The alpha5beta1 integrin mediates elimination of amyloid-beta peptide and protects against apoptosis.

The amyloid-beta peptide (Abeta) can mediate cell attachment by binding to beta1 integrins through an arg-his-asp sequence. We show here that the alpha5beta1 integrin, a fibronectin receptor, is an efficient binder of Abeta, and mediates cell attachment to nonfibrillar Abeta. Cells engineered to express alpha5beta1 internalized and degraded more added Abeta1-40 than did alpha5beta1-negative control cells. Deposition of an insoluble Abeta1-40 matrix around the alpha5beta1-expressing cells was reduced, and the cells showed less apoptosis than the control cells. Thus, the alpha5beta1 integrin may protect against Abeta deposition and toxicity, which is a course of Alzheimer's disease lesions.

Characterization of stable complexes involving apolipoprotein E and the amyloid beta peptide in Alzheimer's disease brain.

Genetic evidence suggests a role for apolipoprotein E (apoE) in Alzheimer's disease (AD) amyloidogenesis. Here, amyloid-associated apoE from 32 AD patients was purified and characterized. We found that brain amyloid-associated apoE apparently exists not as free molecules but as complexes with polymers of the amyloid beta peptide (A beta). Brain A beta-apoE complexes were detected irrespective of the apoE genotype, and similar complexes could be mimicked in vitro. The fine structure of purified A beta-apoE complexes was fibrillar, and immunogold labeling revealed apoE immunoreactivity along the fibrils. Thus, we conclude that A beta-apoE complexes are principal components of AD-associated brain amyloid and that the data presented here support a role for apoE in the pathogenesis of AD.

Endoproteolysis of presenilin 1 and accumulation of processed derivatives in vivo.

The majority of early-onset cases of familial Alzheimer's disease (FAD) are linked to mutations in two related genes, PS1 and PS2, located on chromosome 14 and 1, respectively. Using two highly specific antibodies against nonoverlapping epitopes of the PS1-encoded polypeptide, termed presenilin 1 (PS1), we document that the preponderant PS1-related species that accumulate in cultured mammalian cells, and in the brains of rodents, primates, and humans are approximately 27-28 kDa N-terminal and approximately 16-17 kDa C-terminal derivatives. Notably, a FAD-linked PS1 variant that lacks exon 9 is not subject to endoproteolytic cleavage. In brains of transgenic mice expressing human PS1, approximately 17 kDa and approximately 27 kDa PS1 derivatives accumulate to saturable levels, and at approximately 1:1 stoichiometry, independent of transgene-derived mRNA. We conclude that PS1 is subject to endoproteolytic processing in vivo.

Amyloid beta-peptide polymerization studied using fluorescence correlation spectroscopy.

BACKGROUND: The accumulation of fibrillar deposits of amyloid beta-peptide (Abeta) in brain parenchyma and cerebromeningeal blood vessels is a key step in the pathogenesis of Alzheimer's disease. In this report, polymerization of Abeta was studied using fluorescence correlation spectroscopy (FCS), a technique capable of detecting small molecules and large aggregates simultaneously in solution. RESULTS: The polymerization of Abeta dissolved in Tris-buffered saline, pH 7.4, occurred above a critical concentration of 50 microM and proceeded from monomers/dimers into two discrete populations of large aggregates, without any detectable amount of oligomers. The aggregation showed very high cooperativity and reached a maximum after 40 min, followed by an increase in the amount of monomers/dimers and a decrease in the size of the large aggregates. Electron micrographs of samples prepared at the time for maximum aggregation showed a mixture of an amorphous network and short diffuse fibrils, whereas only mature amyloid fibrils were detected after one day of incubation. The aggregation was reduced when Abeta was incubated in the presence of Abeta ligands, oligopeptides previously shown to inhibit fibril formation, and aggregates were partly dissociated after the addition of the ligands. CONCLUSIONS: The polymerization of Abeta is a highly cooperative process in which the formation of very large aggregates precedes the formation of fibrils. The entire process can be inhibited and, at least in early stages, partly reversed by Abeta ligands.

Activation of protein kinase C inhibits prostaglandin- and potentiates adenosine receptor-stimulated accumulation of cyclic AMP in a human T-cell leukemia line.

Accumulation of cAMP in the human T-cell leukemia cell line Jurkat was stimulated by the adenosine analogue 5'-N-ethylcarboxamidoadenosine (NECA) and by prostaglandin E2 (PGE2). Addition of two phorbol esters, PDiBu and TPA, markedly enhanced the NECA-stimulated accumulation of cAMP whereas the PGE2-stimulated cAMP accumulation was substantially reduced. The non-tumor-promoting phorbol ester, 4 alpha-PDD, had no effect on either NECA- or PGE2-stimulated cAMP accumulation. The ability of PDiBu to inhibit the effect of PGE2 and to stimulate the effect of NECA remained in the presence a low concentration of forskolin (0.3 microM), which per se increased both NECA- and PGE2-stimulated cAMP accumulation. Our results suggest that the effect of PK-C-activating drugs on receptor-mediated cAMP accumulation is entirely dependent on which receptor is being stimulated.

CD3/T-cell receptor coupling to a pertussis and cholera toxin-insensitive G-protein.

We have analyzed the effect of CD3/T-cell receptor stimulation on GTP hydrolysis and GTP binding. We show that stimulation of Jurkat, T-cell, membranes with OKT3 results in a 50% increase in GTP hydrolysis which is specifically inhibited by GDP. Pretreatment of the membranes with neither pertussis toxin nor cholera toxin inhibited the GTP hydrolysis. We also show that stimulation with OKT3 increases the binding of GTP gamma S to Jurkat membranes. These data strongly implicate the involvement of a G-protein in CD3/T-cell receptor signalling.

Beta-amyloid protein protein precursor expression in lacrimal glands and tear fluid.

PURPOSE: Proteases in tear fluid play important roles in the regulation of corneal wound healing. Inhibitors of proteolytic activity are major modulators of the associated events. Although it is known that various enzyme inhibitors exist in tear fluid, it is not known whether certain isoforms of the beta-amyloid protein precursor (beta-APP), a potent inhibitor of serine proteases, are present in tear fluid. The purpose of this study was to investigate whether beta-APP can be detected in human tear fluid and, if so, to determine the isoform composition and cellular origin. METHODS: Tear fluid was collected from healthy volunteers. The beta-APP was identified and characterized by immunoblotting using antibodies specific for domains of the beta-APP. The protein was characterized further by ion exchange chromatography. Expression of the beta-APP gene was studied using in situ hybridization and RNA-RNA solution hybridization assay. RESULTS: beta-APP with protease inhibitory properties was identified in all samples of human tear fluid. Immunologic analysis revealed that it had been processed proteolytically before secretion. Gene expression studies showed that the beta-APP gene was expressed in lacrimal glands, particularly in acinar cells. The gene transcript almost exclusively corresponded to beta-APP containing the protease inhibitor insert. CONCLUSIONS: beta-APP is expressed in lacrimal glands and subsequently is secreted into tear fluid. Because the bulk of the beta-APP contained the protease inhibitor insert, the authors propose that beta-APP is an important regulator of proteolysis in tear fluid and that possibly it plays a role in the events associated with corneal wound healing. This suggests a novel physiological function of beta-APP in addition to those previously described-regulation of blood coagulation and cell growth.

Protein phosphorylation regulates relative utilization of processing pathways for Alzheimer beta/A4 amyloid precursor protein.

The Alzheimer amyloid precursor protein (APP) is a phosphoprotein, and the phosphorylation state of APP at Ser655 can be regulated by protein kinase C, calcium/calmodulin-dependent protein kinase II, and okadaic acid-sensitive protein phosphatases. Other enzymes may also play a role at Ser655 of APP and, perhaps, at other residues. Signal transduction via protein phosphorylation regulates APP metabolism. In particular, APP processing via the nonamyloidogenic secretory cleavage pathway is increased following the activation of protein kinase C or the inactivation of okadaic acid-sensitive protein phosphatases. The mechanism(s) by which protein phosphorylation regulates APP secretory cleavage include (among others): substrate activation, substrate redistribution, protease activation and/or protease redistribution. Current experimental evidence will be discussed, addressing the relative importance of each of these possibilities and the implications for these events in the modulation of beta/A4-amyloidogenesis.

Synergism between phorbol ester and the Ca2+ ionophore A23187 on protein kinase C translocation, [3H]PDBu binding and adenosine A2-receptor activation in Jurkat cells.

Phorbol 12,13-dibutyrate (PDBu) enhances 5'-(N-ethylcarboxamido)-adenosine (NECA) stimulated cyclic adenosine 3',5'-monophosphate (cAMP) production in the human T-cell leukemia line, Jurkat. Addition of the Ca2+ ionophore A23187 lowered the EC50 value for PDBu from 49 to 7.1 nM. In binding experiments, where intact cells were incubated with [3H]PDBu at 37 degrees C, addition of A23187 increased the number of binding sites for the phorbol ester. Pretreatment of cells with A23187 was not sufficient to increase [3H]PDBu binding; A23187 had to be combined with phorbol ester in order to enhance [3H]PDBu binding. PDBu treatment translocated protein kinase from the cytosol to the membrane. This effect of the phorbol ester could be enhanced with A23187 whereas A23187 per se had no effect on protein kinase C distribution. From these data it is concluded that the synergism between A23187 and PDBu, monitored as enhancement of NECA-stimulated cAMP accumulation and increase in [3H]PDBu binding, is paralleled by translocation of the enzyme to the particulate fraction of the cells. The finding that cells where the cellular content of protein kinase C had been translocated to the membrane compartment bound more [3H]PDBu than control cells also suggests that [3H]PDBu binding to intact cells reflects the amount of membrane bound-, rather than the total cellular-enzyme content.

Dual effects of protein kinase-C on receptor-stimulated cAMP accumulation in a human T-cell leukemia line.

In the human T-cell leukemia line Jurkat, cAMP accumulation stimulated by the adenosine receptor agonist 5'-N-ethylcarboxamido adenosine (NECA) was enhanced by tumour-promoting phorbol esters whereas the prostaglandin receptor-stimulated accumulation of cAMP was antagonized. Phorbol esters did not alter the adenosine or prostaglandin receptor-stimulated accumulation of cAMP in cells in which the phospholipid/Ca2+-dependent protein kinase (protein kinase-C) was down-regulated. cAMP stimulation induced by cholera toxin (CT) was enhanced by phorbol esters by 100-300%. The cAMP production induced by forskolin was never enhanced by more than 50% by 4 beta-phorbol-12,13-dibutyrate (PDBu) and there was no stimulation at all after down-regulation of the adenosine receptor by treatment with NECA. Phorbol ester enhanced the NECA-stimulated accumulation of cAMP, even in the presence of concentrations of forskolin that increased the cAMP accumulation several-fold. From these data we conclude that protein kinase-C can interact with receptors coupled to adenylate cyclase in a stimulatory as well as an inhibitory manner. Moreover, protein kinase-C appears to interact with signal transduction at two levels, one highly receptor-specific and one distal to the receptor.

Role of protein kinase C in bradykinin-induced prostaglandin formation in osteoblasts.

UNLABELLED: Bradykinin (1 microM, 5 min) induced translocation of protein kinase C (PKC) to the plasma membrane fraction in osteoblastic MC3T3-E1 cells. Bradykinin also enhanced the binding of phorbol 12,13-dibutyrate (PDBu) to intact cells, a measure of PKC activation. Addition of bradykinin (1 microM) to cells preincubated with [3H]PDBu (10 nM, 20 min) caused an increase in specific PDBu binding that was maximal after 5-10 min. The bradykinin-induced enhancement of PDBu binding was seen at 1 nM and was maximal at 10 nM. The bradykinin B1 receptor agonist des-Arg9-bradykinin (1 microM) did not enhance specific PDBu binding to intact MC3T3-E1 cells. PDBu at and above 3 nM stimulated the formation of prostaglandin E2 (PGE2) in MC3T3-EI cells. This stimulatory effect was seen after 15-20 min incubation. The Ca2+ ionophore A23187 at and above 1 microM induced a rapid (within seconds) burst of PGE2 formation in MC3T3-E1 cells. The effect of PDBu and A23187 on PGE2 formation was synergistic. The PKC inhibitor staurosporine (200 nM) inhibited basal as well as bradykinin-induced prostaglandin-formation in MC3T3-E1 cells. IN CONCLUSION: bradykinin enhances PKC activation in osteoblastic MC3T3-E1 cells. This kinase activation may be involved in bradykinin-induced prostaglandin formation.

Stimulation of the T-cell receptors CD3 and CD2 with OKT3 and OKT11 antibodies activates a common pertussis toxin-insensitive G-protein.

The association of G-proteins with the T-cell-specific receptor structures CD3 and CD2 was investigated. High-affinity GTPase activity in membrane preparations of the human leukemic T-cell line Jurkat could be induced by the monoclonal antibodies OKT3 (anti-CD3) and OKT11 (anti-CD2). When combining maximally active concentrations of OKT3 and OKT11, no additive effect was seen on GTPase activity. In mutant Jurkat cells lacking the CD3 complex but with an intact CD2 receptor, neither OKT3 nor OKT11 could stimulate GTPase activity. Activation of CD3 and CD2 by monoclonal antibodies also stimulated phospholipase C activity as measured by breakdown of membrane phosphoinositides in wild-type but not in mutant Jurkat cells. Neither GTPase nor phospholipase C activation was sensitive to pretreatment with doses of pertussis toxin (PTX) that caused ADP ribosylation of a sensitive G-protein. Our data show that the CD3 complex and the CD2 receptor may activate a common PTX-insensitive G-protein. The CD2 receptor appears to stimulate the G-protein by interacting with the CD3 complex. The data are compatible with, but do not prove, that this G-protein is involved in the activation of phospholipase C by the two receptors.

Levels of cerebrospinal fluid apolipoprotein E in patients with Alzheimer's disease and healthy controls.

One isoform of apolipoprotein E (apoE), a protein primarily involved in transport of lipids, is associated with an increased risk for Alzheimer's disease. Moreover, fragments of apoE are deposited in the amyloid that invariantly are found in brain tissue of disease victims. An intriguing possibility is therefore that increased levels of apoE are involved in the pathogenesis of the disease. Levels of full-length apoE in cerebrospinal fluid from 13 Alzheimer patients and 12 healthy controls were determined using Western blotting technique. Levels of the protein were essentially identical to previously reported findings and no difference between patients and healthy controls was found. Hence, it is concluded that increases in cerebrospinal fluid (CSF) levels of apoE are not involved in the pathogenesis of Alzheimer's disease and that measurement of CSF apoE levels does not seem to be useful as a diagnostic procedure.

Phorbol-12,13-dibutyrate enhances the cyclic AMP accumulation in rat hippocampal slices induced by adenosine analogues.

Phorbol-12,13-dibutyrate (PDiBu) augmented the accumulation of [3H]-cyclic AMP in rat hippocampal slices elicited by the adenosine receptor agonist 5'-(N-ethyl)-carboxamidoadenosine (NECA). A similar, but less pronounced, effect was observed when using tetradecanoylphorbol acetate (TPA) provided that the slices were preincubated with the phorbol ester before addition of NECA. PDiBu also induced cyclic AMP accumulation in the absence of NECA, but this effect could be markedly reduced or even abolished by the adenosine antagonist 8-para sulphophenyl theophylline (8-pst). Whereas no clearcut synergism was observed between isoprenaline and PDiBu under normal circumstances, such synergism was observed after elimination of the cyclic AMP accumulation caused by endogenous adenosine. PDiBu slightly enhanced the stimulatory effect of low (0.1-1 microM) concentrations of forskolin, but did not enhance the effect of 10 microM forskolin. In the presence of forskolin, another adenosine analogue, R-PIA, had a biphasic effect on cAMP accumulation. With PDiBu present the inhibitory phase was entirely eliminated and the stimulatory phase substantially enhanced. A synergistic interaction between PDiBu and NECA was found even after inhibition of the Ni-protein by N-ethyl maleimide. It is concluded that the phorbol ester may enhance the stimulatory effect of adenylate cyclase stimulatory compounds, including endogenous adenosine. The effect may partly depend on protein kinase C-mediated inactivation of N-protein mediated adenylate cyclase inhibition, but other mechanisms also seem to be involved.

Activation of protein kinase C via the T-cell receptor complex potentiates cyclic AMP responses in T-cells.

We have recently shown that activation of protein kinase C by tumour promoting phorbolesters, such as 4 beta-phorbol-12,13-dibutyrate, stimulates adenosine-induced accumulation of cAMP in Jurkat cells, a human T-leukaemia line. Activating the CD3 complex associated with the T-cell receptor by means of the monoclonal antibody OKT3 caused a concentration-dependent accumulation of inositol phosphates and an increase in the phosphorylation of an endogenous protein kinase C substrate. OKT3 also mimicked the previously reported effects of protein kinase C since it potentiated the cAMP stimulation by either an adenosine analogue, NECA, or cholera toxin. Thus, our results indicate that stimulation of a receptor activating phospholipase C and protein kinase C can secondarily enhance the action of agonists that act on adenylate cyclase-coupled receptors.

Role of a pertussis toxin sensitive G-protein in mediating the effects of phorbol esters on receptor activated cyclic AMP accumulation in Jurkat cells.

In the human T-cell line, Jurkat, the accumulation of cyclic AMP induced by adenosine is enhanced by tumor-promoting phorbol esters, whereas prostaglandin E2 receptor-stimulated cAMP accumulation is antagonized (Nordstedt et al. 1989). In the present study we examine the involvement of pertussis toxin sensitive guanine nucleotide binding proteins (G-proteins) in producing the phorbol ester effects. Pertussis toxin pretreatment of the Jurkat cells invariably caused an ADP ribosylation of two G-proteins that inhibit adenylyl cyclase, tentatively identified as Gi2 and Gi3, using Western blots. Pertussis toxin treatment had little effect on basal cAMP accumulation, but sometimes inhibited, sometimes stimulated agonist and cholera toxin induced cAMP accumulation. The latter effect was not mimicked by the B-oligomer. Irrespective of whether pertussis toxin stimulated or inhibited NECA and cholera toxin-induced cAMP accumulation it could not block the effect of phorbol-12,13-dibutyrate (PDBu). The inhibitory effect of PDBu on prostaglandin E2-induced cAMP accumulation was, however, invariably eliminated by pertussis toxin treatment. In conclusion, activation of protein kinase C by phorbol esters reveals a Gi-mediated prostaglandin E receptor-induced inhibition of adenylate cyclase in addition to the prostaglandin E receptor-mediated stimulation of cAMP accumulation in Jurkat cells. The enhancement of adenosine A2 receptor stimulated cAMP accumulation by PDBu, on the other hand, does not involve a PTX sensitive Gi-protein.

Genetics of Alzheimer's disease--routes to the pathophysiology.

Considerable advances have been made the last years in the understanding of the pathogenesis of Alzheimer's disease (AD): Several pathogenic mutations have been found in the amyloid precursor protein gene on chromosome 21. Two other dominantly operating genes on chromosome 14 and 1 were recently cloned, named presenilin 1 and 2, respectively. Mutations in these genes give rise to AD with a very early age of onset. Increased Abeta1-42 is most likely the pathogenic mechanism in all these cases. A susceptibility gene for AD has also been found. There is an association between the epsilon4 allele of the apolipoprotein E (APOE) gene and late-onset AD. The epsilon4 allele increases the risk for AD, although some epsilon4 homozygotes may live a long life without developing AD. The mechanism by which APOE epsilon4 promotes development of AD is most likely increased plaque formation. The new knowledge on pathogenic mechanisms of the disease gives opportunities for alternative strategies for therapeutic intervention.

[Alzheimer's disease--an amyloid disease of the brain]. / Alzheimers sjukdom--en amyloidsjukdom i hjärnan.

During recent years, Alzheimer's disease (AD) has attracted increasing interest among clinicians and neuropathologists throughout the world. The amyloid core of the neuritic plaques found in the brains of individuals with the disease has been shown to be composed of a distinct peptide formed through proteolytic degradation of a large precursor protein, the Alzheimer amyloid precursor protein (APP), which exists in at least three isoforms differing from each other in the splicing of the primary transcript from which they derive. Although the physiological function of APP remains unknown, it has been suggested to function as a protease inhibitor and to be important to the blood coagulation system. The gene encoding APP is located on the long arm of chromosome 21. Individuals with Down's syndrome (trisomy 21) often develop AD in early middle age, suggesting that the 50 percent increase in APP, gene expression may promote the development of the disease. Mutations in the APP gene have also been shown to be associated, probably pathogenetically with familial forms of AD. The conclusions drawn from these studies include (i) that the amyloidosis associated with AD is probably a central pathogenetic factor and (ii) that the development of drugs capable of inhibiting amyloidosis might be an appropriate strategy for the treatment of AD.