Figure 3.

MeCP2 pS421 is upregulated in Alzheimer's disease (AD) mice striatum after AD7c-NTP small-hairpin RNA (shRNA) treatment. Mice striatum was collected at age of 3 months. Cytoplasmic proteins and nuclear proteins were extracted, and protein levels in each fraction were assessed. (A,B) The timeline diagram of this study. Animals received AD7c-NTP shRNA or scrambled shRNA injection into lateral ventricle at age of 3 months and were sacrificed 2 weeks later. (C–E) Altered AD7c-NTP immunoreactivity in AD mice. Immunohistochemical staining showed enhanced expression of AD7c-NTP in (D) AD mice relative to (C) aged control and (E) inhibited by AD7c-NTP shRNA. (F,G) Immunohistochemical double labeling [(F), brown: AD7c-NTP; blue: 4′6-diamidino-2-phenylindole dihydrochloride (DAPI)] and immunofluorescence double labeling [(G), red: AD7c-NTP; blue: DAPI)] showed increased AD7c-NTP in AD striatal neurons (single arrow), as well as in degenerating neurons (double arrows) and abnormal neuritic processes (arrowheads). (H) Western blot analysis showing downregulation of endogenous AD7c-NTP in AD mice. (I–K) Western blot analysis showed that AD7c-NTP shRNA injection has further enhanced the cytoplasmic protein levels of MeCP2 pS421 compared with the scrambled plasmid treatment groups (I). (I) While AD7c-NTP shRNA injection has no impact on the MeCP2 protein levels in nucleus. Quantitative analysis of (J) MeCP2 and (K) MeCP2 pS421 protein levels, respectively (mean ± SEM, *P < 0.05 vs. control group mice, ^#P < 0.05 vs. AD + Ctr shRNA group mice, n = 6 in each group).