Abrupt aggregation of amyloid β1-42 (Aβ1-42) peptide in the frontal lobe is the expected underlying cause of Alzheimer\'s disease (AD). β-Sheet-rich oligomers and fibrils formed by Aβ1-42 exert high cell toxicity. A growing body of evidence indicates that lipids can uniquely alter the secondary structure and toxicity of Aβ1-42 aggregates. At the same time, underlying molecular mechanisms that determine this difference in toxicity of amyloid aggregates remain unclear. Using a set of molecular and biophysical assays to determine the molecular mechanism by which Aβ1-42 aggregates formed in the presence of cholesterol, cardiolipin, and phosphatidylcholine exert cell toxicity. Our findings demonstrate that rat neuronal cells exposed to Aβ1-42 fibrils formed in the presence of lipids with different chemical structure exert drastically different magnitude and dynamic of unfolded protein response (UPR) in the endoplasmic reticulum (ER) and mitochondria (MT). We found that the opposite dynamics of UPR in MT and ER in the cells exposed to Aβ1-42: cardiolipin fibrils and Aβ1-42 aggregates formed in a lipid-free environment. We also found that Aβ1-42: phosphatidylcholine fibrils upregulated ER UPR simultaneously downregulating the UPR response of MT, whereas Aβ1-42: cholesterol fibrils suppressed the UPR response of ER and upregulated UPR response of MT. We also observed progressively increasing ROS production that damages mitochondrial membranes and other cell organelles, ultimately leading to cell death.

Amyloid β (Aβ) oligomers are the most neurotoxic forms of Aβ, and Aβ(1-42) is the prevalent Aβ peptide found in the amyloid plaques of Alzheimer\'s disease patients. Aβ(25-35) is the shortest peptide that retains the toxicity of Aβ(1-42). Aβ oligomers bind to calmodulin (CaM) and calbindin-D28k with dissociation constants in the nanomolar Aβ(1-42) concentration range. Aβ and histidine-rich proteins have a high affinity for transition metal ions Cu2+, Fe3+ and Zn2+. In this work, we show that the fluorescence of Aβ(1-42) HiLyteTM-Fluor555 can be used to monitor hexa-histidine peptide (His6) interaction with Aβ(1-42). The formation of His6/Aβ(1-42) complexes is also supported by docking results yielded by the MDockPeP Server. Also, we found that micromolar concentrations of His6 block the increase in the fluorescence of Aβ(1-42) HiLyteTM-Fluor555 produced by its interaction with the proteins CaM and calbindin-D28k. In addition, we found that the His6-tag provides a high-affinity site for the binding of Aβ(1-42) and Aβ(25-35) peptides to the human recombinant cytochrome b5 reductase, and sensitizes this enzyme to inhibition by these peptides. In conclusion, our results suggest that a His6-tag could provide a valuable new tool to experimentally direct the action of neurotoxic Aβ peptides toward selected cellular targets.

Lipid rafts are a primary target in studies of amyloid β (Aβ) cytotoxicity in neurons. Exogenous Aβ peptides bind to lipid rafts, which in turn play a key role in Aβ uptake, leading to the formation of neurotoxic intracellular Aβ aggregates. On the other hand, dysregulation of intracellular calcium homeostasis in neurons has been observed in Alzheimer\'s disease (AD). In a previous work, we showed that Aβ(1-42), the prevalent Aβ peptide found in the amyloid plaques of AD patients, binds with high affinity to purified calmodulin (CaM), with a dissociation constant ≈1 nM. In this work, to experimentally assess the Aβ(1-42) binding capacity to intracellular CaM, we used primary cultures of mature cerebellar granule neurons (CGN) as a neuronal model. Our results showed a large complexation of submicromolar concentrations of Aβ(1-42) dimers by CaM in CGN, up to 120 ± 13 picomoles of Aβ(1-42) /2.5 × 106 cells. Using fluorescence microscopy imaging, we showed an extensive co-localization of CaM and Aβ(1-42) in lipid rafts in CGN stained with up to 100 picomoles of Aβ(1-42)-HiLyteTM-Fluor555 monomers. Intracellular Aβ(1-42) concentration in this range was achieved by 2 h incubation of CGN with 2 μM Aβ(1-42), and this treatment lowered the resting cytosolic calcium of mature CGN in partially depolarizing 25 mM potassium medium. We conclude that the primary cause of the resting cytosolic calcium decrease is the inhibition of L-type calcium channels of CGN by Aβ(1-42) dimers, whose activity is inhibited by CaM:Aβ(1-42) complexes bound to lipid rafts.