dNAGLU Extends Life Span and Promotes Fitness and Stress Resistance in Drosophila

To identify new factors that promote longevity and healthy aging, we studied Drosophila CG13397, an ortholog of the human NAGLU gene, a lysosomal enzyme overexpressed in centenarians. We found that the overexpression of CG13397 (dNAGLU) ubiquitously, or tissue specifically, in the nervous system or fat body could extend fly life span. It also extended the life span of flies overexpressing human Aβ42, in a Drosophila Alzheimer’s disease (AD) model. To investigate whether dNAGLU could influence health span, we analyzed the effect of its overexpression on AD flies and found that it improved the climbing ability and stress resistance, including desiccation and hunger, suggesting that dNAGLU improved fly health span. We found that the deposition of Aβ42 in the mushroom body, which is the fly central nervous system, was reduced, and the lysosomal activity in the intestine was increased in dNAGLU over-expressing flies. When NAGLU was overexpressed in human U251-APP cells, which expresses a mutant form of the Aβ-precursor protein (APP), APP-p.M671L, these cells exhibited stronger lysosomal activity and and enhanced expression of lysosomal pathway genes. The concentration of Aβ42 in the cell supernatant was reduced, and the growth arrest caused by APP expression was reversed, suggesting that NAGLU could play a wider role beyond its catalytic activity to enhance lysosomal activity. These results also suggest that NAGLU overexpression could be explored to promote healthy aging and to prevent the onset of neurodegenerative diseases, including AD.


Introduction
The effects of aging are complex and diverse and aging causes gradual loss and degeneration of our body's constituent substances, tissue structure integrity, and physical and cognitive functions. It is an unavoidable risk factor for major human diseases, including cancer and cardiovascular and neurodegenerative diseases [1]. Alzheimer's disease (AD) is the most common age-related disease [2,3], characterized by the accumulation of intracellular neurofibrillary tangles composed of hyperphosphorylated tau protein and the extracellular aggregation of β-amyloid (Aβ) plaques [4][5][6]. Excessive Aβ production and insufficient Aβ clearance play a key role in the pathogenesis of AD [7,8].
Centenarians live much longer than the general population, characterized by much delayed onset or absence of aging-related diseases [9][10][11][12], making them excellent subjects to study the mechanism of human longevity and healthy aging. Our previous study found that the autophagy-lysosome pathway was upregulated in centenarians [13]. The product of one of these genes, N-acetyl-alpha-glucosaminidase (NAGLU), degrades heparan sulfate, and is an important glucosidase in the lysosomal pathway. Its deficiency leads to clinically termed mucopolysaccharidosis-type IIIB (MPS IIIB) [14,15], a devastating and currently incurable neurodegenerative disease, characterized by lysosomal accumulation and urinary excretion of heparan sulfate, and causes progressive cognitive decline and behavioral difficulties [16,17].
Model organisms, such as nematodes [18,19], fruit flies [20,21], and rodents [22,23], are extensively used to study mechanisms of aging and longevity. Seventy percent of human disease-related genes are conserved between flies and humans, and given its short life span, Drosophila is well suited to study whether centenarian overexpressed genes have the potential to extend life span or health span. CG13397, the Drosophila homologue of NAGLU, or dNAGLU, is widely expressed in the fat body, the CNS, and the heart of flies [24]. Herein, we used geneswitch (GS), a modified Gal4/UAS inducible expression system, to induce dNAGLU overexpression in specific tissues, and its transcriptional activity within the target tissues depends on the presence of the activator mifepristone (RU486) [25]. The GS-Gal4 flies were used to express steroid-activated GAL4 in the whole body. Then, we investigated whether and how dNAGLU affected the fly life span and health span by overexpressing it in wild type (wt) flies and in the Drosophila model of AD, which expresses human Aβ42 and GS-Gal4 simultaneously [26,27]. We found that when overexpressed either ubiquitously or tissue specifically in the fat body and nervous system, dNAGLU extended the life span in wt flies. In AD flies overexpressing human Aβ42, dNAGLU prolongs both life span and health span, demonstrated by improved climbing ability as well as resistance to stress, including that induced by desiccation and hunger. The overexpression increased lysosomal activity and reduced Aβ42 deposition in the fly CNS. The activity of dNAGLU was confirmed by overexpressing NAGLU in human U251-APP cells, which resulted in stronger lysosomal activity in these cells and a reduction in Aβ42 in the cell supernatant. Thus, NAGLU could extend life span and health span by improving lysosomal function.

NAGLU Is Upregulated in Centenarians
We studied a group of 117 subjects from centenarian families, consisting of 76 centenarians (CENs) and 41 spouses of centenarian children (F1SPs, considered as younger adult controls) to analyze the differentially expressed genes in centenarians' blood samples and found that NAGLU was upregulated in CENs ( Figure 1A, p = 0.0002), while its expression patterns in controls did not show age-related changes ( Figure 1B, p = 0.5768). We also analyzed the dataset of genome-wide RNA-seq profiles of human dermal fibroblasts from 133 people aged 1 to 94 years old [28] and found that NAGLU expression had no correlation with age in the general population ( Figure 1C, p = 0.4064), suggesting that the overexpression in centenarians is unlikely the consequences of extreme aging.

Overexpression of dNAGLU Extends Drosophila Life Span
To study the functional consequences of overexpressing NAGLU, we cloned its Drosophila homologue dNAGLU and used the Gal4-UAS system to induce dNAGLU overexpression by supplementing with RU486. We tested the effects of RU486 on lifespan and fecundity and found that 200 µM RU486 had no significant effect on lifespan and fecundity of wt flies (Supplementary Figure S1). Therefore, we used 200µM RU486 to induce dNAGLU overexpression. We overexpressed dNAGLU ubiquitously and observed its effects on Drosophila lifespan. The induced mRNA level of dNAGLU was confirmed by qRT-PCR to have an increased expression of 30.43%, p = 0.0062 ( Figure 2A) compared to uninduced dNAGLU, and the expression of 3HA-dNAGLU protein was detected by Western blot (Supplementary Figure S2). Statistical analysis of lifespan showed that the overexpression of dNAGLU prolonged the lifespan of Drosophila, and mean lifespan was increased by up 3 of 17 to 3.64% ( Figure 2B, p = 0.0004). When dNAGLU was knocked down in the Drosophila whole body, the lifespan was significantly decreased in comparison with the control group (Supplementary Figure S3).

Overexpression of dNAGLU Extends Drosophila Life Span
To study the functional consequences of overexpressing NAGLU, we clone  (F) survival curves of Drosophila overexpressing dNAGLU in the nervours syste 5% or lower p value is considered to be statistically significant using Student's t-te p<0.001. All survival data were analyzed with the log-rank test.
The fat body plays an important role in intermediary metabolism a endocrine organ that produces a variety of antimicrobial peptides involv dNAGLU in the nervours system, p = 0.0195. A 5% or lower p value is considered to be statistically significant using Student's t-test, ** p < 0.01, *** p < 0.001. All survival data were analyzed with the log-rank test.
The fat body plays an important role in intermediary metabolism and is also an endocrine organ that produces a variety of antimicrobial peptides involved in the humoral immunity of Drosophila [29]. In contrast, the fly central nervous system regulates homeostasis and adapts to environmental changes throughout the organism and is also a target for aging and anti-aging interventions [30]. As dNAGLU is expressed at high levels in the fat body and mushroom body [24], we hypothesize that these tissues are where dNAGLU may exert its main function. We, therefore, induced dNAGLU overexpression in the fat body and mushroom body and detected the overexpressed dNAGLU mRNA in the fat body to be 41.52% (p = 0.0002) higher than that in the control group ( Figure 2C), and the expression of 3HA-dNAGLU protein was detected by Western blot (Supplementary Figure S2). Statistical analysis of lifespan showed that the overexpression of dNAGLU in the fat body prolonged the lifespan of Drosophila, and mean lifespan increased by 2.19% ( Figure 2D, p = 0.0008). When dNAGLU was knocked down in the Drosophila fat body, the lifespan was significantly decreased in comparison with the control group (Supplementary Figure S3). In the CNS, dNAGLU mRNA expression was higher than the control group ( Figure 2E), with an increase of 177.9% (p = 0.0010). Additionally, the overexpression of dNAGLU in the nervous system prolonged the lifespan of Drosophila, and mean lifespan increased by 2.55% ( Figure 2F, p = 0.0195). These results are stable after repeated verification (Supplementary Figure S4). These analyses demonstrate that dNAGLU overexpression prolongs lifespan and suggest that it may exert its function mainly in the fat body and nervous system.

Overexpression of dNAGLU Extends Life Span and Health Span in the Drosophila AD Model
The up-regulation of autophagy and lysosomal signals in centenarians suggests that these pathways may have a role preventing the accumulation of molecules implicated in neurodegenerative diseases, and, therefore, reducing the risk of these diseases in these long-lived individuals. We speculated that genes in this pathway may exert a stronger effect in flies expressing human Aβ42, a Drosophila AD model, than in normal flies and subsequently studied how dNAGLU may function in the Drosophila AD model.
GS-Gal4>Aβ42 flies are homozygous and carry both GS-Gal4 and UAS-Aβ42. Only when RU486 is present could GS-Gal4>Aβ42 flies induce Aβ42 expression in the whole body. In the control group, the F1 generation obtained by crossing of GS-Gal4>Aβ42 flies and w 1118 flies were fed sucrose-yeast (SY) food with 200 µM RU486 to induce ubiquitous Aβ42 expression (i.e., GS-Gal4>Aβ42 >+ +RU flies, this is the Drosophila AD model). While in the experimental group, the F1 generation obtained by crossing of GS-Gal4>Aβ42 flies and dNAGLU transgenic flies were fed SY food with 200 µM RU486 to induce both the expression of Aβ42 and the overexpression of dNAGLU (i.e., GS-Gal4>Aβ42>dNAGLU +RU flies, in other words, we induced dNAGLU overexpression in Drosophila AD model). We fed flies different foods at different stages ( Figure 3A) to measure the effects of dNAGLU overexpression on the lifespan and health span. Again, the mRNA of dNAGLU in induced Drosophila was found to be 42.91% ( Figure 3B, p = 0.0018) higher than uninduced group, and the expression of 3HA-dNAGLU protein was detected by Western blot (Supplementary Figure S2). The overexpression of dNAGLU in AD Drosophila significantly prolonged the lifespan in these flies, and themean lifespan was increased by up to 7.01% ( Figure 3C, p = 0.0004).
A previous study showed that Drosophila exhibited severe age-related deterioration in skeletal muscle function, such as reduced flight time and impaired climbing ability [31]. Studies in yeast have confirmed that activation of an autophagy-lysosomal pathway is critical for maintaining cell viability during extreme nutrient conditions, such as starvation and desiccation [32], and that lysosomal activity decreases with age in most tissues and in most organisms [33]. We, therefore, assessed the fitness of these flies by subjecting 20-day-old flies to climbing test, desiccation, starvation, and H 2 O 2 treatment to evaluate how dNAGLU impacts these functions.  We found that the climbing ability increased in dNAGLU overexpressing flies compared to controls, with an increase of 23.44%, p = 0.0158 ( Figure 3D). When stress resistance was analyzed, we found that the flies overexpressing dNAGLU have better tolerance to starvation ( Figure 3E, p < 0.0001) and desiccation ( Figure 3F, p = 0.0018) than control flies, and the mean survival time was increased by 14.10% and 9.25%, respectively. However, no significant change was seen in tolerance to H 2 O 2 treatment, although the dNAGLU overexpression group showed a tendency of increased survival ( Figure 3G, p = 0.1850). These results are stable after repeated verification (Supplementary Figure S5).
In the negative control group, F1 flies from the above crosses were fed with normal SY food without RU486 but supplemented with the same dose of ethanol (i.e., GS-Gal4>Aβ42>+ −RU flies and GS-Gal4>Aβ42>dNAGLU −RU flies), which induced neither the expression of Aβ42 nor the overexpression of dNAGLU, and no significant difference was observed in the lifespan among these flies. Similarly, their climbing ability and tolerance to desiccation, starvation, and H 2 O 2 showed no significant differences (Supplementary Figure S6). Thus, the changes we observed were due to dNAGLU overexpression. These results demonstrated that overexpression of dNAGLU in AD flies significantly extends the lifespan and improves climbing ability and stress resistance, including resistance to desiccation and hunger.

Overexpression of dNAGLU Reduces Aβ42 Deposition in the Mushroom Body in AD Drosophila
Since lysosomal dysfunction is a common abnormality in neurodegenerative diseases, which leads to impaired Aβ clearance [34], we attempted to determine whether dNAGLU could improve fly fitness and stress resistance by promoting Aβ clearance. We dissected the mushroom bodies of 50-day-old flies for cryo-sectioning and performed immunofluorescence staining to observe the deposition of Aβ42 in the brain tissue of these flies. We used GS-Gal4>Aβ42>+ −RU flies ( Figure 4A), which did not induce Aβ42 expression, as the negative control, and used GS-Gal4>Aβ42>+ +RU flies ( Figure 4B), the Drosophila AD model, which induces ubiquitous Aβ42 expression, as the control. We used GS-Gal4>Aβ42>dNAGLU +RU flies ( Figure 4C), which induce the overexpression of dNAGLU in the Drosophila AD model, as the experimental group.
We observed a dramatic decrease of Aβ42 fluorescence intensity when dNAGLU was overexpressed in AD flies (compare Figure 4B,C). This decrease (52.07% ) was significant when 15 brain slices were analyzed for fluorescence intensity ( Figure 4D, p = 0.0024). In comparison, no significant difference in the mean fluorescence intensity of DAPI was seen in these tissues ( Figure 4E, p = 0.7780). Therefore, overexpression of dNAGLU can significantly reduce the Aβ42 deposition in the AD Drosophila model.

Overexpression of dNAGLU in AD Drosophila Enhances the Intestinal Lysosomal Activity
As lysosomal dysfunction is associated with aging, lysosomal activity decreases with age in most tissues and organisms [33,35]; therefore, detecting the activity of lysosomes is also an important indicator for evaluating the biological health of an organism. Lyso Tracker Red can be used to label and monitor acidic lysosomes in living cells. To reveal whether dNAGLU overexpression increased lysosomal activity, we performed lysosomal probe staining in dissected fly intestines. Here, we used GS-Gal4>Aβ42>+ +RU flies as the control group ( Figure 5A) and used GS-Gal4>Aβ42>dNAGLU +RU flies as the experimental group ( Figure 5B). We found that the overexpression of dNAGLU enhanced the fluorescence intensity of gut lysosomes by 2.48-fold ( Figure 5C, p = 0.0024), while there was no significant difference in the mean fluorescence intensity of DAPI ( Figure 5D, p = 0.6908) in the gut of these 50-day-old AD flies, indicating that the overexpression of dNAGLU resulted in stronger lysosomal activity and promoted lysosomal biogenesis in these flies.  We observed a dramatic decrease of Aβ42 fluorescence intensity when dNAGLU overexpressed in AD flies (compare Figure 4B,C). This decrease (52.07% ) was signi when 15 brain slices were analyzed for fluorescence intensity ( Figure 4D, p = 0.002 comparison, no significant difference in the mean fluorescence intensity of DAP seen in these tissues (Figure4E, p = 0.7780). Therefore, overexpression of dNAGLU experimental group ( Figure 5B). We found that the overexpression of dNAGLU enhan the fluorescence intensity of gut lysosomes by 2.48-fold ( Figure 5C, p = 0.0024), w there was no significant difference in the mean fluorescence intensity of DAPI (Figure p = 0.6908) in the gut of these 50-day-old AD flies, indicating that the overexpressio dNAGLU resulted in stronger lysosomal activity and promoted lysosomal biogenes these flies.

Overexpression of NAGLU Reduces Aβ42 Concentration in Human U251-APP Cells
In order to confirm that NAGLU and dNAGLU are functionally conserved and indeed degrade Aβ42 in both fly and mammalian cells, we tested NAGLU overexpres in a human glioma cell line, U251-APP cells [36], and detected a 105-fold increas NAGLU mRNA expression compared to control ( Figure 6A, p = 0.0001). We also dete the expression of Flag-NAGLU and APP by Western blot and found that when NA was overexpressed in U251-APP cells, there was no significant change in the expres of APP (Supplementary Figure S7). So NAGLU overexpression did not affect the exp sion of APP in the original cell system. We then used ELISA to detect the concentra of Aβ42 in cell supernatant and found that the overexpression of NAGLU led to th covery of cell proliferation ( Figure 6B), and the Aβ42 in cell supernatant was reduce

Overexpression of NAGLU Reduces Aβ42 Concentration in Human U251-APP Cells
In order to confirm that NAGLU and dNAGLU are functionally conserved and can indeed degrade Aβ42 in both fly and mammalian cells, we tested NAGLU overexpression in a human glioma cell line, U251-APP cells [36], and detected a 105-fold increase in NAGLU mRNA expression compared to control ( Figure 6A, p = 0.0001). We also detected the expression of Flag-NAGLU and APP by Western blot and found that when NAGLU was overexpressed in U251-APP cells, there was no significant change in the expression of APP (Supplementary Figure S7). So NAGLU overexpression did not affect the expression of APP in the original cell system. We then used ELISA to detect the concentration of Aβ42 in cell supernatant and found that the overexpression of NAGLU led to the recovery of cell proliferation (Figure 6B), and the Aβ42 in cell supernatant was reduced by 10.41% ( Figure 6C, p = 0.0422). These results demonstrated that when NAGLU was overexpressed in human U251-APP cells, the concentration of Aβ42 in the cell supernatant was reduced, and the growth arrest caused by APP expression was reversed.  When stained lysosomes of U251-APP cells with Lyso Tracker Red ( Figure 6D,E) and counted the average fluorescence intensity of cells, we found that these cells had an enhanced fluorescence intensity of 2.73-fold compared to control ( Figure 6F, p = 0.0209), while there was no significant difference in the mean fluorescence intensity of copGFP ( Figure 6G, p = 0.8375). The tripeptidyl peptidase (TPP) and the Cts family of cysteine proteases, i.e., -CTSB, CTSD, CTSK and CTSL, which are proteolytic enzymes in lysosomes that can degrade Aβ, and TFEB (transcription factor EB)/TFE3 (transcription factor binding to IGHM enhancer 3) are master transcriptional regulators of lysosomal activity [37]. When NAGLU overexpressed in U251-APP cells, transcript levels of CTSB, CTSD, CTSK, CTSL, TPP1, TFEB, and TFE3 were significantly increased, while, other enzymes that can degrade Aβ, such as matrix metalloproteinase (MMP)-2, MMP-9 and angiotensinconverting enzyme (ACE), were also increased ( Figure 6H). These results indicated that when NAGLU is overexpressed in U251-APP cells, it exhibited strong lysosomal activity and promoted lysosomal biogenesis, and NAGLU promoted the degradation of Aβ42 by enhancing the lysosomal pathway.

Discussion
In this study, we investigated the effect of overexpression Drosophila dNAGLU, fly ortholog of human NAGLU, a lysosomal enzyme highly expressed in longevity individuals and found that dNAGLU overexpression leads to life span extension and increased fitness and stress resistance in flies. In addition, we demonstrated that NAGLU overexpression reduced Aβ42 deposition and enhanced lysosomal function in AD flies and in human U251-APP cells. These results suggest that the overexpressed NAGLU level could potentially be a positive factor for health span and life span in humans.
NAGLU is located in lysosomes and is an important glucosidase in the lysosomal pathway. The autophagy-lysosome pathway degrades and recycles cellular components, including toxic protein aggregates and damaged organelles [38]. Growing evidence suggests that lysosomal dysfunction plays a key role in the development of AD. Impaired clearance of Aβ is a potential mechanism of widespread sporadic AD [8]; thus, the health and survival of neurons in the brain depend on the normal lysosomal function [39]. Lysosomal dysfunction is associated with aging, and blockade of autophagic flux may result from lysosomal damage [35]. Our study described herein suggests that NAGLU may have a wider role, as we demonstrated here, that in AD flies, as well as in cultured cells, NAGLU could enhance the lysosomal activity and promote lysosomal biogenesis.
The number of lysosomes decreased during aging and lysosomal dysfunction also lead to decreased muscle mass and function [40]. Furthermore, lysosomes can recycle amino acids required for protein synthesis under extreme nutritional conditions, such as starvation and desiccation, by degrading protein products [41]. Our study demonstrates that overexpression of dNAGLU in AD flies improved climbing ability, as well as starvation and desiccation resistance and ultimately prolonged lifespan. Combined with our findings of enhanced lysosomal activity, we can conclude that dNAGLU could improve health and prolong lifespan by enhancing lysosomal function.
Previous studies suggested that NAGLU deficiency in humans could lead to MPS IIIB [14,15]; thus, an overexpression could at least reduce the risk for MPS IIIB. It is shown that in a mouse model of MPS IIIB, using enzyme replacement therapy by injecting modified NAGLU into the brain could reduce the pathological accumulation of heparan sulfate and other metabolites (e.g., glycine, β-amyloid, P-tau) to normal or near-normal levels [42]. While β-amyloid and P-tau are still the most acknowledged pathological hallmarks of AD [43], it hints that a higher level of NAGLU may play a role in preventing AD. Our study demonstrates that overexpression of NAGLU homologue dNAGLU in AD flies significantly reduced Aβ42 deposition in the brain, and similar results were seen in NAGLU over-expressing U251-APP cells. When NAGLU overexpressed in U251-APP cells, the concentration of Aβ42 in the cell supernatant was reduced, and the cell cycle dysregulation caused by APP expression was reversed resulting in normal cell proliferation.
Moreover, our results also showed that the mRNA levels of many proteolytic enzymes that degrade Aβ were found to be upregulated in lysosomes, e.g., CTSB, CTSD, CTSK, CTSL and TPP1. Importantly, we observed an increase in the mRNA levels of upstream transcription regulators TFEB and TFE3. Thus, NAGLU may promote lysosomal biogenesis through upregulating TFEB/TFE3, which increase the expression of a number of proteolytic enzymes that degrade Aβ in lysosomes and facilitate the degradation of Aβ42 (Figure 7). The mechanism leading to upregulation of TFEB/TFE3 is currently unknown, and the relationship between NAGLU overexpression and autophagy also needs to be explored. These are interesting questions for future studies. Studies have reported an intracerebral gene therapy with recombinant AAV vector serotype 2/5 encoding human NAGLU in four children with MPS IIIB syndrome. At intermediate analysis 30 months after surgery, neurocognition was improved in all patients [44]. After 5.5 years of follow-up, good tolerance, sustained NAGLU production, and milder disease were demonstrated in the patient treated at a very early stage [45]. As MPS IIIB is also a neurodegenerative lysosomal storage disorder, the improved neurocognition is consistent with what our findings suggest: that NAGLU overexpression might alleviate the symptoms of AD. As NAGLU is significantly upregulated in centenarians, and as its deficiency led to MPS IIIB, it is essential to investigate whether its up-regulation is a result of extreme aging or a causal factor for healthy aging and longevity. Our Drosophila study demonstrated that NAGLU is a positive factor for health aging and longevity, and its high expression in centenarians is likely a reason for longevity, rather than a result of extreme aging. Our study also suggests that NAGLU could function as a potential drug target for clinical intervention of aging-related neurodegenerative diseases.
Taken together, we revealed that NAGLU could extend lifespan and health span in flies, and it enhanced β amyloid clearance, as demonstrated in AD flies and in human cells, by enhancing lysosomal pathway. Therefore, NAGLU may be directly or indirectly involved in β-amyloid clearance, suggesting that NAGLU overexpression, or administering its agonist if it exists, could treat AD in experimental animal models, and NAGLU could be a potential intervention target to alleviate AD symptoms in humans. Studies have reported an intracerebral gene therapy with recombinant AAV vector serotype 2/5 encoding human NAGLU in four children with MPS IIIB syndrome. At intermediate analysis 30 months after surgery, neurocognition was improved in all patients [44]. After 5.5 years of follow-up, good tolerance, sustained NAGLU production, and milder disease were demonstrated in the patient treated at a very early stage [45]. As MPS IIIB is also a neurodegenerative lysosomal storage disorder, the improved neurocognition is consistent with what our findings suggest: that NAGLU overexpression might alleviate the symptoms of AD. As NAGLU is significantly upregulated in centenarians, and as its deficiency led to MPS IIIB, it is essential to investigate whether its up-regulation is a result of extreme aging or a causal factor for healthy aging and longevity. Our Drosophila study demonstrated that NAGLU is a positive factor for health aging and longevity, and its high expression in centenarians is likely a reason for longevity, rather than a result of extreme aging. Our study also suggests that NAGLU could function as a potential drug target for clinical intervention of aging-related neurodegenerative diseases.

Drosophila Stocks and Construction of the Transgenic Flies
Taken together, we revealed that NAGLU could extend lifespan and health span in flies, and it enhanced β amyloid clearance, as demonstrated in AD flies and in human cells, by enhancing lysosomal pathway. Therefore, NAGLU may be directly or indirectly involved in β-amyloid clearance, suggesting that NAGLU overexpression, or administering its agonist if it exists, could treat AD in experimental animal models, and NAGLU could be a potential intervention target to alleviate AD symptoms in humans.

Drosophila Stocks and Construction of the Transgenic Flies
The construction of transgenic flies used molecular cloning technology to insert dNAGLU into the UAS promoter-driven vector pUAST-attB-3HA to generate pUAST-attB-3HA-dNAGLU plasmid, which was microinjected into 86F (III) 6110 transgenic strain (gift of Sun Yat-Sen University) with the attP site inserted near 86F. dNAGLU transgenic flies was obtained by standard transgenic. The dNAGLU transgenic flies were then backcrossed with w 1118 flies for at least 6 generations to remove the background. GS-Gal4 flies expressed steroid-activated GAL4 in the whole body.

Husbandry and Life Span Analysis
The larval stage of all strains of Drosophila were fed standard cornmeal-yeast medium (agar 7. The dNAGLU transgenic flies were crossed with driver lines GS-Gal4, S 1 106-Gal4, and 43642 flies, respectively. In the experimental group, the F1 generation flies were fed SY food supplemented with RU486 (w/v, 200 µM; Sigma-Aldrich, St. Louis, MO, USA, CAS 84371-65-3) to induce dNAGLU overexpression in the whole body, fat body, and nervous system, respectively. Additionally, in the control group, F1 generation flies were fed SY food without RU486 but supplemented with the same dose of ethanol.
After hatching, the larvae were mated in standard cornmeal-yeast medium for at least 48 h, and female flies were selected for experimental research. Using Drosophila for lifespan statistics with 10 flies/tube, each tube contained an appropriate amount of SY food, at least 10 tubes per experimental condition. All stocks were maintained at 22 • C and 60% humidity, 12 h light, 12 h dark cycle. The flies were transferred to fresh food every 2 days, during which the number of dead and escaped flies was recorded. All lifespan data were analyzed with the log-rank test.

Health Test Assays
A total of 10 flies/tube were used for the test, with at least 5 tubes per experimental condition. For the desiccation test, the flies were placed in an empty tube without medium, and the number of dead flies was counted every 2 h. For the starvation test, flies were placed in tubes containing 1.5% agarose, just to provide moisture but without any other nutrients. The number of dead flies was counted every day [21]. For the H 2 O 2 oxidative stress test, circular filter paper was placed at the bottom of the culture tube, 50 µL of 6% glucose solution containing 30% H 2 O 2 was added, the flies were put into the tube, and the number of dead flies was counted every 2 h [46]. All survival data were analyzed with the log-rank test. For the climbing test, 6 flies/tube were used for the test, with at least 4 tubes per experimental condition. We calculated the average speed of flies climbing from the bottom of the tube to the top within 5s to evaluate the climbing ability of flies. The climbing data were analyzed by a Student t-test.

Immunofluorescence
The brains of flies were dissected for cryosections, and the brain sections were blocked with blocking buffer (1×PBS/5%BSA/0.3%Triton X-100) at room temperature for 1 h. The slides were incubated with primary antibody: β-Amyloid (CST, 8243S) in the staining buffer (1% BSA and 0.3% Triton X-100 in PBS) at 4 • C overnight and subsequently incubated with Alexa Fluor 594-conjugated secondary antibody (ThermoFisher, Waltham, MA, USA, A-11012) for 2 h at room temperature. The slides were mounted with mounting medium with DAPI (Abcam, Cambridge, UK, ab104139). Imaging was then performed with an ultra-high-resolution laser confocal microscope (Zeiss, Oberkochen, Germany, LSM880), and mean fluorescence intensity analysis was performed with Image J.

Autolysosome Staining
Drosophila guts were dissected and stained with Lyso Tracker Red (Beyotime, C1046), and mounted with mounting medium with DAPI (Abcam, Cambridge, UK, ab104139). For living cells, operations were performed according to the manufacturer's procedures. Imaging was then performed immediately with an ultra-high-resolution laser confocal microscope (Zeiss, Oberkochen, Germany, LSM880), and mean fluorescence intensity analysis was performed with Image J.

qRT-PCR
RNA was extracted with RNAiso Plus (TaKaRa, Kusatsu, Japan, RNAA00250); then, RNA was reverse transcribed into cDNA with Prime Script TMRT reagent kit with gDNA Eraser (TaKaRa, Kusatsu, Japan, RR047Q), and qRT-PCR was performed using the Fast Start Universal SYBR Green Master (ROX) (Roche, Basel, Switzerland, 04913850001) and genespecific primers. All the above experimental operations were performed according to the manufacturer's procedures. Gene expression levels were calculated using the comparative C T method. All primers used are listed in (Supplementary Table S1).

NAGLU Expression Analysis
The differences of NAGLU expression between the CENs and F1SPs were analyzed, as described in our previous study [13]. Pearson's correlation test was used to evaluate the association between gene expression and age through the cor.test function in the R platform.

Cells and Transfection
The U251 cell line, with stable expression of the APP mutant APP-p.M671L (U251-APP cells), was a gift from Yonggang Yao [36]. NAGLU overexpression plasmid was constructed by using the pCDH-CMV-MCS-EF1-copGFP-T2A-Puro lenti-viral vector and was then transduced into U251-APP cells. Stably transfected cells were selected by fluorescence screening.

Cell Proliferation Analysis
We used Enhanced Cell Counting Kit-8 (Beyotime, Haimen, China, C0042) to detect cell proliferation and growth activity. We detected at 12h, 36h, 60h, 84h, and 120h after NAGLU overexpression. The experimental operations were performed according to the manufacturer's procedures.

Aβ42 ELISA Analysis
U251-APP cells supernatant was measured using commercial ELISA kits (Elabscience, Wuhan, China, E-EL-H0543c) to detect human Aβ42. The ELISA was performed to detect Aβ42 according to the manufacturer's instructions.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.