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.     

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.     

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.     

Kinesin-mediated axonal transport of a membrane compartment containing beta-secretase and presenilin-1 requires APP.

Proteolytic processing of amyloid precursor protein (APP) generates amyloid-beta peptide and has been implicated in the pathogenesis of Alzheimer's disease. However, the normal function of APP, whether this function is related to the proteolytic processing of APP, and where this processing takes place in neurons in vivo remain unknown. We have previously shown that the axonal transport of APP in neurons is mediated by the direct binding of APP to the kinesin light chain subunit of kinesin-I, a microtubule motor protein. Here we identify an axonal membrane compartment that contains APP, beta-secretase and presenilin-1. The fast anterograde axonal transport of this compartment is mediated by APP and kinesin-I. Proteolytic processing of APP can occur in the compartment in vitro and in vivo in axons. This proteolysis generates amyloid-beta and a carboxy-terminal fragment of APP, and liberates kinesin-I from the membrane. These results suggest that APP functions as a kinesin-I membrane receptor, mediating the axonal transport of beta-secretase and presenilin-1, and that processing of APP to amyloid-beta by secretases can occur in an axonal membrane compartment transported by kinesin-I.     

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.     

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.     

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.     

Photoactivated gamma-secretase inhibitors directed to the active site covalently label presenilin 1

Cleavage of amyloid precursor protein (APP) by the beta- and gamma-secretases generates the amino and carboxy termini, respectively, of the A beta amyloidogenic peptides A beta40 and A beta42--the major constituents of the amyloid plaques in the brain parenchyma of Alzheimer's disease patients. There is evidence that the polytopic membrane-spanning proteins, presenilin 1 and 2 (PS1 and PS2), are important determinants of gamma-secretase activity: mutations in PS1 and PS2 that are associated with early-onset familial Alzheimer's disease increase the production of A beta42 (refs 4-6), the more amyloidogenic peptide; gamma-secretase activity is reduced in neuronal cultures derived from PS1-deficient mouse embryos; and directed mutagenesis of two conserved aspartates in transmembrane segments of PS1 inactivates the ability of gamma-secretase to catalyse processing of APP within its transmembrane domain. It is unknown, however, whether PS1 (which has little or no homology to any known aspartyl protease) is itself a transmembrane aspartyl protease or a gamma-secretase cofactor, or helps to colocalize gamma-secretase and APP. Here we report photoaffinity labelling of PS1 (and PS2) by potent gamma-secretase inhibitors that were designed to function as transition state analogue inhibitors directed to the active site of an aspartyl protease. This observation indicates that PS1 (and PS2) may contain the active site of gamma-secretase. Interestingly, the intact, single-chain form of wild-type PS1 is not labelled by an active-site-directed photoaffinity probe, suggesting that intact wild-type PS1 may be an aspartyl protease zymogen.     

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.     

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.     

Protease inhibitor domain encoded by an amyloid protein precursor mRNA associated with Alzheimer's disease

Amyloid B-protein/amyloid A4 is a peptide present in the neuritic plaques, neurofibrillary tangles and cerebrovascular deposits in patients with Alzheimer's disease and Down's syndrome (trisomy 21) and may be involved in the pathogenesis of Alzheimer's disease. Recent molecular genetic studies have indicated that amyloid protein is encoded as part of a larger protein by a gene on human chromosome 21 (refs 6-9). The amyloid protein precursor (APP) gene is expressed in brain and in several peripheral tissues, but the specific biochemical events leading to deposition of amyloid are not known. We have now screened complementary DNA libraries constructed from peripheral tissues to determine whether the messenger RNA encoding APP in these tissues is identical to that expressed in brain, and we identify a second APP mRNA that encodes an additional internal domain with a sequence characteristic of a Kunitz-type serine protease inhibitor. The alternative APP mRNA is present in both brain and peripheral tissues of normal individuals and those with Alzheimer's disease, but its pattern of expression differs from that of the previously reported APP mRNA.     

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.     

Structure-based design of non-natural amino-acid inhibitors of amyloid fibril formation

Many globular and natively disordered proteins can convert into amyloid fibrils. These fibrils are associated with numerous pathologies as well as with normal cellular functions, and frequently form during protein denaturation. Inhibitors of pathological amyloid fibril formation could be useful in the development of therapeutics, provided that the inhibitors were specific enough to avoid interfering with normal processes. Here we show that computer-aided, structure-based design can yield highly specific peptide inhibitors of amyloid formation. Using known atomic structures of segments of amyloid fibrils as templates, we have designed and characterized an all-D-amino-acid inhibitor of the fibril formation of the tau protein associated with Alzheimer's disease, and a non-natural L-amino-acid inhibitor of an amyloid fibril that enhances sexual transmission of human immunodeficiency virus. Our results indicate that peptides from structure-based designs can disrupt the fibril formation of full-length proteins, including those, such as tau protein, that lack fully ordered native structures. Because the inhibiting peptides have been designed on structures of dual-β-sheet 'steric zippers', the successful inhibition of amyloid fibril formation strengthens the hypothesis that amyloid spines contain steric zippers.     

Mice cloned from olfactory sensory neurons

Cloning by nuclear transplantation has been successfully carried out in various mammals, including mice. Until now mice have not been cloned from post-mitotic cells such as neurons. Here, we have generated fertile mouse clones derived by transferring the nuclei of post-mitotic, olfactory sensory neurons into oocytes. These results indicate that the genome of a post-mitotic, terminally differentiated neuron can re-enter the cell cycle and be reprogrammed to a state of totipotency after nuclear transfer. Moreover, the pattern of odorant receptor gene expression and the organization of odorant receptor genes in cloned mice was indistinguishable from wild-type animals, indicating that irreversible changes to the DNA of olfactory neurons do not accompany receptor gene choice.     

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.     

Atomic structures of amyloid cross-beta spines reveal varied steric zippers

Amyloid fibrils formed from different proteins, each associated with a particular disease, contain a common cross-beta spine. The atomic architecture of a spine, from the fibril-forming segment GNNQQNY of the yeast prion protein Sup35, was recently revealed by X-ray microcrystallography. It is a pair of beta-sheets, with the facing side chains of the two sheets interdigitated in a dry 'steric zipper'. Here we report some 30 other segments from fibril-forming proteins that form amyloid-like fibrils, microcrystals, or usually both. These include segments from the Alzheimer's amyloid-beta and tau proteins, the PrP prion protein, insulin, islet amyloid polypeptide (IAPP), lysozyme, myoglobin, alpha-synuclein and beta(2)-microglobulin, suggesting that common structural features are shared by amyloid diseases at the molecular level. Structures of 13 of these microcrystals all reveal steric zippers, but with variations that expand the range of atomic architectures for amyloid-like fibrils and offer an atomic-level hypothesis for the basis of prion strains.     

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.     

The most infectious prion protein particles

Neurodegenerative diseases such as Alzheimer's, Parkinson's and the transmissible spongiform encephalopathies (TSEs) are characterized by abnormal protein deposits, often with large amyloid fibrils. However, questions have arisen as to whether such fibrils or smaller subfibrillar oligomers are the prime causes of disease. Abnormal deposits in TSEs are rich in PrP(res), a protease-resistant form of the PrP protein with the ability to convert the normal, protease-sensitive form of the protein (PrP(sen)) into PrP(res) (ref. 3). TSEs can be transmitted between organisms by an enigmatic agent (prion) that contains PrP(res) (refs 4 and 5). To evaluate systematically the relationship between infectivity, converting activity and the size of various PrP(res)-containing aggregates, PrP(res) was partially disaggregated, fractionated by size and analysed by light scattering and non-denaturing gel electrophoresis. Our analyses revealed that with respect to PrP content, infectivity and converting activity peaked markedly in 17-27-nm (300-600 kDa) particles, whereas these activities were substantially lower in large fibrils and virtually absent in oligomers of < or =5 PrP molecules. These results suggest that non-fibrillar particles, with masses equivalent to 14-28 PrP molecules, are the most efficient initiators of TSE disease.     

Review： Protein folding pathology in domestic animals

Fibrillar proteins form structural elements of cells and the extracellular matrix. Pathological lesions of fibrillar microanatomical structures, or secondary fibrillar changes in globular proteins are well known. A special group concerns histologically amorphous deposits, amyloid. The major characteristics of amyloid are: apple green birefringence after Congo red staining of histological sections, and non-branching 7-10nm thick fibrils on electron microscopy revealing a high content of cross beta pleated sheets. About 25 different types of amyloid have been characterised. In animals, AA-amyloid is the most frequent type. Other types of amyloid in animals represent: AIAPP (in cats), AApoAⅠ, AApoAⅡ, localised AL-amyloid, amyloid in odontogenic or mammary tumors and amyloid in the brain. In old dogs Aβ and in sheep APrP^sc-amyloid can be encountered. AA-amyloidosis is a systemic disorder with a precursor in blood, acute phase serum amyloid A (SAA). In chronic inflammatory processes AA-amyloid can be deposited. A rapid crystallization of SAA to amyloid fibrils on small beta-sheeted fragments, the ‘amyloid enhancing factor‘ (AEF), is known and the AEF has been shown to penetrate the enteric barrier. Amyloid fibrils can aggregate from various precursor proteins in vitro in particular at acidic pH and when proteolytic fragments are formed. Molecular chaperones influence this process. Tissue data point to amyloid fibrillogenesis in lysosomes and near cell surfaces. A comparison can be made of the fibrillogenesis in prion diseases and in enhanced AA-amyloidosis. In the reactive form, acute phase SAA is the supply of the precursor protein, whereas in the prion diseases, cell membrane proteins form a structural source. AI3-amyloid in brain tissue of aged dogs showing signs of dementia forms a canine counterpart of senile dementia of the Alzheimer type (ccSDAT) in man. Misfolded proteins remain potential food hazards. Developments concerning prevention of amyloidogenesis and therapy of amyloid deposits are shortly commented.     

Naturally secreted oligomers of amyloid beta protein potently inhibit hippocampal long-term potentiation in vivo

Although extensive data support a central pathogenic role for amyloid beta protein (Abeta) in Alzheimer's disease, the amyloid hypothesis remains controversial, in part because a specific neurotoxic species of Abeta and the nature of its effects on synaptic function have not been defined in vivo. Here we report that natural oligomers of human Abeta are formed soon after generation of the peptide within specific intracellular vesicles and are subsequently secreted from the cell. Cerebral microinjection of cell medium containing these oligomers and abundant Abeta monomers but no amyloid fibrils markedly inhibited hippocampal long-term potentiation (LTP) in rats in vivo. Immunodepletion from the medium of all Abeta species completely abrogated this effect. Pretreatment of the medium with insulin-degrading enzyme, which degrades Abeta monomers but not oligomers, did not prevent the inhibition of LTP. Therefore, Abeta oligomers, in the absence of monomers and amyloid fibrils, disrupted synaptic plasticity in vivo at concentrations found in human brain and cerebrospinal fluid. Finally, treatment of cells with gamma-secretase inhibitors prevented oligomer formation at doses that allowed appreciable monomer production, and such medium no longer disrupted LTP, indicating that synaptotoxic Abeta oligomers can be targeted therapeutically.