NPD1 Enhances Autophagy and Reduces Hyperphosphorylated Tau and Amyloid-β 42 by Inhibiting GSK3β Activation in N2a/APP695swe Cells

Abstract

Background:

The most prevalent kind of dementia, Alzheimer’s disease (AD), is a neurodegenerative disease. Previous research has shown that glycogen synthase kinase-3β (GSK-3β) is involved in the etiology and progression of AD, including amyloid-β (Aβ), phosphorylated tau, and mitochondrial dysfunction. NPD1 has been shown to serve a neuroprotective function in AD, although the mechanism is unclear.

Objective:

The effects of NPD1 on Aβ expression levels, tau protein phosphorylation, apoptosis ratio, autophagy activity, and GSK-3β activity in N2a/APP695swe cells (AD cell model) were studied, as well as the mechanism behind such effects.

Methods:

N2a/APP695swe cells were treated with NPD1, SB216763, or wortmannin as an AD cell model. The associated proteins of hyperphosphorylated tau and autophagy, as well as the activation of GSK3β, were detected using western blot and RT-PCR. Flow cytometry was utilized to analyze apoptosis and ELISA was employed to observe Aβ₄₂. Images of autophagy in cells are captured using transmission electron microscopy.

Results:

In N2a/APP695swe cells, NPD1 decreased Aβ₄₂ and hyperphosphorylated tau while suppressing cell death. NPD1 also promoted autophagy while suppressing GSK-3β activation in N2a/APP695swe cells. The outcome of inhibiting GSK-3β is comparable to that of NPD1 therapy. However, after activating GSK-3β, the opposite experimental results were achieved.

Conclusion:

NPD1 might minimize cell apoptosis, downregulate Aβ expression, control tau hyperphosphorylation, and enhance autophagy activity in AD cell models to promote neuronal survival. NPD1’s neuroprotective effects may be mediated via decreasing GSK-3β.

Keywords

Alzheimer’s disease autophagy glycogen synthase kinase 3 Neuroprotectin D1

INTRODUCTION

Alzheimer’s disease (AD) is a pernicious neurological disease characterized clinically by increasing cognitive impairment, language, behavioral problems, social dysfunction, and, finally, death [1]. There are no viable preventatives or curative therapies for this disease at the moment. The development of intracellular neurofibrillary tangles and extracellular senile plaques containing amyloid-β peptide (Aβ) is the major pathological hallmarks of AD [2]. Neurofibrillary tangles made up of hyperphosphorylated tau are neurotoxic and can induce neuronal apoptosis and cell death in AD [3]. Tau proliferation and toxicity in AD are accelerated by aberrant acumination of Aβ [4]. Research of 50 amnestic moderate cognitive impairment (MCI), AD, and age-matched healthy people discovered that aberrant apoptosis may occur in the early stages of AD [5]. As a result, one of the treatment methods for AD is to prevent Aβ acumination, tau hyperphosphorylation, and apoptosis.

Furthermore, some studies have indicated that an adequate increase in autophagy may preserve the dynamic balance of processes in neurons and decrease the pathological damage associated with AD. Autophagy improvement decreased extracellular amyloid deposition by controlling A metabolism, which also affects the process of excessive and inappropriate phosphorylation of Tau Glycogen synthase kinase-3 (GSK-3) is a serine/threonine kinase that is essential for protein synthesis, axon transport and synaptic plasticity [6]. GSK-3β overexpression or activation is linked to a variety of illnesses. Several studies have demonstrated that the activity of GSK-3β in the brains of AD patients is abnormally elevated, resulting in a rise in Aβ [7]. And the overactivation of GSK-3β is directly related to the hyperphosphorylation of tau protein[8]. Other investigations have found that aberrant GSK-3β activity can impede autophagy [9]. As a result, the hunt for drugs that induce autophagy by targeting GSK-3β may become the focus of contemporary AD research.

Neuroprotectin D1 (NPD1), a pro-resolving mediator (SPM) produced by 15-lipoxygenase activity from omega-3 docosahexaenoic acid (DHA) [10], has demonstrated significant immunoresolvent and neuroprotective benefits in numerous inflammatory disease models [11]. NPD1 is abundant in the neurological system and has a more powerful biological impact than DHA [12]. DHA is highly likely to have a function in the development of the central nervous system, synapse formation and other biological effects in the brain via its derivative NPD1 [13]. Alzheimer’s patients had lower NPD1 content in the CA1 area of the hippocampus. NPD1 increases brain cell survival by inducing antiapoptotic and neuroprotective gene-expression programs that decrease Abeta₄₂ synthesis and neurotoxicity, and it may also repair Aβ-induced neuronal death [14, 15]. NPD1 inhibits Aβ₄₂ peptide release from aged human brain cells and is substantially reduced in AD brain [16]. NPD1 has also been demonstrated to successfully address and minimize the risk of AD-related neuroinflammation as a specialized pro-resolving lipid mediator [17]. It is neuroprotective and may be selectively stored in the neurological system [18].

In reality, few studies have looked at the mechanism through which NPD1 works in AD. However, the effects of NPD1 on autophagy and GSK3β have not been investigated further. This work investigates whether NPD1 induces autophagy in N2a/APP695swe cells by residing on GSK3β, therefore limiting disease progression.

MATERIALS AND METHODS

Cell culture

Mouse neuroblastoma cell line N2a/WT cells and N2a/APP695swe cells were kindly gifted by Professor Xu Hua-xi (Xiamen University). N2a/WT cells were cultured in the solution of 47%Dulbecco’s Modified Eagle Medium (DMEM, Gibco,), 47%Opti-MEN I Reduced-Seum Medium (opti-MEM, Gibco), 5%fetal bovine serum (Hyclone), 1%solution of penicillin and streptomycin (Beyotime). Compared with the N2a/WT cells, N2a/APP695swe cells were additionally supplemented with 200μg/ml G418 (Amresco) to screen for cells stably expressing the APP695swe gene. They were maintained in the incubator containing 5%CO₂ at 37°C.

Medicine treatment

NPD1 was bought from Cayman. Also, SB216763 and wortmannin were purchased from Selleck Chemicals [19, 20]. The cells were plated onto 6-well plates at a density of 1×10⁶ cells/ml. Then, N2a/APP695swe cells were treated with the NPD1, the GSK3β inhibitor SB216763 or the GSK3β activator wortmannin at a final concentration of 100 nM, 5μM, and 1μM for 24 h. After 24 hours of treatment, cells were subjected to western blot analyses, ELISA analyses, flow cytometry analyses, quantitative real-time PCR analysis and transmission electron microscopy detection.

Cell apoptosis assessment by flow cytometry

For cells apoptosis assay, the N2a/WT and N2a/ APP695swe cells were cultured in 6-well plates at a concentration of 1×10⁶ cells/well. The cells were harvested after 48 h with different treatments. Then ice-cold PBS was used to wash cells three times, and AnnexinV-FITC andpropidium iodide (PI, KeyGEN Biotech, Nanjing, China) buffer were chosen to incubate them for 30 min at 37°C in the dark. Finally, cells were analyzed by flow cytometry and were considered to be apoptosis no matter early apoptosis (in the fourth quadrant, Annexin V+/PI-) or late apoptosis (in the first quadrant, Annexin V+/PI+).

Aβ ELISA

Cell cultured was collected as mentioned above. Secreted Aβ₄₂ was measured by a sandwich ELISA kits (Elabscience BioTECH, Wuhan, China) according to the manufacturer’s instructions.

Western blot analysis

The cells were harvested in RIPA buffer (Beyotime, China). Protein concentration was determined by BCA Protein Assay Kit (Beyotime, China) at 570 nm. Equal amounts of protein were loaded in each lane for SDS-PAGE Bis-tris gel and then transferred to polyvinylidene difluoride membranes (Millipore, Billerica, MS, USA). The membranes were washed with blotting buffer (Tris-buffered saline containing 0.1%Tween-20) and then blocked for 120 min in the buffer containing 5%non-fat powdered milk. After washed 3 times with blotting buffer, the membrane was incubated at 4°C overnight with primary antibody. After further washing in blotting buffer, the membrane was incubated with secondary antibody at room temperature for 40 min. Last, the membranes were developed with ECL Western Blotting Detection Reagents, and Image J was used to quantitate the expression of proteins. Primary antibodies were used in the research: Tau (Cell Signaling Technology, 1:1000), Phospho-Tau (Cell Signaling Technology, 1:1000), GSK-3β (Cell Signaling Technology, 1:1000), Phospho-GSK-3β (Cell Signaling Technology, 1:1000), LC3B-Specific (Sigma, 1:1000), P62/SQSTM1 (Cell Signaling Technology, 1:1000), Beclin1 (Santa Cruz Biotechnology, 1:1000).

Transmission electron microscopy

For transmission electron microscopy detection, the N2a/APP695swe cells were cultured in 6-well plates and the concentration is greater than 1×106 cells/well at least. The cells were harvested at 24 h after treat with curcumin. Then 0.1%trypsin-EDTA buffer was used to digest cells, and the cell lysis was centrifuged at 800^*g for 5 min. Phosphate buffer saline (PBS, pH 7.4) was used to resuspend cells before they were centrifuged at 1200 g for 10 min. Furthermore, the cell pellets were fixed in 2.5%electron microscopy-specialized glutaraldehyde for 2 h, washed several times with PBS (0.01 M), stained with 1%osmium tetroxide for 2 h, and then dehydrated in a gradient series of alcohol solutions. The samples were placed in propylene oxide, embedded in the epoxy resin Epon812, and cut into ultrathin sections. After uranyl acetate and lead citrate double staining, cells were observed by a transmission electron microscopy of Philips EM208S.

RNA isolation and quantitative real-time PCR analysis

Total RNA was extracted from cultured cells using RNAiso Plus (TaKaRa) according to the manufacturer’s instructions. For mRNA expression analysis, the synthesis of cDNA was conducted with 1μg of total RNA using PrimeSriptTM RT reagent Kit (TaKaRa) and gene expression quantified using SYBR Premix Ex TaqTM II (TaKaRa). All reactions were performed in triplicate. Primer Sequences used: Beclin1-F: 5^′-AGC CTC TGA AAC TGG ACA CG-3^′; Beclin1-R: 5^′-CTT CCT CCT GGG TCT CTC CT-3^′; GSK-3β-F: 5^′-CAA GCC CAA AGC AAA CAA AG-3^′; GSK-3β-R: 5^′-TCA AGG AAC TAT GCG GTC AA-3^′; β-actin-F: 5^′-GGA AAT CGT GCG TGA CAT C-3^′; β-actin-R: 5^′-CCA AAA GAA GCT GGA A-3^′.

Statistical analysis

Statistical analysis was performed using SPSS 18.0 software. All the data were presented as means±S.E.M. And data were statistically analyzed by one-way ANOVA, followed by Bonferroni post hoc test, or were analyzed with Student’s t test. p < 0.05 was considered to be statistical significance.

RESULTS

NPD1 reduced the Aβ₄₂ and hyperphosphorylated tau, meanwhile suppressed cell apoptosis in N2a/APP695swe cells

N2a/APP695swe cells were treated with 100 nM NPD1 to determine their function. First, we used ELISA to quantify the amount of Aβ₄₂ secreted in cell culture. We discovered that the amount of Aβ₄₂ released in the media was substantially greater in the APP group compared to the WT group (p < 0.001). NPD1 decreased the amount of Aβ₄₂ produced in N2a/APP695swe cells (p < 0.001), but there was no significant difference between the APP and control groups (p > 0.05) (Fig. 1A). The protein level expression of Tau and phosphorylated Tau was measured using western blot. The ratio of Tau-Ser404/Tau proteins was found to be higher in N2a/APP695swe cells (p < 0.01) as compared to N2a/WT cells. The ratio of Tau-Ser404/Tau proteins was decreased after NPD1 treatment (p < 0.01), but no significant change was detected between the APP and control groups (p > 0.05) (Fig. 1B, C). Flow cytometry was used to examine apoptosis in each group. The apoptotic rate of N2a/APP695swe cells was found to be substantially higher than that of N2a/WT cells (p < 0.001). The apoptosis rate of cells treated with NPD1 was lower than that of cells in the APP group (p < 0.001), but there was no significant difference between the APP and control groups (p > 0.05) (Fig. 1D, E). The aforementioned findings showed that the creation of Aβ₄₂, Tau-Ser404/Tau ratio and the apoptotic rate was greater in N2a/APP695swe cells than in N2a/WT cells, indicating that NPD1 may be involved in the development of Aβ₄₂, hyperphosphorylation of tau protein and apoptosis in N2a/APP695swe cells.

Fig. 1

NPD1 reduced the Aβ₄₂ and hyperphosphorylated tau, meanwhile suppressed cell apoptosis in N2a/APP695swe cells. A) The secreted Aβ₄₂ measured by ELISA. B, C) The expression of tau and phosphorylated tau at protein level. D, E) Flow cytometry showed the ratio of apoptotic cells. The data represent as mean±SEM of a typical series of 3 experiments. (# p > 0.05, versus APP group; ^*p < 0.001, versus WT group; ^**p < 0.001, versus APP group).

NPD1 induced autophagy in N2a/APP695swe cells

TEM (Transmission electron microscopy) images indicated that the shape and distribution of organelles and nucleus were mostly normal (Fig. 2A), with a minor number of autophagosomes with monolayer structure visible in the WT group. There were more autophagosomes with bilayer membrane structure including cytoplasmic components and autophagosomes with monolayer structure in the APP and control groups, whereas some mitochondria were enlarged or pyknotic. Autophagy with double-layer membrane or autophagosomes with single-layer membrane can also be found in the NPD1 group, although the morphology of the intracellular organelles is essentially normal. In this investigation, Beclin1 and other autophagy-related proteins were discovered. Using qRT-PCR, we determined the mRNA level of Beclin1 in N2a/WT and N2a/APP695swe cells (Fig. 2B). The results indicated that Beclin1 expression was reduced at both the mRNA and protein levels in N2a/APP695swe cells (p < 0.01) compared to N2a/WT cells. Beclin1 expression was significantly higher in the NPD1 group compared to the APP group (p < 0.01), but there was no significant difference between the APP and control groups (p > 0.05). LC3 (microtubule-associated protein 1 light chain 3 [MAP1A/1BLC3]) is an autophagy marker, while P62 (sequestosome 1) is an autophagy degradation substrate. Beclin1 and LC3II/LC3I expression were also decreased in the N2a/APP695swe group compared to the WT group (p < 0.01, p < 0.01). NPD1 treatment significantly enhanced Beclin1 and LC3II/LC3I expression (p < 0.001, p < 0.001). In contrast, P62 expression was significantly higher in the N2a/aAPP695swe group than in the WT group (p < 0.01), while NPD1 significantly reduced P62 expression (p < 0.001). It has been proposed that NPD1 might promote autophagy in N2a/APP695swe cells (Fig. 2C, D).

Fig. 2

NPD1 induced autophagy in N2a/APP695swe cells. A) Transmission electron microscopy images of each group. B) The mRNA level of Beclin1 in each group. C, D) Western blot analysis of Beclin1, LC3II/LC3I and P62 in each group. The data represent as mean±SEM of a typical series of 3 experiments. (# p > 0.05, versus APP group; ^*p < 0.001, versus WT group; ^**p < 0.001, versus APP group).

NPD1 suppressed the activation of GSK-3β in N2a/APP695swe cells

The results of real-time fluorescence quantitative PCR revealed that there was no significant difference in GSK-3β mRNA expression levels between the WT, APP, control and NPD1 groups (p > 0.05) (Fig. 3A). The GSK-3β-Tyr216/GSK-3β protein ratio was significantly raised in the APP and control groups (p < 0.001, p < 0.001), but significantly decreased in the NPD1 group (p < 0.001). There is no statistically significant difference between the APP and the Control groups (p > 0.05). (Fig. 3B, C). These findings showed that GSK-3 activation was enhanced in N2a/APP695swe cells, whereas NPD1 might suppress it.

Fig. 3

NPD1 suppressed the activation of GSK-3β in N2a/APP695swe cells. A) The mRNA level of GSK-3β in each group. B) Western blot analysis of the GSK-3β-Tyr216/GSK-3β protein in each group. The data represent as mean±SEM of a typical series of 3 experiments. (# p > 0.05, versus APP group; ^*p < 0.001, versus WT group; ^**p < 0.001, versus APP group).

Inhibition of GSK-3β reduced the Aβ₄₂ and hyperphosphorylated tau, meanwhile suppressed cell apoptosis in N2a/APP695swe cells

GSK-3β inhibitor SB216763 (5μM) and GSK-3β activator wortmannin (1μM) were grown with N2a/APP695swe to investigate the impact of GSK3β in an AD cell model. First, we used ELISA to quantify the amount of Aβ₄₂ secreted in cell culture (Fig. 4A). We discovered that the amount of Aβ₄₂ released in the media was substantially lower in the SB216763 group but higher in the wortmannin group compared to the APP group (p < 0.001, p < 0.01). There is no significant difference (p > 0.05) between the APP and control > groups. At the protein level, western blot was utilized to identify the expression of tau, phosphorylated tau and GSK-3β-Tyr216/GSK-3β (Fig. 4B-D). In comparison to the WT group, the ratio of Tau-Ser404/Tau proteins and GSK-3β-Tyr216/GSK-3β proteins was increased in the APP and control groups (p < 0.01, p < 0.01). The ratios of Tau-Ser404/Tau protein and GSK-3β-Tyr216/GSK-3β proteins were lower in the SB216763 group but greater in the wortmannin group (p < 0.01, p < 0.05) compared to the APP and control groups. Flow cytometry study indicated that the apoptosis rate of cells in the APP and control groups was considerably higher than in the N2a/WT cells (p < 0.01, p < 0.01), but no significant difference was detected between the APP and control groups (p > 0.05). The apoptotic rate of cells treated with SB217873 was lower than that of cells in the APP group (p < 0.001), but wortmannin administration increased the apoptotic rate of cells (p < 0.01) (Fig. 4E, F). According to the findings, inhibiting GSK-3β decreased A₄₂ and hyper phosphorylated Tau while suppressing cell death in N2a/APP695swe cells, but activating GSK-3β had the opposite effect.

Fig. 4

Inhibition of GSK-3β reduced the Aβ₄₂ and hyperphosphorylated tau, meanwhile suppressed cell apoptosis in N2a/APP695swe cells. A) The secreted Aβ₄₂ measured by ELISA. B-D) The expression of Tau-Ser404/Tau GSK-3β-Tyr216/GSK-3β at protein level. E, F) Flow cytometry showed the ratio of apoptotic cells in each group. The data represent as mean±SEM of a typical series of 3 experiments. (# p > 0.05, versus APP group; ## p < 0.001, versus APP group; ^*p < 0.05, versus WT group; ^**p < 0.01, versus APP group).

Inhibition of GSK-3β induced autophagy in N2a/APP695swe cells

Beclin1 mRNA levels were determined using qRT-PCR (Fig. 5A). The results indicated that the expression of Beclin1 at the mRNA level was lower in the APP and control groups (p < 0.01, p < 0.01) compared to the WT group. The expression of Beclin1 in the SB216763 group was significantly higher than in the APP group (p < 0.01), but wortmannin administration decreased it (p < 0.05). There was no statistically significant difference between the APP and control groups (p > 0.05). Beclin1 and LC3II/LC3I expression were also decreased in the N2a/APP695swe group compared to the WT group (p < 0.01, p < 0.01). SB216763 treatment significantly enhanced Beclin1 and LC3II/LC3I expression (p < 0.01, p < 0.01). In contrast, P62 expression was significantly higher in the N2a/aAPP695swe group than in the WT group (p < 0.01), while SB216763 significantly decreased P62 expression (p < 0.01). It has been proposed that NPD1 can promote autophagy in N2a/APP695swe cells. Furthermore, as compared to the APP group, the expression of Beclin1 and the ratio of LC3II/LC3I in the wortmannin group were considerably lower (p < 0.05), but P62 expression was significantly higher (p < 0.05) (Fig. 5B, C).

Fig. 5

Inhibition of GSK-3β induced autophagy in N2a/APP695swe cells. A) The mRNA level of Beclin1 in each group. (B, C) Western blot analysis of Beclin1, LC3II/LC3I and P62 in each group. The data represent as mean±SEM of a typical series of 3 experiments. (^#p > 0.05, versus APP group; ^# #p < 0.001, versus APP group; ^*p < 0.05, versus WT group; ^**p < 0.01, versus APP group).

According to the findings, blocking GSK-3 enhanced autophagy in N2a/APP695swe cells whereas activating GSK-3β inhibited it.

DISCUSSION

As the world’s population ages, AD has wreaked havoc on the physical and mental health of the elderly, becoming an increasingly major societal problem [21]. Drugs addressing a single aspect of AD, such as acetyl cholinesterase inhibitors for acetylcholine deficit and N-methyl-D-aspartate receptor antagonists for glutamate excitotoxicity, have failed to produce optimal therapeutic effects [22]. Many single-agent clinical studies have failed to influence disease development or symptoms [23]. As a result, several researchers have hypothesized that the mechanism of therapies for AD should operate on various stages of neurodegenerative cascades, resulting in synergistic effects [24]. As a result, looking for multi-target molecules has become a novel method to prevent and cure AD. In response to inflammation, NPD1 is quickly produced to enhance cell survival, neuroinflammatory signaling and transcription and homeostatic control. Although the role of NPD1 in cell signal transduction has garnered a lot of attention, few studies have found that NPD1 has a multi-target effect in AD. Our research looks at the role of NPD1 in AD cell models from a variety of angles.

Insoluble Aβ, which is produced by repeated cleavages of AβPP at the N- and C-terminal domains via β- and γ-secretases, is thought to represent a critical pathogenic event in AD [25]. Reducing Aβ production or encouraging Aβ degradation reduces the onset and progression of AD, as well as improving the cognitive level and learning and memory capacity of AD patients [26]. In AD, Aβ₄₂ usually forms pathology-associated plaques. In a previous study, we discovered that Aβ₄₂ levels were substantially higher in N2a/APP695swe cells than in N2a/WT cells in this work, we discovered that the amount of Aβ₄₂ secreted in the APP group was considerably greater than in the WT group. NPD1 significantly decreased Aβ₄₂ secretion in N2a/APP695swe cells. This is similar to the findings of Yuhai et al, who found that NPD1 can inhibit the expression of BACE1, a major secretory enzyme in the amyloid cascade, therefore decreasing the production of Aβ [16]. Another study found that NPD1 inhibits AβPP synthesis by activating secretase and inhibiting secretase via PPAR-, converting AβPP from an amyloidogenic route to a non-amyloidogenic one, and therefore NPD1 has anti-inflammation properties [14 , 28].

Tau protein is a microtubule-associated protein that plays a crucial role. Normally, just a few tau protein amino acid residues are phosphorylated. The function of tau protein changed after hyper phosphorylation, and this caused cell damage in a variety of ways, including affecting the activity of intracellular proteases [29], inhibiting the physiological function of tau protein, inhibiting the activity of microtubules and preventing the binding of normal microtubule-associated proteins to microtubules and can also promote Aβ Mitochondrial damage [30 –32]. In AD patients, the phosphorylation level of tau protein is unusually high, even 3–4 times greater than normal [33]. Tau-Ser404 is a key location in the paired helical filament, which is the primary fibrous component of neurofibrillary tangles and is mostly made up of improperly phosphorylated tau [34]. In our study, the ratio of Tau-Ser404/Tau proteins was higher in N2a/APP695swe cells than in N2a/WT cells, indicating that endogenous Aβ increases the hyper phosphorylation of tau protein. This is consistent with Zempel H’s [35] finding that Aβ may promote hyper phosphorylation of tau protein. The ratio of Tau-Ser404/Tau proteins was decreased after NPD1 treatment. The inclusion of DHA in the diet was found to lower levels of early-stage phospho-Tau epitopes, according to the literature [36]. However, this is the first study to show that NPD1 can decrease tau protein phosphorylation.

Apoptosis may be a key step in the neurodegenerative cascade of AD [37]. According to previous research, NPD1 can increase the expression of anti-apoptotic proteins Bcl-2, Bcl-xl and Bfl-1 while inhibiting the expression of pro-apoptotic factors Bax, Bad and Bik, therefore decreasing apoptosis triggered by Aβ₄₂ [24]. Our findings were similar: the apoptotic rate of N2a/APP695swe cells was substantially higher than that of N2a/WT cells, but following NPD1 therapy, it was significantly lower than that of the control group. The findings suggest that NPD1 might be involved in the production of Aβ₄₂, hyperphosphorylation of tau protein and death in N2a/APP695swe cells before playing a neuroprotective effect.

More and more research suggest that autophagy dysfunction has a role in the onset and progression of AD [38]. According to research, both A and phosphorylated tau protein must be efficiently destroyed by autophagy [39, 40]. Autophagy failure results in the build-up of a high quantity of Aβ and hyperphosphorylated tau protein, which causes neurotoxicity. Rapamycin therapy improves cognitive impairments and decreases degenerative damage induced by faulty tau protein in the APP transgenic mice model by boosting autophagy [41, 42]. TEM images show that NPD1 stimulates the development of autophagy. Meanwhile, we discovered an increase in the amount of autophagy-like structures after treating N2a/APP695swe cells with NPD1. This occurrence was further corroborated by western blots of LC3 (Microtubule-associated protein 1 light chain 2) and p62 (sequestosome 1). The protein LC3 is found on the autophagosome membrane. When autophagy is triggered, LC3I undergoes a series of ubiquitination events and converts into LC3II, which is widely employed as a marker to track the development of autophagy [43]. P62 is an autophagic degradation substrate. When LC3II rose and p62 declined, it indicated that autophagy activity was upregulated and substrate degradation could be completed. In this work, we discovered that NPD1 administration enhanced the expression of LC3II while decreasing the expression of p62. Beclin1 is a homologue of the autophagy-related gene Atg6, which is involved in the regulation of autophagy initiation [44]. Torres et al. discovered that 2-hydroxyDHA (HDHA) increases the expression of Beclin1 and other autophagy-related genes (Atg3, Atg5, Atg12, and Atg7), which consequently improves autophagy activity to restore cognitive function in AD model mice [45]. However, the effect of NPD1, another hydroxylated DHA derivative, on autophagy in an AD model has not been documented. Beclin1 expression was observed to be lower in N2a/APP695swe cells than in N2a/WT cells at both the mRNA and protein levels. Beclin1 expression was much higher in the NPD1 group than in the APP group.

To exercise its neuroprotective impact on AD, NPD1 can lower the amount of Aβ₄₂, prevent the excessive and aberrant phosphorylation of tau protein produced by the excessive synthesis of endogenous Aβ, reduce apoptosis and enhance autophagy. So, what mechanism is at work in the neuroprotective impact of npd1 on AD? GSK-3β is a serine/threonine kinase that is constitutively active [8]. Over activation of GSK-3β is associated with a variety of features of neural dysfunction, including deficits in neuronal architecture, plasticity, and survival; GSK-3β inactivation preserves neuronal polarity, survival and activity [46]. GSK-3β activity is abnormally elevated in the brains of AD patients, and the GSK-3β overexpression or aberrant activation may increase Aβ production [47, 48]. GSK-3β may potentially serve as a link between amyloid theory and neurofibrillary tangle hypothesis. Furthermore, the activity of GSK-3β is directly related to the activity of autophagy. Inhibiting the activity of GSK-3β has been demonstrated in studies to enhance autophagy activity and reduce the neuroinflammatory response induced by ischemic brain injury [49]. As a result, the pharmacological research goal of blocking GSK-3β may give a novel method for the treatment of AD. According to one study, DHA can reduce the growth of A549 lung cancer cells by blocking the AKT/GSK-3β/cyclin D1 signaling pathway [50]. However, no studies have been conducted to investigate the connection between NPD1 and GSK-3β activity in AD. The results of real-time fluorescence quantitative PCR revealed that there was no significant difference in GSK-3 mRNA expression levels between the WT, APP, control and NPD1 groups. The protein of GSK-3-Tyr216/GSK-3 was elevated in the APP and control groups but significantly reduced in the NPD1 group. These findings showed that GSK-3 activation was enhanced in N2a/APP695swe cells, whereas NPD1 might suppress it.

To determine if GSK-3β is involved in NPD1’s neuroprotective properties, we investigated the impact of blocking and activating GSK-3β on N2a/APP695swe cells. In the study, we discovered that after treatment with the GSK-3β specific inhibitor SB216863, the apoptosis rate of N2a/APP695swe cells was significantly lower than that of the APP group, the expression of A₄₂ and the level of phosphorylation Tau were also significantly reduced, and the autophagy activity was significantly improved. The opposite findings were observed after treatment with the GSK-3β activator wortmannin. As a result, inhibiting GSK-3β decreased A₄₂ and hyper phosphorylated tau while suppressing cell death and inducing autophagy in N2a/APP695swe cells. NPD1 inhibited GSK-3β activation, as well as A₄₂ and hyper phosphorylated tau, while also suppressing cell death and inducing autophagy in N2a/APP695swe cells. These findings show that NPD1 may have many neuroprotective effects on AD via decreasing GSK-3β activity.

In conclusion, our data show that NPD1 may exhibit several neuroprotective actions by decreasing GSK-3β activity, reducing Aβ₄₂ expression, lowering the degree of excessive and aberrant phosphorylation of Tau protein, invading apoptosis and enhancing autophagy (Fig. 6). Because of its multi-target activity as a natural inhibitor of GSK-3β, NPD1 is predicted to be an ideal medication for the prevention and treatment of AD, but additional study into its mechanism is needed.

Fig. 6

Schematic diagram of the NPD1 possible mechanisms.

Footnotes

ACKNOWLEDGMENTS

Thanks to all the members of our research team for the constructive discussion and suggestions that shaped this work. This work was supported by the National Natural Science Foundation of China (NSFC: 81671261) and the Natural Science Foundation of Chongqing (No. cstc2017jcyjAX0050).

Authors’ disclosures available online ().

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NPD1 Enhances Autophagy and Reduces Hyperphosphorylated Tau and Amyloid-β 42 by Inhibiting GSK3β Activation in N2a/APP695swe Cells

Abstract

Background:

Objective:

Methods:

Results:

Conclusion:

Keywords

INTRODUCTION

MATERIALS AND METHODS

Cell culture

Medicine treatment

Cell apoptosis assessment by flow cytometry

Aβ ELISA

Western blot analysis

Transmission electron microscopy

RNA isolation and quantitative real-time PCR analysis

Statistical analysis

RESULTS

NPD1 reduced the Aβ42 and hyperphosphorylated tau, meanwhile suppressed cell apoptosis in N2a/APP695swe cells

NPD1 induced autophagy in N2a/APP695swe cells

NPD1 suppressed the activation of GSK-3β in N2a/APP695swe cells

Inhibition of GSK-3β reduced the Aβ42 and hyperphosphorylated tau, meanwhile suppressed cell apoptosis in N2a/APP695swe cells

Inhibition of GSK-3β induced autophagy in N2a/APP695swe cells

DISCUSSION

Footnotes

ACKNOWLEDGMENTS

References

NPD1 reduced the Aβ₄₂ and hyperphosphorylated tau, meanwhile suppressed cell apoptosis in N2a/APP695swe cells

Inhibition of GSK-3β reduced the Aβ₄₂ and hyperphosphorylated tau, meanwhile suppressed cell apoptosis in N2a/APP695swe cells