Cyclophilin D (CypD, encoded by Ppif) is an integral part of the mitochondrial permeability transition pore, whose opening leads to cell death. Here we show that interaction of CypD with mitochondrial amyloid-β protein (Aβ) potentiates mitochondrial, neuronal and synaptic stress. The CypD-deficient cortical mitochondria are resistant to Aβ- and Ca2+-induced mitochondrial swelling and permeability transition. Additionally, they have an increased calcium buffering capacity and generate fewer mitochondrial reactive oxygen species. Furthermore, the absence of CypD protects neurons from Aβ- and oxidative stress-induced cell death. Notably, CypD deficiency substantially improves learning and memory and synaptic function in an Alzheimer's disease mouse model and alleviates Aβ-mediated reduction of long-term potentiation. Thus, the CypD-mediated mitochondrial permeability transition pore is directly linked to the cellular and synaptic perturbations observed in the pathogenesis of Alzheimer's disease. Blockade of CypD may be a therapeutic strategy in Alzheimer's disease.

SummaryThe role of two peptides, Aβ40 and Aβ42 in the early pathogenesis of the Alzheimer's disease (AD) is frequently emphasized in the literature. It is known that Aβ42 is more prone to aggregation than Aβ40, even though they only differ in two (IA) amino acid residues at the C-terminal end. A direct comparison of the ensembles of conformations adopted by the monomers in solution has been limited by the inherent flexibility of the unfolded peptides. Here we characterize the conformations of Aβ40 and Aβ42 in water by using a combination of molecular dynamics (MD) and measured scalar 3 J HNHα data from NMR experiments. We perform replica exchange MD (REMD) simulations and find that classical forcefields quantitatively reproduce the NMR data when the sampling is extended to the microseconds time scale. Using the quantitative agreement of the NMR data as a validation of the model, we proceed to compare the conformational ensembles of the Aβ40 and Aβ42 peptide monomers. Our analysis confirms the existence of structured regions within the otherwise flexible Aβ peptides. We find that the C-terminus of Aβ42 is more structured than that of Aβ40. The formation of a β-hairpin in the sequence 31 IIGLMVGGVVIA involving short strands at residues 31−34 and 38−41 reduces the C-terminal flexibility of the Aβ42 peptide and may be responsible for the higher propensity of this peptide to form amyloids. KeywordsAlzheimer's disease; Amyloid-β peptides; conformational ensemble; replica exchange molecular dynamics; J-coupling constants Two peptides have received tremendous interest in modern Alzheimer's disease (AD) research. Aβ40 and 42 are major products of the proteolytic cleavage of a multi-domain integral membrane type I protein, Amyloid-β Precursor Protein (APP), whose functions include cell adhesion, neuronal mobility and transcriptional regulation 1 . APP metabolism includes processing by a group of dedicated proteases, named secretases, in two known pathways to yield intracellular and extracellular fragments with a broad range of functions in synaptic transmission and neuronal plasticity 2 . During the amyloidogenic pathway, the action of β and subsequently γ secretase yields the Aβ peptides. The exact location of the transmembrane cleavage site for γ secretase results in a variability in the length of its Aβ product from 38 to 43 residues, however lengths of 40 and 42 are the dominant species. The physiological role of the Aβ peptides in vivo remains unclear. Aβ40 has been proposed to regulate the activity of K + channels 3 and also to modulate synaptic transmission 4 , however the mechanisms upon it * To whom correspondence should be addressed angel@rpi.edu Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production p...

Graphical abstract

Amyloidoses are diseases characterized by abnormal protein folding and self-assembly, for which no cure is available. Inhibition or modulation of abnormal protein self-assembly therefore is an attractive strategy for prevention and treatment of amyloidoses. We examined Lys-specific molecular tweezers and discovered a lead compound termed CLR01, which is capable of inhibiting the aggregation and toxicity of multiple amyloidogenic proteins by binding to Lys residues and disrupting hydrophobic and electrostatic interactions important for nucleation, oligomerization, and fibril elongation. Importantly, CLR01 shows no toxicity at concentrations substantially higher than those needed for inhibition. We used amyloid β-protein (Aβ) to further explore the binding site(s) of CLR01 and the impact of its binding on the assembly process. Mass-spectrometry and solution-state NMR demonstrated binding of CLR01 to the Lys residues in Aβ at the earliest stages of assembly. The resulting complexes were indistinguishable in size and morphology from Aβ oligomers but were non-toxic and were not recognized by the oligomer-specific antibody A11. Thus, CLR01 binds already at the monomer stage and modulates the assembly reaction into formation of non-toxic structures. The data suggest that molecular tweezers are unique, process-specific inhibitors of aberrant protein aggregation and toxicity, which hold promise for developing disease-modifying therapy for amyloidoses.

Amyloid β (Aβ) peptides are a primary component of fibrils and oligomers implicated in the etiology of Alzheimer’s disease (AD). However, the intrinsic flexibility of these peptides has frustrated efforts to investigate the secondary and tertiary structure of Aβ monomers, whose conformational landscapes directly contribute to the kinetics and thermodynamics of Aβ aggregation. In this work, de novo replica exchange molecular dynamics (REMD) simulations on the μs/replica timescale are used to characterize the structural ensembles of Aβ42, Aβ40, and M35-oxidized Aβ42, three physiologically relevant isoforms with substantially different aggregation properties. J-coupling data calculated from the REMD trajectories were compared to corresponding NMR-derived values acquired through two different pulse sequences, revealing that all simulations converge on the order of hundreds of ns/replica toward ensembles that yield good agreement with experiment. Though all three Aβ species adopt highly heterogeneous ensembles, these are considerably more structured compared to simulations on shorter timescales. Prominent in the C-terminus are antiparallel β-hairpins between L17-A21, A30-L36, and V39-I41, similar to oligomer and fibril intrapeptide models, that expose these hydrophobic side chains to solvent and may serve as hotspots for self-association. Compared to reduced Aβ42, the absence of a second β-hairpin in Aβ40 and the sampling of alternate β topologies by M35-oxidized Aβ42 may explain the reduced aggregation rates of these forms. A persistent V24-K28 bend motif, observed in all three species, is stabilized by buried backbone to side chain hydrogen bonds with D23 and a cross-region salt bridge between E22 and K28, highlighting the role of the familial AD-linked E22 and D23 residues in Aβ monomer folding. These characterizations help illustrate the conformational landscapes of Aβ monomers at atomic resolution and provide insight into the early stages of Aβ aggregation pathways.