Abstract

   Senile plaque, composed of amyloid β protein (Aβ) aggregates, is a
   critical pathological feature in Alzheimer's disease (AD), leading to
   cognitive dysfunction. However, how Aβ aggregates exert age‐dependent
   toxicity and temporal cognitive dysfunction in APP/PS1 mice remains
   incompletely understood. In this study, we investigated AD pathogenesis
   and dynamic alterations in lysosomal pathways within the hippocampus of
   age‐gradient male mice using transcriptome sequencing, molecular
   biology assays, and histopathological analyses. We observed high levels
   of β‐amyloid precursor protein (APP) protein expression in the
   hippocampus at an early stage and age‐dependent Aβ deposition.
   Transcriptome sequencing revealed the enrichment of differential genes
   related to the lysosome pathway. Furthermore, the protein expression of
   ATP6V0d2 and CTSD associated with lysosomal functions exhibited dynamic
   changes with age, increasing in the early stage and decreasing later.
   Similar age‐dependent patterns were observed for the endosome function,
   autophagy pathway, and SGK1/FOXO3a pathway. Nissl and Golgi staining in
   the hippocampal region showed age‐dependent neuronal loss and synaptic
   damage, respectively. These findings clearly define the age‐gradient
   changes in the autophagy–lysosome system, the endosome/lysosome system,
   and the SGK1/FOXO3a pathway in the hippocampus of APP/PS1 mice,
   providing new perspectives and clues for understanding the possible
   mechanisms of AD, especially the transition from compensatory to
   decompensated state.

   Keywords: Alzheimer's disease, autophagy–lysosome system, Aβ,
   endosomal/lysosome system, SGK1/FOXO3a pathway, transcriptome
   sequencing
     __________________________________________________________________

   Schematic diagram of molecular mechanisms of the pathologic process
   from compensatory to decompensated in the hippocampus of age‐gradient
   APP/PS1 mice.

   During the compensation period, ATP6V0d2 protein was significantly
   elevated in the hippocampus of APP/PS1 mice, which increased the fusion
   of autophagosomes and endosomes with lysosomes, thus enhancing the
   degradation of APP protein. As a result, the modest generation of Aβ
   could promote the activation of SGK1, thereby exerting its
   anti‐inflammatory, neuroprotective, and resistance to Aβ toxicity. In
   the decompensation stage, the significant decrease of ATP6V0d2 led to
   the blockage of the fusion of autophagosome–lysosome and
   endosome–lysosome, and a large amount of APP was deposited into the
   early endosome and autophagosome, which was sheared by BACE1 to produce
   a large amount of Aβ, thus inhibiting the expression of SGK1. SGK1
   deficiency induced a decreased phosphorylation of FOXO3a, resulting in
   increased nucleation of nonphosphorylated FOXO3a and elevated
   expression of apoptosis genes and lysosome genes. Further deterioration
   of Aβ pathology resulted in significant loss of neurons and synaptic
   damage in the hippocampus of aged APP/PS1 mice.

   graphic file with name MCO2-5-e540-g007.jpg

1. BACKGROUND

   Alzheimer's disease (AD) is a neurodegenerative disease of
   multifactorial etiology characterized by chronic and progressive
   cognitive decline. Senile plaques, composed primarily of amyloid β (Aβ)
   peptide aggregations, and neurofibrillary tangles, formed by abnormally
   hyperphosphorylated tau (p‐tau) protein, are two major
   neuropathological changes seen in patients with AD. Tau and Aβ are
   mutually causal and promote each other, thereby accelerating AD
   progression. Abnormal Aβ metabolism is among the earliest
   manifestations observed in the brains of AD patients. The Aβ cascade
   hypothesis on the pathogenesis of AD proposes that abnormal Aβ protein
   accumulation is the initiating factor of the disease, subsequently
   leading to neuronal degeneration and dysfunction resulting in cognitive
   decline and mental behavior disorders, leading eventually to death.[54]
   ^1 , [55]^2 , [56]^3 Despite the large number of relevant studies on
   the pathology of AD, the specific mechanism involved in abnormal Aβ
   protein metabolism and the associated toxic effects that lead to AD
   remain to be clarified.

   Autophagy is a lysosomal‐dependent process that serves to recycle
   energy through the degradation of organelles and abnormal proteins,
   thereby maintaining cellular homeostasis. In the brains of AD patients,
   autophagy dysfunction can markedly affect metabolism and is considered
   to underlie the harmful accumulation of Aβ protein in the brain of AD
   patients.[57] ^4 An age‐dependent increase in Aβ and p‐tau contents can
   result in a reduction in the levels of functional proteins in
   autophagosomes and mitochondria, leading to defective autophagy and
   mitochondrial dysfunction.[58] ^5 Aβ can also negatively affect
   lysosomal function, resulting in impaired autophagy and severe cell
   damage.[59] ^6 It has been reported that the autophagy activator
   rapamycin can exert beneficial effects in early‐stage AD but is harmful
   as a late‐stage intervention.[60] ^7 These studies indicated that
   autophagy flux changes dynamically during the progression of AD. Aβ is
   generated via the sequential cleavage of β‐amyloid precursor protein
   (APP), a process that occurs primarily in the endosome following APP
   endocytosis.[61] ^8 Under the acidic conditions of the endosome, APP is
   first cleaved by BACE1, generating the APPβ and C99 fragments, the
   latter of which is further cleaved by γ‐secretase in the endosomal
   membrane, yielding the Aβ peptide.[62] ^9 Endosomal dysfunction is also
   observed in neurons of patients with early‐stage AD,[63] ^8
   Additionally, an electron microscopy‐based study found that the number
   of abnormal endosomes was increased in triploid APP transgenic mice, a
   commonly used animal model of AD,[64] ^10 This finding further
   emphasizes that abnormal APP metabolism resulting from endosomal
   dysfunction may be involved in the pathogenesis of this disease.

   Serum and glucocorticoid‐regulated kinase 1 (SGK1) plays a crucial role
   in cellular stress responses, which is involved in the regulation of
   multiple processes, including hormone release, neuronal excitability,
   inflammation, transport regulation, coagulation, cell proliferation,
   and apoptosis.[65] ^11 Additionally, SGK1 may be involved in the
   pathophysiology of several brain diseases, such as Parkinson's disease,
   schizophrenia, depression, and AD, and may also play a significant role
   in the regulation of neuronal function.[66] ^12 One study reported that
   the expression of SGK1 is decreased in the brain of AD patients and
   that SGK1 can promote nonamyloidogenic APP processing and Aβ
   degradation as well as upregulate the expression of synaptic
   plasticity‐related proteins, playing a notable role in the treatment of
   AD memory disorder.[67] ^13 In vitro, SGK1 protects neurons from injury
   by phosphorylating forkhead box O3a (FOXO3a), resulting in reduced
   transcriptional activity and anti‐apoptotic effects.[68] ^14 Increased
   hippocampal FOXO3a abundance was reported to be positively correlated
   with amyloid plaque density, Aβ42 content, and clinical dementia score
   in AD.[69] ^15 Furthermore, the SGK1/FOXO3a pathway can influence the
   expression of lysosome‐related genes,[70] ^16 thereby potentially
   influencing the functionality of the autophagy–lysosome system and the
   endosomal/lysosome system.[71] ^17 However, the specific impact of the
   SGK1/FOXO3a pathway on the autophagy–lysosome system and
   endosomal/lysosome system remains unclear but is likely to involve a
   unique regulatory mechanism.

   To address this knowledge gap, in this study, we explored the dynamic
   changes occurring in Aβ metabolism in the process of AD using a
   holistic approach. First, we used transcriptome sequencing to identify
   relevant molecular markers in the hippocampus of APP/PS1 mice of
   different age ranges that would allow the determination of the changes
   in the Aβ metabolic pathway during AD. Subsequent bioinformatic,
   biochemical, morphological, and behavioral analysis showed that, in
   APP/PS1 mice, the autophagy–lysosome system, the endosome/lysosome
   system, and the SGK1/FOXO3a pathway underwent
   compensatory‐to‐decompensatory pathophysiological alterations with
   increasing age. These findings suggested that Aβ metabolism is involved
   in the course of AD and shows dynamic changes in the compensatory,
   plateau, and decompensatory stages of the disease, providing new clues
   for elucidating the pathogenesis of this neurodegenerative disorder.

2. RESULTS

2.1. APP/PS1 mice show age‐dependent cognitive dysfunction and accumulation
of brain β‐amyloid plaques

   First, we investigated whether APP/PS1 mice, a classic animal model of
   AD with Aβ toxicity, exhibit age‐dependent cognitive dysfunction. For
   this, APP/PS1 mice were divided into three groups according to age (5,
   9, and 13 months), with age‐matched c57 wild‐type (WT) mice serving as
   controls. Experiments were performed after 1 week of acclimatization in
   the animal room (Figure [72]1A). To explore the relationship between
   memory dysfunction and age, the mice were subjected to the Morris water
   maze (MWM). The results indicated a trend of increased latency to find
   the hidden platform in APP/PS1 mice in all age groups compared to
   age‐matched WT mice during the five consecutive days of the training
   period (Figure [73]S1A‐C). However, no significant difference was
   detected in swimming speed among the six groups (Figure [74]1B),
   indicating that APP/PS1 mice may have decreased spatial learning
   ability, but their motor ability is unaffected. The probe trial was
   conducted on day 6 of the MWM test. For this, the hidden platform was
   removed, and the mice were allowed to freely explore the water maze for
   60 s. The results showed that compared with their WT counterparts,
   APP/PS1 mice at the ages of 9 and 13 months took significantly longer
   to cross the original platform location for the first time and spent
   less time in the target quadrant. Five‐month‐old APP/PS1 mice also took
   longer to reach the original platform location for the first time,
   although the difference was not significant (Figure [75]1C). These
   findings suggested that APP/PS1 mice begin to develop mild memory
   impairment as early as 5 months of age and that the extent of cognitive
   dysfunction is age‐dependent.

FIGURE 1.

   FIGURE 1
   [76]Open in a new tab

   Age‐dependent cognitive impairment and accumulation of brain β‐amyloid
   plaques in APP/PS1 mice. APP/PS1 mice aged 5 months, 9 months and 13
   months were divided into three groups according to age gradient, and
   c57 wild‐type mice aged 5 months, 9 months and 13 months were used as
   controls, and then behavioral tests were conducted. (A) Diagram of the
   experiment. (B–C) The Morris water maze (MWM) test was performed to
   evaluate the spatial memory of mice. (B) The mean swimming speed of
   each group. Data are presented as mean ± SEM (5 M Ctr, n = 10; 5 M
   APP/PS1, n = 10; 9 M Ctr, n = 10; 9 M APP/PS1, n = 10; 13 M Ctr, n = 9;
   13 M APP/PS1, n = 8, *p < 0.05). (C) The first passage time of the
   platform in the target quadrant within 1 min after removing the
   platform and the representative trajectory of mice in the probe trial.
   (D) Levels of APP in the hippocampus were detected by Western
   blotting analysis, and ACTB was used as a loading control. (E)
   Quantitative analysis of the blots. (F) Representative
   immunofluorescence staining images showing amyloid‐β (Aβ) deposits in
   the hippocampus and cortex of wild‐type (Ctr) and APP/PS1 mice.
   Quantification of Aβ deposits in the hippocampus (G) and cortex (H) of
   Ctr and APP/PS1 mice by Image‐Pro Plus 6.0 software. Data are presented
   as mean ± SEM (n = 3 per group. *p < 0.05, **p < 0.01, ***p < 0.001,
   ****p < 0.0001). Aβ, amyloid β protein; EPM, elevated plus maze; MWM,
   Morris water maze; OFT, open‐field test; WB, Western blotting.

   To assess whether APP/PS1 mice show age‐dependent anxiety‐like
   behavior, we first employed the open‐field test and recorded the
   percentage of time spent in the central area of the test apparatus. We
   found that compared with 5‐month‐old WT mice, APP/PS1 mice in the three
   age groups showed a decreasing trend in the percentage of time spent in
   the central area, although the difference did not reach significance
   (Figure [77]S1D‐E). Anxiety‐like behavior in APP/PS1 mice was also
   evaluated using the elevated plus maze (EPM) test. The results showed
   that both the number of entries into the open arms and the time spent
   in the open arms by APP/PS1 mice of the three age groups showed a
   decreasing trend compared with those of 5‐month‐old WT controls;
   however, the difference was again not significant (Figure [78]S1F‐G).
   Altogether, the results of the behavioral experiments indicated that
   APP/PS1 mice may not display age‐dependent anxiety‐like behavior.

   Aβ is produced via the amyloidogenic processing of APP. Western
   blotting was used to detect APP protein levels in APP/PS1 mice of the
   three age groups. The results showed that the hippocampal APP protein
   level was increased in 5‐month‐old APP/PS1 mice compared with that in
   age‐matched WT controls and was further increased at 9 and 13 months of
   age (Figure [79]1D,E). However, there was no difference in age‐gradient
   c57 mice (Figure [80]S2A,B). Next, we used the Aβ peptide‐specific
   antibody 6E10 to detect Aβ plaque accumulation in the hippocampus and
   cortex of the mouse brain using immunofluorescence (Figure [81]1F). Aβ
   plaque accumulation was observed in 5‐month‐old APP/PS1 mice but was
   absent in age‐matched WT controls. Moreover, the observed accumulation
   in the cortex and hippocampus showed significant age dependency,
   particularly in 13‐month‐old APP/PS1 mice (Figure [82]1F–H). However,
   no difference in Aβ plaque accumulation was observed among c57 mice of
   the three age groups (Figure [83]S2C). These findings indicated that
   there is a gradual, age‐dependent increase in Aβ plaque accumulation in
   the APP/PS1 mouse brain.

2.2. Transcriptome sequencing results revealed changes in lysosomal pathways
in the hippocampus area of age gradient APP/PS1 mice brain

   To further explore the mechanisms underlying the dynamic changes in Aβ
   aggregation and the related pathophysiologic mechanisms in the brains
   of APP/PS1 mice of different ages, we undertook a transcriptome
   sequencing analysis of the hippocampus of the animals. As expected
   given that AD is mostly age‐dependent,[84] ^18 we found that the
   differences in gene expression and enriched pathways among the three
   age groups of APP/PS1 mice differed significantly from those observed
   among c57 mice of different ages (Figures [85]S2D and [86]S3). Next,
   differentially expressed genes (DEGs) between 5‐ and 13‐month‐old
   APP/PS1 mice were identified through analysis of transcriptome
   sequencing data (Figure [87]2A) and were submitted to KEGG pathway
   enrichment analysis. The top 20 enriched KEGG pathways included
   osteoclast differentiation, chemokine signaling, cytokine‐cytokine
   receptor interaction, complement cooperative cascade, platelet
   activation, B cell receptor signaling, and natural killer cell‐mediated
   cytotoxicity. The KEGG pathways identified also included phagosome,
   lysosome, and Fc gamma‐R‐mediated phagocytosis pathways
   (Figure [88]2B). In AD, abnormal protein aggregation leads to impaired
   protein degradation, and Aβ aggregation occurs in the brains of APP/PS1
   mice. Thus, we focused on the lysosomal pathway, given its potential
   association with AD pathology, and that significant changes in this
   pathway were identified through KEGG analysis. Most of the DEGs in the
   lysosome pathway were upregulated based on gene set enrichment analysis
   (GSEA) (Figure [89]2C). Accordingly, we next generated a heatmap of
   DEGs related to the lysosome pathway based on RNA‐seq data for
   hippocampal samples from 5‐month‐old WT mice and APP/PS1 mice from the
   three age groups (Figure [90]2D). We found that the products of these
   genes formed a complex protein–protein interaction (PPI) network
   comprising 15 nodes and 30 edges, with an average node degree of 4
   (Figure [91]2E). This demonstrated that the genes encoding these
   lysosome‐related degradationenzymes, such as Cathepsin D (CTSD),
   Cathepsin Z (CTSZ), and Cathepsin S (CTSS), were closely linked with
   other genes in the PPI network, suggesting that impaired activity of
   these enzymes may play a key role in lysosomal dysfunction in APP/PS1
   mice. Next, to elucidate the mechanism underlying the significant
   increase in the expression of lysosome‐associated genes in aged APP/PS1
   mice, a Venn diagram was generated to identify DEGs shared between
   13‐month‐old and 5‐month‐old A PP/PS1 mice and between 13‐month‐old
   APP/PS1 mice and 13‐month‐old WT mice (Figure [92]2F). We analyzed the
   expression of genes, namely, Atp6v0d2 and Ctsd, which may affect
   lysosomal function and the occurrence of AD pathology (for details, see
   Figure [93]3). Meanwhile, we also found the mRNA expression levels of
   ATP6V0d2 (hippocampus [94]GSE36980) and CTSD (hippocampus [95]GSE29378)
   according to the AlzData database ([96]http://www.alzdata.org/) were
   significantly increased, which was consistent with the results of
   transcriptome sequencing and verified in the human hippocampus (Figure
   [97]S4). In summary, our findings suggested that lysosomal physiology
   undergoes significant changes in the hippocampus of APP/PS1 mice in an
   age‐dependent manner and that the ATP6V0d2 and CTSD genes may play an
   important role in lysosomal dysfunction and the occurrence of AD
   pathology.

FIGURE 2.

   FIGURE 2
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   The lysosomal pathway revealed via RNA‐seq data analysis was
   significantly changed in APP/PS1 mice hippocampus with increased age.
   (A) The different expression genes of the hippocampus between
   5‐month‐old (AH5) and 13‐month‐old (AH13) APP/PS1 mice. (B) Top20 KEGG
   pathways most enriched in the different gene expressions of the
   hippocampus between 5‐month‐old (AH5) and 13‐month‐old (AH13) APP/PS1
   mice. (C) Lysosome KEGG term of gene set enrichment analysis. (D) The
   heatmap of differential expression genes in the lysosome pathway of
   four groups of mice. (E) PPI network of targets extracted from (D). (F)
   The Venn diagram to find genes shared between 13‐month‐old (AH13)
   APP/PS1 mice versus 5‐months‐old (AH5) APP/PS1 mice and 13‐month‐old
   (AH13) APP/PS1 mice versus 13‐month‐old (CH13) WT mice.

FIGURE 3.

   FIGURE 3
   [99]Open in a new tab

   The lysosomal function was dynamically changed in the APP/PS1 mice
   hippocampus with increased age. (A) Western blotting was performed to
   detect the expression of ATP6V0d2 and CTSD proteins in the hippocampus.
   (B‐C) Quantitative analysis of the blots. Data are presented as
   mean ± SEM (n = 3 per group. **p < 0.01, ***p < 0.001). (D) KEGG
   pathway analysis of the genes (ATP6V0d2 and CTSD) using Clue GO
   software and the p‐value was set at < 0.05. (E) Gene Ontology (GO)
   analysis of the genes (ATP6V0d2 and CTSD) into three categories:
   biological process (BP), cellular component (CC), and molecular
   function (MF).

2.3. Lysosome function undergoes dynamic changes in the hippocampus of
APP/PS1 mice in an age‐dependent manner

   To better understand the dynamic age‐related changes in lysosome
   function occurring in the hippocampus of APP/PS1 mice, we measured the
   protein expression levels of ATP6V0d2 and CTSD by immunoblotting
   (Figures [100]3A and [101]S5A). The results showed that hippocampal
   ATP6V0d2 protein expression was significantly higher in 9‐month‐old
   APP/PS1 mice than in both 5‐month‐old and 13‐month‐old APP/PS1 mice,
   exhibiting a trend of initially rising and subsequently falling with
   age. However, no difference in hippocampal ATP6V0d2 protein expression
   was detected between 5‐month‐old APP/PS1 mice and age‐matched WT mice
   (Figure [102]3B). Compared with that seen in 5‐month‐old WT mice,
   APP/PS1 mice of the same age had significantly lower levels of the
   mature form of CTSD. However, compared with 5‐month‐old APP/PS1 mice,
   the expression of CTSD was significantly increased in 9‐ and
   13‐month‐old APP/PS1 mice. No difference in CTSD expression was
   observed between 9‐month‐old and 13‐month‐old APP/PS1 mice
   (Figure [103]3C) or among c57 mice of the three age groups (Figure
   [104]S5B‐C). KEGG pathway enrichment analysis showed that the genes
   encoding products were co‐enriched in tuberculosis and lysosome
   pathways, while gene ontology (GO) functional analysis indicated that
   both genes are involved in energy metabolism, proton transport, and
   protein degradation (Figures [105]3D,E, and [106]S5D‐F). These results
   suggested that lysosomal function in the hippocampus of APP/PS1 mice
   first increased and then decreased with increasing age.

2.4. The autophagy–lysosome and endosome/lysosome systems in the hippocampus
of APP/PS1 mice showed dynamic age‐related changes

   The lysosome is the principal site of degradation in the
   autophagy–lysosome and endosome/lysosome systems. Here, we found that
   both systems undergo dynamic changes during AD progression, as can be
   seen in the heatmap of DEGs between APP/PS1 and control mice
   (Figure [107]4A). Accordingly, we first evaluated the expression of the
   endosomal marker proteins Rab5 and Rab7 by Western blot
   (Figure [108]4B). The results showed that compared with 5‐month‐old WT
   mice, APP/PS1 mice showed an age‐dependent increase in Rab7 expression,
   especially in the hippocampus of 9‐month‐old and 13‐month‐old AD model
   mice (Figure [109]4C). Compared with young APP/PS1 mice, the expression
   of Rab5 in the hippocampus of 13‐month‐old APP/PS1 mice showed an
   upward trend (Figure [110]4C). However, no difference in Rab5 or Rab7
   expression was detected among the three groups of c57 mice (Figure
   [111]S6A–C).

FIGURE 4.

   FIGURE 4
   [112]Open in a new tab

   The endosome and autophagy pathways underwent dynamic changes in the
   APP/PS1 mice hippocampus as age increased. (A) The heatmap of
   autophagy–lysosome or endosome–lysosome pathway functional genes in the
   four groups of mice. (B) Western blotting was performed to detect the
   protein in the endosome–lysosome pathway. (C) Quantitative analysis of
   the blots for panel (B). (D) Western blotting was performed to detect
   the protein in the autophagy–lysosome pathway. (E) Quantitative
   analysis of the blots for panel (D). Data are presented as mean ± SEM
   (n = 3 per group. ns: not significant, *p < 0.05, **p < 0.01,
   ***p < 0.001).

   Western blot analysis of autophagy‐related proteins (Figure [113]4D)
   showed that LC3‐II expression exhibited an increasing trend in the
   hippocampus of APP/PS1 mice at 5 and 9 months of age and was
   significantly decreased at 13 months of age (Figure [114]4E). The
   knockout of the p62 gene can lead to AD‐like lesions.[115] ^19 In this
   study, we also found that the expression of p62 in APP/PS1 mice showed
   a decreasing trend with increasing age (Figure [116]4E). However, no
   significant difference in p62 expression was observed among the three
   groups of c57 mice (Figure [117]S6A, S6D). These results suggested that
   autophagy is enhanced in the early stages of AD and degradation is
   increased, while the storage of autophagy marker protein p62 decreased
   in the late stage of the disease due to the consumption of autophagy
   flux. To explore the molecular mechanism underlying autophagy dynamics
   in APP/PS1 mice, we measured the levels of mTOR and beclin1, key
   autophagy‐related proteins. Compared with 5‐month‐old WT mice, the
   p‐mTOR/total mTOR ratio in the hippocampus of APP/PS1 mice showed a
   downward trend at the ages of 5 and 9 months, but increased
   significantly at 13 months of age (Figure [118]4E); meanwhile, no
   difference in beclin1 expression was found among the three groups of
   APP/PS1 mice (Figure [119]4E) or among the three groups of WT mice
   (Figure [120]S6A, [121]6E). These observations suggested that the
   inhibition of autophagy in the hippocampus of 13‐month‐old APP/PS1 mice
   was likely the result of mTOR phosphorylation.

2.5. The SGK1/FOXO3a pathway underwent dynamic changes related to the
survival of hippocampal neurons in APP/PS1 mice

   SGK1 can phosphorylate FOXO3a, which subsequently translocates from the
   nucleus, resulting in decreased FOXO3a‐mediated nuclear transcriptional
   activity and antiapoptotic effects.[122] ^14 Accordingly, we next
   investigated the putative changes in the SGK1/FOXO3a pathway related to
   the survival of hippocampal neurons in APP/PS1 mice (Figure [123]S7A).
   First, we generated a heatmap of DEGs based on SGK1/FOXO3a
   pathway‐related transcriptomic data (Figure [124]S7B). The products of
   these genes formed a complex PPI network comprising nine nodes, with an
   average node degree of 2.44 and 11 edges (Figure [125]S7C). This
   demonstrated that the Sgk1 gene is closely related to other genes in
   the PPI network, suggesting that these genes may play a key role in the
   FOXO3a pathway in APP/PS1 mice. The results further showed that the
   hippocampal expression of the Sgk1 gene in APP/PS1 mice increased with
   age (Figure [126]S7B). Next, to elucidate the mechanism involved in the
   marked increase in the levels of FOXO3a pathway genes in aged APP/PS1
   mice, a Venn diagram was used to identify genes shared between
   13‐month‐old APP/PS1 mice and 5‐month‐old APP/PS1 mice and between
   13‐month‐old APP/PS1 mice and 13‐month‐old WT mice (Figure [127]S7D).
   We analyzed the genes, namely, Sgk1 and Foxo3a, both of which are known
   to affect the FOXO and PI3K‐AKT signaling pathways reported to be
   closely related to AD pathology (Figure [128]S7E). Next, we assessed
   the expression levels of proteins in the SGK1/FOXO3a pathway in the
   hippocampus of APP/PS1 mice using Western blotting (Figure [129]5A).
   The results showed that compared with 5‐month‐old WT mice, the
   p‐FOXO3a/total FOXO3a ratio was significantly increased in APP/PS1 mice
   at the age of 5 months, peaked at the age of 9 months, and decreased at
   the age of 13 months (Figure [130]5B). No difference in the
   p‐FOXO3a/total FOXO3a ratio was seen among the three groups of WT mice
   (Figure [131]S6A, S6F). The expression of SGK1, an upstream kinase of
   FOXO3a, also increased between the ages of 5 and 9 months and then
   decreased sharply at 13 months of age (Figure [132]5C). These results
   implied that in APP/PS1 mice, the SGK1/FOXO3a pathway undergoes dynamic
   changes, which may compromise neuronal survival.

FIGURE 5.

   FIGURE 5
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   Alteration of the SGK1/FOXO3a pathway in the hippocampus of APP/PS1
   mice, which also exhibited an increase in age‐dependent neuron loss and
   synaptic loss. (A) Western blotting was performed to detect the protein
   of the SGK1/FOXO3a pathway. (B‐C) Quantitative analysis of the blots
   about the SGK1/FOXO3a pathway. Data are presented as mean ± SEM (n = 3
   per group. ns: not significant, *p < 0.05, **p < 0.01). (D)
   Representative Nissl staining images showing Nissl bodies in the CA3
   region of the hippocampus in wild‐type (Ctr) and APP/PS1 mice
   (bar = 50 µm). (E) Quantifications of Nissl bodies in the CA3 regions
   of the hippocampus in Ctr and APP/PS1 mice by Image‐Pro Plus 6.0
   software. Data are presented as mean ± SEM (n = 4 per group. ns: not
   significant, **p < 0.01, ****p < 0.0001). (F) Representative Golgi
   staining images (bar = 10 µm). (G) The quantification of spine density
   by Image J software. (H) Common dendritic spine types are found in the
   brain. (I) The quantification of mushroom‐type spines. Data are
   presented as mean ± SEM (n = 9 neuron dendrites/group, sampled from 4
   mice/group. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). (J)
   Schematic diagram of the functional trends of the autophagy pathway,
   lysosomal function, and SGK1/FOXO3a pathway during the pathogenesis of
   AD in APP/PS1 mice.

   Meanwhile, we also used the SGK1 inhibitor GSK‐650394 to study the
   effect of SGK1 in the AD pathological process (Figure [134]S8A). After
   treatment with 5 µM GSK‐650394 in AD model N2a‐APP cells for 48 h, it
   was found that the p‐FOXO3a/total FOXO3a ratio decreased significantly
   compared with the DMSO‐treated control group (Figure [135]S8B),
   suggesting that there may be changes in APP metabolism. Interestingly,
   we found that the protein levels of APP and sAPPβ were increased in the
   SGK1 inhibitor‐treated N2a‐APP cells compared with DMSO‐treated N2a‐APP
   cells (Figure [136]S8C‐D). To further confirm this result, the cleaving
   enzymes in APP metabolism were detected. The Western blot results
   showed no significant difference in the protein levels of BACE1 and PS1
   between the two groups, whereas the ADAM10 protein levels were
   decreased in the SGK1 inhibitor‐treated N2a‐APP cells compared with
   DMSO‐treated N2a‐APP cells (Figure [137]S8E‐G). Besides, the
   enzyme‐linked immunosorbent assay (ELISA) results showed that the
   increased levels of Aβ40 and Aβ42 in N2a‐APP cells treated with SGK1
   inhibitor compared with DMSO‐treated N2a‐APP cells (Figure [138]S8H‐I),
   suggesting that SGK1 inhibitor GSK‐650394 treatment affected Aβ
   production and accumulation. The aggregate findings, therefore,
   indicated that inhibition of the SGK1/FOXO3a pathway aggravates Aβ
   pathology in the AD pathological process.

   Since the SGK1/FOXO3a pathway is involved in neuronal survival,[139]
   ^20 we next quantified the number of neurons in the hippocampi of
   APP/PS1 mice of all age groups using Nissl staining (Figure [140]5D).
   Staining results showed that neuronal loss was significantly more
   extensive in both 9‐month‐old and 13‐month‐old APP/PS1 mice relative to
   that seen in 5‐month‐old APP/PS1 mice (Figure [141]5E), consistent with
   the age‐dependent cognitive dysfunction observed in the AD model mice.
   These results suggested that age‐dependent neuronal loss occurs in the
   hippocampus of APP/PS1 mice. Synaptic damage is well known to be
   closely associated with cognitive decline. Given that the early
   appearance of soluble Aβ can result in early synaptic dysfunction and
   that Aβ can accumulate at synapses,[142] ^21 we next investigated the
   changes in dendritic spine number and morphology in the hippocampus of
   APP/PS1 mice of various age groups using Golgi staining
   (Figure [143]5F). The results showed that compared with 5‐month‐old WT
   mice, the number of dendritic spines and mushroom‐type spines in the
   hippocampus of APP/PS1 mice was significantly decreased at 5 months of
   age and was further decreased at both 9 and 13 months of age
   (Figure [144]5G‐[145]I). Dendritic spine damage appeared concomitantly
   with Aβ accumulation in young APP/PS1 mice, and dendritic spine loss
   became increasingly critical with increasing age.

   Overall, we observed that APP overexpression and abnormal APP
   metabolism promoted Aβ production and senile plaque formation. We
   further found that changes in the autophagy–lysosome system, the
   endosome/lysosome system, and the SGK1/FOXO3a pathway represent
   compensatory‐to‐decompensatory pathophysiological processes in the AD
   process. In particular, lysosomal activity was identified as playing a
   critical role in Aβ clearance in APP/PS1 mice. These phenomena
   manifested as dynamic changes in Aβ metabolism in the compensatory,
   plateau, and decompensatory stages of AD in the hippocampus of APP/PS1
   mice (Figure [146]5J).

3. DISCUSSION

   Compensatory mechanisms are a fundamental physiological phenomenon in
   living organisms. Under pathological conditions, these mechanisms are
   compromised, which often results in vicious cycles that promote the
   continuous aggravation of a pathological state. In this study, we used
   APP/PS1 mice of different ages, thus mimicking the age‐related
   pathogenesis of AD, and evaluated Aβ metabolism, especially the dynamic
   changes in its degradation in the various age groups. We further
   investigated age‐related behavior in APP/PS1 mice using the open field,
   EPM (anxiety‐like behavior), and MWM (cognitive impairment) tests. We
   found that mice exhibited mild, age‐dependent anxiety‐like behavior,
   consistent with a previous report,[147] ^22 while cognitive impairment,
   although mild, could already be detected at the age of 5 months, and
   significantly worsened in an age‐dependent manner. These observations
   indicated that our APP/PS1 mice well mimicked the dementia
   manifestations of AD.

   APP is a precursor molecule of the Aβ peptide. We found that APP
   protein was highly expressed in the brains of APP/PS1 mice at an early
   stage, which may explain the mild cognitive impairment seen in the
   young APP/PS1 mice during the early stage of AD in the absence of
   substantial Aβ plaque accumulation. These findings imply that some
   compensatory mechanism blocks any significant increase of Aβ levels and
   plaque formation in the early stage; in contrast, the Aβ level
   increases significantly in the late stage of AD due to the existence of
   a decompensatory mechanism, which promotes the further increase in the
   number of Aβ toxicity‐exacerbating plaques, finally triggering dementia
   that is characteristic of AD. We also undertook a transcriptome
   sequencing analysis of APP/PS1 mice in the three age groups. The top 20
   KEGG pathways associated with the identified DEGs were mainly involved
   in immune and degradation systems. It has been reported that the
   occurrence of AD is closely related to immune system imbalance.[148]
   ^23 Interestingly, we also observed that the number of DEGs in the
   lysosomal pathway increased markedly with age, suggesting that ATP6V0d2
   and CTSD gene dysfunction may negatively affect the degradation systems
   and may be linked to AD development.

   ATP6V0d2 is a key subunit of the lysosomal proton pump, and its
   functions may be closely related to lysosomal acidification and
   autophagosome/lysosome fusion, among other crucial physiological
   roles.[149] ^24 We found that the expression of ATP6V0d2 in the brains
   of APP/PS1 mice first increased and then decreased with increasing age.
   The significant rise in ATP6V0d2 expression in the hippocampus of
   9‐month‐old APP/PS1 mice may have been due to the enhanced lysosomal
   activity, which favors Aβ degradation. The hippocampal levels of
   ATP6V0d2 were significantly downregulated in 13‐month‐old APP/PS1 mice,
   likely following late decompensation by the lysosome pathway, which
   resulted in weaker lysosome acidification and impaired
   autophagosome/lysosome fusion.

   CTSD is an enzyme responsible for protein degradation in lysosomes.
   Under acidic conditions, the precursor of CTSD in lysosomes can be
   cleaved into its mature form, which has protein‐degrading activity.
   CTSD is reported to have a degradative effect on Aβ as well as serve as
   an indicator of lysosome function.[150] ^25 We found that compared with
   5‐month‐old WT mice, the level of mature CTSD protein in the
   hippocampus of 5‐month‐old APP/PS1 mice was significantly decreased,
   which may have been due to the low basal level of lysosomal
   acidification in the APP/PS1 mouse hippocampus,[151] ^26 leading to
   reduced production of mature CTSD. However, the expression of CTSD in
   the hippocampus in APP/PS1 mice at 9 and 13 months of age was
   significantly increased, which may have been a compensatory effect of
   the lysosomal pathway in clearing abnormal proteins. No difference in
   CTSD expression was observed between 9‐month‐old and 13‐month‐old
   APP/PS1 mice, possibly due to the sharp decline in ATP6V0d2 protein
   levels observed in 13‐month‐old APP/PS1 transgenic animals. Therefore,
   lysosomal acidification disorder does not affect increasing the
   expression of mature CTSD protein. Meanwhile, owing to the lack of an
   appropriately acidic environment, lysosomes may be less capable of
   degrading abnormal proteins. Hippocampal ATP6V0d2 and CTSD gene
   expression levels were significantly higher, whereas the protein
   expression level of ATP6V0d2 was lower in 13‐month‐old than in
   9‐month‐old APP/PS1 mice; no difference in the levels of mature CTSD
   was detected between these two groups. Combined, these observations
   further support the possible existence of late lysosomal degradation
   decompensation.

   Given that the lysosome is the predominant site of degradation for the
   autophagic and endosomal pathways, we evaluated the expression of the
   endosomal marker proteins Rab5 and Rab7. We found that the expression
   of Rab5 showed an upward trend in the hippocampus of APP/PS1 mice at 13
   months of age. Meanwhile, compared with WT controls, the protein levels
   of Rab7 exhibited an age‐dependent increase in APP/PS1 mice. Early APP
   production in APP/PS1 mice leads to increased endosomal activity,
   whereby APP is cleaved by BACE1, yielding Aβ.[152] ^27 Meanwhile,
   endosome–lysosome fusion is enhanced, resulting in the timely removal
   of Aβ peptides. In the hippocampus of 13‐month‐old APP/PS1 mice,
   lysosome acidification and fusion are impaired,[153] ^17 , [154]^28
   implying that a large number of late endosomes accumulate in the
   cytoplasm, representing a key site for massive Aβ production. These
   pathophysiological events may explain Aβ plaque accumulation in the
   hippocampus of aged APP/PS1 mice.

   SGK1, a protein crucial for neuron survival, plays a role in the
   nonamyloidogenic APP processing pathway,[155] ^13 facilitating Aβ
   degradation and enhancing the expression of proteins associated with
   synaptic plasticity.[156] ^29 SGK1 can also phosphorylate FOXO3a,
   leading to its export from the nucleus and the subsequent reduction in
   FOXO3a‐mediated transcriptional activity in this compartment.[157] ^30
   Studies have found that the upregulation of FOXO3a activity in the
   hippocampus is positively correlated with Aβ42 content.[158] ^15 In
   addition, the SGK1/FOXO3a pathway can influence the expression of
   lysosomal‐related genes.[159] ^16 Our RNA‐seq results showed that the
   expression level of the Sgk1 gene in the hippocampus of APP/PS1 mice
   increases with age. Additionally, we found that the SGK1/FOXO3a pathway
   is initially activated, but is subsequently inhibited with increasing
   age in the hippocampus of APP/PS1 mice. The SGK1‐mediated exit of
   FOXO3a from the nucleus blocks FOXO3a from inducing cell cycle arrest
   and apoptosis, thereby promoting cell survival.[160] ^30 The export of
   FOXO3a from the nucleus can also lead to a reduction in the expression
   of the Rhodopsin‐associated protein kinase 1 (ROCK1) gene and the
   promotion of the activity of the nonamyloidogenic APP processing
   pathway, which is beneficial for reducing Aβ accumulation in AD.[161]
   ^31 This observation suggests that the early (at 5 and 9 months of age)
   increase in FOXO3a phosphorylation in the hippocampus of APP/PS1 mice
   may be a compensatory, cell survival‐promoting response to the
   increased activity of the amyloidogenic pathway. The reduction in
   phosphorylated FOXO3a content in the hippocampus in 13‐month‐old
   APP/PS1 mice likely represents decompensatory alterations in the
   amyloidogenic pathway in the late stage of AD, whereby the entry of
   nonphosphorylated FOXO3a into the nucleus induces cell cycle arrest and
   apoptosis, resulting in the formation of numerous Aβ plaques and late
   neuronal apoptosis. The entry of nonphosphorylated FOXO3a into the
   nucleus may also promote the transcription of lysosome‐related genes
   compensatorily, which may explain the rapid increase in the expression
   of lysosome‐related genes in late AD; however, this possibility
   requires further verification. It has also been reported that the
   inhibition of the SGK1/FOXO3a pathway can lead to autophagy
   dysfunction,[162] ^32 which may explain the significant decrease in
   LC3‐II contents in the hippocampus of APP/PS1 mice at 13 months of age.
   FOXO3a‐induced proteolysis further leads to the degradation of the
   lysosomal proton pump and, consequently, impaired lysosome
   acidification.[163] ^33 Studies have also shown that FOXO3a is
   associated with Aβ‐induced neurotoxicity.[164] ^34 Activated FOXO3a
   translocates to the nucleus and binds to the Bim promoter, thereby
   mediating neuronal apoptosis.[165] ^35 Cognitive dysfunction in AD is
   primarily due to neuronal loss. Here, we found that APP/PS1 mice
   exhibited neuronal loss that was both progressive and age‐dependent.
   Additionally, the loss of synapses has been reported to be closely
   associated with cognitive decline. Our results indicated that APP/PS1
   mice display progressively more extensive synaptic damage in an
   age‐dependent manner. The above results also indicate the pathogenesis
   of AD from the pathological observation.

4. CONCLUSION

   In summary, we analyzed various pathophysiological mechanisms
   associated with AD pathogenesis in APP/PS1 mice from a dynamic
   perspective. During the compensation period, elevated ATP6V0d2 in the
   hippocampus of APP/PS1 mice enhanced the fusion of autophagosomes and
   endosomes with lysosomes, facilitating APP protein degradation. This
   led to moderate Aβ production, activating SGK1 for anti‐inflammatory
   and neuroprotective effects. In the decompensation period, reduced
   ATP6V0d2 inhibited fusion processes, causing APP accumulation and
   increased Aβ generation via BACE1. Inhibition of SGK1caused by Aβ
   overload resulted in FOXO3a dephosphorylation, triggering apoptosis and
   lysosomal gene upregulation. This progression culminated in severe
   neuronal loss and synaptic damage in the hippocampus of aged APP/PS1
   mice (Figure [166]6). Our study provides novel ideas for further
   elucidating the pathogenicity of Aβ toxicity as well as identifying
   novel therapeutic targets for the prevention of AD.

FIGURE 6.

   FIGURE 6
   [167]Open in a new tab

   Schematic diagram of molecular mechanisms of the pathologic process
   from compensatory to decompensated in the hippocampus of age‐gradient
   APP/PS1 mice. During the compensation period, the ATP6V0d2 protein was
   significantly elevated in the hippocampus of APP/PS1 mice, which
   increased the fusion of autophagosomes and endosomes with lysosomes,
   thus enhancing the degradation of APP protein. As a result, the modest
   generation of Aβ could promote the activation of SGK1, thereby exerting
   its anti‐inflammatory, neuroprotective, and resistance to Aβ toxicity.
   In the decompensation stage, the significant decrease of ATP6V0d2 led
   to the blockage of the fusion of autophagosome–lysosome and
   endosome–lysosome, and a large amount of APP was deposited into the
   early endosome and autophagosome, which was sheared by BACE1 to produce
   a large amount of Aβ, thus inhibiting the expression of SGK1. SGK1
   deficiency induced a decreased phosphorylation of FOXO3a, resulting in
   increased nucleation of nonphosphorylated FOXO3a and elevated
   expression of apoptosis genes and lysosome genes. Further deterioration
   of Aβ pathology resulted in significant loss of neurons and synaptic
   damage in the hippocampus of aged APP/PS1 mice.

5. MATERIALS AND METHODS

5.1. Reagents

   Nissl staining solution was sourced from the Beyotime Institute of
   Biotechnology (C0117; China). The antibodies used in this study are
   listed in Table [168]S1.

5.2. Animals

   Male APP/PS1 double‐transgenic mice (strain name: B6C3‐Tg [APPswe,
   PSEN1dE9] 85Dbo/J)[169] ^36 were purchased from Nanjing Biomedical
   Research Institute of Nanjing University, and male WT (c57) mice were
   obtained from the Experimental Animal Center of Tongji Medical College,
   Huazhong University of Science and Technology. All mice were housed in
   an air‐conditioned room at a temperature of 23 ± 1°C under a 12‐h/12‐h
   light‐dark cycle and had free access to food and water. The mice were
   divided into six groups, 10 mice/group. Male APP/PS1 mice were divided
   into three age groups (5, 9, and 13 months) for the experiments, with
   age‐matched WT mice serving as controls. Mice were taken to the
   behavioral testing rooms at least 1 day before the tests and were
   euthanized after the final behavioral test. In our experiment, the mice
   were euthanized and their brains were perfused with normal saline. The
   hippocampus of three mice was collected for Western blot analysis. The
   brains of the other three mice were removed after euthanasia, and the
   hippocampal tissue was divided into two portions for immunofluorescence
   assay and transcriptome sequencing, respectively. The remaining four
   mice were also divided into left and right hemispheres for Golgi
   staining and Nissl staining.

5.3. Open‐field test

   The open‐field test was used to assess anxiety‐like behavior in the
   mice. Mice were habituated to the test chamber for 1 h before the test.
   For testing, the animals were placed in a square box (60 × 60 × 50 cm)
   with their heads facing the wall, always in the same direction, and
   were allowed to freely explore for 5 min. The total distance moved and
   the time spent in the central area were recorded with a tracking camera
   installed over the test platform. The platform was cleaned after each
   test.

5.4. Elevated plus maze test

   The EPM test is a standard method for evaluating anxiety‐related
   behaviors. Mice were habituated in the test chamber for 1 h before the
   test. Each mouse was placed at the center of a raised platform shaped
   like a plus sign, 50 cm above the floor, and illuminated by a 60 W bulb
   from above. A video camera recorded their movements, tracking them at
   5‐min intervals to measure the distance traveled, open and closed arm
   entries, and time spent. After the test, the maze was cleaned with 75%
   ethanol. Data from the EPM test were analyzed and presented as
   percentages.

5.5. Morris water maze test

   The mice were familiarized with the MWM testing chamber for 1 day. The
   maze was colored white with harmless flour to aid in tracking mouse
   movement. The water temperature was maintained at around 25°C.
   Geometric patterns on the tank's curtain helped mice navigate spatial
   positions. A platform was placed in one quadrant of the pool, submerged
   2 cm below the water surface. The acquisition phase spanned 5 days,
   with mice trained three times daily in different quadrants. Mice had 60
   s to locate the hidden platform; if unsuccessful, they were guided to
   it and allowed to remain for 20 s. The latency to find the platform was
   recorded digitally. On day 6, a probe trial was conducted with the
   platform removed, allowing mice to swim freely for 60 s. Exploration
   time in the target quadrant and latency to cross the original platform
   location were recorded.

5.6. Immunofluorescence assay

   Following the behavioral tests, mice were anesthetized and perfused
   intracardially with 4% paraformaldehyde followed by normal saline, and
   their brains were extracted. Brain tissues were dehydrated in sucrose
   solutions, embedded in Optimal Cutting Temperature (OCT) compound
   (Tissue‐Tek), and sliced into 30‐µm‐thick sections using a cryostat
   (Leica). For immunofluorescence staining, brain sections were treated
   with phosphate‐buffered saline(PBS), permeabilized with Triton X‐100,
   blocked with bovine serum albumin (BSA), and incubated with primary
   antibody overnight at 4°C. The next day, sections were washed,
   incubated with Alexa Fluor 488‐conjugated secondary antibody,
   counterstained with 4′,6′‐diamidino‐2‐phenylindole (DAPI), mounted on
   glass slides with glycerol, cover‐slipped, and examined using confocal
   microscopy.

5.7. Western blotting

   After homogenization, the concentration of protein in brain tissue or
   N2a‐APP cells was determined. β‐Mercaptoethanol and bromophenol blue
   were then added to the samples, followed by boiling at 100°C for 10 min
   to denature the protein. The extracted protein (15 µg per sample) was
   separated on 8−15% sodium dodecyl sulfate–polyacrylamide gel
   electrophoresis (SDS‐PAGE), transferred to polyvinylidene difluoride
   (PVDF) membranes (Millipore), blocked with 5% BSA in Tris‐buffered
   saline (TBS) with 0.1% Tween 20 (TBST) for 1 h and incubated with the
   appropriate primary antibodies overnight at 4°C. After washing with
   TBST, the membranes were incubated with horseradish peroxidase (HRP)
   conjugated secondary antibody for 1 h at room temperature, and then
   immersed in enhanced chemiluminescence (ECL) solution at a 1:1 ratio,
   and exposed to ECL‐Hyperfilm (Amersham, Germany).

5.8. Nissl staining

   Coronal brain sections (30 µm thick) were washed with PBS, mounted on
   gelatin‐coated slides, allowed to dry for 30 min, and then stained with
   cresyl violet for 5 min and heated in the oven. Subsequently, the
   sections were quickly differentiated in 95% ethanol for 2 min, washed
   in distilled water for 2 min, dehydrated twice in anhydrous ethanol
   (5 min each time), cleared in xylene for 10 min, and sealed with
   neutral balsam. Finally, the brain slices were observed and imaged
   using an optical microscope.

5.9. Golgi staining

   The Golgi‐Cox solution contained 5% potassium dichromate, 5% mercury
   chloride, 5% potassium chromate, and double‐distilled water at a volume
   ratio of 5:5:4:10. The solution was kept in the dark for approximately
   1 week. For staining, the mice were perfused with 0.9% normal saline,
   and the whole brains were harvested and incubated in the Golgi–Cox
   solution for 3 days in the dark. The brain tissues were then
   transferred to a 1% silver nitrate solution and changed daily, soaked
   in darkness, and changed every 3 days. The brains were removed from the
   solution after 7 days and cut into 90‐µm‐thick sections using a
   vibrating microtome (Leica, Wetzlar, Germany).

5.10. Cell culture and treatment

   Mouse neuroblastoma N2a cells with stable expression of APP (N2a‐APP)
   were cultured in DMEM (Keygen, Nanjing, China) containing 10% fetal
   bovine serum (Gibco, USA). N2a‐APP cells were seeded at a density of
   1×10^6 cells per well in 6‐cm plates. Twenty‐four hours postseeding,
   cells were treated with 5 µM SGK1 inhibitor GSK‐650394 (MedChemExpress,
   China) for 48 h. As the inhibitors were dissolved in dimethylsulfoxide
   (DMSO), the DMSO‐treated N2a‐APP cells were used as a control.

5.11. ELISA quantification of Aβ40 and Aβ42

   Aβ40 and Aβ42 levels were quantified using the Human Aβ1‐40 or Aβ1‐42
   ELISA Kit (Elabscience, China). Briefly, cell suspensions were
   homogenized in buffer (PBS with protease inhibitors), centrifuged at
   2000×g for 25 min, and the supernatant added to anti‐Aβ‐coated micro
   ELISA plates. Following a 90‐min incubation, a biotinylated detection
   antibody was applied and incubated at 37°C for 1 h. After washing, HRP
   conjugate solution was added, followed by a 30‐min incubation.
   Subsequently, a substrate reagent was added and incubated for 15 min at
   37°C. Optical density at 450 nm was measured using a microplate reader
   after adding the stop solution.

5.12. RNA extraction and detection

   After dissecting the mouse brain, the brain tissue was quickly placed
   into a pre‐labeled 1.5‐milliliter EP tube and then rapidly frozen into
   tissue blocks in a cooling box containing liquid nitrogen to prevent
   RNA degradation. Subsequently, the EP tube containing the frozen tissue
   was placed on dry ice and transported to Novogene for RNA extraction
   using standard methods from the tissue. The extracted RNA samples
   underwent strict quality control, primarily assessed through the
   Agilent 2100 bioanalyzer to accurately detect RNA integrity before
   library preparation for sequencing.

5.13. Bioinformatics analysis

   The data analysis and visualization were performed using the NovoMagic
   v3.0 platform (Beijing Nuohe Zhiyuan Technology Corp., Beijing, China).
   Differential gene volcano plots, KEGG pathway analysis, GSEA, GO
   enrichment analysis, and heat maps were generated through this
   platform. A PPI network was constructed using the STRING database
   version 11.5 ([170]https://string‐db.org/).[171] ^37 Signaling pathways
   were illustrated using Clue GO in Cytoscape software (version
   3.8.2)[172] ^38 to highlight DEGs. mRNA expression levels of ATP6V0D2
   (from Hippocampus [173]GSE36980) and CTSD (from Hippocampus
   [174]GSE29378) were obtained from the AlzData database
   ([175]http://www.alzdata.org/).[176] ^39

5.14. Statistical analysis

   All data are expressed as means ± standard error of the mean (SEM).
   Statistical significance was analyzed by one‐way analysis of variance
   (ANOVA), two‐way ANOVA, or unpaired Student's t‐tests. A p‐value < 0.05
   was considered significant.

AUTHOR CONTRIBUTIONS

   Xiaochuan Wang designed all experiments and organized all results.
   Zhendong Xu planned and performed all experiments and participated in
   the writing of the manuscript. Jichang Hu, Zhen Wei, Henok Kessete
   Afewerky, Yang Gao, Lu Wan., Longfei Li, Ling Lei, Yi Liu, Fang Huang,
   Tong Yu, Jian‐Zhi Wang, Hong‐Lian Li, and Rong Liu analyzed the data.
   Yu Lei analyzed and interpreted the data. All authors have read and
   approved the final manuscript.

CONFLICT OF INTEREST STATEMENT

   The authors declare that they have no competing interests.

ETHICS APPROVAL AND CONSENT TO PARTICIPATE

   The study protocol was approved by the Ethics Committee of Huazhong
   University of Science and Technology (Wuhan, Hubei, China; IACUC
   number: 3308).

Supporting information

   Supporting information
   [177]MCO2-5-e540-s001.docx^ (3MB, docx)

ACKNOWLEDGMENTS