Aerobic Exercise Alleviates Abnormal Autophagy in Brain Cells of APP/PS1 Mice by Upregulating AdipoR1 Levels

Abnormalities in autophagy are associated with Alzheimer’s disease (AD)-like lesions. Studies have shown that exercise can significantly improve AD autophagy abnormalities, but the mechanism underlying this phenomenon remains unclear. APN not only has an important regulatory effect on AD autophagy abnormalities, but also is affected by exercise. Therefore, this study aims to reveal the pathway by which exercise regulates abnormal autophagy in AD using the APN–AdipoR1 signaling pathway as an entry point. The results of the study showed that APP/PS1 double transgenic AD model mice (24 weeks) showed decreased AdipoR1 levels in the brain, abnormal autophagy, increased Aβ deposition, and increased cell apoptosis, and dendritic spines and cognitive function were reduced. Twelve weeks of aerobic exercise enhanced lysosomes and alleviated abnormal autophagy by activating the AdipoR1/AMPK/TFEB signaling pathway in the brains of AD mice, thereby alleviating Aβ deposition and its associated AD-like abnormalities. These findings suggest that the AdipoR1 plays an important role in aerobic exercise’s alleviation of abnormal autophagy in AD brain cells and alleviation of AD-like lesions.

Introduction
Alzheimer's disease (AD) is a neurodegenerative disease commonly referred to as senile dementia. The amyloid hypothesis states that the abnormal accumulation of amyloid β-protein (Aβ), a transmembrane amyloid precursor proteolytic fragment, in the brain is the main cause of AD [1]. Autophagy is an effective way to clear abnormal proteins such as Aβ in the brain, and abnormal autophagy in AD aggravates the disease [2]. Therefore, the regulation of autophagy is a possible approach to the treatment of AD [3]. Existing studies have shown that exercise can promote the degradation of Aβ by regulating autophagy in AD brain cells, thereby improving cognitive impairment [4][5][6]. However, its specific pathway and mechanism remain unclear.
Adiponectin (APN) is a protein hormone secreted by adipose tissue and has neuroprotective effects [7]. The main types of adiponectin receptors (AdipoR) characterized in vivo are AdipoR1, AdipoR2, and T-cadherin; AdipoR1 and AdipoR2 are the main receptors for APN and are widely expressed in various brain regions such as the cerebral cortex, nucleus basalis of Meynert, and hippocampus [7]. AdipoR1 activates the adenosine 5monophosphate (AMP)-activated protein kinase (AMPK) pathway, and AdipoR2 activates the peroxisome proliferator-activated receptor-α pathway [7]. APN mediates the protective effect of exercise on the central nervous system. Studies have shown that exercise can promote the expression of APN and its receptors [8][9][10]. Many experimental studies of APN knockout have shown that APN is essential for exercise's alleviation of brain damage and cognitive dysfunction caused by chronic diseases [11][12][13][14], and APN plays an important role in alleviating AD. Studies have shown that APN can alleviate AD by reducing the neuronal

Exercise Alleviates Cognitive Impairment in AD Mice
Aerobic exercise is considered to be an effective means of alleviating memory loss and cognitive dysfunction in AD patients [20]. Considered the test of choice for assessing learning and memory in basic and regulatory research, the Morris water maze (MWM) provides accurate and reproducible testing of reference memory, spatial working memory, and learning ability, and benefits include less training, no food deprivation, fast learning, no odor cues, etc. [21]. Therefore, our group used MWM to evaluate the effect of aerobic exercise on cognitive function in AD mice. The experimental results of the MWM learning phase ( Figure 1A,B) showed that the average escape latency and swimming distance of the four groups of mice gradually shortened within five training days. Compared with the mice in the WT + SED group, the mice in the AP + SED group showed longer escape latencies and swimming distances during the five days' training (p < 0.05). Aerobic exercise significantly decreased the escape latencies and swimming distances of the mice in the AP + EX group on days 3-5 compared to those in the AP + SED group (p < 0.05). The average swimming speed of the mice in the WT + SED group and the AP + SED group ( Figure 1C) was only significantly different on the fourth day (p < 0.05), and there was no statistically significant difference between the AP + SED and AP + EX groups in the MWM learning phase (p > 0.05). The experimental results of the retention phase ( Figure 1D,E) showed that compared with the mice in the WT + SED group, the mice in the AP + SED group had fewer platform crossings (p < 0.05). Compared with mice in the AP + SED group, aerobic exercise tended to increase the number of platform crossings and the percentage of time in the target quadrant of mice in the AP + EX group. In addition, there was no statistically significant difference in the total swimming distance ( Figure 1F) among the four groups of mice (p > 0.05). The above results suggest that aerobic exercise can effectively alleviate the spatial learning and memory impairment of APP/PS1 mice without affecting the swimming ability of mice.

Exercise Reduces Aβ Accumulation in the Brains of AD Mice
Aβ is considered to be a key factor leading to memory and cognitive impairment in AD patients, and Aβ aggregates to form Aβ plaques, the main pathological feature of AD [1]. Therefore, our group analyzed the proportional Aβ plaque areas in mouse brain tissue by IHC. As shown in Figure 2B, compared with those for the mice in the AP + SED group, the proportional Aβ plaque areas in the brains of the AP + EX group were significantly reduced (p < 0.01). The red square is the starting position, the blue square is the final position, and the yellow connection between them is the swimming path trajectory. The area enclosed by the red line is the first quadrant, which is the second quadrant, the third quadrant, and the fourth quadrant in counterclockwise order. The green circle in the first quadrant is the platform. n = 10 per group. Data are presented as means ± SDs. # p < 0.05, ## p < 0.01, and ### p < 0.001 vs. WT + SED mice; * p < 0.05, and *** p < 0.001 vs. AP + SED mice.

Exercise Reduces Aβ Accumulation in the Brains of AD Mice
Aβ is considered to be a key factor leading to memory and cognitive impairment in AD patients, and Aβ aggregates to form Aβ plaques, the main pathological feature of AD [1]. Therefore, our group analyzed the proportional Aβ plaque areas in mouse brain tissue by IHC. As shown in Figure 2B, compared with those for the mice in the AP + SED group, the proportional Aβ plaque areas in the brains of the AP + EX group were significantly reduced (p < 0.01). The red square is the starting position, the blue square is the final position, and the yellow connection between them is the swimming path trajectory. The area enclosed by the red line is the first quadrant, which is the second quadrant, the third quadrant, and the fourth quadrant in counterclockwise order. The green circle in the first quadrant is the platform. n = 10 per group. Data are presented as means ± SDs. # p < 0.05, ## p < 0.01, and ### p < 0.001 vs. WT + SED mice; * p < 0.05, and *** p < 0.001 vs. AP + SED mice.

Exercise Inhibits Brain Cell Apoptosis in AD Mice
Aβ induces neuronal apoptosis in AD, leading to cognitive dysfunction [1]. In this study, we measured the antiapoptotic protein B-cell lymphoma-2 (Bcl-2), the proapoptotic protein Bcl-2-associated X protein (Bax), and their ratio to determine the apoptosis in the mouse brain. As shown in Figure 3A, aerobic exercise significantly increased the levels of the Bcl-2 protein in the brains of the mice in the WT + EX group compared to those in the WT + SED group (p < 0.05). Compared with mice in the AP + SED group, aerobic exercise significantly increased the levels of the Bcl-2 protein in the AP + EX group (p < 0.05). As shown in Figure 3B, compared with the AP + SED group, the Bax protein levels in the AP + EX group were significantly decreased (p < 0.05). As shown in Figure 3C, compared with the mice in the WT + SED group, aerobic exercise significantly increased the ratios of the Bcl-2/Bax proteins in the brains of the mice in the WT + EX group (p < 0.05). Compared with mice in the AP + SED group, aerobic exercise significantly increased the ratios of the Bcl-2/Bax proteins in the AP + EX group (p < 0.01). The above results suggest that aerobic exercise can effectively inhibit the apoptosis of brain cells in APP/PS1 mice.

Exercise Increases the Density of Dendritic Spines in AD Mice
Dendritic spines are spinous protrusions on the dendritic branches of nerve cells, and their reduced density is strongly correlated with cognitive impairment in AD [22]. This study quantified the density of dendritic spines in mouse brain tissue by Golgi staining. As shown in Figure 4A,B, compared with the mice in the WT + SED group, the density of apical and basal dendritic spines in the cortices of the mice in the AP + SED group was significantly reduced (p < 0.001). The density of apical dendritic spines in the cortices of the mice in the WT + EX group was significantly increased (p < 0.01). Compared with mice in the AP + SED group, aerobic exercise significantly increased the density of apical and basal dendritic spines in the cortices of the mice in the AP + EX group (apical, p < 0.01; basal, p < 0.05). As shown in Figure 4C,D, compared with the mice in the WT + SED group, the density of apical and basal dendritic spines in the hippocampi of the mice in the AP + SED group was significantly decreased (p < 0.01). Compared with the mice in the AP + SED group, aerobic exercise significantly increased the density of apical and basal dendritic spines in the hippocampi of the mice in the AP + EX group (apical, p < 0.05; basal, p < 0.01). The above results suggest that aerobic exercise can effectively increase the density of dendritic spines in the brain tissue of APP/PS1 mice.  The percentage of Aβ plaque area in mouse brain tissue was quantified to compare the Aβ content in brain tissue. n = 3 per group. Data are presented as means ± SDs. ** p < 0.01 vs. AP + SED mice.

Exercise Inhibits Brain Cell Apoptosis in AD Mice
Aβ induces neuronal apoptosis in AD, leading to cognitive dysfunction [1]. In this study, we measured the antiapoptotic protein B-cell lymphoma-2 (Bcl-2), the proapoptotic protein Bcl-2-associated X protein (Bax), and their ratio to determine the apoptosis in the mouse brain. As shown in Figure 3A, aerobic exercise significantly increased the levels of the Bcl-2 protein in the brains of the mice in the WT + EX group compared to those in the The percentage of Aβ plaque area in mouse brain tissue was quantified to compare the Aβ content in brain tissue. n = 3 per group. Data are presented as means ± SDs. ** p < 0.01 vs. AP + SED mice.  . Exercise inhibited apoptosis in the brain cells of AD mice. Relative protein levels of (A) Bcl-2 and (B) Bax in brain tissue. (C) Bcl-2/Bax protein ratios in brain tissue. n = 6 per group. Data are presented as means ± SDs. # p < 0.05 vs. WT + SED mice, * p < 0.05, ** and p < 0.01 vs. AP + SED mice.

Exercise Increases the Density of Dendritic Spines in AD Mice
Dendritic spines are spinous protrusions on the dendritic branches of nerve cells, and their reduced density is strongly correlated with cognitive impairment in AD [22]. This study quantified the density of dendritic spines in mouse brain tissue by Golgi staining. As shown in Figure 4A,B, compared with the mice in the WT + SED group, the density of apical and basal dendritic spines in the cortices of the mice in the AP + SED group was significantly reduced (p < 0.001). The density of apical dendritic spines in the cortices of the mice in the WT + EX group was significantly increased (p < 0.01). Compared with mice in the AP + SED group, aerobic exercise significantly increased the density of apical and basal dendritic spines in the cortices of the mice in the AP + EX group (apical, p < 0.01; basal, p < 0.05). As shown in Figure 4C,D, compared with the mice in the WT + SED group, the density of apical and basal dendritic spines in the hippocampi of the mice in the AP + SED group was significantly decreased (p < 0.01). Compared with the mice in the AP + SED group, aerobic exercise significantly increased the density of apical and basal dendritic spines in the hippocampi of the mice in the AP + EX group (apical, p < 0.05; basal, p < 0.01). The above results suggest that aerobic exercise can effectively increase the density of dendritic spines in the brain tissue of APP/PS1 mice.

Exercise Alleviates Abnormal Autophagy in the Brain Cells of AD Mice
Autophagy plays an important role in alleviating AD-like lesions, Aβ accumulation, and dendritic spine loss in AD brain cells [2,23,24]. The mammalian target of rapamycin (mTOR) is a "master switch" for cellular synthesis and catabolism, and its overactivation is associated with autophagy inhibition in AD [25]. Therefore, our group detected the mRNA expression levels of mTOR and Beclin1, signaling molecules that regulate autophagy. As shown in Figure 5A, compared with that in the mice in the WT + SED group, the expression level of the mTOR mRNA in the AP + SED group was significantly increased (p < 0.05). Compared with the AP + SED group, aerobic exercise significantly downregulated the mTOR mRNA expression levels in the AP + EX group (p < 0.01). As a marker of autophagy initiation, Beclin1 is decreased in early AD and affects autophagy [26]. As shown in Figure  5B, compared with the mice in the WT + SED group, aerobic exercise significantly increased the expression level of the Beclin1 mRNA in the brain tissue of the mice in the WT + EX group (p < 0.05). On the contrary, in the mice in the AP + SED group, the expression of the Beclin1 mRNA showed a downward trend. Compared with the mice in the AP + SED group, aerobic exercise significantly increased the expression of the Beclin1 mRNA in the brain tissue of the mice in the AP + EX group (p < 0.01). These results suggest that aerobic exercise may regulate abnormal autophagy in the brain cells of APP/PS1 mice. . Exercise inhibited apoptosis in the brain cells of AD mice. Relative protein levels of (A) Bcl-2 and (B) Bax in brain tissue. (C) Bcl-2/Bax protein ratios in brain tissue. n = 6 per group. Data are presented as means ± SDs. # p < 0.05 vs. WT + SED mice, * p < 0.05, ** and p < 0.01 vs. AP + SED mice.

Exercise Alleviates Abnormal Autophagy in the Brain Cells of AD Mice
Autophagy plays an important role in alleviating AD-like lesions, Aβ accumulation, and dendritic spine loss in AD brain cells [2,23,24]. The mammalian target of rapamycin (mTOR) is a "master switch" for cellular synthesis and catabolism, and its overactivation is associated with autophagy inhibition in AD [25]. Therefore, our group detected the mRNA expression levels of mTOR and Beclin1, signaling molecules that regulate autophagy. As shown in Figure 5A, compared with that in the mice in the WT + SED group, the expression level of the mTOR mRNA in the AP + SED group was significantly increased (p < 0.05). Compared with the AP + SED group, aerobic exercise significantly downregulated the mTOR mRNA expression levels in the AP + EX group (p < 0.01). As a marker of autophagy initiation, Beclin1 is decreased in early AD and affects autophagy [26]. As shown in Figure 5B, compared with the mice in the WT + SED group, aerobic exercise significantly increased the expression level of the Beclin1 mRNA in the brain tissue of the mice in the WT + EX group (p < 0.05). On the contrary, in the mice in the AP + SED group, the expression of the Beclin1 mRNA showed a downward trend. Compared with the mice in the AP + SED group, aerobic exercise significantly increased the expression of the Beclin1 mRNA in the brain tissue of the mice in the AP + EX group (p < 0.01). These results suggest that aerobic exercise may regulate abnormal autophagy in the brain cells of APP/PS1 mice.
In order to further study the effect of aerobic exercise on abnormal autophagy in AD, our group detected autophagy-related proteins, microtubule-associated protein 1 light chain 3 (LC3), and sequestosome-1 (P62). As a marker of autophagosome formation, LC3-II is associated with autophagic activity [27,28]. As shown in Figure 5D, compared with the mice in the WT + SED group, the LC3-II/I ratios in the brain cells of the mice in the WT + EX group and the AP + SED group were significantly decreased (WT + EX group, p < 0.01; AP + SED group, p < 0.05). Compared with the mice in the AP + SED group, aerobic exercise significantly increased the LC3-II/I ratios in the brain cells of the mice in the AP + EX group (p < 0.01). P62 localizes autophagic substrates to autophagosomes by interacting with LC3 and is degraded by autophagy together with autophagic substrates, so it can be used as an indicator of autophagic degradation [27,29]. Decreased levels of P62 are associated with autophagy activation [27,29]. As shown in Figure 5E, compared with the mice in the WT + SED group, the level of the P62 protein in the brain cells of the mice in the AP + SED group was significantly increased (p < 0.05). Compared with the mice in the AP + SED group, aerobic exercise significantly decreased the level of the P62 protein in the mice in the AP + EX group (p < 0.05). As shown in Figure 5C, there was no significant difference in the expression of the P62 mRNA in the brain cells of the mice in the WT + EX group and AP + SED group, compared with the WT + SED group.
Compared with the mice in the AP + SED group, the expression of the P62 mRNA in the brain cells of the AP + EX group was significantly increased (p < 0.01). It was suggested that the difference in the expression level of the P62 protein among the groups was not caused by the difference in the transcription level of the P62 gene, but was related to the difference in the level of autophagic degradation. The above results indicated that aerobic exercise alleviated the abnormal autophagy in the brain cells of APP/PS1 mice.    . Exercise regulated autophagy in mouse brain tissue. The relative expression levels of (A) mTOR, (B) Beclin1, and (C) P62 mRNA in brain tissue were measured by RT-PCR (n = 5). Relative protein levels of (D) LC3-II/I and (E) P62 in brain tissue (n = 6). Data are presented as means ± SDs. # p < 0.05, ## p < 0.01 vs. WT + SED mice, * p < 0.05, and ** p < 0.01 vs. AP + SED mice.
In order to further study the effect of aerobic exercise on abnormal autophagy in AD, our group detected autophagy-related proteins, microtubule-associated protein 1 light chain 3 (LC3), and sequestosome-1 (P62). As a marker of autophagosome formation, LC3-II is associated with autophagic activity [27,28]. As shown in Figure 5D, compared with the mice in the WT + SED group, the LC3-II/I ratios in the brain cells of the mice in the WT + EX group and the AP + SED group were significantly decreased (WT + EX group, p < 0.01; AP + SED group, p < 0.05). Compared with the mice in the AP + SED group, aerobic exercise significantly increased the LC3-II/I ratios in the brain cells of the mice in the AP + EX group (p < 0.01). P62 localizes autophagic substrates to autophagosomes by interacting with LC3 and is degraded by autophagy together with autophagic substrates, so it can be used as an indicator of autophagic degradation [27,29]. Decreased levels of P62 are associated with autophagy activation [27,29]. As shown in Figure 5E, compared with the mice in the WT + SED group, the level of the P62 protein in the brain cells of the mice in the AP + SED group was significantly increased (p < 0.05). Compared with the mice in the AP + SED group, aerobic exercise significantly decreased the level of the P62 protein in the mice in the AP + EX group (p < 0.05). As shown in Figure 5C, there was no significant difference in the expression of the P62 mRNA in the brain cells of the mice in the WT + EX group and AP + SED group, compared with the WT + SED group. Compared with the mice in the AP + SED group, the expression of the P62 mRNA in the brain cells of the AP + EX group was significantly increased (p < 0.01). It was suggested that the difference in the expression level of the P62 protein among the groups was Figure 5. Exercise regulated autophagy in mouse brain tissue. The relative expression levels of (A) mTOR, (B) Beclin1, and (C) P62 mRNA in brain tissue were measured by RT-PCR (n = 5). Relative protein levels of (D) LC3-II/I and (E) P62 in brain tissue (n = 6). Data are presented as means ± SDs. # p < 0.05, ## p < 0.01 vs. WT + SED mice, * p < 0.05, and ** p < 0.01 vs. AP + SED mice.

The Effect of Exercise on APN and Its Receptors in the Brains of AD Mice
APN is an adipokine secreted by adipose tissue and is a potential target for the remission of AD [7]. Exercise has been reported to induce increases in circulating APN and AdipoR1 levels [8][9][10]. However, whether aerobic exercise modulates APN and its receptors in the brains of AD mice has not been studied. The research group detected the serum and brain APN and brain AdipoR1 levels in AD mice. As shown in Figure 6A, compared with the mice in the WT + SED group, the serum APN protein levels of the mice in the WT + EX group were significantly increased (p < 0.01). As shown in Figure 6B, compared with the mice in the AP + SED group, the APN protein contents in the brains of the mice in the AP + EX group had a downward trend. As shown in Figure 6C, compared with the mice in the WT + SED group, the expression of the AdipoR1 protein in the brains of the mice in the AP + SED group was significantly decreased (p < 0.05). As shown in Figure 6C,D, compared with the mice in the AP + SED group, aerobic exercise increased the expression of the AdipoR1 protein and mRNA in the brains of the mice in the AP + EX group (protein, p < 0.001; mRNA, p < 0.05). As shown in Figure 6D, compared with the WT + SED group, the brain AdipoR1 mRNA expression levels in the mice in the WT + EX and AP + SED groups showed a downward trend. The above results suggest that aerobic exercise can upregulate the level of AdipoR1 in the AD mouse brain.
the AP + SED group was significantly decreased (p < 0.05). As shown in Figure 6C,D, compared with the mice in the AP + SED group, aerobic exercise increased the expression of the AdipoR1 protein and mRNA in the brains of the mice in the AP + EX group (protein, p < 0.001; mRNA, p < 0.05). As shown in Figure 6D, compared with the WT + SED group, the brain AdipoR1 mRNA expression levels in the mice in the WT + EX and AP + SED groups showed a downward trend. The above results suggest that aerobic exercise can upregulate the level of AdipoR1 in the AD mouse brain.  The relative expression of AdipoR1 mRNA in brain tissue was measured by RT-PCR (n = 5). Data are presented as means ± SDs. # p < 0.05, ## p < 0.01 vs. WT + SED mice, * p < 0.05, and *** p < 0.001 vs. AP + SED mice.

Exercise Promotes the Expression and Phosphorylation of AMPK in the Brain Cells of AD Mice
Exercise upregulates the AdipoR1 levels in AD mouse brains. In order to further study the molecular mechanism by which the AdipoR1 signaling pathway participated in aerobic exercise's alleviation of AD-like lesions, our group detected the expression and phosphorylation of the AMPK protein, a signaling molecule downstream of AdipoR1, and the mRNA expression levels of the signaling molecules between AdipoR1 and AMPK in mouse brain tissue: adaptor protein containing pleckstrin homology domain, phosphotyrosinebinding domain, and leucine zipper motif (APPL1) and liver kinase B1 (LKB1). As shown in Figure 7A,B, compared with the mice in the WT + SED group, aerobic exercise significantly increased the levels of the AMPK and p-AMPK proteins in the brains of the mice in the WT + EX group (p < 0.05). The level of p-AMPK in the brain cells of the mice in the AP + SED group showed a downward trend. Compared with the mice in the AP + SED group, aerobic exercise significantly increased the protein levels of AMPK and p-AMPK in the brains of the mice in the AP + EX group (AMPK, p < 0.01; p-AMPK, p < 0.05). The APPL1/LKB1 signaling pathway mediates the activation of AMPK by AdipoR1 [30][31][32]. As shown in Figure 7C,D, compared with the mice in the AP + SED group, aerobic exercise significantly increased the APPL1 and LKB1 mRNA expression levels in the brain cells of mice in the AP + EX group (p < 0.05). The above results suggest that aerobic exercise promotes the expression and activation of the downstream signaling molecule AMPK to a certain extent through AdipoR1 in the brain cells of APP/PS1 mice.
significantly increased the levels of the AMPK and p-AMPK proteins in the brains of the mice in the WT + EX group (p < 0.05). The level of p-AMPK in the brain cells of the mice in the AP + SED group showed a downward trend. Compared with the mice in the AP + SED group, aerobic exercise significantly increased the protein levels of AMPK and p-AMPK in the brains of the mice in the AP + EX group (AMPK, p < 0.01; p-AMPK, p < 0.05). The APPL1/LKB1 signaling pathway mediates the activation of AMPK by AdipoR1 [30][31][32]. As shown in Figure 7C,D, compared with the mice in the AP + SED group, aerobic exercise significantly increased the APPL1 and LKB1 mRNA expression levels in the brain cells of mice in the AP + EX group (p < 0.05). The above results suggest that aerobic exercise promotes the expression and activation of the downstream signaling molecule AMPK to a certain extent through AdipoR1 in the brain cells of APP/PS1 mice. Figure 7. Exercise enhances AMPK activity in mouse brain tissue. Relative protein levels of (A) AMPK and (B) p-AMPK in brain tissue (n = 6). The relative expression levels of (C) APPL1 and (D) Figure 7. Exercise enhances AMPK activity in mouse brain tissue. Relative protein levels of (A) AMPK and (B) p-AMPK in brain tissue (n = 6). The relative expression levels of (C) APPL1 and (D) LKB1 mRNA in brain tissue were measured by RT-PCR (n = 5). Data are presented as means ± SDs. # p < 0.05 vs. WT + SED mice, * p < 0.05, and ** p < 0.01 vs. AP + SED mice.

Exercise Activates TFEB and Lysosome Function in the Brain Cells of AD Mice
Exercise upregulates the AdipoR1 levels in AD mice brain. AdipoR agonists have been reported to promote TFEB's nuclear translocation and the expression of TFEB-controlled autophagic genes, such as cathepsin D, lysosomal-associated membrane protein 1 (LAMP1), and ATPase H+ transporting V1 subunit H (Atp6v1h), thereby enhancing lysosomal function and autophagic flux [33][34][35]. It is suggested that aerobic exercise may promote the nuclear translocation of TFEB in the brain cells of APP/PS1 mice by upregulating the Adi-poR1 signaling pathway. Therefore, our group detected the expression of TFEB in mouse brain tissue, its nuclear translocation level, and the expression of lysosomal-function-related proteins and genes. As shown in Figure 8B,E, there was no significant difference in the mean fluorescence intensity of TFEB in the cortices and hippocampi of the mice in each group (p > 0.05), but aerobic exercise induced TFEB in the cortices and hippocampi of the mice in the WT + EX and AP + EX groups. The mean fluorescence intensity showed an upward trend. As shown in Figure 8C,F, compared with the mice in the WT + SED group, the mean fluorescence intensity of TFEB in the cortices and hippocampal nuclei of the mice in the AP + SED group was significantly decreased (p < 0.05). Compared with the mice in the AP + SED group, aerobic exercise significantly increased the mean fluorescence intensity of TFEB in the cortices and hippocampal nuclei of the mice in the AP + EX group (cortex, p < 0.05; hippocampus, p < 0.01). As shown in Figure 8G,H, compared with the mice in the WT + SED group, the protein levels of TFEB and cathepsin D in the brain cells of the mice in the WT + EX group were significantly increased (p < 0.05). Compared with mice in the AP + SED group, aerobic exercise significantly increased the protein levels of TFEB and cathepsin D in the brain cells of the mice in the AP + EX group (p < 0.001). As shown in Figure 8J, compared with the mice in the WT + SED group, the Atp6v1h mRNA expression levels of the mice in the AP + SED group were significantly downregulated (p < 0.05). As shown in Figure 8I,J, compared with the mice in the AP + SED group, aerobic exercise significantly increased the expression levels of the LAMP1 and Atp6v1h mRNA in the brain cells of the mice in the AP + EX group (p < 0.001). The above results suggest that aerobic exercise can enhance the lysosomal function of APP/PS1 mouse brain cells by activating TFEB activity, which may be mediated by the AdipoR1 signaling.  (F) TFEB in the nucleus of the hippocampus). Relative protein levels of (G) TFEB and (H) cathepsin D in brain tissue (n = 6). Relative expression levels of (I) LAMP1 and (J) Atp6v1h mRNA in brain tissue (n = 5). Data are presented as means ± SDs. # p < 0.05 vs. WT + SED mice, * p < 0.05, ** p < 0.01, and *** p < 0.001 vs. AP + SED mice.

Exercise Activated PLC/Kcnip3/Mid1/PP2A Signaling Pathway in the Brain Cells of AD Mice
To further study the possible molecular mechanism underlying the AdipoR1-mediated promotion of TFEB nuclear translocation in APP/PS1 mice by aerobic exercise, our group detected the mRNA expression levels of PLC/Kcnip3/Mid1/PP2A, involved in the upstream signaling pathway of TFEB [34]. As shown in Figure 9A-C,E, compared with the mice in the AP + SED group, the mRNA expression levels of phospholipase C gamma (Plcg) 1/2, Kcnip3, and protein phosphatase 2 catalytic subunit alpha (Ppp2cα) in the brain cells of the mice in the AP + EX group were all increased (Plcg1, p < 0.01; Plcg2, p < 0.05; Kcnip3, p < 0.05; Ppp2cα, p < 0.05). As shown in Figure 9D, compared with the mice in the AP + SED group, the expression level of the Mid1 mRNA in the brain cells of the mice in the AP + EX group was significantly downregulated (p < 0.05). The above studies suggest that aerobic exercise activates the PLC/Kcnip3/Mid1/PP2A signaling pathway in the brain cells of APP/PS1 mice.

Discussion
Exercise alleviates AD-like lesions and cognitive impairment in APP/PS1 mice. Previous studies have shown that Aβ in the brain is the main cause of neuronal apoptosis and cognitive impairment in AD [1]. Endurance exercise can delay brain aging, preserve memory and cognitive function, and improve the symptoms of neurodegenerative diseases such as AD [36]. Studies have shown that aerobic exercise can effectively downregulate the expression of Bax and caspase3 in the brain of APP/PS1 mice, upregulate the expression of Bcl-2, and upregulate the ratio of Bcl-2/Bax, thereby inhibiting neuronal apoptosis and improving cognitive function [4,37]. Aerobic exercise increased the number

Discussion
Exercise alleviates AD-like lesions and cognitive impairment in APP/PS1 mice. Previous studies have shown that Aβ in the brain is the main cause of neuronal apoptosis and cognitive impairment in AD [1]. Endurance exercise can delay brain aging, preserve memory and cognitive function, and improve the symptoms of neurodegenerative diseases such as AD [36]. Studies have shown that aerobic exercise can effectively downregulate the expression of Bax and caspase3 in the brain of APP/PS1 mice, upregulate the expression of Bcl-2, and upregulate the ratio of Bcl-2/Bax, thereby inhibiting neuronal apoptosis and improving cognitive function [4,37]. Aerobic exercise increased the number of dendritic spines in APP/PS1 mice and was significantly associated with improved cognitive function [38]. Consistently, the results of this study showed that APP/PS1 mice had severe Aβ deposition in the brain, a loss of dendritic spines, and impaired spatial learning and memory, suggesting that the AD-model mice successfully developed AD-like lesions, while aerobic exercise reversed the above changes; inhibited Aβ deposition, brain cell apoptosis, and dendritic spine loss; and alleviated the spatial learning and memory impairment in APP/PS1 mice.
Autophagy, a catabolic pathway that maintains cellular metabolic balance and realizes cellular self-renewal, involves the autophagosome: a double-layer membrane shed by the intracellular rough endoplasmic reticulum that coats cellular components such as excessively accumulated proteins and damaged organelles in the cytoplasm and then fuses with a lysosome to form an autophagolysosome for degradation [23]. Autophagy is fundamentally active and efficient in normal neurons, whereas autophagy dysfunction occurs in AD [23,39]. Recent studies have shown that dysfunction of the autophagylysosomal pathway in AD precedes AD-related pathologies such as Aβ deposition and induces neuronal apoptosis as well as extracellular plaque formation and expansion [40]. It is suggested that abnormalities in autophagy not only prevent toxic proteins such as Aβ being removed, but also aggravate AD-like lesions [2,23,39]. The results of this study show that the expression of the mTOR gene was upregulated and that Beclin1 and the LC3-II/I ratio was downregulated in APP/PS1 mice, suggesting that autophagic activity was inhibited; at the same time, the expression of the P62 mRNA did not change and even showed a downward trend, while its protein expression was upregulated, suggesting that autophagic degradation was hindered. The above studies suggest that abnormal autophagy in AD is an important factor in aggravating the disease.
Normal autophagy not only contributes to the degradation of toxic proteins such as Aβ but is also critical for maintaining dendritic spine structure and spine pruning [2,23,24]. It was found that the attenuation of dynamin-related protein 1 gene expression caused autophagy activation, increased dendritic spines, and enhanced cognitive function in transgenic Tau mice [41]. The results of this study show that aerobic exercise reversed the above abnormal changes in autophagy, downregulated mTOR gene expression, upregulated Beclin1 and the LC3-II/I ratio, upregulated the P62 mRNA expression level, and downregulated the P62 protein level. It is suggested that aerobic exercise alleviates ADlike lesions, such as the loss of dendritic spines and cognitive dysfunction, by alleviating abnormal autophagy in AD brain cells. Previous studies have also shown that enhanced autophagy plays an important role in inhibiting Aβ levels and neural apoptosis [23]. Exercise can promote the degradation of Aβand inhibit neural apoptosis by regulating autophagy in AD brain cells, thereby reducing the Aβ plaques and alleviating cognitive dysfunction [4][5][6]. However, the specific pathway through which exercise regulates AD autophagy remains unclear.
APN plays an important role in the beneficial effects of physical exercise in protecting brain tissue structure and improving cognitive function [18]. Numerous studies have found that APN knockout abolishes the beneficial effects of physical exercise on hippocampal neurogenesis and cognitive function in mice with chronic disease [11][12][13][14]. It is suggested that exercise can alleviate the damage to brain structure and function caused by chronic diseases through APN. APN plays an important role in relieving AD [7,[15][16][17]42]. Studies have shown that endogenous APN deficiency causes a decrease in dendritic spine density in mouse neurons, while exogenous APN treatment increases dendritic spine density [17,42]. Consistent with this study, aerobic exercise alleviated the loss of dendritic spines. It is suggested that aerobic exercise may alleviate the loss of dendritic spines through APN. APN treatment alleviated spatial learning and memory impairment in 3xTg-AD mice by re-ducing neuroinflammation and the impairment of synaptic plasticity [15]. APN deficiency accelerates Aβ deposition and exacerbates learning and memory impairment; treatment with the APN agonist AdipoRon alleviates Aβ plaque deposition in APP/PS1 mice and cognitive impairment in 5xFAD*APN KO mice [16]. AD patients, APP/PS1 mice, and Adipo −/− mice all showed decreased levels of AdipoR1 in brain tissue and impaired learning and memory functions, but treatment with an adiponectin-mimetic novel nonapeptide reversed the above changes [17]. It is suggested that exercise may relieve AD conditions through APN and is associated with AdipoR1. The alleviating effect of APN on AD is achieved, to a certain extent, through the activation of autophagy by AdipoR1. A study found that APN treatment caused the upregulation of AdipoR1 expression levels and the activation of autophagy in the brain tissue in global cerebral ischemia-reperfusion [43]. The APN agonist AdipoRon inhibits Aβ levels by activating autophagy while increasing AdipoR1 levels; similarly, the overexpression of AdipoR1 resulted in the upregulation of the autophagy-related protein LC3-II and the downregulation of Aβ levels in N2a/APPswe cells [16]. The results of this study show that aerobic exercise can significantly upregulate the expression of AdipoR1 in the brains of APP/PS1 mice, alleviate abnormal autophagy, and inhibit the level of Aβ plaques. In addition, exercise-induced downregulation of AdipoR1 in wild-type mice may be due to the negative feedback between APN and Adi-poR1; under the pathological conditions of AD, aerobic exercise fails to upregulate APN levels, but is enhanced by compensatory upregulation of AdipoR1 levels APN biological effects [43]. However, the specific reasons need to be clarified in our future studies. Based on the above results, aerobic exercise is likely to regulate AD autophagy by upregulating the AdipoR1 levels and to relieve AD-like lesions and cognitive dysfunction in APP/PS1 mice. This view was further confirmed in the follow-up exploration of AdipoR1 downstream signaling molecules.
AMPK, a signaling molecule that acts downstream of AdipoR1, plays an important role in exercise's regulation of autophagy in AD [6,30,[44][45][46]. The previous study of our group showed that aerobic exercise activated autophagy by activating the AMPK/Beclin1 signaling pathway and alleviated the morphological and structural damage of the rat hippocampus [44,45]. Consistently, previous studies have found that long-term exercise induces enhanced AMPK activity in the brain tissue of AD mice, the activation of autophagy, and the remission of AD-like lesions [4,6,46]. AMPK mediates AdipoR1's regulation of autophagy in AD and alleviation of the disease [16,17]. Studies have found that an adiponectin-mimetic novel nonapeptide enhances synaptic plasticity and memory function in AD and Adipo −/− mice by activating the AdipoR1/AMPK signaling pathway [17]. AdipoR agonists upregulate autophagy and reduce Aβ levels in N2a/APPswe cells by activating the AdipoR1/AMPK/mTOR signaling pathway, while AMPK inhibitors reverse the above changes [16]. The results of this study show that aerobic exercise induced an upregulation of the mRNA expression of APPL1/LKB1, a messenger between AdipoR1 and AMPK; enhanced AMPK activity; decreased the mRNA expression of mTOR, a molecule acting downstream of AMPK, in the brain tissue of APP/PS1 mice; and alleviated autophagy abnormalities. In addition, the downward trend of APPL1 mRNA expression in wild-type mice caused by exercise may be related to the increased APN levels and the downward trend of AdipoR1. Further clarification is required in future studies. Based on the above results, aerobic exercise is likely to alleviate autophagy abnormalities in the brain cells of APP/PS1 mice by activating the AdipoR1/AMPK signaling pathway.
In addition, this study found that the LC3-II/I ratio in the brain tissue of wild-type mice was downregulated by aerobic exercise, and the expression of the P62 protein showed a downward trend, but the expression of the P62 mRNA showed an upward trend. Consistently, related studies have shown that drug treatment downregulates cellular LC3-II, p62, and Aβ expression by enhancing lysosomal function, restores autophagic flux, and is associated with TFEB [47,48]. It is suggested that the downregulation of the LC3-II /I ratio and P62 protein level in the brains of wild-type mice and the decrease in the P62 protein levels in the brains of AD mice caused by aerobic exercise may be due to the enhanced lysosomal function that promotes autophagic degradation.
The enhancement of lysosomal function plays an important role in exercise's enhancement of autophagy in AD. Studies have shown that decreased lysosomal acidification accompanied by impaired autophagic substrate clearance is an important factor in the induction of AD-related pathologies such as Aβ deposition [40]. TFEB regulates the expression of lysosomal-function-related genes such as LAMP1 and cathepsin D and autophagy-related genes, thereby enhancing the autophagy-lysosomal pathway, reducing neural apoptosis, and exerting neuroprotective effects [35,49]. Drug treatment enhances the autophagolysosomal pathway by activating TFEB, thereby promoting the degradation of toxic proteins in AD models, and PP2A plays an important role in promoting TFEB's nuclear translocation [33]. The results of this study show that aerobic exercise induced enhanced TFEB protein expression and nuclear translocation in APP/PS1 mice; upregulated the expression of lysosomal-function-related molecules such as cathepsin D, LAMP1, and Atp6V1h; enhanced P62 protein degradation; and alleviated autophagy abnormalities. In addition, the level of LAMP1 mRNA in wild-type mice did not change significantly or even show a downward trend after exercise intervention, which may be related to the downregulation of LC3-II/I ratio and the enhancement of lysosomal function. Further clarification is required in future studies. It is suggested that aerobic exercise enhances lysosomal function by promoting TFEB protein expression and nuclear translocation in APP/PS1 mice, thereby alleviating autophagy abnormalities. Previous studies have also shown that exercise can enhance the expression of lysosomal-function-related proteins such as cathepsin D/L and LAMP1 in AD brain tissue by activating the AMPK/TFEB signaling pathway, improve autophagic degradation, and promote autophagy, thereby promoting Aβ degradation, inhibiting apoptosis, and reducing Aβ plaque [4,6,50]. However, the AMPK/SIRT1 signaling pathway mainly mediates the enhancement of TFEB gene transcription by exercise without affecting the nuclear translocation of TFEB [50]. Therefore, how exercise promotes TFEB nuclear translocation in AD brain cells is unclear [6,50].
It has been reported that both APN-KO and 5xFAD*APN KO mice have decreased lysosomal-function-related proteins such as LAMP1 and cathepsin D [16]. AdipoR agonists promote intracellular TFEB nuclear translocation and TFEB-controlled autophagic gene expression [35]. The results of this study show that aerobic exercise upregulates the Adi-poR1 levels in the brain cells of APP/PS1 mice and enhances TFEB's nuclear translocation and lysosomal function. It is suggested that aerobic exercise may promote the nuclear translocation of TFEB in the brain cells of APP/PS1 mice by upregulating the AdipoR1 levels, thereby enhancing lysosomal function and alleviating abnormal autophagy. This view is explored further below.
Our group preliminarily explored the possible molecular mechanism by which aerobic exercise promotes the nuclear translocation of TFEB in AD brain cells by upregulating AdipoR1 levels. First, AdipoR1 regulates intracellular Ca 2+ levels by activating PLC [30,31,35,51]. Studies have shown that PLC-mediated APN promotes the release of Ca 2+ from the endoplasmic reticulum, which is not affected by extracellular Ca 2+ [30]. Further studies found that AdipoR agonists were dependent on intracellular Ca 2+ for their promotion of TFEB's nuclear translocation, independent of extracellular Ca 2+ , AMPK, protein kinase B (Akt), extracellular signal-regulated kinase (ERK) 1/2, and mTOR [35]. It was also found that the inhibition of AdipoR1 expression abolished the increase in Ca 2+ levels caused by APN [31]. It is suggested that APN regulates intracellular Ca 2+ mainly through AdipoR1, but whether AdipoR1 regulates Ca 2+ in terms of intracellular release or extracellular influx remains controversial, and there may be differences in different cells [30,31,51]. It is suggested that the AdipoR1/PLC signaling pathway plays an important role in promoting the nuclear translocation of TFEB. Second, the regulation of intracellular Ca 2+ by PLC can activate the Kcnip3/Mid1/PP2A signaling pathway, thereby promoting the nuclear translocation of TFEB [34,[52][53][54]. Studies have shown that Ca 2+ inhibits Mid1 transcription by activating Kcnip3, thereby promoting PP2A stabilization and enhancing TFEB's nuclear translocation and the autophagy-lysosomal pathway [34]. AD-related studies have also shown that Mid1 is significantly increased in the brains of AD patients, and that the inhibition of Mid1 expression can enhance the stability of PP2A and promote Tau dephosphorylation [52]. PP2A activity was reduced in APP/PS1 transgenic mice, and the reactivation of PP2A activity effectively suppressed the levels of toxic proteins such as Aβ and protected neurons and synapses, thus alleviating cognitive dysfunction [53]. In a series of human and murine cell experiments, PP2A, similar to APN, dephosphorylated TFEB independently of mTOR and promoted the nuclear translocation of TFEB [54]. Our group hypothesized that the AdipoR1 might promote TFEB's nuclear translocation in AD brain cells by regulating the PLC/Kcnip3/Mid1/PP2A signaling pathway. We found that aerobic exercise upregulated the AdipoR1 levels in the brain cells of APP/PS1 mice. Based on the above, our research group explored the possibility that aerobic exercise affected the mRNA expression of PLC/Kcnip3/Mid1/Ppp2cα in AD mouse brain cells. We found, in this study, that the intervention of aerobic exercise resulted in increased mRNA expression of Plcg1/2, Kcnip3, and Ppp2cα in the brain cells of APP/PS1 mice, while the expression of the Mid1 mRNA was inhibited. In addition, in this study, the mRNA expression levels of PLC/Kcnip3/Mid1/Ppp2cα and other molecules in wild-type mice did not change significantly after exercise intervention, which may be related to their healthy and stable internal environment effectively alleviating stress responses. Further clarification is required in future studies. Based on the above results, the AdipoR1/PLC/PP2A signaling pathway may play an important role in aerobic exercise's promotion of TFEB's nuclear translocation and enhancement of the autophagy-lysosomal pathway in the brain cells of AD mice. This research group will further explore and demonstrate this in future research.

Experimental Animals and Grouping
Twenty 6-week-old male wild-type mice, whose strain number is C57BL/6J, were purchased from Changsha Tianqin Biotechnology Co., Ltd. Twenty wild-type (WT) mice were randomly divided into a sedentary (SED) group (WT + SED) and an exercise (EX) group (WT + EX), with 10 mice in each group. Twenty APP/PS1 double-transgenic (AP) mice were randomly divided into a sedentary (SED) group (AP + SED) and an exercise (EX) group (AP + EX), with 10 mice in each group.

Exercise Protocols
The mouse exercise training device (patent number: ZH-PT/5S) was selected for the experiment. The PT-type mouse animal experimental treadmill (flat treadmill) is mainly used for the running exercise training of small mice. Referring to relevant materials and combining them with the needs of this experiment, a regular aerobic exercise model was established [4,55]. The exercise program was as follows (Figure 10):

Exercise Protocols
The mouse exercise training device (patent number: ZH-PT/5S) was selected for the experiment. The PT-type mouse animal experimental treadmill (flat treadmill) is mainly used for the running exercise training of small mice. Referring to relevant materials and combining them with the needs of this experiment, a regular aerobic exercise model was established [4,55]. The exercise program was as follows (Figure 10): After the mice were raised to 12 weeks of age, the mice in the WT + EX and AP + EX groups underwent adaptive training for three days, exercising for 15 min every day at speeds of 5, 8, and 12 m/min, increasing day by day. After that, random group numbering was carried out. Formal exercise occurred at 7,8,9,10,11,12,13,14, and 15 m/min weekly increments from 1 to 9 weeks, and subsequently at a constant speed for three weeks, with a total of 12 weeks of exercise. Training occurred on every day of the week, except After the mice were raised to 12 weeks of age, the mice in the WT + EX and AP + EX groups underwent adaptive training for three days, exercising for 15 min every day at speeds of 5, 8, and 12 m/min, increasing day by day. After that, random group numbering was carried out. Formal exercise occurred at 7,8,9,10,11,12,13,14, and 15 m/min weekly increments from 1 to 9 weeks, and subsequently at a constant speed for three weeks, with a total of 12 weeks of exercise. Training occurred on every day of the week, except Thursday and Sunday. The training time was from 16:00 to 19:00, and the exercise periods lasted for 45 min.

Morris Water Maze Test
To assess the learning and memory abilities of the mice, the test was performed as previously described [4,11]. Mice were tested in a circular pool with a diameter of 1.5 m and a height of 0.5 m. One day before the test, the mice were acclimated in the pool for 10 min. During the learning phase, the platform (10 cm in diameter) was immersed 1.5 cm below the water surface, keeping the water temperature around 25 • C, adding a nontoxic white dye to make the water opaque and hide the platform. During the learning phase, each mouse performed 4 trials per day, which were pseudo-randomly selected in the four quadrants, and the quadrant where the platform was located was the target quadrant; this lasted a total of 5 days. Each mouse had 60 s to locate the hidden platform. If it failed to reach the hidden platform within 60 s, it was guided to the platform, and 10 s stop learning was conducted. During the retention phase, the platform was removed, and the mice swam freely for 60 s. All the trials were video-recorded, and the data were analyzed using the Xeye Aba animal behavior video analysis system (Beijing Tianming Hongyuan Technology Development Co., Ltd., Beijing, China).
Twenty-four hours after the water maze test, the mice in each group were fasted for 12 h and then anesthetized with isoflurane. Serum was collected for determining the biochemical indices. After the heart was perfused with normal saline, the left hemispheres of 3 mice in each group were fixed in 4% paraformaldehyde and then stored in a solution containing 0.02% sodium azide for immunohistochemical experiments. After the rest of the mice in each group were anesthetized and sacrificed, the brains were quickly removed, frozen in liquid nitrogen, and stored at −80 • C.

Immunohistochemical Detection
Fixed hemibrains were cut into 50 µm coronal sections using a LEICA VT1000S vibratome and stored in a solution containing 0.02% sodium azide. After they were washed with TBS, they were soaked in a 3% hydrogen peroxide methanol solution for 30 min. After TBS cleaning, they were then soaked in 88% formic acid for 7 min, TBS-A (1 L of TBS + 1 mL of Tritonx-100) for 15 min, and TBS-B (100 mL of TBS-A + 2 g of BSA) for 30 min. The sections were incubated with a primary antibody (1:1000, 6E10-Mouse IgG, Biolegend, San Diego, CA, USA) overnight at 4 • C. After they were washed with TBS-A/B, they were incubated with a secondary antibody for 1 h at room temperature. After they were washed with TBS-A, they were incubated in a solution prepared using the ABC Kit (Vector, Tokyo, Japan, Cat: PK-4002) at room temperature for 1 h. After they were washed with TBS, they were soaked in a DAB solution for 2 min 30 s, and the staining was observed at ×40 magnification. The Image-Pro Plus 6.0 software (Media Cybernetics, Inc., Rockville, MD, USA) was used to analyze the percentages of Aβ plaque areas in the field of view of each brain slice.

Enzyme-Linked Immunosorbent Assay
The content of APN protein in the serum and brain tissue was detected according to the instructions of the mouse adiponectin ELISA kit (Quanzhou Ruixin Biotechnology, Cat: RX202420M, Quanzhou, China).

Real-Time PCR
Total RNA from brain tissue was extracted with TRIzol (Ambion, Cat: 15596026, Austin, TX, USA) and reverse-transcribed into cDNA using TransScript ® One-Step gDNA Removal and cDNA Synthesis SuperMix (TransGen Biotech, Cat: AT311-02, Beijing, China). qRT-PCR was performed using the 2×SYBR Green qPCR Master Mix (Servicebio, Cat: G3320, Wuhan, China) and the CFX Connect™ Real-Time System (Bio-Rad, Singapore). Using GAPDH as the internal reference gene, the relative expression of the target gene was calculated by the 2 −∆∆CT method. The sequences of the primers used for the qRT-PCR were as follows (Table 1). Table 1. Sequences of primers for target genes.

Gene
Forward Primer Reverse Primer

Western Blotting
An RIPA (Servicebio, Cat: G2002-100 ML, Wuhan, China) lysis buffer was used to extract the total protein from brain tissue, and various protease inhibitors (Servicebio, Cat: G2006-250UL, G2007-1ML, and G2008-1ML, Wuhan, China) and a 2X protein loading buffer (Servicebio, Cat: G2031-1ML, Wuhan, China) were added. The mixture was heated at 95 • C for 10 min and stored at −20 • C after aliquoting. After gel preparation was completed using an SDS-PAGE gel rapid preparation kit (Servicebio, Cat: G2037, Wuhan, China), the protein lysates were separated by electrophoresis and transferred to a 0.45 µm PVDF membrane (Servicebio, Cat: G6015-0.45, Wuhan, China), with 5% skim milk used for blocking. The membrane was incubated with a primary antibody at 4 • C overnight, and then with a secondary antibody at room temperature for 1 h. Visualization was performed using an ECL kit (Servicebio, Cat: G2014-50 mL, Wuhan, China), and quantification was performed using the ImageJ 1.

Golgi Staining
A Golgi Stain Kit (FD Rapid Golgi Stain Kit PK-401, FD Neuro Technologies, Columbia, MD, USA) was used according to the manufacturer's instructions. The left hemibrains of 4 mice in each group were placed into a mixture of Golgi staining A/B (1:1) and stored at room temperature in the dark for 24 h. They were then placed into a fresh mixture and stored for another 2 weeks. The hemibrains were then put into Solution C and stored in the dark for 12 h. Solution C was then refreshed, and the specimens were stored for another 72 h. The brain tissue was cut into 200 µm coronal sections with a LEICA VT1000S vibrating microtome, and the sections were placed on a gelatin-coated glass slide and dried naturally at room temperature for 1-3 days in the dark. The slides with the sections were washed with double-distilled water for 2 × 3 min and then stained in a staining tank for 10 min (Solution D:Solution E:double-distilled water = 5:5:15 mL). After they were washed with double-distilled water for 2 × 4 min, they were dehydrated using 50%, 75%, and 95% ethanol for 5 min and using anhydrous ethanol for 3 × 5 min, made transparent with a xylene solution for 2 × 5 min, sealed with neutral resin, and air-dried at room temperature. Images were acquired at 1000× magnification with 5-10 neurons each in the cortex and hippocampus of each sample (n = 3-4), with 3-5 segments per neuron, apical and basal. The Image J 1.53q software (Wayne Rasband and contributors National Institutes of Health, USA) was used to calculate the exact length. Dendritic segments larger than 30 µm were selected, and the number of spines on the corresponding dendritic segment was counted. The density of the dendritic spines is expressed as the number of dendritic spines/10 µm [56].

Immunofluorescence Staining
The fixed hemibrains were embedded in paraffin, placed in a paraffin microtome, and cut into 5 µm coronal sections. After the sections were deparaffinized and rehydrated, they were placed in an EDTA antigen retrieval buffer (Servicebio, Cat: G1206, Wuhan, China) for antigen retrieval in a microwave oven (8 min at medium heat, 8 min at intervals, and 7 min at medium-low heat) and then washed with PBS (pH 7.4) for 3 × 5 min. They were then blocked with 3% hydrogen peroxide for 25 min at room temperature and washed with PBS. A 3% BSA solution (Servicebio, Cat: G5001, Wuhan, China) was used to block them for 30 min. The primary antibody was then added and incubated with the sections overnight at 4 • C. After washing with PBS, the corresponding secondary antibody was added and incubated with the sections at room temperature for 50 min. After washing with PBS, CY3-TSA (Servicebio, Cat: G1223, Wuhan, China) or FITC-TSA (Servicebio, Cat: G1222, Wuhan, China) was added, and the mixture was shaken and washed on a destaining shaker (for the second primary antibody, this was repeated, but hydrogen peroxide blocking was avoided). A DAPI staining solution (Servicebio, Cat: G1012, Wuhan, China) was added and incubated with the sections at room temperature for 10 min in the dark. Images at 1000× magnification were acquired using a confocal laser microscope (NIKON Eclipse Ti, Nikon, Tokyo, Japan). The Image J 1.53q software (Wayne Rasband and contributors National Institutes of Health, USA) was used to calculate the average immunofluorescence intensity of the target area, and the threshold was set by the default algorithm.

Statistical Analysis
The obtained data were statistically analyzed and graphed using the GraphPad Prism 8.0.1 software (GraphPad Software, Inc., San Diego, CA, USA), and the data are expressed as the means ± standard deviations (SDs). A Student's t-test or one-way ANOVA was used, and the LSD method was used for post hoc comparisons to test the significance of dif-ferences between data groups. p < 0.05 indicated a significant difference, p < 0.01 indicated a very significant difference, and p < 0.001 indicated an extremely significant difference.

Conclusions
This study was the first to explore the role of the APN-AdipoR1 signaling pathway in exercise's alleviation of AD-like pathophysiological progression through regulating autophagy. The results of this study suggest that aerobic exercise may enhance the autophagy-lysosomal pathway through the activation of the AdipoR1/AMPK/TFEB signaling pathway in APP/PS1 mice and alleviate autophagy abnormalities, thereby reducing Aβ deposition, inhibiting neural apoptosis and dendritic spine loss, and alleviating cognitive dysfunction in AD ( Figure 11). These findings suggest that the AdipoR1 plays an important role in aerobic exercise's alleviation of abnormal autophagy in AD brain cells and alleviation of AD-like lesions. dysfunction in AD ( Figure 11). These findings suggest that the AdipoR1 plays an important role in aerobic exercise's alleviation of abnormal autophagy in AD brain cells and alleviation of AD-like lesions. Figure 11. Proposed model for aerobic exercise's alleviation of abnormal autophagy in brain cells of APP/PS1 mice by upregulating the AdipoR1 levels (created with BioRender.com). Available online: https://app.biorender.com/illustrations/6279e002dbc8411b3f12fe3f (accessed on 22 July 2022) Aerobic exercise upregulates AMPK and TFEB activity by upregulating the AdipoR1 levels in the brains of APP/PS1 mice, enhances lysosomal function, and improves abnormal autophagy, thereby alleviating AD-like lesions, Aβ deposition, and cognitive dysfunction.
The roles of the APN-AdipoR1 signaling pathway in exercise's regulation of autophagy and the alleviation of AD are very complex. Whether it is the effect of different forms of exercise on the APN-AdipoR1 signaling pathway, or the effect and mechanism of the APN-AdipoR1 signaling pathway in AD autophagy abnormalities, there are still many gaps and deficiencies that need to be further studied. This provides new ideas for revealing the mechanism of exercise-regulated autophagy in AD and even the treatment of AD.
Author Contributions: W.L., Y.J. and S.Y. conceived and designed the research. Y.J. and J.Y. performed and analyzed the experiments. Y.J. and Y.L. wrote the manuscript. X.L. and W.L. supervised the study and revised the manuscript. All authors have read and agreed to the published version of the manuscript. . Proposed model for aerobic exercise's alleviation of abnormal autophagy in brain cells of APP/PS1 mice by upregulating the AdipoR1 levels (created with BioRender.com). Available online: https://app.biorender.com/illustrations/6279e002dbc8411b3f12fe3f (accessed on 22 July 2022) Aerobic exercise upregulates AMPK and TFEB activity by upregulating the AdipoR1 levels in the brains of APP/PS1 mice, enhances lysosomal function, and improves abnormal autophagy, thereby alleviating AD-like lesions, Aβ deposition, and cognitive dysfunction.
The roles of the APN-AdipoR1 signaling pathway in exercise's regulation of autophagy and the alleviation of AD are very complex. Whether it is the effect of different forms of exercise on the APN-AdipoR1 signaling pathway, or the effect and mechanism of the APN-AdipoR1 signaling pathway in AD autophagy abnormalities, there are still many gaps and deficiencies that need to be further studied. This provides new ideas for revealing the mechanism of exercise-regulated autophagy in AD and even the treatment of AD.


s2fieldsofstudy: Biology Medicine Psychology

text:
Revisiting regulatory T cells for stroke therapy

Stroke is a leading cause of death and long-term disability. T cells have been extensively studied for their dual role in regulating immunity and inflammation following stroke. In this issue of the JCI, Cai, Shi, et al. demonstrated that CD8+ regulatory-like T cells (CD8+ TRLs) are one of the earliest lymphocyte subtypes to enter the brain after experimental ischemic stroke. Using a mouse model of stroke and comprehensive experimental approaches, the authors found that CD8+ TRLs reduced both brain damage and functional deficits in both young and aged mice. These unique early responding regulatory T cells may also play a role in a wide array of other T cell–mediated neurological disorders.

T cell immunology in stroke
Stroke is a leading cause of disability and death (1). Ischemic stroke is the predominant form of stroke, primarily caused by thrombosis or embolism that leads to a lack of blood flow to the brain. Soon after the onset of ischemia, cells in the brain become damaged and undergo cell death, resulting in long-term behavioral deficits. Clinically, mechanical thrombectomy and recombinant tissue-type plasminogen activator (rtPA; the only FDA-approved drug to treat acute ischemic stroke) are effective therapies for restoring the blood flow and salvaging the penumbra (2, 3); however, additional treatment strategies for stroke are still needed, as many patients are ineligible for current therapies. Importantly, recent studies have emphasized that understanding the immunological and inflammatory mechanisms triggered by stroke is a prerequisite for developing immune-based therapeutics.
Preclinical studies using rodent stroke models have provided us with detailed mechanistic insights into how immune responses contribute to stroke outcome and poststroke recovery. Through characterization of the neuroimmune landscape, it is now well documented that stroke can induce both innate (acute phase) and adaptive (chronic phase) immune responses (4). Among many cell types implicated in stroke-induced inflammation, T cells have received much attention due to their importance in regulating both arms of immunity. In patients, T cells are found in the brain tissue within the first one to three days after stroke (5). Several studies using mouse models have further demonstrated both detrimental and protective roles of T cells after stroke. For instance, γδ T cells have been highlighted due to their detrimental role in neuroinflammation. They can be found in infarcted tissue within days following stroke and contribute to inflammation and neurological deficits. These cells secrete the proinflammatory cytokine IL-17, leading to chemotactic neutrophil infiltration (6, 7). In contrast to γδ T cells, regulatory T (Treg) cells facilitate recovery by reducing inflammation (8).
In mice, manipulation of Treg cells using both adoptive cell transfer and depletion models showed Treg-mediated neuroprotection due to enhanced secretion of antiinflammatory cytokines, including IL-10 (8). Very recent studies have revealed that inflammation in peripheral tissues, such as the gut, can regulate T cell-mediated inflammation after stroke (9, 10). Thus, targeting T cells may be a promising therapeutic option for stroke.

CD8 + regulatory-like T cells as protective cells for stroke
T cells are highly immunomodulatory in the poststroke brain. Previous reports have shown that CD8 + CD122 + T cells perform regulatory functions in autoimmune and inflammatory diseases (11-15). In this issue of the JCI, Cai, Shi, et al. (16) focused on the role of CD8 + CD122 + T cells in stroke. They identified a subtype of CD8 + CD122 + CD49d lo T cells as CD8 + regulatory-like T cells (CD8 + TRLs) that enhanced neuroprotection after stroke. Using a transient middle cerebral artery occlusion (tMCAO) model in mice, they showed that prior to infiltration of CD4 + Treg cells, CD8 + TRLs infiltrated the brain as early as one day after stroke and peaked at poststroke day five. These cells remained in the brain for at least two weeks after injury and highly expressed HELIOS and IL-10, which are essential for the regulatory functions of T cells (17). To directly determine the contribution of these CD8 + TRLs, Cai, Shi, et al. selectively depleted these cells using an anti-CD122 monoclonal antibody that does not affect other immune cells. CD8 + TRL-depleted mice had larger infarcts, acutely and chronically, after ischemia, with worse behavioral outcomes. Importantly, these poststroke characteristics were reversed by CD8 + T cell reconstitution using FACS-isolated CD8 + CD122 + CD49d lo TRLs adoptively transferred into mice depleted of CD8 + TRLs two hours after stroke (16).
Subsequent experiments including RNA sequencing and the use of transgenic mouse models revealed that the interac-

Unanswered questions and conclusions
Certain classes of T cells are detrimental in the early phase of stroke (19,20). Among the regulatory niche of immune cells, Cai, Shi, et al. (16) have elegantly demonstrated that CD8 + TRLs confer neuroprotection, although multiple interesting questions remain: (a) What environmental cues in the periphery or in the injured brain lead to the infiltration of CD8 + TRLs in the early phase of stroke? (b) How do these CD8 + TRLs crosstalk with other immune cells, including microglia? (c) Are there interactions between CXCR3, LIFR, and ETGF, which appear to be master regulators of the function of CD8 + TRLs, in the postischemic brain? (d) Is the change in CD8 + TRLs seen in this study specific to stroke-induced inflammation? (e) Does the enhancement of CD8 + TRLs alter the systemic immune response or the risk of poststroke infections? Examining whether this population of cells shows similar findings in stroke patients and how these cells change in both the periphery and the brain utilizing samples from stroke patients will be an important next step. This study by Cai,Shi,et al. (16) confirms that T cells are important in the response to brain ischemia, and specific subsets of T cells remain players in the orchestrated immune response to stroke. tion between CXCR3 and CXCL10 played an important role in the homing of these CD8 + TRLs into the brain after stroke. Once these cells entered the brain, they upregulated leukemia inhibitory factor receptor (LIFR), epidermal growth factorlike transforming growth factor (ETGF; the protein product of TGFA), and IL-10 within the ischemic hemisphere ( Figure 1). The authors convincingly demonstrate that the neuroprotective effect of CD8 + TRLs was mediated by activation of the ETGF and IL-10 pathways through interactions with LIF/LIFR. Of note, LIF signaling is critical for the regulatory function of T cells (18). Indeed, pretreatment with a LIFR inhibitor abolished the protective effects of CD8 + TRLs. Using an oxygen-glucose deprivation model, they further showed that CD8 + TRL-conditioned media can directly protect primary neurons in an ETGF-dependent manner. Finally, the authors found that adoptive transfer of sorted CD8 + TRLs into poststroke mice is beneficial and improved behavioral outcomes in both young male mice and aged mice of both sexes. This beneficial effect was also seen in young male recipient mice adoptively transferred with CD8 + TRLs from aged (20-month-old) donor mice, suggesting that the beneficial function of these cells is retained with aging. Figure 1. CD8 + TRLs confer early protection in the ischemic brain following stroke. Cai, Shi, et al. (16) showed that brain CD8 + TRLs are remarkably increased in the early phase of stroke, contributing to the amelioration of brain damage and improving functional recovery in mice. CXCR3 was upregulated in circulating CD8 + TRLs and CXCR3-CXCL10 interactions were critical for the infiltration of the ischemic brain tissues by CD8 + TRLs. Days after stroke, CD8 + TRLs from the ischemic site showed increased LIF receptor, ETGF, and IL-10, which critically regulated the neuroprotective effect. Figure  illustrated by Rachel Davidowitz.


s2fieldsofstudy: Biology Engineering

text:
Draft Genome Sequence of a Novel Rhizobium Species Isolated from the Marine Macroalga Codium fragile (Oyster Thief)

ABSTRACT In the process of studying the relationship between marine macroalgae and their bacterial symbionts, we isolated a new species of Rhizobium, which we designated Rhizobium sp. nov. C1 (for “Codium 1”). Here, we report the complete genome sequence of Rhizobium sp. nov. C1.

O yster Thief (Codium fragile) is a green macroalga that is established worldwide and is becoming of increasing interest to researchers due to the identification of the highvalue bioactive compounds it produces (1)(2)(3)(4)(5). We isolated and sequenced the genome of a bacterium associated with C. fragile that we subsequently identified as the Gram-negative Rhizobium sp. nov. C1.
The bacterium was isolated from C. fragile samples collected from a shoreline in Tauranga (6) and inoculated with C. fragile (10% [wt/vol]). The culture was grown in a shaking incubator (200 rpm) at 25°C. Cultures were subsequently subcultured every 2 to 5 days over a period of 2 weeks. Growth from liquid culture was spread onto BD Difco marine agar 2216. Repeated single-colony selection from marine agar was undertaken until a clonal strain was isolated, as determined by its uniform colony appearance and uniform growth rates upon restreaking. A stock of this strain was made by culturing the strain in BD Difco marine broth 2216 at 25°C, adding glycerol (25% [vol/vol]), and storing the strain at 280°C.
Rhizobium bacteria are of interest as important plant symbionts (7,8), but their role as algal symbionts are less understood; therefore, we sequenced the genome of this new species. A single colony was used to inoculate 5 mL of Zobell marine broth, which was incubated for 15 h at 25°C with shaking. From this culture, genomic DNA was extracted using the Wizard genomic DNA purification kit (Promega) with the Gram-negative bacteria protocol. The library was prepared using in-house Tn5-mediated tagmentation (9), followed by PCR enrichment with KAPA HiFi HotStart ReadyMix (Roche). DNA fragments of 400 bp were size selected before 150-bp paired-end reads were generated on an Illumina HiSeq 4000 system by Genewiz (China). Other sequencing and assembly statistics are shown in Table 1.
Genome assembly was achieved using raw reads (no reads were trimmed or discarded on grounds of quality, which was verified with FastQC v.0.11.7 [10]). Reference assembly was carried out using SPAdes v.0.4.7 (11) with the Rhizobium ipomoeae reference genome ASM491216v1 (RefSeq assembly accession number GCF_004912165.1); this produced an assembly containing 22 contigs, with an N 50 value of 469,644 bp. Genome annotation occurred through the NCBI Prokaryotic Genome Annotation Pipeline (PGAP) v.5.13 (12) as part of the GenBank submission process. All programs were run with default parameters unless otherwise stated. Genome features are summarized in Table 1.
The results of genome sequencing confirmed that we had isolated a species of Rhizobium, which we designated C1 (for "Codium 1"). The closest identified organism, based on average nucleotide identity (87.72%) determined using GTDB-Tk v.1.7.0 (13), was the plant-growth-promoting bacterium Rhizobium rhizophilum 7209-2 (RefSeq assembly accession number GCA_004912145.1) isolated from rape (Brassica napus L.) (7). The close relationship between Rhizobium sp. nov. C1 and this plant-growth-promoting bacterium indicates that Rhizobium sp. nov. C1 could be a marine algal symbiont; however, further investigation is required to validate this hypothesis.
Data availability. All data can be found under the NCBI BioProject accession number PRJNA773134, including raw reads (SRA accession number SRX13114500) and genome assembly information (GenBank accession number NZ_JAJGPW010000000).

ACKNOWLEDGMENTS
This work was supported via strategic research funds from the School of Biological Sciences at Victoria University of Wellington and by a grant from the Marsden Fund (grant 18-VUW-050).


s2fieldsofstudy: Biology Chemistry

text:
Trigger of apoptosis in adenocarcinoma gastric cell line (AGS) by a complex of thiosemicarbazone and copper nanoparticles

Seeking novel anticancer agents with minimal side effects against gastric cancer is vitally important. Copper, as an important trace element, takes roles in different physiologic pathways. Also, there is a higher demand for copper in cancer cells than normal ones. Copper complexes containing a therapeutic ligand could be promising candidates for gastric cancer chemotherapy. In this work, copper oxide nanoparticles were synthesized, functionalized with glutamic acid (CuO@Glu) and conjugated with thiosemicarbazone (CuO@Glu/TSC NPs). The NPs were characterized and their antiproliferative potential against AGS cancer cells was investigated using MTT, flow cytometry, Hoechst staining, and caspase 3 activation assays. The FT-IR results showed the proper binding of TSC to CuO@Glu NPs and crystallinity of the prepared NPs was confirmed by the XRD pattern. The EDX analysis confirmed the presence of Cu, N, C, O, and S elements and lack of impurities. The Hydrodynamic size and zeta potential of the CuO@Glu/TSC NPs were 168 nm and 27.5 mV, respectively. The NPs had spherical shape and were in a size range of 10 to 60 nm in diameter. This work revealed that CuO@Glu/TSC NPs efficiently inhibited the proliferation of AGS cells with significantly lower IC50 value (203 µg/mL) than normal HEK293 cells (IC50 = 435 µg/mL). Flow cytometry and Hoechst staining obviously revealed apoptosis induction among CuO@Glu/TSC treated cells, and caspase-3 activity significantly increased by 1.4 folds. This study introduced CuO@Glu/TSC as an efficient anticancer against gastric cancer cells with lower toxicity toward normal cells which could be employed for cancer treatment after further studies.

Background
Gastric cancer is a prevalent and deadly cancer, with about one million annual new cases, in the world [1]. The disease is highly prevalent around the world especially in eastern and central Asian countries. Gastric cancer causes 783.000 deaths annually, which accounts for 8.3% of cancer-associated death [1]. Traditional cancer therapy including chemotherapy, radiotherapy, and surgery are expensive, not always efficient and have sometimes considerable side effects [2]. Therefore, finding novel and inexpensive anticancer agents with minimal side effects is crucially important to treat gastric cancer.
Upon introduction of cisplatin, as a novel and efficient anticancer agent, many efforts have been focused on finding metal-containing drugs [3]. Among metal elements, copper complexes have gained more attention due to their fewer side effects and toxicity. Copper is considered an important trace element in the body which is important for the proper activity of cellular proteins and enzymes [4]. Owing to the higher proliferation rate and physiological activity, there is a higher demand for copper in cancer cells in comparison with normal ones [5]. Therefore, due to the increased copper uptake by cancer cells, the preparation of antiproliferative copper complexes might provide selective toxicity against cancer cells.
Several copper complexes with anticancer activity have been reported [3,6,7]. Among them, the complexes containing therapeutic ligands such as thiosemicarbazones (TSC) have been introduced as novel and efficient anticancer agents [3]. Thiosemicarbazide is able to interact with a variety of metals and to enhance their chemical activity. The anticancer potential metal NPs in conjugation with thiosemicarbazide was reported in the literature. Owing to the versatile coordination to metal ions and also, broad pharmaceutical potentials, including antibacterial, antiviral, and anti-tumor activities, TSC derivatives have been explored to prepare metal-TSC complexes, to be used as pharmaceutical agents [3,6,7]. Several Copper/TSC complexes have been evaluated against a variety of cancer cells including colon adenocarcinoma HT-29, breast cancer cells, lymphoblastic leukemia, and cervical carcinoma [3,6].
Many attempts have been performed to improve the hydrophobicity, anticancer potential, and stability of Copper/ TSC complexes and also to minimize their side effects. The stable Copper/TSC complexes are able to reach the desired targets more efficiently and avoid nonselective interactions of the drug with different parts of cells [3]. Modification of TSCs framework to reduce the toxicity of Copper/TSC complexes toward normal cells was the goal of many studies [3,7,8]. Therefore, in this work we aimed to synthesize copper oxide NPs, functionalize them with glutamic acid (CuO@ Glu), and conjugate with TSC (CuO@Glu/TSC NPs). Then, the anticancer effect of CuO@Glu/TSC NPs on AGS cells was evaluated.

Preparation of CuO and CuO@Glu/TSC NPs
Preparation of CuO NPs was performed by co-precipitation method, as described elsewhere [9]. Briefly, 300 mg of CuCl 2 ⋅2H 2 O was dissolved in 300 mL of distilled water; the pH was adjusted to 11 and the mixture was stirred at 80 °C for two hours. Next, the CuO NPs were centrifuged, washed with distilled water, and dried at 70 °C. A similar protocol was used for the synthesis of CuO@Glu, but 150 mg of l-glutamic acid was also added to the reaction mixture.
To synthesize CuO@Glu/TSC, 300 mg of CuO@Glu and 200 mg of thiosemicarbazone were dissolved in 300 ml of ethanol and stirred at 40 °C for one hour. Then, the mixture was centrifuged; and the resulting NPs were harvested, washed with distilled water, and dried at 70 °C.

Characterization of CuO@Glu/TSC NPs
X-ray diffraction (XRD) pattern of the CuO@Glu/TSC NPs was determined by a Philips X'Pert MPD diffractometer (Co-Ka X-radiation, k = 1.79 Å), and dynamic light scattering (DLS), and Zeta potential analyses were used to characterize the stability and agglomeration state of NPs using a Zeta-sizer instrument (Ver. 6.32, Malvern Instruments Ltd). Also, the morphology, distribution, and size of the prepared CuO@Glu/TSC NPs were determined by scanning electron microscopy (SEM) (KYKY-EM3200) and transmission electron microscopy (TEM) (EM10C). Moreover, to analyze the composition of NPs was energy dispersive X-Ray (EDX) was employed using an X-ray diffraction spectrometer (Delta series KEVEX). Also, a Nicolet IR 100 spectrophotometer was employed to analyze the Fourier-transform infrared (FT-IR) spectra of the NPs.

Cell culture
The adenocarcinoma gastric (AGS) and the human embryonic kidney 293 (HEK293) cells were received from the Pasteur Institute of Iran. The cell culture was performed in 25 cm 2 cell culture flasks using the Dulbecco's modified Eagle (DMEM) medium supplemented with penicillin/streptomycin (100 U/mL) and 100% fetal bovine serum (FBS) (Gibco, Netherland). To prepare cell monolayers, the incubation was performed at 37 °C with 5% CO 2 .

Evaluation of the viability of cells treated with CuO@ Glu/TSC NPs
The antiproliferative potential of CuO@Glu/TSC NPs against the AGS (cancer cell line) and HEK293 cells (as normal human cells) was investigated using the 2-(4,5-dimethythiazol-2-yl) -2,5-diphenyltetrazolium bromide (MTT) assay. Cisplatin was also used as a control drug against AGS cells. The assay was performed in triplicates.
At first, the cell monolayers with a cell population of 1 × 10 4 cell/well were prepared in microtiter plates. The cells were exposed to CuO@Glu/TSC NPs at different concentrations in a range of 0-500 μg/mL for 24 h. As a positive control, similar concentrations of cisplatin were also used against the AGS cells. After that, the MTT solution (0.5 mg/ mL) was added to each well and the plate was incubated for 4 h. After the incubation period, the medium was removed and 200 µL of dimethyl sulfoxide was added to the wells, and the plate was shaken for 30 min. Finally, the optical absorption of the wells was determined at 590 nm using a microplate reader (Bio-Rad, Hercules, CA, USA). The following formula was used to determine the 50% inhibition concentration (IC 50 ) values of CuO@Glu/TSC NPs and cisplatin against the studied cells [10]:

Flow cytometry assay
Flow cytometry analysis was performed to determine the apoptosis/necrosis rate of the AGS cells treated with CuO@ Glu/TSC NPs using a flow cytometry kit (Roche, Germany). The AGS cells were treated with the NPs at IC 50 concentration and incubated for 24 h. Then, the cells were harvested and washed with phosphate-buffered saline (PBS). Then, 10 μL of Annexin-V FLUOS and 5 μL of propidium iodide (PI) were added to the cells and the population of apoptotic/ necrotic cells was determined by a flow cytometry device (Partec, Germany).

Hoechst 33258 staining
The nuclear damages caused by CuO@Glu/TSC NPs in treated AGS cells were evaluated by the Hoechst staining method [11]. At first, the AGS cells (4 × 10 5 cells/well) were prepared and treated with CuO@Glu/TSC NPs at IC 50 concentration. The AGS cells without NPs exposure were regarded as control cells. After 24 h incubation, the cells were stained with Hoechst 33258 dye and visualized using a fluorescent microscope (Incell Analyser 2000, USA).

Caspase-3 activity
The activity of caspase-3 among the AGS cells, treated with CuO@Glu/TSC NPs and also control cells was evaluated using a colorimetric assay kit (Sigma-Aldrich, CASP3C). In brief, the AGS cells with a population of 5 × 10 6 cells/well were prepared in 6-well plates and treated with CuO@Glu/ TSC NPs at IC 50 concentration for 24 h. Then, the cells were harvested, lysed using a cell lysis buffer, and centrifuged. Finally, DEVD-pNA was added to the cell supernatant and the caspase-3 activity was determined by measuring the absorbance of p-nitroaniline (μM) released from the substrate at 405 nm [12].

Statistical analysis
The cellular toxicity assays were conducted in triplicates and the results were presented as mean ± SD. Statistical analyses were performed using SPSS. 16.0 software (SPSS Inc., Chicago, IL). The cellular toxicity of the compounds was expressed as mean ± SD, and the statistical significance of data was analyzed by one-way ANOVA. The p-value less than 0.05 was considered as statistically significant.

Characterization of CuO@Glu/TSC NPs
The X-ray diffraction (XRD) pattern for the CuO@Glu/ TSC nanoparticles was displayed in Fig. 1.  around 800 cm −1 in the IR spectrum is corresponding to ν(C=S) stretching [11,14].
The morphology and size of the CuO@Glu/TSC NPs were evaluated by scanning and transmission electron microscopy. The particles were approximately spherical with the particle size in a range of 10-60 nm. Figure 3 display the SEM and TEM images of the CuO@Glu/TSC NPs.
Energy dispersive X-Ray (EDX) was employed to analyze the Elemental composition of CuO@Glu/TSC NPs. The EDX analysis showed strong signals for the presence of The presence of no other EDX signal clearly indicates the purity of the synthesized NPs (Fig. 4, Table 1). Moreover, the hydrodynamic size and zeta potential of CuO@Glu/TSC NPs were measured 168 nm and 27.5 mV, respectively. The proper zeta potential of the CuO@Glu/TSC NPs could provide sufficient repulsive force between particles to avoid agglomeration of the NPs and provides satisfactory colloidal stability [15] (Fig. 5).

Cytotoxic effect of CuO@Glu/TSC NPs
The anti-proliferative potential of CuO@Glu/TSC NPs against AGS and HEK293 cell lines was evaluated. A considerable anti-proliferative effect of CuO@Glu/TSC NPs against AGS cells was observed with IC 50 value of 203 µg/ mL. Also, the prepared NPs showed a significantly less antiproliferative effect on the HEK293, as normal human cells, with an IC 50 value of 435 µg/mL. However, the anticancer potential of the NPs against AGS cells was significantly less than cisplatin, which exhibited the IC 50 value of 65 µg/mL. The relative viability of the cells exposed to different concentrations of CuO@Glu/TSC NPs and cisplatin was presented in Fig. 6.
As a trace element with specific functions inside the eukaryotic cells, this element has lower toxicity to normal human cells [3]. Also, due to the higher physiological state of cancer cells, they show a higher demand for this element, which could provide selective toxicity of copper-containing drugs against cancer cells [3,6]. Combination of copper with a variety of therapeutic ligands, including imidazoles and phosphines, as well as TSC derivatives showed a promising anticancer potential [3,16].
Several anticancer mechanisms have been proposed to be mediated by copper. The anticancer potential of copper has been mainly associated with the oxidative stress caused by the production of reactive oxygen species (ROS) and also, Glutathione (GSH) oxidation [3,6]. The oxidative imbalance damages cellular molecules, including nucleic acids, lipids, and proteins, which triggers cell apoptosis [6,17,18].
According to the literature, copper complexes have shown proteasome inhibitory potential in different cell lines and copper element had a crucial role in this issue. The proteasome, a large and multi-catalytic protease, is responsible for the degradation of damaged proteins and also regulating the concentrations of cell cycle regulatory proteins, including cyclins and p53 (144 and 145 from 4). Due to the higher load of copper inside cancer cells than normal cells, a higher proteasome inhibitory potential and thus, apoptosis induction by copper complexes was reported, which efficiently inhibited the proliferation of cancer cells [19,20].
Similar to other metal complexes, copper complexes can undergo redox reaction and ligand substitution which enable them to interact with a variety of biological substrates. Thus, conjugation of therapeutic ligands with copper could provide  efficient cytotoxicity against cancer cells by the action of both partners [3,6]. TSCs are considered as efficient cytotoxic agents, which suppress cancer cells proliferation by inhibition of cellular ribonucleotide phosphate reductase, the enzyme necessary for DNA replication [6]. Also, it was reported that the cytotoxicity of copper complexes containing coordinating ligands, including TSCs could be associated to the binding of the ligands to cell DNA which inhibits genome replication and transcription, and finally results in cell apoptosis [6].
Based on the results and above considerations we found that the conjugation of TSC to copper oxide NPs could inhibit the proliferation of cancer cells and results in higher toxicity to AGS cells compared with normal human cells. In addition, the functionalization of CuO@Glu/TSC NPs using glutamic acid seems to improve their stability and bioavailability. In agreement with our finding, several studies aimed to increase the hydrophilicity of Cu/TSC complexes by modification of TSCs framework to improve their stability and reported promising anticancer characteristics [8,21].
Moreover, investigating the antitumor effect of copper-2-keto-3-ethoxybutyraldehyde-bis(thiosemicarbazone) (KTS) revealed that the KTS ligand could stabilize the oxidation state of Cu(II). Following uptake of the complex by cancer cells, the complex is dissociated by interaction with cellular thiols. This dissociation generates Cu(I), which may act as heavy metal poison [6,22]. The similar synergistic potential could be also hypothesized to other CuO-TSC complexes. Figure 7 displays the apoptosis of CuO@Glu/TSC treated and control AGS cells. The results were presented in the diagrams with four regions where Q1 region displays cell necrosis, Q2 and Q3 represent late and early apoptosis, and Q4 for live cells. Our results showed that early apoptosis occurred among the majority of CuO@Glu/TSC treated cells  (69%) and 11.5% of cells experienced late apoptosis, which was significantly higher than control cells. In addition, 96.7% and 19.3% of control and NPs treated cells remained alive. Also, only 0.27% of cell necrosis was found among CuO@Glu/TSC treated cells. The apoptosis induction by copper NPs conjugated to the TSC derivatives against Burkitt's lymphoma cells has been reported previously, which is in agreement with our finding [22]. As described above, the apoptotic effect of CuO@Glu/TSC NPs could be associated with the DNA damages caused by oxidative stress, inhibition of DNA replication, and cellular proteasome inhibition [3,6].

Hoechst 33258 staining
Hoechst staining was performed on CuO@Glu/TSC treated and control cells to evaluate the presence of compact chromatin of apoptotic nuclei. The apoptosis morphological characteristics, including condensed chromatin and chromatin fragmentation, and apoptotic bodies were found among the AGS cells treated with CuO@Glu/TSC NPs. In contrast, the control cells did not show considerable changes (Fig. 8).
As stated above, the increased ROS generation by CuO@ Glu/TSC could results in DNA strand breakage and apoptosis induction [6]. The apoptotic morphological characteristics of CuO@Glu/TSC treated cells comply well with the results from the MTT and flow cytometry assays.

Caspase 3 activity
The activity of caspase-3 among CuO@Glu/TSC treated AGS cells and control cells was investigated. The results showed that treating AGS cells with the NPs resulted in the increased activation of caspase-3. In other words, among NPs treated cells the caspase-3 activity significantly increased by 1.4 folds in comparison with the control cells (Fig. 9). Caspases play a critical role in triggering of apoptosis [23]. In this study a significantly increased activity of caspase-3 was noticed, confirming the apoptosis induction in AGS cells following exposure to CuO@Glu/ TSC NPs. In agreement with our work, previous studies showed increased transcription and activation of caspase-3 in a variety of cancer cells following treatment with copper  NPs or copper complexes [4,6,11,23]. Generation of ROS and subsequently damages to cellular DNA is considered a major signaling for triggering apoptosis pathway [23]. In our opinion, after exposure of AGS cells to CuO@Glu/TSC NPs, generation of ROS by copper and also inhibition of cell proteasome, which regulates the concentration of p53 proteins, are major causes of apoptosis induction following upregulation of caspase proteins. In agreement with this, previous works reported that the activation of p53 among cancer cells treated with copper NPs indirectly promotes caspase-3 activity [24,25].

Conclusion
In this study, CuO@Glu/TSC NPs were synthesized and their anti-proliferative effect on AGS gastric cancer cells was investigated. Our results showed that the prepared NPs efficiently inhibited the cancer cell proliferation with significantly less IC 50 than normal HKE293 cells. Also, the NPs induced early apoptosis, chromatin fragmentation, and increased activity of caspase-3 in AGS cells, which describe the cytotoxic mechanism of CuO@Glu/TSC NPs. This work showed a promising anti-proliferative effect of CuO@Glu/ TSC against AGS cells, but further investigation needed specially in vivo studies on mouse models.