FTO: An Emerging Molecular Player in Neuropsychiatric Diseases

NEUROSCIENCE REVIEW P.K. Annapoorna et al. / Neuroscience 418 (2019) 15–24

FTO: An Emerging Molecular Player in Neuropsychiatric Diseases P.K. Annapoorna, Harish Iyer, Tanvi Parnaik, Harish Narasimhan, Arnav Bhattacharya and Arvind Kumar⁎ Epigenetics and Neuropsychiatric Disorders Laboratory, CSIR-Centre for Cellular and Molecular Biology (CCMB), Hyderabad 500007, India

Abstract—A myriad of chemical modifications of DNA and histones involved in the epigenetic regulation of neural gene expression have been documented and studied in detail since many years. However, more recently, modifications in RNA and their implications for neural gene functions have been progressively investigated. Of these, the most widely studied is the N 6-methyladenosine (m 6A) modification. The discovery of the first m 6A demethylase, known as the fat, mass and obesity (FTO) associated protein, has further fortified the field of epitranscriptomic regulatory mechanisms, owing to FTO's involvement in several biological processes including brain development and function. Concomitantly, multiple lines of evidence have associated FTO with neuropsychiatric disorders. In this review, we discuss how FTO can exert its effect by acting not only on m 6A but also on O, N 6-dimethyladenosine (m 6Am) in different types of RNA and potentially influence the development of some major neuropsychiatric diseases. © 2019 IBRO. Published by Elsevier Ltd. All rights reserved. Key words: FTO, m 6A, RNA methylation, neuropsychiatric disorders, epitranscriptomics.

readers of m 6A. The YTH domain family 2 (YTHDF2) protein causes processing body mediated degradation of its RNA targets (Wang et al., 2014). In addition to these, there are other reader proteins like heterogeneous nuclear ribonucleoprotein C (hnRNP C) and the insulin-like growth factor 2 mRNA binding-proteins (IGF2BPs) which do not recognise m 6 A per se, but instead read the structural changes in the RNA that are caused by m 6 A. These are referred to as m 6A switch readers (Liu et al., 2015; Sun et al., 2019a). The FTO protein was functionally identified as an m 6 A demethylase by Jia et al., wherein they showed that FTO targets m 6 A in both single stranded and double stranded RNA in vitro (Jia et al., 2011). It is an iron and 2oxoglutarate dependent demethylase (Gerken et al., 2007). The Fto gene was first identified in 2002 as one of the six mutated genes in mice with fused toes (ft) phenotype (Peters et al., 2002). Later, genome-wide association studies (GWAS) showed that it is strongly associated with increased body mass index (BMI) and obesity in humans (Frayling et al., 2007; Scuteri et al., 2007). Single nucleotide polymorphisms (SNPs) in the gene have been associated with several other diseases including many neuropsychiatric diseases (Zhao et al., 2014). Two independent studies linked two obesity related Fto variants with attention deficit hyperactivity Disorder (ADHD) in children (Velders et al., 2012; Choudhry et al., 2013). The variants have also been linked with Alzheimer's disease and major depressive disorder (MDD) (Keller et al., 2011; Samaan et al., 2013; Milaneschi et al., 2014).

INTRODUCTION A large number of reversible chemical modifications in RNA have been identified and studied. Modern high throughput sequencing techniques like Methylated RNA ImmunoPrecipitation (MeRIP) and Site-specific Cleavage and Radioactive-labelling followed by Ligation-assisted Extraction and Thin-layer chromatography (SCARLET) have paved the way for determining the nature of these modifications (Maity and Das, 2016). Of the several distinct modifications reported so far, the most common internal modification found in mRNA and long non-coding RNA (lncRNA) is N 6-methyladenosine or m 6A (Roundtree et al., 2017). This modification was first discovered in the 1970s in human cancer cells (Desrosiers et al., 1974; Dubin and Taylor, 1975). Since then, it has been found to affect a number of phenomena like RNA splicing, transport, stability and translation (Niu et al., 2013). Given its abundance and reported involvement in a number of cellular functions and disease states, the m 6A modification has been extensively studied and the molecular players which coordinate its dynamicity and downstream effects have been characterised. The methyltransferase which deposits the methylation mark is a large complex with three main components, METTL3 (methyltransferase like3), METTL14 (methyltransferase like-14) and WTAP (Wilms' tumour 1-associating protein) (Meyer and Jaffrey, 2017). YTH domain family proteins are the canonical *Corresponding author. E-mail address: [email protected] (Arvind Kumar). https://doi.org/10.1016/j.neuroscience.2019.08.021 0306-4522/© 2019 IBRO. Published by Elsevier Ltd. All rights reserved. 15

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These associations fuelled several studies on FTO and its function. Most of the Fto variants reported so far contain SNPs within intron 1 of Fto. The molecular mechanisms of how these SNPs in non-coding regions affect Fto gene expression were not clear. A study reported that the transcript level of an obesity associated Fto variant (rs9939609) was higher in individuals heterozygous for the allele, indicating that the variant might harbour an altered cis-regulation site in the intron 1 of Fto (Berulava and Horsthemke, 2010). However, another study found that this variant does not affect the expression of Fto at all, but instead influences another gene, Iroquois homeobox 3 (IRX3) in cerebellar and adipose tissues. This is facilitated by looping of the DNA and establishment of physical interactions between the intronic region of Fto and the promoter of IRX3 (Smemo et al., 2014). Another SNP (rs1421085) disrupts the binding site for a repressor leading to derepression of an enhancer of genes, IRX3 and IRX5, thus increasing their expression. IRX3 and IRX5 have been functionally implicated in adipocyte differentiation and obesity (Claussnitzer et al., 2015). Allelic dosage effects of Fto variant rs8050136 have been reported to modulate the expression of another neighbouring gene retinoblastoma like-2 (RBL2) in lymphocytes (Jowett et al., 2010). While in a hypothalamic cell line, variants rs8050136 and rs1421085 were shown to affect the expression of Fto and two other genes, retinitis pigmentosa GTPase regulator interacting protein 1-like (RPGRIP1L)

and Akt interacting protein (AKTIP); however, these variants had no effect on IRX3, IRX5 and RBL2 expression (Stratigopoulos et al., 2016). It appears that the effect of each Fto variant is unique and depends on the cell or tissue type. These findings have however raised questions about FTO's involvement in obesity and other diseases that it was associated with earlier. Loss-of-function studies of FTO have provided clues about its function, especially in the nervous system. Fto knockout mice show postnatal growth retardation and reduced brain size (Fischer et al., 2009; Gao et al., 2010). Specific deletion in the central nervous system leads to the same abnormalities that resemble systemic deletion (Gao et al., 2010). In humans, a loss of function mutation in the Fto gene results in symptoms like microcephaly, functional brain deficits, structural changes in the brain, growth retardation and characteristic facial features like cleft palate (Boissel et al., 2009). Specific knockdown of Fto in the medial prefrontal cortex (mPFC) enhances cued fear memory in mice (Widagdo et al., 2016), while knockdown in the dorsal hippocampus increases contextual fear memory (Walters et al., 2017). Deletion in adult neurons also leads to increased fear memory (Engel et al., 2018). These findings indicate that FTO plays crucial roles in brain development and function. In Table 1, we have consolidated a list of studies implicating FTO in brain function. In lieu of its discovery as an m 6A demethylase, the disease phenotypes and functions associated with FTO were

Table 1. . List of studies implicating FTO and its RNA substrates in brain function.

Sr. No.

Model System

Type of Study

Results observed

1

C57BL/6 X C3H mouse

FTO knockout

Postnatal growth retardation, reduction in spontaneous locomotor activity and deregulation of energy homeostasis

NA

(Fischer et al., 2009)

2

Human

Growth retardation and polymalformation syndrome

NA

(Boissel et al., 2009)

3

C57BL/6 mouse (male)

Postnatal growth retardation, deregulation of energy homeostasis

NA

(Gao et al., 2010)

4

C57BL/6 mouse (male and female)

FTO knockout

Dysregulation of dopamine receptor (D2R and D3R) signalling, impairment of D2R and D3R dependent behavioural responses

m 6A in mRNAs

(Hess et al., 2013)

6

C57 BL/6NTac × 129 S6/SvEvTac hybrid mouse (male)

CRISPR/Cas 9 and shRNA mediated knockdown of FTO

Impaired hippocampal memory formation

NA

(Walters et al., 2017)

7

C57BL/6 mouse (male)

FTO double knockout

Reduced proliferation and differentiation of adult neural stem cells, impaired learning and memory formation

Loss of Function mutation R316Q in FTO FTO conditional knockout (both systemic and brain specific)

C57BL/6 mouse tissues and HEK293T cell line C57BL/6 (mouse embryos)

FTO knockout

m 6Am methylation affected and reduced mRNA stability

Inhibitor and siRNA mediated knockdown

Suppression of GAP-43 translation axonal elongation

10

C57BL/6 mouse (male)

FTO conditional knockout

Altered stress response and synaptic plasticity -

11

C57BL/6

FTO conditional knockout

Alteration in gut microbiota, Reduced anxiety and depression-like behaviour

12

C57BL/6 mouse (male)

FTO knockout

Hyperactivation of HPA axis, anxiety-like behaviour and memory impairments

13

C57BL/6 mouse tissues and HEK293T cell line

FTO knockout

snRNA methylation and biogenesis affected

9 8

RNA substrates implicated/ suggested

Reference

(Li et al., 2017a) (Mauer et 6 m Am in mRNAs al., 2017) m 6A in GAP-43 (Yu et al., mRNA 2018) (Engel et al., m 6Am in mRNAs 2018) (Sun et al., NA 2019b) (Spychala m 6A in mRNAs and Ruther, 2019) (Mauer et 6 m Am in snRNAs al., 2019) m 6A in mRNAs

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attributed to its action on m 6A. The FTO-m 6A axis has been implicated in a number of in vitro and in vivo disease models (Karra et al., 2013; Li et al., 2017b; Su et al., 2018; Chen et al., 2019; Mathiyalagan et al., 2019; Zhuang et al., 2019). However, the conclusions from some of these studies should be considered cautiously since many of them used anti-6mA antibodies for MeRIP-Sequencing which can bind to both m 6A and m 6Am (Meyer and Jaffrey, 2017). Structural studies have reported that the preferred substrate of FTO is m 6A (Zhang et al., 2019a). However, in vivo studies weave a different story. In the mid-brain and striatum of Fto knockout mice, the levels of m 6A do not change significantly, only a subset of mRNAs show altered m 6 A levels (Hess et al., 2013). The same research group later found that the altered m 6 A peaks in their MeRIPSequencing data appear only at the 5′ ends of transcripts and in fact correspond to another RNA modification, m 6Am (N 6,2’-O-dimethyladenosine), which usually marks transcription start sites (Mauer et al., 2017). Also, Fto knockout mouse embryos did not show an elevation in m 6A levels. In vitro assays demonstrated that FTO specifically targets m 6 Am instead of m 6A (Mauer et al., 2017). This finding was reproduced by Wei et al. who also showed that FTO has more preference for m 6Am in in vitro experiments. They further reported that the cellular localisation of FTO dictates its substrate specificity; it preferentially acts on m 6A in the nucleus and m 6Am in the cytoplasm (Wei et al., 2018). In addition, FTO binds to different types of RNA like mRNA, tRNA, and snRNA in vitro (Wei et al., 2018). A recent investigation has revealed that the major physiological targets of FTO are snRNAs. RNA methylation marks that showed a significant alteration in miCLIP (m 6A individualnucleotide-resolution cross-linking and immunoprecipitation) reads from Fto knockout liver cells of mice were exclusively present in snRNAs. Structurally, snRNAs exist as two methyl isoforms, m1 and m2 which differ by an m 6Am mark. FTO selectively demethylates the m 6Am in the m2 isoform and converts it to m1, a process which affects the splicing related functions of snRNAs (Mauer et al., 2019). This discovery mandates a rethinking about the mechanism of action of FTO, especially in the context of different diseases that it has been linked with. However, few studies have also shown that mRNAs are targeted by FTO (Hess et al., 2013; Yu et al., 2018; Zhuang et al., 2019). Table 1 also mentions the RNA substrates implicated in different studies. Functional studies in vivo have underlined a role for FTO in brain development and function, as discussed before. In the subsequent sections, we focus on the association of FTO with neuropsychiatric disorders such as Alzheimer's disease, Parkinson's disease, epilepsy, anxiety and depression, and explore the plausible underlying mechanism of its action.

FTO and Alzheimer's disease Alzheimer's disease (AD) is a chronic neurodegenerative disorder characterised by sequential and progressive symptoms like neuronal cell death, loss of synaptic connections and neuronal functions (Bradbury and Brodney, 2007). The three classical hallmarks of AD are perturbed amyloid

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beta (Aβ) metabolism, hyper-phosphorylation of Tau protein, and microgliosis (Querfurth and LaFerla, 2010; Montine et al., 2012; Caccamo et al., 2013; Cai et al., 2015). As the name suggests, Aβ hypothesis is based on the dynamics of Aβ protein. It is derived by the proteolytic action of enzymes like beta and gamma secretases which include, BACE 1, Presenilin 1 (PSEN 1) and Presenilin 2 (PSEN 2), on Amyloid Precursor Protein (APP). Mutations in the APP, PSEN-1 and PSEN-2 change the inherent structure of the Aβ protein to a form with increased β sheet secondary structure which facilitates deformation (Masters et al., 2015). Such deformed proteins cannot be cleared from the cell by the normal protein degradation processes. The resulting aggregation leads to cytotoxicity and death of neuronal cells (Tang et al., 2013). The second hallmark of AD is the presence of hyperphosphorylated tau protein in the brain, which also forms aggregates. The precise reason for the aggregation is not well understood; whether it is the lack of clearance of tau or an elevation in its production (Tang et al., 2013; Park et al., 2018). The third hallmark, microgliosis, refers to the phenomenon in which Aβ activates the resident microglia in the brain by TREM-2 (Triggering Receptor Expressed on Myeloid cells-2)-mediated mechanism that leads to inflammation (Doens and Fernandez, 2014; Leyns and Holtzman, 2017). Studies have also demonstrated that activated microglia cause an increase in tau hyper-phosphorylation and secrete certain factors like TNFα (tumour necrosis factor alpha) and IL-1β (interleukin-1 beta) which harm neuronal cells (Wang et al., 2017a). A link between FTO and tau phosphorylation was established by Pitman et al. using a human neuroblastoma cell line, SH-SY5Y. They observed that FTO knockdown affected the levels of phospho-tau (phosphorylation at Ser396), the form of tau which is extensively observed in AD (Hu et al., 2002). This phosphorylation is mediated by AMPK (AMP-activated protein kinase), an enzyme whose phosphorylation and activation has been linked to the expression levels of FTO (Pitman et al., 2012). This observation led to the hypothesis that FTO can potentially mediate tau phosphorylation via AMPK (Pitman et al., 2013). However, this hypothesis needs to be validated in vivo. The connection between FTO and AD can also be drawn through the mammalian target of rapamycin (mTOR). Dysfunction of mTOR signalling cascade has been considered as one of the major risk factors for AD (Yates et al., 2013; Cai et al., 2015). Multiple studies aimed at understanding the molecular basis of the symptoms of AD have shown that the mTOR signalling cascade is disrupted in neuronal cells of patients with AD (An et al., 2003; Griffin et al., 2005; Pei et al., 2008; Pei and Hugon, 2008; Tramutola et al., 2015; Uddin et al., 2018). Inhibition of mTOR restores the integrity of the blood brain barrier, which is usually disrupted in AD and contributes to its pathology by allowing the entry of neurotoxic blood factors into the brain. Its restoration ameliorated cognitive deficits and Aβ aggregation in murine models of Alzheimer's disease (Spilman et al., 2010; Van Skike et al., 2018). Activation of mTOR leads to increased production of Aβ and also decreases its clearance, thus

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causing aggregation (Cai et al., 2015). mTOR also disrupts Tau protein homeostasis (Tang et al., 2013) by facilitating tau hyper-phosphorylation by a GSK-3β (glucose synthase kinase 3 beta) mediated mechanism (Caccamo et al., 2013). All these studies indicate that mTOR is pivotal in the pathology of AD. There is compelling evidence implicating the FTO-mTOR axis in various biological processes. Cells lacking FTO exhibit decreased basal mTOR signalling and overexpression of FTO increases the activity of mTOR (Gulati et al., 2013). FTO activates mTOR-PGC1α (peroxisome proliferator activated receptor gamma coactivator 1-alpha) mediated mitochondria biogenesis (Wang et al., 2017b). Impairment of mitochondrial biogenesis is exacerbated in PGC1α knockdown conditions and contributes to mitochondrial dysfunction in AD (Sheng et al., 2012). Protein kinase C-beta (PKC-β) activates mTOR in an FTO dependent manner (Tai et al., 2017). PKC-β is a direct regulator of GSK3β which, as mentioned above, mediates tau hyperphosphorylation (Alkon et al., 2007). Recently, using an AD mouse model, Li et al. reported that FTO is involved in AD by its effect on mTOR (Li et al., 2018). Elevated levels of FTO protein were found in the brain and its knockdown led to a decrease in tau hyperphosphorylation. FTO seemed to activate tau phosphorylation by promoting mTOR signalling. Rapamycin, an mTOR inhibitor, blocked FTO-mediated tau phosphorylation. Since an inverse correlation between the levels of FTO and TSC1 (tuberous sclerosis complex 1), an upstream inhibitor of mTOR, was observed, they proposed that TSC1 mediates the regulation of mTOR by FTO. Increased FTO leads to inhibition of TSC1 and thus an elevation in mTOR signalling. This aids in the development of AD phenotype.

FTO and Parkinson's disease Parkinson's disease (PD) is a debilitating and progressive neurodegenerative disease characterised by bradykinesia, resting tremors and rigidity, which result from the loss of dopaminergic neurons within the substantia nigra pars compacta (SNpc) region of the midbrain (Dauer and Przedborski, 2003). In the normal brain, the release of dopamine by the presynaptic neuron triggers signalling in the postsynaptic neuron via D1 and D2 type dopamine receptors. G proteins receive cues from D1 receptors to induce adenylate cyclase, stimulating the synthesis of cAMP and subsequently activating Protein Kinase A. In contrast, D2 receptors hinder the induction of this process by inhibiting adenylate cyclase (Greengard, 2001). Dysregulation of this signalling cascade may precede the death of dopaminergic (DA) neurons, serving as a pathogenic precursor in PD. Inactivation of FTO gene results in the impairment of dopaminergic signalling, indicating a potential role of the demethylase in the pathogenesis of PD (Hess et al., 2013). The functionality of FTO in the DA circuitry was probed using an Fto deficient mice (Fto −/−), and cocaine, which is known to induce behavioural effects via the DA ventral tegmental area and substantia nigra (VTA-SN)–caudate putamen (CPu)–nucleus accumbens (NAc) circuitry (Bello et

al., 2011). Cocaine reduces the firing from DA neurons and induces locomotor activity. This locomotor activity was reduced in Fto −/− mice, suggesting a role for FTO in the DA circuitry-mediated behavioural responses to cocaine. Electrophysiological investigations confirmed reduced inhibition of firing rate of DA neurons by cocaine, implicating dysfunctional D2 receptor signalling in the Fto −/− mice (Hess et al., 2013). DA neurons projecting from the substantia nigra have very high energy demand which renders them vulnerable to degeneration (Mamelak, 2018). Given the role of FTO in mediating inhibition of the rate of firing in DA neurons by cocaine, it is conceivable that the activity of FTO is associated with the extent of DA neuron degeneration and hence the severity of PD. Additionally, the sensitivity of DA neurons to dopaminergic drugs used in PD therapy may be dependent on the activity of FTO. A recent study investigated the prevalence of m 6A modifications in both cellular (PC12) and animal models of PD using 6-hydroxydopamine (6-OHDA) (Chen et al., 2019). Analysis of the m 6A/A ratio in mRNA via LC–MS/MS (Liquid chromatography with tandem mass spectrometry) revealed a significant reduction, particularly in the striatal region as well as in PC12 cells. Within the midbrain and in in vitro model, an increased expression of FTO was observed, but an attenuated expression was seen in the striatum. It is possible that the upregulated FTO in the midbrain may be transmitted to the striatum via the axons of dopaminergic neurons, thereby resulting in the observed reduction of m 6A (Chen et al., 2019). Numerous studies have also implicated mitochondrial dysfunction in the progression of PD, where the accumulation of reactive oxygen species triggers various cellular responses including apoptosis and impaired protein degradation pathways (Imai and Lu, 2011). As FTO overexpression has been observed to exhibit mitochondrial dysfunction (Bravard et al., 2011), it may potentially contribute to the neurodegeneration in PD via the release and accumulation of reactive oxygen species within the dopaminergic neurons. Using an in vitro model, Chen and colleagues also showed that both, the overexpression of FTO and inhibition of m 6A using cycloleucine lead to increased intracellular Ca 2+ and oxidative stress, resulting in death of DA neurons by apoptosis (Chen et al., 2019). There is considerable evidence that mTOR signalling is altered during the progression of PD (Bockaert and Marin, 2015; Dijkstra et al., 2015). However, the association between mTOR pathway and PD is unclear, as both neuroprotective as well as neurotoxic effects have been observed in different PD models. Studies on human samples indicate that increased mTOR signalling is concomitant with higher accumulation of α-synuclein, inducing a neurotoxic effect (Crews et al., 2010; Dijkstra et al., 2015). The study on En1 +/− mouse model reaffirms this observation. En1 (Engrailed 1) is a critical homeobox transcription factor required for the survival of dopaminergic neurons. The En1 +/− model of PD is created by the heterozygous deletion of En1 and exhibits increased levels of mTOR (Nordstroma et al., 2015).

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The neuroprotective effect of mTOR inhibition using rapamycin has also been explored in numerous models (Ravikumar et al., 2006; Pan et al., 2008; Spencer et al., 2009; Tain et al., 2009; Dehay et al., 2010; Malagelada et al., 2010; Cullen et al., 2011; Decressac and Bjorklund, 2013; Jiang et al., 2013). Studies also demonstrate that different PD toxins (rotenone, MPTP, OHDA etc.) result in decreased mTOR signalling as well as cell viability (Chen et al., 2010; Rieker et al., 2011; Rodriguez-Blanco et al., 2012; Selvaraj et al., 2012; Xu et al., 2014). Further substantiating this result, neuron specific deletion of PTEN (phosphatase and tensin homologue), an activator of mTOR, is observed to impart a protective effect on dopaminergic neurons against neurotoxins in a mouse model (Domanskyi et al., 2011). Given the established positive link between FTO and mTOR (under the section ‘FTO and Alzheimer's disease’), FTO might play a significant role in regulating mTOR signalling in PD. It is however difficult to predict a conclusive relation because of the debatable role of mTOR signalling in PD. Considering the other reports of FTO in dysregulating DA neuron signalling and contributing to neuronal death, it could be hypothesised that increased FTO levels contribute to the development of PD.

FTO and epilepsy Epilepsy is a chronic neurological condition caused by excess release of glutamate, an excitatory neurotransmitter within the brain (Waheed et al., 2016). A disturbance in the homeostasis of neurotransmitter levels results in irregular or excessive neuronal activity termed as seizures or fits (Moshé et al., 2015). Many reports have suggested the role of stress, drugs, inflammation and epigenetic factors in the pathophysiology of epilepsy (Moshé et al., 2015). In vivo studies have shown that mutations in genes like tuberous sclerosis complex 1 and 2 (TSC1 and TSC2) cause abnormalities in synaptic protein expression and axonal and dendritic growth. These changes possibly lead to hyperexcitability of neurons, change in neuronal connectivity and reduced seizure threshold (Griffith and Wong, 2018). TSC1 and TSC2 are regulators of mTOR and have been associated with seizure susceptibility and epilepsy. Excessive activation of mTOR signalling has been found in genetic as well as acquired forms of epilepsy (Meng et al., 2013; Citraro et al., 2016). The hitherto discussed FTO-TSC1-mTOR axis can therefore be relevant to epilepsy too, with increased FTO levels potentially contributing to its pathology. The role of an FTO inhibitor as an anticonvulsant has been investigated in an animal model of epilepsy (Zheng et al., 2014). The inhibitor was designed to inhibit 2oxoglutarate (2-OG) dependent enzymes and was found to show specific activity against FTO. Although the study did not test its activity against all 2-OG dependent enzymes, structural analysis showed that FTO is the most likely substrate when compared to three other 2-OG dependent enzymes. The increase in m 6A levels on inhibitor treatment was comparable to the m 6A increase seen after FTO siRNA treatment. The inhibitor could also modulate the levels of

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certain miRNAs in vitro, suggesting that its anticonvulsant activity could be related to this effect, since some miRNAs are known to be involved in epilepsy (Rowles et al., 2012; Risbud and Porter, 2013). The effect of the inhibitor on miRNAs could be through its action on FTO. But the exact mechanism remains elusive and needs further investigation. FTO has been implicated in the processing of pre-mRNAs and nuclear RNAs, serving a regulatory role over the activity of several miRNAs. The highly expressed brain miRNAs, miRNA-134 and miRNA-146a, which are associated with the pathogenesis of epilepsy, possess a potential m 6A site in their sequence, possibly making them susceptible to the action of FTO (Rowles et al., 2012). Loss of FTO has been linked to neural deficits and reduction in brain volume (Fischer et al., 2009; Gao et al., 2010). Changes in the structure and volume of specific brain regions have also been documented in some forms of epilepsy (Whelan et al., 2018), leading to the speculation that complete loss of FTO might contribute to epilepsy. Individuals with a loss of function mutation in Fto indeed develop severe brain malformations and seizures (Boissel et al., 2009). To summarise, FTO potentially influences epileptogenesis in multiple ways. Its effect on mTOR suggests that it promotes epileptogenesis; in contrast, its complete loss has also been associated with seizure susceptibility. Thus, further studies are required to explain this contradiction.

FTO, anxiety and depression Anxiety and depression are distinct neuropsychiatric disorders which show significant co-morbidity (Pizzagalli, 2016; Thibaut, 2017). Anxiety is characterised by excess worry, fear and hyper arousal (Remes et al., 2016). Symptoms of depression include low mood, disturbed sleep and appetite, loss of interest in pleasurable activities, reduced energy and in extreme cases, thoughts of self-harm and suicide (Baldwin and Birtwistle, 2002). Although symptomatically different, there is significant overlap in their causes and the neurobiology, with psychological stress being a major contributor to the development of both. Numerous studies have established links between chronic stress, anxiety and depression (Lovibond and Lovibond, 1995; Graeff et al., 1996; Heilig, 2004; Pizzagalli, 2016). FTO has been functionally associated with stress response and anxiety. FTO knockout mice show increased levels of stress markers like corticosterone in the plasma, which indicates a hyperactive hypothalamic–pituitary–adrenal (HPA) axis, involved in stress response (Spychala and Ruther, 2019). Hyperactivity of the HPA axis is seen in both anxiety and depression (Keller et al., 2017). Intraperitoneal injections of corticosterone alter the levels of FTO in specific brain regions such as the medial prefrontal cortex and the amygdala which are involved in stress response (Engel et al., 2018). The direct effect of FTO on anxiety has also been described. FTO knockout mice show anxiety-associated

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Fig. 1. Schematic illustration of FTO’s action in neuropsychiatric diseases.FTO: fat, mass and obesity-associated protein, m 6Am: N 6, 2- O-dimethyladenosine, m 6A: N 6- methyladenosine, mTOR: mammalian target of rapamycin, TSC1: tuberous sclerosis complex 1, TSC2: tuberous sclerosis complex 2

behaviour according to one study (Spychala and Ruther, 2019), although another study reported no change in anxiety levels (Engel et al., 2018). The effect of FTO on depression has been demonstrated in a study which found that loss of FTO can decrease depression-associated behaviour in mice (Sun et al., 2019b). Contrary to other findings, it also reported a reduced anxiety-associated behaviour in the mice. These results have been attributed to the effect of FTO on the gut microbiota. Loss of FTO generates a signature gut microbial population which potentially contributes lower amount of lipopolysaccharide (LPS) in the serum and thereby stress induced activation of proinflammatory cytokines is suppressed (Sun et al., 2019b). Although this study reported that loss of FTO can prevent depression-associated behaviour, there are studies which indicate otherwise. FTO can affect the development of depression through its action on mTOR. As discussed in the above sections, FTO positively regulates mTOR signalling. Activation of mTOR by fast acting antidepressant ketamine leads to an increase in synaptic signalling proteins and number of synapses in the prefrontal cortex, which alleviates depression in rats (Li et al., 2010).

FTO appears to function in the process of neurogenesis and neuronal differentiation. Its loss leads to reduced proliferation and differentiation of adult neural stem cells in vivo (Li et al., 2017a; Spychala and Ruther, 2019). Neurogenesis is important for antidepressant efficacy and many studies have shown that increase in neurogenesis can alleviate the symptoms of depression (Eisch and Petrik, 2012; Miller and Hen, 2015). This lends support to the finding that increased FTO levels can ameliorate depression. In the field of epitranscriptomics, FTO has made quite a mark. Initially identified as an m 6 A demethylase, it has now been found to preferentially act on m 6 Am over m 6A, and primarily target snRNAs over mRNAs (Mauer et al., 2017; Mauer et al., 2019). Although these findings are compelling, there exist studies which have functionally implicated mRNAs as the target of FTO (Yu et al., 2018; Zhuang et al., 2019). For instance, GAP-43 (Growth Associated Protein 43) which is required for axonal elongation is conducive to axon specific regulation by FTO. Its mRNA has an m 6A modification which when removed by FTO, stabilises it and increases GAP-43 protein levels. Axon specific FTO inhibition reduces the levels of GAP-43. Mutation of its predicted m 6A site makes it unresponsive to FTO regulation, indicating that FTO acts on an m 6A site in the GAP-

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43 mRNA (Yu et al., 2018). Further studies are required to understand the details of FTO's choice of physiological substrates. FTO is involved in the etiopathology of many neuropsychiatric disorders. Its positive association with mTOR signalling, possibly through the reduction of TSC1, is notable since it can affect the outcome of multiple diseases. Increased mTOR signalling is associated with the development of Alzheimer's disease, Parkinson's disease and epilepsy (Pei and Hugon, 2008; Crews et al., 2010; Meng et al., 2013; Dijkstra et al., 2015; Citraro et al., 2016; Li et al., 2018). In contrast, increased mTOR signalling possibly mitigates depression (Li et al., 2010). In PD and epilepsy, this point is contentious. Both increased and decreased mTOR signalling seems to contribute to PD development (Cullen et al., 2011; Decressac and Bjorklund, 2013). A loss-offunction mutation in FTO has been linked to increased seizure susceptibility (Boissel et al., 2009), suggesting that decreased FTO levels could also contribute to epileptogenesis despite causing a decrease in mTOR signalling. It has been hypothesised that FTO affects the stability of TSC1 mRNA by its demethylation activity and thereby reduces its levels (Li et al., 2018). The hypothesis can be rationalised based on the present understanding about the targets of FTO. Since FTO acts on m 6Am which prevents decapping, demethylation of m 6Am could lead to efficient decapping and facilitate miRNA-mediated degradation of the mRNA (Mauer et al., 2017). Given the ambiguity over the preferred substrates of FTO for its demethylation activity, the mechanism may not be so straightforward. It would be interesting to investigate whether the effect of FTO on splicing through snRNA regulation can affect the levels of TSC1 mRNA and subsequently that of mTOR. FTO is also involved in processes like neuronal firing, stress response and neurogenesis (Hess et al., 2013; Li et al., 2017a; Engel et al., 2018). Loss of FTO alters dopaminergic neuronal firing (Hess et al., 2013) which can aid in the development of Parkinson's disease. FTO knockout increases anxiety-associated behaviour and stress markers in blood (Spychala and Ruther, 2019). On the contrary, FTO knockout has also been shown to decrease anxiety-like and depression-like symptoms (Sun et al., 2019b). However, considering that FTO promotes adult neurogenesis and neuronal differentiation (Li et al., 2017a; Spychala and Ruther, 2019), this could serve as a possible mechanism by which it alleviates depression. There is a plethora of pressing questions about FTO. What decides FTO's substrate specificity? Does it act on multiple substrate types like mRNA, snRNA and miRNA parallelly? Does the substrate specificity depend on the cell or tissue type or does it depend on the cellular localisation? Another challenge is to zero in on the RNA modification targeted by FTO. Techniques like miCLIP, m 6A-CLIP and PAm 6 A Seq (photo-crosslinking-assisted m6A sequencing strategy) have good resolution and can differentiate between m 6 A and m 6 Am, but lack reproducibility as they are dependent on m 6 A antibodies (Zhang et al., 2019b). Recently, Guan Luo's group has developed m 6 A-REFSeq, which facilitates the differentiation of m 6A and m 6Am

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in an antibody independent manner (Zhang et al., 2019b). A clear insight into FTO's specific activity can be obtained with such methods. A comprehensive unravelling of the effects of RNA demethylation by FTO at the molecular, cellular and physiological levels merits further research and characterisation. Fig. 1 illustrates some of the downstream effects of FTO that influence the development of AD, PD, epilepsy, depression and anxiety. Chemical inhibitors of FTO have already been developed and tested to some degree (Zheng et al., 2014; Huang et al., 2019). A better understanding of its multifaceted functions using models of diverse neuropsychiatric disorders might carve a path for the development of potential FTO based therapeutics.

ACKNOWLEDGEMENTS Arvind Kumar's lab is funded by the Department of Biotechnology (DBT) National Initiative in Glia Research project (BT/PR4014/MED/30/673/2011) and the Council for Scientific and Industrial Research (CSIR). Annapoorna P K receives junior research fellowship from the Department of Biotechnology, Government of India. We thank Dr. Rachel Jesudasan for professional English editing of the manuscript. We also thank Aishwarya Vedula for critical reading of the manuscript.

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(Received 21 January 2019, Accepted 12 August 2019) (Available online 21 August 2019)