Figure 2. ATF4 immunostaining in neuromelanin-positive SN neurons is elevated in a subset of PD patients. A–I, Hemi-midbrain sections from postmortem brain were immunostained for ATF4. A, Low-power view of adjacent sections from control midbrain. In the right section, the antibody was preabsorbed with blocking peptide. Dotted lines show boundaries of the SN. B, Higher-power view of SN from control, showing predominantly blue–gray cytoplasmic staining in neuromelanin-positive neurons (arrows). C, Serial section incubated with ATF4 antibody preabsorbed with blocking peptide. Arrows demonstrates cytoplasm of neuromelanin-positive cell. D, Control SN, showing one neuromelanin-positive neuron with intense nuclear ATF4 (arrow), surrounded by three neurons with cytoplasmic ATF4. E, PD SN, showing ATF4 staining of proximal neuronal process (arrowheads) and neuropil (arrows). F, PD SN, showing very intense ATF4 in the neuropil. G, Beaded neurites with ATF4 staining (arrows) in PD SN. H, Neuromelanin-positive neuron with four Lewy bodies (indicated by arrows), with varying degrees of ATF4 immunostaining. I, Representative fields from SN are shown from two controls (CTRL; left), one PD patient with low ATF4 levels (top right), and one PD patient with high ATF4 (bottom right). Scale bars: B–F, I, 50 μm; G, H, 25 μm. J, Semiquantitative assessment of ATF4 immunostaining in neuromelanin-positive neurons from controls (n = 9) and PD patients (n = 10). The intensity of ATF4 labeling in each neuron was graded, in a blinded manner, as absent, weak, or strong. For each case, 40–150 cells were analyzed. The results are graphed as the percentage of neuromelanin-positive neurons in each case that exhibited absent, weak, or strong ATF4 staining. PD patients were separated into groups showing low and high ATF4 expression. The mean percentage of neurons from each group with each staining intensity level is indicated by a solid bar.