Accumulating evidence suggests that FTO plays an important role in neural development and synaptic plasticity. Several studies support that FTO plays an important role in learning and memory formation. In addition, downregulation of FTO in the brain leads to psychiatric disorders, including anxiety [37], depression-like behaviors [38], and learning and memory deficits [39]. Therefore, our study focused on the role of FTO in brain function in the MK-SI model. In our study, the MK-SI model showed abnormal expression of the FTO protein, synaptic plasticity protein, apoptosis protein, and key protein of the PI3K/AKT/mTOR signaling pathway in the hippocampus, altered hippocampal neuronal activity, and impaired learning and memory behavior. These results suggested that FTO-mediated m6A modification impairs learning and memory in MK-SI mice. In addition, impaired synaptic plasticity, reduced neuronal activity, hippocampal cell apoptosis and inhibition of the PI3K/AKT/mTOR pathway may be related to impaired learning and memory in MK-SI mice.
Perinatal NMDA receptor antagonists combined with postweaning social isolation induce abnormal behavior, anxiety-like symptoms, and cognitive impairment more consistent with the symptoms of SCZ, which have also been reported in many animal models [10, 22, 40–42]. Therefore, based on previous studies, we constructed an animal model of SCZ neurodevelopment, in which SCZ-like sensory gating disorders, positive symptoms, negative symptoms, anxity-like behaviors, and learning and memory impairment were successfully replicated.
Previous studies have revealed an association between m6A modification and psychotic disorders [30, 38, 43], and its role in learning and memory disorders [44, 45]. In this study, for the first time, we found increased m6A modification and decreased expression of the demethylase FTO in the hippocampus of MK-SI model mice with learning and memory impairment. Interestingly, ALKBH5, another demethylase, showed increased protein and mRNA expression. The methyltransferases METTL3, METTL14 and WTAP were not significantly different. In this study, the reverse expression of two demethylases (FTO and ALKBH5) eventually led to an increase in the m6A modification level. Historically, there has been evidence that FTO expression is related to m6A levels [46], and FTO expression tends to be negatively correlated with m6A modification [47], consistent with our study. Therefore, we speculated that FTO seems to have a stronger regulatory effect on m6A modification, but the detailed reason for this effect is unclear. These results suggested that hippocampal FTO downregulation mediates the increase in m6A modification in the MK-SI model, which may lead to impaired learning and memory in mice. However, there is conflicting evidence on the association between FTO and learning and memory. For example, studies by Widagdo et al. [45] and Walters et al. [39] showed that FTO deletion could increase episodic fear memory in mice. However, Gao et al. [31] suggested that FTO deficiency impairs the learning and memory ability of mice by promoting lipid decomposition in the brain, inhibiting adult neurogenesis and promoting neuronal apoptosis. Li et al. [26] suggested that loss of FTO inhibited the proliferation and differentiation of adult neural stem cells (aNSCs), resulting in impaired learning and memory. Consistent with this, another study [37] revealed that the loss of FTO resulted in impaired working memory in mice.
Studies have confirmed that repeated injections of MK-801 in the neonatal period combined with adolescent social isolation can cause pathological changes in hippocampal morphology and volume and physiological changes in synaptic plasticity, leading to persistent behavioral abnormalities, pathological changes in the brain and cognitive impairment in adulthood [11, 48–50]. In addition, FTO was found to be highly expressed in neuronal cells [51] and to regulate neuronal functions in mice [45, 52], such as neuronal development and maturation [53], adult neurogenesis [26], axon and dendritic plasticity [54], and neuronal proliferation and differentiation [55]. Consistent with this, in our study, we found that specific knockdown of hippocampal FTO in MK-SI mice aggravated the level of hippocampal m6A modification, again demonstrating the association between FTO and m6A modification. In addition, FTO-KD or MK-SI can lead to structural and morphological abnormalities in hippocampal neurons, neuronal pyknosis, and reduced neuronal activity, which further leads to impaired learning and memory in MK-SI mice.
However, hippocampal FTO supplementation significantly reversed the increase in hippocampal m6A modifications, restored morphological and structural abnormalities in hippocampal neurons, reduced neuronal pyknosis, increased neuronal activity, and improved the learning and memory in MK-SI mice. Notably, the DG region of the hippocampus appears to more sensitive to the effects of FTO, than the CA1 and CA2 regions.
In addition, FTO plays an important role in synaptic maturation [31]. Synapses are thought to be the basis of learning and memory. Brain-derived neurotrophic factor (BDNF) is a protein synthesized in the brain that affects the survival, differentiation, growth and development of neurons, effectively prevents neuronal damage, and plays an important role in the process of neural development [56, 57]. Postsynaptic density-95 (PSD-95) is considered an important marker of dendritic branching and dendritic spine density [58]. Synaptophysin (SYN) is an important protein in synaptic vesicles and is thought to be associated with reduced synaptic density [59]. Both PSD-95 and SYN have been shown to be critical markers of both pre-and postsynaptic synapses [60]. In our study, FTO deletion significantly reduced the expression levels of synaptic plasticity proteins (BDNF, PSD-95 and SYN) in the hippocampal neurons of MK-SI mice. In contrast, by upregulating hippocampal FTO expression, we found increased expression of BDNF, PSD-95, and SYN, which are proteins related to synaptic plasticity in the hippocampus. Previously, rodent studies have shown that FTO plays a neuroprotective role by improving hippocampal synaptic plasticity through upregulating the expression of the hippocampal BDNF, SYN and PSD-95 proteins, which is consistent with our study [26, 30]. However, FB23-2 treatment strongly demonstrated that FTO plays an important role in regulating hippocampal synaptic plasticity and ameliorating learning and memory impairment in MK-SI mice. These results suggested that FTO-mediated m6A modification promotes hippocampal learning and memory impairment in MK-SI mice, which may be related to impaired hippocampal neural function and impaired synaptic plasticity.
MeRIP-seq analysis revealed that m6A-modified genes were enriched in two pathways related to cognitive function, the PI3K/AKT signaling pathway and the mTOR signaling pathway. The PI2K/AKT pathway is a key component of synaptic plasticity and neuronal survival [61]. PI3K/AKT is an important signaling pathway for metabolism and proliferation [62–64], cell growth and apoptosis, and the survival and differentiation of various cells. It is widely expressed in the central nervous system and participates in learning and memory. In addition, the PI3K/AKT signaling pathway plays a crucial role in the regulation of apoptosis. PI3K can be divided into three categories according to different structures, among which class I PI3K is divided into two subtypes according to their different adapters, IA and IB, which are composed of 110 kDa catalytic subunit (p110) and 85 kDa catalytic subunit (p85), respectively. IA PI3K is activated by tyrosine kinase receptors, whereas IB is activated by G-protein coupled receptors [65]. AKT, a serine-threonine kinase, is an important downstream signaling molecule for the regulation of cell growth by PI3K[66]. In general, the upstream signal first activates PI3K by promoting the phosphorylation of the PI3K protein, and activated PI3K can promote the phosphorylation and activation of the downstream signaling molecule AKT. Activated AKT can subsequently induce the decomposition of proapoptotic proteins such as Bax and bad, and promote the activation of antiapoptotic proteins such as Bcl-2, thus inhibiting cell apoptosis. In addition, blunted activation of the PI3K/AKT pathway is associated with cognitive impairment [67]. In contrast, elevated PI3K/AKT phosphorylation is associated with neuroprotective effects [68, 69]. In our study, we found that the phosphorylation of PI3K, AKT and mTOR was decresed in the hippocampal neurons of mice induced by MK-SI, and this decrease was more obvious after FTO-KD. Conversely, FTO-OE upregulated the MK-SI-induced decrease in the expression of the PI3K, AKT and mTOR proteins. Moreover, we also found that the downregulation of FTO promoted the MK-SI-induced imbalance in the expression of the apoptotic protein Bax and antiapoptotic protein Bcl-2, while upregulation of FTO inhibited the expression of Bax and promoted the expression of antiapoptotic protein Bcl-2 in the hippocampus of MK-SI mice. These results suggested that FTO might play an antiapoptotic role in the hippocampal neurons of MK-SI mice through the PI3K/AKT/mTOR signaling pathway. Consistent with our study, a review [70] demonstrated that FTO depletion upregulated the m6A modification levels of BDNF signaling pathway-related transcripts, such as BDNF, PI3K, AKT1, AKT2, AKT3, and S6K1, while downregulating their transcript levels, resulting in impaired neurogenesis.