Induction of the Unfolded Protein Response in Familial Amyotrophic Lateral Sclerosis and Association of Protein-disulfide Isomerase with Superoxide Dismutase 1

Julie D Atkin,Manal A Farg,Bradley J Turner,Doris Tomas,Judith A Lysaght,Janelle Nunan,Alan Rembach,Phillip Nagley,Philip M Beart,Surindar S Cheema,Malcolm K Horne

doi:10.1074/jbc.m603393200

Abstract

Mutations in Cu/Zn superoxide dismutase (SOD1) are linked to motor neuron death in familial amyotrophic lateral sclerosis (ALS) by an unclear mechanism, although misfolded SOD1 aggregates are commonly associated with disease. Proteomic analysis of the transgenic SOD1(G93A) ALS rat model revealed significant up-regulation of endoplasmic reticulum (ER)-resident protein-disulfide isomerase (PDI) family members in lumbar spinal cords. Expression of SOD1 mutants (mSOD1) led to an up-regulation of PDI in motor neuron-like NSC-34 cells but not other cell lines. Inhibition of PDI using bacitracin increased aggregate production, even in wild type SOD1 transfectants that do not readily form inclusions, suggesting PDI may protect SOD1 from aggregation. Moreover, PDI co-localized with intracellular aggregates of mSOD1 and bound to both wild type and mSOD1. SOD1 was also found in the microsomal fraction of cells despite being a predominantly cytosolic enzyme, confirming ER-Golgi-dependent secretion. In SOD1(G93A) mice, a significant up-regulation of unfolded protein response entities was also observed during disease, including caspase-12, -9, and -3 cleavage. Our findings therefore implicate unfolded protein response and ER stress-induced apoptosis in the patho-physiology of familial ALS. The possibility that PDI may be a therapeutic target to prevent SOD1 aggregation is also raised by this study.

Highlights

PDI and Erp57 Are Significantly Up-regulated in Spinal Cords of Both Transgenic SOD1G93A Rats and Mice—A proteomic approach was employed to identify differentially expressed proteins associated with the onset and progression of motor neuron disease
Expression of hSOD1-EGFP Induces PDI in the Motor Neuron-like Cell Line NSC-34 but Not in Fibroblast (COS-7) or Nonmotor Neuronal (PC12) Cells—We showed previously that mutant, but not wild type SOD1, forms intracellular fluorescent SOD1 aggregates that correlate with cell death in transfected motor neuron-like NSC-34 cells [26]
The major findings of this study are that UPR-induced chaperones, stress sensor kinases, and apoptotic effectors are up-regulated in transgenic SOD1G93A mouse spinal cords, implying that ER stress plays a role in SOD1-mediated neurodegeneration

Summary

EXPERIMENTAL PROCEDURES

Transgenic rats derived from the SD-TgN (SOD1G93A) L26H line (Taconic Farms, Germantown, NY) were bred on a Sprague-Dawley (SD) background. After 20 min of incubation at 4 °C followed by centrifugation for 5 min at 15,000 ϫ g, the supernatant containing SDS-soluble proteins was collected and frozen at Ϫ20 °C until required. Mouse spinal cord extracts (20 ␮g of total protein) or whole transfected cell lysates (250 ␮l) were incubated with anti-SOD1 or -PDI (1:750) antibodies and 30 ␮l of 50% (w/v) protein A-Sepharose CL-4B (Amersham Biosciences) in Tris buffer (50 mM Tris-HCl, pH 7.5, 0.02% (w/v) NaN3) on a rotating wheel overnight at 4 °C. Untransfected NSC-34 cells (4 ϫ 106 cells) grown in three confluent flasks were pelleted by centrifugation, resuspended in cold 0.25 M sucrose and 0.1 mM EGTA in 10 mM Tris-HCl, pH 7.4, buffer with 1% (v/v) protease inhibitors, and homogenized before differential centrifugation was performed. MALDI-TOF MS data was searched against NCBI data base using the MASCOT search engine

13 Protein-disulfide isomerase gi 91897

RESULTS

DISCUSSION