Amino-terminally elongated Aß peptides are generated by the secreted metalloprotease ADAMTS4 and deposit in a subset of Alzheimer's disease brains.

AIMS: The aggregation and deposition of amyloid-ß (Aß) peptides in the brain is thought to be the initial driver in the pathogenesis of Alzheimer's disease (AD). Aside from full-length Aß peptides starting with an aspartate residue in position 1, both N-terminally truncated and elongated Aß peptides are produced by various proteases from the amyloid precursor protein (APP) and have been detected in brain tissues and body fluids. Recently, we demonstrated that the particularly abundant N-terminally truncated Aß4-x peptides are generated by ADAMTS4, a secreted metalloprotease that is exclusively expressed in the oligodendrocyte cell population. In this study, we investigated whether ADAMTS4 might also be involved in the generation of N-terminally elongated Aß peptides. METHODS: We used cell-free and cell-based assays in combination with matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF) and electrochemiluminescence sandwich immunoassays to identify and quantify N-terminally elongated Aß peptide variants. Antibodies against these Aß variants were characterised by peptide microarrays and employed for the immunohistochemical analyses of human brain samples. RESULTS: In this study, we discovered additional ADAMTS4 cleavage sites in APP. These were located N-terminal to Asp-(1) in the Aß peptide sequence between residues Glu-(-7) and Ile-(-6) as well as Glu-(-4) and Val-(-3), resulting in the release of N-terminally elongated Aß-6-x and Aß-3-x peptides, of which the latter serve as a component in a promising Aß-based plasma biomarker. Aß-6/-3-40 peptides were detected in supernatants of various cell lines and in the cerebrospinal fluid (CSF), and ADAMTS4 enzyme activity promoted the release of Aß-6/-3-x peptides. Furthermore, by immunohistochemistry, a subset of AD cases displayed evidence of extracellular and vascular localization of N-terminally elongated Aß-6/-3-x peptides. DISCUSSION: The current findings implicate ADAMTS4 in both the pathological process of Aß peptide aggregation and in the early detection of amyloid pathology in AD.

Patient-specific Alzheimer-like pathology in trisomy 21 cerebral organoids reveals BACE2 as a gene dose-sensitive AD suppressor in human brain.

A population of more than six million people worldwide at high risk of Alzheimer's disease (AD) are those with Down Syndrome (DS, caused by trisomy 21 (T21)), 70% of whom develop dementia during lifetime, caused by an extra copy of ß-amyloid-(Aß)-precursor-protein gene. We report AD-like pathology in cerebral organoids grown in vitro from non-invasively sampled strands of hair from 71% of DS donors. The pathology consisted of extracellular diffuse and fibrillar Aß deposits, hyperphosphorylated/pathologically conformed Tau, and premature neuronal loss. Presence/absence of AD-like pathology was donor-specific (reproducible between individual organoids/iPSC lines/experiments). Pathology could be triggered in pathology-negative T21 organoids by CRISPR/Cas9-mediated elimination of the third copy of chromosome 21 gene BACE2, but prevented by combined chemical ß and γ-secretase inhibition. We found that T21 organoids secrete increased proportions of Aß-preventing (Aß1-19) and Aß-degradation products (Aß1-20 and Aß1-34). We show these profiles mirror in cerebrospinal fluid of people with DS. We demonstrate that this protective mechanism is mediated by BACE2-trisomy and cross-inhibited by clinically trialled BACE1 inhibitors. Combined, our data prove the physiological role of BACE2 as a dose-sensitive AD-suppressor gene, potentially explaining the dementia delay in ~30% of people with DS. We also show that DS cerebral organoids could be explored as pre-morbid AD-risk population detector and a system for hypothesis-free drug screens as well as identification of natural suppressor genes for neurodegenerative diseases.

Pathogenesis of Alzheimer's Disease.

Alzheimer's disease (AD) is the most common type of dementia, accounting for 60% to 80% of all cases [...].

Association of clusterin with the BRI2-derived amyloid molecules ABri and ADan.

Familial British and Danish dementias (FBD and FDD) share striking neuropathological similarities with Alzheimer's disease (AD), including intraneuronal neurofibrillary tangles as well as parenchymal and vascular amyloid deposits. Multiple amyloid associated proteins with still controversial role in amyloidogenesis colocalize with the structurally different amyloid peptides ABri in FBD, ADan in FDD, and Aß in AD. Genetic variants and plasma levels of one of these associated proteins, clusterin, have been identified as risk factors for AD. Clusterin is known to bind soluble Aß in biological fluids, facilitate its brain clearance, and prevent its aggregation. The current work identifies clusterin as the major ABri- and ADan-binding protein and provides insight into the biochemical mechanisms leading to the association of clusterin with ABri and ADan deposits. Mirroring findings in AD, the studies corroborate clusterin co-localization with cerebral parenchymal and vascular amyloid deposits in both disorders. Ligand affinity chromatography with downstream Western blot and amino acid sequence analyses unequivocally identified clusterin as the major ABri- and ADan-binding plasma protein. ELISA highlighted a specific saturable binding of clusterin to ABri and ADan with low nanomolar Kd values within the same range as those previously demonstrated for the clusterin-Aß interaction. Consistent with its chaperone activity, thioflavin T binding assays clearly showed a modulatory effect of clusterin on ABri and ADan aggregation/fibrillization properties. Our findings, together with the known multifunctional activity of clusterin and its modulatory activity on the complex cellular pathways leading to oxidative stress, mitochondrial dysfunction, and the induction of cell death mechanisms - all known pathogenic features of these protein folding disorders - suggests the likelihood of a more complex role and a translational potential for the apolipoprotein in the amelioration/prevention of these pathogenic mechanisms.

Ion channel formation by N-terminally truncated Aß (4-42): relevance for the pathogenesis of Alzheimer's disease.

Aß deposition is a pathological hallmark of Alzheimer's disease (AD). Besides the full-length amyloid forming peptides (Aß1-40 and Aß1-42), biochemical analyses of brain deposits have identified a variety of N- and C-terminally truncated Aß variants in sporadic and familial AD patients. However, their relevance for AD pathogenesis remains largely understudied. We demonstrate that Aß4-42 exhibits a high tendency to form ß-sheet structures leading to fast self-aggregation and formation of oligomeric assemblies. Atomic force microscopy and electrophysiological studies reveal that Aß4-42 forms highly stable ion channels in lipid membranes. These channels that are blocked by monoclonal antibodies specifically recognizing the N-terminus of Aß4-42. An Aß variant with a double truncation at phenylalanine-4 and leucine 34, (Aß4-34), exhibits unstable channel formation capability. Taken together the results presented herein highlight the potential benefit of C-terminal proteolytic cleavage and further support an important pathogenic role for N-truncated Aß species in AD pathophysiology.

Aß truncated species: Implications for brain clearance mechanisms and amyloid plaque deposition.

Extensive parenchymal and vascular Aß deposits are pathological hallmarks of Alzheimer's disease (AD). Besides classic full-length peptides, biochemical analyses of brain deposits have revealed high degree of Aß heterogeneity likely resulting from the action of multiple proteolytic enzymes. In spite of the numerous studies focusing in Aß, the relevance of N- and C-terminal truncated species for AD pathogenesis remains largely understudied. In the present work, using novel antibodies specifically recognizing Aß species N-terminally truncated at position 4 or C-terminally truncated at position 34, we provide a clear assessment of the differential topographic localization of these species in AD brains and transgenic models. Based on their distinct solubility, brain N- and C-terminal truncated species were extracted by differential fractionation and identified via immunoprecipitation coupled to mass spectrometry analysis. Biochemical/biophysical studies with synthetic homologues further confirmed the different solubility properties and contrasting fibrillogenic characteristics of the truncated species composing the brain Aß peptidome. Aß C-terminal degradation leads to the production of more soluble fragments likely to be more easily eliminated from the brain. On the contrary, N-terminal truncation at position 4 favors the formation of poorly soluble, aggregation prone peptides with high amyloidogenic propensity and the potential to exacerbate the fibrillar deposits, self-perpetuating the amyloidogenic loop. Detailed assessment of the molecular diversity of Aß species composing interstitial fluid and amyloid deposits at different disease stages, as well as the evaluation of the truncation profile during various pharmacologic approaches will provide a comprehensive understanding of the still undefined contribution of Aß truncations to the disease pathogenesis and their potential as novel therapeutic targets.

Sequential Amyloid-ß Degradation by the Matrix Metalloproteases MMP-2 and MMP-9.

Matrix metalloproteases (MMPs) MMP-2 and MMP-9 have been implicated in the physiological catabolism of Alzheimer's amyloid-ß (Aß). Conversely, their association with vascular amyloid deposits, blood-brain barrier disruption, and hemorrhagic transformations after ischemic stroke also highlights their involvement in pathological processes. To better understand this dichotomy, recombinant human (rh) MMP-2 and MMP-9 were incubated with Aß40 and Aß42, and the resulting proteolytic fragments were assessed via immunoprecipitation and quantitative mass spectrometry. Both MMPs generated Aß fragments truncated only at the C terminus, ending at positions 34, 30, and 16. Using deuterated homologues as internal standards, we observed limited and relatively slow degradation of Aß42 by rhMMP-2, although the enzyme cleaved >80% of Aß40 during the 1st h of incubation. rhMMP-9 was significantly less effective, particularly in degrading Aß(1-42), although the targeted peptide bonds were identical. Using Aß(1-34) and Aß(1-30), we demonstrated that these peptides are also substrates for both MMPs, cleaving Aß(1-34) to produce Aß(1-30) first and Aß(1-16) subsequently. Consistent with the kinetics observed with full-length Aß, rhMMP-9 degraded only a minute fraction of Aß(1-34) and was even less effective in producing Aß(1-16). Further degradation of Aß(1-16) by either MMP-2 or MMP-9 was not observed even after prolonged incubation times. Notably, all MMP-generated C-terminally truncated Aß fragments were highly soluble and did not exhibit fibrillogenic properties or induce cytotoxicity in human cerebral microvascular endothelial or neuronal cells supporting the notion that these truncated Aß species are associated with clearance mechanisms rather than being key elements in the fibrillogenesis process.

Oxidative stress and mitochondria-mediated cell death mechanisms triggered by the familial Danish dementia ADan amyloid.

Familial Danish Dementia (FDD), an early-onset non-amyloid-ß (Aß) cerebral amyloidosis, is neuropathologically characterized by widespread cerebral amyloid angiopathy, parenchymal amyloid and preamyloid deposits, as well as neurofibrillary degeneration indistinguishable to that seen in Alzheimer's disease (AD). The main amyloid subunit composing FDD lesions, a 34-amino acid de-novo generated peptide ADan, is the direct result of a genetic defect at the 3'-end of the BRI2 gene and the physiologic action of furin-like proteolytic processing at the C-terminal region of the ADan precursor protein. We aimed to study the impact of the FDD mutation, the additional formation of the pyroglutamate (pE) posttranslational modification as well as the relevance of C-terminal truncations -all major components of the heterogeneous FDD deposits- on the structural and neurotoxic properties of the molecule. Our data indicates that whereas the mutation generated a ß-sheet-rich hydrophobic ADan subunit of high oligomerization/fibrillization propensity and the pE modification further enhanced these properties, C-terminal truncations had the opposite effect mostly abolishing these features. The potentiation of pro-amyloidogenic properties correlated with the initiation of neuronal cell death mechanisms involving oxidative stress, perturbation of mitochondrial membrane potential, release of mitochondrial cytochrome c, and downstream activation of caspase-mediated apoptotic pathways. The amyloid-induced toxicity was inhibited by targeting specific components of these detrimental cellular pathways, using reactive oxygen scavengers and monoclonal antibodies recognizing the pathological amyloid subunit. Taken together, the data indicate that the FDD mutation and the pE posttranslational modification are both primary elements driving intact ADan into an amyloidogenic/neurotoxic pathway while truncations at the C-terminus eliminate the pro-amyloidogenic characteristics of the molecule, likely reflecting effect of physiologic clearance mechanisms.

The carbonic anhydrase inhibitor methazolamide prevents amyloid beta-induced mitochondrial dysfunction and caspase activation protecting neuronal and glial cells in vitro and in the mouse brain.

Mitochondrial dysfunction has been recognized as an early event in Alzheimer's disease (AD) pathology, preceding and inducing neurodegeneration and memory loss. The presence of cytochrome c (CytC) released from the mitochondria into the cytoplasm is often detected after acute or chronic neurodegenerative insults, including AD. The carbonic anhydrase inhibitor (CAI) methazolamide (MTZ) was identified among a library of drugs as an inhibitor of CytC release and proved to be neuroprotective in Huntington's disease and stroke models. Here, using neuronal and glial cell cultures, in addition to an acute model of amyloid beta (Aß) toxicity, which replicates by intra-hippocampal injection the consequences of interstitial and cellular accumulation of Aß, we analyzed the effects of MTZ on neuronal and glial degeneration induced by the Alzheimer's amyloid. MTZ prevented DNA fragmentation, CytC release and activation of caspase 9 and caspase 3 induced by Aß in neuronal and glial cells in culture through the inhibition of mitochondrial hydrogen peroxide production. Moreover, intraperitoneal administration of MTZ prevented neurodegeneration induced by intra-hippocampal Aß injection in the mouse brain and was effective at reducing caspase 3 activation in neurons and microglia in the area surrounding the injection site. Our results, delineating the molecular mechanism of action of MTZ against Aß-mediated mitochondrial dysfunction and caspase activation, and demonstrating its efficiency in a model of acute amyloid-mediated toxicity, provide the first combined in vitro and in vivo evidence supporting the potential of a new therapy employing FDA-approved CAIs in AD.

Mitochondrial dysfunction induced by a post-translationally modified amyloid linked to a familial mutation in an alternative model of neurodegeneration.

Familial British dementia (FBD) is an early-onset non-amyloid-ß (Aß) cerebral amyloidosis that presents with severe cognitive decline and strikingly similar neuropathological features to those present in Alzheimer's disease (AD). FBD is associated with a T to A single nucleotide transition in the stop codon of a gene encoding BRI2, leading to the production of an elongated precursor protein. Furin-like proteolytic processing at its C-terminus releases a longer-than-normal 34 amino acid peptide, ABri, exhibiting amyloidogenic properties not seen in its 23 amino acid physiologic counterpart Bri1-23. Deposited ABri exhibits abundant post-translational pyroglutamate (pE) formation at the N-terminus, a feature seen in truncated forms of Aß found in AD deposits, and co-exists with neurofibrillary tangles almost identical to those found in AD. We tested the impact of the FBD mutation alone and in conjunction with the pE post-translational modification on the structural properties and associated neurotoxicity of the ABri peptide. The presence of pE conferred to the ABri molecule enhanced hydrophobicity and accelerated aggregation/fibrillization properties. ABri pE was capable of triggering oxidative stress, loss of mitochondrial membrane potential and activation of caspase-mediated apoptotic mechanisms in neuronal cells, whereas homologous peptides lacking the elongated C-terminus and/or the N-terminal pE were unable to induce similar detrimental cellular pathways. The data indicate that the presence of N-terminal pE is not in itself sufficient to induce pathogenic changes in the physiologic Bri1-23 peptides but that its combination with the ABri mutation is critical for the molecular pathogenesis of FBD.

Differential contribution of isoaspartate post-translational modifications to the fibrillization and toxic properties of amyloid ß and the Asn23 Iowa mutation.

Mutations within the Aß (amyloid ß) peptide, especially those clustered at residues 21-23, are linked to early-onset AD (Alzheimer's disease) and primarily associated with cerebral amyloid angiopathy. The Iowa variant, a substitution of an aspartic acid residue for asparagine at position 23 (D23N), associates with widespread vascular amyloid and abundant diffuse pre-amyloid lesions significantly exceeding the incidence of mature plaques. Brain Iowa deposits consist primarily of a mixture of mutated and non-mutated Aß species exhibiting partial aspartate isomerization at positions 1, 7 and 23. The present study analysed the contribution of the post-translational modification and the D23N mutation to the aggregation/fibrillization and cell toxicity properties of Aß providing insight into the elicited cell death mechanisms. The induction of apoptosis by the different Aß species correlated with their oligomerization/fibrillization propensity and ß-sheet content. Although cell toxicity was primarily driven by the D23N mutation, all Aß isoforms tested were capable, albeit at different time frames, of eliciting comparable apoptotic pathways with mitochondrial engagement and cytochrome c release to the cytoplasm in both neuronal and microvascular endothelial cells. Methazolamide, a cytochrome c release inhibitor, exerted a protective effect in both cell types, suggesting that pharmacological targeting of mitochondria may constitute a viable therapeutic avenue.

Functionally distinct pericyte subsets differently regulate amyloid-ß deposition in patients with Alzheimer's disease.

Although the concept that the blood-brain barrier (BBB) plays an important role in the etiology and pathogenesis of Alzheimer's disease (AD) has become increasingly accepted, little is known yet about how it actually contributes. We and others have recently identified a novel functionally distinct subset of BBB pericytes (PCs). In the present study, we sought to determine whether these PC subsets differentially contribute to AD-associated pathologies by immunohistochemistry and amyloid beta (Aß) peptidomics. We demonstrated that a disease-associated PC subset (PC2) expanded in AD patients compared to age-matched, cognitively unimpaired controls. Surprisingly, we found that this increase in the percentage of PC2 (%PC2) was correlated negatively with BBB breakdown in AD patients, unlike in natural aging or other reported disease conditions. The higher %PC2 in AD patients was also correlated with a lower Aß42 plaque load and a lower Aß42:Aß40 ratio in the brain as determined by immunohistochemistry. Colocalization analysis of multicolor confocal immunofluorescence microscopy images suggests that AD patient with low %PC2 have higher BBB breakdown due to internalization of Aß42 by the physiologically normal PC subset (PC1) and their concomitant cell death leading to more vessels without PCs and increased plaque load. On the contrary, it appears that PC2 can secrete cathepsin D to cleave and degrade Aß built up outside of PC2 into more soluble forms, ultimately contributing to less BBB breakdown and reducing Aß plaque load. Collectively our data shows functionally distinct mechanisms for PC1 and PC2 in high Aß conditions, demonstrating the importance of correctly identifying these populations when investigating the contribution of neurovascular dysfunction to AD pathogenesis.

Microglia produce the amyloidogenic ABri peptide in familial British dementia.

Mutations in ITM2B cause familial British, Danish, Chinese and Korean dementias. In familial British dementia (FBD) a mutation in the stop codon of the ITM2B gene (also known as BRI2 ) causes a C-terminal cleavage fragment of the ITM2B/BRI2 protein to be extended by 11 amino acids. This fragment, termed amyloid-Bri (ABri), is highly insoluble and forms extracellular plaques in the brain. ABri plaques are accompanied by tau pathology, neuronal cell death and progressive dementia, with striking parallels to the aetiology and pathogenesis of Alzheimer's disease. The molecular mechanisms underpinning FBD are ill-defined. Using patient-derived induced pluripotent stem cells, we show that expression of ITM2B/BRI2 is 34-fold higher in microglia than neurons, and 15-fold higher in microglia compared with astrocytes. This cell-specific enrichment is supported by expression data from both mouse and human brain tissue. ITM2B/BRI2 protein levels are higher in iPSC-microglia compared with neurons and astrocytes. Consequently, the ABri peptide was detected in patient iPSC-derived microglial lysates and conditioned media but was undetectable in patient-derived neurons and control microglia. Pathological examination of post-mortem tissue support ABri expression in microglia that are in proximity to pre-amyloid deposits. Finally, gene co-expression analysis supports a role for ITM2B/BRI2 in disease-associated microglial responses. These data demonstrate that microglia are the major contributors to the production of amyloid forming peptides in FBD, potentially acting as instigators of neurodegeneration. Additionally, these data also suggest ITM2B/BRI2 may be part of a microglial response to disease, motivating further investigations of its role in microglial activation. This has implications for our understanding of the role of microglia and the innate immune response in the pathogenesis of FBD and other neurodegenerative dementias including Alzheimer's disease.

Insights into caspase-mediated apoptotic pathways induced by amyloid-ß in cerebral microvascular endothelial cells.

BACKGROUND: The vascular deposition of amyloid known as cerebral amyloid angiopathy (CAA)--an age-associated condition and a common finding in Alzheimer's disease--compromises cerebral blood flow, causing macro/microhemorrhages and/or cognitive impairment. Very little is known about the mechanisms causing CAA-related degeneration of cerebral vascular cells. The Dutch E22Q familial amyloid-ß (Aß) variant is primarily associated with CAA, and manifests clinically with severe cerebral hemorrhages. OBJECTIVE: We aimed to determine the molecular mechanisms causing apoptosis of cerebral endothelial cells in the presence of wild-type Aß40 or its vasculotropic E22Q variant. METHODS: We challenged human brain microvascular endothelial cells with both Aß variants, and studied the apoptotic pathways triggered by these peptides. RESULTS: Caspase-mediated apoptotic pathways were elicited by both peptides within time frames correlating with their aggregation properties and formation of oligomeric/protofibrillar assemblies. Our data revealed a primary activation of caspase-8 (typically triggered by death receptors) with secondary engagement of caspase-9, with cytochrome C and apoptosis-inducing factor release from the mitochondria, suggesting the independent or synergistic engagement of extrinsic and intrinsic apoptotic mechanisms. CONCLUSION: Our data demonstrate the induction of caspase-8- and caspase-9-dependent mitochondrial-mediated apoptotic pathways by Aß oligomers/protofibrils in vascular cells, likely implicating a primary activation of death receptors.

Identification of Clusterin as a Major ABri- and ADan-Binding Protein Using Affinity Chromatography.

Affinity chromatography has, for many years, been at the research forefront as one of the simplest although highly versatile techniques capable of identifying biologically relevant protein-protein interactions. In the field of amyloid disorders, the use of ligands immobilized to a variety of affinity matrices was the method of choice to individualize proteins with affinity for soluble circulating forms of amyloid subunits. The methodology has also played an important role in the identification of proteins that interact with different amyloidogenic peptides and, as a result, are capable of modulating their physiological and pathological functions by altering solubility, aggregation propensity, and fibril formation proclivity. Along this line, classical studies conducted in the field of Alzheimer's disease (AD) identified clusterin as a major binding protein to both circulating soluble Aß as well as to the brain deposited counterpart. The affinity chromatography-based approach employed herein, individualized clusterin as the major protein capable of binding the amyloid subunits associated with familial British and Danish dementias, two non-Aß neurodegenerative conditions also exhibiting cerebral amyloid deposition and sharing striking similarities to AD. The data demonstrate that clusterin binding ability to amyloid molecules is not restricted to Aß, suggesting a modulating effect on the aggregation/fibrillization propensity of the amyloidogenic peptides that is consistent with its known chaperone activity.

N-terminally truncated Aß4-x proteoforms and their relevance for Alzheimer's pathophysiology.

BACKGROUND: The molecular heterogeneity of Alzheimer's amyloid-ß (Aß) deposits extends well beyond the classic Aß1-40/Aß1-42 dichotomy, substantially expanded by multiple post-translational modifications that increase the proteome diversity. Numerous truncated fragments consistently populate the brain Aß peptidome, and their homeostatic regulation and potential contribution to disease pathogenesis are largely unknown. Aß4-x peptides have been reported as major components of plaque cores and the limited studies available indicate their relative abundance in Alzheimer's disease (AD). METHODS: Immunohistochemistry was used to assess the topographic distribution of Aß4-x species in well-characterized AD cases using custom-generated monoclonal antibody 18H6-specific for Aß4-x species and blind for full-length Aß1-40/Aß1-42-in conjunction with thioflavin-S and antibodies recognizing Aßx-40 and Aßx-42 proteoforms. Circular dichroism, thioflavin-T binding, and electron microscopy evaluated the biophysical and aggregation/oligomerization properties of full-length and truncated synthetic homologues, whereas stereotaxic intracerebral injections of monomeric and oligomeric radiolabeled homologues in wild-type mice were used to evaluate their brain clearance characteristics. RESULTS: All types of amyloid deposits contained the probed Aß epitopes, albeit expressed in different proportions. Aß4-x species showed preferential localization within thioflavin-S-positive cerebral amyloid angiopathy and cored plaques, strongly suggesting poor clearance characteristics and consistent with the reduced solubility and enhanced oligomerization of their synthetic homologues. In vivo clearance studies demonstrated a fast brain efflux of N-terminally truncated and full-length monomeric forms whereas their oligomeric counterparts-particularly of Aß4-40 and Aß4-42-consistently exhibited enhanced brain retention. CONCLUSIONS: The persistence of aggregation-prone Aß4-x proteoforms likely contributes to the process of amyloid formation, self-perpetuating the amyloidogenic loop and exacerbating amyloid-mediated pathogenic pathways.

Matrix metalloproteinase 2 (MMP-2) degrades soluble vasculotropic amyloid-beta E22Q and L34V mutants, delaying their toxicity for human brain microvascular endothelial cells.

Patients carrying mutations within the amyloid-beta (Abeta) sequence develop severe early-onset cerebral amyloid angiopathy with some of the related variants manifesting primarily with hemorrhagic phenotypes. Matrix metalloproteases (MMPs) are typically associated with blood brain barrier disruption and hemorrhagic transformations after ischemic stroke. However, their contribution to cerebral amyloid angiopathy-related hemorrhage remains unclear. Human brain endothelial cells challenged with Abeta synthetic homologues containing mutations known to be associated in vivo with hemorrhagic manifestations (AbetaE22Q and AbetaL34V) showed enhanced production and activation of MMP-2, evaluated via Multiplex MMP antibody arrays, gel zymography, and Western blot, which in turn proteolytically cleaved in situ the Abeta peptides. Immunoprecipitation followed by mass spectrometry analysis highlighted the generation of specific C-terminal proteolytic fragments, in particular the accumulation of Abeta-(1-16), a result validated in vitro with recombinant MMP-2 and quantitatively evaluated using deuterium-labeled internal standards. Silencing MMP-2 gene expression resulted in reduced Abeta degradation and enhanced apoptosis. Secretion and activation of MMP-2 as well as susceptibility of the Abeta peptides to MMP-2 degradation were dependent on the peptide conformation, with fibrillar elements of AbetaE22Q exhibiting negligible effects. Our results indicate that MMP-2 release and activation differentially degrades Abeta species, delaying their toxicity for endothelial cells. However, taking into consideration MMP ability to degrade basement membrane components, these protective effects might also undesirably compromise blood brain barrier integrity and precipitate a hemorrhagic phenotype.

Iowa variant of familial Alzheimer's disease: accumulation of posttranslationally modified AbetaD23N in parenchymal and cerebrovascular amyloid deposits.

Mutations within the amyloid-beta (Abeta) sequence, especially those clustered at residues 21-23, which are linked to early onset familial Alzheimer's disease (AD), are primarily associated with cerebral amyloid angiopathy (CAA). The basis for this predominant vascular amyloid burden and the differential clinical phenotypes of cerebral hemorrhage/stroke in some patients and dementia in others remain unknown. The AbetaD23N Iowa mutation is associated with progressive AD-like dementia, often without clinically manifested intracerebral hemorrhage. Neuropathologically, the disease is characterized by predominant preamyloid deposits, severe CAA, and abundant neurofibrillary tangles in the presence of remarkably few mature plaques. Biochemical analyses using a combination of immunoprecipitation, mass spectrometry, amino acid sequence, and Western blot analysis performed after sequential tissue extractions to separately isolate soluble components, preamyloid, and fibrillar amyloid species indicated that the Iowa deposits are complex mixtures of mutated and nonmutated Abeta molecules. These molecules exhibited various degrees of solubility, were highly heterogeneous at both the N- and C-termini, and showed partial aspartate isomerization at positions 1, 7, and 23. This collection of Abeta species-the Iowa brain Abeta peptidome-contained clear imprints of amyloid clearance mechanisms yet highlighted the unique neuropathological features shared by a non-Abeta cerebral amyloidosis, familial Danish dementia, in which neurofibrillary tangles coexist with extensive pre-amyloid deposition in the virtual absence of fibrillar lesions. These data therefore challenge the importance of neuritic plaques as the sole contributors for the development of dementia.

A strategy for designing a peptide probe for detection of ß-amyloid oligomers.

Aggregation of ß-amyloid (Aß) is implicated in the pathology of Alzheimer's disease. Development of a robust strategy to detect Aß oligomeric intermediates, which have been identified as significant toxic agents, would be highly beneficial in the screening of drug candidates as well as enhancing our understanding of Aß oligomerization. Rapid, specific and quantitative detection, currently unavailable, would be highly preferred for accurate and reliable probing of transient Aß oligomers. Here, we report the development of a novel peptide probe, PG46, based on the nature of Aß self-assembly and the conformation-sensitive fluorescence of the biarsenical dye, FlAsH. PG46 was found to bind to Aß oligomers and displayed an increase in FlAsH fluorescence upon binding. No such event was observed when PG46 was co-incubated with Aß low-molecular-weight species or Aß fibrils. Aß oligomer detection was fast, and occurred within one hour without any additional sample incubation or preparation. We anticipate that the development of a strategy for detection of amyloid oligomers described in this study will be directly relevant to a host of other amyloidogenic proteins.