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Dietary Sargassum fusiforme improves memory and reduces amyloid plaque load in an Alzheimer’s disease mouse model

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  • Hasselt University and Maastricht University

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Activation of liver X receptors (LXRs) by synthetic agonists was found to improve cognition in Alzheimer’s disease (AD) mice. However, these LXR agonists induce hypertriglyceridemia and hepatic steatosis, hampering their use in the clinic. We hypothesized that phytosterols as LXR agonists enhance cognition in AD without affecting plasma and hepatic triglycerides. Phytosterols previously reported to activate LXRs were tested in a luciferase-based LXR reporter assay. Using this assay, we found that phytosterols commonly present in a Western type diet in physiological concentrations do not activate LXRs. However, a lipid extract of the 24(S)-Saringosterol-containing seaweed Sargassum fusiforme did potently activate LXRβ. Dietary supplementation of crude Sargassum fusiforme or a Sargassum fusiforme-derived lipid extract to AD mice significantly improved short-term memory and reduced hippocampal Aβ plaque load by 81%. Notably, none of the side effects typically induced by full synthetic LXR agonists were observed. In contrast, administration of the synthetic LXRα activator, AZ876, did not improve cognition and resulted in the accumulation of lipid droplets in the liver. Administration of Sargassum fusiforme-derived 24(S)-Saringosterol to cultured neurons reduced the secretion of Aβ42. Moreover, conditioned medium from 24(S)-Saringosterol-treated astrocytes added to microglia increased phagocytosis of Aβ. Our data show that Sargassum fusiforme improves cognition and alleviates AD pathology. This may be explained at least partly by 24(S)-Saringosterol-mediated LXRβ activation.
Dietary supplementation with Sargassum fusiforme reduces serum cholesterol, and increases 24(S)/ (R)-Saringosterol in serum and cerebellum. Concentrations of 24(S)/(R)-Saringosterol (a,b), cholesterol (c,d), cholesterol precursors (e,f), cholesterol metabolites (g,h), and phytosterols (i,j) were determined in serum and cerebellum samples of WT and APPswePS1ΔE9 mice (AD) fed either control chow or chow supplemented with Sargassum fusiforme. 24(S)/(R)-Saringosterol concentration was affected by diet in CNS (F(1, 31) = 438.8, p < 0.0001) and serum (F(1, 31) = 30.05, p < 0.0001) as determined by ANOVA, and post-hoc Sidak's test showed that this effect existed in both genotypes (WT CNS: p < 0.0001; WT serum: p = 0.0005; AD CNS: p < 0.0001; AD serum: p = 0.0157). Cholesterol concentration in serum was affected by diet (F(3, 31) = 80.59, p < 0.0001) for both genotypes (WT: p < 0.001; AD: p = 0.0002). Regarding the cholesterol precursors, concentration of lanosterol was affected by diet in CNS (F(1, 32) = 6.463, p = 0.0161) and serum (F(1, 31) = 38.28, p < 0.0001) of WT animals (CNS: p = 0.0265; serum: p = 0.0002) and AD animals (serum: p = 0.0024). Concentration of lathosterol was affected by diet in CNS (F(1, 32) = 19.32, p = 0.0001) and serum (F(1, 31) = 13.42, p = 0.0009) of WT animals (CNS: p = 0.0076; serum: p = 0.0474). Regarding the cholesterol metabolites, concentration of 27-hydroxycholesterol (27OHC) was affected by diet in CNS (F(1, 32) = 20.04, p = 0.0009) of WT animals (p = 0.0004). Concentration of 24(S)-hydroxycholesterol (24(S) OHC) was affected by diet in CNS (F(1, 31) = 8.071, p = 0.0079) of WT animals (p = 0.0079). Regarding the
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Dietary Sargassum fusiforme
improves memory and reduces
amyloid plaque load in an
Alzheimer’s disease mouse model
Jeroen Bogie1, Cindy Hoeks1, Melissa Schepers1,9, Assia Tiane1,9, Ann Cuypers2, Frank Leijten3,
Yupyn Chintapakorn4, Thiti Suttiyut4, Surachai Pornpakakul5, Dicky Struik6, Anja Kerksiek7,
Hong-Bing Liu8, Niels Hellings1, Pilar Martinez-Martinez9, Johan W. Jonker6, Ilse Dewachter1,
Eric Sijbrands
3, Jochen Walter10, Jerome Hendriks1, Albert Groen11, Bart Staels12,
Dieter Lütjohann7, Tim Vanmierlo
1,9 & Monique Mulder3
Activation of liver X receptors (LXRs) by synthetic agonists was found to improve cognition in
Alzheimer’s disease (AD) mice. However, these LXR agonists induce hypertriglyceridemia and hepatic
steatosis, hampering their use in the clinic. We hypothesized that phytosterols as LXR agonists enhance
cognition in AD without aecting plasma and hepatic triglycerides. Phytosterols previously reported
to activate LXRs were tested in a luciferase-based LXR reporter assay. Using this assay, we found that
phytosterols commonly present in a Western type diet in physiological concentrations do not activate
LXRs. However, a lipid extract of the 24(S)-Saringosterol-containing seaweed Sargassum fusiforme
did potently activate LXRβ. Dietary supplementation of crude Sargassum fusiforme or a Sargassum
fusiforme-derived lipid extract to AD mice signicantly improved short-term memory and reduced
hippocampal Aβ plaque load by 81%. Notably, none of the side eects typically induced by full synthetic
LXR agonists were observed. In contrast, administration of the synthetic LXRα activator, AZ876, did
not improve cognition and resulted in the accumulation of lipid droplets in the liver. Administration
of Sargassum fusiforme-derived 24(S)-Saringosterol to cultured neurons reduced the secretion of
Aβ42. Moreover, conditioned medium from 24(S)-Saringosterol-treated astrocytes added to microglia
increased phagocytosis of Aβ. Our data show that Sargassum fusiforme improves cognition and
alleviates AD pathology. This may be explained at least partly by 24(S)-Saringosterol-mediated LXRβ
activation.
1Department of Immunology and Biochemistry, Biomedical research institute, Hasselt University, Martelarenlaan 42,
3500, Hasselt, Belgium. 2Centre for Environmental Sciences, Hasselt University, Martelarenlaan 42, 3500, Hasselt,
Belgium. 3Department of Internal Medicine, Laboratory of Vascular Medicine, Erasmus University Medical Center,
Wytemaweg 80, 3015 CN, Rotterdam, The Netherlands. 4Center of Excellence in Environment and Plant Physiology,
Department of Botany, Faculty of Science, Chulalongkorn University, Bangkok, 10330, Thailand. 5Department
of Chemistry, Faculty of Science, Chulalongkorn University, Bangkok, 10330, Thailand. 6Section of Molecular
Metabolism and Nutrition, Department of Pediatrics, University of Groningen, University Medical Center Groningen,
Hanzeplein 1, 9713 GZ, Groningen, The Netherlands. 7Institute for Clinical Chemistry and Clinical Pharmacology,
Sigmund-Freud-Str. 25, D-53127, Bonn, Germany. 8Key Laboratory of Marine Drugs, Ministry of Education, School
of Medicine and Pharmacy, Ocean University of China, Yushan Road 5, 266003, Qingdao, China. 9School for mental
health and neuroscience, Maastricht University, Universiteitssingel 50, 6229ER, Maastricht, The Netherlands.
10Department of Neurology, Molecular Cell Biology, University of Bonn, Sigmund-Freud-Str. 25, 53127, Bonn,
Germany. 11Department of Medical Biochemistry, Academic Medical Center, University of Amsterdam, Meibergdreef
9, 1105 AZ, Amsterdam, The Netherlands. 12University of Lille - EGID, Inserm, U1011, University Hospital CHU,
Institut Pasteur de Lille, F-59019, Lille, France. Jeroen Bogie, Cindy Hoeks, Tim Vanmierlo and Monique Mulder
contributed equally. Correspondence and requests for materials should be addressed to T.V. (email: tim.vanmierlo@
uhasselt.be)
Received: 1 August 2018
Accepted: 5 March 2019
Published: xx xx xxxx
OPEN
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Alzheimer’s disease (AD) is a progressive neurological disorder characterized by an accumulation of extracellular
amyloid-β (Aβ), intracellular neurobrillary tangles, loss of synapses, neuroinammation, and by a gradual pro-
gression of memory loss1. Accumulating evidence suggests a role for a disturbed cholesterol turnover in the cen-
tral nervous system (CNS) in AD pathogenesis211. In line with this, stimulation of cholesterol turnover improves
disease outcome in animal models of AD2,1218. Liver X receptors (LXR) are master regulators of cholesterol and
triglyceride turnover and suppress an inammatory transcriptional prole via trans-repression of NFκB signa-
ling19. erefore, LXRs are promising well-studied therapeutic targets for increasing cholesterol turnover and
decreasing neuroinammation in AD2024. We and others have reported that synthetic pan LXR agonists improve
the cognitive phenotype in animal models of AD, decrease synaptic compensatory mechanisms, and stimulate
the proteolytic degradation of Aβ by microglia2,13,14,2426. However, synthetic full LXR agonists systematically
cause adverse side eects, such as hypertriglyceridemia and hepatic steatosis, hampering their translation to the
clinic2730.
Phytosterols are structurally similar to cholesterol. However, in contrast to cholesterol, they can cross the
blood-brain barrier (BBB) and accumulate in brain parenchyma11,3133. Several of the more than 260 identied
phytosterols, such as β-sitosterol, fucosterol, stigmasterol, schottenol, 24(S)-Saringosterol, and spinasterol, have
been reported to activate LXRs in vitro3439. Moreover, β-sitosterol and stigmasterol modulate AD pathology in
in vitro models for AD4042. Phytosterols do not induce hypertriglyceridemia and hepatic steatosis, which may be
a consequence of their ABCG5/G8-mediated hepatic excretion into the bile11,43,44. e absence of unwanted side
eects renders phytosterols interesting therapeutic candidates for inducing LXR activation in the CNS.
We aim to identify phytosterols and phytosterol-containing extracts that activate LXRs in vitro, to test their
eect on memory performance and Aβ plaque pathology in an animal model of AD. We found that phytos-
terols typically present in a Western type diet or extracts from a range of Eastern plants hardly activate LXRα
Figure 1. A crude lipid extract of Sargassum fusiforme activates LXRβ. LXRα (a) and LXRβ (b) activation was
assessed with a luciferase-based reporter assay. CHME3 cells were stimulated for 18 hours with vehicle (striped
bar/dotted line), 1, 3, or 5 µg/μl Sargassum fusiforme or 1, 10 or 100 µg/μl of the specied plant extracts. No
dierence was found between groups (LXRα χ2(7) = 11.55, p = 0.1165, LXRβ χ2(7) = 7.689, p = 0.3608; all
datasets analysed using Kruskal-Wallis test). All results are displayed as fold change compared to vehicle control
(striped bar/dotted line). Bars represent mean ± SEM (n 3).
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or LXRβ. In contrast, a lipid extract of the edible brown seaweed Sargassum fusiforme which contains large
amounts of 24(S)-Saringosterol potently activated LXRβ. In a mouse model for AD, dietary supplementation
with Sargassum fusiforme or its lipid extract not only increased the expression of LXR response genes in the CNS,
but also improved cognition without inducing hepatic steatosis. Improved memory performance in these mice
was paralleled by a strong reduction in CNS Aβ plaque load. In contrast, the selective LXRα agonist AZ876 did
not counteract cognitive decline in AD mice or reduce Aβ plaque load, and induced liver steatosis. ese ndings
indicate that Sargassum fusiforme is an attractive option for add-on treatments in the emerging eld of nutritional
neuroscience.
Results
Phytosterols present in a Western diet do not activate LXRs. First, we determined the capacity of
phytosterols typically present in a Western diet to activate LXRs. To dene cellular specicity in LXR activation in
vitro, cells derived from peripheral tissues (HEK293.T and COS7) and the CNS (CHME3, MO3.13, N2a/APPswe)
were tested. Physiologically relevant concentrations of stigmasterol, fucosterol, brassicasterol, β-sitosterol, or a
mix of phytosterols did not activate LXRα or LXRβ in any of the cell lines used (Fig.S2). An increased incubation
period promoted the capacity of the synthetic LXR agonist T0901317, but not of phytosterols, to activate LXRα
and LXRβ (Fig.S3a,b). Although cellular uptake of phytosterols and their capacity to activate nuclear receptors
improves when complexed to proteins45,46, pre-incubation of phytosterols with BSA did not signicantly increase
their ability to activate LXRα and LXRβ (Fig.S3c,d).
An extract of Sargassum fusiforme activates LXRβ in vitro. In addition to phytosterols present in
a Western diet, crude extracts of Eastern plants were screened for their capacity to activate LXRα or LXRβ.
Plants were chosen based on their application in Asian traditional medicine and their presence in an Eastern diet.
Extracts from Asparagus racemosus, Azadirachta indica, Cassia stula, Curcuma aromatica, Datura metel, Piper
retrofractum, Senna tora, and Terminalia chebula did not signicantly activate either LXRα or LXRβ in microglial
CHME3 cells (Fig.1). However, at a dose of 5 µg/ml an extract of Sargassum fusiforme containing the recently
identied LXRβ agonist 24(S)-Saringosterol34 signicantly activated LXRβ but not LXRα (Fig.1). Higher doses of
Sargassum fusiforme induced cell death (data not shown). As Sargassum fusiforme showed the highest capacity to
activate LXRβ, it was selected for further in vivo testing.
Dietary supplementation with Sargassum fusiforme results in 24(S)-Saringosterol accumula-
tion in the cerebellum and activation of LXR-response genes in AD mice. To assess in vivo eects
Figure 2. Dietary supplementation with Sargassum fusiforme activates LXRs in the brain. Gene expression
of Abcg1 (a), Scd1 (b), Srebp-1c (c), Abca1 (d), and ApoE (e) was measured in the brain of WT and
APPswePS1ΔE9 mice (AD) fed normal chow or chow supplemented with Sargassum fusiforme. Two-way
ANOVA revealed a diet eect for Abcg1 (F (1, 17) = 6.244, p = 0.0230)), and Scd1 (F (1, 15) = 7.673, p = 0.0143),
and a genotype eect for Srebp-1c (F (1, 16) = 8.446, p = 0.0103). Gene expression was normalized to Cyca and
Hmbs, and expressed as fold change compared to WT mice fed the control diet. Bars represent mean ± SEM
(n 5).
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Figure 3. Dietary supplementation with Sargassum fusiforme reduces serum cholesterol, and increases 24(S)/
(R)-Saringosterol in serum and cerebellum. Concentrations of 24(S)/(R)-Saringosterol (a,b), cholesterol
(c,d), cholesterol precursors (e,f), cholesterol metabolites (g,h), and phytosterols (i,j) were determined in
serum and cerebellum samples of WT and APPswePS1ΔE9 mice (AD) fed either control chow or chow
supplemented with Sargassum fusiforme. 24(S)/(R)-Saringosterol concentration was aected by diet in CNS
(F(1, 31) = 438.8, p < 0.0001) and serum (F(1, 31) = 30.05, p < 0.0001) as determined by ANOVA, and
post-hoc Sidak’s test showed that this eect existed in both genotypes (WT CNS: p < 0.0001; WT serum:
p = 0.0005; AD CNS: p < 0.0001; AD serum: p = 0.0157). Cholesterol concentration in serum was aected
by diet (F(3, 31) = 80.59, p < 0.0001) for both genotypes (WT: p < 0.001; AD: p = 0.0002). Regarding the
cholesterol precursors, concentration of lanosterol was aected by diet in CNS (F(1, 32) = 6.463, p = 0.0161)
and serum (F(1, 31) = 38.28, p < 0.0001) of WT animals (CNS: p = 0.0265; serum: p = 0.0002) and AD animals
(serum: p = 0.0024). Concentration of lathosterol was aected by diet in CNS (F(1, 32) = 19.32, p = 0.0001)
and serum (F(1, 31) = 13.42, p = 0.0009) of WT animals (CNS: p = 0.0076; serum: p = 0.0474). Regarding
the cholesterol metabolites, concentration of 27-hydroxycholesterol (27OHC) was aected by diet in CNS
(F(1, 32) = 20.04, p = 0.0009) of WT animals (p = 0.0004). Concentration of 24(S)-hydroxycholesterol (24(S)
OHC) was aected by diet in CNS (F(1, 31) = 8.071, p = 0.0079) of WT animals (p = 0.0079). Regarding the
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of Sargassum fusiforme APPswePS1ΔE9 mice were used as a model of AD47. ese mice begin to develop Aβ
plaques at 4 months of age and cognitive decline occurs from 6 months onwards48,49. From the age of 5 months
APPswePS1ΔE9 mice and WT littermates were fed either standard chow or chow supplemented with 50%
(w/w) dried crude Sargassum fusiforme. Ten weeks of Sargassum fusiforme dietary supplementation resulted in
LXR activation in the CNS, evidenced by a cerebral induction of LXR response genes (Abcg1, Scd1) (Fig.2a,b).
ApoE, Srebp-1c, and Abcg1 expression was not altered in animals treated with Sargassum fusiforme (Fig.2c–e).
Interestingly, 24(S)/(R)-Saringosterol was detectable in serum and in the cerebellum of animals that were fed
Sargassum fusiforme, but not in those fed normal chow (Fig.3a,b). As Sargassum fusiforme contains predomi-
nantly 24(S)-Saringosterol34, we postulate that this isoforms is the most abundant isoform present in animals fed
Sargassum fusiforme. Animals fed a diet supplemented with Sargassum fusiforme did not show dierences in total
cholesterol content in the cerebellum (Fig.3c), but showed a marked reduction in circulating cholesterol levels
compared to chow fed animals (Fig.3d). Levels of cholesterol precursors, cholesterol metabolites, and phytoster-
ols were decreased in serum and cerebellum of animals supplemented with Sargassum fusiforme (Fig.3e–j). On a
standard chow diet, WT mice displayed signicantly higher serum cholesterol levels than AD mice (Fig.3d), as
described previously10,50.
Sargassum fusiforme improves memory and reduced Aβ plaque load in AD mice. Next, we inves-
tigated the impact of dietary supplementation with crude Sargassum fusiforme on cognition and neuropathology
in APPswePS1ΔE9 mice. To assess the inuence of selective LXRα activation on cognition, animals were fed
with the synthetic selective LXRα agonist AZ876. e open eld test revealed no eect of genotype or diet on
general locomotor activity, and anxiety (Table1). Dietary supplementation with Sargassum fusiforme signicantly
improved object memory in APPswePS1ΔE9 mice in the object recognition task (ORT) at a 1 h but not a 24 h
inter-trial interval (Fig.4a,b). In contrast, selective LXRα activation did not impact object memory at either the
1 h or 24 h inter-trial interval. Sargassum fusiforme, but not AZ786, markedly decreased Aβ plaque load in the
cortex (70% decrease; Fig.4c,e) and hippocampus (81% decrease; Fig.4d,e) in APPswePS1ΔE9 mice. In con-
cordance, Sargassum fusiforme supplementation reduced Aβ40 protein and APP mRNA expression in the CNS
of AD animals (Fig.4f,h). Sargassum fusiforme did not signicantly reduce insoluble Aβ42 (Fig.4g). Reduced
Aβ plaque load was not associated with an increased expression of phagocytic receptors such as Axl and MerTK
(Fig.S4a,b)51. Yet, the reduced Aβ plaque load was associated with a reduced TREM2 expression, reecting the
expelled need for further Aβ clearance (Fig.S4c).
To determine whether the lipid moiety of Sargassum fusiforme was sucient to modulate the AD-related
pathology, a puried lipid extract of Sargassum fusiforme was administered daily by oral gavage to APPswePS1ΔE9
and WT littermates for 45 days. Working memory was signicantly improved in APPswePS1ΔE9 mice, resulting
in the prevention of the spatial working decit (Fig.5a,b). In line with these ndings, a signicant reduction in
insoluble Aβ42 was observed in the cortex (99% decrease; Fig.5c) and hippocampus (57% decrease; Fig.5d) of the
treated APPswePS1ΔE9 mice. Notably, treatment with the Sargassum fusiforme lipid extract resulted an increased
expression of ApoE in the CNS (Fig.5e–g), which indicates active LXR signaling.
Sargassum fusiforme feeding does not lead to liver steatosis. While synthetic LXR agonists are
well-known to induce liver steatosis in mice52, Sargassum fusiforme supplementation did not induce liver steatosis
in APPswePS1ΔE9 mice, as evidenced by the absence of lipid droplets within the liver of these animals (Fig.6a,b).
Supplementation with AZ876 resulted in a marked increase in lipid droplets in the liver (Fig.6a,b). Likewise,
while AZ876 increased circulating triglyceride levels, Sargassum fusiforme did not impact the level of triglycerides
(Fig.6c). Similar ndings were observed in WT mice fed chow supplemented with Sargassum fusiforme or AZ876
(data not shown). Sargassum fusiforme is known to contain relatively high levels of (in)organic arsenic that can
accumulate in the body53. Although relatively high levels of arsenic were measured in dried Sargassum fusiforme
(40.4 mg/kg), no arsenic accumulation was detectable in lung tissue of the animals that consumed Sargassum
fusiforme (Fig.6d).
24(S)-Saringosterol increases microglial Aβ clearance and reduces neuronal Aβ release. To
obtain insight into the mechanisms by which Sargassum fusiforme-derived 24(S)-Saringosterol impacts AD patho-
genesis we used well-established in vitro models that mimic AD-related pathological processes. Supplementation
of astrocytes with 24(S)-Saringosterol resulted in an increased ApoE secretion (Fig.7a,b). Furthermore, supple-
mentation of microglia with conditioned medium of 24(S)-Saringosterol-treated astrocytes promoted microglial
clearance of Aβ142 (Fig.7c). 24(S)-Saringosterol did not directly impact the capacity of microglia to internalize
Aβ142. (Fig.7d). Finally, 24(S)-Saringosterol was found to reduce the release of Aβ42 using neuronal N2a cells
overexpressing APP (Fig.7e).
plant sterols, concentration of sitosterol was aected by diet in CNS (F(1, 32) = 30.62, p < 0.0001) and serum
(F(1, 31) = 96.88, p < 0.0001) of WT animals (CNS: p = 0.0026; serum: p < 0.0001) and AD animals (CNS:
p = 0.0028; serum: p < 0.0001). Concentration of campesterol was aected by diet in CNS (F(1, 32) = 17.02,
p = 0.0002) and serum (F(1, 31) = 76.4, p < 0.0001) of WT animals (CNS: p = 0.0175; serum: p < 0.0001) and
AD animals (serum: p < 0.0001). Concentration of stigmasterol was aected by diet in serum (F(1, 31) = 99.89,
p < 0.0001) of WT animals (p < 0.0001) and AD animals (p < 0.0001). Bars represent mean ± SEM (n = 5).
Abbreviations:7aOHC 7a-hydroxycholesterol.
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Discussion
Our data show that dietary supplementation with the brown seaweed Sargassum fusiforme improves cognitive
function and reduces Aβ load in APPswePS1ΔE9 mice without inducing liver steatosis. Our data further sug-
gest the involvement of the stereospecic oxyphytosterol 24(S)-Saringosterol, a selective LXRβ activator, in the
observed eects on cognition and Aβ plaque load. Considering that selective LXRα activation did not improve
cognitive decline or Aβ plaque deposition in APPswePS1ΔE9 mice, our ndings suggest that natural and possibly
also synthetic LXRβ agonists are attractive options for the treatment of neurodegenerative disorders such as AD.
Phytosterols, which can cross the blood-brain barrier, have been reported to activate LXRs without inducing
hypertriglyceridemia and hepatic steatosis, rendering them potentially suitable candidates for treatment of neu-
rodegenerative disorders11. Yet, in contrast with reported data on LXR activation by phytosterols common in a
Western diet3439, we observed very little if any eect of stigmasterol, brassicasterol, β-sitosterol, fucosterol, or a
commercial mix of phytosterols, on activation of LXRα and LXRβ in vitro. e discrepancy between our data and
the literature may be explained by dierences in the experimental design, such as phytosterol concentration, incu-
bation time, and cell type and assay used to measure LXR activation. For instance, we used a cell-based luciferase
reporter assay and CNS-derived cell lines, as well as HEK293.T and COS-7 cells, to dene LXR activation and
cellular specicity in LXR activation, while other studies used cell-free assays38. Furthermore, we used phytosterol
concentrations in a physiological range that can be reached through dietary supplementation (1–25 µM), whereas
other studies used concentrations far exceeding this physiological range up to 200 µM36. Of note, compared to
synthetic LXR agonists, the reported ability of phytosterols commonly present in a Western diet to activate LXRs
is relatively limited3439. In line with this, no alterations were observed in the expression of LXR-response genes
in the brain of ABCG5-knockout mice despite high cerebral levels of common phytosterols26. In summary, our
data indicate that phytosterols present in a Western type diet do not activate LXRs at concentrations that can be
reached through dietary supplementation11.
Out of the tested extracts of Asian plants, the extract of Sargassum fusiforme most potently activated LXRβ
in vitro. This is in line with the recently reported LXRβ-activating effects of Sargassum fusiforme-derived
24(S)-Saringosterol34. In Sargassum fusiforme–fed animals, 24(S)-Saringosterol was detected in the cere-
bellum in concentrations equal to common oxysterols, suggesting a good CNS bioavailability. The amount
of 24(S)-Saringosterol detected in the CNS was comparable to previously reported concentrations of other
dietary phytosterols in the CNS26,32,33. With respect to these findings, we found that cerebral expression of
LXR-response genes was upregulated in animals fed Sargassum fusiforme-enriched chow, further supporting the
ability of Sargassum fusiforme to activate LXRβ. e route of entry of 24(S)-Saringosterol to the CNS remains
unclear. Further research is warranted to dene the role of ApoE and SR-B1 in facilitating the transport of
24(S)-Saringosterol into the CNS11,32. Moreover, since 24(S)-Saringosterol is oxydated plant sterol, we hypoth-
esize that 24(S)-Saringosterol can enter the CNS via transcellular diusion, comparable to other oxidized cho-
lesterol derivatives such as 24(S)-Hydroxycholesterol54. Interestingly, in contrast to 24(S)-Saringosterol, all plant
sterol concentrations decreased in serum and CNS upon feeding Sargassum fusiforme. It can be hypothesized that
24(S)-Saringosterol activates intestinal and hepatic Abcg5/g8 transporters and thereby drives the enterohepatic
secretion of plant sterols, thereby lowering circulating levels of sterols55. e latter hypothesis is in line with the
strong reduction in circulating cholesterol levels we found in this study.
Our data indicate that the LXRα selective agonist AZ876 does not signicantly counteract cognitive decline
or reduce Aβ plaque load in APPswePS1ΔE9 mice. However, no active LXR signaling was found in the CNS of
AZ876 treated animals, which suggest poor CNS bioavailability of AZ876 and consequently does not allow us to
conclude that LXRα activation does not impact AD pathology. On the other hand, while both LXRα and LXRβ
are expressed in the murine CNS, expression of LXRβ is 15-fold higher compared to LXRα56,57. Hence, the relative
contribution of LXRα activation upon full-LXR agonist treatment is therefore expected to be limited. e latter
studies may suggest that the benecial impact of pan-LXR agonists on cognitive decline in AD models likely relies
on LXRβ activation.
Synthetic LXR agonists that reduce AD pathology and improve cognitive performance induce hypertriglycer-
idemia and liver steatosis2,13,14,24,25,2730. Sargassum fusiforme supplementation counteracted cognitive decline of
APPswePS1ΔE9 transgenic mice without inducing hepatic steatosis. is may be explained by selective hepatic
LXRβ activation, since animals fed AZ786 (2 mg/kg body weight) displayed a marked increase of liver lipid drop-
lets, indicative of liver steatosis. Alternatively, the presence of other protective compounds present in Sargassum
fusiforme such as fucosterol may prevent hepatic steatosis11. is is supported by the observation that activation
of LXRβ by synthetic agonists was also reported to induce plasma and hepatic hypertriglyceridemia58.
Genotype Diet TIZ
centre (s) TIZ walls and
corners (s) DM (m) N
WT Control 29.5 ± 7.7 570 ± 7.7 36.1 ± 1.6 12
AD Control 29.4 ± 4.2 569.6 ± 4.3 39.0 ± 1.9 13
WT S. fusiforme 30.9 ± 3.4 564.9 ± 4.3 42.0 ± 1.9 9
AD S. fusiforme 33.5 ± 4.1 564.9 ± 5.2 47.6 ± 2.0 7
WT AZ876 28.3 ± 4.3 569.2 ± 4.9 37.3 ± 2.4 11
AD AZ876 31.7 ± 6.4 567.2 ± 6.4 39.9 ± 2.7 13
Table 1. Genotype and diet do not impact general locomotor activity or anxiety levels. Results of open eld test.
Values are displayed as mean ± SEM. Abbreviations: TIZ, time in zone; DM, distance moved.
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Figure 4. Dietary supplementation with Sargassum fusiforme reduces cognitive decline and Aβ plaque
load in APPswePS1ΔE9 mice. (a,b) Cognitive functioning was determined using the object recognition
task. e interval between rst and second trial was set at 1 hour (1 h ITI) or 24 hours (24 h ITI). D2 value is
calculated as the ratio between exploration time spent at the new object and the familiar object in the second
trial, with d2 value > 0 indicating intact object memory. At 1 h ITI the object memory was found to be intact
in WT animals on control diet (t(9) = 4.71, p = 0.0011, one sample t-test), in WT animals on Sargassum
fusiforme-supplemented diet (t(9) = 2.166, p = 0.0585, one sample t-test), and in AD animals on Sargassum
fusiforme-supplemented diet (t(8) = 2.77, p = 0.0243, one sample t-test). Bars represent mean ± SEM from
two independent experiments (n 9 per treatment). (ce) Aβ plaque load was quantied in cortex (d) and
hippocampus (e) of APPswePS1ΔE9 mice using immunohistochemistry (n 5 per treatment). Aβ load is
calculated as percentage of surface coverage, and was found to be decreased in AD animals fed Sargassum
fusiforme-enriched chow in cortex (F (3, 24) = 25.79, p < 0.0001, ANOVA; Tukey’s post-hoc for diet eect in
AD genotype: p = 0.0005) and hippocampus (F (3, 24) = 32.13, p < 0.0001, ANOVA; Tukey’s post-hoc for diet
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We show that dietary supplementation with crude or lipid extract of Sargassum fusiforme reduces Aβ load in
AD mice. Our in vitro data indicate that factors released by 24(S)-Saringosterol-treated astrocytes promote micro-
glial clearance of Aβ1–42. Within the brain, astrocytes are the major source of the LXR response gene Apoe. ApoE
is well-known to increase microglial clearance of Aβ59, possibly via inhibiting cellular uptake of Aβ60. Of interest,
24(S)-Saringosterol and a lipid extract of Sargassum fusiforme increased ApoE protein expression in astrocytes
and the CNS respectively, which suggests a role for ApoE in the observed increase in microglial Aβ clearance. In
addition, we provide evidence that 24(S)-Saringosterol decreases the release of Aβ by neurons in vitro. Together,
these ndings indicate that 24(S)-Saringosterol impacts both the clearance and generation of Aβ42. ese ndings
are of particular interest, as benecial eects of LXR agonists on cognition are not necessarily accompanied by a
reduced Aβ plaque load14,41. Recently, it has been reported that other constituents of Sargassum fusiforme, such as
fucosterol, fucoidan, and fucoxantin improve learning and memory deciencies in pharmacological models for
cognitive impairment6163. Although synergism among constituents of Sargassum fusiforme is likely, future studies
should determine whether 24(S)-Saringosterol is sucient to improve cognitive performance and AD pathology.
Although relatively high levels of arsenic were measured in extracts of Sargassum fusiforme, no arsenic was
detectable in lung tissue, which generally represents a major reservoir for this element64. Arsenic present in
Sargassum fusiforme may therefore be rapidly excreted, degraded or absorbed in low amounts in the intestine.
In summary, our data indicate that phytosterols present in a Western type diet do not activate LXRs at con-
centrations that can be reached through dietary supplementation. However, a crude lipid extract of the edible
seaweed Sargassum fusiforme did selectively activate LXRβ in vitro. Here, we show for the rst time that dietary
supplementation with Sargassum fusiforme signicantly improves memory performance and reduces Aβ plaque
load in a well-established AD model. e accumulation of the natural LXRβ agonist 24(S)-Saringosterol in the
CNS suggests its involvement in the neuroprotective impact of Sargassum fusiforme. Collectively, our ndings
point to 24(S)-Saringosterol-containing Sargassum fusiforme as being a novel candidate for dietary supplementa-
tion to prevent or modulate neurodegenerative disorders such as Alzheimer’s disease.
Materials and Methods
Preparation of plant extracts. Indigenous Asian plants (Asparagus racemosus, Azadirachta indica, Cassia
stula, Curcuma aromatica, Datura metel, Piper retrofractum, Sargassum fusiforme, Senna tora, and Terminalia
chebula), were selected based on their use in Asian traditional medicine as cognition enhancers. Extracts from
all plants, with exception of Sargassum fusiforme, were prepared by the maceration method. All plants were dried
in a hot air oven at 50 °C. e dried samples were nely powdered and soaked in 95% ethanol overnight at room
temperature and ltered with Whatman lter paper No. 3 (Sigma-Aldrich, Bornem, Belgium). ree consecutive
ltrates were pooled and evaporated in a vacuum rotary evaporator at 40 °C. e crude lipid fraction of Sargassum
fusiforme was extracted using an adaptation of the Folch method65. Briey, Sargassum fusiforme was harvested
by hand in early spring, washed in sea water, boiled for three hours and dried in a hot air oven. Next, dried
Sargassum fusiforme (Clearspring Ltd, London, UK) was soaked overnight at room temperature in a 2:1 (v/v)
chloroform and methanol (both VWR, Leuven, Belgium) mixture. e chloroform/methanol extract was evap-
orated under a N2 stream and the remaining lipid fraction was dissolved in ethanol. Using gas chromatography/
mass spectrometry, 24(S)-Saringosterol content of dried Sargassum fusiforme was determined to be 69.4 ng/mg.
Cell culture. Immortalized human embryonic kidney cells (HEK293.T), human microglia (CHME3; a kind
gi from prof. dr. M. Tardieu, Universite Paris-Sud, France66), human oligodendrocytes (MO3.13), mouse neu-
roblastoma expressing APPswe (N2a/APPswe; a kind gi from prof. dr. T.W. Kim, Colombia University, USA67),
and monkey kidney cells (COS7) were used for in vitro experiments. All cell lines were cultured in DMEM
(Sigma-Aldrich) containing 10% heat-inactivated FCS (Invitrogen, Merelbeke, Belgium) and 100 U penicil-
lin/100 µg streptomycin/ml (Invitrogen), at 37 °C/5% CO2. For phytosterol treatment, cells were incubated for
18 hours in culture medium without FCS containing the Eastern plants extracts, brassicasterol (Sigma-Aldrich),
β-sitosterol, (Sigma-Aldrich), fucosterol (Sigma-Aldrich), stigmasterol (analytic conrmed purity of 99,9%), phy-
tosterol mix (containing 60% β-sitosterol, 25% campesterol, and 15% stigmasterol; kindly provided by Ingmar
Wester Raisio, Finland), T0901317 (Cayman Chemicals, Huissen, the Netherlands), ethanol (VWR), or DMSO
(Sigma-Aldrich).
Luciferase-based nuclear receptor reporter assay. To determine the ability of plant extracts and
phytosterols to bind LXRα and LXRβ, a luciferase-based reporter assay was performed using the ONE-GloTM
Luciferase Assay System kit (Promega, Leiden, the Netherlands), according to manufacturer’s instructions. Cell
lines were transfected with bacterial plasmid constructs expressing luciferase under control of the promotor region
of the ligand-binding domain for LXRα or LXRβ68. Cells were grown to 50–60% conuency in 60 mm plates,
transfected with 1.8 μg of plasmid DNA including 0.2 μg pGAL4hLXRα or pGAL4hLXRβ, 1 μg pG5-TK-GL3, and
0.6 μg of pCMV-β-galactosidase, using JetPEI (Polyplus-transfection SA, Illkirch, France) as transfection reagent.
Following treatment, cells were lysed in lysis buer (25 mM Glycyl-Glycine, 15 mM MgSO4, 4 mM EGTA, and
1x Triton; all from Sigma). To correct for transfection ecacy, β-galactosidase activity was measured using lysate
diluted 1:10 in B-gal buer, consisting of 20% 2-Nitrophenyl β-D-galactopyranoside (ONGP; Sigma) and 80%
eect in AD genotype: p < 0.0001). Representative IHC staining of all groups is shown (e). (f,g) Sargassum
fusiforme treated APPswePS1ΔE9 mice show a signicant decrease in Aβ40 ((f); U = 6, nctrl = 11, nextract = 5,
p = 0.0133, Mann-Whitney) but not Aβ42 levels (g). (h) Sargassum fusiforme treated APPswePS1ΔE9 mice show
a signicant decrease in the mRNA expression of APP (U = 0, nctrl = 8, nextract = 4, p = 0.0040, Mann-Whitney).
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(v/v) Buer Z (0.1 M Na2HPO4, 10 mM KCl, 1 mM MgSO4, and 3.4 µl/ml β-mercaptoethanol; all from Sigma).
Luminescence and absorbance (410 nM) was measured using the FLUOstar Optima (BMG Labtech, Ortenberg,
Germany).
Animals and diet. Male APPswePS1ΔE9 mice (Radboud University Medical Center, Nijmegen47) were
backcrossed with female C57Bl6/J mice (Harlan Netherlands B.V., Horst, The Netherlands) to obtain male
APPswePS1ΔE9 and wild-type (WT) littermates. Animals were housed in a conventional animal facility at
Hasselt University. Two series of animal experiments were conducted, the rst using dietary supplementation of
dried Sargassum fusiforme, and the second using gavage of a lipid extract of Sargassum fusiforme. A timeline of
the animal experiments is depicted in Supplemental Fig.1.
For the rst series, experimental diets consisted of either powdered standard chow (Teklad 2018; Harlan
Netherlands B.V.) or chow supplemented with 50% (w/w) pulverized dried Sargassum fusiforme (Clearspring
Ltd, London, UK) or the synthetic LXRα agonist AZ876 (2 mg/kg body weight, kindly provided by AstraZeneca,
Mölndal, Sweden69). e amount of 24(S)-Saringosterol used was 69.4 ng/mg dried Sargassum fusiforme. Based
on an estimated intake of 7 g dry food/day, the amount of daily 24(S)-Saringosterol corresponds to 242 µg/
day. Animals were divided into three groups and received either the Sargassum fusiforme containing diet
(APPswePS1ΔE9 n = 13; C57Bl6/J n = 10), AZ876 containing diet (APPswePS1ΔE9 n = 14; C57Bl6/J n = 13), or
the control diet (APPswePS1ΔE9 n = 13; C57Bl6/J n = 12) from 5 until 7.5 months of age.
e second batch of animals was divided into two groups and received either the Sargassum fusiforme-derived
lipid extract (APPswePS1ΔE9 n = 9; C57Bl6/J n = 8), or the vehicle (APPswePS1ΔE9 n = 11; C57Bl6/J n = 12)
from 6 until 7.5 months of age. Lipids of Sargassum fusiforme were extracted using the Folch-method65. Briey,
dried Sargassum fusiforme was pulverized and soaked in a 2:1 (v/v) chloroform:methanol (VWR, Leuven,
Figure 5. Dietary supplementation with a lipid extract of Sargassum fusiforme reduces cognitive decline and
Aβ plaque load in APPswePS1ΔE9 mice. (a,b) A Sargassum fusiforme lipid extract was administered by gavage
to determine the impact of Sargassum fusiforme-derived lipids on working memory in the spatial alteration
Y maze. All mice showed an intact working memory at baseline ((a), spatial alteration > 50% (chance level);
WT control p = 0.0002; WT Sargassum fusiforme p = 0.0008; AD control p < 0.0001, AD Sargassum fusiforme
p = 0.0006, one-sample t-test). Upon six weeks of daily treatment (b), the untreated AD group displayed an
impaired working memory (p = 0.085) while wild type mice and Sargassum fusiforme extract-treated mice
retained their working memory (WT control p < 0.0001, WT Sargassum fusiforme p = 0.0018, AD Sargassum
fusiforme p = 0.0008, one-sample t-test). (c,d) e extract-treated APPswePS1ΔE9 mice show a signicant
decrease in formic acid-extracted insoluble Aβ42 levels in the cortex ((c); U = 0, nctrl = 8, nextract = 8, p = 0.0045,
Mann-Whitney) and in the hippocampus ((d); U = 3; nctrl = 7, nextract = 5, p = 0.0177, Mann-Whitney). (eg)
Mice treated with a lipid extract of Sargassum fusiforme show an increased mRNA (e) expression of ApoE in
their CNS (F (1, 18) = 4.885, p = 0.0403, two-way ANOVA), although the increase in protein expression (f,g)
does not reach signicance. Data are shown as mean ± SEM.
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Belgium) mixture for 16 hours at room temperature. Next, the mixture was sonicated (10 min), ltered, and
subsequently evaporated by heating the extract until 60 °C. e remaining extract was washed in ethanol, son-
icated (10 min), and evaporated. Finally, the extract was dissolved in corn oil (Vita Dor, the Netherlands) as
vehicle and sonicated prior to in vivo supplementation. Using gas chromatography/mass spectrometry, 24(S)/
(R)-Saringosterol content was measured to be 332 mg/dl (7,744 mM). e extract was administered by daily gav-
age (200 µl/25 g body weight) for a period of 45 days, corresponding to a daily lipid intake of 3.5 g seaweed/day
(664 µg 24(S)/(R)-Saringosterol/day). In a previous study we dened that Sargassum fusiforme contains predom-
inantly 24(S)-Saringosterol34. Hence, we expect that this isoforms is the most abundant isoform present in our
in vivo experiments.
One week prior to the start of the behavioral experiments all animals were housed individually. Animals were
kept at an inverse 12 h light/12 h dark cycle, and behavioral experiments were performed during the dark phase
of the cycle. Cognitive performance was scored blind. All animal procedures were performed in accordance with
institutional guidelines and approved by the ethical committee for animal experiments of Hasselt University.
Behavioral tasks. e object recognition task (ORT) was conducted aer 9 weeks of Sargassum fusiforme
supplementation when the mice were ~8 months old, as described previously70,71. In brief, all animals were habit-
uated to the arena over 2 days, in one trial of 4 min per day. In each habituation trial, two dierent objects were
placed symmetrically in the center of the arena, about 10 cm from the wall. Four dierent objects were available
and at the end of the habituation period each animal had encountered all four objects once. Aer one resting
day, the experiment started. During the rst trial (T1) two identical objects (samples) were placed in the arena.
Aer exploring the samples for 4 min, the animal was placed back in its home cage. Subsequently, aer a pre-
determined delay interval (1 or 24 hours), the second trial of 4 minutes (T2) was performed using one familiar
object from trial 1 and one new object. Exploration time for each object during T1 and T2 was recorded manually
using a personal computer. Sitting on the object or biting in the object was not considered exploratory behavior.
Discrimination index d2 in T2 (d2 = [(exploration time for the novel object) (exploration time for the familiar
object)]/(total exploration time in T2)) was calculated as measure for object memory.
e Y-maze spontaneous alternation test72 was conducted at baseline and aer 6 weeks of extract treatment.
e maze used for the Y-maze spatial alteration task consisted of three arms of grey Perspec of equal length
(40 × 10 × 15 cm) (labeled A, B and C), separated at an angle of 120°. e animals were free to explore the maze
for 6 minutes. Aerwards, the working memory is calculated in order of percentage of alternations dened as:
(number of triads/(total number of entries 2)) * 100. e number of triads was recorded manually and was
dened as subsequently entering the three dierent arms, while entering an arm was dened as placing both hind
paws in that arm. Aer each trial, the maze was cleaned with 70% ethanol to prevent olfactory cues.
Figure 6. Sargassum fusiforme supplementation does not result in liver steatosis, elevated circulating
triglyceride levels, or arsenic accumulation in the lungs. (a) An ORO staining was performed to dene lipid
droplets in liver samples of APPswePS1ΔE9 mice fed normal chow or Sargassum fusiforme/AZ876-enriched
ch ow. ( b) Quantication of the ORO staining shown in (1) calculated as number of lipid droplets per mm2
of liver tissue. (c) AZ876 but not Sargassum fusiforme increases the circulating levels of triglycerides in and
APPswePS1ΔE9 mice (F (2, 31) = 4.056, p = 0.0272, ANOVA; Tukey’s post-hoc for diet eect: p = 0.0305). (d)
Arsenic was assesed in dried Sargassum fusiforme and lung samples of WT and APPswePS1ΔE9 mice (AD) fed
normal chow or chow supplemented with Sargassum fusiforme.
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e open eld test (OF) was conducted as described previously73, aer 9.5 weeks of Sargassum fusiforme
treatment. In short, animals were placed in a square, Perspex box (50 cm × 50 cm × 30 cm), with an open top, grey
walls, and a white oor. Video tracking with a computerized system (Etho VisionTM, Noldus, Wageningen, e
Netherlands) was used to record and analyse movements and position of the animals, in order to determine time
in zone (TIZ) and distance moved (DM) over the 10-minute trial14.
Tissue sample preparation. Mice were anaesthetized with Nembutal (100 mg/kg i.p., CEVA Logistics,
Brussels, Belgium) prior to transcardial perfusion with Ringer’s solution. Blood was collected and centrifuged
for 10 min at 200 g to obtain serum, which was snap-frozen in liquid nitrogen. Liver samples were divided into
two parts and either directly snap-frozen or frozen in Tissue-Tek O.C.T embedding compound (Sakura Finetek,
Berchem, Belgium). Brains were divided into three parts prior to snap-freezing; the cerebellum and two hem-
ispheres. The right hemisphere was preserved for immunohistochemistry and frozen in Tissue-Tek O.C.T.
embedding compound. e rostral half of the le hemisphere was preserved for mRNA expression analyses with
quantitative PCR. e cerebellum was preserved for sterol analyses.
Aβ quantication. e right hemisphere of the crude Sargassum fusiforme-treated mice was cut in the coronal
plane with a Leica CM1900UV cryostat (Leica Microsystems, Wetzlar, Germany) to obtain 10 μm sections. Sections
were mounted on glass slides, air-dried overnight, and stored at room temperature until used. Aer blocking of
endogenous peroxidases by incubation for 30 min with 3% H2O2 in methanol, the sections were incubated for
30 min with blocking solution (5% bovine serum albumin in 1x TBS) followed by overnight 4 °C primary antibody
incubation in blocking solution (clone 3D6, Amyloid-β Antibody 1:8000). e sections were then incubated in
Figure 7. 24(S)-Saringosterol increases astrocytic ApoE production and microglial Aβ clearance and reduces
neuronal Aβ release in vitro. (a,b) Primary astrocytes were treated with 10 µM 24(S)-Saringosterol for 2
consecutive days and medium was collected 48 h aer the last treatment. Western Blot was used to measure
secreted ApoE in the cellular supernatant. 24(S)-Saringosterol increased ApoE secretion (t(5) = 2.531,
p = 0.0524, unpaired t-test). Representative WB bands are displayed in the gure. (c) Primary murine microglia
were examined for their capacity to internalize FAM-labeled Aβ142. Microglia were incubated for 1 h with
uorescently-labeled FAM-Aβ142 and 40% conditioned medium derived from astrocytes treated with vehicle
(ethanol) or 24(S)-Saringosterol (10 µM). Data are depicted as the percentage of cells that phagocytosed
FAM-Aβ142. 24(S)-Saringosterol increased microglial uptake of FAM-Aβ142 (t(14) = 2.218, p = 0.0436,
unpaired t-test). (d) To dene a direct eect on microglial uptake of Aβ142, primary murine microglia were
exposed to 24(S)-Saringosterol prior to dening FAM-labeled Aβ142 uptake. (e) N2aAPPswe cells were
stimulated with vehicle (ethanol) or 24(S)-Saringosterol (10 µM). ELISA was used to dene the release of Aβ42.
24(S)-Saringosterol decreased Aβ42 production (t(5) = 8.126, p = 0.0005, unpaired t-test).
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biotinylated goat anti-human IgG (BA-3000, 1:1000, Vector Laboratories, Burlingame, CA), and the immunore-
activity was developed by the ABC avidin–biotin peroxidase KIT (PK-6100, Vector) for 1 hour, and 5 min with
diaminobenzidine (DAB, ImmPACT SK-4105, Vector). Between every other step, the slides were rinsed (3×) with
TBS/0.3% Triton-X 100. e sections were counterstained with haematoxylin for 30 seconds and coverslipped.
Digital images of the sections were obtained using a Leica DM 2000 LED microscope (Leica Microsystems, Diegem,
Belgium) equipped with hardware and soware from Leica Application suite soware (Leica Microsystems). e
Aβ plaque load was quantied in 3 sections per brain in 5 animals per group using Fiji ImageJ, by dening the pixel
intensity of Aβ plaques in the total cortical or hippocampal area at Bregma 1.5 to 2.5.
Next, le hippocampus and cortex were dissected from the brains of the Sargassum fusiforme extract-treated
APPswePS1ΔE9 mice and homogenated in a 2% SDS TBS-T buer. Aβ42 levels were quantied and related to
the protein content in the starting homogenate using an Aβ42 ELISA (Invitrogen, USA), according to the manu-
facturer’s instructions74. In brief, the homogenates were sonicated twice and mixture centrifuged to generate an
SDS-insoluble fraction (21000 g, 10 min). e SDS-insoluble pellet was sonicated twice in 70% formic acid (FA),
yielding the FA-extracted insoluble Aβ42 fraction.
Mouse neuroblastoma (N2a) cells, stably overexpressing APP, were cultured in high glucose DMEM medium
supplemented with 10%FCS and 100 U pen/100 µg strep/ml (37 °C, 5% CO2) Next, the cells were incubated for
24 h with ethanol (vehicle control) or 10 µM 24(S)-Saringosterol, puried from Sargassum fusiforme as described
earlier34. Aβ42 levels were detected in supernatant using an Aβ42 ELISA (Invitrogen, USA), according to the man-
ufacturer’s instructions.
Determination of astrocytic ApoE secretion. To generate 24(S)-Saringosterol-astrocyte conditioned
medium, mouse primary astrocytes were seeded in a 24-well plate with a density of 250000 cells/well. Next, astro-
cytes were treated with either 10 µM puried 24(S)-Saringosterol or ethanol for 2 consecutive days aer which
medium was replenished and collected 2 days aerwards. Next, ApoE secretion was determined using western
blot. Equal amounts of cellular supernatant (40 µl) were loaded and separated via electrophoresis through a 10%
sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE). Aerwards, proteins were transferred onto a PVDF
membrane (VWR, Belgium), and blots were blocked (5% non-fat dry milk TBS-Tween solution) for 1 h at room
temperature. Next, membranes were incubated with rabbit anti-ApoE primary antibody (ABBIOTEC, USA).
Aer washing the membranes with TBS-Tween, the blots were incubated with swine anti-rabbit HRP conju-
gated antibody (WAKO, Japan). Finally, membranes were washed in TBS and protein bands were detected using
PierceTM ECL Plus Western Blotting Substrated and ImageQuantTM LAS 4000 mini. ImageJ (http://imagej.nih.
gov/ij/) was used to quantify the protein bands.
Aβ phagocytosis assay. Primary murine cells were isolated and cultured as described previously75. In
short, mixed glial cultures were prepared from postnatal d0 mouse cerebral cortices of C57Bl6/J mice (Harlan
Netherlands B.V.) and cultured in high glucose DMEM medium supplemented with 10% FCS and 100 U
pen/100 µg strep/ml. Mixed glial cultures were used to generate microglia-enriched glial cultures by separating
the microglia from the astrocyte monolayer using orbital shaking followed by purication via dierential adhe-
sion to plastic aer 14 days in culture. Puried microglia were seeded on poly-L-lysine (5 µg/ml; Sigma-Aldrich)
coated 96-well plates with a density of 50000 cells/well. Primary microglia were cultured in high glucose DMEM
medium supplemented with 10% FCS, 100 U pen/100 µg strep/ml and 15% L929 conditioned medium.
Gene symbol Gene name Forward and reverse primer
Cyca Cyclin-A F: TGGGATTGTACCACAGCTCCA
R: CTCATGATGACTGCAGCAAACC
Hmbs Hydroxymethylbilane synthase F: GATGAAGCCATTGCTGAACTTG
R: GTCTCCTTGGGTATCCGATGTC
Abca1 ATP-binding cassette, sub-family A, member 1 F: CCCAGAGCAAAAAGCGACTC
R: GGTCATCATCACTTTGGTCCTTG
Abcg1 ATP-binding cassette, sub-family G, member 1 F: CAAGACCCTTT TGAAAGGGATCT
R: GCCAGAATATTCATGAGTGTGGAC
ApoE Apolipoprotein E F: CCTGAACCGCTTCTGGGATT
R: GCTCTTCCTGGACCTGGTCA
App Amyloid precursor protein F: CGA ACC CTA CGA AGA AGC CAC
R: GCT TTC TGG AAA TGG GCA TGT TC
Axl Axl receptor tyrosine kinase F: GGA ACC CAG GGA ATA TCA CAG G
R: AGT TCT AGG ATC TGT CCA TCT CG
Mertk Proto-oncogene tyrosine-protein kinase Mer F: TGC GTT TAA TCA CAC CAT TGG A
R: TGC CCC GAG CAA TTC CTT TC
Scd1 Stearoyl-CoA desaturase 1 F: TGCGATACACTCTGGTGCTCA
R: CTCAGAAGCCCAAAGCTCAGC
Srebp-1c Sterol regulatory element binding protein 1c F: GGAGCCATGGATTGCACATT
R: GCTTCCAGAGAGGAGCCCAG
Trem2 Triggering receptor expressed on myeloid cells 2 F: CTG GAA CCG TCA CCA TCA CTC
R: CGA AAC TCG ATG ACT CCT CGG
Table 2. Quantitative PCR primers. Nucleotide sequence of primers used for quantitative PCR. F denotes
forward primer, R denotes reverse primer.
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The capacity of primary microglia to phagocytose FAM-labeled Aβ142 (Eurogentec, Seraing Belgium)
was assessed with a plate-based assay. Twenty hours after seeding the microglia, cells were treated with
24(S)-Saringosterol-astrocyte conditioned medium (40%) and FAM-labeled Aβ142 was added for 1 h at a nal
concentration of 500 nM. Next, medium was removed and 100 µl 0.2% trypan blue in PBS (pH 4.4) was added to
quench extracellular Aβ. Aer aspiration, uorescence was measured at 485/535 nm excitation/emission. Finally,
to normalize for cell number, 100 µl DAPI in PBS was added, incubated for 10 min and medium was aspirated.
Fluorescence was subsequently measured in a plate reader using 360 nm/465 nm excitation/emission wavelengths.
Sterol prole and triglyceride content determination. Sterol proles were determined in serum and
cerebellum. Preceding the analysis, samples were spun in a speed vacuum dryer (Savant AES 1000) at 12 mbar for
24 h to relate individual sterol concentrations to their dry weight. e sterols were extracted from the dried tissue
by placing them in a mixture of chloroform:methanol (2:1) for 24 h at 4 °C. Sterol levels were determined by gas
chromatography/mass spectrometry (GC/MS) as described previously32,76. Triglyceride contents of serum were
determined using the GOD-PAP method with enzymatic reagent kits according to manufacturer’s instructions
(DiaSys Diagnostic Systems, Holzheim, Germany).
Determination of arsenic concentration. Arsenic halogenide concentrations in lung tissues were ana-
lyzed using inductively coupled plasma-optical emission spectrometry (ICP–OES, Agilent Technologies, 700
Series, Belgium). Tissues were oven-dried and digested with 70–71% HNO3 in a heat block. Prior to analysis, lung
samples and dried Sargassum fusiforme were diluted 1:10 in 1% nitric acid. Concentrations were measured using
a standard calibration curve (NIST Spinach 1570a).
RNA isolation and RT-Q-PCR. Brain tissue was homogenized in Qiazol (Qiagen, Venlo, e Netherlands)
preceding RNA isolation, and total RNA was prepared using the RNeasy mini kit (Qiagen), according to man-
ufacturer’s instructions. RNA concentration and quality was determined with a NanoDrop spectrophotometer
(Isogen Life Science, IJsselstein, e Netherlands). RNA was reverse transcribed to cDNA using the qScriptTM
cDNA synthesis kit (Quanta Biosciences, Gaithersburg, USA). As previously described77,78, quantitative PCR was
subsequently conducted on a StepOnePlus detection system (Applied biosystems, Gaasbeek, Belgium). Relative
quantication of gene expression was performed using the comparative Ct method. Data were normalized to the
most stable reference genes (Cyca and Hmbs). Primers were chosen according to literature or designed using
Primer-Express (http://www.ncbi.nlm.nih.gov/tools/primer-blast), and details of primers used are shown in
Table2.
Experimental design and statistical analyses. All statistical analyses were performed using GraphPad
Prism 7TM and are reported as mean ± SEM. D’Agostino and Pearson omnibus normality test was used to test
normal distribution. Unless specied dierently, one-way ANOVA (post-hoc: Tukey) or two-tailed unpaired
Student t-test was used for normally distributed data sets. e Kruskal-Wallis (Dunns post hoc comparison) or
Mann–Whitney analysis was used for data sets assessed not to be normally distributed. e ORT discrimination
index d2 (compared to the 0) and spatial alteration Y-maze performance (compared to the 50% chance level) was
analyzed using the one-sample t-test. Animals that did not reach the minimum of 4 s exploration in T1 or T2 were
excluded from further analysis71,79. Data from RT-Q-PCR was analyzed using two-way ANOVA with treatment
and genotype as independent variables. In all data sets, extreme values were excluded by means of Dixons princi-
ples of exclusion of extreme values80,81. Signicance levels are denoted as follows: $p < 0.10 *p < 0.05, **p < 0.01,
***p < 0.001 or ****p < 0.0001.
Ethical approval. All animal procedures were performed in accordance with institutional guidelines and
approved by the ethical committee for animal experiments of Hasselt University.
Data Availability
e datasets used and/or analyzed during the current study are available from the corresponding author on rea-
sonable request.
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Acknowledgements
is work was supported by the Internationale Stichting Alzheimer Onderzoek (ISAO)/Alzheimer Nederland
(AN), Scientic Research–Flanders (FWO), Fondation Vaincre Alzheimer (LECMA), and Alzheimer Forschung
Initiative (AFI). e authors like to thank Joke Vanhoof for excellent technical assistance and AstraZeneca for
providing the AZ876 compound through the AstraZeneca Open Innovation program. We sincerely thank Patric
Delhanty for editing the paper and Kenneth Vanbrabant for performing the GC-MS analysis.
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Author Contributions
J.B., C.H., M.S., A.T., A.C., F.L., D.S., A.K., P.M.M., J.W.J., I.D., S.P., D.L. and T.V. performed the experiments and
analyzed the data. J.B., C.H., T.V. and M.M. wrote the manuscript. Y.P., T.S., H.B.L. and B.S. provided experimental
materials. All authors revised the manuscript. J.B., C.H., N.H., J.W., P.M.M.,. E.S., J.W., J.H., A.G., B.S., D.L., T.V.
and M.M. participated in the design and coordination of the project.
Additional Information
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Supplementary resource (1)

... It is a direct precursor of retinoic acid, formed by oxidation of retinol in presence of retinol dehydrogenase (Belyaeva et al., 2019). Stigmasterol reduces neurological deficits and restores endogenous antioxidant defence system of ischemic injury in rat brain by preventing the autophagy activation via AMPK-mTOR, and JNK pathways in brain (Sun et al., 2019;Bogie et al., 2019). β-sitosterol has antioxidant activities of SOD, CAT, GSH and decreased the level of MDA in oxidative stress-induced ROS production. ...
... In SCP-induced groups, rats showed significant (p < 0.001) lower ambulation (peripheral: 23.5 ± (Table 2B). GRHA-400 significantly ameliorated learning and memory deficits in SCP-induced rats due to the collective action of genistin, quercetin-3-D-galactoside, stigmasterol, phytol, 9,12,15-octadecatrienoic acid, methyl ester, retinal, and β-sitosterol (Fig. 5) which imply the cholinergic neurotransmission in the cerebral cortex and hippocampus and restore the loss of memory in AD rats (Sun et al., 2019;Bogie et al., 2019;Maden, 2002;Bhosale et al., 2011). ...
... The current finding signified that GRHA is a potent intracellular radical scavenger to ROS and ameliorate neurological disorders in AD. This may due to the synergistic effect of radical scavengers like genistin, quercetin 3-D-galactoside, stigmasterol, phytol, 9,12,15-octadecatrienoic acid, methyl ester, retinal, and β-sitosterol in GRHA (Sethuraman et al., 2020;Bogie et al., 2019;Islam et al., 2018;Ku et al., 2014;Kim et al., 2011;Maden, 2002). ...
Article
Ethnopharmacological relevance Geophila repens (L.) I.M. Johnst (Rubiaceae) is a small perennial creeper native to India, China, and other countries in Southeast Asia. The hot decoction of leaves is used orally for memory enhancing by the local folk of Andhra Pradesh, India. The ethnomedicinal claim of G. repens as memory enhancer was initially studied by the authors. Results demonstrated the important antioxidant and anticholinesterase activities of isolated molecule Pentylcurcumene and bioactive hydroalcohol extract of leaves of G. repens (GRHA). Aim of the study Based on the previous findings, additional research is needed to examine the efficacy of GRHA for memory enhancing properties. We therefore investigated the modulatory role of prime identified compounds in GRHA in mitigating scopolamine-induced neurotoxicity in experimental rats of Alzheimer's disease (AD) via attenuation of cholinesterase, β-secretase, MAPt levels and inhibition of oxidative stress imparts inflammation. Methods Scopolamine (3 mg/kg) induced experimental rats of AD were treated with GRHA (300, 400 mg/kg) for 14 days. During the experimental period, elevated T-maze and locomotion-activity were performed to assess learning and memory efficacy of GRHA. At the end of the experiment, biochemical, neurochemical, neuroinflammation and histopathological observation of brain cortex were examined. GC-MS/MS analysis reported 31 compounds, among them 8 bioactive compounds possess antioxidant, neuroinflammation, neuroprotective activities, and were considered for docking analysis towards cholinesterase, β-secretase activities in AD. Results GRHA 400 significantly improved learning and memory impairment with the improvement of oxidative stress (MDA, SOD, GSH, CAT), DNA damage (8-OHdG), neurochemical (AChE, BuChE, BACE1, BACE2, MAPt), neuroinflammation (IL-6, TNF-α) markers in neurotoxic rats. Docking studies of 8 compounds demonstrated negative binding energies for cholinesterase and β-secretase indicating high affinity for target enzymes in AD. Test results were corroborated by the improvement of cellular tissue architecture of brain cortex in AD rats. Conclusion Synergistic action of genistin, quercetin-3-D-galactoside, 9,12,15-octadecatrienoic-acid methyl-ester, phytol, retinal, stigmasterol, n-hexadecanoic acid, β-sitosterol in GRHA restores memory-deficits via attenuation of cholinesterase, β-secretase, MAPt level and inhibition of oxidative-stress imparts inflammation in AD.
... Dietary supplementation of S. fusiforme enriched in 24(S)-saringosterol in an AD mouse model indicated a reduction in hippocampal Aβ plaques and improvement in short-term memory. In vitro treatment with the same extract on mouse neuroblastoma (N2a) cells also exhibited a reduced secretion of Aβ plaques [78]. The reduction in the levels of Aβ plaques by dietary supplementation of S. fusiforme extracts enriched with phytosterols including 24(S)-saringosterol may indicate a synergistic model of the phytosterols in the prevention of neurodegenerative diseases. ...
... Minor or no effect on Aβ secretion in vivo (mice) [31] Lanosterol Reduced secretion of Aβ plaques. Improves memory in AD mice model in vivo (mice), in vitro (N2a) [78] α-Amyrin Elevated levels of memory related proteins through the activation of ERKGSK-3β in vivo (mice) [79] β-Amyrin Reduced the βand γ-secretase activity in vitro [44] ...
Article
Full-text available
Phytosterols constitute a class of natural products that are an important component of diet and have vast applications in foods, cosmetics, and herbal medicines. With many and diverse isolated structures in nature, they exhibit a broad range of biological and pharmacological activities. Among over 200 types of phytosterols, stigmasterol and β-sitosterol were ubiquitous in many plant species, exhibiting important aspects of activities related to neurodegenerative diseases. Hence, this mini-review presented an overview of the reported studies on selected phytosterols related to neurodegenerative diseases. It covered the major phytosterols based on biosynthetic considerations, including other phytosterols with significant in vitro and in vivo biological activities.
... Sargassum fusiforme is an important edible brown seaweed that is grown in the lower intertidal zone along the Northwest Pacific (NW-Pacific) coast [1]. This seaweed has a reputation as the "longevity vegetable", as it contains a series of functional bioactive compounds (such as polysaccharides), which are correlated with antioxidants, anti-aging, memory improvement and immune regulation [2][3][4]. S. fusiforme is also used as an essential herb in traditional Chinese medicine to disperse phlegm [5]. However, heavy commercial exploitation of S. fusiforme has led to a significant reduction in population size. ...
Article
Full-text available
Sargassum fusiforme is a commercially important brown seaweed that has experienced significant population reduction both from heavy exploitation and degradation of the environment. Cultivated breed strains are also in a state of population mixing. These population stressors make it necessary to investigate the population genetics to discover best practices to conserve and breed this seaweed. In this study, the genetic diversity and population structure of S. fusiforme were investigated by the genome-wide SNP data acquired from double digest restriction site-associated DNA sequencing (ddRAD-seq). We found a low genetic diversity and a slight population differentiation within and between wild and cultivated populations, and the effective population size of S. fusiforme had experienced a continuous decline. Tajima’s D analysis showed the population contraction in wild populations may be related to copper pollution which showed a consistent trend with the increase of the sea surface temperature. The potential selection signatures may change the timing or level of gene expression, and further experiments are needed to investigate the effect of the mutation on relevant pathways. These results suggest an urgent need to manage and conserve S. fusiforme resources and biodiversity considering the accelerating change of the environment.
... Park et al. [336] found that mice treated with fucoidan extracts from Ecklonia cava had better memory and learning; consequently, the study implies favorable results in future human trials. In comparison to the control group, mice treated with polysaccharide isolated from Sargassum fusiforme demonstrated enhanced memory and cognition [337]. Dieckol and phlorofucofuroeckol, two phlorotannins from Ecklonia cava, are linked to an increase in the main central neurotransmitters in the brain, particularly Acetylcholine (ACh) [338]. ...
Article
Full-text available
Since ancient times, seaweeds have been employed as source of highly bioactive secondary metabolites that could act as key medicinal components. Furthermore, research into the biological activity of certain seaweed compounds has progressed significantly, with an emphasis on their composition and application for human and animal nutrition. Seaweeds have many uses: they are consumed as fodder, and have been used in medicines, cosmetics, energy, fertilizers, and industrial agar and alginate biosynthesis. The beneficial effects of seaweed are mostly due to the presence of minerals, vitamins, phenols, polysaccharides, and sterols, as well as several other bioactive compounds. These compounds seem to have antioxidant, anti-inflammatory, anti-cancer, antimicrobial, and anti-diabetic activities. Recent advances and limitations for seaweed bioactive as a nutraceutical in terms of bioavailability are explored in order to better comprehend their therapeutic development. To further understand the mechanism of action of seaweed chemicals, more research is needed as is an investigation into their potential usage in pharmaceutical companies and other applications , with the ultimate objective of developing sustainable and healthier products. The objective of this review is to collect information about the role of seaweeds on nutritional, pharmacological , industrial, and biochemical applications, as well as their impact on human health.
... It grows chiefly in coastal areas of Asian countries, such as Japan, South Korea, and North Korea (Liu et al., 2020a). There are abundant proteins, polysaccharides, and microelements in S. fusiforme, which has a wide range of biological activity in terms of antioxidant (Li Y. T. et al., 2018), antitumor (Fan et al., 2017), immunity (Sugiura et al., 2016), anti-aging (Bogie et al., 2019), bone growth and blood glucose , anticoagulant, growth, and development. Moreover, Paskaleva et al. showed that S. fusiforme extract inhibited more than 90% of HIV type 1 (HIV-1) infection and replication in T cells, human macrophages, and microglia and more than 70% of pseudotyped HIV-1 [vesicular stomatitis virus (VSV)/NL4-3] infection in human astrocytes (Paskaleva et al., 2006). ...
Article
Full-text available
Human adenovirus (HAdV) has a worldwide distribution and remains a major pathogen that leads to infections of the respiratory tract. No specific treatments or vaccines are yet available for HAdV infection. Sargassum fusiforme , an edible seaweed, has attracted a lot of attention for its various bioactivities. S. fusiforme has been reported to exhibit antiviral activity. However, research studies about its anti-HAdV activity are few. In this research, we found that S. fusiforme had low cytotoxicity and possessed anti-human adenovirus type 7 (HAdV7) activity in vitro , and the most effective ingredient was alginate. The time of addition assay demonstrated inhibitory effects that were observed in all life stages of the virus. In addition, we observed that the antiviral activity of alginate against HAdV7 infection might be closely related to the endoplasmic reticulum stress (ERS) pathway. Taken together, these results suggest that S. fusiforme extracts have potential application in the prevention and treatment of HAdV infection.
... Seaweeds contain large amounts of phytosterols, such as fucosterol, which is the main sterol in brown algae and cholesterol in red algae; however, the sterol composition of green algae is relatively heterogeneous, with a complex mixture of 28-isofucosterol, ergosterol, β-sitosterol, poriferasterol, cholesterol, and others [90,91]. These compounds may have particular biological activities, including antioxidant, antidiabetic, anti-inflammatory, anticancer, hepatoprotective, and anti-Alzheimer's disease activity [92]. ...
Article
Full-text available
Seaweeds or marine macroalgae are known for producing potentially bioactive substances that exhibit a wide range of nutritional, therapeutic, and nutraceutical properties. These compounds can be applied to treat chronic diseases, such as cancer, cardiovascular disease, osteoporosis, neurodegenerative diseases, and diabetes mellitus. Several studies have shown that consumption of seaweeds in Asian countries, such as Japan and Korea, has been correlated with a lower incidence of chronic diseases. In this study, we conducted a review of published papers on seaweed consumption and chronic diseases. We used the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) method for this study. We identified and screened research articles published between 2000 and 2021. We used PubMed and ScienceDirect databases and identified 107 articles. This systematic review discusses the potential use of bioactive compounds of seaweed to treat chronic diseases and identifies gaps where further research in this field is needed. In this review, the therapeutic and nutraceutical properties of seaweed for the treatment of chronic diseases such as neurodegenerative diseases, obesity, diabetes, cancer, liver disease, cardiovascular disease, osteoporosis, and arthritis were discussed. We concluded that further study on the identification of bioactive compounds of seaweed, and further study at a clinical level, are needed.
... Fucosterol (76), a steroid widespread in marine algae, could promote the transactivation of both LXRα (+155% at 200 μM) and LXRβ (+83% at 200 μM) serving as an LXRα/β dual agonist, further indicating nutritional implications in hypercholesterolemia and atherosclerosis (Hoang et al., 2012a). 24S-Saringosterol (77) (maybe same as 75) from an edible seaweed Sargassum fusiforme, promoted the transactivation of LXRα/β, and stimulated LXRβ by 14.4-fold, higher than LXRα by 3.8-fold, acting as a selective LXRβ agonist, the discovery of which also further confirmed that phytosterols in Sargassum fusiforme contributed to the well-known antiarteriosclerosis. Beyond that, 77 was in the observed effects on cognition and Aβ plaque load as an attractive option for the treatment of neurodegenerative disorders such as AD (Chen P. et al., 2014;Bogie et al., 2019;Hannan et al., 2020). Present in marine fish and plant roots is 4-Cholesten-3-one (78), but it is also found in a red marine alga Laurencia papillosa. ...
Article
Full-text available
Nuclear receptors (NRs) are a superfamily of transcription factors induced by ligands and also function as integrators of hormonal and nutritional signals. Among NRs, the liver X receptors (LXRs) and farnesoid X receptor (FXR) have been of significance as targets for the treatment of metabolic syndrome-related diseases. In recent years, natural products targeting LXRs and FXR have received remarkable interests as a valuable source of novel ligands encompassing diverse chemical structures and bioactive properties. This review aims to survey natural products, originating from terrestrial plants and microorganisms, marine organisms, and marine-derived microorganisms, which could influence LXRs and FXR. In the recent two decades (2000–2020), 261 natural products were discovered from natural resources such as LXRs/FXR modulators, 109 agonists and 38 antagonists targeting LXRs, and 72 agonists and 55 antagonists targeting FXR. The docking evaluation of desired natural products targeted LXRs/FXR is finally discussed. This comprehensive overview will provide a reference for future study of novel LXRs and FXR agonists and antagonists to target human diseases, and attract an increasing number of professional scholars majoring in pharmacy and biology with more in-depth discussion.
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Full-text available
Seaweed extracts are considered effective therapeutic alternatives to synthetic anticancer, antioxidant, and antimicrobial agents, owing to their availability, low cost, greater efficacy, eco-friendliness, and non-toxic nature. Since the bioactive constituents of seaweed, in particular, phytosterols, possess plenty of medicinal benefits over other conventional pharmaceutical agents, they have been extensively evaluated for many years. Fortunately, recent advances in phytosterol-based research have begun to unravel the evidence concerning these important processes and to endow the field with the understanding and identification of the potential contributions of seaweed-steroidal molecules that can be used as chemotherapeutic drugs. Despite the myriad of research interests in phytosterols, there is an immense need to fill the void with an up-to-date literature survey elucidating their biosynthesis, pharmacological effects, and other biomedical applications. Hence, in the present review, we summarize studies dealing with several types of seaweed to provide a comprehensive overview of the structural determination of several phytosterol molecules, their properties, biosynthetic pathways, and mechanisms of action, along with their health benefits, which could significantly contribute to the development of novel drugs and functional foods.
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Alzheimer's disease (AD) is the major cause of neurodegeneration worldwide and is characterized by the accumulation of amyloid beta (Aβ) in the brain, which is associated with neuronal loss and cognitive impairment. Liver X receptor (LXR), a critical nuclear receptor, and major regulator in lipid metabolism and inflammation, is suggested to play a protective role against the mitochondrial dysfunction noted in AD. In our study, our established 3D gelatin scaffold model and a well characterized in vivo (APP/PS1) murine model of AD were used to directly investigate the molecular, biochemical and behavioural effects of neuronal stem cell exposure to Aβ to improve understanding of the in vivo etiology of AD. Herein, human neural stem cells (hNSCs) in our 3D model were exposed to Aβ, and had significantly decreased cell viability, which correlated with decreased mRNA and protein expression of LXR, Bcl-2, CREB, PGC1α, NRF-1, and Tfam, and increased caspase 3 and 9 activities. Cotreatment with a synthetic agonist of LXR (TO901317) significantly abrogated these Aβ-mediated effects in hNSCs. Moreover, TO901317 cotreatment both significantly rescues hNSCs from Aβ-mediated decreases in ATP levels and mitochondrial mass, and significantly restores Aβ-induced fragmented mitochondria to almost normal morphology. TO901317 cotreatment also decreases tau aggregates in Aβ-treated hNSCs. Importantly, TO901317 treatment significantly alleviates the impairment of memory, decreases Aβ aggregates and increases proteasome activity in APP/PS1 mice; whereas, these effects were blocked by cotreatment with an LXR antagonist (GSK2033). Together, these novel results improve our mechanistic understanding of the central role of LXR in Aβ-mediated hNSC dysfunction. We also provide preclinical data unveiling the protective effects of using an LXR-dependent agonist, TO901317, to block the toxicity observed in Aβ-exposed hNSCs, which may guide future treatment strategies to slow or prevent neurodegeneration in some AD patients.
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Fucosterol from edible brown seaweeds has various biological activities, including anti-inflammatory, anti-adipogenic, antiphotoaging, anti-acetylcholinesterase, and anti-beta-secretase 1 activities. However, little is known about its effects on soluble amyloid beta peptide (sAβ)-induced endoplasmic reticulum (ER) stress and cognitive impairment. Fucosterol was isolated from the edible brown seaweed Ecklonia stolonifera, and its neuroprotective effects were analyzed in primary hippocampal neurons and in aging rats. Fucosterol attenuated sAβ1-42-induced decrease in the viability of hippocampal neurons and downregulated sAβ1-42-induced increase in glucose-regulated protein 78 (GRP78) expression in hippocampal neurons via activation of tyrosine receptor kinase B-mediated ERK1/2 signaling. Fucosterol co-infusion attenuated sAβ1-42-induced cognitive impairment in aging rats via downregulation of GRP78 expression and upregulation of mature brain-derived neurotrophic factor expression in the dentate gyrus. Fucosterol might be beneficial for the management of cognitive dysfunction via suppression of aging-induced ER stress.
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Current evidence indicates that foods with added plant sterols or stanols can lower serum levels of low-density lipoprotein cholesterol. This review summarizes the recent findings and deliberations of 31 experts in the field who participated in a scientific meeting in Winnipeg, Canada, on the health effects of plant sterols and stanols. Participants discussed issues including, but not limited to, the health benefits of plant sterols and stanols beyond cholesterol lowering, the role of plant sterols and stanols as adjuncts to diet and drugs, and the challenges involved in measuring plant sterols and stanols in biological samples. Variations in interindividual responses to plant sterols and stanols, as well as the personali-zation of lipid-lowering therapies, were addressed. Finally, the clinical aspects and treatment of sitosterolemia were reviewed. Although plant sterols and stanols continue to offer an efficacious and convenient dietary approach to cholesterol management, long-term clinical trials investigating the endpoints of cardiovascular disease are still lacking. Affiliation: P.J.H. Jones and S.B. Myrie are with the Richardson
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Alzheimer’s disease (AD) is a devastating neurodegenerative disease characterized by beta-amyloid (Aβ) accumulation and neurofibrillary tangles formation in the brain which are associated to synaptic deficits and dementia. Liver X receptor (LXR) agonists have been demonstrated to revert of pathologic and cognitive defects in murine models of AD through the regulation of Apolipoprotein E, ATP-Binding Cassette A1 (ABCA1), by dampening neuroinflammation and also by reducing the levels of amyloid-β (Aβ) accumulation in the brain. However, the role of LXR with regard to the regulation of synaptic function remains relatively understudied. In the present paper, we analyzed the in-vitro effect of the LXR agonist GW3965 on synaptic function upon exposure of primary hippocampal cultures to oligomeric amyloid-β (oAβ(1–42)). We showed that oAβ(1–42) exposure significantly decreased the density of mature (mushroom shaped) dendritic spines density and synaptic contacts number. oAβ(1–42) also modulates the expression of pre- (VGlut1, SYT1, SV2A) and post-synaptic (SHANK2, NMDA) proteins, it decreases the expression of PINK1, and increases ROCKII, and activates of caspase-3; these changes were prevented by the pre-treating neuronal cultures with GW3965. These results show further support the role of the LXR agonist GW3965 in synaptic physiology and highlight its potential as an alternative pharmacological strategy for AD.
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Amyloid β (Aβ) is a pathogenic peptide associated with many neurodegenerative diseases such as Alzheimer’s disease, Parkinson's disease. The retinal inflammation in response to Aβ is implicated in the pathogenesis of several ocular diseases including age-related macular degeneration, Alzheimer's-related optic neuropathy and glaucoma. In the present study, we found that a single intravitreal injection of oligomeric Aβ1-40 in mouse activated the NLRP3 inflammasome and the NF-κB signaling, induced the production of inflammatory cytokines including TNF-α and IL-6. In addition, Aβ1-40 caused retinal function impairment while no noticeable morphological changes were observed under light microscope. Furthermore, immunohistochemical results showed that Aβ1-40 enhanced the number of Iba1-positive cells in the inner retina. The mRNA expressions of LXRα and LXRβ decreased in the neuroretina of the Aβ1-40-injected mice. No significant difference was found on the protein expressions of LXRs and ABCA1 in both neuroretina and RPE/choroid complex between the Aβ1-40-injected group and the control group. A synthetic LXR ligand, TO901317 (TO90), enhanced the expressions of LXRα and ABCA1 at both mRNA and protein levels in the Aβ1-40-injected mice, while the LXRβ expression was unchanged. TO90 preserved ERG a- and b-wave amplitudes and reduced the number of Iba1-positive cells in the Aβ1-40-treated retina. Furthermore, TO90 down-regulated the mRNA levels of TNF-α and IL-6, as well as the expressions of p-IκBα, NLRP3, caspase-1 and IL-1β in the Aβ1-40-injected animals. We suggest that activation of LXRα and its target gene ABCA1 exerts potent anti-inflammatory effect in the Aβ-treated retina.
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Glial cells have a variety of functions in the brain, ranging from immune defence against external and endogenous hazardous stimuli, regulation of synaptic formation, calcium homeostasis, and metabolic support for neurons. Their dysregulation can contribute to the development of neurodegenerative disorders, including Alzheimer’s disease (AD). One of the most important functions of glial cells in AD is the regulation of Aβ levels in the brain. Microglia and astrocytes have been reported to play a central role as moderators of Aβ clearance and degradation. The mechanisms of Aβ degradation by glial cells include the production of proteases, including neprilysin, the insulin degrading enzyme (IDE), and the endothelin-converting enzymes, able to hydrolyse Aβ at different cleavage sites. Besides these enzymes, other proteases have been described to have some role in Aβ elimination, such as plasminogen activators, angiotensin-converting enzyme, and matrix metalloproteinases. Other relevant mediators that are released by glial cells are extracellular chaperones, involved in the clearance of Aβ alone or in association with receptors/transporters that facilitate their exit to the blood circulation. These include apolipoproteins, α2macroglobulin, and α1-antichymotrypsin. Finally, astrocytes and microglia have an essential role in phagocytosing Aβ, in many cases via a number of receptors that are expressed on their surface. In this review we examine all of these mechanisms, providing an update on the latest research in this field.
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Background The accumulation, aggregation and deposition of amyloid-β (Aβ) peptides in the brain are central to the pathogenesis of Alzheimer’s disease (AD). Alzheimer’s disease risk increases significantly in individuals carrying one or two copies of APOE ε4 allele compared to individuals with an ε3/ε3 genotype. Growing evidence has demonstrated that apolipoprotein E (apoE) strongly influences AD pathogenesis by controlling Aβ aggregation and metabolism. Heparan sulphate proteoglycans (HSPGs) are abundant cell surface molecules that bind to both apoE and Aβ. HSPGs have been associated with Aβ aggregation and deposition. Although several lines of research have shown that apoE influences Aβ clearance in the brain, it is not clear how apoE influences HSPG-mediated cellular uptake of Aβ. ResultsIn this study, we show that apoE lipoprotein particles from conditioned media of immortalized astrocytes isolated from human APOE-targeted replacement (TR) mice significantly suppress cellular Aβ42 and Aβ40 uptake through cell surface HSPG. ApoE3 and apoE4 particles have similar binding affinity to heparin, while apoE4 particles are likely hypolipidated compared to apoE particles. We also found that the apoE particles antagonize Aβ binding to cell surface, and inhibited Aβ uptake in a concentration-dependent manner in Chinese hamster ovary (CHO) cells. While the effect was not apoE isoform-dependent, the suppressive effect of apoE particles on Aβ uptake was not observed in HSPG-deficient CHO cells. We further demonstrated that apoE particles reduced the internalization of Aβ in mouse primary neurons, an effect that is eliminated by the presence of heparin. Conclusions Taken together, our findings indicate that apoE particles irrespective of isoform inhibit HSPG-dependent cellular Aβ uptake. Modulating the ability of apoE particles to affect Aβ cellular uptake may hold promises for developing new strategies for AD therapy.
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Amyloid beta (Aβ) can aggregate and form plaques, which are considered as one of the major hallmarks of Alzheimer's disease. This study aims to directly compare the neuroprotective activities in vitro of two marine-derived carotenoids astaxanthin and fucoxanthin that have shown a spectrum of biological activities, including neuroprotection. The in vitro neuroprotective activities were investigated against Aβ1-42-mediated toxicity in pheochromocytoma (PC-12) neuronal cells using the MTT cell viability assay, anti-apoptotic, antioxidant and neurite outgrowth activities; as well as inhibition against Aβ1-42 fibrillization in the Thioflavin T (ThT) assay of fibril kinetics and via transmission electron microscopic (TEM) evaluation of fibril morphology. The results demonstrated that both astaxanthin and fucoxanthin exhibited multi-neuroprotective effects favouring fucoxanthin over astaxanthin supporting neuroprotective roles of marine-derived carotenoids as potential novel dementia prevention or therapeutic strategies.
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The development of LXR agonists for the treatment of coronary artery disease has been challenged by undesirable properties in animal models. Here we show the effects of an LXR agonist on lipid and lipoprotein metabolism and neutrophils in human subjects. BMS-852927, a novel LXRβ-selective compound, had favorable profiles in animal models with a wide therapeutic index in cynomolgus monkeys and mice. In healthy subjects and hypercholesterolemic patients, reverse cholesterol transport pathways were induced similarly to that in animal models. However, increased plasma and hepatic TG, plasma LDL-C, apoB, apoE, and CETP and decreased circulating neutrophils were also evident. Furthermore, similar increases in LDL-C were observed in normocholesterolemic subjects and statin-treated patients. The primate model markedly underestimated human lipogenic responses and did not predict human neutrophil effects. These studies demonstrate both beneficial and adverse LXR agonist clinical responses and emphasize the importance of further translational research in this area.
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Herein we describe the development of a functionally selective LXRβ agonist series optimized for Emax selectivity, solubility and physical properties to enable efficacy and safety studies in vivo. Compound 9 showed central pharmacodynamic effects in rodent models, evidenced by statistically significant increases in apoE and ABCA1 levels in the brain, along with a greatly improved peripheral lipid safety profile when compared to full dual agonists. These findings were replicated by subchronic dosing studies in non-human primates, where CSF levels of apoE and Aβ peptides were increased concomitantly with improved peripheral lipid profile relative to that of non-selective compounds. These results suggest that optimization of LXR agonists for Emax selectivity may have the potential to circumvent the adverse lipid-related effects of hepatic LXR activity.