MAP4K4 Activation Mediates Motor Neuron Degeneration in Amyotrophic Lateral Sclerosis  Chen Wu, Michelle E. Watts, Lee L. Rubin  Cell Reports  Volume 26, Issue 5, Pages 1143-1156.e5 (January 2019) DOI: 10.1016/j.celrep.2019.01.019 Copyright © 2019 The Authors Terms and Conditions

Cell Reports 2019 26, 1143-1156.e5DOI: (10.1016/j.celrep.2019.01.019) Copyright © 2019 The Authors Terms and Conditions

Figure 1 The Suppression of Activated MAP4K4 Signaling in Stressed MNs Promotes Survival (A) Western blot analysis and quantification of MAP4K4 pathway activation via p-c-Jun and total c-Jun (c-Jun) levels in Hb9-GFP motor neurons (MNs) in the presence (+TF) or absence (−TF) of trophic factors. (B) Western blot analysis along with quantification showing p-c-Jun and c-Jun levels in Hb9-GFP and SOD1G93A/Hb9-GFP mouse ESC-derived MNs. Samples were collected 3 days after seeding in the presence of trophic factors (TFs). (C) Representative images of Hb9-GFP and SOD1G93A/Hb9-GFP mouse ESC-derived MNs 3 days post-treatment with scrambled siRNA or MAP4K4 siRNA. Scale bar indicates 100 μm. (D) Fold change in the survival of mouse Hb9-GFP MNs and SOD1G93A/Hb9-GFP MNs in the presence and absence of TFs 4 days post-treatment, calculated through high content image quantification. (E) Western blot analysis and quantification showing MAP4K4 signaling levels after treatment of mouse MNs with different MAP4K4 siRNA concentrations (2.5–20 nM). Samples collected 48 h post-transduction. (F) Fold change in the survival of SOD1G93A/Hb9-GFP derived MNs in different siRNA concentrations relevant to (E). Data are presented as mean ± SEM, n (technical repeats) ≥ 3, N (biological repeats) ≥ 3. Cell Reports 2019 26, 1143-1156.e5DOI: (10.1016/j.celrep.2019.01.019) Copyright © 2019 The Authors Terms and Conditions

Figure 2 The Effect of MAP4K4 Inhibition on the Survival of Human ESC and/or iPSC-Derived MNs (A) Representative images of H9/Isl-RFP-derived, BJsiPSC-derived, and SOD1L144FiPSC-derived MNs treated with scramble siRNA or MAP4K4 siRNAs for 20 days. Islet (red), Tuj1 (green), and DAPI (blue) staining shown in images. Scale bar represents 100 μm. (B) Fold change in the survival of human MNs derived from H9/Isl-RFP ESCs, BJsiPSCs, and SOD1L144FiPSC treated with scrambled siRNA and MAP4K4 siRNA, respectively. (C) Dot blot and western blot analysis and quantification results of MAP4K4 and phosphorylated-MAP4K4 (p-MAP4K4) levels after treating with various concentrations of MAP4K4 siRNA (5–20 nM) for 48 h in SOD1L144FiPSC-derived MNs. (D) MAP4K4 mRNA expression in SOD1L144FiPSC-derived MNs treated with various siRNA concentrations (5–20 nM). (E) Fold change in the survival of SOD1L144FiPSC-derived MNs treated with various siRNA concentrations (5–20 nM) relative to scrambled siRNA (10 nM). Data are presented as mean ± SEM, n (technical repeats) ≥ 3, N (biological repeats) ≥ 3. Cell Reports 2019 26, 1143-1156.e5DOI: (10.1016/j.celrep.2019.01.019) Copyright © 2019 The Authors Terms and Conditions

Figure 3 The Rescue Effect of MAP4K4 Inhibition Is Mediated by JNK3 and Partially by Attenuating c-Jun-Induced Apoptosis (A) Fold change in the survival of human MNs derived from SOD1L144FiPSC treated with scrambled siRNA, c-Jun siRNA, and MAP4K4 siRNA, respectively. (B) p-c-Jun and cleaved caspase-3 levels measured in Hb9-GFP MNs treated with TFs, 18 and 40 h of TF withdrawal, and MAP4K4 siRNA treatment. (C) SP600125, a JNK inhibitor, at 1 μM promoted survival in BJsiPSC-derived MNs and SOD1L144FiPSC-derived MNs during TF withdrawal. (D and E) Knockdown of JNK1, JNK2, and JNK3 by siRNAs in H9/Isl-RFP ESCs-derived MNs (D) and SOD1L144FiPSC-derived MNs (E). Three JNK1 siRNAs, three JNK2 siRNAs, and four JNK3 siRNAs were introduced into MNs, respectively. (F) Survival effects of the combination of MAP4K4 and JNK3 siRNAs compared with MAP4K4 or JNK3 siRNAs alone in SOD1L144FiPSC-derived MNs; 20 nM of each siRNA was used to make a final concentration of 10 nM in the combination siRNA mix. Data are presented as mean ± SEM, n (technical repeats) ≥ 3, N (biological repeats) ≥ 3. Cell Reports 2019 26, 1143-1156.e5DOI: (10.1016/j.celrep.2019.01.019) Copyright © 2019 The Authors Terms and Conditions

Figure 4 The Effect of MAP4K4 Suppression Is Cell Autonomous and Its Inhibition Preserves Neurite Outgrowth (A) The survival effect of MAP4K4 suppression by shRNAs in Hb9-GFP-positive MNs derived from both Hb9-GFP and SOD1G93A/Hb9-GFP ESCs. (B) Representative images of wild-type and SOD1 mutant MNs treated with scrambled siRNA, MAP4K4 siRNA, and TFs for 20 days, respectively. Multiple neurite outgrowth properties were measured using imaging analysis software. Arrows represent maximum neurite length in (b), extremities (c), nodes (e), and roots (f). Scale bar represents 100 μm. (C) Quantification of neurite outgrowth properties in Hb9-GFP MNs and SOD1G93A/Hb9-GFP MNs treated with scrambled siRNA, MAP4K4 siRNA, and TFs for 20 days. Blue, red, and green indicate scrambled siRNA, MAP4K4 siRNA, and TFs treated MNs, respectively. Blank and patterned columns represent Hb9-GFP MNs and SOD1G93A/Hb9-GFP MNs. Data are presented as mean ± SEM, n (technical repeats) ≥ 3, N (biological repeats) ≥ 3. See also Figure S5 and Table S1. Cell Reports 2019 26, 1143-1156.e5DOI: (10.1016/j.celrep.2019.01.019) Copyright © 2019 The Authors Terms and Conditions

Figure 5 MAP4K4 Suppression Reduces Mutant SOD1 via Activating FoxO-Dependent Autophagy (A) Western blot analysis and quantification of SOD1G93A levels (SOD1G93A) and endogenous SOD1 levels (wt SOD1) in mouse Hb9-GFP and SOD1G93A/Hb9-GFP MNs after 20 days treatment with scrambled siRNA and MAP4K4 siRNA. (B) Western blot analysis and quantification showing p62 levels in mouse Hb9-GFP MNs and SOD1G93A/Hb9-GFP MNs. Samples collected 48 h after seeding. (C) Western blot analysis of LC3B levels in Hb9-GFP MNs and SOD1G93A/Hb9-GFP MNs after treatment with a lysosomal inhibitor (NH4Cl) and/or MAP4K4 siRNAs for 6 h. (D) p62 and LC3 levels measured by western blot in MNs after 48 h treatment with scrambled siRNA or MAP4K4 siRNAs. (E) Phosphorylated p70S6K (p-p70), total p70S6K (p70), phosphorylated S6 (p-S6), total S6 (S6), phosphorylated FoxO1 (p-FoxO1), and total FoxO1 (FoxO1) evaluated by western blot after 48 h treatment with scrambled siRNA or MAP4K4 siRNAs. (F) Western blot analysis along with quantification showing FoxO1 levels in SOD1G93A/Hb9-GFP mouse ESC-derived MNs after siRNA transfections. (G) Survival effects of the combination of MAP4K4 and FoxO1 siRNAs compared with MAP4K4 siRNAs alone in SOD1L144FiPSC-derived MNs. See also Figure S6 and Table S1. Cell Reports 2019 26, 1143-1156.e5DOI: (10.1016/j.celrep.2019.01.019) Copyright © 2019 The Authors Terms and Conditions

Figure 6 Chemical Inhibition of MAP4K4 Improves MN Survival (A) Dose response of a MAP4K4-specific inhibitor (MAP4K4i) on the survival of human H9/Isl-RFP ESCs-derived MNs and SOD1L144FiPSC-derived MNs during TF withdrawal. Kenpaullone (10 μM) was used as a control. (B) MAP4K4i improves survival of BJsiPSC-derived, TDP43G298S iPSC-derived, and TDP43M377V iPSC-derived MNs during TF withdrawal. Kenpaullone (10 μM) was used as a control. (C) Dose-response survival effect of MAP4K4 selective inhibitor (MAP4K4i) on mouse Hb9-GFP MNs and SOD1G93A/Hb9-GFP MNs in both the presence and absence of TFs. Kenpaullone (10 μM) was used as a control. (D) Survival effect of MAP4K4i treatment for 16 days after TF withdrawal on primary cultured MNs derived from wild-type and SOD1G93A mouse spinal cords. Kenpaullone (10 μM) was used as a control. (E) MAP4K4i treatment for 16 days prevents basal cell death of spinal cord MNs derived from both wild-type and SOD1G93A mice. Data are presented as mean ± SEM, n (technical repeats) ≥ 3, N (biological repeats) ≥ 3. See also Figure S7. Cell Reports 2019 26, 1143-1156.e5DOI: (10.1016/j.celrep.2019.01.019) Copyright © 2019 The Authors Terms and Conditions

Figure 7 Proposed Model of MAP4K4-JNK in Regulating MN Degeneration The MAP4K4-JNK signaling cascade involved in MN degeneration. TF withdrawal or other extracellular stimuli lead to the phosphorylation of MAP4K4. Activated MAP4K4 activates its downstream targets, including JNK and c-Jun. Phosphorylated c-Jun regulates transcription in the nucleus and induces apoptosis. Phosphorylated JNK additionally works to inhibit FoxO1, leading to the inhibition of autophagy. MAP4K4 siRNA, MAP4K4i, JNK3 siRNA, and SP600125 block the signal cascade and rescue MN degeneration. Cell Reports 2019 26, 1143-1156.e5DOI: (10.1016/j.celrep.2019.01.019) Copyright © 2019 The Authors Terms and Conditions