The accurate development of the wiring between the billions of neurons in our brain is fundamental to brain function. Development of this connectivity relies on activity-dependent modification of synapses similar to those that underlie learning and memory. Glutamate is the principal excitatory neurotransmitter in the mammalian brain and several brain disorders result from altered glutamatergic receptor signalling (Catania et al., 2007; Lau and Zukin, 2007). Genes encoding glutamate receptor associated proteins have a high incidence of mutation in cognitive disorders, especially X-linked mental retardation (MR)(Laumonnier et al., 2007). MR has long been associated with altered cortical connectivity, particularly dendritic spine dysgenesis. There is also an emerging view that aberrant local protein synthesis within dendrites and protein trafficking to dendrites underlies some forms of MR (Kelleher and Bear, 2008; Pfeiffer and Huber, 2006; Zalfa and Bagni, 2005). Most studies examining the role of glutamatergic receptors in MR have focused on adults. Little is known about how these MR genes regulate brain development despite their neurodevelopmental aetiology. Fragile X mental retardation (FXS) is the most common form of inherited MR and results from the loss of fragile X mental retardation protein (FMRP). FMRP is a RNA binding protein and is hypothesised to have a role in protein trafficking from nucleus to sites of synapses, and regulating local protein synthesis at sites of synapses (Bagni and Greenough, 2005). A prevalent theory of FXS causation is ‘metabotropic glutamate receptor (mGluR) theory of fragile X’, which postulates that all functional consequences of mGluR (predominantly mGluR5)-dependent protein synthesis maybe exaggerated in FXS (Bear et al., 2004). Primary somatosensory cortex (S1) of rodents provides an excellent model system to study the role of MR genes in development because of its stereotypic, glutamate receptor-dependent, anatomical development (Barnett et al., 2006b; Erzurumlu and Kind, 2001). Hannan et al., (2001) reported that genetic deletion of mGluR5 results in loss of ‘barrels’, the anatomical correlates of rodent whiskers in S1. Chapter 3 extends these findings to show that there is expression of mGluR5 as early as P4 in S1 prior to segregation of layer 4 cells into barrels suggesting a tropic role for glutamate in barrel formation. The expression of mGluR5 is postsynaptic during barrel formation and does not regulate tangential or radial cortical development. Its effects on barrel segregation are dose dependent and are not due to a developmental delay. During late S1 development, loss of mGluR5 results in decreased spine density suggesting a role in synaptogenesis. Supporting this hypothesis in mGluR5 mutant mice there is a general decrease in expression of synaptic markers in early S1 development. Chapter 4 explores the role of FMRP in cortical development. FMRP is expressed early in S1 development with peak expression prior to synaptogenesis at P14. It is expressed postsynaptically at P7 and pre and postsynaptically at P14. FMRP does not regulate cortical arealisation during barrel formation but results in decreased barrel segregation. In the absence of FMRP, biochemical studies show altered expression of glutamatergic receptors in the neocortex P7 and P14 suggesting altered glutamatergic receptor composition at synaptic sites. During late S1 development, loss of FMRP results in increased spine density in layer 4 spiny cells. Together these data indicate a role for FMRP during early and late S1 development. Chapter 5 directly tests the mGluR theory of FXS by examining whether genetic reduction of mGluR5 levels rescues anatomical phenotypes characterised in Fmr1-/y mice. The defect in barrel formation in Fmr1-/y mice is partially rescued by reducing mGluR5 levels. However, layer 4 spine density in Fmr1-/y mice does not appear to be rescued. Chapter 6 explores the expression patterns of three key synaptic MAGUKs (Membrane associated guanylate kinases) PSD95, SAP102 and PSD93, one of which (PSD95) is regulated by FMRP (Zalfa et al., 2007) and the others which have putative binding sites for FMRP. MAGUKs tether glutamatergic receptors to their associated signalling complexes at the postsynaptic membrane and also regulate glutamatergic receptor trafficking (Collins and Grant, 2007; Kim and Sheng, 2004). The immunohistochemical expression profiles of PSD95, SAP102 and PSD93 show dynamic regulation during S1 development that is unaffected by loss of FMRP (at P7), and biochemical data indicates that basal levels of these MAGUKs in neocortex are unaltered at P7 and P14 in Fmr1-/y mice. In Sap102-/y and Psd95-/- mice, there is altered expression of several synaptic proteins biochemically providing evidence for differential roles of SAP102 and PSD95 in regulating expression of glutamatergic receptors at synaptic sites during early S1 development. This thesis demonstrates that synaptic proteins associated with MR are expressed early in development and display regulatory roles in cellular processes governing S1 formation. An understanding of their role in early brain development would be critical in fully appreciating when and where they exert their regulatory effects, and this in turn would be beneficial in designing therapeutic interventions.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:562503 |
| Date | January 2009 |
| Creators | Wijetunge, Lasani Sulochana |
| Contributors | Kind, Peter. : Larkman, Phil |
| Publisher | University of Edinburgh |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/1842/3886 |
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