No / Activity-dependent gene transcription and protein synthesis underlie many forms of learning-related synaptic plasticity. At excitatory glutamatergic synapses, the immediate early gene product Arc/ Arg3.1 couples synaptic activity to postsynaptic endocytosis of AMPA-type glutamate receptors. Although the mechanisms for Arc induction have been described, little is known regarding the molecular machinery that terminates Arc function. Here, we demonstrate that the RING domain ubiquitin ligase Triad3A/RNF216 ubiquitinates Arc, resulting in its rapid proteasomal degradation. Triad3A associates with Arc, localizes to clathrin-coated pits, and is associated with endocytic sites in dendrites and spines. In the absence of Triad3A, Arc accumulates, leading to the loss of surface AMPA receptors. Furthermore, loss of Triad3A mimics and occludes Arc-dependent forms of synaptic plasticity. Thus, degradation of Arc by clathrin-localized Triad3A regulates the availability of synaptic AMPA receptors and temporally tunes Arc-mediated plasticity at glutamatergic synapses. / A final draft copy of this article is not yet available.

Synaptic transmission alters the strength of the postsynaptic potential, through a process called short-term synaptic plasticity (STP). In this study, endplate potentials (EPPs) from the frog neuromuscular junction were used to resolve and quantify the presynaptic components involved in enhancement and depression of transmitter release during repetitive stimulation under normal quantal release conditions (2 mM Ca2+, 1mM Mg2+). During trains of stimulation given between 10 - 200 Hz, the amplitude of the EPPs first increased then decreased; a maximum increase of 77% was produced after 2-4 stimuli. EPP amplitudes began to increase at ~ 20 Hz, were maximal at ~ 55 Hz, and thereafter, decreased as the rate of stimulation increased. The integrated total release after 25 stimuli was little changed across frequencies between 10 - 100 Hz. EPPs ran down in two phases: a fast phase, attributed to the depletion of a readily releasable pool (RRP) of synaptic vesicles, followed by a slow phase, attributed to the depletion of vesicles from a depot pool (DP). Depletion of the readily releasable pool of synaptic vesicles (RRP) was determined by quantifying release under the fast and slow time rundowns and subtracting the number of vesicles associated with mobilization to the RRP from the total number of vesicles released during stimulation trains of 50 impulses. Impulses were delivered at 12 different rates ranging from 50 to 200 /s. Estimates of the number of vesicles released from the RRP increased with frequency of stimulation until maximal depletion levels of 5500 - 6000 vesicles were reached at stimulation rates between 90-130/s, assuming a control quantal content of 200 vesicles released per impulse. Depletion was less at lower frequencies when the number of stimuli delivered was identical. When the RRP maximally depleted, release was inversely related to stimulation rate, as would be expected if mobilization from the depot pool was the sole determinate of release during the slow phase. An equation constructed from four known components of enhancement and two components of depression - the depletion of vesicles from a readily releasable pool (RRP) and from the depot pool (DP) that refills the RRP, was used to fit and then simulate EPPs obtained during trains using different patterns of stimulation and varying amounts of extracellular Ca2+; the decay time constant parameters of enhancement, numerically derived from the observed data, were fixed at tau ~ 46, 220, 1600, and 20000 ms. The number of components of enhancement necessary to approximate the data decreased, from four in low (0.14 - 0.2mM) extracellular Ca2+, to one (tau ~ 46 ms) in 2.0 mM extracellular Ca2+, but four components of enhancement were necessary to fit the data when the amplitude of the EPP was not depressed below the control amplitude. This model was able to predict within ~ 3 % EPP amplitudes over a 10-fold range of frequency and Ca2+ concentration.

Synapse is the fundamental unit of synaptic transmission. Learning, memory and neurodegenerative diseases of the brain are attributed to the maintenance and alteration in synaptic connections. The efficiency for synaptic transmission depends on how well the post synapse receives the signals from the presynapse; this in turn depends on the receptors present in the post synaptic density (PSD). PSD is present in the post synapse right opposite to the neurotransmitter release site in presynapse (active zone) is an indispensable part of the synapse. The PSD is comprised of receptors and scaffold proteins, which is ultimately supported by the actin cytoskeleton of the dendritic spines. Cytoskeletal dynamics is shown to influence the structural plasticity of spine and also PSD, but how it regulates the dynamicity of the synaptic transmission is not completely understood. Here we studied the influence of actin depolymerisation on sub synaptic organization of an excitatory synapse. In order to study the organization of the synapse at molecular resolution, the conventional microscopy cannot be employed due to the limit of diffraction. Super resolution microscopy circumvents this diffraction limitation. In this study we have used custom built fluorescence microscope with Total Internal Reflection Fluorescence (TIRF) modality to observe the nanometre sized structures inside spines of mouse hippocampal primary neurons. The setup was integrated with Metamorph imaging software for both operating the microscope and imaging acquisition purpose with a separate appropriate laser system. This setup was successful in achieving the lateral resolution of ~30nm and axial resolution of ~51nm. Over all we were able to observe the loss of spines and significant reduction in area of nanometer sized protein clusters in postsynaptic density with in the spines of latrunculin A treated mouse hippocampal primary neurons compared to the native neurons. Along with the morphological alterations in neurons we also observed the changes in nanoscale organization of few key molecules in the postsynaptic density.

Le cerveau est formé d’un réseau complexe de neurones responsables de nos fonctions cognitives et de nos comportements. Les neurones reçoivent via des contacts spécialisés nommés « synapses », des signaux d’autres neurones.[...] Le mécanisme par lequel les neurones reçoivent, intègrent et transmettent ces informations est très complexe et n'est toujours pas parfaitement compris. Dans les synapses excitatrices, les récepteurs AMPA (AMPARs) sont responsables de la transmission synaptique rapide. Les récents développements en microscopie de super résolution ont permis à la communauté scientifique de changer la vision de la transmission synaptique. Une première avancée fait suite à l’observation que les AMPARs ne sont pas distribués de façon homogène dans les synapses, mais sont organisés en nanodomaines de ~ 80 nm de diamètre contenant ~ 20 récepteurs. Ce contenu est un facteur important pour déterminer l'amplitude de la réponse synaptique. En raison de la basse affinité des AMPARs pour le glutamate, un AMPAR ne peut être activé que lorsqu'il est situé dans une zone de ~ 150 nm en face du site de libération des neurotransmetteurs. Récemment, il a été montré que les nanodomaines d’AMPARs sont situés en face de ces sites de libération, formant des nano-colonnes trans-synaptiques à l'état basal. Cette organisation précise à l’échelle nanométrique semble être un facteur clé dans l'efficacité de la transmission synaptique. Une autre avancée a été l'observation que les AMPARs diffusent à la surface des neurones et sont immobilisés à la synapse pour participer à la transmission synaptique. L'échange dynamique entre le pool diffusif d’AMPARs et les récepteurs immobilisés dans les nanodomaines participe au maintien de l’efficacité de la réponse synaptique lors de stimulations à hautes fréquences. L'objectif de ma thèse a été de déterminer le rôle des paramètres indiqués ci-dessus sur les propriétés de la transmission synaptique, à l'état basal et au cours de phénomènes dits de plasticité synaptique. Tout d'abord, nous avons identifié le rôle crucial de la Neuroligine dans l'alignement des nanodomaines d’AMPARs avec les sites de libération du glutamate. En plus de cela, nous avons mis en évidence l’impact de cet alignement sur l’efficacité de la transmission synaptique en perturbant celui-ci. En parallèle, nous avons démontré que les AMPARs désensibilisés sont plus mobiles à la membrane plasmatique que les récepteurs ouverts ou fermés, et ce, en raison d'une diminution de leur affinité pour les sites d’immobilisation synaptiques. Nous avons montré que ce mécanisme permettait aux synapses de récupérer plus rapidement de la désensibilisation et d'assurer la fidélité de la transmission synaptique lors de stimulations à hautes fréquences. Enfin, les synapses peuvent moduler leurs intensités de réponse grâce à des mécanismes de plasticité synaptique à long terme, et plus particulièrement, la dépression à long terme (LTD) qui correspond à un affaiblissement durable de ce poids synaptique. [...] À la suite des découvertes précédentes concernant le rôle de la nano-organisation dynamique des AMPARs pour réguler le poids et la fiabilité de la transmission synaptique, j'ai décidé d'étudier leur rôle dans l'affaiblissement et la sélection des synapses. J'ai découvert que la quantité d’AMPAR par nanodomaine diminue rapidement et durablement. Cette première phase semble due à une augmentation de l’internalisation des AMPARs. Dans un deuxième temps, la mobilité des AMPARs augmente suite à une réorganisation moléculaire de la synapse. Ce changement de mobilité des AMPARs permet aux synapses déprimées de maintenir leur capacité à répondre aux signaux neuronaux à hautes fréquences. Ainsi, nous proposons que l'augmentation de la mobilité des AMPARs au cours de la LTD permet de transmettre une réponse fidèle dans les synapses stimulées à hautes fréquences et donc de sélectivement les maintenir tout en éliminant les synapses inactives. / The brain is a complex network of interconnected neurons responsible for all our cognitive functions and behaviors. Neurons receive inputs at specialized contact zones named synapses which convert an all or none electrical signal to a chemical one, through the release of neurotransmitters. This chemical signal is then turned back in a tunable electrical signal by receptors to neurotransmitters. However, a single neuron receives thousands of inputs coming from several neurons in a spatial- and temporal-dependent manner. The precise mechanism by which neurons receive, integrate and transmit this synaptic inputs is highly complex and is still not perfectly understood. At excitatory synapses, AMPA receptors (AMPARs) are responsible for the fast synaptic transmission. With the recent developments in super-resolution microscopy, the community has changed its vision of synaptic transmission. One breakthrough was the discovery that AMPARs are not randomly distributed at synapses but are organized in nanodomains of ~80 nm of diameter containing ~20 receptors. This content is an important factor since it will determine the intensity of the synaptic response. Due to their mM affinity for glutamate, AMPARs can only be activated when located in an area of ~150 nm in front of the neurotransmitter release site. Recently, AMPAR nanodomains have been shown to be located in front of glutamate release sites and to form trans-synaptic nanocolumns at basal state. Thus, the nanoscale organization of AMPARs regarding release sites seems to be a key parameter for the efficiency of synaptic transmission. Another breakthrough in the field was the observation that AMPARs diffuse at the cell surface and are immobilized at synapses to participate to synaptic transmission. The dynamic exchange between AMPAR diffusive pool and the receptors immobilized into the nanodomains participates to maintain the efficiency of synaptic response upon high-frequency stimulation.The overall aim of my PhD has been to determine the role of each above listed parameters on the intimate properties of synaptic transmission both at basal state and during synaptic plasticity. First, we identified the crucial role of Neuroligin in the alignment of AMPAR nanodomains with glutamate release sites. In addition, we managed to break this alignment to understand its impact on synaptic transmission properties. In parallel, we demonstrated that, due to a decrease in their affinity for synaptic traps, desensitized AMPARs diffuse more at the plasma membrane than opened or closed receptors. This mechanism allows synapses to recover faster from desensitization and ensure the fidelity of synaptic transmission upon high-frequency release of glutamate. Finally, synapses can modulate their strength through long-term synaptic plasticity, in particular, Long-Term Depression (LTD) corresponds to a long-lasting weakening of synaptic strength and is thought to be important in some cognitive processes and behavioral flexibility through synapse selective elimination. Following the previous discoveries about the impact of AMPAR dynamic nano-organization at synapses on the regulation of the synaptic transmission strength and reliability, I decided to investigate their role in the weakening of synapses. I found that AMPAR nanodomain content drops down rapidly and this depletion last several minutes to hours. The initial phase seems due to an increase of endocytosis events, but in a second phase, AMPAR mobility is increased following a reorganization of the post-synaptic density. This change in mobility allows depressed synapses to maintain their capacity to answer to high-frequency inputs. Thus, we propose that LTD-induced increase in AMPAR mobility allows to conduct a reliable response in synapses under high-frequency stimulation and thus to selectively maintain them while eliminating the inactive ones.

<p>Mutations in the gene SHANK2, which encodes a synaptic scaffolding protein, have been shown to cause a spectrum of neuropsychiatric disorders including: intellectual disability, autism spectrum disorders (ASDs), bipolar disorder (BD), and schizophrenia. However, many aspects of SHANK2 including the array of isoforms expressed, the expression pattern of the protein, biochemical and regulatory mechanisms, and in vivo protein function remain elusive. This body of work aims to uncover the function of the SHANK2 gene and its role in neuropsychiatric disorders using in vitro and in vivo experimental systems.</p> <p>Using a molecular genetics approach, I revealed the transcript architecture of the mouse Shank2 gene including characterization of promoters, isoforms and protein domains. I then outlined the temporal and spatial pattern of the Shank2 isoform expression throughout development. To further explore the protein’s function, we sought to identify novel SHANK2 interacting proteins using a yeast-2-hybrid screen and characterized the interacting proteins. Lastly, in order to understand how Shank2 deficiencies alter brain function we generated and characterized both Shank2 conventional (∆e24) and conditional mutant mice (e24floxed) by deleting or floxing exon 24 that encodes the Homer binding site and has nonsense mutations in human patients with neuropsychiatric disorders.</p> <p>Collectively, these studies 1) provide insight into the transcriptional regulation of Shank2 during brain development; 2) support the value of using Shank2 to further dissect the pathophysiology and circuitry mechanism underlying manic and autism like behaviors; 3) offers a novel mechanistic link between ubiquitination-mediated protein modification and SHANK2 function that may elucidate the molecular basis underlying SHANK2-related neuropsychiatric disorders. Ultimately, these findings may lead to the development of new therapeutic interventions for SHANK2-related neuropsychiatric disorders.</p> / Dissertation

The mitral cells of the accessory olfactory bulb (AOB) of anuran frogs project their axons directly to the medial amygdala (MeA) along the accessory olfactory tract. An en bloc preparation of the telencephalon of the American bullfrog Lithobates catesbeiana was utilized to study a form of low-frequency inducible long-term potentiation (LTP) expressed at the synapse formed between the terminals of the accessory olfactory tract and the neurons of the MeA. Delivery of repetitive 1Hz-stimulation or sets of 5Hz tetani to the accessory olfactory tract both induced potentiation that was stable for over an hour, as measured by extracellular field recordings. LTP induced by 5Hz tetanus was associated with a decrease in paired-pulse ratio, which would be consistent with an increased probability of release contributing to the increased synaptic strength. Blockade of neither NMDA nor kainate glutamate receptors, with AP5 and UBP310 respectively, prevented LTP induction by 5Hz tetanus; however expression of LTP was partially masked in the presence of UBP310. These results suggest that kainate receptors are involved in the expression of LTP at the AOB-MeA synapse, though the means by which LTP is induced remains unclear. / Graduate / 2016-09-28

Mounting evidence suggests that synaptic connections are early pathological targets in many neurodegenerative diseases, including motor neuron disease. A better understanding of synaptic pathology is therefore likely to be critical in order to develop effective therapeutic strategies. Spinal muscular atrophy (SMA) is a common autosomal recessive childhood form of motor neuron disease. Previous studies have highlighted nerve- and muscle-specific events in SMA, including atrophy of muscle fibres and postsynaptic motor endplates, loss of lower motor neuron cell bodies and denervation of neuromuscular junctions caused by loss of pre-synaptic inputs. Here I have undertaken a detailed morphological investigation of neuromuscular synaptic pathology in the Smn-/- ;SMN2 and Smn-/-;SMN2;Δ7 mouse models of SMA. Results imply that synaptic degeneration is an early and significant event in SMA, with progressive denervation and neurofilament accumulation being present at early symptomatic time points. I have identified selectively vulnerable motor units, which appear to conform to a distinct developmental subtype compared to more stable motor units. I have also identified significant postsynaptic atrophy which does no correlate with pre-synaptic denervation, suggesting that there is a requirement for Smn in both muscle and nerve and pathological events can occur in both tissues independently. Rigorous investigation of lower motor neuron development, connectivity and gene expression at pre-symptomatic time points revealed developmental abnormalities do not underlie neuromuscular vulnerability in SMA. Equivalent gene expression analysis at end-stage time points has implicated growth factor signalling and extracellular matrix integrity in SMA pathology. Using an alternative model of early onset neurodegeneration, I provide evidence that the processes regulating morphologically distinct types of synaptic degeneration are also mechanistically distinct. In summary, in this work I highlight the importance and incidence of synaptic pathology in mouse models of spinal muscular atrophy and provide mechanistic insight into the processes regulating neurodegeneration.

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.

Activity-dependent and synapse-specific translation of mRNAs is required for long-term changes in synaptic strength (or efficacy). However, many of the components mediating repression, transport and activation of mRNAs are unknown. Translational control in neurons is a highly conserved process and mediated by a ribonuclear particle (RNP). This study shows that RNPs in Drosophila neurons are similar not only to mammalian neuronal RNA granules but also to yeast P-bodies, cytoplasmic foci involved in translational repression and RNA decay. The evolutionarily conserved proteins Me31b and Trailer Hitch localize to RNA granules. Me31b and Trailer Hitch are required for normal dendritic growth. Mutations in Me31b and Trailer Hitch suppress phenotypes resulting from overexpression of Fragile X Mental Retardation protein, suggesting that both proteins may act as translational repressors. In addition, this study reports the identification of novel translational repressors in neurons. Using the overexpression phenotype of Fragile X Mental Retardation protein in a candidate-based genetic screen, I identified dominant suppressor mutations in five genes, including Doubletime/Discs Overgrown, Orb2/CPEB, PolyA Binding Protein, Rm62/Dmp68 and SmD3. Like Me31b and Trailer Hitch, all five proteins localize to neuronal RNPs. Overexpression of each proteins affects dendritic branching of sensory neurons in Drosophila. Identification and further characterization of these novel RNP granule components and dFMR1-interacting proteins may provide further insights into the mechanisms controlling translational in dendrites.

Non-competitive antagonists of the glutamate N-methyl-D-aspartate receptor (NMDAR) induce a broad range of schizophrenia-like symptoms in humans. Consequently hypothesis has emerged suggesting that glutamate or NMDAR hypofunction may occur in schizophrenia. The NMDAR is localised at dendritic spines of neurons and is embedded in a multi-protein complex called the post-synaptic density (PSD). The biochemical composition of the postsynaptic membrane and the structure of dendritic spines are continuously modulated by glutamatergic synaptic activity. The activity-dependent interaction between glutamate receptors and proteins of the PSD stimulate intracellular signalling pathways underlying learning and memory processes. These may be disturbed in schizophrenia. In the present study we hypothesised that molecules of the PSD may be disturbed in expression in the premotor cortex of patients with schizophrenia. Postmortem premotor cortex from patients with schizophrenia, major depressive disorder, bipolar disorder and healthy controls were processed for PSD extraction and purification. Protein expression of the PSD fraction was assessed using co-immunoprecipitation (co-IP) and Western blotting (WB) methods. The expression of NMDAR subunit NR2A, PSD-95, Ca2+/calmodulin-dependent protein kinase II subunit β (CaMKIIβ) and truncated isoform of the tropomyosin receptor kinase type B (TrkB-T1) were significantly reduced in schizophrenia. A significant decrease in the expression of NR2A was also observed in patients with major depressive disorder relative to controls. A decrease in the abundance of key PSD proteins in schizophrenia provides strong evidence that PSD function and possibly synaptic plasticity may be disturbed in the premotor cortex in the disease. There may also be more subtle disturbances in PSD function in major depressive disorder.