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The role of MeCP2 in activity-dependent brain processes

Mcleod, Faye Christine (2012) The role of MeCP2 in activity-dependent brain processes. PhD thesis, University of Glasgow.

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Abstract

Rett syndrome (RTT) is a neurodevelopmental disorder that is caused by mutations in the X-linked gene MECP2 and results in cognitive impairment, epilepsy and motor dysfunction. Deletion or silencing of Mecp2 in the brain of mice recapitulates many of the main phenotypes of the disorder and has been fundamental in understanding the actions of MeCP2 although the precise molecular function remains unknown. MeCP2 is classically thought to have a role in repressing gene transcription through recruitment of histone deacetylase proteins. Studies have also shown that Mecp2 can be modified posttranslationally in response to neuronal activity. The aim of this thesis was to investigate the activity-dependent alterations in MeCP2 and the association this has with levels of acetylated histone proteins, a marker of active gene transcription thus gaining a better insight into how neuronal activity affects the function of MeCP2. Furthermore at the network level, a second objective was to characterise the effects loss of functional MeCP2 has on the regulation of network circuitry in the brain and in particular the development of epileptiform activities. To study this I focussed on the hippocampus in wild-type and Mecp2 mutant mice and in vivo administered the convulsant drug kainic acid (25mg/kg; IP) followed by seizure scoring and in vitro by application of various epileptogenic agents (kainic acid, bicuculline and 4-aminopyridine) to acute hippocampal slices. Having characterised the network properties, I then quantified protein alterations in phosphorylation of Mecp2, acetylated histone H3 and H4 and immediate early genes, c-Fos and Egr-1 in the hippocampus using western blot and quantitative immunohistochemistry techniques. Based on a modified seizure scale (eight stages with lower being less severe), administration of kainic acid in vivo to Mecp2-deficient male mice resulted in a higher seizure score (mean = 6 ± 0.7 vs. 4 ± 0.2 units in wild-type) and more rapid onset (77% of mice show seizures after 10 minutes compared to 5% of wild-type mice). Field recording data collected in vitro following application of kainic acid to hippocampal slice from Mecp2-deficient mice show a significant increase in gamma power oscillation (1059 ± 379µV2) compared to slices from WT mice (287 ± 178µV2) which had a lower mean power. Application of bicuculline revealed hippocampal slices from Mecp2-deficient mice had increased frequency of spontaneous epileptiform field events (1µM and 3µM bicuculline) and elevated duration of spontaneous and evoked epileptiform field events (10µM bicuculline). Similarly, 4-aminopyridine (4-AP) administration to hippocampal slices resulted in Mecp2-deficient mice displaying increased frequency of spontaneous field events (50µM 4-AP) and epileptic ictal-like events (88% of slices from Mecp2stop/y mice displayed these events compared to 43% of slices from WT mice). Furthermore there was an increase in spontaneous and evoked field events following application of 30µM and 10µM 4-AP. Western blot experiments using hippocampal extracts from WT and Mecp2-deficient male mice treated with the convulsant drug kainic acid or saline (vehicle control) revealed Mecp2 is highly phosphorylated at serine 421 (3.4 ± 0.5 fold, p< 0.01) upon induction of neuronal activity compared to saline controls but there was no change in histone H3 or H4 acetylated proteins. A complementary quantitative immunohistochemistry approach was used to assess variations in histone H3 or H4 acetylated proteins at the single cell level from heterozygous female mice (displaying a mosaic expression of Mecp2) treated with kainic acid or saline. These results revealed there was no difference in the levels of either acetylated histone H3 of H4 protein between Mecp2 positive and negative nuclei. However there was a clear cell-autonomous effect in terms of a 5% reduction in the nuclear volume of Mecp2-deficient cells. Quantification of immediate early gene signal in Mecp2+/- heterozygous female mice treated with kainic acid or saline using the same immunohistochemistry method showed there was no difference in the distribution of c-Fos intensities between Mecp2 positive and negative nuclei following any treatment. However there was a greater proportion of Mecp2-deficient nuclei (~20%) expressing c-Fos under saline control conditions and following neuronal activity. Furthermore there was a reduction in the percentage of Mecp2 negative nuclei in the top 25% of Egr-1 intensities in the CA1 following three hours of neuronal activity (33.5 ± 2.3% for Mecp2 negative nuclei and 19.1 ± 1.7% for Mecp2 positive nuclei). In summary my results show at the network level there is a reduction in seizure threshold and increase power of gamma oscillations in the hippocampus of Mecp2-deficient mice which could lead to a state of network hyperexcitability and switch activities from physiological oscillatory rhythms to more pathological ones. An imbalance in inhibitory neuron regulation could partially contribute to alterations in network excitability to overall promote epileptogenesis. At the cellular level I report that Mecp2 can become phosphorylated following induction of neuronal activity but this is not associated with alterations in global histone acetylation. Nonetheless Mecp2-deficient cells display a reduction in nuclear volume and have alterations in c-Fos and Egr-1 levels following induction of neuronal activity. Overall my data contribute to the understanding of how the presence or absence of MeCP2 affects network excitability and how in turn neuronal excitability affects MeCP2 phosphorylation, histone acetylation and the activation of immediate early genes.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Keywords: Rett syndrome, MECP2, hippocampus, epilepsy, gamma oscillations, excitability, network, histone modifications, immediate early genes
Subjects: R Medicine > RC Internal medicine > RC0321 Neuroscience. Biological psychiatry. Neuropsychiatry
Colleges/Schools: College of Medical Veterinary and Life Sciences > Institute of Neuroscience and Psychology
Supervisor's Name: Cobb, Dr. Stuart
Date of Award: 2012
Depositing User: Miss Faye Mcleod
Unique ID: glathesis:2012-3747
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 07 Dec 2012
Last Modified: 12 Aug 2013 13:09
URI: http://theses.gla.ac.uk/id/eprint/3747

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