BackgroundMisfolding and pathological aggregation of neuronal proteins has been proposed to play a critical role in the pathogenesis of neurodegenerative disorders. Alzheimer's disease (AD) and Parkinson's disease (PD) are frequent neurodegenerative diseases of the aging population. While progressive accumulation of amyloid β protein (Aβ) oligomers has been identified as one of the central toxic events in AD, accumulation of α-synuclein (α-syn) resulting in the formation of oligomers and protofibrils has been linked to PD and Lewy body Disease (LBD). We have recently shown that Aβ promotes α-syn aggregation and toxic conversion in vivo, suggesting that abnormal interactions between misfolded proteins might contribute to disease pathogenesis. However the molecular characteristics and consequences of these interactions are not completely clear.Methodology/Principal FindingsIn order to understand the molecular mechanisms involved in potential Aβ/α-syn interactions, immunoblot, molecular modeling, and in vitro studies with α-syn and Aβ were performed. We showed in vivo in the brains of patients with AD/PD and in transgenic mice, Aβ and α-synuclein co-immunoprecipitate and form complexes. Molecular modeling and simulations showed that Aβ binds α-syn monomers, homodimers, and trimers, forming hybrid ring-like pentamers. Interactions occurred between the N-terminus of Aβ and the N-terminus and C-terminus of α-syn. Interacting α-syn and Aβ dimers that dock on the membrane incorporated additional α-syn molecules, leading to the formation of more stable pentamers and hexamers that adopt a ring-like structure. Consistent with the simulations, under in vitro cell-free conditions, Aβ interacted with α-syn, forming hybrid pore-like oligomers. Moreover, cells expressing α-syn and treated with Aβ displayed increased current amplitudes and calcium influx consistent with the formation of cation channels.Conclusion/SignificanceThese results support the contention that Aβ directly interacts with α-syn and stabilized the formation of hybrid nanopores that alter neuronal activity and might contribute to the mechanisms of neurodegeneration in AD and PD. The broader implications of such hybrid interactions might be important to the pathogenesis of other disorders of protein misfolding.

In recent years, new hope for understanding the pathogenesis of Parkinson's disease (PD) and Lewy body dementia (LBD) has emerged with the discovery of mutations and duplications in the a-synuclein (a-syn) gene that are associated with rare familial forms of Parkinsonism [1][2][3]. Moreover, it has been shown that a-syn is centrally involved in the pathogenesis of both sporadic and inherited forms of PD and LBD because this molecule accumulates in Lewy bodies (LBs) [4][5][6], synapses, and axons, and its expression in transgenic (tg) mice [7][8][9] and Drosophila [10] mimics several aspects of PD.The mechanisms through which a-syn leads to neurodegeneration and the characteristic symptoms of LBD are unclear. However, recent evidence indicates that abnormal accumulation of misfolded a-syn in the Accumulation of a-synuclein resulting in the formation of oligomers and protofibrils has been linked to Parkinson's disease and Lewy body dementia. In contrast, b-synuclein (b-syn), a close homologue, does not aggregate and reduces a-synuclein (a-syn)-related pathology. Although considerable information is available about the conformation of a-syn at the initial and end stages of fibrillation, less is known about the dynamic process of a-syn conversion to oligomers and how interactions with antiaggregation chaperones such as b-synuclein might occur. Molecular modeling and molecular dynamics simulations based on the micelle-derived structure of a-syn showed that a-syn homodimers can adopt nonpropagating (head-to-tail) and propagating (head-to-head) conformations. Propagating a-syn dimers on the membrane incorporate additional a-syn molecules, leading to the formation of pentamers and hexamers forming a ring-like structure. In contrast, b-syn dimers do not propagate and block the aggregation of a-syn into ring-like oligomers. Under in vitro cell-free conditions, a-syn aggregates formed ring-like structures that were disrupted by b-syn. Similarly, cells expressing a-syn displayed increased ion current activity consistent with the formation of Zn 2+ -sensitive nonselective cation channels. These results support the contention that in Parkinson's disease and Lewy body dementia, a-syn oligomers on the membrane might form pore-like structures, and that the beneficial effects of b-synuclein might be related to its ability to block the formation of pore-like structures.Abbreviations aa, amino acid; a-syn, a-synuclein; b-syn, b-synuclein; GFP, green fluorescent protein; LBD, Lewy body disease; PD, Parkinson's disease; POPC, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine; tg, transgenic.

Parkinson’s disease (PD) and Dementia with Lewy bodies are common disorders of the aging population characterized by the progressive accumulation of α-synuclein (α-syn) in the CNS. Aggregation of α-syn into oligomers with a ring-like appearance has been proposed a role in toxicity. However, the molecular mechanisms and the potential sequence of events involved in the formation of pore-like structures are unclear. We utilized computer modeling and cell-based studies to investigate the process of α-syn (wild type and A53T) oligomerization in membranes. The studies suggest that α-syn rapidly penetrates the membrane, changing its conformation from α-helical toward a coiled structure. This penetration facilitate the incorporation of additional α-syn monomers to the complex, and subsequent displacement of phospholipids, and formation of oligomers in the membrane. This process occurred more rapidly, and with more favorable energy of interaction for mutant A53T compared with wild type α-syn. After 4 ns of simulation for the protein-membrane model α-syn penetrated through two thirds of the membrane. By 9 ns, the penetration of the annular α-syn oligomers can result in the formation of pore-like structures that fully perforate the lipid bilayer. Experimental incubation of recombinant α-syn in synthetic membranes resulted in the formation of similar pore-like complexes. Moreover, mutant (A53T) α-syn had a greater tendency to accumulate in neuronal membrane fractions in cell cultures, resulting in greater neuronal permeability with the calcein efflux assay. These studies provide a sequential molecular explanation for the process of α-syn oligomerization in the membrane, and support the role of formation of pore-like structures in the pathogenesis of the neurodegenerative process in PD.

Dextran-coated superparamagnetic iron oxide nanoparticles (dextran-SPIO conjugates) offer the attractive possibility of enhancing MRI imaging sensitivity so that small or diffuse lesions can be detected. However, systemically injected SPIO are rapidly removed by macrophages. We engineered embryonic cells (HEK293T) to express major macrophage scavenger receptor (SR) subtypes including SR-AI, MARCO, and endothelial receptor collectin-12. These SRs possess a positively charged collagen-like (CL) domain and they promoted SPIO uptake, while the charge neutral lipoprotein receptor SR-BI did not. In silico modeling indicated a positive net charge on the CL domain, and a net negative charge on the cysteine-rich (CR) domain of MARCO and SR-AI. In vitro experiments revealed that CR domain deletion in SR-AI boosted uptake of SPIO 3-fold, while deletion of MARCO's CR domain abolished this uptake. These data suggest that future studies might productively focus on the validation and further exploration of SR charge fields in SPIO recognition.

Human copper transporter 1 (hCTR1) is the major high affinity copper influx transporter in mammalian cells that also mediates uptake of the cancer chemotherapeutic agent cisplatin. A low resolution structure of hCTR1 determined by cryoelectron microscopy was recently published. Several protein structure simulation techniques were used to create an all-atom model of this important transporter using the low resolution structure as a starting point. The all-atom model provides new insights into the roles of specific residues of the N-terminal extracellular domain, the intracellular loop, and C-terminal region in metal ion transport. In particular, the model demonstrates that the central region of the pore contains four sets of methionine triads in the intramembranous region. The structure confirms that two triads of methionine residues delineate the intramembranous region of the transporter, and further identifies two additional methionine triads that are located in the extracellular N-terminal part of the transporter. Together, the four triads create a structure that promotes stepwise transport of metal ions into and then through the intramembranous channel of the transporter via transient thioether bonds to methionine residues. Putative copper-binding sites in the hCTR1 trimer were identified by a program developed by us for prediction of metal-binding sites. These sites correspond well with the known effects of mutations on the ability of the protein to transport copper and cisplatin.

Misfolding and oligomerization of unstructured proteins is involved in the pathogenesis of Parkinson's (PD), Alzheimer's (AD), Huntington's, and other neurodegenerative disorders. Elucidation of possible conformations of these proteins and their interactions with the membrane is necessary to understand the molecular mechanisms of neurodegeneration.We developed a strategy that makes it possible to elucidate the molecular mechanisms of of alphasynuclein aggregation-a key molecular event in the pathogenesis of PD. This strategy can be also useful for the study of other unstructured proteins involved in neurodegeneration. The results of these theoretical studies have been confirmed with biochemical and electrophysiological studies.Our studies provide insights into the molecular mechanism for PD initiation and progression, and provide a useful paradigm for identifying possible therapeutic interventions through computational modeling.

Parkinson’s disease (PD) is associated with the formation of toxic α-synuclein oligomers and their penetration the cell membrane. Familial forms of PD are caused by the point mutations A53T, A30P, E46K, and H50Q. Artificial point mutations E35K and E57K also increase oligomerization and pore formation. We generated structural conformations of α-synuclein and the abovementioned mutants using molecular dynamics. We elucidated four main regions in these conformers contacting the membrane and found that the region including residues 37–45 (Zone2) may have maximum membrane penetration. E57K mutant had the highest rate of interaction with the membrane by Zone2, followed by A53T, E46K, E35K mutants, and wt α-synuclein. The mutant A30P had the smallest percentage of conformers that contact the membrane than all other mutants and wt α-synuclein. These results were confirmed by experiments. We identified the key amino acids that can interact with the membrane (Y38, E62, and N65 (1st hydrophilic layer); E104, E105, and D115 (2nd hydrophilic layer), and V15 and V26 (central hydrophobic layer)) and the residues that are involved in the interprotein contacts (L38, V48, V49, Q62, and T64). Understanding the molecular interactions of α-synuclein mutants is important for the design of compounds blocking the formation of toxic oligomers.

Alzheimer’s disease (AD) is associated with the formation of toxic Aβ42 oligomers and recent evidence supports a role for Aβ dimers as building blocks for oligomers. Molecular dynamics (MD) simulation studies have identified clans for the dominant conformations of Aβ42 forming dimers; however, it is unclear if a larger spectrum of dimers is involved and which set(s) of dimers might evolve to oligomers verse fibrils. Therefore, for this study we generated multiple structural conformations of Aβ42, using explicit all-atom MD, and then clustering the different structures based on key conformational similarities. Those matching a selection threshold were then used to model a process of oligomerization. Remarkably, we showed a greater diversity in Aβ dimers than previously described. Depending on the clan family, different types of Aβ dimers were obtained. While some had the tendency to evolve into oligomeric rings, others formed fibrils of diverse characteristics. Then we selected the dimers that would evolve to membranephilic annular oligomers. Nearly one third of the 28 evaluated annular oligomers had the dimer interfaces between the neighboring Aβ42 monomers with possible salt bridges between the residue K28 from one side and either residue E22 or D23 on the other. Based on these results, key amino acids were identified for point mutations that either enhanced or suppressed the formation and toxicity of oligomer rings. Our studies suggest a greater diversity of Aβ dimers. Understanding the structure of Aβ dimers might be important for the rationale design of small molecules that block formation of toxic oligomers.