Iron-export ferroxidase activity of ß-amyloid precursor protein is inhibited by zinc in Alzheimer's disease.

Alzheimer's Disease (AD) is complicated by pro-oxidant intraneuronal Fe(2+) elevation as well as extracellular Zn(2+) accumulation within amyloid plaque. We found that the AD ß-amyloid protein precursor (APP) possesses ferroxidase activity mediated by a conserved H-ferritin-like active site, which is inhibited specifically by Zn(2+). Like ceruloplasmin, APP catalytically oxidizes Fe(2+), loads Fe(3+) into transferrin, and has a major interaction with ferroportin in HEK293T cells (that lack ceruloplasmin) and in human cortical tissue. Ablation of APP in HEK293T cells and primary neurons induces marked iron retention, whereas increasing APP695 promotes iron export. Unlike normal mice, APP(-/-) mice are vulnerable to dietary iron exposure, which causes Fe(2+) accumulation and oxidative stress in cortical neurons. Paralleling iron accumulation, APP ferroxidase activity in AD postmortem neocortex is inhibited by endogenous Zn(2+), which we demonstrate can originate from Zn(2+)-laden amyloid aggregates and correlates with Aß burden. Abnormal exchange of cortical zinc may link amyloid pathology with neuronal iron accumulation in AD.

Quantitative proteomics of tau and Aß in detergent fractions from Alzheimer's disease brains.

The two hallmarks of Alzheimer's disease (AD) are amyloid-ß (Aß) plaques and neurofibrillary tangles marked by phosphorylated tau. Increasing evidence suggests that aggregating Aß drives tau accumulation, a process that involves synaptic degeneration leading to cognitive impairment. Conversely, there is a realization that non-fibrillar (oligomeric) forms of Aß mediate toxicity in AD. Fibrillar (filamentous) aggregates of proteins across the spectrum of the primary and secondary tauopathies were the focus of recent structural studies with a filament structure-based nosologic classification, but less emphasis was given to non-filamentous co-aggregates of insoluble proteins in the fractions derived from post-mortem human brains. Here, we revisited sarkosyl-soluble and -insoluble extracts to characterize tau and Aß species by quantitative targeted mass spectrometric proteomics, biochemical assays, and electron microscopy. AD brain sarkosyl-insoluble pellets were greatly enriched with Aß42 at almost equimolar levels to N-terminal truncated microtubule-binding region (MTBR) isoforms of tau with multiple site-specific post-translational modifications (PTMs). MTBR R3 and R4 tau peptides were most abundant in the sarkosyl-insoluble materials with a 10-fold higher concentration than N-terminal tau peptides. This indicates that the major proportion of the enriched tau was the aggregation-prone N-terminal and proline-rich region (PRR) of truncated mixed 4R and 3R tau with more 4R than 3R isoforms. High concentration and occupancies of site-specific phosphorylation pT181 (~22%) and pT217 (~16%) (key biomarkers of AD) along with other PTMs in the PRR and MTBR indicated a regional susceptibility of PTMs in aggregated tau. Immunogold labelling revealed that tau may exist in globular non-filamentous form (N-terminal intact tau) co-localized with Aß in the sarkosyl-insoluble pellets along with tau filaments (N-truncated MTBR tau). Our results suggest a model that Aß and tau interact forming globular aggregates, from which filamentous tau and Aß emerge. These characterizations contribute towards unravelling the sequence of events which lead to end-stage AD changes.

Do current therapeutic anti-Aß antibodies for Alzheimer's disease engage the target?

Reducing amyloid-ß peptide (Aß) burden at the pre-symptomatic stages of Alzheimer's disease (AD) is currently the advocated clinical strategy for treating this disease. The most developed method for targeting Aß is the use of monoclonal antibodies including bapineuzumab, solanezumab and crenezumab. We have synthesized these antibodies and used surface plasmon resonance (SPR) and mass spectrometry to characterize and compare the ability of these antibodies to target Aß in transgenic mouse tissue as well as human AD tissue. SPR analysis showed that the antibodies were able to bind Aß with high affinity. All of the antibodies were able to bind Aß in mouse tissue. However, significant differences were observed in human brain tissue. While bapineuzumab was able to capture a variety of N-terminally truncated Aß species, the Aß detected using solanezumab was barely above detection limits while crenezumab did not detect any Aß. None of the antibodies were able to detect any Aß species in human blood. Immunoprecipitation experiments using plasma from AD subjects showed that both solanezumab and crenezumab have extensive cross-reactivity with non-Aß related proteins. Bapineuzumab demonstrated target engagement with brain Aß, consistent with published clinical data. Solanezumab and crenezumab did not, most likely as a result of a lack of specificity due to cross-reactivity with other proteins containing epitope overlap. This lack of target engagement raises questions as to whether solanezumab and crenezumab are suitable drug candidates for the preventative clinical trials for AD.

Changes in plasma amyloid beta in a longitudinal study of aging and Alzheimer's disease.

BACKGROUND: A practical biomarker is required to facilitate the preclinical diagnosis of Alzheimer's disease (AD). METHODS: Plasma amyloid beta (Aß)1-40, Aß1-42, Aßn-40, and Aßn-42 peptides were measured at baseline and after 18 months in 771 participants from the Australian Imaging Biomarkers and Lifestyle (AIBL) study of aging. Aß peptide levels were compared with clinical pathology, neuroimaging and neuropsychological measurements. RESULTS: Although inflammatory and renal function covariates influenced plasma Aß levels significantly, a decrease in Aß1-42/Aß1-40 was observed in patients with AD, and was also inversely correlated with neocortical amyloid burden. During the 18 months, plasma Aß1-42 decreased in subjects with mild cognitive impairment (MCI) and in those transitioning from healthy to MCI. CONCLUSION: Our findings are consistent with a number of published plasma Aß studies and, although the prognostic value of individual measures in any given subject is limited, the diagnostic contribution of plasma Aß may demonstrate utility when combined with a panel of peripheral biomarkers.

Oligomers, fact or artefact? SDS-PAGE induces dimerization of ß-amyloid in human brain samples.

The formation of low-order oligomers of ß-amyloid (Aß) within the brain is widely believed to be a central component of Alzheimer's disease (AD) pathogenesis. However, despite advances in high-throughput and high-resolution techniques such as xMAP and mass spectrometry (MS), investigations into these oligomeric species have remained reliant on low-resolution Western blots and enzyme-linked immunosorbent assays. The current investigation compared Aß profiles within human cortical tissue using sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis (PAGE), xMAP and surface enhanced laser desorption/ionization time-of-flight MS and found that whilst there was significant correlation across the techniques regarding levels of monomeric Aß, only SDS-PAGE was capable of detecting dimeric isoforms of Aß. The addition of synthetic di-tyrosine cross-linked Aß(1-40)Met(35)(O) to the AD tissue demonstrated that the MS methodology was capable of observing dimeric Aß at femto-molar concentrations, with no noticeable effect on monomeric Aß levels. Focus turned to the association between SDS-PAGE and levels of observable dimeric Aß within the AD brain tissue. These investigations revealed that increased levels of dimeric Aß were observed with increasing concentrations of SDS in the sample buffer. This finding was subsequently confirmed using synthetic Aß(1-42) and suggests that SDS was inducing the formation of dimeric Aß. The findings that SDS promotes Aß dimerization have significant implications for the putative role of low-order oligomers in AD pathogenesis and draw into question the utility of oligomeric Aß as a therapeutic target.

Increasing Cu bioavailability inhibits Abeta oligomers and tau phosphorylation.

Cognitive decline in Alzheimer's disease (AD) involves pathological accumulation of synaptotoxic amyloid-beta (Abeta) oligomers and hyperphosphorylated tau. Because recent evidence indicates that glycogen synthase kinase 3beta (GSK3beta) activity regulates these neurotoxic pathways, we developed an AD therapeutic strategy to target GSK3beta. The strategy involves the use of copper-bis(thiosemicarbazonoto) complexes to increase intracellular copper bioavailability and inhibit GSK3beta through activation of an Akt signaling pathway. Our lead compound Cu(II)(gtsm) significantly inhibited GSK3beta in the brains of APP/PS1 transgenic AD model mice. Cu(II)(gtsm) also decreased the abundance of Abeta trimers and phosphorylated tau, and restored performance of AD mice in the Y-maze test to levels expected for cognitively normal animals. Improvement in the Y-maze correlated directly with decreased Abeta trimer levels. This study demonstrates that increasing intracellular copper bioavailability can restore cognitive function by inhibiting the accumulation of neurotoxic Abeta trimers and phosphorylated tau.

Blood-borne amyloid-beta dimer correlates with clinical markers of Alzheimer's disease.

Alzheimer's disease (AD) is the most common age-related dementia. Unfortunately due to a lack of validated biomarkers definitive diagnosis relies on the histological demonstration of amyloid-beta (Abeta) plaques and tau neurofibrillary tangles. Abeta processing is implicated in AD progression and many therapeutic strategies target various aspects of this biology. While Abeta deposition is the most prominent feature of AD, oligomeric forms of Abeta have been implicated as the toxic species inducing the neuronal dysfunction. Currently there are no methods allowing routine monitoring of levels of such species in living populations. We have used surface enhanced laser desorption ionization time of flight (SELDI-TOF) mass spectrometry incorporating antibody capture to investigate whether the cellular membrane-containing fraction of blood provides a new source of biomarkers. There are significant differences in the mass spectra profiles of AD compared with HC subjects, with significantly higher levels of Abeta monomer and dimer in the blood of AD subjects. Furthermore, levels of these species correlated with clinical markers of AD including brain Abeta burden, cognitive impairment and brain atrophy. These results indicate that fundamental biochemical events relevant to AD can be monitored in blood, and that the species detected may be useful clinical biomarkers for AD.

Platinum-based inhibitors of amyloid-beta as therapeutic agents for Alzheimer's disease.

Amelyoid-beta peptide (Abeta) is a major causative agent responsible for Alzheimer's disease (AD). Abeta contains a high affinity metal binding site that modulates peptide aggregation and toxicity. Therefore, identifying molecules targeting this site represents a valid therapeutic strategy. To test this hypothesis, a range of L-PtCl(2) (L = 1,10-phenanthroline derivatives) complexes were examined and shown to bind to Abeta, inhibit neurotoxicity and rescue Abeta-induced synaptotoxicity in mouse hippocampal slices. Coordination of the complexes to Abeta altered the chemical properties of the peptide inhibiting amyloid formation and the generation of reactive oxygen species. In comparison, the classic anticancer drug cisplatin did not affect any of the biochemical and cellular effects of Abeta. This implies that the planar aromatic 1,10-phenanthroline ligands L confer some specificity for Abeta onto the platinum complexes. The potent effect of the L-PtCl(2) complexes identifies this class of compounds as therapeutic agents for AD.

Quantification of N-terminal amyloid-ß isoforms reveals isomers are the most abundant form of the amyloid-ß peptide in sporadic Alzheimer's disease.

Plaques that characterize Alzheimer's disease accumulate over 20 years as a result of decreased clearance of amyloid-ß peptides. Such long-lived peptides are subjected to multiple post-translational modifications, in particular isomerization. Using liquid chromatography ion mobility separations mass spectrometry, we characterized the most common isomerized amyloid-ß peptides present in the temporal cortex of sporadic Alzheimer's disease brains. Quantitative assessment of amyloid-ß N-terminus revealed that > 80% of aspartates (Asp-1 and Asp-7) in the N-terminus was isomerized, making isomerization the most dominant post-translational modification of amyloid-ß in Alzheimer's disease brain. Total amyloid-ß1-15 was ∼85% isomerized at Asp-1 and/or Asp-7 residues, with only 15% unmodified amyloid-ß1-15 left in Alzheimer's disease. While amyloid-ß4-15 the next most abundant N-terminus found in Alzheimer's disease brain, was only ∼50% isomerized at Asp-7 in Alzheimer's disease. Further investigations into different biochemically defined amyloid-ß-pools indicated a distinct pattern of accumulation of extensively isomerized amyloid-ß in the insoluble fibrillar plaque and membrane-associated pools, while the extent of isomerization was lower in peripheral membrane/vesicular and soluble pools. This pattern correlated with the accumulation of aggregation-prone amyloid-ß42 in Alzheimer's disease brains. Isomerization significantly alters the structure of the amyloid-ß peptide, which not only has implications for its degradation, but also for oligomer assembly, and the binding of therapeutic antibodies that directly target the N-terminus, where these modifications are located.

Citrullination of Amyloid-ß Peptides in Alzheimer's Disease.

Protein citrullination (deimination of arginine residue) is a well-known biomarker of inflammation. Elevated protein citrullination has been shown to colocalize with extracellular amyloid plaques in postmortem AD patient brains. Amyloid-ß (Aß) peptides which aggregate and accumulate in the plaques of Alzheimer's disease (AD) have sequential N-terminal truncations and multiple post-translational modifications (PTM) such as isomerization, pyroglutamate formation, phosphorylation, nitration, and dityrosine cross-linking. However, no conclusive biochemical evidence exists whether citrullinated Aß is present in AD brains. In this study, using high-resolution mass spectrometry, we have identified citrullination of Aß in sporadic and familial AD brains by characterizing the tandem mass spectra of endogenous N-truncated citrullinated Aß peptides. Our quantitative estimations demonstrate that ∼ 35% of pyroglutamate3-Aß pool was citrullinated in plaques in the sporadic AD temporal cortex and ∼ 22% in the detergent-insoluble frontal cortex fractions. Similarly, hypercitrullinated pyroglutamate3-Aß (∼ 30%) was observed in both the detergent-soluble as well as insoluble Aß pool in familial AD cases. Our results indicate that a common mechanism for citrullination of Aß exists in both the sporadic and familial AD. We establish that citrullination of Aß is a remarkably common PTM, closely associated with pyroglutamate3-Aß formation and its accumulation in AD. This may have implications for Aß toxicity, autoantigenicity of Aß, and may be relevant for the design of diagnostic assays and therapeutic targeting.

The Caenorhabditis elegans A beta 1-42 model of Alzheimer disease predominantly expresses A beta 3-42.

Transgenic expression of human amyloid beta (A beta) peptide in body wall muscle cells of Caenorhabditis elegans has been used to better understand aspects of Alzheimer disease (AD). In human aging and AD, A beta undergoes post-translational changes including covalent modifications, truncations, and oligomerization. Amino truncated A beta is increasingly recognized as potentially contributing to AD pathogenesis. Here we describe surface-enhanced laser desorption ionization-time of flight mass spectrometry mass spectrometry of A beta peptide in established transgenic C. elegans lines. Surprisingly, the A beta being expressed is not full-length 1-42 (amino acids) as expected but rather a 3-42 truncation product. In vitro analysis demonstrates that A beta(3-42) self-aggregates like A beta(1-42), but more rapidly, and forms fibrillar structures. Similarly, A beta(3-42) is also the more potent initiator of A beta(1-40) aggregation. Seeded aggregation via A beta(3-42) is further enhanced via co-incubation with the transition metal Cu(II). Although unexpected, the C. elegans model of A beta expression can now be co-opted to study the proteotoxic effects and processing of A beta(3-42).

The structure of dopamine induced alpha-synuclein oligomers.

Inclusions of aggregated alpha-synuclein (alpha-syn) in dopaminergic neurons are a characteristic histological marker of Parkinson's disease (PD). In vitro, alpha-syn in the presence of dopamine (DA) at physiological pH forms SDS-resistant non-amyloidogenic oligomers. We used a combination of biophysical techniques, including sedimentation velocity analysis, small angle X-ray scattering (SAXS) and circular dichroism spectroscopy to study the characteristics of alpha-syn oligomers formed in the presence of DA. Our SAXS data show that the trimers formed by the action of DA on alpha-syn consist of overlapping worm-like monomers, with no end-to-end associations. This lack of structure contrasts with the well-established, extensive beta-sheet structure of the amyloid fibril form of the protein and its pre-fibrillar oligomers. We propose on the basis of these and earlier data that oxidation of the four methionine residues at the C- and N-terminal ends of alpha-syn molecules prevents their end-to-end association and stabilises oligomers formed by cross linking with DA-quinone/DA-melanin, which are formed as a result of the redox process, thus inhibiting formation of the beta-sheet structure found in other pre-fibrillar forms of alpha-syn.

Amyloid-beta peptide (Abeta) neurotoxicity is modulated by the rate of peptide aggregation: Abeta dimers and trimers correlate with neurotoxicity.

Alzheimer's disease is an age-related neurodegenerative disorder with its toxicity linked to the generation of amyloid-beta peptide (Abeta). Within the Abeta sequence, there is a systemic repeat of a GxxxG motif, which theoretical studies have suggested may be involved in both peptide aggregation and membrane perturbation, processes that have been implicated in Abeta toxicity. We synthesized modified Abeta peptides, substituting glycine for leucine residues within the GxxxG repeat motif (GSL peptides). These GSL peptides undergo beta-sheet and fibril formation at an increased rate compared with wild-type Abeta. The accelerated rate of amyloid fibril formation resulted in a decrease in the presence of small soluble oligomers such as dimeric and trimeric forms of Abeta in solution, as detected by mass spectrometry. This reduction in the presence of small soluble oligomers resulted in reduced binding to lipid membranes and attenuated toxicity for the GSL peptides. The potential role that dimer and trimer species binding to lipid plays in Abeta toxicity was further highlighted when it was observed that annexin V, a protein that inhibits Abeta toxicity, specifically inhibited Abeta dimers from binding to lipid membranes.

Restored degradation of the Alzheimer's amyloid-beta peptide by targeting amyloid formation.

Accumulation of neurotoxic amyloid-beta (Abeta) is central to the pathology of Alzheimer's disease (AD). Elucidating the mechanisms of Abeta accumulation will therefore expedite the development of Abeta-targeting AD therapeutics. We examined activity of an Abeta-degrading protease (matrix metalloprotease 2) to investigate whether biochemical factors consistent with conditions in the AD brain contribute to Abeta accumulation by altering Abeta sensitivity to proteolytic degradation. An Abeta amino acid mutation found in familial AD, Abeta interactions with zinc (Zn), and increased Abeta hydrophobicity all strongly prevented Abeta degradation. Consistent to all of these factors is the promotion of specific Abeta aggregates where the protease cleavage site, confirmed by mass spectrometry, is inaccessible within an amyloid structure. These data indicate decreased degradation due to amyloid formation initiates Abeta accumulation by preventing normal protease activity. Zn also prevented Abeta degradation by the proteases neprilysin and insulin degrading enzyme. Treating Zn-induced Abeta amyloid with the metal-protein attenuating compound clioquinol reversed amyloid formation and restored the peptide's sensitivity to degradation by matrix metalloprotease 2. This provides new data indicating that therapeutic compounds designed to modulate Abeta-metal interactions can inhibit Abeta accumulation by restoring the catalytic potential of Abeta-degrading proteases.

Inhibition of gamma-secretase causes increased secretion of amyloid precursor protein C-terminal fragments in association with exosomes.

Alzheimer's disease (AD) is the most common form of dementia and is associated with the deposition of the 39- to 43-amino acid beta-amyloid peptide (Abeta) in the brain. C-terminal fragments (CTFs) of amyloid precursor protein (APP) can accumulate in endosomally derived multivesicular bodies (MVBs). These intracellular structures contain intraluminal vesicles that are released from the cell as exosomes when the MVB fuses with the plasma membrane. Here we have investigated the role of exosomes in the processing of APP and show that these vesicles contain APP-CTFs, as well as Abeta. In addition, inhibition of gamma-secretase results in a significant increase in the amount of alpha- and beta-secretase cleavage, further increasing the amount of APP-CTFs contained within these exosomes. We identify several key members of the secretase family of proteases (BACE, PS1, PS2, and ADAM10) to be localized in exosomes, suggesting they may be a previously unidentified site of APP cleavage. These results provide further evidence for a novel pathway in which APP fragments are released from cells and have implications for the analysis of APP processing and diagnostics for Alzheimer's disease.

In vitro characterization of Pittsburgh compound-B binding to Lewy bodies.

Dementia with Lewy bodies (DLB) is pathologically characterized by the presence of alpha-synuclein-containing Lewy bodies within the neocortical, limbic, and paralimbic regions. Like Alzheimer's disease (AD), Abeta plaques are also present in most DLB cases. The contribution of Abeta to the development of DLB is unclear. [11C]-Pittsburgh compound B ([11C]-PIB) is a thioflavin-T derivative that has allowed in vivo Abeta burden to be quantified using positron emission tomography (PET). [11C]-PIB PET studies have shown similar high cortical [11C]-PIB binding in AD and DLB subjects. To establish the potential binding of PIB to alpha-synuclein in DLB patients, we characterized the in vitro binding of PIB to recombinant human alpha-synuclein and DLB brain homogenates. Analysis of the in vitro binding studies indicated that [3H]-PIB binds to alpha-synuclein fibrils but with lower affinity than that demonstrated/reported for Abeta(1-42) fibrils. Furthermore, [3H]-PIB was observed to bind to Abeta plaque-containing DLB brain homogenates but failed to bind to DLB homogenates that were Abeta plaque-free ("pure DLB"). Positive PIB fluorescence staining of DLB brain sections colocalized with immunoreactive Abeta plaques but failed to stain Lewy bodies. Moreover, image quantification analysis suggested that given the small size and low density of Lewy bodies within the brains of DLB subjects, any contribution of Lewy bodies to the [11C]-PIB PET signal would be negligible. These studies indicate that PIB retention observed within the cortical gray matter regions of DLB subjects in [11C]-PIB PET studies is largely attributable to PIB binding to Abeta plaques and not Lewy bodies.

Membrane interactions and the effect of metal ions of the amyloidogenic fragment Abeta(25-35) in comparison to Abeta(1-42).

Abeta(1-42) peptide, found as aggregated species in Alzheimer's disease brain, is linked to the onset of Alzheimer's disease. Many reports have linked metals to inducing Abeta aggregation and amyloid plaque formation. Abeta(25-35), a fragment from the C-terminal end of Abeta(1-42), lacks the metal coordinating sites found in the full-length peptide and is neurotoxic to cortical cortex cell cultures. We report solid-state NMR studies of Abeta(25-35) in model lipid membrane systems of anionic phospholipids and cholesterol, and compare structural changes to those of Abeta(1-42). When added after vesicle formation, Abeta(25-35) was found to interact with the lipid headgroups and slightly perturb the lipid acyl-chain region; when Abeta(25-35) was included during vesicle formation, it inserted deeper into the bilayer. While Abeta(25-35) retained the same beta-sheet structure irrespective of the mode of addition, the longer Abeta(1-42) appeared to have an increase in beta-sheet structure at the C-terminus when added to phospholipid liposomes after vesicle formation. Since the Abeta(25-35) fragment is also neurotoxic, the full-length peptide may have more than one pathway for toxicity.

Modulating Protein Phosphatase 2A Rescues Disease Phenotype in Neurodegenerative Tauopathies.

Alzheimer's disease (AD) is the leading cause of dementia worldwide accounting for around 70% of all cases. There is currently no treatment for AD beyond symptom management and attempts at developing disease-modifying therapies have yielded very little. These strategies have traditionally targeted the peptide Aß, which is thought to drive pathology. However, the lack of clinical translation of these Aß-centric strategies underscores the need for diverse treatment strategies targeting other aspects of the disease. Metal dyshomeostasis is a common feature of several neurodegenerative diseases such as AD, Parkinson's disease, and frontotemporal dementia, and manipulation of metal homeostasis has been explored as a potential therapeutic avenue for these diseases. The copper ionophore glyoxalbis-[N4-methylthiosemicarbazonato]Cu(II) (CuII(gtsm)) has previously been shown to improve the cognitive deficits seen in an AD animal model; however, the molecular mechanism remained unclear. Here we report that the treatment of two animal tauopathy models (APP/PS1 and rTg4510) with CuII(gtsm) recovers the cognitive deficits seen in both neurodegenerative models. In both models, markers of tau pathology were significantly reduced with CuII(gtsm) treatment, and in the APP/PS1 model, the levels of Aß remained unchanged. Analysis of tau kinases (GSK3ß and CDK5) revealed no drug induced changes; however, both models exhibited a significant increase in the levels of the structural subunit of the tau phosphatase, PP2A. These findings suggest that targeting the tau phosphatase PP2A has therapeutic potential for preventing memory impairments and reducing the tau pathology seen in AD and other tauopathies.