Search

Rett syndrome (RTT) is a progressive neurodevelopmental disorder without an available cure. It is a rare disease but is known as one of the most common causes of intellectual disabilities and motor dysfunction in females. Approximately 90-95% of RTT cases result from loss-of-function mutations in the X-linked gene called the Methyl-CpG binding protein 2 (MECP2). MeCP2-T158M is the most common missense mutation found in patients, accounting for about 12% of RTT cases. Interestingly, it was reported that increasing MeCP2-T158M expression improved RTT-like phenotypes in both T158M male and female mice in vivo. For over ten years, researchers have been interested in statins, a family of drugs used to lower plasma LDL-cholesterol, as a potential treatment for RTT due to impaired cholesterol metabolism associated with the syndrome. In the statin family, simvastatin has the highest lipophilicity and can effectively cross the blood-brain barrier to enhance its bioavailability in the brain. Therefore, in this research thesis, I hypothesize that increased translation caused by simvastatin will increase the levels of MeCP2 and MeCP2E1 in the brain of Mecp2T158M/y mice and rescue altered levels of other RTT-related proteins. Additionally, simvastatin improves RTT-like phenotypes in Mecp2T158M/y mice. I address my hypotheses through two research aims: determine the molecular effects of simvastatin at the protein level in a brain-region-dependent manner and examine the therapeutic effects of simvastatin on RTT-like phenotypes in the Mecp2T158M/y mouse model. The results showed that at the protein level, simvastatin significantly increased the levels of pS6 Ser235/236 and pS6 Ser240/244 in the cerebral cortex of T158M male mice, suggesting the molecular effects of simvastatin on increasing translation. Furthermore, preliminary data revealed a trend of improving RTT-like phenotypes in a dose-dependent manner with simvastatin treatment. Nevertheless, simvastatin did not rescue reduc

Neuronal DNA methylation is critical to neuronal cell development, maturation, and function. This was illustrated- in large part- by the discovery of Methyl CpG binding protein 2 (MeCP2) as a reader of DNA methylation in the brain. If this role is impaired in neurons, as is seen in individuals diagnosed with Rett Syndrome (RTT), severe mental disability ensues. A contributing factor to this level of severity could be attributed to the high level of MeCP2 observed in mature neurons, fundamental for normal development in terms of facilitating synaptogenesis and dendrite formation. For normal function of neurons it is also important that MeCP2 splice variants (E1 and E2) maintain their expression levels within a certain threshold. MeCP2 protein and transcript levels vary depending on cell type and organ system in which they reside. In the human brain, various anatomical components possess varying levels of MECP2 transcripts. Interestingly, across both MECP2E1 and E2 transcripts, the expression was highest in the human cerebellum out of four brain regions surveyed including the cortex, amygdala, and hippocampus. In this study, a cell model was created using a proliferative cell line originating from the cerebellum. A stably transduced cell line was created providing a reproducible screening assay for which to quantify Mecp2 promoter activity under various conditions. Two different gene reporter assays were used in the same cell lines in order to investigate the function of the MeCP2 isoforms on the promoter elements. Additional reporter assays were conducted to determine the effects of a potential therapeutic drug that targets metabolic pathways that are deregulated in RTT patients. This study provides characterization of the processes taking place at the promoter by quantifying promoter activity levels using a validated and reliable brain cell model. It also serves as a foundation to identify areas for further investigation into the molecular mechanisms of MeCP2.

Rett Syndrome (RTT) is a severe neurodevelopmental disorder mainly affecting females. After a normal developmental period, symptoms appear at the age of 6-18 months, including developmental regression and loss of learned skills such as speech and purposeful hand movements. De novo mutations in the Methyl CpG Binding Protein 2 (MECP2) gene, which codes the MeCP2 protein are the underlying cause of over 95 % of RTT cases. MeCP2 is an epigenetic reader of methylated DNA, which controls gene expression in various cell types of the brain, mainly neurons. The control of MECP2 gene dosage expression is critical since over-expression and under-expression, or genetic mutations lead to neurological deficits including MECP2 Duplication Syndrome (MDS) and RTT, which currently do not have any cure. The two main RTT-associated molecular signaling abnormalities such as impaired MECP2-BDNF-miR132 homeostasis and compromised mammalian Target of the Rapamycin (mTOR) pathway have been detected. Metformin (an anti-diabetic drug) is an inducer of MECP2E1/BDNF transcripts in brain cells in vitro, and an inhibitor of the hepatic mTOR pathway. However, it is unclear whether these effects of metformin can be detected in murine brain tissues. Furthermore, due to the drug safety profile, low price, and penetrability of metformin through the blood-brain barrier, it has been re-purposed in various neurodevelopmental disorders. As a result, metformin may regulate RTT-associated abnormalities and improve RTT-like phenotypes of an RTT mouse model. Consequently, in my thesis, I aim to reveal the gap in knowledge regarding the Mecp2-deficient abnormalities at the basal level as well as after metformin treatment for therapeutic purposes. To accomplish these aims, metformin, and vehicle treatments in Wild Type mice, monitoring of body weight, measurement of blood glucose level, and tissue collection for molecular analysis were completed. The results confirmed the safety of metformin treatment through

The myelin sheath in the central nervous system (CNS) provides insulation and nutritional support for axons, facilitating rapid and efficient impulse conduction. Dysregulation of CNS myelination can lead to severe neurological disorders, such as leukodystrophies and multiple sclerosis. Microglia, the resident macrophages in the CNS, play crucial roles in myelin development, though the exact mechanisms are not fully understood. This study was conducted to understand the specific involvement of microglia in CNS myelination. Initial experiments involving the culture of neural stem cells showed that myelin sheath formation only occurred when a microglia-conditioned medium was present. This indicates a critical role of microglia secretomes in in vitro myelination. Moreover, the absence of C1q from the microglia secretomes resulted in myelination failure in the neural stem cell culture. This could be rectified by adding the native human C1q protein, suggesting that C1q is both required and sufficient for in vitro myelination. Further in vivo experiments were carried out to better understand the involvement of C1q in developmental myelination. By mapping C1q expression profiles, the results showed that the spatiotemporal pattern of C1q expression was similar to that of myelin proteins during early brain development. The use of a C1q conditional knockout mouse model (C1qaFL/FL: Cx3cr1CreER) revealed that C1q deficiency prior to the onset of myelination led to a significant reduction in myelin thickness and an increase in the g-ratio. Additionally, C1q deficiency resulted in a less precise alignment between myelin and axons, while the total myelin production remained unchanged. This suggests that C1q may be involved in the association between myelin and axons. In summary, these findings underscore the crucial role of microglia-derived C1q in developmental myelination. This discovery could potentially pave the way for new therapeutic strategies for treating demyelinating dise

Rett Syndrome (RTT) and MECP2 Duplication Syndrome (MDS) are caused by mutations in the MECP2 gene encoding “methyl CpG binding protein 2” (MeCP2), a critical epigenetic regulator in the brain. There is no cure available for these diseases, but symptoms have been reversed in mouse models. The two isoforms of the protein, MeCP2E1 and E2 are differentially expressed in the brain and there is increasing evidence that they have unique functions, which is important to consider in developing therapeutic strategies for MeCP2-related disorders. MeCP2 has been shown to function as a transcriptional activator and repressor but the role of each isoform in transcriptional regulation has not been extensively studied. In this study, a new gain-of-function model of MeCP2 isoform-specific overexpression was generated in human brain cells through stable lentiviral transduction. Nascent RNA analysis was performed to test the transcriptional effects of the two isoforms on selected target genes, BDNF and miR-132, which form a homeostasis network with MeCP2, as well as Nucleolin and ribosomal RNAs. Both isoforms were shown to increase nascent BDNF transcripts. The results of the human brain cell line correspond with previous observations in human RTT brain tissues, indicating the model established here could be used in the future for additional screening studies. Small molecule drug screening for RTT therapies has recently focused on metabolic drugs which have the potential to correct metabolic defects in RTT patients as well as cognitive defects. The impact of these drugs on MeCP2 regulation is an important aspect of these studies, since MeCP2 levels are tightly controlled. In this study, the effect of simvastatin and metformin on MECP2 and BDNF transcription was assessed in human brain cells. The results suggested that metformin controls MECP2E1 and BDNF post-transcriptionally, but the effect on MECP2E2 is transcriptional and is a promising drug for RTT. This study highlights the impo

Prenatal alcohol exposure (PAE) is considered as a risk factor for the development of fetal alcohol spectrum disorders (FASD). Mounting evidence suggests that PAE affects epigenetic mechanisms and alters the normal differentiation and development of neural stem cells (NSC) in fetal brain. DNA methylation, as an important epigenetic mechanism, contributes to several biological processes during brain development and undergoes significant changes following PAE. In addition, recent studies suggest that DNA methylation is a considerable biomarker for the early diagnosis of FASD. However, effects of PAE are dependent to a variety of factors such as sex and strain of studied subjects. In this regard, we aimed to investigate whether sex and strain play a determinant role in the effects of chronic ethanol exposure on DNA methylation machinery. In addition, based on our previously studied RNA-Sequencing data, we tested the validity of three potential biomarkers (Dcc, Scn3a, and Sptbn2), which were suggested by IPA (Ingenuity Pathway Analysis) software. To do this, we applied a standard and controlled in vitro model system, in which we treated male and female differentiating NSC (obtained from the forebrain of CD1 and BL6 embryos at the embryonic day 14.5) with chronic ethanol exposure (70 mM) for 8 days. Our results showed that chronic ethanol treatment induced global DNA hypomethylation and altered the components of DNA methylation machinery in a sex and strain-specific manner. These alterations in DNA methylation status were associated with altered expression of glial markers CNPASE, GFAP, and OLIG2 in CD1 (but not BL6) cells. We also found that Dcc, Scn3a, and Sptbn2 and their corresponding proteins were altered by ethanol exposure, but may not be true candidate biomarkers for FASD biomarkers because their alterations were not consistent in all experimental groups. In conclusion, the effects of ethanol on DNA methylation of differentiated NSC depended on sex and strain of