Opioids are gold-standard analgesics for pain relief in chronic pain conditions. Paradoxically, chronic opioid use causes an enhanced pain sensitivity termed ‘Opioid-induced hyperalgesia’ (OIH). OIH is a clinically relevant problem associated with the use of opioids. In addition to decreasing quality of life, increased pain from OIH necessitates increasing dosages of analgesics to effectively control the pain, resulting in an increased risk of opioid epidemics, addiction, and overdose. To prevent this clinically important effect, it is necessary to understand how chronic opioid use causes hyperalgesia. Our preliminary studies revealed that synaptic plasticity in the spinal dorsal horn (SDH) is dependent on neuron type in the OIH model and occurs concurrently with hyperalgesia, suggesting central sensitization as a mechanism of OIH. We found that astrocyte ablation blocked mechanical hyperalgesia and neuron type-dependent synaptic plasticity, indicating that astrocytes are critically involved in OIH. Additionally, morphine treatment upregulated IL-1β expression in the SDH in our preliminary experiments. Inhibition of IL-1β prevented OIH and blocked the repeated morphine-induced synaptic plasticity in the SDH, suggesting IL-1β is a key player in the pathogenesis of OIH. Astrocytes and other glial cells are critical in the development and maintenance of neuroinflammatory conditions, such as OIH, through the release of proinflammatory cytokines (PICs), including IL-1β. The mechanosensitive ion channel, Piezo1, was recently found to be upregulated in astrocytes and microglia under LPS-induced inflammatory conditions, and activation of Piezo1 was found to reduce IL-1β expression in LPS-inflamed primary mouse astrocytes. The goal of this study was to investigate the function of Piezo1 as a potential treatment for neuroinflammatory diseases of the CNS in a model of LPS-induced inflammation. In this study, we created a culture cell model of LPS-induced astrocytic neuroinflammation using the C8-S type II astrocyte culture cell line. We used a multi-disciplinary approach of electrophysiology and imaging to assess changes in calcium flux induced by the selective Piezo1 agonist, Yoda1, and mechanosensitive ion channel activity in the LPS-stimulated C8-S culture astrocytes. We found that calcium flux is increased in LPS stimulation and augmented by additional Yoda1 treatment. We also found that LPS stimulation increases mechanosensitive ion currents and stiffens cell membranes using patch-clamp electrophysiology techniques. These results indicate that Piezo1 is likely upregulated in the LPS model of cultured astrocytes, thus mechanosensitive responses are increased. Results from these experiments reveal key information about the mechanical properties of Piezo1 and poise Piezo1 as a promising therapeutic for OIH and other neuroinflammatory diseases caused by astrocytic IL-1β release.

Neuroinflammation is prevalent in neurodegenerative diseases and plays a significant role in the central nervous system (CNS) innate immunity, which is the body’s first line of defense mechanisms against invading pathogens and injuries to maintain homeostasis. However, in neurodegenerative diseases, neuroinflammation becomes persistent alongside the subsequent damage to nearby neurons and affects CNS-resident immune glial cells, such as microglia and astrocytes. Accumulating evidence suggests that neuroinflammation is mainly characterized by the excessive activation of glial cells, thus causing abnormal changes in their microenvironment and release soluble factors that can promote or inhibit neuroinflammation. Currently, there is no effective treatment to cure these progressive neurological disorders. Therefore, it is critical to understand how neuroinflammation affects astroglia cell function and their biomechanical properties that change their behavior throughout disease progression. Astrocytes are the most predominant glial cell in the CNS and are critical in the development and maintenance of neuroinflammatory disorders. To date, very little is known regarding the role and specific function of Piezo1 mechanosensitive ion channel (MSC) in the CNS. Recently, Piezo1 expression was found to be upregulated in Lipopolysaccharide (LPS)-induced neuroinflammation in mouse astrocyte cultures. However, it is unknown whether the aberrant mechanical environment in astrocytes interplay with the mechanosensory function of Piezo1 and its current activity in neuroinflammatory conditions. In this study, we investigated Piezo1 mechanosensitive ionic currents by performing in vitro patch-clamp electrophysiology and calcium imaging. Our preliminary studies revealed that astrocytes derived from the mouse cerebellum stimulated with LPS or Piezo1 agonist, Yoda1, increased Ca2+ influx and further augmented when treated concurrently. We also found that electrophysiology recordings showed changes in mechanosensitive ionic currents and were comparable with our calcium imaging data indicating that MSCs are involved in neuroinflammation. Therefore, we postulated that Piezo1, a non-selective cation MSC that opens in response to mechanical force is a key mechanosensor involved in neuroinflammation by altered mechanical signals in C8-S astrocytes. Using an in vitro system of Mouse C8-S (Astrocyte type II clone), the goal of this study was to investigate if neuroinflammatory conditions upregulate Piezo1 calcium influx and current activity. We show that astrocytic Piezo1 regulates mechanotransducive release of ATP by controlling the mechanically induced calcium influx and current activation in LPS-induced astrocytes. Additionally, Piezo1 antagonist, GsMTx4 and Piezo1 siRNA significantly reduced the LPS-induced current, indicating that Piezo1 is involved in neuroinflammation. Our findings demonstrate that the activity of Piezo1 stimulated by neuroinflammatory conditions may be significant for the development of therapeutics to prevent or treat neuroinflammatory disorders and diseases.

京都大学 / 0048 / 新制・課程博士 / 博士(工学) / 甲第21372号 / 工博第4531号 / 新制||工||1706(附属図書館) / 京都大学大学院工学研究科合成・生物化学専攻 / (主査)教授 梅田 眞郷, 教授 浜地 格, 教授 秋吉 一成 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DGAM

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Der Herzmuskel muss sich einer dynamischen und sich mechanisch verändernden Umgebung anpassen. Die Mechanosignaltransduktion ermöglicht es Zellen mechanischen Kräfte zu erfassen und durch nachgeschaltete biochemische Signalkaskaden darauf zu reagieren. Obwohl verschiedene Gewebestrukturen und Proteine damit in Verbindung gebracht wurden, wie das Herz die mechanischen Kräfte wahrnimmt, ist unser Verständnis der kardialen Mechanosignaltransduktion unvollständig. Durch Dehnung aktivierte Ionenkanäle spielen eine wichtige Rolle bei der mechanosensitiven Autoregulation des Herzens. Um die funktionelle Rolle von PIEZO1 in Kardiomyozyten zu untersuchen, habe ich daher PIEZO1 in induzierten pluripotenten Stammzellen mittels Genomeditierung deletiert. Die PIEZO1-/- Zellen wurden dann in lebensfähige, herzähnlich schlagende Kardiomyozyten differenziert. In phänotypische Analysen der elektrophysiologischer Eigenschaften, Zellmorphologie und der herzähnlichen Schlagaktivität habe ich den Effekt der PIEZO1-deletion in genomeditierten Kardiomyozyten untersucht. Die Deletion von PIEZO1 zeigte zum ersten Mal, dass es PIEZO1-abhängige dehnungsaktivierte und Kalzium-Ströme in vom Menschen stammenden differenzierten Kardiomyozyten gibt. Dies legt nahe, dass PIEZO1 eine Rolle in der Mechanosignaltransduction in Herzzellen spielt. Darüber hinaus zeigte eine RNA-Sequenz Analyse, dass der Verlust von PIEZO1 in vom Menschen stammenden differenzierten Kardiomyozyten mit der Herunterregulation von Proteinen korreliert, die für die extrazellulärer Matrix von Bedeutung sind. Diese Daten unterstreichen die Rolle von PIEZO1 in Kardiomyozyten und legen seine Bedeutung für die Organisation und Struktur der extrazellulären Matrix nahe. / The cardiac muscle has to adapt in a highly dynamic mechanical environment. Mechanotransduction is the process that allows cells to sense the mechanical forces and respond by downstream biochemical signaling cascades. Although different tissue structures and proteins have been implicated in how the heart senses the mechanical forces, yet our understanding in cardiac mechanotransduction is incomplete. Stretch-activated channels (SACs) have been suggested to play an important role in the mechanosensitive autoregulation of the heart. PIEZO1 is a stretch-activated channel and has been involved in vascularization, erythrocyte volume homeostasis and regulation of the baroreceptor reflex, yet its role in cardiac mechanotransduction has not been described. To study the functional role of PIEZO1 in cardiomyocytes I have generated a PIEZO1 knockout (KO) human induced pluripotent cell (hiPSC) line using genome editing technology. The genome edited cells were then differentiated into viable, beating cardiomyocytes. Different phenotypic analyses were conducted, including the evaluation of electrophysiological characteristics, observation of cell morphology and beating activity of the genome edited hiPSC-derived cardiomyocytes. With this approach the aim was to gain more insight into PIEZO1 function in cardiomyocytes using a reliable, efficient and reproducible human cellular model system. For the first time PIEZO1-dependent calcium transients and stretch-activated currents were observed in hiPSC-derived cardiomyocytes (hiPSC-CMs). This proposes a possible role of PIEZO1 as a cardiac mechanotransducer. Furthermore, RNA-seq analysis revealed that loss of PIEZO1 in hiPSC-CMs is associated with downregulation of the expression of extracellular matrix-associated proteins. These data highlight the role of PIEZO1 in cardiomyocytes and suggest its implication in extracellular matrix organization and structure.

Breast cancer metastasis is one of the reasons why this type of cancer is destructive even after treatment as it tends to move from one organ to another increasing the risk factor for an individual. In the metastatic cascade, the tumour undergoes many different types of stress, including extracellular (ECM) stiffness. Key proteins that have been linked to the change in stiffness of the ECM are YAP and β-catenin. Both functions similarly in the manner that they need to translocate to the nucleus and bind to their respective transcription factors in order to activate their downstream genes. In parallel this seems to be on a stiffness dependent manner. Therefore, the hypothesis is that β-catenin is able to compensate for YAP function when YAP is downregulated in a stiffness dependent manner. In this work, results show a significant increase of YAP and β-catenin translocation to the nucleus of MDA-MB-231 cells when they are subject to the stiffer substrate in comparison to the softer substrate indicating increase gene expression of their respective pathways. The effect of the stiffness was then analyzed by doing single knockdown experiments with siRNA. To investigate the response of β-catenin, knocking down YAP was done, and it was shown that β-catenin translocation significantly increased on the softer matrix, while stiffer matrix showed no significant difference. Downstream gene expression also confirmed this idea with CTGF being downregulated with β-catenin knockdown and AXIN2 being downregulated with YAP knockdown. In the cell behavioural aspect, only when the double knockdown of YAP and β-catenin was done, the migration and proliferation rate had significant lowered. This echoes the idea further of the compensating effects of β-catenin to YAP. In addition, the exploration of the cytoskeleton network was investigated, as this is a key component in protein pathways, by treating the cells using LatA and Blebbistatin, affecting F-actin and myosin-II respectively. Knowing the critical role of cytoskeletal proteins in mechanotransduction, the hypothesis is that actin filaments and myosin-II mediate the YAP & β-catenin nuclear translocation activation. Findings show the direct relationship between F-actin and YAP as actin polymerization state significantly decreased when YAP was knockdown in a similar manner to when LatA was added. When myosin-II was added, both YAP and β-catenin nuclear translocation were affected, indicating its potential role in mechanotransduction. Furthermore, it was found that cell confluency and PIEZO1 activation had significant effects in YAP & β-catenin translocation. By seeding the cells with different densities, the β-catenin signalling could be visualized with IF staining, with the conclusion that at high confluency, the β-catenin translocation was alleviated. For the PIEZO1 studies, results indicate that PIEZO1 is an upstream regulator of YAP by doing single knockdown experiments and subsequently analysing YAP signalling. The findings underscore the potential significance of β-catenin as a modulator of mechanotransduction in the absence of YAP, showcasing the complexity of the protein signalling network orchestrating cellular response due to mechanical cues. Unravelling these protein interplay could offer novel insights into therapeutic targets for breast cancer mechanotransduction. Ultimately, this research adds to the understanding of the intricate protein signalling that governs mechanotransduction in breast cancer cells. The discovery of stiffness dependent YAP & β-catenin signalling, the interplay between YAP and β-catenin pathway mechanotransduction implicated by cell density, the regulation of YAP- β-catenin interplay in mechanotransduction by PIEZO1, the importance of F-actin & myosin-II in YAP & β-catenin translocation, and the YAP & β-catenin effects on cell behaviour, all help lay the groundwork for devising targeted interventions to impede cancer progression. / Thesis / Master of Applied Science (MASc) / Breast cancer is the most prominent type of cancer that exists in women and like other cancers, it can spread to other organs such as the bone, liver, and brain even though the microenvironments are different. With different proteins like yes-associated protein (YAP) regulating this microenvironmental change in the primary and secondary sites, it can flourish and become more aggressive which leads to death for the host. The interactions of these proteins and their pathways which affects the aggressiveness of the cancers are still not well understood. This project investigates the interaction between YAP and β-catenin in response to surface stiffness to understand the mechanical regulation of breast cancer metastasis. Alongside the protein signalling, cytoskeletal components, downstream gene expression, cell confluency, and membrane proteins are explored. Our results show that an increase in stiffness allow for higher nuclear translocation for YAP and β-catenin, enhancing downstream gene expression relating to migration and proliferation. Furthermore, in lower stiffness the crosstalk between YAP and β-catenin results in an inverse relationship. These findings suggest β-catenin compensates YAP function when YAP is inhibited. In terms of the cytoskeletal protein, an integral part of the cell, the intervention saw a significant alteration in the YAP & β-catenin signalling. Additionally, cell confluency played a large role in β-catenin nuclear translocation implicating the role of cell-to-cell contact in mechanotransduction. To see if mechanosensitive membrane proteins fit into the pathway, PIEZO1 studies were done and results show that it is an upstream effector of YAP, and consequently an indirect connection with β-catenin. All in all, this thesis provides insightful information in the role of stiffness matrix, cell confluency, membrane proteins and how that regulate YAP & β-catenin. This research provides the mechanism for the synergistic therapies targeting multiple proteins to prevent cancer growth and metastasis.