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 inhibition induces endogenous neuroregenerative processes toward a dopaminergic phenotype. Here, we show that PDE7 inhibition controls stem cell expansion in the subgranular zone of the dentate gyrus of the hippocampus (SGZ) and the subventricular zone (SVZ) in the adult rat brain. Neurospheres cultures obtained from SGZ and SVZ of adult rats treated with PDE7 inhibitors presented an increased proliferation and neuronal differentiation compared to control cultures. PDE7 inhibitors treatment of neurospheres cultures also resulted in an increase of the levels of phosphorylated cAMP response element binding protein, 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 learning and memory tasks. Stem Cells 2017;35:458–472

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 potent 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 toward 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 findings 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 involves 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 subventricular 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 granular 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 percentage develops into interneurons in the periglomerular 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 regulation 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 14-17. Also, several signaling molecules regulate neurogenesis, including Wnt, Notch, sonic hedge‐hog, and neurotransmitters 18-20. Additionally, adult neurogenesis has also been shown to be influenced by the activation of a number of transcription factors, among which are Sox2, Hes5, Pax6, Neurog2, FoxO3, and cAMP response element binding protein (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 alternative splicing, several isoforms which vary only in the N‐terminal regulatory domain 31. PDE7A and 7B are highly homologous in the C‐terminal catalytic domain 32, in fact, S14 and other PDE7 inhibitors inhibits equally both PDE7A and 7B isoforms 33, 34. Additionally, 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 hippocampus and striatum, being PDE7B much more abundant than PDE7A 35, 36. PDE7 inhibition has been recently 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 nervous system, such as cellular growth, neuronal proliferation, and long‐term memory formation 41. This nucleotide has also been shown to be a neuroprotective agent in different brain disorders, including neurodegenerative diseases such as Huntington, Alzheimer, and Parkinson disease (PD) 38, 42, 43. Recently, several studies have shown that phosphorylated‐CREB (P‐CREB) is involved in hippocampal neurogenesis regulating several steps of this process such as proliferation, differentiation, and survival 44, 45. Diverse studies from our group have shown that different 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 protects dopaminergic neurons and improves motor function in LPS and 6‐OHDA lesioned mice 37. Very recently, we have reported that, in addition to its neuroprotective effect, chemical inhibition of PDE7 induces endogenous neuroregenerative processes toward a dopaminergic phenotype in an animal model of PD 47. On the basis of the previous evidence, we speculated that inhibition of PDE7 could have a role in basal adult neurogenesis in the two well‐characterized neurogenic 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 generate new neurons in the granular zone of the dentate gyrus (DG) of the hippocampus and the OB. Functionally, 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 hippocampus. 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 Investigaciones 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 SGZ of the hippocampus and the SVZ of the lateral ventricle as previously described 48, 49. Briefly, the hippocampus and SVZ were carefully dissected, washed in DMEM (Invitrogen, Madrid, Spain, http://www.thermofisher.com/es/es/home.html), dissociated in DMEM medium with glutamine, gentamicin and fungizone and then digested with 0.1% trypsin‐EDTA + 0.1% DNAase + 0.01% hialuronidase 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, Invitrogen, Madrid, Spain, http://www.thermofisher.com/es/es/home.html) containing 10 ng/ml EGF, 10 ng/ml FGF and N2 medium (Gibco, Madrid, Spain, http://www.thermofisher.com/es/es/home.html). Neurospheres Culture and Treatments Neural precursors 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, Madrid, Spain, https://www.tocris.com/), S14 (10 μM) or vehicle during 7 days. The quinazoline S14 was synthesized 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. Proliferation and growth analysis was determined on these cultures and number and diameter of NS were scored using the Nikon Digital Sight, SD‐L1 software (Nikon, Tokyo, Japan, http://www.nikon.com/). Ten wells per condition tested and experiment were counted. Some of these NS were used for immunoblotting analysis. Remaining NS were then seeded onto poly‐l‐lysine (Sigma, Madrid, Spain, http://www.sigmaaldrich.com/spain/acerca.html) 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 Signaling Technology, Leiden, The Netherlands, https://www.cellsignal.com/) with protease inhibitor cocktail (Roche, Madrid, Spain, http://www.roche.com/index.htm) and incubated for 15‐30 minutes on ice. A total amount of 30 µg of protein was loaded on a 10% or 12% SDS‐PAGE gel and transferred nitrocellulose membranes (Protran, Whatman, GE Healthcare Life Sciences, Barcelona, Spain, http://www.gelifesciences.com/webapp/wcs/stores/servlet/Home/en/GELifeSciences-es/). The membranes were blocked in Tris‐buffered saline with 0.05% Tween‐20 and 5% skimmed milk, incubated with primary and secondary antibodies, and washed according to standard procedures. Primary antibodies were PDE7A (rabbit; Santa Cruz Biotech, Santa Cruz, California, http://www.scbt.com/), PDE7B (rabbit; Proteintech, Manchester, UK, http://www.ptglab.com/), p‐CREB (rabbit; Cell Signaling), CREB (rabbit; Cell Signaling, Danvers, MA, https://www.cellsignal.com/), Musashi1 (rabbit; Abcam, Cambridge, UK, http://www.abcam.com/), β‐III‐tubulin (mouse; Covance, Princetown, NY, http://www.covance.com/), MAP‐2 (mouse; Sigma), GFAP (mouse; Sigma, Madrid, Spain, http://www.sigmaaldrich.com/spain/acerca.html) and α‐tubulin (mouse; Sigma). Secondary peroxidase‐conjugated donkey anti‐rabbit (Amersham Biosciences, GE Healthcare Life Sciences, Barcelona, Spain, http://www.gelifesciences.com/webapp/wcs/stores/servlet/Home/en/GELifeSciences-es/), or rabbit anti‐mouse antibodies (Jackson Immunoresearch, Madrid, Spain, https://jireurope.com/) were used. Values in figures are the average of the quantification of at least three independent experiments corresponding to three different samples. Immunocytochemistry Fluorescence immunocytochemical analysis on differentiated NS was performed as previously described 49. Briefly, NS were incubated at 37°C for 1 hour with primary antibodies directed against β‐III‐tubulin (TuJ‐1 clone; rabbit; Abcam, Cambridge, UK, http://www.abcam.com/), MAP‐2 (mouse; Sigma, Madrid, Spain. http://www.sigmaaldrich.com/spain/acerca.html) and GFAP (mouse; Sigma, Madrid, Spain. http://www.sigmaaldrich.com/spain/acerca.html). 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, Madrid, Spain, http://www.thermofisher.com/es/es/home.html)) for 45 min at 37°C. Staining of nuclei was performed using 4′,6‐diamidino‐2‐phenylindole (DAPI). Finally, images were acquired in a LSM710 laser scanning spectral confocal microscope (Zeiss, Madrid, Spain, http://www.zeiss.com). Confocal microscope settings were adjusted to produce the optimum signal‐to‐noise ratio. PDE7 Inhibitor (S14) Administration In Vivo According with the S14‐treatment time, rats were divided in: (a) Short‐term groups, which received daily intragastrical 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 (Supporting Information Fig. 1A) and (b) 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 (Supporting Information Fig. 1B). For behavioral analysis, rats were first treated with S14 during 27 days and later on tested, as indicated below (Supporting Information Fig. 1C). S14 was administered 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 previously published works 34, 38. Control animals were given the same volume of vehicle. Immunohistochemistry Animals previously anaesthetized were perfused transcardially with 4% paraformaldehyde, and brains were obtained, postfixed in the same solution at 4°C overnight, cryoprotected, frozen, 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 described 48. Briefly, for BrdU detection, samples were first incubated with 2 M HCl for 30 minutes at 37° before blocking 1 hour in PBS containing 5% normal serum, 0.1 M lysine, and 0.1% Triton X‐100. Sections were then incubated with anti‐BrdU mouse monoclonal (DAKO, Barcelona, Spain, http://www.dako.com/) combined with anti‐nestin rabbit (Abcam, Cambridge, UK, http://www.abcam.com/) or anti‐NeuN rabbit (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 hour at room temperature. After rinses, sections were mounted with Vectashield. Images were obtained using a LSM710 laser scanning spectral confocal microscope (Zeiss, Madrid, Spain, http://www.zeiss.com). Confocal microscope settings were adjusted to produce the optimum signal‐to‐noise ratio. For doublecortin (DCX) detection floating sections were immersed in 3% H 2 O 2 to inactivate endogenous peroxidase, blocked for 2 hours at room temperature in 5% normal horse serum in PBS, containing 4% bovine serum albumin, 0.1 M lysine, and 0.1% Triton X‐100. Afterwards, the sections were incubated overnight with an anti‐DCX goat (Santa Cruz, CA, www.scbt.com) antibody. After several rinses, sections were incubated for 1 hour with the corresponding biotinylated secondary antibody and then processed following the avidin‐biotin protocol (ABC, Vectastain kit, Vector Labs, Burlingame, CA, https://vectorlabs.com/). The slides were examined with a Nikon eclipse 90i microscope, equipped with a DS‐Fi1 digital camera. Five animals from each experimental 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, Madrid, Spain, http://www.zeiss.com) 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 coexpression was defined by nuclear colocalization 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 colocalization 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 enhancements and color level adjustments were made; otherwise images were not digitally manipulated. Cell Count Analysis A modified stereological approach 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 immunohistochemistry) of the DG, SVZ and OB were viewed and captured under a x63 objective to avoid oversampling errors. From serial coronal sections (30 µm) from the entire 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 boundaries of these nervous system regions were determined with reference to internal anatomic landmarks 53. For each area of interest, images were analyzed using computer‐assisted image analysis software (Soft Imaging System Corporation, Lakewood, CO, http://www.soft-imaging.com). Positive cells, which intersected the uppermost 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 performed 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 minutes 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 minutes, returned to their home cages for 30 minutes (retention interval) and placed again in the same box with one familiar (sample) and one novel object (counterbalanced across rats) and given 3 minutes to explore the objects (test session). An experimenter blind to the treatment scored the time the rats spent exploring each object, the latency of first approach 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 contacts such as backing into the object 55. Spatial learning and memory tasks were performed in the Morris Water Maze 8 days after the last intragastrical administration of vehicle or S14 (day 1) as previously described in detail 56. From day 1 to 4 (learning curve) the animals were trained to find the hidden scape platform and on day 5 (probe trial) they were tested without platform. Animals were submitted to 4 trials every learning session, with a time limit of 60s/trial and an interval between trials of 4‐5 minutes. 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 1-3 were analyzed using a one‐way ANOVA and data from Figures 4-6A, 6B, 7 were analyzed by Student's t test. Data from Figures 6C, 6D were analyzed using a two‐way mixed ANOVA. After confirming the significance of the primary findings using ANOVA, a significance level of p <.05 was applied to all remaining post hoc statistical analyses. The SPSS statistical software package (version 20.0) for Windows (Chicago, IL) was used for all statistical analyses. Figure 1 Open in figure viewer PowerPoint PDE7 expression on neurosphere derived from the adult SGZ and SVZ. (A): Representative image of immunoblots for PDE7A and PDE7B in cultured SGZ‐ and SVZ‐derived neurospheres. (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 (nontreated cultures) is shown. Data were obtained from three independent experiments and presented as mean ± SD. ***, p ≤ .001 versus nontreated (basal) cultures. Abbreviations: CREB, cAMP response element binding protein; PDE7, phosphodiesterase 7; SGZ, subgranular zone; SVZ, subventricular zone. Figure 2 Open in figure viewer PowerPoint 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 experiments. *, p ≤ .05; **, p ≤.01; ***, p ≤ .001 versus nontreated (basal) cultures. Abbreviations: CREB, cAMP response element binding protein; PDE7, Phosphodiesterase 7; SGZ, subgranular zone; SVZ, subventricular zone. Figure 3 Open in figure viewer PowerPoint In vitro inhibition of PDE7 promotes stem cell differentiation toward a neuronal phenotype. Neural stem cells isolated from the adult SGZ and 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 differentiation 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): Representative Western blots of β‐tubulin, MAP‐2, and GFP. Quantification analyses are also shown. Results are the mean±SD from three independent experiments. **, p ≤ .01; ***, p ≤ .001 versus nontreated (basal) cultures. Abbreviations: CREB, cAMP response element binding protein; PDE7, Phosphodiesterase 7; SGZ, subgranular zone; SVZ, subventricular zone. Figure 4 Open in figure viewer PowerPoint Inhibition of PDE7 promotes in vivo activation of the neurogenic niche located in the 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 GCL. BrdU is shown in green, nestin in red and 4′,6‐diamidino‐2‐phenylindole, DAPI, blue) was used as a nuclear marker. Upper images show the maximum intensity projection. Scale bar = 50 μm. Quantification of the number of BrdU+/Nestin+ cells in the DG is shown. (B): 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 ≤ .01 versus vehicle‐treated animals. Abbreviations: BrdU, 5‐bromo‐2‐deoxyuridine; CREB, cAMP response element binding protein; DCX, doublecortin; DG, dentate gyrus; GCL, granule cell layer; PDE7, phosphodiesterase 7; SGZ, subgranular zone. Figure 5 Open in figure viewer PowerPoint Inhibition of PDE7 promotes in vivo activation of the neurogenic niche in the 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): 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 ≤ .001 versus vehicle‐treated animals. Abbreviations: BrdU, 5‐bromo‐2‐deoxyuridine; DCX, doublecortin; PDE7, phosphodiesterase 7; SVZ, subventricular zone. Figure 6 Open in figure viewer PowerPoint Inhibition of PDE7 promotes in vivo neurogenesis on the SGZ of the DG in the hippocampus and improves performance in learning tasks. (A) 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 experiments and 5 animals/experiment/experimental group. ***, p ≤ .001 versus vehicle‐treated animals. (B) Orthogonal projections showing the colocalization of BrdU (green) and neuN (red) cells in the dentate gyrus of the hippocampus of adult rats. Scale bar = 20 μm. Left images show 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 DG is shown. Values represent the mean ± SD from 3 different experiments and 5 animals/experiment/experimental group. **, p ≤ .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 ≤.05, **, p ≤ .01. Abbreviations: BrdU, 5‐bromo‐2‐deoxyuridine; DCX, doublecortin; DG, dentate gyrus; GCL, granule cell layer; PDE7, phosphodiesterase 7; SGZ, subgranular zone. Figure 7 Open in figure viewer PowerPoint Inhibition of PDE7 promotes in vivo neurogenesis on the OB. (A) Coronal sections of the OB immunostained with DCX showing migrating neuroblasts in the ependymal zone (E) and the 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 ≤ .001 versus vehicle‐treated animals. (B) Confocal imaging analysis showing the colocalization 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 ≤ .001 versus vehicle‐treated animals. Abbreviations: BrdU, 5‐bromo‐2‐deoxyuridine; DCX, doublecortin; GCL, granule cell layer; OB, olfactory bulb; PDE7, phosphodiesterase 7.

Results 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 neurogenic niches of the brain: the SGZ of the dentate gyrus of the hippocampus and the SVZ. Since PDE7 comprises two genes, PDE7A and PDE7B, both isoforms were analyzed by Western blot analysis (Fig. 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 (Fig. 1A). We next examined the effects of S14 and BRL50481 on the phosphorylation levels of the 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 (Fig. 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 (Fig. 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 vehicle‐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 μm in SVZ) or S14 (208 ± 11 µm in SGZ and 170 ± 16 μm in SVZ) treatments, as compared with nontreated 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 undifferentiation. The NS cultured under proliferative conditions were treated for 7 days with the PDE7 inhibitors. After that time proteins were isolated and Western blot performed. Our results show a significant decrease in SGZ‐ and SVZ‐derived NS of the amount of musashi‐1 protein when BRL50481 and S14 were added to the medium (Fig. 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 purpose, 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 (Fig. 3A, 3C) or the SVZ (Fig. 3B, 3D) in basal conditions. After treatment, the number of both Tuj1‐ and MAP‐2‐positive cells was significantly 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 differentiation of astroglial cells. Of note, the S14 compound was more potent than the commercially available PDE7 inhibitor BRL50481 promoting neurogenesis. These results suggest that PDE7 inhibition results in an induction of neuronal differentiation of neural stem cells toward 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 inhibition 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, followed by BrdU administration for 24h (Supporting Information Fig. 1A) or 27 days (Supporting Information Fig. 1B) before sacrifice. Coronal sections from short‐term treated‐animals were stained with specific anti‐Nestin, anti‐BrdU, and anti‐DCX antibodies (Figs. 4 and 5). Nestin is commonly used as a reliable biological marker of neural progenitor cells in vitro and in vivo 58-61. DCX is a microtubule‐associated protein, which is a valuable endogenous marker for dividing neuroblasts and immature neurons 62, 63. Orthogonal view of the subgranular zone of the hippocampus (Fig. 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 increases 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 (Fig. 5A). In addition, the number of DCX‐stained cells was also enhanced in the SVZ of these animals (Fig. 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 neuroblasts originated in the SGZ and the SVZ as a consequence 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 analyzed 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 (Fig. 6). Also, BrdU‐positive cells generated in the SVZ have already reached the olfactory bulb through the RMS, expressing a neuronal phenotype (Fig. 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 neuroblasts in the SGZ of the DG of the hippocampus (Fig. 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 object 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 observed 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 latency time of approach to the sample object (lower interest) in comparison with the controls. In the Morris Water Maze (Fig. 6D), a test generally considered specific for the hippocampus, our results showed an improved spatial memory in S14 treated rats. A significant difference was observed in the learning curve. However, 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 expended more time in target annulus indicating a better memory performance. Further studies may well examine whether animals with learning/memory impairments, which exhibit deficits in their learning curve, would take great benefit from PDE7 inhibition than control animals, which display normal curves of learning. As mentioned above, a significant number of migrating cells was observed in the SVZ of S14‐treated animals after 4 days of treatment (short‐term). These neuroblasts 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 (Fig. 7A). Coronal sections of the olfactory bulb also showed that these migrating neuroblasts have reached the OB and a high number of BrdU+ cells entering 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 differentiate into neurons, as can be observed by the increased number of new born neurons (BrdU+/NeuN+ cells) in the OB (Fig. 7B). Altogether, these observations clearly indicate that PDE7 inhibition in vivo increases the number of new neurons originated in the hippocampus and the olfactory 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 neuroprotection 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 neurogenesis 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, induces neurogenesis toward a dopaminergic phenotype in the substantia nigra pars compacta of adult rats lesioned with 6‐OHDA 47. In the present study we have extended these studies and show that the S14 compound also promotes proliferation and neuronal differentiation 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 primary neurospheres and by S14‐mediated induction of TuJ‐1+ and MAP‐2+ cells, and in vivo by an increased proliferation and a larger population of doublecortin expressing neuroblasts, which migrate to generate new neurons. 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 contain 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 markers 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 (specific transcription factors, epigenetic mechanisms, microRNAs, etc) 21, 65-69 and extracellular signaling cues (environmental, physiological, and pharmacological 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 increased 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 integrated 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 identification of a new target whose inhibition could help to promote neural stem cells proliferation and differentiation in the SGZ and SVZ, which can be relevant in olfactory‐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 agreement with previous works showing that adult hippocampal 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 phosphorylation after S14 treatment of neurospheres, suggest 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 CREB by phosphorylation. Consistent with our results, different studies in the mouse hippocampus, using pharmacological and genetic approaches to decrease 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, human emesis, which was found in clinical development of several PDE4 inhibitors, has endangered their development 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 neurogenic 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 strategy 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 neurogenesis in the substantia nigra pars compacta of 6‐OHDA‐lesioned animals toward a dopaminergic phenotype 47.

Conclusion We have previously shown that PDE7 inhibiton results in a potent neuroprotective and anti‐inflammatory effect in in vitro and in vivo neurodegenerative disorders models. In addition we also demonstrated that PDE7 inhibition induces endogenous neuroregenerative processes towards a dopaminergic phenotype. Here, we show for the first time a novel role for PDE7 inhibition in the process of adult neurogenesis. PDE7 inhibition directly regulates proliferation, migration and differentiation of neural stem cells, improving spatial learning and memory tasks. These findings provide new insights into the mechanism of neurogenesis regulation, suggesting that PDE7 inhibition may represent a novel and potential therapeutic strategy for stem cell activation through the use of small molecules, 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 Sanitaria; 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., and J.A.M.‐G.: Conception and design; J.A.M.‐G., S.A.‐G., M.S‐S., and V.E‐A.: Collection and/or assembly of data; C.G. and A.M.: Provision of Study Materials; J.A.M.‐G., A.P.‐C., A.S., V.E.‐A., J.A.L.‐M., C.G., and A.M.: Data analysis and interpretation; A.P.‐C., J.A.M.‐G., and 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., and A.P.‐C.: Final approval of manuscript.

Disclosure of Potential Conflicts of Interest The authors indicate no potential conflicts of interest.

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