Synapses are highly dynamic structures that undergo continuous functional and structural changes in response to alterations of neuronal activity. This remodeling is critical for brain development, synaptic transmission and neuronal plasticity. At the post-synaptic site two classes of molecules play a crucial role in synaptic organization: scaffolding and cell adhesion proteins. Scaffolds ensure the accurate accumulation of neurotransmitter receptors in precise apposition to pre-synaptic release sites. In addition, they provide the physical constraints for maintaining a high concentration of receptors at synapses, and for regulating the constant flux of receptors and scaffolding elements in and out of post-synaptic sites. Scaffolds also regulate downstream signaling pathways to adjust the molecular composition of the post-synaptic devices necessary to sustain synaptic plasticity. Cell adhesion molecules bridge pre- and postsynaptic specializations through specific interactions of their extracellular domains. Such interactions do not simply provide a mechanical link between pre- and post-synaptic sites but are instrumental in activating transduction signals necessary for the recruitment of various synaptic components. In this context phosphorylation processes are critical for modulating changes in the molecular composition of the post-synaptic device. While the impact of phosphorylation of neurotransmitter receptors has been extensively characterized much less is known about the effect of these post-translational modifications on scaffolding and cell adhesion molecules. At GABAergic synapses specific phosphorylation events targeting the scaffolding molecule gephyrin were shown to alter its oligomerization properties, thus producing concomitant changes in the numbers of receptors trapped by the scaffold and synaptic strength. Most of these phosphorylation events occur at serine or threonine residues preceding a proline, underlying a potential role of proline-directed phosphorylation as modulator of synaptic strength. At excitatory synapses, mass spectrometric analysis performed on isolated postsynaptic density proteins (PSD) has led to the identification of a number of novel serine/proline phosphorylation sites on scaffolding MAGUKs. In addition the prolyl-isomerase activity of Pin1 has been shown to regulate protein synthesis necessary to sustain the late phase of long-term potentiation. Based on these evidences, the aim of my thesis was to study the functional role of proline-directed phosphorylation in remodeling the post-synaptic devise of both inhibitory and excitatory systems by acting on pivotal constituents such as protein scaffolds and cell adhesion molecules. By combining molecular biology, immunocytochemistry and electrophysiological recording I initially investigated the impact of Pin1-dependent signaling on GABAergic transmission. I found that Neuroligin2, the cell adhesion molecule constitutively present at GABAergic synapses, undergoes post-phosphorylation, prolyl-isomerization modulation of its activity. Proline-directed phosphorylation at Serine 714 of NL2 negatively impacts on NL2 ability to complex with gephyrin. As a consequence, an enhanced accumulation of NL2, gephyrin and GABAA receptors was detected at GABAergic synapses in the hippocampus of Pin1-knockout mice (Pin1?/?), which was accompanied by a concomitant increase in amplitude of spontaneous GABAA-mediated post-synaptic currents. These results suggest that Pin1-dependent signalling represents a mechanism to modulate GABAergic transmission by regulating NL2/gephyrin interaction. Then I focused on the impact of Pin1-dependent signaling on excitatory glutamatergic transmission. In particular, I investigated whether the scaffolding molecule PSD-95, a member of the Disc-Large (DGL)-Membrane-associated guanylate kinase, and know to be phosphorylated by several proline-directed kinases, could be a target of Pin1-dependent modulation. I observed that Pin1 is recruited by PSD-95 at specific Serine-Threonine/Proline consensus motifs localized in the linker region connecting PDZ2 to PDZ3 domains and exerts a negative control on PSD-95 ability to complex with NMDARs. Indeed an enhanced PSD-95/NMDA complex formation was detected in brain extracts derived from Pin1-/- mice. In electrophysiological experiments, larger NMDA-mediated synaptic currents were detected in CA1 principal cells in hippocampal slices obtained from Pin1-/- mice as compared to controls, an effect that was associated with an enhancement in spine density and size.
The role of prolyl-isomerase PIN1 in GABAergic and glutamatergic synaptic transmission [Tesi di dottorato]
Tesi di dottorato. | Lingua: en. | Paese: | BID: TD16087839
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