Metab Brain Dis DOI 10.1007/s11011-014-9521-8
RESEARCH ARTICLE
Effects of acidic oligosaccharide sugar chain on amyloid oligomer-induced impairment of synaptic plasticity in rats Lan Chang & Fushun Li & Xiaowei Chen & Shujun Xu & Chuang Wang & Hongzhuan Chen & Qinwen Wang
Received: 22 November 2013 / Accepted: 26 February 2014 # Springer Science+Business Media New York 2014
Abstract Soluble amyloid-β protein (Aβ) oligomers have been recognized to be early and key intermediates in Alzheimer’s disease-related synaptic dysfunction. In this study, using in vitro electrophysiology, we investigated interactions of the acidic oligosaccharide sugar chain (AOSC), a marine-derived acidic oligosaccharide, with oligomeric Aβ. We found that the inhibition of long-term potentiation (LTP) induced by Aβ oligomers can be dose dependently reversed by the application of AOSC, whereas AOSC alone did not alter normal LTP induction. Interestingly, treatment with Aβ monomers with or without AOSC did not affect LTP induction. Additionally, when fresh-made Aβ was co-incubated with AOSC before in vitro testing, there was no impairment of LTP induction. The results from Western blots demonstrated that AOSC prevent the aggregation of Aβ oligomers. These findings indicate that AOSC may reverse Aβ oligomer-mediated cytotoxicity by directly disrupting the amyloid oligomer aggregation, and this action is concentration dependent. Thus, we propose that AOSC might be a potential therapeutic drug for Alzheimer’s disease due to its protection against oligomeric Aβ-induced dysfunction of synaptic plasticity. Keywords Acidic oligosaccharide sugar chain(AOSC) . Amyloid-β protein(Aβ) . N-methyl-D-aspartate (NMDA) . Long-term potentiation(LTP) . Synaptic plasticity Lan Chang and Fushun Li contributed equally to this work. L. Chang (*) : F. Li : X. Chen : S. Xu : C. Wang : Q. Wang (*) Zhejiang Provincial Key Laboratory of Pathophysiology, Medical School, Ningbo University, Ningbo 315211, China e-mail: [email protected] e-mail: [email protected] H. Chen Medical School, Shanghai Jiaotong University, Shanghai 200240, China
Introduction Alzheimer’s disease (AD), the most common cause of senile dementia, is characterized by the presence of senile plaques composed of deposits of amyloid β (Aβ), a cleavage product of beta-amyloid precursor protein (APP) (De-Paula et al. 2012; Singh et al. 2012). In the pathology of the disease, toxicity stems from Aβ oligomers rather than β-amyloid plaques, and over-accumulation of Aβ oligomers has been postulated to induce dysfunction in synaptic plasticity that contributes to the early memory loss that precedes neuronal degeneration in AD (Tam and Pasternak 2012; Klyubin et al. 2012). Indeed, Aβ oligomers have been demonstrated to inhibit the induction of long-term potentiation (LTP) in the hippocampus, both in vivo (Walsh et al. 2002; Barry et al. 2011) and in vitro (Wang et al. 2004), and are known to aggregate in the brains of humans with AD (Takahashi et al. 2002). Treatment strategies that interfere with Aβ aggregation or facilitate clearance have shown initial benefits in AD patients and animal models (Klyubin et al. 2005; Schenk et al. 2005; Demattos et al. 2012). Thus, Aβ-induced neurotoxicity may be involved in the memory deficits observed in the early stages of AD, and the blockade of Aβ toxicity may be a viable strategy for alleviating AD symptoms. Acidic oligosaccharide sugar chain (AOSC) is an acidic oligosaccharide derived from the brown marine algae Ecklonia kurome Okam by enzymatic depolymerization. AOSC is an analog of low-molecular-weight glycosaminoglycans (GAGs) and is rich in mannuronate modules. Preclinical studies have shown that AOSC can attenuate neurotoxicity induced by both Aβ and hydrogen superoxide (H2O2) in vitro (Hu et al. 2004; Wang et al. 2007a). It has been reported that AOSC can alleviate Alzheimer-type behavioral symptoms induced by Aβ in animal subjects, and the cognitionimproving activities have been ascribed to the inhibition of apoptosis and subsequent Aβ-triggered neurotoxicity (Hu
Metab Brain Dis
et al. 2004; Fan et al. 2005). Further evidence was reported that AOSC and its sulfated derivatives can cross the blood– brain barrier (BBB) via a mechanism involving the Glucose transporter 1(GluT-1). AOSC’s oligosaccharide structure (Guo et al. 2006) plays a key role in influencing glial cell line derived-neurotrophic factor (GDNF) activity (Wang et al. 2007b), a regulator of cell proliferation and differentiation, indicating its beneficial value for treating neurodegenerative diseases. However, the efficacy of AOSC as a potential preventative or therapeutic neuroprotector against AD remains to be fully elucidated. In the present study, we have determined whether AOSC can prevent the inhibition of LTP by oligomeric Aβ in the in vitro rat hippocampus . We have further investigated the effects of various concentrations of AOSC on Aβ oligomerinduced LTP disruption. Additionally, we have explored the relationship between AOSC and Aβ monomers and investigated the potential mechanism of interaction between AOSC and Aβ. Together, these experiments provide insight into the interaction of AOSC and Aβ, and implicate AOSC as a potential therapeutic agent in AD.
0.55 CV of the Superdex 75 HR 10/30 column). The combined fractions represented monomeric Aβ. Immunoblotting Detection of total Aβ1–40 peptide(monomer + oligomer) with two commercially available mouse monoclonal antibodies(6E10 for residues 3–8, 4G8 for residues 17–24), was performed as previously described (LeVine 2004). Both of the monoclonal antibodies were purchased from Signet Labs (Dedham, MA). Sandwich ELISAs were performed using the 96-well plate platform following the manufacturer’s protocol. We used 6E10 as capture antibodies and biotinylated 4G8 for detection. The aggregation state of Aβ were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by Western blotting, and transferred onto nitrocellulose membranes using a wet system at 4 °C. Alternatively, 3 mL of sample solution was blotted onto membranes. After being blocked with blocking buffer , antibody immunoreactivity was detected using horseradish peroxidase (HRP)-conjugated anti-rabbit IgG (1:2500), and subsequent fluorescence imaging analysis was performed using an LAS-400 optical system.
Materials and methods Preparation of slices Preparation of agents AOSC, generously provided by the Medical School at Shanghai Jiaotong University, is a white powder that easily dissolves in water. After dissolving the AOSC in doubledistilled water, it was stored at 4 °C. Aβ1–40 oligomers were prepared as described previously (LeVine 2004). Briefly, lyophilized Aβ1–40 peptide (Peptide Institute, Japan) was dissolved in 1,1,1,3,3,3-hexafluoro-2propanol (HFIP) (Wako, Japan) on ice and divided into aliquots to be frozen until ready to use. Aβ was spun under vacuum immediately before the experiment, dissolved in HFIP solution to a final concentration of 10 % (v/v) HFIP, dried under nitrogen, and then kept at room temperature with constant stirring. After 48 h of incubation, the product was maintained at 4 °C and used for Western blot and AOSC studies. In the experiments using Aβ1–40, the concentration was always calculated to the 500 nM of the initial Aβ1–40 peptide. Aβ1–40 monomers were prepared according to a previously described protocol (Jan et al. 2010). Briefly, 1 mg of Aβ was dissolved in 1 ml of 6 M guanidine hydrochloride and mixed until a clear solution was obtained. The solution was then centrifuged, and the supernatant was carefully drawn into a 1 ml syringe for subsequent use in size exclusion chromatography. Using a Superdex 75 HR 10/30 SEC column at a flow rate of 0.5 ml/min to fractionate Aβ, individual fractions of 1 ml were collected under the elution peak at 11–13 ml (0.45–
All experiments were performed on transverse slices of the rat hippocampus (males; age, 3–4 weeks; weight, 40–80 g). The brains were rapidly removed after decapitation and placed in cold oxygenated (95 % O2/5 % CO2) media. Slices were cut at a thickness of 350 μm~400 μm using an Intracell Plus 1000 vibratome and placed in an incubation chamber containing oxygenated medium at room temperature (20–22ºC) for 1 h. The slices were then transferred to a submerged slice recording chamber and were continuously superfused at a rate of 5– 6 ml/min at 30–32ºC. The control media contained the following: 120 mM NaCl, 2.5 mM KCl, 1.25 mM NaH2PO4, 26 mM NaHCO3, 2.0 mM MgSO4, 2.0 mM CaCl2, and 10 mM D-glucose. All solutions contained 100 μM picrotoxin (Sigma) to block GABAA receptor-mediated activity. In vitro electrophysiological techniques Standard electrophysiological techniques were used to record field potentials. Presynaptic stimulation was applied to the medial perforant pathway of the dentate gyrus using an insulated bipolar tungsten wire electrode at a baseline test frequency of 0.033 Hz, and field excitatory postsynaptic potentials (EPSPs) were recorded from the middle one-third of the molecular layer of the dentate gyrus with a glass microelectrode. The inner blade of the dentate gyrus was used in all experiments. In each experiment, an input–output curve (afferent stimulus intensity vs. EPSP amplitude) was plotted at
Metab Brain Dis
the test frequency. For all experiments, the amplitude of the test EPSP was adjusted to one-third of the maximum (~1.0 mV). LTP was evoked by high-frequency stimulation (HFS) consisting of two trains of 100 stimuli at100 Hz with an inter-train interval of 20 s. In experiments involving the application of Aβ (oligomers and monomers), slices were perfused with Aβ for 40 min before HFS. For experiments involving AOSC, slices were exposed to the agents for 90 min before HFS. For all experiments, recordings of the effects of the tested agents and control recordings were made from slices of the same hippocampus.
Statistical analysis Recordings were analyzed using p-CLAMP (Axon Instruments, Sunnyvale, CA, USA). Values are reported as the mean ± standard error of the mean (SEM) for n slices. Analysis of variance (ANOVA) was used for statistical comparison, and P0.05) (Fig. 2b), whereas application of 1.5 μM AOSC partially reversed Aβ1–40 oligomer-mediated LTP inhibition, resulting in a change in field EPSP amplitude of 134.17± 3.09 % at 60 min post-HFS, a value significantly different from control and Aβ1–40 oligomer-treated conditions (n=6, P0.05 compared with control slices). To investigate whether AOSC reduce Aβ oligomer levels, we analysed the composition of the solution by ELISA, the results demonstrated that AOSC significantly blocked the formation of Aβ oligomers
when compared to the controls. The 17–49 kD and 4.5 kD band was shown in with and without AOSC pre-treatment, respectively (Fig. 3c).
Discussion In the present study, we have demonstrated that the inhibitory effects of Aβ oligomers on hippocampal LTP in the adult brain are reversible and that LTP can be restored by AOSC dose dependently in vitro. By contrast, AOSC did not affect Fig. 2 The protective effects of AOSC on Aβ oligomer-mediated inhibition of LTP induction are dose dependent. a LTP induction in the presence of 2.5 μM AOSC alone (open circles) is not significantly different from control LTP (closed circles). b LTP induction in the presence of 500 nM oligomeric Aβ plus 500 nM AOSC (open circles) did not prevent Aβ oligomer inhibition of LTP, whereas co-application of 500 nM oligomeric Aβ and 1.5 μM AOSC (filled circles) partially blocked the inhibition of LTP. c LTP induction in the presence of 500 nM oligomeric Aβ plus 2.5 μM (open circles) or 5 μM (filled circles) AOSC was not significantly reduced compared with controls. The insets are representative traces showing field EPSPs recorded before (a) and after (b) HFS. d Summary bar graph showing the change in field EPSP amplitude (% of control) after HFS for the various AOSC concentrations. *P
RESEARCH ARTICLE
Effects of acidic oligosaccharide sugar chain on amyloid oligomer-induced impairment of synaptic plasticity in rats Lan Chang & Fushun Li & Xiaowei Chen & Shujun Xu & Chuang Wang & Hongzhuan Chen & Qinwen Wang
Received: 22 November 2013 / Accepted: 26 February 2014 # Springer Science+Business Media New York 2014
Abstract Soluble amyloid-β protein (Aβ) oligomers have been recognized to be early and key intermediates in Alzheimer’s disease-related synaptic dysfunction. In this study, using in vitro electrophysiology, we investigated interactions of the acidic oligosaccharide sugar chain (AOSC), a marine-derived acidic oligosaccharide, with oligomeric Aβ. We found that the inhibition of long-term potentiation (LTP) induced by Aβ oligomers can be dose dependently reversed by the application of AOSC, whereas AOSC alone did not alter normal LTP induction. Interestingly, treatment with Aβ monomers with or without AOSC did not affect LTP induction. Additionally, when fresh-made Aβ was co-incubated with AOSC before in vitro testing, there was no impairment of LTP induction. The results from Western blots demonstrated that AOSC prevent the aggregation of Aβ oligomers. These findings indicate that AOSC may reverse Aβ oligomer-mediated cytotoxicity by directly disrupting the amyloid oligomer aggregation, and this action is concentration dependent. Thus, we propose that AOSC might be a potential therapeutic drug for Alzheimer’s disease due to its protection against oligomeric Aβ-induced dysfunction of synaptic plasticity. Keywords Acidic oligosaccharide sugar chain(AOSC) . Amyloid-β protein(Aβ) . N-methyl-D-aspartate (NMDA) . Long-term potentiation(LTP) . Synaptic plasticity Lan Chang and Fushun Li contributed equally to this work. L. Chang (*) : F. Li : X. Chen : S. Xu : C. Wang : Q. Wang (*) Zhejiang Provincial Key Laboratory of Pathophysiology, Medical School, Ningbo University, Ningbo 315211, China e-mail: [email protected] e-mail: [email protected] H. Chen Medical School, Shanghai Jiaotong University, Shanghai 200240, China
Introduction Alzheimer’s disease (AD), the most common cause of senile dementia, is characterized by the presence of senile plaques composed of deposits of amyloid β (Aβ), a cleavage product of beta-amyloid precursor protein (APP) (De-Paula et al. 2012; Singh et al. 2012). In the pathology of the disease, toxicity stems from Aβ oligomers rather than β-amyloid plaques, and over-accumulation of Aβ oligomers has been postulated to induce dysfunction in synaptic plasticity that contributes to the early memory loss that precedes neuronal degeneration in AD (Tam and Pasternak 2012; Klyubin et al. 2012). Indeed, Aβ oligomers have been demonstrated to inhibit the induction of long-term potentiation (LTP) in the hippocampus, both in vivo (Walsh et al. 2002; Barry et al. 2011) and in vitro (Wang et al. 2004), and are known to aggregate in the brains of humans with AD (Takahashi et al. 2002). Treatment strategies that interfere with Aβ aggregation or facilitate clearance have shown initial benefits in AD patients and animal models (Klyubin et al. 2005; Schenk et al. 2005; Demattos et al. 2012). Thus, Aβ-induced neurotoxicity may be involved in the memory deficits observed in the early stages of AD, and the blockade of Aβ toxicity may be a viable strategy for alleviating AD symptoms. Acidic oligosaccharide sugar chain (AOSC) is an acidic oligosaccharide derived from the brown marine algae Ecklonia kurome Okam by enzymatic depolymerization. AOSC is an analog of low-molecular-weight glycosaminoglycans (GAGs) and is rich in mannuronate modules. Preclinical studies have shown that AOSC can attenuate neurotoxicity induced by both Aβ and hydrogen superoxide (H2O2) in vitro (Hu et al. 2004; Wang et al. 2007a). It has been reported that AOSC can alleviate Alzheimer-type behavioral symptoms induced by Aβ in animal subjects, and the cognitionimproving activities have been ascribed to the inhibition of apoptosis and subsequent Aβ-triggered neurotoxicity (Hu
Metab Brain Dis
et al. 2004; Fan et al. 2005). Further evidence was reported that AOSC and its sulfated derivatives can cross the blood– brain barrier (BBB) via a mechanism involving the Glucose transporter 1(GluT-1). AOSC’s oligosaccharide structure (Guo et al. 2006) plays a key role in influencing glial cell line derived-neurotrophic factor (GDNF) activity (Wang et al. 2007b), a regulator of cell proliferation and differentiation, indicating its beneficial value for treating neurodegenerative diseases. However, the efficacy of AOSC as a potential preventative or therapeutic neuroprotector against AD remains to be fully elucidated. In the present study, we have determined whether AOSC can prevent the inhibition of LTP by oligomeric Aβ in the in vitro rat hippocampus . We have further investigated the effects of various concentrations of AOSC on Aβ oligomerinduced LTP disruption. Additionally, we have explored the relationship between AOSC and Aβ monomers and investigated the potential mechanism of interaction between AOSC and Aβ. Together, these experiments provide insight into the interaction of AOSC and Aβ, and implicate AOSC as a potential therapeutic agent in AD.
0.55 CV of the Superdex 75 HR 10/30 column). The combined fractions represented monomeric Aβ. Immunoblotting Detection of total Aβ1–40 peptide(monomer + oligomer) with two commercially available mouse monoclonal antibodies(6E10 for residues 3–8, 4G8 for residues 17–24), was performed as previously described (LeVine 2004). Both of the monoclonal antibodies were purchased from Signet Labs (Dedham, MA). Sandwich ELISAs were performed using the 96-well plate platform following the manufacturer’s protocol. We used 6E10 as capture antibodies and biotinylated 4G8 for detection. The aggregation state of Aβ were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by Western blotting, and transferred onto nitrocellulose membranes using a wet system at 4 °C. Alternatively, 3 mL of sample solution was blotted onto membranes. After being blocked with blocking buffer , antibody immunoreactivity was detected using horseradish peroxidase (HRP)-conjugated anti-rabbit IgG (1:2500), and subsequent fluorescence imaging analysis was performed using an LAS-400 optical system.
Materials and methods Preparation of slices Preparation of agents AOSC, generously provided by the Medical School at Shanghai Jiaotong University, is a white powder that easily dissolves in water. After dissolving the AOSC in doubledistilled water, it was stored at 4 °C. Aβ1–40 oligomers were prepared as described previously (LeVine 2004). Briefly, lyophilized Aβ1–40 peptide (Peptide Institute, Japan) was dissolved in 1,1,1,3,3,3-hexafluoro-2propanol (HFIP) (Wako, Japan) on ice and divided into aliquots to be frozen until ready to use. Aβ was spun under vacuum immediately before the experiment, dissolved in HFIP solution to a final concentration of 10 % (v/v) HFIP, dried under nitrogen, and then kept at room temperature with constant stirring. After 48 h of incubation, the product was maintained at 4 °C and used for Western blot and AOSC studies. In the experiments using Aβ1–40, the concentration was always calculated to the 500 nM of the initial Aβ1–40 peptide. Aβ1–40 monomers were prepared according to a previously described protocol (Jan et al. 2010). Briefly, 1 mg of Aβ was dissolved in 1 ml of 6 M guanidine hydrochloride and mixed until a clear solution was obtained. The solution was then centrifuged, and the supernatant was carefully drawn into a 1 ml syringe for subsequent use in size exclusion chromatography. Using a Superdex 75 HR 10/30 SEC column at a flow rate of 0.5 ml/min to fractionate Aβ, individual fractions of 1 ml were collected under the elution peak at 11–13 ml (0.45–
All experiments were performed on transverse slices of the rat hippocampus (males; age, 3–4 weeks; weight, 40–80 g). The brains were rapidly removed after decapitation and placed in cold oxygenated (95 % O2/5 % CO2) media. Slices were cut at a thickness of 350 μm~400 μm using an Intracell Plus 1000 vibratome and placed in an incubation chamber containing oxygenated medium at room temperature (20–22ºC) for 1 h. The slices were then transferred to a submerged slice recording chamber and were continuously superfused at a rate of 5– 6 ml/min at 30–32ºC. The control media contained the following: 120 mM NaCl, 2.5 mM KCl, 1.25 mM NaH2PO4, 26 mM NaHCO3, 2.0 mM MgSO4, 2.0 mM CaCl2, and 10 mM D-glucose. All solutions contained 100 μM picrotoxin (Sigma) to block GABAA receptor-mediated activity. In vitro electrophysiological techniques Standard electrophysiological techniques were used to record field potentials. Presynaptic stimulation was applied to the medial perforant pathway of the dentate gyrus using an insulated bipolar tungsten wire electrode at a baseline test frequency of 0.033 Hz, and field excitatory postsynaptic potentials (EPSPs) were recorded from the middle one-third of the molecular layer of the dentate gyrus with a glass microelectrode. The inner blade of the dentate gyrus was used in all experiments. In each experiment, an input–output curve (afferent stimulus intensity vs. EPSP amplitude) was plotted at
Metab Brain Dis
the test frequency. For all experiments, the amplitude of the test EPSP was adjusted to one-third of the maximum (~1.0 mV). LTP was evoked by high-frequency stimulation (HFS) consisting of two trains of 100 stimuli at100 Hz with an inter-train interval of 20 s. In experiments involving the application of Aβ (oligomers and monomers), slices were perfused with Aβ for 40 min before HFS. For experiments involving AOSC, slices were exposed to the agents for 90 min before HFS. For all experiments, recordings of the effects of the tested agents and control recordings were made from slices of the same hippocampus.
Statistical analysis Recordings were analyzed using p-CLAMP (Axon Instruments, Sunnyvale, CA, USA). Values are reported as the mean ± standard error of the mean (SEM) for n slices. Analysis of variance (ANOVA) was used for statistical comparison, and P0.05) (Fig. 2b), whereas application of 1.5 μM AOSC partially reversed Aβ1–40 oligomer-mediated LTP inhibition, resulting in a change in field EPSP amplitude of 134.17± 3.09 % at 60 min post-HFS, a value significantly different from control and Aβ1–40 oligomer-treated conditions (n=6, P0.05 compared with control slices). To investigate whether AOSC reduce Aβ oligomer levels, we analysed the composition of the solution by ELISA, the results demonstrated that AOSC significantly blocked the formation of Aβ oligomers
when compared to the controls. The 17–49 kD and 4.5 kD band was shown in with and without AOSC pre-treatment, respectively (Fig. 3c).
Discussion In the present study, we have demonstrated that the inhibitory effects of Aβ oligomers on hippocampal LTP in the adult brain are reversible and that LTP can be restored by AOSC dose dependently in vitro. By contrast, AOSC did not affect Fig. 2 The protective effects of AOSC on Aβ oligomer-mediated inhibition of LTP induction are dose dependent. a LTP induction in the presence of 2.5 μM AOSC alone (open circles) is not significantly different from control LTP (closed circles). b LTP induction in the presence of 500 nM oligomeric Aβ plus 500 nM AOSC (open circles) did not prevent Aβ oligomer inhibition of LTP, whereas co-application of 500 nM oligomeric Aβ and 1.5 μM AOSC (filled circles) partially blocked the inhibition of LTP. c LTP induction in the presence of 500 nM oligomeric Aβ plus 2.5 μM (open circles) or 5 μM (filled circles) AOSC was not significantly reduced compared with controls. The insets are representative traces showing field EPSPs recorded before (a) and after (b) HFS. d Summary bar graph showing the change in field EPSP amplitude (% of control) after HFS for the various AOSC concentrations. *P
