This study sought to test the hypothesis that transient therapeutic intervention can produce long-lasting beneficial effects on cognitive functions in a mouse model of FXS. Our results demonstrate that a transient Nutlin-3 treatment of young adults for 10 days restored impaired hippocampal neurogenesis and related cognitive abilities in
Fmr1 KO mouse for at least 4 months after treatment cessation. Together with our publications [
20,
21], these findings indicate that not only brief Nutlin-3 treatment rescues the neurogenic and cognitive deficits in adult FXS mice, but also these beneficial effects are sustained long after the end of treatment. Our data also suggest that Nutlin-3 treatment during the early adulthood time window might establish the normal adult NSC niche required for intact neurogenesis and cognitive performances in the absence of FMR1.
Numerous therapeutic alternatives including newly developed compounds or repurposed drugs have been proposed for FXS [
20,
57]. There are many advantages of drug repurposing in the treatment of disease, including shortening the time frame and reducing the cost associated with new drug development [
58]. When assessing the feasibility of initiating treatments, an obvious concern is the resulting toxicity from long-term administration. For these reasons, there has been extensive interest in the possibility of repurposing drugs with potentially long-lasting therapeutic effects. However, only very few studies have assessed the persistent effect of treatment long after treatments are stopped. In a recent study, the minocycline treatment effect has lasted for 4 weeks in young FXS mice but not in adult FXS mice [
15]. In another study, transient treatment of FXS rats with lovastatin at 4 weeks of age for 5 weeks prevented the emergence of cognitive deficits in object-place recognition and object-place-context recognition [
12]. The authors show that the corrective effect has been sustained for at least 3 months (the last time point tested) after treatment termination, and the observed restoration of normal cognitive function is associated with sustained rescue of both synaptic plasticity and altered protein synthesis [
12]. One promising candidate for drug repurposing is a group of MDM2 inhibitors, and its prototype is Nutlin-3. Nutlin-3 is a small molecule that specifically inhibits MDM2, an E3 ubiquitin ligase, and the best known MDM2 targets are tumor suppressor TP53; therefore, Nutlin-3 and its derivative have been worked on extensively and used in clinical trials for cancer treatment [
59]. Our lab has found that, in adult NSPCs, FMR1 directly regulates the expression levels and activities of MDM2, which targets TP53 and HDAC1 [
20,
21]. Our published studies have shown that Nutlin-3 administration at a dosage significantly lower than those used for cancer treatment rescues impaired hippocampal neurogenesis and cognitive functions in both 2-month-old young adult FXS mice and 6-month-old mature adult FXS mice analyzed shortly after the treatment [
20,
21]. However, the long-lasting effect of Nutlin-3 was unknown. Our current study has addressed this important question and taken one step further to potential therapeutic applications of MDM2 inhibition for the treatment of FXS.
Understanding the molecular mechanism underlying drug action is important for both therapeutic application and improvement of drug development. To investigate the mechanism underlying the long-lasting effect of Nutlin-3, we first determined whether this effect was due to the persistent changes in intrinsic properties of NSCs by using primary NSPCs isolated from
Fmr1 KO or WT mouse hippocampus. We have previously shown that NSPCs isolated from 2-month-old
Fmr1 KO hippocampus had reduced
TP53 gene expression, increased proliferation, and reduced neuronal differentiation, which can be corrected by Nutlin-3 treatment [
20].
TP53 gene encodes a transcription factor TP53 regulating a network of target genes that play roles in various cellular processes including but limited to apoptosis, cell cycle arrest, genomic integrity, metabolism, redox biology, and stemness [
60]. Tp53 binds DNA in a sequence-specific manner and recruits transcriptional machinery components to activate or suppress the expression of a network of target genes [
61]. TP53 has also been shown to regulate gene expression through epigenetic mechanisms [
62‐
64], which may lead to long-lasting alteration in the gene expression. We therefore hypothesized that Nutlin-3 treatment may exert a sustained therapeutic effect on the FXS mouse model through modulating epigenetic pathways in NSPCs. To our surprise, our results indicate that transient Nutlin-3 treatment did not lead to persistent corrections in active MDM2 levels nor proliferation and differentiation of NSPCs isolated from 6-month-old
Fmr1 KO mice. This suggests that, unlike the immediate response to Nutlin-3 treatment, the long-lasting therapeutic effect of Nutlin-3 on neurogenesis might not act mainly through modulating NSPC intrinsic properties [
20,
21]. Because adult neurogenesis is regulated by both NSC intrinsic pathways and extrinsic stem cell niche [
65], we performed gene expression profile analysis of
Fmr1 KO and WT hippocampal tissue. Nutlin-3 treatment exerted minimal change in the gene expression profile of WT hippocampus which is supported by our previous findings [
20,
21]. On the other hand, Nultlin-3-treated KO mice mounted persistent and significant gene expression changes compared to vehicle-treated KO mice and WT mice. However, Nutlin-3 treatment did not make KO more similar to WT in the gene expression profile. This observation suggests that the low-dosage Nutlin-3 we used was effective in rebalancing cellular pathways in the absence of FMR1 but had no significant effect on WT because the cellular pathways were well regulated by the presence of FMR1. Among DEGs, we found mRNAs of proteins associated to the extracellular matrix, cell membrane, and secreted factors, many of which have been shown to regulate adult neurogenesis [
44‐
46]. For example, genes in TGFß and BMP signaling are upregulated in Nutlin-3-treated KO mice, which we have confirmed using qPCR. It has been shown that TGFß and BMP activation in adult NSC niche can activate adult neurogenesis [
66,
67]. In addition,
Igf2 mRNA expression levels were significantly higher in the Nutlin-3-treated KO hippocampus compared to either vehicle-treated KO hippocampus or WT mice. IGF2 has also been shown to promote adult NSC proliferation and neurogenesis [
48]. It is possible that the Nutlin-3 has an effect on both niche cells and NSCs, but the long-lasting effects of Nutlin-3 on NSCs can only be observed in the presence of niche cells, which explains why we saw rescue in mice with NSC-targeted FMR1 deletion. Furthermore, since Nutlin-3 was given systemically, we do not rule out that the Nutlin-3 effect might in part act through modulating peripheral systems.
One potential limitation of this study is that we have not defined whether an age range or a critical period exists for the initial Nutlin-3 treatment to achieve the long-lasting effectiveness of Nutlin-3 treatment. Future experiments on Nutlin-3 administration time at younger or older ages than 2 months should be considered. In addition, we showed that the beneficial effects of Nutlin-3 on impaired neurogenesis and cognition of FXS mice sustained for at least 4 months. Whether the effect lasts for a longer period or even for the rest of the animal’s life will need to be addressed in future studies. Furthermore, we have assessed adult neurogenesis-dependent behaviors. It is possible that Nutlin-3 also improves other aspects of behavioral deficits in FXS mice which is independent of adult neurogenesis; therefore, it will be beneficial to assess whether the beneficial effects of Nutlin-3 can be generalized to other forms of cognitive and behavior functions found in FXS. Although our transcriptomic analysis has provided an important clue for the long-term effect of Nutlin-3 treatment, a comprehensive assessment of the gene expression and epigenetic profiles of neurogenic niche will be needed to fully understand the molecular basis of the persistent effect of Nutlin-3. Finally, humans and rodents have distinct physiology, lifespan, and genetics [
68]. Whether Nutlin-3 treatment can have therapeutic effects on human FXS remains unknown. Although we observed that the effect of Nutlin-3 lasted 4 months which is a long time for mice with a lifespan of 2 years, human has a much longer lifespan and different metabolic rate. Translating our results into human treatment will need substantial further investigations.