Methyl-CpG-binding proteins (MeCP2, MBD1-MBD3) recruit transcriptional co-repressor molecules to methylated regions and silence transcription. The role of MBD2 in regulating brain function and behavior remains largely unexamined. To begin elucidating whether MBD2 influences neural function, I assessed the behavioral performance of Mbd2 null mice, compared their hippocampal electroencephalographic activity during exploration, and performed protein and mRNA expression assessments. The results indicate that mutant mice display a heightened anxiety-like behavior, diminished explorative activity and reduced sociability compared to wild-type mice. However, these behavioral differences were not paralleled by neurophysiological impairments. Mutant hippocampal and cortical samples display significantly elevated MeCP2 mRNA levels. Yet, MeCP2 protein expression did not mirror the mRNA profile and instead was significantly reduced. Glucocorticoid Receptor mRNA levels were significantly reduced in the hippocampus and cortex regions of Mbd2 null brains. The loss of MBD2 is sufficient to induce behavioral impairments in mice without introducing gross deficits in hippocampal neurophysiology.

Intractable epilepsy remains one of the top issues affecting the quality of living in Rett children. While several MeCP2-deficient mouse models of Rett Syndrome have been established, minimal information exists on how the loss of MeCP2 affects brain network activity. To address this issue, in vivo recordings of the hippocampus and somatosensory cortex of MeCP2-deficient mice were taken during exploration, immobility, and sleep. The frequency of hippocampal theta oscillations was significantly attenuated in MeCP2-deficient mice during exploration. A subset of MeCP2-heterozygotes displayed spontaneous, cortical epileptiform-like discharges in the immobile-awake state. Similar epileptiform-like discharges were observed in one of the four Mecp2-null mice recorded. Aside from these EEG abnormalities, basal network activity was preserved. Further, convulsive seizures were not seen. Collectively, these findings indicate that a deficiency of MeCP2 in mice leads to only subtle alterations in brain wave activity, contrasting the severely abnormal EEG observed in Rett girls.

Methyl-CpG-binding proteins (MeCP2, MBD1-MBD3) recruit transcriptional co-repressor molecules to methylated regions and silence transcription. The role of MBD2 in regulating brain function and behavior remains largely unexamined. To begin elucidating whether MBD2 influences neural function, I assessed the behavioral performance of Mbd2 null mice, compared their hippocampal electroencephalographic activity during exploration, and performed protein and mRNA expression assessments. The results indicate that mutant mice display a heightened anxiety-like behavior, diminished explorative activity and reduced sociability compared to wild-type mice. However, these behavioral differences were not paralleled by neurophysiological impairments. Mutant hippocampal and cortical samples display significantly elevated MeCP2 mRNA levels. Yet, MeCP2 protein expression did not mirror the mRNA profile and instead was significantly reduced. Glucocorticoid Receptor mRNA levels were significantly reduced in the hippocampus and cortex regions of Mbd2 null brains. The loss of MBD2 is sufficient to induce behavioral impairments in mice without introducing gross deficits in hippocampal neurophysiology.

Oligodendrocytes (OL) are the myelinating cells of the central nervous system (CNS). A series of complex cell signaling events in the CNS ensures successful myelination. Various molecular cues including growth factors, transcription factors and cytokines regulate myelination by inducing OL migration, proliferation and differentiation. Plateletderived growth factor A (PDGF-A) and fibroblast growth factor 2 (FGF2) are two of the most well characterized regulators of OP migration. The current study hypothesizes that PDGF-A and FGF2 regulate the migration of OP through transient activation of the extracellular signal-regulated protein kinase (ERK) signaling pathway. The results show that activation of ERK is required for OP migration. It also demonstrates the significance of threshold levels of growth factors and temporal regulation for OP migration. Furthermore, the chemokine CXCL1 has been shown to play a critical role in regulating the dispersal of OP during development, although the mechanisms underlying this regulation are unknown. Previous studies have shown that calcium flux is required for OP migration. CXCL1 induces calcium flux in cells; therefore we hypothesized that CXCL1 inhibition of OP migration was regulated via changes in intracellular calcium flux. However, our results show that CXCL1 inhibition of OP migration is independent of calcium signaling. In addition, we show that CXCL1 inhibition of OP migration is specific to PDGF-A induced migration. Lastly, the current study identifies a transcriptional regulator, methyl-CpG-binding protein 2 (MeCP2) as regulating the expression of myelin specific genes in a transgenic mouse. Interestingly, gene expression of myelin associated proteins myelin basic protein (MBP), myelin associated glycoprotein (MAG)and proteolipid protein (PLP), which play an important role in regulation of OL differentiation and subsequent formation of myelin of the myelin sheath, where found to be dysregulated. Overall, these findings reveal previously unknown roles of various intrinsic factors in successive phases of OL development. It aims to provide a better understanding of complexity to myelin development, function and disease.

Oligodendrocytes (OL) are the myelinating cells of the central nervous system (CNS). A series of complex cell signaling events in the CNS ensures successful myelination. Various molecular cues including growth factors, transcription factors and cytokines regulate myelination by inducing OL migration, proliferation and differentiation. Plateletderived growth factor A (PDGF-A) and fibroblast growth factor 2 (FGF2) are two of the most well characterized regulators of OP migration. The current study hypothesizes that PDGF-A and FGF2 regulate the migration of OP through transient activation of the extracellular signal-regulated protein kinase (ERK) signaling pathway. The results show that activation of ERK is required for OP migration. It also demonstrates the significance of threshold levels of growth factors and temporal regulation for OP migration. Furthermore, the chemokine CXCL1 has been shown to play a critical role in regulating the dispersal of OP during development, although the mechanisms underlying this regulation are unknown. Previous studies have shown that calcium flux is required for OP migration. CXCL1 induces calcium flux in cells; therefore we hypothesized that CXCL1 inhibition of OP migration was regulated via changes in intracellular calcium flux. However, our results show that CXCL1 inhibition of OP migration is independent of calcium signaling. In addition, we show that CXCL1 inhibition of OP migration is specific to PDGF-A induced migration. Lastly, the current study identifies a transcriptional regulator, methyl-CpG-binding protein 2 (MeCP2) as regulating the expression of myelin specific genes in a transgenic mouse. Interestingly, gene expression of myelin associated proteins myelin basic protein (MBP), myelin associated glycoprotein (MAG)and proteolipid protein (PLP), which play an important role in regulation of OL differentiation and subsequent formation of myelin of the myelin sheath, where found to be dysregulated. Overall, these findings reveal previously unknown roles of various intrinsic factors in successive phases of OL development. It aims to provide a better understanding of complexity to myelin development, function and disease.

MeCP2 was identified by its ability to bind DNA in a methylation-specific manner. Yet, how it interprets the DNA methylome remains unclear. Several mechanisms have been proposed, including a role in transcriptional repression. MeCP2 is highly abundant in the brain, and loss-of-function mutations result in a neurological disorder called Rett syndrome (RTT). Strikingly, RTT-causing missense mutations are almost all located in either the methyl-CpG-binding domain (MBD) or a region that has been shown to bind the NCoR/SMRT co-repressor complex (NID). This suggests that the MBD and the NID are the key functional domains in MeCP2, and that the role of MeCP2 is to form a ‘bridge’ between chromatin and the co-repressor complex to regulate gene expression. To test this ‘bridge’ hypothesis, I have made an allelic series of knock-in mice with truncated forms of MeCP2 to determine whether the other regions are dispensable for protein function. The three other regions of MeCP2 (the N-terminus before the MBD, the Intervening region between the MBD and the NID, and the C-terminus after the NID) were deleted in a step-wise manner to produce progressively smaller truncated proteins. Knock-in mice which lack just the N-terminus or both the N- and C-termini are phenotypically normal. Therefore, these regions, which together make up 46% of the protein sequence, are dispensable for MeCP2 function in vivo. Additional deletion of the Intervening region, retaining only 34% of the original sequence, results in mild RTT-like symptoms in the knock in mice. This is likely to be caused by this protein’s decreased stability and reduced ability to bind the NCoR/SMRT complex in the brain. The most severely truncated protein is nevertheless able to reverse the Mecp2-null phenotype when reactivated after the onset of symptoms. Together, these findings strongly support the ‘bridge’ hypothesis.

abstract: Methyl-CpG binding protein 2 (MECP2) is a widely abundant, multifunctional regulator of gene expression with highest levels of expression in mature neurons. In humans, both loss- and gain-of-function mutations of MECP2 cause mental retardation and motor dysfunction classified as either Rett Syndrome (RTT, loss-of-function) or MECP2 Duplication Syndrome (MDS, gain-of-function). At the cellular level, MECP2 mutations cause both synaptic and dendritic defects. Despite identification of MECP2 as a cause for RTT nearly 16 years ago, little progress has been made in identifying effective treatments. Investigating major cellular and molecular targets of MECP2 in model systems can help elucidate how mutation of this single gene leads to nervous system and behavioral defects, which can ultimately lead to novel therapeutic strategies for RTT and MDS. In the work presented here, I use the fruit fly, Drosophila melanogaster, as a model system to study specific cellular and molecular functions of MECP2 in neurons. First, I show that targeted expression of human MECP2 in Drosophila flight motoneurons causes impaired dendritic growth and flight behavioral performance. These effects are not caused by a general toxic effect of MECP2 overexpression in Drosophila neurons, but are critically dependent on the methyl-binding domain of MECP2. This study shows for the first time cellular consequences of MECP2 gain-of-function in Drosophila neurons. Second, I use RNA-Seq to identify KIBRA, a gene associated with learning and memory in humans, as a novel target of MECP2 involved in the dendritic growth phenotype. I confirm bidirectional regulation of Kibra by Mecp2 in mouse, highlighting the translational utility of the Drosophila model. Finally, I use this system to identify a novel role for the C-terminus in regulating the function of MECP in apoptosis and verify this finding in mammalian cell culture. In summary, this work has established Drosophila as a translational model to study the cellular effects of MECP2 gain-of-function in neurons, and provides insight into the function of MECP2 in dendritic growth and apoptosis. / Dissertation/Thesis / Doctoral Dissertation Neuroscience 2015

Methyl-CpG binding protein 2 (MeCP2) was discovered as a protein binding to methylated DNA more than 20 years ago. It is very abundant in the brain and was shown to be able to repress transcription. The mutations in MeCP2 cause Rett syndrome, an autism-spectrum neurological disorder affecting girls. Yet, the exact role of MeCP2 in Rett disease, its function and mechanism of action are not fully elucidated. In order to shed some light on its role in the disease the aim of this project was to identify proteins interacting with MeCP2. Affinity purification of MeCP2 from mouse brains and mass spectrometry analysis revealed new interactions between MeCP2 and protein complexes. Detailed analysis confirmed the findings and narrowed down the top interactions to distinct regions of MeCP2. One of the domains interacts with identified NCoR/SMRT co-repressor complex and is mutated in many patients with Rett syndrome. In vitro assays proved that these mutations abolish the putative transcriptional repressor function of MeCP2. We propose a model in which Rett syndrome is caused by two types of mutations: either disrupting the interaction with DNA or affecting the interaction with the identified complex, which has an effect on the global state of chromatin. The presented findings can help to develop new therapies for Rett syndrome in the future.

No description available.

No description available.