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Self-templating propagation of protein aggregate conformations is increasingly becoming a significant factor in many neurological diseases. In Alzheimer disease (AD), intrinsically disordered amyloid-β (Aβ) peptides undergo aggregation that is sensitive to environmental conditions. High-molecular weight aggregates of Aβ that form insoluble fibrils are deposited as senile plaques in AD brains. However, low-molecular weight aggregates called soluble oligomers are known to be the primary toxic agents responsible for neuronal dysfunction. The aggregation process is highly stochastic involving both homotypic (Aβ-Aβ) and heterotypic (Aβ with interacting partners) interactions. Two of the important members of interacting partners are membrane lipids and surfactants, to which Aβ shows a perpetual association. Aβ–membrane interactions have been widely investigated for more than two decades, and this research has provided a wealth of information. Although this has greatly enriched our understanding, the objective of this review is to consolidate the information from the literature that collectively showcases the unique phenomenon of lipid-mediated Aβ oligomer generation, which has largely remained inconspicuous. This is especially important because Aβ aggregate “strains” are increasingly becoming relevant in light of the correlations between the structure of aggregates and AD phenotypes. Here, we will focus on aspects of Aβ-lipid interactions specifically from the context of how lipid modulation generates a wide variety of biophysically and biochemically distinct oligomer sub-types. This, we believe, will refocus our thinking on the influence of lipids and open new approaches in delineating the mechanisms of AD pathogenesis. This article is part of a Special Issue entitled: Protein Aggregation and Misfolding at the Cell Membrane Interface edited by Ayyalusamy Ramamoorthy. [ABSTRACT FROM AUTHOR]

Alzheimer's disease (AD) is characterized by the presence of large numbers of fibrillar amyloid deposits in the form of senile plaques in the brain. The fibrils in senile plaques are composed of 40- and 42-residue amyloid-β (Aβ) peptides. Several lines of evidence indicate that fibrillar Aβ and especially soluble Aβ aggregates are important in the pathogenesis of AD, and many laboratories have investigated soluble Aβ aggregates generated from monomeric Aβ in vitro. Of these in vitro aggregates, the best characterized are called protofibrils. They are composed of globules and short rods, show primarily β-structure by circular dichroism (CD), enhance the fluorescence of bound thioflavin T, and readily seed the growth of long fibrils. However, one difficulty in correlating soluble Aβ aggregates formed in vitro with those in vivo is the high probability that cellular interfaces affect the aggregation rates and even the aggregate structures. Reports that focus on the features of interfaces that are important in Aβ aggregation have found that amphiphilic interactions and micellar-like Aβ structures may play a role. We previously described the formation of Aβ(1–40) aggregates at polar-nonpolar interfaces, including those generated at microdroplets formed in dilute hexafluoro-2-propanol (HFIP). Here we compared the Aβ(1–40) aggregates produced on sodium dodecyl sulfate (SDS) micelles, which may be a better model of biological membranes with phospholipids that have anionic headgroups. At both HFIP and SDS interfaces, changes in peptide secondary structure were observed by CD immediately when Aβ(1–40) was introduced. With HFIP, the change involved an increase in predominant/3-structure content and in fluorescence with thioflavin T, while with SDS, a partial α-helical conformation was adopted that gave no fluorescence. However, in both systems, initial amorphous clustered aggregates progressed to soluble fibers rich in β-structure over a roughly 2 day period. Fiber formation was much faster than in the absence of an interface, presumably because of the close intermolecular proximity of peptides at the interfaces. While these fibers resembled protofibrils, they failed to seed the aggregation of Aβ(1–40) monomers effectively. [ABSTRACT FROM AUTHOR]

As the primary toxic species in the etiology of Alzheimer disease (AD) are low molecular weight oligomers of Aβ, it is crucial to understand the structure of Aβ oligomers for gaining molecular insights into AD pathology. We have earlier demonstrated that in the presence of fatty acids, Aβ42 peptides assemble as 12-24mer oligomers. These Large Fatty Acid-derived Oligomers (LFAOs) exist predominantly as 12mers at low and as 24mers at high concentrations. The 12mers are more neurotoxic than the 24mers and undergo self-replication, while the latter propagate to morphologically distinct fibrils with succinct pathological consequences. In order to glean into their functional differences and similarities, we have determined their structures in greater detail by combining molecular dynamic simulations with biophysical measurements. We conjecture that the LFAO are made of Aβ units in an S-shaped conformation, with the 12mers forming a double-layered hexamer ring (6 × 2) while the structure of 24mers is a double-layered dodecamer ring (12 × 2). A closer inspection of the (6 × 2) and (12 × 2) structures reveals a concentration and pH dependent molecular reorganization in the assembly of 12 to 24mers, which seems to be the underlying mechanism for the observed biophysical and cellular properties of LFAOs. [ABSTRACT FROM AUTHOR]

Aggregation of Aβ peptides is a key contributor to the etiology of Alzheimer's disease. Being intrinsically disordered, monomeric Aβ is susceptible to conformational excursions, especially in the presence of important interacting partners such as membrane lipids, to adopt specific aggregation pathways. Furthermore, components such as gangliosides in membranes and lipid rafts are known to play important roles in the adoption of pathways and the generation of discrete neurotoxic oligomers. Yet, what roles do carbohydrates on gangliosides play in this process remains unknown. Here, using GM1, GM3, and GD3 ganglioside micelles as models, we show that the sugar distributions and cationic amino acids within Aβ N-terminal region modulate oligomerization of Aβ temporally, and dictate the stability and maturation of oligomers. These results demonstrate the selectivity of sugar distributions on the membrane surface toward oligomerization of Aβ and thus implicate cell-selective enrichment of oligomers. [Display omitted] • Gangliosides are important membrane components linked to the etiology of Alzheimer's disease. • Sugar distributions on gangliosides GM1, GM3, and GD3 differentially affect the oligomerization of Aβ42. • Aβ-sugar interactions are affected by the cationic residues in the N-terminal of Aβ. • Sugar distribution on ganglioside confers specificity of interaction for the formation and stability of oligomers. [ABSTRACT FROM AUTHOR]

Granulins (GRNs 1-7) are cysteine-rich proteolytic products of progranulin (PGRN) that have recently been implicated in neurodegenerative diseases including frontotemporal dementia (FTD) and Alzheimer's disease (AD). Their precise mechanism in these pathologies remains uncertain, but both inflammatory and lysosomal roles have been observed for GRNs. Among the seven GRNs, GRN-3 is well characterized and is implicated within the context of FTD. However, the relationship between GRN-3 and amyloid-β (Aβ), a protein relevant in AD pathology, has not yet been explored. To gain insight into this mechanism, we investigated the effect of both oxidized and reduced GRN-3 on Aβ aggregation and found that both GRN-3 (oxidized) and rGRN-3 (reduced) bind to monomeric and oligomeric Aβ42 to promote rapid fibril formation with subtle rate differences. As low molecular weight oligomers of Aβ are well-established neurotoxins, rapid promotion of fibrils by GRN-3 mitigates Aβ42-induced cellular apoptosis. These data provide valuable insights in understanding GRN-3's ability to modulate Aβ-induced toxicity under redox control and presents a new perspective toward AD pathology. These results also prompt further investigation into the role(s) of other GRNs in AD pathogenesis. [ABSTRACT FROM AUTHOR]

The aggregation of amyloid-β (Aβ) peptide and its deposition in parts of the brain form the central processes in the etiology of Alzheimer disease (AD). The low-molecular weight oligomers of Aβ aggregates (2 to 30 mers) are known to be the primary neurotoxic agents whose mechanisms of cellular toxicity and synaptic dysfunction have received substantial attention in the recent years. However, how these toxic agents proliferate and induce widespread amyloid deposition throughout the brain, and what mechanism is involved in the amplification and propagation of toxic oligomer species, are far from clear. Emerging evidence based on transgenic mice models indicates a transmissible nature of Aβ aggregates and implicates a prion-like mechanism of oligomer propagation, which manifests as the dissemination and proliferation of Aβ toxicity. Despite accumulating evidence in support of a transmissible nature of Aβ aggregates, a clear, molecular-level understanding of this intriguing mechanism is lacking. Recently, we reported the characterization of unique replicating oligomers of Aβ42 (12–24 mers) in vitro called arge atty cid-derived ligomers (LFAOs) (Kumar et al., 2012, J. Biol. Chem). In the current report, we establish that LFAOs possess physiological activity by activating NF-κB in human neuroblastoma cells, and determine the experimental parameters that control the efficiency of LFAO replication by self-propagation. These findings constitute the first detailed report on monomer – oligomer lateral propagation reactions that may constitute potential mechanism governing transmissibility among Aβ oligomers. These data support the previous reports on transmissible mechanisms observed in transgenic animal models. [ABSTRACT FROM AUTHOR]

Aggregates of amyloid-β (Aβ) peptides have been implicated in the etiology of Alzheimer disease. Among the different forms of Aβ aggregates, low molecular weight species ranging between ∼2- and 50-mers, also called "soluble oligomers," have emerged as the species responsible for early synaptic dysfunction and neuronal loss. Emerging evidence suggests that the neurotoxic oligomers need not be formed along the obligatory nucleation-dependant fibril formation pathway. In our earlier work, we reported the isolation of one such "off-pathway" 12–18-mer species of Aβ42 generated from fatty acids called large fatty acid-derived oligomers (LFAOs) (Kumar, A., Bullard, R. L., Patel, P., Paslay, L. C., Singh, D., Bienkiewicz, E. A., Morgan, S. E., and Rangachari, V. (2011) PLoS One 6, e18759). Here, we report the physiochemical aspects of LFAO-monomer interactions as well as LFAO-LFAO associations in the presence of interfaces. We discovered that LFAOs are a replicating strain of oligomers that recruit Aβ42 monomers and quantitatively convert them into LFAO assemblies at the expense of fibrils, a mechanism similar to prion propagation. We also found that in the presence of hexane-buffer or chloroform-buffer interfaces LFAOs are able to associate with themselves to form larger but non-fibrillar aggregates. These results further support the hypothesis that low molecular weight oligomers can be generated via non-fibril formation pathways. Furthermore, the unique replicating property of off-pathway oligomers may hold profound significance for Alzheimer disease pathology. [ABSTRACT FROM AUTHOR]

Amyloid-β (Aβ) peptide aggregation is known to play a central role in the etiology of Alzheimer's disease (AD). Among various aggregates, low-molecular weight soluble oligomers of Aβ are increasingly believed to be the primary neurotoxic agents responsible for memory impairment. Anionic interfaces are known to influence the Aβ aggregation process significantly. Here, we report the effects of interfaces formed by medium-chain (C9-C12), saturated non-esterified fatty acids (NEFAs) on Aβ42 aggregation. NEFAs uniquely affected Aβ42 aggregation rates that depended on both the ratio of Aβ:NEFA as well the critical micelle concentration (CMC) of the NEFAs. More importantly, irrespective of the kind of NEFA used, we observed that two distinct oligomers, 12-18 mers and 4-5 mers were formed via different pathway of aggregation under specific experimental conditions: (i) 12-18 mers were generated near the CMC in which NEFAs augment the rate of Aβ42 aggregation towards fibril formation, and, (ii) 4-5 mers were formed above the CMC, where NEFAs inhibit fibril formation. The data indicated that both 12-18 mers and 4-5 mers are formed along an alternate pathway called 'off-pathway' that did not result in fibril formation and yet have subtle structural and morphological differences that distinguish their bulk molecular behavior. These observations, (i) reflect the possible mechanism of Aβ aggregation in physiological lipid-rich environments, and (ii) reiterate the fact that all oligomeric forms of Aβ need not be obligatory intermediates of the fibril formation pathway. [ABSTRACT FROM AUTHOR]

Background: In all amyloid diseases, protein aggregates have been implicated fully or partly, in the etiology of the disease. Due to their significance in human pathologies, there have been unprecedented efforts towards physiochemical understanding of aggregation and amyloid formation over the last two decades. An important relation from which hydrodynamic radii of the aggregate is routinely measured is the classic Stokes-Einstein equation. Here, we report a modification in the classical Stokes-Einstein equation using a mixture theory approach, in order to accommodate the changes in viscosity of the solvent due to the changes in solute size and shape, to implement a more realistic model for Aβ aggregation involved in Alzheimer's disease. Specifically, we have focused on validating this model in protofibrill lateral association reactions along the aggregation pathway, which has been experimentally well characterized. Results: The modified Stokes-Einstein equation incorporates an effective viscosity for the mixture consisting of the macromolecules and solvent where the lateral association reaction occurs. This effective viscosity is modeled as a function of the volume fractions of the different species of molecules. The novelty of our model is that in addition to the volume fractions, it incorporates previously published reports on the dimensions of the protofibrils and their aggregates to formulate a more appropriate shape rather than mere spheres. The net result is that the diffusion coefficient which is inversely proportional to the viscosity of the system is now dependent on the concentration of the different molecules as well as their proper shapes. Comparison with experiments for variations in diffusion coefficients over time reveals very similar trends. Conclusions: We argue that the standard Stokes-Einstein's equation is insufficient to understand the temporal variations in diffusion when trying to understand the aggregation behavior of Aβ42 proteins. Our modifications also involve inclusion of improved shape factors of molecules and more appropriate viscosities. The modification we are reporting is not only useful in Aβ aggregation but also will be important for accurate measurements in all protein aggregation systems. [ABSTRACT FROM AUTHOR]

Background: The aggregates of a protein called, 'Aβ' found in brains of Alzheimer's patients are strongly believed to be the cause for neuronal death and cognitive decline. Among the different forms of Aβ aggregates, smaller aggregates called 'soluble oligomers' are increasingly believed to be the primary neurotoxic species responsible for early synaptic dysfunction. Since it is well known that the Aβ aggregation is a nucleation dependant process, it is widely believed that the toxic oligomers are intermediates to fibril formation, or what we call the 'on-pathway' products. Modeling of Aβ aggregation has been of intense investigation during the last decade. However, precise understanding of the process, pre-nucleation events in particular, are not yet known. Most of these models are based on curve-fitting and overlook the molecular-level biophysics involved in the aggregation pathway. Hence, such models are not reusable, and fail to predict the system dynamics in the presence of other competing pathways. Results: In this paper, we present a molecular-level simulation model for understanding the dynamics of the amyloid-β (Aβ) peptide aggregation process involved in Alzheimer's disease (AD). The proposed chemical kinetic theory based approach is generic and can model most nucleation-dependent protein aggregation systems that cause a variety of neurodegenerative diseases. We discuss the challenges in estimating all the rate constants involved in the aggregation process towards fibril formation and propose a divide and conquer strategy by dissecting the pathway into three biophysically distinct stages: 1) pre-nucleation stage 2) post-nucleation stage and 3) protofibril elongation stage. We next focus on estimating the rate constants involved in the protofibril elongation stages for Aβ42 supported by in vitro experimental data. This elongation stage is further characterized by elongation due to oligomer additions and lateral association of protofibrils (73) and to properly validate the rate constants involved in these phases we have presented three distinct reaction models. We also present a novel scheme for mapping the fluorescence sensitivity and dynamic light scattering based in vitro experimental plots to estimates of concentration variation with time. Finally, we discuss how these rate constants will be incorporated into the overall simulation of the aggregation process to identify the parameters involved in the complete Aβ pathway in a bid to understand its dynamics. Conclusions: We have presented an instance of the top-down modeling paradigm where the biophysical system is approximated by a set of reactions for each of the stages that have been modeled. In this paper, we have only reported the kinetic rate constants of the fibril elongation stage that were validated by in vitro biophysical analyses. The kinetic parameters reported in the paper should be at least accurate upto the first two decimal places of the estimate. We sincerely believe that our top-down models and kinetic parameters will be able to accurately model the biophysical phenomenon of Aβ protein aggregation and identify the nucleation mass and rate constants of all the stages involved in the pathway. Our model is also reusable and will serve as the basis for making computational predictions on the system dynamics with the incorporation of other competing pathways introduced by lipids and fatty acids. [ABSTRACT FROM AUTHOR]

Selective lowering of Aβ42 levels (the 42-residue isoform of the amyloid-β peptide) with small-molecule γ-secretase modulators (GSMs), such as some non-steroidal anti-inflammatory drugs, is a promising therapeutic approach for Alzheimer’s disease. To identify the target of these agents we developed biotinylated photoactivatable GSMs. GSM photoprobes did not label the core proteins of the γ-secretase complex, but instead labelled the β-amyloid precursor protein (APP), APP carboxy-terminal fragments and amyloid-β peptide in human neuroglioma H4 cells. Substrate labelling was competed by other GSMs, and labelling of an APP γ-secretase substrate was more efficient than a Notch substrate. GSM interaction was localized to residues 28–36 of amyloid-β, a region critical for aggregation. We also demonstrate that compounds known to interact with this region of amyloid-β act as GSMs, and some GSMs alter the production of cell-derived amyloid-β oligomers. Furthermore, mutation of the GSM binding site in the APP alters the sensitivity of the substrate to GSMs. These findings indicate that substrate targeting by GSMs mechanistically links two therapeutic actions: alteration in Aβ42 production and inhibition of amyloid-β aggregation, which may synergistically reduce amyloid-β deposition in Alzheimer’s disease. These data also demonstrate the existence and feasibility of ‘substrate targeting’ by small-molecule effectors of proteolytic enzymes, which if generally applicable may significantly broaden the current notion of ‘druggable’ targets. [ABSTRACT FROM AUTHOR]

Analyses of the biologic effects of mutations in the BRI2 (ITM2b) and the amyloid β precursor protein (APP) genes support the hypothesis that cerebral accumulation of amyloidogenic peptides in familial British and familial Danish dementias and Alzheimer's disease (AD) is associated with neurodegeneration. We have used somatic brain transgenic technology to express the BRI2 and BRI2-Aβ1- 40 transgenes in APP mouse models. Expression of BRI2-Aβ1- 40 mimics the suppressive effect previously observed using conventional transgenic methods, further validating the somatic brain transgenic methodology. Unexpectedly, we also find that expression of wild-type human BRI2 reduces cerebral Aβ deposition in an AD mouse model. Additional data indicate that the 23 aa peptide, Bri23, released from BRI2 by normal processing, is present in human CSF, inhibits Aβ aggregation in vitro and mediates its anti-amyloidogenic effect in vivo. These studies demonstrate that BRI2 is a novel mediator of Aβ deposition in vivo. [ABSTRACT FROM AUTHOR]

Neurofibrillary tangles (NFTs) are a pathological hallmark of Alzheimer's disease and other tauopathies, but recent studies in a conditional mouse model of tauopathy (rTg4510) have suggested that NFT formation can be dissociated from memory loss and neurodegeneration. This suggests that NFTs are not the major neurotoxic tau species, at least during the early stages of pathogenesis. To identify other neurotoxic tau protein species, we performed biochemical analyses on brain tissues from the rTg4510 mouse model and then correlated the levels of these tau proteins with memory loss. We describe the identification and characterization of two forms of tau multimers (140 and 170 kDa), whose molecular weight suggests an oligomeric aggregate, that accumulate early in the pathogenic cascade in this mouse model. Similar tau multimers were detected in a second mouse model of tauopathy (JNPL3) and in tissue from patients with Alzheimer's disease and FTDP-17 (frontotemporal dementia and parkinsonism linked to chromosome 17). Moreover, levels of the tau multimers correlated consistently with memory loss at various ages in the rTg4510 mouse model. Our findings suggest that accumulation of early-stage aggregated tau species, before the formation of NFT, is associated with the development of functional deficits during the pathogenic progression of tauopathy. [ABSTRACT FROM AUTHOR]