NSCs were obtained from mice with a C57BL6 background. Mice were maintained in a 12-h light/dark cycle with free access to food and water ad libitum according to the Animal Care and Ethics Committee of the CSIC. Adult NSCs were isolated from 3-month-old mice after cervical dislocation. The brains were dissected out, and both SVZs from each hemisphere were extracted and cut into small fragments. The pieces were incubated with 0.025% Trypsin-EDTA (Gibco; Cat #25300054) for 30 min at 37°C. The tissue was then transferred to Dulbecco's modified Eagle's medium (DMEM)/F12 (1:1 v/v; Life Technologies, Cat #21331020) and carefully triturated with a fire-polished Pasteur pipette to a single cell suspension. Isolated cells were collected by centrifugation, resuspended in the NSC medium based on DMEM/F12 containing 2 mM Glutamax (Gibco; Cat #35050038), 1X B27 without vitamin A (Gibco; Cat #11500446), 2X antibiotic-antimycotic (Gibco; Cat #15240062), 2 μg/ml heparin (Sigma; Cat #H3393), supplemented with 20 ng/ml epidermal growth factor (EGF; Peprotech, Cat #AF-100-15), and 10 ng/ml fibroblast growth factor (FGF; Peprotech; cat# 100-18B), and maintained in a 95% air−5% CO₂ humidified atmosphere at 37°C (Bizy and Ferron, 2015; Belenguer et al., 2016). Neurospheres were allowed to develop for 7–10 days in these conditions. Each culture was generated using both SVZs from one adult mouse. Thus, each experimental point in the graphs represents the mean value of the replicates of a single independent animal. For culture expansion, primary neurospheres were disaggregated with Accutase (0.5 mM; Sigma; Cat #A6964) for 10 min at room temperature and washed with the NSC medium without mitogens to generate single-cell suspension. Then, 62.5 cells/μl were plated in the fresh mitogen-completed medium in a 95% air−5% CO₂ humidified atmosphere at 37°C and maintained for 6–7 passages maximum. In order to determine the self-renewal capacity of the NSCs, secondary neurospheres were disaggregated, NSCs were plated at low density (5 cells/μl) in the fresh mitogen-completed medium, and the number of neurospheres was counted 5 days later. In the self-renewal experiment, four replicates for each culture were used, and the average value was estimated. All these experiments were repeated four times with different cultures. Images of the neurospheres were taken using the PAULA Smart Cell Imager (Leica), and the diameters of the spheres were estimated by ImageJ.

To estimate proliferation, 62.5 cells/μl were plated after Accutase disaggregation in the fresh mitogen-completed medium in a 95% air−5% CO₂ humidified atmosphere at 37°C. After 3 days, neurospheres were plated onto cover glasses coated with 1X Matrigel (Corning, Cat #356234) for 15 min, allowing NSC attachment and fixed for staining with 2% paraformaldehyde (PFA) 0.1M phosphate buffer saline pH 7.4 (PBS) for 15 min at 37°C. For the differentiation assay, 80,000 cells/cm² were seeded in Matrigel-coated coverslips and incubated for 2 days (2 DIV) in the NSC culture medium without EGF. The medium was then changed with the fresh medium without FGF and supplemented with 2% fetal bovine serum (FBS; Gibco; Cat #10438-026) for 5 more days (7 DIV) to allow terminal differentiation. Cultures were fixed for staining at 7 days of differentiation with 2% PFA 0.1M PBS for 15 min at 37°C. The BDNF treatment was performed by incubating the NSCs with either 10 ng/ml (low concentration) or 50 ng/ml (high concentration) of Recombinant Human/Murine/Rat BDNF (PeproTech; Cat #450-02) since the single cell suspension is plated. When indicated, NSC cultures were treated with 10 μM ANA-12 (MedChemExpress; Cat #HY-12497) (hereafter referred to as TrkB-i) or 10 μM THX-B (MedChemExpress; Cat #HY-137322) (hereafter referred to as p75-i) at the time of plating to inhibit TrkB or p75^NTR, respectively. The specificity and selectivity of both antagonists have been previously evaluated (Bai et al., 2010; Cazorla et al., 2011). Control cultures were exposed to 1:1,000 of DMSO (Sigma; Cat.# D5879). In both proliferation and differentiation assays, 10 random images were taken with ~400 cells analyzed for each culture. These experiments were performed four times with independent cultures.

For immunocytochemical staining, fixed cells were permeabilized and blocked with PBS 0.2% Triton X-100 (Sigma; Cat.#X100) containing 10% normal goat serum and 1% glycine (Thermo Scientific; Cat #A13816.36) for 1 h at RT, incubated with primary antibodies, and prepared in the same blocking solution overnight at 4°C. Cells were washed three times with PBS 1X and incubated with secondary antibodies for 1 h at RT. Primary and secondary antibodies and dilutions used are listed in Tables 1, 2, respectively. DAPI (1 μg/ml) was used to counterstain DNA. The samples were washed three times with PBS 1X and mounted with the ImmunoSelect antifading mounting medium (Dianova; Cat #038447). Images were acquired at 20x or 40x magnification with a Leica SP5 confocal microscope. For fluorescence intensity quantification, maximal projection images were generated, and the mean gray intensities of p-TrkB, TrkB, and p75^NTR were measured with ImageJ/Fiji software and recorded as arbitrary fluorescence units (a.u.). p-TrkB data were normalized to TrkB intensity.

Protein detection by Western blot was performed after protein extraction using 20 mM Tris pH 6.8 (Sigma; Cat.#10708976001) containing 1% Triton X-100 (Sigma), 0.5% sodium dodecyl sulfate (SDS) (Sigma; Cat.#L4509), 1 mM ethylene-dinitrilotetraacetic acid (EDTA) (Merck; Cat.#1.08418.0250), 10 mM β-mercaptoethanol (Sigma; Cat.#M-7154), and 1× cOmplete Mini, EDTA-free protease inhibitor (Roche; Cat.#11836170001). Total protein amount was determined by the Bradford Assay (BioRad; Cat.#500-0006). Proteins were denatured by heat (5 min at 100°C) and loaded in 4–20% precast polyacrylamide gels (BioRad; Cat.#4561095). Proteins were transferred to polyvinylidene difluoride (PVDF) membranes (Merck; Cat.#IPFL00010) and immunoblots were carried out with primary antibodies (Table 1), incubated overnight, and secondary antibodies (Table 2) during 1 h. Antibodies were diluted in Intercept® Blocking Buffer (LI-COR; Cat.#927-60001). After antibodies incubation, the membranes were washed with Tris-buffered saline (TBS) containing 0.1% Tween 20 (Sigma; Cat.#P1379). Finally, protein bands were visualized using the Odyssey CLx Infrared Imaging System (LI-COR).

RNAs were extracted with the RNAeasy mini kit (Qiagen; Cat.# 74104) including DNase treatment, following the manufacturer's guidelines. For quantitative PCR (qPCR), 1 μg of total RNA was reverse transcribed using random primers and SuperScript IV Reverse Transcriptase (ThermoFisher Scientific; Cat# 15317696), following standard procedures. Thermocycling was performed in a final volume of 15 μl, containing 1 μl of cDNA sample (diluted 1:7), and the reverse transcribed RNA was amplified by PCR with appropriate primers from PrimePCR SYBR Green Assay (Cultek; Cat. PB20.11) (see Table 3). qPCR was used to measure gene expression levels normalized to Rpl27, the expression of which did not differ between the groups. qPCR reactions were performed in a 7500 real-time PCR equipment (Applied Biosystems). Raw data from this analysis is shown in Supplementary Table 1.

All statistical tests were performed using the GraphPad Prism Software, version 7.00 for Windows. The significance of the differences between groups was evaluated by the two-tailed paired Student t-test or one-way ANOVA followed by a Tukey post-hoc test. The presence of outlier values was evaluated by Grubb's test. A p-value of < 0.05 was considered statistically significant. Data are presented as the mean ± standard error of the mean (SEM) and the number of independent cultures (n), and p-values are indicated in the figures.

BDNF has proved to act as a pro-neurogenic factor promoting the proliferation and differentiation of NSCs (Lee et al., 2002; Islam et al., 2009; Chen et al., 2013; Liu et al., 2014; Langhnoja et al., 2021). BDNF activity is mediated by high-affinity binding to the TrkB receptor (Naylor et al., 2002), and this neurotrophic factor is able to interact with low-affinity to p75^NTR (Rodriguez-Tebar et al., 1990). Both receptors are expressed in the adult NSCs (Young et al., 2007; Islam et al., 2009; Bath et al., 2012; Faigle and Song, 2013; Vilar and Mira, 2016). A clear positive role for the TrkB pathway has been described in the function of BDNF on the embryonic or P0 NSC proliferation (Islam et al., 2009; Chen et al., 2013), and the proliferative role of p75^NTR in the NSCs located in the adult SVZ (Young et al., 2007) remains to be established. To understand the mechanism behind BDNF's effects on the neurogenic population, adult NSCs were treated with two different doses of this neurotrophic factor (10 and 50 ng/ml). We chose these concentrations as the former mainly activates TrkB, while the latter also activates p75^NTR since the K_d of the interaction of BDNF with p75^NTR is approximately 10⁻⁹ M (~25 ng/ml) (Rodriguez-Tebar et al., 1990). First, self-renewal capacity was tested by determining the number of neurospheres after 5 days of NSCs cultured at low density with low (10 ng/ml) or high (50 ng/ml) concentrations of BDNF (Figure 1A). The presence of BDNF at 10 ng/ml in the NSC cultures significantly increased the number of neurospheres compared to untreated cultures, being this effect potentiated by the addition of BDNF at 50 ng/ml (Figure 1A). This suggests that p75^NTR facilitates NSC self-renewal. Moreover, the diameter of these neurospheres was significantly higher in BDNF-treated NSCs (Figure 1B), suggesting an enhancement of NSC proliferation capacity. Both exposures to 10 and 50 ng/ml of BDNF showed a significant increment in the diameter of the neurospheres compared with untreated cultures, whereas no differences were detected between both concentrations of BDNF (Figure 1B). The proliferative capacity of adult NSCs was analyzed by measuring the percentage of positive cells for the cell cycle marker Ki67 (Figure 1C). Both concentrations of BDNF showed a significant increase in the proliferation ratio compared with untreated NSCs. Again, no differences in the percentage of Ki67+ cells were detected between 10 and 50 ng/ml treated cultures (Figure 1C), indicating that the lowest concentration of the neurotrophic factor was sufficient to activate the proliferation pathway.

Several studies have shown that BDNF exerts a positive effect on the differentiation of NSCs into neurons (Chen et al., 2013; Liu et al., 2014) and oligodendrocytes (Chen et al., 2013; Langhnoja et al., 2021). In accordance, the mRNA levels of relevant differentiation markers were analyzed by qPCR in cDNAs obtained from adult NSCs. This analysis indicated that the expression of the neuronal marker Dcx showed a tendency to increase and the oligodendrocyte marker Olig2 was significantly upregulated in the NSCs treated with 50 ng/ml of BDNF, suggesting that treatment with a high dose of BDNF predisposes NSCs toward a more committed state. Instead, the presence of 10 ng/ml of BDNF in the medium was not sufficient to increase the levels of mRNA of these lineage genes (Figure 2A). Neither the expression of the mRNA encoding the astrocytic marker S100β (S100b) nor the neural precursor gene Nestin (Nes) showed differences between untreated and BDNF-treated NSCs (Figures 2A, B). To test if the upregulation of the neuronal and oligodendrocytic genes in the adult NSCs after 50 ng/ml BDNF treatment drove an increment in the percentage of neurons and oligodendrocytes in differentiating conditions, the number of TUJ1+, O4+, and GFAP+ cells, representing neurons, oligodendrocytes, and astrocytes, respectively, were estimated after seven DIV in NSCs maintained in differentiation conditions. The percentage of neurons and oligodendrocytes were increased in the 50 ng/ml BDNF treated cultures, at the expense of astrocyte generation, which decreased in this condition compared with untreated cells (Figures 2C–F). Moreover, the treatment with the low dose of BDNF (10 ng/ml) did not alter the differentiation capacity of adult NSCs regarding untreated cultures (Figures 2C–F), thus requiring a higher concentration of BDNF to activate the differentiation pathway. These data, together with those from the proliferation analysis shown above, suggest different mechanisms for BDNF to promote proliferation or differentiation in a dose-dependent manner.

The previous results of proliferation and differentiation of the adult NSCs in the presence of low or high doses of BDNF could be explained by the use of different signaling mechanisms to activate each cellular process. Precisely, BDNF presents high-affinity binding to TrkB (Naylor et al., 2002) and low-affinity binding to p75^NTR (Rodriguez-Tebar et al., 1990), two receptors that are expressed by NSCs, showing a dynamic pattern of expression during proliferation and differentiation of these cells (Figure 3A). To understand if the different cellular response of BDNF in a dose-dependent manner could be due to the intervention of different receptors/pathways, adult NSCs were treated with 10 or 50 ng/ml of BDNF, and the gene expression of both receptors, Ntrk2 (TrkB) and Ngfr (p75^NTR), was measured by qPCR (Figures 3B, C). To this aim, BDNF was added after neurosphere disaggregation, and the expression of these receptors was analyzed in the newly formed neurospheres after 5 days in the presence of the neurotrophin. The Ntrk2 gene encodes three receptor isoforms generated by alternative splicing, the full-length isoform (TrkB FL), and two truncated versions of the protein lacking the kinase domain, with TrkB.T1 being the most expressed in the NSCs from the SVZ (Islam et al., 2009; Vilar and Mira, 2016). Thus, the expression of the transcripts encoding both TrkB FL and TrkB.T1 (TrkB FL and TrkB.T1, respectively) was analyzed in adult NSCs grown in the absence or presence of 10 or 50 ng/ml BDNF (Figure 3B). The presence of BDNF in the culture medium resulted in a significant increment of both TrkB FL and TrkB.T1 expressions, regardless of the BDNF concentration (Figure 3B), suggesting that its expression is regulated by the activation of TrkB. As previously shown (Islam et al., 2009), the expression of TrkB.T1 was higher than that of TrkB FL (Figure 3B). In contrast to its mRNA levels, the expression of the TrkB protein using an antibody recognizing the extracellular domain (i.e., recognizing all TrkB isoforms) was not observed to show an increased response to BDNF (Figure 3D), suggesting that post-transcriptional mechanisms regulate TrkB protein expression. As expected, exposure of neurospheres to BDNF resulted in the increase of TrkB phosphorylation in Y516 (Figure 3D), a residue that becomes phosphorylated upon TrkB activation (Mazzaro et al., 2016). This activation of TrkB signaling in NSCs confirms previous published data suggesting TrkB activation in NSCs (Chen et al., 2017). Moreover, the application of the selective TrkB antagonist ANA-12 (TrkB-i) (Cazorla et al., 2011) to neurospheres treated with 10 ng/ml BDNF resulted in the reduction of Y516 TrkB phosphorylation to basal levels (Figure 3D). In contrast to TrkB FL and TrkB.T1 expressions, the expression of Ngfr was significantly upregulated in the NSC cultures only after high-dose exposure to BDNF (Figure 3C), indicating that the presence of high levels of BDNF promotes the activation of a signaling pathway resulting in the expression of Ngfr. The requirement for the dose of BDNF suggests that the upregulation of p75^NTR is modulated by its own activation. To confirm this hypothesis, the expression of Ngfr was measured in NSCs treated with 50 ng/ml BDNF in the presence of TrkB-i or the selective p75^NTR antagonist THX-B (Bai et al., 2010) (p75-i) (Figure 3E). The presence of TrkB-i did not change the Ngfr mRNA levels when NSCs were treated with 50 ng/ml of BDNF, and the expression of Ngfr was not upregulated after 50 ng/ml BDNF treatment in the presence of p75-i (Figure 3E), showing that the increment in the expression of the p75^NTR receptor was regulated by the interaction of BDNF with this receptor. The increment in Ngfr mRNA at 50 ng/ml of BDNF treatment was confirmed at the protein level by Western blot (Figure 3F) and immunocytochemistry (Figure 3G), using a previously characterized antibody (Huber and Chao, 1995).

The expression data of TrkB and p75^NTR in proliferating and differentiating conditions suggest that both receptors are involved in NSC behavior. To determine the implications of TrkB and p75^NTR in these processes, NSCs were treated with TrkB-i and p75-i, respectively (Figure 4A). NSCs were cultured at low density to evaluate self-renewal capacity in the absence or presence of 10 or 50 ng/ml of BDNF as above, using 10 μM of TrkB-i or 10 μM of p75-i to inhibit TrkB or p75^NTR specifically (Figure 4B). Control NSCs were treated with DMSO. The presence of TrkB-i in the medium revealed that the TrkB pathway is essential for NSCs to self-renew, independently of the presence of exogenous BDNF, a finding consistent with the expression of Bdnf by the adult NSCs (Figure 4C). Blocking this receptor significantly decreased the number of neurospheres in 0, 10, and 50 ng/ml of BDNF treatments (Figure 4B). These data were consistent with previous results showing a decrease of newly born neurons in the OB of TrkB heterozygous mice (Bath et al., 2008). In contrast, treatment of NSCs with p75-i in the absence of BDNF showed no effect on the self-renewal capacity of the NSCs (Figure 4B). The presence of 10 ng/ml of BDNF jointly with this antagonist did not alter this ability either (Figure 4B) indicating that lower concentrations of BDNF act through the TrkB pathway. However, treatment with 50 ng/ml of BDNF in the presence of the p75^NTR antagonist resulted in a decrease in the number of neurospheres (Figure 4B), indicating that the higher concentration of BDNF activated a TrkB/p75^NTR-dependent pathway that becomes necessary to control NSC self-renewal. Previous studies demonstrated that TrkA formed complexes with p75^NTR, increasing the affinity and selectivity of NGF binding (Hempstead et al., 1991). Another study showed that BDNF induces TrkB association with p75^NTR in embryonic hippocampal neurons after TrkB activation (Zanin et al., 2019). Importantly, this latter study demonstrated that p75^NTR is necessary for optimal TrkB signaling and function through the PI3K pathway in embryonic neurons (Zanin et al., 2019). In contrast to these studies, where p75^NTR optimizes the signaling capacity of the Trk family receptors, our observation suggests that a novel functional interaction between p75^NTR and TrkB exists in the adult NSCs as the blockade of p75^NTR prevents TrkB function.

The proliferation capacity of adult NSCs was also analyzed in the presence of the receptor antagonists (Figure 4D). NSCs were plated in proliferation-promoting conditions and treated with different doses of BDNF. The percentage of proliferating cells was determined by the number of Ki67+ cells. The treatment with either antagonist in the absence of exogenous BDNF showed no alterations in the percentage of proliferative NSCs (Figure 4D). The presence of the TrkB-i in NSCs treated with low or high concentrations of BDNF prevented the increase in the percentage of Ki67+ cells induced by this neurotrophin, reaching the untreated culture levels (Figure 4D). However, the presence of the p75-i decreased the Ki67 percentage to untreated culture levels only in NSCs treated with 50 ng/ml BDNF (Figure 4D), demonstrating activation of the p75^NTR pathway when BDNF levels are high, leading to increased proliferation.

Since BDNF-mediated differentiation requires high levels of BDNF (Figures 2C–F), we investigated whether the p75^NTR activation observed under proliferative conditions was also required to achieve terminal differentiation of adult NSCs. Thus, NSCs were differentiated in the presence of a high concentration of BDNF and either of the antagonists TrkB-i and p75-i (Figure 4E). In the absence of BDNF, no alterations were detected in the percentage of neurons, oligodendrocytes, and astrocytes after the receptor blockage (Figure 4E). The differentiation of NSCs with 50 ng/ml of BDNF increased the percentage of neurons and oligodendrocytes at the expense of astrocytes, as previously demonstrated. However, only p75^NTR inhibition with p75-i was able to rescue the proportion of oligodendrocytes observed in the control cultures with statistical significance (Figure 4E). No statistically significant alterations were observed in the percentage of astrocytes and neurons with the TrkB-i and p75-i antagonists. However, a decrease in the proportion of oligodendrocytes with TrkB-i antagonist in 50 ng/ml BDNF-treated cultures was detected, not reaching statistical significance (Figure 4E).