AbstractPatients of the fatal neuromuscular disorder Duchenne Muscular Dystrophy (DMD) frequently present with complex intellectual and neuropsychiatric comorbidities that are inadequately explained by our current understanding of the disease in the brain. The broad aim of my PhD was firstly to understand whether isoforms of the anchoring protein dystrophin—the loss of which is responsible for DMD—is expressed at a subset of mouse brain glutamate receptor-containing synapses and whether its deletion impacts upon their expression. Secondly, to understand how dystrophin deletion in these mice impacts brain inflammation and stress-related behaviour, which are secondary hallmarks of DMD. The glutamate-glutamate receptor system is a major mediator of cognitive and behavioural processes, and relates to inflammation and psychosocial stress, but is relatively unexplored in the context of this disease.
To this end I exploited dystrophin-deficient transgenic mice to model DMD-like physiology, and focussed on the cerebellum, a region of the brain which is unique in its expression of dystrophin isoforms and is a locus for many DMD-associated intellectual facets. Using a battery of microscopy, molecular, and behavioural techniques, I explored the association between cerebellar dystrophin isoforms and ionotropic glutamate receptors, inflammatory mediators, and stress-related behaviour. I hypothesised that dystrophin, by contributing to the organisation, and therefore function of, cerebellar excitatory synapses, helps to maintain the intellectual and neuropsychiatric facets impaired in DMD, and that dystrophin deletion results in neuroinflammation and a psychosocial stress phenotype in mice.
My main findings were:
1. Using immunohistochemistry with confocal microscopy on fixed preparations of the cerebellum, I demonstrated that the truncated 71kDa dystrophin isoform (Dp71) is located in the vicinity of Purkinje cell excitatory synapses. This indicates that the two present isoforms of the same protein, Dp427 and Dp71, are differentially targeted to inhibitory and excitatory synapses respectively (Chapter three).
2. Immunohistochemistry further revealed that immunoreactivity corresponding to Dp71 is associated with diverse iGluR subunits from AMPA, NMDA, and Delta type receptors (Chapter three).
3. Using quantitative reverse transcription PCR (qRT-PCR), I discovered that β-geo (lacking all dystrophin), but not mdx (lacking full length dystrophin) mice had significantly reduced levels of specific GluR subunits at the mRNA level in the cerebellum, but not in another brain region known to express dystrophin, namely the hippocampus, thereby demonstrating that the association of dystrophin isoforms with specific GluR subtypes is cell type and brain region dependent (Chapter three).
4. Semi-quantitative immunoreactivity for the GluR-GluD2 subunit, a major receptor protein involved in cerebellar synapse organisation and circuit development, was increased in β-geo mice (Chapter three).
5. Immunohistochemistry revealed that immunoreactivity for a key mediator of dystrophin-dependent inflammation in muscle, the P2X7 purine receptor (P2X7R), is closely associated with Dp71 at cerebellar excitatory synapses (Chapter four).
6. qRT-PCR revealed a tissue-specific change in the expression of P2X7R at the mRNA level, wherein P2X7R levels were increased in the muscle of both mdx and β-geo mice, but decreased in the cerebellum, hippocampus, and cortex of β-geo mice. There was no change in P2X7R levels in the mdx cerebellum, suggesting that muscle and brain P2X7R expression is dependent on different dystrophin isoforms and tissue types (Chapter four).
7. Quantitative immunoreactivity of P2X7R at in the molecular layer of the cerebellum was not significantly altered in β-geo mice (Chapter four).
8. Apart from P2X7R, β-geo mice exhibited a decreased profile of four pro-inflammatory mediators in the cerebellum, but two of these mediators were unaffected in the hippocampus; indicating that dystrophin loss differentially impacts certain inflammatory pathways in the two regions (Chapter four).
9. β-geo mice exhibited a marked increase in basal circulating corticosterone circulations. Corticosterone is the major peripheral neuroendocrine molecule of the stress response, and is indicative of a heightened stress in these mice (Chapter five).
10. At the behavioural level, I found that both mdx and β-geo mice exhibited comparable levels of anxiety-like behaviour in the light-dark box test. Both mdx and β-geo mice exhibited a marked reduction in locomotor behaviour in the open field test (Chapter five).
11. Exposure to acute stress did not alter the levels of anxiogenic-like behaviour in both mdx and β-geo mice, which may be a reflection of the already heightened stress phenotype which prevents any further adaptive responses to future stressors. (Chapter five).
|Date of Award||Aug 2019|