The role of apoptosis in neurodegenerative disorders remains controversial1. Most of the uncertainty derives from the fact that (i) since these diseases progress over years, the possibility of finding a single dying cell in vivo is extremely remote,(ii) tissues are from endstage patients and are therefore already depleted of neurons,(iii) even if present, death changes may be due to premortem agonal status rather than to the disease itself. Such problems may be avoided by using appropriate animal models. Severe loss of motor neurons characterizes amyotrophic lateral sclerosis (ALS), yet there is no conclusive evidence that apoptosis is involved2. The etiology of sporadic ALS is unknown; in 20% of familial cases, point mutations in the superoxide dismutase 1 (SOD1) gene are present, and mutant SOD1 transgenic mice develop an ALS-like disease mediated by a toxic gain-of-function of the mutated enzyme3. Although mutant SOD1 induces apoptosis invitro3, it is unclear whether the same happens in vivo. Overexpression of the antiapoptotic protein Bcl-24 or inhibition of proapoptotic interleukin-1ß-converting enzyme (ICE) 5 modestly slow disease progression in mutant SOD1 mice. These effects, however, do not necessarily imply apoptosis regulation, since Bcl-2 might protect cells from SOD1 toxicity through its antioxidant properties, and ICE has many activities unrelated to apoptosis. Moreover, detailed ultrastructural studies in mutant SOD1 mice did not report on the presence of morphological signs of apoptosis, although they did show a wide array of ongoing degenerative changes, ranging from early vacuolar changes due to mitochondrial damage, to late formation of cytoplasmic inclusions in remaining motor neurons6.

To clarify whether degenerating motor neurons in mutant SOD1 mice undergo apoptosis, we used transgenic mice derived from Jackson Laboratory (Bar Harbor, Maine), that carry about 20 copies of human SOD1 gene with a glycine-to-alanine substitution at position 93 (G93A) 7. These mice become symptomatic at around 70 days of age and die by 142±4.3 days. Sixteen transgenic and six wild-type mice combined in three age groups (9-14-19 weeks) were studied. Following perfusion fixation with 4% paraformaldehyde and paraffin embedding, the cervical and lumbar spinal cord were serially cut. Intraneuronal inclusions were immunohistochemically revealed with antibodies to human SOD1 (Sigma) and ubiquitin (Chemicon). To detect DNA fragmentation, a hallmark of apoptosis, we performed an in situend-labeling (ISEL) assay (Boehringer) 2. Briefly, sections were pretreated with proteinase K (up to 50 µg/ml) and incubated with terminal deoxynucleotidyl transferase and fluorescein-11-dUTP, followed by peroxidase-conjugated anti-fluorescein sheep antisera. To evaluate the expression of apoptosis-related proteins, immunohistochemistry was performed with antibodies to c-Jun/AP-1 (Calbiochem) and PCNA (Dako), two proteins that accumulate in the nuclei of apoptotic neurons8, as well as with an antiserum that selectively recognizes the active form of apoptotic protease caspase-