Amyloid-β Protofibrils: Size,Morphology and Synaptotoxicity of an Engineered Mimic

Structural and biochemical studies of the aggregation of the amyloid-β peptide (Aβ) are important to understand the mechanisms of Alzheimer''s disease, but research is complicated by aggregate inhomogeneity and instability. We previously engineered a hairpin form of Aβ called Aβcc, which forms stable protofibrils that do not convert into amyloid fibrils. Here we provide a detailed characterization of Aβ₄₂ cc protofibrils. Like wild type Aβ they appear as smooth rod-like particles with a diameter of 3.1 (±0.2) nm and typical lengths in the range 60 to 220 nm when observed by atomic force microscopy. Non-perturbing analytical ultracentrifugation and nanoparticle tracking analyses are consistent with such rod-like protofibrils. Aβ₄₂ cc protofibrils bind the ANS dye indicating that they, like other toxic protein aggregates, expose hydrophobic surface. Assays with the OC/A11 pair of oligomer specific antibodies put Aβ₄₂ cc protofibrils into the same class of species as fibrillar oligomers of wild type Aβ. Aβ₄₂ cc protofibrils may be used to extract binding proteins in biological fluids and apolipoprotein E is readily detected as a binder in human serum. Finally, Aβ₄₂ cc protofibrils act to attenuate spontaneous synaptic activity in mouse hippocampal neurons. The experiments indicate considerable structural and chemical similarities between protofibrils formed by Aβ₄₂ cc and aggregates of wild type Aβ₄₂. We suggest that Aβ₄₂ cc protofibrils may be used in research and applications that require stable preparations of protofibrillar Aβ.     

Amyloid-β triggers the release of neuronal hexokinase 1 from mitochondria

Brain accumulation of the amyloid-β peptide (Aβ) and oxidative stress underlie neuronal dysfunction and memory loss in Alzheimer's disease (AD). Hexokinase (HK), a key glycolytic enzyme, plays important pro-survival roles, reducing mitochondrial reactive oxygen species (ROS) generation and preventing apoptosis in neurons and other cell types. Brain isozyme HKI is mainly associated with mitochondria and HK release from mitochondria causes a significant decrease in enzyme activity and triggers oxidative damage. We here investigated the relationship between Aβ-induced oxidative stress and HK activity. We found that Aβ triggered HKI detachment from mitochondria decreasing HKI activity in cortical neurons. Aβ oligomers further impair energy metabolism by decreasing neuronal ATP levels. Aβ-induced HKI cellular redistribution was accompanied by excessive ROS generation and neuronal death. 2-deoxyglucose blocked Aβ-induced oxidative stress and neuronal death. Results suggest that Aβ-induced cellular redistribution and inactivation of neuronal HKI play important roles in oxidative stress and neurodegeneration in AD.     

The Properties of Amyloid-β Fibrils Are Determined by their Path of Formation

Fibril formation of the amyloid-β peptide (Aβ) follows a nucleation-dependent polymerization process and is associated with Alzheimer's disease. Several different lengths of Aβ are observed in vivo, but Aβ1–40 and Aβ1–42 are the dominant forms. The fibril architectures of Aβ1–40 and Aβ1–42 differ and Aβ1–42 assemblies are generally considered more pathogenic. We show here that monomeric Aβ1–42 can be cross-templated and incorporated into the ends of Aβ1–40 fibrils, while incorporation of Aβ1–40 monomers into Aβ1–42 fibrils is very poor. We also show that via cross-templating incorporated Aβ monomers acquire the properties of the parental fibrils. The suppressed ability of Aβ1–40 to incorporate into the ends of Aβ1–42 fibrils and the capacity of Aβ1–42 monomers to adopt the properties of Aβ1–40 fibrils may thus represent two mechanisms reducing the total load of fibrils having the intrinsic, and possibly pathogenic, features of Aβ1–42 fibrils in vivo. We also show that the transfer of fibrillar properties is restricted to fibril-end templating and does not apply to cross-nucleation via the recently described path of surface-catalyzed secondary nucleation, which instead generates similar structures to those acquired via de novo primary nucleation in the absence of catalyzing seeds. Taken together these results uncover an intrinsic barrier that prevents Aβ1–40 from adopting the fibrillar properties of Aβ1–42 and exposes that the transfer of properties between amyloid-β fibrils are determined by their path of formation.     

Curcumin Decreases Amyloid-β Peptide Levels by Attenuating the Maturation of Amyloid-β Precursor Protein

Alzheimer disease (AD) is a devastating neurodegenerative disease with no cure. The pathogenesis of AD is believed to be driven primarily by amyloid-β (Aβ), the principal component of senile plaques. Aβ is an ∼4-kDa peptide generated via cleavage of the amyloid-β precursor protein (APP). Curcumin is a compound in the widely used culinary spice, turmeric, which possesses potent and broad biological activities, including anti-inflammatory and antioxidant activities, chemopreventative effects, and effects on protein trafficking. Recent in vivo studies indicate that curcumin is able to reduce Aβ-related pathology in transgenic AD mouse models via unknown molecular mechanisms. Here, we investigated the effects of curcumin on Aβ levels and APP processing in various cell lines and mouse primary cortical neurons. We show for the first time that curcumin potently lowers Aβ levels by attenuating the maturation of APP in the secretory pathway. These data provide a mechanism of action for the ability of curcumin to attenuate amyloid-β pathology.     

Structure and Dynamics of Amyloid-β Segmental Polymorphisms

It is believed that amyloid-beta (Aβ) aggregates play a role in the pathogenesis of Alzheimer's disease. Aβ molecules form β-sheet structures with multiple interaction sites. This polymorphism gives rise to differences in morphology, physico-chemical property and level of cellular toxicity. We have investigated the conformational stability of various segmental polymorphisms using molecular dynamics simulations and find that the segmental polymorphic models of Aβ retain a U-shaped architecture. Our results demonstrate the importance of inter-sheet side chain-side chain contacts, hydrophobic contacts among the strands (β1 and β2) and of salt bridges in stabilizing the aggregates. Residues in β-sheet regions have smaller fluctuation while those at the edge and loop region are more mobile. The inter-peptide salt bridges between Asp23 and Lys28 are strong compared to intra-chain salt bridge and there is an exchange of the inter-chain salt-bridge with intra-chain salt bridge. As our results suggest that Aβ exists under physiological conditions as an ensemble of distinct segmental polymorphs, it may be necessary to account in the development of therapeutics for Alzheimer's disease the differences in structural stability and aggregation behavior of the various Aβ polymorphic forms.     

Amyloid-β forms fibrils by nucleated conformational conversion of oligomers

Amyloid-β amyloidogenesis is reported to occur via a nucleated polymerization mechanism. If this is true, the energetically unfavorable oligomeric nucleus should be very hard to detect. However, many laboratories have detected early nonfibrillar amyloid-β oligomers without observing amyloid fibrils, suggesting that a mechanistic revision may be needed. Here we introduce Cys-Cys-amyloid-β(1-40), which cannot bind to the latent fluorophore FlAsH as a monomer, but can bind FlAsH as an nonfibrillar oligomer or as a fibril, rendering the conjugates fluorescent. Through FlAsH monitoring of Cys-Cys-amyloid-β(1-40) aggregation, we found that amyloid-β(1-40) rapidly and efficiently forms spherical oligomers in vitro (85% yield) that are kinetically competent to slowly convert to amyloid fibrils by a nucleated conformational conversion mechanism. This methodology was used to show that plasmalogen ethanolamine vesicles eliminate the proteotoxicity-associated oligomerization phase of amyloid-β amyloidogenesis while allowing fibril formation, rationalizing how low concentrations of plasmalogen ethanolamine in the brain are epidemiologically linked to Alzheimer's disease.     

Disruption of Arterial Perivascular Drainage of Amyloid-β from the Brains of Mice Expressing the Human APOE ε4 Allele

Failure of elimination of amyloid-β (Aβ) from the brain and vasculature appears to be a key factor in the etiology of sporadic Alzheimer's disease (AD) and cerebral amyloid angiopathy (CAA). In addition to age, possession of an apolipoprotein E (APOE) ε4 allele is a strong risk factor for the development of sporadic AD. The present study tested the hypothesis that possession of the APOE ε4 allele is associated with disruption of perivascular drainage of Aβ from the brain and with changes in cerebrovascular basement membrane protein levels. Targeted replacement (TR) mice expressing the human APOE3 (TRE3) or APOE4 (TRE4) genes and wildtype mice received intracerebral injections of human Aβ(40). Aβ(40) aggregated in peri-arterial drainage pathways in TRE4 mice, but not in TRE3 or wildtype mice. The number of Aβ deposits was significantly higher in the hippocampi of TRE4 mice than in the TRE3 mice, at both 3- and 16-months of age, suggesting that clearance of Aβ was disrupted in the brains of TRE4 mice. Immunocytochemical and Western blot analysis of vascular basement membrane proteins demonstrated significantly raised levels of collagen IV in 3-month-old TRE4 mice compared with TRE3 and wild type mice. In 16-month-old mice, collagen IV and laminin levels were unchanged between wild type and TRE3 mice, but were lower in TRE4 mice. The results of this study suggest that APOE4 may increase the risk for AD through disruption and impedance of perivascular drainage of soluble Aβ from the brain. This effect may be mediated, in part, by changes in age-related expression of basement membrane proteins in the cerebral vasculature.     

Convergence of Amyloid-β and Tau Pathologies on Mitochondria In Vivo

The histopathological characteristics of Alzheimer’s disease (AD) are amyloid-β (Aβ) containing plaques and neurofibrillary tangles (NFTs) as well as neuronal and synaptic loss. Until today, the underlying mechanisms of the interplay of plaques and tangles remained unresolved. There is increasing evidence that mitochondrial dysfunction might be a possible link, as revealed by studies in several APP and tau transgenic mouse models. Recently, we examined mitochondrial function in a novel triple transgenic mouse model (pR5/APP/PS2)—^tripleAD mice—that combines both pathologic features of the disease in brain. Using comparative, quantitative proteomics (iTRAQ) and mass spectroscopy, we found a massive deregulation of 24 proteins, of which one third were mitochondrial proteins mainly related to complexes I and IV of the oxidative phosphorylation system (OXPHOS). Remarkably, deregulation of complex I was related to tau, whereas deregulation of complex IV was Aβ dependent, both at the protein and activity levels. The ^tripleAD mice showed synergistic effects of Aβ and tau already at the age of 8 months, resulting in a depolarized mitochondrial membrane potential. At 12 months, the strongest defects on OXPHOS, synthesis of ATP and reactive oxygen species, were exhibited in the ^tripleAD mice, again emphasizing synergistic, age-associated effects of Aβ and tau in impairing mitochondria. This review highlights the convergence of Aβ and tau on mitochondria and establishes a molecular link in AD pathology in vivo.     

P38 Activation Mediates Amyloid-β Cytotoxicity

Amyloid-β is a leading candidate factor in the development of Alzheimer disease (AD), however the mechanisms involved are unclear. As such, there has been considerable interest in evidence showing that the neuronal damage caused by amyloid-β is mediated by oxidative stress. Notably, oxidative stress leads to activation of stress-activated protein kinases, which we and others have shown are also involved in AD pathogenesis. One SAPK in particular, p38, appears to be crucial in AD and therefore, in the current study, we investigated the role of p38 activation in amyloid-β cytotoxicity. Our data showed p38 activation was induced by amyloid-β in a concentration-dependent manner in M17 human neuroblastoma cells. Notably, amyloid-β toxicity was significantly decreased by inhibition of p38 activity by overexpressing dominant negative p38. Consistent with this, in primary cortical neurons amyloid-β also induced p38 activation and amyloid-β toxicity was significantly diminished when p38 was inhibited by its specific inhibitor, SB203580. Taken together, these data suggest that p38 is a key downstream effector of amyloid-β-induced neuronal death and blocking this pathway may be of therapeutic value.     

Functional and Structural Effects of Amyloid-β Aggregate on Xenopus laevis Oocytes

Xenopus laevis oocytes exposed to amyloid-β aggregate generated oscillatory electric activity (blips) that was recorded by two-microelectrode voltage-clamp. The cells exhibited a series of “spontaneous” blips ranging in amplitude from 3.8 ± 0.9 nA at the beginning of the recordings to 6.8 ± 1.7 nA after 15 min of exposure to 1 μM aggregate. These blips were similar in amplitude to those induced by the channel-forming antimicrobial agents amphotericin B (7.8 ± 1.2 nA) and gramicidin (6.3 ± 1.1 nA). The amyloid aggregate-induced currents were abolished when extracellular Ca²⁺ was removed from the bathing solution, suggesting a central role for this cation in generating the spontaneous electric activity. The amyloid aggregate also affected the Ca²⁺-dependent Cl⁻ currents of oocytes, as shown by increased amplitude of the transient-outward chloride current (T_out) and the serum-activated, oscillatory Cl⁻ currents. Electron microcopy revealed that amyloid aggregate induced the dissociation of the follicular cells that surround the oocyte, thus leading to a failure in the electro-chemical communication between these cells. This was also evidenced by the suppression of the oscillatory Ca²⁺-dependent ATP-currents, which require proper coupling between oocytes and the follicular cell layer. These observations, made using the X. laevis oocytes as a versatile experimental model, may help to understand the effects of amyloid aggregate on cellular communication.     

Engineered Polymer Nanoparticles Containing Hydrophobic Dipeptide for Inhibition of Amyloid-β Fibrillation

Protein aggregation into amyloid fibrils is implicated in the pathogenesis of many neurodegenerative diseases. Engineered nanoparticles have emerged as a potential approach to alter the kinetics of protein fibrillation process. Yet, there are only a few reports describing the use of nanoparticles for inhibition of amyloid-β 40 (Aβ(40)) peptide aggregation, involved in Alzheimer's disease (AD). In the present study, we designed new uniform biocompatible amino-acid-based polymer nanoparticles containing hydrophobic dipeptides in the polymer side chains. The dipeptide residues were designed similarly to the hydrophobic core sequence of Aβ. Poly(N-acryloyl-l-phenylalanyl-l-phenylalanine methyl ester) (polyA-FF-ME) nanoparticles of 57 ± 6 nm were synthesized by dispersion polymerization of the monomer A-FF-ME in 2-methoxy ethanol, followed by precipitation of the obtained polymer in aqueous solution. Cell viability assay confirmed that no significant cytotoxic effect of the polyA-FF-ME nanoparticles on different human cell lines, e.g., PC-12 and SH-SY5Y, was observed. A significantly slow secondary structure transition from random coil to β-sheets during Aβ(40) fibril formation was observed in the presence of these nanoparticles, resulting in significant inhibition of Aβ(40) fibrillation kinetics. However, the polyA-FF-ME analogous nanoparticles containing the l-alanyl-l-alanine (AA) dipeptide in the polymer side groups, polyA-AA-ME nanoparticles, accelerate the Aβ(40) fibrillation kinetics. The polyA-FF-ME nanoparticles and the polyA-AA-ME nanoparticles may therefore contribute to a mechanistic understanding of the fibrillation process, leading to the development of therapeutic strategies against amyloid-related diseases.     

Racemization of the Aspartic Acid Residue of Amyloid-β Peptide by a Radical Reaction

An E. coli cell extract containing thermostable α-amylase and maltogenic amylase expressed from the clone pTMA322 was used to produce an anomalously linked oligosaccharides (Alo) mixture from starch. The liquefaction and saccharification of starch were done by one-step procedure using the above cell extract. The resulted Alo mixture contained over 40% oligosaccharides having DP 3 or more including branched forms.     

Amyloid-β Receptors: The Good,the Bad,and the Prion Protein

Several different receptor proteins have been identified that bind monomeric, oligomeric, or fibrillar forms of amyloid-β (Aβ). “Good” receptors internalize Aβ or promote its transcytosis out of the brain, whereas “bad” receptors bind oligomeric forms of Aβ that are largely responsible for the synapticloss, memory impairments, and neurotoxicity that underlie Alzheimer disease. The prion protein both removes Aβ from the brain and transduces the toxic actions of Aβ. The clustering of distinct receptors in cell surface signaling platforms likely underlies the actions of distinct oligomeric species of Aβ. These Aβ receptor-signaling platforms provide opportunities for therapeutic intervention in Alzheimer disease.     

Profile for Amyloid-β and Tau Expression in Primary Cortical Cultures from 3xTg-AD Mice

Advances in transgenic technology as well as in the genetics of Alzheimer disease (AD) have allowed the establishment of animal models that reproduce amyloid-beta plaques and neurofibrillary tangles, the main pathological hallmarks of AD. Among these models, 3xTg-AD mice harboring PS1 _M146V, APP _Swe and tau _P301L human transgenes provided the model that most closely mimics human AD features. Although cortical cultures from 3xTg-AD mice have been shown to present disturbances in intracellular [Ca²⁺] homeostasis, the development of AD pathology in vitro has not been previously evaluated. In the current work, we determined the temporal profile for amyloid precursor protein, amyloid-β and tau expression in primary cortical cultures from 3xTg-AD mice. Immunocytochemistry and Western blot analysis showed an increased expression of these proteins as well as several phosphorylated tau isoforms with time in culture. Alterations in calcium homeostasis and cholinergic and glutamatergic responses were also observed early in vitro. Thus, 3x-TgAD cortical neurons in vitro provide an exceptional tool to investigate pharmacological approaches as well as the cellular basis for AD and related diseases.