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International audience; Aggregation of the multifunctional RNA-binding protein TDP-43 defines large subgroups of amyotrophic lateral sclerosis and frontotemporal dementia and correlates with neurodegeneration in both diseases. In disease, characteristic C-terminal fragments of ~25 kDa ("TDP-25") accumulate in cytoplasmic inclusions. Here, we analyze gain-of-function mechanisms of TDP-25 combining cryo-electron tomography, proteomics, and functional assays. In neurons, cytoplasmic TDP-25 inclusions are amorphous, and photobleaching experiments reveal gel-like biophysical properties that are less dynamic than nuclear TDP-43. Compared with full-length TDP-43, the TDP-25 interactome is depleted of low-complexity domain proteins. TDP-25 inclusions are enriched in 26S proteasomes adopting exclusively substrate-processing conformations, suggesting that inclusions sequester proteasomes, which are largely stalled and no longer undergo the cyclic conformational changes required for proteolytic activity. Reporter assays confirm that TDP-25 impairs proteostasis, and this inhibitory function is enhanced by ALScausing TDP-43 mutations. These findings support a pathophysiological relevance of proteasome dysfunction in ALS/FTD.

GGGGCC (G4C2) repeat expansion in the C9orf72 gene has been shown to cause frontotemporal lobar degeneration and amyotrophic lateral sclerosis. Dipeptide repeat proteins produced through repeat-associated non-AUG (RAN) translation are recognized as potential drivers for neurodegeneration. Therefore, selective inhibition of RAN translation could be a therapeutic avenue to treat these neurodegenerative diseases. It was previously known that the porphyrin TMPyP4 binds to G4C2 repeat RNA. However, the consequences of this interaction have not been well characterized. Here, we confirmed that TMPyP4 inhibits C9orf72 G4C2 repeat translation in cellular and in in vitro translation systems. An artificial insertion of an AUG codon failed to cancel the translation inhibition, suggesting that TMPyP4 acts downstream of non-AUG translation initiation. Polysome profiling assays also revealed polysome retention on G4C2 repeat RNA, along with inhibition of translation, indicating that elongating ribosomes stall on G4C2 repeat RNA. Urea-resistant interaction between G4C2 repeat RNA and TMPyP4 likely contributes to this ribosome stalling and thus to selective inhibition of RAN translation. Taken together, our data reveal a novel mode of action of TMPyP4 as an inhibitor of G4C2 repeat translation elongation.

Single nucleotide polymorphisms (SNPs) in TMEM106B encoding the lysosomal type II transmembrane protein 106B increase the risk for frontotemporal lobar degeneration (FTLD) of GRN (progranulin gene) mutation carriers. Currently, it is unclear if progranulin (PGRN) and TMEM106B are synergistically linked and if a gain or a loss of function of TMEM106B is responsible for the increased disease risk of patients with GRN haploinsufficiency. We therefore compare behavioral abnormalities, gene expression patterns, lysosomal activity, and TDP‐43 pathology in single and double knockout animals. Grn −/−/Tmem106b −/− mice show a strongly reduced life span and massive motor deficits. Gene expression analysis reveals an upregulation of molecular signature characteristic for disease‐associated microglia and autophagy. Dysregulation of maturation of lysosomal proteins as well as an accumulation of ubiquitinated proteins and widespread p62 deposition suggest that proteostasis is impaired. Moreover, while single Grn −/− knockouts only occasionally show TDP‐43 pathology, the double knockout mice exhibit deposition of phosphorylated TDP‐43. Thus, a loss of function of TMEM106B may enhance the risk for GRN‐associated FTLD by reduced protein turnover in the lysosomal/autophagic system., Loss of TMEM106B expression aggravates the mild phenotype of Grn knockout mice, suggesting that the FTLD‐associated TMEM106B SNPs might cause partial loss of function.

Contrary to findings in the human brain, 18F-FDG PET shows cerebral hypermetabolism of aged wild-type (WT) mice relative to younger animals, supposedly due to microglial activation. Therefore, we used dual-tracer small-animal PET to examine directly the link between neuroinflammation and hypermetabolism in aged mice. Methods: WT mice (5-20 mo) were investigated in a cross-sectional design using 18F-FDG (n = 43) and translocator protein (TSPO) (18F-GE180; n = 58) small-animal PET, with volume-of-interest and voxelwise analyses. Biochemical analysis of plasma cytokine levels and immunohistochemical confirmation of microglial activity were also performed. Results: Age-dependent cortical hypermetabolism in WT mice relative to young animals aged 5 mo peaked at 14.5 mo (+16%, P < 0.001) and declined to baseline at 20 mo. Similarly, cortical TSPO binding increased to a maximum at 14.5 mo (+15%, P < 0.001) and remained high to 20 mo, resulting in an overall correlation between 18F-FDG uptake and TSPO binding (R = 0.69, P < 0.005). Biochemical and immunohistochemical analyses confirmed the TSPO small-animal PET findings. Conclusion: Age-dependent neuroinflammation is associated with the controversial observation of cerebral hypermetabolism in aging WT mice.

The triggering receptor expressed on myeloid cells 2 (TREM2) is a multifunctional surface protein that affects survival, migration, and phagocytic capacity of myeloid cells. Soluble TREM2 levels were found to be increased in early stages of sporadic and familial Alzheimer's disease (AD) probably reflecting a defensive microglial response to some initial brain damage. The disintegrin and metalloproteases (ADAM) 10 and 17 were identified as TREM2 sheddases. We demonstrate that meprin β is a direct TREM2 cleaving enzyme using ADAM10/17 deficient HEK293 cells. LC‐MS/MS analysis of recombinant TREM2 incubated with meprin β revealed predominant cleavage between Arg136 and Asp137, distant to the site identified for ADAM10/17. We further demonstrate that the metalloprotease meprin β cleaves TREM2 on macrophages concomitant with decreased levels of soluble TREM2 in the serum of Mep1b−/− mice compared to WT controls. Isolated BMDMs from Mep1b−/− mice showed significantly increased full‐length TREM2 levels and enhanced phagocytosis efficiency compared to WT cells. The diminished constitutive shedding of TREM2 on meprin β deficient macrophages could be rescued by ADAM stimulation through LPS treatment. Our data provide evidence that meprin β is a TREM2 sheddase on macrophages and suggest that multiple proteases may be involved in the generation of soluble TREM2.

Signal peptide peptidase (SPP) is an unusual aspartyl protease that mediates clearance of signal peptides by proteolysis within the endoplasmic reticulum (ER). Like presenilins, which provide the proteolytically active subunit of the gamma-secretase complex, SPP contains a critical GXGD motif in its C-terminal catalytic center. Although SPP is known to be an aspartyl protease of the GXGD type, several presenilin homologues/SPP-like proteins (PSHs/SPPL) of unknown function have been identified by data base searches. We now investigated the subcellular localization and a putative proteolytic activity of PSHs/SPPLs in cultured cells and in an in vivo model. We demonstrate that SPPL2b is targeted through the secretory pathway to endosomes/lysosomes, whereas SPP and SPPL3 are restricted to the ER. As suggested by the differential subcellular localization of SPPL2b compared with SPP and SPPL3, we found distinct phenotypes upon antisense gripNA-mediated knockdown in zebrafish. spp and sppl3 knockdowns in zebrafish result in cell death within the central nervous system, whereas reduction of sppl2b expression causes erythrocyte accumulation in an enlarged caudal vein. Moreover, expression of D/A mutations of the putative C-terminal active sites of spp, sppl2, and sppl3 produced phenocopies of the respective knockdown phenotypes. Thus, our data suggest that all investigated PSHs/SPPLs are members of the novel family of GXGD aspartyl proteases. Furthermore, SPPL2b is shown to be the first member of the SPP/PSH/SPPL family that is not located within the ER but in endosomal/lysosomal vesicles.

beta-Secretase (BACE) is a transmembrane aspartyl protease, which generates the N terminus of Alzheimer's disease amyloid beta-peptide. Here, we report that BACE can be phosphorylated within its cytoplasmic domain at serine residue 498 by casein kinase 1. Phosphorylation exclusively occurs after full maturation of BACE by propeptide cleavage and complex N-glycosylation. Phosphorylation/dephosphorylation affects the subcellular localization of BACE. BACE wild type and an S498D mutant that mimics phosphorylated BACE are predominantly located within juxtanuclear Golgi compartments and endosomes, whereas nonphosphorylatable BACE S498A accumulates in peripheral EEA1-positive endosomes. Antibody uptake assays revealed that reinternalization of BACE from the cell surface is independent of its phosphorylation state. After reinternalization, BACE wild type as well as BACE S498D are efficiently retrieved from early endosomal compartments and further targeted to later endosomal compartments and/or the trans-Golgi network. In contrast, nonphosphorylatable BACE S498A is retained within early endosomes. Our results therefore demonstrate regulated trafficking of BACE within the secretory and endocytic pathway.

Gamma-secretase mediates the final proteolytic cleavage, which liberates amyloid beta-peptide (Abeta), the major component of senile plaques in the brains of Alzheimer disease patients. Therefore, gamma-secretase is a prime target for Abeta-lowering therapeutic strategies. gamma-Secretase is a protein complex composed of four different subunits, presenilin (PS), APH-1, nicastrin, and PEN-2, which are most likely present in a 1:1:1:1 stoichiometry. PS harbors the catalytically active site, which is critically required for the aspartyl protease activity of gamma-secretase. Moreover, numerous familial Alzheimer disease-associated mutations within the PSs increase the production of the aggregation-prone and neurotoxic 42-amino acid Abeta. Nicastrin may serve as a substrate receptor, although this has recently been challenged. PEN-2 is required to stabilize PS within the gamma-secretase complex. No particular function has so far been assigned to APH-1. The four components are sufficient and required for gamma-secretase activity. At least six different gamma-secretase complexes exist that are composed of different variants of PS and APH-1. All gamma-secretase complexes can exert pathological Abeta production. Assembly of the gamma-secretase complex occurs within the endoplasmic reticulum, and only fully assembled and functional gamma-secretase complexes are transported to the plasma membrane. Structural analysis by electron microscopy and chemical cross-linking reveals a water-containing cavity, which allows intramembrane proteolysis. Specific and highly sensitive gamma-secretase inhibitors have been developed; however, they interfere with the physiological function of gamma-secretase in Notch signaling and thus cause rather significant side effects in human trials. Modulators of gamma-secretase, which selectively affect the production of the pathological 42-amino acid Abeta, do not inhibit Notch signaling.

Intramembrane proteolysis is now widely recognized as an important physiological pathway required for reverse signaling and membrane protein degradation. Aspartyl intramembrane cleaving proteases of the GXGD-type play an important regulatory role in health and disease. Besides gamma-secretase/presenilin, signal peptide peptidase (SPP) and SPP-like (SPPL) peptidases also belong to the family of GXGD-type aspartyl proteases. Although recently the first SPPL2a/b substrates have been identified, very little is known about substrate requirements, which allow them to be efficiently processed within the membrane. We demonstrate that similar to gamma-secretase substrates, intramembrane proteolysis of Bri2 (Itm2b) is greatly facilitated by an initial shedding event mediated by ADAM-10. Serial deletions revealed that the length of the ectodomain negatively correlates with efficient intramembrane proteolysis. Bri3 (Itm2c), which is highly homologous to Bri2, fails to be shed. Failure of shedding of Bri3 is accompanied by a lack of intramembrane proteolysis by SPPL2b. Surprisingly, a low molecular weight membrane-retained stub of Bri3 also fails to be processed by SPPL2b, indicating that shedding per se is not sufficient for subsequent intramembrane proteolysis. Extensive domain swapping analysis reveals that primary sequence determinants within the intracellular domain and the transmembrane domain together with short luminal juxtamembrane sequences are required for efficient intramembrane proteolysis.

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.

Significant data suggest that soluble Aβ oligomers play an important role in Alzheimer’s disease (AD), but there is great confusion over what exactly constitutes an Aβ oligomer and which oligomers are toxic. Most studies have utilized synthetic Aβ peptides, but the relevance of these test tube experiments to the conditions that prevail in AD are uncertain. A few groups have studied Aβ extracted from human brain, but they employed vigorous tissue homogenization which is likely to release insoluble Aβ that was sequestered in plaques during life. Several studies have found such extracts to possess disease-relevant activity and considerable efforts are being made to purify and better understand the forms of Aβ therein. Here, we compared the abundance of Aβ in AD extracts prepared by traditional homogenization versus using a far gentler extraction, and assessed their bioactivity via real-time imaging of iPSC-derived human neurons plus the sensitive functional assay of long-term potentiation. Surprisingly, the amount of Aβ retrieved by gentle extraction constituted only a small portion of that released by traditional homogenization, but this readily diffusible fraction retained all of the Aβ-dependent neurotoxic activity. Thus, the bulk of Aβ extractable from AD brain was innocuous, and only the small portion that was aqueously diffusible caused toxicity. This unexpected finding predicts that generic anti-oligomer therapies, including Aβ antibodies now in trials, may be bound up by the large pool of inactive oligomers, whereas agents that specifically target the small pool of diffusible, bioactive Aβ would be more useful. Furthermore, our results indicate that efforts to purify and target toxic Aβ must employ assays of disease-relevant activity. The approaches described here should enable these efforts, and may assist the study of other disease-associated aggregation-prone proteins.

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-like 3 (Sppl3) is a Golgi-resident intramembrane-cleaving protease that is highly conserved among multicellular eukaryotes pointing to pivotal physiological functions in the Golgi network which are only beginning to emerge. Recently, Sppl3 was shown to control protein N-glycosylation, when the key branching enzyme N-acetylglucosaminyltransferase V (GnT-V) and other medial/trans Golgi glycosyltransferases were identified as first physiological Sppl3 substrates. Sppl3-mediated endoproteolysis releases the catalytic ectodomains of these enzymes from their type II membrane anchors. Protein glycosylation is a multistep process involving numerous type II membrane-bound enzymes, but it remains unclear whether only few of them are Sppl3 substrates or whether Sppl3 cleaves many of them and thereby controls protein glycosylation at multiple levels. Therefore, to systematically identify Sppl3 substrates we used Sppl3-deficient and Sppl3-overexpression cell culture models and analyzed them for changes in secreted membrane protein ectodomains using the proteomics 'secretome protein enrichment with click sugars (SPECS)' method. SPECS analysis identified numerous additional new Sppl3 candidate glycoprotein substrates, several of which were biochemically validated as Sppl3 substrates. All novel Sppl3 substrates adopt a type II topology. The majority localizes to the Golgi network and is implicated in Golgi functions. Importantly, most of the novel Sppl3 substrates catalyze the modification of N-linked glycans. Others contribute to O-glycan and in particular glycosaminoglycan biosynthesis, suggesting that Sppl3 function is not restricted to N-glycosylation, but also functions in other forms of protein glycosylation. Hence, Sppl3 emerges as a crucial player of Golgi function and the newly identified Sppl3 substrates will be instrumental to investigate the molecular mechanisms underlying the physiological function of Sppl3 in the Golgi network and in vivo. Data are available via ProteomeXchange with identifier PXD001672.

Frontotemporal dementia (FTD) is a heterogeneous clinical syndrome characterized by frontotemporal lobar degeneration (FTLD). Neuropathologically, FTLD is characterized by abnormal protein deposits and almost all cases can now be classified into three major molecular subgroups based on specific accumulating proteins with the most common being FTLD-tau and FTLD-TDP (accounting for ~40% and 50%, respectively) and FTLD-FET (accounting for ~5–10%). In this special issue, the molecular and genetic basics as well as clinical approaches and therapeutics are reviewed in a series of articles. This article is part of the Frontotemporal Dementia special issue.

Sequence variations in the triggering receptor expressed on myeloid cells 2 (TREM2) have been linked to an increased risk for neurodegenerative disorders such as Alzheimer9s disease and frontotemporal lobar degeneration. In the brain, TREM2 is predominantly expressed in microglia. Several disease‐associated TREM2 variants result in a loss of function by reducing microglial phagocytosis, impairing lipid sensing, preventing binding of lipoproteins and affecting shielding of amyloid plaques. We here investigate the consequences of TREM2 loss of function on the microglia transcriptome. Among the differentially expressed messenger RNAs in wild‐type and Trem2 −/− microglia, gene clusters are identified which represent gene functions in chemotaxis, migration and mobility. Functional analyses confirm that loss of TREM2 impairs appropriate microglial responses to injury and signals that normally evoke chemotaxis on multiple levels. In an ex vivo organotypic brain slice assay, absence of TREM2 reduces the distance migrated by microglia. Moreover, migration towards defined chemo‐attractants is reduced upon ablation of TREM2 and can be rescued by TREM2 re‐expression. In vivo , microglia lacking TREM2 migrate less towards injected apoptotic neurons, and outgrowth of microglial processes towards sites of laser‐induced focal CNS damage in the somatosensory cortex is slowed. The apparent lack of chemotactic stimulation upon depletion of TREM2 is consistent with a stable expression profile of genes characterizing the homoeostatic signature of microglia.

Frontotemporal lobar degeneration is associated with cytoplasmic or nuclear deposition of the TAR DNA-binding protein 43 (TDP-43). Haploinsufficiency of progranulin (GRN) is a major genetic risk factor for frontotemporal lobar degeneration associated with TDP-43 deposition. Therefore, understanding the mechanisms that control cellular expression of GRN is required not only to understand disease etiology but also for the development of potential therapeutic strategies. We identified different GRN transcripts with short (38-93 nucleotides) or long (219 nucleotides) 5' UTRs and demonstrate a cellular mechanism that represses translation of GRN mRNAs with long 5' UTRs. The long 5' UTR of GRN mRNA contains an upstream open reading frame (uORF) that is absent in all shorter transcripts. Because such UTRs can be involved in translational control as well as in mRNA stability, we compared the expression of GRN in cells expressing cDNAs with and without 5' UTRs. This revealed a selective repression of GRN translation and a reduction of mRNA levels by the 219-nucleotide-long 5' UTR. The specific ability of this GRN 5' UTR to repress protein expression was further confirmed by its transfer to an independent reporter. Deletion analysis identified a short stretch between nucleotides 76 and 125 containing two start codons within one uORF that is required and sufficient for repression of protein expression. Mutagenesis of the two AUG codons within the uORF is sufficient to reduce translational repression. Therefore initiating ribosomes at the AUGs of the uORF fail to efficiently initiate translation at the start codon of GRN. In parallel the 5' UTR also affects mRNA stability; thus two independent mechanisms determine GRN expression via mRNA stability and translational efficiency.

The β-site APP cleaving enzymes 1 and 2 (BACE1 and BACE2) were initially identified as transmembrane aspartyl proteases cleaving the amyloid precursor protein (APP). BACE1 is a major drug target for Alzheimer's disease because BACE1-mediated cleavage of APP is the first step in the generation of the pathogenic amyloid-β peptides. BACE1, which is highly expressed in the nervous system, is also required for myelination by cleaving neuregulin 1. Several recent proteomic and in vivo studies using BACE1- and BACE2-deficient mice demonstrate a much wider range of physiological substrates and functions for both proteases within and outside of the nervous system. For BACE1 this includes axon guidance, neurogenesis, muscle spindle formation, and neuronal network functions, whereas BACE2 was shown to be involved in pigmentation and pancreatic β-cell function. This review highlights the recent progress in understanding cell biology, substrates, and functions of BACE proteases and discusses the therapeutic options and potential mechanism-based liabilities, in particular for BACE inhibitors in Alzheimer's disease. The protease BACE1 is a major drug target in Alzheimer disease. Together with its homolog BACE2, both proteases have an increasing number of functions within and outside of the nervous system. This review highlights recent progress in understanding cell biology, substrates, and functions of BACE proteases and discusses the therapeutic options and potential mechanism-based liabilities, in particular for BACE inhibitors in Alzheimer disease.

Zebrafish has become a popular model organism to study human diseases. We will highlight the advantages and limitations of zebrafish as a model organism to study neurodegenerative diseases and introduce zinc finger nucleases, transcription activator-like effector nucleases, and the recently established clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeat-associated system for genome editing. The efficiency of the novel genome editing tools now greatly facilitates knock-out and, importantly, also makes knock-in approaches feasible in zebrafish. Genome editing in zebrafish avoids unspecific phenotypes caused by off-target effects and toxicity as frequently seen in conventional knock-down approaches.

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.

Amyloid-β peptide (Aβ) is believed to play a central role in the pathogenesis of Alzheimer's disease. In view of the side effects associated with inhibiting the secretases that produce Aβ, new molecular targets are required to provide alternative therapeutic options. We used RNA interference (RNAi) to systematically screen theDrosophilagenome to identify genes that modulate Aβ production upon knockdown. RNAi of 41 genes inDrosophilacells significantly lowered Aβ without affecting general secretion or viability. After the γ-secretase complex components, the most potent effect was observed for platelet activating factor acetylhydrolase α (Paf-AHα), and, in mammalian cells, the effect was replicated for its ortholog PAFAH1B2. Knockdown of PAFAH1B2 strongly reduced Aβ secretion from human cells, and this effect was confirmed in primary cells derived from PAFAH1B2 knock-out mice. Reduced Aβ production was not attributable to altered β-amyloid precursor protein (APP) ectodomain shedding but was a result of an enhanced degradation of APP C-terminal fragments (CTFs) in the absence of PAFAH1B2 but not its close homolog PAFAH1B3. Enhanced degradation of APP CTFs was selective because no such effects were obtained for Notch or E-/N-cadherin. Thus, we have identified an important protein that can selectively modify Aβ generation via a novel mechanism, namely enhanced degradation of its immediate precursor. In view of the absence of a neurological phenotype in PAFAH1B2 knock-out mice, targeted downregulation of PAFAH1B2 may be a promising new strategy for lowering Aβ.

Impaired protein degradation has been discussed as a cause or consequence of various neurodegenerative diseases, such as Alzheimer's, Parkinson's and Huntington's disease. More recently, evidence accumulated that dysfunctional protein degradation may play a role in frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). Since in almost all neurodegenerative diseases, protein aggregates are disease-defining hallmarks, it is most likely that impaired protein degradation contributes to disease onset and progression. In the majority of FTD cases, the pathological protein aggregates contain either microtubuleassociated protein tau or TAR DNA-binding protein (TDP)-43. Aggregates are also positive for ubiquitin and p62/sequestosome 1 (SQSTM1) indicating that these aggregates are targeted for degradation. FTD-linked mutations in genes encoding three autophagy adaptor proteins, p62/SQSTM1, ubiquilin 2 and optineurin, indicate that impaired autophagy might cause FTD. Furthermore, the strongest evidence for lysosomal impairment in FTD is provided by the progranulin (GRN) gene, which is linked to FTD and neuronal ceroid lipofuscinosis. In this review, we summarize the observations that have been made during the last years linking the accumulation of disease-associated proteins in FTD to impaired protein degradation pathways. In addition, we take resent findings for nucleocytoplasmic transport defects of TDP-43, as discussed for hexanucleotide repeat expansions in C9orf72 into account and provide a hypothesis how the interplay of altered nuclear transport and protein degradation leads to the accumulation of protein deposits.

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.

Regulated intramembrane proteolysis (RIP) controls the communication between cells and the extracellular environment. RIP is essential in the nervous system, but also in other tissues. In the RIP process, a membrane protein typically undergoes two consecutive cleavages. The first one results in the shedding of its ectodomain. The second one occurs within its transmembrane domain, resulting in secretion of a small peptide and the release of the intracellular domain into the cytosol. The proteolytic cleavage fragments act as versatile signaling molecules or are further degraded. An increasing number of membrane proteins undergo RIP. These include growth factors, cytokines, cell adhesion proteins, receptors, viral proteins and signal peptides. A dysregulation of RIP is found in diseases, such as leukemia and Alzheimer's disease. One of the first RIP substrates discovered was the amyloid precursor protein (APP). RIP processing of APP controls the generation of the amyloid β-peptide, which is believed to cause Alzheimer's disease. Focusing on APP as the best-studied RIP substrate, this review describes the function and mechanism of the APP RIP proteases with the goal to elucidate cellular mechanisms and common principles of the RIP process in general.

Numerous loss-of-function mutations in the progranulin (GRN) gene cause frontotemporal lobar degeneration with ubiquitin and TAR–DNA binding protein 43-positive inclusions by reduced production and secretion of GRN. Consistent with the observation that GRN has neurotrophic properties, pharmacological stimulation of GRN production is a promising approach to rescueGRNhaploinsufficiency and prevent disease progression. We therefore searched for compounds capable of selectively increasing GRN levels. Here, we demonstrate that four independent and highly selective inhibitors of vacuolar ATPase (bafilomycin A1, concanamycin A, archazolid B, and apicularen A) significantly elevate intracellular and secreted GRN. Furthermore, clinically used alkalizing drugs, including chloroquine, bepridil, and amiodarone, similarly stimulate GRN production. Elevation of GRN levels occurs via a translational mechanism independent of lysosomal degradation, autophagy, or endocytosis. Importantly, alkalizing reagents rescue GRN deficiency in organotypic cortical slice cultures from a mouse model for GRN deficiency and in primary cells derived from human patients withGRNloss-of-function mutations. Thus, alkalizing reagents, specifically those already used in humans for other applications, and vacuolar ATPase inhibitors may be therapeutically used to prevent GRN-dependent neurodegeneration.

Methylene blue is an FDA approved compound with a variety of pharmacologic activities. It inhibits aggregation of several amyloidogenic proteins known to be deposited in neurodegenerative diseases. Recently, it has been proposed that methylene blue shows significant beneficial effects in a phase 2 clinical trial by slowing cognitive decline in Alzheimer's disease patients. To analyze its therapeutic potential, we investigated the effect of methylene blue on neurotoxicity in a zebrafish model for tauopathies. Transgenic expression of the frontotemporal dementia associated Tau-P301L mutation recapitulates a number of the pathological features observed in humans including abnormal phosphorylation and folding of Tau, tangle formation and Tau dependent neuronal loss. Upon incubation of zebrafish larvae with methylene blue, neither abnormal phosphorylation nor neuronal cell loss, reduced neurite outgrowth or a swimming defect were rescued. Methylene blue is biologically active in zebrafish since it reduced aggregation of a huntingtin variant containing a stretch of 102 glutamine residues. However, although huntingtin aggregation was largely prevented by methylene blue, huntingtin-dependent toxicity was unaffected. Our findings are consistent with the hypothesis that toxicity is not necessarily associated with deposition of insoluble amyloid proteins.

Loss-of-function mutations in the parkin gene (PARK2) and PINK1 gene (PARK6) are associated with autosomal recessive parkinsonism. PINK1 deficiency was recently linked to mitochondrial pathology in human cells and Drosophila melanogaster, which can be rescued by parkin, suggesting that both genes play a role in maintaining mitochondrial integrity. Here we demonstrate that an acute down-regulation of parkin in human SH-SY5Y cells severely affects mitochondrial morphology and function, a phenotype comparable with that induced by PINK1 deficiency. Alterations in both mitochondrial morphology and ATP production caused by either parkin or PINK1 loss of function could be rescued by the mitochondrial fusion proteins Mfn2 and OPA1 or by a dominant negative mutant of the fission protein Drp1. Both parkin and PINK1 were able to suppress mitochondrial fragmentation induced by Drp1. Moreover, in Drp1- deficient cells the parkin/PINK1 knockdown phenotype did not occur, indicating that mitochondrial alterations observed in parkin- or PINK1-deficient cells are associated with an increase in mitochondrial fission. Notably, mitochondrial fragmentation is an early phenomenon upon PINK1/parkin silencing that also occurs in primary mouse neurons and Drosophila S2 cells. We propose that the discrepant findings in adult flies can be explained by the time of phenotype analysis and suggest that in mammals different strategies may have evolved to cope with dysfunctional mitochondria.

Intramembrane-cleaving proteases are required for reverse signaling and membrane protein degradation. A major class of these proteases is represented by the GXGD-type aspartyl proteases. GXGD describes a novel signature sequence that distinguishes these proteases from conventional aspartyl proteases. Members of the family of the GXGD-type aspartyl proteases are the Alzheimer disease-related γ-secretase, the signal peptide peptidases and their homologs, and the bacterial type IV prepilin peptidases. We will describe the major biochemical and functional properties of the signal peptide peptidases and their relatives. We then compare these properties with those of γ-secretase and discuss common mechanisms but also point out a number of substantial differences.

beta-Secretase (beta-site amyloid precursor protein cleaving enzyme 1; BACE1) has been identified as the rate limiting enzyme for amyloid-beta-peptide (Abeta) production. Abeta is the major component of amyloid plaques and vascular deposits in Alzheimer's disease (AD) brains and believed to initiate the deadly amyloid cascade. BACE1 is the principle beta-secretase, since its knock-out completely prevents Abeta generation. BACE1 is likely to process a number of different substrates and consequently several independent physiological functions may be exerted by BACE1. Currently the function of BACE1 in myelination is best understood. BACE1 cleaves and activates Neuregulin-1 and is thus directly involved in myelination of the peripheral nervous system during early postnatal development. However, additional physiological functions specifically within the central nervous system are so far less understood. BACE1 is upregulated in at least some AD brains. Multiple cellular mechanisms for BACE1 regulation are known including post-transcriptional regulation via its 5'-untranslated region, microRNA and non-coding anti-sense RNA. BACE1 is a primary target for Abeta lowering therapies, however the development of high affinity bio-available inhibitors has been a major challenge so far.

More than 150 familial Alzheimer disease (FAD)-associated missense mutations in presenilins (PS1 and PS2), the catalytic subunit of the γ-secretase complex, cause aberrant amyloid β-peptide (Aβ) production, by increasing the relative production of the highly amyloidogenic 42-amino acid variant. The molecular mechanism behind this pathological activity is unclear, and different possibilities ranging from a gain of function to a loss of function have been discussed. γ-Secretase, signal peptide peptidase (SPP) and SPP-like proteases (SPPLs) belong to the same family of GXGD-type intramembrane cleaving aspartyl proteases and share several functional similarities. We have introduced the FAD-associated PS1 G384A mutation, which occurs within the highly conserved GXGD motif of PS1 right next to the catalytically critical aspartate residue, into the corresponding GXGD motif of the signal peptide peptidase-like 2b (SPPL2b). Compared with wild-type SPPL2b, mutant SPPL2b slowed intramembrane proteolysis of tumor necrosis factor α and caused a relative increase of longer intracellular cleavage products. Because the N termini of the secreted counterparts remain unchanged, the mutation selectively affects the liberation of the intracellular processing products. In vitro experiments demonstrate that the apparent accumulation of longer intracellular cleavage products is the result of slowed sequential intramembrane cleavage. The longer cleavage products are still converted to shorter peptides, however only after prolonged incubation time. This suggests that FAD-associated PS mutation may also result in reduced intramembrane cleavage of β-amyloid precursor protein (βAPP). Indeed, in vitro experiments demonstrate slowed intramembrane proteolysis by γ-secretase containing PS1 with the G384A mutation. As compared with wild-type PS1, the mutation selectively slowed Aβ40 production, whereas Aβ42 generation remained unaffected. Thus, the PS1 G384A mutation causes a selective loss of function by slowing the processing pathway leading to the benign Aβ40. © 2008 by The American Society for Biochemistry and Molecular Biology, Inc.

Alzheimer disease amyloid beta-peptide (Abeta) is generated via proteolytic processing of the beta-amyloid precursor protein by beta- and gamma-secretase. Gamma-secretase can be blocked by selective inhibitors but can also be modulated by a subset of non-steroidal anti-inflammatory drugs, including sulindac sulfide. These drugs selectively reduce the generation of the aggregation-prone 42-amino acid Abeta(42) and concomitantly increase the levels of the rather benign Abeta(38). Here we show that Abeta(42) and Abeta(38) generation occur independently from each other. The amount of Abeta(42) produced by cells expressing 10 different familial Alzheimer disease (FAD)-associated mutations in presenilin (PS) 1, the catalytic subunit of gamma-secretase, appeared to correlate with the respective age of onset in patients. However, Abeta(38) levels did not show a negative correlation with the age of onset. Modulation of gamma-secretase activity by sulindac sulfide reduced Abeta(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 Abeta(42) or only rather subtle effects. Strikingly, even the mutations that showed no effect on Abeta(42) levels allowed a robust increase of Abeta(38) upon treatment with sulindac sulfide. Similar observations were made for fenofibrate, a compound known to increase Abeta(42) and to decrease Abeta(38). For mutants that predominantly produce Abeta(42), the ability of fenofibrate to further increase Abeta(42) levels became diminished, whereas Abeta(38) levels were altered to varying extents for all mutants analyzed. Thus, we conclude that Abeta(38) and Abeta(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 Abeta(42)-lowering capacity of gamma-secretase modulators. Furthermore, human patients with certain PS mutations may not respond to gamma-secretase modulators.

Consecutive cleavages of amyloid precursor protein (APP) generate APP intracellular domain (AICD). Its cellular function is still unclear. In this study, we investigated the functional role of AICD in cellular Ca(2+) homeostasis. We could confirm previous observations that endoplasmic reticulum Ca(2+) stores contain less calcium in cells with reduced APP gamma-secretase cleavage products, increased AICD degradation, reduced AICD expression or in cells lacking APP. In addition, we observed an enhanced resting cytosolic calcium concentration under conditions where AICD is decreased or missing. In view of the reciprocal effects of Ca(2+) on mitochondria and of mitochondria on Ca(2+) homeostasis, we analysed further the cellular ATP content and the mitochondrial membrane potential. We observed a reduced ATP content and a mitochondrial hyperpolarisation in cells with reduced amounts of AICD. Blockade of mitochondrial oxidative phosphorylation chain in control cells lead to similar alterations as in cells lacking AICD. On the other hand, substrates of Complex II rescued the alteration in Ca(2+) homeostasis in cells lacking AICD. Based on these observations, our findings indicate that alterations observed in endoplasmic reticulum Ca(2+) storage in cells with reduced amounts of AICD are reciprocally linked to mitochondrial bioenergetic function.

gamma-Secretase is a protease complex, which catalyzes the final of two subsequent cleavages of the beta-amyloid precursor protein (APP) to release the amyloid-beta peptide (Abeta) implicated in Alzheimer's disease (AD) pathogenesis. In human cells, six gamma-secretase complexes exist, which are composed of either presenilin (PS) 1 or 2, the catalytic subunit, nicastrin, PEN-2, and either APH-1a (as S or L splice variants) or its homolog APH-1b. It is not known whether and how different APH-1 species contribute to the pathogenic activity of gamma-secretase complexes with familial AD (FAD)-associated mutant PS. Here we show that all known gamma-secretase complexes are active in APP processing and that all combinations of APH-1 variants with either FAD mutant PS1 or PS2 support pathogenic Abeta(42) production. Since our data suggest that pathogenic gamma-secretase activity cannot be attributed to a discrete gamma-secretase complex, we propose that all gamma-secretase complexes have to be explored and evaluated for their potential as AD drug target.

Ectodomain shedding and intramembrane proteolysis of the amyloid precursor protein (APP) by alpha-, beta- and gamma-secretase are involved in the pathogenesis of Alzheimer disease (AD). Increased proteolytic processing and secretion of another membrane protein, the interleukin-1 receptor II (IL-1R2), have also been linked to the pathogenesis of AD. IL-1R2 is a decoy receptor that may limit detrimental effects of IL-1 in the brain. At present, the proteolytic processing of IL-1R2 remains little understood. Here we show that IL-1R2 can be proteolytically processed in a manner similar to APP. IL-1R2 expressed in human embryonic kidney 293 cells first undergoes ectodomain shedding in an alpha-secretase-like manner, resulting in secretion of the IL-1R2 ectodomain and the generation of an IL-1R2 C-terminal fragment. This fragment undergoes further intramembrane proteolysis by gamma-secretase, leading to the generation of the soluble intracellular domain of IL-1R2. Intramembrane cleavage of IL-1R2 was abolished by a highly specific inhibitor of gamma-secretase and was absent in mouse embryonic fibroblasts deficient in gamma-secretase activity. Surprisingly, the beta-secretase BACE1 and its homolog BACE2 increased IL-1R2 secretion resulting in C-terminal fragments nearly identical to the ones generated by the alpha-secretase-like cleavage. This suggests that both proteases may act as alternative alpha-secretase-like proteases. Importantly, BACE1 and BACE2 did not cleave several other membrane proteins, demonstrating that both proteases do not contribute to general membrane protein turnover but only cleave specific proteins. This study reveals a similar proteolytic processing of IL-1R2 and APP and may provide an explanation for the increased IL-1R2 secretion observed in AD.

Amyloid imaging by small-animal PET in models of Alzheimer disease (AD) offers the possibility to track amyloidogenesis and brain energy metabolism. Because microglial activation is thought to contribute to AD pathology, we undertook a triple-tracer small-animal PET study to assess microglial activation and glucose metabolism in association with amyloid plaque load in a transgenic AD mouse model.Groups of PS2APP and C57BL/6 wild-type mice of various ages were examined by small-animal PET. We acquired 90-min dynamic emission data with (18)F-GE180 for imaging activated microglia (18-kD translocator protein ligand [TSPO]) and static 30- to 60-min recordings with (18)F-FDG for energy metabolism and (18)F-florbetaben for amyloidosis. Optimal fusion of PET data was obtained through automatic nonlinear spatial normalization, and SUVRs were calculated. For the novel TSPO tracer (18)F-GE180, we then calculated distribution volume ratios after establishing a suitable reference region. Immunohistochemical analyses with TSPO antisera, methoxy-X04 staining for fibrillary β-amyloid, and ex vivo autoradiography served as terminal gold standard assessments.SUVR at 60-90 min after injection gave robust quantitation of (18)F-GE180, which correlated well with distribution volume ratios calculated from the entire recording and using a white matter reference region. Relative to age-matched wild-type, (18)F-GE180 SUVR was slightly elevated in PS2APP mice at 5 mo (+9%; P0.01) and distinctly increased at 16 mo (+25%; P0.001). Over this age range, there was a high positive correlation between small-animal PET findings of microglial activation with amyloid load (R = 0.85; P0.001) and likewise with metabolism (R = 0.61; P0.005). Immunohistochemical and autoradiographic findings confirmed the in vivo small-animal PET data.In this first triple-tracer small-animal PET in a well-established AD mouse model, we found evidence for age-dependent microglial activation. This activation, correlating positively with the amyloid load, implies a relationship between amyloidosis and inflammation in the PS2APP AD mouse model.

Amyloid-beta, a peptide derived from the precursor protein APP, accumulates in the brain and contributes to the neuropathology of Alzheimer's disease. Increased generation of amyloid-beta might be caused by axonal transport inhibition, via increased dwell time of APP vesicles and thereby higher probability of APP cleavage by secretase enzymes residing on the same vesicles. We tested this hypothesis using a neuronal cell culture model of inhibited axonal transport and by imaging vesicular transport of fluorescently tagged APP and beta-secretase (BACE1). Microtubule-associated tau protein blocks vesicle traffic by inhibiting the access of motor proteins to the microtubule tracks. In neurons co-transfected with CFP-tau, APP-YFP traffic into distal neurites was strongly reduced. However, this did not increase amyloid-beta levels. In singly transfected axons, APP-YFP was transported in large tubules and vesicles moving very fast (on average 3 microm/s) and with high fluxes in the anterograde direction (on average 8.4 vesicles/min). By contrast, BACE1-CFP movement was in smaller tubules and vesicles that were almost 2x slower (on average 1.6 microm/s) with approximately 18x lower fluxes (on average 0.5 vesicles/min). Two-colour microscopy of co-transfected axons confirmed that the two proteins were sorted into distinct carriers. The results do not support the above hypothesis. Instead, they indicate that APP is transported on vesicles distinct from the secretase components and that amyloid-beta is not generated in transit when transport is blocked by tau.

Several receptor protein tyrosine phosphatases (RPTPs) are cell adhesion molecules involved in homophilic interactions, suggesting that RPTP outside-in signaling is coupled to cell contact formation. However, little is known about the mechanisms by which cell density regulates RPTP function. We show that the MAM family prototype RPTP is cleaved by three proteases: furin, ADAM 10, and -secretase. Cell density promotes ADAM 10-mediated cleavage and shedding of RPTP. This is followed by -secretase-dependent intramembrane proteolysis of the remaining transmembrane part to release the phosphatase intracellular portion (PIC) from the membrane, thereby allowing its translocation to the nucleus. When cells were treated with leptomycin B, a nuclear export inhibitor, PIC accumulated in nuclear bodies. PIC is an active protein tyrosine phosphatase that binds to and dephosphorylates -catenin, an RPTP substrate. The expression of RPTP suppresses -catenin’s transcriptional activity, whereas the expression of PIC increases it. Notably, this increase required the phosphatase activity of PIC. Thus, both isoforms have acquired opposing roles in the regulation of -catenin signaling. We also found that RPTP, another MAM family member, undergoes -secretasedependent processing. Our results identify intramembrane proteolysis as a regulatory switch in RPTP signaling and implicate PIC in the activation of -catenin-mediated transcription. The phosphorylation of cellular proteins on tyrosine residues is reversible and regulated by the coordinated and competing actions of two enzyme families: protein tyrosine kinases and protein tyrosine phosphatases (PTPs). The PTP family is structurally diverse and includes both receptor-like and cytoplasmic enzymes. The majority of the receptor PTPs (RPTPs) contain two catalytic domains: a membrane-proximal domain (D1), which is responsible for mainly catalysis, and a membrane-distal domain (D2), which contains little or no phosphatase activity (45). The extracellular (E) portion exhibits broad structural variation. The MAM (meprin/A5 glycoprotein/PTPmu) family of RPTPs, including RPTP ,- ,- ,- , and PCP-2, are characterized by the presence of the MAM domain at their N termini (3, 6, 18, 29, 46). Additional structural features of the extracellular portions involve one immu

The single-transmembrane receptor SorLA/LR11 contains binding domains typical for lipoprotein receptors and a VPS10 domain, which binds the neuropeptide head-activator. This undecapeptide enhances proliferation of neuronal precursor cells in a SorLA-dependent manner. Using specific inhibitors we found previously that head activator activates shedding of SorLA by the metalloprotease TACE close to the transmembrane domain releasing the large extra-cellular part of the receptor. Here we show that the remaining COOH-terminal membrane fragment of SorLA is processed by gamma-secretase. Inhibition of gamma-secretase by specific inhibitors or overexpression of dominant negative presenilin mutants and knock out of the presenilin genes led to accumulation of the SorLA membrane fragment and also of full-length SorLA in the membrane. In an in vitro assay we observed the gamma-secretase-dependent release of the two soluble cleavage products, the SorLA cytoplasmic domain and the SorLA beta-peptide. These processing steps are reminiscent of a novel signaling pathway that has been described for the notch receptor. Here, the notch cytoplasmic domain is released into the cytoplasm by the gamma-secretase and migrates to the nucleus where it acts as a transcriptional regulator. In parallel we found that a fusion protein of the released cytoplasmic tail of SorLA with EGFP located to the nucleus only if the nuclear localization signal of SorLA was intact. In a reporter gene assay the cytoplasmic domain of SorLA acted as a transcriptional activator indicating that SorLA might directly regulate transcription after activation by gamma-secretase.

The presenilin (PS)/gamma-secretase complex, which contains not only PS but also Aph-1, PEN-2, and nicastrin, mediates proteolysis of the transmembrane domain of beta-amyloid protein precursor (betaAPP). Intramembrane proteolysis occurs at the interface between the membrane and cytosol (epsilon-site) and near the middle of the transmembrane domain (gamma-site), generating the betaAPP intracellular domain (AICD) and Alzheimer disease-associated Abeta, respectively. Both cleavage sites exhibit some diversity. Changes in the precision of gamma-cleavage, which potentially results in secretion of pathogenic Abeta42, have been intensively studied, while those of epsilon-cleavage have not. Although a number of PS-associated factors have been identified, it is unclear whether any of them physiologically regulate the precision of cleavage by PS/gamma-secretase. Moreover, there is currently no clear evidence of whether PS/gamma-secretase function differs according to the subcellular site. Here, we show that endocytosis affects the precision of PS-dependent epsilon-cleavage in cell culture. Relative production of longer AICDepsilon49 increases on the plasma membrane, whereas that of shorter AICDepsilon51 increases on endosomes; however, this occurs without a concomitant major change in the precision of cleavage at gamma-sites. Moreover, very similar changes in the precision of epsilon-cleavage are induced by alteration of the pH. Our findings demonstrate that the precision of epsilon-cleavage by PS/gamma-secretase changes depending upon the conditions and the subcellular location. These results suggest that the precision of cleavage by the PS/gamma-secretase complex may be physiologically regulated by the subcellular location and conditions.

Ectodomain shedding of the amyloid precursor protein (APP) is a key regulatory step in the generation of the Alzheimer disease amyloid beta peptide (Abeta). The molecular mechanisms underlying the control of APP shedding remain little understood but are in part dependent on the low density lipoprotein receptor-related protein (LRP), which is involved in APP endocytosis. Here, we show that the APP homolog APLP1 (amyloid precursor-like protein 1) influences APP shedding. In human embryonic kidney 293 cells expression of APLP1 strongly activated APP shedding by alpha-secretase and slightly reduced beta-secretase cleavage. As revealed by domain deletion analysis, the increase in APP shedding required the NPTY amino acid motif within the cytoplasmic domain of APLP1. This motif is conserved in APP and is essential for the endocytosis of APP and APLP1. Unrelated membrane proteins containing similar endocytic motifs did not affect APP shedding, showing that the increase in APP shedding was specific to APLP1. In LRP-deficient cells APLP1 no longer induced APP shedding, suggesting that in wild-type cells APLP1 interferes with the LRP-dependent endocytosis of APP and there by increases APP alpha-cleavage. In fact, an antibody uptake assay revealed that expression of APLP1 reduced the rate of APP endocytosis. In summary, our study provides a novel mechanism for APP shedding, in which APLP1 affects the endocytosis of APP and makes more APP available for alpha-secretase cleavage.

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 Abeta-like peptide (Nbeta)) should be produced. Here we showed that Nbeta species are indeed secreted during the process of Notch signaling. We identified mainly two distinct molecular species of novel Nbeta, Nbeta21 and C-terminally elongated Nbeta25, which were produced in an approximately 5:1 ratio. This process is reminiscent of the production of Alzheimer disease-associated Abeta. PS pathogenic mutants increased the production of the longer species of Abeta (Abeta42) from beta-amyloid protein precursor. We revealed that several Alzheimer disease mutants also cause a parallel increase in the secretion of the longer form of Nbeta. Strikingly, chemicals that modify the Abeta42 level caused parallel changes in the Nbeta25 level. These results demonstrated that the characteristics of C-terminal elongation of Nbeta and Abeta 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 Abeta- or Nbeta-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 Abeta42 production.

Alzheimer's disease is characterized by brain deposition of extracellular amyloid β-peptide (Aβ)-containing plaques. The cellular site of γ-secretase activity, which releases Aβ and the corresponding amyloid precursor protein intracellular domain (AICD), remains controversial. Proposed cleavage sites range from the endoplasmic reticulum (ER), the Golgi apparatus, and the cell surface to endosomal compartments. We now used C99-green fluorescent protein (GFP), a fluorescent reporter substrate for γ-secretase activity and monitored AICD production in living cells. C99-GFP is efficiently cleaved by γ-secretase, and AICD-GFP is released into the cytosol. Inhibiting γ-secretase results in accumulation of C99-GFP in early endosomes. By blocking selective transport steps along the secretory pathway, we demonstrate that γ-secretase does not cleave its substrates in the ER, the Golgi/trans-Golgi network, or in secretory vesicles. In contrast, inhibition of endocytosis did not inhibit cleavage of C99-GFP. Similar results were obtained for another γ-secretase substrate, NotchΔE. Our results suggest that intracellular domains are generated by γ-secretase at the plasma membrane and/or early endosomes.

Amyloid-β peptide (Aβ) is generated by γ-secretase, a membrane protein complex with an unusual aspartyl protease activity consisting of the four components presenilin, nicastrin, APH-1 and PEN-2. Presenilin is considered the catalytic subunit of this complex since it represents the prototype of the new family of intramembrane-cleaving GxGD-type aspartyl proteases. Recently, five novel members of this family and a nicastrin-like protein were identified. Whereas one of the GxGD-type proteins was shown to be identical with signal peptide peptidase (SPP), the function of the others, now called SPP-like proteins (SPPLs), is not known. We therefore analyzed SPPL2b and SPPL3 and demonstrated that they localize to different subcellular compartments suggesting nonredundant functions. This was supported by different phenotypes obtained in knockdown studies in zebrafish embryos. In addition, these phenotypes could be phenocopied by ectopic expression of putative active site mutants, providing strong evidence for a proteolytic function of SPPL2b and SPPL3. We also identified and characterized the nicastrin-like protein nicalin which, together with the 130-kDa protein NOMO (Nodal modulator), forms a membrane protein complex different from γ-secretase. We found that during zebrafish embryogenesis this complex is involved in the patterning of the axial mesendoderm, a process controlled by the Nodal signaling pathway.

Gamma-secretase catalyzes the final cleavage of the beta-amyloid precursor protein to generate amyloid-beta peptide, the principal component of amyloid plaques in the brains of patients suffering from Alzheimer's disease. Here, we review the identification of gamma-secretase as a protease complex and its assembly and trafficking to its site(s) of cellular function. In reconstitution experiments, gamma-secretase was found to be composed of four integral membrane proteins, presenilin (PS), nicastrin (NCT), PEN-2 and APH-1 that are essential and sufficient for gamma-secretase activity. PS, which serves as a catalytic subunit of gamma-secretase, was identified as a prototypic member of novel aspartyl proteases of the GxGD type. In human cells, gamma-secretase could be further defined as a heterogeneous activity consisting of distinct complexes that are composed of PS1 or PS2 and APH-1a or APH-1b homologues together with NCT and PEN-2. Using green fluorescent protein as a reporter we localized PS and gamma-secretase activity at the plasma membrane and endosomes. Investigation of gamma-secretase complex assembly in knockdown and knockout cells of the individual subunits allowed us to develop a model of complex assembly in which NCT and APH-1 first stabilize PS before PEN-2 assembles as the last component. Furthermore, we could map domains in PS and PEN-2 that govern assembly and trafficking of the complex. Finally, Rer1 was identified as a PEN-2-binding protein that serves a role as an auxiliary factor for gamma-secretase complex assembly.

Gamma-secretase is an aspartyl protease complex that catalyzes the intramembrane cleavage of a subset of type I transmembrane proteins including the beta-amyloid precursor protein (APP) implicated in Alzheimer's disease. Presenilin (PS), nicastrin (NCT), anterior pharynx defective (APH-1) and presenilin enhancer-2 (PEN-2) constitute the active gamma-secretase complex. PEN-2, the smallest subunit, is required for triggering PS endoproteolysis. Stabilization of the resultant N- and C-terminal fragments, which carry the catalytically active site aspartates, but not endoproteolysis itself, requires the C-terminal domain of PEN-2. To functionally dissect the C-terminal domain we created C-terminal deletion mutants and mutagenized several evolutionary highly conserved residues. The PEN-2 mutants were then probed for functional complementation of a PEN-2 knockdown, which displays deficient PS1 endoproteolysis and impaired NCT maturation. Progressive truncation of the C-terminus caused increasing loss of function. This was also observed for an internal deletion mutant as well as for C-terminally tagged PEN-2 with a twofold elongated C-terminal domain. Interestingly, only simultaneous, but not individual substitution of the highly conserved D90, F94, P97 and G99 residues with alanine interfered with PEN-2 function. All loss of function mutants identified allowed PS1 endoproteolysis, but failed to stably associate with the resultant PS1 fragments, which like the PEN-2 loss of function mutants underwent proteasomal degradation. However, complex formation of the PEN-2 mutants with PS1 fragments could be recovered when proteasomal degradation was blocked. Taken together, our data suggest that the PS-subunit stabilizing function of PEN-2 depends on length and overall sequence of its C-terminal domain.

Several proteins linked to neurodegenerative diseases, such as the beta-amyloid precursor protein, amyloid beta-peptide, beta-secretase, and tau, undergo selective polarized sorting. We investigated polarized sorting of the mammalian prion protein (PrP(C)) and its homologue doppel (Dpl). In contrast to Dpl, which accumulates on the apical surface, PrP(C) is targeted selectively to the basolateral side in Madin-Darby canine kidney cells. An extensive deletion and domain swapping analysis revealed that the internal hydrophobic domain (HD) of PrP (amino acids 113-133) confers basolateral sorting in a dominant manner. PrP mutants lacking the HD are sorted apically, while Dpl chimeras containing the HD of PrP are directed to the basolateral membrane. Furthermore, a pathogenic PrP missense mutation within the HD leads to aberrant apical sorting of PrP as well.

gamma-Secretase is an aspartyl protease complex composed of the four core components APH-1, nicastrin (NCT), presenilin (PS), and PEN-2. It catalyzes the final intramembranous cleavage of the beta-secretase-processed beta-amyloid precursor protein to liberate the neurotoxic amyloid beta-peptide. Whereas unassembled complex components appear to be unstable and/or to be retained within the endoplasmic reticulum (ER), the fully assembled complex is known to exert its biological function in late secretory compartments, including the plasma membrane. We thus hypothesized that the gamma-secretase complex undergoes a stepwise assembly within the ER. We demonstrate that gamma-secretase-associated NCT can be actively retained within the ER by the addition of a retention signal. Under these conditions, complex assembly occurred in the absence of maturation of NCT, and ER-retained immature NCT associated with APH-1, PEN-2, and PS fragments. Moreover, a biotinylated transition state gamma-secretase inhibitor allowed the preferential isolation of the fully assembled complex containing immature NCT. Furthermore, we observed a conformational change in immature NCT, which is known to be selectively associated with complete gamma-secretase complex assembly. This was also observed for a small amount of immature endogenous NCT. ER-retained NCT also rescued the biochemical phenotype observed upon RNA interference-mediated NCT knockdown, viz. reduced amyloid beta-peptide production; instability of PS, PEN-2, and APH-1; and accumulation of beta-amyloid precursor protein C-terminal fragments. Finally, we demonstrate that dimeric (NCT/APH-1) and trimeric (NCT/APH-1/PS) intermediates of gamma-secretase complex assembly containing endogenous NCT are retained within the ER and that the incorporation of the fourth and last binding partner (PEN-2) also occurs on immature NCT, suggesting a complete assembly of the gamma-secretase complex within the ER.

Millions of patients suffer from Alzheimer's disease, and intensive efforts to find a cure for this devastating disorder center on the proteases, which release the deadly amyloid β-peptide from its precursor. The cutting procedure is thought to be cholesterol dependent and strategies to lower cholesterol as therapeutic treatment are under intensive investigation. Recent findings suggest that the complete proteolytic machinery required for amyloid β-peptide generation is located within lipid rafts. Data by Dotti and colleagues (Abad-Rodriguez et al., 2004), in this issue, suggest that rafts isolate the cutting machinery away from its deadly substrate. These findings describe a novel mechanism for controlling proteolytic activity by building a lipid boundary between proteases and their substrates.

Beta-site APP-cleaving enzyme (BACE) is a membrane-bound aspartyl protease with no strict primary preference for cleavage. The molecular mechanisms that link the gamma-secretase multicomponent amyloid precursor protein (APP) processing complex to biochemical properties of BACE generating the N terminus of the amyloid beta-peptide have not, as yet, been identified. We found that in human brain tissue, BACE occurred as a dimer. The overall stability of the BACE homodimer was based on intermolecular interactions that were not affected by high salt, nonionic detergents or reducing conditions. BACE homodimers could only partially be separated even under strong denaturing conditions and revealed dramatic differences in the surface charge distribution compared with the monomer. In contrast, the soluble ectodomain of truncated BACE revealed a seemingly lower avidity to the prototypic aspartate protease inhibitor pepstatin and exclusively occurred in the monomeric form. Immunocytochemical studies colocalized APP and BACE in the plasma membrane of cells expressing endogenous levels of BACE and overexpressing APP. In cells that were cotransfected with APP and a putative active site D289A mutant of BACE, colocalization persisted. Remaining enzyme activity was found to be attributable to the mutant protease. Accordingly, inactivation of the carboxyl-terminal active site motif of BACE without an impairment of overall enzyme activity suggests that the enzyme may act as a dimer. Thus, homodimerization of BACE may help the enzyme to acquire specific mechanisms to associate with its substrates to exert catalytic activity.

gamma-Secretase is an intramembrane-cleaving aspartyl protease complex that mediates the final cleavage of beta-amyloid precursor protein to liberate the neurotoxic amyloid-beta peptide implicated in Alzheimer's disease. The four proteins presenilin (PS), nicastrin (NCT), APH-1, and PEN-2 are sufficient to reconstitute gamma-secretase activity in yeast. Although PS seems to contribute the catalytic core of the gamma-secretase complex, no distinct function could be attributed to the other components so far. In Caenorhabditis elegans, mutation of a glycine to an aspartic acid within a conserved GXXXG motif in the fourth transmembrane domain of APH-1 causes a loss of function phenotype. Surprisingly, we now found that the human homologue APH-1a carrying the equivalent mutation G122D is fully active in yeast co-expressing PS1, NCT, and PEN-2. To address this discrepancy, we expressed APH-1a G122D in HEK293 cells. As reported previously, overexpressed APH-1a G122D was not incorporated into the gamma-secretase complex. Separate overexpression of PS1, NCT, or PEN-2 together with APH-1a G122D allowed the formation of heterodimers lacking the other endogenous components. Only the combined overexpression of PS1 and NCT together with APH-1a G122D facilitated the formation of a fully active gamma-secretase complex. Under these conditions, APH-1a G122D supported the production of normal amounts of Abeta. We conclude that cooperative effects may stabilize a trim-eric complex of APH-1a G122D together with PS1 and NCT. Upon successful complex assembly, the GXXXG motif becomes dispensable for gamma-secretase activity.