Adiponectin alleviated Alzheimer‐like pathologies via autophagy‐lysosomal activation

Abstract Adiponectin (APN) deficiency has also been associated with Alzheimer‐like pathologies. Recent studies have illuminated the importance of APN signaling in reducing Aβ accumulation, and the Aβ elimination mechanism remains rudimentary. Therefore, we aimed to elucidate the APN role in reducing Aβ accumulation and its associated abnormalities by targeting autophagy and lysosomal protein changes. To assess, we performed a combined pharmacological and genetic approach while using preclinical models and human samples. Our results demonstrated that the APN level significantly diminished in the plasma of patients with dementia and 5xFAD mice (6 months old), which positively correlated with Mini‐Mental State Examination (MMSE), and negatively correlated with Clinical Dementia Rating (CDR), respectively. APN deficiency accelerated cognitive impairment, Aβ deposition, and neuroinflammation in 5xFAD mice (5xFAD*APN KO), which was significantly rescued by AdipoRon (AR) treatment. Furthermore, AR treatment also markedly reduced Aβ deposition and attenuated neuroinflammation in APP/PS1 mice without altering APP expression and processing. Interestingly, AR treatment triggered autophagy by mediating AMPK‐mTOR pathway signaling. Most importantly, APN deficiency dysregulated lysosomal enzymes level, which was recovered by AR administration. We further validated these changes by proteomic analysis. These findings reveal that APN is the negative regulator of Aβ deposition and its associated pathophysiologies. To eliminate Aβ both extra‐ and intracellular deposition, APN contributes via the autophagic/lysosomal pathway. It presents a therapeutic avenue for AD therapy by targeting autophagic and lysosomal signaling.


| INTRODUC TI ON
Alzheimer's disease (AD) is the most common form of dementia affecting millions of people word widely, predicting to be tripled by 2050 according to the epidemiology studies ("Global, regional, and national burden of Alzheimer's disease and other dementias, 1990-2016: a systematic analysis for the Global Burden of Disease Study 2016," 2019; Kalaria et al., 2008;Nichols et al., 2019). Extracellular accumulation of β-amyloid (Aβ) into amyloid plaques, intraneuronal hyperphosphorylated tau aggregation, neuroinflammation, synaptic loss, and neuronal cell death are standard features of AD. Further, a causative relationship between Aβ accumulation and AD pathology suggests that Aβ aggregation proceeds neurofibrillary tangles (NFTs) formation. However, NFT formation is closely related to the cognitive dysfunction of AD (Di et al., 2016;Nelson et al., 2009Nelson et al., , 2012Sadigh-Eteghad et al., 2015;Zhang et al., 2018). Apart from marked AD pathologies, increasing studies have been seeking early etiological changes and risk factors in AD, which contains at the high ranks of insulin resistance, dysregulated glucose/ lipid metabolism, and oxidative stress (Chen et al., 2014;Willette et al., 2015). Aβ accumulation could impair insulin signaling while insulin modulates Aβ trafficking, release, and Aβ-mediated synaptic loss, supporting a strong association between insulin signaling and Aβ metabolism (Avrahami et al., 2013;De Felice, 2013). Insulin resistance is prominent in AD patients as demonstrated by higher fasting plasma insulin, while reduced insulin-provoked Aβ elevation is reported in AD (Neth & Craft, 2017).
The role of APN in the central nervous system (CNS) is not well and comprehensively studied. However, adiponectin receptors (AdipoR1 and AdipoR2) express in the hippocampus, cortex, and hypothalamus of the brain (Rastegar et al., 2019;Thundyil et al., 2012). Lower levels of APN have been observed in cerebrospinal fluid (CSF) and brain tissues of AD patients (Ng et al., 2020), suggesting a possible involvement of APN in brain function. Further, APNdeficient mice exhibited cognitive impairment (Rizzo et al., 2020) and depressive-like behaviors (Liu et al., 2012), proposing the role of APN in the improvement of cognitive functions. A most recent study crossbred 5xFAD mice with APN KO mice to produce 5xFAD*APN KO mice and found increased Aβ deposition, neuroinflammation, and cognitive impairment, which could be reversed by AdipoRon (AR) treatment (Ng et al., 2020). However, the underlying mechanisms of APN deficiency accelerating AD-like pathologies, how AR rescues these, and their relations with insulin signaling are yet to be investigated. Therefore, in our present investigation, we highlighted the mechanistic relationship between APN and AD-like changes.
Our study demonstrated that APN deficiency accelerates ADlike pathologies; however, AR (AdipoRon) treatment abates the phenomenon by decreasing Aβ deposition in APN-deficient 5xFAD mice. Furthermore, AR treatment reduces cognitive impairments and the dysregulation in autophagy-lysosomal pathways (ALP) protein expression, proposing that AR can be a potential therapeutic drug to treat AD-associated pathologies. (NSFC)  The level of APN in plasma of 2/6-month-old 5xFAD mice and the controls. (f) The relative expression of APP, BACE1, ADAM10, and IDE in the hippocampus region. (g) Quantification of Aβ plaque in the hippocampus, cortex, and amygdala region of 5xFAD and 5xFAD*APN KO mice. Data were expressed as mean ± SEM, * p < 0.05, ** p < 0.01, *** p < 0.001

| Dementia correlates with APN deficiency
Age-and sex-matched healthy individuals and dementia patients (AD and VaD: vascular dementia) were first selected as significant brain atrophy shown with MRI results (Table S1, S2; Figure 1a). Next, we examined plasma APN level, which was significantly reduced in dementia patients compared to healthy controls (Figure 1b; Figure   S6A; Table S1, S2). Further, a significant linear correlation was detected between plasma APN concentration and CDR or MMSE score ( Figure 1c, d), proposing a critical relationship between APN and AD.
Interestingly, these correlations were also present in mice, as demonstrated by a significant decline of plasma APN level of 5xFAD mice compared to that of 2/6-month-old WT mice ( Figure 1e). Notably, it is tentative to show that APN level reduction was accelerated with disease progression, as remarked APN level reduction could be examined in the plasma of 6-month-old 5xFAD mice compared to the 2-month-old 5xFAD mice ( Figure 1e). These results collectively demonstrated a negative correlation of APN level and dementia progression, and these changes were conserved between mice and human pathological status.

| APN deficiency accelerates Aβ deposition, accompanied by cognitive impairment and neuroinflammation
Previous studies have revealed that APN deficiency accelerates Aβ deposition (Ng et al., 2020;Rizzo et al., 2020); however, the mechanism is largely unknown. Here, we eliminate APN from 5xFAD mice, as shown in Figure S1A. No significant difference in APP expression was detected between 5xFAD*APN KO and 5xFAD mice ( Figure 1f; Figure S1C). Similarly, no substantial changes in APP processing enzymes were revealed, including BACE1, ADAM10, and IDE ( Figure 1f; Figure S1D-F). Aβ deposition was validated with immunostaining. Results revealed that Aβ plaques accumulation accelerated in the cortex, hippocampus, and amygdala of 5xFAD*APN KO mice compared with 5xFAD mice (Figure 1g; Figure S1B). Together, these results demonstrated that APN deficiency accelerates Aβ deposition without affecting APP expression and processing in 5xFAD mice.
Next, we sought to determine the effect of APN deficiency on cognitive impairment in AD. 6-month-old mice were employed when impaired spatial memory has been reported in 5xFAD mice (Xiao et al., 2015). The mice were successively subjected to novel object recognition, Y-maze, and morris water maze ( Figure S2). The results showed APN-deficient 5xFAD mice significantly reduced the new object preference compared to the WT mice ( Figure S2A-B). Moreover, APN deficiency aggravated spatial memory deficits of 5xFAD mice in the Y-maze trial, as shown by a reduced percentage of time in the novel arm ( Figure   S2C). Similarly, in the Morris water maze test, APN deficiency accelerated learning and memory deficits in 5xFAD mice, as demonstrated by the increased latency to find the submerged platform during 5 consecutive days of training and the probe trial ( Figure S2D-I).

| AR treatment reduced Aβ pathology and cognitive impairment
The possibility of the APN role in Aβ deposition was further validated by in vitro analysis with AdipoRon (AR, an AdipoR agonist) treatment.
The results showed that "toxic" Aβ 1-42 protein was significantly higher in N2a/APP swe lysates than N2a/WT, which were considerably reduced by 1µM or 5µM AR treatment ( Figure 2a). However, we did not find, Aβ 1-42 expression in the supernatant of the N2a cells. Surprisingly, 5µM AR treatment markedly increased the Aβ 1-40 level in the lysates (but not in the supernatant: Figure S6B) of N2a/APP swe ( Figure 2b); however, the ratio of Aβ 1-42 /Aβ 1-40 had significantly reduced by AR treatment in N2a/APP swe cells (Figure 2c), further supporting the causal effect of APN in Aβ accumulation. Besides, we measured the AR effect on the Aβ plaques accumulation in the cortex and hippocampus of the 6-month-old APP/PS1 mice while treating with AR for two months. Interestingly, AR treatment significantly reduced Aβ plaques deposition in APP/PS1 mice ( Figure S3A). The notion was further validated by immunostaining of the Aβ via 6E10 ( Figure S6C). However, we did not detect any significant changes in the expression of crucial proteins ( Figure S3B) involved in producing Aβ plaques.
As mentioned in the earlier results, APN deficiency increases cognitive impairment. Here, we also measured the experimental mice's cognitive skills after AR treatment ( Figure 2d). By NOR and Y-maze tests, we observed that AR treatment could attenuate the short-term memory deficits of 5xFAD*APN KO mice (Figure 2e-f). Further, the MWM test results also showed that the percentage of distance traveled and time spent in the target quadrant was also obviously increased after AR treatment (Figure 2g-i). These findings suggested that AR treatment could rescue spatial memory deficits in 5xFAD*APN KO mice.

| APN negatively regulates Aβ deposition via autophagy activation
To investigate the possible effects and underlying mechanisms of

1-40 in lysate
into 5 clusters, 3/5-related potential vital proteins were identified, which might be the molecular basis for APN deficiency exacerbating AD cognitive impairment and pathology in 5xFAD mice (Figure 3b-h; Figure S4). These results were further validated by KEGG enrichment analysis and hierarchical heatmap clustering analysis. Overall, higher connectivity was observed among neuron differentiation processes, neurogenesis, proteolysis, cell death, autophagy, and endocytosis in the brain tissue of 5xFAD*APN KO compared to 5xFAD WT mice, suggesting the above-related process may play a crucial role underlying APN deficiency accelerated cognitive impairment.
Former reports revealed that autophagy is essential to eliminate Aβ toxic accumulated (Li et al., 2017;Uddin et al., 2018). Here to associate, autophagy-related gene (ATGs) expression was measured.

| AR promoted Aβ clearance by increasing lysosome activity in microglia
Aβ (extracellular) deposition is mainly cleared through phagocytosis and microglia degradation, indicating that microglial activity is closely related to Aβ metabolism. Here, we measured GFAP and IBA-1 levels, the glial cells activation markers, in the cortex and hippocampus. Significant increases were observed in both GFAP and IBA-1 levels in the brain of the 5xFAD mice, which were remarkably elevated on APN deficiency ( Figure S3C-D). Additionally, AR treatment significantly reversed the hyper-neuroinflammatory state of the APP/PS1 mice ( Figure 5). These findings indicate that APN deficiency aggravated spatial learning and memory deficits in 5xFAD mice. Besides, AR treatment significantly reduced pro-inflammatory cytokine levels in the N2a cells ( Figure S7F).
To elucidate the underlying mechanisms of microglia activation and Aβ deposits, the effect of AR treatment on Aβ accumulation was measured in primary microglia of APN KO mice. As shown in Figure 6a, AR treatment significantly reduced Aβ accumulation within microglia, indicating that AR might promote Aβ degradation or phagocytosis. Thus, we used fluorescent beads to examine the phagocytic ability of microglia derived from WT or APN-KO mice ( Figure 6b). Furthermore, no apparent differences were observed in microglia's phagocytosis between the two groups ( Figure 6b). It indicates that APN deficiency did not affect microglia's phagocytosis.
Interestingly, further results reported that lysosomal markers, including LAMP1 and CTSD (Figure 6c, d), were remarkably reduced in microglia from APN KO mice compared to the WT control, indicating APN deficiency could seriously mitigate the lysosome activity in microglia. These results were further validated with microglia primary culturing of APN KO mice followed by AR treatment; significantly increased lysosomal markers' expression was detected in the presence of CQ or Baf A1 (Figure 6e, g). Besides, we measured the APN receptors (AdipoR1/2) expression and its downstream signaling molecules, including APPL1 and APPL2 ( Figure S7A). Interestingly, AR treatment significantly increased AdipoR1 and APPL1 expression in the hippocampus of the APP/PSI mice. The results were further validated by AR treatment to N2a/APPswe cells ( Figure   S7B). Furthermore, we overexpressed AdipoR1 and AdipoR2 in the 293T cells and measured the expression of LC3B concurrently ( Figure S7E). AdipoR1 overexpressed cells showed an increased level of LC3B, suggesting the involvement of AdipoR1 in the APNlinked autophagic impairment. As AMPK signaling plays a vital role in autophagy regulation (Kim, Kundu, Viollet, & Guan, 2011), we inhibited the AMPK pharmacologically (via Dorsomorphin) the N2a/ APPswe cells ( Figure S7D). Surprisingly, AR pro-autophagy effect was reversed by AMPK antagonism, suggesting AMPK involvement in the APN-associated autophagy impairment.
Notably, we found an increased level of LC3B II in the AdipoR1overexpressed cells ( Figure S8A). Contrarily AdiopR1 overexpression reduced the Aβ 1-42 level in the N2a/APPswe cells. Next, N2a/ APPswe cells were treated with an AMPK inhibitor (Dorsomophin or AdipoR1), and Aβ changes were measured. Surprisingly, Dorsomophin treatment reversed the effects of AdipoR1 on Aβ changes ( Figure S8E-G).
These findings strongly suggested that APN played an essential role in regulating the lysosomal activity of microglia. Dysregulated APN signaling contributes to AD pathology, whereas AR supplementation could rescue the phenomenon by abating the Aβ in microglia via enhancing lysosomal activity.

| DISCUSS ION
The present study demonstrates that APN, an adipokine involved  (Dukic et al., 2016;Gorska-Ciebiada et al., 2016;Khemka et al., 2014). Nevertheless, a recent study strongly supported the idea that the low level of APN significantly increased AD risk (Ng et al., 2020).
In our study, the plasma APN level was dramatically decreased in de- Autophagy is the main conserved pathway for the clearance of abnormal proteins such as toxic Aβ and hyperphosphorylated tau (Li et al., 2017). Several studies showed that autophagy deficits occurred in the early stage of AD and had an essential role in generating and metabolism of Aβ (Uddin et al., 2018). Activation of autophagy was thought to be beneficial in AD treatment (Hamano et al., 2018). Our findings showed that AR treatment could significantly reduce the ratio of Aβ  Wang et al., 2010). Although the ALP mechanism has been discussed in the AD-associated pathologies (Orr & Oddo, 2013), up to our knowledge, we did not find reliable evidence of the association between APN deficiency and ALP defects, which inspire us to explore this mechanism.
We found that AR treatment significantly recovered the lysosomal functional protein levels in the presence of inhibitors (CQ and BafA1) in microglia from APN KO mice. This finding indicates that APN deficiency might be involved in accelerating ALP defects, followed by Aβ accumulation, which participates in AD progression.
On the contrary, in addition to the increase of Aβ accumulation, there were many other factors of exacerbating cognitive impairment in AD, such as chronic neuroinflammation (Kinney et al., 2018), mitochondrial dysfunction (W. , endoplasmic reticulum stress (Salminen et al., 2009), synaptic dysfunction (Kashyap et al., 2019), and so on. Thus, we further employed a TMT-labeled proteomic approach (read Supplementary data for a detailed discussion of proteomic study) to find more possible mechanisms that APN deficiency aggravated cognitive impairment. We discovered that APN deficiency strongly affects ALP signaling and higher connectivity among neuron differentiation processes, neurogenesis, proteolysis, cell death, autophagy, and endocytosis detected in the brain tissue of 5xFAD*APN KO compared to 5xFAD WT mice. Whether APN is also involved in the processes mentioned above that eventually contributed to AD etiology still warrant further investigation. However, it strongly suggests the APN and the above-related proteins may play a crucial role in APN deficiency accelerating cognitive impairment in AD.

| CON CLUS ION
To our knowledge, this is the first study that identified the negative role of APN in the progression of AD-like pathophysiologies via the autophagy-lysosomal pathway. A defect in this signaling could accelerate Aβ deposition in microglia, which is the hallmark of AD pathologies.
Further, AR, a potent APN agonist, could reduce these changes and offer a promising therapeutic avenue for treating dementia, including AD.

| Human plasma samples
Blood samples that were collected followed by plasma separation from AD patients (MCI = 39), and the age-and sex-matched nondemented healthy individuals (n = 41) (Table S1, S2) from the First Hospital of Zibo kindly provided by prof. Shujin Wang (Neurology Department, the First Hospital of Zibo, China). Mini-Mental State F I G U R E 6 AR promoted Aβ clearance by increasing lysosome activity in microglia. The effect of AR on Aβ accumulation in primary microglia from the brain of APN KO mice. (b) The effect of APN deficiency on the phagocytosis of primary microglia from the WT and APN KO mice. (c-d) The relative expression of LAMP1 and CTSD in primary microglia from the WT and APN KO mice. (e-g) The relative expression of LAMP1 and CTSD in primary microglia from the APN KO mice with or without inhibitor treatment. (h) The relative expression of LAMP1, LAMP2, CTSD, and CTSB when the expression abundance of microglia was used as the baseline according to the results from mass spectrometry. Data were expressed as mean ± SEM, * p < 0.05, ** p < 0.01, *** p < 0.001 Examination (MMSE), Clinical Dementia Rating (CDR) Scale, magnetic resonance imaging (MRI), and NINCDS/ADRDA or NINDS/ AIREN criteria (Sarazin et al., 2012;Scheltens & Hijdra, 1998) used to diagnose AD and VaD in the individuals. Notably, the study was approved by the Ethical Committee of the First Hospital of Zibo, and consent from all the subjects was included.

| Behavior test
All tests were conducted in the behavioral room, beginning at 8:00 am every day as performed in our previous studies (Shen et al., 2020;Zhou et al., 2019). The mice were transferred to the specific room 2 h before the test. Novel object recognition (NOR) and Y-maze were performed to evaluate short-term memory, while long-term memory was assessed by the Morris water maze (MWM) test. A 3-day window was arranged between different behavioral experiments to avoid interference effects.
Detailed behaviors assays are mentioned in the Supplementary data.

| Histochemistry
We performed Immunohistochemistry as previously reported . Detailed histological analyses are mentioned in the Supplementary data.

| Sample preparation and protein labeling
The proteomics study was performed as previously reported ( ance was also selected as 20 mmu for all MS/MS spectra obtained.
Quantitative precision was expressed with protein ratio variability.

| Bioinformatic analysis
Multiple approaches were employed to analyze proteomic results including SIMCA 14.1 software for principal component analysis (PCA) and partial least squares-discriminate analysis (PLS-DA).
R-package of "Mfuzz" and "heatmap" was used for Cluster mem- After that, the microglia were plated in 6-well plates at a density of 5 x 10 5 cells per well for Western blot and flow cytometry analysis. For phagocytosis analysis, primary cultured microglia were treated with AR (1 µM or 5 µM) followed by incubated with FAM-Aβ 1-42 (500 nM) for 2 h. Cells were then washed with PBS and collected for analysis by flow cytometry using BD Accuri C6 Plus and subsequently analyzed by flow cytometry.

| Enzyme-linked immunosorbent assay (ELISA) and Western blot
ELISA kits for human and mouse adiponectin, human Aβ 1-40, and Aβ 1-42 were obtained from R&D systems. APN level in the plasma of human dementia patients and experimental animals was quantified.
The level of Aβ 1-40 and Aβ 1-42 in the supernatants from N 2 a/W.T. and N 2 a/APP swe cells with or without different treatments such as AR, 3-MA, Dorsomorphin (Dor), and AdipoR1 overexpression was measured according to the manufacturer's instructions. The level of inflammatory cytokines from the lysate of N 2 a/APP swe cells with or without AR or Dor treatment was measured from Elascience.
Hippocampal and cell lysate supernatant was obtained and quantified by using the BCA protein assay kit. Protein samples from animals or cells were then added loading buffer and boiled at 100℃ for 8 min to denature the protein. Degenerated protein lysates were isolated by 10% SDS-PAGE, transferred onto a 0.22 μm PVDF membrane, blocked with 5% nonfat milk, dissolved in 1 x TBST buffer, and incubated overnight with primary antibodies on ice and then with the corresponding secondary antibodies for 1 h at room temperature. Finally, protein chemiluminescence signal was measured by using the ECL kit and quantified using Quantity One 4.6.2 software.

| Statistical analysis
The data was presented as the mean ± SEM and analyzed using GraphPad Prism 8.0 statistical software (GraphPad Software, Inc., La Jolla, CA, USA). A two-tailed unpaired Student's test was applied to compare two groups statistically. Simultaneously, one-way analysis of variance (ANOVA) and two-way analysis of variance were employed to determine the statistical significance of differences among groups and follow Dunnett's multiple comparison test. A probability value of p < 0.05, p < 0.01, and p < 0.001 was considered statistically significant.

Shenzhen-Hong Kong Institute of Brain Science-Shenzhen
Fundamental Research Institutions, Shenzhen, 518055, China

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest.

AUTH O R CO NTR I B UTI O N S
Kaiwu He and Lulin Nie designed and performed the experiments.
Tahir Ali analyzed data and wrote the manuscript. Zizhen Liu, Weifen Li, Kaiqin Zhang, and Jia Xu helped in the experiment. Shujin Wang, Xiao Chen, and Jianjun Liu helped in manuscript, experimental tools and supported the study. Zhi-Jian Yu, Xifei Yang, and Shupeng Li endorsed the study, corresponding authors, reviewed and approved the manuscript, and held all the responsibilities related to this manuscript. All authors reviewed and approved the manuscript.

E TH I C A L A PPROVA L A N D CO N S E NT TO PA RTI CI PATE
According to the protocols approved by the Animal Care and Use Committee of the Experimental Animal Center at Shenzhen Center for Disease Control and Prevention, all experimental procedures were carried out.

CO N S E NT FO R PU B LI C ATI O N
All involved parties consented to the publication of this work.

DATA AVA I L A B I L I T Y S TAT E M E N T
All data generated or analyzed during this study are included in this published article [and its supplementary information files].