Abstract
Rett syndrome (RTT) is a severe and rare neurological disorder that is caused by mutations in the X-linked MECP2 (methyl CpG-binding protein 2) gene. MeCP2 protein is an important epigenetic factor in the brain and in neurons. In Mecp2-deficient neurons, nucleoli structures are compromised. Nucleoli are sites of active ribosomal RNA (rRNA) transcription and maturation, a process mainly controlled by nucleolin and mechanistic target of rapamycin (mTOR)–P70S6K signaling. Currently, it is unclear how nucleolin–rRNA–mTOR–P70S6K signaling from RTT cellular model systems translates into human RTT brain. Here, we studied the components of nucleolin–rRNA–mTOR–P70S6K signaling in the brain of RTT patients with common T158M and R255X mutations. Immunohistochemical examination of T158M brain showed disturbed nucleolin subcellular localization, which was absent in Mecp2-deficient homozygous male or heterozygote female mice, compared to wild type (WT). We confirmed by Western blot analysis that nucleolin protein levels are altered in RTT brain, but not in Mecp2-deficient mice. Further, we studied the expression of rRNA transcripts in Mecp2-deficient mice and RTT patients, as downstream molecules that are controlled by nucleolin. By data mining of published ChIP-seq studies, we showed MeCP2-binding at the multi-copy rRNA genes in the mouse brain, suggesting that rRNA might be a direct MeCP2 target gene. Additionally, we observed compromised mTOR–P70S6K signaling in the human RTT brain, a molecular pathway that is upstream of rRNA–nucleolin molecular conduits. RTT patients showed significantly higher phosphorylation of active mTORC1 or mTORC2 complexes compared to age- and sex-matched controls. Correlational analysis of mTORC1/2–P70S6K signaling pathway identified multiple points of deviation from the control tissues that may result in abnormal ribosome biogenesis in RTT brain. To our knowledge, this is the first report of deregulated nucleolin–rRNA–mTOR–P70S6K signaling in the human RTT brain. Our results provide important insight toward understanding the molecular properties of human RTT brain.
Highlights
Methyl CpG-binding protein 2 gene was discovered in 1992, encoding for MeCP2 as an important member of the DNA methyl binding proteins (MBP) (Lewis et al, 1992)
In order to study the impact of MeCP2 mutations in nucleoli structures in humans, we analyzed post-mortem cerebellum of a Rett syndrome (RTT) patient with the most common MeCP2 mutation (T158M)
We detected a clear nucleolin staining in the nucleoli of the granular cell layer (GCL) and molecular layer (ML) cells of the control cerebellum, but this appeared to be faint and distributed throughout the nucleus in the RTT T158M cerebellum (Figures 1D,F)
Summary
Methyl CpG-binding protein 2 gene was discovered in 1992, encoding for MeCP2 as an important member of the DNA methyl binding proteins (MBP) (Lewis et al, 1992). This is suggestive of MeCP2 binding to methylated rRNA genes at peri-nucleolar parts of the nucleus (Payen et al, 1998), introducing rRNA genes as potential direct target genes of MeCP2 While these studies highlight a functional importance for MeCP2 in embryonic neuronal nucleoli and Abbreviations: DBD, DNA-binding domain; EEG, electroencephalogram; GCL, granular cell layer; IGV, Integrative Genomics Viewer; MBP, methyl binding proteins; MDS, MECP2 duplication syndrome; MECP2, methyl CpG-binding protein 2) (human gene)/Mecp (murine gene)/MeCP2 (protein); ML, molecular layer; mTOR, mechanistic target of rapamycin; PCL, Purkinje cell layer; PFA, paraformaldehyde; PMI, post-mortem delay; qRT-PCR, quantitative real-time PCR; RPM, reads per million; rRNA, ribosomal RNA; RTT, Rett syndrome; SEM, standard error of the mean; TRD, transcriptional repression domain; WB, Western blot; WGBS, whole-genome bisulfite sequencing; WT, wild type
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