The Role of Heat Shock Protein 70 in the Protective Effect of YC-1 on β-Amyloid-Induced Toxicity in Differentiated PC12 Cells

Neurodegenerative brain disorders such as Alzheimer’s disease (AD) have been well investigated. However, significant methods for the treatment of the progression of AD are unavailable currently. Heat shock protein 70 (Hsp70) plays important roles in neural protection from stress by assisting cellular protein folding. In this study, we investigated the effect and the molecular mechanism of YC-1, an activator of guanylyl cyclase (GC), on Aβ25–35-induced cytotoxicity in differentiated PC12 cells. The results of this study showed that Aβ25–35 (10 µM) significantly increased p25 protein production in a pattern that was consistent with the increase in μ-calpain expression. Moreover, Aβ25–35 significantly increased tau hyperphosphorylation and induced differentiated PC12 cell death. YC-1 (0.5–10 µM) prevented the cell death induced by Aβ25–35. In addition, YC-1 (1, 10 µM) significantly blocked Aβ25–35-induced μ-calpain expression and decreased the formation of p25 and tau hyperphosphorylation. Moreover, YC-1 (5–20 µM) alone or combined with Aβ25–35 (10 µM) significantly increased the expression of Hsp70 in differentiated PC12 cells. The neuroprotective effect of YC-1 was significantly attenuated by an Hsp70 inhibitor (quercetin, 50 µM) or in PC12 cells transfected with an Hsp70 small interfering RNA. However, pretreatment of cells with the GC inhibitor ODQ (10 µM) did not affect the neuroprotective effect of YC-1 against Aβ25–35 in differentiated PC12 cells. These results suggest that the neuroprotective effect of YC-1 against Aβ25–35-induced toxicity is mainly mediated by the induction of Hsp70. Thus, YC-1 is a potential agent against AD.


Introduction
Alzheimer's disease (AD) is the most common cause of dementia in the aged population. AD is characterized by two pathological hallmarks consisting of extracellular plaques of b-amyloid peptide aggregates [1] and intracellular neurofibrillary tangles composed of the hyperphosphorylated microtubular protein tau [2]. The bamyloid deposition that constitutes the plaques is composed of a 39-42 amino-acid peptide (Ab) that is the proteolytic product of the amyloid precursor protein (APP) by b/c secretases. Calpains modulate processes that govern the function and metabolism of key proteins in the pathogenesis of AD, including tau and APP [3]. Cyclin-dependent kinase 5 (cdk5), which promotes the phosphorylation of tau, has been implicated in the pathological processes that contribute to neurodegeneration in AD. p35 is a neuronspecific activator of cdk5, and conversion of p35 into p25 by calpain-dependent proteolysis causes prolonged activation and mislocalization of cdk5. Consequently, the p25/cdk5 kinase hyperphosphorylates tau, disrupts the cytoskeleton, and promotes apoptosis of primary neurons.
Heat shock proteins (Hsps) are the major molecular chaperones that mediate the proper folding of other proteins and ensure that these proteins maintain their native conformations during conditions of stress [4,5]. In addition, Hsps are required for protein trafficking to target organelles and to facilitate the transfer of misfolded proteins to the proteasome, for degradation [4]. Mammalian Hsps have been classified into families on the basis of their molecular weight, including Hsp27, Hsp40, Hsp60, Hsp70, Hsp90, and Hsp110. These molecular chaperones are either constitutively expressed or inducibly synthesized after cellular stress. Hsp70 chaperones are an important part of the cellular protein quality control and degradation systems [6,7]. The Hsp70 family includes the heat shock cognate protein Hsc70 and the heat shock protein Hsp70. Studies demonstrated the presence of elevated levels of Hsp70 synthesis and accumulation in AD brain [8] and neurons with strong staining for Hsp70 that did not contain neurofibrillary tangles [9]. Induction of Hsp70 by heat preconditioning protected against AD-like hyperphosphorylation of tau in PC12 cells [10], and induction of Hsp70 by geldanamycin reduced okadaic acid-induced tau phosphorylation and aggregation in COS-1 cells expressing human tau [9]. These findings suggest that Hsp70 represents an important molecular target for neuroprotective strategies in AD treatment.
YC-1 [3-(50-hydroxymethyl-20-furyl)-1-benzylindazole] is a synthetic benzylindazole compound originally developed as an activator of guanylyl cyclase (GC) to inhibit platelet aggregation and vascular contraction [11]. Several lines of evidence have shown that YC-1 exhibits therapeutic potential for the treatment of a series of vascular diseases, including hypertension, thrombosis, erectile dysfunction, and postangioplasty restenosis [12,13]. Recent studies revealed that YC-1 induces Hsp70 expression and prevents oxidized LDL-mediated apoptosis in vascular smooth muscle cells [14]. Thus, the aim of this study was to determine whether YC-1 can prevent Ab-induced cytotoxicity in PC12 cells and whether the neuroprotective effect of YC-1 is mediated by the induction of Hsp70.

siRNA Transfection
An anti-Hsp70 siRNA was chemically synthesized by Invitrogen (Carlsbad, CA, USA). The following sequences were used: anti-Hsp70 sense, 59-UUA CCU GGC UCU UUG CUG CUG CUC C-39; anti-Hsp70 antisense, 59-GGA GCA GCA GCA AAG AGC CAG GUA A-39. siRNA was complexed with Lipofectamine TM RNAiMAX (Invitrogen) according to the manufacturer's instructions. Cells plated in 6-well culture plates and transfection complexes that contained Neurobasal medium without supplements and the complex of Lipofectamine TM RNAiMAX and 40 nM siRNA. In addition, Tau siRNA (sc-36615; Snata Cruz Biotechnology) is a pool of three target specific siRNAs designed to knock down gene expression. Tau siRNA were transfection to cells according to the manufacturer's instructions. Cells were transfected with siRNA for at least 36 h at 37uC. The cultures treated with agents and subjected to protein analyses or cell viability assay.

Preparation of Total Cellular Proteins
PC12 cell lysates were prepared using 20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% Triton X-100, 2.5 mM EDTA, 2.5 mM EGTA, and 1:200 protease inhibitor cocktail set III (Calbiochem, La Jolla, CA, USA). Lysates were kept on ice for 30 min and centrifuged at 10,0006g for 10 min at 4uC. Protein concentration was determined using the BCA protein assay kit (Pierce, Rockford, IL, USA) according to the manufacturer's instructions.

Western Blot Analysis
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed according to standard procedures. Samples containing 20-40 mg of proteins from PC12 cells were electrophoresed and then transferred to nitrocellulose membranes (Millipore, Bedford, MA, USA). The nitrocellulose membrane was cut according the molecular weight of protein and be incubated with different protein antibody. Therefore, one result of different proteins could get in one nitrocellulose membrane. Meanwhile, these proteins had the same internal standard (b-actin). Immunoblotting was carried out as described previously [19] using a rabbit anti-calpain antibody (1:1000; Cell Signaling Technology), a rabbit anti-p35/25 antibody (1:1000; Cell Signaling Technology), a mouse anti-total tau antibody (1:1000; BD Pharmingen, USA), an anti-tau phospho S199/S202 antibody (1:1000; GeneTex, Inc.), a mouse anti-Hsp70 antibody (1:1000; Stressgen, USA), and a mouse anti-b-actin monoclonal antibody (1:5000; Sigma, St. Louis, MO, USA). The appropriate peroxidase-conjugated secondary antibodies were used, detection was performed using an enhanced chemiluminescence kit (Pierce), and the membranes were exposed to X-ray film (Kodak, Rochester, NY, USA) for 5 min. Relative abundance of different protein compared with beta-actin. All data are means 6 SD of five independent observations with different cell passages and on different days.
The density of the respective bands was quantified by densitometric scanning of the blots using the Image-Pro software (Media Cybermetrics, Inc.).

Statistical Analyses
Data are expressed as the mean 6 SD. Analysis of variance (ANOVA) followed by a Newman-Keuls test was used for statistical comparisons. P,0.05 was considered significant.

Effects of YC-1 on p25 Protein Expression in Differentiated PC12 Cells
The cytosolic p25 protein is generated through the proteolytic cleavage of p35 by activated calpain. We investigated whether p25 was upregulated in Ab 25-35 -treated cells. As illustrated in Figure 3C, p25 protein levels were markedly increased after treatment with Ab 25-35 (10 mM) for 12-24 h in differentiated PC12 cells. In contrast, YC-1 (1, 10 mM) pretreatment dramatically reduced the expression of the p25 protein in differentiated PC12 cells compared with cells treated with Ab 25-35 alone (Fig. 3D).

The Effect of ODQ on the Protective Role of YC-1: Prevention of Ab25-35-induced Signaling in Differentiated PC12 Cells
It is well known that YC-1 is an NO-independent soluble guanylyl cyclase (sGC) activator. Here, we investigated whether sGC is involved in the inhibition of Ab 25-35 -induced m-calpain, p25 expression, and tau hyperphosphorylation in differentiated PC12 cells. As shown in Figure 4, the sGC inhibitor ODQ (10 mM) did not antagonize the inhibitory action of YC-1 on Ab 25-35 -induced m-calpain and p25 expression, as well as tau hyperphosphorylation in differentiated PC12 cells. Therefore, the protective action of YC-1 may not be exerted via an sGCdependent pathway.

Effects of Ab 25-35 and YC-1 on the Expression of Hsp70 in Differentiated PC12 Cells
We evaluated whether YC-1 increased Hsp70 expression in differentiated PC12 cells. As shown in Figure 5A and B, Hsp70 constitutively expressed in vehicle-treated subjects and was upregulated in a concentration-dependent manner after YC-1 (5-20 mM) treatment (Fig. 5A). In addition, we evaluated the variation of Hsp70 expression after YC-1 (10 mM) treatment. Results showed that Hsp70 expression increased at 8 h and reached a peak at 18 and 24 h after YC-1 treatment (Fig. 5B). Furthermore, Ab 25-35 alone induced the expression of Hsp70 in differentiated PC12 cells and YC-1 together with Ab 25-35 led to a more significant increase in the expression of Hsp70 (Fig. 5C).
Numerous studies have shown an abnormal activation of the calpain system in the brain of AD patients [29][30][31]. In addition, calpain activation has been implicated in the cleavage of a number of other proteins that are relevant to AD, including APP, p35, and microtubule-associated proteins [32][33][34][35]. Activated calpain cleaves the normal regulatory subunit p35 to p25, thus forming a p25/ cdk5 complex with an activity profile that is substantially higher compared with that of p35 associated with the kinase [36]. Consequently, the p25/cdk5 kinase hyperphosphorylates tau, disrupts the cytoskeleton, and promotes the apoptosis of neurons. In this study, treatment of differentiated PC12 cells with Ab 25-35 decreased cell viability (Fig. 1C). Moreover, the expression of mcalpain and p25 was significantly increased after a challenge with Ab [25][26][27][28][29][30][31][32][33][34][35] (Fig. 3A, C, and E). In contrast, YC-1 significantly attenuated Ab 25-35 -induced m-calpain activation, p25 protein expression, and cell death (Fig. 3B, D, and F). These data suggest that YC-1 has a great potential as a new therapeutic agent for AD.
It is well known that YC-1 is an activator of GC. To understand better the molecular mechanism underlying this phenomenon, we investigated the GC-cGMP pathway in differentiated PC12 cells after treatment with Ab 25-35 and YC-1. The inhibitory effects of YC-1 on the Ab 25-35 -induced m-calpain and p25 protein expression, as well as on tau hyperphosphorylation, in differentiated PC12 cells were examined in the presence of ODQ, an inhibitor of GC. The results (Fig. 4) revealed that ODQ did not modulate the inhibitory effects of YC-1 in differentiated PC12 cells. In addition, YC-1 did not affect the increase of intracellular calcium level caused by Ab 25-35 (data not shown) . Taken together, these results indicated that YC-1 attenuates Ab-induced cytotoxicity in a cGMP-independent manner.
It is well established that Hsps represent an important cellular protective mechanism against a variety of stresses and insults [37,38]. The cellular protection provided by Hsps is attributed to their molecular chaperone function, as it facilitates nascent protein folding and refolding and the degradation of abnormally folded proteins [38,39]. A large body of evidence indicates that Hsps are potent suppressors of neurodegeneration and are, therefore, promising therapeutic targets for neurodegenerative disorders [40,41]. In this study, YC-1 alone or together with Ab [25][26][27][28][29][30][31][32][33][34][35] significantly increased the expression of the Hsp70 protein in differentiated PC12 cells (Fig. 5). In addition, the inhibitory effect of YC-1 on Ab 25-35 -induced m-calpain activation, p25 protein expression, and tau phosphorylation were significantly attenuated by pretreatment with the Hsp70 inhibitor quercetin and/or Hsp70 siRNA (Figs 6 and 7). These results indicate that the neuroprotective effect of YC-1 against Ab [25][26][27][28][29][30][31][32][33][34][35] occurs via the induction of the expression of Hsp70. Although we have no direct evidence of a physical interaction between Ab 25-35 and Hsp70, it is plausible that Hsp70 is critical in the sequestration of intraneuronal Ab [42]. The potential mechanism underlying the YC-1-mediated induction of Hsp70 expression was not elucidated in this study. However, YC-1 has been found to increase the expression of heat shock factor-1 in vascular smooth muscle cells [14]. Further studies are needed to clarify the mechanism via which YC-1 regulates the expression of Hsp70.
In conclusion, the present study examined the neuroprotective effect of YC-1 in differentiated PC12 cells in an in vitro model of AD. YC-1 suppressed Ab 25-35 toxicity via the inhibition of Ab 25-35 -induced calpain activation, leading to decreased p25 formation and subsequent tau hyperphosphorylation. Moreover, the induction of the expression of Hsp70 may be involved in the neuroprotective effect of YC-1 in differentiated PC-12 cells. These findings suggest that YC-1 may be a potential agent for the treatment of AD. Figure S1 The raw data for the Figure 3D, 3F, and Figure 4. Protein samples from PC12 cells were electrophoresed and then transferred to nitrocellulose membranes. The nitrocellulose membrane was cut according the molecular weight of protein and be incubated with different protein antibody. Therefore, one result of different proteins could get in one nitrocellulose membrane. Meanwhile, these proteins had the same internal standard (b-actin). (PDF)