Mitochondrial import and degradation of amyloid-β peptide

Mitochondrial dysfunctions associated with amyloid-β peptide (Aβ) accumulation in mitochondria have been observed in Alzheimer's disease (AD) patients' brains and in AD mice models. Aβ is produced by sequential action of β- and γ-secretases cleaving the amyloid precursor protein (APP). The γ-secretase complex was found in mitochondria-associated endoplasmic reticulum membranes (MAM) suggesting that this could be a potential site of Aβ production, from which Aβ is further transported into the mitochondria. In vitro, Aβ was shown to be imported into the mitochondria through the translocase of the outer membrane (TOM) complex. The mitochondrial presequence protease (PreP) is responsible for Aβ degradation reducing toxic effects of Aβ on mitochondrial functions. The proteolytic activity of PreP is, however, lower in AD brain temporal lobe mitochondria and in AD transgenic mice models, possibly due to an increased reactive oxygen species (ROS) production. Here, we review the intracellular mechanisms of Aβ production, its mitochondrial import and the intra-mitochondrial degradation. We also discuss the implications of a reduced efficiency of mitochondrial Aβ clearance for AD. Understanding the underlying mechanisms may provide new insights into mitochondria related pathogenesis of AD and development of drug therapy against AD. This article is part of a Special Issue entitled: 18th European Bioenergetic Conference.     

Structural and mechanistic commonalities of amyloid-β and the prion protein

Amyloid beta (Aβ) is a major causative agent of Alzheimer disease (AD). This neurotoxic peptide is generated as a result of the cleavage of the Amyloid-Precursor-Protein (APP) by the action of β-secretase and γ-secretase. The neurotoxicity was previously thought to be the result of aggregation. However, recent studies suggest that the interaction of Aβ with numerous cell surface receptors such as N-methyl-D-aspartate (NMDA), receptor for advanced glycosylation end products (RAGE), P75 neurotrophin receptor (P75^NTR) as well as cell surface proteins such as the cellular prion protein (PrP^c) and heparan sulfate proteoglycans (HSPG) strongly enhances Aβ induced apoptosis and thereby contributes to neurotoxicity. This review focuses on the molecular mechanism resulting in Aβ-shedding as well as Aβ-induced apoptotic processes, genetic risk factors for familial AD and interactions of Aβ with cell surface receptors and proteins, with particular emphasis on the cellular prion protein. Furthermore, comparisons are drawn between AD and prion disorders and the role of laminin, an extracellular matrix protein, glycosaminoglycans and the 37 kDa/67 kDa laminin receptor (LRP/LR) have been highlighted with regards to both neurodegenerative diseases.Key words: Alzheimer disease, amyloid β, apoptosis, 37 kDa/67 kDa laminin receptor, prion proteinsAlzheimer disease (AD), primarily defined by psychiatrist Alois Alzheimer in 1906, is a neurodegenerative disorder and currently exhibits a prevalence that “doubles approximately every five years from 0.5% at the common age of onset-65 years old.”¹ This disease is the most common form of dementia afflicting the elderly and at present affects in excess of 37 million people globally² and it is predicted that 100 million people will be living with the disease by 2050.³AD has received mounting scientific interest and has stimulated tireless research endeavours not only due to the complex mechanism by which it is caused; the multitude of contributing factors and contradictions which have arisen between hypotheses and acquired results, but also due to the rise in life expectancies⁴ owing to the advent of modern medicine, which has socio-economic implications particularly in terms of strain placed upon national health systems.     

Homogeneous and heterogeneous tertiary structure ensembles of amyloid-β peptides

The interplay of modern molecular simulation and high-quality nuclear magnetic resonance (NMR) experiments has reached a fruitful stage for quantitative characterization of structural ensembles of disordered peptides. Amyloid-β 1-42 (Aβ42), the primary peptide associated with Alzheimer's disease, and fragments such as Aβ21-30 are both classified as intrinsically disordered peptides (IDPs). We use a variety of NMR observables to validate de novo molecular dynamics simulations in explicit water to characterize the tertiary structure ensemble of Aβ42 and Aβ21-30 from the perspective of their classification as IDPs. Unlike the Aβ21-30 fragment that conforms to expectations of an IDP that is primarily extended, we find that Aβ42 samples conformations reflecting all possible secondary structure categories and spans the range of IDP classifications from collapsed structured states to highly extended conformations, making it an IDP with a far more heterogeneous tertiary ensemble.     

The role of metallobiology and amyloid-β peptides in Alzheimer's disease

The biggest risk factor for Alzheimer's disease is the process of ageing, but the mechanisms that lead to the manifestation of the disease remain to be elucidated. Why age triggers the disease is unclear but an emerging theme is the inability for a cell to efficiently maintain many key processes such as energy production, repair, and regenerative mechanisms. Metal ions are essential to the metabolic function of every cell. This review will explore the role and reported changes in metal ions in Alzheimer disease, particularly the brain, blood and cerebral spinal fluid, emphasizing how iron, copper and zinc may be involved through the interactions with amyloid precursor protein, the proteolytically cleaved peptide amyloid-beta (Aβ), and other related metalloproteins. Finally, we explore the monomeric makeup of possible Aβ dimers, what a dimeric Aβ species from Alzheimer's disease brain tissue is likely to be composed of, and discuss how metals may influence Aβ production and toxicity via a copper catalyzed dityrosine cross-link.     

Crocetin attenuates inflammation and amyloid-β accumulation in APPsw transgenic mice

Background

Crocetin, an agent derived from saffron, has multiple pharmacological properties, such as neuroprotective, anti-oxidant, and anti-inflammatory actions. These properties might benefit the treatment of Alzheimer’s disease (AD). In the present study, we tested whether crocetin attenuates inflammation and amyloid-β (Aβ) accumulation in APPsw transgenic mice, AD mouse models. Cell viability and the levels of Aβ40 and Aβ42 in HeLa cells stably transfected with Swedish mutant APP751 were evaluated. Mice with Swedish mutant APP751 transgene were used as transgenic mouse models of AD, and were orally administrated with crocetin. Aβ protein and inflammatory cytokines were measured with ELISA. NF-κB and P53 were measured with western blot assay. Learning and memory were analyzed with Morris water maze and novel object recognition tests.

Results

Crocetin significantly reduced Aβ40 and Aβ42 secretion in Hela cells without effecting cell viability. In AD transgenic mice, crocetin significantly reduced the pro-inflammatory cytokines and enhanced anti-inflammatory cytokine in plasma, suppressed NF-κB activation and P53 expression in the hippocampus, decreased Aβ in various brain areas, and improved learning and memory deficits.

Conclusion

Crocetin improves Aβ accumulation-induced learning and memory deficit in AD transgenic mice, probably due to its anti-inflammatory and anti-apoptotic functions.

     

Metal-associated amyloid-β species in Alzheimer's disease

Highly concentrated metals such as Cu, Zn, and Fe are found in amyloid-β (Aβ) plaques within the brain of Alzheimer's disease (AD). In vitro and in vivo studies have suggested that metal binding to Aβ could facilitate Aβ aggregation and generate reactive oxygen species (ROS), which could contribute to the neuropathogenesis of AD. The connection between metal-Aβ interaction/reactivity and AD development, however, has not been clearly revealed owing to the complexity of the disease. In this review, metal-Aβ interaction/reactivity and its relation to neurotoxicity are briefly discussed. Additionally, our review illustrates the recent progress of small molecules, capable of targeting metal-Aβ species and modulating their interaction/reactivity, which could offer a promising approach to interrogate their role in AD.     

Synthesis and characterization of IMPY derivatives that regulate metal-induced amyloid-β aggregation

Metal ions associated with amyloid-β (Aβ) species have been suggested to be involved in neurodegeneration leading to the progression of Alzheimer's disease (AD). The role of metal-involved Aβ species in AD neuropathogenesis, however, is not fully elucidated. In order to advance this understanding and contribute to the therapeutic development for AD, the rational structure-based design of small molecules that specifically target metal ions surrounded by Aβ species has recently received increased attention. To date, only a few compounds have been fashioned for this purpose. Herein, we report the design strategy, synthesis, characterization, and reactivity of new bifunctional IMPY derivatives K1 and K2. Using UV-vis and high-resolution two-dimensional (2D) NMR spectroscopy, the bifunctionality of K1 and K2 (metal chelation and Aβ interaction) was confirmed. These bifunctional IMPY derivatives showed preferential reactivity toward metal-induced Aβ aggregation over metal-free conditions in both in vitro inhibition and disaggregation experiments. Taken together, this study provides another example of a bifunctional small molecule framework that can target metal ions associated with Aβ species.     

Relevance of N-terminal residues for amyloid-β binding to platelet integrin αIIbβ3, integrin outside-in signaling and amyloid-β fibril formation

A pathological hallmark of Alzheimer's disease (AD) is the aggregation of amyloid-β peptides (Aβ) into fibrils, leading to deposits in cerebral parenchyma and vessels known as cerebral amyloid angiopathy (CAA). Platelets are major players of hemostasis but are also implicated in AD. Recently we provided strong evidence for a direct contribution of platelets to AD pathology. We found that monomeric Aβ40 binds through its RHDS sequence to integrin α_IIbβ₃, and promotes the formation of fibrillar Aβ aggregates by the secretion of adenosine diphosphate (ADP) and the chaperone protein clusterin (CLU) from platelets. Here we investigated the molecular mechanisms of Aβ binding to integrin α_IIbβ₃ by using Aβ11 and Aβ16 peptides. These peptides include the RHDS binding motif important for integrin binding but lack the central hydrophobic core and the C-terminal sequence of Aβ. We observed platelet adhesion to truncated N-terminal Aβ11 and Aβ16 peptides that was not mediated by integrin α_IIbβ₃. Thus, no integrin outside-in signaling and reduced CLU release was detected. Accordingly, platelet mediated Aβ fibril formation was not observed. Taken together, the RHDS motif of Aβ is not sufficient for Aβ binding to platelet integrin α_IIbβ₃ and platelet mediated Aβ fibril formation but requires other recognition or binding motifs important for platelet mediated processes in CAA. Thus, increased understanding of the molecular mechanisms of Aβ binding to platelet integrin α_IIbβ₃ is important to understand the role of platelets in amyloid pathology.     

A kinetic aggregation assay allowing selective and sensitive amyloid-β quantification in cells and tissues

The process of amyloid-β (Aβ) fibril formation is genetically and pathologically linked to Alzheimer's disease (AD). Thus, a selective and sensitive method for quantifying Aβ fibrils in complex biological samples allows a variety of hypotheses to be tested. Herein, we report the basis for a quantitative in vitro kinetic aggregation assay that detects seeding-competent Aβ aggregates in mammalian cell culture media, in Caenorhabditis elegans lysate, and in mouse brain homogenate. Sonicated, proteinase K-treated Aβ fibril-containing tissue homogenates or cell culture media were added to an initially monomeric Aβ(1-40) reporter peptide to seed an in vitro nucleated aggregation reaction. The reduction in the half-time (t(50)) of the amyloid growth phase is proportional to the quantity of seeding-competent Aβ aggregates present in the biological sample. An ion-exchange resin amyloid isolation strategy from complex biological samples is demonstrated as an alternative for improving the sensitivity and linearity of the kinetic aggregation assay.     

Zinc-mediated modulation of the configuration and activity of complexes between copper and amyloid-β peptides

A growing body of Alzheimer's disease (AD) research is concerned with understanding the interaction between amyloid-β (Aβ) peptides and metal ions (e.g., Cu, Zn, and Fe) and determining the biological relevance of the metal-Aβ complexes to essential metal homeostasis and neuronal cell loss. Previously, many studies have dealt with the interaction between Aβ and "single" but not "multiple" metal ions in terms of binding affinity and coordination chemistry. In the present work, we found that Zn(II) ions modified the configuration of Aβ-Cu(II) by forming Zn(II)-Aβ-Cu(II) ternary complexes. As a result, the catalytic activity of Aβ-Cu(II) against a biological ascorbic acid species was repressed by Zn(II) binding. The formation of the ternary complex can therefore explain the protective role of Zn(II) in AD.     

Water-soluble ferulic acid derivatives improve amyloid-β-induced neuronal cell death and dysmnesia through inhibition of amyloid-β aggregation

Ferulic acid (FA) has been reported to exhibit protective effects against amyloid-β (Aβ)-induced neurodegeneration in vitro and in vivo. Recently, we developed two water-soluble FA derivatives: 1-feruloyl glycerol and 1-feruloyl diglycerol. In this study, we examined the neuroprotective effects of these water-soluble FA derivatives on Aβ-induced neurodegeneration both in vitro and in vivo. FA and water-soluble FA derivatives inhibited Aβ aggregation and destabilized pre-aggregated Aβ to a similar extent. Furthermore, water-soluble FA derivatives, as well as FA, inhibited Aβ-induced neuronal cell death in cultured neuronal cells. In in vivo experiments, oral administration of water-soluble FA derivatives to mice improved Aβ-induced dysmnesia assessed by contextual fear conditioning test and protected hippocampal neurons against Aβ-induced neurotoxicity. This study provides useful evidence suggesting that water-soluble FA derivatives are expected to be effective neuroprotective agents.     

Solid-state NMR reveals a close structural relationship between amyloid-β protofibrils and oligomers

We have studied tertiary contacts in protofibrils and mature fibrils of amyloid-β (Aβ) peptides using solid-state NMR spectroscopy. Although intraresidue contacts between Glu-22 and Ile-31 were found in Aβ protofibrils, these contacts were completely absent in mature Aβ fibrils. This is consistent with the current models of mature Aβ fibrils. As these intramolecular contacts have also been reported in Aβ oligomers, our measurements suggest that Aβ protofibrils are structurally more closely related to oligomers than to mature fibrils. This suggests that some structural alterations have to take place on the pathway from Aβ oligomers/protofibrils to mature fibrils, in agreement with a model that suggests a conversion of intramolecular hydrogen-bonded structures of Aβ oligomers to the intermolecular stabilized mature fibrils (Hoyer, W., Grönwall, C., Jonsson, A., Ståhl, S., and Härd, T. (2008) Proc. Natl. Acad. Sci. U.S.A. 105, 5099–5104).     

Black tea theaflavins inhibit formation of toxic amyloid-β and α-synuclein fibrils

Causal therapeutic approaches for amyloid diseases such as Alzheimer's and Parkinson's disease targeting toxic amyloid oligomers or fibrils are still emerging. Here, we show that theaflavins (TF1, TF2a, TF2b, and TF3), the main polyphenolic components found in fermented black tea, are potent inhibitors of amyloid-β (Aβ) and α-synuclein (αS) fibrillogenesis. Their mechanism of action was compared to that of two established inhibitors of amyloid formation, (-)-epigallocatechin gallate (EGCG) and congo red (CR). All three compounds reduce the fluorescence of the amyloid indicator dye thioflavin T. Mapping the binding regions of TF3, EGCG, and CR revealed that all three bind to two regions of the Aβ peptide, amino acids 12-23 and 24-36, albeit with different specificities. However, their mechanisms of amyloid inhibition differ. Like EGCG but unlike congo red, theaflavins stimulate the assembly of Aβ and αS into nontoxic, spherical aggregates that are incompetent in seeding amyloid formation and remodel Aβ fibrils into nontoxic aggregates. When compared to EGCG, TF3 was less susceptible to air oxidation and had an increased efficacy under oxidizing conditions. These findings suggest that theaflavins might be used to remove toxic amyloid deposits.     

β-Arrestin1 regulates γ-secretase complex assembly and modulates amyloid-β pathology

Alzheimer''s disease (AD) is a progressive and complex neurodegenerative disease in which the γ-secretase-mediated amyloid-β (Aβ) pathology plays an important role. We found that a multifunctional protein, β-arrestin1, facilitated the formation of NCT/APH-1 (anterior pharynx-defective phenotype 1) precomplex and mature γ-secretase complex through its functional interaction with APH-1. Deficiency of β-arrestin1 or inhibition of binding of β-arrestin1 with APH-1 by small peptides reduced Aβ production without affecting Notch processing. Genetic ablation of β-arrestin1 diminished Aβ pathology and behavioral deficits in transgenic AD mice. Moreover, in brains of sporadic AD patients and transgenic AD mice, the expression of β-arrestin1 was upregulated and correlated well with neuropathological severity and senile Aβ plaques. Thus, our study identifies a regulatory mechanism underlying both γ-secretase assembly and AD pathogenesis, and indicates that specific reduction of Aβ pathology can be achieved by regulation of the γ-secretase assembly.     

Soluble aggregates of the amyloid-β peptide are trapped by serum albumin to enhance amyloid-β activation of endothelial cells

Background  

Self-assembly of the amyloid-β peptide (Aβ) has been implicated in the pathogenesis of Alzheimer's disease (AD). As a result, synthetic molecules capable of inhibiting Aβ self-assembly could serve as therapeutic agents and endogenous molecules that modulate Aβ self-assembly may influence disease progression. However, increasing evidence implicating a principal pathogenic role for small soluble Aβ aggregates warns that inhibition at intermediate stages of Aβ self-assembly may prove detrimental. Here, we explore the inhibition of Aβ_1–40 self-assembly by serum albumin, the most abundant plasma protein, and the influence of this inhibition on Aβ_1–40 activation of endothelial cells for monocyte adhesion.     

Human apolipoprotein A–I binds amyloid-β and prevents Aβ-induced neurotoxicity

Aggregates of the amyloid-β peptide (Aβ) play a central role in the pathogenesis of Alzheimer's disease (AD). Identification of proteins that physiologically bind Aβ and modulate its aggregation and neurotoxicity could lead to the development of novel disease-modifying approaches in AD. By screening a phage display peptide library for high affinity ligands of aggregated Aβ_1–42, we isolated a peptide homologous to a highly conserved amino acid sequence present in the N-terminus of apolipoprotein A–I (apoA-I). We show that purified human apoA-I and Aβ form non-covalent complexes and that interaction with apoA-I affects the morphology of amyloid aggregates formed by Aβ. Significantly, Aβ/apoA-I complexes were also detected in cerebrospinal fluid from AD patients. Interestingly, apoA-I and apoA-I-containing reconstituted high density lipoprotein particles protect hippocampal neuronal cultures from Aβ-induced oxidative stress and neurodegeneration. These results suggest that human apoA-I modulates Aβ aggregation and Aβ-induced neuronal damage and that the Aβ-binding domain in apoA-I may constitute a novel framework for the design of inhibitors of Aβ toxicity.     

Modulating amyloid-β aggregation: The effects of peptoid side chain placement and chirality

Alzheimer’s disease (AD) is characterized by the buildup of insoluble aggregated amyloid-β protein (Aβ) into plaques that accumulate between the neural cells in the brain. AD is the sixth leading cause of death in the United States and is the only cause of death among the top ten that cannot currently be treated or cured (Alzheimer’s Association, 2011; Selkoe, 1996). Researchers have focused on developing small molecules and peptides to prevent Aβ aggregation; however, while some compounds appear promising in vitro, the research has not resulted in a viable therapeutic treatment. We previously reported a peptoid-based mimic (JPT1) of the peptide KLVFF (residues 16–20 of Aβ) that modulates Aβ40 aggregation, specifically reducing the total number of fibrillar, β-sheet structured aggregates formed. In this study, we investigate two new variants of JPT1 that probe the importance of aromatic side chain placement (JPT1s) and side chain chirality (JPT1a). Both JPT1s and JPT1a modulate Aβ40 aggregation by reducing total β-sheet aggregates. However, JPT1a also has a pronounced effect on the morphology of fibrillar Aβ40 aggregates. These results suggest that Aβ40 aggregation may follow a different pathway in the presence of peptoids with different secondary structures. A better understanding of the interactions between peptoids and Aβ will allow for improved design of AD treatments.     

Regulation of amyloid-β production by the prion protein

Alzheimer disease (AD) is characterized by the amyloidogenic processing of the amyloid precursor protein (APP), culminating in the accumulation of amyloid-β peptides in the brain. The enzymatic action of the β-secretase, BACE1 is the rate-limiting step in this amyloidogenic processing of APP. BACE1 cleavage of wild-type APP (APP_WT) is inhibited by the cellular prion protein (PrP^C). Our recent study has revealed the molecular and cellular mechanisms behind this observation by showing that PrP^C directly interacts with the pro-domain of BACE1 in the trans-Golgi network (TGN), decreasing the amount of BACE1 at the cell surface and in endosomes where it cleaves APP_WT, while increasing BACE1 in the TGN where it preferentially cleaves APP with the Swedish mutation (APP_Swe). PrP^C deletion in transgenic mice expressing the Swedish and Indiana familial mutations (APP_Swe,Ind) failed to affect amyloid-β accumulation, which is explained by the differential subcellular sites of action of BACE1 toward APP_WT and APP_Swe. This, together with our observation that PrP^C is reduced in sporadic but not familial AD brain, suggests that PrP^C plays a key protective role against sporadic AD. It also highlights the need for an APP_WT transgenic mouse model to understand the molecular and cellular mechanisms underlying sporadic AD.     

Interactions of amyloid-β peptides on lipid bilayer studied by single molecule imaging and tracking

The amyloid-β peptides (Aβ40 and Aβ42) feature prominently in the synaptic dysfunction and neuronal loss associated with Alzheimer's disease (AD). This has been proposed to be due either to interactions between Aβ and cell surface receptors affecting cell signaling, or to the formation of calcium-permeable channels in the membrane that disrupt calcium homeostasis. In both mechanisms the cell membrane is the primary cellular structure with which Aβ interacts. Aβ concentrations in human bodily fluids are very low (pM-nM) rendering studies of the size, composition, cellular binding sites and mechanism of action of the oligomers formed in vivo very challenging. Most studies, therefore, have utilized Aβ oligomers prepared at micromolar peptide concentrations, where Aβ forms oligomeric species which possess easily observable cell toxicity. Such toxicity has not been observed when nM concentrations of peptide are used in the experiment highlighting the importance of employing physiologically relevant peptide concentrations for the results to be of biological significance. In this paper single-molecule microscopy was used to monitor Aβ oligomer formation and diffusion on a supported lipid bilayer at nanomolar peptide concentrations. Aβ monomers, the dominant species in solution, tightly associate with the membrane and are highly mobile whereas trimers and higher-order oligomers are largely immobile. Aβ dimers exist in a mixture of mobile and immobile states. Oligomer growth on the membrane is more rapid for Aβ40 than for the more amyloidogenic Aβ42 but is largely inhibited for a 1:1 Aβ40:Aβ42 mixture. The mechanism underlying these Aβ40-Aβ42 interactions may feature in Alzheimer's pathology.     

Interactions between amyloid-β and hemoglobin: implications for amyloid plaque formation in Alzheimer's disease

Accumulation of amyloid-β (Aβ) peptides in the brain is one of the central pathogenic events in Alzheimer's disease (AD). However, why and how Aβ aggregates within the brain of AD patients remains elusive. Previously, we demonstrated hemoglobin (Hb) binds to Aβ and co-localizes with the plaque and vascular amyloid deposits in post-mortem AD brains. In this study, we further characterize the interactions between Hb and Aβ in vitro and in vivo and report the following observations: 1) the binding of Hb to Aβ required iron-containing heme; 2) other heme-containing proteins, such as myoglobin and cytochrome C, also bound to Aβ; 3) hemin-induced cytotoxicity was reduced in neuroblastoma cells by low levels of Aβ; 4) Hb was detected in neurons and glial cells of post-mortem AD brains and was up-regulated in aging and APP/PS1 transgenic mice; 5) microinjection of human Hb into the dorsal hippocampi of the APP/PS1 transgenic mice induced the formation of an envelope-like structure composed of Aβ surrounding the Hb droplets. Our results reveal an enhanced endogenous expression of Hb in aging brain cells, probably serving as a compensatory mechanism against hypoxia. In addition, Aβ binds to Hb and other hemoproteins via the iron-containing heme moiety, thereby reducing Hb/heme/iron-induced cytotoxicity. As some of the brain Hb could be derived from the peripheral circulation due to a compromised blood-brain barrier frequently observed in aged and AD brains, our work also suggests the genesis of some plaques may be a consequence of sustained amyloid accretion at sites of vascular injury.