Alzheimer disease amyloid ␤-peptide (A␤) is generated via proteolytic processing of the ␤-amyloid precursor protein by ␤-and ␥-secretase. ␥-Secretase can be blocked by selective inhibitors but can also be modulated by a subset of non-steroidal antiinflammatory drugs, including sulindac sulfide. These drugs selectively reduce the generation of the aggregation-prone 42-amino acid A␤ 42 and concomitantly increase the levels of the rather benign A␤ 38 . Here we show that A␤ 42 and A␤ 38 generation occur independently from each other. The amount of A␤ 42 produced by cells expressing 10 different familial Alzheimer disease (FAD)-associated mutations in presenilin (PS) 1, the catalytic subunit of ␥-secretase, appeared to correlate with the respective age of onset in patients. However, A␤ 38 levels did not show a negative correlation with the age of onset. Modulation of ␥-secretase activity by sulindac sulfide reduced A␤ 42 in the case of wild type PS1 and two FAD-associated PS1 mutations (M146L and A285V). The remaining eight PS1 FAD mutants showed either no reduction of A␤ 42 or only rather subtle effects. Strikingly, even the mutations that showed no effect on A␤ 42 levels allowed a robust increase of A␤ 38 upon treatment with sulindac sulfide. Similar observations were made for fenofibrate, a compound known to increase A␤ 42 and to decrease A␤ 38 . For mutants that predominantly produce A␤ 42 , the ability of fenofibrate to further increase A␤ 42 levels became diminished, whereas A␤ 38 levels were altered to varying extents for all mutants analyzed. Thus, we conclude that A␤ 38 and A␤ 42 production do not depend on each other. Using an independent non-steroidal anti-inflammatory drug derivative, we obtained similar results for PS1 as well as for PS2. These in vitro results were confirmed by in vivo experiments in transgenic mice expressing the PS2 N141I FAD mutant. Our findings therefore have strong implications on the selection of transgenic mouse models used for screening of the A␤ 42 -lowering capacity of ␥-secretase modulators. Furthermore, human patients with certain PS mutations may not respond to ␥-secretase modulators.Alzheimer disease is the most abundant form of dementia, and increasing numbers of patients are to be expected in the near future. Amyloid ␤-peptide (A␤) 5 is a central player in the disease pathology. Originally it was purified as the building block of the disease-defining amyloid plaques. Now it is becoming clear that amyloid plaques are probably not the major neurotoxic entity in the disease rather this is an assembly of soluble oligomeric A␤ species (1). These assemblies initiate the so-called amyloid cascade and finally induce abnormal phosphorylation of tau and subsequent formation of paired helical filaments (2). A␤ is generated by proteolytic processing of the ␤-amyloid precursor protein (APP). Two proteases, ␤-secretase and ␥-secretase, perform the cleavages on the N and C termini of the A␤ domain, respectively (3). ␤-Secretase is a conventional aspartyl protease, whereas ␥-secretase i...

Sequence variations occurring in the gene encoding the triggering receptor expressed on myeloid cells 2 (TREM2) support an essential function of microglia and innate immunity in the pathogenesis of Alzheimer's disease (AD) and other neurodegenerative disorders. TREM2 matures within the secretory pathway, and its ectodomain is shed on the plasma membrane. Missense mutations in the immunoglobulin (Ig)‐like domain such as p.T66M and p.Y38C retain TREM2 within the endoplasmic reticulum and reduce shedding as well as TREM2‐dependent phagocytosis. Using mass spectrometry, we have now determined the cleavage site of TREM2. TREM2 is shed by proteases of the ADAM (a disintegrin and metalloproteinase domain containing protein) family C‐terminal to histidine 157, a position where an AD‐associated coding variant has been discovered (p.H157Y) in the Han Chinese population. Opposite to the characterized mutations within the Ig‐like domain, such as p.T66M and p.Y38C, the p.H157Y variant within the stalk region leads to enhanced shedding of TREM2. Elevated ectodomain shedding reduces cell surface full‐length TREM2 and lowers TREM2‐dependent phagocytosis. Therefore, two seemingly opposite cellular effects of TREM2 variants, namely reduced versus enhanced shedding, result in similar phenotypic outcomes by reducing cell surface TREM2.

Protein N-glycosylation is involved in a variety of physiological and pathophysiological processes such as autoimmunity, tumour progression and metastasis. Signal peptide peptidase-like 3 (SPPL3) is an intramembrane-cleaving aspartyl protease of the GxGD type. Its physiological function, however, has remained enigmatic, since presently no physiological substrates have been identified. We demonstrate that SPPL3 alters the pattern of cellular N-glycosylation by triggering the proteolytic release of active site-containing ectodomains of glycosidases and glycosyltransferases such as N-acetylglucosaminyltransferase V, b-1,3 N-acetylglucosaminyltransferase 1 and b-1,4 galactosyltransferase 1. Cleavage of these enzymes leads to a reduction in their cellular activity. In line with that, reduced expression of SPPL3 results in a hyperglycosylation phenotype, whereas elevated SPPL3 expression causes hypoglycosylation. Thus, SPPL3 plays a central role in an evolutionary highly conserved post-translational process in eukaryotes.

Intramembrane proteases execute fundamental biological processes ranging from crucial signaling events to general membrane proteostasis. Despite the availability of structural information on these proteases, it remains unclear how these enzymes bind and recruit substrates, particularly for the Alzheimer's diseaseassociated c-secretase. Systematically scanning amyloid precursor protein substrates containing a genetically inserted photocrosslinkable amino acid for binding to c-secretase allowed us to identify residues contacting the protease. These were primarily found in the transmembrane cleavage domain of the substrate and were also present in the extramembranous domains. The N-terminal fragment of the catalytic subunit presenilin was determined as principal substrate-binding site. Clinical presenilin mutations altered substrate binding in the active site region, implying a pathogenic mechanism for familial Alzheimer's disease. Remarkably, PEN-2 was identified besides nicastrin as additional substrate-binding subunit. Probing proteolysis of crosslinked substrates revealed a mechanistic model of how these subunits interact to mediate a stepwise transfer of bound substrate to the catalytic site. We propose that sequential binding steps might be common for intramembrane proteases to sample and select cognate substrates for catalysis.

Intramembrane proteolysis by presenilin-dependent ␥-secretase produces the Notch intracellular cytoplasmic domain (NCID) and Alzheimer disease-associated amyloid-␤. Here, we show that upon Notch signaling the intracellular domain of Notch-1 is cleaved into two distinct types of NICD species due to diversity in the site of S3 cleavage. Consistent with the N-end rule, the S3-V cleavage produces stable NICD with Val at the N terminus, whereas the S3-S/S3-L cleavage generates unstable NICD with Ser/Leu at the N terminus. Moreover, intracellular Notch signal transmission with unstable NICDs is much weaker than that with stable NICD. Importantly, the extent of endocytosis in target cells affects the relative production ratio of the two types of NICD, which changes in parallel with Notch signaling. Surprisingly, substantial amounts of unstable NICD species are generated from the Val3Gly and the Lys3Arg mutants, which have been reported to decrease S3 cleavage efficiency in cultured cells. Thus, we suggest that the existence of two distinct types of NICD points to a novel aspect of the intracellular signaling and that changes in the precision of S3 cleavage play an important role in the process of conversion from extracellular to intracellular Notch signaling.

The canonical pathway of Notch signaling is mediated by regulated intramembrane proteolysis (RIP). In the pathway, ligand binding results in sequential proteolysis of the Notch receptor, and presenilin (PS)-dependent intramembrane proteolysis at the interface between the membrane and cytosol liberates the Notch-1 intracellular domain (NICD), a transcription modifier. Because the degradation of the Notch-1 transmembrane domain is thought to require an additional cleavage near the middle of the transmembrane domain, extracellular small peptides (Notch-1 A␤-like peptide (N␤)) should be produced. Here we showed that N␤ species are indeed secreted during the process of Notch signaling. We identified mainly two distinct molecular species of novel N␤, N␤21 and C-terminally elongated N␤25, which were produced in an ϳ5:1 ratio. This process is reminiscent of the production of Alzheimer disease-associated A␤. PS pathogenic mutants increased the production of the longer species of A␤ (A␤42) from ␤-amyloid protein precursor. We revealed that several Alzheimer disease mutants also cause a parallel increase in the secretion of the longer form of N␤. Strikingly, chemicals that modify the A␤42 level caused parallel changes in the N␤25 level. These results demonstrated that the characteristics of C-terminal elongation of N␤ and A␤ are almost identical. In addition, because many other type 1 membrane-bound receptors release intracellular domains by PS-dependent intramembrane proteolysis, we suspect that the release of A␤-or N␤-like peptides is a common feature of the proteolysis during RIP signaling. We anticipate that this study will open the door to searches for markers of RIP signaling and surrogate markers for A␤42 production.

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Alzheimer´s disease (AD) is a devastating neurodegenerative disease of the elderly population. Genetic evidence strongly suggests that aberrant generation and/or clearance of the neurotoxic amyloid-β peptide (Aβ) is triggering the disease. Aβ is generated from the amyloid precursor protein (APP) by the sequential cleavages of β- and γ-secretase. The latter cleavage by γ-secretase, a unique and fascinating four-component protease complex, occurs in the APP transmembrane domain thereby releasing Aβ species of 37-43 amino acids in length including the longer, highly pathogenic peptides Aβ42 and Aβ43. The lack of a precise understanding of Aβ generation as well as of the functions of other γ-secretase substrates has been one factor underlying the disappointing failure of γ-secretase inhibitors in clinical trials, but on the other side also been a major driving force for structural and in depth mechanistic studies on this key AD drug target in the past few years. Here we review recent breakthroughs in our understanding of how the γ-secretase complex recognizes substrates, of how it binds and processes β-secretase cleaved APP into different Aβ species, as well as the progress made on a question of outstanding interest, namely how clinical AD mutations in the catalytic subunit presenilin and the γ-secretase cleavage region of APP lead to relative increases of Aβ42/43. Finally, we discuss how the knowledge emerging from these studies could be used to therapeutically target this enzyme in a safe way.