From the Departments of Molecular Animal Physiology
and ¶ Experimental Morphology, Institute of Zoology, and the
Institute of Molecular Genetics, Johannes Gutenberg University
of Mainz, D-55099 Mainz, Germany
Received for publication, September 26, 2002, and in revised form, October 28, 2002
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ABSTRACT |
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Visual performance of the vertebrate eye requires
large amounts of oxygen, and thus the retina is one of the highest
oxygen-consuming tissues of the body. Here we show that neuroglobin, a
neuron-specific respiratory protein distantly related to hemoglobin and
myoglobin, is present at high amounts in the mouse retina (~100
µM). The estimated concentration of neuroglobin in
the retina is thus about 100-fold higher than in the brain and is in
the same range as that of myoglobin in the muscle. Neuroglobin is
expressed in all neurons of the retina but not in the retinal pigment
epithelium. Neuroglobin mRNA was detected in the perikarya of the
nuclear and ganglion layers of the neuronal retina, whereas the protein was present mainly in the plexiform layers and in the ellipsoid region
of photoreceptor inner segment. The distribution of neuroglobin correlates with the subcellular localization of mitochondria and with
the relative oxygen demands, as the plexiform layers and the inner
segment consume most of the retinal oxygen. These findings suggest that
neuroglobin supplies oxygen to the retina, similar to myoglobin in the
myocardium and the skeletal muscle.
Neuroglobin (Ngb)1 has been
identified as an intracellular respiratory protein expressed mainly in
the brain (1) but also in the peripheral nervous system and endocrine
tissues (2). Ngb displays only limited sequence similarities with
vertebrate hemoglobin and myoglobin (less than 25% amino acid sequence
identity) but resembles the nerve-specific globins known from some
invertebrates, e.g. that of the annelid Aphrodite
aculeata (3). Ngb binds oxygen reversibly via an iron
(Fe2+) ion of the heme group (1, 4, 5). The oxygen affinity (P50) of Ngb was determined to be in the range
of a typical vertebrate myoglobin (~1-2 torr (1, 4)), although the
hexacoordinate structure of the Fe2+ in deoxy-Ngb renders
the oxygen-binding kinetics more complex than in other globins (4-6).
Ngb was suggested to play an important role in oxygen homeostasis of
neuronal tissues (1, 7). In fact, Ngb expression is unregulated under
hypoxic conditions in vitro, and the presence of Ngb
enhances the survival of cultured neuronal cells at low oxygen
levels (8).
The visual process in the vertebrate eye requires large amounts of
metabolic energy, which is provided mainly as ATP that derives from
glycolysis and oxidative phosphorylation (9, 10). High ATP amounts are
consumed, for example, for the functioning of photoreceptor cells
(e.g. for the maintenance of the dark current, for the fast
turnover of photosensitive sensitive membranes, or the
phototransduction process itself (11)) and the transmission at the
synapses of all neurons in the retina. Thus, it is not surprising that
the relative oxygen consumption of the mammalian retina is higher than
that of the brain and other tissues (12, 13). It is assumed that, on a
per gram basis, the retina is one of the highest oxygen demanding
tissues of the human body (10, 14). Lack of oxygen (hypoxia) has
immediate and severe effects to the visual performance of man and other
vertebrates (15, 16).
Oxygen is supplied to the retina by the hemoglobin of the blood. The
degree of vascularization of the retina differs among mammals (10). In
mouse and most other species, the inner parts are nourished by the
central retinal vessels, whereas the outer parts are supplied via the
pigment epithelium by capillaries of the choroidal circulation. Blood
capillaries do not reach the retinal layers of photoreceptor cells
directly. Therefore, oxygen delivered by the blood has to diffuse over
large distances to reach e.g. the photoreceptor
compartments. Nevertheless, the presence of an intracellular
respiratory protein, which could enhance the uptake of oxygen by the
vertebrate retina, has been hitherto unknown. Here we show that Ngb is
highly expressed in the murine retina and that the intra-retinal
distribution of Ngb correlates with the relative oxygen consumption.
Animals--
The procedures concerning animals complied with
German legislation for the protection of animals and were approved by
the county government office (Bezirksregierung Rheinhessen-Pfalz). Balb/C and C57BL/6J mice were maintained under constant conditions (light:dark 12:12 h, room temperature 21 ± 1 °C) with food and water ad libitum. Bovine eyes were obtained from the local
slaughterhouse. Bovine photoreceptor outer segments were purified from
dark-adapted isolated bovine retinas as described previously (17).
Antibody Preparation--
Two different polyclonal antibodies
against neuroglobin were produced using a commercial service
(Eurogentec). The first antibody was raised in chicken against
recombinant mouse Ngb. The IgY fraction was purified from the yolk by
the aid of the EGGstract kit (Promega). Specific anti-Ngb antibodies
were affinity-purified using recombinant mouse Ngb coupled to a
SulfoLink column (Pierce) according to the instructions of the
manufacturer and stored in 50 mM Tris, 100 mM
glycine, pH ~7.4) supplemented with 0.1% NaN3. The
second antibody was raised in rabbits against a synthetic Ngb peptide, which covers the conserved amino acid positions 55-70 of mouse and
human Ngb (H2N-CLSSPEFLDHIRKVML-CONH2).
Affinity purification using the synthetic peptide coupled to a
SulfoLink column was carried out as described above.
Gel Electrophoresis and Western Blotting--
Tissue samples
were homogenized in 1% SDS, 5% Immunostaining--
Immunofluorescence studies were essentially
performed as previously described (17). Briefly, eyes from adult mice
were prefixed in 4% paraformaldehyde in PBS for 1 h at room
temperature, washed, soaked with 30% sucrose in PBS overnight, and
cryofixed in melting iso-pentane. Cryosections were placed on
poly-L-lysine-precoated coverslips (18). Specimens were
incubated with 50 mM NH4Cl and 0.1% Tween 20 in PBS and blocked with blocking solution (0.5% cold-water fish
gelatin (Sigma) plus 0.1% ovalbumin (Sigma) in PBS). The sections were
incubated with anti-Ngb antibodies or, in the case of double labeling,
with a mixture of anti-Ngb and monoclonal anti-arrestin antibodies
(clone 3D1.2, described previously in Ref. 19) in blocking solution
overnight at 4 °C. The specimens were washed and subsequently
incubated with secondary antibodies conjugated to Alexa® 488 or
Alexa® 546 (Molecular Probes) in blocking solution for 1 h at
room temperature in the dark. On some sections, Instead of Alexa®
probes, secondary anti-rabbit antibodies coupled with horseradish
peroxidase (Sigma) were placed and incubated in the dark for 1 h.
Washed sections were mounted in Mowiol 4.88 (Hoechst, Frankfurt,
Germany), containing 2% n-propyl gallate and, in the case
of triple staining, 1 µg/ml 4',6-diamidino-2-phenylindole. Mounted
retinal sections were examined with a Leica DMRP microscope. Images were obtained with a Hamamatsu Orca ER CCD camera (Hamamatsu, Japan) and processed with Adobe Photoshop (Adobe Systems).
In Situ Hybridization--
Digoxigenin-labeled antisense RNA
probes were created by in vitro transcription with T7 RNA
polymerase (Roche Molecular Biochemicals), using PCR-generated
templates covering the 453-base pair mouse Ngb coding region
(EMBL/GenBankTM accession no. AJ245945). A T7 RNA
polymerase promoter sequence was attached to the 5' end of the sense or
antisense PCR primers. In situ hybridization was as
described previously (2). Briefly, the RNA probes were diluted in 2×
SSC, 50% formamide and incubated on the sections at 42 °C over
night. Slides were washed in 2× SSC and subsequently in 0.1× SSC at
55 °C. Tissue sections were then treated with a mixture of RNase A
(25 µg/ml) and RNase T1 (25 units/ml) for 30 min at 37 °C in a wet
chamber and then washed in PBS/Tween 20. After blocking for 15 min in
Roche buffer 2, label was detected by alkaline phosphatase-coupled
anti-digoxigenin antibodies (diluted 1:100 in Roche buffer 2; a 30-min
incubation at room temperature), washing in PBS/Tween 20 and Roche
buffer 3, and subsequent incubation with nitro blue
tetrazolium/5-bromo-4-chloro-3-indolyl phosphate (1:50 diluted in Roche
buffer 3; 15-30 min at room temperature in the dark). The reaction was
stopped by washing in PBS. The sections were covered by PBS/glycerol
(1:1) and processed as described above.
High Ngb Expression in the Mammalian Retina--
Specific anti-Ngb
antibodies were raised in chicken (1) and rabbit and purified by
affinity chromatography. Both types of antibodies detect Ngb in the
protein extracts from the murine brain and the retina at ~17 kDa
(Fig. 1A). The second band of the
double molecular mass (about 34 kDa) likely results from the dimerization of Ngb, as frequently observed for many globins in SDS-PAGE. After preadsorption of the antibodies with the antigen, both
bands disappeared in Western blots of retinal and brain tissue (data
not shown), indicating the specificity of anti-Ngb antibodies. The
apparent molecular mass of native Ngb in brain and retina is ~17 kDa
and thus is about 1 kDa larger than that of the recombinant protein.
The reason for this discrepancy is unknown, although posttranslational
modification may be assumed. Further studies are required to address
this question. No Ngb was detected in protein samples from murine
liver, blood, or skeletal muscle. Ngb is also absent in the fraction of
purified outer segments of bovine photoreceptor cells, whereas the
antibody detects the protein in the retina of this species (Fig.
1B).
The intensity of the Western blot signals was quantified after scanning
with the Scion Image program. The amount of Ngb in the retinal samples
was estimated to be ~50-100-fold higher than in the total brain
extracts. From the comparison with the known amounts of total protein
and recombinant Ngb applied to the lanes, we estimate that Ngb makes up
~2-4% of the total retinal protein. The total concentration of
soluble proteins in the retina was determined to be about 100 mg/ml
(data not shown). Thus, the concentration of Ngb in the total retina is
in the range of 100 to 200 µM.
Localization of Ngb in the Mouse Retina--
The mammalian retina
is composed of distinct layers that are easily distinguishable
in retinal sections by light microscopy. To examine sites of mRNA
expression of Ngb in the mammalian retina, cryosections
through mouse retinae and eyes were analyzed by in situ
hybridization using an in vitro transcribed antisense probe. The specificity and selectivity of this method were demonstrated previously (2). Strong hybridization signals were observed in the inner
segments of the photoreceptor cells, in the outer and inner nuclear
layer, and in the ganglion cell layer of the neuronal retina (Fig.
2A). The perinuclear signal in the
neuronal retina is typical for mRNA hybridization in neurons. Less
intense signals were found in the plexiform layers, whereas in the
layer of photoreceptor outer segments and in the cells of the pigment epithelium, no labeling was detected.
To determine the distribution of the Ngb protein, immunostaining
experiments were carried out with rabbit antibody raised against the
synthetic peptide. Both indirect immunofluorescence and indirect
immunoperoxidase staining methods were applied and provided identical
staining patterns in both fixed and unfixed samples (data not shown).
No staining was observed in the appropriate control experiments. As
shown in Fig. 2B, no anti-Ngb signal was detectable in the cells of the retinal pigment epithelium, whereas prominent indirect anti-Ngb immunofluorescence was visible in the
photoreceptor layer, in the outer and inner plexiform layer, and at the
ganglion cells. In both nuclear layers a less intense, but still
detectable, fluorescence was present in the perinuclear cytoplasm of
the perikarya. In addition, bright anti-Ngb-labeled foci were regularly
detected in the outer nuclear layer, and these may represent stained
domains in the Müller glia cells that project through the outer
nuclear layer to the "external limiting membrane." The localization
of Ngb in the retina does not depend on light; a comparison of
anti-Ngb-labeled dark-adapted (Fig.
3C) with light-adapted (Fig.
2B) mouse retinae did not show any detectable
differences in the staining patterns of Ngb protein.
Localization of Ngb in the Photoreceptor Cell--
Vertebrate
photoreceptor cells are highly polarized neurons with defined
compartments; their outer segments, inner segments, perikarya,
and synapses are organized into distinct layers in retinal sections
(Fig. 3A). This feature makes it relatively simple to
determine the subcellular localization of a polypeptide in retinal
cryosections stained by indirect immunofluorescence. To analyze the
subcellular localization of Ngb in photoreceptor cells, we performed
double immunofluorescence labeling experiments with anti-Ngb and
anti-arrestin antibodies in cryosections through dark-adapted mouse
retinae. As in other G protein-coupled receptor-mediated signal
transduction mechanisms, arrestin is a major component in the
desensitization of the receptor (rhodopsin) and in the process
of molecular adaptation (20) It has been reported previously that
arrestin shows a light-dependent movement from the inner to
the outer segments. Under dark conditions, it is localized in the
entire inner segment and also is distributed throughout the perinuclear
cytoplasm down to the synapses (21, 22) (Fig. 3D). High
magnification analysis of the photoreceptor compartments in these
retinal cryosections revealed that Ngb is localized at the synapses and
in the ellipsoid of the inner segments (Fig. 3C). Although
anti-arrestin weakly stained photoreceptor synapse region, anti-Ngb
immunofluorescence was found to be more intense in foci representing
the presynaptic terminals of photoreceptors but was also present in the
non-photoreceptor domains of the outer plexiform layer (extensions of
bipolar, horizontal, and amacrine cells). In the inner segment of the
photoreceptor cells, in contrast to anti-arrestin, the anti-Ngb
antibody did not stain the entire inner segment but prominently labeled
the ellipsoid region of the apical inner segment. In photoreceptor
outer segments, no Ngb labeling was detectable, which was confirmed
with the Western blot analysis of retina fractions (Fig.
1B).
Because of the high demand for oxygen in the visual process, an
adequate oxygen environment is crucial for the function of the retinal
cells (10). Here we show that Ngb, an oxygen-binding respiratory
protein (1), is synthesized at high levels in the mouse retina. Ngb has
been identified previously mainly in the brain, where it may occur at a
concentration in the range of ~1 µM (1). Such a low
amount was difficult to reconcile with the important role of Ngb in
oxygen supply. For example, myoglobin typically occurs in the cells of
the striated muscle and the myocardium at concentrations of several
hundred µM (23-25). However, the estimated concentration
of Ngb in the mouse retina is >100 µM and thus is within
the range of myoglobin in the muscle (Fig. 1), indicating the important
role of Ngb in retinal function. Our results are in agreement with the
observations by Wittenberg and Wittenberg (26), who identified a heme
protein with an absorption spectrum similar to Ngb that occurs at
apparently high concentrations in the fish eye. Ngb is also present in
fish, although its localization remains to be determined (27).
Ngb Expression Correlates with the Sites of Oxygen
Consumption--
Recent studies in rat have shown that oxygen
consumption by the retina takes place mainly in the inner segments, the
outer plexiform layer, and the inner plexiform layer (10, 28, 29). This
is not surprising, because the energy demand is particularly high in
both types of mammalian photoreceptor cells and the synaptic contact
regions, which are located mainly in the plexiform layers (10, 30). As
demonstrated by immunofluorescence, most of the Ngb protein is present
in these three layers of high oxygen demand (Fig. 3). Subcellular
localization within the photoreceptor cells shows Ngb to be mainly in
the ellipsoid region of the apical inner segment, where mitochondria
are known to be concentrated (9, 31). The same is true for the synaptic
regions of the photoreceptors and the plexiform layers (32), which also
showed strong Ngb signals in our immunocytochemical experiments. These
observations further support the assumption that Ngb is involved in
retinal oxygen consumption.
Intracellular Ngb Transport--
The mammalian retina is a highly
specialized structure that is divided into morphological and functional
distinct layers (Fig. 3A). Immunostaining and in
situ hybridization experiments reveal striking differences in
intracellular distribution of Ngb protein and mRNA within the
retinal layers (Figs. 2 and 3). Whereas the mRNA is present mainly
in the large cell bodies that make up the nuclear and ganglion layers,
as well as in the inner segments of the photoreceptors, the
protein is restricted almost exclusively to the plexiform layers and
the inner segments of the photoreceptors. This redistribution suggests
a massive transport of Ngb protein after translation, at least in the
plexiform layers.
Ngb Is the Respiratory Protein of the Mammalian
Retina--
Although the exact mechanism of myoglobin function is
still a matter of debate, its important role in intracellular oxygen supply is well established (33-35). The observed intracellular oxygen
partial pressures in the retina and muscle are within the same range
(10, 28, 33, 36). In addition, both the intracellular concentration and
the oxygen binding affinities of Ngb and myoglobin are comparable (1,
4). Thus, a similar physiological function in intracellular oxygen
supply can be expected. Therefore, it can be assumed that Ngb either
facilitates the transport of oxygen to the respiratory chain of the
mitochondria or may provide a short-term storage of oxygen that is
required for peaks of retinal activity.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-mercaptoethanol, 10% glycerol, 65 mM Tris, pH 6.8. The samples were heated at 95 °C for 5 min and loaded to a 14% SDS-polyacrylamide gel. Proteins were stained
with Coomassie Brilliant Blue R-250. For Western blot detection, the
heat-denatured protein samples were transferred to nitrocellulose for
2 h at 0.8 mA/cm2. The membranes were blocked for
several hours with TBST (0.3% Tween 20 in 10 mM Tris-HCl,
pH 7.4, 140 mM NaCl) and incubated for 1-2 h with anti-Ngb
antibodies diluted (1:1000 to 1:2000). The membranes were washed four
times for 10 min in TBST and incubated with the appropriate secondary
antibodies (goat anti-rabbit or goat anti-chicken; Promega) coupled
with alkaline phosphatase. The filters were washed in TBST as described
above, and detection was carried out with nitro blue tetrazolium
and 5-bromo-4-chloro-3-indolyl phosphate as substrates. Signals were
quantified after scanning of the blots with the help of the Scion Image
program (version Beta4.02).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (15K):
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Fig. 1.
Western blot analysis of Ngb protein tissue
expression. A, ~30 µg of total protein from
selected mouse tissue extracts were applied per lane. Ngb was detected
in the brain and the retina with an antibody raised against a synthetic
Ngb peptide. 2 µg of recombinant Ngb were applied as a positive
control (lane "Ngb"). B, proteins from the
total bovine retina and isolated outer segments (OS). Ngb is
not present in the outer segment fraction, whereas the antibody stains
Ngb in the bovine retina. On the left, the positions of the
molecular mass standards are indicated.
View larger version (121K):
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Fig. 2.
Localization of Ngb mRNA and protein in
the mouse retina. A, in situ
hybridization of a longitudinal cryosection through the mouse retina.
The mammalian retina is composed of well defined cellular layers:
PL, layer of outer and inner segments of photoreceptor
cells; ONL, outer nuclear layer; OPL, outer
plexiform layer; INL, inner nuclear layer; IPL,
inner plexiform layer; GC, layer of ganglion cells. The
asterisk denotes the retinal pigment epithelium. The Ngb
in situ hybridization signal (dark staining) is
found mainly in the inner segments of the photoreceptor cells, the
nuclear layers (ONL, INL) and the ganglion cells
(GC). B, indirect anti-Ngb immunofluorescence.
Bright indirect immunofluorescence anti-Ngb is visible in the inner
segments of photoreceptors (PL), the plexiform layers
(OPL, IPL), and the ganglion cells
(GC) of the mouse retina but not in the pigment epithelium
(asterisk). Bar: 10 µm. The arrow at
the bottom indicates the direction of the incoming light.
View larger version (56K):
[in a new window]
Fig. 3.
Subcellular localization of Ngb in the mouse
photoreceptor cells. A, schematic representation of a
rod photoreceptor cell. Vertebrate photoreceptors are divided into
distinct compartments: the photosensitive outer segment
(OS), which is the place of visual transduction; the inner
segment (IS), which contains the biosynthetic machinery of
the cell, including numerous mitochondria in the ellipsoid region; the
perikaryon or cell body (P), which is localized in the outer
nuclear layer (ONL in panel B) of the retina and
contains the nucleus; and the synaptic terminal (S) in the
outer plexiform layer (OPL in panel B), which
electrically connects the cell to downstream neurons of the retina.
B, differential interference contrast image of a
longitudinal cryosection through dark-adapted mouse retina.
C, indirect anti-Ngb immunofluorescence in the section shown
in B. Labeling is found predominantly in the inner segment
of photoreceptor cells and the outer plexiform layer. D,
indirect anti-arrestin immunofluorescence is present in the entire
inner segment of the plexiform layer, in the outer nuclear layer, and
in the synaptic terminal (S) of dark-adapted photoreceptor
cells. E, merged images of panels C and
D show partial localization of Ngb and arrestin in the
ellipsoid region of the apical inner segment. The nuclei in the outer
nuclear layer are blue-stained by 4',6-diamidino-2-phenylindole.
Bar: 10 µm. The arrow at the bottom indicates
the incoming light.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
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We thank Stefan Reuss for help with microphotography, Katja Lotz for excellent technical assistance, and Jürgen Markl and Erwin R. Schmidt for excellent working facilities. We also thank Laura S. Lang providing us anti-arrestin antibodies.
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FOOTNOTES |
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* This work was supported by grants from the Deutsche Forschungsgemeinschaft (Ha2103/3, Bu956/5, and Wo548/3) and the FAUN-Stiftung, Nürnberg (to U. W.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ These authors contributed equally to this work.
** To whom correspondence should be addressed: Institute of Zoology, University of Mainz, D-55099 Mainz, Germany. Tel.: 49-6131-39-24477; Fax: 49-6131-39-24652; E-mail: burmeste@mail.uni-mainz.de.
Published, JBC Papers in Press, October 29, 2002, DOI 10.1074/jbc.M209909200
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ABBREVIATIONS |
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The abbreviations used are: Ngb, neuroglobin; PBS, phosphate-buffered saline.
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