Amyloid precursor protein trafficking, processing, and function

Intracellular trafficking and proteolytic processing of amyloid precursor protein (APP) have been the focus of numerous investigations over the past two decades. APP is the precursor to the amyloid beta-protein (Abeta), the 38-43-amino acid residue peptide that is at the heart of the amyloid cascade hypothesis of Alzheimer disease (AD). Tremendous progress has been made since the initial identification of Abeta as the principal component of brain senile plaques of individuals with AD. Specifically, molecular characterization of the secretases involved in Abeta production has facilitated cell biological investigations on APP processing and advanced efforts to model AD pathogenesis in animal models. This minireview summarizes salient features of APP trafficking and amyloidogenic processing and discusses the putative biological functions of APP.     

Proteases and lipoprotein receptors in Alzheimer's disease

Alzheimer's disease (AD) is the leading cause of senile dementia, and is a complex disorder. The pathological hallmarks of AD were discovered by Dr. Alois Alzheimer in 1907, and include deposits of amyloid or senile plaques and neurofibrillar tangles. Plaques are composed of a peptide, termed the Abeta peptide, that is derived by proteolytic processing of the amyloid precursor protein (APP), while neurofibrillar tangles result from a hyperphosphorylation of the tau protein. Mechanisms associated with the formation of plaques and neurofibrillar tangles and their respective contributions to the disease process have been intensely investigated. Proteolytic processing of APP that results in the generation of the Abeta peptide is now well understood and is influenced by several proteins. Recent evidence suggests that the Abeta levels are carefully regulated, and several proteases play an important role in removing the Abeta peptide. Finally, it is becoming apparent that several members of the LDL receptor family play important roles in the brain, and may modulate the course of AD.     

Failure of the interaction between presenilin 1 and the substrate of gamma-secretase to produce Abeta in insect cells

Aggregates of beta-amyloid peptide (Abeta) are the major component of the amyloid core of the senile plaques observed in Alzheimer's disease (AD). Abeta results from the amyloidogenic processing of its precursor, the amyloid precursor protein (APP), by beta- and gamma-secretase activities. If beta-secretase has recently been identified and termed BACE, the identity of gamma-secretase is still obscure. Studies with knock-out mice showed that presenilin 1 (PS1), of which mutations are known to be the first cause of inherited AD, is mandatory for the gamma-secretase activity. However, the proteolytic activity of PS1 remains a matter of debate. Here we used transfected Sf9 insect cells, a cellular model lacking endogenous beta- and/or gamma-secretase activities, to characterize the role of BACE and PS1 in the amyloidogenic processing of human APP. We show that, in Sf9 cells, BACE performs the expected beta-secretase cleavage of APP, generating C99. We also show that C99, which is a substrate of gamma-secretase, tightly binds to the human PS1. Despite this interaction, Sf9 cells still do not produce Abeta. This strongly argues against a direct proteolytic activity of PS1 in APP processing, and points toward an implication of PS1 in trafficking/presenting its substrate to the gamma-secretase.     

Estrogen-induced cell signalling in a cellular model of Alzheimer's disease

Alzheimer's disease (AD) is characterised by deposition of a 4 kDa amyloid-beta peptide (Abeta) into senile plaques of the affected brain. Abeta is a proteolytic product of the membrane protein, amyloid precursor protein (APP). An alternative cleavage pathway involves alpha-secretase activity and results in secretion of a 100 kDa non-amyloidogenic APP (sAPPalpha) and therefore a potential reduction in Abeta secretion. We have shown that estrogen induces alpha-cleavage and therefore results in the secretion of sAPPalpha. This secretion is signalled via MAP-kinase and PI-3 kinase signal-transduction pathways. These pathways also have the potential to inhibit the activation of glycogen synthase kinase 3beta (GSK), a protein involved in cell death. Therefore, the aim of this work was to further elucidate the estrogen-mediated signaling pathways involved in APP processing, with particular emphasis on GSK activity. By stimulating rat hypothalamic neuronal GT1-7 cells with estradiol, we found that estrogen decreases the activation state of GSK via the MAP kinase pathway. Moreover, the inhibition of GSK activity by LiCl causes enhanced sAPPalpha secretion in a pattern similar to that seen in response to estrogen, suggesting a pivotal role for this deactivation in APP processing. Further, inactivation of GSK by estrogen can be confirmed in an in vivo model. Elucidation of the signaling pathways involved in APP processing may help to understand the pathology of AD and may also prove beneficial in developing therapeutic strategies to combat AD.     

The role of amyloid precursor protein processing by BACE1, the beta-secretase, in Alzheimer disease pathophysiology

Amyloid plaques, composed of the amyloid beta-protein (Abeta), are hallmark neuropathological lesions in Alzheimer disease (AD) brain. Abeta fulfills a central role in AD pathogenesis, and reduction of Abeta levels should prove beneficial for AD treatment. Abeta generation is initiated by proteolysis of amyloid precursor protein (APP) by the beta-secretase enzyme BACE1. Bace1 knockout (Bace1(-/-)) mice have validated BACE1 as the authentic beta-secretase in vivo. BACE1 is essential for Abeta generation and represents a suitable drug target for AD therapy, especially because this enzyme is up-regulated in AD. However, although initial data indicated that Bace1(-/-) mice lack an overt phenotype, the BACE1-mediated processing of APP and other substrates may be important for specific biological processes. In this minireview, topics range from the initial identification of BACE1 to the fundamental knowledge gaps that remain in our understanding of this protease. We address pertinent questions such as putative causes of BACE1 elevation in AD and discuss why, nine years since the identification of BACE1, treatments that address the underlying pathological mechanisms of AD are still lacking.     

Flavonols and flavones as BACE-1 inhibitors: structure-activity relationship in cell-free, cell-based and in silico studies reveal novel pharmacophore features

Generation and accumulation of the amyloid beta peptide (Abeta) following proteolytic processing of the amyloid precursor protein (APP) by BACE-1 (Beta-site APP Cleaving Enzyme-1, beta-secretase) and gamma-secretase is a main causal factor of Alzheimer's disease (AD). Consequently, inhibition of BACE-1, a rate-limiting enzyme in the production of Abeta, is an attractive therapeutic approach for the treatment of AD. In this study, we discovered that natural flavonoids act as non-peptidic BACE-1 inhibitors and potently inhibit BACE-1 activity and reduce the level of secreted Abeta in primary cortical neurons. In addition, we demonstrated the calculated docking poses of flavonoids to BACE-1 and revealed the interactions of flavonoids with the BACE-1 catalytic center. We firstly revealed novel pharmacophore features of flavonoids by using cell-free, cell-based and in silico docking studies. These results contribute to the development of new BACE-1 inhibitors for the treatment of AD.     

Regulation of cholesterol and sphingomyelin metabolism by amyloid-beta and presenilin

Amyloid beta peptide (Abeta) has a key role in the pathological process of Alzheimer's disease (AD), but the physiological function of Abeta and of the amyloid precursor protein (APP) is unknown. Recently, it was shown that APP processing is sensitive to cholesterol and other lipids. Hydroxymethylglutaryl-CoA reductase (HMGR) and sphingomyelinases (SMases) are the main enzymes that regulate cholesterol biosynthesis and sphingomyelin (SM) levels, respectively. We show that control of cholesterol and SM metabolism involves APP processing. Abeta42 directly activates neutral SMase and downregulates SM levels, whereas Abeta40 reduces cholesterol de novo synthesis by inhibition of HMGR activity. This process strictly depends on gamma-secretase activity. In line with altered Abeta40/42 generation, pathological presenilin mutations result in increased cholesterol and decreased SM levels. Our results demonstrate a biological function for APP processing and also a functional basis for the link that has been observed between lipids and Alzheimer's disease (AD).     

Partial reduction of BACE1 has dramatic effects on Alzheimer plaque and synaptic pathology in APP Transgenic Mice

The aspartyl protease beta-site amyloid precursor protein cleaving enzyme 1 (BACE1) initiates processing of amyloid precursor protein (APP) into amyloid beta (Abeta) peptide, the major component of Alzheimer disease (AD) plaques. To determine the role that BACE1 plays in the development of Abeta-driven AD-like pathology, we have crossed PDAPP mice, a transgenic mouse model of AD overexpressing human mutated APP, onto mice with either a homozygous or heterozygous BACE1 gene knockout. Analysis of PDAPP/BACE(-/-) mice demonstrated that BACE1 is absolutely required for both Abeta generation and the development of age-associated plaque pathology. Furthermore, synaptic deficits, a neurodegenerative pathology characteristic of AD, were also reversed in the bigenic mice. To determine the extent of BACE1 reduction required to significantly inhibit pathology, PDAPP mice having a heterozygous BACE1 gene knock-out were evaluated for Abeta generation and for the development of pathology. Although the 50% reduction in BACE1 enzyme levels caused only a 12% decrease in Abeta levels in young mice, it nonetheless resulted in a dramatic reduction in Abeta plaques, neuritic burden, and synaptic deficits in older mice. Quantitative analyses indicate that brain Abeta levels in young APP transgenic mice are not the sole determinant for the changes in plaque pathology mediated by reduced BACE1. These observations demonstrate that partial reductions of BACE1 enzyme activity and concomitant Abeta levels lead to dramatic inhibition of Abeta-driven AD-like pathology, making BACE1 an excellent target for therapeutic intervention in AD.     

PAR-4 is involved in regulation of beta-secretase cleavage of the Alzheimer amyloid precursor protein

Mounting evidence indicates that aberrant production and aggregation of amyloid beta-peptide (Abeta)-(1-42) play a central role in the pathogenesis of Alzheimer disease (AD). Abeta is produced when amyloid precursor protein (APP) is cleaved by beta- and gamma-secretases at the N and C termini of the Abeta domain, respectively. The beta-secretase is membrane-bound aspartyl protease, most commonly known as BACE1. Because BACE1 cleaves APP at the N terminus of the Abeta domain, it catalyzes the first step in Abeta generation. PAR-4 (prostate apoptosis response-4) is a leucine zipper protein that was initially identified to be associated with neuronal degeneration and aberrant Abeta production in models of AD. We now report that the C-terminal domain of PAR-4 is necessary for forming a complex with the cytosolic tail of BACE1 in co-immunoprecipitation assays and in vitro pull-down experiments. Overexpression of PAR-4 significantly increased, whereas silencing of PAR-4 expression by RNA interference significantly decreased, beta-secretase cleavage of APP. These results suggest that PAR-4 may be directly involved in regulating the APP cleavage activity of BACE1. Because the increased BACE1 activity observed in AD patients does not seem to arise from genetic mutations or polymorphisms in BACE1, the identification of PAR-4 as an endogenous regulator of BACE1 activity may have significant implications for developing novel therapeutic strategies for AD.     

Regulation of amyloid precursor protein (APP) phosphorylation and processing by p35/Cdk5 and p25/Cdk5

The phosphorylation status of amyloid precursor protein (APP) at Thr668 is suggested to play a critical role in the proteolytic cleavage of APP, which generates either soluble APP(beta) (sAPP(beta)) and beta-amyloid peptide (Abeta), the major component of senile plaques in patient brains inflicted with Alzheimer's disease (AD), or soluble APP(alpha) (sAPP(alpha)) and a peptide smaller than Abeta. One of the protein kinases known to phosphorylate APP(Thr668) is cyclin-dependent kinase 5 (Cdk5). Cdk5 is activated by the association with its regulatory partner p35 or its truncated form, p25, which is elevated in AD brains. The comparative effects of p35 and p25 on APP(Thr668) phosphorylation and APP processing, however, have not been reported. In this study, we investigated APP(Thr668) phosphorylation and APP processing mediated by p35/Cdk5 and p25/Cdk5 in the human neuroblastoma cell line SH-SY5Y. Transient overexpression of p35 and p25 elicited distinct patterns of APP(Thr668) phosphorylation, specifically, p35 increasing the phosphorylation of both mature and immature APP, whereas p25 primarily elevated the phosphorylation of immature APP. Despite these differential effects on APP phosphorylation, both p35 and p25 overexpression enhanced the secretion of Abeta, sAPP(beta), as well as sAPP(alpha). These results confirm the involvement of Cdk5 in APP processing, and suggest that p35- and p25-mediated Cdk5 activities lead to discrete APP phosphorylation.     

Caspase cleavage of the amyloid precursor protein modulates amyloid beta-protein toxicity

The amyloid beta-protein precursor (APP) is proteolytically cleaved to generate the amyloid beta-protein (Abeta), the principal constituent of senile plaques found in Alzheimer's disease (AD). In addition, Abeta in its oligomeric and fibrillar forms have been hypothesized to induce neuronal toxicity. We and others have previously shown that APP can be cleaved by caspases at the C-terminus to generate a potentially cytotoxic peptide termed C31. Furthermore, this cleavage event and caspase activation were increased in the brains of AD, but not control, cases. In this study, we show that in cultured cells, Abeta induces caspase cleavage of APP in the C-terminus and that the subsequent generation of C31 contributes to the apoptotic cell death associated with Abeta. Interestingly, both Abeta toxicity and C31 pathway are dependent on the presence of APP. Both APP-dependent Abeta toxicity and C31-induced apoptotic cell death involve apical or initiator caspases-8 and -9. Our results suggest that Abeta-mediated toxicity initiates a cascade of events that includes caspase activation and APP cleavage. These findings link C31 generation and its potential cell death activity to Abeta cytotoxicity, the leading mechanism proposed for neuronal death in AD.     

Modulation of Abeta generation by small ubiquitin-like modifiers does not require conjugation to target proteins

The sequential processing of the APP (amyloid precursor protein) by the beta- and gamma-secretase and generation of the Abeta (amyloid-beta) peptide is a primary pathological factor in AD (Alzheimer's disease). Regulation of the processing or turnover of these proteins represents potential targets for the development of AD therapies. Sumoylation is a process by which SUMOs (small ubiquitin-like modifiers) are covalently conjugated to target proteins, resulting in a number of functional consequences. These include regulation of protein-protein interactions, intracellular trafficking and protein stability, which all have the potential to impact on several aspects of the amyloidogenic pathway. The present study examines the effects of overexpression and knockdown of the major SUMO isoforms (SUMO1, 2 and 3) on APP processing and the production of Abeta peptides. SUMO3 overexpression significantly increased Abeta40 and Abeta42 secretion, which was accompanied by an increase in full-length APP and its C-terminal fragments. These effects of SUMO3 were independent of its covalent attachment or chain formation, as mutants lacking the motifs responsible for SUMO chain formation or SUMO conjugation led to similar changes in Abeta. SUMO3 overexpression also up-regulated the expression of the transmembrane protease BACE (beta-amyloid-cleaving enzyme), but failed to affect levels of several other unrelated proteins. Suppression of SUMO1 or combined SUMO2+3 by RNA interference did not affect APP levels or Abeta production. These findings confirm a specific effect of SUMO3 overexpression on APP processing and the production of Abeta peptides but also suggest that endogenous sumoylation is not essential and likely plays an indirect role in modulating the amyloid processing pathway.     

Targeted proteomics in Alzheimer's disease: focus on amyloid-beta

Diagnosis and monitoring of sporadic Alzheimer's disease (AD) have long depended on clinical examination of individuals with end-stage disease. However, upcoming anti-AD therapies are optimally initiated when individuals show very mild signs of neurodegeneration. There is a developing consensus for cerebrospinal fluid amyloid-beta (Abeta) as a core biomarker for the mild cognitive impairment stage of AD. Abeta is directly involved in the pathogenesis of AD or tightly correlated with other primary pathogenic factors. It is produced from amyloid precursor protein (APP) by proteolytic processing that depends on the beta-site APP-cleaving enzyme 1 and the gamma-secretase complex, and is degraded by a broad range of proteases. This review summarizes targeted proteomic studies of Abeta in biological fluids and identifies clinically useful markers of disrupted Abeta homeostasis in AD. The next 5 years will see a range of novel assays developed on the basis of these results. From a longer perspective, establishment of the most effective combinations of different biomarkers and other diagnostic modalities may be foreseen.     

gamma-Secretase cleavage and binding to FE65 regulate the nuclear translocation of the intracellular C-terminal domain (ICD) of the APP family of proteins

Regulated intramembrane proteolysis (RIP) of the amyloid precursor protein (APP) produces amyloid beta-protein (Abeta), the probable causative agent of Alzheimer's disease (AD), and is therefore an important target for therapeutic intervention. However, there is a burgeoning consensus that gamma-secretase, one of the proteases that generates Abeta, is also critical for the signal transduction of APP and a growing list of other receptors. APP is a member of a gene family that includes two amyloid precursor-like proteins, APLP1 and APLP2. Although APP and the APLPs undergo similar proteolytic processing, there is little information about the role of their gamma-secretase-generated intracellular domains (ICDs). Here, we show that APLP1 and 2 undergo presenilin-dependent RIP similar to APP, resulting in the release of a approximately 6 kDa ICD for each protein. Each of the ICDs are degraded by an insulin degrading enzyme-like activity, but they can be stabilized by members of the FE65 family and translocate to the nucleus. Given that modulation of APP processing is a therapeutic target and that the APLPs are processed in a manner similar to APP, any strategy aimed at altering APP proteolysis will have to take into account possible effects on signaling by APLP 1 and 2.     

Expression, purification and characterization of a synthetic gene encoding human amyloid beta (Abeta1-42) in Escherichia coli

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by a progressive loss of cognitive function. Existing evidence indicates that abnormal processing and extracellular deposition of the longer form of the amyloid peptide Abeta(1-42), a proteolytic derivative of the amyloid precursor protein (APP), is a key step in the pathogenesis of AD. Active immunization with Abeta(1-42) has been shown to decrease brain beta deposition and improve cognitive performance in mouse models of AD. In the present study, we sought to express the synthetic gene encoding AB in Escherichia coli to enable rapid production of the antigen and its purification. The synthetic gene has been constructed from six oligonucleotides by employing overlapping PCR strategy and expressed in E. coli using the T7 promoter system. The recombinant peptide has been purified to homogeneity by a single step Ni+2 affinity chromatography. Enzyme-linked immunosorbent assay (ELISA) using polyclonal anti-Abeta(1-42) sera confirms that the corresponding linear B-cell epitopic sequences are available for immunorecognition in the recombinant peptide. This methodology enables rapid, continuous production and purification in bulk amounts of human Abeta sequence by employing bacterial expression system     

Amyloid precursor protein and amyloid beta peptide in human platelets. Role of cyclooxygenase and protein kinase C

The main component of Alzheimer's disease (AD) senile plaques is amyloid-beta peptide (Abeta), a proteolytic fragment of the amyloid precursor protein (APP). Platelets contain both APP and Abeta and may contribute to the perivascular amyloid deposition seen in AD. However, no data are available concerning the biochemical mechanism(s) involved in their formation and release by these cells. We found that human platelets released APP and Abeta following activation with collagen or arachidonic acid. Inhibition of platelet cyclooxygenase (COX) reduced APP but not Abeta release following those stimuli. In contrast, activation of platelets by thrombin and calcium ionophore caused release of both APP and Abeta in a COX-independent fashion. Ex vivo studies showed that, despite suppression of COX activity, administration of aspirin did not modify Abeta or APP levels in serum or plasma, suggesting that this enzyme plays only a minor role in vivo. We examined the regulation of APP cleavage and release from activated platelets and found that cleavage requires protein kinase C (PKC) activity and is regulated by the intracellular second messengers phosphatidylinositol 2-phosphate and Ca(2+). Our data provide the first evidence that in human platelets COX is a minor component of APP secretion whereas PKC plays a major role in the secretory cleavage of APP. By contrast, Abeta release may represent secretion of preformed peptide and is totally independent of both COX and PKC activity.     

Downregulation of myosin II-B by siRNA alters the subcellular localization of the amyloid precursor protein and increases amyloid-beta deposition in N2a cells

The Alzheimer's disease (AD) brain pathology is characterized by extracellular deposits of amyloid-beta (Abeta) peptides and intraneuronal fibrillar structures. These pathological features may be functionally linked, but the mechanism by which Abeta accumulation relates to neuronal degeneration is still poorly understood. Abeta peptides are fragments cleaved from the amyloid precursor protein (APP), a transmembrane protein ubiquitously expressed in the nervous system. Although the proteolytic processing of APP has been implicated in AD, the physiological function of APP and the subcellular site of APP cleavages remain unknown. The overall structure of the protein and its fast anterograde transport along the axon support the idea that APP functions as a vesicular receptor for cytoskeletal motor proteins. In the current study, we test the hypothesis that myosin II, important contributor to the cytoskeleton of neuronal cells, may influence the trafficking and/or the processing of APP. Our results demonstrate that downregulation of myosin II-B, the major myosin isoform in neurons, is able to increase Abeta deposition, concomitantly altering the subcellular localization of APP. These new insights might be important for the understanding of the function of APP and provide a novel conceptual framework in which to analyze its pathological role.     

Presenilin-1, nicastrin,amyloid precursor protein,and gamma-secretase activity are co-localized in the lysosomal membrane

Alzheimer's disease (AD) is caused by the cerebral deposition of beta-amyloid (Abeta), a 38-43-amino acid peptide derived by proteolytic cleavage of the amyloid precursor protein (APP). Initial studies indicated that final cleavage of APP by the gamma-secretase (a complex containing presenilin and nicastrin) to produce Abeta occurred in the endosomal/lysosomal system. However, other studies showing a predominant endoplasmic reticulum localization of the gamma-secretase proteins and a neutral pH optimum of in vitro gamma-secretase assays have challenged this conclusion. We have recently identified nicastrin as a major lysosomal membrane protein. In the present work, we use Western blotting and immunogold electron microscopy to demonstrate that significant amounts of mature nicastrin, presenilin-1, and APP are co-localized with lysosomal associated membrane protein-1 (cAMP-1) in the outer membranes of lysosomes. Furthermore, we demonstrate that these membranes contain an acidic gamma-secretase activity, which is immunoprecipitable with an antibody to nicastrin. These experiments establish APP, nicastrin, and presenilin-1 as resident lysosomal membrane proteins and indicate that gamma-secretase is a lysosomal protease. These data reassert the importance of the lysosomal/endosomal system in the generation of Abeta and suggest a role for lysosomes in the pathophysiology of AD.     

APP substitutions V715F and L720P alter PS1 conformation and differentially affect Abeta and AICD generation

The 37-43 amino acid Abeta peptide is the principal component of beta-amyloid deposits in Alzheimer's disease (AD) brain, and is derived by serial proteolysis of the amyloid precursor protein (APP) by beta- and gamma-secretase. gamma-Secretase also cleaves APP at Val50 in the Abeta numbering (epsilon cleavage), resulting in the release of a fragment called APP intracellular domain (AICD). The aim of this study was to determine whether amino acid substitutions in the APP transmembrane domain differentially affect Abeta and AICD generation. We found that the APPV715F substitution, which has been previously shown to dramatically decrease Abeta40 and Abeta42 while increasing Abeta38 levels, does not affect in vitro generation of AICD. Furthermore, we found that the APPL720P substitution, which has been previously shown to prevent in vitro generation of AICD, completely prevents Abeta generation. Using a fluorescence resonance energy transfer (FRET) method, we next found that both the APPV715F and APPL720P substitutions significantly increase the distance between the N- and C-terminus of presenilin 1 (PS1), which has been proposed to contain the catalytic site of gamma-secretase. In conclusion, both APPV715F and APPL720P change PS1 conformation with differential effects on Abeta and AICD production.