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Protective Effects of the Alga Fucoidan Against Amyloid-β-Induced Neurotoxicity in SH-SY5Y Cells

related to Alzheimer's disease (AD). Overaccumulation of amyloid-β protein (Aβ) in the brain forms senile plaques, one of the main features of neurodegeneration in AD. However, there are few drugs avail-able to specifically inhibit senile plaque formation. Fucoidan, a sulfated polysaccharide derived from brown algae, has various bioactivities, such as anti-tumoral and anti-obesity effects. This study aimed to clarify the mechanism underlying the protective effect of fucoidan against Aβ-induced neurotoxicity in human neuroblas-toma SH-SY5Y cells. Cell viability and Aβ-induced cytotoxicity were measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, calcein AM, and ethidium homodimer-1. Aβ-induced oxidative stress was evaluated through reactive oxygen species (ROS), cell membrane phospholipid peroxidation, mitochondrial ROS, and Mn-SOD, a mitochondrial radical scavenger. In addition, mitochondrial membrane permeability transition, and ATP concentration were evaluated. Fucoidan significantly improved Aβ-reduced cell viability. With respect to oxidative stress, Aβ exposure increased ROS, lipid peroxidation, and mitochondrial ROS, while fucoidan significantly suppressed these changes. Fucoidan also suppressed the decline in mitochondrial permeability transition and ATP caused by Aβ. Therefore, through its numerous antioxidant activities, fucoidan might have a neuroprotective role in preventing Aβ-induced neurotoxicity.


INTRODUCTION
Alzheimer's disease (AD) is an age-related progressive neurodegenerative disease characterized by an excess of senile plaques and neurofibrillary changes. These cellular abnormalities lead to neuronal loss in the hippocampus and cerebral cortex, resulting in memory and cognitive impairments. 1) The amyloid cascade hypothesis is the most plausible mechanism of AD, where accumulation of the amyloid-β protein (Aβ) in the brain and phosphorylation of the tau protein cause neurofibrillary changes, leading to neuronal cell death and brain atrophy. Recently, the United States Food and Drug Administration granted fast-track approval to aducanumab, a monoclonal antibody that selectively reduces Aβ aggregates in the brain, as a new treatment for AD; however, this approval is controversial, and its clinical usefulness uncertain. Other approved therapies for AD do not treat the disease but only delay symptom progression; therefore, new therapies for AD are urgently required. Furthermore, since AD is an irreversible disease that takes about 20 years to progress from the beginning of Aβ accumulation to AD onset, it is necessary to treat AD with drugs that inhibit Aβ aggregation and reduce its toxicity from the asymptomatic stage. 2,3) The use of long-term supplements and nutrients to maintain and restore physical function has lately gained attention for preventing AD onset. Various nutrients and supplements, including polyphenols such as curcumin and epigallocatechin gallate, with properties that improve cognitive function have been reported. [4][5][6][7][8] Although polyphenols have a strong antioxidant effect, they have low permeability across the bloodbrain barrier (BBB) and are required in large doses. Recently, polysaccharides derived from seaweed have been reported to be able to cross the BBB 9) and have neuroprotective effects. 10) Accordingly, their potential for long-term prevention of dementia has also been investigated. 11) Fucoidan, an intercellular mucosal polysaccharide, is a slippery component found in brown algae such as kelp, wakame seaweed, and mozuku seaweed. 12) It reaches the different organs via carrier-mediated transport after absorption 13) and has various biological effects, such as improvement of blood circulation, 14) antitumoral and antimetastatic effects, 15,16) improvement of the immune system function, 17) and anti-obesity effects. 18) Recently, fucoidan has been reported to suppress the increase in reactive oxygen species (ROS) production in AD-transgenic C. elegans, 19) and after Aβ-induced neurotoxicity in primary rat neuron cultures. 20) However, the mechanism through which fucoidan reduces ROS production has not been yet elucidated.

Detection of Reactive Oxygen Species
To investigate the effects of Aβ exposure on ROS production, the chloromethyl derivative 2′,7′-dichlorodihydrofluorescein diacetate (CM-H 2 DCFDA: Thermo Fisher Scientific, MA, USA), a useful indicator for ROS detection, was used. SH-SY5Y cells were incubated at 37°C for 24 h, after which these cells were exposed to Aβ and treated with fucoidan for 30 min. ROSmediated fluorescence was measured at Ex: 488 nm and Em: 525 nm using Spectra Max i3 (Molecular Devices).
Assay of Mitochondrial ROS SH-SY5Y cells at 1.0 x 10 6 cells/mL were plated in 96-well plates, incubated at 37°C for 24 h, exposed to Aβ and treated with fucoidan for 30 min. Mitochondrial ROS was detected using the Mitochondrial ROS Detection Kit (Cayman Chemical Company, MI, USA). The fluorescence intensity was measured at Ex: 500 nm and Em: 580 nm using Spectra Max i3 (Molecular Devices).

Assay of Phospholipid Peroxidation in Membranes
To measure phospholipid peroxidation in the membranes of Aβ-exposed cells, SH-SY5Y cells were treated with 5 µM of diphenyl-1-pyrenylphosphine (DPPP: Thermo Fisher Scientific, MA, USA). DPPP reacts with hydroperoxides to produce fluorescent DPPP oxide. 41) SH-SY5Y cells at 1.0x10 6 cells/mL plated in 96-well plates were incubated with DPPP at 37°C for 10 min, then exposed to Aβ, and treated with fucoidan for 30 min. The fluorescence intensity of DPPP oxide was measured at Ex: 351 nm and Em: 380 nm using Spectra Max i3 (Molecular Devices).
Assay of Manganese-Superoxide Dismutase SH-SY5Y cells were incubated at 37°C for 24 h, after which cells were exposed to Aβ and treated with fucoidan for 24 h. The mitochondrial SOD isozyme content in cell lysates was determined and measured by ELISA using a monoclonal antibody (Human SOD2 ELISA Kit; Abcam, Cambridge, UK). These ELISA systems did not show any cross-reactivity with other SOD isozymes. SOD protein level was determined using the OxiSe-lectTM Superoxide Dismutase Activity Assay (Cell Biolabs, Inc., CA, USA), and measured at 450 nm using Spectra Max i3 (Molecular Devices).
Observation of Mitochondrial Membrane Permeability Transition SH-SY5Y cells were incubated at 37°C for 24 h, after which these cells were exposed to Aβ, and treated with fucoidan for 3 h. Changes in mitochondrial membrane permeability transition (MPT) were measured using the Mito Capture Apoptosis Detection Kit (Bio Vision Inc., CA, America). Fluorescence intensities were measured at Ex:488 nm, Em:590 nm (Lm1:red), and Em:530 nm (Lm2:green) using Spectra Max i3 (Molecular Devices), and the Lm1/Lm2 ratio was determined.
ATP Assay ATP levels in cells were measured by the firefly luciferase luminescence method using an intracellular ATP assay kit (TOYO B-NET Co. Ltd, Tokyo, Japan). SH-SY5Y cells were incubated at 37°C for 24 h, after which cells were exposed to Aβ and treated with fucoidan for 3 h. After incubation, the ATP extracted from SH-SY5Y cells was measured for luminescence with Spectra Max i3 (Molecular Devices).
Statistical Analysis All results of the cell experiments are shown as mean ± S.E.M. values. Each measurement was repeated three times. Comparisons with untreated SH-SY5Y cells were performed using one-way analysis of variance (ANOVA) followed by Dunnett's post hoc test. P-values < 0.05 were considered statistically significant.

Effect of Fucoidan on Aβ-Induced Neuronal Injury
To determine the Aβ concentration that induces neuronal toxicity, SH-SY5Y cells were incubated with 1-10 µM Aβ. The changes in cell viability were assessed using the MTT assay. No significant change in cell viability was observed at 1 µM Aβ, but at ≥ 2.5 µM, Aβ significantly reduced cell viability in a concentration-dependent manner (Fig. 1A). Since exposure to 5 µM Aβ decreased cell viability by ~30%, we used this concentration in subsequent experiments. Exposure of SH-SY5Y cells to Aβ (5 µM) for 3 and 24 h resulted in a marked decrease in cell viability compared to control cells without Aβ exposure. Although there was no significant difference in viability depending on incubation time, treatment with fucoidan at 1 and 10 µg/mL significantly reversed the Aβ-induced decrease in cell viability ( Fig. 1B and C).
To further investigate Aβ cytotoxicity, SH-SY5Y cells were stained with calcein AM and ethidium homodimer-1 ( Fig. 2A-F). Calcein AM emits green fluorescence in the cytoplasm of viable cells via esterase activity. When the cell membrane is damaged, ethidium homodimer-1 enters the cell, binds to DNA and RNA, and emits bright red fluorescence. There-fore, double staining with calcein AM and ethidium homodimer-1 allowed the simultaneous analysis of the viability and cytotoxicity of Aβ treated cells and control cells. Fluorescence imaging of control cells only showed green fluorescence, with a few cells having red-stained nuclei (Fig. 2D). Aβ treatment was associated with a change in cell morphology, i.e., shortening of the protrusion length (Fig. 2B), and an increase in the number of red-stained nuclei (Fig. 2E). Co-treatment of SH-SY5Y cells with Aβ and fucoidan (10 µg/mL) decreased the changes to cell morphology (Fig. 2C) and the number of redstained nuclei (Fig. 2F). SH-SY5Y cells incubated with Aβ for 3 h showed significantly reduced viability (Fig. 2G) and increased cytotoxicity compared to control cells (Fig. 2H). Fucoidan (1 and 10 µg/mL) significantly improved cell viability in Aβ-treated cells (Fig. 2G). Additionally, fucoidan (10 µg/mL) significantly reduced Aβ-induced cytotoxicity (Fig. 2H).
Oxidative Stress ROS production was determined by chloromethyl derivative of 2′,7′-dichlorodihydrofluorescein diacetate (CM-H 2 DCFDA) staining using fluorescence microscopy. Since ROS are produced from an early stage, experiments were conducted after a 30 min incubation. ROS production due to Aβ exposure was significantly reduced in cells treat-  (Fig. 3E), but it was attenuated by 10 µg/mL fucoidan treatment (Fig. 3F). ROS production was significantly higher in SH-SY5Y cells exposed to Aβ for 30 min than in control cells (Fig. 3G).
The production of mitochondrial ROS, which consists mainly of superoxide anions, significantly increased in SH-SY5Y cells treated with Aβ for 30 min (Fig. 4). Fucoidan (1 µg/mL) suppressed Aβ-induced mitochondrial ROS production. No significant difference in mitochondrial ROS was observed at the concentration of 10 µg/mL, and no significant difference was observed between the two concentrations of 1 and 10 µg/mL. Therefore, the mitochondrial ROS at both concentrations of 1 and 10 µg/mL maybe the same.
Effects of Fucoidan on Aβ-Induced Mitochondrial Disfunction Aβ-induced cell death is associated with mitochondrial membrane injury. Mitochondrial membrane damage reduces MPT and decreases ATP and scavenger production. MPT and ATP were analysed after incubation for 3 h according to a previous study. 21) SH-SY5Y cells treated with Aβ for 24 h showed a decrease of ~50% in manganese superoxide dismutase (Mn-SOD) levels compared to control cells. Fucoidan significantly increased Mn-SOD levels in a concentration-dependent manner (Fig. 6). Incubation time was set to 24 h to allow Mn-SOD protein expression.
MPT significantly decreased in SH-SY5Y cells treated with Aβ for 3 h to about one-third of that in control cells. Fucoidan significantly ameliorated the Aβ-mediated decrease in MPT (Fig. 7).
A decrease in ATP levels leads to the uncoupling of oxidative phosphorylation, further resulting in ATP depletion. Therefore, we investigated whether ATP production was altered by Aβ treatment and whether fucoidan treatment reversed this effect in the current experimental model. The results showed that ATP levels after Aβ treatment were nearly 80% of those in control cells. Fucoidan (10 µg/mL) significantly recovered the Aβ-induced ATP decrease (Fig. 8).

DISCUSSION
This study demonstrated the protective effect of fucoidan against Aβ-induced neurotoxicity, especially oxidative stress in cell membranes and mitochondrial damage, in SH-SY5Y cells. Fucoidan reduced Aβ-induced cell membrane damage by ameliorating phospholipid peroxidation, maintaining cell membrane integrity, and increasing cell viability. Additionally, fucoidan reduced mitochondrial damage by alleviating the Aβ-mediated decrease in MPT, decreasing mitochondrial ROS production, and increasing ATP levels. Although the protective effects of fucoidan have been previously reported, the mechanism underlying these effects has not been fully elucidated. Fucoidan has been reported to exhibit various biological activities, such as anti-dyslipidemia, anti-arteriosclerosis, 22) and anti-cancer effects, 23) which are related to its anti-inflammatory and anti-oxidative effects. This study showed that fucoidan could have a preventive role against neurodegenerative diseases as well.
A single oral fucoidan administration to both male and female mice reported an lethal dose 50 (LD 50 ) and approximate lethal dose (LD) value of 2000 mg / kg, indicating that the concentration of fucoidan used in this study is safe. 24) Here, SH-SY5Y cells were treated with 1 and 10 µg/mL of fucoidan, far lower concentrations than those used in previous reports (50-800 µg/mL fucoidan). Those reports showed that fucoidan treatment ameliorated 1-methyl-4-phenylpyridinium ion-induced neurotoxicity in SH-SY5Y cells. 25,26) The human plasma concentration of fucoidan was approximately 13 µg/mL after oral administration of 2.25 g of fucoidan for 12 d 27) ; therefore, the fucoidan concentrations used in this study were within the appropriate range.
Furthermore, fucoidan penetrated the BBB at a rate of 10.4 × 10 −6 cm/s in vitro, where the BBB was constructed with astrocytes, vascular endothelial cells, and pericytes. 9) Howev-

Fig. 3. Determination of Reactive Oxygen Species (ROS) Generation
ROS generation in SH-SY5Y cells was evaluated using chloromethyl derivative of 2′,7′-dichlorodihydrofluorescein diacetate. (A-F) SH-SY5Y cells observed using phase-contrast (A-C) and fluorescence microscopy (D-F). The scale bar represents 100 µm. (G) ROS generation (fluorescence intensity [FI] × 10 5 /µg protein) in SH-SY5Y cells treated with 5 µM Aβ and fucoidan (1 and 10 µg/mL). **p < 0.01 vs. control cells. #p < 0.05 vs. Aβ exposed cells. ##p < 0.01 vs. Aβ exposed cells. Values in (G) are expressed as the mean of six independently treated culture wells plus S. E. M. Each measurement was repeated three times with similar results. er, it is unclear whether fucoidan passes through the BBB or transitions into the brain in vivo, thus, further research is needed in this area. This study confirmed that Aβ increases mitochondrial ROS, particularly mitochondrial superoxide anions, decreases Mn-SOD, and induces oxidative stress. Oxidative stress is related with health issues 28) and results in excessive ROS production, which can be mitigated by cellular antioxidative activity. Additionally, free radicals affect the structure and function of neurons and contribute to neurodegenerative diseases. 29) There is increasing evidence that oxidative stress plays a central role in the pathophysiology of AD. 30) Therefore, the development of antioxidative medications that decrease oxidative stress and promote neuronal survival will help in the prevention and early treatment of AD. Seaweed compounds, with their antioxidant effects via ROS scavenging, 31) are one such promising medication. In this study, fucoidan reduced Aβ-induced ROS production and oxidative stress.    The graph shows mitochondrial MPT (ratio/µg protein) in SH-SY5Y cells treated with 5 µM Aβ and fucoidan (1 and 10 µg/mL). The change in membrane potential was measured by comparing the ratio of emission at 585 nm/514 nm. *p < 0.05 vs. control cells. #p < 0.05 vs. Aβ exposed cells. Values are expressed as the mean of six independently treated culture wells plus S. E. M. Each measurement was repeated three times with similar results. ATP levels were measured using luminescence. The graph shows ATP levels (pmol/ mg protein) in SH-SY5Y cells treated with 5 µM Aβ and fucoidan (1 and 10 µg/mL). **p < 0.01 vs. control cells. ##p < 0.01 vs. Aβ exposed cells. Values are expressed as the mean of six independently treated culture wells plus S. E. M. Each measurement was repeated three times with similar results.
Aβ increases phospholipid peroxidation in the cell membrane. Considering the brain is one of the most lipid-rich organs, brain phospholipid changes can lead to various pathogenic processes. 32) Aβ is the main component of senile plaques, a major pathological feature of AD. It is produced by cleavage of the amyloid precursor protein (APP) by β-site amyloid precursor protein cleaving enzyme-1, an endogenous membrane protein in the brain, and γ-secretase. The APP metabolism is closely associated with membrane phospholipids. Furthermore, changes in phospholipid composition have been reported in patients with AD, including a sphingomyelin decrease 33) and high oxidative stress-related changes in erythrocyte phospholipids. 34) The inhibitory effect of fucoidan on Aβ-induced cell membrane damage can be useful in maintaining membrane integrity.
Considering the ameliorating effect of fucoidan on oxidative stress, we investigated mitochondrial function, where ROS are generated. Fucoidan showed a protective effect against the suppression of mitochondrial function by Aβ. Aβ increased production of mitochondrial ROS and reduced Mn-SOD, which removes superoxide produced by mitochondrial electron transport chain complexes. Fucoidan alleviated this Aβ-induced effect by reducing mitochondrial ROS production and restoring MPT and ATP levels.
Fucoidan, a cell wall component of brown algae, is a sulfated polysaccharide whose main component is fucose. Fucoidan can capture radicals. The antioxidation of sulfated polysaccharides is decided by their structural characteristics, such as sulfation degree, molecular weight, major sugar types, glycoside branching, 35,36) and sulfate content. The reducing power of fucoidan increases with increasing sulfate content and molecular weight. 37,38) Fucoidan from Porphyra haitanesis had a high proportion of sulfate, and the derived sulfated polysaccharide fraction showed enhanced SOD, glutathione peroxidase, and antioxidant activity in various tissues of aged mice. 39) Aβ also reduced MPT and ATP production. MPT is affected by mitochondrial Ca 2+ , ATP, and ROS, which decreases with opening of MPT pores. Fucoidan suppressed mitochondrial ROS production and increased ATP production. Additionally, it increased the potential difference reduced by mitochondrial membrane damage. This indicates that fucoidan minimizes Aβ-induced mitochondrial damage.
In summary, fucoidan protects against Aβ-induced neuronal toxicity by directly inhibiting Aβ-induced oxidative stress in the cell membranes and mitochondria (Fig. 9). Therefore, fucoidan is a valuable nutritional supplement for long-term consumption with the potential to slow down the progression or even prevent the onset of AD.