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\begin{document}

\title{Treatments with the specific \selectlanguage{greek}δ-\selectlanguage{english}secretase inhibitor, compound 11, promote
the regeneration of motor and sensory axons after peripheral nerve
injury.\selectlanguage{english}}



\author[1]{Robin Isaacson}%
\author[1]{Dario Carrasco}%
\author[1]{Hannah Holliday}%
\author[1]{Seong Kang}%
\author[1]{Samia Khan}%
\author[1]{David Kim}%
\author[1]{Xia Liu}%
\author[2]{Keqiang Ye}%
\author[1]{Arthur English}%
\affil[1]{Emory University School of Medicine}%
\affil[2]{Shenzhen Institutes of Advanced Technology Chinese Academy of Sciences}%


\vspace{-1em}



  
  \date{June 16, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
Limited axon regeneration following peripheral nerve injury may be
related to activation of the lysosomal protease, asparaginyl
endopeptidase (AEP, \selectlanguage{greek}δ-\selectlanguage{english}secretase), and its degradation of the microtubule
associated protein, Tau. Activity of AEP was increased at the site of
sciatic nerve transection and repair but blocked in mice treated
systemically with a specific AEP inhibitor, compound 11 (CP11).
Treatments with CP11 enhanced axon regeneration in vivo. Amplitudes of
compound muscle action potentials recorded four weeks after nerve
transection and repair and two weeks after daily treatments with CP11
were double those of vehicle-treated mice. At that time after injury,
axons of significantly more motor and sensory neurons had regenerated
successfully and reinnervated the tibialis anterior and gastrocnemius
muscles in CP11-treated mice than vehicle-treated controls. In cultured
adult dorsal root ganglion neurons derived from wild type mice that were
treated in vitro for 24 hours with CP11, neurites were nearly 50\%
longer than in vehicle-treated controls, and similar to neurite lengths
in cultures treated with the TrkB agonist, 7,8-dihydroxyflavone
(7,8-DHF). Combined treatment with CP11 and 7,8-DHF did not enhance
outgrowth more than treatments with either one alone. Enhanced neurite
outgrowth produced by CP11 was found also in the presence of the TrkB
inhibitor, ANA-12, indicating that the enhancement was independent of
TrkB signaling. Longer neurites were found after CP11 treatment in both
TrkB+ and TrkB- neurons. Delta secretase inhibition by CP11 is a
treatment for peripheral nerve injury with great potential.%
\end{abstract}%



\sloppy


Treatments with the specific \selectlanguage{greek}δ-\selectlanguage{english}secretase inhibitor, compound 11, promote
the regeneration of motor and sensory axons after peripheral nerve
injury.

Robin H. Isaacson\textsuperscript{1}, Dario I.
Carrasco\textsuperscript{1}, Hannah Holliday\textsuperscript{1}, Seong
Su Kang\textsuperscript{2}, Samia Khan\textsuperscript{1}, David
Kim\textsuperscript{1}, Xia Liu\textsuperscript{2}, Keqiang
Ye\textsuperscript{3}, and Arthur W. English\textsuperscript{1}

Departments of Cell Biology\textsuperscript{1} and Pathology \&
Laboratory Medicine\textsuperscript{2}, Emory University School of
Medicine, Atlanta, GA, USA

and

Faculty of Life and Health Sciences, and Brain Cognition and Brain
Disease Institute (BCBDI), Shenzhen Institute of Advanced Technology
(SIAT), Chinese Academy of Sciences, Shenzhen, Guangdong 518055,
China\textsuperscript{3}

\textbf{Running title} : Compound 11 and axon regeneration

\selectlanguage{greek}\textbf{Treatments with the specific δ-secretase inhibitor, compound 11,
promote the regeneration of motor and sensory axons after peripheral
nerve injury.}\selectlanguage{english}

\textbf{Abstract}

Limited axon regeneration following peripheral nerve injury may be
related to activation of the lysosomal protease, asparaginyl
endopeptidase (AEP, \selectlanguage{greek}δ-\selectlanguage{english}secretase), and its degradation of the microtubule
associated protein, Tau. Activity of AEP was increased at the site of
sciatic nerve transection and repair but blocked in mice treated
systemically with a specific AEP inhibitor, compound 11 (CP11).
Treatments with CP11 enhanced axon regeneration \emph{in vivo} .
Amplitudes of compound muscle action potentials recorded four weeks
after nerve transection and repair and two weeks after daily treatments
with CP11 were double those of vehicle-treated mice. At that time after
injury, axons of significantly more motor and sensory neurons had
regenerated successfully and reinnervated the tibialis anterior and
gastrocnemius muscles in CP11-treated mice than vehicle-treated
controls. In cultured adult dorsal root ganglion neurons derived from
wild type mice that were treated \emph{in vitro} for 24 hours with CP11,
neurites were nearly 50\% longer than in vehicle-treated controls, and
similar to neurite lengths in cultures treated with the TrkB agonist,
7,8-dihydroxyflavone (7,8-DHF). Combined treatment with CP11 and 7,8-DHF
did not enhance outgrowth more than treatments with either one alone.
Enhanced neurite outgrowth produced by CP11 was found also in the
presence of the TrkB inhibitor, ANA-12, indicating that the enhancement
was independent of TrkB signaling. Longer neurites were found after CP11
treatment in both TrkB+ and TrkB- neurons. Delta secretase inhibition by
CP11 is a treatment for peripheral nerve injury with great potential.

\textbf{Acknowledgements}

This study was conducted with support from grant NS105982 from the
USPHS. The authors have no conflicts of interest to declare.

\textbf{Data Availability:} All data are available on FigShare.com

\textbf{Key words:} axon regeneration, nerve injury, delta secretase,
muscle, dorsal root ganglion neurons

\textbf{Introduction}

Following a peripheral nerve injury (PNI), both sensory and motor axons
are capable of considerable regeneration. However, functional recovery
in nerve-injured patients remains very poor (Scholz \emph{et al.} ,
2009; Bekelis \emph{et al.} , 2015). There is general agreement that the
source of this poor recovery is the slow and often inefficient process
of axon regeneration. Thus, experimental treatments for PNI have largely
aimed at enhancing axon regeneration. The most effective of these
treatments in pre-clinical studies have been those that increase the
activity of injured neurons, such as exercise (Udina \emph{et al.} ,
2011a; Udina\emph{et al.} , 2011b; Boeltz \emph{et al.} , 2013; English
\emph{et al.} , 2014; Gordon \& English, 2016) or low frequency
electrical stimulation (ES) (Al-Majed \emph{et al.} , 2000b; Al-Majed
\emph{et al.} , 2004). The success of these approaches has been tied to
an increase in the binding of brain derived neurotrophic factor (BDNF)
to its high affinity tropomyosin related kinase (TrkB) receptor in the
regenerating axons (Al-Majed \emph{et al.} , 2000a; Wilhelm \emph{et
al.} , 2012). Indeed, systemic treatments with small molecule TrkB
agonists (English \emph{et al.} , 2013) or prodrugs that generate them
(English \emph{et al.} , 2022) largely recapitulate the success of the
activity-dependent therapies.

Asparaginyl endopeptidase (AEP, also known as \selectlanguage{greek}δ-\selectlanguage{english}secretase or legumain),
a lysosomal protease implicated in the pathological features of
Alzheimer's disease (AD) (Zhang \emph{et al.} , 2014; Zhang \emph{et
al.} , 2015), increases rapidly and persistently at the site of nerve
injury. There it degrades the axonal microtubule associated protein,
Tau, as well as amyloid precursor protein (APP) (English \emph{et al.} ,
2021). In mice treated with one hour of ES following sciatic nerve
transection and repair, this degradation of Tau and APP is reduced,
implying that the success of ES in promoting axon regeneration might be
due to an effect on AEP. In mice null for AEP, axon regeneration after
peripheral nerve injury is enhanced markedly and regeneration is not
further enhanced by treatment with ES (English \emph{et al.} , 2021).
Thus, inhibition of AEP might be the principal mechanism by which
activity-dependent therapies like ES or exercise enhance regeneration.
Treatments that produce a direct inhibition of AEP after nerve injury
might be a suitable test of this hypothesis.

In the present study, we used a specific AEP inhibitor, compound 11
(CP11) to evaluate this hypothesis. It was developed using
high-throughput screening as a potential treatment for AD (Zhang\emph{et
al.} , 2017). In mouse models of AD, oral treatments with CP11
dramatically reduced Tau fragmentation and blocked the formation of A\selectlanguage{greek}β
\selectlanguage{english}from APP, both of which are products of AEP activity. We show here
that\emph{in vivo} systemic treatments with CP11 reduce AEP enzymatic
activity at the site of nerve injury and promote the successful
regeneration of motor and sensory axons after PNI. Using primary
cultures of adult dorsal root ganglion neurons, we show further that
treatments with CP11 result in a marked increase in the extension of
neurites that is TrkB-independent.

\textbf{Methods}

\emph{Animals and surgical methods} -- All surgical methods were
approved by the Institutional Animal Care and Use Committee of Emory
University (Protocol No. PROTO201800101). Adult C57B6/J mice of both
sexes were used. These animals were considered wild type (WT).
Transection and repair of the sciatic nerve was performed as described
previously (Akhter \emph{et al.} , 2019). Briefly, in
isoflurane-anesthetized animals, the sciatic nerve was exposed in the
thigh, cut and immediately repaired by end-to-end anastomosis, and
secured in place using fibrin glue. The glue was prepared at the time of
surgery from fibrinogen and thrombin (Akhter \emph{et al.} , 2019) and
nothing was added to the glue. All incisions were then closed and the
mice returned to their cages. On the third day following surgery, the
mice began daily treatments, five days per week for two weeks, either
with CP11 (Santa Cruz Biotechnology, catalog \# sc-319780) (10 mg/kg) or
vehicle (4\% DMSO in sesame oil). In one set of experiments (12 mice:
six CP11-treated and six vehicle-treated, three males and three females
in each treatment group) treatments were administered via
intraperitoneal injection. In an additional cohort of eight mice, the
CP11 or vehicle treatments were given orally. Doses administered were
chosen based on published results (Zhang \emph{et al.} , 2017).

\emph{Measurement of AEP enzymatic activity} -- Under isoflurane
anesthesia, sciatic nerves were cut and repaired in WT mice, as
described above. Beginning on the third post-operative day, mice were
given either CP11 (10mg/kg) or vehicle, orally, each day. One group of
animals was euthanized with Euthasol\selectlanguage{ngerman}® solution (pentobarbital sodium and
phenytoin sodium, 150 mg/Kg) three days after the initial treatment. A
second set of animals were euthanized after seven days of treatments.
The cut and repaired nerves were harvested, including 1 mm proximal and
distal to the injury site. Nerves were harvested from a third group of
intact mice that served as a control.

The AEP activity assay used was a modification of one previously
described (Wang \emph{et al.} , 2018). Freshly made nerve tissue
homogenates (25 \selectlanguage{greek}μ\selectlanguage{english}g) in Lysis buffer (50 mM Tris\selectlanguage{ngerman}·HCl,pH 7.5, 150 mM NaCl,
1\% Nonidet P-40, 5 mM EDTA, 5 mM EGTA, 15 mM MgCl2, 60 mM
\selectlanguage{greek}β-\selectlanguage{english}glycerophosphate, 0.1 mM sodium orthovanadate, 0.1 mM NaF, 0.1 mM
benzamide, 10 mg/mL aprotinin, 10 mg/mL leupeptin, and 1 mM PMSF) were
prepared and incubated with 100 \selectlanguage{greek}μ\selectlanguage{english}L reaction buffer (50 mM Sodium
Citrated, 0.1\% CHAPS, and 1 mM DTT, pH 6.0) containing 10 \selectlanguage{greek}μ\selectlanguage{english}M AEP
substrate, Z-Ala-Ala-Asn-AMC (Bachem). The AMC released by AEP-mediated
substrate cleavage was quantified at 360/460 nm in a fluorescence plate
reader at 37 \selectlanguage{ngerman}°C in kinetic mode. For quantification, densitometry
readings were scaled to the maximum value of all of the specimens
tested.

\emph{Recording of compound muscle action potentials (M responses)} --
Four weeks after sciatic nerve transection and repair, and two weeks
after the end of treatments, the success of motor axon regeneration and
reinnervation to the tibialis anterior (TA) and lateral gastrocnemius
(LG) muscles was evaluated. In isoflurane anesthetized animals, the
sciatic nerve was exposed as it leaves the pelvis and two needle
electrodes (Ambu \#74325-36/40, Columbia, MD, United States) were placed
in contact with it. Bipolar fine wire (Stablohm 800 A, California Fine
Wire) electrodes (Basmajian \& Stecko, 1963) were inserted
transcutaneously into the centers of the TA and LG muscles using a 25G
hypodermic needle. The free ends of the wires were connected to the head
stages of differential amplifiers. Ongoing activity recorded from these
muscles was sampled at 10 KHz by a laboratory computer system running
custom Labview\textsuperscript{®} software and when activity over a 10
ms period was within a user-defined background range, the computer
delivered a single brief (0.3 ms) constant voltage pulse to the nerve
via the needle electrodes and recorded EMG activity for 50 ms. In each
animal, a range of stimulus intensities was applied, extending from
subthreshold to supramaximal. To avoid fatigue, stimuli were delivered
no more frequently than once every five seconds. Amplitudes of the
recorded direct muscle (M) responses were measured as the average full
wave rectified voltage between the onset and duration of the recorded
triphasic action potential. For each muscle tested in each mouse
studied, the amplitude of the largest M response (Mmax) was determined.

\emph{Retrograde labeling experiments} -- The number of motor and
sensory (dorsal root ganglion, DRG) neurons that successfully
regenerated axons and reinnervated the TA and gastrocnemius (GAST)
muscles was investigated using the application of retrograde fluorescent
tracer molecules to these muscle targets, four weeks after bilateral
sciatic nerve transection and repair, and two weeks after the cessation
of daily treatments. This survival time was chosen to be compatible with
those of previous studies (Al-Majed \emph{et al.} , 2000b;
English\emph{et al.} , 2009; English \emph{et al.} , 2011a; English
\emph{et al.} , 2011b; Udina \emph{et al.} , 2011a; Gordon \& English,
2016) evaluating activity-dependent experimental therapies to enhance
peripheral axon regeneration. At the end of the electrophysiological
experiments described above, the TA and gastrocnemius (GAST) muscles
were exposed in the anesthetized animals. Two microliters of a 1\%
solution of wheat germ agglutinin (WGA), conjugated either to Alexa
Fluor 488 (TA) or Alexa Fluor 555 (GAST), was injected into each muscle
using a Hamilton microliter syringe equipped with a 36G needle. Small
amounts of tracer were injected at two locations in each muscle and the
needle was left in place for five minutes between injections to minimize
leakage of the tracer along the needle track. After washing the entire
surgical field three times with normal saline, surgical wounds were
closed in layers before animals were returned to their cages. Three days
later, the mice were euthanized by intraperitoneal injection of Euthasol
and perfused transcardially with saline followed by 4\%
paraformaldehyde, pH 6.9. Lumbar spinal cords and L4 dorsal root ganglia
(DRGs) were harvested and cryoprotected for at least 24 hours in 20\%
sucrose solution. Cryostat sections of spinal cords, in a horizontal
plane at 40 \selectlanguage{greek}µ\selectlanguage{english}m thickness, were mounted onto charged slides and cover
slipped using Vectashield\selectlanguage{ngerman}®. Images of these sections at 20X
magnification, using a Leica DM6000 upright fluorescence microscope, HC
PL APO 0.70 NA objective, and Hamamatsu low-light camera, were made
using HCImage software. Labeled motoneurons were identified if the
retrograde fluorescence filled the soma and extended into the proximal
dendrites and if a clear area of the cell corresponding to the nucleus
could be visualized, as described previously (English, 2005). Profiles
of motoneurons that did not meet these criteria were not counted.
Harvested dorsal root ganglia were sectioned on a cryostat at 40 \selectlanguage{greek}µ\selectlanguage{english}m
thickness, mounted onto charged slides and cover slipped using
Vectashield\selectlanguage{ngerman}®. Imaging of these sections was identical to that used for
spinal cords, above. A DRG neuron was considered labeled if the
fluorescent marker filled the entire soma and a clear nuclear region
could be identified.

\emph{Cell cultures} -- Mouse lumbar dorsal root ganglion cells were
harvested to assay neurite outgrowth after drug treatment. Mice were
decapitated under isoflurane anesthesia. The entire vertebral column was
removed and immediately placed on a cooled surface under a laminar flow
hood. Individual ganglia (L1-L6) were dissected and pooled in a tube
containing room temperature Hanks' Balanced Salt Solution (HBSS).
Following ganglia collection, the HBSS was removed and a
dispase-collagenase solution was added back to the tube, which was then
placed in a 37\selectlanguage{english}@C bead bath for an hour-long incubation. During this
one-hour period, the tube containing ganglia was briefly removed from
the bead bath every 10 minutes and gently agitated by hand to ensure
even enzymatic digestion of tissue. After incubation,
dispase-collagenase was removed and replaced with DNase for 2.5 minutes.
Then, without removing the DNase, pre-warmed (37@\selectlanguage{english}C) HBSS was added to
the tube and tissue was further dissociated into a cell suspension by
repeatedly pipetting with a P1000 pipet. The cell suspension was
centrifuged at 1000 rcf for 3 minutes. The supernatant was subsequently
discarded and the remaining pellet of cells was resuspended in
Neurobasal A medium supplemented with B-27 (2\%), GlutaMAX (1\%), and
penicillin-streptomycin (1\%). Cells (3000/coverslip) were seeded
directly onto laminin- and poly-D-lysine-coated 12 mm glass coverslips
placed at the bottom of each well of a 4-well plate and the volume of
media in each well was brought to 500 \selectlanguage{greek}µ\selectlanguage{english}L. Plates were stored in a
water-jacketed incubator maintained at 37@\selectlanguage{english}C and steadily supplied with
5\% CO\textsubscript{2}. Twenty-four hours after plating, half of the
media from each well was removed and replaced with fresh media with or
without the drugs of interest. After allowing an additional 24 hours of
incubation, cells were fixed in a solution of 4\% paraformaldehyde in
phosphate-buffered saline (PBS), then washed three times for five
minutes in cold PBS, and stored in PBS at 4@\selectlanguage{english}C for up to a week before
immunofluorescent antibody detection. Cultures used cells derived from
both WT mice and AEP knockout (KO) mice (Shirahama-Noda \emph{et al.} ,
2003). All mice used were genotyped from tail samples by Transnetyx,
Inc. prior to use.

\emph{Immunofluorescence Staining} \emph{of Cultured Neurons} --
Paraformaldehyde-fixed dorsal root ganglion cells attached to 12 mm
glass coverslips were incubated with primary antibodies to protein
targets (Table I), and then fluorescent secondary antibodies for
detection (Table I). After blocking nonspecific binding and
permeabilizing cells with a buffer (10\% donkey serum and 0.3\% Triton
X-100 in PBS) at room temperature for one hour, primary antibodies were
diluted in this same buffer, and the plate containing the cells was kept
in continuous agitation at 4@\selectlanguage{english}C overnight. The following morning, cells
were washed in PBS and then incubated with secondary antibodies in
buffer at room temperature for two hours before washing again and
mounting on glass slides with Vectashield with DAPI. Images of cells
were captured at 20X magnification as described above. Neurite lengths
were measured using the FIJI software package.

\emph{Statistical analyses} -- Numbers of animals in experimental
groupings used were deemed adequate based on a post-hoc power analysis
performed using G*Power 3.1 (Power = (1-\selectlanguage{greek}β \selectlanguage{english}err prob) \textgreater{}0.8).
All statistical comparisons were performed using GraphPad Prism
software. If results of analyses of variance were
significant,\emph{post-hoc} paired testing using the two-stage linear
step-up procedure of Benjamini, Krieger and Yekutieli was employed,
unless noted otherwise. Alpha for significance of differences was set at
p\textless{}0.05 throughout.

\textbf{Results}

\emph{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
(F\textsubscript{4, 20} = 9.362, p=0.0002). Based on the results
of\emph{post 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 \emph{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).

\emph{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 (F\textsubscript{1,20}=6.630, p\textless{}0.0203)
and LG (F\textsubscript{1,20}=12.99, p=0.0018), but neither for sex (TA:
F\textsubscript{1,20}=0.186, p=0.671; LG: F\textsubscript{1,20}=3.639,
p=0.071), nor for the interaction of sex and treatment (TA:
F\textsubscript{1,20}=0.058, p=0.812; LG: F\textsubscript{1,20}=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
\emph{post hoc} paired tests, as appropriate. Results of the ANOVA were
significant for both LG (F\textsubscript{3, 37} = 3.98, p=0.0149) and TA
(F\textsubscript{3, 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.

\emph{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: F\textsubscript{1,14}=10.7,
p\textless{}0.0075; GAST:F\textsubscript{1,14}=136.00,
p\textless{}0.0001), but not for sex (TA: F\textsubscript{1,14}=0.059,
p=0.813; GAST: F\textsubscript{1,14}=0.045, p=0.835), or the interaction
of sex and treatment (TA: F\textsubscript{1,14}=4.766, p=0.0516; GAST:
F\textsubscript{1,14}=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 (F\textsubscript{3, 26} = 35.45,
p\textless{}0.0001) and TA (F\textsubscript{3, 26} = 4.724, p=0.0140),
but not for Both (F\textsubscript{3, 24} = 0.6285, p=0.6037).
Using\emph{post 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.

\emph{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 (\emph{+} 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: F\textsubscript{3,20}=
7.588, p\textless{}0.001; GAST: F\textsubscript{3, 20} = 11.89,
p=0.0001; Both: F\textsubscript{3, 20} = 1.445, p\textless{}0.2596).
Applying \emph{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.

\emph{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 \selectlanguage{greek}β\selectlanguage{english}3-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 F\textsubscript{3, 20} = 28.04, p\textless{}0.0001).
Using \emph{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.

\emph{Enhancement of neurite outgrowth produced by CP11 treatment is
TrkB-independent} \emph{--} 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
(F\textsubscript{5, 302.5} = 117.18, p\textless{}0.0001) and Dunnett's
T3 \emph{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 \selectlanguage{ngerman}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\textsuperscript{+} or TrkB\textsuperscript{-}.
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 (\emph{+} 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\textsuperscript{+} DRG neurons
(Fig. 6C: Top), but only treatment with CP11 resulted in longer neurites
in TrkB\textsuperscript{-} neurons (Fig. 6C: Bottom).

\emph{Neurite outgrowth is enhanced in AEP knockout mice}
\emph{--}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 \emph{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 (F\textsubscript{5, 27} = 4.663, p=0.0034). Using \emph{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.

\textbf{Discussion}

Poor functional recovery from injuries to peripheral nerves is a
significant public health issue. Such injuries are relatively common and
despite the well-documented ability of axons to regenerate following
PNI, they do so poorly. The successes of experimental treatments that
promote axon regeneration after peripheral nerve injury in preclinical
studies, such as exercise (Udina \emph{et al.} , 2011a; English \emph{et
al.} , 2014) or electrical stimulation (Al-Majed \emph{et al.} , 2000b;
Gordon \& English, 2016) are dependent on signaling between BDNF and its
TrkB receptor (Al-Majed \emph{et al.} , 2000a; Wilhelm \emph{et al.} ,
2012). Treatments of mice with small molecule TrkB agonists or prodrugs
that generate them also result in enhanced regeneration (English\emph{et
al.} , 2013; English \emph{et al.} , 2022). We hypothesized that at
least one target of all of these treatments is AEP. A prediction of this
hypothesis is that downstream effectors of BDNF-TrkB signaling inhibit
AEP, which then reduce or eliminate its cleavage of Tau and APP, and in
doing so promote axon regeneration.

In the present study, we evaluated our hypothesis using a specific AEP
inhibitor, compound 11 (CP11) (Zhang \emph{et al.} , 2017). The main
findings reported here are: 1) oral treatments with CP11 inhibit AEP
activity at the site of nerve injury; and 2) systemic treatments with
CP11 enhance motor and sensory axon regeneration after PNI. When
administered either orally or by i.p. injection, CP11 treatments
resulted in successful axon regeneration and muscle reinnervation by
significantly more motor and sensory neurons than vehicle treated
controls. Restoration of compound muscle action potentials (M wave
amplitudes) was greater in CP11-treated mice than in controls. These
findings are consistent with our hypothesis that inhibition of AEP is a
prime target of experimental therapies for treating PNI, whether by
activity-dependent or pharmacological approaches.

To begin to investigate the cellular mechanisms that might be involved,
we compared the effects of treatment with 7,8-DHF and CP11 on neurite
outgrowth from cultured DRG neurons. Both the small molecule TrkB
ligand, 7,8-DHF, and the specific AEP inhibitor, CP11, significantly
increased the growth of neurites when added individually to cultures but
when added together they produced no significant increase over that
observed when either was used alone. Similarly, addition of 7,8-DHF or
CP11 to cultures from AEP-KO mice produced no significant effect on
neurite outgrowth relative to untreated cultures of DRG neurons from the
same animals. The extent of enhancement of neurite outgrowth in these
cultures also was not significantly different from the effects of
treatments with 7,8-DHF, CP11, or both on the lengths of neurites from
neurons derived from WT mice. These outcomes all are consistent with our
hypothesis that the effectiveness of treatments that enhance axon
regeneration by stimulating TrkB activation, such as treatments with
7,8-DHF (English \emph{et al.} , 2013) or prodrugs (English \emph{et
al.} , 2022), as well as activity-dependent experimental therapies that
increase BDNF and/or TrkB expression in rats (Al-Majed \emph{et al.} ,
2000a) and mice (Wilhelm \emph{et al.} , 2012), all do so primarily by
inhibiting AEP. Activation of the TrkB receptor in these scenarios
results in its phosphorylation and a downstream inhibition of AEP,
leading to a decrease in cleavage of the microtubule domain of Tau at
asparagine 368 and a promotion of axon regeneration (English \emph{et
al.} , 2021). A similar effect is achieved by direct AEP inhibition via
CP11 treatments.

Of particular interest were the results of experiments in which the
effects of the treatments were evaluated in cells that expressed the
TrkB receptor. Treatments with CP11 stimulated neurite outgrowth in all
cells, regardless of phenotype, but, as might be predicted, treatments
with 7,8-DHF were effective only in neurons that expressed the TrkB
receptor. The mRNA for TrkB is widely expressed in alpha, but not gamma
motoneurons in rats (Buck \emph{et al.} , 2000; Copray \& Kernell,
2000). The robust effects of treatments with 7,8-DHF or its prodrug,
R13, and the results presented here using CP11, on the regeneration of
motor axons or/and restoration of M wave amplitude in reinnervated
muscles are likely due to similar TrkB expression in mouse motoneurons.
The regeneration of axons of a subset of sensory neurons that express
the TrkB receptor, even as modified following PNI in rats
(Karchewski\emph{et al.} , 2002) or mice (English \emph{et al.} , 2007),
also would be expected to respond. However, sensory neurons not
expressing TrkB would not benefit from such a TrkB-dependent therapy. We
believe that these results suggest that direct AEP inhibition, such as
that produced by CP11, might be overall the most effective therapy for
PNI to date. This assertion does not discount the possibility that
pathways alternative to the BDNF-TrkB pathway that result in inhibition
of AEP may exist. Whether they are in play when activity-dependent
treatments are employed remains for future study.

\textbf{Conclusion:} Inhibition of AEP activity is the main focus of
activity-dependent and BDNF-TrkB dependent experimental therapies for
peripheral nerve injury. Direct inhibition of AEP by CP11 is a potential
treatment worthy of further consideration.

\textbf{Table I}

\textbf{Antibodies Used}\selectlanguage{english}
\begin{longtable}[]{@{}llll@{}}
\toprule
Target & Primary Antibody & Secondary Antibody & Dilution\tabularnewline
\midrule
\endhead
\selectlanguage{ngerman}ß3-tubulin & Cell Signaling \#4466 & donkey anti-mouse IgG/Alexa Fluor
488 or 594 & 1:250\tabularnewline
TrkB & R\&D Systems \#AF1494 & donkey anti-goat IgG/Alexa Fluor 647 &
1:20\tabularnewline
\bottomrule
\end{longtable}



\textbf{Figure legends}

\textbf{Figure 1.} Compound 11 treatment reduces AEP activity in injured
nerves. Sciatic nerves were cut and surgically repaired in wild type
mice. Beginning on the third post-injury day, animals were administered
either CP11 or vehicle, orally, once daily. Repaired nerves were
harvested and assayed for AEP activity on the third (3d) or seventh (7d)
after the onset of treatment. A set of intact nerves was assayed
similarly for controls. Mean (\emph{+} SEM) AEP activity is scaled to
the maximum activity found in all samples.

\textbf{Figure 2.} Treatments with CP11 result in larger restored
neuromuscular responses. A. Outline of experimental scheme. Mice were
treated with CP11 or vehicle daily, five days per week for two weeks,
beginning on the third day following sciatic nerve transection and
repair. After two more weeks, neuromuscular reinnervation was tested. B.
Examples of maximal amplitude M responses (Mmax) recorded four weeks
after sciatic nerve transection and repair from a vehicle-treated (top)
and a CP11-treated (bottom) mouse. C. Mean (\emph{+} SEM) amplitudes of
Mmax recorded from tibialis anterior (TA) and lateral gastrocnemius (LG)
in vehicle-treated and CP11-treated mice, either via i.p. injection or
oral gavage, four weeks after sciatic nerve transection and repair.
Significance of differences between CP11-treated and vehicle-treated
mice was evaluated for each muscle using ANOVA and post hoc paired
testing. P values are shown to indicate the significance of differences
in paired comparisons.

\textbf{Figure 3.} Treatments with CP11 increase the number of
motoneurons whose axons have regenerated successfully. A. Image of a
horizontal section through the right side of the lumbar spinal cord of a
mouse in which the sciatic nerve was cut and repaired four weeks
earlier. Motoneurons were labeled by application of different
fluorescent retrograde tracers into the reinnervated gastrocnemius
(GAST, red) and tibialis anterior (TA, green) muscles. Three neurons in
this image contained both tracers (yellow arrows). This mouse was
treated with intraperitoneal injections of CP11. B. Mean (\emph{+} SEM)
number of motoneurons retrogradely labeled from tracer injection into
GAST and TA four weeks after sciatic nerve transection and repair and
two weeks after treatment, either by intraperitoneal injection (i.p.) or
orally (oral), with compound 11 (CP11, 10 mg/Kg), or vehicle. Numeric
values indicate p values from pairwise comparisons (ns = not
significant).

\textbf{Figure 4.} Treatments with compound 11 (CP11) increased the
number of muscle sensory neurons whose axons regenerated successfully.
A. In a section from the L4 DRG of a mouse four weeks after sciatic
nerve transection and repair and two weeks after treatment with CP11,
neurons retrogradely labeled from tracer injections into GAST (red) and
TA (green) are shown. Three cells in this image were labeled by both
tracers (yellow arrows). B. Mean (\emph{+} SEM) numbers of L4 DRG
neurons retrogradely labeled from TA, GAST, or Both in vehicle-treated
and CP11-treated mice are shown. Each symbol in each graph represents
the number of labeled neurons in a single animal in the series.
Significance of differences between groups are displayed as p values
above comparison brackets.

\textbf{Figure 5.} Neurite elongation is enhanced by CP11 treatments. A.
Example of an adult DRG neuron after 48 hours in culture, marked by
immunoreactivity to \selectlanguage{greek}β\selectlanguage{english}3 tubulin. This culture was treated for 24 hours
with CP11. The arrows indicate the longest neurite measured in this
cell. B. Cumulative frequency distributions of neurite lengths in four
treatment groups. Each cumulative histogram represents the average of
the distributions of neurite lengths from cultures of DRG neurons from
six mice. The horizontal dashed line at the
50\textsuperscript{th}percentile marks the median neurite length, shown
at the vertical dashed lines extending to the X axis. C. Average
(\emph{+} SEM) median neurite lengths in six cultures of the four
treatment groups. Each symbol represents the neurite length measured
from cells derived from a single animal. P values are shown only for
significant differences between groups. All other paired comparisons
were not statistically significant.

\textbf{Figure 6.} Enhancement of neurite outgrowth produced by CP11
treatment is TrkB-independent. A. Distributions of neurite lengths of
cultured DRG neurons without treatments (Media) and after treatments
with CP11 or 7,8-DHF in the presence or absence of the TrkB inhibitor,
ANA-12 are displayed as violin plots. The solid white lines in each
violin mark their medians. The horizontal dashed line running through
the figure marks the median neurite length from the control group
(Media). Numbers above brackets indicate p values associated with
statistical comparisons between groups. B. Image of two \selectlanguage{greek}β\selectlanguage{english}3
tubulin-immunoreactive DRG neurons (red) in a culture treated for 24
hours with CP11. Cultures also were reacted with an antibody to the
extracellular domain of the TrkB receptor, and a green fluorescing
secondary antibody. The lower cell in the image was scored as
TrkB\textsuperscript{+} and the upper cell as TrkB\textsuperscript{-}.
C. Cumulative frequency distributions of neurite lengths in cultured
adult DRG neurons identified as TrkB\textsuperscript{+} (top) and
TrkB\textsuperscript{-} (bottom) in control (Media) cultures and in
cultures treated with either CP11 or 7,8-DHF. Neurite lengths in
cultures from different mice were scaled to the median neurite length in
the corresponding control (Media only) group. Each cumulative histogram
represents the average of the distributions of these scaled neurite
lengths from cultures of DRG neurons from six mice. Medians for each
group are represented by the projection of the dashed line at the
50\textsuperscript{th} percentile to the horizontal. D. Average
(\emph{+} SEM) median scaled neurite lengths from the same cultures are
shown for TrkB+ (top) and TrkB- (bottom) neurons. Numbers above brackets
are p values resulting from statistical comparisons made between groups.

\textbf{Figure 7.} Neurite length is not increased by CP11 treatments in
AEP knockout mice. Average median neurite lengths (\emph{+} SEM), scaled
to the median length found in untreated cultures derived from WT mice,
are shown for cultures from AEP KO mice and those derived from WT mice.
Data from untreated cultures (Media), cultures treated with CP11 (red
bars), cultures treated with 7,8-DHF (green bars) are shown. Numbers
above brackets indicate p values for all significant differences between
groups. All other paired comparisons were not statistically significant.



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\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image1/image1}

\end{center}
\end{figure}

\textbf{Figure 1.} Compound 11 treatment reduces AEP activity in injured
nerves. Sciatic nerves were cut and surgically repaired in wild type
mice. Beginning on the third post-injury day, animals were administered
either CP11 or vehicle, orally, once daily. Repaired nerves were
harvested and assayed for AEP activity on the third (3d) or seventh (7d)
after the onset of treatment. A set of intact nerves was assayed
similarly for controls. Mean (\emph{+} SEM) AEP activity is scaled to
the maximum activity found in all samples.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image2/image2}

\end{center}
\end{figure}

\textbf{Figure 2.} Treatments with CP11 result in larger restored
neuromuscular responses. A. Outline of experimental scheme. Mice were
treated with CP11 or vehicle daily, five days per week for two weeks,
beginning on the third day following sciatic nerve transection and
repair. After two more weeks, neuromuscular reinnervation was tested. B.
Examples of maximal amplitude M responses (Mmax) recorded four weeks
after sciatic nerve transection and repair from a vehicle-treated (top)
and a CP11-treated (bottom) mouse. C. Mean (\emph{+} SEM) amplitudes of
Mmax recorded from tibialis anterior (TA) and lateral gastrocnemius (LG)
in vehicle-treated and CP11-treated mice, either via i.p. injection or
oral gavage, four weeks after sciatic nerve transection and repair.
Significance of differences between CP11-treated and vehicle-treated
mice was evaluated for each muscle using ANOVA and post hoc paired
testing. P values are shown to indicate the significance of differences
in paired comparisons.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image3/image3}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image4/image4}

\end{center}
\end{figure}

\textbf{Figure 3.} Treatments with CP11 increase the number of
motoneurons whose axons have regenerated successfully. A. Image of a
horizontal section through the right side of the lumbar spinal cord of a
mouse in which the sciatic nerve was cut and repaired four weeks
earlier. Motoneurons were labeled by application of different
fluorescent retrograde tracers into the reinnervated gastrocnemius
(GAST, red) and tibialis anterior (TA, green) muscles. Three neurons in
this image contained both tracers (yellow arrows). This mouse was
treated with intraperitoneal injections of CP11. B. Mean (\emph{+} SEM)
number of motoneurons retrogradely labeled from tracer injection into
GAST and TA four weeks after sciatic nerve transection and repair and
two weeks after treatment, either by intraperitoneal injection (i.p.) or
orally (oral), with compound 11 (CP11, 10 mg/Kg), or vehicle. Numeric
values indicate p values from pairwise comparisons (ns = not
significant).

\textbf{Figure 4.} Treatments with compound 11 (CP11) increased the
number of muscle sensory neurons whose axons regenerated successfully.
A. In a section from the L4 DRG of a mouse four weeks after sciatic
nerve transection and repair and two weeks after treatment with CP11,
neurons retrogradely labeled from tracer injections into GAST (red) and
TA (green) are shown. Three cells in this image were labeled by both
tracers (yellow arrows). B. Mean (+ SEM) numbers of L4 DRG neurons
retrogradely labeled from TA, GAST, or Both in vehicle-treated and
CP11-treated mice are shown. Each symbol in each graph represents the
number of labeled neurons in a single animal in the series. Significance
of differences between groups are displayed as p values above comparison
brackets.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image5/image5}

\end{center}
\end{figure}\selectlanguage{english}
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\includegraphics[width=0.70\columnwidth]{figures/image6/image6}

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\textbf{Figure 6} . Enhancement of neurite outgrowth produced by CP11
treatment is TrkB-independent. A. Distributions of neurite lengths of
cultured DRG neurons without treatments (Media) and after treatments
with CP11 or 7,8-DHF in the presence or absence of the TrkB inhibitor,
ANA-12 are displayed as violin plots. The solid white lines in each
violin mark their medians. The horizontal dashed line running through
the figure marks the median neurite length from the control group
(Media). Numbers above brackets indicate p values associated with
statistical comparisons between groups. B. Image of two \selectlanguage{greek}β\selectlanguage{english}3
tubulin-immunoreactive DRG neurons (red) in a culture treated for 24
hours with CP11. Cultures also were reacted with an antibody to the
extracellular domain of the TrkB receptor, and a green fluorescing
secondary antibody. The lower cell in the image was scored as TrkB+ and
the upper cell as TrkB-. C. Cumulative frequency distributions of
neurite lengths in cultured adult DRG neurons identified as TrkB+ (top)
and TrkB- (bottom) in control (Media) cultures and in cultures treated
with either CP11 or 7,8-DHF. Neurite lengths in cultures from different
mice were scaled to the median neurite length in the corresponding
control (Media only) group. Each cumulative histogram represents the
average of the distributions of these scaled neurite lengths from
cultures of DRG neurons from six mice. Medians for each group are
represented by the projection of the dashed line at the 50th percentile
to the horizontal. D. Average (+SEM) median scaled neurite lengths from
the same cultures are shown for TrkB+ (top) and TrkB- (bottom) neurons.
Numbers above brackets are p values resulting from statistical
comparisons made between groups.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image7/image7}

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\textbf{Figure 7.} Neurite length is not increased by CP11 treatments in
AEP knockout mice. Average median neurite lengths (\emph{+} SEM), scaled
to the median length found in untreated cultures derived from WT mice,
are shown for cultures from AEP KO mice and those derived from WT mice.
Data from untreated cultures (Media), cultures treated with CP11 (red
bars), cultures treated with 7,8-DHF (green bars) are shown. Numbers
above brackets indicate p values for all significant differences between
groups. All other paired comparisons were not statistically significant.

\textbf{Graphical abstract}\selectlanguage{english}
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\includegraphics[width=0.70\columnwidth]{figures/image8/image8}

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\includegraphics[width=0.70\columnwidth]{figures/Fig1/Fig1}

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\includegraphics[width=0.70\columnwidth]{figures/Fig2/Fig2}

\end{center}
\end{figure}\selectlanguage{english}
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\includegraphics[width=0.70\columnwidth]{figures/Fig3/Fig3}

\end{center}
\end{figure}\selectlanguage{english}
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\includegraphics[width=0.70\columnwidth]{figures/Fig4/Fig4}

\end{center}
\end{figure}\selectlanguage{english}
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\includegraphics[width=0.70\columnwidth]{figures/Fig5/Fig5}

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\includegraphics[width=0.70\columnwidth]{figures/Fig6/Fig6}

\end{center}
\end{figure}\selectlanguage{english}
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\includegraphics[width=0.70\columnwidth]{figures/Fig7/Fig7}

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