Non-fibrillar oligomeric species of the amyloid ABri peptide, implicated in familial British dementia, are more potent at inducing apoptotic cell death than protofibrils or mature fibrils

doi:10.1006/jmbi.2001.4743

Journal of Molecular Biology

Volume 310, Issue 1, 29 June 2001, Pages 157-168

https://doi.org/10.1006/jmbi.2001.4743 Get rights and content

Abstract

Familial British dementia (FBD) is an autosomal dominant neurodegenerative disorder, with biochemical and pathological similarities to Alzheimer’s disease. FBD is associated with a point mutation in the stop codon of the BRI gene. The mutation extends the length of the wild-type protein by 11 amino acids, and following proteolytic cleavage, results in the production of a cyclic peptide (ABri) 11 amino acids longer than the wild-type (WT) peptide produced from the normal gene BRI. ABri was found to be the main component of amyloid deposits in FBD brains. However, pathological examination of FBD brains has shown the presence of ABri as non-fibrillar deposits as well as amyloid fibrils. Taken together, the genetic, pathological and biochemical data support the hypothesis that ABri deposits play a central role in the pathogenesis of FBD. Here we report that ABri, but not WT peptide, can oligomerise and form amyloid-like fibrils. We show for the first time that ABri induces apoptotic cell death, whereas WT is not toxic to cells. Moreover, we report the novel findings that non-fibrillar oligomeric species of ABri are more toxic than protofibrils and mature fibrils. These findings provide evidence that non-fibrillar oligomeric species are likely to play a critical role in the pathogenesis of FBD and suggest that a similar process may also operate in other neurodegenerative diseases.

Introduction

Familial British dementia (FBD), is an autosomal dominant neurodegenerative disorder clinically characterized by progressive spastic tetraparesis, cerebellar ataxia and dementia with an age of onset in the fourth decade,1 which shares common neuropathological features with Alzheimer’s disease (AD), including severe amyloid angiopathy, parenchymal neuritic and non-neuritic plaques as well as neurofibrillary tangles.2, 3 The genetic lesion underlying FBD is a T-A transversion at the termination codon of the BRI gene. The mutant BRI is the precursor protein of a 34 residue peptide named ABri. It has been found that ABri is the main component of amyloid deposits in FBD brain.4 More recently, it has been reported that both the mutant and the wild-type, BRI, are constitutively processed by furin, resulting in the secretion of ∼4 kDa and ∼3 kDa peptides corresponding to cleaved ABri and WT peptide, respectively.5 Notably, elevated levels of peptides were generated from the mutant BRI precursor.5 The deposition of ABri aggregates in the affected regions of FBD brains supports the idea that ABri deposits are of primary importance in the initiation of neurodegeneration in FBD, and is analogous to the accumulation of protein aggregates in other neurodegenerative diseases such as Aβ protein in AD, Huntingtin protein in Huntington’s disease, prion protein in the transmissible spongiform encephalopathies and α-synuclein in Parkinson’s disease and dementia with Lewy bodies (for recent reviews see 6, 7, 8). Most of these amyloid protein aggregates have been shown to be neurotoxic to cells in culture, and may be responsible for the neurodegeneration and neuronal loss seen in these diseases.9, 10, 11

However, the molecular organization of the neurotoxic form of the aggregate has not been defined. Recent attention has focussed on the suggestion that small, non-fibrillar oligomeric species may be toxic, but protofibrils, higher molecular weight aggregates, and mature fibrils have also been implicated.9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 Recent studies have shown that mutant “amyloidogenic” proteins associated with inherited forms of amyloidosis have a greater propensity to form non-fibrillar oligomers. These proteins include the Aβ in early-onset familial Alzheimer’s disease or cerebrovascular amyloidoses,15, 17, 24 and α-synuclein in early-onset familial Parkinson’s disease.25 Furthermore, recent studies using animal models of related neurodegenerative disorders support the idea that non-fibrillar aggregates of the amyloid associated with these diseases are pathogenic.26, 27, 28, 29, 30, 31

In this study we report that oxidized ABri rapidly produces a series of soluble oligomers, and, after incubation, protofibrils and subsequently amyloid-like fibrils. Under the same conditions, no large oligomers, protofibrils or mature fibrils were formed in WT solutions. We have also attempted to find out if ABri aggregates are toxic to neuronal cells. We found that ABri, but not WT, induces apoptotic cell death and that non-fibrillar oligomeric species (referred to as SSNFOS, for SDS-staple non-fibrillar oligomeric species) are more toxic than protofibrils or mature fibrils. This finding provides evidence that SSNFOS are likely to play a critical role in FBD pathogenesis, and has important implications for other neurodegenerative diseases.

Section snippets

Morphological and biochemical characterization of ABri aggregates

We found that at concentrations between 1–5 mg/ml ABri was soluble in Tris-HCl at pH 9 (i.e. gave an optically clear solution as judged by eye), but was less soluble at pH 7.4. Thus, we prepared all stock peptides in 100 mM Tris-HCl (pH 9) at 5 mg/ml (1.26 mM) unless otherwise stated. In order to examine possible self-aggregation and fibril formation of WT and ABri, we aged solutions at 1.26 mM for three weeks and analysed by Congo red staining, electron microscopy (EM), size exclusion

Discussion

Protein conformation-dependent neurotoxicity is an emerging theme in neurodegenerative disorders (reviewed in 6, 7, 8). The enhanced in vitro toxicity of amyloid peptides observed after in vitro aging is correlated with a prominent increase in the β-sheet structure of the aged peptide solution, protofibrils and/or amyloid fibril formation.9, 10, 11, 12, 13, 14, 16, 18, 20, 22 It is not clear if non-fibrillar aggregates or fibrils constitute the toxic species. However, recent studies on amyloid

Peptide synthesis

Reduced forms of ABri and WT peptides were prepared using an Fmoc/tBu methodology, that had been optimized for amyloid sequences, and the oxidized forms were prepared by air oxidation as previously reported.32 In this study we used the oxidized forms of ABri and WT peptides unless otherwise stated. In studies where we used the reduced forms of ABri (red-ABri) or WT (red-WT) peptides, only freshly prepared solutions were used to ensure that only reduced forms were assessed. Aged solutions were

Acknowledgements

This work was supported by funding from the UK Medical Research Council and CeNeS plc. We thank Dr Christina Sidera for assistance with SDS-PAGE. We acknowledge Dr Dominic Walsh for insightful advice and critical evaluation of the manuscript.

References (45)

M.F. Perutz
Glutamine repeats and neurodegenerative diseasesmolecular aspects
Trends Biochem. Sci.
(1999)
O.M.A. El-Agnaf et al.
Reviewformation and properties of amyloid-like fibrils derived from alpha-synuclein and related proteins
J. Struct. Biol.
(2000)
O.M.A. El-Agnaf et al.
Aggregates from mutant and wild-type alpha-synuclein proteins and NAC peptide induce apoptotic cell death in human neuroblastoma cells by formation of beta-sheet and amyloid-like filaments
FEBS Letters
(1998)
A.E. Roher et al.
Morphology and toxicity of Abeta-(1–42) dimer derived from neuritic and vascular amyloid deposits of Alzheimer’s disease
J. Biol. Chem.
(1996)
B. Seilheimer et al.
The toxicity of the Alzheimer’s beta-amyloid peptide correlates with a distinct fiber morphology
J. Struct. Biol.
(1997)
D.M. Walsh et al.
Amyloid beta-protein fibrillogenesis. Detection of a protofibrillar intermediate
J. Biol. Chem.
(1997)
D.M. Walsh et al.
Amyloid beta-protein fibrillogenesis. Structure and biological activity of protofibrillar intermediates
J. Biol. Chem.
(1999)
O.M.A. El-Agnaf et al.
Oligomerization and toxicity of beta-amyloid-42 implicated in Alzheimer’s disease
Biochem. Biophys. Res. Commun.
(2000)
R. Chiesa et al.
Neurological illness in transgenic mice expressing a prion protein with an insertional mutation
Neuron
(1998)
F. Saudou et al.
Huntingtin acts in the nucleus to induce apoptosis but death does not correlate with the formation of intranuclear inclusions
Cell
(1998)

D. Moechars et al.

Early phenotypic changes in transgenic mice that overexpress different mutants of amyloid precursor protein in brain

J. Biol. Chem.

(1999)

C. Worster-Drought et al.

Familial cerebral amyloid angiopathy with nonneuritic plaque formation

J. Neurol. Psychopathol.

(1933)

H. Aikawa et al.

Atypical Alzheimer’s disease with spastic paresis and ataxia

Ann. Neurol.

(1985)

G.T. Plant et al.

Familial cerebral amyloid angiopathy with nonneuritic amyloid plaque formation

Brain

(1990)

R. Vidal et al.

A stop-codon mutation in the BRI gene associated with familial British dementia

Nature

(1999)

S.H. Kim et al.

Furin mediates enhanced production of fibrillogenic ABri peptides in familial British dementia

Nature Neurosci.

(1999)

P.T. Lansbury

Evolution of amyloidwhat normal protein folding may tell us about fibrillogenesis and disease

Proc. Natl Acad. Sci. USA

(1999)

G. Forloni et al.

Neurotoxicity of a prion protein fragment

Nature

(1993)

C.J. Pike et al.

Cotman, C. W. Neurodegeneration induced by beta-amyloid peptides in vitrothe role of peptide assembly state

J. Neurosci.

(1993)

A. Lorenzo et al.

Beta-amyloid neurotoxicity requires fibril formation and is inhibited by congo red

Proc. Natl Acad. Sci. USA

(1994)

M.P. Lambert et al.

Diffusible, nonfibrillar ligands derived from Abeta1-42 are potent central nervous system neurotoxins

Proc. Natl Acad. Sci. USA

(1998)

D.M. Hartley et al.

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

J. Neurosci.

(1999)

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¹: Edited by J. Karn

²: O. M. A. El-Agnaf, Department of Biological Sciences, University of Lancaster, Lancaster LA1 4YQ, UK.

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Journal of Molecular Biology

Regular articleNon-fibrillar oligomeric species of the amyloid ABri peptide, implicated in familial British dementia, are more potent at inducing apoptotic cell death than protofibrils or mature fibrils1

Abstract

Introduction

Section snippets

Morphological and biochemical characterization of ABri aggregates

Discussion

Peptide synthesis

Acknowledgements

Trends Biochem. Sci.

J. Struct. Biol.

FEBS Letters

J. Biol. Chem.

J. Struct. Biol.

J. Biol. Chem.

J. Biol. Chem.

Biochem. Biophys. Res. Commun.

Neuron

Cell

J. Biol. Chem.

Familial cerebral amyloid angiopathy with nonneuritic plaque formation

J. Neurol. Psychopathol.

Atypical Alzheimer’s disease with spastic paresis and ataxia

Ann. Neurol.

Familial cerebral amyloid angiopathy with nonneuritic amyloid plaque formation

Brain

A stop-codon mutation in the BRI gene associated with familial British dementia

Nature

Furin mediates enhanced production of fibrillogenic ABri peptides in familial British dementia

Nature Neurosci.

Evolution of amyloidwhat normal protein folding may tell us about fibrillogenesis and disease

Proc. Natl Acad. Sci. USA

Neurotoxicity of a prion protein fragment

Nature

Cotman, C. W. Neurodegeneration induced by beta-amyloid peptides in vitrothe role of peptide assembly state

J. Neurosci.

Beta-amyloid neurotoxicity requires fibril formation and is inhibited by congo red

Proc. Natl Acad. Sci. USA

Diffusible, nonfibrillar ligands derived from Abeta1-42 are potent central nervous system neurotoxins

Proc. Natl Acad. Sci. USA

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

J. Neurosci.

Regular article
Non-fibrillar oligomeric species of the amyloid ABri peptide, implicated in familial British dementia, are more potent at inducing apoptotic cell death than protofibrils or mature fibrils¹