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Presenilins (PS) are critical components of the gamma-secretase complex that mediates cleavage of type I membrane proteins including the beta-amyloid precursor protein to generate the amyloid beta-peptide. In addition, PS1 interacts with beta-catenin and facilitates its metabolism. We demonstrate that phosphorylation of serines 353 and 357 by glycogen synthase kinase-3beta (GSK3beta) induces a structural change of the hydrophilic loop of PS1 that can also be mimicked by substitution of the phosphorylation sites by negatively charged amino acids in vitro and in cultured cells. The structural change of PS1 reduces the interaction with beta-catenin leading to decreased phosphorylation and ubiquitination of beta-catenin. The decreased interaction of PS1 with beta-catenin leads to stabilization of beta-catenin thereby increasing its nuclear signaling and the transcription of target genes, including c-MYC. Consistent with increased expression of c-myc, a PS1 mutant that mimics phosphorylated PS1 increased cell proliferation as compared with wild-type PS1. These results indicate a regulatory mechanism in which GSK3beta-mediated phosphorylation induces a structural change of the hydrophilic loop of PS1 thereby negatively modulating the formation of a ternary complex between beta-catenin, PS1, and GSK3beta, which leads to stabilization of beta-catenin.

Numerous membrane-bound proteins undergo regulated intramembrane proteolysis. Regulated intramembrane proteolysis is initiated by shedding, and the remaining stubs are further processed by intramembrane-cleaving proteases (I-CLiPs). Neuregulin 1 type III (NRG1 type III) is a major physiological substrate of β-secretase (β-site amyloid precursor protein-cleaving enzyme 1 (BACE1)). BACE1-mediated cleavage is required to allow signaling of NRG1 type III. Because of the hairpin nature of NRG1 type III, two membrane-bound stubs with a type 1 and a type 2 orientation are generated by proteolytic processing. We demonstrate that these stubs are substrates for three I-CLiPs. The type 1-oriented stub is further cleaved by γ-secretase at an ϵ-like site five amino acids N-terminal to the C-terminal membrane anchor and at a γ-like site in the middle of the transmembrane domain. The ϵ-cleavage site is only one amino acid N-terminal to a Val/Leu substitution associated with schizophrenia. The mutation reduces generation of the NRG1 type III β-peptide as well as reverses signaling. Moreover, it affects the cleavage precision of γ-secretase at the γ-site similar to certain Alzheimer disease-associated mutations within the amyloid precursor protein. The type 2-oriented membrane-retained stub of NRG1 type III is further processed by signal peptide peptidase-like proteases SPPL2a and SPPL2b. Expression of catalytically inactive aspartate mutations as well as treatment with 2,2'-(2-oxo-1,3-propanediyl)bis[(phenylmethoxy)carbonyl]-l-leucyl-l-leucinamide ketone inhibits formation of N-terminal intracellular domains and the corresponding secreted C-peptide. Thus, NRG1 type III is the first protein substrate that is not only cleaved by multiple sheddases but is also processed by three different I-CLiPs.

Alzheimer's disease (AD) is characterized by the accumulation of amyloid plaques, which are predominantly composed of amyloid β-peptide (Aβ)1. Two principal physiological pathways either prevent or promote Aβ generation from its precursor (amyloid precursor protein; APP) in a competitive manner1. Modulation of the amyloidogenic pathway is currently exploited by anti-Aβ therapeutic strategies2. Although APP processing has been studied in great detail, unknown proteolytic events appear to hinder stoichiometric analyses of APP metabolism in vivo3. We now identified higher molecular weight C-terminal fragments of APP (CTF-η) in addition to the long-known α- and β-secretase (a disintegrin and metalloproteinase; ADAM10 and β-site APP cleaving enzyme 1; BACE1) generated CTF-α and CTF-β. Generation of CTF-η is mediated in part by membrane bound matrix-metalloproteinases such as MT5-MMP, referred to as η-secretase activity. η-Secretase cleavage occurs primarily at amino acids 504/505 of APP(695) releasing a truncated, soluble APP ectodomain (sAPP-η). Upon shedding of sAPP-η CTF-η is further processed by ADAM10 and BACE1 to release long and short Aη peptides (Aη-α and Aη-β). Aη peptides are therefore distinct from N-terminally extended Aβ variants4,5, since they do not extend to the γ-secretase cleavage sites. η-Secretase produced CTFs are enriched in dystrophic neurites in an AD mouse model and human AD brains6. Genetic and pharmacological inhibition of BACE1 activity results in a robust accumulation of CTF-η and Aη-α. In mice treated with a potent BACE1 inhibitor hippocampal long-term potentiation (LTP) was reduced. Strikingly, when recombinant or synthetic Aη-α was applied on hippocampal slices ex vivo, LTP was lowered. Furthermore, in vivo single cell two-photon calcium imaging revealed that hippocampal neuronal activity was attenuated by Aη-α. These findings not only demonstrate a major physiologically relevant APP processing pathway but may also suggest potential translational relevance for therapeutic strategies targeting APP processing.

Proteolytic shedding of cell surface proteins generates paracrine signals involved in numerous signaling pathways. Neuregulin 1 (NRG1) type III is involved in myelination of the peripheral nervous system, for which it requires proteolytic activation by proteases of the ADAM family and BACE1. These proteases are major therapeutic targets for the prevention of Alzheimer's disease because they are also involved in the proteolytic generation of the neurotoxic amyloid β-peptide. Identification and functional investigation of their physiological substrates is therefore of greatest importance in preventing unwanted side effects. Here we investigated proteolytic processing of NRG1 type III and demonstrate that the ectodomain can be cleaved by three different sheddases, namely ADAM10, ADAM17, and BACE1. Surprisingly, we not only found cleavage by ADAM10, ADAM17, and BACE1 C-terminal to the epidermal growth factor (EGF)-like domain, which is believed to play a pivotal role in signaling, but also additional cleavage sites for ADAM17 and BACE1N-terminal to that domain. Proteolytic processing at N- and C-terminal sites of the EGF-like domain results in the secretion of this domain from NRG1 type III. The soluble EGF-like domain is functionally active and stimulates ErbB3 signaling in tissue culture assays. Moreover, the soluble EGF-like domain is capable of rescuing hypomyelination in a zebrafish mutant lacking BACE1. Our data suggest that NRG1 type III-dependent myelination is not only controlled by membrane-retained NRG1 type III, but also in a paracrine manner via proteolytic liberation of the EGF-like domain.

Deposition of amyloid β peptides (Aβs) in extracellular amyloid plaques within the human brain is a hallmark of Alzheimer's disease (AD). Aβ derives from proteolytic processing of the amyloid precursor protein (APP) by β- and γ-secretases. The initial cleavage by β-secretase results in shedding of the APP ectodomain and generation of APP C-terminal fragments (APP-CTFs), which can then be further processed within the transmembrane domain by γ-secretase, resulting in release of Aβ. Here, we demonstrate that accumulation of sphingolipids (SLs), as occurs in lysosomal lipid storage disorders (LSDs), decreases the lysosome-dependent degradation of APP-CTFs and stimulates γ-secretase activity. Together, this results in increased generation of both intracellular and secreted Aβ. Notably, primary fibroblasts from patients with different SL storage diseases show strong accumulation of potentially amyloidogenic APP-CTFs. By using biochemical, cell biological, and genetic approaches, we demonstrate that SL accumulation affects autophagic flux and impairs the clearance of APP-CTFs. Thus, accumulation of SLs might not only underlie the pathogenesis of LSDs, but also trigger increased generation of Aβ and contribute to neurodegeneration in sporadic AD.