A Multifunctional (-)-Meptazinol-Serotonin Hybrid Ameliorates Oxidative Stress-Associated Apoptotic Neuronal Death and Memory Deficits via Activating the Nrf2/Antioxidant Enzyme Pathway

The pathogenesis of Alzheimer's disease (AD) involves multiple pathophysiological processes. Oxidative stress is a major cause of AD-associated neuronal injury. The current research was designed to examine whether a novel (-)-meptazinol-serotonin hybrid (Mep-S) with potent antioxidant activity and additional inhibitory properties for acetylcholinesterase (AChE) activity could attenuate oxidative neuronal damage and cognitive deficits. In human SH-SY5Y cells, Mep-S suppressed H2O2-induced apoptosis by restoring mitochondrial membrane potential and inhibiting caspase-3 activation. Meanwhile, it attenuated oxidative stress elicited by H2O2 through lessening generation of reactive oxygen species as well as enhancing production of glutathione (GSH) and activity of superoxide dismutase (SOD). Mechanistically, Mep-S promoted nuclear translocation of a transcription factor nuclear factor E2-related factor-2 (Nrf2) in H2O2-challenged cells. This effect was accompanied by reduction in Kelch-like ECH-associated protein-1 (Keap1) levels as well as augmentation of Akt phosphorylation and expression of heme oxygenase-1 (HO-1) and NAD(P)H quinine oxidoreductase-1 (NQO-1). Molecular docking analysis revealed that Mep-S may disrupt the protein-protein interactions between Keap1 and Nrf2. In an in vivo mouse model, Mep-S attenuated scopolamine-caused cognitive deficits with inhibition of apoptotic neuronal death and brain AChE activity. Furthermore, the scopolamine-induced impairment of total antioxidant capacity and reduction in SOD1, SOD2, and γ-glutamate-cysteine ligase expression in the brain were counteracted by Mep-S, accompanied by decreased Keap1 levels, increased Akt catalytic subunit and Nrf2 phosphorylation, and decreased Nrf2, HO-1, and NQO-1 expression. Collectively, our results suggest that Mep-S ameliorates apoptotic neuronal death and memory dysfunction associated with oxidative stress by regulating the Nrf2/antioxidant enzyme pathway through inactivating Keap1 and phosphorylating Nrf2 via Akt activation. Therefore, Mep-S may be a potential lead for multitarget neuroprotective agents to treat AD-like symptoms.


Introduction
Alzheimer's disease (AD), predominantly affecting the elderly people and causing progressive cognitive decline, involves a multifactorial pathogenesis. The primary neuropathological hallmarks in AD brains include the presence of extracellular amyloid-β (Aβ) deposition as senile plaques, intracellular accumulation of hyperphosphorylated tau as neurofibrillary tangles, and loss of cholinergic neurons [1]. Currently, no disease-modifying treatment has been established. To date, FDA-approved drugs for AD therapy include inhibitors of acetylcholinesterase (AChE) or N-methyl-Daspartate receptors [2][3][4]. Although benefiting for cognitive symptoms, they fail to prevent pathological processes of the disease [1,5]. Notably, the Aβ-directed antibody aducanumab received accelerated approval from the FDA in 2021; however, its efficacy for AD is controversial [6]. Since the increasing AD population may pose a significant burden on family and public health, it is urgent to develop innovative disease-modifying medications for this disease.
AD pathogenesis involves multiple pathological processes that are closely linked and interact [1]. Substantial evidence shows that oxidative stress, caused by reactive oxygen species (ROS) overproduction, is an early and sustained event in AD brain [7][8][9]. In addition to directly causing neuronal damage (particularly in cognition-related areas, e.g., the hippocampus), oxidative stress may aggravate Aβ and tau pathology [9,10]. Therefore, oxidative neuronal damage is considered a major factor associated with both the onset and progression of AD pathogenesis. Evidence shows that agents with antioxidant activity could benefit for AD therapy [11][12][13].
The multifactorial nature of AD warrants multitarget intervention for the disease. To this end, the exploration of multi-target-directed ligands (MTDLs) has become a hotspot in the development of new therapies for AD [14][15][16][17]. Considering the critical roles of oxidative stress in AD pathogenesis, MTDLs with antioxidant potency may be favorable for AD therapy. In fact, the development of anti-AD MTDLs with antioxidant activity has recently attracted much attention [15,18]. In a previous research, we reported a number of (-)-meptazinol-melatonin/serotonin hybrids with multiple pharmacological properties [19]. The results of in vitro testing demonstrate that a novel (-)-meptazinol-serotonin hybrid, Mep-S (Figure 1(a)), in addition to inhibiting AChE activity, exhibits high antioxidant potency (7.2-fold of Trolox). Additionally, it displays a favorable effect on neuronal cell viability and shows sufficient blood-brain barrier penetrability. Thus, this compound could serve as a potential MTDL lead for AD therapy.
In this work, we examined whether Mep-S could ameliorate oxidative neuronal injury by in vitro and in vivo testing. The results showed that Mep-S protects against H 2 O 2induced neuronal apoptosis and ameliorates scopolaminecaused cognitive deficits. Further mechanistic studies revealed that the nuclear factor E2-related factor-2 (Nrf2)/ antioxidant enzyme pathway was associated with the neuroprotective effect of Mep-S.
2.2. Animals. ICR mice (male, weighing 25-30 g; Shanghai Laboratory Animal Center, Chinese Academy of Science, Shanghai, China) were kept under a light/dark cycle of 12 h/12 h in a room with constant temperature, with free access to water and food. The experimental procedures were performed following principles of the Declaration of Helsinki. The Ethics Committee of Shanghai Jiao Tong University School of Medicine approved them.

Cell
Culture. SH-SY5Y human neuroblastoma cells were cultured in a DMEM/F-12 mixture containing fetal bovine serum (FBS, 10%; Sigma), L-glutamine (2 mM; Gibco), penicillin (100 U/ml), and streptomycin (100 μg/ml) at 37°C. They were used for experiments after growing to the required confluence, with culture medium changed to serum-free medium prior to addition of drugs.
2.4. Detection of Cell Viability. Cell viability was assessed with a Cell Counting Kit-8 (CCK-8; Dojindo, Kumamoto, Japan). After being seeded in 96-well plates at 2 × 10 4 cells per well and growing to the required confluence, SH-SY5Y cells received 24 h exposure to H 2 O 2 with or without preincubation with Mep-S or NAC (serving as a positive control of antioxidants) for 1 h. Thereafter, the cells were incubated with CCK-8 (20 μl, 5 mg/ml) at 37°C for 2 h. Cell viability, which was evaluated based on the absorbance measured with a test wavelength (570 nm) and a reference wavelength (655 nm), was normalized to the percentage of control group.
2.5. Detection of Lactate Dehydrogenase (LDH) Release. The cell-released LDH content was detected using an LDH cytotoxicity assay kit (Beyotime Institute of Biotechnology, Shanghai, China) following instructions of the manufacturer. In brief, the cells were subjected to 24 h stimulation with H 2 O 2 with or without preincubation with Mep-S or NAC. Then, the culture medium (120 μl) was aspirated to mix with LDH test working solution (60 μl). The absorbance was detected at a wavelength of 490 nm after 30 min incubation at room temperature.
2.6. Annexin V/PI Staining. Neuronal apoptosis was measured by flow cytometry using a commercial FITC Annexin V Apoptosis Detection Kit (BD Pharmingen, Franklin Lakes, NJ, USA). In brief, the cells, which were seeded in 6-well plates at 5 × 10 5    Oxidative Medicine and Cellular Longevity 2.9. Detection of Glutathione (GSH) Content and Superoxide Dismutase (SOD) Activity. A GSH assay kit (Abbkine Scientific Co., Ltd, Wuhan, China) and SOD assay kit (Beyotime) were applied for detection of GSH content and SOD activity, respectively. The cells received 24 h H 2 O 2 stimulation with or without preincubation with Mep-S or NAC. Afterwards, the supernatants of cell homogenates were collected and used for assay of GSH content and SOD activity.
2.10. Molecular Docking. Molecular docking simulation was conducted to explore the potential impact of Mep-S on Keap1-Nrf2 protein-protein interactions (PPI). The X-ray crystal structure of mouse Keap1 Kelch domain (at 1.21 Å resolution), which serves as the recognition module for Nrf2 [20,21], was acquired from the Protein Data Bank (code: 6ZF4). Water molecules and heteroatoms in protein structures were discarded before addition of hydrogen atoms. The 3D coordinates of Mep-S (serving as the ligand) were obtained using CORINA Version 3.0 (Molecular Networks GmbH, Erlangen, Germany). After acquirement of the final ligand conformations by energy minimization with Tripos force field, molecular docking simulation was conducted with AutoDock Vina 1.1.2 to obtain an ensemble of docked conformations. The best conformation was chosen according to the lowest docked energy.
2.11. Design of Animal Experiments. Mice were randomized into 6 groups (n = 10). The control animals were given intraperitoneal (i.p.) injection of saline per day while the scopolamine-treated animals were daily challenged with scopolamine (2 mg/kg) 15 min before the behavioral test. The scopolamine+Mep-S-treated groups received a daily injection of Mep-S at 0.5, 1, 2, and 5 mg/kg, respectively, 15 min prior to the delivery of scopolamine. On days 1-6, the Morris water maze test was performed for behavioral assessment. In a separate experiment, 4 groups of mice received a daily injection of saline and Mep-S (at 0.5, 2, and 5 mg/kg), respectively, prior to the injection of scopolamine for 6 days. The control group received daily saline injection. The mice were transcardially perfused with saline after euthanasia 24 h after the last drug administration. After removal of the brain, one hemisphere was immediately postfixed for 2 h in 10% formalin solution and then subjected to paraffin embedding and sectioning for histological analyses. The other hemisphere was immediately stored at -80°C for subsequent quantitative real-time polymerase chain reaction (qPCR), western blot, and biochemical analyses.
2.12. Morris Water Maze Test. The Morris water maze test was performed as previously described [22]. Briefly, mice were placed in the experimental room to adapt to the environment 24 h before the testing. The testing was conducted in a black circular tank with a diameter of 140 cm, which contained water (22 ± 1:0°C) to a 30 cm depth. During the 5-day acquisition testing, each mouse received 4 trials daily. The interval between trials was 30 s. The test animals that were able to find the submerged platform (9 cm diameter, located on the center of a fixed quadrant) within 60 s were allowed to stay for 30 s on it. Animals failing to find the platform within 60 s were guided to stay for 30 s on it. During the probe test on day 6, retention of spatial memory was assessed, with the platform being removed. The test animals were allowed to swim for 60 s. Swimming traces were analyzed to evaluate spatial learning capacity.

Hematoxylin and Eosin (H&E)
Staining. Serial coronal sections of mouse brains were deparaffinized and rehydrated. After hematoxylin and eosin staining, the histopathological properties were evaluated under a Leica DFC 320 digital camera.
2.18. Detection of Total Antioxidant Capacity. Mouse brain samples were homogenized and centrifuged at 12,000 rpm for 5 min at 4°C. Total antioxidant capacity was detected with a commercial kit (Beyotime) following the instructions of the manufacturer.

Statistical
Analysis. Experimental data were presented as the mean ± standard error of the mean (SEM) and statistically analyzed with analysis of variance (ANOVA; one-way or two-way) and Bonferroni's posttest. A P value of less than 0.05 was regarded as statistically significant.

Protection of Mep-S against H 2 O 2 -Evoked Apoptotic
Neuronal Death. We then examined neuronal apoptosis to further explore the protective effect of Mep-S (at 1 μM, the concentration at which Mep-S showed the most obvious effect) on H 2 O 2 -challenged SH-SY5Y cells. Based on the results of Annexin V/PI staining in SH-SY5Y cells (oneway ANOVA, Fð3, 8Þ = 10:17, P = 0:0042; Figure 2(a)), the apoptotic rate of cells challenged with H 2 O 2 increased by 1.47-fold relative to control group (P < 0:01). Pretreatment with Mep-S or NAC markedly decreased the cellular apoptotic rate compared with H 2 O 2 -treated group (P < 0:05).

Mep-S Activates the Nrf2/Antioxidant Enzyme Signaling
Axis in H 2 O 2 -Challenged Cells. Evidence shows that Keap1/ Nrf2 axis is crucial for protection of neurons against oxidative injury by inducing antioxidant enzyme (e.g., HO-1 and NQO-1) expression to suppress the intracellular ROS level [24]. We therefore explored the effect of Mep-S on this pathway. As shown in Figure 4  Oxidative Medicine and Cellular Longevity nuclear Nrf2 level decreased after H 2 O 2 stimulation compared with control group (P < 0:05 or P <0.01). Mep-S or NAC pretreatment markedly inhibited the H 2 O 2 -induced reduction in HO-1, NQO-1, and nuclear Nrf2 levels as well as elevation of Keap1 and cytoplasmic Nrf2 levels compared to H 2 O 2 -treated group (P < 0:05). Phosphatidylinositol-3-kinase (PI3K)/Akt pathway is an important upstream regulator of Nrf2 signaling that promotes Nrf2 phosphorylation and nuclear translocation [25][26][27]. We found that Akt phosphorylation in H 2 O 2 -stimulated cells was overtly lower (P < 0:01 vs. control group). Mep-S or NAC pretreatment significantly suppressed this effect compared with H 2 O 2 -challenged group (P < 0:05).
It has been suggested that small-molecule regulators can enhance Nrf2 signaling through directly binding with Keap1 or Nrf2 to impair Keap1-Nrf2 PPI [28,29]. We next performed molecular docking analysis to investigate whether Mep-S could affect PPI between Keap1 and Nrf2. As demonstrated in Figure 4 , during the probe test on the 6th day, scopolamine-treated mice displayed markedly decreased proportions of swimming distance and time in target quadrant (P < 0:01 vs. control animals). In mice receiving Mep-S (2 or 5 mg/kg) pretreatment, the proportions of swimming distance and time in target quadrant were significantly higher (P < 0:05 vs. scopolaminechallenged mice). The average swimming speeds of mice on day 6 showed no significant difference between groups ( Figure 5(f)). Mice did not show thigmotaxis during the Morris water maze test (data not shown).

Mep-S Attenuates Neuronal Apoptosis and Inhibits
AChE Activity in Scopolamine-Challenged Mice. We subsequently detected whether Mep-S could attenuate scopolamine-induced neuronal death in brain. H&E staining

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Oxidative Medicine and Cellular Longevity revealed markedly lower amount of surviving neuronal cells in CA1 and CA3 regions of hippocampus in mice receiving scopolamine challenge (P < 0:01 vs. control mice). Pretreatment with Mep-S at 2 and 5 mg/kg markedly inhibited the scopolamine-induced decrease in surviving neuronal number in mouse hippocampus (Figure 6(a)). We then examined the expression of some antiapoptotic and apoptotic markers. It was found that scopolamine reduced expression of the antiapoptotic protein Bcl-2 and enhanced expression of the apoptotic protein Bax, leading to decreased Bcl-2 to Bax ratios in mouse hippocampus and cortex (P < 0:01 vs. the control animals). Mep-S pretreatment resulted in enhanced Bcl-2 levels and reduced Bax levels, leading to increased Bcl-2 to Bax ratios in the hippocampus (2 mg/kg: by 0.81-fold, P < 0:05; 5 mg/kg: by 1.53-fold, P < 0:01) and cortex (2 mg/kg: by 0.76-fold, P < 0:05; 5 mg/kg: by 1.16-fold, P < 0:01) of mice relative to those in mice receiving scopolamine alone (Figure 6(b)). These results indicate that Mep-S attenuated scopolamine-induced neuronal apoptosis in vivo. We also measured AChE activity in mouse brains. Brain AChE activity showed obviously between-group difference (one-way ANOVA, Fð4, 15Þ = 13:00, P < 0:0001; Supplementary material, Figure S2). AChE activity in brain tissues markedly elevated in mice challenged with scopolamine (P < 0:01 vs. control group). Brain AChE activity in mice pretreated with Mep-S was markedly lower compared to that of mice receiving scopolamine alone (P < 0:05).

Antioxidant Effect of Mep-S via Activating the Nrf2/ Antioxidant Enzyme Pathway in Scopolamine-Challenged
Mice. We next detected the effect of Mep-S on brain oxidative status in scopolamine-treated mice. As shown in Figure 7, the total antioxidant capacity along with mRNA levels of SOD1, SOD2, and GCLC in brain tissues markedly declined in scopolamine-treated mice compared with the control mice. Mep-S markedly inhibited the scopolamineinduced effects (P < 0:05). Moreover, levels of HO-1, NQO-1, and phosphorylated Akt and Nrf2 in the hippocampal and cortex regions were lower in scopolaminechallenged mice than in control mice (P < 0:05). Mep-S at 5 mg/kg resulted in elevated levels of these proteins in mouse hippocampal and cortex regions (P < 0:05 vs. mice receiving scopolamine alone). Mep-S at 2 mg/kg led to elevated levels of HO-1 and Nrf2 in the hippocampus and cortex (P < 0:05 vs. mice receiving scopolamine alone). Scopolamine did not obviously affect Keap1 and Nrf2 levels. However, mice receiving Mep-S (2 and 5 mg/kg) treatment showed decreased Keap1 levels and increased Nrf2 expression in mouse hippocampal and cortex regions relative to mice receiving scopolamine alone (Figure 8). The results of immunofluorescence staining showed that scopolamine decreased Nrf2 immunoreactivity in mouse hippocampal region, while Mep-S (5 mg/kg) overtly counteracted this effect (Supplementary material, Figure S3).

Discussion
AD is a neurodegenerative disease associated with multiple etiological factors. As oxidative stress is a key mechanism for AD-associated neuronal injury [7], pharmacological interventions targeting oxidative neurotoxicity may be an important strategy for AD treatment. Here, we demonstrate that Mep-S, a novel multifunctional (-)-meptazinol-serotonin hybrid with the potential for Keap1-Nrf2 PPI inhibition, attenuates apoptotic neuronal death and memory dysfunction associated with oxidative stress. These effects may be exerted through activating Nrf2/antioxidant enzyme axis. Therefore, Mep-S may be a potential MTDL lead with antioxidant activity for the intervention of AD-like symptoms. The brain, with high oxygen consumption, is especially at risk of oxidative stress [30]. ROS (including superoxide, hydroxyl radicals, H 2 O 2 , and singlet oxygen) are regarded as key factors for oxidative pathology [26,31,32]. In The Nrf2/antioxidant enzyme pathway is essential for cells to prevent oxidative injury [27,33]. Physiologically, intracellular Nrf2 level is controlled by the cytosolic repressor Keap1, which can promote ubiquitination and the following proteasomal degradation of Nrf2 via PPI between Keap1 and Nrf2. During the oxidative stress-triggered adaptive responses, Keap1 is inactivated due to a conformational change, facilitating Nrf2 dissociation from the Keap1-Nrf2 complex and nuclear translocation to bind to the antioxidant response element (ARE) and transcriptionally induce expression of antioxidant enzymes [34,35]. However, overproduction of ROS may cause deregulation of redox balance and, thus, hinder nuclear translocation of Nrf2, resulting in impaired antioxidant defense responses [15,36,37]. Accordingly, enhancement of Keap1/Nrf2 signaling is reported to protect against AD-associated neurodegeneration and memory dysfunction by restoring redox homeostasis, and thus, this pathway may be an attractive drug target for AD treatment [24,38,39]. In addition, activation of this pathway contributes to the neuroprotective effect of bioactive agents in animal models of other neurological disorders, such as Parkinson's disease and traumatic brain injury [40,41]. To get a deeper understanding of the mechanisms for neuroprotection of Mep-S, we explored whether Mep-S influences this pathway. We found that in H   Scopolamine can easily enter into the brain to block muscarinic acetylcholine receptors. It has been applied to elicit memory dysfunction in AD-associated animal experiments. It induces cholinergic dysfunction and increases Aβ and tau deposition in the central nervous system [42][43][44]. It also causes mitochondrial dysfunction and neuroinflammation [44]. Moreover, it causes oxidative stress and promotes neuronal apoptosis [15,44,45]. Therefore, scopolamine has been applied in animal models to elicit AD-like symptoms involving both cholinergic dysfunction and oxidative stress [15,18]. We first performed behavioral experiments using the Morris water maze test to detect the effect of Mep-S in scopolamine-challenged mice. It was found that scopolamine-treated mice displayed cognitive dysfunction with impaired acquisition and retention of spatial memory, while Mep-S protected against scopolamineinduced cognitive deficits. Further experiments revealed that Mep-S improved the survival of hippocampal neurons and increased Bcl-2 to Bax ratios (an antiapoptotic index) in the hippocampal and cortex regions of scopolaminechallenged mice, indicating that the effect of Mep-S on scopolamine-induced AD-like symptoms may be associated with its protection against neuronal apoptosis.
We next examined whether the antioxidant activity of Mep-S could have a role in its in vivo effect. The total antioxidant capacity as well as expression of SOD1, SOD2, and GCLC (the rate-limiting enzyme for GSH synthesis [46]) in mouse brains decreased after scopolamine exposure, while Mep-S suppressed the effect of scopolamine. These results indicate that Mep-S could provide protection against scopolamine-induced oxidative neurotoxicity. To investigate the underlying mechanisms, the effect of Mep-S on the Nrf2/

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Oxidative Medicine and Cellular Longevity antioxidant enzyme pathway was tested. As expected, Mep-S increased HO-1 and NQO-1 expression along with the levels of Nrf2 and phosphorylated Akt and Nrf2, accompanied with reduced Keap1 levels in the hippocampal and cortex regions of scopolamine-challenged mice. Collectively, the above findings indicate that the nootropic effect of Mep-S in the scopolamine-elicited cognitive dysfunction model may involve neuroprotection against oxidative stress. We also observed that Mep-S suppressed scopolaminepromoted AChE activity in mouse brains. Notably, evidence suggests an association between brain AChE activity and oxidative stress [17,47]. Therefore, the inhibition of AChE activity by Mep-S could also contribute to neuroprotective and nootropic effects by attenuating oxidative neurotoxicity in the brains of scopolamine-treated mice, in addition to improving neurotransmission by elevating acetylcholine levels in the synaptic cleft. Our research has some limitations. We only used male mice to explore the protection of Mep-S. Notably, both sexes are appropriate in AD research, and female animals may be more prominent since AD shows a higher incidence in women [48,49]. Evidence shows that male and female animals exhibit similar variability of proxies for physiological and neurological outputs across multiple timescales [50]. In contrast, recent studies demonstrate that cognitive impairment of AD animals were detected in males but not in females [51,52]. Therefore, sex differences should also be considered in the exploration of neuroprotective mechanisms. Since our results cannot reflect drug effect in females, future research is needed to investigate the potential of Mep-S for AD therapy in female AD models. In addition, although scopolamine have been used to induce memory dysfunction in animal models [15,18], the pathological changes and cognitive impairment caused by scopolamine may not precisely mimic AD pathology and thus are not specific for AD. In other words, it only induces AD-like pathology and symptoms. Researchers are using transgenic AD animal models to evaluate the therapeutic effect of MTDLs [53]. Therefore, future studies using transgenic AD animal models are warranted.
Collectively, this work demonstrates that Mep-S, a potential Keap1-Nrf2 PPI blocker, attenuates apoptotic neuronal death and scopolamine-elicited memory dysfunction associated with oxidative stress. These effects could be exerted via enhancement of Nrf2/antioxidant enzyme signaling by promoting separation of Nrf2 from Keap1 by inactivating Keap1 and phosphorylation of Nrf2 by activating Akt (Figure 9). Additionally, it inhibits brain AChE activity to improve neurotransmission impairment and oxidative neuronal injury in scopolamine-treated mice. Therefore, Mep-S may be a potential lead for multitarget neuroprotective agents to treat AD-like symptoms.

Data Availability
Data in support of the observations of this work were included in the article and the supplementary files.

Ethical Approval
This study was performed following the principles of the Declaration of Helsinki and approved by the Ethics Committee of Shanghai Jiao Tong University School of Medicine (protocol code A-2019-039).