Alzheimer’s disease (AD) is a progressive neurodegenerative disorder typified by several neuropathological features including amyloid-beta (Aβ) plaque and neurofibrillary tangles (NFTs). Cholesterol retention and oxidative stress (OS) are the major contributors of elevated β- and γ-secretase activities, leading to excessive Aβ deposition, signifying the importance of altered cholesterol homeostasis and OS in the progression of Aβ-mediated neurodegeneration and cognitive deficit. However, the effect of Aβ on cholesterol metabolism is lesser-known. In this study, we evaluated the effect of quinovic acid (QA; 50 mg/kg body weight, i.p.) against the intracerebroventricular (i.c.v.) injection of Aβ (1-42)-induced cholesterol dyshomeostasis, oxidative stress, and neurodegeneration in the cortex and hippocampal brain regions of wild-type male C57BL/6J mice. Our results indicated that Aβ (1-42)-treated mice have increased Aβ oligomer formation along with increased β-secretase expression. The enhanced amyloidogenic pathway in Aβ (1-42)-treated mice intensified brain cholesterol accumulation due to increased expressions of p53 and 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) enzyme. Importantly, we further confirmed the p53-mediated HMGCR axis activation by using pifithrin-α (PFT) in SH-SY5Y cells. Furthermore, the augmented brain cholesterol levels were also associated with increased OS. However, the QA administration to Aβ (1-42)-injected mice significantly ameliorated the Aβ burden, p53 expression, and cholesterol accumulation by deterring the oxidative stress through upregulating the Nrf2/HO-1 pathway. Moreover, the QA downregulated gliosis, neuroinflammatory mediators (p-NF-κB and IL-1β), and the expression of mitochondrial apoptotic markers (Bax, cleaved caspase-3, and cytochrome c). QA treatment also reversed the deregulated synaptic markers (PSD-95 and synaptophysin) and improved spatial learning and memory behaviors in the Aβ-treated mouse brains. These results suggest that Aβ (1-42) induces its acute detrimental effects on cognitive functions probably by increasing brain cholesterol levels through a possible activation of the p53/HMGCR axis. However, QA treatment reduces the cholesterol-induced oxidative stress, neuroinflammation, and neurodegeneration, leading to the restoration of cognitive deficit after Aβ (1-42) i.c.v. injection in mice.
Alzheimer’s disease (AD) is the major progressive neurodegenerative disorder associated with brain atrophy and dementia and is typified by various neuropathological features including the presence of extracellular neuritic plaques of amyloid-beta (Aβ) peptides and hyperphosphorylated tau in intraneuronal neurofibrillary tangles (NFTs) [1, 2]. There is a strong correlation between altered Aβ metabolism and increased p53 expression levels [3–6]. p53 is a tumor-suppressor protein involved in cell cycle control or apoptosis, and its role in cell death and neurodegeneration has been extensively discussed [7, 8]. p53 has diverse biological functions and is known to modulate several pathways , including cholesterol metabolism [10, 11], and regulate the activity of important enzymes of the mevalonate (MVA) pathway, including 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR) to alter cholesterol metabolism . The perturbation in cholesterol metabolism inside the body can be catastrophic to the brain and is manifested by progressive neuronal cell death .
Although most studies illuminated how cholesterol homeostasis affects the production and amyloid precursor protein (APP) processing in promoting AD pathology, very little is known about the influence of Aβ on altering the cellular cholesterol metabolism [14, 15]. Michikawa et al. have previously revealed that oligomeric Aβ in cultured astrocytes and neurons releases cholesterol and phospholipids . Similarly, Aβ can increase synthesis and alter the distribution of free cholesterol in neurons . The oligomeric Aβ (1-40) may also lose its neuroprotective activity  and modulates the synthesis and cellular cholesterol homeostasis in the rat’s primary cultured neurons . The extracellular cholesterol is also known to reside in aggregated Aβ in transgenic (with Swedish Alzheimer mutation APP751) mice and neuritic plaques of AD patients . However, the effect of Aβ (1-42) oligomers on cholesterol dyshomeostasis and metabolism especially in vivo and its concomitant detrimental effects on neurodegeneration are less well defined and may offer novel insights into mechanisms based on the amyloid cascade hypothesis.
Oxidative stress (OS) plays an important role in the pathogenesis of many neurodegenerative diseases  and is considered to be an early event observed in AD disease progression [22, 23]. The cholesterol-mediated alterations deteriorate the critical antioxidant defense system by impairing mitochondrial GSH transport, reducing the cell susceptibility to different stimuli to reduce oxidative stress [24, 25]. The oxidized cholesterol products including oxysterols illustrate a fundamental role in AD progression by increasing oxidative stress linking hypercholesterolemia and altered cholesterol metabolism to neurodegenerative disease [14, 26]. The HMGCR (a key enzyme in the mevalonate pathway) competitive inhibitor statin has been shown in multiple studies in reducing AD pathology . However, some actions of statins are assumed to be associated with the pleiotropic effect on OS, neuroinflammation, and brain oxygenation in particular [28, 29], instead of lowering cholesterol [30–32]. Cholesterol retention is also one of the major reasons for elevated β- and γ-secretase activities, leading to excessive Aβ deposition  that can further induce free radicals , along with different oxidative stress markers , and leads to the abnormal intermediation of the Keap1/Nrf2/ARE pathway [36–38].
Quinovic acid is a triterpene isolated from different plants [39–41]. Quinovic acids and other triterpenes are an important constituent of the cat’s claw (Uncaria tomentosa) extract which is a medicinal plant and is widely used as a strong antioxidant with potent DPPH scavenging and anti-inflammatory activity [42, 43]. Likewise, the quinovic acid glycosides from the Guettarda platypoda and Uncaria tomentosa plants were among the initial studies to show anti-inflammatory activity in rats . Moreover, triterpenes possess potential activity against diabetes-induced β-cell damage that mostly is superseded by OS-mediated inflammation . Previously, we have reported that quinovic acid and its glycoside derivatives have been shown to have strong inhibitory activity against dipeptidyl peptidase-4 (DPP-4) , which is an important enzyme involved in cleaving various neuropeptides and incretin hormones possessing anti-inflammatory activity . Here, we have studied the proteotoxic effect of i.c.v. Aβ (1-42) oligomers on brain cholesterol upsurge associated with OS and its effect on neurodegeneration and behavioral deficits and its possible neuroprotection through antioxidant quinovic acid in reducing AD disease pathology in mice. Our results make evident that oligomeric Aβ (1-42) isoform has an acute effect on cholesterol dyshomeostasis and OS with interactive influence on neuronal loss and synaptic/memory dysfunction and that administering quinovic acid to the mice protects against Aβ-neurotoxicity; alleviates disease pathology by reducing cholesterol-mediated OS, glial activation, and neuroinflammation; and improves memory deficits in the (i.c.v.) β-amyloid mouse model.
2. Materials and Methods
2.1. Drug Preparation
For in vivo intracerebroventricular (i.c.v.) administration, Aβ (1-42) peptide was prepared as formerly mentioned . Briefly, the human Aβ (1-42) peptide (purchased from Sigma Chemicals Co.) was prepared in a sterile saline solution at a stock concentration of 1 mg/ml followed by aggregation via incubation at 37°C and administered at 5 μl/mice. For in vitro studies, the oligomeric Aβ (1-42) (AβO) was prepared as reported previously . Concisely, the Aβ (1-42) peptide was dissolved in 100% hexafluoroisopropanol (HFIP). The HFIP was then evaporated under vacuum and reconstituted in dimethyl sulfoxide (DMSO) to produce 5 mM suspension. The 5 mM HFIP-treated Aβ (1-42) suspension was further diluted to 100 μM in F12 culture media (Gibco by Life Technologies, USA) lacking phenol red and incubated for 24 h at 5°C. The solution containing peptide was centrifuged at 14,000 rpm for 10 min at 4°C, and the supernatant containing AβO was collected. For cell treatment, AβO was used at a final concentration of 5 μM. The quinovic acid (QA) was isolated as previously reported  and was dissolved in a saline solution for in vivo administration and in DMSO for in vitro analysis. Pifithrin-α (Axon Medchem) was dissolved in DMSO and used at a concentration of 10 μM for in vitro analysis.
2.2. Cell Culture, MTT Assay, and Treatment
Human neuroblastoma SH-SY5Y cells (purchased from Korean Cell Line Bank) were grown in MEM+F12 (1 : 1) supplemented with 1% penicillin-streptomycin and 10% fetal bovine serum. Cells were cultured in saturated humidified air containing 5% CO2 at 37°C. The experiments were commenced until cells grew up to 60-70% confluence.
MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was used to evaluate the levels of cell viability and dose optimization as performed previously . SH-SY5Y cells were grown in a 96-well assay plate ( cells/well) in 100 ml/well of MEM+F12 (1 : 1) media augmented with 1% penicillin-streptomycin and 10% FBS. The cells were incubated in humidified air with 5% CO2 at 37°C, and the medium was changed at a regular interval. At 60-70% confluence media were changed, and the cells were either treated alone with different concentrations of quinovic acid (0, 5, 10, 30, 55, 70, 85, 100, and 115 μM) dissolved in DMSO or cotreated with AβO (5 μM) for dose optimization and incubated for 24 h at 37°C. The final concentration of DMSO was kept less than 0.25% in each well. Control culture wells either contained maintenance media or were augmented with 0.2% DMSO (vehicle control). After incubation, 10 μl of MTT (5 mg/ml in PBS)/well was added and incubated for 4 h. The blue formazan formed was solubilized by adding 100 μl of DMSO after media were removed and incubated for another 10-15 min. The absorbance intensity was measured at 550-570 nm using a microplate reader ApoTox-Glo™ (Promega, Madison, WI, USA). The experiments were performed in triplicate.
For western blot analysis, SH-SY5Y cells were seeded in a 100 mm Petri dish. After 70% confluence, the cells were treated with the following: (a) AβO (5 μM) alone, (b) pretreated 60 min with pifithrin-α (10 μM) before exposure to AβO, (c) AβO cotreated with quinovic acid (85 μM), (d) treated with PFT, or (e) quinovic acid alone for 24 h. AβO concentration of 5 μM was used in all culture experiment. Control cells were only incubated with the pure medium. After 24 h of incubation, cells were collected in PBS by scraping and centrifuged at for 10 min at 4°C. The supernatant was removed, and PRO-PREP (a protein extraction solution) was added according to the manufacturer’s approved guidelines (iNtRON Biotechnology) followed by sonication and centrifuged at 4°C at 13,000 rpm for 30 min, and supernatants were collected to obtain cell lysates. The protein concentration in lysates was quantified using the Bio-Rad assay solution.
2.3. Experimental Subjects
Male C57BL/6J wild-type mice were obtained from Samtako Bio Korea and were approximately 25-30 g and 8 weeks old at the start of experiments. The animals were randomly retained in groups of four in each cage and acclimatized for one week at room temperature in a fully controlled university animal facility (12 : 12 h light-dark cycle at ) with free access to water and chow. The animals were assigned to the following 3 different groups ( mice/group): saline-treated control, Aβ+saline, and Aβ+QA. All experiments in the current study followed the guidelines and principles of the Animal Ethics Committee (IACUC) (Approval ID: 125) from the Division of Life Sciences and Applied Life Sciences at Gyeongsang National University (GNU), South Korea.
2.4. Mouse Model and Drug Administration
For Aβ (1-42) i.c.v. administration, the mice were anesthetized with a Rompun : Zoletil ratio of 0.5 ml/100 g body weight and were intracerebroventricular (i.c.v.) injected with aggregated Aβ (1-42) or vehicle control (saline) stereotaxically using a Hamilton microsyringe inserted 1 mm median-to-lateral, -0.2 mm from anterior-to-posterior, and -2.4 mm ventral-to-dorsal to the bregma. The Aβ (1-42) (total 5 μl/mice) was injected at the degree of 1 μl/5 min/mouse. The needle was left in place to avoid leakage or backflow for at least 3 min. The experiments were performed at a controlled temperature () to prevent hypothermia.
Soon after the recovery period (24 h), the mice received quinovic acid at 50 mg/kg dose concentration or saline (0.1% normal) solution, administered intraperitoneally (i.p.) on alternative days for 3 weeks. Schematic representation of experimental procedure and drug administration is indicated in Figure 1.