Mol Neurobiol DOI 10.1007/s12035-014-8702-0

Proteasome Inhibition-Induced Downregulation of Akt/GSK-3β Pathway Contributes to Abnormality of Tau in Hippocampal Slice Min Xie & Ruihong Shi & Ying Pan & Tao Zeng & Qicai Chen & Shaohui Wang & Xiaomei Liao

Received: 18 December 2013 / Accepted: 31 March 2014 # Springer Science+Business Media New York 2014

Abstract Proteasome inhibition can induce abnormal accumulation and phosphorylation of microtubule-associated protein tau. The major function of tau protein is to promote microtubules assembly and stabilization, and abnormal tau protein would disturb its microtubule-binding function. In this study, proteasome inhibitor MG132 was used to treat hippocampal slices to explore the role and mechanism of Akt/ glycogen synthase kinase-3β (GSK-3β) in proteasome inhibition-induced tau abnormality. During the culture period, we measure the lactate dehydrogenase (LDH) content to assay the viability of hippocampal slices. Following 2.5 and 5 μM MG132 treatment for 6 h, we detected the expression, phosphorylation modification, and microtubule-binding function of tau protein of slices. We also analyzed the changed activities of glycogen synthase kinase-3β (GSK-3β) and protein kinase B (PKB/Akt) and the level of heat shock protein 90 (Hsp90) in the process. In addition, co-immunoprecipitation was used to investigate the interaction between Akt and Hsp90, Akt and protein phosphatase-2A (PP2A) in the MG132-treated organotypic hippocampal slices. Our results indicated that proteasome inhibition led to degradation obstacles and abnormal phosphorylation of tau protein. The downregulated Akt/GSK-3β signaling pathway might be responsible for the abnormal phosphorylation of tau protein at multiple sites which further reduced the microtubule-binding function of tau protein. Furthermore, proteasome inhibition decreased

Min Xie, Ruihong Shi and Ying Pan contributed equally to this work. Electronic supplementary material The online version of this article (doi:10.1007/s12035-014-8702-0) contains supplementary material, which is available to authorized users. M. Xie : R. Shi : Y. Pan : T. Zeng : Q. Chen : S. Wang : X. Liao (*) School of Life Sciences and Hubei Key Laboratory of Genetic Regulation and Integrative Biology, Central China Normal University, NO. 152, Luoyu Road, Wuhan 430079, China e-mail: [email protected]

the binding capacity of Akt-Hsp90 while increased the AktPP2A binding ability which mediated Akt inactivity. This current study establishes a hippocampal slice model targeting Akt/GSK-3β signaling pathway to explore the pivotal role of proteasome inhibition in tau pathology. Keywords Organotypic hippocampal slice . Proteasome . Tau protein . Akt/GSK-3β signaling pathway

Introduction Tau is one of the first characterized microtubule-associated proteins (MAPs) whose major function is to promote microtubules assembly and stabilization [1]. Tau protein contains more than 80 serine and threonine phosphorylation sites [2]; the biological function of tau is regulated by phosphorylation modification at certain epitopes [3, 4]. Accumulation and abnormal phosphorylation of tau is found in numerous neurodegenerative diseases called tauopathies, including Alzheimer’s disease (AD), frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17), and so on [1, 5]. In these disorders, tau protein is abnormally hyperphosphorylated and aggregated into paired helical filaments (PHFs) or straight filaments (SFs) [6]. Additionally, hyperphosphorylated and ubiquitinated tau protein observed in PHFs [7] indicates that ubiquitin-proteasome system (UPS) could be involved in the tau pathology [8, 9]. UPS is a major proteolytic system which controls the turnover and biological function of most intracellular proteins [10]. Functional impairment or dysfunction of proteasome accompanies neurodegenerative disease [11] which has been demonstrated in AD and other tauopathies [12–14]. Our previous study in HEK293/tau441 cell lines proved that proteasome inhibition by lactacystin (an irreversible inhibitor of proteasome) caused hyperphosphorylation modification of

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tau protein, and both glycogen synthase kinase-3β (GSK-3β) and protein phosphatase-2A (PP2A) were involved in the process [15]. Moreover, inhibition of GSK-3β activity can reverse proteasome inhibition-induced hyperphosphorylation of tau protein [15]. Intriguingly, although tau was hyperphosphorylated at Ser262, Thr231, Thr205 epitopes, the dramatic dephosphorylation of tau at Ser214 site was observed following lactacystin treatment (unpublished data). These results were consistent with the observation in rats [16], but the underlying molecular mechanisms remain unclear. Phosphorylation at Ser214 site has been demonstrated to protect tau against aggregating into PHFs, a characteristic component of neurofibrillary tangles (NFTs) in AD and other tauopathies [17]. The phosphorylation of tau at Ser214 epitope can be mediated by protein kinase B (PKB, also known as Akt) both in vitro [18] and in vivo [19]. Activation of Akt requires its phosphorylation at Thr308 and Ser473 residues [20] and numerous studies have shown that UPS is closely associated with phosphorylation modification of Akt on these two sites [21, 22]. Moreover, GSK-3β is one of the downstream targets of Akt, and phosphorylation of GSK-3β at Ser9 site by Akt could decrease GSK-3β activity [23]. Thus, we reasoned that Akt must be implicated in the dephosphorylation of tau at Ser214 site induced by proteasome inhibition. In order to explore the role of Akt/GSK-3β pathway in tau phosphorylation following proteasome inhibition, we used MG132 (a proteasome inhibitor which reversibly binds to the proteolytic activity center of the 20S proteasome) [24] to treat organotypic hippocampal slices. Since the organotypic hippocampal slice retains the cytoarchitecture of the tissue which is absent in cell culture lines, it provides an optimum model for studying neuronal signal transduction and neurodegeneration [25]. Our data indicated that MG132 treatment results in downregulation of Akt/GSK-3β pathway, which contributed to the abnormal phosphorylation (including the dephosphorylation of Ser214 site) and decreased microtubulebinding function of tau protein. Moreover, the involvement of heat shock protein (Hsp90) and protein phosphatase-2A (PP2A) in MG132-induced Akt deactivation was also elaborated.

Materials and Methods Organotypic Hippocampal Slice Cultures and Drug Treatment Animals were obtained from the Center for Disease Control and Prevention of Hubei province of China. All animal use procedures were approved by the local Animal Care Committee and were in accordance with the NIH Guide for the Care and Use of Laboratory Animals. Organotypic slice cultures from hippocampus were prepared according to the

method of Stoppini et al [26] with some modifications. Briefly, postnatal day 15 (P15) Kunming mouse (Mus musculus, Km) pups were decapitated, and dissected brains were rapidly placed in ice-cold serum-free DMEM medium. The hippocampi of both sides were isolated and sectioned into 400-μm-thick transverse slices with McIlwain tissue chopper (Mickle Laboratory Engineering Co. Ltd, Goose Green, UK). Subsequently, the slices were carefully separated and transferred onto Millicell cell culture inserts (Millipore, Billerica, MA, USA) in the 6-well culture plate. Each well contained 1.5-ml culture medium consisting of 45 % DMEM, 45 % F12 (Invitrogen, Grand Island, NY, USA), and 10 % heat inactivated horse serum (Thermo Scientific, Rockford, IL, USA) supplemented with 100 U/ml penicillin and 100 μg/ml streptomycin. Slices were maintained at 37 °C and with a 5 % CO2/95 % O2 atmosphere. During culture period, slices were treated with proteasome inhibitor MG132 (Enzo Life Sciences, Farmingdale, NY, USA) and cycloheximide (CHX) for 6 h, the vehicle control was treated with dimethyl sulfoxide (DMSO), and then the slices were harvested for subsequent assay. LDH Activity Assay During the culture period, lactate dehydrogenase (LDH, Nanjing, JS, China) enzymatic activity was assayed to evaluate the cell activity of hippocampal slices. The LDH enzymatic activity was detected by LDH assay kit and used according to the manufacturer’s instructions. Western Blotting After MG132 treatment, slices were homogenized in ice-cold extraction buffer [50 mM NaCl, 10 mM Tris, 1 mM EDTA, 0.5 mM Na3VO4 ·12H2O, 50 mM NaF, supplemented with protease inhibitors phenylmethysulfonyl fluoride (PMSF, 1 mM) and aprotinin (2 mg/ml)]. The protein concentration was determined by the method of BCA (Thermo Fisher Scientific Inc., Rockford, IL, USA). Equal amounts of sample were separated on 10 % SDS-polyacrylamide gel electrophoresis (PAGE) and transferred onto nitrocellulose membrane (Millipore, Billerica, MA, USA). Membranes were incubated with the primary antibodies overnight at 4 °C. The following antibodies were used: anti-phospho-tau Ser214 (1:500), Ser396 (1:500), Thr205 (1:500), Thr231 (1:500), Tau-5 (1:500), anti-β-actin (1:1,000), anti-GSK-3β (1:1,000), antiphospho-GSK-3β (Ser9) (1:1,000), and anti-Hsp90 (1:1,500) (Bioworld Technology, Louis Park, MN, USA); Tau-1 (1:500) and anti-phospho-Akt (Ser473) (1:200) (Millipore, Billerica, MA, USA); anti-Akt (1:800) and anti-phospho-Akt (Thr308) (1:200) (Cell Signaling Technology, Danvers, MA, USA); and R134d (1:400) (a gift from Tongji Medical College). After incubating with IRDye 800CW Conjugated Goat (polyclonal)

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anti-Rabbit IgG and anti-Mouse IgG (LI-COR Biosciences, NE, USA) (1:10,000), the membranes were visualized using the Odyssey Infrared Imaging System (LI-COR Biosciences, NE, USA).

Immunoprecipitation For immunoprecipitation, slices were homogenized in icecold RIPA buffer (10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 5 mM EDTA pH 7.4, 1 % Triton X-100, 1 % DOC, 0.1 % (w/ v) SDS, serine protease inhibitors, 1 mM PMSF, and 1 μg/ml aprotinin). Lysates were centrifuged at 12,000×g for 20 min at 4 °C. Supernatant was incubated with protein G agarose beads for 30 min at 4 °C and centrifuged at 12,000×g for 15 min at 4 °C. About 10 % supernatant was used as input, and the rest was incubated with control IgG and primary antibodies Akt, Hsp90, or PP2A overnight, respectively. Immunoprecipitates were captured by incubation with protein G agarose beads for 3 h at 4 °C, then centrifuged at 12,000×g for 1 min at 4 °C and washed three times with ice-cold PBS; the beads were resuspended in 1× loading buffer and boiled for 10 min. Samples are loaded on the SDS-PAGE gel for Western blotting.

Assay of Microtubule-Binding Activity of Tau The capacity of tau protein binding to microtubule was measured by the microtubule-binding assay [27, 28]. In brief, the slices were harvested in a high-salt reassembly buffer (100 mM Tris, 0.5 mM MgSO4, 1 mM EGTA, 2 mM dithiothreitol, and 750 mM NaCl, pH 6.8) supplemented with 0.1 % Triton X-100, 20 μM Taxol, 2 mM GTP, and a mixture of protease inhibitors (2 mM PMSF and 1 μg/ml aprotinin) at 37 °C and homogenized with 15 strokes in a warm Dounce homogenizer and then centrifuged at 50,000×g for 20 min at 25 °C. The supernatant (S) was removed, and the remaining pellet (P) was resuspended in sample buffer. After measurement of the protein concentration, the samples were subjected to Western blot analysis. The supernatant contains unbound tau, and the pellet contains microtubule-bound tau. The levels of unbound tau (S) and microtubule-bound tau (P) were assessed by the immunoreactivity of tau protein in the supernatant and pellet fractions.

Results The LDH Activity Change During Hippocampal Slices Culture The activity of LDH in medium was measured to monitor the cell viability of organotypic hippocampal slices in cultural process. The results showed that the activity of LDH reached the peak value at 6 h and then decreased gradually. In addition, from 48 to 72 h, the LDH activity remained stable at a relatively low level (Fig. 1). According to the result, the neuropharmacological treatment which was carried on hippocampal slices should be launched after 48 h culture in the subsequent experiments.

Proteasome Inhibition Induces Abnormal Expression and Phosphorylation of Tau Protein Different concentrations of MG132 were used to treat the hippocampal slices for 6 h to inhibition proteasome activity. In order to eliminate the influence of protein synthesis on the level of total and phosphorylated tau, cycloheximide was simultaneously used to inhibit protein synthesis [29]. The increased immunoreactivity of total tau (detected by Tau-5) was observed in a MG132 dose-dependent manner (Fig. 2a, b). Then, we detected the phosphorylated tau by using a series of phosphorylation-specific antibodies, and found the immunoreactivity of phosphorylated tau at Thr231, Ser396, and Thr205 epitopes increased obviously. However, compared to the control group, the immunoreactivity of phosphorylated tau at Ser214 site decreased to 88 and 73 %, respectively, after 2.5 and 5 μM MG132 treatments (Fig. 2a, b). In contrast, treatment of the slices with MG132 did not change the level of tau-1 which represented the dephosphorylation of tau protein at Ser198/199/202 sites.

Statistical Analysis Data were analyzed with SPSS statistical software and expressed as means±SD. Multiple groups’ comparison were done using one-way analysis of variance (ANOVA). Differences were considered significant at p

GSK-3β pathway contributes to abnormality of tau in hippocampal slice.

Proteasome inhibition can induce abnormal accumulation and phosphorylation of microtubule-associated protein tau. The major function of tau protein is...
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