Down-regulation of m6A RNA methylation is involved in dopaminergic

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Down-regulation of m6A RNA methylation is involved in dopaminergic neuronal death Xuechai Chen, Chunyu Yu, Minjun Guo, Xiaotong Zheng, Sakhawat Ali, Hua Huang, Lihua Zhang, Shensen Wang, Yinghui Huang, Shuyan Qie, and Juan Wang ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.8b00657 • Publication Date (Web): 05 Mar 2019 Downloaded from http://pubs.acs.org on March 6, 2019

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ACS Chemical Neuroscience

Down-regulation of m6A mRNA methylation is involved in dopaminergic neuronal death

Xuechai Chen1*, Chunyu Yu1, Minjun Guo1, Xiaotong Zheng1, Sakhawat Ali1, Hua Huang1, Lihua Zhang3, Shensen Wang1, Yinghui Huang1, Shuyan Qie2*, Juan Wang1*

1College

of Life Science and Bioengineering, Beijing University of Technology, 100

Pingleyuan, Chaoyang District, Beijing 100122, China 2Department

of Rehabilitation, Beijing Rehabilitation Hospital affiliated to Capital

Medical University, Xixiazhuang, Badachu Road, Shijingshan District, Beijing 100144, China 3Beijing

Municipal Center for Food Safety Monitoring and Risk Assessment, 64

Shixing Street, Shijingshan District, Beijing 100041, China

1

ACS Paragon Plus Environment

ACS Chemical Neuroscience 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Abstract

N6-methyladenosine (m6A) is the most prevalent internal modification that occurs in the mRNA of eukaryotes and plays a vital role in the post-transcriptional regulation. Recent studies highlighted the biological significance of m6A modification in the nervous system, and its dysregulation has been shown to be related to degenerative and neurodevelopmental diseases. Parkinson's disease (PD) is a common age-related neurological disorder with its pathogenesis still not fully elucidated. Reports have shown that epigenetic mechanisms including DNA methylation and histone acetylation, which alter gene expression, are associated with PD. In this study, we found that global m6A modification of mRNAs is down-regulated in 6-OHDA induced PC12 cells and the striatum of PD rat brain. To further explore the relationship between m6A mRNA methylation and molecular mechanism of PD, we decreased m6A in dopaminergic cells by overexpressing a nucleic acid demethylase, FTO, or by m6A inhibitor. The results showed that m6A reduction could induce the expression of N-methyl-D- aspartate (NMDA) receptor 1, and elevate oxidative stress and Ca2+ influx, resulting in dopaminergic neuron apoptosis. Collectively, m6A modification may play a vital role in the death of dopaminergic neuron, which provides a novel view of mRNA methylation to understand the epigenetic regulation of Parkinson's disease. Keywords: mRNA methylation; 6-methyladenine; Parkinson's disease; NMDAR1; FTO; oxidative stress

Introduction Epigenetic modifications, including DNA methylation and histone modification, play important roles in embryonic and adult neural development. N6-methyladenosine (m6A) is the most abundant chemical modification of RNA, mainly distributed in mRNA and long non-coding RNA (lncRNA). m6A modification is enriched near the stop codon, 3'-untranslated regions and internal long exons of mRNA, and restricted to a consensus multiple RRACH (R = A/G, H = A/C/U) motif (1-4). Similar to DNA and histone 2


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epigenetic modifications, the formation of m6A is also a dynamic and reversible process, which primarily regulated by the methyltransferases (‘writers’) and demethylases (‘erasers’). The methyltransferase complex including METTL3, METTL14, WTAP KIAA1429, and RBM15/B, catalyze m6A formation, whereas two demethylases, Fat mass and obesity-associated protein (FTO) and Alkylated DNA repair protein alkB homolog 5 (ALKBH5), catalyze m6A demethylation (5-8). Recently, it was found that the protein family containing YTH domains can bind m6A (‘readers’), of which YTHDF1, YTHDF2, and YTHDF3 contribute to the translation and degradation of mRNA, while YTHDC1 can regulate RNA splicing (9-12). Substantial evidence suggests that m6A plays a significant regulatory role in the splicing, transport, location, translation, and stability of mRNA. However, the actual regulation by m6A is still unclear (13). Parkinson's disease (PD) is a common age-related neurodegenerative disorder with the early prominent death of dopaminergic neurons in substantia nigra pars compacta (SNpc) (14). Loss of SNpc neurons leads to striatal dopamine deficiency, which is responsible for the major PD symptoms such as slow movements and muscle trembling. Another pathological hallmark of PD is the presence of Lewy bodies （LBs）- an acid-rich inclusion body with abnormal aggregates of α-Synuclein protein as the main component (15, 16). PD is seen to be developed from a complicated interplay of genetics and environment and is accompanied by clinical challenges. Its etiology and pathogenesis largely remain unclear. As reported, five genes including SNCA, PARKIN, PINK1, DJ-1, and LRRK2, are the most studied and considered as critical risk factors for the onset of sporadic PD (17). Epigenetic modification is seen to play an important role in the regulation of gene expression, and its dysregulation has been suggested in the cause of various neurodegenerative disorders such as PD, Alzheimer and other mood disorders (18, 19). Recent studies have found that the DNA methylation of CpG islands in SNCA gene in the brain of PD patients is lower than in healthy subjects. This reduction in DNA methylation leads to an increase in the expression of the α-Synuclein protein (encoded by SNCA gene), which further worsens the condition (20-23). In addition, dysregulated acetylation of histone H3 or H4 in PD patients results in loss of 3



neuronal function. Excessive α-Synuclein can directly bind to H3, inhibiting its acetylation, causing chromatin condensation, leading to abnormal gene expression, which eventually results in cell death (24, 25). This evidence indicates that DNA methylation and histone modifications are closely related to the pathogenesis of PD. For RNA methylation, reports have indicated that the dysregulation of m6A modification might also be related to many neural processes including dopaminergic signaling in the mouse midbrain(26), neurogenesis in adult mice(27), learning and memory(28), suggesting a close relationship between m6A modification and brain activity. In this study, we sought to identify the association between m6A mRNA modification and the potential pathogenesis of PD, which have not been reported before. To begin with, we constructed cellular and rat models of PD using 6-OHDA, to examine the variation of m6A mRNA modification. Then we used lentiviral transfection of FTO and m6A methylation inhibitor to explore whether regulation of mRNA methylation plays a role in the death of dopaminergic neuronal cells. We, therefore, sought to reveal the importance of epitranscriptomic regulation in PD development.

Results m6A level was decreased in PD models In our study, we used 6-OHDA to construct both cellular and rat models of PD, and their characterization was shown in supporting information. For PD rat model, apomorphine, a dopamine agonist, was used to determine the success of nigrostriatal lesion by counting the number of contralateral rotations. Supplementary Figure S1 showed that robust apomorphine-induced contralateral rotations have a >95% loss of tyrosine hydroxylase (TH) immunolabeling. To be consistent with the rat model, we chose PC12 cell line, which was derived from a pheochromocytoma of the rat adrenal medulla, for cellular model establishment. As PC12 cells produce catecholamine and can develop neuron-like properties, it is a good fit for PD studies in vitro(29). As shown in Supplementary Figure S2, compared with control, the activity of lactate dehydrogenase (LDH) was significantly increased, as well as the content of H2O2, indicating that 6-OHDA could cause severe damage to PC12 cells. Subsequently, only 4


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the successful PD models were used for further analysis. Generally, m6A mRNA methylation is enriched throughout the brain(30). To investigate the correlation between mRNA methylation and PD, we detected the m6A/A ratio in mRNA using LC-MS/MS. In the whole brain of the PD rat model, the global m6A was not significantly different compared with SO (Sham operation) group (Figure 1A). We further compared m6A distribution in the cortex, striatum, hippocampus, and midbrain, and found it decreased significantly only in the striatum region (Figure 1B). Interestingly, m6A was also reduced in PD cellular model (Figure 1E). To test whether the decreasing m6A modification of mRNA is interrelated with demethylases, we then identified the distribution of the two demethylases in different brain regions and PC12 cells. In the midbrain of the PD model, the expression of FTO was enhanced, and there was no significant change in ALKBH5. In contrast, ALKBH5 was elevated significantly in the striatum of PD brain, while there was no change in FTO (Figure 1C,D). In the PD cell model, the results showed that FTO was up-regulated, but there was no difference in the demethylase ALKBH5 (Figure 1F,G). In the PD rat model, the striatum showed significant demethylation of mRNA, and this decrease is maybe owing to the high expression of ALKBH5. As the onset of PD is mainly in the SNpc of midbrain which contained lots of dopaminergic neurons, we found no significant difference in the m6A content in this region; however, the expression of demethylase FTO was increased significantly. Reports found that FTO is enriched in axons and can be locally translated (31, 32); we speculated that high expression of FTO in midbrain could transmit to striatum by the axons of dopaminergic neurons, and induce m6A decrease. Furthermore, since the main pathology of PD is the death of dopaminergic neurons in SNpc and dopamine decrease in the striatum, FTO in SNpc may relate to the control of DA transmission (26). Consistent with the rat model, m6A content in the PD cell model also showed a markedly decreasing trend, and the expression of demethylase FTO was increased at both mRNA and protein levels. These results suggest that the global m6A modification of mRNAs is down-regulated in PD models, which may be due to the overexpression of its demethylases. 5



Figure 1. Decreased mRNA m6A modification in PD models. (A) Quantification of the m6A/A ratio in mRNA of the whole rat brain using LC-MS/MS. (B) Quantification of the m6A/A ratio in different regions’ mRNA of rat brain. (C and D) ALKBH5 and FTO expression in different rat brain regions. (E) Quantification of the m6A/A ratio in mRNA of PC12 cells. (F-G) FTO and ALKBH5 expression in PC12 cells.

FTO overexpression harmed PC12 cells The decrease of m6A in the PD model led us to consider whether the down-regulation of m6A causes PD-related pathological features in normal dopaminergic neurons. To examine this hypothesis, we overexpressed FTO in dopaminergic PC12 cells by lentiviral expression system. The efficiency of FTO overexpression was confirmed by quantitative real-time PCR and Western-blot (Figure 2A,B), and the demethylation function of FTO was verified. As shown in Figure 2C, overexpressed-FTO decreased the global mRNA m6A level. In our research, we found that FTO could promote the 6


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apoptosis of PC12, which is similar to the phenomena induced by 6-OHDA (Figure 2F). Furthermore, the damage of PC12 cells over-expressing FTO was determined by detecting the level of oxidative stress and mitochondrial damage. The result showed that intracellular SOD (superoxide dismutase) activity was declined (the level of SOD activity indirectly reflects the ability of the body to scavenge oxygen free radicals), suggesting FTO could increase the level of oxidative stress (Figure 2D). Moreover, the activity of mitochondrial complex II (succinate dehydrogenase, inhibition of its activity leads to severe mitochondrial dysfunction) also decreased in overexpressed cells, indicating that FTO could impair mitochondrial function (Figure 2E). To determine the effect of overexpressed FTO on mitochondrial apoptosis pathway, the expression of an anti-apoptotic protein, B-cell lymphoma-2 protein (Bcl-2), and that of a proapoptotic protein, Bcl-2 associated X protein (Bax), were measured. The result showed that the expression of Bcl-2 decreased and Bax increased (Figure 2G), which further demonstrated that overexpression of FTO could induce apoptosis. Furthermore, we also overexpressed FTO in the human SH-SY5Y neuroblastoma cells and observed similar results which were shown in Supplementary Figure S3. This result indicated that the impairment caused by FTO overexpression is not PC12 cell-specific; it could also harm the other neuronal cells. Combined with the elevated FTO expression and decreased m6A in PD model, these results strengthen our hypothesis that mRNA methylation may be involved in the death of dopaminergic neuron and exacerbate the condition.

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Figure 2. FTO overexpression harmed PC12 cells. (A and B) Characterization of FTO overexpression in PC12 cells. (C) m6A dot-blot in overexpressed FTO cells. (D and E) Enzyme activity of SOD and Complex II. (F) Determination of apoptosis. (G) The expression of Bax and Bcl-2.

Effect of FTO on NMDAR1 expression and Ca2+ influx Hess et al. reported that the increased m6A methylation in a subset of mRNAs is essential for neuronal signaling, including the dopaminergic midbrain circuitry (26). In our research, we also attempted to explore the variation of the neuronal signaling pathway in FTO-overexpressed PC12 cells. As shown in Figure 3C, mRNA levels related to this pathway, such as Grin1, Syn1, and Drd3 genes were found to be elevated in FTO-overexpressing cells. However, in 6-OHDA induced PC12 cells, the mRNA levels of Grin1 and Syn1 were significantly increased, without the change in Drd3 (Figure 3A). Notably, the expression of NMDAR1 protein (encoded by Grin1 gene) was also elevated both in PD cell model and in FTO overexpressing cells (Figure 3B,D). Grin1 is the gene of N-methyl-D- aspartate receptor 1(NMDAR1), an ionotropic 8


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glutamate receptor. NMDA receptor is involved in the regulation of neuron survivals, synaptic plasticity, neuron circuit formation, and learning and memorizing activities, which plays a vital role in the developmental process. The disturbance in activation regulation of NMDA receptors could be the basis of plenty of central nervous system diseases including neurodegenerative diseases, epilepsy, and ischemic cerebral impairments. Reports showed that activation of NMDA receptors could lead to the massive influx of Ca2+, causing overloading of the intracellular Ca2+ level. Elevated intracellular Ca2+ leads to impaired mitochondrial function, which in turn causes the death of dopaminergic neurons (33-35). Therefore, we determined the content of Ca2+ by flow cytometry using specific Ca2+ fluorescence indicator (Fluo-3-Am). The results showed that the Ca2+ level in the PD model was approximately 1.7 times higher than control, and in FTO overexpressed cells, 1.2 times increase was also observed (Figure 3E). It was suggested that FTO overexpression cause an overload of intracellular Ca2+, which may be due to the increased expression of NMDAR1. Consistent with the previous report (36), we suspected that the observed elevated levels of oxidative stress, mitochondrial damage, and apoptosis in FTO overexpressed PC12 cells, may be caused by the up-regulation of Ca2+.

Figure 3. Effect of FTO on NMDAR1 expression and Ca2+ influx. (A and C) Determination of mRNA levels of dopamine signaling related genes in PD cell model and overexpressed FTO cells. (B and D) The expression of NMDAR1 in the PD cell model and overexpressed FTO cells. (E) Changes in intracellular Ca2+ concentration. 9



m6A down-regulation by cycloleucine induced the apoptosis of PC12 cells To investigate whether the effect of FTO was due to its demethylation function, we further treated PC12 cells with cycloleucine (an m6A inhibitor), which also promote mRNA demethylation(37). The cytotoxicity of cycloleucine in PC12 cells was assessed using different concentrations (Supplementary Figure S4), and the 10mM cycloleucine was chosen for the following experiments in the study. As expected, the level of m6A in the 10 mM cycloleucine treated cells was decreased (Figure 4A). Then, the expression of Grin1 and intracellular Ca2+ were also detected. As shown in Figure 4BD, after cycloleucine treatment, mRNA and protein levels of Grin1, and intracellular Ca2+ concentration were up-regulated. It is important to note that the activity of both SOD and Complex II were decreased (Figure 4E,F), indicating that the level of oxidative stress in cells was elevated and mitochondrial function was impaired. We further examined the apoptosis of PC12 cells treated with cycloleucine. The results showed that the level of apoptosis was elevated; also the down-regulation of Bcl-2 and the up-regulation of Bax were observed (Figure 4G,H). As a conclusion, cycloleucine treatment which decreases m6A caused a similar phenotype with overexpression of FTO, suggesting that m6A down-regulation could induce the apoptosis.

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Figure 4. Impairment of PC12 cells treated with cycloleucine. (A) m6A dot-blot of the control and cycloleucine-treated cells. (B and C) qPCR and western-blot analysis of NMDAR1. (D) Detection of intracellular Ca2+ concentration. (E-F) Activity detection of SOD and Complex II. (G) Effect of cycloleucine on apoptosis. (H) Western-blot analysis of Bax and Bcl-2.

Knock-down of FTO exhibited anti-apoptosis In order to confirm the damaging function of m6A down-regulation, we knocked-down FTO by shRNA in PC12 cells for further verification. As shown in Figure 5A and B, FTO was abrogated in shFTO subclone detected by qRT-PCR and Western-blot. Compared with the nontargeting control shRNA (NTC) subclone, knockdown of FTO in PC12 significantly increased m6A level in total mRNA isolation (Figure 5C). At first, we explored the cell damage effect and found that shFTO could decline the apoptosis of PC12 compared with NTC cells (Figure 5D), which was contrary to the 11



result of cycloleucine treatment and FTO overexpression. Then, we also detected the expression of cell death regulator Bcl-2 and Bax, and the result showed that the expression of Bcl-2 increased, and Bax decreased (Figure 5E). This increased Bcl2/Bax ratio inhibits cell apoptosis, suggesting that shFTO may display the function of neuroprotection. Furthermore, our result showed that shFTO could decrease the expression of Grin1 at both mRNA and protein levels (Figure 5F,G), which was contrary to the result of cycloleucine treatment and FTO overexpression. This data also supports that the variation of Grin1 expression is regulated by the demethylate function of cycloleucine (Figure 4B,C), not the result of its side effect. Therefore, we speculated that the increased m6A by shFTO could inhibit Grin1 expression, and reduces glutamate binding to the receptor, thus reducing neurotoxicity.

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Figure 5. Knock-down of FTO exhibited anti-apoptosis. (A and B) Characterization of FTO knockdown in PC12 cells. (C) Quantification of the m6A/A ratio in mRNA using LC-MS/MS. (D) Determination of apoptosis. (E) The expression of Bax and Bcl2. (F and G) Comparison of the expression of NMDAR1.

Discussion Previously, FTO was identified as associated with obesity in diverse human populations, and subsequent studies demonstrated that it is also a member of the Fe2+/αketoglutarate-dependent dioxygenase family with the function of m6A demethylation. 13



FTO is widely distributed in the mammalian brain, indicating that it is involved in the maintenance of neurons (38, 39). Furthermore, several genome-wide association studies showed the importance of FTO in memory processing, which represented that FTO may be related to an increased prevalence of Alzheimer's disease (AD)(40-42). However, the function of m6A in AD is not known. Dysregulation of m6A modification has been shown to be related to cancers, such as acute myeloid leukemia (AML) (43), glioblastoma (44) and colorectal cancer (45). Also, m6A is also related to some degenerative and neurodevelopmental diseases, such as major depressive disorder (MDD) (46). The abnormal expression of FTO in the brain can cause the increase of m6A, leading to down-regulate gene expression, which may end in numerous lesions of the brain. Moreover, we also found that the m6A/A ratio changed in different brain regions, and the ratio in whole brain is much higher than the other four separated brain regions (Figure 1A). It may due to the higher level of m6A in the other brain regions, as reported that there are abundant existence of m6A in the regions of cerebellum (47) and forebrain (31). Nevertheless, the role of m6A and its exact distribution in different rat brain regions are still unclear, and the importance of it is increasingly appreciated. In our research, we found that the m6A modification of mRNAs is down-regulated in PC12 cells induced by 6-OHDA and in the striatum of Parkinson's disease rat brain. Reports have indicated that adenosine methylation in a subset of mRNAs is important for the dopaminergic signaling pathway (26). It urged us to focus on the association between m6A mRNA modification and the potential pathogenesis of PD. Our results showed that the reduction of m6A could increase the expression level of NMDAR1, which could elevate oxidative stress and Ca2+ influx (48). NMDAR1 is an ionotropic glutamate receptor. Glutamic acid is the most abundant excitatory neurotransmitter in the central nervous system and exists in almost all neurons. It has the functions of depolarizing neurons and inducing synchronous discharge. The function of glutamate is exerted mainly through the ionic NMDA receptor. Physiological changes in NMDA receptors have been linked to the development of several brain-injury diseases(49). High expression of NMDAR1 will damage neurons, and subsequent apoptotic death. In support of this, we also found that knockdown of FTO in PC12 cells resulted in the 14


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reduction of NMDAR1 and anti-apoptosis. These results collectively demonstrate that m6A modification via FTO may play a crucial role in the pathogenesis of PD. As reported, the presence of m6A could reduce the stability of the mRNA and promotes its degradation (50). We then speculate that FTO causes the decrease of m6A, and finally stabilize NMDAR1 mRNA, which increases its expression. Naturally, reduction of m6A may regulate other genes’ expression as well, such as Syn1 and Drd3 (Figure 3A). It is well-known that SNCA, PARKIN, PINK1, DJ-1 and LRRK2 genes are closely related to sporadic PD, we therefore further detected their transcription levels in FTO overexpressed PC12 cells. The results showed that there was no significant difference of them compared with control (Supplementary Figure S5), suggesting that the transcription levels of these PD related genes may not be regulated by m6A modification. A recent study highlighted that FTO and mRNA methylation are actively regulated in the dorsal hippocampus (51), and FTO-deficient mice display impaired spatial learning and memory (27). In our research, we also found that FTO in hippocampus and cortex of PD rat model was significantly decreased compared with control (Figure 1D). Together, these findings suggest that FTO may normally regulate memory formation, which needs further exploration. Our work provides a novel perspective of mRNA methylation to understand the molecular mechanism of PD. Further studies on genespecific m6A modifications are needed to explore this new frontier in neuroscience.

Experimental procedures Animals and housing Male Sprague-Dawley rats (Vital River, China) were housed in cages (4 rats per cage) for two weeks before experimentation. All the animals were housed in a room kept at 24±2C temperature and approximately 40% humidity in a 12 h dark/12 h light cycle. Before being divided into experimental groups, all animals had free access to standard food and water. The animals weighed 260-300 g at the time of surgery. All procedures were conducted in authorization by the Animal Ethics Committee of Beijing Institute of Technology. 15



Sham and 6-OHDA injection All surgical procedures were carried out under pentobarbital sodium (70 mg/kg) anesthesia. After shaving the head superior region, the animals were placed into a stereotaxic frame and fixed to the frame by their incisors and ear canals. A longitudinal midline incision was made, and tissues were separated for the bregma visualization. Then, perforations were made in the skulls of the animals with a drill, allowing microinfusion of the suspensions directly into SNpc at the following coordinates (in mm) relative to bregma and midline: AP =﹣5.5, ML =﹢2.2, DV =﹣8.0. Each rat was injected with 2 𝜇L solution containing 8 𝜇g 6-OHDA (Sigma Chem. Co, St. Louis, 8 𝜇g/2 𝜇L of the free base in normal saline containing 0.1% ascorbic acid) into the right brain hemisphere at a rate of 1 𝜇L /min, and these rats (n=3) were named the Parkinson’s disease group (PD group). After infusion, the microsyringe was leaving in situ for a further 3 min to assure the solution-diffusion and prevent reflux. Then the incision was sutured. The animals of the sham-operated group (SO group, n=3) underwent the same surgical procedure of the 6-OHDA rats with the injection of an equal volume of the vehicle only. Afterward, all animals returned to their cages and were monitored for two weeks to ensure the full recovery. After the surgery, additional welfare was provided. Animal health was monitored daily, and all efforts were made to minimize suffering. Behavior testing As reported, after 6-OHDA injection, although SNc neurons start to degenerate within 12h, the most nigrostriatal lesion is reached after two to three weeks post-lesion(52). In our research, the behavioral test was then carried out two weeks after surgery. The administration of apomorphine (Wako, Japan) can induce asymmetric circling behavior between the right (lesioned) and left (unlesioned) brain hemispheres. The number of rotations is a quantifiable motor deficit in PD model. Two weeks after the stereotaxic surgery, all the animals were intraperitoneally injected with apomorphine at a dose of 0.5 mg/kg to induce the contralateral rotations. Ten minutes after the injection, a video was used to record the rotations of each rat for 20 min. Only those 6-OHDA induced rats showing robust contralateral turning (>7 turns/min) that were injected with 616


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OHDA were used in subsequent experiments. Immunohistochemistry of tyrosine hydroxylase (TH) staining After the rotation test, all rats in the corresponding group were deep anesthetized by pentobarbital sodium (70 mg/kg) and then perfused through their heart with saline. After that, the brains were removed quickly and immerse to ice-cold 4% paraformaldehyde for at least 24 h, and then transfer to 30% sucrose for cryoprotection. Brains were cut to 30 μm sections by freezing microtome (CM1900, Leica, Germany). For immunohistochemistry stain, the slices were washed in PBS three times and pretreated with 1% hydrogen peroxidase diluted in 10% methanol PBS solution for 15 min. Then the slices were entirely washed

with the PBS-T (PBS containing 0.3%

Triton X-100) and blocked with the 1% goat serum in PBS-T for 30 min and incubated with polyclone TH antibody raised in rabbit (Sigma, USA) diluted at 1:1000 with 1.5% goat serum in PBS-T for 24 h at 4 °C. After washing three times with PBS-T, the sections were incubated with a secondary antibody raised in goat (ZSGB-BIO, China) diluted at 1:200 in PBS-T for 2 h. Then the ABC and DAB kit (ZSGB-BIO, China) were used for staining. Finally, the slices were rinsed with PBS, dehydrated on the glass slide, and covered with PVP and coverslips. Microscopy was done behind desiccation. Rat brain pretreatment After the rotation test, rats were deep anesthetized by pentobarbital sodium (70 mg/kg) and then perfused through their heart with saline. After decapitation, the cortex, striatum, hippocampus, and midbrain of rat brain were separated on the ice, and stored at -80°C until use. Brain tissues of each rat were weighed and homogenized in a 10fold (w/v) volume of PBS, and the homogenates were centrifuged at 12000 rpm at 4 °C for 15 min. The supernatant was then used for RNA extraction by TRIzol (Invitrogen) according to the manufacturer’s instructions. Subsequently, the total RNA was used for qPCR and mRNA extraction. Cell culture and 6-OHDA induction Human embryonic kidney 293T, rat pheochromocytoma PC12, and human neuroblastoma SH-SY5Y cell lines were purchased from cell center of Chinese Academy of Sciences. Both 293T cells and SH-SY5Y cells were cultured in high 17



glucose-containing DMEM supplemented with 10% fetal bovine serum (Wisent, Canada), and PC12 cells were cultured in 1640 supplemented with 10% horse serum and 5% fetal bovine serum. Both culture mediums contained 1% PenicillinStreptomycin Solution (Solarbio, China). Cells were maintained at 37°C in a maximal humidified atmosphere of 5% CO2. For 6-OHDA induction, PC12 cells were deal with different concentrations (0, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07 mg/mL) of 6-OHDA for 24 h, and cell viability was measured by the 3-[4,5-dimethylthiazol-2-yl]-2,5diphenyltetrazolium bromide (MTT) assay. Ultimately, 0.025 mg/mL 6-OHDA was used to induce PC12 cells for the cellular modeling of PD(53). Lactate dehydrogenase (LDH) and hydrogen peroxide toxicity were used to quantify the damaged cells into the culture medium using kits according to the manufacturer’s instructions (Nanjing Jian Cheng, China). FTO overexpression and knockdown The rat FTO (NM_001039713.1) CDS region was inserted into the MCS region of the plasmid (pCDH-CMV-MCS-EF1-Puro). Expressing non-targeting control shRNA (NTC) and shRNA constructs targeting rat FTO (shFTO) was inserted into the plasmid of pLVX-shRNA2-Puro. Oligonucleotides purchased from Shanghai GenePharma Co., Ltd. All constructed plasmids were transfected into HEK293T cells together with pLP1, pLP2, pLP VSV-G for packaging into lentivirus. Normal PC12 cells were infected with the lentivirus or no virus control, and then puromycin (2 μg/mL) was used to screen the stably transfected cells. Additionally, transient transfection of rat FTO was carried out in PC12 cells or SH-SY5Y cells using Lipofectamine 2000 according to the manufacturer’s instruction (Invitrogen). FTO-shRNA: 5’-GATCC-GATGATGAAGTGGACCTTAAG-CTCGAGCTTAAGGTCCACTTCATCATC-GCTTTTTG-G-3’ NTC:

5’-GATCC-GACGATACAATAACGAGAA-CTCGAG-

TTCTCGTTATTGTATCGTC-GCTTTTTG-G-3’ Quantitative real-time PCR (qRT-PCR) Total RNA was extracted using TRIzol (Invitrogen), and RT-PCR was performed using an RNA PCR kit (Takara Biotechnology, Dalian, China) according to the 18


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manufacturer’s instructions. RNA was reversed transcribed with the AMV reverse transcriptase in the presence of an oligo(dT)15 primer. The cDNA was quantitative realtime PCR amplified on the real-time PCR system using the QuantiTect SYBR Green RT-PCR Kit (QIAGEN). All genes examined are normalized to housekeeping genes encoding β-actin. Using the ΔΔCT method, untreated cells served as controls, and relative expression values are calculated from Ct values. Primer sequences can be found in supplementary Table S1. Determination of the m6A level Total mRNA was extracted from total RNA using Dynabeads™ mRNA DIRECT™ Micro Purification Kit (Invitrogen) according to the manufacturer’s instructions. For dot-blots, the indicated amount of mRNA was applied to the membrane with the help of BIO-DOTTM apparatus and cross-linked by UV. The m6A primary antibody was added at a concentration of 1:500 and incubated for 2 hours at room temperature, then incubated the membrane with a secondary antibody for 1 hour and exposed to an autoradiographic film. Quantitative m6A/A ratio was analyzed by liquid chromatographytandem mass spectrometry (LC-MS/MS). Purified mRNA was lysed into single nucleotides by nuclease P1 and alkaline phosphatase, and then the standard curve method is used to determine the ratio of m6A/A via LC-MS/MS, as described previously(8). Western-Blot analysis Proteins were separated on SDS/polyacrylamide gel and transferred onto PVDF membrane. After blocking with a buffer containing 5% non-fat dry milk, proteins were incubated overnight with primary antibody against GAPDH, FTO, ALKBH5, NMDAR1, Bax, Bcl-2 (Cell Signaling Technology, China) at 4℃ and then washed in Tris-HCl buffered saline/0.1% Tween-20, followed by a secondary antibody incubation for 1.5 hours at room temperature, eventually visualized using an auto-radiographic film. Assessment of apoptosis An Annexin V-FITC/PI apoptosis detection Kit (DOJINDO, Japan) was used to detect apoptotic activity according to the manufacturer’s instructions. The emitted green 19



fluorescence of Annexin-V (FL1) and red fluorescence of PI (FL2) were detected by a FACS Caliber flow cytometer (Becton-Dickinson, USA) with an excitation wavelength of 488 nm and an emission wavelength of 525 and 575 nm, respectively. For each sample, 10,000 events were recorded. Experiments were performed in triplicate. The assay was performed with a detailed procedure as described previously (54). Measurement of intracellular Ca2+ concentration Intracellular Ca2+ levels were monitored as described (55). After washed with assay buffer, PC12 cells were then incubated with 5 μmol/L Fluo-3/AM, for 30 min in the dark at 37°C, and then washed 3 times with PBS. After centrifugation for 5 min, the cells were re-suspended, and the cell concentration was adjusted to 5×108/L, and the fluo-3 fluorescence was then examined by a FACS Caliber flow cytometer (BectonDickinson, USA). The excitation wavelength was 488 nm, and the emission wavelength was 525 nm. Acquisition of rest period fluorescence intensity value of 30 s, and then added anti-CD3 mAb and anti-CD28 mAb, respectively. The real-time signal was continued to acquire till 10 min. Finally, the intracellular Ca2+ concentration was represented by fluorescence intensity. Detection of the activity of SOD and Complex II SOD and Complex II activity were estimated using SOD and Complex II determination kits following the manufacturer’s instructions (Nanjing Jiancheng Bioengineering Company, China). The principle of the assay is that the enzyme catalyzes the substrate to a colored product, then the qualitative or quantitative analysis can be performed according to the depth of the colored reaction. First, all of the cell protein was extracted and added to the substrate after quantification, and the degree of substrate depletion was determined based on the absorbance value, which indirectly reflected the level of enzyme activity.

Supporting Information Primer information for qRT-PCR. Characterization of PD rat and cellular models. FTO overexpression harmed SH-SY5Y cells. The cytotoxicity of cycloleucine in PC12 cells, 20


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and mRNA levels of PD-related genes in overexpressed FTO cells.

Abbreviations m6A, N6-methyladenosine; PD, Parkinson’s disease; NMDAR1, N-methyl-Daspartate receptor 1; lncRNA, long non-coding RNA; FTO, Fat mass and obesityassociated protein; ALKBH5, Alkylated DNA repair protein alkB homolog 5; SNpc, substantia nigra pars compacta; LBs, Lewy bodies; SOD, superoxide dismutase; Bcl-2, B-cell lymphoma-2 protein; Bax, Bcl-2 associated X protein; LDH, lactate dehydrogenase; LC-MS/MS, liquid chromatography-tandem mass spectrometry.

Author information Co-corresponding Author* E-mail: [email protected]; [email protected]; [email protected] Author Contributions X.C. is the first author and the corresponding author, guided and organized all experiments, wrote and revised this manuscript; C.Y. and M.G. did most of the experiments and prepared figures; X.Z. constructed the PD rat model and separate different regions of the brain; S.A. performed the cell culture and helped to revise the manuscript in language editing; H.H. and L.Z. helped to quantify m6A by LC/MS; S.W. helped to guide molecular biology experiments; Y.H. helped to guide the epigenetics protocols; S.Q. and J.W., as the co-corresponding authors, contributed to the research design and revised the manuscript. Notes The authors declare no competing financial interest.

Acknowledgments This work was supported by grants from National Natural Science Foundation of China (No. 81400935, No. 91854115 and No. 31771571), China Postdoctoral Science 21



Foundation (No. 2012M510302), and Beijing Postdoctoral Science Foundation (No. 2012ZZ-02). We thank Prof. Yungui Yang for the FTO plasmid and Dr. Zongjian Liu for critically reading the manuscript. We also thank Prof. Yulin Deng, Beijing Institute of Technology, for providing the support of animal experiments.

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25



“For Table of Contents Use Only” Down-regulation of m6A mRNA methylation is involved in dopaminergic neuronal death Xuechai Chen1*, Chunyu Yu1, Minjun Guo1, Xiaotong Zheng1, Sakhawat Ali1, Hua Huang1, Lihua Zhang3, Shensen Wang1, Yinghui Huang1, Shuyan Qie2*, Juan Wang1*

1College

of Life Science and Bioengineering, Beijing University of Technology, 100

Pingleyuan, Chaoyang District, Beijing 100122, China 26


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2Department

of Rehabilitation, Beijing Rehabilitation Hospital affiliated to Capital

Medical University, Xixiazhuang, Badachu Road, Shijingshan District, Beijing 100144, China 3Beijing

Municipal Center for Food Safety Monitoring and Risk Assessment, 64

Shixing Street, Shijingshan District, Beijing 100041, China

A Table of Contents Graphic (TOC):

27


Down-regulation of m6A RNA methylation is involved in dopaminergic

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