Báo cáo khoa học: Docosahexaenoic acid stabilizes soluble amyloid-b protofibrils and sustains amyloid-b-induced neurotoxicity in vitro

Docosahexaenoic Acid Stabilizes Soluble Amyloid-β Protofibrils and Sustains Amyloid-β-Induced Neurotoxicity In Vitro

Tác giả: Ann-Sofi Johansson, Anita Garlind, Fredrik Berglind-Dehlin, Göran Karlsson, Katarina Edwards, Pär Gellerfors, Frida Ekholm-Pettersson, Jan Palmblad, Lars Lannfelt

Lĩnh vực: Khoa học Y sinh, Hóa sinh

Nội dung tài liệu: Nghiên cứu này khám phá tác động của axit docosahexaenoic (DHA), một axit béo không bão hòa đa (PUFA), lên sự kết tụ của protein amyloid-beta (Aβ) và độc tính do Aβ gây ra trong môi trường in vitro. Các thí nghiệm được thực hiện với các dạng peptide Aβ42wt và Aβ42Arc. Kết quả cho thấy DHA, ở nồng độ nhất định, có khả năng ổn định các protofibril Aβ42wt hòa tan, ngăn chặn chúng chuyển hóa thành các sợi không hòa tan. Sự ổn định này dẫn đến việc duy trì độc tính do Aβ42wt gây ra đối với tế bào PC12 theo thời gian. Ngược lại, khi Aβ42wt không được ổn định bởi DHA và hình thành sợi, độc tính lại giảm. DHA không cho thấy tác dụng ổn định tương tự đối với protofibril Aβ42Arc, thậm chí còn có xu hướng đẩy nhanh quá trình hình thành sợi ở dạng đột biến này. Nghiên cứu cũng chỉ ra rằng tác dụng của DHA phụ thuộc vào nồng độ và sự hình thành micelle của nó. Những phát hiện này cung cấp hiểu biết mới về vai trò của các lipid nội sinh trong việc điều chỉnh sự kết tụ và độc tính của Aβ, có ý nghĩa quan trọng đối với việc nghiên cứu bệnh Alzheimer.

Mục lục chi tiết:

• Keywords
• Correspondence
• Abbreviations
• Enrichment of diet and culture media with the polyunsaturated fatty acid docosahexaenoic acid has been found to reduce the amyloid burden in mice and lower amyloid-β (Aβ) levels in both mice and cultured cells. However, the direct interaction of polyunsaturated fatty acids, such as docosahexaenoic acid, with Aβ, and their effect on Aβ aggregation has not been explored in detail. Therefore, we have investigated the effect of docosahexaenoic acid, arachidonic acid and the saturated fatty acid arachidic acid on monomer oligomerization into protofibrils and protofibril fibrillization into fibrils in vitro, using size exclusion chromatography. The polyunsaturated fatty acids docosahexaenoic acid and arachidonic acid at micellar concentrations stabilized soluble Aβ42 wild-type protofibrils, thereby hindering their conversion to insoluble fibrils. As a consequence, docosahexaenoic acid sustained amyloid-β-induced toxicity in PC12 cells over time, whereas Aβ without docosahexaenoic acid stabilization resulted in reduced toxicity, as Aβ formed fibrils. Arachidic acid had no effect on Aβ aggregation, and neither of the fatty acids had any protofibril-stabilizing effect on Aβ42 harboring the Arctic mutation (AβE22G). Consequently, AβArctic-induced toxicity could not be sustained using docosahexaenoic acid. These results provide new insights into the toxicity of different Aβ aggregates and how endogenous lipids can affect Aβ aggregation.
• Alzheimer’s disease is characterized neuropathologically by two types of protein deposits, extracellular amyloid plaques and intracellular neurofibrillary tangles. Plaques consist mainly of fibrillar amyloid-β (Aβ) protein, whereas tangles are composed of fibrillar tau protein. The aggregation of Aβ is believed to drive the disease process, with extensive neuronal loss as a consequence [1].
• Monomeric Aβ readily aggregates via a population of soluble intermediates, protofibrils, into amyloid fibrils in vitro [2]. Which one of these Aβ species drives the disease process has been debated, as both fibrils and protofibrils have previously been found to be toxic to neurons [3-5] and affect electrophysiologic parameters [3,4].
• Fatty acids are known to affect the aggregation of various polymerizing proteins, including amyloidogenic proteins [6-8]. Arachidonic acid (AA, 20:4, ω6) is, for example, commonly used to induce tau aggregation in vitro. Recently, a diet enriched in docosahexaenoic
• acid (DHA, 22:6, ω3) was found to dramatically reduce the amyloid burden in aged Tg2576 mice, and biochemical studies demonstrated reduced Aβ levels and amyloid precursor protein (APP) processing [9]. However, the direct interaction of polyunsaturated fatty acids (PUFAs) with Aβ, and their effect on Aβ aggregation, have not been explored in detail. Therefore, we have investigated the effect of unsaturated DHA and AA and the saturated fatty acid arachidic acid (20:0) on Aβ monomer oligomerization into protofibrils and protofibril fibrillization into fibrils in vitro, using size exclusion chromatography (SEC) [2]. Two Aβ peptides were used in this study, Aβ42wt, which is implicated in sporadic Alzheimer’s disease (AD), and Aβ42 with the Arctic mutation, Aβ42Arc (E22G). This mutation was found in a family with hereditary AD in northern Sweden, and has been associated with the accelerated formation of Aβ40 protofibrils in vitro [10]. Recently, we have shown that this is the case also for Aβ42Arc, which displays accelerated protofibril formation as well as accelerated protofibril fibrillization into fibrils [2].
• In addition, the effect of DHA on Aβ-induced toxicity was investigated in PC12 cells, a cell line widely used as a neuronal cell model.
• Here, we demonstrate that PUFAs have a profound effect on Aβ aggregation in vitro, stabilizing soluble intermediates, i.e. protofibrils, and thereby maintaining Aβ-induced toxicity in PC12 cells.
• Results
• Unsaturated fatty acids stabilize Aβ42wt protofibrils
• Fatty acids were incubated with Aβ42wt in a physiologic buffer environment at a molar ratio of 1 : 1. The mixture was assayed for monomer and protofibril content, respectively, as a function of incubation time by SEC. Aβ species eluting in the void volume of the Superdex 75 column, and not pelleted in the centrifugation step, were defined as Aβ protofibrils. The gel-included peak eluting at ~20 min was defined as Aβ monomers but could contain dimers as well [11]. No other protein peaks were detected. Fibril formation was measured indirectly as decline in protofibril peak area, i.e. amount of peptide pelleted in the centrifugation step. We have previously shown that the mass of peptide lost in the pellet corresponds to the reduced peak area, using amino acid analysis to quantitate pelleted material, and that this pelleted material is thioflavin T positive [2].
• The PUFAs had a significant effect on Aβ42wt in vitro assembly (Fig. 1). Both DHA (Fig. 1A) and
• AA (Fig. 1B) increased the monomer oligomerization rate. Interestingly, these fatty acids also had an apparent effect on protofibril stability. Soluble protofibrils remained stable for at least 25 h, whereas in the presence
• of fatty acid vehicle only (dimethylsulfoxide), essentially all protofibrils had aggregated into large fibrillar aggregates by that time. In contrast, the saturated fatty acid arachidic acid (Fig. 1C) did not have any effect on either Aβ42wt monomer oligomerization or protofibril stability.
• Neither unsaturated nor saturated fatty acids stabilize Aβ42Arc protofibrils
• When Aβ42Arc peptide was investigated in the same way as Aβ42wt, no stabilizing effect on Aβ42Arc protofibrils was observed. On the contrary, DHA accelerated protofibril assembly into insoluble fibrils, pelleted in a centrifugation step (Fig. 2A). AA also seemed to accelerate Aβ42Arc protofibril fibrillization slightly, even though this effect did not reach statistical significance (Fig. 2B). In agreement with its effect on Aβ42wt, the saturated fatty acid arachidic acid did not result in any change in aggregation kinetics for Aβ42Arc (Fig. 2C). In these experiments, Aβ42Arc monomers were not detected, owing to the high aggregation rate of this peptide, and the presence of Tween-20 in the elution buffer, further increasing the monomer oligomerization rate (unpublished results). Tween-20 was, however, not present during incubation, but only during analysis. In this way, interference by Tween-20 was kept at a minimum.
• SDS/PAGE analysis: soluble Aβ42wt is stabilized by DHA
• In an attempt to determine whether an oligomer of a specific size is stabilized by DHA, SDS/PAGE analysis was performed. Samples were centrifuged to pellet insoluble Aβ species, and supernatants were analyzed with SDS/PAGE. Incubation with 50 µm DHA overnight stabilized soluble Aβ42wt, as demonstrated by the increased amount of soluble species, ranging from monomers to tetramers (Fig. 3). For Aβ42Arc, there was a slight increase in the amount of monomer after incubation with DHA overnight, but this was not as apparent as for Aβ42wt. At 0 h of incubation, DHA seemed to slightly accelerate the aggregation of Aβ42Arc into insoluble species, in line with the SEC data. No high molecular weight oligomers were observed.
• Aβ42wt protofibril stabilization is dependent on the fatty acid concentration and micelle formation
• The magnitude of acceleration of Aβ42wt monomer oligomerization (Fig. 4A) and increase in protofibril
• stability (Fig. 4B) induced by DHA was dependent on DHA concentration and micelle formation. DHA did not affect Aβ42Arc protofibril stability at any concen-
• tration (Fig. 4C). A concentration of 1 µm DHA, i.e. 50x molar excess of Aβ, did not have any effect on Aβ42wt aggregation, whereas 10 µm DHA was enough to stabilize Aβ42wt protofibrils, although not to the same extent as 50 µm DHA (molar ratio 1: 1). Fatty acid micelles, detected in the SEC assay as a gel-included peak eluting just after the void (Fig. 5), were detected at 10 and 50 µm DHA and AA, but not at 1 μμ. Arachidic acid did not stabilize protofibrils or form micelles at any of the concentrations used.
• Cryo-transmission electron microscopy (cryo-TEM)
• Aβ1-42wt was incubated with DHA and compared with a control with fatty acid vehicle only (dimethylsulfoxide). Samples were analyzed with cryo-TEM after 0 and 8 h of incubation at 37 °C. Aβ incubated in the presence of DHA for 8 h formed long, soluble, fibril-like structures with somewhat atypical granular features (Fig. 6D). Control samples formed insoluble aggregates that, in most cases, were too large to be visualized by cryo-TEM (the thickness of the vitrified film is limited to 0.5 µm). One of these large aggregates is shown, however, in Fig. 6B.
• DHA prolongs Aβ42wt-mediated cell toxicity
• To examine the effect of DHA on Aβ-induced neurotoxicity in a neuronal cell model, we set up a kinetic toxicity study, using PC12 cells. Aβ42wt and Aβ42Arc were incubated with or without 50 µm DHA for var-
• ious time points up to 25 h at 37 °C, and then added at the same time to the cell cultures. Toxicity was evaluated by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, and data were shown as percentage cell toxicity (% inhibition of MTT reduction) compared to NaCl/P; alone. The ‘toxicity’ measured by this assay refers to the impaired ability of mitochondria to reduce MTT. This assay reflects cell viability, and makes no distinction between apoptosis and necrosis. Aβ42wt and Aβ42Arc both
• resulted in an initial toxicity of ~30%. Aβ42wt incubated with DHA maintained this toxicity over time, in contrast to Aβ42wt incubated with vehicle, when the toxicity was reduced with prolonged Aβ incubation time (Fig. 7A). Toxicity induced by Aβ42Arc was also reduced with extended Aβ incubation time, but for this peptide, DHA had no effect (Fig. 7B). When the peptides were incubated overnight with different concentrations of DHA, a concentration of DHA shown not to stabilize protofibrils (i.e. 1 µm) did not maintain Aβ42wt-induced toxicity. Aβ42Arc-induced toxicity was not maintained with either a low or a high concentration of DHA (Fig. 7C).
• DHA in itself did not result in increased toxicity, as Aβ42Arc was equally toxic with or without DHA (Fig. 7B,C). Also, neither DHA incubated with BSA as a control protein nor DHA alone induced toxicity as compared to NaCl/P; (data not shown). It is worth
• noting the very high experimental reproducibility with DHA-stabilized Aβ42wt peptide, illustrated by the minimal data variation in three independent experiments (Fig. 7C, third bar from the left).
• Discussion
• PUFAs repesent a significant proportion of the lipids in the brain, with DHA and AA being the most abundant [12]. DHA and AA are essential fatty acids, i.e. they cannot be synthesized de novo, but have to be ingested through the diet. Fatty acids are normally bound to different lipid-binding proteins, but they also exist as free fatty acids.
• PUFAs have proven effects on the aggregation of proteins, both amyloidogenic, such as a-synuclein [7] and tau [8], and nonamyloidogenic polymerizing proteins, such as synexin [13]. Previously, Wilson et al. showed that the presence of PUFAs increased Aβ40wt and Aβ42wt fluorescence to a higher extent than the presence of saturated fatty acids in a thioflavin T-binding assay [8]. However, it has not been determined in which stage of the aggregation process this occurs. We have shown that PUFAs accelerate the early aggregation process of Aβ42wt, and stabilize soluble aggregates. This only happens when the concentration of fatty acid is over 10 µm, probably due to micelle formation at these concentrations. It thus appears that fatty acid micelles interact with the Aβ peptide and facilitate nucleation, thereby accelerating the early aggregation phase. Both a-synuclein [14] and tau [15] aggregation have previously been demonstrated to be induced by anionic micellar detergents and fatty acids. The need for micellar structures explains why arachidic acid did not stabilize protofibrils in our study. The critical melting temperature for this fatty acid is 75 °C [16], and no micelle formation can occur below this temperature. Cryo-TEM revealed granular structures, which may represent DHA micelles. These granular structures are located in close proximity to the soluble Aβ aggregates, indicating a direct interaction. Possibly, DHA and AA micelles work as a protective agent and prevent intermolecular interactions and consequent aggregation into insoluble fibrils.
• The concentrations of free DHA and AA in human cerebrospinal fluid (CSF) have been determined to be 185 nm and 86 nm, respectively [17]. In comparison, the levels of Aβ1-42 in CSF are in the range 100-200 рм [18]. This means that DHA and AA are present in 1000-fold excess compared to Aβ1-42, and could thus affect the aggregation situation for this Aβ peptide in vivo. However, it is not clear if the low DHA and AA concentrations found in CSF are suffi-
• cient to form micelles. Critical micelle concentrations for the pure fatty acids are substantially higher; a critical micelle concentration (CMC) in the range of 200 µm has been determined for AA [15]. It is in this context important to note, however, that the CMC for surfactants is well known to decrease in the presence of polymers [19]. In line with this, other amyloidogenic proteins [14,15] have been shown to decrease the CMC for fatty acids dramatically. Moreover, DHA and AA can form mixed micelles with other fatty acids, lowering the CMC even further ([20] and references therein).
• In addition, brain trauma and ischemia have been associated with up to six-fold elevated concentrations of free DHA and AA in human CSF [21,22]. Interest-ingly, these conditions are also associated with an increased risk of AD ([23,24] and references therein).
• DHA did not stabilize soluble Aβ in experiments with Aβ42 containing the Arctic mutation, where the glutamic acid in position 22 is substituted by glycine. DHA actually slightly accelerated Aβ42Arc protofibril assembly into insoluble fibrils, as demonstrated by SEC. One explanation for this could be that the rapid
• aggregation of Aβ42Arc prevents PUFAs from intervening quickly or potently enough. Alternatively, the negative charge in glutamic acid could be essential for the fatty acid interaction. This seems unlikely, however, as aggregation of both the negatively charged a-synuclein [14] and the positively charged tau [15] is induced by anionic, not cationic, micelles and vesicles. In fact, an artificial negative surface, such as carboxylate-modified polystyrene microspheres [25] or negatively charged mica [26], is enough to induce tau and IgG light chain aggregation, respectively. The net charge of the protein is thus probably insignificant. More likely, the anionic surface interacts with clustered positive charges; in the case of Aβ, the arginine at position 5, and the lysines at positions 16 and 28. Interestingly, the formation of fibrils by association of protofilaments has been