Methyllycaconitine Alleviates Amyloid-β Peptides-Induced Cytotoxicity in SH-SY5Y Cells

Alzheimer's disease (AD) is a chronic progressive neurodegenerative disorder. As the most common form of dementia, it affects more than 35 million people worldwide and is increasing. Excessive extracellular deposition of amyloid-β peptide (Aβ) is a pathologic feature of AD. Accumulating evidence indicates that macroautophagy is involved in the pathogenesis of AD, but its exact role is still unclear. Although major findings on the molecular mechanisms have been reported, there are still no effective treatments to prevent, halt, or reverse Alzheimer's disease. In this study, we investigated whether Aβ25–35 could trigger an autophagy process and inhibit the growth of SH-SY5Y cells. Furthermore, we examined the effect of methyllycaconitine (MLA) on the cytotoxity of Aβ25–35. MLA had a protective effect against cytotoxity of Aβ, which may be related to its inhibition of Aβ-induced autophagy and the involvement of the mammalian target of rapamycin pathway. Moreover, MLA had a good safety profile. MLA treatment may be a promising therapeutic tool for AD.


Introduction
Alzheimer's disease (AD), the most prevalent form of dementia in older adults, is a chronic progressive neurodegenerative disorder [1]. AD patients have severe progressive cognitive dysfunction, memory impairment, behavioral symptoms and loss of independence [2]. According to Alzheimer's Disease Intemational (ADI), at least 35.6 million people had dementia in 2010, with the numbers nearly doubling every 20 years [3]. Many factors contribute to the etiology of AD, elevated amyloid-b peptide (Ab) and loss of nicotinic acetylcholine receptors (nAChRs) being prominent [4]. Extracellular amyloid plaques, predominantly consisting of Ab, and intracellular neurofibrillar tangles, formed by hyperphosphorylated tau, are the major pathological hallmarks in the brain of AD patients [5]. Abnormal Ab protein accumulation represents a key feature and is the triggering mechanism of subsequent cerebral degradation in AD [6].
Ab is generated predominantly as a 40-or 42-amino acid peptide from amyloid precursor protein (APP) on sequential cleavage by b-secretase and the c-secretase complex [7]. Ab  has a strong ability to oligomerize to form diffusible dimers and trimers as well as larger oligomers, which induces early synaptotoxic effects and progressive dendritic-spine loss in AD [8]. Ab [25][26][27][28][29][30][31][32][33][34][35] is neurotoxic when forming oligomers, which is similar to Ab 1-42 [9]. Ab plays a critical role in the pathogenesis of AD and is associated with energy failure, neuronal apoptosis and neuron loss in the AD brain [10]. The mechanism of Ab in AD pathogenesis is still unclear. However, suppressing Ab-induced cytotoxicity has become the focus of much AD research.
Macroautophagy (hereafter referred to as autophagy) is an evolutionarily conserved lysosomal-dependent pathway degrading long-lived or misfolded proteins and damaged organelles. It is an intracellular self-defense process by providing an adaptive strategy for cell survival in eukaryotes [11]. Specific membrane segments elongate, encapsulate part of the cytoplasm, and form doublemembrane structures to generate an autophagosome. Autophagosomes become autolysosomes by fusion with endosome or lysosome containing proteases (autophagic maturation), and their inner-membrane and contents undergo clearance [12]. In autophagy studies, LC3 is proposed to act during elongation and expansion of the phagophore membrane. LC3 is cleaved to generate the cytosolic LC3-I with a C-terminal glycine residue, which is conjugated to phosphatidylethanolamine. The lipidated form of LC3 (LC3-II) is attached to both faces of the phagophore membrane but is ultimately removed from the autophagosome outer membrane, which is followed by fusion of the autophagosome with a late endosome/lysosome [13]. Mammalian target of rapamycin (mTOR) is a master controller of autophagy. Activated mTORC1 enhances protein translation by directly phosphorylating 4E-binding protein 1 and p70S6K to negatively regulate autophagy, which is involved in normal physiological processes, including aging, and the pathogenesis of diverse diseases, such as certain types of neuronal degeneration and cancer [14]. Autophagy pathology has been observed in AD. A massive accumulation of autophagic vacuoles was observed in dystrophic neurites in an animal model of AD and in postmortem brains from AD patients, which colocalized intimately with b-secretase complexes, APP, and c-secretase-derived C-terminal fragment (c-CTF). Here, autophagy seems to be abnormal because of alteration in the endolysosomal pathway, which impairs fusion of autophagosomes with lysosomes [15]. It is indicated that abnormal Ab-related autophagic vacuoles accumulation may closely cause neuron dysfunction and neuron loss, thereby leading to Alzheimer's neurodegeneration [16,17].
Neuronal nicotinic acetylcholine receptors (nAChRs) are a family of ligand gated ion channels widely distributed in the human brain. Multiple subtypes of these receptors are involved in a wide range of physiological and behavioral processes, including cognitive enhancement, increased arousal and decreased anxiety and neuroprotection [18]. AD is characterized by a loss of neurons, particularly those expressing nAChRs. The loss of nAChRs has been detected in several regions of the brains of patients with AD, which is thought to underlie memory impairment and cognitive deficits in AD [19]. In our previous study, we found that granulocyte colony-stimulating factor could improve the learning and memory deficits of APP transgenic mice by up-regulating a7nAChR in the brain [20]. Ab 1-42 coimmunoprecipitated with a7nAChR in postmortem samples of hippocampal tissue from patients with AD [21]. This suggests that Ab and a7nAChR may have a high affinity and binding interaction. All the evidences have revealed that a7nAChR may play an important role in the Ab-induced pathogenic process of AD.
Methyllycaconitine (MLA), a norditerpenoid alkaloid isolated from the seeds of Delphinium brownii, is one of the most potent and specific a7nAChR ligands that bind to neuronal a-bungarotoxin sites. Because of its specific, concentration-dependent, reversible, and voltage-independent antagonism, it could inhibit acetylcholine-and anatoxin-induced whole-cell currents in cultured fetal rat hippocampal neurons [22]. One recent report showed that MLA and the weak (,10%) agonist NS6740 reduced lipopolysaccharide-induced tumor necrosis factor a release, so a7nAChR antagonism may confer anti-inflammatory properties on microglia. As well, antagonism of a7nAChRs may reduce neuroinflammation, which is beneficial to AD [23]. Observations of the crystal structure of a complex between MLA and an AChBP isolated from the salt-water snail, Aplysia californica, revealed that MLA interacted with AChBP at the molecular level [24]. Thus, MLA might affect the pathogenic process of AD caused by Ab.

Cell culture and drug treatment
The human neuroblastoma cell line SH-SY5Y was purchased from the Cell Resource Center, IBMS, CAMS/PUMC. Cells were cultured in RPMI-1640 supplemented with 10% FBS at 37uC. Cells at 60-70% confluence were treated with concentrations of Ab 25-35 , MLA, rapamycin or Ab 25-35 with or without MLA. Control cells were cultured under normal conditions.

Cell viability assay
Cells were plated in 96-well plates containing complete medium and cultured for 24 h. Then cells were treated with compounds at the indicated concentrations for specified times. After drug treatment, cell viability was measured by MTT assay [26]. Briefly, 10 ml of the MTT solution (5 mg/mL) was added to each well and incubated for 4 h at 37uC. After removing the supernatant, 100 mL DMSO was added into each well. The absorbance was measured at 570 nm with a microplate reader (Thermo, MULTISKAN MK3, USA). All experiments were repeated 3 times.

Monodansylcadaverine staining (MDC)
To detect autophagy in SH-SY5Y cells, cells were plated on coverslips in 6-well plates. After 24 h, cells were treated with compounds at the indicated concentrations, fixed with 4% paraformaldehyde for 15 min at room temperature, then stained with MDC (1 mg/mL in phosphate buffered saline [PBS]) at 37uC in the dark, and observed immediately with fluorescence microscopy. To quantify the number of cells with acidic vesicles, cells were seeded into 6-well plates and cultured overnight, then stained with 1 mg/mL MDC at 37uC for 15 min. After incubation, cells were washed with PBS and removed with trypsin-EDTA, resuspended, and analyzed by flow cytometry.

Apoptosis detection by Hoechst 33258 staining
Hoechst 33258 staining was used to detect apoptotic nuclei. Cells were plated in 24-well plates. After drug treatment, cells were stained with 10 mg/mL Hoechst 33258 for 15 min. After being gently washed with PBS once, cells were observed and photographed under a fluorescence microscopy (NIKON ECLIPSE 90i, LH-M100CB-1, Japan).

Apoptosis detection by flow cytometry
Cells were plated in six-well plates and incubated for 24 h, exposed to desired concentrations of Ab 25-35 for 24 h, then harvested by trypsinization, and washed twice in PBS. After staining with a combination of AnnexinV/fluorescein isothiocyanate (FITC) and propidium iodide (PI) (Annexin V: FITC Apoptosis Detection Kit, BD Pharmingen), cells were immediately analyzed by flow cytometry (FACS Calibur, Becton Dickinson).

Western blot analysis
After treatment, cells were collected and washed gently with PBS twice, then lysed with protein lysis buffer (1% SDS in 25 mM Tris-HCl, pH 7.5, 4 mM EDTA, 100 mM NaCl, 1 mM PMSF, 1% cocktail protease inhibitor). Samples were centrifuged at 12,000 g for 15 min at 4uC, and supernatants were collected. The concentration of the protein was determined by Coomassie brilliant blue protein assay. Equal amounts of protein (50 mg) were resolved by SDS-PAGE and transferred onto nitrocellulose membrane, which was blocked with 5% non-fat dry milk in TBS for 1 h at room temperature, and then incubated with primary antibodies (1:1000) overnight at 4uC. Membranes were washed and treated with appropriate secondary antibodies for 1 h at room temperature. The immunocomplexes were detected with an enhanced chemiluminescence plus kit.

Electron microscopy (EM)
Cells were postfixed with 2% osmium tetroxide, followed by an increasing gradient dehydration step with ethanol and propylene oxide. Cells were then embedded in LX-112 medium (Ladd), and sections were cut ultrathin (90 nm), placed on uncoated copper grids, and stained with 0.2% lead citrate and 1% uranyl acetate. Images were examined under a JEOL-1010 electron microscope (JEOL) at 80 kV.

Data analysis
Data were analyzed by use of SPSS 15.0 (SPSS Inc.). Data were expressed as mean 6 SE. Differences between groups were analyzed by t test. P,0.05 was considered statistically significant.

Ab25-35-induced growth inhibition of SH-SY5Y cells is mediated by autophagy
To identify the causes of Ab-induced growth inhibition of SH-SY5Y cells, we tested the effect of Ab 25-35 on apoptosis of SH-SY5Y cells by flow cytometry and Hoechst 33258 staining. The SH-SY5Y cells administrated with Ab 25-35 for 24 h didn't induce obvious apoptosis (Fig. 3A and 3B). Therefore, cytotoxicity induced by Ab could not be attributed to apoptosis.
The role of autophagy in Ab-mediated growth inhibition was further studied by siRNA knockdown of the expression of beclin 1, a component of the class III phosphatidy-linositol 3-kinase complex essential for autophagosome formation. The expression of beclin 1 was markedly suppressed in SH-SY5Y cells transfected with beclin 1 siRNA but not scramble siRNA (Fig. 3C). Accordingly, siRNA knockdown of beclin 1 expression reduced LC3-II accumulation after Ab treatment as compared with the siRNA control (Fig. 3C). Although the result did not reach statistical significance, it showed that the growth inhibition effect of Ab could be decreased by siRNA knockdown of beclin 1 expression as compared with the siRNA control (Fig. 3D, P = 0.0522). These data suggest that autophagy is required for Ab-induced growth inhibition.

Discussion
One of the main histopathological features of AD is the presence of extracellular proteinaceous deposits in the brain, identified as senile plaques, which are enriched in Ab. It is widely accepted that AD onset can be initially triggered by interaction of Ab oligomers with the brain parenchyma [33]. In agreement with this, the levels of soluble Ab oligomers appear to correlate well with the severity of AD dysfunction [34,35]. In vitro [28] and in vivo [29,30] studies have shown that these soluble oligomers produced toxicity leading to neuron dysfunction or loss. Consistent with previous studies, we also observed the neurotoxicity of Ab oligomers on the SH-SY5Y cells. Cell growth was remarkably inhibited by Ab oligomers treatment. MLA, a norditerpenoid alkaloid isolated from the seeds of Delphinium brownii, had a protective effect against the cytotoxity of Ab, which may be related to its inhibition of Ab-induced autophagy and the involvement of the mTOR pathway. Moreover, it had a good safety profile. MLA treatment may be a promising therapeutic tool for AD.
Although AD has been discovered for 100 years, the disease continues to affect millions of patients. Although multiple drugs have now been approved, their expected benefits are modest. Therefore, numerous efforts have been made to develop more potent AD drugs. To date, emphasis has been on strategies to reduce the pathogenicity of amyloid-b (Ab) peptides [36], widely believed to play a key role in AD. As an antagonist selective for abungarotoxin-sensitive a7nAChR, MLA alleviated Ab-induced cytotoxicity in our SH-SY5Y cells. Pretreatment with MLA could significantly inhibit the decreased cell viability induced by Ab [25][26][27][28][29][30][31][32][33][34][35] , which indicates a protective effect of MLA against Ab-induced cytotoxicity. Of note, MLA from 5 to 20 mM alone did not have any significant anti-proliferative effect on SH-SY5Y cells. MLA is a relatively small reversible-binding compound that can easily across the blood-brain barrier in vivo [37,38]. Considering the low cytotoxicity and the ability to pass the blood-brain barrier, MLA may be a potent drug in the treatment of AD.
Autophagy is a lysosome degradation process that turns over cytoplasmic materials and helps the cell maintain homeostasis. It is usually maintained at low levels under normal conditions for cell survival but can be augmented rapidly as a cytoprotective response when cells undergo starvation or damaging components, such as oxidative stress, infection, or protein aggregate accumulation [11,12]. Dysregulated or excessive autophagy can lead to cell death. Autophagosomes accumulate abnormally in the brain in several neurodegenerative disorders including AD [39]. Here, we found that Ab could induce autophagy in SH-SY5Y cells. Ab treatment could increase LC3II expression, punctate fluorescent signals in SH-SY5Y cells, the formation of acidic vesicular organelles and the accumulation of autophagosomes. These results are consistent with our previous study of PC12 cells in vitro [40].
To investigate the possible mechanism by which MLA pretreatment alleviated the cytotoxity of Ab in SH-SY5Y cells, we examined the effect of MLA on Ab-induced autophagy. Abinduced upregulation of LC3BII levels and accumulation of autophagosomes or autolysosomes was inhibited by administration of MLA, which suggests that MLA could inhibit Ab-induced autophagy in SH-SY5Y cells. Autophagy can be a pro-survival response or contribute to cell death; whether it is detrimental or protective remains unclear in AD [41]. It was reported that beclin-1, a protein with a key role in autophagy, was decreased in level in patients with AD and in APP transgenic mice early in the disease process, beclin 1 deficiency disrupted neuronal autophagy and promoted neurodegeneration in mice [42]. Another report showed that rapamycin administration could reduce Ab levels in neurons and improve cognitive deficits by enhancing autophagy [43]. This evidence indicates that increasing autophagy may be helpful to AD. However, excessive Ab can activate autophagy, thus resulting in cell dysfunction or death in vitro [44] and in vivo [45,46], and suppression of autophagy may alleviate Ab-induced cell death or cognitive deficits. Ab generation was found linked to autophagy, which is activated and abnormal in AD, and suppressing autophagy by 3-MA could decrease Ab 1-40 secretion [47]. Furthermore, an AD drug, galanthamine hydrochloride, and an AD drug candidate, Ghrelin, could inhibit autophagy, which suggests that decreasing input into the lysosomal system may help reduce cellular stress in AD [48]. This evidence suggests that augmented autophagy in AD may be harmful; suppressing augmented autophagy could be an effective therapy for AD. Here, we found that MLA could inhibit Ab-induced autophagy in SH-SY5Y cells. The suppression of autophagy by MLA may contribute to its protective effect against the cytotoxity of Ab.
Several signaling pathways regulate the autophagy process with the mTOR pathway playing a key role. We paid attention to the downstream targets. 4E-binding protein 1 and p70S6K are directly phosphorylated by activated mTORC1 to negatively regulate autophagy [14]. In our previous study, we showed that Ab could induce autophagy in PC12 cells through an mTORdependent pathway [40]. Here, we also found that Ab induced autophagy in SH-SY5Y cells via mTOR signaling as evidenced by the downregulation of phosphorylated p70S6K levels. Moreover, Ab-decreased p70S6K phosphorylation was attenuated by administration of MLA. The upregulation of mTOR signaling by MLA may inhibit Ab-induced autophagy and contribute to its protective effect against Ab-related cytotoxicity.