SPOT14-positive neural stem/progenitor cells in the hippocampus respond dynamically to neurogenic regulators

Abstract

Proliferation of neural stem/progenitor cells (NSPCs) in the adult brain is tightly controlled to prevent exhaustion and to ensure proper neurogenesis. Several extrinsic stimuli affect NSPC regulation. However, the lack of unique markers led to controversial results regarding the in vivo behavior of NSPCs to different stimuli. We recently identified SPOT14, which controls NSPC proliferation through regulation of de novo lipogenesis, selectively in low-proliferating NSPCs. Whether SPOT14-expressing (SPOT14+) NSPCs react in vivo to neurogenic regulators is not known. We show that aging is accompanied by a marked disappearance of SPOT14+ NSPCs, whereas running, a positive neurogenic stimulus, increases proliferation of SPOT14+ NSPCs. Furthermore, transient depletion of highly proliferative cells recruits SPOT14+ NSPCs into the proliferative pool. Additionally, we have established endogenous SPOT14 protein staining, reflecting transgenic SPOT14-GFP expression. Thus, our data identify SPOT14 as a potent marker for adult NSPCs that react dynamically to positive and negative neurogenic regulators.

Graphical abstract

Figure 1

Endogenous SPOT14 Protein Is Expressed in the Adult Hippocampus (A) Endogenous SPOT14 protein revealed by antibody staining is confined to the SGZ and colocalizes with the NSPC markers SOX2 (red) and NESTIN (blue). Shown are representative images of a wt mouse at 2 m (left panel) and a magnification of the boxed area (right panel). Arrows point to SPOT14/SOX2/NESTIN-positive radial NSPCs, and arrowheads point to SPOT14/SOX2-positive nonradial NSPCs. (B) SPOT14-GFP-expressing cells colabel with the immunohistochemical signal from endogenous SPOT14 protein. Shown are representative images of a SPOT14 reporter mouse at 2 m (left panel) and a magnification of the boxed area (right panel). Arrows point to SPOT14-GFP/SPOT14-antibody positive radial NSPCs, and arrowheads point to SPOT14-GFP/SPOT14 antibody-positive nonradial NSPCs. Scale bars represent 50 μm (A and B, left) and 20 μm (A and B, right). GCL, granular cell layer.

Figure 2

The Number of SPOT14+ NSPCs Decreases with Aging (A) Analysis of SPOT14 reporter mice at postnatal day 7 (p7), day 21 (p21), 2 months (2 m), and 7 months (7 m) of age shows consistent expression of the transgene within the DG but robust reduction of SPOT14+ NSPCs with age. Shown are representative pictures of DAB stainings against GFP. (B) Quantification of SPOT14+ NSPCs at 2 m (n = 4) and 7 m (n = 3) illustrating the substantial decrease in the total number of SPOT14+ cells with age (left bars). This decrease is dependent on a loss of both radial and nonradial cells (middle bars); thus, the ratio is not changed with age (right bars). An example image of a SPOT14+ radial (arrow) and nonradial (arrowhead) cell is shown on the right. (C) In situ hybridization using a riboprobe against Spot14 mRNA shows expression in the DG at p7, p21, 2 m, and 7 m. In parallel to the SPOT14-GFP expression shown in (A), Spot14 mRNA is markedly decreasing with aging. (D) Radial and nonradial morphology as well as expression of the NSPC markers SOX2 (red) and NESTIN (blue) in SPOT14+ cells (green) is present from early postnatal times through adulthood. Shown are representative images at p7, p21, 2 m, and 7 m. Scale bars represent 100 μm (A and C) and 20 μm (D). Error bars represent mean ± SEM. ^∗p < 0.05

Figure 3

Running Increases Proliferative Activity of SPOT14+ NSPCs (A) Running enhances proliferation in the DG, as measured by EdU pulse labeling, and leads to a mobilization of SPOT14+ NSPCs into the proliferative pool. Shown are representative confocal images of SPOT14 reporter mice with access to a running wheel (Run) and control mice (Con). Arrows point to GFP/EdU-colabeled cells. Top panels show single channels for SPOT14-GFP (green), middle panels EdU signal (red), and lower panels an overlay of SPOT14-GFP/EdU. (B) Quantification of SPOT14+/EdU+ double-labeled cells (n = 3 per group). (C) Quantification of total numbers of EdU+ cells and SPOT14+ cells. Note the slight but significant increase in SPOT14+ NSPCs in running mice. (D) Quantification of SPOT14+/EdU+ cells subdivided into radial and nonradial morphology. The majority of double-positive cells in both running and control mice have nonradial morphology. Running does not change the ratio of proliferating radial to nonradial SPOT14+ NSPCs (n = 3 per group). Scale bar represents 100 μm. Error bars represent mean ± SEM. ^∗p < 0.05

Figure 4

Transient Ablation of Highly Proliferating Cells Recruits SPOT14+ NSPCs into the Proliferative Pool (A) Treatment with TMZ reduces the total number of proliferative cells in the DG of adult SPOT14 reporter mice. However, as a consequence of the ablation, more SPOT14+ cells are recruited into the proliferative pool. Shown are representative images (EdU in red, SPOT14-GFP in green) of control mice (left panel) and mice immediately or 3 days after TMZ (middle and right panel). Arrows point to GFP/EdU-colabeled cells. (B) Quantifications show the drop in total EdU+ cells after TMZ treatment. The total number of SPOT14+ cells is not affected, in line with the fact that cytostatic drugs mainly affect highly proliferating cells (n = 3 per group). (C) TMZ-induced transient depletion of highly proliferative cells recruits SPOT14+ cells into proliferation 3 days after the end of TMZ treatment. (D) The majority of double-positive cells in all three groups have nonradial morphology and TMZ treatment does not change the ratio of proliferating radial to nonradial SPOT14+ NSPCs (n = 3 per group). Scale bar represents 100 μm. Error bars represent mean ± SEM. ^∗p < 0.05