Regulation of Amyloid Precursor Protein Processing by Serotonin Signaling

Proteolytic processing of the amyloid precursor protein (APP) by the β- and γ-secretases releases the amyloid-β peptide (Aβ), which deposits in senile plaques and contributes to the etiology of Alzheimer''s disease (AD). The α-secretase cleaves APP in the Aβ peptide sequence to generate soluble APPα (sAPPα). Upregulation of α-secretase activity through the 5-hydroxytryptamine 4 (5-HT₄) receptor has been shown to reduce Aβ production, amyloid plaque load and to improve cognitive impairment in transgenic mouse models of AD. Consequently, activation of 5-HT₄ receptors following agonist stimulation is considered to be a therapeutic strategy for AD treatment; however, the signaling cascade involved in 5-HT₄ receptor-stimulated proteolysis of APP remains to be determined. Here we used chemical and siRNA inhibition to identify the proteins which mediate 5-HT_4d receptor-stimulated α-secretase activity in the SH-SY5Y human neuronal cell line. We show that G protein and Src dependent activation of phospholipase C are required for α-secretase activity, while, unexpectedly, adenylyl cyclase and cAMP are not involved. Further elucidation of the signaling pathway indicates that inositol triphosphate phosphorylation and casein kinase 2 activation is also a prerequisite for α-secretase activity. Our findings provide a novel route to explore the treatment of AD through 5-HT₄ receptor-induced α-secretase activation.     

Mutations in Nicastrin Protein Differentially Affect Amyloid β-Peptide Production and Notch Protein Processing

The γ-secretase complex is responsible for intramembrane processing of over 60 substrates and is involved in Notch signaling as well as in the generation of the amyloid β-peptide (Aβ). Aggregated forms of Aβ have a pathogenic role in Alzheimer disease and, thus, reducing the Aβ levels by inhibiting γ-secretase is a possible treatment strategy for Alzheimer disease. Regrettably, clinical trials have shown that inhibition of γ-secretase results in Notch-related side effects. Therefore, it is of great importance to find ways to inhibit amyloid precursor protein (APP) processing without disturbing vital signaling pathways such as Notch. Nicastrin (Nct) is part of the γ-secretase complex and has been proposed to be involved in substrate recognition and selection. We have investigated how the four evenly spaced and conserved cysteine residues in the Nct ectodomain affect APP and Notch processing. We mutated these cysteines to serines and analyzed them in cells lacking endogenous Nct. We found that two mutants, C213S (C2) and C230S (C3), differentially affected APP and Notch processing. Both the formation of Aβ and the intracellular domain of amyloid precursor protein (AICD) were reduced, whereas the production of Notch intracellular domain (NICD) was maintained on a high level, although C230S (C3) showed impaired complex assembly. Our data demonstrate that single residues in a γ-secretase component besides presenilin are able to differentially affect APP and Notch processing.     

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.Key words: Amyloid β precursor protein, APP, TAG1, AICD, Fe65, neurogenesis, Alzheimer''s disease     

Inhibition of Amyloid Precursor Protein Processing Enhances Gemcitabine-mediated Cytotoxicity in Pancreatic Cancer Cells

Pancreatic adenocarcinoma or pancreatic cancer is often diagnosed at a very late stage at which point treatment options are minimal. Current chemotherapeutic interventions prolong survival marginally, thereby emphasizing the acute need for better treatment options to effectively manage this disease. Studies from different laboratories have shown that the Alzheimer disease-associated amyloid precursor protein (APP) is overexpressed in various cancers but its significance is not known. Here we sought to determine the role of APP in pancreatic cancer cell survival and proliferation. Our results show that pancreatic cancer cells secrete high levels of sAPPα, the α-secretase cleaved ectodomain fragment of APP, as compared with normal non-cancerous cells. Treatment of cells with batimastat or GI254023X, inhibitors of the α-secretase ADAM10, prevented sAPPα generation and reduced cell survival. Additionally, inhibition of sAPPα significantly reduced anchorage independent growth of the cancer cells. The effect of batimastat on cell survival and colony formation was enhanced when sAPPα downregulation was combined with gemcitabine treatment. Moreover, treatment of batimastat-treated cells with recombinant sAPPα reversed the inhibitory effect of the drug thereby indicating that sAPPα can indeed induce proliferation of cancer cells. Down-regulation of APP and ADAM10 brought about similar results, as did batimastat treatment, thereby confirming that APP processing is important for growth and proliferation of these cells. These results suggest that inhibition of sAPPα generation might enhance the effectiveness of the existing chemotherapeutic regimen for a better outcome.     

Octyl Gallate Markedly Promotes Anti-Amyloidogenic Processing of APP through Estrogen Receptor-Mediated ADAM10 Activation

Our previous studies showed that the green tea-derived polyphenolic compound (−)-epigallocatechin-3 gallate (EGCG) reduces amyloid-β (Aβ) production in both neuronal and mouse Alzheimer’s disease (AD) models in concert with activation of estrogen receptor-α/phosphatidylinositide 3-kinase/protein kinase B (ERα/PI3K/Akt) signaling and anti-amyloidogenic amyloid precursor protein (APP) α-secretase (a disintegrin and metallopeptidase domain-10, ADAM10) processing. Since the gallate moiety in EGCG may correspond to the 7α position of estrogen, thereby facilitating ER binding, we extensively screened the effect of other gallate containing phenolic compounds on APP anti-amyloidogenic processing. Octyl gallate (OG; 10 µM), drastically decreased Aβ generation, in concert with increased APP α-proteolysis, in murine neuron-like cells transfected with human wild-type APP or “Swedish” mutant APP. OG markedly increased production of the neuroprotective amino-terminal APP cleavage product, soluble APP-α (sAPPα). In accord with our previous study, these cleavage events were associated with increased ADAM10 maturation and reduced by blockade of ERα/PI3k/Akt signaling. To validate these findings in vivo, we treated Aβ-overproducing Tg2576 mice with OG daily for one week by intracerebroventricular injection and found decreased Aβ levels associated with increased sAPPα. These data indicate that OG increases anti-amyloidogenic APP α-secretase processing by activation of ERα/PI3k/Akt signaling and ADAM10, suggesting that this compound may be an effective treatment for AD.     

γ-Secretase Modulators and APH1 Isoforms Modulate γ-Secretase Cleavage but Not Position of ε-Cleavage of the Amyloid Precursor Protein (APP)

The relative increase in Aβ42 peptides from familial Alzheimer disease (FAD) linked APP and PSEN mutations can be related to changes in both ε-cleavage site utilization and subsequent step-wise cleavage. Cleavage at the ε-site releases the amyloid precursor protein (APP) intracellular domain (AICD), and perturbations in the position of ε-cleavage are closely associated with changes in the profile of amyloid β-protein (Aβ) species that are produced and secreted. The mechanisms by which γ-secretase modulators (GSMs) or FAD mutations affect the various γ-secretase cleavages to alter the generation of Aβ peptides have not been fully elucidated. Recent studies suggested that GSMs do not modulate ε-cleavage of APP, but the data were derived principally from recombinant truncated epitope tagged APP substrate. Here, using full length APP from transfected cells, we investigated whether GSMs modify the ε-cleavage of APP under more native conditions. Our results confirmed the previous findings that ε-cleavage is insensitive to GSMs. In addition, fenofibrate, an inverse GSM (iGSM), did not alter the position or kinetics of ε-cleavage position in vitro. APH1A and APH1B, a subunit of the γ-secretase complex, also modulated Aβ42/Aβ40 ratio without any alterations in ε-cleavage, a result in contrast to what has been observed with PS1 and APP FAD mutations. Consequently, GSMs and APH1 appear to modulate γ-secretase activity and Aβ42 generation by altering processivity but not ε-cleavage site utilization.     

New Insights into Epigenetic and Pharmacological Regulation of Amyloid-Degrading Enzymes

Currently, deficit of amyloid β-peptide (Aβ) clearance from the brain is considered as one of the possible causes of amyloid accumulation and neuronal death in the sporadic form of Alzheimer’s disease (AD). Aβ clearance can involve either specific proteases present in the brain or Aβ-binding/transport proteins. Among amyloid-degrading enzymes the most intensively studied are neprilysin (NEP) and insulin-degrading enzyme (IDE). Since ageing and development of brain pathologies is often accompanied by a deficit in the levels of expression and activity of these enzymes in the brain, there is an urgent need to understand the mechanisms involved in their regulation. We have recently reported that NEP and also an Aβ-transport protein, transthyretin are epigenetically co-regulated by the APP intracellular domain (AICD) and this regulation depends on the cell type and APP₆₉₅ isoform expression in a process that can be regulated by the tyrosine kinase inhibitor, Gleevec. We have now extended our work and shown that, unlike NEP, another amyloid-degrading enzyme, IDE, is not related to over-expression of APP₆₉₅ in neuroblastoma SH-SY5Y cells but is up-regulated by APP₇₅₁ and APP₇₇₀ isoforms independently of AICD but correlating with reduced HDAC1 binding to its promoter. Studying the effect of the nuclear retinoid X receptor agonist, bexarotene, on NEP and IDE expression, we have found that both enzymes can be up-regulated by this compound but this mechanism is not APP-isoform specific and does not involve AICD but, on the contrary, affects HDAC1 occupancy on the NEP gene promoter. These new insights into the mechanisms of NEP and IDE regulation suggest possible pharmacological targets in developing AD therapies.     

Novel β-Secretase Cleavage of β-Amyloid Precursor Protein in the Endoplasmic Reticulum/Intermediate Compartment of NT2N Cells

Previous studies have demonstrated that NT2N neurons derived from a human embryonal carcinoma cell line (NT2) constitutively process the endogenous wild-type β-amyloid precursor protein (APP) to amyloid β peptide in an intracellular compartment. These studies indicate that other proteolytic fragments generated by intracellular processing must also be present in these cells. Here we show that the NH₂-terminal fragment of APP generated by β-secretase cleavage (APPβ) is indeed produced from the endogenous full length APP (APP_FL). Pulse–chase studies demonstrated a precursor–product relationship between APP_FL and APPβ as well as intracellular and secreted APPβ fragments. In addition, trypsin digestion of intact NT2N cells at 4°C did not abolish APPβ recovered from the cell lysates. Furthermore, the production of intracellular APPβ from wild-type APP appears to be a unique characteristic of postmitotic neurons, since intracellular APPβ was not detected in several non-neuronal cell lines. Significantly, production of APPβ occurred even when APP was retained in the ER/ intermediate compartment by inhibition with brefeldin A, incubation at 15°C, or by expression of exogenous APP bearing the dilysine ER retrieval motif.     

Genetic Dissection of the Amyloid Precursor Protein in Developmental Function and Amyloid Pathogenesis

Proteolytic processing of the amyloid precursor protein (APP) generates large soluble APP derivatives, β-amyloid (Aβ) peptides, and APP intracellular domain. Expression of the extracellular sequences of APP or its Caenorhabditis elegans counterpart has been shown to be sufficient in partially rescuing the CNS phenotypes of the APP-deficient mice and the lethality of the apl-1 null C. elegans, respectively, leaving open the question as what is the role of the highly conserved APP intracellular domain? To address this question, we created an APP knock-in allele in which the mouse Aβ sequence was replaced by the human Aβ. A frameshift mutation was introduced that replaced the last 39 residues of the APP sequence. We demonstrate that the C-terminal mutation does not overtly affect APP processing and amyloid pathology. In contrast, crossing the mutant allele with APP-like protein 2 (APLP2)-null mice results in similar neuromuscular synapse defects and early postnatal lethality as compared with mice doubly deficient in APP and APLP2, demonstrating an indispensable role of the APP C-terminal domain in these development activities. Our results establish an essential function of the conserved APP intracellular domain in developmental regulation, and this activity can be genetically uncoupled from APP processing and Aβ pathogenesis.     

Regulation of amyloid-β production by the prion protein

Alzheimer disease (AD) is characterized by the amyloidogenic processing of the amyloid precursor protein (APP), culminating in the accumulation of amyloid-β peptides in the brain. The enzymatic action of the β-secretase, BACE1 is the rate-limiting step in this amyloidogenic processing of APP. BACE1 cleavage of wild-type APP (APP_WT) is inhibited by the cellular prion protein (PrP^C). Our recent study has revealed the molecular and cellular mechanisms behind this observation by showing that PrP^C directly interacts with the pro-domain of BACE1 in the trans-Golgi network (TGN), decreasing the amount of BACE1 at the cell surface and in endosomes where it cleaves APP_WT, while increasing BACE1 in the TGN where it preferentially cleaves APP with the Swedish mutation (APP_Swe). PrP^C deletion in transgenic mice expressing the Swedish and Indiana familial mutations (APP_Swe,Ind) failed to affect amyloid-β accumulation, which is explained by the differential subcellular sites of action of BACE1 toward APP_WT and APP_Swe. This, together with our observation that PrP^C is reduced in sporadic but not familial AD brain, suggests that PrP^C plays a key protective role against sporadic AD. It also highlights the need for an APP_WT transgenic mouse model to understand the molecular and cellular mechanisms underlying sporadic AD.     

Retention in Endoplasmic Reticulum 1 (RER1) Modulates Amyloid-β (Aβ) Production by Altering Trafficking of γ-Secretase and Amyloid Precursor Protein (APP)

The presence of neuritic plaques containing aggregated amyloid-β (Aβ) peptides in the brain parenchyma is a pathological hallmark of Alzheimer disease (AD). Aβ is generated by sequential cleavage of the amyloid β precursor protein (APP) by β- and γ-secretase, respectively. As APP processing to Aβ requires transport through the secretory pathway, trafficking of the substrate and access to the secretases are key factors that can influence Aβ production (Thinakaran, G., and Koo, E. H. (2008) Amyloid precursor protein trafficking, processing, and function. J. Biol. Chem. 283, 29615–29619). Here, we report that retention in endoplasmic reticulum 1 (RER1) associates with γ-secretase in early secretory compartments and regulates the intracellular trafficking of γ-secretase. RER1 overexpression decreases both γ-secretase localization on the cell surface and Aβ secretion and conversely RER1 knockdown increases the level of cell surface γ-secretase and increases Aβ secretion. Furthermore, we find that increased RER1 levels decrease mature APP and increase immature APP, resulting in less surface accumulation of APP. These data show that RER1 influences the trafficking and localization of both γ-secretase and APP, thereby regulating the production and secretion of Aβ peptides.     

Specific antibody binding to the APP672–699 region shifts APP processing from α- to β-cleavage

Alzheimer''s disease (AD), a progressive neurodegenerative disorder that is the most common cause of dementia in the elderly, is characterized by the accumulation of amyloid-β (Aβ) plaques and neurofibrillary tangles, as well as a progressive loss of synapses and neurons in the brain. The major pertinacious component of amyloid plaques is Aβ, a variably sized peptide derived from the integral membrane protein amyloid precursor protein (APP). The Aβ region of APP locates partly within its ecto- and trans-membrane domains. APP is cleaved by three proteases, designated as α-, β-, and γ-secretases. Processing by β- and γ-secretase cleaves the N- and C-terminal ends of the Aβ region, respectively, releasing Aβ, whereas α-secretase cleaves within the Aβ sequence, releasing soluble APPα (sAPPα). The γ-secretase cleaves at several adjacent sites to yield Aβ species containing 39–43 amino acid residues. Both α- and β-cleavage sites of human wild-type APP are located in APP_672–699 region (ectodomain of β-C-terminal fragment, ED-β-CTF or ED-C99). Therefore, the amino acid residues within or near this region are definitely pivotal for human wild-type APP function and processing. Here, we report that one ED-C99-specific monoclonal antibody (mAb_ED-C99) blocks human wild-type APP endocytosis and shifts its processing from α- to β-cleavage, as evidenced by elevated accumulation of cell surface full-length APP and β-CTF together with reduced sAPPα and α-CTF levels. Moreover, mAb_ED-C99 enhances the interactions of APP with cholesterol. Consistently, intracerebroventricular injection of mAb_ED-C99 to human wild-type APP transgenic mice markedly increases membrane-associated β-CTF. All these findings suggest that APP_672–699 region is critical for human wild-type APP processing and may provide new clues for the pathogenesis of sporadic AD.Abnormal functioning and/or processing of amyloid precursor protein (APP), a type I membrane protein, has a pivotal role in the pathogenesis of Alzheimer''s disease (AD).^{1, 2, 3} APP is cleaved by three proteases, designated as α-, β-, and γ-secretases (Supplementary Figure S1). The major fraction (>90%) of wild-type APP is proteolyzed by α-secretase that cleaves wild-type APP between residues APP₆₈₇ and APP₆₈₈ within the amyloid-β (Aβ) sequence, releasing soluble APPα (sAPPα) and α-C-terminal fragment (α-CTF, C83). Only a minority (<10%) of all wild-type APP molecules undergo β-cleavage at the β-cleavage site (between residues APP₆₇₁ and APP₆₇₂) generating sAPPβ and β-CTF (C99), the latter of which is subsequently processed by γ-secretase complex to generate a mixture of Aβ peptides primarily 40 or 42 residues in length (Aβ_1-40/42).^{4, 5} The β-secretase cleaves APP in addition at a β′-site (between residues APP₆₈₁ and APP₆₈₂) to generate C89 that is further processed by γ-secretase to produce truncated Aβ_11–40/42 species.⁶Both α- and β-cleavage sites of wild-type APP are located in APP_672–699 region (the ectodomain of β-CTF, ED-β-CTF, or ED-C99; Supplementary Figure S1). Therefore, the amino acid residues within or near this region are definitely pivotal for wild-type APP function and processing. Previous studies have identified that mutation in ED-C99 region can affect the physiological processing of APP and contribute to pathological features of familial AD (fAD). For example, Swedish APP carrying APP_670/671 mutation (KM→NL) is cleaved by β-secretase over 50-fold more efficiently than wild-type APP.⁷ APP₆₇₃ mutation (A→V) and APP₆₉₃ mutation (E→G) can enhance Aβ production and accelerate formation of amyloid fibrils.^{8, 9, 10} APP₆₈₂ mutation (E→K) blocks APP β′-site and shifts cleavage to β-site, thus increasing Aβ_1–40/42 production.⁶ Although sporadic AD (sAD), the more common type of AD comprising 90 to 95% of all AD cases, lacks mutations in the APP gene, region-specific protein modifications within the ED-C99 region may affect wild-type APP processing similarly to APP gene mutations. For example, phosphorylation of ED-C99 at the threonine 687 (of APP₇₇₀ isoform, or corresponding threonine 668 of APP₇₅₁ isoform; Supplementary Figure S1) facilitates APP processing by γ-secretase.¹¹ Therefore, the elucidation of potential influences of region-specific modifications, induced by either endogenous or exogenous molecules, on wild-type APP processing would be especially critical for clarifying the mechanisms underlying the pathogenesis of sAD.To confirm this hypothesis, we used one mouse monoclonal antibody specifically recognizing ED-C99 (mAb_ED-C99) with its epitope at APP_674–679 (Supplementary Figure S1). The influences of mAb_ED-C99 binding on human wild-type APP processing were evaluated in vitro using Chinese hamster ovary cells expressing human wild-type APP (CHO/APP_wt cells) and cortical neurons derived from human wild-type APP transgenic (TgAPP_wt) mice. The in vitro effects of ED-C99 binding with mAb_ED-C99 on wild-type APP processing were further evaluated and confirmed in vivo using TgAPP_wt mice and 5 × FAD transgenic mice (Tg6799 line).     

LRRTM3 Interacts with APP and BACE1 and Has Variants Associating with Late-Onset Alzheimer’s Disease (LOAD)

Leucine rich repeat transmembrane protein 3 (LRRTM3) is member of a synaptic protein family. LRRTM3 is a nested gene within α-T catenin (CTNNA3) and resides at the linkage peak for late-onset Alzheimer’s disease (LOAD) risk and plasma amyloid β (Aβ) levels. In-vitro knock-down of LRRTM3 was previously shown to decrease secreted Aβ, although the mechanism of this is unclear. In SH-SY5Y cells overexpressing APP and transiently transfected with LRRTM3 alone or with BACE1, we showed that LRRTM3 co-localizes with both APP and BACE1 in early endosomes, where BACE1 processing of APP occurs. Additionally, LRRTM3 co-localizes with APP in primary neuronal cultures from Tg2576 mice transduced with LRRTM3-expressing adeno-associated virus. Moreover, LRRTM3 co-immunoprecipitates with both endogenous APP and overexpressed BACE1, in HEK293T cells transfected with LRRTM3. SH-SY5Y cells with knock-down of LRRTM3 had lower BACE1 and higher CTNNA3 mRNA levels, but no change in APP. Brain mRNA levels of LRRTM3 showed significant correlations with BACE1, CTNNA3 and APP in ∼400 humans, but not in LRRTM3 knock-out mice. Finally, we assessed 69 single nucleotide polymorphisms (SNPs) within and flanking LRRTM3 in 1,567 LOADs and 2,082 controls and identified 8 SNPs within a linkage disequilibrium block encompassing 5′UTR-Intron 1 of LRRTM3 that formed multilocus genotypes (MLG) with suggestive global association with LOAD risk (p = 0.06), and significant individual MLGs. These 8 SNPs were genotyped in an independent series (1,258 LOADs and 718 controls) and had significant global and individual MLG associations in the combined dataset (p = 0.02–0.05). Collectively, these results suggest that protein interactions between LRRTM3, APP and BACE1, as well as complex associations between mRNA levels of LRRTM3, CTNNA3, APP and BACE1 in humans might influence APP metabolism and ultimately risk of AD.     

High Fat Diet Enhances β-Site Cleavage of Amyloid Precursor Protein (APP) via Promoting β-Site APP Cleaving Enzyme 1/Adaptor Protein 2/Clathrin Complex Formation

Obesity and type 2 diabetes are risk factors of Alzheimer’s disease (AD). We reported that a high fat diet (HFD) promotes amyloid precursor protein (APP) cleavage by β-site APP cleaving enzyme 1 (BACE1) without increasing BACE1 levels in APP transgenic mice. However, the detailed mechanism had remained unclear. Here we demonstrate that HFD promotes BACE1/Adaptor protein-2 (AP-2)/clathrin complex formation by increasing AP-2 levels in APP transgenic mice. In Swedish APP overexpressing Chinese hamster ovary (CHO) cells as well as in SH-SY5Y cells, overexpression of AP-2 promoted the formation of BACE1/AP-2/clathrin complex, increasing the level of the soluble form of APP β (sAPPβ). On the other hand, mutant D495R BACE1, which inhibits formation of this trimeric complex, was shown to decrease the level of sAPPβ. Overexpression of AP-2 promoted the internalization of BACE1 from the cell surface, thus reducing the cell surface BACE1 level. As such, we concluded that HFD may induce the formation of the BACE1/AP-2/clathrin complex, which is followed by its transport of BACE1 from the cell surface to the intracellular compartments. These events might be associated with the enhancement of β-site cleavage of APP in APP transgenic mice. Here we present evidence that HFD, by regulation of subcellular trafficking of BACE1, promotes APP cleavage.     

The Resveratrol Trimer Miyabenol C Inhibits β-Secretase Activity and β-Amyloid Generation

Accumulation and deposition of amyloid-β peptide (Aβ) in the brain is a primary cause of the pathogenesis of Alzheimer’s disease (AD). Aβ is generated from amyloid-β precursor protein (APP) through sequential cleavages first by β-secretase and then by γ-secretase. Inhibiting β-secretase activity is believed to be one of the most promising strategies for AD treatment. In the present study, we found that a resveratrol trimer, miyabenol C, isolated from stems and leaves of the small-leaf grape (Vitisthunbergii var. taiwaniana), can markedly reduce Aβ and sAPPβ levels in both cell cultures and the brain of AD model mice. Mechanistic studies revealed that miyabenol C affects neither protein levels of APP, the two major α-secretases ADAM10 and TACE, and the γ-secretase component Presenilin 1, nor γ-secretase-mediated Notch processing and TACE activity. In contrast, although miyabenol C has no effect on altering protein levels of the β-secretase BACE1, it can inhibit both in vitro and in vivo β-secretase activity. Together, our results indicate that miyabenol C is a prominent β-secretase inhibitor and lead compound for AD drug development.     

Cholesterol-lowering drugs reduce APP processing to Aβ by inducing APP dimerization

Amyloid beta (Aβ) is a major component of amyloid plaques, which are a key pathological hallmark found in the brains of Alzheimer’s disease (AD) patients. We show that statins are effective at reducing Aβ in human neurons from nondemented control subjects, as well as subjects with familial AD and sporadic AD. Aβ is derived from amyloid precursor protein (APP) through sequential proteolytic cleavage by BACE1 and γ-secretase. While previous studies have shown that cholesterol metabolism regulates APP processing to Aβ, the mechanism is not well understood. We used iPSC-derived neurons and bimolecular fluorescence complementation assays in transfected cells to elucidate how altering cholesterol metabolism influences APP processing. Altering cholesterol metabolism using statins decreased the generation of sAPPβ and increased levels of full-length APP (flAPP), indicative of reduced processing of APP by BACE1. We further show that statins decrease flAPP interaction with BACE1 and enhance APP dimerization. Additionally, statin-induced changes in APP dimerization and APP-BACE1 are dependent on cholesterol binding to APP. Our data indicate that statins reduce Aβ production by decreasing BACE1 interaction with flAPP and suggest that this process may be regulated through competition between APP dimerization and APP cholesterol binding.