Biomédicas (CSIC-UAM), Arturo Duperier, 4. 28029-Madrid, Spain.; 2 Centro de Investigación Biomédi-ca en Red sobre Enfermedades Neurodegenerativas (CIBERNED), 28031-Madrid, Spain.; 3 Departa-mento de Psicobiologia, Facultad de Psicología, Universidad Complu-tense de Madrid, 28223-Madrid, Spain.; 4 Centro de Investigaciones Biológicas (CSIC). Ramiro de Maez-tu 9, 28040-Madrid, Spain.; 5 De-partamento de Bioquímica y Biolo-gía Molecular, Facultad de Medici-na, Universidad Complutense de Madrid, 28040-Madrid, Spain.
*Address correspondence to: Ana Pérez-Castillo (E-mail: [email protected]) or to Jose A. Morales-Garcia (E-mail: [email protected]). Instituto de Investigaciones Biomédicas, (CSIC-UAM), Arturo Duperier, 4. 28029-Madrid, Spain. Phone: +34 91 585 44 61; Fax: +34 91 585 44 01.
Received March 01, 2016; ac-cepted for publication July 29, 2016; available online without sub-scription through the open access option.
for publication and undergone full peer review but has not been through the copyediting, typeset-ting, pagination and proofreading process which may lead to differ-ences between this version and the Version of Record. Please cite this article as doi: 10.1002/stem.2480
Phosphodiesterase7 Inhibition Activates Adult Neurogenesis in Hippocampus and Subventric-ular Zone in vitro and in vivo JOSE A. MORALES-GARCIA1,2*, VICTOR ECHEVERRY-ALZATE3, SANDRA
ALONSO-GIL1,2, MARINA SANZ-SANCRISTOBAL1,2, JOSE A. LOPEZ-MORENO3, CARMEN GIL4, ANA MARTINEZ4, ANGEL SANTOS2,5
AND ANA PEREZ-CASTILLO1,2* Key words. adult neurogenesis • hippocampus • phosphodiesterase7 • sub-
ventricular zone. ABSTRACT
The phosphodiesterase 7 (PDE7) enzyme is one of the enzymes responsible for controlling intracellular levels of cyclic adenosine 3’,5’-monophosphate in the immune and central nervous system. We have previously shown that inhibitors of this enzyme are potent neuroprotective and anti-inflammatory agents. In addition we also demonstrated that PDE7 inhibi-tion induces endogenous neuroregenerative processes towards a dopa-minergic phenotype. Here, we show that PDE7 inhibition controls stem cell expansion in the subgranular zone of the dentate gyrus of the hippocam-pus (SGZ) and the subventricular zone (SVZ) in the adult rat brain. Neuro-spheres cultures obtained from SGZ and SVZ of adult rats treated with PDE7 inhibitors presented an increased proliferation and neuronal differ-entiation compared to control cultures. PDE7 inhibitors treatment of neu-rospheres cultures also resulted in an increase of the levels of phosphory-lated CREB, suggesting that their effects were indeed mediated through the activation of the cAMP/PKA signaling pathway. In addition, adult rats orally treated with S14, a specific inhibitor of PDE7, presented elevated numbers of proliferating progenitor cells, and migrating precursors in the SGZ and the SVZ. Moreover, long-term treatment with this PDE7 inhibitor shows a significant increase in newly generated neurons in the olfactory bulb and the hippocampus. Also a better performance in memory tests was observed in S14 treated rats, suggesting a functional relevance for the S14-induced increase in SGZ neurogenesis. Taken together, our results indicate for the first time that inhibition of PDE7 directly regulates proliferation, migration and differentiation of neural stem cells, improving spatial learn-ing and memory tasks. Stem Cells 2016; 00:000–000 SIGNIFICANCE STATEMENT
Phosphodiesterase 7 (PDE7) is an enzyme responsible for the hydrolysis of cyclic adenosine monophosphate (cAMP) on different tissues. PDE7 is very abundant in the brain and PDE7 mRNA has been found in the cerebellum, olfactory bulb, dentate gyrus of the hippocampus and striatum. Diverse studies from our group have shown that different inhibitors of PDE7 are po-tent neuroprotective and anti-inflammatory agents in some animal models of neurodegenerative disorders and, more recently, we have demonstrated that a specific inhibitor of this enzyme named S14 induces neurogenesis to-ward a dopaminergic phenotype in an animal model of Parkinson disease. This study reports that this compound stimulates the generation of new
neurons in the granular zone of the dentate gyrus of the hippocampus and the olfactory bulb. These find-ings indicate a novel role for PDE7
inhibition in the neurogenic process in the adult and provide new insights into the mechanism of neurogenesis regulation. Thus, PDE7 inhibition may represent a novel and potential therapeutic strategy for stem cell activation.
INTRODUCTION
Neurogenesis is a major feature of the brain, which in-volves proliferation and differentiation of neural stem cells (NSCs) and their migration and integration into functional circuitries [1-3]. In mammals, the majority of neurons are born by the prenatal period, but it is well-established that neurogenesis persists in the adults. The most prominent adult neurogenic niches are the sub-ventricular zone (SVZ) adjacent to the lateral ventricles and the subgranular zone (SGZ) of the hippocampal dentate gyrus (DG) [4-8]. Progenitor cells in the SVZ are responsible for new neurons being added to the granu-lar and the periglomerular layers of the olfactory bulb (OB). These cells migrate to the olfactory bulb through the rostral migratory stream (RMS), where the majority scatters throughout the granule layer and a small per-centage develops into interneurons in the periglomeru-lar layer [9-11]. In the hippocampus, NSCs migrate into the granule layer and differentiate into new dentate granule cell neurons [7, 12].
Both cell-extrinsic and cell-intrinsic factors have been shown to influence the maintenance and regula-tion of the neurogenic system in vivo [13]. Among the extrinsic factors a number of growth factors have been shown to affect the proliferation and differentiation of precursor cell populations, including insulin-like growth factor-1, epidermal growth factor, and basic fibroblast growth factor [14] [15] [16, 17]. Also, several signaling molecules regulate neurogenesis, including Wnt, Notch, sonic hedge-hog, and neurotransmitters [18-20]. Addi-tionally, adult neurogenesis has also been shown to be influenced by the activation of a number of transcrip-tion factors, among which are Sox2, Hes5, Pax6, Neu-rog2, FoxO3, and CREB [5, 21]. With regard to CREB, the cAMP/PKA/CREB pathway has been suggested to play an essential role in adult neurogenesis in the SVZ and the SGZ [22].
Phosphodiesterases (PDEs) comprise a family of 21 members, which have been so far classified into 11 groups, according to their sequence homology, cellular distribution, and sensitivity to different PDE inhibitors [23, 24], being some of them expressed in the central nervous system [25]. Specifically PDE7 hydrolyzes cAMP and is highly expressed in endothelial cells, immune system, and brain [26-30]. PDE7 is encoded by two genes, PDE7A and PDE7B. Each gene generates, by al-ternative splicing, several isoforms which vary only in the N-terminal regulatory domain [31]. PDE7A and 7B are highly homologous in the C-terminal catalytic do-main [32], in fact, S14 and other PDE7 inhibitors inhibits equally both PDE7A and 7B isoforms [33, 34]. Addition-ally, S14 also inhibits PDE4B and PD10A although at a much higher concentration [34].
Within the brain, PDE7 mRNA has been found in the cerebellum, olfactory bulb, dentate gyrus of the hippo-campus and striatum, being PDE7B much more abun-dant than PDE7A [35, 36]. PDE7 inhibition has been re-cently reported to be as a good therapeutic option for the treatment of different neurodegenerative diseases [37-40]. In this regard, cAMP signaling pathway has been involved in different functions in the central nerv-ous system, such as cellular growth, neuronal prolifera-tion, and long-term memory formation [41]. This nucle-otide has also been shown to be a neuroprotective agent in different brain disorders, including neuro-degenerative diseases such as Huntington, Alzheimer, and Parkinson disease (PD) [38, 42, 43]. Recently, sev-eral studies have shown that phosphorylated-CREB (P-CREB) is involved in hippocampal neurogenesis regulat-ing several steps of this process such as proliferation, differentiation, and survival [44, 45].
Diverse studies from our group have shown that dif-ferent inhibitors of PDE7 are potent neuroprotective and anti-inflammatory agents in some animal models of neurodegenerative disorders, including PD [34, 38-40, 46]. We have also shown that PDE7 depletion in the SNpc, using specific shRNAs for PDE7B significantly pro-tects dopaminergic neurons and improves motor func-tion in LPS and 6-OHDA lesioned mice [37]. Very recent-ly, we have reported that, in addition to its neuropro-tective effect, chemical inhibition of PDE7 induces en-dogenous neuroregenerative processes towards a do-paminergic phenotype in an animal model of PD [47].
On the basis of the previous evidence, we speculat-ed that inhibition of PDE7 could have a role in basal adult neurogenesis in the two well-characterized neu-rogenic niches (SGZ and SVZ) of the adult rodent brain. Our in vitro studies show that the chemical inhibition of PDE7 enhances the number and differentiation of adult rat neurospheres. In vivo, the PDE7 inhibitor S14 acts a potent inducer of neuroblasts formation in the SGZ and SVZ of adult rats, which are able to migrate and gener-ate new neurons in the granular zone of the dentate gyrus (DG) of the hippocampus and the OB. Functional-ly, and in agreement with the neurogenic effect in the SGZ, we showed that S14 treated rats performed better in behavioral memory tasks that require the hippocam-pus. Altogether these findings suggest that inhibitors of PDE7 regulate neural progenitor cell proliferation and may influence their neuronal differentiation in the two main neurogenic niches of the adult brain.
MATERIALS AND METHODS
Animals. Adult male Wistar rats (8–12 weeks old) were used throughout the study. All procedures with animals were specifically approved by the “Ethics Committee for
Animal Experimentation” of the Instituto de Investi-gaciones Biomédicas and carried out in accordance with the European Communities Council, directive 2010/63/EEC and National regulations, normative 53/2013. Special care was taken to minimize pain or discomfort of animals.
Adult precursors isolation. Neural stem cells were isolated from the two main neurogenic niches in the adult rat: the subgranular zone (SGZ) of the hippocam-pus and the subventricular zone (SVZ) of the lateral ven-tricle as previously described [48, 49]. Briefly, the hip-pocampus and SVZ were carefully dissected, washed in DMEM (Invitrogen), dissociated in DMEM medium with glutamine, gentamicin and fungizone and then digested with 0.1% trypsin-EDTA + 0.1% DNAase + 0.01% hialu-ronidase for 15 min at 37 °C. Neural stem cells isolated from both niches were seeded into 12-well dishes at a density of ~40,000 cells per cm2 in DMEM/F12 (1:1, Invi-trogen) containing 10 ng/mL EGF, 10 ng/mL FGF and N2 medium (Gibco).
Neurospheres culture and treatments. Neural pre-cursors were allowed to proliferate on culture until neural progenitors-enriched spheres (neurospheres, NS) were visible (~ 3 days). At this moment cultures were treated with BRL-50481 (30 μM, Tocris), S14 (10 μM) or vehicle during 7 days. The quinazoline S14 was synthe-sized following described procedures [50]. BRL50481 is a selective substrate-competitive inhibitor for the PDE7 subtype (Ki = 180 nM) [51]. The quinazoline S14 is a selective substrate-competitive inhibitor for PDE7 (both isoforms A and B) with an IC50 value of 4.7 and 8.8 µM, respectively [32, 34]. The effective dose of compounds was chosen based on previous studies [38]. Prolifera-tion and growth analysis was determined on these cul-tures and number and diameter of NS were scored us-ing the Nikon Digital Sight, SD-L1 software (Nikon, Ja-pan). Ten wells per condition tested and experiment were counted. Some of these NS were used for im-munoblotting analysis. Remaining NS were then seeded onto poly-L-lysine (Sigma) precoated 6-well plates and/or coverslips for another 3 days in the absence of exogenous growth factors in medium containing 1% fetal bovine serum to promote differentiation and in the presence of BRL-50481 or S14. Differentiated NS in coated 6-well plates were used for immunoblotting and coverslips previously fixed in 4% paraformaldehyde for immunocytochemical analysis.
Immunoblot analysis. Differentiated cultured NS were resuspended in ice-cold cell lysis buffer (Cell Sig-naling Technology) with protease inhibitor cocktail (Roche) and incubated for 15-30 min on ice. A total amount of 30 µg of protein was loaded on a 10% or 12% SDS-PAGE gel and transferred nitrocellulose mem-branes (Protran, Whatman). The membranes were blocked in Tris-buffered saline with 0.05% Tween-20 and 5% skimmed milk, incubated with primary and sec-ondary antibodies, and washed according to standard procedures. Primary antibodies were PDE7A (rabbit; Santa Cruz Biotech), PDE7B (rabbit; ProteinTech), p-
CREB (rabbit; Cell Signaling), CREB (rabbit; Cell Signal-ing), Musashi1 (rabbit; Abcam), β-III-tubulin (mouse; Covance), MAP-2 (mouse; Sigma), GFAP (mouse; Sigma) and α-tubulin (mouse; Sigma). Secondary peroxidase-conjugated donkey anti-rabbit (Amersham Biosciences, GE Healthcare), or rabbit anti-mouse antibodies (Jack-son Immunoresearch) were used. Values in figures are the average of the quantification of at least three inde-pendent experiments corresponding to three different samples.
Immunocytochemistry. Fluorescence immunocyto-chemical analysis on differentiated NS was performed as previously described [49]. Briefly, NS were incubated at 37ºC for 1h with primary antibodies directed against β-III-tubulin (TuJ-1 clone; rabbit; Abcam), MAP-2 (mouse; Sigma) and GFAP (mouse; Sigma). After several rinses in PBS, samples were then incubated with Alexa-488 goat anti-rabbit, Alexa-488 goat anti-mouse and Alexa-647 goat anti-mouse antibodies (Molecular Probes) for 45 min at 37 °C. Staining of nuclei was per-formed using 4′,6-diamidino-2-phenylindole (DAPI). Finally images were acquired in a LSM710 laser scan-ning spectral confocal microscope (Zeiss). Confocal mi-croscope settings were adjusted to produce the opti-mum signal-to-noise ratio.
PDE7 inhibitor (S14) administration in vivo. Accord-ing with the S14-treatment time, rats were divided in: 1) Short-term groups, which received daily intra-gastrical administration of S14 during 4 consecutive days. First administration was considered as day 1. On day 4 rats were intraperitoneally injected with 5-bromo-2-deoxyuridine (BrdU, 50 mg/kg) and sacrificed on day 5 (Supplemental Figure 1A) and 2) Long-term groups, which received daily intragastrical administration of S14 during 27 consecutive days. To label proliferating cells for long-term studies on survival and differentiation, rats were intraperitoneally injected with BrdU (50 mg/kg) on day 2 and sacrificed on day 28 (Supplemental Figure 1B). For behavioral analysis, rats were first treat-ed with S14 during 27 days and later on tested, as indi-cated below (Supplemental Figure 1C). S14 was admin-istered at a dose of 10 mg/kg body weight, in a sodium carboxy methyl cellulose suspension. This dose was chosen based on their effectiveness in different previ-ously published works [34, 38]. Control animals were given the same volume of vehicle.
Immunohistochemistry. Animals previously anaes-thetized were perfused transcardially with 4% para-formaldehyde, and brains were obtained, postfixed in the same solution at 4 °C overnight, cryoprotected, fro-zen, and finally 30 μm coronal sections were obtained in a cryostat. Free-floating sections were immunostained using immunofluorescence analysis or 3,3-diaminobenzidine (DAB) method as previously de-scribed [48]. Briefly, for BrdU detection, samples were first incubated with 2 M HCl for 30 min at 37° before blocking 1 h in PBS containing 5% normal serum, 0.1 M lysine, and 0.1% Triton X-100. Sections were then incu-bated with anti-BrdU mouse monoclonal (DAKO) com-
bined with anti-nestin rabbit (Abcam) or anti-NeuN rab-bit (Millipore) antibodies at 4°C overnight, washed three times and incubated with AlexaFluor 488 goat anti-mouse and Alexa 647 goat anti-rabbit secondary antibodies for 1 h at room temperature. After rinses, sections were mounted with Vectashield. Images were obtained using a LSM710 laser scanning spectral confo-cal microscope (Zeiss). Confocal microscope settings were adjusted to produce the optimum signal-to-noise ratio. For doublecortin (DCX) detection floating sections were immersed in 3% H2O2 to inactivate endogenous peroxidase, blocked for 2 h at room temperature in 5% normal horse serum in PBS, containing 4% bovine se-rum albumin, 0.1 M lysine, and 0.1% Triton X-100. Af-terwards, the sections were incubated overnight with an anti-DCX goat (Santa Cruz, CA) antibody. After sever-al rinses, sections were incubated for 1 h with the cor-responding biotinylated secondary antibody and then processed following the avidin-biotin protocol (ABC, Vectastain kit, Vector Labs). The slides were examined with a Nikon eclipse 90i microscope, equipped with a DS-Fi1 digital camera. Five animals from each experi-mental group were analyzed.
Orthogonal image acquisition. To confirm double staining confocal imaging was performed on coronal brain sections with a LSM710 laser scanning spectral confocal microscope (Zeiss) connected to a PC running the ZEN imaging software. The objective used was Zeiss 63x Plan-Apochromat. Sections (30 μm) containing the DG region of the hippocampus and/or the olfactory bulb were used for the analysis. BrdU/Nestin and BrdU/neuN co-expression was defined by nuclear co-localization of the two markers over the extent of the nucleus in consecutive 0.5 µm z-stacks, when green (BrdU) and red (Nestin or NeuN) signals coincided, and when co-localization was confirmed in x-y, x-z and y-z cross-sections produced by orthogonal reconstructions from z-series. Images were processed with the image processing package Fiji [52]. Only contrast enhance-ments and color level adjustments were made; other-wise images were not digitally manipulated.
Cell count analysis. A modified stereological ap-proach was used to estimate the total numbers of cells stained with a particular marker (BrdU/Nestin- and BrdU/NeuN-double immunofluorescence or DCX-immunohistochemistry) as previously described [47]. Confocal images (BrdU/Nestin and BrdU/NeuN) or DAB-stained light microscope images (DCX immunohisto-chemistry) of the DG, SVZ and OB were viewed and cap-tured under a x63 objective to avoid oversampling er-rors. From serial coronal sections (30µm) from the en-tire rostrocaudal extent of the DG, the SVZ or the OB, every sixth section was selected to count the number of immunoreactive cells for a given marker. The bounda-ries of these nervous system regions were determined with reference to internal anatomic landmarks [53]. For each area of interest, images were analyzed using com-puter-assisted image analysis software (Soft Imaging System Corporation, Lakewood, CO, http://www.soft-
imaging.com). Positive cells, which intersected the up-permost focal plane (exclusion plane) and the lateral exclusion boundaries of the counting frames, were not counted. Six rats per group were used. The results were expressed as the total number of labeled cells in the DG of the hippocampus, the SVZ or the OB by multiplying the average number of labeled cells/structure section by the total number of 30 µm thick-sections containing the related structure (DG, SVZ or OB).
Behavioral studies. Behavioral tests were per-formed in a dimly lit room (20 luxes) and monitored by a video camera above the apparatus. Novel object recognition test was performed, 72 hours after the last intragastrical administration of vehicle or S14 (day 1), as previously described [54]. Basically, on day 1 each rat was allowed to habituate during 10 min in the test box (40 × 35 × 35 cm) (habituation session). On day 2 rats were placed in the box and allowed to explore two identical sample objects during 3 min, returned to their home cages for 30 min (retention interval) and placed again in the same box with one familiar (sample) and one novel object (counterbalanced across rats) and giv-en 3 min to explore the objects (test session). An exper-imenter blind to the treatment scored the time the rats spent exploring each object, the latency of first ap-proach to explore them and the frequency of approach. A valid object approach was any directed contact with the mouth, nose or paw not including accidental con-tacts such as backing into the object [55]. Spatial learn-ing and memory tasks were performed in the Morris Water Maze 8 days after the last intragastrical admin-istration of vehicle or S14 (day 1) as previously de-scribed in detail [56]. From day 1 to 4 (learning curve) the animals were trained to find the hidden scape plat-form and on day 5 (probe trial) they were tested with-out platform. Animals were submitted to 4 trials every learning session, with a time limit of 60s/trial and an interval between trials of 4-5min. An experimenter blind to the treatment scored the latency time to reach the target site (the previous platform location), the number of platform-site crosses and the time spent within the target annulus, around the former platform.
Statistics analysis. Data from Figures 1, 2, and 3 were analyzed using a one-way ANOVA and data from Figures 4, 5, 6A-B, and 7 were analyzed by Student’s t-test. Data from Figures 6C-D were analysed using a two-way mixed ANOVA. After confirming the significance of the primary findings using ANOVA, a significance level of p<0.05 was applied to all remaining post-hoc statisti-cal analyses. The SPSS statistical software package (ver-sion 20.0) for Windows (Chicago, IL, USA) was used for all statistical analyses.
Effect of PDE7 inhibition on the levels of P-CREB in neurosphere cultures. First, we analyzed the levels of expression of PDE7 in neurosphere cultures isolated from the two main neu-rogenic niches of the brain: the SGZ of the dentate gy-rus of the hippocampus and the SVZ. Since PDE7 com-prises two genes, PDE7A and PDE7B, both isoforms were analyzed by Western blot analysis (Figure 1A). Our results confirmed that neural stem cells express both isoforms of PDE7, although the levels of PDE7A were less prominent in comparison with PDE7B. The level of both proteins was not altered by the treatment of the neurosphere cultures with the two PDE7 inhibitors used, BRL50481 and S14 (Figure 1A). We next examined the effects of S14 and BRL50481 on the phosphorylation levels of the cAMP response element binding protein (CREB), a well-known target of the cAMP signaling pathway. As can be observed in Figure 1B, NS cultures treated during 7 days with BRL50481 or S14 (Figure 1B) showed a significant increase in the levels of p-CREB, confirming that the S14 compound is acting through inhibition of PDE7 and the subsequent induction of the cAMP pathway.
PDE7 inhibition induces proliferation and growth in neurosphere cultures. Next, we investigated whether inhibition of PDE7 would affect the proliferation ability of in vitro cultured NS established from adult SGZ and SVZ. To that end, free floating NS growing in non-adhesive conditions were treated with BRL50481 or S14 during 7 days. Treatment with both PDE7 inhibitors significantly increased the rate of formation and the size of NS derived from the adult SGZ and SVZ (Figure 2). After 7 days of growth in suspension in the presence of BRL50481 or S14, the number of SGZ-derived NS were 380±12 and 372±20, respectively, in comparison with that found in the vehi-cle-treated cultures, 208±11. In the case of SVZ-derived NS the number of NS growing in the presence of BRL50481 or S14 were 320±12 and 340±9, respectively, in contrast with 196±14 in control cultures. The size of NS was also increased after BRL50481 (195±13 µm in SGZ and 130±11 �m in SVZ) or S14 (208±11 µm in SGZ and 170±16 �m in SVZ) treatments, as compared with non-treated cultures (90±10 µm in SGZ and 70±12 in SVZ).
In order to study the stemness of cultured NS, we analyzed the expression of musashi-1, a marker for un-differentiation. The NS cultured under proliferative conditions were treated for 7 days with the PDE7 inhibi-tors. After that time proteins were isolated and West-ern blot performed. Our results shows a significant de-crease in SGZ- and SVZ-derived NS of the amount of musashi-1 protein when BRL50481 and S14 were added to the medium (Figure 2B). These results suggest that PDE7 inhibition promotes a loss of stemness in the NS
derived from SGZ and SVZ. Taking together, the results here described suggest that PDE7 inhibition stimulates the proliferation and growth of neurospheres derived from adult SGZ and SVZ controlling the activity of neural progenitors.
PDE7 inhibition induces differentiation of neural stem cells. Next, we examined whether treatment of NS cultures with BRL50481 or S14 could result in a regulation of cell differentiation after adhesion of the NS. For this pur-pose, NS cultures established from the SGZ and SVZ were cultured in the absence of growth factors, and the percentage of Tuj1-, MAP-2- and GFAP-positive cells was analyzed by immunocytochemistry and Western blot. NS were allowed to adhere to the substrate and then incubated for 72 hours in the presence or absence of the PDE7 inhibitors. As shown in Figure 3, only a few cells stained with Tuj-1 or MAP-2 were observed in NS cultures obtained from the SGZ (Figure 3A,C) or the SVZ (Figure 3B,D) in basal conditions. After treatment, the number of both Tuj1- and MAP-2-positive cells was sig-nificantly enhanced. In the case of GFAP-positive cells, a high number was observed in basal conditions and this number was increased by PDE7 inhibition suggesting that inhibition of PDE7 also promotes the differentia-tion of astroglial cells. Of note, the S14 compound was more potent than the commercially available PDE7 in-hibitor BRL50481 promoting neurogenesis. These re-sults suggest that PDE7 inhibition results in an induction of neuronal differentiation of neural stem cells towards mature neurons.
Effect of PDE7 inhibition on proliferation of adult progenitor cells in vivo. Given the in vitro results showing a neurogenic effect of PDE7 inhibition, we finally analyzed whether this inhibi-tion affected the proliferation kinetics of progenitor cells in the SGZ and SVZ in vivo. For this study, rats were orally treated during 4 (short-term) or 27 days (long-term) with the PDE7 inhibitor, S14, which is known to cross the blood brain barrier [34, 57] or vehicle, fol-lowed by BrdU administration for 24h (Supplemental Figure 1A) or 27 days (Supplemental Figure 1B) before sacrifice. Coronal sections from short-term treated-animals were stained with specific anti-Nestin, anti-BrdU, and anti-DCX antibodies (Figures 4 and 5). Nestin is commonly used as a reliable biological marker of neu-ral progenitor cells in vitro and in vivo [58-61]. DCX is a microtubule-associated protein, which is a valuable en-dogenous marker for dividing neuroblasts and imma-ture neurons [62, 63]. Orthogonal view of the subgranu-lar zone of the hippocampus (Figure 4A) shows that 24 hours after BrdU injection there was a considerable increase in the number of double stained BrdU/Nestin cells in the SGZ of those animals treated with S14, in comparison with the vehicle-treated control group. PDE7 inhibition significantly increased the number of
BrdU/Nestin-stained cells in comparison with control values. These results indicate that PDE7 inhibition in-creases the number of new progenitor cells in the SGZ.
To test the hypothesis that S14 is also able to induce neurogenesis in vivo, brain section from control and S14-treated rats were stained for doublecortin (DCX). The results shown in Figure 4B show a higher number of DCX-positive cells in the SGZ of S14-treated animals, relative to the vehicle-treated controls. Besides, DCX-stained cells in S14-treated animals exhibited extensive dendritic arborizations.
Regarding the other adult neurogenic niche, the SVZ, similar to what happened in the hippocampus, an increase in the number of Nestin/BrdU double-stained cells was observed in the SVZ of animals treated with S14 (Figure 5A). In addition, the number of DCX-stained cells was also enhanced in the SVZ of these animals (Figure 5B), suggesting an effect of this compound on neurogenesis in vivo. We also observed an increase in the migrating chain of cells in this area.
In order to know whether the new migrating neuro-blasts originated in the SGZ and the SVZ as a conse-quence of the short-treatment with the PDE7 inhibitor were able to properly reach the granular cell layer of the hippocampus or the OB, respectively, long-term treated animals were used. For this purpose animals were treated with the PDE7 inhibitor during 27 days and neuronal differentiation of newborn cells were ana-lyzed by immunofluorescence, labeling double BrdU/NeuN positive cells, as indicated in Material and Methods. After this time most of BrdU immunoreactive cells produced in the SGZ of the hippocampus have reached the granular zone and have differentiated into neurons (Figure 6). Also, BrdU-positive cells generated in the SVZ have already reached the olfactory bulb through the RMS, expressing a neuronal phenotype (Figure 7).
Regarding the hippocampus, the results shown in figure 6 clearly demonstrate that after 27 days, those animals treated orally with the PDE7 inhibitor showed a significant increase in the number of migrating neuro-blasts in the SGZ of the DG of the hippocampus (figure 6A), in comparison with vehicle treated animals. At this time also a noticeable increase in the amount of newly generated neurons (BrdU+/NeuN+ cells) was seen in the granular cell layer (Fig. 6B) as a consequence of PDE7 inhibition. In view of these results, we analyzed the functional consequences of PDE7 inhibition by analyzing memory and learning. As shown in Figure 6C in the ob-ject recognition test, both groups of animals learned the task as shown by the increased time spent exploring the new object. However, a better performance was ob-served in the S14-treated rats since they increased the time exploring the new object (35%), along with the number of approaches to the new object and the laten-cy time of approach to the sample object (lower inter-est) in comparison with the controls. In the Morris Wa-ter Maze (Figure 6D), a test generally considered specif-ic for the hippocampus, our results showed an im-
proved spatial memory in S14 treated rats. A significant difference was observed in the learning curve. Howev-er, no significant difference was observed in the latency time to reach the platform-site in the test day or in the swim speed (data ranged between 23.7 ± 0.73 and 24.1 ± 0.65 cm/s for vehicle and S14 groups, respectively). The lack of a significant effect on latency is probably due to the fact that already the control rats performed very well and the latency time was already very short (i.e., a floor effect). In contrast the S14 treated rats ex-pended more time in target annulus indicating a better memory performance. Further studies may well exam-ine whether animals with learning/memory impair-ments, which exhibit deficits in their learning curve, would take great benefit from PDE7 inhibition than con-trol animals, which display normal curves of learning.
As mentioned above, a significant number of migrat-ing cells was observed in the SVZ of S14-treated animals after 4 days of treatment (short-term). These neuro-blasts integrate into the RMS to finally reach the OB. In fact, when long-term S14 treated rats were analyzed (Fig. 7), we observed a numerous amount of DCX+ cells that reached OB through the RMS spreading across the ependymal zone and mainly across the granular cell layer (Figure 7A). Coronal sections of the olfactory bulb also showed that these migrating neuroblasts have reached the OB and a high number of BrdU+ cells enter-ing the OB from the RMS through the ependymal zone was observed. We also detected that, in S14-treated animals, most of these new migrating cells finally differ-entiate into neurons, as can be observed by the in-creased number of new born neurons (BrdU+/NeuN+ cells) in the OB (Figure 7B).
Altogether, these observations clearly indicate that PDE7 inhibition in vivo increases the number of new neurons originated in the hippocampus and the olfacto-ry bulb of adult rats, improving spatial learning and memory tasks.
DISCUSSION
Although our group has recently demonstrated that PDE7 inhibition plays an important role on neuroprotec-tion in different animal models of neurodegenerative disorders [34, 37, 38, 40, 46], so far there is almost no information about the effect of PDE7 inhibition on neu-rogenesis in the adult brain. More recently, our group has reported for the first time that the PDE7 inhibitor S14, which is able to cross the blood brain barrier, in-duces neurogenesis towards a dopaminergic phenotype in the substantia nigra pars compacta of adult rats le-sioned with 6-OHDA [47]. In the present study we have extended these studies and show that the S14 com-pound also promotes proliferation and neuronal differ-entiation in the two main neurogenic niches of the adult brain, the SVZ and the SGZ. The results of this study demonstrate that PDE7 inhibition plays a role in regulating the expansion and differentiation of the stem cell population in these two regions of the adult brain.
This is manifest in vitro by enhanced numbers of prima-ry neurospheres and by S14-mediated induction of TuJ-1+ and MAP-2+ cells, and in vivo by an increased prolif-eration and a larger population of doublecortin express-ing neuroblasts, which migrate to generate new neu-rons.
The adult mammalian forebrain has two specialized niches, the SGZ and the SVZ, holding stem cells that are primarily involved in generating neurons throughout life [6, 7].These niches contains neural progenitors which express nestin and GFAP, exhibit proliferative activity and are capable of self-renewal [64]. They give rise to more rapidly dividing progenitors, which express mark-ers of immature neurons (PSA-NCAM and doublecortin) and then exit the cell cycle to differentiate into mature neurons. Accumulated data indicate that self-renewal, proliferation, migration and differentiation of these neural stem cells are under a number of intrinsic (spe-cific transcription factors, epigenetic mechanisms, mi-croRNAs, etc...) [21, 65-69] and extracellular signaling cues (environmental, physiological, and pharmacologi-cal stimuli) [5, 70, 71]. Among the transcription factors, the cAMP response element binding protein, CREB, is considered to act as a central integrator in the control of adult neurogenesis [22]. Our data showing that the enhancement in proliferation and differentiation of SGZ- and SVZ-derived neural stem cells triggered by PDE7 inhibition is accompanied by an increase in CREB phosphorylation are in agreement with a role of cAMP in adult neurogenesis.
Besides the role of PDE7 inhibition on neural stem cells proliferation in both niches, as shown by the in-creased number of BrdU/Nestin-positive cells after S14 administration, we also present evidence that PDE7 inactivation increases the number of newly generated neurons in the DG of the hippocampus and the OB, as suggested by the enhancement in the number of inte-grated neuroblasts (DCX-positive cells) in the granular zone of the DG and the increase in the number of newly generated neurons (BrdU/NeuN-immunoreactive cells) in the OB found in those animals treated with S14. Thus, our results suggest that inhibition of PDE7 can be an important regulator for the neural stem cells to become neurons in the hippocampus and the olfactory bulb of the rodent adult brain, then identifying a new function for this enzyme in this area of the brain. These results are especially important since they represent the identi-fication of a new target whose inhibition could help to promote neural stem cells proliferation and differentia-tion in the SGZ and SVZ, which can be relevant in olfac-tory-associated behavior, certain forms of learning, memory and mood [72, 73]. In fact, here we show that S14 treatment during 27 days in addition to increase SGZ neurogenesis, clearly improved rat performance in learning and memory tasks in which the hippocampus is considered to play an essential role. This is in agree-ment with previous works showing that adult hippo-campal neurogenesis plays an important role in these cognitive functions [74, 75].
Our in vitro results showing that PDE7A and B are expressed in SGZ- and SVZ-derived neural stem cells, together with the observed increase in CREB phos-phorylation after S14 treatment of neurospheres, sug-gest that the mechanism of action of this compound is an inhibition of PDE7, the subsequent activation of cAMP/PKA signaling pathway, and the activation of the transcription factor cAMP response element-binding protein (CREB) by phosphorylation. Consistent with our results, different studies in the mouse hippocampus, using pharmacological and genetic approaches to de-crease the activity of PDE4, another cAMP specific phosphodiesterase, have shown that p-CREB plays a significant role in adult neurogenesis [75-77]. These studies show that rolipram, a PDE4 inhibitor, increases hippocampal neurogenesis and promotes survival of newborn hippocampal neurons in different conditions. Also it has been shown that cilostazol, an inhibitor of the dual type 3 phosphodiesterase , PDE3, increases neurogenesis in the subventricular zone of adult mice in a model of focal cerebral ischemia[78]. However, hu-man emesis, which was found in clinical development of several PDE4 inhibitors, has endangered their develop-ment and has rule out some promising candidates from reaching the pharmaceutical market. Therefore, our study adds new and important data suggesting that activation of CREB following PDE7 inhibition by S14, with does not show any emetogenic activity, results in a generation of new neurons in the two specialized neu-rogenic niches of the adult brain.
Given the fact that alterations in the specialized niches of the adult brain underlay many different brain disorders including neurodegenerative disease, our work suggest that PDE inhibition could be a novel strat-egy for the treatment of these disorders. This idea is further substantiated by our previous work showing an important neuroprotective role of PDE7 inhibition in different brain disorders [37-40] and by our previous report in which we demonstrated that PDE7 inhibition after S14 treatment in vivo induces a strong neurogene-sis in the substantia nigra pars compacta of 6-OHDA-lesioned animals towards a dopaminergic phenotype [47].
Collectively, our findings clearly demonstrate a nov-el role for PDE7 inhibition in the process of adult neuro-genesis, providing new insights into the mechanism of neurogenesis regulation and suggesting that PDE7 inhi-bition may represent a novel and potential therapeutic strategy for stem cell activation through the use of small molecules, such as S14, that inhibits this enzyme.
ACKNOWLEDGMENTS
This work was supported by the MINECO (Grants SAF2010-16365 and SAF2014-52940-R to A.P-C and SAF2012-33600 to C.G), by Fondo de Investigación Sani-taria; Red de Trastornos Adictivos (RD12/0028/0015 to J.A.L.M, the European Union program; Grant IPT-2012-0762-300000 to C.G and partially financed with FEDER
funds. CIBERNED is funded by the Instituto de Salud Carlos III. J.A.M-G. is a post-doctoral fellow from CIBERNED. Special thanks to Lucía Sánchez-Ruiloba for her assistance with the orthogonal image acquisition at confocal microscopy facility. AUTHOR CONTRIBUTIONS
A.P-C., A.S., J.A.M-G: Conception and design; J.A.M-G., S.A-G., M.S-S. V.E-A.: Collection and/or assembly of da-
ta; C.G., A.M.: Provision of Study Materials; J.A.M-G., A.P-C., A.S., V.E-A., J.A.L-M., C.G., A.M: Data analysis and interpretation; A.P-C., J.A.M-G., A.S: manuscript writing. J.A.M-G., V.E-A., S.A-G., M.S-S., J.A.L-M., C.G., A.M., A.S., A.P-C.: Final approval of manuscript. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
All authors declare no competing financial interests.
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See www.StemCells.com for supporting information available online. STEM CELLS ; 00:000–000
Figure 1.- PDE7 expression on neurosphere derived from the adult subgranular (SGZ) and subventricular zones (SVZ). A) Representative image of immunoblots for PDE7A and PDE7B in cultured SGZ- and SVZ-derived neuro-spheres. B) Western blot showing the levels of CREB-phosphorylated, after incubation of SGZ and SVZ neurospheres with BRL50481 (BRL; 30 µM) and S14 (10 µM). Quantitative analysis expressed as protein content relative to basal (non-treated cultures) is shown. Data were obtained from three independent experiments and presented as mean ± SD. ***p ≤ 0.001 versus non-treated (basal) cultures.
Figure 2.- Effects of PDE7 inhibition on adult neurosphere formation. A) Representative phase-contrast micrographs showing the number and size of neurospheres after 7 days in culture in the presence of BRL50481 (BRL; 30 µM) and S14 (10 µM). The number and diameter of at least 50 neurospheres was determined in control and treated cultures. Scale bar = 100 μm. B) Representative Western blot and quantification showing expression levels of the precursor cell marker Musashi-1 after treatment with PDE7 inhibitors. Results are mean values ± SD from three independent exper-iments. *P ≤ 0.05; **P ≤ 0.01; ***p ≤ 0.001 versus non-treated (basal) cultures.
Figure 3.- In vitro inhibition of PDE7 promotes stem cell differentiation towards a neuronal phenotype. Neural stem cells isolated from the adult subgranular (SGZ) and subventricular (SVZ) zone were cultured as neurospheres (NS) in the presence of BRL50481 (BRL; 30 µM) and S14 (10 µM) for 7 days and later on adhered for 3 days to allow differen-tiation in the presence of inhibitors. A-B) Representative immunofluorescence images showing the expression of the neuronal markers β-III-Tubulin (TuJ-1 clone, green) and MAP-2 (red) inside the NS and the expression of the astroglial marker GFAP in the distal portion of the NS. DAPI was used for nuclear staining. Scale bar = 20 μm. C-D) Representa-tive Western blots of β-tubulin, MAP-2, and GFP. Quantification analyses are also shown. Results are the mean±SD from three independent experiments. **P ≤ 0.01; ***p ≤ 0.001 versus non-treated (basal) cultures.
Figure 4.- Inhibition of PDE7 promotes in vivo activation of the neurogenic niche located in the subgranular zone (SGZ) of the dentate gyrus in the hippocampus. A) Orthogonal projections of BrdU/Nestin co-staining on coronal sections of the SGZ of adult rats, showing the granule cell layer (GCL). BrdU is shown in green, nestin in red and DAPI was used as a nuclear marker. Upper images show the maximum intensity projection. Scale bar = 50 μm. Quantifica-tion of the number of BrdU+/Nestin+ cells in the dentate gyrus (DG) is shown. B) Doublecortin (DCX)-expressing cells in the SGZ. Insets show higher magnifications of representatives selected areas. Scale bar = 250 μm. Insets scale bar = 50 μm. Quantification of the number of DCX+ cells in the DG is shown. All quantification values represent the mean ± SD from 3 different experiments and 5 animals/experiment/experimental group. **p ≤ 0.01 versus vehicle-treated ani-mals.
Figure 5.- Inhibition of PDE7 promotes in vivo activation of the neurogenic niche in the subventricular zone (SVZ) of the lateral ventricle. A) Immunofluorescence analysis of coronal sections of the SVZ of adult rats stained with specific antibodies against BrdU (green) and nestin (red). Scale bar = 50 μm. B) Doublecortin (DCX)-expressing cells in the SVZ. Insets show higher magnifications of representatives selected areas. Scale bar = 75 μm. Quantification of the number of BrdU+/Nestin+ cells in A and DCX+ cells in B evaluated in the SVZ is shown. Values represent the mean ± SD from 3 different experiments and 5 animals/experiment/experimental group. ***p ≤ 0.001 versus vehicle-treated animals.
Figure 6.- Inhibition of PDE7 promotes in vivo neurogenesis on the subgranular zone (SGZ) of the dentate gyrus (DG) in the hippocampus and improves performance in learning tasks . A) Doublecortin (DCX)-expressing cells in the DG. Insets show higher magnifications of representatives selected areas. Scale bar = 250 μm. Insets scale bar = 50 μm. Quantification of the number of DCX+ cells in the DG is shown. Values represent the mean ± SD from 3 different ex-periments and 5 animals/experiment/experimental group. ***p ≤ 0.001 versus vehicle-treated animals. B) Orthogo-nal projections showing the co-localization of BrdU (green) and neuN (red) cells in the dentate gyrus of the hippo-campus of adult rats. Scale bar = 20 μm. Left images show the maximum intensity projection. Right images are or-thogonal views of a z-stack showing dual BrdU+NeuN+ cells. Quantification of the number of BrdU+NeuN+ cells in DG is shown. Values represent the mean ± SD from 3 different experiments and 5 animals/experiment/experimental group. **p ≤ 0.01. GCL, granule cell layer. C) Object recognition test was performed in vehicle and S14 treated animals, as described in Materials and methods. D) Morris Water Maze test was performed in vehicle and S14 treated animals, as described in Materials and methods. Values represent the mean ±SD from at least 12 animals/group *p ≤ 0.05,**p ≤ 0.01.
Figure 7.- Inhibition of PDE7 promotes in vivo neurogenesis on the olfactory bulb (OB). A) Coronal sections of the OB immunostained with doublecortin (DCX) showing migrating neuroblasts in the ependymal zone (E) and the gran-ule cell layer (GCL). Insets show higher magnifications of representatives selected areas.Scale bar = 250 μm. Insets scale bar = 50 μm. Quantification of the number of DCX+ cells in the OB is shown. Values represent the mean ± SD from 3 different experiments and 5 animals/experiment/experimental group. ***p ≤ 0.001 versus vehicle-treated animals. B) Confocal imaging analysis showing the co-localization of BrdU (green) and neuN (red) cells in the OB of adult rats. Scale bar = 20 μm. Left image shows the maximum intensity projection. Right images are orthogonal views of a z-stack showing dual BrdU+NeuN+ cells. Quantification of the number of BrdU+NeuN+ cells in OB is shown. Scale bar = 20 μm. Values represent the mean ± SD from 3 different experiments and 5 animals/experiment/experimental group. ***p ≤ 0.001 versus vehicle-treated animals.
Graphical abstract The phosphodiesterase 7 (PDE7) enzyme is one of the enzymes responsible for controlling intracellular levels of cAMP in central nervous system. Here we described that PDE7 inhibition controls, in vitro and in animal models, stem cell expansion in the two main adult neurogenic niches: the subgranular zone of the dentate gyrus of the hippocampus (SGZ) and the subventricular zone (SVZ) in the adult rat brain. Adult rats orally treated with an inhibitor of PDE7, pre-sented in the SGZ and the SVZ elevated numbers of proliferating progenitor cells, migrating precursors and finally a significant increase in newly generated neurons in the olfactory bulb and the hippocampus together with a better performance in memory tests. Our results indicate for the first time that inhibition of PDE7 directly regulates prolifer-ation, migration and differentiation of neural stem cells, improving spatial learning and memory tasks.
