Comparative analysis of the mitochondrial proteome in a mouse model of Rett syndrome

Rett syndrome is a neural development disorder. The gene, for culprit protein, MeCP2 (methylated CpG binding protein) is located on chromosome X and the majority of patients are young girls. Rett syndrome patients do not suffer from neurodegenerative processes, instead, due to the neurons´ abnormal small size and denser packaging, the brain´s general volume is reduced and synapses as well as dendrite structure are weakened. Rett syndrome patient's postnatal development appears normal during the first 6 to 16 month of life. After that, phenotype signs such as mental problems and lack of communication are observed, which are followed by a number of additional severe symptoms: irregular sleep and breathing, labored motor movements, frequent epileptic seizures, body weight loss, scoliosis, and stereotypic hand movements. Post mortem tissue analyses of Rett syndrome patients show that mitochondrial ultra-structure is damaged and organelle functions are hindered. Cells suffer from higher concentrations of free radicals, oxidative stress, lack of ATP, and increased susceptibility to hypoxia. Mitochondrial changes were first discovered in muscle tissue. Damaged mitochondria contain vacuoles, small granular inclusions, distorted inner membranes, are leaky to protons, and general mitochondrial mass is increased. Mitochondrial impairments obviously lead to insufficient respiratory chain protein activity, cellular ATP deficits, and increased oxidative stress on the systemic level. Frequent apneas in Rett syndrome patients give rise to transient drops in blood oxygen levels, and cells with malfunctioning mitochondria were proposed to suffer from an oxidative burden. Main goals of this thesis were (i) broad-range comparative proteomic analyses of mitochondria obtained from the brains of wild type and Mecp2 gene knockout mice, specifically focusing on neocortex and hippocampus and (ii) untargeted metabolomic studies of the neocortex on the same group of animals. So far such a research approach was not carried out on the brains of Mecp2 knockout Rett syndrome mouse models. For the experiments, flash frozen brain tissues of 50 days old Mecp2-deficent and wild type variant male mice were used. The broad range proteomic study involved analyzing samples with two-dimensional electrophoresis and identifying peptide contents of differently expressed spots by mass spectrometry. The observed differences for some proteins were further confirmed by western blotting and specific antibodies. Findings show an upregulation of cytochrome b-c1 complex subunit 1 and prohibitin-1, as well as a downregulation of gamma-enolase and cAMP-dependent protein xi kinase catalytic subunit alpha in both, Mecp2-/y neocortex and hippocampus. The other series of mitochondrial proteome experiments involved an ad hoc approach and were aiming at mitochondrial fission and fusion regulatory proteins and mitochondrial oxidative phosphorylation chain components. Here it became apparent that mitochondrial dynamics regulation proteins were decreased in RTT mice, specifically mitofusin 1 was found at lower levels in cortical tissue and DRP-1 was expressed less in hippocampus. For the rating of physiological aspects of Rett syndrome and wild type mice, general phenotypic parameters – such as body weight, size and blood parameters – were assessed. Also, brain mitochondria purification was performed on freshly extirpated brain tissues and biochemical assays were conducted on live and still functioning mitochondria to measure their reactive oxygen species accumulation by recording the emitted fluorescence signal of an oxidation sensitive dye in a spectrometer-based cuvette assay. Untargeted metabolomic analysis required higher amounts of tissue material and was therefore limited to the cortices of Mecp2-deficent and wild type male mice. Flash frozen cortices were sent to a service provider, who performed the analyses and provided a large amount of valuable raw data for further in-depths bioinformatics analyses. Metabolomic data reveals 101 significantly altered metabolites in the MeCP2-deficient neocortex of adult male mice; 68 of them were increased whilst 33 were decreased compared to wild types. These differences cover more than 31 metabolic pathways, including pivotal aspects of cellular metabolism, such as pyruvate metabolism, glycolysis, citrate cycle and oxidative phosphorylation. Data collected from different experiments show various molecular and metabolic abnormalities in the brains of Rett syndrome mice. The mitochondrial proteome of RTT mice clearly differs from wild type mice and this difference is also brain-region specific. Mitochondrial fusion/fission dynamics as well as their physiological performance seem negatively affected. Mecp2-knockout mice are suffering from increased oxidative stress, which in part might be due to poor mitochondrial function, and these alterations might have additional tolling influence on the already impaired conditions of metabolic and mitochondrial activities as well as key features of cellular metabolism. All these results shed further light onto the mitochondrial and the metabolic alterations that appear as part of the pathogenesis of Rett syndrome in MeCP2 deficient brain tissue. Accordingly, these aspects should be taken into account when therapeutic approaches in RTT are developed and/or when further treatment concepts are formulated.