Differential Membrane Toxicity of Amyloid-β Fragments by Pore Forming Mechanisms

Abstract

A major characteristic of Alzheimer’s disease (AD) is the presence of amyloid-β peptide (Aβ) oligomers and aggregates in the brain. It is known that Aβ oligomers interact with the neuronal membrane and induce perforations that cause an influx of calcium ions and enhance the release of synaptic vesicles leading to a delayed synaptic failure by vesicle depletion. To better understand the mechanism by which Aβ exerts its effect on the plasma membrane, we evaluated three Aβ fragments derived from different regions of Aβ_1 - 42; Aβ_1 - 28 from the N-terminal region, Aβ_25 - 35 from the central region, and Aβ_17 - 42 from the C-terminal region. The neuronal activities of these fragments were examined with patch clamp, immunofluorescence, transmission electron microscopy, aggregation assays, calcium imaging, and MTT reduction assays. The present results indicate that the fragment Aβ_1 - 28 contributes to aggregation, an increase in intracellular calcium and synaptotoxicity, but is not involved in membrane perforation; Aβ_25 - 35 is important for membrane perforation, calcium increase, and synaptotoxicity; and Aβ_17 - 42 induced mitochondrial toxicity similar to the full length Aβ_1 - 42, but was unable to induce membrane perforation and calcium increase, supporting the idea that it is less toxic in the non-amyloidogenic pathway.

Keywords

Alzheimer’s disease amyloid membranes peptides pore

INTRODUCTION

Alzheimer’s disease (AD) is a progressive neurodegenerative pathology that affects the human brain and causes cognitive and behavioral disorders. A major characteristic of AD is the accumulation of amyloid-β peptide (Aβ) aggregates in the brain that lead to synaptic dysfunctions [1]. A number of original studies have shown clearly that Aβ can form ion pores in lipid membranes and increase intracellular calcium levels, contributing significantly to the understanding of Aβ toxicity at the membrane level [2 –5]. Moreover, we have previously shown that Aβ oligomers from 1–40, 1–42, and arctic forms of amyloid associate with neuronal membranes and induce the formation of large pores (the so called membrane perforations), increasing calcium ion influx and the release of synaptic vesicles that lead to a delayed synaptic failure produced by vesicle depletion [6 –9].

It is believed that the length and the physicochemical properties of the amino acids of Aβ are important in the aggregation and toxicity processes. For example, the longer form of Aβ, Aβ_1 - 42, can aggregate more readily than Aβ_1 - 40 and seems to be the most toxic specie [10, 11]. In addition, it is accepted that residues F19 and F20 play a critical role in the aggregation process [12], and that G33 and G37 are important for membrane perforation induced by Aβ [8, 13]. Previous studies suggested that Aβ fragments, such as Aβ_25 - 35, produced in the brain by enzymatic processing, can produce toxic effects similar to those of Aβ_1 - 42 [14 –17].

Various studies have suggested that fragment Aβ_25 - 35 contributes to aggregation and toxicity [16 –20]. Interestingly, this fragment is augmented in the brain of AD patients, possibly produced as the natural process of aging [14]. Indeed, Aβ undergoes a process of racemization and proteolytic processing as the brain ages producing fragments like Aβ_25 - 35 [14, 21], which could play a role in the toxic effects induced by Aβ in AD. On the other hand, a recent study suggested that while Aβ_1 - 28 binds to hydrophilic regions in the lipid membrane, Aβ_25 - 40 localizes in the hydrophobic core of the bilayer, where it can affect the phospholipid order [22].

In the present study, we examined three main Aβ fragments from different regions of Aβ_1 - 42: Aβ_1 - 28 from the N-terminal region, Aβ_25 - 35 from the central region and Aβ_17 - 42 from the C-terminal region (Fig. 1A). Our aim was to determine the role of these Aβ fragments on membrane perforation and to propose a scheme that could explain our working hypothesis that neuronal toxicity involves the sequence of aggregation, membrane association, perforation, changes in intracellular calcium and finally synaptic failure.

MATERIALS AND METHODS

Primary cultures of rat hippocampal neurons

Hippocampal neurons were obtained from 18-day pregnant Sprague-Dawley rats and maintained for 10–14 days in vitro (DIV) as previously described [23]. All animals were handled in strict accordance with NIH guidelines and approved by the Ethics Committee of the Universidad de Concepción (Concepción, Chile).

Peptide preparation and storage

For the experiments we used Aβ_1 - 42 (GenicBio, Shanghai, China) and the Aβ fragments: Aβ_1 - 28, Aβ_25 - 35, and Aβ_17 - 42 (Anaspec, CA, USA). Aβ and Aβ fragments were dissolved in hexafluoroisopropanol (HFIP), then aliquoted, evaporated at room temperature, and stored at –20°C. For the preparation of oligomers, ultrapure water was added to the aliquots in an Eppendorf tube to a final concentration of 80 μM. After 10–20-min incubation at room temperature, the samples were stirred at 500 rpm using a Teflon-coated micro-stir bar for 24–48 h at room temperature in order to form oligomers (∼22°C) and subsequently stored at 4°C until required [8, 9]. All the experiments were performed using oligomeric species, unless otherwise stated. For Aβ fiber formation, the oligomeric species were incubated at 37°C for 24 h.

Thioflavin T assay

Aggregation curve experiments were performed using Thioflavin T (ThT). 40 μM of ThT was added to a 32 μM concentration of freshly prepared or oligomeric species (previously aggregated) of Aβ and Aβ fragments (1-28, 25-35, and 17-42). The fluorescence intensities for the mixed solutions were recorded at 482 nm, after an excitation of 440 nm, in a Novostar fluorescence microplate reader (BMG labtech, Germany). The experiments were performed in triplicate in a 96-well black clear-bottom plates (Corning, USA).

Transmission electron microscopy

Ten microliters of Aβ at a concentration of 80 μM was applied to carbon-coated Formvar grids. Samples were fixed with a 2% glutaraldehyde solution for 5 min. Aβ aggregates were stained with 5 μl of 0.2% (w/v) phosphotungstic acid (PTA) and the grid was air-dried. Samples were examined using a JEOL 1200 EX II electron microscope.

Electrophysiological recordings

Recordings were carried out using the patch clamp technique as previously described [24]. Briefly, culture media was changed for an external solution containing (in mM): 150 NaCl, 5.4 KCl, 2.0 CaCl₂, 1.0 MgCl₂, 10 glucose and 10 HEPES (pH 7.4). The internal solution consisted of (in mM): 120 KCl, 2.0 MgCl₂, 2 ATP-Na₂, 10 BAPTA, 0.5 GTP, 10 HEPES (pH 7.4). The holding potential was fixed at –60 mV and single channel measurements were performed in cell attached mode and acquired using a Digidata 1200 board. The cells were visualized using an Eclipse TE200-U phase contrast inverted microscope (Nikon, Japan). Recording pipettes were pulled from borosilicate glass (WPI, Sarasota, FL) in a horizontal puller (Sutter Instruments, Novato, CA) having a resistance between 10 and 15 MrmOmega. Perforated recordings were obtained as previously described [8, 25] and recorded using the pClamp10 software (Axon Instruments, Inc.). Briefly, Aβ was added to the pipette internal solution and a 5 mV pulse was used to monitor the formation of the seal and subsequent membrane perforation. For microscopic recordings, we used the cell-attached configuration with the same conditions of perforated patch clamp, but measuring the channel activity induced by Aβ [9, 25].

Immunofluorescence

Hippocampal neurons were washed in PBS (pH 7.4) and fixed with 4% paraformaldehyde for 15 min. Subsequently, the neurons were washed in PBS, and then the cells were permeabilized and blocked for 30 min with PBST (PBS + triton X-100 0.1%) and horse serum 10%, respectively. The cells were then incubated with the following primary antibodies for 16 h: anti-MAP2, 1:400 (Santa Cruz Biotechnology, CA, USA) and anti-SV2, 1:200 (Hybridoma Bank, IA, USA). Secondary antibodies conjugated with FITC, Cy3, and/or Cy5 at a dilution of 1:200 for 2 h were used for fluorescent staining (Jackson ImmunoResearch Laboratories, PA, USA). Finally, samples were mounted in fluorescent mounting medium (DAKO, CA, USA) and images were obtained using a Zeiss LSM700 confocal microscope (Zeiss, Germany). The immunoreactivity of the proteins was quantified in the primary processes with ImageJ software (NIH). Fluorescent signal was quantified as relative units (RU) using a region of interest (ROI) that comprised the first 20 μm of the primary processes.

Calcium imaging

Neurons were loaded with Fluo-4 AM (1 μM in pluronic acid/DMSO, Molecular Probes, Eugene, OR, USA) for 20 min at 37°C and then washed twice with external solution and incubated an additional 20 min at 37°C. Neurons were mounted in a perfusion chamber that was placed on the stage of an inverted fluorescent microscope (Eclipse TE, Nikon, Japan) equipped with a xenon lamp and a 40× objective (22–24°C). Cells were subsequently illuminated for 200 ms using a computer-controlled Lambda 10-2 filter wheel (Sutter Instruments), and ROIs were simultaneously selected on neuronal soma containing Fluo-4 fluorescence (Ex/Em: 480/510 nm). Images were collected at 5-s intervals during a continuous 200-s period of recording with a 12 bit cooled SensiCam camera (PCO, Germany). Finally, calcium imaging was acquired and analyzed off line with Axon Instruments Workbench 2.2 software. For Aβ application, Aβ oligomers were delivered directly into the external solution at a final concentration of 1 μM. The basal fluorescence was recorded for 80 s before Aβ application. Three independent experiments were done for each condition.

Data analysis

Non-lineal analysis was performed using Prism (Graph Pad). Single channel recordings were analyzed using Clampfit software (Axon Instruments, Inc.) and QuB (University at Buffalo, USA). The values are expressed as mean±SEM (standard error mean). Statistical differences were determined using Student’s t test or ANOVA. The experiments were performed in triplicate.

RESULTS

We evaluated three Aβ fragments representing the N-terminal (1-28), central (25-35), and C-terminal region (17-42) of Aβ that are found in the brain of older adults (Fig. 1A) [14 , 26–28]. First, we performedaggregation experiments using a Thioflavin T (ThT) dye that binds to β sheet-rich structures, such as those in amyloid aggregates, and displays enhanced fluorescence. Aβ_1 - 42 oligomers or the distinct fragments were incubated with ThT for 24 h at 37°C (Fig. 1B). The data revealed a fast increase in ThT fluorescence, with the exception of the Aβ_17 - 42 fragment (Fig. 1C). This result suggests that the oligomers are acting as seeds for further aggregation, forming amyloid fibers. Moreover, in order to better characterize the aggregation of these fragments, we performed transmission electron microscopy experiments evaluating the oligomeric and fibrillar ultrastructure of Aβ_1 - 42 and the fragments, in similar conditions to those in the presence of ThT (Fig. 2). The data showed that the soluble oligomers of Aβ_1 - 42 and Aβ_25 - 35 presented similar amorphous structures after 24 h at room temperature (Fig. 2A). Interestingly, when these oligomers were incubated for an additional time (24 h) at 37°C, they were able to proceed toward fibers, although with distinct characteristics between them (Fig. 2B). For example, Aβ_25 - 35 formed thick cross-linked fibers rather than thin fibers like those observed with Aβ_1 - 42 (Fig. 2B). On the other hand, Aβ_1 - 28 oligomers appeared larger than Aβ_1 - 42 oligomers (Fig. 2A), but formed fibers that were structurally similar to Aβ_1 - 42 (Fig. 2B). Aβ_17 - 42 displayed the most distinct structures after incubating the oligomers for 24 h at 37°C, being unable to form fibers like the other fragments (Fig. 2B).

Subsequently, we evaluated mitochondrial dysfunction after chronic treatment (24 h) with the oligomeric species of Aβ_1 - 42, Aβ_1 - 28, Aβ_25 - 35, and Aβ_17 - 42 (5 μM) in hippocampal neurons. For this study, we used an MTT (3-[4,5-dimethylthiazol-2-yl, 4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) assay that is based on the conversion of MTT into formazan crystals by mitochondrial enzymes, thus determining mitochondrial activity. The data showed that all species decreased mitochondrial function with values of 40±0.7% with Aβ_1 - 42, 60±1.5% with Aβ_1 - 28, 65±1.2% with Aβ_25 - 35, and 50±1.4% with Aβ_17 - 42 (n = 3, ^*** p < 0.001, ^** p < 0.01, ^* p < 0.05) (Fig. 3). The results demonstrated that although Aβ_1 - 42 was the more toxic species, all the fragments were able to produce mitochondrial dysfunction.

It is known that Aβ can increase intracellular calcium levels after acute application [6, 29]. Thus, we performed experiments with the Fluo-4 probe to analyze intracellular calcium levels after acute applications with the different Aβ fragments (Fig. 4). The data revealed that only Aβ_1 - 42, Aβ_1 - 28, and Aβ_25 - 35 increased the calcium levels. Aβ_17 - 42, on the other hand, was unable to change the basal level of calcium (Fig. 4A,B). Although other mechanisms can explain the increase in intracellular calcium, previous data support the formation of pore-like structures that allow the flow of calcium ions [8, 25]. Thus, we evaluated pore formation using a microscopic current approach in HEK cells, which are a good cell model because they are devoid of spontaneous single channels [7 , 25]. After acquiring the cell-attached configuration, Aβ_1 - 42 (1 μM) was added into the internal solution in the patch pipette. After a few minutes, unlike the control condition, Aβ perforated the plasma membrane and induced microscopic currents (Fig. 5A), gradually increasing the open probability in a time dependent manner (Fig. 5B). Moreover, the amplitude distribution of the events after 20 min in control cells and with Aβ_1 - 42 (1 μM) in the pipette, showed that Aβ_1 - 42 produced high conductance currents up to -58 pA (Fig. 5C,D). This membrane perforation induced by Aβ appears responsible for the increase in intracellular calcium levels [7 , 25]. To evaluate each of the Aβ fragments on this phenomenon of membrane perforation, we performed microscopic current recordings with the fragments in the patch pipette (1 μM). The results showed that only Aβ_25 - 35 increased the currents after 20 min in the cell attached configuration, indicating that this fragment was the only one able to perforate the plasma membrane similar to Aβ_1 - 42 (Fig. 6A), while fragments Aβ_17 - 42 and Aβ_1 - 28 did not change the microscopic currents even at 20 min of application (Fig. 6A). Graphs displaying the amplitude distribution of the events after 20 min in control cells and with Aβ_1 - 28, Aβ_17 - 42, and Aβ_25 - 35 (1 μM) in the pipette, showed that only Aβ_25 - 35 was able to produce high conductance currents up to -7 pA (Fig. 6B–E). On the other hand, the membrane currents in presence of Aβ_1 - 28 and Aβ_17 - 42 were ∼–2 pA, which is very close to the baseline of the recordings(Fig. 6B–E).

Finally, we evaluated if these fragments were able to induce synaptotoxicity similar to Aβ_1 - 42. Previous studies showed that chronic treatment with Aβ_1 - 42 (1 μM, 24–72 h) decreased synaptic SV2 protein levels, which reflects a delayed synaptic failure [6, 8]. We performed immunofluorescence experiments on hippocampal neurons treated for 72 h with Aβ_1 - 42, Aβ_1 - 28, Aβ_25 - 35, and Aβ_17 - 42 (1 μM), and the data revealed that the three fragments decreased the fluorescence intensity and the number of SV2 puncta in primary processes (20 μm) of hippocampal neurons (Fig. 7A). Nevertheless, Aβ_17 - 42 did not decrease SV2 levels as strongly as Aβ_1 - 28 and Aβ_25 - 35 (Fig. 7B,C). This data is in agreement with our results obtained from intracellular calcium and membrane perforation experiments where Aβ_17 - 42 did not produce any effect, supporting the idea that Aβ_17 - 42 isless toxic.

DISCUSSION

Although there is still some debate about how full length Aβ may increase or decrease selective components of synaptic transmission, previous studies have shown that Aβ affects long-term potentiation, calcium homeostasis, and NMDA/AMPA neurotransmission [6 , 31]. In line with the complexity of these effects, a single mechanism cannot explain all these actions, making it very difficult to develop effective anti-Aβ therapies. Our working hypothesis to describe the pleiotropic effects of Aβ is that it perforates the plasma membrane causing calcium ion influx into the cell that leads to a sustained release of synaptic vesicles [4 , 25]. Finally, this produces a vesicular depletion explaining the delayed synaptic failure [6], in addition to other multiple cellular toxic effects. The significance of the present study is that it provides an understanding for the roles of the different regions (N-terminal, C-terminal, central region) of Aβ_1 - 42 involved in the amyloid cascade on the cell membrane. Thus, the N terminal region is important for aggregation and association to membranes, and the central region for ion conduction or perforation(Fig. 8A).

Notably, the use of oligomeric Aβ_1 - 42 species as starting points of aggregation produced a similar degree of aggregation for Aβ_1 - 42, Aβ_1 - 28 and Aβ_25 - 35 (Fig. 1B,C), suggesting that oligomers can act as seeds for further aggregation similar to other amyloidogenic proteins such as prions [32]. The only fragment that was unable to aggregate, either as a monomer or oligomer, was Aβ_17 - 42 resulting in the least toxic fragment. The latter suggests that the state of aggregation is critical for the many toxic processes induced by Aβ_1 - 42 and its fragments.

On the other hand, Aβ_25 - 35 presented a similar behavior to Aβ_1 - 42 with regards to aggregation, membrane perforation, intracellular calcium and synaptotoxicity in hippocampal neurons. This result indicates that the residues in the 25–35 region of the Aβ sequence are important for the toxic properties of the peptide. Although the length of Aβ_25 - 35 as a monomer may not be enough to penetrate themembrane by itself, a complex with a higher structural level could easily damage the membrane (Fig. 8). Also, it is well known that other small peptides, like gramicidin a small decapeptide, can effectively perforate the plasma membrane [25 , 34]. Interestingly, the Aβ_25 - 35 fragment was found to be increased in the brain of AD patients [14]. Thus, it is likely that Aβ is racemized and proteolyzed by brain proteases [21] generating the fragment Aβ_25 - 35, which could contribute to the toxic effects of Aβ in AD.

Our finding that Aβ_1 - 28 increased the level of intracellular calcium in the absence of membrane leakage could be explained by various mechanisms. Although some previous studies showed a lack of effects on calcium influx induced by Aβ_1 - 28, these studies were performed in GT1-7 cells, an immortalized hypothalamic neurosecretory clone [35], and in human platelets [36], while the present study was done in primary hippocampal cultures. Our data suggest that the increase in intracellular calcium caused by Aβ_1 - 28 could be associated with the capacity of Aβ to interact with several membrane proteins, like cellular prion, mGluR5, NMDA and nicotinic receptors, increasing the intracellular calcium as previously described [9 , 37–39]. In fact, previous data suggest that Aβ_1 - 28 remains on the membrane surface without penetration [22]. Moreover, other plausible explanations for this increase in calcium is that, as some studies show, Aβ_1 - 28 potentiates the release of glutamate induced by high K⁺ concentration, thus activating ligand-gated ion channels such as NMDA receptors, allowing the flow of calcium ions into the cell [15]. For Aβ_25 - 35, on the other hand, the increase in intracellular calcium is likely caused by membrane perforation (Fig. 6). Thus, the Aβ_1 - 28 fragment could be acting on neuronal membrane constituents, proteins or lipids, causing intracellular calcium increase (Fig. 8B). For example, recent studies suggest that Aβ_1 - 28 would interact with hydrophilic zones in lipid membranes, while fragments like Aβ_25 - 40 would be localized in the hydrophobic core of the membrane bilayer [22]. While the fragments were able to decrease SV2 levels following long-term treatment (72 h), it is interesting to note that Aβ_1 - 42, Aβ_1 - 28, and Aβ_25 - 35 caused similar levels of synaptic toxicity. Aβ_17 - 42, on the other hand, was less effective than Aβ_1 - 42, suggesting a much lower toxicity for this fragment.

The present study is a comprehensive analysis of different Aβ_1 - 42 regions using fragments that could be participating in the toxicity induced by Aβ in AD (Fig. 8A). Thus, the present results provide important information for understanding if Aβ fragments can exert their effects using different mechanisms and alter important processes like the regulation of intracellular calcium, mitochondrial function, membrane stability, and the release of synaptic vesicles.

In conclusion, the data indicate that the aggregation process, initiated at the N terminal of Aβ, is critical for synaptic toxicity in the amyloid cascade, and also shows that species that can facilitate further oligomerization will proceed into fibers (see 1 in Fig. 8B). In addition, the scheme in Fig. 8 shows that Aβ_1 - 42, Aβ_1 - 28, and Aβ_25 - 35 form oligomers and then fibers that are enriched in β sheets. The Aβ_17 - 42 structure forms oligomers and fibers that lack β sheets and are structurally different from Aβ_1 - 42. Therefore, the resulting structure of Aβ_17 - 42 does not allow for membrane perforation, which appears critical for some toxic actions of Aβ [8, 25]. Thus, only Aβ_1 - 42 and Aβ_25 - 35 perforate the plasma membrane, while Aβ_1 - 28 associates to the surface of the membrane without perforation. Finally, the perforation of the membrane increases the level of intracellular calcium in a sustained fashion [5, 7]. A prolonged change in calcium will lead to synaptic failure, which is represented as a decrease in synaptic proteins like SV2(Fig. 7) [6].

Although the contribution or relevance of the fragments Aβ_1 - 28, Aβ_25 - 35, and Aβ_17 - 42 in AD is still not clear because of their low presence in situ, a better comprehension for the role of each Aβ region is critical to understand the mechanism behind the main toxic agent of this pathology. Nevertheless, the present study assesses the contribution of each Aβ region in several steps such as aggregation, membrane association and perforation, alteration on intracellular pathways and synaptic failure in order to establish a more defined picture of their role in the amyloid cascade(Fig. 8).

Footnotes

ACKNOWLEDGMENTS

We thank Lauren Aguayo for revising the manuscript and for technical assistance. We also thank M. P. Espinoza for technical assistance and Cecilia González for graphic design. This work was supported by FONDECYT 1140473 (LGA). CP was a CONICYT fellow.

Authors’ disclosures available online ().

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