Figure 1. PNNs are increased in human RTT CA2 and develop prematurely in CA2 of a mouse model of RTT.

(A) Staining for the CA2-enriched protein RGS14 labels CA2 pyramidal neurons in control human hippocampal tissue in the stratum pyramidale (SP) and stratum radiatum (SR), but less in the stratum oriens (SO). Scale bars: 1 mm (top), 100 μm (bottom). (B) HAPLN1, a PNN link protein, is localized to area CA2 in a healthy human hippocampus (relative to background: CA1, 0.57; CA2, 1.67; CA3, 0.082). HAPLN1 appears greater in hippocampal tissue from an age- and sex-matched individual with RTT (relative to background: CA1, 1.06; CA2, 2.87; CA3, 0.52). Scale bars: 1 mm (top), 100 μm (bottom). (C) Left: Staining for the PNN marker WFA (green) is greater in Mecp2-null CA2 compared with WT littermates throughout early postnatal development. Scale bars: 200 μm. (D) Normalized WFA fluorescence intensity was significantly greater in Mecp2-null CA2 (n = 4, 4, 5 for ages P14, P21, P45, respectively) compared with WT littermates (n = 4, 3, 5 for ages P14, P21, P45, respectively); **P = 0.004, ****P < 0.0001, 2-way ANOVA, Bonferroni’s post hoc test, significant main effects of age F_2,19 = 194.8, condition F_1,19 = 110.5, and interaction F_2,19 = 4.913. Indicated are means ± SEM. In addition, WFA staining surrounding PNN⁺ neurons in CA1 and CA3 did not differ between Mecp2-null males and WT littermates; P > 0.05, 2-way ANOVA. Indicated are means ± SEM. (E) Staining intensity for WFA (green) is greater in area CA2 of Mecp2-null at P11 compared with WT littermate. The CA2 pyramidal neuron borders with areas CA1 and CA3 are represented with white lines. Scale bars: 200 μm. (F) Normalized WFA fluorescence intensity was significantly greater in CA2 of P11 of Mecp2-null; ***P = 0.0005, 2-tailed unpaired t test (n = 9 and 5, WT littermate and Mecp2-null, respectively). Indicated are means ± SEM. Fluorescence intensity was amplified by camera exposure settings relative to exposure settings in C for quantification purposes.