Amyloid-beta-protein isoforms in brain of subjects with PS1-linked, beta APP-linked and sporadic Alzheimer disease

To determine whether similar abnormalities of various soluble full-length and N-terminal truncated Abeta peptides occur in postmortem cerebral cortex of affected PS1 mutation carriers, we examined the amounts of two amyloid species ending at residue 40 or at residues 42(43) using sandwich ELISA systems. Our results indicate that PS1 mutations effect a dramatic accumulation in brain of the highly insoluble potentially neurotoxic long-tailed isoforms of the Abeta peptide such as Abeta1-42(43) and Abetax-42(43). This enhancing effect of PS1 mutation on Abetax-42(43) deposition was highly similar to that of a betaAPP mutation (Val717Ile) but the effects on Abetax-40 production were significantly different between these two causal genes. In contrast to previous studies of soluble Abeta in plasma and in supernatants from cultured fibroblasts of subjects with PS1 mutations, our studies also show that there is an increase in insoluble Abetax-40 peptides in brain of subjects with PS1 mutations.     

HOP-1, a Caenorhabditis elegans presenilin, appears to be functionally redundant with SEL-12 presenilin and to facilitate LIN-12 and GLP-1 signaling

Mutant presenilins have been found to cause Alzheimer disease. Here, we describe the identification and characterization of HOP-1, a Caenorhabditis elegans presenilin that displays much more lower sequence identity with human presenilins than does the other C. elegans presenilin, SEL-12. Despite considerable divergence, HOP-1 appears to be a bona fide presenilin, because HOP-1 can rescue the egg-laying defect caused by mutations in sel-12 when hop-1 is expressed under the control of sel-12 regulatory sequences. HOP-1 also has the essential topological characteristics of the other presenilins. Reducing hop-1 activity in a sel-12 mutant background causes synthetic lethality and terminal phenotypes associated with reducing the function of the C. elegans lin-12 and glp-1 genes. These observations suggest that hop-1 is functionally redundant with sel-12 and underscore the intimate connection between presenilin activity and LIN-12/Notch activity inferred from genetic studies in C. elegans and mammals.     

Two distinct populations of primary cytotoxic cells infiltrating into allografted tumor rejection sites: infiltration of macrophages cytotoxic against allografted tumor precedes that of multiple sets of cytotoxic T lymphocytes with distinct specificity to alloantigens

The majority of cases with familial Alzheimer's disease (FAD) are linked to mutations of the presenilin (PS) genes. These genes show considerable sequence similarity to the sel-12 gene of Caenorhabditis elegans, which has been postulated to function in the facilitated signalling by lin-12 and glp-1. In order to analyse the functional conservation of the presenilins, we introduced the human PS-1 cDNA, as well as clinical and deletion mutant proteins, into sel-12 mutant animals and tested their potential to rescue the egg-laying defect. Human PS-1 expressed from the sel-12 promoter fully rescued the sel-12 phenotype, whereas two missense mutations, C410Y and A246E, identified in pedigrees with FAD, exhibited a strongly decreased rescuing activity. The large hydrophilic loop and transmembrane domain 7 are required for the biological activity of PS-1. PS-1 protein was proteolytically cleaved in C. elegans as it is in human cells. A PS-1 splice variant (FAD mutation deltaexon9) that does not undergo proteolytic cleavage also substituted for sel-12. The conservation of function of human PS-1 and C. elegans sel-12 suggests that presenilin proteins are required, directly or indirectly, for the proper operation of the Notch signalling pathway. FAD-associated mutant proteins tested showed different rescuing activities, indicating that they might affect different functional or regulatory aspects of PS-1. Proteolytic processing is not a prerequisite for PS-1 function in C. elegans.     

Presenilin 1 regulates the processing of beta-amyloid precursor protein C-terminal fragments and the generation of amyloid beta-protein in endoplasmic reticulum and Golgi

Progressive cerebral deposition of the amyloid beta-protein (Abeta) is believed to play a pivotal role in the pathogenesis of Alzheimer's disease (AD). The highly amyloidogenic 42-residue form of Abeta (Abeta42) is the first species to be deposited in both sporadic and familial AD. Mutations in two familial AD-linked genes, presenilins 1 (PS1) and 2 (PS2), selectively increase the production of Abeta42 in cultured cells and the brains of transgenic mice, and gene deletion of PS1 shows that it is required for normal gamma-secretase cleavage of the beta-amyloid precursor protein (APP) to generate Abeta. To establish the subcellular localization of the PS1 regulation of APP processing to Abeta, fibroblasts from PS1 wild-type (wt) or knockout (KO) embryos as well as Chinese hamster ovary (CHO) cells stably transfected with wt or mutant PS1 were subjected to subcellular fractionation on discontinuous Iodixanol gradients. APP C-terminal fragments (CTF) were markedly increased in both endoplasmic reticulum- (ER-) and Golgi-rich fractions of fibroblasts from KO mice; moreover, similar increases were documented directly in KO brain tissue. No change in the subcellular distribution of full-length APP was detectable in fibroblasts lacking PS1. In CHO cells, a small portion of APP, principally the N-glycosylated isoform, formed complexes with PS1 in both ER- and Golgi-rich fractions, as detected by coimmunoprecipitation. When the same fractions were analyzed by enzyme-linked immunosorbent assays for Abetatotal and Abeta42, Abeta42 was the major Abeta species in the ER fraction (Abeta42:Abetatotal ratio 0.5-1.0), whereas absolute levels of both Abeta42 and Abeta40 were higher in the Golgi fraction and the Abeta42:Abetatoal ratio was 0.05-0.16 there. Mutant PS1 significantly increased Abeta42 levels in the Golgi fraction. Our results indicate PS1 and APP can interact in the ER and Golgi, where PS1 is required for proper gamma-secretase processing of APP CTFs, and that PS1 mutations augment Abeta42 levels principally in Golgi-like vesicles.     

Presenilins and early-onset familial Alzheimer's disease

Thirty-seven missense mutations and a splice-site mutation in the presenilin gene PS1 on chromosome 14 and two missense mutations PS2 on chromosome 1 co-segregate with early-onset familial Alzheimer's disease (AD). The presenilins belong to a family of conserved integral membrane proteins which include Caenorhabditis elegans SPE4 and SEL12 and the rat apoptosis-linked gene, ALG3. This review summarizes the genetics of presenilins in AD and indicators of putative function based on cellular localization and the functions of non-human homologues. Findings to date suggest an important role of presenilins in beta-amyloid (A beta) production: in vitro and in vivo studies have shown that presenilin mutations are associated with relatively increased production of the longer, and highly fibrillogenic A beta 42(43) peptide, and a marked elevation in the number of A beta 42-immunoreactive plaques in the brains of individuals with familial AD who carry PS1 and PS2 mutations. There is growing evidence that the deposition of A beta 42(43) could in some cases be an early and key event in the AD pathogenic cascade. The genetic and molecular biological data discussed in this review describe mechanisms by which presenilin mutations could lead to the development of AD. Also, mutant presenilins may be more proapoptotic. It is argued that the understanding of the processes by which presenilin mutations lead to the development of AD will help in devising a coherent framework for therapeutic strategies.     

The role of A beta 42 in Alzheimer's disease

Our recent studies of plasma, fibroblasts, transfected cells and transgenic mice show that a fundamental effect of the mutations linked to familial Alzheimer's disease (FAD) is to increase the extracellular concentration of A beta 42. This effect of the FAD-linked mutations is likely to be directly related to the pathogenesis of Alzheimer's disease (AD) because A beta 42 is deposited early and selectively in the senile plaques that are an invariant feature of all forms of AD. Thus our results provide strong evidence that the FAD-linked mutations all cause AD by increasing the extracellular concentration of A beta 42 (43), thereby fostering A beta deposition, and they support the hypothesis that cerebral A beta deposition is an essential early event in the pathogenesis of all forms of AD. Interactions between the basal forebrain cholinergic system and A beta that could influence AD pathogenesis are discussed.     

The alternatively spliced Kunitz protease inhibitor domain alters amyloid beta protein precursor processing and amyloid beta protein production in cultured cells

The insoluble amyloid deposited extracellularly in the brains of patients with Alzheimer's disease (AD) is composed of amyloid beta protein, a approximately 4-kDa secreted protein that is derived from a set of large proteins collectively referred to as the amyloid beta protein precursor (betaAPP). During normal processing the betaAPP is cleaved by beta secretase, producing a large NH2-terminal secreted derivative (sAPPbeta) and a COOH-terminal fragment beginning at Abeta1, which is subsequently cleaved by gamma secretase releasing secreted Abeta. Most secreted Abeta is Abeta1-40, but approximately 10% of secreted Abeta is Abeta1-42. Alternative betaAPP cleavage by alpha secretase produces a slightly longer NH2-terminal secreted derivative (sAPPalpha) and a COOH-terminal fragment beginning at Abeta17, which is subsequently cleaved by gamma secretase releasing a approximately 3-kDa secreted form of Abeta (P3). Several of the betaAPP isoforms that are produced by alternative splicing contain a 56-amino acid Kunitz protease inhibitor (KPI) domain known to inhibit proteases such as trypsin and chymotrypsin. To determine whether the KPI domain influences the proteolytic cleavages that generate Abeta, we compared Abeta production in transfected cells expressing human KPI-containing betaAPP751 or KPI-free betaAPP695. We focused on Abetas ending at Abeta42 because these forms appear to be most relevant to AD. Using specific sandwich enzyme-linked immunosorbent assays, we analyzed full-length Abeta1-42 and total Abeta ending at Abeta42 (Abeta1-42 + P3(42)). In addition, we analyzed the large secreted derivatives produced by alpha secretase (sAPPalpha) and beta secretase (sAPPbeta). In mouse teratocarcinoma (P19) cells expressing betaAPP695 or betaAPP751, expression of the KPI-containing betaAPP751 resulted in the secretion of a lower percentage of P3(42) and sAPPalpha and a correspondingly higher percentage of Abeta1-42 and sAPPbeta. Similar results were obtained in human embryonic kidney (293) cells. These results indicate that expression of the KPI domain reduces alpha secretase cleavage so that less P3 and relatively more full-length Abeta are produced. Thus, in human brain and in animal models of AD, the amount of KPI-containing betaAPP produced may be an important factor influencing Abeta deposition.     

Amyloid beta-protein reduces acetylcholine synthesis in a cell line derived from cholinergic neurons of the basal forebrain

The characteristic features of a brain with Alzheimer disease (AD) include the presence of neuritic plaques composed of amyloid beta-protein (Abeta) and reductions in the levels of cholinergic markers. Neurotoxic responses to Abeta have been reported in vivo and in vitro, suggesting that the cholinergic deficit in AD brain may be secondary to the degeneration of cholinergic neurons caused by Abeta. However, it remains to be determined if Abeta contributes to the cholinergic deficit in AD brain by nontoxic effects. We examined the effects of synthetic Abeta peptides on the cholinergic properties of a mouse cell line, SN56, derived from basal forebrain cholinergic neurons. Abeta 1-42 and Abeta 1-28 reduced the acetylcholine (AcCho) content of the cells in a concentration-dependent fashion, whereas Abeta 1-16 was inactive. Maximal reductions of 43% and 33% were observed after a 48-h treatment with 100 nM of Abeta 1-42 and 50 pM of Abeta 1-28, respectively. Neither Abeta 1-28 nor Abeta 1-42 at a concentration of 100 nM and a treatment period of 2 weeks was toxic to the cells. Treatment of the cells with Abeta 25-28 (48 h; 100 nM) significantly decreased AcCho levels, suggesting that the sequence GSNK (aa 25-28) is responsible for the AcCho-reducing effect of Abeta. The reductions in AcCho levels caused by Abeta 1-42 and Abeta 1-28 were accompanied by proportional decreases in choline acetyltransferase activity. In contrast, acetylcholinesterase activity was unaltered, indicating that Abeta specifically reduces the synthesis of AcCho in SN56 cells. The reductions in AcCho content caused by Abeta 1-42 could be prevented by a cotreatment with all-trans-retinoic acid (10 nM), a compound previously shown to increase choline acetyltransferase mRNA expression in SN56 cells. These results demonstrate a nontoxic, suppressive effect of Abeta on AcCho synthesis, an action that may contribute to the cholinergic deficit in AD brain.     

Intracellular generation and accumulation of amyloid beta-peptide terminating at amino acid 42

Amyloid beta-peptide (Abeta) is known to accumulate in senile plaques of Alzheimer's disease (AD) patients and is now widely believed to play a major role in the disease. Two populations of peptides occur terminating either at amino acid 40 or at amino acid 42 (Abeta1-40 and Abeta1-42). Alternative N-terminal cleavages produce additional heterogeneity (Abetax-40 and Abetax-42). Peptides terminating at amino acid 42 are believed to be the major player in sporadic AD as well as familial AD (FAD). Whereas the cellular mechanism for the generation of Abeta terminating at amino acid 40 is well understood, very little is known about the cleavage of Abeta after amino acid 42. By using two independent methods we demonstrate intracellular Abeta1-42 as well as Abetax-42 but less Abetax-40 and Abeta1-40 in kidney 293 cells stably transfected with wild type beta-amyloid precursor protein (betaAPP) or the FAD-associated Val/Gly mutation. Moreover, retention of betaAPP within the endoplasmic reticulum (ER) by treatment with brefeldin A does not block the cleavage at amino acid 42 but results in an increased production of all species of Abeta terminating at amino acid 42. This indicates that the cleavage after amino acid 42 can occur within the ER. Treatment of cells with monensin, which blocks transport of (betaAPP) within the Golgi causes a marked accumulation of intracellular Abetax-42 and Abetax-40. Therefore these experiments indicate that the gamma-secretase cleavage of Abeta after amino acid 42 can occur within the ER and later within the secretory pathway within the Golgi. Moreover inhibition of reinternalization by cytoplasmic deletions of betaAPP as well as inhibition of intracellular acidification by NH4Cl does not block intracellular Abeta1-42 or Abetax-42 production.     

Increased amyloid-beta42(43) in brains of mice expressing mutant presenilin 1

Mutations in the genes encoding amyloid-beta precursor protein (APP), presenilin 1 (PS1) and presenilin 2 (PS2) are known to cause early-onset, autosomal dominant Alzheimer's disease. Studies of plasma and fibroblasts from subjects with these mutations have established that they all alter amyloid beta-protein (beta APP) processing, which normally leads to the secretion of amyloid-beta protein (relative molecular mass 4,000; M(r) 4K; approximately 90% A beta1-40, approximately 10% A beta1-42(43)), so that the extracellular concentration of A beta42(43) is increased. This increase in A beta42(43) is believed to be the critical change that initiates Alzheimer's disease pathogenesis because A beta42(43) is deposited early and selectively in the senile plaques that are observed in the brains of patients with all forms of the disease. To establish that the presenilin mutations increase the amount of A beta42(43) in the brain and to test whether presenilin mutations act as true (gain of function) dominants, we have now constructed mice expressing wild-type and mutant presenilin genes. Analysis of these mice showed that overexpression of mutant, but not wild-type, PS1 selectively increases brain A beta42(43). These results indicate that the presenilin mutations probably cause Alzheimer's disease through a gain of deleterious function that increases the amount of A beta42(43) in the brain.     

Contrasting role of presenilin-1 and presenilin-2 in neuronal differentiation in vitro

Presenilin-1 (PS1) and presenilin-2 (PS2), the major genes of familial Alzheimer's disease, are homologous to sel-12, a Caenorhabditis elegans gene involved in cell fate decision during development. Recently, wild-type and mutant presenilins have been associated also with apoptotic cell death. By using stable transfection of antisense cDNAs, we studied the functions of PS1 and PS2 during neuronal differentiation in the NTera2 human teratocarcinoma (NT2) cell line. Expression of antisense PS1 resulted in a failure of the clones to differentiate into neurons after retinoic acid induction, whereas cells transfected with antisense PS2 differentiated normally. Concomitantly, antisense PS1 clones were associated with increased apoptosis both under basal conditions and during the early period of neuronal differentiation after retinoic acid treatment. Overexpression of bcl-2 in antisense PS1 clones reduced cell death and resulted in a recovery of neuronal differentiation. These studies suggest that PS1 plays a role in differentiation and cell death and that PS1 and PS2 have differing physiological roles in this experimental paradigm.     

Paediatric pyogenic granuloma presenting as a unilateral nasal polyp

The 42/43-residue amyloid beta-peptide (Abeta) is widely believed to play a major role in Alzheimer's disease. The present study shows that the rat brain contains a carboxypeptidase that efficiently deletes three amino acids from Abeta1-43. The carboxypeptidase activity in the brain was completely inhibited by 1 mM phenylmethylsulfonyl fluoride, suggesting the protease is a serine carboxypeptidase. The carboxy-terminal truncation of Abeta1-43 was moderately inhibited by carbobenzoxy-Leu-leucinal, carbobenzoxy-Leu-Leu-leucinal, and carbobenzoxy-Leu-Leu-norvalinal, and weakly by antipain. The present data suggest that the serine carboxypeptidase contributes to the generation of short-tailed Abeta peptides and is important in the intracellular clearance of Abeta1-42/43 in brains.     

Evidence that levels of presenilins (PS1 and PS2) are coordinately regulated by competition for limiting cellular factors

Mutations in two related genes, PS1 and PS2, account for the majority of early onset cases of familial Alzheimer's disease. PS1 and PS2 are homologous polytopic membrane proteins that are processed endoproteolytically into two fragments in vivo. In the present report we examine the fate of endogenous PS1 and PS2 after overexpression of human PS1 or PS2 in mouse N2a neuroblastoma cell lines and human PS1 in transgenic mice. Remarkably, in N2a cell lines and in brains of transgenic mice expressing human PS1, accumulation of human PS1 derivatives is accompanied by a compensatory, and highly selective, decrease in the steady-state levels of murine PS1 and PS2 derivatives. Similarly, the levels of murine PS1 derivatives are diminished in cultured cells overexpressing human PS2. To define the minimal sequence requirements for "replacement" we expressed familial Alzheimer's disease-linked and experimental deletion variants of PS1. These studies revealed that compromised accumulation of murine PS1 and PS2 derivatives resulting from overexpression of human PS1 occurs in a manner independent of endoproteolytic cleavage. Our results are consistent with a model in which the abundance of PS1 and PS2 fragments is regulated coordinately by competition for limiting cellular factor(s).     

Effects of SEL-12 presenilin on LIN-12 localization and function in Caenorhabditis elegans

Presenilins have been implicated in the development of Alzheimer's disease and in facilitating LIN-12/Notch activity. Here, we use genetic methods to explore the relationship between C. elegans LIN-12 and SEL-12 presenilin. Reducing sel-12 activity can suppress the effects of elevated lin-12 activity when LIN-12 is activated by missense mutations but not when LIN-12 is activated by removal of the extracellular and transmembrane domains. These results suggest that SEL-12 does not function downstream of activated LIN-12. An active SEL-12::GFP hybrid protein accumulates in the perinuclear region of the vulval precursor cells (VPCs) of living hermaphrodites, consistent with a localization in endoplasmic reticulum/Golgi membranes; when sel-12 activity is reduced, less LIN-12 protein accumulates in the plasma membranes of the VPCs. Together with the genetic interactions between lin-12 and sel-12, these observations suggest a role for SEL-12 in LIN-12 processing or trafficking. However, SEL-12 does not appear to be a general factor that influences membrane protein activity, since reducing sel-12 activity does not suppress or enhance hypomorphic mutations in other genes encoding membrane proteins. We discuss potential parallels for the role of SEL-12/presenilin in facilitating LIN-12/Notch activity and in amyloid precursor protein (APP) processing.     

Essential role of E2-25K/Hip-2 in mediating amyloid-beta neurotoxicity

The ubiquitin/proteasome system has been proposed to play an important role in Alzheimer's disease (AD) pathogenesis. However, the critical factor(s) modulating both amyloid-beta peptide (Abeta) neurotoxicity and ubiquitin/proteasome system in AD are not known. We report the isolation of an unusual ubiquitin-conjugating enzyme, E2-25K/Hip-2, as a mediator of Abeta toxicity. The expression of E2-25K/Hip-2 was upregulated in the neurons exposed to Abeta(1-42) in vivo and in culture. Enzymatic activity of E2-25K/Hip-2 was required for both Abeta(1-42) neurotoxicity and inhibition of proteasome activity. E2-25K/Hip-2 functioned upstream of apoptosis signal-regulating kinase 1 (ASK1) and c-Jun N-terminal kinase (JNK) in Abeta(1-42) toxicity. Further, the ubiquitin mutant, UBB+1, a potent inhibitor of the proteasome which is found in Alzheimer's brains, was colocalized and functionally interacted with E2-25K/Hip-2 in mediating neurotoxicity. These results suggest that E2-25K/Hip-2 is a crucial factor in regulating Abeta neurotoxicity and could play a role in the pathogenesis of Alzheimer's disease.     

Presenilin 1 associates with glycogen synthase kinase-3beta and its substrate tau

Families bearing mutations in the presenilin 1 (PS1) gene develop Alzheimer's disease. Previous studies have shown that the Alzheimer-associated mutations in PS1 increase production of amyloid beta protein (Abeta1-42). We now show that PS1 also regulates phosphorylation of the microtubule-associated protein tau. PS1 directly binds tau and a tau kinase, glycogen synthase kinase 3beta (GSK-3beta). Deletion studies show that both tau and GSK-3beta bind to the same region of PS1, residues 250-298, whereas the binding domain on tau is the microtubule-binding repeat region. The ability of PS1 to bring tau and GSK-3beta into close proximity suggests that PS1 may regulate the interaction of tau with GSK-3beta. Mutations in PS1 that cause Alzheimer's disease increase the ability of PS1 to bind GSK-3beta and, correspondingly, increase its tau-directed kinase activity. We propose that the increased association of GSK-3beta with mutant PS1 leads to increased phosphorylation of tau.     

Intravascular infusions of soluble beta-amyloid compromise the blood-brain barrier, activate CNS glial cells and induce peripheral hemorrhage

Vascular wall levels of soluble beta-amyloid1-40 (Abeta1-40) are elevated in Alzheimer's disease (AD). Moreover, plasma Abeta levels are increased in familial AD, as well as in some cases of sporadic AD. To determine the histopathologic and behavioral consequences of elevated vascular Abeta levels, Abeta1-40 (50 micrograms in distilled water) or vehicle was intravenously infused twice daily into 3-month old male Sprague-Dawley rats for 2 weeks. Intravenous Abeta infusions impaired blood-brain barrier integrity, as indicated by substantial perivascular and parenchyma IgG immunostaining within the brain. Also evident in Abeta-infused animals was an increase in GFAP immunostaining around cerebral blood vessels, and an enhancement of OX-42 microglial immunostaining in brain white matter. Gross pulmonary hemorrhage was noted in most Abeta-infused animals. All the observed changes occurred in the absence of Congo red birefringence. No significant cognitive deficits were present in Abeta-infused animals during water maze acquisition and retention testing, which was conducted during the second week of treatment. These results indicate that circulating Abeta can: (1) induce vessel dysfunction/damage in both the brain and the periphery without complex Abeta fibril formation/deposition, and (2) induce an activation of brain astrocytes and microglia. Taken together, our results suggest that if circulating Abeta is elevated in AD, it is likely to have a pathophysiologic role.     

Amyloid precursor protein processing in sterol regulatory element-binding protein site 2 protease-deficient Chinese hamster ovary cells

Amyloid peptides of 39-43 amino acids (Abeta) are the major constituents of amyloid plaques present in the brains of Alzheimer's (AD) patients. Proteolytic processing of the amyloid precursor protein (APP) by the yet unidentified beta- and gamma-secretases leads to the generation of the amyloidogenic Abeta peptides. Recent data suggest that all of the known mutations leading to early onset familial AD alter the processing of APP such that increased amounts of the 42-amino acid form of Abeta are generated by a gamma-secretase activity. Identification of the beta- and/or gamma-secretases is a major goal of current AD research, as they are prime targets for therapeutic intervention in AD. It has been suggested that the sterol regulatory element-binding protein site 2 protease (S2P) may be identical to the long sought gamma-secretase. We have directly tested this hypothesis using over-expression of the S2P cDNA in cells expressing APP and by characterizing APP processing in mutant Chinese hamster ovary cells that are deficient in S2P activity and expression. The data demonstrate that S2P does not play an essential role in the generation or secretion of Abeta peptides from cells, thus it is unlikely to be a gamma-secretase.