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. 2009 Dec 1;18(23):4552-64.
doi: 10.1093/hmg/ddp421. Epub 2009 Sep 24.

Mutant SOD1 in neuronal mitochondria causes toxicity and mitochondrial dynamics abnormalities

Jordi Magrané  1 , Isabel Hervias, Matthew S Henning, Maria Damiano, Hibiki Kawamata, Giovanni Manfredi
Affiliations

Mutant SOD1 in neuronal mitochondria causes toxicity and mitochondrial dynamics abnormalities

Jordi Magrané et al. Hum Mol Genet. .

Abstract

Amyotrophic lateral sclerosis (ALS) is a fatal neurological disorder characterized by motor neuron degeneration. Mutations in Cu,Zn-superoxide dismutase (SOD1) are responsible for 20% of familial ALS cases via a toxic gain of function. In mutant SOD1 transgenic mice, mitochondria of spinal motor neurons develop abnormal morphology, bioenergetic defects and degeneration, which are presumably implicated in disease pathogenesis. SOD1 is mostly a cytosolic protein, but a substantial portion is associated with organelles, including mitochondria, where it localizes predominantly in the intermembrane space (IMS). However, whether mitochondrial mutant SOD1 contributes to disease pathogenesis remains to be elucidated. We have generated NSC34 motor neuronal cell lines expressing wild-type or mutant SOD1 containing a cleavable IMS targeting signal to directly investigate the pathogenic role of mutant SOD1 in mitochondria. We show that mitochondrially-targeted SOD1 localizes to the IMS, where it is enzymatically active. We prove that mutant IMS-targeted SOD1 causes neuronal toxicity under metabolic and oxidative stress conditions. Furthermore, we demonstrate for the first time neurite mitochondrial fragmentation and impaired mitochondrial dynamics in motor neurons expressing IMS mutant SOD1. These defects are associated with impaired maintenance of neuritic processes. Our findings demonstrate that mutant SOD1 localized in the IMS is sufficient to determine mitochondrial abnormalities and neuronal toxicity, and contributes to ALS pathogenesis.

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Figures

Figure 1.
Figure 1.
Targeting of SOD1 to mitochondria in cultured cells. (A) Schematic representation of the fusion protein containing human SOD1 (hSOD1) appended in-frame at its N-terminus to the mitochondrial import signal of cytochrome b2 (CytB2). MMP, matrix metalloproteases; IMP, intermembrane space proteases. (B) Transient transfection of WT or mutant (G93A and G85R) IMS-hSOD1 in COS cells. Recombinant SOD1 is immuno-labeled in green and mitochondria stained with the fluorescent dye Mitotracker red. The overlay of the two images indicates that IMS-h SOD1 localizes within mitochondria. Colocalization of the two fluorochromes is further confirmed by z-section reconstruction (insets).
Figure 2.
Figure 2.
Expression and localization of IMS-targeted hSOD1 in NSC34 cells. (A) Western blot of cellular homogenates from cells stably expressing WT and G93A IMS-hSOD1 using a polyclonal anti-SOD1 antibody that recognizes both the human and the mouse protein. mSOD1, mouse SOD1; P1 and P2, precursor products of incomplete protein cleavage; M, mature, fully processed hSOD1. (B) Western blot of enriched mitochondrial fractions from NSC34 cells stably transfected with WT or mutant IMS-hSOD1. Each lane contains 50 µg of protein. The mitochondrial outer membrane protein VDAC1 is used as a loading control. Note that mature G85R mutant IMS-hSOD1 is masked by endogenous mSOD1. (C) Western blot of mitochondria from cells stably expressing G85R IMS-hSOD1 using a human specific anti-SOD1 antibody (first lane). Proteinase K (PK) treatment does not completely digest mutant hSOD1 (second lane), but digestion is almost complete when mitochondrial membranes are solubilized with detergents (TX, third lane). (D) Western blot of mitochondria form G85R IMS-hSOD1 cells after hypo-osmotic swelling. PK treatment fully digested mutant hSOD1 only when the outer mitochondrial membrane was disrupted. (E) SOD1 native activity gel assay of enriched mitochondrial fractions from cells stably expressing IMS-hSOD1. hSOD1, purified human SOD1; mock, cells transfected with empty plasmid. (F) Western blot of supernatant (SN) and pellet (P) fractions of cells expressing WT untargeted or IMS-hSOD1 after digitonin (DG) treatment. A human specific anti-SOD1 antibody demonstrates the presence of hSOD1 only in the pellet fraction of cells expressing IMS-hSOD1. Longer exposure of the same blot shows small amounts of hSOD1 in the supernatant after DG treatment when IMS-hSOD1 is expressed. Cytochrome c and Akt antibodies are used as controls for IMS and soluble cytosolic protein fractions, respectively.
Figure 3.
Figure 3.
Metabolic stress in NSC34 cells expressing IMS-targeted hSOD1. (A) Representative phase contrast micrographs of NSC34 cells either mock transfected or stably expressing IMS-targeted hSOD1. Untreated, normal growth conditions; galactose, galactose medium induces morphologic changes in all cells, including extrusion of cell processes, similar to neurites (arrows). (B, C, D) Quantification of cell death by LDH release in cells grown in galactose medium (n = 7), serum-deprived medium (n = 8) and xantine oxidase (X/XO) medium (n = 8). (E) Cell viability by WST-1 assay (n = 8) in cells treated with the combination of rotenone (50 nm) for 72 h, plus ethacrynic acid (100 µm). *P < 0.05 compared with mock-transfected controls. Error bars represent the SEM.
Figure 4.
Figure 4.
Mitochondrial abnormalities in neurites of differentiated mutant hSOD1 motor neuronal cells. (A) Representative images of mitochondria along neurites in differentiated NSC34 clones stably expressing IMS- or untargeted hSOD1. Mitochondria are identified by mitoGFP labeling. Note a cluster of mitochondria in G85R IMS-hSOD1 neurite. Scale bar, 10 µm. (B) Clustering of mitochondria in neurites. n (neurites) = 20–50 from three to six independent experiments. *P < 0.005 versus WT IMS-hSOD1; #P < 0.00005 versus mock. Error bars represent the SEM. (C) Size (length) and (D) density (mass per neurite segment) of mitochondria was quantified in neuritic segments. Data were referred to an arbitrary 50 µm segment. From 17 to 45 neurites were analyzed in three to four independent experiments. n (mitochondria) = 223–754. *P < 0.0005 versus WT-IMS; **P < 0. 05 × 10−4 versus WT. (E) Length of mitochondria in the cell bodies of differentiated NSC34 cells. From 16 to 18 somas were analyzed in three independent experiments. *P < 0.05 × 10−8 versus mock controls.
Figure 5.
Figure 5.
Altered mitochondrial dynamics in differentiated mutant SOD1 NSC34 cells. (A) Representative kymographs of mitoGFP-labeled mitochondria from control (mock and WT IMS-hSOD1) and mutant (G93A and G85R IMS-hSOD1) cells. Total imaging time, 5 min. Scale bar in all panels, 10 µm. Note the reduced motility in mutant SOD1 cells indicated by a prevalence of vertical fluorescent lines corresponding to immobile mitochondria. (B) Relative motility of neuritic mitochondria from IMS-hSOD1 cells. n = 10–13 neurites, *P < 0.05, **P < 0.005 referred to mock-transfected cells. (C) Relative motility of neurite mitochondria in untargeted hSOD1 cells. n = 9–13 neurites, *P < 0.05, **P < 0.005 referred to mock control cells. Error bars represent the SEM.
Figure 6.
Figure 6.
Targeting of mutant SOD1 to mitochondria causes a failure in neurite maintenance. (A) Representative images of NSC34 cells after 12 days in differentiation medium. Neurites are labeled using a MAP2 antibody. Scale bar, 50 µm. (B) Quantification of neurite length plotted as the average change between day 8 and day 12 after inducing differentiation. n (neurites at 8/12 days) = 52–346 from three independent experiments. *P < 0.0005 and **P < 0.000005 versus mock; #P < 0.05 and ##P < 0.0005 versus WT IMS-SOD1. Error bars represent the SEM.
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