Age-dependent and differential effects of Smad7ΔEx1 on neural progenitor cell proliferation and on neurogenesis

Abstract

We recently reported that young (3 to 4months old) mice lacking Exon 1 of the Smad7 gene (S7ΔEx1 mice) show enhanced proliferation of neural stem and progenitor cells (NPCs) in the hippocampal dentate gyrus (DG) and in the subventricular zone (SVZ) of the lateral ventricles. It remained unclear, however, whether this phenotype would persist along aging, the latter typically being associated with a profound decrease in neurogenesis. Analysis of NPCs' proliferation based on the cell cycle marker PCNA in 12month-old S7ΔEx1 mice revealed a reversal of the phenotype. Hence, in contrast to their younger counterparts, 12month-old S7ΔEx1 mice had a reduced number of proliferating cells, compared to wildtype (WT) mice. At the same time, the survival of newly generated cells was enhanced in the aged transgenic animals. 12month-old S7ΔEx1 mice further displayed a reduced level of neurogenesis based on the numbers of cells expressing doublecortin (DCX), a marker for newborn neurons. The reduced neurogenesis in aged S7ΔEx1 mice was not due to a stem cell depletion, which might have occurred as a consequence of hyperproliferation in the young mice, since the number of Nestin and Sox2 positive cells was similar in WT and S7ΔEx1 mice. Instead, Nestin positive cells in the DG as well as primary neurosphere cultures derived from 12month-old S7ΔEx1 mice had a reduced capability to proliferate. However, after passaging, when released from their age- and niche-associated proliferative block, neurospheres from aged S7ΔEx1 mice regained the hyperproliferative property. Further, pSmad2 antibody staining intensity was elevated in the DG and SVZ of 12-month old transgenic compared to WT mice, indicating increased intracellular TGF-beta signaling in the aged S7ΔEx1 mice. In summary, this points toward differential effects of S7ΔEx1 on neurogenesis: (i) a hyperproliferation in young animals caused by a cell autonomous mechanism, and (ii) a TGF-beta dependent modulation of neurogenesis in aged S7ΔEx1 animals that abrogates the cell-intrinsic hyperproliferative properties and results in reduced proliferation, increased stem cell quiescence, and enhanced survival of newly generated cells.

Keywords: Aging; Neural stem cell; Neurogenesis; Proliferation; Smad; TGF-beta signaling.

Fig. 1

Age-dependent modulation of cell proliferation, cell differentiation, and neurogenesis in S7ΔEx1 mutant mice. (A) Numbers of PCNA⁺ cells within the DG (A′) and SVZ (A″) are significantly increased in 4 month-old S7ΔEx1 mice compared to age-matched WT. In contrast, in 12 month-old animals, numbers of PCNA⁺ cells are significantly lower in transgenic compared to WT animals. (B) Numbers of DCX⁺ cells do not differ between the 4 month old S7ΔEx1 and WT mice in the DG (B′) and the SVZ (B″). At 12 months, S7ΔEx1 mice show a significant reduction of DCX⁺ cells. (C) In the DG of 4 month-old S7ΔEx1 mice, Nestin and Sox2 cell numbers are significantly elevated compared to WT (C′), and Sox2 cell numbers are significantly higher in the SVZ in the young transgenic mice (C″). At 12 months, the numbers of Nestin⁺ and Sox2⁺ cells are similar in WT and knockout mice in the DG and the SVZ. (D) PCNA expression within the Nestin⁺ cell population is decreased in 12 month-old S7ΔEx1 mice compared to WT mice (p = 0.075). In contrast, the proliferative activity (PCNA⁺) of DCX⁺ neuronal progenitor cells does not differ between 12 month-old WT and S7ΔEx1 mice. (E) Numbers of BrdU positive cells in the DG (E′) and SVZ (E″) were significantly higher in the young transgenic compared to WT mice, but did not differ in the 12 month-old mice. In the DG, the survival index of newly generated cells, based on BrdU cell number divided by PCNA cell number, was significantly higher in the 12 month-old S7ΔEx1 animals compared to the WT mice (E‴). (F) Cell fate analyses by BrdU co-labeling with specific markers revealed no differences in the percentage of BrdU double labeled cells in the DG (F′) and the SVZ (F″) between old transgenic and WT mice. In young mice, the fraction of BrdU⁺/NeuN⁺ cells was significantly lower in the S7ΔEx1 mice. In addition, independent of their genotype, young mice possessed a significant higher percentage of BrdU⁺/Sox2⁺ cells in the SVZ than aged mice (F″) Scale bars: 100 μm.

Fig. 2

NPC cultures derived from old S7ΔEx1-mice regain their hyperproliferative activity in vitro. (A) Quantification of primary spheres forming units from the SVZ of 4 and 12 month-old mice plated directly into a collagen gel (A′). While in the 4 month age group the S7ΔEx1-mice exhibit a significant higher number of primary spheres, less spheres could be obtained from 12 month-old S7ΔEx1-mice in respect to age-matched WT mice (A″). (B) A proliferation assay on cultured NPCs derived from the SVZ of 12 month-old WT and S7ΔEx1 mice (passages 2–6) revealed increased sphere size (B′) and increased cell numbers (B″) in the S7ΔEx1-mice compared to WT mice. Further, the percentages of NPCs labeled with the cell-cycle-marker Ki67 were higher in 12 month-old S7ΔEx1-mice than in age-matched WT mice (B″). Counting was performed in triplicates (> 300 total cells per sample).

Fig. 3

pSmad2 staining intensity is elevated within the neurogenic regions of 12 month old S7ΔEx1 mice. Immunohistochemical staining of pSmad2 is more intense in the dentate gyrus of 12 month old S7ΔEx1 mice (A) compared to age-matched WT mice (B). Similarly, pSmad2 staining is more apparent in the SVZ of 12 month old S7ΔEx1 mice (C) compared to WT (D).