Nicotinamide Ameliorates Amyloid Beta-Induced Oxidative Stress-Mediated Neuroinflammation and Neurodegeneration in Adult Mouse Brain

Alzheimer’s disease (AD) is the most predominant age-related neurodegenerative disease, pathologically characterized by the accumulation of aggregates of amyloid beta Aβ1–42 and tau hyperphosphorylation in the brain. It is considered to be the primary cause of cognitive dysfunction. The aggregation of Aβ1–42 leads to neuronal inflammation and apoptosis. Since vitamins are basic dietary nutrients that organisms need for their growth, survival, and other metabolic functions, in this study, the underlying neuroprotective mechanism of nicotinamide (NAM) Vitamin B3 against Aβ1–42 -induced neurotoxicity was investigated in mouse brains. Intracerebroventricular (i.c.v.) Aβ1–42 injection elicited neuronal dysfunctions that led to memory impairment and neurodegeneration in mouse brains. After 24 h after Aβ1–42 injection, the mice were treated with NAM (250 mg/kg intraperitoneally) for 1 week. For biochemical and Western blot studies, the mice were directly sacrificed, while for confocal and “immunohistochemical staining”, mice were perfused transcardially with 4% paraformaldehyde. Our biochemical, immunofluorescence, and immunohistochemical results showed that NAM can ameliorate neuronal inflammation and apoptosis by reducing oxidative stress through lowering malondialdehyde and 2,7-dichlorofluorescein levels in an Aβ1–42-injected mouse brains, where the regulation of p-JNK further regulated inflammatory marker proteins (TNF-α, IL-1β, transcription factor NF-kB) and apoptotic marker proteins (Bax, caspase 3, PARP1). Furthermore, NAM + Aβ treatment for 1 week increased the amount of survival neurons and reduced neuronal cell death in Nissl staining. We also analyzed memory dysfunction via behavioral studies and the analysis showed that NAM could prevent Aβ1–42 -induced memory deficits. Collectively, the results of this study suggest that NAM may be a potential preventive and therapeutic candidate for Aβ1–42 -induced reactive oxygen species (ROS)-mediated neuroinflammation, neurodegeneration, and neurotoxicity in an adult mouse model.


Introduction
Alzheimer's disease (AD), a dementing neurodegenerative disease, is a global health problem that has affected millions of individuals globally, and its prevalence continues to grow [1]. The disease is neuropathologically characterized by the accumulation of insoluble hyperphosphorylated tau proteins (intracellularly, resulting in tangle formation) and amyloid-β (Aβ) peptides (extracellularly, resulting in neuritic-plaque formation) [2]. Amyloid beta (Aβ), with 40-42 amino acid peptides, is produced by the proteolytic processing of amyloid precursor protein (APP), which is considered to be the main cause of AD [3].
of Biology, Gyeongsang National University, Korea (Approval ID: 125). We used male mice in this study because they are resistant to stress, tough ecological conditions, and hormonal alteration [23].
Rompun TM (xylazine) and 0.1 mL/100 g body weight Zoletil TM (ketamine). The surgical procedure was stereotaxically arranged in a separate heated room by designing the heating system in such a way as to control body temperature (maintained at 36-37 • C). Using a thermometer, temperature was regularly examined because the anesthesia reduced the animals' body temperature. Mice were divided after 24 h from Aβ 1-42 and vehicle i.c.v. injection into groups: (1) control (C) mice injected i.c.v. with 0.9% saline as a vehicle, (2) mice injected i.c.v. with Aβ 1-42 (Aβ 1-42 group), and (3) mice injected with Aβ 1-42 and NAM (via I.P route) NAM + Aβ 1-42 (250 mg/kg for 1 week); dosages of NAM were selected following previously published studies [17]. The NAM-alone group was not examined in the current study, as no ill effects of NAM have previously been reported in the brain [17,23]. Fresh NAM was prepared on daily basis in a normal saline solution followed by the needed volume of injection, and it was injected into the mice for treatment. The treatment schedule is explained in Figure 1.
protocols of the Animal Ethics Committee of the Division of Applied Life Sciences, Gyeongsang National University, Korea (approval ID: 125, animal ethics code: 200331-M0020, 3 June 2020). Animals were adjusted in the university animal house to a 12 h light/dark cycle at 23-25 °C with 60 ± 10% humidity, and food and water were provided in a standard way. Experimental procedures were conducted in accordance with the Animal Ethics Committee (IACUC) of the Division of Applied Life Science, Department of Biology, Gyeongsang National University, Korea (Approval ID: 125). We used male mice in this study because they are resistant to stress, tough ecological conditions, and hormonal alteration [23].

Drug Treatment Protocol
A stock solution of human Aβ1-42 peptide was prepared with a specific concentration of 1 mg/mL in sterile saline solution, followed by aggregation via incubation at 37 °C for 4 days, where Intracerebroventricular (i.c.v.) administration of the Aβ1-42 peptide or vehicle (0.9% NaCl, 3 µL/5 min/mouse) was stereotaxically performed using a Hamilton microsyringe (0.2 mm anteroposterior (AP), 1 mm mediolateral (ML), and 2.4 mm dorsoventral (DV) to Bregma) in anesthetic state, homogenizing with 0.05 mL/100 g body weight.
Rompun TM (xylazine) and 0.1 mL/100 g body weight Zoletil TM (ketamine). The surgical procedure was stereotaxically arranged in a separate heated room by designing the heating system in such a way as to control body temperature (maintained at 36-37 °C). Using a thermometer, temperature was regularly examined because the anesthesia reduced the animals' body temperature. Mice were divided after 24 h from Aβ1-42 and vehicle i.c.v. injection into groups: (1) control (C) mice injected i.c.v. with 0.9% saline as a vehicle, (2) mice injected i.c.v. with Aβ1-42 (Aβ1-42 group), and (3) mice injected with Aβ1-42 and NAM (via I.P route) NAM + Aβ1-42 (250 mg/kg for 1 week); dosages of NAM were selected following previously published studies [17]. The NAM-alone group was not examined in the current study, as no ill effects of NAM have previously been reported in the brain [17,23]. Fresh NAM was prepared on daily basis in a normal saline solution followed by the needed volume of injection, and it was injected into the mice for treatment. The treatment schedule is explained in Figure 1.

Morris Water Maze (MWM) Test
The behavioral pattern of mice was examined by dividing the mice into 3 groups, n = 6 for each. The experimental apparatus was a circular water tank (100 cm in diameter, 40 cm in height) with water (23 ± 1 °C) to a depth of 15.5 cm, and white ink was dissolved to make it opaque. The transparent escape platform (10 cm in diameter, 20 cm in height) was made invisible at 1 cm from the water surface and kept at the midpoint of one quadrant. Every mouse had training once per day for 5 consecutive days using a single hidden platform in one quadrant with three rotating quadrants. For each trial, latency to escape

Morris Water Maze (MWM) Test
The behavioral pattern of mice was examined by dividing the mice into 3 groups, n = 6 for each. The experimental apparatus was a circular water tank (100 cm in diameter, 40 cm in height) with water (23 ± 1 • C) to a depth of 15.5 cm, and white ink was dissolved to make it opaque. The transparent escape platform (10 cm in diameter, 20 cm in height) was made invisible at 1 cm from the water surface and kept at the midpoint of one quadrant. Every mouse had training once per day for 5 consecutive days using a single hidden platform in one quadrant with three rotating quadrants. For each trial, latency to escape from the water maze (finding the invisible escape platform) was examined after 24 h. On Day 5, for the assessment of memory consolidation, a probe test was performed by removing the invisible platform and freely allowing each mouse to swim for 60 s. The total time that the mice spent in the target quadrant and the number of crossings over the platform site (where the platform was kept during hidden platform training) were measured. Time spent in the target quadrant signified the degree of memory consolidation. Data were documented using video tracking software (SMART, Panlab Harvard Apparatus; Bioscience Company, Holliston, MA, USA).

Y-Maze Test
The Y maze was composed of black-painted wood, with each arm being 50 cm long, 20 cm high, and 10 cm wide at the bottom and 10 cm wide at the top. The mice were kept at the center of the apparatus one by one and were allowed to move freely through the maze during three 8 min sessions. Visual observation of the series of arm entries was examined. The successive entrance of the mice into the three arms in overlapping triplet sets is called spontaneous alteration. The alteration behavior percentage (%) was measured as (successive triplet sets (entrance into three different arms consecutively)/total number of arm entries-2) × 100. Spontaneous alternation behavior with a higher percentage was thought to enhance cognitive performance.

Protein Extraction from Mouse Brain
After behavioral studies, the mice were anesthetized and euthanized using a combination of ketamine and xylazine. Brains were directly removed, the cortex and hippocampus were carefully dissected, and the tissue was stored at −80 • C. Both hippocampus and cortical tissue samples were homogenized in PRO-PREP TM protein extraction solution according to the manufacturer's instructions (iNtRON Biotechnology, Inc., Sungnam, Korea). Centrifugation was performed at 10,000 rpm at 4 • C for 25 min, and supernatants were collected and stored at −80 • C.

Western Blot Analysis
Western blotting was conducted to measure different protein expressions in the hippocampal and cortical regions. Protein concentration was calculated using a Bio-Rad protein assay kit (Bio-Rad Laboratories, Irvine, CA, USA). Proteins (20-25 µg) were electrophoresed in equal amounts using 4-12% Bolt TM Mii Gels (Thermo Fisher Scientific Inc., Waltham, MA, USA) and SDS (sodium dodecyl sulphate (Merck KGaA, Darmstadt, Germany) running buffer 1 × (Novex, Life Technologies). Protein ladders (GangNam-STAIN™, iNtRON Biotechnology Inc., Sungnam, Korea) with a broad range of molecular-weight levels were applied for the detection of proteins' molecular weights. All membranes were blocked in 5% (w/v) skim milk and further incubated with primary antibodies (1:1000 dilution) overnight at 4 • C to avoid nonspecific bindings. Membranes having primary antibodies were allowed to react with horseradish peroxidase-conjugated (HRP) secondary antibodies. Using ECL (Enhanced chemiluminescent) detection reagents (ATTO Corporation, Tokyo, Japan), proteins were identified to avoid extra bindings, which were further followed by scanning the X-ray films, and the optical densities of the bands were examined with densitometry using Sigma gel software, version 1.0 (SPSS, Chicago, IL, USA).

ROS Detection Assay
The ROS assay was completed with some alteration on the basis of the oxidation of DCFH-DA (2 ,7 -dichlorofluorescin diacetate) to 2',7'-dichlorodihydrofluorescein. The homogenate of all treated groups was diluted using Lock's buffer at 1:20 time to obtain a final concentration of 5 mg tissue/mL. The reaction mixture (1 mL) with Locke's buffer had a pH of 7.4, 0.2 mL homogenate, and 10 mL of DCFH-DA (5 mM). To acquire a fluorescent product DCF (dichlorofluorescin), the mixture was further incubated at room temperature (15 min) and calculated using a spectrofluorometer with excitation at 484 nm and emission at 530 nm. For background fluorescence (conversion of DCFH-DA in the absence of homogenate), a parallel blank was used. Data are shown as per mol DCF formed/min/mg of protein in the tissue homogenate.

Lipid Peroxidation Determination
For the assessment of oxidative stress, lipid peroxidation (LPO) was carried out. A marker of LPO free malondialdehyde (MDA) was calculated in the tissue homogenate of the hippocampus and cortical parts using a lipid peroxidation (MDA) colorimetric/fluorometric assay kit (BioVision, Milpitas, CA, USA, Cat. #K739-100), followed by the manufacturer's protocol.

Immunofluorescence Analysis
An immunofluorescence study was carried out. Slides with brain tissue samples were washed twice for 10 min in 0.01 M PBS (Phosphate-Buffered Saline) by adding a proteinase K solution. Tissue samples were incubated for 60 min in blocking solution covering 2% normal goat/mouse serum and 0.3% Triton X-100 in PBS. Slides were further incubated overnight in primary antibodies at 4 • C, and further incubated at room temperature for 2 h in secondary antibodies (goat anti-mouse, fluorescein isothiocyanate (FITC)-labeled secondary antibodies (1:50 in PBS) Santa Cruz Biotechnology), after incubating the primary antibodies. The mounting medium was applied to cover the slides with glass coverslips after counterstaining with DAPI (4,6-diamidino-2-phenylindole) (for 10 min). Immunofluorescence was studied using a confocal laser-scanning microscope (Flouview FV 1000MPE, Olympus, Japan); for immunohistological quantitative analysis, ImageJ software was used (v. 1.50).

Histological Examination
To assess the histological study and degree of neuronal cell death, cresyl violet (Nissl) staining was carried out. Slides containing 14 µm tissue sections were washed twice for 15 min in 0.01 M PBS, and further stained with a 0.5% cresyl violet solution (with a few drops of glacial acetic acid) for about 10-15 min. Sections were then further washed with distilled water and dehydrated in graded ethanol (70%, 95%, and 100%), immersed in xylene, cover-slipped using a mounting medium, and slides were examined with a fluorescent light microscope. An assessment of the results was performed using Image J software.

Antibodies
All antibodies (primary and secondary) used in the present study are shown in Table 1.

Statistical Analysis
To evaluate the scanned Western blots, Sigma gel software (SPSS Inc., Chicago, IL, USA) was used, and Image J software was used to analyze immunohistological findings. One-way analysis of variance (ANOVA) followed by Student's t-test was used to find out the mean ± SEM, and Graph Pad Prism 6 software San Diego, CA, USA was used to desi gn the graphs. "Asterisk sign (*) indicated significant difference between control and Aβ injection group; hash sign (#) indicated significant difference between Aβ injection group and Aβ + NAM treated group. Significance: (*) # = p ≤ 0.05, (**) ## = p ≤ 0.01."

Nicotinamide Treatment Attenuated Aβ 1-42 -Elevated Oxidative Stress and p-JNK Levels in Mouse Brain
Several studies reported that oxidative stress plays an important role in the pathophysiology of numerous neurodegenerative diseases, including AD [24][25][26]. Similarly, Aβ 1-42 -induced oxidative stress was demonstrated to alter the brain's antioxidant pathways [5]. Therefore, to analyze the effect of NAM on Aβ 1-42 -induced oxidative stress, we first performed reactive oxygen species (ROS) and lipid peroxidation (LPO) assays. Our results indicated a significant increase in DCF (ROS levels) and MDA (LPO levels) in Aβ 1-2 -injected mouse brains in comparison with saline-injected normal mouse brains (Figure 2A,B). NAM + Aβ 1-42 -treatment significantly reduced the increased ROS and LPO levels in both regions (cortex and hippocampus) compared with in Aβ 1-42 -injected mouse brains (Figure 2A,B). Moreover, Aβ peptides were reported to initiate the JNK signaling pathway in Alzheimer's brains [27]. Therefore, to analyze the effect of NAM on Aβ-induced oxidative stress markers and related p-JNK level, we performed Western blotting and immunofluorescence analysis. Our Western blot results showed a significant effect on the protein expression level of Nrf2/HO, which was reduced, and p-JNK, which was increased in different brain regions in Aβ 1-42 -injected mouse brains compared to in saline-injected normal mouse brains. However, NAM + Aβ 1-42 treatment significantly upregulated the protein expression level of Nrf2/HO-1 and reduced p-JNK expression in comparison with Aβ 1-42 -injected mouse brains ( Figure 2C). in Alzheimer's brains [27]. Therefore, to analyze the effect of NAM on Aβ-induced oxidative stress markers and related p-JNK level, we performed Western blotting and immunofluorescence analysis. Our Western blot results showed a significant effect on the protein expression level of Nrf2/HO, which was reduced, and p-JNK, which was increased in different brain regions in Aβ1-42-injected mouse brains compared to in saline-injected normal mouse brains. However, NAM + Aβ1-42 treatment significantly upregulated the protein expression level of Nrf2/HO-1 and reduced p-JNK expression in comparison with Aβ1-42-injected mouse brains ( Figure 2C). Immunofluorescence analysis also confirmed the immunoblotting results for HO-1; HO-1 reactivity was increased together with a significant reduction in p-JNK reactivity in both the indicated regions of NAM + Aβ1-42 treatment mouse brains in comparison with the Aβ1-42-injected mouse brains ( Figure 2D,E). These findings indicate that NAM is effective in preventing Aβ1-42-induced oxidative stress-mediated elevated p-JNK expression Immunofluorescence analysis also confirmed the immunoblotting results for HO-1; HO-1 reactivity was increased together with a significant reduction in p-JNK reactivity in both the indicated regions of NAM + Aβ 1-42 treatment mouse brains in comparison with the Aβ 1-42 -injected mouse brains ( Figure 2D,E). These findings indicate that NAM is effective in preventing Aβ 1-42 -induced oxidative stress-mediated elevated p-JNK expression level in mouse brains.

Nicotinamide Treatment Abrogates in Aβ 1-42 -Elevated Astrocyte and Neuroinflammatory Cytokines in Mouse Brains
Early reported studies documented that Aβ-induced glial cell activation with the subsequent release of neuroinflammatory cytokines to initiate inflammatory responses in AD pathophysiology [28][29][30]. Therefore, to analyze the effect of NAM on Aβ 1-42 -induced astrocyte activation and associated neuroinflammatory markers, we performed Western blotting and immunofluorescence analysis.
Our immunoblot results indicated a significant increase in the protein expression level of GFAP (an astrocyte marker), nuclear factor kappa-light-chain-enhancer of activated B cells (p-NF-KB), and its associated downstream neuroinflammatory mediators, including tumor necrosis factor alpha (TNFα) and interleukin 1 beta (IL-1β) in Aβ 1-42 -injected mouse brains. NAM + Aβ 1-42 treatment significantly reduced astrocyte activation and its associated neuroinflammatory mediators in comparison with in Aβ 1-42 -injected mouse brains ( Figure 3A).

Nicotinamide Treatment Abrogates in Aβ1-42-Elevated Astrocyte and Neuroinflammatory Cytokines in Mouse Brains
Early reported studies documented that Aβ-induced glial cell activation with the subsequent release of neuroinflammatory cytokines to initiate inflammatory responses in AD pathophysiology [28][29][30]. Therefore, to analyze the effect of NAM on Aβ1-42-induced astrocyte activation and associated neuroinflammatory markers, we performed Western blotting and immunofluorescence analysis.
Our immunoblot results indicated a significant increase in the protein expression level of GFAP (an astrocyte marker), nuclear factor kappa-light-chain-enhancer of activated B cells (p-NF-KB), and its associated downstream neuroinflammatory mediators, including tumor necrosis factor alpha (TNFα) and interleukin 1 beta (IL-1β) in Aβ1-42-injected mouse brains. NAM + Aβ1-42 treatment significantly reduced astrocyte activation and its associated neuroinflammatory mediators in comparison with in Aβ1-42-injected mouse brains ( Figure 3A).  To assess these immunoblot results further, we performed confocal microscopy. Accordingly, our immunofluorescence results also suggested that p-NF-kB and IL-1β immunoreactivity was significantly increased (cortex and dentate gyrus (DG) region of hippocampus) in Aβ 1-42 -injected mouse brains in comparison with the saline-injected normal mouse brains. NAM + Aβ 1-42 treatment significantly reduced the immunoreactivity of p-NF-kB and IL-1β in both the indicated regions in comparison with Aβ 1-42 -injected mouse brains ( Figure 3B,C).
These results indicate that NAM is effective in abrogating Aβ 1-42 -induced neuroinflammation in the cortex and hippocampal regions of the mouse brains.

Nicotinamide Treatment Halts Aβ 1-42 -Induced Neurodegeneration in Mouse Brains
Previous studies determined that Aβ-induced neurodegeneration involved the initiation of apoptotic pathway in neurodegeneration [31][32][33]. Therefore, to analyze the effect of NAM on Aβ 1-42 induced neuro-apoptosis, we performed Western blotting and immunofluorescence analysis. Our immunoblot results showed a significant increase in the protein expression level of BCL2-associated X protein (Bax, a proapoptotic marker), caspase-3, and poly (ADP-ribose) polymerase 1 (PARP-1, a DNA damage marker) accompanied with a significant reduction in B cell lymphoma 2 (Bcl-2, an antiapoptotic marker) in different regions in Aβ 1-42 -injected mouse brains. NAM+ Aβ 1-42 treatment significantly reduced the elevated expression of Bax, caspase-3, and PARP-1, and significantly upregulated Bcl-2 in comparison with Aβ 1-42 -injected mouse brains ( Figure 4A). To assess these immunoblot results further, we performed confocal microscopy. Accordingly, our immunofluorescence results also suggested that p-NF-kB and IL-1β immunoreactivity was significantly increased (cortex and dentate gyrus (DG) region of hippocampus) in Aβ1-42-injected mouse brains in comparison with the saline-injected normal mouse brains. NAM + Aβ1-42 treatment significantly reduced the immunoreactivity of p-NF-kB and IL-1β in both the indicated regions in comparison with Aβ1-42-injected mouse brains ( Figure 3B,C).
These results indicate that NAM is effective in abrogating Aβ1-42-induced neuroinflammation in the cortex and hippocampal regions of the mouse brains.

Nicotinamide Treatment Halts Aβ1-42-Induced Neurodegeneration in Mouse Brains
Previous studies determined that Aβ-induced neurodegeneration involved the initiation of apoptotic pathway in neurodegeneration [31][32][33]. Therefore, to analyze the effect of NAM on Aβ1-42 induced neuro-apoptosis, we performed Western blotting and immunofluorescence analysis. Our immunoblot results showed a significant increase in the protein expression level of BCL2-associated X protein (Bax, a proapoptotic marker), caspase-3, and poly (ADP-ribose) polymerase 1 (PARP-1, a DNA damage marker) accompanied with a significant reduction in B cell lymphoma 2 (Bcl-2, an antiapoptotic marker) in different regions in Aβ1-42-injected mouse brains. NAM+ Aβ1-42 treatment significantly reduced the elevated expression of Bax, caspase-3, and PARP-1, and significantly upregulated Bcl-2 in comparison with Aβ1-42-injected mouse brains ( Figure 4A).  To assess these immunoblot results further, we performed confocal microscopy. Accordingly, our immunofluorescence results also indicated that caspase-3 and PARP-1 immunoreactivity was significantly increased (cortex and hippocampus, DG region) in Aβ 1-42injected mouse brains in comparison with saline-injected normal mouse brains. NAM+ Aβ 1-42 treatment significantly reduced the immunoreactivity of caspase-3 and PARP-1 in both the indicated regions in comparison with Aβ 1-42 -injected mouse brains ( Figure 4B,C).
Furthermore, Nissl staining indicated that NAM + Aβ 1-42 treatment significantly increased the amount of surviving neurons (cortex and DG region of hippocampus) in comparison with Aβ 1-42 -injected mouse brains ( Figure 4D), suggesting that the extent of neuronal death was significantly reduced. These results indicate that NAM is effective in halting AB-induced apoptotic neurodegeneration in different regions of mouse brains.

Nicotinamide Treatment Reversed Aβ 1-42 -Induced Memory Dysfunction in Mouse Brains
Recently, it was reported that Aβ 1-42 -induced memory impairment [9]. Therefore, to examine the effects of NAM on learning and memory dysfunction, we performed behavioral analysis (Morris water maze (MWM) and Y-maze tests). In the MWM test, mean latency (time required in seconds (s) to find the hidden platform) was gradually reduced in all experiment groups during the training days except for the Aβ 1-42 -injected mouse group, which showed longer latency than that of the saline-injected normal mouse group, indicating impaired learning and memory abilities. However, compared with the Aβ 1-42injected mouse group, this effect was significantly reduced by NAM+ Aβ 1-42 treatment, as the experiment mice taking less time (in seconds) to reach the hidden platform indicated improved memory performance ( Figure 5A). Furthermore, we performed a probe test in which the hidden platform was removed. The time spent in the target quadrant ( Figure 5B) and number of platform crossings ( Figure 5C) was significantly increased in the NAM+ Aβ 1-42 treatment group compared with the Aβ 1-42 -injected mouse group, showing that NAM reduced Aβ 1-42 -induced memory impairment.
After the MWM test, we performed the Y-maze test to evaluate spatial working memory. A higher percentage (%) of spontaneous-alteration behavior was considered to be an indication of increased cognitive performance. The Aβ 1-42 -injected mouse group exhibited significantly lower percentage (%) of spontaneous alterations than the saline-injected normal mouse group did, indicating impaired working memory. However, compared to the Aβ 1-42 -injected mouse group, NAM+ Aβ 1-42 treatment showed a significant increase in spontaneous-alteration behavior, indicating that NAM attenuated short-term memory deficits in the Aβ 1-42 -injected mouse group ( Figure 5D).

Discussion
Oxidative stress [34], (Aβ1-42-induced oxidative stress), has been documented in various (in vitro and in vivo) models of neurological disorders [35][36][37]. Neuroprotective agents [38] and emerging antioxidants as therapeutics still provide hope and are receiving increased attention [39,40]. Accordingly, in this preclinical study, we examined and explored the neuroprotective role of NAM against Aβ1-42-induced neurotoxicity in mouse brains. NAM significantly reversed Aβ1-42-induced oxidative stress, neuroinflammation, neurodegeneration, and memory impairment in Aβ1-42 treated in the mouse AD model ( Figure 6). maze test to evaluate spatial working memory. A higher percentage (%) of spontaneous-alteration behavior was considered to be an indication of increased cognitive performance. Compared to the Aβ 1-42 -injected mouse group, NAM+ Aβ 1-42 treatment showed a significant increase in spontaneous-alteration behavior.8 animals were kept per group. Asterisk sign (*) indicated significant difference between control and Aβ injection group; hash sign (#) indicated significant difference between Aβ injection group and Aβ + NAM treated group. Significance: (*) # = p ≤ 0.05.

Discussion
Oxidative stress [34], (Aβ 1-42 -induced oxidative stress), has been documented in various (in vitro and in vivo) models of neurological disorders [35][36][37]. Neuroprotective agents [38] and emerging antioxidants as therapeutics still provide hope and are receiving increased attention [39,40]. Accordingly, in this preclinical study, we examined and explored the neuroprotective role of NAM against Aβ 1-42 -induced neurotoxicity in mouse brains. NAM significantly reversed Aβ 1-42 -induced oxidative stress, neuroinflammation, neurodegeneration, and memory impairment in Aβ 1-42 treated in the mouse AD model ( Figure 6). Biomedicines 2021, 9, x FOR PEER REVIEW 12 of 18 Inside our body, oxidative stress (OxS), an abnormal phenomenon [41,42] ROS representing a set of large battery free radical and non-radical species are constantly formed, play an important role in cell signaling to maintain cellular homeostasis [43]. However, increased ROS production is considered to be a major cause in the development of several human diseases [44,45]. More specifically, among all body organs, the brain is the most metabolically active organ, approximately using 20% of total oxygen uptake and is, therefore, more vulnerable to oxidative damage [42]. Free radicals are highly reactive with biomolecules (carbohydrate, proteins, lipids, and DNA) [42,43] that attack brain-cell membranes, cause lipid peroxidation, and damage plasma membrane and cytosolic proteins [46]. Increased oxidative stress biomarkers were detected in AD brains [41,42,47]. Many in vitro and in vivo studies showed that Aβ treatment increased oxidative stress or ROS generation in both neuronal and non-neuronal cells in Aβ-mediated neuronal cell death [41,48]. On the other hand, many studies reported that the administration of different antioxidant vitamins and their combinations or derivatives act as a free radical scavenger, inhibits the oxidation of biomolecules, and thus prevents the development of Aβ1-42-induced ROS formation; therefore, it is effective in decreasing the incidence of disease progression [36,[49][50][51][52][53][54]. Additionally, Vitamins C were shown to attenuate Aβ oligomerization and Aβ oligomer-mediated oxidative stress as one of the potential mechanisms in disease prevention [55]. Likewise, in accordance with previous findings, our results also indicated that NAM+ Aβ1-42 treatment significantly reduced Aβ1-42-induced increase in ROS and LPO levels in both the indicated regions of the mouse brain. Furthermore, upon exposure to oxidative stress, cells respond via the antioxidant response element (ARE) pathway. Several genes encoding antioxidant proteins are simultaneously induced [56]. This response is regulated upon the activation of nuclear factor E2-related factor 2 (Nrf2), Inside our body, oxidative stress (OxS), an abnormal phenomenon [41,42] ROS representing a set of large battery free radical and non-radical species are constantly formed, play an important role in cell signaling to maintain cellular homeostasis [43]. However, increased ROS production is considered to be a major cause in the development of several human diseases [44,45]. More specifically, among all body organs, the brain is the most metabolically active organ, approximately using 20% of total oxygen uptake and is, therefore, more vulnerable to oxidative damage [42]. Free radicals are highly reactive with biomolecules (carbohydrate, proteins, lipids, and DNA) [42,43] that attack brain-cell membranes, cause lipid peroxidation, and damage plasma membrane and cytosolic proteins [46]. Increased oxidative stress biomarkers were detected in AD brains [41,42,47]. Many in vitro and in vivo studies showed that Aβ treatment increased oxidative stress or ROS generation in both neuronal and non-neuronal cells in Aβ-mediated neuronal cell death [41,48]. On the other hand, many studies reported that the administration of different antioxidant vitamins and their combinations or derivatives act as a free radical scavenger, inhibits the oxidation of biomolecules, and thus prevents the development of Aβ 1-42 -induced ROS formation; therefore, it is effective in decreasing the incidence of disease progression [36,[49][50][51][52][53][54]. Additionally, Vitamins C were shown to attenuate Aβ oligomerization and Aβ oligomer-mediated oxidative stress as one of the potential mechanisms in disease prevention [55]. Likewise, in accordance with previous findings, our results also indicated that NAM+ Aβ 1-42 treatment significantly reduced Aβ 1-42 -induced increase in ROS and LPO levels in both the indicated regions of the mouse brain. Furthermore, upon exposure to oxidative stress, cells respond via the antioxidant response element (ARE) pathway. Several genes encoding antioxidant proteins are simultaneously induced [56]. This response is regulated upon the activation of nuclear factor E2-related factor 2 (Nrf2), key transcriptional factor that binds with AREs, activating cytoprotective genes (heme oxygenase-1 (HO-1)) [56,57]. Thus, the function of Nrf2 and its downstream target genes indicates that the Nrf2-ARE pathway is essential in the regulation of the cellular antioxidant defense system [58]. However, studies showed that signaling via Nrf2 is attenuated in both mouse models [59,60]. Recent studies, including ours and those of other research groups, also showed an altered Nrf2/HO-1 signaling pathway in both transgenic (APP/PS1 mouse model of AD) [59,60] and non-transgenic (Aβ-induced AD model) models [60,61]. In an attempt to protect neural cells, many other studies found that neuroprotectants involve the activation of Nrf2/HO-1 signaling pathways as a part of their underlying neuroprotective molecular mechanisms against Aβ-induced cell death [3,62]. On the other hand, Vitamins with antioxidant potentials proved to stimulate the antioxidant (Nrf2/HO-1) pathway, indicating its pivotal role in oxidative stress [63,64]. Likewise, in accordance with previous findings, our results also showed that NAM + Aβ 1-42 treatment significantly upregulated Aβ 1-42 -suppressed Nrf2/HO-1 levels in both the cortex and hippocampus of the mouse brain, indicating the antioxidant effects of NAM. Furthermore, mitogen-activated protein kinases (MAPKs), for example, c-Jun N-terminal kinase (JNK), also known as stress-activated protein kinase (SAPK), is activated in response to a wide range of cellular stress types (genotoxic, proinflammatory cytokines, etc.), including Aβ oligomers [65]. Increasing evidence reported abnormal JNK activation in aged transgenic mouse and human AD brains as one of a potential mechanism in Aβ-induced neurotoxicity [66]. On the other hand, many studies reported that the administration of antioxidant vitamins markedly prevent the activation of MAPK [67]. Likewise, our results also showed that NAM + Aβ 1-42 treatment suppressed Aβ 1-42 -induced elevated p-JNK expression in both the cortex and hippocampus of mouse brains. On the basis of the above results, NAM could be an antioxidant Vitamin that enhances antioxidant response, and limits Aβ 1-42 -induced oxidative stress and its associated elevated p-JNK protein expression in mouse brains (Figure 2). A growing number of neuropathological studies have shown that neuroinflammation is an early event or feature in the pathophysiology of numerous neurodegeneration [68]. The brain is mainly populated by two broad categories of cells, neurons and neuroglial cells (astrocytes, oligodendrocytes, and ependymal and microglial cells) [69]. Glial cells were characterized as a major brain-derived sources of inflammation, as seen in AD brains [70]. Astrocytes are key elements in maintaining homeostasis and regulators of multiple physiological functions in brain [71]. However, they are responsive to various stimuli, and astrocyte dysfunction may impair neuronal function and thus lead to neurodegeneration [72]. Many studies showed that activated astrocytes are a common neuropathological feature of brain disorders that respond to Aβ metabolically, morphologically, and/or functionally [73]. Previous work from us and other research groups showed that Aβ peptides act as stimuli that significantly contribute to reactive astrocytes (activated for astrocytes [5]), which, in turn, can lead to the production of several potentially toxic inflammatory cytokines or mediators [74]. These reactive astrocytes, with the increased expression of inflammatory cytokines or molecules (IL-1β, TNF-α, COX-2, and iNOS), were well detected [75] in the brains of both AD patients and AD animal models [73]. Additionally, in various central neuroinflammation signaling pathways, Aβ peptides induce the activation of nuclear factor-kappa B (p-NF-κB; a transcription factor), which, in turn, enhances the production and expression of several inflammatory cytokines (IL-6, IL-1β, iNOS, and TNF-α) and causes neuroinflammation, as reported in neuroinflammatory neurodegeneration [76]. Many studies showed that the administration of vitamins or their derivatives inhibited the activation of the NF-κB signaling pathway and its effect on related downstream inflammatory cytokines (IL-6, IL-1β, iNOS, and TNF-α) [77]. Likewise, our results also showed that NAM+ Aβ 1-42 treatment significantly inhibits Aβ 1-42 -activated astrocytosis (GFAP, a specific marker of activated astrocytes) and the activation of transcription factor/p-NF-κB, along with its various related proinflammatory cytokines (TNF-αand IL-1β) in the cortex and hippocampal regions of mouse brains. On the basis of the above results, NAM could be effective in reversing Aβ 1-42 -activated astrocytosis and the elevated expression of p-NF-κB associated with neuroinflammatory cytokines in mouse brains [5].
In recent years, evidence has indicated that neuronal apoptosis (a form of nerve cell death) and cognitive impairment are central features of many neurodegenerative diseases [78], as seen in human AD brains. ROS serve as physiological modulators of mitochondrial functions, but can damage the mitochondria, which ultimately results in apoptosis [79]. Apoptosis is a fundamental-cell death process that occurs through the activation of specific signal transduction, including the mitochondria, mitochondrial regulatory proteins, and caspase activation. In cellular apoptosis, mitochondria-associated Bcl-2 family proteins are essential modulators that are commonly categorized into two groups: one class promoting cell survival (e.g., Bcl-XL, Bcl-2, and Bcl-w) and the other facilitating cell death (e.g., Bad, Bid, Bax, and Bim). Previous studies suggested that the expression level of antiapoptotic proteins (Bcl-2 and Bcl-XL) was decreased and/or proapoptotic protein (Bax and Bim) levels were increased during Aβ-mediated apoptosis [80]. During Aβ-induced neuroapoptosis, the subsequent disturbance of or decrease in mitochondrial transmembrane potential is also clearly evident, and disrupted mitochondrial membranes may promote the apoptotic process through the leakage of apoptotic-regulated signaling molecules through mitochondria into cytosol [81]. In addition, caspases, particularly the activation of caspase-3 (a cysteine-dependent aspartic protease) by the Aβ peptide, were reported as final effector molecules in apoptotic cell death. For example, in Aβ 1-42 -induced neuronal apoptosis, many laboratories showed the involvement and activation of different caspases (caspase-9 and caspase-3) in neuronal populations or cell types and in animal models of AD [31]. Others showed that the activated neuronal caspase-3 causes the cleavage of poly (ADP-ribose) polymerase-1 (PARP-1, an essential enzyme important in DNA repair), thereby promoting apoptosis [82]. Indeed, both in in vivo and in vitro studies, and even in postmortem human brain tissue, apoptosis is commonly observed in Aβ-induced toxicity [83]. On the other hand, many studies reported that the administration of different antioxidant vitamins significantly inhibited neuronal apoptosis via the regulation of pro-and antiapoptotic proteins, thereby preventing brain damage [84,85]. Our results also showed that NAM+ Aβ 1-42 treatment significantly reversed Aβ 1-42 -induced increases in Bax, caspase-3, and PARP-1 levels in both the cortex and hippocampal regions of the mouse brain, indicating the antiapoptotic effects of NAM. Additionally, Nissl staining (used for the measurement of neuronal loss) indicated that NAM+ Aβ 1-42 treatment significantly reversed Aβ 1-42 -induced neuronal cell death, as it significantly increased the amount of surviving neurons in the cortex and hippocampal regions of the mouse brain ( Figure 4C,D).
With regard to Aβ 1-42 -induced neuronal apoptosis, our group and others reported that Aβ 1-42 -induced memory or cognitive dysfunctions in mouse models of AD [31]. On the other hand, the administration of different antioxidant vitamins, either alone or in combination, were well-studied, and proven to improve learning and memory performance in rodent models [84]. Our results also showed that NAM + Aβ 1-42 treatment reduced Aβ 1-42 -induced memory dysfunction, as evidenced from behavioral analysis (MWM and Y-maze tests), thus improving cognition, spatial learning, and memory processing ( Figure 5). These results showed that NAM is significantly effective in reversing Aβ 1-42 -induced apoptosis neurodegeneration and memory impairment in mouse brains.

Conclusions
In conclusion, our study provides considerable evidence that NAM can abrogate Aβ 1-42 induced oxidative stress, neuroinflammation, and neuronal apoptosis. NAM+ ABtreatment maintains the cellular antioxidant system and regulates Nrf2/HO-1 protein levels. This shows the therapeutic potential of NAM against Aβ 1-42 -accelerated neurotoxicity, and may also open the door for new therapeutic preclinical research work to be carried out ( Figure 6).