Download Signalling organelle for retrograde axonal transport of

Immunology and Cell Biology (2000) 78, 430–435
Curtin Conference
Signalling organelle for retrograde axonal transport of internalized
neurotrophins from the nerve terminal
S L S A N D OW, K H E Y D O N, M W W E I B L E I I , A J R E Y N O L D S , S E BA RT L E T T a n d
I A H E N D RY
Developmental Neurobiology Group, Division of Neuroscience, John Curtin School of Medical Research, Australian
National University, Canberra, Australian Capital Territory, Australia
Summary The retrograde axonal transport of neurotrophins occurs after receptor-mediated endocytosis into
vesicles at the nerve terminal. We have been investigating the process of targeting these vesicles for retrograde
transport, by examining the transport of [ I]-labelled neurotrophins from the eye to sympathetic and sensory
ganglia. With the aid of confocal microscopy, we examined the phenomena further in cultures of dissociated
sympathetic ganglia to which rhodamine-labelled nerve growth factor (NGF) was added. We found the label in
large vesicles in the growth cone and axons. Light microscopic examination of the sympathetic nerve trunk in vivo
also showed the retrogradely transported material to be sporadically located in large structures in the axons. Ultrastructural examination of the sympathetic nerve trunk after the transport of NGF bound to gold particles showed
the label to be concentrated in relatively few large organelles that consisted of accumulations of multivesicular
bodies. These results suggest that in vivo NGF is transported in specialized organelles that require assembly in the
nerve terminal.
125
Key words: confocal microscopy, electron microscopy, nerve growth factor, retrograde axonal transport, vesicle
recycling.
Introduction
Neurons require contact with their target tissue in order to
survive and make correct connections.1 In order to achieve
this result, developing neurons derive survival information
from the target tissue. This survival, differentiation and maintenance information for responsive neurons is mediated by
neurotrophic factors,2 which are secreted by target tissues and
interact with receptors on the axon tip.3 These neurotrophins
are members of the superfamily containing nerve growth
factor (NGF),4 brain-derived neurotrophic factor (BDNF),5
neurotrophic factor 3 (NT-3),6 neurotrophin 4 (NT-4),7
neurotrophin 6 (NT-6)8 and neurotrophin 7 (NT-7).9 However,
the mechanism by which the neurotrophin signal is propagated from axon terminal to cell body, which can be more
than 1 m away, to influence biochemical events critical for
growth and survival of neurons remains unclear. Retrograde
axonal transport of neurotrophins is the simplest explanation.4 In this model, neurotrophins are taken up at the nerve
terminal using the process of receptor-mediated endocytosis.10 A complex of the neurotrophin together with its activated receptor and associated second messenger molecules is
transported back to the cell body.11 It has been suggested
that, in the adult mammalian nervous system, a cascade of
activated tyrosine receptor kinase (Trk) receptors may
Correspondence: Prof. IA Hendry, Developmental Neurobiology
Group, Division of Neuroscience, The John Curtin School of
Medical Research, The Australian National University, GPO Box
334, Canberra, ACT 2601, Australia. Email: [email protected]
Received 10 April 2000; accepted 10 April 2000.
function as rapid retrograde signal carriers.12,13 However, it is
more likely that the neurotrophins stimulate retrograde transport of the activated Trks, bound to their cognate ligand, to
transmit this information14 by delivering an activated receptor to the cell body.15
Neurotrophins have two types of receptor: the high-affinity Trk family of tyrosine kinase receptors16 and the lowaffinity p75 neurotrophin receptor (p75).17 All neurotrophins
bind to the low-affinity receptor with similar affinity. Nerve
growth factor binds to TrkA,18 NT-3 binds to TrkC19 and to
some extent to TrkA,20 while BDNF and NT-4 bind to TrkB.21
What is not clear is which population of receptors is responsible for retrograde axonal transport of various neurotrophins.
The retrograde axonal transport of TrkA is stimulated by
treatment with NGF in the mammalian nervous system.14 The
retrograde transport of TrkB has been shown in the chick
visual system.22 Retrograde transport of p75 has been demonstrated23 and transport of the TrkB ligands, NT-4 and BDNF,
to sensory neurons is dependent on p75.24 This has been
shown by pharmacological manipulation of p75 in vivo using
a p75 antibody, which completely blocked retrograde transport of NT-4 and BDNF to sensory neurons despite the presence of trkB, while having minimal effects on the transport of
NGF in either sensory or sympathetic neurons. In addition, in
mice with a null mutation of p75, the transport of NT-4 and
BDNF, but not NGF, is dramatically reduced. Thus, NGF
appears to be able to be retrogradely transported by both
TrkA and p75.24
Nerve growth factor induces rapid and extensive endocytosis of TrkA in the pheocytochroma cell line PC12,25 possibly being internalized by clathrin-mediated endocytosis.26
Neurotrophin retrograde transport
Nerve growth factor remains bound to TrkA in endocytic
organelles. This TrkA is tyrosine-phosphorylated and bound
to phospholipase C (PLC)-γ1, suggesting that these receptors
are competent to initiate signal transduction.10 Internalization
of NGF and its receptor tyrosine kinase TrkA and their transport to the cell body are required for transmission of this
signal. Tyrosine kinase activity of TrkA is required to maintain it in an autophosphorylated state on its arrival in the cell
body and for propagation of the signal to cAMP response
element-binding protein (CREB) within neuronal nuclei.
Thus, an NGF-TrkA complex is a messenger that delivers the
NGF signal from axon terminals to cell bodies of sympathetic neurons.11 We have shown that there is a phase in the
process of taking the neurotrophins from the extracellular
space, before their transport, when the sequestered neurotrophin is easily exchanged with unbound neurotrophin.
The unexpected finding was that at a time when neurotrophins should be trapped inside vesicles within the nerve
terminal, the contents of these vesicles could be displaced by
an excess of the neurotrophin.
The present paper seeks to examine more closely the
physical processes that occur at the nerve terminal, of
binding, internalization and targeting of neurotrophins for
transport, using a combination of light and electron
microscopy. We have shown that NGF is transported in relatively few very large organelles made up of clusters of multivesicular bodies. This appears to be the physical correlate of
the signalling endosome described by Beattie et al.27
Materials and Methods
β-Nerve growth factor was purified from male mouse salivary glands
in our laboratory as previously described.28 Sodium bicarbonate,
HEPES and Dulbecco’s modified Eagle’s medium (DMEM) were
purchased from Sigma (St Louis, MO, USA). Unless otherwise
stated, all reagents were of the highest available purity and purchased
from commercial suppliers.
431
trigeminal ganglia (TGG) was measured using a method previously
described.33 Briefly, adult male CBA mice were anaesthetized using
88 µg/g ketamine and 16 µg/g rompun (i.p.) and 1 µL containing 148
kBq [125I]-labelled βNGF (22 ng), 111 kBq [125I]-labelled NT-3
(16 ng) and 148 kBq [125I]-labelled NT-4 (25 ng) was injected into
the right anterior eye chamber. Unlabelled neurotrophins were
injected immediately before the 125I-neurotrophin, using the same
injection site, and were placed in the same region within the anterior
chamber as the 125I-neurotrophin. Thus, terminals of the sensory and
sympathetic neurons were exposed to the same concentrations of
compounds. After 20 h, the animals were killed, both SCG and TGG
were dissected out and accumulated radioactivity was determined
using a Packard γ-counter (Packard Instrument Company, Meriden,
CT, USA). The amount of 125I-neurotrophins retrogradely transported
within the neurons was calculated by subtracting the amount of
radioactivity present on the contralateral side from that present on
the injected side. 125I-β-Nerve growth factor was not degraded after
retrograde transport and retained its antigenicity and electrophoretic
mobility following SDS-PAGE.33 A minimum of five animals was
used in each experiment.
Rhodamine- and gold-labelled NGF were injected at a concentration of 1 µg/µL into the anterior eye chamber of mice. The postganglionic sympathetic nerve trunk was ligated adjacent to the SCG
after the eye injection and the animals were left for 16 h for transport
to occur. The ligated nerves were dissected and mounted in buffered
saline for viewing with the confocal microscope or fixed and
processed for electron microscopy using standard methods.
Dissociated neuronal cultures
Newborn (P0) mouse SCG were dissected and dissociated as
described previously.34,35 Following dissociation, the cell suspension
was plated out into rose chambers36 coated with poly-L-lysine
(0.1 mg/mL) and laminin (0.5 µg/mL). The cell cultures were maintained with a constant concentration of NGF (100 ng/mL) in
40 mmol/L HEPES/DMEM medium containing 0.1% heat-inactivated FCS and were incubated for 24 h at 37°C in a humidified
atmosphere. Cultures were washed in NGF-free medium and incubated with rhodamine-labelled NGF at 20 ng/mL and fluorescence
observed using a Leitz confocal microscope at 30°C.
Labelling of neurotrophins
β-Nerve growth factor was labelled with rhodamine B isothiocyanate
(RITC; Molecular probes, Eugene, OR, USA) using the method
described by the manufacturers. In brief, 400 µg NGF in 200 µL 0.5
mol/L NaHCO3 pH 8 was mixed with 10 mg RITC in 100 µL DMSO
for 3 h at room temperature and the excess rhodamine was removed
by dialysis against three changes of 5 L of 0.2% acetic acid for 24 h
at 4°C. The final product was free from unbound rhodamine and
retained full biological activity as tested in its survival-promoting
activity for newborn mouse sympathetic neurons.
Neurotrophins were labelled with Na125I (Amersham Pharmacia
Biotech AB, Uppsala, Sweden) to a specific activity of between 60
and 130 GBq/mol using the iodogen method.29,30 The 15 nm gold
beads were made as previously described31 and these were coupled
to NGF using the method described by Slot and Geuze.32
Retrograde axonal transport of rhodamine, gold and
[125I]-labelled βNGF
Experimental protocols followed were approved by the Australian
National University Animal Ethics Committee. The retrograde
axonal transport of 125I-neurotrophins in sympathetic neurons to the
superior cervical ganglia (SCG) and in sensory neurons to the
Results
Interaction of neurotrophins during retrograde transport
In order to ascertain the interactions between the neurotrophins and their various receptors, we carried out a series of
cross-inhibition studies (Table 1). The retrograde transport of
125
I-NGF, which binds to both TrkA and p75 in the sympathetic system, was completely inhibited by a 50-fold excess
of unlabelled NGF, while it was only poorly inhibited by
BDNF, NT-3 and NT-4. The retrograde transport of 125I-NT-3,
which binds to both TrkC and p75, was completely inhibited
by a 50-fold excess of unlabelled NT-3. 125I-NT-3 transport
was inhibited 30–50% by NGF, BDNF and NT-4 in the
sensory system, but NGF and BDNF only slightly reduced
its transport in the sympathetic system. The transport of
125
I-NT-4, which binds to TrkB and p75, was also blocked by
a 50-fold excess of unlabelled NT-4 and was the most
strongly affected by the other neurotrophins, being reduced
below 20% by NT-3 and below 40% by BDNF and NGF.
Because there is no TrkB in the sympathetic system, it would
appear that the transport of NT-4 is mediated only by p75
binding.
432
Table 1
SL Sandow et al.
Cross-inhibition of retrograde transport of neurotrophins in sympathetic and sensory systems
125
NGF
BDNF
NT-3
NT-4
I-NGF
8±1
51 ± 7
60 ± 9
44 ± 8
Superior cervical ganglion
125
125
I-NT-3
I-NT-4
73 ± 11
75 ± 8
12 ± 4
31 ± 5
32 ± 6
39 ± 7
15 ± 5
14 ± 3
I-NGF
Trigeminal ganglion
125
I-NT-3
13 ± 2
51 ± 17
60 ± 11
82 ± 9
40 ± 9
30 ± 5
15 ± 6
55 ± 4
125
125
I-NT-4
24 ± 5
46 ± 11
17 ± 2
20 ± 6
An aliquot (1 µL) containing 148 kBq 125I-labelled β-nerve growth factor (NGF, 22 ng), 111 kBq 125I-labelled neurotrophic factor (NT)-3 (16
ng) and 148 kBq 125I-labelled NT-4 (25 ng) was injected into the right anterior eye chamber. Unlabelled neurotrophins NGF, brain-derived neurotrophic factor (BDNF), NT-3 and NT-4 were injected immediately before the 125I-neurotrophin, using the same injection site, and placed in the
same region within the anterior chamber as the 125I-neurotrophin. Values are the percentage of neurotrophin transport in the absence of unlabelled
neurotrophin and are the mean ± SEM for groups of five or six animals.
In vivo transport of rhodamine-labelled NGF
The rhodamine-labelled NGF was injected to the anterior eye
chamber and the ipsilateral SCG dissected out 6 h later and
examined using the confocal microscope. The fluorescent
material was seen to accumulate in axons in the nerve trunk,
showing that it is successfully transported in vivo (Fig. 2a).
At higher power, it can be seen that the labelled material is in
large vesicle-like structures, arranged quite sparsely along
the axons (Fig. 2b,c).
In vivo transport of gold-labelled NGF
Figure 1 Time-lapse confocal micrograph of newborn mouse
superior cervical ganglion neurons grown in culture in the
presence of 20 ng/mL rhodamine-nerve growth factor (NGF).
Vesicles (arrowed) can be seen in the neurite moving away from
the growth cone. The frames are at 30 s intervals. It can be seen
that the vesicles have a saltatory movement, with the fastest
moving vesicle at this temperature (30°C) reaching a speed of
approximately 10 µm/min.
Visualization of internalized rhodamine NGF by confocal
microscopy
We examined cultures of sympathetic ganglia labelled with
rhodamine-NGF so as to visualize the uptake process in the
nerve terminal and growth cone. Immediately after addition
of labelled NGF, the membrane of the axons and growth cone
were uniformly fluorescent, with the brightness of this fluorescence increasing over the first 15–30 min. After the cells
had been in contact with NGF for approximately 1 h, vesiclelike structures could be seen inside the terminal regions. A
series of time-lapse pictures was taken at 30 s intervals to
show the movement of the organelles. Figure 1 shows that
there are few large labelled vesicle-like structures in the neurites that move towards the cell body.
A similar injection regime was used to examine the ultrastructure of the organelles transporting NGF. There was an
accumulation of the gold-labelled NGF in organelles in the
axon at a site near the SCG (Fig. 3). These multivesicular
organelles consisted of accumulations of multivesicular
bodies, which contained many gold particles in each cluster.
The size of the bolus was considerably larger than the diameter of the axon containing it. Figure 3 shows four serial
sections through such a bolus of multivesicular bodies, with
the axon at either end of the structure (Fig. 3b,d). However,
the number of such structures was quite infrequent and no
gold label was seen in other structures at this site in the axon.
Discussion
The process of retrograde axonal transport can be separated
into distinct phases. First there is receptor binding followed
by internalization of the ligand, then the internalized
organelle must be targeted for retrograde transport.37 The retrograde transport process itself follows this. When the active
complex arrives at the cell body, it must generate the appropriate second messenger cascade to signal to the nucleus.15
This paper looks in more detail at the steps of receptor
binding and internalization and the resultant structural correlate of the retrograde signal.
Much of the binding data presented here can be explained
by the kinetics of the various receptors involved. Kinetics of
the p75 receptor indicate fast on and off rates, with an association constant of 2.3 × 10–2 /s and a dissociation constant of
approximately 10–9 mol/L.38 While the Trk receptor also
expresses a fast on rate, it has a slower off rate, with an
association constant of 5.8 × 10–4 /s, and a much lower disassociation constant of approximately 10–11 mol/L.38–40
Neurotrophin retrograde transport
433
Figure 2 Confocal
images
showing the accumulation of
rhodamine-labelled nerve growth
factor (NGF) in ligated postganglionic nerve trunk after injection into the anterior chamber of
the eye. Scale bars are (a) 100 µm,
(b) 50 µm, (c) 5 µm.
Figure 3 Nerve growth factor
(NGF) transport occurs in clusters
of multivesicular bodies. Goldlabelled NGF injected into the
anterior eye chamber was visualized in serial sections of the postganglionic trunk adjacent to the
superior cervical ganglion, 16 h
after ligation, using conventional
electron microscopy. Such particles were localized to multivesicular bodies approximately
100–250 nm in diameter (small
dense particles; (a) and (c), for
example) and were localized in
clusters (e.g. asterixed region in
panel c). The axon (Ax), which is
approximately 100 nm in diameter, can be seen on either side of
the multivesicular body (b,d).
Areas within the cluster of multivesicular bodies also contained
loosely aggregated small vesicles
(SV) 50 nm in diameter (b,c).
Scale bars represent 1 µm in (a–d)
and 250 nm for the inset in (b).
As such, the initial binding of NGF is distributed between the
two receptors and the other neurotrophins will be able to displace the NGF that is bound to the p75 receptor. This displacement will lead to a reduction of approximately 50% with
regards to NGF transport when labelled NGF is coinjected
with unlabelled neurotrophins. At later times, the proportion of
NGF bound to the TrkA receptor will increase and that to the
p75 receptor will decrease, thus preventing the other neurotrophins being able to displace the NGF bound to Trk-A.
The internalization phase of NGF has been well described
in the PC12 cell line, with the TrkA receptor subsequently
being localized to small organelles located near the plasma
membrane.10 These organelles may be the site where the rapid
exchange of the 125I-NGF with extracellular neurotrophin can
occur. At later times, the receptor and the neurotrophin
become associated with a large endosome, which may be the
signalling organelle.25 This organelle may represent the
change from a local vesicle to one that has been targeted for
retrograde transport. Sympathetic neurons take 15–18 h to be
irreversibly committed to apoptosis after the removal of
NGF.41 This suggests that there is a pool of NGF available to
the neuron, which continues to be transported to the cell
body. A targeting mechanism must thus be present to remove
the neurotrophins from the recycling pool at the nerve
434
SL Sandow et al.
Figure 4 Two potential models
for the formation of retrogradely
transported vesicles. Model 1
represents the currently accepted
view that internalized vesicles
are targeted for retrograde transport and accumulate into multivesicular bodies in their path to
lysosomes. Model 2 suggests that
there may be a process of internalizing the neurotrophins, such that
it is the formation of the multivesicular body that targets the
signalling organelle for retrograde
transport.
terminal and direct them for transport. Transport has been
described in multivesicular bodies for horseradish peroxidase
(HRP)-NGF,42 but the morphology of this transport is
obscured by the HRP reaction product. We have two unique
labels to visualize the transport organelles in vivo. Rhodamine NGF demonstrates the occurrence of large organelles
within axons, with these occurring at a low frequency along
the axon fibres. The accumulation of label occurs via retrograde transport of these structures because the site of observation is about 2 cm from the injection site.
The displacement of the labelled neurotrophins is
specific, because the unlabelled neurotrophin is able to
inhibit its own transport while non-identical neurotrophins
have a reduced ability to block the transport. This suggests
that a similar process of binding and internalization occurs
for all the neurotrophins. Therefore, the difference in their
ability to cross-react is not due to the displacing neurotrophin
being unable to access the site of the bound [125I]-labelled
neurotrophin.
We propose the following two models to explain the formation of the multivesicular bodies in the nerve terminal
(Fig. 4). The first model expands on the recycling hypothesis
for synaptic vesicles43 and suggests that there is a rapid recycling of endocytosed vesicles to the plasma membrane (Fig.
4 model 1). This may be via the clathrin-coated vesicles normally seen in neurotransmitter recycling. A subpopulation of
these vesicles is targeted for retrograde transport by the formation of multivesicular bodies, which are rapidly removed
from the terminal region. The second model explores the possibility that the neurotrophins are sequestered at the plasma
membrane into organelles, which may be the neuronal equivalent of calveoli,44 that remain in communication with the
extracellular fluid. The step that targets them for retrograde
transport comes about when these plasma membrane-located
organelles are internalized by the endocytosis of a large area
of plasma membrane containing a number of these structures
(Fig. 4 model 2). This would lead to the formation of the
multivesicular bodies in which the transport of NGF has been
observed to occur in the present paper and as previously
described.42 The formation of such an internalized organelle
signals the need for retrograde transport. The organelle would
then immediately be targeted for rapid retrograde transport
and removed from the terminal environment.
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