Julian F. B. Mercer

Copper levels are increased in the cerebral cortex and liver of APP and APLP2 knockout mice.

The pathological process in Alzheimers disease (AD) involves amyloid beta (Abeta) deposition and neuronal cell degeneration. The neurotoxic Abeta peptide is derived from the amyloid precursor protein (APP), a member of a larger gene family including the amyloid precursor-like proteins, APLP1 and APLP2. The APP and APLP2 molecules contain metal binding sites for copper and zinc. The zinc binding domain (ZnBD) is believed to have a structural rather than a catalytic role. The activity of the copper binding domain (CuBD) is unknown, however, APP reduces copper (II) to copper (I) and this activity could promote copper-mediated neurotoxicity. The expression of APP and APLP2 in the brain suggests they could have an important direct or indirect role in neuronal metal homeostasis. To examine this, we measured copper, zinc and iron levels in the cerebral cortex, cerebellum and selected non-neuronal tissues from APP (APP(-/-)) and APLP2 (APLP2(-/-)) knockout mice using atomic absorption spectrophotometry. Compared with matched wild-type (WT) mice, copper levels were significantly elevated in both APP(-/-) and APLP2(-/-) cerebral cortex (40% and 16%, respectively) and liver (80% and 36%, respectively). Copper levels were not significantly different between knockout and WT cerebellum, spleen or serum samples. There were no significant differences observed between APP(-/-), APLP2(-/-) and WT mice zinc or iron levels in any tissue examined. These findings indicate APP and APLP2 expression specifically modulates copper homeostasis in the liver and cerebral cortex, the latter being a region of the brain particularly involved in AD. Perturbations to APP metabolism and in particular, its secretion or release from neurons may alter copper homeostasis resulting in increased Abeta accumulation and free radical generation. These data support a novel mechanism in the APP/Abeta pathway which leads to AD.

Correction of the Copper Transport Defect of Menkes Patient Fibroblasts by Expression of the Menkes and Wilson ATPases

Menkes’ disease is a fatal, X-linked, copper deficiency disorder that results from defective copper efflux from intestinal cells and inadequate copper delivery to other tissues, leading to deficiencies of critical copper-dependent enzymes. Wilson’s disease is an autosomally inherited, copper toxicosis disorder resulting from defective biliary excretion of copper, which leads to copper accumulation in the liver. TheATP7A and ATP7B genes that are defective in patients with Menkes’ and Wilson’s diseases, respectively, encode transmembrane, P-type ATPase proteins (ATP7A or MNK and ATP7B or WND, respectively) that function to translocate copper across cellular membranes. In this study, the cDNAs derived from a normal humanATP7A gene and the murine ATP7B homologue,Atp7b, were separately transfected into an immortalized fibroblast cell line obtained from a Menkes’ disease patient. Both MNK and WND expressed from plasmid constructs were able to correct the copper accumulation and copper retention phenotype of these cells. However, the two proteins responded differently to elevated extracellular copper levels. Although MNK showed copper-induced trafficking from the trans-Golgi network to the plasma membrane, in the same cell line the intracellular location of WND did not appear to be affected by elevated copper.

Expression of the Menkes gene homologue in mouse tissues lack of effect of copper on the mRNA levels.

The expression of the homologue of the Menkes disease gene (Mnk) in mice was studied using RNA blots. The highest level of expression of the 8.0 kb mRNA was found in placenta, substantial expression was noted in lung, heart, brain, testis and kidney and gut mucosa, but very low levels were found in spleen and adult liver. In fetal liver, the amount of Mnk mRNA is similar to that found in kidney, however, it declines soon after birth. Results with copper‐loaded normal mice and mutant mice with genetic defects in copper transport suggested that Mnk mRNA levels are not regulated by tissue copper concentrations.

Stability of protein and mRNA in human postmortem liver--analysis by two-dimensional gel electrophoresis.

The method of two-dimensional polyacrylamide gel electrophoresis has been used to investigate the post mortem stability of protein and mRNA in human liver. The electrophoretic mobility of proteins and of the in vitro translation products of the mRNA were found to be essentially unaffected by incubation of the liver at 37 degrees C for up to 2 h or at 4 degrees C for up to 16 h. This study indicates that the major protein and mRNA species in liver are stable enough following death to allow meaningful studies on tissue collected under standard autopsy conditions.

Animal models of Menkes disease.

There are three recognized X-linked copper deficiency disorders in humans: classical Menkes disease (MD), mild Menkes disease and occipital horn syndrome (OHS, also known as X-linked cutis laxa). Since the features of these diseases are so distinct, it was not clear until recently whether the phenotypes were due to mutations in the same gene, or whether OHS in particular is due to mutations in a gene on the X-chromosome, closely linked to the Menkes locus (Danks, 1995). Molecular analysis has now demonstrated that MD, mild Menkes and OHS are indeed due to allelic mutations of the gene affected in Menkes disease (MNK or ATP7A), however, the basis for the phenotypic differences is still not fully understood. A similar range of phenotypes is also found in the mottled mice mutants, and these are discussed below.

A total extract dot blot hybridization procedure for mRNA quantitation in small samples of tissues or cultured cells

A simple method for the estimation of specific mRNA concentrations in small tissue samples (as little as 1 mg) or cultured cells (lower limit 10(5) cells) is described. Guanidine hydrochloride extracts of whole cells or tissues are applied directly onto nitrocellulose and hybridized with the appropriate nick-translated probe. Loading according to DNA content allows expression of the result as concentration per cell. Hybridizing with a ribosomal RNA probe allows expression of results relative to rRNA and estimation of the RNA/DNA ratio in the sample. We describe the application of this procedure to the measurement of ceruloplasmin mRNA in tissues and cultured hepatocytes.

A highly basic N-terminal extension of the mitochondrial matrix enzyme ornithine transcarbamylase from rat liver

We have deduced the amino acid sequence of the N‐terminal leader peptide of the mitochondrial enzyme ornithine transcarbamylase from a cDNA clone obtained from a rat liver cDNA library. The sequence is remarkable in being highly basic, having 4 arginine, 3 lysine and 1 histidine with no acidic residues in a total of 32 residues. The leader sequence has no extensive hydrophobic stretches, has 72% homology with the leader peptide of human ornithine transcarbamylase [1], and in terms of its basic character resembles the N‐terminal extensions on a number of fungal mitochondrial [2‐5] and pea chloroplast [6] proteins. Thus the basic nature of these leader peptides may constitute the signal for mitochondrial import.

Analysis of the distribution of Cu, Fe and Zn and other elements in brindled mouse kidney using a scanning proton microprobe

An analysis of the distribution of trace metals in the kidney cortex in normal and brindled male mice has been carried out with a scanning proton microprobe. Enzyme histochemical staining techniques were used to distinguish between proximal and distal tubules. Average copper levels were increased in brindled kidney tissue sections, with the above-normal Cu accumulation found to occur entirely within the proximal tubules. Therefore, the proximal tubule is now regarded as the location where the defect in Cu transport in brindled mice is manifested the most clearly. The distribution of Fe was found to be non-uniform with some tubule cross-sections exhibiting high concentrations in both genotypes. The distribution of Zn was found to be uniform, and the concentration was similar for each genotype.

The Cell Biology of the Menkes Disease Protein

Copper is a trace element which is readily converted between cuprous and cupric forms under physiological conditions. This redox property has been harnessed in biological systems where copper forms an integral component of enzymes whose catalytic function involves electron exchange. However, this same property of copper also makes it toxic when present within the cell at elevated levels. Consequently, intracellular copper levels must be carefully controlled, presumably by regulated transport mechanisms. Disruptions of some of these mechanisms can have a genetic basis as illustrated in the case of both Menkes disease and Wilson disease (Danks, 1995).

Copper induction of translatable metallothionein messenger RNA

Copper chloride injection of rats resulted in a 4.5- to 9-Iold increase in translatable metallothionein messenger RNA in the liver. Metallothionein in the translation products was identified on the basis of high cysteine and serine incorporation and absence of leucine incorporation as well as comigration with authentic zinc-induced rat-liver metallothionein on SDS-polyacrylamide gels.