Challenges in Experimental Methods

Gąsior-Głogowska, Marlena; Szulc, Natalia; Szefczyk, Monika

doi:10.1007/978-1-0716-1546-1_13

“…29 This spectral signature is characteristic of amyloids, which are known to form long rigid fibrils. 30,31 The distinctive structural characteristics of peptide 1_c can be explained by the structural transition from an α-helix to a β-sheet, which has also been observed in other studies of coiled-coil peptides. 32 The variations in the helical packing of amino acid chains caused slight differences in the position of the local maxima of the amide I band in the spectra of the studied peptides.…”

Section: Resultssupporting

confidence: 66%

The application of the hierarchical approach for the construction of foldameric peptide self-assembled nanostructures

Szefczyk,

Szulc,

Gąsior-Głogowska

et al. 2023

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“…These two mechanisms have been widely investigated by various groups using many different approaches [30-34]. Nevertheless, most of these investigations have been carry out using synthetic Aβs peptides, living some ambiguity whether the proposed mechanisms of action can be translated to the effect of endogenous Aβ oligomers found in neurons of AD-affected brains [35]. Here, we report the ability of brain extract samples, displaying high content of Aβ oligomers, to evoke cytosolic Ca 2+ release upon intracellular injection, closely mirroring the effect of synthetic Aβ42 oligomers in analogous experiments [8].…”

Section: Resultsmentioning

confidence: 99%

Intracellular injection of brain extracts from Alzheimer’s disease patients trigger unregulated Ca²⁺ release from intracellular stores that hinders cellular bioenergetics

Pensalfini

¹

,

Umar

²

,

Glabe

³

et al. 2022

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0

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Strong evidence indicates that amyloid beta (Aβ) inflicts its toxicity in Alzheimer’s disease (AD) by promoting uncontrolled elevation of cytosolic Ca2+ in neurons. We have previously shown that synthetic Aβ42 oligomers stimulate abnormal intracellular Ca2+ release from the endoplasmic reticulum stores, suggesting that a similar mechanism of Ca2+ toxicity may be common to the endogenous Aβs oligomers. To investigate this possibility, we use human postmortem brain extracts from control and AD-affected patients and test their ability to trigger Ca2+ fluxes when injected intracellularly into Xenopus oocytes. Immunological characterization of samples from AD patients revealed elevated content of soluble Aβ oligomers, detected by the conformation-dependent OC-antibody, whereas no immunoreactivity was detected in the normal samples. Intracellular injection of brain extracts from control patients failed to trigger detectable changes in intracellular Ca2+. Conversely, brain extracts from AD patients triggered Ca2+ events consisting of local and global Ca2+ fluorescent transients rising within few seconds after injection and persisting for several seconds. Pre-incubation of brain extracts with the conformation specific OC antibody completely suppressed brain extract ability to trigger cytosolic Ca2+ events. Comparison of the elementary events triggered by brain extracts and synthetic Aβ42 oligomer showed comparable temporal evolution and amplitudes to events triggered by direct injection of IP3. Moreover, bath application of caffeine reversibly inhibited local and global Ca2+ signals in all the samples confirming the involvement of Ca2+ release from the ER. Analysis of the recorded Ca2+ fluorescence signals by computational modeling allowed quantification of the IP3 and Ca2+ generated by each sample. The model further shows that the abnormal increase of Ca2+ and IP3 may affect mitochondrial bioenergetics. These results, supports the hypothesis that endogenous amyloid oligomer contained in neurons of AD-affected brains may represent the toxic agents responsible for neurons malfunctioning and death, associated with the disruption of neuronal Ca2+ homeostasis.

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“…These two mechanisms have been widely investigated by various groups using many different approaches [ 29 , 30 , 31 , 32 , 33 ]. Nevertheless, most of these investigations have been carried out using synthetic Aβ peptides, leading to ambiguity as to whether the proposed mechanisms of action are also applicable to endogenous Aβ oligomers found in neurons of AD-affected brains [ 34 ]. Here, we report the ability of brain extract samples displaying a high content of Aβ oligomers to evoke cytosolic Ca 2+ release upon intracellular injection, closely mirroring the effect of synthetic Aβ42 oligomers in analogous experiments.…”

Section: Discussionmentioning

confidence: 99%

Intracellular Injection of Brain Extracts from Alzheimer’s Disease Patients Triggers Unregulated Ca2+ Release from Intracellular Stores That Hinders Cellular Bioenergetics

Pensalfini

¹

,

Umar²,

Glabe³

et al. 2022

Cells

2

0

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Strong evidence indicates that amyloid beta (Aβ) inflicts its toxicity in Alzheimer’s disease (AD) by promoting uncontrolled elevation of cytosolic Ca2+ in neurons. We have previously shown that synthetic Aβ42 oligomers stimulate abnormal intracellular Ca2+ release from the endoplasmic reticulum stores, suggesting that a similar mechanism of Ca2+ toxicity may be common to the endogenous Aβs oligomers. Here, we use human postmortem brain extracts from AD-affected patients and test their ability to trigger Ca2+ fluxes when injected intracellularly into Xenopus oocytes. Immunological characterization of the samples revealed the elevated content of soluble Aβ oligomers only in samples from AD patients. Intracellular injection of brain extracts from control patients failed to trigger detectable changes in intracellular Ca2+. Conversely, brain extracts from AD patients triggered Ca2+ events consisting of local and global Ca2+ fluorescent transients. Pre-incubation with either the conformation-specific OC antiserum or caffeine completely suppressed the brain extract’s ability to trigger cytosolic Ca2+ events. Computational modeling suggests that these Ca2+ fluxes may impair cells bioenergetic by affecting ATP and ROS production. These results support the hypothesis that Aβ oligomers contained in neurons of AD-affected brains may represent the toxic agents responsible for neuronal malfunctioning and death associated with the disruption of Ca2+ homeostasis.

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Challenges in Experimental Methods

Cited by 7 publications

References 135 publications

The application of the hierarchical approach for the construction of foldameric peptide self-assembled nanostructures

The application of the hierarchical approach for the construction of foldameric peptide self-assembled nanostructures

Intracellular injection of brain extracts from Alzheimer’s disease patients trigger unregulated Ca²⁺ release from intracellular stores that hinders cellular bioenergetics

Intracellular Injection of Brain Extracts from Alzheimer’s Disease Patients Triggers Unregulated Ca2+ Release from Intracellular Stores That Hinders Cellular Bioenergetics

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Challenges in Experimental Methods

Cited by 7 publications

References 135 publications

The application of the hierarchical approach for the construction of foldameric peptide self-assembled nanostructures

The application of the hierarchical approach for the construction of foldameric peptide self-assembled nanostructures

Intracellular injection of brain extracts from Alzheimer’s disease patients trigger unregulated Ca2+ release from intracellular stores that hinders cellular bioenergetics

Intracellular Injection of Brain Extracts from Alzheimer’s Disease Patients Triggers Unregulated Ca2+ Release from Intracellular Stores That Hinders Cellular Bioenergetics

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Intracellular injection of brain extracts from Alzheimer’s disease patients trigger unregulated Ca²⁺ release from intracellular stores that hinders cellular bioenergetics