- Email: [email protected]
Geniposide effectively reverses cognitive impairment and inhibits pathological cerebral damage by regulating the mTOR Signal pathway in APP∕PS1 mice
Journal Pre-proof Geniposide Effectively Reverses Cognitive Impairment and Inhibits Pathological Cerebral Damage by Regulating the mTOR Signal Pathway in APPnullPS1 Mice Zhihua Zhang, Wenping Gao, Xiaojian Wang, Di Zhang, YueZe Liu, Lin Li
PII:
S0304-3940(20)30019-7
DOI:
https://doi.org/10.1016/j.neulet.2020.134749
Reference:
NSL 134749
To appear in:
Neuroscience Letters
Received Date:
17 October 2019
Revised Date:
3 January 2020
Accepted Date:
7 January 2020
Please cite this article as: Zhang Z, Gao W, Wang X, Zhang D, Liu Y, Li L, Geniposide Effectively Reverses Cognitive Impairment and Inhibits Pathological Cerebral Damage by Regulating the mTOR Signal Pathway in APPx2215;PS1 Mice, Neuroscience Letters (2020), doi: https://doi.org/10.1016/j.neulet.2020.134749
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Geniposide Effectively Reverses Cognitive Impairment and Inhibits Pathological Cerebral Damage by Regulating the mTOR Signal Pathway in APP∕PS1 Mice Zhihua Zhang1,2, Wenping Gao3, Xiaojian Wang1,4, Di Zhang5, YueZe Liu6, Lin Li1*
Highlights
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PI3-K/Akt/mTOR signaling pathway performs the effect of longevity and health span as a nutrient and growth factor sensing and inhibition of the mTOR had being developed into a novel for AD therapy. Geniposide reverse AD pathophysiological process as a glucagon-like-1 receptor agonist. Geniposide protect against AD by inhibiting PI3-K/Akt/mTOR signaling pathway and enhancing autophagy.
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Authors’s information Zhihua Zhang, Email:[email protected] Wenping Gao, Email:[email protected] Xiaojian Wang, Email:[email protected] Di Zhang, Email:[email protected] Yueze Liu, Email:[email protected] Lin Li, Email:[email protected]
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1Key Laboratory of Cellular Physiology, Shanxi Medical University, Taiyuan, Shanxi, China. 2 Shanxi Health Vocational College, Taiyuan, Shanxi, China. 3 Shanxi Provincial Rongjun's Hospital, Taiyuan, Shanxi, China. 4 Shanxi Provincial People's Hospital, Taiyuan, Shanxi, China. 5Chemistry department, 6 Second hospital, Shanxi Medical University, Taiyuan, Shanxi, China. *Corresponding author: [email protected]
Abstract Objective: The aim of this study is to investigate the protective effects as well as the underlying molecular mechanisms of geniposide in APP/PS1 transgenic mice. Method: APP/PS1 mice were subjected to intragastric administration of geniposide (50mg/kg/d) for 8 weeks (including a 2-week behavior test). The novel object recognition (NOR) 1
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and the Morris water maze (MWM) tests were used for behavioral assessments. Aβ1-40 plaques in mice cortices and hippocampi are visualized with immunohistochemistical staining. ELISA was used to quantify the levels of soluble Aβ1-40 and Aβ1-42 in the hippocampus. Western blot was used to detect p-Akt/Akt, p-mTOR/mTOR and p-4E-BP1/4E-BP1 levels. The relative mRNA levels of Akt, mTOR and 4E-BP1 were quantified using real-time PCR (RT-PCR). Results: Geniposide alleviated cognitive impairment by improving the ability of novel object exploration, spatial memory, and reduced the level of Aβ in the brain of APP/PS1 mice. Geniposide possibly regulates mTOR-related proteins through modification of phosphorylation. Geniposide markedly lowered p-mTOR and p-Akt expressions while elevating p-4E-BP1 expression. Geniposide obviously reduced the relative mRNA levels of Akt and mTOR and increased the relative mRNA level of 4E-BP1. Conclusion: Geniposide is able to alleviate cognitive impairments and cerebral damage in APP/PS1 mice, with its neuroprotective effects likely mediated via modulation of the mTOR signaling pathway. Keywards: Alzheimer’s disease; mTOR; Geniposide; APP/PS1 mice; Aβ plaques
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INTRODUCTION
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The Chinese herb Gardenia is commonly utilized for treating inflammatory, cerebral and hepatic disorders [1,2]. Geniposide is the primary active compound extracted from the Gardenia jasminoidesellis fruit. Liu et al. [3] has previously isolated geniposide as a GLP-1 receptor agonist via high-throughput screening methods. Geniposide was also demonstrated to work in a GLP-1 dependent mechanism [4]. Geniposide has been proven to be able to easily cross the blood-brain barrier [5], leading to several studies focused on exploring its utility in treating neurodegenerative diseases [6]. Geniposide has been shown to reverse memory impairment and τ phosphorylation in streptozotocin (STZ)-injected rats [6]. However, the mechanisms underlying these effects have yet to be fully clarified. The risk of AD increases significantly with age. The process of aging results in the accumulation of several damaging effects in cells that culminates in disease and death [16,17]. Rapamycin (mTOR), a serine/threonine protein kiniase, is responsible for regulating cellular growth as well as cellular homeostasis. The mTOR signaling pathway plays a significant role in aging, lifespan and aged-related diseases including diabetes and AD [18]. Huang et al. [19] showed that geniposide was able to protect neurons against post-ischemic neurovascular injury through the mTOR pathway, highlighting the potential benefits of GLP-1 therapy in neurodegenerative diseases. Additionally, Yin et al. [20] put forward that phosphatidylinositol 3-kinase (PI3K)/Akt activation may also be essential in mediating geniposide’s protective effects. We hypothesize that the mTOR pathway is central in facilitating geniposide-mediated neuroprotection. In the present study, we attempt to verify this hypothesis by assessing behavioral changes as well as quantifying Aβ expressions and the proteins related to mTOR signaling pathway after geniposide treatment in APP/PS1 mice. 2
MATERIAL AND METHODS Chemicals Geniposide (purity> 98%) was bought from Med Che Express. All antibodies including anti-Aβ1-40, anti-p-Akt, anti-Akt, anti-p-mTOR, anti-mTOR,anti-p-4E-BP1, andanti-4E-BP1were obtained from Abcam Technology Inc. RIPA lysis buffer was obtained from Beyotime Institute of Biotechnology.4% Paraformaldehyde, ELISA kits for Aβ1-40 and Aβ1-42 as well as cDNA Synthesis kit were procured from Boster Biotechnology Co., Ltd.
Animals and drug administration
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APP/PS1 double-transgenic mice and C57BL background mice (6 months, male) were obtained from Beijing HFK Bio-Technology Co., Ltd. The animals were housed individually in plastic cages with a temperature of 23±1℃ and a humidity level of 55% ± 5%. The mice were exposed to a 12-hour light-dark cycle. All mice were given ad libitum access to water and food. All mice were housed for three days in the above conditions prior to the experiment to allow adaptation to the environment. Mice were randomly grouped as follows: (i) vehicle-treated WT group, 15 C57BL/6 wild-type (WT) mice given water, (ii) vehicle-treated APP/PS1 group, 15 APP/PS1 double-transgenic mice given water, (iii) geniposide-treated APP/PS1 group, 15 APP/PS1 double-transgenic mice given geniposide(50mg/kg/d). Geniposide was solubilized in water (10ml/Kg) and administrated in an intragastric manner for 8 weeks (including 2 weeks to carry out behavioral tests) [21]. After the behavioral tests, mice were anaesthetized and brains were immediately extracted for IHC, ELISA, Western blot, and PCR. The time line of the experimental design is shown in Fig. 1.
Behavioral assessment with the novel object recognition (NOR) test
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and Morris water maze (MWM) test
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NOR test Behavioral assessment was performed to estimate the ability of mice to recognize novel objects and to memorize space position between the different groups. The NOR test is based on the spontaneous tendency of mice to interact more with novel rather than familiar objects. The test is performed as described previously [12]. In the habituation phase, each mouse was allowed to freely explore the open-field area (a black box 60 cm long × 60 cm wide × 40 cm deep) in the absence of objects. During the familiarization period, each mouse was placed in the box, which contained two identical objects (A1 and A2) for 10 minutes. Recognition memory was tested after 24h by exposing the mice to one familiar (A1 or A2) and one novel object (B). The time spent exploring and sniffing each object was recorded. 3
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MWM test The MWM assay was performed to further assess the spatial memory ability and the test was performed as described previously[13].The MWM test comprised of a stainless steel circular water tank, which was 120 cm in diameter and 50 cm in height with its inner wall painted white. An escape platform (14cm in diameter) was placed about 1.5cm underwater. The escape platform was made invisible by adding titanium dioxide. The temperature of water was kept at the 25±2℃ during the test. Movements of all experimental animals were recorded by video camera systems (Ethovision 3.0, Noldus Information Technology, Wageningen, Holland).The experiment lasted 6 days. During the first 5 days, the navigation test was done to estimate the learning ability of mice by analyzing the time to find the escape platform. The water tank was divided into four quadrants and four fixed points were selected as starting points. The mouse was put into water from one of the four points in turn and allowed to swim until reaching the hidden escape platform. Escape latency, i.e., the time that mouse used to locate the platform was recorded. If the mouse did not locate the platform within 60s, the experimenter guided it onto the platform for 5s and the escape latency was recorded as 60s. The spatial probe test was done on the 6th day of the experiment, which was carried out by first removing the hidden platform. The mouse was put into the water facing the wall of the tank in a randomly selected quadrant. The number of times the mouse crossed the area where the platform used to be in 60 seconds was used as an estimate of the memory ability of mouse. In order to exclude the influence of mice motor ability and visual impairment on the experimental results, the visible platform test was performed following the spatial probe test. The average swimming speed of each mouse was recorded.
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Visualisation of Aβ1-40 plaques in mice cortices and hippocampi by immunohistochemical (IHC) staining
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After the behavioral tests, all mice were anaesthetized with chloral hydrate 0.9% saline before given intracardiac injections of 4% paraformaldehyde (350mg/kg). Brains were immediately extracted and fixed in 4% paraformadehyde. Tissue dehydration was performed with ethanol and tissues subsequently treated with paraffin for immunohistochemical staining. Paraffin was removed from each tissue with graded ethanol solutions and xylene. Endogenous peroxidase was inactivated via a 10 minute treatment with 3% H2O2. All sections were then incubated at 4℃ overnight with mouse anti-Aβ1-40 primary antibody (1:200). The next day, a second incubation took place with biotinylated goat anti-mouse secondary antibody for 20 minutes at room temperature and a final incubation with avidin-biotin peroxidase complex reagent incubation for 20 minutes at room temperature. 3,3-diaminobenzidine (DAB) was used to visualize each sample, with restaining done using hematoxylin. An optical microscope was used to capture all photomicrographs.
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Soluble Aβ1-40 and Aβ1-42 in mice hippocampi were measured using ELISA Mice hippocampus was homogenized in 5 m guanidine hydrochloride using a hand-held motor, and the homogenates were then centrifuged at 20,000 × g at 4°C for 30 min. The supernatants were collected, and the levels ofAβ1-40 and Aβ1-42 were quantified with their respective ELISA kits in accordance to the manufacturer’s protocols.
p-Akt/Akt,
p-mTOR/mTOR
and
p-4E-BP1/4E-BP1
in
mice
hippocampi were quantified using Western blot
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Western blot tests utilized hippocampal tissues that were previously extracted and preserved at -80°C. Cold RIPA buffer that contained complete phosphatase and protease inhibitors was used to lyse the brain tissue before the mixture was centrifuged for 10 minutes at 15,000×g at 4℃. BCA protein assays were used to determine protein concentrations while protein analysis was done via Western blot. Protein samples (40-60μg) were then run on 8%, 10% or 12% SDS-PAGE gels before being applied onto PVDF membranes. The membranes were blocked for an hour in 5% bovine serum albumin with TBST (Tris-buffered saline containing 0.05% Tween-20) before being incubated overnight at 4℃ with the following primary antibodies: Akt (1:1000), p-Akt (1:2000), mTOR (1:1000), p-mTOR (1:1000), 4E-BP1 (1:1000) and p-4E-BP1 (1:2000), followed by the secondary antibodies (goat-anti-rabbit IgG-horseradish peroxidase, HRP) (Boster, Wuhan, China) incubation at 4℃ for 2h.The Sage creation chemilluminescent imaging system (Beijing, China) allowed us to capture relative immunoreactive bands which were then visualized with the ECL-enhanced chemilluminescence system (Boster Biotechnology Co., Ltd. Wuhan, China). All images were digitalized with the Quantity One 4.31 equipment (Bio-Rad, Hercules, CA, USA).
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The relative mRNA levels of Akt, mTOR, and 4E-BP1 in
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micehippocampi were measured using real-time PCR (RT-PCR) TRIZOL Reagent was used to extract total mice brain mRNA based on kit instructions. Oligo-DT primers were used to synthesize complementary DNA using the Boster Bio reverse-PCR kit. The specific primer pairs were designed and manufactured by Invitrogen Trading, Shanghai, China. The CFX97 real time system was used to carry out real-time PCR. The cycling conditions were as follows: initial denaturation at 95°C for 30s, followed by 40 reaction cycles (95°C for 30s, 60°C for 10s, and 72°C for 10s). Housekeeping β-actin gene was used as the reference gene. The 2−ΔΔCt method was used to determine relative gene expression levels. PCR primer pairs are as follows: 5
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Akt： (F)：5’-GGCAGGATGTGTATGAGAAGAAG-3’ (R)：5’-GAGTAGGAGAACTGGGGGAAGTC-3’ mTOR： (F)：5’-ATAGCAGCGAAAACGAGGACTC-3’ (R)：5’-CATTGAGGGCTTTGGTAGGGAG-3’ 4E-BP1： (F)：5’-GAAGTTGCTCTACCCAGTGTCC-3’ (R)：5’-GATAGCCGTTCCTTTCATTTGG-3’ β-actin： (F)：5’-TCCTGTGGCATCCACGAAACT-3’ (R)：5’-GAAGCATTTGCGGTGGACGAG-3’
Statistical analysis
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All data was calculated and presented as mean ± SEM. GraphPad Prism 5(Graph-Pad software Inc., San Diego, CA, USA) was used for statistical calculations. The data obtained from the MWM experiment was subjected to repeated two-way ANOVA analysis. The data was normally distributed. Comparisons among different groups were analyzed by the one-way ANOVA and Tukey’s Post Test. P< 0.05 was considered to be statistically significant. A p of less than 0.05 was taken to indicate statistical significance.
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RESULTS
Geniposide improved the ability of APP/PS1 mice to identify new
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objects
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The NOR test was used to estimate the intelligence of mice. The recognition index (RI）for A1 or A2 is calculated as the ratio of time taken to explore either A1 or A2 (TA1 or TA2) to the time taken to explore both A1 and A2（TA1+TA2）[RI = TA1 or TA2/(TA1+TA2)]. RI of mice in each group was compared. There was no significant difference in RI for A1 and A2 across all three groups (Fig. 2A). RI for new objects was taken as the ratio of the time used to explore a new object (B) (TN) to the total exploring time for either A1 or A2 and B (TF+TN) [RI = TN/ （TF+TN)]. The ability of the vehicle-treated APP/PS1 group to explore new objects was diminished in contrast to the WT group. This loss in exploring ability was recovered after geniposide treatment. Fig. 2B illustrates that initially while the vehicle-treated APP/PS1 group demonstrated lower RI in contrast to the WT group(p< 0.01), this effect was reversed upon geniposide treatment(p< 0.05). This supports the hypothesis that geniposide has the ability to improve new object identification in APP/PS1 mice.
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Geniposide
effectively
ameliorated
learning
and
memory
impairments in APP/PS1 mice
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The MWM test was done to determine the ability of geniposide to counter memory impairment present in double transgenic APP/PS1 mice, a condition triggered by age-related cerebral Aβ accumulation and deposition. Vehicle-treated APP/PS1 group had significantly longer escape latency to locate the platform compared to those in the vehicle-treated WT group on day 3, 4 and 5 (all p < 0.001) (Fig. 3A). However, the prolonged escape latency was significantly reversed by geniposide administration on day 5 (p< 0.05). In the spatial probe test, geniposide-treated APP/PS1 group was found to move across the location of the hidden platform at a higher frequency in contrast to vehicle-treated APP/PS1 group(p< 0.05) (Fig. 3B). Swimming tracks of mice was shown in Fig. 3C. The swimming track of vehicle-treated APP/PS1 group was disorganized, indicating that mice sought for the hidden platform randomly. The APP/PS1 mice treated by geniposide were noted to spend more time in the removed-platform area and demonstrated a more selective search track. The swimming speeds of all three groups of mice did not differ in the visible platform test (Fig. 3D). In conclusion, our results suggest that geniposide may alleviate memory and learning deficits in APP/PS1 transgenic mice.
Geniposide reduced cerebral Aβ levels in APP/PS1 mice
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Aβ is the major component of senile plaques (SPs) and is a characteristic hallmark of AD. We first confirmed the absence or presence of Aβ1-40 plaques with immunohistochemistical staining in all three groups. As shown in Fig 3A, the cortex and hippocampus of vehicle-treated WT group was free of Aβ1-40 plaques (Fig 4Aa), whereby abundant Aβ1-40 plaques were seen in cortex and hippocampus in vehicle-treated APP/PS1 group (Fig 4Ab). Geniposide partially reduced the quantity of Aβ1-40 plaques (Fig 4Ac). The quantities of Aβ1-40 plaques in cortex and hippocampus were significantly lowered after geniposide treatment. This effect was quantitatively shown by analyzing the Aβ1-40 plaques number/mm2as well as the percentage of the area occupied by Aβ1-40 plaques. 6 slices were selected from every animal in all 6 APP/PS1 mice treated by geniposide and water. As seen in Fig. 4B, geniposide remarkably decreased the number of Aβ1-40 plaques in APP/PS1 mice (p< 0.01). The covered areas of Aβ1-40 plaques in geniposide-treated APP/PS1 group was also reduced as compared with that in vehicle-treated APP/PS1 group (p< 0.05)(Fig. 4C). We further measured soluble Aβ1-40 and Aβ1-42 in hippocampus with ELISA. In accordance to our immunolabelling results, ELISA testing also revealed that hippocampal solubleAβ1-40 and Aβ1-42 in geniposide-treated APP/PS1 group were notably attenuated in contrast to vehicle-treated APP/PS1 group(all p< 0.05)(Fig. 4D and 4E). 7
Geniposide inhibited mTOR signaling in APP/PS1 mice
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DISCUSSION
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Akt is an upstream signal molecule of mTOR and can activate mTOR, while 4E-BP1 is an mTOR substrate. Therefore, Akt, mTOR, and 4E-BP1 are key molecules in the mTOR pathway. All three molecules and their phosphorylated forms were measured to explore whether inhibition of mTOR was crucial in mediating the protective effect of geniposide in AD prevention p-Akt/Akt, p-mTOR/mTOR and p-4E-BP1/4E-BP1 quantities were determined with western blot. The results indicated that p-Akt expression was remarkably elevated in vehicle-treated APP/PS1 group in contrast to vehicle-treated WT group(p< 0.01). Geniposide obviously reduced the increment of p-Akt (p< 0.05) ( Fig.5A and B). Similarly, geniposide markedly attenuated p-mTOR expression in APP/PS1 mice (p< 0.05) (Fig. 5A and C). Conversely, p-4E-BP1 was significantly decreased invehicle-treated APP/PS1 group in comparison to vehicle-treated WT group (p< 0.001), with geniposide obviously increasing its expression (p< 0.05) (Fig. 5A and D). The relative mRNA levels of Akt, mTOR and 4E-BP1 were measured using real-time PCR. Both Akt and mTOR mRNA expressions were remarkably increased in vehicle-treated APP/PS1 group in contrast to vehicle-treated WT group (all p< 0.001). Geniposide obviously reduced Akt mRNA and mTOR mRNA expressions (all p< 0.05) (Fig. 6A and 6B). Conversely, the expression of 4E-BP1 mRNA was significantly decreased in vehicle-treated APP/PS1 group when contrasted to vehicle-treated WT group (p< 0.001), with geniposide obviously increasing the expression of 4E-BP1 mRNA (p< 0.05) (Fig. 6C) .
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Our previous study verified that geniposide revised memory impairments and τ phosphorylation in streptozotocin (STZ)-induced rat models of AD [6]. Our current investigations further demonstrates that geniposide treatment can improve the ability of new object identification, ameliorate learning and memory impairments as well as reduce Aβ1-40plaques in cortex and hippocampus along with soluble Aβ1-40 and Aβ1-42 in hippocampus of APP/PS1 mice through inhibition the mTOR signaling pathway. In the present study, 6-month-old double transgenic APP/PS1 mice were used to determine if geniposide administration conferred neurological benefits in an animal model of AD. These transgenic mice models containing dual PS1 and APP gene mutations are frequently utilized in researching AD[14]. Geniposide has been proven to be able to easily cross the blood-brain barrier and is protective against the pathophysiological hallmarks of AD [5]. In this mouse model, we showed that intragastric administration of geniposide protected against behavioral decline and neuropathologic changes of AD. Geniposide can improve the ability of new object identification, and learning and memory ability as well as reducing the levels of soluble Aβ1-40 and Aβ1-42 in the hippocampi of APP/PS1 mice. Our experiments also attempted to validate mTOR as a molecule crucial in 8
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allowing geniposide to confer its protective effects against AD. mTOR possesses extensive bioactivity, including regulation of cell metabolism and growth [15]. In the brain, mTOR functions to regulate neuroplasticity, neurodevelopment and aging [16-18]. Delaying the aging process may help decrease the incidence of AD while also slowing its progression in patients who are already diagnosed with AD [19-21]. mTOR inhibitors are promising pharmacologic agents that may slow the aging process and prevent against the onset of age-related disorders such as neurodegenerative diseases, cancer, cardiovascular disease and type 2 diabetes mellitus[22,23]. The interaction between PI3K-Akt-mTOR signaling pathway and Aβ has previously been studied [24]. Accumulating evidence suggests that Aβ activates mTOR via enhancing the PI3K-Akt pathway [25]. Aβ oligomers increase PI3K-AKT-mTOR signaling in primary neurons. Conversely, increased PI3K-AKT-mTOR signaling increases level of Aβ and suppressed PI3K-AKT-mTOR signaling reduces level of Aβ, thereby attenuating the pathophysiological process of AD. Caccamo [26] knocked-down one copy of the mTOR gene from the forebrain of Tg2576 mice through a crossbreeding strategy and noted that this modification lead to improvement of cognition and decreased levels of Aβ. One explanation for the effect of mTOR inhibition on Aβ is related to an increase in autophagy induction and decrease in translation of BACE-1[27]. Despite the strong evidence linking Aβ and mTOR, the mechanisms leading to mTOR hyperactivity in AD remain elusive. It is speculated that Aβ accumulation increases PRAS40 phosphorylation, which in turn leads to chronic hyperactivation of mTOR and S6K1 signaling [28]. In turn, hyperactive mTOR increases Aβ and tau production by altering the expression of BACE-1, a key enzyme in the production of Aβ, and tau and by inhibiting autophagy [27]. Conversely, inhibition of mTOR attenuates Aβ by enhancing autophagy.
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Several lines of evidence suggest that cognitive dysfunction is mediated by mTOR. Interaction between mTOR and Aβ has also been proven. Aβ accumulation augments the mTOR signal pathway while mTOR signal suppression decreases Aβ levels [28,29]. Caccamo et al. [30] showed that rapamycin-induced inhibition of the mTOR signaling pathway ameliorated Aβ pathology while reversing cognitive decline. Other studies suggest that mTOR inhibiting compounds may enhance Aβ clearance while reversing memory impairment in AD mouse models [31-33]. In the present study we showed that geniposide significantly inhibited the mTOR signal pathway. Geniposide regulated the activity of proteins related to mTOR by phosphorylation modification. Geniposide inhibited the overexpression of p-mTOR and p-Akt, and enhanced expression of p-4E-BP1 in APP/PS1 mice. Similarly, geniposide obviously reduced the relative mRNA levels of Akt and mTOR and increased the relative mRNA level of 4E-BP1.
Conclusion In conclusion, our results suggest that geniposide significantly inhibits the mTOR signaling pathway in APP/PS1 mice. This signaling pathway may be responsible in 9
allowing geniposide to confer its beneficial effects in AD.
Funding Research project was supported by Shanxi Scholarship Council of China (2017important 4), and by the Fund for Shanxi “1331 Project” Key Subjects Construction. Ethics approval and consent to participate This study was approved by the Shanxi Medical University ethic committee and consent to participate was provided by all participants.
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Consent for publication Not applicable Availability of data and materials
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The datasets analyzed during the current study are available from the corresponding author on reasonable request. Competing interests
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The authors declare that they have no competing interests. Funding
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Author contributions
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Research project was supported by Shanxi Scholarship Council of China (2017important 4), and by the Fund for Shanxi “1331 Project” Key Subjects Construction.
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Zhang Zhihua, Gao Wenping and Wang Xiaojian were involved in the design and execution of the study. They contributed to the development of the manuscript and reviewed and approved the final version of the manuscript. Zhang Di directed the statistical analysis of the data, contributed to the development of the manuscript, and reviewed and approved the final version of the manuscript. Yueze Liu and Li Lin were involved in the design and execution of the study, contributed to the development of the manuscript, and reviewed and approved the final version of the manuscript.
Competing interests The authors declare no competing interests. Acknowledgements 10
I am grateful to Professor jinshun Qi (employees of Shanxi Medical University) and Doctor yanwei Li (employees of Shaoyang medical college) who provided me a lot of assistance in the process of experiment implementation. References
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[1] WeiZ, SuK, JiangP, ShiM, WangJ, XieG. Geniposide reduces Staphylococcus aureus internalization into bovine mammary epithelial cells by inhibiting NF-κB activation. Microb Pathog125: 443-7 (2018). [2] Zhao C, Lv C, Li H, Du S, Liu X, Li Z, Xin W, Zhang W. (2016) Geniposide protects primary cortical neurons against oligomeric Aβ1-42-induced neurotoxicity through a mitochondrial pathway. PLoS ONE 11(4):e0152551 (2016). [3] Liu J, Zheng X, Yin F, Hu Y, Guo L, Deng X, Chen G, Jia J, Zhang H. Neurotrophic property of geniposide for inducing the neuronal differentiation of PC12 cells. Int J Dev Neurosci 24(7):419-24 (2006). [4] Gong N, Fan H, Ma AN, Xiao Q, Wang YX. Geniposide and its iridoid analogs exhibit antinociception by acting at the spinal GLP-1 receptors. Neuropharmacology 84:31-45 (2014). [5] Che X, Wang M, Wang T, Fan H, Yang M, Wang W, Xu H. Evaluation of the antidepressant activity, hepatotoxicity and blood brain barrier permeability of methyl genipin. Molecules 16:21(7) (2016). [6] Gao C, Liu Y, Jiang Y, Ding J, Li L. Geniposide ameliorates learning memory deficits, reduces τ phosphorylation and decreases apoptosis via GSK3β pathway in streptozotocin-induced Alzheimer rat model. Brain Pathol 24(3):261-9 (2014). [7] Kaeberlein M. Translational Geroscience: Targeting mTOR Signaling to Promote Healthy Longevity. Innov Aging1(Suppl 1): 743 (2017). [8] Li L. The molecular mechanism of glucagon-like peptide-1 therapy in Alzheimer’s disease, based on a mechanistic target of rapamycin pathway. CNS drugs31(7):535-49 (2017). [9] Huang B, Chen P, Huang L, Li S, Zhu R, Sheng T, Yu W, Chen Z, Wang T. Geniposide attenuates post-ischaemic neurovascular damage via GluN2A/AKT/ERK-dependent mechanism. Cell Physiol Biochem 43(2):705-16 (2017). [10] Yin F, Liu J, Zheng X, Guo L, Xiao H. Geniposide induces the expression of heme oxygenase-1 via PI3K/Nrf2-signaling to enhance the antioxidant capacity in primary hippocampal neurons. Biol Pharm Bull33(11):1841-6 (2010). [11] Zhao C, Zhang H, Li H, Lv C, Liu X, Li Z, Xin W, Wang Y, Zhang W. Geniposide ameliorates cognitive deficits by attenuating the cholinergic defect and amyloidosis in middle-aged Alzheimer model mice. Neuropharmacology116:18-29 (2017). [12] Valvassori SS, Borges C, Bavaresco DV, Varela RB, Resende WR, Peterle BR, Arent CO, Budni J, Quevedo J. Hypericum perforatum chronic treatment affects cognitive parameters and brain neurotrophic factor levels. Rev Bras Psiquiatr40(4):367-75 (2018). [13] WenkGL. Assessment of spatial memory using the radial arm maze and Morris water maze. CurrProtocNeurosciChapter8:Unit 8.5A (2004). [14] Bilkei G. Genetic mouse models of brain ageing and Alzheimer’s disease. Pharmacol Ther142(2):244-57 (2014). 11
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[15] Chang YY, Juhasz G, Goraksha-Hicks P, Arsham AM, Mallin DR, Muller LK, Neufeld TP. Nutrient-dependent regulation of autophagy through the target of rapamycin pathway. Biochem SocTrans 37(Pt 1):232-6 (2009). [16] Bishop NA, Lu T, Yankner BA. Neural mechanisms of ageing and cognitive decline. Nature464(7288):529-35 (2010). [17] Howell JJ, Manning BD. mTOR couples cellular nutrient sensing to organismal metabolic homeostasis. Trends Endocrinol Metab 22(3):94-102 (2011). [18] Zoncu R, Efeyan A, Sabatini DM. mTOR: from growth signal integration to cancer, diabetes and ageing. Nat Rev Mol Cell Biol 12(1):21-35 (2011). [19] Jirillo E, Candore G, Magrone T, Caruso C. A scientific approach to anti-ageing therapies: state of the art. Curr Pharm Des 14(26): 2637-42 (2008). [20] Pickford F, Masliah E, Britschgi M, Lucin K, Narasimhan R, Jaeger PA, Small S, Spencer B, Rockenstein E, Levine B, Wyss-Coray T. The autophagy-related protein beclin 1 shows reduced expression in early Alzheimer disease and regulates amyloid beta accumulation in mice. Clin Invest118(6):2190-9 (2008). [21] Yang DS，Stavrides P，Mohan PS，Kaushik S，Kumar A，Ohno M，Schmidt SD，Wesson DW，Bandyopadhyay U，Jiang Y，Pawlik M，Peterhoff CM，Yang AJ，Wilson DA， St George-Hyslop P，Westaway D，Mathews PM，Levy E，Cuervo AM，Nixon RA. Therapeutic effects of remediating autophagy failure in a mouse model of Alzheimer disease by enhancing lysosomal proteolysis. Autophagy7(7):788-9 (2011). [22] Ravikumar B, Vacher C, Berger Z, Davies JE, Luo S, Oroz LG, Scaravilli F, Easton DF, Duden R, O’Kane CJ, Rubinsztein DC. Inhibition of mTOR: induces autophagy and reduces toxicity of polyglutamine expansions in fly and mouse models of Huntington disease: Nature Genetics36(6):585-95 (2004). [23] Cai Z, Chen G, He W, Xiao M, Yan LJ. Activation of mTOR: a culprit of Alzheimer’s disease. Neuropsychiatr Dis Treat11:1015-30 (2015). [24] Caccamo A, Majumder S, Richardson A, Strong R, Oddo S. Molecular interplay between mammalian target of rapamycin (mTOR), amyloid-beta, and Tau: effects on cognitive impairments. J Biol Chem. 2010 23;285(17):13107-20. [25] Bhaskar K1, Miller M, Chludzinski A, Herrup K, Zagorski M, Lamb BT. The PI3K-Akt-mTOR pathway regulates Abeta oligomer induced neuronal cell cycle events. Mol Neurodegener. 2009 Mar 16;4:14. [26] Caccamo A1, Belfiore R2, Oddo S3. Genetically reducing mTOR signaling rescues central insulin dysregulation in a mouse model of Alzheimer's disease. Neurobiol Aging. 2018 Aug;68:59-67) [27] Caccamo A, Branca C, Talboom JS, Shaw DM, Turner D, Ma L, Messina A, Huang Z, Wu J, Oddo S. Reducing Ribosomal Protein S6 Kinase 1 Expression Improves Spatial Memory and Synaptic Plasticity in a Mouse Model of Alzheimer’s Disease. J Neurosci. 2015;35(41):14042–14056) [28] Tian Y, Bustos V, Flajolet M, Greengard P. A small-molecule enhancer of autophagy decreases levels of Abeta and APP-CTF via Atg5-dependent autophagy pathway. FASEB25(6):1934-42 (2011). [29] Sun Q, Wei LL, Zhang M, Li TX, Yang C, Deng SP, Zeng QC. Rapamycin inhibits activation of AMPK-mTOR: signaling pathway-induced Alzheimer’s disease lesion in 12
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hippocampus of rats with type 2 diabetes mellitus. Int J Neurosci2:1-22 (2018). [30] Caccamo A, Magrì A, Medina DX, Wisely EV, López-Aranda MF, Silva AJ, OddoS.mTOR: regulates tau phosphorylation and degradation: implications for Alzheimer’s disease and other tauopathies. Aging Cell 12(3):370-80 (2013). [31] Spilman P, Podlutskaya N, Hart MJ, Debnath J, Gorostiza O, Bredesen D, Richardson A, Strong R, Galvan V. Inhibition of mTOR by rapamycin abolishes cognitive deficits and reduces amyloid-beta levels in a mouse model of Alzheimer’s disease. PLoS One 5(4):e9979 (2010). [32] Vingtdeux V, Chandakkar P, Zhao H, d’Abramo C, Davies P, MarambaudP.Novel synthetic small-molecule activators of AMPK as enhancers of autophagy and amyloid-β peptide degradation. FASEB 25(1):219-31 (2011). [33] Li T, TangY, LiuH, Yang W, LeW. Autophagy enhancer carbamazepine alleviates memory deficits and cerebral amyloid-β pathology in a mouse model of Alzheimer’s disease. Curr Alzheimer Res10: 433-41 (2013).
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Fig 1. The time line of the experimental design.
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Fig 2. Effects of geniposide on the ability of identifying new object in the three groups. (A) RI for A1 and A2 in familiar stage. No significant differences in RI for A1 and A2 in the three groups. (B) RI for new object in test stage in the three groups. Data presented as mean±SEM (n= 13-15/ group). ** p<0.01 vs. WT+Vehiclegroup; #p<0.05 vs. APP/PS1+Vehicle group. WT: wild-type mice. GP: geniposide.
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Fig 3. Effects of geniposide on the ability of learning and memory in the three groups. (A) Escape latency in the hidden-platform test. (B) Number of crossing in the spatial probe test. (C) Swimming tracks in the spatial probe test. (D) Swimming speed in the visible platform test.Data presented as mean±SEM (n= 13-15/group). ***p <0.001 vs. WT+Vehicle group; #:p<0.05 vs. APP/PS1+Vehicle group. WT: wild-type mice. GP: geniposide.
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Fig 4. Effects of geniposide on Aβ in the cortex and hippocampus in the three groups. Representative images of Aβ1-40-stained brain sections were shown in A. Scale bars: 500 μm. Aβ plaque number/mm2 (B) and the percentage of the area occupied by Aβ plaque (C). The level of Aβ1-40 in the hippocampus (D). The level of Aβ1-42 in the hippocampus (E):.Data presented as mean±SEM (n = 6).##p <0.01, #p<0.05vs.APP/PS1+Vehicle group. WT: wild-type mice. GP: geniposide. 16
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Fig 5. Effects of geniposide on the expressions of the proteins related to mTOR signaling pathway in the hippocampus of the three groups. (A) Representative western blot analysis of p-Akt/Akt, p-mTOR/mTOR and p-4E-BP1/4E-BP1. (B), (C) and (D) Quantification of western blot from (A) in the three groups. Data presented as mean±SEM (n = 6).***p< 0.001, **p< 0.01, * p< 0.05vs.WT+Vehiclegroup; #p < 0.05 vs.APP/PS1+Vehicle group. WT: wild-type mice. GP: geniposide. 17
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Fig 6. Effects of geniposide on the relative mRNA levels of the proteins related to mTOR signaling pathway in the hippocampus of the three groups. RT-PCR was used to determine the Akt (A), mTOR (B) and 4E-BP1(C) mRNA expression levels in the three groups. Data presented as mean±SEM (n = 6).***p< 0.001vs. WT+Vehicle group; #p < 0.05 vs.APP/PS1+Vehicle group.WT: wild-type mice. GP: geniposide.
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PII:
S0304-3940(20)30019-7
DOI:
https://doi.org/10.1016/j.neulet.2020.134749
Reference:
NSL 134749
To appear in:
Neuroscience Letters
Received Date:
17 October 2019
Revised Date:
3 January 2020
Accepted Date:
7 January 2020
Please cite this article as: Zhang Z, Gao W, Wang X, Zhang D, Liu Y, Li L, Geniposide Effectively Reverses Cognitive Impairment and Inhibits Pathological Cerebral Damage by Regulating the mTOR Signal Pathway in APPx2215;PS1 Mice, Neuroscience Letters (2020), doi: https://doi.org/10.1016/j.neulet.2020.134749
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier.
Geniposide Effectively Reverses Cognitive Impairment and Inhibits Pathological Cerebral Damage by Regulating the mTOR Signal Pathway in APP∕PS1 Mice Zhihua Zhang1,2, Wenping Gao3, Xiaojian Wang1,4, Di Zhang5, YueZe Liu6, Lin Li1*
Highlights
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PI3-K/Akt/mTOR signaling pathway performs the effect of longevity and health span as a nutrient and growth factor sensing and inhibition of the mTOR had being developed into a novel for AD therapy. Geniposide reverse AD pathophysiological process as a glucagon-like-1 receptor agonist. Geniposide protect against AD by inhibiting PI3-K/Akt/mTOR signaling pathway and enhancing autophagy.
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Authors’s information Zhihua Zhang, Email:[email protected] Wenping Gao, Email:[email protected] Xiaojian Wang, Email:[email protected] Di Zhang, Email:[email protected] Yueze Liu, Email:[email protected] Lin Li, Email:[email protected]
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1Key Laboratory of Cellular Physiology, Shanxi Medical University, Taiyuan, Shanxi, China. 2 Shanxi Health Vocational College, Taiyuan, Shanxi, China. 3 Shanxi Provincial Rongjun's Hospital, Taiyuan, Shanxi, China. 4 Shanxi Provincial People's Hospital, Taiyuan, Shanxi, China. 5Chemistry department, 6 Second hospital, Shanxi Medical University, Taiyuan, Shanxi, China. *Corresponding author: [email protected]
Abstract Objective: The aim of this study is to investigate the protective effects as well as the underlying molecular mechanisms of geniposide in APP/PS1 transgenic mice. Method: APP/PS1 mice were subjected to intragastric administration of geniposide (50mg/kg/d) for 8 weeks (including a 2-week behavior test). The novel object recognition (NOR) 1
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and the Morris water maze (MWM) tests were used for behavioral assessments. Aβ1-40 plaques in mice cortices and hippocampi are visualized with immunohistochemistical staining. ELISA was used to quantify the levels of soluble Aβ1-40 and Aβ1-42 in the hippocampus. Western blot was used to detect p-Akt/Akt, p-mTOR/mTOR and p-4E-BP1/4E-BP1 levels. The relative mRNA levels of Akt, mTOR and 4E-BP1 were quantified using real-time PCR (RT-PCR). Results: Geniposide alleviated cognitive impairment by improving the ability of novel object exploration, spatial memory, and reduced the level of Aβ in the brain of APP/PS1 mice. Geniposide possibly regulates mTOR-related proteins through modification of phosphorylation. Geniposide markedly lowered p-mTOR and p-Akt expressions while elevating p-4E-BP1 expression. Geniposide obviously reduced the relative mRNA levels of Akt and mTOR and increased the relative mRNA level of 4E-BP1. Conclusion: Geniposide is able to alleviate cognitive impairments and cerebral damage in APP/PS1 mice, with its neuroprotective effects likely mediated via modulation of the mTOR signaling pathway. Keywards: Alzheimer’s disease; mTOR; Geniposide; APP/PS1 mice; Aβ plaques
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INTRODUCTION
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The Chinese herb Gardenia is commonly utilized for treating inflammatory, cerebral and hepatic disorders [1,2]. Geniposide is the primary active compound extracted from the Gardenia jasminoidesellis fruit. Liu et al. [3] has previously isolated geniposide as a GLP-1 receptor agonist via high-throughput screening methods. Geniposide was also demonstrated to work in a GLP-1 dependent mechanism [4]. Geniposide has been proven to be able to easily cross the blood-brain barrier [5], leading to several studies focused on exploring its utility in treating neurodegenerative diseases [6]. Geniposide has been shown to reverse memory impairment and τ phosphorylation in streptozotocin (STZ)-injected rats [6]. However, the mechanisms underlying these effects have yet to be fully clarified. The risk of AD increases significantly with age. The process of aging results in the accumulation of several damaging effects in cells that culminates in disease and death [16,17]. Rapamycin (mTOR), a serine/threonine protein kiniase, is responsible for regulating cellular growth as well as cellular homeostasis. The mTOR signaling pathway plays a significant role in aging, lifespan and aged-related diseases including diabetes and AD [18]. Huang et al. [19] showed that geniposide was able to protect neurons against post-ischemic neurovascular injury through the mTOR pathway, highlighting the potential benefits of GLP-1 therapy in neurodegenerative diseases. Additionally, Yin et al. [20] put forward that phosphatidylinositol 3-kinase (PI3K)/Akt activation may also be essential in mediating geniposide’s protective effects. We hypothesize that the mTOR pathway is central in facilitating geniposide-mediated neuroprotection. In the present study, we attempt to verify this hypothesis by assessing behavioral changes as well as quantifying Aβ expressions and the proteins related to mTOR signaling pathway after geniposide treatment in APP/PS1 mice. 2
MATERIAL AND METHODS Chemicals Geniposide (purity> 98%) was bought from Med Che Express. All antibodies including anti-Aβ1-40, anti-p-Akt, anti-Akt, anti-p-mTOR, anti-mTOR,anti-p-4E-BP1, andanti-4E-BP1were obtained from Abcam Technology Inc. RIPA lysis buffer was obtained from Beyotime Institute of Biotechnology.4% Paraformaldehyde, ELISA kits for Aβ1-40 and Aβ1-42 as well as cDNA Synthesis kit were procured from Boster Biotechnology Co., Ltd.
Animals and drug administration
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APP/PS1 double-transgenic mice and C57BL background mice (6 months, male) were obtained from Beijing HFK Bio-Technology Co., Ltd. The animals were housed individually in plastic cages with a temperature of 23±1℃ and a humidity level of 55% ± 5%. The mice were exposed to a 12-hour light-dark cycle. All mice were given ad libitum access to water and food. All mice were housed for three days in the above conditions prior to the experiment to allow adaptation to the environment. Mice were randomly grouped as follows: (i) vehicle-treated WT group, 15 C57BL/6 wild-type (WT) mice given water, (ii) vehicle-treated APP/PS1 group, 15 APP/PS1 double-transgenic mice given water, (iii) geniposide-treated APP/PS1 group, 15 APP/PS1 double-transgenic mice given geniposide(50mg/kg/d). Geniposide was solubilized in water (10ml/Kg) and administrated in an intragastric manner for 8 weeks (including 2 weeks to carry out behavioral tests) [21]. After the behavioral tests, mice were anaesthetized and brains were immediately extracted for IHC, ELISA, Western blot, and PCR. The time line of the experimental design is shown in Fig. 1.
Behavioral assessment with the novel object recognition (NOR) test
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and Morris water maze (MWM) test
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NOR test Behavioral assessment was performed to estimate the ability of mice to recognize novel objects and to memorize space position between the different groups. The NOR test is based on the spontaneous tendency of mice to interact more with novel rather than familiar objects. The test is performed as described previously [12]. In the habituation phase, each mouse was allowed to freely explore the open-field area (a black box 60 cm long × 60 cm wide × 40 cm deep) in the absence of objects. During the familiarization period, each mouse was placed in the box, which contained two identical objects (A1 and A2) for 10 minutes. Recognition memory was tested after 24h by exposing the mice to one familiar (A1 or A2) and one novel object (B). The time spent exploring and sniffing each object was recorded. 3
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MWM test The MWM assay was performed to further assess the spatial memory ability and the test was performed as described previously[13].The MWM test comprised of a stainless steel circular water tank, which was 120 cm in diameter and 50 cm in height with its inner wall painted white. An escape platform (14cm in diameter) was placed about 1.5cm underwater. The escape platform was made invisible by adding titanium dioxide. The temperature of water was kept at the 25±2℃ during the test. Movements of all experimental animals were recorded by video camera systems (Ethovision 3.0, Noldus Information Technology, Wageningen, Holland).The experiment lasted 6 days. During the first 5 days, the navigation test was done to estimate the learning ability of mice by analyzing the time to find the escape platform. The water tank was divided into four quadrants and four fixed points were selected as starting points. The mouse was put into water from one of the four points in turn and allowed to swim until reaching the hidden escape platform. Escape latency, i.e., the time that mouse used to locate the platform was recorded. If the mouse did not locate the platform within 60s, the experimenter guided it onto the platform for 5s and the escape latency was recorded as 60s. The spatial probe test was done on the 6th day of the experiment, which was carried out by first removing the hidden platform. The mouse was put into the water facing the wall of the tank in a randomly selected quadrant. The number of times the mouse crossed the area where the platform used to be in 60 seconds was used as an estimate of the memory ability of mouse. In order to exclude the influence of mice motor ability and visual impairment on the experimental results, the visible platform test was performed following the spatial probe test. The average swimming speed of each mouse was recorded.
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Visualisation of Aβ1-40 plaques in mice cortices and hippocampi by immunohistochemical (IHC) staining
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After the behavioral tests, all mice were anaesthetized with chloral hydrate 0.9% saline before given intracardiac injections of 4% paraformaldehyde (350mg/kg). Brains were immediately extracted and fixed in 4% paraformadehyde. Tissue dehydration was performed with ethanol and tissues subsequently treated with paraffin for immunohistochemical staining. Paraffin was removed from each tissue with graded ethanol solutions and xylene. Endogenous peroxidase was inactivated via a 10 minute treatment with 3% H2O2. All sections were then incubated at 4℃ overnight with mouse anti-Aβ1-40 primary antibody (1:200). The next day, a second incubation took place with biotinylated goat anti-mouse secondary antibody for 20 minutes at room temperature and a final incubation with avidin-biotin peroxidase complex reagent incubation for 20 minutes at room temperature. 3,3-diaminobenzidine (DAB) was used to visualize each sample, with restaining done using hematoxylin. An optical microscope was used to capture all photomicrographs.
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Soluble Aβ1-40 and Aβ1-42 in mice hippocampi were measured using ELISA Mice hippocampus was homogenized in 5 m guanidine hydrochloride using a hand-held motor, and the homogenates were then centrifuged at 20,000 × g at 4°C for 30 min. The supernatants were collected, and the levels ofAβ1-40 and Aβ1-42 were quantified with their respective ELISA kits in accordance to the manufacturer’s protocols.
p-Akt/Akt,
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p-4E-BP1/4E-BP1
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hippocampi were quantified using Western blot
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Western blot tests utilized hippocampal tissues that were previously extracted and preserved at -80°C. Cold RIPA buffer that contained complete phosphatase and protease inhibitors was used to lyse the brain tissue before the mixture was centrifuged for 10 minutes at 15,000×g at 4℃. BCA protein assays were used to determine protein concentrations while protein analysis was done via Western blot. Protein samples (40-60μg) were then run on 8%, 10% or 12% SDS-PAGE gels before being applied onto PVDF membranes. The membranes were blocked for an hour in 5% bovine serum albumin with TBST (Tris-buffered saline containing 0.05% Tween-20) before being incubated overnight at 4℃ with the following primary antibodies: Akt (1:1000), p-Akt (1:2000), mTOR (1:1000), p-mTOR (1:1000), 4E-BP1 (1:1000) and p-4E-BP1 (1:2000), followed by the secondary antibodies (goat-anti-rabbit IgG-horseradish peroxidase, HRP) (Boster, Wuhan, China) incubation at 4℃ for 2h.The Sage creation chemilluminescent imaging system (Beijing, China) allowed us to capture relative immunoreactive bands which were then visualized with the ECL-enhanced chemilluminescence system (Boster Biotechnology Co., Ltd. Wuhan, China). All images were digitalized with the Quantity One 4.31 equipment (Bio-Rad, Hercules, CA, USA).
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The relative mRNA levels of Akt, mTOR, and 4E-BP1 in
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micehippocampi were measured using real-time PCR (RT-PCR) TRIZOL Reagent was used to extract total mice brain mRNA based on kit instructions. Oligo-DT primers were used to synthesize complementary DNA using the Boster Bio reverse-PCR kit. The specific primer pairs were designed and manufactured by Invitrogen Trading, Shanghai, China. The CFX97 real time system was used to carry out real-time PCR. The cycling conditions were as follows: initial denaturation at 95°C for 30s, followed by 40 reaction cycles (95°C for 30s, 60°C for 10s, and 72°C for 10s). Housekeeping β-actin gene was used as the reference gene. The 2−ΔΔCt method was used to determine relative gene expression levels. PCR primer pairs are as follows: 5
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Akt： (F)：5’-GGCAGGATGTGTATGAGAAGAAG-3’ (R)：5’-GAGTAGGAGAACTGGGGGAAGTC-3’ mTOR： (F)：5’-ATAGCAGCGAAAACGAGGACTC-3’ (R)：5’-CATTGAGGGCTTTGGTAGGGAG-3’ 4E-BP1： (F)：5’-GAAGTTGCTCTACCCAGTGTCC-3’ (R)：5’-GATAGCCGTTCCTTTCATTTGG-3’ β-actin： (F)：5’-TCCTGTGGCATCCACGAAACT-3’ (R)：5’-GAAGCATTTGCGGTGGACGAG-3’
Statistical analysis
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All data was calculated and presented as mean ± SEM. GraphPad Prism 5(Graph-Pad software Inc., San Diego, CA, USA) was used for statistical calculations. The data obtained from the MWM experiment was subjected to repeated two-way ANOVA analysis. The data was normally distributed. Comparisons among different groups were analyzed by the one-way ANOVA and Tukey’s Post Test. P< 0.05 was considered to be statistically significant. A p of less than 0.05 was taken to indicate statistical significance.
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RESULTS
Geniposide improved the ability of APP/PS1 mice to identify new
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The NOR test was used to estimate the intelligence of mice. The recognition index (RI）for A1 or A2 is calculated as the ratio of time taken to explore either A1 or A2 (TA1 or TA2) to the time taken to explore both A1 and A2（TA1+TA2）[RI = TA1 or TA2/(TA1+TA2)]. RI of mice in each group was compared. There was no significant difference in RI for A1 and A2 across all three groups (Fig. 2A). RI for new objects was taken as the ratio of the time used to explore a new object (B) (TN) to the total exploring time for either A1 or A2 and B (TF+TN) [RI = TN/ （TF+TN)]. The ability of the vehicle-treated APP/PS1 group to explore new objects was diminished in contrast to the WT group. This loss in exploring ability was recovered after geniposide treatment. Fig. 2B illustrates that initially while the vehicle-treated APP/PS1 group demonstrated lower RI in contrast to the WT group(p< 0.01), this effect was reversed upon geniposide treatment(p< 0.05). This supports the hypothesis that geniposide has the ability to improve new object identification in APP/PS1 mice.
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The MWM test was done to determine the ability of geniposide to counter memory impairment present in double transgenic APP/PS1 mice, a condition triggered by age-related cerebral Aβ accumulation and deposition. Vehicle-treated APP/PS1 group had significantly longer escape latency to locate the platform compared to those in the vehicle-treated WT group on day 3, 4 and 5 (all p < 0.001) (Fig. 3A). However, the prolonged escape latency was significantly reversed by geniposide administration on day 5 (p< 0.05). In the spatial probe test, geniposide-treated APP/PS1 group was found to move across the location of the hidden platform at a higher frequency in contrast to vehicle-treated APP/PS1 group(p< 0.05) (Fig. 3B). Swimming tracks of mice was shown in Fig. 3C. The swimming track of vehicle-treated APP/PS1 group was disorganized, indicating that mice sought for the hidden platform randomly. The APP/PS1 mice treated by geniposide were noted to spend more time in the removed-platform area and demonstrated a more selective search track. The swimming speeds of all three groups of mice did not differ in the visible platform test (Fig. 3D). In conclusion, our results suggest that geniposide may alleviate memory and learning deficits in APP/PS1 transgenic mice.
Geniposide reduced cerebral Aβ levels in APP/PS1 mice
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Aβ is the major component of senile plaques (SPs) and is a characteristic hallmark of AD. We first confirmed the absence or presence of Aβ1-40 plaques with immunohistochemistical staining in all three groups. As shown in Fig 3A, the cortex and hippocampus of vehicle-treated WT group was free of Aβ1-40 plaques (Fig 4Aa), whereby abundant Aβ1-40 plaques were seen in cortex and hippocampus in vehicle-treated APP/PS1 group (Fig 4Ab). Geniposide partially reduced the quantity of Aβ1-40 plaques (Fig 4Ac). The quantities of Aβ1-40 plaques in cortex and hippocampus were significantly lowered after geniposide treatment. This effect was quantitatively shown by analyzing the Aβ1-40 plaques number/mm2as well as the percentage of the area occupied by Aβ1-40 plaques. 6 slices were selected from every animal in all 6 APP/PS1 mice treated by geniposide and water. As seen in Fig. 4B, geniposide remarkably decreased the number of Aβ1-40 plaques in APP/PS1 mice (p< 0.01). The covered areas of Aβ1-40 plaques in geniposide-treated APP/PS1 group was also reduced as compared with that in vehicle-treated APP/PS1 group (p< 0.05)(Fig. 4C). We further measured soluble Aβ1-40 and Aβ1-42 in hippocampus with ELISA. In accordance to our immunolabelling results, ELISA testing also revealed that hippocampal solubleAβ1-40 and Aβ1-42 in geniposide-treated APP/PS1 group were notably attenuated in contrast to vehicle-treated APP/PS1 group(all p< 0.05)(Fig. 4D and 4E). 7
Geniposide inhibited mTOR signaling in APP/PS1 mice
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DISCUSSION
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Akt is an upstream signal molecule of mTOR and can activate mTOR, while 4E-BP1 is an mTOR substrate. Therefore, Akt, mTOR, and 4E-BP1 are key molecules in the mTOR pathway. All three molecules and their phosphorylated forms were measured to explore whether inhibition of mTOR was crucial in mediating the protective effect of geniposide in AD prevention p-Akt/Akt, p-mTOR/mTOR and p-4E-BP1/4E-BP1 quantities were determined with western blot. The results indicated that p-Akt expression was remarkably elevated in vehicle-treated APP/PS1 group in contrast to vehicle-treated WT group(p< 0.01). Geniposide obviously reduced the increment of p-Akt (p< 0.05) ( Fig.5A and B). Similarly, geniposide markedly attenuated p-mTOR expression in APP/PS1 mice (p< 0.05) (Fig. 5A and C). Conversely, p-4E-BP1 was significantly decreased invehicle-treated APP/PS1 group in comparison to vehicle-treated WT group (p< 0.001), with geniposide obviously increasing its expression (p< 0.05) (Fig. 5A and D). The relative mRNA levels of Akt, mTOR and 4E-BP1 were measured using real-time PCR. Both Akt and mTOR mRNA expressions were remarkably increased in vehicle-treated APP/PS1 group in contrast to vehicle-treated WT group (all p< 0.001). Geniposide obviously reduced Akt mRNA and mTOR mRNA expressions (all p< 0.05) (Fig. 6A and 6B). Conversely, the expression of 4E-BP1 mRNA was significantly decreased in vehicle-treated APP/PS1 group when contrasted to vehicle-treated WT group (p< 0.001), with geniposide obviously increasing the expression of 4E-BP1 mRNA (p< 0.05) (Fig. 6C) .
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Our previous study verified that geniposide revised memory impairments and τ phosphorylation in streptozotocin (STZ)-induced rat models of AD [6]. Our current investigations further demonstrates that geniposide treatment can improve the ability of new object identification, ameliorate learning and memory impairments as well as reduce Aβ1-40plaques in cortex and hippocampus along with soluble Aβ1-40 and Aβ1-42 in hippocampus of APP/PS1 mice through inhibition the mTOR signaling pathway. In the present study, 6-month-old double transgenic APP/PS1 mice were used to determine if geniposide administration conferred neurological benefits in an animal model of AD. These transgenic mice models containing dual PS1 and APP gene mutations are frequently utilized in researching AD[14]. Geniposide has been proven to be able to easily cross the blood-brain barrier and is protective against the pathophysiological hallmarks of AD [5]. In this mouse model, we showed that intragastric administration of geniposide protected against behavioral decline and neuropathologic changes of AD. Geniposide can improve the ability of new object identification, and learning and memory ability as well as reducing the levels of soluble Aβ1-40 and Aβ1-42 in the hippocampi of APP/PS1 mice. Our experiments also attempted to validate mTOR as a molecule crucial in 8
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allowing geniposide to confer its protective effects against AD. mTOR possesses extensive bioactivity, including regulation of cell metabolism and growth [15]. In the brain, mTOR functions to regulate neuroplasticity, neurodevelopment and aging [16-18]. Delaying the aging process may help decrease the incidence of AD while also slowing its progression in patients who are already diagnosed with AD [19-21]. mTOR inhibitors are promising pharmacologic agents that may slow the aging process and prevent against the onset of age-related disorders such as neurodegenerative diseases, cancer, cardiovascular disease and type 2 diabetes mellitus[22,23]. The interaction between PI3K-Akt-mTOR signaling pathway and Aβ has previously been studied [24]. Accumulating evidence suggests that Aβ activates mTOR via enhancing the PI3K-Akt pathway [25]. Aβ oligomers increase PI3K-AKT-mTOR signaling in primary neurons. Conversely, increased PI3K-AKT-mTOR signaling increases level of Aβ and suppressed PI3K-AKT-mTOR signaling reduces level of Aβ, thereby attenuating the pathophysiological process of AD. Caccamo [26] knocked-down one copy of the mTOR gene from the forebrain of Tg2576 mice through a crossbreeding strategy and noted that this modification lead to improvement of cognition and decreased levels of Aβ. One explanation for the effect of mTOR inhibition on Aβ is related to an increase in autophagy induction and decrease in translation of BACE-1[27]. Despite the strong evidence linking Aβ and mTOR, the mechanisms leading to mTOR hyperactivity in AD remain elusive. It is speculated that Aβ accumulation increases PRAS40 phosphorylation, which in turn leads to chronic hyperactivation of mTOR and S6K1 signaling [28]. In turn, hyperactive mTOR increases Aβ and tau production by altering the expression of BACE-1, a key enzyme in the production of Aβ, and tau and by inhibiting autophagy [27]. Conversely, inhibition of mTOR attenuates Aβ by enhancing autophagy.
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Several lines of evidence suggest that cognitive dysfunction is mediated by mTOR. Interaction between mTOR and Aβ has also been proven. Aβ accumulation augments the mTOR signal pathway while mTOR signal suppression decreases Aβ levels [28,29]. Caccamo et al. [30] showed that rapamycin-induced inhibition of the mTOR signaling pathway ameliorated Aβ pathology while reversing cognitive decline. Other studies suggest that mTOR inhibiting compounds may enhance Aβ clearance while reversing memory impairment in AD mouse models [31-33]. In the present study we showed that geniposide significantly inhibited the mTOR signal pathway. Geniposide regulated the activity of proteins related to mTOR by phosphorylation modification. Geniposide inhibited the overexpression of p-mTOR and p-Akt, and enhanced expression of p-4E-BP1 in APP/PS1 mice. Similarly, geniposide obviously reduced the relative mRNA levels of Akt and mTOR and increased the relative mRNA level of 4E-BP1.
Conclusion In conclusion, our results suggest that geniposide significantly inhibits the mTOR signaling pathway in APP/PS1 mice. This signaling pathway may be responsible in 9
allowing geniposide to confer its beneficial effects in AD.
Funding Research project was supported by Shanxi Scholarship Council of China (2017important 4), and by the Fund for Shanxi “1331 Project” Key Subjects Construction. Ethics approval and consent to participate This study was approved by the Shanxi Medical University ethic committee and consent to participate was provided by all participants.
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Consent for publication Not applicable Availability of data and materials
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The datasets analyzed during the current study are available from the corresponding author on reasonable request. Competing interests
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The authors declare that they have no competing interests. Funding
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Author contributions
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Research project was supported by Shanxi Scholarship Council of China (2017important 4), and by the Fund for Shanxi “1331 Project” Key Subjects Construction.
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Zhang Zhihua, Gao Wenping and Wang Xiaojian were involved in the design and execution of the study. They contributed to the development of the manuscript and reviewed and approved the final version of the manuscript. Zhang Di directed the statistical analysis of the data, contributed to the development of the manuscript, and reviewed and approved the final version of the manuscript. Yueze Liu and Li Lin were involved in the design and execution of the study, contributed to the development of the manuscript, and reviewed and approved the final version of the manuscript.
Competing interests The authors declare no competing interests. Acknowledgements 10
I am grateful to Professor jinshun Qi (employees of Shanxi Medical University) and Doctor yanwei Li (employees of Shaoyang medical college) who provided me a lot of assistance in the process of experiment implementation. References
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[1] WeiZ, SuK, JiangP, ShiM, WangJ, XieG. Geniposide reduces Staphylococcus aureus internalization into bovine mammary epithelial cells by inhibiting NF-κB activation. Microb Pathog125: 443-7 (2018). [2] Zhao C, Lv C, Li H, Du S, Liu X, Li Z, Xin W, Zhang W. (2016) Geniposide protects primary cortical neurons against oligomeric Aβ1-42-induced neurotoxicity through a mitochondrial pathway. PLoS ONE 11(4):e0152551 (2016). [3] Liu J, Zheng X, Yin F, Hu Y, Guo L, Deng X, Chen G, Jia J, Zhang H. Neurotrophic property of geniposide for inducing the neuronal differentiation of PC12 cells. Int J Dev Neurosci 24(7):419-24 (2006). [4] Gong N, Fan H, Ma AN, Xiao Q, Wang YX. Geniposide and its iridoid analogs exhibit antinociception by acting at the spinal GLP-1 receptors. Neuropharmacology 84:31-45 (2014). [5] Che X, Wang M, Wang T, Fan H, Yang M, Wang W, Xu H. Evaluation of the antidepressant activity, hepatotoxicity and blood brain barrier permeability of methyl genipin. Molecules 16:21(7) (2016). [6] Gao C, Liu Y, Jiang Y, Ding J, Li L. Geniposide ameliorates learning memory deficits, reduces τ phosphorylation and decreases apoptosis via GSK3β pathway in streptozotocin-induced Alzheimer rat model. Brain Pathol 24(3):261-9 (2014). [7] Kaeberlein M. Translational Geroscience: Targeting mTOR Signaling to Promote Healthy Longevity. Innov Aging1(Suppl 1): 743 (2017). [8] Li L. The molecular mechanism of glucagon-like peptide-1 therapy in Alzheimer’s disease, based on a mechanistic target of rapamycin pathway. CNS drugs31(7):535-49 (2017). [9] Huang B, Chen P, Huang L, Li S, Zhu R, Sheng T, Yu W, Chen Z, Wang T. Geniposide attenuates post-ischaemic neurovascular damage via GluN2A/AKT/ERK-dependent mechanism. Cell Physiol Biochem 43(2):705-16 (2017). [10] Yin F, Liu J, Zheng X, Guo L, Xiao H. Geniposide induces the expression of heme oxygenase-1 via PI3K/Nrf2-signaling to enhance the antioxidant capacity in primary hippocampal neurons. Biol Pharm Bull33(11):1841-6 (2010). [11] Zhao C, Zhang H, Li H, Lv C, Liu X, Li Z, Xin W, Wang Y, Zhang W. Geniposide ameliorates cognitive deficits by attenuating the cholinergic defect and amyloidosis in middle-aged Alzheimer model mice. Neuropharmacology116:18-29 (2017). [12] Valvassori SS, Borges C, Bavaresco DV, Varela RB, Resende WR, Peterle BR, Arent CO, Budni J, Quevedo J. Hypericum perforatum chronic treatment affects cognitive parameters and brain neurotrophic factor levels. Rev Bras Psiquiatr40(4):367-75 (2018). [13] WenkGL. Assessment of spatial memory using the radial arm maze and Morris water maze. CurrProtocNeurosciChapter8:Unit 8.5A (2004). [14] Bilkei G. Genetic mouse models of brain ageing and Alzheimer’s disease. Pharmacol Ther142(2):244-57 (2014). 11
Jo
ur
na
lP
re
-p
ro of
[15] Chang YY, Juhasz G, Goraksha-Hicks P, Arsham AM, Mallin DR, Muller LK, Neufeld TP. Nutrient-dependent regulation of autophagy through the target of rapamycin pathway. Biochem SocTrans 37(Pt 1):232-6 (2009). [16] Bishop NA, Lu T, Yankner BA. Neural mechanisms of ageing and cognitive decline. Nature464(7288):529-35 (2010). [17] Howell JJ, Manning BD. mTOR couples cellular nutrient sensing to organismal metabolic homeostasis. Trends Endocrinol Metab 22(3):94-102 (2011). [18] Zoncu R, Efeyan A, Sabatini DM. mTOR: from growth signal integration to cancer, diabetes and ageing. Nat Rev Mol Cell Biol 12(1):21-35 (2011). [19] Jirillo E, Candore G, Magrone T, Caruso C. A scientific approach to anti-ageing therapies: state of the art. Curr Pharm Des 14(26): 2637-42 (2008). [20] Pickford F, Masliah E, Britschgi M, Lucin K, Narasimhan R, Jaeger PA, Small S, Spencer B, Rockenstein E, Levine B, Wyss-Coray T. The autophagy-related protein beclin 1 shows reduced expression in early Alzheimer disease and regulates amyloid beta accumulation in mice. Clin Invest118(6):2190-9 (2008). [21] Yang DS，Stavrides P，Mohan PS，Kaushik S，Kumar A，Ohno M，Schmidt SD，Wesson DW，Bandyopadhyay U，Jiang Y，Pawlik M，Peterhoff CM，Yang AJ，Wilson DA， St George-Hyslop P，Westaway D，Mathews PM，Levy E，Cuervo AM，Nixon RA. Therapeutic effects of remediating autophagy failure in a mouse model of Alzheimer disease by enhancing lysosomal proteolysis. Autophagy7(7):788-9 (2011). [22] Ravikumar B, Vacher C, Berger Z, Davies JE, Luo S, Oroz LG, Scaravilli F, Easton DF, Duden R, O’Kane CJ, Rubinsztein DC. Inhibition of mTOR: induces autophagy and reduces toxicity of polyglutamine expansions in fly and mouse models of Huntington disease: Nature Genetics36(6):585-95 (2004). [23] Cai Z, Chen G, He W, Xiao M, Yan LJ. Activation of mTOR: a culprit of Alzheimer’s disease. Neuropsychiatr Dis Treat11:1015-30 (2015). [24] Caccamo A, Majumder S, Richardson A, Strong R, Oddo S. Molecular interplay between mammalian target of rapamycin (mTOR), amyloid-beta, and Tau: effects on cognitive impairments. J Biol Chem. 2010 23;285(17):13107-20. [25] Bhaskar K1, Miller M, Chludzinski A, Herrup K, Zagorski M, Lamb BT. The PI3K-Akt-mTOR pathway regulates Abeta oligomer induced neuronal cell cycle events. Mol Neurodegener. 2009 Mar 16;4:14. [26] Caccamo A1, Belfiore R2, Oddo S3. Genetically reducing mTOR signaling rescues central insulin dysregulation in a mouse model of Alzheimer's disease. Neurobiol Aging. 2018 Aug;68:59-67) [27] Caccamo A, Branca C, Talboom JS, Shaw DM, Turner D, Ma L, Messina A, Huang Z, Wu J, Oddo S. Reducing Ribosomal Protein S6 Kinase 1 Expression Improves Spatial Memory and Synaptic Plasticity in a Mouse Model of Alzheimer’s Disease. J Neurosci. 2015;35(41):14042–14056) [28] Tian Y, Bustos V, Flajolet M, Greengard P. A small-molecule enhancer of autophagy decreases levels of Abeta and APP-CTF via Atg5-dependent autophagy pathway. FASEB25(6):1934-42 (2011). [29] Sun Q, Wei LL, Zhang M, Li TX, Yang C, Deng SP, Zeng QC. Rapamycin inhibits activation of AMPK-mTOR: signaling pathway-induced Alzheimer’s disease lesion in 12
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hippocampus of rats with type 2 diabetes mellitus. Int J Neurosci2:1-22 (2018). [30] Caccamo A, Magrì A, Medina DX, Wisely EV, López-Aranda MF, Silva AJ, OddoS.mTOR: regulates tau phosphorylation and degradation: implications for Alzheimer’s disease and other tauopathies. Aging Cell 12(3):370-80 (2013). [31] Spilman P, Podlutskaya N, Hart MJ, Debnath J, Gorostiza O, Bredesen D, Richardson A, Strong R, Galvan V. Inhibition of mTOR by rapamycin abolishes cognitive deficits and reduces amyloid-beta levels in a mouse model of Alzheimer’s disease. PLoS One 5(4):e9979 (2010). [32] Vingtdeux V, Chandakkar P, Zhao H, d’Abramo C, Davies P, MarambaudP.Novel synthetic small-molecule activators of AMPK as enhancers of autophagy and amyloid-β peptide degradation. FASEB 25(1):219-31 (2011). [33] Li T, TangY, LiuH, Yang W, LeW. Autophagy enhancer carbamazepine alleviates memory deficits and cerebral amyloid-β pathology in a mouse model of Alzheimer’s disease. Curr Alzheimer Res10: 433-41 (2013).
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Fig 1. The time line of the experimental design.
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Fig 2. Effects of geniposide on the ability of identifying new object in the three groups. (A) RI for A1 and A2 in familiar stage. No significant differences in RI for A1 and A2 in the three groups. (B) RI for new object in test stage in the three groups. Data presented as mean±SEM (n= 13-15/ group). ** p<0.01 vs. WT+Vehiclegroup; #p<0.05 vs. APP/PS1+Vehicle group. WT: wild-type mice. GP: geniposide.
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Fig 3. Effects of geniposide on the ability of learning and memory in the three groups. (A) Escape latency in the hidden-platform test. (B) Number of crossing in the spatial probe test. (C) Swimming tracks in the spatial probe test. (D) Swimming speed in the visible platform test.Data presented as mean±SEM (n= 13-15/group). ***p <0.001 vs. WT+Vehicle group; #:p<0.05 vs. APP/PS1+Vehicle group. WT: wild-type mice. GP: geniposide.
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Fig 4. Effects of geniposide on Aβ in the cortex and hippocampus in the three groups. Representative images of Aβ1-40-stained brain sections were shown in A. Scale bars: 500 μm. Aβ plaque number/mm2 (B) and the percentage of the area occupied by Aβ plaque (C). The level of Aβ1-40 in the hippocampus (D). The level of Aβ1-42 in the hippocampus (E):.Data presented as mean±SEM (n = 6).##p <0.01, #p<0.05vs.APP/PS1+Vehicle group. WT: wild-type mice. GP: geniposide. 16
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Fig 5. Effects of geniposide on the expressions of the proteins related to mTOR signaling pathway in the hippocampus of the three groups. (A) Representative western blot analysis of p-Akt/Akt, p-mTOR/mTOR and p-4E-BP1/4E-BP1. (B), (C) and (D) Quantification of western blot from (A) in the three groups. Data presented as mean±SEM (n = 6).***p< 0.001, **p< 0.01, * p< 0.05vs.WT+Vehiclegroup; #p < 0.05 vs.APP/PS1+Vehicle group. WT: wild-type mice. GP: geniposide. 17
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Fig 6. Effects of geniposide on the relative mRNA levels of the proteins related to mTOR signaling pathway in the hippocampus of the three groups. RT-PCR was used to determine the Akt (A), mTOR (B) and 4E-BP1(C) mRNA expression levels in the three groups. Data presented as mean±SEM (n = 6).***p< 0.001vs. WT+Vehicle group; #p < 0.05 vs.APP/PS1+Vehicle group.WT: wild-type mice. GP: geniposide.
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