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First published online January 13, 2009
doi: 10.1242/10.1242/dev.018234
Montreal Neurological Institute, Department of Neurology and Neurosurgery, McGill University, Montreal, Quebec H3A 2B4, Canada.
* Author for correspondence (e-mail: timothy.kennedy{at}mcgill.ca)
Accepted 21 November 2008
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: Oligodendroglia, Myelination, Myelin, Netrin, Integrin, Laminin, Autocrine, Mouse, Rat, FAK (Ptk2), N-WASP (Wasl)
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INTRODUCTION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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;
Kirby et al., 2006). Upon
contact with a target axon, oligodendrocyte processes coalesce to form a
spreading sheet of membrane that begins to wrap the axon. The extracellular
cues that govern this process and the intracellular mechanisms involved are
poorly understood.
Laminin 2, which promotes oligodendrocyte maturation by signaling through
6β1 integrins (Baron et al.,
2005), has been suggested to regulate process elaboration.
Laminin-2-deficient mice have dysmyelinated and hypomyelinated axons
(Chun et al., 2003). However,
laminin 2 is not ubiquitous in myelinated CNS axon tracts
(Colognato et al., 2002) and
transgenic mice lacking β1 integrin expression in oligodendrocytes
exhibit no defects in CNS myelination
(Benninger et al., 2006). Thus,
other ligand-receptor interactions must also direct the changes in
oligodendrocyte morphology required for myelination.
The axon-guidance cue netrin 1 (Ntn1) is a chemorepellent for migrating
oligodendrocyte precursors (OPCs) in the embryonic spinal cord
(Jarjour et al., 2003;
Tsai et al., 2003). These
cells express the netrin 1 receptors Dcc, Unc5a and Unc5b, but not netrin 1
itself. In the adult CNS, netrin 1 is expressed by myelinating
oligodendrocytes and is associated with non-compacted oligodendroglial
membranes (Manitt et al.,
2001). We therefore investigated the possibility that netrin 1,
expressed in the developing CNS and later by the oligodendrocytes themselves,
might influence late stages of oligodendrocyte differentiation.
The lamella elaborated by the tip of an extending oligodendrocyte process
has been compared to a neuronal growth cone
(Fox et al., 2006;
Jarjour and Kennedy, 2004;
Sloane and Vartanian, 2007).
Interestingly, a number of the same intracellular signaling proteins have been
implicated in netrin-1-mediated axon guidance and the development of
oligodendrocyte processes required for myelination
(Fox et al., 2006;
Jarjour and Kennedy, 2004). In
both cases, the reorganization of the actin cytoskeleton requires activation
of the Src family kinase (SFK) Fyn
(Meriane et al., 2004;
Osterhout et al., 1999;
Umemori et al., 1994) and
involves Wiscott-Aldrich syndrome protein (N-WASP; Wasl - Mouse Genome
Informatics) and Rho GTPases (Bacon et al.,
2007; Liang et al.,
2004; Shekarabi et al.,
2005).
Here we provide evidence that netrin 1 and Dcc promote the extension of oligodendrocyte processes in vivo. We used in vitro assays to demonstrate that netrin 1 increases oligodendrocyte process branching. Furthermore, expression of Dcc and netrin 1 by oligodendrocytes promotes the formation of myelin-like membrane sheets. Addressing the signaling mechanisms involved, we show that the SFK Fyn is required for netrin-1-induced process branching, that netrin 1 recruits Fyn to Dcc in oligodendrocytes, increases SFK activity, and promotes process extension and branching associated with a decrease in RhoA activity. Our findings reveal a novel role for netrin 1 and Dcc in activating a signaling cascade in oligodendrocytes that directs process remodeling.
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MATERIALS AND METHODS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Cell culture
OPCs were derived from mixed glial cultures from the cerebral cortices of
postnatal day 0 (P0) rat pups and grown in oligodendrocyte defined medium
(OLDEM) as described previously (Armstrong,
1998; Jarjour et al.,
2003), with 0.1% fetal bovine serum (FBS) to initiate
differentiation. Cells were seeded at 1.5x104 cells/chamber
in 8-well chamber slides coated with 10 µg/ml poly-L-lysine
(Nalge Nunc, Rochester, NY, USA). For immunoprecipitation, western blots and
GST-rhotekin pulldowns, cells were plated at 1.5x106
cells/well in 6-well tissue culture dishes.
Mouse oligodendrocyte cultures
Culturing of mouse OPCs was similar to that of rat OPCs, but 10% horse
serum was used instead of 10% FBS in mixed glial culture media. Each T75 flask
of mixed glial culture required two to three pups. Newborn
Ntn1-/- and Dcc-/- mice were
identified by distinct behaviors. The genotype of individual pups was
confirmed by PCR.
Antibodies
Primary antibodies used in this study were: rabbit polyclonal anti-netrin 1
PN3 (Manitt et al., 2001),
mouse monoclonal anti-Dcc (G97-449; BD Biosciences Pharmingen, San Jose, CA,
USA), goat polyclonal anti-Dcc (Santa Cruz Biotech, Santa Cruz, CA, USA),
rabbit polyclonal anti-myelin basic protein (Mbp, Chemicon, Temecula, CA,
USA), mouse monoclonal anti-Mbp (Chemicon), mouse monoclonal RIP antibody
(Chemicon), mouse monoclonal anti-2',3'-cyclic nucleotide 3'
phosphodiesterase (Cnp, Sternberger Monoclonals, Lutherville, MD, USA), rabbit
polyclonal anti-Mag (Chemicon), rabbit polyclonal anti-Nfm (Nefm - Mouse
Genome Informatics) (Chemicon), rabbit polyclonal anti-Fyn (Upstate Cell
Signaling, Charlottesville, VA, USA) used for western blots, rabbit polyclonal
anti-Fyn [gift of Dr Andre Veillete and described previously
(Davidson et al., 1992)] used
for immunoprecipitation and western blots, mouse monoclonal anti-FAK (BD
Biosciences), rabbit polyclonal anti-N-WASP (Santa Cruz), and rabbit
polyclonal anti-phospho-Src (Cell Signaling), which recognizes the pY416
epitope in all SFK members.
For analyses of oligodendrocyte morphology in vivo, we crossed Ntn1 or Dcc heterozygous mice, obtained embryonic day 18 (E18) embryos (plug date taken as E1), fixed them in 4% paraformaldehyde and cut 18 µm sections of the spinal brachial enlargement with a cryostat. Sections were then stained with anti-Cnp (1:250), anti-Nfm (1:250), anti-netrin PN3 (1:100), visualized using Alexa 546- or Alexa 488-conjugated secondary antibodies (Jackson ImmunoResearch, West Grove, PA, USA), and nuclei stained with Hoechst. Images were obtained with a Magnafire CCD camera (Optronics, Goleta, CA, USA) and a Zeiss Axiovert 100 microscope (Toronto, Ontario, Canada).
Analysis of oligodendrocyte morphology
Analyses of immature oligodendrocytes in vitro and in vivo were performed
using the NeuronJ plugin for ImageJ (NIH, Bethesda, MD, USA). The length of
the longest process was measured from the base of the process to its tip
(Fig. 3D;
Fig. 4B). For Sholl analysis,
the grid function in Northern Eclipse (Empix) was used to draw concentric
circles 15 µm apart around the cell body of mature RIP-positive
oligodendrocytes. The number of intersections made by processes with each
successive circle was counted. For studies using the β1 integrin subunit
function-blocking antibody and mouse oligodendrocyte cultures, a Sholl
analysis plugin was used with ImageJ (starting radius, 1.02 cm; step size,
1.02 cm; end, 5.08 cm; thickness, 0.02 cm). The Mbp-positive myelin-like
membrane sheets were outlined in ImageJ and the surface area reported in
arbitrary units.
The pharmacological inhibitors PP2 and PP3 (Calbiochem) were used at 2 µM to inhibit SFK activity. Purified hamster anti-rat CD29 (β1 integrin) monoclonal and purified hamster anti-IgM monoclonal antibodies were used at 2 µM to investigate β1 integrin function.
Analysis of phospho-Src puncta
The number of phospho-Src-positive puncta was measured using ImageJ. The
brightness and contrast of the images of individual oligodendrocytes were
modified to enhance puncta associated with extending processes; modifications
made were consistent across all images. Images were converted to a binary
format and then the number of particles counted automatically. Staining in the
cell body and major processes was not punctate and thus counted as one
particle, making variations under different conditions a direct indicator of
changes in the number of distally located puncta. The number of puncta was
divided by oligodendrocyte area to control for variance in oligodendrocyte
size.
Quantification and statistical analyses
Statistical significance was calculated by ANOVA followed by a post-hoc
Tukey test using Systat Software (San Jose, CA, USA). Analyses of
oligodendrocyte morphology in vitro used three independent experiments with a
minimum of 30 cells per condition. In vivo analysis of process extension and
purification of oligodendrocytes from transgenic mice were performed using at
least four pups of each genotype, derived from at least two different
litters.
Immunoprecipitation
Cells in 6-well dishes were treated with netrin 1 for 5 minutes, then lysed
in RIPA lysis buffer (10 mM sodium phosphate pH 7.2, 150 mM NaCl, 1% NP40,
0.1% SDS, 0.5% deoxycholate) and centrifuged at 13,000 rpm (13,800
g) for 7 minutes. Supernatant was pre-cleared with 30 µl
protein A/G beads (Santa Cruz) for 30 minutes, incubated with 1 µg/ml
anti-Dcc (monoclonal) or anti-Fyn (rabbit polyclonal) for 1 hour, followed by
addition of 30 µl protein A/G beads for 45 minutes.
GST pulldown assays
Fusion proteins comprising the RhoA-binding domain of rhotekin (a
downstream substrate of RhoA) or Pak-CRIB and glutathione-S-transferase (GST)
were purified as described (Reid et al.,
1996). Oligodendrocytes were treated with netrin 1 for 24 hours.
Cells were then lysed and protein purified as described
(Ren and Schwartz, 2000;
Shekarabi et al., 2005).
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RESULTS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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;
Kennedy et al., 1994;
Tsai et al., 2006;
Tsai et al., 2003). Although
not expressed by OPCs, netrin 1 is widely expressed by spinal interneurons,
motoneurons and by most, if not all, mature myelinating oligodendrocytes in
dorsal and ventral white matter in the adult rat and mouse CNS
(Manitt et al., 2001).
To determine when netrin 1 is expressed by differentiating
oligodendrocytes, we performed a time-course analysis. Premyelinating,
postmigratory oligodendrocytes within the corticospinal tract at brachial,
thoracic and lumbar levels were examined. Netrin 1 expression was not detected
in oligodendrocytes at embryonic stages of development in mice (data not
shown). For postnatal stages, netrin-1-expressing cells were identified
immunohistochemically. Oligodendrocytes in the developing dorsolateral white
matter tracts were identified by expression of 2',3'-cyclic
nucleotide 3' phosphodiesterase (Cnp), a marker that labels
oligodendrocyte cell bodies and processes in vivo, and by expression of the
mature oligodendrocyte marker myelin basic protein (Mbp). At P8, before
myelination begins in the corticospinal tract, netrin 1 was not expressed by
premyelinating oligodendrocytes (Fig.
1A-C). Netrin-1-expressing neuronal cell bodies and
neural-epithelial cells (Fig.
1D,E), but not astrocytes (Fig.
1I), were detected immediately adjacent to differentiating
oligodendrocytes. At P12, following the initiation of myelination in the rat
corticospinal tract (Schwab and Schnell,
1989), netrin 1 was detected in the cell bodies of a subset of
oligodendrocytes double labeled with Cnp
(Fig. 1F,G). At this stage of
development, netrin 1 immunoreactivity was associated with axons
(Fig. 1I). By P22, large
numbers of mature myelinating oligodendrocytes expressing netrin 1 were
readily detectable throughout the nascent white matter of the spinal cord
(Fig. 1J,K). These findings
indicate that netrin 1 begins to be expressed by oligodendrocytes during early
stages of myelination.
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),
extend highly branched processes that coalesce to form cytoplasmic sheets
(Fox et al., 2006). These
sheets, which resemble unwrapped non-compacted myelin membrane
(Knapp et al., 1987), were
immunopositive for netrin 1 (Fig.
2A). This staining was present on cells that were not
permeabilized, consistent with netrin 1 protein associated with the
extracellular face of the plasma membrane. To verify that the cells were not
permeabilized, the absence of Mbp staining was used as a negative control (not
shown). We conclude that in the absence of neuronal contact in vitro,
oligodendrocytes express netrin 1, and that netrin 1 protein is associated
with the surface of myelin-like membrane sheets. The netrin 1 receptor Dcc was detected along the processes of immature and mature oligodendrocytes. In mature oligodendrocytes, Dcc was associated with major branches and present in small puncta at the leading edge of myelin-like membrane sheets (Fig. 2B). Addition of recombinant Myc-tagged netrin 1 to mature oligodendrocytes revealed a preferential localization of exogenous ligand at the branches and edges of sheets formed by the cells, similar to the distribution of Dcc (Fig. 2A',B').
Netrin 1 and Dcc promote process extension by premyelinating oligodendrocytes in vivo
In the developing spinal cord, as premyelinating oligodendrocytes mature
into myelinating oligodendrocytes, netrin 1 is widely expressed by neurons and
neuroepithelial cells, but not by oligodendrocytes
(Fig. 1D,I;
Fig. 3C). Mice lacking either
netrin 1 or Dcc die within hours of birth
(Fazeli et al., 1997;
Serafini et al., 1996). We
therefore used E18 littermates to compare the morphology of postmigratory,
premyelinating oligodendrocytes in tissue sections from the spinal cords of
wild-type and Ntn1 and Dcc heterozygotes and knockout mouse
embryos (Fig. 3A-C). At E18,
Cnp-immunoreactive premyelinating oligodendrocytes in the dorsolateral spinal
cord typically extend one or more processes
(Fig. 3D, dashed lines). We
found the length of these processes in both Ntn1-/- and
Dcc -/- mice to be significantly shorter than in wild-type
littermates (Fig. 3E,F). These
findings provide evidence that at the end of precursor migration and at the
initiation of oligodendrocyte differentiation, netrin 1 in the local
environment of a postmigratory, premyelinating oligodendrocyte promotes
Dcc-dependent process extension in vivo, a phenomenon closely associated with
the capacity of an oligodendrocyte to contact target axons
(Hardy and Friedrich, 1996;
Kirby et al., 2006).
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Netrin 1 does not affect the differentiation of immature or mature oligodendrocytes in vitro
Distinct changes in oligodendrocyte morphology accompany the
differentiation of oligodendrocytes in vitro. Immature oligodendrocytes are
characteristically multipolar cells that express Cnp but not Mbp
(Fig. 4A,B). These cells
differentiate into mature oligodendrocytes that elaborate myelin-like membrane
sheets and express both Mbp and Cnp (Fig.
4A,D), and eventually myelin-associated glycoprotein (Mag).
Immature cells (4-5 DIV) grown in the presence of netrin 1 (100 ng/ml) for 24
hours showed a modest (4%) decrease in the ratio of Cnp- to Mbp-positive
cells. In more-mature Mbp/Mag-positive cultures (6-8 DIV), the ratio of
Mbp-positive cells to Mag-positive cells was not affected by the addition of
netrin 1 (Fig. 4A). We conclude
that the addition of netrin 1 in vitro does not alter the acquisition of a
mature phenotype, as assessed by the expression of standard markers.
Netrin 1 induces Dcc-dependent process extension by immature oligodendrocytes in vitro
The effect of netrin 1 on process extension, as assayed by measuring the
length of the longest process, was investigated in Cnp-immunopositive immature
oligodendrocytes in culture (Fig.
4B). Application of 100 ng/ml netrin 1 for 24 hours to immature
oligodendrocytes increased process length compared with control cells
(Fig. 4C). To determine whether
Dcc is required for netrin-1-induced extension of oligodendrocyte processes in
vitro, a Dcc function-blocking antibody (Dccfb) was added for 24 hours to
immature oligodendrocytes treated with netrin 1. Consistent with the findings
obtained in vivo and described above (Fig.
3F), disruption of Dcc function blocked the netrin-1-induced
increase in process length, but did not significantly alter process extension
when added alone (Fig. 4C). We
conclude that the application of netrin 1 promotes Dcc-dependent
oligodendrocyte process extension, and that these changes occur independently
of defects in migration and axon growth.
Netrin 1 promotes Dcc-dependent increases in oligodendrocyte branching and myelin-like sheet formation
To investigate roles for netrin 1 during later stages of oligodendrocyte
development, we characterized changes in oligodendrocyte process branching and
in the capacity of these cells to elaborate myelin-like membrane sheets in
vitro. Mature oligodendrocytes cultured for 6-8 DIV were double labeled with
RIP antibody (against Cnp) to identify major processes, and Mbp antibody to
visualize extended myelin-like membrane sheets
(Fig. 4D, left).
Oligodendrocytes grown in the presence of 100 ng/ml netrin 1 for 24 hours
exhibited a significant increase in the area of Mbp-positive sheets
(Fig. 4E). The morphological
complexity of oligodendrocyte processes was quantified using Sholl analysis
(Ricard et al., 2001)
(Fig. 4D). Cells treated with
100 ng/ml netrin 1 for 24 hours exhibited a significant increase in branching
compared with the control (Fig.
4F,G). A dose-response analysis determined 100 ng/ml netrin 1 to
be optimal (Fig. 4F).
As described above, immunocytochemical analyses detected Dcc, but little, if any, netrin 1 associated with the branches of oligodendrocyte processes. Both Nfb (netrin function-blocking antibody) and Dccfb antibodies blocked the increase in myelin-like membrane sheet formation and process branching induced by the addition of exogenous netrin 1 (Fig. 4E,G), indicating that the netrin-1-induced changes in oligodendrocyte morphology are Dcc-dependent. Substantial netrin 1, but not Dcc, immunoreactivity was detected in association with the myelin-like membrane sheets (Fig. 2A); however, function-blocking antibodies applied in the absence of netrin 1 did not alter sheet formation or process branching (Fig. 4E,G). We therefore tested the hypotheses that netrin 1 made by oligodendrocytes does not exert an autocrine influence on the formation of myelin-like membrane sheets, and, alternatively, that the relatively short-term (24 hours) loss-of-function assays described above are too brief to reveal a role for endogenous netrin 1.
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By contrast, oligodendrocytes lacking either netrin 1 or Dcc exhibited a significantly reduced surface area of the myelin-like membrane sheets compared with cells derived from wild-type or heterozygote littermates (Fig. 5D,E), thereby identifying an autocrine role for netrin 1 in sheet formation. This is consistent with our demonstration that the addition of exogenous netrin 1 increases myelin-like membrane sheet formation through a Dcc-dependent mechanism (Fig. 4E). These findings reveal differences between the response of oligodendrocytes to netrin 1 made by the cells themselves, which regulates myelin-like membrane sheet formation, and netrin 1 encountered in the local environment, which primarily influences process branching.
Process elaboration induced by netrin 1 does not require β1 subunit-containing integrins
Oligodendrocytes express vβ1, vβ3, vβ5
and 6β1 integrins. Engagement of integrins, specifically
6β1, by extracellular matrix (ECM) components activates SFKs to
regulate changes in oligodendrocyte morphology
(Baron et al., 2005). Netrins
are members of the laminin family of ECM proteins, and netrin 1 has been
proposed to function as a ligand for 3β1 and 6β4
integrins in pancreatic cells (Yebra et
al., 2003). We therefore investigated whether netrin-1-mediated
changes in the elaboration of oligodendrocyte processes involve an interaction
between netrin 1 and integrins.
|
), did not significantly alter the netrin-1-induced increase
in oligodendrocyte process branching (Fig.
6A). By contrast, this antibody blocked HEK293T cell spreading on
a fibronectin substrate (Fig.
6B), verifying its efficacy. Yebra and colleagues identified a 25
amino acid region within the C-terminus of netrin 1 that binds 6β4
and 3β1 integrins, and hypothesized that potential interactions
between netrin 1 and other integrins might also occur through this region
(Yebra et al., 2003). To
determine whether such an interaction might contribute to netrin-1-induced
changes in oligodendroglial morphology, we incubated cells with a peptide
comprising the putative integrin-binding sequence that functions as a
competitive inhibitor of integrins binding netrin 1
(Yebra et al., 2003). The
netrin 1 peptide (20 µg/ml, a gift from Dr V. Cirulli, UCSD, CA, USA) did
not affect process complexity (Fig.
6A), nor did it disrupt the netrin-1-dependent increase in
branching (Fig. 6A). We
conclude that the novel role for netrin 1 in regulating oligodendrocyte
morphology occurs through Dcc independently of β1-containing integrins
and the integrin-binding region at the C-terminus of netrin 1.
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). Of
these, only mice lacking Fyn show defects in myelination
(Sperber et al., 2001).
Furthermore, Fyn activation has been implicated in the regulation of process
branching during the morphological maturation of oligodendrocytes
(Osterhout et al., 1999;
Umemori et al., 1994). In
axonal growth cones, application of netrin 1 recruits Fyn to the Dcc
intracellular domain, where it is activated by focal adhesion kinase (FAK;
Ptk2 - Mouse Genome Informatics) (Liu et
al., 2004). To determine whether Fyn might function downstream of
Dcc in oligodendrocytes, cells derived from newborn (P0) rats were allowed to
mature in culture for 3-4 days or for 5-6 days until they expressed Mbp.
Co-immunoprecipitation studies carried out using two different antibodies
against Fyn revealed an interaction between Fyn and Dcc in both immature (4
DIV) and mature (6 DIV) oligodendrocytes
(Fig. 7A). Five minutes
following application of netrin 1, an increased amount of Fyn
co-immunoprecipitated with Dcc (Fig.
7A,D). A relatively minor Fyn-immunoreactive band of slightly
higher molecular weight was consistently detected following
immunoprecipitation and might reflect Fyn phosphorylation as a result of
activation by netrin 1. Our findings also revealed a constitutive interaction
between Dcc and FAK (Fig. 7A),
suggesting that application of netrin 1 to oligodendrocytes recruits Fyn into
a complex with FAK that is bound to the intracellular domain of Dcc, as has
been reported for neurons (Li et al.,
2004; Ren et al.,
2004). Application of netrin 1 led to increased phosphorylation of
SFK tyrosine 416 (Y416), an event associated with kinase activation
(Smart et al., 1981), in the
SFK associated with Dcc (Fig.
7B,D). Analysis of SFK phosphorylation in whole-cell lysates did
not detect a global change in phospho-Y416, indicating that the
netrin-1-induced change is specific to SFK recruited into a complex with Dcc
(Fig. 7C). Using an antibody
specific for the SFK Src, an interaction with Dcc was not detected (not
shown). This provides evidence for specific recruitment of Fyn to Dcc;
however, we do not rule out that other SFKs might be involved in netrin 1
signaling in oligodendrocytes.
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SFK activity is required for the netrin-1-induced increase in oligodendrocyte process length and branching
Immunocytochemistry revealed SFK phospho-Y416 immunoreactivity distributed
within the oligodendrocyte cell body and proximal branches, and punctate
staining within the distal branches (Fig.
8C,D). Colocalization with Dcc was observed, consistent with our
immunoprecipitation results (Fig.
7B). To determine whether changes in SFK phosphorylation occurred
upon netrin 1 stimulation, the relative number of SFK
phospho-Y416-immunoreactive puncta was measured per unit area of the cell
(Fig. 8E). Treatment of mature
oligodendrocytes with netrin 1 (100 ng/ml) for 24 hours significantly
increased the number of SFK phospho-Y416-positive puncta per unit area,
consistent with an association between netrin 1 stimulation and increased SFK
activity (Fig. 8F). Treatment
with the SFK inhibitor PP2 (2 µM)
(Hanke et al., 1996) blocked
the netrin-1-induced increase in SFK phospho-Y416-positive puncta
(Fig. 8F), whereas the inactive
SFK inhibitor analog PP3 (2 µM) had no significant effect on the number of
puncta per unit area (Fig. 8F).
Application of PP2 alone led to a decrease in the relative number of puncta
per unit area, consistent with constitutive SFK activity contributing to the
basal level of puncta observed (Fig.
8F). To exclude the possibility that the increase in SFK
phospho-Y416-positive puncta was secondary to increased branch formation, the
same quantification was performed with cells immunostained for Fyn. No
difference was found in the number of Fyn-immunopositive puncta per unit area
in control and netrin-1-treated cells (Fig.
8G), consistent with the increase in SFK phospho-Y416-positive
puncta resulting from an increase in SFK activity and not a netrin-1-induced
increase in the number of branches.
Our findings indicate that netrin 1 recruits Fyn to a complex with Dcc, increasing SFK activity in oligodendrocytes. We then tested the hypothesis that SFK activation is required for the morphological changes induced by netrin 1. SFK activity was assessed in both immature (4 DIV, Cnp-positive) and mature (6 DIV, Mbp-positive) oligodendrocytes. Treatment of oligodendrocytes with PP2 blocked netrin-1-induced process extension in Cnp-immunoreactive immature cells and the netrin-1-dependent increase in branching in Mbp-expressing mature oligodendrocytes, whereas the inactive analog PP3 had no effect (Fig. 8H,I). Immature oligodendrocytes treated with PP2 alone showed a small but significant decrease in process length (Fig. 8H), which was not seen with PP3 treatment. Mature oligodendrocytes appeared less sensitive to the inhibition of basal SFK activity than immature cells, as PP2 alone did not affect branching (Fig. 8I). We conclude that netrin 1 binding to Dcc results in the recruitment of the SFK Fyn, and that subsequent activation of Fyn is required for the netrin-1-induced changes in oligodendrocyte morphology.
Netrin 1 inhibits RhoA but does not affect Cdc42 or Rac1 in oligodendrocytes
We next asked what signals might act downstream of SFKs to trigger the
morphological changes induced by netrin 1 in oligodendrocytes. Members of the
Rho family of small GTPases, including RhoA, Rac1 and Cdc42, regulate the
elaboration and branching of oligodendrocyte processes
(Liang et al., 2004). The
effect of netrin 1 on Cdc42 and Rac1 activity was investigated using a
GST-Pak-CRIB pulldown assay (Sander et
al., 1998). Cell lysates of cultured mature oligodendrocytes were
incubated with GST-Pak-CRIB fusion protein to quantify the levels of GTP-bound
Cdc42 and Rac1. No significant change in the levels of GTP-bound Rac1 or Cdc42
was detected in oligodendrocytes following application of netrin 1
(Fig. 9B,C). We have reported
that the Cdc42 effector protein N-WASP is recruited into a protein complex
with the intracellular domain of Dcc following the addition of netrin 1 to
embryonic spinal commissural neurons
(Shekarabi et al., 2005).
Unlike in oligodendrocytes, netrin 1 activates Cdc42 and Rac1 in commissural
neurons. In mature oligodendrocytes, N-WASP also constitutively
co-immunoprecipitated with Dcc; however, addition of netrin 1 did not
significantly alter the amount of N-WASP associated with Dcc
(Fig. 9D), which is consistent
with netrin 1 not increasing the activation of Cdc42 in oligodendrocytes.
|
;
Miron et al., 2007;
Wolf et al., 2001).
Interestingly, activation of Fyn in oligodendrocytes leads to inactivation of
RhoA (Wolf et al., 2001), and
we therefore investigated the possibility that netrin 1 might regulate RhoA
activity in oligodendrocytes. Levels of GTP-bound RhoA in mature Mbp-positive
oligodendrocytes were assessed using a GST-rhotekin pulldown assay
(Reid et al., 1996). Following
a 5 minute treatment of mature oligodendrocytes with netrin 1, a significant
decrease in RhoA-GTP was detected compared with controls
(Fig. 9A). We conclude that
netrin-1-induced inhibition of RhoA, in the absence of altered Rac1 or Cdc42
activity, promotes process elaboration by oligodendrocytes. |
DISCUSSION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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; Kirby et al.,
2006). As the cell matures, these processes branch and eventually
form lamellae that ensheath target axons. Our findings indicate that netrin 1
and Dcc regulate oligodendrocyte process extension, branching and myelin-like
membrane sheet formation, which are all essential events for initiating
myelination.
Exogenous and autocrine netrin 1 contribute to oligodendrocyte maturation
Our results support the conclusion that netrin 1, as expressed by neurons
and neuroepithelial cells, when encountered by a premyelinating
oligodendrocyte evokes Dcc-dependent extension of the motile processes of the
cell. In more mature cells, exogenous netrin 1 promotes Dcc-dependent process
branching and myelin-like membrane sheet formation. Based on our findings in
vitro and in vivo, we hypothesize that by promoting the morphological
maturation of oligodendrocytes, netrin 1 facilitates the search for
appropriate axonal targets. Interestingly, although exogenous netrin 1
promotes changes in oligodendrocyte morphology, at later stages of maturation
oligodendrocytes themselves begin to express netrin 1. We identify a
selective, Dcc-dependent autocrine role for netrin 1 in promoting the
formation of myelin-like membrane sheets. Oligodendrocytes only begin to
express netrin 1 in the developing spinal cord after myelination has begun.
Our findings indicate that contact with axons is not essential for the
initiation of netrin 1 expression by these cells, but that it occurs
coincident with myelin-like membrane sheet formation. We hypothesize that
autocrine expression of netrin 1 specifically promotes later stages of
maturation, facilitating the formation of large myelin-like membrane sheets by
these cells. Netrin 1 expression by oligodendrocytes might also facilitate
axon remodeling or prevent aberrant axonal sprouting at later stages of
development.
|
; Jarjour and Kennedy,
2004), and increasing evidence suggests that common regulators of
actin nucleation function in neuronal growth cones and at the leading edge of
oligodendroglial processes and sheets
(Sloane and Vartanian, 2007).
Here, we report that the similarities between growing axons and
oligodendrocyte processes include the recruitment of similar intracellular
signaling proteins downstream of netrin 1: Fyn, FAK, N-WASP and Rho GTPases
(Li et al., 2004;
Liu et al., 2004;
Ren et al., 2004;
Shekarabi and Kennedy, 2002).
We demonstrate that Fyn is required for the netrin-1-dependent increase in
oligodendrocyte process branching. Netrin 1 recruits Fyn to a complex that
includes Dcc and FAK, resulting in SFK phosphorylation and activation.
Importantly, previous studies have implicated Fyn, Rac1, Cdc42, RhoA and
N-WASP in the mechanism governing the cytoskeletal changes in oligodendrocytes
that lead to myelination (Simons and
Trotter, 2007).
|
). We
demonstrate reduced levels of GTP-bound RhoA upon netrin 1 stimulation, but
found no evidence for the activation of Cdc42 or Rac1. Interestingly,
transgenic mice specifically lacking Cdc42 and Rac1 in oligodendrocytes
exhibit normal oligodendroglial differentiation, but show eventual defects in
myelin compaction (Thurnherr et al.,
2006). Our findings support the hypothesis that in a background of
unchanging Rac1 and Cdc42 activity, modulation of RhoA function plays a key
role in regulating the morphological differentiation of oligodendrocyte
processes.
Netrin 1 activates a canonical signaling mechanism required for oligodendrocyte maturation
To date, the candidate extracellular signals that might regulate the
elaboration of oligodendroglial processes immediately preceding myelination
have been limited to ECM proteins, of which laminin 2 and its receptor
integrin 6β1 have been well characterized. Studies in vitro have
shown that integrin-dependent activation of Fyn activates Cdc42 and Rac1 and
deactivates RhoA, leading to process outgrowth
(Liang et al., 2004;
Osterhout et al., 1999).
However, oligodendrocytes lacking the β1 integrin subunit mature and
myelinate normally, demonstrating that this pathway is not essential in vivo
(Benninger et al., 2006).
Furthermore, laminin 2 is not ubiquitously present in myelinating axon tracts
in the CNS, indicating that other ligand-receptor complexes must trigger these
signaling mechanisms independently of β1 integrin function. Our results
show that netrin 1, acting through Dcc, activates an intracellular signaling
pathway that is required for the morphological maturation of oligodendrocytes.
Crucially, the effects of netrin 1 do not require β1 integrin function
and do not appear to act through netrin-binding integrins.
Our findings identify a novel mechanism regulating oligodendrocyte
morphology during later stages of differentiation. Interestingly, many
oligodendroglial cells detected in multiple sclerosis lesions appear to have
differentiated, but remain unable to elaborate myelin
(Chang et al., 2002). A better
understanding of the mechanisms that promote myelination will advance the
development of therapeutics that aim to promote the recovery of nervous system
function.
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Footnotes |
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REFERENCES |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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