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<p>The primary cilium is a singular signaling organelle found on most mammalian cell types. Dysfunction of the primary cilium or associated structures form a group of genetic disorders called ciliopathies. Recently, Huntington’s disease (HD), a monogenetic neurodegenerative disorder, was classified, at least in part, as a ciliopathy. How the primary cilium contributes to the pathogenesis of HD is the focus of this work. We demonstrate that huntingtin localization to the basal body or primary cilium is dependent on the phosphorylation status of serine residues 13 and 16. Furthermore, we demonstrate that, compared to controls, HD cell models have an increased number of cells with a primary cilium and that these cells have higher presence of huntingtin within the ciliary compartment. The primary cilia that form in HD cell lines demonstrate abnormal dynamics and morphology with bulging tips, characteristic of defective retrograde trafficking. We also demonstrate that alpha tubulin acetyltransferase 1 (αTAT1) expression and localization is increased in the primary cilium of HD cell lines. Subsequently, the primary cilium of HD cell lines are highly acetylated when compared to controls. These data support that primary cilia structure, ciliogenesis and ciliome are altered in HD.</p> / Master of Science (MSc)

京都大学 / 新制・課程博士 / 博士(薬科学) / 甲第23474号 / 薬科博第144号 / 新制||薬科||16(附属図書館) / 京都大学大学院薬学研究科薬科学専攻 / (主査)教授 中山 和久, 教授 井垣 達吏, 教授 土居 雅夫 / 学位規則第4条第1項該当 / Doctor of Pharmaceutical Sciences / Kyoto University / DFAM

BACKGROUND:Primary cilia are microtubule-based organelles that protrude from the cell surface. Primary cilia play a critical role in development and disease through regulation of signaling pathways including the Hedgehog pathway. Recent mouse models have also linked ciliary dysfunction to cancer. However, little is known about the role of primary cilia in breast cancer development. Primary cilia expression was characterized in cancer cells as well as their surrounding stromal cells from 86 breast cancer patients by counting cilia and measuring cilia length. In addition, we examined cilia expression in normal epithelial and stromal cells from reduction mammoplasties as well as histologically normal adjacent tissue for comparison.RESULTS:We observed a statistically significant decrease in the percentage of ciliated cells on both premalignant lesions as well as in invasive cancers. This loss of cilia does not correlate with increased proliferative index (Ki67-positive cells). However, we did detect rare ciliated cancer cells present in patients with invasive breast cancer and found that these express a marker of basaloid cancers that is associated with poor prognosis (Cytokeratin 5). Interestingly, the percentage of ciliated stromal cells associated with both premalignant and invasive cancers decreased when compared to stromal cells associated with normal tissue. To understand how cilia may be lost during cancer development we analyzed the expression of genes required for ciliogenesis and/or ciliary function and compared their expression in normal versus breast cancer samples. We found that expression of ciliary genes were frequently downregulated in human breast cancers.CONCLUSIONS:These data suggest that primary cilia are lost early in breast cancer development on both the cancer cells and their surrounding stromal cells.

An anti-G"11 antibody was used to label neuronal cilia throughout the rat central nervous system. Immunoreactive cilia were observed in every examined region of the rat CNS, but not in monkey or mouse tissue. Antibodies to G"q and G"q/11 failed to label cilia. Immunoreactive cilia were observed as early as postnatal day 0 in spinal tissue, and postnatal day 3 in hypothalamic tissue. There was a statistically significant negative correlation between a region's mean cilium length and that region's distance to the nearest ventricle; regions nearest ventricles were those with the longest cilia. This correlation suggests neuronal cilia may function as chemosensors, detecting substances as they move out from the cerebrospinal fluid and into the extracellular space of the brain.

Résumé confidentiel / Confidential abstract

Résumé confidentiel / Confidential abstract

Le cil primaire est une antenne sensorielle présente à la surface de la plupart des cellules qui contrôle des voies de signalisation clés au cours du développement et de l’homéostasie tissulaire. Des défauts de formation ou de fonctionnement des cils sont responsables de maladies génétiques complexes appelées ciliopathies. La néphronophtise (NPH) est une ciliopathie caractérisée par une néphropathie tubulo-interstitielle chronique évoluant généralement vers l’insuffisance rénale terminale (IRT) avant l’âge adulte. La NPH peut être isolée ou associée à des signes extra-rénaux tels que la rétinite pigmentaire et des défauts du squelette permettant de définir des syndromes comme celui de Saldino-Mainzer (MZSDS). La NPH est une maladie à transmission autosomique récessive très hétérogène sur le plan génétique et les protéines codées par les gènes identifiés ont quasiment toutes été impliquées dans des fonctions ciliaires. Le séquençage d’exome de patients, ciblant plus de 1300 gènes ciliaires (ciliome), a permis de mettre en évidence des mutations dans deux nouveaux gènes candidats pour la NPH : CEP83 et TEKT1. Mon travail de thèse a consisté à caractériser l’effet des mutations et à valider leur implication dans les phénotypes des patients. CEP83 a été retrouvé muté chez plusieurs patients non-apparentés présentant une NPH avec IRT précoce (< 5 ans). CEP83 est un composant des appendices distaux du centriole père qui joue un rôle clé dans les étapes précoces de la formation du cil. J’ai montré que les mutations identifiées entraînaient une désorganisation des appendices distaux qui pourrait expliquer les défauts de ciliogénèse observés dans les fibroblastes et les biopsies rénales de patients. Ces résultats ont permis de démontrer l’implication d’une nouvelle protéine centriolaire dans la physiopathologie des formes sévères de NPH. TEKT1 présente des mutations hétérozygotes composites chez un patient ayant un tableau clinique complexe associant un MZSDS et une dyskinésie ciliaire primitive (PCD) due à des défauts de cils motiles. Une analyse génétique détaillée a mis en évidence des mutations sévères dans un second gène, WDR19, déjà caractérisé dans les formes de NPH associées à des défauts osseux. TEKT1 code la protéine Tektine-1, un membre encore non caractérisé de la famille des tektines impliquées dans les cils motiles. L’analyse de cellules nasales multiciliées a montré que Tektine-1 était localisée le long de l’axoneme des cils motiles contrôles et absent des cils des cellules du patient qui présentaient aussi des anomalies sévères de battement. En parallèle, des défauts de ciliogénèse, typiques de mutations de WDR19, ont été observés dans les fibroblastes du patient. Ces résultats suggèrent que ce phénotype complexe est dû aux effets complémentaires des mutations des deux gènes TEKT1 et WDR19, responsables des défauts dans les cils motiles et primaires, respectivement. / The primary cilium is a sensory antenna present on the surface of most of the cells. It controls key signaling pathways during development and tissue homeostasis. Defects in cilia growth or activity are responsible for complex genetic diseases called ciliopathies. Nephronophthisis (NPH) is a ciliopathy characterized by chronic tubulointerstitial nephritis which usually progresses to end-stage renal disease (ESRD) before adulthood. NPH may be isolated or associated with extra-renal defects such as retinitis pigmentosa and skeleton involvement. The combination of these symptoms defines syndromes such as Saldino-Mainzer (MZSDS). NPH is an autosomal recessive disorder highly genetically heterogeneous and almost all of proteins encoded by the identified genes have been involved in ciliary function. The exome sequencing in patients, targeting up to 1300 ciliary genes (ciliome), highlighted new mutations in 2 NPH candidate genes: CEP83 and TEKT1. My work was to characterize the effects of the mutations and validate their involvement in patient phenotypes. CEP83 was found mutated in several unrelated patients with early-onset of NPH (IRT<5 years). CEP83 is a component of distal appendages on the mother centriole which play a crucial role in the early steps of cilia formation. I have shown that the identified mutations perturbed the distal appendages formation which might explain the defects in ciliogenesis observed in fibroblasts and kidney biopsies from patients. These results have demonstrated the involvement of a new centriolar protein in the pathophysiology of NPH severe forms. TEKT1 presents compound heterozygous mutations in a patient with a complex phenotype combining a MZSDS and primary ciliary dyskinesia (PCD) due to defects in motile cilia. The genetic analysis showed mutations in a second gene, WDR19, already characterized in NPH associated with bone defects. TEKT1 encodes the Tektin-1 protein, an uncharacterized member of the tektin family involved in motile cilia. The nasal multiciliated cells analysis showed that Tektin-1 was localized along the axoneme of control motile cilia and absent from the cilia in patient cells, which also had severe beating impairment. In parallel, defects in ciliogenesis, typical of WDR19 mutations, were observed in the fibroblasts from the patient. These results suggest that this dual ciliary phenotype is rather due to the additional effect of mutations in both TEKT1 and WDR19, responsible for the defects in motile and primary cilia, respectively.

Thesis (Ph. D.)--University of Virginia, 2008. / Title from title page. Includes bibliographical references. Also available online through Digital Dissertations.

Primary cilia are sensory organelles that are important for initiating cell division in the brain, especially through sonic hedgehog (Shh) signaling. Several lines of evidence suggest that the mitogenic effect of Shh requires primary cilia. Proliferation initiated by Shh signaling plays key roles in brain development, in neurogenesis in the adult hippocampus, and in the generation of glial cells in response to cortical injury. In spite of the likely involvement of cilia in these events, little is known about their characteristics. Centrosomes, which are associated with primary cilia, also have multiple influences on the cell cycle, and they are important in assembling microtubules for the maintenance of the cell’s cytoskeleton and cilia. The cilia of terminally differentiated neurons have been previously examined with respect to length, incidence, and receptors present. However, almost nothing is known about primary cilia in stem cells, progenitors, or differentiated glial cells. Moreover, it is not known how the properties of cilia and centrosomes may vary with cell cycle or proliferative potential, in brain or other tissues. This dissertation focuses first on neurogenesis in the hippocampal subgranular zone (SGZ). The SGZ is one of the few brain regions in mammals that gives rise to a substantial number of new neurons throughout adulthood. The neuron lineage contains a progression of identifiable precursor cell types with different proliferation rates. This present study found that primary cilia were present in every cell type in the neuronal lineage in SGZ. Cilium length and incidence were positively correlated among these cell types. Ciliary levels of adenylyl cyclase type III (ACIII) levels relative to ADP-ribosylation factor-like protein 13b (Arl13b) was higher in neurons than in precursor cells and glia, and also changed with the cell cycle. G-protein coupled receptors, SstR3, MCHR1, and Gpr161 receptors were only found in neuronal cilia. The levels and distribution of three centrosomal proteins, γ-tubulin, pericentrin and cenexin in neurons was different from the distributions in precursors and glia. The second focus of study is glial responses to injury in the neocortex, which has been widely studied as an injury model. This study found that in the normal adult somatosensory cortex, primary cilia were present in astrocytes and polydendrocytes but not in microglia. Following injury, the incidence of primary cilia decreased in astrocytes. Also, a new cell type expressing GFAP, NG2 and Olig2 was seen 3 days following injury, but was not present in normal mice. The characteristics of primary cilia and centrosome described here suggest that in stem cells and progenitors their characteristics may be well suited for proliferation, whereas in neurons, the cilia and centrosomes are important for other sensory functions.