Results
CP11 treatments inhibit AEP activity at nerve injury sites –
Asparaginyl endopeptidase activity was assayed at the site of sciatic
nerve transection and repair at three and seven days after the start of
treatments. Measures of activity at the two post-injury times from five
nerves from WT mice treated with CP11 and five nerves from WT mice
treated with vehicle, at two different times after injury, were compared
to the activity measured in five intact nerves from control mice (Fig.
1). Results of a one-way ANOVA of these data were significant
(F4, 20 = 9.362, p=0.0002). Based on the results ofpost hoc paired testing, AEP activity was increased significantly
after nerve injury, relative to intact nerves, as was anticipated from
the results of immunoblotting reported previously (English et
al. , 2021). At both post-injury times studied, treatments with CP11
significantly reduced the magnitude of this increase in activity. After
seven days of daily CP11 treatments, the reduction in AEP enzymatic
activity was to a level that was not significantly different from that
measured in the nerves of intact mice (Fig. 1).
CP11 treatments facilitate recovery of neuromuscular function –
Compound muscle action potentials (M responses) were evoked in the LG
and TA muscles by sciatic nerve stimulation four weeks after sciatic
nerve transection and repair in mice treated for two weeks with either
CP11 or vehicle (Fig. 2A). Examples of maximum M responses recorded from
a vehicle-treated mouse and from a CP11-treated mouse four weeks after
sciatic nerve transection and repair are shown in Figure 2B. Two types
of treatments were evaluated: daily intraperitoneal (i.p.) injections
and daily administration by oral gavage. For the mice receiving i.p
injections, average Mmax amplitudes recorded in TA and LG from males and
females were compared using a two-way (sex and treatment) analysis of
variance (ANOVA). In these analyses, the effect of treatment was
significant in both TA
(F1,20=6.630, p<0.0203) and LG
(F1,20=12.99, p=0.0018), but neither for sex (TA:
F1,20=0.186, p=0.671;
LG: F1,20=3.639, p=0.071), nor for the interaction of
sex and treatment (TA: F1,20=0.058, p=0.812; LG:
F1,20=3.114, p=0.0929). Data from males and females were
combined for further analysis. We assumed that a similar lack of sex
difference was present in the mice treated orally with CP11. We then
evaluated the significance of differences between the two groups of
CP11-treated mice and vehicle-treated mice for each muscle using a
one-way ANOVA and post hoc paired tests, as appropriate. Results
of the ANOVA were significant for both
LG (F3, 37 =
3.98, p=0.0149) and TA (F3, 37 = 4.567, p=0.0081). For
both muscles, both i.p. and oral administration of CP11 resulted in a
significant increase in Mmax amplitude relative to identically
administered vehicle treatments (Fig. 2C). Differences in Mmax amplitude
between animals treated with CP11 via different routes of administration
were not significant for both muscles. Four weeks after nerve injury,
restoration of neuromuscular activity was improved more than twofold in
mice treated with CP11, either orally or via i.p. injection, relative to
vehicle-treated controls.
Axons of more motoneurons regenerate successfully after CP11
treatments – Following injection of the retrograde tracers WGA 555
into GAST and WGA 488 into TA, motoneurons were identified in horizontal
sections of the lumbar spinal cord containing these fluorescent markers
(Fig. 3A), indicating that their motor axons had regenerated and
successfully reinnervated those muscles. A small number of motoneurons
contained both retrograde tracers (Fig. 3A: yellow arrows), suggesting
that their regenerating axons may have branched and were exposed to both
tracers. As above, two different routes of administration of CP11 were
used in different groups of mice. In the mice receiving CP11 or vehicle
via i.p injections, we conducted a two-way (sex and treatment) ANOVA to
evaluate if a sex difference existed. Results for both GAST and TA
indicated a significant difference among treatment groups with regard to
number of labeled motoneurons
(TA: F1,14=10.7,
p<0.0075; GAST:F1,14=136.00,
p<0.0001), but not for sex (TA: F1,14=0.059,
p=0.813; GAST: F1,14=0.045, p=0.835), or the interaction
of sex and treatment (TA: F1,14=4.766, p=0.0516; GAST:
F1,14=0.0018, p=0.967). We then combined the data from
males and females and evaluated the significance of differences in
numbers of retrogradely labeled motoneurons reinnervating TA, GAST or
containing both tracers between CP11-treated (both oral and i.p
injected) and vehicle-treated mice using a one-way ANOVA. Results were
significant for GAST
(F3, 26 = 35.45,
p<0.0001) and TA (F3, 26 = 4.724, p=0.0140),
but not for Both (F3, 24 = 0.6285, p=0.6037). Usingpost hoc paired testing, the number of retrogradely labeled
motoneurons encountered was significantly greater in mice treated with
CP11 than in mice treated with vehicle for both GAST and TA. The number
of labeled motoneurons in mice treated with CP11 via intraperitoneal
injection was not significantly different from the number in mice
treated via oral gavage (Fig. 3B). Both oral and injectable treatments
with CP11 nearly doubled the number of motoneurons whose axons
regenerated and successfully reinnervated the TA and GAST muscles while
not changing the number of motoneurons whose axons were exposed to both
retrograde tracers.
CP11 treatments enhance muscle sensory axon regeneration – The
same protocol was used to evaluate the effects of CP11 treatments on
motor axon regeneration and study the regeneration of sensory axons
reinnervating the TA and GAST muscles (Fig. 4A). The numbers of
retrogradely labeled neurons in L4 DRGs were compared in mice four weeks
after transection and repair of the sciatic nerve and two weeks of
treatment with either CP11 or vehicle. Mean (+ SEM) numbers of
cells labeled from tracer injections into the different muscles and with
different treatments are shown for mice treated with CP11 via
intraperitoneal injection and from mice treated orally (Figure 4B). For
each data set, a one-way ANOVA was conducted on these data and proved
significant for TA and GAST, but not Both
(TA: F3,20=
7.588, p<0.001; GAST: F3, 20 = 11.89,
p=0.0001; Both: F3, 20 = 1.445, p<0.2596).
Applying post hoc paired testing, we found significantly more
sensory neurons whose axons had regenerated and reinnervated the TA and
GAST muscles in mice treated with CP11 than in mice treated with
vehicle. Among the relatively small number of sensory neurons containing
both retrograde labels, no significant difference was found between the
vehicle-treated and CP11-treated animals.
CP11 treatments promote neurite elongation in cultured DRG cells
– We cultured adult DRG neurons for 24 hours and then exposed the
cells to different treatments for another 24 hours. To evaluate the
effects of these treatments on neurite outgrowth, we measured the
lengths of the longest neurites in each of at least 50 neurons per
culture, identified by immunoreactivity to β3-tubulin, in cultures from
six WT mice. In each well of a four-well plate, half of the initial
plating media was removed and replaced with fresh media containing
7,8-DHF (500 nM), CP11 (5 uM), both 7,8-DHF and CP11, or no drug, termed
Media. In the Media group, 0.02% DMSO was added, as this was the final
concentration of DMSO in media containing diluted drug stocks. An
example of a DRG neuron and the neurite measured from it is shown in
Figure 5A. Cumulative frequency distributions of neurite lengths were
constructed for each run in each of the four treatment groups and the
data were fitted with a non-linear (Gaussian percentages) function.
Fitted distributions for the six runs (cultures from different mice)
were then averaged for each treatment group. These average frequency
distributions are shown for the four groups by the solid lines in Figure
5B. The curves fitted to the frequency distributions for the cultures
treated with CP11, 7,8-DHF, and the combined treatment are clearly
shifted to the right of controls (Media), indicating that neurite
lengths were increased by these treatments. Average median neurite
lengths in each of these groups (Fig. 5C) were significantly longer than
controls (ANOVA
F3, 20 = 28.04, p<0.0001). Using post
hoc (Tukey) paired testing, average median neurite lengths were
significantly longer in the cultures treated with 7,8-DHF, CP11, or
7,8-DHF and CP11 (Both) than those in the control cultures, but no
significant difference in median length was found between neurites in
the three groups of treated cultures. Treatments with either 7,8-DHF or
CP11 enhance neurite length by the same amount. Combining the two
treatments did not increase this enhancement.
Enhancement of neurite outgrowth produced by CP11 treatment is
TrkB-independent – An additional set of cultures from WT mice
was treated with CP11 or 7,8-DHF, as above, but some of the treated
cultures also were exposed to the specific TrkB inhibitor, ANA-12. The
distributions of neurite lengths measured in these cultures are shown in
Figure 6A. Because the standard deviations of the groups compared were
found to be significantly different, we performed a Brown-Forsythe ANOVA
(F5, 302.5 =
117.18, p<0.0001) and Dunnett’s T3 post hoc paired
testing to evaluate the significance of differences in neurite lengths
in the different culture conditions. Neurite lengths were increased
significantly by exposure to CP11, relative to Media controls, whether
or not the treatment also included ANA-12. As anticipated, a significant
increase in neurite elongation produced by 7,8-DHF treatment alone was
blocked completely by administration of ANA-12. Treatment of cultures
with ANA-12 alone produced no significant effect on neurite length.
Enhancement of neurite elongation produced by CP11 is thus
TrkB-independent.
We also investigated whether the effects of CP11 treatments on neurite
elongation were effective in cultured DRG neurons expressing or lacking
the TrkB receptor. Cultures from wild type mice were treated as above
and neurons were identified by immunoreactivity to ß3-tubulin to mark
neurites. Cells were also reacted with an antibody to the extracellular
domain of the TrkB receptor. For each neurite measured, the cell of
origin was scored as TrkB+ or TrkB-.
Examples of such cells are shown in Figure 6B. Five sets of cultures
were studied. Neurite lengths measured in each culture were scaled to
the average median neurite length in the untreated control cultures
(Media). Results of these scaled neurite lengths from TrkB+ and TrkB-
neurons in control, CP11-treated and 7,8-DHF-treated cultures are shown
in Figure 6C as cumulative histograms. Average (+ SEM) median
scaled neurite lengths are shown in Figure 6D. Significance of
differences between groups was evaluated using ANOVA and post hoc paired
testing. Treatments with CP11 or 7,8-DHF were equally effective in
promoting outgrowth of neurites from TrkB+ DRG neurons
(Fig. 6C: Top), but only treatment with CP11 resulted in longer neurites
in TrkB- neurons (Fig. 6C: Bottom).
Neurite outgrowth is enhanced in AEP knockout mice –Finally, we compared the elongation of neurites from neurons cultured
from wild type (WT) mice to similar treatments in cultures from AEP
knockout (AEP KO) mice (Shirahama-Noda et al. , 2003). All of the
data from DRG neurons lacking AEP were compared to results of similar
measurements from treated and untreated cultures from wild type mice.
For analysis, all neurite lengths were scaled to the average median
length of neurites measured in untreated cultures derived from WT mice.
Results of a one-way ANOVA of these scaled neurite lengths were
significant (F5,
27 = 4.663, p=0.0034). Using post hoc paired testing, neurite
lengths in untreated DRG cells from AEP KO mice were significantly
longer than those in cultures from untreated WT mice (Fig. 7). Lengths
of neurites from neurons from AEP KO mice that had been treated with
CP11 or 7,8-DHF also were significantly longer than those in the
untreated cultures from wild type mice, but not significantly longer
than neurites from untreated cells from AEP KO mice (Fig. 7). In the
absence of AEP, neurite elongation was increased by about 50% over that
observed in cells from WT mice without any treatment. This enhancement
was not further increased by addition of the AEP inhibitor, CP11, or by
treatment with 7,8-DHF. In addition, the lengths of neurites in all
groups of DRG neurons from AEP KO mice were not significantly different
from those of neurons derived from WT mice that had been exposed to
either 7,8-DHF or CP11. Thus, inhibition of AEP by CP11 treatment
resulted in an effect on neurite outgrowth similar to knocking out the
AEP gene.