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Human prion protein-mediated calcineurin activation induces neuron cell death via AMPK and autophagy pathway
International Journal of Biochemistry and Cell Biology 119 (2020) 105680
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Human prion protein-mediated calcineurin activation induces neuron cell death via AMPK and autophagy pathway
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Jeong-Min Hong, Ji-Hong Moon, Sang-Youel Park* Biosafety Research Institute, College of Veterinary Medicine, Chonbuk National University, Iksan, Jeonbuk 54596, South Korea
ARTICLE INFO
ABSTRACT
Keywords: Prion Calcineurin AMPK Autophagy Neurotoxicity
It is usually accepted that prion proteins induce apoptosis in nerve cells. However, the mechanisms of PrPScneurotoxicity are not completely clear. Calcineurin is a Ca2+/calmodulin-dependent phosphatase. It activates autophagy, and may represent a link between deregulation of Ca2+ homeostasis and neuronal cell death. In this study, the effect of calcineurin activation mediated by human prion protein induced neuronal cell death via AMPK dephosphorylation and autophagy, was investigated. Synthetic peptides of PrP (PrP 106–126) increased calcineurin activity, without changing the levels of this protein phosphatase. Furthermore, these peptides reduced the levels of AMPK phosphorylation at threonine residue 172 and in autophagy activation. Calcineurin inhibitor, FK506, prevented this effect. The data showed that PrP-treated neurons had lower levels of AMPK than control neurons. This decrease in AMPK levels was matched via activation of autophagy. FK506 prevented the changes in AMPK and autophagy levels induced by PrP peptides. Taken together, the data demonstrated that prion peptides triggered an apoptotic cascade via calcineurin activation, which mediated AMPK dephosphorylation and autophagy activation. Therefore, these data suggest that therapeutic strategies targeting calcineurin inhibition might facilitate the management of neurodegenerative disorders including prion disease.
1. Introduction Prion diseases or transmissible spongiform encephalopathies (TSEs) constitute a group of fatal degenerative neurologic disorders that include Creutzfeldt–Jakob disease (CJD) and fatal familial insomnia (FFI) in humans, and scrapie and bovine spongiform encephalopathy (BSE) in animals (Collinge, 2001). Prion diseases are characterized by conformational transformation of the normal cellular prion protein (PrPc) into a pathogenic isoform (PrPSc). A key event in prion diseases is the accumulation and aggregation of an infectious and neurotoxic PrPSc protein (Aguzzi and Calella, 2009). PrPSc is characterized by a high βsheet content and relatively poor level of α-helices, which are detergent insoluble and usually partially resistant to proteases (Prusiner, 1998). The major conformational change of PrPc into PrPSc is localized in residues 90–112. In addition, residues 113–126 constitute the conserved hydrophobic region that also displays structural plasticity (Jackson et al., 1999). A synthetic peptide corresponding to the 106–126 region of PrPc has been used to elicit cellular effects in vitro and in vivo (Brown, 2000; Ettaiche et al., 2000). The PrP peptide 106–126 carries a high inherent ability for polymerization into amyloid-like fibrils in vitro. Cerebral accumulation of PrPsc and its degradation products may play a role in ⁎
neuronal degeneration in prion-related encephalopathies (Forloni et al., 1993). The N-terminal fragment of PrP corresponding to the sequence of PrP 106–126 exhibits physicochemical and biophysical properties similar to those of PrPSc, such as induction of neuronal apoptosis, antiproteinase K digestion, fiber formation, and mediation of conversion of PrPC into PrPSc (Salmona et al., 1999; Walsh et al., 2009). Residues 106–126 of PrP are targeted in the research model generally used to investigate neural degeneration of prion disease (Fioriti et al., 2005). Calcineurin is a calcium/calmodulin-dependent serine/threonine phosphatase composed of catalytic and regulatory subunits. Calcineurin is a heterodimer comprising a catalytic calmodulin-binding subunit (calcineurin A) and a regulatory Ca2+-binding subunit (calcineurin B) (Zhao et al., 1995). Moreover, calcineurin has been shown to play a critical role in apoptosis and pro-inflammatory cytokines of neuronal cells (Fernandez et al., 2007; Hara and Snyder, 2007; Mansuy, 2003). Cyclosporine A, a pharmacological inhibitor of calcineurin, has been reported to increase AMPK phosphorylation in the hippocampus of rat (Park et al., 2011). The toxic effects of PrP peptides and their relation to calcineurin activity were demonstrated via inhibition by FK506 (Agostinho and Oliveira, 2003). AMP-activated protein kinase (AMPK) is a sensor of cellular energy status. It is composed of a heterotrimeric complex consisting of three
Corresponding author at: College of Veterinary Medicine, Chonbuk National University, Gobong ro, Iksan, Jeonbuk 54596, South Korea. E-mail address: [email protected] (S.-Y. Park).
https://doi.org/10.1016/j.biocel.2019.105680 Received 20 September 2019; Received in revised form 28 November 2019; Accepted 16 December 2019 Available online 19 December 2019 1357-2725/ © 2019 Elsevier Ltd. All rights reserved.
International Journal of Biochemistry and Cell Biology 119 (2020) 105680
J.-M. Hong, et al.
was used with the following equation: [Ca2+]i = Kd(F − Fmin)/ (Fmax − F), where Kd is 345 nM for fluo-4, and F is the observed fluorescence level. Each tracing was calibrated for the maximal (Fmax) by addition of ionomycin (2 μM) and for the minimal intensity (Fmin) by addition of EGTA 5 mM at the end of each measurement.
subunits: a catalytic α-subunit and two regulatory β- and γ -subunits, each of which occurs in different isoforms (α1, α2, β1, β2, γ 1, γ 2, and γ 3). AMPK is activated by phosphorylation at threonine-172 of the catalytic subunit. Phosphorylation of the α-subunit at Thr172 by upstream kinases in the activation loop is a prerequisite for AMPK activation (Momcilovic et al., 2006). Recent studies have shown that AMPK increases the autophagy activation via direct activation of Atg proteins, such as Ulk1, rather than through mTOR inhibition (Kim et al., 2011). Autophagy is one of the major pathways for the degradation of cytoplasmic constituents, such as proteins and various organelles within lysosomes/vacuoles. Autophagy was originally described as a cellular response to nutrient starvation, thus facilitating cell survival via degradation and recycling of cytoplasmic contents (Klionsky and Emr, 2000). LC3, the microtubule-associated protein 1A light chain 3, is a central protein in the autophagy pathway. LC3 is known to exist in two forms: a cytosolic form (LC3-I) and a membrane-bound form (LC3-II). The conversion of LC3-I protein to LC3- II (PE-conjugated form) is closely correlated with the formation of autophagosomes (Kabeya et al., 2000). The p62 protein, also called sequestosome 1 (SQSTM1), is a selective substrate of autophagy (Zhang et al., 2013), which induces incorporation into autophagosomes (Komatsu et al., 2007) via direct binding to LC3 and selective degradation by autophagy. Our previous study showed that prion protein induced neuronal cell damage via autophagy flux (Moon et al., 2016). However, the effect of prions on neuronal cell damage by calcineurin through AMPK-autophagy flux has not been reported. In this study, calcineurin activation by prion peptide induced apoptosis via AMPK dephosphorylation and autophagy activation in neurons.
2.5. Immunocytochemistry Immunocytochemical analyses of neuroblastoma cells were performed with anti-calcineurin (ab137335, abcam), p-AMPK (#2535, cell signaling technology) and anti-P62 (#5114, Cell Signaling Technology). Cells were cultured on a Slide Glass (Nalge Nunc International, Naperville, IL). Cells were washed in sterilized TBST for 10 min, and blocked for 15 min with 5 % FBS in TBST, followed by incubation overnight at 4 °C with the primary antibodies diluted with 5 % FBS in TBST. Alexa Fluor 488-labeled donkey anti-rabbit IgG antibody diluted 1:1000 (Molecular Probes, A21206) was used to visualize the channel expression using fluorescence microscopy. 2.6. Confocal microscopy After treatment, coverslips were fixed with 4 %PFA in PBS for 15–20 min at room temperature (RT) and permeabilized in 0.3 %TritonX100 in PBS supplemented with 5 % horse serum for 10 min. Subsequent incubations were carried out in the permeabilization buffer. Coverslips were incubated with appropriate primary antibodies for 60 min at RT, washed 4 times in PBS and incubated with AlexaFluor488, AlexaFluor568- and AlexaFluor647-conjugated secondary antibodies at a concentration of 0.3 μg/ml each for 60 min at RT. Coverslips were then mounted in mounting medium (Southern) and imaged on a Zeiss LSM710 microscope equipped with a standard set of lasers through a 63× oil objective installed at the Center for University-Wide Research Facilities (CURF) at Chonbuk National University. Excitation wavelengths were 488, 543 nm and 633 nm. Bandpass filters were set at 500–550 (AlexaFluor488), 560–615 nm (Cy3, AlexaFluor568) and 650–750 nm (AlexaFluor647). Image acquisition was carried out at the 12-bit rate. Settings were optimized to ensure appropriate dynamic range, low background and sufficient signal/noise ratio.
2. Materials and methods 2.1. Cell culture The human neuroblastoma cell line SK-N-SH was obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA). Cells were cultured in Minimum Essential Medium (MEM, Hyclone Laboratories, Logan, UT, USA) containing 10 % fetal bovine serum (Invitrogen-GIBCO, Grand Island, NY, USA) and gentamycin (0.1 mg/ mL) in a humidified incubator maintained at 37 °C and 5 % CO2.
2.7. Western blot analysis
2.2. PrP (106–126) treatment
SK-N-SH cells were lysed in a lysis buffer [25 mM HEPES at pH 7.4, 100 mM NaCl, 1 mM EDTA, 5 mM MgCl2, 0.1 mM DTT (dithiothreitol), and a protease inhibitor mixture]. Whole cell proteins were electrophoretically resolved on a 10 %–15 % sodium dodecyl sulfate polyacrylamide gel and transferred to a nitrocellulose membrane. Immunoreactivity was detected via sequential incubation with primary antibodies, horseradish peroxidase-conjugated secondary antibodies, and enhanced chemiluminescence reagents i.e. west save gold detection kit (AbFrontier Inc.). The primary antibodies used for immunoblotting were anti-calcineurin (ab137335, abcam), p-AMPK (#2535, cell signaling technology), anti-P62 (#5114, Cell Signaling Technology), LC3 (Novus Biologicals, Littleton, CO, USA) and anti-β-actin (A5441, Sigma Aldrich). Images were examined using a Fusion FX7 imaging system (Vilber Lourmat, Torcy Z.I. Sud, France). Densitometry was used to analyze the signal bands with the Bio-1D software (Vilber Lourmat, Marne La Vallee, France).
Synthetic PrP (106–126) peptides (sequence, Lys-Thr-Asn-Met-LysHis-Met-Ala-Gly-Ala-Ala-Ala-Ala-Gly-Ala-Val-Val-Gly-Gly-Leu-Gly) were synthesized by Peptron (Seoul, Korea). The peptides were dissolved in sterile dimethyl sulfoxide at a stock concentration of 10 mM and stored at −20 °C. 2.3. Annexin V/PI test Apoptosis in detached cells was assessed using an annexin V Assay kit (Santa Cruz Biotechnology, Santa Cruz, CA, USA) according to the manufacturer's protocol. Annexin V levels were determined by measuring fluorescence at 488-nm excitation and 525/30 emission using a Guava EasyCyte HT System (Millipore, Bedford, MA, USA). 2.4. Measurement of [Ca2+]i
2.8. Calcineurin activity assay
Neuronal cells on collagen-coated confocal dish were incubated with 5 μ M Fluo-4 AM (Invitrogen) in media containing 1 % FBS at 37 °C for 40 min. The cells were washed three times with HBSS (Hank’s Balanced Salt Solution). Changes of [Ca2+]i were determined at 488 nm excitation/530 nm emission by air-cooled argon laser system. The emitted fluorescence at 530 nm was collected using a photomultiplier. The image was scanned using a confocal microscope (Zeiss). For the calculation of [Ca2+]i, the method of Tsien et al. (Tsien et al., 1982)
The calcineurin cellular activity assay kit (Enzo Life Sciences #BML‐AK816‐0001, USA) was used to determine the phosphatase activity of calcineurin in neuronal cells, using the manufacturer's instructions. In brief, the cells were lysed on ice in lysis buffer containing protease inhibitors. Phosphatase activity was quantified by detection of free phosphate released from the reaction by measuring the absorbance 2
International Journal of Biochemistry and Cell Biology 119 (2020) 105680
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of malachite green (OD 620 nm).
determine the translocation of activated calcineurin, the nuclear and cytosol fractions were subjected to western blot. The data showed that prion peptide treatment dose-dependently induced calcineurin translocation from cytosol to nuclei (Fig. 1C and D). These results demonstrated that prion protein treatment increased calcineurin expression, activity and translocation in human neuroblastoma.
2.9. TEM (transmission electron microscopy) analysis Following fixation of the cells in 2 % glutaraldehyde (Electron Microscopy Sciences, Hatfield, PA, USA) and 2 % paraformaldehyde (Electron Microscopy Sciences) in 0.05 M sodium cacodylate (pH 7.2; Electron Microscopy Sciences) for 2 h at 4 °C specimens were fixed in 1 % osmium tetroxide (Electron Microscopy Sciences) for 1 h at 4 °C, dehydrated with increasing ethanol (25, 50, 70, 90 and 100 %) for 5 min each and embedded in epoxy resin (Embed 812; Electron Microscopy Sciences) for 48 h at 60 °C according to the manufacturers' instructions. Ultrathin sections (60 nm) were prepared using an LKB-III ultratome (Leica Microsystems GmbH, Wetzlar, Germany) and were stained with 0.5 % uranyl acetate (Electron Microscopy Sciences) for 20 min and 0.1 % lead citrate (Electron Microscopy Sciences) for 7 min at room temperature. Images were recorded on a Hitachi H7650 electron microscope (Hitachi, Ltd., Tokyo, Japan; magnification, x10,000) installed at the Center for University-Wide Research Facilities (CURF) at Chonbuk National University.
3.2. Inhibition of calcineurin activation alleviated prion protein-induced neurotoxicity The induction of neuronal cell death by calcineurin activation by prion protein was investigated. The results showed that prion protein induced neuronal cell death and the prion protein-mediated neurotoxicity was reduced by pharmacological inhibition of calcineurin (Fig. 2A and B). Calcineurin inhibition was confirmed via calcineurin protein expression (Fig. 2C), activity (Fig. 2D) and translocation (Fig. 2E, F). To determine the effect of prion peptide on neuronal cells, we investigated the influence of Prion peptide on Ca2+ alteration in neuronal cells. PrP (106–126) treatment resulted in a rapid increase in Ca2+, and the increased Ca2+ was temporary. And calcineurin inhibitor did not affect that prion protein increase Ca2+ (Fig. 2G). Next, the effect of calcineurin inhibition on prion peptide-induced neurotoxicity was investigated via knockdown of gene levels. The results showed that prion protein induced neuron cell death and PPP3CB siRNA reduced neurotoxicity induced by prion peptide (Fig. 3A, B). Genetically, calcineurin inhibition by PPP3CB siRNA decreased calcineurin protein (Fig. 3C) and calcineurin activity (Fig. 3D). Taken together, this study demonstrated that inhibition of prion protein-mediated calcineurin activation attenuated prion peptide–induced neuronal apoptosis.
2.10. RNA interference SK-N-SH cells were transfected with Calcineurin small interfering RNA (siRNA: siRNA ID s11002: Ambion, Austin, USA) using Lipofectamine 2000 according to manufacturer’s instructions. After a 48-hr culture, knockdown efficiency was measured at the protein level by immunoblot analysis. Nonspecific siRNA (oligoID 12,935–300: Invitrogen) was used as the negative control. 2.11. Statistical analysis
3.3. Abrogation of calcineurin activation induced p-AMPK up-regulation and autophagy inactivation
All data are expressed as mean ± standard error, and the data were compared using the one-way ANOVA followed by the Tukey test. All statistical analysis was performed using GraphPad Prism software. Results were considered significant at * p < 0.05, ** p < 0.01 or *** p < 0.001, as appropriate.
To identify the role of calcineurin on AMPK signaling and autophagy by prion peptide, the effects of calcineurin inhibitor and calcineurin siRNA on PrP (106–126)-induced AMPK and autophagy were investigated. First, we investigated the influence of prion peptide on AMPK in neuronal cells. AMPK phosphorylation was blocked by PrP (106–126) in a dose-dependent manner (Fig. 4A). Calcineurin inhibition recovered the prion peptide-induced decreased AMPK activity. Red fluorescence, which suggested p-AMPK, was restored by calcineurin inhibitor (Fig. 4B). Calcineurin inhibitor recovered AMPK activity that was decreased by prion peptide, as shown in the western blot (Fig. 4C). These results were also supported by additional experimental data using the western blot. Calcineurin inhibition via knockdown of gene levels also affected prion peptide-induced P-AMPK. Knockdown of PPP3CB using PPP3CB small interfering RNA (PPP3CB siRNA) restored prion peptide-induced AMPK activity decreased by prion peptide, as shown in the western blot analysis (Fig. 4D). We further investigated
3. Results 3.1. Prion peptide induced calcineurin activation in neuronal cells We investigated the effect of prion protein on calcineurin levels and activity. Red fluorescence, which suggests calcineurin protein expression, was dose-dependently increased by prion peptide treatment in neuronal cells (Fig. 1A). The activity of calcineurin was evaluated by measuring the formation of free phosphate (PO4 ), using a specific kit assay. The results showed that calcineurin activity was significantly elevated in neuronal cells exposed to prion protein (Fig. 1B). To
Fig. 1. Prion protein increased calcineurin activity. (A) Immunocytochemistry for the calcineurin was analyzed from SKN-SH cells. (B) Calcineurin activity levels increased significantly at 12 h following PrP (106–126). (C, D) SK-N-SH cells were treated with 25, 50, and 100 μM of PrP (106–126) for 12 h. The treated cells were assessed for calcineurin by Western blot analysis. Bar graph was generated using mean ± standard error of the mean (n = 3). *** p < 0.001; significant differences when compared with control and each treatment group. PrP, PrP (106–126).
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International Journal of Biochemistry and Cell Biology 119 (2020) 105680
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Fig. 2. Calcineurin inhibitor alleviated prion protein-induced neurotoxicity. (A) SK-N-SH were treated with PrP (106–126) for 12 h. Cell viability was measured by annexin V assay. Cells were treated with FITC-annexin V and PI, which binds to phosphatidylserine to the plasma membrane and nuclei during apoptosis. (B) Bar graph indicating the average number of annexin V negative cells. (C) Immunocytochemistry for the calcineurin was analyzed from neuroblastoma cells. (D) Calcineurin activity levels increased significantly at 12 h following PrP (106–126). (E, F) SK-N-SH cells were treated with PrP (106–126) for 12 h. The treated cells were assessed for calcineurin by Western blot analysis. (G) SK-N-SH cells were loaded with fluo-4 AM and the changes in Ca2+ levels were measured using confocal microscope. The time point of 100 u M of FK506 addition is indicated by the ① arrow and 100 μM of PrP (106–126) addition is indicated by the ② arrow. Data are mean ± SEM of [Ca2+]i at 60 s from three independent experiments. Bar graph was generated using mean ± standard error of the mean (n = 3). *** p < 0.001, # p < 0.05, ### p < 0.001; significant differences when compared with control and each treatment group. PrP, PrP (106–126).
Fig. 3. Calcineurin siRNA alleviated prion protein-induced neurotoxicity. (A) SK-N-SH cells were treated with PrP (106–126) for 12 h. Cell viability was measured by annexin V assay. Cells were treated with FITC-annexin V and PI, which binds to phosphatidylserine to the plasma membrane and nuclei during apoptosis. (B) Bar graph indicating the average number of annexin V negative cells. (C) SK-N-SH cells were treated with PrP (106–126) for 12 h. The treated cells were assessed for calcineurin by Western blot analysis. (D) Calcineurin activity levels increased significantly at 12 h following PrP (106–126). Bar graph was generated using mean ± standard error of the mean (n = 3). *** p < 0.001, #p < 0.05, ### p < 0.001; significant differences when compared with control and each treatment group. PrP, PrP (106–126).
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Fig. 4. Inhibition of calcineurin activity induced PrP (106–126)mediated p-AMPK up-regulation and autophagy inactivation. (A) SK-N-SH cells were treated with 25, 50, and 100 μM of PrP (106–126) for 12 h. The treated cells were assessed for p-AMPK by Western blot analysis. (B) Immunocytochemistry for the calcineurin was analyzed from SK-N-SH cells. (C, D) SK-N-SH cells were treated with PrP (106–126) for 12 h. The treated cells were assessed for P62, LC3 by Western blot analysis. (E) SK-N-SH cells were treated with 25, 50, and 100 μM of PrP (106–126) for 12 h. The treated cells were assessed for P62 by Western blot analysis. (F) Immunocytochemistry for the P62 was analyzed from SK-N-SH cells. (G, H) SK-N-SH cells were treated with PrP (106–126) for 12 h. The treated cells were assessed for P62, LC3 by Western blot analysis.. (I) SK-N-SH cells were incubated with PrP (106–126) at 100 μM for 12 h and analyzed by TEM. Arrowheads indicate autophagosomes and arrows indicate autolysosomes.
the influence of prion peptide on autophagy in neuronal cells. We detected a dose-dependent decrease in the late autophagosome markers LC3-II and SQSTM1/p62 in the PrP-treated group compared with the control group in human neuroblastoma (Fig. 4E). The effect of calcineurin inhibitor on prion peptide-induced autophagy induction was also investigated. Red fluorescence, suggesting the presence of P62 was recovered by calcineurin inhibitor (Fig. 4F). Prion peptide-induced autophagy flux was abolished by calcineurin inhibitor and was confirmed by the up-regulation of SQSTM1/p62 protein and LC3-II (Fig. 4G). We further tested whether calcineurin inhibition via knockdown of gene levels also affected prion peptide-induced autophagy. Prion peptide-induced autophagy was inhibited by PPP3CB siRNA and was confirmed by the up-regulation of SQSTM1/p62 protein and LC3-II (Fig. 4H). We also conduct Transmission electron microscopy (TEM) analysis to certainly determine the effect of lysosomal inhibition on autophagy flux by calcineurin inhibitor. As shown in Fig. 4I, a lot of vesicles including double-membraned autophagosomes (arrowheads) were induced by treatment of cells with FK506, which indicated inhibition of lysosomal degradation (Fig. 4I). These results indicate the inhibition of calcineurin-induced p-AMPK up-regulation and autophagy inactivation.
3.4. Phospho-AMPK reduction induced autophagy activation by prion peptides in neuronal cells We investigated the role of AMPK and autophagy in PrP (106–126)induced neurotoxicity and protective effect mediated by AMPK activator and autophagy inhibitor. We also analyzed whether autophagy was inhibited by autophagy inhibitor chloroquine (CQ). The AMPK agonist, AICAR restored AMPK phosphorylation that was decreased by prion peptide. However, the effects of CQ were similar to those found in cells treated with PrP (106–126) group (Fig. 5A). Inhibition of prion peptide-induced autophagy flux by AICAR and CQ was confirmed by the up-regulation of SQSTM1/p62 protein and LC3-II (Fig. 5B). The effect of AMPK activator and autophagy inhibitor on PrP (106–126)induced neurotoxicity was investigated. The results showed that prion peptide induced neuron cell death and AICAR and CQ reduced neurotoxicity induced by prion peptide (Fig. 5C, D). Our study demonstrated that p-AMPK reduction induced autophagy activation by prion peptideinduced neurotoxicity, and treatment with AMPK activator and autophagy inhibitor may attenuate prion peptide–induced neuronal apoptosis.
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Fig. 5. Phospho-AMPK reduction induced autophagy activation by PrP (106–126) (A) SK-N-SH cells were treated with 25, 50, and 100 μM of PrP (106–126) for 12 h. The treated cells were assessed for p-AMPK by Western blot analysis. (B) SK-NSH cells were treated with PrP (106–126) for 12 h. The treated cells were assessed for P62, LC3 by Western blot analysis. (C) SK-N-SH cells were treated with PrP (106–126) for 12 h. Cell viability was measured by annexin V assay. Cells were treated with FITC-annexin V and PI, which binds to phosphatidylserine to the plasma membrane and nuclei during apoptosis. (D) Bar graph indicating the average number of annexin V negative cells. Bar graph was generated using mean ± standard error of the mean (n = 3). *** p < 0.001, # p < 0.05, ## p < 0.01; significant differences when compared with control and each treatment group. PrP, PrP (106–126).
3.5. Autophagy inhibitor and AMPK activator had no effect on prion peptide-induced calcineurin
4. Discussion The purpose of this study is to investigate the role of calcineurin on human prion protein-mediated AMPK/autophagy signals and neurotoxicity in neuron cells. The results showed that increased calcineurin activity mediated by prion protein induced neuronal cell damage and also provided an insight into the fundamental mechanism underlying the AMPK and autophagy flux in prion disease. Autophagy play an important role in cell survival and cell death in various pathological and physiological conditions (Fimia et al., 2007). Autophagy protects cells against death under conditions of starvation and neurodegeneration, but is also important contributing factor in some types of cell death (Dang et al., 2015). Autophagy has several pathways depending on the circumstances, and the biochemical basis for the diverse functions is not well understood (Cuervo, 2004; CzyzykKrzeska et al., 2012). We identified human prion peptide induces autophagy flux in neuronal cells (Moon et al., 2016). In this study, we found that human prion peptide induced autophagic cell death in neuronal cell. Autophagy inhibitor reduced prion protein induced autophagic cell death. AMP-activated protein kinase (AMPK) is a sensor of energy status including energy deficiency based on the increased AMP/ATP ratio (Poels et al., 2009). Calcineurin prolonged the oxidative stress-induced activation of calcineurin resulting in the attenuation of AMPK signaling and inhibition of autophagy in cardiomyocytes (He et al., 2014). In this study, the results showed that prion protein-mediated calcineurin activation decreased the AMPK activity via dephosphorylation and suggest that AMPK inactivation may lead to autophagy activation and neuronal toxicity. This study demonstrated that prion protein-calcineurin activation was involved not only in prion protein-mediated neuronal cell death but also AMPK and autophagy signaling pathways. It regulates metabolic homeostasis (Hardie, 2007) by controlling autophagy (Vingtdeux et al., 2011). However, our present study demonstrated that prion peptide decreased the AMPK activity via dephosphorylation but increases autophagic cell death (Fig. 5). The role of AMPK and autophagy flux in neurodegeneration is not clear so that further studies are required. Our results indicate that prion protein increases calcineurin activity, resulting in decreased AMPK phosphorylation that induces autophagic cell death. The current study defines a novel interaction between calcineurin and the AMPK/mTOR pathway in the control of autophagy. This study demonstrated that calcineurin plays a critical biologic role in the survival of neuronal cells exposed to prions. Inhibition of calcineurin and autophagy may represent a novel therapeutic approach with a broad clinical potential to prevent neurodegenerative diseases including prion disease.
We investigated whether autophagy induction and AMPK activator affected prion peptide-induced calcineurin. We demonstrated that autophagy inhibitor did not affect prion peptide-induced calcineurin (Fig. 6A). We found that AMPK activator also had no effect on PrP (106–126)-induced calcineurin (Fig. 6B). The activity of calcineurin was evaluated by measuring the formation of free phosphate (PO4 ), using a specific kit assay. The results show that calcineurin activity was significantly decreased by exposure to autophagy inhibitor and AMPK activator (Fig. 6C, D). These results indicate that autophagy inhibitor and AMPK activator may not affect calcineurin in prion peptide-induced neurotoxicity.
Fig. 6. Autophagy inhibitor and AMPK activator have no influence in prion peptide-induced calcineurin. (A) Immunocytochemistry for the P62 was analyzed from SK-N-SH cells (B) Immunocytochemistry for the P-AMPK was analyzed from SK-N-SH cells. (C, D) Calcineurin activity levels increased significantly at 12 h following PrP (106–126). Bar graph was generated using mean ± standard error of the mean (n = 3). *** p < 0.001, ## p < 0.01, ### p < 0.001; significant differences when compared with control and each treatment group. PrP, PrP (106–126). 6
International Journal of Biochemistry and Cell Biology 119 (2020) 105680
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Ethical approval
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Ethical approval for the project was granted by the institutional review board of the Chonbuk National University. Author contributions J.M.H., J.H.M. and S.Y.P. designed, executed the study, analyzed data and J.M.H. and J.H.M. wrote the manuscript. All authors have read and approved the final manuscript. CRediT authorship contribution statement Jeong-Min Hong: Conceptualization, Methodology, Data curation, Writing - original draft. Ji-Hong Moon: Visualization, Investigation, Supervision. Sang-Youel Park: Writing - review & editing. Declaration of Competing Interest The authors declare no conflicts of interest. Acknowledgments This study was supported by the National Research Foundation of the Korea Grant (NRF) funded by the Ministry of Education (2019R1A6A1A03033084). References Agostinho, P., Oliveira, C.R., 2003. Involvement of calcineurin in the neurotoxic effects induced by amyloid‐beta and prion peptides. Eur. J. Neurosci. 17 (6), 1189–1196. Aguzzi, A., Calella, A.M., 2009. Prions: protein aggregation and infectious diseases. Physiol. Rev. 89 (4), 1105–1152. Brown, D.R., 2000. Prion protein peptides: optimal toxicity and peptide blockade of toxicity. Mol. Cell. Neurosci. 15 (1), 66–78. Collinge, J., 2001. Prion diseases of humans and animals: their causes and molecular basis. Annu. Rev. Neurosci. 24 (1), 519–550. Cuervo, A.M., 2004. Autophagy: many paths to the same end. Mol. Cell. Biochem. 263 (12), 55–72. Czyzyk-Krzeska, M.F., Meller, J., Plas, D.R., 2012. Not all autophagy is equal. Autophagy 8 (7), 1155–1156. Dang, S., Yu, Z.-m., Zhang, C.-y., Zheng, J., Li, K.-l., Wu, Y., Qian, L.-l., Yang, Z.-y., Li, X.r., Zhang, Y., 2015. Autophagy promotes apoptosis of mesenchymal stem cells under inflammatory microenvironment. Stem Cell Res. Ther. 6 (1), 247. Ettaiche, M., Pichot, R., Vincent, J.-P., Chabry, J., 2000. In vivo cytotoxicity of the prion protein fragment 106–126. J. Biol. Chem. 275 (47), 36487–36490. Fernandez, A.M., Fernandez, S., Carrero, P., Garcia-Garcia, M., Torres-Aleman, I., 2007. Calcineurin in reactive astrocytes plays a key role in the interplay between proinflammatory and anti-inflammatory signals. J. Neurosci. 27 (33), 8745–8756. Fimia, G.M., Stoykova, A., Romagnoli, A., Giunta, L., Di Bartolomeo, S., Nardacci, R., Corazzari, M., Fuoco, C., Ucar, A., Schwartz, P., 2007. Ambra1 regulates autophagy and development of the nervous system. Nature 447 (7148), 1121.
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Human prion protein-mediated calcineurin activation induces neuron cell death via AMPK and autophagy pathway
T
Jeong-Min Hong, Ji-Hong Moon, Sang-Youel Park* Biosafety Research Institute, College of Veterinary Medicine, Chonbuk National University, Iksan, Jeonbuk 54596, South Korea
ARTICLE INFO
ABSTRACT
Keywords: Prion Calcineurin AMPK Autophagy Neurotoxicity
It is usually accepted that prion proteins induce apoptosis in nerve cells. However, the mechanisms of PrPScneurotoxicity are not completely clear. Calcineurin is a Ca2+/calmodulin-dependent phosphatase. It activates autophagy, and may represent a link between deregulation of Ca2+ homeostasis and neuronal cell death. In this study, the effect of calcineurin activation mediated by human prion protein induced neuronal cell death via AMPK dephosphorylation and autophagy, was investigated. Synthetic peptides of PrP (PrP 106–126) increased calcineurin activity, without changing the levels of this protein phosphatase. Furthermore, these peptides reduced the levels of AMPK phosphorylation at threonine residue 172 and in autophagy activation. Calcineurin inhibitor, FK506, prevented this effect. The data showed that PrP-treated neurons had lower levels of AMPK than control neurons. This decrease in AMPK levels was matched via activation of autophagy. FK506 prevented the changes in AMPK and autophagy levels induced by PrP peptides. Taken together, the data demonstrated that prion peptides triggered an apoptotic cascade via calcineurin activation, which mediated AMPK dephosphorylation and autophagy activation. Therefore, these data suggest that therapeutic strategies targeting calcineurin inhibition might facilitate the management of neurodegenerative disorders including prion disease.
1. Introduction Prion diseases or transmissible spongiform encephalopathies (TSEs) constitute a group of fatal degenerative neurologic disorders that include Creutzfeldt–Jakob disease (CJD) and fatal familial insomnia (FFI) in humans, and scrapie and bovine spongiform encephalopathy (BSE) in animals (Collinge, 2001). Prion diseases are characterized by conformational transformation of the normal cellular prion protein (PrPc) into a pathogenic isoform (PrPSc). A key event in prion diseases is the accumulation and aggregation of an infectious and neurotoxic PrPSc protein (Aguzzi and Calella, 2009). PrPSc is characterized by a high βsheet content and relatively poor level of α-helices, which are detergent insoluble and usually partially resistant to proteases (Prusiner, 1998). The major conformational change of PrPc into PrPSc is localized in residues 90–112. In addition, residues 113–126 constitute the conserved hydrophobic region that also displays structural plasticity (Jackson et al., 1999). A synthetic peptide corresponding to the 106–126 region of PrPc has been used to elicit cellular effects in vitro and in vivo (Brown, 2000; Ettaiche et al., 2000). The PrP peptide 106–126 carries a high inherent ability for polymerization into amyloid-like fibrils in vitro. Cerebral accumulation of PrPsc and its degradation products may play a role in ⁎
neuronal degeneration in prion-related encephalopathies (Forloni et al., 1993). The N-terminal fragment of PrP corresponding to the sequence of PrP 106–126 exhibits physicochemical and biophysical properties similar to those of PrPSc, such as induction of neuronal apoptosis, antiproteinase K digestion, fiber formation, and mediation of conversion of PrPC into PrPSc (Salmona et al., 1999; Walsh et al., 2009). Residues 106–126 of PrP are targeted in the research model generally used to investigate neural degeneration of prion disease (Fioriti et al., 2005). Calcineurin is a calcium/calmodulin-dependent serine/threonine phosphatase composed of catalytic and regulatory subunits. Calcineurin is a heterodimer comprising a catalytic calmodulin-binding subunit (calcineurin A) and a regulatory Ca2+-binding subunit (calcineurin B) (Zhao et al., 1995). Moreover, calcineurin has been shown to play a critical role in apoptosis and pro-inflammatory cytokines of neuronal cells (Fernandez et al., 2007; Hara and Snyder, 2007; Mansuy, 2003). Cyclosporine A, a pharmacological inhibitor of calcineurin, has been reported to increase AMPK phosphorylation in the hippocampus of rat (Park et al., 2011). The toxic effects of PrP peptides and their relation to calcineurin activity were demonstrated via inhibition by FK506 (Agostinho and Oliveira, 2003). AMP-activated protein kinase (AMPK) is a sensor of cellular energy status. It is composed of a heterotrimeric complex consisting of three
Corresponding author at: College of Veterinary Medicine, Chonbuk National University, Gobong ro, Iksan, Jeonbuk 54596, South Korea. E-mail address: [email protected] (S.-Y. Park).
https://doi.org/10.1016/j.biocel.2019.105680 Received 20 September 2019; Received in revised form 28 November 2019; Accepted 16 December 2019 Available online 19 December 2019 1357-2725/ © 2019 Elsevier Ltd. All rights reserved.
International Journal of Biochemistry and Cell Biology 119 (2020) 105680
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was used with the following equation: [Ca2+]i = Kd(F − Fmin)/ (Fmax − F), where Kd is 345 nM for fluo-4, and F is the observed fluorescence level. Each tracing was calibrated for the maximal (Fmax) by addition of ionomycin (2 μM) and for the minimal intensity (Fmin) by addition of EGTA 5 mM at the end of each measurement.
subunits: a catalytic α-subunit and two regulatory β- and γ -subunits, each of which occurs in different isoforms (α1, α2, β1, β2, γ 1, γ 2, and γ 3). AMPK is activated by phosphorylation at threonine-172 of the catalytic subunit. Phosphorylation of the α-subunit at Thr172 by upstream kinases in the activation loop is a prerequisite for AMPK activation (Momcilovic et al., 2006). Recent studies have shown that AMPK increases the autophagy activation via direct activation of Atg proteins, such as Ulk1, rather than through mTOR inhibition (Kim et al., 2011). Autophagy is one of the major pathways for the degradation of cytoplasmic constituents, such as proteins and various organelles within lysosomes/vacuoles. Autophagy was originally described as a cellular response to nutrient starvation, thus facilitating cell survival via degradation and recycling of cytoplasmic contents (Klionsky and Emr, 2000). LC3, the microtubule-associated protein 1A light chain 3, is a central protein in the autophagy pathway. LC3 is known to exist in two forms: a cytosolic form (LC3-I) and a membrane-bound form (LC3-II). The conversion of LC3-I protein to LC3- II (PE-conjugated form) is closely correlated with the formation of autophagosomes (Kabeya et al., 2000). The p62 protein, also called sequestosome 1 (SQSTM1), is a selective substrate of autophagy (Zhang et al., 2013), which induces incorporation into autophagosomes (Komatsu et al., 2007) via direct binding to LC3 and selective degradation by autophagy. Our previous study showed that prion protein induced neuronal cell damage via autophagy flux (Moon et al., 2016). However, the effect of prions on neuronal cell damage by calcineurin through AMPK-autophagy flux has not been reported. In this study, calcineurin activation by prion peptide induced apoptosis via AMPK dephosphorylation and autophagy activation in neurons.
2.5. Immunocytochemistry Immunocytochemical analyses of neuroblastoma cells were performed with anti-calcineurin (ab137335, abcam), p-AMPK (#2535, cell signaling technology) and anti-P62 (#5114, Cell Signaling Technology). Cells were cultured on a Slide Glass (Nalge Nunc International, Naperville, IL). Cells were washed in sterilized TBST for 10 min, and blocked for 15 min with 5 % FBS in TBST, followed by incubation overnight at 4 °C with the primary antibodies diluted with 5 % FBS in TBST. Alexa Fluor 488-labeled donkey anti-rabbit IgG antibody diluted 1:1000 (Molecular Probes, A21206) was used to visualize the channel expression using fluorescence microscopy. 2.6. Confocal microscopy After treatment, coverslips were fixed with 4 %PFA in PBS for 15–20 min at room temperature (RT) and permeabilized in 0.3 %TritonX100 in PBS supplemented with 5 % horse serum for 10 min. Subsequent incubations were carried out in the permeabilization buffer. Coverslips were incubated with appropriate primary antibodies for 60 min at RT, washed 4 times in PBS and incubated with AlexaFluor488, AlexaFluor568- and AlexaFluor647-conjugated secondary antibodies at a concentration of 0.3 μg/ml each for 60 min at RT. Coverslips were then mounted in mounting medium (Southern) and imaged on a Zeiss LSM710 microscope equipped with a standard set of lasers through a 63× oil objective installed at the Center for University-Wide Research Facilities (CURF) at Chonbuk National University. Excitation wavelengths were 488, 543 nm and 633 nm. Bandpass filters were set at 500–550 (AlexaFluor488), 560–615 nm (Cy3, AlexaFluor568) and 650–750 nm (AlexaFluor647). Image acquisition was carried out at the 12-bit rate. Settings were optimized to ensure appropriate dynamic range, low background and sufficient signal/noise ratio.
2. Materials and methods 2.1. Cell culture The human neuroblastoma cell line SK-N-SH was obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA). Cells were cultured in Minimum Essential Medium (MEM, Hyclone Laboratories, Logan, UT, USA) containing 10 % fetal bovine serum (Invitrogen-GIBCO, Grand Island, NY, USA) and gentamycin (0.1 mg/ mL) in a humidified incubator maintained at 37 °C and 5 % CO2.
2.7. Western blot analysis
2.2. PrP (106–126) treatment
SK-N-SH cells were lysed in a lysis buffer [25 mM HEPES at pH 7.4, 100 mM NaCl, 1 mM EDTA, 5 mM MgCl2, 0.1 mM DTT (dithiothreitol), and a protease inhibitor mixture]. Whole cell proteins were electrophoretically resolved on a 10 %–15 % sodium dodecyl sulfate polyacrylamide gel and transferred to a nitrocellulose membrane. Immunoreactivity was detected via sequential incubation with primary antibodies, horseradish peroxidase-conjugated secondary antibodies, and enhanced chemiluminescence reagents i.e. west save gold detection kit (AbFrontier Inc.). The primary antibodies used for immunoblotting were anti-calcineurin (ab137335, abcam), p-AMPK (#2535, cell signaling technology), anti-P62 (#5114, Cell Signaling Technology), LC3 (Novus Biologicals, Littleton, CO, USA) and anti-β-actin (A5441, Sigma Aldrich). Images were examined using a Fusion FX7 imaging system (Vilber Lourmat, Torcy Z.I. Sud, France). Densitometry was used to analyze the signal bands with the Bio-1D software (Vilber Lourmat, Marne La Vallee, France).
Synthetic PrP (106–126) peptides (sequence, Lys-Thr-Asn-Met-LysHis-Met-Ala-Gly-Ala-Ala-Ala-Ala-Gly-Ala-Val-Val-Gly-Gly-Leu-Gly) were synthesized by Peptron (Seoul, Korea). The peptides were dissolved in sterile dimethyl sulfoxide at a stock concentration of 10 mM and stored at −20 °C. 2.3. Annexin V/PI test Apoptosis in detached cells was assessed using an annexin V Assay kit (Santa Cruz Biotechnology, Santa Cruz, CA, USA) according to the manufacturer's protocol. Annexin V levels were determined by measuring fluorescence at 488-nm excitation and 525/30 emission using a Guava EasyCyte HT System (Millipore, Bedford, MA, USA). 2.4. Measurement of [Ca2+]i
2.8. Calcineurin activity assay
Neuronal cells on collagen-coated confocal dish were incubated with 5 μ M Fluo-4 AM (Invitrogen) in media containing 1 % FBS at 37 °C for 40 min. The cells were washed three times with HBSS (Hank’s Balanced Salt Solution). Changes of [Ca2+]i were determined at 488 nm excitation/530 nm emission by air-cooled argon laser system. The emitted fluorescence at 530 nm was collected using a photomultiplier. The image was scanned using a confocal microscope (Zeiss). For the calculation of [Ca2+]i, the method of Tsien et al. (Tsien et al., 1982)
The calcineurin cellular activity assay kit (Enzo Life Sciences #BML‐AK816‐0001, USA) was used to determine the phosphatase activity of calcineurin in neuronal cells, using the manufacturer's instructions. In brief, the cells were lysed on ice in lysis buffer containing protease inhibitors. Phosphatase activity was quantified by detection of free phosphate released from the reaction by measuring the absorbance 2
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of malachite green (OD 620 nm).
determine the translocation of activated calcineurin, the nuclear and cytosol fractions were subjected to western blot. The data showed that prion peptide treatment dose-dependently induced calcineurin translocation from cytosol to nuclei (Fig. 1C and D). These results demonstrated that prion protein treatment increased calcineurin expression, activity and translocation in human neuroblastoma.
2.9. TEM (transmission electron microscopy) analysis Following fixation of the cells in 2 % glutaraldehyde (Electron Microscopy Sciences, Hatfield, PA, USA) and 2 % paraformaldehyde (Electron Microscopy Sciences) in 0.05 M sodium cacodylate (pH 7.2; Electron Microscopy Sciences) for 2 h at 4 °C specimens were fixed in 1 % osmium tetroxide (Electron Microscopy Sciences) for 1 h at 4 °C, dehydrated with increasing ethanol (25, 50, 70, 90 and 100 %) for 5 min each and embedded in epoxy resin (Embed 812; Electron Microscopy Sciences) for 48 h at 60 °C according to the manufacturers' instructions. Ultrathin sections (60 nm) were prepared using an LKB-III ultratome (Leica Microsystems GmbH, Wetzlar, Germany) and were stained with 0.5 % uranyl acetate (Electron Microscopy Sciences) for 20 min and 0.1 % lead citrate (Electron Microscopy Sciences) for 7 min at room temperature. Images were recorded on a Hitachi H7650 electron microscope (Hitachi, Ltd., Tokyo, Japan; magnification, x10,000) installed at the Center for University-Wide Research Facilities (CURF) at Chonbuk National University.
3.2. Inhibition of calcineurin activation alleviated prion protein-induced neurotoxicity The induction of neuronal cell death by calcineurin activation by prion protein was investigated. The results showed that prion protein induced neuronal cell death and the prion protein-mediated neurotoxicity was reduced by pharmacological inhibition of calcineurin (Fig. 2A and B). Calcineurin inhibition was confirmed via calcineurin protein expression (Fig. 2C), activity (Fig. 2D) and translocation (Fig. 2E, F). To determine the effect of prion peptide on neuronal cells, we investigated the influence of Prion peptide on Ca2+ alteration in neuronal cells. PrP (106–126) treatment resulted in a rapid increase in Ca2+, and the increased Ca2+ was temporary. And calcineurin inhibitor did not affect that prion protein increase Ca2+ (Fig. 2G). Next, the effect of calcineurin inhibition on prion peptide-induced neurotoxicity was investigated via knockdown of gene levels. The results showed that prion protein induced neuron cell death and PPP3CB siRNA reduced neurotoxicity induced by prion peptide (Fig. 3A, B). Genetically, calcineurin inhibition by PPP3CB siRNA decreased calcineurin protein (Fig. 3C) and calcineurin activity (Fig. 3D). Taken together, this study demonstrated that inhibition of prion protein-mediated calcineurin activation attenuated prion peptide–induced neuronal apoptosis.
2.10. RNA interference SK-N-SH cells were transfected with Calcineurin small interfering RNA (siRNA: siRNA ID s11002: Ambion, Austin, USA) using Lipofectamine 2000 according to manufacturer’s instructions. After a 48-hr culture, knockdown efficiency was measured at the protein level by immunoblot analysis. Nonspecific siRNA (oligoID 12,935–300: Invitrogen) was used as the negative control. 2.11. Statistical analysis
3.3. Abrogation of calcineurin activation induced p-AMPK up-regulation and autophagy inactivation
All data are expressed as mean ± standard error, and the data were compared using the one-way ANOVA followed by the Tukey test. All statistical analysis was performed using GraphPad Prism software. Results were considered significant at * p < 0.05, ** p < 0.01 or *** p < 0.001, as appropriate.
To identify the role of calcineurin on AMPK signaling and autophagy by prion peptide, the effects of calcineurin inhibitor and calcineurin siRNA on PrP (106–126)-induced AMPK and autophagy were investigated. First, we investigated the influence of prion peptide on AMPK in neuronal cells. AMPK phosphorylation was blocked by PrP (106–126) in a dose-dependent manner (Fig. 4A). Calcineurin inhibition recovered the prion peptide-induced decreased AMPK activity. Red fluorescence, which suggested p-AMPK, was restored by calcineurin inhibitor (Fig. 4B). Calcineurin inhibitor recovered AMPK activity that was decreased by prion peptide, as shown in the western blot (Fig. 4C). These results were also supported by additional experimental data using the western blot. Calcineurin inhibition via knockdown of gene levels also affected prion peptide-induced P-AMPK. Knockdown of PPP3CB using PPP3CB small interfering RNA (PPP3CB siRNA) restored prion peptide-induced AMPK activity decreased by prion peptide, as shown in the western blot analysis (Fig. 4D). We further investigated
3. Results 3.1. Prion peptide induced calcineurin activation in neuronal cells We investigated the effect of prion protein on calcineurin levels and activity. Red fluorescence, which suggests calcineurin protein expression, was dose-dependently increased by prion peptide treatment in neuronal cells (Fig. 1A). The activity of calcineurin was evaluated by measuring the formation of free phosphate (PO4 ), using a specific kit assay. The results showed that calcineurin activity was significantly elevated in neuronal cells exposed to prion protein (Fig. 1B). To
Fig. 1. Prion protein increased calcineurin activity. (A) Immunocytochemistry for the calcineurin was analyzed from SKN-SH cells. (B) Calcineurin activity levels increased significantly at 12 h following PrP (106–126). (C, D) SK-N-SH cells were treated with 25, 50, and 100 μM of PrP (106–126) for 12 h. The treated cells were assessed for calcineurin by Western blot analysis. Bar graph was generated using mean ± standard error of the mean (n = 3). *** p < 0.001; significant differences when compared with control and each treatment group. PrP, PrP (106–126).
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Fig. 2. Calcineurin inhibitor alleviated prion protein-induced neurotoxicity. (A) SK-N-SH were treated with PrP (106–126) for 12 h. Cell viability was measured by annexin V assay. Cells were treated with FITC-annexin V and PI, which binds to phosphatidylserine to the plasma membrane and nuclei during apoptosis. (B) Bar graph indicating the average number of annexin V negative cells. (C) Immunocytochemistry for the calcineurin was analyzed from neuroblastoma cells. (D) Calcineurin activity levels increased significantly at 12 h following PrP (106–126). (E, F) SK-N-SH cells were treated with PrP (106–126) for 12 h. The treated cells were assessed for calcineurin by Western blot analysis. (G) SK-N-SH cells were loaded with fluo-4 AM and the changes in Ca2+ levels were measured using confocal microscope. The time point of 100 u M of FK506 addition is indicated by the ① arrow and 100 μM of PrP (106–126) addition is indicated by the ② arrow. Data are mean ± SEM of [Ca2+]i at 60 s from three independent experiments. Bar graph was generated using mean ± standard error of the mean (n = 3). *** p < 0.001, # p < 0.05, ### p < 0.001; significant differences when compared with control and each treatment group. PrP, PrP (106–126).
Fig. 3. Calcineurin siRNA alleviated prion protein-induced neurotoxicity. (A) SK-N-SH cells were treated with PrP (106–126) for 12 h. Cell viability was measured by annexin V assay. Cells were treated with FITC-annexin V and PI, which binds to phosphatidylserine to the plasma membrane and nuclei during apoptosis. (B) Bar graph indicating the average number of annexin V negative cells. (C) SK-N-SH cells were treated with PrP (106–126) for 12 h. The treated cells were assessed for calcineurin by Western blot analysis. (D) Calcineurin activity levels increased significantly at 12 h following PrP (106–126). Bar graph was generated using mean ± standard error of the mean (n = 3). *** p < 0.001, #p < 0.05, ### p < 0.001; significant differences when compared with control and each treatment group. PrP, PrP (106–126).
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Fig. 4. Inhibition of calcineurin activity induced PrP (106–126)mediated p-AMPK up-regulation and autophagy inactivation. (A) SK-N-SH cells were treated with 25, 50, and 100 μM of PrP (106–126) for 12 h. The treated cells were assessed for p-AMPK by Western blot analysis. (B) Immunocytochemistry for the calcineurin was analyzed from SK-N-SH cells. (C, D) SK-N-SH cells were treated with PrP (106–126) for 12 h. The treated cells were assessed for P62, LC3 by Western blot analysis. (E) SK-N-SH cells were treated with 25, 50, and 100 μM of PrP (106–126) for 12 h. The treated cells were assessed for P62 by Western blot analysis. (F) Immunocytochemistry for the P62 was analyzed from SK-N-SH cells. (G, H) SK-N-SH cells were treated with PrP (106–126) for 12 h. The treated cells were assessed for P62, LC3 by Western blot analysis.. (I) SK-N-SH cells were incubated with PrP (106–126) at 100 μM for 12 h and analyzed by TEM. Arrowheads indicate autophagosomes and arrows indicate autolysosomes.
the influence of prion peptide on autophagy in neuronal cells. We detected a dose-dependent decrease in the late autophagosome markers LC3-II and SQSTM1/p62 in the PrP-treated group compared with the control group in human neuroblastoma (Fig. 4E). The effect of calcineurin inhibitor on prion peptide-induced autophagy induction was also investigated. Red fluorescence, suggesting the presence of P62 was recovered by calcineurin inhibitor (Fig. 4F). Prion peptide-induced autophagy flux was abolished by calcineurin inhibitor and was confirmed by the up-regulation of SQSTM1/p62 protein and LC3-II (Fig. 4G). We further tested whether calcineurin inhibition via knockdown of gene levels also affected prion peptide-induced autophagy. Prion peptide-induced autophagy was inhibited by PPP3CB siRNA and was confirmed by the up-regulation of SQSTM1/p62 protein and LC3-II (Fig. 4H). We also conduct Transmission electron microscopy (TEM) analysis to certainly determine the effect of lysosomal inhibition on autophagy flux by calcineurin inhibitor. As shown in Fig. 4I, a lot of vesicles including double-membraned autophagosomes (arrowheads) were induced by treatment of cells with FK506, which indicated inhibition of lysosomal degradation (Fig. 4I). These results indicate the inhibition of calcineurin-induced p-AMPK up-regulation and autophagy inactivation.
3.4. Phospho-AMPK reduction induced autophagy activation by prion peptides in neuronal cells We investigated the role of AMPK and autophagy in PrP (106–126)induced neurotoxicity and protective effect mediated by AMPK activator and autophagy inhibitor. We also analyzed whether autophagy was inhibited by autophagy inhibitor chloroquine (CQ). The AMPK agonist, AICAR restored AMPK phosphorylation that was decreased by prion peptide. However, the effects of CQ were similar to those found in cells treated with PrP (106–126) group (Fig. 5A). Inhibition of prion peptide-induced autophagy flux by AICAR and CQ was confirmed by the up-regulation of SQSTM1/p62 protein and LC3-II (Fig. 5B). The effect of AMPK activator and autophagy inhibitor on PrP (106–126)induced neurotoxicity was investigated. The results showed that prion peptide induced neuron cell death and AICAR and CQ reduced neurotoxicity induced by prion peptide (Fig. 5C, D). Our study demonstrated that p-AMPK reduction induced autophagy activation by prion peptideinduced neurotoxicity, and treatment with AMPK activator and autophagy inhibitor may attenuate prion peptide–induced neuronal apoptosis.
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Fig. 5. Phospho-AMPK reduction induced autophagy activation by PrP (106–126) (A) SK-N-SH cells were treated with 25, 50, and 100 μM of PrP (106–126) for 12 h. The treated cells were assessed for p-AMPK by Western blot analysis. (B) SK-NSH cells were treated with PrP (106–126) for 12 h. The treated cells were assessed for P62, LC3 by Western blot analysis. (C) SK-N-SH cells were treated with PrP (106–126) for 12 h. Cell viability was measured by annexin V assay. Cells were treated with FITC-annexin V and PI, which binds to phosphatidylserine to the plasma membrane and nuclei during apoptosis. (D) Bar graph indicating the average number of annexin V negative cells. Bar graph was generated using mean ± standard error of the mean (n = 3). *** p < 0.001, # p < 0.05, ## p < 0.01; significant differences when compared with control and each treatment group. PrP, PrP (106–126).
3.5. Autophagy inhibitor and AMPK activator had no effect on prion peptide-induced calcineurin
4. Discussion The purpose of this study is to investigate the role of calcineurin on human prion protein-mediated AMPK/autophagy signals and neurotoxicity in neuron cells. The results showed that increased calcineurin activity mediated by prion protein induced neuronal cell damage and also provided an insight into the fundamental mechanism underlying the AMPK and autophagy flux in prion disease. Autophagy play an important role in cell survival and cell death in various pathological and physiological conditions (Fimia et al., 2007). Autophagy protects cells against death under conditions of starvation and neurodegeneration, but is also important contributing factor in some types of cell death (Dang et al., 2015). Autophagy has several pathways depending on the circumstances, and the biochemical basis for the diverse functions is not well understood (Cuervo, 2004; CzyzykKrzeska et al., 2012). We identified human prion peptide induces autophagy flux in neuronal cells (Moon et al., 2016). In this study, we found that human prion peptide induced autophagic cell death in neuronal cell. Autophagy inhibitor reduced prion protein induced autophagic cell death. AMP-activated protein kinase (AMPK) is a sensor of energy status including energy deficiency based on the increased AMP/ATP ratio (Poels et al., 2009). Calcineurin prolonged the oxidative stress-induced activation of calcineurin resulting in the attenuation of AMPK signaling and inhibition of autophagy in cardiomyocytes (He et al., 2014). In this study, the results showed that prion protein-mediated calcineurin activation decreased the AMPK activity via dephosphorylation and suggest that AMPK inactivation may lead to autophagy activation and neuronal toxicity. This study demonstrated that prion protein-calcineurin activation was involved not only in prion protein-mediated neuronal cell death but also AMPK and autophagy signaling pathways. It regulates metabolic homeostasis (Hardie, 2007) by controlling autophagy (Vingtdeux et al., 2011). However, our present study demonstrated that prion peptide decreased the AMPK activity via dephosphorylation but increases autophagic cell death (Fig. 5). The role of AMPK and autophagy flux in neurodegeneration is not clear so that further studies are required. Our results indicate that prion protein increases calcineurin activity, resulting in decreased AMPK phosphorylation that induces autophagic cell death. The current study defines a novel interaction between calcineurin and the AMPK/mTOR pathway in the control of autophagy. This study demonstrated that calcineurin plays a critical biologic role in the survival of neuronal cells exposed to prions. Inhibition of calcineurin and autophagy may represent a novel therapeutic approach with a broad clinical potential to prevent neurodegenerative diseases including prion disease.
We investigated whether autophagy induction and AMPK activator affected prion peptide-induced calcineurin. We demonstrated that autophagy inhibitor did not affect prion peptide-induced calcineurin (Fig. 6A). We found that AMPK activator also had no effect on PrP (106–126)-induced calcineurin (Fig. 6B). The activity of calcineurin was evaluated by measuring the formation of free phosphate (PO4 ), using a specific kit assay. The results show that calcineurin activity was significantly decreased by exposure to autophagy inhibitor and AMPK activator (Fig. 6C, D). These results indicate that autophagy inhibitor and AMPK activator may not affect calcineurin in prion peptide-induced neurotoxicity.
Fig. 6. Autophagy inhibitor and AMPK activator have no influence in prion peptide-induced calcineurin. (A) Immunocytochemistry for the P62 was analyzed from SK-N-SH cells (B) Immunocytochemistry for the P-AMPK was analyzed from SK-N-SH cells. (C, D) Calcineurin activity levels increased significantly at 12 h following PrP (106–126). Bar graph was generated using mean ± standard error of the mean (n = 3). *** p < 0.001, ## p < 0.01, ### p < 0.001; significant differences when compared with control and each treatment group. PrP, PrP (106–126). 6
International Journal of Biochemistry and Cell Biology 119 (2020) 105680
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Ethical approval
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Ethical approval for the project was granted by the institutional review board of the Chonbuk National University. Author contributions J.M.H., J.H.M. and S.Y.P. designed, executed the study, analyzed data and J.M.H. and J.H.M. wrote the manuscript. All authors have read and approved the final manuscript. CRediT authorship contribution statement Jeong-Min Hong: Conceptualization, Methodology, Data curation, Writing - original draft. Ji-Hong Moon: Visualization, Investigation, Supervision. Sang-Youel Park: Writing - review & editing. Declaration of Competing Interest The authors declare no conflicts of interest. Acknowledgments This study was supported by the National Research Foundation of the Korea Grant (NRF) funded by the Ministry of Education (2019R1A6A1A03033084). References Agostinho, P., Oliveira, C.R., 2003. Involvement of calcineurin in the neurotoxic effects induced by amyloid‐beta and prion peptides. Eur. J. Neurosci. 17 (6), 1189–1196. Aguzzi, A., Calella, A.M., 2009. Prions: protein aggregation and infectious diseases. Physiol. Rev. 89 (4), 1105–1152. Brown, D.R., 2000. Prion protein peptides: optimal toxicity and peptide blockade of toxicity. Mol. Cell. Neurosci. 15 (1), 66–78. Collinge, J., 2001. Prion diseases of humans and animals: their causes and molecular basis. Annu. Rev. Neurosci. 24 (1), 519–550. Cuervo, A.M., 2004. Autophagy: many paths to the same end. Mol. Cell. Biochem. 263 (12), 55–72. Czyzyk-Krzeska, M.F., Meller, J., Plas, D.R., 2012. Not all autophagy is equal. Autophagy 8 (7), 1155–1156. Dang, S., Yu, Z.-m., Zhang, C.-y., Zheng, J., Li, K.-l., Wu, Y., Qian, L.-l., Yang, Z.-y., Li, X.r., Zhang, Y., 2015. Autophagy promotes apoptosis of mesenchymal stem cells under inflammatory microenvironment. Stem Cell Res. Ther. 6 (1), 247. Ettaiche, M., Pichot, R., Vincent, J.-P., Chabry, J., 2000. In vivo cytotoxicity of the prion protein fragment 106–126. J. Biol. Chem. 275 (47), 36487–36490. Fernandez, A.M., Fernandez, S., Carrero, P., Garcia-Garcia, M., Torres-Aleman, I., 2007. Calcineurin in reactive astrocytes plays a key role in the interplay between proinflammatory and anti-inflammatory signals. J. Neurosci. 27 (33), 8745–8756. Fimia, G.M., Stoykova, A., Romagnoli, A., Giunta, L., Di Bartolomeo, S., Nardacci, R., Corazzari, M., Fuoco, C., Ucar, A., Schwartz, P., 2007. Ambra1 regulates autophagy and development of the nervous system. Nature 447 (7148), 1121.
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