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CB2ExStress: a neurogenic strategy to tackle mood disorders
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Catalysing brain plasticity through adult neural stem cell modulation : the role of cannabinoids and neurotrophic factors
Publication . Rodrigues, Rui Miguel Silva; Xapelli, Sara Alves; Fitzsimons, Carlos; Sebastião, Ana Maria Ferreira
The adult mammalian brain is endowed with a considerable amount of regenerative potential through the generation of new cells, particularly neurons, throughout adulthood from adult neural stem cells (NSCs). This adult neurogenesis phenomenon mainly operates in two regions – the neurogenic niches – in the subventricular zone (SVZ) of the lateral ventricles and in the subgranular zone (SGZ) of the hippocampal dentate gyrus (DG). In the latter, in a process of adult hippocampal neurogenesis (AHN), adultborn neurons participate in hippocampal-dependent functions such as cognitive processing and emotional flexibility by promoting circuitry remodelling and endogenous brain plasticity. This process is tightly regulated by several factors present in the surrounding extracellular milieu that shape the dynamics of NSCs and progenitors. Among these, neurotrophic factors are key molecules that support neural growth and survival and contribute to NSC regulation. On the other hand, cannabinoids are known modulators of homeostasis, neuroplasticity and proliferation, differentiation and maturation of NSCs and progenitor cells. Nevertheless, there is still a lack of knowledge about the mechanisms by which these molecules control adult neurogenesis and NSC activity.
This work aimed to evaluate and dissect the individual and combined actions of both neurotrophic factors and cannabinoids in the regulation of adult NSC dynamics in physiological and pathological contexts.
In the first study of this thesis (chapter 4), we show that cannabinoid type 1 (CB1R) and type 2 (CB2R) receptors and brain-derived neurotrophic factor (BDNF) are key players responsible for fine-tuning early SVZ and DG postnatal neurogenesis. Furthermore, we demonstrate that BDNF is essential for cannabinoid-mediated neurogenesis. Importantly, in DG neurogenesis, we show that BDNF-mediated actions in cell proliferation are dependent on CB2Rs while CB2R-mediated effects in neuronal differentiation require endogenous BDNF, depicting an interdependence between these two systems.
These findings led us to investigate the actions of other exercise-associated neurotrophic factors, namely vascular endothelial growth factor (VEGF) and insulin-like growth factor 1 (IGF-1) as well as the role of CB2Rs in the regulation of DG postnatal neurogenesis. In the second study of this thesis (chapter 5), we found that a cocktail of these neurotrophic factors, composed of BDNF+VEGF+IGF-1, significantly enhanced DG cell proliferation, an effect that was partially abrogated by CB2R blockage. Moreover, the actions of this exercise-mimicking cocktail in early neuronal commitment, but not late neuronal differentiation, were abrogated by CB2R blockage, suggesting that CB2Rs have a determinant role in the regulation of early stages of DG neurogenesis.
Taking this into consideration, and aiming at a translational rationale with a putative stress-countering strategy, in a third study (chapter 7) we evaluated the combined actions of CB2Rs together with physical exercise (PE), which is a known inductor of neurotrophic factor upregulation, in the context of depression. For this, we have submitted adult mice to an unpredictable chronic mild stress (uCMS) protocol to induce core symptoms of depressive-like behaviour. At the end of uCMS, animals were submitted to a PE regimen and treatment with CB2R modulators, independently or in combination with PE, for 2 weeks. We found that CB2R blockage, but not CB2R activation, together with PE treatment significantly ameliorated chronic stress-evoked emotional changes (anxious- and anhedonic-like behaviours) as well as cognitive deficits (long-term memory). Importantly, we show that this combined strategy critically influences stress-induced changes in AHN dynamics, leading to a significant increase in the rates of proliferation and differentiation of newborn neurons. Moreover, CB2R blockage together with PE reduced the overall neuroinflammatory tone, counteracting the deleterious effects exerted by chronic stress. This reveals a synergistic effect of CB2R blockage and PE treatment that is critical to counter the detrimental effects of chronic stress and sheds light on the potential of multitargeted approaches for stressrelated pathologies.
Altogether, the data herein revealed a novel layer of interaction between different adult NSC modulators, namely neurotrophic factors and cannabinoids, showing for the first time how CB2Rs fine-tune adult neurogenesis in collaboration with exercise-associated neurotrophic factors. These results also contribute to a better understanding of NSC dynamics and the importance of these neurogenic modulators in physiological and pathological contexts. Finally, this work provides new insights on the potential of CB2Rs and PE as promising targets for the development of brain repair and regeneration strategies for stress-related disorders such as depression.
Brain-derived neurotrophic factor (BDNF) role in cannabinoid-mediated neurogenesis
Publication . Ferreira, Filipa Fiel; Ribeiro, Filipa; Rodrigues, Rui S.; Sebastião, Ana M; Xapelli, Sara
The adult mammalian brain can produce new neurons in a process called adult neurogenesis, which occurs mainly in the subventricular zone (SVZ) and in the hippocampal dentate gyrus (DG). Brain-derived neurotrophic factor (BDNF) signaling and cannabinoid type 1 and 2 receptors (CB1R and CB2R) have been shown to independently modulate neurogenesis, but how they may interact is unknown. We now used SVZ and DG neurosphere cultures from early (P1-3) postnatal rats to study the CB1R and CB2R crosstalk with BDNF in modulating neurogenesis. BDNF promoted an increase in SVZ and DG stemness and cell proliferation, an effect blocked by a CB2R selective antagonist. CB2R selective activation promoted an increase in DG multipotency, which was inhibited by the presence of a BDNF scavenger. CB1R activation induced an increase in SVZ and DG cell proliferation, being both effects dependent on BDNF. Furthermore, SVZ and DG neuronal differentiation was facilitated by CB1R and/or CB2R activation and this effect was blocked by sequestering endogenous BDNF. Conversely, BDNF promoted neuronal differentiation, an effect abrogated in SVZ cells by CB1R or CB2R blockade while in DG cells was inhibited by CB2R blockade. We conclude that endogenous BDNF is crucial for the cannabinoid-mediated effects on SVZ and DG neurogenesis. On the other hand, cannabinoid receptor signaling is also determinant for BDNF actions upon neurogenesis. These findings provide support for an interaction between BDNF and endocannabinoid signaling to control neurogenesis at distinct levels, further contributing to highlight novel mechanisms in the emerging field of brain repair.
The neurosphere assay : an effective in vitro technique to study neural stem cells
Publication . Xapelli, Sara; Soares, Rita; Ribeiro, Filipa; Lourenço, Diogo M.; Rodrigues, Rui S.; Moreira, João B.; Sebastião, Ana M.; Morais, Vanessa A.
Neural stem cells (NSCs) are known to be present in the adult mammalian brain where they constitutively differentiate into the neuronal, astroglial, and oligodendroglial lineages, in defined processes termed n e u ro ge n e s i s , a st ro g l i o ge n e s i s a n d oligodendrogenesis, respectively (reviewed in Braun and Jessberger, 2014). During brain development, NSCs are present throughout the brain, becoming progressively restricted to defined brain regions. In the adult brain, NSCs are mainly present in areas classically known as neurogenic niches, i.e. the subventricular zone (SVZ), along the lateral walls of the lateral ventricles, and the subgranular zone, located in the dentate gyrus (DG) of the hippocampus. These areas are particularly enriched with NSCs, which not only are multipotent cells but also proliferative cells with the ability to selfrenew, thus maintaining their own pool of cells. In fact, neurogenesis, astrogliogenesis and oligodendrogenesis are highly intricate processes comprising several steps, including proliferation, differentiation, migration, and functional integration of the newly formed cells in the existing circuitry, which are regulated by a plethora of factors. These newly differentiated adult-born cells have the capacity to continuously modulate brain function and plasticity, by constantly reacting to external or internal stimuli (reviewed in Braun and Jessberger, 2014).
Sustained hippocampal neural plasticity questions the reproducibility of an amyloid-β-induced Alzheimer’s disease model
Publication . Paulo, Sara L; Ribeiro Rodrigues, Leonor; Rodrigues, Rui S.; Mateus, Joana; Fonseca-Gomes, João; Soares, Rita; Diógenes, Maria José; Solá, Susana; Sebastião, Ana M; Ribeiro, Filipa; Xapelli, Sara
Background: The use of Alzheimer's disease (AD) models obtained by intracerebral infusion of amyloid-β (Aβ) has been increasingly reported in recent years. Nonetheless, these models may present important challenges.
Objective: We have focused on canonical mechanisms of hippocampal-related neural plasticity to characterize a rat model obtained by an intracerebroventricular (icv) injection of soluble amyloid-β42 (Aβ42).
Methods: Animal behavior was evaluated in the elevated plus maze, Y-Maze spontaneous or forced alternation, Morris water maze, and open field, starting 2 weeks post-Aβ42 infusion. Hippocampal neurogenesis was assessed 3 weeks after Aβ42 injection. Aβ deposition, tropomyosin receptor kinase B levels, and neuroinflammation were appraised at 3 and 14 days post-Aβ42 administration.
Results: We found that immature neuronal dendritic morphology was abnormally enhanced, but proliferation and neuronal differentiation in the dentate gyrus was conserved one month after Aβ42 injection. Surprisingly, animal behavior did not reveal changes in cognitive performance nor in locomotor and anxious-related activity. Brain-derived neurotrophic factor related-signaling was also unchanged at 3 and 14 days post-Aβ icv injection. Likewise, astrocytic and microglial markers of neuroinflammation in the hippocampus were unaltered in these time points.
Conclusion: Taken together, our data emphasize a high variability and lack of behavioral reproducibility associated with these Aβ injection-based models, as well as the need for its further optimization, aiming at addressing the gap between preclinical AD models and the human disorder.
Cannabinoid actions on neural stem cells: implications for pathophysiology
Publication . Rodrigues, Rui S.; Lourenço, Diogo M.; Paulo, Sara L; Mateus, Joana; Ferreira, Miguel F.; Mouro, Francisco; Moreira, João B.; Ribeiro, Filipa; Sebastião, Ana M; Xapelli, Sara
With the increase of life expectancy, neurodegenerative disorders are becoming not only a health but also a social burden worldwide. However, due to the multitude of pathophysiological disease states, current treatments fail to meet the desired outcomes. Therefore, there is a need for new therapeutic strategies focusing on more integrated, personalized and effective approaches. The prospect of using neural stem cells (NSC) as regenerative therapies is very promising, however several issues still need to be addressed. In particular, the potential actions of pharmacological agents used to modulate NSC activity are highly relevant. With the ongoing discussion of cannabinoid usage for medical purposes and reports drawing attention to the effects of cannabinoids on NSC regulation, there is an enormous, and yet, uncovered potential for cannabinoids as treatment options for several neurological disorders, specifically when combined with stem cell therapy. In this manuscript, we review in detail how cannabinoids act as potent regulators of NSC biology and their potential to modulate several neurogenic features in the context of pathophysiology.
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SFRH/BD/129710/2017