FTO (Fat-Mass and Obesity-Associated Protein) Participates in Hemorrhage-Induced Thalamic Pain by Stabilizing Toll-Like Receptor 4 Expression in Thalamic Neurons

Background and Purpose: Hemorrhage-caused gene changes in the thalamus likely contribute to thalamic pain genesis. RNA N6 -methyladenosine modification is an additional layer of gene regulation. Whether FTO (fat-mass and obesity-associated protein), an N6 -methyladenosine demethylase, participates in hemorrhage-induced thalamic pain is unknown.Methods: Expression ofFto mRNA and protein was assessed in mouse thalamus after hemorrhage caused by microinjection of Coll IV (type IV collagenase) into unilateral thalamus. Effect of intraperitoneal administration of meclofenamic acid (a FTO inhibitor) or microinjection of adeno-associated virus 5 (AAV5) expressing Cre into the thalamus ofFto fl/fl mice on the Coll IV microinjection–induced TLR4 (Toll-like receptor 4) upregulation and nociceptive hypersensitivity was examined. Effect of thalamic microinjection of AAV5 expressingFto (AAV5-Fto ) on basal thalamic TLR4 expression and nociceptive thresholds was also analyzed. Additionally, level of N6 -methyladenosine inTlr4 mRNA and its binding to FTO or YTHDF2 (YTH N6 -methyladenosine RNA binding protein 2) were observed.Results: FTO was detected in neuronal nuclei of thalamus. Level of FTO protein, but not mRNA, was time-dependently increased in the ipsilateral thalamus on days 1 to 14 after Coll IV microinjection. Intraperitoneal injection of meclofenamic acid or adeno-associated virus-5 expressing Cre microinjection intoFto fl/fl mouse thalamus attenuated the Coll IV microinjection–induced TLR4 upregulation and tissue damage in the ipsilateral thalamus and development and maintenance of nociceptive hypersensitivities on the contralateral side. Thalamic microinjection of AAV5-Fto increased TLR4 expression and elicited hypersensitivities to mechanical, heat and cold stimuli. Mechanistically, Coll IV microinjection produced an increase in FTO binding toTlr4 mRNA, an FTO-dependent loss of N6 -methyladenosine sites inTlr4 mRNA and a reduction in the binding of YTHDF2 toTlr4 mRNA in the ipsilateral thalamus.Conclusions: Our findings suggest that FTO participates in hemorrhage-induced thalamic pain by stabilizing TLR4 upregulation in thalamic neurons. FTO may be a potential target for the treatment of this disorder.