Molecular characterisation of the Alzheimer's amyloid precursor protein secretases.
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Alzheimer Disease↗
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Presenilin 1 mediates protein kinase C dependent alpha-secretase derived amyloid precursor protein secretion and mitogen-activated protein kinase activation in presenilin 1 transfected human embryonic kidney 293 cell.
We investigated the role of presenilin 1 (PS1) in the secretion of alpha-secretase derived amyloid precursor protein (sAPP alpha) and associated intracellular signaling pathways. Human embryonic kidney (HEK) 293 cells were transfected with exon 9 deletion (deltaE9) mutant PS1 cDNA in an ecdyson-inducible system. sAPPalpha secretion was lower in the mutant PS1 expressing cells compared with non-expressing cells. When activated by PDBu, secretion of sAPPalpha and the level of phosphorylated mitogen activated protein kinase (MAPK) were greatly enhanced in deltaE9 PS1 uninduced cells, but not in the mutant PS1 induced cells. PD98059, a MAPK inhibitor, blocked PDBu induced sAPPalpha secretion from deltaE9 PS1 uninduced cells but had no effect on the mutant PS1 induced cells. These data indicate that PS1 mediates PDBu-induced sAPPalpha secretion and MAPK activation.
Alzheimer Disease↗
Angiotensin-converting enzyme and the amyloid precursor protein secretases.
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Amyloid Precursor Protein Secretases↗
Levels of alpha- and beta-secretase cleaved amyloid precursor protein in the cerebrospinal fluid of Alzheimer's disease patients.
Alternative cleavage of the amyloid precursor protein (APP) results in generation and secretion of both soluble APP (sAPP) and beta-amyloid (Abeta). Abeta is the main component of the amyloid depositions in the brains of Alzheimer's disease (AD) patients. Using Western blotting, we compared the levels of alpha-secretase cleaved sAPP, beta-secretase cleaved sAPP and total sAPP, in cerebrospinal fluid (CSF) from 13 sporadic AD patients and 13 healthy controls. Our findings show significant amounts of beta-secretase cleaved sAPP in CSF. There was no statistically significant difference in the levels of beta-secretase cleaved sAPP between AD patients and controls. The levels of alpha-secretase cleaved sAPP and total sAPP were, however, found to be significantly lower in the AD patients than in the controls.
Aged↗
Reduced cerebrospinal fluid levels of alpha-secretase-cleaved amyloid precursor protein in aged rats: correlation with spatial memory deficits.
The amyloid precursor protein undergoes proteolysis at several sites to yield a number of functionally relevant peptides, including beta-amyloid and the soluble amyloid precursor protein derivatives alpha-soluble amyloid precursor protein and beta-soluble amyloid precursor protein. beta-Amyloid is the primary constituent of senile plaques associated with Alzheimer's disease, while a-soluble amyloid precursor protein promotes synaptogenesis and plays a role in neuroprotective processes. We tested for age-related alterations in these amyloid precursor protein proteolytically derived peptides by measuring the levels of alpha-soluble amyloid precursor protein, total soluble amyloid precursor proteins (alpha- and beta-soluble amyloid precursor protein combined) and beta-amyloid in cerebrospinal fluid from three-, 13- and 23-month-old Fischer-344 rats. Western blot analysis using selective antibodies revealed 50% less total soluble amyloid precursor protein and a-soluble amyloid precursor protein in cisternal cerebrospinal fluid from 23-month-old rats compared with three- and 13-month-old animals. Mass spectrometric analysis indicated, however, that beta-amyloid in cerebrospinal fluid was not different between the three age groups. In a second group of young (five to six months of age) and aged (24-25 months of age) rats, spatial working and reference memory were assessed in a water maze followed by collection of cerebrospinal fluid. As a group, the aged rats consistently performed below the young rats in both working and reference memory tests. The aged rats also had 49% less cerebrospinal fluid alpha-soluble amyloid precursor protein than did their younger counterparts. There was a positive correlation (r= 0.52-0.57, P < 0.001) between performance in spatial memory tasks and cerebrospinal fluid alpha-soluble amyloid precursor protein in these young and aged rats. These results suggest that there is a positive association between cerebrospinal fluid levels of alpha-soluble amyloid precursor protein and cognitive performance in rats, and that alpha-soluble amyloid precursor protein may be involved in the spatial learning and memory changes that accompany ageing.
Aging↗
Beta-secretase-cleaved amyloid precursor protein accumulates at actin inclusions induced in neurons by stress or amyloid beta: a feedforward mechanism for Alzheimer's disease.
Rod-like inclusions (rods), composed of actin saturated with actin depolymerizing factor (ADF)/cofilin, are induced in hippocampal neurons by ATP depletion, oxidative stress, and excess glutamate and occur in close proximity to senile plaques in human Alzheimer's disease (AD) brain (Minamide et al., 2000). Here, we show rods are found in brains from transgenic AD mice. Soluble forms of amyloid beta (Abeta(1-42)) induce the formation of rods in a maximum of 19% of cultured hippocampal neurons in a time- and concentration-dependent manner. Approximately one-half of the responding neurons develop rods within 6 h or with as little as 10 nM Abeta(1-42). Abeta(1-42) induces the activation (dephosphorylation) of ADF/cofilin in neurons that form rods. Vesicles containing amyloid precursor protein (APP), beta-amyloid cleavage enzyme, and presenilin-1, a component of the gamma-secretase complex, accumulate at rods. The beta-secretase-cleaved APP (either beta-C-terminal fragment of APP or Abeta) also accumulates at rods. These results suggest that rods, formed in response to either Abeta or some other stress, block the transport of APP and enzymes involved in its processing to Abeta. These stalled vesicles may provide a site for producing Abeta(1-42), which may in turn induce more rods in surrounding neurons, and expand the degenerative zone resulting in plaque formation.
Actins↗
[Effect of an inhibitor of alpha-secretase metabolizing amyloid precursor protein on memory in rats].
Intracortical administration of 10(-4) M batimastat, a specific inhibitor of one of metalloproteinases metabolizing amyloid precursor protein, namely alpha-secretase, to adult rats resulted in a decrease in the number of correct runs in a one-level 8-arm maze down to 92.78 +/- 1.03% of the control values (p < 0.01) already 60 min after an injection. The effect of a single injection of the inhibitor to adult rats did not have a prolonged character. However, injections of batimastat into the cortex of brain hemispheres of rats during early postnatal ontogenesis (5th and 7th days after birth) resulted in considerable deterioration of 8-arm maze orientation of these animals at adult age (90.92 +/- 2.21% of correct runs, p < 0.001) compared to control animals. The findings suggest an important role of alpha-secretase in memorization. A possible role of alpha-secretase in memory and pathogenesis of Alzheimer's disease is discussed.
Amyloid Precursor Protein Secretases↗
Calcium ionophore A23187 specifically decreases the secretion of beta-secretase cleaved amyloid precursor protein during apoptosis in primary rat cortical cultures.
Alzheimer's disease (AD) is characterized by the degeneration and loss of neurons, intracellular neurofibrillary tangles and the accumulation of extracellular senile plaques consisting mainly of beta-amyloid (A beta). A beta is generated from the amyloid precursor protein (APP) by sequential beta- and gamma-secretase cleavage. Alternatively, APP may be cleaved within the A beta region by alpha-secretase, preventing A beta formation. Here we investigated APP processing and secretion in primary neurons, using either colchicine or the calcium ionophore A23187 to induce apoptosis. Cell viability was determined by MTT measurements and apoptosis was further confirmed by annexin V and propidium iodide staining. We found that exposure to A23187 significantly decreased the secretion of soluble beta-secretase cleaved APP (beta-sAPP) in a caspase-dependent manner, although the secretion of total soluble APP beta sAPP) did not change. In addition, caspase inhibition restored cell viability to control levels. Exposure to colchicine did not change the amount of either secreted beta-sAPP or total sAPP and caspase inhibition was only partially able to restore cell viability. We conclude that calcium homeostasis is an important apoptotic effector specifically affecting the beta-secretase cleavage of APP.
Amino Acid Chloromethyl Ketones↗
Beta-secretase-cleaved amyloid precursor protein in Alzheimer brain: a morphologic study.
beta-amyloid (Abeta) is the main constituent of senile plaques seen in Alzheimer's disease. Abeta is derived from the amyloid precursor protein (APP) via proteolytic cleavage by proteases beta- and gamma-secretase. In this study, we examined content and localization of beta-secretase-cleaved APP (beta-sAPP) in brain tissue sections from the frontal, temporal and occipital lobe. Strong granular beta-sAPP staining was found throughout the gray matter of all three areas, while white matter staining was considerably weaker. beta-sAPP was found to be localized in astrocytes and in axons. We found the beta-sAPP immunostaining to be stronger and more extensive in gray matter in Alzheimer disease (AD) cases than controls. The axonal beta-sAPP staining was patchy and unevenly distributed for the AD cases, indicating impaired axonal transport. beta-sAPP was also found surrounding senile plaques and cerebral blood vessels. The results presented here show altered beta-sAPP staining in the AD brain, suggestive of abnormal processing and transport of APP.
Alzheimer Disease↗
Decreased alpha-secretase-cleaved amyloid precursor protein as a diagnostic marker for Alzheimer's disease.
The neuropathologic hallmarks of Alzheimer's disease (AD) are extracellular plaques and intracellular neurofibrillary tangles. A constituent of senile plaques in AD is beta-amyloid, a hydrophobic peptide of 39-43 amino acids and a fragment of the amyloid precursor protein (APP). APP can be metabolized by at least two pathways, one of which involves generation of soluble APP by an unidentified enzyme named alpha-secretase. This cleavage generates alpha-secretase-cleaved, soluble APP (alpha-sAPP), which in this investigation was measured by a new assay in cerebrospinal fluid (CSF) from members of a Swedish AD family with a pathogenic mutation at APP670/671 (ref. 2). Family members who carry the mutation and are diagnosed with AD had low levels of alpha-sAPP (160 +/- 48 ng ml-1), with no overlap compared with non-carriers (257 +/- 48 ng ml-1). Carriers of the presymptomatic mutation showed intermediate alpha-sAPP levels. Today there exists no antemortem marker in AD with sufficient sensitivity and specificity, but measurement of alpha-sAPP represents a new and promising diagnostic marker.
Adult↗
Modeling of substrate specificity of the Alzheimer's disease amyloid precursor protein beta-secretase.
The enzyme BACE (beta-site APP-cleaving enzyme) has recently been identified as the beta-secretase that cleaves the amyloid precursor protein (APP) to produce the N terminus of the Abeta peptide found in plaques in the brains of Alzheimer's disease patients. BACE is an aspartic protease similar to pepsin and renin. Comparative modeling of the three-dimensional structure of BACE in complex with its substrate shows that several residues confer specificity of the enzyme for APP. In particular, Arg296 forms a salt-bridge with the P1' Asp of the APP substrate, explaining the unusual preference of BACE among aspartic proteases for a P1' residue that is negatively charged. Several hydrophobic residues in the enzyme form a pocket for the P1 hydrophobic residue (Met in wild-type APP and Leu in APP with the "Swedish mutation" associated with early-onset of Alzheimer's disease). Inhibitors that can bind to the BACE active site may prove useful for drugs to treat and prevent Alzheimer's disease.
Alzheimer Disease↗
Cholesterol modulates alpha-secretase cleavage of amyloid precursor protein.
Amyloid precursor protein (APP) and cholesterol metabolism are genetically linked to Alzheimer's disease, the latter through apolipoprotein E, a lipid and cholesterol transport protein. We have examined the hypothesis that the processing of APP is disrupted by elevated cholesterol, which is known to modulate the activity of several transmembrane proteins. In the current study, cholesterol, solubilized by methyl- beta-cyclodextrin or ethanol, was added to the culture media of APP 751 stably transfected HEK 293 cells. Radiolabeled APP and APPsol (the soluble N-terminal derivative following alpha-secretase cleavage) were precipitated from lysates and conditioned media of stably transfected HEK 293 cells; the relative levels were determined by quantitative densitometry following separation by SDS-polyacrylamide gel electrophoresis. The data show that cholesterol, solubilized by methyl-beta-cyclodextrin, greatly reduced the levels of APPsol. Low doses of ethanol-solubilized cholesterol similarly caused a dramatic reduction of APPsol. By contrast, levels of APP holoprotein remained the same or increased. The large decrease seen in APPsol production was not due to nonspecific inhibition of secretion because several secreted proteins increased in level. Cholesterol, which impedes membrane fluidity, may lower APPsol production by impeding the interaction of the substrate with its protease(s). If APPsol were to function trophically, as suggested by other studies, the current conclusion suggests that changes in cellular cholesterol levels in Alzheimer's disease could contribute to neuronal degeneration by decreasing the production of APPsol.
Amyloid Precursor Protein Secretases↗
Autophagic vacuoles are enriched in amyloid precursor protein-secretase activities: implications for beta-amyloid peptide over-production and localization in Alzheimer's disease.
In Alzheimer's disease (AD), the neuropathologic hallmarks of beta-amyloid deposition and neurofibrillary degeneration are associated with early and progressive pathology of the endosomal-lysosomal system. Abnormalities of autophagy, a major pathway to lysosomes for protein and organelle turnover, include marked accumulations of autophagy-related vesicular compartments (autophagic vacuoles or AVs) in affected neurons. Here, we investigated the possibility that AVs contain the proteases and substrates necessary to cleave the amyloid precursor protein (APP) to A beta peptide that forms beta-amyloid, a key pathogenic factor in AD. AVs were highly purified using a well-established metrizamide gradient procedure from livers of transgenic YAC mice overexpressing wild-type human APP. By Western blot analysis, AVs contained APP, beta CTF - the beta-cleaved carboxyl-terminal domain of APP, and BACE, the protease-mediating beta-cleavage of APP. beta-Secretase activity measured against a fluorogenic peptide was significantly enriched in the AV fraction relative to whole-liver lysate. Compared to other recovered subcellular fractions, AVs exhibited the highest specific activity of gamma-secretase based on a fluorogenic assay and inhibition by a specific inhibitor of gamma-secretase, DAPT. AVs were also the most enriched subcellular fraction in levels of the gamma-secretase components presenilin and nicastrin. Immunoelectron microscopy demonstrated selective immunogold labeling of AVs with antibodies specific for the carboxyl termini of human A beta 40 and A beta 42. These data indicate that AVs are a previously unrecognized and potentially highly active compartment for A beta generation and suggest that the abnormal accumulation of AVs in affected neurons of the AD brain contributes to beta-amyloid deposition.
Alzheimer Disease↗
Blood brain barrier endothelial cells express candidate amyloid precursor protein-cleaving secretases.
Proteolytic cleavage of the amyloid precursor protein (A beta PP) results in the generation of the amyloidogenic fragment known as amyloid beta peptide (A beta). Deposition of A beta in the brain parenchyma and cerebrovasculature is a feature of Alzheimer's disease (AD). To date, the process whereby A beta is generated and deposited remains unclear. We have previously established that activated platelets from AD patients retain more A beta PP on their surface than control platelets. We report here that an endothelial cell-derived enzyme can cleave this surface platelet A beta PP. Human blood brain barrier endothelial cells from brains of AD patients were assayed for potential A beta PP-cleaving enzymes using synthetic peptide substrates encompassing the A beta N-terminus cleavage site. A protease activity capable of cleaving A beta PP on the surface of AD platelets was noted. The A beta PP cleavage is partially inhibited by EDTA, by ZincOV, as well as by a specific inhibitor of the Zn metalloprotease E.C.3.4.24.15. Furthermore, the protease is recognized by an antibody directed against it, using immunohistochemistry, Western blot analysis and flow cytometry. The protease is not secreted, but rather resides intracellularly as well as on the surface of the endothelial cells. The data suggest that E.C.3.4.24.15 synthesized by brain endothelial cells may process the platelet-derived A beta PP, yielding fragments which could contribute to cerebrovascular A beta deposits.
Alzheimer Disease↗
P2Y2 nucleotide receptors enhance alpha-secretase-dependent amyloid precursor protein processing.
The amyloid precursor protein (APP) is proteolytically processed by beta- and gamma-secretases to release amyloid beta, the main component in senile plaques found in the brains of patients with Alzheimer disease. Alternatively, APP can be cleaved within the amyloid beta domain by alpha-secretase releasing the non-amyloidogenic product sAPP alpha, which has been shown to have neuroprotective properties. Several G protein-coupled receptors are known to activate alpha-secretase-dependent processing of APP; however, the role of G protein-coupled nucleotide receptors in APP processing has not been investigated. Here it is demonstrated that activation of the G protein-coupled P2Y2 receptor (P2Y2R) subtype expressed in human 1321N1 astrocytoma cells enhanced the release of sAPP alpha in a time- and dose-dependent manner. P2Y2 R-mediated sAPP alpha release was dependent on extracellular calcium but was not affected by 1,2-bis(2-aminophenoxy)ethane-N,N,N,-trimethylammonium salt, an intracellular calcium chelator, indicating that P2Y2R-stimulated intracellular calcium mobilization was not involved. Inhibition of protein kinase C (PKC) with GF109203 or by PKC down-regulation with phorbol ester pre-treatment had no effect on UTP-stimulated sAPP alpha release, indicating a PKC-independent mechanism. U0126, an inhibitor of the mitogen-activated protein kinase pathway, partially inhibited sAPPalpha release by UTP, whereas inhibitors of Src-dependent epidermal growth factor receptor transactivation by P2Y2Rs had no effect. The metalloprotease inhibitors phenanthroline and TAPI-2 and the furin inhibitor decanoyl-Arg-Val-Lys-Arg-chloromethylketone also diminished UTP-induced sAPP alpha release. Furthermore, small interfering RNA silencing of an endogenous adamalysin, ADAM10 or ADAM17/TACE, partially suppressed P2Y2R-activated sAPP alpha release, whereas treatment of cells with both ADAM10 and ADAM17/TACE small interfering RNAs completely abolished UTP-activated sAPP alpha release. These results may contribute to an understanding of the non-amyloidogenic processing of APP.
ADAM Proteins↗
Inhibition of prolylendopeptidase does not affect gamma-secretase processing of amyloid precursor protein in a human neuroblastoma cell line.
Abeta peptides are major components of the amyloid plaques that characterize Alzheimer's disease. The enzyme activities (beta- and gamma-secretases) involved in generating Abeta from amyloid precursor protein (APP) are unidentified. It has been suggested that prolylendopeptidase (PEP), an oligopeptidase that normally cleaves after proline residues, could also cleave after the alanine at position 42 of Abeta to generate Abeta42. We investigated whether inhibition of PEP activity in human neuroblastoma cells affected Abeta levels in cell culture media. An SH-SY5Y cell line expressing SPA4CT, encoding the C-terminal 100 residues of APP and the signal sequence, was used. Only gamma-secretase activity is required for Abeta production in this cell line. The PEP inhibitor Fmoc-AlaPro-CN (10 microM) reduced PEP activity in these cells by approximately 95% in the absence of significant toxicity, but had no effect on Abeta40 or Abeta42 levels in cell culture media. We conclude that PEP is unlikely to be involved in gamma-secretase processing of APP.
Amino Acids↗
Presenilin 1 controls gamma-secretase processing of amyloid precursor protein in pre-golgi compartments of hippocampal neurons.
Mutations of presenilin 1 (PS1) causing Alzheimer's disease selectively increase the secretion of the amyloidogenic betaA4(1-42), whereas knocking out the gene results in decreased production of both betaA4(1-40) and (1-42) amyloid peptides (De Strooper et al. 1998). Therefore, PS1 function is closely linked to the gamma-secretase processing of the amyloid precursor protein (APP). Given the ongoing controversy on the subcellular localization of PS1, it remains unclear at what level of the secretory and endocytic pathways PS1 exerts its activity on APP and on the APP carboxy-terminal fragments that are the direct substrates for gamma-secretase. Therefore, we have reinvestigated the subcellular localization of endogenously expressed PS1 in neurons in vitro and in vivo using confocal microscopy and fine-tuned subcellular fractionation. We show that uncleaved PS1 holoprotein is recovered in the nuclear envelope fraction, whereas the cleaved PS fragments are found mainly in post-ER membranes including the intermediate compartment (IC). PS1 is concentrated in discrete sec23p- and p58/ERGIC-53-positive patches, suggesting its localization in subdomains involved in ER export. PS1 is not found to significant amounts beyond the cis-Golgi. Surprisingly, we found that APP carboxy-terminal fragments also coenrich in the pre-Golgi membrane fractions, consistent with the idea that these fragments are the real substrates for gamma-secretase. Functional evidence that PS1 exerts its effects on gamma-secretase processing of APP in the ER/IC was obtained using a series of APP trafficking mutants. These mutants were investigated in hippocampal neurons derived from transgenic mice expressing PS1wt or PS1 containing clinical mutations (PS1(M146L) and PS1(L286V)) at physiologically relevant levels. We demonstrate that the APP-London and PS1 mutations have additive effects on the increased secretion of betaA4(1-42) relative to betaA4(1-40), indicating that both mutations operate independently. Overall, our data clearly establish that PS1 controls gamma(42)-secretase activity in pre-Golgi compartments. We discuss models that reconcile this conclusion with the effects of PS1 deficiency on the generation of betaA4(1-40) peptide in the late biosynthetic and endocytic pathways.
Amyloid Precursor Protein Secretases↗
L-685,458, an aspartyl protease transition state mimic, is a potent inhibitor of amyloid beta-protein precursor gamma-secretase activity.
Progressive cerebral amyloid beta-protein (A beta) deposition is believed to play a central role in the pathogenesis of Alzheimer's disease (AD). Elevated levels of A beta(42) peptide formation have been linked to early-onset familial AD-causing gene mutations in the amyloid beta-protein precursor (A beta PP) and the presenilins. Sequential cleavage of A beta PP by the beta- and gamma-secretases generates the N- and C-termini of the A beta peptide, making both the beta- and gamma-secretase enzymes potential therapeutic targets for AD. The identity of the A beta PP gamma-secretase and the mechanism by which the C-termini of A beta are formed remain uncertain, although it has been suggested that the presenilins themselves are novel intramembrane-cleaving gamma-secretases of the aspartyl protease class [Wolfe, M. S., Xia, W., Ostaszewski, B. L., Diehl, T. S., Kimberly, W. T., and Selkoe, D. J. (1999) Nature 398, 513-517]. In this study we report the identification of L-685,458 as a structurally novel inhibitor of A beta PP gamma-secretase activity, with a similar potency for inhibition of A beta(42) and A beta(40) peptides. This compound contains an hydroxyethylene dipeptide isostere which suggests that it could function as a transition state analogue mimic of an aspartyl protease. The preferred stereochemistry of the hydroxyethylene dipeptide isostere was found to be the opposite to that required for inhibition of the HIV-1 aspartyl protease, a factor which may contribute to the observed specificity of this compound. Specific and potent inhibitors of A beta PP gamma-secretase activity such as L-685,458 will enable important advances toward the identification and elucidation of the mechanism of action of this enigmatic protease.
Amino Acid Sequence↗