Cre Adeno-associated virus(AAV Serotype 5)

Genetic changes caused by thalamic hemorrhage may contribute to the development of thalamic pain. RNA N⁶-methyladenosine (m⁶A) modification is an additional layer of gene regulation. Whether fat mass and obesity-associated protein (FTO), an m⁶A demethylase, is involved in hemorrhage-induced thalamic pain is unknown. In this study, FTO was detected in neuronal nuclei of the thalamus. FTO protein levels, but not mRNA, increased over time in the ipsilateral thalamus 1-14 days after microinjection of type IV collagenase (Coll IV). Intraperitoneal injection of MA or AAV5-Cre microinjection into the thalamus of Fto^fl/fl mice attenuated Coll IV microinjection-induced ipsilateral thalamic TLR4 upregulation and tissue damage as well as the generation and maintenance of contralateral nociceptive hypersensitivity. Thalamic microinjection of AAV5-Fto increased TLR4 expression and induced hypersensitivity to mechanical, heat, and cold stimuli. Mechanistically, Coll IV microinjection produced an increase in FTO binding to Tlr4 mRNA, an FTO-dependent loss of m⁶A sites in Tlr4 mRNA and a reduction in the binding of YTHDF2 to Tlr4 mRNA in the ipsilateral thalamus. These findings suggest that FTO is involved in hemorrhage-induced thalamic pain by stabilizing TLR4 upregulation in thalamic neurons.

The researchers knocked down thalamic FTO in male Fto^fl/fl mice by microinjecting AAV5-Cre into the unilateral thalamic VPL and VPM 5 weeks before Coll IV or by microinjecting saline into the same area. AAV5-Gfp was used as a negative control. In the entire ipsilateral thalamus of male Fto^fl/fl mice microinjected with AAV5-Cre at Coll IV, the increase in FTO protein levels caused by Coll IV microinjection was significantly blocked 7 days after microinjection of AAV5-Cre (Figure 1A). Unexpectedly, male Fto^fl/fl mice microinjected with AAV5-Cre failed to change the basal level of FTO protein in the ipsilateral thalamus 7 days after microinjection of saline (Figure 1A). Consistent with the above behavioral observations, significant mechanical allodynia, heat hyperalgesia, and cold hyperalgesia were observed on days 1, 3, and 7 after Coll IV microinjection in AAV5-Gfp pre-microinjected male Fto^fl/fl mice contralaterally (but not ipsilaterally) (Figure 1B-F). However, these hyperalgesias were significantly reduced in Coll IV male Fto^fl/fl mice pre-microinjected with AAV5-Cre (Figure 1B-D). Similar behavioral responses were observed in female Fto^fl/fl mice.

Figure 1. Effect of thalamic pre-microinjection of AAV5-Cre on Coll IV microinjection-induced FTO protein expression and thalamic pain genesis in male Fto^fl/fl mice. (Fu G, et al., 2021)