Cellular and Molecular Life Sciences

, Volume 70, Issue 21, pp 4181–4195 | Cite as

Extracellular wildtype and mutant SOD1 induces ER–Golgi pathology characteristic of amyotrophic lateral sclerosis in neuronal cells

  • Vinod Sundaramoorthy
  • Adam K. Walker
  • Justin Yerbury
  • Kai Ying Soo
  • Manal A. Farg
  • Vy Hoang
  • Rafaa Zeineddine
  • Damian Spencer
  • Julie D. Atkin
Research Article


Amyotrophic lateral sclerosis (ALS) is a fatal and rapidly progressing neurodegenerative disorder and the majority of ALS is sporadic, where misfolding and aggregation of Cu/Zn-superoxide dismutase (SOD1) is a feature shared with familial mutant-SOD1 cases. ALS is characterized by progressive neurospatial spread of pathology among motor neurons, and recently the transfer of extracellular, aggregated mutant SOD1 between cells was demonstrated in culture. However, there is currently no evidence that uptake of SOD1 into cells initiates neurodegenerative pathways reminiscent of ALS pathology. Similarly, whilst dysfunction to the ER–Golgi compartments is increasingly implicated in the pathogenesis of both sporadic and familial ALS, it remains unclear whether misfolded, wildtype SOD1 triggers ER–Golgi dysfunction. In this study we show that both extracellular, native wildtype and mutant SOD1 are taken up by macropinocytosis into neuronal cells. Hence uptake does not depend on SOD1 mutation or misfolding. We also demonstrate that purified mutant SOD1 added exogenously to neuronal cells inhibits protein transport between the ER–Golgi apparatus, leading to Golgi fragmentation, induction of ER stress and apoptotic cell death. Furthermore, we show that extracellular, aggregated, wildtype SOD1 also induces ER–Golgi pathology similar to mutant SOD1, leading to apoptotic cell death. Hence extracellular misfolded wildtype or mutant SOD1 induce dysfunction to ER–Golgi compartments characteristic of ALS in neuronal cells, implicating extracellular SOD1 in the spread of pathology among motor neurons in both sporadic and familial ALS.


Extracellular SOD1 ALS Endoplasmic reticulum Golgi 



We thank Professor Neil Cashmann for the gift of NSC-34 cells, and we thank Dr Jennifer Lippincott-Schwartz and Dr George Patterson for the VSVG-ts045-mCherry construct. This work was supported by National Health and Medical Research Council of Australia Project Grants [# 1006141, 1030513 to J.A.], Bethlehem Griffiths Research Foundation, Motor Neuron Disease Research Institute of Australia, Suzie Harris Memorial Fund MND Research Grant and Rosalind Nicholson MND Research Grant [to J.A.], a National Health and Medical Research Council CJ Martin Biomedical Early Career fellowship [1036835 to A.W.] and an Australian Postgraduate Award and Australian Rotary Health scholarship [to A.W.].

Supplementary material

18_2013_1385_MOESM1_ESM.tif (2.8 mb)
Fig.S1. SOD1 uptake is mediated by macropinocytosis. Scanning confocal micrographs of SOD1 protein internalisation by NSC-34 cells. Cells were incubated with 20 μg/mL of either SOD1WT or G93A SOD1 for 30 min. At 30 min cells were fixed and stained for human SOD1. In some cases cells were pre- and co-treated with endocytosis inhibitors rottlerin, 5-(N-Ethyl-N-isopropyl)amiloride (EIPA), genistein or chlorpromazine hydrochloride (CPZ). Scale bar represent 10 μm.(TIFF 2886 kb)
18_2013_1385_MOESM2_ESM.tif (2.9 mb)
Fig.S2. Baculovirus expressed GST-labeled SOD1WT and mutant SOD1 proteins are enzymically active in conditioned medium of insect Sf9 cells. (A) Insect Sf9 cells were transfected with pIEX-3 alone and pIEX-3 SOD1 WT and G93A constructs for 48 h. The conditioned cell culture medium was harvested, centrifuged at 1200 rpm for 5 min to remove cell debris and then analysed by immunoblotting with an anti-GST antibody (50 μg/lane). The blot confirms the presence of GST, GST-SOD1WT and GST-SOD1G93A proteins, indicating secretion into Sf9 conditioned medium. (B) Zymography reveals SOD1 dismutase activity is present in the Sf9 conditioned medium of GST-SOD1WT and GST-SOD1G93A but not GST-only transfected cells.(TIFF 2921 kb)
18_2013_1385_MOESM3_ESM.tif (2.5 mb)
Fig.S3. ER to Golgi trafficking is inhibited in SH-SY5Y cells expressing intracellular mutant SOD1. Cells were transfected with VSVG-ts045 mCherry and either WT, A4 V, or G85R SOD1-EGFP constructs for 24 h. After trapping VSVG-ts045 mCherry in the ER by incubation at 40 °C for 24 h after transfection, the temperature was shifted to 32 °C for 30 min or fixed immediately at 0 min. The cells were then fixed with 4 % PFA and stained with calnexin (blue) or GM130 (blue). (A) Representative fluorescent images after 30 min incubation at 32 °C. VSVG is co-localised with GM130 in cells expressing SOD1 WT and control untransfected (UT) cells. In contrast, in cells expressing mutant SOD1 A4 V or G85R, VSVG-ts045 is still predominantly co-localised with calnexin. All scale bars 10 μm. (C) Quantification of the degree of co-localisation of VSVG with calnexin and GM130 at 60 min as in (B) using Mander’s coefficient. Data are presented as mean ± SEM, *** P < 0.001, ** P < 0.01 versus SOD1WT expressing cells or untransfected cells by one-way ANOVA with Tukey’s post-test, n = 3. (TIFF 2566 kb)
18_2013_1385_MOESM4_ESM.tif (3.8 mb)
Fig.S4. Extracellular mutant G93A SOD1 does not localize in the ER after uptake. NSC-34 cells treated with SOD1G93A for 48 h was fixed with 4 % PFA and stained with anti-calnexin antibody (red), ER marker. Series of Z stack images of NSC-34 cell bearing SOD1G93A inclusions (green) are shown. Scale bar represent 10 μm. (TIFF 3894 kb)


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Copyright information

© Springer Basel 2013

Authors and Affiliations

  • Vinod Sundaramoorthy
    • 1
  • Adam K. Walker
    • 1
    • 2
  • Justin Yerbury
    • 3
  • Kai Ying Soo
    • 1
  • Manal A. Farg
    • 1
  • Vy Hoang
    • 1
  • Rafaa Zeineddine
    • 3
  • Damian Spencer
    • 1
  • Julie D. Atkin
    • 1
    • 4
  1. 1.Department of Biochemistry, Latrobe Institute for Molecular ScienceLa Trobe UniversityMelbourneAustralia
  2. 2.Center for Neurodegenerative Disease Research, School of MedicineUniversity of PennsylvaniaPhiladelphiaUSA
  3. 3.School of Biological SciencesUniversity of WollongongWollongongAustralia
  4. 4.Department of Florey NeuroscienceUniversity of MelbourneParkvilleAustralia

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