Appendix. Table of Content

Transcription

1 Appendix Table of Content Page Appendix Table S1 1 Appendix Table S2 2 Appendix Table S3 3 Appendix Figure S1 4 Appendix Figure S2 5 Appendix Figure S3 6 Appendix Figure S4 7 Appendix Figure S5 8 Antibody List 9

2 Appendix Table S1. Distribution of phenotypes in zebrafish injected with PDIA1 and ERp57 mrna. Table showing global phenotypes of control (Mock injected), wildtype and mutant PDIA1 or ERp57 mrna overexpression in 5 independent experiments. Phenotypes were: Not affected (normal development), curly tail, short AP axis (anteriorposterior axis shorter) and SDD (serious developmental defect). The number of embryos with phenotype and percentage of total is shown. 1

3 Appendix Table S2. Average KD values of ERp57 mutants. Four independent experiments were performed. Student s test was performed. Mean ± SD with p values: *, p 0.05; **, p 0.01 are shown. 2

4 Appendix Table S3. Electromyography of ERp57 deficient mice. In all mice completely deficient for ERp57 (ERp57 Nes/, n = 5) we observed fibrillations and positive sharp waves (PSW) scoring 1 (n = 4) or 2 (n = 1), whereas 6 of 12 heterozygous, ERp57 Nes/, mice displayed a score of 1. As a positive control for this test, a symptomatic SOD1 G86R transgenic mouse was monitored. All ERp57 WT mice (n = 16) and all presymptomatic SOD1 G86R transgenic mice (n = 4) were normal with a score of 0. Genotype Observation PSW n ERp57 Nes/ 1/2 4/1 ERp57 Nes/ 1/0 6/6 ERp57 WT 0 16 SOD1 G86R presymptomatic 0 4 SOD1 G86R endstage of disease 3 1 3

5 A B C D V5 E Mock shpdia1 * Actin PDIA1 V5 Mock sherp57 ERp57 Actin Appendix Figure S1: Motoneuron morphology and viability upon expression of ALSlinked PDI variants. A Cell viability was determined after expression of PDIA1 or ERp57 after 48 h of transient transfection followed by propidium iodide (PI) staining. Mean and SEM of 3 independently experiments are presented. B Primary rat motoneuron survival. GFP, MAP2 and SMI32positive neurons were counted in 20 random fields. The percentage of GFP and SMI32positive motor neurons within the total number of MAP2positive cells was calculated under the different conditions. Mean and SEM are shown from 3 independent experiments. C Rat ventral spinal cord neurons were transfected at 4DIV with GFP alone or together with the indicated PDI constructs. Cells were fixed at 10 DIV and ICC with antibody against SMI32 was used to identify motoneurons. Pictures were taken and length as well as number of primary, secondary and tertiary branches of cells positive for GFP and SMI32 was quantified. Results are compiled from 3 independent experiments analyzing 7 SMI32 positive cells in each experiment to a total of 21 cells per condition. D HEK293T cells were transduced with lentivirus expressing the indicated PDIs. The V5tag was detected in transduced cells compared to mock condition, indicating that the virus transduction is working. E NSC34 cells were stably transduced with lentivirus expressing shrna constructs targeting Pdia1 or Erp57. The knockdown of PDIs was confirmed by semiquantitative PCR. Actin was used as a reference. Asterisks indicates an unspecific band observed in the PCR for PDIA1. Mean and SEM are shown. For statistical analysis oneway ANOVA with Bonferroni s post hoc test was performed. p values: n.s., nonsignificant, p > 0.05; *, p 0.05; **, p 0.01; ***, p

6 V5 KDELRFP Merge V5 KDELRFP Merge PDIA1 R300H ERp57 Q418K PDIA1 D292N ERp57 D217N PDIA1 WT ERp57 WT Mock Mock Appendix Figure S2: ALSrelated mutant PDIs do not display altered localization. Subcellular localization of transiently overexpressed V5tagged PDIs in NSC34 cells was monitored by immunofluorescence (antiv5 in green). Localization to the ER of PDIA1 WT, PDIA1 D292N, PDIA1 R300H or ERp57 WT, ERp57 D217N and ERp57 Q481K was investigated by coexpressing RFPKDEL to label the ER. Bottom panel: the Manders coefficient (M1) of colocalization was quantified, using the Interactive Data Language (IDL). 5

7 A Appendix Figure S3: ERp57 deficiency in the nervous system has no obvious effect on cerebellum structure. A Rotarod performance was performed ERp57 WT (n=20), ERp57 Nes/ (n=15) and ERp57 Nes/ (n=8) mice starting at age 30 until 150 days of age. B a a ERp57 WT b ERp57 Nes/ b B Histological analysis of the cerebellum from ERp57 WT (n = 3) and ERp57 Nes/ (n = 3) mice. At 6 months of age, mice were perfused and the brain was processed for immunohistochemistry for the Purkinje cell marker Calbindin. Representative images of two animals per genotype are shown. Scale bar represent 480 μm. Right panel, quantifications of cerebellar convultions, Purkinje cell number and layers of ERp57 WT (n = 3), and ERp57 Nes/ (n = 3) mice. No significant differences were found. Mean ± SEM with p values: n.s., nonsignificant, p > 0.05; *, p 0.05; **, p 0.01; ***, p

8 A MW Cortex ERp57 WT ERp57 Nes/ ERp57 Nes/ MW SOD1 (DTT) C DTT Cortex ERp57 WT ERp57 Nes/ ERp57 Nes/ C1 C2 E MW 25 Spinal cord ERp57 WT ERp57 Nes/ ERp57 Nes/ TDP43 (43kD) TDP43 (kd) TDP43 (43kD) Lower exposure SOD1 HSP B 25 MW Cortex ERp57 WT ERp57 Nes/ ERp57 Nes/ SOD1 βactin MW TDP43 HSP90 TDP43 (DTT) D DTT Cortex ERp57 WT ERp57 Nes/ ERp57 Nes/ C1 C2 Appendix Figure S4: ERp57 deficiency in the CNS has no effects on SOD1 or TDP43 expression. A and B Western blot analysis of SOD1 and TDP43 levels and aggregation in the cortex of ERp57 WT (n = 4), ERp57 Nes/ (n = 5) and ERp57 Nes/ (n = 4) mice, respectively. HSP90 and β Actin were determined as loading controls. To assess total levels of SOD1 or TDP43 samples were run under reducing conditions (DTT), whereas samples were run under nonreducing (DTT) conditions to determine aggregation levels. As a positive control for aggregation a cortex sample from either a SOD1 G93A (A) or TDP43 A315T (B) transgenic animal is shown. C and D Filter trap analysis of SOD1 and TDP43 aggregation in the cortex of ERp57 WT (n = 4), ERp57 Nes/ (n = 5) and ERp57 Nes/ (n = 4) mice, respectively. As a positive controls for aggregation a cortex sample from either a SOD1 G93A (C1 in A) or TDP43 A315T (C1 in B) transgenic animal is shown. C2 refers to a positive control of either SOD1 G93A aggregates (A) or TDP43 A315T aggregates (B) derived from NSC34 cell extracts after transient overexpression. E Western blot analysis of SOD1 and TDP43 levels in the spinal cord of ERp57 WT (n = 4), ERp57 Nes/ (n = 5) and ERp57 Nes/ (n = 4) mice. HSP90 was determined as loading control. 7

9 MW Spinal cord ERp57 WT ERp57 Nes/ ERp57 Nes/ SV2 βactin NMDAR2a PSD95 Synaptophysin ERp57 βactin Appendix Figure S5: ERp57 deficiency in the spinal cord has no effect on the levels of synaptic markers. The levels of synaptic proteins SV2, NMDAR2A, PSD95 and Synaptophysin in the spinal cord of ERp57 WT (n = 4), ERp57 Nes/ (n = 5) and ERp57 Nes/ (n = 4) mice were monitored by Western blot. βactin was determined as a loading control. 8

10 Antibody Company Catalog Number MAP2 (H300) Santa Cruz sc20172 SMI32 Convance SMI32P GFP Molecular Probes A64 ChAT Millipore AB143 V5 Invitrogen R96025 ERO1Lα Novus Biologicals NB2525 PDIA1 (1D3) Enzo ADISPA891 PDIA1 (H160) Santa Cruz SC20132 ERp57 Santa Cruz SC28823 NMDAR2A Convance PRB513P Synaptophysin Millipore MAB368 PSD95 Millipore SV2 Developmental Studies AB_ Hybridoma Bank SOD1 Calbiochem TDP43 Proteintech AP βactinhrp conjugate Cell Signaling 5125 Actin Santa Cruz SC1615 HSP90 Santa Cruz SC7947 BIP Abcam ab21685 ATF4 Santa Cruz SC200 XBP1 Santa Cruz SC7160 Calbindin Cell Signaling C26D12 9