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Secretases are a group of proteases that are major drug targets considered for the prevention and treatment of Alzheimer's disease (AD). Secretases do not only process the AD-linked neuronal amyloid precursor protein (APP) but also the triggering receptor expressed on myeloid cells 2 (TREM2), thereby controlling microglial functions. This review highlights selected recent discoveries for the α-secretases a disintegrin and metalloprotease 10 (ADAM10) and a disintegrin and metalloprotease 17 (ADAM17), the β-secretase β-site APP cleaving enzyme 1 (BACE1) and γ-secretase and their link to AD. New genetic evidence strengthens the role of α-secretases in AD through cleavage of APP and TREM2. Novel proteins were linked to AD, which control α- and β-secretase activity through transcriptional and post-translational mechanisms. Finally, new opportunities but also challenges are discussed for pharmacologically targeting β- and γ-secretase cleavage of APP and α-secretase cleavage of TREM2 with the aim to prevent or treat AD.

Alzheimer disease (AD) is the most common cause of dementia in older individuals (>65 years) and has a long presymptomatic phase. Preventive therapies for AD are not yet available, and potential disease-modifying therapies targeting amyloid-β plaques in symptomatic stages of AD have only just been approved in the United States. Small-molecule inhibitors of β-site amyloid precursor protein (APP)-cleaving enzyme 1 (BACE1; also known as β-secretase 1) reduce the production of amyloid-β peptide and are among the most advanced drug candidates for AD. However, to date all phase II and phase III clinical trials of BACE inhibitors were either concluded without benefit or discontinued owing to futility or the occurrence of adverse effects. Adverse effects included early, mild cognitive impairment that was associated with all but one inhibitor; preliminary results suggest that the cognitive effects are non-progressive and reversible. These discontinuations have raised questions regarding the suitability of BACE1 as a drug target for AD. In this Perspective, we discuss the status of BACE inhibitors and suggest ways in which the results of the discontinued trials can inform the development of future clinical trials of BACE inhibitors and related secretase modulators as preventative therapies. We also propose a series of experiments that should be performed to inform 'go-no-go' decisions in future trials with BACE inhibitors and consider the possibility that low levels of BACE1 inhibition could avoid adverse effects while achieving efficacy for AD prevention. ispartof: NATURE REVIEWS NEUROLOGY vol:17 issue:11 pages:703-714 ispartof: location:England status: published

The protease beta-site APP cleaving enzyme 1 (BACE1) has fundamental functions in the nervous system. Its inhibition is a major therapeutic approach in Alzheimer's disease, because BACE1 cleaves the amyloid precursor protein (APP), thereby catalyzing the first step in the generation of the pathogenic amyloid beta (Aβ) peptide. Yet, BACE1 cleaves numerous additional membrane proteins besides APP. Most of these substrates have been identified in vitro, but only few were further validated or characterized in vivo. To identify BACE1 substrates with in vivo relevance, we used isotope label-based quantitative proteomics of wild type and BACE1-deficient (BACE1 KO) mouse brains. This approach identified known BACE1 substrates, including Close homolog of L1 and contactin-2, which were found to be enriched in the membrane fraction of BACE1 KO brains. VWFA and cache domain-containing protein 1 (CACHD)1 and MAM domain-containing glycosylphosphatidylinositol anchor protein 1 (MDGA1), which have functions in synaptic transmission, were identified and validated as new BACE1 substrates in vivo by immunoblots using primary neurons and mouse brains. Inhibition or deletion of BACE1 from primary neurons resulted in a pronounced inhibition of substrate cleavage and a concomitant increase in full-length protein levels of CACHD1 and MDGA1. The BACE1 cleavage site in both proteins was determined to be located within the juxtamembrane domain. In summary, this study identifies and validates CACHD1 and MDGA1 as novel in vivo substrates for BACE1, suggesting that cleavage of both proteins may contribute to the numerous functions of BACE1 in the nervous system.

The intramembrane proteases SPPL2a/b control pro-atherogenic signaling of membrane-bound proteolytic fragments derived from the oxLDL receptor LOX-1. In mice deficient for these proteases, plaque development and fibrosis is enhanced. This highlights SPPL2a/b as crucial players of a novel athero-protective mechanism, which is conserved in humans., The lectin-like oxidized LDL receptor 1 (LOX-1) is a key player in the development of atherosclerosis. LOX-1 promotes endothelial activation and dysfunction by mediating uptake of oxidized LDL and inducing pro-atherogenic signaling. However, little is known about modulators of LOX-1–mediated responses. Here, we show that the function of LOX-1 is controlled proteolytically. Ectodomain shedding by the metalloprotease ADAM10 and lysosomal degradation generate membrane-bound N-terminal fragments (NTFs), which we identified as novel substrates of the intramembrane proteases signal peptide peptidase–like 2a and b (SPPL2a/b). SPPL2a/b control cellular LOX-1 NTF levels which, following self-association via their transmembrane domain, can activate MAP kinases in a ligand-independent manner. This leads to an up-regulation of several pro-atherogenic and pro-fibrotic targets including ICAM-1 and the connective tissue growth factor CTGF. Consequently, SPPL2a/b-deficient mice, which accumulate LOX-1 NTFs, develop larger and more advanced atherosclerotic plaques than controls. This identifies intramembrane proteolysis by SPPL2a/b as a novel atheroprotective mechanism via negative regulation of LOX-1 signaling., Graphical Abstract

BACE1-mediated cleavage of APP is a pivotal step in the production of the Alzheimer related Aβ peptide and inhibitors of BACE1 are currently in clinical development for the treatment of Alzheimer disease (AD). While processing and trafficking of APP has been extensively studied in non-neuronal cells, the fate of APP at neuronal synapses and in response to reduced BACE1 activity has not been fully elucidated. Here we examined the consequence of reduced BACE1 activity on endogenous synaptic APP by monitoring N- and C-terminal APP epitopes by immunocytochemistry. In control rodent primary hippocampal neuron cultures, labeling with antibodies directed to N-terminal APP epitopes showed a significant overlap with synaptic vesicle markers (SV2 or synaptotagmin). In contrast, labeling with antibodies directed to C-terminal epitopes of APP showed only a limited overlap with these proteins. In neurons derived from BACE1-deficient mice, and in control neurons treated with a BACE1 inhibitor, both the N-terminal and the C-terminal APP labeling overlapped significantly with synaptic vesicle markers. Moreover, BACE1 inhibition increased the proximity between the APP C-terminus and SV2 as shown by a proximity ligation assay. These results, together with biochemical observations, indicate that BACE1 can regulate the levels of full-length APP at neuronal synapses.

Inhibition of the protease β-site amyloid precursor protein-cleaving enzyme 1 (BACE1) is a promising treatment strategy for Alzheimer’s disease, and a number of BACE inhibitors are currently progressing through clinical trials. The strategy aims to decrease production of amyloid-β (Aβ) peptide from the amyloid precursor protein (APP), thus reducing or preventing Aβ toxicity. Over the last decade, it has become clear that BACE1 proteolytically cleaves a number of substrates in addition to APP. These substrates are not known to be involved in the pathogenesis of Alzheimer’s disease but have other roles in the developing and/or mature central nervous system. Consequently, BACE inhibition and knockout in mice results in synaptic and other neuronal dysfunctions and the key substrates responsible for these deficits are still being elucidated. Of the BACE1 substrates that have been validated to date, a number may contribute to the synaptic deficits seen with BACE blockade, including neuregulin 1, close homologue of L1 and seizure-related gene 6. It is important to understand the impact that BACE blockade may have on these substrates and other proteins detected in substrate screens and, if necessary, develop substrate-selective BACE inhibitors.

The protease β-site amyloid precursor protein (APP)-cleaving enzyme 1 (BACE1) is required for the production of the amyloid-β (Aβ) peptide, which is central to the pathogenesis of Alzheimer's disease (AD). Chronic inhibition of this protease may temper amyloid production and cure or prevent AD. However, while BACE1 inhibitors are being pushed forward as drug candidates, a remarkable gap in knowledge on the physiological functions of BACE1 and its close homolog BACE2 becomes apparent. Here we discuss the major discoveries of the past 3 years concerning BACE1 biology and to what extent these could limit the use of BACE1 inhibitors in the clinic.

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.

Signal peptide peptidase (SPP) and the four homologous SPP-like proteases SPPL2a, SPPL2b, SPPL2c and SPPL3 are GxGD-type intramembrane-cleaving proteases (I-CLIPs). In addition to divergent subcellular localisations, distinct differences in the mechanistic properties and substrate requirements of individual family members have been unravelled. SPP/SPPL proteases employ a catalytic mechanism related to that of the γ-secretase complex. Nevertheless, differential targeting of SPP/SPPL proteases and γ-secretase by inhibitors has been demonstrated. Furthermore, also within the SPP/SPPL family significant differences in the sensitivity to currently available inhibitory compounds have been reported. Though far from complete, our knowledge on pathophysiological functions of SPP/SPPL proteases, in particular based on studies in mice, has been significantly increased over the last years. Based on this, inhibition of distinct SPP/SPPL proteases has been proposed as a novel therapeutic concept e.g. for the treatment of autoimmunity and viral or protozoal infections, as we will discuss in this review. This article is part of a Special Issue entitled: Proteolysis as a Regulatory Event in Pathophysiology edited by Stefan Rose-John.

Proteolytic cleavage of the amyloid precursor protein (APP) by the two proteases α- and β-secretases controls the generation of the amyloid β peptide (Aβ), a key player in Alzheimer's disease pathogenesis. The α-secretase ADAM10 and the β-secretase BACE1 have opposite effects on Aβ generation and are assumed to compete for APP as a substrate, such that their cleavages are inversely coupled. This concept was mainly demonstrated in studies using activation or overexpression of α- and β-secretases. Here, we report that this inverse coupling is not seen to the same extent upon inhibition of the endogenous proteases. Genetic and pharmacological inhibition of ADAM10 and BACE1 revealed that the endogenous, constitutive α-secretase cleavage of APP is largely uncoupled from β-secretase cleavage and Aβ generation in neuroglioma H4 cells and in neuronally differentiated SH-SY5Y cells. In contrast, inverse coupling was observed in primary cortical neurons. However, this coupling was not bidirectional. Inhibition of BACE1 increased ADAM10 cleavage of APP, but a reduction of ADAM10 activity did not increase the BACE1 cleavage of APP in the neurons. Our analysis shows that the inverse coupling of the endogenous α- and β-secretase cleavages depends on the cellular model and suggests that a reduction of ADAM10 activity is unlikely to increase the AD risk through increased β-secretase cleavage.

Melanocytes of the hair follicle produce melanin and are essential in determining the differences in hair color. Pigment cell-specific MELanocyte Protein (PMEL17) plays a crucial role in melanogenesis. One of the critical steps is the amyloid-like functional oligomerization of PMEL17. Beta Site APP Cleaving Enzyme-2 (BACE2) and γ-secretase have been shown to be key players in generating the proteolytic fragments of PMEL17. The β-secretase (BACE1) is responsible for the generation of amyloid-β (Aβ) fragments in the brain and is therefore proposed as a therapeutic target for Alzheimer’s disease (AD). Currently BACE1 inhibitors, most of which lack selectivity over BACE2, have demonstrated efficacious reduction of amyloid-β peptides in animals and the CSF of humans. BACE2 knock-out mice have a deficiency in PMEL17 proteolytic processing leading to impaired melanin storage and hair depigmentation. Here, we confirm BACE2-mediated inhibition of PMEL17 proteolytic processing in vitro in mouse and human melanocytes. Furthermore, we show that wildtype as well as bace2+/− and bace2−/− mice treated with a potent dual BACE1/BACE2 inhibitor NB-360 display dose-dependent appearance of irreversibly depigmented hair. Retinal pigmented epithelium showed no morphological changes. Our data demonstrates that BACE2 as well as additional BACE1 inhibition affects melanosome maturation and induces hair depigmentation in mice.

The presenilin homologue signal peptide peptidase-like 2a (SPPL2a) is an intramembrane protease of lysosomes/late endosomes which cleaves type II transmembrane proteins. We recently identified CD74, the invariant chain of the MHCII complex, as the first in vivo validated substrate of this protease. In endosomal compartments, CD74 undergoes sequential proteolysis leading to the generation of a membrane-bound N-terminal fragment (NTF) that requires cleavage by SPPL2a for its turnover. In SPPL2a−/− mice, this fragment accumulates in B-cells and significantly disturbs their maturation and functionality. To date, the substrate requirements of the protease SPPL2a have not been investigated. In the present study, we systematically analysed the molecular determinants of CD74 with regard to the intramembrane cleavage by SPPL2a. Using domain-exchange experiments, we demonstrate that the intracellular domain (ICD) of CD74 can be substituted without affecting cleavability by SPPL2a. Based on IP-MS analysis of the cleavage product, we report identification of the primary SPPL2a cleavage site between Y52 and F53 within the CD74 transmembrane segment. Furthermore, systematic alanine-scanning mutagenesis of the transmembrane and membrane-proximal parts of the CD74 NTF has been performed. We show that none of the analysed determinants within the CD74 NTF including the residues flanking the primary cleavage site are absolutely essential for SPPL2a cleavage. Importantly, we found that alanine substitution of helix-destabilizing glycines within the transmembrane segment and distinct residues within the luminal membrane-proximal segment led to a reduced efficiency of SPPL2a-mediated processing. Therefore we propose that elements within the transmembrane segment and the luminal juxtamembrane domain facilitate intramembrane proteolysis of CD74 by SPPL2a.

Background BACE1 (beta site amyloid precursor protein cleaving enzyme 1) is the rate limiting protease in amyloid β production, hence a promising drug target for the treatment of Alzheimer's disease. Inhibition of BACE1, as the major β-secretase in vivo with multiple substrates, however is likely to have mechanism-based adverse effects. We explored the impact of long-term pharmacological inhibition of BACE1 on dendritic spine dynamics, synaptic functions, and cognitive performance of adult mice. Methods Sandwich enzyme-linked immunosorbent assay was used to assess Aβ40 levels in brain and plasma after oral administration of BACE1 inhibitors SCH1682496 or LY2811376. In vivo two-photon microscopy of the somatosensory cortex was performed to monitor structural dynamics of dendritic spines while synaptic functions and plasticity were measured via electrophysiological recordings of excitatory postsynaptic currents and hippocampal long-term potentiation in brain slices. Finally, behavioral tests were performed to analyze the impact of pharmacological inhibition of BACE1 on cognitive performance. Results Dose-dependent decrease of Aβ40 levels in vivo confirmed suppression of BACE1 activity by both inhibitors. Prolonged treatment caused a reduction in spine formation of layer V pyramidal neurons, which recovered after withdrawal of inhibitors. Congruently, the rate of spontaneous and miniature excitatory postsynaptic currents in pyramidal neurons and hippocampal long-term potentiation were reduced in animals treated with BACE1 inhibitors. These effects were not detected in Bace1 −/− mice treated with SCH1682496, confirming BACE1 as the pharmacological target. Described structural and functional changes were associated with cognitive deficits as revealed in behavioral tests. Conclusions Our findings indicate important functions to BACE1 in structural and functional synaptic plasticity in the mature brain, with implications for cognition.

Analysis of murine cerebrospinal fluid (CSF) by quantitative mass spectrometry is challenging because of low CSF volume, low total protein concentration, and the presence of highly abundant proteins such as albumin. We demonstrate that the CSF proteome of individual mice can be analyzed in a quantitative manner to a depth of several hundred proteins in a robust and simple workflow consisting of single ultra HPLC runs on a benchtop mass spectrometer. The workflow is validated by a comparative analysis of BACE1−/− and wild-type mice using label-free quantification. The protease BACE1 cleaves the amyloid precursor protein (APP) as well as several other substrates and is a major drug target in Alzheimer's disease. We identified a total of 715 proteins with at least 2 unique peptides and quantified 522 of those proteins in CSF from BACE1−/− and wild-type mice. Several proteins, including the known BACE1 substrates APP, APLP1, CHL1 and contactin-2 showed lower abundance in the CSF of BACE1−/− mice, demonstrating that BACE1 substrate identification is possible from CSF. Additionally, ectonucleotide pyrophosphatase 5 was identified as a novel BACE1 substrate and validated in cells using immunoblots and by an in vitro BACE1 protease assay. Likewise, receptor-type tyrosine-protein phosphatase N2 and plexin domain-containing 2 were confirmed as BACE1 substrates by in vitro assays. Taken together, our study shows the deepest characterization of the mouse CSF proteome to date and the first quantitative analysis of the CSF proteome of individual mice. The BACE1 substrates identified in CSF may serve as biomarkers to monitor BACE1 activity in Alzheimer patients treated with BACE inhibitors.

The invariant chain (CD74) mediates targeting of the MHCII complex to endosomal compartments, where CD74 undergoes degradation allowing MHCII to acquire peptides. We demonstrated recently that intramembrane proteolysis of the final membrane-bound N-terminal fragment (NTF) of CD74 is catalyzed by Signal-peptide-peptidase-like 2a (SPPL2a) and that this process is indispensable for development and function of B lymphocytes in mice. In SPPL2a(-/-) mice, homeostasis of these cells is disturbed by the accumulation of the unprocessed CD74 NTF. So far, evidence for this essential role of SPPL2a is restricted to mice. Nevertheless, inhibition of SPPL2a has been suggested as novel approach to target B cells for treating autoimmunity. Here, we characterize human B cell lines with a homozygous microdeletion on chromosome 15. We demonstrate that this deletion disrupts the SPPL2a genomic locus and leads to loss of SPPL2a transcript. Lymphoblastoid cell lines from patients with this deletion exhibit absence of SPPL2a at the protein level and show an accumulation of the CD74 NTF comparable to B cells from SPPL2a(-/-) mice. By this means, we present evidence that the role of SPPL2a in CD74 proteolysis is conserved in human B cells and provide support for modulation of SPPL2a activity as a therapeutic concept.

We reported recently that the presenilin homologue signal peptide peptidase-like 2a (SPPL2a) is essential for B cell development by cleaving the N-terminal fragment (NTF) of the invariant chain (li, CD74). Based on this, we suggested that pharmacological modulation of SPPL2a may represent a novel approach to deplete B cells in autoimmune disorders. With regard to reported overlapping substrate spectra of SPPL2a and its close homologue, SPPL2b, we investigated the role of SPPL2b in CD74 NTF proteolysis and its impact on B and dendritic cell homeostasis. In heterologous expression experiments, SPPL2b was found to cleave CD74 NTF with an efficiency similar to that of SPPL2a. For in vivo analysis, SPPL2b single-deficient and SPPL2a/SPPL2b double-deficient mice were generated and examined for CD74 NTF turnover/accumulation, B cell maturation and functionality, and dendritic cell homeostasis. We demonstrate that in vivo SPPL2b does not exhibit a physiologically relevant contribution to CD74 proteolysis in B and dendritic cells. Furthermore, we reveal that both proteases exhibit divergent subcellular localizations in B cells and different expression profiles in murine tissues. These findings suggest distinct functions of SPPL2a and SPPL2b and, based on a high abundance of SPPL2b in brain, a physiological role of this protease in the central nervous system.

Alzheimer’s disease (AD), the most common form of dementia in the elderly, is characterized by two neuropathological hallmarks: senile plaques, which are composed of Aβ peptides, and neurofibrillary tangles, which are composed of hyperphosphorylated TAU protein. Diabetic patients with dysregulated insulin signalling are at increased risk of developing AD. Further, several animal models of diabetes show increased Aβ expression and hyperphosphorylated tau. As we have shown recently, the anti-diabetic drug metformin is capable of dephosphorylating tau at AD-relevant phospho-sites. Here, we investigated the effect of metformin on the main amyloidogenic enzyme BACE1 and, thus, on the production of Aβ peptides, the second pathological hallmark of AD. We find similar results in cultures of primary neurons, a human cell line model of AD and in vivo in mice. We show that treatment with metformin decreases BACE1 protein expression by interfering with an mRNA-protein complex that contains the ubiquitin ligase MID1, thereby reducing BACE1 activity. Together with our previous findings these results indicate that metformin may target both pathological hallmarks of AD and may be of therapeutic value for treating and/or preventing AD.

The protease β-secretase 1 (Bace1) was identified through its critical role in production of amyloid-β peptides (Aβ), the major component of amyloid plaques in Alzheimer's disease. Bace1 is considered a promising target for the treatment of this pathology, but processes additional substrates, among them Neuregulin-1 (Nrg1). Our biochemical analysis indicates that Bace1 processes the Ig-containing β1 Nrg1 (IgNrg1β1) isoform. We find that a graded reduction in IgNrg1 signal strength in vivo results in increasingly severe deficits in formation and maturation of muscle spindles, a proprioceptive organ critical for muscle coordination. Further, we show that Bace1 is required for formation and maturation of the muscle spindle. Finally, pharmacological inhibition and conditional mutagenesis in adult animals demonstrate that Bace1 and Nrg1 are essential to sustain muscle spindles and to maintain motor coordination. Our results assign to Bace1 a role in the control of coordinated movement through its regulation of muscle spindle physiology, and implicate IgNrg1-dependent processing as a molecular mechanism., Bace1 and Neuregulin-1 cooperate to control formation and maintenance of muscle spindles Bace1 is required for Nrg1 processing for muscle spindle development. Bace1 inhibition leads to loss of motor coordination even in adult mice, suggesting potentially serious side effects for drugs targeting Bace1 as a treatment for Alzheimer's disease.

The intramembrane protease SPPL2a cleaves the NTF of invariant chain (CD74), which is essential for normal trafficking of MHC class II–containing endosomes and thus for B cell development and function., Regulated intramembrane proteolysis is a central cellular process involved in signal transduction and membrane protein turnover. The presenilin homologue signal-peptide-peptidase-like 2a (SPPL2a) has been implicated in the cleavage of type 2 transmembrane proteins. We show that the invariant chain (li, CD74) of the major histocompatability class II complex (MHCII) undergoes intramembrane proteolysis mediated by SPPL2a. B lymphocytes of SPPL2a−/− mice accumulate an N-terminal fragment (NTF) of CD74, which severely impairs membrane traffic within the endocytic system and leads to an altered response to B cell receptor stimulation, reduced BAFF-R surface expression, and accumulation of MHCII in transitional developmental stage T1 B cells. This results in significant loss of B cell subsets beyond the T1 stage and disrupted humoral immune responses, which can be recovered by additional ablation of CD74. Hence, we provide evidence that regulation of CD74-NTF levels by SPPL2a is indispensable for B cell development and function by maintaining trafficking and integrity of MHCII-containing endosomes, highlighting SPPL2a as a promising pharmacological target for depleting and/or modulating B cells.

Signal peptide peptidase (SPP) and the homologous SPP-like (SPPL) proteases SPPL2a, SPPL2b, SPPL2c and SPPL3 belong to the family of GxGD intramembrane proteases. SPP/SPPLs selectively cleave transmembrane domains in type II orientation and do not require additional co-factors for proteolytic activity. Orthologues of SPP and SPPLs have been identified in other vertebrates, plants, and eukaryotes. In line with their diverse subcellular localisations ranging from the ER (SPP, SPPL2c), the Golgi (SPPL3), the plasma membrane (SPPL2b) to lysosomes/late endosomes (SPPL2a), the different members of the SPP/SPPL family seem to exhibit distinct functions. Here, we review the substrates of these proteases identified to date as well as the current state of knowledge about the physiological implications of these proteolytic events as deduced from in vivo studies. Furthermore, the present knowledge on the structure of intramembrane proteases of the SPP/SPPL family, their cleavage mechanism and their substrate requirements are summarised. This article is part of a Special Issue entitled: Intramembrane Proteases.

The aspartyl protease BACE1 cleaves neuregulin 1 and is involved in myelination and is a candidate drug target for Alzheimer's disease, where it acts as the β-secretase cleaving the amyloid precursor protein. However, little is known about other substrates in vivo. Here, we provide a proteomic workflow for BACE1 substrate identification from whole brains, combining filter-aided sample preparation, strong-anion exchange fractionation, and label-free quantification. We used bace1-deficient zebrafish and quantified differences in protein levels between wild-type and bace1 -/- zebrafish brains. Over 4500 proteins were identified with at least two unique peptides and quantified in both wild-type and bace1 -/- zebrafish brains. The majority of zebrafish membrane proteins did not show altered protein levels, indicating that Bace1 has a restricted substrate specificity. Twenty-four membrane proteins accumulated in the bace1 -/- brains and thus represent candidate Bace1 substrates. They include several known BACE1 substrates, such as the zebrafish homologs of amyloid precursor protein and the cell adhesion protein L1, which validate the proteomic workflow. Additionally, several candidate substrates with a function in neurite outgrowth and axon guidance, such as plexin A3 and glypican-1 were identified, pointing to a function of Bace1 in neurodevelopment. Taken together, our study provides the first proteomic analysis of knock-out zebrafish tissue and demonstrates that combining gene knock-out models in zebrafish with quantitative proteomics is a powerful approach to address biomedical questions.

The successive events of shedding and regulated intramembrane proteolysis are known to comprise a fundamental biological process of type I and II membrane proteins (e.g. amyloid precursor protein, Notch receptor and pro-tumor necrosis factor-α). Some of the resulting fragments were shown to be involved in important intra- and extracellular signalling events. Although shedding of the human transferrin receptor-1 (TfR1) has been known for > 30 years and soluble TfR1 is an accepted diagnostic marker, the fate of the remaining N-terminal fragment (NTF) remains unknown. In the present study, we demonstrate for the first time that TfR1-NTF is subject to regulated intramembrane proteolysis and, using MALDI-TOF-TOF-MS, we have identified the cleavage site as being located C-terminal from Gly-84. We showed that the resulting C-terminal peptide is extracellularly released after regulated intramembrane proteolysis and it was detected as a monomer with an internal disulfide bridge. We further identified signal peptide peptidase-like 2a and mainly signal peptide peptidase-like 2b as being responsible for the intramembrane proteolysis of TfR1-NTF.

Cell surface proteolysis is essential for communication between cells and results in the shedding of membrane-protein ectodomains. However, physiological substrates of the contributing proteases are largely unknown. We developed the secretome protein enrichment with click sugars (SPECS) method, which allows proteome-wide identification of shedding substrates and secreted proteins from primary cells, even in the presence of serum proteins. SPECS combines metabolic glycan labelling and click chemistry-mediated biotinylation and distinguishes between cellular and serum proteins. SPECS identified 34, mostly novel substrates of the Alzheimer protease BACE1 in primary neurons, making BACE1 a major sheddase in the nervous system. Selected BACE1 substrates-seizure-protein 6, L1, CHL1 and contactin-2-were validated in brains of BACE1 inhibitor-treated and BACE1 knock-out mice. For some substrates, BACE1 was the major sheddase, whereas for other substrates additional proteases contributed to total substrate shedding. The new substrates point to a central function of BACE1 in neurite outgrowth and synapse formation. SPECS is also suitable for quantitative secretome analyses of primary cells and may be used for the discovery of biomarkers secreted from tumour or stem cells.

Myelin sheath thickness is precisely adjusted to axon caliber, and in the peripheral nervous system, neuregulin 1 (NRG1) type III is a key regulator of this process. It has been proposed that the protease BACE1 activates NRG1 dependent myelination. Here, we characterize the predicted product of BACE1-mediated NRG1 type III processing in transgenic mice. Neuronal overexpression of a NRG1 type III-variant, designed to mimic prior cleavage in the juxtamembrane stalk region, induces hypermyelination in vivo and is sufficient to restore myelination of NRG1 type III-deficient neurons. This observation implies that the NRG1 cytoplasmic domain is dispensable and that processed NRG1 type III is sufficient for all steps of myelination. Surprisingly, transgenic neuronal overexpression of full-length NRG1 type III promotes hypermyelination also in BACE1 null mutant mice. Moreover, NRG1 processing is impaired but not abolished in BACE1 null mutants. Thus, BACE1 is not essential for the activation of NRG1 type III to promote myelination. Taken together, these findings suggest that multiple neuronal proteases collectively regulate NRG1 processing. © 2011 Wiley Periodicals, Inc.

Item does not contain fulltext AIMS: Intracellular amyloid beta (Abeta) oligomers and extracellular Abeta plaques are key players in the progression of sporadic Alzheimer's disease (AD). Still, the molecular signals triggering Abeta production are largely unclear. We asked whether mitochondrion-derived reactive oxygen species (ROS) are sufficient to increase Abeta generation and thereby initiate a vicious cycle further impairing mitochondrial function. RESULTS: Complex I and III dysfunction was induced in a cell model using the respiratory inhibitors rotenone and antimycin, resulting in mitochondrial dysfunction and enhanced ROS levels. Both treatments lead to elevated levels of Abeta. Presence of an antioxidant rescued mitochondrial function and reduced formation of Abeta, demonstrating that the observed effects depended on ROS. Conversely, cells overproducing Abeta showed impairment of mitochondrial function such as comprised mitochondrial respiration, strongly altered morphology, and reduced intracellular mobility of mitochondria. Again, the capability of these cells to generate Abeta was partly reduced by an antioxidant, indicating that Abeta formation was also ROS dependent. Moreover, mice with a genetic defect in complex I, or AD mice treated with a complex I inhibitor, showed enhanced Abeta levels in vivo. INNOVATION: We show for the first time that mitochondrion-derived ROS are sufficient to trigger Abeta production in vitro and in vivo. CONCLUSION: Several lines of evidence show that mitochondrion-derived ROS result in enhanced amyloidogenic amyloid precursor protein processing, and that Abeta itself leads to mitochondrial dysfunction and increased ROS levels. We propose that starting from mitochondrial dysfunction a vicious cycle is triggered that contributes to the pathogenesis of sporadic AD.

Regulated intramembrane proteolysis is a widely accepted concept describing the processing of various transmembrane proteins via ectodomain shedding followed by an intramembrane cleavage. The resulting cleavage products can be involved in reverse signaling. Presenilins, which constitute the active center of the γ-secretase complex, signal peptide peptidase (SPP), and its homologues, the SPP-like (SPPL) proteases are members of the family of intramembrane-cleaving aspartyl proteases of the GXGD-type. We recently demonstrated that Bri2 (itm2b) is a substrate for regulated intramembrane proteolysis by SPPL2a and SPPL2b. Intramembrane cleavage of Bri2 is triggered by an initial shedding event catalyzed by A Disintegrin and Metalloprotease 10 (ADAM10). Additionally primary sequence determinants within the intracellular domain, the transmembrane domain and the luminal juxtamembrane domain are required for efficient cleavage of Bri2 by SPPL2b. Using mutagenesis and circular dichroism spectroscopy we now demonstrate that a high α-helical content of the Bri2 transmembrane domain (TMD) reduces cleavage efficiency of Bri2 by SPPL2b, while the presence of a GXXXG dimerization motif influences the intramembrane cleavage only to a minor extent. Surprisingly, only one of the four conserved intramembrane glycine residues significantly affects the secondary structure of the Bri2 TMD and thereby its intramembrane cleavage. Other glycine residues do not influence the α-helical content of the transmembrane domain nor its intramembrane processing.

To successfully treat Alzheimer’s disease (AD), pathophysiological events in preclinical stages need to be identified. Preclinical AD refers to the stages that exhibit amyloid deposition in the brain but have normal cognitive function, which are replicated in young adult APPswe/PS1deltaE9 (deltaE9) mice. By long-term in vivo two-photon microscopy, we demonstrate impaired adaptive spine plasticity in these transgenic mice illustrated by their failure to increase dendritic spine density and form novel neural connections when housed in enriched environment (EE). Decrease of amyloid plaques by reducing BACE1 activity restores the gain of spine density upon EE in deltaE9 mice, but not the remodeling of neural networks. On the other hand, anti-inflammatory treatment with pioglitazone or interleukin 1 receptor antagonist in deltaE9 mice successfully rescues the impairments in increasing spine density and remodeling of neural networks during EE. Our data suggest that neuroinflammation disrupts experience-dependent structural plasticity of dendritic spines in preclinical stages of AD. Electronic supplementary material The online version of this article (doi:10.1007/s00401-015-1527-8) contains supplementary material, which is available to authorized users.