ROS Dynamics Delineate Functional States of Hippocampal Neural Stem Cells and Link to Their Activity-Dependent Exit from Quiescence

Abstract

Cellular redox states regulate the balance between stem cell maintenance and activation. Increased levels of intracellular reactive oxygen species (ROS) are linked to proliferation and lineage specification. In contrast to this general principle, we here show that in the hippocampus of adult mice, quiescent neural precursor cells (NPCs) maintain the highest ROS levels (hiROS). Classifying NPCs on the basis of cellular ROS content identified distinct functional states. Shifts in ROS content primed cells for a subsequent state transition, with lower ROS content marking proliferative activity and differentiation. Physical activity, a physiological activator of adult hippocampal neurogenesis, recruited hiROS NPCs into proliferation via a transient Nox2-dependent ROS surge. In the absence of Nox2, baseline neurogenesis was unaffected, but the activity-induced increase in proliferation disappeared. These results provide a metabolic classification of NPC functional states and describe a mechanism linking the modulation of cellular ROS by behavioral cues to the activation of adult NPCs.

Keywords: adult neurogenesis; adult stem cells; physical activity; quiescent neural stem cells; reactive oxygen species; stem cell heterogeneity.

Graphical abstract

Figure 1

Physical Activity Stimulates Quiescent Cells into an Invariable Proliferation Scheme (A) Three nights of physical activity are required to significantly increase the number of proliferating (BrdU⁺) cells in the DG. (B) Experimental design for the dual thymidine labeling and running paradigm. (C) Although there is no change in baseline proliferating cells (CldU⁺), the number of IdU⁺ cells significantly increase in the 2R and 5R groups. (D) The small proportion of CldU⁺IdU⁺ cells indicates that most proliferating cells had exited the cell cycle within the 5 day paradigm. (E and F) The proportion of the IdU⁺ cells that are in early (E) or late (F) stages of the neurogenic trajectory. The horizontal line in the boxplots indicates the median, and the + sign denotes the mean. Bar plots show mean ± SEM. ^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.001. For further details pertaining to replicate numbers, see Table S5.

Figure 2

Nes-GFP⁺ Cells of the DG and SVZ Can Be Distinguished on the Basis of Their Differential Regulation of ROS (A) A principal-component analysis showed that Nes-GFP⁺ samples from DG and SVZ cluster distinctly. (B) About 30% and ~26% of the detected transcripts are uniquely expressed in the NPCs of the DG and SVZ respectively (“signatures”). (C) A Gene Ontology (GO) analysis of the signatures revealed that the most enriched pathways in the SVZ precursor cells were related to cell division and transcriptional regulation. (D) In contrast, redox regulation was identified as the most enriched pathway in the DG.

Figure 3

Nes-GFP⁺ Cells Can Be Classified into Distinct Functional Subsets on the Basis of Their Cellular ROS Content (A and E) Flow cytometry revealed that cells from the DG (A) and SVZ (E) have distinct ROS profiles (see Figures S2A–S2E for gating strategy). (B, C, F, and G) Plots showing the distribution (B and F) and cellular ROS content (C and G) of cells in each of the four manually gated ROS classes. (D and H) NS formation is restricted to the midROS and hiROS classes, with more neurospheres formed from hiROS cells from both the DG (D) and SVZ (H). (I) The ROS content in SVZ cells is higher than in DG cells (all cells). Nes-GFP⁺ cells of the DG have higher ROS than those of the SVZ. (J and K) Gating of Nes-GFP⁺ cells from the DG (J) and SVZ (K) on the basis of ROS content. (L) The DG and SVZ show unique distribution patterns across the four ROS classes, with more cells from the DG in the hiROS class. (M) The Nes-GFP⁺ cells from the DG have higher levels of cellular ROS than those from the SVZ. The horizontal line within the boxplots indicates the median and the + sign denotes the mean. ^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗∗p < 0.001.

Figure 4

ROS Levels Decrease along the Neurogenic Trajectory (A) PCA shows that the different ROS groups have distinct transcriptional profiles. (B) Venn diagram showing similarities in gene expression between the different ROS classes. Note that the 0.2% transcripts co-enriched in hiROS and loROS are omitted in this panel. (C) Transcripts upregulated in the DG were enriched in hiROS cells, whereas loROS transcripts showed more overlap with the SVZ signature. (D) With decreasing ROS, expression of ROS-related genes decreased, while cell cycle activity and neural differentiation genes increased. (E) In the pseudotime trajectory of Shin et al. (2015), hiROS signature transcripts peaked early, with later peaks in expression for the lower ROS groups. (F) Decreasing ROS content is associated with increased expression of transcription factors associated with activated stem cells (as identified by Shin et al., 2015) as well as a decrease in expression of quiescent stem cell transcription factors. (G) Genes upregulated in adult Hopx⁺ cells (Berg et al., 2019) showed enrichment for hiROS and midROS signatures and genes co-expressed in hiROS and midROS. (H) Heatmap showing expression changes of selected genes at the hiROS to midROS transition (left) and midROS to loROS transition (right).

Figure 5

ROS Levels in Different Neurogenic DG Cell Types and In Vitro Monolayer Culture (A) Relative cellular ROS content in major cell types within the DG (see also Figures S4A–S4D). (B) FACS gating for Dcx-GFP⁺ sub-clusters. (C) Cellular ROS content in Dcx-GFP⁺ sub-clusters (see also Figures S4E and S4F). (D–F) Relative expression levels of Ascl1 (D), Calb2 (E), and Rbfox3 (F) in the different Dcx-GFP⁺ subsets normalized to the expression levels of Actb. (G) EdU labeling scheme for assaying cellular ROS content in proliferating cells in vivo. (H) Relative cellular ROS content in Nes-GFP⁺ cells and the subset of Nes-GFP⁺EdU⁺ cells. (I) Pseudohistogram of Nes-GFP⁺ cells and their EdU subsets. Each bin contains 2% of all cells. (J) Relative cellular ROS content in monolayer culture following BMP4 treatment. For boxplots, the horizontal line indicates the median. Other data represent mean ± SEM.

Figure 6

Nox2-Mediated ROS Fluctuations in the hi-ROS Fraction of Nes-GFP+ Cells Are Critical for Physical Activity-Mediated Precursor Activation (A–D) Distribution of Nes-GFP⁺ cells (A and C) and normalized ROS content (B and D) across the four ROS classes in response to physical activity in wild-type or Nox^−/y mice (see also Figures S5E and S5F). (E and F) Relative levels of pAkt (Ser473, E; Thr308, F) following the indicated bouts of physical activity (normalized to standard-housed mice). (G) Number of Ki67⁺ cells increases in the wild-type but not the Nox2 mutant animals after 10 day physical activity paradigm. (H and I) Relative fractions of proliferating NPCs (H) and relative ROS levels (I) following BMP4 treatment and after re-plating cells with growth factors. The horizontal line within the boxplots indicates the median, and the + sign denotes the mean. Other data represent mean ± SEM.