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G Multhaup

Publications and source records attributed to G Multhaup.

At least 55 records · Page 3Linked to original sources
PubMed1998

Copper-binding amyloid precursor protein undergoes a site-specific fragmentation in the reduction of hydrogen peroxide.

The extracellular domain of transmembrane Abeta amyloid precursor protein (APP) has a Cu(II) reducing activity upon Cu(II) binding associated with the formation of a new disulfide bridge. The complete assignment of the disulfide bond revealed the involvement of cysteines 144 and 158 around copper-binding histidine residues. The vulnerability of APP-Cu(I) complexes to reactive oxygen species was elaborated as a site-specific and random fragmentation of APP in a time-dependent manner and at low concentrations of H2O2. Analysis of the specific reaction revealed the generation of C-terminal polypeptides, containing the Abeta domain. APP catalyzed the reduction of H2O2 and oxidation of Cu(I) to Cu(II) in a "peroxidative" reaction in vitro. The resulting bound copper-hydroxyl radical intermediate [APP-Cu(II)(.OH)] then likely participated in a Fenton type of reaction with radical formation as a prerequisite for protein degradation. Evidence from two observations suggests that the reaction takes place in two phases. Bathocuproine, a trapping agent for Cu(I), abolished the initial fragmentation, and chelation of Cu(II) by DTPA (diethylenetriaminepentaacetic acid) interrupted the reaction cascade induced by H2O2 at later stages. Consequently, the results suggest that a cytotoxic gain-of-function of APP-Cu(I) complexes might result in a perturbation of free radical homeostasis. What significance such a perturbation may have for the pathogenesis of Alzheimer's disease remains to be determined.

Amino Acid Sequence↗
PubMed1998

Relative increase in Alzheimer's disease of soluble forms of cerebral Abeta amyloid protein precursor containing the Kunitz protease inhibitory domain.

Although a number of studies have examined amyloid precursor protein (APP) mRNA levels in Alzheimer's disease (AD), no clear consensus has emerged as to whether the levels of transcripts for isoforms containing a Kunitz protease inhibitory (KPI)-encoded region are increased or decreased in AD. Here we compare AD and control brain for the relative amounts of APP protein containing KPI to APP protein lacking this domain. APP protein was purified from the soluble subcellular fraction and Triton X-100 membrane pellet extract of one hemisphere of AD (n = 10), normal (n = 7), and neurological control (n = 5) brains. The amount of KPI-containing APP in the purified protein samples was determined using two independent assay methods. The first assay exploited the inhibitory action of KPI-containing APP on trypsin. The second assay employed reflectance analysis of Western blots. The proportion of KPI-containing forms of APP in the soluble subcellular fraction of AD brains is significantly elevated (p < 0.01) compared with controls. Species containing a KPI domain comprise 32-41 and 76-77% of purified soluble APP from control and AD brains, respectively. For purified membrane-associated APP, 72-77 and 65-82% of control and AD samples, respectively, contain a KPI domain. Since KPI-containing species of APP may be more amyloidogenic (Ho, L., Fukuchi, K., and Yonkin, S. G. (1996) J. Biol. Chem. 271, 30929-30934), our findings support an imbalance of isoforms as one possible mechanism for amyloid deposition in sporadic AD.

Alternative Splicing↗
PubMed1998

Inhibition of platelet activation by the Alzheimer's disease amyloid precursor protein.

The amyloid precursor protein (APP) of Alzheimer's disease is abundantly expressed in the platelet alpha-granule where its role remains unclear. This study describes a novel function for APP in regulating human platelet activation. Preincubation of platelet-rich plasma with recombinant secreted APP (sAPP) isoforms dose-dependently inhibited platelet aggregation and secretion induced by ADP or adrenaline. Similarly, sAPP potently inhibited low-dose thrombin-induced activation in washed platelet suspensions, indicating that the activity does not require plasma cofactors. There were no functional differences between sAPP forms with or without the Kunitz protease inhibitor domain or derived from either alpha- or beta-secretase cleavage. In fact, the N-terminal cysteine-rich region of APP (residues 18-194) was as effective as the entire sAPP region in the inhibition of platelet activation. The inhibitory activity of sAPP correlated with a significant reduction in the agonist-induced production of the arachidonic acid (AA) metabolites thromboxane B2 and prostaglandin E2. However, sAPP did not affect AA-induced platelet aggregation or secretion, indicating the enzymatic conversion of AA was not inhibited. The addition of a threshold dose of AA reversed the sAPP-inhibition of agonist-induced platelet activation. This suggests that sAPP decreases the availability of free AA, although the mechanism is not yet known. These data provide evidence that the release of sAPP upon platelet degranulation may result in negative feedback regulation during platelet activation.

Adenosine Diphosphate↗
PubMed1997

Mutations in the transmembrane domain of APP altering gamma-secretase specificity.

Alzheimer's disease (AD) beta-amyloid peptide (Abeta and betaA4) is derived from the amyloid precursor protein (APP) by the subsequent action of the so-far unidentified beta- and gamma-secretases. gamma-secretase, which generates the C-terminus of Abeta, cleaves within the transmembrane domain of APP, preferentially after Abeta-residue 40 (Abeta 40) but also after residue 42 (Abeta 42). This Abeta 42 represents the major subunit of the plaques in AD. Since the position of gamma-secretase cleavage is crucial for understanding the pathogenic pathway, we investigated the effect of different point mutations at Thr43 on gamma-secretase specificity in SPA4CT (SPC99)-expressing COS7 cells. These constructs only require gamma-cleavage for Abeta release. We observed that all Thr43 mutations altered the specificity of gamma-secretase. Small hydrophobic residues favored the generation of Abeta 42, leading to an increase in the 42/40 ratio of Abeta (1.6-2.8-fold). The increase was even stronger (5.6-5.8-fold) when combined with the familial mutation Val46Phe. Thus, these constructs might be highly valuable for the generation of animal models for AD. Processing of full-length APP or SPA4CT yielded the same 42/40 ratio of Abeta (4. 7%). Both constructs, bearing the familial AD mutation Val46Phe, led to a similar increase in the 42/40 ratio (3.3- versus 3.6-fold). The p3 fragment, produced by alpha- and gamma-secretase, showed 42/40 ratios similar to Abeta when derived from wild-type and mutant proteins. These results suggest that the different Abeta- and p3-species are generated by gamma-cleavage activities with a similar enzymatic mechanism.

Amyloid Precursor Protein Secretases↗
PubMed1997

Human amyloid precursor-like protein 1--cDNA cloning, ectopic expression in COS-7 cells and identification of soluble forms in the cerebrospinal fluid.

Amyloid precursor-like protein 1 (APLP1) represents an integral membrane type 1 protein of unknown function which was originally cloned from a mouse cDNA library on the basis of sequence similarity with the Alzheimer's amyloid precursor protein (APP). Here we report on the molecular cloning and expression of the human APLP1 (hAPLP1). hAPLP1 consists of 650 amino acids, displays 89% identity on the amino acid level to its mouse homologue and has a calculated molecular mass of 72 kDa. hAPLP1 synthesized in a cell-free system displays an apparent molecular mass of approximately 80 kDa in SDS-containing gels and becomes N-glycosylated when the in vitro translation is performed in the presence of microsomes. The hAPLP1 cDNA was also expressed ectopically in COS-7 cells and the protein expression was analyzed by immunoprecipitation and western blotting. We have demonstrated that hAPLP1 represents a novel glycoprotein which carries both N- and O-linked glycans. Moreover, hAPLP1 undergoes limited proteolysis which results in the secretion of the carboxy-terminal truncated molecule into the cells conditioned medium. Examination of cells transfected with hAPLP1 cDNA by confocal laser microscopy reveals an intense perinuclear and Golgi staining, a pattern resembling the subcellular distribution of APP. Using a novel hAPLP1-specific antiserum, we identified soluble hAPLP1 in the human cerebrospinal fluid, which suggests that secretion of hAPLP1 from brain cells also takes place in vivo.

Amino Acid Sequence↗
PubMed1997

Reactive oxygen species and Alzheimer's disease.

Although a consensus that Alzheimer's disease (AD) is a single disease has not been reached yet, the involvement of the amyloid precursor protein (APP) and betaA4 (A beta) in the pathologic changes advances our understanding of the underlying molecular alterations. Increasing evidence implicates oxidative stress in the neurodegenerative process of AD. This hypothesis is based on the toxicity of betaA4 in cell cultures, and the findings that aggregation of betaA4 can be induced by metal-catalyzed oxidation and that free oxygen radicals may be involved in APP metabolism. Another neurological disorder, familial amyotrophic lateral sclerosis (FALS), supports our view that AD and FALS may be linked through a common mechanism. In FALS, SOD-Cu(I) complexes are affected by hydrogen peroxide and free radicals are produced. In AD, the reduction of Cu(II) to Cu(I) by APP involves an electron-transfer reaction and could also lead to a production of hydroxyl radicals. Thus, copper-mediated toxicity of APP-Cu(II)/(I) complexes may contribute to neurodegeneration in AD.

Aging↗
PubMed1997

Proteolytic processing of the Alzheimer disease-associated presenilin-1 generates an in vivo substrate for protein kinase C.

The majority of familial Alzheimer disease mutations are linked to the recently cloned presenilin (PS) genes, which encode two highly homologous proteins (PS-1 and PS-2). It was shown that the full-length PS-2 protein is phosphorylated constitutively within its N-terminal domain by casein kinases, whereas the PS-1 protein is not. Full-length PS proteins undergo endoproteolytic cleavage within their hydrophilic loop domain resulting in the formation of approximately 20-kDa C-terminal fragments (CTF) and approximately 30-kDa N-terminal fragments [Thinakaran, G., et al. (1996) Neuron 17, 181-190]. Here we describe the surprising finding that the CTF of PS-1 is phosphorylated by protein kinase C (PKC). Stimulation of PKC causes a 4- to 5-fold increase of the phosphorylation of the approximately 20-kDa CTF of PS-1 resulting in reduced mobility in SDS gels. PKC-stimulated phosphorylation occurs predominantly on serine residues and can be induced either by direct stimulation of PKC with phorbol-12,13-dibutyrate or by activation of the m1 acetylcholine receptor-signaling pathway with the muscarinic agonist carbachol. However, phosphorylation of full-length PS-1 and PS-2 is not altered upon PKC stimulation. In addition, a mutant form of PS-1 lacking exon 10, which does not undergo endoproteolytic cleavage [Thinakaran, G., et al. (1996) Neuron 17, 181-190] is not phosphorylated by PKC, although it still contains all PKC phosphorylation sites conserved between different species. These results show that PKC phosphorylates the PS-1 CTF. Therefore, endoproteolytic cleavage of full-length PS-1 results in the generation of an in vivo substrate for PKC. The selective phosphorylation of the PS-1 CTF indicates that the physiological and/or pathological properties of the CTF are regulated by PKC activity.

Alkaline Phosphatase↗
PubMed1997

Intracellular and secreted Alzheimer beta-amyloid species are generated by distinct mechanisms in cultured hippocampal neurons.

Cerebral plaques containing beta-amyloid (beta A4) represent an invariant pathological feature of Alzheimer disease (AD). beta A4 is proteolytically generated from its parent molecule, amyloid precursor protein (APP). In non-neuronal cells beta A4 has been shown to be secreted via a pH-sensitive and endocytosis-dependent pathway, and this process, when occurring in the brain, is considered to play an important role in AD. In neurons the mechanisms of beta A4 production are not known. Here we have analyzed these mechanisms by expressing human APP and its mutant versions in hippocampal neurons using the Semliki forest virus system. We show that these cells initially generate two pools of beta A4, an extracellular and an intracellular, and only the extracellular pool is produced via a pH-sensitive and endocytosis-dependent pathway. Thus, hippocampal neurons are able to utilize an alternate pathway to produce intracellular beta A4. We also show that a common feature of two types of APP mutations ("Swedish" and "London") implicated in early-onset AD is their increased production of C-terminally elongated beta A4 (beta 42), both intra- and extracellularly. Since neurons are the only cells that produce substantial levels of intracellular beta A4 and also the main victims in AD, these findings may provide an important link between beta A4 and neurodegeneration.

Amyloid beta-Peptides↗
PubMed1997

Amyloid precursor-like protein 1 accumulates in neuritic plaques in Alzheimer's disease.

The Alzheimer's disease (AD) beta-amyloid precursor protein (APP) and the amyloid precursor-like protein 1 (APLP1) and 2 (APLP2) are members of a super-family of proteins that appear functionally related. Although APLPs are highly homologous to APP in the N- and C-terminal domains, they lack the beta A4/amyloid peptide, i.e., the main constituent of neuritic plaques in AD. To assess a potential role of APLP1 in AD, we have determined its immunohistochemical distribution in human hippocampal formation, a structure which is strongly affected in AD, and compared it with APP immunoreactivity. There was a considerable overlap of APP and APLP1 regional expression patterns. Significant APLP1 immunoreactivity was observed in neuritic plaques. Large pyramidal neurons of the subiculum showed an accumulation of APLP1 protein in their dendritic compartment. Some astrocytes elicited perinuclear APLP1 staining, but this was observed in both AD and control brains. These findings raise the possibility that APLP1 may contribute to the pathogenesis of AD-associated neurodegeneration.

Aged↗
PubMed1997

Amyloid precursor protein, copper and Alzheimer's disease.

Although a consensus that Alzheimer's disease (AD) is a single disease has not yet been reached, the involvement of the amyloid precursor protein (APP) and beta A4 (A beta) in the pathologic changes advances our understanding of the underlying molecular alterations. Increasing evidence implicates oxidative stress in the neurodegenerative process of AD. This hypothesis is based on the toxicity of beta A4 in cell cultures, and the findings that aggregation of beta A4 can be induced by metal-catalyzed oxidation and that free oxygen radicals might be involved in APP metabolism. Another neurological disorder, familial amyotrophic lateral sclerosis (FALS), supports our view that AD and FALS might be linked through a common mechanism. In FALS, SOD-Cu(I) complexes are affected by hydrogen peroxide and free radicals are produced. In AD, the reduction of Cu(II) to Cu(I) by APP involves an electron-transfer reaction and could also lead to a production of hydroxyl radicals. Thus, copper-mediated toxicity of APP-Cu(II)/(I) complexes may contribute to neurodegeneration in AD.

Alzheimer Disease↗
PubMed1996

The beta-amyloid domain is essential for axonal sorting of amyloid precursor protein.

We have analysed the axonal sorting signals of amyloid precursor protein (APP). Wild-type and mutant versions of human APP were expressed in hippocampal neurons using the Semliki forest virus system. We show that wild-type APP and mutations implicated in Alzheimer's disease and another brain beta-amyloidosis are sorted to the axon. By analysis of deletion mutants we found that the membrane-inserted APP ectodomain but not the cytoplasmic tail is required for axonal sorting. Systematic deletions of the APP ectodomain identified two regions required for axonal delivery: one encoded by exons 11-15 in the carbohydrate domain, the other encoded by exons 16-17 in the juxtamembraneous beta-amyloid domain. Treatment of the cells with the N-glycosylation inhibitor tunicamycin induced missorting of wild-type APP, supporting the importance of glycosylation in axonal sorting of APP. The data revealed a hierarchy of sorting signals on APP: the beta-amyloid-dependent membrane proximal signal was the major contributor to axonal sorting, while N-glycosylation had a weaker effect. Furthermore, recessive somatodendritic signals, most likely in the cytoplasmic tail, directed the protein to the dendrites when the ectodomain was deleted. Analysis of detergent solubility of APP and another axonally delivered protein, hemagglutinin, demonstrated that only hemagglutinin formed CHAPS-insoluble complexes, suggesting distinct mechanisms of axonal sorting for these two proteins. This study is the first delineation of sorting requirements of an axonally targeted protein in polarized neurons and indicates that the beta-amyloid domain plays a major role in axonal delivery of APP.

Alzheimer Disease↗
PubMed1996

Alzheimer's disease betaA4 protein release and amyloid precursor protein sorting are regulated by alternative splicing.

We show here that alternative splicing influences the polarized secretion of amyloid precursor protein (APP) as well as the release of its proteolytic 3-4-kDa fragments betaA4 and p3. In Madin-Darby canine kidney II cells stably transfected with various APP isoforms and APP mutants, APPsec was consistently secreted basolaterally. In contrast, Madin-Darby canine kidney II cells transfected with L-APP677, which occurs naturally by alternative splicing of exon 15, secreted this isoform both apically and basolaterally, while maintaining the basolateral sorting of endogenous APPsec. This suggests that the alternative splicing of APP exon 15 modulates the polarized sorting of secretory APP. The same alternative splicing event also decreased the production of betaA4 relative to p3. This is the first example of alternative splicing regulating polarized trafficking of a secretory protein.

Alternative Splicing↗
PubMed1996

The amyloid precursor protein of Alzheimer's disease in the reduction of copper(II) to copper(I)

The transition metal ion copper(II) has a critical role in chronic neurologic diseases. The amyloid precursor protein (APP) of Alzheimer's disease or a synthetic peptide representing its copper-binding site reduced bound copper(II) to copper(I). This copper ion-mediated redox reaction led to disulfide bond formation in APP, which indicated that free sulfhydryl groups of APP were involved. Neither superoxide nor hydrogen peroxide had an effect on the kinetics of copper(II) reduction. The reduction of copper(II) to copper(I) by APP involves an electron-transfer reaction and could enhance the production of hydroxyl radicals, which could then attack nearby sites. Thus, copper-mediated toxicity may contribute to neurodegeneration in Alzheimer's disease.

Alzheimer Disease↗
PubMed1996

Amyloidogenic processing of the human amyloid precursor protein in primary cultures of rat hippocampal neurons.

The aim of this study was to investigate the proteolytic processing of the amyloid precursor protein (APP) in polarized primary cultures of hippocampal neurons. We have used the Semliki Forest virus (SFV) vector to express human APP695 in hippocampal neurons, sympathetic ganglia, and glial cells. The latter two cells secrete little or no APP, whereas hippocampal neurons secrete two forms of APP695, which differ in sialic acid content and in their kinetic appearance in the culture medium. In addition, rat hippocampal neurons expressing human APP produced significant amounts of the 4 kDa peptide beta A4. After 3 hr of metabolic labeling, the relative amount of beta A4 peptide to total cellular APP was 5.3%. Fibroblasts expressing APP695 using the same SFV vector mainly produced a related 3 kDa p3 peptide, a nonamyloidogenic fragment. Remarkably, the hippocampal neurons also produced significant amounts of beta A4-containing C-terminal fragments (10-12 kDa) intracellularly. Radiosequencing showed that these fragments were created at a previously described beta-secretase cleavage site and at a cleavage site 12 residues from the N terminus of the beta A4 domain (Thr584 of APP695), which we named delta-cleavage. Based on the observation that mature hippocampal neurons produce two potentially amyloidogenic fragments and secrete substantial amounts of beta A4 when expressing human APP, our results strengthen the hypothesis that neurons play a central role in the process of beta A4 deposition in cases of Alzheimer's disease and in aged primates.

Amino Acid Sequence↗
PubMed1996

A cycle of binding and release of the DnaK, DnaJ and GrpE chaperones regulates activity of the Escherichia coli heat shock transcription factor sigma32.

The chaperone system formed by DnaK, DnaJ and GrpE mediates stress-dependent negative modulation of the Escherichia coli heat shock response, probably through association with the heat shock promoter-specific sigma32 subunit of RNA polymerase. Interactions of the DnaK system with sigma32 were analysed. DnaJ and DnaK bind free, but not RNA polymerase-bound, sigma32 with dissociation constants of 20 nM and 5 muM respectively. Association and dissociation rates of DnaJ-sigma32 complexes are 5900- and 20-fold higher respectively than those of DnaK-sigma32 complexes in the absence of ATP. ATP destabilizes DnaK-sigma32 interactions. DnaJ, through rapid association with sigma32 and stimulation of hydrolysis of DnaK-bound ATP, mediates efficient binding of DnaK to sigma32 in the presence of ATP, resulting in DnaK-DnaJ-sigma32 complexes containing ADP. GrpE binding to these complexes stimulates nucleotide release and subsequent complex dissociation by ATP. We propose that the principles of this cycle also operate in other chaperone activities of the DnaK system. DnaK and DnaJ cooperatively inhibit sigma32 activity in heat shock gene transcription and GrpE partially reverses this inhibition. These data indicate that reversible inhibition of sigma32 activity through transient association of DnaK and DnaJ is a central regulatory element of the heat shock response.

Adenosine Diphosphate↗
PubMed1996

Regulation of amyloid protein precursor (APP) binding to collagen and mapping of the binding sites on APP and collagen type I.

The specific binding of the amyloid precursor protein (APP) to extracellular matrix molecules suggests that APP regulates cell interactions and has a function as a cell adhesion molecule and/or substrate adhesion molecule. On the molecular level APP has binding sites for collagen, laminin, and glycosaminoglycans which is a characteristic feature of cell adhesion molecules. We have examined the interactions between the APP and collagen types I and IV and identified the corresponding binding sites on APP and collagen type I. We show that APP bound most efficiently to collagen type I in a concentration-dependent and specific manner in the native and heat-denatured states, suggesting an involvement of a contiguous binding site on collagen. This binding site was identified on the cyanogen bromide fragment alpha 1(I)CB6 of collagen type I, which also binds heparin. APP did not bind to collagen type I-heparin complexes, which suggests that there are overlapping binding sites for heparin and APP on collagen. We localized the site of APP that mediates collagen binding within residues 448-465 of APP695, which are encoded by the ubiquitously expressed APP exon 12, whereas the high affinity heparin binding site of APP is located in exon 9. Since a peptide encompassing this region binds to collagen type I and inhibits APP-collagen type I binding in nanomolar concentrations, this region may comprise the major part of the collagen type I binding site of APP. Moreover, our data also indicate that the collagen binding site is involved in APP-APP interaction that can be modulated by Zn(II) and heparin. Taken together, the data suggest that the regulation of APP binding to collagen type I by heparin occurs through the competitive binding of heparin and APP to collagen.

Amino Acid Sequence↗
PubMed1996

Alzheimer's disease: genesis of amyloid.

Much of the present knowledge on the genes and genetic processes involved in the genesis of amyloid formation in Alzheimer's disease (AD) has come directly or indirectly from the retrospective molecular and genetic analysis of amyloid beta-protein (A beta or beta A4) deposits and from the identification of genes involved in inherited susceptibility to the disease. This analysis shows that the release and aggregation of the A beta fragment from the amyloid precursor protein (APP) is involved in APP (AD1), chromosome 14 (AD3), 1 (AD4) and 19(AD2) families as well as in the sporadic forms of AD, suggesting that AD is a single disease with a common APP/A beta amyloid pathogenesis. Synthetic A beta protein readily forms beta sheets, filaments and amyloid at micromolar concentrations. The principle to inhibit this process has been worked out by our groups with A beta variants. The N-terminal and C-terminal A beta sequences, oxidative radicals, membrane integrity and metal ions also affect the aggregation of A beta. Amino acid substitutions within the A beta sequence, as occur in rodents, alter A beta release and change the degree to which oxidation of the peptides occurs. Transgenic approaches resulting in overexpression of human APP have confirmed that A beta sequence and concentration are critical prerequisites to amyloid deposition in vivo.

Alzheimer Disease↗