Fig 2: Representative immunofluorescence staining of Npy. Representative immunofluorescence images (A) and relative green fluorescence intensity showing rate of Npy by immunofluorescence staining (B). Brain cells of mandarin fish were cultured in lysine-deprived medium alone or with 1 µM GCN2iB and 200 nM ISRIB treatments for 120 or 60 min, respectively. Npy (green), DAPI (blue) (scale bar = 100 µm). Results were expressed from at least three independent experiments. Labeled means without a common lowercase letter differ significantly (p < 0.05).

Fig 3: 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 4: A proposed model.(A) In a fasted state, leptin signaling and TET3 levels are low, and there is no association of the chromatin-modifying complex with the Agrp promoter. Histones are acetylated and the chromatin is in an open state, Agrp transcription is on, and the neurons are active. The lack of inhibition of expression of Agrp, Npy, and Slc32a1 by TET3 enables sustained production and synaptic release of AGRP, NPY, and GABA. The physiological outcomes are increased food intake and decreased energy expenditure. (B) In a fed state, a rise in leptin levels promotes binding of p-STAT3 to the Agrp promoter, which in turn recruits TET3 and the chromatin-modifying complex. Binding of TET3 induces 5hmC modification, which is required for a stable association of STAT3 and the chromatin-modifying complex with the promoter. The chromatin-modifying complex promotes histone deacetylation, thereby inducing a closed chromatin state and inhibition of Agrp transcription. Neuronal activity is also suppressed, in part, by a yet-unknown TET3-mediated mechanism. The expression of all 3 genes is reduced. The physiological outcomes are decreased food intake and increased energy expenditure. (C) In a fed state without TET3 expression, there is no 5hmC modification, activated STAT3 is unable to stably associate with the Agrp promoter to allow recruitment of the chromatin-modifying complex, histones remain acetylated, the chromatin is open, and Agrp transcription is not inhibited. In addition, the neuron remains active due to the lack of inhibition from a yet-unidentified TET3-dependent mechanism. The lack of inhibition of expression of all 3 genes enables sustained production and synaptic release of AGRP, NPY, and GABA. The physiological outcomes are increased food intake and decreased energy expenditure. The sustained neuronal activity and synaptic release of AGRP, NPY, and GABA also promote anti-stress effects.

Fig 5: 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.