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Volume 17, Number 12,
Issue of June 15, 1997
pp. 4623-4632
Copyright ©1997 Society for Neuroscience
Expression of Specific Tubulin Isotypes Increases during
Regeneration of Injured CNS Neurons, But Not after the Application of
Brain-Derived Neurotrophic Factor (BDNF)
Alyson E. Fournier and
Lisa McKerracher
Faculté de Médecine, Département de Pathologie,
Université de Montréal, and McGill University, C.P. 6128, Succursale Centre-ville Montréal, Québec H3C 3J7, Canada
 ABSTRACT
- INTRODUCTION
- MATERIALS AND METHODS
- RESULTS
- DISCUSSION
- FOOTNOTES
- REFERENCES
ABSTRACT 
Axonal regrowth after injury is accompanied by changes in the
expression of tubulin, but the contributions of substrate molecules and
neurotrophic factors in regulating these changes in vivo
are not known. Adult rat retinal ganglion cells (RGCs) were examined after intraorbital axotomy, after application of a peripheral nerve
(PN) graft to stimulate regeneration, and after axotomy and treatment
with brain-derived neurotrophic factor (BDNF). After these treatments
we used in situ hybridization to study mRNA levels for
 I, II, III, IVa, and T 1 tubulin isotypes.
Levels of mRNA for all isotypes were downregulated after intraorbital
axotomy. During regrowth of injured RGC axons, mRNA levels for II,
III, and T1 isotypes were upregulated specifically and
dramatically, suggesting that elevated expression of these isotypes is
correlated specifically with axonal regrowth. A corresponding increase
in III protein levels was detected by immunocytochemistry. The I and IVa mRNAs were not increased during regeneration.
BDNF did not elicit a specific increase in the mRNA levels for the
III and T1 isotypes and had only a small effect on mRNA levels
for the II isotype. Therefore, despite the ability of BDNF to
support the survival of injured RGCs and to enhance neurite outgrowth of retinal neurons in vitro, the in vivo
application of BDNF alone is unable to induce the program of changes in
growth-associated tubulins that accompany regeneration of RGC axons
into PN grafts. We speculate that, in addition to BDNF, cooperative
signaling with substrate molecules is required to allow RGCs to
regenerate and exhibit tubulin isotype switching.
Key words:
BDNF;
tubulin isotypes;
microtubules;
rat;
regeneration;
axotomy
INTRODUCTION
Axonal injury of adult neurons results in a number
of changes that are detectable in the cell body, including marked
changes in the levels of various types of mRNAs. Changes in mRNA levels for cytoskeletal proteins are of interest because they are correlated tightly with neurite outgrowth, and cytoskeletal proteins support the
regenerating axon and growth cone. Similarly, the factors that modify
expression levels of these proteins may play critical roles in
regeneration. Some cytoskeletal mRNAs show similar changes after axonal
injury in the CNS and PNS, such as the decrease in mRNAs that encode
neurofilaments (Hoffman and Cleveland, 1988 ; Tetzlaff et al., 1991;
McKerracher et al., 1993a). However, the regulation of tubulin mRNAs
after injury differs according to the neuronal cell type and the
distance of axotomy from the cell body (for review, see Bisby and
Tetzlaff, 1992; Tetzlaff et al., 1994). After axotomy in the CNS where
there is no sustained regeneration, there is a long-term decrease in
total tubulin mRNAs, but sometimes an early transient increase in
tubulin mRNAs occurs (Tetzlaff et al., 1991; McKerracher et al.,
1993b). In the PNS an overall increase in total tubulin mRNA is
observed after peripheral nerve injury when neurons regenerate
spontaneously; this is attributable to increases in specific tubulin
isotype mRNAs. Isotypes are distinct members of the large tubulin gene
family that encode both and tubulin subunits, and these
isotypes are regulated differentially during development (Lewis et al.,
1985; Jiang and Oblinger, 1992). The II, III, and T1 isotype
levels are elevated after peripheral nerve injury, whereas I, IV,
and T26 levels are not (Miller et al., 1987, 1989; Hoffman and
Cleveland, 1988; Tetzlaff et al., 1991; Moskowitz et al., 1993). It is
not known if the different tubulin isotypes are affected differentially
by injury and during regeneration in the CNS as they are in peripheral
nerves.
It also is not known if tubulin expression in injured neurons may be
affected by the presence of neurotrophic factors present in the
non-neuronal extracellular environment after injury. An understanding
of the different factors that regulate the complex changes in tubulin
will be important to our understanding of the regenerative process.
Examination of adult rat retinal ganglion cells (RGCs) has helped to
identify a number of extrinsic signals important for regrowth of
injured axons. Brain-derived neurotrophic factor (BDNF), a major
neurotrophic factor for RGCs, is able to augment RGC survival (Mey and
Thanos, 1993; Mansour-Robaey et al., 1994) and influence neurite
outgrowth in cell culture (Johnson et al., 1986; Rodriguez-Tebar et
al., 1989; Thanos et al., 1989; Cohen et al., 1994). Furthermore, BDNF
is produced by Schwann cells that are present in PN grafts, which can
support regeneration of some injured RGCs (David and Aguayo, 1981).
Although the in vivo application of BDNF in the eye does not
enhance the number of RGC axons able to penetrate peripheral nerve (PN)
grafts (Mansour-Robaey et al., 1994), it is not clear if this is
attributable to a failure of BDNF to stimulate regeneration-associated
changes in gene expression or attributable to blocked neurite outgrowth
because of inhibitory molecules present at the optic nerve head. We
sought to test directly the effect of BDNF on intrinsic determinants of
regeneration in RGCs by following changes in mRNA levels for some
growth-associated tubulin genes in response to BDNF.
By following mRNA levels in regenerating RGCs identified by retrograde
labeling, we document here that the II, III, and T1 isotypes
are upregulated specifically only in those RGCs that successfully
regrow their axons. Elevation of these mRNAs therefore specifically
correlates with regrowth of injured RGCs in vivo. We also
document that BDNF was not sufficient by itself to induce changes in
tubulin mRNA levels.
MATERIALS AND METHODS
Surgical procedure and BDNF applications. Adult
female Sprague Dawley rats (180-220 gm) were anesthetized with chloral
hydrate (0.42 mg/gm body weight, i.p.), and the left optic nerve was
exposed and transected 0.5 mm from the optic nerve head (Fig.
1b; Villegas-Perez et al., 1993). For sham
operations the left optic nerve was exposed but not cut. To graft a
peripheral nerve to the optic nerve stump, we attached an autologous
segment of sciatic nerve with 10.0 sutures (Fig. 1c;
Vidal-Sanz et al., 1987). Crystals of the retrograde tracer DiI
(Vidal-Sanz et al., 1988) were inserted at multiple sites along the
length of the graft before closing the skin with silk sutures.
BDNF-treated animals had their left eye injected with 5 µg of BDNF in
5 µl of PBS, 0.5% BSA immediately after optic nerve cut (Fig.
1d). A posterior approach for injection was used because the
local release of endogenous factors into the eye is minimized, as
compared with an anterior approach (Mansour-Robaey et al., 1994). For
injection the eye was rotated forward, the sclera was punctured with a
26 gauge needle, and BDNF was injected with a Hamilton syringe. For
controls, the vehicle of 5 µl of PBS, 0.5% BSA was injected (called
PBS treatment). Rats were killed 3 d (n = 4) or
14 d (n = 4) after axotomy and 14 d after
sham axotomy (n = 2). Animals with a PN graft
(n = 3) were examined from 14 to 20 d after
grafting, a time when RGC axons are growing actively along the graft
(McKerracher et al., 1990). Animals that received axotomy and BDNF
treatment were examined at 3 d (n = 3) and 14 d (n = 4). Animals treated by axotomy and PBS
injections were processed at 3 d (n = 3) and
14 d (n = 2). These times were chosen because BDNF
has maximal effects on gene expression 3 d after injection into
the eye (Fournier et al., 1997), whereas 14 d is a time when
axotomy alone is known to affect gene expression and is also
appropriate for detecting regenerating RGCs in PN-grafted animals
(McKerracher et al., 1990).
Fig. 1.
Schematic representation of the surgical
procedures and the known morphological response of injured retinal
ganglion cells. A, Normal adult rat retinal ganglion
cells project their axons into the optic nerve. B, After
axotomy some RGCs die (Villegas-Perez et al., 1993), and RGC axons
retract into the optic nerve head. C, Application of a
peripheral nerve graft to injured RGCs elicits long distance regrowth
of a small number of RGCs (a regenerating RGC is shown in
black) (Vidal-Sanz et al., 1987). D, A
single application of BDNF prevents all death (Mansour-Robaey et al., 1994), and there is an increase in axonal branch length (Sawai et al.,
1996).
[View Larger Version of this Image (26K GIF file)]
When the animals were killed, their eyes were removed and
immersion-fixed in 4% paraformaldehyde and 0.1 M phosphate
buffer, pH 7.2, for 3-4 hr. The lens and cornea were removed, and the eyes were placed in an RNase-free solution of 10% sucrose in PBS overnight. Left and right eyes were mounted and frozen together in
Tissue Tek O.C.T. (Canlab, Montreal, Quebec). The blocks were stored at
 80°C until cryostat sections of 8-12 µm were cut. Multiple slides were examined for each animal (n = 3-10 slides,
typically 5 slides).
Preparation of RNA and Northern blots. Total RNA was
prepared from postnatal day 1 (P1) rat brain, adult brain, and retina. For the retinal samples six retinas from three animals were pooled before homogenization and prepared by the acid phenol method
(Chomczynski and Sacchi, 1987). Total RNA was separated
electrophoretically on formaldehyde gels and transferred to Nytran
filters. Equal amounts of RNA were loaded in each lane, and this was
verified by ethidium bromide staining of formaldehyde gels. Probes of
~300 base pairs in length and recognizing the unique 3 regions of the I, II, III, and IVa -tubulin isotypes
were generated by oligolabeling with 32P-dCTP (Pharmacia,
Montreal, Quebec). The mouse -tubulin cDNAs were obtained from Dr.
Nicholas Cowan (New York University Medical Center, New York). The
T1 probe was 174 base pairs and was derived from a pGEM-4 plasmid
provided by Dr. Freda Miller (McGill University, Montreal) (Miller et
al., 1987). For this, the T1 cDNA was linearized with
HindIII before in vitro transcription with SP6
polymerase. Sense probes were generated by linearizing with
EcoRI and by using T7 polymerase.
Blots were prehybridized for 1 hr with 2× SSC, 50% deionized
formamide, 1 mM DTT, 250 µg/ml denatured tRNA, 250 µg/ml denatured fish sperm DNA, 1× Denhardt's solution, 5 mM EDTA, pH 8, and 0.2% SDS and then hybridized overnight.
Washes were in 4× SSC, 0.1% SDS for 2 × 30 min and then 2×
SSC, 0.1% SDS for 2 × 30 min. Hybridization and washing were at
45°C for cDNA probes or 65°C for cRNA probes. Blots were exposed to
autoradiographic film and were stripped and reprobed up to five
times.
In situ hybridization. Slides were removed from
80°C and warmed on a slide warmer; sections from grafted animals
were examined under the fluorescence microscope to identify and
document the position of DiI-labeled cells. For in situ
hybridization slides were acetylated, washed in PBS, and pre-hybridized
as for Northern blots for 1 hr at 42°C. Slides subsequently were
hybridized overnight at 42°C with 1 × 106 cpm of
labeled probe per two pairs of retinal sections.
35S-labeled -tubulin cDNA and T1 cRNA probes were
generated as described for Northern blots and used in preliminary
experiments. To increase sensitivity for in situ
hybridization, we subcloned II, III, and IVa
isotypes into the EcoRI/HindIII site of pGEM 72+ vectors to generate cRNA probes by in vitro
transcription. Antisense and sense probes were generated from
linearized plasmids and transcription from the SP6 or T7 promoter as
appropriate. After hybridization, slides were washed sequentially in
4× SSC, 1 mM DTT for 2 × 10 min and 4× SSC for
2 × 10 min at 65°C for cRNA probes and 45°C for cDNA probes.
For cRNA probes single-stranded RNA was digested with RNase A (50 µg/ml RNase A, 0.5 M NaCl, 10 mM Tris, and
0.1 mM EDTA) at 37°C for 30 min. Final washes were with
SSC, gradually increasing the stringency to a final wash of 0.1× SSC
for 20 min at 65°C for cRNA probes and 45°C for cDNA probes. The
same hybridization patterns were observed with cDNA and cRNA probes for
a given isotype. The cRNA probes were used for experiments with
quantitative analysis because of a cleaner hybridization signal.
Emulsion autoradiography and quantitative analysis.
After in situ hybridization slides were air-dried and
dipped in Kodak NTB2 autoradiographic emulsion diluted 1:1 with water
and stored in a light-tight box. Exposure times for the tubulin probes
varied from 2-4 weeks. Slides were developed for 5 min in Kodak D19
mixed 1:1 with water at 20°C, rinsed, and fixed for 5 min in Kodak
fix. Sections were stained with 0.25% thionin stain and permanently mounted in Entellan (VWR, Canada).
An Image 1 (Universal Imaging, West Chester, PA) image analysis
system was used to count the number of autoradiographic silver grains
over RGCs of treated and intact retinas after in situ
hybridization. Treated and control sections were cut together and thus
were hybridized and dipped in emulsion on the same slide. This control
for experimental variability allowed for remarkably consistent results
when the average number of grains per RGC was determined and expressed as a percentage of that in the same slide control retina. Cells smaller
than 70 µm, which would include all of the displaced amacrine cells,
were excluded from the analysis. Also, cells larger than 130 µm,
which rarely were observed, were excluded from the analysis. Hybridization "hotspots" were identified visually, and quantitation was performed. Occasionally, hotspots that did not correlate with DiI-labeled cells were identified most likely because the regenerating RGC failed to take up or retrogradely transport DiI from the graft. Statistics were performed with the Sigma Stat statistical program (Jandel, Corte Madera, CA). Data that met the criteria for parametric tests were analyzed by a Student's t test. Groups of data
that failed tests for normality were analyzed by the Mann-Whitney
test.
Immunocytochemistry. Tissue was prepared as described
for in situ hybridization. DiI back-labeled cells were
identified by fluorescence microscopy, and their positions were
documented. Then slides were washed in PBS and blocked for 1 hr at room
temperature in 3% BSA, 0.1% Triton X-100 in sterile PBS. Slides were
incubated overnight at room temperature with the primary antibody. The
monoclonal anti- tubulin isotype I and II antibody (Sigma, St.
Louis, MO) was used at a dilution of 1:1000. The monoclonal anti-III
antibody (Sigma) was used at 1:200. Slides were incubated with goat
anti-mouse IgG FITC antibody (Calbiochem, San Diego, CA) in PBS, used
at a 1:100 dilution. Slides were washed in PBS and mounted in SlowFade (Molecular Probes, Eugene, OR).
RESULTS
Each tubulin isotype mRNA decreases after axotomy
Four different -tubulin isotypes and two -tubulin isotypes
are expressed in rodent brain with a distinctive developmental profile
(Lewis et al., 1985; Miller et al., 1987). To determine which isotypes
are expressed in adult rat retina and to confirm that the mouse
-tubulin isotypes are found in rat brain, we prepared Northern blots
with RNA extracted from P1 brain, adult brain, and adult retina. Each
of the four -tubulin isotypes recognized distinct bands of the
predicted sizes (Fig. 2). The I, II, and III
isotypes were expressed at higher levels in P1 brain than in adult
brain, and the IVa isotype showed a reciprocal pattern of expression, with the highest levels in adult brain. These results, which are consistent with previous reports (Lewis et al., 1985; Jiang
and Oblinger, 1992) show that the -tubulin isotypes are regulated
differentially during rat brain development. Further, I, II,
III, and IVa isotypes were each detected in the adult rat retina (Fig. 2). The rat T1 -tubulin isotype also has been characterized fully. T1 is a neuron-specific isotype expressed at
high levels in developing rat brain and in regenerating peripheral neurons (Miller et al., 1987, 1989). In previous in situ
hybridization studies of the retina, we have documented that T1
(Fournier et al., 1997) and I (Fournier and McKerracher, 1995)
isotypes are expressed in the retina, and the individual -tubulin
isotypes studied here, II, III, and IVa, all
showed similar patterns of expression in the different retinal layers
(Fig. 3). Therefore, many types of adult retinal neurons
express at least four different -tubulin mRNAs (Fournier and
McKerracher, 1995). With sense probes used as controls, only very light
labeling was distributed equally over all layers (Fig.
3d).
Fig. 2.
Northern blots of postnatal day 1 (P1) rat brain, adult rat brain (Ad), and
adult rat retina (R). Blots were probed with
32P-labeled cDNA antisense probes recognizing the
I, II, III, and
IVa tubulin isotypes. The I,
II, and III isotype mRNA levels were
highest in the developing rat brain, whereas the
IVa isotype mRNA was highest in the
adult. The mRNAs for all isotypes were detected in the adult rat
retina.
[View Larger Version of this Image (64K GIF file)]
Fig. 3.
-Tubulin isotype mRNA levels decrease after
axotomy. Dark-field micrographs of radial sections from uninjured
contralateral control (left panel) or 2 week
axotomized (right panel) retinas probed with
35S-labeled cRNA probes recognizing the II
(a), III (b), and IVa (c) isotypes. All isotype mRNA levels were reduced 2 weeks after injury. d, Dark-field micrograph of a radial
retinal section probed with a control II sense probe. Scale bar
(a), 85 µm; (b-d), 100 µm. The
retinal ganglion cell layer is indicated in each micrograph in the
left panel by an arrow.
[View Larger Version of this Image (140K GIF file)]
After intraorbital transection of the optic nerve (Fig. 1), the
hybridization signal for each isotype decreased in the RGC layer of the
retina (Fig. 3). Such a change was not observed in the other cellular
layers that were not influenced directly by the optic nerve cut. It is
known that RGCs begin to die 3 d after axotomy and that by 2 weeks
only 12% of RGCs survive (Villegas-Perez et al., 1993). To determine
whether the loss in the hybridization signal resulted exclusively from
cell death or whether changes in the mRNA levels in the surviving RGCs
contributed to the loss of signal, we counted the grains over
individual cells to estimate relative mRNA levels, as compared with
contralateral controls processed on the same slide. This analysis
demonstrated that the mRNA levels decreased in the individual cells.
The grain counts for the II, IVa, and T1 isotypes
were decreased to 81, 79, and 75% of control levels, respectively,
3 d after axotomy. The III isotype was more resistant to this
downregulation, and its mRNA level was maintained at 100% of control
levels at this early time (Fig. 4). However, by 14 d there was a drop in the signal for this isotype to 61% of the
control level, and the mRNA levels for all of the other isotypes
remained low, as compared with controls or with sham-operated animals
(Fig. 4). At 2 weeks the II, IVa, and T1 mRNA
levels were 72, 72, and 77% of contralateral control levels,
respectively. Decreased expression levels for the III, IVa, and T1 isotypes were significant 2 weeks after
axotomy. Decreases in III and IVa were analyzed by
the Student's t test, whereas the decrease in T1 was
analyzed by the Mann-Whitney test. Therefore, in contrast to responses
in injured peripheral nerves where isotype-specific changes are
induced, RGC injury results in a similar change in mRNA levels for all
tubulin isotypes.
Fig. 4.
Quantitative analysis of tubulin isotype mRNA
levels 3 d and 2 weeks after intraorbital axotomy. Counts from the
experimental retinas were compared with the contralateral retinas
mounted on the same slide, and the contralateral control was expressed
as 100%. Each bar represents data from multiple slides
from multiple animals (see Materials and Methods). The II,
IVa, and T1 levels decreased
by 3 d after injury and remained low at 2 weeks. The III isotype levels decreased by 2 weeks after injury.
Sham-operated rats did not show significant changes.
[View Larger Version of this Image (24K GIF file)]
Specific tubulins mRNAs are elevated in regenerating RGCs
We next investigated how a peripheral nerve graft might modify the
drop in tubulin isotype levels after axotomy. For these experiments
RGCs that regenerated their axons were detected by retrograde labeling
with DiI. DiI was applied to the peripheral nerve graft at the time of
grafting, and regenerating RGC axons took up the tracer as they grew in
the PN graft and transported it back to the cell body. Then the mRNA
levels were examined in RGCs with and without DiI labeling. The RGCs
were examined 14-20 d after surgery, times when some RGCs have
extended axons in the graft (McKerracher et al., 1990). After axotomy
with a PN graft attached to the optic nerve stump, hybridization of the
retina with isotype-specific probes revealed that most RGCs had reduced mRNA levels for each of the separate isotypes, as was observed 14 d after axotomy alone (Fig. 5). However, dramatically
enhanced mRNA levels were detected in a few RGCs in sections hybridized with the II (Fig. 5c), III (Fig. 5d), and
T1 (Fig. 5a,b) isotype probes. These RGC "hotspots"
were not observed after hybridization with probes for the I or
IVa isotypes. Further examination revealed that the
hybridization hotspots correlated with the DiI back-labeled RGCs (Fig.
5b-d). Therefore, the T1 isotype and two of the three -tubulin isotypes, the expression levels of which are high during development, are upregulated specifically in those RGCs that
regenerated their axons into the PN grafts. Quantitative analysis of
RGCs in the PN-grafted samples revealed that most RGCs decreased their mRNA levels for all isotypes, a result that correlates with the fact
that a majority of RGCs do not regenerate in PN-grafted retinas. Only
~3% of RGCs regenerate (Vidal-Sanz et al., 1987), and those RGCs
that do not regrow do not increase their tubulin levels. Isotype mRNA
levels were 73, 80, and 63% of control levels for the II, III,
and T1 isotypes, respectively (Fig. 6). These results
are similar to those retinas that received an axotomy without a PN
graft. Only IVa tubulin levels were rescued by the graft, with average levels of 110% relative to intact contralateral controls. However, no hybridization hotspots were detected with the
IVa isotype probe. For the II, III, and T1
isotypes mRNA levels in RGCs that appeared as hotspots increased
dramatically over the control values. Grain counts of visually
identified hotspots were 260, 369, and 401% of the control levels for
the II, III, and T1 isotypes, respectively (Fig. 6). All
increases were statistically significant (Student's t
test).
Fig. 5.
II, III, and T1 mRNA levels are
upregulated specifically in regenerating RGCs. a,
Dark-field micrographs of radial sections from an uninjured
contralateral control retina (a, left
panel) and a retina 2 weeks after injury and peripheral
nerve graft application (a, right
panel). Retinas were probed with a T1 antisense
probe. T1 mRNA levels were decreased in most RGCs but were
upregulated specifically in a small number of RGCs
(arrow). b-d, Radial retinal sections
visualized by fluorescence microscopy (right
panel) showing RGCs that have been retrogradely labeled
with DiI (arrowheads). The left panel shows
the same section after in situ hybridization with T1,
II, or III antisense probes. II, III, and T1 mRNA levels
were enhanced specifically in regenerating RGCs that had taken up the
DiI marker. Scale bars: in a, 85 µm; in
b-d, 55 µm. rgc, Retinal ganglion cell
layer.
[View Larger Version of this Image (83K GIF file)]
Fig. 6.
Quantitative analysis of isotype mRNA levels in
regenerating and nonregenerating RGCs 2 weeks after injury and
peripheral nerve graft application. The II, III,
and T1 mRNA levels were decreased in most RGCs but
were enhanced dramatically in some RGCs. The I and
IV isotypes were not increased in RGCs in PN-grafted retinas.
[View Larger Version of this Image (14K GIF file)]
To determine whether the increases in mRNA levels were indicative
of changes in the level of tubulin protein, we used isotype-specific antibodies to immunolabel the sections. Such examination revealed that
the DiI back-labeled cells tended to be stained more intensely with the
anti-III -tubulin antibody (Fig. 7). Normal
retinal sections typically were well stained with the anti-I/II
antibody, but in some cases a correlation between high
immunofluorescence and a DiI back-labeled cell could be detected.
Failure to detect intensely immunopositive regenerating RGCs likely was
attributable to high levels of expression of I and II tubulin
isotypes in adjacent injured RGCs. Specific I and IV antibodies
were not available at the time of these experiments.
Fig. 7.
The III protein levels are enhanced
specifically in regenerating RGCs (arrow). A radial
retinal section visualized by fluorescence microscopy shows a
retrogradely labeled RGC with DiI from a PN graft (a).
The same RGC is labeled with FITC after immunocytochemistry with an
anti-III antibody (b). Note that the adjacent
axotomized RGCs that do not extend axons in the graft (and are not
labeled with DiI) are not immunoreactive. Immunolabeling with the
III antibody also was observed in the outer nuclear layer. Scale
bar, 55 µm.
[View Larger Version of this Image (150K GIF file)]
Application of BDNF fails to elicit marked changes in tubulin
expression in injured RGCs
BDNF is a potent survival factor for injured RGCs (Mansour-Robaey
et al., 1994), and studies examining the effect of BDNF on GAP-43
expression indicate that most, if not all, RGCs respond to BDNF
(Fournier et al., 1997). To examine if BDNF would modify the expression
of the tubulin isotypes in injured RGCs, we injected BDNF into the
vitreous of the eye at the time of axotomy. As a control, vehicle alone
was injected into the eye. It is known that intraretinal injections of
vehicle alone can enhance cell survival; however, this effect is
minimal after posterior injections (Mansour-Robaey et al., 1994).
Similarly, in this study we used a posterior approach and detected
small changes in the PBS injection controls, because there was no
decrease in the II, IVa, and T1 mRNA levels 3 d after axotomy and PBS injection, as was observed with axotomy alone.
Therefore, the control posterior injections exerted small but
detectable effects on RGCs.
BDNF injection at the time of axotomy did not result in any general,
dramatic changes in tubulin expression (Fig. 8). Three days after BDNF injection, a time when BDNF application has maximal effects on the expression of another growth-associated protein, GAP-43
(Fournier et al., 1997), only II isotype mRNA levels were increased
significantly. Moreover, this increase to 122% of the intact
contralateral level was modest, as compared with the changes observed
when RGCs regenerated their axons in a peripheral nerve graft (Figs. 6,
8). BDNF injection had no significant effects on any of the other
isotypes when compared with control PBS injections.
Fig. 8.
Tubulin isotype levels are not enhanced
dramatically by BDNF application. Shown is the quantitation of tubulin
isotype mRNA levels 3 d (left) or 2 weeks
(right) after the application of BDNF to injured RGCs.
BDNF elicited a modest enhancement of II levels 3 d after
treatment but did not affect the other tubulin isotypes. This effect
was diminished by 2 weeks.
[View Larger Version of this Image (21K GIF file)]
By 2 weeks after BDNF treatment, all of the tubulin isotypes dropped
below the intact contralateral control levels (Fig. 8). The small
effect of BDNF on II tubulin 3 d after axotomy was not
sustained at 2 weeks.
DISCUSSION
Tubulin isotypes are regulated differentially during
RGC regeneration
We have followed the expression of five tubulin isotypes in adult
rat RGCs after injury, after injury and BDNF treatment, or after
replacement of the optic nerve with a peripheral nerve graft. We have
documented that each of these isotypes is expressed in adult rat RGCs.
We find that mRNA levels for all isotypes are decreased in a similar
manner after intraorbital RGC injury. Therefore, in contrast to neurons
in the peripheral nervous system, RGCs do not spontaneously upregulate
expression of growth-associated tubulin isotypes after injury close to
the eye. We further demonstrate that long distance regrowth of injured
RGCs is accompanied by a specific increase in the mRNA levels of the
T1, II, and III isotypes. Unlike other growth-associated
proteins, such as GAP-43, the expression of which can be induced by
injury alone in the CNS (Doster et al., 1991; Fournier et al., 1997),
the expression of these tubulin isotypes is correlated tightly with
long distance growth of RGC axons (Fig. 1c). The expression
of T1 is upregulated after injury of rubrospinal neurons (Tetzlaff
et al., 1991), but the morphological correlate of this response is
unclear. The expression of all neuronal tubulin isotypes by RGCs,
including the growth-associated isotypes, may allow for some local
growth or plasticity in adult animals, but increased amounts of
specific isotypes may be required to sustain regeneration of injured
axons.
BDNF enhances early regenerative events but does not mediate long
distance growth in vivo
It is known that BDNF stimulates intraretinal branching events of
some injured RGCs (Sawai et al., 1996) and that the expression of
GAP-43 is upregulated during these early events (Fournier et al.,
1997). However, we have documented here that expression of the III
and T1 growth-associated tubulin isotypes is not affected dramatically by BDNF application, and only small effects on the II
isotype were observed (Fig. 8). The inability of BDNF to enhance the
expression of these isotypes suggests that BDNF alone does not
stimulate molecular events required for long distance axon growth
because increases in these mRNAs correlated with the regeneration of
RGC axons into PN grafts. If BDNF alone does not stimulate all the
changes in gene expression that correlate with regeneration, additional
factors in the PN graft, such as substrate cues, may be critical for
successful regeneration. Although it is possible that the intravitreal
injection of BDNF did not mimic the effects of a PN graft because it
was not applied at the cut axon stump, we do not think this is the
case. First, after intraorbital axotomy most RGC axons retract into the
eye where they should have adequate access to the exogenously applied
BDNF. Furthermore, BDNF applied to the vitreous increases the axonal
branch length of injured RGCs, a finding that indicates that injured
RGC axons do respond to BDNF applied by injection into the eye (Sawai
et al., 1996). Our finding that the application of BDNF in
vivo does not lead to an increase in the mRNA levels of the
growth-associated isotypes is surprising, because BDNF is able to
enhance RGC neurite outgrowth in vitro (Cohen et al., 1994).
In culture, however, the RGCs also are exposed to additional factors
from serum or to growth-permissive substrates, such as laminin or the
surface of non-neuronal cells. We predict that in vivo the
RGCs that regenerate are those for which the growth cones successfully
interact with the peripheral nerve graft substrate.
Both in vitro experiments (Hopkins and Bunge, 1991) and
experiments in which peripheral nerve grafts were freeze-thawed before being grafted into the visual system in rats (Berry et al., 1988) determined that living Schwann cells are essential for PN grafts to
support regeneration. Although it is possible that the cells must be
living to synthesize neurotrophic factors, the results reported here on
BDNF application suggest that this is not their only role. We found
that additional soluble factors released by the injection injury
prevented the decrease in mRNA levels for tubulin isotypes but did not
mimic the effects of the peripheral nerve graft on tubulin isotype
expression. These results suggest that other diffusible factors likely
to be produced by non-neuronal cells in PN grafts (Rappolee et al.,
1988; Funakoshi et al., 1993; Elkabes et al., 1996) also may not be
sufficient to increase mRNA levels of growth-associated tubulin
isotypes. We speculate that the migration of Schwann cells into the
optic nerve head where injured axons might contact their cell surface
may provide critical substrate cues needed for regeneration.
Cooperative signaling events may be required for successful
regenerative growth
Cell adhesion molecules present on Schwann cell surfaces,
such as L1 and N-cadherin (Bixby et al., 1988; Kleitman et al., 1988a,b; Seilheimer and Schachner, 1988; Haussman et al., 1989) as well
as extracellular matrix molecules (Bixby et al., 1988; Fawcett and
Keynes, 1990), all may contribute to the growth-permissive environment
of PN grafts. Recent studies suggest that there is convergence between
neurotrophin and extracellular matrix-mediated intracellular signaling
pathways (Clark and Brugge, 1995; Rozengurt, 1995), and perhaps
neurotrophins and substrate molecules act in a cooperative manner to
signal regenerative axon growth. Cooperative signaling may be needed to
regulate intrinsic molecules, such as microtubules.
Although BDNF is not sufficient to elicit tubulin isotype switching in
regeneration, it is also possible that growth-inhibitory molecules play
a role (McKerracher and David, 1997). After axotomy RGC axons
degenerate back toward their cell bodies, leaving their growth cones in
the vicinity of the optic nerve head (Mansour-Robaey et al., 1994;
Sawai et al., 1996). An intriguing possibility is that most RGCs
attempt to regrow after neurite retraction but that their growth cones
are diverted by the myelin-derived inhibitory proteins (McKerracher and
David, 1997) or by inhibitory molecules, such as chondroitin sulfate
proteoglycan, at the optic nerve head (Geisert et al., 1992). Although
the specific factors within the graft that affect tubulin isotype
switching during regeneration are unknown, it is possible that
growth-inhibitory molecules can repress the upregulation of
growth-associated mRNAs that should follow injury.
Specific regulation of individual tubulin isotypes
The common downregulation of all isotypes after axotomy is likely
to be regulated post-translationally by the soluble monomer pool of
tubulin. An MREI sequence present in all of the -tubulin isotypes
(Lewis et al., 1985; Bachurski et al., 1994) is involved in detecting
the monomer pool, and when monomers increase, isotype mRNA levels
decrease (Cleveland et al., 1981). Therefore an increase in the tubulin
monomer pool after axotomy would result in a decrease in all isotype
mRNAs such as we report here. During regeneration of both RGCs and
neurons of the peripheral nervous system, the soluble monomer pool of
tubulin is likely to be depleted as new microtubules form to support
the growing axon. Although this could account for a general
upregulation in mRNA levels for all isotypes, the mechanism eliciting a
selective increase in the expression of individual isotypes remains to
be determined. Further, it is unclear why specific isotypes are
recruited during both development and axonal regeneration. It has been
hypothesized that the selective recruitment of individual isotypes for
specific functions may be attributable to unique functional aspects of
microtubules of varying isotype composition (Fulton and Simpson, 1976).
The isotypes undergo unique post-translational modifications that
modify tubulin properties and may affect their assembly characteristics
and their ability to bind various microtubule-associated proteins
(MAPs) (Serrano et al., 1984, 1985; Luduena et al., 1988; Luduena,
1993). Of particular interest, the III tubulin isotype, the
expression levels of which increase during regeneration, is the only
isotype to be phosphorylated at the unique C-terminal end (Diaz-Nido et al., 1990). This modification correlates with neurite extension in
neuroblastoma cells (Gard and Kirschner et al., 1985), and phosphorylated tubulin is found primarily in the assembled microtubule fraction (Diaz-Nido, 1990). Further, this phosphorylation of III tubulin at Serine 444 resides in a region important for MAP binding, and certain MAPs are know to favor process elongation (Leclerc et al.,
1996). We speculate that the increased expression of the III isotype
in regenerating axons reflects a requirement for a tubulin that can be
modified by extracellular cues such as those that affect intracellular
signal transduction mechanisms by protein phosphorylation.
FOOTNOTES
Received Dec. 18, 1996; revised Feb. 14, 1997; accepted April 2, 1997.
We gratefully acknowledge support from Natural Science and Engineering
Research Council of Canada. L.M. is supported by Fonds de la Recherche
en Santé du Québec and A.E.F. holds an Fonds pour la
Formations de Chercheurs et l'Aide à la Recherche student scholarship. We thank Yi-Chun Wang and Soran Singel for surgical assistance and Gaston Lambert for help with the figures.
Correspondence should be addressed to Dr. Alyson Fournier,
Faculté de Médecine, Département de Pathologie,
Université de Montréal, C.P. 6128, Succursale Centre-ville,
Montréal, Québec H3C 3J7, Canada.
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