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The inner ear contains cells with ciliary bundles that have been identified as sites of mechanoelectrical transduction; they take a mechanical stimuli and convert it to an electrical response. The ciliary bundles vary structurally within the organs of the inner ear; this structural difference may play a role in the mechanical properties of each bundle. A relationship between the structure and the mechanics of the ciliary bundle can be found by studying structurally diverse bundles. To explore this relationship, a system was designed to mechanically stimulate the ciliary bundles in normal physiological range and measure their displacement. An extrinsic Fabry-Perot interferometer (EFPI) was developed to measure the response of a ciliary bundle that is subjected to a force applied by a glass whisker. 'Imitation bundles', similar in stiffness to living ciliary bundles, were made to test the system. The stiffness of an 'imitation bundle' was first determined by suspending styrene beads from its tip and optically measuring the resultant displacement. Then the EFPI was also used to determine the stiffness. The EFPI compared well to the stiffness found using the styrene beads; the largest difference between the two methods was 130/0. The EFPI was also tested in water to ensure its operation in the tissue environment; this test was successful in that it was able to measure displacements in a bundle's normal physiological range. With both of the tests showing good results, we conclude that our system can be used to measure the stiffness of the ciliary bundles located in the inner ear. / Master of Science

京都大学 / 新制・課程博士 / 博士(医学) / 甲第23571号 / 医博第4785号 / 新制||医||1054(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 齊藤 博英, 教授 大森 孝一, 教授 大鶴 繁 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

The prostaglandins comprise a group of bioactive lipids generated from arachidonic acid by cyclooxygenases and cell type-specific prostaglandin and thromboxane synthases. Prostaglandins mediate local cell signaling interactions by activation of G-protein coupled prostanoid receptors. Because the prostaglandins and their receptors are active in all tissues, they have an extraordinarily broad spectrum of physiological and pathophysiological functions that have hampered the development of safe prostanoid-based medications. This situation has emphasized the importance of understanding the functional properties of the prostanoid receptors and developing selective ligands capable of being used in patient care.The aims of this project were to identify novel regulatory functions of endogenous EP and FP prostanoid receptors in cultured human cells. Our results show that activation of EP₂ receptors in human microglia and astrocytes led to increased secretion of BDNF, a growth factor that regulates the survival of neurons. In the same cell lines, FP receptors regulate the induction of TNF-α gene expression through a classic G_q-PKC pathway. In microglia these FP receptors also stimulate a novel signaling crosstalk mechanism involving the up-regulation of TCF transcriptional function by Raf kinases, which culminates in the expression of the angiogenic inducer Cyr61. FP receptors also regulate the induction of angiogenic immediate early genes in cultured ciliary muscle cells, which may constitute the early steps in a mechanism by which commercial FP agonists reduce intraocular pressure in glaucoma therapy.The up-regulation of BDNF through glial EP₂ receptors constitutes a mechanism by which elevated PGE₂ in the inflamed brain might elicit either healing processes in the brain or neuronal apoptosis. On the other hand, induction of TNF-α and Cyr61 by glial FP receptors may mediate neuroinflammation and may also contribute to glioma tumor growth. Stimulation of FP receptors in the ciliary muscle leads to the induction of immediate early genes capable of coordinating tissue remodeling processes that have been previously documented. The results of these studies suggest novel regulatory functions of the prostanoid receptors in the brain and eye. Furthermore, these findings provide insight on how the selective modulation of the EP₂ and FP receptors might be therapeutically advantageous.

The primary cilium is an organelle that serves as a signaling center of the cell and is involved in the hedgehog signaling, cAMP pathway, Wnt pathways, etc. Ciliary function relies on the transportation of molecules between the primary cilium and the cell, which is facilitated by intraflagellar transport (IFT). IFT88, one of the important IFT proteins in complex B, is known to play a role in the formation and maintenance of cilia in various types of organisms. The ciliary transition zone (TZ), which is part of the gating apparatus at the ciliary base, is home to a large number of ciliopathy molecules. Recent studies have identified important regulating elements for TZ gating in cilia. However, the architecture of the TZ region and its arrangement relative to intraflagellar transport (IFT) proteins remain largely unknown, hindering the mechanistic understanding of the regulation processes. One of the major challenges comes from the tiny volume at the ciliary base packed with numerous proteins, with the diameter of the TZ close to the diffraction limit of conventional microscopes. Using a series of stimulated emission depletion (STED) superresolution images mapped to electron microscopy images, we analyzed the structural organization of the ciliary base. Subdiffraction imaging of TZ components defines novel geometric distributions of RPGRIP1L, MKS1, CEP290, TCTN2 and TMEM67, shedding light on their roles in TZ structure, assembly, and function. We found TCTN2 at the outmost periphery of the TZ close to the ciliary membrane, with a 227±18 nm diameter. TMEM67 was adjacent to TCTN2, with a 205±20 nm diameter. RPGRIP1L was localized toward the axoneme at the same axial level as TCTN2 and TMEM67, with a 165±8 nm diameter. MKS1 was situated between TMEM67 and RPGRIP1L, with an 186±21 nm diameter. Surprisingly, CEP290 was localized at the proximal side of the TZ close to the distal end of the centrin-labeled basal body. The lateral width was unexpectedly close to the width of the basal body, distant from the potential Y-links region of the TZ. Moreover, IFT88 was intriguingly distributed in two distinct patterns, forming three puncta or a Y shape at the ciliary base found in human retinal pigment epithelial cells (RPE), human fibroblasts (HFF), mouse inner medullary collecting duct (IMCD) cells and mouse embryonic fibroblasts (MEFs). We hypothesize that the two distribution states of IFT88 correspond to the open and closed gating states of the TZ, where IFT particles aggregate to form three puncta when the gate is closed, and move to form the branches of the Y-shape pattern when the gate is open. Two reservoirs of IFT particles, correlating with phases of ciliary growth, were localized relative to the internal structure of the TZ. These subdiffraction images reveal unprecedented architectural details of the TZ, providing a basic structural framework for future functional studies. To visualize the dynamic movement of IFT particles within primary cilia, we further conducted superresolution live-cell imaging of IFT88 fused to EYFP in IMCD cells. Our findings, in particular, show IFT88 particles pass through the TZ at a reduced speed by approximately 50%, implying the gating mechanism is involved at this region to slow down IFT trafficking. Finally, we report the distinct transport pathways of IFT88 and Smo (Smoothened), an essential player to hedgehog signaling, to support our hypothesis that two proteins are transported in different mechanisms at the ciliary base, based on dual-color superresolution imaging.

Mechanosensation, the ability for cells to sense and respond to physical cues, is a ubiquitous process among living organisms and its dysfunction can lead to devastating diseases, including atherosclerosis, osteoporosis, and cancer. The primary cilium is a solitary, immotile organelle that projects from the surface of virtually every cell in the human body and can function as a mechanosensor across diverse biological contexts, deflecting in response to fluid flow, pressure, touch and vibration. It can detect urinary flow rate in the kidney, monitor bile flow in the liver, and distinguish the direction of nodal flow in embryos. In this thesis, we examined the interplay of biology and mechanics in the context of this multifunctional sensory organelle from the tissue to subcellular scale. In the first part of this work, we examined the cilium at the tissue level. Primary cilia are just beginning to be appreciated in bone with studies recently reporting loss of cilia results in defects in skeletal development and adaptation. We disrupted primary cilia in osteocytes, the principal mechanosensing cells in bone, and demonstrated that loss of primary cilia in osteocytes impairs load-induced bone formation. Over the course of our work with primary cilia, we also identified the need for more standardized imaging approaches to the cilium and presented an improvement to distinguishing proteins within the cilium from the rest of the cell. In the later part of this work, we examined the primary cilium at the subcellular level. While deflection is integral to the cilium's mechanosensory function, it remains poorly understood and characterized. Using a novel experimental and computational approach to capture and determine the mechanical properties of the cilium, we demonstrated cilium deflection can be mechanically and chemically modulated. We revealed a mechanism, acetylation, through which this mechanosensor can adapt and regulate overall cellular mechanosensing. By modifying our combined experimental and computational approach, we analyzed cilium deflection in vivo for the first time. Collectively, this work uncovers new insights across biological scales in the primary cilium as an extracellular nexus integrating mechanical stimuli and cellular signaling. Understanding the mechanisms driving cilium mechanosensing has broad reaching implications and unlocks the cilium's potential as a therapeutic target to treat impaired cellular mechanosensing critical to a multitude of diseases.

Osteoporosis is a devastating condition characterized by decreased bone mass, and affects over 50% of the population over 50 years old. Progression of osteoporosis results in significantly heightened risk of fracture leading to loss of mobility, prolonged rehabilitation, and even mortality due to extended hospitalization. Current therapeutic options exist to combat low bone mass, but these treatments are being met with increasing concern as reports emerge of atypical fractures and necrosis. Thus, new therapeutic strategies are required. Bone is highly dynamic, and it has long been known that physical load is a potent stimulus of bone formation. Despite this, none of the current treatments for bone disease leverage the inherent mechanosensitivity of bone – the ability of bone cells to sense and respond to mechanical forces such as exercise. One potential therapeutic target is the primary cilium. Primary cilia are solitary antenna-like organelles, and over the last 20 years have been identified as a critical cellular mechanosensor. Primary cilia and cell mechanotransduction are critical to the function of numerous cells and tissues. Thus, understanding primary cilia-mediated mechanotransduction has potential applications in treating kidney and liver disease, atherosclerosis, osteoarthritis, and even certain cancers. Previous work from our group has demonstrated that disruption of the cilium impairs bone cell mechanosensitivity, resulting in abrogated whole bone adaptation in response to physical load. In this thesis we examine the potential of targeting the primary cilium to enhance bone cell mechanosensitivity and promote whole bone formation. First, we demonstrate the pharmacologically increasing primary cilia length significantly enhances cell mechanotransduction. Next, we expand our list of candidate compounds to manipulate ciliogenesis through the use of high-throughput drug screening. We developed an automated platform for culturing, staining, imaging, and analyzing nearly 7000 small molecules with known biologic activity, and classify them based on mechanism of action. One of these compounds is then used in a co-culture model to study the effects of manipulating osteocyte primary cilia-mediated mechanosensing on pro-osteogenic paracrine signaling to promote the activity of bone-forming osteoblasts and osteogenic differentiation of mesenchymal stem cells. Finally, we translate our in vitro findings into an in vivo model of load-induced bone formation using the same compound to enhance cell mechanotransduction. We demonstrate that we can sensitize bones to mechanical stimulation to enhance load-induced bone formation in healthy and osteoporotic animals, with minimal adverse effects. Together, this work demonstrates the therapeutic potential and viability of targeting primary cilia-mediated mechanotransduction for treating bone diseases.

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Neurotrophic factor signaling modulates differentiation, axon growth and maintenance, synaptic plasticity and regeneration of neurons after injury. Ciliary neurotrophic factor (CNTF), a Schwann cell derived neurotrophic factor, has an exclusive role in axon maintenance, sprouting and synaptic preservation. CNTF, but not GDNF, has been shown to alleviate motoneuron degeneration in pmn mutant mice carrying a missense mutation in Tbce gene, a model for Amyotrophic Lateral Sclerosis (ALS). This current study elucidates the distinct signaling mechanism by which CNTF rescues the axonal degeneration in pmn mutant mice. ... / Neurotrophe Faktoren beeinflussendie die neuronale Differenzierung, das Wachstum und die Stabilisierung von Axonen sowie Synaptische Plastizität und die Regeneration von Neuronen nach Verletzung. Der von Schwannzellen synthetisierte neurotrophe Faktor Ciliary neurotrophic factor (CNTF) spielt eine wichtige Rolle bei der axonalen Erhaltung sowie bei der Induktion und Reduktion von axonalen Verzweigungen. Die Behandlung der pmn Mausmutante mit CNTF, aber nicht mit GDNF führt zu einem späteren Krankheitsbeginn und verminderten Fortschreiten der Motoneuronendegeneration. Diese Mausmutante, die eine Punktmutation im Tbce Gen trägt, dient als Modell für die Amyotrophe Lateralsklerose. Ziel der vorliegenden Arbeit war es, die zugrunde liegenden Signalkaskaden aufzudecken, die den CNTF-vermittelten Effekt auf den Krnakheitsverlauf bei der pmn Maus verursachen. ...