Ac4C Enhances the Translation Efficiency of Vegfa mRNA and Mediates Central Sensitization in Spinal Dorsal Horn in Neuropathic Pain

Abstract

N4-Acetylcytidine (ac4C), a highly conserved post-transcriptional machinery with extensive existence for RNA modification, plays versatile roles in various cellular processes and functions. However, the molecular mechanism by which ac4C modification mediates neuropathic pain remains elusive. Here, it is found that the enhanced ac4C modification promotes the recruitment of polysome in Vegfa mRNA and strengthens the translation efficiency following SNI. Nerve injury increases the expression of NAT10 and the interaction between NAT10 and Vegfa mRNA in the dorsal horn neurons, and the gain and loss of NAT10 function further confirm that NAT10 is involved in the ac4C modification in Vegfa mRNA and pain behavior. Moreover, the ac4C-mediated VEGFA upregulation contributes to the central sensitivity and neuropathic pain induced by SNI or AAV-hSyn-NAT10. Finally, SNI promotes the binding of HNRNPK in Vegfa mRNA and subsequently recruits the NAT10. The enhanced interaction between HNRNPK and NAT10 contributes to the ac4C modification of Vegfa mRNA and neuropathic pain. These findings suggest that the enhanced interaction between HNRNPK and Vegfa mRNA upregulates the ac4C level by recruiting NAT10 and contributes to the central sensitivity and neuropathic pain following SNI. Blocking this cascade may be a novel therapeutic approach in patients with neuropathic pain.

Keywords: NAT10; VEGFA; ac4C modification; neuropathic pain; spinal dorsal horn; translation efficiency.

Figure 1

SNI induced the upregulation of ac4C modification of Vegfa mRNA. A) Paw withdrawal threshold of rats was significantly reduced on days 4, 7, and 14 following SNI (^** p < 0.01 vs corresponding sham group, n = 12 in each group). B) Heat hyperalgesia was induced on days 4, 7, and 14 after SNI (^** p < 0.01 vs corresponding sham group, n = 8 ‐ 9 in each group). C) Dot blot showed that the level of total RNA ac4C modification with different sample loading amounts increased on days 4, 7, and 14 following SNI in spinal dorsal horn tissue. Methylene blue staining was used as a loading control (^* p < 0.05, ^** p < 0.01 vs corresponding sham group, n = 3 in each group). D) Schematic of acRIP‐seq for mRNA of spinal dorsal horn tissue in rats. E) Metagene plot revealed the distribution of ac4C‐containing peaks across mRNA in sham and SNI groups. F) The motif of ac4C modification in the sham group and SNI group was identified by HOMER. G) VEGFA signaling pathway generated from GSEA. The top portion of the plot showed the enrichment score for the gene set of VEGF signaling pathway. The middle portion of the plot showed the members of the gene set of VEGF signaling pathway. The bottom portion showed the value of gene ranking in VEGF signaling pathway. The ranking value indicated an individual gene's correlation with the mechanical allodynia. H) The ac4C level in Vegfa mRNA exhibited the highest fold change following SNI in VEGF signaling pathway. I) Integrative Genomics Viewer (IGV) tracks displayed ac4C peak distributions across Vegfa mRNA from acRIP‐seq data. The ac4C modification sequence was CTTCCCCAT in the 3′UTR region in the SNI group. J) AcRIP‐qPCR showed that SNI significantly increased the ac4C level of Vegfa mRNA 3′UTR on day 14 (^** p < 0.01 vs corresponding sham group, n = 4 in each group).

Figure 2

Ac4C modification improved the translation efficiency of Vegfa mRNA in neuropathic pain. A) Western blot showed the upregulation of VEGFA on day 14 following SNI (^** p < 0.01 vs corresponding sham group, n = 7 in each group). B) qPCR analysis showed the relative expression of Vegfa mRNA in the spinal dorsal horn on day 14 following SNI (n = 7 in each group). C) Compared with the sham group, the ratio of VEGFA protein to its mRNA was significantly increased following SNI (^* p < 0.05 vs corresponding sham group, n = 7 in each group). D) Schematic diagram of Ribo‐seq for spinal dorsal horn tissue in the rats. E) Venn diagram showed the integration results of acRIP‐seq, Ribo‐seq, and RNA‐seq. F) Friends analysis showed the key genes associated with neuropathic pain and highly correlated with each other. Vegfa showed a strong correlation with other genes. G) By calculating the ratio of ribosome number (Ribo‐up)/unchanged mRNA (RNA‐non‐sig), the genes with increased translation efficiency (Log₂ fold change>0.5) were obtained. H) Venn diagram showed the intersection obtained from Friends analysis (F) and genes with increased translation efficiency (G). I) AcRIP‐qPCR was performed to explore the ac4C level of five mRNA on day 14 after SNI (^* p < 0.05, ^** p < 0.01 vs corresponding sham group, n = 4 in each group). J) The ac4C level of Timp3 mRNA was examined at different time points following SNI (^* p < 0.05 vs corresponding sham group, n = 4 in each group). K) The representation of polysome profiling of PC‐12 cells following glutamate incubation, and relative levels of Vegfa mRNA in each ribosome fraction were quantified (right side). L) The actinomycin D assay showed that glutamate incubation did not change the level of Vegfa mRNA at different time points when compared with the vehicle n = 4 in each group.

Figure 3

NAT10 contributed to the upregulation of ac4C modification in Vegfa mRNA and neuropathic pain following SNI. A) The expression of NAT10 protein was significantly increased on days 4, 7, and 14 following SNI (^* p < 0.05 vs sham group, n = 3 in each group). B) Immunofluorescence staining showed the NAT10 integrated density in the spinal dorsal horn on day 14 after sham and SNI (scale bar = 100 µm, n = 3 in each group). C) Colocalization of NAT10‐positive cells with NeuN‐positive cells (a marker for neurons), GFAP‐positive cells (a marker for astrocytes), or Iba1‐positive cells (a marker for microglia) in spinal dorsal horn were quantified (scale bar  =  25 µm, n = 4 or 5 in each group). D) Intrathecal injection of NAT10 siRNA alleviated the mechanical allodynia induced by SNI (^** p < 0.01 vs corresponding sham group, ^# p < 0.05, ^## p < 0.01 vs corresponding SNI group, n = 12 in each group). E) Intrathecal injection of NAT10 siRNA alleviated the heat hyperalgesia induced by SNI (^** p < 0.01 vs corresponding sham group, ^# p < 0.05, ^## p < 0.01 vs corresponding SNI group, n = 8 in each group). F) Twenty one days after intraspinal injection of AAV‐NAT10‐Flag, the withdrawal threshold was significantly reduced in naïve rats (^* p < 0.05, ^** p < 0.01 vs corresponding AAV‐Flag group, n = 10 in each group). G) Overexpression of NAT10 by injecting AAV‐NAT10‐Flag decreased the withdrawal latency in naïve rats (^** p < 0.01 vs corresponding AAV‐Flag group, n = 8 in each group). H) Application of remodelin or NAT10 siRNA inhibited the ac4C upregulation in the spinal dorsal horn on day 14 following SNI (^* p < 0.05, ^** p < 0.01 vs corresponding sham group, n = 3 in each group). I) The interaction between NAT10 and Vegfa mRNA ac4C sites was increased on day 14 following SNI (^** p < 0.01 vs corresponding sham group, n = 3 in each group). J) Application of NAT10 siRNA inhibited the increase of ac4C level at Vegfa mRNA ac4C sites induced by SNI on day 14 (^** p < 0.01 vs corresponding sham group, ^## p < 0.01 vs corresponding SNI, n = 4 in each group).

Figure 4

The VEGFA upregulation contributed to the central sensitivity and neuropathic pain following SNI. A) The level of VEGFA was significantly increased on days 4, 7, and 14 after SNI (^* p < 0.05 vs corresponding sham group, n = 4 in each group). B) Colocalization of VEGFA‐positive cells with NeuN‐positive cells (a marker for neurons), GFAP‐positive cells (a marker for astrocytes), or Iba1‐positive cells (a marker for microglia) in spinal dorsal horn was quantified (scale bar = 50 µm, n = 3 or 4 in each group). C) Vegfa siRNA treatment ameliorated the increase in the amplitude and frequency of mEPSCs on day 14 induced by SNI (^** p < 0.01 vs the sham group, ^# p < 0.05, ^## p < 0.01 vs the corresponding SNI group, n = 11–14 cells from 7–9 rats in each group). D) The number of depolarization‐induced neuronal firing was reduced in Vegfa siRNA‐treated rats following SNI (^** p < 0.01 vs the sham group, ^## p < 0.01 vs the corresponding SNI group, n = 11–13 cells from 7–8 rats in each group). E,F) Intrathecal injection of Vegfa siRNA alleviated the SNI‐induced mechanical allodynia and heat hyperalgesia (^** p < 0.01 vs corresponding sham group, ^# p < 0.05, ^## p < 0.01 vs corresponding SNI group, n = 7–10 in each group). G) Intraspinal injection of recombinant AAV‐hySn‐Vegfa‐Flag increased the amplitude and frequency of mEPSCs (^* p < 0.05, ^** p < 0.01 vs the AAV‐Flag group, n = 13‐14 cells from 8 rats in each group). H) Overexpression of VEGFA increased the number of depolarization‐induced neuronal firing in naïve rats (^* p < 0.05 vs the AAV‐Flag group, n = 12‐15 cells from 8 rats in each group). I) Local application of VEGFA onto the spinal dorsal horn significantly enhanced the C‐fiber–evoked field potential in naive rats. The trace at the top was recorded before (left) and 210 min after (right) VEGFA application. (^** p < 0.01 relative to the vehicle group, n = 4 in each group). J,K) Overexpression of VEGFA by intraspinal injection of AAV‐Vegfa significantly decreased the mechanical withdrawal threshold and heat hyperalgesia in the naïve rats (^** p < 0.01 vs corresponding naive group, n = 8–10 in each group).

Figure 5

NAT10 mediated VEGFA upregulation and contributed to central hypersensitivity and neuropathic pain following SNI. A) The confocal image showed that NAT10 was expressed on the nucleus of VEGFA‐positive cells in the spinal dorsal horn (scale bar = 10 µm, n = 3). B) Intrathecal injection of remodelin inhibited the VEGFA upregulation induced by SNI (^** p < 0.01 relative to the sham group, ^# p < 0.05 relative to the corresponding SNI group, n = 4 in each group). C) The level of VEGFA protein in the spinal dorsal horn was decreased after intrathecal injection of NAT10 siRNA in SNI rats (^** p < 0.01 relative to the sham group; ^# p < 0.05 relative to the corresponding SNI group, n = 4 in each group). D) Intraspinal injection of AAV‐hySn‐NAT10‐Flag increased the level of VEGFA protein in the spinal dorsal horn in naïve rats (^** p < 0.01 relative to the corresponding naive group, n = 3 in each group). E) Application of anti‐VEGFA neutralizing antibody inhibited the increase in the amplitude and frequency of mEPSCs in the AAV‐hySn‐NAT10‐Flag rats (^** p < 0.01 vs the naïve group, ^# p < 0.05, ^## p < 0.01 vs the AAV‐NAT10 group, n = 11‐15 cells from 8–9 rats in each group). F) Application of anti‐VEGFA neutralizing antibody inhibited the increase in the number of depolarization‐induced neuronal firing induced by the AAV‐hySn‐NAT10‐Flag treatment (^** p < 0.01 vs the naive group, ^## p < 0.01 vs the AAV‐NAT10 group, n = 10‐14 cells from 7–8 rats in each group). G,H) Intrathecal injection of anti‐VEGFA neutralizing antibody also alleviated the mechanical allodynia and heat hyperalgesia induced by AAV‐hySn‐NAT10‐Flag in rats (^* p < 0.05, ^** p < 0.01 vs naive group, ^# p < 0.05, ^## p < 0.01 vs AAV‐NAT10 group, n = 7–9 in each group).

Figure 6

The enhanced interaction between HNRNPK and Vegfa mRNA contributed to the ac4C modification in rats by recruiting NAT10. A) Schematic diagram of RNA pulldown‐LC/MS/MS with Vegfa probe for spinal dorsal horn tissue in rats. B) Results from RNA pulldown‐LC/MS/MS assay showed an interaction between HNRNPK and Vegfa mRNA. C) Friends Analysis showed that the interaction of HNRNPK with other proteins was the most obvious. D) RNA pulldown‐WB assay showed an enhanced interaction between Vegfa mRNA and HNRNPK following SNI (n = 3 in each group). E) The Vegfa mRNA level immunoprecipitated by the HNRNPK antibody was significantly increased (n = 3 in each group). F) Co‐immunoprecipitation results showed that the interaction between NAT10 and HNRNPK was significantly increased in the spinal dorsal horn following SNI (n = 3 in each group). G) High‐resolution images showed that the binding of NAT10 to HNRNPK was significantly upregulated following SNI (scale bar = 50 µm in the left and scale bar = 5 µm in the right, n = 3 in each group). H) Intrathecal injection of HNRNPK siRNA inhibited the enhanced recruitment of NAT10 on Vefga mRNA in SNI rats (^* p < 0.05 relative to the corresponding SNI group, n = 4 in each group). I) Intrathecal injection of NAT10 siRNA did not change the interaction of HNRNPK and Vegfa mRNA in SNI rats (n = 3 in each group). J) HNRNPK siRNA treatment prevented the increase of Vegfa mRNA ac4C modification induced by SNI (^** p < 0.05 relative to the corresponding SNI group, n = 3 in each group). K) Knockdown of HNRNPK by siRNA inhibited the SNI‐induced VEGFA upregulation in the spinal dorsal horn of rats (^** p < 0.05 relative to the corresponding SNI group, n = 4 in each group). L,M) Knockdown of HNRNPK by siRNA alleviated the SNI‐induced mechanical allodynia and heat hyperalgesia (^** p < 0.01 vs corresponding sham group, ^# p < 0.05, ^## p < 0.01 vs corresponding SNI group, n = 10 in each group for mechanical allodynia test and n = 8 in each group for heat hyperalgesia test). N,O) Overexpression of HNRNPK by intraspinal injection of AAV‐Hnrnpk significantly decreased the mechanical withdrawal threshold and heat hyperalgesia in the naïve rats (^** p < 0.01 relative to the corresponding naïve group, n = 10 in each group for mechanical allodynia test and n = 8 in each group for heat hyperalgesia test).

Figure 7

Summary graph for the hypothesis: the mechanism involving NAT10‐mediated VEGFA upregulation contributed to neuropathic pain following SNI.