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Aβ25–35 induces presynaptic changes in organotypic hippocampal slice cultures
NeuroToxicology 29 (2008) 691–699
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NeuroToxicology
Ab25–35 induces presynaptic changes in organotypic hippocampal slice cultures Eun Cheng Suh a, Yeon Joo Jung a,b, Yul A. Kim a, Eun Mi Park a, Kyung Eun Lee a,* a b
Department of Pharmacology, Medical Research Institute, School of Medicine, Ewha Womans University, 911-1, Mok-6-Dong, Yangcheon-Gu, Seoul 158-710, South Korea Department of Biochemistry, School of Medicine, Chungnam National University, Daejeon 301-747, South Korea
A R T I C L E I N F O
A B S T R A C T
Article history: Received 22 September 2007 Accepted 7 April 2008 Available online 27 May 2008
Memory loss in Alzheimer’s disease (AD) may be related to synaptic defects in damaged hippocampal neurons. We investigated the relationship between amyloid peptide Ab25–35-induced neuronal death pattern and presynaptic changes in organotypic hippocampal slice cultures. In propidium iodide (PI) uptake and annexin V labeling, Ab25–35-induced neuronal damage dramatically increased in a concentration dependent manner, indicating both types of cell death. In ultrastructural analysis, apoptotic features in CA1 and CA3 area and synaptic disruption in stratum lucidum were detected in Ab25–35-treated slices. Immunofluorescence and Western blot analysis for caspase-3 showed Ab25–35 concentration dependently induced caspase-3 activation. Immunofluorescence and Western blot analysis to determine changes in presynaptic marker proteins demonstrated that expression of synaptosomal-associated protein-25 (SNAP-25) and synaptophysin were reduced by Ab25–35 in CA1, CA3 and DG area at concentrations >2.5 mM. In conclusion, Ab25–35-induced apoptotic cell death and caspase-3 activation at relatively low concentration, and induced synaptic disruption and loss of synaptic marker protein at concentrations >2.5 mM in organotypic hippocampal slice cultures. These suggest that Ab25–35-induced apoptosis via triggering caspase-3 activation and lead to synaptic dysfunction in organotypic hippocampal slice cultures. ß 2008 Elsevier Inc. All rights reserved.
Keywords: Ab25–35 Apoptosis Presynaptic dysfunction Organotypic hippocampal slice culture
1. Introduction Alzheimer’s disease (AD) is characterized by neuronal degeneration, which leads to dysfunction and loss of the synapses that are involved in learning and memory (Cirrito et al., 2005; Greber et al., 1999; Minger et al., 2001; Yankner, 1996). The pathological hallmark of AD includes accumulation of amyloid b peptide (Ab) deposits (i.e., senile plaques) in the brain (Hardy, 1997; Hardy and Selkoe, 2002; Mattson, 2004; Selkoe, 1991). Increased Ab deposition probably contributes to the demise of neurons because Ab is toxic to neurons and greatly increases neuronal vulnerability to oxidative and metabolic stress and excitotoxicity (Dickson, 2004; Mattson, 1997; Mattson et al., 1998). Previous studies have shown that many neurons in the AD brain undergo apoptosis and Ab toxicity can be mediated by triggering intracellular apoptotic cascade including caspase-3 (Chan and Mattson, 1999; Fan et al., 2005). Activation of caspase-3 triggered apoptotosis in hippocampal neurons by Ab-induced neurotoxicity
* Corresponding author. Tel.: +82 2 2650 5744; fax: +82 2 2653 5076. E-mail address: [email protected] (K.E. Lee). 0161-813X/$ – see front matter ß 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.neuro.2008.04.001
and blocked by estrogen (Park et al., 2007; Sur et al., 2003). Activity of caspases is increased in vulnerable regions of the AD brain even before synaptic loss, tau phosphorylation and neuronal degeneration. Active caspases may be involved in the cytoskeletal disorganization and degeneration of neurons in AD (Cotman et al., 2005; Cribbs et al., 2004). Increasing evidence indicates that dysfunction and loss of nerve terminals might represent one of the earliest modifications in the course of neurodegenerative diseases (Wishart et al., 2006). Synaptic activity and synaptic vesicle release are related to extracellular Ab level (Chauhan and Siegel, 2002; Cirrito et al., 2005; Craft et al., 2004; Dickson, 1997; Greber et al., 1999; Masliah, 1995; Masliah et al., 1993, 1994; Pike et al., 1995). In AD, the loss of synaptic markers, such as SNAP-25 or synaptophysin is better parameter that correlates with memory dysfunction than neuronal loss (Selkoe, 2002). SNAP25 (‘‘synaptosome-associated protein of 25 kDa’’) is a presynaptic nerve terminal protein involved in vesicle exocytosis (DelgadoMartinez et al., 2007) and synaptophysin is a synaptic vesicle glycoprotein that is involved in the release of neurotransmitter vesicles (Fuentes-Santamaria et al., 2007; Calabrese et al., 2007) have demonstrated that Ab induces rapid cellular synaptic changes including reduction of synaptophysin in hippocampal neurons.
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Fig. 1. Annexin V labeling and propidium iodide (PI) uptake in organotypic hippocampal slice cultures (OHSCs) treated with Ab25–35. (A–C) Control slices. (D–F) 10 nM Ab25–35-treated OHSCs. (G–I) 100 nM Ab25–35-treated OHSCs. (J–L) 1 mM Ab25–35-treated OHSCs. (M–O) 2.5 mM Ab25–35-treated OHSCs. (P–R) 5 mM Ab25–35-treated OHSCs. Representative images of annexin V labeling (green, left), PI uptake (red, middle), and the merged images (right). Annexin V and PI fluorescence increased in a concentration-dependent manner. In contrast to PI uptake, annexin V labeling was observed after low concentrations (10, 100 nM) of Ab25–35 were applied to OHSCs. Scale bar = 200 mm.
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Even though some reports show that synapses, which are highly dependent on intact cytoskeletal structure and protein phosphorylation, are particularly vulnerable in AD, it is difficult to ascertain the relationship between neuronal death and presynaptic dysfunction. In this study, we hypothesized that Ab25–35 induces neuronal damage via apoptotic cell death and presynaptic changes in rat organotypic hippocampal slice culture system. 2. Materials and methods 2.1. Preparation of organotypic hippocampal slice cultures Techniques for culturing hippocampal slices have been described in detail (Gahwiler et al., 1997; Jung et al., 2004; Stoppini et al., 1991). The brains of 10-day-old Sprague–Dawley rats were removed and immersed in ice-cold dissecting medium. Hippocampi were isolated and cut into 300 mm transverse slices with a McIlwain tissue chopper (Mickle Laboratory Engineering, Surrey, UK). The slices were placed on semiporous Millicell membranes in plastic inserts (0.4 mm, Millicell-CM, Millipore, Bedford, MA) and transferred to a 37 8C 5% CO2 incubator in six-well culture trays (Falcon, Becton Dickinson, Franklin Lakes, NJ) with 1.2 mL serum-containing culture medium in each well. The medium was composed of 50% Opti-MEM, 25% Hanks’ balanced salt solution, 25% horse serum, and supplemented with glucose to a final concentration of 25 mM. After 2–4 days, the medium was replaced with serum-free Neurobasal medium (Invitrogen, Carlsbad, CA). The medium was changed twice weekly for the subsequent 3 weeks until the experiments commenced (Noraberg et al., 1999).
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2.4. Ultrastructural analysis with transmission electron microscopy (TEM) The cultured slices were fixed with 2.5% glutaraldehyde and 2% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). Slices were postfixed in 1% osmium tetroxide, dehydrated gradually in ethanol, and embedded with Epon 812. Semi-thin sections (100 nm) were cut from tissue blocks and stained with 0.5% toluidine blue. The most superficial stratum lucidum and mossy fiber layers were identified by meningeal tissue for subsequent thin sectioning. The ultrathin sections were stained with 0.25% lead citrate and 2% uranyl acetate in 50% methanol and were observed with an electron microscope (Hitachi H7650, Hitachi, Tokyo, Japan). 2.5. Protein extraction and Western blot analysis Cultured slices were lysed in 80 mL of ice-cold lysis buffer (50 mM Tris–HCl, 1% IGEPAL, 0.25% deoxycholic acid, 150 mM
2.2. Treatment of Ab25–35 to organotypic hippocampal slice cultures Ab25–35 (Sigma, St. Louis, MO) was dissolved in sterile deionized water to a concentration of 500 mM and incubated at 37 8C for 24 h to obtain the aggregated form. Varying concentrations (10, 100 nM, 1, 2.5, 5 mM) of the Ab25–35 solution were applied to culture medium for 3 days. 2.3. Assessment of cell death by Annexin V-FITC and PI staining Annexin V was detected with the Annexin V-FITC Apoptosis Detection Kit (Calbiochem, La Jolla, CA) according to the manufacturer’s instructions. 20 mL media binding regent, 2 mL Annexin V-FITC, and 20 mg/mL propidium iodide (PI) were added to the culture medium and incubated in the dark for 20 min at 37 8C. After wash with binding buffer, images were obtained with inverted fluorescence microscopy (Zeiss Axiovert 200, Zeiss, Go¨ttingen, Germany). Image intensity was analyzed using Image J program (National Institutes of Health, Bethesda, MD). For quantification, a region of interest (ROI) was selected from bright field images of each slice. Relative cell death was calculated from each ROI as follows: relative percent cell death = (Fexp Fmin)/(Fmax Fmin) 100, where Fexp is the fluorescence in the test condition, Fmax the maximum fluorescence in cultured slices that were immersed in 4 8C phosphate-buffered saline (PBS) to kill all cells, and Fmin is the background fluorescence prior to preconditioning and excluding the pyramidal cell layer (Xu et al., 2002). Data were analyzed by ANOVA using Statview version 5 softwares (SAS Institute, Cary, NC). A Fisher’s post hoc test was used to determine the significance. p-Values <0.05 were considered to be statistically significant.
Fig. 2. Region-specific quantitative analysis of annexin V and PI fluorescence. Annexin h labeling and PI uptake increased in a dose-dependent manner at selected areas, including CA1 (A), CA3 (B), and DG (C). The symbols indicate significant differences compared to control values (*) and to different Ab25–35 concentrations (+). p < 0.05, one-way ANOVA on ranks followed by Fisher’s PLSD method; n = 8.
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NaCl, 1 mM EGTA, 1 mM NaF, pH 7.4) with protease inhibitor (Roche, Penzberg, Germany) and briefly sonicated. Aliquots of the homogenate were used to determine protein concentration. Samples were boiled for 5 min, and 20 mg of protein was electrophoresed on a 12% SDS-PAGE gel. Protein was transferred onto blotting membranes (0.45 mm, PVDF, Millipore, Billerica, MA), blocked for 1 h with Tris-buffered saline Tween (TBST; 20 nM Tris, 500 nM NaCl, 0.05% Tween 20) containing 10% skim milk and probed overnight at 4 8C with mouse anti-caspase-3 (monoclonal IgG2a, 1:200; Santa Cruz Biotechnology, Santa Cruz, CA), mouse anti-SNAP-25 (monoclonal IgG1, 1:500; Oncogene Research Products, San Diego, CA) and mouse anti-synaptophysin (monoclonal IgG1, 1:1000; Chemicon International, Temecula, CA).
Immunoreactive bands were visualized on X-ray film (Super RX Fuji Film; Hanimax, Stafford, Qld.) using chemiluminescence reagent (Santa Cruz Biotechnology, Santa Cruz, CA). For the loading control, membranes were stripped and re-probed with an anti-actin antibody (1:1000; Santa Cruz Biotechnology, Santa Cruz, CA). Total protein values for caspase-3, SNAP-25 and synaptophysin measured with aliquots from the same slice sample, were normalized to actin. The results were expressed as a percentage to control value (mean S.E.M.). Data were analyzed by ANOVA using Statview version 5 softwares (SAS Institute, Cary, NC). A Fisher’s post hoc test was used to determine which means were significantly different from the control mean. p-Values <0.05 were considered to be statistically significant.
Fig. 3. Transmission electron microscopic images of CA1 and CA3 area in control and 2.5 mM Ab25–35-treated slices. (A) Stratum pyramidale of CA1 in control slice. (B) Stratum pyramidale of CA1 in 2.5 mM Ab25–35-treated slice. (C) Stratum pyramidale of CA3 in control slice. (D) Stratum pyramidale of CA3 in 2.5 mM Ab25–35-treated slice. (E) Stratum lucidum of CA3 in control slice. (F) Stratum lucidum of CA3 in 2.5 mM Ab25–35-treated slice. Control slices were morphologically intact. In Ab25–35-treated slices, neurofilament aggregation was observed in stratum pyramidale of CA1 and CA3 (white arrow head). Some apoptotic cells were observed (*). Some disrupted dendrites and synapses were noticed in stratum lucidum of CA3 area (white arrow) compared to the synapses in control slices (black arrow).
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2.6. Immunofluorescence staining Cultured slices were fixed for 2 h with 4% paraformaldehyde (Merck, Darmstadt, Germany) in 0.1 M phosphate buffer (PB). The slices were then incubated at 4 8C for 1 h in blocking solution containing 0.2% Triton X-100 in 10% bovine serum albumin in PBS. Slices were incubated for 24 h at 4 8C in the following primary antibodies: mouse caspase-3 (monoclonal IgG2a, 1:500; Santa Cruz Biotechnology, Santa Cruz, CA), mouse anti-synaptophysin (monoclonal IgG1, 1:1000; Chemicon International Billerica, MA), mouse anti-SNAP-25 (monoclonal IgG1, 1:500; Oncogene Research Products, Cambridge, MA). After three washes with PBS, anti-mouse or anti-rabbit secondary antibodies conjugated to either fluorescein anti-mouse IgG (FITC, Vectorlab, Burlingame, CA) or Alexa 568 goat anti-mouse IgG2a (Molecular Probes, Eugene, OR) were applied to the slices. Slices were mounted with Elvanol mounting medium (Waterborne, New Orleans, LA), and images were obtained with a confocal microscopy (Zeiss LSM 510, Zeiss, Jena, Germany) or inverted fluorescence microscopy (Axiovert 200, Zeiss Go¨ttingen, Germany). 3. Results 3.1. Induction of neuronal death by Ab25–35 in organotypic hippocampal slice cultures We pre-tested Ab25–35-induced neuronal toxicity at 3 and 7 days according to PI staining method. Because neuronal damage was too extensive in case of 7 day-treatment of Ab25–35 (data not shown), all the experiments were undertaken at 3 day-treatment model. Annexin V and PI staining was hardly observed in control slices (Fig. 1A–C). Ab25–35 caused a concentration-dependent increase in cell death (Fig. 1D–R). Annexin V fluorescence was detected in CA1 after application of 10 nM Ab25–35 (Fig. 1D and F), and PI staining was observed after application of 100 nM Ab25–35 (Fig. 1H) in CA1. Cell death was massive in CA1, CA3, and DG at 2.5 mM (Fig. 1M–O) and 5 mM Ab25–35 (Fig. 1P–R). Quantitative assessment of the concentration–effect relationship for Ab25–35-induced cell death showed that Ab25–35 increased cell death in CA1 at concentrations >1 mM. Cell death in CA3 (Fig. 2B) and DG (Fig. 2C) varied in severity but was always less pronounced than cell death in CA1 (Fig. 2A). 3.2. Analysis of ultrastructural changes by Ab25–35 in organotypic hippocampal slice cultures In control slices, pyramidal neurons in CA1 (Fig. 3A) and CA3 (Fig. 3C) had round nuclei with evenly distributed chromatin and clear nucleoli, well-developed rough endoplasmic reticulum, mitochondria, polyribosomes, and Golgi apparatus. The ultrastructure of stratum pyramidale in CA3 area was similar to that in CA1 area except larger cell bodies of CA3 neurons. After 2.5 mM Ab25–35 treated, neurons with high electron density, neurofilament aggregation, many vacuoles, chromatin aggregation and shrinkage of cytoplasm were observed in CA1 (Fig. 3B) and CA3 area (Fig. 3D), indicating apoptosis. Microglia were frequently found in close contact with these shrinking neurons. The ultrastructure of synaptic membrane in stratum lucidum of control slices was intact and vesicles were centered on presynaptic active zone (Fig. 3E). In 2.5 mM Ab25–35-treated slices, some disrupted synaptic membranes and less postsynaptic density were observed (Fig. 3F). 3.3. Induction of caspase-3 by Ab25–35 in organotypic hippocampal slice cultures To determine whether Ab25–35-induced apoptosis in hippocampal slice cultures occurs via activation of caspase-3, we
Fig. 4. Western blot analysis of caspase-3 in Ab25–35-treated and control slices. (A) Representative image of caspase-3 in Western blot. The immunoblot showed a visible increase in protein after application of Ab25–35. (B) Quantitative analysis of caspase-3 (35 kDa, n = 4) with the Image J program. Densitometric analysis was based on b-actin levels. Data are expressed as a percentage to control value (mean S.E.M.). The symbols indicate significant differences compared to control values (*). p < 0.05, one-way ANOVA on ranks followed by Fisher’s PLSD method.
examined Western blot analysis and immunofluorescence staining with confocal imaging. Data are expressed as the percent increase of caspase-3 in Ab25–35-treated organotypic hippocampal slice cultures (Fig. 4). Total protein of activated caspase-3 in cultured hippocampal slices increased in a concentration dependent manner (Fig. 4). The immunoreactivity of caspase-3 in CA1, CA3, and DG area of control slices was present in a small subset of pyramidal and granular neurons as small punctuates (Fig. 5A–C). Ab25–35 increased immunoreactivity of caspase-3 in CA1, CA3, and DG area in a concentration dependent manner (Fig. 5D–L). 3.4. Alteration of presynaptic proteins by Ab25–35 in organotypic hippocampal slice cultures Western blots had typical bands of the appropriate molecular weights in control and Ab25–35-treated slices. Data are presented as the percent decrease of synaptic protein in Ab25–35-treated organotypic hippocampal slice cultures. Decreased levels of SNAP25 and synaptophysin in Ab25–35-treated compared to control slices were observed (Fig. 6). Ab25–35-induced decrease was significant at 2.5 and 5 mM Ab25–35. In control slices, immunoreactivity of SNAP-25 and synaptophysin were well detected in CA1, CA3, and DG area (Fig. 7A–C, G– I). Stratum lucidum of CA3 (Fig. 7B) and hilar region of DG (Fig. 7C) or granular cell layer of DG (Fig. 7I) were lightly stained to SNAP-25 or synaptophysin, respectively. After treatment of Ab25–35, the immunoreactivity of SNAP-25 and synaptophysin was attenuated in CA1, CA3, and DG area (Fig. 7D–F, J–L). A mild decrease of SNAP25 and synaptophysin immunoreactivity was observed after application of 1 mM Ab25–35, and the decrease in SNAP-25 and synaptophysin immunoreactivity was apparent at 2.5 and 5 mM Ab25–35. 4. Discussion 4.1. Treatment with Ab25–35 causes apoptotic cell death in organotypic hippocampal slice cultures Organotypic hippocampal slice culture (OHSC) has become an increasingly popular tool to study neurodegenerative disorders including AD (Bonde et al., 2002; Gahwiler et al., 1997; Holopainen
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Fig. 5. Confocal image of caspase-3 in Ab25–35-treated and control slices. (A–C) Control slices. (D–F) 1 mM Ab25–35-treated slices. (G–I) 2.5 mM Ab25–35-treated slices. (J–L) 5 mM Ab25–35-treated slices. (A, D, G, J) CA1 area. (B, E, H, K) CA3 area. (C, F, I, L) DG area. Immunoreactivity of caspase-3 increased in CA1, CA3, and DG area in a concentration dependent manner. Scale bar = 20 mm.
et al., 2004; Ito et al., 2003; Kim et al., 2002; Laake et al., 1999; Li et al., 1997; Noraberg et al., 1999), because OHSC offer several advantages over primary cultures, which makes them attractive for investigating the anatomical and physiological integration of newly generated granule cells into the hippocampal network (Jung et al., 2004; Kim et al., 2002; Xu et al., 2002). We established OHSC model for studying neuronal death and synaptic function. In AD brain, the proteolytic processing of APP is changed, resulting in accumulation of neurotoxic forms of Ab in brain (Hardy, 1997; Mattson, 2004). Ab25–35 is a short synthetic peptide of Ab which is suitable to use in studying Ab toxicity. It is highly
toxic and forms fibrillar aggregates typical of b-amyloid. (Hughes et al., 2000). It is known to have properties similar to Ab1–40 and Ab1–42 (Pike et al., 1995). In our pre-experiment with primary hippocampal neuronal cell culture, Ab25–35 showed similar neuronal cell death pattern to Ab1–40 and Ab1–42 (data not shown). Annexin V is widely used for detection of apoptosis, because it binds to phosphatidylserine transposed to the outer membrane leaflet during the apoptotic process (van Engeland et al., 1998; Vincent and Maiese, 1999). PI is a membrane impermeable fluorescent dye known to bind to nucleic acid (Bonde et al., 2002; Jung et al., 2004; Laake et al., 1999; Macklis and Madison,
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Fig. 6. Western blot analysis of SNAP-25 and synaptophysin reactivity in Ab25–35-treated and control slices. (A) Representative image of SNAP-25 and synaptophysin in Western blot. The immunoblot showed a visible reduction in protein after application of Ab25–35. (B) Quantitative analysis of SNAP-25 (25 kDa, n = 4) and synaptophysin (38 kDa, n = 4) with the Image J program. Densitometric analysis was based on b-actin levels. Data are expressed as a percentage to control value (mean S.E.M.). The symbols indicate significant differences compared to control values (*). p < 0.05, one-way ANOVA on ranks followed by Fisher’s PLSD method.
Fig. 7. Immunofluorescence image of SNAP-25 and synaptophysin in Ab25–35-treated and control slices. (A–C) Immunoreactivity of SNAP-25 in control slices, (D–F) immunoreactivity of SNAP-25 in 2.5 mM Ab25–35-treated slices, (G–I) immunoreactivity of synaptophysin in control slices and (J–L) immunoreactivity of synaptophysin in 2.5 mM Ab25–35-treated slices. Scale bar of CA1 (A, D, G, J) = 200 mm; scale bar of CA3 and DG = 100 mm. After 2.5 mM Ab25–35 treatment, the immunoreactivity of SNAP-25 and synaptophysin decreased in CA1, CA3, and DG area.
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1990). But it rapidly enters cells with damaged membranes, rendering them brightly fluorescent (Brana et al., 2002). In this study, the distribution and intensity of annexin V and PI fluorescence showed that Ab25–35 dose-dependently increased cell death in CA1, CA3 and DG area, indicating both types of cell death. Neuronal damage in CA3 and DG varied in severity, but was always less profound than CA1, suggesting regional vulnerability (Figs. 1 and 2). Ultrastructural features in stratum pyramidale of CA1 and CA3 also showed that Ab25–35-induced chromatin aggregation and shrinkage of cytoplasm, indicating apoptosis (Fig. 3). Ab peptides by triggering intracellular apoptotic cascade can induce toxicity to a variety of cell lines and primary rat and human cultured neurons (Cotman et al., 2005; Cribbs et al., 2004), and many neurons undergoing apoptosis in AD shows high levels of activated apoptotic proteins, such as caspase-3 and Bax (Mattson, 2000, 2004; Mukaetova-Ladinska et al., 2000; Satou et al., 1995). Caspase activation is a relatively early event in apoptosis (Mattson, 2000; Perez-Navarro et al., 2005; Yuan, 1997). Activity of caspases may increase in vulnerable regions of the AD brain even before synaptic loss, tau phosphorylation and neuronal degeneration (Cotman et al., 2005; Cribbs et al., 2004). The toxic potency of the peptide is related to its ability to form specific oligomeric forms and/or insoluble aggregates which depend to some extent on the concentration of Ab peptide used (Fuentealba et al., 2004; Kim et al., 2003; Smith et al., 2006). Our data showed that Ab25–35-induced activation in caspase-3 was evident at 1 mM, increased at 2.5 mM, and prominent at 5 mM (Figs. 4 and 5). Taken together, these suggested that caspase-3 could be induced even at low concentration of Ab25–35 stimulation. 4.2. Treatment with Ab25–35 causes presynaptic protein alteration by triggering apoptosis In our ultrastructural study of stratum lucidum, 2.5 mM Ab25–35-induced synaptic disruption (Fig. 3E and F). One of the key features of AD is loss of synapses and neurons in hippocampus and cortex, leading to cognitive dysfunction (Lopez and DeKosky, 2003; Selkoe and Schenk, 2003). Brain regions with plaques typically exhibit reduced numbers of synapses, and neurites associated with the plaques are often damaged, suggesting that Ab induces damage to synapses and neurites (Mattson, 2004; Palop et al., 2005; Satou et al., 1995; Sun and Alkon, 2002). In AD, the loss of synaptic markers, such as SNAP-25 or synaptophysin is better parameter that correlates with memory dysfunction than neuronal loss (Selkoe, 2002). The presynaptic plasma membrane-associated protein, SNAP25, labels synapses and is transported to axonal terminals by fast axoplasmic flow (Masliah et al., 1994; Masliah, 1995; Greber et al., 1999) have demonstrated that decreased levels of SNAP-25 in Down syndrome or AD patients cause a secondary neuronal damage, resulting in functional loss or impaired synaptogenesis. Our data demonstrated that total protein level and immunoreactivity of SNAP-25 and synaptophysin decreased at 2.5 and 5 mM Ab25–35 in a dose-dependent manner (Figs. 6 and 7). This study showed that Ab25–35-induced apoptotic cell death and caspase-3 activation at relatively low concentration, and induced synaptic disruption and loss of synaptic marker protein at concentrations >2.5 mM in organotypic hippocampal slice cultures. In conclusion, Ab25–35-induced apoptosis via triggering caspase-3 activation and lead to synaptic dysfunction in organotypic hippocampal slice cultures.
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Contents lists available at ScienceDirect
NeuroToxicology
Ab25–35 induces presynaptic changes in organotypic hippocampal slice cultures Eun Cheng Suh a, Yeon Joo Jung a,b, Yul A. Kim a, Eun Mi Park a, Kyung Eun Lee a,* a b
Department of Pharmacology, Medical Research Institute, School of Medicine, Ewha Womans University, 911-1, Mok-6-Dong, Yangcheon-Gu, Seoul 158-710, South Korea Department of Biochemistry, School of Medicine, Chungnam National University, Daejeon 301-747, South Korea
A R T I C L E I N F O
A B S T R A C T
Article history: Received 22 September 2007 Accepted 7 April 2008 Available online 27 May 2008
Memory loss in Alzheimer’s disease (AD) may be related to synaptic defects in damaged hippocampal neurons. We investigated the relationship between amyloid peptide Ab25–35-induced neuronal death pattern and presynaptic changes in organotypic hippocampal slice cultures. In propidium iodide (PI) uptake and annexin V labeling, Ab25–35-induced neuronal damage dramatically increased in a concentration dependent manner, indicating both types of cell death. In ultrastructural analysis, apoptotic features in CA1 and CA3 area and synaptic disruption in stratum lucidum were detected in Ab25–35-treated slices. Immunofluorescence and Western blot analysis for caspase-3 showed Ab25–35 concentration dependently induced caspase-3 activation. Immunofluorescence and Western blot analysis to determine changes in presynaptic marker proteins demonstrated that expression of synaptosomal-associated protein-25 (SNAP-25) and synaptophysin were reduced by Ab25–35 in CA1, CA3 and DG area at concentrations >2.5 mM. In conclusion, Ab25–35-induced apoptotic cell death and caspase-3 activation at relatively low concentration, and induced synaptic disruption and loss of synaptic marker protein at concentrations >2.5 mM in organotypic hippocampal slice cultures. These suggest that Ab25–35-induced apoptosis via triggering caspase-3 activation and lead to synaptic dysfunction in organotypic hippocampal slice cultures. ß 2008 Elsevier Inc. All rights reserved.
Keywords: Ab25–35 Apoptosis Presynaptic dysfunction Organotypic hippocampal slice culture
1. Introduction Alzheimer’s disease (AD) is characterized by neuronal degeneration, which leads to dysfunction and loss of the synapses that are involved in learning and memory (Cirrito et al., 2005; Greber et al., 1999; Minger et al., 2001; Yankner, 1996). The pathological hallmark of AD includes accumulation of amyloid b peptide (Ab) deposits (i.e., senile plaques) in the brain (Hardy, 1997; Hardy and Selkoe, 2002; Mattson, 2004; Selkoe, 1991). Increased Ab deposition probably contributes to the demise of neurons because Ab is toxic to neurons and greatly increases neuronal vulnerability to oxidative and metabolic stress and excitotoxicity (Dickson, 2004; Mattson, 1997; Mattson et al., 1998). Previous studies have shown that many neurons in the AD brain undergo apoptosis and Ab toxicity can be mediated by triggering intracellular apoptotic cascade including caspase-3 (Chan and Mattson, 1999; Fan et al., 2005). Activation of caspase-3 triggered apoptotosis in hippocampal neurons by Ab-induced neurotoxicity
* Corresponding author. Tel.: +82 2 2650 5744; fax: +82 2 2653 5076. E-mail address: [email protected] (K.E. Lee). 0161-813X/$ – see front matter ß 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.neuro.2008.04.001
and blocked by estrogen (Park et al., 2007; Sur et al., 2003). Activity of caspases is increased in vulnerable regions of the AD brain even before synaptic loss, tau phosphorylation and neuronal degeneration. Active caspases may be involved in the cytoskeletal disorganization and degeneration of neurons in AD (Cotman et al., 2005; Cribbs et al., 2004). Increasing evidence indicates that dysfunction and loss of nerve terminals might represent one of the earliest modifications in the course of neurodegenerative diseases (Wishart et al., 2006). Synaptic activity and synaptic vesicle release are related to extracellular Ab level (Chauhan and Siegel, 2002; Cirrito et al., 2005; Craft et al., 2004; Dickson, 1997; Greber et al., 1999; Masliah, 1995; Masliah et al., 1993, 1994; Pike et al., 1995). In AD, the loss of synaptic markers, such as SNAP-25 or synaptophysin is better parameter that correlates with memory dysfunction than neuronal loss (Selkoe, 2002). SNAP25 (‘‘synaptosome-associated protein of 25 kDa’’) is a presynaptic nerve terminal protein involved in vesicle exocytosis (DelgadoMartinez et al., 2007) and synaptophysin is a synaptic vesicle glycoprotein that is involved in the release of neurotransmitter vesicles (Fuentes-Santamaria et al., 2007; Calabrese et al., 2007) have demonstrated that Ab induces rapid cellular synaptic changes including reduction of synaptophysin in hippocampal neurons.
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Fig. 1. Annexin V labeling and propidium iodide (PI) uptake in organotypic hippocampal slice cultures (OHSCs) treated with Ab25–35. (A–C) Control slices. (D–F) 10 nM Ab25–35-treated OHSCs. (G–I) 100 nM Ab25–35-treated OHSCs. (J–L) 1 mM Ab25–35-treated OHSCs. (M–O) 2.5 mM Ab25–35-treated OHSCs. (P–R) 5 mM Ab25–35-treated OHSCs. Representative images of annexin V labeling (green, left), PI uptake (red, middle), and the merged images (right). Annexin V and PI fluorescence increased in a concentration-dependent manner. In contrast to PI uptake, annexin V labeling was observed after low concentrations (10, 100 nM) of Ab25–35 were applied to OHSCs. Scale bar = 200 mm.
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Even though some reports show that synapses, which are highly dependent on intact cytoskeletal structure and protein phosphorylation, are particularly vulnerable in AD, it is difficult to ascertain the relationship between neuronal death and presynaptic dysfunction. In this study, we hypothesized that Ab25–35 induces neuronal damage via apoptotic cell death and presynaptic changes in rat organotypic hippocampal slice culture system. 2. Materials and methods 2.1. Preparation of organotypic hippocampal slice cultures Techniques for culturing hippocampal slices have been described in detail (Gahwiler et al., 1997; Jung et al., 2004; Stoppini et al., 1991). The brains of 10-day-old Sprague–Dawley rats were removed and immersed in ice-cold dissecting medium. Hippocampi were isolated and cut into 300 mm transverse slices with a McIlwain tissue chopper (Mickle Laboratory Engineering, Surrey, UK). The slices were placed on semiporous Millicell membranes in plastic inserts (0.4 mm, Millicell-CM, Millipore, Bedford, MA) and transferred to a 37 8C 5% CO2 incubator in six-well culture trays (Falcon, Becton Dickinson, Franklin Lakes, NJ) with 1.2 mL serum-containing culture medium in each well. The medium was composed of 50% Opti-MEM, 25% Hanks’ balanced salt solution, 25% horse serum, and supplemented with glucose to a final concentration of 25 mM. After 2–4 days, the medium was replaced with serum-free Neurobasal medium (Invitrogen, Carlsbad, CA). The medium was changed twice weekly for the subsequent 3 weeks until the experiments commenced (Noraberg et al., 1999).
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2.4. Ultrastructural analysis with transmission electron microscopy (TEM) The cultured slices were fixed with 2.5% glutaraldehyde and 2% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). Slices were postfixed in 1% osmium tetroxide, dehydrated gradually in ethanol, and embedded with Epon 812. Semi-thin sections (100 nm) were cut from tissue blocks and stained with 0.5% toluidine blue. The most superficial stratum lucidum and mossy fiber layers were identified by meningeal tissue for subsequent thin sectioning. The ultrathin sections were stained with 0.25% lead citrate and 2% uranyl acetate in 50% methanol and were observed with an electron microscope (Hitachi H7650, Hitachi, Tokyo, Japan). 2.5. Protein extraction and Western blot analysis Cultured slices were lysed in 80 mL of ice-cold lysis buffer (50 mM Tris–HCl, 1% IGEPAL, 0.25% deoxycholic acid, 150 mM
2.2. Treatment of Ab25–35 to organotypic hippocampal slice cultures Ab25–35 (Sigma, St. Louis, MO) was dissolved in sterile deionized water to a concentration of 500 mM and incubated at 37 8C for 24 h to obtain the aggregated form. Varying concentrations (10, 100 nM, 1, 2.5, 5 mM) of the Ab25–35 solution were applied to culture medium for 3 days. 2.3. Assessment of cell death by Annexin V-FITC and PI staining Annexin V was detected with the Annexin V-FITC Apoptosis Detection Kit (Calbiochem, La Jolla, CA) according to the manufacturer’s instructions. 20 mL media binding regent, 2 mL Annexin V-FITC, and 20 mg/mL propidium iodide (PI) were added to the culture medium and incubated in the dark for 20 min at 37 8C. After wash with binding buffer, images were obtained with inverted fluorescence microscopy (Zeiss Axiovert 200, Zeiss, Go¨ttingen, Germany). Image intensity was analyzed using Image J program (National Institutes of Health, Bethesda, MD). For quantification, a region of interest (ROI) was selected from bright field images of each slice. Relative cell death was calculated from each ROI as follows: relative percent cell death = (Fexp Fmin)/(Fmax Fmin) 100, where Fexp is the fluorescence in the test condition, Fmax the maximum fluorescence in cultured slices that were immersed in 4 8C phosphate-buffered saline (PBS) to kill all cells, and Fmin is the background fluorescence prior to preconditioning and excluding the pyramidal cell layer (Xu et al., 2002). Data were analyzed by ANOVA using Statview version 5 softwares (SAS Institute, Cary, NC). A Fisher’s post hoc test was used to determine the significance. p-Values <0.05 were considered to be statistically significant.
Fig. 2. Region-specific quantitative analysis of annexin V and PI fluorescence. Annexin h labeling and PI uptake increased in a dose-dependent manner at selected areas, including CA1 (A), CA3 (B), and DG (C). The symbols indicate significant differences compared to control values (*) and to different Ab25–35 concentrations (+). p < 0.05, one-way ANOVA on ranks followed by Fisher’s PLSD method; n = 8.
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NaCl, 1 mM EGTA, 1 mM NaF, pH 7.4) with protease inhibitor (Roche, Penzberg, Germany) and briefly sonicated. Aliquots of the homogenate were used to determine protein concentration. Samples were boiled for 5 min, and 20 mg of protein was electrophoresed on a 12% SDS-PAGE gel. Protein was transferred onto blotting membranes (0.45 mm, PVDF, Millipore, Billerica, MA), blocked for 1 h with Tris-buffered saline Tween (TBST; 20 nM Tris, 500 nM NaCl, 0.05% Tween 20) containing 10% skim milk and probed overnight at 4 8C with mouse anti-caspase-3 (monoclonal IgG2a, 1:200; Santa Cruz Biotechnology, Santa Cruz, CA), mouse anti-SNAP-25 (monoclonal IgG1, 1:500; Oncogene Research Products, San Diego, CA) and mouse anti-synaptophysin (monoclonal IgG1, 1:1000; Chemicon International, Temecula, CA).
Immunoreactive bands were visualized on X-ray film (Super RX Fuji Film; Hanimax, Stafford, Qld.) using chemiluminescence reagent (Santa Cruz Biotechnology, Santa Cruz, CA). For the loading control, membranes were stripped and re-probed with an anti-actin antibody (1:1000; Santa Cruz Biotechnology, Santa Cruz, CA). Total protein values for caspase-3, SNAP-25 and synaptophysin measured with aliquots from the same slice sample, were normalized to actin. The results were expressed as a percentage to control value (mean S.E.M.). Data were analyzed by ANOVA using Statview version 5 softwares (SAS Institute, Cary, NC). A Fisher’s post hoc test was used to determine which means were significantly different from the control mean. p-Values <0.05 were considered to be statistically significant.
Fig. 3. Transmission electron microscopic images of CA1 and CA3 area in control and 2.5 mM Ab25–35-treated slices. (A) Stratum pyramidale of CA1 in control slice. (B) Stratum pyramidale of CA1 in 2.5 mM Ab25–35-treated slice. (C) Stratum pyramidale of CA3 in control slice. (D) Stratum pyramidale of CA3 in 2.5 mM Ab25–35-treated slice. (E) Stratum lucidum of CA3 in control slice. (F) Stratum lucidum of CA3 in 2.5 mM Ab25–35-treated slice. Control slices were morphologically intact. In Ab25–35-treated slices, neurofilament aggregation was observed in stratum pyramidale of CA1 and CA3 (white arrow head). Some apoptotic cells were observed (*). Some disrupted dendrites and synapses were noticed in stratum lucidum of CA3 area (white arrow) compared to the synapses in control slices (black arrow).
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2.6. Immunofluorescence staining Cultured slices were fixed for 2 h with 4% paraformaldehyde (Merck, Darmstadt, Germany) in 0.1 M phosphate buffer (PB). The slices were then incubated at 4 8C for 1 h in blocking solution containing 0.2% Triton X-100 in 10% bovine serum albumin in PBS. Slices were incubated for 24 h at 4 8C in the following primary antibodies: mouse caspase-3 (monoclonal IgG2a, 1:500; Santa Cruz Biotechnology, Santa Cruz, CA), mouse anti-synaptophysin (monoclonal IgG1, 1:1000; Chemicon International Billerica, MA), mouse anti-SNAP-25 (monoclonal IgG1, 1:500; Oncogene Research Products, Cambridge, MA). After three washes with PBS, anti-mouse or anti-rabbit secondary antibodies conjugated to either fluorescein anti-mouse IgG (FITC, Vectorlab, Burlingame, CA) or Alexa 568 goat anti-mouse IgG2a (Molecular Probes, Eugene, OR) were applied to the slices. Slices were mounted with Elvanol mounting medium (Waterborne, New Orleans, LA), and images were obtained with a confocal microscopy (Zeiss LSM 510, Zeiss, Jena, Germany) or inverted fluorescence microscopy (Axiovert 200, Zeiss Go¨ttingen, Germany). 3. Results 3.1. Induction of neuronal death by Ab25–35 in organotypic hippocampal slice cultures We pre-tested Ab25–35-induced neuronal toxicity at 3 and 7 days according to PI staining method. Because neuronal damage was too extensive in case of 7 day-treatment of Ab25–35 (data not shown), all the experiments were undertaken at 3 day-treatment model. Annexin V and PI staining was hardly observed in control slices (Fig. 1A–C). Ab25–35 caused a concentration-dependent increase in cell death (Fig. 1D–R). Annexin V fluorescence was detected in CA1 after application of 10 nM Ab25–35 (Fig. 1D and F), and PI staining was observed after application of 100 nM Ab25–35 (Fig. 1H) in CA1. Cell death was massive in CA1, CA3, and DG at 2.5 mM (Fig. 1M–O) and 5 mM Ab25–35 (Fig. 1P–R). Quantitative assessment of the concentration–effect relationship for Ab25–35-induced cell death showed that Ab25–35 increased cell death in CA1 at concentrations >1 mM. Cell death in CA3 (Fig. 2B) and DG (Fig. 2C) varied in severity but was always less pronounced than cell death in CA1 (Fig. 2A). 3.2. Analysis of ultrastructural changes by Ab25–35 in organotypic hippocampal slice cultures In control slices, pyramidal neurons in CA1 (Fig. 3A) and CA3 (Fig. 3C) had round nuclei with evenly distributed chromatin and clear nucleoli, well-developed rough endoplasmic reticulum, mitochondria, polyribosomes, and Golgi apparatus. The ultrastructure of stratum pyramidale in CA3 area was similar to that in CA1 area except larger cell bodies of CA3 neurons. After 2.5 mM Ab25–35 treated, neurons with high electron density, neurofilament aggregation, many vacuoles, chromatin aggregation and shrinkage of cytoplasm were observed in CA1 (Fig. 3B) and CA3 area (Fig. 3D), indicating apoptosis. Microglia were frequently found in close contact with these shrinking neurons. The ultrastructure of synaptic membrane in stratum lucidum of control slices was intact and vesicles were centered on presynaptic active zone (Fig. 3E). In 2.5 mM Ab25–35-treated slices, some disrupted synaptic membranes and less postsynaptic density were observed (Fig. 3F). 3.3. Induction of caspase-3 by Ab25–35 in organotypic hippocampal slice cultures To determine whether Ab25–35-induced apoptosis in hippocampal slice cultures occurs via activation of caspase-3, we
Fig. 4. Western blot analysis of caspase-3 in Ab25–35-treated and control slices. (A) Representative image of caspase-3 in Western blot. The immunoblot showed a visible increase in protein after application of Ab25–35. (B) Quantitative analysis of caspase-3 (35 kDa, n = 4) with the Image J program. Densitometric analysis was based on b-actin levels. Data are expressed as a percentage to control value (mean S.E.M.). The symbols indicate significant differences compared to control values (*). p < 0.05, one-way ANOVA on ranks followed by Fisher’s PLSD method.
examined Western blot analysis and immunofluorescence staining with confocal imaging. Data are expressed as the percent increase of caspase-3 in Ab25–35-treated organotypic hippocampal slice cultures (Fig. 4). Total protein of activated caspase-3 in cultured hippocampal slices increased in a concentration dependent manner (Fig. 4). The immunoreactivity of caspase-3 in CA1, CA3, and DG area of control slices was present in a small subset of pyramidal and granular neurons as small punctuates (Fig. 5A–C). Ab25–35 increased immunoreactivity of caspase-3 in CA1, CA3, and DG area in a concentration dependent manner (Fig. 5D–L). 3.4. Alteration of presynaptic proteins by Ab25–35 in organotypic hippocampal slice cultures Western blots had typical bands of the appropriate molecular weights in control and Ab25–35-treated slices. Data are presented as the percent decrease of synaptic protein in Ab25–35-treated organotypic hippocampal slice cultures. Decreased levels of SNAP25 and synaptophysin in Ab25–35-treated compared to control slices were observed (Fig. 6). Ab25–35-induced decrease was significant at 2.5 and 5 mM Ab25–35. In control slices, immunoreactivity of SNAP-25 and synaptophysin were well detected in CA1, CA3, and DG area (Fig. 7A–C, G– I). Stratum lucidum of CA3 (Fig. 7B) and hilar region of DG (Fig. 7C) or granular cell layer of DG (Fig. 7I) were lightly stained to SNAP-25 or synaptophysin, respectively. After treatment of Ab25–35, the immunoreactivity of SNAP-25 and synaptophysin was attenuated in CA1, CA3, and DG area (Fig. 7D–F, J–L). A mild decrease of SNAP25 and synaptophysin immunoreactivity was observed after application of 1 mM Ab25–35, and the decrease in SNAP-25 and synaptophysin immunoreactivity was apparent at 2.5 and 5 mM Ab25–35. 4. Discussion 4.1. Treatment with Ab25–35 causes apoptotic cell death in organotypic hippocampal slice cultures Organotypic hippocampal slice culture (OHSC) has become an increasingly popular tool to study neurodegenerative disorders including AD (Bonde et al., 2002; Gahwiler et al., 1997; Holopainen
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Fig. 5. Confocal image of caspase-3 in Ab25–35-treated and control slices. (A–C) Control slices. (D–F) 1 mM Ab25–35-treated slices. (G–I) 2.5 mM Ab25–35-treated slices. (J–L) 5 mM Ab25–35-treated slices. (A, D, G, J) CA1 area. (B, E, H, K) CA3 area. (C, F, I, L) DG area. Immunoreactivity of caspase-3 increased in CA1, CA3, and DG area in a concentration dependent manner. Scale bar = 20 mm.
et al., 2004; Ito et al., 2003; Kim et al., 2002; Laake et al., 1999; Li et al., 1997; Noraberg et al., 1999), because OHSC offer several advantages over primary cultures, which makes them attractive for investigating the anatomical and physiological integration of newly generated granule cells into the hippocampal network (Jung et al., 2004; Kim et al., 2002; Xu et al., 2002). We established OHSC model for studying neuronal death and synaptic function. In AD brain, the proteolytic processing of APP is changed, resulting in accumulation of neurotoxic forms of Ab in brain (Hardy, 1997; Mattson, 2004). Ab25–35 is a short synthetic peptide of Ab which is suitable to use in studying Ab toxicity. It is highly
toxic and forms fibrillar aggregates typical of b-amyloid. (Hughes et al., 2000). It is known to have properties similar to Ab1–40 and Ab1–42 (Pike et al., 1995). In our pre-experiment with primary hippocampal neuronal cell culture, Ab25–35 showed similar neuronal cell death pattern to Ab1–40 and Ab1–42 (data not shown). Annexin V is widely used for detection of apoptosis, because it binds to phosphatidylserine transposed to the outer membrane leaflet during the apoptotic process (van Engeland et al., 1998; Vincent and Maiese, 1999). PI is a membrane impermeable fluorescent dye known to bind to nucleic acid (Bonde et al., 2002; Jung et al., 2004; Laake et al., 1999; Macklis and Madison,
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Fig. 6. Western blot analysis of SNAP-25 and synaptophysin reactivity in Ab25–35-treated and control slices. (A) Representative image of SNAP-25 and synaptophysin in Western blot. The immunoblot showed a visible reduction in protein after application of Ab25–35. (B) Quantitative analysis of SNAP-25 (25 kDa, n = 4) and synaptophysin (38 kDa, n = 4) with the Image J program. Densitometric analysis was based on b-actin levels. Data are expressed as a percentage to control value (mean S.E.M.). The symbols indicate significant differences compared to control values (*). p < 0.05, one-way ANOVA on ranks followed by Fisher’s PLSD method.
Fig. 7. Immunofluorescence image of SNAP-25 and synaptophysin in Ab25–35-treated and control slices. (A–C) Immunoreactivity of SNAP-25 in control slices, (D–F) immunoreactivity of SNAP-25 in 2.5 mM Ab25–35-treated slices, (G–I) immunoreactivity of synaptophysin in control slices and (J–L) immunoreactivity of synaptophysin in 2.5 mM Ab25–35-treated slices. Scale bar of CA1 (A, D, G, J) = 200 mm; scale bar of CA3 and DG = 100 mm. After 2.5 mM Ab25–35 treatment, the immunoreactivity of SNAP-25 and synaptophysin decreased in CA1, CA3, and DG area.
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1990). But it rapidly enters cells with damaged membranes, rendering them brightly fluorescent (Brana et al., 2002). In this study, the distribution and intensity of annexin V and PI fluorescence showed that Ab25–35 dose-dependently increased cell death in CA1, CA3 and DG area, indicating both types of cell death. Neuronal damage in CA3 and DG varied in severity, but was always less profound than CA1, suggesting regional vulnerability (Figs. 1 and 2). Ultrastructural features in stratum pyramidale of CA1 and CA3 also showed that Ab25–35-induced chromatin aggregation and shrinkage of cytoplasm, indicating apoptosis (Fig. 3). Ab peptides by triggering intracellular apoptotic cascade can induce toxicity to a variety of cell lines and primary rat and human cultured neurons (Cotman et al., 2005; Cribbs et al., 2004), and many neurons undergoing apoptosis in AD shows high levels of activated apoptotic proteins, such as caspase-3 and Bax (Mattson, 2000, 2004; Mukaetova-Ladinska et al., 2000; Satou et al., 1995). Caspase activation is a relatively early event in apoptosis (Mattson, 2000; Perez-Navarro et al., 2005; Yuan, 1997). Activity of caspases may increase in vulnerable regions of the AD brain even before synaptic loss, tau phosphorylation and neuronal degeneration (Cotman et al., 2005; Cribbs et al., 2004). The toxic potency of the peptide is related to its ability to form specific oligomeric forms and/or insoluble aggregates which depend to some extent on the concentration of Ab peptide used (Fuentealba et al., 2004; Kim et al., 2003; Smith et al., 2006). Our data showed that Ab25–35-induced activation in caspase-3 was evident at 1 mM, increased at 2.5 mM, and prominent at 5 mM (Figs. 4 and 5). Taken together, these suggested that caspase-3 could be induced even at low concentration of Ab25–35 stimulation. 4.2. Treatment with Ab25–35 causes presynaptic protein alteration by triggering apoptosis In our ultrastructural study of stratum lucidum, 2.5 mM Ab25–35-induced synaptic disruption (Fig. 3E and F). One of the key features of AD is loss of synapses and neurons in hippocampus and cortex, leading to cognitive dysfunction (Lopez and DeKosky, 2003; Selkoe and Schenk, 2003). Brain regions with plaques typically exhibit reduced numbers of synapses, and neurites associated with the plaques are often damaged, suggesting that Ab induces damage to synapses and neurites (Mattson, 2004; Palop et al., 2005; Satou et al., 1995; Sun and Alkon, 2002). In AD, the loss of synaptic markers, such as SNAP-25 or synaptophysin is better parameter that correlates with memory dysfunction than neuronal loss (Selkoe, 2002). The presynaptic plasma membrane-associated protein, SNAP25, labels synapses and is transported to axonal terminals by fast axoplasmic flow (Masliah et al., 1994; Masliah, 1995; Greber et al., 1999) have demonstrated that decreased levels of SNAP-25 in Down syndrome or AD patients cause a secondary neuronal damage, resulting in functional loss or impaired synaptogenesis. Our data demonstrated that total protein level and immunoreactivity of SNAP-25 and synaptophysin decreased at 2.5 and 5 mM Ab25–35 in a dose-dependent manner (Figs. 6 and 7). This study showed that Ab25–35-induced apoptotic cell death and caspase-3 activation at relatively low concentration, and induced synaptic disruption and loss of synaptic marker protein at concentrations >2.5 mM in organotypic hippocampal slice cultures. In conclusion, Ab25–35-induced apoptosis via triggering caspase-3 activation and lead to synaptic dysfunction in organotypic hippocampal slice cultures.
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