Mitochondrial Disruption by Amyloid Beta 42 Identified by Proteomics and Pathway Mapping.
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ABSTRACT:
SUBMITTER: Sinclair P
PROVIDER: S-EPMC8468661 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Misfolding and aggregation of proteins is strongly linked to several neurodegenerative diseases, but how such species bring about their cytotoxic actions remains poorly understood. Here we used specifically-designed optical reporter probes and live fluorescence imaging in primary hippocampal neurons to characterise the mechanism by which prefibrillar, oligomeric forms of the Alzheimer's-associated peptide, Aβ42, exert their detrimental effects. We used a pH-sensitive reporter, Aβ42-CypHer, to track Aβ internalisation in real-time, demonstrating that oligomers are rapidly taken up into cells in a dynamin-dependent manner, and trafficked via the endo-lysosomal pathway resulting in accumulation in lysosomes. In contrast, a non-assembling variant of Aβ42 (vAβ42) assayed in the same way is not internalised. Tracking ovalbumin uptake into cells using CypHer or Alexa Fluor tags shows that preincubation with Aβ42 disrupts protein uptake. Our results identify a potential mechanism by which amyloidogenic aggregates impair cellular function through disruption of the endosomal-lysosomal pathway.
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Project description:Two carrier-free peptides were synthesized and used in experiments: amyloid beta 42 (DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA), and a scrambled variant with the same chemical constituency as amyloid beta 42 (AIAEGDSHVLKEGAYMEIFDVQGHVFGGKIFRVVDLGSHNVA). To perform blockade current measurements, first, the sub-nanopore was wetted by immersion in de-gassed 250 mM NaCl electrolyte for 1-3 days. Subsequently, to measure the blockade current, a transmembrane voltage bias (< 700 mV) was applied to the reservoir relative to the ground in the channel using Ag/AgCl electrodes and the corresponding pore current was measured using either an Axopatch 700B or an Axopatch 200B amplifier with an open bandwidth. The actual bandwidth was inferred from the rise-time to a sharp (10 ps rise-time) input pulse to be about 75 kHz to 100 kHz, depending on the amplifier and the feedback. The analog data were digitized by a 16-bit DigiData 1550B data acquisition system (DAQ, Molecular Devices, Sunnyvale, CA) at a sampling rate of 500 kS/s and recorded in 3 minute-long acquisition windows. A total of 12 Axon binary files (ABF) were collected for amyloid beta 42, and 71 ABF files for scrambled amyloid beta 42.
2022-06-30 | MSV000089779 | MassIVE
Project description:IntroductionCerebrospinal fluid (CSF) amyloid-beta 38 (A?38), 40 (A?40), 42 (A?42) and total tau (T-tau) are finding increasing utility as biomarkers of Alzheimer's disease (AD). The purpose of this study was to determine whether measured CSF biomarker concentrations were affected by transfer of CSF between tubes, and whether addition of a non-ionic surfactant mitigates any observed effects.MethodsAD and control CSF was transferred consecutively between polypropylene tubes. A? peptides and T-tau were measured with and without addition of Tween 20 (0.05%).ResultsMeasured concentrations of A?42 decreased by approximately 25% with each consecutive transfer. Measured concentrations of A?38 and A?40 were also observed to decrease significantly with each consecutive transfer (approximately 16% loss per transfer). Measured concentrations of T-tau also decreased significantly, but at much smaller magnitude than the A? peptides (approximately 4% loss per transfer). The addition of Tween 20 mitigated this effect in all samples.ConclusionsConsecutive CSF transfer between tubes has a significant impact on the measured concentration of all A? peptides, and significant effect of lesser magnitude on T-tau. This would be sufficient to alter biomarker ratios enough to mislead diagnosis. The introduction of Tween 20 at the initial aliquoting stage was observed to significantly mitigate this effect.
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Project description:Beta-amyloid (Abeta) degrading endopeptidases are thought to protect against Alzheimer's disease (AD) and are potentially therapeutic. Of particular interest are endopeptidases that are blocked by thiorphan and phosphoramidon (T/P), as these inhibitors rapidly induce Abeta deposition in rodents. Neprilysin (NEP) is the best known target of T/P; however neprilysin knockout results in only modest Abeta increases insufficient to induce deposition. Therefore, other endopeptidases targeted by T/P must be critical for Abeta catabolism. Another candidate is the T/P sensitive membrane metallo-endopeptidase-like protein (MMEL), a close homolog of neprilysin. The endopeptidase properties of beta and gamma splice forms of human MMEL were determined in HEK293T cells transduced with the human cDNAs for the two splice forms; this showed degradation of both Abeta(42) and Abeta(40) by hMMEL-beta but not hMMEL-gamma. hMMEL-beta activity was found at the extracellular surface with no significant secreted activity. hMMEL-gamma was not expressed at the extracellular surface. Finally, it was found that hMMEL cleaves Abeta near the alpha-secretase site (producing Abeta(1-17)>>Abeta(1-16)). These data establish hMMEL as a mediator of Abeta catabolism and raise the possibility of its involvement in the etiology of AD and as a target for intervention.
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Project description:RelevanceMolecular network changes are the hallmark of the pathogenesis of ovarian cancers (OCs). Network-based biomarkers benefit for the effective treatment of OC.PurposeThis study sought to identify key pathway-network alterations and network-based biomarkers for clarification of molecular mechanisms and treatment of OCs.MethodsIngenuity Pathway Analysis (IPA) platform was used to mine signaling pathway networks with 1198 human tissue mitochondrial differentially expressed proteins (mtDEPs) and compared those pathway network changes between OCs and controls. The mtDEPs in important cancer-related pathway systems were further validated with qRT-PCR and Western blot in OC cell models. Moreover, integrative analysis of mtDEPs and Cancer Genome Atlas (TCGA) data from 419 patients was used to identify hub molecules with molecular complex detection method. Hub molecule-based survival analysis and multiple multivariate regression analysis were used to identify survival-related hub molecules and hub molecule signature model.ResultsPathway network analysis revealed 25 statistically significant networks, 192 canonical pathways, and 5 significant molecular/cellular function models. A total of 52 canonical pathways were activated or inhibited in cancer pathogenesis, including antigen presentation, mitochondrial dysfunction, GP6 signaling, EIF2 signaling, and glutathione-mediated detoxification. Of them, mtDEPs (TPM1, CALR, GSTP1, LYN, AKAP12, and CPT2) in those canonical pathway and molecular/cellular models were validated in OC cell models at the mRNA and protein levels. Moreover, 102 hub molecules were identified, and they were regulated by post-translational modifications and functioned in multiple biological processes. Of them, 62 hub molecules were individually significantly related to OC survival risk. Furthermore, multivariate regression analysis of 102 hub molecules identified significant seven hub molecule signature models (HIST1H2BK, ALB, RRAS2, HIBCH, EIF3E, RPS20, and RPL23A) to assess OC survival risks.ConclusionThese findings provided the overall signaling pathway network profiling of human OCs; offered scientific data to discover pathway network-based cancer biomarkers for diagnosis, prognosis, and treatment of OCs; and clarify accurate molecular mechanisms and therapeutic targets. These findings benefit for the discovery of effective and reliable biomarkers based on pathway networks for OC predictive and personalized medicine.
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Project description:Alzheimer's disease is the most fatal neurodegenerative disorder wherein the process of amyloid-beta (Abeta) amyloidogenesis appears causative. Here, we present the 3D structure of the fibrils comprising Abeta(1-42), which was obtained by using hydrogen-bonding constraints from quenched hydrogen/deuterium-exchange NMR, side-chain packing constraints from pairwise mutagenesis studies, and parallel, in-register beta-sheet arrangement from previous solid-state NMR studies. Although residues 1-17 are disordered, residues 18-42 form a beta-strand-turn-beta-strand motif that contains two intermolecular, parallel, in-register beta-sheets that are formed by residues 18-26 (beta1) and 31-42 (beta2). At least two molecules of Abeta(1-42) are required to achieve the repeating structure of a protofilament. Intermolecular side-chain contacts are formed between the odd-numbered residues of strand beta1 of the nth molecule and the even-numbered residues of strand beta2 of the (n - 1)th molecule. This interaction pattern leads to partially unpaired beta-strands at the fibrillar ends, which explains the sequence selectivity, the cooperativity, and the apparent unidirectionality of Abeta fibril growth. It also provides a structural basis for fibrillization inhibitors.
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Project description:Using a predictive coarse-grained protein force field, we compute and compare the free energy landscapes and relative stabilities of amyloid-? protein (1-42) and amyloid-? protein (1-40) in their monomeric and oligomeric forms up to the octamer. At the same concentration, the aggregation free energy profile of A?42 is more downhill, with a computed solubility that is about 10 times smaller than that of A?40. At a concentration of 40 ?M, the clear free energy barrier between the pre-fibrillar tetramer form and the fibrillar pentamer in the A?40 aggregation landscape disappears for A?42, suggesting that the A?42 tetramer has a more diverse structural range. To further compare the landscapes, we develop a cluster analysis based on the structural similarity between configurations and use it to construct an oligomerization map that captures the paths of easy interconversion between different but structurally similar states of oligomers for both species. A taxonomy of the oligomer species based on ?-sheet stacking topologies is proposed. The comparison of the two oligomerization maps highlights several key differences in the landscapes that can be attributed to the two additional C-terminal residues that A?40 lacks. In general, the two terminal residues strongly stabilize the oligomeric structures for A?42 relative to A?40, and greatly facilitate the conversion from pre-fibrillar trimers to fibrillar tetramers.
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Project description:Amyloid beta 42 (Abeta42) is the principal trigger of neurodegeneration during Alzheimer's disease (AD). However, the etiology of its noxious cellular effects remains elusive. In a combinatory genetic and proteomic approach using a yeast model to study aspects of intracellular Abeta42 toxicity, we here identify the HSP40 family member Ydj1, the yeast orthologue of human DnaJA1, as a crucial factor in Abeta42-mediated cell death. We demonstrate that Ydj1/DnaJA1 physically interacts with Abeta42 (in yeast and mouse), stabilizes Abeta42 oligomers, and mediates their translocation to mitochondria. Consequently, deletion of YDJ1 strongly reduces co-purification of Abeta42 with mitochondria and prevents Abeta42-induced mitochondria-dependent cell death. Consistently, purified DnaJ chaperone delays Abeta42 fibrillization in vitro, and heterologous expression of human DnaJA1 induces formation of Abeta42 oligomers and their deleterious translocation to mitochondria in vivo. Finally, downregulation of the Ydj1 fly homologue, Droj2, improves stress resistance, mitochondrial morphology and memory performance in a Drosophila melanogaster AD model. These data reveal an unexpected and detrimental role for specific HSP40s in promoting hallmarks of Abeta42 toxicity.
| S-SCDT-EMM-2021-13952 | biostudies-other