The pathways leading specifically to the toxic Aβ42 peptide production, a key event in Alzheimer's disease (AD), are unknown. While searching for pathways that mediate pathological increases of Aβ42, we identified Aftin-4, a new compound that selectively and potently increases Aβ42 compared to DMSO (N2a cells: 7-fold; primary neurons: 4-fold; brain lysates: 2-fold) with an EC(50) of 30 μM. These results were confirmed by ELISA and IP-WB. Using affinity chromatography and mass spectrometry, we identified 3 proteins (VDAC1, prohibitin, and mitofilin) relevant to AD that interact with Aftin-4, but not with a structurally similar but inactive molecule. Electron microscopy studies demonstrated that Aftin-4 induces a reversible mitochondrial phenotype reminiscent of the one observed in AD brains. Sucrose gradient fractionation showed that Aftin-4 perturbs the subcellular localization of γ-secretase components and could, therefore, modify γ-secretase specificity by locally altering its membrane environment. Remarkably, Aftin-4 shares all these properties with two other "AD accelerator" compounds. In summary, treatment with three Aβ42 raising agents induced similar biochemical alterations that lead to comparable cellular phenotypes in vitro, suggesting a common mechanism of action involving three structural cellular targets.