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

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

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

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

Microglia produce the amyloidogenic ABri peptide in familial British dementia.

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

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

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

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

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

Pathogenesis of Alzheimer's Disease.

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

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

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

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

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

Alzheimer's amyloid ß heterogeneous species differentially affect brain endothelial cell viability, blood-brain barrier integrity, and angiogenesis.

Impaired clearance in the Alzheimer's Disease (AD) brain is key in the formation of Aß parenchymal plaques and cerebrovascular deposits known as cerebral amyloid angiopathy (CAA), present in >80% of AD patients and ~50% of non-AD elderly subjects. Aß deposits are highly heterogeneous, containing multiple fragments mostly derived from catabolism of Aß40/Aß42, which exhibit dissimilar aggregation properties. Remarkably, the role of these physiologically relevant Aß species in cerebrovascular injury and their impact in vascular pathology is unknown. We sought to understand how heterogeneous Aß 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 Aß 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 Aß species tested inhibited angiogenesis in vitro. These data link the biological effects of the heterogeneous Aß 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.

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

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

Correction to: Nrf2 activation through the PI3K/GSK-3 axisprotects neuronal cells from Aß-mediatedoxidative and metabolic damage.

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

Nrf2 activation through the PI3K/GSK-3 axis protects neuronal cells from Aß-mediated oxidative and metabolic damage.

BACKGROUND: Mounting evidence points to a crucial role of amyloid-ß (Aß) 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 Aß, 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 oligAß in SH-SY5Y and primary neurons testing their pharmacologic protection by small molecules. RESULTS: Our findings indicate that oligomeric Aß induces a dramatic increase in ROS production and severely affects neuronal metabolism and bioenergetics. Assessment of global energy metabolism in real time demonstrated Aß-mediated reduction in oxygen consumption affecting basal and maximal respiration and causing decreased ATP production. Pharmacologic targeting of Aß-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 Aß protective action. CONCLUSIONS: Our study provides insights into the complex molecular mechanisms triggered by oligAß 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.

Unveiling Brain Aß Heterogeneity Through Targeted Proteomic Analysis.

Amyloid ß (Aß) is the major constituent of the brain deposits found in parenchymal plaques and cerebral blood vessels of patients with Alzheimer's disease (AD). Besides classic full-length peptides, biochemical analyses of brain deposits have revealed high degree of Aß heterogeneity likely resulting from the action of multiple proteolytic enzymes. This chapter describes a sequential extraction protocol allowing the differential fractionation of soluble and deposited Aß species taking advantage of their differential solubility properties. Soluble Aß is extracted by water-based buffers like phosphate-buffered saline-PBS-whereas pre-fibrillar and fibrillar deposits, usually poorly soluble in PBS, are extractable in detergent containing solutions or more stringent conditions as formic acid. The extraction procedure is followed by the biochemical identification of the extracted Aß species using Western blot and a targeted proteomic analysis which combines immunoprecipitation with MALDI-ToF mass spectrometry. This approach revealed the presence of numerous C- and N-terminal truncated Aß species in addition to Aß1-40/42. Notably, the more soluble C-terminal cleaved fragments constitute a main part of PBS homogenates. On the contrary, N-terminal truncated species typically require more stringent conditions for the extraction in agreement with their lower solubility and enhanced aggregability. Detailed assessment of the molecular diversity of Aß species composing interstitial fluid and amyloid deposits at different disease stages, as well as the evaluation of the truncation profile during various pharmacologic approaches will provide a comprehensive understanding of the still undefined contribution of Aß truncations to AD pathogenesis and their potential as novel therapeutic targets.

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

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

Proteomic Analysis Shows Constitutive Secretion of MIF and p53-associated Activity of COX-2-/- Lung Fibroblasts.

The differential expression of two closelyassociated cyclooxygenase isozymes, COX-1 and COX-2, exhibited functions beyond eicosanoid metabolism. We hypothesized that COX-1 or COX-2 knockout lung fibroblasts may display altered protein profiles which may allow us to further differentiate the functional roles of these isozymes at the molecular level. Proteomic analysis shows constitutive production of macrophage migration inhibitory factor (MIF) in lung fibroblasts derived from COX-2-/- but not wild-type (WT) or COX-1-/- mice. MIF was spontaneously released in high levels into the extracellular milieu of COX2-/- fibroblasts seemingly from the preformed intracellular stores, with no change in the basal gene expression of MIF. The secretion and regulation of MIF in COX-2-/- was "prostaglandin-independent." GO analysis showed that concurrent with upregulation of MIF, there is a significant surge in expression of genes related to fibroblast growth, FK506 binding proteins, and isomerase activity in COX-2-/- cells. Furthermore, COX-2-/- fibroblasts also exhibit a significant increase in transcriptional activity of various regulators, antagonists, and co-modulators of p53, as well as in the expression of oncogenes and related transcripts. Integrative Oncogenomics Cancer Browser (IntroGen) analysis shows downregulation of COX-2 and amplification of MIF and/or p53 activity during development of glioblastomas, ependymoma, and colon adenomas. These data indicate the functional role of the MIF-COX-p53 axis in inflammation and cancer at the genomic and proteomic levels in COX-2-ablated cells. This systematic analysis not only shows the proinflammatory state but also unveils a molecular signature of a pro-oncogenic state of COX-1 in COX-2 ablated cells.

In vivo Differential Brain Clearance and Catabolism of Monomeric and Oligomeric Alzheimer's Aß protein.

Amyloid ß (Aß) 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 Aß 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 Aß influencing the delicate balance between rate of synthesis, dynamics of aggregation, and clearance kinetics. Emerging new data suggest that reduced Aß 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 Aß1-40 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 Aß 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 Aß efflux. Immunofluorescence confocal microscopy studies provide insight into the cellular pathways involved in the brain removal and cellular uptake of Aß. 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.

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

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

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

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