Synapses are specialised contacts between neurons. At postsynaptic terminals of glutamatergic synapses, protein complexes process and transmit the information received from the presynaptic terminal. Scaffolding proteins, among which members of the disc large homologue (DLG) family are the most abundant, assemble the molecular machinery in the postsynaptic terminal. Recently, two members of the DLG family, postsynaptic density protein 95 (PSD95) and synapse associated protein 102 (SAP102), have been shown to form different types of complexes, thus giving the synapse different signalling capabilities. However, the spatial distribution of these synaptic markers in different synapses remains elusive due to technical challenges. This thesis presents the first applications of a new method, the Genes to Cognition Synaptome Mapping pipeline (G2CSynMapp), to map individual synapses at the whole-brain level, in a quantitative and unbiased manner. This method was used to generate PSD95 and SAP102 synaptome maps – i.e. comprehensive maps of PSD95 and SAP102 positive synapses – in the mouse brain and to achieve three aims: i) characterise PSD95 and SAP102 synapse diversity, ii) measure the trajectory of PSD95 and SAP102 synapse changes during the postnatal lifespan and iii) determine whether PSD95 synaptome is reorganised by mutation. First, I have used G2CSynMapp to generate the first synaptome maps in the adult mouse brain. This reference map of PSD95 and SAP102 positive synapses revealed a highly organised distribution pattern of glutamatergic synapses between anatomical regions. Moreover, it uncovered that synapse populations are very diverse within anatomical regions and can form patches, gradients and input-specific glomeruli. Second, the trajectories of PSD95 and SAP102 synaptomes were mapped across the mouse postnatal lifespan. At birth, synapse densities are low and increase rapidly during the first month of life. During ageing, the density of SAP102 and PSD95 positive synapses decrease gradually. Interestingly, different anatomical regions show different trajectories of synapse density and parameters across the lifespan. Moreover, the packing of PSD95 and SAP102 at synapses have specific pattern of changes. Third, the PSD95 synaptome was found to be reorganised differently in two disease models, PSD93 and SAP102 knock-out mice. In humans, mutations in the genes encoding PSD93 or SAP102 have been involved in schizophrenia and mental retardation, respectively. Of particular interest, opposite changes were identified in the neocortex of the two mutant lines that are reminiscent of their inverse behavioural phenotypes.

No description available.

A major area of interest is the identification of proteins that play a role in hormone dependent cancers and in collaboration with the MRC Centre for Reproductive Health we studied the gonadotropin releasing hormone receptor (GnRH-R). Other targets described in the thesis are the SH3 domain of PSD-95 and the protein BLyS. In order to identify potential inhibitory small molecules we have used a variety of computational data base mining approaches as well as using and developing experimental binding assays. It has become increasingly challenging to evaluate the most representative drug like small molecule compounds when using traditional high throughput screening methods. This thesis assesses the use of in silico tools to probe key protein-protein and protein-peptide interactions. These tools provide a means to identify enriched compound datasets which can be purchased and tested in vitro in a time and cost efficient way. The transmembrane protein GnRH-R provides an interesting opportunity to identify small molecules that could inhibit the binding of its peptide ligand GnRH. This is a challenging project as there are few examples in the literature of drug-like molecules that bind to such protein-peptide interfaces. The first step involved receptor modelling using solved crystal structures of homologous proteins. The model was then validated by developing structure activity relationships for established high affinity ligands. We also performed crystallographic and biophysical studies on the native GnRH decapeptide. Two other protein-protein systems were also examined using the same virtual screening and experimental ligand binding methodology. SH3 domains play an important role in cell signalling and we used the PSD-95 protein as our target for study as a crystal structure has been published. As well as identifying potential ligands we characterised structural properties of PSD-95 fusion proteins and also developed the basis for compound assay. The third system studied was B Lymphocyte Stimulator (BLyS) which is a target for treatment of a number of autoimmune diseases. This presented an interesting target for study as the protein binds to multiple receptors depending on its multimeric state. BLyS protein was characterised using electron microscopy and other biophysical techniques.

Indiana University-Purdue University Indianapolis (IUPUI) / Stimulation of N-­methyl-­D-­aspartic acid receptors (NMDARs) and the resulting activation of neuronal nitric oxide synthase (nNOS) are critical for fear memory formation. A variety of previously studied NMDAR antagonists and NOS inhibitors can disrupt fear memory, but they also affect many other CNS functions. Following NMDAR stimulation, efficient activation of nNOS requires linking nNOS to a scaffolding protein, the postsynaptic density protein 95 (PSD95). We hypothesized that PSD95-­nNOS interaction in critical limbic regions (such as amygdala and hippocampus) during fear conditioning is important in regulating fear memory formation, and disruption of this protein-­protein binding may cause impairments in conditioned fear memory. Utilizing co-­immunoprecipitation, electrophysiology and behavioral paradigms, we first showed that fear conditioning results in significant increases in PSD95-­nNOS binding within the basolateral amygdala (BLA) and the ventral hippocampus (vHP) in a time-­dependent manner, but not in the medial prefrontal cortex (mPFC). Secondly, by using ZL006, a small molecule disruptor of PSD95-­ nNOS interaction, it was found that systemic and intra-­BLA disruption of PSD95-­ nNOS interaction by ZL006 impaired the consolidation of cue-­induced fear. In contrast, disruption of PSD95-­nNOS interaction within the vHP did not affect the consolidation of cue-­induced fear, but significantly impaired the consolidation of context-­induced fear. At the cellular level, disruption of PSD95-­nNOS interaction with ZL006 was found to impair long-­term potentiation (LTP) in the BLA neurons. Finally, unlike NMDAR antagonist MK-­801, ZL006 is devoid of adverse effects on many other CNS functions, such as motor function, social activity, cognitive functions in tasks of object recognition memory and spatial memory. These findings collectively demonstrated that PSD95-­nNOS interaction within the conditioned fear network appears to be a key molecular step in regulating synaptic plasticity and the consolidation of conditioned fear. Disruption of PSD95-­nNOS interaction holds promise as a novel treatment strategy for fear-­ motivated disorders, such as post-­traumatic stress disorder and phobias.

Indiana University-Purdue University Indianapolis (IUPUI) / Fear is an evolutionarily conserved response that can facilitate avoidance learning and promote survival, but excessive and persistent fear responses lead to development of phobias, generalized fear, and post-traumatic stress disorder. The primary goal of experiments in this dissertation is to determine the molecular mechanisms underlying formation of fear memories. The acquisition and consolidation of fear is dependent upon activation of N-methyl-D-aspartic acid receptors (NMDARs). Stimulation of NMDARs recruits neuronal nitric oxide synthase (nNOS) to the synaptic scaffolding protein, postsynaptic density protein 95 (PSD95), to produce nitric oxide (NO). Our laboratory has previously shown that disruption of the PSD95-nNOS interaction attenuates fear consolidation and impairs long-term potentiation of basolateral amygdala (BLA) neurons in a rodent model of auditory fear conditioning. However, the molecular mechanisms by which disrupting the PSD95-nNOS interaction attenuates fear consolidation are not well understood. Here, we used pharmacological and genetic approaches to study the effects underlying nNOS activity in the BLA during fear consolidation. During the early stage of fear memory consolidation (4-6 hours after fear acquisition), we observed increased α- Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR)-mediated current and synaptosomal AMPAR GluR1 subunit trafficking in the BLA; while during the late stage (24h after fear acquisition), we detected a combination of enhanced AMPAR- and NMDAR-mediated currents, increased synaptosomal NMDAR NR2B subunit expression, and phosphorylation of synaptosomal AMPAR GluR1 and NMDAR NR2B subunits in the BLA. Importantly, we showed that pharmacological and genetic blockade of nNOS activity inhibits all of these glutamatergic synaptic plasticity changes in the BLA. Additionally, we discovered whole transcriptome changes in the BLA following fear consolidation. In the group with pharmacological inhibition of nNOS activity, however, gene expression levels resembled control-like levels. We also observed altered expression of multiple genes and identified the insulin-like growth factor system, D3/D4 dopamine receptor binding, and cGMP effects as key pathways underlying nNOSmediated consolidation of fear. Our results reveal nNOS-mediated, sequentially orchestrated synaptic plasticity changes facilitated by AMPA and NMDA receptors in the BLA during early and late stages of fear memory consolidation. We also report novel genetic targets and pathways in the BLA underlying NMDAR-PSD95-nNOS axis-mediated formation of fear memories.

Les interactions protéine-protéine (IPPs), complexes et dynamiques, sont le cœur des réseaux protéiques cellulaires. Au niveau des synapses excitatrices, la densité post-synaptique (PSD) est un exemple typique de réseau protéique dont la structure et la composition à l’échelle nanoscopique détermine la fonction cellulaire. Ainsi, la régulation dynamique de la composition de la PSD et des mouvements des récepteurs au glutamate dans ou hors de la PSD constitue la base des théories moléculaires actuelles sur l’apprentissage et la mémoire. Dans ce contexte, durant ma thèse, j’ai étudié une classe d’IPPs faisant intervenir les domaines PDZ. En effet, durant ces dernières années, de nombreuses études ont démontré l’implication de ces interactions impliquant les domaines PDZ de la famille de PSD95 dans le ciblage synaptique et l’ancrage des récepteurs au glutamate. Cependant, en partie dû au manque d’outils adaptés, les mécanismes moléculaires sous-jacents qui contrôlent de façon dynamique leur rétention à la synapse restent mal compris. Dans le but d’étudier ces interactions impliquant des domaines PDZ, j’ai développé plusieurs stratégies de sélection par phage display basées sur l’utilisation du dixième domaine de type III de la fibronectine humaine (10Fn3) dans le but de cibler les motifs d’interaction aux domaines PDZ des récepteurs (Stargazin pour les rAMPA et GluN2A pour les rNMDA) ou les domaines PDZ eux-mêmes. En utilisant une approche multidisciplinaire, mes objectifs principaux ont été de concevoir de petits anticorps synthétiques qui nous permettront de rompre ou de stabiliser spécifiquement ces complexes protéiques, ainsi que d’observer les interactions endogènes. / Complex and dynamic protein-protein interactions are the core of protein-based networks in cells. At excitatory synapses, the postsynaptic density (PSD) is a typical example of protein-based network whose nanoscale structure and composition determines the cellular function. For instance, the dynamic regulation of PSD composition and glutamate receptors movements into or out of the PSD are the base of current molecular theories of learning and memory. In this context, during my PhD, I focused on a class of protein-protein interactions mediated by PDZ domains. Indeed, over the last decade, numerous studies have shown the critical implication of PDZ domain-mediated interactions from the PSD95 scaffolding protein family in the synaptic targeting and anchoring of glutamate receptors. However, in part due to the lack of adapted tools, the molecular mechanisms that dynamically govern their respective synaptic retention remain poorly understood. In order to investigate these PDZ domain-mediated interactions, I developed several selection strategies by phage-display based on the fibronectin type III (FN3) scaffold in order to either target the PDZ domain-binding motifs of the receptors complexes (e.g., stargazin for AMPARs and GluN2A for NMDARs) or the PDZ domains themselves. Using a multidisciplinary approach, my main objectives were to engineer small synthetic antibodies that will allow us to acutely and specifically disrupt or stabilize these protein complexes, as well as monitor endogenous interactions.

PSD95 is the canonical member of the Membrane Associated Guanylate Kinase class of scaffold proteins. PSD95 is a five-domain major scaffolding protein abundant in the postsynaptic density (PSD) of the neuronal excitatory synapse. Within PSD95 three PDZ domains modulate protein-protein interactions by selectively binding to short peptide motifs of target proteins. Under the direction of the multivalent PDZ domain interactions, the interacting proteins tend to cluster at the PSD, a phenomenon that is critical for synaptic signalling regulation. Earlier studies have shown that the N-terminal PDZ domains of PSD95 are obligatory for the clustering to occur. This thesis focuses on the strong inwardly rectifying potassium channel, Kir2.1 as the PSD95 binding partner. Kir2.1 is known to maintain membrane resting potential and control cell excitability. Previous studies have reported that Kir2.1 clustered into ordered tetrad complexes upon association with PSD95.This study investigates the detailed clustering mechanisms of Kir2.1 by PDZ domains. To achieve this, components that are involved in the formation of a complex namely PSD95 sub-domains comprising single PDZ and the tandem N terminal PDZ double domain (PDZ1-2), and Kir2.1 cytoplasmic domains(Kir2.1NC) are studied in detail via different structural and biophysical approaches; 1) PDZ1-2 is examined in apo- and bound ligand form with a Kir2.1 Cterminal peptide in crystal and solution via X-ray crystallography and small angle X-ray scattering; 2) the tandem and the single PDZ domain interaction with ligand are measured thermodynamically via isothermal calorimetry (ITC); 3) the complex of full length PSD95 with Kir2.1NC is analyzed with electron microscopy (EM). The protein components are produced in high quality by protein expression and multiple-step protein purification techniques. PDZ1-2 crystallographic structures were solved at 2.02A and 2.19A in theapo- and the liganded forms respectively. The solution state analysis showed domain separation and structural extension of the tandem domain when incorporated with the ligand. The ITC experiment revealed PDZ1-2 to have greater affinity towards the peptide ligand relative to the single PDZ domains. These combinatorial outcomes lead to the conclusion that PSD95 clusters Kir2.1 by adopting an enhanced binding interaction which is associated with increased PDZ1-2 inter-domain separation. The preliminary analysis of PSD95-Kir2.1NC complex with cryo-EM showed the establishment of a tetrad and led to a reconstruction at 40A resolution. The work in obtaining a higher resolution complex structure is promising with further data collection required to allow the employment of more sophisticated model reconstruction processes.