Volume 23, Issue 3, Pages 716-724 (April 2018) Sarm1/Myd88-5 Regulates Neuronal Intrinsic Immune Response to Traumatic Axonal Injuries  Qi Wang, Shan Zhang, Tingting Liu, Huanhuan Wang, Kaili Liu, Qiujun Wang, Wenwen Zeng  Cell Reports  Volume 23, Issue 3, Pages 716-724 (April 2018) DOI: 10.1016/j.celrep.2018.03.071 Copyright © 2018 The Author(s) Terms and Conditions

Cell Reports 2018 23, 716-724DOI: (10.1016/j.celrep.2018.03.071) Copyright © 2018 The Author(s) Terms and Conditions

Figure 1 Neuronal Intrinsic Immune Response to Traumatic Axonal Injuries (A) Diagram of the in vivo model of traumatic nerve injuries. (B–E) Wild-type mice were subject to the sciatic nerve injury, and their L4 DRGs were harvested at the indicated time points post-injury. (B and C) CD11b+ immune cells were examined by the volume fluorescence-imaging technique. (B) Representative 3D projections of L4 DRGs in the uninjured condition or 72 hr post-injury, coimmunolabeled by anti-CD11b (green) or anti-Tuj1 (magenta, neurons) and imaged at a 6.4× magnification on the lightsheet microscope. The highlighted regions are shown in the insets. (C) CD11b+ immune cells in L4 DRGs were quantified. (D and E) mRNA levels of chemokines (D) and cytokines (E) were determined by the qPCR analysis. ND, not detected. (F) Diagram of the in vitro model of traumatic axonal injuries. (G) Removal of non-neuronal cells by the mitotic inhibitors. (H–J) Cultured neurons were subject to traumatic axonal injuries. (H) Representative images of uninjured neurons (upper) or injured neurons with their distal axons removed (lower). (I and J) mRNA levels of chemokines (I) and cytokines (J) were determined at the indicated time points post-injury by the qPCR analysis. n = 4, mean ± SEM, ∗p < 0.01. See also Figures S1 and S2 and Videos S1 and S2. Cell Reports 2018 23, 716-724DOI: (10.1016/j.celrep.2018.03.071) Copyright © 2018 The Author(s) Terms and Conditions

Figure 2 Sarm1 Regulates the Neuronal Immune Response to Traumatic Axonal Injuries (A) Lentiviral-shRNA knockdown of indicated genes was performed in cultured neurons. The neurons were then subject to traumatic axonal injuries and harvested 24 hr post-injury. mRNA levels of Ccl12 were determined by the qPCR analysis. n = 3. (B and C) Sarm1 is required for the neuronal immune response to traumatic axonal injuries. (B) Representative images of cultured Sarm1+/− or Sarm1−/− neurons. (C) Cultured Sarm1+/− or Sarm1−/− neurons were subject to traumatic axonal injuries and harvested at the indicated time points post-injury. mRNA levels of chemokines and cytokines were determined by the qPCR analysis. n = 3. (D) Domain arrangement of Sarm1 protein. (E–G) Activation of the Sarm1 signal triggers the neuronal immune response. (E) Diagram of the inducible dimerization of FKBP(F36V)-TIR by AP20187. (F) Lentiviral expression of FKBP(F36V)-TIR in cultured neurons was examined by immunoblot analysis. (G) FKBP(F36V)-TIR-expressing or control GFP-expressing neurons were treated with the FKBP dimerization inducer AP20187. mRNA levels of chemokines and cytokines were determined by the qPCR analysis. n = 3. (H) Neuronal expression of Sarm1 mRNA in DRGs was revealed by in situ hybridization. Sense probe was included as control for the specificity of the in situ hybridization procedure. (I) Neuronal expression of Sarm1 protein in DRGs was revealed by immunohistochemistry. (J and K) Sarm1 regulates the neuronal immune response to traumatic nerve injuries. Sarm1−/− or control Sarm1+/+ mice were subject to the sciatic nerve injury, and their L4 DRGs were harvested at the indicated time points post-injury. (J) mRNA levels of chemokines and cytokines were determined by the qPCR analysis. n = 4. (K) Representative 3D projections of Sarm1−/− L4 DRGs at the indicated time points post-injury, coimmunolabeled by anti-CD11b (green) or anti-Tuj1 (magenta, neurons) and imaged at a 6.4× magnification on the lightsheet microscope. Mean ± SEM, ∗p < 0.01, n.s., not significant. See also Figure S3. Cell Reports 2018 23, 716-724DOI: (10.1016/j.celrep.2018.03.071) Copyright © 2018 The Author(s) Terms and Conditions

Figure 3 JNKs Function Downstream of Sarm1 in the Neuronal Immune Response to Traumatic Axonal Injuries (A) Activation of Jnk proteins (p-JNKs) in cultured neurons in response to traumatic axonal injuries was examined by immunoblot analysis. (B and C) Jnk2 and Jnk3 are required for the neuronal immune response to traumatic axonal injuries. (B) Representative images of cultured Jnk2+/−; Jnk3+/− or Jnk2−/−; Jnk3−/− neurons. (C) Cultured Jnk2+/−; Jnk3+/− or Jnk2−/−; Jnk3−/− neurons were subject to traumatic axonal injuries and harvested at the indicated time points post-injury. mRNA levels of chemokines and cytokines were determined by the qPCR analysis. (D) Activation of the Sarm1 signal promotes Jnk activation. FKBP(F36V)-TIR was expressed in cultured neurons by lentiviral transduction. FKBP(F36V)-TIR-expressing neurons were treated with the FKBP dimerization inducer AP20187, and activation of Jnk proteins (p-JNKs) was examined by immunoblot analysis. (E) Jnk2 and Jnk3 are required for Sarm1-mediated neuronal immune response. FKBP(F36V)-TIR was expressed in cultured Jnk2+/−; Jnk3+/− or Jnk2−/−; Jnk3−/− neurons by lentiviral transduction, and the neurons were then treated with AP20187. mRNA levels of chemokines and cytokines were determined by the qPCR analysis. (F and G) Jnk2 and Jnk3 regulate the neuronal immune response to traumatic nerve injuries. Jnk2−/−; Jnk3−/− or control Jnk2+/−; Jnk3+/− mice were subject to the sciatic nerve injury, and their L4 DRGs were harvested at the indicated time points post-injury. (F) mRNA levels of chemokines and cytokines were determined by the qPCR analysis. (G) CD11b+ immune cells in DRGs were quantified. n = 3, mean ± SEM, ∗p < 0.01. See also Figure S4. Cell Reports 2018 23, 716-724DOI: (10.1016/j.celrep.2018.03.071) Copyright © 2018 The Author(s) Terms and Conditions

Figure 4 c-Jun Is Directly Involved in the Neuronal Immune Response to Traumatic Axonal Injuries (A and B) Sarm1 regulates the neuronal activation of transcription factor c-Jun in response to traumatic nerve injuries. Sarm1−/− or control Sarm1+/+ mice were subject to the sciatic nerve injury, and their L4 DRGs were harvested at the indicated time points post-injury. Ser73 phosphorylation of c-Jun (p-c-Jun) in the neurons of DRGs was examined by immunohistochemistry (A) and quantified (B). (C) Jnk signaling is required for the neuronal activation of c-Jun in response to traumatic nerve injuries. Jnk2−/−; Jnk3−/− and control Jnk2+/−; Jnk3+/− mice were subject to the sciatic nerve injury, and their L4 DRGs were harvested at the indicated time points post-injury. Ser73 phosphorylation of c-Jun (p-c-Jun) in the neurons of DRGs was quantified. (D) c-Jun is required for the neuronal immune response to traumatic axonal injuries. Lentiviral expression of Cre recombinase or control GFP was performed in cultured c-junfl/fl neurons. The neurons were then subject to traumatic axonal injuries and harvested at the indicated time points post-injury. mRNA levels of chemokines and cytokines were determined by the qPCR analysis. (E) c-Jun directly regulates the specific expression of chemokines and cytokines in the neurons in response to traumatic axonal injuries. c-Jun-targeting sites in the neurons at 24 hr post-injury were profiled by the ChIP-seq analysis, and the ChIP-seq tracks at the indicated loci are shown. n = 3, mean ± SEM, ∗p < 0.01. See also Figure S4. Cell Reports 2018 23, 716-724DOI: (10.1016/j.celrep.2018.03.071) Copyright © 2018 The Author(s) Terms and Conditions