1. Volume 18, Issue 6, Pages 1484-1498 (February 2017)
Single-Cell Analysis of SMN Reveals Its Broader Role in Neuromuscular Disease 
Natalia Rodriguez-Muela, Nadia K. Litterman, Erika M. Norabuena, Jesse L. Mull, Maria José Galazo, Chicheng Sun, Shi-Yan Ng, Nina R. Makhortova, Andrew White, Maureen M. Lynes, Wendy K. Chung, Lance S. Davidow, Jeffrey D. Macklis, Lee L. Rubin 
Cell Reports 
Volume 18, Issue 6, Pages (February 2017)
DOI: /j.celrep
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2. Cell Reports 2017 18, 1484-1498DOI: (10.1016/j.celrep.2017.01.035)
Copyright © 2017 The Author(s) Terms and Conditions

3. Figure 1
Motor Neurons In Vitro and In Vivo Show Remarkable Heterogeneity in SMN Levels
(A) Lysates of WT and Smn−/−;SMN2+/+;Hb9-GFP (SMA A2) MN cultures at dissociation were immunoblotted with SMN and Actin antibodies. SMN levels are extremely reduced in Smn−/−;SMN2+/+;Hb9::GFP cells.
(B) Quantification of MN number 24 hr after dissociation in WT and Smn−/−;SMN2+/+;Hb9-GFP (A2) cultures reveals that SMN-deficient MNs have reduced survivability in this early period (∗∗p < 0.01, Student’s t test; n = 11).
(C) Mouse Hb9-GFP (WT and A2) MNs fixed and immunostained with the SMN antibody (red). DNA dye bisbenzimide (Hoechst 33258) was used to stain the nucleus (blue), and GFP marks MNs (green). Dotted lines outline MNs. SMN expression is indicated with closed triangles marking high, open triangles marking medium, and the V-shape marking low levels in individual cells. Scale bar, 50 μm.
(D) Histogram analysis showing the percentage of MNs falling into each SMN intensity bin.
(E and F) Representative western blot of lysates from MN cultures differentiated from iPSCs derived from a control and type III, II, and I SMA patients showing SMN levels (E). The quantification of four biological repeats from independent sets of MN differentiations is shown in (F). (One-way ANOVA test followed by Dunnett’s analysis, ∗∗p < 0.01, ∗∗∗p < 0.001; n = 4).
(G) Histogram analysis from MNs derived from iPSCs from SMA patients with different disease severities.
(H) Histogram analysis from MNs derived from ALS iPSC patient lines with different mutations.
(I) Representative immunostaining on cervical cryosections from a P10 WT untreated mouse showing MNs labeled with Hb9-GFP and SMN in red in the ventral horns of the spinal cords (nuclei stained with DAPI are blue). Scale bar, 20 μm.
(J) Histogram analysis from three P10 WT and SMNΔ7 untreated mice showing the MN populations based on their SMN levels.
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4. Figure 2 MNs Expressing Low SMN Protein Levels Are More Prone to Die
(A) Mouse MNs expressing mCherry under the Hb9 promoter (Hb9::mCherry) infected with pGIPZ lentivirus expressing GFP and SMN shRNA or NS shRNA control were imaged each day as indicated. Representative images are shown. Scale bar, 100 μm.
(B) Quantification of the percentage of MN survival reveals MN death induced by SMN knockdown (∗∗∗p < 0.001, two-way ANOVA with Dunnett’s multiple comparison test; n = 5).
(C) Histogram analysis reveals that SMN levels across the MN population are quite variable. SMN knockdown reduces SMN levels, and cells with sub-threshold SMN levels die.
(D) Quantification of the number of Hb9-GFP mouse WT ESC-derived MNs after dissociation showing their survival profile. The MN cultures were live-imaged, taking advantage of the Hb9-GFP endogenous labeling at the indicated time points. (One-way ANOVA test followed by Dunnett’s analysis, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001; n = 3).
(E) Histogram analysis from WT mouse ESC-derived MNs shows the percentage of MNs falling into each SMN intensity bin after the indicated number of days in culture.
(F) Human SMA type II MNs derived from iPSCs were treated for 3 days with the indicated compounds. Quantification of the SMN protein levels in the Islet1+ MNs related to the DMSO-treated cells is shown. (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p <0.01 between each treatment compared to the DMSO. Two-way ANOVA with Dunnett’s multiple comparison test, n = 3).
(G and H) The total number of cells per well after treatment with the indicated compounds is quantified and shown in (G), and the number of Islet1+ MNs related to DMSO-treated wells is shown in (H).
(I) Based on the DMSO-treated cells, arbitrary SMN thresholds to bin the Islet1+ MNs into three categories were established: high, the 10% higher SMN expressors; low, the 40% expressing the lowest SMN amount; and medium, the remaining 50%. Quantification of the percentage of Islet1+ MNs derived from type II SMA iPSCs harboring high, medium, and low SMN levels after the 3-day treatment is shown. (∗p < 0.05 between the “High SMN” population found for each treatment compared with the DMSO, two-way ANOVA with Dunnett’s multiple comparison test; n = 3).
(J) Quantification of the number of Islet1+ MNs derived from type II SMA iPSC harboring high, medium, and low SMN levels after the treatment. (∗∗∗p < between the “High SMN” population found for each treatment compared with the DMSO, ###p < between the “Medium SMN” population found for each treatment compared with the DMSO, §§p < 0.01 and §§§p < between the “Low SMN” population found for each treatment compared with the DMSO, two-way ANOVA with Dunnett’s multiple comparison test. n = 3).
(K) Representative images of the type II MN cultures treated with the indicated compounds. Scale bar, 50 μm.
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5. Figure 3
SMN Overexpression Promotes MN Survival, but Does Not Affect the Survival of iPSC-Derived Cortical Neurons
(A) Representative images of mouse WT MNs (green) fixed and immunostained with an SMN antibody (red). Cells were infected with a doxycycline (DOX)-inducible lentivirus carrying SMN or RFP as control, then treated with increasing concentrations of DOX or vehicle (water) (no DOX and 30 ng/mL DOX-treated cells are shown) and subjected to trophic factor withdrawal for the last 7 days of the culture. Scale bar, 50 μm.
(B) Histogram analysis showing the percentage of MNs falling into each SMN intensity category after SMN overexpression induced by different DOX concentrations.
(C) Quantification of the surviving MNs after SMN overexpression relative to the number of MNs remaining after RFP overexpression (∗∗p < 0.01, one-way ANOVA followed by Dunnett’s multiple comparison test, n = 4).
(D) Quantification of SMN protein levels after SMN overexpression relative to RFP overexpression (∗p < 0.05, ANOVA followed by Dunnett’s multiple comparison test, n = 4).
(E) Human MNs differentiated from iPSCs derived from ALS patients with the indicated mutations were infected with a DOX-inducible lentivirus carrying SMN or RFP at day 1 after plating, and 0.5 μg/ml DOX added 1 day after the infection and kept for 5 days, when the cultures were fixed and stained for Islet1. Scale bar, 50 μm.
(F–H) In each case the number of Islet1+ MNs remaining at the end of the culture was related to each RFP control infected, with no DOX cells (“RFP + DOX”, “SMN − DOX”, and “SMN + DOX” related to “RFP − DOX”) (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, one-way ANOVA followed by Dunnett’s multiple comparison test; n = 3).
(I–M) Healthy control (1016A and BJ) and type III, II, and I SMA iPSCs were differentiated into cortical neurons by forcing the expression of neurogenin-2 (Zhang et al., 2013). Subsequently, cells were transduced or SMN-overexpressing DOX-inducible lentivirus or RFP. Six days later, trophic factors (TFs) were removed for 9 days followed by fixation and staining of the cultures with anti-Brn2 and SMN antibodies. The quantification of the number of Brn2+ cortical neurons remaining after TF withdrawal (−TF) for each overexpressing virus (RFP or SMN) was related to the “plus TF” (+TF) condition and shown for each line: (I) 1016A, (J) BJ, (K) I-39C, (L) I-51N, and (M) I-38G. No statistical significance was found by one-way ANOVA. n = 3.
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6. Figure 4
Therapeutic Implications Associated with SMN Having a Broader Role in Regulating MN Survival
(A) Lysates of 293T cells transfected with expression plasmids encoding HA-tagged SMN-FL or SMNΔ7 and treated with 0.3 μM MLN4924 or DMSO for 3 days in 0.5% serum-containing media were immunoblotted with the HA and tubulin (Tub) antibodies. Light and dark exposures are shown for HA.
(B) Quantification of fold intensity change of HA-SMN-FL and HA-SMNΔ7 protein levels normalized to tubulin protein levels analyzed as in (A). Numbers reflect the fold-change effect of MLN4924 compared with the DMSO control for each SMN form (∗p < 0.05, ∗∗p < 0.01, Student’s t test; n = 4).
(C) Lysates of 293T cells transfected with the expression plasmid including HA-SMN-FL and treated with 0.3 μM MLN4924 or DMSO for 3 days were incubated with 2% SDS to disrupt non-covalent interactions, diluted to 0.2% SDS, and immunoprecipitated with the HA antibodies. Ten percent of the total lysates and immunoprecipitates were immunoblotted with the HA, Tub, and ubiquitin (ub) antibodies, respectively. MLN4924 prevents ubiquitination of SMN protein.
(D) Quantification of average SMN levels reveals that MLN4924 increases SMN protein in SMA A2 (Smn−/−;SMN2+/+;Hb9-GFP) MNs (∗∗p < 0.01, one-way ANOVA followed by Dunnett’s multiple comparison test; n = 11).
(E) Histogram analysis showing the distribution of Hb9-GFP MNs according to their SMN protein levels after MLN4924 treated.
(F) Quantification of average SMN protein levels after MLN4924 treatment in MNs derived from SMA type I iPSCs (∗∗p < 0.01, one-way ANOVA followed by Dunnett’s multiple comparison test; n = 8).
(G) Histogram analysis showing the distribution of type I Islet1+ MNs according to their SMN protein levels after MLN4924 treatment.
(H) Mouse SMA A2 MNs (green) treated with increasing doses of MLN4924 or DMSO for 4 days were subjected to immunocytochemistry with the SMN antibody (red) and Hoechst (blue). Selected images are shown for 0.3 μM MLN4924 and DMSO. Dotted lines represent tracing of the MNs. Scale bar, 50 μm.
(I) Quantification of the percentage of surviving MNs treated as in (G) and expressed as a comparison of MNs surviving after 4 days relative to 1 day reveals that MLN4924 treatment significantly promotes survival of SMA A2 MNs (∗p < 0.05, ∗∗p < 0.01, one-way ANOVA followed by Dunnett’s multiple comparison test; n = 11).
(J) Representative images of Islet1+ MNs differentiated from iPSCs derived from SMA type I patients and treated with 1.25 μM DMSO, MLN4924. Scale bar, 50 μm.
(K) Quantification of Islet1+ SMA type I MNs treated with DMSO or increasing concentrations of MLN4924 for 3 days, fixed, and subjected to immunocytochemistry (∗p < 0.05, ∗∗p < 0.01, one-way ANOVA followed by Dunnett’s multiple comparison test; n = 4).
(L) Quantification of the increase of SMN levels in SMNΔ7 mouse MNs infected with lentivirus expressing the dominant-negative (DN) form of the indicated Cullin for 8 days as compared with the empty vector-infected cells (∗p < 0.05, one-way ANOVA followed by Dunnett’s multiple comparison test; n = 6).
(M) Hb9::GFP SMNΔ7 mouse MNs fixed and immunostained 8 days after being infected with empty vector or Cul5DN lentivirus (SMN in red; scale bar, 50 μm).
(N) Quantification of the number of SMNΔ7 MNs 8 days after infection with a lentivirus carrying the Cul5DN compared with empty vector-treated ones (∗∗p < 0.01, two-tailed t test; n = 6).
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7. Figure 5
MLN4924 Increases SMN Levels and Promotes Robust Survival in ALS Patient iPSC-Derived MNs, Whereas an SMN2 Splicing Modulator Does Not
(A–D) Human MNs differentiated from iPSCs derived from ALS patients with the indicated mutations were treated with DMSO, MLN4924, or C3 at indicated concentrations for 3 days, fixed, and subjected to immunocytochemistry with SMN and Islet1 antibodies. SMN protein levels measured in Islet1+ MNs are shown as a fold change related to the DMSO-treated MNs (represented as 0 μM).
(E and F) Representative images from DMSO, 1.25 μM MLN4924, or 1.25 μM C3-treated ALS MNs carrying the indicated mutations (SMN in red, Islet1 in green, and Hoechst in blue). Scale bar, 50 μm.
(G and H) Quantification of the number of Islet1+ MNs derived from (H) TDP43-47A and (G) SOD1 ALS iPSC patient lines after MLN4924 or C3 treatment.
Concentration-dependent data were fitted to a non-linear regression equation using Prism (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, two-way ANOVA followed by Dunnett’s multiple comparison test; n = 4).
Cell Reports  , DOI: ( /j.celrep )
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