Journal: Cell stem cell

Article Title: Oligodendrocyte Death in Pelizaeus-Merzbacher Disease Is Rescued by Iron Chelation.

doi: 10.1016/j.stem.2019.09.003

Figure Lengend Snippet: Figure 7. Iron Chelation Rescues Phenotypes of Multiple PLP1 Mutations In Vitro and In Vivo (A) Treatment strategy to assess effects of DFO in additional human PMD mutations. (B–G) Differentiation phenotype of additional human PMD mutations after treatment with DFO (E–G) compared to control culture condition (B–D) (n = 3–4, biological replicates). B&E, T75P point mutation; C&F, Exon6 deletion; D&G, duplication. (H) Jimpy mouse OPCs isolated at postnatal days 5–8 were tested for differentiation phenotype and transiently treated with iron chelation. (I–L) Jimpy OPCs (I) completely failed to differentiate upon removal of growth factors that was associated with increased cell death. Transient (4 day) DFO treatment (J) rescued both differentiation and cell death as measured by caspase-3 and MBP expression (n = 10 fields/treatment from 2 biological replicates) (K and L). (M) In vivo iron chelation strategy in Jimpy mice. Subcutaneous injections with the brain-permeable iron chelator deferiprone (DFP) were given from postnatal days 7–14 and 21–death. (N and O) Corpus callosum of vehicle-treated Jimpy mice (N) at postnatal day 28 showed caspase-3+ apoptotic cells compared to the DFP-treated Jimpy mice (O). (P–Q0) Electron microscopic analysis of corpus callosum in DFP- (Q) and vehicle-treated (P) Jimpy mice showed surviving oligodendrocytes in the DFP-treated mice. Higher magnification of (P) and (Q) are depicted in (P0) and (Q0), respectively. (R–W) Fluorescent in situ hybridization (RNA scope) showed a significant reduction in Atf4 (R, S, and V) and Chop (T, U, and W) gene expressions in Mbp+ mature oligodendrocytes after a 7-day in vivo DFP treatment from P7 in Jimpy mice (n = 3–4). (legend continued on next page) 538 Cell Stem Cell 25, 531–541, October 3, 2019

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Olig2 Courtesy of Charles Stiles N/A NKX2.2 Developmental Studies Hybridoma Bank Cat# 74.5A5 SOX1 R&D Cat# AF3369 PAX6 Developmental Studies Hybridoma Bank Cat# Pax6 O4 Mouse Hybridoma N/A PLP1 Abcam Cat# 28486 Tra1-60 Cell Signaling 4746T Oct3/4 Santa Cruz sc-5279 MBP AbD Serotec Cat# MCA409S Cleaved Caspase-3 Cell Signaling Cat# 9664S KDEL Abcam Cat# ab50601 GFP Aveslab Cat# GFP-1020 Human cytoplasmic marker Clontech Cat# STEM121 NeuN EMD Millipore Cat# MAB377 PDGFRa BD Bioscience Cat# 556001 Transferrin Abcam Cat# ab137744 Ferritin Novus Biologicals Cat# NB600-920 IRP1 Cell Signaling Cat# 20272 IRP2 Cell Signaling Cat# 37135S Transferrin receptor BD Bioscience Cat# 561939 Ferroportin Novus Biologicals Cat# NBP1-21502 beta Actin Sigma Cat# A5441 GFAP Fisher Cat# 13-0300 Iba1 Wako Cat# 019-19741 EIf2a Cell Signaling Cat# 9722 Phospho-EIf2a Fisher Cat# MA5-15133 ATF4 Aviva Systems Biology Cat# ARP37017 CHOP Fisher Cat# MA1-250 O4-APC Miltenyi Biotec Inc 130-099-211 TFRC-GFP BD Biosciences 561939 Bacterial and Virus Strains AAV serotype DJ This paper Melo et al., 2014 Lenti-GFP Addgene Tet-O-FUW-EGFP Biological Samples Dermal fibroblasts from PMD patients This paper N/A Human brain tissues from fetal gestational week 20 to 23 This paper N/A Chemicals, Peptides, and Recombinant Proteins T3 Sigma Cat# T6397 SB431542 Tocris Cat# 1614 LDN193189 Stemgent Cat# 04-0074 SAG Shenzhen Shengjie Chemical Cat# 912545-86-9 Thiazovivin Santa Cruz Cat# sc-361380 basic FGF Thermo Fisher Cat# PHG0024 PDGF-A Peprotech Cat# 100-13A (Continued on next page) e1 Cell Stem Cell 25, 531–541.e1–e6, October 3, 2019

Techniques: In Vitro, In Vivo, Control, Mutagenesis, Isolation, Expressing, In Situ Hybridization, RNAscope