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ABSTRACT Aceruloplasminemia is an autosomal recessivedisorder characterized by progressive neurodegeneration of theretina and basal ganglia associated with specific inherited muta-tions in the ceruloplasmin gene. Clinical and pathologic studiesin patients with aceruloplasminemia revealed a marked accumu-lation of iron in affected parenchymal tissues, a finding consis-tent with early work identifying ceruloplasmin as a ferroxidaseand with recent findings showing an essential role for a homolo-gous copper oxidase in iron metabolism in yeast. The presence

of neurologic symptoms in aceruloplasminemia is unique amongthe known inherited and acquired disorders of iron metabolism;recent studies revealed an essential role for astrocyte-specificexpression of ceruloplasmin in iron metabolism and neuronalsurvival in the central nervous system. Recognition of acerulo-plasminemia provides new insights into the genetic and environ-mental determinants of copper metabolism and has importantimplications for our understanding of the role of copper inhuman neurodegenerative diseases. Am J Clin Nutr 1998;67(suppl):972S7S.

KEY WORDS Ceruloplasmin, copper, iron, neurodegenera-tion, human genetics, aceruloplasminemia

PHYSIOLOGIC FUNCTION OF CERULOPLASMIN

Ceruloplasmin is an abundant 2-glycoprotein that contains>95% of the copper found in the plasma of all vertebrate species.This protein is synthesized and secreted as a single polypeptidechain of 1046 amino acids with six atoms of copper incorporatedper molecule during biosynthesis. Copper does not affect the rateof synthesis or secretion of apoceruloplasmin but a failure toincorporate copper during biosynthesis results in secretion of anapoprotein that is devoid of oxidase activity and rapidlydegraded (13). In the inherited disorder of copper metabolismknown as Wilson disease, an inability to transfer copper into acommon intracellular pool for holoceruloplasmin biosynthesisand biliary copper excretion results in a marked decrease in the

concentration of ceruloplasmin in the plasma secondary to therapid turnover of the secreted apoprotein. Consistent with thisconcept, the Wilson disease gene has been cloned and shown toencode a cation transport P-type ATPase essential for coppertransfer into the secretory pathway of the cell (46).

Ceruloplasmin is a multicopper oxidase and three distincttypes of copper ions within the protein can be distinguishedspectroscopically. Three of these copper atoms form a trinuclearcluster that is responsible for oxygen activation during the cat-

alytic cycle of the enzyme (7). Recent determination of the crys-tal structure of ceruloplasmin supports these biophysical studiesand delineates the amino acids in the amino and carboxyl termi-nal domains that are the essential copper ligands of the trinuclearcluster (8). Although ceruloplasmin can oxidize several sub-strates in vitro, early studies suggested a distinct role for cerulo-plasmin as a ferroxidase. In these experiments, ceruloplasminwas shown to play a role in the mobilization and oxidation ofiron from tissue stores with subsequent incorporation of ferric

iron into transferrin (9). Further studies showed that animalsmade deficient in copper by dietary restriction develop anabsence of circulating holoceruloplasmin and are thus unable torelease tissue iron to the plasma (10). Support for a role of ceru-loplasmin as a ferroxidase has also come from studies in Sac-charomyces cerevisiae in which high-affinity iron uptakerequires the presence of a homologous multicopper oxidasetermed Fet3 (11, 12). This protein oxidizes iron in the extracel-lular environment, permitting high-affinity iron uptake in con-

junction with a membrane permease (13). Consistent with thepreviously described role for the Wilson ATPase in the deliveryof copper to ceruloplasmin, a homologous P-type ATPase inyeast (CCC2) delivers copper to Fet3, revealing a remarkableevolutionary conservation of the role of these copper proteins in

iron homeostasis (14).

ACERULOPLASMINEMIA

The precise physiologic role of ceruloplasmin has beendefined by the recognition of individuals with diabetes, retinaldegeneration, and neurologic symptoms in association with atotal absence of serum ceruloplasmin (1517). Molecular geneticanalysis of these patients identified specific inherited mutationsin the ceruloplasmin gene (1823). A typical pedigree from afamily with aceruloplasminemia is shown in Figure 1. Alsoshown are the results of DNA amplification from exon 7 of theceruloplasmin gene, which contains a fivebase pair insertion inthe affected individuals in this kindred. As can be seen from this

analysis, aceruloplasminemia is inherited in a classic autosomal

Aceruloplasminemia: an inherited neurodegenerative disease with

impairment of iron homeostasis1-3

Z Leah Harris, Leo WJ Klomp, and Jonathan D Gitlin

1 From the Edward Mallinckrodt Department of Pediatrics, WashingtonUniversity School of Medicine, St Louis.

2 Supported by grant HL41536 from the National Institutes of Health.3 Address reprint requests to JD Gitlin, Department of Pediatrics, St Louis

Childrens Hospital, One Childrens Place, St Louis, MO 63130. E-mail:gitlin@kidsa1.wustl.edu.

Am J Clin Nutr1998;67(suppl):972S7S. Printed in USA. 1998 American Society for Clinical Nutrition972S

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recessive fashion. Thus far, five distinct mutations have beendefined in the ceruloplasmin gene in six separate kindreds withthis disease (Table 1). Consistent with the autosomal recessivenature of this disorder, consanguinity has been observed in mostcases. In all cases reported, these mutations result in an alter-

ation of the ceruloplasmin open reading frame such that theamino acid ligands in the carboxyl terminal region essential forformation of the trinuclear copper cluster are eliminated.Because ceruloplasmin synthesized without this region would beunable to incorporate copper during biosynthesis, these data areconsistent with the absence of detectable oxidase activity in theplasma of affected patients.

Clinical and pathologic studies in patients with aceruloplas-minemia have shown both the histology of the hepaticparenchyma and hepatic copper content to be normal but liveriron concentrations to be markedly elevated (1517). In addition,the iron content of the pancreas and other parenchymal organs isincreased, with specific accumulation of iron in the islets ofLangerhans and selective loss of pancreatic cells. T2-weighted

magnetic resonance imaging reveals an increased iron content inbasal ganglia and examination of brain tissue reveals abundantiron deposition in microglia and neurons as well as selective neu-ronal loss (Figure 2). Similar findings are observed in the eye,with iron deposition and photoreceptor loss in the peripheralregion of the retina. Taken together, these findings reveal a directrole for ceruloplasmin in human iron metabolism consistent withthe early studies noted above.

The pathophysiology of aceruloplasminemia is best under-stood by examining ceruloplasmin within the context of the ironcycle, where ceruloplasmin functions to oxidize ferrous iron fortransfer into transferrin; iron is subsequently delivered by trans-

ferrin to the bone marrow for hematopoiesis (Figure 3). Theabsence of ceruloplasmin leads to an accumulation of ferrous ironwithin both the reticuloendothelial system and the plasma. In thisrespect the pathophysiology of iron accumulation in parenchymalorgans is analogous to that observed in atransferrinemia,

hemochromatosis, and transfusion-related iron overload. Any sit-uation in which transferrin is unavailable or unable to be satu-rated with iron or is oversaturated will lead to the rapid removalof ferrous iron from the circulation and its accumulation in liverand other tissues (26). Patients with aceruloplasminemia havehepatic iron and serum ferritin concentrations equivalent to thoseobserved in persons with hemochromatosis, but are only mildlyanemic because the nonenzymatic oxidation of iron permits sometransfer of iron into transferrin. Symptoms of aceruloplasmine-mia are not observed in patients with Wilson disease because theextrahepatic production of holoceruloplasmin is sufficient to pro-vide the 5% of the normal serum concentration necessary to sus-tain plasma iron turnover rates (10). The results of studies of cop-per metabolism in patients with aceruloplasminemia are normal,

indicating that ceruloplasmin has no essential role in coppertransport or metabolism in humans. Because copper metabolismis also normal in the offspring of these patients, it can be con-cluded that there is no essential role for maternal ceruloplasminin placental or fetal copper transport or metabolism.

NEUROLOGIC CONSEQUENCES OF

ACERULOPLASMINEMIA

Despite evidence of systemic iron overload, the clinical fea-tures of aceruloplasminemia are neurologic, resulting from pro-gressive neurodegeneration of the retina and basal ganglia inassociation with iron accumulation in these tissues. This uniqueinvolvement of the central nervous system distinguishes acerulo-

plasminemia from other inherited and acquired disorders of ironmetabolism and suggests that ceruloplasmin plays an essentialrole in normal brain iron homeostasis. Under normal circum-stances, little, if any, ceruloplasmin crosses the blood-brain bar-rier and, thus, any proposed direct role for this protein in ironhomeostasis in the central nervous system implies extrahepaticexpression at this site. Consistent with this theory, in situhybridization of murine and human central nervous system tis-sues reveals abundant cell-specific ceruloplasmin gene expres-sion (27). As can be seen in Figure 4, ceruloplasmin gene expres-sion in the murine brain is observed abundantly in astrocytes

ACERULOPLASMINEMIA 973S

TABLE 1Mutations in the human ceruloplasmin gene in aceruloplasminemia

Mutation Exon Predicted effect Reference

Insertion or deletion607insA 3 Frameshift 231285 ins TACAC 7 Frameshift 182389delG 13 Frameshift 22

NonsenseTrp858ter 15 Truncates protein 20, 21

Splice site3019-1G-A 18 Frameshift, truncates protein 19

FIGURE 1. Pedigree and agarose gel electrophoresis of polymerase chain reaction (PCR) products corresponding to exon 7 of the ceruloplasmin gene.Circles indicates females and squares indicate males, a slash through a circle or square indicates that the family member is deceased, fully filled symbolsrepresent homozygotes, and vertically lined symbols represent heterozygotes. C, control subject. Reproduced with permission from reference 24.

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surrounding the cerebral microvasculature. Additional studiesreveal ceruloplasmin gene expression in glia surrounding specificneurons within the substantia nigra and other basal ganglia tis-sues. The expression of the ceruloplasmin gene in astrocytesobserved by in situ hybridization is confirmed by in vitro studies

that have identified ceruloplasmin gene expression in cultured

astrocytes but not in neurons. Ceruloplasmin is synthesized andsecreted from these astrocytes with size characteristics and kinet-ics identical to those observed in hepatocytes (Figure 5B).

The observation that ceruloplasmin is expressed in central ner-vous system tissues that are affected in patients with aceruloplas-

minemia suggests a direct role for the absence of this protein in the

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FIGURE 2. Photomicrograph of a section of putamen from the brain of a patient who died of aceruloplasminemia. The section is stained withPrussian blue and abundant iron is evident within microglial cells. In addition, abnormal accumulation of iron is observed in large populations of neu-rons (opened arrows) as well as significant neuronal loss with spongiform degeneration (arrowheads).

FIGURE 3. Schematic demonstration of the iron cycle, illustrating the role of ceruloplasmin (Cp) as a ferroxidase in mediating ferrous ironoxidation and subsequent transfer into transferrin (Tf). Hb, hemoglobin; RBC, red blood cell. Modified and reproduced with permission from refer-ence 25.

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accumulation of iron and tissue injury (Figure 6). Under normal

circumstances, iron exits the cerebral microvasculature afterreceptor-mediated endocytosis of transferrin and release of ironacross the blood-brain barrier. As suggested by the data discussedabove, ferrous iron is then oxidized by astrocyte-secreted cerulo-

plasmin and incorporated into transferrin synthesized by oligo-

dendrocytes. In the absence of ceruloplasmin, ferrous iron is takenup rapidly by the brain parenchymal cells, analogous to whatoccurs in the periphery. The accumulation of ferrous iron withinglia may then lead to cell-specific injury with resultant loss ofglial-derived neurotrophic factors essential for neuronal survival.

Alternatively, the accumulation of ferrous iron may resultdirectly in oxidant-mediated tissue injury. The accumulation ofiron in the brain in association with neurologic symptoms hasbeen observed in other neurologic diseases, but the mechanismsof iron accumulation in the basal ganglia of such patients and therelation of this to clinical symptoms is unclear (28, 29). Never-theless, a direct role for iron-mediated tissue injury in the brainis supported by studies on the role of this metal in oxidant-mediated ischemia-reperfusion injury after stroke and cerebral

hemorrhage and by studies that revealed a marked increase inplasma lipid peroxidation in patients with aceruloplasminemia(24, 30). Last, it is possible that the inability to transfer iron intotransferrin results in an iron-deficient state in neurons and thatdespite the accumulation of brain iron the neuronal loss is due toiron deficiency. Clarification of these issues must await thedevelopment of an animal model of aceruloplasminemia byhomologous recombination.

TREATMENT OF ACERULOPLASMINEMIA

Aceruloplasminemia is a fatal disease and its early diagnosisand early treatment of patients are issues of paramount impor-tance. Deferoxamine is a high-affinity iron chelator that com-

bines with ferric iron in a 1:1 molar ratio. Although the precisecellular location of iron chelation by this drug is unknown, it hasbeen shown to cross the blood-brain barrier and to promote theexcretion of excess iron in patients with inherited and acquiredforms of iron overload (31). Preliminary studies of this drug inpatients with aceruloplasminemia indicate a reduction in bodyiron stores as well as amelioration of diabetes and a decrease inneurologic symptoms (32). If supported by further studies, theuse of this drug may be an approach for preventive therapy inasymptomatic individuals before the onset of clinical symptomsor substantial iron accumulation in the central nervous system.

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FIGURE 4. In situ hybridization of murine brain with ceruloplasmin-specific complementary RNA probes. In situ hybridization was performed andslides were examined with bright-field (A and C) and corresponding dark-field (B) microscopy. Exposure of a sagittal section of adult mouse brain;magnification 64 (A and B) and 400 (C). Arrows indicate parenchymal sites of ceruloplasmin gene expression, which on magnification are shownto localize to perivascular astrocytes (pva). An individual neuron (n) is noted by the arrowheads. Hybridization is also observed in the pia mater (pm).

FIGURE 5. Expression of ceruloplasmin in primary cell cultures.Glial and neuronal cell cultures were prepared and analyzed by RNAblot analysis with a ceruloplasmin-specific complementary RNA probe(A) or a murine -actin complementary RNA probe (B). Newly synthe-sized proteins were analyzed from glia (lanes 1 and 2 in A) or neurons(lane 2 in A) after metabolic labeling by immunoprecipitation with con-trol serum (lane 1 in B) or antibody to ceruloplasmin (lanes 2 and 3 inB). Immunoprecipitates were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Reproduced with permission fromreference 27.

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CONCLUSIONS

The presence of mutations in the ceruloplasmin gene in con-junction with clinical and pathologic findings suggest an essen-tial role for ceruloplasmin in human biology and identify aceru-loplasminemia as an autosomal recessive disorder of ironmetabolism (25). The genetic characterization of aceruloplas-minemia has provided initial insight into the specific role ofceruloplasmin in iron metabolism in the brain. Recent studies onthe biochemical mechanisms of copper incorporation into thisprotein as well as data indicating a loss of ceruloplasmin oxidase

activity with aging suggest that further analysis of the functionof ceruloplasmin within the central nervous system will be ofvalue in elucidating the mechanisms of neuronal loss in severalneurodegenerative disorders in which abnormalities in ironmetabolism have been shown (33, 34).

We gratefully acknowledge the collaboration of our many colleagues,Yoshitoma Takahashi, Hiroaki Miyajima, Ross MacGillivray, Anne Hughes,John Logan, and John Walshe, for sharing information and referring patients.We thank Hiroshi Morita for the micrograph of the putamen from a patientwith aceruloplasminemia.

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FIGURE 6. Illustration of the proposed sites of synthesis and function of ceruloplasmin (Cp) and transferrin (Tf) within the central nervous

system. The role of ceruloplasmin as a ferroxidase and the possible sites of iron-mediated damage are illustrated. Tfr, transferrin receptor; NOS, nitricoxide synthase.

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