Fig 1.
FAM173B knockdown abrogated persistent inflammatory and neuropathic pain.
(A) Intrathecal mFam173b antisense ODN (mFam173b-AS) injection at day 5, 6, 7, 9, and 10 after intraplantar injection of CFA reduces mFam173b mRNA expression (corrected for housekeeping genes GAPDH and HPRT) in the DRG 24 hours after the last mFam173b-AS injection (n = 8 mice). (B–D) Time course of (B) thermal and (C, D) mechanical hyperalgesia following (B, C) intraplantar injection of CFA (n = 8 mice), veh (n = 4 mice), or (D) after SNI (n = 4 mice). Mice received intrathecal injections of mFam173b-AS or MM-ODN at days 5, 6, 7, 9, and 10 during inflammatory pain or day 1–9 after SNI. Data are represented as mean ± SEM. * = P < 0.05; ** = P < 0.01; *** = P < 0.001. Statistical analyses were performed by unpaired two-tailed t tests (A) or by two-way repeated measures ANOVA followed by a post-hoc Holm-Sidak multiple comparison test (B–D). Underlying data can be found in S1 Data. CFA, complete Freund’s adjuvant; contra, contralateral; DRG, dorsal root ganglia; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; HPRT, Hypoxanthine Phosphoribosyltransferase 1; ipsi, ipsilateral; MM-ODN, mismatch ODN; ODN, oligodeoxynucleotide; SEM, standard error of the mean; SNI, spared nerve injury; veh, vehicle.
Fig 2.
Sensory neuron FAM173B promotes chronic pain.
(A) Intraplantar injections with HSV amplicons (day 1 and 3) encoding either for hFAM173B-GFP or GFP alone (HSV-EV) induced hFAM173B expression selectively in sensory neurons at day 4. Left panel depicts rabbit IgG control. Nuclei are visualized with DAPI (blue). Scale bar is 50 μm. For larger magnification of FAM173B staining of the DRG after HSV-hFAM173b, see S1 Fig. (B, C) Intraplantar HSV-hFAM173B injection at day 5 and 7 rescued mFam173b-AS–mediated attenuation of (B) thermal and (C) mechanical hypersensitivity in the CFA model of persistent inflammatory pain. Mice received intrathecal ODN at day 5, 6, 7, 9, and 10 after CFA (mFam173b-AS n = 8; MM-ODN n = 4 mice). (D, E) Intraplantar HSV-hFAM173B injections at 3 and 1 day prior to intraplantar carrageenan injection prolonged transient inflammatory (D) thermal and (E) mechanical hypersensitivity (carrageenan: n = 10, vehicle: n = 6 mice). (F) Intraplantar HSV-hFAM173B injections at 3 and 1 day prior to a unilateral intraplantar carrageenan injection reduced weight bearing of the affected paw that persisted at least until day 19 in HSV-FAM173B–injected but not HSV-EV–injected mice (n = 6 mice). (G) Ongoing spontaneous pain measured with gabapentin-induced place preference in HSV-hFAM173B– but not HSV-EV–treated mice 1 month after intraplantar carrageenan injection (n = 6 mice). Data are represented as mean ± SEM. * = P < 0.05; ** = P < 0.01; *** = P < 0.001. Statistical analyses were performed by unpaired two-tailed t tests (G) or by two-way repeated measures ANOVA followed by a post-hoc Holm-Sidak multiple comparison test (B–F). Underlying data can be found in S1 Data. CFA, complete Freund’s adjuvant; DAPI, 4′,6-diamidino-2-phenylindole; DRG, dorsal root ganglia; GFP, green fluorescent protein; HSV, herpes simplex virus; HSV-EV, HSV empty vector; IgG, immunoglobulin G; MM-ODN, mismatch ODN; ODN, oligodeoxynucleotide; SEM, standard error of the mean.
Fig 3.
FAM173B is a lysine-specific methyltransferase.
(A) Topology diagram of archetypical 7BS methyltransferase with alpha-helices (boxes) and β-strands (arrows). (B) Protein sequence alignment of FAM173A/B from Homo sapiens (h), Mus musculus (m), and the aKMT homolog of FAM173B (FAM173hom) from Sulfolobus islandicus (Si). Predicted secondary structure of mFam173b above alignment, coded as in A. Red bars indicate predicted N- and C-terminus of mFam173b. Motif I and Post I (boxed) are involved in binding of SAM. Asp94 (*) was mutated to generate an enzymatically inactive protein. The first residue (Thr56) in recombinant truncated hFAM173B (FAM173BΔ55) is also indicated (vertical arrow). (C) Fluorography of HEK293-extracts incubated with [3H]-SAM and recombinant hFAM173BΔ55. (D, E) WT FAM173BΔ55 (D, E) but not FAM173BΔ55-D94A (E) methylated lysine-homopolymers (n = 3 MTase reactions). Data are represented as mean ± SEM. * = P < 0.05; ** = P < 0.01. Statistical analyses were performed by one-way ANOVA followed by a post-hoc Holm-Sidak multiple comparison test (D) or by an unpaired two-tailed t test (E). Underlying data can be found in S1 Data. 7BS, 7 β-strands; HEK293, human embryonic kidney 293 cells; MTase, methyltransferase; SAM, S-adenosyl-L-methionine; WT, wild-type.
Fig 4.
FAM173B is localized in mitochondria.
(A) hFAM173B-GFP (green) colocalized with the mitochondrial dye MitoTrackerRedCMXROS but not with endoplasmic reticulum (PDI) or Golgi (PGM130) (all red). Scale bar 10 μm. (B) Electron microscopy of GFP-tagged, hFAM173B-expressing HEK293. Dotted line: boundary between nontransfected (left) and transfected cell (right). Scale bar 500 nm. (C) Cultured primary sensory neurons were stained for endogenous mFam173b and the mitochondrial marker COXIV. Right panel is the colocalization profile at the white line shown in panel 3 of the double immunostaining for mFam173b and COXIV. Scale bar 10 μm. COXIV, cytochrome c oxidase IV; HEK293, human embryonic kidney 293 cells; M, mitochondrion; N, nucleus; PDI, protein disulfide-isomerase.
Fig 5.
FAM173B and mitochondrial function.
MitoTrackerRedCMXROS fluorescence, as a measure of mitochondrial potential, 48 hours after (A) mFam173b knockdown (MM-ODN n = 14; mFAM173b-AS n = 16 wells) or (B) hFAM173B overexpression (n = 7 wells) in N2A cells. (C) ΔTMRM fluorescence in HSV-mediated hFAM173B expression in cultured primary sensory neurons increased (n = 100–145 cells, 7 cultures). (D) HSV-mediated hFAM173B expression in cultured primary sensory neurons increased ROS production (DHE) after vehicle or 6 hours stimulation with 100 ng/ml TNFα (n = 90–130 cells, 9 cultures). (E–F) In vivo expression of hFAM173B in sensory neurons with HSV-hFAM173B prior to intraplantar carrageenan increased (E) DHE fluorescence intensity at day 5 (n = 7 mice) and (F) MitoTrackerRedCMH2-XROS fluorescence intensity at day 3 (n = 9 mice) and day 6 (EV n = 4, hFAM173B n = 6 mice) in small-diameter neurons. (G) Intraperitoneal injection of the ROS scavenger PBN attenuated the hFAM173B-mediated prolongation of carrageenan-induced mechanical hypersensitivity (n = 5 mice; HSV-FAM173B + PBN n = 6 mice). Data are represented as mean ± SEM. * = P < 0.05; ** = P < 0.01. Statistical analyses were performed by unpaired two-tailed t tests (A-C, E/F), by one-way (D) or two-way (G) repeated measures ANOVA followed by a post-hoc Holm-Sidak multiple comparison test. For exemplar pictures of A, B, and F, see S3 Fig. Blue bars indicate mFam173b knockdown and red bars/lines hFAM173B overexpression. Underlying data can be found in S1 Data. DHE, dihydroethidium; EV, empty vector; HSV, herpes simplex virus; MM-ODN, mismatch ODN; N2A, Neuro2a; ODN, oligodeoxynucleotide; PBN, phenyl-N-t-butylnitrone; ROS, reactive oxygen species; SEM, standard error of the mean; TNFα, tumor necrosis factor alpha.
Fig 6.
FAM173B-induced microglia activation.
(A) IL6 release by spinal microglia after stimulation for 24 hours with supernatants of hFAM173B-expressing sensory neurons that were previously stimulated with 100 ng/ml TNFα +/- PBN (2 mM; ROS scavenger) for 6 hours, washed, and cultured for a subsequent 15 hours to capture sensory neuron–derived factors (EV-PBN n = 11; FAM173B-PBN n = 7; EV+PBN and FAM173B+PBN n = 3 wells; 100% = 59.4 pg/ml based on 3 independent experiments). (B) Intrathecal anti-TNFα (100 μg/mouse, HSV-FAM173B: n = 6; HSV-EV: n = 6 mice) or (C) minocycline (30 μg/mouse, minocycline: n = 12, vehicle: n = 6 mice) injection 7 days after intraplantar carrageenan attenuates hFAM173B-mediated prolongation of carrageenan-induced hyperalgesia. (D–E) Intraplantar HSV-hFAM173B injection prior to induction of paw inflammation increased Iba1-positive area in (D) spinal cord and (E) DRG at day 5 (n = 4 mice) and day 10 (n = 6 mice) after intraplantar carrageenan injection. (F) Exemplar images of quantified Iba1 staining in D and E. Scale bars 50 μm. Data are represented as mean ± SEM. * = P < 0.05; ** = P < 0.01; *** = P < 0.001. Statistical analyses were performed by one-way ANOVA (A) or by two-way repeated measures ANOVA (B–E) followed by a post-hoc Holm-Sidak multiple comparison test. Underlying data can be found in S1 Data. DRG, dorsal root ganglia; EV, empty vector; HSV, herpes simplex virus; Iba1, ionized calcium binding adaptor molecule 1; IL6, interleukin 6; PBN, phenyl-N-t-butylnitrone; ROS, reactive oxygen species; SEM, standard error of the mean; TNFα, tumor necrosis factor α.
Fig 7.
FAM173B knockdown prevents microglia activation.
(A–H) Mice received intraplantar CFA to induce persistent hyperalgesia and received intrathecal mFam173b-AS to knockdown mFam173b or MM-ODN as control at day 5, 6, 7, 9, and 10. At day 11 after CFA injection, microglia activation in (A–C) DRG and (D–F) spinal cord was assessed by analysis of (A, D) fluorescent Iba1-positive area (n = 4 mice) and (B, E) Iba1 mRNA (n = 8 mice) in the dorsal horn of spinal cord or DRG. (C, F) Exemplar images of Iba1 staining of (C) DRG and (F) spinal cord as quantified in A and D. Scale bars 50 μm. The specific area quantified in the spinal cord is shown in S4D Fig. (G, H) Inflammatory mediator mRNA expression 24 hours after the last intrathecal injection of mFam173b-AS to knockdown mFam173b (day 11 after CFA) in (G) spinal cord and (H) DRG (n = 8 mice). Data are represented as mean ± SEM. * = P < 0.05; ** = P < 0.01; *** = P < 0.001. Statistical analyses were performed by unpaired two-tailed t tests ([A, B], [D, E]), by one-way ANOVA (G/H) followed by a post-hoc Holm-Sidak multiple comparison test. Underlying data can be found in S1 Data. CFA, complete Freund’s adjuvant; DRG, dorsal root ganglia; Iba1, ionized calcium binding adaptor molecule 1; MM-ODN, mismatch ODN; ODN, oligodeoxynucleotide; SEM, standard error of the mean.
Fig 8.
Methyltransferase activity requirement.
WT hFAM173B but not hFAM173B-D94A expression in sensory neurons prolonged carrageenan-induced (A) mechanical and (B) thermal hypersensitivity (n = 6 mice). (C–F) Compared to WT hFAM173B, the methyltransferase-inactive mutant hFAM173B-D94A did not increase Iba1-positive area in (C) DRG and (D) dorsal horn of the spinal cord (n = 5 mice) at day 5 after intraplantar carrageenan injection, or enhance (E) ΔTMRM fluorescence in N2A in vitro (n = 115–150 cells) and (F) ROS production in small-diameter neurons in vivo (hFAM173B n = 7; hFAM173B-D94A n = 5 mice) at 5 days after intraplantar carrageenan. (G) Supernatants of 15 h–cultured sensory neurons expressing hFAM173B-D94A that were stimulated with 100 ng/ml TNFα for 6 hours and subsequently washed did not enhance IL6 release in spinal microglia in vitro (hFAM173B n = 4; hFAM173B-D94A n = 8 wells). Data are represented as mean ± SEM. * = P < 0.05; ** = P < 0.01; *** = P < 0.001. Statistical analyses were performed by unpaired two-tailed t tests (F, G), one-way ANOVA (C–E), or by a two-way ANOVA (A, B) followed by a post-hoc Holm-Sidak multiple comparison test. Underlying data can be found in S1 Data. DRG, dorsal root ganglia; Iba1, ionized calcium binding adaptor molecule 1; IL6, interleukin 6; N2A, Neuro2a; ROS, reactive oxygen species; SEM, standard error of the mean; TMRM, tetramethylrhodamine methyl ester; TNFα, tumor necrosis factor α; WT, wild-type.
Fig 9.
Inflammation increases FAM173B expression. Mitochondrial FAM173B methylates lysine(s) on a yet unknown substrate(s), causes mitochondrial hyperpolarization (ΔΨm↑), and increases superoxide and ROS production, which activates glial cells, thereby promoting persistent inflammatory pain. ΔΨm, mitochondrial membrane potential; ROS, reactive oxygen species.