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Biomedical subjects

Gerd Multhaup

Publications and source records attributed to Gerd Multhaup.

At least 19 recordsLinked to original sources

Copper and clioquinol treatment in young APP transgenic and wild-type mice: effects on life expectancy, body weight, and metal-ion levels.

There is mounting evidence that the amyloid precursor protein (APP), the key protein in Alzheimer's disease (AD) is involved in the copper (Cu) homeostasis in the brain. Conflicting results about the potential use of dietary Cu and clioquinol (CQ), a known Cu chelator, have been reported using APP transgenic mice. Previously, in vitro studies have demonstrated that CQ can act as a Cu transporter. To analyze the potential function of CQ as a Cu transporter in vivo, the nutritional effect of Cu and CQ was analyzed in young APP transgenic mice and nontransgenics with food pellets containing either Cu, CQ, Cu plus CQ (Cu + CQ), or without addition of supplements (control). The offspring were fed with corresponding food pellets until the age of 14 weeks. We observed an increased lethality of APP transgenics upon CQ treatment, which could be rescued by a co-treatment with Cu. The exposure of Cu + CQ led to a modest but significant increase in cerebral Cu levels, most likely due to an enhanced transport of CQ-Cu complexes. In CQ or Cu + CQ treatment groups, the plasma levels of Cu, zinc, and iron were reduced in all animals; moreover, Cu treatment alone reduced only plasma iron levels. We conclude not only that CQ has certain toxicity but also that the chelating effect, perhaps, plays a secondary role with respect to its properties as an intracellular Cu transporter, thus, counteracting the supposed therapeutic effects of CQ as an agent for chelating therapy in AD.

Alzheimer Disease↗

Transcriptome response to heavy metal stress in Drosophila reveals a new zinc transporter that confers resistance to zinc.

All organisms are confronted with external variations in trace element abundance. To elucidate the mechanisms that maintain metal homeostasis and protect against heavy metal stress, we have determined the transcriptome responses in Drosophila to sublethal doses of cadmium, zinc, copper, as well as to copper depletion. Furthermore, we analyzed the transcriptome of a metal-responsive transcription factor (MTF-1) null mutant. The gene family encoding metallothioneins, and the ABC transporter CG10505 that encodes a homolog of 'yeast cadmium factor' were induced by all three metals. Zinc and cadmium responses have similar features: genes upregulated by both metals include those for glutathione S-transferases GstD2 and GstD5, and for zinc transporter-like proteins designated ZnT35C and ZnT63C. Several of the metal-induced genes that emerged in our study are regulated by the transcription factor MTF-1. mRNA studies in MTF-1 overexpressing or null mutant flies and in silico search for metal response elements (binding sites for MTF-1) confirmed novel MTF-1 regulated genes such as ferritins, the ABC transporter CG10505 and the zinc transporter ZnT35C. The latter was analyzed in most detail; biochemical and genetic approaches, including targeted mutation, indicate that ZnT35C is involved in cellular and organismal zinc efflux and plays a major role in zinc detoxification.

ATP-Binding Cassette Transporters↗

Effect of copper and manganese on the de novo generation of protease-resistant prion protein in yeast cells.

The prion protein (PrP) is the key protein implicated in diseases known as transmissible spongiform encephalopathies. PrP has been shown to bind manganese and copper, the latter being involved in the normal function of the protein. Indeed, upon expression in yeast we noted a major increase in intracellular copper and a decrease in manganese. Interestingly, protease-resistant PrP(Sc)-like protein (PrP(res)) formation was induced when PrP-expressing yeast cells were grown in copper- and/or manganese-supplemented media. The pattern of PrP banding in SDS-PAGE was dominantly determined by manganese. This conformational transition was stable against EDTA treatment but not in the presence of the copper chelators bathocuproinedisulfonic acid or clioquinol. Conclusively, PrP itself influences manganese and copper metabolism, and a replacement of copper in PrP complexes with manganese is highly likely under the condition of copper depletion or if excess amounts of copper and manganese are present. Taken together, our present study demonstrates the involvement of PrP in the regulation of intracellular metal ion homeostasis and uncovers copper and, more severely, manganese ions as in vivo risk factors for the conversion into PrP(Sc).

Amino Acid Sequence↗

The Mechanism of nucleotide-assisted molybdenum insertion into molybdopterin. A novel route toward metal cofactor assembly.

The molybdenum cofactor (Moco) is synthesized by an ancient and conserved biosynthetic pathway. In plants, the two-domain protein Cnx1 catalyzes the insertion of molybdenum into molybdopterin (MPT), a metal-free phosphorylated pyranopterin carrying an ene-dithiolate. Recently, we identified a novel biosynthetic intermediate, adenylated molybdopterin (MPT-AMP), which is synthesized by the C-terminal G domain of Cnx1. Here, we show that MPT-AMP and molybdate bind in an equimolar and cooperative way to the other N-terminal E domain (Cnx1E). Tungstate and sulfate compete for molybdate, which demonstrates the presence of an anion-binding site for molybdate. Cnx1E catalyzes the Zn(2+)-/Mg(2+)-dependent hydrolysis of MPT-AMP but only when molybdate is bound as co-substrate. MPT-AMP hydrolysis resulted in stoichiometric release of Moco that was quantitatively incorporated into plant apo-sulfite oxidase. Upon Moco formation AMP is release as second product of the reaction. When comparing MPT-AMP hydrolysis with the formation of Moco and AMP a 1.5-fold difference in reaction rates were observed. Together with the strict dependence of the reaction on molybdate the formation of adenylated molybdate as reaction intermediate in the nucleotide-assisted metal transfer reaction to molybdopterin is proposed.

Adenosine Monophosphate↗

Axonopathy in an APP/PS1 transgenic mouse model of Alzheimer's disease.

While axonopathy is a prominent feature in a variety of neurodegenerative diseases, it has been largely neglected in Alzheimer's disease (AD), despite the observation of frequent motoric deficits in AD patients. In the present report we used transgenic mice overexpressing human mutant beta-amyloid precursor protein (APP(751SL)) and presenilin-1 (PS1(M146L)) that exhibit elevated intraneuronal Abeta42 levels. We observed abundant age-dependent axonopathy in the spinal cord: axons immunopositive for ubiquitin in the dorsal column; axonal swellings (spheroids) which accumulated APP, neurofilament, and ubiquitin; as well as myelin ovoid structures, which serve as markers for nerve fiber degeneration in both white and gray matter. Both descending and ascending axonal tracts in white matter were affected. Neuritic plaques also developed in an age-dependent manner starting in the cervical region. Furthermore, early intraneuronal Abeta was detected in some but not all motor neurons before plaque formation. In the present APP/PS1 transgenic mouse model we could show for the first time that elevated intracellular Abeta levels lead to an axonopathy characterized by the formation of axonal spheroids and myelin ovoids. The same pathological alterations are known from AD patients or transgenic models overexpressing Tau or ApoE, however, these disturbances in axonal transport occur in the absence of any signs of concomitant Tau pathology. This strengthens the prevailing amyloid hypothesis as a primary trigger of AD-typical pathological alterations.

Alzheimer Disease↗

A family knockout of all four Drosophila metallothioneins reveals a central role in copper homeostasis and detoxification.

Metallothioneins are ubiquitous, small, cysteine-rich proteins with the ability to bind heavy metals. In spite of their biochemical characterization, their in vivo function remains elusive. Here, we report the generation of a metallothionein gene family knockout in Drosophila melanogaster by targeted disruption of all four genes (MtnA to -D). These flies are viable if raised in standard laboratory food. During development, however, they are highly sensitive to copper, cadmium, and (to a lesser extent) zinc load. Metallothionein expression is particularly important for male viability; while copper load during development affects males and females equally, adult males lacking metallothioneins display a severely reduced life span, possibly due to copper-mediated oxidative stress. Using various reporter gene constructs, we find that different metallothioneins are expressed with virtually the same tissue specificity in larvae, notably in the intestinal tract at sites of metal accumulation, including the midgut's "copper cells." The same expression pattern is observed with a synthetic minipromoter consisting only of four tandem metal response elements. From these and other experiments, we conclude that tissue specificity of metallothionein expression is a consequence, rather than a cause, of metal distribution in the organism. The bright orange luminescence of copper accumulated in copper cells of the midgut is severely reduced in the metallothionein gene family knockout, as well as in mutants of metal-responsive transcription factor 1 (MTF-1), the main regulator of metallothionein expression. This indicates that an in vivo metallothionein-copper complex forms the basis of this luminescence. Strikingly, metallothionein mutants show an increased, MTF-1-dependent induction of metallothionein promoters in response to copper, cadmium, silver, zinc, and mercury. We conclude that free metal, but not metallothionein-bound metal, triggers the activation of MTF-1 and that metallothioneins regulate their own expression by a negative feedback loop.

Aging↗

Amyloid precursor protein and BACE function as oligomers.

Processing of the amyloid precursor protein (APP) by beta- and gamma-secretases leads to the generation of amyloid-beta (Abeta) peptides, which are the toxic agents in the pathogenesis of Alzheimer's disease. The molecular reasons for the sequential Abeta generation by secretase activities have remained unclear. Our studies support an oligomerization-dependent mechanism for the conversion of APP into Abeta. By different lines of evidence, we showed that APP is capable of forming homodimers and tetramers. Oligomerization of APP occurs in a zipper-like mechanism primarily mediated by two highly conserved sites of the ectodomain. We also found that in human brain tissue beta-secretase (BACE) occurred as a dimer, whereas the soluble ectodomain of truncated BACE exclusively occurred in the monomeric form. A mutational analysis of the active sites supports the idea that BACE might have acquired a specific catalytic activity by oligomerization, which is stabilized through the transmembrane and the cytoplasmic domains. Our results predict that APP homodimers are functionally active within the plasma membrane and most likely represent substrates for BACE oligomers. Understanding the molecular tasks of homophilic binding of substrates and secretases will allow to find secretase inhibitors which specifically bind to contact sites of dimers and thus inhibit Abeta formation.

Alzheimer Disease↗

The amyloid precursor protein (APP) of Alzheimer disease and its paralog, APLP2, modulate the Cu/Zn-Nitric Oxide-catalyzed degradation of glypican-1 heparan sulfate in vivo.

Processing of the recycling proteoglycan glypican-1 involves the release of its heparan sulfate chains by copper ion- and nitric oxide-catalyzed ascorbate-triggered autodegradation. The Alzheimer disease amyloid precursor protein (APP) and its paralogue, the amyloid precursor-like protein 2 (APLP2), contain copper ion-, zinc ion-, and heparan sulfate-binding domains. We have investigated the possibility that APP and APLP2 regulate glypican-1 processing during endocytosis and recycling. By using cell-free biochemical experiments, confocal laser immunofluorescence microscopy, and flow cytometry of tissues and cells from wild-type and knock-out mice, we find that (a) APP and glypican-1 colocalize in perinuclear compartments of neuroblastoma cells, (b) ascorbate-triggered nitric oxidecatalyzed glypican-1 autodegradation is zinc ion-dependent in the same cells, (c) in cell-free experiments, APP but not APLP2 stimulates glypican-1 autodegradation in the presence of both Cu(II) and Zn(II) ions, whereas the Cu(I) form of APP and the Cu(II) and Cu(I) forms of APLP2 inhibit autodegradation, (d) in primary cortical neurons from APP or APLP2 knock-out mice, there is an increased nitric oxide-catalyzed degradation of heparan sulfate compared with brain tissue and neurons from wild-type mice, and (e) in growth-quiescent fibroblasts from APLP2 knock-out mice, but not from APP knock-out mice, there is also an increased heparan sulfate degradation. We propose that the rate of autoprocessing of glypican-1 is modulated by APP and APLP2 in neurons and by APLP2 in fibroblasts. These observation identify a functional relationship between the heparan sulfate and copper ion binding activities of APP/APLP2 in their modulation of the nitroxyl anion-catalyzed heparan sulfate degradation in glypican-1.

Alzheimer Disease↗

Detection of prion epitopes on PrP and PrP of transmissible spongiform encephalopathies using specific monoclonal antibodies to PrP.

Amino acid residues 90-120 of the prion protein (PrP) are likely to be critical for the conversion of PrP(c) to PrP(sc) in the transmissible spongiform encephalopathies. We raised 10 monoclonal antibodies against the 90-120 amino acid region, mapped the epitope specificity of these anti-PrP antibodies, and investigated the expression of epitopes recognized by the antibodies in both PrP(c) and PrP(sc). Four out of five of the anti-PrP antibodies raised in a prion knockout mouse immunized with the linear peptide of PrP90-120 could detect PrP(sc) in 'native' and denatured forms and PrP(c) in normal cells, as well as recognize epitopes within PrP93-112 residues. In contrast, the other six anti-PrP reagents, including five raised from the two knockout mice immunized with conformationally modified PrP90-120 peptide, could detect PrP(c) and recognize epitopes within PrP93-107 residues. Four of these reagents could also detect denatured PrP(sc) on western blots but not PrP(sc) plaques in brain tissue. The results indicate that residues PrP93-102 are exposed in PrP(c) but are buried upon conversion to the PrP(sc) isoform. Furthermore, PrP103-107 residues are partially buried in PrP(sc) while only the PrP107-112 epitope remains exposed, suggesting that the region PrP93-112 undergoes conformational changes during its conversion to PrP(sc).

Animals↗

Cognitive decline correlates with low plasma concentrations of copper in patients with mild to moderate Alzheimer's disease.

Alzheimer's disease (AD) is a devastating brain disorder clinically characterised by progressive loss of characteristic cognitive abilities. Increasing evidence suggests a disturbed copper (Cu) homeostasis to be associated with the pathological processes. In the present study we analysed the plasma Cu levels and cognitive abilities using the Alzheimer's disease Assessment Scale-cognitive subscale (ADAS-cog) in 32 patients with mild to moderate AD. Statistical analysis revealed a negative correlation between plasma Cu levels and cognitive decline (r=-0.49; P<0.01). Patients with low plasma Cu (mean 82 +/- SD 9) had significant higher ADAS-cog values (mean 23 +/- SD 7), than patients with medium plasma Cu (mean 110 +/- SD 7), who exhibited lower ADAS-cog scores (mean 16 +/- SD 4; ANOVA, P<0.0001). Despite the fact that all patients had plasma Cu levels within the physiological range between 65 microg and 165 microg/dL, 87.5% of the patients revealed a significant negative correlation between plasma Cu and ADAS-cog. This finding supports the hypothesis of a mild Cu deficiency in most AD patients.

Aged↗

Involvement of amyloid beta precursor protein (AbetaPP) modulated copper homeostasis in Alzheimer's disease.

AbetaPP is involved in Cu homeostasis in mouse and man. In vitro observations and in vivo data obtained from AbetaPP mouse models at least provide strong evidence that AbetaPP and Abeta overproduction enables intracellular Cu to be transported out of the cell. The increased Cu efflux seems to lead to a Cu deficiency and a subsequently reduced SOD-1 activity. Studies have shown that a disturbed metal-ion homeostasis with elevated serum Cu levels occurs in Alzheimer and Down's patients and lowered levels in post-mortem AD brain. We conclude that bioavailable Cu has beneficial and specific effects in Alzheimer's disease transgenic mice, and suggest that our observation can be regarded as a proof-of-concept for a prophylactic approach to overcome the observed CNS Cu deficiency in the brain of Alzheimer's disease patients.

Alzheimer Disease↗

Dimerization of beta-site beta-amyloid precursor protein-cleaving enzyme.

Cleavage of the beta-amyloid precursor protein (APP) by the aspartyl protease beta-site APP-cleaving enzyme (BACE) is the first step in the generation of the amyloid beta-peptide, which is deposited in the brain of Alzheimer's disease patients. Whereas the subsequent cleavage by gamma-secretase was shown to originate from the cooperation of a multicomponent complex, it is currently unknown whether in a cellular environment BACE is enzymatically active as a monomer or in concert with other proteins. Using blue native gel electrophoresis we found that endogenous and overexpressed BACE has a molecular mass of 140 kDa instead of the expected mass of 70 kDa under denaturing conditions. This suggests that under native conditions BACE exists as a homodimer. Homodimerization was confirmed by co-immunoprecipitation of full-length BACE carrying different epitope tags. In contrast, the soluble active BACE ectodomain was exclusively present as a monomer both under native and denaturing conditions. A domain analysis revealed that the BACE ectodomain dimerized as long as it was attached to the membrane, whereas the cytoplasmic domain and the transmembrane domain were dispensable for dimerization. By adding a KKXX-endoplasmic reticulum retention signal to BACE, we demonstrate that dimerization of BACE occurs already before full maturation and pro-peptide cleavage. Furthermore, kinetic analysis of the purified native BACE dimer revealed a higher affinity and turnover rate in comparison to the monomeric soluble BACE. Dimerization of BACE might, thus, facilitate binding and cleavage of physiological substrates.

Amino Acid Motifs↗

Clioquinol mediates copper uptake and counteracts copper efflux activities of the amyloid precursor protein of Alzheimer's disease.

The key protein in Alzheimer's disease, the amyloid precursor protein (APP), is a ubiquitously expressed copper-binding glycoprotein that gives rise to the Abeta amyloid peptide. Whereas overexpression of APP results in significantly reduced brain copper levels in three different lines of transgenic mice, knock-out animals revealed increased copper levels. A provoked rise in peripheral levels of copper reduced concentrations of soluble amyloid peptides and resulted in fewer pathogenic Abeta plaques. Contradictory evidence has been provided by the efficacy of copper chelation treatment with the drug clioquinol. Using a yeast model system, we show that adding clioquinol to the yeast culture medium drastically increased the intracellular copper concentration but there was no significant effect observed on zinc levels. This finding suggests that clioquinol can act therapeutically by changing the distribution of copper or facilitating copper uptake rather than by decreasing copper levels. The overexpression of the human APP or APLP2 extracellular domains but not the extracellular domain of APLP1 decreased intracellular copper levels. The expression of a mutant APP deficient for copper binding increased intracellular copper levels several-fold. These data uncover a novel biological function for APP and APLP2 in copper efflux and provide a new conceptual framework for the formerly diverging theories of copper supplementation and chelation in the treatment of Alzheimer's disease.

Alzheimer Disease↗

Human BACE forms dimers and colocalizes with APP.

Beta-site APP-cleaving enzyme (BACE) is a membrane-bound aspartyl protease with no strict primary preference for cleavage. The molecular mechanisms that link the gamma-secretase multicomponent amyloid precursor protein (APP) processing complex to biochemical properties of BACE generating the N terminus of the amyloid beta-peptide have not, as yet, been identified. We found that in human brain tissue, BACE occurred as a dimer. The overall stability of the BACE homodimer was based on intermolecular interactions that were not affected by high salt, nonionic detergents or reducing conditions. BACE homodimers could only partially be separated even under strong denaturing conditions and revealed dramatic differences in the surface charge distribution compared with the monomer. In contrast, the soluble ectodomain of truncated BACE revealed a seemingly lower avidity to the prototypic aspartate protease inhibitor pepstatin and exclusively occurred in the monomeric form. Immunocytochemical studies colocalized APP and BACE in the plasma membrane of cells expressing endogenous levels of BACE and overexpressing APP. In cells that were cotransfected with APP and a putative active site D289A mutant of BACE, colocalization persisted. Remaining enzyme activity was found to be attributable to the mutant protease. Accordingly, inactivation of the carboxyl-terminal active site motif of BACE without an impairment of overall enzyme activity suggests that the enzyme may act as a dimer. Thus, homodimerization of BACE may help the enzyme to acquire specific mechanisms to associate with its substrates to exert catalytic activity.

Amyloid Precursor Protein Secretases↗

Interaction kinetics of the copper-responsive CopY repressor with the cop promoter of Enterococcus hirae.

In Enterococcus hirae, copper homeostasis is controlled by the cop operon, which encodes the copper-responsive repressor CopY, the copper chaperone CopZ, and two copper ATPases, CopA and CopB. The four genes are under control of CopY, which is a homodimeric zinc protein, [Zn(II)CopY]2. It acts as a copper-responsive repressor: when media copper is raised, CopY is released from the DNA, allowing transcription to proceed. This involves the conversion of [Zn(II)CopY]2 to [Cu(I)2CopY]2, which is no longer able to bind to the promoter. Binding analysis of [Zn(II)CopY]2 to orthologous promoters and to control DNA by surface plasmon resonance analysis defined the consensus sequence TACAnnTGTA as the repressor binding element, or " cop box", of Gram-positive bacteria. Association and dissociation rates for the CopY-DNA interaction in the absence and presence of added copper were determined. The dissociation rate of [Zn(II)CopY]2 from the promoter was 7.3 x 10(-6) s(-1) and was increased to 5 x 10(-5) s(-1) in the presence of copper. This copper-induced change may be the underlying mechanism of copper induction. Induction of the cop operon was also assessed in vivo with a biosensor containing a lux reporter system under the control of the E. hirae cop promoter. Half-maximal induction of this biosensor was observed at 5 microM media copper, which delineates the ambient copper concentration to which the cop operon responds in vivo.

Bacterial Proteins↗

Copper depletion down-regulates expression of the Alzheimer's disease amyloid-beta precursor protein gene.

Alzheimer's disease is characterized by the accumulation of amyloid-beta peptide, which is cleaved from the amyloid-beta precursor protein (APP). Reduction in levels of the potentially toxic amyloid-beta has emerged as one of the most important therapeutic goals in Alzheimer's disease. Key targets for this goal are factors that affect the regulation of the APP gene. Recent in vivo and in vitro studies have illustrated the importance of copper in Alzheimer's disease neuropathogenesis and suggested a role for APP and amyloid-beta in copper homeostasis. We hypothesized that metals and in particular copper might alter APP gene expression. To test the hypothesis, we utilized human fibroblasts overexpressing the Menkes protein (MNK), a major mammalian copper efflux protein. MNK deletion fibroblasts have high intracellular copper, whereas MNK overexpressing fibroblasts have severely depleted intracellular copper. We demonstrate that copper depletion significantly reduced APP protein levels and down-regulated APP gene expression. Furthermore, APP promoter deletion constructs identified the copper-regulatory region between -490 and +104 of the APP gene promoter in both basal MNK overexpressing cells and in copper-chelated MNK deletion cells. Overall these data support the hypothesis that copper can regulate APP expression and further support a role for APP to function in copper homeostasis. Copper-regulated APP expression may also provide a potential therapeutic target in Alzheimer's disease.

Adenosine Triphosphatases↗

Hippocampal neuron loss exceeds amyloid plaque load in a transgenic mouse model of Alzheimer's disease.

According to the "amyloid hypothesis of Alzheimer's disease," beta-amyloid is the primary driving force in Alzheimer's disease pathogenesis. Despite the development of many transgenic mouse lines developing abundant beta-amyloid-containing plaques in the brain, the actual link between amyloid plaques and neuron loss has not been clearly established, as reports on neuron loss in these models have remained controversial. We investigated transgenic mice expressing human mutant amyloid precursor protein APP751 (KM670/671NL and V717I) and human mutant presenilin-1 (PS-1 M146L). Stereologic and image analyses revealed substantial age-related neuron loss in the hippocampal pyramidal cell layer of APP/PS-1 double-transgenic mice. The loss of neurons was observed at sites of Abeta aggregation and surrounding astrocytes but, most importantly, was also clearly observed in areas of the parenchyma distant from plaques. These findings point to the potential involvement of more than one mechanism in hippocampal neuron loss in this APP/PS-1 double-transgenic mouse model of Alzheimer's disease.

Aging↗

A modified beta-amyloid hypothesis: intraneuronal accumulation of the beta-amyloid peptide--the first step of a fatal cascade.

Accumulating evidence points to an important role of intraneuronal A beta as a trigger of the pathological cascade of events leading to neurodegeneration and eventually to Alzheimer's disease (AD) with its typical clinical symptoms, like memory impairment and change in personality. In the present article, we review recent findings on intracellular monomeric and oligomeric beta-amyloid (A beta) generation and its pathological function in cell culture, transgenic AD mouse models and post mortem brain tissue of AD and Down syndrome patients, as well as its interaction with oxidative stress and its relevance in apoptotic cell death. Based on these results, a modified A beta hypothesis is formulated, that integrates biochemical, neuropathological and genetic observations with AD-typical neuron loss and plaque formation.

Alzheimer Disease↗