Menkes Disease

Menkes disease (MD) is a multisystemic lethal disorder of copper metabolism, inherited as an X-linked recessive trait. Progressive neurodegeneration and connective tissue manifestations together with peculiar "kinky" hair are the main manifestations (2,13). Although many patients present a severe clinical course, variable forms can be distinguished, and the occipital horn syndrome (OHS) is the mildest recognized form. Clinical and physiopathological features of MD are attributable to deficiency of one or more important copper-requiring enzymes secondary to a defect in ATP7A.

3.1. Intracellular Copper Transport in Menkes Disease

Elimination of copper from cells is the basic disturbance in MD and almost all of the tissues except for the liver and the brain will accumulate copper to abnormal levels. When functional defects in MD are discussed, the body tissues and organs therefore can be viewed in three categories: liver, brain, and other tissues.

In the liver, the copper level is low, but can be corrected by copper supplementation. This indicates that the low copper level is not a result of disturbed hepatic copper metabolism, but a secondary effect resulting from the requirement of the metal in other tissues. In the normal liver, ATP7A mRNA expression is very low (14-16), suggesting that its role is taken over by ATP7B. A defect in ATP7B will result in copper accumulation in the liver, with subsequent overflow to other organs as seen in Wilson's disease (17).

The reason for the low copper content in the brain of MD patients is, however, different. The mammalian brain is one of the richest copper-containing organs in the body (18,19). The regulation of brain copper level is not well understood, but because MD leads to low copper levels in the brain, ATP7A probably participates in this process. In MD patients, copper is likely to be trapped in both barriers of the brain, whereas the neurons and glial cells are deprived of copper (20-23). The brain is therefore unique in that it is the only organ deprived of copper, whereas for the other organs and tissues, copper deficiency is functional.

In tissues and organs other than liver and brain, the ATP7A defect leads to intracellular copper accumulation (24). Excess copper induces synthesis of metallothionein, a small and cystein-rich intracellular copper-binding protein, which functions as a copper scavenger. The disturbance of copper metabolism is also reflected in easily accessible cell types like cultured skin fibroblasts (25) and Epstein-Barr-virus transformed lymphocytes (cultured lymphoblasts) (26) of MD patients, making these cell types useful in vitro models. Furthermore, copper accumulation in cultured fibroblasts is also used as a reliable and definitive diagnostic test for MD (2,13).

3.2. Copper Enzymes

Menkes disease is a multisystemic disorder involving several copper-dependent enzymes (2,27). Understanding the pathological pathways in different systems in MD requires knowledge of these metalloenzymes and their function, which will be summarized in the following subsection. In the brain the activity of all copper-dependent enzymes is low because of deprivation of copper in this organ. However, systemic changes are caused by the lack of incorporation of copper into the secreted or vesicular enzymes secondary to malfunction of ATP7A.

3.2.1. Dopamine p-Hydroxylase

Dopamine P-hydroxylase (DHB) is a critical enzyme in the catecholamine biosynthetic pathway converting dopamine into noradrenaline and (subsequently) adrenaline. Deficient activity of DBH will expectedly lead to autonomic failure and this will contribute to central nervous system (CNS) degeneration and other symptoms like ataxia, hypotension, hypothermia, and diarrhea observed in MD patients.

In addition to copper, the enzyme requires ascorbate as a cofactor (28). DBH is localized in secretory vesicles in nerve terminals and in the adrenal medulla, but it is not expressed in fibroblasts. Because of its vesicular location, the enzyme has to pass through the secretory pathway of the cells, and it is likely that copper is added at this stage. Biosynthesis of DBH does not appear to be copper dependent, and reduced enzyme activity as a result of low copper levels in the brain can be corrected by the addition of copper (29,30).

Recently, a novel monooxygenase (MOX) that is homologous to DBH has been identified in senescent fibroblasts (31). MOX maintains the active copper site, but because it lacks a signal peptide sequence, it is probably not secreted. The substrate of this enzyme is yet unknown.

3.2.2. Peptidyl a-Amidating Monooxygenase

Peptidyl a-amidating monooxygenase (PAM) is involved in the maturation of several growth factors and neural peptide hormones involved in hypothalamic-pituitary regulation, as well as in intestinal function (32). Dysfunction of this complex system will expectedly lead to numerous neurological disturbances although the specific nature has not been delineated yet. The deficiency of one of the peptide hormones, vasopressin, may, for example, contribute to the development of hypotension in MD patients. PAM has only been studied in three milder Menkes patients. The plasma activities were found to be normal, but the activity could be stimulated by addition of copper (33).

Peptidyl a-amidating monooxygenase is structurally similar to DBH, and it also requires copper and ascorbic acid as cofactors (28). PAM is found in both secreted and membrane-associated forms, and its biosynthesis is likely to require copper loading in the secretory pathway in analogy with DBH. Copper can easily be removed from PAM and restored again (32).

Peptidyl a-amidating monooxygenase is widely expressed including fibroblasts. Because of the strong molecular and mechanistic similarities between DBH and PAM, information obtained about PAM by studying fibroblasts may be extrapolated to DBH to some extent.

3.2.3. Lysyl Oxidase Gene Family

Lysyl oxidase (LOX) is a secreted enzyme that catalyzes crosslinking of elastin and collagen, and it is found extracellularly attached to its substrates. A deficiency of LOX will virtually affect all organs containing connective tissue. In Menkes disease, skin, bones, and blood vessels are profoundly affected, leading to symptoms like loose skin and joints, osteoporosis, abnormal facies, hernias, bladder diverticula, arterial aneurysms, petechial hemorrhage. Apart from its role in the connective tissue disturbance, LOX deficiency also has an indirect contribution to the CNS degeneration through arterial changes (2). Decreased LOX activity has been demonstrated in cultured skin fibroblasts of MD patients by several researchers (34-41).

In addition to its well-known role in extracellular matrix formation, several new biological functions have also been attributed to LOX, ranging from developmental regulation to tumor suppression and cell growth control. Identification of other lysyl-oxidase-like genes indicates that they may be responsible for some of these diverse functions. In addition to LOX, three distinct genes have been isolated, lysyl-oxidase-like gene, LOXL1 (42,43), lysyl-oxidase-like gene 2, LOXL2 (44), and lysyl-oxidase-related gene, LOR1 (45). The predicted amino acid sequences of all of the members of this gene family contain a copper-binding site and the two conserved residues (Lys and Tyr), which are known to participate in the formation of the intramolecular quinone cofactor. LOX and LOXL1 are both acting extracellularly and evidence exists to indicate that they crosslink different types of collagen (46). The LOR gene probably also functions extracellularly and it is overexpressed in senescent fibroblasts (45). LOXL2 lacks a hydrophobic export signal sequence necessary for extracellular transport and, hence, it is likely to function within the cell.

Lysyl oxidase (and hence also the other members of this gene family) is particularly sensitive to impaired delivery of copper, because activation of the enzyme is a two-step process requiring copper at both steps. Copper catalyzes the formation of the cofactor, but it is also part of the active catalytic center. After the synthesis of prolysyl oxidase, the enzyme is copper loaded and the cofactor is formed before secretion as part of the Golgi or trans-Golgi processing. Copper deficiency will affect the formation of the cofactor and, hence, the enzymatic activity of LOX. Thus, LOX activity in the body is solely dependent on the amount of copper available during the biosynthesis of the enzyme. Copper added after secretion of the enzyme is unable to catalyze the intramolecular cofactor formation and restore the enzymatic activity of LOX. Although the mechanisms by which copper is delivered to lysyl oxidase are not well studied, it is very likely that ATP7A is involved in this process, as LOX secretion and copper efflux use the same pathway. This hypothesis is supported by studies in rat, which showed that the relative levels of LOX and ATP7A mRNA transcripts were quantitatively similar throughout embryonic and early fetal life (47).

Lysyl oxidase is present in high concentrations in dense connective tissue and the fibroblast is one of the principal cell types expressing lysyl oxidase. The study of MD fibroblasts may also provide information about the function and regulation of the new homologous genes in relation to copper homeostasis.

3.2.4. Copper-Containing Amine Oxidase

Copper-containing amine oxidase (CAO) comprises a heterogeneous group of tissue-specific amine oxidases that participate in wound healing, growth regulation, differentiation, and, possibly, apoptosis (48). The preferred substrates are polyamines and diamines and CAO may hereby also influence neurological functions. In the brain, CAO is required for the deamination of putrescine and is necessary for the formation of the neurotransmitter GABA (y-aminobutyric acid) (49). Disturbance of CAO may therefore add to the neurological symptoms observed in MD patients, but this enzyme has not been studied in MD yet.

Similar to LOX, CAO uses a quinone cofactor that is formed by a copper-catalyzed modification of a single amino acid in the protein and this probably occurs in the secretory pathway, as the enzyme is either secreted or membrane anchored. Unlike LOX, enzymatic activity of a form of CAO can be restored by copper addition after the protein has been synthesized and secreted (50). CAO is found in numerous tissues, including vascular smooth muscle, lung, eye, adipose tissue, placenta, and, in particular, intestine and plasma. A form is also expressed in fibroblast (51).

3.2.5. Cytochrome-c Oxidase

Cytochrome-c oxidase (COX) is the terminal oxidase in the respiratory chain and important for energy formation. Adequate energy supply is crucial for normal nerve conduction as well as other metabolic processes. COX is located in the mitochondrial inner membrane and requires copper for the correct assembly of the enzyme complex. COX is obviously affected in the copper-deplete brain tissue of Menkes patients (52-54) adding to the neurodegenerative processes. A restricted ATP production because of COX deficiency in the CNS can cause seizures as observed frequently in MD patients.

Muscle weakness is a common symptom in MD and a compromised mitochondrial function has also been observed in muscle tissue of patients (52,55-57), even though copper levels are elevated in this tissue. It has recently been shown that in cultured hepatoma cells, copper regulated expression of cytochrome-^, a mitochondrially encoded membrane-bound protein that is part of the mitochondrial respiratory chain (58). Copper may therefore affect the respiratory chain and, hence, the energy production also through this protein. Identification of differentially expressed genes in fibroblasts may therefore help in defining the physiopathological mechanisms giving rise to these abnormalities.

3.2.6. Superoxide Dismutase

In several metabolic processes, highly reactive superoxide anions (free radicals) are produced and they have to be eliminated immediately because of their toxic nature. An important class of antioxi-dant enzymes is the SODs, which converts superoxide anions into oxygen and hydrogen peroxide and exists in three distinct forms. Two of these forms (SOD1 and SOD3) contain copper, and the third one, the mitochondrial SOD, contains manganese (SOD2).

SOD1 (Cu,Zn-SOD) is located in the cytoplasm and peroxisomes of all cell types, showing high activity in metabolically active organs such as the liver and kidney and low activity in, for example, skeletal muscle (59). Copper is inserted into the enzyme in the cytosol by means of the chaperone CCS after the enzyme has been synthesized (11). A diminished function of SOD1 in the brain of MD patients will result in further accumulation of free radicals, leading to peroxidation of lipids and neuronal degeneration. On the other hand, in the peripheral tissues (erythrocytes and cultured lym-phoblasts), SOD1 activity is not reduced (24,60), suggesting that copper delivery to SOD1 is independent of ATP7A and its diminished function in the brain is the result of copper deficiency. Furthermore, SOD1 activity is significantly increased in cultured lymphoblasts of MD patients, and the increased synthesis of SOD1 may play a protective role against copper toxicity (60).

The second copper-containing superoxide dismutase, SOD3, is also known as extracellular SOD (EC-SOD). It is secreted from a few well-dispersed cell types, such as fibroblasts and glia cells, but the principal source is vascular smooth-muscle cells (61-63). It is found in the interstitial matrix of several tissues, but SOD3 content is very high in some organs such as lungs (59,64). The biological significance is still poorly described. As SOD3 is a secreted enzyme, the metal is likely to be inserted in the secretory pathway and, hence, dependent on a normal ATP7A function. We are currently studying this enzyme in Menkes fibroblasts (unpublished results).

3.2.7. Ceruloplasmin

Ceruloplasmin is a ferroxidase and catalyzes the oxidation of highly reactive ferrous ions to less toxic ferric ions, which, in turn, are bound to transferrin. During this oxidation process, ferrous ions can react with molecular oxygen and form superoxide radicals. Ceruloplasmin is synthesized as a holoprotein and copper ions are incorporated during the biosynthesis. The availability of copper does not influence the rate of synthesis or secretion of apoceruloplasmin, but failure of copper incorporation during biosynthesis will result in secretion of an apoprotein that is devoid of oxidase activity and is easily degradable (65). Ceruloplasmin is synthesized primarily in the liver, and ATP7B transports copper into the hepatocyte secretory pathway for incorporation into ceruloplasmin.

A membrane-anchored form of ceruloplasmin is synthesized in various tissues and cell types, including cultured fibroblasts (66). Most importantly, the same form is also synthesized within the brain (8,9,67). Ceruloplasmin production within the CNS is necessary for iron homeostasis. As observed in aceruloplasminemia, an abolished activity of ceruloplasmin will lead to iron accumulation at certain brain regions and cause progressive neurodegeneration and lipid peroxidation (68). Low activity of brain ceruloplasmin secondary to low copper levels may also lead to similar consequences in MD.

3.2.8. Hephaestin

The gene encoding for the so-called hephaestin protein has recently been identified and the protein product is predicted to play an important role in intestinal iron absorption (69). It is homologous to ceruloplasmin and contains similar copper-binding sites. In addition, it contains a transmembrane domain, suggesting that the ceruloplasmin-like domain is located in an extracytosolic compartment or in the extracellular space. Based on its homology to ceruloplasmin, it has been proposed that hephaestin is a ferroxidase necessary for iron release from intestinal epithelial cells. Studies in mouse show that hephaestin expression is high in the small intestine and colon and contrasts that of cerulo-plasmin, which is not expressed in the intestine, but highly expressed in liver. How copper is delivered to hephaestin has not been delineated yet, but ATP7A might be involved in this process. Reduced iron absorption may partly explain the anemia observed in some MD patients.

3.2.9. Clotting Factors V and VIII

The blood-clotting factors V and VIII contain copper and the copper-binding site is structurally related to that of ceruloplasmin. This suggests a role for copper in these coagulation factors, although the specific nature is yet unknown. It has been suggested that copper may play a role in the correct folding of these proteins (70) and failure of incorporation of the metal might impair their synthesis or function. Both proteins are secreted and they are primarily synthesized in the liver. Copper is likely to be delivered to these proteins in the secretory pathway by ATP7B.

3.2.10. Copper-Dependent Sulfhydryl Oxidase

Disulfide bridges between the cysteine residues are required for the stability and function of a large number of proteins, including keratin (71,72). Several enzymes are involved in disulfide metabolism. Sulfhydryl oxidases contribute to disulfide bridge formation by catalyzing the oxidation of sulfhydryl groups to disulfides. Sulfhydryl oxidases have been purified from a number of mammalian sources (73-75), but skin is the only tissue where a possibly copper-dependent form has been described (76).

Abnormal hair structure in MD patients indicates that keratinization is a copper-dependent process. It is likely that lack of copper impairs the function of copper-containing sulfhydryl oxidase resulting in deficient crosslinking of keratin, which normally provides a more durable structure to hair and skin. The hair from MD patients have a normal sulfur content, but free-sulfhydryl groups are increased and disulfide bonds are reduced grossly (77). Copper therapy can normalize the hair structure in MD patients.

3.2.11. Tyrosinase

Tyrosinase is a copper-dependent enzyme that catalyzes several steps in the biosynthesis of melanin pigment, which neutralizes the harmful effect of the sun. Dysfunction of tyrosinase will lead to hypopigmentation of the skin and hair as observed in MD patients. Tyrosinase is expressed in mel-anocytes and is stored in melanosomes. Tyrosinase is not expressed in fibroblasts, but, very recently, Petris and colleagues (78) have expressed the enzyme in normal and Menkes fibroblasts using a cDNA construct. In this elegant study, they have shown that ATP7A was required for tyrosinase activation, suggesting that ATP7A had a role in delivering copper to the secretory pathway and to tyrosinase.

3.2.12. Copper-Dependent Ceramide Hydroxylase

In yeast, sphingolipid biosynthesis is a copper-dependent process controlled by the activity of the ATP7A/ATP7B ortholog (CCC2) (79). A key component of sphingolipid is ceramide that may be hydroxylated at one or more sites of the fatty acid side chains. This process may also occur in humans, providing an explanation for the dysmyelination observed in MD patients (80). The copper-dependent hydroxylation clearly occurs in the Golgi apparatus, but the molecular mechanisms remain to be identified and characterized in both man and yeast. It is not known at what stage of the biosynthesis the hydroxylation occurs.

3.2.13. Other Copper Proteins

Dysfunction of copper-containing proteins like the prion protein and the amyloid precursor protein (APP) may also add to the neurological disturbances in MD, but further investigations are necessary to support this hypothesis.

4. DIFFERENTIAL DISPLAY 4.1. Overview of the Procedure

Differential mRNA display by polymerase chain reaction (DD-PCR) is an mRNA fingerprinting technique described in early 1990s for detecting gene-expression levels and identifying differentially expressed genes through arbitrary amplification and comparison of different mRNA resources (1). A major area of application for DD has been the identification of genes differentially expressed in tumor tissues compared to the corresponding normal tissues.

The general strategy of DD is to amplify partial cDNA fragments with PCR using subsets of reverse transcribed mRNAs and displaying these fragments on a denaturing polyacrylamide gel. In

Menkes Syndrome Models

Fig. 1. Outline of the differential display of mRNA technique. (I) Reverse transcription: mRNAs (gray bar) are reverse transcribed using Superscript II RNase H- Reverse Transcriptase (Life Technologies), dNTPs, and an anchored oligo-dT primer (HTnV) to produce single-stranded DNA complements (black bar) of of mRNA. H 5' HindIII restriction site overhang (AAGC); V denotes G, A, or C; and Tn 11 thymidine nucleotides. The 3' nucleotide (V) anchor the HT11V oligonucleotide to the 5' end of the poly(A) tail of the mRNA. An stands for the end of the mRNA strand. With this procedure three different subpopulations of mRNAs are generated. (II) PCR amplification: One of 26 arbitrary decamer primers (AP) is then used in combination with one of the [a-32P]-ATP labeled HT11V primers to amplify a subset of mRNA 3' termini from the cDNAs generated. (III) Display: The labeled cDNA fragments are then displayed on a 6% denaturing polyacrylamide gel. (IV) A band of interest (indicated by an arrow), such as one overexpressed in Menkes fibroblasts (lanes 1-3) compared to the control fibroblasts (lanes 4 and 5) is investigated further as described in the text.

Fig. 1. Outline of the differential display of mRNA technique. (I) Reverse transcription: mRNAs (gray bar) are reverse transcribed using Superscript II RNase H- Reverse Transcriptase (Life Technologies), dNTPs, and an anchored oligo-dT primer (HTnV) to produce single-stranded DNA complements (black bar) of of mRNA. H 5' HindIII restriction site overhang (AAGC); V denotes G, A, or C; and Tn 11 thymidine nucleotides. The 3' nucleotide (V) anchor the HT11V oligonucleotide to the 5' end of the poly(A) tail of the mRNA. An stands for the end of the mRNA strand. With this procedure three different subpopulations of mRNAs are generated. (II) PCR amplification: One of 26 arbitrary decamer primers (AP) is then used in combination with one of the [a-32P]-ATP labeled HT11V primers to amplify a subset of mRNA 3' termini from the cDNAs generated. (III) Display: The labeled cDNA fragments are then displayed on a 6% denaturing polyacrylamide gel. (IV) A band of interest (indicated by an arrow), such as one overexpressed in Menkes fibroblasts (lanes 1-3) compared to the control fibroblasts (lanes 4 and 5) is investigated further as described in the text.

our DD studies, we have used a slightly modified form of the original description (81), as summarized below and schematized in Fig. 1. The mRNAs were reverse transcribed using one of three anchored oligo-dT primers (designated as HTnA, HTnC, or HTnG) annealing to the poly(A) tails present on most eukaryotic mRNAs and generating three subpopulations of mRNA. The cDNA species were subsequently amplified with PCR, where a short primer (a decamer) with arbitrary sequence served as the 5' primer and the 3' primer was the anchored oligo-dT labeled with [a-32P]-ATP (82). The PCR products were separated by electrophoresis on a 6% denaturing polyacrylamide gel, the gels were dried, and the fragments were visualized by autoradiography. Individual bands that indicate differentially regulated mRNAs were recovered from dried polyacrylamide gels by eluting in water and purifying with ethanol precipitation. The fragments were subsequently reamplified with PCR using the same primer set. The reamplified fragments were separated by agarose gel electro-phoresis, recovered by eluting in water and directly sequenced using Cy5-labeled oligo-dT primers in an ALFexpress automatic DNA sequencer (Pharmacia Biotech AB). Search for sequence similarities has been carried out using public databases such as BLAST (83) and FASTA (84). To verify the expression patterns of the bands of interest, specific primers were designed for each fragment and RT-PCR and/or Northern blot hybridization was conducted.

This procedure generates up to 500-bp-long fragments representing mainly the 3' ends of mRNA species. In this study, we have used 26 different decamers for each of the three mRNA subpopulations and an estimated population of 20,000 expressed mRNA should be effectively screened by this approach (82).

Differential display is a widely used technique because it is fast, inexpensive, sensitive, and relatively simple. Alhough powerful, DD has several drawbacks, including reproducibility, presence of false positives, and confirmation of differential expression of the candidate clones. Several reports have described improvements of the initial method dealing with these problems. As to our experience, isolation of high-quality and intact RNA, careful sample treatment, quality of the enzymes used for reverse transcription and PCR amplification, elimination of DNA contamination, and replicate and duplicate experiments are some of the important points that should be taken into consideration when carrying out DD.

4.2. Preliminary Results

In the first attempt to identify known/unknown proteins that might be involved in the pathogenesis of Menkes disease, we have compared mRNA expression profiles of cultured fibroblasts of three MD patients with two controls. One of the patients had the OHS phenotype and a splice-site mutation affecting the donor site of intron 15 (unpublished result). The two other patients had the classical severe form of Menkes disease and one of them had a partial deletion of ATP7A including exons 3-23 and the other patient had a nonsense mutation in exon 4 (Arg409Ter) (unpublished results). Biochemical studies did not reveal any significant difference in the amount of copper accumulated or retained in fibroblasts with different mutations.

Using 26 different decamers for each of the 3 subsets of mRNA, we have generated 78 displays corresponding to approx 5000-7000 fragments for each individual cell. A total of 147 fragments ranging between 50 and 500 bp have been selected for sequencing and 119 of them could be sequenced successfully. For each fragment, approx 30-300 base pairs were sequenced. Of the 119 fragments sequenced, 38 fragments matched genes coding for known proteins; 36 showed sequence similarity to expressed sequence tags (ESTs) or known proteins found in the databases; 29 did not show any sequence similarity to already known expressed sequence (ESTs); and 16 fragments showed sequence similarity to theoretical proteins or genomic sequences (as of November 2000). One of the fragments of interest was matching to lysyl oxidase gene, which will be discussed in Section 5.2.

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