Proteolysis Of APP Research Articles

Early Animal Origin of BACE1 APP/Aβ Proteolytic Function.

Alzheimer's disease is characterized, in part, by the accumulation of β-amyloid (Aβ) in the brain. Aβ is produced via the proteolysis of APP by BACE1 and γ-secretase. Since BACE1 is the rate-limiting enzyme in the production of Aβ, and a target for therapeutics, it is of interest to know when its proteolytic function evolved and for what purpose. Here, we take a functional evolutionary approach to show that BACE1 likely evolved from a gene duplication event near the base of the animal clade and that BACE1 APP/Aβ proteolytic function evolved during early animal diversification, hundreds of millions of years before the evolution of the APP/Aβ substrate. Our examination of BACE1 APP/Aβ proteolytic function includes cnidarians, ctenophores, and choanoflagellates. The most basal BACE1 ortholog is found in cnidarians, while ctenophores, placozoa, and choanoflagellates have genes equally orthologous to BACE1 and BACE2. BACE1 from a cnidarian (Hydra) can cleave APP to release Aβ, pushing back the date of the origin of its function to near the origin of animals. We tested more divergent BACE1/2 genes from a ctenophore (Mnemiopsis) and a choanoflagellate (Monosiga), and neither has this activity. These findings indicate that the specific proteolytic function of BACE1 evolved during the very earliest diversification of animals, most likely after a gene-duplication event.

Alternative Theory of AD Pathogenesis: Membrane Delimitation of the Histone Acetyltransferase Tip60/Kat5

AbstractBackgroundFor decades, mounting genetic and physiological data have pointed to the APP proteolysis as a seminal event in AD pathogenesis. This data has been viewed as supporting the amyloid hypothesis. However, growing data from multiple disciplines disputes that amyloid is a causal mechanism of neurodegeneration. Evidence suggests that FAD mutations are loss of function, inhibiting normal proteolytic processing of APP. APP extracellular and intracellular domains exhibit a broad array of binding interactions. Tip60 (a histone acetyltransferase, also known as Kat5) is an intracellular binding partner that supports the long‐standing speculation that APP functions as a signaling receptor. Recently, a conditional knockout (cKO) of Tip60 demonstrated neurodegenerative pathology and massive reactive gliosis, supporting a role for Tip60 in neurodegeneration. Reports that Tip60 is restricted from the nucleus in AD patient brains bolsters this idea.MethodTo assess the overlap of the cKO Tip60 model and human AD pathogenesis, we compared the dysregulated gene sets (DEGs) in the cKO Tip60 mouse and late‐onset AD postmortem brains. We break DEGs from mouse and human models into AD “biological domains” (BDs), and perform GSEA of GO enrichment within each BD. We then perform downstream analyses using network reconstruction applications and by examining the enriched BDs with pseudotime measures of disease progression in LOAD patients.ResultWe find significant overlap between DEGs in LOAD patients and the Tip60 cKO in multiple BDs, including, mitochondrial metabolism, immune response, structural stabilization, synaptic function, and endolysosomal trafficking. GSEA of LOAD and cKO Tip60 further implicates similar deficits in both models. The network analysis shows a convergence of gene dysregulation at core nodes, further implicating similar processes of dysregulation may impact particularly significant genes in human and mouse models. The pseudotime analysis shows that this dysfunction is an early signature of AD, suggesting Tip60 dysregulation may trigger early‐stage homeostatic destabilization.ConclusionWe propose that aberrant APP proteolysis traps Tip60 at the membrane, preventing the nuclear localization of Tip60, and altering both positive and negative gene regulation that is necessary for neuronal and glial homeostasis, and hypothesize that this is an explanation for the genetic linkage of APP proteolytic processing and AD.

The orphan drug dichloroacetate reduces amyloid beta-peptide production whilst promoting non-amyloidogenic proteolysis of the amyloid precursor protein.

The amyloid cascade hypothesis proposes that excessive accumulation of amyloid beta-peptides is the initiating event in Alzheimer's disease. These neurotoxic peptides are generated from the amyloid precursor protein via sequential cleavage by β- and γ-secretases in the 'amyloidogenic' proteolytic pathway. Alternatively, the amyloid precursor protein can be processed via the 'non-amyloidogenic' pathway which, through the action of the α-secretase a disintegrin and metalloproteinase (ADAM) 10, both precludes amyloid beta-peptide formation and has the additional benefit of generating a neuroprotective soluble amyloid precursor protein fragment, sAPPα. In the current study, we investigated whether the orphan drug, dichloroacetate, could alter amyloid precursor protein proteolysis. In SH-SY5Y neuroblastoma cells, dichloroacetate enhanced sAPPα generation whilst inhibiting β-secretase processing of endogenous amyloid precursor protein and the subsequent generation of amyloid beta-peptides. Over-expression of the amyloid precursor protein partly ablated the effect of dichloroacetate on amyloidogenic and non-amyloidogenic processing whilst over-expression of the β-secretase only ablated the effect on amyloidogenic processing. Similar enhancement of ADAM-mediated amyloid precursor protein processing by dichloroacetate was observed in unrelated cell lines and the effect was not exclusive to the amyloid precursor protein as an ADAM substrate, as indicated by dichloroacetate-enhanced proteolysis of the Notch ligand, Jagged1. Despite altering proteolysis of the amyloid precursor protein, dichloroacetate did not significantly affect the expression/activity of α-, β- or γ-secretases. In conclusion, dichloroacetate can inhibit amyloidogenic and promote non-amyloidogenic proteolysis of the amyloid precursor protein. Given the small size and blood-brain-barrier permeability of the drug, further research into its mechanism of action with respect to APP proteolysis may lead to the development of therapies for slowing the progression of Alzheimer's disease.

Nanoscale rearrangement of APP organization as a therapeutic target for Alzheimer’s disease

Despite the importance of canonical processing of Amyloid Precursor Protein at synapses as a major risk factor for the development of Alzheimer’s Disease, there have been very little progress on designing effective therapeutic paradigms targeting it. Majority of the drugs developed or under clinical evaluation focus on the clearance of the detrimental proteoforms or secretases involved in the proteolysis of APP. The lack of interventions targeting APP is in part due to the lack of information in understanding the fine organization of APP and the chemical map of its association with subsynaptic functional zones of the synapse. The recent advances to evaluate the molecular organization of synapses allows us to readdress the need for designing tools to target the full-length APP. Here, we describe the potential role of nanoscale segregation of synaptic APP and how this organization influences the local processing of APP in different subsynaptic compartments opening avenues for early intervention strategies. We envision the need to design smart molecules which would interfere with the real-time chemical composition and physical properties of APP at nanoscale. These tools could alter the balance of proteoforms generated and/or enhance the proteolysis by selective secretases to reduce the toxic products formed through amyloidogenic pathway. We believe that such an approach would be rational to treat or delay the onset of neurodegenerative diseases like AD.

Complex formation of APP with GABAB receptors links axonal trafficking to amyloidogenic processing

GABAB receptors (GBRs) are key regulators of synaptic release but little is known about trafficking mechanisms that control their presynaptic abundance. We now show that sequence-related epitopes in APP, AJAP-1 and PIANP bind with nanomolar affinities to the N-terminal sushi-domain of presynaptic GBRs. Of the three interacting proteins, selectively the genetic loss of APP impaired GBR-mediated presynaptic inhibition and axonal GBR expression. Proteomic and functional analyses revealed that APP associates with JIP and calsyntenin proteins that link the APP/GBR complex in cargo vesicles to the axonal trafficking motor. Complex formation with GBRs stabilizes APP at the cell surface and reduces proteolysis of APP to Aβ, a component of senile plaques in Alzheimer’s disease patients. Thus, APP/GBR complex formation links presynaptic GBR trafficking to Aβ formation. Our findings support that dysfunctional axonal trafficking and reduced GBR expression in Alzheimer’s disease increases Aβ formation.

Toward β-Secretase-1 Inhibitors with Improved Isoform Selectivity.

BACE1 is responsible for the first step in APP proteolysis, leading to toxic Aβ production, and has been indicated to play a key role in the pathogenesis of Alzheimer's disease. The related isoform BACE2 is thought to be involved in processing of the pigment cell-specific melanocyte protein. To avoid potential effects on pigmentation, we investigated the feasibility for developing isoform-selective BACE1 inhibitors. Cocrystal structures of 47 compounds were analyzed and clustered according to their selectivity profiles. Selective BACE1 inhibitors were found to exhibit two distinct conformational features proximal to the flap and the S3 subpocket. Several new molecules were designed and tested to make use of this observation. The combination of a pyrimidinyl C-ring and a methylcyclohexyl element resulted in lead molecule 28, which exhibited ∼50-fold selectivity. Compared to a nonselective BACE1/2 inhibitor, 28 showed significantly less inhibition of PMEL processing in human melanocytes, indicating good functional selectivity of this inhibitor class.

MAPPING PROTEIN-PROTEIN INTERACTIONS BY PEPTIDE ARRAY FOR DISRUPTOR PEPTIDE DEVELOPMENT AND TRANSLATIONAL IMPACT!

Aberrant cellular signalling is central to the pathogenesis of a number of neurodegenerative disorders. Our group focuses in particular on the role of Wnt signalling in β-amyloid toxicity, and have demonstrated not only that aberrant Wnt activation, APP proteolysis and synapse loss are intimately linked, but have identified the key protein-protein interactions responsible. A fundamental aspect of our pathway-driven approach is the mapping and disruption of protein-protein interactions using peptide array technology. Peptide arrays are created from peptide libraries consisting of overlapping peptides spanning the entire sequence of the protein of interest and spotted onto cellulose coated slides. These peptide arrays can be overlaid with putative binding partners to identify potential binding regions or novel post-translational modification sites. Key amino acid residues can be rapidly identified by alanine/ single substitution scanning. Crucially, we can delineate the minimal “core” binding regions, which can be synthesised and used as cell permeable “disruptor peptides” capable of selectively blocking specific protein-protein interactions. Furthermore, disruptor peptides can be used as the basis of high throughput biochemical and cell based assays for drug discovery. We wish to showcase the power of this technology, using specific examples from our own work on Alzheimer's disease, and demonstrate its capabilities, which are readily amenable across a broad range of applications, to provide useful research tools in dissecting specific signalling events. Mapping and disrupting protein-protein interactions by peptide array has significant potential to provide translational outcomes for neurodegenerative disease.

C-Terminal Threonine Reduces Aβ43 Amyloidogenicity Compared with Aβ42

Aβ43, a product of the proteolysis of the amyloid precursor protein APP, is related to Aβ42 by an additional Thr residue at the C-terminus. Aβ43 is typically generated at low levels compared with the predominant Aβ42 and Aβ40 forms, but it has been suggested that this longer peptide might have an impact on amyloid-β aggregation and Alzheimer's disease that is out of proportion to its brain content. Here, we report that both Aβ42 and Aβ43 spontaneously aggregate into mature amyloid fibrils via sequential appearance of the same series of oligomeric and protofibrillar intermediates, the earliest of which appears to lack β-structure. In spite of the additional β-branched amino acid at the C-terminus, Aβ43 fibrils have fewer strong backbone H-bonds than Aβ42 fibrils, some of which are lost at the C-terminus. In contrast to previous reports, we found that Aβ43 spontaneously aggregates more slowly than Aβ42. In addition, Aβ43 fibrils are very inefficient at seeding Aβ42 amyloid formation, even though Aβ42 fibrils efficiently seed amyloid formation by Aβ43 monomers. Finally, mixtures of Aβ42 and Aβ43 aggregate more slowly than Aβ42 alone. Both in this Aβ42/Aβ43 co-aggregation reaction and in cross-seeding by Aβ42 fibrils, the structure of the Aβ43 in the product fibrils is influenced by the presence of Aβ42. The results provide new details of amyloid structure and assembly pathways, an example of structural plasticity in prion-like replication, and data showing that low levels of Aβ43 in the brain are unlikely to favorably impact the aggregation of Aβ42.

Activation of peroxisome proliferator-activated receptor α stimulates ADAM10-mediated proteolysis of APP

Amyloid precursor protein (APP) derivative β-amyloid (Aβ) plays an important role in the pathogenesis of Alzheimer's disease (AD). Sequential proteolysis of APP by β-secretase and γ-secretase generates Aβ. Conversely, the α-secretase "a disintegrin and metalloproteinase" 10 (ADAM10) cleaves APP within the eventual Aβ sequence and precludes Aβ generation. Therefore, up-regulation of ADAM10 represents a plausible therapeutic strategy to combat overproduction of neurotoxic Aβ. Peroxisome proliferator-activated receptor α (PPARα) is a transcription factor that regulates genes involved in fatty acid metabolism. Here, we determined that the Adam10 promoter harbors PPAR response elements; that knockdown of PPARα, but not PPARβ or PPARγ, decreases the expression of Adam10; and that lentiviral overexpression of PPARα restored ADAM10 expression in Ppara(-/-) neurons. Gemfibrozil, an agonist of PPARα, induced the recruitment of PPARα:retinoid x receptor α, but not PPARγ coactivator 1α (PGC1α), to the Adam10 promoter in wild-type mouse hippocampal neurons and shifted APP processing toward the α-secretase, as determined by augmented soluble APPα and decreased Aβ production. Accordingly, Ppara(-/-) mice displayed elevated SDS-stable, endogenous Aβ and Aβ1-42 relative to wild-type littermates, whereas 5XFAD mice null for PPARα (5X/α(-/-)) exhibited greater cerebral Aβ load relative to 5XFAD littermates. These results identify PPARα as an important factor regulating neuronal ADAM10 expression and, thus, α-secretase proteolysis of APP.

Retinoic Acid-Elicited RARα/RXRα Signaling Attenuates Aβ Production by Directly Inhibiting γ-Secretase-Mediated Cleavage of Amyloid Precursor Protein

Retinoic acid (RA)-elicited signaling has been shown to play critical roles in development, organogenesis, and the immune response. RA regulates expression of Alzheimer's disease (AD)-related genes and attenuates amyloid pathology in a transgenic mouse model. In this study, we investigated whether RA can suppress the production of amyloid-β (Aβ) through direct inhibition of γ-secretase activity. We report that RA treatment of cells results in significant inhibition of γ-secretase-mediated processing of the amyloid precursor protein C-terminal fragment APP-C99, compared with DMSO-treated controls. RA-elicited signaling was found to significantly increase accumulation of APP-C99 and decrease production of secreted Aβ40. In addition, RA-induced inhibition of γ-secretase activity was found to be mediated through significant activation of extracellular signal-regulated kinases (ERK1/2). Treatment of cells with the specific ERK inhibitor PD98059 completely abolished RA-mediated inhibition of γ-secretase. Consistent with these findings, RA was observed to inhibit secretase-mediated proteolysis of full-length APP. Finally, we have established that RA inhibits γ-secretase through nuclear retinoic acid receptor-α (RARα) and retinoid X receptor-α (RXRα). Our findings provide a new mechanistic explanation for the neuroprotective role of RA in AD pathology and add to the previous data showing the importance of RA signaling as a target for AD therapy.

β - but not γ-secretase proteolysis of APP causes synaptic and memory deficits in a mouse model of dementia

β - but not γ-secretase proteolysis of APP causes synaptic and memory deficits in a mouse model of dementia

Alterations in phosphatidylethanolamine levels affect the generation of Aβ

Several studies suggest that the generation of Aβ is highly dependent on the levels of cholesterol within membranes' detergent-resistant microdomains (DRM). Indeed, the β-amyloid precursor protein (APP) cleaving machinery, namely β- and γ-secretases, has been shown to be present in DRM and its activity depends on membrane cholesterol levels. Counterintuitive to the localization of the cleavage machinery, the substrate, APP, localizes to membranes' detergent-soluble microdomains enriched in phospholipids (PL), indicating that Aβ generation is highly dependent on the capacity of enzyme and substrate to diffuse along the lateral plane of the membrane and therefore on the internal equilibrium of the different lipids of DRM and non-DRM domains. Here, we studied to which extent changes in the content of a main non-DRM lipid might affect the proteolytic processing of APP. As phosphatidylethanolamine (PE) accounts for the majority of PL, we focused on its impact on the regulation of APP proteolysis. In mammalian cells, siRNA-mediated knock-down of PE synthesis resulted in decreased Aβ owing to a dual effect: promoted α-secretase cleavage and decreased γ-secretase processing of APP. In vivo, in Drosophila melanogaster, genetic reduction in PL synthesis results in decreased γ-secretase-dependent cleavage of APP. These results suggest that modulation of the membrane-soluble domains could be a valuable alternative to reduce excessive Aβ generation.

Additive effects of fatty acid mixtures on the levels and ratio of amyloid β40/42 peptides differ from the effects of individual fatty acids

Several studies have shown the protective and/or deleterious effects of dietary enrichment of single fatty acids (FAs) in several animal and cell-culture models of Alzheimer's disease (AD). However, potential interactions among dietary fatty acids are traditionally ignored. None of these studies has examined and compared the differential effects of FAs in combination, as well as alone, for their effects on amyloid β production or AD. Here we investigated the effects of omega-9 (oleic acid) and omega-6 (linoleic and arachidonic acids) fatty acids, either alone or combined, on Aβ production by APP-695 and SP-C99 transfected COS-7 cells. Overall, our results are the first to demonstrate that mixtures of FAs alter the production of Aβ40 and Aβ42 peptides and consequently the Aβ40:42 ratio differently from individual FAs. Here we show that the effects of a single lipid on Aβ production are not attributed to that single FA alone. Rather, the overall lipid composition influences the specificity and level of the regulated intramembranous proteolysis of APP by the γ-secretase complex. Our results reinforce the importance of studying composite lipids/nutrients rather than single lipids or nutrients.

Phenylbutyric acid reduces amyloid plaques and rescues cognitive behavior in AD transgenic mice

Trafficking through the secretory pathway is known to regulate the maturation of the APP-cleaving secretases and APP proteolysis. The coupling of stress signaling and pathological deterioration of the brain in Alzheimer's disease (AD) supports a mechanistic connection between endoplasmic reticulum (ER) stress and neurodegeneration. Consequently, small molecular chaperones, which promote protein folding and minimize ER stress, might be effective in delaying or attenuating the deleterious progression of AD. We tested this hypothesis by treating APPswePS1delta9 AD transgenic mice with the molecular chaperone phenylbutyric acid (PBA) for 14 months at a dose of 1 mg PBA g(-1) of body weight in the drinking water. Phenylbutyric acid treatment increased secretase-mediated APP cleavage, but was not associated with any increase in amyloid biosynthesis. The PBA-treated AD transgenic mice had significantly decreased incidence and size of amyloid plaques throughout the cortex and hippocampus. There was no change in total amyloid levels suggesting that PBA modifies amyloid aggregation or pathogenesis independently of biogenesis. The decrease in amyloid plaques was paralleled by increased memory retention, as PBA treatment facilitated cognitive performance in a spatial memory task in both wild-type and AD transgenic mice. The molecular mechanism underlying the cognitive facilitation of PBA is not clear; however, increased levels of both metabotropic and ionotropic glutamate receptors, as well as ADAM10 and TACE, were observed in the cortex and hippocampus of PBA-treated mice. The data suggest that PBA ameliorates the cognitive and pathological features of AD and supports the investigation of PBA as a therapeutic for AD.

Calmodulin regulates the non-amyloidogenic metabolism of amyloid precursor protein in platelets

A balance between the proteolytic processing of amyloid precursor protein APP through the amyloidogenic and the non-amyloidogenic pathways controls the production and release of amyloid β-protein, whose accumulation in the brain is associated to the onset of Alzheimer Disease. APP is also expressed on circulating platelets. The regulation of APP processing in these cells is poorly understood. In this work we show that platelets store considerable amounts of APP fragments, including sAPPα, that can be released upon stimulation of platelets. Moreover, platelet stimulation also promotes the proteolysis of intact APP expressed on the cell surface. This process is supported by an ADAM metalloproteinase, and causes the release of sAPPα. Processing of intact platelet APP is promoted also by treatment with calmodulin antagonist W7. W7-induced APP proteolysis occurs through the non-amyloidogenic pathway, is mediated by a metalloproteinase, and causes the release of sAPPα. Co-immunoprecipitation and pull-down experiments revealed a physical association between calmodulin and APP. These results document a novel role of calmodulin in the regulation of non-amyloidogenic processing of APP.

The inhibitory effects of different curcuminoids on β-amyloid protein, β-amyloid precursor protein and β-site amyloid precursor protein cleaving enzyme 1 in swAPP HEK293 cells

The hallmark of Alzheimer's disease (AD) is the accumulation of β-amyloid protein (Aβ). Aβ is generated from the β-amyloid precursor protein (APP) through the proteolysis of β-site APP cleaving enzyme 1 (BACE1) and γ-secretase. Aβ 42 isoform is more easily aggregate and more toxic to neurons than any other Aβ isoforms, thus being regarded as the primary toxic specie in AD. Curcumin mix has potent anti-amyloidogenic effect and shows great promise for AD treatment and prevention. The present study was conducted to examine the effects of curcumin mix and its different curcuminoids including curcumin (Cur), demethoxycurcumin (DMC) and bisdemethoxycurcumin (BDMC) on Aβ 42, APP and BACE1. We found that Cur was the most active curcuminoid fraction in suppressing Aβ 42 production and the order of inhibitory potency of other curcuminoids was DMC > curcumin mix > BDMC. Cur, but not other curcuminoids, could reduce APP protein expression and none of curcuminoids affected APP mRNA level. BDMC could reduce BACE1 mRNA and protein levels, while DMC only affected BACE1 mRNA expression. Our data indicate that the anti-amyloidogenic effect of Cur may be mediated through the modulation of APP, while the anti-amyloidogenic effect of BDMC may be mediated through the modulation of BACE1.

In vivo effects of APP are not exacerbated by BACE2 co-overexpression: behavioural characterization of a double transgenic mouse model

Down syndrome, the most common genetic disorder leading to mental retardation, is caused by the presence of all or part of an extra copy of chromosome 21. At relatively early ages, Down syndrome patients develop progressive formation and extracellular aggregation of amyloid-β peptide, considered as one of the causal factors for the pathogenesis of Alzheimer's disease. This neuropathological hallmark has been attributed to the overexpression of APP but could also be contributed by other HSA21 genes. BACE2 maps to HSA21 and is homologous to BACE1, a β-secretase involved in the amyloidogenic pathway of APP proteolysis, and thus it has been hypothesized that the co-overexpression of both genes could contribute to Alzheimer's like neuropathology present in Down syndrome. The aim of the present study has been to analyse the impact of the co-overexpression of BACE2 and APP, using a double transgenic mouse model. Double transgenic mice did not present any neurological or sensorimotor alterations, nor genotype-dependent anxiety-like behaviour or age-associated cognitive dysfunction. Interestingly, TgBACE2-APP mice showed deregulation of BACE2 expression levels that were significantly increased with respect to single TgBACE2 mice. Co-overexpression of BACE2 and APP did not increase amyloid-β peptide concentration in brain. Our results suggest that the in vivo effects of APP are not exacerbated by BACE2 co-overexpression but may have some protective effects in specific behavioural and cognitive domains in transgenic mice.

Phospholipids and a phospholipid-rich diet alter the in vitro amyloid-beta peptide levels and amyloid-beta 42/40 ratios

Amyloid-β peptides (Aβ) generated by proteolysis of the β-amyloid precursor protein (APP) by β- and γ-secretases play an important role in the pathogenesis of Alzheimer's disease (AD). There is mounting evidence that the lipid matrix of neuronal cell membranes plays an important role in the accumulation of Aβ peptides into senile plaques, one of the hallmarks of AD. With the aim to clarify the molecular basis of the interaction between Aβ and cellular membranes, we investigated the effects of various phospholipids (PLs) and a PL-rich diet on Aβ production. Here we show that modulation of Aβ production and Aβ42:40 ratio is not limited to individual fatty acids, rather it is the composition of the PLs of the membrane bilayer, that influences the specificity and level of the regulated intramembranous proteolysis of APP by the γ-secretase complex. We show that Aβ levels in the conditioned media, in response to some of the PL supplements, is increased in the center and decreased on either side of a graph that resembles bell-shaped distribution. This means that the PLs have less of a tendency to produce unusually extreme effects on Aβ production in SP-C99 transfected Cos-7 cultured cells. We proposed a mechanism-based hypothesis to rationalize PLs’ effects on Aβ production.

Phenylbutyric Acid Rescues Endoplasmic Reticulum Stress-Induced Suppression of APP Proteolysis and Prevents Apoptosis in Neuronal Cells

BackgroundThe familial and sporadic forms of Alzheimer's disease (AD) have an identical pathology with a severe disparity in the time of onset [1]. The pathological similarity suggests that epigenetic processes may phenocopy the Familial Alzheimer's disease (FAD) mutations within sporadic AD. Numerous groups have demonstrated that FAD mutations in presenilin result in ‘loss of function’ of γ-secretase mediated APP cleavage [2], [3], [4], [5]. Accordingly, ER stress is prominent within the pathologically impacted brain regions in AD patients [6] and is reported to inhibit APP trafficking through the secretory pathway [7], [8]. As the maturation of APP and the cleaving secretases requires trafficking through the secretory pathway [9], [10], [11], we hypothesized that ER stress may block trafficking requisite for normal levels of APP cleavage and that the small molecular chaperone 4-phenylbutyrate (PBA) may rescue the proteolytic deficit. Methodology/Principal FindingsThe APP-Gal4VP16/Gal4-reporter screen was stably incorporated into neuroblastoma cells in order to assay γ-secretase mediated APP proteolysis under normal and pharmacologically induced ER stress conditions. Three unrelated pharmacological agents (tunicamycin, thapsigargin and brefeldin A) all repressed APP proteolysis in parallel with activation of unfolded protein response (UPR) signaling—a biochemical marker of ER stress. Co-treatment of the γ-secretase reporter cells with PBA blocked the repressive effects of tunicamycin and thapsigargin upon APP proteolysis, UPR activation, and apoptosis. In unstressed cells, PBA stimulated γ-secretase mediated cleavage of APP by 8–10 fold, in the absence of any significant effects upon amyloid production, by promoting APP trafficking through the secretory pathway and the stimulation of the non-pathogenic α/γ-cleavage.Conclusions/SignificanceER stress represses γ-secretase mediated APP proteolysis, which replicates some of the proteolytic deficits associated with the FAD mutations. The small molecular chaperone PBA can reverse ER stress induced effects upon APP proteolysis, trafficking and cellular viability. Pharmaceutical agents, such as PBA, that stimulate α/γ-cleavage of APP by modifying intracellular trafficking should be explored as AD therapeutics.

An Overview of APP Processing Enzymes and Products

The generation of amyloid beta-peptide (A beta) by enzymatic cleavages of the beta-amyloid precursor protein (APP) has been at the center of Alzheimer's disease (AD) research. While the basic process of beta- and gamma-secretase-mediated generation of A beta is text book knowledge, new aspects of A beta and other cleavage products have emerged in recent years. Also our understanding of the enzymes involved in APP proteolysis has increased dramatically. All of these discoveries contribute to a more complete understanding of APP processing and the physiologic and pathologic roles of its secreted and intracellular protein products. Understanding APP processing is important for any therapeutic strategy aimed at reducing A beta levels in AD. In this review, we provide a concise description of the current state of understanding the enzymes involved in APP processing, the cleavage products generated by different processing patterns, and the potential functions of those cleavage products.