Asp664 cleavage of amyloid precursor protein induces tau phosphorylation by decreasing protein phosphatase 2A activity

Caspase cleavage of amyloid precursor protein (APP) has been reported to be important in amyloid beta protein (Aβ)‐mediated neurotoxicity. However, the underlying mechanisms are not clearly understood. In this study, we explored the effect of caspase cleavage of APP on tau phosphorylation in relation to Aβ. We found that Asp664 cleavage of APP increased tau phosphorylation at Thr212 and Ser262 in N2A cells and primary cultured hippocampal neurons. Compared with wild‐type APP, protein phosphatase 2A (PP2A) activity was significantly increased when Asp664 cleavage was blocked by the D664A point mutation. Furthermore, we found that over‐expression of C31 reduced PP2A activity. C31 binds directly to the PP2A catalytic subunit, through the asparagine, proline, threonine, tyrosine (NPTY) motif, which is essential for C31‐induced tau hyperphosphorylation. However, it appears that the other fragment produced by Asp664 cleavage, Jcasp, modulates neither PP2A activity nor tau hyperphosphorylation. Asp664 cleavage and accompanying tau hyperphosphorylation were remarkably diminished by blockage of Aβ production using a γ‐secretase inhibitor. Taken together, our results suggest that Asp664 cleavage of APP leads to tau hyperphosphorylation at specific epitopes by modulating PP2A activity as a downstream of Aβ. Direct binding of C31 to PP2A through the C31‐NPTY domain was identified as a mechanism underlying this effect.     

Phosphorylation of amyloid precursor protein (APP) at Tyr687 regulates APP processing by alpha- and gamma-secretase

Abnormal proteolytic processing of amyloid precursor protein (APP) is a pathologic feature of Alzheimer’s disease. Recent studies have demonstrated that serine/threonine phosphorylation specifically at amino-acid residue Thr668 (APP695 numbering) regulates APP processing. In this study, we investigated the possibility that tyrosine phosphorylation of APP regulates APP processing. A tyrosine kinase inhibitor decreased expression of the C83 fragment which is a cleaved product of APP by α-secretase. By overexpressing APP mutant proteins, Tyr687 was found to be the major tyrosine kinase phosphorylation site. Expression of the C83 fragment was decreased in APPY687A-expressing cells relative to APP wild-type (APPWT)-expressing cells, which likely reflects the different cellular localization patterns of these two proteins. Expression of APP intracellular domain (AICD) which is a cleaved product of the C83 fragment by γ-secretase was decreased in C83Y687A-expressing cells. These results suggest that phosphorylation of APP at Tyr687 regulates APP processing by α- and γ-secretases, determining the expression level of AICD.     

Turnover of C99 is Controlled by a Crosstalk between ERAD and Ubiquitin-Independent Lysosomal Degradation in Human Neuroglioma Cells

Alzheimer’s disease (AD) is characterized by the buildup of amyloid-β peptides (Aβ) aggregates derived from proteolytic processing of the β-amyloid precursor protein (APP). Amyloidogenic cleavage of APP by β-secretase/BACE1 generates the C-terminal fragment C99/CTFβ that can be subsequently cleaved by γ-secretase to produce Aβ. Growing evidence indicates that high levels of C99/CTFβ are determinant for AD. Although it has been postulated that γ-secretase-independent pathways must control C99/CTFβ levels, the contribution of organelles with degradative functions, such as the endoplasmic reticulum (ER) or lysosomes, is unclear. In this report, we investigated the turnover and amyloidogenic processing of C99/CTFβ in human H4 neuroglioma cells, and found that C99/CTFβ is localized at the Golgi apparatus in contrast to APP, which is mostly found in endosomes. Conditions that localized C99/CTFβ to the ER resulted in its degradation in a proteasome-dependent manner that first required polyubiquitination, consistent with an active role of the ER associated degradation (ERAD) in this process. Furthermore, when proteasomal activity was inhibited C99/CTFβ was degraded in a chloroquine (CQ)-sensitive compartment, implicating lysosomes as alternative sites for its degradation. Our results highlight a crosstalk between degradation pathways within the ER and lysosomes to avoid protein accumulation and toxicity.     

Ligand-Dependent Activation of EphA4 Signaling Regulates the Proteolysis of Amyloid Precursor Protein Through a Lyn-Mediated Pathway

Alzheimer's disease is the most common dementia afflicting the elderly in modern society. This disease arises from the neurotoxicity elicited by abnormal aggregates of amyloid-β (Aβ) protein. Such aggregates form through the cleavage of amyloid precursor protein (APP) by β-secretase and the subsequent proteolysis of the APP C-terminal fragment (APP-βCTF or C99) by γ-secretase to yield Aβ and APP intracellular domain (AICD). Recent evidence suggests that C99 and AICD may exert harmful effects on cells, suggesting that the proteolytic products of APP, including Aβ, C99, and AICD, could play a pivotal role in neuronal viability. Here, we demonstrate that ligand-activated EphA4 signaling governs the proteostasis of C99, AICD, and Aβ, without significantly affecting γ-secretase activity. EphA4 induced accumulation of C99 and AICD through a Lyn-dependent pathway; activation of this pathway triggered phosphorylation of EphA4, resulting in positive feedback of C99 and AICD proteostasis. Inhibition of EphA4 by dasatinib, a receptor tyrosine kinase inhibitor, effectively suppressed C99 and AICD accumulation. Furthermore, EphA4 signaling controlled C99 and AICD proteolysis through the ubiquitin–proteasome system. In conclusion, we have identified an EphA4–Lyn pathway that is essential for the metabolism of APP and its proteolytic derivatives, thereby providing novel pharmacological targets for the development of anti-Aβ therapeutics for AD.     

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.     

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.     

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.     

Lysine 624 of the amyloid precursor protein (APP) is a critical determinant of amyloid β peptide length: support for a sequential model of γ-secretase intramembrane proteolysis and regulation by the amyloid β precursor protein (APP) juxtamembrane region

γ-Secretase is a multiprotein intramembrane cleaving aspartyl protease (I-CLiP) that catalyzes the final cleavage of the amyloid β precursor protein (APP) to release the amyloid β peptide (Aβ). Aβ is the primary component of senile plaques in Alzheimer's disease (AD), and its mechanism of production has been studied intensely. γ-Secretase executes multiple cleavages within the transmembrane domain of APP, with cleavages producing Aβ and the APP intracellular domain (AICD), referred to as γ and ε, respectively. The heterogeneous nature of the γ cleavage that produces various Aβ peptides is highly relevant to AD, as increased production of Aβ 1-42 is genetically and biochemically linked to the development of AD. We have identified an amino acid in the juxtamembrane region of APP, lysine 624, on the basis of APP695 numbering (position 28 relative to Aβ) that plays a critical role in determining the final length of Aβ peptides released by γ-secretase. Mutation of this lysine to alanine (K28A) shifts the primary site of γ-secretase cleavage from 1-40 to 1-33 without significant changes to ε cleavage. These results further support a model where ε cleavage occurs first, followed by sequential proteolysis of the remaining transmembrane fragment, but extend these observations by demonstrating that charged residues at the luminal boundary of the APP transmembrane domain limit processivity of γ-secretase.     

The Swedish APP mutation alters the effect of genetically reduced BACE1 expression on the APP processing

Inhibition of β-secretase (BACE1) is a key therapeutic approach in Alzheimer's disease (AD), as BACE1 initiates amyloid-β (Aβ) cleavage from the β-amyloid precursor protein (APP). As Aβ reductions in mice lacking one BACE1 allele diverged considerably between studies we investigated the effect of BACE1 knock-out in more detail. With both BACE1 alleles the Swedish mutation (APP23 mice) increased APP processing and shifted it towards the β-secretase pathway as compared with non-mutated APP expressed at a similar level (APP51/16 mice). This effect was much smaller then observed in cell culture. An about 50% decrease in BACE1 enzyme activity resulted in a sub-proportional Aβ reduction with the Swedish mutation (-20%) and even less for non-mutated APP (-16%). In wild-type mice, the Aβ reduction may be even further diminished. Other metabolites of the β-secretase pathway decreased accordingly while the alternative α-secretase pathway increased. Complete BACE1 deletion strongly enhanced these changes. The remaining Aβ signal also described by others can be explained by assay cross-reactivity with other APP metabolites supporting BACE1 as the major β-secretase. Our data indicate that BACE1 is in excess over APP at the cleavage site(s). Alterations in APP expression or substrate properties, therefore, quantitatively change its cleavage and Aβ generation.     

Biogenesis of gamma-secretase early in the secretory pathway

γ-Secretase is responsible for proteolytic maturation of signaling and cell surface proteins, including amyloid precursor protein (APP). Abnormal processing of APP by γ-secretase produces a fragment, Aβ₄₂, that may be responsible for Alzheimer's disease (AD). The biogenesis and trafficking of this important enzyme in relation to aberrant Aβ processing is not well defined. Using a cell-free reaction to monitor the exit of cargo proteins from the endoplasmic reticulum (ER), we have isolated a transient intermediate of γ-secretase. Here, we provide direct evidence that the γ-secretase complex is formed in an inactive complex at or before the assembly of an ER transport vesicle dependent on the COPII sorting subunit, Sec24A. Maturation of the holoenzyme is achieved in a subsequent compartment. Two familial AD (FAD)–linked PS1 variants are inefficiently packaged into transport vesicles generated from the ER. Our results suggest that aberrant trafficking of PS1 may contribute to disease pathology.     

γ-Secretase complexes containing caspase-cleaved presenilin-1 increase intracellular Aβ(42) /Aβ(40) ratio

Markers for caspase activation and apoptosis have been shown in brains of Alzheimer's disease (AD) patients and AD-mouse models. In neurons, caspase activation is associated with elevated amyloid β-peptide (Aβ) production. Caspases cleave numerous substrates including presenilin-1 (PS1). The cleavage takes place in the large cytosolic loop of PS1-C-terminal fragment (PS1CTF), generating a truncated PS1CTF lacking half of the loop domain (caspCTF). The loop has been shown to possess important regulatory functions with regard to Aβ(40) and Aβ(42) production. Previously, we have demonstrated that γ-secretase complexes are active during apoptosis regardless of caspase cleavage in the PS1CTF-loop. Here, a PS1/PS2-knockout mouse blastocyst-derived cell line was used to establish stable or transient cell lines expressing either caspCTF or full-length CTF (wtCTF). We show that caspCTF restores γ-secretase activity and forms active γ-secretase complexes together with Nicastrin, Pen-2, Aph-1 and PS1-N-terminal fragment. Further, caspCTF containing γ-secretase complexes have a sustained capacity to cleave amyloid precursor protein (APP) and Notch, generating APP and Notch intracellular domain, respectively. However, when compared to wtCTF cells, caspCTF cells exhibit increased intracellular production of Aβ(42) accompanied by increased intracellular Aβ(42) /Aβ(40) ratio without changing the Aβ secretion pattern. Similarly, induction of apoptosis in wtCTF cells generate a similar shift in intracellular Aβ pattern with increased Aβ(42) /Aβ(40) ratio. In summary, we show that caspase cleavage of PS1 generates a γ-secretase complex that increases the intracellular Aβ(42) /Aβ(40) ratio. This can have implications for AD pathogenesis and suggests caspase inhibitors as potential therapeutic agents.     

Rac1 changes the substrate specificity of gamma-secretase between amyloid precursor protein and Notch1

Beta amyloid peptide is generated from amyloid precursor protein (APP) by proteolytic cleavage of β- and γ-secretases, and plays a critical role in the pathogenesis of Alzheimer’s disease. Since γ-secretase cleaves several proteins including APP and Notch in a number of cell types, it is important to understand the conditions determining γ-secretase substrate specificity. In the present study, inhibition of Rac1 attenuated γ-secretase activity for APP, resulting in decreased production of the APP intracellular domain but accumulated C-terminal fragments (APP-CTF). In contrast, Rac1 inhibitor, NSC23766 increased production of the Notch1 intracellular domain but slightly decreased the ectodomain-shed form of Notch1 (NotchΔE). To elucidate the mechanism underlying these observations, we performed co-immunoprecipitation experiments to analyze the interaction between Rac1 and presenilin1 (PS1), a component of the γ-secretase complex. Inhibition of Rac1 enhanced its interaction with PS1. Under the same condition, PS1 interacted more strongly with NotchΔE than with APP-CTF. Our results suggested that PS1 determines the preferred substrate for γ-secretase between APP and Notch1, depending on the activation status of Rac1.     

Etazolate, a neuroprotective drug linking GABA(A) receptor pharmacology to amyloid precursor protein processing

Pharmacological modulation of the GABA_A receptor has gained increasing attention as a potential treatment for central processes affected in Alzheimer disease (AD), including neuronal survival and cognition. The proteolytic cleavage of the amyloid precursor protein (APP) through the α-secretase pathway decreases in AD, concurrent with cognitive impairment. This APP cleavage occurs within the β-amyloid peptide (Aβ) sequence, precluding formation of amyloidogenic peptides and leading to the release of the soluble N-terminal APP fragment (sAPPα) which is neurotrophic and procognitive. In this study, we show that at nanomolar-low micromolar concentrations, etazolate, a selective GABA_A receptor modulator, stimulates sAPPα production in rat cortical neurons and in guinea pig brains. Etazolate (20 nM–2 μM) dose-dependently protected rat cortical neurons against Aβ-induced toxicity. The neuroprotective effects of etazolate were fully blocked by GABA_A receptor antagonists indicating that this neuroprotection was due to GABA_A receptor signalling. Baclofen, a GABA_B receptor agonist failed to inhibit the Aβ-induced neuronal death. Furthermore, both pharmacological α-secretase pathway inhibition and sAPPα immunoneutralization approaches prevented etazolate neuroprotection against Aβ, indicating that etazolate exerts its neuroprotective effect via sAPPα induction. Our findings therefore indicate a relationship between GABA_A receptor signalling, the α-secretase pathway and neuroprotection, documenting a new therapeutic approach for AD treatment.     

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.     

Distinct secretases, a cysteine protease and a serine protease, generate the C termini of amyloid beta-proteins Abeta1-40 and Abeta1-42, respectively

The carboxy-terminal ends of the 40- and 42-amino acids amyloid beta-protein (Abeta) may be generated by the action of at least two different proteases termed gamma(40)- and gamma(42)-secretase, respectively. To examine the cleavage specificity of the two proteases, we treated amyloid precursor protein (APP)-transfected cell cultures with several dipeptidyl aldehydes including N-benzyloxycarbonyl-Leu-leucinal (Z-LL-CHO) and the newly synthesized N-benzyloxycarbonyl-Val-leucinal (Z-VL-CHO). All dipeptidyl aldehydes tested inhibited production of both Abeta1-40 and Abeta1-42. Changes in the P1 and P2 residues of these aldehydes, however, indicated that the amino acids occupying these positions are important for the efficient inhibition of gamma-secretases. Peptidyl aldehydes inhibit both cysteine and serine proteases, suggesting that the two gamma-secretases belong to one of these mechanistic classes. To differentiate between the two classes of proteases, we treated our cultures with the specific cysteine protease inhibitor E-64d. This agent inhibited production of secreted Abeta1-40, with a concomitant accumulation of its cellular precursor indicating that gamma(40)-secretase is a cysteine protease. In contrast, this treatment increased production of secreted Abeta1-42. No inhibition of Abeta production was observed with the potent calpain inhibitor I (acetyl-Leu-Leu-norleucinal), suggesting that calpain is not involved. Together, these results indicate that gamma(40)-secretase is a cysteine protease distinct from calpain, whereas gamma(42)-secretase may be a serine protease. In addition, the two secretases may compete for the same substrate. Dipeptidyl aldehyde treatment of cultures transfected with APP carrying the Swedish mutation resulted in the accumulation of the beta-secretase C-terminal APP fragment and a decrease of the alpha-secretase C-terminal APP fragment, indicating that this mutation shifts APP cleavage from the alpha-secretase site to the beta-secretase site.     

Activation of α-secretase cleavage

Alpha-secretase-mediated cleavage of the amyloid precursor protein (APP) releases the neuroprotective APP fragment sαAPP and prevents amyloid β peptide (Aβ) generation. Moreover, α-secretase-like cleavage of the Aβ transporter 'receptor for advanced glycation end products' counteracts the import of blood Aβ into the brain. Assuming that Aβ is responsible for the development of Alzheimer's disease (AD), activation of α-secretase should be preventive. α-Secretase-mediated APP cleavage can be activated via several G protein-coupled receptors and receptor tyrosine kinases. Protein kinase C, mitogen-activated protein kinases, phosphatidylinositol 3-kinase, cAMP and calcium are activators of receptor-induced α-secretase cleavage. Selective targeting of receptor subtypes expressed in brain regions affected by AD appears reasonable. Therefore, the PACAP receptor PAC1 and possibly the serotonin 5-HT(6) receptor subtype are promising targets. Activation of APP α-secretase cleavage also occurs upon blockade of cholesterol synthesis by statins or zaragozic acid A. Under physiological statin concentrations, the brain cholesterol content is not influenced. Statins likely inhibit Aβ production in the blood by α-secretase activation which is possibly sufficient to inhibit AD development. A disintegrin and metalloproteinase 10 (ADAM10) acts as α-secretase on APP. By targeting the nuclear retinoic acid receptor β, the expression of ADAM10 and non-amyloidogenic APP processing can be enhanced. Excessive activation of ADAM10 should be avoided because ADAM10 and also ADAM17 are not APP-specific. Both ADAM proteins cleave various substrates, and therefore have been associated with tumorigenesis and tumor progression.     

Mechanism of Intramembrane Cleavage of Alcadeins by γ-Secretase

Background

Alcadein proteins (Alcs; Alcα, Alcβand Alcγ) are predominantly expressed in neurons, as is Alzheimer''s β-amyloid (Aβ) precursor protein (APP). Both Alcs and APP are cleaved by primary α- or β-secretase to generate membrane-associated C-terminal fragments (CTFs). Alc CTFs are further cleaved by γ-secretase to secrete p3-Alc peptide along with the release of intracellular domain fragment (Alc ICD) from the membrane. In the case of APP, APP CTFβ is initially cleaved at the ε-site to release the intracellular domain fragment (AICD) and consequently the γ-site is determined, by which Aβ generates. The initial ε-site is thought to define the final γ-site position, which determines whether Aβ40/43 or Aβ42 is generated. However, initial intracellular ε-cleavage sites of Alc CTF to generate Alc ICD and the molecular mechanism that final γ-site position is determined remains unclear in Alcs.

Methodology

Using HEK293 cells expressing Alcs plus presenilin 1 (PS1, a catalytic unit of γ-secretase) and the membrane fractions of these cells, the generation of p3-Alc possessing C-terminal γ-cleavage site and Alc ICD possessing N-terminal ε-cleavage site were analysed with MALDI-TOF/MS. We determined the initial ε-site position of all Alcα, Alcβ and Alcγ, and analyzed the relationship between the initially determined ε-site position and the final γ-cleavage position.

Conclusions

The initial ε-site position does not always determine the final γ-cleavage position in Alcs, which differed from APP. No additional γ-cleavage sites are generated from artificial/non-physiological positions of ε-cleavage for Alcs, while the artificial ε-cleavage positions can influence in selection of physiological γ-site positions. Because alteration of γ-secretase activity is thought to be a pathogenesis of sporadic Alzheimer''s disease, Alcs are useful and sensitive substrate to detect the altered cleavage of substrates by γ-secretase, which may be induced by malfunction of γ-secretase itself or changes of membrane environment for enzymatic reaction.     

Caspase cleavage of members of the amyloid precursor family of proteins

The synapse loss and neuronal cell death characteristic of Alzheimer's disease (AD) are believed to result in large part from the neurotoxic effects of beta-amyloid peptide (Abeta), a 40-42 amino acid peptide(s) derived proteolytically from beta-amyloid precursor protein (APP). However, APP is also cleaved intracellularly to generate a second cytotoxic peptide, C31, and this cleavage event occurs in vivo as well as in vitro and preferentially in the brains of AD patients (Lu et al. 2000). Here we show that APPC31 is toxic to neurons in primary culture, and that like APP, the APP family members APLP1 and possibly APLP2 are cleaved by caspases at their C-termini. The carboxy-terminal peptide derived from caspase cleavage of APLP1 shows a degree of neurotoxicity comparable to APPC31. Our results suggest that even though APLP1 and APLP2 cannot generate Abeta, they may potentially contribute to the pathology of AD by generating peptide fragments whose toxicity is comparable to that of APPC31.     

Independent Relationship between Amyloid Precursor Protein (APP) Dimerization and γ-Secretase Processivity

Altered production of β-amyloid (Aβ) from the amyloid precursor protein (APP) is closely associated with Alzheimer’s disease (AD). APP has a number of homo- and hetero-dimerizing domains, and studies have suggested that dimerization of β-secretase derived APP carboxyl terminal fragment (CTFβ, C99) impairs processive cleavage by γ-secretase increasing production of long Aβs (e.g., Aβ1-42, 43). Other studies report that APP CTFβ dimers are not γ-secretase substrates. We revisited this issue due to observations made with an artificial APP mutant referred to as 3xK-APP, which contains three lysine residues at the border of the APP ectodomain and transmembrane domain (TMD). This mutant, which dramatically increases production of long Aβ, was found to form SDS-stable APP dimers, once again suggesting a mechanistic link between dimerization and increased production of long Aβ. To further evaluate how multimerization of substrate affects both initial γ-secretase cleavage and subsequent processivity, we generated recombinant wild type- (WT) and 3xK-C100 substrates, isolated monomeric, dimeric and trimeric forms of these proteins, and evaluated both ε-cleavage site utilization and Aβ production. These show that multimerization significantly impedes γ-secretase cleavage, irrespective of substrate sequence. Further, the monomeric form of the 3xK-C100 mutant increased long Aβ production without altering the initial ε-cleavage utilization. These data confirm and extend previous studies showing that dimeric substrates are not efficient γ-secretase substrates, and demonstrate that primary sequence determinants within APP substrate alter γ-secretase processivity.     

Generation of Alzheimer disease-associated amyloid β42/43 peptide by γ-secretase can be inhibited directly by modulation of membrane thickness

Pathogenic generation of amyloid β-peptide (Aβ) by sequential cleavage of β-amyloid precursor protein (APP) by β- and γ-secretases is widely believed to causally underlie Alzheimer disease (AD). β-Secretase initially cleaves APP thereby generating a membrane-bound APP C-terminal fragment, from which γ-secretase subsequently liberates 37-43-amino acid long Aβ species. Although the latter cleavages are intramembranous and although lipid alterations have been implicated in AD, little is known of how the γ-secretase-mediated release of the various Aβ species, in particular that of the pathogenic longer variants Aβ(42) and Aβ(43), is affected by the lipid environment. Using a cell-free system, we have directly and systematically investigated the activity of γ-secretase reconstituted in defined model membranes of different thicknesses. We found that bilayer thickness is a critical parameter affecting both total activity as well as cleavage specificity of γ-secretase. Whereas the generation of the pathogenic Aβ(42/43) species was markedly attenuated in thick membranes, that of the major and rather benign Aβ(40) species was enhanced. Moreover, the increased production of Aβ(42/43) by familial AD mutants of presenilin 1, the catalytic subunit of γ-secretase, could be substantially lowered in thick membranes. Our data demonstrate an effective modulation of γ-secretase activity by membrane thickness, which may provide an approach to lower the generation of the pathogenic Aβ(42/43) species.