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Abstract

Sporadic Alzheimer’s disease (AD) is associated with an inefficient clearance of the β-amyloid (Aβ) peptide from the central nervous system. The protein levels and activity of the Zn2+-dependent endopeptidase neprilysin (NEP) inversely correlate with brain Aβ levels during aging and in AD. The present study considered the ability of Cu2+ ions to inhibit human recombinant NEP and the role for NEP in generating N-truncated Aβ fragments with high-affinity Cu2+ binding motifs that can prevent this inhibition. Divalent copper noncompetitively inhibited NEP (Ki = 1.0 μM), while proteolysis of Aβ yielded the soluble, Aβ4–9 fragment that can bind Cu2+ with femtomolar affinity at pH 7.4. This provides Aβ4–9 with the potential to act as a Cu2+ carrier and to mediate its own production by preventing NEP inhibition. Enzyme inhibition at high Zn2+ concentrations (Ki = 20 μM) further suggests a mechanism for modulating NEP activity, Aβ4–9 production, and Cu2+ homeostasis.

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... 296 Incubation of Ab(1-16) with human NEP yields cleavage products, including the Ab(3-x), (4-x), and (11-x) fragments. 297 Interestingly, NEP activity is impacted by metal ions, such as copper, displaying a noncompetitive inhibition in vitro (K i ¼ 1.0 mM). 297 In the following sections, how Ab N-truncations impact Cu 2+ -coordination is discussed. ...
... 297 Interestingly, NEP activity is impacted by metal ions, such as copper, displaying a noncompetitive inhibition in vitro (K i ¼ 1.0 mM). 297 In the following sections, how Ab N-truncations impact Cu 2+ -coordination is discussed. ...
... 301 Indeed, metallothionein-3 (MT-3), a protein involved in neuroprotection against antioxidant injuries, cannot compete for copper ions with the Ab(4-40/42) isoforms. 301 The generation of Ab(4-x) fragments with a femtomolar affinity for Cu 2+ would also prevent NEP inhibition by copper ions, 297 and it has been demonstrated that the presence of the Ab(4-40) fragment has an impact on the aggregation profile of the full-length Ab . 302 The second copper-binding site appears only after saturation of the ATCUN site. ...
Chapter
d-block metal ions (Cu¹⁺, Cu²⁺, Fe²⁺, Fe³⁺, Mn²⁺, and Zn²⁺) are essential for the brain; however, disruption of metal ion homeostasis is closely linked to neurodegenerative diseases. Interestingly, many of the proteins that play a key role in neurodegeneration can bind metal ions and, in some cases, impact metal homeostasis. This chapter reviews the role of d-block metal ions in different neurodegenerative diseases, including Prion, Alzheimer’s, Parkinson’s, and Huntington's diseases. For each pathology, the metal-binding properties of the proteins involved are discussed, attempting to link the bioinorganic chemistry of these proteins with the role of metal ions in function and disease. Therapeutic approaches that target metal-protein interactions for each disease are also discussed.
... During the proteolysis of Aβ 1−16 , the Aβ 10−16 fragment also underwent further cleavage to the final products, Aβ 10−11 and Aβ 12−16 . 17 We further demonstrated that NEP undergoes noncompetitive inhibition by Cu(II) ions. 17 Similar observations were reported for IDE, which was competitively inhibited by Cu(II) and irreversibly inhibited by Cu(I) ions. ...
... 17 We further demonstrated that NEP undergoes noncompetitive inhibition by Cu(II) ions. 17 Similar observations were reported for IDE, which was competitively inhibited by Cu(II) and irreversibly inhibited by Cu(I) ions. 44,45 A key feature of Aβ 4−9 , Aβ 12−16 , and Aβ 11−16 , which may be formed by NEP, IDE, and BACE1 enzymes, is the amino-terminal (H 2 N-Xaa-Yaa-His-) Cu(II) binding motif (ATCUN), also referred to as the N-terminal site (NTS), characterized by the His residue at the third position from the N-terminus. ...
... 50−52 The proteolysis of CuAβ 1−16 by NEP leads to a transfer of Cu(II) from Aβ 1−16 to Aβ 4−9 and Aβ 12−16 as these fragments are formed. 17 The Aβ 12−x and Aβ 11−x peptides contain another His residue at the second (H 2 N-Xaa-His-His-) or fourth (H 2 N-Xaa-Yaa-His-His-) position, respectively. Additionally, Aβ 11−x contains a glutamic acid residue at the first position, which can spontaneously cyclize into pyroglutamic acid, a process that also occurs for the Aβ 3−x peptide. ...
Article
The catabolism of β-amyloid (Aβ) is carried out by numerous endopeptidases including neprilysin, which hydrolyzes peptide bonds preceding positions 4, 10, and 12 to yield Aβ4-9 and a minor Aβ12-x species. Alternative processing of the amyloid precursor protein by β-secretase also generates the Aβ11-x species. All these peptides contain a Xxx-Yyy-His sequence, also known as an ATCUN or NTS motif, making them strong chelators of Cu(II) ions. We synthesized the corresponding peptides, Phe-Arg-His-Asp-Ser-Gly-OH (Aβ4-9), Glu-Val-His-His-Gln-Lys-am (Aβ11-16), Val-His-His-Gln-Lys-am (Aβ12-16), and pGlu-Val-His-His-Gln-Lys-am (pAβ11-16), and investigated their Cu(II) binding properties using potentiometry, and UV-vis, circular dichroism, and electron paramagnetic resonance spectroscopies. We found that the three peptides with unmodified N-termini formed square-planar Cu(II) complexes at pH 7.4 with analogous geometries but significantly varied Kd values of 6.6 fM (Aβ4-9), 9.5 fM (Aβ12-16), and 1.8 pM (Aβ11-16). Cyclization of the N-terminal Glu11 residue to the pyroglutamate species pAβ11-16 dramatically reduced the affinity (5.8 nM). The Cu(II) affinities of Aβ4-9 and Aβ12-16 are the highest among the Cu(II) complexes of Aβ peptides. Using fluorescence spectroscopy, we demonstrated that the Cu(II) exchange between the Phe-Arg-His and Val-His-His motifs is very slow, on the order of days. These results are discussed in terms of the relevance of Aβ4-9, a major Cu(II) binding Aβ fragment generated by neprilysin, as a possible Cu(II) carrier in the brain.
... 205,206 In our hands, NEP proved to be an excellent producer of ATCUN peptides indeed, of Aβ 4−x and Aβ 12−x types, but not really of long Aβ 4−x species. 182 This was because the elimination of the Tyr-Glu dipeptide (Aβ 10−11 ) was among the fastest processes catalyzed by NEP in both Aβ 1−x substrates. Hence, Aβ 4−9 and Aβ 12−16 peptides emerged as significant products (the latter also in Aβ 1−40 , due to the cleavage behind Lys16). ...
... We found that the excess of Cu(II) and Zn(II) ions inhibited NEP, with K i values of 1 and 20 μM, respectively. 182 The micromolar Cu 2+ concentrations are easily available in the synaptic cleft, 207 and so Aβ 4−9 has the potential to enhance its own production by sequestering Cu(II) released during neurotransmission, thereby preventing NEP inhibition. 208 In the follow-up work, we described the Cu(II) binding properties of Aβ 4−9 , Aβ 11−16 , and Aβ 12−16 peptides. ...
... Our concept is very important for the idea of therapeutic chelation, because such chelator should be sufficiently weak to avoid interference with CuAβ 4−x complexes, both the long ones presumably residing in synapses and the short ones, possibly participating in copper clearance. 182,183 There is no doubt that Aβ 4−42 apopeptide can be neurotoxic when in excess, even more neurotoxic than the Aβ 1−x species, due to its rapid aggregation and the ability to seed the Aβ 1−x coaggregates. 56 The key process related to the former issue is the cellular copper uptake. ...
Article
As life expectancy increases, the number of people affected by progressive and irreversible dementia, Alzheimer’s Disease (AD), is predicted to grow. No drug designs seem to be working in humans, apparently because the origins of AD have not been identified. Invoking amyloid cascade, metal ions, and ROS production hypothesis of AD, herein we share our point of view on Cu(II) binding properties of Aβ4–x, the most prevalent N-truncated Aβ peptide, currently known as the main constituent of amyloid plaques. The capability of Aβ4–x to rapidly take over copper from previously tested Aβ1–x peptides and form highly stable complexes, redox unreactive and resistant to copper exchange reactions, prompted us to propose physiological roles for these peptides. We discuss the new findings on the reactivity of Cu(II)Aβ4–x with coexisting biomolecules in the context of synaptic cleft; we suggest that the role of Aβ4–x peptides is to quench Cu(II) toxicity in the brain and maintain neurotransmission.
... An imbalance in the levels of copper and zinc has been linked to Alzheimer's disease (AD) (Mital et al., 2018;Sensi et al., 2018) [54,55]. Pathogenesis of Alzheimer's disease involves accumulation of the β-amyloid (Aβ) peptide in the brain. ...
... An imbalance in the levels of copper and zinc has been linked to Alzheimer's disease (AD) (Mital et al., 2018;Sensi et al., 2018) [54,55]. Pathogenesis of Alzheimer's disease involves accumulation of the β-amyloid (Aβ) peptide in the brain. ...
Article
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The aim of this paper was to review recent literature (from 2000 onwards) and summarize the newest findings on fluctuations in the concentration of some essential macro- and microelements in those patients with a history of chronic alcohol abuse. The focus was mainly on four elements which the authors found of particular interest: Iron, magnesium, copper, and manganese. After independently reviewing over 50 articles, the results were consistent with regard to iron and magnesium. On the other hand, data were limited, and in some cases contradictory, as far as copper and manganese were concerned. Iron overload and magnesium deficiency are two common results of an excessive and prolonged consumption of alcohol. An increase in the levels of iron can be seen both in the serum and within the cells, hepatocytes in particular. This is due to a number of factors: Increased ferritin levels, lower hepcidin levels, as well as some fluctuations in the concentration of the TfR receptor for transferrin, among others. Hypomagnesemia is universally observed among those suffering from alcoholism. Again, the causes for this are numerous and include malnutrition, drug abuse, respiratory alkalosis, and gastrointestinal problems, apart from the direct influence of excessive alcohol intake. Unfortunately, studies regarding the levels of both copper and manganese in the case of (alcoholic) liver disease are scarce and often contradictory. Still, the authors have attempted to summarize and give a thorough insight into the literature available, bearing in mind the difficulties involved in the studies. Frequent comorbidities and mutual relationships between the elements in question are just some of the complications in the study of this topic.
... Besides IDE, other Zn 2+ -dependent endopeptidase like NEP previously described as hIAPP-degrading enzymes have been shown to be inhibited by excess Cu 2+ ions. 500 Indeed, Zn 2+ ions present in metallopeptidases are known to significantly contribute to enzyme folding and stability. Moreover, the distorted tetrahedral geometry adopted by zinc centers in metalloprotease enhances the Lewis acidity of the catalytic site and of the coordinated water molecules as well which are both essential for the peptidase activity. ...
... 541 Therefore, the use of metal ion chelators appear an attractive possibility to delay of the onset of various amyloid-associated disorders. 500,542,543 Bush and colleagues made pioneering contributions in the study of the ability of metal ion chelators to interfere with amyloid formation. 544 Although these metal chelators were promising potential drugs to fight Alzheimer's, they were abandoned due to their side effects. ...
Article
The possible link between hIAPP accumulation and β-cell death in diabetic patients has inspired numerous studies focusing on amyloid structures and aggregation pathways of this hormone. Recent studies have reported on the importance of early oligomeric intermediates, the many roles of their interactions with lipid membrane, pH, insulin, and zinc on the mechanism of aggregation of hIAPP. The challenges posed by the transient nature of amyloid oligomers, their structural heterogeneity, and the complex nature of their interaction with lipid membranes have resulted in the development of a wide range of biophysical and chemical approaches to characterize the aggregation process. While the cellular processes and factors activating hIAPP-mediated cytotoxicity are still not clear, it has recently been suggested that its impaired turnover and cellular processing by proteasome and autophagy may contribute significantly toward toxic hIAPP accumulation and, eventually, β-cell death. Therefore, studies focusing on the restoration of hIAPP proteostasis may represent a promising arena for the design of effective therapies. In this review we discuss the current knowledge of the structures and pathology associated with hIAPP self-assembly and point out the opportunities for therapy that a detailed biochemical, biophysical, and cellular understanding of its aggregation may unveil.
... Additionally, Fe could be related to decreased NEP activity. Once a chelator, deferasirox, chelates an Fe out from treated 18-month-old rats for 4 months, the A β42 -degrading activity of NEP was recovered compared to the same aged rats [374]. www.videleaf.com ...
... The spectra of Cu II :Aβ 4−9/12/16 are comparable with those of the previously characterized Cu II :Aβ 4−9 −OH. 60 Chemical reduction of the Cu II :Aβ complexes by Asc and GSH was monitored by the change in intensity of the EPR signals with time. Baseline correction of all spectra was performed by weighted subtraction of the spectrum obtained using a water blank. ...
Article
This report establishes that despite the pM binding of CuII to the N-truncated Aβ4−y peptides, the reduction chemistry of Aβ4-9,12,16 is critically dependent on retention of the CuI(bis-H13H14) binding site. The slow kinetics for the reduction requires the use of methods which allow quantification of the relative concentrations of the oxidized and reduced forms of the metal. This is provided by the novel technique of ambient temperature X-ray absorption spectroscopy under in situ electrochemical control.
... This has been shown by high-performance liquid chromatography analysis yielding several product peaks after incubation of Aβ 1-40 with either recombinant soluble NEP produced in Sf9 cells or NEP purified from rabbit kidney cortex (46). More recent studies using synthetic Aβ peptides and recombinant human NEP confirmed the generation of Aβ peptide fragments starting with Phe-4 (such as Aβ [4][5][6][7][8][9] or Aβ 4-16 but also the existence of several other cleavage sites, at least under the given in vitro conditions (48,49). Therefore, it is currently unclear whether NEP might contribute to the generation of longer Aβ peptides such as Aβ 4-40 and Aβ 4-42 . ...
Chapter
Full-text available
The accumulation and aggregation of amyloid-β (Aβ) peptides in the brain is believed to be the initial trigger in the molecular pathology of Alzheimer’s disease (AD). In addition to the widely studied full-length Aβ peptides (mainly Aβ1–40 and Aβ1–42), a variety of amino-terminally truncated (N-truncated) peptides, such as AβpE3-x and Aβ4-x, have been detected in high abundance in autopsy samples from sporadic and familial AD patients. N-truncated Aβ species adopt specific physicochemical properties resulting in a higher aggregation propensity and increased peptide stability, which likely account for their neurotoxic potential. The presence of N-truncated Aβ peptides in transgenic mouse models of AD and the selective overexpression of specific N-truncated variants in the murine brain have facilitated their investigation in relevant in vivo settings. In this chapter, we address the pathological relevance of N-truncated Aβ peptide species and summarize the current knowledge about the enzymatic activities that might be involved in their generation.
... Upon incubation of synthetic Aβ 1-40 with recombinant soluble NEP produced in Sf9 cells, several product peaks could be identified [135]. More recent studies utilizing mass spectrometry were able to confirm the generation of short Aβ peptide fragments starting with Phe-4 (such as Aβ [4][5][6][7][8][9] or Aβ 4-16 ) under the given in vitro conditions, in addition to several other peptide fragments [136][137][138]. Due to the fact that cleavage of the Gly-9 -Tyr-10 bond seems to be the critical rate-limiting step in Aβ proteolysis, as shown under in vivo conditions, a major contribution of NEP to Aβ 4-x generation seems unlikely, as this cleavage would preclude the generation of full-length Aβ 1-40 and Aβ 1-42 peptides [139]. ...
Article
Introduction: Alzheimer’s disease (AD) is characterized by a cerebral accumulation and aggregation of amyloid-β (Aβ) peptides, which mainly accumulate in the form of extracellular deposits. In addition to the well-described full-length peptides Aβ1-40 and Aβ1-42, a variety of amino- and carboxy-terminally truncated Aβ variants has been identified in brain samples from sporadic and familial AD cases. Areas covered: This review gives an overview on the role of truncated Aβ species in human AD, as well as in transgenic AD mouse models. We outline the relevance of the most abundant N- and C-truncated Aβ species, highlight potential mechanisms with regard to their generation and discuss their suitability as targets for pharmacological interventions. Expert opinion: A variety of recent clinical trials aiming either at a reduced Aβ production by the use of secretase inhibitors or at increased Aβ clearance by the use of immunotherapy were terminated unsuccessful. Truncated or post-translationally modified Aβ peptides are becoming increasingly recognized as important players in the etiology of AD and a more thorough comprehension of their cellular origin and biochemical peculiarities might break new ground for therapeutic strategies.
... Moreover, Aβ12-x and Aβ11-x contain another His residue at the second or fourth position, respectively, which can affect their coordination properties. It has been reported that copper plays an important role in the formation of AD as it promotes the Aβ aggregation, inhibits the activity of Aβ degradation enzymes [7], and triggers the production of free radicals [8]. The so-called metal hypothesis proposes that this process contributes to disease progression and that the Aβ aggregation process can be prevented by "therapeutic chelation" [9,10]. ...
Chapter
Dishomeostasis of Cu(II) ions in the human body is connected with several serious diseases such as Alzheimer’s disease or Wilson’s disease. Therefore, a deep understanding of Cu(II)-binding properties to metal ions carriers, together with the knowledge about how they can interact with other copper-binding partners, e.g., amyloid-β (Aβ), is required to assess their relevance to the brain metal homeostasis. Ultraviolet-visible spectrometry (UV-Vis) and circular dichroism (CD) were used to study the coordination characteristics of Cu(II) with peptide containing the amino-terminal (H2N–Xaa–Yaa–His–) copper-binding (ATCUN) motif (Aβ12–16—VHHQK-NH2) derived from Aβ peptide.
... 21 Therefore, these complexes may well contribute to the biological activity of Ab 4-42 , and of putative short peptidic fragments generated by neprilysin cleavage, such as Ab 4-9 . 22,23 There is only one way in which four nitrogen ligands of the ATCUN motif can be arranged around the Cu II ion, and so it is reasonable to assume that the intermediate species contain the coordinatively unsaturated Cu II . Such species have been implicated in the reverse reaction of Cu II dissociative transfer from Cu II Ab 4-16 to MT3, to explain the catalytic effect of glutamate, 24 but it has not been observed directly. ...
Article
Multiple intermediates were found in Cu( ii ) binding to Aβ 4–16 before the formation of a tight complex.
... However, the excessive intake of copper can also cause gastrointestinal disorders and damage to the liver, kidneys and neurodegenerative diseases. By the way, the industry development, such as electronics, thermal conductor and metal alloys in today's societies, has led to increased consumption of copper, and environmental and biological contaminations are followed [3]. The maximum level of copper in drinking water is about 2.0 ppm (30 nM) designated by the World Health Organisation [4]. ...
Article
Full-text available
Copper is an essential mineral that plays a vital role in the body, which in the event of an increase it can be toxic to the body. The concentration of copper in the blood is very important. In the present study, a surface-modified glass electrode bead on Metal–organic frameworks (MOFs) and p-aminobenzoic acid (PABA) is presented and used for rapid, simple, selective and highly sensitive determination of copper using differential pulse adsorptive stripping voltammetry. A novel Zn-based metal-organic framework, (Zn-TMU-24) through a solvothermal method, is constructed and investigated by different spectra such as FT-IR, SEM and TGA. Then, the Zn-TMU-24 is conjugated with PABA via electrochemical polymerisation to form PABA-MOF on surface glass electrode. The amide-functionalised groups in the structure of PABA-MOF have provided a potential for chemical interaction with a metal ion Cu²⁺, and used for the fast detection of Cu²⁺ in milk and milk powder as real samples. The interaction between the proposed Zn- TMU-24 and Cu²⁺ was characterised by electrochemical impedance spectroscopy (EIS), cyclic voltammetry (CV) and differential pulse voltammetry (DPV). The calibration plot is linear in the concentration range of 0.05 to 250 nM with detection limit of 0.014 nM. The excellent stability of the proposed electrode was observed. The proposed sensor was successfully applied to the analysis of milk and milk powder samples.
... While the actual role of Aβ 4−x peptides in brain copper metabolism remains speculative, we postulate that the longer species serve to mop up the excess of Cu(II) ions from the synaptic cleft, 18 and Aβ 4−9 , as product of Aβ 4−40 cleavage by neprilysin may participate in the export of copper across the blood−brain barrier. 48,49 Aβ 5−x peptides are normally only very minor contributors to the overall β-amyloid pool but our results indicate that these peptides, when multiplicated as a result of therapeutic intervention, may interfere with the synaptic and extrasynaptic Cu(II) handling. The affinity of the Cu(II)-Aβ 5−9 /Aβ 1−16 complex, much higher than that of imidazole, points at a possibility of strong, specific interactions with larger ligands that can be recruited from the Aβ family or other synaptic proteins. ...
Article
Full-text available
The Aβ5–x peptides (x = 38, 40, 42) are minor Aβ species in normal brains but elevated upon the application of inhibitors of Aβ processing enzymes. They are interesting from the point of view of coordination chemistry for the presence of an Arg-His metal binding sequence at their N-terminus capable of forming a 3-nitrogen (3N) three-coordinate chelate system. Similar sequences in other bioactive peptides were shown to bind Cu(II) ions in biological systems. Therefore, we investigated Cu(II) complex formation and reactivity of a series of truncated Aβ5–x peptide models comprising the metal binding site: Aβ5–9, Aβ5–12, Aβ5–12Y10F, and Aβ5–16. Using CD and UV–vis spectroscopies and potentiometry, we found that all peptides coordinated the Cu(II) ion with substantial affinities higher than 3 × 1012 M–1 at pH 7.4 for Aβ5–9 and Aβ5–12. This affinity was elevated 3-fold in Aβ5–16 by the formation of the internal macrochelate with the fourth coordination site occupied by the imidazole nitrogen of the His13 or His14 residue. A much higher boost of affinity could be achieved in Aβ5–9 and Aβ5–12 by adding appropriate amounts of the external imidazole ligand. The 3N Cu-Aβ5–x complexes could be irreversibly reduced to Cu(I) at about −0.6 V vs Ag/AgCl and oxidized to Cu(III) at about 1.2 V vs Ag/AgCl. The internal or external imidazole coordination to the 3N core resulted in a slight destabilization of the Cu(I) state and stabilization of the Cu(III) state. Taken together these results indicate that Aβ5–x peptides, which bind Cu(II) ions much more strongly than Aβ1–x peptides and only slightly weaker than Aβ4–x peptides could interfere with Cu(II) handling by these peptides, adding to copper dyshomeostasis in Alzheimer brains.
... [42,58] This concept was further supported by a finding that the short Aβ4-9 peptide, the prominent product of the neprilysin cleavage of Aβ1-40, formed a Cu(II) complex with the inertness and affinity even higher than that of Aβ4-16 (log K at pH 7.4 = 14.2). [59,60] ...
Article
Alzheimer’s Disease (AD) is one of the most common multifactorial diseases characterized by a range of abnormal molecular processes such as the accumulation of extracellular plaques containing the amyloid‐β (Aβ) peptides and dyshomeostasis of copper in the brain. Herein, we investigate the effect of Cu(II) ions on the aggregation of Aβ1‐40, and Aβ4‐40, representing two most prevalent families of Aβ peptides, full length and N‐truncated ones. Both families are similarly abundant in healthy and AD brains. In either of the studied peptides, substoichiometric Cu(II) concentrations accelerated aggregation, while superstoichiometric Cu(II) inhibited the fibril formation, likely by stabilizing oligomers. The addition of either Aβ4‐40 or substoichiometric Cu(II) ions affected the aggregation profile of Aβ1‐40, by yielding shorter and thicker fibrils. The similarity of these two effects can be attributed to the increase of positive charge at the Aβ N‐terminus, which is caused either by Cu(II) complexation or N‐truncation at position 4. Our findings provide a better understanding of the biological Aβ aggregation process as these two Aβ species and Cu(II) ions coexist and interact under physiological conditions.
... Post-mortem studies of Cu concentration within the CNS showed increased Cu concentrations mainly in the caudate nucleus in schizophrenic patients [212]. Disturbed distribution and homeostasis of Cu within the CNS contributes to the promotion of many neurodegenerative diseases: Alzheimer and Parkinson diseases, Huntington disease, amyotrophic lateral sclerosis, as well as psychiatric conditions such as depressive disorder, ASD, epilepsy, or schizophrenia primarily due to the impaired neuronal myelination, catecholamine imbalances, and disturbed brain architecture [251][252][253][254][255]. Neurobehavioral abnormalities might be a consequence of maternal Cu deficiency as well [186]. ...
Article
Full-text available
The alterations in serum trace element levels are common phenomena observed in patients with different psychiatric conditions such as schizophrenia, autism spectrum disorder, or major depressive disorder. The fluctuations in the trace element concentrations might act as potential diagnostic and prognostic biomarkers of many psychiatric and neurological disorders. This paper aimed to assess the alterations in serum trace element concentrations in patients with a diagnosed schizophrenia. The authors made a systematic review, extracting papers from the PubMed, Web of Science, and Scopus databases according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. Among 5009 articles identified through database searching, 59 of them were assessed for eligibility. Ultimately, 33 articles were included in the qualitative synthesis. This review includes the analysis of serum levels of the following trace elements: iron, nickel, molybdenum, phosphorus, lead, chromium, antimony, uranium, magnesium, aluminum, zinc, copper, selenium, calcium, and manganese. Currently, there is no consistency regarding serum trace element levels in schizophrenic patients. Thus, it cannot be considered as a reliable prognostic or diagnostic marker of schizophrenia. However, it can be assumed that altered concentrations of those elements are crucial regarding the onset and exaggeration of either psychotic or negative symptoms or cognitive dysfunctions.
... Increased Aβ levels were observed in IDE knock-out animals [215]. In another study by Mital et al. [216], the ability of Cu ions to inhibit human recombinant NEP and the resulting generation of N-truncated Aβ fragments with high-affinity Cu-binding motifs are worthy of note. Drew and colleagues observed that Cu noncompetitively inhibited NEP (Ki =1.0 μM), while proteolytic degradation of Aβ peptide produced a more soluble, Aβ 4− 9 fragment possessing very high affinity to bind Cu. ...
Article
Background Alzheimer's disease (AD) is the most prevalent cause of cognitive impairment and dementia worldwide. The pathobiology of the disease has been studied in the form of several hypotheses, ranging from oxidative stress, amyloid-beta (Aβ) aggregation, accumulation of tau forming neurofibrillary tangles (NFT) through metal dysregulation and homeostasis, dysfunction of the cholinergic system, and to inflammatory and autophagic mechanism. However, none of these hypotheses has led to confirmed diagnostics or approved cure for the disease. Objective This review is aimed as a basic and an encyclopedic short course into metals in AD and discusses the advances in chelation strategies and developments adopted in the treatment of the disease. Since there is accumulating evidence of the role of both biometal dyshomeostasis (iron (Fe), copper (Cu), and zinc (Zn)) and metal-amyloid interactions that lead to the pathogenesis of AD, this review focuses on unraveling therapeutic chelation strategies that have been considered in the treatment of the disease, aiming to sequester free and protein-bound metal ions and reducing cerebral metal burden. Promising compounds possessing chemically modified moieties evolving as multi-target ligands used as anti-AD drug candidates are also covered. Results and Conclusion Several multidirectional and multifaceted studies on metal chelation therapeutics show the need for improved synthesis, screening, and analysis of compounds to be able to effectively present chelating anti-AD drugs. Most drug candidates studied have limitations in their physicochemical properties; some enhance redistribution of metal ions, while others indirectly activate signaling pathways in AD. The metal chelation process in vivo still needs to be established and the design of potential anti-AD compounds that bi-functionally sequester metal ions as well as inhibit the Aβ aggregation by competing with the metal ions and reducing metal-induced oxidative damage and neurotoxicity may signal a bright end in chelation-based therapeutics of AD.
... Similar results were obtained using a transgenic drosophila experiment that has a silent Ctr1 protein, along with a dissociation constant of NEP for Cu(II) determined as 1.04 (±0.07) µM. Even 100 µM of Zn(II) was not able to restore the activity of NEP [372,373]. Downregulation of NEP activity by Cu(II) was also observed in mouse neuroblastoma N2a cells. NEP activity was not inhibited at the transcription level. ...
Article
Full-text available
Redox-active metal ions, Cu(I/II) and Fe(II/III), are essential biological molecules for the normal functioning of the brain, including oxidative metabolism, synaptic plasticity, myelination, and generation of neurotransmitters. Dyshomeostasis of these redox-active metal ions in the brain could cause Alzheimer’s disease (AD). Thus, regulating the levels of Cu(I/II) and Fe(II/III) is necessary for normal brain function. To control the amounts of metal ions in the brain and understand the involvement of Cu(I/II) and Fe(II/III) in the pathogenesis of AD, many chemical agents have been developed. In addition, since toxic aggregates of amyloid-β (Aβ) have been proposed as one of the major causes of the disease, the mechanism of clearing Aβ is also required to be investigated to reveal the etiology of AD clearly. Multiple metalloenzymes (e.g., neprilysin, insulin-degrading enzyme, and ADAM10) have been reported to have an important role in the degradation of Aβ in the brain. These amyloid degrading enzymes (ADE) could interact with redox-active metal ions and affect the pathogenesis of AD. In this review, we introduce and summarize the roles, distributions, and transportations of Cu(I/II) and Fe(II/III), along with previously invented chelators, and the structures and functions of ADE in the brain, as well as their interrelationships.
... Besides general clearance of full-length Aβ peptides, NEP is also involved in the generation of N-truncated Aβ peptides Aβ 4-X (Bayer and Wirths, 2014), and further clearing of Aβ 4-42 in vivo and in vitro (Hornung et al., 2019). Aβ 4-9 , a main degradation product of NEP, is a major Cu 2+ binding and has been suggested as a possible Cu 2+ carrier in the brain (Bossak-Ahmad et al., 2019) and NEP modulation (Mital et al., 2018). Modulating Cu metabolism is discussed as a relevant therapeutic target (Lei et al., 2020). ...
Article
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The discussion of whether amyloid plaque Aβ is a valid drug target to fight Alzheimer’s disease (AD) has been a matter of scientific dispute for decades. This question can only be settled by successful clinical trials and the approval of disease-modifying drugs. However, many clinical trials with antibodies against different regions of the amyloid Aβ peptide have been discontinued, as they did not meet the clinical endpoints required. Recently, passive immunization of AD patients with Donanemab, an antibody directed against the N-terminus of pyroglutamate Aβ, showed beneficial effects in a phase II trial, supporting the concept that N-truncated Aβ is a relevant target for AD therapy. There is long-standing evidence that N-truncated Aβ variants are the main variants found in amyloid plaques besides full-length Aβ 1–42 , t, therefore their role in triggering AD pathology and as targets for drug development are of interest. While the contribution of pyroglutamate Aβ 3–42 to AD pathology has been well studied in the past, the potential role of Aβ 4–42 has been largely neglected. The present review will therefore focus on Aβ 4–42 as a possible drug target based on human and mouse pathology, in vitro and in vivo toxicity, and anti-Aβ 4-X therapeutic effects in preclinical models.
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Amyloid beta (Aβ) peptides are notorious for their involvement in Alzheimer’s disease (AD), by virtue of their propensity to aggregate to form oligomers, fibrils, and eventually plaques in the brain. Nevertheless, they appear to be essential for correct neurophysiology on the synaptic level and may have additional functions including antimicrobial activity, sealing the blood–brain barrier, promotion of recovery from brain injury, and even tumor suppression. Aβ peptides are also avid copper chelators, and coincidentally copper is significantly dysregulated in the AD brain. Copper (Cu) is released in significant amounts during calcium signaling at the synaptic membrane. Aβ peptides may have a role in maintaining synaptic Cu homeostasis, including as a scavenger for redox-active Cu and as a chaperone for clearing Cu from the synaptic cleft. Here, we employed the Aβ1–16 and Aβ4–16 peptides as well-established non-aggregating models of major Aβ species in healthy and AD brains, and the Ctr1–14 peptide as a model for the extracellular domain of the human cellular copper transporter protein (Ctr1). With these model peptides and a number of spectroscopic techniques, we investigated whether the Cu complexes of Aβ peptides could provide Ctr1 with either Cu(II) or Cu(I). We found that Aβ1–16 fully and rapidly delivered Cu(II) to Ctr1–14 along the affinity gradient. Such delivery was only partial for the Aβ4–16/Ctr1–14 pair, in agreement with the higher complex stability for the former peptide. Moreover, the reaction was very slow and took ca. 40 h to reach equilibrium under the given experimental conditions. In either case of Cu(II) exchange, no intermediate (ternary) species were present in detectable amounts. In contrast, both Aβ species released Cu(I) to Ctr1–14 rapidly and in a quantitative fashion, but ternary intermediate species were detected in the analysis of XAS data. The results presented here are the first direct evidence of a Cu(I) and Cu(II) transfer between the human Ctr1 and Aβ model peptides. These results are discussed in terms of the fundamental difference between the peptides’ Cu(II) complexes (pleiotropic ensemble of open structures of Aβ1–16 vs the rigid closed-ring system of amino-terminal Cu/Ni binding Aβ4–16) and the similarity of their Cu(I) complexes (both anchored at the tandem His13/His14, bis-His motif). These results indicate that Cu(I) may be more feasible than Cu(II) as the cargo for copper clearance from the synaptic cleft by Aβ peptides and its delivery to Ctr1. The arguments in favor of Cu(I) include the fact that cellular Cu export and uptake proteins (ATPase7A/B and Ctr1, respectively) specifically transport Cu(I), the abundance of extracellular ascorbate reducing agent in the brain, and evidence of a potential associative (hand-off) mechanism of Cu(I) transfer that may mirror the mechanisms of intracellular Cu chaperone proteins.
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Aβ4-42 is the major subspecies of Aβ peptides characterized by avid Cu(II) binding via the ATCUN/NTS motif. It is thought to be produced in vivo proteolytically by neprilysin, but in vitro experiments in the presence of Cu(II) ions indicated preferable formation of C-terminally truncated ATCUN/NTS species including CuIIAβ4-16, CuIIAβ4-9, and also CuIIAβ12-16, all with nearly femtomolar affinities at neutral pH. Such small complexes may serve as shuttles for copper clearance from extracellular brain spaces, on condition they could survive intracellular conditions upon crossing biological barriers. In order to ascertain such possibility, we studied the reactions of CuIIAβ4-16, CuIIAβ4-9, CuIIAβ12-16, and CuIIAβ1-16 with reduced glutathione (GSH) under aerobic and anaerobic conditions using absorption spectroscopy and mass spectrometry. We found CuIIAβ4-16 and CuIIAβ4-9 to be strongly resistant to reduction and concomitant formation of Cu(I)-GSH complexes, with reaction times ∼10 h, while CuIIAβ12-16 was reduced within minutes and CuIIAβ1-16 within seconds of incubation. Upon GSH exhaustion by molecular oxygen, the CuIIAβ complexes were reformed with no concomitant oxidative damage to peptides. These finding reinforce the concept of Aβ4-x peptides as physiological trafficking partners of brain copper.
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Copper(II) forms well-known and stable complexes with peptides having histidine at position 2 (Xxx-His) or 3 (Xxx-Zzz-His). Their properties differ considerably due to the histidine positioning. Here we report that...
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In sporadic Alzheimer's disease (AD), an imbalance between production and clearance of amyloid-β (Aβ) peptides seems to account for enhanced Aβ accumulation. The metalloprotease neprilysin (NEP) is an important Aβ degrading enzyme as shown by a variety of in vitro and in vivo studies. While the degradation of full-length Aβ peptides such as Aβ 1-40 and Aβ 1-42 is well established, it is less clear whether NEP is also capable of degrading N-terminally truncated Aβ species such as the common variant Aβ 4-42. In the present report, we confirmed the degradation of Aβ 4-x species by neprilysin using in vitro digestion and subsequent analysis using gel-based assays and mass spectrometry. By crossing Tg4-42 mice expressing only Aβ 4-42 peptides with homozygous NEP-knock-out mice (NEP -/-), we were able to demonstrate that NEP deficiency increased hippocampal intraneuronal Aβ levels and aggravated neuron loss in the Tg4-42 transgenic mouse model of AD.
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In an aging society in the world, dementia that leads to pain in patients and their families has become a common disease in our lives. Among dementia, Alzheimer's disease (AD) is the most commonly shown disease. Various causes of the disease have been proposed and amyloid hypothesis insists that the toxic amyloid‐β (Aβ) species could be the major risk factor of the onset and progression of AD. In this perspective, clearance of Aβ species from the brain by regulating the activity of amyloid degrading enzymes (ADE), including neprilysin and matrix metalloproteinases, could be a potent treatment for AD. Therefore, the structures and functions of these enzymes along with biological molecules in the brain would be important to understand the pathogenesis of AD and develop an effective medication for the disease. In this review, multiple ADE with biological molecules which could affect the activities and/or expression of the enzymes are summarized. The amyloid degrading enzymes (ADE) could control the levels of amyloidogenic proteins in brain consequently affect the pathogenesis of Alzheimer's disease (AD). Therefore, understanding the expression and activity of ADE along with other biological molecules in the brain is essential to develop therapeutic agents to treat AD.
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Herein, we have developed a novel electrochemical sensor for the detection of Cu2+ with silver nanoparticles modified glassy carbon electrode and a tripeptide to selectively recognize target ions. Electrochemical impedance spectroscopy technique is applied to probe the electron transfer resistance of the sensing system. This method is demonstrated to be able to selectively detect Cu2+ in nanomolar range, which meets the requirements of the United States Environmental Protection Agency. It has great potential use for biological studies and environment surveys.
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The “therapeutic chelation” approach to treating Alzheimer’s disease (AD) evolved from the metals hypothesis, with the premise that small molecules can be designed to prevent transition metal-induced amyloid deposition and oxidative stress within the AD brain. Over more than 20 years, countless in vitro studies have been devoted to characterizing metal binding, its effect on Ab aggregation, ROS production, and in vitro toxicity. Despite a lack of evidence for any clinical benefit, the conjecture that therapeutic chelation is an effective approach for treating AD remains widespread. Here, the author plays the devil’s advocate, questioning the experimental evidence, the dogma, and the value of therapeutic chelation, with a major focus on copper ions.
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The interactions between the Aβ1–40 molecules species and the copper ions (Cu(II)) were intensively investigated due to their potential role in the development of the Alzheimer Disease (AD). The rate and the mechanism of the Cu(II)—Aβ complexes formation determines the aggregation pathway of the Aβ species, starting from smaller but more cytotoxic oligomers and ending up in large Aβ plaques, being the main hallmark of the AD. In our study we exploit the existing knowledge on the Cu(II)—Aβ interactions and create the theoretical model of the initial phase of the copper- driven Aβ aggregation mechanism. The model is based on the direct solution of the Chemical Master Equations, which capture the inherent stochastics of the considered system. In our work we argue that due to a strong Cu(II) affinity to Aβ and temporal accessibility of the Cu(II) ions during normal synaptic activity the aggregation driven by Cu(II) dominates the pure Aβ aggregation. We also demonstrate the dependence of the formation of different Cu(II)—Aβ complexes on the concentrations of reagents and the synaptic activity. Our findings correspond to recent experimental results and give a sound hypothesis on the AD development mechanisms.
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The amyloid-b peptide (Ab) is a key protein in Alzheimer’s disease (AD) pathology. We previously reported in vitro evidence suggesting that Ab is an antimicrobial peptide. We present in vivo data showing that Ab expression protects against fungal and bacterial infections in mouse, nematode, and cell culture models of AD. We show that Ab oligomerization, a behavior traditionally viewed as intrinsically pathological, may be necessary for the antimi- crobial activities of the peptide. Collectively, our data are consistent with a model in which soluble Ab oligomers first bind to microbial cell wall carbohydrates via a heparin-binding domain. Developing protofibrils inhibited path- ogen adhesion to host cells. Propagating b-amyloid fibrils mediate agglutination and eventual entrapment of un- atttached microbes. Consistent with our model, Salmonella Typhimurium bacterial infection of the brains of transgenic 5XFAD mice resulted in rapid seeding and accelerated b-amyloid deposition, which closely colocalized with the invading bacteria. Our findings raise the intriguing possibility that b-amyloid may play a protective role in innate immunity and infectious or sterile inflammatory stimuli may drive amyloidosis. These data suggest a dual protective/damaging role for Ab, as has been described for other antimicrobial peptides.
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Alzheimer's disease (AD) is a neurodegenerative process primarily characterized by amyloid-β (Aβ) agglomeration, neuroinflammation, and cognitive dysfunction. The prominent cause for dementia is the deposition of Aβ plaques and tau-neurofibrillary tangles that hamper the neuronal organization and function. Aβ pathology further affects numerous signaling cascades that disturb the neuronal homeostasis. For instance, Aβ deposition is responsible for altered expression of insulin encoding genes that lead to insulin resistance, and thereby affecting insulin signaling pathway and glucose metabolism in the brain. As a result, the common pathology of insulin resistance between Type-2 diabetes mellitus and AD has led AD to be proposed as a form of diabetes and termed 'Type-3 diabetes'. Since accumulation of Aβ is the prominent cause of neuronal toxicity in AD, its clearance is the prime requisite for therapeutic prospects. This purpose is expertly fulfilled by the potential role of Aβ degrading enzymes such as insulin degrading enzyme (IDE) and Neprilysin (NEP). Therefore, their molecular study is important to uncover the proteolytic and regulatory mechanism of Aβ degradation. Herein, (i) In silico sequential and structural analysis of IDE and NEP has been performed to identify the molecular entities for proteolytic degradation of Aβ in the AD brain, (ii) to analyze their catalytic site to demonstrate the enzymatic action played by IDE and NEP, (iii) to identify their structural homologues that could behave as putative partners of IDE and NEP with similar catalytic action and (iv) to illustrate various IDE- and NEP-mediated therapeutic approaches and factors for clearing Aβ in AD.
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Alzheimer's disease is characterized by the extracellular deposition in the brain and its blood vessels of insoluble aggregates of the amyloid beta-peptide (A beta), a fragment, of about 40 amino acids in length, of the integral membrane protein beta-amyloid precursor protein (beta-APP). The mechanism of extracellular accumulation of A beta in brain is unknown and no simple in vitro or in vivo model systems that produce extracellular A beta have been described. We report here the unexpected identification of the 4K (M(r) 4,000) A beta and a truncated form of A beta (approximately 3K) in media from cultures of primary cells and untransfected and beta-APP-transfected cell lines grown under normal conditions. These peptides were immunoprecipitated readily from culture medium by A beta-specific antibodies and their identities confirmed by sequencing. The concept that pathological processes are responsible for the production of A beta must not be reassessed in light of the observation that A beta is produced in soluble form in vitro and in vivo during normal cellular metabolism. Further, these findings provide the basis for using simple cell culture systems to identify drugs that block the formation or release of A beta, the primary protein constituent of the senile plaques of Alzheimer's disease.
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N-truncated Aβ4-42 is highly abundant in Alzheimer disease (AD) brain and was the first Aβ peptide discovered in AD plaques. However, a possible role in AD aetiology has largely been neglected. In the present report, we demonstrate that Aβ4-42 rapidly forms aggregates possessing a high aggregation propensity in terms of monomer consumption and oligomer formation. Short-term treatment of primary cortical neurons indicated that Aβ4-42 is as toxic as pyroglutamate Aβ3-42 and Aβ1-42. In line with these findings, treatment of wildtype mice using intraventricular Aβ injection induced significant working memory deficits with Aβ4-42, pyroglutamate Aβ3-42 and Aβ1-42. Transgenic mice expressing Aβ4-42 (Tg4-42 transgenic line) developed a massive CA1 pyramidal neuron loss in the hippocampus. The hippocampus-specific expression of Aβ4-42 correlates well with age-dependent spatial reference memory deficits assessed by the Morris water maze test. Our findings indicate that N-truncated Aβ4-42 triggers acute and long-lasting behavioral deficits comparable to AD typical memory dysfunction.
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The amyloid β-protein (Aβ) is subject to proteolytic degradation by a diverse array of peptidases and proteinases, known collectively as Aβ-degrading proteases (AβDPs). A growing number of AβDPs have been identified, which, under physiological and/or pathophysiological conditions, contribute significantly to the determination of endogenous cerebral Aβ levels. Despite more than a decade of investigation, the complete set of AβDPs remains to be established, and our understanding of even well-established AβDPs is incomplete. Nevertheless, the study of known AβDPs has contributed importantly to our understanding of the molecular pathogenesis of Alzheimer disease (AD) and has inspired the development of several novel therapeutic approaches to the regulation of cerebral Aβ levels. In this article, we discuss the general features of Aβ degradation and introduce the best-characterized AβDPs, focusing on their diverse properties and the numerous conceptual insights that have emerged from the study of each.
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Progressive cerebral deposition of the amyloid β-protein (Aβ) in brain regions serving memory and cognition is an invariant and defining feature of Alzheimer disease. A highly similar but less robust process accompanies brain aging in many nondemented humans, lower primates, and some other mammals. The discovery of Aβ as the subunit of the amyloid fibrils in meningocerebral blood vessels and parenchymal plaques has led to innumerable studies of its biochemistry and potential cytotoxic properties. Here we will review the discovery of Aβ, numerous aspects of its complex biochemistry, and current attempts to understand how a range of Aβ assemblies, including soluble oligomers and insoluble fibrils, may precipitate and promote neuronal and glial alterations that underlie the development of dementia. Although the role of Aβ as a key molecular factor in the etiology of Alzheimer disease remains controversial, clinical trials of amyloid-lowering agents, reviewed elsewhere in this book, are poised to resolve the question of its pathogenic primacy.
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Alzheimer’s disease is hypothesized to be caused by an imbalance between β-amyloid (Aβ) production and clearance that leads to Aβ accumulation in the central nervous system (CNS). Aβ production and clearance are key targets in the development of disease-modifying therapeutic agents for Alzheimer’s disease. However, there has not been direct evidence of altered Aβ production or clearance in Alzheimer’s disease. By using metabolic labeling, we measured Aβ42 and Aβ40 production and clearance rates in the CNS of participants with Alzheimer’s disease and cognitively normal controls. Clearance rates for both Aβ42 and Aβ40 were impaired in Alzheimer’s disease compared with controls. On average, there were no differences in Aβ40 or Aβ42 production rates. Thus, the common late-onset form of Alzheimer’s disease is characterized by an overall impairment in Aβ clearance.
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The amyloid beta-protein (Abeta) is believed to be the key mediator of Alzheimer's disease (AD) pathology. Abeta is most often characterized as an incidental catabolic byproduct that lacks a normal physiological role. However, Abeta has been shown to be a specific ligand for a number of different receptors and other molecules, transported by complex trafficking pathways, modulated in response to a variety of environmental stressors, and able to induce pro-inflammatory activities. Here, we provide data supporting an in vivo function for Abeta as an antimicrobial peptide (AMP). Experiments used established in vitro assays to compare antimicrobial activities of Abeta and LL-37, an archetypical human AMP. Findings reveal that Abeta exerts antimicrobial activity against eight common and clinically relevant microorganisms with a potency equivalent to, and in some cases greater than, LL-37. Furthermore, we show that AD whole brain homogenates have significantly higher antimicrobial activity than aged matched non-AD samples and that AMP action correlates with tissue Abeta levels. Consistent with Abeta-mediated activity, the increased antimicrobial action was ablated by immunodepletion of AD brain homogenates with anti-Abeta antibodies. Our findings suggest Abeta is a hitherto unrecognized AMP that may normally function in the innate immune system. This finding stands in stark contrast to current models of Abeta-mediated pathology and has important implications for ongoing and future AD treatment strategies.
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Copper plays a central role in conserved processes such as respiration, and in highly specialized processes, such as protein modification. The metalloprotease neprilysin (NEP) degrades a variety of bioactive peptides and is involved in many physiological processes. However, very little is known about the regulation of NEP activity. In the current study, we focused on the effect of Cu2+ on the enzymatic activity and protein stability of NEP. Using mouse neuroblastoma N2a cells, we found that the enzymatic activity of NEP was decreased by treatment with Cu2+ in a dose- and time-dependent manner. In our investigation of the mechanism by which Cu2+ downregulates NEP enzyme activity, we found that treatment with Cu2+ caused a decrease in the level of NEP as determined by Western blot analysis. Quantitative analysis of NEP mRNA with RT-PCR excluded the possibility that Cu2+ downregulates NEP protein at the gene transcription level. Moreover, specific proteasome inhibitors, MG132 and lactacystin, blocked the turnover of NEP, whereas inhibitors of lysosome had no significant effect, suggesting that Cu2+-induced degradation of NEP is via a proteasome pathway. Taken together, our data suggest that copper downregulates NEP activity through modulation of NEP protein degradation.
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The amyloid beta protein is deposited in the brains of patients with Alzheimer's disease but its pathogenic role is unknown. In culture, the amyloid beta protein was neurotrophic to undifferentiated hippocampal neurons at low concentrations and neurotoxic to mature neurons at higher concentrations. In differentiated neurons, amyloid beta protein caused dendritic and axonal retraction followed by neuronal death. A portion of the amyloid beta protein (amino acids 25 to 35) mediated both the trophic and toxic effects and was homologous to the tachykinin neuropeptide family. The effects of the amyloid beta protein were mimicked by tachykinin antagonists and completely reversed by specific tachykinin agonists. Thus, the amyloid beta protein could function as a neurotrophic factor for differentiating neurons, but at high concentrations in mature neurons, as in Alzheimer's disease, could cause neuronal degeneration.
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The protein component of Alzheimer's disease amyloid [neurofibrillary tangles (NFT), amyloid plaque core and congophilic angiopathy] is an aggregated polypeptide with a subunit mass of 4 kd (the A4 monomer). Based on the degree of N-terminal heterogeneity, the amyloid is first deposited in the neuron, and later in the extracellular space. Using antisera raised against synthetic peptides, we show that the N terminus of A4 (residues 1-11) contains an epitope for neurofibrillary tangles, and the inner region of the molecule (residues 11-23) contains an epitope for plaque cores and vascular amyloid. The non-protein component of the amyloid (aluminum silicate) may form the basis for the deposition or amplification (possible self-replication) of the aggregated amyloid protein. The amyloid of Alzheimer's disease is similar in subunit size, composition but not sequence to the scrapie-associated fibril and its constituent polypeptides. The sequence and composition of NFT are not homologous to those of any of the known components of normal neurofilaments.
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Brain beta-amyloid plaques are principal targets for the development of treatments designed to slow the progression of Alzheimer's disease. Intracranial injections of synthetic beta-amyloid peptide (Abeta(42)) in transgenic mice expressing the Alzheimer's disease-causing Swedish APP double mutations increased neuronal levels of neprilysin, a metalloendopeptidase that degrades Abeta(42) in vivo, on mRNA and protein level. This increase was associated with significant reductions in brain levels of Abeta and with almost complete prevention of amyloid plaque formation throughout the brain. In addition, astrogliosis normally associated with amyloidosis was significantly reduced. Our results suggest that up-regulation of neprilysin in brain may represent an opportunity to reduce or prevent amyloid plaque formation in vivo.
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Proteases that degrade the amyloid β-protein (Aβ) are important regulators of brain Aβ levels in health and in Alzheimer's disease, yet few practical methods exist to study their detailed kinetics. Here, we describe robust and quantitative Aβ degradation assays based on the novel substrate, fluorescein-Aβ-(1–40)-Lys-biotin (FAβB). Liquid chromatography/mass spectrometric analysis shows that FAβB is hydrolyzed at closely similar sites as wild-type Aβ by neprilysin and insulin-degrading enzyme, the two most widely studied Aβ-degrading proteases. The derivatized peptide is an avid substrate and is suitable for use with biological samples and in high throughput compound screening. The assays we have developed are easily implemented and are particularly useful for the generation of quantitative kinetic data, as we demonstrate by determining the kinetic parameters of FAβB degradation by several Aβ-degrading proteases, including plasmin, which has not previously been characterized. The use of these assays should yield additional new insights into the biology of Aβ-degrading proteases and facilitate the identification of activators and inhibitors of such enzymes.
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The accumulation of amyloid beta (Abeta) in the walls of small vessels in the cerebral cortex is associated with diseases characterized by dementia or stroke. These include Alzheimer's disease, Down syndrome, and sporadic and hereditary cerebral amyloid angiopathies (CAAs) related to mutations within the Abeta sequence. A higher tendency of Abeta to aggregate, a defective clearance to the systemic circulation, and insufficient proteolytic removal have been proposed as mechanisms that lead to Abeta accumulation in the brain. By using immunoprecipitation and mass spectrometry, we show that insulin-degrading enzyme (IDE) from isolated human brain microvessels was capable of degrading (125)I-insulin and cleaved Abeta-(1-40) wild type and the genetic variants Abeta A21G (Flemish), Abeta E22Q (Dutch), and Abeta E22K (Italian) at the predicted sites. In microvessels from Alzheimer's disease cases with CAA, IDE protein levels showed a 44% increase as determined by sandwich enzyme-linked immunosorbent assay and Western blot. However, the activity of IDE upon radiolabeled insulin was significantly reduced in CAA as compared with age-matched controls. These results support the notion that a defect in Abeta proteolysis by IDE contributes to the accumulation of this peptide in the cortical microvasculature. Moreover they raise the possibility that IDE inhibition or inactivation is a pathogenic mechanism that may open novel strategies for the treatment of cerebrovascular Abeta amyloidoses.
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Brain deposition of amyloid-beta (A beta) is a pathological hallmark of Alzheimer disease (AD) but A beta is also detected in non-demented elderly individuals. Neprilysin has been shown to be an important enzyme to degrade A beta in brain. We investigated whether decreased neprilysin levels contributes to the accumulation of A beta in AD and in normal aging. No difference in neprilysin protein and mRNA levels were found between AD subjects and age-matched controls. Protein levels of neprilysin were reduced with age in the temporal and frontal cortex of AD and normal brain. A significant positive correlation between insoluble A beta 40 and A beta 42 with age was found in cortex of normal brain whereas in AD brain the correlation between age and A beta was weaker. Our findings of an inverse correlation between neprilysin and insoluble A beta levels in both groups suggest that neprilysin is involved in the clearance of A beta. The observed age-dependent decline in neprilysin may be related to the increased A beta levels during normal aging. The similar rate of decline in neprilysin with age may not be the major cause of the high levels of A beta associated with AD but is likely to be a trigger of AD pathology.
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The N-truncated β-amyloid (Aβ) isoform Aβ4–x is known to bind Cu2+ via a redox-silent ATCUN motif with a conditional Kd = 30 fM at pH 7.4. This study characterizes the Cu2+ interactions and redox activity of Aβx–16 (x = 1, 4) and 2-[(dimethylamino)-methyl-8-hydroxyquinoline, a terdentate 8-hydroxyquinoline (8HQ) with a conditional Kd(CuL) = 35 pM at pH 7.4. Metal transfer between Cu(Aβ1–16), CuL, CuL2, and ternary CuL(NImAβ) was rapid, while the corresponding equilibrium between L and Aβ4–16 occurred slowly via a metastable CuL(NImAβ) intermediate. Both CuL and CuL2 were redox-silent in the presence of ascorbate, but a CuL(NIm) complex can generate reactive oxygen species. Because the NImAβ ligand will be readily exchangeable with NIm ligands of ubiquitous protein His side chains in vivo, this class of 8HQ ligand could transfer Cu2+ from inert Cu(Aβ4–x) to redox-active CuL(NIm). These findings have implications for the use of terdentate 8HQs as therapeutic chelators to treat neurodegenerative disease.
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Aβ4-42 is a major species of Aβ peptide in the brains of both healthy individuals and those affected by Alzheimer's disease. It has recently been demonstrated to bind CuII with an affinity approximately 3000 times higher than the commonly studied Aβ1-42 and Aβ1-40 peptides, which are implicated in the pathogenesis of Alzheimer's disease. Metallothionein-3, a protein considered to orchestrate copper and zinc metabolism in the brain and provide antioxidant protection, was shown to extract CuII from Aβ1-40 when acting in its native Zn7MT-3 form. This reaction is assumed to underlie the neuroprotective effect of Zn7MT-3 against Aβ toxicity. In this work, we used the truncated model peptides Aβ1-16 and Aβ4-16 to demonstrate that the high-affinity CuII complex of Aβ4-16 is resistant to Zn7MT-3 reactivity. This indicates that the analogous complex of the full-length peptide Cu(Aβ4-42) will not yield copper to MT-3 in the brain, thus supporting the concept of a physiological role for Aβ4-42 as a CuII scavenger in the synaptic cleft.
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Accumulation of the β-amyloid (Aβ) peptide in extracellular senile plaques rich in copper and zinc is a defining pathological feature of Alzheimer's disease (AD). The Aβ1-x (x=16/28/40/42) peptides have been the primary focus of Cu(II) binding studies for more than 15 years; however, the N-truncated Aβ4-42 peptide is a major Aβ isoform detected in both healthy and diseased brains, and it contains a novel N-terminal FRH sequence. Proteins with His at the third position are known to bind Cu(II) avidly, with conditional log K values at pH 7.4 in the range of 11.0-14.6, which is much higher than that determined for Aβ1-x peptides. By using Aβ4-16 as a model, it was demonstrated that its FRH sequence stoichiometrically binds Cu(II) with a conditional Kd value of 3×10(-14) M at pH 7.4, and that both Aβ4-16 and Aβ4-42 possess negligible redox activity. Combined with the predominance of Aβ4-42 in the brain, our results suggest a physiological role for this isoform in metal homeostasis within the central nervous system. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
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Decades after the discovery that ionic zinc is present at high levels in glutamatergic synaptic vesicles, where, when, and how much zinc is released during synaptic activity remains highly controversial. Here we provide a quantitative assessment of zinc dynamics in the synaptic cleft and clarify its role in the regulation of excitatory neurotransmission by combining synaptic recordings from mice deficient for zinc signaling with Monte Carlo simulations. Ambient extracellular zinc levels are too low for tonic occupation of the GluN2A-specific nanomolar zinc sites on NMDA receptors (NMDARs). However, following short trains of physiologically relevant synaptic stimuli, zinc transiently rises in the cleft and selectively inhibits postsynaptic GluN2A-NMDARs, causing changes in synaptic integration and plasticity. Our work establishes the rules of zinc action and reveals that zinc modulation extends beyond hippocampal mossy fibers to excitatory SC-CA1 synapses. By specifically moderating GluN2A-NMDAR signaling, zinc acts as a widespread activity-dependent regulator of neuronal circuits.
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We investigated the subcellular distribution of NEP protein and activity in brains of human individuals with no cognitive impairment (NCI), mild cognitive impairment (MCI) and AD dementia, as well as double transgenic mice and human neuronal cell line treated with Aβ and 4-hydroxy-2-nonenal (HNE). Total cortical neuronal-related NEP was significantly increased in MCI compared to NCI brains. NeuN was decreased in both MCI and AD, consistent with neuronal loss occurring in MCI and AD. Negative relationship between NEP protein and NeuN in MCI brains, and positive correlation between NEP and pan-cadherin in NCI and MCI brains, suggesting the increased NEP expression in NCI and MCI might be due to membrane associated NEP in non-neuronal cells. In subcellular extracts, NEP protein decreased in cytoplasmic fractions in MCI and AD, but increased in membrane fractions, with a significant increase in the membrane/cytoplasmic ratio of NEP protein in AD brains. By contrast, NEP activity was decreased in AD. Similar results were observed in AD-mimic transgenic mice. Studies of SH-SY5Y neuroblastoma showed an up-regulation of NEP protein in the cytoplasmic compartment induced by HNE and Aβ; however, NEP activity decreased in cytoplasmic fractions. Activity of NEP in membrane fractions increased at 48 hours and then significantly decreased after treatment with HNE and Aβ. The cytoplasmic/membrane ratio of NEP protein increased at 24 hours and then decreased in both HNE and Aβ treated cells. Both HNE and Aβ up-regulate NEP expression, but NEP enzyme activity did not show the same increase, possibly indicating immature cytoplasmic NEP is less active than membrane associated NEP. These observations indicate that modulation of NEP protein levels and its subcellular location influence the net proteolytic activity and this complex association might participate in deficiency of Aβ degradation that is associated with amyloid deposition in AD.
Article
Background: A promising approach for treating Alzheimer's disease relies on the net efflux of the amyloid-β (Aβ) peptide from the brain to peripheral plasma, as a result of plasma Aβ clearance promoted by plasma removal and therapeutic albumin replacement. Objective: To assess the binding of therapeutic albumin (Albutein, Grifols) to monomeric and aggregated Aβ according to methods previously tested on the interactions between Aβ and research-grade albumin. Methods: Albumin integrity and the interactions with albumin stabilizers (octanoic acid and N-Ac-Trp) were assessed through one-dimensional (1D) 1H-NMR and saturation transfer difference (STD) NMR spectra. The interactions between monomeric Aβ1-40 and albumin were probed by 2D 1H-15 N HSQC spectra of labeled Aβ1-40. The formation of cross-β structured Aβ1-42 assemblies was monitored by ThT fluorescence. The interactions between self-assembled Aβ1-42 and albumin were probed by Trp fluorescence. Results: NMR spectra indicated that both therapeutic and research-grade albumin are similarly well-folded proteins. No significant changes in either HSQC peak position or intensity were observed upon addition of albumin to 15N-labeled Aβ1-40, which rules out binding of albumin to monomeric Aβ with dissociation constant in the μM or lower range. When aggregated Aβ1-42 was added to albumin, quenching of Trp fluorescence was observed, which indicates albumin binding to Aβ1-42 aggregates. The relative potency of therapeutic albumin as an Aβ self-association inhibitor was in the same order of magnitude as research-grade albumin. Conclusions: Albutein inhibited Aβ self-association by selectively binding Aβ aggregates rather than monomers and by preventing further growth of the Aβ assemblies.
Article
Cu(II) binding to the amyloid-β peptide has been proposed to be a key event in the cascade leading to Alzheimer disease. As a direct consequence, the strength of the Cu(II) to Aβ interaction, i.e. the Cu(II) affinity of Aβ, is a very important parameter to determine. Because Aβ peptide contain one Tyr fluorophore in its sequence and because Cu(II) does quench Tyr fluorescence, fluorescence measurements appears to be a straightforward way to obtain this parameter. However, this proved to be wrong, mainly due to misinterpretation of fluorescence experiments in some previous studies that leads to a conflicting situation. In the present paper, we have investigated in details a large new set of fluorescence data that were analyzed with a new method taking into account the presence of two Cu(II) site and the inner-filter effect. This leads to re-interpretation of the published data and to the determination of a unified affinity value in the 1010 M-1 range.
Article
The potential importance of copper (Cu) in neurosecretion can be inferred from the demonstration that extracellular Cu modulates the secretory function of peptidergic neurons (in vitro studies) and from the presence of high Cu concentrations in nerve terminals and secretory vesicles, primarily within the soluble matrix of the latter. We have previously hypothesized that vesicular Cu is released from neurons undergoing exocytosis and that such extracellular Cu plays an important modulatory role in the central nervous system. To test this Cu release hypothesis, rat hypothalami were incubated under in vitro conditions for 1 or 2 hr with 20 nM radiolabeled Cu (67Cu), and then 67Cu release was stimulated by a depolarizing concentration (60 mM) of K+. K+ markedly (P < 0.001) stimulated 67Cu release in a Ca2+-dependent manner (stimulated release was 95 fmol/10 min/mg protein after 1 hr 67Cu loading and 160 after 2 hr). These amounts of released 67Cu account for about 10% of the total 67Cu taken up by the tissue. These results indicate that part of the 67Cu taken up by hypothalamic explants is directed into an intracellular compartment from where it can be released by a Ca2+-dependent mechanism, thus providing strong support to our hypothesis that release of copper is operative in situ in the brain.
Article
Accumulation of neurotoxic amyloid-β peptide (Aβ) and alteration of metal homeostasis (metallostasis) in the brain are two main factors that have been very often associated with neurodegenerative diseases, such as Alzheimer's disease (AD). Aβ is constantly produced from the amyloidprecursor-protein APP precursor and immediately catabolized under normal conditions, whereas dysmetabolism of Aβ and/or metal ions seems to lead to a pathological deposition. Although insulin-degrading enzyme (IDE) is the main metalloprotease involved in Aβ degradation in the brain being up-regulated in some areas of AD brains, the role of IDE for the onset and development of AD is far from being understood. Moreover, the biomolecular mechanisms involved in the recognition and interaction between IDE and its substrates are still obscure. In spite of the important role of metals (such as copper, aluminum, and zinc), which has brought us to propose a "metal hypothesis of AD", a targeted study of the effect of metallostasis on IDE activity has never been carried out. In this work, we have investigated the role that various metal ions (i.e., Cu(2+), Cu(+), Zn(2+), Ag(+), and Al(3+)) play in modulating the interaction between IDE and two Aβ peptide fragments, namely Aβ(1-16) and Aβ(16-28). It was therefore possible to identify the direct effect that such metal ions have on IDE structure and enzymatic activity without interferences caused by metal-induced substrate modifications. Mass spectrometry and kinetic studies revealed that, among all the metal ions tested, only Cu(2+), Cu(+), and Ag(+) have an inhibitory effect on IDE activity. Moreover, the inhibition of copper(II) is reversed by adding zinc(II), whereas the monovalent cations affect the enzyme activity irreversibly. The molecular basis of their action on the enzyme is also discussed on the basis of computational investigations.
Article
A promising strategy to control the aggregation of the Alzheimer's Aβ peptide in the brain is the clearance of Aβ from the central nervous system into the peripheral blood plasma. Among plasma proteins, human serum albumin plays a critical role in the Aβ clearance to the peripheral sink by binding to Aβ oligomers and preventing further growth into fibrils. However, the stoichiometry and the affinities of the albumin-Aβ oligomer interactions are still to be fully characterized. For this purpose, here we investigate the Aβ oligomer-albumin complexes through a novel and generally applicable experimental strategy combining saturation transfer and off-resonance relaxation NMR experiments with ultrafiltration, domain deletions, and dynamic light scattering. Our results show that the Aβ oligomers are recognized by albumin through sites that are evenly partitioned across the three albumin domains and that bind the Aβ oligomers with similar dissociation constants in the 1-100 nM range, as assessed based on a Scatchard-like model of the albumin inhibition isotherms. Our data not only explain why albumin is able to inhibit amyloid formation at physiological nM Aβ concentrations, but are also consistent with the presence of a single high affinity albumin-binding site per Aβ protofibril, which avoids the formation of extended insoluble aggregates.
Article
A proposed key event in the pathogenesis of Alzheimer's disease (AD) is the formation of neurotoxic amyloid beta (Abeta) oligomers and amyloid plaques in specific brain regions that are affected by the disease. The main plaque component is the 42 amino acid isoform of Alphabeta (Abeta1-42), which is thought to initiate plaque formation and AD pathogenesis. Numerous isoforms of Abeta, e.g., Abeta1-42, Abeta1-40 and the 3-pyroglutamate derivate of Abeta3-42 (pGluAbeta3-42), have been detected in the brains of sporadic AD (SAD) and familial AD (FAD) subjects. However, the relative importance of these isoforms in the pathogenesis of AD is not fully understood. Here, we report a detailed study using immunoprecipitation in combination with mass spectrometric analysis to determine the Abeta isoform pattern in the cerebellum, cortex and hippocampus in AD, including subjects with a mutation in the presenilin (M146V) or amyloid precursor protein (KM670/671NL) genes, SAD subjects and non-demented controls. We show that the dominating Abeta isoforms in the three different brain regions analyzed from control, SAD, and FAD are Abeta1-42, pGluAbeta3-42, Abeta4-42 and Abeta1-40 of which Abeta1-42 and Abeta4-42 are the dominant isoforms in the hippocampus and the cortex in all groups analyzed, controls included. No prominent differences in Abeta isoform patterns between FAD and SAD patients were seen, underscoring the similarity in the amyloid pathology of these two disease entities.
Article
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.
Article
Membrane vesicles from rat cerebral cortex were prepared and the functional response of the GABAA receptor was followed by monitoring GABA-activated influx of the radiotracer 36Cl- ion. CuCl2 decreased GABA-activated 36Cl- influx into synaptosomal membrane vesicles. The effect of Cu2+ was concentration dependent (5-500 microM CuCl2) and occurred with saturating (1 mM) as well as low (30 microM) GABA concentrations. A similar inhibition of the responses to muscimol (30 microM) was also observed with 50 microM CuCl2. In addition, release of copper from cortical synaptosomes and median eminence was followed by atomic absorption technique. An increased release of copper into the extracellular space was observed upon depolarization with 50 mM K+. A minimal concentration of copper was estimated to be 100 microM in the synaptic cleft. These findings suggest that copper may play a role in regulating neuronal excitability.
Article
The beta-amyloid protein is progressively deposited in Alzheimer's disease as vascular amyloid and as the amyloid cores of neuritic plaques. Contrary to its metabolically inert appearance, this peptide may have biological activity. To evaluate this possibility, a peptide ligand homologous to the first 28 residues of the beta-amyloid protein (beta 1-28) was tested in cultures of hippocampal pyramidal neurons for neurotrophic or neurotoxic effects. The beta 1-28 appeared to have neurotrophic activity because it enhanced neuronal survival under the culture conditions examined. This finding may help elucidate the sequence of events leading to plaque formation and neuronal damage in Alzheimer's disease.
Article
We have purified and characterized the cerebral amyloid protein that forms the plaque core in Alzheimer disease and in aged individuals with Down syndrome. The protein consists of multimeric aggregates of a polypeptide of about 40 residues (4 kDa). The amino acid composition, molecular mass, and NH2-terminal sequence of this amyloid protein are almost identical to those described for the amyloid deposited in the congophilic angiopathy of Alzheimer disease and Down syndrome, but the plaque core proteins have ragged NH2 termini. The shared 4-kDa subunit indicates a common origin for the amyloids of the plaque core and of the congophilic angiopathy. There are superficial resemblances between the solubility characteristics of the plaque core and some of the properties of scrapie infectivity, but there are no similarities in amino acid sequences between the plaque core and scrapie polypeptides.
Article
The role of divalent transition metal ions in neural function is poorly understood. In excess, these ions are associated with neurological disorders such as Wilson's disease, Pick's disease and epileptic seizures. We suggest that zinc ions, which are contained in nerve terminals, are extruded into the extracellular space during neuronal activity. Excessive levels of zinc may be released during intense neuronal activation, and contribute to the paroxysm and toxic damage observed. Zinc ions are contained in high concentrations in mossy fibres of the hippocampal formation, and it is the postsynaptic neurones of these fibres which are most susceptible to the toxic effects of kainic acid, a potent convulsant, or to chronic exposure to organometallic compounds. Here we demonstrate for the first time that Zn2+ is released into the extracellular space during excitation of hippocampal slices.
Article
High performance liquid chromatographic analyses of incubations of beta-amyloid(1-40) with neutral endopeptidase revealed at least nine product peaks, indicating that neutral endopeptidase can cleave beta-amyloid at multiple sites. Mass spectroscopic analysis of hydrolyzed beta-amyloid identified at least five cleavage sites, between residues Glu3-Phe4, Gly9-Trp10, Phe19-Phe20, Ala30-Ile31, and Gly33-Leu34. In contrast, amyloid precursor protein metabolism in Neuro2A cells was unaffected by the expression of recombinant neutral endopeptidase in the same cells or by the addition of a secreted form of neutral endopeptidase to spent Neuro2A cell media.
Article
The pathological findings of Alzheimer's disease include amyloid deposition in cerebral blood vessels and in senile plaques. Both deposits are known to include peptides that contain a common sequence. Both forms of amyloid were isolated and their peptide compositions were determined. The peptides were resolved by size-exclusion chromatography in 70% formic acid, and reverse-phase chromatography in 60% formic acid, 0-40% acetonitrile. Senile plaque amyloid cores contain about 25% protein, about 70% of which is composed of peptides containing the beta-amyloid sequence. Amino-terminal sequencing of the core amyloid peptides (CAPs) revealed extensive amino-terminal heterogeneity, with variable amounts of blocked amino termini. Matrix-assisted, laser-desorption-time-of-flight mass spectrometry of the CAP mixture revealed an array of peptides the molecular weights of which corresponded to peptides beginning with each of the first 11 amino acids of the beta-peptide sequence and ending with Ala-42 of that sequence. The carboxyl-terminal residues were identified by tandem mass spectrometry of chymotrypsin digests. CAP possessed a minor degree of carboxyl-terminal heterogeneity. Cerebrovascular amyloid peptides (CVAPs) possessed minor degrees of both amino- and carboxyl-terminal heterogeneity. The major CVAP commenced at Asp-1 and ended at Val-40. Minor components of CAP possessed masses of 8000-9000 Da and the same amino-terminal residues as the major components of CAP. They may be precursors to the smaller CAPs. The differences in amino termini and carboxyl termini of CAPs and CVAPs suggest that the two types of amyloid form by different pathways, on which they encounter different proteases.
Article
Neprilysin is an enzyme capable of degrading beta-amyloid protein. We measured neprilysin mRNA and protein levels in brain and peripheral organs of Alzheimer disease (AD) and control cases. Neprilysin mRNA levels were lowest in the hippocampus and temporal gyrus, which are vulnerable to senile plaque development. They were highest in the caudate and peripheral organs which are resistant to senile plaque development. Levels in AD were significantly lower than controls in the hippocampus and midtemporal gyrus but not in other brain areas or peripheral organs. We also measured levels of the mRNA for the neuronal marker microtubule-associated protein-2. They were remarkably constant in all brain areas and were not lowered in AD, indicating that the neprilysin mRNA reduction in the hippocampus and temporal gyrus was not correlated with simple neuronal loss. Relative levels of neprilysin protein generally paralleled those of the mRNA. These results suggest that deficient degradation of beta-amyloid protein caused by low levels of neprilysin may contribute to AD pathogenesis.
Article
Amyloid beta-peptide (Abeta) is widely believed to play a central role in Alzheimer's disease (AD). Coordinate regulation of cerebral Abeta level is important in the pathogenesis of AD since either increased production of Abeta from amyloid precursor protein or decreased degradation causes elevated levels of Abeta, leading to accumulation of cerebral plaque formation or amyloid angiopathy. Here we studied neprilysin, a putative proteolytic enzyme for Abeta, and found that it degraded not only monomeric but also oligomeric forms of Abeta1-40. Moreover, neprilysin was found to be capable of degradation of the oligomeric form of Abeta1-42, a significant Abeta species in early pathogenesis. Neprilysin to decrease cerebral Abeta is suggested to be inevitable factor as a vital therapeutic target.
Article
Neutral endopeptidase (NEP) is the major enzyme involved in the metabolic inactivation of a number of bioactive peptides including the enkephalins, substance P, endothelin, bradykinin and atrial natriuretic factor. Owing to the physiological importance of NEP in the modulation of nociceptive and pressor responses, there is considerable interest in inhibitors of this enzyme as novel analgesics and antihypertensive agents. Here, the crystal structures of the soluble extracellular domain of human NEP (residues 52-749) complexed with various potent and competitive inhibitors are described. The structures unambiguously reveal the binding mode of the different zinc-chelating groups and the subsite specificity of the enzyme.
Article
The proteolysis of beta-amyloid (Abeta) requires neprylisin, an enzyme that has been shown as reduced in Alzheimer's disease (AD). We investigated whether a decrease in neprilysin levels contributes to the accumulation of amyloid deposits not only in AD but also in the normal aging. We analyzed neprilysin mRNA and protein levels in cerebral cortex from 10 cognitively normal elderly subjects with amyloid plaques (NA), 10 cases of AD, and 10 control cases free of amyloid plaques. We found a significant decrease in neprilysin mRNA levels in both AD and NA compared to control cases. Thereby, the defect of neprilysin appears to correlate with Abeta deposition but not with degeneration and dementia.
Article
The copper complex [KGHK-Cu]+ demonstrates catalytic inactivation of human angiotensin converting enzyme at sub-saturating concentrations, under oxidative conditions, with an observed rate constant k approximately 2.9 +/- 0.5 x 10(-2) min(-1).
Article
Cerebral deposition of the amyloid beta protein (Abeta), an invariant feature of Alzheimer's disease, reflects an imbalance between the rates of Abeta production and clearance. The causes of Abeta elevation in the common late-onset form of Alzheimer's disease (LOAD) are largely unknown. There is evidence that the Abeta-degrading protease neprilysin (NEP) is down-regulated in normal aging and LOAD. We asked whether a decrease in endogenous NEP levels can prolong the half-life of Abeta in vivo and promote development of the classic amyloid neuropathology of Alzheimer's disease. We examined the brains and plasma of young and old mice expressing relatively low levels of human amyloid precursor protein and having one or both NEP genes silenced. NEP loss of function 1) elevated whole-brain and plasma levels of human Abeta(40) and Abeta(42), 2) prolonged the half-life of soluble Abeta in brain interstitial fluid of awake animals, 3) raised the concentration of Abeta dimers, 4) markedly increased hippocampal amyloid plaque burden, and 5) led to the development of amyloid angiopathy. A approximately 50% reduction in NEP levels, similar to that reported in some LOAD brains, was sufficient to increase amyloid neuropathology. These findings demonstrate an important role for proteolysis in determining the levels of Abeta and Abeta-associated neuropathology in vivo and support the hypothesis that primary defects in Abeta clearance can cause or contribute to LOAD pathogenesis.
Article
Human serum albumin (HSA) is the major carrier of Abeta peptides in blood plasma. 1:1 interaction stoichiometries were established in previous indirect antibody-based studies for both Abeta40 and Abeta42, but corresponding binding constants were not provided. In this study we applied direct titrations of HSA with Abeta40 monitored using circular dichroism spectroscopy and obtained a dissociation constant (K(d)) of 5+/-1 microM for a HSA complex with Abeta40. The interaction resulted in an increase of the alpha-helical contents in the complex, compared to its components, which is quantitatively consistent with the known ability of Abeta40 to adopt a partially alpha-helical conformation in a hydrophobic environment. The relevance of these findings for the role of HSA in Abeta physiology is discussed.
Proteolytic degradation of amyloid βprotein. Cold Spring Harbor Perspect
  • T Saido
  • M A Leissring
Saido, T.; Leissring, M. A. Proteolytic degradation of amyloid βprotein. Cold Spring Harbor Perspect. Med. 2012, 2, a006379.