|dc.description.abstract||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.||en