Conditional deficiency of m6A methyltransferase Mettl14 in substantia nigra alters dopaminergic neuron function

Abstract N6‐Methyladenosine (m6A) is the most prevalent internal modification in messenger RNAs (mRNAs) of eukaryotes and plays a vital role in post‐transcriptional regulation. Recent studies demonstrated that m6A is essential for the normal function of the central nervous system (CNS), and the deregulation of m6A leads to a series of CNS diseases. However, the functional consequences of m6A deficiency within the dopaminergic neurons of adult brain are elusive. To evaluate the necessity of m6A in dopaminergic neuron functions, we conditionally deleted Mettl14, one of the most important part of m6A methyltransferase complexes, in the substantia nigra (SN) region enriched with dopaminergic neurons. By using rotarod test, pole test, open‐field test and elevated plus maze, we found that the deletion of Mettl14 in the SN region induces impaired motor function and locomotor activity. Further molecular analysis revealed that Mettl14 deletion significantly reduced the total level of m6A in the mRNA isolated from SN region. Tyrosine hydroxylase (TH), an essential enzyme for dopamine synthesis, was also down‐regulated upon Mettl14 deletion, while the activation of microglia and astrocyte was enhanced. Moreover, the expression of three essential transcription factors in the regulation of TH including Nurr1, Pitx3 and En1, with abundant m6A‐binding sites on their RNA 3’‐untranslated regions (UTR), was significantly decreased upon Mettl14 deletion in SN. Our finding first confirmed the significance of m6A in maintaining normal dopaminergic function in the SN of adult mouse.


| INTRODUC TI ON
N6-Methyladenosine (m6A) is one of the most abundant RNA modifications that is involved in the control of sophisticated gene expression under physiological and pathophysiological conditions. m6A of mRNA is edited by a conserved methyltransferase complex that includes Mettl3 (methyltransferase-like 3), Mettl14 (methyltransferase-like 14) and WTAP (Wilms tumour 1-associated protein). 1,2 Dopamine (DA), the most abundant neurotransmitter in the brain, is involved in various physiological functions of the central nervous system (CNS), such as motor functions, motivation and reward-related learning. There are three important dopaminergic pathways in the CNS, the most important of which is the nigrostriatal pathway, which originates in the Substantia nigra pars compacta (SNpc) and projects to the caudate and putamen. This pathway mainly participates in the regulation of movement. 3,4 Recently, a growing number of studies demonstrated the important role of m6A in the development and functions of the nervous system. Abnormal m6A has been reported to be involved in the regulation of several behaviour functions, including sensorimotor function, locomotor activity and leaning. 1,2,5,6 Dopamine, as a major regulatory neurotransmitter of these behaviour functions, its relationship with m6A is still indistinct.
In the present study, we used lentivirus-mediated deletion of Mettl14 in SN region, to study the necessity of m6A in maintaining the survival and relative function in dopaminergic neurons.

| Mice
As previously described, 7 C57BL/6J background Mettl14-loxp mice were generated by using the CRISPR/cas9-based genome-editing system. Animal procedures were approved by the Institutional Animal Care and Use Committee of School of Medicine, University of electronic science and technology of China. The details of all experimental process were provided in Appendix S1.

| Immunohistochemistry (IHC)
Immunohistochemistry assay was operated by using DAB detection kit (Streptavidin-Biotin, ZSGB-BIO, SP-9000-D) according to the manufacturer's manual. TH primary antibody (ProteinTech, 25859-I-AP) was used to detect the expression of TH in brain sections.

| Dot blot
Anti-m6A primary antibody (Millipore, ABE572) was used, and m6A level was detected by HRP chemiluminescence kit under chemiluminescence imaging analysis system.

| Statistical analysis
Data were expressed as mean values ± standard deviation (SD).
Statistical significance was assessed using unpaired Student's t test.
F I G U R E 1 Viral-mediated deletion of Mettl14 in Substantia nigra impaired mice motor function and locomotor activity. (A) Mettl14 deficiency was made by using loxp-cre system, and the region from second exon to tenth exon would be deleted if Cre recombinase is existed. (B) Example of genotyping result. The PCR products by using 5'loxp-F and 5'loxp-R primers were separated by agar gel electrophoresis, and a 230 bp band will be detected if loxp sequence was inserted while 198 bp will be observed in wild type. We marked homozygote of the floxed Mettl14 gene as Mettl14 (f/f) and heterozygote as Mettl14 (f/+) . (C) Experimental plan of the present study. . Time in open arm (K) and total distance in total plus maze zone (L) were calculated. * P < .05, ** P < .01 and *** P < .001 vs Mettl14 (f/f) Ctrl (n = 6) P < .05 was considered that there were significant differences between the groups.

| Viral-mediated deletion of Mettl14 in Substantia nigra impaired mice motor function and locomotor activity
To explore the effect of m6A in dopaminergic neuron function, we generated SN conditional Mettl14 knockout mice by using loxp-cre system through in vivo stereotactic injection of lentivirus ( Figure 1A Figure 1D). Both the rotarod ( Figure 1E and Video S1) and pole test ( Figure 1F

| Viral-mediated deletion of Mettl14 in SN reduced TH expression and enhanced the activation of microglia and astrocytes
Tyrosine hydroxylase (TH) is a monooxygenase that acts as a ratelimiting enzyme in the formation of L-dopamine, loss of function or reduced expression of TH directly affects the synthesis and secretion of dopamine. 4 We found an obvious reduction in TH expression

CO N FLI C T O F I NTE R E S T
The authors confirm that there are no conflicts of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.