Investigating the Structural Impact of the Glutamine Repeat in Huntingtin Assembly

Acquiring detailed structural information about the various aggregation states of the huntingtin-exon1 protein (Htt-exon1) is crucial not only for identifying the true nature of the neurotoxic species responsible for Huntington’s disease (HD) but also for designing effective therapeutics. Using time-resolved small-angle neutron scattering (TR-SANS), we followed the conformational changes that occurred during fibrillization of the pathologic form of Htt-exon1 (NtQ₄₂P₁₀) and compared the results with those obtained for the wild-type (NtQ₂₂P₁₀). Our results show that the aggregation pathway of NtQ₂₂P₁₀ is very different from that of NtQ₄₂P₁₀, as the initial steps require a monomer to 7-mer transition stage. In contrast, the earliest species identified for NtQ₄₂P₁₀ are monomer and dimer. The divergent pathways ultimately result in NtQ₂₂P₁₀ fibrils that possess a packing arrangement consistent with the common amyloid sterical zipper model, whereas NtQ₄₂P₁₀ fibrils present a better fit to the Perutz β-helix structural model. The structural details obtained by TR-SANS should help to delineate the key mechanisms that underpin Htt-exon1 aggregation leading to HD.     

Alzheimer’s Protective A2T Mutation Changes the Conformational Landscape of the Aβ1–42 Monomer Differently Than Does the A2V Mutation

The aggregation of amyloid-β (Aβ) peptides plays a crucial role in the etiology of Alzheimer’s disease (AD). Recently, it has been reported that an A2T mutation in Aβ can protect against AD. Interestingly, a nonpolar A2V mutation also has been found to offer protection against AD in the heterozygous state, although it causes early-onset AD in homozygous carriers. Since the conformational landscape of the Aβ monomer is known to directly contribute to the early-stage aggregation mechanism, it is important to characterize the effects of the A2T and A2V mutations on Aβ_1–42 monomer structure. Here, we have performed extensive atomistic replica-exchange molecular dynamics simulations of the solvated wild-type (WT), A2V, and A2T Aβ_1–42 monomers. Our simulations reveal that although all three variants remain as collapsed coils in solution, there exist significant structural differences among them at shorter timescales. A2V exhibits an enhanced double-hairpin population in comparison to the WT, similar to those reported in toxic WT Aβ_1–42 oligomers. Such double-hairpin formation is caused by hydrophobic clustering between the N-terminus and the central and C-terminal hydrophobic patches. In contrast, the A2T mutation causes the N-terminus to engage in unusual electrostatic interactions with distant residues, such as K16 and E22, resulting in a unique population comprising only the C-terminal hairpin. These findings imply that a single A2X (where X = V or T) mutation in the primarily disordered N-terminus of the Aβ_1–42 monomer can dramatically alter the β-hairpin population and switch the equilibrium toward alternative structures. The atomistically detailed, comparative view of the structural landscapes of A2V and A2T variant monomers obtained in this study can enhance our understanding of the mechanistic differences in their early-stage aggregation.     

Sequestration of the Aβ Peptide Prevents Toxicity and Promotes Degradation In Vivo

Protein aggregation, arising from the failure of the cell to regulate the synthesis or degradation of aggregation-prone proteins, underlies many neurodegenerative disorders. However, the balance between the synthesis, clearance, and assembly of misfolded proteins into neurotoxic aggregates remains poorly understood. Here we study the effects of modulating this balance for the amyloid-beta (Aβ) peptide by using a small engineered binding protein (Z_Aβ3) that binds with nanomolar affinity to Aβ, completely sequestering the aggregation-prone regions of the peptide and preventing its aggregation. Co-expression of Z_Aβ3 in the brains of Drosophila melanogaster expressing either Aβ₄₂ or the aggressive familial associated E22G variant of Aβ₄₂ abolishes their neurotoxic effects. Biochemical analysis indicates that monomer Aβ binding results in degradation of the peptide in vivo. Complementary biophysical studies emphasize the dynamic nature of Aβ aggregation and reveal that Z_Aβ3 not only inhibits the initial association of Aβ monomers into oligomers or fibrils, but also dissociates pre-formed oligomeric aggregates and, although very slowly, amyloid fibrils. Toxic effects of peptide aggregation in vivo can therefore be eliminated by sequestration of hydrophobic regions in monomeric peptides, even when these are extremely aggregation prone. Our studies also underline how a combination of in vivo and in vitro experiments provide mechanistic insight with regard to the relationship between protein aggregation and clearance and show that engineered binding proteins may provide powerful tools with which to address the physiological and pathological consequences of protein aggregation.     

In silico and in vitro studies to elucidate the role of Cu2+ and galanthamine as the limiting step in the amyloid beta (1–42) fibrillation process

The formation of fibrils and oligomers of amyloid beta (Aβ) with 42 amino acid residues (Aβ_1–42) is the most important pathophysiological event associated with Alzheimer''s disease (AD). The formation of Aβ fibrils and oligomers requires a conformational change from an α-helix to a β-sheet conformation, which is encouraged by the formation of a salt bridge between Asp 23 or Glu 22 and Lys 28. Recently, Cu²⁺ and various drugs used for AD treatment, such as galanthamine (Reminyl®), have been reported to inhibit the formation of Aβ fibrils. However, the mechanism of this inhibition remains unclear. Therefore, the aim of this work was to explore how Cu²⁺ and galanthamine prevent the formation of Aβ_1–42 fibrils using molecular dynamics (MD) simulations (20 ns) and in vitro studies using fluorescence and circular dichroism (CD) spectroscopies. The MD simulations revealed that Aβ_1–42 acquires a characteristic U-shape before the α-helix to β-sheet conformational change. The formation of a salt bridge between Asp 23 and Lys 28 was also observed beginning at 5 ns. However, the MD simulations of Aβ₁₋₄₂ in the presence of Cu²⁺ or galanthamine demonstrated that both ligands prevent the formation of the salt bridge by either binding to Glu 22 and Asp 23 (Cu²⁺) or to Lys 28 (galanthamine), which prevents Aβ₁₋₄₂ from adopting the U-characteristic conformation that allows the amino acids to transition to a β-sheet conformation. The docking results revealed that the conformation obtained by the MD simulation of a monomer from the 1Z0Q structure can form similar interactions to those obtained from the 2BGE structure in the oligomers. The in vitro studies demonstrated that Aβ remains in an unfolded conformation when Cu²⁺ and galanthamine are used. Then, ligands that bind Asp 23 or Glu 22 and Lys 28 could therefore be used to prevent β turn formation and, consequently, the formation of Aβ fibrils.     

Intrinsic Determinants of Neurotoxic Aggregate Formation by the Amyloid β Peptide

The extent to which proteins aggregate into distinct structures ranging from prefibrillar oligomers to amyloid fibrils is key to the pathogenesis of many age-related degenerative diseases. We describe here for the Alzheimer's disease-related amyloid β peptide (Aβ) an investigation of the sequence-based determinants of the balance between the formation of prefibrillar aggregates and amyloid fibrils. We show that by introducing single-point mutations, it is possible to convert the normally harmless Aβ₄₀ peptide into a pathogenic species by increasing its relative propensity to form prefibrillar but not fibrillar aggregates, and, conversely, to abolish the pathogenicity of the highly neurotoxic E22G Aβ₄₂ peptide by reducing its relative propensity to form prefibrillar species rather than mature fibrillar ones. This observation can be rationalized by the demonstration that whereas regions of the sequence of high aggregation propensity dominate the overall tendency to aggregate, regions with low intrinsic aggregation propensities exert significant control over the balance of the prefibrillar and fibrillar species formed, and therefore play a major role in determining the neurotoxicity of the Aβ peptide.     

Effects of Familial Mutations on the Monomer Structure of Aβ42

Amyloid beta (Aβ) peptide plays an important role in Alzheimer’s disease. A number of mutations in the Aβ sequence lead to familial Alzheimer’s disease, congophilic amyloid angiopathy, or hereditary cerebral hemorrhage with amyloid. Using molecular dynamics simulations of ∼200 μs for each system, we characterize and contrast the consequences of four pathogenic mutations (Italian, Dutch, Arctic, and Iowa) for the structural ensemble of the Aβ monomer. The four familial mutations are found to have distinct consequences for the monomer structure.Amyloid beta (Aβ) peptides have long been thought to play a central role in Alzheimer’s disease (AD). Usually 40 or 42 residues in length, Aβ peptides are proteolytic products of the Aβ precursor protein and they aggregate to form the fibrillar plaques in AD patients’ brains. Besides fibrillar plaques, Aβ oligomers are also neurotoxic. The significance and nature of Aβ oligomerization has recently become a focus of intensive research studies and debates (1,2). Notably, numerous pathogenic mutations have been identified in the Aβ precursor protein sequence and in the enzymes involved in Aβ processing (3). These mutations generally lead to early onset of AD or cerebral amyloid angiopathy. Understanding how the pathogenic mutations alter Aβ oligomerization/aggregation is essential to our understanding of the disease mechanism.Four of these pathogenic mutations (Italian E22K, Dutch E22Q, Arctic E22G, and Iowa D23N) cluster in the region of E22 and D23 in the Aβ sequence (distal from proteolytic cleavage sites) and they have higher neurotoxicity compared to wild-type (WT) Aβ (4). These mutations are thought to modify the physicochemistry of the peptide. For example, kinetic studies (4) show that the E22K and E22Q mutations lead to faster peptide aggregation, whereas the E22G and D23N mutations result in slightly slower aggregation than WT Aβ₄₂ (although the E22G mutation shows increased protofibril formation (5)). Recent solid-state NMR studies also suggest that rather than the in-register β-sheet conformation adopted by WT Aβ, the Iowa D23N mutant forms amyloid fibrils with antiparallel β-sheet structure (6).To understand how the mutations modify the peptide oligomerization/aggregation it is critical to characterize the starting point of the process, the monomers. Unfortunately, investigating the early phase of the oligomerization process experimentally is a challenging task due to the high aggregation propensity of Aβ and its intrinsic disorder. Therefore, a number of computational approaches have been adopted to investigate the consequences of mutations for the monomer structure (7–16). However, due to the high computational demands of explicit-solvent molecular dynamics (MD) simulations to simulate full-length Aβ peptides, most of these computational studies are either on Aβ fragments (to decrease the system size) using explicit-solvent simulations (8–12) or on full-length Aβ using implicit-solvent simulations (which are less computationally demanding and enable longer simulation times, but lack explicit water molecules in the simulations to fully describe water-peptide interactions) (13–15). In a very recent report, explicit-solvent simulations were used to study the effects of the E22Q mutation on full-length Aβ; however, rather limited data (<10 μs) were collected (16). Thus, characterizing full-length Aβ monomers remains quite a daunting task even with simulations.To characterize the effects of mutations on full-length Aβ monomer using explicit-solvent MD simulations, we employed distributed computing (17) to simulate the WT Aβ₄₂, Aβ₄₂-E22K, Aβ₄₂-E22Q, Aβ₄₂-E22G, and Aβ₄₂-D23N monomers. MD simulations of >200 μs were performed for each system and AMBER ff99sb (18) and the tip3p water model (19) were used for force field parameters. Peptide configurations in the MD trajectories were clustered with the root mean-square deviation metric to identify representative conformations (i.e., states) and transitions between these states were counted. Markov state model analysis was then performed where the master equations were solved and the equilibrium population of each state deduced (20). Details of the MD simulation procedures and Markov state model analysis can be found in the Supporting Material.Each of the five Aβ monomer systems exhibits great structural diversity and can only be characterized in an ensemble fashion (rather than described by a handful of representative configurations). This is in accord with the notion that full-length Aβ peptides are intrinsically disordered (21,22). Using the Dictionary of Secondary Structure of Proteins program (23) to assign secondary structure, it is clear that the five Aβ monomer systems are found overall not well structured, although small β-hairpins and α-helices are observed. In Fig. 1 we plot the residue-dependent extended β propensity and α-helix propensity, in the top and bottom panels, respectively, for each Aβ monomer system. Although we are reasonably confident of the convergence behavior of the α-helix propensity, we note that the convergence of the extended β-propensity might be more challenging and demand a much longer sampling time than the current aggregate simulation time of ∼200 μs (24).Open in a separate windowFigure 1Ensemble-averaged %population of β-strand (top) and α-helix (bottom) propensity for all five monomer systems. The sequence of the WT Aβ₄₂ is given on the x axis.We observe in Fig. 1 that all five Aβ monomer systems share a rather similar residue-dependent tendency to form an extended β-structure, although minor differences are present. On the other hand, these pathogenic mutations alter the α-helix propensity quite significantly. The E22K and E22Q mutations increase the α-helix propensity in the region of residues 20–23. All four mutations (E22K, E22Q, E22G, and D23N) decrease the α-helix propensity in the region of residues 33–36.Notably, we find that in all five systems only short stretches of α-helices are formed. That is, when a residue is involved in α-helix formation, it participates in forming mostly short helical segments (consisting of only four helical residues). To provide more insight into the changes of α-helix propensity due to the mutations, in Fig. S1 we plot the tendency of forming short α-helices along the sequence for all five systems. Each data point in Fig. S1 represents the propensity to form an α-helix of four residues in length, ending at the specific residue. For example, in the structural ensemble adopted by the WT peptide, ∼5.5% of the conformations have a short α-helix of size four, involving residues 15–18. We see from Fig. S1 that the E22K and E22Q mutations induce the formation of two short helices in residues 19–22 and 20–23. The higher α-helix propensity in this region for the E22K mutant compared to the WT was previously attributed to the elimination of the electrostatic repulsion between E22 and D23 in the WT by the mutation and the longer aliphatic chain of K22 in the mutant compared to E22 in the WT (9,22). This is consistent with the observation that the E22Q mutation also induces helix formation in this region (by eliminating the electrostatic repulsion between E22 and D23 in the WT) but to a lesser extent, possibly due to the shorter aliphatic chain of Q22 compared to K22.In the E22G mutant, although the mutation eliminates the electrostatic repulsion between E22 and D23 in the WT peptide, glycine is known to be a helix breaker (25), leading to diminished α-helix propensity in the region around residue G22 seen in Fig. S1.In the D23N mutant, although the mutation eliminates the electrostatic repulsion between E22 and D23 in the WT peptide, it does not induce (or rather even slightly decreases) helix formation around residue 23. This may be due to the short aliphatic chain of N23 but it is possible that the mutation induces some nonlocal effects on the peptide structure, disfavoring helix formation in this region.It is worth noting that all four mutations (E22K, E22Q, E22G, and D23N) virtually eliminate the α-helix propensity in the region of residues 33–36. This region is rather far away from the mutation sites in sequence but its α-helix propensity is nonetheless affected. The origin of such a nonlocal effect is less straightforward to explain and further analysis will aid untangling this behavior. Nonetheless, the diminished α-helix propensity in the region of residues 33–36 appears to be a consistent feature across all four mutants.The four mutations studied here (E22K, E22Q, E22G, and D23N) have been thought to modify the physicochemistry of the peptide and alter the oligomerization/aggregation process, leading to higher neurotoxicity. In predicting intrinsic aggregation propensities using peptide sequences, all four mutants are suggested to be more aggregation prone (26). On the other hand, kinetic studies show that only the E22K and E22Q mutants aggregate more quickly, whereas the E22G and D23N mutations result in slightly slower aggregation than WT Aβ₄₂ (4). Our simulation results suggest these pathogenic mutations have complicated effects on the monomer structure—all four mutations decrease helix propensity in residues 33–36, whereas only the E22K and E22Q mutations increase helix propensity in residues 20–23. It is interesting to note that α-helix propensity is generally thought to anticorrelate with aggregation propensity; however, recent studies have suggested an important role of α-helical intermediates in amyloid oligomerization (27–29). Our studies suggest that it would be of great value to investigate how the distinct patterns of α-helix propensity in these five systems may propagate to give rise to different oligomerization kinetics or even mechanisms. The pathogenic mutations studied here have complex effects on the oligomerization of the peptide. The characterization of the monomer structural ensembles reported here should aid understanding of such an important and complicated process.     

Oligomeric and fibrillar species of amyloid-beta peptides differentially affect neuronal viability

Genetic evidence predicts a causative role for amyloid-beta (A beta) in Alzheimer's disease. Recent debate has focused on whether fibrils (amyloid) or soluble oligomers of A beta are the active species that contribute to neurodegeneration and dementia. We developed two aggregation protocols for the consistent production of stable oligomeric or fibrillar preparations of A beta-(1-42). Here we report that oligomers inhibit neuronal viability 10-fold more than fibrils and approximately 40-fold more than unaggregated peptide, with oligomeric A beta-(1-42)-induced inhibition significant at 10 nm. Under A beta-(1-42) oligomer- and fibril-forming conditions, A beta-(1-40) remains predominantly as unassembled monomer and had significantly less effect on neuronal viability than preparations of A beta-(1-42). We applied the aggregation protocols developed for wild type A beta-(1-42) to A beta-(1-42) with the Dutch (E22Q) or Arctic (E22G) mutations. Oligomeric preparations of the mutations exhibited extensive protofibril and fibril formation, respectively, but were not consistently different from wild type A beta-(1-42) in terms of inhibition of neuronal viability. However, fibrillar preparations of the mutants appeared larger and induced significantly more inhibition of neuronal viability than wild type A beta-(1-42) fibril preparations. These data demonstrate that protocols developed to produce oligomeric and fibrillar A beta-(1-42) are useful in distinguishing the structural and functional differences between A beta-(1-42) and A beta-(1-40) and genetic mutations of A beta-(1-42).     

Design,synthesis, and evaluation of chalcone-Vitamin E-donepezil hybrids as multi-target-directed ligands for the treatment of Alzheimer’s disease

A novel series of chalcone-Vitamin E-donepezil hybrids was designed and developed based on multitarget-directed ligands (MTDLs) strategy for treating Alzheimer’s disease (AD). The biological results revealed that compound 17f showed good AChE inhibitory potency (ratAChE IC₅₀ = 0.41 µM; eeAChE IC₅₀ = 1.88 µM). Both the kinetic analysis and docking study revealed that 17f was a mixed type AChE inhibitor. 17f was also a good antioxidant (ORAC = 3.3 eq), selective metal chelator and huMAO-B inhibitor (IC₅₀ = 8.8 µM). Moreover, it showed remarkable inhibition of self- and Cu²⁺-induced Aβ_1–42 aggregation with a 78.0 and 93.5% percentage rate at 25 µM, respectively, and disassembled self-induced and Cu²⁺-induced aggregation of the accumulated Aβ_1–42 fibrils with 72.3 and 84.5% disaggregation rate, respectively. More importantly, 17f exhibited a good neuroprotective effect on H₂O₂-induced PC12 cell injury and presented good blood-brain barrier permeability in vitro. Thus, 17f was a promising multi-target-directed ligand for treating AD.     

Variations in the FRA10AC1 Fragile Site and 15q21 Are Associated with Cerebrospinal Fluid Aβ1-42 Level

Proteolytic fragments of amyloid and post-translational modification of tau species in Cerebrospinal fluid (CSF) as well as cerebral amyloid deposition are important biomarkers for Alzheimer’s Disease. We conducted genome-wide association study to identify genetic factors influencing CSF biomarker level, cerebral amyloid deposition, and disease progression. The genome-wide association study was performed via a meta-analysis of two non-overlapping discovery sample sets to identify genetic variants other than APOE ε4 predictive of the CSF biomarker level (Aβ_1–42, t-Tau, p-Tau_181P, t-Tau:Aβ_1–42 ratio, and p-Tau_181P:Aβ_1–42 ratio) in patients enrolled in the Alzheimer’s Disease Neuroimaging Initiative (ADNI) study. Loci passing a genome-wide significance threshold of P < 5 x 10⁻⁸ were followed-up for replication in an independent sample set. We also performed joint meta-analysis of both discovery sample sets together with the replication sample set. In the discovery phase, we identified variants in FRA10AC1 associated with CSF Aβ_1–42 level passing the genome-wide significance threshold (directly genotyped SNV rs10509663 P _FE = 1.1 x 10⁻⁹, imputed SNV rs116953792 P _FE = 3.5 x 10⁻¹⁰), rs116953792 (P _one-sided = 0.04) achieved replication. This association became stronger in the joint meta-analysis (directly genotyped SNV rs10509663 P _FE = 1.7 x 10⁻⁹, imputed SNV rs116953792 P _FE = 7.6 x 10⁻¹¹). Additionally, we identified locus 15q21 (imputed SNV rs1503351 P _FE = 4.0 x 10⁻⁸) associated with CSF Aβ_1–42 level. No other variants passed the genome-wide significance threshold for other CSF biomarkers in either the discovery sample sets or joint analysis. Gene set enrichment analyses suggested that targeted genes mediated by miR-33, miR-146, and miR-193 were enriched in various GWAS analyses. This finding is particularly important because CSF biomarkers confer disease susceptibility and may be predictive of the likelihood of disease progression in Alzheimer’s Disease.     

Stability of Transmembrane Amyloid β-Peptide and Membrane Integrity Tested by Molecular Modeling of Site-Specific Aβ 42 Mutations

Interactions of the amyloid β-protein (Aβ) with neuronal cell membranes, leading to the disruption of membrane integrity, are considered to play a key role in the development of Alzheimer’s disease. Natural mutations in Aβ ₄₂, such as the Arctic mutation (E22G) have been shown to increase Aβ ₄₂ aggregation and neurotoxicity, leading to the early-onset of Alzheimer’s disease. A correlation between the propensity of Aβ ₄₂ to form protofibrils and its effect on neuronal dysfunction and degeneration has been established. Using rational mutagenesis of the Aβ ₄₂ peptide it was further revealed that the aggregation of different Aβ ₄₂ mutants in lipid membranes results in a variety of polymorphic aggregates in a mutation dependent manner. The mutant peptides also have a variable ability to disrupt bilayer integrity. To further test the connection between Aβ ₄₂ mutation and peptide–membrane interactions, we perform molecular dynamics simulations of membrane-inserted Aβ ₄₂ variants (wild-type and E22G, D23G, E22G/D23G, K16M/K28M and K16M/E22G/D23G/K28M mutants) as β-sheet monomers and tetramers. The effects of charged residues on transmembrane Aβ ₄₂ stability and membrane integrity are analyzed at atomistic level. We observe an increased stability for the E22G Aβ ₄₂ peptide and a decreased stability for D23G compared to wild-type Aβ ₄₂, while D23G has the largest membrane-disruptive effect. These results support the experimental observation that the altered toxicity arising from mutations in Aβ is not only a result of the altered aggregation propensity, but also originates from modified Aβ interactions with neuronal membranes.     

The binding of resveratrol to monomer and fibril amyloid beta

As currently understood, Alzheimer’s disease (AD) is a chronic neurodegenerative disorder that is driven by the aggregation of amyloid beta (Aβ) protein. It has been shown that resveratrol (RES) may attenuate amyloid β peptide-induced toxicity, promote Aβ clearance and reduce senile plaques. However, it remains to be determined whether RES could interact directly with Aβ. The aim of the present study was to examine the direct binding of RES to monomer and fibril Aβ. Using surface plasmon resonance (SPR) and proton nuclear magnetic resonance (¹H NMR), our results identified the direct binding of RES to Aβ. The ability of RES to bind to both fibril and monomer Aβ(1–40 and 1–42) was further analyzed by SPR. The binding response of RES to fAβ(1–42) was higher than that to monomer Aβ(1–42), whereas the binding response of RES to fAβ(1–40) was lower than that to monomer Aβ(1–40). The K_D of RES for fibril Aβ(1–40 or 1–42) was higher than that for the corresponding monomer Aβ. Compared to the control compound Congo red (CR), the binding responses of RES to monomer Aβ(1–42) and Aβ(1–40) were stronger, but binding to fibril Aβ(1–42) was weaker, and the K_Ds of RES with both monomer and fibril Aβ(1–40) and Aβ(1–42) were higher than that of CR. When Aβ(1–40 or 1–42) was co-incubated with RES (50 μM), the thioflavin T fluorescence of the mixture was weakened, and the number and length of amyloid fibrils were decreased. Furthermore, the results of staining in consecutive brain slices from AD patients showed that RES (10⁻⁴ M) could stain senile plaques. These results indicated that RES could bind directly to Aβ in different states, which may provide new insight into the protective properties of RES against AD.     

On the Characterization of Intermediates in the Isodesmic Aggregation Pathway of Hen Lysozyme at Alkaline pH

Protein aggregation leading to formation of amyloid fibrils is a symptom of several diseases like Alzheimer’s, type 2 diabetes and so on. Elucidating the poorly understood mechanism of such phenomena entails the difficult task of characterizing the species involved at each of the multiple steps in the aggregation pathway. It was previously shown by us that spontaneous aggregation of hen-eggwhite lysozyme (HEWL) at room temperature in pH 12.2 is a good model to study aggregation. Here in this paper we investigate the growth kinetics, structure, function and dynamics of multiple intermediate species populating the aggregation pathway of HEWL at pH 12.2. The different intermediates were isolated by varying the HEWL monomer concentration in the 300 nM—0.12 mM range. The intermediates were characterized using techniques like steady-state and nanosecond time-resolved fluorescence, atomic force microscopy and dynamic light scattering. Growth kinetics of non-fibrillar HEWL aggregates were fitted to the von Bertalanffy equation to yield a HEWL concentration independent rate constant (k = (6.6±0.6)×10⁻⁵ s⁻¹). Our results reveal stepwise changes in size, molecular packing and enzymatic activity among growing HEWL aggregates consistent with an isodesmic aggregation model. Formation of disulphide bonds that crosslink the monomers in the aggregate appear as a unique feature of this aggregation. AFM images of multiple amyloid fibrils emanating radially from amorphous aggregates directly confirmed that on-pathway fibril formation was feasible under isodesmic polymerization. The isolated HEWL aggregates are revealed as polycationic protein nanoparticles that are robust at neutral pH with ability to take up non-polar molecules like ANS.     

Time-Resolved Surface-Enhanced IR-Absorption Spectroscopy of Direct Electron Transfer to Cytochrome c Oxidase from R.?sphaeroides

The aggregation of α-synuclein is thought to play a role in the death of dopamine neurons in Parkinson’s disease (PD). Alpha-synuclein transitions itself through an aggregation pathway consisting of pathogenic species referred to as protofibrils (or oligomer), which ultimately convert to mature fibrils. The structural heterogeneity and instability of protofibrils has significantly impeded advance related to the understanding of their structural characteristics and the amyloid aggregation mystery. Here, we report, to our knowledge for the first time, on α-synuclein protofibril structural characteristics with cryo-electron microscopy. Statistical analysis of annular protofibrils revealed a constant wall thickness as a common feature. The visualization of the assembly steps enabled us to propose a novel, to our knowledge, mechanisms for α-synuclein aggregation involving ring-opening and protofibril-protofibril interaction events. The ion channel-like protofibrils and their membrane permeability have also been found in other amyloid diseases, suggesting a common molecular mechanism of pathological aggregation. Our direct visualization of the aggregation pathway of α-synuclein opens up fresh opportunities to advance the understanding of protein aggregation mechanisms relevant to many amyloid diseases. In turn, this information would enable the development of additional therapeutic strategies aimed at suppressing toxic protofibrils of amyloid proteins involved in neurological disorders.     

A Food and Drug Administration-approved Asthma Therapeutic Agent Impacts Amyloid β in the Brain in a Transgenic Model of Alzheimer Disease

Interfering with the assembly of Amyloid β (Aβ) peptides from monomer to oligomeric species and fibrils or promoting their clearance from the brain are targets of anti-Aβ-directed therapies in Alzheimer disease. Here we demonstrate that cromolyn sodium (disodium cromoglycate), a Food and Drug Administration-approved drug already in use for the treatment of asthma, efficiently inhibits the aggregation of Aβ monomers into higher-order oligomers and fibrils in vitro without affecting Aβ production. In vivo, the levels of soluble Aβ are decreased by over 50% after only 1 week of daily intraperitoneally administered cromolyn sodium. Additional in vivo microdialysis studies also show that this compound decreases the half-life of soluble Aβ in the brain. These data suggest a clear effect of a peripherally administered, Food and Drug Administration-approved medication on Aβ economy, supporting further investigation of the potential long-term efficacy of cromolyn sodium in Alzheimer disease.     

Molecular insights into the primary nucleation of polymorphic amyloid β dimers in DOPC lipid bilayer membrane

Alzheimer''s disease (AD) pathology is characterized by loss of memory cognitive and behavioral deterioration. One of the hallmarks of AD is amyloid β (Aβ) plaques in the brain that consists of Aβ oligomers and fibrils. It is accepted that oligomers, particularly dimers, are toxic species that are produced extracellularly and intracellularly in membranes. It is believed that the disruption of membranes by polymorphic Aβ oligomers is the key for the pathology of AD. This is a first study that investigate the effect of polymorphic “α‐helix/random coil” and “fibril‐like” Aβ dimers on 1,2‐dioleoyl‐sn‐glycero‐3‐phosphocholine (DOPC) membrane. It has been found that the DOPC membrane promotes Aβ_1–42 “fibril‐like” dimers and impedes Aβ_1–42 “α‐helix/random coil” dimers. The N‐termini domains within Aβ_1–42 dimers play a role in Aβ aggregation in membrane milieus. In addition, the aromatic π–π interactions (involving residues F19 and F20 in Aβ_1–42) are the driving forces for the hydrophobic interactions that initiate the primary nucleation of polymorphic Aβ_1–42 dimers within DOPC membrane. Finally, the DOPC bilayer membrane thickness is locally decreased, and it is disrupted by an embedded distinct Aβ_1–42 dimer, due to relatively large contacts between Aβ_1–42 monomers and the DOPC membrane. This study reveals insights into the molecular mechanisms by which polymorphic early‐stage Aβ_1–42 dimers have distinct impacts on DOPC membrane.     

Insights into the inhibitory mechanism of a resveratrol and clioquinol hybrid against Aβ42 aggregation and protofibril destabilization: A molecular dynamics simulation study

Amyloid-β (Aβ) peptide instinctively aggregate and form plaques in the brain of Alzheimer’s disease (AD) patients. At present, there is no cure or treatment for AD, and significant effort has, therefore, been made to discover potent drugs against AD. Previous studies reported that a resveratrol and clioquinol hybrid compound [(E)-5-(4-hydroxystyryl)quinolone-8-ol], C1, strongly inhibit Aβ₄₂ aggregation and disassemble preformed fibrils. However, the atomic level details of the inhibitory mechanism of C1 against Aβ₄₂ aggregation and protrofibril disassembly remains elusive. In this regard, molecular docking and molecular dynamics (MD) simulation of Aβ₄₂ monomer, Aβ₄₂ monomer–C1 complex, Aβ₄₂ protofibril, and Aβ₄₂ protofibril–C1 complex were performed in the present study. MD simulations highlighted that C1 bind in the central hydrophobic core (CHC) region, i.e., KLVFF (16–20) of Aβ₄₂ monomer, which plays a critical role in Aβ₄₂ aggregation. C1 promote the formation of native helical conformation in the Aβ₄₂ monomer and decrease the probability of D23–K28 salt bridge interaction that is critical in the formation of aggregation-prone β-sheet conformation. Further, C1 destabilize Aβ₄₂ protofibril structure by increasing the interchain distance between chains A–B, disrupting the salt–bridge interaction between D23–K28, and decreasing the number of backbone hydrogen bonds between chains A–B of the Aβ₄₂ protofibril structure. The insights into the underlying inhibitory mechanism of small molecules that display potential in vitro anti–aggregation activity against Aβ₄₂ will be beneficial for the rational design of more potent drug molecules against AD.

Communicated by Ramaswamy H. Sarma     

A Huntingtin Peptide Inhibits PolyQ-Huntingtin Associated Defects

Background

Huntington’s disease (HD) is caused by the abnormal expansion of the polyglutamine tract in the human Huntingtin protein (polyQ-hHtt). Although this mutation behaves dominantly, huntingtin loss of function also contributes to HD pathogenesis. Indeed, wild-type Huntingtin plays a protective role with respect to polyQ-hHtt induced defects.

Methodology/Principal Findings

The question that we addressed here is what part of the wild-type Huntingtin is responsible for these protective properties. We first screened peptides from the Huntingtin protein in HeLa cells and identified a 23 aa peptide (P42) that inhibits polyQ-hHtt aggregation. P42 is part of the endogenous Huntingtin protein and lies within a region rich in proteolytic sites that plays a critical role in the pathogenesis process. Using a Drosophila model of HD, we tested the protective properties of this peptide on aggregation, as well as on different polyQ-hHtt induced neuronal phenotypes: eye degeneration (an indicator of cell death), impairment of vesicular axonal trafficking, and physiological behaviors such as larval locomotion and adult survival. Together, our results demonstrate high protective properties for P42 in vivo, in whole animals. These data also demonstrate a specific role of P42 on Huntington’s disease model, since it has no effect on other models of polyQ-induced diseases, such as spinocerebellar ataxias.

Conclusions/Significance

Altogether our data show that P42, a 23 aa-long hHtt peptide, plays a protective role with respect to polyQ-hHtt aggregation as well as cellular and behavioral dysfunctions induced by polyQ-hHtt in vivo. Our study also confirms the correlation between polyQ-hHtt aggregation and neuronal defects. Finally, these results strongly suggest a therapeutic potential for P42, specific of Huntington’s disease.     

Kinetics of fibril formation of bovine kappa-casein indicate a conformational rearrangement as a critical step in the process

S-carboxymethylated (SCM) κ-casein forms in vitro fibrils that display several characteristics of amyloid fibrils, although the protein is unrelated to amyloid diseases. In order to get insight into the processes that prevent the formation of amyloid fibrils made of κ-caseins in milk, we have characterized in detail the reaction and the roles of its possible effectors: glycosylation and other caseins. Given that native κ-casein occurs as a heterogeneous mixture of carbohydrate-free and carbohydrate-containing chains, kinetics of fibril formation were performed on purified glycosylated and unglycosylated SCM κ-caseins using the fluorescent dye thioflavin T in conjunction with transmission electron microscopy and Fourier transform infrared spectroscopy for morphological and structural analyses. Both unglycosylated and glycosylated SCM κ-caseins have the ability to fibrillate. Kinetic data indicate that the fibril formation rate increases with SCM κ-casein concentration but reaches a plateau at high concentrations, for both the unglycosylated and glycosylated forms. Therefore, a conformational rearrangement is the rate-limiting step in fibril growth of SCM κ-casein. Transmission electron microscopy images indicate the presence of 10- to 12-nm spherical particles prior to the appearance of amyloid structure. Fourier transform infrared spectroscopy spectra reveal a conformational change within these micellar aggregates during the fibrillation. Fibrils are helical ribbons with a pitch of about 120-130 nm and a width of 10-12 nm. Taken together, these findings suggest a model of aggregation during which the SCM κ-casein monomer is in rapid equilibrium with a micellar aggregate that subsequently undergoes a conformational rearrangement into a more organized species. These micelles assemble and this leads to the growing of amyloid fibrils. Addition of α_s1-and β-caseins decreases the growth rate of fibrils. Their main effect was on the elongation rate, which became close to that of the limiting conformation change, leading to the appearance of a lag phase at the beginning of the kinetics.     

Curcumin Alters the Salt Bridge-containing Turn Region in Amyloid β(1–42) Aggregates

Amyloid β (Aβ) fibrillar deposits in the brain are a hallmark of Alzheimer disease (AD). Curcumin, a common ingredient of Asian spices, is known to disrupt Aβ fibril formation and to reduce AD pathology in mouse models. Understanding the structural changes induced by curcumin can potentially lead to AD pharmaceutical agents with inherent bio-compatibility. Here, we use solid-state NMR spectroscopy to investigate the structural modifications of amyloid β(1–42) (Aβ₄₂) aggregates induced by curcumin. We find that curcumin induces major structural changes in the Asp-23–Lys-28 salt bridge region and near the C terminus. Electron microscopy shows that the Aβ₄₂ fibrils are disrupted by curcumin. Surprisingly, some of these alterations are similar to those reported for Zn²⁺ ions, another agent known to disrupt the fibrils and alter Aβ₄₂ toxicity. Our results suggest the existence of a structurally related family of quasi-fibrillar conformers of Aβ₄₂, which is stabilized both by curcumin and by Zn^2+.