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Impaired clearance in the Alzheimer's Disease (AD) brain is key in the formation of Aβ parenchymal plaques and cerebrovascular deposits known as cerebral amyloid angiopathy (CAA), present in >80% of AD patients and ~50% of non‐AD elderly subjects. Aβ deposits are highly heterogeneous, containing multiple fragments mostly derived from catabolism of Aβ40/Aβ42, which exhibit dissimilar aggregation properties. Remarkably, the role of these physiologically relevant Aβ species in cerebrovascular injury and their impact in vascular pathology is unknown. We sought to understand how heterogeneous Aβ species affect cerebral endothelial health and assess whether their diverse effects are associated with the peptides aggregation propensities. We analyzed cerebral microvascular endothelial cell (CMEC) viability, blood‐brain barrier (BBB) permeability, and angiogenesis, all relevant aspects of brain microvascular dysfunction. We found that Aβ peptides and fragments exerted differential effects on cerebrovascular pathology. Peptides forming mostly oligomeric structures induced CMEC apoptosis, whereas fibrillar aggregates increased BBB permeability without apoptotic effects. Interestingly, all Aβ species tested inhibited angiogenesis in vitro. These data link the biological effects of the heterogeneous Aβ peptides to their primary structure and aggregation, strongly suggesting that the composition of amyloid deposits influences clinical aspects of the AD vascular pathology. As the presence of predominant oligomeric structures in proximity of the vessel walls may lead to CMEC death and induction of microhemorrhages, fibrillar amyloid is likely responsible for increased BBB permeability and associated neurovascular dysfunction. These results have the potential to unveil more specific therapeutic targets and clarify the multifactorial nature of AD. [ABSTRACT FROM AUTHOR]

Background: Mounting evidence points to a crucial role of amyloid-β (Aβ) in the pathophysiology of Alzheimer's disease (AD), a disorder in which brain glucose hypometabolism, downregulation of central elements of phosphorylation pathways, reduced ATP levels, and enhanced oxidative damage coexist, and sometimes precede, synaptic alterations and clinical manifestations. Since the brain has limited energy storage capacity, mitochondria play essential roles in maintaining the high levels of energy demand, but, as major consumers of oxygen, these organelles are also the most important generators of reactive oxygen species (ROS). Thus, it is not surprising that mitochondrial dysfunction is tightly linked to synaptic loss and AD pathophysiology. In spite of their relevance, the mechanistic links among ROS homeostasis, metabolic alterations, and cell bioenergetics, particularly in relation to Aβ, still remain elusive. Methods: We have used classic biochemical and immunocytochemical approaches together with the evaluation of real-time changes in global energy metabolism in a Seahorse Metabolic Analyzer to provide insights into the detrimental role of oligAβ in SH-SY5Y and primary neurons testing their pharmacologic protection by small molecules. Results: Our findings indicate that oligomeric Aβ induces a dramatic increase in ROS production and severely affects neuronal metabolism and bioenergetics. Assessment of global energy metabolism in real time demonstrated Aβ-mediated reduction in oxygen consumption affecting basal and maximal respiration and causing decreased ATP production. Pharmacologic targeting of Aβ-challenged neurons with a set of small molecules of known antioxidant and cytoprotective activity prevented the metabolic/bioenergetic changes induced by the peptide, fully restoring mitochondrial function while inducing an antioxidant response that counterbalanced the ROS production. Search for a mechanistic link among the protective small molecules tested identified the transcription factor Nrf2—compromised by age and downregulated in AD and transgenic models—as their main target and the PI3K/GSK-3 axis as the central pathway through which the compounds elicit their Aβ protective action. Conclusions: Our study provides insights into the complex molecular mechanisms triggered by oligAβ which profoundly affect mitochondrial performance and argues for the inclusion of small molecules targeting the PI3K/GSK-3 axis and Nrf2-mediated pathways as part of the current or future combinatorial therapies. [ABSTRACT FROM AUTHOR]

Familial Danish Dementia (FDD), an early-onset non-amyloid-β (Aβ) 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 β-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. [ABSTRACT FROM AUTHOR]

Substantial genetic, biochemical, and in vivo data indicate that progressive accumulation of amyloid-β (Aβ) plays a central role in the pathogenesis of Alzheimer's disease (AD). Historically centered in the importance of parenchymal plaques, the role of cerebral amyloid angiopathy (CAA)-a frequently neglected amyloid deposit present in >80% of AD cases-for the mechanism of disease pathogenesis is now starting to emerge. CAA consistently associates with microvascular modifications, ischemic lesions, micro- and macro-hemorrhages, and dementia, progressively affecting cerebral blood flow, altering blood-brain barrier permeability, interfering with brain clearance mechanisms and triggering a cascade of deleterious pro-inflammatory and metabolic events that compromise the integrity of the neurovascular unit. New evidence highlights the contribution of pre-fibrillar Aβ in the induction of cerebral endothelial cell dysfunction. The recently discovered interaction of oligomeric Aβ species with TRAIL DR4 and DR5 cell surface death receptors mediates the engagement of mitochondrial pathways and sequential activation of multiple caspases, eliciting a cascade of cell death mechanisms while unveiling an opportunity for exploring mechanistic-based therapeutic interventions to preserve the integrity of the neurovascular unit. [ABSTRACT FROM AUTHOR]

R4023 --> 495.5 --> The deposition of amyloid‐β (Aβ) in brain parenchyma and cerebral vasculature together with intraneuronal neurofibrillary tangles and the gradual loss of synapsis are central neuropathological hallmarks of Alzheimer's disease (AD). Although it remains unclear what primarily triggers and drives the progression of AD, different lines of investigation point out to a central role of oligomeric Aβ conformations for the disease pathogenesis. Recent studies revealed that the molecular heterogeneity of Aβ deposits is significantly more complex than originally anticipated, extending well beyond the classic Aβ1‐40/Aβ1‐42 dichotomy and being substantially expanded by the presence of multiple post‐translational modifications that increase the proteome diversity. Although N‐terminal truncations at glutamates 3 and 11 and their subsequent cyclation to pyroglutamate are perhaps the most extensively studied modifications, many others have been reported, with species beginning at phenylalanine 4 and bearing an intact C‐terminus being especially relevant. Indeed, Aβ4‐42 peptides were reported as major components of amyloid plaque cores and the limited studies available seem to indicate their relative abundance in patients with AD, Down's syndrome, and vascular dementia. In this work we assessed the topographic distribution of these species with in‐house‐generated anti‐Aβ4‐x monoclonal 18H6 – specific for Aβ species starting at position 4 and blind for the full‐length Aβ40 and Aβ42 – and used in conjunction with thioflavin S and antibodies specific for the C‐terminus 40 and 42 in confirmed AD cases. All types of Aβ deposits – parenchymal plaques, pre‐amyloid lesions and CAA – contained the Aβ epitopes tested, albeit expressed in different proportions. These co‐localizations were further stressed by combining double immunohistochemical analysis with Thioflavin S staining, showing the preferential location of Aβ4‐x species in CAA and cored plaques with fibrillar amyloid conformation, strongly suggesting poor clearance characteristics. To investigate the brain clearance of Aβ4‐x species in comparison with full‐length Aβ40 and Aβ42 homologues, peptides were synthesized, biochemically and biophysically characterized, radiolabeled with [125I]Na and utilized for in vivo brain clearance experiments in 5–6‐week‐old C57Bl/6 mice. Brain efflux of all monomeric Aβ species tested was fast, with >80% cleared within the 60 min duration of the experiment whereas retention of oligomeric preparations was consistently higher than that of monomeric forms. Notably, clearance of Aβ4‐40 and Aβ4‐42 was significantly lower (<45%) than the full‐length counterparts (61% ‐ 68%), further supporting a pathogenic role for Aβ oligomerization in AD and specifically highlighting the importance of oligomeric Aβ4‐x species exhibiting higher brain retention compared to their full‐length counterparts. Since the process of multimerization is concentration dependent, failing to remove these oligomeric forms of Aβ from the brain will further contribute to exacerbate the amyloidogenic loop and the persistence of amyloid deposits. [ABSTRACT FROM AUTHOR]

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-β (Aβ) 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 Aβ40 or its vasculotropic E22Q variant. Methods: We challenged human brain microvascular endothelial cells with both Aβ 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 Aβ oligomers/protofibrils in vascular cells, likely implicating a primary activation of death receptors. Copyright © 2011 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Abstract: Familial British and Familial Danish Dementia (FBD and FDD) are two dominantly inherited neurodegenerative diseases that present striking similarities with Alzheimer''s disease. The genetic defects underlying those dementias are mutations in the gene that encodes for BRI2 protein. Cleavage of mutated BRI2 by furin releases the peptides ABri or ADan, which accumulate in the brains of patients. BRI2 normal function is yet unknown. To unwind aspects of its cellular role, we investigated the possibility that BRI2 forms dimers, based on structural elements of the protein, the GXXXG motif within its transmembrane domain and the odd number of cysteine residues. We found that BRI2 dimerizes in cells and that dimers are held via non-covalent interactions and via disulfide bridges between the cysteines at position 89. Additionally, we showed that BRI2 dimers are formed in the ER and appear at the cell surface. Finally, BRI2 dimers were found to exist in mouse brain. Revealing the physiological properties of BRI2 is critical in the elucidation of the deviations that lead to neurodegeneration. [Copyright &y& Elsevier]

After the publication of this article [1], we became aware that there were errors in Figs. 4 and 31. [ABSTRACT FROM AUTHOR]

To better understand the physiologic excretion and/or catabolism of circulating peripheral amyloid β (Aβ), we labeled human Aβ40 (monomeric, with predominant unordered structure) and Aβ42 (mixture of monomers and oligomers in ∼50:50 ratio, rich in β-sheet conformation) with either Na125I or 125I-tyramine cellobiose, also known as the cell-trapping ligand procedure, testing their blood clearance and organ uptake in B6SJLF1/J mice. Irrespective of the labeling protocol, the peptide conformation, and the degree of oligomerization, both Aβ40 and Aβ42 showed a short half-life of 2.5-3.0 min. The liver was the major organ responsible for plasma clearance, accounting for >60% of the peptide uptake, followed by the kidney. In vivo, hepatocytes captured >90% of the radiolabeled peptides which, after endocytosis, were preferentially catabolized and excreted into the bile. Biliary excretion of intact as well as partially degraded AB species became obviously relevant at doses above 10 µg. The use of biotin-labeled Aβ allowed the visualization of the interaction with HepG2 cells in culture, whereas competitive inhibition experiments with unlabeled Aβ demonstrated the specificity of the binding. The capability of the liver to uptake, catabolize, and excrete large doses of Aβ, several orders of magnitude above its physiologic concentration, may explain not only the femtomolar plasma levels of AB but the little fluctuation observed with age and disease stages. [ABSTRACT FROM AUTHOR]

Alzheimer’s disease (AD) is the most frequent type of amyloidosis in humans and the commonest form of dementia. Extracellular Aβ amyloid deposits in the form of amyloid plaques and cerebral amyloid angiopathy as well as intraneuronal neurofibrillary tangles co-exist in the brain parenchyma of AD patients, the cognitive areas being the most severely affected. This review focuses on the potential role of amyloid in the development of neurodegeneration and presents studies of AD and other unrelated inherited dementia syndromes associated with neuronal loss and amyloid deposition in the brain. [Copyright &y& Elsevier]

Cerebral amyloid angiopathy (CAA) is the common term used to define the deposition of amyloid in the walls of medium- and small-size leptomeningeal and cortical arteries, arterioles and, less frequently, capillaries and veins. CAA is an important cause of cerebral hemorrhages although it may also lead to ischemic infarction and dementia. It is a feature commonly associated with normal aging, Alzheimer disease (AD), Down syndrome (DS), and Sporadic Cerebral Amyloid Angiopathy. Familial conditions in which amyloid is chiefly deposited as CAA include hereditary cerebral hemorrhage with amyloidosis of Icelandic type (HCHWA-I), familial CAA related to Abeta variants, including hereditary cerebral hemorrhage with amyloidosis of Dutch origin (HCHWA-D), the transthyretin-related meningocerebrovascular amyloidosis of Hungarian and Ohio kindreds, the gelsolin-related spinal and cerebral amyloid angiopathy, familial PrP-CAA, and the recently described chromosome 13 familial dementia in British and Danish kindreds. This review focuses on the various molecules and genetic variants that target the cerebral vessel walls producing clinical features related to stroke and/or dementia, and discusses the potential role of amyloid in the mechanism of neurodegeneration. [ABSTRACT FROM AUTHOR]

The formation of amyloid-β (Aβ) aggregates in brain is a neuropathological hallmark of Alzheimer's disease (AD). However, there is mounting evidence that Aβ also plays a pathogenic role in other types of dementia and that specific post-translational Aβ modifications contribute to its pathogenic profile. The objective of this study was to test the hypothesis that distinct types of dementia are characterized by specific patterns of post-translationally modified Aβ variants. We conducted a comparative analysis and quantified Aβ as well as Aβ with pyroglutamate (pGlu3-Aβ and pGlu11-Aβ), N-truncation (Aβ(4-X)), isoaspartate racemization (isoAsp7-Aβ and isoAsp27-Aβ), phosphorylation (pSer8-Aβ and pSer26-Aβ) or nitration (3NTyr10-Aβ) modification in post mortem human brain tissue from non-demented control subjects in comparison to tissue classified as pre-symptomatic AD (Pre-AD), AD, dementia with Lewy bodies and vascular dementia. Aβ modification-specific immunohistochemical labelings of brain sections from the posterior superior temporal gyrus were examined by machine learning-based segmentation protocols and immunoassay analyses in brain tissue after sequential Aβ extraction were carried out. Our findings revealed that AD cases displayed the highest concentrations of all Aβ variants followed by dementia with Lewy bodies, Pre-AD, vascular dementia and non-demented controls. With both analytical methods, we identified the isoAsp7-Aβ variant as a highly abundant Aβ form in all clinical conditions, followed by Aβ(4-X), pGlu3-Aβ, pGlu11-Aβ and pSer8-Aβ. These Aβ variants were detected in distinct plaque types of compact, coarse-grained, cored and diffuse morphologies and, with varying frequencies, in cerebral blood vessels. The 3NTyr10-Aβ, pSer26-Aβ and isoAsp27-Aβ variants were not found to be present in Aβ plaques but were detected intraneuronally. There was a strong positive correlation between isoAsp7-Aβ and Thal phase and a moderate negative correlation between isoAsp7-Aβ and performance on the Mini Mental State Examination. Furthermore, the abundance of all Aβ variants was highest in APOE 3/4 carriers. In aggregation assays, the isoAsp7-Aβ, pGlu3-Aβ and pGlu11-Aβ variants showed instant fibril formation without lag phase, whereas Aβ(4-X), pSer26-Aβ and isoAsp27-Aβ did not form fibrils. We conclude that targeting Aβ post-translational modifications, and in particular the highly abundant isoAsp7-Aβ variant, might be considered for diagnostic and therapeutic approaches in different types of dementia. Hence, our findings might have implications for current antibody-based therapies of AD. [ABSTRACT FROM AUTHOR]

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. The ABri peptide was detected in patient iPSC-derived microglial lysates and conditioned media but was undetectable in patient-derived neurons and control microglia. The pathological examination of post-mortem tissue supports the presence of ABri 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 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. These data have 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. [ABSTRACT FROM AUTHOR]

The importance of amyloid plaques in the pathogenesis of dementia is usually centered on β-amyloid (Aβ) and its role in Alzheimer’s disease (AD). However, since fibrillar plaques correlate poorly with neurodegeneration, challenging their importance in the mechanism(s) of dementia, investigators turned their focus to the importance of soluble oligomers and the role of preamyloid and cerebrovascular deposits. Two non-Aβ cerebral amyloidoses, familial British and Danish dementias (FBD and FDD), share many aspects of AD, including cognitive impairment and the presence of neurofibrillary tangles in limbic areas. The lack of compact plaques in FDD and in many areas in FBD further questions the importance of these lesions in the mechanism of dementia. The main components of the deposits – ABri and ADan – are structurally unrelated to Aβ and yet they all have a high tendency to oligomerize and assemble into amyloid fibrils in vitro and form ion-like channels in lipid membranes. Thus, different amyloid species have the capability to induce similar neuropathological changes, which are neither exclusive for Aβ nor dependent on the presence of compact plaques. These findings reaffirm the notion that non-Aβ amyloidoses constitute alternative models to study the role of preassembled amyloid subunits in neuronal death. Copyright © 2008 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

The article presents information on hereditary cerebral hemorrhage with amyloidosis-Dutch type (HCHWA-D). HCHWA-D, a genetic disorder first identified in four large families from the Netherlands, strikes individuals as young as 40 years old and causes blood vessels in the brain to hemorrhage. About half of affected individuals die, and patients who survive these recurrent strokes develop cognitive impairment and dementia a few years later. The HCHWA-D disease is associated with the gene, located on chromosome 21, that codes for the Aβ precursor protein (APP) where a single mutation, the first ever reported in the human APP gene, replaces a guanine with a cytosine at codon 693.

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 (Aβ) 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 Aβ42 plaque load and a lower Aβ42:Aβ40 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 Aβ42 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 Aβ built up outside of PC2 into more soluble forms, ultimately contributing to less BBB breakdown and reducing Aβ plaque load. Collectively our data shows functionally distinct mechanisms for PC1 and PC2 in high Aβ conditions, demonstrating the importance of correctly identifying these populations when investigating the contribution of neurovascular dysfunction to AD pathogenesis. [ABSTRACT FROM AUTHOR]

Aims: The aggregation and deposition of amyloid‐β (Aβ) peptides in the brain is thought to be the initial driver in the pathogenesis of Alzheimer's disease (AD). Aside from full‐length Aβ peptides starting with an aspartate residue in position 1, both N‐terminally truncated and elongated Aβ 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 Aβ4–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 Aβ 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 Aβ peptide variants. Antibodies against these Aβ 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 Aβ 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 Aβ‐6‐x and Aβ‐3‐x peptides, of which the latter serve as a component in a promising Aβ‐based plasma biomarker. Aβ‐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 Aβ‐6/‐3‐x peptides. Furthermore, by immunohistochemistry, a subset of AD cases displayed evidence of extracellular and vascular localization of N‐terminally elongated Aβ‐6/‐3‐x peptides. Discussion: The current findings implicate ADAMTS4 in both the pathological process of Aβ peptide aggregation and in the early detection of amyloid pathology in AD. [ABSTRACT FROM AUTHOR]

Substantial genetic, biochemical, and in vivo data indicate that progressive accumulation of amyloid-β (Aβ) plays a central role in the pathogenesis of Alzheimer's disease (AD). Historically centered in the importance of parenchymal plaques, the role of cerebral amyloid angiopathy (CAA)--a frequently neglected amyloid deposit present in >80% of AD cases--for the mechanism of disease pathogenesis is now starting to emerge. CAA consistently associates with microvascular modifications, ischemic lesions, micro- and macro-hemorrhages, and dementia, progressively affecting cerebral blood flow, altering blood-brain barrier permeability, interfering with brain clearance mechanisms and triggering a cascade of deleterious pro-inflammatory and metabolic events that compromise the integrity of the neurovascular unit. New evidence highlights the contribution of pre-fibrillar Aβ in the induction of cerebral endothelial cell dysfunction. The recently discovered interaction of oligomeric Aβ species with TRAIL DR4 and DR5 cell surface death receptors mediates the engagement of mitochondrial pathways and sequential activation of multiple caspases, eliciting a cascade of cell death mechanisms while unveiling an opportunity for exploring mechanistic-based therapeutic interventions to preserve the integrity of the neurovascular unit. [ABSTRACT FROM AUTHOR]

Familial British dementia (FBD) is an early-onset non-amyloid-β (Aβ) 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 Aβ 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. [ABSTRACT FROM AUTHOR]

Background: The molecular heterogeneity of Alzheimer's amyloid-β (Aβ) deposits extends well beyond the classic Aβ1-40/Aβ1-42 dichotomy, substantially expanded by multiple post-translational modifications that increase the proteome diversity. Numerous truncated fragments consistently populate the brain Aβ peptidome, and their homeostatic regulation and potential contribution to disease pathogenesis are largely unknown. Aβ4-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 Aβ4-x species in well-characterized AD cases using custom-generated monoclonal antibody 18H6—specific for Aβ4-x species and blind for full-length Aβ1-40/Aβ1-42—in conjunction with thioflavin-S and antibodies recognizing Aβx-40 and Aβx-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 Aβ epitopes, albeit expressed in different proportions. Aβ4-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 Aβ4-40 and Aβ4-42—consistently exhibited enhanced brain retention. Conclusions: The persistence of aggregation-prone Aβ4-x proteoforms likely contributes to the process of amyloid formation, self-perpetuating the amyloidogenic loop and exacerbating amyloid-mediated pathogenic pathways. [ABSTRACT FROM AUTHOR]

Background: The molecular heterogeneity of Alzheimer's amyloid-β (Aβ) deposits extends well beyond the classic Aβ1-40/Aβ1-42 dichotomy, substantially expanded by multiple post-translational modifications that increase the proteome diversity. Numerous truncated fragments consistently populate the brain Aβ peptidome, and their homeostatic regulation and potential contribution to disease pathogenesis are largely unknown. Aβ4-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 Aβ4-x species in well-characterized AD cases using custom-generated monoclonal antibody 18H6—specific for Aβ4-x species and blind for full-length Aβ1-40/Aβ1-42—in conjunction with thioflavin-S and antibodies recognizing Aβx-40 and Aβx-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 Aβ epitopes, albeit expressed in different proportions. Aβ4-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 Aβ4-40 and Aβ4-42—consistently exhibited enhanced brain retention. Conclusions: The persistence of aggregation-prone Aβ4-x proteoforms likely contributes to the process of amyloid formation, self-perpetuating the amyloidogenic loop and exacerbating amyloid-mediated pathogenic pathways. [ABSTRACT FROM AUTHOR]

Abstract: Numerous studies have suggested that estradiol (E) improves spatial memory as female rats with E perform better than those without E. However there is an inverse relationship between E and luteinizing hormone (LH) levels and LH could play a role. We examined whether treatment with the LH homologue human chorionic gonadotropin (hCG), would impair spatial memory of adult E-treated female rats. In the object location memory task, ovariectomized (ovxed) rats treated with E and either a single high dose (400 IU/kg) or a lower repeated dose of hCG (75 IU/kg hourly for 8 h) showed spatial memory disruption compared to ovxed rats treated with estradiol alone. Impairment was attributed to memory disruption as performance improved with shortened delay between task exposure and testing. Tests on another spatial memory task, the Barnes maze, confirmed that hCG (400 IU/kg) can impair memory: although E+veh treated animals made significantly fewer hole errors across time, E+hCG-treated did not. In humans, high LH levels have been correlated with Alzheimer''s disease (AD). Because brain amyloid-beta (Aβ) species have been implicated as a toxic factor thought to cause memory loss in AD, we analyzed whether hCG-treated animals had increased Aβ levels. Levels of Aβ from whole brains or hippocampi were assessed by Western blot. hCG treatment to E-implanted females significantly increased soluble Aβ40 and Aβ42 levels. These results indicate that high levels of LH/hCG can impair spatial memory, and an increase in brain Aβ species may account for the memory impairment in hCG-treated rats. [Copyright &y& Elsevier]

Abstract: Neprilysin (NEP) is a zinc metalloproteinase that degrades enkephalins, endothelins, and the Alzheimer''s disease amyloid β (Aβ) peptides. NEP-deficient mice possess increased levels of brain Aβ1–40 and Aβ1–42. The objective of this study was to determine whether tissue NEP specific activity differs according to age and/or across mouse strains, especially those strains predisposed toward formation of Aβ-amyloid plaques following overexpression of the human Alzheimer amyloid precursor protein (APP). The C57Bl/6J mouse strain appears to be relatively susceptible to cerebral amyloidosis, whereas the Swiss Webster (SW) strain appears more resistant. We investigated whether NEP specific activity in brain and kidney homogenates from SW and C57 mice of 6, 40, and 80 weeks old varied according to mouse strain, age, and gender. Among the variables tested, NEP specific activity varied most dramatically across mouse strain, with the kidney and brain of SW mice displaying the highest activities. Aging was associated with a reduction in brain NEP specific activity in both strains. Gender-specific differences were identified in kidney but not in brain. We conclude that aging- and strain-dependent differences in NEP specific activity may play a role in the differential susceptibility of some mouse strains for developing cerebral amyloidosis following human APP overexpression. [Copyright &y& Elsevier]

1. We studied cerebrovascular sequestration and blood–brain barrier (BBB) permeability to [125I]- or [123I]-labeled amyloid-β peptides (Aβ) in aged rhesus and aged squirrel monkey, the nonhuman primate models of cerebral β-amyloidosis and cerebrovascular amyloid angiopathy (CAA), respectively.2. In aged rhesus, the half-time of elimination of [125I]Aβ1–40, t1/2e, was faster by 1.34 h, the systemic clearance, Clss, increased by 4.21 ml/min/kg and the mean residence time of intact peptide in the circulation shortened by 2 h.3. Cerebrovascular sequestration of [125I]Aβ1−40 was significant in aged squirrel monkey (20.8 ml/g×102), but undetectable in the rhesus.4. The permeability surface area product, PS, for [14C]inulin was low in both species (0.11–0.18 ml/g/s×106) indicating an intact barrier.5. The BBB permeability to Aβ1−40 was 34.8- and 13.7-fold higher than for [14C]inulin in aged squirrel and rhesus, respectively, suggesting a specialized Aβ transport across the BBB.6. The single photon computed emission tomography studies confirmed a saturable [123I]Aβ1–40 transport at the BBB in primates (Km=40 nM).7. Brain autoradiographic analysis of [125I]Aβ1–42 or [125I]Aβ1−40 after intracarotid infusions of radiotracers confirmed co-localization of the signal with Aβ-immunoreactive plaques in rhesus monkeys.8. Metabolism of [125I]Aβ1−40 in brain and plasma was slower in aged squirrel compared to aged rhesus, by 2.9- and 2.6-fold, respectively.9. Thus, transport of circulating Aβ across the BBB contributes to brain parenchymal accumulation of amyloid in aged nonhuman primates. Negligible capillary binding, rapid systemic and brain degradation, and accelerated body elimination of blood-borne Aβ, may prevent the development of CAA in rhesus in contrast to squirrel monkeys. [Copyright &y& Elsevier]

Extensive parenchymal and vascular Aβ deposits are pathological hallmarks of Alzheimer's disease (AD). Besides classic full-length peptides, biochemical analyses of brain deposits have revealed high degree of Aβ heterogeneity likely resulting from the action of multiple proteolytic enzymes. In spite of the numerous studies focusing in Aβ, the relevance of N- and C-terminal truncated species for AD pathogenesis remains largely understudied. In the present work, using novel antibodies specifically recognizing Aβ 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 Aβ peptidome. Aβ 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 Aβ 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 Aβ truncations to the disease pathogenesis and their potential as novel therapeutic targets. [ABSTRACT FROM AUTHOR]

Background: The deposition of neurotoxic amyloid-β (Aβ) peptides in plaques in the brain parenchyma and in cerebral blood vessels is considered to be a key event in Alzheimer's disease (AD) pathogenesis. Although the presence and impact of full-length Aβ peptides such as Aβ1-40 and Aβ1-42 have been analyzed extensively, the deposition of N-terminally truncated Aβ peptide species has received much less attention, largely because of the lack of specific antibodies. Methods: This paper describes the generation and characterization of novel antibodies selective for Aβ4-x peptides and provides immunohistochemical evidence of Aβ4-x in the human brain and its distribution in the APP/PS1KI and 5XFAD transgenic mouse models. Results: The Aβ4-x staining pattern was restricted mainly to amyloid plaque cores and cerebral amyloid angiopathy in AD and Down syndrome cases and in both AD mouse models. In contrast, diffuse amyloid deposits were largely negative for Aβ4-x immunoreactivity. No overt intraneuronal staining was observed. Conclusions: The findings of this study are consistent with previous reports demonstrating a high aggregation propensity of Aβ4-x peptides and suggest an important role of these N-truncated Aβ species in the process of amyloidogenesis and plaque core formation. [ABSTRACT FROM AUTHOR]

The misfolding and accumulation of the protein fragment β-amyloid (Aβ) is an early and essential event in the pathogenesis of Alzheimer's disease (AD). Despite close biological similarities among primates, humans appear to be uniquely susceptible to the profound neurodegeneration and dementia that characterize AD, even though nonhuman primates deposit copious Aβ in senile plaques and cerebral amyloid-β angiopathy as they grow old. Because the amino acid sequence of Aβ is identical in all primates studied to date, we asked whether differences in the properties of aggregated Aβ might underlie the vulnerability of humans and the resistance of other primates to AD. In a comparison of aged squirrel monkeys ( Saimiri sciureus ) and humans with AD, immunochemical and mass spectrometric analyses indicate that the populations of Aβ fragments are largely similar in the 2 species. In addition, Aβ-rich brain extracts from the brains of aged squirrel monkeys and AD patients similarly seed the deposition of Aβ in a transgenic mouse model. However, the epitope exposure of aggregated Aβ differs in sodium dodecyl sulfate-stable oligomeric Aβ from the 2 species. In addition, the high-affinity binding of 3 H Pittsburgh Compound B to Aβ is significantly diminished in tissue extracts from squirrel monkeys compared with AD patients. These findings support the hypothesis that differences in the pathobiology of aggregated Aβ among primates are linked to post-translational attributes of the misfolded protein, such as molecular conformation and/or the involvement of species-specific cofactors. [ABSTRACT FROM AUTHOR]

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 (Aβ) toxicity, which replicates by intra-hippocampal injection the consequences of interstitial and cellular accumulation of Aβ, 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 Aβ 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 Aβ 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 Aβ-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. [ABSTRACT FROM AUTHOR]

Matrix metalloproteases (MMPs) MMP-2 and MMP-9 have been implicated in the physiological catabolism of Alzheimer's amyloid-β (Aβ). Conversely, their association with vascular amyloid deposits, blood-brain barrier disruption, and hemor-rhagic 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 Aβ40 and Aβ42, and the resulting proteolytic fragments were assessed via immunoprecipitation and quantitative mass spectrometry. Both MMPs generated Aβ 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 Aβ42 by rhMMP-2, although the enzyme cleaved > 80% of Aβ40 during the 1st h of incubation. rhMMP-9 was significantly less effective, particularly in degrading Aβ(1-42), although the targeted peptide bonds were identical. Using Aβ(l-34) and Aβ(1-30), we demonstrated that these peptides are also substrates for both MMPs, cleaving Aβ(1-34) to produce Aβ(1-30) first and Aβ(1-16) subsequently. Consistent with the kinetics observed with full-length Aβ, rhMMP-9 degraded only a minute fraction of Aβ(1-34) and was even less effective in producing Aβ(1-16). Further degradation of Aβ(1-16) by either MMP-2 or MMP-9 was not observed even after prolonged incubation times. Notably, all MMP-generated C-terminally truncated Aβ fragments were highly soluble and did not exhibit fibrillogenic properties or induce cytotoxicity in human cerebral micro-vascular endothelial or neuronal cells supporting the notion that these truncated Aβ species are associated with clearance mechanisms rather than being key elements in the fibrillo-genesis process. [ABSTRACT FROM AUTHOR]

R4076 --> 495.6 --> Clusterin, a multifunctional glycoprotein also known as apolipoprotein J, has been implicated in several physiological processes and is an important contributor to many pathological conditions, including Alzheimer's disease (AD) in which genetic variants and plasma levels of the apolipoprotein were identified as risk factors for the disorder. To investigate whether clusterin exerted a more general role in the pathophysiology of cerebral amyloidoses, we turned to two unrelated hereditary conditions, familial British and Danish dementias (FBD and FDD), which share striking neuropathological similarities with AD, including neurofibrillary degeneration in limbic areas as well as parenchymal and vascular amyloid deposition. Two different 4 kDa molecules – ABri in FBD and ADan in FDD – totally unrelated to the 4 kDa Alzheimer's Aβ, are the main constituents of the amyloid deposits in these forms of cerebral amyloidoses. Mirroring findings in AD, a panel of immunohistochemical studies demonstrated 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. Solid‐phase enzyme‐linked immunosorbent assays highlighted a specific saturable binding of clusterin to ABri and ADan. Non‐linear regression analysis of the data revealed Kd values within the same low nanomolar range as those demonstrated for the clusterin‐Aβ interaction. Consistent with its reported chaperone activity, clusterin showed a modulatory effect on ABri, ADan, and Aβ aggregation/fibrillization properties evaluated using thioflavin T, a dye that displays enhanced fluorescence upon binding fibrillar and protofibrillar amyloid conformations. 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 protein folding disorders, including AD, FBD, and FDD – suggests the likelihood of a more complex role and a translational potential of clusterin in the amelioration/prevention of these pathogenic mechanisms. [ABSTRACT FROM AUTHOR]

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-mutatedAß 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 deathmechanisms. 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. [ABSTRACT FROM AUTHOR]

Abstract: Familial British and familial Danish dementia (FDD) are progressive neurodegenerative disorders characterized by cerebral deposition of the amyloidogenic peptides ABri and ADan, respectively. These amyloid peptides start with an N-terminal glutamate residue, which can be posttranslationally converted into a pyroglutamate (pGlu) modified form, a mechanism which has been extensively described to be relevant for amyloid-beta (Aβ) peptides in Alzheimer''s disease. Like pGlu-Aβ peptides, pGlu-ABri peptides have an increased aggregation propensity and show higher toxicity on human neuroblastoma cells as their nonmodified counterparts. We have generated novel N-terminal specific antibodies detecting the pGlu-modified forms of ABri and ADan peptides. With these antibodies we were able to identify abundant extracellular amyloid plaques, vascular, and parenchymal deposits in human familial British dementia and FDD brain tissue, and in a mouse model for FDD. Double-stainings using C-terminal specific antibodies in human samples revealed that highly aggregated pGlu-ABri and pGlu-ADan peptides are mainly present in plaque cores and central vascular deposits, leading to the assumption that these peptides have seeding properties. Furthermore, in an FDD-mouse model ADan peptides were detected in presynaptic terminals of the hippocampus where they might contribute to impaired synaptic transmission. These similarities of ABri and ADan to Aβ in Alzheimer''s disease suggest that the posttranslational pGlu-modification of amyloid peptides might represent a general pathological mechanism leading to increased aggregation and toxicity in these forms of degenerative dementias. [Copyright &y& Elsevier]

Two different mutated forms of BRI2 protein are linked with familial British and Danish dementias, which present neuropathological similarities with Alzheimer's disease. BRI2 is a type II transmembrane protein that is trafficked through the secretory pathway to the cell surface and is processed by furin and ADAM10 (a disintegrin and metalloproteinase domain 10) to release secreted fragments of unknown function. Its apparent molecular mass (42–44 kDa) is significantly higher than that predicted by the number and composition of amino acids (30 kDa) suggesting that BRI2 is glycosylated. In support, bioinformatics analysis indicated that BRI2 bears the consensus sequence Asn-Thr-Ser (residues 170–173) and could be N-glycosylated at Asn170. Given that N-glycosylation is considered essential for protein folding, processing and trafficking, we examined whether BRI2 is N-glycosylated. Treatment of HEK293 (human embryonic kidney) cells expressing BRI2 with the N-glycosylation inhibitor tunicamycin or mutation of Asn170 to alanine reduced its molecular mass by ∼2 kDa. These data indicate that BRI2 is N-glycosylated at Asn170. To examine the effect of N-glycosylation on BRI2 trafficking at the cell surface, we performed biotinylation and 35S methionine pulse-chase experiments. These experiments showed that mutation of Asn170 to alanine reduced BRI2 trafficking at the cell surface and its steady state levels at the plasma membrane. Furthermore, we obtained data indicating that this mutation did not affect cleavage of BRI2 by furin or ADAM10. Our results confirm the theoretical predictions that BRI2 is N-glycosylated at Asn170 and show that this post-translational modification is essential for its expression at the cell surface but not for its proteolytic processing. [ABSTRACT FROM PUBLISHER]

The ℇ4 allele of apolipoprotein E (APOE) is currently the major genetic risk factor identified for Alzheimer's disease (AD). Previous in vivo data from our laboratory has demonstrated that amyloid-β (Aβ) is rapidly removed from the plasma by the liver and kidney and that the rate of its clearance is affected by ApoE in C57BL/6J and APOE-/- mice. To expand upon these findings, we assessed the peripheral clearance of human synthetic Aβ42 in APOE ℇ2, ℇ3, and ℇ4 knock-in and APOE knock-out mice injected with lipidated recombinant apoE2, E3, and E4 protein. Our results show that APOE does influence the rate at which the mice are able to clear Aβ42 from their bloodstream. Both APOE ℇ4 mice and APOE knock-out mice treated with lipidated recombinant apoE4 demonstrated increased retention of plasma Aβ42 over time compared to APOE ℇ2/APOE knock-out rE2 and APOE ℇ3/APOE knock-out rE3 mice. These findings suggest that the peripheral clearance of Aβ42 is significantly altered by APOE genotype. Given that APOE ℇ4 is a risk factor for AD, then these novel findings provide some insight into the role of ApoE isoforms on the peripheral clearance of Aβ which may impact on clearance from the brain. [ABSTRACT FROM AUTHOR]

Patients carrying mutations within the amyloid-β (Aβ) 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 Aβ synthetic homologues containing mutations known to be associated in vivo with hemorrhagic manifestations (AβE22Q and AβL34V) 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 Aβ peptides. Immunoprecipitation followed by mass spectrometry analysis highlighted the generation of specific C-terminal proteolytic fragments, in particular the accumulation of Aβ-(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 Aβ degradation and enhanced apoptosis. Secretion and activation of MMP-2 as well as susceptibility of the Aβ peptides to MMP-2 degradation were dependent on the peptide conformation, with fibrillar elements of AβE22Q exhibiting negligible effects. Our results indicate that MMP-2 release and activation differentially degrades Aβ 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. [ABSTRACT FROM AUTHOR]

It is known that apolipoprotein E (ApoE) is essential for normal lipid metabolism. ApoE is the major apolipoprotein in the central nervous system and plays a key role in neurobiology by mediating the transport of cholesterol, phospholipids, and sulfatides. We therefore examined APOE ℇ2, ℇ3, and ℇ4 knock-in mice, using electrospray ionization mass spectrometry to determine if APOE genotype or age leads to altered levels in the brain of a number of glycerophospholipids (phosphatidylinositol, PI; phosphatidylethanolamine, PE; phosphatidic acid, PA, phosphatidylserine, PS; phosphatidylcholine, PC), sphingolipids (sphingomyelin, SM; ceramide, Cer), cholesterol, and triacylglycerols. We observed slight changes within individual PI, PE, PC, Cer, and SM lipid levels in APOE ℇ2 and ℇ4 mice compared to APOE ℇ3 mice. However, overall, we did not observe any major effects in APOE ℇ4 knock-in mice for the levels of the glycerophospholipids measured, as compared to APOE ℇ2 and ℇ3 mice. Our findings indicate that variations in ApoE isoforms do not per se affect bulk lipid homeostasis in the brain. These findings indicate that APOE ℇ4 is not associated with disturbances in brain sterol or sphingolipids in the absence of environmental factors. [ABSTRACT FROM AUTHOR]

Familial Danish dementia (FDD) is a progressive neurodegenerative disease with cerebral deposition of Dan-amyloid (ADan), neuro-inflammation, and neurofibrillary tangles, hallmark characteristics remarkably similar to those in Alzheimer's disease (AD). We have generated transgenic (tg) mouse models of familial Danish dementia that exhibit the age-dependent deposition of ADan throughout the brain with associated amyloid angiopathy, microhemorrhage, neuritic dystrophy, and neuroinflammation. Tg mice are impaired in the Morris water maze and exhibit increased anxiety in the open field. When crossed with TauP301S tg mice, ADan accumulation promotes neurofibrillary lesions, in all aspects similar to the Tau lesions observed in crosses between β-amyIoid (Aβ)-depositing tg mice and TauP301S tg mice. Although these observations argue for shared mechanisms of downstream pathophysiology for the sequence-unrelated ADan and AD peptides, the lack of codeposition of the two peptides in crosses between ADan- and An-depositing mice points also to distinguishing properties of the peptides. Our results support the concept of the amyloid hypothesis for AD and related dementias, and suggest that different proteins prone to amyloid formation can drive strikingly similar pathogenic pathways in the brain. [ABSTRACT FROM AUTHOR]

Cerebral amyloid diseases are part of a complex group of chronic and progressive entities bracketed together under the common denomination of protein folding disorders and characterized by the intra- and extracellular accumulation of fibrillar aggregates. Of the more than 25 unrelated proteins known to produce amyloidosis in humans only about a third of them are associated with cerebral deposits translating in cognitive deficits, dementia, stroke, cerebellar and extrapyramidal signs, or a combination thereof. The familial forms reviewed herein, although infrequent, provide unique paradigms to examine the role of amyloid in the mechanism of disease pathogenesis and to dissect the link between vascular and parenchymal amyloid deposition and their differential contribution to neurodegeneration. [ABSTRACT FROM AUTHOR]

Radiolabeled Pittsburgh compound B (PIB) is a benzothiazole imaging agent that usually binds with high affinity, specificity, and stoichiometry to cerebral β-amyloid (Aβ) in patients with Alzheimer’s disease. Among a cohort of ten AD subjects examined postmortem, we describe a case of idiopathic, end-stage Alzheimer’s disease with heavy Aβ deposition yet substantially diminished high-affinity binding of 3H-PIB to cortical homogenates and unfixed cryosections. Cortical tissue samples were analyzed by immunohistochemistry, electron microscopy, ELISA, immunoblotting, MALDI-TOF mass spectrometry, in vitro 3H-PIB binding and 3H-PIB autoradiography. The PIB-refractory subject met the histopathological criteria for AD. However, cortical tissue from this case contained more vascular β-amyloidosis, higher levels of insoluble Aβ40 and Aβ42, and a higher ratio of Aβ40:Aβ42 than did tissue from the nine comparison AD cases. Furthermore, cerebral Aβ from the PIB-refractory subject displayed an unusual distribution of low- and high-molecular weight Aβ oligomers, as well as a distinct pattern of N- and C-terminally truncated Aβ peptides in both the soluble and insoluble cortical extracts. Genetically, the patient was apolipoprotein-E3/4 heterozygous, and exhibited no known AD-associated mutations in the genes for the β-amyloid precursor protein, presenilin1 or presenilin2. Our findings suggest that PIB may differentially recognize polymorphic forms of multimeric Aβ in humans with Alzheimer’s disease. In addition, while the prevalence of PIB-refractory cases in the general AD population remains to be determined, the paucity of high-affinity binding sites in this AD case cautions that minimal PIB retention in positron-emission tomography scans of demented patients may not always rule out the presence of Alzheimer-type Aβ pathology. [ABSTRACT FROM AUTHOR]

In cerebral amyloid angiopathy (CAA), amyloid fibrils deposit in walls of arteries, arterioles and less frequently in veins and capillaries of the central nervous system, often resulting in secondary degenerative vascular changes. Although the amyloid-β peptide is by far the commonest amyloid subunit implicated in sporadic and rarely in hereditary forms of CAA, a number of other proteins may also be involved in rare familial diseases in which CAA is also a characteristic morphological feature. These latter proteins include the ABri and ADan subunits in familial British dementia and familial Danish dementia, respectively, which are also known under the umbrella term BRI2 gene-related dementias, variant cystatin C in hereditary cerebral haemorrhage with amyloidosis of Icelandic-type, variant transthyretins in meningo-vascular amyloidosis, disease-associated prion protein (PrPSc) in hereditary prion disease with premature stop codon mutations and mutated gelsolin (AGel) in familial amyloidosis of Finnish type. In this review, the characteristic morphological features of the different CAAs is described and the implication of the biochemical, genetic and transgenic animal data for the pathogenesis of CAA is discussed. [ABSTRACT FROM AUTHOR]

Abstract: Single point mutations of the amyloid precursor protein generate Aβ variants bearing amino acid substitutions at positions 21–23. These mutants are associated with distinct hereditary phenotypes of cerebral amyloid angiopathy, manifesting varying degrees of tropism for brain vessels, and impaired microvessel remodeling and angiogenesis. We examined the differential effects of E22Q (Dutch), and E22G (Arctic) variants in comparison to WT Aβ on brain endothelial cell proliferation, angiogenic phenotype expression triggered by fibroblast growth factor (FGF-2), pseudo-capillary sprouting, and induction of apoptosis. E22Q exhibited a potent anti-angiogenic profile in contrast to E22G, which had a much weaker effect. Investigations on the FGF-2 signaling pathway revealed the greatest differences among the peptides: E22Q and WT peptides suppressed FGF-2 expression while E22G had barely any effect. Phosphorylation of the FGF-2 receptor, FGFR-1, and the survival signal Akt were abolished by E22Q and WT peptides, but not by E22G. The biological dissimilar effect of the mutant and WT peptides on cerebral EC cannot be assigned to a particular Aβ structure, suggesting that the toxic effect of the Aβ assemblies goes beyond mere multimerization. [Copyright &y& Elsevier]

Abstract: Two hereditary forms of cerebrovascular amyloidosis, familial British and Danish dementias (FBD and FDD), share striking similarities with Alzheimer''s disease (AD) despite structural differences among their amyloid subunits (ABri in FBD, ADan in FDD, and Aβ in AD). Neuropathological lesions in these disorders include neurofibrillary tangles, parenchymal amyloid and pre-amyloid deposits and overwhelming cerebral amyloid angiopathy co-localizing with reactive microglia and multiple amyloid associated proteins including activation products of the complement cascade. Immunohistochemical analysis of FBD and FDD brain lesions unveiled the presence of serum amyloid P-component (SAP) primarily associated with thioflavin positive amyloid deposits in spite of the significant pre-amyloid burden existing in both disorders. Using affinity chromatography and ELISA binding assays we demonstrated specific, calcium-dependent, saturable, high affinity binding interactions between SAP and ABri/ADan peptides, with dissociation constant values in the sub-nanomolar range and within the same order of magnitude as those resulting from the interaction of SAP with Alzheimer''s Aβ1-40 and Aβ1-42. The preferential association of SAP with fibrillar amyloid lesions and not with non-fibrillar pre-amyloid deposits is puzzling, suggesting that SAP modulates the assembly and stability of the final fibril rather than participating in the early steps of protein misfolding and oligomerization. [Copyright &y& Elsevier]

Protein aggregation is an established pathogenic mechanism in Alzheimer's disease, but little is known about the initiation of this process in vivo. Intracerebral injection of dilute, amyloid-β (Aβ)-containing brain extracts from humans with Alzheimer's disease or β-amyloid precursor protein (APP) transgenic mice induced cerebral β-amyloidosis and associated pathology in APP transgenic mice in a time- and concentration-dependent manner. The seeding activity of brain extracts was reduced or abolished by Aβ immunodepletion, protein denaturation, or by Aβ immunization of the host. The phenotype of the exogenously induced amyloidosis depended on both the host and the source of the agent, suggesting the existence of polymorphic Aβ strains with varying biological activities reminiscent of prion strains. [ABSTRACT FROM AUTHOR]

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 β-amyloid (Aβ)), 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 Aβ-(1–42) and Aβ-(4–42) in ∼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 Aβ was mainly Aβ-(4–42). In all cases, the presence of Aβ-(X-40) was negligible, a surprising finding in view of the prevalence of Aβ40 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 Aβ molecules was demonstrated in vitro by ligand blot analysis using synthetic peptides. The absence of compact plaques in the presence of extensive neurofibrillar degeneration strongly suggests that compact plaques, fundamental lesions for the diagnosis of AD, are not essential for the mechanism of dementia. [ABSTRACT FROM AUTHOR]

Transmembrane proteins BRI2 and amyloid precursor protein (APP) co-localize with amyloid β (Aβ) lesions in sporadic Alzheimer disease and mutations in both precursor proteins are linked to early-onset familial cases of cerebral amyloidosis associated with dementia and/or cerebral hemorrhage. A specific interaction between BRI2 and APP was unveiled by immunoprecipitation experiments using transfected and non-transfected cells. The use of deletion mutants further revealed that stretches 648–719 of APP751 and 46–106 of BRI2, both inclusive of the full transmembrane domains, are sufficient for the interaction. Removal of most of the APP and BRI2 extracellular domains without affecting the interaction implies that both proteins interact when are expressed on the same cell membrane (cis) rather than on adjacent cells (trans). The presence of BRI2 had a modulatory effect on APP processing, specifically increasing the levels of cellular APP as well as β-secretase-generated COOH-terminal fragments while decreasing the levels of a-secretase-generated COOH-terminal fragments as well as the secretion of total APP and Aβ peptides. Determining the precise molecular pathways affected by the specific binding between APP and BRI2 could result in the identification of common therapeutic targets for these sporadic and familial neurodegenerative disorders. [ABSTRACT FROM AUTHOR]

Alzheimer disease (AD), the most common senile dementia, is characterized by amyloid plaques, vascular amyloid, neurofibrillary tangles, and progressive neurodegeneration. Amyloid is mainly composed by amyloid-β (Aβ) peptides, which are derive from processing of the β-amyloid precursor protein (APP), better named amyloid-β precursor protein (AβPP), by secretases. The AβPP intracellular domain (AID), which is released together with Aβ, has signaling function, since it modulates apoptosis and transcription. Despite its biological and pathological importance, the mechanisms regulating AβPP processing are poorly understood. As cleavage of other γ-secretase substrates is regulated by membrane bound proteins, we have postulated the existence of integral membrane proteins that bind AβPP and regulate its processing. Here, we show that BRI2, a type II membrane protein, interacts with AβPP. Interestingly, 17 amino acids corresponding to the NH2-terminal portion of Aβ are necessary for this interaction. Moreover, BRI2 expression regulates AβPP processing resulting in reduced Aβ and AID levels. Altogether, these findings characterize the BRI2-AβPP interaction as a regulatory mechanism of AβPP processing that inhibits Aβ production. Notably, BRI2 mutations cause familial British (FBD) and Danish dementias (FDD) that are clinically and pathologically similar to AD. Finding that BRI2 pathogenic mutations alter the regulatory function of BRI2 on AβPP processing would define dysregulation of AβPP cleavage as a pathogenic mechanism common to AD, FDD, and FBD. [ABSTRACT FROM AUTHOR]

Protein conformational diseases, including Alzheimer's, Hunting- ton's, and Parkinson's diseases, result from protein misfolding. giving a distinct fibrillar feature termed amyloid. Recent studies show that only the globular (not fibrillar) conformation of amyloid proteins is sufficient to induce cellular pathophysiology. However, the 3D structural conformations of these globular structures, a key missing link in designing effective prevention and treatment remain undefined as of yet. By using atomic force microscopy, circular dichroism, gel electrophoresis, and electrophysiological recordings, we show here that an array of amyloid molecules, including amyloid-β(1-40), α-synuclein, ABri, ADan, serum amyloid A, and amylin undergo supramolecular conformational change. In reconstituted membranes, they form morphologically compatible ion-channel-like structures and elicit single ion-channel currents. These ion channels would destabilize cellular ionic homeostasis and hence induce cell pathophysiology and degeneration in amyloid diseases. [ABSTRACT FROM AUTHOR]

Abstract: 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 β-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. [Copyright &y& Elsevier]

Mechanisms for the regulation of intracellular cholesterol levels in various types of brain and vascular cells are of considerable importance in our understanding of the pathogenesis of a variety of diseases, particularly atherosclerosis and Alzheimer’s disease (AD). It is increasingly clear that conversion of brain cholesterol into 24-hydroxycholesterol and its subsequent release into the periphery is important for the maintenance of brain cholesterol homeostasis. Recent studies have shown elevated plasma concentrations of 24-hydroxycholesterol in patients with AD and vascular dementia, suggesting increased brain cholesterol turnover during neurodegeneration. The oxygenases involved in the degradation and excretion of cholesterol, including the cholesterol 24-hydroxylase and the 27-hydroxylase, are enzymes of the cytochrome P-450 family. This review focuses on the newly recognized importance of cholesterol and its oxygenated metabolites in the pathogenesis of ischemic stroke and AD. The reduction in stroke and AD risk in patients treated with cholesterol-lowering statins is also discussed. [Copyright &y& Elsevier]