Figure 4.

Ultrastructural and biochemical changes in G93A^high-mSOD1 tg mice are consistent with permeability transition pore activation.

A and B. EM shows major cristae remodeling and matrix swelling and vesiculation (black asterisks) in spinal cord ventral horn dendrites of G93A^high-mSOD1 mice. Normal mitochondria are marked by white asterisks. Mitochondria delineated by white-dotted boxes are shown at higher magnification in insets. Mitochondria with vacuoles (A, lower left and right; B, inset) have apparent OMM and IMM contacts (darker spots without narrow space separating the OMM and IMM, hatched arrows). The OMM appears breached at some locations (A, lower left and right, open arrows). Some mitochondria with vesiculation of the matrix have buds extending from the surface (A, lower right, double arrow), suggesting a non-replicative fission or fragmentation process. Some dendrites (B, open arrow) have mitochondria with extreme cristae remodeling and matrix vesiculation. A nearby mitochondrion exhibits early swelling (B, hatched arrow). Scale bars = 0.63 μm (A); 0.3 μm (A, insets); 0.3 μm (B); 0.15 μm (B, inset).

C. Graph showing the estimated number (mean ± SD) of contact sites per mitochondria in spinal cord ventral horn dendrites (identified by presynaptic contacts) of wtSOD1 and G93A^high-mSOD1 mice. Asterisk denotes significantly different (p < 0.05).

D. Apparent fragmentation of mitochondria was seen in spinal cord ventral horn dendrites of G93A^high-mSOD1 mice. Shown is a cross-sectional profile of a dendrite with four identifiable mitochondria (1-4). All of the mitochondria contain matrix vacuoles. Mitochondrion 4 has fragmented into several smaller mitochondrial buds with vacuoles (open arrows). Scale bar = 4 μm.

E. Apparent fission of mitochondria was seen in spinal MN cell bodies of G93A^high-mSOD1 mice. Contact sites between mitochondria are distinct (arrows). One mitochondrion is abnormal with pale matrix, but not showing vacuolation, while the other three mitochondria appear normal. Scale bar = 0.2 μm.

F. Western blot analysis for protein carbonyls (Oxyblot) in the mitochondrial membrane-enriched fraction and the soluble fraction of spinal cord from wtSOD1 tg mice and G93A^high-mSOD1 tg mice at different stages of disease. Molecular weight standards are indicated at right.

G. Increased nitration of CyPD and ANT in G93A^high-mSOD1 mouse spinal cord. Nitrated proteins from soluble and mitochondrial membrane-enriched fractions from wtSOD1 and mSOD1 tg mice at different stages of disease were IP with monoclonal mouse 3-nitrotyrosine antibody followed by western blotting with monoclonal CyPD or polyclonal ANT antibodies.