THE JOURNAL OF COMPARATIVE NEUROLOGY 382:260–271 (1997)

Distribution of Transferrin Binding

Protein Immunoreactivity in the Chicken
Central and Peripheral Nervous Systems
SA SUN CHO,1* JOHN J. LUCAS,2 EUN JUNG ROH,1 YOUNG BOK YOO,1
KYUNG HOON LEE,3 KYEONG HAN PARK,1 DOUK HO HWANG,1 AND SANG HO BAIK1
1Department of Anatomy, Seoul National University, College of Medicine,

Seoul 110-799, Korea

2Department of Biochemistry and Molecular Biology, State University of New York

Health Science Center, Syracuse, New York 13210

3Department of Anatomy, Dankook University, College of Medicine,

Cheon-An 330-714, Korea

ABSTRACT
Transferrin binding protein (TfBP) is a glycoprotein originally purified from chicken
oviduct that exhibits transferrin binding activity. Recent work has shown that TfBP is a
post-translationally modified form of the heat shock protein (HSP108), the avian homologue of
a glucose regulated protein, GRP94. The function of this protein, however, has not yet been
clearly defined. Antiserum to TfBP was found to selectively stain oligodendrocytes of the avian
brain. In this study, we further describe these oligodendrocytes and other cell types positive to
anti-TfBP in the chick nervous system. In accordance with previous studies, the most
prominent cell type that labels with antiserum to TfBP is the oligodendrocyte. At the electron
microscopic level, the immunoreactive product is confined to the perinuclear cytoplasm and
fine processes of the oligodendrocytes, whereas myelin and axoplasm are devoid of staining.
The immunoreactive product is found both in the cytoplasmic matrix and bound to the
endoplasmic reticulum and plasma membrane, suggesting that TfBP may have properties of
both a soluble and an integral membrane protein. There is great variability in the number of
TfBP-oligodendrocytes in different areas of the central nervous system (CNS); large numbers
of cells are associated with the white matter regions and are found in the myelinated tracts,
whereas few cells are present in the gray matter regions. In the retina, TfBP is localized
specifically in the cells that are morphologically oligodendrocytic and is present in the optic
nerve fiber layer and the ganglion cell layer. Obvious staining is also seen in the Bergmann
glial cells of the cerebellum and in the Schwann cells of the sciatic nerve. Furthermore, the
choroid plexus cells similarly exhibit a strong reaction. The association of TfBP in these
specific cell types responsible for myelination and sequestering iron and transferrin implies
that TfBP may be involved in myelination and iron metabolism of the chick nervous system,
perhaps through a role in transferrin concentration in these cells. A putative role of TfBP, as
HSP108, is considered. J. Comp. Neurol. 382:260–271, 1997. r 1997 Wiley-Liss, Inc.

Indexing terms: oligodendrocytes; Schwann cells; heat shock protein; myelin; iron

Transferrin binding protein (TfBP) is an estrogen- fied as a heat shock protein, HSP108, by microsequencing
inducible membrane glycoprotein purified from the chicken of reverse phase-high performance liquid chromatography
oviduct that exhibits transferrin binding activity and (RP-HPLC) purified tryptic peptides (Hayes et al., 1994).
shares a number of physical properties with the human
transferrin receptor (Poola and Lucas, 1988). However, Contract grant sponsor: Korean Ministry of Education; Contract grant
based on peptide map analysis and immunologic studies number: 1995-078.
(Poola et al., 1990; Feurnkranz et al., 1991) as well as *Correspondence to: Dr. Sa Sun Cho, Department of Anatomy, Seoul
National University, College of Medicine, Yongon-Dong, Chongno-Gu, Seoul
sequence analysis of the cloned cDNA (Gerhardt et al., 110-799, Korea. E-mail: chossn@plaza.snu.ac.kr
1991), TfBP is clearly distinct from the avian erythrocyte Received 2 August 1996; Revised 15 January 1997; Accepted 22 January
transferrin receptor. Furthermore, TfBP has been identi- 1997

r 1997 WILEY-LISS, INC.

TfBP IMMUNOREACTIVITY IN CHICKEN NERVOUS SYSTEM 261

Because TfBP has been shown to be HSP108, it belongs Immunohistochemical procedures

in the family of glucose regulated proteins, which include
ERp99 (Mazzarella and Green, 1987), GRP94 (Sorger and Chicks were deeply anesthetized by etherization and
Pelham, 1987; Kang and Welch, 1991), gp96 (Srivastava et perfused through the heart with ethanol-acetic acid fixa-
al., 1986; Maki et al., 1990), and HSP100 (Koyasu et al., tive (Clark, 1984) or 4% paraformaldehyde. Perfusion was
1989). The functions of these proteins, especially HSP108, initiated with 200 ml of saline followed by 250–500 ml of
fixative. The brain, spinal cord, retina, and sciatic nerve
have not yet been clearly defined. TfBP appears, however,
were removed 1 hour after the perfusion was completed
to be present in tissues with high iron requirements
and placed overnight in the same fixative at 4°C. The
(Feurnkranz et al., 1991), suggesting a role for this protein
tissues were washed three times in cold phosphate-
in iron metabolism.
buffered saline (PBS; 0.1 M sodium PBS, pH 7.4) and then
Iron is the most abundant metal in the body (Youdim,
cryoprotected by infiltration with sucrose solutions, first
1988). The brain, like the liver, contains a substantially
with 10% sucrose for 2–3 hours and then with 30% sucrose
higher concentration of iron than of any other metal
overnight. The tissues were embedded in Optimal Cutting
because of its high rate of oxidative metabolism (Yehuda
Temperature (OCT) compound and then frozen rapidly in
and Youdim, 1988). Insufficient iron, particularly during
2-methyl butane precooled to its freezing point with liquid
development, may result in mental and motor impair-
nitrogen. Tissue specimens were cut into 10-µm sagittal or
ments (Youdim et al., 1989; Pollite and Metallinos- horizontal sections on a Reichert-Jung Frigocut model
Katsaras, 1990). In contrast, high levels of iron are highly 2800 cryostat (Heidelberg, Germany). Sections were thaw
toxic and a prime initiator of lipid peroxidative damage mounted on gelatin-coated microscopic slides and stored at
(Gerlach et al., 1994). The high iron requirement of the 270°C until processing for immunohistochemistry. Immu-
brain coupled with its susceptibility to iron-induced peroxi- nohistochemical staining was performed on the ethanol-
dative damage requires stringent regulation of the avail- acetic acid-fixed tissue section using the avidin-biotin
ability of iron. The iron binding protein responsible for iron peroxidase complex (ABC) method described previously
mobilization and storage thus appears to be important for (Cho and Hyndman, 1991).
the maintenance of brain iron homeostasis and for protec- Appropriate controls for the immunohistochemical stain-
tion of the brain from oxidative damage. The mechanism of ing procedure included the omission of the primary antise-
transferrin-mediated iron uptake and recycling has been rum, or goat anti-rabbit IgG, the substitution of normal
well established in non-neural tissue (May and Cuatreca- rabbit serum for the primary antiserum, and preincuba-
sas, 1985; Huebers and Finch, 1987). Little is known, tion of primary antiserum with the purified oviduct TfBP
however, about iron regulation in the brain and the (10 mg/ml of diluted antiserum) for 24 hours before its
mechanism by which transferrin crosses the blood-brain application to tissue sections.
barrier. Myelin staining was performed on the 4% paraformalde-
In the course of studying the distribution of TfBP in hyde fixed tissue section by the mordant-dye method using
tissues with high iron requirements, we found an enrich- a ferric salt and chromoxane cyanine R (Kiernan, 1990).
ment of TfBP in avian oligodendrocytes (Cho and Lucas,
1995). This finding points to a role of this protein in
connection with transferrin in oligodendrocytes and shows Immunoelectron microscopy
the usefulness of anti-TfBP antiserum as a possible marker Perfusion fixation with freshly prepared 4% paraformal-
for avian oligodendrocytes. To elucidate the role of TfBP in dehyde and 0.1% glutaraldehyde in neutral 0.1 M phos-
the nervous system, information on the precise anatomical phate buffer was performed transcardially. The brains
distribution of TfBP is needed. Therefore, our investiga- were removed, cut into small pieces, and postfixed in the
tion was extended to provide a more comprehensive view of same fixative for 4 hours. Tissues were sectioned at 50-µm
the localization of TfBP in the central nervous system thickness by using a microslicer (DTK 2000; Dosaka EM,
(CNS) and peripheral nervous system. Kyoto, Japan). The pre-embedding immunocytochemical
technique was used for free-floating sections with the
method described previously (Cho and Lucas, 1995). In
MATERIALS AND METHODS brief, the sections were incubated sequentially in 1) pri-
mary antiserum diluted 1:5,000 overnight at 4°C, 2)
Animals and antisera biotinylated goat anti-rabbit IgG (Vector Laboratory, Bur-
All animal experiments were approved by the local lingame, CA 94010) diluted 1:200 for 2 hours at room
Institutional Animal Care and Use Committee. Five white temperature, and 3) avidin and biotinylated horseradish
leghorn chicks aged 3–4 weeks were used in this study. The peroxidase complex prepared by incubation of a 1:100
primary antibody was rabbit antiserum directed against dilution of Vectastain A and B reagents in PBS at room
the chick oviduct TfBP (aOV-TfBP). The preparation and temperature for 30 minutes before use. The peroxidase
characterization of this antibody has been published in activity was visualized in the usual way using a freshly
detail elsewhere (Poola and Lucas, 1988; Hayes et al., prepared solution of 38,3-diaminobenzidine (0.025%) in
1994). Briefly, the TfBP was purified by electroelution from PBS containing 0.0006% hydrogen peroxide.
sodium dodecyl-sulfate polyacrylamide gel electrophoresis All antibodies were diluted in PBS containing 1% nor-
(SDS-PAGE) tube gels. The purified protein was mixed mal goat serum (Vector Laboratory). The incubation was
with Freund’s adjuvant and injected intradermally into performed in microcentrifuge tubes with continuous agita-
New Zealand albino rabbits. The antiserum is specific for tion. After each step, free-floating sections were rinsed four
the TfBP; only a single radiolabeled band is immunoprecipi- times for 1 hour in cold PBS. After examination by light
tated from oviduct primary cell culture labeled with 35S- microscope, stained sections were postfixed in 1% (W/V)
methionine, 32P-, and 3H-mannose. No immunoreactivity osmic acid dissolved in PBS for 1 hour, dehydrated in
against transferrin was observed on Western blots. graded ethanols, and embedded in Epon 812 (EMS, Fort
262 S.S. CHO ET AL.

Washington, PA 19034). Ultrathin sections were cut tangen- antibody or with the mordant-dye method for myelin. The
tially to the surface of sections and mounted on nickel distributional pattern of TfBP was similar to that of
grids coated with Formvar film. Grids were observed with myelin; TfBP-rich areas such as the optic chiasm coincided
a JEOL 1200 EX-II electron microscope (Tokyo, Japan) with the heavily myelinated fibers (Fig. 1E,F).
with and without uranyl-lead counterstaining. In the cerebellum, TfBP-positive oligodendrocytes were
found in abundance in the white matter where heavily
Preparation of retinal whole mount myelinated fibers occurred (Fig. 2A,B). The interfascicular
Eyes were removed from chicks under ether anesthesia oligodendrocytes in the white matter were elongated or
and placed in PBS. The cornea, lens, sclera, and choroid rectangular and frequently aligned in rows, whereas TfBP-
and pigment layers were gently dissected away from the oligodendrocytes in the gray matter were round and
neural retina. The rest of the eye was fixed by immersion occurred separately (Fig. 2C). The number of TfBP-
for 3 hours in 4% paraformaldehyde and 0.05% glutaralde- positive oligodendrocytes and myelinated fibers decreased
hyde in 100 mM phosphate buffer (pH 7.4). gradually toward the granular and molecular layers. Thus,
Twenty minutes after commencement of fixation, the eye only a few positive oligodendrocytes were visible in the
was rinsed in PBS; the pecten and optic nerve were then molecular layer (Fig. 2A). The Purkinje cell layer showed a
cut along the retinal attachment, and the neural retina peculiar localization of TfBP; the immunoreaction was not
was detached from the vitreous and the ciliary bodies. restricted to oligodendrocytes but was also seen in the
After fixation, the retina was thoroughly washed in PBS Bergmann glial cells. The immunoreactive product was
and then incubated for 15 minutes in neutralized 0.1 M clearly localized in the perinuclear cytoplasm of these glia,
sodium periodate. The tissue was immersed for 10 minutes which clothed the unstained Purkinje cell somata, and in
in a solution of sodium borohydride (5 mg/ml) and washed their long apical processes extending into the molecular
again in PBS. Immunohistochemical staining was per- layer (Fig. 2D).
formed using the free-floating method described in the The choroid plexus exhibited a strong positive immuno-
section on immunoelectron microscopy, except that all staining for TfBP (Fig. 3A). At higher magnification, an
antibodies were diluted in PBS containing 0.5% Triton intense staining was evident in the cytoplasm of cells
X-100. After immunohistochemical staining, the retinae lining the choroid plexus. In contrast, the ependymal cells
were radially cut four or five times and flat mounted on lining the ventricular cavity were devoid of reaction prod-
gelatinized slides with the ganglionic cell layer lying uct (Fig. 3B). TfBP immunoreactivity was also associated
uppermost. A fine brush was used to spread the retina, with blood vessels throughout the CNS, although no
allowing maximum contact with the gelatin. Retinal whole particular regional pattern of reactivity was evident. Immu-
mount preparations were dehydrated through an alcohol nostaining was seen at a lower level in the walls of
series, cleared in xylene, and coverslipped using Per- capillaries (Fig. 3C,D). However, blood components such as
mount. red blood cells were negative. These results are consistent
with those of Fuernkranz et al. (1991).

RESULTS Spinal cord

General characteristics Large numbers of TfBP-oligodendrocytes were distrib-
uted quite regularly between the fascicles of the white
Intense immunostaining was seen in the central and matter, whereas the numbers of TfBP-labeled cells were
peripheral nervous tissue sections stained immunohisto- significantly diminished in the gray matter (Fig. 4A,B).
chemically with TfBP antibody. Immunostaining was ob- The TfBP-oligodendrocytes found in the gray matter were
served in the brain, spinal cord, retina, and sciatic nerves frequently in the perineuronal satellite position, especially
(Figs. 1–6). Control tissue sections that had been incu- in the region of the anterior motor horn cells (Fig. 4C). In
bated in normal or preabsorbed serum were devoid of any the white matter, short processes of TfBP-oligodendro-
immunoreactive products. Although the prominent cell cytes were associated with fascicles of axons. Seen in
type labeled with TfBP antibody was oligodendrocyte, an longitudinal sections, TfBP-positive cells were arranged in
obvious staining was also found in the Bergmann glia, the rows characteristic of interfascicular oligodendrocytes
choroid plexus, blood vessels, and Schwann cells. (Fig. 4D).

Brain Retina
A typical TfBP immunostaining pattern of the brain in Strong TfBP immunostaining was evident in the small
sagittal section is revealed by a series of photomicrographs cells of the retina, which were confined to the ganglion cell
at low magnification (Figs. 1, 2). The numbers of TfBP- layer and the optic nerve fiber layer (Fig. 5A). These
positive oligodendrocytes were highly variable in the differ- immunoreactive cells were interposed sporadically among
ent regions of the chick CNS. The brainstem revealed a ganglion cells in the eighth layer and occurred along the
rather uniform density of TfBP-oligodendrocytes (Fig. 1A), nerve fibers in the ninth layer of the retina. In whole
but these cells diminished markedly toward the diencepha- mount preparations (Fig. 5B), TfBP-labeled cells displayed
lon (Fig. 1B) and the cerebral cortex (Fig. 1C), where an the unique morphologic features of oligodendrocytes; the
abundance of cells were seen only in well-myelinated areas fine processes that extended radially from the perikarya
such as the anterior commissure, septomesencephalic tract could be traced to the terminals located around or along
(Fig. 1B), and optic chiasm. As shown in Figure 1D, the optic nerve fibers. These immunoreactive cells were distrib-
TfBP-labeled cells in the cerebral cortex showed typical uted evenly in the central portion but diminished toward
oligodendrocytic morphology and were frequently in a the periphery of the retina. Diffuse immunostaining at a
perineuronal position. Two representative sections of the lower level was also visible in the inner and the outer
diencephalon were compared by staining with either TfBP nuclear layers (Fig. 5A).
TfBP IMMUNOREACTIVITY IN CHICKEN NERVOUS SYSTEM 263

Fig. 1. Sagittal sections of the chicken brainstem and forebrain anterior commissure (ac) and septomesencephalic tract (sm).
stained with transferrin binding protein (TfBP) antibody (A–E) and C: Cerebral cortex (hyperstriatum). A limited number of TfBP-labeled
mordant-dye method (F) to show the cellular localization and the cells are seen. D: Higher magnification of the boxed area in C. The
regional distribution of TfBP immunoreactivity compared with myelin- TfBP-labeled cells exhibit the typical morphology of oligodendrocytes,
ated fibers. A: Brainstem (medulla oblongata). Large numbers of some of which are in a perineuronal position (arrows). E,F: Ventral
TfBP-labeled cells are distributed all over the area. Inset (higher portions of the diencephalon (hypothalamus) stained for TfBP (E) and
magnification of boxed area in A) shows the reaction product confined myelin (F) to show a parallelism in distribution of TfBP-oligodendro-
to small cells with a few proximal processes. B: Diencephalon. The cytes and myelinated fibers. Note the optic chiasm (oc) that stains
high density of cells are seen only in well-myelinated tracts such as the most strongly for both TfBP (E) and myelin (F). Scale bars 5 100 µm.
264 S.S. CHO ET AL.

Fig. 2. Sagittal sections of the chicken cerebellum showing TfBP- oligodendrocytes in the white matter are elongated or rectangular and
positive cells (A,C,D) and myelinated fibers (B). A,B: Folia of the frequently occur in rows, whereas oligodendrocytes in the granular
cerebellum stained for TfBP-oligodendrocytes (A) and myelin fibers layer have rounded somata and occur separately. D: Purkinje cell
(B). TfBP-labeled oligodendrocytes and myelinated fibers show similar- layer. TfBP-positive Bergmann glial cells are located around un-
ity in the distributional patterns, in which both are most abundant in stained Purkinje cell bodies (P), and their long processes extend
the white matter (wm) and diminished gradually toward the molecu- vertically through the molecular layer (arrows). Scale bars 5 100 µm.
lar layer (ml). C: White matter (wm) and granular layer (gl). TfBP-
TfBP IMMUNOREACTIVITY IN CHICKEN NERVOUS SYSTEM 265

Fig. 3. Sagittal sections of chicken brain stained by immunohisto- ventricular cavity are not stained (arrows). C,D: Blood vessels of the
chemistry to show the choroid plexus and blood vessels. A: Choroid cerebral (C) and cerebellar (D) cortices. A lower level of TfBP immuno-
plexus of the lateral ventricle. B: Higher magnification of a portion of staining is seen in the wall of blood vessels that are often associated
A. Intense TfBP immunoreactivity is evident in the cytoplasm of cells with more intensely stained perivascular oligodendrocytes (arrows).
lining the choroid plexus. In contrast, the ependymal cells lining the Scale bars 5 50 µm.

Sciatic nerve light type oligodendrocytes were characterized by a large,

pale nucleus. In the nucleus, there was little or no clump-
TfBP was localized specifically to Schwann cells en-
ing of the chromatin, and a large nucleolus was occasion-
sheathing axons in the sciatic nerve. The immunoreactive
ally present. The perinuclear cytoplasm contained many
product was confined to the perinuclear cytoplasm and
mitochondria, cisterna of rough endoplasmic reticulum,
processes of the Schwann cells. However, the nucleus of
and distinct Golgi apparatus (Fig. 7A). The medium type
the Schwann cells and the myelinated fibers remained
oligodendrocytes had abundant chromatin masses at the
unstained (Fig. 6A). The staining intensity of Schwann
cells was as great as that of oligodendrocytes in the optic periphery and in the center of the nucleus (Fig. 7B).
nerve (Fig. 6B). TfBP-immunoreactive product was confined to the peri-
nuclear cytoplasm and processes, whereas the nucleus and
organelles—such as mitochondria and Golgi apparatus—
Immunoelectron microscopy were devoid of reaction products (Fig. 7A,B). At higher
Based on ultrastructural features of the oligodendrocyte magnification, immunoreactive product was distributed
nucleus (Mori and Leblond, 1970), two subtypes of TfBP- throughout the cytoplasmic matrix and bound to the rough
oligodendrocytes were identified: light and medium. The endoplasmic reticulum and outer surface of the nuclear
266 S.S. CHO ET AL.

Fig. 4. Immunohistochemical localization of TfBP in chicken spi- oligodendrocytes are scattered in the gray matter and are often
nal cord. A,B: Transverse (A) and longitudinal (B) sections of the perineuronal in position (arrows). D: Higher magnification of the
upper cervical cord. Dark immunoreactive cells are found predomi- boxed area in B. TfBP-positive oligodendrocytes in the white matter
nantly in the white matter, whereas fewer cells are present in the gray occur in clusters and frequently in rows. Scale bars 5 100 µm.
matter. C: Higher magnification of the boxed area in A. TfBP-

membrane (Fig. 7C). In contrast, neither myelin nor Schwann cells of the sciatic nerve. The distribution of
axoplasm were immunostained. TfBP-positive oligodendrocytes and Schwann cells is closely
related to myelin density. For example, the greatest num-
ber of TfBP-positive cells are present in the heavily
DISCUSSION myelinated white matter of the cerebellum, whereas very
The present investigation demonstrates the strong im- few labeled cells are found in the molecular layer where no
munoreactivity of TfBP in the chick nervous system. At the stained myelin exists. In rodents and humans, transferrin-
cellular level, TfBP is specifically localized in oligodendro- labeled oligodendrocytes and Schwann cells are mostly
cytes, Bergmann glia, choroid plexus cells, and capillary associated with myelinated fibers (Connor and Fine, 1986;
endothelial cells of the brain as well as in myelinating Lin et al., 1990; Morris et al., 1992a). During development,
TfBP IMMUNOREACTIVITY IN CHICKEN NERVOUS SYSTEM 267

Fig. 5. Retinal preparations of the chicken stained with TfBP layer (in) and the external nuclear layer (en). B: Whole mount.
antibody. A: Cross-section. TfBP-positive cells are evident in the TfBP-positive cells display typical morphology of oligodendrocytes.
ganglion cell (gl) and the optic nerve fiber (on) layers. Somewhat The long secondary processes of TfBP-positive cells run in the same
diffuse staining is also found in the outer portion of internal nuclear direction as the optic nerve fibers. Scale bars 5 20 µm.

Fig. 6. Longitudinal sections of chicken optic and sciatic nerves cells of the sciatic nerve (B). Note reaction product confined to somata
stained with TfBP antibody. TfBP reaction product is associated with and staining intensity of Schwann cells (arrows) comparable to that of
interfascicular oligodendrocytes of the optic nerve (A) and Schwann oligodendrocytes. Scale bars 5 20 µm.

transferrin is transiently expressed in the neurons of 1975; Rager, 1976; Inoue et al., 1980); the identity of
various species including chicken and rats (Oh et al., 1986; myelin-forming cells has, however, been uncertain. In a
Connor and Fine, 1987; Dion et al., 1988). In the brain of histologic study of the pigeon retina, Hughes et al. (1972)
adult animals, however, the pattern is typically one in found that glial cells accounted for a considerable portion
which transferrin immunoreactivity in neurons is either (more than 15%) of the total cell numbers in the ganglion
absent or quite weak, whereas transferrin is positive in cell layer. On the basis of electron microscopic studies of
oligodendrocytes, choroid plexus, and endothelial cells the chick and pigeon, Smith (1982) reported that the
(Connor et al., 1990; Connor and Menzies, 1995; Morris et presence of myelinating gliocytes exhibited the ultrastruc-
al., 1992a). tural characteristics of CNS oligodendrocytes in the eighth
The presence of myelin in the avian retina has been and ninth layers of the retina. In the rabbit and human,
documented by many investigators (Hughes and LaVelle, the presence of retinal myelin is always associated with
268 S.S. CHO ET AL.

Fig. 7. Immunoelectron micrographs from chicken cerebellar white chromatin masses at the periphery and sometimes in the center of the
matter showing subtypes of TfBP-labeled oligodendrocytes and intra- nucleus. C: Higher magnification of the TfBP-oligodendrocytic cyto-
cytoplasmic localization of TfBP. A: Light oligodendrocytes. Note the plasm. Immunoreactivity products are present in the cytoplasmic
large, pale nucleus and scanty perinuclear cytoplasm filled with the matrix of the perikaryon and fine processes (arrows) as well as bound
immunoreactivity products. The Golgi apparatus (g) and mitochondria to the endoplasmic reticulum (er). Myelin and axoplasm are devoid of
(m) are devoid of reaction products. B: Medium oligodendrocytes. reaction product. Scale bars 5 2 µm.
Compared to the light type (A), this cell is characterized by distinct
TfBP IMMUNOREACTIVITY IN CHICKEN NERVOUS SYSTEM 269

the existence of oligodendrocytes in the retina (Schnitzer, al., 1990; Connor and Menzies, 1995; Moos, 1996). On the
1985; Jeon and Masland, 1993). However, immunohisto- other hand, ferritin and iron are found predominantly in
chemical efforts using murine oligodendrocytic markers to oligodendrocytes of the gray and white matter (Connor and
identify retinal oligodendrocytes in birds have been unsuc- Menzies, 1995). The implication from data of the transfer-
cessful (Linser and Moscona, 1981; Kohsaka et al., 1980, rin receptor and iron binding proteins is that although iron
1983). In the present study, we have demonstrated for the uptake and utilization in neurons are high, there is little
first time the presence of TfBP-immunoreactive cells that storage of iron. Thus, storage of iron in a replaceable glial
have the appearance of CNS oligodendrocytes and are pool may be necessary for the removal of potentially toxic
present in the eighth and ninth layers of the chick retina. iron from neurons vulnerable to free radical damage.
The localization and distribution of TfBP-labeled cells Common features shared by Bergmann glia and perineu-
observed in this study parallel the presence of the retinal ronal oligodendrocytes identified in the present and previ-
myelin, which reportedly exists mostly in the ninth layer ous studies are that they are satellites around neuronal
and partially in the eighth layer throughout the whole cell bodies and that they contain iron and iron regulatory
avian retina (Smith, 1982; Yamada, 1989). Astrocytic proteins. The anatomical and immunocytochemical find-
labeling is unlikely because the avascular chick retina is ings support the notion that glial cells play a role in the
not thought to contain astrocytes (Stone and Dreher, nutrition and maintenance of neurons (Hyden, 1962, 1967).
1987). The distribution and morphology of TfBP-immuno- Given this notion, special enrichment of transferrin in
reactive cells observed in this study are most characteris- iron-containing glia may be a necessary factor for iron
tic of CNS oligodendrocytes. This strongly suggests that mobilization from ferritin found in these glia (Griot and
TfBP-positive cells are retinal oligodendrocytes respon- Vandevelde, 1988; Connor and Benkovic, 1992; Morris et
sible for myelin formation in the chick retina. al., 1992b). There is a need for further study of the
The Bergmann glia have been found to play an impor- molecular mechanism by which intracellular iron is mobi-
tant role in cytoarchitectural development by providing lized within the CNS.
guidance for neurons migrating through the molecular The mechanism by which these cells accumulate trans-
layer (Sidman and Rakic, 1973). However, the functional ferrin is not known, although the presence of transferrin
role of these cells in the adult has not been established. mRNA has been shown in both oligodendrocytes and
The finding of Bergmann glia labeled with TfBP antibody choroid plexus cells of the mammalian brain (Bloch et al.,
is not surprising in the chick. Unlike the rat, in which 1985, 1987; Aldred et al., 1987; Tu et al., 1991). The
Bergmann glia express the well-characterized astrocytic overlapping distribution patterns of TfBP and transferrin
marker, glial fibrillary acid protein (GFAP), the chick does in these cell types suggest that TfBP may be linked to the
not exhibit GFAP immunoreactivity in Bergmann glia accumulation of transferrin. Ultrastructurally, the TfBP
(Bignami and Dahl, 1973, 1974; Dahl and Bignami, 1973). immunoreaction product is dispersed throughout the peri-
Such variation between species in the distribution of glial nuclear cytoplasm and thin processes, as are transferrin
proteins is not unprecedented; carbonic anhydrase II (CA (Connor and Fine, 1986) and CAII reaction products
II), the oligodendrocytic marker, is present in Bergmann (Cammer, 1984). The coincidence of TfBP and transferrin
glia in the chick (Rogers and Hunt, 1987) but not in the rat in their intracellular locale is an intriguing morphologic
(Ghandour et al., 1979, 1980; Kumpulainen and Nystrom, correlate of our previous report that TfBP has transferrin-
1981). In a recent study using a rabbit model of superficial binding activity (Poola and Lucas, 1988; Hayes et al.,
siderosis (Koeppen and Borke, 1991; Koeppen et al., 1995), 1994).
the presence of transferrin, iron, and ferritin was demon- At high magnification, the immunoreaction product
strated in Bergmann glia of the normal and siderotic appears granular; it is found in the cytoplasmic matrix and
cerebellum, suggesting a role for these radial glia in the bound to endoplasmic reticulum and outer surface of the
transport and storage of iron. nuclear membrane. Thus, TfBP may have properties of
The reason for the selective accumulation of transferrin both a soluble and integral membrane protein. Although
in these brain cells has not been well established, although the mammalian homologue of TfBP, GRP94, is generally
it has been suggested that transferrin accumulation in stated to be a lumenal rough endoplasmic reticulum
myelinating oligodendrocytes and Schwann cells is a neces- protein, there is substantial evidence in the literature that
sary condition for myelination (Lin et al., 1990). Because of a portion, including the plasma membrane, is membrane
the high oxidative metabolic demands of lipid metabolism bound (Maki et al., 1990; Kang and Welch, 1991; Takemoto
and synthesis (Bourre et al., 1984), myelinating oligoden- et al., 1992). Furthermore, there is ample evidence for
drocytes and Schwann cells are likely to require high multiple isoforms of each of the proteins in this closely
levels of iron. Metabolic activity associated with iron, related family (Fuernkranz et al., 1991; Kang and Welch,
however, is not restricted to the myelinating cells. Further- 1991; Poola and Kiang, 1994; Feldweg and Srivastava,
more, the presence of transferrin in gray matter, perineu- 1995).
ronal oligodendrocytes, and Bergmann glia suggests a role Given the fact that TfBP is a post-translationally modi-
of transferrin other than as support for myelin formation. fied form of HSP108, it is conceivable that the presence of
It has been suggested that although transferrin in the TfBP may help to protect these cells against potential
white matter oligodendrocytes could be primarily involved damage. For example, myelinating cells are under extreme
with maintaining myelin, perineuronal oligodendrocytes metabolic stress, and minor metabolic insults during my-
might be involved in mobilizing iron for neurons in the elin synthesis can induce irreversible damage (Selmaj et
brain (Connor and Fine, 1986). The transferrin receptor, a al., 1992). Furthermore, iron in these cells can lead to
transmembrane glycoprotein responsible for the binding oxidative stress through the formation of oxygen free
and internalizing of iron-transferrin into cells, has been radicals (Gerlach et al., 1994). At present there is little
reportedly expressed by neurons of all areas of the chicken evidence to suggest a clear relationship between TfBP and
and mammalian CNS (Markelonis et al., 1985; Connor et stress. However, stressed oligodendrocytes in multiple
270 S.S. CHO ET AL.

sclerosis lesions have been shown to express HSP65 (Sel- Clark, G. (1984) Staining Procedures. 4th ed. Baltimore: Williams &
maj et al., 1992). Wilkins, p. 12.
There is now increasing evidence that HSPs have a key Connor, J.R., and R.E. Fine (1986) The distribution of transferrin-
immunoreactivity in the rat nervous system. Brain Res. 368:319–328.
role in maintaining the health of cells in the nervous
Connor, J.R., and R.E. Fine (1987) Development of transferrin-positive
system (Marcuccilli and Miller, 1994). For example, the oligodendrocytes in the rat CNS. J. Neurosci. Res. 17:51–59.
HSP70 family expressed in both the constitutive and Connor, J.R., and S.A. Benkovic (1992) Iron regulation in the brain:
stress-inducible form in the glial cells and neurons (Mas- Histochemical, biochemical, and molecular considerations. Ann. Neu-
ing and Brown, 1989; Marini et al., 1990; Welch, 1992) can rol. 32:S51–61.
prevent protein denaturation or assist in refolding dam- Connor, J.R., and S.L. Menzies (1995) Cellular management of iron in the
aged proteins following stress (Masing and Brown, 1989; brain. J. Neurol. Sci. Suppl. 134:33–44.
Marini et al., 1990). Glia-axon transfer of HSP70 has been Connor, J.R., S.L. Menzies, S.M. St. Martin, and E.J. Mufson (1990)
observed in the giant squid axon in response to heat shock Cellular distribution of transferrin, ferritin, and iron in normal and
aged human brains. J. Neurosci. Res. 27:595–611.
(Tytell et al., 1986), thus providing a model for glia-to-
neuron transfer of HSPs. Furthermore, in some HSPs, the Dahl, D., and A. Bignami (1973) Immunochemical and immunofluorescence
studies of the GFAP in vertebrates. Brain Res. 61:279–293.
degree of their constitutive expression appears relatively
Dion, T.L., G.J. Markelonis, T.H. Oh, B.S. Bregmann, M.A. Pugh, S.L.
dependent on the metabolic activities of the cells (Marcuc- Hobbs, and Y.C. Kim (1988) Immunocytochemical localization of trans-
cilli and Miller, 1994). We have also previously localized ferrin and mitochondrial malate dehydrogenase in the developing
TfBP specific to light and medium types of oligodendro- nervous system of the rat. Dev. Neurosci. 10:152–164.
cytes reportedly involved in the production and mainte- Feldweg, A.M., and P.K. Srivastava (1995) Molecular heterogeneity of
nance of myelin (Sturrock, 1974). tumor rejection antigen/heat shock protein GP96. Int. J. Cancer
In addition to its constitutive expression, HSP108 is 63:310–314.
regulated by heat shock (Sargan et al., 1986) and also by Feurnkranz, H.A., J.E. Schwob, and J.J. Lucas (1991) Differential tissue
localization of oviduct and erythroid transferrin receptors. Proc. Natl.
steroid hormones (Baez et al., 1987; Poola and Lucas, Acad. Sci. USA 88:7505–7508.
1988). This suggests that it may have more than one Gerhardt, E.M., L.N.L. Chan, S. Jing, M. Qi, and I.S. Trowbridge (1991) The
function, whose expression is dependent on the specific DNA sequence and primary structure of the chicken transferrin recep-
modulator present in the cell. It is possible that like other tor. Gene 102:249–254.
HSPs, HSP108 is involved in protein synthesis, serving as Gerlach, M., D. Ben-Shachar, P. Riederer, and M.B.H. Youdim (1994)
a chaperone. On the other hand, the correlation with iron Altered brain metabolism of iron as a cause of neurodegenerative
levels and transferrin in neural tissue suggests a role in diseases? J. Neurochem. 63:793–807.
iron metabolism. But HSP108/TfBP could be functioning Ghandour, M.S., O.K. Langley, G. Vincendon, and G. Gombos (1979) Double
labeling immunohistochemical technique provides evidence of the
as a stress-induced protein to minimize oxidative stress.
specificity of glial cell markers. J. Histochem. Cytochem. 27:1634–1637.
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Ghandour, M.S., O.K. Langley, G. Vincendon, and G. Gombos (1980)
requires further investigation. Immunochemical and immunohistochemical study of carbonic anhy-
drase II in adult rat cerebellum: A marker for oligodendrocytes.
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