Role of mitochondrial dynamics and metabolism in postnatal neural stem cells differentiation

Neural stem cells (NSCs) are found in discrete regions of the adult mammalian brain, the so called neurogenic niches. These niches are localized in the subventricular zone (SVZ) of the lateral ventricles and in the subgranular zone of the hippocampal dentate gyrus (DG). During adulthood, NSCs present the capacity of self-renewal, proliferation and differentiation into neurons, astrocytes and oligodendrocytes, making them a powerful tool to treat disease-related neural loss. Several efforts have been made to elucidate the mechanisms involved in NSC fate regulation. Importantly, mitochondria are at the center of these findings. In fact, several studies suggest that these organelles have an important role in regulating NSC differentiation and lineage determination. Impairments in mitochondrial dynamics and metabolism were shown to negatively impact on NSC properties. Nevertheless, a major aspect that remains unclear is whether mitochondrial dynamics and metabolism have a role in directing NSC fate towards neuronal, oligodendroglial and astroglial lineages. Therefore, this PhD thesis aimed to dissect how mitochondria biogenesis, fusion/fission cycles, morphology and bioenergetics are involved in NSC differentiation. To address these questions, we used the NSA model which is an in vitro model widely used to evaluate the NSC properties. For this, NSCs were obtained by isolating SVZ and DG cells from P1-3 C57BL/6 mice. The isolated cells were grown in neurospheres, and consequently passaged to guarantee higher yields of NSCs. Thereafter, neurospheres were plated under specific differentiation conditions giving rise to a mixed culture composed of immature cells and of neurons, astrocytes and oligodendrocytes. Through the NSA, we demonstrated that the neurospheres are mainly composed by NSCs, and that these cells present self-renewal, proliferation and differentiation capacities. Additionally, SVZ-derived NSCs have a higher proliferative capacity compared to DG-derived NSCs. Having a suitable model to study NSC fate, we then investigated the role of mitochondrial dynamics during NSC differentiation, by assessing the levels of proteins involved in mitochondrial biogenesis and fusion/fission cycles in SVZ cells from passage 0 (P0) to P2 and in DG cells at P1-P2. Importantly, with this analysis we could also analyse whether the neurosphere passage interfere with the expression of those proteins. Overall, expression of mitochondrial biogenesis-related proteins did not significantly change with NSC differentiation, in both neurogenic niches. Importantly, the levels of proteins involved in mitochondrial fusion such as Mitofusin (Mfn) 1 and 2 significantly increased while proteins involved in fission namely dynamin-related protein 1 (DRP1) significantly decreased along differentiation, independently of mitochondrial mass alterations, in SVZ cells at P2. To further comprehend the fusion/fission cycles in the SVZ cells at P2, mitochondrial morphology was evaluated in both NSCs and differentiated cells, using the Mitochondrial Network Analysis (MiNA) macro. Mitochondrial number, length and area differed in the different cell types. Mitochondrial number significantly increased during astroglial and neuronal differentiation. Moreover, both NSCs and oligodendrocyte precursor cells presented a more elongated mitochondria, compared to the other cells. Interestingly, mitochondrial area did not change in neuronal cells, increased significantly during astrocytic differentiation while there was a significant reduction along oligodendroglial maturation. Since mitochondrial dynamics are at a certain extent linked to mitochondrial bioenergetics, the differences obtained could be due to differences in the metabolic profile. Therefore, we then decided to explore the bioenergetic profile during NSC differentiation, and between cells from different lineages, in SVZ cells obtained from tertiary neurospheres dissociation. To sort astrocytes, oligodendrocytes and neurons, magnetic-activated cell sorting was used. We observed that with differentiation the cells seem to become more reliant on oxidative phosphorylation. Although neurons presented a significantly lowest basal respiration, these cells are the most energetically flexible cells. In addition to this, neurons presented a similar respiratory profile when compared with the control mixed population of cells. Altogether, the data herein contribute to further elucidate the role of mitochondrial dynamics during NSC differentiation, reinforcing the metabolic differences throughout this process and within cell lineages. These findings will serve as a groundwork to direct NSC fate by modulating mitochondrial properties, which in the future can lead to the development of therapeutic targets for the regeneration of the Central Nervous System (CNS).

IMM/BI/8-2021