**7. m6A and Neurological Disorders**

Consistent with important functions in neural development [18,32], neurogenesis [11,12,15,16], learning and memory [12,13,15,42] and stress response [44,45], the present evidence also indicates that m6A modification is involved in several neurological disorders, including Alzheimer's disease (AD), and Parkinson's disease (PD), schizophrenia, and attention-deficit/hyperactivity disorder (ADHD) via the regulation of gene expression and RNA metabolism [10,11,46–50]. Next, we discuss the function of m6A modification in neurodegenerative diseases, including Alzheimer's disease and Parkinson's disease.

A temporal feature of m6A modification has been revealed during postnatal brain development and aging [6,12]. In the brain of amyloid precursor protein (APP)/presenilin-1 (PS1) (APP/PS1) transgenic AD mouse models, m6A levels increased in the cortex and hippocampus, and the expressions of Mettl3 and Fto increased and decreased, respectively, compared with control mice [48]. Very recently, Shafik et al. found that m6A peaks decreased during the maturation stage of postnatal brain development (postnatal 2 weeks to 6 weeks), whereas these peaks increased during the process of aging (26 weeks and 52 weeks) [21]. In addition, this study also showed increased Fto expression and decreased Mettl3 expression. The differentially methylated transcripts were enriched in the signaling pathways related to Alzheimer's disease, and differential m6A methylation is associated with decreased protein expression in an AD mouse model, which was further validated

in a Drosophila transgenic AD model [21]. In agreement with this study, the Fto protein level increased in the brain tissues of transgenic AD mice, and Fto depletion did not affect the level of amyloid β 42 (Aβ42) but significantly increased the level of phosphorylated Tau in the neurons from an AD mice model [51]. They further found that Fto regulates Tau phosphorylation by activating mTOR signaling. Yoon et al. performed MeRIP, followed by next-generation sequencing with forebrain organoids, and the ontology analysis of human-specific m6A-targeted transcripts showed an enrichment in neurodegenerative disorders, including Alzheimer's disease [11]. Taken together, these findings suggest that m6A modification could play a pivotal function in the progression of AD (Figure 2C).

Acute knockdown of *Mettl14* in substantia nigra reduced m6A levels and impaired motor function and locomotor activity [52]. Nuclear receptor-related protein 1 (Nurr1), pituitary homeobox 3 (Pitx3) and engrailed1 (En1) are related to tyrosine hydroxylase expression and dopaminergic function, and their expression was remarkably reduced by *Mettl14* depletion [52]. The specific knockout of *Fto* in dopaminergic neurons impairs the dopamine neuron-dependent behavioral response by regulating dopamine transmission, which implies the important role of Fto-mediated m6A demethylation in regulating dopaminergic midbrain circuitry [34]. In a Parkinson's disease (PD) rat model, the overall level of m6A in the striatum decreased, and the Fto level significantly increased [53]. Either ectopic Fto or treatment with m6A inhibitors reduces m6A levels and induces oxidative stress and apoptosis of dopamine neurons, partially by promoting the expression of N-methyl-D-aspartate (NMDA) receptor 1 [53]. Consistently, *Fto* knockdown increases m6A levels and reduces apoptosis in vitro [53]. In addition, a large cohort study with 1647 Han Chinese individuals with Parkinson's disease (PD) has identified 214 rare variants in 10 genes with m6A modification; however, no significant association was observed between these variants and the risk for PD according to their analysis [54]. Therefore, the roles of m6A modification still need more comprehensive investigation (Figure 2C).

#### **8. Conclusions and Perspectives**

As the most abundant modification in mRNAs, previous studies have revealed the dynamic features of m6A modification and have uncovered its important function in a variety of biological processes and diseases. It seems that the more we explore m6A modification, the more complicated it becomes. First, m6A modification is reversible and includes multiple key "players": writers, erasers, and readers. The interaction between these key players and other epigenetic modifications, such as histone modifiers, makes the field more complicated. Second, the complexity of m6A modification also lies in the fact that it is hard to define a promoting or repressing function of m6A modification in a set of diseases. The deficiency of m6A writers and erasers could show similar effects on the diseases but could not exhibit contrary effects as routinely thought. Third, m6A modification can regulate a defined biological process, i.e., the maintenance, renewal, and differentiation of neural stem cells by modulating diverse gene expression and signaling pathways. In addition, multiple players of m6A modification exhibit effects on the same biological process, such as neurogenesis. It is hard to distinguish whether the effect is independent of each other, and it remains unclear whether they crosstalk. Therefore, how m6A writers, erasers, and readers cooperate to regulate adult neurogenesis still needs more investigation.

Although dramatic progress has been made in understanding the function of m6A modification, future studies should devote more effort to uncovering the multi-faceted nature of the associated mechanisms. The interaction between m6A modification and histone modifiers suggests a colorful landscape wherein m6A modification interacts with other epigenetic machinery, i.e., DNA modifications and non-coding RNAs. In addition, considering a substantial enrichment of m6A in the 5<sup>0</sup> and 3<sup>0</sup> UTRs of transcripts, do multiple writers, erasers, and readers have binding specificity for distinct regions? Finally, establishing a more precise spatiotemporal landscape of m6A in the pathological context could be of clinical significance. With the technical advances of sequencing, we anticipate

the identification of key m6A site(s) that can contribute to the diagnosis and treatment of specific diseases.

**Author Contributions:** All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was supported by the National Key Research and Development Program of China (2017YFE0196600 to X.L.) and the National Natural Science Foundation of China (grants 92049108 to X.L.).

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

