An investigation of respiratory abnormalities in a male and female mouse model of Rett Syndrome

• Main Author: Brockett, Emma Grace
• Published: University of Glasgow 2013
• Subjects: 612.8; QP Physiology
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.567980

Rett Syndrome (RTT) is a severe neurodevelopmental disorder affecting 1 in 10,000 girls and is often associated with respiratory abnormalities. RTT is almost exclusively caused by loss-of-function mutations in the human MECP2 gene. It remains unknown as to whether the respiratory abnormalities seen in RTT patients and MeCP2 deficient animals may be due to problems with respiratory rhythmogenesis or the result of an inappropriate chemosensitive response. The main aim of this thesis was to investigate the respiratory abnormalities presented in a male and female mouse model of RTT syndrome. In a male mouse model, the endogenous Mecp2 gene was silenced by insertion of a Lox-Stop cassette, which mimicked a number of RTT symptoms, including disordered breathing (Guy et al., 2007). The Mecp2 gene was reactivated by Tamoxifen(TM)-induced deletion of the Lox-Stop cassette. As such, the progression and development of respiratory disturbances were monitored in the early stages of MeCP2 deficiency and also assessed during and after gene reactivation. Respiratory parameters were recorded using whole body plethysmography, a non-invasive method of recording respiratory behaviour. Compared to WT, MeCP2 deficient male mice had an increased respiratory frequency and increased number of sighs prior to gene reactivation. The fact that animals were still viable suggests that neuronal development can occur in the absence of MeCP2, but signs of respiratory instability e.g. the increased number of sighs, indicate that MeCP2 may be required for neuronal maturation as the animal develops. Gene reactivation reduced respiratory frequency and the number of sighs in MeCP2 deficient mice to a level comparable with WT suggesting that TM-induced activation of Mecp2 can reverse some of the respiratory abnormalities. Since RTT syndrome is a predominantly female based disorder it was of benefit to observe the progression of the respiratory phenotype in a female model. Female mice which were heterozygous for the Mecp2-null mutation were also studied using plethysmography. It was observed that at 23 wks of age, following a period of normal development, the Mecp2+/- mice began to display an increased number of respiratory abnormalities compared to WT animals which suggested an inability of the respiratory network to maintain optimal function. Also, the respiratory response to hypoxia was significantly greater in Mecp2+/- mice compared to WT, yet the hypercapnic response of the two genotypes was comparable. This indicates that the response to hypoxia and hypercapnia are mediated by two different mechanisms and that the hypoxic response may be affected by a reduction in the level of MeCP2. Neuromodulators such as noradrenaline and serotonin are important in modulation of the respiratory pattern and the chemosensitive response. Previous research has implicated changes in bioamine content in the pathophysiology of RTT patients and MeCP2 deficient animals (Riederer et al., 1985; Ide et al., 2005; Viemari et al., 2005; Roux et al., 2010) Thus, cells expressing tyrosine hydroxylase (TH), a marker for noradrenergic cells, and cells expressing 5-HT were quantified in the brainstems of MeCP2 deficient male mice to observe how the absence and subsequent reactivation of Mecp2 may affect noradrenergic and serotonergic cell number. Compared to WT, MeCP2 deficient mice exhibited a trend towards a decrease in the number of TH and 5-HT expressing cells in various noradrenergic and serotonergic regions of the brainstem, which may account for the development of the respiratory abnormalities in the mutant mice. It was also found that reactivation of Mecp2 did not restore the number of MeCP2 expressing cells to WT level. MeCP2 was often found to be co-expressed with 5-HT and TH, yet many MeCP2 positive neurons were not found to be 5-HT or TH positive indicating that these MeCP2 expressing neurons may be co-localised with another as yet unidentified neuromodulator. Interestingly, reactivation of Mecp2 appeared to occur at a greater rate in noradrenergic TH expressing neurons than 5-HT expressing neurons. Whilst studying MeCP2 deficient male mice it was noted that some of the mutant animals began to develop rale-like rattling within the chest and also began to foam at the mouth. Much of the research in RTT syndrome focuses on the neurological aspect, whereas this phenotype indicated a problem within the lungs. Lungs of WT and MeCP2 deficient mice were removed and processed with various histological stains to highlight various aspects of the lung morphology. Comparison of WT and MeCP2 deficient tissue revealed that there was a trend towards an increase in the amount of elastin surrounding airways and an increase in the thickness of the interalveolar septum in MeCP2 deficient mice compared to WT. An increase in interalveolar septum may interfere with ventilation and may account for the increased occurrence of sighing observed in the male MeCP2 deficient mice. These morphological changes in the lung may indicate that the respiratory abnormalities of RTT may not be solely neurological. Since changes in the morphology of the lung were clearly present in the male with evidence of increased elastin deposits surrounding the airways, investigation into the presence of pulmonary arterial hypertension (PAH) was carried out in the female model. Results indicated that there was a trend towards a higher right ventricular pressure in Mecp2+/- animals compared to WT, along with a trend towards right ventricular hypertrophy, indices of the presence of PAH. In conclusion, Mecp2 has been shown in vivo to be involved in both the development and maintenance of neurons involved in the respiratory network, both neuromodulatory and chemosensitive, and the absence or reduction of MeCP2 is also proposed to have a novel role in the development of lung pathology in MeCP2 deficient mice.