Type II diabetes is a major cause of blindness according to the World Health Organization (WHO, 2018). Diabetics are at risk of developing corneal diseases such as recurrent abrasions, ulcers, and erosions due to dysfunctional wound healing. Corrective surgeries or corneal transplants may be considered as a treatment in some, but not all, cases. The purinoreceptor P2X7 has been shown to be involved in cell-cell communication and in the restructuring of cytoskeletal actin, a necessary process for cell migration in wound healing. P2X7 relies on the binding of extracellular ATP for activation. Panx1 is a transmembrane protein whose primary role is for the release of intracellular ATP into the extracellular space. In healthy corneal epithelium, Panx1 localizes to the wound edge and forms clusters with the P2X7, which augments the wound healing response. This thesis looks at the localization of Panx1 in pre-diabetic murine corneal tissue. It was found that Panx1 is less expressed and does not localize to the wound edge to the extent as control corneas, therefore, creating less clusters with P2X7. Furthermore, preliminary studies that inhibit Panx1 with probenecid reduce the communication between cells, which is hypothesized as critical for migration of the tissue sheet and proper wound healing. / 2019-12-17T00:00:00Z

Pannexins represent a recently discovered second family of gap junction proteins in vertebrates. However, instead of forming intercellular gap junction channels like connexins, pannexins operate as unpaired pannexons, allowing the flux of molecules from the cytoplasm to the extracellular space and vice versa. Pannexins appear to play a vital role in the local control loop of blood perfusion and oxygen delivery. The properties of Panx1 channels indicate that this protein is the most probable candidate for an ATP release channel and is involved in the propagation of intercellular calcium waves. It is also proposed to mediate the large pore formation of the P2X7 receptor death complex. Prolonged activation of this receptor can lead to cell death. There must be some mechanisms to stop this ATP-induced ATP release and opening of the lethal pore. Here we describe a negative feedback loop controlling pannexin 1 channel activity. ATP, permeant to pannexin 1 channels, was found to inhibit its permeation pathway when applied extracellularly. ATP analogues, including BzATP, suramine, and BBG were even more effective inhibitors of pannexin 1 currents than ATP. These compounds also attenuated the uptake of dyes by erythrocytes, which express pannexin 1. The rank order of the compounds in attenuation of pannexin 1 currents was similar to their binding affinities to the P2X7 receptor, except that receptor agonists and antagonists both were inhibitory to the channel. The ATP inhibitory effect is largely decreased when R75 on the first extracellular loop of Pannexin1 is mutated to alanine, strongly indicating that the ATP regulates this channel through binding. To further investigate the structural property of the ATP binding, we did alanine scanning mutagenesis of the extracellular loops and found that mutations on W74, S237, S240, I247 and L266 on the extracellular loops severely impair the BzATP inhibitory effect indicating that they might be direct binding partners for the ligands. Mutations on R75, S82, S93, L94, D241, S249, P259 and I267 have largely decreased BzATP sensitivity. Mutations on other residues didn't change the BzATP sensitivity compared to the wild type except for some nonfunctional mutants. All these data demonstrate that some amino acid residues on the extracellular loop of Pannexin 1 mediate ATP sensitivity. However, how these residues form the ATP-binding pocket remains to be elucidated.

The ATP-gated P2X7 receptors (P2X7Rs) play a key role in the release of pro-inflammatory cytokines in response to immunological challenges. Pannexin 1 (Panx1), conventionally described as a hemichannel forming protein, was suggested to be involved in the formation of the P2X7 large pore, which provides a conduit for large molecules such as fluorescent dyes. Firstly, this thesis demonstrated that the P2X7R-mediated dye uptake, a phenomenon attributed to the activation of Panx1, was suppressed by acidic pH and this inhibition was abolished in a P2X7 mutant (aspartic acid 197 to alanine) that was insensitive to extracellular pH. Then, the functional properties of human or mouse Panx1 in HEK293 cells were analysed in the absence of P2X7. The Panx1 currents were not affected by extracellular/intracellular calcium, but were reversibly inhibited by adenosine triphosphate (ATP) and non-specific anion channel blockers. Ion substitution experiments showed that Panx1 was permeable only to monovalent anions and single channel studies revealed a medium sized unitary conductance of Panx1 (~65 pS). Based on the evidence, this thesis concluded that Panx1 is an anion channel but not a hemichannel as originally proposed.

Neuroblastoma (NB) is the most common extracranial solid tumour in childhood. NB is thought to arise from the failed differentiation of neural crest progenitor cells that would normally form tissues of the adrenal gland and sympathetic nervous system. These neural crest progenitors then uncontrollably proliferate forming a tumour. Despite aggressive surgery and chemotherapy, the cure rate of high-risk NB patients remains below 30%. Our laboratory has shown that human NB tumour specimens and high-risk patient derived cell lines express pannexin 1 (PANX1), and that treatment with the PANX1 channel blockers carbenoxolone or probenecid constitute reduce NB progression in vitro and in vivo. PANX1 is a glycoprotein that forms single membrane channels best known to serve as conduits for ATP release. Interestingly, while PANX1 was also detected in control neurons by western blotting, its banding pattern was strikingly different as a band at around 50 kDa was found in all NB cell lines, but not in neurons. Using shRNA targeting PANX1 and deglycosylation enzymes, I have shown that this band corresponds to a PANX1 glycosylated species. PANX1 has been reported to be phosphorylated in NB at amino acid Y10. PANX1 is also predicted to be glycosylated at N255. In order to study the role of these post-translational modifications, myc-tagged Y10F and N255A PANX1 mutants were engineered by site-directed mutagenesis. Immunolocalization and cell surface biotinylation assays suggest that the localization both mutants at the cell surface is reduced compared to that of myc-PANX1. Dye uptake assays revealed that myc-Y10F has significantly reduced channel activity. Expression of myc-Y10F and myc-N255A in NB cells inhibited cell proliferation and decreased metastatic potential in vitro. Further analysis of NB tumour specimens revealed that there is a missense mutation in PANX1 resulting in the formation of truncated peptide (amino acid 1-99). Interestingly, I have found that when co-expressed with myc-PANX1, PANX11-99, reduced PANX1 channel activity. Taken together, these findings indicate that phosphorylation on Y10 and glycosylation on N255 regulate PANX1 channel activity and exacerbate NB malignancy, while the expression of PANX11-99 in NB may be beneficial.

Carotid body (CB) receptor type I cells transduce blood-borne chemical stimuli into electrical signals and release the excitatory neurotransmitter ATP onto afferent terminals that project to the breathing centre located in the brainstem. Within the CB, type I cells are ensheathed by glial-like processes of type II cells. Recently, it was hypothesized that type II cells have a paracrine function in CB chemotransduction by acting as an ATP amplifier and enhancing chemoexcitation (Zhang et al. 2012). Given this recent development, the primary goal of this thesis was to further elucidate the paracrine function of type II cells and characterize the signalling mechanisms involved in type I and type II cell interactions. Ratiometric calcium imaging was used to investigate type II cell sensitivity to two prominent CB neuromodulators, angiotensin II (ANG II) and 5-HT, in rat CB cultures. Both ANG II and 5-HT elicited large rises in intracellular Ca<sup>2+<sup> that were present in the absence of extracellular Ca<sup>2+<sup> and were inhibited by intracellular store depletion agents. ANG II and 5-HT acted on their respective G-protein coupled receptors, AT<sub>1<sub> receptor and 5-HT<sub>2A<sub> receptor, to initiate these Ca<sup>2+<sup> responses presumably via a PLC-IP<sub>3<sub> mediated mechanism. Interestingly, these Ca<sup>2+<sup> responses were required to activate pannexin-1 channels (Panx-1), a channel that has been previously shown to be a conduit for ATP in type II cells (Zhang et al. 2012). We were also interested in determining whether type II cells were capable of indirectly responding to a chemostimulus such that the stimulus would elicit neurosecretion from type I cells and result in a secondary Ca<sup>2+<sup> responses in type II cells. Isohydric hypercapnia and a depolarizing stimulus (30 mM KCl saline) were capable of eliciting indirect Ca<sup>2+<sup> responses in type II cells. These secondary Ca<sup>2+<sup> responses in type II cells were partially inhibited by suramin, a purinergic P2Y2 receptor antagonist, suggesting that ATP was the predominant neurotransmitter responsible for type I to type II crosstalk. Similarly, a selective agonist for type II cells, UTP, evoked indirect Ca<sup>2+<sup> responses in nearby type I cells. Type II to type I cell communication was dependent on Panx-1 channels since the secondary Ca<sup>2+<sup> responses in type I cells were inhibited by the Panx-1 blocker, carbenoxolone (5 µM). UTP-evoked indirect Ca<sup>2+<sup> in type I cells were partially inhibited by adenosine A<sub>2<sub> receptor antagonists suggesting that the neuromodulator, adenosine, governs cross-talk between type II and type I cells. This study elucidates the importance of purinergic signalling in the bi-directional cross-talk between receptor type I cells and glial-like type II cells. / Thesis / Master of Science (MSc)

No description available.

Stem cells, with the ability to self-renew and differentiate into intended cell types, are a valuable tool for disease modeling and mechanistic study. CRISPR-Cas9 has been widely used for genome editing due to its high efficiency and convenience. However, CRISPR-Cas9 has large-deletion safety issues that dramatically restrict its applications. Wiskott-Aldrich syndrome (WAS) is an inborn immunological disorder caused by WASP deficiency. WASP functions in the nucleus, which may help to understand WAS pathology, are poorly defined. Pannexin 1 (PANX1) forms large plasma membrane pores to exchange intracellular small molecules with the extracellular environment and functions in inflammatory processes. The regulatory mechanisms of the PANX1 channel remain obscure. In this dissertation, I focused on mechanistic studies of CRISPR-Cas9 genome editing, and two immune disease relevant genes, WASP and PANX1 using stem cell-derived immune cells. We first found that CRISPR-induced large deletions (LDs) are predominantly mediated by the MMEJ repair pathway through statistical studies. Further, we found POLQ and RPA play vital roles in CRISPR-induced LDs. Modulation of POLQ and RPA can decrease CRISPR-induced LDs and increase HDR efficiency. Using three isogenic WAS iPSC models generated via gene editing, we successfully recapitulated WAS phenotypes, and for the first time, revealed that WASP regulates RNA splicing via epigenetically controlling the transcription of splicing factors and directly participating in the splicing machinery through a liquid-liquid phase separation process. We established a full-length human PANX1 (hPANX1) channel model via cryo-electron microscopy experiments and molecular dynamics simulation study, and found that hPANX1 channel is a homo-heptamer with both the N- and C-termini stretching deeply into the pore funnel. Functional studies of three selected residues support the new hPANX1 channel model and suggest the potential regulatory role of hPANX1 in pyroptosis upon immune responses. Overall, the mechanistic studies of WASP, PANX1 and CRISPR genome editing revealed new roles of WASP in regulating RNA splicing, new functional insights of PANX1 in pyroptosis, and uncovered two critical players POLQ and RPA in CRISPR-induced LDs.

Pendant très longtemps, les traitements contre les cancers se sont basés sur la cytotoxicité des chimiothérapies, sur leur capacité à tuer directement les cellules malignes ou à induire leur senescence. Mais cette cytotoxicité accrue et non ciblée a également pour effet de tuer les cellules du système immunitaire du patient. Cependant, il a été montré que la radiothérapie, les anthracyclines ainsi que l’oxaliplatine étaient capables d’induire une apoptose décrite comme étant une mort cellulaire immunogène. De ce fait, les cellules tumorales mourantes agiront comme vaccin thérapeutique.La mort cellulaire immunogène se caractérise par trois grands marqueurs : un stress du réticulum endoplasmique pré-mortem qui va induire la translocation de la calréticuline de la lumière du réticulum endoplasmique vers la surface cellulaire, la libération d’ATP dans le milieu extracellulaire permettant le recrutement des cellules dendritiques et l’activation de l’inflammasome NLRP3 via le récepteur P2RX7, et enfin la libération de la protéine HMGB1 dans le milieu extracellulaire, qui va aller interagir avec TLR4 à la surface des cellules dendritiques pour stimuler leur fonction présentatrice d’antigène. La première partie de ce travail a consisté à comprendre les mécanismes moléculaires précis par lesquels l’ATP est sécrétée activement lors d’une mort cellulaire immunogène. En utilisant une combinaison de techniques impliquant des criblages pharmacologiques, des techniques de monitorage de la localisation intracellulaire de l’ATP entre autres, nous avons montré qu’après un traitement par les inducteurs de la mort immunogène, l’ATP était redistribué des lysosomes aux autolysosomes et que sa sécrétion requiert la protéine lysosomale LAMP1. Nous avons également montré qu’il existait d’autres voies de libération d’ATP telles que la voie de signalisation Rho, et également l’ouverture des hémicanaux pannexine 1 (PANX1). De façon surprenante, nous avons observé une implication de PANX1 dans la translocation de LAMP1 à la surface cellulaire. Ces résultats ont permis de comprendre un peu plus précisément les mécanismes de sécrétion d’ATP dans la mort cellulaire immunogène, mettant en évidence l’importance de l’exocytose lysosomale caspases dépendante et PANX1 dépendante.La seconde partie de ce travail s’est portée sur l’étude d’une autre caractéristique de la mort cellulaire immunogène, à savoir l’exposition de la calréticuline à la surface cellulaire. En partant du constat qu’après un traitement par la mitoxantrone, la calréticuline était relocalisée en périphérie à la fois dans les cellules humaines et les cellules de levure, il a été suggéré que la voie d’exposition de la calréticuline était conservée phylogénétiquement. Nous avons montré que les phéromones pouvaient agir comme inducteurs physiologiques de l’exposition de la calréticuline dans les cellules de levure. Un criblage d’ARN interférant et des analyses de transcriptome nous ont permis de montré que les chimiokines, en particulier CXCL8 chez l’humain (appelé également interleukine-8) et son orthologue Cxcl2 chez la souris étaient impliquées dans la translocation de la calréticuline à la surface cellulaire. En traitant les cellules cancéreuses par la mitoxantrone, nous observons une production de CXCL8 par les cellules cancéreuses humaines in vitro et de Cxcl2 par les cellules cancéreuses murines in vivo. Un « knockdown » des récepteurs pour CXCL8/Cxcl2 réduit de manière significative l’exposition de la calréticuline à la surface cellulaire. Ces résultats ont donc montré l’importance des chimiokines dans la voie d’exposition de la calréticuline.L’ensemble de ce travail a permis de comprendre plus en détails deux des trois grandes caractéristiques de la mort cellulaire immunogène. / Cytotoxic anti-neoplastic agents were considered for a long time to mediate their therapeutic effects via their capacity to directly kill malignant cells. Nevertheless, this high cytotoxicity is non-targeted and will eventually diminish immune cells. During the last years, it has been shown that radiotherapy and some anticancer agents, such as anthracyclines and oxaliplatin, can stimulate actively anti-tumor immune responses. In fact, they can induce an immunogenic type of apoptosis, which we termed immunogenic cell death (ICD). Thereby, dying cells can act as therapeutic vaccine against residual cancer cells that overcame the initial treatment.ICD is characterized by three major hallmarks: a pre-mortem stress of the endoplasmic reticulum (ER), which triggers the translocation of the ER chaperone protein called calreticulin (CRT) to the cell surface, the secretion of ATP from apoptotic cells, which acts as a signal for the recruitment of dendritic cells and for the activation of the NLRP3 inflammasome via its receptor P2RX7, and the release of HMGB1 into the extracellular space, allowing it to interact with TLR4 and thus stimulate the antigen-presenting functions of the DCs.The first part of my work focused on the precise molecular mechanisms by which ATP is actively secreted during ICD. Using a large panel of techniques, including chemical compounds screens and monitoring the subcellular localization of ATP, we showed that following treatment of various tumor cells with ICD inducers, ATP is redistributed from lysosomes to autolysosomes and the lysosomal protein LAMP1 is required for active ATP secretion. We also showed that Rho and pannexin 1 (PANX1) are indispensable for efficient ATP release in response to ICD inducers. Surprisingly, we observed an unexpected link between PANX1 and the exposure of LAMP1 at the cell surface. These results will help to understand the mechanisms necessary for ATP secretion during ICD.In the second part of this work we further studied the surface exposure of CRT during ICD. We observed that mitoxantrone (MTX), which belongs to the group of anthracyclines, can induce a peripheral relocalisation of CRT, both in human cells and yeast cells. In addition, we showed that pheromones can act as a physiological inducer of CRT translocation in yeast. Focused siRNA screening combined with transcriptome analyses revealed that human CXCL8 (also called interleukin-8) and its mouse ortholog Cxcl2 play an essential role in the translocation of CRT to the cell surface. Interestingly, MTX-treated human cancer cells displayed an elevated production of CXCL8 in vitro. These results were confirmed in vivo, with MTX treated murine tumors, which also displayed elevated Cxcl2 levels. The MTX-induced CRT exposure was significantly reduced when we performed a knockdown of CXCL8/Cxcl2 receptors. Altogether, these results showed the importance of chemokine signaling circuitries in immunogenic CRT exposure.This work allows for the detailed understanding of the mechanisms of ICD and might thus be useful for further targeted drug development.