Neurochem Res DOI 10.1007/s11064-014-1410-3

ORIGINAL PAPER

Protective Effects of Humanin on Okadaic Acid-Induced Neurotoxicities in Cultured Cortical Neurons Jinfeng Zhao • Dan Wang • Lingmin Li Wenhui Zhao • Ce Zhang

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Received: 26 January 2014 / Revised: 5 August 2014 / Accepted: 6 August 2014 Ó Springer Science+Business Media New York 2014

Abstract Neurofibrillary tangles are pathological hallmarks of Alzheimer’s disease (AD), which are mostly composed of hyperphosphorylated tau and directly correlate with dementia in AD patients. Okadaic acid (OA), a toxin extracted from marine life, can specifically inhibit protein phosphatases (PPs), including PP1 and Protein phosphatase 2A (PP2A), resulting in tau hyperphosphorylation. Humanin (HN), a peptide of 24 amino acids, was initially reported to protect neurons from AD-related cell toxicities. The present study was designed to test if HN could attenuate OA-induced neurotoxicities, including neural insults, apoptosis, autophagy, and tau hyperphosphorylation. We found that administration of OA for 24 h induced neuronal insults, including lactate dehydrogenase released, decreased of cell viability and numbers of living cells, neuronal apoptosis, cells autophagy and tau protein hyperphosphorylation. Pretreatment of cells with HN produced significant protective effects against OAinduced neural insults, apoptosis, autophagy and tau hyperphosphorylation. We also found that OA treatment inhibited PP2A activity and HN pretreatment significantly attenuated the inhibitory effects of OA. This study demonstrated for the first time that HN protected cortical

neurons against OA-induced neurotoxicities, including neuronal insults, apoptosis, autophagy, and tau hyperphosphorylation. The mechanisms underlying the protections of HN may involve restoration of PP2A activity. Keywords Okadaic acid
Neurotoxicity
Protein phosphatase 2A
Tau protein hyperphosphorylation
Humanin
Neuroprotection Abbreviations AD NFTs PHF Ab AVs PPs PP2A OA HN UP LDH MTT Ac-DEVD-pNA TUNEL

Jinfeng Zhao and Dan Wang have contributed equally to this work. J. Zhao
D. Wang
L. Li
W. Zhao
C. Zhang (&) Department of Neurobiology, Shanxi Medical University, 56# Xin Jian South Road, Taiyuan 030001, Shanxi, People’s Republic of China e-mail: [email protected] Present Address: J. Zhao College of Physical Education, Shanxi University, Taiyuan 030006, People’s Republic of China

Alzheimer’s disease Neurofibrillary tangles Paired helical filaments Amyloid-b Autophagic vacuoles Protein phosphatases Protein phosphatase 2A Okadaic acid Humanin Unrelated peptide Lactate dehydrogenase 3-(4,5-Dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide Acetyl-Asp-Glu-Val-Asp p-nitroaniline Terminal-deoxynucleoitidyl transferase mediated nick end labeling

Introduction Alzheimer’s disease (AD) is the most common human neurodegenerative disorder characterized by the progressive deterioration of cognition and memory in associated with the presence of senile plaques, neurofibrillary tangles (NFTs), and massive loss of neurons primarily in the

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cerebral cortex and hippocampus [1]. NFTs contain paired helical filaments (PHF) which are mostly composed of hyperphosphorylated tau. Tau is a microtubule-associated protein enriched in the axon of neurons and plays a key role in microtubule stabilization, axonal transportation, and neurite outgrowth under physiological conditions [2, 3]. The extent of tau protein phosphorylation is low in normal brain, however, wide-bound hyperphosphorylation of tau occurs in AD brain, which is the critical pathological change of AD [4, 5]. It is well known that the phosphorylation of tau protein is regulated by protein kinases and protein phosphatases (PPs). Consequently, an imbalance of protein kinases and PPs is essential to tau abnormal hyperphosphorylation [6, 7]. Protein kinases include GSK-3, cAMP-dependent protein kinase, and so on [8]. PPs also include different members, and PP2A is one of the major serine/threonine PPs that regulate tau dephosphorylation in the brain [9]. Therefore, down-regulation of PP2A activity is partly responsible for abnormal tau phosphorylation in the AD brain [10, 11]. Okadaic acid (OA), extracted from the common black sponge Halichondria okaddai, exerts inhibitory effects on PPs, including PP2A and PP1 [12]. Several studies have identified that expression and activity of PP2A are reduced in select areas of the AD brain [13, 14]. Inhibition of PP2A by OA thus can induce microtubule-associated protein tau hyperphosphorylation leading to the pathological process of analogic AD [7, 15]. Studies reported autophagic vacuoles (AVs) were abundant in AD brains particularly within neuritic processes, in other words, autophagy increased in human AD brains [16].Yoon’s studies showed OA could increase autophagy in cortical neurons [17]. Authophagy is an essential, conserved lysosomal degradation pathway that maintains cytoplasmic homeostasis by eliminating protein aggregates and damaged organelles [18, 19]. Excess autophagy,however, can induce cell death called type II cell programmed death compared with apoptosis (typeIcell programmed death) [20]. Autophagy is orchestrated by lots of highly conserved autophagy-related genes (ATGs), which were originally identified in yeast and in mammalian cells [21, 22]. Autophagy includes macroautophagy, chaperone-mediated autophagy (CMA) and microautophagy. They are primarily different in the way of transporting cytosolic components to lysosomes. The most familiar is macroautophagy [23]. LC3 and Beclin-1 are ATGs required for macroautophagy. In the process of mature of autophagysome, LC3 I is phosphorylated to LC3 II and the expression of Beclin-1 is enhanced, so the proportion of LC3 II and the expression of Beclin-1 are always used to assess autophagy in cells [20, 24]. Humanin (HN) is a polypeptide of 24 amino acids and was first discovered upon isolation from the occipital lobe of a patient with AD [26, 38]. Studies showed that HN can inhibit

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amyloid-b (Ab) peptide activities, including full length Ab peptide-and Ab segment-induced neurotoxicity, as well as mutational APP, PS1, and PS2-induced neuronal death [25, 26]. Recent studies, however, showed that HN not only protected neurons against AD-related insults but also prevented cell death induced by AD-unrelated damage [27]. It was reported that HN could attenuate renal microvascular remodeling, inflammation, and apoptosis in the early stages of kidney disease in hypercholesterolemic ApoE-/- mice [28]. Previous studies in our lab showed that HN attenuated excitotoxicity induced by NMDA and inhibited neuronal insults induced by hypoxia in cortical neurons [15]. The present study was investigated whether HN could attenuate neurotoxicities, including neuronal insults, apoptosis, autophagy and tau hyperphosphorylation induced by OA.

Materials and Methods Reagents Anti-phospho-tau (pSer199/202), coomassie brilliant blue, OA, 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) regent and Calcein-AM were purchased from Sigma (USA). Rabbit polyclonal to tau (pS396, pT231, Tau5), Beclin-1, LC3 and b-actin were bought from Abcam and Cell Signaling (USA). Protein phosphatase 2A (PP2A) assay kit was purchased from Promega (USA). Kits of terminal-deoxynucleoitidyl transferase mediated nick end labeling (TUNEL) was gained from Boehringer Mannheim GmbH (Germany). Caspase 3 apoptosis detection kit was bought from Usbiologiocal (USA). Kits of lactate dehydrogenase (LDH) was gained from nNjjcbio (Nanking, China). HN and unrelated peptide (UP) in this study were chemically synthesized by tetras multi-channel peptide synthesizer (CreoSalus USA) from Sangon biotech (Shanghai Chain), and their purity reached 92.2 % and 93 %. The sequences of HN and UP were MAPRGFSCLLLLTSEIDLPVKRRA and IYMCILTVYPAEAISQWGRDLAVD, respectively. New born SD rats were provided by the Experimental Animal Center of Shanxi Medical University. Primary Cortical Neuron Cultures All experiments were performed on primary cortical neurons. In brief, cortical neurons derived from newborn SD rat (\1 day) were dissociated in DMEM supplemented with 10 % fetal bovine serum, glutamine (4 mmol/l), 4.5 g/l D-glucose, and mycillin (100 U/ml), and then seeded in plates which pre-coated with poly-L-lysine (serum-free media were used when detected LDH.). Cultures were kept at 37 °C in a humidified CO2 incubator. Non-neuronal cell division was halted by exposure to 10 lmol/l cytosine arabinoside after

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incubation neurons for 24 h, half the medium was changed for 24–48 h. After 8–10 days in culture, observation through a phase-contrast microscope demonstrated that the cells were predominantly neuronal cells. Only mature cells (8–10 days) were used for the experiments.

process was [15]: (1) the cells were incubated with 20 lmol/l Calcein-AM solution at 37 °C for 30 min; (2) washed cells twice with PBS; (3) observed the cells using a fluorescence microscope. Apoptosis Assay

OA and HN Treatment To investigate the effects of OA-induced neurotoxicities, cultured cortical neurons were treated with different concentrations of OA (5, 10 and 20 nmol/l) for 24 h, respectively. To investigate whether HN has protective effect on cortical neurons against OA induced injury, the cultured cortical neurons were pre-incubated with different concentration of HN (5, 10 and 20 lmol/l) for 24 h, then added OA (10 nmol/l) for 24 h at 37 °C. Neurotoxicities Assay

Measurement of Caspase 3 Activity Caspase 3 activity was measured by using a United States biological assay kit following the instruction, acetyl-Asp-Glu-Val-Asp p-nitroaniline (Ac-DEVD-pNA) was used as a colorimetric substrate. Primary cultured cortical neurons were plated at a density of 1 9 106 cells per culture dish. After different treatments as designed, the cells were harvested by centrifugation. The pellets were washed with PBS and lysed in 50 ll of chilled cell lysis buffer and incubated in ice for 10 min. Lysate was centrifuged at 10,000 rpm for 1 min at 4 °C, and the supernatant was used for the caspase-3 assay. The samples were read on a microplate reader at 405 nm.

LDH Assay TUNEL Assay The LDH cytotoxicity detection kit measures plasma membrane damage, based on the release of lactate dehydrogenase, a stable cytoplasmic enzyme present in most cells. In this study, we used serum-free media when detected LDH [46, 47]. The formula was Neurobasal 96.75 ml, B27 2 ml, L-glutamine 0.25 ml and mycillin 1 ml to configure 100 ml serum-free media. This combination had been shown to reduce glia to less than 0.5 %. The cortical neurons were pretreated with HN, and then added OA, 500 ll of supernatant was then collected from each well and mixed with 1.3 ml of NADH (0.217 mmol/l) and 1.3 ml of sodium pyruvate (1.77 mmol/l) in the modiWed Krebs–Henseleit buffer for 30 s at 37 °C. The activity was measured by microplate reader at 450 nm [15]. MTT Assay MTT cell proliferation assay is now widely accepted as a reliable way to examine the reduction in cell viability when external factors lead to apoptosis or necrosis. Brief process was [15, 29]: (1) cells used for MTT assay were plated on 96 well plates, (2) washed cultured cells with PBS twice, then added MTT working solution (5 mg/ml) 20 ll into each well, incubated the plate for 4 h at 37 °C; (3) then removed supernatant carefully, added 150 ll DMSO and oscillated for 10 min in order to dissolve crystal; (4) measured absorbance at 490 nm. Calcein-AM Assay Calcein-AM readily passes through the cell membrane of viable cells because of its enhanced hydrophobicity. Brief

Apoptotic cell was quantified by DNA strand breaks which were detected in situ by the TUNEL method. In brief, cultures were fixed in fresh 4 % paraformaldehyde solution (PBS, pH 7.4), permeabilized with 0.1 % Triton X-100 at room temperature for 15 min. The cultures were incubated with TdT reaction mixture in a humidified chamber in the dark for 1 h, then washed with PBS. The cultures were counterstained with DAPI for 20 min and analyzed with a fluorescence microscope. Western Blotting The process of protein extraction:Cultures were scraped with a cell scraper, washed in cold PBS and then centrifuged. The cell pellet was resuspended, then homogenized in an ice-cold lysis buffer (1 mM PMSF, 10 lg/ml aprotinin, 1 mM Na3 VO4, 10 lg/ml leupeptin and 1 mM NaF). The homogenates were centrifuged at 12,000 rpm for 30 min at 4 °C. The supernatants were collected and total protein concentrations were measured using a standard Bradford method. The protein were subjected to separate using 10 % SDS-PAGE gel and then electrophoretically transferred to nitrocellulose membranes. After blocking non-specific binding sites with 5 % low-fat milk in Trisbased buffer containing 0.1 % Tween 20 for 1 h in room temperature, the membranes were probed with antibodies overnight at 4 °C. After washing, the antibody–protein complexes were probed with appropriate secondary antibodies labeled with horseradish peroxidase for 1 h at room temperature, and detected with chemiluminescnet reagents. The nitrocellulose membranes were stripped with strip

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Statistical Analysis

buffer and reblotted with different antibodies. The antibody used to detect the level of autophagy including LC3 and Beclin-1, phospho-tau protein including Ser199/202, Ser396, Thr231, Tau-5 (Tau-5, which can react with phosphorylated as well as non-phosphorylated forms of tau, and represents total tau.) and b-actin.

All data were expressed as the mean ± SEM. In our study, the difference between means was determined by one-way ANOVA followed by a Student–Newman–Keuls test for multiple comparisons. Values of P \ 0.05 were considered statistically significant.

PP2A Assay Results

PP2A activity was assayed with the serine/threonine phosphatase assay kit. Briefly, cells were collected from six well plates and washed before lysis on ice in lysis buffer with protease inhibitor complex for 40 min, with vortex every 10 min. The cell lysate was filtered through a Sephadex G25 column to remove free phosphate. Protein concentration was determined using the Bradford method. 2.5 lg of cell protein were distributed equally among the wells of a 96 well plates with the specific peptide substrate RRA (pT) VA in PP2A specific reaction buffer at 37 °C for 15 min. After incubation, 50 ll of molybdate dye/additive mixture was added, and the color was allowed to develop for 20 min. The optical density of the samples was read using a 96 well plate reader at 630 nm.

We initially examined the effects of OA on neuronal cytotoxicity by treating the cultured cortical neurons for 24 h with different concentrations of OA (5, 10 and 20 nM). The results showed that LDH increased in supernatant after added 10 and 20 nM OA (P \ 0.05), administration of 5 nM OA didn’t produce significant effects compared with control group (Fig. 1a). Serious injury was produced by 20 nM OA, which was not conducive to subsequent experiments. Thus, an OA concentration of 10 nM was selected as an insult-induced model for further

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Fig. 1 Effects of HN on cell viability including LDH, MTT assay. LDH increased in supernatant after added OA (10, 20 nM) (P \ 0.05) (a) and pretreatment of HN (10, 20 lM) can reduce the LDH in cell supernatant (P \ 0.05) (b). OA (10, 20 nM) (P \ 0.05) decreased the

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cell viability (c) and pretreatment of HN (10, 20 lM) improved cell viability compared with OA treatment group (P \ 0.05) (d). *P \ 0.05 versus control group, #P \ 0.05 versus OA treatment group

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experiments. To investigate the effects of HN on OAinduced LDH release, HN was pretreatment prior to OA administration. The results showed that 5 lM HN did not protect neurons from OA-induced toxicity (10 nM). However, as the concentration of HN increased, both 10 lM and 20 lM significantly reduced LDH levels (P \ 0.05). Addition of 20 lM produced strong effects, although no significant statistical difference was observed (Fig. 1b). MTT assay is a reliable method widely accepted for the determination of cellular proliferation, viability and activation. Treatment with 10 nM OA significantly decreased cell viability (P \ 0.05). Pretreatment with HN (10 lM) protected neurons by increasing cell viability from 69.25 ± 4.37 to 80.14 ± 5.63 % (P \ 0.05). HN (20 lM) produced strong effects and improved cell viability from 69.25 ± 4.37 to 91.26 ± 7.38 % (P \ 0.05) (Fig. 1c, d). We then examined the effects of HN on OA-induced insults by dyeing Calcein AM, which represents living cells. The similar results were obtained as previous observations (Fig. 2). In order to further confirm the protections of HN, we designed an unrelated peptide (UP) as a control for HN. The experimental results showed that

Effects of HN on OA-Induced Neuronal Apoptosis To examine the effects of OA on neuronal apoptosis, cultured neurons were treated with different concentrations of OA for 24 h. The activity of caspase 3 increased after addition of 10 nM and 20 nM OA (P \ 0.05) (Fig. 3a). Pretreatment of 5 lM HN showed no effects on OA-indueced increase of caspase 3 activity, 10 lM HN partially reduced the activity of caspase 3 (P \ 0.05), and 20 lM HN strongly inhibited caspase 3 activity (P \ 0.05) (Fig. 3b). We also examined the effects of HN on OA-induced apoptosis by TUNEL assay, the similar results were obtained as caspase 3 (Fig. 4a, b). We also found that pretreatment of UP (20 lM) did not inhibit OA-induced neuronal apoptosis Effects of HN on OA-Induced Neuronal Autophagy We examined the autophagy system during OA-induced neurodegeneration by Western blotting with the marker of

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Fig. 2 Effects of HN on Calcein-AM-positive living cells treated with OA. Neurons were treated with OA (10 nM), UP (20 lM) or HN (5, 10, and 20 lM) were pretreatment of OA. Calcein-AM assay was conducted and the cells were observed under fluorescence microscope (OLYMPUS) (a), the living cells were counted by IPP, 10 nM OA significantly decreased calcein AM-positive cells, pretreatment of HN (10, 20 lM) increased the number of living cells (P \ 0.05) (b)

pretreatment of 20 lM UP had no neuroprotective effects against OA-induced neuronal insults.

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Fig. 3 Effects of HN on OA-induced increase of caspase 3 activity. The activity of casapase 3 increased after added OA (10, 20 nM) (P \ 0.05) (a), and pretreatment of HN (10, 20 lM) reduced the

Fig. 4 Effects of HN on OAinduced neuronal apoptosis by TUNEL. TUNEL labeling neurons for apoptosis assay showed in Fig. 4 (a), in which apoptosis neurons are flavogreen and no apoptotic cell nuclei are blue. Apoptotic index was calculated by the apoptosis cell nuclei/whole cell nuclei. Pretreatment of HN (10, 20 lM) protected OA induced apoptosis (b). *P \ 0.05 versus control group, #P \ 0.05 versus OA treatment group

activity of caspase 3 (P \ 0.05) (b). *P \0.05 versus control group, # P \ 0.05 versus OA treatment group

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Beclin-1 and LC3. Western blot analysis revealed very low level of LC3 II in control neurons. However, the level of LC3 II was increased after 24 h of 10 nM OA treatment. Pretreatment with 20 lM HN significantly decreased LC3 II (P \ 0.05) (Fig. 5a, b). The autophagy marker of Beclin-

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1 was also obtained similar results as LC3 II. Beclin-1 increased after addition of 10 nM OA, pretreatment of 20 lM HN reduced the level of Beclin-1 (P \ 0.05) (Fig. 5a, c). Pretreatment of UP (20 lM) had no effects on OA-induced autophagy.

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Fig. 5 Effects of HN attenuated OA-induced autophagy. Western blotting analysis was performed for autophagy marker of LC3 IIand Beclin-1 (a). LC3 II increased after added OA (10 nM) (P \ 0.05) and pretreatment of HN (20 lM) reduced the level of LC3

II(P \ 0.05) (b). Beclin-1 was enhanced after addition of OA (10 nM) (P \ 0.05) and pretreatment of HN (20 lM) declined the Beclin-1 (P \ 0.05) (c). *P \ 0.05 versus control group, #P \ 0.05 versus OA treatment group

Effects of HN on OA-Induced Tau Hyperphosphorylation

Discussion

Western blot analysis was carried out to investigate the phosphorylation level of tau protein. Tau phosphorylations were significantly increased when neurons were treated with 10 nM OA (P \ 0.05). The phosphorylation sites induced by OA included Ser-199/202, Ser-396, and Thr-231. Pretreatment with 5 lM HN for 24 h showed no effects on the phosphorylation level of tau (P [ 0.05). However, pretreatments with 10 lM and 20 lM HN significantly decreased tau phosphorylation at these different sites (P \ 0.05). Pretreatment UP (20 lM) also showed no protections against OAinduced tau hyperphosphorylation (Fig. 6a, b). Effects of HN on OA-Induced Decrease of PP2A Activity OA has been reported to inhibit PP2A activity. Our experiments initially confirmed that OA induced decrease of PP2A activity and then determined the effects of HN on OA-induced change of PP2A activity. OA (10 nM and 20 nM) produced significant decreases of PP2A activity (P \ 0.05) (Fig. 7a). Pretreatment with 10 lM and 20 lM HN significantly inhibited OA-induced decreases in PP2A activity (P \ 0.05). Administration of UP (20 lM) showed no significant effects (Fig. 7b).

The present study systematically investigated the effects of HN on OA-induced neurotoxicities, including OA-induced neural insults, apoptosis, autophagy and tau hyperphosphorylation. OA extracted from the common black sponge H. okaddai has been reported to selectively inhibit protein PPs, namely PP2A and PP1 [12], resulting in tau hyperphosphorylation. It is well known that tau hyperphosphorylation causes microtubule-integrity damage, loss of microtubule function, and impaired axonal transport and mitochondrial trafficking [30], eventually leading to neuronal insults. We found that treatment of neurons with OA induced apoptosis, in which the activity of caspase 3 increased and neuronal apoptosis rates increased by TUNEL examination. The mechanisms of apoptosis induced by tau hyperphosphorylation may be related to the following factors: (1) Tau hyperphosphorylation may increase bcl-2 protein phosphorylation, which diminishes it’s effect of antiapoptosis [31]. (2) Tau hyperphosphorylation may enhance the activity of caspase 3, the key executor of apoptosis, and induce many proteins and kinases inactivation, thus to promote apoptosis [32]. (3) Microtubule activity may influence Ca2? homeostasis, whereas tau hyperphosphorylation undermines the structure and function of microtubule, and further affects the function of Ca2? channels or excitability of neurons to promote apoptosis [33, 34].

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increase of lead exposure, the autophagy level was increased by the Beclin-1 and LC3 II [20]. Yoon’s studies [17] were also found that OA increased Beclin-1 and LC3 II in cortical neurons, and 3-methyladenine (3-MA), an autophagy inhibitor, reduced autophagy and cytotoxicity in OAinduced neurons. The possible mechanism is that the basal level of AVs formation is regulated by upstream signaling pathways under normal conditions, and AVs eventually fuse with lysosomes and are degraded. In OA-treated neurons, however, the fusion of AVs with lysosomes might be disturbed because of disruption of microtubules, which caused AVs accumulation and led to increase the level of autophagy [17]. However, the role of autophagy in neurodegeneration is controversial, with some reports concluding that inhibiting autophagy was neuroprotective [17, 20, 24], and other studies showing that activating autophagy, reduced aggregated proteins and improved neuronal survival [35, 36]. In addition, we found that OA induced tau hyperphosphorylation at different sites. Tau phosphorylation is regulated by protein kinases and protein PPs. Hyperphosphorylation of tau occurs as a result of imbalance in phosphorylation processes, including increases in protein kinase activity and, especially, decreases in protein PPs [6, 7]. As previously described, OA can selectively inhibit the protein PPs, including PP2A and PP1 [12], thus, OA was used as an insult-inducing model to investigate the neurotoxicities of tau hyperphosphorylation. To date, approximately 30 sites of tau phosphorylation-related AD have been examined. These sites principally locate the area of tau microtubular binding zone, however, which are the most important sites still to be disputed. Given the restrictions of our experiment, we did not analyze all of the phosphorylation sites. Instead, we selected four sites of tau phosphorylation, namely, Ser-199/202, Ser-396, and Thr231, which had been reported to have higher disease incidences in AD brain [37].

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Fig. 6 Effects of HN attenuated OA-induced tau protein hyperphosphorylation. Western blotting analysis was performed for phosphorylated tau protein (at Ser199/202, Ser396 and Thr231 sites) (a). The phosphorylation level of tau was significantly increased when neurons treated with 10 nM OA, pretreatment with HN (10, 20 lM) significantly decreased the tau phosphorylation respectively (b). *P \ 0.05 versus control group, #P \ 0.05 versus OA treatment group

Western blotting experiments showed that OA induced autophagy with increase of levels of LC3 II and Beclin-1 in cortical neurons. Consistently with our results, marked accumulation of AVs in dystrophic neuritis was reported in human AD brains [16], acute NMDA exposure induced autophagy in cultured granule neurons [24], and with

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Fig. 7 HN restored OA-induced decrease of PP2A activity. The activity of PP2A decreased after added OA (10 nM) (P \ 0.05), pretreatment of HN (10, 20 lM) significantly increased the activity of

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PP2A, respectively (P \ 0.05). *P \ 0.05 versus control group, # P \ 0.05 versus OA treatment group

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HN was initially found in the surviving neurons of the human AD brain [38]. Evidences showed that HN protected neurons against Ab-induced neurotoxicities, including full-length Ab peptide-, Ab fragment-, as well as mutational APP, PS1, and PS2-induced neuronal death [25, 26]. The mechanisms underlying the neuroprotective effects of HN against neurotoxicities may involve the following: (1) HN could bind with interleukin-6 receptorrelated receptor (s), such as CNTFRalpha, WSX-1, and gp130, on the cell surface [39, 40] and activated the JAK2/ STAT3 signaling pathway [41]. (2) HN could inhibit Bax activation and translocation from the cytosol to mitochondria to prevent the release of cytochrome C and other apoptogenic proteins from mitochondria [42, 43]. (3) HN could attenuate the activity of caspase 3 and block the production of caspase 3 [38]. Finally, HN could prevent Ca2? overloading to maintain Ca2? homeostasis [44] and increased ATP levels in different types of cells [45]. Since the initial study on HN, subsequent studies, as well as this study, suggested that HN may have broader neuroprotective effects [27]. The present study demonstrated that HN could antagonize all of the neuronal toxicities induced by OA, including neural insults, apoptosis, autophagy and tau hyperphosphorylation. It is the first time to report the protective effects of HN on OA-induced neurotoxicities. The significance of these results not only proved neuroprotective effect of HN on OA-induced neuronal toxicities, but also provided a new evidence confirming that HN may be a broader neuroprotective factor rather than just for specific protection of AD-related insults. Given the basis of OA-induced neurotoxicities may involve tau hyperphosphorylation, the major mechanisms underlying the neuroprotective effects of HN against OAinduced toxicities should be antagonism of OA-induced tau hyperphosphorylation. It is well known that tau hyperphosphorylation is induced by the imbalance between the activity of protein kinases and PPs, especially decrease of PPs [6, 7]. In the present study, treatment of OA significantly decreased the activity of PP2A, and this inactivation of PP2A was dramatically attenuated by HN pretreatment, indicating the regulatory effects of HN on PP2A activity may have critical roles in anti-OA-induced neurotoxicities, including neural insults, apoptosis, autophagy, and tau hyperphosphorylation. However, we can not rule out there may be other phosphatases involved in OA-induced neurotoxicities. In conclusion, we systemically observed OA-induced neurotoxicities, and demonstrated for the first time that HN had significant neuroprotective effects against OA-induced neurotoxicities, including neural insults, apoptosis, autophagy, and tau hyperphosphorylation. The possible mechanisms might due to improving the activity of PP2A and then inhibiting tau protein hyperphosphorylation although there might be other targets for the neuroprotections of HN.

Acknowledgments The study was supported by National Natural Science Foundation of China (30572085).

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