The generation of new cells in the human adult brain is still today a matter of intense debate (2, 5, 58). If this aspect remains contested, establishing the importance of newly born cells in overall brain networks and function is an even bigger challenge. In this context, our group and others have demonstrated that a defined group of cells (neural stem cells) continuously proliferate within restricted brain regions in adult rats (59, 60), and when those cells are specifically targeted, several behavioral domains are affected (37). However, most of the studies have been focused on the generation of new neurons, neglecting the relevance of newly born glial cells. In fact, just considering the higher proportion of glial cells in the mammalian brain, it is logical to foresee a critical role for newly generated glia. Furthermore, glial cells are involved with neurons in the arrangement of molecular signals that regulate the function of neuronal networks in the developing brain, possessing a fundamental role in regulating brain homeostasis. In this aspect, there is limited knowledge about the specific contribution of newly born glia to brain function, as well as a lack of information on their potential role in the development of new therapeutic routes for brain disorders. Hence, in this work, we aimed to specifically boost gliogenesis by introducing a population of glial progenitors (GRPs) into one of the most important cytogenic niches in the adult brain, the hippocampal DG, in a model in which the capacity of generating new cells was transiently compromised (37, 38)By doing so, we would be specifically increasing the generation of new glial cells, dissecting their role from the contribution of newly born neurons. This rationale was established based on recently published results (37), where we demonstrated that, following GCV administration, the GFAP-tk rat model presented a stable depletion of newly born cells for about two and half-weeks. Therefore, by transplanting GRPs or injecting their secreted factors within that time window, we assured a major contribution from transplanted glial progenitor cells (excluding the role of endogenous newborn cells).
The treatments were administered in the dDG and injections were confirmed to be given in the appropriate coordinates (Fig. 1E), as GRPs were mainly concentrated in the dDG (no cells were found in vDG sections), and no major cell migration patterns were observed. Additionally, we have not only confirmed that the transplanted cells survived (at least 40 days post-grafting), but they also proliferated and differentiated into glial-derived cells. These results go along with work from Hattiangady and colleagues in which GRPs transplanted into the hippocampus of aged animals remained clustered at the site of grafting, with minimal migration from the transplant core, and readily differentiated into astrocytes and oligodendrocytes (61). Then, regarding the induction of the GFAP-tk model, we have confirmed a significant suppression of BrdU + cells in the DG, which resulted in long-term pronounced anxiety- and depressive-like behaviors in transgenic animals. Interestingly, we have shown that a one-time transplantation of GRPs was capable of reverting not only these behavioral deficits, but also neurogenic and gliogenic levels, in particular in the vDG. Once again, these results are similar to the ones observed by Hattiangady and colleagues, in which GRP transplants elicited a boost of endogenous neurogenesis in aged WT rats (61). In our study, the administration of factors secreted by GRPs (8h conditioning protocol) was also sufficient to revert anxiety-like behaviors, but not depressive-like ones. Importantly, secretome-treated animals did not present significant alterations in neurogenesis or gliogenesis in comparison to GFAP-tk sham rats. These results indicate that transplanted glial progenitors boosted cytogenic levels in the vDG, and these new cells might have had a pivotal role in preventing the behavioral deficits characteristic of this animal model.
It is relevant to highlight that in this work, the GFAP-tk rat model presented depressive-like impairments, which contrasts with our previous findings using the same model (37). However, it should be stressed that in our previous results depressive-like behaviors were assessed at four weeks post-cytogenesis abrogation, while here the same tests (FST) were conducted between six-to-seven weeks after ending GCV treatment, which could account for the differences observed. In fact, Snyder et al. (62) have already shown that GFAP-tk mice present increased immobility times in the FST, indicative of a depressive-like phenotype. Apart from the animal model chosen, these mice were assessed 12 weeks post-cytogenesis ablation, once again accounting time as a decisive factor in the establishment of specific emotional deficits. Curiously, and as already mentioned, GRPs were transplanted into the dDG, with no APP + cells detected in vDG sections, suggesting that these two regions effectively communicate and are able to modulate each other. Traditionally, these regions have been regarded as functionally distinct, with the dorsal hippocampus being more associated with both learning and memory tasks (63–66), while the ventral hippocampus revealed to be a key structure of the emotional brain, responsible for regulating affective behaviors and modulating anxiety states (67–69). However, it has already been shown that both regions are in fact interdependent during spatial navigation tasks (64) or in novelty-induced contextual memory formation (70), corroborating the anatomical connections between them, which was already described decades ago (71–73). Hence, further studies are needed to understand this possible connection in the context of emotional behaviors, such as the ones affected in this work. Nevertheless, herein we have analyzed both regions individually, trying to dissect the impact of each treatment.
When analyzing all differentially expressed proteins, the pathways enriched in the dDG and vDG were very similar between GRP-treated and secretome-treated animals, with higher significance in the GRP-treated group, even though there are different players involved. No major differences were seen in the main clusters of biological processes (Fig. S2), with all regions analyzed (from both treatments) presenting at least one cluster related to neural processes, either neuronal communication or neurogenesis, among others. However, taking into consideration the proteins that were uniquely altered in each treatment, there is a clear difference in the profile of enriched proteins (and associated pathways) in the dDG. Furthermore, there is also a restricted group of proteins that were differentially expressed between GRP-treated and secretome-treated groups. For instance, neuroligin-2, which is enriched in the dDG of GRP-treated rats, is a transmembrane scaffolding protein involved in cell-cell communication via interaction with neurexin family members. In fact, several studies have already demonstrated the importance of this family of proteins for inhibitory synapse formation and function (74, 75), and the specific deletion of neuroligin-2 has resulted in anxiety-like behavior in mice (76, 77). This fits our data, where GRP-treated animals present higher levels of neuroligin-2 expression and, simultaneously, a recovery of the anxiety-like phenotype. Another protein specifically enriched in GRP-treated animals was the aryl hydrocarbon receptor-interacting protein, which was enhanced in the vDG. The aryl hydrocarbon receptor (AhR) is known to be present in nestin-expressing neural progenitor cells (78), and its experimental deletion adversely impacts neurogenesis (79). AhR-deficient mice have reduced cell birth, neuronal differentiation, and fewer mature neurons in the DG (79). Hence, its increased expression is logical, considering the augmented neurogenic and gliogenic levels registered in the vDG of GRP-treated rats. More recently, AhR has also been implicated in the regulation of granule neuron morphology and synaptic maturation (80). Finally, there was a group of proteins whose expression was enriched in both GRP and secretome groups compared to GFAP-tk sham animals. The expression levels of these proteins were not statistically different between GRP-treated and secretome-treated animals; however, this finding does not necessarily mean that biological significance could be disregarded. Notably, GRP-treated rats presented higher levels of the protein S100β in the vDG. Primarily expressed in astrocytes, it is logical that its expression is increased in regions with higher gliogenic levels. Moreover, and knowing that astrocytes can secrete S100β, it has been demonstrated that its intraventricular infusion enhanced hippocampal neurogenesis following traumatic brain injury (81). Another protein increased in the vDG was the heat shock protein HSP105, whose increased expression was previously associated with augmented hippocampal cell proliferation and recovery of depressive-like behavior in mice (82). Overall, some of these highlighted proteins might have played a role in the effects observed following GRP transplantation.
Nevertheless, the injection of the factors secreted by GRPs also resulted in the reversion of anxiety-related deficits in GFAP-tk rats. An unbiased reactome analysis focused on GRP secretome protein content revealed a myriad of different enriched pathways (Fig. S3A), from which positive regulation of neuron projection development and regulation of synapse organization can be highlighted. Furthermore, a total of 118 proteins related to neuronal development processes, neurogenesis and gliogenesis, regulation and behavioral processes were identified, possibly explaining the results observed (Fig. S3B). In fact, GRP secretome presented pro-cytogenic proteins such as nestin (83), fatty acid binding protein 7 (fabp7) (84) and brevican core protein (bcan) (85). In addition, other proteins associated with neuronal maturation, growth-associated protein 43 (gap43) (86) and neurocan core protein (cspg3) (87) were found. Proteins related to glial structure (GFAP) (88) and function [myelin-associated glycoprotein (Mag)] (89) were also detected in the proteomic analysis, which might be involved in gliogenesis. Considering this protein content, a protocol of repeated secretome injections, potentially through alternative routes of administration, would be interesting to study in future experiments.
The importance of newly born cells in health and disease is still far from being fully understood. To the best of our knowledge, this is one of the first studies to demonstrate that boosting levels of glial progenitors in vivo results in a faster recovery of cytogenic impairments in a transgenic rat model, thereby avoiding associated emotional deficits (anxiety and depressive-like behaviors). Transplanted cells elicited a cascade of events that resulted in increased cytogenic levels in the vDG, based on a mechanistic component that is yet to be identified. Further studies should aim to determine the exact role of transplanted glial progenitors and how they affect the communication between brain regions, such as the dorsal-ventral axis of the hippocampus. The genesis of mood disorders is indeed a result of delicate alterations in orchestrated brain neuronal and glial networks, to which more insights about the role of new neurons and new glia are of the utmost importance.