Figure 11.

Dnmts and 5-mC are upregulated in human sporadic ALS motor cortex. A, Westerns blots for Dnmt1 and Dnmt3a in human control and sporadic ALS primary motor cortex homogenates fractionated into nuclear-enriched, soluble, and mitochondrial-enriched protein compartments. The blots were reprobed for GAPDH to show protein loading. B, Graphs showing the analysis of Dnmt1 and Dnmt3a Western blots on human control and ALS primary motor cortex tissue fractions. Values represent mean ± SD of optical density (OD) units. Dnmt1 was barely detectable or undetectable in control mitochondrial fractions and was undetectable in control and ALS soluble fractions. Dnmt3a was barely detectable or undetectable in control soluble fractions. Asterisks denote significant differences (p < 0.05) compared with control. C, D, Immunohistochemical localization of 5-mC in human control (C) and ALS (D) motor cortex. Panoramic images of deep layers and higher-magnification images (insets) are shown. C, In control motor cortex, Dnmt1 immunoreactivity is very low to not detectable in neurons, including large Betz cells (open arrows), but is present in small non-neuronal cells (inset, hatched arrows). D, In ALS motor cortex, numerous neurons are distinctly positive for Dnmt1 (open arrows). Dnmt1 immunoreactivity is enriched in the nucleus of some neurons (inset, open arrows and asterisk), but nearby neurons have scant immunoreactivity (inset hatched arrow). Scale bars: (in C, same for D–H), 22 μm; (in A, inset; same for D–H, insets), 10 μm. E, F, Immunohistochemical localization of Dnmt3a in human control (E) and ALS (F) motor cortex. E, In control motor cortex, Dnmt3a immunoreactivity is present in the cytoplasm of neurons (open arrows), including Betz cells, and is present in non-neuronal cells (inset, hatched arrow) and in the neuropil as fine puncta. The nucleus of control neurons has low Dnmt3a immunoreactivity (inset asterisk). F, In ALS motor cortex, numerous neurons, including Betz cells, are distinctly positive for Dnmt3a (open arrows). Dnmt3a immunoreactivity is enriched in the nucleus of some neurons (inset, open arrow and asterisk), but nearby cells are negative (inset, hatched arrow). G, H, Immunohistochemical localization of 5-mC in human control (G) and ALS (H) motor cortex. 5-mC immunoreactivity was visualized with diaminobenzedine (brown), and sections were counterstained with cresyl violet. G, In control motor cortex, most pyramidal neurons (open arrows) have low 5-mC immunoreactivity. 5-mC immunoreactivity was present faintly in pyramidal neurons (inset, open arrow) as fine particles in the nucleus (asterisk) and cytoplasm, but in nearby glial cells (inset, hatched arrows), 5-mC immunoreactivity was enriched in the nucleus. H, In ALS motor cortex, many pyramidal neurons (open arrows) show obvious 5-mC immunoreactivity. 5-mC immunoreactivity was enriched in the nucleus (inset, asterisk) of pyramidal neurons (open arrow), whereas nearby glial cells (inset, hatched arrows) showed scant 5-mC immunoreactivity.