Ablation of Fmrp in adult neural stem cells disrupts hippocampus-dependent learning

Abstract

Deficiency in fragile X mental retardation protein (FMRP) results in fragile X syndrome (FXS), an inherited form of intellectual disability. Despite extensive research, it is unclear how FMRP deficiency contributes to the cognitive deficits in FXS. Fmrp-null mice show reduced adult hippocampal neurogenesis. As Fmrp is also enriched in mature neurons, we investigated the function of Fmrp expression in neural stem and progenitor cells (aNSCs) and its role in adult neurogenesis. Here we show that ablation of Fmrp in aNSCs by inducible gene recombination leads to reduced hippocampal neurogenesis in vitro and in vivo, as well as markedly impairing hippocampus-dependent learning in mice. Conversely, restoration of Fmrp expression specifically in aNSCs rescues these learning deficits in Fmrp-deficient mice. These data suggest that defective adult neurogenesis may contribute to the learning impairment seen in FXS, and these learning deficits can be rectified by delayed restoration of Fmrp specifically in aNSCs.

Figure 1

Fmrp deletion in Nestin-expressing cells resulted in fewer YFP+ cells in the DG. (a, b) Immunohistological analyses of brain sections from cKO:Cre:YFP mice (a) and Cre:YFP Control mice (b) at 1 d post-Tam. Red, Fmrp; green, YFP; white, GFAP; blue, DAPI. Left scale bar = 20 μm; Right scale bar = 10 μm. (c, d) Immunohistological analyses of brain sections from cKO:Cre:YFP mice (c) and Cre:YFP Control mice (d) at 56 d post-Tam. Red, Fmrp; green, YFP; white, NeuN; blue, DAPI. Left scale bar = 20 μm; Right scale bar = 10 μm. (e) Sample images of YFP⁺ cells in the DG at 56 d post-Tam. Green, YFP; blue, DAPI. Scale bar = 50 μm. (f) Quantification of the number of YFP⁺ cells in cKO:Cre:YFP and Cre:YFP Control mice. ML, molecular layer; GCL, granule cell layer.

Figure 2

Selective deletion of Fmrp in Nestin-expressing cells alters cell proliferation and fate specification of aNSCs. (a) Schematic diagram showing the cell lineage-specific markers across stages of neurogenesis, which were used for fate mapping. (b–d) Sample confocal images used for fate mapping of YFP⁺ (green) cells in the DG. (b) Red, GFAP; green, YFP; white, S100β. (c) Red, DCX; green, YFP; white, Ki67. (d) Red, NeuN; green, YFP. Scale bars, 20 μm. (e) Quantitative comparison of the numbers of YFP⁺GFAP⁺S100β⁻ type 1 aNSCs in the DG of cKO:Cre:YFP mice and Cre:YFP Control mice. (f) Quantitative comparison of the numbers of YFP⁺Ki67⁺DCX⁻ transient amplifying (TAP) cells in the DG of cKO:Cre:YFP mice and Cre:YFP Control mice. (g) Quantitative comparison of the numbers of YFP⁺Ki67⁺DCX⁺ neuroblasts in the DG of cKO:Cre:YFP mice and Cre:YFP Control mice. (h) Quantitative comparison of the numbers of YFP⁺Ki67⁻DCX⁺ immature neurons in the DG of cKO:Cre:YFP mice and Cre:YFP Control mice. (i) Quantitative comparison of the numbers of YFP⁺NeuN⁺ mature neurons in the DG of cKO:Cre:YFP mice and Cre:YFP Control mice. (j) Quantitative comparison of the numbers of YFP⁺S100β⁺ astrocyte in the DG of cKO:Cre:YFP mice and Cre:YFP Control mice.

Figure 3

Selective deletion of Fmrp in primary aNSCs isolated from adult DG results in altered proliferation and differentiation of aNSCs and reduced neurite extension of aNSC-differentiated neuron. (a, b) Proliferation analysis. (a) Sample image of aNSCs with (+) or without (−) Cre-GFP retrovirus infection, followed by BrdU pulse labeling and immunocytochemistry analysis. Red, BrdU; green, Cre-GFP; blue, Dapi. Scale bar, 20 μm. (b) Quantitative comparison of the percentage BrdU-labeled cells in both cKO cells and WT control cells either without (left) or with (right) Cre-GFP viral infection. (c–e) Differentiation analysis. (c) Sample image of differentiated aNSCs with (+) or without (−) retrovirus-Cre-GFP infection, analyzed by immunocytochemistry. Red, Tuj1; green, Cre-GFP; white, GFAP; blue, Dapi. Scale bar, 20 μm. (d, e) Quantitative comparison of the percentage Tuj1⁺ neurons (d) and GFAP⁺ astrocytes (e) in both cKO cells and WT control cells either without (left) or with (right) Cre-GFP viral infection. (f) Sample images of neurons differentiated from WT and cKO aNSCs infected with Cre-GFP-virus. Scale bar, 20 μm. (g–j) Neurite complexity analysis of neurons differentiated from cKO or WT aNSCs either with (+) or without (−) Cre-GFP virus-infection. (g) Scholl analysis for dendritic complexity; (h) neurite length; (i) number of dendritic nodes (branching points); (j) number of ends. *, p < 0.05; **, p < 0.01. Fmrp ON, Fmrp is expressed; Fmrp OFF, Fmrp is not expressed.

Figure 4

Deletion of Fmrp from Nestin-positive aNSCs results in hippocampus-dependent learning deficits. (a, b) Context (a) and Tone (b) trace learning analyses of Fmr1 KO mice and WT control littermates as determined by the percentage of the time that the animal spent freezing to either training context (a) or training tone (b). (c, d) Context (c) and Tone (d) trace learning analyses of cKO:Cre:YFP mice and Cre:YFP Control littermates as determined by the percentage time of freezing to either training context (c) or training tone (d). (e) DNMP-RAM analyses of Fmr1 KO mice and WT control littermates as determined by the percentage of correct entry in both separation 2 test and separation 4 test. (f) DNMP-RAM analyses of cKO:Cre:YFP mice and Cre:YFP Control littermates as determined by the percentage of correct entry in both separation 2 test and separation 4 test. ***, p < 0.001.

Figure 5

Restoration of Fmrp in primary aNSCs rescues proliferation and differentiation deficits of Fmrp-deficient aNSCs and neurite extension deficits of aNSC-differentiated neurons. (a, b) Proliferation analysis. (a) Sample image of aNSCs with (+) or without (−) Cre-GFP retrovirus infection, followed by BrdU pulse labeling and immunocytochemistry analysis. Red, BrdU; green, Cre-GFP; blue, Dapi. Scale bar, 20 μm. (b) Quantitative comparison of the percentage BrdU-labeled cells in both cON cells and WT control cells either without (left) or with (right) Cre-GFP viral infection. (c–e) Differentiation analysis. (c) Sample image of differentiated aNSCs with or without retrovirus-Cre-GFP infection, analyzed by immunocytochemistry. Red, Tuj1; green, Cre-GFP; white, GFAP; blue, Dapi. Scale bar, 20 μm. (d, e) Quantitative comparison of the percentage Tuj1⁺ neurons (d) and GFAP⁺ astrocytes (e) in both cON cells and WT control cells either without (left) or with (right) Cre-GFP viral infection. (f) Sample images of neurons differentiated from WT and cON aNSCs infected with Cre-GFP-virus. Scale bar, 20 μm. (g–j) Neurite complexity analysis of neurons differentiated from cON or WT aNSCs with (+) or without (−) Cre-GFP virus-infection. (g) Scholl analysis for dendritic complexity; (h) neurite length; (i) number of dendritic nodes (branching points); (j) number of ends. *, p < 0.05; **, p < 0.01. Fmrp ON, Fmrp is expressed; Fmrp OFF, Fmrp is not expressed.

Figure 6

Restoration of Fmrp in Nestin-expressing aNSCs and their progenies rescues hippocampus-dependent learning deficits. (a, b) Immunohistological analyses of brain sections from cON:Cre:YFP mice at 56 d post-Tam. (a) Red, Fmrp; green, YFP; white, GFAP; blue, DAPI. (b) Red, Fmrp; green, YFP; white, NeuN; blue, DAPI. Left scale bar = 20 μm; Right scale bar = 10 μm. (c, d) Context (c) and Tone (d) trace learning analyses of Cre:YFP Control mice (express Fmrp), cON:Cre:YFP mice (Fmrp restored), and cON:YFP Control (no Fmrp) littermates as determined by the percentage time of freezing to either training context (c) or training tone (d). (e) DNMP-RAM analyses of Cre:YFP Control (has Fmrp), cON:Cre:YFP mice (Fmrp restored), and cON:YFP Control (no Fmrp) littermates as determined by the percentage of correct entry in both separation 2 test and separation 4 test.