Fucoidan from Fucus vesiculosus prevents the loss of dopaminergic neurons by alleviating mitochondrial dysfunction through targeting ATP5F1a

Parkinson''s disease is a neurodegenerative disease that is characterized by the loss of dopaminergic neurons. Fucoidan, which has emerged as a neuroprotective agent, is a marine-origin sulfated polysaccharide enriched in brown algae and sea cucumbers.

Carbohydrate Polymers 303 (2023) 120470 Contents lists available at ScienceDirect Carbohydrate Polymers journal homepage: www.elsevier.com/locate/carbpol Fucoidan from Fucus vesiculosus prevents the loss of dopaminergic neurons by alleviating mitochondrial dysfunction through targeting ATP5F1a Meimei Xing a, 1, Guoyun Li b, c, 1, Yang Liu a, 1, Luyao Yang b, Youjiao Zhang a, Yuruo Zhang a, Jianhua Ding d, Ming Lu d, *, Guangli Yu b, c, **, Gang Hu a, d, *** a Department of Pharmacology, School of Medicine & Holistic Integrative Medicine, Nanjing University of Chinese Medicine, Nanjing, 210023, Jiangsu, China Key Laboratory of Marine Drugs of Ministry of Education, Shandong Provincial Key Laboratory of Glycoscience and Glycotechnology, School of Medicine and Pharmacy, Ocean University of China, Qingdao 266003, China c Laboratory for Marine Drugs and Bioproducts, Pilot National Laboratory for Marine Science and Technology (Qingdao), Qingdao 266237, China d Jiangsu Key Laboratory of Neurodegeneration, Department of Pharmacology, Nanjing Medical University, Nanjing, Jiangsu 211116, China b A R T I C L E I N F O A B S T R A C T Keywords: Fucoidan Neuroprotection Parkinson's disease Mitochondrial dysfunction ATP5F1a Parkinson's disease is a neurodegenerative disease that is characterized by the loss of dopaminergic neurons Fucoidan, which has emerged as a neuroprotective agent, is a marine-origin sulfated polysaccharide enriched in brown algae and sea cucumbers However, variations in structural characteristics exist among fucoidans derived from different sources, resulting in a wide spectrum of biological effects It is urgent to find the fucoidan with the strongest neuroprotective effect, and the mechanism needs to be further explored We isolated and purified four different fucoidan species with different chemical structures and found that Type II fucoidan from Fucus ves­ iculosus (FvF) significantly improved mitochondrial dysfunction, prevented neuronal apoptosis, reduced dopa­ minergic neuron loss, and improved motor deficits in an 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)induced PD mouse model Further mechanistic investigation revealed that the ATP5F1a protein is a key target responsible for alleviating mitochondrial dysfunction of FvF to exert neuroprotective effects This study high­ lights the favorable properties of FvF for neuroprotection, making FvF a promising candidate for the treatment of PD Introduction Parkinson's disease (PD) is a degenerative disease of the nervous system that is characterized by the loss of dopaminergic (DA) neurons in the substantia nigra (Grayson, 2016; Simon, Tanner, & Brundin, 2020) The cause of PD can be related to multiple factors, including aging, genetics, and environmental factors The major motor symptoms of PD include bradykinesia, rigidity, and resting tremors (Ascherio & Schwarzschild, 2016; Xu, Fu, & Le, 2019) PD is also associated with many nonmotor symptoms, including olfactory impairment, cognitive impairment, psychiatric symptoms, and autonomic dysfunction All of these symptoms add up to overall disability (Schapira, Chaudhuri, & Jenner, 2017) The existing drugs to treat PD, such as levodopa and carbidopa, only focus on increasing the concentration of dopamine in the brain to relieve motor symptoms (Vijiaratnam, Simuni, Bandmann, Morris, & Foltynie, 2021) These drugs not provide neuroprotection Abbreviations: MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; HpF, Holothuria polii; LjF, Laminaria japonica; AnF, Ascophyllum nodosum; FvF, Fucus ves­ iculosus; PD, Parkinson's disease; MPP+, 1-methyl-4-phenylpyridine; DHE, dihydroethidium; TH, tyrosine hydroxylase; SNpc, substantia nigra pars compacta; DA, dopamine; DOPAC, dihydroxy-phenylacetic acid; 5-HT, 5-hydroxytryptamine; LC-MS/MS, liquid chromatography tandem mass spectrometry; KEGG, Kyoto Ency­ clopedia of Genes and Genomes; ROS, reactive oxygen species; MAO-B, monoamine oxidase B; DAT, dopamine transporter; Oli A, oligomycin A * Correspondence to: M Lu, Jiangsu Key Laboratory of Neurodegeneration, Department of Pharmacology, Nanjing Medical University, 101 Longmian Avenue, Nanjing, Jiangsu 211116, China ** Correspondence to: G Yu, Laboratory of Marine Drugs of Ministry of Education, School of Medicine and Pharmacy, Ocean University of China, Yushan Road, Shandong, Qingdao 266003, China *** Correspondence to: G Hu, Jiangsu Key Laboratory of Neurodegeneration, Department of Pharmacology, Nanjing Medical University, 101 Longmian Avenue, Nanjing, Jiangsu 211166, China E-mail addresses: lum@njmu.edu.cn (M Lu), glyu@ouc.edu.cn (G Yu), ghu@njmu.edu.cn (G Hu) These authors contributed equally to this work https://doi.org/10.1016/j.carbpol.2022.120470 Received October 2022; Received in revised form 11 December 2022; Accepted 12 December 2022 Available online 15 December 2022 0144-8617/© 2022 The Authors Published by Elsevier Ltd This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/bync-nd/4.0/) M Xing et al Carbohydrate Polymers 303 (2023) 120470 or prevent or delay the degeneration of DA neurons They also have serious adverse effects, including nausea and vomiting, upright hypo­ tension, sedation, confusion, sleep disturbances, hallucinations, dyski­ nesia, chorea, and progressive dystonia There is an urgent need to develop drugs with neuroprotective effects and few side effects to treat PD Although the underlying pathology of PD is still not well understood, oxidative stress-induced mitochondrial dysfunction is thought to be an important cause of DA neuron loss in PD mice (Ascherio & Schwarzs­ child, 2016; Jankovic & Tan, 2020) Thus, antioxidants are often studied as potential compounds for the protective effects of PD (Schapira & Jenner, 2011) The great potential of marine-derived glycans to treat neurodegenerative diseases has emerged because marine natural prod­ ucts are rich in the biological activity of antioxidants (Karthikeyan, Joseph, & Nair, 2022) For example, GV-971, a compound derived from acidic oligosaccharides in brown algae (Wang et al., 2019), was the first drug developed in China to treat Alzheimer's disease Therefore, we have been focusing on finding a potential marine-derived compound as a possible treatment for PD Fucoidans are sulfated polysaccharides rich in L-fucose, usually found in brown algae and sea cucumbers Fucoidan normally has two types of backbones: type (I) encompasses repeated (1→3)-L-fucopyr­ anose, and type (II) encompasses alternating and repeated (1→3)- and (1→4)-L-fucopyranose (Usoltseva et al., 2019; Usoltseva et al., 2021) Owing to its beneficial biological activities, fucoidan is studied exten­ sively as a multifunctional and nontoxic polysaccharide (Zhang et al., 2020) A variety of classic bioactivities have been reported for fucoidan, including antitumor, antibacterial, antiviral, antioxidant, anticoagulant, antiobesity, and immune-modulating properties In addition, fucoidan has also been shown to play a role in neuroprotection (Kim et al., 2019; Wang et al., 2021) However, variations in structural characteristics exist among different species of seaweeds or sea cucumbers (Li, Lu, Wei, & Zhao, 2008) The molecular weight also varies between different species (Fitton, Stringer, & Karpiniec, 2015), and the relationship be­ tween molecular weight and bioactivity is not clear (Yang et al., 2018) Previous studies showed that fucoidan from Laminaria japonica pro­ tected dopaminergic neurons from rotenone-induced PD in rats and reduced behavioral deficits and increased striatal dopamine (Luo et al., 2009; Zhang et al., 2018) Sulfated fucoidan isolated from Saccharina japonica showed better neuroprotective activity than low molecular weight fucoidan (Liu, Wang, Zhang, & Zhang, 2018) The bioactivities of fucoidan from Fucus vesiculosus gradually depolymerized fractions were also proven to decrease with decreasing molecular weight (Han, Lee, & Lee, 2019; Lahrsen, Schoenfeld, & Alban, 2018) However, He et al showed that low-molecular-weight fucoidan reduces mitochondrial dysfunction in aged mice compared to higher molecular weight de­ rivatives (Wang, Zhu, & He, 2016) These results confirmed that diverse sources of fucoidans result in diverse structures and a broad spectrum of bioactivities, indicating that the structure-effect relationship deserves further study Target fishing technology is a method combining active small molecule probes and pull-down technology, which can accurately detect protein targets of small molecule compounds Tu et al identified precise targets of small molecule compounds in neuroinflammation, diabetes and fatty liver using this method, which illustrates the feasibility of targets for fishing applications in the mechanistic study of carbohydratebased pharmaceutical molecules (Dai et al., 2022; Ma et al., 2022; Yang et al., 2021; Zheng et al., 2022) In this study, we isolated and purified four kinds of fucoidans with different chemical structures from Holothuria polii (HpF), Laminaria japonica (LjF), Ascophyllum nodosum (AnF) and Fucus vesiculosus (FvF) We found that type II fucoidan (FvF) had the best neuroprotective effect in the MPTP-PD mouse model Finally, we identified ATP5F1a as the target of FvF to protect DA neurons by improving mitochondrial func­ tion, suggesting that FvF may be a pluripotent and promising drug for PD therapy Materials and methods 2.1 Materials Holothuria polii (HpF), Laminaria japonica (LjF), Ascophyllum nodosum (AnF) and Fucus vesiculosus (FvF) were provided by the Key Laboratory of Glycoscience and Glycotechnology, School of Medicine and Phar­ macy, Ocean University of China Oligomycin A (Selleck, #S1478); MPTP (Selleck, #S4732); 1-methyl-4-phenylpyridine (MPP+) iodide (Sigma, #D048); anti-tyrosine hydroxylase (TH) antibody (Sigma, #T1299); Hoechst 33342 (Sigma, #B2261); JC-1 (Invitrogen, #T3168); MitoSOX (Invitrogen, #M36008); streptavidin agarose resin (Invi­ trogen, #20347); Anti-MAP2 antibody (Santa Cruz, #Sc-32791); Annexin V/PI (Vazyme Biotech Co., Ltd., #A21-02); and dihydrothi­ dium (Beyotime Biotechnology; #M36008) 2.2 Preparation and characterization of fucoidans from different sources Crude polysaccharides were extracted from the body wall of the sea cucumber Holothuria polii or seaweeds by methods reported previously (Li et al., 2020; Shan et al., 2016) and then separated by a Q Sepharose Fast Flow anion-exchange column to obtain pure high-sulfated fucoi­ dans The chemical composition and physical properties of fucoidans were analyzed The monosaccharide composition was determined by using the 1-phenyl-3-methyl-5-pyrazolone (PMP)-HPLC method The composition and sulfate content were determined by the BaCl2-Gelatin method Purity and relative molecular weight (Mw) were determined by gel filtration columns (Shodex OHpak SB-804 HQ and SB802.5 HQ) connected to an HPLC system In addition, we also carried out 1H NMR at 298 K on an Agilent DD2 500 MHz to assign the structural type of fucoidans Table S1 shows the structural information and molecular mass of the four kinds of fucoidan 2.3 Animals Three-month-old male C57BL/6 mice were purchased from Nanjing Medical University Animal Centre Mice were housed in SPF grade laboratories and were allowed to drink and feed ad libitum All exper­ iments were carried out in accordance with the Guide for the Care and Use of Laboratory Animals All animal experiments in the present study were approved by the Ethical Committee of Nanjing Medical University (Permission No 1903038) 2.4 Cell culture Culture of primary midbrain and cortical neurons was performed according to previous studies (Han et al., 2018) Pregnant C57BL/6 fe­ male mice were rapidly cervically dislocated and disinfected with iodophor and 75 % medical alcohol The midbrain or cortical tissue of fetal mice was carefully dissected, and the meninges and blood vessels were removed The midbrain or cortical tissues were next digested by trypsin Subsequently, the cells were incubated in neurobasal medium containing % B27 (Gibco, 17504044) SH-SY5Y cells were purchased from the Cell Bank of the Chinese Academy of Sciences and cultured in 10 % FBS in a constant temperature incubator 2.5 Cell treatment Neurons were administered FvF (10 μM) and MPP+ (10 μM) for 48 h SH-SY5Y cells were administered FvF (5, 10 and 25 μM) and MPP+ (500 μM) for 48 h 2.6 CCK-8 assay Neurons or SHSY5Y cells were seeded in a 96-well plate and then M Xing et al Carbohydrate Polymers 303 (2023) 120470 pretreated with LjF, HpF, AnF and FvF (5 μM) for 24 h and treated with MPP+ (500 μM) for another 24 h Then, CCK-8 was added to each well for h Finally, the absorbance was detected at 450 nm surfaced, 50 cm high and cm diameter iron rod The time required for the mouse to reach the bottom of the floor (T-TLA) and the time required for the mouse to be fully head down (T-turn) were recorded Motor function for all mice was trained during the lights-on cycle from 9:00 to 14:00 All tests were similar to those of previous research (Yun et al., 2018) 2.7 LDH assay Culture media was collected according to the manufacturer's in­ structions to assess LDH levels using an assay kit (Nanjing Jiancheng Bioengineering Institute) 2.14 Brain sample collection Mice were rapidly anesthetized after behavioral testing The midbrain and stratum tissues of whole brains were quickly dissected, and all samples were stored at − 80 ◦ C The isolated brains were fixed in % paraformaldehyde (Aladdin, #F111941) for days and dehydrated for days using 20 % and 30 % sucrose (Saint-Bio, #T16373) Next, the brains were embedded in OCT (Tissue-Tek) and sliced into 25 μm slices using a freezing microtome (Leica CM1950, Nussloch, Germany) Slices in 50 % glycerol PBS solution could be stored at − 80 ◦ C for long-term storage (Han et al., 2021) 2.8 Hoechst staining Fluorescence microscopy (Olympus, Tokyo, Japan) was used to observe cells that had been stained with Hoechst 33342 for 10 2.9 Quantitative real-time PCR TRIzol reagent (Invitrogen, #10296028) was used to extract total RNA from the midbrains and neurons A NanoDrop 5500 (Thermo) was used to perform total RNA quantification analysis Reverse transcription was carried out using Hiscript III RT SuperMix for qPCR (Vazyme, #R323-01) SYBR Green Master Mix (Roche, #04913914001) was used to determine the relative expression of mRNAs Table S2 shows the primer sequences used for real-time qPCR 2.15 Neurotransmitter measurement using HPLC Each striatum was weighed and then homogenized in ice-cold buffer comprising 0.01 % DHBA Following that, centrifugation was performed (15,000 ×g, 30 min, ◦ C), and collection of the supernatant was used for determination of monoamine neurotransmitters 2.10 Flow cytometry analysis 2.16 Immunofluorescence and immunohistochemistry According to the manufacturer's instructions, Annexin V/propidium iodide (PI) staining (Vazyme Biotech, China) was used to assess cellular apoptosis using flow cytometry (Qiao et al., 2017) In brief, neurons were washed in ice-cold PBS, resuspended in binding buffer, and incu­ bated with PI and Annexin V-fluorescein isothiocyanate for 10 A flow cytometry instrument was immediately used to perform a flow cytometric analysis (Millipore, USA) For immunofluorescence staining, the neurons were incubated in PBS buffer with 0.2 % Triton X-100 (Sigma, #T9284) for 20 before being cultured with % BSA (Yi Fei Biotechnology, #YB0006-100) for h Next, the neurons were incubated with anti-MAP2 antibodies (Santa Cruz, #SC-32791; 1:500) at ◦ C for 24 h Furthermore, the neurons were washed and incubated with Goat anti-mouse IgG (Alexa Fluor 555conjugated Invitrogen, A21422; 1:700) for h Finally, the neurons were photographed by fluorescence microscopy (Olympus, Tokyo, Japan) For immunohistochemistry staining, slides encompassing the entire SNpc were incubated in PBS buffer with 0.2 % Triton X-100 (Sigma, #T9284) for 20 before being cultured with % BSA for h The slides were then incubated at ◦ C overnight with the primary antibodies anti-TH (Sigma, #T1299; 1:500), washed, and incubated with secondary antibody (Thermo, #SA5-10275, 1:500) for h at room temperature, and then the slides were stained with DAB (Shanghai Gene Company, #GK500705) for 30 s and washed three times Finally, slides were visualized and photographed under a fluorescence microscope (Olympus, Tokyo, Japan) The immunostaining signals were analyzed quantitatively by using Micro bright field Stereo-Investigator software (Stereo Investigator software; Micro bright field) (Han et al., 2021) 2.11 ROS detection in intracellular and mitochondrial environments Dihydroethidium (DHE) was used to measure intracellular ROS In brief, SH-SY5Y cells were treated with 10 μM FvF and 500 μM MPP+ for 24 h Next, the cells were incubated with μM DHE for 30 in a 37 ◦ C thermostatic incubator Finally, the cells were washed three times with PBS, and the fluorescence intensity was observed by flow cytometry (Millipore, USA) MitoSOX was used to measure mitochondrial ROS in SH-SY5Y cells In brief, cells were incubated with μM MitoSOX for 30 in a 37 ◦ C thermostatic incubator and washed three times with PBS Finally, the fluorescence intensity was measured by fluorescence mi­ croscopy (Olympus, Tokyo, Japan) 2.12 Administration of FvF in MPTP-induced PD mouse models 2.17 Target fishing In total, thirty four-month-old male C57BL/6 mice weighing 22–28 g were separated into groups (n = in each group): control, MPTP (20 mg/kg), FvF (40 mg/kg), MPTP + FvF (10 mg/kg), and MPTP + FvF (40 mg/kg) FvF was administered daily starting one day before MPTP in­ jection Then, both MPTP and FvF were administered together daily for days FvF was then provided for another two days Mice in the control group were injected with an equal volume of saline Neurons were collected and homogenized in % NP-40 lysis buffer (GenSar, #E124-01), and the extracted protein was measured with a BCA protein quantification kit (KeyGen BioTech, #KGP903) The pro­ tein was incubated with 10 μM FvF-biotin for 12 h at ◦ C and incubated with streptavidin agarose resin for h at ◦ C Next, the protein was centrifuged for at 3000 rpm The precipitate was washed times, added to 25 μL 5× loading buffer and boiled for Finally, the protein was removed by 10 % SDS-PAGE and then analyzed by highperformance liquid chromatography tandem mass spectrometry (LCMS/MS) The potential target information of FvF is shown in Table S3 2.13 Behavior analysis Behavioral tests were performed after the last administration of MPTP to mice (Sampson et al., 2016; Yun et al., 2018) For the opened field test, the crawling track and distance of mice in the activity box of 50 cm × 50 cm × 42 cm in were logged For the rotarod test, the Rotarod Analysis System recorded the time of latency to fall at 20 rpm for 300 s For the pole test, mice were placed on the top of a rough- 2.18 Docking studies The RCSB Protein Data Bank was used to obtain the threedimensional structure of ATP5F1a (PDB ID: 1BMF) The docking M Xing et al Carbohydrate Polymers 303 (2023) 120470 application was AutoDock Vina PyMOL version Open-Source 1.6.x was used to make the graphics variance, or one-way analysis of variance (ANOVA) followed by Tukey's post hoc test was used to determine the significance of differences Differences were considered significant at P < 0.05 2.19 Knockdown of ATP5F1a Results ATP5F1a siRNA was synthesized by Sangon Biotech Negative con­ trol siRNA and ATP5F1a siRNA were also transiently transfected into neurons or SH-SY5Y cells by the utilization of Lipofectamine 3000 (Invitrogen, #L3000-15) in agreement with the manufacturer's protocols 3.1 Neuroprotective activity screening of carbohydrate compounds of marine origin Based on previous work in our laboratory (Cui et al., 2018; Jiao, Yu, Zhang, & Ewart, 2011; Li et al., 2018; Wang et al., 2012), we constructed a library of marine carbohydrates From this library, forty-two marineorigin carbohydrates belonging to categories (fucosylated chondroitin sulfate, alginate derivative, fucoidan, chitosan derivative, agar deriva­ tive, homoxylan and marine glycosaminoglycans) were selected to 2.20 Statistical analysis GraphPad Prism 9.0 was used to analyze the results The data are presented as the mean ± SD Student's t-test, two-way analysis of Fig Neuroprotective activity of carbohydrate compounds of marine origin (A) Cell viability of SH-SY5Y cells after treatment with 42 carbohydrate compounds at a concentration of 10 μM (B) Cell viability of SH-SY5Y cells treated with MPP+ (500 μM) and carbohydrate compounds (C) 1H NMR spectra of four kinds of fucoidan (D) Structure of four types of fucoidan (E) Cell viability of neurons treated with HpF, LjF, AnF, and FvF for 48 h (F) LDH release was detected after treatment with HpF, LjF, AnF, and FvF (G) Effect of HpF, LjF, AnF, and FvF on neuronal viability with MPP+ stimulation (H) Effect of HpF, LjF, AnF, and FvF on LDH release with MPP+ stimulation (I–J) Immunostaining for MAP2 after MPP+ and fucoidan treatment Red color defines neuronal axons, showing the protection of neurons from MPP+ by AnF and FvF Scale bar is 20 μm Data are analyzed as the means ± SD using one-way ANOVA and then combined with Dunnett's test to assess the dif­ ferences between groups #P < 0.05, ##P < 0.01, ###P < 0.001 vs Control group; *P < 0.05, **P < 0.01, ***P < 0.001 vs MPP+ group M Xing et al Carbohydrate Polymers 303 (2023) 120470 evaluate their neuroprotective effect Marine carbohydrates are soluble in water and insoluble in organic solvents Therefore, all marine car­ bohydrates were dissolved in PBS, and PBS was used as a negative control in the cell experiments Each carbohydrate compound was incubated with SH-SY5Y cells at a concentration of 10 μM for 24 h, and then cell viability of each group was detected by the CCK-8 method (Fig 1A) Subsequently, thirty-six compounds with cell viability >90 % was exposed to 500 μM MPP+ for another 24 h (Fig 1B) In MPP+induced SH-SY5Y cells, we noticed that one compound, fucoidan from Fucus vesiculosus (FvF), markedly improved the cell viability Fucoidan includes a large family with different chemical structures, and exists in a variety of marine organisms, which may result in a wide spectrum of bioactivity To investigate the potential protective effect of different fucoidans, we then isolated and purified fucoidans from three other sources: Holothuria polii (HpF), Laminaria japonica (LjF) and Ascophyllum nodosum (AnF) (Li et al., 2020; Shan et al., 2016) All structural infor­ mation of four fucoidans is shown in Fig 1C–D All of these were highly sulfated (35.6 %– 40.5 %) fucans with trace amounts of galactose or xylose residues As shown in Fig 1C, strong signals at about 1.1–1.4 ppm in the 1H NMR spectra of HpF, LjF, AnF and FvF can be readily assigned to the methyl protons of fucose residues (CH3) In addition, the chemical shifts of the envelope of anomeric signals at 4.9–5.6 ppm are consistent with the presence of α-L-fucopyranosyl units Differently, the spectra of HpF and LjF show only one broad peak at 5.31 ppm in their anomeric regions, which suggest the anomeric proton signals of 3-linked α-Lfucose A feature at around 5.10 ppm can be tentatively assigned to their H1 of non-sulfated fucose residues according to literature comparison The spectra of AnF and FvF show two broad peaks at 5.27 ppm, 5.04 ppm and 5.35 ppm, 5.13 ppm respectively in their anomeric regions, which suggest the anomeric proton signals of both 3-linked and 4-linked α-L-fucose In conclusion, HpF and LjF were classified as Type I fucoidan, with the main chain composed of 1,3-linked α-L-fucopyranose residues AnF and FvF was classified as Type II fucoidan, which encompassed alternating and repeated (1→3)- and (1→4)-L-fucopyranose To further evaluate the potential protective effect of fucoidans, CCK8 and LDH assays were used We first demonstrated that the four types of Fig FvF attenuated MPP+-induced cytotoxicity in SH-SY5Y cells and neurons (A–B) Cell viability and LDH release after SH-SY5Y cells were treated with FvF (5, 10, 25 and 100 μM) for 24 h (C–D) Cell viability and LDH release after SH-SY5Y cells were treated with FvF (5, 10 and 25 μM) and MPP+ (500 μM) for 24 h (E–F) Apoptosis by Annexin V-FITC/PI and quantitative analysis in neurons treated with FvF (10 μM) and MPP+ (500 μM) for 24 h (G–H) Representative images of Hoechst staining; the scale bar is 20 μm (H) Quantitative analysis of Hoechst-positive neurons (I–J) Representative images of MAP2 and (J) the average neurite length; the scale bar is 20 μm (K–L) Representative images of TH, and (L) the average neurite length; the scale bar is 40 μm Data are analyzed as the means ± SD using one-way ANOVA and then combined with Dunnett's test to assess the differences between groups #P < 0.05, ##P < 0.01, ###P < 0.001 vs Control group; *P < 0.05, **P < 0.01, ***P < 0.001 vs MPP+ group M Xing et al Carbohydrate Polymers 303 (2023) 120470 fucoidans have no significant cytotoxicity (Fig 1E–F) In primary neuron culture, cell viability was greatly reduced with damage from MPP+, but with the two type-II fucoidans AnF and FvF, cell viability decreased less, showing the neuroprotective effect of these two fucoi­ dans (Fig 1G) LDH release was greatly increased with damage from MPP+ but with the two type-II fucoidans AnF and FvF (Fig 1H) Microscopic observation confirmed that with 10 μM MPP+ stimulation for 48 h, few neurons remained, which decreased in size, and cell pro­ trusions were also shortened The AnF- and FvF-treated cells, on the other hand, did not decrease in number and size as much as the un­ treated culture (Fig S1) Fig 1I–J shows the overall survival of neurons by immunostaining with the pan-neuronal marker MAP2 and further confirmed that AnF and FvF treatment could protect neurons from being damaged by MPP+ Moreover, the neuroprotective effect of FvF was superior to that of AnF In previous studies, MAO-B inhibitor selegiline was used as a positive tool drug in cell and animal models (Anastassova et al., 2021; Zhang et al., 2022) However, the protection mechanism of selegiline is not the same as that of the experimental compounds in our study, so we did not include a positive control in our following experi­ ments (Fig S2) 3.2 FvF attenuated MPP+-induced cytotoxicity in SH-SY5Y cells and primary neurons To verify the cytotoxic effects of FvF on SH-SY5Y cells and neurons, cell viability was assessed with CCK-8 and LDH assays FvF (5, 10, 25 and 100 μM, for 24 h) had no significant cytotoxicity in SH-SY5Y cells (Fig 2A–B) In the MPP+-induced SH-SY5Y cell model, 5, 10 and 25 μM FvF markedly enhanced the viability of SH-SY5Y cells (Fig 2C–D) A concentration of 10 μM was chosen to investigate the mechanism behind Fig Neuroprotective effects of FvF in the MPTP-induced PD mouse model (A) Schematic diagram of the experimental design FvF (10/40 mg/kg) or vehicle (saline) was administered intraperitoneally for consecutive days beginning on day − 1, and mice received MPTP (20 mg/kg) or vehicle (saline) each day for days before tissues were taken for molecular analysis on Day following the behavior test Behavior tests, such as the OFT (B–C), latency to fall (D), T-turns (E) and T-TLA (F), were conducted (G) Immunostaining of TH-positive neurons in the SNpc Scale bars is 40 μm (H) Stereological counts of TH-positive neurons in G (I) Con­ centrations of DA, DOPAC and 5-HT in the striatum of mice were measured using HPLC Data are analyzed as the means ± SD using one-way ANOVA and then combined with Dunnett's test to assess the differences between groups #P < 0.05, ##P < 0.01, ###P < 0.001 vs Control group; *P < 0.05, **P < 0.01, ***P < 0.001 vs MPTP group M Xing et al Carbohydrate Polymers 303 (2023) 120470 the effects of FvF As shown in Fig S3, MPP+ (10 μM, 48 h) induced morphological damage in neurons, and FvF (10 μM) significantly attenuated morphological damage Furthermore, MPP+ triggered cell death, as demonstrated by an increase in the proportion of Annexin Vand PI-stained cells, which was protected by FvF (Fig 2E–F) According to the Hoechst staining data, FvF (10 μM) dramatically decreased the proportion of apoptotic cells (Fig 2G–H) Immunostaining with MAP2 was used to examine the survival of non-DA neurons As shown in Fig 2I–J, non-DA neurons were much more susceptible to MPP+induced axon loss, and FvF (10 μM) therapy significantly reduced the effect of MPP+ Additionally, FvF (10 μM) notably reversed MPP+induced damage in dopamine neurons (Fig 2K–L) These findings sug­ gest that FvF (10 μM) can reverse the detrimental effects of MPP+ on neurons 3.4 FvF is a neuroprotective agent associated with signaling pathways involved in oxidative stress and mitochondrial function Given that FvF exerted significant neuroprotective activity both in vitro and in vivo, it is worthwhile to discover its macromolecular binding partners and fully define the molecular mechanism We syn­ thesized biotin probes to target the protein target of FvF that is responsible for its neuroprotective impact The workflow chart of pro­ tein target identification is shown in Fig 4A FvF was conjugated with biotin and then enhanced on streptavidin beads To identify proteins targeted by FvF, the digested peptides were examined using liquid chromatography tandem mass spectrometry (LC-MS/MS) Kyoto Ency­ clopedia of Genes and Genomes (KEGG) analysis using Metascape was performed to identify the pathways associated with the 512 proteins captured by FvF (Fig 4B) Pathways associated with Parkinson's disease, mitochondrial function, apoptosis and energy metabolism were found Strikingly, we found that FvF recovered the downregulation of gene expression related to mitochondrial function (Mfn1, Mfn2, Drp1 and Opa1), anti-apoptosis and anti-oxidative stress (Dj1, Nqo1, Sod1, and Gpx3) in primary neurons and midbrains and lowered gene expression levels related to oxidative stress and apoptosis However, FvF had no effects on genes associated with ER stress (Fig 4C, D) In conclusion, these results suggested that the PD-protective effect of FvF could be mediated through signaling pathways related to oxidative stress and mitochondrial function 3.3 FvF exerted neuroprotective effects in the MPTP-induced PD model To assess the neuroprotective effect of FvF in PD, the MPTP-induced PD mouse model was used We treated MPTP-PD mice with 10 and 40 mg/kg doses of FvF for days to evaluate the neuroprotective effects of FvF in vivo (Fig 3A) The open field test, rotarod test, and pole test were used to evaluate the motor and behavioral performance of the mice As shown in Fig 3B–F, the impaired motor coordination and balance ability were relieved by FvF FvF treatment significantly slowed the loss of TH markers in the substantia nigra pars compacta (SNpc) region of MPTPPD mice (Fig 3G, H) The decrease in dopamine (DA) and dihydroxyphenylacetic acid (DOPAC) content in the striatum was mitigated by FvF but had no effect on the level of 5-hydroxytryptamine (5-HT) (Fig 3I) In conclusion, in MPTP-induced PD mice, FvF slowed the progression of dopamine neuron loss, protected the function of the nigral striatum, and alleviated motor dysfunction 3.5 FvF rescued mitochondrial dysfunction in SH-SY5Y cells Owing to our findings on the restoration in gene expression associ­ ated with oxidative stress and mitochondrial function following FvF treatment, we next examined mitochondrial function using dihy­ droethidium and MitoSOX superoxide dye Reactive oxygen species (ROS) production was increased in MPP+-treated SH-SY5Y cells compared to the control group but was recovered with the addition of Fig FvF is a neuroprotective agent associated with signaling pathways involved in oxidative stress and mitochondrial function (A) Schematic illustration of target identification, biotin probe design and target fishing of FvF (B) KEGG analysis of the 512 protein targets identified by LC-MS/MS revealed their association with Parkinson's disease (C) Gene heatmap of the midbrain in MPTP-induced PD mice by q-PCR analysis (D) Gene heatmap of neurons in MPP+-induced neuronal injury by q-PCR analysis M Xing et al Carbohydrate Polymers 303 (2023) 120470 FvF (Fig 5A–B) MitoSOX mitochondrial superoxide indicator was used to detect mitochondrial ROS Superoxide increased in mitochondria of MPP+-treated SH-SY5Y cells as predicted, compared to the control group, and FvF therapy restored this accumulation ROS accumulation is a result of mitochondrial dysfunction, and therefore the above results suggested a rescue in mitochondria (Fig 5C–D) Transmission electron microscopy showed that MPP+ induced a decrease in mitochondrial density and an increase in the number of abnormal mitochondria but was recovered with the addition of FvF in SH-SY5Y cells (Fig 5E–G) These findings imply that FvF can protect SH-SY5Y cells against MPP+induced mitochondrial dysfunction at approximately 60, 110 and 190 kDa Among the potential targets shown in Table S3, the bands at approximately 110 or 190 kDa did not have any matching results The band at approximately 60 kDa matched ATP5F1a after excluding other targets at similar molecular weights but did not have biological functions related to the pathogenesis of PD (Fig S4C, Table S3) Next, we discovered the potential protein binding with FvF by LC-MS/MS (Fig 6A) and verified the target protein by Western blotting (Fig 6B) Then, we performed a molecular docking (MD) analysis to investigate the binding site of the ATP5F1a/FvF com­ plex based on the published crystal structure of human ATP5F1a (PDB ID: 1BMF) The corresponding docking conformation suggested that FvF fits into the active pocket, forming multiple important interactions with surrounding amino acid residues, including Asp252, Thr255, Asp312, Lys316, Lys218, Thr19, Gly217, Glu371, and Gln215 (Fig 6C) To investigate whether the target of FvF, ATP5F1a, played a role in the neuroprotective effects of MPP+ on neurological damage, we used ATP5F1a siRNA to test the function of FvF on ATP5F1a (Fig S5) The knockdown of ATP5F1a stopped the anti-apoptosis effects that FvF should exert on MPP+-treated primary neurons (Fig 6D–F) FvF also lost its potency to alleviate the production of mitochondrial ROS (Fig 6G–H) 3.6 ATP5F1a was recognized as a potential target of FvF To study which proteins FvF binds to exert its neuroprotective bio­ logical activity, we first evaluated the effects of FvF-biotin on neuron survival The results indicated that FvF-biotin (10 μM, 48 h) had no significant cytotoxicity (Fig S4A–B) The proteins labeled with biotin probes were separated by SDS-PAGE and visualized by Coomassie bril­ liant blue As shown in Fig S4C, there were three distinct protein bands Fig FvF recovered mitochondrial dysfunction in SH-SY5Y cells (A) ROS levels and (B) mean fluorescence intensity analysis of dihydroethidium by flow cytometry (C) Fluorescence intensity of cells stained with MitoSOX analyzed by fluorescence microscopy, and (D) mean fluorescence intensity analysis of MitoSOX Scale bar is 20 μm (E) Mitochondrial morphology was observed via transmission electron microscopy, and (F–G) the mitochondrial density and abnormal mito­ chondria were analyzed Data are analyzed as the means ± SD using one-way ANOVA and then combined with Dunnett's test to assess the differences between groups #P < 0.05, ##P < 0.01, ###P < 0.001 vs Control group; *P < 0.05, **P < 0.01, ***P < 0.001 vs MPP+ group M Xing et al Carbohydrate Polymers 303 (2023) 120470 Fig IP-LC/MS identified ATP5F1a as a target of FvF for alleviating mitochondrial dysfunction (A) A representative peptide of ATP5F1a was identified by LC-MS/ MS (B) Pull-down/Western blotting for target validation of ATP5F1a with biotin probes (C) The hydrogen bonds formed between FvF and neighboring amino acid residues in ATP5F1a (D) Fluorescence microscopy of primary neurons labeled with Hoechst 33342 Red arrow: apoptotic cells (E) Quantitative analysis of Hoechstpositive neurons Scale bar is 20 μm (F) Cell viability of SH-SY5Y cells after treatment with FvF, MPP+ and ATP5F1a siRNA (G) Fluorescence images of cells stained with MitoSOX and (H) mean fluorescence intensity analysis of MitoSOX The scale bar is 20 μm Data are analyzed as the means ± SD using one-way ANOVA and then combined with Dunnett's test to assess the differences between groups #P < 0.05, ##P < 0.01, ###P < 0.001 vs Control group; *P < 0.05, **P < 0.01, ***P < 0.001 vs MPP+ group induced by MPP+ in SH-SY5Y cells when ATP5F1a was knocked down Taken together, the neuroprotective effect of FvF was reversed by ATP5F1a siRNA, which confirmed that FvF exerts its neuroprotective effects through ATP5F1a to protect against DAergic neuron loss induced by MPTP (Fig 7G–H) Taken together, these results suggested that the pharmacological in­ hibitor of ATP5F1a oligomycin A reversed the neuroprotective effect in the MPTP PD mouse model induced by FvF 3.7 The neuroprotective effects of FvF could be attenuated by a pharmacological inhibitor of ATP5F1a, oligomycin A Discussion Oxidative stress and mitochondrial dysfunction play an important role in the etiology of Parkinson's disease (Dai et al., 2018; Trist, Hare, & Double, 2019) Evidence previously found suggests that oxidative stress, which occurs in the case of neurotoxicity, has been an important mechanism of impaired mitochondria that drives degenerative neurology, making it an important therapeutic target for PD (Mahoney´nchez et al., 2021) Marked oxidative stress, which is increased Sa mitochondrial ROS and intracellular ROS, was also found in neurons in PD pathology (Burbulla et al., 2017; Han et al., 2021) 1-Methyl-4phenyl-1,2,3,6-tetrahydropyridine (MPTP) is a highly lipophilic mole­ cule that can rapidly penetrate the blood–brain barrier (Langston, 2017) Oligomycin A is widely used as a pharmacological inhibitor of ATP5F1a After treatment with oligomycin A, motor performance improved by FVF was significantly reduced In open field tests, mice treated with Oligomycin A plus FvF did not show significant improve­ ment in speed of movement compared to MPTP mice, as FvF only would have (Fig 7A–C) Similarly, in rotarod tests, FvF treatment increased the time on the rod of MPTP mice, while FvF plus oligomycin A treatment did not (Fig 7D) The same result of the time of turning and climbing in pole tests was also observed: Oligomycin A canceled the effect of decreasing the time FvF had (Fig 7E–F) Furthermore, FvF lost its ability M Xing et al Carbohydrate Polymers 303 (2023) 120470 Fig Neuroprotective effects of FvF could be attenuated by a pharmacological inhibitor of ATP5F1a, oligomycin A FvF (40 mg/kg) or Oli (0.5 mg/kg) was administered intraperitoneally for consecutive days beginning on day − and received MPTP (20 mg/kg) each day for days before tissues were taken for mo­ lecular analysis on Day following the behavior test Representative image (A), average speed (B) and distance (C) of movement were recorded simultaneously during the open field test Latency to fall (D), T-turns (E), and T-TLA (F) were conducted (G) Representative photomicrographs of TH staining in the SNpc (H) Unbiased stereological counts of TH Scale bar is 40 μm Data are analyzed as the means ± SD using one-way ANOVA and then combined with Dunnett's test to assess the differences between groups n = #P < 0.05, ##P < 0.01, ###P < 0.001 vs Control group; *P < 0.05, **P < 0.01, ***P < 0.001 vs MPTP group with type І Fucoidan (HpF and LjF) in the primary neuron (Fig 1E–J) And the differences in neuroprotective effects between type І and type II Fucoidan may be probably due to the differences in link and sulfated modification For the first time, we found that the type II fucoidan FvF had neuroprotective and anti-PD effects in both a primary neuronal model and an MPTP-induced PD mouse model In view of the key role of mitochondrial dysfunction in the patho­ genesis of PD, we hypothesized that the neuroprotective effect of FvF is mediated by the alleviation of mitochondrial dysfunction (Zhang et al., 2018) Oxidative stress produced by reactive oxygen species (ROS) is a major contributor to the pathogenesis of PD Mitochondria are the main source of ROS, and mitochondrial dysfunction increases the formation of ROS Increased oxidation products were consistently observed in the SNpc of PD animal models (Zhang et al., 2018) In Alzheimer's diseaserelated studies, fucoidan reduced the production of ROS stimulated by Aβ in Caenorhabditis elegans (C elegans) (Jin et al., 2013; Liu et al., 2018; Wang et al., 2021; Wang, Yi, & Zhao, 2018) In addition, fucoidan reversed the reduction in intracellular superoxide dismutase (SOD) and glutathione (GSH) induced by MPP+ in a dopaminergic nerve precursor cell line (MN9D) (Liang et al., 2018) The results of our in vitro assays also indicate that FvF prevents the generation of excessive mitochon­ drial ROS and recovers abnormalities in mitochondrial membrane po­ tential, indicating that FvF has a neuroprotective effect by improving mitochondrial function Utilizing target fishing technology, we detected ATP5F1a as a po­ tential target of FvF ATP5F1a is a mitochondrial complex V component and is one of the subunits of the ATPase complex ATP synthase (ATPase) is responsible for the majority of ATP production(Zech et al., 2022), but only a few studies have shown that ATPase plays a role in ROS and then be metabolized into MPP+ by monoamine oxidase B (MAO-B) MPP+ is selectively absorbed and accumulates in dopaminergic neurons through the dopamine transporter (DAT) MPP+ can interfere with the function of mitochondrial complex I, mediate a variety of harmful oxidative stress responses, and lead to the death of dopamine neurons (Dionísio, Amaral, & Rodrigues, 2021; Schildknecht, Di Monte, Pape, Tieu, & Leist, 2017) Among the two type-I fucoidans we tested, HpF has been shown to have an anticoagulant effect (Li et al., 2020; Mansour et al., 2019) However, there has been no report on its neuroprotective effects Our results further confirmed that HpF does not protect against neuronal damage in primary neuron culture LjF, on the other hand, has been extensively studied for its endothelial protective, immune activating, antiviral, antithrombotic, anti-inflammatory and antioxidant activities (Ahmad et al., 2021; An et al., 2022; Chen et al., 2017; Kim et al., 2020; Liang et al., 2018; Zhang et al., 2021; Zhao et al., 2016) It has also been reported to protect dopaminergic neurons in a rotenone-induced rat model of PD (Zhang et al., 2018) AnF has been reported to suppress postprandial hyperglycemia, ameliorate atherosclerosis and alleviate gut microbiota dysbiosis (Shan et al., 2020; Wang et al., 2020; Yin et al., 2019) We observed similar neuroprotective effect of LiF and AnF in our study Previous studies on FvF have only reported on its bioactivities not related to the nervous system, such as reduced HBV DNA, hBsAg, and HBeAg levels in the blood (Li et al., 2017), a bacteriostatic effect (Ayr­ apetyan et al., 2021) and a protective role in DOX-induced acute car­ diotoxicity (Zhang et al., 2020) We explored the structure-activity relationship between type І and type II Fucoidan It was found that type II Fucoidan (AnF and FvF) had superior effects in lowering the release of lactate dehydrogenase, improving cell viability compared 10 Carbohydrate Polymers 303 (2023) 120470 M Xing et al production Quintana-Cabrera et al showed that the OPA1 gene could decrease mitochondrial ROS accumulation, but ATPase activity is required in this process (Quintana-Cabrera et al., 2021) ATP5F1a itself has been linked to various human diseases, including cancer, amyo­ trophic lateral sclerosis and frontotemporal dementia (Chin et al., 2014; Choi et al., 2019; Feichtinger et al., 2018) A growing number of sci­ entists are noticing the role of ATP5F1a in mitochondrial function and metabolism (Chin et al., 2014; Goldberg et al., 2018; Quintana-Cabrera et al., 2021; Xiao et al., 2021) Previous information shows that the loss of ATP5F1a participates in neurotoxicity and has a significant impact on amyotrophic lateral sclerosis (ALS) In addition, induction of ectopic ATP5F1a expression in poly (GR)-expressing neurons or reduction in poly (GR) levels in adult mice after they have been rescued from poly (GR)-toxicity (Choi et al., 2019) In this study, our findings reveal that ATP5F1a may play a significant role in the pleiotropic effects of FvF in Parkinson's disease therapy FvF may provide protection against PD by targeting ATP5F1a and mitigating mitochondrial dysfunction Oligo­ mycin A abolishes the neuroprotective effect of FvF in MPTP PD mice This is also proof that ATP5F1a played a role in ROS reduction Our data suggested that the neuroprotective effects of FvF may be mediated via ATP5F1a The significance of our study is to enrich the anti-PD effects of different sources of fucoidan based on previous studies and to confirm the potential value of marine glycoconjugate fucoidan in the develop­ ment of anti-PD drugs We used target fishing technology for the first time to assess the potential targets of FvF and to elucidate the mecha­ nism of its neuroprotection Although we elucidated the role of ATP5F1a in FvF alleviating mitochondrial dysfunction and apoptosis in primary neurons, there might be other targets in FvF against PD that still need to be explored Furthermore, the main role of ATP5F1a is to regulate ATP production and participate in energy metabolism Therefore, it is worth exploring in depth the role of FvF in the energy metabolism of neuronal cells Acknowledgments This work was supported by the National Natural Science Foundation of China (81991523, 81991522, 31971210), National Key Research and Development Program of China (2021ZD0202901), Taishan Scholar Climbing Project (TSPD20210304) and Key Scientific and Technological Projects of Shandong Province (2021ZDSYS22, 2021KJ012) Appendix A Supplementary data Supplementary data to this article can be found online at https://doi org/10.1016/j.carbpol.2022.120470 References Ahmad, T., Eapen, M S., Ishaq, M., Park, A Y., Karpiniec, S S., Stringer, D N., Sohal, S S., Fitton, J H., Guven, N., Caruso, V., & Eri, R (2021) Anti-inflammatory activity of fucoidan extracts in vitro Marine Drugs, 19(12) An, E K., Hwang, J., Kim, S J., Park, H B., Zhang, W., Ryu, J H., You, S., & Jin, J O (2022) Comparison of the immune activation capacities of fucoidan and laminarin extracted from Laminaria japonica International Journal of Biological Macromolecules, 208, 230–242 Anastassova, N., Aluani, D., Kostadinov, A., Rangelov, M., Todorova, N., HristovaAvakumova, N., Argirova, M., Lumov, N., Kondeva-Burdina, M., Tzankova, V., & Yancheva, D (2021) Evaluation of the combined 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in the neuroprotective effects of MPP+ on neurological damage, we used ATP5F1a siRNA to test the function of FvF on ATP5F1a (Fig S5) The knockdown... key role of mitochondrial dysfunction in the patho­ genesis of PD, we hypothesized that the neuroprotective effect of FvF is mediated by the alleviation of mitochondrial dysfunction (Zhang et al.,... for the first time to assess the potential targets of FvF and to elucidate the mecha­ nism of its neuroprotection Although we elucidated the role of ATP5F1a in FvF alleviating mitochondrial dysfunction