Ceruloplasmin is a member of the highly conserved family of blue multicopper oxidases and is encoded in a 19 exon, single copy, 40 kB gene in the haploid human genome (1). Two transcripts are produced, 3.7 and 4.2 kB, which differ not in the coding region but by differential polyadenylation. Each of these transcripts are translated into a protein of 132 kD or 1046 amino acids. It is synthesized and secreted primarily by hepatocytes as a single polypeptide chain. The active holoprotein has its' ferroxidase activity conferred through the incorporation of six copper atoms (2). The Wilson disease P-type ATPase is essential for the movement of copper into the secretory pathway where it is available for incorporation into ceruloplasmin during its' biosynthesis or for excretion into the bile (3). The absence of copper within this pathway either as a result of a defect in copper trafficking associated with a mutation/deletion in the Wilson disease gene or copper deficiency result in the production of a ceruloplasmin lacking copper. This apoprotein is produced at the same synthetic rate as the holoprotein but in the absence of incorporated copper is rapidly degraded (4,5). Hence patients with Wilson disease and copper deficiency have low circulating ceruloplasmin.

First described in 1987 by Miyajima et al., a family was refered to us for evaluation of Wilson disease (6). Despite having near absent serum ceruloplasmin levels and neurologic degeneration manifest as dysarthria, cogwheel rigidity, memory loss and dementia, there were unique features that clearly distinguished this disorder from Wilson disease. The family pedigree suggested an autosomal recessive disorder. Affected homozygotes had virtually absent serum ceruloplasmin levels while obligate heterozygotes had half-normal values ( normal serum ceruloplasmin - 25-40 mg/dL). Wilson disease heterozygote patients would have normal ceruloplamsin levels for a single functional allele provides adequate available copper for incorporation during normal ceruloplasmin holoprotein biosynthesis. Neuroimaging studies revealed the second unique feature. T2 weighted magnetic resonance imaging (MRI) studies were remarkable for the presence of iron deposition in the basal ganglia not copper deposition. Finally, liver biopsy samples revealed normal hepatocyte architecture and histology without evidence of fibrosis or cirrhosis. Serial sections treated with a Perl stain to identify iron were remarkable for significant iron accumulation in both hepatocytes and kupfer cells. Thus, to distinguish these patients from Wilson disease patients, a new disease was coined - aceruloplasminemia.

The findings of elevated tissue iron in conjunciton with a defect in a copper-containing protein was particularly exciting. As early as 1928, E.B. Hart discovered that nutritional anemia in animals not responsive to iron administration could be corrected when copper administration was coupled with the iron delivery. Only in the presence of copper could these animals utilize the iron they were receiving. Frieden and Cartwright had demonstrated that copper deficient pigs developed an iron deficiency anemia despite iron supplementation that was only corrected when the pigs were given copper (7-10). In 1994 a ceruloplasmin homologue - FET3 - a blue copper oxidase was discovered in yeast (11). Within the yeast membrane resides a ferroreductase, responsible for the reduction of ferric iron (Fe3+) to ferrous iron (Fe2+). In order for iron to be stored within yeast it must be oxidized back to the ferric state by FET3 - a ferroxidase dependent on copper incorporation to function. FET3 is intimately dependent on copper incoporation to oxidize substrates and defects in copper incorporation result in absent holo-FET3 (12-14).

Molecular characterization of genomic DNA isolated from red cells from the family initially referred to us revealed a five base pair insertion at amino acid 410 in exon 7 of the ceruloplasmin gene (15). This five base pair insertion resulted in a frame shift mutation and a predicted open reading frame termination resulting in a truncated protein. Given that there is no prosthetic ligand for the copper atoms in ceruloplasmin and that the ligand for the copper is the protein itself, mutations at multiple sites would be predicted to affect the conformational integrity of the protein. Details of the ligand structure essential for the function of ceruloplasmin have been further provided by the elucidation of the human ceruloplasmin structure by x-ray crystallography (16). Ceruloplasmin is composed of six domains: three of the copper atoms occupy mononuclear sites in domains 2, 4,and 6. The remaining three copper atoms exist in a trinuclear copper cluster. It is this trinuclear copper cluster, capable of transferring electrons, that is essential for ferroxidase function (17). Indeed, five subsequent mutations have been identified to date within the ceruloplasmin gene that abrogate ceruloplasmin ferroxidase function and culminate in aceruloplasminemia (18-23).

The mutations in the patients and their associated clinical findings have allowed us to hypothesize the following biological role for ceruloplasmin in iron metabolism (24). Ceruloplasmin is synthesized as a holoprotein and secreted into the plasma. There, in some manner as of yet unknown, it acts as an essential ferroxidase, oxidizing ferrous (Fe2+) to ferric (Fe3+) iron for binding to transferrin. Given that aceruloplasminemic patients are not anemic, other oxidants are able to assist in the mobilization of newly digested iron, via transferrin, to the bone marrow. Ceruloplasmin is thus responsible for moving iron out of the storage compartment of the reticuloendothelial system to the functional compartment of the bone marrow following recycling of senescent red blood cell iron mass. A defect in ceruloplasmin synthesis that results in aceruloplasminemia causes cellular damage due to (1) storage damage from excessive iron deposition, (2) oxyradical damage secondary to the presence of a free transition metal and H2O2 within the cell or (3) a lack of substrate and a failure of the functional compartment.

One unsatisfying aspect of this hypothesis is that the patients present with and succumb to neurodegeneration. Pathologic examination of brain from aceruloplamsinemic patients at autopsy reveal pigmentary discoloration and cavitary degeneration of the basal ganglia and substantia nigra. Microscopic examination of the basal ganglia is remarkable for marked neuronal drop-out and loss with associated spongioform degeneration. A 132 kD plasma protein would not be able to penetrate the blood-brain barrier and hence, ceruloplasmin must be synthesized and secreted within the central nervous system. Indeed, in-situ hybridization revealed ceruloplasmin expression confined to astrocytes associated with the cerebral microvasculature (25). This astrocyte specific expression was unique for ceruloplasmin and distinct from other cells. In-situ hybridization of adjacent tissue sections with a human transferrin cRNA probe revealed oligodendrocyte specific transferrin expression (25).

These observations direct adjustment of the previous hypothesis of the role of ceruloplasmin in iron metabolism. Iron, bound to tranferrin, enters two separate iron cycles - the systemic iron cycle and the CNS iron cycle. In the systemic iron cycle, iron is delivered to the bone marrow for incorporation into red blood cells. As those cells die they pass through the reticuloendothelial system(predominantly the liver and spleen) where iron is released as the red cell is degraded. Through a process still to be identified, ceruloplasmin plays a role in the return of iron to the systemic iron cycle. Parients with aceruloplasminemia therefore have an iron efflux disorder and develop a slow iron accumulation. Within the central nervous system, iron delivered by transferrin is probably released through a transferrin receptor-mediated endocytosis and iron enters the CNS where it binds oligodendrocyte synthesized transferrin and is transported to neurons. Presumably, the process favoring iron binding transferrin requires functional ceruloplasmin ferroxidase activity. It is the role of ceruloplasmin in the CNS iron cycle that is the most fascinating and which as of yet still needs to be determined.

The characterization of aceruloplasminemia recognizes a novel disease of iron metabolism and distinguishes a role for ceruloplasmin in the process. Further studies of the role of ceruloplasmin in both the systemic and central nervous system iron cycles, elucidating the specific mechanisms of this process, will provide an opportunity to test our hypothesis and may have broader implications for our understanding of the role of iron in human neuropathology.

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