Fig 2: A recapitulating scheme of the proposed molecular mechanism of Npy expression upon lysine deprivation. Lysine deprivation activates GCN2 signaling by accumulating uncharged tRNA. Then, GCN2 signaling phosphorylates the a subunit of eIF2 on serine 51. Next, the phosphorylated eIF2a restrains translation initiation to lessen global protein synthesis, whereas still allowing continued translation of specific transcripts, ATF4, that coordinate cellular stress feedback. ATF4, as a master transcription factor, induces Npy expression, which further exerts the orexigenic effect to maintain energy and metabolism homeostasis.

Fig 3: TET3 negatively regulates the expression of NPY and VGAT.(A) GT1-7 cells transfected with NT siRNA were maintained in Lept L (NT siRNA/Lept L) or Lept H (NT siRNA/Lept H) or were transfected with Tet3 siRNA and maintained in Lept H (Tet3 siRNA/Lept H). RNAs were extracted at 12 hours (for Tet3) or 36 hours (for Npy and Slc32a1) following the switch and analyzed by qPCR. n = 3 per group in technical replicates. **P < 0.01 and ***P < 0.001, by 1-way ANOVA with Tukey’s post test. (B) Representative immunoblots for TET3, NPY, and VGAT from GT1-7 cells treated as in A. Proteins were isolated at the 36-hour time point. (C) SH-SY5Y cells transfected with NT siRNA were maintained in Lept L (NT siRNA/Lept L) or Lept H (NT siRNA/Lept H) or were transfected with TET3 siRNA and maintained in Lept H (TET3 siRNA/Lept H). RNA was extracted at 12 hours (for TET3) or 36 hours (for NPY and SLC32A1) following the switch and analyzed by qPCR. n = 3 per group in technical replicates. **P < 0.01 and ***P < 0.001, by 1-way ANOVA with Tukey’s post test. (D) Representative immunoblots for TET3, NPY, and VGAT from SH-SY5Y cells treated as in C. Proteins were isolated at the 36-hour time point. (E and F) Representative micrographs of NPY (green) and VGAT (green) in the ARCs of Cas9+ mice injected with AAV or AAV-sgTet3. AGRP neurons from the injected viruses are labeled red. Scale bars: 50 µm. All data represent the mean ± SEM.

Fig 4: FLT3 is critical for upregulation of markers associated with peripheral nerve injury. a Changes in PNP-related biomarkers mRNA expression, measured by q-PCR 3 days after sham or CCI surgery in wild-type (WT) or Flt3-knockout (Flt3KO) mice. Values are means ± s.e.m of data from 4 animals. One-way ANOVA and Bonferroni’s test, *P < 0.05; **P < 0.01 vs. respective Sham, #P < 0.05; ##P < 0.01 vs. CCI WT. b ATF3, NPY, and GFAP immunoreactivity in DRG from WT and Flt3KO CCI mice, 14 days post-CCI. Scale bars = 100 µm except for GFAP: 50 µm. c Quantification of experiments as in b. d GFAP and IBA1 immunoreactivity in DSC from WT and Flt3KO CCI mice. Scale bars = 100 µm. e Quantification of experiments as in d. f Normalized gene expressions of Csf1 and Csf1R measured by q-PCR in DRG and DSC from WT and Flt3KO sham or 3 days post-CCI mice. Means ± s.e.m. of data from 4 animals. One-way ANOVA and Bonferroni’s test, *P < 0.05; **P < 0.01 vs. respective Sham, #P < 0.05; ##P < 0.01 vs. CCI WT. g CSF1 and ATF3 immunoreactivity in DRG from WT and Flt3KO CCI mice at 3 days post-injury. Bars = 100 µm. h Quantification of experiments as in g. i CSF1-induced mechanical pain hypersensitivity in WT and Flt3KO mice. Means ± s.e.m. of data from 8 animals. c, e, f, h, i NS non-significant; unpaired Mann–Whitney t-test (c, d, f, h); two-way ANOVA and Bonferroni’s test (i), *P < 0.05; **P < 0.01 vs. saline

Fig 5: Curative effects of Flt3 downregulation on PNP-related genes, and nerve injury-induced and FL-induced pain hypersensitivity. a Mechanical pain hypersensitivity after intrathecal infusion for 4 days of scrambled- or Flt3-siRNA in CCI mice. Means ± s.e.m. of data from 8 animals. Two-way ANOVA and Dunnett’s test, *P < 0.05 vs. Scrambled siRNA animals. b ATF3, NPY, and GFAP immunoreactivity in lumbar DRG after intrathecal infusion for 4 days of scrambled- or Flt3-siRNA in CCI mice. Bars = 100 µm except for GFAP: 50 µm. c Quantification of experiments as in b. Means ± s.e.m. of data from 4 animals. Mann–Whitney t-test, *P < 0.05. d Localization of the AAV9 virus expressing anti-Flt3 shRNA and GFP in neurons (TUJ1-positive cells), but not in CD45- or S100ß-positive cells in DRG of WT mice. Scale bars = 100 µm. e, f Effects of Flt3-shRNA on FL-induced mechanical pain hypersensitivity (e), and on pain-related behaviors (f). FL (500 ng/10 µl) was injected intrathecally 17 days after the virus. Means ± s.e.m. of data from 8 animals. One-way ANOVA and Dunnett’s test, *P < 0.05; **P < 0.01; ***P < 0.001 vs. Bsl; two-way ANOVA with repeated measures and Dunnett’s test, #P < 0.05 vs. non-targeting shRNA. g Effects of Flt3-shRNA on mechanical pain hypersensitivity in CCI mice. The virus was injected intrathecally 48 h before CCI. Means ± s.e.m. of data from 8 animals. Two-way ANOVA with repeated measures and Dunnett’s test, *P < 0.05 vs. non-targeting shRNA