Alzheimer's disease. Molecular consequences of presenilin-1 mutation

Alzheimer's disease is characterized by accumulation in the brain of a family of insoluble amyloid peptides (Abeta peptides), which are produced as a result of the normal processing of beta-amyloid precursor protein (beta-APP). Russo et al. claim that a truncated Abeta peptide that lacks the first ten amino acids accumulates in the brains of patients carrying a mutant form of pre-senilin 1 (PS1), a protein that is involved in cleavage of beta-APP. However, we have found that this same species is also overrepresented in Alzheimer's patients with mutations in beta-APP itself. Our findings do not support the conclusion of Russo et al. that pathogenic PS1 mutations may control cleavage of beta-APP by beta-secretase.     

Mutation of the beta-amyloid precursor protein in familial Alzheimer's disease increases beta-protein production.

Progressive cerebral deposition of the 39-43-amino-acid amyloid beta-protein (A beta) is an invariant feature of Alzheimer's disease which precedes symptoms of dementia by years or decades. The only specific molecular defects that cause Alzheimer's disease which have been identified so far are missense mutations in the gene encoding the beta-amyloid precursor protein (beta-APP) in certain families with an autosomal dominant form of the disease (familial Alzheimer's disease, or FAD). These mutations are located within or immediately flanking the A beta region of beta-APP, but the mechanism by which they cause the pathological phenotype of early and accelerated A beta deposition is unknown. Here we report that cultured cells which express a beta-APP complementary DNA bearing a double mutation (Lys to Asn at residue 595 plus Met to Leu at position 596) found in a Swedish FAD family produce approximately 6-8-fold more A beta than cells expressing normal beta-APP. The Met 596 to Leu mutation is principally responsible for the increase. These data establish a direct link between a FAD genotype and the clinicopathological phenotype. Further, they confirm the relevance of the continuous A beta production by cultured cells for elucidating the fundamental mechanism of Alzheimer's disease.     

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.     

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.     

Role of the prolyl isomerase Pin1 in protecting against age-dependent neurodegeneration

The neuropathological hallmarks of Alzheimer's disease and other tauopathies include senile plaques and/or neurofibrillary tangles. Although mouse models have been created by overexpressing specific proteins including beta-amyloid precursor protein, presenilin and tau, no model has been generated by gene knockout. Phosphorylation of tau and other proteins on serine or threonine residues preceding proline seems to precede tangle formation and neurodegeneration in Alzheimer's disease. Notably, these phospho(Ser/Thr)-Pro motifs exist in two distinct conformations, whose conversion in some proteins is catalysed by the Pin1 prolyl isomerase. Pin1 activity can directly restore the conformation and function of phosphorylated tau or it can do so indirectly by promoting its dephosphorylation, which suggests that Pin1 is involved in neurodegeneration; however, genetic evidence is lacking. Here we show that Pin1 expression is inversely correlated with predicted neuronal vulnerability and actual neurofibrillary degeneration in Alzheimer's disease. Pin1 knockout in mice causes progressive age-dependent neuropathy characterized by motor and behavioural deficits, tau hyperphosphorylation, tau filament formation and neuronal degeneration. Thus, Pin1 is pivotal in protecting against age-dependent neurodegeneration, providing insight into the pathogenesis and treatment of Alzheimer's disease and other tauopathies.     

Amyloid beta-peptide is produced by cultured cells during normal metabolism.

Alzheimer's disease is characterized by the extracellular deposition in the brain and its blood vessels of insoluble aggregates of the amyloid beta-peptide (A beta), a fragment, of about 40 amino acids in length, of the integral membrane protein beta-amyloid precursor protein (beta-APP). The mechanism of extracellular accumulation of A beta in brain is unknown and no simple in vitro or in vivo model systems that produce extracellular A beta have been described. We report here the unexpected identification of the 4K (M(r) 4,000) A beta and a truncated form of A beta (approximately 3K) in media from cultures of primary cells and untransfected and beta-APP-transfected cell lines grown under normal conditions. These peptides were immunoprecipitated readily from culture medium by A beta-specific antibodies and their identities confirmed by sequencing. The concept that pathological processes are responsible for the production of A beta must not be reassessed in light of the observation that A beta is produced in soluble form in vitro and in vivo during normal cellular metabolism. Further, these findings provide the basis for using simple cell culture systems to identify drugs that block the formation or release of A beta, the primary protein constituent of the senile plaques of Alzheimer's disease.     

Reversing EphB2 depletion rescues cognitive functions in Alzheimer model

Amyloid-β oligomers may cause cognitive deficits in Alzheimer's disease by impairing neuronal NMDA-type glutamate receptors, whose function is regulated by the receptor tyrosine kinase EphB2. Here we show that amyloid-β oligomers bind to the fibronectin repeats domain of EphB2 and trigger EphB2 degradation in the proteasome. To determine the pathogenic importance of EphB2 depletions in Alzheimer's disease and related models, we used lentiviral constructs to reduce or increase neuronal expression of EphB2 in memory centres of the mouse brain. In nontransgenic mice, knockdown of EphB2 mediated by short hairpin RNA reduced NMDA receptor currents and impaired long-term potentiation in the dentate gyrus, which are important for memory formation. Increasing EphB2 expression in the dentate gyrus of human amyloid precursor protein transgenic mice reversed deficits in NMDA receptor-dependent long-term potentiation and memory impairments. Thus, depletion of EphB2 is critical in amyloid-β-induced neuronal dysfunction. Increasing EphB2 levels or function could be beneficial in Alzheimer's disease.     

Apolipoprotein E controls cerebrovascular integrity via cyclophilin A

Human apolipoprotein E has three isoforms: APOE2, APOE3 and APOE4. APOE4 is a major genetic risk factor for Alzheimer's disease and is associated with Down's syndrome dementia and poor neurological outcome after traumatic brain injury and haemorrhage. Neurovascular dysfunction is present in normal APOE4 carriers and individuals with APOE4-associated disorders. In mice, lack of Apoe leads to blood-brain barrier (BBB) breakdown, whereas APOE4 increases BBB susceptibility to injury. How APOE genotype affects brain microcirculation remains elusive. Using different APOE transgenic mice, including mice with ablation and/or inhibition of cyclophilin A (CypA), here we show that expression of APOE4 and lack of murine Apoe, but not APOE2 and APOE3, leads to BBB breakdown by activating a proinflammatory CypA-nuclear factor-κB-matrix-metalloproteinase-9 pathway in pericytes. This, in turn, leads to neuronal uptake of multiple blood-derived neurotoxic proteins, and microvascular and cerebral blood flow reductions. We show that the vascular defects in Apoe-deficient and APOE4-expressing mice precede neuronal dysfunction and can initiate neurodegenerative changes. Astrocyte-secreted APOE3, but not APOE4, suppressed the CypA-nuclear factor-κB-matrix-metalloproteinase-9 pathway in pericytes through a lipoprotein receptor. Our data suggest that CypA is a key target for treating APOE4-mediated neurovascular injury and the resulting neuronal dysfunction and degeneration.     

Reduction of choline acetyltransferase activities in APP770 transgenic mice

Transgenic mice overexpressing the 770-amino acid isoform of human Alzheimer amyloid precursor protein exhibit extracellular b -amyloid deposits in brain regions including cerebral cortex and hippocampus, which are severely affected in Alzheimer's disease patients. Significant reduction in choline acetyltransferase (ChAT) activities has been observed in both cortical and hippocampal brain regions in the transgenic mice at the age of 10 months compared with the age-matched non-transgenic mice, but such changes have not been observed in any brain regions of the transgenic mice under the age of 5 months. These results suggest that deposition of b -amyloid can induce changes in the brain cholinergic system of the transgenic mice.     

A beta peptide immunization reduces behavioural impairment and plaques in a model of Alzheimer's disease

Much evidence indicates that abnormal processing and extracellular deposition of amyloid-beta peptide (A beta), a proteolytic derivative of the beta-amyloid precursor protein (betaAPP), is central to the pathogenesis of Alzheimer's disease (reviewed in ref. 1). In the PDAPP transgenic mouse model of Alzheimer's disease, immunization with A beta causes a marked reduction in burden of the brain amyloid. Evidence that A beta immunization also reduces cognitive dysfunction in murine models of Alzheimer's disease would support the hypothesis that abnormal A beta processing is essential to the pathogenesis of Alzheimer's disease, and would encourage the development of other strategies directed at the 'amyloid cascade'. Here we show that A beta immunization reduces both deposition of cerebral fibrillar A beta and cognitive dysfunction in the TgCRND8 murine model of Alzheimer's disease without, however, altering total levels of A beta in the brain. This implies that either a approximately 50% reduction in dense-cored A beta plaques is sufficient to affect cognition, or that vaccination may modulate the activity/abundance of a small subpopulation of especially toxic A beta species.     

Two transmembrane aspartates in presenilin-1 required for presenilin endoproteolysis and gamma-secretase activity

Accumulation of the amyloid-beta protein (Abeta) in the cerebral cortex is an early and invariant event in the pathogenesis of Alzheimer's disease. The final step in the generation of Abeta from the beta-amyloid precursor protein is an apparently intramembranous proteolysis by the elusive gamma-secretase(s). The most common cause of familial Alzheimer's disease is mutation of the genes encoding presenilins 1 and 2, which alters gamma-secretase activity to increase the production of the highly amyloidogenic Abeta42 isoform. Moreover, deletion of presenilin-1 in mice greatly reduces gamma-secretase activity, indicating that presenilin-1 mediates most of this proteolytic event. Here we report that mutation of either of two conserved transmembrane (TM) aspartate residues in presenilin-1, Asp 257 (in TM6) and Asp 385 (in TM7), substantially reduces Abeta production and increases the amounts of the carboxy-terminal fragments of beta-amyloid precursor protein that are the substrates of gamma-secretase. We observed these effects in three different cell lines as well as in cell-free microsomes. Either of the Asp --> Ala mutations also prevented the normal endoproteolysis of presenilin-1 in the TM6 --> TM7 cytoplasmic loop. In a functional presenilin-1 variant (carrying a deletion in exon 9) that is associated with familial Alzheimer's disease and which does not require this cleavage, the Asp 385 --> Ala mutation still inhibited gamma-secretase activity. Our results indicate that the two transmembrane aspartate residues are critical for both presenilin-1 endoproteolysis and gamma-secretase activity, and suggest that presenilin 1 is either a unique diaspartyl cofactor for gamma-secretase or is itself gamma-secretase, an autoactivated intramembranous aspartyl protease.     

A specific amyloid-beta protein assembly in the brain impairs memory

Memory function often declines with age, and is believed to deteriorate initially because of changes in synaptic function rather than loss of neurons. Some individuals then go on to develop Alzheimer's disease with neurodegeneration. Here we use Tg2576 mice, which express a human amyloid-beta precursor protein (APP) variant linked to Alzheimer's disease, to investigate the cause of memory decline in the absence of neurodegeneration or amyloid-beta protein amyloidosis. Young Tg2576 mice (< 6 months old) have normal memory and lack neuropathology, middle-aged mice (6-14 months old) develop memory deficits without neuronal loss, and old mice (> 14 months old) form abundant neuritic plaques containing amyloid-beta (refs 3-6). We found that memory deficits in middle-aged Tg2576 mice are caused by the extracellular accumulation of a 56-kDa soluble amyloid-beta assembly, which we term Abeta*56 (Abeta star 56). Abeta*56 purified from the brains of impaired Tg2576 mice disrupts memory when administered to young rats. We propose that Abeta*56 impairs memory independently of plaques or neuronal loss, and may contribute to cognitive deficits associated with Alzheimer's disease.     

Neurogenic phenotypes and altered Notch processing in Drosophila Presenilin mutants

Presenilin proteins have been implicated both in developmental signalling by the cell-surface protein Notch and in the pathogenesis of Alzheimer's disease. Loss of presenilin function leads to Notch/lin-12-like mutant phenotypes in Caenorhabditis elegans and to reduced Notch1 expression in the mouse paraxial mesoderm. In humans, presenilins that are associated with Alzheimer's disease stimulate overproduction of the neurotoxic 42-amino-acid beta-amyloid derivative (Abeta42) of the amyloid-precursor protein APP. Here we describe loss-of-function mutations in the Drosophila Presenilin gene that cause lethal Notch-like phenotypes such as maternal neurogenic effects during embryogenesis, loss of lateral inhibition within proneural cell clusters, and absence of wing margin formation. We show that presenilin is required for the normal proteolytic production of carboxy-terminal Notch fragments that are needed for receptor maturation and signalling, and that genetically it acts upstream of both the membrane-bound form and the activated nuclear form of Notch. Our findings provide evidence for the existence of distinct processing sites or modifications in the extracellular domain of Notch. They also link the role of presenilin in Notch signalling to its effect on amyloid production in Alzheimer's disease.     

A beta peptide vaccination prevents memory loss in an animal model of Alzheimer's disease

Vaccinations with amyloid-beta peptide (A beta) can dramatically reduce amyloid deposition in a transgenic mouse model of Alzheimer's disease. To determine if the vaccinations had deleterious or beneficial functional consequences, we tested eight months of A beta vaccination in a different transgenic model for Alzheimer's disease in which mice develop learning deficits as amyloid accumulates. Here we show that vaccination with A beta protects transgenic mice from the learning and age-related memory deficits that normally occur in this mouse model for Alzheimer's disease. During testing for potential deleterious effects of the vaccine, all mice performed superbly on the radial-arm water-maze test of working memory. Later, at an age when untreated transgenic mice show memory deficits, the A beta-vaccinated transgenic mice showed cognitive performance superior to that of the control transgenic mice and, ultimately, performed as well as nontransgenic mice. The A beta-vaccinated mice also had a partial reduction in amyloid burden at the end of the study. This therapeutic approach may thus prevent and, possibly, treat Alzheimer's dementia.     

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.     

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.     

Rapid appearance and local toxicity of amyloid-beta plaques in a mouse model of Alzheimer's disease

Senile plaques accumulate over the course of decades in the brains of patients with Alzheimer's disease. A fundamental tenet of the amyloid hypothesis of Alzheimer's disease is that the deposition of amyloid-beta precedes and induces the neuronal abnormalities that underlie dementia. This idea has been challenged, however, by the suggestion that alterations in axonal trafficking and morphological abnormalities precede and lead to senile plaques. The role of microglia in accelerating or retarding these processes has been uncertain. To investigate the temporal relation between plaque formation and the changes in local neuritic architecture, we used longitudinal in vivo multiphoton microscopy to sequentially image young APPswe/PS1d9xYFP (B6C3-YFP) transgenic mice. Here we show that plaques form extraordinarily quickly, over 24 h. Within 1-2 days of a new plaque's appearance, microglia are activated and recruited to the site. Progressive neuritic changes ensue, leading to increasingly dysmorphic neurites over the next days to weeks. These data establish plaques as a critical mediator of neuritic pathology.     

Functional significance of the Kunitz-type inhibitory domains of lipoprotein-associated coagulation inhibitor

Blood coagulation can be initiated when factor VII or VIIa, a plasma protease, binds to its essential cofactor, tissue factor (TF), and proteolytically activates factors IX and X, triggering a cascade of events which eventually leads to the formation of thrombin and a fibrin clot. Plasma contains a lipoprotein-associated coagulation inhibitor (LACI) which inhibits activated factor X (Xa) directly and, in a Xa-dependent way, inhibits VII(a)/TF activity, presumably by forming a quaternary Xa/LACI/VII(a)/TF complex. Sequence analysis of complementary DNA clones has shown that LACI contains three tandemly repeated Kunitz-type serine protease inhibitory domains. To investigate the relationship between these Kunitz structures and LACI function, we have used site-directed mutagenesis to produce altered forms of LACI in which the residue at the active-site cleft of each Kunitz domain has been individually changed. The second Kunitz domain is required for efficient binding and inhibition of Xa, and both Kunitz domains 1 and 2 are required for the inhibition of VIIa/TF activity; but alteration of the active-site residue of the third Kunitz domain has no significant effect on either function. We propose that in the putative inhibitory complex, Kunitz domain 1 is bound to the active site of VII(a)/TF and that Kunitz domain 2 is bound to Xa's active site.     

Early-onset Alzheimer's disease caused by mutations at codon 717 of the beta-amyloid precursor protein gene

A mutation at codon 717 of the beta-amyloid precursor protein gene has been found to cosegregate with familial Alzheimer's disease in a single family. This mutation has been reported in a further five out of approximately 100 families multiply affected by Alzheimer's disease. We have identified another family, F19, in which we have detected linkage between the beta-amyloid precursor protein gene and Alzheimer's disease. Direct sequencing of exon 17 in affected individuals from this family has revealed a base change producing a Val----Gly substitution, also at codon 717. The occurrence of a second allelic variant at codon 717 linked to the Alzheimer's phenotype supports the hypothesis that they are pathogenic mutations.     

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.