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.

Keywords: Mettl14; dopaminergic neuron; m6A; tyrosine hydroxylase.

FIGURE 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. One group of mice (Mettl14^(f/f)Cre) was injected with lentivirus packaging the Cre recombinase (Lenti‐Cre) into SN region (AP: −3.4 mm, ML: ±1.25 mm, DV: −4.5 mm) to induce SN conditional Mettl14 deletion. The control group (Mettl14^(f/f)Ctrl) received lentivirus without Cre recombinase (Lenti‐Ctrl). Five months later, the behavioural functions of mice were examined by rotarod test, pole test, open‐field test and elevated plus maze. Subsequently, mice brains were harvested. (D) Visual observation of mice. (E) A computer‐controlled rotarod apparatus with a rod (7 cm diameter) was set to accelerate from 0 to 40 revolutions per minute (rpm) for 30 sec and sustain 40 rpm for 5 min, and the time to fall was recorded (latency to fall). (F) Mice were placed on the bottom or top of the pole (40 cm length, at a 45‐degree angle to the ground), the time used from bottom to top (t1) or from top to bottom (t2) was recorded, and the sum of t1 and t2 was calculated as time in pole test. (G) An open‐field chamber with tetrahedral enclosing walls (total zone: diameter of 40 × 40 cm) was divided into three parts: border (green), periphery (red) and centre (blue). The time in border (H) and total distance in open‐field chamber zone (I) were calculated. (J) Mice were placed on the centre of elevated plus maze with an open arm (red, 40cm length) and a closed arm (green, 40cm length). 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)

FIGURE 2

Viral‐mediated deletion of Mettl14 in SN reduced TH expression, enhanced the activation of microglia and astrocytes, and decreased the expression of Nurr1, Pitx3 and En1. (A)The RNA expression of Mettl14 in SN was detected by RT‐qPCR. (B and C) The protein level of METTL14 was ascertained by Western blot. (D) IF assay showed the METTL14 expression in neurons of SN. Images were captured at 20× (red: Mettl14, green: NeuN, blue: DAPI, bar=40μm). (E and F) m6A level of total RNA from SN was determined by dot blot assay. (G) IHC assay was utilized to stain tyrosine hydroxylase (TH) in SN. (H) Quantification of TH‐positive cell number from (G) by cell count. The RNA expression (I) and the protein level (J and K) of TH were ascertained by RT‐qPCR and Western blot. (J, L and M) The protein levels of Iba1 and GFAP were ascertained by Western blot. (N) Schematic diagram of m6A‐binding targets on mRNA of Nurr1, Pitx3 and En1. The green box represents the coding sequence (CDS), and the blue line represents the 3’ untranslated region (3’UTR) of mRNA. The red dot and related number indicate the position of m6A‐binding sites. (O‐R) The protein levels of Nurr1, Pitx3 and En1 were measured by Western blot. “ns” represents that there is no statistical significance, * P < .05, ** P < .01 and *** P < .001 vs Mettl14^(f/f)Ctrl. (n = 4 for Mettl14^(f/f)Ctrl and n = 5 for Mettl14^(f/f)Cre)