GSK3β Overexpression in Dentate Gyrus Neural Precursor Cells Expands the Progenitor Pool and Enhances Memory Skills

Abstract

In restricted areas of the adult brain, like the subgranular zone of the dentate gyrus (DG), there is continuous production of new neurons. This process, named adult neurogenesis, is involved in important cognitive functions such as memory and learning. It requires the presence of newborn neurons that arise from neuronal stem cells, which divide and differentiate through successive stages in adulthood. In this work, we demonstrate that overexpression of glycogen synthase kinase (GSK) 3β in neural precursor cells (NPCs) using the glial fibrillary acidic protein promoter during DG development produces an increase in the neurogenic process, increasing NPCs numbers. Moreover, the transgenic mice show higher DG volume and increased number of mature granule neurons. In an attempt to compensate for these alterations, glial fibrillary acidic protein/GSK3β-overexpressing mice show increased levels of Dkk1 and sFRP3, two inhibitors of the Wnt-frizzled complex. We have also found behavioral differences between wild type and transgenic mice, indicating a higher rating in memory tasks for GSK3β-overexpressing mice compared with wild type mice. These data indicate that GSK3β is a crucial kinase in NPC physiology and suggest that this molecule plays a key role in the correct development of DG and adult neurogenesis in this region.

Keywords: astrocyte; glial cell; glycogen synthase kinase 3 (GSK-3); neural stem cell (NSC); transgenic mice.

FIGURE 1.

Generation and pattern of transgene expression in GFAP/GSK3β. A, generation of conditional double transgenic mice GFAP/GSK3β is achieved by breeding Bi-TetO GSK-3β mice, carrying GSK3β transgene, with GFAP-tTa mice expressing tTa under control of a truncated GFAP promoter. B, diagram representation of GSK3β transgene activation. Bi-TetO promoter is formed by seven copies of the palindromic tet operator sequence containing two cytomegalovirus minimal promoter sequences (P), one in each end, in divergent orientations. This bidirectional promoter is followed by a GSK3β cDNA sequence (fused with a myc epitope at its 5′-end) in one direction and β-galactosidase (LacZ) sequence encoding a nuclear localization signal in the other. The minimal promoter is active, and therefore the transgene is expressed only when it has joined the transactivator. This allows muting the expression with tetracycline administration. C and D, Western blotting detection of transgene expression using β-gal antibody in hippocampus (C) and cortex (D) of GFAP/GSK3β mice and WT mice. Actin and GFAP has been used as a load control. Lines show molecular masses in kDa. E–G, β-Galactosidase immunofluorescence on sagittal brain sections from GFAP/GSK3β mice, showing DG (E), cortex (F), and subventricular zone (G). The white arrows in F show mature astrocytes positive for β-gal. H–K, triple immunofluorescence showing a β-gal-positive cell (H) which also express myc epitope corresponding to transgenic GSK3β (I). As expected, this cell is a neural precursor cell, positive for BLBP marker (J). K shows merge of all channels. DG, dentate gyrus; Hil, hilus; I–VI, cortical layers; LV, lateral ventricle; St, striatum; RMS, rostral migratory stream.

FIGURE 2.

Overexpression of GSK3β in neural progenitors of GFAP/GSK3β. A–C, double immunofluorescence for GFAP (A, green channel) and β-galactosidase (B, red channel). C, merge panel with granule neurons in DG counterstained with DAPI (blue channel). The white arrow shows a double positive NPC located in the SGZ. The white arrowhead shows a double positive astrocyte in the hilus. D and E, orthogonal view of double positive NPC, which overexpress GSK3β. F–I, triple immunolabeling for GFAP (F, green), β-galactosidase (G, red), and S100β (H, gray). I, merge panel with DAPI staining in blue. The white arrows show a NPCs located in the SGZ, positive for β-galactosidase and negative for S100β. The white arrowhead shows a double positive non-neurogenic astrocyte. The thin white arrows show a mature astrocytes in the hilus and in the DG, which do not express the transgene. J and K, orthogonal view of double positive NPC, which overexpress GSK3β. L–N, double immunolabeling for BLBP (L, green) and β-galactosidase (M, red) in hippocampus of GFAP/GSK3β. N, merge panel with DAPI staining in blue. O and P, orthogonal view of double positive NPC, which overexpress GSK3β. Q–S, double immunolabeling for Sox2 (Q, green) and β-galactosidase (R, red) in hippocampus of GFAP/GSK3β. S, merge panel with DAPI staining in blue. T and U, orthogonal view of double positive NPC, which overexpress GSK3β.

FIGURE 3.

Transgene expression in different maturation stages cells in DG. The left column (A, D, and G; green channel) corresponds to β-galactosidase immunofluorescence in GFAP/GSK3β mice. The middle column (B, E, and H; red channel) shows Ki67 (B, proliferation marker), DCX (E, differentiation and migration marker), and NeuN (H, adult neurons) immunostaining. The right column shows merge images (C, F, and I) with nuclei stained with DAPI (blue channel). The white arrows in the middle and right columns show positive cells for Ki67 colocalizing with β-gal (B and C) and the lack of costaining with DCX (E and F) and NeuN (H and I) of β-gal-positive cells.

FIGURE 4.

GSK3β transgenic mice show an increase in DG volume and number of mature granule neurons. A and B, Nissl staining to visualize cells nuclei in sagittal section of WT (A) and GFAP/GSK3β (B) mice. C, quantification of total DG volume expressed in mm³ using Cavalieri method (p = 0.0001). D and E, mature granule neurons nuclei stained with DAPI sagittal section of WT (D) and GFAP/GSK3β (E) mice. F, quantification of total mature granule neurons in both genotypes (p = 0.015). ***, p < 0.001; *, p < 0.05. n = number of animals analyzed.

FIGURE 5.

Overexpression of GSK3β produces an increase of neural progenitors. A and B, BLBP immunolabeling in DG of sagittal brain sections of WT mice (A) and GFAP/GSK3β mice (B). C and D, Sox2 immunolabeling in DG of sagittal brain sections of WT mice (C) and GFAP/GSK3β mice (D). E and F, DCX immunolabeling in DG of sagittal brain sections of WT mice (E) and GFAP/GSK3β mice (F). G, quantification of total BLBP neural progenitors (p = 0.004). H, quantification of total Sox2 neural progenitors (p = 0.019). I, quantification of total DCX neural progenitors (p = 0.0001). ***, p < 0.001; **, p < 0.01; *, p < 0.05. n = number of animals analyzed.

FIGURE 6.

Neural precursor cells from GSK3β-overexpressing mice do not show any alteration in cell cycle re-entry and survival. IdU was injected, and after 12 h, CldU was injected. A and B, double immunostaining against thymidine analogs, IdU (red channel) and CldU (green channel) in WT (A) and GSK3β mice (B). The white arrows show positive cells for both markers. C, quantification of the percentage of cells labeled for both markers of the total IdU cells analyzed (p = 0.488). D and E, detection of CldU+ cells after 24 h postinjection in WT mice (D) and GFAP/GSK3β mice (E). F, quantification of the total CldU+ cells in both genotypes (p = 1). G and H, immunofluorescence to detect IdU+ cells after 1 week postinjection in WT mice (G) and GFAP/GSK3β mice (H). I, quantification of the total IdU+ cells with 1 week of age in both genotypes (p = 0.619). J, quantification of the total IdU+ cells with 4 weeks of age in both genotypes (p = 1). K, quantification of 4-week-old newborn neurons expressing DCX (p = 0.173). L, quantification of 4-week-old newborn neurons expressing Calbindin (p = 0.427). M, quantification of the total number of apoptotic cells expressing fractin. n.s., no significant. n = number of animals analyzed.

FIGURE 7.

The developing DG in P14 transgenic GSK3β mice present an increased number of neural progenitors. A and B, BLBP immunolabeling of sagittal brain sections of P14 WT mice DG (A) and P14 GFAP/GSK3β mice DG (B). C and D, Sox2 immunolabeling in DG of sagittal brain sections of P14 WT mice DG (C) and P14 GFAP/GSK3β mice DG (D). E and F, PHisH3 immunostaining of sagittal brain sections of P14 WT mice DG (E) and P14 GFAP/GSK3β mice DG (F). G, quantification of total BLBP neural progenitors (p = 0.007). H, quantification of total Sox2 neural progenitors (p = 0.003). I, quantification of total PHisH3 neural progenitors (p = 0.023). **, p < 0.01; *, p < 0.05. n = number of animals analyzed.

FIGURE 8.

Increase of Wnt pathway inhibitors in GFAP/GSK3β mice. A, diagram of the inhibitors action on Wnt-Frizzled complex, which drives to GSK3β activity regulation. B, mRNA levels quantification of two Wnt-Frizzled complex inhibitors, Dkk1 and sFRP3, in WT and GFAP/GSK3β mice by quantitative RT-PCR. The levels were normalized by 18S gene levels as a control and expressed as percentages of variation respect to WT mice (Dkk1 p = 0.029; sFRP3 p = 0.002). *, p < 0.05; **, p < 0.01. n = number of animals analyzed.

FIGURE 9.

Behavioral alteration in GFAP/GSK3β mice. A, quantification of open field behavioral test perform in 15-week-old WT and GFAP/GSK3β mice. The results are expressed as percentages (vertical counts p = 0.083; jumps p = 0.668; average velocity p = 0,990). B, quantification of fear conditioning test carried out in 15-week-old WT and GFAP/GSK3β mice. The results are expressed as percentages of immobility time (“freezing”) (p = 0.043). *, p < 0.05. n.s., no significant. n = number of animals analyzed.