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Human Molecular Genetics Pages 1989-1997 © Oxford University Press
Expression of the ceruloplasmin gene in the human retina and brain: implications for a pathogenic model in aceruloplasminemia
Introduction
Results
Discussion
Materials And Methods
   RNA isolation and analysis
   Biosynthetic labeling of tissue samples and immunoprecipitation
   In situ hybridization and immunohistochemistry
   ACKNOWLEDEMENTS
References

Expression of the ceruloplasmin gene in the human retina and brain: implications for a pathogenic model in aceruloplasminemia

Expression of the ceruloplasmin gene in the human retina and brain: implications for a pathogenic model in aceruloplasminemia Leo W. J. Klomp and Jonathan D. Gitlin*

Edward Mallinckrodt Department of Pediatrics, Washington University School of Medicine, St Louis Children's Hospital, One Children's Place, St Louis, MO 63110, USA

Received July 29, 1996; Revised and Accepted October 4, 1996

Aceruloplasminemia is an autosomal recessive disorder of iron metabolism characterized by progressive neurodegeneration of the retina and basal ganglia in association with inherited mutations of the ceruloplasmin gene. To begin to elucidate the pathogenesis of this disease, ceruloplasmin gene expression was examined in human brain and retinal tissue. RNA blot analysis and RNAse protection studies demonstrate ceruloplasmin-specific transcripts in multiple regions of the human brain, and biosynthetic studies reveal ceruloplasmin synthesis and secretion in these same regions. Consistent with these observations, in situ hybridization of central nervous system tissue utilizing ceruloplasmin cRNA probes reveals abundant ceruloplasmin gene expression in specific populations of glial cells associated with the brain microvasculature, surrounding dopaminergic melanized neurons in the substantia nigra and within the inner nuclear layer of the retina. Taken in the context of the clinical and pathological features observed in patients with aceruloplasminemia, these data reveal that glial cell-specific ceruloplasmin gene expression is essential for iron homeostasis and neuronal survival in the human central nervous system.

INTRODUCTION

The human brain has a high rate of oxidative metabolism and the availability of iron to the central nervous system is essential for neuronal function and survival. As free ionic iron results in the rapid formation of highly toxic reactive oxygen species, the interdependence of brain oxygen and iron metabolism has necessitated the evolution of distinct pathways of cellular iron trafficking within the central nervous system (1 ). Although the molecular mechanisms determining brain iron homeostasis are largely unknown, the essential nature of this process is underscored by the number of inherited and acquired neurodegenerative diseases in which abnormalities of iron metabolism are a prominent feature (2 -3 ). As alterations in transition metal metabolism resulting in oxyradical production may be a common mechanism underlying cell death in the nervous system, additional insight into the proteins involved in brain iron homeostasis is clearly warranted.

Ceruloplasmin is an abundant [alpha]2-glycoprotein which contains >95% of the copper found in the plasma of all vertebrate species. This protein is synthesized and secreted as a single polypeptide chain of 1046 amino acids with six atoms of copper oxidase, and, although this protein can oxidize a number of substrates in vitro, early studies demonstrated a specific role for ceruloplasmin as a ferroxidase (4 -5 ). This concept is further supported by recent findings which indicate an essential role for a homologous copper oxidase in iron metabolism in Saccharomyces cerevisiae (6 -8 ). Recently the physiological role of ceruloplasmin has been clarified by the recognition of aceruloplasminemia, an autosomal recessive disorder characterized by absent serum ceruloplasmin and neurodegeneration of the retina and basal ganglia in association with iron accumulation in these tissues (9 -11 ). Molecular genetic analysis of affected individuals has revealed specific inherited mutations in the ceruloplasmin gene (12 -17 ).

Aceruloplasminemia is a fatal neurologic disease presenting in the fourth or fifth decade of life with signs of dystonia, dysarthria and dementia and radiologic evidence of iron accumulation in the basal ganglia (9 -11 ). This unique involvement of the central nervous system distinguishes aceruloplasminemia from other inherited and acquired iron storage disorders and suggests that ceruloplasmin may play a direct role in brain iron homeostasis. Nevertheless, under normal circumstances, little or no ceruloplasmin crosses the blood brain barrier making it unclear how a deficiency of this plasma protein results in either iron accumulation or neurodegenerative disease in the central nervous system (18 ). One potential explanation for these findings would be the local expression of ceruloplasmin within the human brain and retina, and we have conducted this current study to directly test this hypothesis.

RESULTS

Initially, RNA was isolated from specific regions of human brain and subjected to RNAse protection using a human ceruloplasmin-specific cRNA probe. As can be seen in Figure 1 A, a protected fragment of appropriate size was detected in RNA samples from human liver and spleen as well as multiple regions of human brain. Analysis of similar samples by RNA blot hybridization demonstrated ceruloplasmin-specific transcripts of 4.2 and 3.7 kb in multiple brain regions with no differences in transcript size between these regions (Fig. 1 B). Similar studies using RNA isolated from human peripheral blood cells did not detect ceruloplasmin mRNA, indicating that the transcripts observed in brain tissue were not the result of contamination with residual blood at the time of RNA isolation (data not shown). To determine if the ceruloplasmin transcripts detected in brain tissue were translated into protein, tissue samples were metabolically labeled followed by immunoprecipitation of ceruloplasmin from the culture media. Consistent with the RNA analysis, a single 135 kDa band was detected following immunoprecipitation in the media from all brain regions examined which was identical in electrophoretic mobility to ceruloplasmin immunoprecipitated from HepG2 cell media (Fig. 1 C).


Figure 1. Expression of the ceruloplasmin gene in the central nervous system. (A) RNAse protection analysis of RNA isolated from the indicated tissues using a 32P-labeled human ceruloplasmin specific cRNA probe. The arrow indicates a protected fragment of 357 nucleotides. (B) Northern blot analysis of polyadenylated RNA from the indicated tissues using a 32P-labeled ceruloplasmin specific cDNA-probe. Arrows indicate ceruloplasmin specific transcripts of 4.2 and 3.7 kb. (C) Ceruloplasmin was immunoprecipitated from the conditioned media of metabolically labelled HepG2 cells or from the indicated brain tissue samples and resolved on 7.5% SDS-PAGE. The gel was processed for fluorography and exposed for 1 day (lane 1) or 4 weeks (lanes 2-6). Molecular sizes are indicated and the arrow illustrates the 135 kDa immunoprecipitated ceruloplasmin.


Figure 2. In situ hybridization of human liver and spleen with ceruloplasmin specific cRNA probes. Tissue sections were prepared and analyzed following in situ hybridization with bright (A, C, D and F) and corresponding darkfield (B and E) microscopy. Liver (A-C) with hepatic parenchyma (hp), portal space (ps) and sinusoids (s). The arrows indicate hepatocytes and the arrowhead points to biliary duct epithelial cells. Original magnifications: *125 (A and B), *800 (C). Spleen (D-F) with white pulp (wp), marginal zone (mz) and trabecular vein (tv). Arrows point to macrophages (m) of the reticuloendothelial system concentrated in the marginal zones. Arrowheads indicate erythrocytes (e). Original magnifications: *125 (D and E), *2000 (F).

To determine the cell types responsible for ceruloplasmin gene expression in the central nervous system, in situ hybridization was performed using ceruloplasmin-specific cRNA probes. The specificity of these probes was confirmed by detection of abundant ceruloplasmin mRNA in hepatocytes throughout the liver parenchyma (Fig. 2 A-C). Ceruloplasmin mRNA in the liver was cell-specific with no signal detected in vascular endothelium, biliary epithelium or mesenchymal cells in the portal spaces. Hybridization with sense strand cRNA probes or predigestion of tissue with RNAse A resulted in negligible background hybridization (data not shown). Ceruloplasmin gene expression was also detected by RNAse protection in human spleen (Fig. 1 A, lane 2), and in situ hybridization revealed that this expression was detected in macrophages of the reticuloendothelial system concentrated in the marginal zones of the splenic lymphoid follicles (Fig. 2 D-F).

As these studies confirmed the ability of the probes to detect cell-specific ceruloplasmin gene expression, a similar analysis was next performed on sections of human brain. In the caudate nucleus, ceruloplasmin-specific transcripts were detected in ependymal cells lining the lateral ventricle and in glial cells in the subependymal glial membrane (Fig. 3 A and B). Endothelial cells and neurons readily identified by histologic characteristics were devoid of detectable ceruloplasmin mRNA (Fig. 3 A and B). Analysis of ceruloplasmin gene expression in the globus pallidus revealed a punctate appearance which upon higher magnification was found to be confined to a specific population of astrocytes intimately associated with the brain microvasculature (Fig. 3 C-E). This astrocyte-specific pattern of ceruloplasmin expression was similarly observed in several other regions of the human brain, presumably accounting for the transcripts detected by RNAse protection in each of these regions (data not shown).


Figure 3. In situ hybridization of human basal ganglia with ceruloplasmin and transferrin specific cRNA probes. In situ hybridization using ceruloplasmin specific cRNA probes (A-E) or a transferrin specific probe (F) was performed and analyzed by bright (A, C, E and F) and corresponding darkfield (B and D) illumination. (A and B) Section of caudate nucleus immediately adjacent to the lateral ventricle (lv) showing subependymal glial membrane (sgm) and a blood vessel (bv). Arrows indicate ependymal cells (e), the arrowhead points to an endothelial cell and the open arrow indicates ceruloplasmin gene expression in a glial cell in the sgm. Original magnifications: *500. (C and D) Section of globus pallidus with myelinized fibre bundles (mfb). Note the punctate labeling-pattern in blood vessel-associated cells (arrows). Original magnification: *100. High magnification composite micrographs (E and F) of microvasculature associated astrocytes (arrows), oligodendrocytes (arrowheads) and neurons (n). Original magnifications: *2000 (E), *1600 (F).

To directly compare the cell-types involved in ceruloplasmin and transferrin gene expression in the human brain, serial sections of caudate nucleus and globus pallidus were hybridized with a transferrin specific cRNA probe. As can be seen in Figure 3 F, specific expression of the transferrin gene was observed in a glial cell population with the histologic characteristics of oligodendrocytes. No transferrin mRNA was detected in the microvasculature-associated astrocytes shown to express the ceruloplasmin gene in this same tissue.

In addition to the population of astrocytes surrounding the microvasculature, cell-specific ceruloplasmin expression was also observed in glia surrounding neurons within specific basal ganglia regions known to be affected in aceruloplasminemia. This is illustrated in Figure 4 where ceruloplasmin mRNA is detected in glial cells dispersed between and in close apposition to the large melanized dopaminergic neurons of the substantia nigra. Glial cells expressing the ceruloplasmin gene in this region were easily distinguishable from oligodendrocytes based on morphological criteria and this was confirmed by hybridization of adjacent sections with a transferrin cRNA probe (Fig. 4 C).


Figure 4. In situ hybridization of human substantia nigra pars compacta with ceruloplasmin and transferrin specific cRNA probes. In situ hybridization using ceruloplasmin specific cRNA probes (A and B) or a transferrin specific probe (C) was performed on sections of substantia nigra and analyzed by brightfield microscopy. Melanized dopaminergic neurons (mdn) are indicated, arrows in (A) and (B) point to astrocytes. Original magnifications: *1000 (A), *2000 (B). Transferrin mRNA positive oligodendrocyte (C) is identified by an arrow and transferrin mRNA negative cells are indicated by arrowheads. Original magnification: *1250.

The retina is consistently affected in patients with aceruloplasminemia and because this central nervous system tissue is also separated from the systemic circulation by the blood-brain barrier, ceruloplasmin gene expression was examined in normal human retina using in situ hybridization. Utilizing this method, ceruloplasmin mRNA was readily detected within the retina confined to the inner nuclear layer. Ceruloplasmin-specific transcripts were detected in pale staining glial cells within this inner nuclear layer whereas neuronal cells in this same layer were devoid of any signal (Fig. 5 A and B). No ceruloplasmin mRNA was detected in any of the multiple neuronal cell-types within the retinal layers which were easily identified by histologic analysis (Fig. 5 A and B). To confirm that ceruloplasmin is present in this tissue, immunohistochemistry was performed. By this analysis, ceruloplasmin was readily detected as the brown reaction product within the retina in a distribution similar to that observed for the glial cell marker S-100 (Fig. 5 C and D).


Figure 5. In situ hybridization and immunohistochemistry of human retina with ceruloplasmin specific cRNA probes and antibody. Tissue sections were prepared and analyzed following in situ hybridization with bright (A) and corresponding darkfield (B) microscopy. Human retina (A and B) with retinal pigment epithelium (RPE), rods and cones (R&C), outer limiting membrane (OLM), outer nuclear layer (ONL), outer plexiform layer (OPL), inner nuclear layer (INL), inner plexiform layer (IPL), ganglion cell layer (GCL), afferent fibers (AF) and inner limiting membrane (ILM). Arrows indicate nuclei of glial cells, arrowheads indicate nuclei of bipolar neurons in the INL. Original magnification: *400. Immunohistochemical staining of ceruloplasmin (C) and S-100[beta] (D) in human retina. Specific layers are designated for each section. Original magnification *400.

DISCUSSION

The recognition of aceruloplasminemia as a neurodegenerative disease underscored the essential role of ceruloplasmin in iron homeostasis in the brain. The data in this paper demonstrate synthesis and secretion of ceruloplasmin within the central nervous system, providing a potential explanation for the neurologic features of this disease. Ceruloplasmin is a secreted protein and, thus, in situ hybridization of mRNA was utilized to ensure detection of the cells expressing this protein. These in situ hybridization experiments, taken together with the biosynthetic experiments, indicate that ceruloplasmin is synthesized in a specific subpopulation of astrocytes in close contact with the brain microvasculature and in glial cells surrounding large neurons throughout the brain parenchyma. Cell-specific expression of ceruloplasmin was also detected in the retina where immunohistochemical data confirm the production of this protein in a pattern similar to that of S-100, a marker of perivascular glial cells in this tissue (19 ). These data are consistent with recent studies of in situ hybridization of ceruloplasmin in the murine brain and retina and with experiments demonstrating ceruloplasmin biosynthesis and secretion in primary cultures of murine and rat astrocytes (20 ,21 ).

Previous studies have shown that iron metabolism within the central nervous system is largely independent of systemic iron metabolism (22 -24 ). Nevertheless, the detection of ceruloplasmin and transferrin gene expression in the human brain suggests that iron trafficking in the central nervous system involves a similar mechanism to that in the periphery (1 ). The detection of transferrin gene expression in oligodendrocytes is consistent with previous in situ data in the rat brain and indicates that, in contrast to hepatocytes, distinct cell-types synthesize transferrin and ceruloplasmin in the human central nervous system (25 ). As neither ceruloplasmin or transferrin cross the blood-brain barrier, the synthesis and secretion of these proteins by glia provides a mechanism for iron mobilization and oxidation within the CNS extracellular fluid thus ensuring adequate iron delivery to neurons. Since in the absence of ceruloplasmin ferrous iron cannot be oxidized for uptake by transferrin, central nervous system iron accumulation in aceruloplasminemia thus results from a mechanism analogous to that in other iron-overload disorders where a loss or oversaturation of transferrin iron binding capacity results in rapid removal of excess ferrous iron with accumulation in systemic tissues (26 ). While it is reasonable to implicate the ferroxidase activity of ceruloplasmin in this process, the molecular mechanisms of ceruloplasmin iron mobilization and oxidative transfer to transferrin remain unknown (27 ).

Despite evidence of ceruloplasmin expression throughout the brain, selective neuronal loss and iron accumulation in aceruloplasminemia is observed predominantly in the basal ganglia. This finding may reflect either the increased baseline level of iron in this region of the brain or an increased sensitivity of these tissues to iron-mediated damage. Similar abnormalities in brain iron metabolism and oxidative injury have been implicated in neuronal loss in the substantia nigra in patients with Parkinsons disease (3 ,28 ). The melanized neurons in the substantia nigra are particularly sensitive to oxidative stress and the intimate association of ceruloplasmin synthesis with the cells observed here suggests that ceruloplasmin may participate in antioxidant protection of these cells by limitation of ferrous iron. Consistent with this concept, recent studies indicate a marked increase in plasma lipid peroxidation in patients with aceruloplasminemia (29 ). Alternatively, it is also possible that in aceruloplasminemia excessive ferrous iron uptake by glial cells may lead to cell injury with reduced production of trophic factors essential for neuronal survival (30 ).

Recently, abnormalities in transition metal metabolism have been implicated in the biochemical pathways leading to neuronal cell death in several neurodegenerative disorders including Alzheimer's disease and amyotrophic lateral sclerosis (31 -34 ). The studies in this report taken together with the clinical and pathological findings in aceruloplasminemia reveal a direct link between iron metabolism and neurodegeneration and provide the first molecular characterization of a protein essential for this process in the central nervous system. Further study of the specific role of ceruloplasmin in brain iron homeostasis may provide a useful framework to advance our knowledge of the pathogenesis of neurodegenerative disease and may permit the development of novel therapeutic approaches to prevent or ameliorate neuronal injury in a variety of human neurologic diseases associated with oxidative injury.

MATERIALS AND METHODS

RNA isolation and analysis

Human tissue samples were obtained at autopsy from individuals with diseases unrelated to the organs obtained and in all cases histologic examination revealed no significant abnormalities. Tissues were snap-frozen in liquid nitrogen and RNA was isolated by CsCl density gradient centrifugation following tissue dissolution in guanidinium isothiocyanate (35 ). RNAse protection was performed using an RPA II-kit (Ambion) in accordance with the manufacturer's instructions. For this analysis, a 357 bp fragment encompassing nucleotides 2661-3018 of the human ceruloplasmin sequence was amplified by polymerase chain reaction (PCR), subcloned into PCR II (Invitrogen) and used as a template for the generation of 32P-labeled antisense cRNA probes (36 ). RNA blot analysis was performed utilizing a nitrocellulose membrane containing RNA samples from different regions of human brain (Clontech) which was hybridized with this same 32P-labeled human ceruloplasmin probe and washed under stringent conditions as described (37 ).

Biosynthetic labeling of tissue samples and immunoprecipitation

Brain tissue samples obtained within 6 h post mortem were minced and metabolically labeled with [35S]methionine and [35S]cysteine for 90 min at 37oC (38 ). Protein synthesis was determined as trichloroacetic acid precipitable radioactivity in the tissue lysates and was equivalent in all these samples. Following labeling, media were clarified by centrifugation at 14 000 g for 10 min and ceruloplasmin was quantitatively immunoprecipitated from the supernatants using a rabbit anti-human ceruloplasmin antiserum. For control experiments, HepG2 cells were maintained at confluence, metabolically labeled for 90 min and ceruloplasmin was subsequently immunoprecipitated from media as described (38 ). In all cases immunoprecipitates were resolved on 7.5% SDS-PAGE under reducing conditions followed by autoradiography.

In situ hybridization and immunohistochemistry

Tissues were rinsed in phosphate-buffered saline and placed in 10% formalin for 72 h. Paraffin embedded sections were then prepared from these or previously stored specimens and in situ hybridization was performed as described (37 ,39 ). For these studies, ceruloplasmin specific probes corresponding to nucleotides 107-701, 1232-1739 and 2661-3018 of the human ceruloplasmin cDNA sequence and a transferrin probe corresponding to nucleotides 1801-2160 of the human transferrin cDNA were amplified by PCR, subcloned, sequenced and used to synthesize 35S-labeled sense and antisense cRNA probes. Hybridization was performed overnight at 60oC followed by treatment of slides with RNAse A and washing in decreasing concentrations of SSC prior to exposure to liquid autoradiography emulsion for 4-14 days. Following development, slides were counterstained using hematoxylin and eosin and examined using bright and darkfield light microscopy.

For immunohistochemistry, paraffin embedded sections were deparafinized, rinsed in decreasing concentrations of ethanol followed by quenching in 0.02 M glycine in PBS and 0.01% sodium borohydride (40 ). Slides were then incubated in PBS and 2% bovine serum albumin with either preimmune sera, rabbit polyclonal antisera to human ceruloplasmin or murine monoclonal antibody to bovine S-100 (Sigma) at a dilution of 1:1000 for 1 h at 22oC. Following rinsing in PBS, slides were incubated with horseradish peroxidase coupled antibodies to IgG (Jackson Labs), developed with diaminobenzadine counterstained with hematoxilin and examined by light microscopy.

ACKNOWLEDEMENTS

We thank Joel Price and Dennis Choi for useful discussions and David Wilson for critical review of the manuscript. Human studies were conducted under approved protocol in accordance with the guidelines of the Human Studies Committee of the Washington University School of Medicine (IRB# 94-0909). This work was supported by funds from National Institutes of Health grant HL 41536 (to JDG).

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*To whom correspondence should be addressed

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