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Original Article
Amelioration of functional and histopathological consequences after spinal
cord injury through phosphodiesterase 4D (PDE4D) inhibition
Melissa Schepers
a
,
b
,
c
,
1
, Sven Hendrix
d
,
1
, Femke Mussen
a
,
b
,
e
,
1
, Elise van Breedam
f
,
Peter Ponsaerts
f
, Stefanie Lemmens
e
, Niels Hellings
c
,
e
, Roberta Ricciarelli
g
,
h
, Ernesto Fedele
g
,
i
,
Olga Bruno
j
, Chiara Brullo
j
, Jos Prickaerts
k
, Jana Van Broeckhoven
c
,
e
,
2
, Tim Vanmierlo
a
,
b
,
c
,
*
,
2
a
Department of Neuroscience, Biomedical Research Institute, Faculty of Medicine and Life Sciences, Hasselt University, 3500 Hasselt, Belgium
b
Department of Psychiatry and Neuropsychology, School for Mental Health and Neuroscience, Maastricht University, 6229ER Maastricht, the Netherlands
c
University MS Centre (UMSC) Hasselt –Pelt, Belgium
d
Institute for Translational Medicine, Medical School Hamburg, 20457 Hamburg, Germany
e
Department of Immunology and Infection, Biomedical Research Institute, Faculty of Medicine and Life Sciences, Hasselt University, 3500 Hasselt, Belgium
f
Laboratory of Experimental Hematology, Vaccine and Infectious Disease Institute (Vaxinfectio), University of Antwerp, 2610 Wilrijk, Belgium
g
IRCCS Ospedale Policlinico San Martino, 16100 Genoa, Italy
h
Department of Experimental Medicine, Section of General Pathology, University of Genova, 16100 Genoa, Italy
i
Department of Pharmacy, Section of Pharmacology and Toxicology, University of Genoa, 16100 Genoa, Italy
j
Department of Pharmacy, Section of Medicinal Chemistry, University of Genoa, 16100 Genoa, Italy
k
Peitho Translational, 6229ER Maastricht, the Netherlands
ARTICLE INFO
Keywords:
Spinal cord injury
Phosphodiesterase 4
cAMP
Neuroinflammation
Regeneration
ABSTRACT
Spinal cord injury (SCI) is a life-changing event that severely impacts the patient's quality of life. Modulating
neuroinflammation, which exacerbates the primary injury, and stimulating neuro-regenerative repair mechanisms
are key strategies to improve functional recovery. Cyclic adenosine monophosphate (cAMP) is a second messenger
crucially involved in both processes. Following SCI, intracellular levels of cAMP are known to decrease over time.
Therefore, preventing cAMP degradation represents a promising strategy to suppress inflammation while stim-
ulating regeneration. Intracellular cAMP levels are controlled by its hydrolyzing enzymes phosphodiesterases
(PDEs). The PDE4 family is most abundantly expressed in the central nervous system (CNS) and its inhibition has
been shown to be therapeutically relevant for managing SCI pathology. Unfortunately, the use of full PDE4 in-
hibitors at therapeutic doses is associated with severe emetic side effects, hampering their translation toward
clinical applications. Therefore, in this study, we evaluated the effect of inhibiting specific PDE4 subtypes (PDE4B
and PDE4D) on inflammatory and regenerative processes following SCI, as inhibitors selective for these subtypes
have been demonstrated to be well-tolerated. We reveal that administration of the PDE4D inhibitor Gebr32a, even
when starting 2 dpi, but not the PDE4B inhibitor A33, improved functional as well as histopathological outcomes
after SCI, comparable to results obtained with the full PDE4 inhibitor roflumilast. Furthermore, using a lumi-
nescent human iPSC-derived neurospheroid model, we show that PDE4D inhibition stabilizes neural viability by
preventing apoptosis and stimulating neuronal differentiation. These findings strongly suggest that specific
PDE4D inhibition offers a novel therapeutic approach for SCI.
Introduction
Spinal cord injury (SCI) is characterized by a complex secondary
injury phase that drives further permanent damage and causes neuro-
logical dysfunction [1,2]. To date, regeneration and recovery of function
remain limited after SCI [3]. The provoked neuroinflammation and the
limited endogenous regeneration potential of neural tissue are the critical
bottlenecks. Despite multiple efforts, current treatments suppress in-
flammatory processes (e.g. corticosteroids) but remain ineffective in
promoting repair. Therefore, there is an urgent need to develop new
* Corresponding author.
E-mail address: t.vanmierlo@maastrichtuniversity.nl (T. Vanmierlo).
1
Equally contributing first authors.
2
Equally contributing last authors.
Contents lists available at ScienceDirect
Neurotherapeutics
journal homepage: www.sciencedirect.com/journal/neurotherapeutics
https://doi.org/10.1016/j.neurot.2024.e00372
Received 15 December 2023; Received in revised form 15 April 2024; Accepted 1 May 2024
1878-7479/©2024 The Author(s). Published by Elsevier Inc. on behalf of American Society for Experimental NeuroTherapeutics. This is an open access article under
the CC BY license (http://creativecommons.org/licenses/by/4.0/).
Neurotherapeutics 21 (2024) e00372
therapeutic strategies that tackle both neuroinflammatory and regener-
ative processes.
Cyclic adenosine monophosphate (cAMP) is a crucial molecule
involved as a second messenger in multiple signaling pathways and ex-
erts broad modulatory effects in various cell types [4,5]. In the context of
central nervous system (CNS) injury, cAMP has been shown to exhibit
both anti-inflammatory and neuroregenerative functions. Upon SCI,
cAMP levels in both neurons and glial cells decrease dramatically [6].
Therefore, maintaining or elevating the intracellular cAMP levels to
regulate the immune responses or to stimulate neuroregeneration can be
considered a valuable approach to temper SCI pathogenesis. Phospho-
diesterases (PDEs) are a class of enzymes responsible for the degradation
of cyclic nucleotides, such as cAMP. In the CNS, PDE4 is primarily
responsible for the breakdown of cAMP [4,7]. As such, PDE4 inhibition,
initiated after SCI induction, demonstrated a wide range of beneficial
actions in a SCI mouse model [8–12]. The golden standard,
first-generation PDE4 inhibitor rolipram was found to act as
anti-inflammatory, neuroprotective, and regenerative agent [9–11].
Continuous and controlled mini-osmotic pump-mediated release of roli-
pram (0.4
μ
mol/kg/h) attenuated astrogliosis and enhanced axonal
outgrowth following hemisection SCI, while administration of a single
dose of 0.5 or 1 mg/kg rolipram per day appeared to be sufficient to
enhance neuronal survival and simultaneously protect oligodendrocytes,
thus preserving CNS myelination following contusion SCI [9–11]. Addi-
tionally, the PDE4 inhibitor IC486051 further confirmed the
anti-inflammatory properties observed upon rolipram administration as
bolus doses of 0.5 or 1.0 mg/kg IC486051 decreased oxidative stress
markers and leukocyte infiltration into the lesion size, thereby reducing
the resulting tissue damage following compression SCI in Wistar rats [8].
However, despite these promising findings, the clinical translation of pan
PDE4 inhibitors has been hampered due to tolerability problems (e.g.,
emesis). Therefore, to mitigate the side effects and increase tolerability,
PDE4 subtype-selective inhibitors have been developed. The PDE4 family
consists of four genes yielding different PDE4 subtypes (PDE4A-PDE4D).
PDE4A, B, and D are widely expressed in the CNS of rodents and humans,
while PDE4C has limited CNS expression. In particular, the PDE4B and
PDE4D subtypes recently gained more interest in neurological disorders
because of their anti-inflammatory and neuroplastic effects, respectively
[13–15]. More specifically, the inhibition of PDE4B suppresses the neu-
roinflammatory responses of macrophages and microglia, while PDE4D
blockage has been shown to successfully boost myelin regeneration and
enhance neuroplasticity [14–18]. Targeting these individual subtypes
circumvented the emetic side-effects accompanied by pan PDE4 in-
hibitors such as roflumilast and rolipram, and is predicted as a valuable
strategy to target neuroinflammation [19].
In this study, we aimed to disentangle the effect of PDE4B and PDE4D
inhibition on SCI pathology. Using the SCI hemisection model, we show
in mice that, in contrast to the PDE4B inhibitor A33, the PDE4D inhibitor
Gebr32a improved functional and histopathological outcomes after SCI
to a similar extent as the pan PDE4 inhibitor roflumilast. Gebr32a could
still improve SCI outcomes even when administered 2 dpi. In vitro,
neuronal apoptosis was prevented by inhibiting PDE4D, as demonstrated
with primary neuronal mouse cultures, human iPSC-derived neuronal
precursor cultures, and luminescent iPSC-derived neurospheroids.
In addition, increased neuronal differentiation was observed in these
iPSC-derived neurospheroids. Overall, these findings underline the
therapeutic potential of specific PDE4D inhibition to act neuroprotective
and consequently improve neural plasticity, leading to functional re-
covery after SCI.
Methods
Animals
In vivo experiments were performed with 10- to 12-week-old female
WT C57BL/6j mice (Janvier Labs). Mice were housed in groups at the
conventional animal facility of Hasselt University under stable conditions
(temperature-controlled room, 12 h light/dark cycle, food, and water ad
libitum). Experiments were approved by the local ethical committee of
Hasselt University (ethical ID 202060) and were performed according to
the guidelines of Directive 2010/63/EU on the protection of animals
used for scientific purposes.
Spinal cord injury model
A standardized T-cut spinal cord hemisection injury was performed as
previously described [20–22]. In brief, mice were anesthetized with
2–3% isoflurane and a partial laminectomy was performed at thoracic
level 8. A bilateral hemisection injury was done using iridectomy scissors
to transect the dorsomedial and ventral corticospinal tract. Afterward,
the back muscles were sutured, and the skin was closed with wound clips
(Bioconnect). Post-operative treatment included blood-loss compensa-
tion by glucose solution (20%, intraperitoneal [i.p.]) and pain relief by
buprenorphine (0.1 mg/kg body weight, Temgesic, subcutaneous [s.c.]).
In addition, mice were placed in a heated recovery chamber (33 C) until
they regained consciousness. Bladders were voided daily until the
micturition reflex was restored spontaneously. The in vivo experiments
were conducted in two independent cohorts. Table 1 provides a sample
size overview for each experimental animal group for both cohorts.
Animal treatments
Starting 1 h after SCI, mice were injected twice daily s.c. for 28 days
with either (1) vehicle (0.1% DMSO (VWR prolabo) þ0.5% methylcel-
lulose þ2% Tween80), (2) the pan PDE4 inhibitor roflumilast (3 mg/kg,
Xi'an leader biochemical engineering co., LTD), (3) the PDE4B inhibitor
A33 (3 mg/kg, Sigma-Aldrich), or (4) the PDE4D inhibitor Gebr32a (0.3
mg/kg, University of Genoa [23]). We included (5) a sequential treat-
ment group who first received A33 (3 mg/kg) until 10 days post injury
(dpi), followed by Gebr32a (0.3 mg/kg) until the end of the experiment
(injection volume: 10
μ
l/g body weight). This treatment was used to first
diminish the acute inflammatory responses with PDE4B inhibition in the
early phase, followed by PDE4D inhibition later to boost endogenous
repair mechanisms and hence, promote SCI recovery by tackling both the
inflammatory and repair processes. Lastly, we elucidated the therapeutic
potential of Gebr32a when administered in a more clinically relevant
therapeutic window. To this purpose, mice were injected twice daily s.c.
for 28 days with (1) vehicle (0.1% DMSO) or Gebr32a (0.3 mg/kg)
starting from (2) 2 dpi or (3) 10 dpi onwards.
Locomotion test
Following SCI, functional recovery was assessed using the standard-
ized Basso Mouse Scale (BMS) score for locomotion [24]. This 10-point
scale ranges from 0, indicating complete hind limb paralysis, to 9, rep-
resenting normal motor function. These scores are based on hind limb
movements in an open field during a 4 min testing window. The
Table 1
Overview of the number of animals receiving a hemisection SCI which were
included in cohort 1 and cohort 2 for functional and histopathological analysis
based on locomotor function. The A33 and Gebr32a sequential treatment group
was not included in cohort 2 due to the lack of additional efficacy compared to
continuous PDE4D inhibition.
Treatment group Number of animals
Included cohort 1 Included cohort 2 Total
Vehicle 6 9 15
Roflumilast (3 mg/kg) 7 7 14
A33 (3 mg/kg) 6 10 16
Gebr32a (0.3 mg/kg) 8 6 14
A33 followed by Gebr32a 10 / 10
M. Schepers et al. Neurotherapeutics 21 (2024) e00372
2
evaluation was done by an investigator blinded to treatment groups and
was performed daily from 1 until 7 dpi, followed by a scoring every 2–3
days until the end of the experiment (28 dpi). The mean BMS score of the
left and right hind limb was used per animal. Mice were excluded from
the analysis if 1) they had a BMS score higher than 1 at 1 dpi or 2) they
did not show an increase in BMS score of at least 1 point at 28 dpi.
Mouse primary neuronal cultures
Fetal mice brains (E16-19) were used to obtain primary cortical
neuron cultures [25]. Meninges-free cerebral cortices were chemically
dissociated for 15 min using trypsin. Next, chemical dissociation was
stopped by washing cortices with minimal essential medium (MEM)
supplemented with 1% heat-inactivated horse serum (Thermo Fisher),
0.6% glucose (Sigma-Aldrich) and 100 U/ml penicillin/streptomycin
(Life Technologies). The cortical tissue was subsequentially mechanically
dissociated with a P1000 pipette to generate single-cell suspensions.
Primary mouse neurons were seeded (8 10
4
cells/well) in a poly--
L-lysine (PLL, Sigma) coated 96-well plate (flat bottom, Greiner) in MEM
supplemented medium. After allowing attachment for 4 h, plating MEM
medium was replaced by neurobasal medium supplemented with 1B27
supplement (Thermo Fisher), 2 mM L-glutamine (Thermo Fisher), and
100 U/ml penicillin/streptomycin (Life Technologies). Cells were
maintained at 37 C with 5% CO
2
culture conditions. Treatment (vehicle:
0.1% DMSO; roflumilast: 1
μ
M; A33: 1
μ
M; Gebr32a: 1
μ
M) was started
24 h after isolation, under growth factor B27 deprivation to induce
neuronal cell death (an additional 48 h).
Propidium iodide (PI) viability assay
The viability of mouse primary neurons was assessed using a propi-
dium iodide (PI) viability assay as described previously [26,27]. Briefly,
48 h after B27 growth factor deprivation and PDE inhibitor treatment,
culture medium was replaced with Lysis buffer A100 (ChemoMetec) and
lysis reaction was then halted by adding equal amounts of stabilization
buffer B (ChemoMetec), supplemented with PI (10
μ
g/ml, Sigma). After
15 min incubation in the dark, fluorescent emission was measured using
the FluoStart OPTIMA plate reader (Bottom-up, excitation: 540 nm;
emission: 612 nm).
Luminescent iPSC-derived neurospheroid cultures
Neurospheroids were formed as described previously [28]. Briefly,
eGFP/Luc human iPSC-NSCs were seeded at equal densities of 1.6 10
4
cells per well (ULA 96-well plate (Corning)) in neural expansion medium
(NEM, Gibco). Neurospheroids were maintained at 37 C, 5% CO
2
culture
conditions under constant orbital shaking (85 rpm). Two days after
plating, fresh NEM was added. A 50% medium change was conducted
every other day. Additionally, the luminescent signal was measured
weekly by adding 1.5 mg/ml Beetle luciferin (E1601, Promega) for 48 h
to the neurospheroid cultures. The luminescent signal was measured
using the Clariostar Plus plate reader, after which a complete medium
change was performed to eliminate remaining luciferin. On the same day
of bioluminescence evaluation, phase contrast pictures (4magnifica-
tion) of every neurospheroid were taken using the Incucyte system to
evaluate overall neurospheroid size. The size of each neurospheroid was
determined using Image J by manually delineating the spheroids. PDE
inhibition treatment (vehicle: 0.1% DMSO; roflumilast: 1
μ
M; A33: 1
μ
M;
Gebr32a: 1
μ
M) was initiated following 1 week of culturing (after the first
luminescent signal measurement). At the end of the experiment (6 weeks
of culturing), neurospheroids were fixed with 4% paraformaldehyde
(PFA) for 150 min at room temperature (RT), incubated overnight in 20%
sucrose (w/v in 1phosphate-buffered saline [PBS]) and consecutively
used for cryosectioning and immunocytochemical analysis.
Immunofluorescence
Post-mortem spinal cord tissue
At 28 dpi, mice received an overdose of i.p. dolethal (200 mg/kg)
(Vetiquinol B.V.) and were transcardially perfused with Ringer solution
containing heparin (50 units/l), followed by a 4% PFA in 1xPBS perfu-
sion [29]. Longitudinal spinal cord cryosections of 10
μ
m thickness were
obtained. Immunofluorescent staining was performed as described pre-
viously [2,3]. In brief, sections were blocked using 10% protein block
(Dako) in PBS containing 0.5% Triton-X-100 for 1 h at RT. For evaluating
oligodendrocyte differentiation using Olig2 and CC1, an additional an-
tigen retrieval step using a sodium citrate buffer (10 mM Sodium citrate,
0.05% Tween20, pH 6.0) was conducted. Primary antibodies were
diluted in PBS with 1% protein block and 0.5% Triton-X-100 and were
incubated overnight at 4 C(Table 2). Following washing, secondary
antibody incubation was done for 1 h at RT. Antibodies used were: goat
anti-rat Alexa fluor 488 (1:250, A11006, ThermoFisher Scientific), goat
anti-mouse Alexa fluor 568 (1:250, A11004, ThermoFisher Scientific),
goat anti-rat Alexa fluor 568 (1:250, A11077, Invitrogen), and goat
anti-rabbit Alexa 488 (1:250, A11008, Invitrogen). Specificity of sec-
ondary antibodies was tested by omitting the primary antibody. Coun-
terstaining with DAPI (1:1000, Sigma-Aldrich) was performed for 10
min. Pictures were taken using a LEICA DM4000 B LED microscope and
LAS X software (Leica).
Neurospheroids
After fixation, neurospheroids were processed as described previously
to allow high-throughput staining [28]. Briefly, a silicone mold with 66
wells corresponding to the size of the neurospheroids was filled with
Tissue-Tek-OCT (VWR). Single neurospheroids were loaded into separate
wells of the silicone mold. Next, the mold was snap-frozen in isopentane
at a fixed temperature (50 C), after which the resulting OCT-block was
removed from the silicon mold, turned upside down and covered with
additional OCT before freezing a second time. Cryosections of 10
μ
m
were obtained on PLL-coated glass slides. For immunocytochemical
Table 2
List of primary antibodies used in immunofluorescence experiments.
Immunofluorescence Antibody Host Source Dilution factor
Immunohistochemistry GFAP Mouse Sigma Aldrich (G3893) 1/500
MBP Rat Merck Millipore (MAB386) 1/250
CD4 Rat BD Biosciences (553043) 1/250
Cleaved caspase 3 Rabbit Cell signaling (9661) 1/100
NeuN Mouse Merck
Millipore (MAB377)
1/1000
Olig2 Goat Biotechne R&D(AF2418) 1/50
APC (CC1) Mouse Calbiochem (OP80) 1/50
5HT Rabbit Immunostar (20080) 1/1000
Immunocytochemistry NeuN Guinea-pig Merck
Millipore (ABN90P)
1/100
Cleaved caspase 3 Rabbit Cell signaling (9661) 1/400
Doublecortin (DCX) Rabbit Abcam (ab18723) 1/500
M. Schepers et al. Neurotherapeutics 21 (2024) e00372
3
analysis, neurospheroid sections were permeabilized for 30 min (10%
milk solution [Sigma] in tris-buffered saline [TBS]). Primary antibodies
were diluted in 10% milk solution (Sigma) in TBS and were incubated
overnight at 4 C(Table 2). Following washing, secondary antibody in-
cubation was done for 1 h at RT. Antibodies used were: donkey
anti-rabbit Alexa fluor 488 (1:600, A11006, ThermoFisher Scientific),
donkey anti-rabbit Alexa fluor 555 (1:600, A11004, ThermoFisher Sci-
entific), and goat anti-guinea pig Alexa fluor 555 (1:600, A11077, Invi-
trogen). DAPI was used to counterstain cellular nuclei. Pictures were
taken using an Axioscan 7 microscope slide scanner (Zeiss).
Fluorescence quantification
The original, unedited pictures were used for quantification. Repre-
sentative images were digitally enhanced to improve readability
(contrast and brightness). Quantification of histopathological parameters
was performed as described previously by investigators blinded to
experimental groups [2,3[. To quantify lesion size (GFAP
area) and
demyelinated area (MBP
area), 5–710
μ
m thick sections per animal
were obtained, whereby the lesion center and consecutive rostral and
caudal area were analyzed. An intensity analysis was performed to
determine astrogliosis (GFAP expression) using ImageJ [2]. To assess
neuronal cell death at the lesion site, we quantified the number of cells
positive for both cleaved caspase 3 and NeuN markers. This counting was
performed in both the rostral and caudal regions relative to the lesion
epicenter. The obtained values were normalized by the total number of
NeuN
þ
cells. Similarly, to quantify oligodendrocyte differentiation, cells
positive for Olig2 and CC1 were counted using ImageJ in both rostral and
caudal regions relative to the injury. The results were normalized by the
total number of Olig2
þ
cells. For evaluating serotonergic 5-HT regrowth,
the rostral and caudal white matter regions of the ventral funiculus were
analyzed for the amount of descending 5-HT
þ
fibers. T helper cells,
identified as CD4
þ
Iba-1
-
, were quantified by counting their number in
one microscope field both rostral and caudal of the lesion site [30].
Differences in cleaved caspase, NeuN, and DCX positive cells within the
neurospheroids were determined by intensity analysis using ImageJ,
after which the positive area for each marker was corrected for the
number of nuclei (based on the DAPI count) present in the pictures.
IncuCyte live-cell imaging of cleaved caspase 3/7
eGFP/Luc human iPSC-NSCs were seeded at a density of 1 10
4
cells
per well in a Geltrex (Life Technologies) coated 96-well plate (flat bottom,
Greiner). After allowing attachment for 24 h, cells were treated with the
PDE4 inhibitors (vehicle: 0.1% DMSO; roflumilast: 1
μ
M; A33: 1
μ
M;
Gebr32a: 1
μ
M), and apoptosis was induced by oxygen deprivation using a
hypoxic chamber (1% O
2
for 4 h). Simultaneously, the IncuCyte Caspase-
3/7 Red apoptosis reagent (1.5
μ
M; #4704, Sartorius) was supplemented
to the cultures. Cell plates were placed into the IncuCyte live-cell analysis
system, and 5 images were taken per well. End-point apoptosis was
measured 6 h after oxygen deprivation. The IncuCyte integrated analysis
software was used to quantify the total level of apoptosis.
Statistics
Data were analyzed using GraphPad Prism version 9 (GraphPad
Software). All data were checked for normality using the Shapiro-Wilk
test. The BMS scores, GFAP intensity and neurospheroid biolumines-
cent results were analyzed using a two-way ANOVA for repeated mea-
surements with a Bonferroni post hoc test. Normally distributed data
were subsequently analyzed with a one-way ANOVA with Dunnett's
multiple comparisons (compared to vehicle). Not normally distributed
data were tested using the non-parametric Kruskal-Wallis test with
Dunn's multiple comparisons (compared to vehicle). Data are presented
as mean standard error of the mean (SEM). Differences with P values <
0.05 were considered significant.
Results
Pan PDE4 and selective PDE4D, but not PDE4B, inhibition improve
functional recovery and histopathological outcomes after SCI
Initially, we examined whether selective PDE4B or PDE4D inhibitors
could improve functional recovery in a hemisection model of SCI. As a
positive control, we included roflumilast, a second-generation pan PDE4
inhibitor. Starting 1 h post SCI injury, mice received either vehicle
(DMSO) or the different PDE4 inhibitors: the pan PDE4 inhibitor roflu-
milast, the selective PDE4B inhibitor A33, the selective PDE4D inhibitor
Gebr32a, or a sequential administration of A33 (1–9 dpi) followed by
Gebr32a (10–28 dpi) to evaluate possible additive effects. Functional
recovery was measured for 4 weeks using the BMS score. Both roflumilast
and Gebr32a significantly improved functional recovery compared to
vehicle-treated mice, whereas A33 treatment did not show any signifi-
cant effect (Fig. 1A and B). Noteworthy, Gebr32a had a similar recovery
profile as roflumilast (Fig. 1A and B). The sequential treatment with A33,
followed by Gebr32a showed the same trend in functional recovery as
Gebr32a treatment alone, namely starting from day 10, functional re-
covery of these mice mimicked the curve of the continuous Gebr32a
treatment (Fig. 1A and B).
To further elucidate the clinical translatability of PDE4D inhibition,
we administered Gebr32a starting 2 dpi which is considered a more
clinically relevant treatment window. In addition, as the sequential
treatment (A33 followed by Gebr32a after 10 dpi) resembled the results
of Gebr32a treatment alone (Fig. 1A), we included a new treatment group
where Gebr32a was administered alone starting 10 dpi to elucidate
whether therapeutic efficacy is maintained without the preceding A33
treatment. Inhibition of PDE4D with Gebr32a, starting 2 dpi significantly
improved functional recovery compared to vehicle-treated mice. How-
ever, starting Gebr32a treatment 10 dpi had no significant therapeutic
benefit compared to vehicle treatment observed in the BMS scores but
showed a significantly higher area under the curve compared to vehicle
(Fig. 2A and B). Notably, the administration of Gebr32a 1 h after injury
appeared to have a more pronounced improvement in the BMS score
compared to Gebr32a treatment starting 2 dpi (Gebr32a start 1 h pi 3.93
0.43 vs Gebr32a start 2 dpi 3.13 0.29).
Histological GFAP and MBP analyses indicated significantly reduced
lesion size and demyelinated areas, respectively, in roflumilast- and
Gebr32a-treated mice compared to the vehicle group whereas the
treatment with A33 was ineffective (Fig. 3A–J). Next, the level of
astrogliosis was determined by analyzing GFAP intensity at varying dis-
tances to the lesion center. Whereas roflumilast and Geb32a treatment
did not alter GFAP intensities, A33 exacerbated astrogliosis, especially at
the rostral lesion site (Fig. 4A–E). As an additional neuroinflammatory
outcome measurement, we determined the number of infiltrated CD4
þ
T
lymphocytes in the perilesional area. However, no differences between
vehicle and treatment groups were observed (Supplementary Fig. S1).
Pan PDE4 and selective PDE4D inhibition increase the number of
differentiated oligodendrocytes at the (peri)lesion site following SCI, unlike
PDE4B inhibition
Due to the reduced demyelinated area observed at the lesion site upon
both PDE4 and PDE4D inhibition, we next investigated the presence of
differentiated oligodendrocytes (CC1
þ
) at the lesion site. The total
number of oligodendrocyte lineage cells was first determined based on
sole Olig2 positivity at the lesion site, which was unaltered upon either
pan PDE4 or PDE4 subtype-selective inhibition compared to vehicle-
M. Schepers et al. Neurotherapeutics 21 (2024) e00372
4
treated animals (Supplementary Fig. S2). However, mice treated with
roflumilast or Gebr32a did display a significant higher number of mature
oligodendrocytes (CC1
þ
Olig2
þ
) compared to vehicle- or A33-treated
animals (Fig. 5A–E).
Pan PDE4 and selective PDE4D inhibition is neuroprotective and stimulates
5-HT serotonergic regrowth after SCI, unlike PDE4B inhibition
Next, we investigated whether the abovementioned decreased lesion
size was accompanied by neuroprotection and neuroregeneration. First,
the neuroprotective effects of pan PDE4 or PDE4 subtype-selective inhi-
bition were determined based on Cleaved Caspase 3 and NeuN double
positivity at the lesion site to evaluate the number of apoptotic neurons.
Mice treated with the pan PDE4 inhibitor roflumilast or the selective
PDE4D inhibitor Gebr32a displayed a reduced number of Cleaved Caspase
3
þ
NeuN
þ
double positive cells compared to vehicle-treated animals at the
(peri-)lesion (Fig. 6A–E). Furthermore, A33-treated animals displayed a
trend (p ¼0.08) toward reduced neuronal cell death (Fig. 6A–E).
Next, to determine the spinal dendritic plasticity of serotoninergic
fiber projections, the number of descending 5-HT positive tracts was
determined. In comparison with A33, both roflumilast- and Gebr32a-
treated animals showed significantly increased mean number of
descending 5-HT dendrites, indicating serotonergic neuroregeneration or
protection (Fig. 7A and B).
Pan PDE4 and selective PDE4D inhibition prevents apoptosis of primary
murine neurons and human iPSC-derived neural stem cells, unlike PDE4B
inhibition
To evaluate whether the neuroprotection by roflumilast and Gebr32a
observed in vivocould be attributed to direct neural protection, we assessed
the effects of these PDE4 inhibitors on neural apoptosis in both primary
mouse-derived neurons and human iPSC-derived NSCs. Fig. 8A shows the
level of neuronal apoptosis of mouse neurons following 48 h of B27 growth
factor deprivation. Both roflumilast-mediated PDE4 inhibition and
Gebr32a-mediated PDE4D inhibition partly prevented the stress-induced
neuronal apoptosis as observed by a higher PI signal compared to
vehicle-treated cultures (Fig. 8A). Similarly, human iPSC-derived neural
stem cell cultures subjected to oxygen deprivation for 4 h and treated with
either roflumilast or Gebr32a displayed a significant reduction of Cleaved
Caspase 3/7 positivity 6 h post stress-induced neural apoptosis (Fig. 8B).
Real-time bioluminescence monitoring of human neurospheroids
demonstrates the neuroprotective feature of both pan PDE4 and selective
PDE4D inhibition, which is accompanied by increased neuronal
differentiation
To enable real-time read-out of neurospheroid viability, we used an
eGFP/Luc human iPSC-derived neural stem cells stably expressing the
Fig. 2. Treatment with the PDE4D inhibitor
Gebr32a starting 2 dpi, but not from 10 dpi, im-
proves functional recovery after a spinal cord
injury.(A–B) Starting directly after injury, mice were
treated with vehicle. The Gebr32a treated animals
changed from vehicle to Gebr32a (0.3 mg/kg) at 2 dpi
or 10 dpi, while the vehicle croup continued DMSO
(0.1%) treatment until 28 dpi. (A) Starting Gebr32a
treatment 2 dpi, but not 10 dpi, significantly
improved functional outcomes, measured by the BMS
score. The black, and gray arrows indicate the start of
Gebr32a treatment 2 dpi and 10 dpi, respectively. (B)
Starting at 2 dpi and 10 dpi with Gebr32a signifi-
cantly improved the area under the curve, compared
to vehicle-treated mice over time. Data were analyzed
using a two-way ANOVA with Bonferroni multiple
comparison test (compared to vehicle). Data are dis-
played as mean SEM. n¼5–9 mice/group. (A) *p
0.05 Gebr32a start 2 dpi vs vehicle; (B) *p <0.05,
**p <0.01 compared to vehicle.
Fig. 1. Treatment with the PDE4 inhibitor
roflumilast or the PDE4D inhibitor
Gebr32a improve functional recovery
after spinal cord injury, whereas the
PDE4B inhibitor A33 has no effect.(A–B)
Starting 1 h after injury, mice were treated
with vehicle, a general PDE4 inhibitor
roflumilast (3 mg/kg), or gene-specific PDE4
inhibitors, A33 (3 mg/kg) and Gebr32a (0.3
mg/kg). In contrast to A33, roflumilast and
Gebr32a significantly improved (A) func-
tional outcomes, measured by the BMS score
and (B) area under the curve, compared to
vehicle-treated mice over time. Data were
analyzed using a two-way ANOVA with
Bonferroni multiple comparison test
(compared to vehicle). Data are displayed as
mean SEM. n¼10–16 mice/group. #p
0.05 vehicle versus roflumilast; **p <0.01
vehicle versus Gebr32a.
M. Schepers et al. Neurotherapeutics 21 (2024) e00372
5
firefly luciferase reporter. Over time, decreased viability was observed in
the neurospheroids due to oxygen/nutrient deprivation in the core
(Fig. 9A). In contrast to the selective PDE4B inhibitor A33, treatment
with either the pan PDE4 inhibitor roflumilast or the selective PDE4D
inhibitor Gebr32a stabilized neurospheroid viability when stress-induced
core cell loss occurred (Fig. 9A). Of note, the overall size of the neuro-
spheroids was not different between treatment groups (Fig. 9B). At the
end of the 6-week culture period, neurospheroids were characterized for
the level of apoptosis (Cleaved Caspase 3), neuronal differentiation
(NeuN), and neurogenesis (DCX). Quantification of the Cleaved Caspase
3
þ
area revealed a significant reduction in apoptosis when inhibiting
PDE4 or PDE4D (Fig. 9C–F). Furthermore, both roflumilast and Gebr32a
treatment significantly increased neuronal differentiation as more
NeuN
þ
cells were present at the end of the experiment (Fig. 9D–F). At end
Fig. 3. Roflumilast or Gebr32a treatments
reduce the lesion size and demyelinated
area after spinal cord injury, whereas A33
has no effect.(A–J) Starting 1 h after injury,
mice were treated with vehicle, the pan
PDE4 inhibitor roflumilast (3 mg/kg), or the
subtype-selective PDE4 inhibitors, A33 (3
mg/kg) and Gebr32a (0.3 mg/kg). (A)
Quantification of lesion size, determined by
the GFAP negative area, showed that this
was reduced in mice treated with roflumilast
or Gebr32a compared to the vehicle group.
No difference between the vehicle and A33
groups was observed. Data were normalized
to vehicle and are shown as mean SEM. n
¼7–8 mice/group. (B–E) Representative
images from the spinal cord sections are
shown. Lesion size (GFAP
area) was deter-
mined as depicted by the dotted white line.
Scale bar ¼250
μ
m. (F) Quantification of the
demyelinated area, determined by the MBP
negative area, showed that this was reduced
in mice treated with roflumilast or Gebr32a
compared to the vehicle group. No difference
between vehicle and A33 groups was
observed. Data were normalized to vehicle
and are shown as mean SEM. n¼7–8
mice/group. (G–J) Representative images
from the spinal cord sections are shown.
Demyelinated area (MBP
area) was deter-
mined as depicted by the dotted white line.
Scale bar ¼250
μ
m. Demyelination area and
lesion size were analyzed using a one-way
ANOVA with Dunnett's multiple comparison
test (compared to vehicle), *p <0.05, ***p <
0.005. Data are displayed as mean SEM.
Fig. 4. Roflumilast and Gebr32a treat-
ment do not affect astrogliosis after spinal
cord injury, whereas A33 administration
exacerbates astrocyte reactivity.(A–E)
Starting 1 h after injury, mice were treated
with vehicle, a pan PDE4 inhibitor roflumi-
last (3 mg/kg), or subtype-selective PDE4
inhibitors, A33 (3 mg/kg) and Gebr32a (0.3
mg/kg). (A) Quantification of astrogliosis by
GFAP intensity analysis showed that, in
contrast to other treatment groups, A33
application exacerbated astrogliosis
compared to vehicle-treated mice. Data are
shown as mean SEM. n¼4–6 mice/group.
GFAP intensity was analyzed using a two-
way ANOVA with a Bonferroni post hoc
test. *p <0.05 A33 versus vehicle, **p <
0.01 A33 versus vehicle.(B–E) Representa-
tive images from the spinal cord sections are
shown. All analyses were quantified within
square areas of 100
μ
m100
μ
m perile-
sional placed as indicated in the figure,
extending 600
μ
m rostral to 600
μ
m caudal
from the lesion center (white line). Scale bar
¼500
μ
m.
M. Schepers et al. Neurotherapeutics 21 (2024) e00372
6
stage, no significant differences were found in DCX
þ
area, nor for A33
treatment within any marker evaluated (Fig. 9E and F).
Discussion
In SCI research, PDE4 inhibition has yielded promising results due to
its broad effects on different secondary injury-related outcomes, such as
immune cell infiltration, inflammation, and axonal sprouting [8–11,31].
However, the clinical translation of pan-PDE4 inhibitors remains limited
due to their poor tolerability in patients at doses required for clinical
effectiveness [32]. In order to circumvent this pitfall, we investigated
the potential of specific non-emetic PDE4B and PDE4D inhibitors,
respectively A33 and Gebr32a. We now show that the PDE4D inhibitor
Gebr32a significantly improved functional recovery after SCI similarly to
the pan PDE4 inhibitor roflumilast, whereas the PDE4B inhibitor A33 did
not. Moreover, starting Gebr32a at a more clinically relevant therapeutic
window (2 dpi) still promoted functional recovery. In addition, both
roflumilast and Gebr32a reduced the lesion size, demyelinated area, and
Fig. 5. Roflumilast and Gebr32a treat-
ments increase the amount of mature ol-
igodendrocytes at the peri-lesion site
after spinal cord injury.(A–E) Starting 1 h
after injury, mice were treated with vehicle,
the pan PDE4 inhibitor roflumilast (3 mg/
kg), or the subtype-selective PDE4 inhibitors,
A33 (3 mg/kg) and Gebr32a (0.3 mg/kg).
(A) Using a double staining for Olig2 (oli-
godendrolineage marker) and CC1 (mature
oligodendrocyte marker), we showed a
significantly increased percentage of mature
oligodendrocytes at the lesion site following
PDE4 (roflumilast) and PDE4D (Gebr32a)
inhibition. n¼7–8 mice/group. Data were
normalized to vehicle and are shown as
mean SEM. Results were analyzed using a
one-way ANOVA with Dunnett's multiple
comparison test (compared to vehicle), **p
<0.01, ****p <0.001. (B–E) Representative
images of the Olig2-CC1 double staining at
the lesion site. Single stainings are shown
above the merged image. Scale bar ¼100
μ
m.
Fig. 6. Roflumilast and Gebr32a treat-
ment act neuroprotective at the lesion
site after spinal cord injury.(A–E) Starting
1 h after injury, mice were treated with
vehicle, the pan PDE4 inhibitor roflumilast
(3 mg/kg), or the subtype-selective PDE4
inhibitors, A33 (3 mg/kg) and Gebr32a (0.3
mg/kg). (A) Quantification of the number of
Cleaved Caspase 3
þ
NeuN
þ
neurons at the
(peri)lesion site indicated a neuroprotective
effect of both PDE4 and PDE4D inhibition as
reduced neuronal apoptosis was observed. n
¼7–8 mice/group. Data are normalized to
vehicle and are shown as mean SEM. Re-
sults were analyzed using a one-way ANOVA
with Dunnett's multiple comparison test
(compared to vehicle), ***p <0.005. (B–E)
Representative images of the Cleaved Cas-
pase 3 NeuN staining at the lesion site. Single
stainings are shown above the merged
image. The white boxed regions are shown at
higher magnification (40) underneath the
merged image. Scale bar ¼100
μ
m.
M. Schepers et al. Neurotherapeutics 21 (2024) e00372
7
neuronal apoptosis while increasing the number of mature oligoden-
drocytes, and 5-HT
þ
serotonergic fibers. The neuroprotective feature of
both pan PDE4 inhibition and PDE4D subtype inhibition can be partially
attributed to a direct neur(on)al effect as we showed a decreased
neur(on)al apoptosis in vitro using murine neurons and iPSC-derived 3D
neurospheroid cultures, which is in line with the previously described
neuroplasticity enhancing properties of PDE4D inhibition [33]. These
results support the use of the PDE4D inhibitor Gebr32a for SCI therapy.
We confirm that the second-generation PDE4 inhibitor roflumilast
promoted SCI recovery, as demonstrated previously [34]. This
second-generation PDE4 inhibitor is accompanied with less emetic side
effects compared to first-generation pan-PDE4 inhibitors (e.g., rolipram)
[35,36]. Noteworthy, the second-generation PDE3, PDE4, and PDE5 in-
hibitor Ibudilast is currently being evaluated for degenerative cervical
myelopathy, a non-traumatic SCI [37]. However, translation of the
roflumilast dose to promote CNS repair from rodents to humans, results
in a dose approximately 500 times higher than the approved dose for
roflumilast in chronic obstructive pulmonary disease. Hence, the CNS
repair-inducing dose is still emetic [38]. Therefore, research to safer and
more targeted options are crucial to influence discrete cell processes and
promote SCI outcomes. Previous studies showed that PDE4B orchestrates
the inflammatory immune response, while PDE4D contributes to adult
neurogenesis, neuroplasticity, and myelin regeneration [39]. As SCI is
characterized by a robust neuroinflammatory response and limited
Fig. 7. Roflumilast and Gebr32a induce 5-HT serotonergic regrowth following SCI as indicated by the increased number of descending 5-HT tracts over the
lesion site.(A–B) Starting 1 h after injury, mice were treated with vehicle, the pan PDE4 inhibitor roflumilast (3 mg/kg), or the subtype-specific PDE4 inhibitors, A33
(3 mg/kg) and Gebr32a (0.3 mg/kg). (A) Quantification of the 5-HT serotonergic staining showed an increase in the number (A) of descending 5-HT tracts over the SCI
lesion site upon PDE4 (roflumilast) and PDE4D (Gebr32a) inhibition. Data were normalized to vehicle and are shown as mean SEM. n¼4–9 mice/group. (B)
Representative images of the 5-HT staining at the lesion site. The white arrows indicate examples of 5-HT descending tracts. Results were analyzed using a one-way
ANOVA with Dunnett's multiple comparison test (compared to vehicle), ***p <0.005.
Fig. 8. Apoptosis of primary mouse neu-
rons and human iPSC-derived NSCs was
prevented by both roflumilast and
Gebr32a treatment.(A) Primary mouse
neurons deprived of the growth factor B27
for 48 h showed a decreased neuronal
viability at the end of the experiment, which
was partly prevented by inhibiting PDE4
(roflumilast, 1
μ
M) or PDE4D (Gebr32a, 1
μ
M). Data were normalized to vehicle and
are shown as mean SEM. n¼6–7/group
with an ‘n’representative for one well. PI
measurements of primary mouse neurons
were analyzed using a one-way ANOVA with
Dunnett's multiple comparison test
(compared to vehicle). (B) Human iPSC-
derived neural stem cells showed increased
levels of Cleaved Caspase 3/7 upon oxygen
deprivation, which was significantly reduced
upon PDE4 (roflumilast, 1
μ
M) and PDE4D
(Gebr32a, 1
μ
M) inhibition. n¼8–9/group
with an ‘n’representative for one well.
Cleaved Caspase 3/7 signal measurements
were analyzed using a non-parametric Krus-
kal-Wallis test with Dunn's multiple com-
parison test (compared to vehicle), *p <
0.05, **p <0.01, ****p <0.001. Data are
displayed as mean SEM.
M. Schepers et al. Neurotherapeutics 21 (2024) e00372
8
axonal regeneration, we focused on PDE4B and PDE4D inhibition.
Important to note is that the inhibitors used in this study (A33 and
Gebr32a) both lack an emetic response up to 100 mg/kg in animals,
highlighting the clinical relevance of the evaluated compounds [38].
After SCI, PDE4B is acutely upregulated in the damaged spinal cord,
especially in phagocytes [40]. The PDE4B subfamily is an important
modulator of the intracellular cAMP levels in inflammatory cells,
including macrophages, microglia, and astrocytes [5,14]. In a mouse
model of multiple sclerosis, the PDE4B expression in antigen-presenting
cells, such as phagocytes, was correlated with the disease severity [41].
Pharmacological inhibition of PDE4B by 0.3 mg/kg A33 i.p. administered
30 min after traumatic brain injury induction, induced anti-inflammatory
markers (e.g., Arginase-1) in phagocytes and limited lesion size [42–44].
Complete PDE4B knockdown had beneficial effects on recovery in a
contusion SCI model [40]. Based on these data, it was somewhat sur-
prising that PDE4B inhibition by 3 mg/kg A33 (s.c) did not improve
functional or histopathological recovery in our study. Moreover, PDE4B
inhibition did exaggerate astrogliosis, which is considered detrimental
for SCI outcomes since astrocytes contribute to the formation of the
regenerative-limiting glial scar 45–48. Therefore, the absence of func-
tional outcomes upon A33 treatment in our study could be attributed to
the excessive astrogliosis. Importantly, it has been previously shown that
treatment protocol and dose are important determinants for beneficial
effects [49]. For example, intravenous (i.v.) or s.c. administration of the
PDE4 inhibitor rolipram has been shown to be more effective to treat SCI
compared to oral administration. Additionally, finetuning the PDE4
concentration showed to be crucial. More specifically, a low rolipram
dose of 0.5 mg/kg/day had no beneficial effect for SCI treatment, while a
higher dose of 1 mg/kg with the same treatment protocol could signifi-
cantly improve SCI recovery [9]. However, an even higher dose of 0.8
mmol/kg/day demonstrated to not improve SCI recovery, postulated
through off-target effects [10]. Therefore, performing a dose response
study, testing another administration route, or different treatment tim-
ings can potentially result in ameliorated SCI outcomes upon A33
treatment. Hence, we cannot exclude that A33 could still provide
long-term benefits after SCI. It is additionally crucial to highlight that the
hemisection SCI model is highly effective for studying neuronal regen-
eration, but does not fully represent the human SCI pathology due to the
lack of the complex neuroinflammatory responses. Conversely, the
contusion SCI model provides a more faithful representation of neuro-
inflammation and axonal preservation evident in human SCI [50].
Consequently, it remains essential to consider the prospect that the
anti-inflammatory attributes associated with the inhibition of PDE4B
could potentially confer an indirect neuroprotective influence within the
contusion SCI model. This warrants a thorough exploration when
assessing the therapeutic viability of PDE4B inhibition within the
Fig. 9. Roflumilast and Gebr32a treatment protected human iPSC-derived neurospheroids from neural apoptosis and stimulated neuronal differentiation,
while not affecting neurogenesis.(A) Weekly luminescence measurement of neurospheroids showed a stabilized viability over time, which decreased at 6 weeks of
culture. However, this decrease was counteracted by treatment with roflumilast (1
μ
M) or Gebr32a (1
μ
M). (B) The size of the neurospheroids was not different
between groups. (A, B) Data are shown as mean SEM. n¼24 spheroids/group. Data were analyzed using a two-way ANOVA with Dunnets multiple comparison test
(compared to vehicle), *p <0.05, ***p <0.005. (C–F) At the end of the culture experiment, the 6-week-old neurospheroids were stained and quantified for (C)
Cleaved Caspase 3 (apoptosis), (D) NeuN (neuronal differentiation), or (E) DCX (neurogenesis) positive cells with respect to the total number of nuclei. Data were
normalized to vehicle and are shown as mean SEM. n¼6–8 spheroids/group. The amount of cleaved caspase positive cells was analyzed using a non-parametric
Kruskal-Wallis test with Dunn's multiple comparisons (compared to vehicle). The amount of NeuN or DCX positive cells was analyzed using a one-way ANOVA with
Dunnett's multiple comparison test (compared to vehicle), **p <0.01. (F) Representative immunofluorescent images of the human iPSC-derived neurospheroids. Scale
bar ¼500
μ
m.
M. Schepers et al. Neurotherapeutics 21 (2024) e00372
9
framework of the contusion SCI model.
Pan PDE4 inhibition using rolipram treatment after SCI has previ-
ously been shown to attenuate oligodendrocyte apoptosis and promote
axonal growth and plasticity [4,49]. We have found that selective PDE4D
inhibition by Gebr32a boosted oligodendrocyte precursor cell differen-
tiation in vitro and stimulated remyelination in an ex vivo model [38]. In
the current study, we show that Gebr32a administration, even when
started 2 dpi, improved functional recovery after SCI. Previous research
showed that earlier intervention results in better SCI outcomes [51,52].
Noteworthy, Gebr32a improved SCI outcomes significantly when
administered in a clinically relevant therapeutic window but indeed, our
results suggest that starting earlier with Gebr32a leads to better SCI
outcomes (Gebr32a start 1 h pi 3.93 0.43 vs Gebr32a start 2 dpi 3.13
0.29). In mice treated 1 h post injury, we observed reduced lesion size
and decreased demyelinated area, which was accompanied by increased
numbers of mature oligodendrocytes, pointing toward reduced demye-
lination, mature oligodendrocyte protection or increased remyelination.
Moreover, Gebr32a acts neuroprotective following SCI as demonstrated
by the decreased number of apoptotic neurons. Previously, Gebr32a was
shown to regulate neuronal morphology as demonstrated by the
increased neurite outgrowth in both N2a and HT22 cells [13]. Due to this
neuroregenerative feature of Gebr32a, combined with the observation
that the loss of locomotor function following SCI correlates to the damage
of 5-HT serotonergic projections in the spinal cord, we aimed to evaluate
in vivo neuroregeneration by quantifying the descending 5-HT tracts over
the lesion site [53]. Treatment of Gebr32a increased the number of
descending 5-HT fibers, indicating either axonal sparing or neuro-
regenerative features of PDE4D inhibition. The effects observed after
Gebr32a treatment were comparable to roflumilast. Although we cannot
exclude any indirect neuroprotective effects of Gebr32a so far, we
demonstrated here, at least partially, that the observed decrease in
neuronal apoptosis can be attributed to direct neuronal protection. In
both murine and human iPSC-derived neural stem cells, Gebr32a treat-
ment diminished neur (on)al cell death. Similarly, Gebr32a stabilized the
human neurospheroid viability, accompanied by decreased apoptosis
and increased neuronal differentiation. Spinal cord neurons differ in
calcium load, protection against reactive oxygen species, gene expres-
sion, and mitochondrial metabolism compared to brain-derived neurons,
which suggests that spinal cord-derived neurons are more prone to
trauma-induced injury [54–56]. Hence, validation of the neuroprotective
and differentiation-stimulating effects of Gebr32a on spinal cord-derived
neurons could be interesting for future research. Notably, previous
research already demonstrated the neuroprotective effects of
cAMP-elevating agents in spinal cord and brain-derived neurons [13,57,
58]. Therefore, the neuroprotective effects and enhanced differentiation
capacity upon Gebr32a administration in brain-derived cultures are
proposed to translate toward spinal cord neurons.
Due to the hypothesized anti-inflammatory properties of PDE4B in-
hibition, and the previously observed regenerative properties of PDE4D
inhibition, we evaluated whether a sequential treatment regimen could
further improve SCI outcomes compared to monotreatment strategies.
Thus, the PDE4B inhibitor A33 was administered during the initial phase
of SCI and subsequently substituted by the PDE4D inhibitor Gebr32a
from day 10 onwards. While inflammatory processes are essential for
removing pathogens and cell debris, their benefits are overshadowed by
the accumulation of inflammatory cytokines in the CNS upon inflam-
matory immune cell infiltration and activation [59,60]. These secondary
inflammatory-mediated damage processes severely impair regenerative
processes and glial functioning [61]. Therefore, by diminishing the
neuroinflammatory response with PDE4B inhibition, we aimed to create
a favorable micro-environment thereby allowing regeneration to occur
more efficiently. By inhibiting PDE4D in a later phase, we hypothesized
that the regenerative process could be further enhanced, and functional
outcome would be improved even more compared to continuous PDE4B
or PDE4D subtype inhibition throughout the disease course. However, in
our model, inhibiting PDE4B by means of A33 during the early phase of
the disease did not provide any additional benefit on functional outcome
following hemisection SCI compared to continuous PDE4D inhibition by
Gebr32a. However, when PDE4D inhibition was started 10 dpi, without
preceding A33 administration, Gebr32a did not significantly ameliorate
functional recovery, observed in the BMS scores. We propose that
Gebr32a provides a therapeutic benefit when started 10 dpi, upon A33
treatment, due to the priming of A33 in the SCI environment. A33 can
potentially create a more favorable CNS environment for neuronal repair,
resulting in the therapeutic efficacy of Gebr32a when administered 10
dpi. Based on post-mortem analysis, A33 alone lacked efficacy which
could be due to the dose, timing, route of administration or the harsh
neurodegenerative conditions accompanied with a hemisection SCI
lesion. Therefore, it cannot be excluded that sequential PDE4
subtype-specific treatment can be even more efficient and clinically
relevant compared to only PDE4D inhibition to treat SCI.
Despite the promising preclinical findings on PDE4 inhibitors, the
accompanied severe side effects at the therapeutic dose have hindered
their clinical translation so far. In this study, we analyzed the impact of
the PDE4B inhibitor A33 and the PDE4D inhibitor Gebr32a in a mouse
model of SCI. In contrast to A33, Gebr32a improved functional recovery,
even when treatment was initiated 2 dpi. In addition Gebr32a amelio-
rated histopathological outcomes to a comparable level as the pan PDE4
inhibitor roflumilast. Whereas roflumilast is associated with emetic-like
side effects at its repair-inducing dose, Gebr32a is not. These data
strongly support the notion that the selective PDE4D inhibitor Gebr32a
holds great potential as a novel therapeutic approach for SCI treatment.
Funding
This study was supported by grants from Fonds Wetenschappelijk
Onderzoek (FWO-Vlaanderen) to TVM, MS, SH, JVB, and FM
(12G0817N, 1S57521N, G041421N, 1272324N, 1209123N, and
1SH2E24N). We also acknowledge partial funding from the University of
Antwerp IOF-SBO brain organoid project granted to PP.
Author contributions
MS, SH, SL, NH, PP, JVB, and TV participated in the conceptualiza-
tion, data interpretation and supervised the project. MS, FM, EvB, JVB
carried out the investigation, and participated in data collection. MS, FM,
and JVB wrote the manuscript. SH, EvB, PP, SL, NH, RR, EF, OB, CB, and
JP reviewed the manuscript. All authors read and approved the final
manuscript.
Declaration of competing interest
The authors declare the following financial interests/personal re-
lationships which may be considered as potential competing interests:
MS, JP, and TV have a proprietary interest in selective PDE4D inhibitors
for the treatment of demyelinating disorders and neurodegenerative
disorders. RR, EF, OB, CB, and JP have a proprietary interest in the use of
Gebr32a. If there are other authors, they declare that they have no known
competing financial interests or personal relationships that could have
appeared to influence the work reported in this paper.
Acknowledgments
The authors would like to thank Dr. Leen Timmermans for her
excellent technical assistance with the in vivo experiments.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https
://doi.org/10.1016/j.neurot.2024.e00372.
M. Schepers et al. Neurotherapeutics 21 (2024) e00372
10
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