Amyloid-β-Acetylcholinesterase complexes potentiate neurodegenerative changes induced by the Aβ peptide. Implications for the pathogenesis of Alzheimer's disease
- Margarita C Dinamarca†1,
- Juan P Sagal†1,
- Rodrigo A Quintanilla†1,
- Juan A Godoy1,
- Macarena S Arrázola1 and
- Nibaldo C Inestrosa1Email author
© Dinamarca et al; licensee BioMed Central Ltd. 2010
Received: 1 July 2009
Accepted: 18 January 2010
Published: 18 January 2010
The presence of amyloid-β (Aβ) deposits in selected brain regions is a hallmark of Alzheimer's disease (AD). The amyloid deposits have "chaperone molecules" which play critical roles in amyloid formation and toxicity. We report here that treatment of rat hippocampal neurons with Aβ-acetylcholinesterase (Aβ-AChE) complexes induced neurite network dystrophia and apoptosis. Moreover, the Aβ-AChE complexes induced a sustained increase in intracellular Ca2+ as well as a loss of mitochondrial membrane potential. The Aβ-AChE oligomers complex also induced higher alteration of Ca2+ homeostasis compared with Aβ-AChE fibrillar complexes. These alterations in calcium homeostasis were reversed when the neurons were treated previously with lithium, a GSK-3β inhibitor; Wnt-7a ligand, an activator for Wnt Pathway; and an N-methyl-D-aspartate (NMDA) receptor antagonist (MK-801), demonstrating protective roles for activation of the Wnt signaling pathway as well as for NMDA-receptor inhibition. Our results indicate that the Aβ-AChE complexes enhance Aβ-dependent deregulation of intracellular Ca2+ as well as mitochondrial dysfunction in hippocampal neurons, triggering an enhanced damage than Aβ alone. From a therapeutic point of view, activation of the Wnt signaling pathway, as well as NMDAR inhibition may be important factors to protect neurons under Aβ-AChE attack.
Alzheimer's disease (AD) is an age-related neurodegenerative disease characterized by selective neuronal cell death that affects brain areas related to memory and learning . The neuropathological hallmarks of AD patients are the presence of senile plaques and neurofibrillary tangles in the brain . Senile plaques are aggregates of deposited amyloid-β peptide (Aβ), surrounded by dystrophic neurites and reactive glial cells . Aβ-peptide is the main constituent of senile plaques and a major neurotoxic agent . Other proteins associated to amyloid deposits, known as "chaperone molecules"  include laminin, apolipoprotein E and acetylcholinesterase (AChE) [3–5]. In fact, AChE has been found to co-localize with Aβ deposits such at those present in pre-amyloid diffuse deposits, mature senile plaques and cerebral blood vessels [6, 7]. Most of the cortical AChE activity present in AD brain is predominantly associated to the amyloid core of senile plaques rather than with the neuritic component found in the periphery . More than 10 years ago, we found that AChE a key enzyme in the degradation of the neurotransmitter acetylcholine, present in cholinergic terminals accelerates Aβ aggregation , promoting the formation of a stable complex with the enzyme (Aβ-AChE complex) . We showed for the first time that a macromolecule found in the synapse interacts with Aβ to form a complex which alters the normal synaptic function in hippocampal neurons. In vivo studies showed that AChE infused stereotaxically into the CA1 region of the rat hippocampus promotes novel plaque-like structures [9, 10]. More recently, independent studies support our initial observation indicating that AChE accelerates Aβ deposition, in fact a double transgenic mouse over expressing both the human APP containing the Swedish mutation and the human AChE has been developed. Such double transgenic mice start to form amyloid plaques around 3 months, earlier than mice expressing only the APP transgene. Moreover, the double AChE-APP transgenic mouse presents more and larger plaques than the control animals, as well as some behavioural deterioration, as demonstrated by a working memory test . Indeed, injection of the complex into the rat hippocampus produces neuronal cell loss and astrocyte hypertrophy .
The early events triggered in neurons in response to Aβ peptide have been largely studied [12–16]. It has been described that Aβ oligomers/fibrils induce intracellular calcium deregulation that leads to apoptosis through mitochondria dysfunction, whether by direct interaction with isolated mitochondria or by indirect association with the neuronal membrane [12–16]. We report here the early effects that Aβ-AChE complexes induce in rat hippocampal neurons using live-cell imaging techniques. Results show that Aβ-AChE complexes are more toxic than the Aβ fibrils alone on rat hippocampal neurons. In fact, neurons treated with Aβ-AChE complexes showed a much disrupted neurite network compared to neurons treated with Aβ. One the earliest effect of Aβ-AChE complexes is an increase in intracellular calcium, which leads to the loss of the mitochondrial membrane potential, this being in agreement with the notion that calcium homeostasis and mitochondrial function are the main targets of these complexes.
Aβ-AChE complexes disrupt neuronal morphology and induce intracellular calcium increase in hippocampal neurons
Aβ-AChE complex induces a neurite network loss in hippocampal neurons
Neurite length (μm)
65 ± 7
Aβ (5 μM)
30 ± 4*
Aβ-AChE (5 μM)
28 ± 5*
To determine whether Aβ-AChE is more toxic than Aβ, we performed cell viability studies using MTT, as an indicator of cell viability. Aβf alone decreased cell viability by 35 ± 7% when compared with control, while Aβ-AChEf under the same conditions and concentration reduced viability by 50 ± 6%, suggesting that the complexes are significantly more toxic than Aβ aggregates alone at equal concentrations (Fig. 1B).
An increase in intracellular calcium has been observed in Aβ treated neurons as well as during glutamate excitotoxicity . Therefore, we characterized calcium homeostasis after exposure of hippocampal neurons to Aβ-AChEf. Neurons were loaded for 30 min with Fluo-3 AM and then cytosolic calcium levels were evaluated in cells exposed to 5 nM AChE (Fig. 1C, silver circles), 5 μM Aβf (Fig. 1C, open circles) and 5 μM Aβ-AChEf (Fig. 1C, closed circles). Neurons treated with Aβf and Aβ-AChEf showed a marked and consistent calcium increase, while treatment with AChE enzyme alone showed no effect on the mobilization of intracellular calcium. However, the final fluorescence levels reached after 1 h treatment with Aβ-AChEf were significantly higher than Aβf, indicating that Aβ-AChEf complexes induces a sustained higher increase in intracellular calcium, in comparison with Aβ-treated neurons (Fig. 1C, inset). Then, we evaluated whether the calcium increase observed by treatments trigger the apoptotic cascade after 1 h treatment. The caspase-3 activity measurements showed that Aβ-AChEf significant increase the caspase-3 activity compared with Aβf or Aβo. On the other hand, AChE treatment did not show any effect (Fig. 1D). These results indicate that Aβ-AChE complexes showed a greater modification of intracellular calcium homeostasis than Aβ alone triggering the activation of the apoptotic pathway.
Aβ-AChE complexes induce neurotoxicity by alteration of intracellular calcium
Aβ-AChE complexes induce mitochondrial membrane potential loss in hippocampal neurons
Role of mitochondria in the intracellular calcium influx induced by Aβ-AChE complexes in hippocampal neurons
Wnt-7a, lithium chloride and a glutamate receptor antagonist, prevent the Aβ-AChE induced neuronal calcium deregulation in hippocampal neurons
Presence of AChE increases Aβ oligomer formation and neurotoxicity in hippocampal neurons
AChE is an enzyme involved in diverse functions in both central and peripheral nervous systems and it has been demonstrated to be associated with senile plaques . On the other hand, a renewed interest in calcium as part of the pathology of AD began with the recent observation of polymorphism in a CALHM1 gene that may influence calcium mutations and increase the risk of AD . Indeed, several studies indicate that familial AD mutations in presenilins affect calcium homeostasis, releasing intracellular calcium from ryanodine or IP3 channels .
We had reported here that Aβ-AChE complex trigger more neurodegeneration than the Aβ alone, acting as a chaperone protein which increases the toxicity of Aβ peptide, confirming previous studies both in vitro and in vivo [9, 10]. We have shown that a short exposure of hippocampal neurons to Aβ-AChE complexes caused an influx of calcium with higher levels than the amyloid fibrils alone and this increase was reversible and concentration-dependent; in addition the calcium source for such increase was the extracellular calcium present in the media, which probably release some intracellular calcium reservoirs, as mitochondria. Our results suggest that Aβ-AChE complexes are exerting their actions at the plasma membrane level, possibly acting on a calcium channel or on some yet unidentified cell surface receptor-channel that could activate calcium entry upon Aβ complex interaction . Disruption of intracellular homeostasis of Ca2+ and K+ by opening of these channels, has been extensively proposed as a mechanism of Aβ neurotoxicity [14, 18]. It has been reported that Aβ-induced activation of AChE in P19 cells was mediated through the opening of L-type calcium channels . Also, it has been found that Aβ induces production of an amphiphilic monomeric isoform of AChE through a mechanism that involves at L-type calcium channels. This specific increase in a minor isoform of the enzyme has also been seen to be increasing in transgenic mice that overexpress AβPP , as well as in the brains of rats that received intracerebral injections of Aβ peptides . It is also increased in the brain and cerebrospinal fluid of patients with AD . These studies suggest an active role of the Aβ-AChE complexes in the AD pathology. Aβ-AChE complexes and Aβ treatment had different effects over the mitochondrial membrane potential (ΔΨmit) observed with the TMRM+ dye. Our experiments indicated that Aβ-AChE complexes affected ΔΨmit more than Aβ; also, we observed that the mitochondrial membrane potential was compromised in a non-reversible manner, even when the calcium increase was reverted after wash out. These events could be related with previously described mitochondrial dysfunction effects, which included the mitochondria Permeability Transition Pore (mPTP) opening . This structure is involved in cytochrome c release, apoptotic mechanisms on high levels of cytosolic calcium and oxidative stress excess contribute to this structure opening and finally affect mitochondrial membrane potential, ATP levels, and mitochondrial calcium uptake as we observed in Fig. 4C[35–37]. Our results indicate that the increased neurotoxicity of Aβ-AChE complexes with regard to Aβ aggregates is probably related to a more rapid and non-reversible increase of mitochondrial dysfunction or to some direct effects at the mitochondrial viability level.
It has been described that Aβ oligomers increase intracellular calcium . We showed that Aβ in the presence of AChE produces Aβ oligomers faster than Aβ aggregates incubated without the enzyme, an effect that could explain why the alterations triggered by Aβ-AChE complexes were stronger at Ca2+ homeostasis level.
Previous studies from our laboratory indicated that lithium protects hippocampal neurons against Aβ peptide and Aβ-AChE complex neurotoxicity [22, 38]. Additionally, we found that pre-incubation with Wnt-7a ligand prevents the increase in cytosolic calcium induced by Aβ . Hence, in order to study whether Wnt signaling had any effect over the observed calcium increase, we performed time-lapse experiments co-treating neurons with Aβ-AChE complexes in the presence of Wnt-7a or lithium. These experiments proved that Wnt-7a and lithium prevent the cytoplasmic calcium influx induced by these complexes. These results are also consistent with the idea that lithium induces NMDA receptor inactivation with a concomitant decrease in the cytosolic calcium increase induced by glutamate . Moreover, lithium treatment reduced the level of NR2B subunit phosphorylation at Tyr1472 which results in inactivation of NMDA receptors contributing to neuroprotection against glutamate excitotoxicity . Furthermore, it has been reported that lithium robustly protected individual brain mitochondria loaded with Rhodamine 123 (mitochondrial potential dye) against Ca2+-induced depolarization, an event that was mediated by inhibition of mPTP activation by lithium . Taken together, this evidence and our studies allow us to suggest a novel and interesting role of lithium protection against mitochondrial dysfunction triggered by calcium dyshomeostasis.
Excessive glutamate, an abundant neurotransmitter in the brain, is toxic to neurons and can lead to intracellular calcium overload and neuron death. Overstimulation of the glutamatergic system, also known as glutamate excitotoxicity is observed in a number of neurodegenerative diseases including AD . In fact, memantine, an open-channels blocker of the NMDA receptor is use to treat moderate and severe AD .
We have studied here, the role of the NMDA receptor in neurotoxic effects induced by Aβ-AChE complexes using MK-801, a glutamate receptor antagonist, our studies indicates that the NMDA receptor antagonist, totally prevents the cytoplasmic calcium influx induced by the complex. Therefore, it may be possible that the decrease observed in calcium cytosolic levels may be due in part to inactivation of the NMDA receptors by lithium. Further studies are necessary to clarify this issue.
We report here new findings about the Aβ-AChE complex mechanism, which includes a massive cytoplasmic calcium influx and severe mitochondrial dysfunction in rat hippocampal neurons. The pathologic entry of calcium determines that mitochondria participate in the buffering of cation excess, and this process leading to its membrane depolarization and loss of viability. The increased Aβ-AChE complexes neurotoxicity observed with regard to Aβ aggregates could be explained by a rapid loss of the mitochondrial membrane potential. These events may be related to key neuropathological mechanism of AD, and Aβ-AChE complexes might cause further damage through changes in calcium homeostasis and mitochondrial function, as well as at other neuronal levels.
Synthetic Aβ1-42 and Aβ42-1 peptides corresponding to the human Aβ wild-type and reverse sequence, respectively and both were obtained from Genemed Synthesis, Inc. (San Francisco, CA). Chemicals, culture media and sera were obtained from Sigma (St. Louis, MO), Roche (Alameda, CA), Merck (Darmstadt, Germany), Gibco BRL (Paisley, UK). Fluo-3 AM, Calcein-AM, Fura Red AM, and Bapta AM from Molecular Probes (Eugene, OR). Rhod-2AM, and TMRM+ (Xanthylium,3,6-bis(dimethylamino)-9-(2-(methoxycarbonyl)phenyl)-, perchlorate) were a kind gift of Dr. Luis Felipe Barros (CECS, Valdivia, Chile).
Tetrameric G4 AChE form (sedimentation coefficient, 10.7 S) was purified from bovine caudate nucleus, using acridine affinity chromatography as previously described . Both specific activity (6,000 U/mg protein) and staining intensity after sodium dodecyl sulfate-polyacrylamide gel electrophoresis were used to verify purity.
Formation of Amyloid Species
Aβ aggregates were formed in a turbidity assay as previously described . Briefly, For Aβf and Aβ-AChEf, the Aβ peptide stock solution was prepared by dissolving freeze-dried aliquots of Aβ in dimethyl sulfoxide (DMSO) at 15 mg/ml (3.5 mmol/L). An aliquot of this stock solution equivalent to 15 μg of Aβ peptide was added to aqueous buffer (725 μl total volume; 100 μM PSB, pH 7.4). For the aggregation assay in the presence of AChE, an identical aliquot of the stock solution was added to a buffer containing AChE (100 nM). The solutions were stirred continuously (1,350 rpm) at room temperature for 24 h and then left at 4°C for another 48 h, then an aliquot was taken and observed by electron microscopy. Aggregation was measured by turbidity at 405 nm against a buffer blank. Amyloid aggregates obtained were characterized by Thioflavin-T (Th-T) binding .
Preparation of Aβ oligomers
Aβoligomers were formed using Aβ peptide in the presence of AChE. Briefly, a 50 μM Aβ stock solution was prepared in the presence of 50 nM AChE. The solution was kept at 37°C for 1 h and then used immediately. An aliquot of Aβo and Aβ-AChEo was separated by centrifugation at 14,000 rpm (15 min) for electron microscopy. The supernatant was saved for non-denaturing SDS-PAGE gels using SDS-free and β-mercaptoethanol-free loading buffer and then regular western blot.
Fresh aliquots of samples were diluted 1:3 in water and 5 βl placed on Parlodion/carbon-coated 300-mesh copper grids for 60s. Excess sample was removed and 5 μl of 2% aqueous uranyl acetate was placed onto the grid for 10s, followed by removal of excess staining solution with filter paper and air-drying. Observations were carried out using a Philips Tecnai 12 electron microscope operated at 80 kV. Photographs were taken at original magnifications of 49,000×. Copies from the negatives were made with a further 3× magnification of given areas and used for figure presentation.
Culture of rat hippocampal neurons
Hippocampi from Sprague-Dawley rats at embryonic day 18 were dissected and primary rat hippocampal cultures were prepared as described previously [39, 45]. Hippocampal cells were seeded in polylysine-coated wells and cultivated in Neurobasal medium supplemented with B27, on day 3 of culture; cells were treated with 2 μM 1-β-D-arabinofuranosylcytosine (AraC) for 24 h to reduce most of the glial cells present in the culture. Seven days later, cultured hippocampal neurons were used for various experiments. The average number of neurons in each experiment corresponded approximately to 98% of total cells present in the cultures.
Cell survival assay
Cell viability was measured by the modified 3-[4,5-dimethylthiazol-2yl]-2,5-diphenyltetrazolium bromide (MTT) assay as described previously .
Hippocampal neurons plated on polylysine-coated covers (25,000 cells/cover) were treated and then fixed with 4% paraformaldehyde/4% sucrose in PBS for 20 min, permeabilized with 0.2% Triton-X100 for 5 min, blocked with 0.2% gelatine and immunostained using an anti- Map-1B, neurofilament protein, synapsin-1 or PSD-95 antibody (1:500). Coverslips were mounted and then analyzed under a Zeiss confocal microscope.
The number and length of neurites were quantified using an Image-Pro plus software as described previously .
Hippocampal neurons 15 DIV were treated with Aβ-AChEf, Aβo and Aβf for 1 h at 37°C, then the cells were homogenized with RIPA buffer and then lysates were centrifuged at 15,000 g for 20 min at 4°C, the supernatant were collected to determinate Caspase activity. Caspase-3, substrate chromogenic (Upstate Biotechnology Inc., Lake Placid, NY, USA) was prepared in 12.5 mM buffer Hepes; 31.25 w/v Sucrose; 0,3125 w/v CHAPS pH 7.4. The reaction was measured at 405 nm in a microtiter plate reader.
Aβ-AChE in plasmatic membrane
Hippocampal neurons 15 DIV were treated with Aβ-AChEo (1 or 5 μM) or AChE alone (1 or 5 nM) for 1 h and membrane surface proteins were biotinylated with sulfo-NHS-LC-biotin (Pierce) in a final concentration of 0.5 mg/mL for 45 min. After biotinylation step, the free biotin was quenched by incubation with 50 mM NH4Cl for 10 min. Cells were lysed in ice-cold SA buffer (150 mM NaCl; 20 mM Tris pH 8.0; 5 mM EDTA; 1% Triton-X-100; 0.2% BSA and protease inhibitors). Nuclear and cellular debris was removed by centrifugation at 14,000 x g for 5 min at 4 °C and the biotinylated cell-surface proteins were then adsorbed to streptavidin agarose beads for 16 h at 4 °C. Beads were washed and bound proteins were analyzed by SDS-PAGE followed by immunoblotting against AChE. The values for biotinylated cargo proteins were normalized to total cargo proteins expressed in the cells.
Mitochondria membrane potential
Changes in mitochondrial membrane potential was determined by specific mitochondrial probe TMRM+ and detected in a confocal microscope [19, 46]. Neurons were grown on poly-L-lysine-coated glass coverslips and cultured for 5 days. The cells were then loaded for 30 min with 30 nM TMRM+ in Krebs-Ringer-Hepes (136 mM NaCl; 20 mM Hepes; 4.7 mM KCl; 1.25 mM MgSO4; 1.25 mM CaCl2) (KRH)-glucose containing 0.02% pluronic acid, then washed, and allowed to equilibrate for 30 min. Coverslips were then mounted in a chamber on the stage of a confocal laser scanning microscope (LSM Pascal Zeiss model 510, Carl Zeiss Ltd.). The fluorescence changes determined by TMRM fluorescence indicated the mitochondria potential changes. Images were acquired using a 543-nm He-Ne laser to excite TMRM+ and the signals were collected at 570 nm [18–20]. Signal from control neurons and neurons treated with Aβ-AChE complexes were compared using identical settings for laser power, confocal thickness and detector sensitivity. The images were analyzed with Zeiss confocal software, the mean TMRM+ fluorescence signal was measured per live cell. Estimation of fluorescence intensity of TMRM+ was presented like the pseudoratio (ΔF/Fo) indicated by: ΔF/Fo = (F-Fbase)/(Fbase-B), where F is the measured fluorescence intensity of the indicator, Fbase is the fluorescence intensity before the stimulation, and B is the background signal determined from the average of areas adjacent to the cells. For mitochondrial membrane potential by MitoTracker fluorescence, hippocampal neurons 15 DIV were treated with Aβ-AChEf, Aβo and Aβf for 1 h at 37°C. Then, cells were stained with MitoTrackers-fx (Molecular Probes, Eugene, OR). In all treatments, individual coverslips were rinsed twice in PBS and loaded with 10-500 nM MitoTracker for 15 min at 37°C. Dyes were diluted in PBS from a 1 mM stock in anhydrous dimethyl sulfoxide. Coverslips were rinsed for 15 min in PBS at room temperature. Loading parameters were tested and optimized for assessing fluorescence intensity and localization in Confocal Zeis Microscope and the quantification was made using Image J Program.
Cytoplasmic Calcium Imaging
Neurons grown on glass coverslip were loaded for 30 min (37°C) with the following fluorescent probes as their acetoxymethyl (AM) ester forms: 5 μM Fluo-3 AM and 10 μM Rhod-2 AM in Krebs-Ringer-Hepes (KRH)-glucose containing 0.02% pluronic acid. The fluorescence changes determined by Fluo-3 represent the cytoplasmic calcium [Ca2+]cyt changes. Coverslips were washed three times with PBS and left in KRH-glucose for 10 min until cell fluorescence had reach plateau. Fluorescence was imaged with a confocal laser scanning microscope as described previously . Images were acquired using a 488-nm Argon laser to excite Fluo-3 and Fura-Red fluorescence. The signals were collected at 505-530 nm (Fluo-3). Background was measured in parts of the field devoid of cells and found to be not significantly different from the signal recorded in cells depleted of dye with 100 μM digitonin. This value was subtracted from cell measurements. The fluorescence intensity variation was recorded from 15-20 neurons in average per experiment. Estimation of fluorescence intensity of Fluo-3 was presented like the pseudoratio (ΔF/Fo), as indicated before [18–20].
Cell membrane integrity
Hippocampal neurons were loaded 30 min with Calcein-AM (Molecular Probes, Leiden, Netherlands) as an indicator of cell integrity .
Mitochondrial Calcium Measurements
Hippocampal neurons were grown on 35-mm dishes and loaded with 5 ìM Rhod-2 AM in KRH-glucose buffer containing 0.02% pluronic acid. The fluorescence changes determined by Rhod-2 indicate calcium changes in the mitochondria . To estimate Rhod-2 fluorescence pattern in live mitochondria, we used MitoTracker Green™ (MTG) to mark the mitochondria. Cells were washed 3 times and left in KRH-glucose buffer for 10 min until cell fluorescence equilibrated. Fluorescence was imaged with a confocal laser scanning microscope (Leica TCS SP1) using a 40× water immersion lens. Images were acquired using a 488-nm argon laser to excite MTG fluorescence and a 563-nm He-Ne laser to excite Rhod-2 fluorescence. The signals were collected at 505-530 nm (MTG) and at 590 nm (Rhod-2). Fluorescence background signal was subtracted from cell fluorescence measurements in every experiment. The fluorescence intensity variation was recorded from 5-10 cells on average per experiment. Estimation of fluorescence intensities were presented as the pseudoratio (ΔF/Fo), which was calculated using the formula ΔF/Fo = (F - Fbase)/(Fbase - B), where F is the measured fluorescence intensity of the indicator, Fbase is the fluorescence intensity before the stimulation, and B is the background signal determined from the average of areas adjacent to the cells .
Results were expressed as mean ± standard error. Student's t test and the Mann-Whitney test were used for analyzing data for fluorescence measurements and image analysis. P < 0.05 was regarded as statistically significant.
This research was funded by Center for Aging and Regeneration (CARE) & FONDAP-Biomedicine grant N° 13980001 (N.C.I.). Support from the Millennium Institute of Fundamental and Applied Biology (MIFAB) is gratefully acknowledged. MCD and MSA are predoctoral fellows from CONICYT.
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