脊髓损伤
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Figure 2 ｜ Mettl14 is highly expressed in the sensorimotor cortex after SCI.
m6 A modification is common in CNS disease (Lei and Wang, 2022). To detect the expression of m6 A-related genes and proteins in the sensorimotor cortex after SCI, qPCR, westernblotting, and immunofluorescence were conducted. As shown in Figure 2A, Mettl14 expression increased on 3 dpi, peaked on 7 dpi, and remained elevated. The western blot results indicated the same for METTL14 protein (Figure 2B and C). Other m6 Arelated genes were also detected. The expression of Mettl3, Ythdf1, Ythdf3, and Wtap increased, while that of Fto decreased (Figure 2D–J). Western blot analysis demonstrated that METTL3, YTHDF1, and YTHDF3 were highly expressed on 7 dpi (Figure 2K– N). Immunofluorescence staining showed that METTL14 was upregulated and mainly expressed in neurons (NeuN+ cells); it was less expressed in astrocytes (GFAP+ cells) and microglia/ macrophages (F4/80+ cells) (Figure 2O, P, and Additional Figure 1A and B). The m6 A quantification assay showed that RNA extracted from the sensorimotor cortex had an elevated m6 A level (Figure 2Q).
Figure 6 ｜ METTL14 promotes axon regeneration by activating the Trib2/MAPK pathway.
To further investigate whether METTL14 affects axon regeneration by regulating the TRIB2/MAPK pathway, we transfected primary cultured neurons with lentivirus to knockdown Mettl14. As shown in Figure 6A–D, Mettl14 knockdown downregulated TRIB2, p-JNK, and p-P38 protein expression, while OE-Trib2 treatment promoted their expression. Mettl14 knockdown decreased the expression of Ntrk3, Cntf, Igf1r, NeuroD1, and Nrg1 (positive regulation of axon regeneration) and increased the expression of Pten, Cers2, Ptprs (negative regulation of axon regeneration), while OE-Trib2 reduced the effect of Mettl14 knockdown (Figure 6E–L). Immunofluorescence staining showed that Mettl14 knockdown inhibited neurite growth while OE-Trib2 treatment promoted it (Figure 6M and N). The microfluidic plate experiments also indicated that Mettl14 knockdown reduced axonal length, while treating with OE-Trib2 increased it (Figure6O and P). These results demonstrated that METTL14 appear to be an indispensable factor for axon regeneration via activation of the TRIB2/MAPK signaling pathway.
Figure 8 ｜ Syringin promotes CST regeneration and functional recovery after SCI.
To investigate the potential effect of syringin on promoting CST regeneration and neurological recovery after SCI, syringin or vehicle was administered to mice (100 mg/kg per day) by gavage for 1 month (Figure 8A; Li et al., 2017). First, we assessed whether syringin administration had any obvious adverse effects. As shown in Additional Figure 5A and B, syringin administration did not affect body weight or fur color, nor did it result in appreciable cardiotoxicity, hepatotoxicity, spleen cell toxicity, pulmonary toxicity, or nephrotoxicity based on H&E staining (Additional Figure 5C). Immunofluorescence staining showed that syringin upregulated METTL14 expression in sensorimotor neurons (Additional Figure 6). BMS score analysis showed that syringin improved functional recovery from the 2nd week to the observation endpoint (Figure 8B). Compared with the vehicle group, the syringin group showed a lower frequency of hind paw slipping under the grid at 3 weeks and thereafter (Figure 8C). The swimming test suggested that syringin administration improved swimming ability as well (Figure 8D and E). As shown in Figure 8F–H, the mice in the syringin-treated group showed higher motor evoked potentials with no significant change in the latent period compared to the vehicle group. Next, we investigated the effect of syringin on CST regeneration and effective synapticformation. Compared with the vehicle group, the axon density near the lesion was higher in the syringin-treated group; some axons even crossed the lesion, indicating that the regeneration ability of CST was enhanced (Figure 8I–K). Immunofluorescence staining showed that syringin did not affect synaptophysin density in the injured area; however, it increased the number of BDA+ SYN+ dots, indicating that syringin promoted effective synaptic formation (Figure 8L–N). These results suggest that syringin is an ideal small molecule that promoted CST regeneration and improved neurological recovery after SCI.