BACKGROUND: At the postsynaptic membrane of neurons there is a dense network of proteins called the postsynaptic density (PSD). One such protein is the postsynaptic density protein 95 (PSD-95), which functions as a molecular scaffold for forming protein complexes at the PSD. PSD-95 is composed of three PDZ domains, which studies have shown to be sequentially and structurally similar. Studies have shown that PSD-95 plays a role in regulating signaling of glutamatergic neurons, as well as the induction of longterm potentiation through an association with TrkB receptors. PSD-95 may be a promising target for treatment of a number of neurological disorders such as depression, epilepsy, and cognitive dysfunction. The cyclic peptidomimetic drug CN2097 was designed based on the PDZ-binding motif of the CRIPT protein that binds to PDZ3. While CN2097 has been shown to affect the binding of PSD-95 to different synaptic proteins, no NMR studies have been performed to characterize the binding of CN2097 to PDZ3. Furthermore, few studies have characterized the inter-domain interactions between PDZ domains or whether the binding of calmodulin (CaM) to the N-terminal region of PSD-95 has any effect on the binding between the PDZ domains and CN2097. OBJECTIVE: To use isothermal titration calorimetry and nuclear magnetic resonance spectroscopy to analyze and characterize how CN2097 binds to PDZ domains and whether inter-domain interactions exist between PDZ domains. METHODS: The gene sequences for the PDZ domains were inserted into the pET28a(+) vector by subcloning. E. coli bacteria were then transformed with the different PDZ plasmids. The bacterial cells were grown and induced to express the proteins of interest, followed by lysis and purification using affinity chromatography and fast protein liquid chromatography (FPLC). Isothermal titration calorimetry (ITC) was used to measure the dissociation constant and thermodynamic binding parameters between the peptidomimetic drug CN2097 and each isolated PDZ domain. Nuclear magnetic resonance (NMR) spectroscopy was used to study how CN2097 binds to PDZ1 and PDZ3, and how the PDZ domains interact with each other. RESULTS: The ITC data showed the dissociation constant between CN2097 and PDZ3 to be 5.12 ± (1.65) μM, and that of PDZ2+3 to be 42.63 ± (6.11)μM. ITC data on other domains was inconclusive. The NMR data showed no interaction between N-terminal region and PDZ1, and between PDZ2 and PDZ3 but significant interaction was seen between PDZ1 and PDZ2, as well as between PDZ3 and the inter-domain linker connecting it to PDZ2. NMR data showed that CN2097 binding perturbs PDZ3 more strongly than PDZ1 and that CN2097 does not bind to PDZ1 in the presence of CaM. Significant NMR chemical shift perturbations are seen on the second α-helix, second β- sheet, and β2-β3 loop. CONCLUSIONS: There are no significant contacts between the N-terminal α-helix and PDZ1. There is inter-domain conformational exchange and interaction between PDZ1 and PDZ2. PDZ3 interacts with the second inter-domain linker. CN2097 binds tighter to PDZ3 than to PDZ1, and does not bind to PDZ1 in the presence of CaM. The β2-β3 loop is a prime target for future development of CN2097.

Synapses are the communication junctions of the nervous system and contain protein machinery necessary for cognitive functions such as learning and memory. Postsynaptic density protein-95 (PSD-95) is a key scaffolding molecule at the PSD of synapses, yet its sub-synaptic organisation in the mammalian brain remains poorly understood. This thesis presents the use of genetically labelled PSD-95 with super-resolution imaging to resolve its nano-architecture in the mouse brain. To visualize PSD-95, two knock-in mouse lines were generated where the fluorescent proteins eGFP or mEos2 was fused to the carboxyl terminus of the endogenous PSD- 95 protein (PSD-95-eGFP or PSD-95-mEos2). Methods were developed by which fixed tissue sections of PSD-95-eGFP mice were examined using gated-stimulated emission depletion (g-STED) microscopy and PSD-95-mEos2 sections were examined with photoactivatable localisation microscopy (PALM) and quantitative image analysis was developed for both methods. From these platforms it was demonstrated that PSD-95 has a two tiered organisation: it is assembled into nanoclusters (NCs) approximately 140 nm diameter, which form part of the greater envelope of the PSD within synapses. Synapse subtypes were observed as characterised by the number of NCs per PSD. Using double colour g- STED microscopy. It was then asked whether PSD-95 nano-architecture remained the same across different sub-regions of the brain. A survey of PSD-95 was performed from seven different sub-regions of the hippocampus, quantifying ~110,000 NCs within ~70,000 PSDs from across the two super-resolution platforms. It was found that synapses displayed structural diversity both within and between different brain subregions as a function of the number of NCs per PSD. PSD-95 NCs were structurally conserved across the hippocampus, but showed molecular diversity in the abundance of PSD-95 molecules within. The findings of this thesis are: 1) genetic labelling of endogenous proteins combined with super-resolution microscopy is a powerful tool to study synaptic protein organisation in tissue. 2) Synaptic structural diversity in the brain is underlined by the number of PSD-95 NC units per synapse 3) PSD-95 NCs are structurally conserved but molecularly diverse synaptic units of synapses throughout the brain. These findings suggest that cognitive processing at the synapse is based upon a conserved, fundamental, molecular architecture.

共著者あり。共著者名:Takamiya Kogo(高宮考悟), Sun Jie(孫傑), Kitabatake Akira(北畠顕), Sobue Kenji(祖父江憲治). / Hokkaido University (北海道大学) / 博士 / 医学

No description available.

No description available.

PSD-95 is an abundant scaffolding protein found in the postsynaptic densities (PSDs) of excitatory synapses throughout the mammalian brain, and plays a critical role in innate and learned behaviours. PSD-95 assembles with numerous other proteins, including glutamate receptors, adhesion molecules and signalling proteins, into postsynaptic supercomplexes that are then organised into nanoclusters that comprise the postsynaptic density of excitatory synapses. While the subcellular localisation of PSD-95 has been widely studied, much less is known about its turnover. In this thesis, I present novel insights into PSD-95 synthesis and degradation at synapses of cultured primary neurons gained using the HaloTag technology. The HaloTag consists of an engineered bacterial protein domain that covalently binds synthetic ligands labelled with fluorescent and affinity moieties. Hence, cells expressing proteins fused to the HaloTag can be used to study protein levels, complexes and turnover using these different ligands. This project was based upon a knock-in mouse line expressing the HaloTag fused to endogenous PSD-95 using gene targeting. After demonstrating that these mice were phenotypically normal and that PSD95-HaloTag fusion proteins normally assembled into supercomplexes in the PSD, hippocampal primary cultures were grown from this mouse line. Fluorescent HaloTag ligands were then used to label live neurons, allowing for the visualisation of PSD-95 at synapses by confocal microscopy. Next, I established a pulse-chase labelling method, where one ligand is used to label all existing PSD-95 first, before a second ligand can then be used to label any newly synthesised PSD-95. This allows for the identification and characterisation of subpopulations of PSD-95, which can be separately analysed. I find that PSD-95 has a half-life of 36 hours at synapses, consistent with previous literature. I was also able to observe synaptic heterogeneity in PSD-95 turnover, and classify synapses into types according to their PSD-95 expression profile. Finally, a range of chemical compounds known to modulate protein turnover and neuronal activity was applied over a 24-hour period, and their effects on PSD-95 turnover analysed. It was found that inhibiting either the proteasome or protein synthesis led to significant reductions in PSD-95 degradation as well as inhibiting PSD-95 synthesis. Thus, this project established a method offering a unique way of investigating the turnover of a specific, tagged protein, as well as gaining novel insights into the turnover of PSD-95 at individual synapses.

The dissertation is divided into two separate experiments that explore the effects of visual-spatial learning on PSD-95 dorsal hippocampal expression. Specifically, the aim of these studies was to explore the effect of learning an assay, the Hebb-Williams mazes, on the protein expression of PSD-95 in Fmr1 KO mice. PSD-95 is an important scaffolding protein hypothesized to be involved in learning and memory. In cellular models of Fragile X Syndrome it has been shown to be dysregulated but it has never been measured following behavioural learning. Establishment of a deficit using an ecologically valid behavioural assay could lead to the development of novel interventions. Study one employed a subset of the Hebb-Williams mazes of various levels of difficulty to evaluate PSD-95 protein expression in Fmrp intact and Fmr1 KO mice following learning. The results revealed significant increases in PSD-95 protein expression in control runners when compared to Fmr1 KO mice. There was a negative correlation between PSD-95 protein levels and mean total errors on the mazes meaning that as expression was increased, errors were decreased. The goals of study two were to reverse the molecular and behavioural deficits using pharmacological antagonist treatment shown to be effective in cellular models of Fragile X Syndrome. Fmr1 KO mice were treated with either saline or 20 mg/kg of a metabotropic glutamate receptor antagonist, 2-Methyl-6-(phenylethynyl) pyridine (MPEP). Relative to saline treated controls, drug treated Fmr1 KO mice made fewer errors on the same subset of Hebb-Williams mazes used in study one. Latency to complete these mazes did not differ between groups, indicating that MPEP treatment does not adversely affect motor functioning. Protein assessment revealed that PSD-95 was selectively rescued in MPEP treated mice and not saline controls. Similar to study one, a negative correlation between PSD-95 protein levels and mean total errors was observed. When taken together, these studies indicate that protein deficits are associated with a deficit of learning that can be reversed with a selective glutamate receptor antagonist. One of the strengths of the Hebb-Williams mazes is that performance is measurable without floor or ceiling effects, which plague other common behavioural assays. These data further suggest that pharmacological antagonist treatments may be promising in correcting the learning deficits in human Fragile X Syndrome patients.

It is estimated that one in 59 children in the US are affected by autism spectrum disorder (ASD). ASD is distinguished by social and communication deficits that can be displayed throughout a wide range of severity. This resulting spectrum of behaviors observed in ASD suggests that a complex etiology is involved. Previous studies have shown a genetic susceptibility to autism including paternal age, twin and sibling concordance. Genetic sequencing of those affected as well as first order relatives have identified alterations in genes associated with neuronal synaptic communication. However, very little information is available regarding the pathophysiology of synapses in ASD. Neuronal communication between anterior cingulate cortical neurons via synapses with other brain regions is vital in the execution of social behaviors in individuals. The aim of this study was to evaluate the protein expression of the synaptic marker spinophilin and post-synaptic density protein-95 (PSD-95) in postmortem ASD gray matter brain tissue from the anterior cingulate and frontal cortex to compare to typically developing (TD) control brain tissue. Postmortem brain tissue of ASD and TD subjects was acquired from nationally funded brain repositories previously matched by brain area, age and gender. Immunoblotting for spinophilin and PSD-95 was performed using anterior cingulate and frontal cortical gray matter brain tissue from matched ASD and TD brain tissue. Spinophilin and PSD-95 protein amounts for all donors were normalized using GAPDH. Frontal cortical tissue demonstrated no significant differences in spinophilin protein expression between TD and ASD groups (N=6). Anterior cingulate tissue demonstrated no significant differences in spinophilin protein expression between TD and ASD groups (N=5). PSD-95 protein expression levels did not result in any significant differences between ASD donors and their control pairs for either brain tissue region. Although no changes were detected in the frontal cortex or anterior cingulate cortex, more brain areas and subjects must be evaluated to determine if spinophilin or PSD-95 can be reliable markers for synaptic alterations in ASD. These data are critical in determining synaptic pathology in ASD which may lead to future treatments.

Dans le système nerveux central, la synaptogenèse est un processus complexe multi-étapes qui se déroule aux contacts axones/dendrites. Les molécules d’adhérence neurexines/neuroligines jouent un rôle essentiel dans ce processus, en créant un lien physique entre les compartiments pré- et post-synaptiques et en participant au recrutement des complexes macromoléculaires essentiels à la fonction synaptique. Plus spécifiquement, le complexe neurexine-1β/neuroligine-1 induit la formation de post-synapses excitatrices, en recrutant des molécules d’échafaudage telles que PSD-95 et des récepteurs du glutamate.Mon travail de thèse a consisté à étudier les mécanismes moléculaires mis en jeu par les adhésions neurexines/neuroligines lors de la formation des post-synapses glutamatergiques. En utilisant des systèmes biomimétiques (neurexine purifiée fixée sur des billes, ou agrégée par des anticorps réticulés), nous avons induit des adhésions spécifiques neurexine-1β/neuroligine-1 sur des neurones d’hippocampe en culture. Nous avons ainsi étudié la distribution dynamique des composants post-synaptiques (récepteurs AMPA, PSD-95) endogènes ou étiquetés avec des protéines fluorescentes, par vidéo-microscopie. Dans un premier article, nous avons montré que la formation de ces contacts induisait un recrutement rapide de PSD-95 ainsi que des récepteurs NMDA et AMPA fonctionnels. En utilisant des récepteurs AMPA recombinants, j’ai montré que ce recrutement était dicté par la sous-unité GluA2. Dans une deuxième étude, en comparant le recrutement de PSD-95 induit par la neurexine avec des anticorps non–activants, nous avons mis en évidence un mécanisme d’activation spécifique de neuroligine-1 induit par la liaison de neurexine-1β. L’utilisation de mutants ponctuels de neuroligine-1 a permis de montrer que cette activation passe probablement par la déphosphorylation d’une tyrosine unique située dans le domaine C-terminal de la neuroligine-1.Enfin, en étudiant la diffusion latérale des rAMPA de surface par suivi de particules uniques fluorescentes (Quantum dots), ainsi qu’une batterie d’outils moléculaires pour moduler les adhésions neurexine/neuroligine (sur-expression, siRNA, souris KO), nous avons montré que les rAMPA sont recrutés aux adhésions neurexine-1β/neuroligine-1 via l’échafaudage PSD-95 et que ce recrutement nécessite la diffusion des récepteurs dans la membrane plasmique. Nous proposons ainsi que les récepteurs AMPA soient recrutés aux contacts naissants via un mécanisme original de diffusion/piégeage. / In the central nervous system, synaptogenesis is a multi step process occuring at axo-dendritic contacts. Neurexins/neuroligins adhesions are particularly involved in this process, making a bridge between the pre- and the post-synapse, and participating to the recruitment of macromolecular complexes essential for synaptic function. More precisely neurexin-1β/neuroligin-1 complex is specifically involved in the formation of excitatory synapses, inducing the recruitment of glutamatergic post-synapses components, such as PSD-95, and glutamate receptors.During my PhD, I focused on the molecular mechanisms involved in glutamatergic post-synapses formation triggered by neurexin-1β/neuroligin-1 adhesions. Using biomimetic models (beads coated with purified neurexin, or purified neurexin cross-linked with aggregated antibodies) we induced specific neurexin-1β/neuroligin-1 adhesions on cultured hippocampal neurons. We then studied the dynamic distribution of either endogenous or recombinant post-synaptic components (PSD-95, AMPARs) with live-imaging techniques. First, we showed that the formation of these contacts induced a rapid recruitment of PSD-95 and functional NMDA and AMPA receptors. Using recombinant AMPA receptors, I showed that this recruitment was mediated by GluA2 subunit.In a second study, using systematic comparison between the recruitment of PSD-95 induced either by neurexin-1β or by “non activating” antibody binding on neuroligin-1, we revealed a specific activation mechanism of neuroligin-1 induced by neurexin-1β binding. Using point mutations on neuroligin-1, we showed that this activation mechanism is mediated by a tyrosine dephosphorylation on neuroligin-1 intracellular tail.Finally, we studied AMPA receptor surface diffusion with single particle tracking experiments, using different molecular tools to perturb neurexin-1β/neuroligin-1 adhesions (overexpression, RNA interference, KO mice). We showed that AMPA receptors recruitment at new-formed neurexin-1β/neuroligin-1 adhesions occurs through PSD-95, and involves surface diffusion of AMPA receptors. We proposed an original diffusion/trap mechanism of AMPA receptors at nascent contacts.

No description available.