Alzheimer’s disease (AD) is divided into two main subtypes, early-oneset AD and late-onset AD, which are defined according to the patient’s age at disease onset. Early-onset AD or familial AD (FAD) comprises less than 2% of the AD cases and affects the patients before they reach the age of 65 years. The remaining 98% of the patients suffer from late-onset AD, which occurs at older ages. Several genetic mutations that promote FAD have been identified and helped to characterize the molecular processes that contribute to the development of AD. Most of these mutations can be directly associated with the processing of amyloid precursor protein (APP), either directly affecting the molecular characteristics of APP itself or affecting the presenilin subunits of the γ-secretase complex (Presenilin 1 or 2). The amyloidogenic peptide Aβ, which is causally linked to AD, is released from the APP through sequential proteolytic cleavage by β- and γ-secretases. The APP is a type I transmembrane protein that can undergo sequential proteolytic cleavage in two distinct routes. In the non-amyloidogenic pathway, APP is cleaved by α-secretase, giving rise to the N-terminal soluble-APP alpha fragment (sAPPα) and the 83-amino-acid-long C‑terminal fragment (C83). C83 can undergo further proteolytic cleavage by γ-secretase releasing the short transmembrane domain p3 and the APP intracellular domain (AICD or C59). In the amyloidogenic pathway, APP is first cleaved by β-secretase giving rise to the N-terminal-soluble APP beta fragment (sAPPβ) and the 99-amino-acid-long C‑terminal fragment (C99). C99 can undergo further proteolytic cleavage by γ-secretase releasing the amyloid beta peptide (Aβ) and C59 (AICD). α-Secretase cleaves APP within the Aβ domain, hence precluding the production of Aβ and is, therefore, termed as the non-amyloidogenic pathway. α-Secretase activity on APP has been shown to be conferred mainly by ADAM-10, a member of the protein family of disintegrin and metalloproteases (ADAM). Members of the ADAM family are transmembrane proteins locating to the plasma membrane, where α-cleavage of APP occurs. They are implicated in ectodomain shedding of several substrates. Because α-secretase cleavage of APP evades Aβ formation, enhancing its activity was considered for therapeutic intervention in AD. But the resulting side effects include metastasis formation in tumor patients. Hence α-secretase was abandoned as a potential therapeutic target. The generation of Aβ from APP is initiated through cleavage by a transmembrane aspartyl protease termed as BACE1 (β-site APP-cleaving enzyme 1). BACE1/β-secretase is a type I transmembrane protein whose active site is located in the ectodomain that cleaves APP between the amino acids Met-671 and Asp-672. This cleavage results in the release of the extracellular APP domain (sAPPβ) and a membrane-bound, C99. BACE1 is mainly localized to the TGN, endosomes, and lysosomes, but β-secretase cleavage of APP occurs predominantly in endosomes where the acidic pH (4.0–5.0) is optimal for β‑secretase activity. Agents that disrupt the intracellular pH will, therefore, also inhibit β‑secretase activity. Trafficking of BACE1 to the endosomes, which is necessary for Aβ production, can happen either via internalization from the plasma membrane or by direct sorting from the TGN. Furthermore, interference of this trafficking in order to prevent BACE1 to reach the endosomes might be a potential therapeutic strategy to reduce β-cleavage of APP. Alternatively, accelerating its trafficking away from endosomes reduces β-cleavage of APP and, therefore, Aβ release. γ-Secretase is a multimeric protein complex that is composed of four different transmembrane components: Presenilin-1(PS1)/Presenilin-2(PS2), Anterior pharynx defective-1 (in humans, Aph-1a or Aph-1b), Nicastrin, and Presenilin enhancer2 (Pen-2). In addition, numerous other proteins have been shown to bind and interact with this protein complex to modulate its function, but whether they also affect γ-secretase-mediated processing of APP remains to be resolved. The γ-secretase complex is the last of the proteolytic enzymes in APP processing and, therefore, directly contributes to Aβ levels. γ-Secretase components are synthesized in the endoplasmatic reticulum (ER), but the assembly of the mature and functional complex requires the coordinated regulation of the ER-Golgi recycling circuit. Not only at the synthesis level but also in order to access its substrate APP, the γ-secretase complex relies on intracellular trafficking as cleavage of C-terminal APP occurs in post‑Golgi compartments, i.e., in endosomes. APP cleavage by γ‑secretase happens at different positions within the APP sequence, leading to the release of amyloid peptides of various lengths (Aβ1-38, Aβ1-40, Aβ1-42). The reason why γ-secretase acts after α-or β-cleavage has been attributed to the fact that the large ectodomain of the substrates probably sterically hinders the substrate binding to γ-secretase and needs to be shedded. This is probably one reason for ectodomain shedding so that the C-terminal fragments can now be accommodated in the active site of γ-secretase complex. Thus, ectodomain shedding by α-/β-secretases is a prerequisite for γ-cleavage and γ-secretase activity is needed only after the cleavages by α-/β-secretases have occurred. However, here we report that γ-secretase inhibition, either by pharmacological inhibition or by silencing γ-secretase components, increases α-secretase cleavage of APP. Our results uncover a novel feedback regulation of α-secretase via γ-secretase. Since γ-secretase inhibitors are considered for AD therapy, and since sAPPα plays a role in neuroprotection, our study reveals an important side effect of γ-secretase inhibitor therapy and also suggests the elevation of sAPPa levels to be a theragnostic marker for γ-secretase inhibition.