Biocompatible, hyperbranched nanocarriers for the transport and release of copper ions.

Core-shell and core-multishell nanocarriers were designed to transport copper ions into cells. Herein, we present their synthesis and physicochemical characterization and demonstrate the high influence of their architectures on the loading and release of copper. Their low toxicity may open a new way to balance the Cu-homeostasis in neurodegenerative diseases.

Molecular mechanisms and therapeutic application of NSAIDs and derived compounds in Alzheimer's disease.

Alzheimer's disease (AD) is the most common form of neurodegenerative dementias worldwide. Amyloid-ß deposition, neurofibrillary tangle formation and Neuroinflammation are the major pathogenetic mechanisms that in concert lead to memory dysfunction and decline of cognition. To date, there is no curative treatment for AD. Epidemiological analysis support the notion that sustained intake of non-steroidal anti-inflammatory drugs (NSAIDs) reduce the risk and delay the onset of AD. In contrast, therapeutic studies testing NSAID efficacy in AD patients have not yielded positive results. This suggests that either the investigated drugs have not addressed the mechanism of action required for mediating beneficial effects or that NSAIDs are effective at stages way before clinical onset of symptoms. The NSAIDs concerned are pleiotrophic in nature and interact with more than one pathomechanism. Therefore evidence for more than one neuroprotective action of NSAIDs has been put forward and it seems likely that some of the drugs act at multiple levels through more than one molecular mechanism. Some, even may not only be beneficial, but negative actions may be overruled by protective effects. Within these mechanisms, modulation of γ-secretase activity, the activation of the peroxisome proliferator-activated receptor-γ, binding to prostaglandin receptors or interactions at the blood-brain barrier may account for the observed protection from AD. This article reviews the current knowledge and views on the above mechanisms and critically discusses current obstacles and the potential as future AD therapeutics.

Gender dependent APP processing in a transgenic mouse model of Alzheimer's disease.

Epidemiological studies have reported a higher prevalence and incidence of Alzheimer's disease (AD) in women. The biochemical basis for this gender-disparate susceptibility is unknown. A gender effect on AD-typical plaque pathology has been shown in APP transgenic mouse models of AD. Female mice elicit higher plaque load than male mice. In an effort to analyze gender-dependent APP processing during postnatal development, we examined APP transgenic mice at time points prior to plaque deposition. At 14 weeks of age there was a significant elevation of C99 and Abeta in female mice compared to males. Furthermore we observed a slight decrease of BACE-activity in male mice as well as higher cerebral manganese levels in females. Although the decline in estrogen levels due to menopause in female patients is still discussed to be a risk factor for AD our results implicates that additional factors like modified BACE-activity or metal levels may contribute to the higher prevalence and incidence of AD in females.

A vicious circle: role of oxidative stress, intraneuronal Abeta and Cu in Alzheimer's disease.

Recent evidence indicates that both intraneuronal Abeta and Cu are involved in the pathological processes in Alzheimer's disease (AD). This perspective shows a possible interrelation of these factors. AbetaPP, the precursor of Abeta which represents the main constituent of amyloid plaques, is involved in Cu homeostasis in mammals. In vitro observations and in vivo data obtained from AbetaPP mouse models provide strong evidence that AbetaPP and the resulting Abeta overproduction facilitate intracellular Cu to leave the cell. An increased Cu efflux seems to lead to Cu deficiency and, subsequently, reduced SOD-1 activity. The Cu-dependent SOD-1 activity is the main enzyme involved in detoxifying free radicals. Several reports have shown that oxidative stress is an invariable age-dependent feature in the brain of AD patients. Increased oxidative stress leads to an increase in intraneuronal Abeta accumulation, which has been shown to be the main trigger for neuronal loss in transgenic mouse models. Thus, we conclude that bioavailability of Cu is a crucial point for the pathogenesis of AD.

Cerebrospinal fluid diagnostic markers correlate with lower plasma copper and ceruloplasmin in patients with Alzheimer's disease.

Increasing evidence links Alzheimer's disease (AD) with misbalanced Cu homeostasis. Recently, we have shown that dietary Cu supplementation in a transgenic mouse model for AD increases bioavailable brain Cu levels, restores Cu, Zn-super oxide-1 activity, prevents premature death, and lowers A beta levels. In the present report we investigated AD patients with normal levels of A beta 42, Tau and Phospho-Tau in the cerebrospinal fluid (CSF) in comparison with AD patients exhibiting aberrant levels in these CSF biomarkers. The influence of these cerebrospinal fluid (CSF) diagnostic markers with primary dependent variables blood Cu, Zn and ceruloplasmin (CB) and secondary with CSF profiles of Cu, Zn and neurotransmitters was determined. Multivariate tests revealed a significant effect of factor diagnostic group (no AD diagnosis in CSF or AD diagnosis in CSF) for variables plasma Cu and CB (F=4.80; df=2, 23; p=0.018). Subsequent univariate tests revealed significantly reduced plasma Cu (-12.7%; F=7.05; df=1, 25; p=0.014) and CB (-14.1%; F=9.44; df=1, 24; p=0.005) levels in patients with aberrant CSF biomarker concentrations. Although only AD patients were included, the reduced plasma Cu and CB levels in patients with a CSF diagnosis of advanced AD supports previous observations that a mild Cu deficiency might contribute to AD progression.

[The role of copper in the pathophysiology of Alzheimer's disease]. / Zur Bedeutung von Kupfer für die Pathophysiologie der Alzheimer-Krankheit.

Alzheimer's dementia (AD) is a chronically progressive neurodegenerative disease. The key protein in the pathophysiology of AD is the amyloid precursor protein (APP) which releases the amyloid-beta peptide (Abeta) by proteolytic cleavage. APP is probably involved in the homeostasis of cellular copper (Cu) metabolism, because significantly changed Cu levels in the brain were found in AD patients as well as in mouse models. In vivo studies with transgenic mice showed that oral Cu supplements can restore lowered Cu levels in the brain to normal, can reduce Abeta production, and can reduce mortality of the animals. Currently, the influence of oral Cu supplementation (in addition to an established acetylcholinesterase inhibitor) on the progression of the disease is being studied in a prospective, double-blind, randomized and placebo-controlled longitudinal clinical trial in patients with mild AD.

The LPS receptor (CD14) links innate immunity with Alzheimer's disease.

To rapidly respond to invading microorganisms, humans call on their innate immune system. This occurs by microbe-detecting receptors, such as CD14, that activate immune cells to eliminate the pathogens. Here, we link the lipopolysaccharide receptor CD14 with Alzheimer's disease, a severe neurodegenerative disease resulting in dementia. We demonstrate that this key innate immunity receptor interacts with fibrils of Alzheimer amyloid peptide. Neutralization with antibodies against CD14 and genetic deficiency for this receptor significantly reduced amyloid peptide induced microglial activation and microglial toxicity. The observation of strongly enhanced microglial expression of the LPS receptor in brains of animal models of Alzheimer's disease indicates a clinical relevance of these findings. These data suggest that CD14 may significantly contribute to the overall neuroinflammatory response to amyloid peptide, highlighting the possibility that the enormous progress currently being made in the field of innate immunity could be extended to research on Alzheimer's disease.

Hohenheim consensus workshop: copper.

Copper (Cu) is an essential trace element with many physiological functions. Homeostatic mechanisms exist to allow Cu to act as a cofactor in enzymatic processes and to prevent accumulation of Cu to toxic levels. The aim of this commentary is to better understand the role of dietary Cu supply in deficiency and under physiological and pathological conditions. The essentiality of Cu can be attributed to its role as a cofactor in a number of enzymes that are involved in the defence against oxidative stress. Cu, however, has a second face, that of a toxic compound as it is observed with accumulating evidence in hepatic, neurodegenerative and cardiovascular diseases. The destructive potential of Cu can be attributed to inherent physico-chemical properties. The main property is its ability to take part in Fenton-like reactions in which the highly reactive and extremely deleterious hydroxyl radical is formed. Diseases caused by dietary Cu overload could be based on a genetic predisposition. Thus, an assessment of risk-groups, such as infants with impaired mechanisms of Cu homeostasis regarding detoxification, is of special interest, as their Cu intake with resuspended formula milk may be very high. This implies the need for reliable diagnostic markers to determine the Cu status. These topics were introduced at the workshop by the participants followed by extensive group discussion. The consensus statements were agreed on by all members. One of the conclusions is that a re-assessment of published data is necessary and future research is required.

Reelin in plaques of beta-amyloid precursor protein and presenilin-1 double-transgenic mice.

There is circumstantial evidence that the reelin signaling pathway may contribute to neurodegeneration in the adult brain and could be linked to Alzheimer's disease (AD). In the present immunohistochemical report we studied the reelin expression profile in double-transgenic mice that express both human mutant beta-amyloid precursor protein (APP) and human mutant presenilin-1. We were able to demonstrate that reelin immunostaining was found together with human APP in the neuritic component of many AD-typical plaques in both hippocampus and neocortex. This observation gives the first evidence for the association of reelin with amyloid deposits.

Interaction of the CopZ copper chaperone with the CopA copper ATPase of Enterococcus hirae assessed by surface plasmon resonance.

Intracellular copper routing in Enterococcus hirae can be accomplished by the CopZ metallochaperone. Using surface plasmon resonance analysis, we show here that CopZ interacts with the CopA copper ATPase. The binding affinity of CopZ for CopA was increased in the presence of copper, due to a 15-fold lower dissociation rate constant. Mutating the N-terminal copper binding motif of CopA from CxxC to SxxS abolished this copper-induced effect. Moreover, CopZ failed to show an interaction with an unrelated copper binding protein used as a control. These results show that (i) the CopA copper ATPase specifically interacts with the CopZ chaperone, (ii) this interaction is based on protein-protein interaction, and (iii) surface plasmon resonance is a novel tool for quantitative analysis of metallochaperone-target interactions.

Presenilin-dependent gamma-secretase processing of beta-amyloid precursor protein at a site corresponding to the S3 cleavage of Notch.

The presenilin (PS)-dependent site 3 (S3) cleavage of Notch liberates its intracellular domain (NICD), which is required for Notch signaling. The similar gamma-secretase cleavage of the beta-amyloid precursor protein (betaAPP) results in the secretion of amyloid beta-peptide (Abeta). However, little is known about the corresponding C-terminal cleavage product (CTFgamma). We have now identified CTFgamma in brain tissue, in living cells, as well as in an in vitro system. Generation of CTFgamma is facilitated by PSs, since a dominant-negative mutation of PS as well as a PS gene knock out prevents its production. Moreover, gamma-secretase inhibitors, including one that is known to bind to PS, also block CTFgamma generation. Sequence analysis revealed that CTFgamma is produced by a novel gamma-secretase cut, which occurs at a site corresponding to the S3 cleavage of Notch.

Homodimerization of amyloid precursor protein and its implication in the amyloidogenic pathway of Alzheimer's disease.

We reported previously that the carbohydrate domain of the amyloid precursor protein is involved in amyloid precursor protein (APP)-APP interactions. Functional in vitro studies suggested that this interaction occurs through the collagen binding site of APP. The physiological significance remained unknown, because it is not understood whether and how APP dimerization occurs in vivo. Here we report that cellular APP exists as homodimers matching best with a two-site model. Consistent with our published crystallographic data, we show that a deletion of the entire sequence after the kunitz protease inhibitor domain did not abolish APP homodimerization, suggesting that two domains are critically involved but that neither is essential for homodimerization. Finally, we generated stabilized dimers by expressing mutant APP with a single cysteine in the ectodomain juxtamembrane region. Mutation of Lys(624) to cysteine produced approximately 6-8-fold more A beta than cells expressing normal APP. Our results suggest that amyloid A beta production can in principle be positively regulated by dimerization in vivo. We suggest that dimerization could be a physiologically important mechanism for regulating the proposed signal activity of APP.

Copper in disorders with neurological symptoms: Alzheimer's, Menkes, and Wilson diseases.

Copper is an essential element for the activity of a number of physiologically important enzymes. Enzyme-related malfunctions may contribute to severe neurological symptoms and neurological diseases: copper is a component of cytochrome c oxidase, which catalyzes the reduction of oxygen to water, the essential step in cellular respiration. Copper is a cofactor of Cu/Zn-superoxide-dismutase which plays a key role in the cellular response to oxidative stress by scavenging reactive oxygen species. Furthermore, copper is a constituent of dopamine-beta-hydroxylase, a critical enzyme in the catecholamine biosynthetic pathway. A detailed exploration of the biological importance and functional properties of proteins associated with neurological symptoms will have an important impact on understanding disease mechanisms and may accelerate development and testing of new therapeutic approaches. Copper binding proteins play important roles in the establishment and maintenance of metal-ion homeostasis, in deficiency disorders with neurological symptoms (Menkes disease, Wilson disease) and in neurodegenerative diseases (Alzheimer's disease). The Menkes and Wilson proteins have been characterized as copper transporters and the amyloid precursor protein (APP) of Alzheimer's disease has been proposed to work as a Cu(II) and/or Zn(II) transporter. Experimental, clinical and epidemiological observations in neurodegenerative disorders like Alzheimer's disease and in the genetically inherited copper-dependent disorders Menkes and Wilson disease are summarized. This could provide a rationale for a link between severely dysregulated metal-ion homeostasis and the selective neuronal pathology.

Intraneuronal Abeta accumulation precedes plaque formation in beta-amyloid precursor protein and presenilin-1 double-transgenic mice.

beta-Amyloid peptides are key molecules that are involved in the pathology of Alzheimer's disease (AD). The source and place of the neurotoxic action of Abeta, however, is still a matter of controversial debates. In the present report, we studied the neuropathological events in a transgenic mouse model expressing human mutant beta-amyloid precursor protein and human mutant presenilin-1 in neurons. Western blot and immunohistochemical analysis revealed that intracellular Abeta staining preceded plaque deposition, which started in the hippocampal formation. At later stages, many neuritic Abeta positive plaques were found in all cortical, hippocampal and many other brain areas. Interestingly, intraneuronal Abeta staining was no longer detected in the brain of aged double-transgenic mice, which correlates with the typical neuropathology in the brain of chronic AD patients.

Phosphorylation regulates intracellular trafficking of beta-secretase.

beta-Secretase (BACE) is a transmembrane aspartyl protease, which generates the N terminus of Alzheimer's disease amyloid beta-peptide. Here, we report that BACE can be phosphorylated within its cytoplasmic domain at serine residue 498 by casein kinase 1. Phosphorylation exclusively occurs after full maturation of BACE by propeptide cleavage and complex N-glycosylation. Phosphorylation/dephosphorylation affects the subcellular localization of BACE. BACE wild type and an S498D mutant that mimics phosphorylated BACE are predominantly located within juxtanuclear Golgi compartments and endosomes, whereas nonphosphorylatable BACE S498A accumulates in peripheral EEA1-positive endosomes. Antibody uptake assays revealed that reinternalization of BACE from the cell surface is independent of its phosphorylation state. After reinternalization, BACE wild type as well as BACE S498D are efficiently retrieved from early endosomal compartments and further targeted to later endosomal compartments and/or the trans-Golgi network. In contrast, nonphosphorylatable BACE S498A is retained within early endosomes. Our results therefore demonstrate regulated trafficking of BACE within the secretory and endocytic pathway.

A splice variant of beta-secretase deficient in the amyloidogenic processing of the amyloid precursor protein.

beta-Secretase (BACE) initiates the amyloidogenic processing of the amyloid precursor protein leading to the generation of the beta-amyloid, the main component of Alzheimer's disease senile plaques. BACE is a type I transmembrane aspartyl protease of 501 amino acids. Here we describe a novel BACE mRNA lacking 132 base pairs that is expressed in the pancreas but not in the brain. Sequence alignment indicates that the deleted fragment matches the terminal two-thirds of exon 3. The new BACE variant is short of a 44-amino acid region located between the two catalytic aspartyl residues. Accordingly, a 50-kDa form of BACE (BACE457) is detected in the human pancreas. When expressed in cells, BACE457 colocalizes with the marker for the endoplasmic reticulum BiP. Moreover, BACE457 remains in a proenzymatic and endoglycosidase H-sensitive state, suggesting that its transport along the secretory pathway is blocked at the level of the endoplasmic reticulum. Notably, this novel form of BACE does not contribute to the processing of the amyloid precursor protein. Our findings suggest that tissue-specific splicing of the BACE mRNA may explain the observation that in the human pancreas robust transcription of the BACE gene does not translate into recovered enzymatic activity.

Key factors in Alzheimer's disease: beta-amyloid precursor protein processing, metabolism and intraneuronal transport.

During the last years it has become evident that the beta-amyloid (Abeta) component of senile plaques may be the key molecule in the pathology of Alzheimer's disease (AD). The source and place of the neurotoxic action of Abeta, however, is still a matter of controversy. The precursor of the beta-amyloid peptide is the predominantly neuronal beta-amyloid precursor protein. We, and others, hypothesize that intraneuronal misregulation of APP leads to an accumulation of Abeta peptides in intracellular compartments. This accumulation impairs APP trafficking, which starts a cascade of pathological changes and causes the pyramidal neurons to degenerate. Enhanced Abeta secretion as a function of stressed neurons and remnants of degenerated neurons provide seeds for extracellular Abeta aggregates, which induce secondary degenerative events involving neighboring cells such as neurons, astroglia and macrophages/microglia. Beta-amyloid precursor protein has a pivotal role in Alzheimer's disease.

Processing of beta-secretase by furin and other members of the proprotein convertase family.

The amyloid peptide is the main constituent of the amyloid plaques in brain of Alzheimer's disease patients. This peptide is generated from the amyloid precursor protein by two consecutive cleavages. Cleavage at the N terminus is performed by the recently discovered beta-secretase (Bace). This aspartyl protease contains a propeptide that has to be removed to obtain mature Bace. Furin and other members of the furin family of prohormone convertases are involved in this process. Surprisingly, beta-secretase activity, neither at the classical Asp(1) position nor at the Glu(11) position of amyloid precursor protein, seems to be controlled by this maturation step. Furthermore, we show that Glu(11) cleavage is a function of the expression level of Bace, that it depends on the membrane anchorage of Bace, and that Asp(1) cleavage can be followed by Glu(11) cleavage. Our data suggest that pro-Bace could be active as a beta-secretase in the early biosynthetic compartments of the cell and could be involved in the generation of the intracellular pool of the amyloid peptide. We conclude that modulation of the conversion of pro-Bace to mature Bace is not a relevant drug target to treat Alzheimer's disease.

Lewy body variant of Alzheimer's disease: alpha-synuclein in dystrophic neurites of A beta plaques.

The contribution of alpha-synuclein accumulation in Alzheimer's disease (AD) plaques is currently a matter of scientific debate. In the present study antisera against the N- and C-terminus, the full-length protein and the central so-called non-amyloid component (NAC) domain of the alpha-synuclein protein were used to address this question in brains of cases with typical AD and of cases with the Lewy body (LB) variant of AD. In typical AD cases, none of the antisera revealed evidence for co-accumulation of alpha-synuclein with extracellular A beta peptides in plaques or in dystrophic neurites decorating the plaque core. Interestingly, cases with mixed pathology of the LB variant of AD revealed accumulation of alpha-synuclein in LBs and in dystrophic neurites of A beta plaques.

Chelation and intercalation: complementary properties in a compound for the treatment of Alzheimer's disease.

Selective application of metal chelators to homogenates of human Alzheimer's disease (AD) brain has led us to propose that the architecture of aggregated beta-amyloid peptide, whether in the form of plaques or soluble oligomers, is determined at least in part by high-affinity binding of transition metals, especially copper and zinc. Of the two metals, copper is implicated in reactive oxygen species generating reactions, while zinc appears to be associated with conformational and antioxidant activity. We tested the copper chelators trientine, penicillamine, and bathophenanthroline for their ability to mobilize brain Abeta as measured against our benchmark compound bathocuproine (BC). All of these agents were effective in solubilizing brain Abeta, although BC was the most consistent across the range of AD brain tissue samples tested. Similarly, all of the copper chelators depleted copper in the high-speed supernatants. BC alone had no significant effect upon zinc levels in the soluble fraction. BC extraction of brain tissue from C100 transgenic mice (which express human Abeta but do not develop amyloid) revealed SDS-resistant dimers as Abeta was mobilized from the sedimentable to the soluble fraction. NMR analysis showed that, in addition to its copper chelating properties, BC interacts with Abeta to form a complex independent of the presence of copper. Such hybrid copper chelating and "chain breaking" properties may form the basis of a rational design for a therapy for Alzheimer's disease.