J Mol Neurosci (2014) 52:124–130 DOI 10.1007/s12031-013-0216-0
Amyloid Beta-Derived Diffusible Ligands (ADDLs) Induce Abnormal Expression of Insulin Receptors in Rat Hippocampal Neurons Xin Liu & Zongyan Teng & Can Cui & Ruitao Wang & Meiling Liu & Yina Zhang
Received: 13 October 2013 / Accepted: 18 December 2013 / Published online: 10 January 2014 # Springer Science+Business Media New York 2014
Abstract Amyloid beta (Aβ) is an important pathogenic factor in Alzheimer’s disease (AD). In this study, we investigated the hypothesis that administration of amyloid-derived diffusible ligands (ADDLs) prepared from a synthetic Aβ1-42 amyloid peptide can cause defective expression of insulin receptors (IRs). To this end, primary rat hippocampal neurons were treated with various concentrations of ADDLs and expression levels of IRs were measured using real-time PCR and western blots. In these experiments, the expression of IRs significantly increased following treatment with low concentrations of Aβ142. In contrast, when higher concentrations of Aβ1-42 were applied, the number of apoptotic cells present increased, and expression of IRs significantly decreased. In combination, these results suggest that ADDLs is able to induce abnormal expression of IRs and interrupt normal insulin signaling, thereby potentially contributing to central insulin resistance that can occur during progression of AD.
Keywords Alzheimer’s disease . Aβ1-42 . Insulin receptor . Insulin resistance
Abbreviations ADDLs Aβ AD IRs
Amyloid beta-derived diffusible ligands Amyloid beta Alzheimer’s disease Insulin receptors
Xin Liu and Zongyan Teng contributed to this work equally. X. Liu : Z. Teng : C. Cui : R. Wang : M. Liu : Y. Zhang (*) Department of Geriatrics, the Second Hospital of Harbin Medical University, Harbin, Heilongjiang 150081, China e-mail: [email protected]
Introduction Alzheimer’s disease (AD) is a neurodegenerative disease that is characterized by progressive cognitive dysfunction and memory damage. Moreover, it is well-recognized that the deposition of amyloid-beta (Aβ) plaques in the brain is a significant event in the development of AD (Joachim and Selkoe 1992; Querfurth and LaFerla 2010; Yankner and Lu 2009). For example, deposition of Aβ plaques can cause a series of biochemical reactions which result in the generation of neurofibrillary tangles and neuron degradation due to apoptosis (Hernandez et al. 2010; Jin et al. 2011; Schrag et al. 2008). However, the exact mechanisms responsible for these processes remain unclear. In recent years, a great deal of evidence has suggested that AD is strongly associated with abnormal energy metabolism (Rhein and Eckert 2007). In particular, aberrant glucose metabolism and disrupted insulin signaling (Mosconi et al. 2009; Wang et al. 2010) have been strongly associated with the deposition of amyloid protein (Ling et al. 2002). Based on the complexity of insulin signaling in the brain, Aβ may potentially interfere with various molecular functions via different signaling pathways (Rhein and Eckert 2007; Takeda et al. 2011; Youssef et al. 2008). Insulin receptors (IRs) are members of a transmembrane, tetrameric (2α, 2β) tyrosine kinase receptor family and are widely expressed in the central nervous system, especially in the cortex and hippocampus (Kar et al. 1993; Marks et al. 1990). When brain autopsies of AD patients have been performed, a decrease in expression of IRs has been detected (Steen et al. 2005). Moreover, Hoyer et al. found that when IR disensitization occurred in response to lower insulin levels, a cascade of pathological processes was triggered. However, it remains unknown whether changes in IR expression occurs in the early stages of AD.
J Mol Neurosci (2014) 52:124–130
Therefore, in this study, expression of IRs by hippocampal neurons treated with amyloid-derived diffusible ligands (ADDLs) prepared from a synthetic Aβ1-42 amyloid peptide was examined.
Materials and Methods
125
were used to visualize NSE; FITC-conjugated secondary antibodies and confocal laser scanning microscopy (Nikon) were used to detect MAP2- and GAP-43-positive cells. Under a high-power microscopic view, five fields were randomly selected and the number of positive neurons per 100 cells per field were counted and recorded. Mean values were used to calculate hippocampal cell purity.
Animals Preparation of Amyloid-Derived Diffusible Ligands Both male and female Wistar neonate rats (between 1 and 3 days old) were provided by the Experimental Animal Center of the Second Affiliated Hospital of Harbin Medical University. All animal procedures were performed according to the guidelines of the local ethical committee of Health and the National Institutes of Health, and were approved by the Harbin Medical University, Heilongjiang Institutional Animal Care and Use Committee. Isolation and Culture of Rat Neonate Hippocampal Neurons Primary cultures of rat hippocampal neurons were obtained as previously described with minor modifications (Jordan et al. 1998). Briefly, neonate rats were sacrificed using carbon dioxide inhalation, and the hippocampal was removed and dissociated with trypsin. When a single-cell suspension was achieved, it was subsequently incubated in DMEM/F12 medium (Hyclone, Shanghai, China) containing 10 % fetal bovine serum (Invitrogen, Shanghai, China) in a cell culture flask pre-coated with 0.01 % poly-L-lysine (Sigma-Aldrich, Shanghai, China) at a density of 5−10×105 cells/mL. After 24 h, the medium was completely replaced with serum-free culture medium containing 97 % Neurobasal and 2 % B27 (Invitrogen, Shanghai, China). This medium was replaced every 2–3 days. Cell growth was confirmed using an inverted phase contrast microscope (Olympus, Japan), and cells were cultured up to 7 days for experiments. Identification of Hippocampal Neurons Briefly, cells were fixed with 4 % paraformaldehyde for 1 h at room temperature (RT), then were treated with 1 % TritonX100 for 30 min. Cells were blocked with 10 % sheep serum in PBS for 20 min at 37 °C, followed by incubation with primary antibodies at 4 °C for 18 h. The primary antibodies used included neuron-specific enolase (NSE; Boster, Wuhan, China) (1:100), growth-associated protein 43 (GAP-43; Abcam, Shanghai, China) (1:250), and microtubuleassociated protein (MAP2; Santa Cruz, Peking, China) (1:100). After being washed three times with 0.01 % sheep serum/PBS, the corresponding secondary antibodies and chromogenic reagents were added. Horseradish peroxidaselabeled secondary antibodies and diaminobenzidine (DAB)
ADDLs were prepared using a synthetic Aβ1-42 peptide (Sigma-Aldrich, Shanghai, China). Briefly, the Aβ1-42 peptide was dissolved in 1,1,1,3,3,3-Hexafluoro-2-propanol to 1 mM, then the solvent was evaporated and the peptide was stored on a dried film at −80 °C. When needed, this film was resuspended in DMSO to a final concentration of 5 mM, vortexed thoroughly, and sonicated for 10 min. The resulting solution was diluted with ice-cooled, phenol red-free Ham’s F12 medium to 200 μM and stored at 4 °C overnight to allow Aβ oligomers to form. The oligomer solution was subsequently centrifuged briefly, and the supernatant containing soluble Aβ oligomers (e.g., ADDLs) was collected.
Treatment of Hippocampal Neurons with ADDLs and Determination of Cell Viability Primary hippocampal neurons were cultured for 7 days then divided into five groups to be treated with various concentrations of ADDLs (e.g., 20, 30, 60, and 100 μM). Cells were incubated for 24 h, and untreated hippocampal neurons were used as a control. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assays were used to detect cell viability. Briefly, cells were cultured in 96-well plates (104 cells/well), with five replicates plated for each treatment. Each well received 20 μl MTT (5 mg/mL) and plates were incubated at 37 °C for 4 h. Cells were then dissolved in 150 μL DMSO, and OD values at 570 nm were recorded using a spectrophotometer. Cell survival rates were calculated as the ODexp/ODcontrol ×100 %.
Flow Cytometric Analysis Apoptosis was detected following the double staining of cells with FITC-labeled Annexin Vand propidium iodide (PI) (BD, Shanghai, China). Analysis of cell populations by flow cytometry (Beckman) included the detection of AnnexinV-/PI+ cells (e.g., machine-damaged cells), AnnexinV-/PI- cells (e.g., normal cells), AnnexinV+/PI- cells (e.g., early apoptotic cells), and AnnexinV+/PI+cells (e.g., late apoptotic cells or secondary necrotic cells). Cells were stained in triplicate.
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Real-Time PCR Real-time PCR (RT-PCR) was carried out using a reverse transcription kit (Takara DRRO37). The reaction conditions included 37 °C for 15 min and 85 °C for 5 s. SYBR Green RTPCR amplification was performed using the SYBR Premix Ex Taq kit (Takara DRR081) in a volume of 20 μL with the following reaction conditions: pre-denaturing at 95 °C for 30 s, denaturing at 95 °C for 5 s, annealing (β-actin at 54 °C, IR at 56 °C) for 15 s, and elongation at 72 °C for 10 s. A total of 40 cycles were completed. Melting curve analysis involved incubations at 72 °C for 3 min and 25 °C for 2 s. Primers used to amplify IRs included 5′-AAGGCGAG AAGACCATTGATT-3′ (forward) and 5′-CACCAGAGCA TAGGAGCGAC-3′ (reverse). These primers generated a fragment of 185 bp. For the housekeeping gene, β-actin, the primers for amplification included 5′-CCCATCTATGAGGG TTACGC-3′ (forward) and 5′-TTTAATGTCACGCACGAT TTC-3′ (reverse). These primers generated a fragment of 150 bp. Both combinations of primer sequences were designed by the Shanghai Shanjing Research Institute of Molecular Biology. All amplified DNA products were electrophoresed on 2.5 % agarose gels, and were compared with DNA markers using an ultraviolet transilluminator to confirm the specificity of the PCR reactions completed. To assure the amplification efficiency of target and reference genes, all cDNAs were diluted according to a ten-fold concentration gradient. Dilutions of 10°, 101, 102, 103, 104, and 105 were then amplified, and a standard curve was generated based on differences between both CT values. All R2 values were ≥0.99. When the slope was closer to zero, the amplification efficiency of the two genes was consistent and the results had comparability. Calculations of relative quantification were performed using 2−△△CT (Livak and Schmittgen 2001), and gene expression levels determined for the control group were set to one. Relative gene expression levels for the experimental group were also calculated using 2−△△CT. Western Blotting Proteins were extracted from cells using lysis buffer containing phenylmethanesulfonyl fluoride (PMSF; 100:1) on ice. Following centrifugation, supernatants were collected and proteins were denatured at 95 °C for 5 min. Protein samples were then separated using 10 % SDS-PAGE electrophoresis (Amerisco, Shanghai, China) and transferred to 0.45 μm PVDF membranes. Ponceau stain was used to evaluate transfer efficiency. Membranes were blocked with TBST containing 5 % dried skim milk, and then were incubated with monoclonal mouse anti-rat IR antibodies (Santa Cruz, Peking, China; 1:200) at 4 °C overnight (e.g., ≥18 h). After the membranes were washed, alkaline phosphatase-labeled
J Mol Neurosci (2014) 52:124–130
secondary antibodies (1:500) were incubated with the membranes at RT for 1 h. The BCIP/NBT Alkaline Phosphatase Color Development (Promega, Madison, WI) was used as a chromogen reagent and detection of β-actin was used as an internal reference. Imaging of membranes were processed using Quantity One software, with the results semiquantitatively analyzed using the lane/strip track quantitative method. Statistical Analysis All data were expressed as mean ± standard deviation (SD). Homogeneity was tested for variance, and one-way analysis of the variance and Student-Newman-Keuls test were used to compare between each group. A P value less than 0.05 was considered statistically significant. Statistical treatments were performed using SPSS 13.0 software.
Results Identification of Cultured Hippocampal Neurons To ensure our experimental results would be reliable, the purity of primary cultures of rat hippocampal neurons was examined. After 7 days of culturing, cells exhibited rich processes and well-developed neural networks (Fig. 1a). Immunohistochemistry staining further detected brownyellow granules present in the cytoplasm of NSE-positive cells (Fig. 1b). GAP-43 was detected in cell bodies and axons (Fig. 1c), while MAP2 expression was detected in dendrites (Fig. 1d). The percentages of NSE-positive, GAP-43-positive, and MAP2-positive cells was 96.13±6.4, 95.20±5.2, and 96.78±4.9 %, respectively. In combination, these data indicate that the cultured cells obtained were hippocampal neurons. ADDLs Reduced the Viability of Hippocampal Neurons To examine the effect of ADDLs on hippocampal neurons, cell viability assays were performed. As shown in Fig. 2, treatment of hippocampal neurons with ADDLs resulted in a significant dose-dependent decrease in cell viability. For example, except for the 10-μM group, the cell viability of hippocampal neurons treated with 20-100-μM ADDLs was observed to decrease significantly compared to the control group (P
Amyloid Beta-Derived Diffusible Ligands (ADDLs) Induce Abnormal Expression of Insulin Receptors in Rat Hippocampal Neurons Xin Liu & Zongyan Teng & Can Cui & Ruitao Wang & Meiling Liu & Yina Zhang
Received: 13 October 2013 / Accepted: 18 December 2013 / Published online: 10 January 2014 # Springer Science+Business Media New York 2014
Abstract Amyloid beta (Aβ) is an important pathogenic factor in Alzheimer’s disease (AD). In this study, we investigated the hypothesis that administration of amyloid-derived diffusible ligands (ADDLs) prepared from a synthetic Aβ1-42 amyloid peptide can cause defective expression of insulin receptors (IRs). To this end, primary rat hippocampal neurons were treated with various concentrations of ADDLs and expression levels of IRs were measured using real-time PCR and western blots. In these experiments, the expression of IRs significantly increased following treatment with low concentrations of Aβ142. In contrast, when higher concentrations of Aβ1-42 were applied, the number of apoptotic cells present increased, and expression of IRs significantly decreased. In combination, these results suggest that ADDLs is able to induce abnormal expression of IRs and interrupt normal insulin signaling, thereby potentially contributing to central insulin resistance that can occur during progression of AD.
Keywords Alzheimer’s disease . Aβ1-42 . Insulin receptor . Insulin resistance
Abbreviations ADDLs Aβ AD IRs
Amyloid beta-derived diffusible ligands Amyloid beta Alzheimer’s disease Insulin receptors
Xin Liu and Zongyan Teng contributed to this work equally. X. Liu : Z. Teng : C. Cui : R. Wang : M. Liu : Y. Zhang (*) Department of Geriatrics, the Second Hospital of Harbin Medical University, Harbin, Heilongjiang 150081, China e-mail: [email protected]
Introduction Alzheimer’s disease (AD) is a neurodegenerative disease that is characterized by progressive cognitive dysfunction and memory damage. Moreover, it is well-recognized that the deposition of amyloid-beta (Aβ) plaques in the brain is a significant event in the development of AD (Joachim and Selkoe 1992; Querfurth and LaFerla 2010; Yankner and Lu 2009). For example, deposition of Aβ plaques can cause a series of biochemical reactions which result in the generation of neurofibrillary tangles and neuron degradation due to apoptosis (Hernandez et al. 2010; Jin et al. 2011; Schrag et al. 2008). However, the exact mechanisms responsible for these processes remain unclear. In recent years, a great deal of evidence has suggested that AD is strongly associated with abnormal energy metabolism (Rhein and Eckert 2007). In particular, aberrant glucose metabolism and disrupted insulin signaling (Mosconi et al. 2009; Wang et al. 2010) have been strongly associated with the deposition of amyloid protein (Ling et al. 2002). Based on the complexity of insulin signaling in the brain, Aβ may potentially interfere with various molecular functions via different signaling pathways (Rhein and Eckert 2007; Takeda et al. 2011; Youssef et al. 2008). Insulin receptors (IRs) are members of a transmembrane, tetrameric (2α, 2β) tyrosine kinase receptor family and are widely expressed in the central nervous system, especially in the cortex and hippocampus (Kar et al. 1993; Marks et al. 1990). When brain autopsies of AD patients have been performed, a decrease in expression of IRs has been detected (Steen et al. 2005). Moreover, Hoyer et al. found that when IR disensitization occurred in response to lower insulin levels, a cascade of pathological processes was triggered. However, it remains unknown whether changes in IR expression occurs in the early stages of AD.
J Mol Neurosci (2014) 52:124–130
Therefore, in this study, expression of IRs by hippocampal neurons treated with amyloid-derived diffusible ligands (ADDLs) prepared from a synthetic Aβ1-42 amyloid peptide was examined.
Materials and Methods
125
were used to visualize NSE; FITC-conjugated secondary antibodies and confocal laser scanning microscopy (Nikon) were used to detect MAP2- and GAP-43-positive cells. Under a high-power microscopic view, five fields were randomly selected and the number of positive neurons per 100 cells per field were counted and recorded. Mean values were used to calculate hippocampal cell purity.
Animals Preparation of Amyloid-Derived Diffusible Ligands Both male and female Wistar neonate rats (between 1 and 3 days old) were provided by the Experimental Animal Center of the Second Affiliated Hospital of Harbin Medical University. All animal procedures were performed according to the guidelines of the local ethical committee of Health and the National Institutes of Health, and were approved by the Harbin Medical University, Heilongjiang Institutional Animal Care and Use Committee. Isolation and Culture of Rat Neonate Hippocampal Neurons Primary cultures of rat hippocampal neurons were obtained as previously described with minor modifications (Jordan et al. 1998). Briefly, neonate rats were sacrificed using carbon dioxide inhalation, and the hippocampal was removed and dissociated with trypsin. When a single-cell suspension was achieved, it was subsequently incubated in DMEM/F12 medium (Hyclone, Shanghai, China) containing 10 % fetal bovine serum (Invitrogen, Shanghai, China) in a cell culture flask pre-coated with 0.01 % poly-L-lysine (Sigma-Aldrich, Shanghai, China) at a density of 5−10×105 cells/mL. After 24 h, the medium was completely replaced with serum-free culture medium containing 97 % Neurobasal and 2 % B27 (Invitrogen, Shanghai, China). This medium was replaced every 2–3 days. Cell growth was confirmed using an inverted phase contrast microscope (Olympus, Japan), and cells were cultured up to 7 days for experiments. Identification of Hippocampal Neurons Briefly, cells were fixed with 4 % paraformaldehyde for 1 h at room temperature (RT), then were treated with 1 % TritonX100 for 30 min. Cells were blocked with 10 % sheep serum in PBS for 20 min at 37 °C, followed by incubation with primary antibodies at 4 °C for 18 h. The primary antibodies used included neuron-specific enolase (NSE; Boster, Wuhan, China) (1:100), growth-associated protein 43 (GAP-43; Abcam, Shanghai, China) (1:250), and microtubuleassociated protein (MAP2; Santa Cruz, Peking, China) (1:100). After being washed three times with 0.01 % sheep serum/PBS, the corresponding secondary antibodies and chromogenic reagents were added. Horseradish peroxidaselabeled secondary antibodies and diaminobenzidine (DAB)
ADDLs were prepared using a synthetic Aβ1-42 peptide (Sigma-Aldrich, Shanghai, China). Briefly, the Aβ1-42 peptide was dissolved in 1,1,1,3,3,3-Hexafluoro-2-propanol to 1 mM, then the solvent was evaporated and the peptide was stored on a dried film at −80 °C. When needed, this film was resuspended in DMSO to a final concentration of 5 mM, vortexed thoroughly, and sonicated for 10 min. The resulting solution was diluted with ice-cooled, phenol red-free Ham’s F12 medium to 200 μM and stored at 4 °C overnight to allow Aβ oligomers to form. The oligomer solution was subsequently centrifuged briefly, and the supernatant containing soluble Aβ oligomers (e.g., ADDLs) was collected.
Treatment of Hippocampal Neurons with ADDLs and Determination of Cell Viability Primary hippocampal neurons were cultured for 7 days then divided into five groups to be treated with various concentrations of ADDLs (e.g., 20, 30, 60, and 100 μM). Cells were incubated for 24 h, and untreated hippocampal neurons were used as a control. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assays were used to detect cell viability. Briefly, cells were cultured in 96-well plates (104 cells/well), with five replicates plated for each treatment. Each well received 20 μl MTT (5 mg/mL) and plates were incubated at 37 °C for 4 h. Cells were then dissolved in 150 μL DMSO, and OD values at 570 nm were recorded using a spectrophotometer. Cell survival rates were calculated as the ODexp/ODcontrol ×100 %.
Flow Cytometric Analysis Apoptosis was detected following the double staining of cells with FITC-labeled Annexin Vand propidium iodide (PI) (BD, Shanghai, China). Analysis of cell populations by flow cytometry (Beckman) included the detection of AnnexinV-/PI+ cells (e.g., machine-damaged cells), AnnexinV-/PI- cells (e.g., normal cells), AnnexinV+/PI- cells (e.g., early apoptotic cells), and AnnexinV+/PI+cells (e.g., late apoptotic cells or secondary necrotic cells). Cells were stained in triplicate.
126
Real-Time PCR Real-time PCR (RT-PCR) was carried out using a reverse transcription kit (Takara DRRO37). The reaction conditions included 37 °C for 15 min and 85 °C for 5 s. SYBR Green RTPCR amplification was performed using the SYBR Premix Ex Taq kit (Takara DRR081) in a volume of 20 μL with the following reaction conditions: pre-denaturing at 95 °C for 30 s, denaturing at 95 °C for 5 s, annealing (β-actin at 54 °C, IR at 56 °C) for 15 s, and elongation at 72 °C for 10 s. A total of 40 cycles were completed. Melting curve analysis involved incubations at 72 °C for 3 min and 25 °C for 2 s. Primers used to amplify IRs included 5′-AAGGCGAG AAGACCATTGATT-3′ (forward) and 5′-CACCAGAGCA TAGGAGCGAC-3′ (reverse). These primers generated a fragment of 185 bp. For the housekeeping gene, β-actin, the primers for amplification included 5′-CCCATCTATGAGGG TTACGC-3′ (forward) and 5′-TTTAATGTCACGCACGAT TTC-3′ (reverse). These primers generated a fragment of 150 bp. Both combinations of primer sequences were designed by the Shanghai Shanjing Research Institute of Molecular Biology. All amplified DNA products were electrophoresed on 2.5 % agarose gels, and were compared with DNA markers using an ultraviolet transilluminator to confirm the specificity of the PCR reactions completed. To assure the amplification efficiency of target and reference genes, all cDNAs were diluted according to a ten-fold concentration gradient. Dilutions of 10°, 101, 102, 103, 104, and 105 were then amplified, and a standard curve was generated based on differences between both CT values. All R2 values were ≥0.99. When the slope was closer to zero, the amplification efficiency of the two genes was consistent and the results had comparability. Calculations of relative quantification were performed using 2−△△CT (Livak and Schmittgen 2001), and gene expression levels determined for the control group were set to one. Relative gene expression levels for the experimental group were also calculated using 2−△△CT. Western Blotting Proteins were extracted from cells using lysis buffer containing phenylmethanesulfonyl fluoride (PMSF; 100:1) on ice. Following centrifugation, supernatants were collected and proteins were denatured at 95 °C for 5 min. Protein samples were then separated using 10 % SDS-PAGE electrophoresis (Amerisco, Shanghai, China) and transferred to 0.45 μm PVDF membranes. Ponceau stain was used to evaluate transfer efficiency. Membranes were blocked with TBST containing 5 % dried skim milk, and then were incubated with monoclonal mouse anti-rat IR antibodies (Santa Cruz, Peking, China; 1:200) at 4 °C overnight (e.g., ≥18 h). After the membranes were washed, alkaline phosphatase-labeled
J Mol Neurosci (2014) 52:124–130
secondary antibodies (1:500) were incubated with the membranes at RT for 1 h. The BCIP/NBT Alkaline Phosphatase Color Development (Promega, Madison, WI) was used as a chromogen reagent and detection of β-actin was used as an internal reference. Imaging of membranes were processed using Quantity One software, with the results semiquantitatively analyzed using the lane/strip track quantitative method. Statistical Analysis All data were expressed as mean ± standard deviation (SD). Homogeneity was tested for variance, and one-way analysis of the variance and Student-Newman-Keuls test were used to compare between each group. A P value less than 0.05 was considered statistically significant. Statistical treatments were performed using SPSS 13.0 software.
Results Identification of Cultured Hippocampal Neurons To ensure our experimental results would be reliable, the purity of primary cultures of rat hippocampal neurons was examined. After 7 days of culturing, cells exhibited rich processes and well-developed neural networks (Fig. 1a). Immunohistochemistry staining further detected brownyellow granules present in the cytoplasm of NSE-positive cells (Fig. 1b). GAP-43 was detected in cell bodies and axons (Fig. 1c), while MAP2 expression was detected in dendrites (Fig. 1d). The percentages of NSE-positive, GAP-43-positive, and MAP2-positive cells was 96.13±6.4, 95.20±5.2, and 96.78±4.9 %, respectively. In combination, these data indicate that the cultured cells obtained were hippocampal neurons. ADDLs Reduced the Viability of Hippocampal Neurons To examine the effect of ADDLs on hippocampal neurons, cell viability assays were performed. As shown in Fig. 2, treatment of hippocampal neurons with ADDLs resulted in a significant dose-dependent decrease in cell viability. For example, except for the 10-μM group, the cell viability of hippocampal neurons treated with 20-100-μM ADDLs was observed to decrease significantly compared to the control group (P
