(Investigative Ophthalmology and Visual Science. 2001;42:1907-1914.)
© 2001
by The Association for Research in Vision and Ophthalmology, Inc.
Distribution of Transcript and Protein Isoforms of the Synaptic Glycoprotein Neuroplastin in Rat Retina
Michael R. Kreutz1,
Kristina Langnaese1,2,
Daniela C. Dieterich1,
Constanze I. Seidenbecher1,
Werner Zuschratter1,
Philip W. Beesley3 and
Eckart D. Gundelfinger1
1 From the AG Molecular Mechanisms of Plasticity, Department of Neurochemistry/Molecular Biology, Leibniz Institute for Neurobiology, Magdeburg, Germany; and the
3 School of Biological Sciences, Division of Biochemistry, Royal Holloway University of London, Egham, United Kingdom.
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Abstract
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PURPOSE. To examine the expression and localization of the neuroplastins (np),
two synapse-enriched members of the immunoglobulin (Ig) superfamily of
cell-adhesion molecules, in the developing and adult retina and optic
nerve.
METHODS. Expressions of the two isoforms np55 and np65 and carboxyl-terminal
splice variants were investigated by immunocytochemistry, Western blot
analysis, RT-PCR, and in situ hybridization.
RESULTS. Immunoreactivity for both neuroplastins was confined to the two
synaptic layers of the retina: the inner (IPL) and outer plexiform
layer (OPL). Significant overlap was found in staining at synaptic
structures with synaptophysin. A large proportion of immunoreactivity
for both isoforms, however, was of perisynaptic origin. In situ
hybridization studies were suggestive of a pre- and postsynaptic
localization of np65 in the OPL. Transcripts for np55 were already
present at birth in the inner retina, but the hybridization signals
increased during postnatal development. Np65 transcripts and
immunosignals appeared at later developmental ages, concomitant with
synapse formation in the OPL. Several C-terminal neuroplastin cDNA
clones harbor an insert of 12 bp, coding for four amino acids (DDEP) in
the intracellular domain of neuroplastins. Splice isoforms containing
the insert exhibited a developmental expression pattern similar to that
of np55; however, both neuroplastins could harbor the C-terminal
insert. Neuroplastins were also detected in optic nerve homogenates.
RT-PCR and blockade of axonal transport by nerve crush confirmed
transcript and protein expression in optic nerve tissue.
CONCLUSIONS. The findings suggest a role for neuroplastins in cell adhesion in the
plexiform layers during histogenesis, as well as in maintenance of
connections between specific cellular
structures.
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Introduction
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Cell adhesion is a fundamental prerequisite for the
genesis, maintenance, and plasticity of synaptic contacts throughout
the nervous system. These processes are mediated by a variety of neural
cell adhesion molecules (CAMs), which are expressed in a highly
segregated spatial and temporal manner. Np65 and np55 are isoforms of
neuroplastin, a glycoprotein of the synaptic membrane protein fraction
of rat brain with apparent molecular masses of 65 and 55 kDa,
respectively.1
2
Deglycosylation studies have
revealed that the protein cores of np55 and np65 have molecular masses
of 28 and 40 kDa, respectively.1
They were originally
identified as gp65 and gp55 and biochemically characterized using the
monoclonal antibody (mAb) Smgp65.1
3
Both molecules belong
to the immunoglobulin (Ig) superfamily of CAMs and behave as integral
membrane proteins. Np65 contains three Ig domains and np55
two.2
They arise by alternative splicing from the same
primary transcript and differ by one N-terminal Ig domain, which
contains no N-linked glycosylation site.2
Fractionation of
brain protein homogenates has shown that np65 is enriched in the
postsynaptic density fraction of synaptic junctional proteins, whereas
np55 is virtually absent from this fraction.1
3
Although
not exclusively localized at the synapse, it is likely that np65 is
involved in adhesion of synaptic membranes.
Whereas np65 is primarily expressed in the brain, np55 is widely
distributed in many tissues and occurs in multiple
glycoforms.2
4
In situ hybridization experiments also
reveal a more restricted expression of np65 in rat brain than of
np55.2
The functions of the neuroplastins are largely
unknown. Members of several CAM families have been localized to
synaptic junctions and are thought to mediate synapse formation and
stabilization during development and to be required for
activity-dependent long-term restructuring of synapses.5
Our recent evidence suggests that np65 plays a role in cell adhesion
mechanisms involved in long-term, activity-dependent synaptic
plasticity.6
A detailed analysis of the cellular and
subcellular localization of neuroplastins in the central nervous
system, however, is still needed.
The retina is particularly well suited for this type of study, because
its cell types and synaptic connections are organized into different
layers and its cytoarchitecture is well understood.7
Moreover, in the rat retina nerve cell maturation and interneuronal
connectivity occur postnatally, which makes this model particularly
useful for developmental studies.8
9
Therefore, we
analyzed the developmental and adult localization of np65 and np55
transcripts and protein in rat retina. In addition we investigated the
expression pattern of a processing variant of neuroplastins that was
detected originally in np55 transcripts and codes for a four-amino-acid
(DDEP) insertion into the C-terminal cytoplasmic domain of the
protein.2
We investigated whether only np55 or both
neuroplastins contain this insertion and the pattern of expression of
these splice variants in brain and retina.
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Methods
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Animals and Surgery
Male Sprague-Dawley rats from the local breeding facilities of
the Leibniz Institute were used for all experiments. Animals were kept
on a 12-hour dark–light cycle with food and water available ad
libitum. All rats were killed 4 hours after onset of the light period,
and retinas of at least three animals for each developmental time point
were sectioned and processed for in situ hybridization, Western blot
analysis, and immunohistochemistry. Optic nerve crush was performed as
described previously.10
Animal experiments were conducted
in accordance with the ARVO Statement for the Use of Animals in
Ophthalmic and Vision Research and the regulations for animal
experiments of the federal government of Germany.
In Situ Hybridization
In situ hybridization of rat retinal and brain cryostat sections
was performed as described previously.10
11
The position
of the oligonucleotide probes and their complementary location on the
neuroplastin cDNA is shown in Figure 1
. Various rat brain cDNA libraries were screened for overlapping
neuroplastin cDNA clones.2
Sequence analysis of these
clones revealed that numerous clones contained an insertion of 12 bp
encoding the amino acids DDEP in the cytoplasmic tail of
neuroplastin.2
Specific oligonucleotides for isoforms
either harboring or without the C-terminal insertion were synthesized,
and in situ hybridization studies of rat brain and retina cryostat
sections were performed. The nucleotide sequences of the probes were as
follows: np65: 5'-gctgtctgaaagactctgcccggttgacttctgcgtaccac-3';
np65/55: 5'-cctcagctcttggcttattgatcctgtattccatgttgct-3'; np55:
5'-gctggtgacaattcttggttcgttctgagcagcgcctgg-3'; DDEP+:
5'-cattggcccggctggttcatcatcatcaggaacctc-3'; and DDEP-:
5'-ggttttcattggcccggcatcaggaacctcatctgg-3'.
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Figure 1. Schematic representation of the Ig and cDNA structures of np65 and
np55. Positions of PCR primers and oligonucleotides used for in situ
hybridization are indicated. In addition, the epitopes for the
monospecific (ASIg1), the monoclonal (Smgp65), and rabbit polyclonal
(ASIg2-3) antisera are depicted.
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Specificity of labeling was checked by a variety of control procedures
including pretreatment with RNase A (Promega, Mannheim, Germany) and
use of a sense probe. Some alternate sections were stained with 0.125%
cresyl violet to reveal the cellular pattern and layering of the
retina.
Isoform-Specific Neuroplastin Antibodies and Western Blot Analysis
of Brain, Retinal, and Optic Nerve Protein Homogenates
Production and characterization of rabbit polyclonal antisera AS
Ig1 and AS Ig2-3 directed against the np65-specific and the two common
Ig domains, respectively, have been described previously.6
The mouse mAb SMgp65 was generated against a ConA-binding glycoprotein
fraction prepared from rat brain synaptic membranes and recognizes both
neuroplastin isoforms.3
Retinal homogenization and Western
blot analysis were performed essentially as described
previously.2
10
Homogenates from retinal sublayers were
prepared from cryostat sections of retinal flatmounts.12
The first two sections (40 µm each) were found to contain mainly the
optic nerve fiber layer, retinal ganglion cell layer (GCL), and the
inner plexiform layer (IPL). The next two sections contained mainly the
inner nuclear layer (INL) and the outer plexiform layer (OPL), whereas
the last three sections predominantly contained cells from the outer
nuclear layer (ONL).
Immunostaining of Rat Retinal Paraffin and Cryostat Sections
Staining of retinal paraffin sections was performed as described
previously.13
The slides were incubated for 1 hour with
5% normal goat serum (Sigma, Deisenhofen, Germany) in 10 mM PBS (pH
7.4), followed by an overnight incubation with mAb SMgp65 (dilution
1.800). The slides were washed three times for 5 minutes in 10 mM PBS
and incubated with the secondary antibody by the ABC method
(Vectastain; Camon, Wiesbaden, Germany), according to the
manufacturer’s protocol.
Immunostaining of cryostat sections was performed as described
previously.14
For immunofluorescence experiments, vertical
retinal sections (10 µm) were incubated for 1 hour in blocking
solution, containing 10% normal goat serum (NGS) and 1% bovine serum
albumin (BSA) in 0.01 M PBS (pH 7.4), followed by an overnight
incubation with the primary antibody, either AS Ig1 (dilution 1:500) or
As Ig2-3 (dilution 1:1000), diluted in incubation solution containing
3% NGS and 1% BSA in PBS. After several washes in PBS, the sections
were incubated for 2 hours with secondary antibody (goat anti-mouse IgG
and goat anti-rabbit IgG, either conjugated to CY2 or to CY5
(carboxymethylindocyanine; Dianova, Hamburg, Germany) diluted in
incubation solution. Mouse monoclonal anti-synaptophysin (Sigma) was
diluted 1:2000. Confocal laser scanning microscopy was performed as
described previously.15
Control experiments were performed
by omission of the primary antibody and incubation with the rabbit
preimmune serum.
RNA Extraction and RT-PCR
Total RNA was extracted from adult rat retina and optic nerve
using a commercially available kit (RNAeasy; Qiagen, Hilden, Germany).
The following oligonucleotides were used as PCR primers (see also Fig. 1
): np65 forward (np65F), 5'-gaagcgccgtgtcaccgtaaac-3'; np55 forward
(np55F), 5'-cgctgctcagaacgaaccaagaa-3'; np65 and np55 common reverse
(npCR), 5'-ttatggccagtgatgtcagga-3'; np65/55DDEP- reverse (npD-R),
5'- cattggcccggcatcaggaac-3'; np65/55DDEP+ reverse (npD+R), 5'-ggc tgg
ttc atc atc atc-3'. PCR was initiated by adding 30 picomoles of each
forward primer and 60 picomoles of each reverse primer to 2.5 µl 10x
PCR buffer (Clontech, Heidelberg, Germany), 200 µM dNTPs, and 2.5
units Taq polymerase (Advantage; Clontech) to 1, 10, or 100
ng cDNA. The reaction mixture was brought to 25 µl with
H2O before the cDNA was denatured at 95°C for 1
minute. Annealing and extension temperatures were 55°C and 72°C,
respectively, for 1 minute each. Denaturing temperature was set at
95°C for 1 minute. After 38 cycles, 8 µl of the sample was
separated on a 1.5% agarose gel including ethidium bromide to
visualize the bands.
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Results
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Expression of Transcripts of Neuroplastin Isoforms with or without
the DDEP Insertion
Transcripts encoding the DDEP insertion in the cytoplasmic tail of
neuroplastin (Fig. 1)
have been detected in np55 cDNA clones. A PCR
strategy was designed to detect whether this insertion is present also
in the 65-kDa isoform of neuroplastin. To this end PCR primers were
designed that specifically recognize DDEP+ or DDEP- isoforms at the 3'
end and np55 or np65 at the 5' end. The localization of primer
oligonucleotides is indicated in Figure 1
. As shown in Figure 2
, transcripts for all four isoforms—np55 DDEP-, np55 DDEP+, np65
DDEP-, and np65 DDEP+—were expressed in both brain and retina. It
should be noted that the strategy was not designed to quantify the
transcript levels of the individual isoforms.
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Figure 2. RT-PCR of retina and brain total RNA with splice isoform–specific
primers. PCR products were loaded on 1.5% agarose gels and DNA bands
were stained with ethidium bromide. A 100-bp DNA ladder was used to
identify the size of the products.
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In situ hybridization studies were performed to assess the distribution
of the different isoforms in the brain. As reported
previously,2
np65 is more abundant in forebrain regions
and less abundant in the cerebellum than np55 (Fig. 2)
. These different
transcript levels are also reflected by different protein levels of
both isoforms as evidenced by Western blot analysis of cerebellum and
basal ganglia total protein homogenates.1
3
The expression
patterns of DDEP+ and DDEP- isoforms also showed some variation (Fig. 3) . DDEP+ splice variants were more prominently found in the cerebellum.
The high level of np55 relative to np65 in the cerebellum3
suggests expression is primarily np55DDEP+ in this region. DDEP-
isoforms were found throughout the brain including hippocampus and
forebrain regions (Fig. 3)
. Both np65 and np55 transcripts are thought
to contribute to labeling obtained with these oligonucleotides.
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Figure 3. In situ hybridization of neuroplastin isoforms in the adult brain.
Horizontal cryostat sections were hybridized with np65-, np55-,
DDEP--, and DDEP+-specific probes (see Fig. 1
for the position of the
probes).
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Using the common probe that detects all splice variants of
neuroplastin, transcripts were detected in all three cell layers of the
adult retina (Fig. 4)
. Hybridization signals were homogenously distributed, indicating
expression of np65 and np55 in the majority of retinal cell types. In
situ hybridization with the np55-specific probe produced hybridization
signals only in the INL and the GCL. Silver grains covered all
sublaminae of the INL, suggesting that amacrine, bipolar, and
horizontal cells express np55 (Fig. 4) . Experiments using the
np65-specific probe showed that cells in all three cell layers were
labeled (Fig. 4)
. The localization of transcripts with probes for the
two N-terminal splice variants was in agreement with the signal found
with the common probe. Transcripts for the C-terminal processing
variants were localized to all retinal nuclear layers with slightly
stronger hybridization signals for DDEP- variants (Fig. 4)
.
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Figure 4. In situ hybridization of neuroplastin isoforms in the adult rat retina.
Retinal cryostat sections were hybridized with np65-, np55-, DDEP--,
and DEEP+- specific probes (see Fig. 1
for the position of the probes)
and the np65 and np55 core probe, which detects all splice isoforms.
Scale bar, 50 µm.
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Developmental Regulation of Neuroplastin Transcripts in the Retina
To elucidate whether the expression of neuroplastin isoforms
correlates with specific stages of retinal development, hybridization
experiments were performed with tissue obtained at postnatal day (P)1
to P14 (Fig. 5)
. Np55 was found to be expressed already at P1 in the cytoblast layer
(CBL; also referred to as ventricular zone) and primordial GCL (Fig. 5)
. At this stage, no np65 hybridization signals above background were
observed. In the INL and ONL a faint label with the np65 specific probe
first appeared at P4 (Fig. 5)
. At P6 this label was much stronger,
covering mainly the ONL and the outer half of the INL (Fig. 5)
. At this
time point, the synaptic connections in the OPL have formed, and
synaptogenesis of ribbon synapses between photoreceptors and bipolar
cells is occurring in albino rats. Ribbon synapses in the IPL are
formed at P11 to P13. Faint in situ label for np65 was present in the
GCL at this stage of retinal development, coinciding with maturation of
synapses in the INL and eye opening (Fig. 5)
. Expression of DDEP+ and
DDEP- splice variants was essentially similar to those of np65 and
np55 at all stages of retinal development (data not shown).
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Figure 5. In situ hybridization of neuroplastin isoforms during retinal
development are depicted in the left panels. Np55
hybridization signals were found to be diffusely distributed early in
development but became restricted to the INL and GCL in the second
postnatal week. Hybridization signals similar to those of the np65 and
np55 core probe were seen with DDE-- and DDEP+-specific probes (not
shown). In the right panels, immunostaining of retinal
cryostat sections with ASIg1 (np65) and ASIg2-3 (np65/55) is depicted
at different stages of postnatal development. In contrast to the
diffuse distribution of immunosignals with ASIg2-3 the appearance of
np65 immunoreactivity largely correlated with the histogenesis and
synapse formation in OPL and IPL. In the first and fourth rows,
sections counterstained with cresyl violet were inserted to facilitate
morphologic orientation at each time point during retinal development.
Scale bar, 50 µm.
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Neuroplastin Protein Expression in the Adult and Early Postnatal
Retina
Western blot analysis using mAb SMgp65 and a polyclonal antiserum
AS Ig2-3 directed against the common region of np65 and np55 confirmed
that both glycoproteins were expressed in the adult rat retina (Fig. 6) . Moreover, the np65-specific antiserum AS Ig1 detected only one
protein band migrating at the molecular weight of np65 (Fig. 6) . Thus,
all antibodies used in this study were specific for either one or both
neuroplastin isoforms.
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Figure 6. Western blot analysis with different neuroplastin antibodies in rat
retina. Protein homogenates from total retina were separated by
SDS-PAGE. Equal amounts of protein were loaded in each lane (50 µg).
Immunodetection was performed with the antibodies indicated:
monospecific (ASIg1), mouse monclonal (SMgp65), and rabbit polyclonal
(ASIg2-3).
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With monoclonal and polyclonal antibodies recognizing both neuroplastin
isoforms, immunoreactivity was found in both synaptic layers, IPL and
OPL, in rat retinal paraffin sections. A similar staining pattern was
found with the monospecific np65 polyclonal antiserum (Fig. 7) . Immunofluorescence studies with AS Ig2-3 and AS Ig1 confirmed the
presence of intensely labeled structures in the OPL and IPL (Fig. 7)
.
In the OPL, immunoreactivity was concentrated in one thin layer (Fig. 7)
, surrounded by more diffuse label. Double-immunofluorescence
confocal laser scanning microscopy with an antibody against the
presynaptic marker protein synaptophysin and AS Ig1 showed that
immunofluorescence for np65 was colocalized to a large degree with
immunoreactivity of this synaptic marker in the OPL and INL (Fig. 7)
.
It should be noted, however, that np65 and np65/np55 immunoreactivities
were broader and more diffusely distributed in the OPL. This probably
indicates an additional perisynaptic localization. Unfortunately,
although we tried several different protocols, all neuroplastin
antibodies used in this study were found to be unsuitable for
ultrastructural studies using electron microscopy.
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Figure 7. Immunostaining of adult retina with different neuroplastin antibodies.
(A, B) Immunostaining with the mAb Smnp65
(A) and rabbit polyclonal ASIg2-3 (B) of rat
retinal paraffin-embedded sections. Strong labeling was found in the
OPL and IPL. Two prominent immunoreactive bands were detected in
the IPL. (C) At higher magnification the labeling
was found to be concentrated at one membranous substructure in the OPL
(arrows). (D–I) Double immunofluorescence
labeling in the IPL and OPL with a synaptophysin (D,
G) and the rabbit monospecific ASIg1 (E,
H) antibody. Labeling with the monospecific np65 antibody
indicated localization of this splice isoform in both synaptic layers
(E, F). Double-immunofluorescence confocal laser
scanning microscopy show a large overlap of synaptophysin and np65
fluorescence signals in both synaptic layers (F). At higher
magnification a perisynaptic localization of np65 immunoreactivity also
became evident (I, arrows). Scale bar,
(A, B, D–F) 50 µm; (C)
10 µm; (G–I) 5 µm._art>
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To further substantiate the immunostaining data, retinal flatmounts
were cut on a cryostat, and the retina was divided into three parts:
sections containing mainly GCL and IPL, or INL and OPL, or ONL. Western
blot analysis of these preparations showed abundant expression of both
isoforms in the inner retina and the OPL with very low levels in the
ONL (Fig. 8)
. Most interesting, np65 was found to be much more abundant in the
preparation containing the OPL photoreceptor synapse fraction than np55
(Fig. 8)
. Synaptophysin was used as a synaptic marker protein to
compare the content of synaptic structures with that of neuroplastins
in all three preparations (Fig. 8)
.
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Figure 8. Western blot analysis of protein preparations from the inner and outer
retina with a polyclonal neuroplastin antibody. (A) The
relative intensity of immunoreactive bands for np65 compared with np55
were found to be stronger in mainly protein preparations containing the
INL+OPL part of the retina than in those containing mainly GCL+IPL.
(B) The enrichment of synaptic structures was demonstrated
by the presence of synaptophysin immunoreactivity in GCL+IPL and
INL+OPL and only negligible amounts in the part containing mainly the
ONL. Equal amounts of protein (50 µg) were loaded in each lane.
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In agreement with the in situ hybridization experiments we found np65
and np55 immunoreactivity, as detected with AS Ig2-3, to be diffusely
distributed in the CBL and GCL (Fig. 5)
. This distribution was altered
in the second postnatal week with prominent label in both synaptic
layers (Fig. 5)
, although cellular staining was still present at this
stage and in mature retinas (Figs. 5 7)
. Stainings with the
monospecific AS Ig1 antiserum confirmed the presence of np65
immunoreactivity in the OPL and IPL during retinal development and
demonstrated a correlation of histogenesis of the OPL at P6 and synapse
formation in the IPL in the second postnatal week with the first
appearance of np65 immunosignals restricted to these layers (Fig. 5)
.
Neuroplastin Expression in the Optic Nerve
Immunoblot analysis revealed neuroplastin-immunoreactive bands in
optic nerve protein preparations, indicating that both isoforms were
present in the optic nerve. To identify the origin and potential
regulation of neuroplastin expression, a crush of the optic nerve was
performed to interrupt axonal transport. After nerve crush,
immunoreactivity for both neuroplastins did not accumulate in the
proximal part of the optic nerve, which was still in continuity with
retinal ganglion cells. The immunoreactivity in the distal part of the
nerve was also not affected (Fig. 9) . RT-PCR experiments showed that transcripts for both neuroplastins
were present in the optic nerve (Fig. 9)
.
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Figure 9. Western blot analysis and RT-PCR from optic nerve protein preparations
and total RNA, respectively. (A) Np65 and np55 were found to
be present in the optic nerve, although protein levels were clearly
lower than those in retina and brain. Optic nerve crush led to no clear
difference in protein levels in the parts either proximal or distal
from the crush site. Equal amounts of protein were loaded in each lane
(50 µg). (B) PCR products were loaded on 1.5% agarose
gels, and DNA bands were stained with ethidium bromide. A 100-bp DNA
ladder was used to identify the size of the products.
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Discussion
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In this study we localized immunoreactivity for the 65- and 55-kDa
isoforms of neuroplastin to the synaptic layers of the retina.
Corresponding transcripts were found to be differentially distributed
with predominant expression of np65 transcripts in the outer retina,
whereas both np55 and np65 transcripts were present in the inner
retina. In particular, np65 appeared to be associated with synaptic
structures in the OPL—that is, it colocalized with the presynaptic
marker protein synaptophysin. Immunoreactivity of the neuroplastins in
the INL was more diffusely distributed. Thus, although not restricted
to synapses, neuroplastins are most probably found at both conventional
and ribbon synapses of the retina. Moreover, we provide evidence for
the first time for the expression of neuroplastins in central nervous
system glia. Previous studies have shown no evidence for glial
localization of the neuroplastins in rat brain, and neither np55 nor
np65 is expressed by cultured astrocytes.3
6
The immunoreactivity for both neuroplastins was confined to the two
synaptic layers of the retina. Although, due to methodologic
limitations, a direct localization at the synapse could not be shown
with immunoelectron microscopy, several lines of evidence suggested
that at least np65 is localized at ribbon and at conventional synapses.
First, we found a clear colocalization of neuroplastins with a synaptic
marker protein in both synaptic layers. Second, the appearance of np65
transcripts and immunosignals during retinal development coincided with
the period of formation of ribbon synapses in the OPL. In addition, the
overlap of synaptophysin with np65 immunoreactivity in the OPL strongly
suggests that neuroplastins are CAMs situated in the synaptic cleft of
ribbon synapses. Neuroplastin immunoreactivity was broadly distributed,
which probably indicates that either np55 or both neuroplastins are
also found in horizontal cell synapses and perisynaptic regions. In the
IPL, neuroplastin immunoreactivity was more diffuse. Thus, it seems
that np65 is a component enriched in synaptic structures of the retina,
which is in accordance with the previous finding that the 65-kDa, but
not the 55-kDa isoform is enriched in rat forebrain postsynaptic
density preparations.3
The localization of np65 transcripts in the ONL and INL suggests both a
pre- and postsynaptic localization of the protein in ribbon synapses of
the OPL. Because most of the np65 immunoreactivity was found in the OPL
it has to be assumed that the translated transcript in photoreceptor
cells is localized at presynaptic structures. Of interest in this
respect are recent binding experiments in which neuroplastin-Fc
chimeric proteins were used to show that constructs containing the Ig
domains 1 to 3 or only the np65-specific Ig domain 1, but not
constructs containing the two common Ig domains, mediate homophilic
adhesion.6
It is therefore conceivable that np65 at ribbon
synapses binds to its postsynaptic counterpart expressed by bipolar
cells. Taken together, the available evidence is therefore suggestive
of a role of np65 in adhesion between the pre- and postsynaptic nerve
endings in the OPL, possibly by a homophilic mechanism.
Although cell differentiation and layer formation of the retina appear
to advance in an inside-to-outside sequence with cell birth of ganglion
cells first and appearance of photoreceptor cells last,16
the maturation of synaptic structures proceeds from the outer to the
inner layers of the retina.8
9
Synaptogenesis of
conventional amacrine synapses is first apparent at the end of the
first postnatal week17
and ribbon synapses formed between
bipolar and ganglion cells in the IPL are found at P5,18
with increasing numbers at P12 or P13.19 Ribbon synapses
in the OPL, however, are already present at P5 in pigmented
rats9
and at P6 in albino rats, which coincides with the
formation of this synaptic layer.8
In contrast to np55,
the transcript levels of np65 are regulated during retinal development
in a cell-specific manner.
It is striking that the transcript expression of np65 in the ONL and
INL correlated with the formation of ribbon synapses in the OPL,
whereas no correlation with retinal synaptogenesis was found for the
expression of np55. Moreover, np65 immunoreactivity was also prominent
in both synaptic layers during development and, most interesting,
immunosignals were first detected at developmental stages correlating
with synaptogenesis in the OPL and IPL. Thus, it seems that
specifically np65 could be an early cell adhesion component during
synapse formation. Moreover, it is tempting to speculate that a
homophilic interaction of pre- and postsynaptically situated np65 is
involved in the formation of these synapses.
At present few of the molecules involved in adhesion events at
conventional and ribbon synapses in the retina have been identified. Of
the CAMs localized to synaptic junctions to date, only NCAM-180 and
cadherins have been immunolocalized in the retina.20
The
distribution of NCAM-180 was found to be largely similar to those of
np65 and np55, whereas cadherin expression was found to be more
confined to certain retinal cell populations.21
In
addition, the glycoprotein ß-dystroglycan, which is suggested to play
a role in cell–cell contacts, has been identified in the OPL and nerve
fiber layer of the retina.22
23
24
Similarly,
ß-dystroglycan seems to have a much more restricted expression
pattern compared with the neuroplastins. It is reported to be
associated with the more proximal lateral wall of the photoreceptor
synaptic cavity that projects into the postsynaptic dendritic complex.
The more widespread and perisynaptic localization of a significant
fraction of np65 and np55 in the OPL suggests that its function is
probably less restricted to specific aspects of stabilization of
synaptic morphology or clustering of proteins to microdomains, which
have been proposed for ß-dystroglycan.22
Thus, a
broader function in histogenesis and maintenance of cellular contact in
synaptic regions, possibly comparable to those of NCAM-180, has to be
assumed for neuroplastins.
A C-terminal splice variant of neuroplastins was identified in retina
and brain. The broad expression pattern of both isoforms and the
results of the RT-PCR experiments indicated the presence of the DDEP
insertion in both np65 and np55. It is therefore unlikely that this
insertion is specific for neuroplastins that are localized to synapses.
The function of the four-amino-acid insertion remains elusive, but
because they are situated in the intracellular domain of neuroplastins,
it is conceivable that they are involved in mediating a
protein–protein interaction that may be important in intracellular
signal transduction or protein localization. Further studies are
warranted to identify such protein interactions and to determine
whether the C-terminal insertion renders this interaction specific for
one splice isoform.
With RT-PCR and Western blot analysis, we confirmed the expression of
both neuroplastins in rat optic nerve. The expression was found to be
unaltered after nerve crush, indicating that the protein is not
transported in the optic nerve but is synthesized by optic nerve glia.
The levels of protein were substantially lower than in retina and
brain. Because the antibodies used in this study were found to be
unsuitable for electron microscopy, the functional role of these
molecules could not be addressed. However, it would be of interest to
elucidate whether a cell adhesion contact between glia cells and optic
nerve axons is mediated in part by neuroplastins.
In summary, we have provided evidence for a synaptic localization of
neuroplastins in rat retina. In particular, np65 could play a role in
cell adhesion between photoreceptors and the postsynaptic complex
formed by horizontal and bipolar cells. Thus, it seems likely that
neuroplastins belong to the group of molecules that have been
implicated in cell adhesion in the plexiform layers during histogenesis
as well as in maintenance of connections between specific cellular
structures.
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Acknowledgements
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The authors thank Corinna Borutzki, Bettina Kracht, and Monika
Marunde for professional technical assistance throughout the project.
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Footnotes
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2 Present affiliation: Institute for Medical Neurobiology,
Medical Faculty, Otto-von-Guericke University, Magdeburg, Germany. 
Supported by the Fonds der Chemischen Industrie (EDG), the Fritz
Thyssen Stiftung (MRK), the Land Sachsen-Anhalt (EDG, MRK), the
Wellcome Trust (PWB), a North Atlantic Treaty Organization travel grant
(PWB, EDG), and Deutsche Forschungsgemeinschaft (EDG, MRK).
Submitted for publication September 20, 2000; revised February 20,
2001; accepted March 9, 2001.
Commercial relationships policy: N.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be marked
"advertisement" in accordance with 18 U.S.C. 
1734
solely to indicate this fact.
Corresponding author: Michael R. Kreutz, AG Molecular Mechanisms of
Plasticity, Department of Neurochemistry/Molecular Biology, Leibniz
Institute for Neurobiology, Brenneckestr. 6, 39118 Magdeburg, Germany.
kreutz{at}ifn-magdeburg.de
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Abstract
Introduction
