Mutant SOD1G93A triggers mitochondrial fragmentation in spinal cord motor neurons: neuroprotection by SIRT3 and PGC-1α

Abstract

Mutations in the Cu/Zn Superoxide Dismutase (SOD1) gene cause an inherited form of ALS with upper and lower motor neuron loss. The mechanism underlying mutant SOD1-mediated motor neuron degeneration remains unclear. While defects in mitochondrial dynamics contribute to neurodegeneration, including ALS, previous reports remain conflicted. Here, we report an improved technique to isolate, transfect, and culture rat spinal cord motor neurons. Using this improved system, we demonstrate that mutant SOD1(G93A) triggers a significant decrease in mitochondrial length and an accumulation of round fragmented mitochondria. The increase of fragmented mitochondria coincides with an arrest in both anterograde and retrograde axonal transport and increased cell death. In addition, mutant SOD1(G93A) induces a reduction in neurite length and branching that is accompanied with an abnormal accumulation of round mitochondria in growth cones. Furthermore, restoration of the mitochondrial fission and fusion balance by dominant-negative dynamin-related protein 1 (DRP1) expression rescues the mutant SOD1(G93A)-induced defects in mitochondrial morphology, dynamics, and cell viability. Interestingly, both SIRT3 and PGC-1α protect against mitochondrial fragmentation and neuronal cell death by mutant SOD1(G93A). This data suggests that impairment in mitochondrial dynamics participates in ALS and restoring this defect might provide protection against mutant SOD1(G93A)-induced neuronal injury.

Conflict of interest statement

None of the authors declare a financial or any other conflict of interest.

Published by Elsevier Inc.

Figures

Figure 1

A representative fluorescence wide-field microscopic image of one 7 DIV rat motor neuron co-cultured with astrocytes. The motor neuron was transfected with DsRed2-Mito to visualize mitochondria. Scale bar: 100µm.

Figure 2

Mitochondrial fragmentation occurs in mutant SOD1G93A motor neurons. A) Fluorescence micrographs (top panels) and close up views (bottom panels) of mitochondria in 2 DIV motor neurons expressing DsRed2-Mito and either SOD1WT or SOD1G93A co-cultured with astrocytes. Scale bar: 50µm. B) Mean length of mitochondria in motor neurons expressing DsRed2-Mito and either SOD1WT or SOD1G93A (n = 10). C) Number of mitochondria in 100 µm neurites of motor neurons expressing DsRed2-Mito and either SOD1WT or SOD1G93A (n = 10). D) Mitochondrial fragmentation of motor neurons expressing DsRed2-Mito and either SOD1WT or SOD1G93A in mixed cultures. E) Cell death of 2 DIV motor neurons expressing DsRed2-Mito and either SOD1WT or SOD1G93A. P value: two-tail paired Student’s t-test.

Figure 3

Mutant SOD1 G93A triggers a decrease in axonal anterograde and retrograde transport of mitochondria in 2 DIV motor neurons. A) Kymographs of motor neurons expressing DsRed2-Mito and either SOD1WT or SOD1G93A (Supplement movie 1 and 2). B) Mitochondrial directional movement in motor neurons expressing DsRed2-Mito and either SOD1WT or SOD1G93A. C) Mean velocity of mitochondria in the axons of motor neurons expressing DsRed2-Mito and either SOD1WT or SOD1G93A. D) Mitochondrial mobility in motor neurons expressing DsRed2-Mito and either SOD1WT or SOD1G93A. P value: two-tail paired Student’s t-test, n = 10.

Figure 4

Mutant SOD1 G93A triggers defective neurite branching and abnormal mitochondrial accumulation in growth cones. A) Representative 3D fluorescence wide-field microscopic images and close up views below of motor neurons expressing DsRed2-Mito, MAP2-EGFP, and either SOD1WT or SOD1G93A. Scale bar: 50 µm. B) The total neurite length of motor neurons expressing DsRed2-Mito, MAP2-EGFP, and either SOD1WT or SOD1G93A. C) The scheme for the neurite branching quantification. D) The number of branches of motor neurons expressing DsRed2-Mito, MAP2-EGFP, and either SOD1WT or SOD1G93A. P value: two-tail paired Student’s t-test, n = 10.

Figure 5

Inhibiting mitochondrial fission with the GTPase-defective DRP1K38A mutant rescues mitochondrial fragmentation and neuronal cell death by mutant SOD1G93A. A) Fluorescence micrographs and close up views of cortical neurons expressing DsRed2-Mito and either SOD1WT or SOD1G93A alone, or in combination with DRP1K38A at day 2 post transfection. Scale bar, 50 µm. Mitochondrial length (Lmito) is indicated as mean ± S.E.M. (n = 10). B) Mitochondrial fragmentation of cortical neurons expressing DsRed2-Mito and either SOD1WT or SOD1G93A alone, or in combination with DRP1K38A. C) Cell death of cortical neurons expressing DsRed2-Mito and either SOD1WT or SOD1G93A alone, or in combination with DRP1K38A. P value: two-tail paired Student’s t-test.

Figure 6

DRP1K38A rescues neurons from mitochondrial trafficking defects. A) Kymographs of mitochondrial transport in cortical neurons expressing DsRed2-Mito and either SOD1WT or SOD1G93A alone, or in combination with DRP1K38A. B) Mitochondrial directional movement in cortical neurons expressing DsRed2-Mito and either SOD1WT or SOD1G93A alone, or in combination with DRP1K38A. C) Mitochondrial velocity in cortical neurons expressing DsRed2-Mito and either SOD1WT or SOD1G93A alone, or in combination with DRP1K38A. D) Mitochondrial mobility in cortical neurons expressing DsRed2-Mito and either SOD1WT or SOD1G93A alone, or in combination with DRP1K38A. P value: two-tail paired Student’s t-test, n = 10.

Figure 7

Both SIRT3 and PGC-1α rescue SOD1G93A-induced mitochondrial fragmentation. A) Sirtuin deacetylase activity of spinal cord tissue lysates isolated from non-transgenic and transgenic mutant SOD1G93A mice. B) Fluorescence micrographs and close up views of mitochondria in cortical neurons expressing DsRed2-Mito and SOD1WT or SOD1G93A alone, or in combination with SIRT3 or PGC-1α. Scale bar: 50µm. C) Mitochondrial fragmentation of cortical neurons expressing DsRed2-Mito and SOD1WT or SOD1G93A alone, or in combination with SIRT3. D) Cell death of cortical neurons expressing DsRed2-Mito and SOD1WT or SOD1G93A alone, or in combination with SIRT3. E) Mitochondrial fragmentation of cortical neurons expressing DsRed2-Mito and SOD1WT or SOD1G93A alone or in combination with PGC-1α. F) Cell death of cortical neurons expressing DsRed2-Mito and SOD1WT or SOD1G93A, alone or in combination with PGC-1α. P value: two-tail paired Student’s t-test.