Using TRPV1-transiently transfected F11 cells and embryonic dorsal root ganglia explants, we show that activation of TRPV1 results in growth cone retrac-tion, and collapse and formation
Trang 1morphology and movement through cytoskeleton
reorganization
C Goswami1,*, H Schmidt2and F Hucho1
1 Freie Universita¨t Berlin, Institut fu¨r Chemie und Biochemie, Berlin, Germany
2 Department of Developmental Neurobiology, Max Delbru¨ck Centrum for Molecular Medicine, Berlin, Germany
Nerve endings and their growth cones are neuronal
structures where microtubule dynamics and complex
signalling events determine various biological
func-tions Though the importance of Ca2+ in such
func-tions is established to a certain extent, the role of
individual Ca2+ channels and the regulatory function
of Ca2+ are not clear [1–6] Recently, members of the
transient receptor potential, canonical type (TRPC)
channel family were reported to be present at growth
cones and to play an important role in axonal guid-ance [7–12] However, the mechanisms by which mem-bers of the TRPC family regulates axonal guidance is not clear
Transient receptor potential vanilloid receptor 1 (TRPV1), a nonselective cation channel, is involved in pain signalling TRPV1 detects various noxious phys-ical and chemphys-ical stimuli resulting in influx of Ca2+ A high level of TRPV1 expression was reported in dorsal
Keywords
capsaicin; cytoskeleton; dorsal root ganglia
(DRG); growth cone; TRPV1
Correspondence
F Hucho, Freie Universita¨t Berlin, Institut
fu¨r Chemie und Biochemie, Thielallee 63,
14195 Berlin, Germany
Fax: +49 30 83853753
Tel: +49 30 83855545
E-mail: hucho@chemie.fu.berlin.de
C Goswami, Department of Human
Molecular Genetics, Max Planck Institute for
Molecular Genetics, Ihnestrasse 63-73,
14195 Berlin, Germany
Fax: +49 30 84131383
Tel: +49 30 84131243
E-mail: goswami@molgen.mpg.de
*Present address
Department of Human Molecular Genetics,
Max Planck Institute for Molecular Genetics,
Berlin, Germany
(Received 19 August 2006, revised 29
Sep-tember 2006, accepted 1 December 2006)
doi:10.1111/j.1742-4658.2006.05621.x
While the importance of Ca2+ channel activity in axonal path finding is established, the underlying mechanisms are not clear Here, we show that transient receptor potential vanilloid receptor 1 (TRPV1), a member of the TRP superfamily of nonspecific ion channels, is physically and functionally present at dynamic neuronal extensions, including growth cones These nonselective cation channels sense exogenous ligands, such as resenifera toxin, and endogenous ligands, such as N-arachidonoyl-dopamine (NADA), and affect the integrity of microtubule cytoskeleton Using TRPV1-transiently transfected F11 cells and embryonic dorsal root ganglia explants, we show that activation of TRPV1 results in growth cone retrac-tion, and collapse and formation of varicosities along neurites These chan-ges were due to TRPV1-activation-mediated disassembly of microtubules and are partly Ca2+-independent Prolonged activation with very low doses (1 nm) of NADA results in shortening of neurites in the majority of isolec-tin B4-positive dorsal root ganglia neurones We postulate that TRPV1 activation plays an inhibitory role in sensory neuronal extension and motil-ity by regulating the disassembly of microtubules This might have a role
in the chronification of pain
Abbreviations
CB1, cannabinoid receptor 1; DRG, dorsal root ganglia; GAP43, growth cone-associated protein 43; IB4, isolectin B4; 5¢-IRTX,
5¢-iodoresiniferatoxin; NADA, N-arachidonoyl-dopamine; RTX, resiniferatoxin; TRPC, transient receptor potential, canonical type; TRPV1, transient receptor potential vanilloid receptor 1.
Trang 2root ganglia (DRG) [13] However, the distribution of
TRPV1 with respect to tissue is under debate Among
the neuronal tissues, TRPV1 was also detected in
many parts of the brain and spinal cord [14–16],
sug-gesting that TRPV1’s distribution is widespread and
not restricted to peripheral neuronal structures
Previously, we reported that the C-terminus of
TRPV1 interacts with tubulin, and provides stability
to microtubules under certain conditions [17]
How-ever, we also demonstrated that activation of TRPV1
results in rapid disassembly of dynamic microtubules
[18] Based on these observations, we hypothesized that
TRPV1 may regulate some specific neuronal functions
where fine regulation of microtubule dynamics is
important In this work we extend our observations
and demonstrate that TRPV1 is physically and
func-tionally present at dynamic neuronal extensions, and
their growth cones Using DRG-derived F11 cells,
embryonic DRG explants and dissociated DRG
neu-rones, we show that TRPV1 modulates the cytoskeletal
organization of neurite extensions and regulates neurite
morphology and extension
Results
TRPV1 is localized in the central and peripheral
zones of growth cones
Growth cones, the specialized structures at nerve
endings, are known to regulate axonal growth We
explored if TRPV1 is localized at growth cones As
F11 cells reflect many properties of DRG neurones
[19], we used this cell line as a model to express
TRPV1 This cell line offers a better neuronal
environ-ment suitable for monitoring the localization and
func-tion of TRPV1 (therefore, in the following, we term
the neurite-like extensions of F11 cells as ‘neurites’)
Previously, we also used this cell line to explore how
TRPV1 and the neuronal cytoskeleton are interrelated
[17,18,20,21] After transient expression in F11 cells,
TRPV1 was detected intracellularly, at the plasma
membrane, and throughout the neurites
(supplement-ary Fig S1)
By immunofluorescence analysis, we could detect
TRPV1 at neurite endings that extend into growth
cone-like structures By co-immunostaining for the
growth cone-associated protein 43 (GAP43⁄
neuromod-ulin), a well-characterized growth cone marker [22–25],
we confirmed the identity of these structures as
con-ventional axonal growth cones (Fig 1A)
Extending growth cones display distinct structural
features, a central zone (C-zone), a peripheral zone
(P-zone) and a transition zone (T-zone), which
correlate with the presence of different cytoskeletal proteins [26–28] The C-zone of growth cone is known
to be enriched with vesicles of various sizes and with microtubules, in particular their plus ends In contrast, the P-zone contains the plasma membrane and the actin cytoskeleton beneath it In extending growth cones we observed a differential distribution of TRPV1
in these areas (Fig 1A) Negligible TRPV1 immunore-activity was detected in the T-zone whereas a signifi-cant TRPV1 immunoreactivity was observed in the P- and C-zones (Fig 1A) To confirm further that this differential localization of TRPV1 at growth cones is not appearing during fixation, we expressed TRPV1-GFP in F11 cells and performed live cell imaging We observed a similar distribution of TRPV1-GFP at the growth cones, i.e TRPV1 is mainly present in the P- and C-zones (Fig 1B)
Activation of TRPV1 results in retraction
of growth cones The importance of Ca2+ channels in the regulation of growth cone movements has been reported previously [1–6] Earlier, we observed that activation of TRPV1 has a destabilizing effect on microtubules in the cell body [18] Therefore, we investigated if activation of TRPV1 can also alter or regulate the cytoskeleton in growth cones and neurite extensions For that purpose
we expressed TRPV1-GFP in F11 cells
We observed that the majority of the TRPV1-GFP positive growth cones responded to the application of the agonists resiniferatoxin (RTX) or capsaicin, caus-ing retraction and⁄ or collapse of growth cones We observed that these cones (56 out of 60), which are dynamic, immature, not connected to each other and have an extending morphology, retracted quickly (Fig 1C; see also supplementary Video S1) This pro-cess of retraction, due to TRPV1 activation, could be completely blocked by the antagonist 5¢-iodoresinifera-toxin (5¢-IRTX, data not shown), indicating that the retraction of growth cones was indeed due to TRPV1 activation This not only indicates that TRPV1 can regulate the growth cone motility, but also suggests that TRPV1 activation leads to a rapid change in the cytoskeleton organization This result was further con-firmed with a true neuronal system (see later)
Activation of TRPV1 results in disassembly
of microtubules and thus induces the formation
of varicosities
We observed that in contrast to the above-mentioned growth cone, the extending cone at the end of long
Trang 3axon-like neurites does not retract upon TRPV1
acti-vation, but rapidly turns to a pause state (growth
cones at pause exhibit a distinct morphology) [28]
Their neurites develop multiple varicosities (not
shown) Formation of varicosities is believed to
repre-sent a change in the microtubule cytoskeleton within
neurites [29,30] To confirm that TRPV1 activation
mediated varicosity formation in long neurites is
indeed due to disassembly of microtubules, we
transi-ently expressed TRPV1 in F11 cells and compared the
status of microtubules within neurites from TRPV1
activated and nonactivated cells In the absence of
acti-vation, immunoreactivity against b-tubulin subtype III
appears continuous along the neurites of
TRPV1-expressing cells In contrast, upon activation, it was no
longer visible as a continuous pattern and often
appeared as distinct discontinuous accumulations
within the varicosities The majority of these varicosi-ties reveal TRPV1 immunoreactivity, indicating that these neurites indeed developed from transfected cells (supplementary Fig S2a) In contrast, nontransfected F11 cells reveal a normal and continuous immunoreac-tivity for tubulin and do not form varicosities even after addition of RTX (supplementary Fig S2b)
Functional TRPV1 is expressed in embryonic (murine) DRG explants and are located at growth cones and neurites
To confirm that activation of TRPV1 results in similar changes not only in transfected F11 cells but also in a truly neuronal system, we used DRG explants from E12 and E13 mouse embryos At this stage (E13) of development, we detected TRPV1-specific mRNA only
Phase A
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B
P T
10 min
6 min
5 min
4 min
C
5 µm
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Fig 1 TRPV1 regulates growth cone movements (A) Localization of TRPV1 at a growth cone developed from a TRPV1 transiently
transfect-ed F11 cell A mergtransfect-ed image of TRPV1 (green) and GAP43 (rtransfect-ed) is superimpostransfect-ed on the phase contrast image The central zone (C), the transition zone (T) and the peripheral zone (P) of the growth cone is indicated Black arrows indicate filopodial structures Majority of anti-TRPV1 immunoreactivity appears at the C- and P-zone (B) Examples of growth cones developed from anti-TRPV1-GFP transiently transfected F11 cell Live cell images reveal the presence of TRPV1-GFP (green) predominantly at the P- and C-zones of growth cone Scale bar ¼
10 lm (C) Time-lapse confocal images of a growth cone (the same one as shown in (B); see also supplementary Video S1) just after adding RTX The red line and the black arrow indicate the borderline and the centre of the main shaft of the growth cone, respectively, at time 0 The blue arrow indicates the position of the main shaft at the centre of the growth cone at a particular time point during the retraction phase In the last frame, the green arrow indicates a further extension of the main shaft.
Trang 4in DRGs, but not in the spinal cord (Fig 2A).
However, we could not detect any specific TRPV1
im-munoreactivity in these DRG explants by
immuno-staining (see Discussion) We presumed that expression
of TRPV1 at this stage of development might be too
low to be detected by immunostaining Therefore, we
probed the mice DRG explants by means of more
sen-sitive methods, such as Ca2+-imaging, in response to
pharmacologically specific components We observed
that some, but not all Fluo-4-AM loaded neurites and
growth cones showed an increase in Ca2+ intensity
after adding RTX (Fig 2B) The increase in the Ca2+
influx was simultaneous throughout the ‘responding
neurites’ and correlates very well with the formation of
multiple varicosities all along the neurites
(supple-mentary Video S2)
As both RTX and capsaicin are exogenous natural
agonists, we tested if endogenous ligands of TRPV1 can
cause similar changes We tested
N-arachidonoyl-dopamine (NADA), which is present in neuronal tissues and is as potent as capsaicin [31] We observed that application of NADA also resulted in influx of Ca2+in some but not in all growth cones and neurites (Fig 2C) Response to NADA (indicated by the arrow) is observed at the growth cone, throughout the neurites and also at neurite branching points This confirms the presence of functional TRPV1 in ‘responding’ neurites This observation is in agreement with the report that TRPV1 is expressed in the embryonic stage E13 ([32], also see Discussion)
TRPV1 activation leads to the collapse of murine growth cones and formation of varicosities
As embryonic DRG explants expressed functional TRPV1, we tested if activation of TRPV1 can also affect the microtubule cytoskeleton Under control conditions all growth cones revealed a normal
a
A
C
b
Before
100 µ M
2 min after RTX
Fig 2 Functional TRPV1 is expressed in murine DRG neurones at embryonic stage E12 (A) RT-PCR analysis of the mRNA isolated from meurine E12 dorsal root ganglia (DRG, middle lane) and spinal cord (SC, last lane) (B) Functional TRPV1 is present in growth cones and neurites Mouse DRG explants (E12) loaded with Fluo 4-AM was treated with 50 n M RTX Fluorescence images before (a) and 2 min after RTX application (b) are shown in false colour (scale shown at right) ‘Responding’ neurites are indicated by arrows and nonresponding neurites by arrowheads Significant TRPV1 activity is observed at growth cones and regions forming varicosities Scale bar ¼ 20 lm (C) Mouse DRG explants (E12) loaded with fluo 4-AM were treated with 1 l M NADA Fluorescence images before (a) and 20 min after (b) NADA application are shown.
An enlarged (indicated by a white box) image of a NADA-responding neurite is shown in right (c) Scale bar ¼ 100 lm.
Trang 5extended phenotype, and no neurites with varicosities
were found (a total of 437 neurites from five explants
were counted) (Fig 3Aa,b) As expected, we observed
that application of RTX to the murine DRG explant
revealed distinct changes of growth cones and neurites
(Fig 3Ac,d) In response to RTX, 36.54% of growth
cones (129 out of 353) revealed a ‘collapsed’
mor-phology, and neurites developed many varicosities
(Fig 3Ac,d; see also supplementary Video S3) Tubulin
immunoreactivity was discontinuous in the responding
neurites and was found only in the varicosities However, nonresponding growth cones remained unaffected and retained normal neurites, even in the presence of RTX
To prove that this phenotype is specific for TRPV1 activation, we preincubated the DRG explants with 5¢-IRTX, the antagonist of TRPV1 Application of RTX
in these 5¢-IRTX preincubated explants resulted in the formation of varicosities or growth cone collapse with only 0.9% (only four out of 433, from a total of five DRG explants from mice) of the neurites (Fig 3Ae,f) This not only proves that some neurites developed from embryonic DRG explants at this stage (E12) are sensitive to RTX, but also proves the functional presence of TRPV1 there
Activation of a naturally occurring mutant form of TRPV1 does not result in the formation
of varicosities
To confirm further the specificity of the TRPV1 activa-tion-mediated varicosity formation in a different system,
we used the DRG explants from E7 chicken as a negat-ive control It is known that the chicken homologue of TRPV1 does not respond to capsaicin or RTX due to a point mutation at the region of the capsaicin-binding site [33] We observed that DRG explants isolated from chicken developed many growth cones within a day (Fig 3B) Upon treatment with the agonist RTX, DRG explants from E7 chicken revealed a normal phenotype, and formation of varicosities was not observed at all (Fig 3B) This demonstrates that the observed growth cone collapse and varicosity formation in mice DRG explant is indeed due to TRPV1 activation by RTX
TRPV1 activation-mediated varicosity formation
is partially Ca2+independent Next we tested the influence of direct Ca2+ influx on the TRPV1 activation-mediated varicosity formation
To test this, we added EGTA to the culture media before activation of TRPV1 with RTX EGTA treat-ment alone does not result in any varicosity formation (Fig 4Aa,b) However, we observed that in a medium with low Ca2+ (2 mm EGTA), application of RTX resulted in the formation of varicosities (Fig 4Ac–d) However, in this condition, the tubulin immunoreactiv-ity between two varicosities appears as a thin con-necting line This indicates that disassembly of microtubules was not complete and suggests that a partial conservation of microtubules occurs Even at
20 mm EGTA, activation of TRPV1 by RTX caused varicosity formation (Fig 4Bc,d) However, tubulin
Zoom A
d c
B
b a
Fig 3 Activation of TRPV1 by RTX results in growth cone collapse
and varicosity formation (A) Activation of TRPV1 by RTX causes
formation of ‘varicosities’ due to disassembly of the microtubule
cytoskeleton Neurites and growth cones originating from E12
mouse DRGs are shown (left side) Enlarged areas (indicated by
white boxes) of the corresponding explants are shown at right side.
Untreated explants (top panel, a and b), treated with agonist RTX in
absence (middle panel, c and d) or presence of the antagonist IRTX
(lower panel, e and f) are fixed and stained for tubulin Arrowheads
indicate neurites with collapsed growth cones and discontinuous
immunoreactivity for tubulin at the varicosities Scale bar ¼ 20 lm.
(B) Chicken DRG explants are insensitive to RTX, and do not form
varicosities The control (a) and RTX-treated (b) E7 chicken DRG
explants are shown Scale bar ¼ 20 lm.
Trang 6immunoreactivity between two varicosities again
appeared as a thin connecting line This indicates that
while TRPV1 activation by RTX leads to varicosity
formation, this process is partially independent of
Ca2+influx (see Discussion)
We observed that depletion of Ca2+ from the
endo-plasmic reticulum by incubating the explants with
thapsigargin (1 lm for 35 min), a specific inhibitor of
endoplasmic reticulum Ca2+-ATPase, does not cause
varicosity formation (Fig 4Ca,b) In contrast, addition
of 50 nm RTX for 15 min to cultures that are
preincu-bated with 1 lm thapsigargine led to the formation of
varicosities (Fig 4Cc,d) This indicates that varicosity
formation is independent of Ca2+ mobilization from
the endoplasmic reticulum
NADA, an endogenous ligand of TRPV1, regulates growth cone morphology and motility
As application of NADA results in specific influx of
Ca2+ in some neurites, we tested if NADA can also regulate growth cone morphology and movement We observed that application of NADA also resulted in col-lapse of growth cones in murine DRG explants (Fig 5A) Application of NADA also results in retraction of growth cones in the majority but not all neurites (supplementary Video S4) Some neurites were observed to also develop varicosities However, NADA had a much weaker effect than RTX Furthermore, NADA-mediated growth cone retraction is delayed, beginning about 20 min after application (supplementary Video S4)
As TRPV1 activation has a negative effect on neur-ite extension, we explored if prolonged but low-level activation of TRPV1 by NADA affects neurite exten-sion and growth Therefore, we tested the effect of a very low dose of NADA (1 nm) on dissociated DRG neurones A significant fraction of TRPV1-positive small DRG neurones (>50%, by using tissue section) are isolectin B4 (IB4)-positive [34] Therefore, in order
to characterize the effect of NADA, we used IB4 as a marker for a subpopulation of DRG neurones
We observed that this subpopulation of mainly small IB4-positive DRG neurones developed very small neurites when NADA was added for 24 h (Fig 5Ba,b) No effect of NADA was observed in the majority of IB4-negative neurones under these condi-tions (1 nm) or even at higher concentracondi-tions (1 lm)
of NADA (Fig 5B) Under the same conditions, but
2 m M EGTA
A
d b
2 m M EGTA+RTX
B
C
c a
Fig 4 RTX-mediated varicosity formation is partly Ca 2+ independ-ent and due to the activation of TRPV1 located at the cell surface (A) Varicosity formation is partly independent of Ca 2+ influx Mouse DRG explant treated with 2 m M EGTA for 15 min (a) and an enlarged section of the same (below, b) is shown A DRG explant pre-treated with 2 m M EGTA for 15 min followed by activation with
50 n M RTX (c) and an enlarged section of the same explant (below, d) is shown All explants are immunostained for tubulin and the arrowheads indicate the thin connection between two subsequent varicosities Scale bar ¼ 20 lm (B) Tubulin immunostaining of a mouse DRG explant treated with 20 m M EGTA (a) is shown A sim-ilar explant further treated with 50 n M RTX is shown on the right (b) Scale bar ¼ 20 lm (C) RTX-mediated varicosity formation is independent of Ca 2+ influx from internal Ca 2+ stores Mouse DRG explants are treated with either thapsigargin alone (1 l M , 15 min) (left side, a) or with thapsigargin for 15 min and subsequently with RTX for 15 min (right side, c), then fixed and immunostained Tubu-lin immunostaining of the explants (top panel, a and c) and an enlarged section (indicated by white box) of the corresponding explants (bottom panel, b and d) are shown Thapsigargin treatment does not prevent TRPV1 activation-mediated varicosity (indicated
by arrow) formation Scale bar ¼ 20 lm.
Trang 7in the absence of NADA, these IB4-positive, small
neurones grow normally, develop many neurites and
are comparable to the IB4-negative neurones
(Fig 5Bd) This indicates that in response to the
endogenous ligand, TRPV1 can regulate both
mor-phology and length of neurites in a subpopulation of
DRG neurones
In summary, TRPV1 seems to be present and
func-tional in sensory neurones including growth cones at
embryonic stages Overall, our results suggest that
TRPV1 regulates the microtubule cytoskeleton and
affects growth cone morphology and motility
Discussion
Growth cones are well-organized dynamic structures involved in neurite pathway finding Frequencies of
Ca2+ waves generated by Ca2+ channels present in neurites and growth cones have a strong regulatory influence on neurite initiation, extension, branching and direction [1–6] Here we provide evidence that TRPV1 is localized at specific areas of the growth cone and influences morphology and motility of neurites We also show that the presence of TRPV1 at certain neurites and growth cones modulates the
d
c
b
a
d
c
A
1 µm NADA
No NADA
Fig 5 NADA regulates growth cone morphology and movement (A) NADA treatment (30 min) results in the collapse of a growth cone Control E12 mouse DRG explants (a) and NADA-treated explant (c) were stained for tubulin (green) and actin (red) The majority of growth cones treated with NADA display collapsed morphology (b,d) Enlarged areas (white box) of the untreated (b) and NADA-treated growth cones (d) are shown inside Scale bar ¼ 100 lm (B) Prolonged activation of TRPV1 by low level of NADA negatively affects the neurite extension of IB4-positive small-sized DRG neurones Dissociated DRG neurones from adult rat were stained for neurone-specific b-III tubulin (green), IB4 (red) and DAPI (a) Prolonged exposure with NADA (1 n M for 24 h) inhibits the growth of IB4 positive small-sized DRG neurones (indicated by arrows) Under this condition, IB4 negative neurones grow normally Scale bar ¼ 50 lm (b) An example of an IB4-positive small DRG neurone grown under the above conditions Note that these small IB4-positive neurones are fully viable in NADA-containing med-ium, even after 24 h of exposure, but develop very small neurites Scale bar ¼ 20 lm (c) The majority of IB4 negative neurones do not respond to NADA (24 h) These neurones develop multiple extended neuritis, even in a medium containing 1 l M NADA Scale bar ¼ 50 lm (d) In the absence of NADA, both IB4-positive and IB4-negative neurones grow normally and develop extended neurites Scale bar ¼ 50 lm.
Trang 8cytoskeletal reorganization by destabilizing them upon
activation
As the TRPV1 activation-mediated retraction of
growth cones is fast (initiated within a few minutes)
and results in disassembly of microtubules (18 and this
study), it is unlikely that activation of TRPV1 will
alter the ratio of the different cytoskeleton proteins
This suggests that the retraction of neurites is most
likely to be independent of transcription or translation
of a new gene product(s), but depends on the
reorgani-zation of structural proteins affecting both
morphol-ogy and movement of neurites Previously we had
observed that activation of TRPV1 significantly affects
the polymeric organization of the microtubule
cytoske-leton Activation of TRPV1 results in disassembly of
dynamic microtubules but not of the actin cytoskeleton
[18] Growth cone retraction is a process involving
both the actin and microtubule cytoskeletons More
specifically, the force generated by motor proteins is
involved in this process [29,35] We also observed that
Taxol, a microtubule cytoskeleton stabilizer,
signifi-cantly delayed RTX activation-mediated varicosity
for-mation, but could not block it completely (data not
shown) Based on all these observations and previous
evidence, we postulate that growth cone retraction and
varicosity formation upon TRPV1 activation is a
multi-step process that leads to sequential disassembly
of microtubules (Fig 6) In support of this hypothesis,
we provide several lines of evidence Firstly, after
TRPV1 activation, immunoreactivity for tyrosinated
tubulin (a marker for dynamic microtubules [36]) is
diffusely present at the P- and T-zones of activated
growth cones, including in the filopodial structures
(supplementary Fig S2C) This indicates a loss of
dynamic microtubules due to TRPV1 activation
Sec-ondly, long neurites frequently showed substantial
formation of varicosities, a classic morphological
symptom of microtubule depolymerization [29]
Finally, using embryonic DRG explants, we prove that
activation of TRPV1 by RTX can result in formation
of many varicosities along the neurites, most probably
due to a very rapid disassembly of microtubules
How-ever, some neurites are unchanged upon RTX
applica-tion and do not develop varicosities This suggests that
TRPV1 is present in some but not in all neurites This
result is in agreement with the fact that a major
frac-tion of DRG neurones is TRPV1 negative
Interest-ingly TRPV1 is not exclusively present at the growth
cones of the ‘responding neurites’ For example, RTX
application results in rapid increase in Ca2+ influx
throughout the ‘responding neurites’ (Fig 2),
indica-ting that TRPV1 is also present all along the neurite
shaft
Using time-lapse microscopy, we observed that upon RTX application, responding neurites first retract for a short time and then develop varicosities (supplement-ary Video S3) In most cases, within a single neurite, varicosity formation was almost instant and did not occur one by one, suggesting a simultaneous global change all over the neurites and a dynamic instability This is in agreement with our previous observation that TRPV1 activation by RTX results in rapid disas-sembly of dynamic microtubules, but not of the actin cytoskeleton [18] During the morphological transition from smooth neurites to varicosities due to RTX appli-cation, we frequently observed a short retrograde movement of the growing varicosities (supplementray Videos S1 and S3) This short retrograde movement within the retracting neurite suggests the involvement
of functional actin cytoskeleton
Growth cones are capable of recognizing different external stimuli and of integrating different cues in terms of Ca2+ transients [37] Our data also indicate that chemical stimuli specific for TRPV1 can regulate
A
C
B
Fig 6 Proposed model of growth cone motility regulation by TRPV1 (A) Both anterograde force from the microtubule cytoskele-ton (blue, upside arrow) and a retrograde force provided by actin cytoskeleton (red, down side arrow) determine the net axonal growth and movement (B) TRPV1 activation-mediated growth cone retraction and varicosity formation is dependent on the status of the microtubule cytoskeleton Stage 1: Activation of TRPV1 (indica-ted by arrow) results in partial disassembly of microtubules, leading
to the retraction of growth cone Stage 2: Further disassembly of microtubules leads to further retraction and initiates varicosity for-mation Stage 3: Complete disassembly of microtubules results in a stage where further retraction is no longer possible Stage 4: Retro-grade force from actin cytoskeleton and complete disassembly of microtubules results in the varicosity formation.
Trang 9the morphology and movement of TRPV1-positive
growth cones We demonstrate that NADA, an
endo-genous ligand of TRPV1, not only affects growth cone
morphology and movement, but also the neurite length
(Fig 5B) Apart from NADA, there are few other
puta-tive endogenous ligands, such as N-oleoyldopamine
known to activate TRPV1 [38] Some other
endovanil-loids and fatty acid metabolites are also known to act
on TRPV1 [39] We did not test all of these endogenous
ligands because they are not selective for TRPV1 and
are also subjected to metabolic changes NADA is also
known to activate cannabinoid receptor 1 (CB1),
although at higher doses [31,40,41] Therefore
involve-ment of CB1 receptors in the NADA-mediated process
can not be ruled out completely
TRPV1 activation by other noxious physical stimuli
like low pH and high temperature are also expected to
exert a similar phenotype The combination of
differ-ent physiological stimuli might produce a synergistic
effect on the growth cone retraction and varicosity
for-mation through TRPV1, but this is difficult to prove
due to the presence of many other receptors sensing
low pH and temperature changes In agreement with
our results, the presence of TRPV1 was recently
detec-ted at varicosities within embryonic spinal cords [42]
In this regard, it is important to mention that
varicosi-ties are often considered as synaptic terminals
There-fore, it might be possible that TRPV1 is involved in
the synapse formation too
Growth cone retraction and collapse due to RTX
application is fast and seems to be irreversible, as
addi-tion of 5¢-IRTX shortly after RTX applicaaddi-tion does
not result in any recovery of growth cones In contrast
to RTX-mediated growth cone collapse and retraction,
the NADA-mediated effect in primary neurones is
much weaker and delayed However, NADA exerts
more physiological effects without affecting the cell’s
viability The growth cones and neurites remain motile
in NADA-containing medium, even 1 h after
applica-tion Using time-lapse imaging, we observed that after
NADA treatment, retracted growth cones remained
motile and started to re-grow after a short period of
time (data not shown) Moreover, we could also show
that at low concentrations of NADA (both with 1 nm
and 1 lm), the majority of the IB4-positive dissociated
DRG neurones survive for 24 h or more, but in these
cases, the neurites grow very little (Fig 5B)
We observed that growth cone retraction in response
to TRPV1 activation is dependent on the nature and
amount of the ligand Strong agonists like RTX cause
retraction for a short duration and subsequent
forma-tion of many varicosities, indicating that disassembly
of microtubules is rapid, complete and most likely to
be irreversible In contrast, an endogenous ligand such
as NADA causes retraction for a longer time but rarely forms varicosities Application of NADA most probably results in incomplete and slow disassembly of microtubules Thus effects of NADA are most likely
to be reversible and physiological
Our work also establishes another important aspect: TRPV1 is of functional importance in a subpopulation
of DRG neurones already at an early developmental stage (E12–13 in mice) Recently, expression of TRPV1 has also been reported at embryonic stage (E13) by Fun-akoshi et al [32] Using tissue sections from embryonic mice, Funakoshi et al [32] demonstrated that TRPV1 is present in certain nerve fibres In contrast, we could not detect specific TRPV1 staining in our explant cultures or
in dissociated DRG neurones by immunofluorescence analysis These differences in the immunostaining might
be due to the different antibodies used However, it seems that the expression of TRPV1 in embryonic DRG neurones is subjected to different external conditions and is reduced upon culturing Nevertheless, our results indicate that expression of TRPV1 at early embryonic stages can regulate the growth cone motility in certain neurones This might have a developmental role, especi-ally in response to endogenous ligands like endovanil-loids, compounds belonging to the lipoxygenase pathway [31,38,39] Further research must be conducted
to confirm this aspect of TRPV1
It is known that Ca2+ has a depolymerizing effect
on microtubules in vitro [43,44] This effect of Ca2+on microtubules has also been demonstrated in vivo, and
it has been shown that Ca2+ leads to two distinct processes: the ‘dynamic destabilization’ (a direct depo-lymerizing effect of Ca2+ on microtubules) and ‘sig-nal-cascade-induced fragmentation’ of microtubules [45] In the present work, we have attempted to deter-mine the influence of Ca2+ion and⁄ or Ca2+-signalling
on the TRPV1 activation-mediated varicosity forma-tion We demonstrate that varicosity formation and growth cone retraction is dependent on TRPV1 activa-tion, but partly independent of Ca2+influx For exam-ple, RTX application results in varicosity formation even in the presence of 20 mm EGTA We have dem-onstrated that varicosity formation is independent of
Ca2+ release from the endoplasmic reticulum and involves TRPV1 receptors located at the plasma mem-brane Additionally, we have observed that in TRPV1 transfected F11 cells, microtubule disassembly due to RTX application at low temperatures does not result
in disassembly of microtubuli (data not shown) This may suggest the requirement of an additional step(s) (other than a direct effect of Ca2+) involving
enzymat-ic or metabolenzymat-ic activity Although the menzymat-icrotubule
Trang 10cytoskeleton acts as a downstream effector of TRPV1,
the exact molecular mechanism underlying the changes
due to TRPV1 activation remains to be determined
Growth cones have a remarkable ability for
chemo-attraction and chemorepulsion Recently, several
mem-bers of TRPC channels were reported to be present at
growth cones, and to regulate growth-cone
morphol-ogy and⁄ or functions [7–12,46] For example, TRPC
channels are important for brain-derived neurotrophic
factor and netrin-1-induced growth cone turning [9,10]
Xenopus TRPC1 (XTRPC1) is also involved in growth
cone motility and turning in response to growth
fac-tors [11] The TRPC5 receptor has been reported to
control neurite length: its activation leads to a decrease
in the length of neurites [12,46] Though we did not
test the effect of different growth factors and all the
known endogenous ligands on the TRPV1-positive
growth cone movement, our results point to the
simi-larities between TRPV1 and other TRPC channels,
and indicate a common function of TRP channels in
regulation of growth cone motility in response to
endogenous compounds during axonal path finding
In summary, our results suggest a functional
impli-cation of TRPV1 in the regulation of growth cone
morphology and neurite movement These may have
relevance in some pathological and neurological
disor-ders including chronification of pain
Experimental procedures
Reagents and antibodies
RTX, capsaicin, 5¢-IRTX, thapsigargin and Taxol were
purchased from Sigma Aldrich (Deisenhofen, Germany)
NADA was purchased from Biomol (Hamburg, Germany)
Mouse monoclonal anti-b-tubulin class III-specific
antibod-ies (clone SDL.3D10), mouse monoclonal anti-a-tubulin
spe-cific antibodies (clone DM1A) and tetramethylrhodamine
isothiocyanate-labelled IB4 from Griffonia simplicifolia plant
were purchased from Sigma Aldrich Rat monoclonal
anti-bodies YL1⁄ 2, often used as microtubule marker was
purchased from AbCam Ltd (Cambridge, UK) Rabbit
poly-clonal anti-N-terminal TRPV1 Ig were purchased from
Affinity Bioreagents (Golden, CO, USA) Mouse
monoclo-nal antibodies against neuromodulin, commonly known as
growth cone-associated protein 43 or GAP-43 (clone 31)
were purchased from BD-transduction Ltd (Heidelberg,
Ger-many) Calcium sensor dye fluo 4-AM, Alexa-594-labelled
phalloidin, Alexa-594-labelled anti-rat secondary IgG,
Alexa-594-labelled anti-mouse secondary IgG were
pur-chased from Molecular Probes (Invitrogen, Karlsruhe,
Ger-many) Cy2-labelled-antigoat and Cy2-labelled anti-rabbit
IgG were purchased from Dianova (Hamburg, Germany)
Constructs
For heterologous expression in mammalian cells, the full-length rat TRPV1 cDNA subcloned in a pcDNA3.1 vector was used [17,18,20] For expression of the C-terminally GFP-fused TRPV1, a cDNA fragment encoding rat TRPV1 was amplified by PCR using 5¢-ATGGAACAACGGG CTAGCTT-3¢ and 5¢-TCTCCCCTGGGACCATGGAA-3¢ primers and subcloned into pCDNA3.1⁄ CT-GFP-TOPO vector (Invitrogen) [21]
RT-PCR
RT-PCR amplification of DNA coding for a TRPV1-frag-ment was carried out using cDNA libraries separately isola-ted from DRG and spinal cord of embryonic mouse (E13) Specific primer sets (forward primer TGTACTTCAGCCA TCGCAAG and reverse primer CCAGGATGGTGATGG CTC) were used for amplifying a 534 basepair fragment of TRPV1 For control purposes, amplification of a natriuretic peptide receptor 3 fragment was carried out using the same cDNA isolated from DRG as well as from spinal cord
Cell culture and transfection
F11 cells were cultured in Ham’s F12 medium (Invitrogen) supplemented with 20% fetal bovine serum (Invitrogen) For transient transfection, lipofectamine (Invitrogen) was used
TRPV1 activation assay and live cell imaging
For visualizing the effect of TRPV1 activation, F11 cells were seeded on glass cover slips TRPV1 was expressed by transient transfection Two days after transfection, F11 cells were incubated with Hank’s balanced salt solution buffer at room temperature (25C) supplemented with 1 mm CaCl2
and RTX (100 nm) for 1 min, followed by immediate fixing with paraformaldehyde (PFA) (2%) Immunocytochemistry
of fixed cells was performed as described previously [17,18,20,21] For live cell imaging, F11 cells transiently expressing TRPV1-GFP were maintained in complete medium and RTX (100 nm) and Ca2+(1 mm) was added Antagonist 5’-IRTX (1 lm) was used for blocking TRPV1 All live-cell images and fixed-cell images were taken with a confocal laser-scanning microscope (Zeiss Axiovert 100 M) with a 63· objective and analysed using the Zeiss LSM image examiner software
Immunohistochemistry
For immunohistochemistry analysis, the cells were fixed with 2% PFA for 5 min and fixed cells were permeabilized with 0.1% triton X-100 in NaCl⁄ Pi(5 min) The cells were subsequently quenched with 2% glycine in NaCl⁄ Piand the