Groucho/transducin-like Enhancer of split (Gro/TLE) family proteins are corepressors found as part of multiple transcriptional complexes that play significant roles during many developmental processes, including neurogenesis. This thesis sought to characterize the molecular mechanisms underlying the biological activity of Gro/TLE1. More specifically, the aim was to clarify the contribution of different transcriptional cofactors, as well as phosphorylation events induced by cofactor binding, to Gro/TLE1 ability to inhibit neuronal differentiation from proliferating neural progenitor cells. / By characterizing specific point mutations within the C-terminal domain of Gro/TLE1, we were able to selectively impair binding of Gro/TLE1 to different classes of DNA-binding proteins and then assess the effect of those mutations on Gro/TLE1 anti-neurogenic function. These studies showed that the inhibition of cerebral cortex (cortical) neuron differentiation by Gro/TLE1 requires interaction with transcription factors that use short tetrapeptide sequences, WRP(W/Y), to recruit Gro/TLE1. In contrast, interactions with proteins that either interact with the C-terminal domain of Gro/TLE1 using a different type of binding sequence, termed engrailed homology 1 (Eh1) motif, or bind to the N-terminal part of the protein, are not required for Gro/TLE1 anti-neurogenic function. / Using a similar strategy based on mutation analysis, we characterized point mutations that block the hyperphosphorylation of Gro/TLE1 induced by transcription cofactor binding ("cofactor-activated phosphorylation") without impairing cofactor binding and transcriptional corepression ability. These mutations map at phosphorylatable serine residues, Ser-286, Ser-289, and Ser298. Mutation of those residues to alanine blocks/reduces both cofactor-activated phosphorylation and anti-neurogenic activity of Gro/TLE1, demonstrating that cofactor-activated phosphorylation is required for that function. Tandem mass spectroscopy analysis showed further that Ser-286 is phosphorylated. Taken together, these findings characterize the role of cofactor-activated phosphorylation and identify residues important for this mechanism. / Our studies also showed that homeodomain-interacting protein kinase 2 (HIPK2) mediates phosphorylation of Gro/TLE1 when the latter is complexed with transcriptional partners of the WRP(W/Y) motif family. However, HIPK2 is not involved in Gro/TLE1 cofactor-activated phosphorylation. Rather, HIPK2--mediated phosphorylation is antagonistic to the latter and decreases the ability of Gro/TLE1 to interact and repress transcription with WRP(W/Y) motif proteins. / Taken together, these results improve significantly our understanding of the mechanisms underlying the anti-neurogenic function of Gro/TLE1. This information provides new insight into the regulation of mammalian neuronal development and, possibly, other developmental processes controlled by Gro/TLE proteins.

No description available.

This thesis focused on the spatiotemporal patterning of classical excitatory and inhibitory synaptic contacts accounting for the majority of cerebral cortical connections, in relation to ageing and cognitive status. These investigations tested the hypothesis that higher CNS functions depend on the balance between excitatory and inhibitory synaptic connections. Glutamatergic and GABAergic presynaptic bouton densities were determined in aged animals segregated according to their cognitive status into aged and cognitively unimpaired (AU) and aged and cognitively impaired (AI), using the Morris water maze. These two groups were compared in terms of behaviour and the pattern of excitatory and inhibitory synapses. It was evident that an excitatory and inhibitory presynaptic decline is associated with age-related cognitive impairments; whereby both glutamatergic and GABAergic boutons gradually diminish from young to AU to AI. Nevertheless, the balance between excitatory and inhibitory presynaptic inputs was maintained. To determine whether postsynaptic sites differed with respect to ageing and cognitive impairments, excitatory and inhibitory postsynaptic scaffold proteins were investigated in the same cohort of segregated aged animals. There was an imbalance in density ratio between immunoreactive sites of excitatory versus inhibitory postsynaptic scaffold proteins in AI animals. This resulted from a marked decrease in the density of excitatory postsynaptic sites. To further investigate ultrastructural aspects of excitatory synapses I carried out electron microscopical studies of cerebral cortex to measure the abundance of NR2 receptor subunits of the NMDA receptor- a receptor site directly associated with excitatory postsynaptic scaffold proteins. This study revealed that NR2 immunoreactive sites were largely preserved during age-related cognitive decline with an uneven profile distribution. Finally, protein expression of specific receptor subunits and key proteins representative of excitatory and inhibitory postsynaptic sites was investigated by semi-quantitative Western blot analyses in selected cortical areas. It was clear that many of these postsynaptic proteins are affected by age and cognitive status. The most striking change was a marked up-regulation in neuroligin-1 in AI animals, which may affect the delicate balance between excitatory versus inhibitory synaptic inputs. Another notable finding was the down-regulated expression of GluR2 receptor subunits in AI animals, which should have implications for neuronal Ca2+ regulation. In conclusion, we have demonstrated the greater vulnerability of excitatory postsynaptic sites in aged and cognitively impaired animals.

No description available.