Transcriptional Inhibition of the Mecp2 Promoter by MeCP2E1 and MeCP2E2 Isoforms Suggests Negative Auto-Regulatory Feedback that can be Moderated by Metformin.

The epigenetic factor Methyl-CpG-Binding Protein 2 (MeCP2) is a nuclear protein that binds methylated DNA molecules (both 5-methylcytosine and 5-hydroxymethylcytosine) and controls gene transcription. MeCP2 is an important transcription factor that acts in a dose-dependent manner in the brain; thus, its optimal expression level in brain cells is important. As such, its deregulated expression, as well as gain- or loss-of-function mutation, lead to impaired neurodevelopment, and compromised structure and function of brain cells, particularly in neurons. Studies from others and us have characterized two well-recognized MeCP2 isoforms: MeCP2E1 and MeCP2E2. We have reported that in Daoy medulloblastoma brain cells, MeCP2E2 overexpression leads to MeCP2E1 protein degradation. Whether MeCP2 isoforms regulate the Mecp2 promoter regulatory elements remains unexplored. We previously showed that in Daoy cells, metformin (an anti-diabetic drug) induces MECP2E1 transcripts. However, possible impact of metformin on the Mecp2 promoter activity was not studied. Here, we generated stably transduced Daoy cell reporters to express EGFP driven by the Mecp2 promoter. Transduced cells were sorted into four EGFP-expressing groups (R4-to-R7) with different intensities of EGFP expression. Our results confirm that the Mecp2 promoter is active in Daoy cells, and that overexpression of either isoform inhibits the Mecp2 promoter activity, as detected by flow cytometry and luciferase reporter assays. Interestingly, metformin partially relieved the inhibitory effect of MeCP2E1 on the Mecp2 promoter, detected by flow cytometry. Taken together, our data provide important insight towards the regulation of MeCP2 isoforms at the promoter level, which might have biological relevance to the neurobiology of the brain.

Determining affected memory domains in patients with amnestic mild cognitive impairment using computerized and interactive tests.

This study explores different episodic memory domains, namely object, temporal and spatial memory, affected in patients with a clinical diagnosis of single domain amnestic mild cognitive impairment (aMCI). 15 aMCI patients and 25 healthy controls were recruited and tested. Object, spatial, and temporal memory were tested using computerized tasks and again in interactive, real-world tasks. Controls outperformed patients on the object computerized task and showed a trend toward significance for the computerized spatial and temporal tasks, but there was no difference in spatial and temporal memory when using the interactive tasks, indicating the employment of compensatory mechanisms in patients to overcome some of the memory impairments associated with aMCI. These findings highlight that aMCI patients might delay seeking help due to compensatory mechanisms which mask their deficits in real-world situations.

Simvastatin Induces Apoptosis in Medulloblastoma Brain Tumor Cells via Mevalonate Cascade Prenylation Substrates.

Medulloblastoma is a common pediatric brain tumor and one of the main types of solid cancers in children below the age of 10. Recently, cholesterol-lowering "statin" drugs have been highlighted for their possible anti-cancer effects. Clinically, statins are reported to have promising potential for consideration as an adjuvant therapy in different types of cancers. However, the anti-cancer effects of statins in medulloblastoma brain tumor cells are not currently well-defined. Here, we investigated the cell death mechanisms by which simvastatin mediates its effects on different human medulloblastoma cell lines. Simvastatin is a lipophilic drug that inhibits HMG-CoA reductase and has pleotropic effects. Inhibition of HMG-CoA reductase prevents the formation of essential downstream intermediates in the mevalonate cascade, such as farnesyl pyrophosphate (FPP) and gernaylgerany parophosphate (GGPP). These intermediates are involved in the activation pathway of small Rho GTPase proteins in different cell types. We observed that simvastatin significantly induces dose-dependent apoptosis in three different medulloblastoma brain tumor cell lines (Daoy, D283, and D341 cells). Our investigation shows that simvastatin-induced cell death is regulated via prenylation intermediates of the cholesterol metabolism pathway. Our results indicate that the induction of different caspases (caspase 3, 7, 8, and 9) depends on the nature of the medulloblastoma cell line. Western blot analysis shows that simvastatin leads to changes in the expression of regulator proteins involved in apoptosis, such as Bax, Bcl-2, and Bcl-xl. Taken together, our data suggests the potential application of a novel non-classical adjuvant therapy for medulloblastoma, through the regulation of protein prenylation intermediates that occurs via inhibition of the mevalonate pathway.

MECP2 Mutation Interrupts Nucleolin-mTOR-P70S6K Signaling in Rett Syndrome Patients.

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.