Tesis sobre el tema "Dendritic Spine Plasticity"

Les changements des connections neuronales interviendraient dans la formation de la mémoire. J’ai développé de nouvelles approches basées sur l’imagerie photonique pour étudier (i) les interactions entre les microglies et les épines dendritiques, et (ii) le renouvellement des épines dans l’hippocampe in vivo. Ces deux phénomènes contribueraient au remodelage des circuits synaptiques intervenant dans la mémoire. (i) Les microglies sont impliquées dans de nouvelles fonctions en condition saine. J’ai examiné l’effet de la plasticité synaptique sur la dynamique morphologique des microglies, et sur leur interaction avec les épines. En combinant l’électrophysiologie et l’imagerie bi-photonique dans des tranches aigües de souris transgéniques, je démontre que la microglie intensifie son interaction physique avec les épines. Ainsi pour continuer l’étude de ces interactions et leur impact fonctionnel plus précisément, j’ai optimisé l’imagerie STED dans des tranches aigües. (ii) La plasticité structurale des épines est cruciale pour la mémoire, mais les connaissances à ce sujet dans l’hippocampe in vivo restent limitées. J’ai donc établi une technique d’imagerie chronique STED in vivo pour visualiser les épines dans l’hippocampe. Cette approche a révélé une densité double de celle reportée précédemment à l’aide de la microscopie bi-photonique. De plus j’ai observé un renouvellement des épines de 40% en 5 jours, représentant un taux important de remodelage synaptique dans l’hippocampe. Les approches d’imagerie super-résolutive permettent l’étude des interactions microglie-épine, et du renouvellement des épines hippocampiques avec une résolution inédite chez la souris vivante
Activity-dependent changes in neuronal connectivity are thought to underlie learning and memory. I developed and applied novel high-resolution imaging-based approaches to study (i) microglia-spine interactions and (ii) the turnover of dendritic spines in the mouse hippocampus, which are both thought to contribute to the remodeling of synaptic circuits underlying memory formation. (i) Microglia have been implicated in a variety of novel tasks beyond their classic immune defensive roles. I examined the effect of synaptic plasticity on microglial morphological dynamics and interactions with spines, using a combination of electrophysiology and two-photon microscopy in acute brain slices. I demonstrated that microglia intensify their physical interactions with spines after the induction of hippocampal synaptic plasticity. To study these interactions and their functional impact in greater detail, I optimized and applied time-lapse STED imaging in acute brain slices. (ii) Spine structural plasticity is thought to underpin memory formation. Yet, we know very little about it in the hippocampus in vivo, which is the archetypical memory center of the mammalian brain. I established chronic in vivo STED imaging of hippocampal spines in the living mouse using a modified cranial window technique. The super-resolution approach revealed a spine density that was two times higher than reported in the two-photon literature, and a spine turnover of 40% over 5 days, indicating a high level of structural remodeling of hippocampal synaptic circuits. The developed super-resolution imaging approaches enable the examination of microglia-synapse interactions and dendritic spines with unprecedented resolution in the living brain (tissue)

Autism is one of the leading causes of human intellectual disability (ID). More than 1% of the human population has autism spectrum disorders (ASDs), and it has been estimated that over 50% of those with ASDs also have ID. Fragile X syndrome (FXS) is the most common inherited form of mental retardation and is the leading known genetic cause of autism, affecting approximately 1 in 4000 males and 1 in 8000 females. Approximately 30% of boys with FXS will be diagnosed with autism in their later lives. The cause of FXS is through an over-expansion of the CGG trinucleotide repeat located at the 5’ untranslated region of the FMR1 gene, leading to hypermethylation of the surrounding sequence and eventually partially or fully silencing of the gene. Therefore, the protein product of the gene, fragile X mental retardation protein (FMRP), is reduced or missing. As a single-gene disorder, FXS offers a scientifically tractable way to examine the underlying mechanism of the disease and also shed some light on understanding ASD and ID. The mouse model of FXS (Fmr1−/y mice) is widely accepted and used as a good model, offering good structural and face validity. Since a primary deficit of FXS is believed to be altered neuronal communication, in this thesis I examined white matter tract and dendritic spine abnormalities in the mouse model of FXS. Loss of FMRP does not alter the gross morphology of the white matter. However, recent brain imaging studies indicated that loss of FMRP could lead to some minute abnormalities in different major white matter tracts in the human brain. The gross white matter morphology and myelination was unaltered in the Fmr1−/y mice, however, a small but significant increase of axon diameter in the corpus callosum (CC) was found compared to wild-type (WT) controls. Our computation model suggested that the increase of axon diameter in the Fmr1−/y mice could lead to an increase of conduction velocity in these animals. One of the key phenotypes reported previously in the loss of FMRP is the increase of “immature” dendritic spines. The increase of long and thin spines was reported in several brain regions including the somatosensory cortex and visual cortex in both FXS patients and the mouse model of FXS. Although recent studies which employed state-of-the-art microscopy techniques suggested that only minute differences were noticed between the WT and Fmr1−/y mice. In agreement with previous findings, I found an increase of dendritic spine density in the visual cortex in the Fmr1−/y mice, and spine morphology was also different between the two genotypes. We found that the spine head diameter is significantly increased in the CA1 area of the apical dendrites of the Fmr1−/y mice compared to WT controls. Dendritic spine length is also significantly increased in the same region of the Fmr1−/y mice. However, apical spine head size does not alter between the two genotypes in the V1 region of the visual cortex, and spine length is significantly decreased in the Fmr1−/y mice compared to WT animals in this region. Lovastatin, a drug known as one of the 3-hydroxy-3-methyl-glutaryl-CoA (HMG-CoA) reductase inhibitors, functions as a modulator of the mitogen-activated protein kinases (MAPK) pathway through inhibiting Ras farnesylation, was used in an attempt to rescue the dendritic spine abnormalities in the Fmr1−/y mice. Mice lacking FMRP are susceptible to audiogenic seizure (AGS). Previous work has shown that 48 hr of lovastatin treatment reduced the incidence of AGS in the Fmr1−/y mice. However, chronic lovastatin treatment failed to rescue the spine density and morphology abnormalities in the Fmr1−/y mice. Mouse models are invaluable tools for modelling human diseases. However inter-strain differences have often confounded results between laboratories. In my final Chapter of this thesis, I compared two commonly used C57BL/6 substrains of mice by recording their electrophysiological responses to visual stimuli in vivo. I found a significant increase of high-frequency gamma power in adult C57BL/6JOla mice, and this phenomenon was reduced during the critical period. My results suggested that the C57BL/6JOla substrain has a significant stronger overall inhibitory network activity in the visual cortex than the C57BL/6J substrain. This is in good agreement with previous findings showing a lack of open-eye potentiation to monocular deprivation in the C57BL/6JOla substrain, and highlights the need for appropriate choice of mouse strain when studying neurodevelopmental models. They also give valuable insights into the genetic mechanisms that permit experience-dependent developmental plasticity. In summary, these findings give us a better understanding of the fine structure abnormalities of the Fmr1−/y mice, which in turn can benefit future discoveries of the underlying mechanisms of neurodevelopmental disorders such as ID and ASDs.

On short timescales, the brain represents, transmits, and processes information through the electrical activity of its neurons. On long timescales, the brain stores information in the strength of the synaptic connections between its neurons. This thesis examines the surprising implications of two separate, well documented microscopic processes — the stochastic gating of ion channels and the plasticity of dendritic spines — for neural information processing and storage. Electrical activity in neurons is mediated by many small membrane proteins called ion channels. Although single ion channels are known to open and close stochastically, the macroscopic behaviour of populations of ion channels are often approximated as deterministic. This is based on the assumption that the intrinsic noise introduced by stochastic ion channel gating is so weak as to be negligible. In this study we take advantage of newly developed efficient computer simulation methods to examine cases where this assumption breaks down. We find that ion channel noise can mediate spontaneous action potential firing in small nerve fibres, and explore its possible implications for neuropathic pain disorders of peripheral nerves. We then characterise the magnitude of ion channel noise for single neurons in the central nervous system, and demonstrate through simulation that channel noise is sufficient to corrupt synaptic integration, spike timing and spike reliability in dendritic neurons. The second topic concerns neural information storage. Learning and memory in the brain has long been believed to be mediated by changes in the strengths of synaptic connections between neurons — a phenomenon termed synaptic plasticity. Most excitatory synapses in the brain are hosted on small membrane structures called dendritic spines, and plasticity of these synapses is dependent on calcium concentration changes within the dendritic spine. In the last decade, it has become clear that spines are highly dynamic structures that appear and disappear, and can shrink and enlarge on rapid timescales. It is also clear that this spine structural plasticity is intimately linked to synaptic plasticity. Small spines host weak synapses, and large spines host strong synapses. Because spine size is one factor which determines synaptic calcium concentration, it is likely that spine structural plasticity influences the rules of synaptic plasticity. We theoretically study the consequences of this observation, and find that different spine-size to synaptic-strength relationships can lead to qualitative differences in long-term synaptic strength dynamics and information storage. This novel theory unifies much existing disparate data, including the unimodal distribution of synaptic strength, the saturation of synaptic plasticity, and the stability of strong synapses.

The plasticity of dendritic spine structure is important for neural development and synaptic function and is altered in many pathological conditions. In this study, we investigated the mechanisms underlying spine structural plasticity during development and the pathological changes in spine structure during ischemic stroke by using confocal and two-photon microscopy. We first investigated spine structural dynamics during development and the role of intracellular Ca²⁺ in determining basal spine motility in cultured hippocampal neurons. We found that young cultured neurons displayed significantly more spine motility than older neurons. In addition, we found that global buffering of intracellular Ca²⁺ failed to alter the basal motility of developing spines. Thus basal spine motility may represent an intrinsic feature of developing neurons and is not necessarily choreographed by ongoing changes in intracellular Ca²⁺ levels. We then examined spine structure changes during cerebral ischemia in vivo and investigated the relationships between cortical microcirculation and spine structure and function. We found moderate ischemia did not significantly affect spines within a 5-h time span; however, severe ischemia caused a rapid loss of spines and induced beading of dendrite structure within as little as 10 min following stroke. Surprisingly this damage was found to be reversible if reperfusion occurred within 20-60 min. By monitoring both cortical microcirculation and dendritic spine structure, we found that dendritic integrity deteriorated proportionally with the fraction of blocked vessels and the volume of affected brain during stroke. In ischemic border regions, we demonstrated that intact dendritic structure could be stably maintained for hours by blood flow from vessels that were on average 81 μm away. Functional imaging of intrinsic optical signals indicated that signal changes induced by limb movement were blocked in areas with blebbed dendrites, but were present at ~225 μm and beyond from the border of dendritic damage, suggesting peri-infarct tissues could function under acute ischemia in the core. In summary, our findings indicate that basal spine motility is maintained in a Ca²⁺ independent manner, and changes in spine structure during ischemia can now be directly linked to alterations in synaptic function and reductions in the cortical microcirculation.
Medicine, Faculty of
Graduate

Thesis (Ph.D. in Pharmacology) -- University of Colorado Denver, 2008.
Typescript. Includes bibliographical references (leaves 135-162). Free to UCD affiliates. Online version available via ProQuest Digital Dissertations;

Neuroplasticity has been researched in many different ways, from the growing neonatal brain to neural responses to trauma and injury. According to recent research, neuroplasticity is also prevalent in the ability of the brain to repurpose areas that are not of use, like in the case of a loss of a sense. Specifically, behavioral studies have shown that deaf humans (Bavalier and Neville, 2002) and cats have increased visual ability, and that different areas of the auditory cortex enhance specific kinds of sight. One such behavioral test demonstrated that the dorsal zone (DZ) of the auditory cortex enhances sensitivity to visual motion through cross-modal plasticity (Lomber et. al., 2010). Current research seeks to examine the anatomical structures responsible for these changes through analysis of excitatory neuron dendritic spine density and spine head diameter. This present study focuses on the examination of DZ neuron spine density, distribution, and size in deaf and hearing cats to corroborate the visual changes seen in behavioral studies. Using Golgi-stained tissue and light microscopy, our results showed a decrease in overall spine density but slight increase in spine head diameter in deaf cats compared to hearing cats. These results, along with several other studies, support multiple theories on how cross-modal reorganization of the auditory cortex occurs after deafening

Una delle funzioni principali della memoria risiede nella sua capacità di conservare le informazioni nel tempo. Il modello più accreditato si basa sulla premessa che le informazioni vengono temporaneamente immagazzinate nell’ippocampo dove rimangono vulnerabili alle interferenze provocate da contingenze esterne. Attraverso un processo molto lento, la traccia mnestica è trasferita in altre strutture del cervello dove la memoria è resa stabile e non più vulnerabile alle interferenze. Questo processo richiede tempo per essere portato a termine ed è chiamato consolidamento (Mueller and Pilzecher, 1900). Varie teorie ipotizzano che i meccanismi attraverso i quali le memorie possono essere acquisite e consolidate nel cervello dei mammiferi implichino modificazioni nella plasticità strutturale (Cajal, 1891). Lo scopo principale di questo lavoro è quello di ricercare le modificazioni morfologiche necessarie alla formazione e all’estinzione della memoria. Nel primo lavoro esposto mostriamo la presenza di cambiamenti plastici (sotto forma di aumento in densità di spine dendritiche) nell’ippocampo, immediatamente dopo un addestramento in un condizionamento avversivo al contesto. Questi cambiamenti sono temporanei poiché scompaiono 36 giorni dopo. Contemporaneamente in corteccia anteriore cingolata è visibile un aumento di spine dendritiche solo dopo aver testato gli animali per la loro memoria a lungo termine ma non dopo il test di memoria a breve termine, mostrando un pattern inverso rispetto a quello trovato in ippocampo. Nel secondo lavoro esposto, blocchiamo la possibilità di aumentare il numero di spine in corteccia anteriore cingolata e troviamo una finestra temporale durante la quale i rimodellamenti sinaptici che avvengono in questa regione sono fondamentali per un corretto consolidamento delle memorie a lungo termine. Nel terzo lavoro presentato mostriamo come l’estinzione di un comportamento indotto dal consolidamento di una memoria precedentemente acquisita modifichi nuovamente la rete sinaptica che si era lentamente formata nella corteccia anteriore cingolata. Contemporaneamente troviamo un aumento in connettività sinaptica nei neuroni della corteccia infralimbica indotto dal consolidamento che persiste dopo l’estinzione. I nostri risultati puntano sulla versatilità e plasticità delle reti neuronali che sottostanno ai processi di memoria.
It is generally believed that in order to enable long-term episodic memory, the information is temporarily stored in the hippocampus where it remains vulnerable to interference. Via a slow read-out process, the information is transferred into other brain structures where the memory is established and no longer vulnerable to interference. This slow read-out is termed consolidation (Mueller and Pilzecker, 1900). The mechanisms by which memories can be acquired and consolidated in the mammalian brain are assumed to involve modifications in structural plasticity (Cajal, 1891). The main goal of this work is to discover the morphological modification requested in memory formation and extinction. In study I we shown that plastic changes (i.e. dendritic spine density increase) immediately develop in CA1 field of the hippocampus after a training in the contextual fear conditioning. These modifications are only transient because they disappear 36 days later, while an inverse pattern of spine density in recent and remote memory recall were found in the anterior cingulate cortex. In study II we block the possibility to increase the number of spines in the aCC after training and we found an early temporal window in which synaptic remodelling occurring in this region is fundamental for the correct consolidation of memory. In study III we presented a new and conflicting memory (extinction) after the consolidation of an old one, founding a disruption of the synaptic network in the aCC field. At the same time, we found an increase of connectivity in the Infra limbic cortex induced by consolidation that persist after extinction. Our results point on a dynamic view of memory consolidation: a regulated balance of synaptic stability and synaptic plasticity is required for optimal memory retention to allow the incorporation of new memories in neuronal circuits.

Le cerveau contient environ cent milliards de cellules nerveuses, ou neurones. Ces neurones communiquent entre eux par des structures fonctionnellement distinctes – l’axone et la dendrite – capables d’émettre et recevoir des signaux électriques ou chimiques à partir d’un compartiment présynaptique vers un compartiment, dit post-synaptique. Nous avons focalisé notre étude sur les synapses des neurones hippocampiques, qu’on estime responsables de fonctions cérébrales dites supérieures, comme la mémoire et l’apprentissage. Plus particulièrement, on s’est intéressé au développement et au maintien des épines dendritiques, dont les changements morphologiques sont intimement liés à la plasticité synaptique, autrement dit, capacité de réponse à l’activité synaptique. Les épines dendritiques ont pour origine les filopodes qui évoluent en épines lors du contact axonal. La transition entre filopode et épine implique une myriade de molécules, dont des récepteurs glutamatergiques, des protéines d’échafaudage et du cytosquelette d’actine capables de recevoir, transmettre et intégrer le signal présynaptique. Cependant, la coordination spatiale et temporelle de tous ces composants moléculaires au long de la formation et maturation d’une synapse reste largement méconnue.Scribble1 (Scrib1) est une protéine de polarité cellulaire (PCP) classiquement impliquée dans l’homéostasie de tissues épithéliaux ainsi que dans la croissance et progression des tumeurs. Scrib1 est aussi une protéine d’échafaudage critique pour le développement et le bon fonctionnement du cerveau. L’objectif de cette étude a donc été d’étudier les mécanismes moléculaires sous-jacents à un rôle potentiel de Scrib1 dans la formation et le maintien des synapses. Dans un premier temps, on a décrit l’importance d’interactions dépendantes des domaines PDZ sur le trafic des récepteurs glutamatergiques ainsi que sur la voie de signalisation de plasticité synaptique sous-jacente à la mémoire spatiale. Dans un second temps, nous avons évalué les conséquences fonctionnelles d’une mutation de Scrib1 récemment identifiée chez un patient humain atteint des troubles du spectre autistique (TSA) dans la morphologie et fonction des neurones. On a démontré que Scrib1 régule l’arborisation dendritique ainsi que la formation et le maintien fonctionnel des épines dendritiques via un mécanisme dépendent du cytosquelette d’actine. Le dérèglement de ces mécanismes pourrait être à l’origine du phénotype TSA. L’ensemble de ce travail met en évidence que Scrib1, protéine d’échafaudage clé dans le développement et la fonction du cerveau, joue une multitude de rôle du niveau subcellulaire au niveau cognitif
The brain is made up of billions of nerve cells, or neurons. Neurons communicate with each other through functionally distinct structures - the axon and the dendrite - which are able to release and receive an electrical or chemical signal from a pre- to a post-synaptic compartment, respectively. We focused our study on hippocampal neurons synapses, which ultimately underlie high-order brain functions, such as learning and memory. In particular, we studied the development and maintenance of dendritic spines, whose changes in morphology are intimately correlated with synaptic plasticity, or the ability to respond to synaptic activity. Dendritic spines originate from motile dendritic filopodia, which mature into spines following axonal contact. The filopodia-to-spine transition involves a plethora of molecular actors, including glutamate receptors, scaffold proteins and the actin cytoskeleton, able to receive, transmit and integrate the pre-synaptic signal. The spatial and temporal coordination of all these molecular components throughout the formation and maturation of a synapse remains, however, unclear. Scribble1 (Scrib1) is planar cell polarity protein (PCP) classically implicated in the homeostasis of epithelial tissues and tumour growth. In the mammalian brain, Scrib1 is a critical scaffold protein in brain development and function. The main goal of this work was, therefore, to investigate the molecular mechanisms underlying Scrib1 role in synapse formation and maintenance. In a first part, we depict the importance of Scrib1 PDZ-dependent interactions on glutamate receptors trafficking as well as bidirectional plasticity signalling pathway underying spatial memory. In a second part, we focus on the functional consequences of a recently identified autism spectrum disorder (ASD) mutation of Scrib1 on neuronal morpholgy and function. We demonstrated that Scrib1 regulates dendritic arborization as well as spine formation and functional maintenance via an actin-dependent mechanism, whose disruption might underlie the ASD phenotype. Taken altogether, this thesis highlights the PCP protein Scrib1 as key scaffold protein in brain development and function, playing a plethora of roles from the subcelular to the cognitive level

Engrailed (En) est un facteur de transcription important pour la mise en place de la segmentation de l’embryon et du plan d’organisation antéro-postérieur. Comme d’autres membres de la famille des homéoprotéines, Engrailed peut aussi agir comme une molécule de signalisation extracellulaire, internalisable grâce à son domaine « pénétratine » et stimulant dans la cellule cible la transcription ou la traduction des ARNm. De cette façon, Engrailed guide les axones en modifiant la traduction dans le cône de croissance axonal et l’infusion cérébrale d’Engrailed protège les neurones dopaminergiques dans un modèle de la maladie de Parkinson en augmentant la traduction de protéines mitochondriales. Des troubles cognitifs et un déficit des interactions sociales ont été observés chez les souris En1+/- et les souris En2-/-. Une augmentation de l’expression d’En2 a aussi été observée chez des patients atteints de troubles du spectre autistique. Néanmoins, le lien entre les modifications de l’expression d’Engrailed et l’autisme ne sont pas compris. L’objectif de cette thèse a été d’étendre notre connaissance des fonctions d’Engrailed dans une région télencéphalique où elle est a priori peu exprimée (l’hippocampe). Nos résultats confirment l’expression d’En1 et En2 dans l’hippocampe mature et décrivent les variations de l’expression de ces gènes au cours du développement de cette structure. En1 et En2 présentent des patrons d’expression différents pendant la première semaine postnatale et chez l’adulte suggérant que des variations du dosage génique d’Engrailed sont liées à certaines phases du développement, en particulier au début de la synaptogenèse. Nous avons également découvert que dans les cultures de cellules d’hippocampe Engrailed est exprimé dans les neurones et que son expression est plus forte dans les neurones GABA-ergiques, notamment dans leurs prolongements dendritiques et axonaux. Nous avons constaté qu’un excès d’Engrailed (décrit dans certains cas de TSA) augmente la complexité dendritique ainsi que la densité des épines dendritiques plastiques mais sans augmenter parallèlement la formation de synapses matures excitatrices. Nous avons observé des variations de densité des épines dendritiques chez les souris En2-/- et les souris En1+/-, ce qui confirme l’implication d’Engrailed dans leur formation ou leur stabilisation. Si dans nos conditions expérimentales l’excès d’Engrailed ne modifie pas la densité des synapses, un mutant d’Engrailed qui présente une interaction réduite avec eIF4E est moins efficace qu’Engrailed pour augmenter la densité des épines et diminue la densité des boutons présynaptiques et le synaptic matching. Ces résultats indiquent que l’interaction avec eIF4E régule au moins en partie les effets d’Engrailed sur la spinogenèse et suggèrent également une implication d’Engrailed dans la formation ou la stabilisation des boutons présynaptiques. Le rôle clef d’eIF4E dans la traduction permet de postuler que certains effets d’Engrailed observés dans notre étude pourraient dépendre de la synthèse protéique. Nos résultats montrent à cet égard qu’Engrailed augmente la synthèse protéique dans les neurones d’hippocampe. Cette traduction est différente de celle induite par la LTP chimique (LTPc) car insensible à l’action des oligomères synthétiques d’AβO, responsables sous leur forme naturelle de synaptopathies dans le contexte de la maladie d’Alzheimer. Engrailed permet également de restaurer la traduction défaillante de neurones issus de souris TG2576, modèles de la maladie d’Alzheimer. Dans leur ensemble, nos résultats identifient Engrailed comme un nouvel acteur de la plasticité dendritique. Ils révèlent qu’un excès d’Engrailed au cours de la synaptogenèse modifie les caractéristiques des dendrites, une situation susceptible d’altérer les caractéristiques fonctionnelles du réseau dendritique dans une situation de surexpression pathologique de la protéine. (...)
Engrailed (En) is an important transcription factor in embryo’s segmentation and anterior-posterior axis establishment during early embryogenesis. As several homeoproteins, Engrailed can act as an extracellular signalling molecule which can be internalized by target cells thanks to its penetratin domain and act through transcriptional and/or translation dependent mechanisms. Engrailed has for instance, translation-dependent effects on axonal guidance and cerebral infusion of Engrailed protects dopaminergic neurons in a Parkinson disease model by increasing mitochondrial protein translation. Also, cognitive defects were observed in En1+/+ and En2-/- and En2 expression is increased in ASD patients. This work consisted in extending the knowledge of Engrailed expression and functions. We explored the links with a telencephalic structure where it is a priori fewly expressed (hippocampus). Our results confirm En1 and En2 expression in the mature hippocampus and describe their respective expression along the development of this structure. En1 and En2 have different expression patterns during the first post-natal week as well as in the adulthood suggesting a genetic dosage of Engrailed during the development, specifically with the beginning of synaptogenesis. We also reveal that Engrailed, expressed in hippocampal neurons, is more expressed in GABA-ergic neurons, notably in their dendritic and axonal neurites. We observe that an excess of Engrailed (described in some ASD cases) increases dendritic complexity as well as plastic dendritic spine density, without affecting mature excitatory synapses. We show that En2-/- and heterozygote En1 mice have variations in dendritic spine density, which confirms that Engrailed is involved either in their formation or stabilization. Even though our experiments show no modification of synapse density with an excess of Engrailed, a mutant showing a decreased eIF4E interaction and less efficient than wild type Engrailed to increase dendritic spine density, decreases presynaptic button density and synaptic matching. Those results indicate that eIF4E interaction with Engrailed is, at least in part, responsible for its effects on spinogenesis and suggest a role of Engrailed in presynaptic button formation/stabilization. Key-role of eIF4E in translation allow to hypothesize that some of Engrailed effects we report could be translation dependent. In this sense, our results show that Engrailed is able to increase proteic synthesis in hippocampal neurons. This translation is different from the one induced by chemical LTP (LTPc): it is not altered by synthetic AβO, which are the main toxic agent when produced at abnormally high levels in Alzheimer disease. Engrailed is also able to restore defaulting translation in neurons from Alzheimer disease mice model (TG2576). As a whole, our results identify Engrailed as a novel actor in dendritic plasticity. They reveal that an excess of Engrailed during synaptogenesis can modify dendrite characteristics. This can lead to dendritic network dysfunction in a context of pathologic surexpression of Engrailed. Our observations open to new perspectives contributing to a better understanding of the relationship between Engrailed and ASD. Finally, this work lays the foundation to potentially fruitful links between Engrailed and AβOligomers signalling pathways, where modulation of protein synthesis could be a therapeutic lever in physiopathologic conditions

The majority of fast excitatory neurotransmission in the brain occurs at glutamatergic synapses. The extensive dendritic arborisations of pyramidal neurons in the neocortex and hippocampus harbor thousands of synaptic connections, each formed on tiny protrusions called dendritic spines. Spine synapses are rapidly established during early postnatal development – a key period in neural circuit assembly – and are subject to dynamic activity-dependent plasticity mechanisms that are believed to underlie neural information storage and processing for learning and memory. Recent decades have seen remarkable progress in identifying diverse plasticity mechanisms responsible for regulating synapse structure and function, and in understanding the processes underlying computation of synaptic inputs in the dendrites of individual neurons. These advances have strengthened our understanding of the biological mechanisms underlying brain function but, not surprisingly, they have also raised many new questions. Using a combination of whole-cell electrophysiology, 2-photon imaging and glutamate uncaging in rodent brain slice preparations, I have helped to document the subtype-specific regulation of glutamate receptors during a homeostatic form of synaptic plasticity at CA1 pyramidal neurons of the hippocampus, and have discovered novel synaptic calcium dynamics during a critical period of neural circuit formation. First, we found that during a homeostatic response to prolonged inactivity, both AMPA and NMDA subtypes of glutamate receptors undergo a switch in subunit composition at synapses, but exhibit a divergence in their subcellular localization at extrasynaptic regions of the plasma membrane (this work was published in the Journal of Neuroscience in 2013). In separate series of experiments using 2-photon calcium imaging, I discovered a functional coupling between NMDA receptor activation and intracellular calcium release at dendritic spines and dendrites that is selectively expressed during a critical period of synapse formation. This synaptic calcium signaling mechanism enabled the transformation of distinct spatiotemporal patterns of synaptic input into salient biochemical signals, and is thus apt to locally regulate synapse development along individual dendritic branches. Consistent with this hypothesis, I found evidence for non-random clustering of synapse development between neighboring dendritic spines. Together, these experimental results expand the current understanding of the dynamics of synapse function during homeostatic plasticity and early postnatal development. --- Les synapses glutamatergiques soutiennent la majorité de la neurotransmission excitatrice rapide du cerveau. Des milliers de ces synapses, localisées sur de minuscules saillies appelées épines dendritiques, décorent les vastes arborisations dendritiques des neurones pyramidaux du néocortex et de l'hippocampe. Ces synapses sont formées tôt lors du développement postnatal et sont soumises à des mécanismes dynamiques de plasticité qui sous-tendent, croit-on, les capacités d'apprentissage et de mémoire du cerveau. Les dernières décennies ont vu des progrès remarquables dans l'identification de divers mécanismes de régulation de la structure et de la fonction des synapses sur différentes échelles de temps, et dans la compréhension des processus qui régissent l’intégration des inputs synaptiques au niveau des dendrites individuelles. Ces progrès ont renforcé notre compréhension des éléments fondamentaux régissant la fonction cérébrale et ont ouvert de nouvelles voies d’investigations neurophysiologiques. En utilisant une combinaison d’électrophysiologie cellulaire, d'imagerie à deux-photons et de photolibération de glutamate sur des neurones pyramidaux de la région CA1 de l'hippocampe de rats, j’ai contribué à la découverte et à la caractérisation de nouvelles régulations des récepteurs du glutamate durant la plasticité synaptique homéostatique. J’ai également découvert un nouveau type de dynamique de calcium synaptique relié à une organisation spatiale du développement des synapses pendant une période critique de l’ontogénie des circuits neuronaux. Dans la première étude, nous avons constaté que lors d'une plasticité de type homéostatique induite par une inactivité prolongée, les récepteurs de glutamate de types AMPA et NMDA sont soumis à un changement important dans la composition de leurs sous-unités. De plus, nous avons observé un ciblage différentiel de ces récepteurs vers des compartiments subcellulaires spécifiques des neurones. Dans une série d'expériences séparée utilisant l’imagerie calcique à deux-photons, j’ai découvert un couplage fonctionnel durant le développent entre l'activation des récepteurs NMDA et une libération de calcium intracellulaire qui envahit tant les épines dendritiques que les dendrites. J’ai également trouvé que ce mécanisme de signalisation de calcium synaptique transforme des motifs spatiotemporels d’activités synaptiques spécifiques en signaux biochimiques post-synaptiques de manière à potentiellement réguler l’organisation spatiale des synapses durant le développement. Conformément à cette hypothèse, j’ai observé des manifestations fonctionnelles claires de regroupement dans l’espace de synapses de forces similaires le long de branches dendritiques individuelles. Ensemble, ces résultats expérimentaux élargissent notre compréhension actuelle de de la fonction des synapses durant la plasticité homéostatique ainsi que durant le développement postnatal du cerveau. En étudiant les mécanismes neurophysiologiques de base, il sera possible d'avoir un aperçu plus profond du fonctionnement du cerveau et de ses pathologies.

L'efficacité de la transmission synaptique GABAergique est influencée par la concentration intracellulaire en ions chlorure. Dans les neurones matures, l'extrusion de ces ions par le transporteur neuronal potassium chlore de type 2 (KCC2) permet l'influx d'ions chlorure lors de l'activation des récepteurs du GABA de type A. Néanmoins, KCC2 est aussi enrichi à proximité des synapses excitatrices portées par les épines dendritiques qui correspondent à des protrusions dendritiques enrichies en actine. Alors que l'effet d'une suppression de KCC2 sur l'homéostasie des ions chlorure et la transmission GABAergique est largement documenté, peu de choses sont connues sur l'impact qu'une telle suppression peut avoir sur la transmission glutamatergique. Lors de ma thèse, j'ai exploré le rôle de KCC2 dans la potentialisation à long terme (LTP) de la transmission glutamatergique à l'origine des phénomènes d'apprentissage et de mémorisation. Ce travail a révélé que la suppression de KCC2 compromet les modifications fonctionnelles et structurales sous-tendant la LTP. Cet effet est associé à une inhibition de la cofilin, protéine responsable de la dépolymérisation de l'actine, qui corrèle avec une augmentation de la quantité d'actine filamenteuse dans les épines dendritiques. En empêchant l'inhibition de la cofilin liée à l'absence de KCC2, il m'a alors été possible de restaurer la LTP suggérant que KCC2 pourrait influencer cette forme de plasticité en régulant la dynamique de polymérisation du cytosquelette d'actine. Mes résultats démontrent que la fonction de KCC2 va au-delà du contrôle de l'homéostasie des ions chlorure et influence les mécanismes de plasticité de la synapse excitatrice
The polarity and efficacy of GABAergic synaptic transmission are both influenced by the intra-neuronal chloride concentration. In mature neurons, chloride extrusion through the neuronal K/Cl cotransporter KCC2 allows an inhibitory influx of chloride upon activation of GABAA receptors. Nevertheless, KCC2 is enriched in the vicinity of excitatory synapses within the dendritic spines that are actin-rich protrusions emerging from dendritic shafts. While it has become clear that KCC2 suppression alters chloride homeostasis and GABA signaling, little is known on its impact on glutamatergic transmission. In the laboratory, we have previously demonstrated that KCC2 suppression in mature neurons leads to decreased glutamatergic transmission efficacy through an ion-transport independent function of KCC2. During my PhD, I have explored how KCC2 may also impact LTP of glutamatergic synapses. My work reveals that KCC2 suppression compromises both functional and structural LTP at these synapses. This effect is associated with inhibition of the actin-severing protein cofilin and enhanced mobilization of F-actin in dendritic spines. Since LTP can be rescued by preventing cofilin inhibition upon KCC2 suppression, I suggest KCC2 might influence LTP through altered actin cytoskeleton dynamics. My results demonstrate that KCC2 function extends beyond the mere control of neuronal chloride homoeostasis and suggest regulation of KCC2 membrane stability may act as a metaplastic switch to gate long term plasticity at excitatory synapses in cortical neurons

Calpains, a highly conserved family of calcium-dependent cysteine proteases, are divided in two groups; classical and non-conventional calpains. Calpain-1 and calpain-2, the classical ones, are ubiquitously expressed and abundant in the CNS. Findings through different experimental approaches, predominantly pharmacological calpain inhibitors, proposed the necessity of the proteases for the modulation of various biological events particularly in the CNS, or a functional link between calpain and neurodegeneration. Significant functions associated with calpain activity are neuronal proliferation/differentiation, signal transduction, apoptosis, and synaptic plasticity; or neuronal death in Alzheimer’s disease, Huntington’s disease, Parkinson’s disease, and ischemic stroke. However, due to limited insights of the approaches taken, such as non-specificity of the inhibitors, the exact roles of calpains in the CNS and the key mechanisms underlying them remain controversial. Calpain-1/calpain-2 germline knock-out are embryonic lethal at a very early stage hindering the use of these lines as mouse models for CNS studies. Accordingly, this thesis research introduced a unique brain-specific calpain-1/calpain-2 knock-out and explored the role of the proteases in brain development/function and in neuronal death. The first set of analyses examined how the elimination of calpain-1/calpain-2 activities in mouse brain impacts CNS development in general and synaptic plasticity in CA1 neurons of hippocampus. CNS-specific elimination of CAPNS1, the common small subunit, abolished calpain-1/calpain-2 activities in mouse brain. In contrast to Calpain-1/calpain-2 germ line knock-outs, the brain-specific knock-outs are viable and the general development of mouse brain is normal. However, morphology of dendrites in pyramidal neurons of the hippocampal CA1 region showed significantly decreased dendritic branching complexity and spine density. Consistent with dendrite morphological abnormalities, electrophysiological analyses revealed a significant decrease in field excitatory postsynaptic potentials, long term potentiation, and learning and memory in the hippocampal CA1 neurons of the mutants. In the second part of this research we investigated the direct role of the calpains in neuronal death and their potential downstream targets in in vitro models of PD and ischemic stroke. Our findings indicated that ablation of calpains activity improves survival of different types of neurons against mitochondrial toxin 1-methyl-4-phenylpyridinium (MPP+), glutamate, and hypoxia. Importantly, we demonstrated an increase in p35-cleavage to p25, a cyclin dependent kinase 5 (Cdk5) activator, and that restoration of p25 significantly suppresses the neuronal survival associated with calpain deficiency. Taken together, this work unequivocally establishes two central roles of calpain-1/calpain-2 in CNS function in plasticity and neuronal death.

Les événements vécus peuvent laisser une trace durable au niveau des réseaux cérébraux. Ces réseaux sont constitués de neurones connectés par des synapses, dont l'efficacité de transmission est régulée sur le plan fonctionnel et structural. Les drogues d'abus détournent les circuits neuronaux impliqués dans l'apprentissage régulé par la récompense, induisant une plasticité des neurones striataux de projection (SPN) du noyau Accumbens (NAc), notamment via l'activation de la voie de signalisation Extracellular Regulated Kinase (ERK) et l'augmentation de la densité en épines dendritiques -qui sont les protrusions portant l'élément post-synaptique glutamatergique-. L'objectif de ma thèse était d'étudier l'impact de l'exposition répétée ou unique à la cocaïne sur le mode formation des synapses des SPN du NAc et d'élucider les rôles précis de la voie ERK dans ce phénomène. J'ai pu montrer qu'une ou plusieurs injections de cocaïne chez la souris induisaient la formation de synapses glutamatergiques persistantes au sein des SPN in vivo. Par des expériences d'imagerie en temps-réel sur tranches striatales, j'ai dissocié les phases de pousse et de stabilisation de nouvelles épines dendritiques. J'ai pu mettre en évidence que la voie ERK joue un rôle prépondérant dans ces deux phases via des processus moléculaires distincts. Ainsi, la phase de pousse des épines est directement régulée par ERK, tandis que le maintien est régulé par MNK-1, une kinase cytoplasmique en aval de ERK, et par la synthèse protéique. Ce travail apporte des données nouvelles sur le mode de formation de ces synapses et les mécanismes moléculaires associés
Brief life occurrences can leave durable changes at the level of neuronal networks. These networks consist of neurons connected by synapses, which transmission efficacy is regulated at the functional and structural levels. Drugs of abuse highjack neuronal circuits involved in reward-driven learning by activating the Extracellular Regulated Kinase (ERK) pathway and induce an increase in the dendritic spines density –protrusions which host the glutamatergic pre-synaptic element- of SPN. The goal of my thesis work was to study the consequences of acute and chronic cocaine exposures on the mode of synapse formation in SPN from the NAc and to decipher the precise roles of ERK pathway in this phenomenon. I demonstrated that acute and chronic cocaine treatments induced the formation of persisting glutamatergic synapses in SPN in vivo. Time-lapse imaging using two-photon microscopy in acute striatal slices allowed me to dissociate the phases of growth and stabilization of the new dendritic spines. I could indeed demonstrate a key role for ERK in those two phases, although through distinct molecular mechanisms. Firstly, the growth phase is dependent on ERK. Secondly, the stabilization of newly grown spines is controlled by MNK-1, a cytosolic kinase downstream ERK, and by protein synthesis. This work brings new results on the mode of synapse formation as well as on the associated molecular mechanisms

2010/2011
The long-term potentiation (LTP) represents a widely studied form of synaptic plasticity related to learning and memory processes, which involves a long-lasting strengthening of synaptic connections through changes of their transmission and cytoarchitecture. The induction of LTP is classically achieved by tetanic stimulation of presynaptic components but it is also possible to in- duce chemically a long-term potentiation of the synaptic efficacy, thus enhancing a larger number of synapses compared to electrical stimulation and facilitating the biochemical and morphological study. The first part of this thesis is a methodological study of glycine and tetraethylammonium (TEA) induced chemical LTP (cLTP) in cultured hippocampal cells. Brief glycine (in Mg2+-free) application activate NMDA receptors, whereas TEA blocks of K+ channels inducing a depolariza- tion responsible for Ca2+ influx. Both drugs were briefly superfused and mEPSCs were monitored for all the duration of the experiments (≃60 min). This was considered as a necessary step to detect later the role of the Brain Derived Neurophic Factor (BDNF) in cLTP. Healthy hippocampal cells were dissected from rats of postnatal day 1-2. After a period of 10-12 days in vitro the cells reached optimal density, a typical mature pyramidal neuron morphology, and an extended dendritic arborization which facilitates synaptic contacts. At this stage patch-clamp technique in the whole-cell configuration was used to study the electrophysiological properties of pyramidal hippocampal neurons, able to produce spontaneous electrical activity. cLTP was tested recording miniature excitatory postsynaptic currents (mEPSCs) in voltage-clamp mode focusing on changes in their amplitude and frequency. A significant decrease in mEPSCs inter-event intervals was observed after glycine and TEA application, without significant changes in aptitudes. Therefore 20 min after glycine application an increase (≃ 61.6 %) in mEPSCs frequency was observed. A similar result was obtained also after TEA application (≃ 66 %). Following cLTP we observed also morphological changes such as an increase in density and a remodeling of different classes of dendritic spines. The role of BDNF in this cLTP model was assessed testing by ELISA assay the total BDNF expres- sion on cell lysate and by blocking Tropomyosin Receptor Kinase (Trk) with K252A. A significant increase in BDNF levels (≃ 120 %) was observed 50 min after cLTP induction. A switch from cLTP into cLTD was observed blocking Trk receptors. Moreover, confocal images collected before and after chemical potentiation in the presence of K252A showed a significant reduction (≃10%) in the average spine density both at the proximal and distal level. A significant reduction of the p-TrkB/TrkB ratio, after both gly- and TEA-LTP, was observed in distal dendrites compared to the soma. This therefore suggests a translocation of the activated receptor from periphery to the soma.
XXIV Ciclo
1983

Une des principales caractéristiques de la maladie d'Alzheimer (MA) est l'accumulation intracérébrale du peptide neurotoxique Amyloïde β (Aβ) sous forme oligomérique et sous forme agrégée en plaques amyloïdes. Ce peptide est le produit du clivage de l'Amyloid Precursor Protein (APP) selon la voie amyloïdogène, voie pathologique suractivée dans la MA. La majorité des recherches, au cours des 25 dernières années, se sont concentrées sur les conséquences pathologiques de cette dérégulation, mettant au second plan la compréhension des fonctions physiologiques de l’APP. Cependant, de nombreuses études montrent que ses fonctions physiologiques pourraient être médiées par ses formes solubles (APPs). Dans la voie de clivage physiologique, la voie non-amyloïdogène, l’APP est clivé par l’α secrétase pour libérer l’APPsα, peptide disposant de propriétés neuroprotectrices et synaptotrophiques, essentielles au bon fonctionnement cérébral. Dans le contexte de la MA, la suractivation de la voie amyloïdogène va aboutir à la production de l’APPsβ au détriment de celle d’APPsα. Les conséquences fonctionnelles associées à la maladie d’Alzheimer pourraient ainsi être dues à la diminution de la production d’APPsα associée à une augmentation de la production d’APPsβ. Mon projet de thèse porta sur les conséquences mnésiques et fonctionnelles de la surexpression de ces deux formes et à leur potentiel thérapeutique dans la maladie d’Alzheimer. Nous avons tout d’abord surexprimé l’APPsα dans les neurones de l’hippocampe de souris transgéniques APP/PS1ΔE9, modèle de la MA, qui présentent des déficits cognitifs et synaptiques. L’expression continue d’APPsα, à l’aide de vecteurs AAV, permet la restauration des performances mnésiques des souris, de la potentialisation à long terme (LTP) ainsi que du nombre d’épines dendritiques dans l’hippocampe. Ce sauvetage phénotypique s’accompagne de la diminution conjointe des niveaux d’Aβ et des plaques amyloïdes. Ceci serait en partie la conséquence de l’activation de la microglie, type cellulaire ayant la capacité d’internaliser et de dégrader l’Aβ. Mon second axe de recherche a consisté à étudier l’APPsβ dont l’implication dans la MA reste méconnue. Sa surexpression dans le modèle murin APP/PS1ΔE9 n’induit pas de restauration de la LTP ni de la mémoire spatiale. Néanmoins, l’injection d’APPsβ aboutit à la diminution de la concentration en Aβ solubles sans cependant réduire le nombre de plaques amyloïdes. Ce défaut pourrait-être la conséquence de l’absence d’activation microgliale. En résumé, mon travail de thèse montre que, contrairement à l’APPsβ, la surexpression d’APPsα pourrait contrecarrer l’évolution inéluctable de la maladie et en particulier en réduisant l’atteinte synaptique et mnésique caractéristique de la MA. Ces résultats renforcent une nouvelle voie d’action pour lutter contre la progression de la MA. L’utilisation de l’APPsα en tant qu’agent thérapeutique pourrait ainsi s’avérer être un élément important dans l’arsenal clinique de ces prochaines années
One of the main characteristic of Alzheimer’s Disease (AD) is the intracerebral accumulation of the neurotoxic Amyloid β peptide (Aβ) either as oligomeric or aggregated forms known as the amyloid plaques. This peptide is produced via the Amyloid Precursor Protein (APP) processing following the amyloidogenic pathway, pathological pathway overactivated in AD. Most of the research performed during the last 25 years focused on pathogenic consequences of this dysregulation, deprioritizing the understanding of the APP physiological functions. Nonetheless, numerous studies show that these physiological functions might be mediated via APP soluble forms (APPs). In the physiological APP processing pathway, the non-amyloidogenic pathway, APP is cleaved by the α secretase, releasing the APPsα which display neuroprotective and synaptotrophic properties, essential for brain normal functions. In the context of AD, the amyloidogenic pathway overactivation leads to APPsβ overproduction at the expense of APPsα. Therefore, AD harmful consequences could be due to the decrease of APPsα concentration associated with an increase of APPsβ. My thesis project aimed to characterize mnemonic and functional properties following the overexpression of these two soluble forms of APP and their therapeutic potential in AD. We firstly overexpressed APPsα in hippocampal neurons of APP/PS1ΔE9 mice, animal model of AD, which display cognitive and synaptic deficits. The continual expression of APPsα, mediated via AAV viruses, enabled restoration of spatial memory, long-term potentiation and dendritic spines density in the hippocampus. This phenotypic rescue was accompanied with the decrease of both Aβ levels and amyloid plaques. This might be due to the activation of microglia, cell type able to internalize and degrade Aβ. In a second hand, I studied the involvement of APPsβ in AD, which remains poorly known. Its overexpression in APP/PS1ΔE9 did not induce neither LTP nor spatial memory restoration. However, APPsβ injection lead to the decrease of Aβ levels without reducing amyloid plaques. This default might be due to the lack of microglial activation. In conclusion, my thesis work show that, unlike APPsβ, APPsα overexpression might overcome the AD inevitable evolution by reducing synaptic and memory alterations, typical of AD. These results reinforce a new way of treatment to cope with AD progression. The use of APPsα as therapeutic agent might be an important tool for future AD therapies

La maladie d’Alzheimer est caractérisée par un déclin progressif des capacités cognitives. Les Aßo induisent des dysfonctionnements de la transmission via une altération des récepteurs au glutamate et une perte de synapses.Nos récents résultats démontrent que le VEGF facilite la plasticité synaptique et la mémoire chez des souris via son action sur son récepteur VEGFR2. Nous avons montré que le VEGF stimule l’insertion synaptique des récepteurs glutamatergiques et la formation de synapses, suggérant ainsi un rôle dans la modulation des altérations synaptiques observées dans la maladie d’Alzheimer.Notre objectif est d’étudier le rôle du VEGF, spécifiquement dans la maladie d’Alzheimer. Tout d’abord, nous avons examiné son expression en relation avec les plaques séniles chez des patients et dans un modèle de la maladie d’Alzheimer. Nos résultats ont démontré une colocalisation entre le VEGF et ces plaques.Afin d’examiner plus finement l’interaction Aß-VEGF, nous avons analysé la liaison entre les Aßo et le VEGF en test ELISA et puces à peptides. Nous avons ainsi démontré un potentiel blocage de l’interaction entre le VEGF et son récepteur, menant à des défauts de son activation.Enfin, nous avons examiné si le VEGF prévient les altérations synaptiques par des approches électrophysiologiques, biochimiques et immunocytochimiques. Nos résultats démontrent que lors d’un traitement aux Aßo, le VEGF restaure la LTP, l’expression des récepteurs au glutamate et limite la perte synaptique.Dans l’ensemble, nos résultats suggèrent que l’interaction Aß-VEGF altère la voie du VEGF chez les patients. De plus, le VEGF réduit la toxicité induite par les Aßo sur les synapses
Alzheimer disease (AD) is characterized by a progressive decline in cognitive abilities. Amyloid-ß oligomers (Aßo) trigger synapse dysfunction through defects in glutamate receptor function and subsequent dendritic spine loss. These synaptic impairments compromise memory and contribute to cognitive deficits.Our recent findings revealed that VEGF facilitates synaptic plasticity and memory in mice through its VEGFR2 receptor in neurons. We showed that VEGF promotes glutamate receptor synaptic insertion and stimulates dendritic spine formation, suggesting it may be a key candidate for alleviating synapse damage in AD.Our objective is to study the role of VEGF in synapse protection in AD models and unravel the underlying mechanisms.First, we examined the VEGF expression pattern in postmortem brain tissue from AD patients and APPPS1 model of AD. Our results showed a partial colocalization between VEGF and Aß plaques in AD patients and APPPS1 brains.To further investigate the Aß-VEGF interaction, we used Elisa assay and peptide arrays and demonstrated that Aßo binds several domains of VEGF, impedding VEGFR2 activation.Finally, we examined whether VEGF can prevent synapse damage induced by Aßo using electrophysiological, biochemical and 3D modelling approaches. Our results demonstrated that VEGF treatments can restore LTP in Aßo-treated hippocampal slices, glutamate receptor content at synapses and increase dendritic spine density.All together, our results suggest that Aß-VEGF interaction may alter VEGF pathway in AD and that VEGF reduces Aßo-induced toxicity at synapses by modulating glutamate receptor expression and promoting spine formation and/or stabilization

Les protéines d’adhésions homophiles N-cadhérine jouent un rôle majeur dans le développement du cerveau, notamment en agissant sur la croissance et la plasticité synaptique. Au cours de ma thèse, j’ai étudié le rôle de la N-cadhérine dans ces deux processus en utilisant des neurones issus de cultures primaires déposés sur des substrats micropatternés. Ces substrats sont recouverts de N-cadhérine purifiée afin d’induire des adhésions N-cadhérines sélectives au niveau de micro-motifs régulièrement espacés. Mes deux premières études sont basées sur le modèle d’embrayage moléculaire, décrivant le processus par lequel la motilité du cytosquelette d’actine se couple aux adhésions au niveau de la membrane cellulaire afin de générer des forces de traction aux zones de contact avec le substrat, permettant ainsi l’avancée cellulaire (Giannone et al., 2009). Plusieurs études ont mis en avant l’existence d’un tel modèle (Mitchison et Kirschner, 1988 ; Suter et Forscher, 1998), cependant le mécanisme exact permettant d’expliquer ce couplage mécanique de l’actine aux protéines d’adhésions reste mal connu. Via des techniques de pinces optiques, des travaux précédemment menés dans l’équipe ont prouvé l’existence d’un couplage entre le flux d’actine et les adhésions N-cadhérine permettant la migration du cône de croissance (Bard et al., 2008). Cette technique n’a cependant pas permis la visualisation directe de l’engagement d’un tel mécanisme. Nous avons donc couplé l’utilisation des substrats micro-patternés à la microscopie haute résolution sptPALM/TIRF afin de visualiser directement la dynamique des protéines impliquées dans l’embrayage moléculaire. Dans le premier article, j’ai montré pour la première fois l’existence d’interactions transitoires entre le flux d’actine et les adhésions N-cadhérines au niveau du cône de croissance, reflétant un embrayage glissant à l’échelle de la molécule unique (Garcia et al., en préparation). Dans le second article, en travaillant sur des neurones plus matures, nous avons pu montrer l’engagement d’un embrayage moléculaire trans-synaptique entre adhésions N-cadhérines et flux d’actine permettant la stabilisation du filopode dendritique et ainsi sa transition en épine mature (Chazeau/Garcia et al., en préparation). J’ai également participé à une troisième étude dans laquelle j’ai observé l’effet des substrats micropatternés recouverts de N-cadhérine, sur la synaptogenèse. J’ai ainsi pu prouver que la N-cadhérine déposée sur les micro-motifs, stimule la croissance dendritique et axonale et joue un rôle prépondérant dans la maturation morphologique des neurones. Cependant, la N-cadhérine est incapable d’induire la formation de synapses contrairement aux protéines d’adhésion neurexine/neuroligine ou SynCam (Czöndör et al., 2013)
The homophilic adhesion molecule N-cadherin plays major roles in brain development, notably affecting axon outgrowth and synaptic plasticity. During my PhD work, I addressed the role of N-cadherin in these two processes, using primary neurons cultured on micro-patterned substrates. These substrates are coated with purified N-cadherin to trigger selective N-cadherin adhesions in a spatially controled manner. My two first studies are based on the “molecular clutch” paradigm, by which the actin motile machinery is coupled to adhesion at the cell membrane to generate forces on the substrate and allow cells to move forward (Giannone et al., 2009). Many publications have provided evidence for such a mechanism (Mitchison et Kirschner, 1988 ; Suter et Forscher, 1998), but the exact mechanisms underlying the molecular coupling between the actin retrograde flow and adhesion proteins remain elusive. The team previously inferred, using optical tweezers, that a molecular clutch between the actin flow and N-cadherin adhesions drives growth cone migration (Bard et al., 2008), but could not achieve a direct visualization of the engagement process with this technique. Here, we combined the use of micropattern substrates with high resolution microscopy sptPALM/TIRF to visualize directly the dynamics of the main proteins involved in the molecular clutch. In my first paper, I reveal for the first time transient interactions between the actin flow and N-cadherin adhesions in growth cones, reflecting a slipping clutch process at the individual molecular level (Garcia et al., in preparation). In a second study, working with more mature neurons, we revealed that engagement of a molecular clutch between trans-synaptic N-cadherin adhesions and the actin flow underlies the stabilization of dendritic filopodia into mature spines (Chazeau/Garcia et al., in preparation). I also participated to a third study, where I observed the effect of N-cadherin coated substrates on synaptogenesis. I showed that, although N-cadherin on micro-patterned substrates stimulated axonal and dendritic elongation and played a major role in morphological maturation, it was not able to induce synapse formation like neurexin/neuroligin or SynCAM adhesions (Czöndör et al., 2013)

Chronic exposure to high dose of glucocorticoids (GC) is a key risk factor for the development of Alzheimer’s Disease (AD), as recently described by clinical and genetic studies. Furthermore, hyper-activation of glucocorticoids receptors (GR) induces, in brain, alterations comparable to those produced by AD. In a transgenic mice model for AD, GC induces the increasing production of Aβ40, Aβ42 and Tau total, the most important and typical hallmarks of this dementia. Two of the key roles of GC in brain are the regulation of dendritic spine turnover and the inflammation state, two phenomena strongly altered in AD. The aim of my project was to investigate the correlation between glucocorticoids and Alzheimer’s Disease. In particular, I focused my attention on how dendritic spine plasticity and microglia activation in CA1 region of hippocampus of 3xTg-AD mice are modified by modulation of glucocorticoid receptor with agonist and antagonist. Using an innovative combined Golgi Cox and immunofluorescence technique, we found that 5 days of treatment with 8mg/kg of dexamethasone, an agonist of GR, was able to vigorously reduce dendritic spine density in CA1 region of 3xTg-AD mice, both at 6 and 10 months of age and induced proliferation and activation of microglia. The activation of microglia could contribute to spine damage. On the contrary, the treatment with 20mg/kg of mifepristone, an antagonist of GR, strongly enhanced dendritic spine density in CA1 region, at both ages, results confirmed also by electron microscopy analyses. Moreover, the antagonist was able to improve the 3xTg-AD mice performance in Y-maze task at 10 months of ages and the proliferation of microglia, but it was not able to reduce the activation of microglia. I speculated that these apparently ambiguous results could be explained by the well-known biphasic behavior of GC in brain, as already observed for spine plasticity and memory. Additionally, in vitro experiments, using immunofluorescence and immunoblotting techniques, revealed that dexamethasone, clearly, induced activation of microglia in vitro, a result never described before. On the contrary, mifepristone promoted both activation and inhibition of microglia inflammatory state, suggesting the existence of a biphasic behavior of GC also on inflammation regulation. In conclusion, my data demonstrates that stress induced by dexamethasone exacerbate AD and promote a more rapid progression of the pathology through a premature reduction of dendritic spine density and enhancement of inflammation. Consequently, the use of antagonist, like mifepristone, could represent a promising therapeutic strategy to delay the onset and slow down the progression of AD. Taking in account the biphasic behavior of GC, the right dose and time of treatment need to be found, in order to obtain the best improvement: the increasing of spine turnover together with the reduction of inflammation and improvement of behavioral performances.

RESUMO: Os circuitos dos núcleos da base estão crucialmente envolvidos na aquisição, aprendizagem e consolidação de habilidades motoras. O corpo estriado é a principal região de input dos núcleos da base e é composto principalmente por neurónios médios do estriado (medium spiny neurons, MSNs). As espículas dendríticas dos MSNs representam um importante local de plasticidade sináptica nos núcleos da base. Embora tenha sido demonstrado que a plasticidade sináptica funcional do MSN ocorre num contexto de aprendizagem motora, não se sabe se esta é acompanhada por plasticidade estrutural, ou seja, por mudanças no número, tamanho e/ou padrão espacial das espículas dendríticas. Neste estudo, o nosso objetivo consistiu em caracterizar o desempenho motor e a atividade do corpo estriado na aprendizagem de habilidades motoras, com o objetivo final de estudar a plasticidade estrutural na aprendizagem motora. Treinamos ratinhos numa tarefa operante individualizada, em que uma recompensa é obtida após pressionar uma alavanca quatro vezes. À medida que o treino avança, a posição e retração da alavanca impõem lateralidade, permitindo o estudo das sequências de movimento realizadas por um único membro anterior. Durante o treino, os ratinhos melhoraram o seu desempenho e aprenderam a realizar a tarefa com apenas uma das patas dianteiras, aumentando o número total de movimentos de pressão sobre a alavanca por minuto e organizando o comportamento em sequências de movimentos. Através da deteção da trajetória da pata baseada em vídeo, examinamos ainda mais a cinemática do movimento durante a aprendizagem da tarefa e descobrimos que a variabilidade da trajetória da pata durante a pressão sobre a alavanca não diminuiu significativamente durante o treino. Para identificar as células que estiveram ativas na última sessão da tarefa motora e, portanto, com maior probabilidade de terem sofrido plasticidade sináptica, realizamos uma imunohistoquímica contra c-Fos, um gene de expressão rápida frequentemente usado como marcador de plasticidade neuronal. Usando um pipeline de deteção de células no cérebro inteiro, conseguimos detetar células que expressam c-Fos e mapeá-las no atlas de uma maneira imparcial, usando fatias de cérebro fixadas de ratinhos sacrificados 1h após a última sessão de treino. Os nossos resultados preliminares sugerem que animais treinados na tarefa apresentam mudanças ligeiras na expressão de c-Fos no hemisfério contralateral à pata treinada. Estas mudanças parecem ser consistentes em regiões envolvidas na aprendizagem e desempenho motor nos animais treinados, e incluem o corpo estriado, a camada 5 do córtex motor primário e a região do membro superior do córtex somatossensorial primário. Finalmente, procuramos estabelecer as ferramentas e técnicas necessárias para estudar a plasticidade estrutural em fatias fixadas do cérebro. Otimizamos uma nova abordagem viral para obter uma marcação pouco densa de MSNs, permitindo a visualização e reconstrução de toda a árvore dendrítica de MSNs individuais, incluindo espículas dendríticas. Usando microscopia confocal de alta resolução, em conjunto com softwares de deconvolução e análise de espículas dendríticas, adquirirmos imagens e reconstruimos as espículas dendríticas do MSN e caracterizamos a morfologia das mesmas. Descobrimos que os D1-MSNs têm uma preponderância de espículas do tipo mushroom, seguidas por espículas do tipo thin e stubby, com os filopodia constituindo apenas uma pequena fração do número total das protuberâncias dendríticas. O nosso trabalho estabeleceu as técnicas e metodologias básicas que permitirão estudos futuros sobre a densidade, volume e distribuição das espículas dendríticas em neurónios que recentemente sofreram plasticidade após aprendizagem motora. Essas experiências, por sua vez, irão avançar a nossa compreensão de como a computação de inputs pelos MSNs muda durante a aprendizagem motora, com implicações importantes para o estudo e tratamento de doenças motoras.
ABSTRACT The basal ganglia circuits are critically involved in the acquisition, learning and consolidation of motor skills. The striatum is the major input region of the basal ganglia and is mainly composed of medium spiny neurons (MSNs). MSN dendritic spines represent a major site of synaptic plasticity in the basal ganglia. While MSN functional synaptic plasticity has been shown to occur in the context of motor learning, it remains unknown whether it is accompanied by structural plasticity, i.e., by changes in the number, size and/or spatial pattern of dendritic spines. In this study, we aimed to characterize motor performance and striatal activity upon motor skill learning, with the ultimate goal of studying structural plasticity upon motor learning. We trained mice in a self-paced operant task where a reward is obtained after pressing a lever four times. As training progresses, the position and retractability of the lever impose laterality, allowing the study of movement sequences performed by a single forelimb. During training, mice improved their performance and learned to perform the task with only one forepaw, increasing the total number of lever presses per minute and organizing their behavior in sequences of lever presses. Using video-based paw trajectory detection, we further dissected movement kinematics during task learning, and found that paw trajectory variability during lever press did not significantly decrease throughout training. To identify cells that were active in the last session of the motor task, and therefore more likely to have undergone synaptic plasticity, we performed immunostaining against c-Fos, an immediate early gene commonly used as a neuronal plasticity marker. Using a whole-brain cell detection pipeline, we were able to achieve unbiased cell detection and atlas mapping of c-Fos expressing cells, using fixed brain slices of mice sacrificed 1h after the last training session. Our preliminary results suggest that task-trained animals have subtle changes in c-Fos expression in the contralateral hemisphere to the trained paw. These changes appeared to be consistent across regions involved in motor learning and performance in task-trained animals that include the striatum, primary motor cortex layer 5, and upper limb region of the primary somatosensory cortex. Finally, we sought to establish the tools and techniques needed to study structural plasticity in fixed brain slices. We optimized a novel viral approach to achieve sparse labelling of MSNs, allowing the visualization and reconstruction of the whole dendritic arbor of single MSNs, including dendritic spines. Using high-resolution confocal microscopy, together with deconvolution and spine analysis software, we were able to image and reconstruct MSN dendritic spines and characterize spine morphology. We found that D1-MSNs have a preponderance of mushroom spines, followed by thin and stubby spines, with filopodia making up just a small fraction of overall dendritic protrusions. Our work has established the basic techniques and methodologies that will allow future studies on dendritic spine density, volume and distribution in neurons that recently underwent plasticity upon motor learning. Those experiments will, in turn, advance our understanding of how MSN input computation changes during motor learning, with important implications for the study and treatment of movement disorders.

Synaptic plasticity is the leading candidate for the cellular/molecular basis of learning and memory. One of the key molecules involved in synaptic plasticity is Calcium/calmodulin-dependent Kinase II (CaMKII). Synaptic plasticity can be expressed at a single dendritic spine independent of its neighboring dendritic spines. Here, we investigated how long the activity of CaMKII lasts during synaptic plasticity of single dendritic spines. We found that CaMKII activity lasted ~2 minutes during synaptic plasticity and was restricted to the dendritic spines undergoing synaptic plasticity while nearby dendritic spines did not show any change in the level of CaMKII activity. Our experimental data argue against the persistent activation of CaMKII in dendritic spines undergoing synaptic plasticity and suggest that the activity of CaMKII is a spine-specific biochemical signal necessary for synapse-specificity of synaptic plasticity. We provide a biophysical explanation of how spine-specific CaMKII activation can be achieved during synaptic plasticity. We also found that CaMKII is activated by highly localized calcium influx in the proximity of Voltage-dependent Calcium Channels (VDCCs) and a different set of VDCCs and their respective Ca2+ nanodomains are responsible for the differential activation of CaMKII between dendritic spines and dendritic shafts.

Dissertation

This thesis examines the rapid, non-genomic effects of estrogens on neural plasticity and learning. Estrogens are classically known to affect gene transcription events, however they have more recently been found to also rapidly activate second messenger systems within 1hr of administration. Therefore, we first examined the rapid effects of 17β-estradiol, and an estrogen receptor (ER) α and ERβ agonist on three different learning paradigms: object placement, object recognition, and social recognition. We found that both systemic injections and intrahippocampal delivery of 17β-estradiol and the ERα agonist improved performance on all 3 learning paradigms within 40min of hormone administration. However, the ERβ agonist administered systemically or intrahippocampally, improved performance only on the object placement learning paradigm, while having no effect on object recognition, and impairing social recognition at high doses. To elucidate how estrogens might rapidly affect learning, we examined how estrogens rapidly affect the neural plasticity of CA1 hippocampal neurons. We found that 17β-estradiol and the ERα agonist increased dendritic spine density in CA1 hippocampal neurons within 40min of administration, suggesting that estrogens rapidly increase the density of synapses within this brain region. Conversely, the ERβ agonist did not affect spine density, or decreased spine density. In addition, by using whole-cell patch clamp recordings of CA1 pyramidal neurons, we were able to determine that 17β-estradiol and the ERα agonist rapidly reduced AMPA receptor (but not NMDA receptor) mediated membrane depolarizations after 15min of hormone application. Similar to above, the ERβ agonist had no effect on AMPA or NMDA receptor mediated membrane depolarizations. These data suggest that estrogens rapidly promote the development of immature synapses (which contain low levels of synaptic AMPA receptors) within the CA1 hippocampus. Immature spines provide synaptic sites at which new memories can be stored and are thought of as “learning spines” (Kasai et al, 2003). Therefore, estrogens (through ERα) may rapidly induce the formation of hippocampal immature spines to promote learning.
Funded by NSERC

La mémoire et l’apprentissage sont des phénomènes complexes qui demeurent encore incertains quant aux origines cellulaire et moléculaire. Il est maintenant connu que des changements au niveau des synapses, comme la plasticité synaptique, pourraient déterminer la base cellulaire de la formation de la mémoire. Alors que la potentialisation à long-terme (LTP) représente un renforcement de l’efficacité de transmission synaptique, la dépression à long-terme (LTD) constitue une diminution de l’efficacité des connexions synaptiques. Des études ont mis à jour certains mécanismes qui participent à ce phénomène de plasticité synaptique, notamment, les mécanismes d’induction et d’expression, ainsi que les changements morphologiques des épines dendritiques. La grande majorité des synapses excitatrices glutamatergiques se situe au niveau des épines dendritiques et la présence de la machinerie traductionnelle près de ces protubérances suggère fortement l’existence d’une traduction locale d’ARNm. Ces ARNm seraient d’ailleurs acheminés dans les dendrites par des protéines pouvant lier les ARNm et assurer leur transport jusqu’aux synapses activées. Le rôle des protéines Staufen (Stau1 et Stau2) dans le transport, la localisation et dans la régulation de la traduction de certains ARNm est bien établi. Toutefois, leur rôle précis dans la plasticité synaptique demeure encore inconnu. Ainsi, cette thèse de doctorat évalue l’importance des protéines Staufen pour le transport et la régulation d’ARNm dans la plasticité synaptique. Nous avons identifié des fonctions spécifiques à chaque isoforme; Stau1 et Stau2 étant respectivement impliquées dans la late-LTP et la LTD dépendante des récepteurs mGluR. Cette spécificité s’applique également au rôle que chaque isoforme joue dans la morphogenèse des épines dendritiques, puisque Stau1 semble nécessaire au maintien des épines dendritiques matures, alors que Stau2 serait davantage impliquée dans le développement des épines. D’autre part, nos travaux ont permis de déterminer que la morphogenèse des épines dendritiques dépendante de Stau1 était régulée par une plasticité synaptique endogène dépendante des récepteurs NMDA. Finalement, nous avons précisé les mécanismes de régulation de l’ARNm de la Map1b par Stau2 et démontré l’importance de Stau2 pour la production et l’assemblage des granules contenant les transcrits de la Map1b nécessaires pour la LTD dépendante des mGluR. Les travaux de cette thèse démontrent les rôles spécifiques des protéines Stau1 et Stau2 dans la régulation de la plasticité synaptique par les protéines Stau1 et Stau2. Nos travaux ont permis d’approfondir les connaissances actuelles sur les mécanismes de régulation des ARNm par les protéines Staufen dans la plasticité synaptique. MOTS-CLÉS EN FRANÇAIS: Staufen, hippocampe, plasticité synaptique, granules d’ARN, traduction, épines dendritiques.
Learning and memory are complex processes that are not completly understood at the cellular and molecular levels. It is however accepted that persistent modifications of synaptic connections, like synaptic plasticity, could be responsible for the encoding of new memories. Whereas long-term potentiation (LTP) is classically defined as a persistent and stable enhancement of synaptic connections, long-term depression (LTD) is a reduction in the efficacy of neuronal synapses. Numerous studies have identified some of the mechanisms of this phenomenon, in particular, the induction and expression mechanisms, as well as the changes in dendritic spine morphology. The most abundant type of synapse in the hippocampus is the excitatory glutamatergic synapse made on dendritic spines; the presence of the translational machinery in dendrites near spines strongly supports the concept of local mRNA translation. Moreover, those mRNA are transported in dendrites to activated synapses by RNA binding-proteins (RBP). Staufen proteins (Stau1 and Stau2) function in transport, localization and translational regulation of mRNA are now established. However, their precise roles in synaptic plasticity are still unknown. Thus, this Ph.D. thesis evaluates the importance of Staufen proteins in mRNA transport and regulation in synaptic plasticity. We have identified specific functions for each isoform; while Stau1 is implicated in late-LTP, Stau2 is required for mGluR-LTD. This specificity is also relevant for dendritic spine morphogenesis since Stau1 is involved in mature dendritic spine maintenance while Stau2 participates in dendritic spine morphogenesis at a developmental stage. Moreover, our studies have indicated that Stau1 involvement in spine morphogenesis is dependent on ongoing NMDA receptor-mediated plasticity. Finally, our results suggest that Stau2 is implicated in a particular form of synaptic plasticity through transport and regulation of specific mRNA granules required for mGluR-LTD such as Map1b. Our work uncovers specific roles of Stau1 and Stau2 in regulation of synaptic plasticity. These studies help to better understand mechanisms involving mRNA regulation by Staufen in long-term synaptic plasticity and memory. ENGLISH KEY WORDS: Staufen, hippocampus, synaptic plasticity, RNA granules, translation, dendritic spines

Multifunctional calcium/calmodulin dependent protein kinases (CaMKs) are key regulators of spine structural plasticity and long-term potentiation (LTP) in neurons. CaMKs have promiscuous and overlapping substrate recognition motifs, and are distinguished in their regulatory role based on differences in the spatiotemporal dynamics of activity. While the function and activity of CaMKII in synaptic plasticity has been extensively studied, that of CaMKI, another major class of CaMK required for LTP, still remain elusive.

Here, we develop a Förster’s Resonance Energy Transfer (FRET) based sensor to measure the spatiotemporal activity dynamics of CaMK1. We monitored CaMKI activity using 2-photon fluorescence lifetime imaging, while inducing LTP in single dendritic spines of rat (Rattus Norvegicus, strain Sprague Dawley) hippocampal CA1 pyramidal neurons using 2-photon glutamate uncaging. Using RNA-interference and pharmacological means, we also characterize the role of CaMKI during spine structural plasticity.

We found that CaMKI was rapidly and transiently activated with a rise time of ~0.3 s and decay time of ~1 s in response to each uncaging pulse. Activity of CaMKI spread out of the spine. Phosphorylation of CaMKI by CaMKK was required for this spreading and for the initial phase of structural LTP. Combined with previous data showing that CaMKII is restricted to the stimulated spine and required for long-term maintenance of structural LTP, these results suggest that CaMK diversity allows the same incoming signal – calcium – to independently regulate distinct phases of LTP by activating different CaMKs with distinct spatiotemporal dynamics.

Dissertation

Synapses are the sites at which learning is proposed to occur through changes in the strength of neuronal connections. Utilizing 2-photon mediated glutamate uncaging and imaging, the size of a dendritic spine and the amount of current which that synapse conducts has been shown to be linearly correlated and thus allows for structural changes in spine volumes to serve as a proxy for measuring plasticity. In order to e ciently and accurately quantify such structural dynamics, we developed a Matlab-based toolbox, named SpineS, which automatically analyses dendritic spine volume changes more rapidly, and with greater precision, based on a learned library of representative images. Regularly spaced stimulations, such as the high- and low-frequency patterns traditionally used to induce plasticity in the hippocampus, are not the most common forms of activity which occur in the brain.(...)
TÜBiTAK_Grant Nº 113E603

Most stroke survivors recover some degree of lost function after an ischemic event. Recovery however, is negatively affected by comorbid conditions such as diabetes. Successful recovery is dependent on the ability of adjacent surviving cortical tissue and functionally related areas to take over functions lost by the stroke. Recently our lab has shown that diabetes interferes with the remapping of sensory function to peri-infarct areas after photothrombotic stroke. Given this result, it is crucial to understand how diabetes affects the structure of neurons following stroke, particularly at the level of dendritic spines, which receive the vast majority of excitatory synaptic inputs. Type I diabetes was pharmacologically induced in transgenic mice expressing yellow fluorescent protein (YFP) in a subset of cortical neurons 4 weeks prior to receiving unilateral photothrombotic stroke in the forelimb area of the primary somatosensory cortex (FLS1). Spine density measurements were made on the apical and basilar dendrites of layer-5 pyramidal neurons at 1 and 6 weeks after stroke. Our analysis indicated that diabetes was associated with fewer apical and basilar dendritic spines in the peri-infarct region 1 week after stroke. At 6 weeks of recovery, peri-infarct dendritic spine density in both control and diabetic animals returned to baseline levels. These changes were specific to the peri-infarct cortex, as spine density in distant cortical areas such as the forelimb sensorimotor region of the contralateral hemisphere, were not affected by stroke. In order to relate changes in spine density to the recovery of forepaw function, we re-analyzed data from a previous study that employed the forepaw adhesive-tape-removal test (Sweetnam et al 2012). This analysis revealed that diabetes significantly increased the latency of tape removal from the impaired forepaw (when normalized to the unaffected paw) at 1 but not 6 weeks of recovery. Collectively, these findings indicate that diabetes exacerbates forepaw impairments and basilar spine loss initially after stroke, but does not affect the ability of the brain to replace lost spines over weeks of recovery.
Graduate
0317

Membrane trafficking in dendritic spines is critical for regulating the number of channels and spine structure during synaptic plasticity. Here I report two small Rab GTPases, Rab4 and Rab10, oppositely regulate AMPA receptors (AMPARs) trafficking and structural plasticity of dendritic spines. Combining two-photon glutamate uncaging with two-photon fluorescence lifetime imaging microscopy (2pFLIM), I found that Rab4 is transiently activated whereas Rab10 is persistently inactivated in the stimulated spines during structural long-term potentiation (sLTP). Inhibition of Rab4 signaling has no effect on GluA1 endocytosis but inhibits activity-dependent GluA1 exocytosis. Conversely, disruption of Rab10 signaling inhibits GluA1 endocytosis while enhancing activity-dependent GluA1 exocytosis. In summary, these results uncover a new mechanism to establish the specificity and directionality of AMPARs trafficking and sLTP via distinct regulations of Rab4 and Rab10 signaling.

Dissertation

Dans la cellule, chaque ARNm se doit d’être régulé finement au niveau transcriptionnel, bien entendu, mais également au niveau de sa traduction, de sa dégradation ainsi que de sa localisation intracellulaire, et ce, afin de permettre l’expression de chaque produit protéique au moment et à l’endroit précis où son action est requise. Lorsqu’un mécanisme physiologique est mis de l’avant dans la cellule, il arrive souvent que plusieurs ARNm se doivent d’être régulés simultanément. L’un des moyens permettant d’orchestrer un tel processus est de réguler l’action d’une protéine commune associée à chacun de ces ARNm, via un mécanisme post-traductionnel par exemple. Ainsi l’expression d’un groupe précis d’ARNm peut être régulée finement dans le temps et dans l’espace selon les facteurs protéiques auxquels il est associé. Dans l’optique d’étudier certains de ces complexes ribonucléoprotéiques (mRNP), nous nous sommes intéressés aux isoformes et paralogues de Staufen, une protéine à domaine de liaison à l’ARN double-brin (dsRBD) impliquée dans de nombreux aspects de la régulation post-transcriptionnelle, tels la dégradation, la traduction ou encore la localisation d’ARNm. Chez la drosophile, un seul gène Staufen est exprimé alors que chez les mammifères, il existe deux paralogues de la protéine, soit Stau1 et Stau2, tous deux possédant divers isoformes produits suite à l’épissage alternatif de leur gène. Stau1 et Stau2 sont identiques à 50%. Les deux isoformes de Stau2, Stau259 et Stau262 ne diffèrent qu’en leur extrémité N-terminale. En effet, alors que Stau259 arbore un dsRBD1 tronqué, celui de Stau262 est complet. Ces observations introduisent une problématique très intéressante à laquelle nous nous sommes attaqué : ces différentes protéines, quoique très semblables, font-elles partie de complexes ribonucléoprotéiques distincts ayant des fonctions propres à chacun ou, au contraire, vu cette similarité de séquence, travaillent-elles de concert au sein des mêmes complexes ribonucléoprotéiques? Afin d’adresser cette question, nous avons entrepris d’isoler, à partir de cellules HEK293T, les différents complexes de Stau1 et Stau2 par la technique d’immunoprécipitation. Nous avons isolé les ARNm associés à chaque protéine, les avons identifiés grâce aux micropuces d’ADN et avons confirmé nos résultats par RT-PCR. Malgré la présence d’une population commune d’ARNm associée à Stau1 et Stau2, la majorité des transcrits identifiés furent spécifiques à chaque orthologue. Cependant, nous avons remarqué que les diverses populations d’ARNm participaient aux mêmes mécanismes de régulation, ce qui suggère que ces deux protéines possèdent des rôles complémentaires dans la mise en œuvre de divers phénomènes cellulaires. Au contraire, les transcrits associés à Stau259 et Stau262 sont davantage similaires, indiquant que celles-ci auraient des fonctions plutôt semblables. Ces résultats sont très intéressants, car pour la première fois, nous avons identifié des populations d’ARNm associées aux isoformes Stau155, Stau259 et Stau262. De plus, nous les avons analysées en parallèle afin d’en faire ressortir les populations spécifiques à chacune de ces protéines. Ensuite, connaissant l’importance de Stau2 dans le transport dendritique d’ARNm, nous avons cherché à caractériser les complexes ribonucléoprotéiques neuronaux associés à celle-ci. Dans un premier temps et à l’aide de la technique d’immunoprécipitation, nous avons identifié une population d’ARNm neuronaux associés à Stau2. Plus de 1700 ARNm montraient une présence d’au moins huit fois supérieure dans le précipité obtenu avec l’anticorps anti-Stau2 par rapport à celui obtenu avec le sérum pré-immun. Ces ARNm codent pour des protéines impliquées dans des processus de modifications post-traductionnelles, de traduction, de transport intracellulaire et de métabolisme de l’ARN. De façon intéressante, cette population d’ARNm isolée du cerveau de rat est relativement différente de celle caractérisée des cellules humaines HEK293T. Ceci suggère que la spécificité d’association Stau2-ARNm peut diffèrer d’un tissu à un autre. Dans un deuxième temps, nous avons isolé les protéines présentes dans les complexes ribonucléoprotéiques obtenus de cerveaux de rat et les avons identifiées par analyse en spectrométrie de masse. De cette façon, nous avons identifié au sein des particules de Stau2 des protéines liant l’ARN (PABPC1, hnRNPH1, YB1, hsc70), des protéines du cytosquelette (α- et β-tubuline), de même que la protéine peu caractérisée RUFY3. En poussant davantage la caractérisation, nous avons établi que YB1 et PABPC1 étaient associées à Stau2 grâce à la présence de l’ARN, alors que la protéine hsc70, au contraire, interagissait directement avec celle-ci. Enfin, cette dernière association semble être modulable par l’action de l’ATP. Ce résultat offre de nombreuses possibilités quant à la régulation de la fonction de Stau2 et/ou de son mRNP. Entre autres, cette étude suggère un mécanisme de régulation de la traduction au sein de ces particules. Pour faire suite à la caractérisation des mRNP de Stau, nous avons voulu déterminer au niveau neurophysiologique l’importance de ceux-ci. Comme l’étude de Stau2 avait déjà été entreprise préalablement par un autre laboratoire, nous avons décidé de concentrer notre étude sur le rôle de Stau1. Ainsi, nous avons démontré que celle-ci était nécessaire à la mise en place d’une forme de plasticité synaptique à long terme, la forme tardive de potentialisation à long terme ou L-LTP, dépendante de la transcription et de l’activité des récepteurs NMDA. La transmission de base, de même que la faculté de ces épines à faire de la E-LTP, la forme précoce de potentialisation à long terme, et la dépression à long terme ou LTD sont conservées. Ceci indique que les épines conservent la capacité d’être modulées. Ainsi, l’inhibition de la L-LTP, suite à la sous-expression de Stau1, n’est pas simplement due à la perte d’éléments fonctionnels, mais réside plutôt dans l’incapacité de ceux-ci à induire les changements synaptiques spécifiquement nécessaires à la mise en place de la L-LTP. De plus, au niveau synaptique, la sous-expression de Stau1 réduit à la fois l’amplitude et la fréquence des mEPSC. Ces résultats concordent avec l’observation que la sous-expression de Stau1 augmente significativement la proportion d’épines allongées et filopodales, des épines formant des synapses dites silencieuses. Par le fait même, elle diminue le nombre d’épines fonctionnelles, de forme dite normale. Ainsi, nous avons été en mesure de démontrer que l’absence, au niveau neuronal, de la protéine Stau1 induisait un déficit probable dans la localisation et/ou la traduction d’ARNm responsable de la restructuration de l’épine et de facteurs nécessaires à la mise en place de la L-LTP. En conclusion, nous avons participé à lever le voile sur la composition et l’importance des complexes ribonucléoprotéiques de Stau1 et Stau2. Nous avons identifié des populations distinctes et communes d’ARNm associées aux différents isoformes de Stau, à partir des mRNP présents au sein des cellules HEK293. De plus, nous avons réussi à mettre à l’avant plan certaines composantes des mRNP neuronaux de Stau2, dont un partenaire protéique direct, hsc70, partenaire dont l’association est modulable par l’action de l’ATP, ainsi qu’une population neuronale de transcrits d’ARNm. Enfin, nous avons mis en lumière l’importance de Stau1 dans la morphologie des épines dendritiques ainsi que dans le phénomène de la plasticité synaptique.
In the cell, the expression of each mRNA is finely tuned transcriptionally, but also, at the level of translation, degradation and intracellular localisation. These mechanisms of regulation are important in order to control the expression of each translational product at the right time and place. When a physiological phenomenon is activated, the expression of multiple functionally-related mRNAs must be simultaneously regulated. To orchestrate the coordinated expression of all the transcripts that respond to specific cell needs, it is advantageous to regulate the function of a common trans-acting factor that associates with them. Such a mechanism permits to control the fate of a sub-population of mRNAs according to the factors to which they are bound. As a means to learn more about the regulation of mRNAs in ribonucleoprotein complexes (mRNP), we decided to focus our study on the characterisation of Staufen-associated mRNPs. In mammalian cells, two Staufen paralogs, Stau1 and Stau2, are expressed and each gene generates different isoforms through differential splicing. Staufen proteins are double-stranded RNA binding proteins implicated in numerous aspects of the post-transcriptional regulation of mRNAs such as degradation, translation and localisation. Stau1 and Stau2 are similar proteins with an overall percentage identity of around 50%. This percentage increases to near 75% when only the functional double-stranded RNA-binding domains (dsRBD3) are compared. Similarly, Stau2 isoforms, Stau259 and Stau262, are perfectly identical except at the N-terminal extremity where the sequence of Stau262 is extended as compared to that of Stau259. Therefore, their RNA-binding domain 3 are perfectly identical. These observations bring in an interesting problematic. Are those almost identical proteins part of the same mRNP, acting in conjunction or, despite their high similarities, are they part of distinct mRNP participating in specific function? In order to address this question, we decided to immunoprecipitated from HEK293 cells, Stau155, Stau259 and Stau262-associated mRNPs and identify bound mRNAs. Resulting mRNAs isolated from each complex were identified by microarray analysis. There is a predominance of mRNAs involved in cell metabolism, transport, transcription and regulation of cell processes. The presence of at least some of these transcripts in specific mRNP was confirmed by RT-PCR. Despite the presence of a common population of mRNA associated with both Stau1 and Stau2, the majority of the transcripts were specific to each paralog. Interestingly, we observed that transcripts associated with either Stau1 or Stau2 were nevertheless involved in the same pathways of cell regulation, suggesting that both proteins have complementary roles in the same cellular processes. On the other hand, mRNAs associated with Stau259 and Stau262 were more similar. This suggests that these two isoforms might have more overlapping functions. Consistent with a model of post-transcriptional gene regulation, our results show that Stau1- and Stau2-mRNPs associate with distinct but overlapping sets of cellular mRNAs and that these mRNAs are nevertheless involved in common pathways. It is consistent with the high degree of sequence similarity between Stau1 and Stau2 that predicts that they may have conserved convergent functions and with the observation that they are distributed in distinct mRNP complexes in neurites. Knowing the importance of Stau2 in the transport of dendritic mRNA and to further understand the molecular mechanisms by which it modulates synaptic function, we decided to characterise Stau2-containing mRNPs in neurons. Using anti-Stau2 antibody to immunoprecipitate the mRNPs, we have identified a population of more than 1700 transcripts associated with Stau2 in embryonic rat brain. These mRNAs code for proteins involved in cellular processes such as post-translational modification, translation, intracellular transport and RNA metabolism. Interestingly, Stau2-associated mRNAs isolated form rat brains are relatively different from those isolated from HEK293 cells. This result suggests that the specificity of Stau2-mRNA association can differ from one tissue to the other. Similarly, we have identified the proteins presents in Stau2-containing complexes isolated from embryonic rat brains by a proteomic approach. We were able to determine the presence of mRNA-binding proteins (PABPC1, hnRNP H1, YB1 and hsc70), proteins of the cytoskeleton (α- and β -tubulin) and RUFY3 a poorly characterized protein. While PABPC1 and YB1 associate with Stau2-containing mRNPs through RNAs, hsc70 is directly bound to Stau2 and this interaction is regulated by ATP. The presence of the RNA-binding proteins YB1 and PABPC1, both involved in translation regulation, suggests that the expression of Stau2-bound mRNAs may be regulated at the level of translation initiation. Finally, it is well known that synaptic plasticity requires mRNA transport in dendrites and their local translation. Since the study of the neurophysiological role of Stau2 was already in progress we decided to concentrate our energy on the function of Stau1. Therefore, we studied the importance of Stau1 protein at the neurophysiological level, especially looking for a role in synaptic plasticity. We were able to demonstrate that Stau1 is required for the late form of long term synaptic potentiation, L-LTP, a plasticity dependent not only on local translation of mRNAs, but also on newly transcribed and transported mRNAs. Using hippocampal slices, we showed that Stau1 down-regulation by RNA interference prevents the development and/or maintenance of L-LTP. However, neurons displayed normal early-LTP, mGluR1/5-mediated long-term depression, or basal evoked synaptic transmission. In addition, at the cellular level, Stau1 down-regulation shifted spine shape from regular to elongated spines, without changes in spine density. The change in spine shape could be rescued by an RNA interference-resistant Stau1 isoform. Therefore, Stau1 is important for processing and/or transporting in dendrites mRNAs that are critical in regulation of synaptic strength and maintenance of functional connectivity changes underlying hippocampus dependent learning and memory. In conclusion we were able to further reveal the composition and the importance of the Stau1 and Stau2 mRNP. First, we have identified distinct and overlapping population of mRNAs associated to the diverse isoform of Stau, form HEK293 cells. Second, we were able to identify a population of neuronal transcript as well as some proteins factors present in the Stau2 particles. One of which, hsc70, is directly bound to Stau2 and its interaction is regulated by the presence of ATP. Finally, we have demonstrated the importance of Stau1 in the morphology of the dendritic spine as well as its fundamental implication in synaptic plasticity.

Kora baryłkowa gryzoni, gdzie reprezentowane są wibrysy i droga nerwowa wiodąca od wibrys do baryłek jest użytecznym modelem badania plastyczności neuronalnej towarzyszącej asocjacyjnemu uczeniu. Warunkowanie klasyczne, w którym stymulacja wibrys jest sprzężona ze słabym bodźcem elektrycznym w ogon, powoduje "metaboliczną" ekspansję obszaru kory odpowiadającej stymulowanym wibrysom. Wyniki wcześniejszych badań wskazują, że zmianom metabolicznym towarzyszy wzrost ilości mRNA dla białka GAD67, jak również samego GAD67 (izoformy enzymu syntetyzującego kwas gamma-aminomasłowy - GABA) w badanym rejonie kory. Celem niniejszej pracy było znalezienie strukturalnych korelatów zmian plastycznych zachodzących w procesie uczenia asocjacyjnego.W pracy analizowano gęstość symetrycznych (hamujących) i asymetrycznych (pobudzeniowych) synaps oraz ilościowe i morfologiczne zmiany kolców dendrytycznych w baryłkach odpowiadających stymulowanym wibrysom przy użyciu transmisyjnego mikroskopu elektronowego. Dodatkowo przy użyciu metody immunocytochemicznej połączonej z EM badano poziom neuroprzekaźnika GABA w hamujących zakończeniach presynaptycznych reprezentujących stymulowane wibrysy. Uzyskane w niniejszej pracy wynik i pokazują, że krótkotrwałe warunkowanie klasyczne prowadzi do tworzenia dwusynaptycznych kolców dendrytycznych de novo lub w wyniku dodawania synaps hamujących do już istniejących kolców dendrytycznych z pojedynczą synapsą pobudzeniową. Stwierdzono ponadto, że uczenie asocjacyjne zmienia morfologię dwusynaptycznych kolców dendrytycznych skracając ich długość i zwiększając pole przekroju poprzecznego ich szyjek. Poza tym warunkowanie prowadzi do podwyższenia poziomu GABA w zakończeniach presynaptycznych "trenowanych" baryłek. Uczenie asocjacyjne wywołuje więc szybkie i wyraźnie zaznaczone zmiany strukturalne związane z przekaźnictwem hamującym. Zmiany te obserwowane są w ściśle określonym regionie mózgu i dotyczą konkretnego rodzaju synaps i kolców dendrytycznych.
The barrel cortex of rodents, where vibrissae are represented, and its afferent pathway from the vibrissae is a very useful model for studying associative learning-dependent neuronal plasticity. Classical conditioning, in which stimulation of a row of whiskers is paired with mild electric shock to the tail, produces 'metabolic' expansion of cortical representations of the stimulated vibrissae. Previeus data indicate that the metabolic changes are accompanied by an up-regulation of mRNA of GAD67 and also GAD67 protein within the affected barrels (isoform of enzyme synthesizing gamma amino-butyric acid - GABA) within the affected barrels. The aim of this study was to detect the structural correlates of the plastici changes induced by associative learning. The density of symmetric (inhibitory) and asymmetric (excitatory) synapses and as well as quantitative and the morphological changes of dendritic spines in the barrels representing the stimulated vibrissae were analyzed using transmission electron microscopy. In addition, using the immunocytochemical method, the level of GABA in the inhibitory presynaptic terminals were examined. The results indicate that short-lasting classical conditioning either leads to the formation of the double-synapse spines de novo or through selective addition of inhibitory synapses to the pre-existing single-excitatory synapse spines. It was also found that associative learning modulates morphology of the double synapse spines by decreasing their lenght and increasing the cross-section area of their necks. Moreover, classical conditioning leads to the increase of GABA in the inhibitory presynaptic terminals of ‘trained’ barrels. In conclusion, associative learning induced rapid and pronounced structural changes in the inhibitory transmission. These changes are localized in a strictly definite region of brain and were connected to a strictly definite type of synapses and dendritic spines.

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The ability of neurons to modulate synaptic strength underpins synaptic plasticity, learning and memory, and adaptation to sensory experience. Despite the importance of synaptic adaptation in directing, reinforcing, and revising the behavioral response to environmental influences, the cellular and molecular mechanisms underlying synaptic adaptation are far from clear. Brain-derived neurotrophic factor (BDNF) is a prime initiator of structural and functional synaptic adaptation. However, the signaling cascade activated by BDNF to initiate these adaptive changes has not been elucidated. We have previously shown that BDNF activates mitogen- and stress-activated kinase 1 (MSK1), which regulates gene transcription via the phosphorylation of both CREB and histone H3. Using mice with a kinase-dead knock-in mutation of MSK1, we now show that MSK1 is necessary for the upregulation of synaptic strength in response to environmental enrichment in vivo. Furthermore, neurons from MSK1 kinase-dead mice failed to show scaling of synaptic transmission in response to activity deprivation in vitro, a deficit that could be rescued by reintroduction of wild-type MSK1. We also show that MSK1 forms part of a BDNF- and MAPK-dependent signaling cascade required for homeostatic synaptic scaling, which likely resides in the ability of MSK1 to regulate cell surface GluA1 expression via the induction of Arc/Arg3.1. These results demonstrate that MSK1 is an integral part of a signaling pathway that underlies the adaptive response to synaptic and environmental experience. MSK1 may thus act as a key homeostat in the activity- and experience-dependent regulation of synaptic strength.