MECP2 Mutation Interrupts Nucleolin–mTOR–P70S6K Signaling in Rett Syndrome Patients

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.

Differential brain region‐specific expression of MeCP2 and BDNF in Rett Syndrome patients: a distinct grey‐white matter variation

“…Lower transcript level of MECP2 that we showed in the human RTT cerebrum also confirms previous studies [24,84]. However, as a rare disease, small sample size is one of the limitations of our study, similarly experienced in previous studies using human RTT brain tissues [43,50,85]. Accessing human RTT brain samples, and comparable controls is part of the challenge when we move from studying RTT animal models and cell lines to human brain samples.…”

“…Formalin‐fixed autopsy brain samples of four RTT patients (three females and one male) were analysed in this study and compared with samples of age‐ and sex‐matched controls. The samples included: (i) a 13‐year‐old female RTT patient (T158M), for whom the brain tissue was received by direct organ donation (clinical information has been recently reported) [50], (ii) a 19‐year‐old female RTT patient (A201V), for whom the brain was also received through organ donation, (iii) a 20‐year‐old female RTT patient (R255X) whose brain tissue was received from NIH Neurobiobank (NIH # 4516), and a 44‐year‐old male RTT patient (P152H) whose brain was directly donated to our laboratory for research purposes. Available frozen tissue from the frontal cerebrum of the three female RTT patients was used for RNA extraction and RT‐PCR.…”

“…Total RNA was extracted from the frontal cerebrum by using Trizole (Life Technologies Inc. 15596‐062) as per manufacturer’s guidelines, as reported [50]. Extracted RNA was then treated with DNase I to remove probable genomic DNA contaminations, using the Ambion TURBO DNA‐free™ kit (ThermoFisher Scientific), and subjected to cDNA synthesis by Superscript III Reverse Transcriptase (Invitrogen), as described previously [53,54].…”

The MeCP2E1/E2-BDNF-miR132 Homeostasis Regulatory Network Is Region-Dependent in the Human Brain and Is Impaired in Rett Syndrome Patients

“…Tissue samples and related data for R255X and frozen control tissues were obtained from the "University of Maryland Brain and Tissue Bank, which is a Brain and Tissue Repository of the NIH Biobank (at NIH NeuroBioBank Program: neurobiobank.nih.gov). The authors would like to sincerely thank the family members of the T158M and A201V patients for organ brain donation for research, as previously described in our publications (Olson et al, 2018;Pejhan et al, 2020).…”

DNA Methylation Contributes to the Differential Expression Levels of Mecp2 in Male Mice Neurons and Astrocytes

“…MeCP2 is a major transcriptional regulator in the brain with solid links to neurodevelopmental disorders [1]. Altered expression and function of MECP2 / Mecp2 /MeCP2 have been linked to Rett syndrome (RTT) [2,3,4], MECP2 duplication syndrome (MDS) [5,6,7], autism spectrum disorders (ASD) [8,9], and fetal alcohol spectrum disorders (FASD) [10,11]. MeCP2-associated diseases have a strong sex correlation (especially in the case of RTT and MDS), possibly due to the X-linked nature of the MECP2 gene [12] and/or X chromosome inactivation (XCI) in females [13].…”