Prospects of Marine Sterols against Pathobiology of Alzheimer’s Disease: Pharmacological Insights and Technological Advances

Alzheimer’s disease (AD) is a degenerative brain disorder characterized by a progressive decline in memory and cognition, mostly affecting the elderly. Numerous functional bioactives have been reported in marine organisms, and anti-Alzheimer’s agents derived from marine resources have gained attention as a promising approach to treat AD pathogenesis. Marine sterols have been investigated for several health benefits, including anti-cancer, anti-obesity, anti-diabetes, anti-aging, and anti-Alzheimer’s activities, owing to their anti-inflammatory and antioxidant properties. Marine sterols interact with various proteins and enzymes participating via diverse cellular systems such as apoptosis, the antioxidant defense system, immune response, and cholesterol homeostasis. Here, we briefly overview the potential of marine sterols against the pathology of AD and provide an insight into their pharmacological mechanisms. We also highlight technological advances that may lead to the potential application of marine sterols in the prevention and therapy of AD.


Introduction
Alzheimer's disease (AD) is a devastating chronic neurodegenerative disorder characterized by intracellular aggregations of tau protein in neurofibrillary tangles (NFTs) formation and extracellular amyloid β-protein (Aβ) accumulation as the formation of a senile plaque in the specific brain regions [1,2]. About 70% of AD risk is found to be based on genetic predisposition, although numerous genes participate and its real causes in addition to molecular mechanisms have not been clearly elucidated [2][3][4]. However, aggregation of misfolded proteins could result in AD pathogenesis [5], and the extracellular domain along with a small cytosolic domain present in amyloid β-protein precursor (APP) is the key molecular driver of AD pathogenesis [6].
Despite the failure of recent clinical trials in antibody-based AD therapy [7], there is still hope for targeting AD-associated pathobiology by means of pharmacological agents. The therapeutic strategy of AD requires a multi-targeted approach because of its multifaceted pathobiology. Oxidative stress, neuroinflammation, and cholesterol dyshomeostasis constitute primary contributing factors in the pathogenesis of AD, and can, therefore, Mar. Drugs 2021, 19, 167 2 of 19 be potential targets for the development of anti-AD agents. Although synthetic and semi-synthetic drugs are the primary source of therapeutics against neurological diseases, including AD, their adverse side effects have led researchers to search for therapeutic leads in natural resources, such as the marine environment [8]. Approximately 70% of the Earth's surface is covered by oceans, and diverse marine organisms offer a wonderful source of natural compounds [9]. Accordingly, recent observations have paid attention to the use of marine natural products that are relevant to treat AD [10]. Marine sterols, a class of sterol compounds, are such a group of natural molecules that are structurally and functionally similar to cholesterol, and their involvement in human health benefit and nutrition are imperative. Due to structural similarity and the sharing of the same absorption route, dietary sterols cause a reduction in intestinal cholesterol absorption and thereby play a significant role in maintaining cholesterol homeostasis, the disturbance of which is implicated in the pathobiology of various neurological diseases.
Beyond their cholesterol-lowering potentials, marine sterols are shown to have therapeutic promise against AD by protecting against apoptosis, oxidative stress, and neuroinflammation through modulating cell survival pathways, such as brain-derived neurotrophic factor (BDNF), nuclear factor erythroid 2-2-related factor 2 (Nrf2), and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling systems [11]. Despite the tremendous impact on neuropharmacology, much effort is required to achieve the use of marine sterols against AD in clinics. Here, we reviewed the neuropharmacological potentials of marine sterols against the pathobiology of AD and highlight technological advances towards the application of marine sterols in AD management.

Distribution and Pharmacokinetics of Marine Sterols
Marine sterols are distributed across several marine phyla (Table 1), and their pattern is influenced by geographic origin and ecological variation. Algae are among the marine organisms that contain an abundance of phytosterols, such as fucosterol, with significant pharmacological benefits [12]. Other marine organisms such as sponge [13], coral [14], and mollusk [15] differ in sterol contents; however, only a few of these sterols are important in neuropharmacology.
Over the last few decades, pharmaceutical scientists have invested considerable interest in the modeling of in silico absorption, distribution, metabolism, excretion, and toxicity (ADME/T) as a rational drug design tool that plays an emerging role in drug development. The ADME/T profile of marine sterols was predicted using Schrodinger's QikProp module, which provides ADME/T at a reliable level, describing drug likeliness and different pharmacokinetic parameters of compounds as shown in Table 1. Marine sterols were predicted to be potential drug-like molecules based on the comparison and range given at the bottom of Table 1. As reported here, fucosterol, the most abundant sterol of marine algae, conforms to Lipinski's rule of five and Jorgensen's rule of three, presenting its drug-likeliness. In addition, as the brain-blood partition coefficient (QPlogBB) of fucosterol is within the recommended range (−3.0-1.2), this sterol is likely able to cross the blood-brain barrier. Since marine sterols lack experimental data on pharmacokinetics, the in silico data that were incorporated in the review could provide future direction on studying pharmacokinetics and form a basis for the selection of a potential candidate in drug development.

Pathobiology of Alzheimer's Disease
Alzheimer's disease (AD) is the most prevalent neurodegenerative disorder, contributing to dementia in the elderly. The amyloid plaque and neurofibrillary tangles (NFT) constitute the major pathological features of AD [33]. Oxidative stress and neuroinflammation are known to be among the primary causal factors in the pathobiology of AD [34,35]. When the generation of reactive oxygen species (ROS) exceeds the capacity of the cellular antioxidant defense system, a pathological condition called oxidative stress develops. Excitotoxicity, the exhaustive cellular antioxidant system, and brain susceptibility to lipid peroxidation contribute to OS [36]. ROS potentially causes damage by compromising the structure and function of cellular biomolecules that, in turn, cause neurodegeneration [37]. Neuroinflammation initiated by microglial activation culminates into chronic neurodegeneration [38]. Upon activation through toxicity, infection, and hypoxia, microglia secrete several pro-inflammatory and inflammatory cytokines [39] that stimulate neurons leading to neurodegeneration [40]. Imbalance in cholesterol homeostasis also may provoke OS and inflammation, thereby contributing to the pathobiology of AD [41]. Brain cholesterol metabolism is tightly regulated by the cholesterol transport mechanism. Upon activation, liver X receptor beta (LXR-β) upregulates multiple genes that encode proteins involved in the regulation of reverse cholesterol transport and thereby ensures neuroprotection [42,43]. For example, LXR-β agonist augmented amyloid β (Aβ) clearance [44]. Having association with pathobiology of AD, oxidative stress, inflammation, and cholesterol dyshomeostasis can be potential targets for therapeutic development.

Effects of Marine Sterols against Pathobiology of AD
Marine sterols, including fucosterol and saringasterol, were shown to be promising against AD by targeting oxidative stress, inflammation, cholinergic deficit, amyloidogenesis, cholesterol homeostatic pathway, and signaling systems that are linked with neuronal survival ( Table 2).

Protection against Oxidative Stress
Fighting off oxidative stress, cells are equipped with antioxidant defense systems, comprising antioxidant enzymes such as catalase (CAT), glutathione peroxidase (GPx), and superoxide dismutase (SOD), and non-enzymatic antioxidants, such as glutathione and ascorbate. Dietary consumption of natural compounds can also strengthen the cellular antioxidant defense system through their adaptogenic potential [45]. Natural compounds can also target signaling pathways, including Nrf2/heme oxygenase-1 (HO-1), and thereby, potentiate intrinsic defense system [46]. Marine sterols were shown to protect against oxidative injury in various experimental models through their antioxidant property. Fucosterol and two other sterols, 3,6,17-trihydroxy-stigmasta-4,7,24(28)-triene and 14,15,18,20-diepoxyturbinarin, isolated from Pelvetia siliquosa protected against carbon tetrachloride (CCl 4 )-induced oxidative stress by enhancing SOD, CAT, and GPx1 levels in CCl 4 -challenged rats [20]. Fucosterol isolated from Eisenia bicyclis inhibited ROS production in tert-butyl hydroperoxide (t-BHP)-induced RAW264.7 macrophages [21]. In tert-BHPand tacrine-challenged HepG2cell, fucosterol treatment caused a reduction in ROS and thereby attenuated oxidative stress by increasing glutathione level [22]. Fucosterol from Sargassum binderi protected against oxidative stress in particulate matter-induced injury and inflammation model of A549 human lung epithelial cells by accumulating SOD, CAT, and HO-1 in the cytosol, and Nrf2 levels in the nucleus [23]. A steroidal antioxidant, 7-dehydroerectasteroid F, isolated from the soft coral Dendronephthya gigantea was shown to protect against H 2 O 2 -induced oxidative damage in PC12 cells by enhancing nuclear translocation of Nrf2 and subsequent activation of HO-1 expression [16]. These protective effects of marine sterols against oxidative injury suggest their potential efficacy against oxidative stress-associated neurological disorders, including AD (Figure 1).

Figure 2.
Effects of marine sterols on inflammation. Various stress stimuli, growth factors, and cytokines bind with diversified cell surface receptors (such as TLRs) and mediate different downstream signaling pathways, such as p38 MAPK, JNK, ERK, and NF-κB. These enter into the nucleus for transcription of various pro-inflammatory cytokines, including iNOS, TNFα, COX2, IL-6, and IL1β. All of these ultimately help in the formation of Aβ plaque in brain. Various sterols including fucosterol have been reported to disturb the cell surface receptors as well as major signaling systems leading to inhibition of inflammatory response.

Marine Sterols as Cholinesterase Inhibitors
The cholinergic deficit has been established as a clinical consequence of AD pathology. Cholinesterase inhibitors that can temporarily slow down cholinergic neurotransmission can improve AD outcomes. Marine sterols have also been shown to inhibit the activity of cholinesterase. Fucosterol and 24-hydroperoxy 24-vinylcholesterol showed inhibition against butyrylcholinesterase (BChE) with IC 50 values of 421.72 ± 1.43 and 176.46 ± 2.51 µM, respectively [27]. In another study, fucosterol exhibited dose-dependent inhibition against acetylcholinesterase (AChE) and BChE activities [24]. Enzyme kinetics and structural analysis demonstrated that fucosterol acts as a non-competitive inhibitor to AChE [47].

Marine Sterols as β-Secretase Inhibitors
The aggregation of Aβ represents a characteristic hallmark of AD. β-secretase, which catalyzes the initial breakdown of amyloid precursor protein (APP) to generate Aβ, may represent a promising target for the development of an anti-AD agent [57]. However, evidence suggests that complete inhibition of β-secretase activity might have unintended sequelae with behavioral deficits [58]. Natural products that bear reversible and non-competitive binding patterns with β-secretase may therefore bear therapeutic promise against AD. Natural products, including marine sterols, possess anti-amyloidogenic potential. Fucosterol can be such a potential candidate due to its anti-β-secretase activity [48]. The mode of inhibition is of noncompetitive type, indicating that fucosterol could be an effective and safer inhibitor. Additionally, as shown in computational analysis, fucosterol can be docked on the active site of β-secretase via hydrogen bonding and hydrophobic interactions [59]. Moreover, fucosterol shows competitive binding energies of −10.1 [48] and −19.88 kcal/mol [59], respectively, indicating that hydrogen bonding may ensure close association with enzyme active site, leading to a more effective β-secretase inhibition. Moreover, hecogenin and cholest-4-en-3-one isolated from fat innkeeper worm Urechis unicinctus exhibited anti-β-secretase activity with EC50 of 390.6 µM and 116.3 µM, respectively [29]. With this evidence, these marine sterols can be a potent anti-amyloidogenic agent for use against AD (Figure 3). Figure 3. Effects of marine sterols on APP processing pathways in AD. In the amyloidogenic pathway, APP is cleaved by β-secretase, which produces a soluble amyloid precursor protein β (sAPP β) and a C-terminal fragment β (CTFβ) or C99 fragment. The C99 fragment is cleaved by γ-secretase to generate Aβ and C-terminal fragment γ (CTFγ) or AICD. Further, Aβ constructs Aβ oligomers which ultimately form fibrils and Aβ plaques. Interestingly, fucosterol and other marine sterols inhibit β-secretase, protect against Aβ-mediated inflammation and promote Aβ-clearance.

Marine Sterols as Regulators of Cholesterol Homeostasis
Cholesterol is known to regulate cell-to-cell communication and transmembrane signaling [60], and is critical in the development and maintenance of central nervous system (CNS) neurons. A defect in cholesterol metabolism results in synaptic dysfunction, oxidative stress and inflammation, triggering the onset of AD pathology [61]. Activation of LXR-β upregulates several genes of reverse cholesterol transport, including apolipoprotein E (ApoE), ATP-binding cassette transporter (ABCA1), ATP binding cassette subfamily G member 1 (ABCG1), and sterol regulatory element-binding protein 1 (SREBP1), and thereby this nuclear receptor plays a significant role in the protection against neurodegeneration [42,43]. Upon ligand activation, LXR-β attenuated dopaminergic loss [62] and reduced the toxic burden of mutant huntingtin [63], and also accelerated Aβ clearance [44]. Experimentally, acting as a selective LXR-β agonist, fucosterol augmented the expression of LXR target genes encoding ABCA1, ABCG1, and ApoE [31,52]. This evidence demonstrates that fucosterol may produce similar LXR-β-mediated effects to aid in brain cholesterol homeostasis and play a pivotal role against AD pathology involving Aβ clearance via ABC/SHREBP1/ApoE-dependent pathways (Figure 3). Saringasterol is also a selective LXRβ agonist and promoted the transcriptional activation of ABCA1, ABCG1, and SREBP-1c in multiple cell lines and thus is suggested to be a potent natural cholesterol-lowering agent [31].

Pharmacological Mechanism of Protective Actions of Marine Sterols against AD Pathology
Marine sterols confer neuroprotection by attenuating various factors implicated in the pathobiology of AD, including oxidative stress, inflammation, Aβ 1−42 -induced apoptosis, and cholesterol dyshomeostasis. Antioxidant activity of marine sterols has been manifested by their capacity to promote expression of enzymatic (such as SOD, GPx, CAT, and HO-1) and non-enzymatic (such as GSH) antioxidants, and normalize various oxidative markers (such as ROS; malondialdehyde, MDA; lipid hydroperoxide, LPO and 4-Hydroxynonenal, 4-HNE) ( Figure 1). As activation of Nrf2 results in the upregulation of over 250 genes that encode proteins of antioxidant defense systems [64], overexpression of this transcription factor in marine sterols-treated cultures [16,23] indicates the involvement of the Nrf2 signaling system.
Another potential mechanism of sterol-mediated neuroprotection involves anti-inflammation, which is indicated by their capacity to inhibit the release of proinflammatory and inflammatory mediators (such as IL-1β, IL-6, TNF-α, NO, and PGE2) and the expression of inflammatory enzymes (such as NOS, and COX2) and to downregulate the activation and subsequent nuclear translocation of transcription factor NF-κB, and phosphorylation of MAPK, ERK1/2 and JNK [17,21,23,24] (Figure 2). Yet, another potential mechanism is that the reverse cholesterol transport system under the influence of marine sterols that induces expression of LXR target genes such as ABCA1, ABCG1, and ApoE regulates cholesterol homeostasis in the brain and can prevent AD progression by playing an important role in Aβ clearance ( Figure 3). Furthermore, the cell survival system, such as the TrkB-mediated ERK1/2 signaling pathway, is implicated in sterol-mediated antiapoptotic effects in Aβ-induced hippocampal neurons ( Figure 4). In addition, BDNF expression by sterol treatment also plays a crucial role in ameliorating memory impairment in Aβ-induced aging rats (Figure 4).

Technological Advances toward Sterol Therapy
After the discovery of cholesterol-lowering potentiality, dietary sterols have taken their place in the global market as nutraceuticals supplements, available either in tablet or capsule forms [65]. When administrated, sterols integrate into the mixed micelles in the intestinal chyme and compete with cholesterol to be transported to the enterocyte. Once transported, sterols, however, elated back out from enterocytes into the lumen with the help of ABCG5/G8 system [66]. The ABCG5/G8 system is also responsible for the excretion of sterols that are available in the circulatory system and chylomicrons via the liver biliary system [67]. Therefore, an optimal delivery system or formulation of sterols is necessary to enhance subsequent pharmacological activities.
Sterols are slightly soluble in oil, insoluble in water, and can exist as a crystalline powder. To increase the water solubility, phytosterol esterification was first introduced and used in the first commercial functional food product, margarine [68]. Esterification allows phytosterol to be dissolved in the oil to a ten-fold greater degree than usual and also shows no effect in food texture and test. It was postulated that smaller particle size sterols are more soluble in water than the large size one [69]. However, Keller et al. [70] found no difference in tissue distribution between the customary and nanoscale size of free phytosterol in the hamsters, and also no significant decrease in total cholesterol level was observed. In addition, several methods to date have been adopted to enhance the solubility of sterols, by incorporating free sterols into functional foods and center around reducing crystallization. As an example, Leong et al. constructed sterol nanodispersions by using the emulsification-evaporation technique in the various organic solvents, where they found that larger phytosterol nanoparticles can be produced through a higher organic: aqueous phase ratio and higher homogenization pressure. Furthermore, hexane allowed for obtaining the smallest particle size [71]. Likewise, several methods such as supersaturation using crystallization inhibitors [72], emulsion with lecithin [73], the rapid expansion of supercritical solution into an aqueous solution [74], and microemulsion by solvent displacement [75] are beingly considered. Ling and Lin showed that the bioavailability of sterols can be improved by using the microencapsulation method using in vitro release analysis [76]. In the respective study, they used oven-dried kenaf seed oil containing microencapsulated sterols, where chitosan and alginate with high methoxy pectin were used as shell materials. Ubeyitogullari et al. developed a novel approach to produce low crystallinity phytosterol nanoparticles, which improved both bioaccessibility and bioavailability of phytosterol. In the study, phytosterol nanoparticles were formulated by nanoporous starch aerogels, in combination with supercritical carbon dioxide, wheat starch, and corn starch aerogels. This combination improves sterols' bioavailability by 20 fold when impregnated into wheat starch aerogels monolith [77]. Meng et al. proposed a method to enhance the stability and bioavailability of sterols by formulating hydroxypropyl β-cyclodextrin sterols inclusion complex. Their study showed that the inclusion complex enhanced water solubility of sterols to 8.68 mg mL −1 and resulted in free form 0.02 mg mL −1 [78]. Likewise, many studies have recently been conducted to enhance the bioavailability of sterols, but no studies have focused on brain delivery [79][80][81][82]. Sterol-loaded nanocarriers seem promising to increase more bioavailability in blood; however, more extensive studies are required to investigate tissue and organ distributions and the toxicity risks.

Concluding Remarks and Future Perspectives
This review highlights the neuroprotective potential of marine sterols against AD pathobiology and provides an insight into the underlying molecular mechanisms. Substantial evidence shows that marine sterols protect against AD-associated pathological factors such as apoptosis, oxidative stress, and neuroinflammation by adapting cell survival pathways, such as BDNF, Nrf2, and NF-κB signaling systems and attenuate cholesterol imbalance by activating LXR-mediated reverse cholesterol transport mechanism, and thereby can prevent, or at least slow down, AD progression, suggesting that these marine natural products can be potential candidates in the development of anti-AD agents.
Despite significant progress, marine sterols, such as common phytosterols, are still far from clinical applications. Additional investigations are highly recommended to further elucidate the exact mechanisms of action of marine sterols. Since the existing evidence on the neuroprotective efficacy is based on preclinical studies, human clinical trials with appropriate study protocols are crucial to further characterize the beneficial roles of marine sterols as well as to recommend for future clinical use against AD.
The possible advantages of considering marine sterols in clinical application stand by their multitargeted actions in the pathobiology of AD. Moreover, marine sterols share common features and functionality of cholesterol and other biological sterols, in particular, stigmasterol and β-sitosterol, which have shown promise in clinical trials against various chronic diseases [83]. With technological advances, including microencapsulation or nanoparticle-based drug delivery, marine sterols may offer potential lead chemicals in developing viable anti-AD therapeutics.