PMC

Cell death markers in cortical motor neurons. Motor cortex tissues from 18 ALS and 10 non-neurological disease controls were immunostained for Fas (A), caspase-8 (B) and caspase-3 (C). There was negligible staining for each protein in motor neurons of the cortex. Each panel is at ×200 magnification with the insets from the same cases at ×400.

Fas and Caspase-8 co-localize in spinal motor neurons. Lumbar spinal cord sections from 11 ALS and 6 age-matched controls were labeled with anti-caspase-8 (Alexa green conjugated secondary antibody) and anti-Fas (Cy5 conjugated secondary antibody) antibodies. In ALS motor neurons, Fas (red signal) is present in the nucleus and co-localized with caspase-8.

Co-localization of ppRb and p53 in motor neurons of ALS spinal cord. Tissue sections were immunostained for p53 and ppRb by double-label confocal microscopy. The secondary antibodies were either conjugated to a red dye (Cy5) or a green dye (Alexa green). While p53 co-localized with ppRb in the nucleus of spinal motor neurons (A), nuclei of cortical motor neurons lacked p53 but contain abundant ppRb (B). All panels are at 7times;400 magnification.

E2F-1 and p53 co-localize with caspase-3 in ALS spinal motor neurons. Tissue sections of lumbar spinal cord (A and B) and motor cortex (C and D) from 11 ALS and 6 age-matched controls were labeled with the combinations of p53 or E2F-1 (Alexa green conjugated secondary antibody) and active caspase-3 (Cy5 conjugated secondary antibody). Arrows in the control panels indicate representative motor neurons. E2F-1 and p53 co-localize with caspase-3 in spinal motor neurons of ALS patients. All panels are at ×400 magnification.

Increased p53 immunoreactivity in ALS spinal cord motor neurons. Lumbar spinal cord (A-C) and motor cortex (D-F) tissues from 18 ALS and 9 non-neurological disease controls were immunostained for p53 using a phospho-specific anti-p53 antibody. Each panel is at ×200 magnification with the insets at ×400. Relative to controls (panels A and D), there is abundant nuclear p53 in ALS spinal cord (B) but not motor cortex (E). Panels C and F are the internal controls for the respective regions.

Increased immunoreactivity of cell death markers in spinal cord motor neurons. Lumbar spinal cord tissues from 18 ALS and 9 non-neurological disease controls were immunostained for BAX (A-C), Fas (D-F), caspase-8 (G-I) and caspase-3 (J-L). Each panel is at ×200 magnification with the insets at ×400. Relative to controls, there is increased BAX (B), nuclear Fas (E), caspase-8 (H) and caspase-3 (K) in ALS spinal cord motor neurons. Panels C, F, I and L represent immunostaining of sensory neurons of the ALS spinal cords.

Increased DNA fragmentation in spinal but not cortical motor neurons. Lumbar spinal cord (A and B) and motor cortex (C and D) sections from 11 ALS and 6 age-matched controls were double-labeled for TUNEL (FITC-conjugated) and p53 or ppRb (Cy-5 conjugated). Sections from all control subjects exhibited negligible co-localization of ppRb or p53 and TUNEL within neurons. Arrows in A and B indicate TUNEL positive spinal motor neurons. TUNEL positive motor neurons were also ppRb or p53 immunoreactive in the spinal cord (Merge panels; arrowheads). The asterisk in the inset depicts a ppRb(+) but TUNEL(-) ALS motor neuron. The cortical motor neurons in ALS cases were ppRb positive (C) and p53 negative (D). Motor neurons in panels C and D appear negative for TUNEL (arrows indicate control motor neurons and arrowheads indicate ALS motor neurons).

Increased p53 and cell death markers in ALS spinal cord. Immunoblot analysis of lumbar spinal cord tissue extracts. A) Top panel: Lane assignments for p53, p73 and Bax are as follows: “+” indicates positive control, lanes 1 to 5 are control cases and lanes 6 to 12 are ALS cases. The arrows on the BAX blot indicate immunoreactive bands of 19- and 21-kDa. Bottom panel: Lanes for Fas and Caspase immunoblots are as follows: “+” indicates positive control, lanes 1 to 3 are control cases and lanes 4 to 9 are ALS cases. B) Quantification (mean +/- SEM) of the immunoblots. Black bars represent control cases (n=10) and the gray bars are ALS cases (n=18). Statistical analysis was performed using single-factor ANOVA and asterisks (**) indicate p ≤ 0.05.

No change in p53 DNA binding activity in ALS motor neurons. Nuclear extracts from lumbar spinal cord and motor cortex were used for electrophoretic mobility shift assays (EMSA). (A): Competition EMSA using radiolabeled p53 oligonucleotide and either extract from NIH3T3 cells as a positive control (lanes 1 to 3) or spinal cord nuclear extracts (25μg; lanes 4 to 7) indicating a concentration dependent competition (lanes 2 – 3 and 5 – 7) when excess cold oligonucleotides were used in the binding reaction. The complex remains unaltered when excess unrelated oligonucleotides (lanes 8 – 10) were utilized in the binding reaction. Lane assignments for competition assays were as follows: Lanes 2 and 3: 3T3 extract with excess unlabeled p53 oligonucleotide (3ng and 100ng, respectively); Lanes 5 to 7: spinal cord extract with excess unlabeled p53 oligonucleotide (3ng, 30ng and 100ng, respectively); Lanes 8 to 10: spinal cord extract with excess unlabeled unrelated oligonucleotide (30ng, 100ng and 300ng, respectively); Lane 11: Free radiolabeled probe alone (FP). (B): EMSA of control or ALS spinal cord nuclear extract and radiolabeled p53 oligonucleotide. Lane 1 is free probe alone; Lanes 2 to 4 are Control subjects; Lanes 5 to 10 are ALS cases. (C): EMSA of control or ALS motor cortex nuclear extract and radiolabeled p53 oligonucleotide. Lanes 1 to 3 are Control subjects; Lanes 4 to 9 are ALS subjects; Lanes 10 to 12 are 3T3 extracts with 100ng excess unlabeled p53 oligonucleotide (lane 10), 3ng excess unlabeled p53 oligonucleotide (lane 11), and radiolabeled p53 oligonucleotide without competitor (lane 12). Lane 13 is free probe alone. (D): Densitometry measurement of the p53:DNA complex in each lane using NIH 1.58 software indicates unaltered levels of DNA binding activity in either the spinal cord or motor cortex. Controls are denoted in black bars (n=3) and ALS cases in stippled bars (n=6). There is no statistically significant alteration in DNA binding activity.