- Email: [email protected]
Effect of transition metals in synaptic damage induced by amyloid beta peptide
Neuroscience 170 (2010) 381–389
EFFECT OF TRANSITION METALS IN SYNAPTIC DAMAGE INDUCED BY AMYLOID BETA PEPTIDE R. M. URANGA, N. M. GIUSTO AND G. A. SALVADOR*
Alzheimer’s disease (AD) is clinically characterized by a progressive loss of cognitive skills and dementia (DeKosky and Scheff, 1990; Terry et al., 1991; Selkoe, 1996; Scheff and Price, 2003). AD brains typically present an extensive oxidative stress with augmented protein oxidation and lipid peroxidation, overproduction of reactive oxygen species (ROS), mitochondrial impairment, and oxidation of nucleic acids (Keller et al., 1997; Nunomura et al., 2001; Zhu et al., 2004). The increase in free radicals, whose major source is cellular energy metabolism, generates the activation of stress-response signaling mechanisms (Mattson and Liu, 2002). These changes occur very early in the onset of the disease and represent a very interesting target for prevention. Amyloid- peptide (A) has long been thought of as the major cause of AD, with A being considered as the triggering insult. Nowadays, there are two central and contrasting hypotheses regarding A and oxidative stress: on the one hand, it has been shown that A together with transition metals give rise to oxidative injury, but on the other hand, the peptide seems to have antioxidant properties in some conditions (Kontush, 2001; Smith et al., 2002; Atwood et al., 2003). The hypothesis that proposes A as the promoter of oxidative damage holds that oxidative stress is one of the first pathologic events in AD, and it would precede and even hasten the formation of senile plaques (Sayre et al., 2001). The contrasting hypothesis states that the age-related increase of A would be able to diminish the levels of oxidative stress, and it is based on several studies which have shown that the peptide is able to exert, in certain circumstances, neuroprotective effects (Atwood et al., 2003; Plant et al., 2003). A third hypothesis has gained leading role and proposes a failure in biometal homeostasis (e.g. iron, copper), together with A/metal interaction, as the key factor in triggering AD pathology (Huang et al., 2004). A high correlation between synapse loss and cognitive impairment has been described (DeKosky and Scheff, 1990; Terry et al., 1991; Selkoe, 1996; Scheff and Price, 2003) and synapses are held to be the sites where AD and AD-related neurodegenerative disorders are likely to be initiated. Signaling pathways have been found to be highly concentrated in the synaptic terminals so an abnormal signaling in this particular region of the neuron, the accretion of transition metals and an increased susceptibility to oxidative stress would be suspected to trigger this disorder. Cumulative evidence points to the effects of A and metal ions on neuronal cultures, but their real effect on the synapses and the precise synaptic signaling pathways
Instituto de Investigaciones Bioquímicas de Bahía Blanca, Universidad Nacional del Sur and Consejo Nacional de Investigaciones Científicas y Técnicas, Camino La Carrindanga Km 7, CC 857, B8000FWB Bahía Blanca, Argentina
Abstract—The amyloid -peptide (A), which is thought to be the major cause of Alzheimer’s disease (AD), is known to be capable of aggregating in different states: soluble monomers and oligomers, and insoluble aggregates. The A aggregation state as well as its toxicity has been related to the interaction between the peptide and transition metals such as iron and copper. However, this relationship, as well as the effects of A on the synaptic endings, is not fully understood. The aggregation states of A in the presence of iron and copper, as well as their effects on synaptic viability and signaling were investigated in this work. During acute incubation treatments (5 min– 4 h), A/metal impaired mitochondrial function to the same extent as has been observed with the metal alone. However, in the presence of A/iron (10 and 50 M), plasma membrane integrity was disrupted to a greater extent than when generated by either iron or A alone, indicating that the membrane constitutes the first target of synaptic injury. Akt activation by A/iron was evident after 5 min of incubation and was higher than that observed in the presence of the metal alone. This activation was barely detected after 4 h of incubation, demonstrating that there is no correlation between the extent of synaptic damage and the activation of this kinase. Extracellular signal-regulated kinases 1 and 2 (ERK1/2) activation profile was different from that observed for Akt. Accordingly, the presence of A/metal could differentially modulate the activity of these kinases. This work shows evidence of the initial events locally triggered at the synapse by A and transition metals. As synapses have been proposed as the starting point of A/metaltriggered events, the characterization of early mechanisms occurring in models that mimic AD could be important for the search of unexplored therapeutics tools. © 2010 IBRO. Published by Elsevier Ltd. All rights reserved. Key words: iron, copper, synaptosomes, Akt, ERK1/2, A peptide. *Corresponding author. Tel: ⫹54-291-4861201; fax: ⫹54-2914861200. E-mail address: [email protected] (G. A. Salvador). Abbreviations: AD, Alzheimer’s disease; ADDLs, amyloid-beta derived diffusible ligands; A, amyloid-beta peptide; DMSO, dimethyl sulfoxide; EDTA, ethylenediaminetetraacetic acid; EM, electron microscopy; ERK1/2, extracellular signal-regulated kinases 1 and 2; HEPES, 4-(2hydroxyethyl)-1-piperazine ethanesulfonic acid; HRP, horse raddish peroxidise; LDH, lactate dehydrogenase; MTT, 3-(4,5-dimethylthiazol2-yl)-2,5-diphenyltetrazolium bromide; NAD⫹, oxidized nicotinamide adenine dinucleotide; NADH, reduced nicotinamide adenine dinucleotide; PBS, phosphate-buffered saline; PI3K, phosphatidylinositol 3-kinase; PMSF, phenylmethylsulfonyl fluoride; PVDF, polyvinylidene fluoride; ROS, reactive oxygen species; SD, standard deviation; SDS, sodium dodecyl sulfate; SDS-PAGE, SDS-polyacrylamide gel electrophoresis; TBST, tris-buffered saline Tween-20.
0306-4522/10 $ - see front matter © 2010 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2010.07.044
381
382
R. M. Uranga et al. / Neuroscience 170 (2010) 381–389
activated by AD-simulating conditions have not yet been elucidated. Taking into account that many signaling pathways observed in intact cells can be observed in cells lacking nuclei (Jacobson et al., 1994), we used rat cortical synaptosomes to investigate signaling mechanisms activated locally in synapses by oxidative insult. The aim of this work is to study the effects of transition metals and/or A on phosphatidylinositol 3-kinase (PI3K)/Akt and extracellular signal-regulated kinases 1 and 2 (ERK1/2) pathways in synaptic endings from adult animals. Elucidation of synaptic mechanisms altered in these conditions could be important to delay the onset and/or the progression of neurodegenerative disorders which involve oxidative processes.
EXPERIMENTAL PROCEDURES Animals Wistar-strain adult (4 months of age) rats housed under controlled conditions (constant room temperature, 12-h light/12-h dark cycle), bred in our own colony and fed a standard rat chow diet with free access to water, were used. All the procedures were in strict accordance with the guidelines published in the National Institutes of Health Guide for the care and use of laboratory animals.
Materials
Beckman J2-21 centrifuge. Washed synaptosomal fraction was used for the experiments detailed below. Protein content of the synaptosomal fraction was determined by the method of Lowry et al., (1951).
Incubation of synaptosomes Synaptosomes were diluted in Locke’s buffer (154 mM NaCl, 5.6 mM KCl, 2.3 mM CaCl2, 1.0 mM MgCl2, 3.6 mM NaHCO3, 5 mM glucose, 5 mM HEPES, pH 7.2) for all experiments except where stated otherwise. Synaptosomal suspensions were aliquoted (2 mg protein/ml) into tubes and incubated at 37 °C under an O2:CO2 (95:5, v:v) atmosphere during experimental treatments.
Metals and A1-40 FeSO4 and CuSO4 were prepared as 10 mM stocks in water immediately prior to use. Rat amyloid- peptide 1– 40 (A1-40, Calbiochem, CA, USA) was resuspended in dimethyl sulfoxide (DMSO) at a concentration of 10 mg/ml and immediately stored in aliquots at ⫺20 °C. A oligomers/aggregates (80 M) were prepared according to previously published methods (Sepulveda et al., 2009; Parodi et al., 2010). Briefly, aliquots of peptide stock (250 g in 25 l of DMSO) were added to 700 l of phosphatebuffered saline (PBS, pH 7.4) and stirred continuously (300 rpm, at 37 °C) for 120 min and stored at 4 °C until use. Aliquots of the preparation obtained were analyzed by electron microscopy (EM) either alone or combined with one of the metal ions.
EM of A1-40 preparations
The kit (LDH-P UV AA) for measuring lactate dehydrogenase (LDH) activity was generously supplied by the Wiener Laboratory (Santa Fe, Argentina). Rabbit polyclonal anti-phospho-Ser473-Akt and anti-Akt antibodies were from Cell Signaling Technology (Beverly, MA, USA). Mouse anti-phospho-Tyr204-ERK1/2, rabbit anti-ERK2, polyclonal horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG, and polyclonal HRP-conjugated goat antimouse IgG were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). All other reagents were of analytical grade, purchased from Sigma-Aldrich (St. Louis, MO, USA) unless otherwise stated.
EM studies were carried out according to a method used by several laboratories with slight modifications (Santos et al., 2005; Wei et al., 2008; Parodi et al., 2010). Briefly, 1 M A1-40 was incubated in PBS at 37 °C, with or without the metals, for 5 and 30 min, and 1 and 4 h. After incubation, 10 L of media containing the peptide was placed on carbon-coated grids and incubated for 60 s. Ten microlitres of 0.5% glutaraldehyde was added to each grid and incubated for an additional 60 s. The grid was then washed with drops of water and dried. Finally, the grids were stained for 2 min with 2% uranyl acetate and then air-dried. Grids were subsequently examined in a Jeol 100 Cx II electron microscope.
Preparation of synaptosomal fraction and experimental treatments
MTT reduction assay
The purified synaptosomal fraction was obtained as previously described by Cotman, (1974), with slight modifications. Briefly, rats were sacrificed by decapitation, brains were removed on a cold plate, and the cerebral cortex was immediately dissected (2– 4 min after decapitation) and placed in 0.32 M sucrose isolation buffer containing 2 g/ml leupeptin, 1 g/ml pepstatin, 1 g/ml aprotinin, 1 mM dithiothreitol (DTT), 0.1 mM phenylmethylsulfonyl fluoride (PMSF), 1 mM ethylenediaminetetraacetic acid (EDTA), and 10 mM 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid (HEPES), pH 7.4. The cerebral cortex was homogenized by 10 strokes with a Thomas tissue homogenizer. The homogenate was then centrifuged at 1800 g for 7.5 min at 4 °C using a JA-21 rotor in a Beckman J2-21 centrifuge. The pellet was discarded, and the supernatant was retained and centrifuged at 14,000 g for 20 min at 4 °C. The resulting pellet was washed and resuspended in 3 ml of 0.32 M sucrose isolation buffer and layered over a discontinuous Ficoll gradient (8.5% pH 7.4, 13% pH 7.4 Ficoll solutions, each prepared in isolation buffer) and spun at 85,500 g for 30 min at 4 °C using an SW 28.1 rotor in a Beckman L5-50 ultracentrifuge. Synaptosomes in the 8.5–13% Ficoll interface were removed, resuspended in isolation buffer, and centrifuged at 33,000 g for 20 min at 4 °C using a JA-21 rotor in a
Neuronal viability was measured by determining cellular reducing capacity via the extent of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction to the insoluble intracellular Formazan, which depends on the activity of intracellular dehydrogenases and is independent of changes in the integrity of the plasma membrane. The methods employed in the present study were similar to those described previously by Keller et al., (1997). In brief, MTT was dissolved in PBS at a concentration of 5 mg/ml. The MTT solution was mixed with synaptosomes (1:10 MTT: synaptosomes, v:v) and allowed to incubate for 2 h at 37 °C. At the end of incubation with MTT, solubilization buffer [20% sodium dodecyl sulfate (SDS), pH 4.7] was added and mixed thoroughly to dissolve the crystals of Formazan. The extent of MTT reduction then was measured spectrophotometrically at 570 nm. Results are expressed as a percentage of control.
Measurement of LDH release After treatments, synaptosomes were centrifuged at 33,000 g for 20 min at 4 °C. The resulting supernatant was used to determine LDH activity, measured spectrophotometrically by using an LDH-P UV AA kit following the manufacturer’s instructions. Briefly, the rate of conversion of reduced nicotinamide adenine dinucleotide
R. M. Uranga et al. / Neuroscience 170 (2010) 381–389 (NADH) to oxidized nicotinamide adenine dinucleotide (NAD⫹) was followed at 340 nm. Results are expressed as a percentage of the control value.
SDS-PAGE and Western blot assays Samples were denatured with Laemmli sample buffer at 100 °C for 5 min (Laemmli, 1970). Equivalent amounts of synaptosomal proteins were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) on 10% polyacrylamide gels and then transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore, Bedford, MA, USA). Membranes were blocked with 5% nonfat dry milk in Tris-buffered saline Tween-20 (TBST) buffer [20 mM Tris–HCl (pH 7.4), 100 mM NaCl, and 0.1% (w/v) Tween 20] for 2 h at room temperature for all the Western blots assayed. Membranes were then incubated with primary antibodies [anti-phosphoSer473-Akt, anti-Akt, anti-phosphoTyr204-ERK1/2, anti-ERK2, (1:1000) overnight at 4 °C], washed three times with TBST, and then exposed to the appropriate HRP-conjugated secondary antibody (anti-rabbit or anti-mouse) for 1 h at room temperature. Membranes were again washed three times with TBST, and immunoreactive bands were detected by enhanced chemiluminescence (ECL; Amersham Biosciences) using standard X-ray film (Kodak X-Omat AR). Several different exposure times were used for each blot to ensure linearity of band intensities. Immunoreactive bands were quantified using image analysis software (ImageJ, a freely available application in the public domain for image analysis and processing, developed and maintained by Wayne Rasband at the Research Services Branch, National Institute of Mental Health).
383
Data analysis Quantitative results were expressed as the mean⫾standard deviation (SD) of the N indicated in the corresponding figures. Data were analyzed by one-way ANOVA followed by the Tukey multiple comparison test. P-values lower than 0.05 were considered statistically significant. Western blots shown are representative of at least three analyses performed on samples from at least three separate experiments.
RESULTS Aggregation of A1-40. Effect of the coincubation with metals Numerous studies have shown that neuronal response to A exposure depends exclusively on the aggregation state of the peptide. To assess the status of A1-40 aggregation by EM, 1 M A1-40 was incubated in Locke’s buffer at 37 °C for the same periods of time used for the toxicity model. Our EM photomicrographs show that incubation of A1-40 for 5 min and 1 h lead to spheroidal structures individually or in small groups (Fig. 1A, E). These ultrastructural forms were similar to a heterogeneous array of forms defined as oligomers, protofibrils and amyloid-beta derived diffusible ligands (ADDLs). The coincubation with transition metal ions such as copper (10 M) and iron (10 and 50 M) did not modify the aggregation state of the
Fig. 1. High power (⫻140,000) electron photomicrographs showing A and A/metal preparations. A was dissolved and prepared as described in Experimental procedures. 1 M A1-40 was incubated in PBS at 37 °C for 5 min (A), 1 h (E) and 4 h (I). A1-40 was also coincubated with 10 M copper, or 10 or 50 M iron in PBS at 37 °C for 5 min (B–D, respectively), and 1 (F–H, respectively) and 4 h (J–L, respectively). After incubation, 10 L of media containing the peptide was placed on carbon-coated grids and fixed with of 0.5% glutaraldehyde. The grids were stained with 2% uranyl acetate and then air-dried. The grids were subsequently examined in a Jeol 100 Cx II electron microscope. White arrows show spheroidal structures observed after 5 and 1 h of incubation (A–H). Black arrows show more organized structures present in the preparations after 4 h of incubation (I–L). Black arrows point at the amorphous clumps formed after incubating the A for 4 h either alone or with the metal ions (I–L). For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.
384
R. M. Uranga et al. / Neuroscience 170 (2010) 381–389
Table 1. MTT reduction and LDH leakage, time course in control conditions Time
MTT (arbitrary units)
LDH (arbitrary units)
0 min 5 min 30 min 1h 4h
1.060⫾0.035 1.062⫾0.046 1.055⫾0.027 1.016⫾0.039 0.986⫾0.037
0.053⫾0.006 0.055⫾0.004 0.058⫾0.003 0.058⫾0.003 0.113⫾0.004***
The reduction of MTT as well as LDH leakage was assayed in synaptosomes (2 mg protein/ml) exposed to vehicle (control) for 0, 5 and 30 min, and 1 and 4 h. Results are expressed in arbitrary units and represent the mean⫾SD; n⫽3– 6. *** P⬍0.001 with respect to the control.
peptide (Fig. 1B–D, 1F–H). However, Fig. 1I–L show that after 4 h of incubation, A1-40 appears in some sort of amorphous clumps or more organized structures but still without any evidence of fibril formation. Exposure of synaptic endings to A1-40 and transition metals: effects on viability A great body of evidence has shown that transition metals act as neurotoxic agents by inducing lipid peroxidation as well as mitochondrial dysfunction. However, information regarding the effects and mechanisms of action of A1-40 is quite controversial. Our first goal was to characterize the effect of FeSO4 (10 and 50 M), CuSO4 (10 M) and A1-40 (1 M) on synaptosomal viability after 5- and 30min, 1-h and 4-h incubations. To determine the consequence of exposing rat cerebral cortex synaptic endings to
either transition metals or A1-40, MTT reduction was evaluated as a measure of mitochondrial function. Control conditions were also assessed, replacing metals or A1-40 by an equal volume of water (vehicle) (Table 1). As shown in Fig. 2A, after 5 min of incubation, only iron (50 M) slightly but significantly decreased mitochondrial viability. A1-40 did not cause any significant change in MTT reduction when incubated with synaptosomes alone or when incubated in combination with the metals. Fig. 2B shows that after a 30-min exposure iron in both concentrations caused a marked decrease in mitochondrial viability, being the greatest damage caused by 50 M iron. When A1-40 was coincubated with iron or copper, the damage observed was essentially the same as that caused by each metal separately. However, A did not produce any damage at all by itself. The MTT reduction after 1 and 4 h of incubation (Fig. 2C, D, respectively) shows basically the same pattern of damage than that observed after a 30-min treatment, but the longer the time of incubation the higher the levels of injury generated by the metal ions (either alone or combined with A). In either case A did not cause any damage at all by itself. The evaluation of plasma membrane integrity is a useful tool for determining the degree of injury provoked by several stressors. Plasma membrane integrity was determined by monitoring the leakage of LDH to the extrasynaptosomal medium. As shown in Fig. 3A, after 5 min of exposure, the presence of iron was the only condition that caused a slight but significant increase in LDH leakage. A did not increase the damage to the membrane with respect to the control condition when incubated either alone or
Fig. 2. MTT reduction assay. The reduction of MTT was assayed in synaptosomes (2 mg protein/ml) exposed to vehicle (control), 10 and 50 M iron, 10 M copper, 1 M A1-40, 1 M A1-40 plus 10 M iron, 1 M A1-40 plus 50 M iron and 1 M A1-40 plus 10 M copper for 5 min (A), 30 min (B), 1 h (C), and 4 h (D). Results are expressed as percentage of control and represent the mean⫾SD; n⫽3– 6. *** P⬍0.001 and ** P⬍0.01 with respect to the control.
R. M. Uranga et al. / Neuroscience 170 (2010) 381–389
385
Fig. 3. LDH leakage. The release of LDH to the medium was measured in synaptosomes (2 mg protein/ml) exposed to vehicle (control), 10 and 50 M iron, 10 M copper, 1 M A1-40, 1 M A1-40 plus 10 M iron, 1 M A1-40 plus 50 M iron and 1 M A1-40 plus 10 M copper for 5 min (A), 30 min (B), 1 h (C), and 4 h (D). Results are expressed as percentage of control and represent the mean⫾SD; n⫽3– 6. *** P⬍0.001, ** P⬍0.01, and * P⬍0.05 with respect to the control; ### P⬍0.001 and # P⬍0.05 when comparing “1 M A1-40⫹metal” versus the “metal” condition.
combined with the metals. Fig. 3B, C show that also after 30 min and 1 h of incubation, respectively, only iron induced a significant damage to the membrane (higher than that observed after 5 min). No damage caused by A itself was observed. However, the coincubation of A with iron (50 M) for 1 h generated a slightly higher (but significant) damage, compared to that induced by 50 M iron alone. Copper did not introduce any change in synaptosomal membrane integrity, irrespective of being incubated alone or together with A. After 4 h, the level of damage caused by iron (both concentrations) was essentially the same as that observed after 1 h (Fig. 3D). However, the presence of A potentiated the effect of iron, increasing the leakage of LDH more than when iron was alone. It was only after 4 h of incubation that copper induced a slight damage to the membrane, and A did not modify that effect when coincubated with the metal. As reported for MTT assays, control conditions were also assessed, replacing metals or A1-40 by an equal volume of water (vehicle) (Table 1). State of Akt phosphorylation after metal and A/metal exposure To assess the state of Akt phosphorylation after metal and A/metal exposure, Akt phosphorylation was determined by Western blot in adult rat synaptosomes. Fig. 4A demonstrates that 5 min of iron exposure (both concentrations) induced an increase in Akt phosphorylation. A exposure also generated an augmented phosphorylation in Akt, irrespective of being alone or together with iron. Neither copper alone nor combined with A showed any effect on Akt phosphorylation. However, when synaptosomes were
exposed to A alone or coincubated with the metal ions, Akt phosphorylation was increased with respect to the control condition, observing the highest phosphorylation for the A (alone) condition. Similarly, Fig. 4B shows that metals did not cause any change in Akt phosphorylation, but the exposure to A together with the metals increased the phosphorylation of the kinase, being the greatest effect observed in the presence of A plus copper. The 4-h treatment did not show any dramatic activation of Akt, only a slight change in the presence of copper (Fig. 4C). State of ERK1/2 phosphorylation after metal and A/ metal treatment When ERK1/2 phosphorylation was assessed, the most striking changes were observed after 5 min (Fig. 5A) and 1 h (Fig. 5B) of incubation. The 5-min iron treatment (50 M) caused an increase in the phosphorylation of ERK1/2 both alone or together with A. Additionally, copper induced an augmented level of phosphor-ERK1/2 only when combined with A (Fig. 5A). After the 1-h treatment, A caused the increase in ERK1/2 phosphorylation both alone and when coincubated with both metal ions, being the greatest effect observed in the presence of iron (50 M) and copper.
DISCUSSION As previously stated, the A hypothesis proposes that the continuous disruption of normal synaptic physiology by A contributes to the development of AD (Shankar et al., 2007; Parodi et al., 2010). Moreover, a new hypothesis
386
R. M. Uranga et al. / Neuroscience 170 (2010) 381–389
Fig. 4. Akt phosphorylation. Western blot analysis of Akt phosphorylation in synaptosomes (50 g protein per lane) exposed to vehicle (control), 10 and 50 M iron, 10 M copper, 1 M A1-40, 1 M A1-40 plus 10 M iron, 1 M A1-40 plus 50 M iron and 1 M A1-40 plus 10 M copper for 5 min (A), 1 h (B), and 4 h (C). Each Western blot is representative of three different experiments. Bands of proteins were quantified using scanning densitometry, and phospho-Akt levels were normalized to total Akt levels and expressed as a percentage of the control condition (mean⫾SD of three different experiments). *** P⬍0.001 with respect to the control condition; ### P⬍0.001 when comparing “1 M A1-40⫹metal” versus the “metal” condition.
involves transition metals (such as iron and copper) as one of the main contributors to synaptic toxicity in AD and AD-related disorders (Syme et al., 2004; Dong et al., 2007; Barnham and Bush, 2008; Liu et al., 2010). Several laboratories have suggested that the ability to generate A/ metal ion complexes enables the metal ion to retain its full redox capacity and catalyze synergistic reactions on signaling cascades thus potentiating neuronal damage and cell death (Barnham and Bush, 2008). In the last few years, the interaction between A and transition metals has undoubtedly become the focus of research efforts as they appear to play a key role in the pathogenesis of this devastating disease (Barnham and Bush, 2008). In addition, A has been shown to trap the excess of intracellular
free copper ions buffering their cytotoxic effects, and also to participate in the Fe(II)/Fe(III) redox-cycling (Minniti et al., 2009; Khan et al., 2006). Moreover, the N-terminal region of A can access a range of metal-coordination structures which are able to alter not only the peptide self-assembly but also its toxicity (Dong et al., 2007). In spite of all the evidence about the role of transition metals in neurodegeneration, there is little consensus about how these A/iron, copper complexes mediate synaptic damage and participate in the onset and progression of AD (Maynard et al., 2005; Bush and Tanzi, 2008). In this connection, the main goal of this work was to investigate how A in combination with bioactive metals (such as iron and copper) affects the synaptic viability and modulates
R. M. Uranga et al. / Neuroscience 170 (2010) 381–389
387
Fig. 5. ERK1/2 phosphorylation. Western blot analysis of ERK1/2 phosphorylation in synaptosomes (50 g protein per lane) exposed to vehicle (control), 10 and 50 M iron, 10 M copper, 1 M A1-40, 1 M A1-40 plus 10 M iron, 1 M A1-40 plus 50 M iron and 1 M A1-40 plus 10 M copper for 5 min (A), 1 h (B), and 4 h (C). Each Western blot is representative of three different experiments. Bands of proteins were quantified using scanning densitometry, and phospho-ERK1/2 levels were normalized to total ERK2 levels and expressed as a percentage of the control condition (mean⫾SD of three different experiments). *** P⬍0.001 and ** P⬍0.01 with respect to the control condition; ### P⬍0.001, ## P⬍0.01 and # P⬍0.05 when comparing “1 M A1-40⫹metal” versus the “metal” condition.
signaling pathways in isolated synaptic endings. Specifically, the state of Akt and ERK1/2 signaling was assessed because these kinases are known to play essential roles in synaptic plasticity and neuronal survival (Brazil et al., 2004; Dwivedi et al., 2009; Uranga et al., 2009). As previously demonstrated by our group, the presence of free iron alone in the medium provokes a time- and concentration-dependent diminution in mitochondrial function together with an increase in plasma membrane damage (Uranga et al., 2007, 2009). In the present work, the extent of synaptic damage in the presence of A peptide in combination with iron or copper was characterized. For this purpose, we used either free iron or free copper in the synaptosomal incubation medium as control conditions. A/metal impaired mitochondrial function to the same extent as metal alone, thus demonstrating that metal-induced
mitochondrial damage was not further increased by the simultaneous incubation with A and metal ions. Other studies have reported that A/iron or A/copper complexes cause increased damage when compared to the damage provoked by A or metal alone. These discrepancies with the results presented here could be mainly due to the fact that our assays were carried out in the isolated synaptic endings treated in an acute manner (5 min – 4 h), while most works show results obtained in whole neurons treated for more than 12 h (Bush, 2003; Dai et al., 2006). However, in the presence of A/iron (10 and 50 M), plasma membrane integrity was significantly disrupted demonstrating an additive effect with respect to the values found in the presence of metal alone after long times of incubation (4 h). Previous work from other laboratories demonstrated that chronic exposure (more than 24 h) to
388
R. M. Uranga et al. / Neuroscience 170 (2010) 381–389
A alone causes membrane perforations in the plasma membrane of hippocampal neurons leading to alteration in ionic homeostasis noticeable as calcium inward currents measured by patch clamp (Parodi et al., 2010). Our results, however, show that acute exposure to A/iron is able to generate membrane pores of such a size as to allow the leakage of LDH. As previously discussed, in spite of the damage observed in the synaptic membrane, mitochondrial function was not affected to the same extent. This could be indicating that the membrane constitutes the first target of A/iron, and that it is the starting sign of synaptic damage. The affection of other synaptic parameters which determine the severity of the damage, like mitochondrial function, could depend on the balance between the levels of transition metals, the availability of A, the production of free radicals and the persistence of stress. The fact that A/copper did not provoke any additional damage to the membrane (although copper is also a divalent transition metal as well as iron) strongly supports that both the structure of the coordination complexes and their interaction with the membrane depend on the chemical features of the metal. We have previously demonstrated that PI3K/Akt activation by free iron (5 min of exposure) occurs when the deleterious effects of the metal are slightly observed, and that this activation in the isolated synaptic ending is not able to prevent the oxidative damage induced by the metal (Uranga et al., 2007, 2009). Here we show that a short exposure to A/iron has an additional effect on Akt phosphorylation, thus demonstrating that the presence of the peptide is able to enhance iron-induced PI3K/Akt activation in an acute manner. Akt activation by A/iron was barely detected after 4 h of incubation, demonstrating that the activation pattern of this kinase does not correlate with the levels of synaptic damage. It is worth to note that while the A activation is long lived, the metal-induced Akt activation is transient. This early and transient activation could represent an attempt by the synaptic ending to activate a neuroprotective mechanism; however, the final outcome of this activation appears to depend on the orchestration of several signaling pathways whose evaluation would involve the entire neuron. Here we show that ERK1/2 presented a profile of activation different from that observed for Akt. The level of ERK1/2 phosphorylation in the presence of A/iron was the same as that observed for the free metal (iron) after short times of incubation (5 min). ERK1/2 activation by free iron has been demonstrated to be a PI3K/Akt-dependent event (Uranga et al., 2009). However, after 1-h exposure to A/iron (50 M) an enhancing effect on ERK1/2 phosphorylation with respect to the metal alone was observed. This synergistic effect was transient (1-h treatment) and previous to the major membrane damage observed after 4 h of exposure. Here we show that the differential profile in the activation of Akt and ERK1/2 in the presence of A/iron argues in favor of the independent activation of both kinase pathways in the synapse. These observations are in accordance with reports from other laboratories which show that A/metal complexes are able to independently mod-
ulate PI3K/Akt and ERK1/2 pathways in the neuron (Bica et al., 2009). Intriguingly, Akt and ERK1/2 activation were coincident after 1 h of incubation in the presence of A/ copper, thus demonstrating that the presence of A together with different metals could regulate differentially the activity of these kinases. There is accumulating evidence highlighting the local action of signaling pathways in the preservation of synaptic endings through gene-transcription independent mechanisms (Guo and Mattson, 2000; Gilman et al., 2003). The results presented here certainly demonstrate that synaptic Akt and ERK1/2 signaling are differentially activated by the presence of A and metal ions. While Akt activation is independent of synaptic damage, ERK1/2 activation seems to be transiently dependent. This work presents evidence about the initial events locally triggered at the synapse by A and transition metals. The overall mechanism as well as the physiological meaning of these findings should be further studied in a neuronal model. Acknowledgments—The authors specially thank Dr. Carlos Opazo for his valuable help in providing and discussing the protocol for A preparations. This work was supported by grants from the Universidad Nacional del Sur, the Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET) and the Agencia Nacional de Promoción Científica (FONCYT). Dr. Norma M. Giusto and Dr. Gabriela A. Salvador are research members of the CONICET. Romina M. Uranga is a research fellow of the CONICET.
REFERENCES Atwood CS, Obrenovich ME, Liu T, Chan H, Perry G, Smith MA, Martins RN (2003) Amyloid-beta: a chameleon walking in two worlds: a review of the trophic and toxic properties of amyloid-beta. Brain Res Brain Res Rev 43:1–16. Barnham KJ, Bush AI (2008) Metals in Alzheimer’s and Parkinson’s diseases. Curr Opin Chem Biol 12:222–228. Bica L, Crouch PJ, Cappai R, White AR (2009) Metallo-complex activation of neuroprotective signalling pathways as a therapeutic treatment for Alzheimer’s disease. Mol Biosyst 5:134 –142. Brazil DP, Yang ZZ, Hemmings BA (2004) Advances in protein kinase B signalling: AKTion on multiple fronts. Trends Biochem Sci 29:233–242. Bush AI (2003) The metallobiology of Alzheimer’s disease. Trends Neurosci 26:207–214. Bush AI, Tanzi RE (2008) Therapeutics for Alzheimer’s disease based on the metal hypothesis. Neurotherapeutics 5:421– 432. Cotman CW (1974) Isolation of synaptosomal and synaptic plasma membrane fractions. Methods Enzymol 31:445– 452. Dai XL, Sun YX, Jiang ZF (2006) Cu(II) potentiation of Alzheimer Abeta1-40 cytotoxicity and transition on its secondary structure. Acta Biochim Biophys Sin (Shanghai) 38:765–772. DeKosky ST, Scheff SW (1990) Synapse loss in frontal cortex biopsies in Alzheimer’s disease: correlation with cognitive severity. Ann Neurol 27:457– 464. Dong J, Canfield JM, Mehta AK, Shokes JE, Tian B, Childers WS, Simmons JA, Mao Z, Scott RA, Warncke K, Lynn DG (2007) Engineering metal ion coordination to regulate amyloid fibril assembly and toxicity. Proc Natl Acad Sci U S A 104:13313–13318. Dwivedi Y, Rizavi HS, Zhang H, Roberts RC, Conley RR, Pandey GN (2009) Aberrant extracellular signal-regulated kinase (ERK)1/2 signalling in suicide brain: role of ERK kinase 1 (MEK1). Int J Neuropsychopharmacol 12:1337–1354.
R. M. Uranga et al. / Neuroscience 170 (2010) 381–389 Gilman CP, Chan SL, Guo Z, Zhu X, Greig N, Mattson MP (2003) p53 is present in synapses where it mediates mitochondrial dysfunction and synaptic degeneration in response to DNA damage, and oxidative and excitotoxic insults. Neuromolecular Med 3:159 –172. Guo ZH, Mattson MP (2000) Neurotrophic factors protect cortical synaptic terminals against amyloid and oxidative stress-induced impairment of glucose transport, glutamate transport and mitochondrial function. Cereb Cortex 10:50 –57. Huang X, Moir RD, Tanzi RE, Bush AI, Rogers JT (2004) Redox-active metals, oxidative stress, and Alzheimer’s disease pathology. Ann N Y Acad Sci 1012:153–163. Jacobson MD, Burne JF, Raff MC (1994) Programmed cell death and Bcl-2 protection in the absence of a nucleus. EMBO J 13:1899 –1910. Keller JN, Mark RJ, Bruce AJ, Blanc E, Rothstein JD, Uchida K, Waeg G, Mattson MP (1997) 4-Hydroxynonenal, an aldehydic product of membrane lipid peroxidation, impairs glutamate transport and mitochondrial function in synaptosomes. Neuroscience 80:685– 696. Khan A, Dobson JP, Exley C (2006) Redox cycling of iron by Abeta42. Free Radic Biol Med 40:557–569. Kontush A (2001) Amyloid-beta: an antioxidant that becomes a prooxidant and critically contributes to Alzheimer’s disease. Free Radic Biol Med 31:1120 –1131. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680 – 685. Liu G, Men P, Perry G, Smith MA (2010) Nanoparticle and iron chelators as a potential novel Alzheimer therapy. Methods Mol Biol 610:123–144. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193:265–275. Mattson MP, Liu D (2002) Energetics and oxidative stress in synaptic plasticity and neurodegenerative disorders. Neuromolecular Med 2:215–231. Maynard CJ, Bush AI, Masters CL, Cappai R, Li QX (2005) Metals and amyloid-beta in Alzheimer’s disease. Int J Exp Pathol 86:147–159. Minniti AN, Rebolledo DL, Grez PM, Fadic R, Aldunate R, Volitakis I, Cherny RA, Opazo C, Masters C, Bush AI, Inestrosa NC (2009) Intracellular amyloid formation in muscle cells of Abeta-transgenic Caenorhabditis elegans: determinants and physiological role in copper detoxification. Mol Neurodegener 4:2. Nunomura A, Perry G, Aliev G, Hirai K, Takeda A, Balraj EK, Jones PK, Ghanbari H, Wataya T, Shimohama S, Chiba S, Atwood CS, Petersen RB, Smith MA (2001) Oxidative damage is the earliest event in Alzheimer disease. J Neuropathol Exp Neurol 60: 759 –767. Parodi J, Sepulveda FJ, Roa J, Opazo C, Inestrosa NC, Aguayo LG (2010) Beta-amyloid causes depletion of synaptic vesicles leading to neurotransmission failure. J Biol Chem 285:2506 –2514.
389
Plant LD, Boyle JP, Smith IF, Peers C, Pearson HA (2003) The production of amyloid beta peptide is a critical requirement for the viability of central neurons. J Neurosci 23:5531–5535. Santos MJ, Quintanilla RA, Toro A, Grandy R, Dinamarca MC, Godoy JA, Inestrosa NC (2005) Peroxisomal proliferation protects from beta-amyloid neurodegeneration. J Biol Chem 280:41057– 41068. Sayre LM, Smith MA, Perry G (2001) Chemistry and biochemistry of oxidative stress in neurodegenerative disease. Curr Med Chem 8:721–738. Scheff SW, Price DA (2003) Synaptic pathology in Alzheimer’s disease: a review of ultrastructural studies. Neurobiol Aging 24: 1029 –1046. Selkoe DJ (1996) Amyloid beta-protein and the genetics of Alzheimer’s disease. J Biol Chem 271:18295–18298. Sepulveda FJ, Opazo C, Aguayo LG (2009) Alzheimer beta-amyloid blocks epileptiform activity in hippocampal neurons. Mol Cell Neurosci 41:420 – 428. Shankar GM, Bloodgood BL, Townsend M, Walsh DM, Selkoe DJ, Sabatini BL (2007) Natural oligomers of the Alzheimer amyloidbeta protein induce reversible synapse loss by modulating an NMDA-type glutamate receptor-dependent signaling pathway. J Neurosci 27:2866 –2875. Smith MA, Casadesus G, Joseph JA, Perry G (2002) Amyloid-beta and tau serve antioxidant functions in the aging and Alzheimer brain. Free Radic Biol Med 33:1194 –1199. Syme CD, Nadal RC, Rigby SE, Viles JH (2004) Copper binding to the amyloid-beta (Abeta) peptide associated with Alzheimer’s disease: folding, coordination geometry, pH dependence, stoichiometry, and affinity of Abeta-(1–28): insights from a range of complementary spectroscopic techniques. J Biol Chem 279:18169 –18177. Terry RD, Masliah E, Salmon DP, Butters N, DeTeresa R, Hill R, Hansen LA, Katzman R (1991) Physical basis of cognitive alterations in Alzheimer’s disease: synapse loss is the major correlate of cognitive impairment. Ann Neurol 30:572–580. Uranga RM, Giusto NM, Salvador GA (2009) Iron-induced oxidative injury differentially regulates PI3K/Akt/GSK3beta pathway in synaptic endings from adult and aged rats. Toxicol Sci 111:331–344. Uranga RM, Mateos MV, Giusto NM, Salvador GA (2007) Activation of phosphoinositide-3 kinase/Akt pathway by FeSO4 in rat cerebral cortex synaptic endings. J Neurosci Res 85:2924 –2932. Wei Z, Song MS, MacTavish D, Jhamandas JH, Kar S (2008) Role of calpain and caspase in beta-amyloid-induced cell death in rat primary septal cultured neurons. Neuropharmacology 54:721–733. Zhu X, Raina AK, Lee HG, Casadesus G, Smith MA, Perry G (2004) Oxidative stress signalling in Alzheimer’s disease. Brain Res 1000:32–39.
(Accepted 22 July 2010) (Available online 29 July 2010)
EFFECT OF TRANSITION METALS IN SYNAPTIC DAMAGE INDUCED BY AMYLOID BETA PEPTIDE R. M. URANGA, N. M. GIUSTO AND G. A. SALVADOR*
Alzheimer’s disease (AD) is clinically characterized by a progressive loss of cognitive skills and dementia (DeKosky and Scheff, 1990; Terry et al., 1991; Selkoe, 1996; Scheff and Price, 2003). AD brains typically present an extensive oxidative stress with augmented protein oxidation and lipid peroxidation, overproduction of reactive oxygen species (ROS), mitochondrial impairment, and oxidation of nucleic acids (Keller et al., 1997; Nunomura et al., 2001; Zhu et al., 2004). The increase in free radicals, whose major source is cellular energy metabolism, generates the activation of stress-response signaling mechanisms (Mattson and Liu, 2002). These changes occur very early in the onset of the disease and represent a very interesting target for prevention. Amyloid- peptide (A) has long been thought of as the major cause of AD, with A being considered as the triggering insult. Nowadays, there are two central and contrasting hypotheses regarding A and oxidative stress: on the one hand, it has been shown that A together with transition metals give rise to oxidative injury, but on the other hand, the peptide seems to have antioxidant properties in some conditions (Kontush, 2001; Smith et al., 2002; Atwood et al., 2003). The hypothesis that proposes A as the promoter of oxidative damage holds that oxidative stress is one of the first pathologic events in AD, and it would precede and even hasten the formation of senile plaques (Sayre et al., 2001). The contrasting hypothesis states that the age-related increase of A would be able to diminish the levels of oxidative stress, and it is based on several studies which have shown that the peptide is able to exert, in certain circumstances, neuroprotective effects (Atwood et al., 2003; Plant et al., 2003). A third hypothesis has gained leading role and proposes a failure in biometal homeostasis (e.g. iron, copper), together with A/metal interaction, as the key factor in triggering AD pathology (Huang et al., 2004). A high correlation between synapse loss and cognitive impairment has been described (DeKosky and Scheff, 1990; Terry et al., 1991; Selkoe, 1996; Scheff and Price, 2003) and synapses are held to be the sites where AD and AD-related neurodegenerative disorders are likely to be initiated. Signaling pathways have been found to be highly concentrated in the synaptic terminals so an abnormal signaling in this particular region of the neuron, the accretion of transition metals and an increased susceptibility to oxidative stress would be suspected to trigger this disorder. Cumulative evidence points to the effects of A and metal ions on neuronal cultures, but their real effect on the synapses and the precise synaptic signaling pathways
Instituto de Investigaciones Bioquímicas de Bahía Blanca, Universidad Nacional del Sur and Consejo Nacional de Investigaciones Científicas y Técnicas, Camino La Carrindanga Km 7, CC 857, B8000FWB Bahía Blanca, Argentina
Abstract—The amyloid -peptide (A), which is thought to be the major cause of Alzheimer’s disease (AD), is known to be capable of aggregating in different states: soluble monomers and oligomers, and insoluble aggregates. The A aggregation state as well as its toxicity has been related to the interaction between the peptide and transition metals such as iron and copper. However, this relationship, as well as the effects of A on the synaptic endings, is not fully understood. The aggregation states of A in the presence of iron and copper, as well as their effects on synaptic viability and signaling were investigated in this work. During acute incubation treatments (5 min– 4 h), A/metal impaired mitochondrial function to the same extent as has been observed with the metal alone. However, in the presence of A/iron (10 and 50 M), plasma membrane integrity was disrupted to a greater extent than when generated by either iron or A alone, indicating that the membrane constitutes the first target of synaptic injury. Akt activation by A/iron was evident after 5 min of incubation and was higher than that observed in the presence of the metal alone. This activation was barely detected after 4 h of incubation, demonstrating that there is no correlation between the extent of synaptic damage and the activation of this kinase. Extracellular signal-regulated kinases 1 and 2 (ERK1/2) activation profile was different from that observed for Akt. Accordingly, the presence of A/metal could differentially modulate the activity of these kinases. This work shows evidence of the initial events locally triggered at the synapse by A and transition metals. As synapses have been proposed as the starting point of A/metaltriggered events, the characterization of early mechanisms occurring in models that mimic AD could be important for the search of unexplored therapeutics tools. © 2010 IBRO. Published by Elsevier Ltd. All rights reserved. Key words: iron, copper, synaptosomes, Akt, ERK1/2, A peptide. *Corresponding author. Tel: ⫹54-291-4861201; fax: ⫹54-2914861200. E-mail address: [email protected] (G. A. Salvador). Abbreviations: AD, Alzheimer’s disease; ADDLs, amyloid-beta derived diffusible ligands; A, amyloid-beta peptide; DMSO, dimethyl sulfoxide; EDTA, ethylenediaminetetraacetic acid; EM, electron microscopy; ERK1/2, extracellular signal-regulated kinases 1 and 2; HEPES, 4-(2hydroxyethyl)-1-piperazine ethanesulfonic acid; HRP, horse raddish peroxidise; LDH, lactate dehydrogenase; MTT, 3-(4,5-dimethylthiazol2-yl)-2,5-diphenyltetrazolium bromide; NAD⫹, oxidized nicotinamide adenine dinucleotide; NADH, reduced nicotinamide adenine dinucleotide; PBS, phosphate-buffered saline; PI3K, phosphatidylinositol 3-kinase; PMSF, phenylmethylsulfonyl fluoride; PVDF, polyvinylidene fluoride; ROS, reactive oxygen species; SD, standard deviation; SDS, sodium dodecyl sulfate; SDS-PAGE, SDS-polyacrylamide gel electrophoresis; TBST, tris-buffered saline Tween-20.
0306-4522/10 $ - see front matter © 2010 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2010.07.044
381
382
R. M. Uranga et al. / Neuroscience 170 (2010) 381–389
activated by AD-simulating conditions have not yet been elucidated. Taking into account that many signaling pathways observed in intact cells can be observed in cells lacking nuclei (Jacobson et al., 1994), we used rat cortical synaptosomes to investigate signaling mechanisms activated locally in synapses by oxidative insult. The aim of this work is to study the effects of transition metals and/or A on phosphatidylinositol 3-kinase (PI3K)/Akt and extracellular signal-regulated kinases 1 and 2 (ERK1/2) pathways in synaptic endings from adult animals. Elucidation of synaptic mechanisms altered in these conditions could be important to delay the onset and/or the progression of neurodegenerative disorders which involve oxidative processes.
EXPERIMENTAL PROCEDURES Animals Wistar-strain adult (4 months of age) rats housed under controlled conditions (constant room temperature, 12-h light/12-h dark cycle), bred in our own colony and fed a standard rat chow diet with free access to water, were used. All the procedures were in strict accordance with the guidelines published in the National Institutes of Health Guide for the care and use of laboratory animals.
Materials
Beckman J2-21 centrifuge. Washed synaptosomal fraction was used for the experiments detailed below. Protein content of the synaptosomal fraction was determined by the method of Lowry et al., (1951).
Incubation of synaptosomes Synaptosomes were diluted in Locke’s buffer (154 mM NaCl, 5.6 mM KCl, 2.3 mM CaCl2, 1.0 mM MgCl2, 3.6 mM NaHCO3, 5 mM glucose, 5 mM HEPES, pH 7.2) for all experiments except where stated otherwise. Synaptosomal suspensions were aliquoted (2 mg protein/ml) into tubes and incubated at 37 °C under an O2:CO2 (95:5, v:v) atmosphere during experimental treatments.
Metals and A1-40 FeSO4 and CuSO4 were prepared as 10 mM stocks in water immediately prior to use. Rat amyloid- peptide 1– 40 (A1-40, Calbiochem, CA, USA) was resuspended in dimethyl sulfoxide (DMSO) at a concentration of 10 mg/ml and immediately stored in aliquots at ⫺20 °C. A oligomers/aggregates (80 M) were prepared according to previously published methods (Sepulveda et al., 2009; Parodi et al., 2010). Briefly, aliquots of peptide stock (250 g in 25 l of DMSO) were added to 700 l of phosphatebuffered saline (PBS, pH 7.4) and stirred continuously (300 rpm, at 37 °C) for 120 min and stored at 4 °C until use. Aliquots of the preparation obtained were analyzed by electron microscopy (EM) either alone or combined with one of the metal ions.
EM of A1-40 preparations
The kit (LDH-P UV AA) for measuring lactate dehydrogenase (LDH) activity was generously supplied by the Wiener Laboratory (Santa Fe, Argentina). Rabbit polyclonal anti-phospho-Ser473-Akt and anti-Akt antibodies were from Cell Signaling Technology (Beverly, MA, USA). Mouse anti-phospho-Tyr204-ERK1/2, rabbit anti-ERK2, polyclonal horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG, and polyclonal HRP-conjugated goat antimouse IgG were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). All other reagents were of analytical grade, purchased from Sigma-Aldrich (St. Louis, MO, USA) unless otherwise stated.
EM studies were carried out according to a method used by several laboratories with slight modifications (Santos et al., 2005; Wei et al., 2008; Parodi et al., 2010). Briefly, 1 M A1-40 was incubated in PBS at 37 °C, with or without the metals, for 5 and 30 min, and 1 and 4 h. After incubation, 10 L of media containing the peptide was placed on carbon-coated grids and incubated for 60 s. Ten microlitres of 0.5% glutaraldehyde was added to each grid and incubated for an additional 60 s. The grid was then washed with drops of water and dried. Finally, the grids were stained for 2 min with 2% uranyl acetate and then air-dried. Grids were subsequently examined in a Jeol 100 Cx II electron microscope.
Preparation of synaptosomal fraction and experimental treatments
MTT reduction assay
The purified synaptosomal fraction was obtained as previously described by Cotman, (1974), with slight modifications. Briefly, rats were sacrificed by decapitation, brains were removed on a cold plate, and the cerebral cortex was immediately dissected (2– 4 min after decapitation) and placed in 0.32 M sucrose isolation buffer containing 2 g/ml leupeptin, 1 g/ml pepstatin, 1 g/ml aprotinin, 1 mM dithiothreitol (DTT), 0.1 mM phenylmethylsulfonyl fluoride (PMSF), 1 mM ethylenediaminetetraacetic acid (EDTA), and 10 mM 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid (HEPES), pH 7.4. The cerebral cortex was homogenized by 10 strokes with a Thomas tissue homogenizer. The homogenate was then centrifuged at 1800 g for 7.5 min at 4 °C using a JA-21 rotor in a Beckman J2-21 centrifuge. The pellet was discarded, and the supernatant was retained and centrifuged at 14,000 g for 20 min at 4 °C. The resulting pellet was washed and resuspended in 3 ml of 0.32 M sucrose isolation buffer and layered over a discontinuous Ficoll gradient (8.5% pH 7.4, 13% pH 7.4 Ficoll solutions, each prepared in isolation buffer) and spun at 85,500 g for 30 min at 4 °C using an SW 28.1 rotor in a Beckman L5-50 ultracentrifuge. Synaptosomes in the 8.5–13% Ficoll interface were removed, resuspended in isolation buffer, and centrifuged at 33,000 g for 20 min at 4 °C using a JA-21 rotor in a
Neuronal viability was measured by determining cellular reducing capacity via the extent of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction to the insoluble intracellular Formazan, which depends on the activity of intracellular dehydrogenases and is independent of changes in the integrity of the plasma membrane. The methods employed in the present study were similar to those described previously by Keller et al., (1997). In brief, MTT was dissolved in PBS at a concentration of 5 mg/ml. The MTT solution was mixed with synaptosomes (1:10 MTT: synaptosomes, v:v) and allowed to incubate for 2 h at 37 °C. At the end of incubation with MTT, solubilization buffer [20% sodium dodecyl sulfate (SDS), pH 4.7] was added and mixed thoroughly to dissolve the crystals of Formazan. The extent of MTT reduction then was measured spectrophotometrically at 570 nm. Results are expressed as a percentage of control.
Measurement of LDH release After treatments, synaptosomes were centrifuged at 33,000 g for 20 min at 4 °C. The resulting supernatant was used to determine LDH activity, measured spectrophotometrically by using an LDH-P UV AA kit following the manufacturer’s instructions. Briefly, the rate of conversion of reduced nicotinamide adenine dinucleotide
R. M. Uranga et al. / Neuroscience 170 (2010) 381–389 (NADH) to oxidized nicotinamide adenine dinucleotide (NAD⫹) was followed at 340 nm. Results are expressed as a percentage of the control value.
SDS-PAGE and Western blot assays Samples were denatured with Laemmli sample buffer at 100 °C for 5 min (Laemmli, 1970). Equivalent amounts of synaptosomal proteins were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) on 10% polyacrylamide gels and then transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore, Bedford, MA, USA). Membranes were blocked with 5% nonfat dry milk in Tris-buffered saline Tween-20 (TBST) buffer [20 mM Tris–HCl (pH 7.4), 100 mM NaCl, and 0.1% (w/v) Tween 20] for 2 h at room temperature for all the Western blots assayed. Membranes were then incubated with primary antibodies [anti-phosphoSer473-Akt, anti-Akt, anti-phosphoTyr204-ERK1/2, anti-ERK2, (1:1000) overnight at 4 °C], washed three times with TBST, and then exposed to the appropriate HRP-conjugated secondary antibody (anti-rabbit or anti-mouse) for 1 h at room temperature. Membranes were again washed three times with TBST, and immunoreactive bands were detected by enhanced chemiluminescence (ECL; Amersham Biosciences) using standard X-ray film (Kodak X-Omat AR). Several different exposure times were used for each blot to ensure linearity of band intensities. Immunoreactive bands were quantified using image analysis software (ImageJ, a freely available application in the public domain for image analysis and processing, developed and maintained by Wayne Rasband at the Research Services Branch, National Institute of Mental Health).
383
Data analysis Quantitative results were expressed as the mean⫾standard deviation (SD) of the N indicated in the corresponding figures. Data were analyzed by one-way ANOVA followed by the Tukey multiple comparison test. P-values lower than 0.05 were considered statistically significant. Western blots shown are representative of at least three analyses performed on samples from at least three separate experiments.
RESULTS Aggregation of A1-40. Effect of the coincubation with metals Numerous studies have shown that neuronal response to A exposure depends exclusively on the aggregation state of the peptide. To assess the status of A1-40 aggregation by EM, 1 M A1-40 was incubated in Locke’s buffer at 37 °C for the same periods of time used for the toxicity model. Our EM photomicrographs show that incubation of A1-40 for 5 min and 1 h lead to spheroidal structures individually or in small groups (Fig. 1A, E). These ultrastructural forms were similar to a heterogeneous array of forms defined as oligomers, protofibrils and amyloid-beta derived diffusible ligands (ADDLs). The coincubation with transition metal ions such as copper (10 M) and iron (10 and 50 M) did not modify the aggregation state of the
Fig. 1. High power (⫻140,000) electron photomicrographs showing A and A/metal preparations. A was dissolved and prepared as described in Experimental procedures. 1 M A1-40 was incubated in PBS at 37 °C for 5 min (A), 1 h (E) and 4 h (I). A1-40 was also coincubated with 10 M copper, or 10 or 50 M iron in PBS at 37 °C for 5 min (B–D, respectively), and 1 (F–H, respectively) and 4 h (J–L, respectively). After incubation, 10 L of media containing the peptide was placed on carbon-coated grids and fixed with of 0.5% glutaraldehyde. The grids were stained with 2% uranyl acetate and then air-dried. The grids were subsequently examined in a Jeol 100 Cx II electron microscope. White arrows show spheroidal structures observed after 5 and 1 h of incubation (A–H). Black arrows show more organized structures present in the preparations after 4 h of incubation (I–L). Black arrows point at the amorphous clumps formed after incubating the A for 4 h either alone or with the metal ions (I–L). For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.
384
R. M. Uranga et al. / Neuroscience 170 (2010) 381–389
Table 1. MTT reduction and LDH leakage, time course in control conditions Time
MTT (arbitrary units)
LDH (arbitrary units)
0 min 5 min 30 min 1h 4h
1.060⫾0.035 1.062⫾0.046 1.055⫾0.027 1.016⫾0.039 0.986⫾0.037
0.053⫾0.006 0.055⫾0.004 0.058⫾0.003 0.058⫾0.003 0.113⫾0.004***
The reduction of MTT as well as LDH leakage was assayed in synaptosomes (2 mg protein/ml) exposed to vehicle (control) for 0, 5 and 30 min, and 1 and 4 h. Results are expressed in arbitrary units and represent the mean⫾SD; n⫽3– 6. *** P⬍0.001 with respect to the control.
peptide (Fig. 1B–D, 1F–H). However, Fig. 1I–L show that after 4 h of incubation, A1-40 appears in some sort of amorphous clumps or more organized structures but still without any evidence of fibril formation. Exposure of synaptic endings to A1-40 and transition metals: effects on viability A great body of evidence has shown that transition metals act as neurotoxic agents by inducing lipid peroxidation as well as mitochondrial dysfunction. However, information regarding the effects and mechanisms of action of A1-40 is quite controversial. Our first goal was to characterize the effect of FeSO4 (10 and 50 M), CuSO4 (10 M) and A1-40 (1 M) on synaptosomal viability after 5- and 30min, 1-h and 4-h incubations. To determine the consequence of exposing rat cerebral cortex synaptic endings to
either transition metals or A1-40, MTT reduction was evaluated as a measure of mitochondrial function. Control conditions were also assessed, replacing metals or A1-40 by an equal volume of water (vehicle) (Table 1). As shown in Fig. 2A, after 5 min of incubation, only iron (50 M) slightly but significantly decreased mitochondrial viability. A1-40 did not cause any significant change in MTT reduction when incubated with synaptosomes alone or when incubated in combination with the metals. Fig. 2B shows that after a 30-min exposure iron in both concentrations caused a marked decrease in mitochondrial viability, being the greatest damage caused by 50 M iron. When A1-40 was coincubated with iron or copper, the damage observed was essentially the same as that caused by each metal separately. However, A did not produce any damage at all by itself. The MTT reduction after 1 and 4 h of incubation (Fig. 2C, D, respectively) shows basically the same pattern of damage than that observed after a 30-min treatment, but the longer the time of incubation the higher the levels of injury generated by the metal ions (either alone or combined with A). In either case A did not cause any damage at all by itself. The evaluation of plasma membrane integrity is a useful tool for determining the degree of injury provoked by several stressors. Plasma membrane integrity was determined by monitoring the leakage of LDH to the extrasynaptosomal medium. As shown in Fig. 3A, after 5 min of exposure, the presence of iron was the only condition that caused a slight but significant increase in LDH leakage. A did not increase the damage to the membrane with respect to the control condition when incubated either alone or
Fig. 2. MTT reduction assay. The reduction of MTT was assayed in synaptosomes (2 mg protein/ml) exposed to vehicle (control), 10 and 50 M iron, 10 M copper, 1 M A1-40, 1 M A1-40 plus 10 M iron, 1 M A1-40 plus 50 M iron and 1 M A1-40 plus 10 M copper for 5 min (A), 30 min (B), 1 h (C), and 4 h (D). Results are expressed as percentage of control and represent the mean⫾SD; n⫽3– 6. *** P⬍0.001 and ** P⬍0.01 with respect to the control.
R. M. Uranga et al. / Neuroscience 170 (2010) 381–389
385
Fig. 3. LDH leakage. The release of LDH to the medium was measured in synaptosomes (2 mg protein/ml) exposed to vehicle (control), 10 and 50 M iron, 10 M copper, 1 M A1-40, 1 M A1-40 plus 10 M iron, 1 M A1-40 plus 50 M iron and 1 M A1-40 plus 10 M copper for 5 min (A), 30 min (B), 1 h (C), and 4 h (D). Results are expressed as percentage of control and represent the mean⫾SD; n⫽3– 6. *** P⬍0.001, ** P⬍0.01, and * P⬍0.05 with respect to the control; ### P⬍0.001 and # P⬍0.05 when comparing “1 M A1-40⫹metal” versus the “metal” condition.
combined with the metals. Fig. 3B, C show that also after 30 min and 1 h of incubation, respectively, only iron induced a significant damage to the membrane (higher than that observed after 5 min). No damage caused by A itself was observed. However, the coincubation of A with iron (50 M) for 1 h generated a slightly higher (but significant) damage, compared to that induced by 50 M iron alone. Copper did not introduce any change in synaptosomal membrane integrity, irrespective of being incubated alone or together with A. After 4 h, the level of damage caused by iron (both concentrations) was essentially the same as that observed after 1 h (Fig. 3D). However, the presence of A potentiated the effect of iron, increasing the leakage of LDH more than when iron was alone. It was only after 4 h of incubation that copper induced a slight damage to the membrane, and A did not modify that effect when coincubated with the metal. As reported for MTT assays, control conditions were also assessed, replacing metals or A1-40 by an equal volume of water (vehicle) (Table 1). State of Akt phosphorylation after metal and A/metal exposure To assess the state of Akt phosphorylation after metal and A/metal exposure, Akt phosphorylation was determined by Western blot in adult rat synaptosomes. Fig. 4A demonstrates that 5 min of iron exposure (both concentrations) induced an increase in Akt phosphorylation. A exposure also generated an augmented phosphorylation in Akt, irrespective of being alone or together with iron. Neither copper alone nor combined with A showed any effect on Akt phosphorylation. However, when synaptosomes were
exposed to A alone or coincubated with the metal ions, Akt phosphorylation was increased with respect to the control condition, observing the highest phosphorylation for the A (alone) condition. Similarly, Fig. 4B shows that metals did not cause any change in Akt phosphorylation, but the exposure to A together with the metals increased the phosphorylation of the kinase, being the greatest effect observed in the presence of A plus copper. The 4-h treatment did not show any dramatic activation of Akt, only a slight change in the presence of copper (Fig. 4C). State of ERK1/2 phosphorylation after metal and A/ metal treatment When ERK1/2 phosphorylation was assessed, the most striking changes were observed after 5 min (Fig. 5A) and 1 h (Fig. 5B) of incubation. The 5-min iron treatment (50 M) caused an increase in the phosphorylation of ERK1/2 both alone or together with A. Additionally, copper induced an augmented level of phosphor-ERK1/2 only when combined with A (Fig. 5A). After the 1-h treatment, A caused the increase in ERK1/2 phosphorylation both alone and when coincubated with both metal ions, being the greatest effect observed in the presence of iron (50 M) and copper.
DISCUSSION As previously stated, the A hypothesis proposes that the continuous disruption of normal synaptic physiology by A contributes to the development of AD (Shankar et al., 2007; Parodi et al., 2010). Moreover, a new hypothesis
386
R. M. Uranga et al. / Neuroscience 170 (2010) 381–389
Fig. 4. Akt phosphorylation. Western blot analysis of Akt phosphorylation in synaptosomes (50 g protein per lane) exposed to vehicle (control), 10 and 50 M iron, 10 M copper, 1 M A1-40, 1 M A1-40 plus 10 M iron, 1 M A1-40 plus 50 M iron and 1 M A1-40 plus 10 M copper for 5 min (A), 1 h (B), and 4 h (C). Each Western blot is representative of three different experiments. Bands of proteins were quantified using scanning densitometry, and phospho-Akt levels were normalized to total Akt levels and expressed as a percentage of the control condition (mean⫾SD of three different experiments). *** P⬍0.001 with respect to the control condition; ### P⬍0.001 when comparing “1 M A1-40⫹metal” versus the “metal” condition.
involves transition metals (such as iron and copper) as one of the main contributors to synaptic toxicity in AD and AD-related disorders (Syme et al., 2004; Dong et al., 2007; Barnham and Bush, 2008; Liu et al., 2010). Several laboratories have suggested that the ability to generate A/ metal ion complexes enables the metal ion to retain its full redox capacity and catalyze synergistic reactions on signaling cascades thus potentiating neuronal damage and cell death (Barnham and Bush, 2008). In the last few years, the interaction between A and transition metals has undoubtedly become the focus of research efforts as they appear to play a key role in the pathogenesis of this devastating disease (Barnham and Bush, 2008). In addition, A has been shown to trap the excess of intracellular
free copper ions buffering their cytotoxic effects, and also to participate in the Fe(II)/Fe(III) redox-cycling (Minniti et al., 2009; Khan et al., 2006). Moreover, the N-terminal region of A can access a range of metal-coordination structures which are able to alter not only the peptide self-assembly but also its toxicity (Dong et al., 2007). In spite of all the evidence about the role of transition metals in neurodegeneration, there is little consensus about how these A/iron, copper complexes mediate synaptic damage and participate in the onset and progression of AD (Maynard et al., 2005; Bush and Tanzi, 2008). In this connection, the main goal of this work was to investigate how A in combination with bioactive metals (such as iron and copper) affects the synaptic viability and modulates
R. M. Uranga et al. / Neuroscience 170 (2010) 381–389
387
Fig. 5. ERK1/2 phosphorylation. Western blot analysis of ERK1/2 phosphorylation in synaptosomes (50 g protein per lane) exposed to vehicle (control), 10 and 50 M iron, 10 M copper, 1 M A1-40, 1 M A1-40 plus 10 M iron, 1 M A1-40 plus 50 M iron and 1 M A1-40 plus 10 M copper for 5 min (A), 1 h (B), and 4 h (C). Each Western blot is representative of three different experiments. Bands of proteins were quantified using scanning densitometry, and phospho-ERK1/2 levels were normalized to total ERK2 levels and expressed as a percentage of the control condition (mean⫾SD of three different experiments). *** P⬍0.001 and ** P⬍0.01 with respect to the control condition; ### P⬍0.001, ## P⬍0.01 and # P⬍0.05 when comparing “1 M A1-40⫹metal” versus the “metal” condition.
signaling pathways in isolated synaptic endings. Specifically, the state of Akt and ERK1/2 signaling was assessed because these kinases are known to play essential roles in synaptic plasticity and neuronal survival (Brazil et al., 2004; Dwivedi et al., 2009; Uranga et al., 2009). As previously demonstrated by our group, the presence of free iron alone in the medium provokes a time- and concentration-dependent diminution in mitochondrial function together with an increase in plasma membrane damage (Uranga et al., 2007, 2009). In the present work, the extent of synaptic damage in the presence of A peptide in combination with iron or copper was characterized. For this purpose, we used either free iron or free copper in the synaptosomal incubation medium as control conditions. A/metal impaired mitochondrial function to the same extent as metal alone, thus demonstrating that metal-induced
mitochondrial damage was not further increased by the simultaneous incubation with A and metal ions. Other studies have reported that A/iron or A/copper complexes cause increased damage when compared to the damage provoked by A or metal alone. These discrepancies with the results presented here could be mainly due to the fact that our assays were carried out in the isolated synaptic endings treated in an acute manner (5 min – 4 h), while most works show results obtained in whole neurons treated for more than 12 h (Bush, 2003; Dai et al., 2006). However, in the presence of A/iron (10 and 50 M), plasma membrane integrity was significantly disrupted demonstrating an additive effect with respect to the values found in the presence of metal alone after long times of incubation (4 h). Previous work from other laboratories demonstrated that chronic exposure (more than 24 h) to
388
R. M. Uranga et al. / Neuroscience 170 (2010) 381–389
A alone causes membrane perforations in the plasma membrane of hippocampal neurons leading to alteration in ionic homeostasis noticeable as calcium inward currents measured by patch clamp (Parodi et al., 2010). Our results, however, show that acute exposure to A/iron is able to generate membrane pores of such a size as to allow the leakage of LDH. As previously discussed, in spite of the damage observed in the synaptic membrane, mitochondrial function was not affected to the same extent. This could be indicating that the membrane constitutes the first target of A/iron, and that it is the starting sign of synaptic damage. The affection of other synaptic parameters which determine the severity of the damage, like mitochondrial function, could depend on the balance between the levels of transition metals, the availability of A, the production of free radicals and the persistence of stress. The fact that A/copper did not provoke any additional damage to the membrane (although copper is also a divalent transition metal as well as iron) strongly supports that both the structure of the coordination complexes and their interaction with the membrane depend on the chemical features of the metal. We have previously demonstrated that PI3K/Akt activation by free iron (5 min of exposure) occurs when the deleterious effects of the metal are slightly observed, and that this activation in the isolated synaptic ending is not able to prevent the oxidative damage induced by the metal (Uranga et al., 2007, 2009). Here we show that a short exposure to A/iron has an additional effect on Akt phosphorylation, thus demonstrating that the presence of the peptide is able to enhance iron-induced PI3K/Akt activation in an acute manner. Akt activation by A/iron was barely detected after 4 h of incubation, demonstrating that the activation pattern of this kinase does not correlate with the levels of synaptic damage. It is worth to note that while the A activation is long lived, the metal-induced Akt activation is transient. This early and transient activation could represent an attempt by the synaptic ending to activate a neuroprotective mechanism; however, the final outcome of this activation appears to depend on the orchestration of several signaling pathways whose evaluation would involve the entire neuron. Here we show that ERK1/2 presented a profile of activation different from that observed for Akt. The level of ERK1/2 phosphorylation in the presence of A/iron was the same as that observed for the free metal (iron) after short times of incubation (5 min). ERK1/2 activation by free iron has been demonstrated to be a PI3K/Akt-dependent event (Uranga et al., 2009). However, after 1-h exposure to A/iron (50 M) an enhancing effect on ERK1/2 phosphorylation with respect to the metal alone was observed. This synergistic effect was transient (1-h treatment) and previous to the major membrane damage observed after 4 h of exposure. Here we show that the differential profile in the activation of Akt and ERK1/2 in the presence of A/iron argues in favor of the independent activation of both kinase pathways in the synapse. These observations are in accordance with reports from other laboratories which show that A/metal complexes are able to independently mod-
ulate PI3K/Akt and ERK1/2 pathways in the neuron (Bica et al., 2009). Intriguingly, Akt and ERK1/2 activation were coincident after 1 h of incubation in the presence of A/ copper, thus demonstrating that the presence of A together with different metals could regulate differentially the activity of these kinases. There is accumulating evidence highlighting the local action of signaling pathways in the preservation of synaptic endings through gene-transcription independent mechanisms (Guo and Mattson, 2000; Gilman et al., 2003). The results presented here certainly demonstrate that synaptic Akt and ERK1/2 signaling are differentially activated by the presence of A and metal ions. While Akt activation is independent of synaptic damage, ERK1/2 activation seems to be transiently dependent. This work presents evidence about the initial events locally triggered at the synapse by A and transition metals. The overall mechanism as well as the physiological meaning of these findings should be further studied in a neuronal model. Acknowledgments—The authors specially thank Dr. Carlos Opazo for his valuable help in providing and discussing the protocol for A preparations. This work was supported by grants from the Universidad Nacional del Sur, the Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET) and the Agencia Nacional de Promoción Científica (FONCYT). Dr. Norma M. Giusto and Dr. Gabriela A. Salvador are research members of the CONICET. Romina M. Uranga is a research fellow of the CONICET.
REFERENCES Atwood CS, Obrenovich ME, Liu T, Chan H, Perry G, Smith MA, Martins RN (2003) Amyloid-beta: a chameleon walking in two worlds: a review of the trophic and toxic properties of amyloid-beta. Brain Res Brain Res Rev 43:1–16. Barnham KJ, Bush AI (2008) Metals in Alzheimer’s and Parkinson’s diseases. Curr Opin Chem Biol 12:222–228. Bica L, Crouch PJ, Cappai R, White AR (2009) Metallo-complex activation of neuroprotective signalling pathways as a therapeutic treatment for Alzheimer’s disease. Mol Biosyst 5:134 –142. Brazil DP, Yang ZZ, Hemmings BA (2004) Advances in protein kinase B signalling: AKTion on multiple fronts. Trends Biochem Sci 29:233–242. Bush AI (2003) The metallobiology of Alzheimer’s disease. Trends Neurosci 26:207–214. Bush AI, Tanzi RE (2008) Therapeutics for Alzheimer’s disease based on the metal hypothesis. Neurotherapeutics 5:421– 432. Cotman CW (1974) Isolation of synaptosomal and synaptic plasma membrane fractions. Methods Enzymol 31:445– 452. Dai XL, Sun YX, Jiang ZF (2006) Cu(II) potentiation of Alzheimer Abeta1-40 cytotoxicity and transition on its secondary structure. Acta Biochim Biophys Sin (Shanghai) 38:765–772. DeKosky ST, Scheff SW (1990) Synapse loss in frontal cortex biopsies in Alzheimer’s disease: correlation with cognitive severity. Ann Neurol 27:457– 464. Dong J, Canfield JM, Mehta AK, Shokes JE, Tian B, Childers WS, Simmons JA, Mao Z, Scott RA, Warncke K, Lynn DG (2007) Engineering metal ion coordination to regulate amyloid fibril assembly and toxicity. Proc Natl Acad Sci U S A 104:13313–13318. Dwivedi Y, Rizavi HS, Zhang H, Roberts RC, Conley RR, Pandey GN (2009) Aberrant extracellular signal-regulated kinase (ERK)1/2 signalling in suicide brain: role of ERK kinase 1 (MEK1). Int J Neuropsychopharmacol 12:1337–1354.
R. M. Uranga et al. / Neuroscience 170 (2010) 381–389 Gilman CP, Chan SL, Guo Z, Zhu X, Greig N, Mattson MP (2003) p53 is present in synapses where it mediates mitochondrial dysfunction and synaptic degeneration in response to DNA damage, and oxidative and excitotoxic insults. Neuromolecular Med 3:159 –172. Guo ZH, Mattson MP (2000) Neurotrophic factors protect cortical synaptic terminals against amyloid and oxidative stress-induced impairment of glucose transport, glutamate transport and mitochondrial function. Cereb Cortex 10:50 –57. Huang X, Moir RD, Tanzi RE, Bush AI, Rogers JT (2004) Redox-active metals, oxidative stress, and Alzheimer’s disease pathology. Ann N Y Acad Sci 1012:153–163. Jacobson MD, Burne JF, Raff MC (1994) Programmed cell death and Bcl-2 protection in the absence of a nucleus. EMBO J 13:1899 –1910. Keller JN, Mark RJ, Bruce AJ, Blanc E, Rothstein JD, Uchida K, Waeg G, Mattson MP (1997) 4-Hydroxynonenal, an aldehydic product of membrane lipid peroxidation, impairs glutamate transport and mitochondrial function in synaptosomes. Neuroscience 80:685– 696. Khan A, Dobson JP, Exley C (2006) Redox cycling of iron by Abeta42. Free Radic Biol Med 40:557–569. Kontush A (2001) Amyloid-beta: an antioxidant that becomes a prooxidant and critically contributes to Alzheimer’s disease. Free Radic Biol Med 31:1120 –1131. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680 – 685. Liu G, Men P, Perry G, Smith MA (2010) Nanoparticle and iron chelators as a potential novel Alzheimer therapy. Methods Mol Biol 610:123–144. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193:265–275. Mattson MP, Liu D (2002) Energetics and oxidative stress in synaptic plasticity and neurodegenerative disorders. Neuromolecular Med 2:215–231. Maynard CJ, Bush AI, Masters CL, Cappai R, Li QX (2005) Metals and amyloid-beta in Alzheimer’s disease. Int J Exp Pathol 86:147–159. Minniti AN, Rebolledo DL, Grez PM, Fadic R, Aldunate R, Volitakis I, Cherny RA, Opazo C, Masters C, Bush AI, Inestrosa NC (2009) Intracellular amyloid formation in muscle cells of Abeta-transgenic Caenorhabditis elegans: determinants and physiological role in copper detoxification. Mol Neurodegener 4:2. Nunomura A, Perry G, Aliev G, Hirai K, Takeda A, Balraj EK, Jones PK, Ghanbari H, Wataya T, Shimohama S, Chiba S, Atwood CS, Petersen RB, Smith MA (2001) Oxidative damage is the earliest event in Alzheimer disease. J Neuropathol Exp Neurol 60: 759 –767. Parodi J, Sepulveda FJ, Roa J, Opazo C, Inestrosa NC, Aguayo LG (2010) Beta-amyloid causes depletion of synaptic vesicles leading to neurotransmission failure. J Biol Chem 285:2506 –2514.
389
Plant LD, Boyle JP, Smith IF, Peers C, Pearson HA (2003) The production of amyloid beta peptide is a critical requirement for the viability of central neurons. J Neurosci 23:5531–5535. Santos MJ, Quintanilla RA, Toro A, Grandy R, Dinamarca MC, Godoy JA, Inestrosa NC (2005) Peroxisomal proliferation protects from beta-amyloid neurodegeneration. J Biol Chem 280:41057– 41068. Sayre LM, Smith MA, Perry G (2001) Chemistry and biochemistry of oxidative stress in neurodegenerative disease. Curr Med Chem 8:721–738. Scheff SW, Price DA (2003) Synaptic pathology in Alzheimer’s disease: a review of ultrastructural studies. Neurobiol Aging 24: 1029 –1046. Selkoe DJ (1996) Amyloid beta-protein and the genetics of Alzheimer’s disease. J Biol Chem 271:18295–18298. Sepulveda FJ, Opazo C, Aguayo LG (2009) Alzheimer beta-amyloid blocks epileptiform activity in hippocampal neurons. Mol Cell Neurosci 41:420 – 428. Shankar GM, Bloodgood BL, Townsend M, Walsh DM, Selkoe DJ, Sabatini BL (2007) Natural oligomers of the Alzheimer amyloidbeta protein induce reversible synapse loss by modulating an NMDA-type glutamate receptor-dependent signaling pathway. J Neurosci 27:2866 –2875. Smith MA, Casadesus G, Joseph JA, Perry G (2002) Amyloid-beta and tau serve antioxidant functions in the aging and Alzheimer brain. Free Radic Biol Med 33:1194 –1199. Syme CD, Nadal RC, Rigby SE, Viles JH (2004) Copper binding to the amyloid-beta (Abeta) peptide associated with Alzheimer’s disease: folding, coordination geometry, pH dependence, stoichiometry, and affinity of Abeta-(1–28): insights from a range of complementary spectroscopic techniques. J Biol Chem 279:18169 –18177. Terry RD, Masliah E, Salmon DP, Butters N, DeTeresa R, Hill R, Hansen LA, Katzman R (1991) Physical basis of cognitive alterations in Alzheimer’s disease: synapse loss is the major correlate of cognitive impairment. Ann Neurol 30:572–580. Uranga RM, Giusto NM, Salvador GA (2009) Iron-induced oxidative injury differentially regulates PI3K/Akt/GSK3beta pathway in synaptic endings from adult and aged rats. Toxicol Sci 111:331–344. Uranga RM, Mateos MV, Giusto NM, Salvador GA (2007) Activation of phosphoinositide-3 kinase/Akt pathway by FeSO4 in rat cerebral cortex synaptic endings. J Neurosci Res 85:2924 –2932. Wei Z, Song MS, MacTavish D, Jhamandas JH, Kar S (2008) Role of calpain and caspase in beta-amyloid-induced cell death in rat primary septal cultured neurons. Neuropharmacology 54:721–733. Zhu X, Raina AK, Lee HG, Casadesus G, Smith MA, Perry G (2004) Oxidative stress signalling in Alzheimer’s disease. Brain Res 1000:32–39.
(Accepted 22 July 2010) (Available online 29 July 2010)