Sustained correction of hippocampal neurogenic and cognitive deficits after a brief treatment by Nutlin-3 in a mouse model of fragile X syndrome

Abstract

Background: Fragile X syndrome (FXS), the most prevalent inherited intellectual disability and one of the most common monogenic forms of autism, is caused by a loss of fragile X messenger ribonucleoprotein 1 (FMR1). We have previously shown that FMR1 represses the levels and activities of ubiquitin ligase MDM2 in young adult FMR1-deficient mice, and treatment by a MDM2 inhibitor Nutlin-3 rescues both hippocampal neurogenic and cognitive deficits in FMR1-deficient mice when analyzed shortly after the administration. However, it is unknown whether Nutlin-3 treatment can have long-lasting therapeutic effects.

Methods: We treated 2-month-old young adult FMR1-deficient mice with Nutlin-3 for 10 days and then assessed the persistent effect of Nutlin-3 on both cognitive functions and adult neurogenesis when mice were 6-month-old mature adults. To investigate the mechanisms underlying the persistent effects of Nutlin-3, we analyzed the proliferation and differentiation of neural stem/progenitor cells isolated from these mice and assessed the transcriptome of the hippocampal tissues of treated mice.

Results: We found that transient treatment with Nutlin-3 of 2-month-old young adult FMR1-deficient mice prevents the emergence of neurogenic and cognitive deficits in mature adult FXS mice at 6 months of age. We further found that the long-lasting restoration of neurogenesis and cognitive function might not be mediated by changing intrinsic properties of adult neural stem cells. Transcriptomic analysis of the hippocampal tissue demonstrated that transient Nultin-3 treatment leads to significant expression changes in genes related to the extracellular matrix, secreted factors, and cell membrane proteins in the FMR1-deficient hippocampus.

Conclusions: Our data indicates that transient Nutlin-3 treatment in young adults leads to long-lasting neurogenic and behavioral changes likely through modulating adult neurogenic niche that impact adult neural stem cells. Our results demonstrate that cognitive impairments in FXS may be prevented by an early intervention through Nutlin-3 treatment.

Keywords: Adult neurogenesis; FMR1; Fragile X syndrome; MDM2; Neural stem cells; Nutlin-3.

Fig. 1

Transient treatment with Nutlin-3 has a long-lasting rescue effect on impaired hippocampal neurogenesis in FMR1-deficient mice. a Experimental scheme for assessing the hippocampal neurogenesis in Fmr1 KO and WT mice treated with Nutlin-3 (Nut3) or vehicle (Veh). b Sample confocal images used for identifying NSCs (GFP+GFAP+) and proliferating NSCs (GFP+GFAP+MCM2+) in the DG of adult Fmr1 KO and WT mice bred into a Nestin-GFP mouse background. Scale bar, 20 μm. c Comparison of the percentage of activated NSCs among total NSCs in the DG of Fmr1 KO and WT mice with or without Nutlin-3 treatment (n = 3 or 4 per group). d Sample confocal images to identify new mature neurons (NeuN+BrdU+) in the dentate gyrus of Fmr1 KO and WT mice. Scale bar, 20 μm. e Comparison of the percentage of mature neurons among BrdU+ cells in DG of Fmr1 KO and WT mice with or without Nutlin-3 treatment (n = 3 per group). *P < 0.05; **P < 0.01; ***P < 0.001. Data are presented as means ± SEM

Fig. 2

Transient treatment with Nutlin-3 has a long-lasting rescue effect on cognitive deficits in FMR1-deficient mice. a Experimental scheme for analyzing cognitive performances in Fmr1 KO and WT mice treated with Nutlin-3 (Nut3) or vehicle (Veh). b Schematic of novel location recognition (NLR) test for assessing spatial learning. c Beneficial effects of Nutlin-3 treatment on spatial memory deficits in Fmr1 KO mice sustained at least 4 months after injection (n = 8 to 13 mice per group). d Schematic of the novel object recognition (NOR) test. e Therapeutic effects of Nutlin-3 treatment on deficits in the NOR test in Fmr1 KO mice last at least for 4 months after treatment cessation (n = 8 to 13 mice per group). *P < 0.05; **P < 0.01; ***P < 0.001. Data are presented as means ± SEM

Fig. 3

Transient treatment with Nutlin-3 does not have a persistent effect on intrinsic properties of adult neural stem/progenitor cells. a Experimental scheme for analyzing proliferation and differentiation of hippocampal NSPCs isolated from Fmr1 KO and WT mice treated with Nutlin-3 (Nut3) or vehicle (Veh). b Sample images of proliferating NSPCs pulse-labeled with thymidine analog, BrdU followed by immunohistology for in vitro quantification assay. Red, BrdU; blue, DAPI; scale bar, 20 μm. c Sample images of differentiating NSPCs assessed by immunohistological detection of a neuronal marker Tuj1⁺ for in vitro quantification of NSPC neuronal differentiation. Red, Tuj1; blue, DAPI; scale bar, 20 μm. d, e Nutlin-3 treatment did not rescue impaired proliferation (d) and neuronal differentiation (e) of hippocampal NSPCs isolated from Fmr1 KO and WT mice 4 months after injection (n = 3). f, g Western blot analysis of P-MDM2 levels in isolated NSPCs isolated from Fmr1 KO and WT 4 months after Nutlin-3 or vehicle treatment (n = 3). h–k Western blot analysis of the total histone H3 (T-H3), acetylated histone H3 (acetyl-H3), and HDAC1 levels in NSPCs isolated from Fmr1 KO and WT mice 4 months after Nutlin-3 or vehicle treatment (n = 3). l, m Western blot analysis of P53 levels in isolated NSPCs isolated from Fmr1 KO and WT 4 months after Nutlin-3 or vehicle treatment (n = 3). n, o Western blot analysis of EP300 levels in isolated NSPCs isolated from Fmr1 KO and WT 4 months after Nutlin-3 or vehicle treatment (n = 3). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as loading controls. Due to the large size of EP300, loading controls were run on a separate gel. *P < 0.05; **P < 0.01; ***P < 0.001. Data are presented as means ± SEM

Fig. 4

Transient treatment with Nutlin-3 leads to long-lasting gene expression changes in the hippocampus of FMR1-deficient mice. a Experimental timeline for sample collection and transcriptomic profiling of the hippocampal tissue of Fmr1 KO and WT mice injected with Nutlin-3 (Nut3) or vehicle (Veh) (n = 3 per group). b M-A plot of M (log ratio) and A (mean average) displaying log₂ fold change of genes compared with the mean expression levels of all genes with log₂ fold change thresholds between − 3 and 3. The genes identified differential expression (adjusted P < 0.05) are indicated as blue dots. c Volcano plot showing the gene fold changes and adjusted P-values between KO-Veh and KO-Nutl3 groups. The most upregulated genes are towards the right, the most downregulated genes are towards the left, and the most statistically significant genes are towards the top. The red points are the genes with adjusted P-value < 0.05. d Venn diagram showing the overlap patterns of differentially expressed genes between the different experimental groups. e The numbers of up (blue) and down (red) regulated genes between KO-Veh and WT-Veh and between KO-Nut3 and KO-Veh

Fig. 5

Differentially expressed genes in FMR1-deficient mice treated with Nutlin-3 were enriched for genes associated with regulation of adult neural stem cell niche. a Bubble plots for Gene Ontology (GO) analysis showing enriched terms identified with Enricher for DEGs between Fmr1 KO treated with either Nutlin-3 or vehicle. The results of three different categories of GO analysis are shown. The size of the bubbles indicates the number of genes. The x-axis indicates the z score (negative = downregulated in Nutlin-3-treated Fmr1 KO mice; positive = upregulated in Nutlin-3-treated Fmr1 KO mice). The y-axis indicates the negative logarithm of adjusted P-value from GO analysis (higher = more significant). ECM organization, membrane proteins, and secreted factors are the top hits in each GO category. b Heat map of the transcriptional changes of selected DEGs between Nutlin-3 and vehicle-treated Fmr1 KO mice, revealed by RNA-seq (n = 3) and quantitative PCR (qPCR) analysis (n= 4). Red and green represent upregulation and downregulation, respectively. c–k Quantitative PCR analysis to validate a subset of DEGs in each GO category including Angptl2 (c), Aqp1 (d), Bmp6 (e), Col8a1 (f), Enpp2 (g), Erdr1 (h), Igf2 (i), Tmp3 (j), and Tmem72 (k) (n = 3/condition). The mRNA levels of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were used as the internal control. *P < 0.05; **P < 0.01; ***P < 0.001. Data are presented as means ± SEM

Fig. 6

Top TFs ranked by TF enrichment analysis were associated to ECM and stem cell fate. a Transcription factor target enrichment analysis of differentially expressed genes in Fmr1 KO treated with Nutlin-3 vs vehicle using average integrated ranks across all libraries through ChEA3 (n = 3). FET, Fisher’s exact test. Orange bars indicate TFs associated with ECM. Green bars indicate TFs associated with stem cell fate. Orange and green patterned bars indicate TFs associated to both ECM and stem cell function, respectively. b top TF network generated by Chea3 using the average integrated ranks across all libraries