Membrane-anchored aspartyl protease with Alzheimer's disease beta-secretase activity

Mutations in the gene encoding the amyloid protein precursor (APP) cause autosomal dominant Alzheimer's disease. Cleavage of APP by unidentified proteases, referred to as beta- and gamma-secretases, generates the amyloid beta-peptide, the main component of the amyloid plaques found in Alzheimer's disease patients. The disease-causing mutations flank the protease cleavage sites in APP and facilitate its cleavage. Here we identify a new membrane-bound aspartyl protease (Asp2) with beta-secretase activity. The Asp2 gene is expressed widely in brain and other tissues. Decreasing the expression of Asp2 in cells reduces amyloid beta-peptide production and blocks the accumulation of the carboxy-terminal APP fragment that is created by beta-secretase cleavage. Solubilized Asp2 protein cleaves a synthetic APP peptide substrate at the beta-secretase site, and the rate of cleavage is increased tenfold by a mutation associated with early-onset Alzheimer's disease in Sweden. Thus, Asp2 is a new protein target for drugs that are designed to block the production of amyloid beta-peptide peptide and the consequent formation of amyloid plaque in Alzheimer's disease.     

Amyloid beta-protein deposition in tissues other than brain in Alzheimer's disease

Alzheimer's disease is the most common cause of progressive intellectual failure in aged humans. The filamentous brain lesions which define the disease occur within neurons (neurofibrillary tangles), in extracellular cerebral deposits (amyloid plaques) and in meningocerebral blood vessels (amyloid angiopathy). They are found in lesser numbers in the brains of virtually all old humans. A protein with a relative molecular mass (Mr) of approximately 4,000, designated amyloid beta-protein or amyloid A4 protein, is the subunit of the vascular and plaque amyloid filaments in individuals with Alzheimer's disease, normal ageing and trisomy 21 (Down's syndrome). The amyloid beta-protein is a small fragment of a membrane-associated glycoprotein, encoded by a gene on human chromosome 21 which is telomeric to a genetic defect that causes at least some cases of familial Alzheimer's disease. Until now, the pathological lesions of the disease have been found only in the brain, although reports of phenotypic abnormalities in non-neural tissues have suggested that Alzheimer's disease may be a widespread, systemic disorder. Here we report the detection of amyloid beta-protein deposits in non-neural tissues and blood vessels of Alzheimer's disease patients, including skin, subcutaneous tissue and intestine. The protein was also present in non-neural tissues in a proportion of aged, normal subjects. Our findings indicate that a principal feature of the disease process is expressed subclinically in tissues other than brain. The occurrence of amyloid beta-protein deposits in multiple tissues suggests that the protein may be produced locally in numerous organs or may, as in other human amyloidoses, be derived from a common circulating precursor. These observations affect the rationale for many experiments analysing the amyloid beta-protein precursor and its messenger RNAs in Alzheimer's disease brain tissue and have major implications for the pathogenesis and treatment of the disease.     

Failure of familial Alzheimer's disease to segregate with the A4-amyloid gene in several European families

The gene coding for the amyloid protein, a component of neuritic plaques found in brain tissue from patients with Alzheimer's disease, has been localized to chromosome 21, and neighbouring polymorphic DNA markers segregate with Alzheimer's disease in several large families. These data, and the association of Alzheimer's disease with Down's syndrome, suggest that overproduction of the amyloid protein, or production of an abnormal variant of the protein, may be the underlying pathological change causing Alzheimer's disease. We have identified a restriction fragment length polymorphism of the A4-amyloid gene, and find recombinants in two Alzheimer's disease families between Alzheimer's disease and the A4-amyloid locus. This demonstrates that the gene for plaque core A4-amyloid cannot be the locus of a defect causing Alzheimer's disease in these families. These data indicate that alterations in the plaque core amyloid gene cannot explain the molecular pathology for all cases of Alzheimer's disease.     

Novel precursor of Alzheimer's disease amyloid protein shows protease inhibitory activity

Alzheimer's disease is characterized by cerebral deposits of amyloid beta-protein (AP) as senile plaque core and vascular amyloid, and a complementary DNA encoding a precursor of this protein (APP) has been cloned from human brain. From a cDNA library of a human glioblastoma cell line, we have isolated a cDNA identical to that previously reported, together with a new cDNA which contains a 225-nucleotide insert. The sequence of the 56 amino acids at the N-terminal of the protein deduced from this insert is highly homologous to the basic trypsin inhibitor family, and the lysate from COS-1 cells transfected with the longer APP cDNA showed an increased inhibition of trypsin activity. Partial sequencing of the genomic DNA encoding APP showed that the 225 nucleotides are located in two exons. At least three messenger RNA species, apparently transcribed from a single APP gene by alternative splicing, were found in human brain. We suggest that protease inhibition by the longer APP(s) could be related to aberrant APP catabolism.     

Formation of amyloid-like fibrils in COS cells overexpressing part of the Alzheimer amyloid protein precursor

A pathological hallmark of Alzheimer's disease is the deposition of amyloid fibrils in the brain. The principal component of the amyloid fibril is beta/A4 protein, which is derived from a large membrane-bound glycoprotein, Alzheimer amyloid protein precursor (APP). Although the deposition of amyloid is thought to result from the aberrant processing of APP, the detailed molecular mechanisms of amyloidogenesis remain unclear. A C-terminal fragment of APP which spans the beta/A4 and cytoplasmic domains has a tendency to self-aggregate. In an attempt to establish a cultured-cell model for amyloid fibril formation, we have transfected COS-1 cells with complementary DNA encoding the C-terminal 100 residues of APP. In the perinuclear regions of a small population of DNA-transfected cells, we observed inclusion-like deposits which showed a strong immunohistochemical reaction towards an anti-C-terminal APP antibody or an anti-beta/A4 amyloid core-specific antibody. Electron microscope observations of the inclusion-carrying cells revealed an accumulation of amyloid-like fibrils of 8-22 nm diameter near and on the nuclear membrane. The fibrils showed a beaded or helical structure, and reacted positively with the anti-C-terminus antibody by immunoelectron microscopy. These results suggest that the formation of amyloid fibrils is an inherent characteristic of the C-terminal peptide of APP. The present system provides a suitable model for the molecular dissection of the process of brain amyloidogenesis.     

Segregation of a missense mutation in the amyloid precursor protein gene with familial Alzheimer's disease.

A locus segregating with familial Alzheimer's disease (AD) has been mapped to chromosome 21, close to the amyloid precursor protein (APP) gene. Recombinants between the APP gene and the AD locus have been reported which seemed to exclude it as the site of the mutation causing familial AD. But recent genetic analysis of a large number of AD families has demonstrated that the disease is heterogeneous. Families with late-onset AD do not show linkage to chromosome 21 markers. Some families with early-onset AD show linkage to chromosome 21 markers, but some do not. This has led to the suggestion that there is non-allelic genetic heterogeneity even within early onset familial AD. To avoid the problems that heterogeneity poses for genetic analysis, we have examined the cosegregation of AD and markers along the long arm of chromosome 21 in a single family with AD confirmed by autopsy. Here we demonstrate that in this kindred, which shows linkage to chromosome 21 markers, there is a point mutation in the APP gene. This mutation causes an amino-acid substitution (Val----Ile) close to the carboxy terminus of the beta-amyloid peptide. Screening other cases of familial AD revealed a second unrelated family in which this variant occurs. This suggests that some cases of AD could be caused by mutations in the APP gene.     

Amyloid plaques, neurofibrillary tangles and neuronal loss in brains of transgenic mice overexpressing a C-terminal fragment of human amyloid precursor protein.

Alzheimer's disease (AD) affects more than 30% of people over 80 years of age. The aetiology and pathogenesis of this progressive dementia is poorly understood, but symptomatic disease is associated histopathologically with amyloid plaques, neurofibrillary tangles and neuronal loss primarily in the temporal lobe and neocortex of the brain. The core of the extracellular plaque is a derivative of the amyloid precursor protein (APP), referred to as beta/A4, and contains the amino-acid residues 29-42 that are normally embedded in the membrane-spanning region of the precursor. The cellular source of APP and the relationship of its deposition to the neuropathology of AD is unknown. To investigate the relationship between APP overexpression and amyloidogenesis, we have developed a vector to drive expression specifically in neurons of a C-terminal fragment of APP that contains the beta/A4 region, and have used a transgenic mouse system to insert and express this construct. We report here that overexpression of this APP transgene in neurons is sufficient to produce extracellular dense-core amyloid plaques, neurofibrillary tangles and neuronal degeneration similar to that in the AD brain.     

Purification and cloning of amyloid precursor protein beta-secretase from human brain

Proteolytic processing of the amyloid precursor protein (APP) generates amyloid beta (Abeta) peptide, which is thought to be causal for the pathology and subsequent cognitive decline in Alzheimer's disease. Cleavage by beta-secretase at the amino terminus of the Abeta peptide sequence, between residues 671 and 672 of APP, leads to the generation and extracellular release of beta-cleaved soluble APP, and a corresponding cell-associated carboxy-terminal fragment. Cleavage of the C-terminal fragment by gamma-secretase(s) leads to the formation of Abeta. The pathogenic mutation K670M671-->N670L671 at the beta-secretase cleavage site in APP, which was discovered in a Swedish family with familial Alzheimer's disease, leads to increased beta-secretase cleavage of the mutant substrate. Here we describe a membrane-bound enzyme activity that cleaves full-length APP at the beta-secretase cleavage site, and find it to be the predominant beta-cleavage activity in human brain. We have purified this enzyme activity to homogeneity from human brain using a new substrate analogue inhibitor of the enzyme activity, and show that the purified enzyme has all the properties predicted for beta-secretase. Cloning and expression of the enzyme reveals that human brain beta-secretase is a new membrane-bound aspartic proteinase.     

GSK-3α/β kinases and amyloid production in vivo

Arising from C. J. Phiel, C. A. Wilson, V. M.-Y. Lee & P. S. Klein 423, 435-439 (2003)A major unresolved issue in Alzheimer's disease is identifying the mechanisms that regulate proteolytic processing of amyloid precursor protein (APP)-glycogen synthase kinase-3 (GSK-3) isozymes are thought to be important in this regulation. Phiel et al. proposed that GSK-3α, but not GSK-3β, controls production of amyloid. We analysed the proteolytic processing of mouse and human APP in mouse brain in vivo in five different genetic and viral models. Our data do not yield evidence for either GSK-3α-mediated or GSK-3β-mediated control of APP processing in brain in vivo.     

Degeneration in vitro of post-mitotic neurons overexpressing the Alzheimer amyloid protein precursor.

A pathological hallmark of Alzheimer's disease is the deposition of amyloid fibrils in the brain. The principal component of amyloid fibrils is beta/A4 amyloid protein, which can be generated by the aberrant processing of a large membrane-bound glycoprotein, the beta/A4 amyloid protein precursor (APP)3. To test whether overexpression of APP generates abnormally processed derivatives that affect the viability of neurons, we stably transfected full-length human APP complementary DNA into murine embryonal carcinoma P19 cells. These cells differentiate into post-mitotic neurons and astrocytes after exposure to retinoic acid. When differentiation of the APP cDNA-transfected P19 cells was induced, all neurons showed severe degenerative changes and disappeared within a few days. The degenerating neurons contained large amounts of APP derivatives that were truncated at the amino terminus and encompassed the entire beta/A4 domain. These results suggest that post-mitotic neurons are vulnerable to overexpressed APP, which undergoes aberrant processing to generate potentially amyloidogenic fragments.     

Protease nexin-II, a potent antichymotrypsin, shows identity to amyloid beta-protein precursor

Protease nexin-II (PN-II) is a protease inhibitor that forms SDS-resistant inhibitory complexes with the epidermal growth factor (EGF)-binding protein, the gamma-subunit of nerve growth factor, and trypsin. The properties of PN-II indicate that it has a role in the regulation of certain proteases in the extracellular environment. Here we describe more of the amino-acid sequence of PN-II and its identity to the deduced sequence of the amyloid beta-protein precursor (APP). Amyloid beta-protein is present in neuritic plaques and cerebrovascular deposits in individuals with Alzheimer's disease and Down's syndrome. A monoclonal antibody against PN-II (designated mAbP2-1) recognized PN-II in immunoblots of serum-free culture medium from human glioblastoma cells and neuroblastoma cells, as well as in homogenates of normal and Alzheimer's disease brains. In addition, mAbP2-1 stained neuritic plaques in Alzheimer's disease brain. PN-II was a potent inhibitor of chymotrypsin with an inhibition constant Ki of 6 x 10(-10)M. Together, these data demonstrate that PN-II and APP are probably the same protein. The regulation of extracellular proteolysis by PN-II and the deposition of at least parts of the molecule in senile plaques is consistent with previous reports that implicate altered proteolysis in the pathogenesis of Alzheimer's disease.     

The precursor of Alzheimer's disease amyloid A4 protein resembles a cell-surface receptor

Alzheimer's disease is characterized by a widespread functional disturbance of the human brain. Fibrillar amyloid proteins are deposited inside neurons as neurofibrillary tangles and extracellularly as amyloid plaque cores and in blood vessels. The major protein subunit (A4) of the amyloid fibril of tangles, plaques and blood vessel deposits is an insoluble, highly aggregating small polypeptide of relative molecular mass 4,500. The same polypeptide is also deposited in the brains of aged individuals with trisomy 21 (Down's syndrome). We have argued previously that the A4 protein is of neuronal origin and is the cleavage product of a larger precursor protein. To identify this precursor, we have now isolated and sequenced an apparently full-length complementary DNA clone coding for the A4 polypeptide. The predicted precursor consists of 695 residues and contains features characteristic of glycosylated cell-surface receptors. This sequence, together with the localization of its gene on chromosome 21, suggests that the cerebral amyloid deposited in Alzheimer's disease and aged Down's syndrome is caused by aberrant catabolism of a cell-surface receptor.     

Targeting of cell-surface beta-amyloid precursor protein to lysosomes: alternative processing into amyloid-bearing fragments.

Progressive cerebral deposition of the amyloid beta-peptide is an early and invariant feature of Alzheimer's disease. The beta-peptide is released by proteolytic cleavages from the beta-amyloid precursor protein (beta APP), a membrane-spanning glycoprotein expressed in most mammalian cells. Normal secretion of beta APP involves a cleavage in the beta-peptide region, releasing the soluble extramembranous portion and retaining a 10K C-terminal fragment in the membrane. Because this secretory pathway precludes beta-amyloid formation, we searched for an alternative proteolytic processing pathway that can generate beta-peptide-bearing fragments from full-length beta APP. Incubation of living human endothelial cells with a beta APP antibody revealed reinternalization of mature beta APP from the cell surface and its targeting to endosomes/lysosomes. After cell-surface biotinylation, full-length biotinylated beta APP was recovered inside the cells. Purification of lysosomes directly demonstrated the presence of mature beta APP and an extensive array of beta-peptide-containing proteolytic products. Our results define a second processing pathway for beta APP and suggest that it may be responsible for generating amyloid-bearing fragments in Alzheimer's disease.     

A new A4 amyloid mRNA contains a domain homologous to serine proteinase inhibitors

The amyloid proteins isolated from neuritic plaques and the cerebrovasculature of Alzheimer's disease are self-aggregating moieties termed A4 protein and beta-protein, respectively. A putative A4 amyloid precursor (herein termed A4(695] has been characterized by analysis of a human brain complementary DNA. We report here the sequence of a closely related amyloid cDNA, A4(751), distinguished from A4(695) by the presence of a 168 base-pair (bp) sequence which adds 57 amino acids to, and removes one residue from, the predicted A4(695) protein. The peptide predicted from this insert is very similar to the Kunitz family of serine proteinase inhibitors. The two A4-specific messenger RNAs are differentially expressed: in a limited survey, A4(751) mRNA appears to be ubiquitous, whereas A4(695) mRNA has a restricted pattern of expression which includes cells from neuronal tissue. These data may have significant implications for understanding amyloid deposition in Alzheimer's disease.     

GSK-3alpha regulates production of Alzheimer's disease amyloid-beta peptides

Alzheimer's disease is associated with increased production and aggregation of amyloid-beta (Abeta) peptides. Abeta peptides are derived from the amyloid precursor protein (APP) by sequential proteolysis, catalysed by the aspartyl protease BACE, followed by presenilin-dependent gamma-secretase cleavage. Presenilin interacts with nicastrin, APH-1 and PEN-2 (ref. 6), all of which are required for gamma-secretase function. Presenilins also interact with alpha-catenin, beta-catenin and glycogen synthase kinase-3beta (GSK-3beta), but a functional role for these proteins in gamma-secretase activity has not been established. Here we show that therapeutic concentrations of lithium, a GSK-3 inhibitor, block the production of Abeta peptides by interfering with APP cleavage at the gamma-secretase step, but do not inhibit Notch processing. Importantly, lithium also blocks the accumulation of Abeta peptides in the brains of mice that overproduce APP. The target of lithium in this setting is GSK-3alpha, which is required for maximal processing of APP. Since GSK-3 also phosphorylates tau protein, the principal component of neurofibrillary tangles, inhibition of GSK-3alpha offers a new approach to reduce the formation of both amyloid plaques and neurofibrillary tangles, two pathological hallmarks of Alzheimer's disease.     

Immunization with amyloid-beta attenuates Alzheimer-disease-like pathology in the PDAPP mouse.

Amyloid-beta peptide (Abeta) seems to have a central role in the neuropathology of Alzheimer's disease (AD). Familial forms of the disease have been linked to mutations in the amyloid precursor protein (APP) and the presenilin genes. Disease-linked mutations in these genes result in increased production of the 42-amino-acid form of the peptide (Abeta42), which is the predominant form found in the amyloid plaques of Alzheimer's disease. The PDAPP transgenic mouse, which overexpresses mutant human APP (in which the amino acid at position 717 is phenylalanine instead of the normal valine), progressively develops many of the neuropathological hallmarks of Alzheimer's disease in an age- and brain-region-dependent manner. In the present study, transgenic animals were immunized with Abeta42, either before the onset of AD-type neuropathologies (at 6 weeks of age) or at an older age (11 months), when amyloid-beta deposition and several of the subsequent neuropathological changes were well established. We report that immunization of the young animals essentially prevented the development of beta-amyloid-plaque formation, neuritic dystrophy and astrogliosis. Treatment of the older animals also markedly reduced the extent and progression of these AD-like neuropathologies. Our results raise the possibility that immunization with amyloid-beta may be effective in preventing and treating Alzheimer's disease.     

The prolyl isomerase Pin1 regulates amyloid precursor protein processing and amyloid-beta production

Neuropathological hallmarks of Alzheimer's disease are neurofibrillary tangles composed of tau and neuritic plaques comprising amyloid-beta peptides (Abeta) derived from amyloid precursor protein (APP), but their exact relationship remains elusive. Phosphorylation of tau and APP on certain serine or threonine residues preceding proline affects tangle formation and Abeta production in vitro. Phosphorylated Ser/Thr-Pro motifs in peptides can exist in cis or trans conformations, the conversion of which is catalysed by the Pin1 prolyl isomerase. Pin1 has been proposed to regulate protein function by accelerating conformational changes, but such activity has never been visualized and the biological and pathological significance of Pin1 substrate conformations is unknown. Notably, Pin1 is downregulated and/or inhibited by oxidation in Alzheimer's disease neurons, Pin1 knockout causes tauopathy and neurodegeneration, and Pin1 promoter polymorphisms appear to associate with reduced Pin1 levels and increased risk for late-onset Alzheimer's disease. However, the role of Pin1 in APP processing and Abeta production is unknown. Here we show that Pin1 has profound effects on APP processing and Abeta production. We find that Pin1 binds to the phosphorylated Thr 668-Pro motif in APP and accelerates its isomerization by over 1,000-fold, regulating the APP intracellular domain between two conformations, as visualized by NMR. Whereas Pin1 overexpression reduces Abeta secretion from cell cultures, knockout of Pin1 increases its secretion. Pin1 knockout alone or in combination with overexpression of mutant APP in mice increases amyloidogenic APP processing and selectively elevates insoluble Abeta42 (a major toxic species) in brains in an age-dependent manner, with Abeta42 being prominently localized to multivesicular bodies of neurons, as shown in Alzheimer's disease before plaque pathology. Thus, Pin1-catalysed prolyl isomerization is a novel mechanism to regulate APP processing and Abeta production, and its deregulation may link both tangle and plaque pathologies. These findings provide new insight into the pathogenesis and treatment of Alzheimer's disease.     

The genetic defect in familial Alzheimer's disease is not tightly linked to the amyloid beta-protein gene

Amyloid beta-protein (AP) is a peptide of relative molecular mass (Mr) 42,000 found in the senile plaques, cerebrovascular amyloid deposits, and neurofibrillary tangles of patients with Alzheimer's disease and Down's syndrome (trisomy 21). Recent molecular genetic evidence has indicated that AP is encoded as part of a larger protein by a gene on chromosome 21 (refs 5-7). The defect in the inherited autosomal dominant form of Alzheimer's disease, familial Alzheimer's disease (FAD), has been mapped to the same approximate region of chromosome 21 by genetic linkage to anonymous DNA markers, raising the possibility that this gene product, which could be important in the pathogenesis of Alzheimer's disease, is also the site of the inherited defect in FAD (ref. 5). We have determined the pattern of segregation of the AP gene in FAD pedigrees using restriction fragment length polymorphisms. The detection of several recombination events with FAD suggests that the AP gene is not the site of the inherited defect underlying this disorder.     

The secreted form of the Alzheimer's amyloid precursor protein with the Kunitz domain is protease nexin-II

The A4 protein (or beta-protein) is a 42- or 43-amino-acid peptide present in the extracellular neuritic plaques in Alzheimer's disease and is derived from a membrane-bound amyloid protein precursor (APP). Three forms of APP have been described and are referred to as APP695, APP751 and APP770, reflecting the number of amino acids encoded for by their respective complementary DNAs. The two larger APPs contain a 57-amino-acid insert with striking homology to the Kunitz family of protease inhibitors. Here we report that the deduced amino-terminal sequence of APP is identical to the sequence of a cell-secreted protease inhibitor, protease nexin-II (PN-II). To confirm this finding, APP751 and APP695 cDNAs were over-expressed in the human 293 cell line, and the secreted N-terminal extracellular domains of these APPs were purified to near homogeneity from the tissue-culture medium. The relative molecular mass and high-affinity binding to dextran sulphate of secreted APP751 were consistent with that of PN-II. Functionally, secreted APP751 formed stable, non-covalent, inhibitory complexes with trypsin. Secreted APP695 did not form complexes with trypsin. We conclude that the secreted form of APP with the Kunitz protease inhibitor domain is PN-II.     

Probing sporadic and familial Alzheimer's disease using induced pluripotent stem cells

Our understanding of Alzheimer's disease pathogenesis is currently limited by difficulties in obtaining live neurons from patients and the inability to model the sporadic form of the disease. It may be possible to overcome these challenges by reprogramming primary cells from patients into induced pluripotent stem cells (iPSCs). Here we reprogrammed primary fibroblasts from two patients with familial Alzheimer's disease, both caused by a duplication of the amyloid-β precursor protein gene (APP; termed APP(Dp)), two with sporadic Alzheimer's disease (termed sAD1, sAD2) and two non-demented control individuals into iPSC lines. Neurons from differentiated cultures were purified with fluorescence-activated cell sorting and characterized. Purified cultures contained more than 90% neurons, clustered with fetal brain messenger RNA samples by microarray criteria, and could form functional synaptic contacts. Virtually all cells exhibited normal electrophysiological activity. Relative to controls, iPSC-derived, purified neurons from the two APP(Dp) patients and patient sAD2 exhibited significantly higher levels of the pathological markers amyloid-β(1-40), phospho-tau(Thr?231) and active glycogen synthase kinase-3β (aGSK-3β). Neurons from APP(Dp) and sAD2 patients also accumulated large RAB5-positive early endosomes compared to controls. Treatment of purified neurons with β-secretase inhibitors, but not γ-secretase inhibitors, caused significant reductions in phospho-Tau(Thr?231) and aGSK-3β levels. These results suggest a direct relationship between APP proteolytic processing, but not amyloid-β, in GSK-3β activation and tau phosphorylation in human neurons. Additionally, we observed that neurons with the genome of one sAD patient exhibited the phenotypes seen in familial Alzheimer's disease samples. More generally, we demonstrate that iPSC technology can be used to observe phenotypes relevant to Alzheimer's disease, even though it can take decades for overt disease to manifest in patients.