Effects of Ghrelin on the Proteolytic Pathways of Alzheimer’s Disease Neuronal Cells Valentina Cecarini, Laura Bonfili, Massimiliano Cuccioloni, Jeffrey N. Keller, Annadora J. Bruce-Keller & Anna Maria Eleuteri Molecular Neurobiology ISSN 0893-7648 Mol Neurobiol DOI 10.1007/s12035-015-9227-x
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Author's personal copy Mol Neurobiol DOI 10.1007/s12035-015-9227-x
Effects of Ghrelin on the Proteolytic Pathways of Alzheimer’s Disease Neuronal Cells Valentina Cecarini 1 & Laura Bonfili 1 & Massimiliano Cuccioloni 1 & Jeffrey N. Keller 2 & Annadora J. Bruce-Keller 2 & Anna Maria Eleuteri 1
Received: 16 February 2015 / Accepted: 21 May 2015 # Springer Science+Business Media New York 2015
Abstract Ghrelin is an orexigenic hormone with a role in the onset and progression of neurodegenerative disorders. It has been recently associated to Alzheimer’s disease (AD) for its neuroprotective and anti-apoptotic activity. In the present study, we dissected the effect of ghrelin treatment on the two major intracellular proteolytic pathways, the ubiquitin-proteasome system (UPS) and autophagy, in cellular models of AD (namely SHSY5Y neuroblastoma cells stably transfected with either the wild-type AβPP gene or the 717 valine-to-glycine AβPP-mutated gene). Ghrelin showed a growthpromoting effect on neuronal cells inducing also timedependent modifications of the growth hormone secretagogue receptor type 1 (GHS-R1) expression. Interestingly, we demonstrated for the first time that ghrelin was able to activate the proteasome in neural cells playing also a role in the interplay between the UPS and autophagy. Our data provide a novel mechanism by which circulating hormones control neural homeostasis through the regulation of proteolytic pathways implicated in AD.
Keywords Ghrelin . Alzheimer’s disease . Proteasome . Autophagy
* Valentina Cecarini
[email protected] 1
School of Biosciences and Veterinary Medicine, University of Camerino, 62032 Camerino, Italy
2
Pennington Biomedical Research Center, Louisiana State University System, Baton Rouge, LA, USA
Abbreviations UPS Ubiquitin-proteasome system GHS-R1 Growth hormone secretagogue receptor type 1 GOAT Ghrelin O-acyltransferase Aβ Amyloid-β AβPP Amyloid-β precursor protein
Introduction Ghrelin is a multifunctional 28-amino acid hormone originally purified from the rat stomach [1]. It is mainly produced in the gastrointestinal tract but also identified in a wide variety of tissues including the brain, in particular in the hypothalamus and to a less extent in the cerebral cortex and hippocampus [2, 3]. The native, intact ghrelin molecule is known as desacyl-ghrelin (DAG) that, undergoing esterification of the serine-3 residue with n-octanoic acid, results in acyl-ghrelin (AG), the natural ligand of the growth hormone secretagogue receptor 1a isoform (GHS-R1a) [4–6]. This modification, mediated by the ghrelin O-acyltransferase (GOAT) enzyme, confers to the molecule an increased lipophilicity [5]. Levels of plasma ghrelin inversely correlate with nutritional status and body mass index: a rise is observed before a meal, during fasting, and conditions of cachexia and anorexia, whereas a decline after food ingestion and under conditions of obesity in rodents and humans [5]. Ghrelin was originally identified for its role in stimulating the GH release, but recently, the ghrelin system was associated to a large number of biological functions: cell proliferation, development of obesity and metabolic syndrome, inflammation, and neuromodulation [3, 7]. In particular, considering that ghrelin and its receptors are detected in regions involved in memory and learning and that the peptide is able to modulate neuronal activities and neurotransmitter release, it is reasonable to hypothesize for this peptide a role in the onset and progression of
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neurodegenerative disorders [4, 7, 8]. On this regard, an increasing number of studies links ghrelin with Alzheimer’s disease (AD) pathogenesis highlighting its neuroprotective and antiapoptotic activity [9]. AD is an age-related neurodegenerative disorder characterized by loss of weight, cognitive deficits, neuroinflammation, and neuronal cells death [10]. In brain tissues of AD subjects, vast areas with deposition of protein aggregates including neurofibrillary tangles of hyperphosphorylated tau protein and oligomeric and fibrillar amyloid-β (Aβ) protein can be detected [11]. Interestingly, plasma levels of ghrelin naturally decrease with age both in AD and in age-matched nondementia subjects [12]. Additionally, the ghrelin system is markedly altered in AD brain with a reduction of ghrelin, GOAT, and GHS-R1a mRNA levels in the temporal lobe of AD patients [13]. Exogenous ghrelin was shown to rescue memory deficits in intrahippocampal Aβ(1-42) oligomer-injected mice with a simultaneous reduction of microgliosis and neuronal loss and prevention of Aβ oligomer-associated synaptic degeneration [14]. Ghrelin can also protect cells from apoptosis that favors AD neuronal loss by inhibiting activation of caspase-3 and also targeting the Bcl-2 family [15]. Additionally, it also limits neuronal oxidative stress stabilizing mitochondria via reduction of ROS production, preservation of mitochondrial inner transmembrane potential, and prevention of cytochrome-c release [16]. Besides plaque deposition, neuronal apoptosis, and oxidative stress, AD presents impairments in the functionality of the two major cellular proteolytic pathways, the ubiquitinproteasome system (UPS) and autophagy [17–20]. The UPS is the major instrument cells use to remove short-lived and misfolded proteins and displays also a major role in regulating cell cycle progression, DNA repair, apoptosis, gene transcription, signal transduction, senescence, and immune response [21]. Autophagy is a stepwise intracellular process that mediates the recycling of dysfunctional organelles or aggregated proteins [22]. We recently demonstrated that the two proteolytic systems are intimately correlated both in AD and cancer, with the inhibition of one pathway stimulating the activity of the other [23–25]. We also showed that ghrelin was able to trigger the intrinsic apoptosis pathway in HCT116 cells via proteasome inhibition and concomitant autophagy induction [25]. Despite all the available data strongly suggesting a close connection between the ghrelin system and the onset and/or progression of AD, the definitive relationship is still under discussion. In the light of the neuroprotective role of ghrelin, in this work, we investigated its ability to modulate the interplay between the UPS and autophagy and their proteolytic activities in neuronal cells. In details, human SHSY5Y neuroblastoma cells stably transfected with either the wild-type AβPP gene (APPwt) or the 717 valine-toglycine AβPP-mutated gene (APPmut) and control untransfected cells were treated with ghrelin for 6 and 24 h, and the effects on the main components of both proteolytic systems were examined.
Materials and Methods Reagents and Chemicals Substrates for assaying the chymotrypsin-like (ChT-L), trypsin-like (T-L), peptidylglutamyl-peptide hydrolyzing (PGPH), and aminopeptidase-N (AP-N) activities were purchased from Sigma-Aldrich S.r.L. (Milano, Italy). The substrate for the branched chain amino acids preferring (BrAAP) activity was obtained from Biomatik (Cambridge, Ontario, Canada). Aminopeptidase-N (EC 3.4.11.2), used to detect BrAAP activity [26], was purified from pig kidney [27]. SH-SY5Y cells transfected with APPwt or APPmut gene were a kind gift from Prof. Uberti Daniela, University of Brescia, Italy. Membranes for Western blotting analyses were purchased from Millipore (Milano, Italy). Human ghrelin was purchased from AnaSpec (Fremont, CA, USA) reconstituted in water and stored at −20 °C. Cathepsin B substrate, Z-ArgArg-AMC, and inhibitor, CA074Me, were obtained from Sigma-Aldrich S.r.L. (Milano, Italy). Media and chemical used for cell cultures were purchased from Euroclone (Milano, Italy). Proteins on films were detected with the enhanced chemiluminescence (ECL) system (Amersham Pharmacia Biotech, Milano, Italy). The anti-p62 antibody was purchased from Sigma-Aldrich S.r.L. (Milano, Italy), and the anti-p27 antibody was obtained from Merck S.p.a. (Vimodrone, Italy). Other primary and secondary antibodies used in the present work were all purchased from Santa Cruz Biotechnology, Inc. (Heidelberg, Germany). Cell Cultures Cell transfection was performed as previously reported [23]. SH-SY5Y cells were cultured in 1:1 Dulbecco’s modified Eagle’s medium and Nutrient Mixture F12 containing 10 % fetal bovine serum (FBS), 2 mM glutamine, 100 units/mL penicillin, and 100 μg/mL streptomycin at 37 °C in a 5 % CO 2 -containing atmosphere. Stably transfected cells expressing either the APPwt or the APPmut construct were maintained in the same medium added with G418 at a final concentration of 800 μg/mL. Confluent cells were treated with 0.1 and 1 μM ghrelin for 6 and 24 h. Ghrelin was dissolved in water and then added to cell culture media. Control cells were included in each time point. After removing the medium and washing with cold phosphate buffered saline (PBS), cells were harvested in 4 mL of PBS and centrifuged at 1600×g for 5 min. The pellet was resuspended in a lysis buffer (20 mM Tris, pH 7.4, 250 mM sucrose, 1 mM EDTA, and 5 mM β-mercaptoethanol) and passed through a 29gauge needle at least ten times. Lysates were centrifuged at 12,000×g for 15 min and the supernatants were stored at −80 °C. Protein concentration was determined by the
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method of Bradford [28] using bovine serum albumin (BSA) as standard.
antibody (Santa Cruz Biotechnology, Heidelberg, Germany). The bands were quantified as reported elsewhere [30].
Cell Viability Assay
Cathepsin B Activity
Cell viability was evaluated using 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide assay (MTT) [29]. After ghrelin treatment, cells were washed in PBS, pH 7.5, and then MTT (final concentration 0.5 mg/mL) was added to the culture medium without FBS and incubated for 2 h at 37 °C. The medium was then removed and replaced with 100 μL of DMSO. The optical density was measured at 550 nm in a microtiter plate reader. At least six cultures were utilized for each time point.
Cathepsin B proteolytic activity was measured following the protocol described by Tchoupè et al. [31] using the fluorogenic peptide Z-Arg-Arg-AMC (final concentration of 50 μM). The mixture, containing 1 μg of protein lysate, was incubated in 100 mM phosphate buffer pH 6.0, 1 mM EDTA, and 2 mM dithiothreitol for 1 h at 30 °C. The fluorescence of the hydrolyzed AMC (λexc =365 nm, λem =449 nm) was recorded on a SpectraMax Gemini XPS microplate reader. The effective cathepsin contribution to the proteolysis was evaluated through control experiments performed using the specific inhibitor CA074Me. Fluorescence values obtained by analyzing the lysates were then subtracted of the values of control assays in the presence of the inhibitor.
Proteasome Activity The effects of ghrelin treatment on the proteasome system were evaluated through fluorimetric assays using the following synthetic substrates: Leu-Leu-Val-Tyr-AMC for ChT-L, Leu-Ser-Thr-Arg-AMC for T-L, Leu-Leu-Glu-AMC for PGPH, and Gly-Pro-Ala-Leu-Ala-AMC for BrAAP, whose test is performed with the addition of the aminopeptidase-N (AP-N). The incubation mixture contained 1 μg of cell lysate, the appropriate substrate, and 50 mM Tris–HCl pH 8.0, up to a final volume of 100 μL. Incubation was performed at 37 °C, and after 60 min, the fluorescence of the hydrolyzed 7-amino4-methyl-coumarin (AMC) was recorded (AMC, λexc = 365 nm, λem =449 nm) on a SpectraMax Gemini XPS microplate reader. The 26S proteasome ChT-L activity was tested using Suc-Leu-Leu-Val-Tyr-AMC as substrate and 50 mM Tris–HCl pH 8.0 buffer containing 10 mM MgCl2, 1 mM dithiothreitol, and 2 mM ATP. The effective 20S proteasome contribution to short peptide cleavage was evaluated with control experiments performed using specific proteasome inhibitors, Z-Gly-Pro-Phe-Leu-CHO and lactacystin (5 μM in the reaction mixture). The fluorescence values of lysates were subtracted of the values of control assays in the presence of the two inhibitors.
Caspase Activity Assay Caspase-3 and caspase-7 (DEVDase) activity assays were performed in cell lysates (5 μg of total proteins in the mixture) using the Ac-Asp-Glu-Val-Asp-AMC substrate (SigmaAldrich S.r.L., Milan, Italy) in 50 mM Tris–HCl, 50 mM NaCl, 5 mM CaCl2, 1 mM EDTA, 0.1 % CHAPS, 5 mM βmercaptoethanol, pH 7.5. The incubation was carried out at 37 °C for 60 min, and the hydrolysis product was detected (AMC: λexc =365 nm, λem =449 nm) on a SpectraMax Gemini XPS microplate reader. Statistical Analysis Results are expressed as mean values±standard deviation of results obtained from five separate experiments. Statistical analysis was performed with one-way ANOVA, followed by the Bonferroni test using Sigma-stat 3.1 software (SPSS, Chicago, IL, USA). p values
