Hydrogen Sulfide Selectively Inhibits γ-Secretase Activity and Decreases Mitochondrial Aβ Production in Neurons from APP/PS1 Transgenic Mice

Hydrogen sulfide (H₂S) is now considered to be a gasotransmitter and may be involved in the pathological process of Alzheimer’s disease (AD). A majority of APP is associated with mitochondria and is a substrate for the mitochondrial γ-secretase. The mitochondria-associated APP metabolism where APP intracellular domains (AICD) and Aβ are generated locally and may contribute to mitochondrial dysfunction in AD. Here, we aimed to investigate the ability of H₂S to mediate APP processing in mitochondria and assessed the possible mechanisms underlying H₂S-mediated AD development. We treated neurons from APP/PS1 transgenic mice with a range of sodium hydrosulfide (NaHS) concentrations. NaHS attenuated APP processing and decreased Aβ production in mitochondria. Meanwhile, NaHS did not changed BACE-1 and ADAM10 (a disintegrin and metalloprotease 10) protein levels, but NaHS (30 μM) significantly increased the levels of presenilin 1(PS1), PEN-2, and NCT, as well as improved the γ-secretase activity, while NaHS (50 μM) exhibits the opposing effects. Furthermore, the intracellular ATP and the COX IV activity of APP/PS1 neurons were increased after 30 μM NaHS treatment, while the ROS level was decreased and the MMP was stabilized. The effect of NaHS differs from DAPT (a non-selective γ-secretase inhibitor), and it selectively inhibited γ-secretase in vitro, without interacting with Notch and modulating its cleavage. The results indicated that NaHS decreases Aβ accumulation in mitochondria by selectively inhibiting γ-secretase. Thus, we provide a mechanistic view of NaHS is a potential anti-AD drug candidate and it may decrease Aβ deposition in mitochondria by selectively inhibiting γ-secretase activity and therefore protecting the mitochondrial function during AD conditions.

     

Minor contribution of presenilin 2 for γ-secretase activity in mouse embryonic fibroblasts and adult mouse brain

γ-Secretase plays an important function in the development of Alzheimer disease, since it participates in the production of the toxic amyloid β-peptide (Aβ) from the amyloid precursor protein (APP). Besides APP, γ-secretase cleaves many other substrates resulting in adverse side effects when γ-secretase inhibitors are used in clinical trials. γ-Secretase is a membrane bound protein complex consisting of at least four subunits, presenilin (PS), nicastrin, Aph-1 and Pen-2. PS and Aph-1 exist as different homologs (PS1/PS2 and Aph-1a/Aph-1b, respectively), which generates a variation in complex composition. PS1 and PS2 appears to have distinct roles since PS1 is essential during embryonic development whereas PS2 deficient mice are viable with a mild phenotype. The molecular mechanism behind this diversity is, however, largely unknown. In order to investigate whether PS1 and PS2 show different substrate specificity, we used PS1 or PS2 deficient mouse embryonic fibroblasts to study the processing on the γ-secretase substrates APP, Notch, N-cadherin, and ephrinB. We found that whereas depletion of PS1 severely affected the cleavage of all substrates, the effect of PS2 depletion was minor. In addition, less PS2 was found in active γ-secretase complexes. We also studied the effect of PS2 depletion in adult mouse brain and, in concordance with the results from the mouse embryonic fibroblasts, PS2 deficiency did not alter the cleavage of the two most important substrates, APP and Notch. In summary, this study shows that the contribution of PS2 on γ-secretase activity is of less importance, explaining the mild phenotype of PS2-deficient mice.     

Niemann–Pick type C cells show cholesterol dependent decrease of APP expression at the cell surface and its increased processing through the β-secretase pathway

The link between cholesterol and Alzheimer's disease has recently been revealed in Niemann–Pick type C disease. We found that NPC1^?/? cells show decreased expression of APP at the cell surface and increased processing of APP through the β-secretase pathway resulting in increased C99, sAPPβ and intracellular Aβ40 levels. This effect is dependent on increased cholesterol levels, since cholesterol depletion reversed cell surface APP expression and lowered Aβ/C99 levels in NPC1^?/? cells to the levels observed in wt cells. Finding that overexpression of C99, a direct γ-secretase substrate, does not lead to increased intracellular Aβ levels in NPC1^?/? cells vs. CHOwt suggests that the effect on intracellular Aβ upon cholesterol accumulation in NPC1^?/? cells is not due to increased APP cleavage by γ-secretase. Our results indicate that cholesterol may modulate APP processing indirectly by modulating APP expression at the cell surface and thus its cleavage by β-secretase.     

In vitro reconstitution of gamma-secretase activity using yeast microsomes

γ-Secretase is composed of at least four transmembrane proteins, presenilin (PS) 1/2, nicastrin, anterior pharynx-1 (Aph-1) and presenilin enhancer-2 (Pen-2), and cleaves amyloid precursor protein (APP) to produce amyloid β peptides (Aβ) that is deposited in the brains of Alzheimer disease. However, the mechanism of γ-secretase-mediated cleavage remains unclear. To examine the enzymatic properties of γ-secretase, we established an in vitro assay system using Saccharomyces cerevisiae, which does not possess homologs of human PS1/2, nicastrin, Aph-1, or Pen-2. We transformed these subunits and the substrate in pep4Δ cells with vacuole proteases inactivated, and microsome was isolated for in vitro assay. In the assay, Aβ40, Aβ42, and Aβ43 were produced with an optimal pH of ∼7.0. We also detected Aβ-production by yeast endogenous protease(s), which was abolished by the addition of phosphatidyl choline. This novel system will facilitate the analysis of substrate recognition by γ-secretase.     

Rab21, a Novel PS1 Interactor,Regulates γ-Secretase Activity via PS1 Subcellular Distribution

γ-Secretase has been a therapeutical target for its key role in cleaving APP to generate β-amyloid (Aβ), the primary constituents of senile plaques and a hallmark of Alzheimer’s disease (AD) pathology. Recently, γ-secretase-associating proteins showed promising role in specifically modulating APP processing while sparing Notch signaling; however, the underlying mechanism is still unclear. A co-immunoprecipitation (Co-IP) coupled with mass spectrometry proteomic assay for Presenilin1 (PS1, the catalytic subunit of γ-secretase) was firstly conducted to find more γ-secretase-associating proteins. Gene ontology analysis of these results identified Rab21 as a potential PS1 interacting protein, and the interaction between them was validated by reciprocal Co-IP and immunofluorescence assay. Then, molecular and biochemical methods were used to investigate the effect of Rab21 on APP processing. Results showed that overexpression of Rab21 enhanced Aβ generation, while silencing of Rab21 reduced the accumulation of Aβ, which resulted due to change in γ-secretase activity rather than α- or β-secretase. Finally, we demonstrated that Rab21 had no effect on γ-secretase complex synthesis or metabolism but enhanced PS1 endocytosis and translocation to late endosome/lysosome. In conclusion, we identified a novel γ-secretase-associating protein Rab21 and illustrate that Rab21 promotes γ-secretase internalization and translocation to late endosome/lysosome. Moreover, silencing of Rab21 decreases the γ-secretase activity in APP processing thus production of Aβ. All these results open new gateways towards the understanding of γ-secretase-associating proteins in APP processing and make inhibition of Rab21 a promising strategy for AD therapy.     

Altered expression of γ-secretase components in animal model of major depressive disorder induced by reserpine administration

Altered expression of neurotrophic factors as well as neuroinflammation is commonly associated with Major depressive disorder (MDD) and Alzheimer's disease (AD). To investigate whether or not reserpine-induced MDD affects the expression of AD-related proteins, the expression of γ-secretase components and substrate were measured in brains of ICR mice following reserpine treatment for 15 days. In active avoidance test, total response time and peak slightly increased in the 2 mg/kg reserpine (RSP2)-treated group compared to vehicle-treated group (P<0.05). Expression and phosphorylation of MKP-1, which is a key factor in MDD pathology, were both higher in the RSP2-treated group than the vehicle- and 1 mg/kg reserpine (RSP1)-treated groups (P<0.02). Furthermore, full-length expression of amyloid precursor protein (APP) was enhanced in the RSP1 and RSP2-treated groups compared to the vehicle-treated group, whereas expression of γ-secretase components decreased (P<0.03). Among the three components of the γ-secretase complex, nicastrin protein underwent the largest decrease in expression, as detected by Western blotting (P<0.03). Therefore, the data presented here provide additional evidence about the pathological correlation between MDD and AD.     

Mutation analysis of the presenilin 1 N-terminal domain reveals a broad spectrum of gamma-secretase activity toward amyloid precursor protein and other substrates

The γ-secretase protein complex executes the intramembrane proteolysis of amyloid precursor protein (APP), which releases Alzheimer disease β-amyloid peptide. In addition to APP, γ-secretase also cleaves several other type I membrane protein substrates including Notch1 and N-cadherin. γ-Secretase is made of four integral transmembrane protein subunits: presenilin (PS), nicastrin, APH1, and PEN2. Multiple lines of evidence indicate that a heteromer of PS-derived N- and C-terminal fragments functions as the catalytic subunit of γ-secretase. Only limited information is available on the domains within each subunit involved in the recognition and recruitment of diverse substrates and the transfer of substrates to the catalytic site. Here, we performed mutagenesis of two domains of PS1, namely the first luminal loop domain (LL1) and the second transmembrane domain (TM2), and analyzed PS1 endoproteolysis as well as the catalytic activities of PS1 toward APP, Notch, and N-cadherin. Our results show that distinct residues within LL1 and TM2 domains as well as the length of the LL1 domain are critical for PS1 endoproteolysis, but not for PS1 complex formation with nicastrin, APH1, and PEN2. Furthermore, our experimental PS1 mutants formed γ-secretase complexes with distinct catalytic properties toward the three substrates examined in this study; however, the mutations did not affect PS1 interaction with the substrates. We conclude that the N-terminal LL1 and TM2 domains are critical for PS1 endoproteolysis and the coordination between the putative substrate-docking site and the catalytic core of the γ-secretase.     

Effects of Aluminium on β-Amyloid (1–42) and Secretases (APP-Cleaving Enzymes) in Rat Brain

Chronic administration of aluminium has been proposed as an environmental factor that may affect some pathological changes related to neurotoxicity and Alzheimer’s disease (AD). The abnormal generation and deposition of β-amyloid (Aβ) in senile plaques are hallmark features in the brains of AD patients. Furthermore, Aβ is generated by the sequential cleavage of the amyloid precursor protein (APP) via the APP cleaving enzyme (α-secretase, or β-secretase) and γ-secretase. In the present study, we investigated the modulation of Aβ deposition and neurotoxicity in aluminium-maltolate-treated (0, 15, 30, 45 mmol/kg body weight via intraperitoneal injection) in experimental rats. We measured Aβ1–40 and Aβ1–42 in the cortex and hippocampus in rat brains using ELISA. Subtypes of α-secretase, β-secretase, and γ-secretase, including ADAM9, ADAM10, ADAM17 (TACE), BACE1, presenilin 1 (PS1) and nicastrin (NCT), were determined using western blotting analyses. These results indicated that aluminium-maltolate induced an AD-like behavioural deficit in rats at 30 and 45 mmol/kg body weight. Moreover, the Aβ1–42 content increased significantly, both in the cortex and hippocampus, although no changes were observed in Aβ1–40. Furthermore, ADAM9, ADAM10, and ADAM17 decreased significantly; in contrast, BACE1, PS1, and NCT showed significant increase. Taken together, these results suggest that the changes in secretases may correlate to the abnormal deposition of Aβ by aluminium in rat brains.     

7-Deoxy-trans-dihydronarciclasine Reduces β-Amyloid and Ameliorates Memory Impairment in a Transgenic Model of Alzheimer’s Disease

The critical pathological feature of Alzheimer’s disease (AD) is the accumulation of β-amyloid (Aβ), the main constituent of amyloid plaques. β-amyloid precursor protein (APP) undergoes amyloidogenic cleavage by β- and γ-secretase generating Aβ at endosomes or non-amyloidogenic processing by α-secretase precluding the production of Aβ at the plasma membrane. Recently, several natural products have been widely researched on the prevention of Aβ accumulation for AD treatment. We previously reported that Lycoris chejuensis K. Tae et S. Ko (CJ), which originated from Jeju Island in Korea, improved the disrupted memory functions and reduced Aβ production in vivo. Here, we further explored the effect of its active component, 7-deoxy-trans-dihydronarciclasine (coded as E144), on Aβ generation and the underlying mechanism. Our results showed that E144 reduced the level of APP, especially its mature form, in HeLa cells overexpressing human APP with the Swedish mutation. Concomitantly, E144 decreased the levels of Aβ, sAPPβ, sAPPα, and C-terminal fragment. In addition, administration of E144 normalized the behavioral deficits in Tg2576 mice, an APP transgenic mouse model of AD. E144 also decreased the Aβ and APP levels in the cerebral cortex of Tg2576 mice. Thus, we propose that E144 could be a potential drug candidate for an anti-amyloid disease-modifying AD therapy.     

Oxidative lipid modification of nicastrin enhances amyloidogenic γ-secretase activity in Alzheimer's disease

The cause of elevated level of amyloid β-peptide (Aβ42) in common late-onset sporadic [Alzheimer's disease (AD)] has not been established. Here, we show that the membrane lipid peroxidation product 4-hydroxynonenal (HNE) is associated with amyloid and neurodegenerative pathologies in AD and that it enhances γ-secretase activity and Aβ42 production in neurons. The γ-secretase substrate receptor, nicastrin, was found to be modified by HNE in cultured neurons and in brain specimens from patients with AD, in which HNE-nicastrin levels were found to be correlated with increased γ-secretase activity and Aβ plaque burden. Furthermore, HNE modification of nicastrin enhanced its binding to the γ-secretase substrate, amyloid precursor protein (APP) C99. In addition, the stimulation of γ-secretase activity and Aβ42 production by HNE were blocked by an HNE-scavenging histidine analog in a 3xTgAD mouse model of AD. These findings suggest a specific molecular mechanism by which oxidative stress increases Aβ42 production in AD and identify HNE as a novel therapeutic target upstream of the γ-secretase cleavage of APP.     

An alternative spliced mouse presenilin-2 mRNA encodes a novel γ-secretase inhibitor

The γ-secretase, composed of presenilin-1 (PS1) or presenilin-2 (PS2), nicastrin (NCT), anterior pharynx-defective phenotype 1 (APH-1), and PEN-2, is critical for the development of Alzheimer’s disease (AD). PSs are autoproteolytically cleaved, producing an N-terminal fragment (NTF) and a hydrophilic loop domain-containing C-terminal fragment. However, the role of the loop domain in the γ-secretase complex assembly remains unknown. Here, we report a novel PS2 isoform generated by alternative splicing, named PS2β, which is composed of an NTF with a hydrophilic loop domain. PS2β disturbed the interaction between NCT and APH-1, resulting in the inhibition of amyloid-β production. We concluded that PS2β may inhibit γ-secretase activity by affecting the γ-secretase complex assembly.

Structured summary

MINT-7025654: APH1 (uniprotkb:Q96BI3) physically interacts (MI:0218) with PEN2 (uniprotkb:Q9NZ42), PS2 beta (uniprotkb:Q61144-2) and PS1 (uniprotkb:P49769) by anti tag coimmunoprecipitation (MI:0007)MINT-7025631: APH1 (uniprotkb:Q96BI3) physically interacts (MI:0218) with NCT (uniprotkb:Q92542), PEN2 (uniprotkb:Q9NZ42) and PS1 (uniprotkb:P49769) by anti tag coimmunoprecipitation (MI:0007)     

S-Palmitoylation of ��-Secretase Subunits Nicastrin and APH-1

Proteolytic processing of amyloid precursor protein (APP) by β- and γ-secretases generates β-amyloid (Aβ) peptides, which accumulate in the brains of individuals affected by Alzheimer disease. Detergent-resistant membrane microdomains (DRM) rich in cholesterol and sphingolipid, termed lipid rafts, have been implicated in Aβ production. Previously, we and others reported that the four integral subunits of the γ-secretase associate with DRM. In this study we investigated the mechanisms underlying DRM association of γ-secretase subunits. We report that in cultured cells and in brain the γ-secretase subunits nicastrin and APH-1 undergo S-palmitoylation, the post-translational covalent attachment of the long chain fatty acid palmitate common in lipid raft-associated proteins. By mutagenesis we show that nicastrin is S-palmitoylated at Cys⁶⁸⁹, and APH-1 is S-palmitoylated at Cys¹⁸² and Cys²⁴⁵. S-Palmitoylation-defective nicastrin and APH-1 form stable γ-secretase complexes when expressed in knock-out fibroblasts lacking wild type subunits, suggesting that S-palmitoylation is not essential for γ-secretase assembly. Nevertheless, fractionation studies show that S-palmitoylation contributes to DRM association of nicastrin and APH-1. Moreover, pulse-chase analyses reveal that S-palmitoylation is important for nascent polypeptide stability of both proteins. Co-expression of S-palmitoylation-deficient nicastrin and APH-1 in cultured cells neither affects Aβ40, Aβ42, and AICD production, nor intramembrane processing of Notch and N-cadherin. Our findings suggest that S-palmitoylation plays a role in stability and raft localization of nicastrin and APH-1, but does not directly modulate γ-secretase processing of APP and other substrates.Alzheimer disease is the most common among neurodegenerative diseases that cause dementia. This debilitating disorder is pathologically characterized by the cerebral deposition of 39–42 amino acid peptides termed Aβ, which are generated by proteolytic processing of amyloid precursor protein (APP)² by β- and γ-secretases (1, 2). The β-site APP cleavage enzyme 1 cleaves full-length APP within its luminal domain to generate a secreted ectodomain leaving behind a C-terminal fragment (β-CTF). γ-Secretase cleaves β-CTF within the transmembrane domain to release Aβ and APP intracellular C-terminal domain (AICD). γ-Secretase is a multiprotein complex, comprising at least four subunits: presenilins (PS1 and PS2), nicastrin, APH-1, and PEN-2 for its activity (3). PS1 is synthesized as a 42–43-kDa polypeptide and undergoes highly regulated endoproteolytic processing within the large cytoplasmic loop domain connecting putative transmembrane segments 6 and 7 to generate stable N-terminal (NTF) and C-terminal fragments (CTF) by an uncharacterized proteolytic activity (4). This endoproteolytic event has been identified as the activation step in the process of PS1 maturation as it assembles with other γ-secretase subunits (3). Nicastrin is a heavily glycosylated type I membrane protein with a large ectodomain that has been proposed to function in substrate recognition and binding (5), but this putative function has not been confirmed by others (6). APH-1 is a seven-transmembrane protein encoded by two human or three rodent genes that are alternatively spliced (7). Although PS1 (or PS2), nicastrin, APH-1, and PEN-2 are sufficient for γ-secretase processing of APP, a type I membrane protein, termed p23 (also referred toTMP21), was recently identified as a γ-secretase component that modulates γ-secretase activity and regulates secretory trafficking of APP (8, 9).A growing number of type I integral membrane proteins has been identified as γ-secretase substrates within the last few years, including Notch1 homologues, Notch ligands, Delta and Jagged, cell adhesion receptors N- and E-cadherins, low density lipoprotein receptor-related protein, ErbB-4, netrin receptor DCC, and others (10). Mounting evidence suggests that APP processing occurs within cholesterol- and sphingolipid-enriched lipid rafts, which are biochemically defined as detergentresistant membrane microdomains (DRM) (11, 12). Previously we reported that each of the γ-secretase subunits localizes in lipid rafts in post-Golgi and endosome membranes enriched in syntaxin 6 (13). Moreover, loss of γ-secretase activity by gene deletion or exposure to γ-secretase inhibitors results in the accumulation of APP CTFs in lipid rafts indicating that cleavage of APP CTFs likely occurs in raft microdomains (14). In contrast, CTFs derived from Notch1, Jagged2, N-cadherin, and DCC are processed by γ-secretase in non-raft membranes (14). The mechanisms underlying association of γ-secretase subunits with lipid rafts need further clarification to elucidate spatial segregation of amyloidogenic processing of APP in membrane microdomains.Post-translational S-palmitoylation is increasingly recognized as a potential mechanism for regulating raft association, stability, intracellular trafficking, and function of several cytosolic and transmembrane proteins (15–17). S-palmitoylation refers to the addition of 16-carbon palmitoyl moiety to certain cysteine residues through thioester linkage. Cysteines close to transmembrane domains or membrane-associated domains in non-integral membrane proteins are preferred S-palmitoylation sites, although no conserved motif has been identified (18). Palmitoylation modifies numerous neuronal proteins, including postsynaptic density protein PSD-95 (19), a-amino-3-hydroxyl-5-methyl-4-isoxazole propionic acid receptors (20), nicotinic α7 receptors (21), neuronal t-SNAREs SNAP-25, synaptobrevin 2 and synaptogagmin (22, 23), neuronal growth-associated protein GAP-43 (24), protein kinase CLICK-III (CL3)/CaMKIγ (25), β-secretase (26), and Huntingtin (27). Although palmitoylation can occur in vitro without the involvement of an enzyme, a family of palmitoyltransferases that specifically catalyze S-palmitoylation has been identified (28, 29).In this study, we have identified S-palmitoylation of γ-secretase subunits nicastrin and APH-1, and characterized its role on DRM association, protein stability, and γ-secretase enzyme activities. We show that nicastrin is S-palmitoylated at Cys⁶⁸⁹, and APH-1 at Cys¹⁸² and Cys²⁴⁵. Mutagenesis of palmitoylation sites results in increased degradation of nascent nicastrin and APH-1 polypeptides and reduced association with DRM. Nevertheless, in cultured cells overexpression of S-palmitoylation-deficient nicastrin and APH-1 does not modulate γ-secretase processing of APP or other substrates.     

Identification of presenilin 1-selective γ-secretase inhibitors with reconstituted γ-secretase complexes

Accumulation of the β-amyloid (Aβ) peptides is one of the major pathologic hallmarks in the brains of Alzheimer's disease (AD) patients. Aβ is generated by sequential proteolytic cleavage of the amyloid precursor protein (APP) catalyzed by β- and γ-secretases. Inhibition of Aβ production by γ-secretase inhibitors (GSIs) is thus being pursued as a target for treatment of AD. In addition to processing APP, γ-secretase also catalyzes proteolytic cleavage of other transmembrane substrates, with the best characterized one being the cell surface receptor Notch. GSIs reduce Aβ production in animals and humans but also cause significant side effects because of the inhibition of Notch processing. The development of GSIs that reduce Aβ production and have less Notch-mediated side effect liability is therefore an important goal. γ-Secretase is a large membrane protein complex with four components, two of which have multiple isoforms: presenilin (PS1 or PS2), aph-1 (aph-1a or aph-1b), nicastrin, and pen-2. Here we describe the reconstitution of four γ-secretase complexes in Sf9 cells containing PS1--aph-1a, PS1--aph-1b, PS2--aph-1a, and PS2--aph-1b complexes. While PS1--aph-1a, PS1--aph-1b, and PS2--aph-1a complexes displayed robust γ-secretase activity, the reconstituted PS2--aph-1b complex was devoid of detectable γ-secretase activity. γ-Secretase complexes containing PS1 produced a higher proportion of the toxic species Aβ42 than γ-secretase complexes containing PS2. Using the reconstitution system, we identified MRK-560 and SCH 1500022 as highly selective inhibitors of PS1 γ-secretase activity. These findings may provide important insights into developing a new generation of γ-secretase inhibitors with improved side effect profiles.     

Structural optimization of a CXCR2-directed antagonist that indirectly inhibits γ-secretase and reduces Aβ

Amyloid β (Aβ), a key molecule in the pathogenesis of Alzheimer’s disease (AD), is derived from the amyloid precursor protein (APP) by sequential proteolysis via β- and γ-secretases. Because of their role in generation of Aβ, these enzymes have emerged as important therapeutic targets for AD. In the case of γ-secretase, progress has been made towards designing potent inhibitors with suitable pharmacological profiles. Direct γ-secretase inhibitors are being evaluated in clinical trials and new strategies are being explored to block γ-secretase activity indirectly as well. In this regard, we have previously reported an indirect regulation of γ-secretase through antagonism of CXCR2, a G-protein coupled receptor (GPCR). We demonstrated that N-(2-hydroxy-4-nitrophenyl)-N′-(2-bromophenyl)urea (SB225002), a selective inhibitor of CXCR2 also plays a role in an indirect inhibition of γ-secretase. Furthermore, we reported a ～5-fold difference in the selective inhibition of APP versus Notch processing via γ-secretase following treatment with SB225002. Herein we describe the synthesis and optimization of SB225002. By determination of the structure–activity relationship (SAR), we derived small molecules that inhibit Aβ40 production with IC₅₀ values in the sub-micromolar range in a cell-based assay and also validated the potential of CXCR2 as a new target for therapeutic intervention in AD.     

DC2 and Keratinocyte-associated Protein 2 (KCP2), Subunits of the Oligosaccharyltransferase Complex,Are Regulators of the γ-Secretase-directed Processing of Amyloid Precursor Protein (APP)

The oligosaccharyltransferase complex catalyzes the transfer of oligosaccharide from a dolichol pyrophosphate donor en bloc onto a free asparagine residue of a newly synthesized nascent chain during the translocation in the endoplasmic reticulum lumen. The role of the less known oligosaccharyltransferase (OST) subunits, DC2 and KCP2, recently identified still remains to be determined. Here, we have studied DC2 and KCP2, and we have established that DC2 and KCP2 are substrate-specific, affecting amyloid precursor protein (APP), indicating that they are not core components required for N-glycosylation and OST activity per se. We show for the first time that DC2 and KCP2 depletion affects APP processing, leading to an accumulation of C-terminal fragments, both C99 and C83, and a reduction in full-length mature APP. This reduction in mature APP levels was not due to a block in secretion because the levels of sAPPα secreted into the media were unaffected. We discover that DC2 and KCP2 depletion affects only the γ-secretase complex, resulting in a reduction of the PS1 active fragment blocking Aβ production. Conversely, we show that the overexpression of DC2 and KCP2 causes an increase in the active γ-secretase complex, particularly the N-terminal fragment of PS1 that is generated by endoproteolysis, leading to a stimulation of Aβ production upon overexpression of DC2 and KCP2. Our findings reveal that components of the OST complex for the first time can interact with the γ-secretase and affect the APP processing pathway.     

A TAG on to the neurogenic functions of APP

The proteolytic processing of amyloid β precursor protein (APP) has long been studied because of its association with the pathology of Alzheimer's disease (AD). The ectodomain of APP is shed by α- or β-secretase cleavage. The remaining membrane bound stub can then undergo regulated intramembrane proteolysis (RIP) by γ-secretase. This cleavage can release amyloid β (Aβ) from the stub left by β-secretase cleavage but also releases the APP intracellular domain (AICD) after α- or β-secretase cleavage. The physiological functions of this proteolytic processing are not well understood. We compare the proteolytic processing of APP to the ligand-dependent RIP of Notch. In this review, we discuss recent evidence suggesting that TAG1 is a functional ligand for APP. The interaction between TAG1 and APP triggers γ-secretase-dependent release of AICD. TAG1, APP and Fe65 colocalise in the neurogenic ventricular zone and in fetal neural progenitor cells in vitro. Experiments in TAG1, APP and Fe65 null mice as well as TAG1 and APP double-null mice demonstrate that TAG1 induces a γ-secretase- and Fe65-dependent suppression of neurogenesis.     

CD40/CD40L interaction induces Aβ production and increases γ-secretase activity independently of tumor necrosis factor receptor associated factor (TRAF) signaling

CD40, a member of tumor necrosis factor receptor superfamily, and its cognate ligand CD40L are both elevated in the brain of Alzheimer's disease (AD) patients compared to controls. We have shown that pharmacological or genetic interruption of CD40/CD40L interaction results in mitigation of AD-like pathology in vivo in transgenic AD mouse models, and in vitro. Recently, we showed that CD40L stimulation could increase Aβ levels via NFκB signaling, presumably through TRAFs. In the present work, using CD40 mutants, we show that CD40L can increase levels of Aβ(1-40), Aβ(1-42), sAPPβ, sAPPα and CTFβ independently of TRAF signaling. We report an increase in mature/immature APP ratio after CD40L treatment of CD40wt and CD40-mutant cells, reflecting alterations in APP trafficking. In addition, results from CD40L treatment of a neuroblastoma cell line over-expressing the C-99 APP fragment suggest that CD40L has an effect on γ-secretase. Furthermore, inhibition of γ-secretase activity significantly reduces sAPPβ levels in the CD40L treated HEK/APPsw CD40wt and the CD40-mutant cells. The latter suggests CD40/CD40L interaction primarily acts on γ-secretase and affects β-secretase via a positive feedback mechanism. Taken together, our data suggest that CD40/CD40L interaction modulates APP processing independently of TRAF signaling.     

Inhibiting β-secretase activity in Alzheimer's disease cell models with single-chain antibodies specifically targeting APP

The Amyloid-β (Aβ) peptide is produced from the amyloid precursor protein (APP) by sequential proteolytic cleavage of APP first by β-secretase and then by γ-secretase. β-Site APP cleaving enzyme-1 (BACE-1) is the predominant enzyme involved in β-secretase processing of APP and is a primary therapeutic target for treatment of Alzheimer's disease. While inhibiting BACE-1 activity has obvious therapeutic advantages, BACE-1 also cleaves numerous other substrates with important physiological activity. Thus, blanket inhibition of BACE-1 function may have adverse side effects. We isolated a single chain variable fragment (scFv) from a human-based scFv yeast display library that selectively inhibits BACE-1 activity toward APP by binding the APP substrate at the proteolytic site. We selected the iBSEC1 scFv, since it recognizes the BACE-1 cleavage site on APP but does not bind the adjacent highly antigenic N-terminal of Aβ, and thus it will target APP but not soluble Aβ. When added to 7PA2 cells, a mammalian cell line that overexpresses APP, the iBSEC1 scFv binds APP on the cell surface, reduces toxicity induced by APP overexpression, and reduces both intracellular and extracellular Aβ levels by around 50%. Since the iBSEC1 scFv does not contain the antibody F_c region, this construct does not pose the risk of exacerbating inflammation in the brain as faced with full-length monoclonal antibodies for potential therapeutic applications.