Citrus junos Tanaka peel ameliorates hepatic lipid accumulation in HepG2 cells and in mice fed a high-cholesterol diet

Eun Ju Shin
Jae Ho Park
Mi Jeong Sung
Min-Yu Chung
Jin-Taek Hwang
Citrus junos Tanaka peel ameliorates hepatic lipid accumulation in HepG2 cells and in mice fed a high-cholesterol diet
10.1186/s12906-016-1460-yR E S E A R C H A R T I C L E Open Access
Background: Citrus junos Tanaka (yuja), a yellow-coloured citrus fruit has traditionally been consumed in Korea, Japan, and China and has been found effective in preventing certain diseases. However, the inhibitory effect of yuja on hepatic lipid accumulation has not been clearly elucidated thus far. Methods: The inhibitory effect of yuja on hepatic lipid accumulation was investigated in both cell culture and mouse models. We investigated the inhibitory effect of ethanol extract of yuja peel (YE) using HepG2 cells. We next confirmed the effect of YE in mice fed a high cholesterol diet. Animals were divided into 4 groups (n = 8): a normal diet group (ND), a high-cholesterol diet group (HC), high-cholesterol diet plus 1% YE (YL), high-cholesterol diet plus 5% YE (YH).Result: Seventy percent ethanolic extracts of yuja peel (YE) reduced oleic acid-induced hepatic lipid accumulation in HepG2 cells. Treatment with YE at 100, 200 μg/mL up-regulated expression levels of cholesterol metabolism-related proteins such as AMPK, ACC, PPAR-α, and CPT1 and down-regulated the expression of 3-hydroxy-3-methylglutaryl coenzyme A reductase. The hypocholesterolemic effect of YE was further confirmed in mice fed a high-cholesterol diet. Compared to ND (normal diet) mice, HC (high-cholesterol diet) mice showed increased body weight, liver fat content, liver weight, and content of total cholesterol and low-density lipoprotein (LDL) cholesterol. On the contrary, administrations of YL (HC + 1% YE) or YH (HC + 5% YE) significantly reduced body weight, liver fat content, liver weight, total cholesterol, and LDL cholesterol compared to those of only HC fed mice group. As a result of in vitro data, protein expressions of PPAR-α and CPT1 were induced in mice fed YE diet compared to HC diet but HMGCR expression was decreased.Conclusions: Yuja peel ameliorates hepatic lipid accumulation in both cell culture and mouse models and therefore, could serve as a useful supplement for hypercholesterolemia.

Background

Lifestyle-related diseases including hepatic lipid accumulation are associated with irregular lifestyle and diet. Hepatic lipid accumulation is accompanied by excessive accumulation of cholesterol and triglycerides, thereby causing atherosclerosis, which affects the lipid-laden blood vessels, or hepatic steatosis, or both. It is well established that lowdensity lipoproteins (LDLs) play a central role in the development of atherosclerosis. LDL promotes formation of fatty streaks, a key event in early atherosclerosis and induces the uptake of oxidized LDL by macrophages and smooth muscle cells. A negative correlation has been demonstrated between LDL and high-density lipoproteins (HDLs). High level of HDL can inhibit LDL oxidation by various mechanisms [1]. Several studies have shown that natural ingredients are effective in preventing hypercholesterolemia by suppressing LDL oxidation [2]. For example, cocoa polyphenols inhibit LDL oxidation, thereby suppressing the formation of atherosclerosis. The intake of dairy cocoa powder was shown to reduce LDL oxidation in humans [2]. It was also reported that vitamin E supplementation inhibits LDL accumulation in arterial diseases. A clinical study reported that almonds significantly reduced total cholesterol (range 8-12%) and LDL cholesterol (range 9-15%) [3]. Therefore, reduction of total cholesterol and LDL cholesterol levels may be a significant strategy for preventing atherosclerosis.

Another mechanism of preventing hypercholesterolemia includes targeting enzymes including AMPK and 3hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR). AMPK is a metabolic protein, which plays a central role in lipid metabolism and inhibits anabolic pathways, including cholesterol synthesis by targeting HMGCR [4]. HMGCR is a rate-limiting enzyme in cholesterol synthesis pathway; it is suppressed by cholesterol derived from the degradation of LDL via the LDL receptor [5]. HMGCR is inhibited by statins, which upregulate the expression of LDL receptors in the liver, resulting in increased catabolism of plasma LDL and decreased plasma cholesterol [5]. Thus, AMPK and HMGCR enzymes are the targets of various cholesterollowering natural ingredients.

Citrus junos Tanaka, also known as yuja is a yellowcoloured citrus fruit that has been reported to exhibit beneficial health effects against oxidative stress and inflammation [6,7]. In our previously paper, several active compounds found in yuja exist as rutin, quercetin, tangeretin, naringin and hesperidin [6]. In addition, our studies have also reported the beneficial health effects of yuja, such as potent antidiabetic, anticancer, and antiinflammatory effects, both in in vitro and in vivo [6,7]. The present study investigated the effects of yuja on hepatic lipid accumulation. A 70% ethanolic extract of yuja peel was examined for its effects on oleic acidinduced hepatic lipid accumulation, AMPK activation, and HMGCR expression in HepG2 cells. Further, the same ethanolic extract was evaluated in mice fed a highcholesterol diet, because mice fed high-cholesterol diet is widely used to study hepatic lipid metabolism.

Methods

Reagents

Preparation of Yuja extracts

Yuja was provided by Goheung Country Office (Goheung, Republic of Korea), where a voucher specimen was deposited. Yuja peel extracts (YE) were prepared as described previously [7]. Briefly, yuja peels were dissolved in a 10-fold volume of 70% ethanol by shaking for 24 h at 25°C, and precipitates were removed by centrifugation at 8000xg for 30 min. Supernatants were dried using freeze dryer. Yuja extract was dissolved in DMSO and used to treat HepG2 cells and mice fed high fat diet. The cells were incubated with 1% BSA-supplemented low-glucose DMEM (None), 0.5 mM oleic acid (OA) in 1% BSA-supplemented lowglucose DMEM, and 0.5 mM OA in DMEM with 50, 100, and 200 μg/mL YE as a treatment group for 24 h.

Cell culture and sample treatment

HepG2 cells were purchased from the American Type Culture Collection (Mannassas, VA, USA). These cells were grown in high-glucose Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and antibiotics, which were purchased from Welgene Inc. (Daegu, Republic of Korea). The cells were maintained at 37°C in a humidified atmosphere under 5% CO 2 . When processing the sample on the cells, cells were treated with 1% BSA in low-glucose DMEM (None), 0.5 mM oleic acid (OA) in 1% BSA-supplemented lowglucose DMEM, and 0.5 mM OA in DMEM with 50, 100, and 200 μg/mL YE as a treatment group for 24 h.

Cytotoxicity (MTT assay)

HepG2 cells were cultured in 24-well plates and treated with YE at the indicated concentrations. Then, 10 μL of MTT solution (5 mg/mL in PBS) was added and incubated for 3 h. After removing the medium, the cells were dissolved in dimethylsulfoxide (DMSO), and 100 μL of supernatant was transferred to 96-well plates. Absorbance was measured at 540 nm using microplate reader (Molecular Device Co., Sunnyvale, CA, USA).

Oil Red O staining

HepG2 cells were cultured in 24-well plates and then treated with YE for 24 h. After treatment, they were stained with Oil Red O to measure lipid droplet accumulation, washed with 200 μL of PBS, and fixed with 200 μL of 4% formaldehyde for 15 min, at room temperature (RT). The cells were then washed three times with PBS, incubated with 200 μL of 60% isopropanol for 5 min, and then stained with 200 μL of 0.1% Oil Red O staining solution for 60 min at RT. The cells were further washed 3 times with 1 mL of water. The images of these cells were captured by microscopy (Olympus, Tokyo, Japan). To measure lipid accumulation, the cells were dissolved in isopropanol for 10 min. The dissolved dye was transferred to 96-well plates and the absorbance was measured at 510 nm.

Western blot assay

The proteins were harvested in RIPA buffer (Elpis, Daejeon, Republic of Korea) containing a protease inhibitor and a phosphatase inhibitor (Roche, Basel, Switzerland). After this treatment, the protein was quantified using Bicinchoninic Acid (BCA) methods. The protein samples (20 μg) were loaded onto 10% Bis-Tris gel and transferred to nitrocellulose membranes. The membrane was blocked using 5% skim milk solution for 1 h and probed with each antibody at 4°C overnight. After triplicate washing with PBS, the membrane was incubated with horseradish peroxidaseconjugated secondary antibodies for 1 h. It was washed again with PBS and the protein expression was detected by chemiluminescence methods.

Animal fed a high-cholesterol diet

Male C57BL/6 J mice (3 weeks old) were housed at the Korea Food Research Institute (KFRI) in a climatecontrolled environment (24°C at 50% relative humidity) with a 12 h light/dark cycle. After 1 week of adaptation, the mice were randomly divided into four groups (n = 8): N (Normal diet), HC (high-cholesterol diet), YL (HC + 1% YE), and YH (HC + 5% YE). D12336 diet (high-cholesterol diet, Purified diet to match Paigen's atherogenic rodent diet, Research Diets, Inc.) was used as HC. The composition of the high cholesterol diet was as follow: 46% carbohydrate, 16% fat, 21% protein, 12.5% cholesterol, 0.5% cholic acid, mineral mixture and vitamin mixture (source of carbohydrate = maltodextrin, sucrose and corn starch; source of fat = soybean oil, cocoa butter and coconut oil; source of protein = casein, soy protein and DL-methionine, source of mineral mixture = calcium carbonate, sodium chloride, potassium citrate; vitamin mixture = choline bitartrate). The mice were fed these diets for 10 weeks with free access to autoclaved tap water in cages. The total daily intake and weight of mice were recorded every week for up to 10 weeks. At the end of the experiment, animals were sacrificed to collect their blood and liver tissue samples, which were then stored at −70°C. Our experimental protocol was approved by the Institutional Animal Care and Use Committee of the Korea Food Research Institute.

Hematoxylin and eosin (H&E) staining

Liver tissues were fixed using 4% formaldehyde solution and cut into 4-μm-thick portions, which were stained with hematoxylin and eosin, and images were captured using a microscope (Olympus, Tokyo, Japan).

Measurement of enzyme and lipid change in blood

The collected blood samples were centrifuged at 12000 rpm for 15 min at 4°C. After centrifugation, the serum was transferred to a new centrifuge tube and then stored at −70°C. Blood serum levels of TG, TC, HDL-cholesterol, GOT, GPT, and ALT were analysed according to the manufacturer's protocol.

Statistical analysis

Results obtained from at least three independent in vitro studies were expressed as mean ± standard deviation (SD). The results were determined by nonparametric methods using the SPSS computer-based statistics programs (Ver. 20; SPSS Inc., Chicago, IL, USA). In case of in vivo studies using mice, the results are expressed as mean ± standard error of mean (SE). Statistical differences between mean values were evaluated by one-way analysis of variance (ANOVA) followed by Bonferroni test. A P value of <0.05 was considered statistically significant.

Results

Inhibition of OA-induced hepatic lipid accumulation by YE without toxicity in HepG2 cells

To determine the inhibitory effect of YE on hepatic lipid accumulation, lipid accumulation was evaluated using Oil red O staining of HepG2 cells. As shown in Fig. 1a and b, treatment with 0.5 mM OA significantly induced lipid accumulation by about 16% compared to control (untreated HepG2 cells). Co-treatment with 0.5 mM OA and YE (100, 200 and 400 μg/ml) significantly inhibited up to 23% OAinduced lipid accumulation in a dose-dependent manner. Under similar conditions, the effect of YE on cell viability was also determined by MTT assay. There were no significant differences in cell viability, indicating that the tested concentrations of YE did not affect cell viability (Fig. 1c). Similar results were obtained for treatment with only YE (Fig. 1d). These results suggest that YE has preventive effects against OA-induced hepatic lipid accumulation without cell toxicity.

Prevention of hepatic lipid accumulation by YE via modulation of AMPK signalling HMGCR, PPAR-α, and CPT-1 in HepG2 cells AMPK is a potential modulator of metabolism of lipid accumulations via up-regulation of fatty acid oxidation and down-regulation of lipogenesis, glucose production, and protein synthesis [4]. In view of this information, the effect of YE on AMPK signalling was investigated. HepG2 cells were incubated with OA, as well with OA and YE for 1 h. As shown in Fig. 2a, YE stimulated the phosphorylation (Thr172) of AMPK and its major downstream targeting enzyme, ACC. These results indicated that the AMPK signalling pathway is the potential target of YE, thereby preventing hepatic lipid accumulation in HepG2 cells. To investigate the specific downstream of AMPK activation, the effects of YE on the expression of lipid metabolism-related proteins were evaluated, such as HMGCR, PPAR-α, and CPT1, which are known for fatty acid β-oxidation [8,9]. Treatment with OA alone did not affect HMGCR, PPAR-α, and CPT1 expression. However, co-treatment with OA and YE reduced HMGCR expression. In contrast, co-treatment with OA and YE induced PPAR-α and CPT1 expression (Fig. 2b). These results indicated that YE inhibited OA-induced hepatic lipid accumulation via AMPK signalling, in particular, by stimulating PPAR-α and CPT1, β-oxidation-related proteins and reducing HMGCR proteins involved in lipogenesis.

Inhibition of increases in body weight and liver weight by YE in HC-fed mice

To further examine the hypocholesterolaemic effects of YE, studies were carried out using HC-fed mice. High cholesterol diet caused fatty liver in this mice model. While the food intake in normal group is higher than in HC group (Fig. 3b), As Fig. 3a, the body weight between the normal and HC groups did not differ significantly (28.4 ± 0.9 vs. 28.3 ± 0.7 g, normal vs. HC group). YL (HC + 1% YE) and YH (HC + 5% YE) group mice had significantly lower body weights than normal or HC group mice (26.2 ± 0.3, 25.1 ± 0.4, respectively, vs. 28.3 ± 0.7 g, YL, YH vs. HC group). In addition, YL and YH group showed inhibited fat droplet accumulation compared with HC group in the liver (Fig. 3c). And YL group mice showed reduced liver weight compared to HC group mice (Fig. 3d). These result suggested that administration of YE protected the mice from HC-induced fatty liver.

Attenuation of serum biomarkers increased HC by YE in mice

To examine the effects of YE on serum biomarker, the serum levels of GPT, GOT, and ALP were evaluated. The levels of GPT, GOT, and ALT, which are indicative of liver activity, increased in the HC group. In contrast, the levels of GPT, GOT and ALT significantly decreased in the YL Fig. 1 Effects of 70% ethanolic extracts of yuja peel (YE) on hepatic lipid accumulation in and viability of HepG2 cells. HepG2cells were treated for 24 h with YE in the presence or absence of oleic acid (OA), and then lipid accumulation was measured by Oil- Red O staining (a, b). Cytotoxicity was measured by the MTT assay (c, d). All results are expressed as the mean ± SD. * p < 0.05 vs. control (None); # p < 0.05 vs. OA and YH groups (Fig. 4a, b, c). Next, we analysed serum cholesterol, HDL, and LDL levels. Along with hepatic lipid accumulation, cholesterol and LDL levels of HC group dramatically enhanced compared with normal diet group. Supplementation with YL and YH attenuated the HCinduced blood levels of TC and LDL, with little effect on HDL cholesterol levels (Fig. 4d, e, f). These results indicated that YE exerts hypocholesterolaemic effects against HC-induced lipid accumulation by improving the blood lipid levels and liver activity.

Inhibition of FAS, HMGCR and induction of PPAR-α hepatic protein expressions by YE in mice

We further examined protein expressions related to lipid metabolism in liver tissue. Consistent with the vitro protein results in HepG2 cells, administration of HC with YL and YH groups inhibited HMGCR and FAS protein expressions compared to HC group (Fig. 5a, b, c). On the other hands, only administration of YL increased expressions of PPARα which are involved in β-oxidation (Fig. 5a, d), but not in YH groups.

Taken together, both in vitro and in vivo data showed that YE attenuated hepatic lipid accumulation by multiple mechanisms such as fatty acid oxidation, lipogenesis.

Discussion

In the present study, the hypocholesterolemic effect of YE was evaluated in a cell culture system and in mice fed an HC. It was also demonstrated that YE reduces hepatic lipid accumulation, HMGCR expression, and induces the phosphorylation of AMPK without cytotoxicity in HepG2 cells. Furthermore, 1% YE (YL) or 5% YE (YH) reduces the body weight, liver fat content, liver weight, total cholesterol, and LDL cholesterol in mice fed an HC.

HepG2 cells have been proposed to be a valuable model for screening of hypocholesterolemic agents [10]. To determine the safety of plant extracts, a cytotoxicity assay was performed before evaluating physiological activities in vivo studies. Therefore, the cytotoxicity of YE in HepG2 cells was evaluated using the MTT assay. No significant cytotoxicity was observed (Fig. 1). Treatment with YE was also shown to reduce OA-stimulated hepatic lipid accumulation in HepG2 cells. In addition, treatment with YE induces protein expressions of AMPK, PPAR-α, CPT1 and inhibits HMGCR expression in OA-mediated HepG2 cells. Lovastatin and simvastin have been used as inhibitors of HMGCR, a major target for cholesterol synthesis [11]. HMGCR catalyses the conversion of HMG-CoA to mevalonic acid, which is a rate-limiting step in cholesterol biosynthesis [5,11]. Therefore, YE may inhibit the conversion of HMG-CoA to mevalonic acid by HMGCR without any cytotoxicity. In addition, AMPK was phosphorylated by YE in HepG2 cells. AMPK is an upstream regulator of HMGCR and many investigators have focused on the role of AMPK for the development hypocholesterolemic compounds [4]. For example, several plant extracts and ingredients have been reported to be effective in inhibiting HMGCR and AMPK, such as caffeine, berberine, and Artemisia scoparia extract [12][13][14]. This is in agreement with our result that the hypocholesterolemic effect of YE is modulated via the AMPK/HMGCR pathways.

Based on the above results from HepG2 cell culture, an animal study was conducted using mice fed an HC. 1% YE (YL) or 5% YE (YH) ameliorates hypercholesterolemia by decreasing weight gain, liver fat content, liver weight, total cholesterol, and LDL cholesterol content. Liver weight and ALT values of YL groups was more effective than that of YH group. On the other hand, the overall results of YH groups were better than that of YL group. Several recent studies demonstrated that consumption of plant extracts decreases the level of LDL, an important factor for cardiovascular diseases. Increased level of cholesterol results in atherosclerosis due to the increased lipid peroxidation [14][15][16]. Thus, LDL peroxidation is an important target for the prevention of atherosclerosis and can be prevented by antioxidants. Owing to minimal side effects, natural products are reported as an alternative for the prevention of atherosclerosis. Several plants have antioxidative properties. In previous studies, Biebersteinia multifida DC (Geraniaceae) was shown to have an antioxidant effect and significantly decreased the level of LDL [17]. The antioxidant ability of this plant possibly facilitates its hypocholesterolemic effect. Our previous study has shown that yuja peel has antidiabetic activity and has several phenolic compounds, including rutin, quercetin, tangeretin, naringin, and hesperidin [6,7]. Most of these compounds have excellent anti-oxidant abilities. Citrus flavonoids have been reported to have the hypocholesterolemic effects [18]. Our previous study showed that tangeretin could reduce the levels of circulating lipid mediators, including total cholesterol, in obese mice [19]. In the present study, YE reduced weight gain, liver fat content, liver weight, total cholesterol, and LDL cholesterol content in mice fed an HC. Besides, YE also inhibited HMGCR, FAS and stimulated PPARα expressions in liver Fig. 3 Effects of 70% ethanolic extracts of yuja peel (YE) on body weight and liver weight in high-cholesterol diet (HC)-fed mice. Experimental mice were fed normal diet, HC, and HC supplemented with 1% (YL) or 5% (YH) YE for 10 weeks. Body weight (a), food intake (b), liver fat accumulation (c), and liver weight (d) were measured, as described in materials and methods. Results are expressed as mean ± SE. * p < 0.05 vs. HC tissue compared to HC-fed groups like in vitro results. These results indicate that YE have the potential to improve hypercholesterolemia, which could be attributed to the antioxidant effect of polyphenol compounds. These compounds have an aromatic-ring structure with various substitutions [20]. The 3-hydroxyl group on the aromatic ring is a hydrogen donor and has an antioxidant effect [20]. Therefore, it is speculated that the 3-hydroxyl group of flavonoids that are abundant in YE is important for the hypocholesterolemic effect. Fig. 4 Effects of 70% ethanolic extracts of yuja peel (YE) on biochemical parameters related to cholesterol metabolism. Experimental mice were fed normal diet, high-cholesterol-diet (HC), and HC supplemented with 1% (YL) or 5% (YH) YE for 10 weeks. Serum levels of (a); glutamic pyruvic transaminase (GPT), (b); glutamic oxaloacetic transaminase (GOT), (c); alanine phosphatase (ALP-S), (d); total cholesterol (TC), (e); low-density lipoprotein (LDL), (f); and high-density lipoprotein (HDL) were measured, as described in materials and methods. Results are expressed as mean ± SE (n = 8). * p < 0.05 vs. ND; # p < 0.05 vs. HC

Conclusions

In this study, we demonstrate the hypocholesterolemic effect of 70% ethanolic extract of yuja peel in HepG2 cells. A 70% ethanolic extract of yuja peel also reduced weight gain, liver fat content, liver weight, total cholesterol, and LDL cholesterol content. Therefore Citrus junos Tanaka (yuja) may be useful for preventing atherosclerosis. Experimental mice were fed normal diet, high-cholesterol-diet (HC), and HC supplemented with 1% (YL) or 5% (YH) YE for 10 weeks. Three livers were randomly chosen in each group. Hepatic protein expressions of HMGCR, FAS, PPARα and actin were determined by immunoblotting (a). And relative densities of each bands were quantified (b, c, d). Results are expressed as mean ± SD. # p < 0.05 vs. HC

bromide) and Oil Red O were purchased from Sigma-Aldrich (St. Louis, MO, USA). Triglyceride (TG), Total Cholesterol (TC), Glutamic Oxaloacetic Transaminase (GOT), Glutamic Pyruvic Transaminase (GPT), and Alkaline Phosphatase (ALP) kits were purchased from Asan Pharmaceutical company (Seoul, Republic of Korea). Fatty Acid Synthase (FAS), Carnitine Palmitoyl Transferase-1 (CPT-1), and Peroxisome Proliferator-Activated Receptor-α (PPAR-α) antibodies were purchased from santacruz Biotechnology Inc. (Santa Cruz, CA, USA), 3-Hydroxy-3-Methylglutaryl-Coenzyme A Reductase (HMGCR) was purchased from Cell Signaling (Beverly, MA, USA). β-Actin was purchased from Bethyl Laboratories (Montgomery, TX, USA).

Fig. 2
2Effects of 70% ethanolic extracts of yuja peel (YE) on AMPK signalling and expression of proteins associated with cholesterol metabolism. HepG2cells were treated for 1 h with YE in the presence or absence of oleic acid, and then western blot analysis was performed with phospho-specific ACC, AMPK, normal AMPK, and beta-actin antibodies (a). HepG2cells were treated for 24 h with YE in the presence or absence of oleic acid, followed by western blot analysis with 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR), peroxisome proliferator-activated receptor (PPAR)-α, carnitine palmitoyltransferase (CPT)-1, and beta-actin antibodies (b)

Fig. 5
5Effects of 70% ethanolic extracts of yuja peel (YE) on protein expressions associated with cholesterol metabolism in liver tissue.
AcknowledgementsThis research was supported by grants from the Yuja Cluster Agency, Goheung County Office and the Korea Food Research Institute, Republic of KoreaAvailability of data and materialsThe datasets supporting the conclusions of this article are included within the article.Authors' contributions EJS and J-TH designed the study, performed the research, and prepared the manuscript; JHP, MJS, M-YC were involved in the experiments. All authors read and approved the final manuscript.Competing interestsThe authors declare that they have no competing interests.Consent for publicationNot applicable. Submit your next manuscript to BioMed Central and we will help you at every step:
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Juan M Maler
Henrik Zetterberg
Kaj Blennow
Piotr Lewczuk
Characterization of the postsynaptic protein neurogranin in paired cerebrospinal fluid and plasma samples from Alzheimer's disease patients and healthy controls
10.1186/s13195-015-0124-3R E S E A R C H Open Access
Introduction: Synaptic dysfunction and degeneration are central events in Alzheimer's disease (AD) pathophysiology that are thought to occur early in disease progression. Synaptic pathology may be studied by examining protein biomarkers specific for different synaptic elements. We recently showed that the dendritic protein neurogranin (Ng), including the endogenous Ng peptide 48 to 76 (Ng 48-76 ), is markedly increased in cerebrospinal fluid (CSF) in AD and that Ng 48-76 is the dominant peptide in human brain tissue. The aim of this study was to characterize Ng in plasma and CSF using mass spectrometry and to investigate the performance of plasma Ng as an AD biomarker.Methods: Paired plasma and CSF samples from patients with AD (n = 25) and healthy controls (n = 20) were analyzed in parallel using an immunoassay developed in-house on the Meso Scale Discovery platform and hybrid immunoaffinity-mass spectrometry (HI-MS). A second plasma material from patients with AD (n = 13) and healthy controls (n = 17) was also analyzed with HI-MS. High-resolution mass spectrometry was used for identification of endogenous plasma Ng peptides.Results: Ng in human plasma is present as several endogenous peptides. Of the 16 endogenous Ng peptides identified, seven were unique for plasma and not detectable in CSF. However, Ng 48-76 was not present in plasma. CSF Ng was significantly increased in AD compared with controls (P < 0.0001), whereas the plasma Ng levels were similar between the groups in both studies. Plasma and CSF Ng levels showed no correlation. CSF Ng was stable during storage at −20°C for up to 2 days, and no de novo generation of peptides were detected. Conclusions: For the first time, to our knowledge, we have identified several endogenous Ng peptides in human plasma. In agreement with previous studies, we show that CSF Ng is significantly increased in AD as compared with healthy controls. The origin of Ng in plasma and its possible use as a biomarker need to be further investigated. The results suggest that CSF Ng, in particular Ng 48-76 , might reflect the neurodegenerative processes within the brain, indicating a role for Ng as a potential novel clinical biomarker for synaptic function in AD.

Introduction

Alzheimer's disease (AD) is a neurodegenerative disease characterized by neuropathological changes in the brain, including extracellular amyloid-β (Aβ) deposits called plaques as well as neurofibrillary tangles consisting of hyperphosphorylated tau protein (p-tau) [1,2]. Lower cerebrospinal fluid (CSF) levels of the 42-amino acid-long Aβ peptide Aβ  , reflecting the plaque pathology, together with increased concentrations of total tau (t-tau) and p-tau, reflecting neurodegeneration and tangle pathology, respectively, are today considered the core CSF biomarkers for AD [3].

A central event in AD pathology is synaptic dysfunction and degeneration [4]. Synaptic loss correlates with cognitive decline and is more associated with the degree of dementia compared to plaques and tangles [5][6][7][8], especially within certain areas of the brain, such as the hippocampus [7,9]. Furthermore, synaptic loss has been identified as an early event in the disease progression, as well as the underlying cause of the progressive cognitive deterioration as the disease advances [4]. Thus, synaptic markers are promising tools for early AD diagnosis. Moreover, such biomarkers could also be used to monitor disease progression and play an important role in the evaluation of novel disease modifying therapeutics.

Neurogranin (Ng) is a postsynaptic protein with a critical role in long-term potentiation, where it regulates the levels of calmodulin in response to intracellular calcium levels after neuronal excitation [10][11][12]. In the brain, Ng is localized to dendritic spines of neurons within associative cortical areas, including the hippocampus, amygdala and cerebral cortex [11,13]. Studies on rodents have shown that both the Ng mRNA and total protein levels are age-dependently decreased in several regions of the brain, including the hippocampus [14]. In addition, in situ hybridization studies on the human brain have shown that the Ng mRNA is selectively translocated to dendrites and that such translocation is impaired in the cortex of patients with AD [15].

Recently, we generated three monoclonal anti-Ng antibodies with epitopes near the C-terminal part of the protein [16]. By immunoprecipitation using the antibodies Ng2 and Ng3, followed by high-resolution mass spectrometry (MS), we identified several endogenous Ng peptides in both brain tissue and CSF. A third monoclonal antibody, Ng7, was used as a capturing antibody in an enzyme-linked immunosorbent assay (ELISA) developed in-house for measuring CSF Ng. In three independent cohorts, we showed increased CSF Ng levels in AD CSF as compared with healthy controls. Using hybrid finity MS (HI-MS), we also showed that the endogenous Ng peptide 48 to 76 (Ng 48-76 ) was significantly increased in AD CSF as compared with controls [16].

If a synaptic marker reflecting the ongoing synaptic degeneration in the central nervous system (CNS) could be identified in blood or plasma, it would be a very valuable clinical tool because CSF sampling is considered to be a somewhat more invasive procedure. In addition, simple and repeated sampling would also be made possible, allowing for dynamic changes to be measured over time at a regular clinic. Therefore, in this study of paired plasma and CSF samples, we aimed to first characterize Ng in plasma using MS and then investigate whether plasma Ng is a marker of synaptic degeneration in AD.

Using MS, we show-for the first time, to our knowledge-that Ng is expressed as a variety of endogenous Ng peptides in human plasma. Although plasma Ng was not found to differ between AD patients and healthy controls, we replicated the previous finding of increased CSF Ng in the AD group using an in-house assay developed on the Meso Scale Discovery (MSD) platform. Finally, we also show that CSF Ng is stable during storage and that no peptides are generated de novo. The endogenous peptide Ng 48-76 , which in our previous study was found to be significantly increased in both patients with AD and individuals with prodromal AD, was not found in plasma. The findings presented in the present study imply that CSF Ng might reflect the ongoing neurodegenerative processes within the CNS, indicating a role for Ng, and in particular Ng 48-76 , as a potential novel clinical biomarker for synaptic function in AD.

Methods

Anonymized plasma samples collected in the routine workflow at the Neurochemistry Laboratory at Sahlgrenska University Hospital, Mölndal, Sweden, were used for method development, as approved by the ethics committee at the University of Gothenburg.

Patients in paired cerebrospinal fluid and plasma study

The paired CSF and plasma study included 25 AD patients (mean age: 76 ± 8 years) and 20 control subjects without dementia (mean age: 54 ± 14 years) (see Table 1 for demographics and biomarker characteristics). The AD group consisted of patients with probable AD with a high level of evidence of an AD pathophysiologic process and patients with mild cognitive impairment with a high likelihood of an underlying AD process. In the rest of this article, this group is called AD. The patients were recruited in Erlangen, Germany, and each underwent a thorough clinical investigation, including a medical history as well as a physical, neurological and psychiatric examinations. The diagnosis of AD was made according to the National Institute of Neurological and Communicative Disorders and Stroke-Alzheimer's Disease and Related Disorders association (NINCDS-ADRDA) criteria [17]. No patient with AD had a family history of dementia suggestive of autosomal dominant AD. The control group consisted of patients with other neuropsychiatric conditions without dementia. The clinical diagnoses (including neuroimaging and neuropsychology) were combined with the results of the following CSF biomarkers: Aβ 1-42 , Aβ 1-40 (to calculate the Aβ 42/40 concentration ratio), t-tau and p-tau181. The subjects were included in the study only if the clinical, neuroimaging and neuropsychological diagnoses were in accordance with the neurochemical findings as described elsewhere [18]. There were no patients included in the study with a completely negative AD biomarker pattern. Control subjects had all biomarkers within normal ranges. CSF t-tau, p-tau, Aβ 1-40 and Aβ 1-42 concentrations were determined using established ELISA methods as described elsewhere [18]. All analyses were performed at the Universitätsklinikum Erlangen. The study was conducted according to the provisions of the Declaration of Helsinki. All subjects gave their written informed consent for the use of their clinical data for research purposes, as approved by the ethics committee of the Universitätsklinikum Erlangen.

Patients and plasma samples included in the verification study

Plasma samples were collected from 13 patients with AD (mean age: 78 ± 7 years) and 17 individuals without dementia (controls; mean age: 58 ± 9 years) at the Memory Clinic, Skåne University Hospital, Sweden (see Table 1 for full biomarker characteristics and demographics). The individuals underwent brain imaging, routine laboratory testing and neurological, psychiatric and cognitive examinations. Patients diagnosed with AD fulfilled the dementia criteria of the Diagnostic and Statistical Manual of Mental Disorders, Third Edition, Revision [19], and the criteria for probable AD as defined by NINCDS-ADRDA [17]. The control individuals without dementia experienced subjective cognitive symptoms at baseline, but thorough clinical investigation as well as clinical follow-up revealed that the patients were not affected by a dementia disorder or a neurological disease. CSF t-tau, p-tau and Aβ 1-42 concentrations were determined using established ELISA methods as described elsewhere [16]. All analyses were performed at the Clinical Neurochemistry Laboratory at the Sahlgrenska University Hospital, Mölndal, Sweden. All individuals gave their informed consent to participate in research before their samples were stored in a biobank. A passive consent procedure was then used whereby consent for retrospective use of banked samples and basic data was assumed if individuals did not actively retract permission, as instructed in local press advertisements. This study procedure was approved by the local ethics committee at Lund University, Sweden.

Meso Scale Discovery neurogranin assay

The monoclonal mouse antibody Ng7, with an epitope including amino acids 52 to 65 [16], was used as a capturing antibody and was coated on QUICKPLEX 96-well plates (Meso Scale Discovery (MSD), Rockville, MD, USA) at a final concentration of 2.0 μg/ml (40 μl/well) in phosphate-buffered saline (PBS), followed by incubation overnight at room temperature (RT). Before being coated with the capturing antibody, the plates were washed once with 150 μl of PBS. After the plates were washed with 300 μl of PBS containing 0.05% Tween 20 (PBS-Tween) four times for 30 seconds each time, the remaining protein-binding sites were blocked with 5% MSD Blocker A solution (150 μl/well; Meso Scale Discovery) for 1 hour at RT and subsequently washed again with 300 μl of PBS-Tween four times for 30 seconds each time. Then the full-length Ng calibrators with concentrations ranging between 31.3 and 4,000 pg/ml (1:2 dilution; CSF samples) or 31.3 to 23,000 pg/ml (1:3 dilution; plasma samples), blanks and samples (50 μl/well) were incubated in duplicate together with the detecting antibody, polyclonal Ng anti-rabbit antibody (ab23570; Upstate Biotechnology, Lake Placid, NY, USA) diluted 1:20,000 in 0.1% bovine serum albumin (BSA) in PBS-Tween overnight at RT (50 μl/well). Plasma samples were diluted 1:40 in PBS. After washing four times for 30 seconds each time in PBS-Tween, a MSD SULFO-TAG goat anti-rabbit antibody (25 μl/well; Meso Scale Discovery), concentration 0.5 μg/ml diluted in 0.1% BSA in PBS-Tween, was added and incubated for 2 hours at RT while being shaken at 700 rpm. After washing with 300 μl of PBS-Tween four times for 30 seconds each time, 150 μl of 2× MSD Read Buffer T with surfactant (Meso Scale Discovery) were used for immediate reading with a QUICKPLEX SQ 120 reader (Meso Scale Discovery). A fitted four-parameter logistic model with relative weighting (1/y 2 ) was used as the calibration curve, and the blank was included as zero concentration of Ng. All samples were analyzed without knowledge of clinical diagnosis.

Immunoprecipitation/matrix-assisted laser desorption/ ionization time-of-flight/time-of-flight analysis

To minimize nonspecific binding of other proteins during the immunoprecipitation, all plasma samples were pretreated twice with 20 μl of Novex Dynabeads Protein G (Life Technologies, Carlsbad, CA, USA) per milliliter of plasma and incubated for 1 hour at RT before the beads were magnetically removed as described previously, with some modifications [20]. HI-MS analysis of Ng peptides was performed as described elsewhere [16]. In brief, 4 μg of the monoclonal anti-Ng antibodies Ng2 and Ng3 were separately added to 25 μl of Dynabeads M-280 sheep anti-mouse immunoglobulin G (Life Technologies) and cross-linked as previously described [21]. Beads coated with Ng2 and Ng3 were used for immunoprecipitation of plasma to which n-octyl-β-D-glucopyranoside (10%, end concentration 0.1%; Fluka Chemie, Buchs, Switzerland) had been added. The beads and sample were transferred to a KingFisher magnetic particle processor (polypropylene tubes; Thermo Scientific, Waltham, MA, USA) for automatic washing and elution of the Ng peptides and protein. Eluted Ng was collected and dried in a vacuum centrifuge and redissolved in 5 μl of 0.1% formic acid (FA) in 20% acetonitrile and subsequently analyzed using an ultrafleXtreme matrixassisted laser desorption/ionization (MALDI) time-of-flight/ time-of-flight (TOF/TOF) MS (Bruker Daltonics, Bremen, Germany). All solvents used were of high-performance liquid chromatography (HPLC) quality.

In both the paired cohort and validation study where MALDI-TOF/TOF MS was used, the custom Ng peptide RKKIKSGERGRKGPGPGGPGGAGVARGGAGGGP (corresponding to Ng 43-75 ), with glycine labeled with double 13 C (theoretical molecular mass: 3,011 Da; CASLO, Lyngby, Denmark), was added to the plasma prior to sample preparation and was used as an internal standard. Samples were analyzed blinded (that is, without knowledge of clinical diagnosis).

High-resolution tandem mass spectrometry

The identities of the Ng peptides were confirmed using a top-down approach as previously described [22], but with updated instrumentation. Briefly, analysis was performed using nanoflow liquid chromatography (Dionex UltiMate 3000; Thermo Scientific) coupled to an electrospray ionization Q Exactive Hybrid Quadrupole Orbitrap tandem mass spectrometer (Thermo Scientific). An Acclaim PepMap 100 C8 trap column (length: 20 mm; inner diameter: 75 μm; particle size: 3 μm; pore size: 100 Å (Thermo Scientific)) was used for online desalting, and a reversed-phase Acclaim PepMap rapid separation liquid chromatography column (length: 500 mm, inner diameter: 75 μm; particle size: 2 μm; pore size: 100 Å (Thermo Scientific)) was used for high-resolution separation. Mobile phases were 0.1% aqueous FA (v/v) (A) and 0.1% FA in 84% acetonitrile in water (v/v) (B). The separation was performed at a flow rate of 150 nl/min by applying a linear gradient of 3% to 40% B for 50 minutes. A column oven was used and set to 40°C. All solvents used were of HPLC quality. The Q Exactive system was used in positive ion mode to acquire full-scan spectra in the 300 to 1,200 mass-to-charge (m/z) ratio range at a resolution setting of 70,000. Subsequent tandem mass spectra (MS/MS) were produced using the higher-energy collisional dissociation fragmentation cell and acquired at the same resolution setting. An inclusion list was employed to ensure that we obtained MS/MS data of the compounds of interest. Mass spectral raw files were then charge-deconvoluted by using Mascot Distiller software (Matrix Science, Boston, MA, USA) and subsequently submitted to a Mascot database search (Matrix Science).

Degradation of neurogranin in plasma and cerebrospinal fluid at room temperature

Plasma and CSF samples were depleted of native Ng by three consecutive immunoprecipitations before full-length recombinant Ng (1,000 pg/ml) was added to the samples, followed by 24-hour incubation at RT. After the incubation, samples were analyzed by HI-MS to screen for degradation products from the full-length Ng protein in the form of newly formed Ng peptides.

Cerebrospinal fluid neurogranin storage stability assessment

Pooled CSF samples with or without a stabilizing agent (sodium azide at a final concentration of 0.1% in samples), were kept at RT, 4°C or −20°C for 1 to 7 days before they were placed in long-term storage at −80°C pending analysis. As a reference sample, a freshly prepared aliquot, with or without stabilizer, was stored at −80°C on the day of the preparation (that is, day 0). All other samples were normalized against the Ng content of the respective reference sample for either group. Ng content was analyzed by MSD.

Statistical analysis

For statistical analysis, Prism 6 for Windows software (GraphPad Software, La Jolla, CA, USA) was used. Because biomarker values were skewed, nonparametric tests were used. Differences between groups were assessed using the Mann-Whitney U test, and significance was determined at P < 0.05. Correlations were determined using Spearman's correlation.

Results

Characterization of plasma neurogranin

Plasma Ng was characterized using MALDI-TOF/TOF and high-resolution MS/MS. Several short C-terminal peptides were repeatedly detected by MALDI-TOF/TOF, and 16 of these were independently verified and identified by high-resolution MS/MS ( Figure 1A and Table 2). Of these, seven had not previously been detected in brain tissue or CSF [16]. A summary of all Ng peptides that have been identified using MS in plasma, CSF and brain tissue can be found in Additional file 1: Table S1. A cluster of peaks at the m/z ratio corresponding roughly to full-length Ng (theoretical mass: 7,618 Da) was detected ( Figure 1B), indicating that the protein most likely is subjected to several posttranslational modifications; however, these have yet to be identified.

Clinical validation of paired plasma and cerebrospinal fluid samples

Paired plasma and CSF samples from patients with AD and controls were analyzed for Ng levels to investigate the potential of plasma Ng as a novel biomarker. Plasma samples were analyzed in parallel by using an MSD platform assay and HI-MS targeting both the shorter endogenous peptides and full-length Ng. Using HI-MS, several plasma Ng peptides were reliably detected and quantified, but none were found to differ significantly between the groups. Concentrations of the most abundant plasma Ng peptide, Ng 43-75 , which was present in all samples, are displayed in Figure 2A. This finding was replicated by HI-MS analysis in a second independent plasma material, where, once again, none of the peptides differed between patients with AD and healthy controls. Concentrations of Ng 43-75 obtained in the validation study are displayed in Figure 2B. The peaks at the mass range of full-length Ng ( Figure 1B) were also investigated, but no significant difference was obtained between patients with AD and healthy controls (data not shown). In agreement with this, MSD analysis of plasma Ng did not reveal any quantitative differences between the groups ( Figure 2C).

In contrast, MSD analysis of the CSF samples showed a marked increase of Ng in patients with AD (P < 0.0001) ( Figure 2D). As the control group was significantly younger than the AD group in both studies (see Table 1), we also tested for a correlation between CSF Ng and age, but no such association was found in the paired cohort (AD group: P = 0.667, r s = −0.1; control group: P = 0.11, r s = −0.37) or in the validation study (AD group: P = 0.258, r s = 0.34; control group: P = 0.77, r s = −0.08). No correlation between CSF and plasma Ng was found in the paired cohort (P = 0.83, r s = 0.0334). Owing to the large CSF volume needed for HI-MS analysis (1 ml), the CSF samples were analyzed only by MSD.

Neurogranin storage stability and degradation in cerebrospinal fluid and plasma

With the purpose of investigating the stability of CSF Ng during storage, CSF was kept at RT, 4°C or −20°C with or without the addition of a stabilizer for between 1 and 7 days before it was stored at −80°C pending analysis. The samples were analyzed using the MSD Ng assay, and all values were normalized against a reference sample, which was stored at −80°C on the day of preparation. The CSF Ng levels were stable at RT for 2 days, after which the concentration dropped to around 70% compared with the day of preparation. Samples kept at either 4°C or −20°C were both stable for all time points investigated ( Figure 3A). Similar results were obtained when a stabilizing agent was added ( Figure 3B).

To address the question whether any degradation of Ng occurs in CSF and plasma, CSF and plasma samples that had been depleted of all native Ng by immunoprecipitation using Ng2 and Ng3 were spiked with full-length Ng (1,000 pg/ml) and incubated at RT for 24 hours. The samples were then analyzed for newly generated Ng peptides using HI-MS. In CSF, no Ng peptides generated de novo could be detected in the spiked samples after the incubation (data not shown). In contrast, 14 Ng peptides were detected in the spiked plasma samples, indicating that Ng is degraded in plasma (Additional file 2: Table S2).

Discussion

In this study, we characterized the Ng content in plasma and investigated the potential of plasma Ng as a biomarker for AD. We also addressed the stability of CSF Ng during Mass deviation is displayed in parts per million (ppm). b Peptides found only in plasma and not in cerebrospinal fluid or brain tissue.

storage and investigated any potential degradation of Ng in both CSF and plasma. In total, we identified 16 endogenous Ng peptides in plasma. We also show that full-length Ng most likely is present, but mostly in posttranslationally modified forms. Of the endogenous plasma peptides, seven had not been detected previously in either brain tissue or CSF [16]. Thus, they seem to be plasma-specific. As we have now performed HI-MS analysis of Ng peptides in more than 75 plasma samples in total and the plasma-specific peptides have been present in all of these, we can conclude that these are consistently found in plasma and are thus most likely not caused by the preanalytical handling,

In a previous study, we showed increased CSF Ng levels in AD using ELISA [16]. In the same study, using MS, we also found that, of the endogenous Ng peptides examined, Ng 48-76 showed the most pronounced increase in AD CSF compared with controls. Importantly, Ng 48-76 was also found to be the dominant peptide in AD brain tissue [16], but this peptide was not found in plasma (Table 2). These data suggest that this particular peptide most likely is brain-specific. Furthermore, there are also peptides that are unique for plasma as well as for CSF and brain tissue (Additional file 1: Table S1). Thus, Ng peptides detected in CSF may specifically reflect ongoing neurodegenerative processes within the brain and are most likely not influenced by Ng peptides produced in the periphery, indicating that they may have valid clinical relevance. Further supporting CSF Ng as an AD marker are the discoveries that the plasma Ng levels did not differ between AD and controls (Figure 2A, B and C) and that there was a lack of correlation between CSF and plasma Ng content.

In a series of experiments designed to test the stability of Ng in plasma, we show that recombinant full-length protein incubated in plasma, depleted of native Ng, is metabolized into a variety of fragments (Additional file 2: Table S2), probably by some of the proteases present in plasma. Five of the detected degradation fragments most likely correspond to endogenous peptides that were identified in native plasma samples, and, in addition to those, we also detected a number of peaks that most likely correspond to other Ng peptides that have not been seen in their native counterparts (Additional file 2: Table S2). Also, four of the Ng peptides found only in plasma were not generated after incubation of full-length Ng in Ng-depleted plasma, thus indicating that they are not an artefact of sample handling. This indicates that there are several enzymes present in plasma that are capable of cleaving Ng. As the peptide patterns of native plasma Ng and degraded full-length Ng differ, this might point toward differences regarding the enzymes in freeze-thawed plasma samples and enzymes in plasma in vivo with regard to activity and/or specificity. These differences could be due to a number of different factors, such as pH or storage stability. However, a characterization of the active enzymes in plasma after −80°C storage that are capable of cleaving full-length Ng to generate the peptides observed in such samples is beyond the scope of this article.

Ng 48-76 was neither naturally occurring (Table 2) nor generated after incubation of full-length Ng in the plasma samples investigated (Additional file 2: Table S2). Therefore, it is unlikely that this specific peptide is produced in the periphery. These findings indicate that the enzyme responsible for the formation of Ng 48-76 is present only in the brain or at least is not active in either CSF or plasma. This hypothesis is further supported by the fact that, though there are three Ng peptides in CSF that end with amino acid 76 (Additional file 1: Table S1), neither these endogenous peptides (Table 2) nor fragments generated from recombinant full-length protein (Additional file 2: Table S2) were found in plasma. The enzymes responsible for the different cleavages of Ng remains to be identified.

Compared with CSF, plasma Ng concentration in paired samples was around 100 times higher ( Figure 2C and D).

Studies have shown that Ng is expressed in the periphery in the spleen, bone marrow, platelets and lung tissue [11], which may contribute to the high concentration in plasma.

Limitations of both studies are their small sample size and that healthy control subjects were significantly younger than the AD patient groups (P < 0.001) ( Table 1). However, considering that no difference in plasma Ng was found between the groups in either cohort and that increased CSF Ng in AD has been shown before in age-matched groups [16], we believe that the age difference between the groups did not affect the results of this study. Also, we did not find any correlation between age and Ng concentration in either plasma or CSF.

Conclusions

The present study shows that Ng is present in plasma at high concentrations, both as posttranslationally modified full-length protein and as a number of endogenous peptides. The previously reported difference in Ng levels in CSF between controls and AD patients is a consistent finding that is not reflected in the plasma. The most important discoveries are that there is no degradation of full-length Ng or de novo formation of peptides in CSF and that CSF Ng is stable for storage at -20°C, as well as that there are no native plasma Ng peptides ending with amino acid 76. This suggests that CSF Ng, especially Ng 48-76 , might reflect ongoing neurodegenerative processes within the brain, indicating a role for Ng as a potential novel clinical biomarker for synaptic function in AD.

Additional files

Additional 1: Table S1. Summary of all Ng peptides identified by MS/ MS in human brain tissue, CSF and plasma. Microsoft Excel 97-2003 worksheet (.xls). A complete list of all endogenous Ng peptides identified by MS/MS in human brain tissue, CSF and plasma.

Additional 2: Table S2. Newly formed Ng peptides in plasma incubated with recombinant full-length Ng. Microsoft Excel 97-2003 worksheet (.xls). Plasma samples were depleted of native Ng by three consecutive immunoprecipitations before full-length recombinant Ng (1,000 pg/ml) was added to samples, followed by 24-hour incubation at RT. After the incubation, the samples were analyzed by HI-MS to screen for newly formed Ng peptides.  Samples were kept at room temperature (RT), 4°C or −20°C for between 1 and 7 days before transfer to storage at −80°C pending analysis. Samples were normalized to day 0, with or without stabilizer.

Figure 1
1Hybrid immunoaffinity-mass spectrometry characterization of plasma neurogranin. (A) Hybrid immunoaffinity-mass spectrometry analysis of human plasma repeatedly detected several short C-terminal peptides. *Peptides found only in plasma and not in cerebrospinal fluid or brain tissue. ‡ Nonspecific binding of other plasma proteins to magnetic beads. (B) A cluster of peaks roughly corresponding to the mass of full-length neurogranin (Ng) is present in plasma. *Peak corresponding to the theoretical mass of full-length Ng (7,618 Da). m/z, Mass-to-charge ratio.

Figure 2
2Scatterplots displaying the results from hybrid immunoaffinity-mass spectrometry and Meso Scale Discovery analysis of plasma and cerebrospinal fluid samples. (A) Scatterplot of plasma concentrations of the most abundant neurogranin (Ng) peptide 43 to 75 (Ng 43-75 ), in samples from the paired cohort obtained by hybrid immunoaffinity-mass spectrometry (HI-MS). (B) Scatterplot of plasma Ng 43-75 concentrations in samples from the verification study obtained by HI-MS. (C) Scatterplot of plasma Ng levels obtained by Meso Scale Discovery (MSD) in samples from the paired cohort. (D) Scatterplot of cerebrospinal fluid Ng levels obtained by MSD. The data presented are medians and interquartile ranges. AD, Alzheimer's disease; Co, Control.

Abbreviations
Aβ: Amyloid-β; AD: Alzheimer's disease; BSA: Bovine serum albumin; CNS: Central nervous system; CSF: Cerebrospinal fluid; ELISA: Enzyme-linked immunosorbent assay; FA: Formic acid; HI-MS: Hybrid immunoaffinity-mass spectrometry; HPLC: High-performance liquid chromatography; MALDI: Matrix-assisted laser desorption/ionization; MS: Mass spectrometry; MS/MS: Tandem mass spectrometry; m/z: Mass-to-charge ratio; Ng: Neurogranin; NINCDS-ADRDA: National Institute of Neurological and Communicative Disorders and Stroke/Alzheimer's Disease and Related Disorders Association; PBS: Phosphate-buffered saline; ppm: Parts per million; p-tau: Phosphorylated tau; RT: Room temperature; TOF/TOF: Time-of-flight/ time-of-flight; t-tau: Total tau.

Figure 3
3Analysis of cerebrospinal fluid neurogranin storage stability. Normalized cerebrospinal fluid neurogranin (Ng) concentrations in samples without (A) or with (B) the addition of a stabilizing agent.

Table 1
1Demographic and clinical characteristics of subjects included in the study 1 and 2 a Aβ, Amyloid-β; AD, Alzheimer's disease; CSF, Cerebrospinal fluid; HI-MS, Hybrid immunoaffinity-mass spectrometry; LP, lumbar puncture; MSD, Meso Scale Discovery; Ng, Neurogranin; p-tau, Phosphorylated tau; t-tau, Total tau. The values presented are median (interquartile range). b P < 0.001 vs. controls.Control
AD

Study 1
(paired plasma + CSF)

n = 20
n = 25

Sex, n, female/male
(% female)

12/8 (60)
14/11 (56)

Age at LP, yr
54 (41 to 63)
76 (71 to 78) b

CSF Aβ 1-42 , pg/ml
1,399 (1189 to 1702) 709 (588 to 897) b

CSF Aβ 1-42 /Aβ 1-40 ratio 0.094 (0.079 to 0.104) 0.033 (0.028 to 0.042) b

CSF t-tau, pg/ml
185 (136 to 254)
580 (425 to 655) b

CSF p-tau, pg/ml
35 (27 to 45)
89 (78 to 105 to 126) b

CSF Ng, pg/ml (MSD)
291 (251 to 438)
620 (521 to 818) b

Plasma Ng, pg/ml (MSD) 47,451 (21,904 to
90,320)

36,525 (25,324 to
57,715)

Plasma, pg/ml (HI-MS) 21,698 (13,401 to
106,954)

25,644 (17,262 to
53,900)

Study 2 (plasma)
n = 17
n = 13

Sex, n, female/male
(% female)

9/17 (53)
9/13 (69)

Age at LP, yr
58 (53 to 68)
78 (69 to 80) b

CSF Aβ 1-42 , pg/ml
875 (580 to 940)
330 (268 to 415) b

CSF t-tau, pg/ml
300 (210 to 348)
690 (605 to 980) b

CSF p-tau, pg/ml
48 (40 to 61)
110 (77 to 160) b

a

Table 2
2Identified plasma neurogranin peptides by high-resolution mass spectrometry aCalculated mass (Da)
Theoretical mass (Da)
Deviation (ppm)
Confirmed sequence
Number of y-ions
Number of b-ions

1,899.98
1,899.98
−1.3
52 to 75 b
1
1 1

2,203.24
2,203.24
1.7
43 to 65 b
1
1 4

2,217.24
2,217.25
−1.8
44 to 67 b
2
8

2,242.14
2,242.14
−0.1
49 to 75
5
12

2,329.18
2,329.17
1.0
48 to 75
10
20

2,373.34
2,373.35
−1.0
43 to 67 b
0
6

2,570.36
2,570.35
1.8
46 to 75
10
17

2,698.45
2,698.45
0.1
45 to 75
12
19

2,826.54
2,826.54
−2.4
44 to 75
19
21

2,982.64
2,982.64
−1.7
43 to 75
21
8

3,053.69
3,053.68
2.9
42 to 75 b
18
6

3,085.61
3,085.62
−4.3
44 to 78
6
12

3,184.73
3,184.72
2.7
41 to 75
14
4

3,241.72
3,241.73
−1.6
43 to 78
19
13

3,378.81
3,378.80
1.8
39 to 75 b
8
2

3,534.91
3,534.90
1.7
38 to 75 b
11
10

a

Competing interests PL received consultation and lecture honoraria from Innogenetics, IBL International, AJ Roboscreen, Beckman Coulter and Roche and holds the position of visiting professor at the Medical University of Białystok, Białystok, Poland. HK, EP, UA, GB, KH, NL, JK, OH, LM, JMM, PS, HZ and KB declare that they have no competing interests.Authors' contributions HK developed the HI-MS and MSD methods, acquired MS and MSD data, analyzed and interpreted data and drafted the manuscript. EP developed the HI-MS method, planned the experimental design and analyzed and interpreted data. UA developed the MSD method and analyzed and interpreted data. GB acquired Q Exactive MS data and analyzed and interpreted data. KH was involved in MSD data collection and analyzed and interpreted data. NL, JK, JMM and PS were responsible for subjects in the clinical study and analyzed and interpreted data. OH and LM were responsible for subjects in the validation study, analyzed and interpreted data. HZ analyzed and interpreted data. KB designed the production of anti-Ng antibodies and analyzed and interpreted data. PL planned the experimental design and analyzed and interpreted data. All authors agree with the presented findings and read and approved the final manuscript.
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Toward a Pedagogy of Educated Hope under Casino Capitalism Hacia una pedagogía de la esperanza educada bajo el capitalismo de casino Para uma pedagogia da esperança educada sob o capitalismo de casino

Henry A Giroux firstname.lastname@example.com
Artículo de reflexión

Toward a Pedagogy of Educated Hope under Casino Capitalism Hacia una pedagogía de la esperanza educada bajo el capitalismo de casino Para uma pedagogia da esperança educada sob o capitalismo de casino
147 Fecha de recepción: 13 de noviembre de 2017 Fecha de aprobación: 27 de mayo de 2018 To cite this article: Giroux, H. (2019). Toward a Pedagogy of Educated Hope under Casino Capitalism. Pedagogía y Saberes, 50, 147-151. * Profesor de McMaster University. Correo electrónico: Pedagogía y Saberes No. 50 Universidad Pedagógica Nacional Facultad de Educación. 2019. pp. 147-151
The purpose of this reflection paper is to analyze education and critical thinking as fundamental strategies for the consolidation of radical democracy. Social and political change, together with educated hope, are proposed as the main pillars to allow the construction of a non-authoritarian future. It is argued that critical pedagogy is the possibility for education to be recognized as a civic commitment that favors civic literacy, as well as the capacity for agency, action and change that allows for inclusion and social responsibility. Finally, it is affirmed that democracy becomes deficient when pedagogy is not the center of politics and that informed hope is the possibility of facing the obstacles and concrete realities of domination.

Resumen

La educación y el pensamiento crítico como estrategias fundamentales para consolidación de la democracia radical son el objeto de análisis del este artículo de reflexión. El cambio social y político junto a la esperanza educada se proponen como los ejes que permitirían construir un futuro no autoritario. Se argumenta que la pedagogía crítica es la posibilidad para que la educación se reconozca como un compromiso cívico que favorece la alfabetización cívica, así como la capacidad de agencia, acción y cambio que permite la inclusión y responsabilidad social. Finalmente, se afirma que la democracia se empobrece cuando la pedagogía no es el centro de la política y que la esperanza informada es la posibilidad de enfrentar los obstáculos y las realidades concretas de dominación.

Palabras clave

Resumo

A educação e o pensamento crítico como estratégias fundamentais na consolidação da democracia radical são o objeto de análises desse artigo de reflexão. O cambio social e político junto à esperança educada se propõem como eixos que permitiríam construir um futuro não autoritário. Argumenta-se que a pedagogia crítica é a possibilidade para que a educação se reconheça como um compromisso cívico que favorece a alfabetização cívica, assim como a capacidade de agência, ação e mudança que permite a inclusão e responsabilidade social. Finalmente, afirma-se que a democracia empobrece-se quando a pedagogia não é o centro da política e que a esperança informada é a possibilidade de enfrentar os obstáculos e as realidades concretas de dominação.

Palavras-chave

pedagogia; esperança educada; democracia; ética; moral; política Henry A. Giroux Artículo de reflexión E ducation is the oxygen for democracy because, without an infomed citizenry, the formative cultures necessary for a radical democracy wither along with the institutions that make it possible. In North and Latin America, the forces of neoliberal authoritarianism are on the march. From Brazil to the United States, despots have seized political power and are transforming education into a feeder for the military, the workplace, and, in many cases, for the carceral state. In the United States, Donald Trump has assumed the presidency and has ushered in a culture of fear, humiliation, bigotry, and white supremacy coupled with a deep disdain for education and critical thinking. Ignorance is now valorized as critical education is viewed with disdain. This is all the more reason to once again connect learning to social and political change and to embrace a notion of educated hope in which in becomes possible to imagine the unimaginable, a future unwilling to mimic the authoritarian present.

Increasingly, neoliberal regimes across Europe and North America have waged a major assault on critical pedagogy and the public spheres in which it takes place. Public and higher education are being defunded, turned into accountability factories, and now largely serve as adjuncts of an instrumental logic that mimics the values of the market. But this is not only true for spaces in which formal schooling takes place; it is also true for those public spheres and cultural apparatuses actively engaged in producing knowledge, values, subjectivities, and identities. This applies to a range of creative spaces, including art galleries, museums, movie houses, and various elements of mainstream media (Giroux, 2011). What the apostles of neoliberalism have learned is that this art and science of education can be dangerous and can not only create critically engaged students, intellectuals, and artists, but can expand the capacity of the imagination to think otherwise in order to act otherwise, hold power accountable, and imagine the unimaginable.

Reclaiming pedagogy as a form of educated hope begins with the crucial recognition that education is not just about job training and product manufacturing, but also about matters of civic engagement, critical thinking, civic literacy, and the capacity for democratic agency, action, and change. It is also connected to the issues of power, inclusion, and social responsibility. 1 If young people, artists, and cultural workers are to develop a deep respect for others and a keen sense of social responsibility and civic engagement, pedagogy must be viewed as a cultural, political, and moral force. That collective force provides the knowledge, values, and social relations that make such democratic practices possible in order to connect human agency to the idea of social responsibility and the politics of possibility. In this instance, pedagogy needs to be rigorous, self-reflective and committed to the practice of freedom, to advancing the parameters of knowledge, addressing crucial social issues, and connecting private troubles and public issues. At stake here are pedagogical practices that create militant dreamers, people who are willing to struggle for a more just and democratic world. In this instance, pedagogy becomes central to politics and critical to the practice of art-a performance addressed to changing the way people think-one that awakens passion and energizes forms of identification that speak to the conditions in which people find themselves.

Critical pedagogy must be meaningful in order to be critical and transformative. It should be cosmopolitan, imaginative and demand a thoughtful engaged interaction with the world we live in, mediated by a responsibility for challenging structures of domination and for alleviating human suffering. This is a pedagogy that addresses the needs of multiple publics. As an ethical and political practice, a public pedagogy of wakefulness rejects modes of education that are removed from political, social, historical, and unjust concerns. This is a pedagogy that includes "lifting complex ideas into the public space" (Said, 2000, p. 7), recognizes human injury inside and outside of the academy, and uses theory as a form of criticism to change things. This is a pedagogy in which educators and cultural workers are neither afraid of controversy nor the connections between private issues and the broader elements of society's problems that are otherwise hidden.

Critical pedagogy arises from the conviction that artists, educators and other cultural workers have a responsibility to unsettle power, trouble consensus, and challenge common sense. This is a view of pedagogy that should disturb, inspire, and energize a vast array of individuals and publics. Critical pedagogy comes with the responsibility to view intellectual work as public, raise political awareness, make connections to those elements of power and politics often hidden from public view, and remind "the audience of the moral questions that may be hidden in the clamor and din of the public debate" (Said, 2001, p. 504).

Pedagogy is not a method but a moral and political practice, one that recognizes the relationship between knowledge and power. At the same time, it realizes that inside what is central to all pedagogical practices lies a struggle over agency, power, politics, and the formative cultures that make a radical democracy possible. This view of pedagogy does not mold, but inspires, directs energizes, and is capable of imagining a better world and the need to reimagine a democracy that is never finished. Critical pedagogy is a form of educated hope committed to producing young people capable and willing to expand and deepen their sense of themselves, to think of the "world" critically, to imagine something beyond their their own self interest and well-being," to serve the public good, take risks, and struggle for a substantive democracy. 2 Pedagogy is always the outcome of struggles, especially in terms of how pedagogical practices produce particular notions of citizenship and an inclusive democracy. Pedagogy looms large in this instance, not as a technique or a priori set of methods, but as a political and moral practice. As a political practice, pedagogy illuminates the relationship between power, knowledge, and ideology, while selfconsciously, if not self-critically, recognizing the role it plays as a deliberate attempt to influence how and what knowledge and identities are produced within particular sets of social relations. As a moral practice, pedagogy recognizes that what cultural workers, artists, activists, media workers and others teach cannot be abstracted from what it means to invest in public life, presuppose some notion of the future, or locate oneself in a public discourse.

The moral implications of pedagogy suggest that our responsibility as cultural workers cannot be separated from the consequences of our knowledge, our social relations, and the ideologies and identities we offer up to students. Refusing to separate politics from pedagogy means that teaching should not simply honor the experiences people bring to such sites, but should also connect experiences to problems that come from everyday life. Pedagogy, in this sense, is not just about deconstructing texts, but about politics in a broader sense. Such a project recognizes the political nature of pedagogy and calls for artists, intellectuals, and others to assume responsibility for their actions. As Susan Sontag (2003) once suggested, this summons these people to link their teaching to those moral principles that allow them to do something about human suffering. Part of this task necessitates that cultural workers anchor their own work, however diverse, in a radical project that seriously engages the promise of an unrealized democracy against its radically incomplete forms. Of crucial importance to such a project is rejecting the 2 See, especially, Newfield (2008). assumption that theory can understand social problems without contesting their appearance in public life. Yet, any viable cultural politics needs a socially committed notion of injustice if we are to take seriously what it means to fight for the idea of the good society. Zygmunt Bauman (2002) argues that "if there is no room for the idea of wrong society, there is hardly much chance for the idea of good society to be born, let alone make waves" (p. 170).

A society must constantly nurture the possibilities for self-critique, collective agency, and forms of citizenship in which people play a fundamental role in critically discussing, administrating and shaping the material relations of power and ideological forces that impact their everyday lives. At stake here is the task, as Jacques Derrida (2000) insists, of viewing the project of democracy as a promise, a possibility rooted in an ongoing struggle for economic, cultural, and social justice. Democracy in this instance is not a sutured or formalistic regime. It is the site of struggle itself. The struggle over creating an inclusive and just democracy can take many forms. That struggle offers no political guarantees but provides an important dimension to politics as an ongoing process of democratization that never ends. Such a project is based on the realization that a democracy open to exchange, question, and self-criticism never reaches the limits of justice.

Theorists like Raymond Williams and Cornelius Castoriadis recognized that the crisis of democracy was not only about the crisis of culture, but also the crisis of pedagogy and education. Cultural workers would do well to take account of the profound transformations taking place in the public sphere and reclaim pedagogy as a central category of politics itself. Pierre Bourdieu (1998) was right when he stated that cultural workers have too often "underestimated the symbolic and pedagogical dimensions of struggle and have not always forged appropriate weapons to fight on this front" (Bourdieu, 1998, p. 11). He goes on to say in a later conversation with Gunter Grass that left intellectuals must recognize that the most important forms of domination are not only economic but also intellectual and pedagogical, and lie on the side of belief and persuasion. It is important to recognize that intellectuals bear an enormous responsibility for challenging this form of domination. (Bourdieu, 2002, p. 2). These are important pedagogical interventions and imply rightly that critical pedagogy in the broadest sense is not just about understanding, but also about assuming the responsibilities we have as citi-zens to expose human misery and to eliminate the conditions that produce it. Matters of responsibility, social action, and political intervention do not simply develop out of social critique but also self-critique. The relationship between knowledge and power, on the one hand, and creativity and politics, on the other, should always be self-reflexive about its effects and how it relates to the larger world. In short, this project points to the need for cultural workers to address critical pedagogy not only as a mode of educated hope and a crucial element of an insurrectional educational project, but also as a practice that addresses the possibility of interpretation as intervention in the world. I want to end by insisting that democracy begins to fail and civic life becomes impoverished when pedagogy is no longer central to politics. The failure to recognize the educative nature of how agency is constructed, moral witnessing legitimated, and the politics of social responsibility renewed, empties democracy of any meaning. Democracy should be a way of thinking about education, one that thrives on connecting equity to excellence, learning to ethics, and agency to the imperatives of the public good (Giroux, 2015). The question regarding what role education and pedagogy should play in democracy becomes all the more urgent at a time when the dark forces of authoritarianism are on the march all over the globe. As public values, trust, solidarities, and modes of education are under siege, the discourses of hate, racism, rabid self-interest, and greed are exercising a poisonous influence in many Western societies, now most evident in the discourse of the right-wing extremists vying for the American presidency. Democracy is on life support, but rather than being a rationale for cynicism, it should create moral and political outrage, a new understanding of politics, and the pedagogical projects needed to allow democracy to breathe once again.

As Ernst Bloch (as cited in Benjamin, 1997) once insisted, "reason, justice, and change cannot blossom without hope" because educated hope taps into our deepest experiences and longing for a life of dignity with others, a life in which it becomes possible to imagine a future that does not mimic the present. I am not referring to a romanticized and empty notion of hope, but to a notion of informed hope that faces the concrete obstacles and realities of domination yet continues the ongoing pedagogical and political task of "holding the present open and thus unfinished" (Benjamin, 1997, p. 10).

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