Transferrin gene expression visualized in ... - Europe PMC

Proc. Natl. Acad. Sci. USA Vol. 82, pp. 6706-6710, October 1985 Neurobiology

Transferrin gene expression visualized in oligodendrocytes of the rat brain by using in situ hybridization and immunohistochemistry (growth factor/glial cell/iron binding protein)

BERTRAND BLOCH*, THEODORA POPOVICIt, MARIANO J. LEVINt, DAVID TUILt, AND AXEL KAHN: *Laboratoire d'Histologie, Unite Associde 040561 du Centre National de la Recherche Scientifique, Facultd de MWdecine, 4 place Saint Jacques, 25030 Besanqon Cedex, France; tUnit6 198 de l'Institut National de la Sante et de la Recherche MWdicale, route de Dole, 25000 Besancon, France; and *Institut de Pathologie Mol6culaire, Unit6 129 de l'Institut National de la Sante et de la Recherche MWdicale, 24, rue du Faubourg Saint Jacques, 75674 Paris, Cedex 14, France

Communicated by W. Maxwell Cowan, June 7, 1985

ABSTRACT The presence and production of transferrin in the adult rat brain have been investigated using both immunohistochemistry and in situ hybridization in tissue sections. Indirect immunofluorescence with four distinct antisera against rat and human transferrin and one monoclonal antibody against human transferrin demonstrated labeling of the cytoplasm of oligodendrocytes (a category of glial cells) in most parts of the brain, especially in the white matter. In situ hybridization using rat transferrin 32P-labeled cDNA as a probe revealed the presence of transferrin mRNA in glial cells whose appearance, distribution, and organization exactly matched those of the cells decorated with the transferrin antibodies. These results provide evidence that the transferrin gene is expressed in the central nervous system and that transferrin is synthesized by and stored within oligodendrocytes in the adult rat brain. These data suggest that this molecule could have a specific function in nervous system

ranville, PA; Dako Laboratories, Copenhagen, Denmark), and one (10) kindly provided by J. Foucrier, (it) one rabbit antiserum against human transferrin (Nordic Laboratories, Tilburg, The Netherlands), and (iii) nine mouse monoclonal antibodies against human transferrin (11) (kindly provided by V. Viklick9). All antisera and antibodies were shown to exhibit monospecificity for transferrin by immunoelectrophoresis. Sections were treated by indirect immunofluorescence as described (9). They were incubated for 16 hr with primary antibody (at various dilutions), then for 30 min with fluorescein isothiocyanate-labeled sheep anti-rabbit or goat anti-mouse immunoglobulins (Wellcome Reagents, Beckenham, England) (diluted 1:20). Controls for specificity included analysis of sections treated with preimmune sera, or with antisera raised against molecules distinct from transferrin (albumin, neuropeptides). Furthermore, a comparison was made of sections treated either with transferrin antibodies alone or with transferrin antibodies adsorbed with human transferrin (Sigma; 0.1-4 mg/ml of undiluted serum). All experiments included treatment of rat liver sections as a positive control. In Situ Hybridization. In situ hybridization was performed on 6-tkm or 12- to 14-tum sections of brain, liver, kidney, and myocardium of six adult male rats using as a probe a rat transferrin cDNA 1.4-kilobase long (7, 12) labeled with [32P]dCTP by nick-translation. The procedures used for nick-translation, processing of the tissue, and autoradiography have been described previously (13, 14). Cryostat tissue sections were fixed for 10 min at 40C using Carnoy's fluid, dried, and incubated with 5 ng of 32P-labeled rat transferrin cDNA in appropriate conditions for 16 hr at 400C. After incubation, the sections were placed in contact with x-ray film for 16 hr at -700C with an intensifying screen, then coated with Ilford K-5 liquid emulsion and exposed in the dark 7-17 days. The use of x-ray film provides a macroscopic image of transferrin mRNA distribution in the brain. The autoradiograms were then developed and the sections were stained with toluidine blue and viewed under bright- and dark-field conditions. Controls included cDNA probes unrelated to transferrin (pro-opiocortin and prolactin cDNAs), buffer devoid of probe, and processing of control tissues in which the cells are known either to express the transferrin gene (i.e., liver) or not to express it (i.e., kidney cortex, myocardium).

activity. Transferrin, the iron binding protein produced by the liver, is known to transport iron through the bloodstream to various tissues in the body, including erythroid cells (1). Interest in the study of transferrin has recently increased because of its profound effect in stimulating the proliferation and differentiation of many cell populations (2, 3) and because of its possible involvement as a neurotrophic factor (4, 5). There are several lines of evidence which suggest that transferrin can be produced in tissues other than the liver (6, 7). In particular, transferrin mRNA has been detected in the central nervous system of the chicken (8) and of the rat (7). In the latter, the amount oftransferrin progressively increases from birth until the 30th postnatal day. This has led us to search for morphological evidence for the presence and the location of synthesis of transferrin in the adult rat brain using immunohistochemistry to detect transferrin and in situ hybridization to detect transferrin mRNA. The present study demonstrates that a category of glial cells, the oligodendrocytes, synthesize and store transferrin.

MATERIAL AND METHODS Immunohistochemical Analysis. Immunohistochemical analysis was performed in brain and liver sections of 10 adult male rats (200- to 400-g body weight). Tissues were fixed by intracardiac perfusion of the animal with 4% (wt/vol) chilled paraformaldehyde, and frontal or sagittal cryostat sections were processed as described (9). The antibodies used were as follows: (i) three rabbit antisera against rat transferrin, two of them commercially available (Cappel Laboratories, Coch-

RESULTS Detection of Transferrin. The four rabbit polyclonal antisera against human and rat transferrin as well as one monoclonal antibody against human transferrin (referred to as HTF 14 in ref. 11) gave immunolabeling of both rat liver and brain sections. This monoclonal antibody was shown to crossreact with transferrin of several species (11). The other

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Proc. Natl. Acad. Sci. USA 82 (1985)

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FIG. 1. Indirect immunofluorescence using rabbit antiserum against human transferrin (1:400) in adult rat liver (A and B) and brain (C and D). (A) Rat liver treated with anti-human transferrin antiserum shows labeling of the hepatocyte cytoplasm. (x500.) (B) An adjacent section treated with anti-human transferrin antiserum adsorbed with human transferrin (0.5 mg/ml of undiluted serum) demonstrates a complete lack of the specific immunolabeling. (C) Basal hypothalamus of rat brain treated with an'ti-human transferrin. The labeling occurs in oligodendrocytes in gray matter of the hypothalamus and in the optic tract (OT). (x150.) (D) An adjacent section treated identically to B showing absence of decoration. Nonspecific fluorescence is due to autofluorescence of lipofuschins.

FIG. 2. Indirect immunofluorescence of adult rat brain treated with anti-rat transferrin antiserum (1:200). (A) A general view of the parietal cortex shows labeled oligodendrocytes. The deep layers of the cortex (bottom) contain numerous cells while the superficial layers (top) contain very few. (x450.) (B) Oligodendrocytes in white matter (cerebellum). Labeled cells are frequently arranged in rows and show small processes. Myelinated fibers are undecorated. (C) Oligodendrocytes in gray matter (lateral hypothalamus). Several oligodendrocytes appear in close contact with capillaries (stars). (x 1125.)

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FIG. 3. Detection of transferrin mRNA by in situ hybridization with rat transferrin 32P-labeled cDNA. Liver, kidney, and myocardium sections (A) and sagittal section of rat brain (B) were fixed with Carnoy's fluid and incubated 16 hr at 40'C with 5 ng of 32P-labeled cDNA. Then sections were exposed 72 hr at -70'C in contact with Cronex MRF31 x-ray film with an intensifying screen. Dark areas of the x-ray film correspond to the presence of labeled areas of the sections. (x 3.) (A) The area over the liver section was heavily darkened while the areas over myocardium (m) and kidney (k) sections were unlabeled. (B) White matter of the cerebellum (arrow), the corpus callosum (C), and the internal capsule (IC) is the most intensely labeled. Gray matter of the cerebellar folia (*) is unlabeled.

monoclonal antibodies against human transferrin did not bind to liver or brain sections. The same pattern of immunoreactivity was obtained whatever the antibody used. The cytoplasm of most liver parenchymal cells was decorated (Fig. 1). In the brain sections, binding was restricted to the choroid plexus and to the cytoplasm and initial processes of oligodendrocytes in both the gray and white matter (see Figs. 1, 2, 4, and 5). Such cells appeared small in size with very little cytoplasm and had only short, unbranched processes. Their appearance differs slightly according to the area in which they were located. In gray matter, they were generally round with a slim rim of cytoplasm. In white matter, they were frequently stellate-shaped and had more abundant cytoplasm (Fig. 2). They were arranged either singly or in small clusters, frequently in the vicinity of capillaries. They were prominent in bundles of white matter such as the corpus callosum, the internal capsule, and the optic tract where several were frequently arranged in rows among the myelin-

ated fibers (Fig. 5). They were also abundant in the globus pallidus, the hypothalamus, and the deeper layers of the cerebral cortex. In some areas, such as the upper layers of the neocortex (Fig. 2), the amygdaloid complex, the median eminence, and the molecular layer of the cerebellum (Fig. 4), there were very few, if any, labeled cells. Antisera raised against certain other molecules, and preimmune sera did not bind to these glial cells. Preincubation of antibodies against human transferrin with human transferrin (0.5 mg of transferrin/ml of undiluted serum) led to a total disappearance of labeling in both the liver and brain (Fig. 1). Decoration obtained with antisera against rat transferrin was not inhibited by human transferrin, which suggests that while there was obvious crossreactivity, these antisera recognized distinct antigenic determinants in the rat transferrin. Detection of Transferrin mRNA. In situ hybridization showed a labeling in numerous areas of the brain, the most conspicuous labeling being located within bundles of white

FIG. 4. Indirect immunofluorescence of sagittal sections of rat cerebellum using antiserum against rat transferrin (A) and in situ hybridization using transferrin 32P-labeled cDNA (B). (x225.) (A) Section of formaldehyde-fixed tissue showing oligodendrocytes containing transferrin immunoreactivity located in the central axis of white matter of the cerebellar folia. The granule cell layer (gl) contains very few labeled cells. The Purkinje cell layer and the molecular layer (ml) are devoid of labeling. Capillary endothelial cells exhibit unspecific fluorescence not blocked by addition of transferrin to the antisera. (B) In situ hybridization of a similar section of Carnoy's fluid fixed tissue exposed 14 days with Ilford K-5 emulsion and viewed under dark-field conditions localizes transferrin mRNA (as an accumulation of bright silver grains) in the same regions as transferrin.



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FIG. 5. Sections examined by in situ hybridization with transferrin [32P]cDNA (A and C), and by indirect immunofluorescence with antiserum against rat transferrin (B). Autoradiograms were photographed under bright field after toluidine blue staining. The corpus callosum shows transferrin mRNA (A) and transferrin (B) in oligodendrocytes. Note the similar arrangement of oligodendrocytes in rows among myelinated fibers. (C) The junction between the optic chiasma (QC) and the supraoptic nucleus (SON) shows labeled oligodendrocytes in the optic chiasma, and unlabeled neurons. (x845.)

matter. Some areas such as the corpus callosum and the white matter of the cerebellum were heavily labeled, as was the globus pallidus (Fig. 3B). The cerebellar gray matter and the amygdala were conspicuously free of labeling. The topography of the areas labeled in the autoradiograms closely paralleled that of the regions which showed transferrin

immunoreactivity. Analysis of histological sections by autoradiography using liquid emulsion confirmed the labeling of such areas and demonstrated an accumulation of transferrin mRNA- in individual cells or clusters of cells whose appearance, number, and distribution were identical to those of the cells identified with transferrin antisera (Figs. 4 and 5). This was especially conspicuous in brain areas where the oligodendrocytes have a defined distribution such as the cerebellum (Fig. 4B) and in such regions of white matter as the optic tract and the corpus callosum (Fig. 5A). Neurons, ependyma, and endothelial cells did not exhibit labeling (Fig. SC). Liver sections also showed heavy labeling while sections of the kidney and myocardium did not (Fig. 3A). In situ hybridization, using cDNA probes for prolactin and proopiQcortin in these tissues, was negative except labeling of pro-opiocortin neurons in the hypothalamus.

DISCUSSION The present immunohistochemical results demonstrate the presence of transferrin immunoreactivity in what appears to be oligodendrocytes in the rat brain. Essentially, identical results have been obtained using four distinct antisera and one monoclonal antibody. These results show a close parallel between liver and brain and strongly suggest that the molecule immunologically detected in oligodendrocytes is either transferrin or a very closely related molecule. Indirect evidence for this conclusion is provided by the fact that, in the brain, iron is found most commonly in oligodendrocyte cytoplasm (15-17). The uniformity of the results obtained with immunohistochemistry and in situ hybridization also demonstrates that transferrin and transferrin mRNA are present within the same cell population and implies that transferrin is synthesized in the brain by oligodendrocytes. The local production of transferrin, of course, does not preclude the possibility that transferrin is also delivered to the brain from the

bloodstream. Indeed, this is suggested by the presence of transferrin and other plasma proteins in the choroid plexus and also in neurons of the developing brain (18-20). It is conceivable that the development of the blood brain barrier during ontogeny limits access of the circulating transferrin to the brain, requiring the synthesis of transferrin inside the blood brain barrier for normal brain cell activity. It also follows from our results that the expression of the transferrin gene may be used as a specific marker for oligodendrocytes in the adult rat brain. However, this specificity may suggest that transferrin plays some unique role in oligodendrocyte function and conceivably in the life of neurons, especially those with myelinated axons. Some recent data (unpublished observations) indicate that transferrin gene expression in the brain is regulated independently from its expression in the liver. While the role of transferrin in the brain is unclear, its function could be related to the general properties of transferrin as an iron binding protein (21), or it could play a more specific role such as a neurotrophic factor (5). The latter possibility is suggested by the facts that the transferrin mRNA content of the rat brain progressively increases during postnatal brain development (7) and that brain capillaries are the only blood vessels in the body that have receptors for transferrin (22). We thank Prof. C. Bugnon (Unite Associee 040561 du Centre National de la Recherche Scientifique) and Prof. G. Adessi (Unite 198 de l'Institut National de la Sante et de la Recherche Medicale) for their support and for the use of facilities and Prof. J. C. Dreyfus (Unite 129 de l'Institut National de la Sante et de la Recherche Medicale) for his permanent interest and stimulating discussions. Dr. J. Foucrier (Laboratoire de Biologie du Developpement, Unite d'Enseignement et de Recherche Biomedicale, Bobigny, France) and Dr. V. Viklicky (Institute of Molecular Genetics, Prague, Czechoslovakia) kindly provided us with antibodies against transfenin. We also thank Drs. G. Roussel and J. L. Nussbaum (Centre de Neurochimie, Strasbourg) for their expert opinion. This work was supported by funds from the Fondation pour la Recherche Melicale and the Centre National de la Recherche Scientifique (Action Thdmatique Programm&e Gdndtique Moleculaire et Systeme Nerveux). 1. Huebers, H. A. & Finch, C. A. (1984) Blood 64, 763-767. 2. Barnes, D. & Sato, G. (1980) Cell 22, 649-655. 3. Ekblom, P., Thesleff, I., Saxdn, L., Miettinen, A. & Timpl, R.

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