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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]

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]

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]

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]

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]

Amyloid β (Aβ) is the major constituent of the brain deposits found in parenchymal plaques and cerebral blood vessels of patients with Alzheimer's disease (AD). Several lines of investigation support the notion that synaptic pathology, one of the strongest correlates to cognitive impairment, is related to the progressive accumulation of neurotoxic Aβ oligomers. Since the process of oligomerization/fibrillization is concentration-dependent, it is highly reliant on the homeostatic mechanisms that regulate the steady state levels of Aβ influencing the delicate balance between rate of synthesis, dynamics of aggregation, and clearance kinetics. Emerging new data suggest that reduced Aβ clearance, particularly in the aging brain, plays a critical role in the process of amyloid formation and AD pathogenesis. Using well-defined monomeric and low molecular mass oligomeric Aβ 1-40 species stereotaxically injected into the brain of C57BL/6 wild-type mice in combination with biochemical and mass spectrometric analyses in CSF, our data clearly demonstrate that Aβ physiologic removal is extremely fast and involves local proteolytic degradation leading to the generation of heterogeneous C-terminally cleaved proteolytic products, while providing clear indication of the detrimental role of oligomerization for brain Aβ efflux. Immunofluorescence confocal microscopy studies provide insight into the cellular pathways involved in the brain removal and cellular uptake of Aβ. The findings indicate that clearance from brain interstitial fluid follows local and systemic paths and that in addition to the blood-brain barrier, local enzymatic degradation and the bulk flow transport through the choroid plexus into the CSF play significant roles. Our studies highlight the diverse factors influencing brain clearance and the participation of various routes of elimination opening up new research opportunities for the understanding of altered mechanisms triggering AD pathology and for the potential design of combined therapeutic strategies. [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]

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]

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]

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]

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]

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

Amyloid-β peptide (Aβ) interacts with the vasculature to influence Aβ levels in the brain and cerebral blood flow, providing a means of amplifying the Aβ-induced cellular stress underlying neuronal dysfunction and dementia. Systemic Aβ infusion and studies in genetically manipulated mice show that Aβ interaction with receptor for advanced glycation end products (RAGE)-bearing cells in the vessel wall results in transport of Aβ across the blood-brain barrier (BBB) and expression of proinflammatory cytokines and endothelin-1 (ET-1), the latter mediating Aβ-induced vasoconstriction. Inhibition of RAGE-ligand interaction suppresses accumulation of Aβ in brain parenchyma in a mouse transgenic model. These findings suggest that vascular RAGE is a target for inhibiting pathogenic consequences of Aβ-vascular interactions, including development of cerebral amyloidosis. [ABSTRACT FROM AUTHOR]

Abstract Increased levels of a 40–42 amino-acid peptide called the amyloid β protein (Aβ) and evidence of oxidative damage are early neuropathological markers of Alzheimer's disease (AD). Previous investigations have demonstrated that melatonin is decreased during the aging process and that patients with AD have more profound reductions of this hormone. It has also been recently shown that melatonin protects neuronal cells from Aβ-mediated oxidative damage and inhibits the formation of amyloid fibrils in vitro. However, a direct relationship between melatonin and the biochemical pathology of AD had not been demonstrated. We used a transgenic mouse model of Alzheimer's amyloidosis and monitored over time the effects of administering melatonin on brain levels of Aβ, abnormal protein nitration, and survival of the mice. We report here that administration of melatonin partially inhibited the expected time-dependent elevation of β-amyloid, reduced abnormal nitration of proteins, and increased survival in the treated transgenic mice. These findings may bear relevance to the pathogenesis and therapy of AD. [ABSTRACT FROM AUTHOR]

Tumoral monoclonal immunoglobulin (Ig) light chain non-fibrillar deposits ('aggregomas'), which can be considered analogous to solitary light chain amyloidomas, are a rare presenting feature of B-cell dyscrasias. It is not certain if they are truly localized or if in reality they represent an initial expression of a silent systemic non-amyloid light chain deposition disease (LCDD). This report describes three patients, two of whom presented with cervical masses and the third with a solitary lung nodule, each comprising granular aggregates of monoclonal kappa light chain. Extracted deposits from the lymph node of one patient were shown by N-terminal amino acid sequence analysis to belong to the variable-region kappa I (Vκ I) light chain subgroup, the first reported κ-LCDD protein encoded by the L9 gene and the first report of an expressed protein related to this gene. Extracted deposits from the lung nodule of the second patient belonged to the Vκ IV light chain subgroup encoded by the B3 germ line gene. The N-terminal amino acid sequences of the light chains from the aggregomas were compared with the related germ line sequences and to the N-terminal amino acid sequences of the nine other known κ-LCDD light chains reported thus far from patients with systemic LCDD. [ABSTRACT FROM AUTHOR]

Squirrel monkey is a valuable model to study pathogenesis of cerebrovascular amyloid angiopathy (CAA). Previous studies suggested that circulating amyloid-β40 peptide (Aβ40) crosses the blood-brain barrier (BBB) and may therefore enhance cerebrovascular amyloidosis in aged squirrel monkeys. In the present study, we used single photon emission computed tomography (SPECT) to determine elimination of 123 I-Aβ40 and 99m Tc-DTPA, an extracellular marker, from the brain in squirrel monkeys at different age. Following intracerebral microinfusions, the time-activity brain clearance curves indicated bi-exponential removal of 123 I-Aβ40 with an initial rapid washout (1.1≤ t 1/2 ≤2.7 h). This, plus the observed appearance of 123 I-radioactivity in plasma suggest significant brain-to-blood transport. In contrast, 99m Tc-DTPA was removed slowly by brain interstitial fluid bulk flow (monoexponential decay with 6.8≤ t 1/2 ≤16.8 h ). A comparison of three middle aged (11-16 years old) vs. two old (22 yrs old) monkeys was consistent with an age-related decline in the BBB capacity to remove 123 I-Aβ from the brain. This correlated with an age-dependent increase in Aβ40/42 cerebrovascular immunoreactivity and amyloid deposition. Thus, vascular clearance plays an important role in reducing Aβ levels in the squirrel monkey brain and impaired Aβ40 elimination across the BBB may contribute to the development of CAA. [ABSTRACT FROM AUTHOR]

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]

Amyloid β protein deposition in cortical and leptomeningeal vessels, causing the most common type of cerebral amyloid angiopathy, is found in sporadic and familial Alzheimer’s disease (AD) and is the principal feature in the hereditary cerebral hemorrhage with amyloidosis, Dutch type. The presence of the Apolipoprotein E (APOE)-ℇ4 allele has been implicated as a risk factor for AD and the development of cerebral amyloid angiopathy in AD. We report clinical, pathological and biochemical studies on two APOE-ℇ4 homozygous subjects, who had senile dementia and whose main neuropathological feature was a severe and diffuse amyloid angiopathy associated with perivascular tau neurofibrillary pathology. Amyloid β protein and ApoE immunoreactivity were observed in leptomeningeal vessels as well as in medium-sized and small vessels and capillaries in the parenchyma of the neocortex, hippocampus, thalamus, cerebellum, midbrain, pons, and medulla. The predominant peptide form of amyloid β protein was that terminating at residue Val40, as determined by immunohistochemistry, amino acid sequence and mass spectrometry analysis. A crown of tau-immunopositive cell processes was consistently present around blood vessels. DNA sequence analysis of the Amyloid Precursor Protein gene and Presenilin-1 (PS-1) gene revealed no mutations. In these APOE-ℇ4 homozygous patients, the pathological process differed from that typically seen in AD in that they showed a heavy burden of perivascular tau-immunopositive cell processes associated with severe amyloid β protein angiopathy, neurofibrillary tangles, some cortical Lewy bodies and an absence of neuritic plaques. These cases emphasize the concept that tau deposits may be pathogenetically related to amyloid β protein deposition. [ABSTRACT FROM AUTHOR]

Disorders of immunoglobulin (Ig) synthesis that occur in malignant plasma-cell proliferation may result in either granular (LCDD) or fibrillar (AL) tissue deposition of light-chain monoclonal components. The structural features that govern the transition from soluble polypeptides to either fibrillar or granular conformational states remain undefined. Among the many factors presumed to play a role in these transitions the net charge of the molecule has been associated with folding conformation changes. The majority of the proteins involved in AL amyloidosis show acidic isoelectric points (pI 3.8–5.2), whereas most L chains with basic pIs deposit in granular patterns. In our studies a 12 kD VκIII fragment was purified as the main component of the fibrils isolated from myocardium and adipose tissue of the pericardium obtained post-mortem from an individual with systemic AL amyloidosis. An apparently identical 12 kD VL fragment with the same N-terminal sequence constituted the BJ protein present in the urine. This urinary protein exhibited strikingly cathodic electrophoretic mobility on agarose gels and lacked retention by anionic exchange chromatography matrices, indicative of a highly basic pI (>10). When it was subjected to in vitro fibril-formation experiments, the BJ protein adopted a fibrillar conformation only at acidic pHs, remaining aggregated but not fibrillar at physiological pH. The data indicate that a specific tissue deposition pattern involves not only structural properties of the protein but rather more complex mechanisms in which acidic micro-environments may contribute to the stabilization of amyloidogenic conformations. [ABSTRACT FROM AUTHOR]

Presents research which identified a 4K protein subunit named ABri from isolated amyloid fibrils associated with familial British dementia (FBD). Symptoms of FBD; FBD's common pathological lesions; Encoding of ABri by the gene BRI; Effects of mutating BRI; Impact of the release of the 34 carboxy-terminal amino acids from the mutated gene; Behavior of antibodies against the amyloid or homologous synthetic peptides; Generation of the ABri peptide.

The carboxy-terminal ends of the 40- and 42-amino acids amyloid β-protein (Aβ) may be generated by the action of at least two different proteases termed γ(40)- and γ(42)-secretase, respectively. To examine the cleavage specificity of the two proteases, we treated amyloid precursor protein (APP)-transfected cell cultures with several dipeptidyl aldehydes including N-benzyloxycarbonyl-Leu-leucinal (Z-LL-CHO) and the newly synthesized N-benzyloxycarbonyl-Val-leucinal (Z-VL-CHO). All dipeptidyl aldehydes tested inhibited production of both Aβ1-40 and Aβ1-42. Changes in the P1 and P2 residues of these aldehydes, however, indicated that the amino acids occupying these positions are important for the efficient inhibition of γ-secretases. Peptidyl aldehydes inhibit both cysteine and serine proteases, suggesting that the two γ-secretases belong to one of these mechanistic classes. To differentiate between the two classes of proteases, we treated our cultures with the specific cysteine protease inhibitor E-64d. This agent inhibited production of secreted Aβ1-40, with a concomitant accumulation of its cellular precursor indicating that γ(40)-secretase is a cysteine protease. In contrast, this treatment increased production of secreted Aβ1-42. No inhibition of Aβ production was observed with the potent calpain inhibitor I (acetyl-Leu-Leu-norleucinal), suggesting that calpain is not involved. Together, these results indicate that γ(40)-secretase is a cysteine protease distinct from calpain, whereas γ(42)-secretase may be a serine protease. In addition, the two secretases may compete for the same substrate. Dipeptidyl aldehyde treatment of cultures transfected with APP carrying the Swedish mutation resulted in the accumulation of the β-secretase C-terminal APP fragment and a decrease of the α-secretase C-terminal APP fragment, indicating that this mutation shifts APP cleavage from the α-secretase site to the β-secretase site. [ABSTRACT FROM AUTHOR]

Amyloidosis is a generic term for a group of clinically and biochemically diverse diseases that are characterized by the deposition of an insoluble fibrillar protein in the extracellular space. Over 16 biochemically distinct amyloids are known. Despite this diversity, all amyloids have a particular ultrastructural and tinctorial appearance, a β-pleated sheet structure, and are codeposited with a group of amyloid-associated proteins. The most common amyloidosis is Alzheimer's disease (AD), where Aβ is the main component of the amyloid. Recently it has been found that Aβ exists as a normal soluble protein (sAβ) in biological fluids. This links AD more closely to some of the systemic amyloidoses, where the amyloid precursor is found in the circulation normally. Numerous mutations have been found in the Aβ precursor (βPP) gene, associated with familial AD. Many mutations are also found in some of the hereditary systemic amyloidoses. For example, over 40 mutations in the transthyretin (TTR) gene are associated with amyloid. However, both Aβ and TTR related amyloid deposition can occur with no mutation. The pathogenesis of amyloid is complex, and appears to be associated with genetic and environmental risk factors that can be similar in the systemic and cerebral amyloidoses. [ABSTRACT FROM AUTHOR]