Figure 5.

Forced Expression of p38α Restores the Proliferative Capacity of NPCs and Neurogenesis in Aged Mice without Exhaustion of NSCs

(A) EdU incorporation in the neurospheres was significantly increased by p38α overexpression (OE) (n = 3 independent cultures; at least 700 cells were analyzed).

(B and C) Representative confocal images of EdU⁺ cells (magenta) in neurospheres from aged mice (6 months old) transduced with lentiviral vectors expressing VENUS or p38α. VENUS-expressing (green) control vector. HA staining (green) for detecting the exogenous expression of p38α (B). P-p38 staining (green) for confirmation of p38 activation (C). Nuclei were stained with Hoechst (blue). Scale bars, 100 μm.

(D and E) p38α OE via infusion of a lentiviral vector significantly increased the number of ASCL1⁺ cells in the SVZ of aged mice (n = 6 mice) (D). Representative confocal images of ASCL1⁺ cells (magenta) in the SVZ of aged (6-month-old) mice infected with lentiviruses (E). VENUS-expressing (green) control vector. p38α OE was detected by HA staining (green). Only VENUS⁺ or HA⁺ cells are counted. Scale bars, 50 μm.

(F and G) p38α OE via infusion of a lentiviral vector significantly increased EdU incorporation into the SVZ of aged (6-month-old) mice (F). EdU labeling was performed as described in Figure 2A (n = 6 mice). Representative confocal images of EdU⁺ cells (magenta) in the SVZ of aged (6-month-old) mice infected with lentiviruses (G). VENUS-expressing control vector (green). p38α OE was detected by HA staining (green). Only VENUS⁺ or HA⁺ cells are counted. Scale bars, 100 μm.

(H and I) p38α OE did not alter the number of SOX2⁺/EdU⁺/GFAP⁺ cells in the SVZ of aged mice (H). EdU labeling was performed as described in Figure 2A (n = 6 mice). Representative confocal images of activated NSCs defined as SOX2⁺ (pseudocolored green)/EdU⁺ (magenta)/GFAP⁺ (cyan) cells in the SVZ of aged mice infected with control and p38α OE lentiviruses (I). Only VENUS⁺ or HA⁺ cells are counted. Scale bars, 50 μm.

(J and K) p38α OE restored neurogenesis in the SVZ of aged mice. The number of DCX⁺ immature neurons in the SVZ of the aged mice was increased to the level of that in young mice by p38α OE (n = 6 mice) (J). Representative confocal images of DCX⁺ cells (red) in the SVZ of aged mice infected with lentiviruses and in the SVZ of 10-week-old wild-type mice (K). Only VENUS⁺ or HA⁺ cells are counted. Scale bars, 200 μm.

(L and M) A significant increase in the number of ASCL1⁺ TACs in the SVZ of 18-month-old mice by p38α OE (n = 4 mice) (L). Representative confocal images of ASCL1⁺ (magenta) cells in the SVZ of 18-month-old mice (M). Control VENUS-expressing lentiviruses or p38α OE lentiviruses were infused into the lateral ventricles of 6-month-old mice, followed by analysis at 18 months of age. Scale bars, 50 μm.

(N and O) A significant increase in EdU incorporation into the SVZ of 18-month-old mice by p38α OE (n = 4 mice) (N). Representative confocal images of EdU⁺ (red) cells in the SVZ of 18-month-old mice (O). Control VENUS-expressing lentiviruses or p38α OE lentiviruses were infused into the lateral ventricles of 6-month-old mice, followed by analysis at 18 months of age. Scale bars, 100 μm.

(P and Q) A significant increase in the number of DCX⁺ immature neurons in the SVZ of 18-month-old mice by p38α OE (n = 4 mice) (P). Representative confocal images of DCX⁺ (green) cells in the SVZ of 18-month-old mice (Q). Control VENUS-expressing lentiviruses or p38α OE lentiviruses were infused into the lateral ventricles of 6-month-old mice, followed by analysis at 18 months of age. Scale bars: 100 μm.

Statistical analysis was performed with an unpaired two-tailed Student's t test (A, D, F, H, L, N, and P) or one-way ANOVA, Tukey-Kramer post hoc test (J). Values in the bar graphs represent the mean ± SD. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001. NS, not significant; LV, lateral ventricle.