Dissertations / Theses on the topic 'Knockin mouse model'

Les troubles du spectre autistique (TSA) sont des troubles neurodéveloppementaux définis par des déficits d'interaction sociale et des comportements restreints ou répétitifs. Les TSA présentent une forte héritabilité, déterminée par de rares mutations monogéniques, ainsi que par des variations dans de nombreux gènes de susceptibilité. Le gène CNTNAP2, qui code pour la protéine Caspr2, est considéré comme l'un des principaux gènes de risque pour les TSA, un grand nombre de variants hétérozygotes faux-sens ayant été identifiés chez les patients. Les souris knock-out Cntnap2 présentent des déficits comportementaux de type TSA, ce qui supporte les données de génétique humaine. Cependant, la signification clinique des variants hétérozygotes n'a pas encore été démontrée et reste débattue. Le projet de thèse visait à élucider cette question en évaluant la pathogénicité in vivo d'un variant CNTNAP2 hétérozygote faux-sens identifié chez un patient français atteint de TSA, I236S, qui a été prédit comme pathogène et pourrait être représentatif d'une large classe de variants CNTNAP2. Nous avons généré un nouveau modèle de souris knockin, les souris KI-I236S, et mené une étude comparant les souris de type sauvage et les souris hétérozygotes (HET) KI-I236S. Caspr2 est une glycoprotéine transmembranaire d'adhésion cellulaire neuronale identifiée à l'origine dans les régions juxtaparanodales des nœuds de Ranvier dans les neurones myélinisés matures. Récemment, en étudiant des souris knock-out Cntnap2, le laboratoire a montré que Caspr2 agit également comme un régulateur majeur du développement de l'axone et de la myélinisation. Dans le cerveau, Caspr2 contrôle le diamètre des axones aux premiers stades du développement postnatal, l'excitabilité intrinsèque des neurones corticaux au début de la myélinisation, ainsi que le diamètre des axones et l'épaisseur de la myéline à l'âge adulte. Caspr2 module également le diamètre des axones, l'épaisseur de la myéline et la morphologie du nœud de Ranvier dans les nerfs périphériques. Nous avons donc évalué l'impact du variant I235S sur le développement des axones, la myélinisation et l'organisation des nœuds de Ranvier dans le système nerveux central et périphérique. Nous avons également caractérisé en détail le comportement des souris HET KI-I236S, en utilisant une batterie de tests qui peuvent indiquer des déficits cognitifs, moteurs et sensorimoteurs. De façon intéressante, les souris HET KI-I236S présentent des déficits cognitifs et somatosensoriels dépendants du sexe par rapport aux souris de type sauvage (interaction sociale légèrement réduite chez les femelles ; sensibilité à la chaleur et force musculaire légèrement réduites chez les mâles). Elles présentent également des altérations sexe-dépendantes des axones myélinisés et des fibres C sensorielles non myélinisées du système nerveux périphérique. Les analyses du cerveau ne montrent pas de défauts majeurs de myélinisation chez les souris mutantes adultes, mais suggèrent que le variant pourrait perturber les fonctions de Caspr2 au début de la myélinisation, conduisant probablement à une accélération des processus de myélinisation à des stades précoces. Ainsi, nos résultats indiquent que les variants faux-sens hétérozygotes de CNTNAP2 tels que I236S peuvent affecter la fonction de Caspr2 de manière dépendante du sexe in vivo, et suggèrent que les variants de CNTNAP2 de la même classe pourraient être pathogènes et contribuer au développement des TSA chez les patients, et/ou contribuer à la variabilité interindividuelle dans les conditions physiologiques
Autism spectrum disorders (ASD) are neurodevelopmental disorders, defined by deficits in social interaction and restricted or repetitive behavior. ASD show high heritability, shaped by rare monogenic mutations, as well as variations in numerous susceptibility genes. Intriguingly, CNTNAP2, encoding the protein Caspr2, is considered to be one of the major ASD-risk genes, with a large number of heterozygous missense variants identified in patients. Cntnap2 knockout mice display ASD-related behavioral deficits supporting human genetic data. However, the clinical significance of the heterozygous variants has not yet been demonstrated and is still debated. The PhD project aimed to unravel this question by evaluating the pathogenicity of an inherited heterozygous missense CNTNAP2 variant identified in a French ASD patient, I236S, which was predicted to be disease-causing and may be representative of a large class of CNTNAP2 variants. We generated a novel knockin mouse model, the KI-I236S mice, and conducted a study comparing wild-type and KI-I236S heterozygous (HET) mice. Caspr2 is a neuronal cell-adhesion transmembrane glycoprotein originally identified in the juxtaparanodal regions of the nodes of Ranvier in mature myelinated neurons. Recently, studying Cntnap2 knockout mice the lab showed that Caspr2 also acts as a major regulator of axon development and myelination. In the brain, Caspr2 controls axon diameter at early postnatal developmental stages, cortical neuron intrinsic excitability at the onset of myelination, and axon diameter and myelin thickness at adulthood. Caspr2 also modulates axon diameter, myelin thickness and node of Ranvier morphology in peripheral nerves. We thus assessed the impact of the variant I235S on axon development, myelination, and node of Ranvier organization in both the central and peripheral nervous system, as well as thoroughly characterizing the behavior of HET KI-I236S mice, using a battery of tests that may indicate cognitive, motor, and sensorimotor deficits. Interestingly, KI-I236S HET mice display sex-dependent cognitive and somatosensory behavioral deficits as compared to wild-type mice (social interaction slightly decreased in females; heat sensitivity and muscular strength slightly decreased in males). They also show sex-dependent alterations in myelinated axons and unmyelinated sensory C-fibers of the peripheral nervous system. Brain analyses do not show major myelination defects in adult mutant mice, but suggest that the variant could perturb the functions of Caspr2 at the onset of myelination, leading likely to an acceleration of the myelination processes at early stages. Thus, our results indicate that CNTNAP2 heterozygous missense variants such as I236S can affect Caspr2 function in a sex-dependent manner in vivo and suggest that the CNTNAP2 variants of the same class could indeed be pathogenic and contribute to the development of ASD patients, and/or contribute to inter-individual variability in physiological conditions

Neurotrophins are a family of growth factors known for their pleiotropic effects on neuronal survival, maturation and plasticity. Brain-derived neurotrophic factor (BDNF) is the most expressed in the brain. The activation of specific BDNF downstream pathways hinges on BDNF binding to its receptor TrkB. Kidins220 is a scaffold protein that interacts with neurotrophin receptors and is directly involved in the activation of neurotrophin signaling. It is also required for neuron differentiation, survival and plasticity. This protein has been linked to several diseases including psychiatric and neurodegenerative pathologies and for this reason, several KO animal models have been generated. So far none of them was viable after birth, making it impossible to investigate the role of this protein in postnatal/adult brain development. Astrocytes are fundamental in maintaining nervous system homeostasis. They are capable of perceiving a wide variety of extracellular cues and transducing them via the activation of specific intracellular signaling pathways into responses that may be protective or disruptive toward neighboring neurons. Moreover, astrocytes are key regulators of neuronal circuit formation and synaptic transmission. Several aspects of astrocyte physiology are controlled by neurotrophins. However, the role of Kidins220 in astrocytes, as well as in the adult brain remains largely unknown. Thus, in this thesis we aimed to deeply understand the role of Kidins220 using both in vivo and in vitro models. First we compared the signaling competence of embryonic and postnatal primary cortical astrocytes exposed to BDNF, and observed a shift from a kinase-based response in embryonic cells to a predominantly Ca2+-based response in postnatal cultures. We demonstrated that Kidins220 ablation is accompanied by a decreased expression of both BDNF receptor TrkB isoforms. We also described the role of Kidins220 in BDNF-induced signaling in astrocytes, showing that it contributes to both kinase and Ca2+-activated pathways. To evaluate the effect of Kidins220 ablation in the adult brain we used a floxed line that expresses only the full-length isoform, which we crossed with mice expressing Cre under the CamKII promoter, leading to a conditional knockout (cKO) line where Kidins220 is absent only in the excitatory neurons of the forebrain, starting at the second postnatal week. In this animal model, we have observed altered dendritic arborization and spine number in the cortico-hippocampal regions. The deletion of Kidins220 also leads to behavioral changes, such as reduced anxiety-like traits due to to alterations in TrkB-BDNF signaling. Our data increase the knowledge of the complex role played by Kidins220 both in astrocytes and in adult brains, reveal a previously unidentified role of this protein in astrocytes, controlling the response to BDNF and to Ca2+ dynamics during development. Finally, our data confirm the fundamental role of Kidins220 in adult mice, where its ablation leads to both behavioral and biochemical impairments.

The discovery of leptin and other humoral signals which regulate food intake and energy expenditure has greatly contributed to our understanding of molecular pathways controlling energy homeostasis. Leptin produced by adipocytes, insulin produced by the pancreas, and ghrelin produced by the stomach all contribute to the body’s energy balance. One question remaining is whether the lipid transport system also plays a role. Our hypothesis is that lipid clearance is important in the maintenance of energy homeostasis. The low-density lipoprotein receptor (Ldlr) is a key molecule involved with lipid clearance. The experiments presented in this thesis used the Ldlr-/- mouse to study the Ldlr’s role in energy balance. One aim of this thesis was to provide a detailed analysis of the energy balance phenotype of the Ldlr-/- mouse. Another aim of this thesis was to use the Ldlr-/- mouse to study the potential interaction between Ldlr and the leptin signaling pathway. Adult Ldlr-/- mice and Ldlr+/+ controls on a C57BL/6J background were fed either a chow or a high-fat, high-sucrose Western-type diet (WTD) for eight weeks. Physiological studies of food intake, energy expenditure, activity, heat production, insulin sensitivity, and leptin responsiveness were performed. As well, the effect of these diet interventions on circulating leptin and on leptin gene expression was examined. On the chow diet, Ldlr-/- mice had lower energy expenditure and higher activity levels relative to controls. On the WTD, Ldlr-/- mice gained less weight relative to Ldlr+/+ mice, specifically gaining less fat mass. Increased thermogenesis in Ldlr-/- mice fed the WTD was detected. Additionally, leptin responsiveness was blunted in chow-fed Ldlr-/- mice, suggesting a novel role for the Ldlr pathway that extends to leptin’s regulation of energy balance. In addition to its known role in lipid transport, these results from the Ldlr-/- mouse demonstrate the importance of the Ldlr in regulating energy homeostasis and suggest a direct physiological link between dyslipidemia and energy balance.

The sense of smell enables animals to detect myriads of different odors carrying information about the quality of food, the presence of pathogens, prey, predators, or potential mates. Olfactory sensory neurons (OSNs) in the nasal cavity are the interface of the main olfactory system with the external environment. The binding of an odorant molecule to specific olfactory receptors (OR) located in the cilia of these neurons triggers a transduction cascade that transduces the chemical signal into action potentials which travel along the axon of the OSNs to the olfactory bulb. Here, the odor information is processed and conveyed to higher brain centers, ultimately leading to the perception of smell. In this Thesis I studied the effect of two genetic manipulations on the firing activity of the OSNs: the ectopic overexpression of the inward rectifier potassium channel Kir2.1 and the deletion of the TMEM16b/Ano2 gene, that codes for the Ca2+- activated chloride channel TMEM16B. The overexpression of Kir2.1 reduces the excitability of the neurons, and when expressed in OSNs, mice show a general disorganization of the glomerular map in the olfactory bulb. Since spontaneous and sensory-evoked electrical activity play important roles in the formation of several sensory circuits, including the olfactory system, in the first part of this Thesis, I investigated how the spiking activity of mouse OSNs is influenced by the Kir2.1 overexpression, using loose-patch recordings from the OSNs knobs. I found that the overexpression of Kir2.1 caused a decrease in the spontaneous firing activity of OSNs but did not influence the evoked firing properties induced by odorant stimulation, indicating that the olfactory bulb disorganization was caused by a reduced spontaneous firing activity. Ca2+-activated Cl¯ current (CaCC) is an important component of the transduction current evoked by odor stimulation in OSNs. Binding of odorants to their specific receptor on the cilia of OSNs causes the activation of adenylyl cyclase with a relative increase of intracellular cAMP, activating cyclic nucleotide-gated (CNG) channels. Ca2+ entry through CNG channels increases the open probability of Ca2+-activated Cl- channels. The molecular identity of these channels has been elusive for a long time, but recently it has been shown that the olfactory CaCC are mediated by the membrane protein TMEM16B/Anoctamin2. However, the physiological role of olfactory CaCC is still unclear, and the first description of TMEM16B knockout (KO) mice reported no clear olfactory deficits. In the second part of this Thesis, I studied basal firing properties and stimulus-evoked responses with loose-patch recordings in OSNs from TMEM16B KO or WT mice. OSNs responded to a stimulus with a transient burst of action potentials. Responses of OSNs from TMEM16B KO mice showed an increased number of action potentials compared to responses from WT mice, both in OSNs expressing a random or I7 OR. The basal spiking activity of individual OSNs is correlated with the expressed OR that drives basal transduction activity. I measured a reduced basal activity in TMEM16B KO OSNs expressing the I7 OR compared to WT OSNs. Moreover, axonal targeting was altered and TMEM16B KO had supernumerary I7 glomeruli compared to WT. These results show that the expression of TMEM16B affects OSNs firing properties and contributes to the glomerular formation and refinement of I7-expressing OSNs in the olfactory bulb, suggesting a crucial role for TMEM16B in normal olfaction.

Chez les mammifères, les protéines SIRT, appelées aussi sirtuines, appartiennent à la famille des histones désacétylases (HDAC) de classe III et ont été nommées ainsi pour leur homologie avec le gène de la levure Saccharomyces cerevisiae Sir2. Il est clairement établi que le gène Sir2 joue un rôle primordial dans l’extension de la durée de vie de la levure induite par la restriction calorique et que des copies supplémentaires de ce gène retardent le vieillissement chez la levure. Contrairement aux HDAC de classe I et II, les protéines SIRT nécessitent un cofacteur, le NAD+, pour désacetyler les histones ainsi que d’importants facteurs régulateurs de la transcription. Le chef de file des sirtuines, SIRT1, semble impliqué dans des pathologies variées telles le diabète, l’obésité, l’insuffisance cardiaque, le SIDA et les maladies neurodégénératives. Même si le rôle de SIRT1 n’a pas encore été clairement établi in vivo, il apparaît indissociable de la régulation de différents processus biologiques allant de l’apoptose à la différentiation adipocytaire et musculaire ainsi que dans le métabolisme glucidique en régulant la néoglucogenèse. La fonction des autres SIRT n’est pas encore élucidée. Le sujet de ma thèse de doctorat est d’essayer de comprendre les fonctions biologiques des gènes SIRT en les inactivant de façon spatio-temporelle par l’utilisation de la stratégie du « knock-out conditionnel » (inactivation conditionnelle) et le système Cre/Lox. Cette technologie permet d’inactiver de façon contrôlée le gène d’intérêt dans un organe donné d’une souris adulte afin d’éviter les problèmes liés aux anomalies que l’absence du gène pourrait provoquer au cours du développement de l’animal lors d’un « knock-out classique ». Pour parvenir à notre objectif, nous avons réalisé des constructions modifiées des différents gènes SIRT. La particularité de ces constructions consiste à introduire des séquences Lox autour du domaine du gène codant pour la partie catalytique de l’enzyme. Les Lox sont des séquences nucléotidiques de 34 paires de bases qui sont excisées par l’enzyme Cre-recombinase lors de l’inactivation du gène. Les vecteurs contenant les gènes SIRT modifiés ont été électroporés dans des cellules souches embryonnaires (ES) de souris 129/Sv afin de s’intégrer au génome par recombinaison homologue. Les clones positifs ont été injectés par la suite dans des blastocystes. Nous avons obtenu ainsi des souris transgéniques pour le gène d’intérêt. Ces souris doivent par la suite être croisées avec des souris exprimant une protéine de fusion Cre-ERT sous le contrôle d’un promoteur spécifique d’un organe donné. La protéine Cre-ERT est une fusion entre la Cre-recombinase et le récepteur aux oestrogènes ayant une affinité élevée pour le ligand tamoxifène. En cas d’injection de tamoxifène, il y a activation de la Cre qui induit l’inactivation du gène d’intérêt à un moment donné de la vie de la souris et permet d’étudier ainsi les effets biologiques qui en résultent. A l’heure actuelle, les constructions pour les gènes SIRT1-7 ont été réalisées. Nous disposons des souris transgéniques pour le gène SIRT2 « floxé » et les croisements sont en cours avec des souris transgéniques CMV-Cre-ERT2 (inactivation de SIRT2 dans tous les tissus) et Syn-Cre-ERT2 (inactivation de SIRT2 dans les cellules nerveuses). Les autres constructions ont été électroporées dans les cellules ES et sont en cours d’analyse. Partie II : Etude de la fonction du gène Rb dans la différentiation adipocytaire et le métabolisme énergétique. Le gène du rétinoblastome Rb est le premier gène suppresseur de tumeur identifié et a été largement étudié. Son rôle a bien été démontré dans différents processus biologiques tels le contrôle du cycle cellulaire, l’apoptose, la prolifération et la différenciation cellulaires. Récemment, des études in vitro ont montré que la protéine pRb avait un rôle important dans la différentiation adipocytaire et qu’un déficit en pRb provoque un changement dans le programme de différentiation d’adipocytes blancs de souris en adipocytes bruns. Dans notre étude, nous avons utilisé le système Cre-lox pour inactiver spécifiquement le gène Rb dans le tissu adipeux. Soumises à un régime riche en graisses, des souris déficientes en pRb restent maigres par rapport à leurs congénères qui voient leurs poids augmenter de manière significative. L’analyse histologique et en microscopie électronique du tissu adipeux blanc (WAT) des souris déficientes en pRb montre une transformation d’une partie du WAT en tissu adipeux brun (BAT) avec une augmentation significative du nombre de mitochondries suggérant une activité métabolique élevée. L’analyse moléculaire a démontré une augmentation de l’expression des gènes impliqués dans la dépense énergétique tels UCP-1, PGC-1a et Dio2. Ceci se traduit physiologiquement par une augmentation de la consommation en O2 et de la production de CO2 démontrant une élévation de la dépense énergétique chez les souris pRb-déficientes. Ces données confirment que l’absence de pRb provoque la conversion d’une partie du WAT en BAT, un tissu connu pour son rôle actif dans la dépense énergétique. Le gène Rb devient ainsi une cible thérapeutique potentielle dans le but d’induire une perte de poids chez les patients obèses
In mammals, the sirtuin family of histone deacetylases (HDACs) family was named after their homology to the yeast Saccharomyces cerevisiae gene Sir2. In yeast, it has been shown that Sir2 mediates the effects of calorie restriction on the extension of lifespan and that high levels of Sir2 activity promote longevity. Like their yeast homologs, the mammalian sirtuins (SIRT1-7) are class III HDACs and require NAD+ as a cofactor to deacetylate substrates such as histones and transcription regulators. Through this activity, sirtuins are shown to regulate important biological processes ranging from apoptosis, adipocyte and muscle differentiation, and energy expenditure to gluconeogenesis. SIRT1, the most studied sirtuin, seems to be implicated in several pathologies such as diabetes, obesity, heart failure and neurodegenerative disorders. The aim of this Ph. D. Thesis was to help understand the biological function of SIRT genes through their inactivation in mice in a spatial and temporal controlled manner, using the Cre/Lox technology. This system allows the controlled inactivation of a gene of interest in a given organ of an adult mouse to avoid abnormalities that could occur during the development when using a “classical knock-out”. We have generated genetically modified constructs for all SIRT genes by introducing 2 LoxP sequences flanking the catalytic domain of the enzyme. LoxP sites are sequences of 34 nucleotides that can be recognized and excised by the Cre-recombinase enzyme. Vectors containing the modified SIRT gene constructs were electroporated in embryonic stem cells (ES) of 129/Sv mice in order to be integrated in their genome by homologous recombination. Positif clones were then injected in blastocysts of C57BL/6J pseudopregnant mice. We obtained transgenic mice for the gene of interest. These mice will be crossed with mice expressing the Cre recombinase fused to a modified estrogen receptor with high affinity for the synthetic ligand tamoxifen, under the control of a cell specific promoter that targets Cre expression in a specific organ or cell type (promoter-Cre-ERT2). After tamoxifen injection, the Cre recombinase is activated and subsequently the SIRT gene of interest will be inactivated in a specific cell type (e. G. Adipocytes), whilst its activity remains in other cells, allowing the study of the biological effects that result from such gene inactivation. At present, we have completed the constructs for all SIRT(1-7) genes. We obtained mice with a floxed SIRT2 allele that were crossed with a CMV-Cre-ERT2 and Synapsin-Cre-ERT2 transgenic mice to generate cohorts of double transgenic mice expressing the floxed SIRT2 allele and the Cre-ERT2 either in all cell types or specifically in neurons. Constructs for other SIRT genes have been electroporated in ES cells and the generation of mice is underway. PART 2: Phenogenomics of adipocyte-specific retinoblastoma deficient miceThe role of the tumor suppressor retinoblastoma protein (pRb) has been firmly established in the control of cell cycle, apoptosis, and differentiation. Recently, it was demonstrated that lack of pRb promotes a switch from white to brown adipocyte differentiation in vitro. We used the Cre-Lox system to specifically inactivate pRb in adult adipose tissue. Under a high-fat diet, pRb-deficient (pRbad-/-) mice failed to gain weight because of increased energy expenditure. This protection against weight gain was caused by the activation of mitochondrial activity in white and brown fat as evidenced by histologic, electron microscopic, and gene expression studies. Moreover, pRb(-/-) mouse embryonic fibroblasts displayed higher proliferation and apoptosis rates than pRb(+/+) mouse embryonic fibroblasts, which could contribute to the altered white adipose tissue morphology. Taken together, our data support a direct role of pRb in adipocyte cell fate determination in vivo and suggest that pRb could serve as a potential therapeutic target to trigger mitochondrial activation in white adipose tissue and brown adipose tissue, favoring an increase in energy expenditure and subsequent weight loss

Mutations on the leucine-rich repeat kinase 2 (LRRK2) gene are the most common variants responsible for idiopathic Parkinson’s disease (PD). Historically, PD is thought of as a late-stage neurodegenerative disease resulting from the loss of dopaminergic neurons in the substantia nigra pars compacta with the presence of Lewy pathology. Current treatments focus on the dopaminergic aspect of this disease, without addressing or successfully halting the underlying causes of this disease. The LRRK2 G2019S mutation is the single most common genetic risk factor for Parkinson’s disease and leads to increased kinase activity with subtle effects on the timing of nigrostriatal dopaminergic transmission. Here, using a genetically faithful G2019S knock-in (GKI) mouse model at multiple age points, although no differences were seen in monoamine, glutamate, or GABA release by in vivo microdialysis of the dorsolateral striatum, more temporally sensitive investigation revealed subtle release augmentation in mutants. Using fast-scan cyclic voltammetry to examine dopamine release and reuptake on a millisecond timescale in acute striatal slices at early (<3 months) and later age points (~12 months), alterations in dopamine release were observed with repeated stimulation and increased decay constant in mutants. In GKI mice dopamine release is augmented and responds differently across age points to pharmacological D2 receptor agonism and dopamine transporter (DAT) inhibition. LRRK2 is thought to be involved in synaptic vesicle storage and recycling and GKI mutation in mice leads to augmented dopamine release from an early age that is not attenuated by DAT inhibition, indicating other mechanisms (e.g. altered release / vesicle cycling) are affected and play a role in this alteration from early stages (<3 months, potentially earlier) in response to this mutation. Elucidation of the function of LRRK2 and the deleterious effects caused by the G2019S mutation will help target neuroprotective therapies to delay or halt disease progression.
Medicine, Faculty of
Graduate

Opitz G/BBB syndrome (OS) is a multiple congenital anomaly disorder characterized by developmental defects of midline structures. Mutations in the MID1 gene are responsible of the X-linked form of the syndrome and lead to loss-of-function of the protein. MID1 is an E3 ubiquitin ligase of the tripartite motif (TRIM) subfamily of RING proteins and associates with microtubules. The mouse line carrying a nonfunctional ortholog of the human MID1 gene, Mid1, (Mid1-/Y) recapitulates the brain morphological abnormalities observed in patients, i.e. hypoplasia of the anterior cerebellar vermis, although the role of Mid1 in cerebellar development is still unclear. Previous analysis demonstrated that the alteration of the cerebellum in Mid1-/Y mice originates prenatally, delineating the embryonic day (E) 13.5 as the time of the first sign of the cerebellar defect. We started to explore the molecular pathways affected by the lack of Mid1 at E13, thus placing immediately before the appearance of the defect. Starting from an unbiased proteomics study using differential mass spectrometry approach, we explored altered pathways in Mid1-/Y developing cerebella. The network analysis has suggested that Mid1-/Y cerebella might present impairment in vesicular trafficking and intercellular communication, later confirmed by immunoblot experiments. In parallel, the association of Mid1 with some regulators of RNA processing and stability has been investigated in cells upon MID1-overexpression. All the collected results may be helpful in disclosing potential molecular mechanisms involved in the establishment of the neurological defects observed in mouse embryos and in OS patients. Further investigations are required to provide new evidence confirming our data and the results might increase our knowledge of the OS pathogenesis not only in the cerebellar compartments but also in the other midline-affected structures.

Of the thousands of dividing progenitor cells (PCs) generated daily in the adult brain only a very small proportion survive to become mature neurons through the process of neurogenesis. Identification of the mechanisms that regulate cell death associated with neurogenesis would aid in harnessing the potential therapeutic value of PCs. Apoptosis, or programmed cell death, is suggested to regulate death of PCs in the adult brain as overexpression of B-cell lymphoma 2 (Bcl-2), an anti-apoptotic protein, enhances the survival of new neurons. To directly assess if Bcl-2 is a regulator of apoptosis in PCs, this study examined the outcome of removal of Bcl-2 from the developing PCs in the adult mouse brain. Retroviral mediated gene transfer of Cre into adult floxed Bcl-2 mice eliminated Bcl-2 from developing PCs and resulted in the complete absence of new neurons at 30 days post viral injection. Similarly, Bcl-2 removal through the use of nestin-induced conditional knockout mice resulted in reduced number of mature neurons. The function of Bcl-2 in the PCs was also dependent on Bcl-2-associated X (BAX) protein, as demonstrated by an increase in new neurons formed following viral-mediated removal of Bcl-2 in BAX knockout mice. Together these findings demonstrate that Bcl-2 is an essential regulator of neurogenesis in the adult hippocampus.

Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disorder due to upper and lower motor neurons (MNs) death. Recognized as a non-cell autonomous disease, ALS is also characterized by damage and degeneration of glial cells, such as astrocytes, microglia, and oligodendrocytes. In particular, astrocytes acquire a reactive and toxic phenotype defined by an abnormal proliferation and by the release of neurotoxic factors. One major cause for MN degeneration in ALS is represented by the glutamate-mediated excitotoxicity, due to the alteration of glutamate transmission mechanisms, including glutamate receptor function. In this context, the Group I metabotropic glutamate receptor 5 (mGluR5) has been proposed to play an important role in ALS, since it is largely overexpressed during disease progression and is involved in the altered neuronal and glial cellular processes. We previously demonstrated that mGluR5 produces abnormal glutamate release in the spinal cord of the SOD1G93A mouse model of ALS and that halving its expression has a positive impact on in-vivo disease progression, including motor neuron survival, astrogliosis and microgliosis. We here investigated the consequences of reducing the mGluR5 expression in SOD1G93A mice on the reactive phenotype of spinal cord astrocytes cultured from late symptomatic (120 days old) SOD1G93A, age matched WT and SOD1G93AGrm5-/+ mice. SOD1G93A astrocytes displayed a higher cytoplasmic calcium concentration respect to WT cells and knocking down of mGluR5 reduced calcium level, both under basal and 3,5-DHPG-stimulated conditions. GFAP and S100β, two markers of astrogliosis, were increased in SOD1G93A astrocytes, whereas their overexpression was reduced in SOD1G93AGrm5-/+ cells. The same positive effect was obtained in the case of NLRP3, a marker strictly related to inflammation, which was upregulated in SOD1G93A astrocytes and less expressed in double mutant astrocytes. The partial ablation of mGluR5 also resulted in a lower cellular presence of misfolded SOD1. Both the expression and secretion of pro-inflammatory cytokines were strongly reduced in SOD1G93AGrm5-/+ respect to SOD1G93A astrocytes. The uncoupling between oxygen consumption and ATP synthesis and the impairment of mitochondria function, present in SOD1G93A astrocytes, were recovered in double mutant astrocytes. Notably, the viability of spinal MNs co-cultured with SOD1G93AGrm5-/+ astrocytes was significantly increased respect to MNs co-cultured with SOD1G93A astrocytes. The acute in-vitro treatment of SOD1G93A astrocytes with an antisense nucleotide (ASO) specific for mGluR5 decreased the mRNA and protein expression of mGluR5 in these cells and led to the reduction of GFAP and S100β. The in-vitro pharmacological treatment with the negative allosteric modulator of mGluR5, CTEP, also reduced the expression of GFAP and S100β in SOD1G93A astrocytes. Altogether, these results indicate that mGluR5 ablation has a positive impact on astrocytes in SOD1G93A mice, supporting the idea that the in-vivo amelioration of the disease progression, registered after mGluR5 genetical or pharmacological silencing, involve astrocyte phenotype improvement. As a whole, it may be outlined that mGluR5 may represent a potential therapeutic target able to preserve MNs from death, also by modulating the reactive astroglia phenotype in ALS. Due to the active contribution of microglia to ALS pathogenesis, the effect of mGluR5 partial ablation in SOD1G93A mice on the balance between the pro- and anti-inflammatory profile of microglia acutely purified from the brain and spinal cord of WT, Grm5-/+, SOD1G93A and SOD1G93AGrm5-/+ mice has been investigated at the early (90 days) and late symptomatic (120 days) stages of ALS by detecting the mRNA and protein levels of some relevant markers involved in neuroinflammation, such as IL-1β, CD86, iNOS, TNF-α (pro-inflammatory), Arginase 1, IL-10, CD206 and IL-4 (anti-inflammatory). Experiments are in progress to complete this part of the project.

Le syndrome des Néoplasies Endocriniennes Multiples de type 1 (NEM1) est une maladie à transmission autosomique dominante liée à l’inactivation du gène MEN1. Le but de mon travail de thèse était d’étudier le rôle biologique et oncosuppressif du gène Men1 dans le pancréas endocrine. La caractérisation d’un nouveau modèle m’a permis de démontrer que l’invalidation du gène Men1 spécifiquement dans les cellules alpha conduit à la fois au développement de glucagonomes et d’insulinomes par un mécanisme de transdifférenciation de cellules exprimant le glucagon en cellules exprimant l’insuline. Parallèlement, en explorant les modèles murins où le gène Men1 est invalidé respectivement dans les cellules alpha et beta Pancréatiques, j’ai pu identifier l’expression altérée de certains facteurs de transcription ayant des fonctions vitales dans ces cellules, notamment Foxa2 et MafB, dans les lésions précoces des cellules endocrines pancréatiques correspondantes. En conclusion, mon travail de thèse a permis de mieux clarifier la fonction biologique du gène Men1 dans les cellules pancréatiques endocrines et de mieux comprendre les mécanismes impliqués dans la survenue du syndrome MEN1
Multiple endocrine neoplasia type 1 (MEN1) is an autosomal dominant inherited syndrome caused by mutations of the MEN1 gene. The aim of my work is to investigate the biological and oncosuppressive roles of the Men1 gene in the pancreatic endocrine cells. The analyses carried out in a new mouse model showed that Men1 ablation in alpha cells trigged the development of both glucagonoma and insulinoma by the transdifferentiation from glucagon-producing cells to insulin-expressing cells. Furthermore, the data obtained from the characterization of both alpha- and beta-cell-specific Men1 mutant mice allowed to identify the altered expression of several important endocrine specific transcriptional factors, including Foxa2 and MafB, found in the early lesions of the corresponding pancreatic endocrine cells. Overall, my thesis work provides interesting clues for better understanding the mechanisms involved in the tumorigenesis of MEN1 syndrome

Les maladies auto-immunes, qui touchent plus de 5% de la population, sont induites par une perte de la tolérance aux antigènes du Soi. Ces pathologies, généralement multifactorielles, résultent de l’effet combiné de plusieurs allèles de susceptibilité et de différents facteurs environnementaux. Les agents infectieux ont été tout particulièrement incriminés, mais les mécanismes en jeu restent encore mal élucidés. Les lymphocytes B, qui jouent un rôle central dans la pathogénie de nombreuses maladies auto-immunes, sont susceptibles d’être activés selon différents mécanismes au cours d’un processus infectieux et cette activation peut englober des cellules autoréactives. On ne sait cependant pas si cette activation peut entraîner la production d’auto-anticorps pathogènes de forte affinité et d’isotype IgG à partir du pool de cellules productrices d’auto-anticorps naturels de faible affinité, qui sont présentes de façon constitutive dans le répertoire B de l’individu sain. Nous avons mis au point un nouveau modèle murin knock-in pour des lymphocytes B présentant une affinité intermédiaire pour leur auto-antigène, la protéine HEL2X mutée (Hen-Egg Lysozyme). Ce modèle autoréactif d’affinité intermédiaire SWHEL X HEL2X, élaboré sur un fond génétique non autoimmun, permet de suivre le processus de maturation d’affinité des cellules B anti-HEL en présence de leur auto-antigène HEL2X au cours de l’infection chronique par la bactérie Borrelia burgdorferi. L’infection induit au niveau ganglionnaire une prolifération ainsi qu’une activation lymphocytaire B incluant des cellules anergiques. Certains clones autoréactifs sont capables de gagner les centres germinatifs ganglionnaires, de commuter vers l’isotype IgG et présentent des mutations somatiques au niveau de la région variable de la chaîne lourde de leur immunoglobuline, dans la zone d’interaction avec HEL2X, indiquant un processus de sélection par l’auto-antigène. Malgré un taux augmenté d’auto-anticorps d’isotype IgM, ces animaux ne produisent pas de plasmocytes capables de sécréter des auto-anticorps d’isotype IgG. Nos observations suggèrent l’existence de mécanismes de tolérance périphérique intrinsèques mis en place en particulier au niveau du centre germinatif. Un premier point de contrôle va éliminer les lymphocytes B autoréactifs ayant commuté de classe et présentant des mutations somatiques leur conférant une affinité augmentée pour l’auto-antigène tandis qu’un second point de contrôle va empêcher la différenciation en plasmocytes IgG+.Chez l’individu non prédisposé génétiquement, des mécanismes pourraient ainsi permettre de prévenir le développement d’une auto-immunité pathogène au cours d’un épisode infectieux
Autoimmune diseases, affecting more than 5% of the population, reflect a loss of tolerance to selfantigens. These multifactorial diseases result from the combined effect of several susceptibility alleles and different environmental factors. Infectious agents have been particularly incriminated but there is no clear understanding of the underlying mechanisms. B lymphocytes, that appear central to the pathogenesis of several autoimmune diseases, may be activated by several mechanisms during infectious processes and this activation can encompass autoreactive cells. Whether or not the lattercan induce the production of high-affinity pathogenic IgG isotype auto-antibodies from the naturally present low-affinity self-reactive B cells is still unknown. To gain further insight into this question, we created a new intermediate affinity autoreactive mouse model called SWHEL X HEL2X. In these mice, knock-in B cells express a B cell receptor highly specific for Hen-Egg Lysozyme (HEL) that recognizes HEL2X mutated auto-antigen with intermediate affinity. This model, generated on a non-autoimmune-prone genetic background, allows the following of anti-HEL B cells affinity maturation process in presence of their auto-antigen during Borrelia burgdorferi chronic bacterial infection. The infection leads to lymph nodes lymphoproliferation and B cell activation including anergic cells. Some autoreactive clones are able to form germinal centers, toswitch their immunoglobulin heavy chain and to introduce somatic mutations in the heavy chain variable regions on amino-acids forming direct contacts with HEL2X, suggesting an auto-antigen-driven selection process. Despite increased levels of IgM autoantibodies, infected mice are unable to generate IgG autoantibody secreting plasma-cells. These observations suggest the existence of intrinsic peripheral tolerance mechanisms operating mainly at the level of germinal centers. The first checkpoint eliminates switched autoreactive B cells with increasing affinity mutations while a secondcheckpoint avoids IgG+ plasma-cell differentiation. Thus, in genetically non predisposed individuals, tolerance mechanisms may be set-up to prevent the development of pathogenic autoimmunity during the course of an infection

A l’aide d’études génétiques, notre équipe, a identifié chez des patients avec des malformations du développement cortical (MDC), des mutations dans le gène KIF2A, une kinésine impliquée dans la dynamique des microtubules. Afin d’étudier dans un contexte physiologique l’impact de ces mutations sur le développement cortical, nous avons développé un modèle murin exprimant la mutation p.His321Asp de KIF2A. Les analyses neuro-anatomiques et neuro-développementales des souris exprimant la mutation ont permis de mettre en évidence une microcéphalie, et des anomalies de positionnement neuronale dans le cortex et l’hippocampe. Les explorations phénotypiques, nous ont permis de montrer une susceptibilité accrue à l’épilepsie chez la souris mutante. De plus, des analyses fonctionnelles sur les fibroblastes de patient et par purification de la protéine mutante ont montré l’incapacité de la protéine mutante à dépolymériser les microtubules. Nous pensons que l’ensemble des résultats obtenus lors de ce projet de thèse pourra apporter une meilleure compréhension des mécanismes physiopathologiques impliqués dans les MDC lié aux mutations dans KIF2A
By using genetic studies, our team have identified in patient with malformations of cortical development, missense mutations in the KIF2A gene, a kinesin involved in microtubules depolymerization. In order to study in a physiological context the impact of these mutations on the cortical development, we have developed expressing the KIF2A p.His321Asp mutation. The first neuro-anatomical and neuro-developmental analyzes of the mice expressing the mutation during embryonic development allowed us to highlight microcephaly and neuronal positioning abnormalities in the cortex and the hippocampus. Phenotypic explorations allowed us to highlight increased susceptibility to epilepsy in the mutant mouse. In addition, functional analyzes using patient fibroblasts and purification of the mutant protein have shown that the mutant protein can not depolymerize microtubules. We believe that all the results obtained during this thesis project will provide a better understanding of the pathophysiologic mechanisms involved in malformations of cortical development related to mutations in the KIF2A gene

Des mutations hétérozygotes faux-sens dans le gène de la tubuline gamma TUBG1, ont été identifiées dans le contexte des malformations du développement cortical, associées à une déficience intellectuelle et à l'épilepsie. Ici, nous avons étudié par la technique d’électroporation in-utero et par des études in vivo, l’effet de quatre de ces variantes sur le développement cortical. Nous montrons que les mutations dans TUBG1 affectent le positionnement neuronal dans la plaque corticale, en perturbant la locomotion des neurones nouvellement nés, mais sans affecter la neurogenèse. Nous proposons que la γ-tubuline mutante affecte le fonctionnement global de ses complexes, et en particulier leur rôle dans la régulation de la dynamique des microtubules. De plus, nous avons développé un modèle de souris knock-in Tubg1Y92C/+ et évalué les conséquences de la mutation sur le développement cortical, les caractéristiques neuroanatomiques et le comportement. Les souris mutantes présentent une microcéphalie globale, des anomalies du néocortex et de l'hippocampe, des altérations du comportement et une susceptibilité épileptique. Ainsi, nous montrons que les souris Tubg1Y92C/+ miment au moins partiellement le phénotype humain et représentent donc un modèle pertinent pour d'autres investigations de la physiopathologie des malformations du développement cortical
Missense heterozygous variants in the gamma tubulin gene TUBG1 have been linked to malformations of cortical development, associated with intellectual disability and epilepsy. Here, we investigated through in-utero electroporation and in-vivo studies, how four of these variants affect cortical development. We show that TUBG1 mutants affect neuronal positioning within the cortical wall, by a disrupting the locomotion of newly born neurons but without affecting neurogenesis. We propose that mutant γ-tubulin affects overall functioning of γ-tubulin complexes, and in particular their role in the regulation of microtubule dynamics. Additionally, we developed a knock-in Tubg1Y92C/+ model and assessed consequences of the mutation on cortical development, neuroanatomical features and behaviour. Mutant mice present with global microcephaly, neocortical and hippocampal abnormalities, behavioural alterations and epileptic susceptibility. Thus, we show that Tubg1Y92C/+ mice partially mimic the human phenotype and therefore represent a relevant model for further investigations of the physiopathology of malformations of cortical development

Sleep disturbances are common in patients with schizophrenia. Mice lacking the cytoskeletal-associated protein Stable Tubule Only Polypeptide (STOP) display cognitive, behavioural and neurobiological deficits that mimic those seen in schizophrenia, but there is little evidence of sleep changes in these mice. To investigate their sleep patterns, electroencephalogram (EEG) and electromyogram were recorded under a 12:12 light:dark cycle in adult male STOP-null (KO; n=7) and wild-type (WT; n=8) mice, during a 24 h baseline period, followed by 6 h of sleep deprivation, and a 24 h recovery period. In the baseline period, KO mice spent more time awake and less time in non-rapid eye movement (NREM) and REM sleep compared to WT mice. Particularly in the dark phase, KO mice had more wake and NREM sleep episodes, and shorter NREM and REM sleep episodes relative to WT mice. Following sleep deprivation, during the first 12 h of recovery (i.e. dark phase), both groups showed similar increases in NREM and REM sleep amounts and NREM EEG delta power relative to corresponding baseline periods. These findings indicate that the STOP-null mice sleep less and their sleep is more fragmented compared to WT mice. These features are consistent with the sleep abnormalities found in individuals with schizophrenia.

Activating mutations in the PI3K/AKT pathway are present in majority of breast cancer. The gain of function mutation E17K of AKT1, was found in 8% of breast cancers, especially ductal carcinomas, but several studies performed so far have failed to define the real role of this mutation in the breast tissue trnsformation. To investigate the role of the AKT1E17K in breast tumorigenesis, we explored the phenotype of a new mouse model which express the mutant transgene in mammary epithelium. The expression of AKT1E17K enhances the activity of the kinase and the phosphorilation status of downstream substrates, such as FOXO1 and GSK3α/β. In addition, transgenic mice showed an increased cellularity 8-10 times higher than control mice breast tissues. Moreover 70% of transgenic mice expressing the mutant form of AKT1 develop ductal carcinomas from medium to high grade. We have identified also the contribution of AKT1E17K in the generation and maintenance of putative breast cancer stem cells. Finally, using a pharmacological study, we were able to slow down tumor formation by inhibiting downstream effect of AKT1 pathway. All together these data have allowed us to demonstrate that AKT1E17K is itself capable to induct the onset of ductal carcinoma in transgenic mice.

碩士
國立陽明大學
口腔生物研究所
95
Tid1 is the mammalian counterpart of the Drosophila tumor suppressor Tid56 and is also a DnaJ protein containing a conserved J domain through which it interacts with the heat shock protein 70 (Hsp70) family of chaperone proteins. Several reports have indicated that Tid1 functions as a regulator in intracellular signaling pathways related to cell survival, senescence, and stress-induced cellular responses. Other reports raised the interesting possibility that it may exert tumor suppressor activity through cell-based assays. However, the biological function of Tid1 and how it acts as a tumor suppressor in vivo remain unclear because of lack of an instrumental in vivo analysis system. This research is focused on constructing a conditional knockout (CKO) targeting vector for mouse tid1 gene. We have employed a highly efficient recombineering-based method to make a tid1 CKO targeting vector in a specific Escherichia coli strain. The deleted region of tid1 gene is from putative promoter to exon 2, spanning approximate 5.8 kb genomic sequences, to not only disrupt the expression of tid1 transcript but also to cause a frameshift mutation with the consequence of early appearance of stop codon to generate null mutation. The constructed CKO targeting vector was subsequently linearized with restriction enzymes and electroporated into 129S6/SvEv-Tc1 ES cells to allow homologous recombination. The transformants were selected for their G418 and Ganciclovir (Ganc) resistance. Following electroporation, we screened 165 G418/Ganc resistant clones for correctly recombined clones by using Southern blot hybridization. We have up to now successfully obtained one clone of targeted ES cells with correct 5’ and 3' homologous recombination.

Autism Spectrum Disorders (ASDs) are neurodevelopmental syndromes, in which several environmental risk factors act on a vulnerable genetic background. Among genes whose mutations have been associated with ASDs, the R451C substitution in the synaptic protein Neuroligin3 (NLGN3) has been highly characterized. It is known from in vitro studies, that the mutation affects folding of the extracellular domain of the protein, causing its retention in the Endoplasmic Reticulum (ER) and the activation of the Unfolded Protein Response (UPR). It has been shown both in vitro and in vivo, that only ~10% of the mutant protein reach the synapse, causing loss of NLGN3 on the cell surface and leading to alterations in synaptic neurotransmission. In this work, we have evaluated whether UPR was activated in vivo, in the brain of the knock-in mouse model carrying the R451C mutation in the endogenous NLGN3. We showed a selective increase of UPR markers levels in the cerebellum of the R451C mice, along with an increase in the frequency of the miniature excitatory currents in the Purkinje cells, that resulted to be UPR-dependent. At the same time, in order to find a strategy to rescue NLGN3 folding and expression on the cell surface, we have generated and characterized a new cell-based model system that allowed studying NLGN3 protein trafficking. By using this system, we have screened an FDA-approved library of compounds for improving impaired protein folding. Among the compounds that have been tested, several members of the glucocorticoid family showed efficacy in increasing mutant protein trafficking and restoring membrane localization. Collectively, our data indicated that the ER-retention of R451C NLGN3 in vivo, caused UPR activation and alterations of synaptic function in the cerebellum of a mouse model of a monogenic form of autism. Furthermore, we identified compounds improving NLGN3 folding and rescuing impaired trafficking.

La stimulation du récepteur de la rénine/prorénine [(P) RR], un membre récemment découvert du système rénine-angiotensine (SRA), augmente l'activité du SRA et des voies de signalisation angiotensine II-indépendante. Pour étudier l'impact potentiel du (P)RR dans le développement de l`obésité, nous avons émis l'hypothèse que les souris déficientes en (P)RR uniquement dans le tissus adipeux (KO) auront une diminution du poids corporel en ciblant le métabolisme du tissu adipeux, l'activité locomoteur et/ou la prise alimentaire. Ainsi, des souris KO ont été générées en utilisant la technologie Cre/Lox. Le gain de poids et la prise alimentaire ont été évalués hebdomadairement dans les mâles et femelles KO et de type sauvage (WT) pendant 4 semaines alors qu’ils étaient maintenu sur une diète normal. De plus, un groupe de femelles a été placé pour 6 semaines sur une diète riche en gras et en glucides (HF/HC). La composition corporelle et l'activité ambulatoire ont été évaluées par l’EchoMRI et à l’aide de cages Physioscan, respectivement. Les tissus adipeux ont été prélevés et pesés. De plus, les gras péri-gonadaux ont été utilisés pour le microarray. Finalement, le niveaux d'expression d'ARNm du (P)RR ont été évalués. Comme le gène du (P)RR est situé sur le chromosome X, les mâles étaient des KOs complets et les femelles étaient des KOs partielles. Les souris KO avaient un poids corporel significativement plus petit par rapport à WT, les différences étant plus prononcées chez les mâles. De plus, les femelles KOs étaient résistantes à l'obésité lorsqu'elles ont été placées sur la diète HF/HC et donc elles avaient significativement moins de masse grasse par rapport aux WTs. L’analyse histologique des gras péri-gonadaux des KOs nous ont dévoilés qu’il avait une réduction du nombre d'adipocytes mais de plus grande taille. Bien qu'il n'y ait eu aucun changement dans la consommation alimentaire, une augmentation de près de 3 fois de l'activité ambulatoire a été détectée chez les mâles. De plus, nous avons observé que leurs tibias étaient de longueur réduite ce qui suggère fortement l'affection de leur développement. Les gras péri-gonadaux des souris KO avaient une expression réduite de l`ABLIM2 (Actin binding LIM protein family, member 2) qui est associé avec le diabète de type II chez l'humain. Ainsi, les données recueillies suggèrent fortement que le (P)RR est impliquée dans la régulation du poids corporelle.
Stimulation of the (pro)renin receptor [(P)RR], a recently discovered member of the renin-angiotensin system (RAS), increases the activity of the RAS and stimulates angiotensin II-independent signaling pathways. To investigate the possible impact of the (P)RR on obesity development, we hypothesized that mice deficient in the (P)RR specifically in their adipose tissue (KO) would have a decrease in body weight by targeting adipose tissue metabolism, locomotor activity and/or food intake. As such, KO mice were generated using the Cre/Lox technology. Weekly weight gain and food intake were assessed in both male and female KO and wild-type (WT) littermates for 4 weeks on a normal diet. A group of females were also placed for an additional 6 weeks on a high-fat/high-carbohydrate diet (HF/HC). Body composition and physical activity were evaluated using EchoMRI and Physioscan cages, respectively. Adipose tissues were collected and weighed at sacrifice. Moreover, perigonadal fat was used for Gene assay and histological analysis. (P)RR mRNA expression levels were evaluated using real-time PCR. Different circulating metabolites and proteinuria were measured by ELISA kits. As the (P)RR gene is located on the X chromosome, males were complete KOs and females were partial KOs. KO body weights were significantly lower compared to WTs, the differences being more pronounced in males. Female KOs were resistant to obesity development when placed on a HF/HC diet and as such, had significantly smaller fat mass as well as lower circulating leptin levels compared to WTs. All KO perigonadal fat had a reduced number of adipocytes but of bigger size. Although there were no changes in food intake, an almost 3-fold increase in activity was detected in males. Moreover, they presented with shorter tibial length which strongly suggests that they may have developmental issues. Gonadal fat of KO mice showed a reduced expression of ABLIM2 gene (Actin binding LIM protein family, member 2) which is associated with type II diabetes in humans. Conversely, no obvious changes in glycemia were detected while tendencies for lower proteinuria could be observed. The data collected thus strongly suggests that the (P)RR is implicated in body weight regulation.

Results of recent studies indicate that dendritic cells are capable of transporting commensal intestinal bacteria into the mammary glands, which ultimately leads to their occurrence in breast milk. We have therefore decided to evaluate the phenotype of immunologically relevant antigen presenting cells (APCs) present in the mammary glands and the small intestine, respectively and perform a comparison study. We also studied plasticity of these populations during lactation. In situ immunodetection and flow cytometry methods were used to determine phenotype. We succeeded in optimising the methods for preparation of samples for flow cytometry and microscopy. We thoroughly tested protocols for 3D visualisation of APC populations and quantitative image analysis for correlation with flow cytometry, further optimization is nevertheless needed. We found out that during lactation large numbers of MHC II+ cells cluster around the alveoli and milk ducts. These cells are of a distinctly dendritic shape and their phenotype does not correspond to the APCs in the surrounding tissue. A pronounced increase of APC cells in the mammary glands between the fourth and sixth days of lactation was observed, with the majority of these cells expressing the CD103 antigen typical for cell populations of immune cells of the...

The immune system is essential for keeping the integrity of multicellular organisms. We were able to make a step forward in studying the complex immune reactions in mammals in vivo and/ or in situ using the major histocompatibility complex (MHC) class II/ enhanced green fluorescent protein (EGFP) knock-in mouse model. Due to the EGFP visualization of MHC II expressing cells we were able to observe antigen presenting cells, which are essential for the onset of immune responses, in their natural environment. Thus, we report some original features of the immune system. We have identified MHC II+ cell clusters with unknown, probably unique function, in the intestine. We have also described MHC II+ cell migration to the lactating mammary gland and tested few hypotheses about the role of this phenomenon for the development of the mammary gland, milk secretion or infant immune system establishment. Lastly, we observed residential macrophages in the cornea. The presence of APCs in the cornea is a very contradictory issue due to the fact that cornea is an immunologically privileged tissue and therefore harbors special immune features. key words: antigen presenting cells (APC), major histocompatibility complex class II (MHC II), enhanced green fluorescent protein (EGFP), immune system, knock-in mouse model

Several forms of monogenic autism spectrum disorders are associated to mutations in the genes encoding for the postsynaptic cell adhesion molecules, Neuroligins. The autism-linked substitution of the arginine at position 451 by a cysteine (R451C) in Neuroligin3 induces local misfolding of the extracellular domain, causing partial retention in the endoplasmic reticulum. The accumulation of misfolded proteins in the endoplasmic reticulum can eventually result in stress conditions and ultimately in the activation of the unfolded protein response. We have generated a PC12 Tet-On cell system with inducible expression of either wild type or R451C Neuroligin3. In this system, we show that the over-expression of R451C NLGN3 leads to the activation of the unfolded protein response, both in proliferative and neuronal-differentiating conditions. The knockin mouse strain expressing R451C Neuroligin3 is currently considered a model for studying a monogenic form of autism spectrum disorders. We have characterized the effect of the mutation on Neuroligin3 protein levels during development from embryonic stage E12 to postnatal day P60, observing in the knockin mice lower Neuroligin3 protein levels and a delay in the expression of the protein. We showed that the endogenous mutant Neuroligin3 is partially retained in the endoplasmic reticulum as observed in vitro. In the R451C Neuroligin3 migrates in two different bands, representing the incompletely glycosylated ER-retained protein and the mature glycosylated form that traffics to the cell membrane. We have investigated the activation of the unfolded protein response in vivo, in the R451C Neuroligin3 mouse model and showed the Unfolded protein response and autism spectrum disorders upregulation of the two main targets, BiP and CHOP, in total brain and cerebellum extracts from both adult and embryonic knockin mice. BiP protein levels and the phosphorylation of eIF2α were significantly increased only in the cerebellum of adult knockin mice in comparison to wild type, in agreement with the mRNA data. Unfolded protein response signaling has been reported to regulate synaptic function and plasticity. The AMPA-mediated glutamatergic currents were studied in the cerebellum, where we observed a significant increase in miniature excitatory synaptic currents in Purkinje cells of the knockin in comparison to the wild type mice. The final aim of this thesis was focused on selecting molecules from a library of chemical compounds, acting in correcting the defective trafficking of mutant Neuroligin3. The cellbased screening used HEK293 stably transfected with a truncated and fluorescent form of R451C. We have identified one compound active on improving selectively the trafficking of the R451C along the secretory pathway. The effects caused by this compound are promising for evaluating in vivo the rescue of the behavioral and functional phenotype described for the R451C Knockin mouse. This molecule, or molecules structurally correlated, could be used for designing therapeutic strategies for monogenic forms of autism characterized by the retention of misfolded Neuroligins within the endoplasmic reticulum. In conclusion, we provide a link, both in vitro and in vivo, between UPR activation and a form of monogenic ASD caused the R451C misfolding mutation in Neuroligin3. We have identified in the cerebellum of the knockin mouse model expressing R451C Neuroligin3, the specific brain region where UPR targets and modulators are regulated. Lately this region has been implicated in cognitive and emotional traits typical of the autistic Unfolded protein response and autism spectrum disorders phenotype. Since UPR mediators are involved in neuronal plasticity, the activation of UPR in cerebellum can lead to neuronal circuits alterations and consequently have a role in the autistic phenotype.