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The Journal of Neuroscience, November 15, 2001, 21(22):8915-8930
Sorting of Internalized Neurotrophins into an Endocytic
Transcytosis Pathway via the Golgi System: Ultrastructural Analysis in
Retinal Ganglion Cells
Rafal
Butowt and
Christopher S.
von Bartheld
Department of Physiology and Cell Biology, University of Nevada
School of Medicine, Reno, Nevada 89557
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ABSTRACT |
Subcellular pathways and accumulation of internalized
radiolabeled neurotrophins NGF, BDNF, and NT-3 were examined in retinal ganglion cells (RGCs) of chick embryos by using quantitative electron microscopic autoradiography. All three neurotrophins accumulated in
endosomes and multivesicular bodies. BDNF and NGF also concentrated at
the plasma membrane, whereas NT-3 accumulated transiently in the Golgi
system. The enhanced targeting of NT-3 to the Golgi system correlated
with the anterograde axonal transport of this neurotrophin. Anterograde
transport of NT-3, but not its internalization, was significantly
attenuated by the tyrosine kinase (trk) inhibitor K252a.
Abolishment of trk activity with K252a shifted NT-3 (and BDNF) away
from the Golgi system and into a lysosomal pathway, indicating that trk
activity regulated sorting of the ligand-receptor complex.
Cross-linking of neurotrophins and immunoprecipitation with antibodies
to the neurotrophin receptors p75, trkA, trkB, and trkC showed that the
large majority of exogenous, receptor-bound NT-3 was bound to trkC in
RGC somata, but during anterograde transport in the optic nerve most
receptor-bound NT-3 was associated with p75, and after arrival and
release in the optic tectum transferred to presumably postsynaptic
trkC. These results reveal remarkable and unexpected differences in the
intracellular pathways and fates of different neurotrophins within the
same cell type. They provide first evidence for an endocytic pathway of
internalized neurotrophic factors via the Golgi system before
anterograde transport and transcytosis. The results challenge the
belief that after internalization all neurotrophins are rapidly
degraded in lysosomes.
Key words:
anterograde transport; BDNF; NT-3; NGF; trkB; trkC; p75
neurotrophin receptor; neurotrophic factor; visual system; internalization; degradation; K252a; Golgi; lysosome; sorting; retina
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INTRODUCTION |
Nothing is known about the
subcellular pathways and organelles that contain the internalized
neurotrophins BDNF and NT-3, despite significant advances in the
identification of downstream targets of neurotrophins and cascades of
signal transduction (Segal and Greenberg, 1996
; Riccio et al., 1997;
Kaplan and Miller, 2000). Most of the internalized NGF appears to be
targeted to lysosomes and is rapidly degraded (Schwab and Thoenen,
1977; Johnson et al., 1978; Claude et al., 1982; Hogue-Angeletti
et al., 1982; Rohrer et al., 1982; Layer and Shooter, 1983; Stieber et
al., 1984; Eveleth and Bradshaw, 1992; Ure and Campenot, 1997) (but see
Bernd and Greene, 1983). These studies suggested a rather simple model
of termination of trophic action, namely the rapid degradation of the
neurotrophin after delivery of the trophic signal.
Recent studies have suggested a more complex fate of internalized
neurotrophins. The neurotrophins BDNF and NT-3 can avoid the
degradation pathway and can be targeted for release or for packaging in
vesicles for anterograde axonal transport after internalization (von
Bartheld et al., 2001). The subcellular trafficking of trophic factor-receptor complexes in neurons is unknown but likely includes sorting in endosomes, an endocytic pathway to the Golgi system (Green
and Kelly, 1992; Sandvig and van Deurs, 1996; Mukherjee et al., 1997),
followed by packaging in vesicles for anterograde axonal transport
(Hurtley, 1993; Fawcett et al., 1997; Michael et al., 1997).
Retinal ganglion cells (RGCs) are an excellent model system for
trafficking studies, because their environment can be well controlled
in vivo, yet they are typical CNS neurons, unlike other model system neurons such as dorsal root ganglia, which lack normal dendrites and afferent input. Developing chick RGCs express the neurotrophin receptors p75, trkB, and trkC but not trkA (von Bartheld, 1998a), and they internalize NGF, BDNF, and NT-3. Internalized NT-3 is
transported anterogradely much more efficiently than BDNF in this
system (von Bartheld et al., 1996a). Likewise, endogenous neurotrophins are differentially sorted in chick RGCs, with NT-3, but
very little BDNF, destined for anterograde transport (von Bartheld and
Butowt, 2000). Taken together, these studies indicate that
neurotrophins are targeted to different domains within neurons and that
internalized neurotrophins are not necessarily degraded after internalization.
Here we show that NGF, BDNF, and NT-3 follow different intracellular
pathways in RGCs after internalization and that their sorting is
regulated at least in part by tyrosine kinase activity. Consistent with
its anterograde transport, NT-3 accumulated in the Golgi system,
whereas BDNF was prevalent at the plasma membrane. When trk kinase
activity was blocked with K252a, anterograde transport of NT-3, but not
its internalization, was reduced, and its distribution was shifted from
the Golgi to lysosomal pathways. A significant fraction of the
anterogradely transported NT-3 bound to neurotrophin receptors during
transport, with both trkC and p75 playing roles as presumptive
transporters. Internalized neurotrophins are differentially sorted,
presumably reflecting their complex functions as factors promoting
survival, differentiation, dendritic growth, and synaptic plasticity.
Preliminary data have been published previously in abstract form (von
Bartheld, 1996; Butowt and von Bartheld, 2000).
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MATERIALS AND METHODS |
Sources of materials. Polyclonal antibodies to
chicken trkA, trkB, and trkC (against the extracellular domain) were
from Louis Reichardt, Frances Lefcort and Doug Clary (Lefcort et
al., 1996; von Bartheld et al., 1996b), and antibodies to chicken p75
neurotrophin receptor (ChEX, Weskamp and Reichardt, 1991) were from
Gisela Weskamp and Louis Reichardt (Howard Hughes Medical Institute, San Francisco, CA). For RGC purification, monoclonal antibodies to Thy1
(IgG SB1-20.1) (French and Jeffrey, 1986) were obtained from Peter
Jeffrey (Sydney, Australia). Human recombinant BDNF, NT-3, and NT-4
were provided by Ron Lindsay (Regeneron, Tarrytown, NY). Mouse
NGF was a gift from Mark Bothwell (University of Washington, Seattle,
WA) or purchased from Alomone Labs (Jerusalem, Israel). Glial cell
line-derived neurotrophic factor (GDNF) and cardiotrophin-1 (CT-1) were
from PeproTech (Rocky Hill, NJ). Brefeldin A, cytochrome c,
monensin, wortmannin, and secondary anti-chicken antibody (M8645) were
from Sigma (St. Louis, MO), Ilford L4 emulsion was from Polysciences (Warrington, PA), K252a was from Alomone Labs, and the
PI3-kinase inhibitor LY294,002 was from Alexis Corp (San Diego,
CA). The cross-linkers 1-ethyl-3 (3-dimethylaminopropyl)
carbodiimide-HCl (EDC) and disuccinimidyl suberate (DSS) were from
Pierce (Rockford, IL). Chicken eggs were obtained from California
Golden Eggs (Sacramento, CA) and were incubated in humidified
incubators at 37.5-38°C. Approximately 1050 chicken eggs were used.
The ages of chick embryos were verified at the time they were
killed according to Hamburger and Hamilton (1951). Experimental
procedures were conducted in compliance with the Policy on the Use of
Animals in Neuroscience Research and were approved by the local animal
care committee.
Radio-iodination and intraocular injections. Neurotrophins
and cytochrome c were iodinated with lactoperoxidase as
described (Vale and Shooter, 1985; von Bartheld, 2001).
Na-I125 was purchased from NEN Life
Science Products (Boston, MA). Incorporations were 47-86.5% (NGF),
91-95.9% (BDNF), 79.7-92.7% (NT-3), 41.3-60.7% (cytochrome
c), 64.5-89.8% (GDNF), and 95.0% (CT-1). Specific activities were 61-109 cpm/pg NGF, 90-196.6 cpm/pg BDNF, 70.6-197.3 cpm/pg NT-3, 42-118.6 cpm/pg cytochrome c, 168-333 cpm/pg
GDNF, and 32.4 cpm/pg CT-1. For transport studies, free iodide was
removed from the preparation when the incorporation was 
90%, and for ultrastructural analysis of internalization it was removed when incorporation was 95%. Iodinated neurotrophins were used within 12 d after iodination. They migrated in 15% SDS-PAGE as single bands at ~13-14 kDa and were transported in a receptor-mediated fashion (von Bartheld et al., 1996a). Twenty hours after injections, the embryos were anesthetized with Nembutal, perfused with 4% paraformaldehyde (PFA), staged, and post-fixed. The amount of radioactivity in the dissected eyes and midbrains [embryonic day (E)
15-17] was measured in a gamma counter. The ratios of the activity in the midbrain/eye were determined for each animal and averaged for the different treatment groups. SEs were calculated and
the data plotted as a function of the doses injected. The brains and
the eyes were embedded in paraffin, and four series of sections (10 µm thickness) through the midbrain at the level of the isthmo-optic
nucleus were collected on glass slides and exposed on x-ray film for
2-5 d, and the sections were coated with NTB-2 emulsion (Kodak,
Rochester, NY). Sections were exposed for 3-8 weeks at 4°C, were
lightly counterstained with thionin after development, and
coverslipped. After injections of iodinated proteins into the retina or
into the tectum, the amount of radioactivity in the eyes was measured
in a gamma counter.
Compounds were injected into chick embryo eyes in volumes of 5-10 µl
(von Bartheld, 1998b). Coinjection procedures with radiolabeled neurotrophins or cytochrome c included monensin (9 µg),
excess cold NT-3 (1-3 µg), excess cold NGF (2-4 µg), excess cold
BDNF (2-4 µg), p75 antibody (10 µg), trkC antibody (10 µg),
normal IgG (10 µg), K252a (1.8 µg), DMSO (2 µl), wortmannin (200 ng, every 3 hr), LY294,002 (5 µg), brefeldin A (5-15 µg), and
excess cold cytochrome c (2-4 µg). Some of the embryos
injected with 125I-NGF were coinjected
with an equal amount of cold NT-3 (50-60 ng) or cold BDNF (50-60 ng).
Autoradiography and ultrastructural analysis.
Autoradiography, localization, and quantification of
125I-neurotrophins was performed as
described previously (von Bartheld et al., 1996a; von Bartheld, 2001).
Embryos were anesthetized with Nembutal 3, 10, or 20 hr after injection
and intracardially perfused with 2.0% glutaraldehyde/1%
paraformaldehyde in PBS. Retinas were dissected and post-fixed in 2.5%
glutaraldehyde/0.5% paraformaldehyde in PBS, pH 7.4, for 6 hr at
4°C, rinsed with PBS, and post-fixed in 1%
OsO4 for 2 hr at room temperature. Retinas were
dehydrated in graded alcohols to 100% ethanol, then washed with 100%
propylene oxide and embedded in Spurr resin (Electron Microscopy
Sciences, Fort Washington, PA). Thin sections on grids were coated with
a monolayer of high-resolution Ilford L4 emulsion using the "loop
method" (Caro and van Tubergen, 1962). After 1-2 months of exposure,
the autoradiograms were developed in D19 (Kodak), fixed, and stained
with 2.5% aqueous lead citrate. Sections were examined and
photographed or scanned in a Philips CM 10 transmission electron
microscope equipped with a Gatan 792 BioScan digital imaging system.
RGCs and amacrine cells in the retinal ganglion cell layer were
distinguished using established ultrastructural criteria (Prada et al.,
1989).
To ensure adequate sampling of each retina, we sectioned blocks for
each experiment from two to three different areas containing RGCs.
Sections from each block were collected on several grids. The
background level of silver grains was determined by photographing six
to nine areas containing resin only (at a magnification of 4000-5000×). Background levels were consistently negligible over individual grids. From each grid, two to three squares were analyzed. In each square, all RGCs with silver grains were photographed at a
magnification of 11,000-20,000×. Autoradiography of sections through
the optic tectum loaded with 125I-NT-3 by
anterograde transport from the retina followed the protocol of von
Bartheld et al. (1996a). In short, samples of the superficial tectum
were embedded in Spurr's, thin-sectioned, coated with Ilford L4
emulsion, and exposed for 8-12 weeks for autoradiography. Grains over
cell bodies in superficial layers were photographed and printed at 20,000-40,000 magnification, and the probable source was analyzed as described below. The fractional area of organelles from all labeled
tectal cell bodies containing silver grains was measured as described below.
The circle probability method with a half-distance (HD) of 90 nm
(Salpeter et al., 1978) was used to analyze and quantify the source of
grains. The half-distance is the distance from a line source of
radioactivity within which 50% of the grains are predicted to
originate (Salpeter et al., 1978). Quantification was restricted to the
RGC cell body; neurites and growth cones were not considered in this
study, because correlation of neurite fragments with particular cell
types is impossible without identifying labels. The average area
occupied by particular organelles was determined by morphometric
analysis (referred to as fraction area). Electron micrographs were
overlaid with a transparent grid with a diameter of 1.5-2 HD, and the
frequency of organelles intersecting the grid was scored. To ascertain
that treatment with K252a or DMSO did not change the organelle fraction
area, treated retinas were compared with normal retinas; no differences
were found.
The "labeling density" of organelles was determined by dividing the
percentage of silver grains found over organelles by the percentage of
the fraction area of that organelle. A labeling density of 1
indicates that silver grains were distributed randomly, whereas a
labeling density much more than 1 indicates a specific association of
silver grains with this organelle. To determine at which threshold a
labeling density demonstrates specific accumulation, all data were
subjected to statistical analysis, using the distribution of free
iodide and denatured NT-3 as controls. The total numbers of silver
grains for each category (100-200) were assigned randomly to two to
four groups, and both t test and one-way ANOVA were used to
determine whether labeling densities in the experimental groups were
significantly (p 0.05) different from control
groups (random distribution). This analysis revealed that all labeling densities of >3.3 were significant, whereas those <2.4 were not. It
should be noted that such analyses depend on the number of silver
grains analyzed, and the variability in both the control and the
experimental groups, and thus will differ between studies.
Denaturation of NT-3. Previously radiolabeled NT-3 was
incubated in 100 mM Tris-HCl, pH 8.7, and 50 mM dithiothreitol. The mixture was boiled for 5 min and cooled down to room temperature, and the generated free
sulfhydryl groups were alkylated with iodoacetamide (200 mM). The preparation was reacted for 2 hr at room
temperature and then desalted by membrane filtration (Ultrafree MC
filters, Millipore, Bedford, MA; 10 kDa cutoff) using at least 25 vol
of sodium acetate buffer (10 mM sodium acetate,
100 mM sodium chloride, pH 4.0). The
incorporation and specific activity of the denatured 125I-NT-3 were determined by TCA
precipitation. Thus denatured NT-3 does not bind to receptors in assays
of internalization and anterograde transport (von Bartheld et al.,
1996a).
Purification of retinal ganglion cells and internalization of
NT-3. RGCs from E20 chick embryos were purified by using a simple immunopanning step with Thy1 antibodies (Butowt et al., 2000). This
procedure yields ~100,000-200,000 RGCs per retina with a purity of
~97%. Retinas of E18-E21 chick embryos were pooled, and cell
suspensions were divided equally onto 55 mm plates (in duplicate) for
immunopanning. All plates were visually inspected on an inverted
microscope containing a counting box, and the number of panned cells
was estimated (100,000-300,000 cells per plate) to ensure that amounts
of immunopanned cells were comparable. After immunopanning, cells were
incubated for 3 hr in 10-20 ng/ml 125I-NT-3 (1-2 × 106 cpm per plate) in "binding buffer"
(PBS, pH 7.4, containing 1 mg/ml bovine serum albumin and 1 mg/ml
glucose) (Vale and Shooter, 1985), at either 4°C (control) or 37°C.
Duplicate plates at 37°C were incubated with either 1 µg/ml DMSO
(control) or 1 µg/ml K252a with the same concentration of DMSO. All
solutions of this and the subsequent steps were collected for gamma
counting. After the 3 hr incubation period at 4 or 37°C, the binding
buffer was removed, cells were washed with PBS and stripped with an
ice-cold acid wash for 5 min (0.5 M NaCl, 0.2 M acetic acid, pH 2.5) (Bernd and Greene, 1984).
Again, it was verified by visual inspection on the inverted microscope
that comparable amounts of cells were retained at this step. Cells were
washed with PBS and then collected in single detergent lysis buffer (50 mM TrisCl, pH 7.6, 150 mM NaCl, 1% Triton X-100). It was verified for each plate by inspection on the inverted scope that virtually all cells were removed from the
plates. All solutions (incubation buffer, first wash buffer, acid wash,
second wash buffer, lysis buffer) for each plate were counted
separately in a gamma counter, and the percentage of internalization (counts in lysis buffer divided by counts in incubation buffer + counts
in lysis buffer) was determined as well as the contribution of
nonspecific association (counts in lysis buffer at 4°C divided by
counts in incubation buffer at 4°C + counts in lysis buffer). The
results from four independent experiments (each in duplicate) were
subjected to unpaired t test for statistical analysis.
Because the large majority (>80%) of NT-3 binding to RGCs was
specific (i.e., was competed by 50- to 100-fold excess cold NT-3; data not shown), the binding seen at 37°C must be largely specific binding.
SDS-PAGE gel electrophoresis. To determine whether the
radioactivity contained within cells in the in vivo
experiments represented intact neurotrophins or their breakdown
products, 18- to 20-d-old chick embryos were injected in the eye with
radiolabeled neurotrophins NGF, BDNF, or NT-3, and after 10 hr, RGCs
were purified as described above. After intraocular injection of
50-100 ng radiolabeled NGF, BDNF, or NT-3, or free iodide as a
control, retinas were collected 10 hr later and pooled, and the cells
were dissociated and immunopanned. Purified RGCs were used for analysis
by SDS-PAGE or for lysis, cross-linking, and immunoprecipitation with
neurotrophin receptor antibodies (see below). After immunopanning,
purified RGCs or samples of whole retina (without immunopanning) were
washed five times in PBS (containing 0.2% BSA), pH 7.4, dissolved in
2× Laemmli electrophoresis sample buffer, homogenized by repetitive
pipetting, and centrifuged at 10,000 × g for 10 min at
4°C. The supernatant was boiled for 5 min and centrifuged, and its
radioactivity was determined by gamma counting. Between 1,000 and
10,000 cpm were loaded per lane on 15% SDS-PAGE gels. Low molecular
weight protein standard (Life Technologies, Gaithersburg, MD) or
cytochrome c was used as molecular weight indicators. Gels
were stained with Coomassie blue, dried, and exposed to Kodak XR-5
x-ray film for 3-6 d. As a control for trypsin digestion of BDNF
during the dissociation procedure, trypsin-digested BDNF was examined
on SDS-PAGE.
Neurotrophin receptor in situ hybridization. A
chicken p75 cDNA, subcloned into pGEM4Z, was used to generate
riboprobes (436 bp fragment) labeled with
35S-UTP (Heuer et al., 1990). Animals of
E15/16 were anesthetized and perfused with 4% PFA. The eyes were
either frozen for cryosectioning (10 µm) and thaw mounted onto
poly-L-lysine-coated slides or dehydrated, embedded in
paraffin, and sectioned at 10 µm, and sections through the central
retinas were collected on silane-coated glass slides. Adjacent sections
were hybridized with sense control probes. Hybridization conditions
were as described previously (von Bartheld et al., 1991). Emulsion
(NTB-2)-coated paraffin sections were exposed for 14-28 d, developed,
and lightly counterstained with thionin. Chicken trkA, trkB, and trkC
oligonucleotide probes recognizing the kinase domain (von Bartheld et
al., 1996b) were hybridized to cryosections from E16 and E18 retinas,
coated in NTB-2 emulsion, and developed after 4-6 weeks. These
sections were lightly counterstained with cresyl violet. The
autoradiographic label of cells in the central ganglion cell layer
(GCL) was quantified in both series of slides by using a reticule in
the eye piece. The level of background grains was measured in adjacent
sense control sections, and the values were subtracted to normalize for
specific label intensities.
Cross-linking and immunoprecipitation. Chick embryos at the
age of 16-18 d of incubation (E16-18) were injected with 20-40 ng
radiolabeled NT-3 in the eye in ovo. Twenty hours later, the animals were anesthetized with Nembutal and transcardially perfused with cold PBS. The retina, the optic chiasm, and the contralateral optic tectum were dissected and collected in cold PBS, chopped into
small pieces, and homogenized in lysis buffer using 22 gauge needles.
For analysis of internalized neurotrophins in RGC bodies, immunopanning
was used for purification (Butowt et al., 2000). RGCs were lysed after
immunopanning, and 2-10% of RGCs (1000-5000 cpm) were recovered.
Lysis buffer consisted of 50 mM TrisCl (pH 6.5 for EDC, pH 7.6 for DSS), 150 mM NaCl, 100 µg/ml aprotinin, 1% Triton X-100, 1 µg/ml leupeptin, and 0.5 mM PMSF. One of two cross-linkers was added to
the homogenates for 30 min at room temperature in a rotating device
(nutator): EDC (Pierce Chemical, Rockford, IL) at 6 mM, pH 6.5, or DSS (Pierce) at 0.1 mM, pH 7.6. The cross-linking reaction was
terminated by incubation in cold PBS containing 50 mM lysine for 30 min. The preparation was
centrifuged for 5-10 min at 4°C, and pellets and supernatants were
counted separately in a gamma counter. Usually 70-80% of the total
counts were retained in the supernatant. The supernatant was incubated overnight at 4°C with 1.4-1.7 µg/ml polyclonal antibodies against chick p75, trkB, or trkC, or the same concentration of normal rabbit
IgG as control. The next day, 10-20 µl of washed Pansorbin cells
(Calbiochem, La Jolla, CA) were added to the preparation for 1 hr in
the cold room, the immunocomplex was pelleted by centrifugation for 5 min in the cold room, and the pellets and supernatants were counted
separately in a gamma counter. All determinations were done in
triplicate. Nonspecific precipitation was subtracted by using the
counts from the control (IgG-incubated) pellets. We are using the
terminology of "specific precipitation" to indicate that the
antibodies were presumably precipitating their antigens specifically,
but not to indicate that the binding of the neurotrophin was
"specific" (i.e., competed by excess cold = receptor bound). Nevertheless, it can be assumed that the antibody precipitates only
those neurotrophins that are bound to receptors, thus presumably precipitating indeed only specifically bound neurotrophins. Control experiments included (1) incubation of normal lysed tissues with similar amounts of radiolabeled NT-3 to assess whether free NT-3 may
bind to different receptors when they become available during lysis,
(2) omission of the cross-linking step, and (3) repeated immunoprecipitation of the same supernatants to determine the efficiency with which the antibodies pulled down the receptors. Statistical significance was determined by unpaired t test.
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RESULTS |
Anterograde transport of NT-3 is receptor-mediated
We have shown previously that the anterograde axonal transport of
exogenous NT-3 in RGCs is receptor mediated (von Bartheld et al.,
1996a). To determine which receptors mediate the internalization and
anterograde transport of NT-3, excess amounts of cold homologous factor
were coinjected. Background radioactivity (attributable to systemic
circulation) was assessed by coinjection of the Golgi-blocking agent
monensin (Hammerschlag et al., 1982). Excess cold NT-3 (20- to
200-fold) significantly reduced transport of radiolabeled NT-3 (Fig.
1A). To test the
contribution of the p75 neurotrophin receptor for NT-3 transport,
excess cold NGF (20- to 100-fold) was coinjected. This would also
examine trkA binding of NT-3, but trkA is not expressed in chick RGCs
(Karlsson et al., 1998). To test the contribution of p75 and trkB
receptors for NT-3 internalization, excess cold BDNF (50- to 100-fold)
was coinjected. Coinjection of heterologous neurotrophins that should
prevent the binding of NT-3 to the p75 receptor (NGF) or the trkB
receptor (BDNF) (DiStefano et al., 1992; Rodriguez-Tebar et al., 1992)
did not reduce the internalization or the anterograde transport of NT-3
(Fig. 1A). Coinjection of blocking antibodies against
p75 at doses that significantly reduce retrograde axonal transport of
NT-3 (von Bartheld et al., 1996b) only slightly reduced anterograde
NT-3 transport. Together, these data indicate that neither the p75
receptor nor the trkB receptor is required for internalization of NT-3
by RGCs. To directly test whether trkC is essential for
internalization, we sought to use a trkC antibody that had been shown
to interfere with trkC signaling (Lefcort et al., 1996). However, at
the doses used (10 µg per eye), there was no effect on the
anterograde transport of NT-3 by RGCs (Fig. 1A).
Normal IgG was used as a control for these experiments (Fig.
1A). Because it has not been established that this
trkC antibody can prevent the binding of NT-3 to the trkC receptor (as
opposed to inhibiting trkC function by allosteric interactions)
(Taniuchi and Johnson, 1985; Bothwell, 1995), these data, collectively,
are consistent with the notion that RGCs internalize NT-3 primarily via
trkC receptors rather than p75.
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Figure 1.
A-C, Anterograde
axonal transport of neurotrophic factors and cytochrome
c in chick RGCs. A, B, The
relative amount of anterograde transport of radiolabeled NT-3 was
plotted by dividing the amount measured by gamma counting in the tectum
(counts per minute per specific activity in picograms) by the
amount measured in the eye (counts per minute per specific activity in
nanograms) at the time chick embryos were killed (20 hr after
injection in the eye). A, The effects of coinjection of
monensin (MON) (von Bartheld et al., 1996a),
excess cold NT-3 (cold NT-3), excess cold NGF
(cold NGF), or excess cold BDNF (cold
BDNF), blocking p75 antibody (aP75),
blocking trkC antibody (atrkC), and normal rabbit IgG
(IgG). B, The effects of coinjection of
monensin (MON), tyrosine kinase inhibitor K252a
(K252a), vehicle (DMSO), wortmannin
(WOR), LY294,002 (LY), and
brefeldin A (BFA) (von Bartheld et al., 1996a) are
indicated. C, The relative amount of anterograde
transport of radiolabeled glial cell line-derived neurotrophic factor
(GDNF), cardiotrophin-1 (CT1), and
cytochrome c (CytC) was plotted by
dividing the amount measured by gamma counting in the midbrain
(picograms) by the amount measured in the eye (nanograms) at the time
chick embryos were killed (20 hr after injection in the eye). The
effects of coinjection of monensin (MON), excess
cold GDNF, or CytC, and K252a are indicated. Error bars indicate SEM.
The number of independent experiments is indicated.
***p 0.005; **p 0.01;
*p 0.05.
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Anterograde transport requires tyrosine kinase but not
PI3-kinase activity
To determine whether tyrosine kinase activity may play a role in
the anterograde transport of NT-3, as reported previously for the
sorting of other receptor ligands (Felder et al., 1990; Bos et al.,
1993; Opresko et al., 1995; Mukherjee et al., 1997), radiolabeled NT-3
was coinjected with K252a, a relatively specific tyrosine kinase
inhibitor (Knusel and Hefti, 1992). K252a does not reduce
internalization of NGF (Kahle et al., 1994) or BDNF (von Bartheld et
al., 1996b), yet this compound (1.8 µg per eye) dramatically reduced
anterograde transport of NT-3 (Fig. 1B). Because
K252a has to be dissolved in DMSO, control embryos received intraocular
injections of similar amounts (2 µl) of DMSO with the radiolabeled
NT-3. DMSO alone had no significant effect on the NT-3 transport. The
PI3-kinase inhibitors wortmannin and LY294,002 have been implicated in
protein trafficking and NGF internalization (Vlahos et al., 1994;
Bartlett et al., 1997; Gallo and Letourneau, 1998; Shitara et al.,
1998; Mallet and Maxfield, 1999; York et al., 2000). Therefore, we
tested whether coinjection of these compounds with NT-3 affected
anterograde transport. Neither wortmannin (200 ng; single or three
times repeated doses over 10 hr) nor LY294,002 (0.5 µl of 100 µM) had an effect on anterograde transport of
internalized NT-3 (Fig. 1B). This is surprising,
because internalization of NGF via trkA in PC12 cells is substantially
reduced by PI3-kinase inhibitors (York et al., 2000), but the data may
be explained by differences between neurotrophins, between in
vivo/in vitro approaches, or the time course examined
(minutes vs hours). The inhibitor of endoplasmic reticulum
(ER)-to-Golgi traffic, brefeldin A, has been implicated in transcytosis
of receptor ligands in some cell types but not in others (Hunziker et
al., 1991; Lippincott-Schwartz et al., 1991). Brefeldin A (5-15 µg)
slightly, but significantly, reduced NT-3 transport (Fig.
1B), indicating that this drug does not dramatically
impair endosome trafficking required for NT-3 anterograde transport. We
conclude that the activity of tyrosine kinase, but not PI3-kinase, has
a significant role in the internalization, sorting, and targeting of
NT-3.
Cytochrome c as a control for nonspecific
anterograde transport
To determine whether other proteins with similar size and charge
as neurotrophins can be anterogradely transported, radiolabeled GDNF,
CT-1, and cytochrome c were injected into the eye of chick embryos. Anterograde transport of GDNF was relatively sparse, and
although it was blocked by monensin, it could not be blocked with
excess cold GDNF and thus was not receptor mediated (Fig. 1C). Anterograde transport of CT-1 was even less and barely
measurable (Fig. 1C). By contrast, significant amounts of
cytochrome c were transported to the tectum, with an
efficiency only slightly less than NT-3 (Fig. 1C). The
transport of cytochrome c was blocked almost entirely by
monensin, but the cytochrome c transport could not be
reduced with excess cold cytochrome c (Fig. 1C),
indicating that its transport was not receptor mediated. To determine
whether K252a generally reduces axonal transport, or whether K252a has specific effects on transport of NT-3 (Fig. 1B),
radiolabeled cytochrome c was coinjected with K252a. The
anterograde transport of cytochrome c was not diminished by
K252a (Fig. 1C), showing that blockade of trk tyrosine
kinase activity does not reduce axonal transport in general, but rather
has a specific effect on internalization, sorting, or axonal transport
of NT-3.
Internalization of NT-3 does not require tyrosine
kinase activity
Because anterograde axonal transport of exogenous NT-3 requires
internalization and sorting, as well as axonal transport, K252a could
affect either of these steps or more than one step. It has been shown
that neither NGF (Kahle et al., 1994) nor BDNF (von Bartheld et al.,
1996b) requires tyrosine kinase activity for internalization, but it is
not known whether this holds for NT-3 as well. To determine whether
internalization of NT-3 is diminished in K252a-treated cells and thus
to more precisely localize the effect of K252a on anterograde
transport, we tested whether NT-3 internalization is reduced in
K252a-treated purified RGCs. Internalization of 10-20 ng/ml
125I-NT-3 was not diminished by treatment
with 1 µg/ml K252a (Fig. 2), a dose
that reliably blocks tyrosine kinases including trk receptors (Knusel
and Hefti, 1992). Approximately two-thirds of the NT-3 in the RGCs was
specific internalization, as shown by control experiments at 4°C that
prevent neurotrophin internalization but not binding (Vale and Shooter,
1985) and by cold competition experiments which showed that >80% of
NT-3 binding to RGCs is specific (data not shown). Because it could be
argued that internalization of neurotrophins in vitro and in
cells purified by a step that involves trypsinization may alter the
receptor makeup at the cell surface membranes, we verified that NT-3
internalization in vivo does not require tyrosine kinase
activity by using the isthmo-optic nucleus as a model system as in
previous studies (von Bartheld et al., 1996b). When
125I-NT-3 was coinjected with K252a in the
eye, the retrograde axonal transport of
125I-NT-3 from the eye to the isthmo-optic
nucleus was not significantly diminished (data not shown), indicating
that tyrosine kinase activity is not required for internalization at
axon terminals and retrograde axonal transport of NT-3 in
vivo. Taken together, these data confirm that none of the
neurotrophins so far investigated (NGF, BDNF, or NT-3) require tyrosine
kinase activity for internalization. In a broader perspective, these
data add to the list of trophic receptor functions that do not require
tyrosine kinase activity (Kullander et al., 2001). Furthermore, our
data show that the effect of K252a on anterograde axonal transport of
exogenous NT-3 involves a step subsequent to internalization that is
likely a sorting step (see below).
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Figure 2.
Internalization of 125I-NT-3 (10-20
ng/ml) in purified retinal ganglion cells from E18-21 chick embryos
does not require tyrosine kinase activity. The amount of
internalization in the presence of 1 µg/ml K252a (tyrosine kinase
inhibitor) is plotted as the percentage relative to the values for
vehicle (1 µg/ml DMSO), which were averaged to 100% internalization
(~40,000 cpm per plate). Nonspecific association of NT-3 (at 4°C)
is indicated (dotted line). Error bars indicate SEM. The
number of independent experiments (each in duplicate) is indicated. The
p level for confidence (t test) is
indicated, showing no significant effect of K252a on NT-3
internalization.
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Relative expression levels of p75, trkB, and trkC in the GCL
Cells in the GCL of chick embryos express p75 mRNA (von Bartheld
et al., 1991; Karlsson et al., 1998). Chick GCL cells also express trkB
and trkC (Garner et al., 1996; Hallböök et al., 1996; Das
et al., 1997), but unlike mammalian RGCs (von Bartheld, 1998a), chick
RGCs do not express trkA at the protein or mRNA level (Karlsson et al.,
1998). The relative contributions of RGCs and amacrine cells to
expression levels of neurotrophin receptors are not known. To
semiquantitatively compare levels of expression of p75, trkB, and trkC
in RGCs, E18 retinas were hybridized with neurotrophin receptor probes,
and autoradiographic grains were counted over cells in sections from
the central regions of the retina, where the large majority of cells
comprise RGCs (Ehrlich, 1981). Probes for p75, kinase-specific trkB, or
kinase-specific trkC were used, and background grains (measured in
adjacent control sections hybridized with sense probes) were
subtracted. With this analysis, approximately twice the amount of trkB
than trkC or p75 mRNA was seen in presumptive RGCs (Fig.
3). Because most cells in the central GCL
are RGCs, but with the caveat that message levels do not always reflect
protein levels, we conclude that RGCs probably express significantly
larger amounts of trkB than trkC or p75 at this age (E18).
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Figure 3.
A-D, In
situ hybridization for neurotrophin receptors in the central
region of the ganglion cell layer (GCL) of 16- to
18-d-old chick embryos. This area contains ~85-90% retinal ganglion
cells (Ehrlich, 1981). A, Cells labeled for p75
neurotrophin receptor mRNA. B, Cells labeled with probes
specific for the tyrosine kinase domain of the chick trkB neurotrophin
receptor. C, Cells labeled with probes specific for the
tyrosine kinase domain of the chick trkC receptor. Scale bars, 5 µm.
D, The intensity of labeling
(Grains/Cell) is plotted against the relative
frequency (percentage of cells with this labeling intensity). Note that
trkB-labeled cells contain nearly twice the number of grains as p75- or
trkC-labeled cells (after subtraction of background). The number of
cells sampled is indicated.
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Ultrastructural distribution of NT-3 in RGCs: comparison with NGF
and BDNF
On the basis of the apparent expression of p75, trkB, and trkC
receptors by many RGCs, one would expect that RGCs may internalize NGF
(via p75), BDNF (via p75 or trkB), and NT-3 (via p75, trkB, or trkC).
Thus, binding to different receptors may determine intracellular pathways. To examine whether different members of the neurotrophin family accumulate in the same or in different organelles within RGC
cell bodies, radiolabeled neurotrophins were injected into the vitreous
body in E18-19 embryos, and after 10 hr the tissues were processed to
quantify the distribution of autoradiographic grains at the
ultrastructural level.
For the identification of RGCs within the GCL (which contains RGCs as
well as amacrine cells), we used the fact that RGCs differ considerably
from amacrine cells at the ultrastructural level (Prada et al., 1989;
H. Kolb, personal communication). RGCs have a much more
extensive and characteristic rough ER as compared with amacrine cells
(Fig. 4). For the following analysis,
only those RGCs that could be unambiguously identified and
distinguished from amacrine cells were used.
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Figure 4.
Ultrastructure of a retinal ganglion cell
(RGC) and an amacrine cell (AC) in the
ganglion cell layer of an 18-d-old chick embryo. The plasma membrane of
the RGC is marked by black dots; the plasma membrane of
the AC is marked with asterisks. Note the abundance of
rough endoplasmic reticulum (ER) in the RGC, and the
paucity of such organelles in the AC. G, Golgi system;
M, mitochondrion. This retina was injected with NT-3 and
processed after 10 hr. Scale bar, 1 µm.
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For the quantitative analysis of grain accumulation in RGCs, a
half-distance analysis of source probability (Salpeter et al., 1978)
was applied to a total of ~1700 silver grains from ~650 micrographs
printed at 20,000-40,000× magnification, with at least 100 grains
from two to three retinas analyzed for each treatment group. Examples
of silver grains over five different types of organelles in RGCs are
shown in Figure
5A-E. For control
purposes (to verify that the grains truly represent the respective
neurotrophins), embryos were injected into the vitreous with similar
amounts of denatured radiolabeled NT-3 or with free iodine. There was
no preferential accumulation of grains over particular organelles in
these control experiments (i.e., silver grains precisely reflected the
fractional area of each organelle), consistent with a random background
distribution (Table 1). The distribution
of denatured NT-3 and free iodine matched the percentiles of fractional
areas; accordingly, their labeling density (LD = % grains/%
fractional area) was ~1.0 or less (Table 1). LDs of >3.3 were
statistically significant in our material, whereas LDs of <2.4 were
not significantly different from control groups.
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Figure 5.
A-F, Examples of
accumulation of radiolabeled internalized neurotrophins over organelles
in E18 retinal ganglion cells (A-E) and
tectal cells (F). A, Accumulation
at the plasma membrane (PM); BDNF after 10 hr.
Three silver grains touch the PM. B, Accumulation in
multivesicular bodies (MVB); NT-3 after 20 hr.
C, Accumulation in dense endosomes (ES);
NT-3 after 10 hr. D, Accumulation in the endoplasmic
reticulum (ER); NT-3 after 20 hr. E,
Accumulation in the Golgi apparatus (G) and
Golgi-associated vesicles; NT-3 after 10 hr. F,
Accumulation in a lysosome (LS); NT-3 after 20 hr and
anterograde transport in RGC axons and axo-dendritic transfer to a
tectal cell in a 16-d-old chick embryo. Scale bars (shown in
A for A-D), 500 nm;
(shown in F for E,
F), 500 nm.
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Table 1.
Distribution of autoradiographic silver grains and labeling
densities in organelles of retinal ganglion cells 10 hr after
intraocular injection in chick embryos
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The most striking differences in distribution between the neurotrophins
were the accumulation of NGF and BDNF, but not NT-3, at the plasma
membrane (Table 1), the accumulation of BDNF > NT-3 > NGF
in multivesicular bodies (MVBs) (Table 1), and the accumulation of NT-3
(and, to a lesser extent, of BDNF) in the Golgi apparatus (including
those vesicles that were clearly Golgi-associated) (Fig. 5E,
Table 1). Nearly 20% of the internalized NT-3 in RGC somata was
located within the Golgi system. The Golgi passage of internalized NT-3
is novel, because it has not been described previously for trophic
factors internalized in neurons. The lack of NGF in the Golgi system is
consistent with previous studies on internalized NGF (see Discussion).
Also, the accumulation of all neurotrophins in endosomes (including
lysosomes) is consistent with previous reports. In our analysis, we did
not attempt to distinguish between light endosomes, dark endosomes, and
lysosomes, because these categories have ill-defined criteria (Nixon
and Cataldo, 1995; Mukherjee et al., 1997), resulting in significant variability between investigators. None of the experimental or control
groups showed label in the nucleus or in the nuclear membrane; therefore, these two categories were combined in Tables 1-4. Twenty percent of the NT-3 silver grains were found over the Golgi system, and
the same percentage was previously estimated to be transported to the
optic tectum (von Bartheld et al., 1996a). We conclude that ~20% of
the NT-3 within RGCs follows the Golgi and potential anterograde
transport pathway. We calculate that on average each RGC contained
1000-2000 NT-3 dimers and transported 20% = 200-400 NT-3 dimers to
the tectum. Collectively, these data show that neurotrophins have
different patterns of accumulation, probably reflecting different
intracellular pathways of internalized receptor-ligand complexes.
Time course of NGF, BDNF, and NT-3 accumulation: 3, 10, and
20 hr
Because the 10 hr time point after internalization presents only a
snapshot of the distribution and does not reveal kinetics of
processing, the 10 hr time point was compared with an early (3 hr) and
a late (20 hr) time point after injection of the radiolabeled neurotrophins NGF, BDNF, and NT-3 in the eye. NGF accumulated 3 hr
after injection in the plasma membrane, with a subsequent decrease of
labeling in the plasma membrane and an increased targeting to endosomes
and lysosomes. Much of the BDNF was found 3 and 10 hr after injection
at the plasma membrane, then it accumulated at 10-20 hr in the ER and
endosomes (Table 2). NT-3 was strikingly absent from the plasma membrane at all time points examined (3, 10, and
20 hr), but accumulated at 3 and 10 hr in the Golgi system, and at
10-20 hr in MVBs and endosomes/lysosomes, whereas NT-3 decreased in
the Golgi system at 20 hr (Table 2). These dynamic patterns of
distribution confirm the hypothesis that different neurotrophins follow
different intracellular pathways after internalization in RGCs,
especially for the plasma membrane and the Golgi system.
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Table 2.
Distribution of autoradiographic silver grains representing
NGF, BDNF, and NT-3 and their labeling densities in organelles of
retinal ganglion cells 3, 10, and 20 hr after intraocular injection in
chick embryos
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Effect of K252a on the distribution of BDNF and NT-3 in RGCs
Tyrosine kinase activity has been implicated in sorting of
internalized receptor-ligand complexes (Felder et al., 1990; Bos et
al., 1993; Opresko et al., 1995). Because coinjection of K252a (but not
DMSO vehicle alone) substantially reduced the anterograde transport of
exogenous NT-3 (Fig. 1B), but not its internalization (Fig. 2), we tested the hypothesis that blockade of trk activity may
alter the pattern of NT-3 accumulation and specifically that K252a
may reduce the accumulation of NT-3 in the Golgi system, which is
known to be required for anterograde axonal transport of newly
synthesized proteins (Hammerschlag et al., 1982).
Coinjection of K252a significantly altered the pattern of accumulation
of BDNF and NT-3 in RGCs after 10 hr. The accumulation of NT-3 and BDNF
in the Golgi apparatus was abolished. The labeling densities dropped
from 4.56 to 0.76 (NT-3) and 2.39 to 0.51 (BDNF) (Fig. 6A, Table
3), whereas the accumulation of BDNF and
NT-3 in endosomes and lysosomes was much increased (Fig.
6B; Table 3). In addition to the effect on the
endosomes and lysosomes, K252a also increased the labeling density of
NT-3 at the plasma membrane, making the NT-3 pattern more similar to
that seen with NGF and BDNF (compare Tables 1 and 3). Other patterns of
distribution were not much altered, with the exception of a decrease of
the BDNF labeling density for MVBs (reduction from 6.8 to 1.5). These data confirm that tyrosine kinase activity (presumably trkC activity) sorts NT-3 into the Golgi-anterograde pathway, whereas its absence directs the neurotrophins into the degradative lysosomal pathway or
increases their retention at, or recycling to, the plasma membrane.
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Figure 6.
A-D, Effect of
K252a and trkC activity on the distribution of internalized
neurotrophins in retinal ganglion cells (RGC)
(A-C) and SDS-PAGE analysis of
internalized neurotrophins in purified RGCs (D).
A, Labeling densities of internalized BDNF and NT-3 in
the Golgi system of RGCs with and without K252a. The analysis was done
in triplicate; significance was determined by unpaired t
test. B, Labeling densities of internalized BDNF and
NT-3 in lysosomes and endosomes of RGCs with and without K252a. The
analysis was done in triplicate; significance was determined by
unpaired t test. C, Quantification of
anterograde transport of 50-80 ng radiolabeled NGF when coinjected in
the eye with 50-60 ng cold NT-3 or BDNF. The number of experiments is
indicated. Significance was determined by unpaired t
test. Error bars indicate SEM. D, SDS-PAGE (15%) of
internalized neurotrophins NGF, BDNF, and NT-3 recovered from purified
immunopanned RGCs after 10 hr. Each sample was run with an adjacent
sample of native same factor (Na). The molecular weight
is indicated. Arrow indicates the dye front. Note that
much of the BDNF recovered from RGCs is cleaved, whereas virtually all
the NGF and NT-3 is intact protein by this analysis.
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Table 3.
Distribution of autoradiographic silver grains and labeling
densities in organelles of retinal ganglion cells 10 hr after
intraocular injection of BDNF or NT-3 in chick embryos with or without
the tyrosine kinase inhibitor K252a
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Comparison with accumulation after axo-dendritic transfer
in tectum
Different cell types may traffic internalized neurotrophins into
different compartments. To determine subcellular routes of internalized
NT-3 in neurons other than RGCs, we examined the pattern of
accumulation of NT-3 after axo-dendritic transfer from RGC axons and
internalization into tectal neurons.
125I-NT-3 was injected into the eye, and
20 hr later samples of the contralateral tectum were processed for
autoradiography. When silver grains over neuronal cell bodies in the
tectum were analyzed, NT-3 was found to be similarly concentrated over
MVBs and lyso/endosomes as in RGCs (Tables 1,
4), but NT-3 did not accumulate in the Golgi system of tectal neurons (Table 4). It is not known whether these
neurons anterogradely transport internalized NT-3. Nevertheless, these
data indicate that there are differences between cell types in the
trafficking of the same neurotrophin.
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Table 4.
Distribution of autoradiographic silver grains and labeling
densities in organelles of tectal neurons after intraocular injection
and anterograde axonal transport and axo-dendritic transfer of NT-3 in
chick embryos
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NT-3 directs NGF into the anterograde pathway
If our hypothesis is correct that trkC activity
enhances the sorting of internalized NT-3 into the anterograde axonal
pathway, then one would predict that coinjection of a small amount of
NT-3 (to activate trkC) with NGF (which binds to p75, but not trkC) may
facilitate the anterograde transport of NGF, provided that they are
internalized into the same vesicle or endosome. A positive result would
also indicate that the membranes of the internalized vesicles contain
both types of receptors, trkC as well as p75. To test this hypothesis,
50-100 ng radiolabeled NGF was coinjected with 50-60 ng cold NT-3, or
50-60 ng cold BDNF as a negative control, into the eye of 15- to
16-d-old chick embryos, and the amount of anterogradely transported NGF
relative to the amount retained in the eye at the time embryos were
killed was calculated. Consistent with our prediction, coinjection of
cold NT-3 increased the anterograde transport of radiolabeled NGF
significantly, by over 27%, whereas coinjection of cold BDNF had no
effect (Fig. 6C). These data support the notion that
activated trkC, but not activated trkB, directs the internalized
neurotrophin-receptor complex into an anterograde transport pathway.
These data also indicate that a significant fraction of neurotrophins
are internalized in, or at least transiently merge with, vesicles with
membranes that contain both p75 and trkC receptors.
Cleavage and degradation of internalized neurotrophins in
purified RGCs
Rapid degradation of internalized neurotrophins has been reported
for NGF in sympathetic neurons, sensory ganglion cells, enteric cells,
and PC12 cells (Johnson et al., 1978; Siminoski et al., 1986; Eveleth
and Bradshaw, 1992; Ure and Campenot, 1997), but not for BDNF
internalized in cultured astrocytes and Schwann cells (Rubio, 1997;
Alderson et al., 2000), and relatively slow "clipping" of
transcytosing bFGF (Ferguson et al., 1990). Compared with
control experiments using free iodine and denatured NT-3, internalized
intact NT-3 distributes differently and thus likely represents ligand
that still can bind to its receptor. Autoradiography of tissue
sections, however, does not assess the extent of possible degradation
after internalization. To determine the extent to which internalized
neurotrophins are degraded in RGCs, RGCs were purified 10-12 hr after
intraocular injection of radiolabeled neurotrophins, and the recovered
radioactive samples were subjected to either TCA precipitation or
SDS-PAGE analysis. Because we know the yield of immunopanned RGCs
(~100,000-200,000 per retina at this age) and the amount of
radioactivity and the specific activity of radiolabeled NT-3, we can
calculate that on average, each RGC internalized ~0.1 fg = 2000 dimers NT-3 in the cell body (excluding dendrites/axons, and possibly
excluding surface binding that may have been removed by the extensive
washes). TCA precipitation showed that incorporation was unchanged
compared with the native radiolabeled neurotrophin (data not shown),
indicating that very little, if any, ligand was degraded into free
iodine, or that such iodine was rapidly released from the RGCs. When
the lysed cell extracts of purified RGC somata were analyzed on 15%
SDS-PAGE, the NGF and NT-3 migrated as bands that were
indistinguishable from the native radiolabeled protein, whereas BDNF
showed multiple bands, with a major band at ~10 kDa, a minor band at
14 kDa, and a faint band close to the dye front at ~3-5 kDa (Fig.
6D), where small degradation products (but not
necessarily free iodide) can be expected. Cleavage was not apparent
when 125I-BDNF was recovered from whole
retinal extracts, but only when RGCs were purified, and the same
pattern of cleavage was seen when native BDNF was incubated with
trypsin (data not shown). One possible explanation for differences in
neurotrophin degradation is that a major fraction of the BDNF
internalized in RGCs was exposed to trypsin during the 10 min digestion
for dissociation. Such data may point to a rapid recycling of BDNF (and
its receptor, presumably trkB) between endosomes and the surface
membrane. This cleavage does not seem to render the major BDNF fragment
functionally impaired [consistent with previous reports for NGF
(Mercanti et al., 1977) and bFGF (Ferguson et al., 1990)], because
125I-BDNF cleaved with trypsin was
axonally transported in a retrograde transport assay (data not shown).
These results indicate that major fractions of each of the three
neurotrophins may be differentially processed after internalization in
RGCs, presumably by targeting into different compartments or
intracellular pathways, so that NGF is predominantly targeted to
lysosomes, BDNF is recycled to the surface membrane, and NT-3 is
targeted to the Golgi-anterograde pathway.
Cross-linking and immunoprecipitation of internalized neurotrophins
in purified RGCs: internalization of NT-3 via trkC receptors
Because many different cell types in the retina express
neurotrophin receptors (von Bartheld, 1998a), neurotrophins may bind to
many different cell types after injection in the eye. To determine which receptors bind radiolabeled neurotrophins after intraocular injections specifically in RGCs, we purified RGCs, and then
cross-linked the neurotrophins with EDC or DSS and immunoprecipitated
with antibodies specific for chicken p75, chicken trkA, chicken trkB, or chicken trkC (Weskamp and Reichardt, 1991; von Bartheld et al.,
1996b; Lefcort et al., 1996). These antibodies are thought to have
roughly similar precipitation efficiencies (Herzog and von Bartheld,
1998) and allow a tentative qualitative comparison of receptor binding.
Normal IgG was included in each experiment to determine nonspecific
precipitation, which was subtracted from each data set to calculate
specific precipitation. Data obtained with this technique have to be
interpreted with caution and allow one to make conclusions only about
relative changes in receptor association, rather than absolute levels
of receptor binding, because the efficiencies of cross-linking and
immunoprecipitation may vary. Nonspecific precipitation, generally
~1-6%, was subtracted from the total to calculate specific
precipitation. Typically, the total amount precipitated by
receptor-specific antibodies (after subtraction of nonspecific
precipitation) was 12-30% of the total amount of radiolabeled
neurotrophins in the preparation.
At 12 hr after injection, internalized BDNF bound to trkB and p75
receptors with a ratio of ~1:1, whereas NT-3 bound trkC almost
exclusively with a ratio of 12:1 (trkC/p75) (Fig.
7C). There was no specific
precipitation of BDNF or NT-3 with trkA antibodies, indicating that
BDNF and NT-3 did not bind to trkA (data not shown). The lack of
binding of NT-3 to p75 does not appear to be attributable to technical
reasons, because the p75 antibody immunoprecipitated much of the
receptor-bound BDNF (~40%) (Fig. 7C), and some NGF was
precipitated with p75 antibodies (data not shown). There was no
significant difference between the two cross-linkers used, EDC and DSS.
The binding of NT-3 to the trkC receptor in RGCs was transient, because
its precipitation was much reduced at 44 hr after injection compared
with 12 hr after injection (Fig. 7C). The data do not allow
us to rule out that NT-3 may bind p75 receptors at the cell surface,
but if it does, the NT-3 does not seem to be internalized by this
receptor type. We conclude that trkC, rather than p75 or trkB, binds
NT-3 during the initial internalization and sorting steps within RGCs,
consistent with the lack of effects of either excess cold NGF or BDNF
competition or p75 blocking antibody in the coinjection experiments
described above.
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Figure 7.
A-C, Cross-linking
and immunoprecipitation of radiolabeled neurotrophins internalized and
anterogradely transported by retinal ganglion cells
(RGCs). A, Binding of NT-3 to p75, trkB,
and trkC receptors cross-linked with EDC or DSS and immunoprecipitated
with chicken-specific neurotrophin receptor antibodies. The relative
amount of total specific precipitation (after subtraction of
nonspecific precipitation) is shown separately for each tissue
(purified RGCs, optic chiasm, and optic tectum) and for each receptor.
Totals for p75, trkB, and trkC (from top to
bottom) add up to 100% for each tissue and
cross-linker. B, Binding of NT-3 to receptors when
radiolabeled NT-3 was added to the same tissues and then cross-linked
and immunoprecipitated. Note the much more extensive precipitation with
trkB antibodies (binding to trkB) when NT-3 was added to normal lysates
of the same tissues rather than introduction of NT-3 in
vivo. C, Immunoprecipitation of internalized
radiolabeled BDNF and NT-3 binding to receptors in RGCs purified by
immunopanning. Note that BDNF binds to p75 as well as trkB, whereas
NT-3 binds almost exclusively to trkC receptors at 12 hr; binding of
NT-3 to trkC in RGCs is transient, because it is much reduced 44 hr
after injection (C). Error bars indicate SEM. The
number of experiments is indicated.
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Cross-linking and immunoprecipitation of NT-3 during anterograde
transport: roles of trkC and p75
To determine whether NT-3 remains bound to trkC during anterograde
axonal transport and after arrival in the optic tectum, we dissected
the retina, optic chiasm, and contralateral optic tectum 20 hr after
injection of the radiolabeled NT-3 in the eye, cross-linked with either
EDC or DSS, and immunoprecipitated with chick p75, trkB, or trkC
antibodies. Because it can be assumed that cross-linking and
precipitation efficiencies do not change between preparations from
retina, chiasm, and tectum for the same neurotrophin, this analysis
should reveal major potential changes in the ratios of receptor
binding. Therefore, the data are presented as percentages of the ratios
of receptor binding, with the total binding to trkB, trkC, and p75
combined = 100% (Fig.
7A,B).
The cross-linking and immunoprecipitation experiments revealed that, in
the retina, ~60% of immunoprecipitated NT-3 bound to p75, and
~35% to trkC (data not shown). The difference compared with the
purified RGC cell bodies is likely attributable to the substantial
binding of neurotrophins in the inner plexiform layer, which contains
mostly neuropil. Here, p75 binding appears to predominate, consistent
with the distribution of this receptor by immunolabeling (Das et al.,
1997; Herzog and von Bartheld, 1998). In the optic chiasm, a
significant fraction (60%) bound to p75 and less (25%) to trkC, but
in the tectum the ratio between p75 (~30%) and trkC (50-60%)
shifted back in favor of trkC (Fig. 7A). The percentages for
cross-linking with either EDC or DSS were very similar, with DSS
showing slightly higher ratios for the trk receptor, and EDC showing
slightly higher ratios for the p75 receptor. This is in keeping with the known higher efficiencies of cross-linking for DSS
(trks) and EDC (p75) (Escandon et al., 1993). Taken together, these
data are consistent with the notion that NT-3 is internalized by
trkC, then dissociates from trkC and gains access to p75, presumably as
a "transporter" in RGC fibers within the optic nerve and tract (von
Bartheld and Butowt, 2000). After arrival in the tectum, the p75-bound
NT-3 is released and presumably binds to trkC on postsynaptic tectal
target neurons (von Bartheld et al., 1996a). P75 has previously been
identified as a trkB-ligand-specific retrograde transporter in
projection neurons (Curtis et al., 1995).
Control experiments for cross-linking and immunoprecipitation
Control experiments were performed to verify that neurotrophins do
not massively dissociate from their receptors and bind to other,
previously not occupied, receptors that become available in the lysis
step. When NT-3 was added to the untreated retina, chiasm, or tectum,
the ratios of binding were significantly different. Notably, added NT-3
bound much more to trkB than it did when it was introduced into the RGC
axons by intraocular injection, internalization, and anterograde axonal
transport (Fig. 7B), indicating that many trkB receptors are
present in fibers within the optic nerve that are unoccupied. When the
cross-linking step was omitted, much neurotrophin binding to p75 was
lost, whereas binding to trk receptors was maintained (data not shown),
consistent with the notion that p75 receptors are "fast" receptors
that bind ligands with a fast rate of dissociation, whereas trk
receptors are thought to bind ligands with high affinity and release
the neurotrophins slowly (Barker and Murphy, 1992; Bothwell, 1995).
Because NT-3 also binds to isthmo-optic terminals and is transported
retrogradely in the optic nerve (von Bartheld et al., 1996b), it was
necessary to rule out a significant contribution of neurotrophin
binding to the 10,000 isthmo-optic nerve fibers (Clarke et al., 1976),
as opposed to the 2.5 × 106 RGC
fibers in the optic nerve (Rager, 1980). Injection of monensin in the
eye abolishes anterograde transport of neurotrophins but leaves
retrograde axonal transport via isthmo-optic fibers undisturbed (von
Bartheld et al., 1996b). When monensin was coinjected, NT-3 binding to
receptors in the isthmo-optic fibers was similar to the NT-3 binding
for combined RGC fibers + isthmo-optic fibers in the chiasm (data not
shown). Because the isthmo-optic nucleus contributes <10% of the
total axonal transport of neurotrophins from the retina to the brain,
as revealed by coinjection experiments with monensin (von Bartheld et
al., 1996b), the ratios observed would be minimally altered because of
"contamination" by ~0.5% isthmo-optic fibers among 99.5% RGC
fibers in the optic nerve.
In conclusion, the receptor binding data suggest intriguing dynamics of
receptor association and dissociation, namely the initial binding of
NT-3 to trkC in the RGC cell body, followed by trafficking in which
trkC binding is diminished and p75 binding is increased. This likely
represents the fusion of trkC endosomes with p75-containing membranes
in the Golgi. The subsequently increased binding of NT-3 to trkC in the
tectum is likely caused by the binding of NT-3 to trkC after its
release from RGC terminals, uptake into tectal neurons, and
accumulation in MVBs (von Bartheld et al., 1996a). The proposed model
of NT-3 trafficking in RGCs and in the retinotectal projection is
schematically summarized in Figure 8.
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Figure 8.
Diagram summarizing proposed
subcellular pathways of internalized NT-3 (black dots)
in a retinal ganglion cell. At least two pathways of internalized NT-3
can be distinguished. A lysosomal pathway of NT-3 may be common to all
neurotrophins and may involve binding to the p75 receptor
(U) or binding of NT-3 to trkC receptor
(Y) in the presence of the tyrosine kinase
inhibitor K252a. The neurotrophin is degraded in lysosomes
(LYS). Alternatively, a novel pathway of NT-3
internalized in endosomes fuses with membranes of the Golgi apparatus:
Golgi Pathway. This sorting requires tyrosine kinase
activity (presumably trkC, Y), and
this pathway may join that of newly synthesized neurotrophins as well
as p75 receptor (U) from the endoplasmic
reticulum (ER) via the Golgi into an anterograde axonal
path (von Bartheld et al., 2001). After passage through the Golgi
system, internalized NT-3 is packaged in presumptive large dense-core
vesicles (LDCV) (Wang et al., 2001) for
anterograde axonal transport. In this pathway, internalized NT-3 binds
preferentially to p75. Anterogradely transported NT-3 is released from
RGC axon terminals in the tectum, and after release it binds
predominantly to trkC in tectal cells where it accumulates in
multivesicular bodies (MVB) (von Bartheld et al.,
1996a).
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DISCUSSION |
The subcellular distribution and pathways of internalized
neurotrophins, NGF, BDNF, and NT-3, were examined and compared in RGCs.
Internalized BDNF and NT-3 can follow a novel pathway into the Golgi
apparatus, not previously described for trophic factors in neurons, and
this pathway correlates with anterograde axonal transport. Tyrosine
kinase activity is required for sorting into this pathway, but not for
internalization. Rather than being rapidly degraded after
internalization, a significant fraction of exogenous NT-3 is targeted
within neurons and recycled for further uses.
Internalization of neurotrophins: roles of receptors
All three internalized neurotrophins accumulated in MVBs and
endosomes. This is remarkable for NGF, because chick RGCs do not
possess trkA receptors (Karlsson et al., 1998), indicating that NGF was
internalized by the p75 receptor, as shown previously for glial cells
(Kahle and Hertel, 1992). There were significant subcellular
differences in the labeling densities between neurotrophins. BDNF and
NGF accumulated at the plasma membrane, indicating retention, a slower
internalization, or possibly recycling of these neurotrophins after
internalization (Eveleth and Bradshaw, 1988, 1992). By contrast, NT-3
was conspicuously absent from the plasma membrane (Tables 1, 2). NT-3
may be more efficiently or rapidly internalized than BDNF and NGF, or
may lack a recycling pathway to the cell surface membrane. Consistent
with previous studies, internalized NGF did not accumulate in the Golgi
system (Schwab and Thoenen, 1977; Claude et al., 1982; Bernd and
Greene, 1983; Stieber et al., 1984
TOP
ABSTRACT
INTRODUCTION
