MeCP2 was initially identified as an abundant protein in the brain, with an affinity for methylated DNA in vitro. Interestingly, both deficiency and excess of the protein leads to severe neurological problems, such as Rett syndrome, which is the result of mutations in the MECP2 gene. Subsequent transfection experiments showed that MeCP2 can recruit corepressor complexes and inhibit gene expression in vivo. MeCP2 was therefore thought to repress specific gene targets and the aetiology of Rett syndrome was proposed to result from aberrant gene expression in the MeCP2-deficient brain. Although gene expression is perturbed in the Mecp2-null mouse brain, few specific targets have been verified and alternative hypotheses for MeCP2 function have been put forward. Previous binding studies have also failed to clearly identify MeCP2 targets. To shed light on these matters, a novel technique was generated to isolate neuronal and glial nuclei and established that the amount of MeCP2 is unexpectedly high in neurons, with an abundance approaching that of the histone octamer. Chromatin immunoprecipitation experiments on mature mouse brain showed widespread binding of MeCP2, consistent with its high abundance, tracking the methyl-CpG density of the genome. MeCP2 deficiency results in global changes in neuronal chromatin structure, including elevated histone acetylation and a doubling of histone H1. The mutant brain also shows elevated transcription of repetitive elements, which are distributed throughout the mouse genome. Based on this data, we propose that MeCP2 binds genome wide and suppresses spurious transcription through binding in a DNA methylation dependent manner.

The common neurotrophin receptor, p75NTR, has been implicated in diverse responses of sensory neurons including a role in nociception following nerve injury, suggesting that it may serve a similar role in intact sensory neurons and their satellite glial cells (SGCs). To examine the impact of suppressing neuronal p75NTR expression on known molecular modulators/regulators of the nociceptive state namely, the sodium channels NaV1.8 and NaV1.9, the nerve growth factor receptor TrkA, the potassium channel Kir4.1, glial fibrillary acidic protein (GFAP), SGC p75NTR, connexin 43, we intrathecally infused p75NTR anti-sense oligonucleotides (AS OGN), previously shown by Obata et al. (2006) to effectively suppress p75NTR expression in intact neurons. Male, Wistar rats were divided into three groups, receiving either no treatment (non-infused), seven day intrathecal infusion of p75NTR AS OGN or sense control (SC OGN) via an osmotic pump. Serial L4 and L5 DRG sections were processed for immunohistochemistry to detect alterations in NaV1.8, NaV1.9, TrkA, Kir4.1, p75NTR, GFAP and connexin-43 protein expression. Sciatic nerve sections were also processed for immunohistochemistry to detect NaV1.8, NaV1.9, TrkA and GFAP protein expression. Infusion of p75NTR AS OGNs resulted in a significant decrease in neuronal p75NTR expression, however no significant change was observed in neuronal NaV1.8, NaV1.9 or TrkA expression relative to SC OGN treated or non-infused controls. On the contrary, SGC expression of phenotypic markers normally associated with the reactive state that is induced in these cells in response to peripheral nerve axotomy was dramatically altered. More specifically, in response to p75NTR AS OGN infusion, there was a significant increase in SGC protein expression of the cytoskeletal protein GFAP and p75NTR, along with a significant decrease in expression of the inward rectifying potassium channel Kir4.1. Preliminary data also revealed this induced reactive state in SGCs to be associated with an increase in the number of SGCs surrounding individual neurons as well as increased SGC expression of the gap junction protein, connexin 43. In conclusion, reductions in neuronal p75NTR expression and potentially reduced neurotrophin signaling lead to alterations in neuron/glial or axon/glial communication that results in induction of a reactive phenotype in the associated SGCs. With our ever increasing understanding of the role of SGCs modulating pain states, elucidation of the pathways leading to adoption of pathological phenotypes can help in the identification of novel therapeutic targets.

The Quaking (qk) gene has been implicated in the development of oligodendroglial cells which are the primary source of myelin in the mammalian central nervous system (CNS). Qk encodes three alternatively spliced variants, QKI-5, QKI-6 and QKI-7, all of which are RNA binding proteins. Loss of QKI-6 and QKI-7 results in a dysmyelination phenotype that is present shortly after birth while loss of QKI-5 results in embryonic lethality. CG4 oligodendroglial cells were transfected with either pIRES2-QKI5 to up regulate QKI-5 expression or a QKI-5 specific siRNA to down regulate QKI-5. Cells were cultured for 6d in differentiation medium (DM) following which total RNA and protein was collected from the cell cultures, and coverslips with attached cells were processed for immunofluorescence. Increased QKI-5 expression following transfection with pIRES2-QKI5 resulted in increased Sirt2 and Plp mRNA expression, but did not affect SIRT2 and PLP protein expression. Down regulation of QKI-5 expression had no significant effect on mRNA or protein levels for QKI-6, QKI-7, Plp or Sirt2. Immunocytochemistry revealed that up regulation of QKI-5 resulted in significantly higher percentage of A2B5+ cells and a lower percentage of GalC+ cells, whereas siRNA treatment resulted in an increase in the percentage of GalC+ cells. Our results suggest QKI-5 regulates oligodendrocyte differentiation and modulates the transcription and availability of target mRNAs, such as Sirt2 and Plp, for translation. In order to gain a more complete understanding of the relationship between qk and both Sirt2 and Plp, future studies would include RNA coimmunoprecipitation, miRNA studies, and expanding the list of target genes to include various cell cycle components.

Primary cultured astrocytes were treated with Mn in the absence and presence of proinflammatory cytokines to determine their effect upon stimulation of nitric oxide (NO) production. Treatments of manganese and cytokines raised NO production to intermediate levels, whereas combined treatment raised NO creation to much greater levels. Furthermore, this combined treatment differed from control only in its ability to elevate cellular NO levels at 24 hours, but not at earlier time points. Combined exposure in astrocytes derived from mice lacking the nos2 gene prevented any increase in production of NO. Thus, manganese and cytokines enhance NO production through activation of the nos2 gene. Additionally, pharmacologic ligands of the peroxisome proliferator-activated receptor gamma (PPARγ) were used to test the role of this orphan nuclear receptor in modulating Mn-dependent production of NO. The agonist, 1,1-Bis(3’-indolyl)-1-(p-trifluormethylphenyl) methane (cDIM1) diminished NO in a dose-dependent manner, whereas addition of the PPARγ antagonist, GW 9662, amplified cellular NO production, also in a dose-dependent fashion. Moreover, it was observed that NO production was both attenuated and augmented at similar rates, suggesting the agonist and antagonist work through similar mechanisms. To clarify the means by which NO levels are manipulated by PPARγ, we measured activation levels of the transcription factor NF-κB, a primary factor resulting in expression of NOS2. We found that NF-κB was slightly activated in cells treated solely with manganese or cytokines, whereas cells treated with both manganese and cytokines showed the highest levels of activation. Also, we found that these ligands function through an NF-κB dependent mechanism. Treatment of cDIM1 to astrocytes already treated with manganese and cytokines caused decreased activation of NF-κB, while addition of GW9662 to similarly treated cells resulted in increased activation of NF-κB. While these compounds were effective at manipulating induction of the nos2 gene, they had no effect on induction of guanosine tri-phosphate cyclohydrolase (GTPCH) the rate limiting enzyme for the production of tetrahydrobiopterin (BH4), a cofactor essential to the conversion of arginine to NO, Thus, these novel PPARγ ligands can influence manganese- and cytokine-induced production of NO by an NF-κB dependent mechanism.

No description available.

Bitter taste signaling in humans is mediated by a group of 25 bitter receptors (T2Rs) that belong to the G-protein coupled receptor (GPCR) family. Previously, several bitter peptides were isolated and characterized from bitter tasting food protein derived extracts, such as pea protein and soya bean extracts. However, their molecular targets in humans were poorly characterized. In this study, we tested the ability of the bitter tasting tri- and di-peptides to activate the human bitter receptor, T2R1. Using a heterologous expression system, T2R1 gene was transiently expressed in C6-glioma cells and changes in intracellular calcium were measured following addition of the peptides. We found that the bitter tasting tri-peptides are more potent in activating T2R1 than the di-peptides tested. Furthermore, to elucidate the potential ligand binding pocket of T2R1 we used homology molecular modeling. The ligand binding pocket in T2R1 is present on the extracellular surface of the receptor, and is formed by the transmembrane helices 1, 2, 3 and 7 and with extracellular loops 1 and 2.

Bitter taste signaling in humans is mediated by a group of 25 bitter receptors (T2Rs) that belong to the G-protein coupled receptor (GPCR) family. Previously, several bitter peptides were isolated and characterized from bitter tasting food protein derived extracts, such as pea protein and soya bean extracts. However, their molecular targets in humans were poorly characterized. In this study, we tested the ability of the bitter tasting tri- and di-peptides to activate the human bitter receptor, T2R1. Using a heterologous expression system, T2R1 gene was transiently expressed in C6-glioma cells and changes in intracellular calcium were measured following addition of the peptides. We found that the bitter tasting tri-peptides are more potent in activating T2R1 than the di-peptides tested. Furthermore, to elucidate the potential ligand binding pocket of T2R1 we used homology molecular modeling. The ligand binding pocket in T2R1 is present on the extracellular surface of the receptor, and is formed by the transmembrane helices 1, 2, 3 and 7 and with extracellular loops 1 and 2.

Aromatase is the only enzyme that converts androgens into estrogens, which is found in the brain, testes and ovaries. In teleosts, brain aromatase is exclusively expressed in radial glial cells, which are the abundant stem-like non-neuronal progenitors involved in neuroendocrine functions and neurogenesis in the central nervous system. With little information about radial glial cell regulation by neurotransmitters and neurohormones available, the overall goal of this thesis is to investigate the interactions between dopamine neurons and radial glial cells in the adult goldfish (Carassius auratus) forebrain. Immunocytochemistry and confocal imaging revealed a close anatomical relationship between dopamine neurons and radial glial cells along the ventricular surface in the telencephalon. Transcriptional regulation of brain aromatase by dopamine indicated a brain region-specific pattern and suggested the involvement of other regulators in the goldfish forebrain. A novel goldfish primary radial glial cell culture model was established and characterized for brain aromatase regulation studies. Pharmacological studies demonstrated that specific activation of dopamine D1 receptors up-regulates brain aromatase through a cAMP-dependent molecular mechanism, which can be enhanced or attenuated by the product of aromatase action, 17β-estradiol. Proteome profiling and the response following treatment with the specific dopamine D1 receptor agonist SKF 38393 revealed that proteins involved in cell proliferation and growth are regulated through small molecules- and transcription factors-mediated signaling pathways. Analysis of genes related to radial glial cell and dopamine neuron functions demonstrated that glial activation and dopamine neuron recovery are estrogen-dependent in a neurotoxin MPTP-induced goldfish model of Parkinson’s disease. This thesis illustrates novel molecular mechanisms underlying brain aromatase regulation as well as radial glial cell function regulation and provides a framework for future investigation of existing endocrine disruptors modulating neurosteroid levels in the teleost brain.

No description available.

No description available.