Caspase-1 cleavage appears early in the lifespan of G37R- and G85R-transgenic mice. Immunoblots of spinal cord extracts from G37R (a) and G85R (b) mice probed with a polyclonal antibody against both the inactive (p45) pro-enzyme and the active (p20) caspase-1. In mice over-expressing the wt human SOD1 (hSOD1) protein, the antibody only recognized the p45 inactive and the p30 intermediate form. (c) Caspase-1 cleavage in G37R mice detected with the polyclonal antibody used for the cell culture experiments. wt indicates mice overexpressing wt hSOD1.

Caspase-3 immunoreactivity appears ≈2 months before onset and increases with age in the spinal cord anterior horn of all mutant SOD1 mouse lines tested. Immunostaining for active caspase-3 (brown precipitate) in G37R (a and b) and G85R (c and d) mice and their nontransgenic littermate controls. (b and d) Increased magnification of a and c (end stage). (e) Active caspase-3 immunoreactivity in G93A mouse, nontransgenic littermate control, and wt hSOD1-expressing mouse spinal cord. Black arrows indicate active caspase-3-positive inclusions. White arrows indicate caspase-3-positive motor neurons. Arrowheads indicate vacuoles. (Bars = 25 μm.)

Activated caspase-3 appears within the neuronal and glial inclusions present in mutant SOD1 mouse spinal cords. Double immunofluorescence of spinal cord anterior horn using CM1 antibody (Casp 3 in the Left panels) and either GFAP antibody to stain astrocytes, ubiquitin antibody (UBIQ) to stain inclusions, or an antibody recognizing nonphosphorylated neurofilaments (NF) to visualize neurons. Right panels depict the merged image from the first two channels with DAPI to show the position of nuclei. Activated caspase-3-positive inclusions (arrowheads) within astrocytes (A) and/or extracellular debris (*) are common features within mutant G85R (a) and G93A-mutant (b) spinal cords. (c) Activated caspase-3 colocalizes with ubiquitin-positive inclusions in G93A-mutant mice. Activated caspase-3 immunoreactivity is present within motor neurons (MN) (d) and motor neuron inclusions (e) (arrow) of mutant G85R mice. (f) Activated caspase-3 is absent from 10-mo-old wt hSOD1-transgenic mice whereas astrocytic GFAP staining is normal. (Bar = 12.5 μm.)

Caspase-3 is activated late in mutant SOD1-transgenic mouse spinal cord and cortex but not in cerebellum or sciatic nerve. Immunoblots of spinal cord and cortex extracts from G37R (a and b) and G85R (e and f) mice probed with the CM1 antibody. No immunostaining is detected early in life. Staining for the p18 fragment appears at 4 months in the G37R mice and at 10 months in the G85R mice but not in littermate nontransgenic controls (wt). Activation of caspase-3 is tissue-specific; no active fragment appears in cerebellum or sciatic nerve of either G37R (c and d) or G85R (g and h) mice. Each gel was loaded with 30 μg/lane.

Caspase-1 and caspase-3 activation precedes loss of large motor axons and the appearance of apoptotic morphology in all mutant SOD1 mouse lines tested. Axon counts from the L5 motor root of the spinal cord for G85R (a), G37R (b), and G93A (c) mice (dotted lines) and their littermate nontransgenic controls (solid lines) at various ages. The appearance of activated caspase-1 in immunoblot is indicated by the leftmost arrowhead in a and b. The appearance of activated caspase-3 in immunoblot is indicated by solid triangles. The appearance of activated caspase-3 in immunohistochemistry is indicated by open triangles in a, b, and c. Axon counts are averages from three to five animals of each genotype and age. Horizontal brackets indicate the age of disease onset (DO). Error bars are the SD of the data. (d and e) Electron microscopy images show apoptotic changes within the ventral horn of 8-mo-old G93A-mutant mice. Arrows indicate condensed chromatin within nuclei. Vacuoles are marked with a V. Filaments within neurons are indicated with an F. (Bars in d and e = 2 μm.)

Caspase-1 is activated early in differentiated mouse neuroblastoma N2a cells. Immunoblots showing caspase-1 activation in differentiated G37R (a) or G85R (b) positive N2a exposed to X/XO for 0, 1, 3, or 4 h. (c) Immunoblot of lysates from wt hSOD1-expressing cells (left) or G85R-positive cells (right) 4 h after X/XO treatment. (d) YVAD-AMC cleavage was measured as described and is reported relative to the cleavage induced by lysates of untreated (time 0) wt hSOD1-positive cultures. Data are the mean ± SD of three independent experiments for the G85R-positive cells and of two independent experiments assayed in duplicate for the G37R and wt hSOD1 expressing cells. Asterisks (*, P < 0.05; **, P < 0.01) indicate significant differences between groups. (e) Immunoblot showing levels of wt and mutant SODs in N2a cell lines by using an antibody that recognizes human and mouse SOD1 equally. The hSOD1 protein migrates more slowly than the mouse, only the G85R mutant comigrates with the mouse SOD1. (f) Caspase-3 activity recorded as DEVD-AMC cleavage by cell lysates relative to the cleavage induced by lysates of untreated wt hSOD1-positive cells. Data are the mean ± SD of three independent experiments. Asterisks (*, P < 0.05) indicate a statistical difference between groups. (g) Percentage of cells that stained positive for activated caspase-3 at different times after X/XO treatment. (h) YVAD-AMC cleavage and DEVD-AMC cleavage measured in lysates from G85R cells treated with X/XO in the presence of caspase-1-and-3 inhibitors. Data are the mean ± SD of six replicates. Fluorescence emitted from the fluorogenic substrate is expressed relative to that emitted from untreated cells.