Cortical origin of functional recovery in the somatosensory cortex of the adult mouse after thalamic lesion

Résumé L'accident vasculaire cérébral sensoriel pur est un des syndromes lacunaires, dû à l'occlusion de petits vaisseaux cérébraux, souvent dans le cadre d'une lésion intéressant le noyau ventro-caudal du thalamus. Il produit un hémisyndrome sensitif pur, et parfois un syndrome douloureux se développe à distance de l'événement aigu. Afin d'étudier la récupération fonctionnelle dans le cortex somatosensoriel (SI) après une telle lésion dans le thalamus, un modèle de lésion excitotoxique a été développé dans le système somatosensoriel de la souris adulte, caractérisé par la présence de formations cytoarchitectoniques dans SI appelées "tonneaux". Chacun de ces tonneaux correspond à la représentation corticale d'une vibrisse du museau. L'activité métabolique a été mesurée dans SI à différents intervalles après la lésion, à l'aide de déoxyglucose marqué radioactivement. Dans les deux premiers jours suivant celle-ci, l'activité métabolique diminue de manière importante dans toutes les couches corticales, avec une atteinte plus marquée dans la couche IV, principale projection des axones thalamo-corticaux. Une récupération de l'activité métabolique se produit ensuite, d'autant plus marquée que le délai après la lésion est grand. Cette récupération s'observe dans toutes les couches coticales, les couches I et Vb récupérant plus rapidement que les couches II, III, IV, Va et VI. Cinq semaines après la lésion, l'absence des vibrisses correspondant à la partie déafférentée de SI diminue l'activité métabolique corticale de 32% et démontre l'activation par la périphérie de cette partie de l'écorce, malgré la perte des axones thalamo-corticaux provenant du noyau ventro-caudal. Des expériences de traçage rétrograde ont montré une augmentation des projections intracorticales sur la partie déafférentée de l'écorce, en particulier de longue distance, ainsi que des projections interhémisphériques, mais n'ont pas permis de mettre en évidence de nouvelle projection thalamique, indiquant une origine corticale à la récupération fonctionnelle observée. Abstract To study the degree and time course of the functional recovery in the somatosensory cortex (SI) after an excitotoxic lesion in the adult mouse thalamus, metabolic activity was determined in SI at various times points post lesion. Immediately after the lesion, metabolic activity in the thalamically deafferented part of SI was at its lowest value but increased progressively at subsequent time points. This was seen in all cortical layers, however, layers I and Vb recover more rapidly than layers II, III, IV, Va and VI. Removal of the mystacial whiskers corresponding to the deafferented area, 5 weeks after cortical recovery, produced a subsequent 32% drop in metabolic activity, demonstrating peripheral sensory activation of this part of the cortex. Tracing experiments revealed that the deafferented cortex did not receive a novel thalamic input, but cortico-cortical and contralateral barrel cortex projections to this area were reinforced. We conclude that the cortical functional recovery after a thalamic lesion is, at least partially, due to modified cortico-cortical and callosal projections to the deafferented cortical area.

Innate Immune Sensing of Fusarium culmorum by Mouse Dendritic Cells.

Chronic inhalation of grain dust is associated with asthma and chronic bronchitis in grain worker populations. Exposure to fungal particles was postulated to be an important etiologic agent of these pathologies. Fusarium species frequently colonize grain and straw and produce a wide array of mycotoxins that impact human health, necessitating an evaluation of risk exposure by inhalation of Fusarium and its consequences on immune responses. Data showed that Fusarium culmorum is a frequent constituent of aerosols sampled during wheat harvesting in the Vaud region of Switzerland. The aim of this study was to examine cytokine/chemokine responses and innate immune sensing of F. culmorum in bone-marrow-derived dendritic cells and macrophages. Overall, dendritic cells and macrophages responded to F. culmorum spores but not to its secreted components (i.e., mycotoxins) by releasing large amounts of macrophage inflammatory protein (MIP)-1α, MIP-1β, MIP-2, monocyte chemoattractant protein (MCP)-1, RANTES, and interleukin (IL)-12p40, intermediate amounts of tumor necrosis factor (TNF), IL-6, IL-12p70, IL-33, granulocyte colony-stimulating factor (G-CSF), and interferon gamma-induced protein (IP-10), but no detectable amounts of IL-4 and IL-10, a pattern of mediators compatible with generation of Th1 or Th17 antifungal protective immune responses rather than with Th2-dependent allergic responses. The sensing of F. culmorum spores by dendritic cells required dectin-1, the main pattern recognition receptor involved in β-glucans detection, but likely not MyD88 and TRIF-dependent Toll-like receptors. Taken together, our results indicate that F. culmorum stimulates potently innate immune cells in a dectin-1-dependent manner, suggesting that inhalation of F. culmorum from grain dust may promote immune-related airway diseases in exposed worker populations.

When orthologs diverge between human and mouse.

Despite the common assumption that orthologs usually share the same function, there have been various reports of divergence between orthologs, even among species as close as mammals. The comparison of mouse and human is of special interest, because mouse is often used as a model organism to understand human biology. We review the literature on evidence for divergence between human and mouse orthologous genes, and discuss it in the context of biomedical research.

Role of cytokines in secondary lymphoid organ development

Summary Secondary lymphoid organs are sites of antigen presentation, clonal expansion of B and lymphocytes, and affinity maturation of B lymphocytes. In the intestine, these immune functions occur mainly in Peyer's patches (PP). PP develop through the interplay of two main cell types, haematopoietic cells and meserichyrnal cells. One particular haematopoietic cell type was identified as the inductive cell type in the formation of both PP and lymph nodes and was therefore designated as lymphoid tissue inducer cell. For a successful PP organogenesis, the crucial molecular components involved in the crosstalk of inducer cells and their mesenchymal target cells are adhesion molecules, lymphotoxin (LT) family members, and cytokines. In particular, the interleukin 7 receptor (IL-7R) expressed on inducer cells is absolutely required. To investigate the contribution of the ligand for the IL-7R. the cytokine IL-7, in the process of PP formation, we analyzed double transgenic (TG) mice. These mice resulted from an interbreeding of an IL-7TG mouse strain where the transgene is under the control of the MHC class II promoter with a second transgenic mouse strain, which overexpresses a transactivator for MHC class II genes. Double TG offsprings revealed higher levels of IL-7 mRNA occuring earlier in embryogenesis. Consequently, double TG mice showed a striking phenotype with a 3- to 5-fold increase in PP numbers compared to single IL-7TG or control littermates. Analysis of embryonic double TG intestines demonstrated that the process of PP development was already elevated during development as early as the embryonic day 16.5. Importantly, inducer cells were significantly increased in numbers in these embryonic intestines. Furthermore, the expression of LT? mRNA, which at this early time point is exclusively expressed by inducer cells, was also increased in double TG animals. These data clearly indicate a direct influence of IL-7 on the expansion of lymphoid tissue inducer cells and on the availability of LT? leading to a higher frequency of developing PP in fetal life. Interestingly, in addition to an enhanced frequency of PP development, in double TG mice, three additional phenotypic differences were observed. i) Lymphocyte infiltration in various non-lymphoid organs, such as stomach, salivary gland, and liver. Subsequent analysis demonstrated that B lymphocytes were predominant within these tertiary lymphoid structures. ii) Ectopic lymph node-like structures containing both B and T lymphocytes were found near the inguinal lymph node. iii) Double TG mice had a severe bone resorption syndrome most likely as a consequence of the pro-osteoclastic effect of IL-7. Taken together, these results show that IL-7 plays a key role in the homeostasis of inducer cells, in the generation of PP in the gut, in the formation of ectopic lymphoid tissue, and in bone resorption. Résumé Les organes lymphoïdes secondaires sont les lieux de présentation des antigènes aux lymphocytes, permettant l'expansion des lymphocytes B et T et la maturation d'affinité des lymphocytes B. Dans l'intestin, ces fonctions immunitaires se déroulent dans les plaques de Peyer (PP). Ces plaques se développent grâce à l'interaction des cellules hématopoïétiques avec des cellules mésenchymales. Un type particulier de cellules hématopoïétiques a été identifié comme cellule inductrice dans la formation des PP et des ganglions lymphatiques et de ce fait a été désigné cellule inductrice des tissus lymphoïdes. Durant l'organogénèse des PP, les composants moléculaires cruciaux impliqués dans l'interaction des cellules inductrices et des cellules mésenchymales sont les molécules d'adhésion, les membres de la famille des lymphotoxines (LT) et les cytokines. En particulier, le récepteur de l'interleukine 7 (IL-7R) exprimé par les cellules inductrices est absolument nécessaire. Pour étudier le rôle du ligand de l'IL-7R, l'interleukine IL-7, dans la formation des PP, nous avons croisé une lignée de souris transgénique (TG) surexprimant IL-7 sous contrôle du promoteur MHC class Il avec une lignée de souris transgénique surexprimant un transactivateur des genes MHC class II. Les souris doubles TG présentent une concentration élevée d'ARNm de l'IL-7 durant l'embryogénèse, ce qui résulte en une augmentation du nombre de PP de 3 à 5 fois en comparaison aux souris ayant seul le transgène IL-7 et aux souris contrôles. L'analyse des intestins des souris doubles TG démontre que le processus de développement des PP était élevé dès le jour 16.5 du développement embryonnaire. L'augmentation du nombre des cellules inductrices dans ces intestins embryonnaires est signilicative. De plus l'expression de l'ARNm LT?, qui à ce stade précoce est exclusivement exprimé dans les cellules inductrices, est également augmenté dans les doubles TG. Ces résultats indiquent clairement une influence directe d'IL-7 sur l'expansion des cellules inductrices des tissues lymphoïdes et sur la synthèse de LT? induisant une augmentation des PP se développant durant la vie foetale. En plus du développement accru des PP dans les souris doubles TG, trois différences phénotypiques ont été observées. i) L'infiltration lymphocytaire dans différents organes non-lymphoïdes, comme l'estomac, les glandes salivaires et le foie. Des analyses complémentaires ont demontré que les lymphocytes B étaient prédominants dans ces structures lymphoïdes tertiaires. ii) Des structures de ganglions lymphatiques ectopiques contenant des lymphocytes B et T ont été trouvées près des ganglions lymphatiques inguinaux. iii) Les souris doubles TG présentent un syndrome de résorption osseuse sévère probablement dû à l'effet pro-osteoclaste d'IL-7. Globalement, ces résultats montrent que IL-7 joue un rôle clé dans l'homéostasie des cellules inductrices dans la génèse de PP de l'intestin, dans la formation des tissus lymphoïdes ectopiques et dans la résorption osseuse.

Proteomic and Metabolomic Analyses of Mitochondrial Complex I-deficient Mouse Model Generated by Spontaneous B2 Short Interspersed Nuclear Element (SINE) Insertion into NADH Dehydrogenase (Ubiquinone) Fe-S Protein 4 (Ndufs4) Gene.

Eukaryotic cells generate energy in the form of ATP, through a network of mitochondrial complexes and electron carriers known as the oxidative phosphorylation system. In mammals, mitochondrial complex I (CI) is the largest component of this system, comprising 45 different subunits encoded by mitochondrial and nuclear DNA. Humans diagnosed with mutations in the gene NDUFS4, encoding a nuclear DNA-encoded subunit of CI (NADH dehydrogenase ubiquinone Fe-S protein 4), typically suffer from Leigh syndrome, a neurodegenerative disease with onset in infancy or early childhood. Mitochondria from NDUFS4 patients usually lack detectable NDUFS4 protein and show a CI stability/assembly defect. Here, we describe a recessive mouse phenotype caused by the insertion of a transposable element into Ndufs4, identified by a novel combined linkage and expression analysis. Designated Ndufs4(fky), the mutation leads to aberrant transcript splicing and absence of NDUFS4 protein in all tissues tested of homozygous mice. Physical and behavioral symptoms displayed by Ndufs4(fky/fky) mice include temporary fur loss, growth retardation, unsteady gait, and abnormal body posture when suspended by the tail. Analysis of CI in Ndufs4(fky/fky) mice using blue native PAGE revealed the presence of a faster migrating crippled complex. This crippled CI was shown to lack subunits of the "N assembly module", which contains the NADH binding site, but contained two assembly factors not present in intact CI. Metabolomic analysis of the blood by tandem mass spectrometry showed increased hydroxyacylcarnitine species, implying that the CI defect leads to an imbalanced NADH/NAD(+) ratio that inhibits mitochondrial fatty acid β-oxidation.

Acute flare induced by the calcific tendonitis of the rotator cuff: inhibition of IL-1 a potential therapeutic target?

Introduction: Calcific tendonitis of rotator cuff is observed on plainradiographs in 10% of adults, but remains asymptomatic in half thesecases. Sometimes, these calcifications induce acute flares withmassive inflammation similar to gout or CPPD crisis. Analgesics/anti-inflammatory medications are usually not sufficient to controlssymptoms in these situations. Local steroid infiltration with or withoutremoval of the calcific deposition with a needle aspiration may beuseful. A new approach could be IL-1 inhibitors. Indeed, basic calciumphosphate crystals are capable of stimulating the release of activeIL-1β in vitro. These crystals trigger IL-1β release, in an analogousmanner to MSU crystals in acute gout, suggesting that IL-1β blockademay be clinically useful.Case presentation: This report describes a 70-year old woman withacute rest pain of the right shoulder since 48 hours. On examination,we found massive limitations of active and passive movements. Thepatient evaluated, on the visual scale, her symptoms at 10/10 the nightand 5/10 the day. The radiography and showed a rounded, 8 mmcalcification in the subscapularis tendon. The ultrasound aspectrevealed a heterogeneous calcification partially non solid, surroundedby massive inflammation on Doppler. C-reactive protein anderythrocyte sedimentation rate were high (74 mg/ml, 54 mm/hour).The patient received subcutaneous injections of anakinra: 100 mgdaily for 3 days (D1-D3). We evaluated the patient in our consult at dayD1, D2, D3, D7, D16 and by phone at D70.This treatment rapidly relieved the inflammatory symptoms (within afew hours with no relapse). The mobility of the shoulder, the biologicsparameters improved and the size of the calcification as well thedegree of inflammation regressed on ultrasound after 3 days.Conclusion: This is the first report of a woman with an acute flareinduced by calcific tendonitis who received anakinra. IL-1 inhibitionmay be a therapeutic target in calcific tendonitis. To analyse thisresponse more precisely and elaborate definitive conclusions, aprospective pilot study is on-going in our ambulatory institute.

Functional and histopathological identification of the respiratory failure in a DMSXL transgenic mouse model of myotonic dystrophy.

Acute and chronic respiratory failure is one of the major and potentially life-threatening features in individuals with myotonic dystrophy type 1 (DM1). Despite several clinical demonstrations showing respiratory problems in DM1 patients, the mechanisms are still not completely understood. This study was designed to investigate whether the DMSXL transgenic mouse model for DM1 exhibits respiratory disorders and, if so, to identify the pathological changes underlying these respiratory problems. Using pressure plethysmography, we assessed the breathing function in control mice and DMSXL mice generated after large expansions of the CTG repeat in successive generations of DM1 transgenic mice. Statistical analysis of breathing function measurements revealed a significant decrease in the most relevant respiratory parameters in DMSXL mice, indicating impaired respiratory function. Histological and morphometric analysis showed pathological changes in diaphragmatic muscle of DMSXL mice, characterized by an increase in the percentage of type I muscle fibers, the presence of central nuclei, partial denervation of end-plates (EPs) and a significant reduction in their size, shape complexity and density of acetylcholine receptors, all of which reflect a possible breakdown in communication between the diaphragmatic muscles fibers and the nerve terminals. Diaphragm muscle abnormalities were accompanied by an accumulation of mutant DMPK RNA foci in muscle fiber nuclei. Moreover, in DMSXL mice, the unmyelinated phrenic afferents are significantly lower. Also in these mice, significant neuronopathy was not detected in either cervical phrenic motor neurons or brainstem respiratory neurons. Because EPs are involved in the transmission of action potentials and the unmyelinated phrenic afferents exert a modulating influence on the respiratory drive, the pathological alterations affecting these structures might underlie the respiratory impairment detected in DMSXL mice. Understanding mechanisms of respiratory deficiency should guide pharmaceutical and clinical research towards better therapy for the respiratory deficits associated with DM1.

The role of the hypothalamic kisspeptin/GPR54 system in the control of GnRH neurons

Summary : The hypothalamus represents less than 1 % of the total volume of the brain tissue, yet it plays a crucial role in endocrine regulations. Puberty is defined as a process leading to physical, sexual and psychosocial maturation. The hypothalamus is central to this process, via the activation of GnRH neurons. Pulsatile GnRH secretion, minimal during childhood, increases with the onset of puberty. The primary function of GnRH is to regulate the growth, development and function of testes in boys and ovaries in girls, by stimulating the pituitary gland secretion of luteinizing hormone (LH) and follicle-stimulating hormone (FSH). Several factors contribute to the timing of puberty, including sex and ethnicity, genetics, dietary intake and energy expenditure. Kisspeptins constitute a family of small peptides arising from the proteolytic cleavage of metastin, a peptide with 54 amino acids initially purified from human placenta. These kisspeptins were the subject of much attention following their discovery because of their antimetastatic properties, but it was more recently that their determining role in the reproductive function was demonstrated. It was shown that kisspeptins are ligands of a receptor, GPR54, whose natural inactivating mutation in humans, or knockout in the mouse, lead to infertility. GnRH neurons play a pivotal role in the central regulation of fertility. Kisspeptin greatly increases GnRH release and GnRH neuron firing activity, but the neurobiological mechanisms for these actions are unknown. Gprotein-coupled receptor 54, the receptor for kisspeptin, is expressed by GnRH neurons as well as other hypothalamic neurons, suggesting that both direct and indirect effects are possible. In the first part of my thesis, we investigated a possible connection between the acceleration of sexual development induced by leptin and hypothalamic metastin neurons. However, the data generated by our preliminary experiments confirmed that the commercially available antibodies are non-specific. This finding constituted a major drawback for our studies, which relied heavily upon the neuroanatomical study of the hypothalamic metastinergic pathways to elucidate their sensitivity to exogenous leptin. Therefore, we decided to postpone any further in vivo experiment until a better antibody becomes available, and focused on in vitro studies to better understand the mechanisms of action of kisspeptins in the modulation of the activity of GnRH neurons. We used two GnRH-expressing neuronal cell lines to investigate the cellular and molecular mechanisms of action of metastin in GnRH neurons. We demonstrated that kisspeptin induces an early activation of the MAP kinase intracellular signaling pathway in both cell lines, whereas the SAP/JNK or the Akt pathways were unaffected. Moreover, we found an increase in GnRH mRNA levels after 6h of metastin stimulation. Thus, we can conclude that kisspeptin regulates GnRH neurons both at the secretion and the gene expression levels. The MAPK pathway is the major pathway activated by metastin in GnRH expressing neurons. Taken together, these data provide the first mechanism of action of kisspeptin on GnRH neurons. Résumé : L'hypothalamus est une zone située au centre du cerveau, dont il représente moins de 1 du volume total. La puberté est la période de transition entre l'enfance et l'age adulte, qui s'accompagne de transformations somatiques, psychologiques, métaboliques et hormonales conduisant à la possibilité de procréer. La fonction principale de la GnRH est la régulation de la croissance, du développement et de la fonction des testicules chez les hommes, et des ovaires chez les femmes en stimulant la sécrétion de l'hormone lutéinisante (LH) et de l'hormone folliculostimulante (FSH) par la glande hypophysaire. Plusieurs facteurs contribuent au déclanchement de la puberté, y compris le sexe et l'appartenance ethnique, la génétique, l'apport alimentaire et la dépense énergétique. Les Kisspeptines constituent une famille de peptides résultant de la dissociation proteolytique de la métastine, un peptide de 54 acides aminés initialement purifié à partir de placenta humain. Ces kisspeptines ont fait l'objet de beaucoup d'attention à la suite de leur découverte en raison de leurs propriétés anti-metastatiques, et c'est plus récemment que leur rôle déterminant dans la fonction reproductive a été démontré. Les kisspeptines sont des ligands du récepteur GPR54, dont la mutation inactivatrice chez l'homme, ou le knockout chez la souris, conduisent à l'infertilité par hypogonadisme hypogonadotrope. Les neurones à GnRH jouent un rôle central dans le règlement des fonctions reproductrices et la kisspeptine stimule l'activité des neurones à GnRH et la libération de GnRH par ces neurones. Toutefois, les mécanismes neurobiologiques de ces actions ne sont pas connus. Dans la première partie de ma thèse, nous avons étudié le lien potentiel entre l'accélération du développement sexuel induite par la leptine et les neurones hypothalamiques à metastine. Les données générées dans cette première série d'expériences ont malheureusement confirmé que les anticorps anti-metastine disponibles dans le commerce sont aspécifiques. Ceci a constitué un inconvénient majeur pour nos études, qui devaient fortement s'appuyer sur l' étude neuroanatomique des neurones hypothalamiques à metastine pour évaluer leur sensibilité à la leptine exogène. Nous avons donc décidé de focaliser nos travaux sur une étude in vitro des mécanismes d'action de la kisspeptine pour moduler l'activité des neurones à GnRH. Nous avons utilisé deux lignées de cellules neuronales exprimant la GnRH pour étudier les mécanismes d'action cellulaires et moléculaires de la metastine dans des neurones. Nous avons ainsi pu démontrer que la kisspeptine induit une activation précoce de la voie f de signalisation de la MAP kinase dans les deux lignées cellulaires, alors que nous n'avons observé aucune activation de la voie de signalisation de la P13 Kinase et de la SAP/JNK. Nous avons en outre démontré une augmentation de l'expression de la GnRH par la stimulation avec la Kisspeptine. L'ensemble de ces données contribue à élucider le mécanisme d'action avec lequel la kisspeptine agit dans les neurones à GnRH, en démontrant un effet sur l'expression génique de la GnRH. Nous pouvons également conclure que la voie de la MAPK est la voie principale activée par la metastine dans les neurones exprimant la GnRH.

Redirection of T cells by delivering a transgenic mouse-derived MDM2 tumor antigen-specific TCR and its humanized derivative is governed by the CD8 coreceptor and affects natural human TCR expression.

Retroviral transfer of T cell antigen receptor (TCR) genes selected by circumventing tolerance to broad tumor- and leukemia-associated antigens in human leukocyte antigen (HLA)-A*0201 (A2.1) transgenic (Tg) mice allows the therapeutic reprogramming of human T lymphocytes. Using a human CD8 x A2.1/Kb mouse derived TCR specific for natural peptide-A2.1 (pA2.1) complexes comprising residues 81-88 of the human homolog of the murine double-minute 2 oncoprotein, MDM2(81-88), we found that the heterodimeric CD8 alpha beta coreceptor, but not normally expressed homodimeric CD8 alpha alpha, is required for tetramer binding and functional redirection of TCR- transduced human T cells. CD8+T cells that received a humanized derivative of the MDM2 TCR bound pA2.1 tetramers only in the presence of an anti-human-CD8 anti-body and required more peptide than wild-type (WT) MDM2 TCR+T cells to mount equivalent cytotoxicity. They were, however, sufficiently effective in recognizing malignant targets including fresh leukemia cells. Most efficient expression of transduced TCR in human T lymphocytes was governed by mouse as compared to human constant (C) alphabeta domains, as demonstrated with partially humanized and murinized TCR of primary mouse and human origin, respectively. We further observed a reciprocal relationship between the level of Tg WT mouse relative to natural human TCR expression, resulting in T cells with decreased normal human cell surface TCR. In contrast, natural human TCR display remained unaffected after delivery of the humanized MDM2 TCR. These results provide important insights into the molecular basis of TCR gene therapy of malignant disease.

Phenotypic consequences of copy number variation: insights from Smith-Magenis and Potocki-Lupski syndrome mouse models.

A large fraction of genome variation between individuals is comprised of submicroscopic copy number variation of genomic DNA segments. We assessed the relative contribution of structural changes and gene dosage alterations on phenotypic outcomes with mouse models of Smith-Magenis and Potocki-Lupski syndromes. We phenotyped mice with 1n (Deletion/+), 2n (+/+), 3n (Duplication/+), and balanced 2n compound heterozygous (Deletion/Duplication) copies of the same region. Parallel to the observations made in humans, such variation in gene copy number was sufficient to generate phenotypic consequences: in a number of cases diametrically opposing phenotypes were associated with gain versus loss of gene content. Surprisingly, some neurobehavioral traits were not rescued by restoration of the normal gene copy number. Transcriptome profiling showed that a highly significant propensity of transcriptional changes map to the engineered interval in the five assessed tissues. A statistically significant overrepresentation of the genes mapping to the entire length of the engineered chromosome was also found in the top-ranked differentially expressed genes in the mice containing rearranged chromosomes, regardless of the nature of the rearrangement, an observation robust across different cell lineages of the central nervous system. Our data indicate that a structural change at a given position of the human genome may affect not only locus and adjacent gene expression but also "genome regulation." Furthermore, structural change can cause the same perturbation in particular pathways regardless of gene dosage. Thus, the presence of a genomic structural change, as well as gene dosage imbalance, contributes to the ultimate phenotype.

Overexpression of mutant ataxin-3 in mouse cerebellum induces ataxia and cerebellar neuropathology.

Machado-Joseph disease (MJD), also known as spinocerebellar ataxia type 3 (SCA3), is a fatal, dominant neurodegenerative disorder caused by the polyglutamine-expanded protein ataxin-3. Clinical manifestations include cerebellar ataxia and pyramidal signs culminating in severe neuronal degeneration. Currently, there is no therapy able to modify disease progression. In the present study, we aimed at investigating one of the most severely affected brain regions in the disorder-the cerebellum-and the behavioral defects associated with the neuropathology in this region. For this purpose, we injected lentiviral vectors encoding full-length human mutant ataxin-3 in the mouse cerebellum of 3-week-old C57/BL6 mice. We show that circumscribed expression of human mutant ataxin-3 in the cerebellum mediates within a short time frame-6 weeks, the development of a behavioral phenotype including reduced motor coordination, wide-based ataxic gait, and hyperactivity. Furthermore, the expression of mutant ataxin-3 resulted in the accumulation of intranuclear inclusions, neuropathological abnormalities, and neuronal death. These data show that lentiviral-based expression of mutant ataxin-3 in the mouse cerebellum induces localized neuropathology, which is sufficient to generate a behavioral ataxic phenotype. Moreover, this approach provides a physiologically relevant, cost-effective and time-effective animal model to gain further insights into the pathogenesis of MJD and for the evaluation of experimental therapeutics of MJD.

Expression analysis of the AtPHO1 gene family in Arabidopsis thaliana and the involvement of AtPHO1 in the regulation of stomatal movements

Résumé Le transfert du phosphate des racines vers les feuilles s'effectue par la voie du xylème. Il a été précédemment démontré que la protéine AtPHO1 était indispensable au transfert du phosphate dans les vaisseaux du xylème des racines chez la plante modèle Arabidopsis thaliana. Le séquençage et l'annotation du génome d'Arabidopsis ont permis d'identifier dix séquences présentant un niveau de similarité significatif avec le gène AtPHO1 et constituant une nouvelle famille de gène appelé la famille de AtPHO1. Basée sur une étude moléculaire et génétique, cette thèse apporte des éléments de réponse pour déterminer le rôle des membres de ia famille de AtPHO1 chez Arabidopsis, inconnue à ce jour. Dans un premier temps, une analyse bioinformatique des séquences protéiques des membres de la famille de AtPHO1 a révélé la présence dans leur région N-terminale d'un domaine nommé SPX. Ce dernier est conservé parmi de nombreuses protéines impliquées dans l'homéostasie du phosphate chez la levure, renforçant ainsi l'hypothèse que les membres de la famille de AtPHO1 auraient comme AtPHO1 un rôle dans l'équilibre du phosphate dans la plante. En parallèle, la localisation tissulaire de l'expression des gènes AtPHO dans Arabidopsis a été identifiée par l'analyse de plantes transgéniques exprimant le gène rapporteur uidA sous le contrôle des promoteurs respectifs des gènes AtPHO. Un profil d'expression de chaque gène AtPHO au cours du développement de la plante a été obtenu. Une expression prédominante au niveau des tissus vasculaires des racines, des feuilles, des tiges et des fleurs a été observée, suggérant que les gènes AtPHO pourraient avoir des fonctions redondantes au niveau du transfert de phosphate dans le cylindre vasculaire de ces différents organes. Toutefois, plusieurs régions promotrices des gènes AtPHO contrôlent également un profil d'expression GUS non-vasculaire, indiquant un rôle putatif des gènes AtPHO dans l'acquisition ou le recyclage de phosphate dans la plante. Dans un deuxième temps, l'analyse de l'expression des gènes AtPHO durant une carence en phosphate a établi que seule l'expression des gènes AtPHO1, AtPHO1; H1 et AtPHO1; H10 est régulée par cette carence. Une étude approfondie de leur expression en réponse à des traitements affectant l'homéostasie du phosphate dans la plante a ensuite démontré leur régulation par différentes voies de signalisation. Ensuite, une analyse détaillée de la régulation de l'expression du gène AtPHO1; H1O dans des feuilles d'Arabidopsis blessées ou déshydratées a révélé que ce gène constitue le premìer gène marqueur d'une nouvelle voie de signalisation induite par l'OPDA, pas par le JA et dépendante de la protéine COI1. Ces résultats démontrent pour la première fois que l'OPDA et le JA peuvent activer différents gènes via des voies de signalisation dépendantes de COI1. Enfin, cette thèse révèle l'identification d'un nouveau rôle de la protéine AtPHO1 dans la régulation de l'action de l'ABA au cours des processus de fermeture stomatique et de germination des graines chez Arabidopsis. Bien que les fonctions exactes des protéines AtPHO restent à être déterminées, ce travail de thèse suggère leur implication dans la propagation de différents signaux dans la plante via la modulation du potentiel membranaire et/ou l'affectation de la composition en ions des cellules comme le font de nombreux transporteurs ou régulateur du transport d'ions. Summary Phosphate is transferred from the roots to the shoot via the xylem. The requirement for AtPHO1 protein to transfer phosphate to the xylem vessels of the root has been previously demonstrated in Arabidopsis thaliana. The sequencing and the annotation of the Arabidopsis genome had allowed the identification of ten sequences that show a significant level of similarity with the AtPHO1 gene. These 10 genes, of unknown functions, constitute a new gene family called the AtPHO1 gene family. Based on a molecular and genetics study, this thesis reveals some information needed to understand the role of the AtPHO1 family members in the plant Arabidopsis. First, a bioinformatics study revealed that the AtPHO sequences contained, in the N-terminal hydrophilic region, a motif called SPX and conserved among multiple proteins involved in phosphate homeostasis in yeast. This finding reinforces the hypothesis that all AtPHO1 family members have, as AtPHO1, a role in phosphate homeostasis. In parallel, we identified the pattern of expression of AtPHO genes in Arabidopsis via analysis of transgenic plants expressing the uidA reporter gene under the control of respective AtPHO promoter regions. The results exhibit a predominant expression of AtPHO genes in vascular tissues of all organs of the plant, implying that these AtPHO genes could have redundant functions in the transfer of phosphate to the vascular cylinder of various organs. The GUS expression pattern for several AtPHO promoter regions was also detected in non-vascular tissue indicating a broad role of AtPHO genes in the acquisition or in the recycling of phosphate in the plant. In a second step, the analysis of the expression of AtPHO genes during phosphate starvation established that only the expression of the AtPHO1, AtPHO1; H1 and AtPHO1; H10 genes were regulated by Pi starvation. Interestingly, different signalling pathways appeared to regulate these three genes during various treatments affecting Pi homeostasis in the plant. The third chapter presents a detailed analysis of the signalling pathways regulating the expression of the AtPHO1; H10 gene in Arabidopsis leaves during wound and dehydrated stresses. Surprisingly, the expression of AtPHO1; H10 was found to be regulated by OPDA (the precursor of JA) but not by JA itself and via the COI1 protein (the central regulator of the JA signalling pathway). These results demonstrated for the first time that OPDA and JA could activate distinct genes via COI1-dependent pathways. Finally, this thesis presents the identification of a novel role of the AtPHO1 protein in the regulation of ABA action in Arabidopsis guard cells and during seed germination. Although the exact role and function of AtPHO1 still need to be determined, these last findings suggest that AtPHO1 and by extension other AtPHO proteins could mediate the propagation of various signals in the plant by modulating the membrane potential and/or by affecting cellular ion composition, as it is the case for many ion transporters or regulators of ion transport.