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Protofibrillar Intermediates of Amyloid β-Protein Induce Acute Electrophysiological Changes and Progressive Neurotoxicity in Cortical Neurons

Hartley, Dean M., Walsh, Dominic M., Ye, Chianping P., Diehl, Thekla, Vasquez, Sara, Vassilev, Peter M., Teplow, David B., Selkoe, Dennis. J.·1999·The Journal of Neuroscience
DOI 10.1523/jneurosci.19-20-08876.1999
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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 β-protein (Aβ). The presence of neuritic plaques containing abundant Aβ-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 Aβ 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 Aβ (LMW Aβ) 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 Aβ and PF cannot be explained by conversion to fibrils. Importantly, protofibrils, but not LMW Aβ, 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 Aβ assembly intermediates formed during the process of fibrillogenesis.

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Crossref · 10.1523/jneurosci.19-20-08876.1999

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Figure 2 Western blotting reveals electrophoretic differences between LMW A b , PF, and fibril preparations. Freshly collected fractions of LMW A b ( a ) and PF ( b ) from the size-exclusion column and preformed fibrils ( c ) were subjected to SDS-PAGE. Proteins were immunoblotted with an anti-A b 1-40 polyclonal antibody, R1282, and immunopositive bands were visualized by chemiluminescence. The numbers under each lane represent the concentrations of A b in individual SEC fractions for LMW A b and PF (and similar amounts of fibrils), as determined by amino acid analysis. Molecular weight of protein standards ( 3 1000) are at right . This gel depicts one of three independent experiments that gave similar results.
Figure 2 Western blotting reveals electrophoretic differences between LMW A b , PF, and fibril preparations. Freshly collected fractions of LMW A b ( a ) and PF ( b ) from the size-exclusion column and preformed fibrils ( c ) were subjected to SDS-PAGE. Proteins were immunoblotted with an anti-A b 1-40 polyclonal antibody, R1282, and immunopositive bands were visualized by chemiluminescence. The numbers under each lane represent the concentrations of A b in individual SEC fractions for LMW A b and PF (and similar amounts of fibrils), as determined by amino acid analysis. Molecular weight of protein standards ( 3 1000) are at right . This gel depicts one of three independent experiments that gave similar results.CC-BY-NC-SA · From the PDF
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Image 3
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Image 4
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Figure 3 Immunocytochemistry of neurons and of A b in primary mixed cortical cultures exposed to various A b preparations. Mixed cortical cultures were exposed to medium only ( a ), LMW A b ( b ), PF ( c ), or fibrils ( d ) for 5 d, fixed and double-labeled for A b deposition (R1282) ( red staining ) and neurons (MAP2) ( blue staining ). Significant neuron loss is observed with each of the A b preparations (LMW A b , 18 m M; PF, 21 m M; fibrils, 28 m M). Scale bar, 50 m m.
Figure 3 Immunocytochemistry of neurons and of A b in primary mixed cortical cultures exposed to various A b preparations. Mixed cortical cultures were exposed to medium only ( a ), LMW A b ( b ), PF ( c ), or fibrils ( d ) for 5 d, fixed and double-labeled for A b deposition (R1282) ( red staining ) and neurons (MAP2) ( blue staining ). Significant neuron loss is observed with each of the A b preparations (LMW A b , 18 m M; PF, 21 m M; fibrils, 28 m M). Scale bar, 50 m m.CC-BY-NC-SA · From the PDF
Figure 4 Comparison of neurotoxicity induced by LMW A b , PF, and fibrils. Various concentrations of LMW A b , PF, and fibrils [ m M 5 concentration of A b applied ( top numbers , xaxis)] 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 A b 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 m M A b ), 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).
Figure 4 Comparison of neurotoxicity induced by LMW A b , PF, and fibrils. Various concentrations of LMW A b , PF, and fibrils [ m M 5 concentration of A b applied ( top numbers , xaxis)] 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 A b 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 m M A b ), 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).CC-BY-NC-SA · From the PDF
Image 7
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Figure 7 Increased frequency of action potentials and membrane depolarizations caused by PF but not LMW A b . 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 A b ( 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.
Figure 7 Increased frequency of action potentials and membrane depolarizations caused by PF but not LMW A b . 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 A b ( 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.CC-BY-NC-SA · From the PDF

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Cites

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Metadata

Authors
Hartley, D. M., Walsh, D. M., Ye, C. P., Diehl, T., Vasquez, S., Vassilev, P. M., Teplow, D. B., & Selkoe, D. J.
Venue
The Journal of Neuroscience
Publisher
Society for Neuroscience
Published
1999-10-15
Volume
19
Issue
20
Pages
8876-8884
ISSN
  • 0270-6474
  • 1529-2401
DOI
10.1523/jneurosci.19-20-08876.1999
Cited by
817
References
9
Language
en
Licence
creativecommons.org/licenses/by-nc-sa/4.0/ (vor, 183 d)

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