Protofibrillar intermediates of amyloid beta-protein induce acute electrophysiological changes and progressive neurotoxicity in cortical neurons

Abstract

Alzheimer's disease (AD) is a progressive neurodegenerative disorder that is thought to be caused in part by the age-related accumulation of amyloid beta-protein (Abeta). The presence of neuritic plaques containing abundant Abeta-derived amyloid fibrils in AD brain tissue supports the concept that fibril accumulation per se underlies neuronal dysfunction in AD. Recent observations have begun to challenge this assumption by suggesting that earlier Abeta assemblies formed during the process of fibrillogenesis may also play a role in AD pathogenesis. Here, we present the novel finding that protofibrils (PF), metastable intermediates in amyloid fibril formation, can alter the electrical activity of neurons and cause neuronal loss. Both low molecular weight Abeta (LMW Abeta) and PF reproducibly induced toxicity in mixed brain cultures in a time- and concentration-dependent manner. No increase in fibril formation during the course of the experiments was observed by either Congo red binding or electron microscopy, suggesting that the neurotoxicity of LMW Abeta and PF cannot be explained by conversion to fibrils. Importantly, protofibrils, but not LMW Abeta, produced a rapid increase in EPSPs, action potentials, and membrane depolarizations. These data suggest that PF have inherent biological activity similar to that of mature fibrils. Our results raise the possibility that the preclinical and early clinical progression of AD is driven in part by the accumulation of specific Abeta assembly intermediates formed during the process of fibrillogenesis.

Fig. 1.

Flow diagram depicting generation of LMW Aβ and PF and their structural differences. a, Aβ was dissolved in NaOH:PBS, incubated for 2–3 d, centrifuged, and then injected onto a size-exclusion column. As the PF or LMW Aβ peak emerged from the column, it was collected in fractions, to which tissue culture reagents were added and then applied to mixed brain cultures. Neuronal viability was monitored by phase-contrast microscopy and by measuring the release of LDH into the medium. b,Protofibrils (PF) were present as individual, dispersed, curvilinear structures of 4–11 nm diameter and <200 nm length. Mature fibrils (F) were 6–10 nm in diameter, much longer (indeterminate length; mean diameter, ≅8 nm), and often occurred in dense mats of multiple fibrils.

Fig. 2.

Western blotting reveals electrophoretic differences between LMW Aβ, PF, and fibril preparations. Freshly collected fractions of LMW Aβ (a) and PF (b) from the size-exclusion column and preformed fibrils (c) were subjected to SDS-PAGE. Proteins were immunoblotted with an anti-Aβ_1–40 polyclonal antibody, R1282, and immunopositive bands were visualized by chemiluminescence. The numbers under each lane represent the concentrations of Aβ in individual SEC fractions for LMW Aβ and PF (and similar amounts of fibrils), as determined by amino acid analysis. Molecular weight of protein standards (×1000) are atright. This gel depicts one of three independent experiments that gave similar results.

Fig. 3.

Immunocytochemistry of neurons and of Aβ in primary mixed cortical cultures exposed to various Aβ preparations. Mixed cortical cultures were exposed to medium only (a), LMW Aβ (b), PF (c), or fibrils (d) for 5 d, fixed and double-labeled for Aβ deposition (R1282) (red staining) and neurons (MAP2) (blue staining). Significant neuron loss is observed with each of the Aβ preparations (LMW Aβ, 18 μm; PF, 21 μm; fibrils, 28 μm). Scale bar, 50 μm.

Fig. 4.

Comparison of neurotoxicity induced by LMW Aβ, PF, and fibrils. Various concentrations of LMW Aβ, PF, and fibrils [μm = concentration of Aβ applied (top numbers, x-axis)] were applied to mixed cortical cultures established 3–4 weeks earlier. Fraction number refers to the order the fractions were collected from either the LMW Aβ or PF peak (bottom numbers,x-axis)(also see Fig. 1). After each day of treatment (z-axis), an aliquot of conditioned medium was analyzed for LDH release (y-axis). Values were normalized to blanks (0 μm Aβ), which were given a value of 1. The graph represents one experiment, with each bar being the mean of duplicate assays; this graph is representative of four independent experiments in which four separate brain dissections were used (see Table 1 for comparison of the relative neurotoxicity of the preparations using LDH values from day 3).

Fig. 5.

Lack of detectable fibril formation in mixed brain cultures treated for 5–6 d with LMW Aβ or PF. a,Mixed cortical cultures were exposed to LMW, PF, or preformed fibrils for 6 d, at which time the medium was removed and analyzed for fibril formation by Congo red binding. Each column is the mean of duplicate assays. Graph represents one of three identical experiments yielding similar results. b–d, Lack of fibril formation as determined by immuno-EM in 5 d conditioned media of LMW Aβ (b), PF (c), or fibril (d) preparation. PF-treated media (c) predominantly contained large electron-dense mats of distinct protofibrils. In contrast, distinct fibrils (d) could readily be detected in the media of cultures treated with preformed fibrils. Results represent one of two experiments, in both of which few or no fibrils were detected in the media of LMW Aβ and PF cultures.

Fig. 6.

Sustained increases in EPSCs caused by PF and fibrils but not LMW Aβ. Cell-attached patch-clamp mode was used to record inward EPSCs in voltage-clamp mode. a,Examples of current traces are shown in left panels. Baseline activities were recorded for 4 min (the ends of which periods are shown as dotted lines). Addition of LMW Aβ (a, LMW, solid line) at a concentration of 3 μm caused only a brief, transient increase in EPSCs compared with the preceding control period. In contrast, addition of PF (3–5 μm) (a, PF) or fibrils (3–5 μm) (a, Fibrils, solid lines) caused a rapid, sustained increase in EPSCs. b, The number of EPSCs per minute plotted versus time for the experiment shown in a. Each plot inb graphs data from a representative single cell exposed to one of the three Aβ preparations. Data from multiple experiments (c) were then compiled by normalizing the EPSCs per minute to the basal activity, which was given a value of one. The normalized mean EPSC values (5–8 experiments; mean ± SEM) for the basal, first, and eighth minute revealed a significant increase in EPSCs with PF and fibril application compared with the basal or LMW Aβ activity (c). PF and fibrils were found to be statistically different from both medium alone and LMW Aβ (p < 0.05); no significant difference between the PF and fibril preparations was observed.

Fig. 7.

Increased frequency of action potentials and membrane depolarizations caused by PF but not LMW Aβ. Whole-cell recordings in current-clamp mode were used to measure APs (single sharp deflections) and MDs (arrows). Baseline activities were recorded for 3–5 min (dotted lines). Addition of LMW Aβ (solid line) produced no significant increase in APs and MDs, whereas PF and fibrils (solid lines) increased the frequency of APs and the amplitude of MDs.