP/PS1 mice showed enhanced reactive astrogliosis, accompanied with widespread loss of perivascular AQP4 polarization, when compared to 5-month-old APP/PS1 mice. Voluntary exercise improved AQP4 polarity and glymphatic transport, reduced glial inflammation and brain Aβ plaque load, plus alleviated synapse protein loss and cognitive deficits in 5-month-old, but not 9-month-old, APP/PS1 mice. Exercise intervention did not mitigate glymphatic transport dysfunction, Aβ accumulation and cognitive impairment in AQP4 −/− /APP/PS1 mice at 5 or 9 months of age. Conclusion: These results have revealed that AQP4-dependent glymphatic transport is an influential factor in the timeliness of voluntary exercise ability to alleviate Aβ pathology, potentially offering a new target for the early prevention of the disease. Neurology Alzheimer’s disease astrocytes aquaporin-4 glymphatic system voluntary exercise Figures Figure 1 Figure 1 Figure 1 Figure 2 Figure 2 Figure 2 Figure 3 Figure 3 Figure 3 Figure 4 Figure 4 Figure 4 Figure 5 Figure 5 Figure 5 Figure 6 Figure 6 Figure 6 Background Alzheimer's disease (AD) is a common neurodegenerative disease, characterized by extracellular amyloid-β (Aβ) plaques in the brain [ 1 ]. Currently, non-pharmacological interventions are receiving substantial interest due to the inability to develop AD therapeutic drugs and vaccines [ 2 ]. Epidemiological and clinical studies show that physical activity improves cognition during healthy aging [ 4 – 6 ], as well as individuals with mild cognitive impairment [ 7 – 9 ]. However, exercise therapy has not been shown to exert explicitly beneficial outcomes on patients with AD [ 10 , 11 ]. Exploring the underlying mechanisms for an effective exercise intervention time window is crucial for the prevention of AD. Experimental animal studies indicate that exercise plays a variety of neuroprotective roles, including promoting neurogenesis, reducing neuroinflammation, improving mitochondrial energy metabolism and promoting neurotrophic factor secretion [ 12 , 13 ]. Notably, both voluntary exercise and forced exercise have been shown to reduce extracellular Aβ levels in the brains of several transgenic mouse models of AD [ 14 – 17 ]. However, there is considerable evidence that exercise interventions do not reduce Aβ plague load in mice with mid- to late-stage AD-like pathology [ 18 – 21 ]. The mechanism for exercise promoting Aβ clearance within an effective time window remains unclear. A variety of clearance pathways are implicated in removal of extracellular Aβ from the brain, which include enzymatic degradation, cellular uptake and transport across the brain barriers [ 22 ]. Recent findings suggest that the glymphatic system may be a large contributor in extracellular Aβ clearance [ 23 ]. The glymphatic system, also known as the perivascular space, is surrounded by incomplete continuous astrocyte endfeet, at which large amounts of aquaporin 4 (AQP4) is located [ 24 ]. Such structural features facilitate rapid removal of brain interstitial fluid and soluble macromolecular substances from the brain [ 25 ]. Aged mice, as well as mouse models of AD, show loss of specific localization of AQP4 in perivascular endfeet due to reactive astrogliosis, which in turn impairs glymphatic clearance of Aβ [ 26 , 27 ]. Our previous studies reported that deletion of AQP4 increases Aβ plaque deposit and cerebral amyloid angiopathy in 12-month-old APP/PS1 mice [ 28 ]. Moreover, recent studies have shown that voluntary exercise promotes glymphatic clearance of Aβ, which is associated with improved astrocytic AQP4 polarization in aged mice [ 29 ]. Together, these findings suggest that AQP4-mediated glymphatic clearance of Aβ may act as a key factor in determining a therapeutic time window for exercise in AD. In particular, under healthy aging or the onset stage of AD, exercise facilitates clearance of Aβ from the brain parenchyma via increased AQP4 dependent bulk flow along the glymphatic pathway. However, with the progress of AD, the polarity of AQP4 is totally impaired because of reactive astrogliosis. Therefore, the structural basis for exercise improving glymphatic clearance is elapsed, subsequently failing to prevent Aβ-related pathological cascades. APP695swe/PS1-dE9 (APP/PS1) mice are extensively used mouse model of AD with occurrence of Aβ-associated long-term memory malfunction from approximately 6–7 months of age, which serves as a hallmark of moderate stage AD [ 30 ]. At 3 months old, despite no Aβ plaques, this AD mouse line exhibits a mild activation of astrocytes with slight impairments of AQP4 polarity and glymphatic transport [ 31 , 32 ]. Therefore, we chose the 3- and 7-month-old APP/PS1 mice and AQP4 −/− /APP/PS1 mice to test whether the time-dependent benefit of exercise on Aβ related pathology is dependent on AQP4-mediated glymphatic transport. Materials And Methods Animals AQP4 −/− /APP/PS1 mice were established via AQP4 −/− mice crossing with APP695/PS1-dE9 mice as described previously [ 28 ]. Both 3-month-old and 7-month-old male AQP4 −/− /APP/PS1 mice, APP/PS1 mice and their wild-type (WT) littermates were randomly divide into exercise group and sedentary group, respectively, and received 2-month voluntary exercise treatment followed by behavioral tests (Fig. 1 A). The animal experiments were approved by the Animal Ethical and Welfare Committee of Nanjing Medical University. Voluntary exercise Mice habituated for one week prior to the beginning of voluntary exercise training, referred to as the “pre-intervention” period. Exercise mice were placed in the running wheel for 4 h every day, 5 days per week, for 2 months. Control mice were placed in the same room as the exercise mice. Y-maze test The Y-maze test was carried out to evaluate short-term working memory [ 28 ]. It consisted of 2 trials with a 1-h interval. Three identical arms of the maze were randomly designated start arm, novel arm, and other arm. In the first trial, one arm of the Y-maze was blocked by a removable door, and the animal was placed in the start arm and allowed to explore the two arms for 5 min. In the second trial, the blocking baffle was removed and mice could now have access to all three arms for 5 min. The number of entries into the novel arm, and the percentage of novel arm entries made during the second trial, were analyzed. Novel object recognition test (NORT) The NORT was performed to assess object recognition memory [ 33 ]. This task consisted of two trials, with an interval of 2 hours. In the first trial, mice were placed in an arena with two of the same objects and allowed to explore for 5 min. Following a two-hour delay, mice were placed back in the familiar arena where a novel object was replaced by one of the identical objects (the other object remained the same), and mice were allowed to freely re-explore for 5 min. Time spent exploring the familiar and novel objects were recorded. The discrimination index was derived by calculating the time spent exploring the novel object/the time spent exploring novel object + the time spent exploring a familiar object. Morris water maze (MWM) test The MWM task was performed to assess spatial learning and memory function [ 28 ]. Briefly, the mice received 6 days of training and a probe test on the 7th day. In the training trail, mice were given as long as 60 s to find the dark-colored cylindrical platform submerged 1 cm beneath the surface of water which was made opaque with milk and invisible to the mice, and required to remain seated on the platform for 5 s. Mice underwent four trials per day, starting from each of four different locations in the pool. The escape latency and swimming speed were analyzed. During the probe trail, the platform was removed from the pool and mice were allowed to swim in the pool for 60 s in order to determine the percentage of total time spent in each quadrant and the number of crossing where the platform was located. Cisterna magna injection of fluorescent tracer Cisterna magna injection of fluorescent tracer was performed as described previously [ 24 , 31 ]. The mice were anesthetized and positioned in a stereotaxic apparatus while the posterior atlanto-occipital membrane was surgically exposed. 5 µl Texas Red-dextran-3 (TR-d3, 3000 MW; Invitrogen, Cat. D3328) was injected at a concentration of 0.5 mg/mL into the cisterna magna by a microsyringe needle at a flow rate of 1 µl/min. Forty minutes after the start of infusion, animals were anesthetized again. The brains were removed and post-fixed overnight in 4% paraformaldehyde (PFA) at 4 °C. Tissue preparation Following behavioral testing, mice were anesthetized and perfused transcardially with 0.9% saline from the left ventricle by a perfusion pump (Longer Pump, China, Cat # BT100-2J). The entire brain was carefully removed from the skull and divided into two hemispheres. One hemisphere was postfixed in 4% PFA at 4 °C overnight and subsequently dehydrated in a series of graded ethanol solutions. The brains were embedded in paraffin and cut into 5-µm thick sagittal sections using a microtome (Leica, Germany Cat. # RM2135). Serial sections containing the hippocampus and cerebral cortex were placed on gelatin-coated slides for immunohistochemistry. The other hemisphere was immediately frozen in liquid nitrogen and then stored at -80 °C until Western blot or ELISA analyses. For CSF tracer experiments, PFA postfixed forebrain tissues were sliced with a vibrating microtome (Word Precision Instruments Inc, WPI, USA; Cat. NVSLM1), at a thickness of 100 µm, and mounted onto gelatin-coated slides in sequence. Immunohistochemistry Brain sections were deparaffinized, hydrated and microwaved in citric acid buffer to achieve antigen retrieval, then treated with 3% H 2 O 2 for 20 min in order to reduce endogenous peroxidase activity. Brain sections were incubated with following primary antibodies directed against glial fibrillary acidic protein (GFAP) (1:1000; Millipore; Cat. MAB360), AQP4 (1:400; Millipore; Cat. AB3594), 6E10 (1:1000; Covance, Cat. 803001), ionized calcium binding adaptor molecule 1 (Iba-1) (1:1000; Wako, Cat. 019-19741), postsynaptic density protein 95 (PSD-95) (1:200; Abcam, Cat. ab18258) and synaptophysin (SYN) (1:200, Abcam, Cat. ab64581) at 4 °C overnight. The next day, sections were incubated for 1 hour at room temperature with secondary antibodies labeled with horseradish peroxidase and developed with a diaminobenzidine (DAB) horseradish peroxidase color development kit (Sigma-Aldrich, Cat. D8001). Partial AQP4 stained sections were counterstained with Congo red (Sigma-Aldrich, Cat. C6767). Immunofluorescence Brain sections were blocked for 1 hour at room temperature with 5% BSA and incubated with the primary antibodies including mouse anti-GFAP (1:1000) and rabbit anti-AQP4 (1:400) at 4 °C overnight. The next day, all sections were rinsed 3 times and incubated for 2 hours at room temperature in a mixture of Alexa Flour™ 555 donkey anti-mouse IgG (1:1000; Thermo Fisher; Cat. A31570) and Alexa Flour™ 488 donkey anti-rabbit IgG (1:1000; Thermo Fisher; Cat. A21206). After rinsing, the sections were incubated for 6 min in 4’,6-diamidino-2-phenylindole (DAPI) (1:1000; Thermo Fisher; Cat. D21490) and coverslipped with anti-fluorescent quencher. Thioflavine-S staining Deparaffinized sections were incubated with 1% thioflavine-S (Sigma-Aldrich) for 5 min. Next, 70% ethanol was used to differentiate for 5 min, followed by rinsing with distilled water. The brain sections coverslipped with anti-fluorescent quencher. Image analysis Brain sections were captured by a digital microscope (Leica Microsystems, Wetzlar, Germany) with constant exposure time, offset, and gain for each staining marker. The positive signal area was measured with Image J (NIH). The positive signal percentage area of GFAP, Ibal, 6E10 and AQP4 was calculated by dividing the area of positive signal to the total area in the hippocampus and adjacent cortex. The mean integrated optical density (MIOD = IOD/total area) was measured to assess immunostaining intensity of SYN and PSD-95 in the hippocampus. For analysis of AQP4 polarization, images at 400 × magnification were randomly captured from the superficial layers of the frontal cortex. The mean immunofluorescent or immunohistochemical intensity of AQP4 at the regions immediately abutting vessels or pia maters and adjacent parenchyma domains was measured. AQP4 polarization was calculated by comparing expression ratios of AQP4 at perivascular domains or abutting pia maters versus adjacent parenchymal domains [ 23 , 28 ]. For the analysis of intracisternally injected TR-d3 diffused into the brain parenchyma, positive signal percentage area of TR-d3 was measured on 3 coronal sections at + 0.86, 0 and − 1.06 mm from anterior to posterior, relative to the bregma. TR-d3 penetration along perivascular spaces was quantified on 400 × magnification images of the frontal cortex (Fig. 2 A). The fluorescence intensity within the perivascular space was measured along microvessels (diameter = 30–50 µm) extending 250 µm below the brain surface. All quantification was done blind to animal genotype and treatment. Western blotting Hippocampal tissues were homogenized at 4 °C, then centrifuged at 12,000 rpm for 15 min. The extracts were loaded onto 10%-12% Tris SDS gels and transferred onto polyvinylidene fluoride membranes. After blocking by 5% skim milk diluted with TBST buffer for 1 h, these membranes were incubated at 4 °C overnight with one of the following primary antibodies: Aβ 1−40 (1:1000, Abcam, Cat. ab20068), Aβ 1−42 (1:1000, Abcam, Cat. ab201060), SYN (1:1500, Abcam, Cat. ab64581), PSD-95 (1:1000; Abcam, Cat. ab18258), brain-derived neurotrophic factor (BDNF) (1:1000; Abcam, Cat. Ab108319), tropomyosin receptor kinase B (TrkB) (1:1000; Abcam, ab187041), cyclic adenosine monophosphate response element binding protein (CREB) (1:1000; CST, Cat. 9197S), p-CREB (1:1000; CST, Cat. 9198S) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (1:3000; Bioworld, Cat. BS60630). Following TBST washing, the membranes were incubated with horseradish peroxidase-labeled secondary antibodies (1:2000; ZSGB-BIO; Cat. ZB2301, Cat. ZB2302) for 1 h at room temperature. The protein bands were visualized via ECL plus detection system. GAPDH was used as loading control. ELISA The frontal cortex were homogenized in TBS with protease inhibitors and 1% Triton X-100, followed by centrifugation at 20,000 × g for 1 h. Supernatants were set aside for measurements utilizing soluble Aβ 1−40 (R&D Systems; Cat. DAB140B), Aβ 1−42 (R&D Systems; Cat. DAB142), interleukin 1 beta (IL-1β) (Excell Biotech Corporation; Cat. EM001-96), interleukin 6 (IL-6) (Excell Biotech Corporation; Cat. EM004-96) and tumor necrosis factor-α (TNF-α) (Excell Biotech Corporation; Cat. EM008-96). The above indexes were quantified with corresponding ELISA kits according to the manufacturer’s instructions. Statistical analysis All data were expressed as means ± SEM. Using GraphPad Prism, version 5.02 software (GraphPad software, San Diego, CA, USA), data for the MWM platform training and TR-d3 diffusion within the perivascular space, were analyzed by repeated-measures analysis of variance (ANOVA). Other data were analyzed by Student's t test, two-way ANOVA and multi-way ANOVA with Tukey's post hoc test as indicated in the figure legends. p < 0.05 was considered to have statistical significance. Results AQP4 deficiency eliminates timeliness of voluntary exercise improving cognitive functions of APP/PS1 mice We assessed the spatial learning and memory of mice using the MWM test. For 5-month-old groups, voluntary exercise intervention reduced the escape latency to find the hidden platform in WT mice (F 5,137 = 18.72, p < 0.0001) and APP/PS1 mice (F 5,132 = 37.04, p < 0.0001), but not AQP4 −/− /APP/PS1 mice (F 5,134 = 0.03363, p = 0.9994) (Fig. 1 b). For 9-month-old groups, exercise shorted the escape latency in WT mice (F 5,143 = 37.1, p < 0.0001), but not APP/PS1 mice (F 5,139 = 0.2621, p = 0.933) or AQP4 −/− /APP/PS1 mice (F 5,136 = 0.105, p = 0.9910) (Fig. 1 c). Time spent in the target quadrant and number of platform crossings during the probe trials on day 7, were high in 5-month-old WT mice and APP/PS1 mice received voluntary exercise ( p = 0.0213, p = 0.02, WT-Exe vs WT-Con; p = 0.0429, p = 0.0442, APP/PS1-Exe vs APP/PS1-Con, respectively). For 9-month groups, the above parameters were only increased in WT mice with exercise intervention ( p = 0.0126, p = 0.027, respectively). Notably, exercise did not improve spatial memory performance in 5-month-old or 9-month-old AQP4 −/− /APP/PS1 mice (Fig. 1 d-e). We performed the Y-maze test to measure mouse short-term working memory. For 5-month groups, both WT and APP/PS1 exercise mice exhibited a higher percentage of time spent in the novel arm, when compared with the corresponding sedentary controls ( p = 0.0225, WT-Exe vs WT-Con; p = 0.0235, APP/PS1-Exe vs APP/PS1-Con). Nevertheless the performance of AQP4 −/− /APP/PS1 mice was comparable between exercise and sedentary groups. For 9-month groups, exercise increased the residence time of WT mice in the novel arm ( p = 0.03), but failed to reverse working memory deficits in APP/PS1 mice and AQP4 −/− /APP/PS1 mice (Fig. 1 f). A novel object recognition test was also performed to assess the effect of exercise on recognition memory of mice. For 5-month groups, exercise improved the recognition index of WT mice and APP/PS1 mice ( p = 0.0051, p = 0.0392, respectively), but not AQP4 −/− /APP/PS1 mice. For 9-month groups, exercise was sufficient to improve recognition memory in WT mice ( p = 0.0467), but not in APP/PS1 mice and AQP4 −/− /APP/PS1 mice (Fig. 1 g). AQP4 deficiency in APP/PS1 mice abolishes timeliness of voluntary exercise promoting glymphatic transport We assessed glymphatic transport by quantifying TR-d3 penetration into the brain parenchyma following injection into the cisterna magna (Fig. 2 a). APP/PS1 mice displayed lower of TR-d3 positive area percentage and paravascular fluorescence intensity than WT mice at age of 5 months ( p = 0.0343, F 4,30 = 25.09, p < 0.0001, respectively) and 9 months ( p = 0.007, F 4,29 = 23.7, p < 0.0001, respectively). Both 5-month-old and 9-month-old WT mice received voluntary exercise increased fluorescence penetration area ( p = 0.0036, p = 0.0067, respectively) and intensity (F 1,28 = 39.05, p < 0.0001, F 1,29 = 21.12, p < 0.0001, respectively). However, for APP/PS1 mice, the improving effect of exercise on penetration of TR-d3 into the brain parenchyma was observed only in the 5-month group (Area, p = 0.01; Intensity, F 1,30 = 39.27, p < 0.0001). Exercise did not increase TR-d3 positive area percentage and paravascular fluorescence intensity in either 5- or 9-month-old AQP4 −/− /APP/PS1 mice (Fig. 2 b-f). These results suggest that voluntary exercise ameliorates glymphatic transport malfunction in the onset stage rather than mid-stage of APP/PS1 mice, and AQP4 deletion eliminates the promoting effect of exercise on glymphatic transport. AQP4 deficiency in APP/PS1 mice negates timeliness of voluntary exercise alleviating brain Aβ load We further evaluated whether the timeliness of voluntary exercise improves glymphatic transport, thereby affecting aggregation of Aβ in the brain of APP/PS1 mice. Compared with age-matched APP/PS1 mice, both 5-month-old and 9-month-old AQP4 −/− /APP/PS1 mice show more brain Aβ load in the hippocampus and frontal cortex, as revealed quantification of the area percentage and number of thioflavine-S positive fibrillary plaques and 6E10-immunopositive diffuse plaques (all p < 0.05). Exercise decreased Aβ plaque accumulation in the above two brain regions of APP/PS1 mice at 5 months (all p < 0.05 for the above indexes). The ameliorating effect of exercise on Aβ load was not observed in AQP4 −/− /APP/PS1 mice at the two different ages (Fig. 3 a-f). Consistently, both Western blot and ELISA analysis revealed that voluntary exercise only decreased Aβ 1−40 and Aβ 1−42 levels in the forebrain samples of 5-month-old APP/PS1 mice (all p < 0.05) rather than 9-month-old APP/PS1 mice. Voluntary exercise failed to decrease brain Aβ 1−40 and Aβ 1−42 levels in either 5-or 9-month-old AQP4 −/− /APP/PS1 mice (Fig. 3 g-i; Fig. S1a-b). Voluntary exercise ameliorates reactive gliosis and neuroinflammation in the onset stage rather than mid-stage of APP/PS1 mice In addition to Aβ deposition, reactive gliosis is a characteristic pathologic hallmark of AD [ 34 , 35 ]. We determined effects of voluntary exercise and AQP4 deletion on activation of astrocytes and microglia in APP/PS1 mice at different stages of AD-like progression. Both GFAP-positive astrocytes and Ibal-positive microglia were activated in the frontal cortex and hippocampus of 5-month-old APP/PS1 mice and further apparent in 9 months old. AQP4 deletion in 5-month-old APP/PS1 mice mildly increased activation of microglia and astrocytes in the frontal cortex and hippocampus (Fig. 4 a; Fig. 5 a; Fig. S2a). Microglia activation was also more pronounced in AQP4 −/− /APP/PS1 mice at aged 9 months, revealed by high percentages of Iba1 positive area in the frontal cortex ( p = 0.0107 ) and hippocampus ( p = 0.0168) as compared with age-matched APP/PS1 mice. However, a considerable proportion of astrocytes underwent atrophy in 9-month-old AQP4 −/− /APP/PS1 mice, causing no significant difference in the percentage of GFAP positive area between the two AD mouse lines (Fig. 4 a-c, Fig. S2a-d). There was no overall change in immunocytochemical labeling patterns of GFAP and Ibal in the frontal cortex and hippocampus in 5- and 9-month-old WT-exercise mice when compared with age-matched sedentary controls. But it is worth noting that, there were GFAP strong positive glia limitans abutting the pia maters of WT-exercise mice (Fig. 4 a and Fig. S2a). As for APP/PS1 mice, exercise reduced the percentages of positive area for GFAP (Cortex, p = 0.0128; Hippocampus, p = 0.0331) and Iba1 (Cortex, p = 0.0215; Hippocampus, p = 0.0107) in the 5-month-old group, but not 9-month-old group. Exercise did not mitigate reactivation of astrocytes and microglia in AQP4 −/− /APP/PS1 mice in the both age groups (Fig. 4 a-c and Fig. S2a-d). Consistently, voluntary exercise partially reduced neuroinflammatory factor levels in APP/PS1 mice in 5 months old (IL-1β, p = 0.0343; IL-6, p = 0.045; TNF-α, p = 0.031), but not 9 months old. The Aqp4 knock-out in APP/PS1 mice totally abolished the time-effect of aerobic exercise on neuroinflammation (Fig. 4 d). Voluntary exercise ameliorates impaired polarity of AQP4 in the onset stage rather than mid-stage of APP/PS1 mice Substantial evidence suggests that reactive astrogliosis results in mislocalization of AQP4 from the endfeet to the entire cell surface of astrocytes, subsequently contributes to glymphatic clearance dysfunction [ 26 , 27 , 36 ]. Both immunofluorescence and immunohistochemical staining revealed that AQP4 expression was selectively bordering the subarachnoid space and microvessels with very low intensity in the adjacent brain parenchyma in WT mice (Fig. 5 a, Fig. S3a). AQP4 was abnormally expressed at the non-vascular domains, especially surrounding Aβ plaques, in 5-month-old APP/PS1 mice. The mislocalization of AQP4 was further obvious in the brain parenchyma of 9-month-old APP/PS1 mice, causing total loss of polarity distribution of AQP4. Voluntary exercise reduced astrocyte activation in 5-month-old APP/PS1 mice, in turn improving AQP4 polarity abutting pia mater (Immunofluorescence, p = 0.005; Immunohistochemistry, p = 0.0148) and microvessels (Immunofluorescence, p = 0.0302; Immunohistochemistry, p = 0.0398). By contrast, in 9-month-old APP/PS1-exercise mice, this improvement was not apparent because reactive astrogliosis was not reduced (Fig. 5 a-c; Fig. S2a-d). WT exercise mice showed increases in AQP4 polarization abutting pia maters (Immunofluorescence: 5-month, p = 0.0032, 9-month, p < 0.0001, respectively) and microvessels (Immunofluorescence: 5-month, p = 0.0161, 9-month, p = 0.0002; respectively) (Fig. 5 a, Fig. S3a). AQP4 deficiency eliminates timeliness of voluntary exercise activating BDNF signaling pathway and improving synaptic protein levels in APP/PS1 mice BDNF signaling pathway is associated with synaptic function and regulates the expression of synaptic proteins such as presynaptic protein SYN and postsynaptic protein PSD-95 [ 37 , 38 ]. Down regulation of BDNF signaling pathway and synaptic protein loss occur in AD patients [ 39 , 40 ] as well as animal models of AD [ 41 , 42 ]. Moreover, BDNF signaling pathway is involved in neuroproection of physical exercise in AD [ 43 , 44 ], stroke [ 45 , 46 ] and depression [ 47 , 48 ]. In this way, the present study measured the activation of BDNF-TrkB-CREB pathway and expression levels of SYN and PSD-95 in the forebrain. The expression levels of mature BDNF, TrkB and p-CREB generally decreased in APP/PS1 mice compared to WT controls aged at 5 months ( p < 0.0001, p = 0.005, p = 0.009, respectively) and 9 months ( p = 0.004, p = 0.08043, p = 0.0174, respectively). Voluntary activated this signaling pathway in WT mice aged at 5 months (BDNF: p = 0.0352, TrkB: p = 0.0997, p-CREB: p = 0.0029, respectively) and 9 months (BDNF: p = 0.012, TrkB: p = 0.0308, p-CREB: p = 0.0402, respectively), but only in 5-month-old APP/PS1 mice (BDNF: p = 0.0153, TrkB: p = 0.018, p-CREB: p = 0.0379, respectively) (Fig. 6 a-b). Consistently, voluntary exercise significantly upregulated SYN and PSD-95 levels in the hippocampus of 5-month-old WT and APP/PS1 mice (all p < 0.05), but not AQP4 −/− /APP/PS1 mice, as revealed by immunohistochemical staining and Western blot analysis (Fig. 6 c-d, Fig. S4a-b). For 9-month-old groups, an improving effect of voluntary exercise on the synaptic protein levels was only observed in WT mice (Immunohistochemistry SYN: p = 0.0231, PSD-95: p = 0.0121; Western blot: SYN: p = 0.0113, PSD-95: p = 0.0108). Discussion At present, there is a lack of effective pharmacological treatments and other methods for AD. Exploring ways of preventing, or delaying the onset this disease is a pragmatic strategy. Regular exercise can reduce the occurrence of AD in the healthy elderly population and delay the progression of MCI, although the beneficial effect is not apparent in the clinical treatment of AD [ 6 – 11 ]. The underlying mechanism has not yet to be determined. The current study was designed to address this issue. The results show that the beneficial effect of voluntary exercise intervention on AD-like pathology of APP/PS1 mice is time-dependent. Early exercise intervention improves AQP4 polarity, subsequently promoting glymphatic transport while reducing brain Aβ load. However, during the mid-stage of AD-like pathology, AQP4 polarity is disrupted due to reactive astrogliosis. Therefore, exercise intervention is unable to improve glymphatic transport, and has no improving effect on Aβ clearance. Voluntary exercise does not mitigate Aβ accumulation and cognitive impairment of AQP4 −/− /APP/PS1 mice, further suggesting that AQP4-dependent glymphatic clearance is a key factor for exercise therapy to delay the onset of AD. Increasing evidence supports the idea that the glymphatic system plays a central role in the clearance of macromolecular substances from the brain [ 22 , 23 ]. This model of glymphatic system has opened up new perspectives in understanding the pathogenesis of AD [ 49 , 50 ], stroke [ 51 , 52 ], brain injury [ 53 ] and other neurological diseases [ 54 , 55 ]. Glymphatic system visualization is not only implemented in animal studies [ 56 , 57 ], but also being used for clinical imaging diagnosis of hydrocephalus [ 58 , 59 ] and AD [ 60 ]. It is suggested that the driving force of glymphatic transport comes from arterial pulsation, and is facilitated by AQP4 mediated bulk fluid flow from the para-arterial space entering the interstitial space, and finally into the para-venous space [ 23 ]. AQP4 gene knockout significantly delays clearance of exogenous Aβ from the brain following intrastriatal injection [ 23 ]. Our present and previous studies consistently demonstrate that AQP4 deletion in APP/PS1 mice exacerbates glymphatic clearance impairment and Aβ plaque load [ 28 , 31 ]. Together, these results indicate that AQP4 is vital for glymphatic clearance, thus playing a critical role in regulating the pathological progress of AD. The current results further suggest that glymphatic clearance not only depends on the presence of AQP4, but also on its specific localization at the perivascular endfeet of astrocytes. Early studies have shown that glymphatic clearance of exogenous Aβ in aged mice is significantly less than that of young mice [ 28 ]. The primary cause is activated astrocytes abnormally expressing AQP4 in the non-perivascular membrane. Aged mice taking part in voluntary exercise exhibit decreased astrocyte activation, improvement of AQP4 polarity, and low brain Aβ concentration, compared to sedentary controls. Consistently, the present results have confirmed that in the onset stage of APP/PS1 mice, voluntary exercise intervention attenuates reactive astrogliosis and mislocalization of AQP4 in the nonvascular domains of the brain parenchyma of astrocytes, subsequently reducing Aβ plaque accumulation. These results imply that specific expression of AQP4 on the perivascular endfeet of astrocytes is the molecular structural basis for maintaining glymphatic transport. Additionally, previous studies report that alterations in AQP4 expression and localization in the frontotemporal lobe are associated with AD status and pathology, which is related to noncoding aqp4 single-nucleotide polymorphisms [ 61 , 62 ]. Notably, perivascular AQP4 localization is preserved among individuals older than 85 years who remains cognitively intact. Lymphatic transport ability of AD patients has been found to be lower than that of healthy controls of the same age, via assessment of perivascular fluid movement with diffusion tensor MRI [ 60 ]. Our recent studies reported that a moderate-intensity aerobic dance improves cognitive function, especially episodic memory and processing speed, in MCI patients [ 63 ]. The future studies are necessary for determining whether this protective effect is linked to improving glymphatic transport capacity. Consistent with the present findings, previous studies report an age-dependent beneficial effect of a continuous non-shock treadmill exercise paradigm on the pathogenic characteristics of APP/PS1 transgenic mice. Five-week of exercise reduces Aβ 1−40 and Aβ 1−42 levels in the hippocampus of adult (7–8 month-old) APP/PS1 mice but not aged (24-month-old) APP/PS1 mice, and does not reduce plaque loading in either adult or aged APP/PS1 mice [ 21 ]. By contrast, a 4-month treadmill exercise intervention significantly improves spatial learning and memory ability, reduces amyloid plaques in the hippocampus, and induces neurogenesis in the dentate gyrus of 12-month-old APP/PS1 mice [ 64 ]. This indicates that a longer exercise intervention may have a more positive effect in delaying the pathological process of AD mice. In addition, exercise of different intensity may bring different effects on APP/PS1 mice. Moderate exercise intensity (60–70% of max oxygen uptake) seems to be more effective in increasing lipid metabolism and reducing soluble Aβ levels, compared with low-intensity exercise (45–55% of maximal oxygen uptake) [ 65 ]. Based on this, future studies are also needed to clarify the effects of exercise intensity and duration on glymphatic clearance. In conclusion, this study has revealed that voluntary exercise promotes the removal of Aβ by the glymphatic system, attenuating its aggregation, and subsequently reducing astrocyte activation, which is conducive to maintain AQP4 polarity. This effect, in turn, maintains glymphatic transport capability, thereby effectively promoting cognitive function of adult WT mice and delaying the onset of AD-like pathogenesis in APP/PS1 mice. Long-term voluntary exercise intervention fails to improve Aβ clearance and alleviate AD-like pathology under AQP4 deficiency or AQP4 polarity damage condition. These results highlight that AQP4-dependent glymphatic transport is the neural basis for exercise against AD. The finding has revealed the molecular pathology mechanism that exercise helps prevent AD, but cannot treat AD, which is a long-standing dilemma in the field of AD research. Keeping a certain amount of exercise vitality is a convenient, effective and simple way to prevent the onset of AD. Abbreviations AD: Alzheimer’s disease; ANOVA: Analysis of variance; AQP4: aquaporin-4; BDNF: brain-derived neurotrophic factor; CREB: cyclic adenosine monophosphate response element binding protein; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GFAP: glial fibrillary acid protein; Iba-1: ionized calcium binding adaptor molecule 1; IL-1β: interleukin 1β; IL-6: interleukin 6; MWM: Morris water maze; NORT: novel object recognition test; PSD-95: postsynaptic density protein 95; SYN: synaptophysin; TrkB: tropomyosin receptor kinase B; TNF-α: tumor necrosis factor-α; TR-d3: Texas Red-dextran-3; WT: Wild-type. Declarations Ethics approval and consent to participate All procedures were approved by Institutional Animal Care and Use Committee of Nanjing Medical University (IACUC-1601106). Consent for publication Not applicable. Availability of data and material All data mentioned in this article are available on published article. Competing interests Authors have no conflict of interest to declare. Funding This study was supported by grants from the National Natural Science Foundation of China (No. 81772454 and No. 81671070). Authors’ contributions YL, XW, RZ, PH and SZ performed experiments and statistical analyses. YL, XW and QL were involved in interpretation of data. TW made substantial contributions to design experiment. YL and MX wrote the manuscript. CM revised the article for important intellectual content. All authors read and approved the final version of the article. Acknowledgments Not applicable. References Querfurth HW, LaFerla FM. Alzheimer's disease. N Engl J Med. 2010;362(4):329-44. Caselli RJ, Beach TG, Knopman DS, Graff-Radford NR. Alzheimer Disease: Scientific Breakthroughs and Translational Challenges. Mayo Clin Proc. 2017;92(6):978-94. Kivipelto M, Mangialasche F, Ngandu T. Lifestyle interventions to prevent cognitive impairment, dementia and Alzheimer disease. 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Data are analyzed by the three-way factor ANOVA with Tukey’s post hoc test and represent mean ± SEM from 3 independent experiments and 4 mice per group. *p < 0.05, Exercise versus Control; ##p < 0.01, ###p < 0.001, APP/PS1 versus WT; &p < 0.05, &&p < 0.01, &&&p < 0.001, AQP4−/−/APP/PS1 versus APP/PS1. FigureS1.tif Additional file 1: Figure S1. The effect of voluntary exercise on brain Aβ levels in 5- and 9-month-old APP/PS1 mice with or without AQP4. Data are analyzed by the three-way factor ANOVA with Tukey’s post hoc test and represent mean ± SEM from 3 independent experiments and 4 mice per group. *p < 0.05, Exercise versus Control; ##p < 0.01, ###p < 0.001, APP/PS1 versus WT; &p < 0.05, &&p < 0.01, &&&p < 0.001, AQP4−/−/APP/PS1 versus APP/PS1. FigureS1.tif Additional file 1: Figure S1. The effect of voluntary exercise on brain Aβ levels in 5- and 9-month-old APP/PS1 mice with or without AQP4. Data are analyzed by the three-way factor ANOVA with Tukey’s post hoc test and represent mean ± SEM from 3 independent experiments and 4 mice per group. *p < 0.05, Exercise versus Control; ##p < 0.01, ###p < 0.001, APP/PS1 versus WT; &p < 0.05, &&p < 0.01, &&&p < 0.001, AQP4−/−/APP/PS1 versus APP/PS1. FigureS2.tif Additional file 2: Figure S2. The effect of voluntary exercise on reactive gliosis in the hippocampus of 5- and 9-month-old APP/PS1 mice with or without AQP4. (A-B) Immunohistochemical staining for GFAP and Iba1 in the hippocampal radiatum layer, respectively. (C-D) Quantification of the percentage of GFAP and Iba1 positive area in the hippocampus, respectively. Data are analyzed by the three-way factor ANOVA with Tukey’s post hoc test and represent mean ± SEM from 5 sections per mouse and 4 mice per group. *p < 0.05, Exercise versus Control; #p < 0.05, ##p < 0.01, ###p < 0.001, APP/PS1 versus WT; &p < 0.05, &&p < 0.01, &&&p < 0.001, AQP4−/−/APP/PS1 versus APP/PS1. FigureS2.tif Additional file 2: Figure S2. The effect of voluntary exercise on reactive gliosis in the hippocampus of 5- and 9-month-old APP/PS1 mice with or without AQP4. (A-B) Immunohistochemical staining for GFAP and Iba1 in the hippocampal radiatum layer, respectively. (C-D) Quantification of the percentage of GFAP and Iba1 positive area in the hippocampus, respectively. Data are analyzed by the three-way factor ANOVA with Tukey’s post hoc test and represent mean ± SEM from 5 sections per mouse and 4 mice per group. *p < 0.05, Exercise versus Control; #p < 0.05, ##p < 0.01, ###p < 0.001, APP/PS1 versus WT; &p < 0.05, &&p < 0.01, &&&p < 0.001, AQP4−/−/APP/PS1 versus APP/PS1. FigureS2.tif Additional file 2: Figure S2. The effect of voluntary exercise on reactive gliosis in the hippocampus of 5- and 9-month-old APP/PS1 mice with or without AQP4. (A-B) Immunohistochemical staining for GFAP and Iba1 in the hippocampal radiatum layer, respectively. (C-D) Quantification of the percentage of GFAP and Iba1 positive area in the hippocampus, respectively. Data are analyzed by the three-way factor ANOVA with Tukey’s post hoc test and represent mean ± SEM from 5 sections per mouse and 4 mice per group. *p < 0.05, Exercise versus Control; #p < 0.05, ##p < 0.01, ###p < 0.001, APP/PS1 versus WT; &p < 0.05, &&p < 0.01, &&&p < 0.001, AQP4−/−/APP/PS1 versus APP/PS1. FigureS3.tif Additional file 3: Figure S3. The effect of voluntary exercise on AQP4 expression and polarity in 5- and 9-month-old APP/PS1 mice as revealed by AQP4 and Congo Red double staining. (A) Representative AQP4 immunoreactive images of the frontal cortex showed localization of AQP4 abutting the pia maters (black arrows) and microvessels (grey arrows). Note that AQP4 was expressed at parenchymal domains surrounding Aβ plaques. (B-C) The percentage of AQP4 positive area in the frontal cortex of mice. (C) The polarization of AQP4 in the frontal cortex of mice. Data are analyzed by the three-way factor ANOVA with Tukey’s post hoc test and represent mean ± SEM from 4 sections (16-20 microvessels) per mouse and 4 mice per group. *p < 0.05, **p < 0.01, Exercise versus Control; #p < 0.05, ###p < 0.001, APP/PS1 versus WT. FigureS3.tif Additional file 3: Figure S3. The effect of voluntary exercise on AQP4 expression and polarity in 5- and 9-month-old APP/PS1 mice as revealed by AQP4 and Congo Red double staining. (A) Representative AQP4 immunoreactive images of the frontal cortex showed localization of AQP4 abutting the pia maters (black arrows) and microvessels (grey arrows). Note that AQP4 was expressed at parenchymal domains surrounding Aβ plaques. (B-C) The percentage of AQP4 positive area in the frontal cortex of mice. (C) The polarization of AQP4 in the frontal cortex of mice. Data are analyzed by the three-way factor ANOVA with Tukey’s post hoc test and represent mean ± SEM from 4 sections (16-20 microvessels) per mouse and 4 mice per group. *p < 0.05, **p < 0.01, Exercise versus Control; #p < 0.05, ###p < 0.001, APP/PS1 versus WT. FigureS3.tif Additional file 3: Figure S3. The effect of voluntary exercise on AQP4 expression and polarity in 5- and 9-month-old APP/PS1 mice as revealed by AQP4 and Congo Red double staining. (A) Representative AQP4 immunoreactive images of the frontal cortex showed localization of AQP4 abutting the pia maters (black arrows) and microvessels (grey arrows). Note that AQP4 was expressed at parenchymal domains surrounding Aβ plaques. (B-C) The percentage of AQP4 positive area in the frontal cortex of mice. (C) The polarization of AQP4 in the frontal cortex of mice. Data are analyzed by the three-way factor ANOVA with Tukey’s post hoc test and represent mean ± SEM from 4 sections (16-20 microvessels) per mouse and 4 mice per group. *p < 0.05, **p < 0.01, Exercise versus Control; #p < 0.05, ###p < 0.001, APP/PS1 versus WT. FigureS4.tif Additional file 4: Figure S4. The effect of voluntary exercise on SYN and PSD-95 expression in the hippocampus of 5- and 9-month-old APP/PS1 mice with or without AQP4. (A) Immunohistochemical staining for SYN and PSD-95 in the hippocampal CA1. (B-C) Quantification of SYN and PSD-95 immunoreactive intensity in the hippocampus. Data are analyzed by the three-way factor ANOVA with Tukey’s post hoc test and represent mean ± SEM from 4 sections per mouse and 4 mice per group. *p < 0.05, Exercise versus Control; #p < 0.05, APP/PS1 versus WT; &p < 0.05, APP/PS1 versus AQP4−/−/APP/PS1. FigureS4.tif Additional file 4: Figure S4. The effect of voluntary exercise on SYN and PSD-95 expression in the hippocampus of 5- and 9-month-old APP/PS1 mice with or without AQP4. (A) Immunohistochemical staining for SYN and PSD-95 in the hippocampal CA1. (B-C) Quantification of SYN and PSD-95 immunoreactive intensity in the hippocampus. Data are analyzed by the three-way factor ANOVA with Tukey’s post hoc test and represent mean ± SEM from 4 sections per mouse and 4 mice per group. *p < 0.05, Exercise versus Control; #p < 0.05, APP/PS1 versus WT; &p < 0.05, APP/PS1 versus AQP4−/−/APP/PS1. FigureS4.tif Additional file 4: Figure S4. The effect of voluntary exercise on SYN and PSD-95 expression in the hippocampus of 5- and 9-month-old APP/PS1 mice with or without AQP4. (A) Immunohistochemical staining for SYN and PSD-95 in the hippocampal CA1. (B-C) Quantification of SYN and PSD-95 immunoreactive intensity in the hippocampus. Data are analyzed by the three-way factor ANOVA with Tukey’s post hoc test and represent mean ± SEM from 4 sections per mouse and 4 mice per group. *p < 0.05, Exercise versus Control; #p < 0.05, APP/PS1 versus WT; &p < 0.05, APP/PS1 versus AQP4−/−/APP/PS1. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-108136","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research","associatedPublications":[],"authors":[{"id":4757198,"identity":"5ff2353d-b67f-4748-b22b-bdc44c6b61ba","order_by":0,"name":"Yun Liu","email":"","orcid":"","institution":"Nanjing Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yun","middleName":"","lastName":"Liu","suffix":""},{"id":4757199,"identity":"2feadaa2-829f-40c5-bcc7-160faf8aa4d0","order_by":1,"name":"Weixi Feng","email":"","orcid":"","institution":"Nanjing Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Weixi","middleName":"","lastName":"Feng","suffix":""},{"id":4757200,"identity":"f8f639dc-1f78-46ab-b514-646a055ee486","order_by":2,"name":"Panpan Hu","email":"","orcid":"","institution":"Nanjing Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Panpan","middleName":"","lastName":"Hu","suffix":""},{"id":4757201,"identity":"af041cfe-059e-4f96-945d-de2109c56a2c","order_by":3,"name":"Shuang Zhai","email":"","orcid":"","institution":"Nanjing Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Shuang","middleName":"","lastName":"Zhai","suffix":""},{"id":4757202,"identity":"f7cd7deb-0617-403c-9476-5d0d98d410ae","order_by":4,"name":"Rui Zhang","email":"","orcid":"","institution":"Nanjing Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Rui","middleName":"","lastName":"Zhang","suffix":""},{"id":4757203,"identity":"b34803ce-633c-4d46-bfec-7d8841c1321c","order_by":5,"name":"Qian Li","email":"","orcid":"","institution":"Nanjing Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Qian","middleName":"","lastName":"Li","suffix":""},{"id":4757204,"identity":"20553e30-6668-43db-bf5e-5e0e59fce393","order_by":6,"name":"Charles Marsahall","email":"","orcid":"","institution":"Nanjing Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Charles","middleName":"","lastName":"Marsahall","suffix":""},{"id":4757205,"identity":"e10b0d03-c30d-49a7-9dcf-dd9fa687fd07","order_by":7,"name":"Ming Xiao","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAvElEQVRIiWNgGAWjYDACZhBRAaEYeIjXcoYkLSDA2AZlEKVFt5338GveeXfYdWckMD5428Ygb05Ii9lhvjTLmdueMZvdSGA2nNvGYLizgaAWHjODj9sOg7SwSfO2MSQYHCBGS+IcsBb238RqMX7wsQFiCzPRtjDOOAbUcuZhs+SccxKGGwhqOX/G+DNPzeFks+PJBz+8KbORJ2gLELBJAIlkYOw0AGkJwuqBgPkDkLAjSukoGAWjYBSMTAAACBA9+eKzejoAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0001-5528-9102","institution":"Nanjing Medical University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Ming","middleName":"","lastName":"Xiao","suffix":""},{"id":4757206,"identity":"874375d1-2dc6-4f14-988d-b218067548fb","order_by":8,"name":"Ting Wu","email":"","orcid":"","institution":"Nanjing Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Ting","middleName":"","lastName":"Wu","suffix":""}],"badges":[],"createdAt":"2020-11-13 19:14:36","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-108136/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-108136/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":3618337,"identity":"021ea806-1434-4fd1-9b18-cc66c2acf5f2","added_by":"auto","created_at":"2020-11-16 23:13:32","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":291865,"visible":true,"origin":"","legend":"The effect of voluntary exercise on cognitive function in 5- and 9-month-old APP/PS1 mice with or without AQP4. a Schematic representation of the experimental design. Two and several months old WT mice, APP/PS1 mice and AQP4-/-/APP/PS1 mice were randomly assigned to voluntary exercise group or sedentary control group. The mice in each group were given the corresponding treatment for 2 months and then tested by the Y-maze, novel object recognition test (NORT) and Morris water maze (MWM) followed by pathological and biochemical analyses. b-c The mean escape latency during the hidden platform training period of the MWM of 5-month-old groups (b) and 9-month-old groups (c). d The percentage of time spent in the target quadrant. e The number of crossing the platform area. f The percentage of time spent in the novel arm of Y-maze. g The recognition index in the NORT. Data in b and c were analyzed by the repeated-measures ANOVA with post hoc Student-Newman-Keuls test. Data in d-g were analyzed by the three-way factor ANOVA with Tukey’s post hoc test. Data represent mean ± SEM from 12-15 mice per group. *p \u003c 0.05, **p \u003c 0.01, Exercise versus Control; #p \u003c 0.05, ##p \u003c 0.01, ###p \u003c 0.001, APP/PS1 versus WT; \u0026p \u003c 0.05, \u0026\u0026p \u003c 0.01, \u0026\u0026\u0026p \u003c 0.001, AQP4−/−/APP/PS1 versus APP/PS1.","description":"","filename":"OnlineFigure1.Png","url":"https://assets-eu.researchsquare.com/files/rs-108136/v1/5eb48326aa11a3fd844ee3a2.Png"},{"id":3618326,"identity":"012b5aea-efbe-4b1c-8bd3-e7386e61f1ed","added_by":"auto","created_at":"2020-11-16 23:13:08","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":291865,"visible":true,"origin":"","legend":"The effect of voluntary exercise on cognitive function in 5- and 9-month-old APP/PS1 mice with or without AQP4. a Schematic representation of the experimental design. Two and several months old WT mice, APP/PS1 mice and AQP4-/-/APP/PS1 mice were randomly assigned to voluntary exercise group or sedentary control group. The mice in each group were given the corresponding treatment for 2 months and then tested by the Y-maze, novel object recognition test (NORT) and Morris water maze (MWM) followed by pathological and biochemical analyses. b-c The mean escape latency during the hidden platform training period of the MWM of 5-month-old groups (b) and 9-month-old groups (c). d The percentage of time spent in the target quadrant. e The number of crossing the platform area. f The percentage of time spent in the novel arm of Y-maze. g The recognition index in the NORT. Data in b and c were analyzed by the repeated-measures ANOVA with post hoc Student-Newman-Keuls test. Data in d-g were analyzed by the three-way factor ANOVA with Tukey’s post hoc test. Data represent mean ± SEM from 12-15 mice per group. *p \u003c 0.05, **p \u003c 0.01, Exercise versus Control; #p \u003c 0.05, ##p \u003c 0.01, ###p \u003c 0.001, APP/PS1 versus WT; \u0026p \u003c 0.05, \u0026\u0026p \u003c 0.01, \u0026\u0026\u0026p \u003c 0.001, AQP4−/−/APP/PS1 versus APP/PS1.","description":"","filename":"OnlineFigure1.Png","url":"https://assets-eu.researchsquare.com/files/rs-108136/v1/2ec192b107d653924f2ee7cd.Png"},{"id":3618311,"identity":"d99fbef3-87e3-4547-b20b-a4f21b548feb","added_by":"auto","created_at":"2020-11-16 23:13:01","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":291865,"visible":true,"origin":"","legend":"The effect of voluntary exercise on cognitive function in 5- and 9-month-old APP/PS1 mice with or without AQP4. a Schematic representation of the experimental design. Two and several months old WT mice, APP/PS1 mice and AQP4-/-/APP/PS1 mice were randomly assigned to voluntary exercise group or sedentary control group. The mice in each group were given the corresponding treatment for 2 months and then tested by the Y-maze, novel object recognition test (NORT) and Morris water maze (MWM) followed by pathological and biochemical analyses. b-c The mean escape latency during the hidden platform training period of the MWM of 5-month-old groups (b) and 9-month-old groups (c). d The percentage of time spent in the target quadrant. e The number of crossing the platform area. f The percentage of time spent in the novel arm of Y-maze. g The recognition index in the NORT. Data in b and c were analyzed by the repeated-measures ANOVA with post hoc Student-Newman-Keuls test. Data in d-g were analyzed by the three-way factor ANOVA with Tukey’s post hoc test. Data represent mean ± SEM from 12-15 mice per group. *p \u003c 0.05, **p \u003c 0.01, Exercise versus Control; #p \u003c 0.05, ##p \u003c 0.01, ###p \u003c 0.001, APP/PS1 versus WT; \u0026p \u003c 0.05, \u0026\u0026p \u003c 0.01, \u0026\u0026\u0026p \u003c 0.001, AQP4−/−/APP/PS1 versus APP/PS1.","description":"","filename":"OnlineFigure1.Png","url":"https://assets-eu.researchsquare.com/files/rs-108136/v1/5492d78455505928ef629189.Png"},{"id":3618339,"identity":"5f299063-5f94-424b-a258-8dffc0bbbbcf","added_by":"auto","created_at":"2020-11-16 23:13:32","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":769604,"visible":true,"origin":"","legend":"The effect of voluntary exercise on glymphatic transport in 5- and 9-month-old APP/PS1 mice with or without AQP4. a Schematic diagram for intracisternal injection of TR-d3 tracer and experiment schedule. b Representative images of coronal brain sections of each group at bregma 0 mm showing TR-d3 influx into the brain at 40 min after cisterna magna injection. c High magnification micrographs of the frontal cortex showing TR-d3 distribution on the brain surface (arrowheads) and within the perivascular space (stars). d Quantification of the area percentage of TR-d3 fluorescence in the brain section. e-f Quantification of the mean fluorescence intensity of TR-d3 along the perivascular space. Data in d were analyzed by the three-way factor ANOV with Tukey’s post hoc test. Data in e and f were analyzed by the repeated-measures ANOVA with post hoc Student-Newman-Keuls test. Data represent mean ± SEM from 4 sections (in d) or 6-8 microvessels (in e and f) per mouse, and four mice per group. *p \u003c 0.05, **p \u003c 0.01, Exercise versus Control; #p \u003c 0.05, ###p \u003c 0.001, APP/PS1 versus WT; \u0026\u0026p \u003c 0.01, \u0026\u0026\u0026p \u003c 0.001, AQP4−/−/APP/PS1 versus APP/PS1.","description":"","filename":"OnlineFigure2.Png","url":"https://assets-eu.researchsquare.com/files/rs-108136/v1/d8467e55c83b748d82983f07.Png"},{"id":3618328,"identity":"f1df43e7-6f37-496c-9ffe-71d04445e427","added_by":"auto","created_at":"2020-11-16 23:13:08","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":769604,"visible":true,"origin":"","legend":"The effect of voluntary exercise on glymphatic transport in 5- and 9-month-old APP/PS1 mice with or without AQP4. a Schematic diagram for intracisternal injection of TR-d3 tracer and experiment schedule. b Representative images of coronal brain sections of each group at bregma 0 mm showing TR-d3 influx into the brain at 40 min after cisterna magna injection. c High magnification micrographs of the frontal cortex showing TR-d3 distribution on the brain surface (arrowheads) and within the perivascular space (stars). d Quantification of the area percentage of TR-d3 fluorescence in the brain section. e-f Quantification of the mean fluorescence intensity of TR-d3 along the perivascular space. Data in d were analyzed by the three-way factor ANOV with Tukey’s post hoc test. Data in e and f were analyzed by the repeated-measures ANOVA with post hoc Student-Newman-Keuls test. Data represent mean ± SEM from 4 sections (in d) or 6-8 microvessels (in e and f) per mouse, and four mice per group. *p \u003c 0.05, **p \u003c 0.01, Exercise versus Control; #p \u003c 0.05, ###p \u003c 0.001, APP/PS1 versus WT; \u0026\u0026p \u003c 0.01, \u0026\u0026\u0026p \u003c 0.001, AQP4−/−/APP/PS1 versus APP/PS1.","description":"","filename":"OnlineFigure2.Png","url":"https://assets-eu.researchsquare.com/files/rs-108136/v1/e4b4189e1d5dfed8dd5e3e43.Png"},{"id":3618313,"identity":"998909c7-4f5c-4a68-8fd0-d455e230e2dd","added_by":"auto","created_at":"2020-11-16 23:13:02","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":769604,"visible":true,"origin":"","legend":"The effect of voluntary exercise on glymphatic transport in 5- and 9-month-old APP/PS1 mice with or without AQP4. a Schematic diagram for intracisternal injection of TR-d3 tracer and experiment schedule. b Representative images of coronal brain sections of each group at bregma 0 mm showing TR-d3 influx into the brain at 40 min after cisterna magna injection. c High magnification micrographs of the frontal cortex showing TR-d3 distribution on the brain surface (arrowheads) and within the perivascular space (stars). d Quantification of the area percentage of TR-d3 fluorescence in the brain section. e-f Quantification of the mean fluorescence intensity of TR-d3 along the perivascular space. Data in d were analyzed by the three-way factor ANOV with Tukey’s post hoc test. Data in e and f were analyzed by the repeated-measures ANOVA with post hoc Student-Newman-Keuls test. Data represent mean ± SEM from 4 sections (in d) or 6-8 microvessels (in e and f) per mouse, and four mice per group. *p \u003c 0.05, **p \u003c 0.01, Exercise versus Control; #p \u003c 0.05, ###p \u003c 0.001, APP/PS1 versus WT; \u0026\u0026p \u003c 0.01, \u0026\u0026\u0026p \u003c 0.001, AQP4−/−/APP/PS1 versus APP/PS1.","description":"","filename":"OnlineFigure2.Png","url":"https://assets-eu.researchsquare.com/files/rs-108136/v1/17f421c0410a1dc9ad96761e.Png"},{"id":3618341,"identity":"faa226e3-73fd-4bd3-816a-5b88949156b6","added_by":"auto","created_at":"2020-11-16 23:13:33","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1487518,"visible":true,"origin":"","legend":"The effect of voluntary exercise on Aβ accumulation in 5- and 9-month-old APP/PS1 mice with or without AQP4. a-b 6E10 immunostaining and thioflavine-S staining showing Aβ plaques in the hippocampus and adjacent cortex, respectively. c-d The area percentage and number of 6E10-immunopositive diffuse plaques. e-f The area percentage and number of thioflavine-S positive fibrillary plaques. g-i Representative bands of Western bolt and densitometry analysis of Aβ1-40 and Aβ1-42 expression levels in the hippocampus. Data are analyzed by the three-way factor ANOVA with Tukey’s post hoc test and represent mean ± SEM from 5 sections per mouse (in c-f) or three independent experiments (in h-i) and four mice per group. *p \u003c 0.05, **p \u003c 0.01, Exercise versus Control; #p \u003c 0.05, ###p \u003c 0.001, APP/PS1 versus WT; \u0026p \u003c 0.05, \u0026\u0026p \u003c 0.01, \u0026\u0026\u0026p \u003c 0.001, AQP4−/−/APP/PS1 versus APP/PS1.","description":"","filename":"OnlineFigure3.Png","url":"https://assets-eu.researchsquare.com/files/rs-108136/v1/2ce9e1d10742dc8cc5320c9b.Png"},{"id":3618330,"identity":"6d01bad5-5a1f-42a8-ae3b-bc2ac86c3304","added_by":"auto","created_at":"2020-11-16 23:13:09","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1487518,"visible":true,"origin":"","legend":"The effect of voluntary exercise on Aβ accumulation in 5- and 9-month-old APP/PS1 mice with or without AQP4. a-b 6E10 immunostaining and thioflavine-S staining showing Aβ plaques in the hippocampus and adjacent cortex, respectively. c-d The area percentage and number of 6E10-immunopositive diffuse plaques. e-f The area percentage and number of thioflavine-S positive fibrillary plaques. g-i Representative bands of Western bolt and densitometry analysis of Aβ1-40 and Aβ1-42 expression levels in the hippocampus. Data are analyzed by the three-way factor ANOVA with Tukey’s post hoc test and represent mean ± SEM from 5 sections per mouse (in c-f) or three independent experiments (in h-i) and four mice per group. *p \u003c 0.05, **p \u003c 0.01, Exercise versus Control; #p \u003c 0.05, ###p \u003c 0.001, APP/PS1 versus WT; \u0026p \u003c 0.05, \u0026\u0026p \u003c 0.01, \u0026\u0026\u0026p \u003c 0.001, AQP4−/−/APP/PS1 versus APP/PS1.","description":"","filename":"OnlineFigure3.Png","url":"https://assets-eu.researchsquare.com/files/rs-108136/v1/bec2fe713aa627d3e810007f.Png"},{"id":3618315,"identity":"6cd3eac7-de58-4745-b686-01751944d6c3","added_by":"auto","created_at":"2020-11-16 23:13:03","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1487518,"visible":true,"origin":"","legend":"The effect of voluntary exercise on Aβ accumulation in 5- and 9-month-old APP/PS1 mice with or without AQP4. a-b 6E10 immunostaining and thioflavine-S staining showing Aβ plaques in the hippocampus and adjacent cortex, respectively. c-d The area percentage and number of 6E10-immunopositive diffuse plaques. e-f The area percentage and number of thioflavine-S positive fibrillary plaques. g-i Representative bands of Western bolt and densitometry analysis of Aβ1-40 and Aβ1-42 expression levels in the hippocampus. Data are analyzed by the three-way factor ANOVA with Tukey’s post hoc test and represent mean ± SEM from 5 sections per mouse (in c-f) or three independent experiments (in h-i) and four mice per group. *p \u003c 0.05, **p \u003c 0.01, Exercise versus Control; #p \u003c 0.05, ###p \u003c 0.001, APP/PS1 versus WT; \u0026p \u003c 0.05, \u0026\u0026p \u003c 0.01, \u0026\u0026\u0026p \u003c 0.001, AQP4−/−/APP/PS1 versus APP/PS1.","description":"","filename":"OnlineFigure3.Png","url":"https://assets-eu.researchsquare.com/files/rs-108136/v1/c6da8ecbe8c3ee622dc2080b.Png"},{"id":3618343,"identity":"7bfb7a7e-f13c-47b0-a6be-26ea874e0075","added_by":"auto","created_at":"2020-11-16 23:13:33","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":3447455,"visible":true,"origin":"","legend":"The effect of voluntary exercise on reactive gliosis and inflammatory factor levels in the brain of 5- and 9-month-old APP/PS1 mice with or without AQP4. a-b Immunohistochemical staining for GFAP and Iba1 in the frontal cortex. c Quantification of the percentage of GFAP and Iba1 positive area in the frontal cortex, respectively. Data represent mean ± SEM from 5 sections per mouse and four mice per group. d ELISA analysis of inflammatory factors IL-1β, IL-6 and TNF-α levels in the frontal cortex. Data represent mean ± SEM from 3 independent experiments and 4 mice per group. Three-way factor ANOVA with Tukey’s post hoc test. *p \u003c 0.05, Exercise versus Control; #p \u003c 0.05, ##p \u003c 0.01,###p \u003c 0.001, APP/PS1 versus WT; \u0026p \u003c 0.05, \u0026\u0026p \u003c 0.01, \u0026\u0026\u0026p \u003c 0.001, AQP4−/−/APP/PS1 versus APP/PS1.","description":"","filename":"OnlineFigure4.Png","url":"https://assets-eu.researchsquare.com/files/rs-108136/v1/7d25d8eb685613e5152cf027.Png"},{"id":3618332,"identity":"6cbd87e9-cafe-4239-88f2-f75ab3f619ab","added_by":"auto","created_at":"2020-11-16 23:13:09","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":3447455,"visible":true,"origin":"","legend":"The effect of voluntary exercise on reactive gliosis and inflammatory factor levels in the brain of 5- and 9-month-old APP/PS1 mice with or without AQP4. a-b Immunohistochemical staining for GFAP and Iba1 in the frontal cortex. c Quantification of the percentage of GFAP and Iba1 positive area in the frontal cortex, respectively. Data represent mean ± SEM from 5 sections per mouse and four mice per group. d ELISA analysis of inflammatory factors IL-1β, IL-6 and TNF-α levels in the frontal cortex. Data represent mean ± SEM from 3 independent experiments and 4 mice per group. Three-way factor ANOVA with Tukey’s post hoc test. *p \u003c 0.05, Exercise versus Control; #p \u003c 0.05, ##p \u003c 0.01,###p \u003c 0.001, APP/PS1 versus WT; \u0026p \u003c 0.05, \u0026\u0026p \u003c 0.01, \u0026\u0026\u0026p \u003c 0.001, AQP4−/−/APP/PS1 versus APP/PS1.","description":"","filename":"OnlineFigure4.Png","url":"https://assets-eu.researchsquare.com/files/rs-108136/v1/3968e27d414e92200e60d133.Png"},{"id":3618317,"identity":"82c33666-b31f-483c-aada-8526d05c8ccc","added_by":"auto","created_at":"2020-11-16 23:13:04","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":3447455,"visible":true,"origin":"","legend":"The effect of voluntary exercise on reactive gliosis and inflammatory factor levels in the brain of 5- and 9-month-old APP/PS1 mice with or without AQP4. a-b Immunohistochemical staining for GFAP and Iba1 in the frontal cortex. c Quantification of the percentage of GFAP and Iba1 positive area in the frontal cortex, respectively. Data represent mean ± SEM from 5 sections per mouse and four mice per group. d ELISA analysis of inflammatory factors IL-1β, IL-6 and TNF-α levels in the frontal cortex. Data represent mean ± SEM from 3 independent experiments and 4 mice per group. Three-way factor ANOVA with Tukey’s post hoc test. *p \u003c 0.05, Exercise versus Control; #p \u003c 0.05, ##p \u003c 0.01,###p \u003c 0.001, APP/PS1 versus WT; \u0026p \u003c 0.05, \u0026\u0026p \u003c 0.01, \u0026\u0026\u0026p \u003c 0.001, AQP4−/−/APP/PS1 versus APP/PS1.","description":"","filename":"OnlineFigure4.Png","url":"https://assets-eu.researchsquare.com/files/rs-108136/v1/f1b8df1ae56794572ea41058.Png"},{"id":3618345,"identity":"af41d53a-a59d-45eb-93a9-70d7686ffd98","added_by":"auto","created_at":"2020-11-16 23:13:33","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1933123,"visible":true,"origin":"","legend":"Immunofluorescence staining showing the effect of voluntary exercise on AQP4 expression and polarity in 5- and 9-month-old APP/PS1 mice. a AQP4 and GFAP double immunofluorescence in the frontal cortex. b Quantitative analyses of immunofluorescence intensity of AQP4 at the regions immediately abutting pia maters (large arrowheads) and microvessels (small arrowheads) and as well as correspondingly adjacent parenchymal domains (stars). c Quantitative analyses of AQP4 polarization abutting pia maters and microvessels. Data are analyzed by the three-way factor ANOVA with Tukey’s post hoc test and represent mean ± SEM from 4 sections (16-20 microvessels) per mouse and 4 mice per group. *p \u003c 0.05, **p \u003c 0.01; ***p \u003c 0.001, Exercise versus Control; #p \u003c 0.05, ###p \u003c 0.001, APP/PS1 versus WT.","description":"","filename":"OnlineFigure5.Png","url":"https://assets-eu.researchsquare.com/files/rs-108136/v1/305baa9c929e460183221c33.Png"},{"id":3618334,"identity":"0b03b1cc-6e3b-4d0f-acd4-75acba18fa09","added_by":"auto","created_at":"2020-11-16 23:13:10","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1933123,"visible":true,"origin":"","legend":"Immunofluorescence staining showing the effect of voluntary exercise on AQP4 expression and polarity in 5- and 9-month-old APP/PS1 mice. a AQP4 and GFAP double immunofluorescence in the frontal cortex. b Quantitative analyses of immunofluorescence intensity of AQP4 at the regions immediately abutting pia maters (large arrowheads) and microvessels (small arrowheads) and as well as correspondingly adjacent parenchymal domains (stars). c Quantitative analyses of AQP4 polarization abutting pia maters and microvessels. Data are analyzed by the three-way factor ANOVA with Tukey’s post hoc test and represent mean ± SEM from 4 sections (16-20 microvessels) per mouse and 4 mice per group. *p \u003c 0.05, **p \u003c 0.01; ***p \u003c 0.001, Exercise versus Control; #p \u003c 0.05, ###p \u003c 0.001, APP/PS1 versus WT.","description":"","filename":"OnlineFigure5.Png","url":"https://assets-eu.researchsquare.com/files/rs-108136/v1/5274365c7810c5e1c5dc3885.Png"},{"id":3618319,"identity":"ed4c9801-9c89-48de-9c6c-9cfcc5806d1f","added_by":"auto","created_at":"2020-11-16 23:13:04","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1933123,"visible":true,"origin":"","legend":"Immunofluorescence staining showing the effect of voluntary exercise on AQP4 expression and polarity in 5- and 9-month-old APP/PS1 mice. a AQP4 and GFAP double immunofluorescence in the frontal cortex. b Quantitative analyses of immunofluorescence intensity of AQP4 at the regions immediately abutting pia maters (large arrowheads) and microvessels (small arrowheads) and as well as correspondingly adjacent parenchymal domains (stars). c Quantitative analyses of AQP4 polarization abutting pia maters and microvessels. Data are analyzed by the three-way factor ANOVA with Tukey’s post hoc test and represent mean ± SEM from 4 sections (16-20 microvessels) per mouse and 4 mice per group. *p \u003c 0.05, **p \u003c 0.01; ***p \u003c 0.001, Exercise versus Control; #p \u003c 0.05, ###p \u003c 0.001, APP/PS1 versus WT.","description":"","filename":"OnlineFigure5.Png","url":"https://assets-eu.researchsquare.com/files/rs-108136/v1/fa33cb3ea7ca72787effe40e.Png"},{"id":3618346,"identity":"62892924-44cd-4974-9c86-611d3cdd63f2","added_by":"auto","created_at":"2020-11-16 23:13:34","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":2728163,"visible":true,"origin":"","legend":"The effect of voluntary exercise on BDNF-TrkB signaling activation and synaptic protein levels in the brain of 5- and 9-month-old APP/PS1 mice with or without AQP4. a-b Representative bands of Western blot and densitometry analysis of BDNF, TrkB and p-CREB levels. c-d Representative bands of Western bolt and densitometry analysis of SYN and PSD-95 expression levels in the hippocampus. Data are analyzed by the three-way factor ANOVA with Tukey’s post hoc test and represent mean ± SEM from 3 independent experiments and 4 mice per group. *p \u003c 0.05, Exercise versus Control; #p \u003c 0.05, ##p \u003c 0.01, ###p \u003c 0.001, APP/PS1 versus WT; \u0026p \u003c 0.05, \u0026\u0026p \u003c 0.01, \u0026\u0026\u0026p \u003c 0.001, AQP4−/−/APP/PS1 versus APP/PS1.","description":"","filename":"OnlineFigure6.Png","url":"https://assets-eu.researchsquare.com/files/rs-108136/v1/2d45d7a4b4676ce1d1f131b2.Png"},{"id":3618335,"identity":"72919232-7690-4e44-b2eb-352cc3ab5a07","added_by":"auto","created_at":"2020-11-16 23:13:10","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":2728163,"visible":true,"origin":"","legend":"The effect of voluntary exercise on BDNF-TrkB signaling activation and synaptic protein levels in the brain of 5- and 9-month-old APP/PS1 mice with or without AQP4. a-b Representative bands of Western blot and densitometry analysis of BDNF, TrkB and p-CREB levels. c-d Representative bands of Western bolt and densitometry analysis of SYN and PSD-95 expression levels in the hippocampus. Data are analyzed by the three-way factor ANOVA with Tukey’s post hoc test and represent mean ± SEM from 3 independent experiments and 4 mice per group. *p \u003c 0.05, Exercise versus Control; #p \u003c 0.05, ##p \u003c 0.01, ###p \u003c 0.001, APP/PS1 versus WT; \u0026p \u003c 0.05, \u0026\u0026p \u003c 0.01, \u0026\u0026\u0026p \u003c 0.001, AQP4−/−/APP/PS1 versus APP/PS1.","description":"","filename":"OnlineFigure6.Png","url":"https://assets-eu.researchsquare.com/files/rs-108136/v1/7e432e393a3543c5a882afb2.Png"},{"id":3618320,"identity":"80778238-7cea-44dc-9f43-5f713f9b25af","added_by":"auto","created_at":"2020-11-16 23:13:05","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":2728163,"visible":true,"origin":"","legend":"The effect of voluntary exercise on BDNF-TrkB signaling activation and synaptic protein levels in the brain of 5- and 9-month-old APP/PS1 mice with or without AQP4. a-b Representative bands of Western blot and densitometry analysis of BDNF, TrkB and p-CREB levels. c-d Representative bands of Western bolt and densitometry analysis of SYN and PSD-95 expression levels in the hippocampus. Data are analyzed by the three-way factor ANOVA with Tukey’s post hoc test and represent mean ± SEM from 3 independent experiments and 4 mice per group. *p \u003c 0.05, Exercise versus Control; #p \u003c 0.05, ##p \u003c 0.01, ###p \u003c 0.001, APP/PS1 versus WT; \u0026p \u003c 0.05, \u0026\u0026p \u003c 0.01, \u0026\u0026\u0026p \u003c 0.001, AQP4−/−/APP/PS1 versus APP/PS1.","description":"","filename":"OnlineFigure6.Png","url":"https://assets-eu.researchsquare.com/files/rs-108136/v1/4ba23d935de4ae67dfda776f.Png"},{"id":13615175,"identity":"ad8a39b1-1e4e-48ce-91ab-06dfa82155f7","added_by":"auto","created_at":"2021-09-17 06:44:20","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":10692117,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-108136/v1/f106cd44-1021-4270-b450-94a09f5df632.pdf"},{"id":3618338,"identity":"37dc30a9-aecd-44b3-a16d-1191bca6aba9","added_by":"auto","created_at":"2020-11-16 23:13:32","extension":"tif","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":716556,"visible":true,"origin":"","legend":"Additional file 1: Figure S1. The effect of voluntary exercise on brain Aβ levels in 5- and 9-month-old APP/PS1 mice with or without AQP4. Data are analyzed by the three-way factor ANOVA with Tukey’s post hoc test and represent mean ± SEM from 3 independent experiments and 4 mice per group. *p \u003c 0.05, Exercise versus Control; ##p \u003c 0.01, ###p \u003c 0.001, APP/PS1 versus WT; \u0026p \u003c 0.05, \u0026\u0026p \u003c 0.01, \u0026\u0026\u0026p \u003c 0.001, AQP4−/−/APP/PS1 versus APP/PS1.","description":"","filename":"FigureS1.tif","url":"https://assets-eu.researchsquare.com/files/rs-108136/v1/f30c9317f86578bce698a927.tif"},{"id":3618327,"identity":"fca38134-a284-4760-a165-c64d906be80f","added_by":"auto","created_at":"2020-11-16 23:13:08","extension":"tif","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":716556,"visible":true,"origin":"","legend":"Additional file 1: Figure S1. The effect of voluntary exercise on brain Aβ levels in 5- and 9-month-old APP/PS1 mice with or without AQP4. Data are analyzed by the three-way factor ANOVA with Tukey’s post hoc test and represent mean ± SEM from 3 independent experiments and 4 mice per group. *p \u003c 0.05, Exercise versus Control; ##p \u003c 0.01, ###p \u003c 0.001, APP/PS1 versus WT; \u0026p \u003c 0.05, \u0026\u0026p \u003c 0.01, \u0026\u0026\u0026p \u003c 0.001, AQP4−/−/APP/PS1 versus APP/PS1.","description":"","filename":"FigureS1.tif","url":"https://assets-eu.researchsquare.com/files/rs-108136/v1/c2b8965602e75aba7f60372b.tif"},{"id":3618312,"identity":"af48ae87-24e4-496b-ac64-1968a7b090c2","added_by":"auto","created_at":"2020-11-16 23:13:02","extension":"tif","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":716556,"visible":true,"origin":"","legend":"Additional file 1: Figure S1. The effect of voluntary exercise on brain Aβ levels in 5- and 9-month-old APP/PS1 mice with or without AQP4. Data are analyzed by the three-way factor ANOVA with Tukey’s post hoc test and represent mean ± SEM from 3 independent experiments and 4 mice per group. *p \u003c 0.05, Exercise versus Control; ##p \u003c 0.01, ###p \u003c 0.001, APP/PS1 versus WT; \u0026p \u003c 0.05, \u0026\u0026p \u003c 0.01, \u0026\u0026\u0026p \u003c 0.001, AQP4−/−/APP/PS1 versus APP/PS1.","description":"","filename":"FigureS1.tif","url":"https://assets-eu.researchsquare.com/files/rs-108136/v1/9e72e93a146a49194cd8c184.tif"},{"id":3618340,"identity":"173b100f-7d0a-47be-b42d-aabfde553880","added_by":"auto","created_at":"2020-11-16 23:13:32","extension":"tif","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":8519740,"visible":true,"origin":"","legend":"Additional file 2: Figure S2. The effect of voluntary exercise on reactive gliosis in the hippocampus of 5- and 9-month-old APP/PS1 mice with or without AQP4. (A-B) Immunohistochemical staining for GFAP and Iba1 in the hippocampal radiatum layer, respectively. (C-D) Quantification of the percentage of GFAP and Iba1 positive area in the hippocampus, respectively. Data are analyzed by the three-way factor ANOVA with Tukey’s post hoc test and represent mean ± SEM from 5 sections per mouse and 4 mice per group. *p \u003c 0.05, Exercise versus Control; #p \u003c 0.05, ##p \u003c 0.01, ###p \u003c 0.001, APP/PS1 versus WT; \u0026p \u003c 0.05, \u0026\u0026p \u003c 0.01, \u0026\u0026\u0026p \u003c 0.001, AQP4−/−/APP/PS1 versus APP/PS1.","description":"","filename":"FigureS2.tif","url":"https://assets-eu.researchsquare.com/files/rs-108136/v1/4e40f4b3d623d24c9c9d8db7.tif"},{"id":3618329,"identity":"e1b5f3b6-5663-4517-ab1d-dcc361f50ae5","added_by":"auto","created_at":"2020-11-16 23:13:08","extension":"tif","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":8519740,"visible":true,"origin":"","legend":"Additional file 2: Figure S2. The effect of voluntary exercise on reactive gliosis in the hippocampus of 5- and 9-month-old APP/PS1 mice with or without AQP4. (A-B) Immunohistochemical staining for GFAP and Iba1 in the hippocampal radiatum layer, respectively. (C-D) Quantification of the percentage of GFAP and Iba1 positive area in the hippocampus, respectively. Data are analyzed by the three-way factor ANOVA with Tukey’s post hoc test and represent mean ± SEM from 5 sections per mouse and 4 mice per group. *p \u003c 0.05, Exercise versus Control; #p \u003c 0.05, ##p \u003c 0.01, ###p \u003c 0.001, APP/PS1 versus WT; \u0026p \u003c 0.05, \u0026\u0026p \u003c 0.01, \u0026\u0026\u0026p \u003c 0.001, AQP4−/−/APP/PS1 versus APP/PS1.","description":"","filename":"FigureS2.tif","url":"https://assets-eu.researchsquare.com/files/rs-108136/v1/43c5ee4220343c099fa5580d.tif"},{"id":3618314,"identity":"9d0973b8-0d0f-416b-9b1d-a4d19d201157","added_by":"auto","created_at":"2020-11-16 23:13:03","extension":"tif","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":8519740,"visible":true,"origin":"","legend":"Additional file 2: Figure S2. The effect of voluntary exercise on reactive gliosis in the hippocampus of 5- and 9-month-old APP/PS1 mice with or without AQP4. (A-B) Immunohistochemical staining for GFAP and Iba1 in the hippocampal radiatum layer, respectively. (C-D) Quantification of the percentage of GFAP and Iba1 positive area in the hippocampus, respectively. Data are analyzed by the three-way factor ANOVA with Tukey’s post hoc test and represent mean ± SEM from 5 sections per mouse and 4 mice per group. *p \u003c 0.05, Exercise versus Control; #p \u003c 0.05, ##p \u003c 0.01, ###p \u003c 0.001, APP/PS1 versus WT; \u0026p \u003c 0.05, \u0026\u0026p \u003c 0.01, \u0026\u0026\u0026p \u003c 0.001, AQP4−/−/APP/PS1 versus APP/PS1.","description":"","filename":"FigureS2.tif","url":"https://assets-eu.researchsquare.com/files/rs-108136/v1/d98dbb91fda86c6b289b5f2a.tif"},{"id":3618342,"identity":"aef1071e-eb8e-4672-bc63-4266bb3bef8b","added_by":"auto","created_at":"2020-11-16 23:13:33","extension":"tif","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":4884932,"visible":true,"origin":"","legend":"Additional file 3: Figure S3. The effect of voluntary exercise on AQP4 expression and polarity in 5- and 9-month-old APP/PS1 mice as revealed by AQP4 and Congo Red double staining. (A) Representative AQP4 immunoreactive images of the frontal cortex showed localization of AQP4 abutting the pia maters (black arrows) and microvessels (grey arrows). Note that AQP4 was expressed at parenchymal domains surrounding Aβ plaques. (B-C) The percentage of AQP4 positive area in the frontal cortex of mice. (C) The polarization of AQP4 in the frontal cortex of mice. Data are analyzed by the three-way factor ANOVA with Tukey’s post hoc test and represent mean ± SEM from 4 sections (16-20 microvessels) per mouse and 4 mice per group. *p \u003c 0.05, **p \u003c 0.01, Exercise versus Control; #p \u003c 0.05, ###p \u003c 0.001, APP/PS1 versus WT. ","description":"","filename":"FigureS3.tif","url":"https://assets-eu.researchsquare.com/files/rs-108136/v1/9246ec13761bf2cbe7c1ee4f.tif"},{"id":3618331,"identity":"6370ce28-c9d5-43ce-b203-ba9d096046a4","added_by":"auto","created_at":"2020-11-16 23:13:09","extension":"tif","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":4884932,"visible":true,"origin":"","legend":"Additional file 3: Figure S3. The effect of voluntary exercise on AQP4 expression and polarity in 5- and 9-month-old APP/PS1 mice as revealed by AQP4 and Congo Red double staining. (A) Representative AQP4 immunoreactive images of the frontal cortex showed localization of AQP4 abutting the pia maters (black arrows) and microvessels (grey arrows). Note that AQP4 was expressed at parenchymal domains surrounding Aβ plaques. (B-C) The percentage of AQP4 positive area in the frontal cortex of mice. (C) The polarization of AQP4 in the frontal cortex of mice. Data are analyzed by the three-way factor ANOVA with Tukey’s post hoc test and represent mean ± SEM from 4 sections (16-20 microvessels) per mouse and 4 mice per group. *p \u003c 0.05, **p \u003c 0.01, Exercise versus Control; #p \u003c 0.05, ###p \u003c 0.001, APP/PS1 versus WT. ","description":"","filename":"FigureS3.tif","url":"https://assets-eu.researchsquare.com/files/rs-108136/v1/d7a8d868b9182959a84980e3.tif"},{"id":3618316,"identity":"60d74de5-c549-4d1e-be51-f715ce1cec13","added_by":"auto","created_at":"2020-11-16 23:13:03","extension":"tif","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":4884932,"visible":true,"origin":"","legend":"Additional file 3: Figure S3. The effect of voluntary exercise on AQP4 expression and polarity in 5- and 9-month-old APP/PS1 mice as revealed by AQP4 and Congo Red double staining. (A) Representative AQP4 immunoreactive images of the frontal cortex showed localization of AQP4 abutting the pia maters (black arrows) and microvessels (grey arrows). Note that AQP4 was expressed at parenchymal domains surrounding Aβ plaques. (B-C) The percentage of AQP4 positive area in the frontal cortex of mice. (C) The polarization of AQP4 in the frontal cortex of mice. Data are analyzed by the three-way factor ANOVA with Tukey’s post hoc test and represent mean ± SEM from 4 sections (16-20 microvessels) per mouse and 4 mice per group. *p \u003c 0.05, **p \u003c 0.01, Exercise versus Control; #p \u003c 0.05, ###p \u003c 0.001, APP/PS1 versus WT. ","description":"","filename":"FigureS3.tif","url":"https://assets-eu.researchsquare.com/files/rs-108136/v1/7de1e026e13218c3775b87c1.tif"},{"id":3618344,"identity":"de41fa5d-dcdc-452e-b0af-225b6720e774","added_by":"auto","created_at":"2020-11-16 23:13:33","extension":"tif","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":3073540,"visible":true,"origin":"","legend":"Additional file 4: Figure S4. The effect of voluntary exercise on SYN and PSD-95 expression in the hippocampus of 5- and 9-month-old APP/PS1 mice with or without AQP4. (A) Immunohistochemical staining for SYN and PSD-95 in the hippocampal CA1. (B-C) Quantification of SYN and PSD-95 immunoreactive intensity in the hippocampus. Data are analyzed by the three-way factor ANOVA with Tukey’s post hoc test and represent mean ± SEM from 4 sections per mouse and 4 mice per group. *p \u003c 0.05, Exercise versus Control; #p \u003c 0.05, APP/PS1 versus WT; \u0026p \u003c 0.05, APP/PS1 versus AQP4−/−/APP/PS1.","description":"","filename":"FigureS4.tif","url":"https://assets-eu.researchsquare.com/files/rs-108136/v1/241f0cb695126f5660ccd14e.tif"},{"id":3618333,"identity":"f60ebc68-4aa3-434d-b669-0d9788ee51c3","added_by":"auto","created_at":"2020-11-16 23:13:09","extension":"tif","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":3073540,"visible":true,"origin":"","legend":"Additional file 4: Figure S4. The effect of voluntary exercise on SYN and PSD-95 expression in the hippocampus of 5- and 9-month-old APP/PS1 mice with or without AQP4. (A) Immunohistochemical staining for SYN and PSD-95 in the hippocampal CA1. (B-C) Quantification of SYN and PSD-95 immunoreactive intensity in the hippocampus. Data are analyzed by the three-way factor ANOVA with Tukey’s post hoc test and represent mean ± SEM from 4 sections per mouse and 4 mice per group. *p \u003c 0.05, Exercise versus Control; #p \u003c 0.05, APP/PS1 versus WT; \u0026p \u003c 0.05, APP/PS1 versus AQP4−/−/APP/PS1.","description":"","filename":"FigureS4.tif","url":"https://assets-eu.researchsquare.com/files/rs-108136/v1/8f37f4c05a1bc7b94f47f1ef.tif"},{"id":3618318,"identity":"f4ec8462-72c1-4aaa-9ce9-22e31a955dc6","added_by":"auto","created_at":"2020-11-16 23:13:04","extension":"tif","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":3073540,"visible":true,"origin":"","legend":"Additional file 4: Figure S4. The effect of voluntary exercise on SYN and PSD-95 expression in the hippocampus of 5- and 9-month-old APP/PS1 mice with or without AQP4. (A) Immunohistochemical staining for SYN and PSD-95 in the hippocampal CA1. (B-C) Quantification of SYN and PSD-95 immunoreactive intensity in the hippocampus. Data are analyzed by the three-way factor ANOVA with Tukey’s post hoc test and represent mean ± SEM from 4 sections per mouse and 4 mice per group. *p \u003c 0.05, Exercise versus Control; #p \u003c 0.05, APP/PS1 versus WT; \u0026p \u003c 0.05, APP/PS1 versus AQP4−/−/APP/PS1.","description":"","filename":"FigureS4.tif","url":"https://assets-eu.researchsquare.com/files/rs-108136/v1/8140df44309c5e306a15d84b.tif"}],"financialInterests":"","formattedTitle":"Beneficial effects of voluntary exercise therapy on an Alzheimer’s mouse model are dependent on aquaporin 4-mediated glymphatic transport","fulltext":[{"header":"Background","content":"\u003cp\u003eAlzheimer's disease (AD) is a common neurodegenerative disease, characterized by extracellular amyloid-\u0026beta; (A\u0026beta;) plaques in the brain [\u003cspan class=\"CitationRef\"\u003e1\u003c/span\u003e]. Currently, non-pharmacological interventions are receiving substantial interest due to the inability to develop AD therapeutic drugs and vaccines [\u003cspan class=\"CitationRef\"\u003e2\u003c/span\u003e]. Epidemiological and clinical studies show that physical activity improves cognition during healthy aging [\u003cspan class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e6\u003c/span\u003e], as well as individuals with mild cognitive impairment [\u003cspan class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e9\u003c/span\u003e]. However, exercise therapy has not been shown to exert explicitly beneficial outcomes on patients with AD [\u003cspan class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e11\u003c/span\u003e]. Exploring the underlying mechanisms for an effective exercise intervention time window is crucial for the prevention of AD.\u003c/p\u003e\n\u003cp\u003eExperimental animal studies indicate that exercise plays a variety of neuroprotective roles, including promoting neurogenesis, reducing neuroinflammation, improving mitochondrial energy metabolism and promoting neurotrophic factor secretion [\u003cspan class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e13\u003c/span\u003e]. Notably, both voluntary exercise and forced exercise have been shown to reduce extracellular A\u0026beta; levels in the brains of several transgenic mouse models of AD [\u003cspan class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e17\u003c/span\u003e]. However, there is considerable evidence that exercise interventions do not reduce A\u0026beta; plague load in mice with mid- to late-stage AD-like pathology [\u003cspan class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e21\u003c/span\u003e]. The mechanism for exercise promoting A\u0026beta; clearance within an effective time window remains unclear.\u003c/p\u003e\n\u003cp\u003eA variety of clearance pathways are implicated in removal of extracellular A\u0026beta; from the brain, which include enzymatic degradation, cellular uptake and transport across the brain barriers [\u003cspan class=\"CitationRef\"\u003e22\u003c/span\u003e]. Recent findings suggest that the glymphatic system may be a large contributor in extracellular A\u0026beta; clearance [\u003cspan class=\"CitationRef\"\u003e23\u003c/span\u003e]. The glymphatic system, also known as the perivascular space, is surrounded by incomplete continuous astrocyte endfeet, at which large amounts of aquaporin 4 (AQP4) is located [\u003cspan class=\"CitationRef\"\u003e24\u003c/span\u003e]. Such structural features facilitate rapid removal of brain interstitial fluid and soluble macromolecular substances from the brain [\u003cspan class=\"CitationRef\"\u003e25\u003c/span\u003e]. Aged mice, as well as mouse models of AD, show loss of specific localization of AQP4 in perivascular endfeet due to reactive astrogliosis, which in turn impairs glymphatic clearance of A\u0026beta; [\u003cspan class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e27\u003c/span\u003e]. Our previous studies reported that deletion of AQP4 increases A\u0026beta; plaque deposit and cerebral amyloid angiopathy in 12-month-old APP/PS1 mice [\u003cspan class=\"CitationRef\"\u003e28\u003c/span\u003e]. Moreover, recent studies have shown that voluntary exercise promotes glymphatic clearance of A\u0026beta;, which is associated with improved astrocytic AQP4 polarization in aged mice [\u003cspan class=\"CitationRef\"\u003e29\u003c/span\u003e]. Together, these findings suggest that AQP4-mediated glymphatic clearance of A\u0026beta; may act as a key factor in determining a therapeutic time window for exercise in AD. In particular, under healthy aging or the onset stage of AD, exercise facilitates clearance of A\u0026beta; from the brain parenchyma via increased AQP4 dependent bulk flow along the glymphatic pathway. However, with the progress of AD, the polarity of AQP4 is totally impaired because of reactive astrogliosis. Therefore, the structural basis for exercise improving glymphatic clearance is elapsed, subsequently failing to prevent A\u0026beta;-related pathological cascades.\u003c/p\u003e\n\u003cp\u003eAPP695swe/PS1-dE9 (APP/PS1) mice are extensively used mouse model of AD with occurrence of A\u0026beta;-associated long-term memory malfunction from approximately 6\u0026ndash;7 months of age, which serves as a hallmark of moderate stage AD [\u003cspan class=\"CitationRef\"\u003e30\u003c/span\u003e]. At 3 months old, despite no A\u0026beta; plaques, this AD mouse line exhibits a mild activation of astrocytes with slight impairments of AQP4 polarity and glymphatic transport [\u003cspan class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e32\u003c/span\u003e]. Therefore, we chose the 3- and 7-month-old APP/PS1 mice and AQP4\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e/APP/PS1 mice to test whether the time-dependent benefit of exercise on A\u0026beta; related pathology is dependent on AQP4-mediated glymphatic transport.\u003c/p\u003e"},{"header":"Materials And Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n\u003cp\u003e\u003cstrong\u003eAnimals\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAQP4\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e/APP/PS1 mice were established via AQP4\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice crossing with APP695/PS1-dE9 mice as described previously [\u003cspan class=\"CitationRef\"\u003e28\u003c/span\u003e]. Both 3-month-old and 7-month-old male AQP4\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e/APP/PS1 mice, APP/PS1 mice and their wild-type (WT) littermates were randomly divide into exercise group and sedentary group, respectively, and received 2-month voluntary exercise treatment followed by behavioral tests (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA). The animal experiments were approved by the Animal Ethical and Welfare Committee of Nanjing Medical University.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eVoluntary exercise\u003c/strong\u003e\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\n\u003cp\u003eMice habituated for one week prior to the beginning of voluntary exercise training, referred to as the \u0026ldquo;pre-intervention\u0026rdquo; period. Exercise mice were placed in the running wheel for 4\u0026nbsp;h every day, 5 days per week, for 2 months. Control mice were placed in the same room as the exercise mice.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eY-maze test\u003c/strong\u003e\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\n\u003cp\u003eThe Y-maze test was carried out to evaluate short-term working memory [\u003cspan class=\"CitationRef\"\u003e28\u003c/span\u003e]. It consisted of 2 trials with a 1-h interval. Three identical arms of the maze were randomly designated start arm, novel arm, and other arm. In the first trial, one arm of the Y-maze was blocked by a removable door, and the animal was placed in the start arm and allowed to explore the two arms for 5\u0026nbsp;min. In the second trial, the blocking baffle was removed and mice could now have access to all three arms for 5\u0026nbsp;min. The number of entries into the novel arm, and the percentage of novel arm entries made during the second trial, were analyzed.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNovel object recognition test (NORT)\u003c/strong\u003e\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\n\u003cp\u003eThe NORT was performed to assess object recognition memory [\u003cspan class=\"CitationRef\"\u003e33\u003c/span\u003e]. This task consisted of two trials, with an interval of 2 hours. In the first trial, mice were placed in an arena with two of the same objects and allowed to explore for 5\u0026nbsp;min. Following a two-hour delay, mice were placed back in the familiar arena where a novel object was replaced by one of the identical objects (the other object remained the same), and mice were allowed to freely re-explore for 5\u0026nbsp;min. Time spent exploring the familiar and novel objects were recorded. The discrimination index was derived by calculating the time spent exploring the novel object/the time spent exploring novel object\u0026thinsp;+\u0026thinsp;the time spent exploring a familiar object.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMorris water maze (MWM) test\u003c/strong\u003e\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\n\u003cp\u003eThe MWM task was performed to assess spatial learning and memory function [\u003cspan class=\"CitationRef\"\u003e28\u003c/span\u003e]. Briefly, the mice received 6 days of training and a probe test on the 7th day. In the training trail, mice were given as long as 60\u0026nbsp;s to find the dark-colored cylindrical platform submerged 1\u0026nbsp;cm beneath the surface of water which was made opaque with milk and invisible to the mice, and required to remain seated on the platform for 5\u0026nbsp;s. Mice underwent four trials per day, starting from each of four different locations in the pool. The escape latency and swimming speed were analyzed. During the probe trail, the platform was removed from the pool and mice were allowed to swim in the pool for 60\u0026nbsp;s in order to determine the percentage of total time spent in each quadrant and the number of crossing where the platform was located.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCisterna magna injection of fluorescent tracer\u003c/strong\u003e\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\n\u003cp\u003eCisterna magna injection of fluorescent tracer was performed as described previously [\u003cspan class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e31\u003c/span\u003e]. The mice were anesthetized and positioned in a stereotaxic apparatus while the posterior atlanto-occipital membrane was surgically exposed. 5\u0026nbsp;\u0026micro;l Texas Red-dextran-3 (TR-d3, 3000\u0026nbsp;MW; Invitrogen, Cat. D3328) was injected at a concentration of 0.5\u0026nbsp;mg/mL into the cisterna magna by a microsyringe needle at a flow rate of 1\u0026nbsp;\u0026micro;l/min. Forty minutes after the start of infusion, animals were anesthetized again. The brains were removed and post-fixed overnight in 4% paraformaldehyde (PFA) at 4\u0026nbsp;\u0026deg;C.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTissue preparation\u003c/strong\u003e\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\n\u003cp\u003eFollowing behavioral testing, mice were anesthetized and perfused transcardially with 0.9% saline from the left ventricle by a perfusion pump (Longer Pump, China, Cat # BT100-2J). The entire brain was carefully removed from the skull and divided into two hemispheres. One hemisphere was postfixed in 4% PFA at 4\u0026nbsp;\u0026deg;C overnight and subsequently dehydrated in a series of graded ethanol solutions. The brains were embedded in paraffin and cut into 5-\u0026micro;m thick sagittal sections using a microtome (Leica, Germany Cat. # RM2135). Serial sections containing the hippocampus and cerebral cortex were placed on gelatin-coated slides for immunohistochemistry. The other hemisphere was immediately frozen in liquid nitrogen and then stored at -80\u0026nbsp;\u0026deg;C until Western blot or ELISA analyses. For CSF tracer experiments, PFA postfixed forebrain tissues were sliced with a vibrating microtome (Word Precision Instruments Inc, WPI, USA; Cat. NVSLM1), at a thickness of 100\u0026nbsp;\u0026micro;m, and mounted onto gelatin-coated slides in sequence.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmunohistochemistry\u003c/strong\u003e\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\n\u003cp\u003eBrain sections were deparaffinized, hydrated and microwaved in citric acid buffer to achieve antigen retrieval, then treated with 3% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e for 20\u0026nbsp;min in order to reduce endogenous peroxidase activity. Brain sections were incubated with following primary antibodies directed against glial fibrillary acidic protein (GFAP) (1:1000; Millipore; Cat. MAB360), AQP4 (1:400; Millipore; Cat. AB3594), 6E10 (1:1000; Covance, Cat. 803001), ionized calcium binding adaptor molecule 1 (Iba-1) (1:1000; Wako, Cat. 019-19741), postsynaptic density protein 95 (PSD-95) (1:200; Abcam, Cat. ab18258) and synaptophysin (SYN) (1:200, Abcam, Cat. ab64581) at 4\u0026nbsp;\u0026deg;C overnight. The next day, sections were incubated for 1 hour at room temperature with secondary antibodies labeled with horseradish peroxidase and developed with a diaminobenzidine (DAB) horseradish peroxidase color development kit (Sigma-Aldrich, Cat. D8001). Partial AQP4 stained sections were counterstained with Congo red (Sigma-Aldrich, Cat. C6767).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmunofluorescence\u003c/strong\u003e\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n\u003cp\u003eBrain sections were blocked for 1 hour at room temperature with 5% BSA and incubated with the primary antibodies including mouse anti-GFAP (1:1000) and rabbit anti-AQP4 (1:400) at 4\u0026nbsp;\u0026deg;C overnight. The next day, all sections were rinsed 3 times and incubated for 2 hours at room temperature in a mixture of Alexa Flour\u0026trade; 555 donkey anti-mouse IgG (1:1000; Thermo Fisher; Cat. A31570) and Alexa Flour\u0026trade; 488 donkey anti-rabbit IgG (1:1000; Thermo Fisher; Cat. A21206). After rinsing, the sections were incubated for 6\u0026nbsp;min in 4\u0026rsquo;,6-diamidino-2-phenylindole (DAPI) (1:1000; Thermo Fisher; Cat. D21490) and coverslipped with anti-fluorescent quencher.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThioflavine-S staining\u003c/strong\u003e\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\n\u003cp\u003eDeparaffinized sections were incubated with 1% thioflavine-S (Sigma-Aldrich) for 5\u0026nbsp;min. Next, 70% ethanol was used to differentiate for 5\u0026nbsp;min, followed by rinsing with distilled water. The brain sections coverslipped with anti-fluorescent quencher.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImage analysis\u003c/strong\u003e\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\n\u003cp\u003eBrain sections were captured by a digital microscope (Leica Microsystems, Wetzlar, Germany) with constant exposure time, offset, and gain for each staining marker. The positive signal area was measured with Image J (NIH). The positive signal percentage area of GFAP, Ibal, 6E10 and AQP4 was calculated by dividing the area of positive signal to the total area in the hippocampus and adjacent cortex. The mean integrated optical density (MIOD\u0026thinsp;=\u0026thinsp;IOD/total area) was measured to assess immunostaining intensity of SYN and PSD-95 in the hippocampus. For analysis of AQP4 polarization, images at 400\u0026thinsp;\u0026times;\u0026thinsp;magnification were randomly captured from the superficial layers of the frontal cortex. The mean immunofluorescent or immunohistochemical intensity of AQP4 at the regions immediately abutting vessels or pia maters and adjacent parenchyma domains was measured. AQP4 polarization was calculated by comparing expression ratios of AQP4 at perivascular domains or abutting pia maters versus adjacent parenchymal domains [\u003cspan class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e28\u003c/span\u003e]. For the analysis of intracisternally injected TR-d3 diffused into the brain parenchyma, positive signal percentage area of TR-d3 was measured on 3 coronal sections at +\u0026thinsp;0.86, 0 and \u0026minus;\u0026thinsp;1.06\u0026nbsp;mm from anterior to posterior, relative to the bregma. TR-d3 penetration along perivascular spaces was quantified on 400\u0026thinsp;\u0026times;\u0026thinsp;magnification images of the frontal cortex (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA). The fluorescence intensity within the perivascular space was measured along microvessels (diameter\u0026thinsp;=\u0026thinsp;30\u0026ndash;50\u0026nbsp;\u0026micro;m) extending 250\u0026nbsp;\u0026micro;m below the brain surface. All quantification was done blind to animal genotype and treatment.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWestern blotting\u003c/strong\u003e\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\n\u003cp\u003eHippocampal tissues were homogenized at 4\u0026nbsp;\u0026deg;C, then centrifuged at 12,000\u0026nbsp;rpm for 15\u0026nbsp;min. The extracts were loaded onto 10%-12% Tris SDS gels and transferred onto polyvinylidene fluoride membranes. After blocking by 5% skim milk diluted with TBST buffer for 1\u0026nbsp;h, these membranes were incubated at 4\u0026nbsp;\u0026deg;C overnight with one of the following primary antibodies: A\u0026beta;\u003csub\u003e1\u0026minus;40\u003c/sub\u003e (1:1000, Abcam, Cat. ab20068), A\u0026beta;\u003csub\u003e1\u0026minus;42\u003c/sub\u003e (1:1000, Abcam, Cat. ab201060), SYN (1:1500, Abcam, Cat. ab64581), PSD-95 (1:1000; Abcam, Cat. ab18258), brain-derived neurotrophic factor (BDNF) (1:1000; Abcam, Cat. Ab108319), tropomyosin receptor kinase B (TrkB) (1:1000; Abcam, ab187041), cyclic adenosine monophosphate response element binding protein (CREB) (1:1000; CST, Cat. 9197S), p-CREB (1:1000; CST, Cat. 9198S) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (1:3000; Bioworld, Cat. BS60630). Following TBST washing, the membranes were incubated with horseradish peroxidase-labeled secondary antibodies (1:2000; ZSGB-BIO; Cat. ZB2301, Cat. ZB2302) for 1\u0026nbsp;h at room temperature. The protein bands were visualized via ECL plus detection system. GAPDH was used as loading control.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eELISA\u003c/strong\u003e\u003c/p\u003e\n\u003cdiv id=\"Sec15\" class=\"Section3\"\u003e\n\u003cp\u003eThe frontal cortex were homogenized in TBS with protease inhibitors and 1% Triton X-100, followed by centrifugation at 20,000\u0026thinsp;\u0026times;\u0026thinsp;g for 1\u0026nbsp;h. Supernatants were set aside for measurements utilizing soluble A\u0026beta;\u003csub\u003e1\u0026minus;40\u003c/sub\u003e (R\u0026amp;D Systems; Cat. DAB140B), A\u0026beta;\u003csub\u003e1\u0026minus;42\u003c/sub\u003e (R\u0026amp;D Systems; Cat. DAB142), interleukin 1 beta (IL-1\u0026beta;) (Excell Biotech Corporation; Cat. EM001-96), interleukin 6 (IL-6) (Excell Biotech Corporation; Cat. EM004-96) and tumor necrosis factor-\u0026alpha; (TNF-\u0026alpha;) (Excell Biotech Corporation; Cat. EM008-96). The above indexes were quantified with corresponding ELISA kits according to the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\n\u003cp\u003eAll data were expressed as means\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM. Using GraphPad Prism, version 5.02 software (GraphPad software, San Diego, CA, USA), data for the MWM platform training and TR-d3 diffusion within the perivascular space, were analyzed by repeated-measures analysis of variance (ANOVA). Other data were analyzed by Student's t test, two-way ANOVA and multi-way ANOVA with Tukey's \u003cem\u003epost hoc\u003c/em\u003e test as indicated in the figure legends. \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered to have statistical significance.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\n\u003cp\u003e\u003cstrong\u003eAQP4 deficiency eliminates timeliness of voluntary exercise improving cognitive functions of APP/PS1 mice\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe assessed the spatial learning and memory of mice using the MWM test. For 5-month-old groups, voluntary exercise intervention reduced the escape latency to find the hidden platform in WT mice (F\u003csub\u003e5,137\u003c/sub\u003e = 18.72, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) and APP/PS1 mice (F\u003csub\u003e5,132\u003c/sub\u003e = 37.04, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), but not AQP4\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e/APP/PS1 mice (F\u003csub\u003e5,134\u003c/sub\u003e = 0.03363, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.9994) (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eb). For 9-month-old groups, exercise shorted the escape latency in WT mice (F\u003csub\u003e5,143\u003c/sub\u003e = 37.1, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), but not APP/PS1 mice (F\u003csub\u003e5,139\u003c/sub\u003e = 0.2621, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.933) or AQP4\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e/APP/PS1 mice (F\u003csub\u003e5,136\u003c/sub\u003e = 0.105, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.9910) (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ec). Time spent in the target quadrant and number of platform crossings during the probe trials on day 7, were high in 5-month-old WT mice and APP/PS1 mice received voluntary exercise (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0213, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.02, WT-Exe vs WT-Con; \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0429, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0442, APP/PS1-Exe vs APP/PS1-Con, respectively). For 9-month groups, the above parameters were only increased in WT mice with exercise intervention (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0126, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.027, respectively). Notably, exercise did not improve spatial memory performance in 5-month-old or 9-month-old AQP4\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e/APP/PS1 mice (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ed-e).\u003c/p\u003e\n\u003cp\u003eWe performed the Y-maze test to measure mouse short-term working memory. For 5-month groups, both WT and APP/PS1 exercise mice exhibited a higher percentage of time spent in the novel arm, when compared with the corresponding sedentary controls (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0225, WT-Exe vs WT-Con; \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0235, APP/PS1-Exe vs APP/PS1-Con). Nevertheless the performance of AQP4\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e/APP/PS1 mice was comparable between exercise and sedentary groups. For 9-month groups, exercise increased the residence time of WT mice in the novel arm (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.03), but failed to reverse working memory deficits in APP/PS1 mice and AQP4\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e/APP/PS1 mice (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ef).\u003c/p\u003e\n\u003cp\u003eA novel object recognition test was also performed to assess the effect of exercise on recognition memory of mice. For 5-month groups, exercise improved the recognition index of WT mice and APP/PS1 mice (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0051, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0392, respectively), but not AQP4\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e/APP/PS1 mice. For 9-month groups, exercise was sufficient to improve recognition memory in WT mice (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0467), but not in APP/PS1 mice and AQP4\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e/APP/PS1 mice (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eg).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\n\u003cp\u003e\u003cstrong\u003eAQP4 deficiency in APP/PS1 mice abolishes timeliness of voluntary exercise promoting glymphatic transport\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe assessed glymphatic transport by quantifying TR-d3 penetration into the brain parenchyma following injection into the cisterna magna (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ea). APP/PS1 mice displayed lower of TR-d3 positive area percentage and paravascular fluorescence intensity than WT mice at age of 5 months (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0343, F\u003csub\u003e4,30\u003c/sub\u003e = 25.09, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, respectively) and 9 months (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.007, F\u003csub\u003e4,29\u003c/sub\u003e = 23.7, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, respectively). Both 5-month-old and 9-month-old WT mice received voluntary exercise increased fluorescence penetration area (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0036, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0067, respectively) and intensity (F\u003csub\u003e1,28\u003c/sub\u003e = 39.05, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, F\u003csub\u003e1,29\u003c/sub\u003e = 21.12, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, respectively). However, for APP/PS1 mice, the improving effect of exercise on penetration of TR-d3 into the brain parenchyma was observed only in the 5-month group (Area, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.01; Intensity, F\u003csub\u003e1,30\u003c/sub\u003e = 39.27, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). Exercise did not increase TR-d3 positive area percentage and paravascular fluorescence intensity in either 5- or 9-month-old AQP4\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e/APP/PS1 mice (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eb-f). These results suggest that voluntary exercise ameliorates glymphatic transport malfunction in the onset stage rather than mid-stage of APP/PS1 mice, and AQP4 deletion eliminates the promoting effect of exercise on glymphatic transport.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\n\u003cp\u003e\u003cstrong\u003eAQP4 deficiency in APP/PS1 mice negates timeliness of voluntary exercise alleviating brain A\u0026beta; load\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe further evaluated whether the timeliness of voluntary exercise improves glymphatic transport, thereby affecting aggregation of A\u0026beta; in the brain of APP/PS1 mice. Compared with age-matched APP/PS1 mice, both 5-month-old and 9-month-old AQP4\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e/APP/PS1 mice show more brain A\u0026beta; load in the hippocampus and frontal cortex, as revealed quantification of the area percentage and number of thioflavine-S positive fibrillary plaques and 6E10-immunopositive diffuse plaques (all \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Exercise decreased A\u0026beta; plaque accumulation in the above two brain regions of APP/PS1 mice at 5 months (all \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 for the above indexes). The ameliorating effect of exercise on A\u0026beta; load was not observed in AQP4\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e/APP/PS1 mice at the two different ages (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ea-f). Consistently, both Western blot and ELISA analysis revealed that voluntary exercise only decreased A\u0026beta;\u003csub\u003e1\u0026minus;40\u003c/sub\u003e and A\u0026beta;\u003csub\u003e1\u0026minus;42\u003c/sub\u003e levels in the forebrain samples of 5-month-old APP/PS1 mice (all \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) rather than 9-month-old APP/PS1 mice. Voluntary exercise failed to decrease brain A\u0026beta;\u003csub\u003e1\u0026minus;40\u003c/sub\u003e and A\u0026beta;\u003csub\u003e1\u0026minus;42\u003c/sub\u003e levels in either 5-or 9-month-old AQP4\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e/APP/PS1 mice (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eg-i; Fig. S1a-b).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eVoluntary exercise ameliorates reactive gliosis and neuroinflammation in the onset stage rather than mid-stage of APP/PS1 mice\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn addition to A\u0026beta; deposition, reactive gliosis is a characteristic pathologic hallmark of AD [\u003cspan class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e35\u003c/span\u003e]. We determined effects of voluntary exercise and AQP4 deletion on activation of astrocytes and microglia in APP/PS1 mice at different stages of AD-like progression. Both GFAP-positive astrocytes and Ibal-positive microglia were activated in the frontal cortex and hippocampus of 5-month-old APP/PS1 mice and further apparent in 9 months old. AQP4 deletion in 5-month-old APP/PS1 mice mildly increased activation of microglia and astrocytes in the frontal cortex and hippocampus (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ea; Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ea; Fig. S2a). Microglia activation was also more pronounced in AQP4\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e/APP/PS1 mice at aged 9 months, revealed by high percentages of Iba1 positive area in the frontal cortex (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0107 ) and hippocampus (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0168) as compared with age-matched APP/PS1 mice. However, a considerable proportion of astrocytes underwent atrophy in 9-month-old AQP4\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e/APP/PS1 mice, causing no significant difference in the percentage of GFAP positive area between the two AD mouse lines (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ea-c, Fig. S2a-d). There was no overall change in immunocytochemical labeling patterns of GFAP and Ibal in the frontal cortex and hippocampus in 5- and 9-month-old WT-exercise mice when compared with age-matched sedentary controls. But it is worth noting that, there were GFAP strong positive glia limitans abutting the pia maters of WT-exercise mice (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ea and Fig. S2a). As for APP/PS1 mice, exercise reduced the percentages of positive area for GFAP (Cortex, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0128; Hippocampus, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0331) and Iba1 (Cortex, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0215; Hippocampus, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0107) in the 5-month-old group, but not 9-month-old group. Exercise did not mitigate reactivation of astrocytes and microglia in AQP4\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e/APP/PS1 mice in the both age groups (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ea-c and Fig. S2a-d). Consistently, voluntary exercise partially reduced neuroinflammatory factor levels in APP/PS1 mice in 5 months old (IL-1\u0026beta;, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0343; IL-6, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.045; TNF-\u0026alpha;, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.031), but not 9 months old. The Aqp4 knock-out in APP/PS1 mice totally abolished the time-effect of aerobic exercise on neuroinflammation (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ed).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eVoluntary exercise ameliorates impaired polarity of AQP4 in the onset stage rather than mid-stage of APP/PS1 mice\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSubstantial evidence suggests that reactive astrogliosis results in mislocalization of AQP4 from the endfeet to the entire cell surface of astrocytes, subsequently contributes to glymphatic clearance dysfunction [\u003cspan class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e36\u003c/span\u003e]. Both immunofluorescence and immunohistochemical staining revealed that AQP4 expression was selectively bordering the subarachnoid space and microvessels with very low intensity in the adjacent brain parenchyma in WT mice (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ea, Fig. S3a). AQP4 was abnormally expressed at the non-vascular domains, especially surrounding A\u0026beta; plaques, in 5-month-old APP/PS1 mice. The mislocalization of AQP4 was further obvious in the brain parenchyma of 9-month-old APP/PS1 mice, causing total loss of polarity distribution of AQP4. Voluntary exercise reduced astrocyte activation in 5-month-old APP/PS1 mice, in turn improving AQP4 polarity abutting pia mater (Immunofluorescence, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.005; Immunohistochemistry, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0148) and microvessels (Immunofluorescence, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0302; Immunohistochemistry, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0398). By contrast, in 9-month-old APP/PS1-exercise mice, this improvement was not apparent because reactive astrogliosis was not reduced (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ea-c; Fig. S2a-d). WT exercise mice showed increases in AQP4 polarization abutting pia maters (Immunofluorescence: 5-month, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0032, 9-month, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, respectively) and microvessels (Immunofluorescence: 5-month, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0161, 9-month, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0002; respectively) (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ea, Fig. S3a).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAQP4 deficiency eliminates timeliness of voluntary exercise activating BDNF signaling pathway and improving synaptic protein levels in APP/PS1 mice\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBDNF signaling pathway is associated with synaptic function and regulates the expression of synaptic proteins such as presynaptic protein SYN and postsynaptic protein PSD-95 [\u003cspan class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e38\u003c/span\u003e]. Down regulation of BDNF signaling pathway and synaptic protein loss occur in AD patients [\u003cspan class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e40\u003c/span\u003e] as well as animal models of AD [\u003cspan class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e42\u003c/span\u003e]. Moreover, BDNF signaling pathway is involved in neuroproection of physical exercise in AD [\u003cspan class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e44\u003c/span\u003e], stroke [\u003cspan class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e46\u003c/span\u003e] and depression [\u003cspan class=\"CitationRef\"\u003e47\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e48\u003c/span\u003e]. In this way, the present study measured the activation of BDNF-TrkB-CREB pathway and expression levels of SYN and PSD-95 in the forebrain. The expression levels of mature BDNF, TrkB and p-CREB generally decreased in APP/PS1 mice compared to WT controls aged at 5 months (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.005, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.009, respectively) and 9 months (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.004, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.08043, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0174, respectively). Voluntary activated this signaling pathway in WT mice aged at 5 months (BDNF: \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0352, TrkB: \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0997, p-CREB: \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0029, respectively) and 9 months (BDNF: \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.012, TrkB: \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0308, p-CREB: \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0402, respectively), but only in 5-month-old APP/PS1 mice (BDNF: \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0153, TrkB: \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.018, p-CREB: \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0379, respectively) (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003ea-b). Consistently, voluntary exercise significantly upregulated SYN and PSD-95 levels in the hippocampus of 5-month-old WT and APP/PS1 mice (all \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), but not AQP4\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e/APP/PS1 mice, as revealed by immunohistochemical staining and Western blot analysis (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003ec-d, Fig. S4a-b). For 9-month-old groups, an improving effect of voluntary exercise on the synaptic protein levels was only observed in WT mice (Immunohistochemistry SYN: \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0231, PSD-95: \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0121; Western blot: SYN: \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0113, PSD-95: \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0108).\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eAt present, there is a lack of effective pharmacological treatments and other methods for AD. Exploring ways of preventing, or delaying the onset this disease is a pragmatic strategy. Regular exercise can reduce the occurrence of AD in the healthy elderly population and delay the progression of MCI, although the beneficial effect is not apparent in the clinical treatment of AD [\u003cspan class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e11\u003c/span\u003e]. The underlying mechanism has not yet to be determined. The current study was designed to address this issue. The results show that the beneficial effect of voluntary exercise intervention on AD-like pathology of APP/PS1 mice is time-dependent. Early exercise intervention improves AQP4 polarity, subsequently promoting glymphatic transport while reducing brain A\u0026beta; load. However, during the mid-stage of AD-like pathology, AQP4 polarity is disrupted due to reactive astrogliosis. Therefore, exercise intervention is unable to improve glymphatic transport, and has no improving effect on A\u0026beta; clearance. Voluntary exercise does not mitigate A\u0026beta; accumulation and cognitive impairment of AQP4\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e/APP/PS1 mice, further suggesting that AQP4-dependent glymphatic clearance is a key factor for exercise therapy to delay the onset of AD.\u003c/p\u003e\n\u003cp\u003eIncreasing evidence supports the idea that the glymphatic system plays a central role in the clearance of macromolecular substances from the brain [\u003cspan class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e23\u003c/span\u003e]. This model of glymphatic system has opened up new perspectives in understanding the pathogenesis of AD [\u003cspan class=\"CitationRef\"\u003e49\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e50\u003c/span\u003e], stroke [\u003cspan class=\"CitationRef\"\u003e51\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e52\u003c/span\u003e], brain injury [\u003cspan class=\"CitationRef\"\u003e53\u003c/span\u003e] and other neurological diseases [\u003cspan class=\"CitationRef\"\u003e54\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e55\u003c/span\u003e]. Glymphatic system visualization is not only implemented in animal studies [\u003cspan class=\"CitationRef\"\u003e56\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e57\u003c/span\u003e], but also being used for clinical imaging diagnosis of hydrocephalus [\u003cspan class=\"CitationRef\"\u003e58\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e59\u003c/span\u003e] and AD [\u003cspan class=\"CitationRef\"\u003e60\u003c/span\u003e]. It is suggested that the driving force of glymphatic transport comes from arterial pulsation, and is facilitated by AQP4 mediated bulk fluid flow from the para-arterial space entering the interstitial space, and finally into the para-venous space [\u003cspan class=\"CitationRef\"\u003e23\u003c/span\u003e]. AQP4 gene knockout significantly delays clearance of exogenous A\u0026beta; from the brain following intrastriatal injection [\u003cspan class=\"CitationRef\"\u003e23\u003c/span\u003e]. Our present and previous studies consistently demonstrate that AQP4 deletion in APP/PS1 mice exacerbates glymphatic clearance impairment and A\u0026beta; plaque load [\u003cspan class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e31\u003c/span\u003e]. Together, these results indicate that AQP4 is vital for glymphatic clearance, thus playing a critical role in regulating the pathological progress of AD.\u003c/p\u003e\n\u003cp\u003eThe current results further suggest that glymphatic clearance not only depends on the presence of AQP4, but also on its specific localization at the perivascular endfeet of astrocytes. Early studies have shown that glymphatic clearance of exogenous A\u0026beta; in aged mice is significantly less than that of young mice [\u003cspan class=\"CitationRef\"\u003e28\u003c/span\u003e]. The primary cause is activated astrocytes abnormally expressing AQP4 in the non-perivascular membrane. Aged mice taking part in voluntary exercise exhibit decreased astrocyte activation, improvement of AQP4 polarity, and low brain A\u0026beta; concentration, compared to sedentary controls. Consistently, the present results have confirmed that in the onset stage of APP/PS1 mice, voluntary exercise intervention attenuates reactive astrogliosis and mislocalization of AQP4 in the nonvascular domains of the brain parenchyma of astrocytes, subsequently reducing A\u0026beta; plaque accumulation. These results imply that specific expression of AQP4 on the perivascular endfeet of astrocytes is the molecular structural basis for maintaining glymphatic transport.\u003c/p\u003e\n\u003cp\u003eAdditionally, previous studies report that alterations in AQP4 expression and localization in the frontotemporal lobe are associated with AD status and pathology, which is related to noncoding \u003cem\u003eaqp4\u003c/em\u003e single-nucleotide polymorphisms [\u003cspan class=\"CitationRef\"\u003e61\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e62\u003c/span\u003e]. Notably, perivascular AQP4 localization is preserved among individuals older than 85\u0026nbsp;years who remains cognitively intact. Lymphatic transport ability of AD patients has been found to be lower than that of healthy controls of the same age, via assessment of perivascular fluid movement with diffusion tensor MRI [\u003cspan class=\"CitationRef\"\u003e60\u003c/span\u003e]. Our recent studies reported that a moderate-intensity aerobic dance improves cognitive function, especially episodic memory and processing speed, in MCI patients [\u003cspan class=\"CitationRef\"\u003e63\u003c/span\u003e]. The future studies are necessary for determining whether this protective effect is linked to improving glymphatic transport capacity.\u003c/p\u003e\n\u003cp\u003eConsistent with the present findings, previous studies report an age-dependent beneficial effect of a continuous non-shock treadmill exercise paradigm on the pathogenic characteristics of APP/PS1 transgenic mice. Five-week of exercise reduces A\u0026beta;\u003csub\u003e1\u0026minus;40\u003c/sub\u003e and A\u0026beta;\u003csub\u003e1\u0026minus;42\u003c/sub\u003e levels in the hippocampus of adult (7\u0026ndash;8 month-old) APP/PS1 mice but not aged (24-month-old) APP/PS1 mice, and does not reduce plaque loading in either adult or aged APP/PS1 mice [\u003cspan class=\"CitationRef\"\u003e21\u003c/span\u003e]. By contrast, a 4-month treadmill exercise intervention significantly improves spatial learning and memory ability, reduces amyloid plaques in the hippocampus, and induces neurogenesis in the dentate gyrus of 12-month-old APP/PS1 mice [\u003cspan class=\"CitationRef\"\u003e64\u003c/span\u003e]. This indicates that a longer exercise intervention may have a more positive effect in delaying the pathological process of AD mice. In addition, exercise of different intensity may bring different effects on APP/PS1 mice. Moderate exercise intensity (60\u0026ndash;70% of max oxygen uptake) seems to be more effective in increasing lipid metabolism and reducing soluble A\u0026beta; levels, compared with low-intensity exercise (45\u0026ndash;55% of maximal oxygen uptake) [\u003cspan class=\"CitationRef\"\u003e65\u003c/span\u003e]. Based on this, future studies are also needed to clarify the effects of exercise intensity and duration on glymphatic clearance.\u003c/p\u003e\n\u003cp\u003eIn conclusion, this study has revealed that voluntary exercise promotes the removal of A\u0026beta; by the glymphatic system, attenuating its aggregation, and subsequently reducing astrocyte activation, which is conducive to maintain AQP4 polarity. This effect, in turn, maintains glymphatic transport capability, thereby effectively promoting cognitive function of adult WT mice and delaying the onset of AD-like pathogenesis in APP/PS1 mice. Long-term voluntary exercise intervention fails to improve A\u0026beta; clearance and alleviate AD-like pathology under AQP4 deficiency or AQP4 polarity damage condition. These results highlight that AQP4-dependent glymphatic transport is the neural basis for exercise against AD. The finding has revealed the molecular pathology mechanism that exercise helps prevent AD, but cannot treat AD, which is a long-standing dilemma in the field of AD research. Keeping a certain amount of exercise vitality is a convenient, effective and simple way to prevent the onset of AD.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eAD: Alzheimer\u0026rsquo;s disease; ANOVA: Analysis of variance; AQP4: aquaporin-4; BDNF: brain-derived neurotrophic factor; CREB: cyclic adenosine monophosphate response element binding protein; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GFAP: glial fibrillary acid protein; Iba-1: ionized calcium binding adaptor molecule 1; IL-1\u0026beta;: interleukin 1\u0026beta;; IL-6: interleukin 6; MWM: Morris water maze; NORT: novel object recognition test; PSD-95: postsynaptic density protein 95; SYN: synaptophysin; TrkB: tropomyosin receptor kinase B; TNF-\u0026alpha;: tumor necrosis factor-\u0026alpha;; TR-d3: Texas Red-dextran-3; WT: Wild-type.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll procedures were approved by Institutional Animal Care and Use Committee of Nanjing Medical University (IACUC-1601106).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and material\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data mentioned in this article are available on published article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAuthors have no conflict of interest to declare.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by grants from the National Natural Science Foundation of China (No. 81772454 and No. 81671070).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eYL, XW, RZ, PH and SZ performed experiments and statistical analyses. YL, XW and QL were involved in interpretation of data. TW made substantial contributions to design experiment. YL and MX wrote the manuscript. CM revised the article for important intellectual content. All authors read and approved the final version of the article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eQuerfurth HW, LaFerla FM. Alzheimer's disease. N Engl J Med. 2010;362(4):329-44.\u003c/li\u003e\n\u003cli\u003eCaselli RJ, Beach TG, Knopman DS, Graff-Radford NR. Alzheimer Disease: Scientific Breakthroughs and Translational Challenges. Mayo Clin Proc. 2017;92(6):978-94.\u003c/li\u003e\n\u003cli\u003eKivipelto M, Mangialasche F, Ngandu T. Lifestyle interventions to prevent cognitive impairment, dementia and Alzheimer disease. 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Neuroscience. 2020;430:73-81.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Alzheimer’s disease, astrocytes, aquaporin-4, glymphatic system, voluntary exercise ","lastPublishedDoi":"10.21203/rs.3.rs-108136/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-108136/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground: \u003c/strong\u003eEpidemiological and clinical evidence suggest there is an effective time window for the ability of exercise to slow the progression of Alzheimer's disease (AD). Astrocyte aquaporin-4 (AQP4) dependent glymphatic transport is necessary for clearance of extracellular amyloid-β (Aβ) from the brain. The purpose of this study is to explore whether this Aβ clearance mechanism is involved in the time-dependent benefit of exercise on Aβ related pathology. \u003c/p\u003e\u003cp\u003e\u003cstrong\u003eMethod: \u003c/strong\u003e3- and 7-month-old male wild type (WT) mice, APP695\u003csub\u003eswe\u003c/sub\u003e/PS1-dE9 (APP/PS1) mice and AQP4 knockout (AQP4\u003csup\u003e\u003cem\u003e−/−\u003c/em\u003e\u003c/sup\u003e)/APP/PS1 mice received voluntary wheel exercise intervention for 2 months, followed by behavioral and pathological analyses.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eResults: \u003c/strong\u003e9-month-old APP/PS1 mice showed enhanced reactive astrogliosis, accompanied with widespread loss of perivascular AQP4 polarization, when compared to 5-month-old APP/PS1 mice. Voluntary exercise improved AQP4 polarity and glymphatic transport, reduced glial inflammation and brain Aβ plaque load, plus alleviated synapse protein loss and cognitive deficits in 5-month-old, but not 9-month-old, APP/PS1 mice. Exercise intervention did not mitigate glymphatic transport dysfunction, Aβ accumulation and cognitive impairment in AQP4\u003csup\u003e−/−\u003c/sup\u003e/APP/PS1 mice at 5 or 9 months of age. \u003c/p\u003e\u003cp\u003e\u003cstrong\u003eConclusion: \u003c/strong\u003eThese results have revealed that AQP4-dependent glymphatic transport is an influential factor in the timeliness of voluntary exercise ability to alleviate Aβ pathology, potentially offering a new target for the early prevention of the disease.\u003c/p\u003e","manuscriptTitle":"Beneficial effects of voluntary exercise therapy on an Alzheimer’s mouse model are dependent on aquaporin 4-mediated glymphatic transport","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2020-11-16 23:13:00","doi":"10.21203/rs.3.rs-108136/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"0c22a970-1ea5-40ab-9d21-109aee523f8f","owner":[],"postedDate":"November 16th, 2020","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":1096023,"name":"Neurology"}],"tags":[],"updatedAt":"2020-11-17T22:58:56+00:00","versionOfRecord":[],"versionCreatedAt":"2020-11-16 23:13:00","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-108136","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-108136","identity":"rs-108136","version":["v1"]},"buildId":"7rjqhiLT3MXkJMwkYKINL","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}