Figure 1.
Axonal degeneration in the spinal cord of Irp2-null mice.
A; Toluidine blue staining of Epon-embedded spinal cord cross-sections at 12 months. Red-circled areas indicate where myelin dense body (MDBs) were found. The yellow star represents the area enlarged in Figure 1B. Scale bar = 200 µm. B; Ventral white matter (L4) from wildtype (a), Irp1+/+;Irp2-/- (b), Irp1+/-;Irp2-/- (c) mice at 12 months. Arrows indicate MDBs. Scale bars = 50 µm, 20 µm (inset). C; Quantification of MDBs per spinal cord sections at 4, 7.5, 12 months. The bar chart show average±SEM, n = 4-5, **; p<0.001, analyzed by two-way ANOVA. A summary of the pairwise comparisons is presented in Table S1. D; Progressive accumulation of MDBs in ventral white matter of spinal cords at 4, 7.5 months. Scale bars = 10 µm.
Figure 2.
Atrophy and loss of lower motor neurons in the Irp2-null mice.
Aa; Toluidine blue staining of Epon-embedded sections of ventral nerve root. Arrows show MDBs and arrowhead shows occasional swelling of axon. DRG; dorsal root ganglia. Scale bar = 25 µm. Ab; Cross section pictures of ventral nerve root. Arrows show MDBs. Scale bar = 10 µm. B; Motor neuronal axon exit area of the ventral white matter (dotted line). Scale bar = 30 µm. Ca; Cresyl violet staining showed distorted motor neuronal cell bodies (arrows) and motor neurons with chromatolysis (arrowhead) in the Irp2-null mice ventral horn. Scale bar = 25 µm. Cb; Lower magnification of ventral horn stained with cresyl violet. Arrows indicate motor neurons. Scale bar = 50 µm. Da; Quantification of number of myelinated fibers in the venral nerve root per 5000 µm2. n = 3,. Db; Quantification of motor neuronal axon bundles diameters per spinal cord. n = 5,. Dc; Quantification of motor neurons in the ventral horn stained with cresyl violet. n = 4, All data in D show average value±SEM per genotype, *; p<0.05, **; p<0.001, analyzed by one-way ANOVA.
Figure 3.
Status of primary motor cortex neurons in Irp2-null mice.
A; H&E staining of the primary motor cortex (PMC). Arrows indicate large neurons in this area. Scale bar = 10 µm (both) B; Quantification of cell bodies that are larger than 10 µm in diameter / 20,000 µm2. n = 3. *; p<0.05, **; p<0.001, analyzed by one-way ANOVA.
Figure 4.
Increased expression of stress markers in the Irp2-null mice.
A; Immunoreactivity for SMI32 (non-phosphoneurofilament antibody) was increased in Irp2-null mice. Scale bar = 30 µm. B; Infiltration of macrophages and/or microglia (F4/80 positive, arrows) was detected by confocal microscopy. Scale bar = 10 µm.
Figure 5.
Abnormal expression of TfR1 and ferritin in motor neurons and glia of Irp2-null mice and diminished iron concentrations in spinal cord.
Aa; Anti-TfR1 staining was decreased in Irp2-null mice motor neurons (arrows) and the endothelial cells of blood-brain barrier (arrowheads). Scale bar = 20 µm. Ab; Lower magnification picture of spinal cord ventral horn stained with anti-TfR1 antibody. Scale bar = 100 µm. B; Ferritin expression was prominent in both white (a) and grey matter (b) of the spinal cord. Motor neurons exhibited high ferritin expression (arrows) as did in glial cells (arrowheads). Scale bars = 100, 30 µm (respectively). C; Western blot analysis of TfR1 and Ferritin light chain from total spinal cord lysate. Beta-actin was used as a loading control. n = 5. D; Total tissue metal concentrations from different areas of spinal cord by ICP-MS reveal diminished Fe concentrations in the Irp2-null mice *; p<0.05, compared to wildtype, as analyzed by one-way ANOVA.
Figure 6.
Mitochondrial dysfunction and atrophy in the Irp2-null mice.
A; Complex I activity was decreased in Irp2-null mice whereas there were no changes in the amount of GRIM-19, a subunit of Complex I that was quantified. n = 6. Ba; SDH activity (Complex II) was markedly decreased in Irp2-null mice. Bb; There was no change in Complex IV activity which contains heme as a cofactor. *; p<0.05, **; p<0.001, analyzed by one-way ANOVA. C; The mitochondrial proteins SDH-A, SDH-B and ferrochelatase were significantly decreased in Irp2-null mice, whereas citrate synthase, which does not contain a [Fe-S] cluster, did not change and was used as a mitochondrial loading control. n = 5 **; p<0.001 compared to wildtype, analyzed by one-way ANOVA, D; TEM showed mitochondrial vacuolization (arrows) in axons in the ventral white matter of Irp2-null mice and abnormal bundling of neurofilaments within axons was also noted (arrowhead). Scale bar = 0.5 µm.
Figure 7.
Dietary Tempol supplementation partially prevented axonal degeneration.
A; Toluidine blue staining of Epon-embedded sections showed decreased MDBs (arrows) in the ventral white matter of Tempol treated mice. Scale bar = 50 µm. B; Quantification of MDBs per spinal cord section. n = 4, **; p<0.001, analyzed by two-way ANOVA. C; Gel-shift assay (top panel) show activation of IRP1 by Tempol treatment (100 µM) in wildtype mouse embryonic fibroblasts. The iron chelator, DFO, was used to show both IRP1 and IRP2 bands. Tempol treatment did not change total IRP1 (middle panel) by Western blot. Anti-tubulin antibody was used as a loading control. Da; Immunoreactivity for TfR1 was increased in the Tempol treated in the motor neurons of Irp1+/+;Irp2-/- mice (arrows). Db; Western blotting for TfR1 also showed increased expression of TfR1. E; Dietary Tempol treatment increased mitochondrial Complex I activity in Irp1+/+; Irp2-/- mice. n = 5, *; p<0.05, analyzed by two-way ANOVA.
Figure 8.
Decreased expression of ferritin H chain is beneficial for axonal survival.
A; Anti-ferritin staining showed decreased expression of total ferritin in the spinal cord of Irp1+/-;Irp2-/-;Fth+/- mice. Scale bar = 50 µm. B; Toluidine blue staining of the ventral white matter showed decreased number of MDBs in the Irp1+/-;Irp2-/-;Fth+/- mice. Scale bar = 30 µm. C; Quantification of MDBs per spinal cord section. The bar chart show average±SEM, n = 3, *; p<0.05, student's t-test. D; Toluidine blue staining of the cross section of ventral nerve root. Irp1+/-;Irp2-/-;Fth+/- mice showed decreased number of MDBs (arrows). Scale bar = 20 µm. E; Quantification of MDBs per nerve root section. The bar chart show average±SEM, n = 3, **; p<0.001, student's t-test.