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.

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.     

Insulin-degrading enzyme degrades amyloid peptides associated with British and Danish familial dementia

Familial British dementia (FBD) and familial Danish dementia (FDD) are autosomal dominant disorders characterized by cerebrovascular and parenchymal amyloid deposition and neurofibrillary degeneration. In both conditions, the genetic defects cause the loss of the normal stop codon in the precursor BRI, generating novel 34-residue peptides named ABri and ADan in FBD and FDD, respectively. ABri and ADan show a strong tendency to aggregate into non-fibrillar and fibrillar structures at neutral pH and this property seems to be directly related to neurotoxicity. Here we report that a recombinant insulin-degrading enzyme (rIDE) was capable of degrading monomeric ABri and ADan in vitro more efficiently than oligomeric species. These peptides showed high beta-structure content and were more resistant to proteolysis as compared to the BRI wild-type product of 23 amino acids. Specific sites of cleavage within the C-terminal pathogenic extensions raise the possibility that proteolysis of monomeric soluble precursors by IDE may delay ABri and ADan aggregation in vivo.     

Kinetic partitioning between aggregation and vesicle permeabilization by modified ADan

The neurodegenerative illness Familial Danish Dementia (FDD) is linked to formation and aggregation of the 34-residue ADan peptide, whose cytotoxicity may be mediated by membrane interactions. Here we characterize the derived peptide SerADan, in which the two cysteines found in ADan have been changed to serines to emulate the reduced peptide. SerADan aggregates rapidly at pH 5.0 and 7.5 in a series of conformational transitions to form beta-sheet rich fibril-like structures, which nevertheless do not bind amyloid-specific dyes, probably due to the absence of organized beta-sheet contacts. Aggregation is prevented at neutral/acidic pH and low ionic strength by anionic lipid vesicles. These vesicles are permeabilized by monomeric SerADan assembling on the membrane to form stable beta-sheet structures which are different from the solution aggregates. In contrast, solution ageing of SerADan first reduces and then abolishes permeabilization properties. The competition between lipid binding and aggregation may reflect bifurcating pathways for the ADan peptide in vivo between accumulation of inert aggregates and formation of cytotoxic permeabilizing species. Our work demonstrates that non-fibrillar aggregates can assemble in a series of steps to form a hierarchy of higher-order assemblies, where rapid formation of stable local beta-sheet structure may prevent rearrangement to amyloid proper.     

Proteolytic processing of familial British dementia-associated BRI variants: evidence for enhanced intracellular accumulation of amyloidogenic peptides.

Different mutations in the BRI(2) gene cause rare neurodegenerative conditions, termed familial British dementia (FBD) and familial Danish dementia (FDD). The mutant genes encode BRI-L and BRI-D, the precursors of fibrillogenic ABri and ADan peptides, respectively. We previously reported that furin processes both BRI-L and its wild type counterpart, BRI, resulting in the secretion of C-terminal peptides; elevated levels of peptides were generated from BRI-L. In the present study, we show that inducible expression of alpha1-antitrypsin Portland, a furin inhibitor, inhibits the endoproteolysis of BRI and BRI-L in a dose-dependent manner. Moreover, comparison of the activities of several proprotein convertases reveals that furin is most efficient in endoproteolysis of BRI and BRI-L; PACE4, PC6A, PC6B, and LPC show much lower activities. Interestingly, LPC also exhibits enhanced cleavage of BRI-L compared with BRI. Finally, we demonstrate that BRI-D is also processed by furin and, like BRI-L, the cleavage of BRI-D is more efficient than that of BRI. Interestingly, while the ABri peptide is detected both intracellularly and in the medium, the ADan peptide accumulates predominantly in intracellular compartments. We propose that intracellular accumulation of amyloidogenic ADan or ABri peptides results in the neuronal damage leading to FDD and FBD, respectively.     

Properties of neurotoxic peptides related to the Bri gene

Familial British dementia, a rare autosomal dominant neurodegenerative disorder, shares features with Alzheimer's disease, including amyloid plaque deposits, neurofibrillary tangles, neuronal loss,progressive dementia, but clinically presents with additional physical defects [1,2]. A mutation in the termination codon of the BRI gene produces a BRI precursor protein 11 amino acids longer than the wild-type protein [3,4]. Mutant and wild-type precursor proteins both may undergo furin cleavage between residues 243 and 244, producing a peptide of 34 amino acids in the case of ABri and 23 amino acids long in the case of the wild type peptide. The ABri 4kDa peptide is the main component of the amyloid deposits found in familial British dementia brains. A decamer duplication in the 3- region of the BRI gene originates the peptide Adan that is associated with dementia in Familial Danish dementia (FDD), similar to BDD clinically, but with additional hearing and eyesight loss [5]. The resulting reading frame is extended to 277 amino acid residues, and cleavage by furin releases a peptide of 34 residues, which is identical to Abri and WT in its N-terminal 22-residues, but contains a distinct C-terminal 10 residues composed of mainly hydrophobic residues. Here we demonstrate that C-terminal extensions of Abri and Adan are required to elongate initially-formed dimers to neurotoxic soluble oligomers and fibrils. In contrast, the shorter wild-type peptide does not aggregate under the same conditions and is not toxic. Conformational analyses indicate triple-beta-sheet structures. Soluble nonfibrillar oligomers of oxidised ABri and reduced Adan were observed in solution (pH7.4) of peptides prior to the appearance of mature fibrils.     

Properties of neurotoxic peptides related to the BRI gene

Mutations in the BRI gene are thought to cause dementias in members of families. The clinical symptoms are similar to those of Alzheimer's disease, but with additional ocular and hearing deficits, and spasticity. The mutations lead to the release of the 34-residue peptides, ABri and ADan, in the brains of afflicted individuals. We have synthesized the peptides in their straight-chain and oxidized cyclic forms and shown that the oxidized form of ABri and reduced form of ADan are toxic to human neuronal cell lines in culture. Neurotoxicity correlates with the extent of formation of SDS-stable non-fibrillar low-molecular-mass oligomers (SSNFOs).     

Familial Danish dementia: co-existence of Danish and Alzheimer amyloid subunits (ADan AND A{beta}) in the absence of compact plaques

Familial Danish dementia is an early onset autosomal dominant neurodegenerative disorder linked to a genetic defect in the BRI2 gene and clinically characterized by dementia and ataxia. Cerebral amyloid and preamyloid deposits of two unrelated molecules (Danish amyloid (ADan) and beta-amyloid (Abeta)), the absence of compact plaques, and neurofibrillary degeneration indistinguishable from that observed in Alzheimer disease (AD) are the main neuropathological features of the disease. Biochemical analysis of extracted amyloid and preamyloid species indicates that as the solubility of the deposits decreases, the heterogeneity and complexity of the extracted peptides exponentially increase. Nonfibrillar deposits were mainly composed of intact ADan-(1-34) and its N-terminally modified (pyroglutamate) counterpart together with Abeta-(1-42) and Abeta-(4-42) in approximately 1:1 mixture. The post-translational modification, glutamate to pyroglutamate, was not present in soluble circulating ADan. In the amyloid fractions, ADan was heavily oligomerized and highly heterogeneous at the N and C terminus, and, when intact, its N terminus was post-translationally modified (pyroglutamate), whereas Abeta was mainly Abeta-(4-42). In all cases, the presence of Abeta-(X-40) was negligible, a surprising finding in view of the prevalence of Abeta40 in vascular deposits observed in sporadic and familial AD, Down syndrome, and normal aging. Whether the presence of the two amyloid subunits is imperative for the disease phenotype or just reflects a conformational mimicry remains to be elucidated; nonetheless, a specific interaction between ADan oligomers and Abeta molecules was demonstrated in vitro by ligand blot analysis using synthetic peptides. The absence of compact plaques in the presence of extensive neuro fibrillar degeneration strongly suggests that compact plaques, fundamental lesions for the diagnosis of AD, are not essential for the mechanism of dementia.     

Complement activation in chromosome 13 dementias. Similarities with Alzheimer's disease

Chromosome 13 dementias, familial British dementia (FBD) and familial Danish dementia (FDD), are associated with neurodegeneration and cerebrovascular amyloidosis, with striking neuropathological similarities to Alzheimer's disease (AD). Despite the structural differences among the amyloid subunits (ABri in FBD, ADan in FDD, and Abeta in AD), these disorders are all characterized by the presence of neurofibrillary tangles and parenchymal and vascular amyloid deposits co-localizing with markers of glial activation, suggestive of local inflammation. Proteins of the complement system and their pro-inflammatory activation products are among the inflammation markers associated with AD lesions. Immunohistochemistry of FBD and FDD brain sections demonstrated the presence of complement activation components of the classical and alternative pathways as well as the neo-epitope of the membrane attack complex. Hemolytic experiments and enzyme-linked immunosorbent assays specific for the activation products iC3b, C4d, Bb, and C5b-9 indicated that ABri and ADan are able to fully activate the complement cascade at levels comparable to those generated by Abeta1-42. ABri and ADan specifically bound C1q with high affinity and formed stable complexes in physiological conditions. Activation proceeds approximately 70-75% through the classical pathway while only approximately 25-30% seems to occur through the alternative pathway. The data suggest that the chronic inflammatory response generated by the amyloid peptides in vivo might be a contributing factor for the pathogenesis of FBD and FDD and, in more general terms, to other neurodegenerative conditions.     

Amyloid in neurodegenerative diseases: friend or foe?

Accumulation of amyloid-like aggregates is a hallmark of numerous neurodegenerative disorders such as Alzheimer's and polyglutamine disease. Yet, whether the amyloid inclusions found in these diseases are toxic or cytoprotective remains unclear. Various studies suggest that the toxic culprit in the amyloid folding pathway is actually a soluble oligomeric species which might interfere with normal cellular function by a multifactorial mechanism including aberrant protein-protein interactions. Molecular chaperones suppress toxicity of amyloidogenic proteins by inhibiting aggregation of non-native disease substrates and targeting them for refolding or degradation. Paradoxically, recent studies also suggest a protective action of chaperones in their promotion of the assembly of large, tightly packed, benign aggregates that sequester toxic protein species.     

Non-fibrillar components of amyloid deposits mediate the self-association and tangling of amyloid fibrils

Amyloid deposits are proteinaceous extra-cellular aggregates associated with a diverse range of disease states. These deposits are composed predominantly of amyloid fibrils, the unbranched, beta-sheet rich structures that result from the misfolding and subsequent aggregation of many proteins. In addition, amyloid deposits contain a number of non-fibrillar components that interact with amyloid fibrils and are incorporated into the deposits in their native folded state. The influence of a number of the non-fibrillar components in amyloid-related diseases is well established; however, the mechanisms underlying these effects are poorly understood. Here we describe the effect of two of the most important non-fibrillar components, serum amyloid P component and apolipoprotein E, upon the solution behavior of amyloid fibrils in an in vitro model system. Using analytical ultracentrifugation, electron microscopy, and rheological measurements, we demonstrate that these non-fibrillar components cause soluble fibrils to condense into localized fibrillar aggregates with a greatly enhanced local density of fibril entanglements. These results suggest a possible mechanism for the observed role of non-fibrillar components as mediators of amyloid deposition and deposit stability.     

Hierarchy and the mechanism of fibril formation in ADan peptides

Familial Danish dementia is a neurodegenerative disease which is a consequence of alterations in the BRI gene. The pathological signatures of the disease are cerebral amyloidolysis, parenchymal protein deposits and neuronal degeneration. Synthetic Danish dementia (ADan) peptides are capable of forming fibrillar assemblies in vitro at pH 4.8. However, the morphology of the aggregates formed depends greatly on the form of the peptides (oxidized or reduced). In addition to long slender assemblies (2-5 nm in diameter and several micrometers in length) we report ring-like or annular masses (8-9 nm in diameter and 1-2 mm in perimeter) in the case of the oxidized form of the peptides. The reduced forms mainly aggregate to produce granular heaps. The biophysical and kinetic characterization of the process of aggregation was carried out using different spectroscopic and imaging techniques. Neurotoxicity assays performed on both the forms reveal that the toxicity bears proportionality with the aggregate size.     

N-Terminal pyroglutamate formation of Aβ38 and Aβ40 enforces oligomer formation and potency to disrupt hippocampal long-term potentiation

Pyroglutamate (pGlu)-modified amyloid peptides have been identified in sporadic and familial forms of Alzheimer's disease (AD) and the inherited disorders familial British and Danish Dementia (FBD and FDD). In this study, we characterized the aggregation of amyloid-β protein Aβ37, Aβ38, Aβ40, Aβ42 and ADan species in vitro, which were modified by N-terminal pGlu (pGlu-Aβ3-x, pGlu-ADan) or possess the intact N-terminus (Aβ1-x, ADan). The pGlu-modification confers rapid formation of oligomers and short fibrillar aggregates. In accordance with these observations, the pGlu-modified Aβ38, Αβ40 and Αβ42 species inhibit hippocampal long term potentiation of synaptic response, but pGlu-Aβ3-42 showing the highest effect. Among the unmodified Aβ peptides, only Aβ1-42 exhibites such propensity, which was similar to pGlu-Aβ3-38 and pGlu-Aβ3-40. Likewise, the amyloidogenic peptide pGlu-ADan impaired synaptic potentiation more pronounced than N-terminal unmodified ADan. The results were validated using conditioned media from cultivated HEK293 cells, which express APP variants favoring the formation of Aβ1-x, Aβ3-x or N-truncated pGlu-Aβ3-x species. Hence, we show that the ability of different amyloid peptides to impair synaptic function apparently correlates to their potential to form oligomers as a common mechanism. The pGlu-modification is apparently mediating a higher surface hydrophobicity, as shown by 1-anilinonaphtalene-8-sulfonate fluorescence, which enforces potential to interfere with neuronal physiology.     

Molecular chaperones antagonize proteotoxicity by differentially modulating protein aggregation pathways

The self-association of misfolded or damaged proteins into ordered amyloid-like aggregates characterizes numerous neurodegenerative disorders. Insoluble amyloid plaques are diagnostic of many disease states. Yet soluble, oligomeric intermediates in the aggregation pathway appear to represent the toxic culprit. Molecular chaperones regulate the fate of misfolded proteins and thereby influence their aggregation state. Chaperones conventionally antagonize aggregation of misfolded, disease proteins and assist in refolding or degradation pathways. Recent work suggests that chaperones may also suppress neurotoxicity by converting toxic, soluble oligomers into benign aggregates. Chaperones can therefore suppress or promote aggregation of disease proteins to ameliorate the proteotoxic accumulation of soluble, assembly intermediates.Key words: chaperone, heat shock protein, protein aggregation, amyloid, Hsp70, Hsp40, prion     

Aβ Oligomers and Fibrillar Aggregates Induce Different Apoptotic Pathways in LAN5 Neuroblastoma Cell Cultures

Fibril deposit formation of amyloid β-protein (Aβ) in the brain is a hallmark of Alzheimer's disease (AD). Increasing evidence suggests that toxicity is linked to diffusible Aβ oligomers, which have been found in soluble brain extracts of AD patients, rather than to insoluble fibers. Here we report a study of the toxicity of two distinct forms of recombinant Aβ small oligomers and fibrillar aggregates to simulate the action of diffusible Aβ oligomers and amyloid plaques on neuronal cells. Different techniques, including dynamic light scattering, fluorescence, and scanning electron microscopy, have been used to characterize the two forms of Aβ. Under similar conditions and comparable incubation times in neuroblastoma LAN5 cell cultures, oligomeric species obtained from Aβ peptide are more toxic than fibrillar aggregates. Both oligomers and aggregates are able to induce neurodegeneration by apoptosis activation, as demonstrated by TUNEL assay and Hoechst staining assays. Moreover, we show that aggregates induce apoptosis by caspase 8 activation (extrinsic pathway), whereas oligomers induce apoptosis principally by caspase 9 activation (intrinsic pathway). These results are confirmed by cytochrome c release, almost exclusively detected in the cytosolic fraction of LAN5 cells treated with oligomers. These findings indicate an active and direct interaction between oligomers and the cellular membrane, and are consistent with internalization of the oligomeric species into the cytosol.     

Microcin amyloid fibrils A are reservoir of toxic oligomeric species

Microcin E492 (Mcc), a low molecular weight bacteriocin produced by Klebsiella pneumoniae RYC492, has been shown to exist in two forms: soluble forms that are believed to be toxic to the bacterial cell by forming pores and non-toxic fibrillar forms that share similar biochemical and biophysical properties with amyloids associated with several human diseases. Here we report that fibrils polymerized in vitro from soluble forms sequester toxic species that can be released upon changing environmental conditions such as pH, ionic strength, and upon dilution. Our results indicate that basic pH (≥8.5), low NaCl concentrations (≤50 mm), and dilution (>10-fold) destabilize Mcc fibrils into more soluble species that are found to be toxic to the target cells. Additionally, we also found a similar conversion of non-toxic fibrils into highly toxic oligomers using Mcc aggregates produced in vivo. Moreover, the soluble protein released from fibrils is able to rapidly polymerize into amyloid fibrils under fibril-forming conditions and to efficiently seed aggregation of monomeric Mcc. Our findings indicate that fibrillar forms of Mcc constitute a reservoir of toxic oligomeric species that is released into the medium upon changing the environmental conditions. These findings may have substantial implications to understand the dynamic process of interconversion between toxic and non-toxic aggregated species implicated in protein misfolding diseases.     

BRI2 as a central protein involved in neurodegeneration

BRI2 is a protein that when mutated causes familial British and familial Danish dementias. Upon cleavage, the mutated BRI2 proteins release the peptides ABri and ADan, which are amyloidogenic and accumulate in the brains of patients. Although BRI2 has an unknown function, several reports indicate that it could play multiple roles. For example, the fact that it exists at the cell surface as a homodimer indicates that it could be involved in cell signaling events by acting as a receptor. BRI2 also interacts with amyloid precursor protein (APP), involved in Alzheimer's disease (AD). In cell cultures and mouse models of AD, BRI2 inhibits APP processing and reduces amyloid β peptide deposition. The interaction between the two proteins could be responsible for the neuropathological similarities between familial British/Danish dementias and AD. The study of BRI2, which is central in familial British and Danish dementia, could unravel underlying molecular mechanisms of neurodegeneration.     

Anti-oligomeric Abeta single-chain variable domain antibody blocks Abeta-induced toxicity against human neuroblastoma cells

The Amyloid-β (Aβ) peptide is a major component of the amyloid plaques associated with Alzheimer's disease (AD). Recent studies suggest that the most toxic forms of Aβ are small, soluble oligomeric aggregates. Here, we report the isolation and characterization of a single-chain variable domain (scFv) antibody isolated against oligomeric Aβ using a protocol developed in our laboratory that combines phage display technology and atomic force microscopy (AFM). Starting with a randomized, single framework phage display library, after three rounds of selection against oligomeric Aβ, we identified an scFv that bound oligomeric Aβ specifically, but not monomeric or fibrillar forms. The anti-oligomeric scFv inhibits Aβ aggregation and toxicity, and reduces the toxicity of preformed oligomeric Aβ towards human neuroblastoma cells. When used to probe samples of human brain tissue, the scFv reacted with AD tissue but not a healthy control or Parkinson's disease brain samples. The anti-oligomeric Aβ scFv therefore has potential therapeutic and diagnostic applications in specifically targeting or identifying the toxic morphologies of Aβ in AD brains.     

Effect of the disulfide bridge and the C-terminal extension on the oligomerization of the amyloid peptide ABri implicated in familial British dementia

Familial British dementia (FBD) is a rare neurodegenerative disorder and shares features with Alzheimer's disease, including amyloid plaque deposits, neurofibrillary tangles, neuronal loss, and progressive dementia. Immunohistochemical and biochemical analysis of plaques and vascular amyloid of FBD brains revealed that a 4 kDa peptide named ABri is the main component of the highly insoluble amyloid deposits. In FBD patients, the ABri peptide is produced as a result of a point mutation in the usual stop codon of the BRI gene. This mutation produces a BRI precursor protein 11 amino acids longer than the wild-type protein. Mutant and wild-type precursor proteins both undergo furin cleavage between residues 243 and 244, producing a peptide of 34 amino acids in the case of ABri and 23 amino acids in the case of the wild-type (WT) peptide. Here we demonstrate that the intramolecular disulfide bond in ABri and the C-terminal extension are required to elongate initially formed dimers to oligomers and fibrils. In contrast, the shorter WT peptide did not aggregate under the same conditions. Conformational analyses indicate that the disulfide bond and the C-terminal extension of ABri are required for the formation of beta-sheet structure. Soluble nonfibrillar ABri oligomers were observed prior to the appearance of mature fibrils. A molecular model of ABri containing three beta-strands, and two beta-hairpins annealed by a disulfide bond, has been constructed, and predicts a hydrophobic surface which is instrumental in promoting oligomerization.     

Conversion of non-fibrillar beta-sheet oligomers into amyloid fibrils in Alzheimer's disease amyloid peptide aggregation

Abeta(1-40) is one of the main components of the fibrils found in amyloid plaques, a hallmark of brains affected by Alzheimer's disease. It is known that prior to the formation of amyloid fibrils in which the peptide adopts a well-ordered intermolecular beta-sheet structure, peptide monomers associate forming low and high molecular weight oligomers. These oligomers have been previously described in electron microscopy, AFM, and exclusion chromatography studies. Their specific secondary structures however, have not yet been well established. A major problem when comparing aggregation and secondary structure determinations in concentration-dependent processes such as amyloid aggregation is the different concentration range required in each type of experiment. In the present study we used the dye Thioflavin T (ThT), Fourier-transform infrared spectroscopy, and electron microscopy in order to structurally characterize the different aggregated species which form during the Abeta(1-40) fibril formation process. A unique sample containing 90microM peptide was used. The results show that oligomeric species which form during the lag phase of the aggregation kinetics are a mixture of unordered, helical, and intermolecular non-fibrillar beta-structures. The number of oligomers and the amount of non-fibrillar beta-structures grows throughout the lag phase and during the elongation phase these non-fibrillar beta-structures are transformed into fibrillar (amyloid) beta-structures, formed by association of high molecular weight intermediates.     

Solution state characterization of amyloid beta-derived diffusible ligands

A growing body of evidence suggests that soluble oligomeric forms of the amyloid beta peptide known as amyloid-derived diffusible ligands (ADDLs) are the toxic species responsible for neurodegeneration associated with Alzheimer's disease. Accurate biophysical characterization of ADDL preparations is hampered by the peptide's strong tendency to self-associate and the effect of factors such as ionic strength, temperature, and pH on its behavior. In addition, amyloid peptides are known to interact with common laboratory excipients, specifically detergents, further complicating the results from standard analytical methods such as denaturing polyacrylamide gel electrophoresis. We have studied the solution behavior of various amyloid peptide preparations using analytical ultracentrifugation and size exclusion chromatography coupled with multiangle laser light scattering. Our results indicate that ADDL preparations exist in solution primarily as a binary mixture of a monomeric peptide and high-molecular mass oligomers. We relate our findings to previously described characterizations utilizing atomic force microscopy and electrophoretic methods and demonstrate that low-molecular mass oligomers identified by gel electrophoresis likely represent artifacts induced by the peptide's interaction with detergent, while atomic force microscopy results are likely skewed by differential binding of monomeric and oligomeric peptide species. Finally, we confirm that only the high-molecular mass oligomeric components of an ADDL preparation are capable of binding to subpopulations of primary hippocampal neurons in vitro.