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Thesis advisor: Charles Hoffman / In both mammals and fission yeast, control of cAMP levels is maintained by adenylyl cyclases (ACs), which synthesize cyclic nucleotide, and by cyclic nucleotide phosphodiesterases (PDEs), which are responsible for its degradation. AC activity is regulated by G proteins, which respond to signals from G protein-coupled receptors (GPCRs) that detect extracellular signaling factors such as hormones. cAMP is a second messenger that has several effectors, with protein kinase A (PKA) being a primary target of activation that phosphorylates several downstream targets and results in modulation of pathways such as cell growth and gluconeogenesis. Aberrant cAMP regulation has been linked to several human disease states, such as McCune-Albright Syndrome, which is the result of elevated cAMP levels. Whereas the targeting of PDEs with drugs and selective inhibitors has been very successful, the AC-inhibiting compounds identified to date are unfavorable for clinical use. Inhibitors may not necessarily bind to and inhibit a given AC directly but instead act on a regulatory pathway such as calmodulin signaling. Theoretically, they also may bind to the G protein, interfere with the AC-G protein stimulatory complex, or regulate a factor of AC transcription. Since more than one AC species is expressed in many human cell types, it is difficult to selectively reduce cAMP levels. Therefore, for an AC inhibitor to be favored as a candidate for drug development, it is likely that the compound should directly bind to and inhibit the AC. This thesis describes my studies on a scaffold of 41 structurally related BCAC compounds, called the BCAC51 scaffold, that was identified in a high-throughput screen (HTS) with Schizosaccharomyces pombe strains transformed with GNAS and either mammalian AC4 or AC7. I carried out a series of experiments to examine whether the compounds bind to and inhibit mammalian ACs directly. The most active compounds were further characterized for potency and specificity against a panel of ACs. Several compounds significantly reduced cAMP production, but it could not be determined if the compounds directly or indirectly altered AC activity. I also cloned and constructed strains expressing the human wild-type AC5 gene and the AC5 R418W mutant, which has shown an increased sensitivity to GNAS. cAMP assays on these strains using various BCAC compounds showed that while most compounds had similar effects on both forms of AC5, BCAC62 was significantly more effective on the wild-type enzyme than on the mutant AC5, although the reason for this is unclear. To test whether the compounds could reduce AC activity in the absence of GNAS (basal activity), a flow cytometry study was carried out using a PKA-repressed GFP reporter. Results suggested that BCAC compounds do reduce basal-AC activity and therefore do not act by binding to and inhibiting GNAS, by interfering with the AC-GNAS stimulatory complex, nor by stimulating PDE. Finally, I developed a molecular genetic screen for mutant alleles of an AC gene that confer compound-resistance. One cycle of the screen is near completion, and the screen provides a foundation for future examination of compound-resistant AC candidates. The results presented in this thesis serve as a basis for further research into members of the BCAC51 compound series being putative direct inhibitors of mammalian ACs. / Thesis (BS) — Boston College, 2022. / Submitted to: Boston College. College of Arts and Sciences. / Discipline: Scholar of the College. / Discipline: Biology.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20663号 / 医博第4273号 / 新制||医||1024(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 藤渕 航, 教授 木村 剛, 教授 岩田 想 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Diss. (sammanfattning) Stockholm : Karol. inst., 2005. / Härtill 5 uppsatser.

The discovery of cyclic AMP (cAMP), nearly 50 years ago by Sutherland radically altered the appreciation of metabolic regulation. Since then the presence of cAMP and its tremendous physiological impact has been demonstrated in many prokaryotic systems. In fact, virulence mechanisms of several pathogens known today exploit cAMP dependent pathways. Interestingly the genome of Mycobacterium tuberculosis H37Rv, the causative agent of tuberculosis, encodes as many as 16 adenylyl cyclases (enzymes that convert ATP to 3’, 5’-cAMP) and 10 cyclic-nucleotide binding proteins. Recent reports show that bacterial-derived cAMP manipulates host signaling for bacterial survival, suggesting an important role for cAMP in the pathogenesis of M. tuberculosis. A large number of non-pathogenic species of mycobacteria also share and conserve several of these cAMP metabolism genes, suggesting that cAMP is not only important for pathogenesis but also may play a critical physiological role in the genus. The work carried out in this thesis aims at a better understanding of the role of cAMP by studying the adenylyl cyclases and cyclic AMP receptor proteins (CRPs) from Mycobacterium smegmatis, a non-pathogenic member of the genus. Intracellular cAMP levels in a cell are precisely maintained by modulating the activities of the adenylyl cyclases (cAMP synthesising enzymes), the phosphodiesterases (cAMP hydrolysing enzymes) and the secretion machinery, if any. To assess the role of cAMP in mycobacteria, cAMP levels were measured in M. smegmatis during growth and under various stress conditions. The results show that cAMP levels peak at log phase of growth and decline thereafter. Under acidic conditions or on perturbing the cell-wall, cellular cAMP levels are altered, which indicate a possible role of cAMP in stress adaptation. Earlier work in our laboratory has led to the identification of multiple adenylyl cyclases in the mycobacterial genomes. These cyclases are similar in sequence to the mammalian enzymes and several of them have been shown to be active in vitro displaying a diverse range of biochemical properties. The M. smegmatis genome encodes 10 adenylyl cyclase-like genes. In order to understand the role of cAMP in M. smegmatis, individual cyclases were analysed for their biochemical properties and physiological functions. The work presented in this thesis is concerned with the functional characterization of MSMEG_3578 and MSMEG_3780, two of the several adenylyl cyclases from M. smegmatis. MSMEG_3578 encodes for a protein that comprises two transmembrane domains, an extracellular receptor-like domain, a membrane anchoring HAMP domain and an intracellular cyclase domain. The cyclase domain is closely related to mammalian cyclases but lacks the canonical residues that are critical for the catalysis of class III cyclases. Interestingly, the stop codon of this gene overlaps with the start codon of the downstream gene, MSMEG_3579 (a putative cyclic nucleotide gated mechanosensitive ion channel), suggesting a functional link between the two genes. The conservation of this gene pair across the mycobacterial genus indicates the importance of this putative receptor-effector pair in the physiology of mycobacteria. Additionally, microarray analysis by various groups have shown that this gene pair in Mycobacterium tuberculosis is differentially regulated in conditions that mimic stress the bacteria may experience during infection. In order to ascertain the physiological role of MSMEG_3578, a knock-out M. smegmatis strain was generated and tested for growth and cAMP defects. The knock-out strain showed growth and cAMP profiles similar to the wild-type strain. Over-expression of MSMEG_3578 in M. smegmatis resulted in a significant rise in cAMP levels. Interestingly, over-expression of the MSMEG_3578 adenylyl cyclase in E. coli did not lead to an elevation in cAMP levels indicating that other mycobacterial proteins may be required for the activity of MSMEG_3578 in vivo. In agreement with this, the purified adenylyl cyclase domain of MSMEG_3578 was found to be biochemically inactive in vitro. Additionally, the over-expressing strain has altered colony morphology as compared to the wild type strain. Perturbation of cAMP levels by over-expression of other cyclases also leads to a similar colony morphology phenotype, indicating the phenotype to be controlled by cAMP in general rather than by a specific cyclase in the cell. MSMEG_3780 is a highly conserved, biochemically active adenylyl cyclase, speculated to play a house-keeping function in M. smegmatis. Its orthologs from M. tuberculosis (Rv1647) and M. leprae (ML1399) have been biochemically characterized earlier in our laboratory. To unravel the role of this gene in vivo, the ∆MSMEG_3780 strain was tested for growth and cAMP defects under various conditions. The deletion strain did not show any difference in growth rate or morphology when compared to the wild-type strain. However it showed a reduction in intracellular cAMP levels at the log-phase of growth. Reintroduction of the MSMEG_3780 gene in the deletion strain restored cAMP to wild-type levels, thus indicating a crucial role for this adenylyl cyclase in the maintenance of intracellular cAMP levels during logarithmic growth. In order to investigate the regulation of the MSMEG_3780 gene, its promoter activity was tested under various stress-conditions. Acid-stress specifically resulted in the repression of the MSMEG_3780 promoter activity, a condition which also leads to a decrease in cAMP levels in the cells. Further evidence for the susceptibility of the MSMEG_3780 gene to acid-stress was obtained when the ∆MSMEG_3780 strain failed to reduce intracellular cAMP content upon sustained acid-stress conditions. Since Rv1647 shares similar biochemical properties with MSMEG_3780 and can also heterodimerize with the MSMEG_3780 protein in vitro, it was interesting to test whether the M. tuberculosis ortholog could functionally complement MSMEG_3780. To assess this, a complement strain was generated that contained the Rv1647 gene under the control of MSMEG_3780 promoter, integrated into the genome of ∆MSMEG_3780 strain. Rv1647 protein was able to restore the cAMP phenotype seen on acid stress as well as the cAMP levels in the mutant strain at the log phase of growth. This study indicated the role of cAMP and MSMEG_3780 in acid adaptation and also suggested a non-redundancy of adenylyl cyclases in mycobacteria, where different individual cyclases may have unique functions in the cells. Since Rv1647 could complement the cAMP defective phenotype in ∆MSMEG_3780, this suggests functional parallels between the proteins from the two species. Bacterial adaptation to environmental stress is brought about by a rapid change in its gene expression profile. Cyclic AMP plays an important role by binding to and activating a transcriptional factor, cAMP receptor protein or CRP. We have identified two CRPs from M. smegmatis, viz., MSMEG_0539 and MSMEG_6189 that possess high similarity at the amino acid level (78% overall sequence identity). The CRP ortholog from M. tuberculosis, Rv3676 has been characterized structurally, biochemically and functionally earlier. Western blot and RT-PCR analyses showed that CRPs in M. smegmatis were present during all phases of growth. Both the CRPs were cloned, expressed and shown to bind cAMP. Since the DNA binding domains of Rv3676 and the two M. smegmatis CRPs are nearly identical, the CRPs from M. smegmatis were predicted to bind similar target sequences. Interestingly, a CRP site was identified in the promoter of the MSMEG_3780 gene, suggesting a possible feed-forward or feed-back loop, where the enzymatic product of the adenylyl cyclase now governs its own gene expression. We performed Electrophoretic Mobility Gel Shift Assays (EMSAs) with M. smegmatis lysates to show that CRP binds to the MSMEG_3780 promoter at the CRP site. Subsequent Chromatin Immunoprecipitation (ChIP) assays confirmed that CRP binding to the MSMEG_3780 promoter occurred in vivo. To investigate the role of CRP in the regulation of the MSMEG_3780 gene, luciferase reporter assays with the wild-type and CRP site mutant promoters were carried out. Results suggest that CRP regulates the MSMEG_3780 gene under osmotic stress. However, CRP did not play any role in basal expression of MSMEG_3780 during growth. To assess which CRP of the two is functionally linked to the MSMEG_3780 promoter, we carried out a footprint assay with purified CRPs. It was intriguing to note that both the CRPs were in fact able to bind the promoter albeit under different conditions. Whereas MSMEG_6189 bound the promoter both in the presence and absence of cAMP, MSMEG_0539 bound the promoter only in the presence of cAMP. MSMEG_6189 thus deviates from the accepted CRP paradigm that seeks an absolute requirement of cAMP for specific DNA binding. The present study identifies cAMP as an important stress signal in M. smegmatis. Using MSMEG_3780 as a model gene, the role of cAMP in mycobacteria was studied. The two divergent CRPs that were characterized may function and dictate cAMP-mediated and perhaps independent functions in cells. Taken together, our results provide compelling evidence for the important role of cAMP in the general physiology and stress adaptation in M. smegmatis.

Tuberculosis continues to be one of the major causes of morbidity and mortality worldwide. Several mycobacterial species such as M. tuberculosis and M. africanum are responsible for causing this disease in humans. Reports of high cAMP levels in mycobacterial species (as compared to other bacteria such as E. coli) suggested that this second messenger may play an important role in the biology of mycobacteria. Further, it was reported that infection with mycobacteria led to an increase in the cAMP levels within the host macrophage. More recent studies have shown that this cAMP increase may be due to bacterially derived cAMP, hinting at a role for cAMP in mycobacterial pathogenesis. Given this background, the study of cAMP in mycobacteria proves to be an interesting field of research. Signalling through cAMP involves an interaction of this cyclic nucleotide with a cAMP-binding protein. These proteins typically contain a cyclic nucleotide-binding domain (CNB domain) linked to another (effector) domain. The CNB domain is thought to allosterically control the activity of the effector domain, thus mediating cellular responses to altered cAMP levels. For example, in the case of eukaryotic protein kinase A (PKA), binding of cAMP to the CNB domain results in relieving the inhibitory effects of the regulatory subunit on the catalytic subunit. The catalytic subunit then phosphorylates its target substrates, eliciting a variety of cellular responses. This work involves the characterisation of novel cAMP-binding proteins from mycobacteria, in an attempt to better understand cAMP signalling mechanisms in these organisms. The genome of M .tuberculosis H37Rv is predicted to code for ten CNB domain-containing proteins. One of these genes is Rv0998 (KATmt). KATmt was found to contain a GCN5 related N-acetyltransferase (GNAT) domain linked to a CNB domain. KATmt finds orthologues throughout the genus Mycobacterium, thereby suggesting its role in the basic physiology of these organisms. In addition, such a domain fusion is unique to mycobacteria and hence promises to deliver insights into the biology of this medically important genus. Presented here are the biochemical and functional characterisation of KATmt and its orthologue from M. smegmatis, MSMEG_5458 (KATms). Recombinant KATms bound cAMP with high affinity, validating the functionality of its CNB domain. Mutational and analogue-binding studies showed that the biochemical properties of the CNB domain were similar to mammalian protein kinase A and G-like CNB domains. The substrate for the GNAT acetyltransferase domain was identified to be a universal stress protein from M. smegmatis (MSMEG_4207). MSMEG_4207 was acetylated at a single lysine residue (Lys 104) by KATms in vitro. Further, cAMP binding to KATms increased the initial rate of acetylation of MSMEG_4207 by 2.5-fold, suggesting allosteric control of acetyltransferase activity by the CNB domain. To ascertain that KATms acetylated MEMEG_4207 in vivo, an in-frame deletion of the KATms gene was generated in M. smegmatis (ΔKATms). MSMEG_4207 was immunoprecipitated from wild-type M. smegmatis and the ΔKATms strains, followed by mass spectrometric analysis. Acetylated MSMEG_4207 was only present in the wild-type strain, confirming that KATms and MSMEG_4207 is an in vivo enzyme-substrate pair. Key biochemical differences were observed between KATms and KATmt. KATmt had an affinity for cAMP in the micromolar range, close to three log orders lower than that of KATms. In addition, KATmt showed strictly cAMP-dependent acetylation of MSMEG_4207. This demonstrates that orthologous proteins often evolve under varied selective pressures, resulting in divergent properties. Using a combination of bioluminescence resonance energy transfer (BRET) and amide hydrogen/deuterium exchange mass spectrometry (HDXMS), the conformational changes that occur upon cAMP binding to the CNB domain of KATms were monitored. A BRET-based conformation sensor was constructed for KATms by inserting KATms between GFP2 (green fluorescent protein) and Rluc (Renilla luciferase). An increase in BRET upon cAMP binding to the sensor was observed. HDXMS analysis revealed that besides the CNB domain, the only other region that showed conformational changes in KATms upon cAMP-binding was the linker region. To confirm that the linker region was important in propagating the effects of cAMP-binding to the acetyltransferase domain, an additional construct for BRET analysis encompassing the CNB domain and the linker region was generated. The magnitude of the increase in BRET was similar to the full length BRET-based sensor, validating the crucial role of the linker region in propagating cAMP-mediated conformational changes. A ‘PXXP’ motif found in the linker region, showed maximum exchange in HDXMS analysis. Mutation of both these proline residues to alanine in KATms, as well as KATmt, resulted in decoupling of cAMP-binding and allosteric potentiation of acetyltransferase activity. In contrast to the intricate parallel allosteric relays observed in other CNB domain-containing proteins, the CNB domain in KATms functions as a simpler cyclic nucleotide binding-induced switch involving stabilization of the CNB and linker domain alone. Therefore, KATms is an example of a primordial CNB domain where conformational changes are a consequence of binding-induced ordering alone. Using a computational approach, putative substrate proteins of KATmt from M. tuberculosis were identified. The substrate specificity of lysine acetyltransferases is determined loosely by a consensus sequence around the lysine residue which is acetylated. Using this property of protein acetyltransferases, the genome of M. tuberculosis H37Rv was mined for proteins harboring lysine residues in a similar sequence context as seen in MSMEG_4207. In vitro biochemical analysis of some of the predicted substrates helped confirm a subset of enzymes belonging to the fatty acyl CoA synthetase (FadD) class as substrates of KATmt. The acetylation of FadDs by KATmt was cAMP-dependent. In each of the four proteins tested, acetylation was found to occur at a single conserved lysine residue. To confirm that FadDs were acetylated by KATmt in vivo, BCG_1055, the orthologue of KATmt in M. bovis BCG, was deleted using the specialised transduction method. FadD13, one of the FadDs acetylated by KATmt in vitro, was immunoprecipitated from wild-type M. bovis and the ΔBCG_1055 strains using a FadD13-specific polyclonal antibody. Acetylated FadD13 was almost completely absent in ΔBCG_1055 but substantial amounts of acetylated FadD13 were present in the wild-type strain, indicating that FadD13 was indeed an in vivo substrate of KATmt. The functional consequences of acetylation of FadDs were analysed using an in vitro fatty acyl CoA synthetase assay. The activities of FadD2 and FadD13 were inhibited on acetylation with KATmt, while acetylation of FadD5 resulted in the formation of a novel product. Therefore, modification of the highly conserved lysine residue in these enzymes by acetylation led to loss or alteration of their enzymatic activity, suggesting that acetylation may be used as a regulatory mechanism to modulate the activities of some of the FadDs by KATmt in a cAMP-dependent manner. Given the extensive role of FadDs in cell wall biosynthesis and lipid degradation in mycobacteria, it seems possible that post-translational control by KATmt in a cAMP-dependent manner constitutes a novel mechanism utilised by these bacteria to regulate these pathways. This direct regulation of protein lysine acetylation by cAMP appears to be unique to mycobacteria, as orthologues of KATmt are not found outside this genus. In addition, the biochemical differences between KATmt and its orthologue from M. smegmatis KATms, indicate species specific variation, on a common theme. This study is the first report of protein lysine acetylation in mycobacteria. In addition to the identification of several proteins subject to this post-translational modification, the effect of acetylation on the enzymatic activities of some of them has been elucidated.

Le glaucome est la principale cause de cécité irréversible dans le monde. Chez les patients atteints de cette pathologie, la perte de la vue résulte de la mort sélective des cellules ganglionnaires (CGR) de la rétine ainsi que de la dégénérescence axonale. La pression intraoculaire élevée est considérée le facteur de risque majeur pour le développement de cette maladie. Les thérapies actuelles emploient des traitements pharmacologiques et/ou chirurgicaux pour diminuer la pression oculaire. Néanmoins, la perte du champ visuel continue à progresser, impliquant des mécanismes indépendants de la pression intraoculaire dans la progression de la maladie. Il a été récemment démontré que des facteurs neuroinflammatoires pourraient être impliqués dans le développement du glaucome. Cette réponse est caractérisée par une régulation positive des cytokines pro-inflammatoires, en particulier du facteur de nécrose tumorale alpha (TNFα). Cependant, le mécanisme par lequel le processus neuroinflammatoire agit sur la mort neuronale reste à clarifier. L’hypothèse principale de ce doctorat propose que les facteurs pro-inflammatoires comme le TNFα et la phosphodiestérase 4 (PDE4) interagissent avec les mécanismes moléculaires de la mort neuronale, favorisant ainsi la survie et la protection des CGRs au cours du glaucome. Dans la première partie de ma thèse, J’ai utilisé un modèle in vivo de glaucome chez des rats Brown Norway pour montrer que l’expression du TNFα est augmentée après l'induction de l'hypertension oculaire. L'hypothèse spécifique de cette étude suggère que les niveaux élevés de TNFα provoquent la mort des CGRs en favorisant l'insertion de récepteurs AMPA perméables au calcium (CP-AMPAR) à la membrane cytoplasmique. Pour tester cette hypothèse, j’ai utilisé un inhibiteur sélectif de la forme soluble du TNFα, le XPro1595. L'administration de cet agent pharmacologique a induit une protection significative des somas et des axones des neurones rétiniens. L'évaluation de la perméabilité au cobalt a montré que le TNFα soluble est impliqué dans l'insertion de CP-AMPAR à la membrane des CGRs lors du glaucome. L’exposition des neurones à une pression oculaire élevée est à l’origine de la hausse de la densité membranaire des CP-AMPARs, grâce à une diminution de l’expression de la sous-unité GluA2. La présence de GluA2 au sein du récepteur ne permet pas l’entrée du calcium à l’intérieur de la cellule. L'administration intraoculaire d’antagonistes spécifiques des CP-AMPARs promeut la protection des somas et des axones des CGRs. Ces résultats montrent que les CP-AMPARs jouent un rôle important dans la pathologie du glaucome. Dans la deuxième partie de ma thèse, j’ai caractérisé l'effet neuroprotecteur d’un inhibiteur de la PDE4, l’ibudilast, dans notre modèle de glaucome. L'hypothèse spécifique s’oriente vers une atténuation de la réponse neuroinflammatoire et de la gliose par l’administration d’ibudilast, favorisant ainsi la protection neuronale. Les résultats montrent que dans les rétines glaucomateuses, l’ibudilast diminue la gliose et l'expression de plusieurs facteurs tels que le TNFα, l'interleukine-1β (IL-1β), l’interleukine-6 (IL-6) et le facteur inhibiteur de la migration des macrophages (MIF). Chez les rats glaucomateux, nous avons observé une expression notable de PDE4A dans les cellules de Müller, qui est en corrélation avec l'accumulation de l’AMP cyclique (AMPc) dans ces cellules après un traitement d’ibudilast. Finalement, nous avons démontré que la protection des CGRs via l’administration d’ibudilast est un mécanisme dépendent de l’AMPc et de la protéine kinase A (PKA). En conclusion, les résultats présentés dans cette thèse identifient deux mécanismes différents impliqués dans la perte des CGRs au cours du glaucome. Ces mécanismes pourraient fournir des perspectives potentielles pour le développement de nouvelles stratégies de traitement du glaucome. / Glaucoma is the leading cause of irreversible blindness worldwide. Loss of vision in glaucoma results from the selective death of retinal ganglion cells (RGCs) and axonal degeneration. Elevated intraocular pressure (IOP) is the major risk factor for developing glaucoma, and current therapies have focused on pharmacological or surgical strategies to lower IOP. However, visual field loss continues to progress in spite of effective pressure control, indicating that mechanisms other than elevated IOP contribute to disease progression. Recent data demonstrate a neuroinflammatory component in glaucoma, characterized by upregulation of proinflammatory cytokines, most notably tumor necrosis factor α (TNFα). However, the mechanism by which the neuroinflammatory response acts on RGC death needs to be clarified. The main hypothesis of this thesis is that targeting pro-inflammatory factors including TNFα and phosphodiesterase-type 4 (PDE4), interferes with molecular mechanisms that contribute to RGC death and this will thus successfully promote neuronal protection. In the first part of my thesis, I used an in vivo glaucoma model in Brown Norway rats to show that TNFα is upregulated early after induction of ocular hypertension. The specific hypothesis of this study is that high levels of TNFα promote RGC death by mediating the membrane insertion of Ca2+-permeable AMPA receptors (CP-AMPARs). I blocked TNFα function with XPro1595, a selective inhibitor of soluble TNFα. Administration of XPro1595 effectively protected RGC soma and axons. The cobalt permeability assay was used to show that soluble TNFα triggers the membrane insertion of CP-AMPAR in RGCs of glaucomatous retinas. This CP-AMPAR activation is caused by the downregulation of GluA2 which occurs when neurons are exposed to elevated IOP. Finally, intraocular administration of specific CP-AMPAR antagonists promoted RGC soma and axon protection. Taken together, these results show that CP-AMPARs play an important role in in the pathology of glaucoma. In the second part of my thesis, I characterized the neuroprotective effect of ibudilast, an inhibitor of PDE4, in the Brown Norway glaucoma model. We hypothesized that ibudilast promotes neuron protection by attenuating gliosis and the neuroinflammatory response. The results show that in glaucomatous retinas, ibudilast attenuates gliosis and the expression of TNFα, interleukin-1β (IL-1β), interleukin-6 (IL-6) and macrophage migration inhibitory factor (MIF). Interestingly, elevated IOP leads to substantial expression of PDE4A in Müller cells, which correlates with the accumulation of cAMP in these cells after ibudilast treatment. Lastly, ibudilast promoted RGC soma and axons protection through the activation of the cAMP/PKA pathway. In conclusion, the findings presented in this thesis identify two different mechanisms underlying RGC loss in glaucoma. These mechanisms can potentially provide new insights to develop novel strategies for the treatment of glaucoma

Cystic fibrosis (CF) is the most common genetic disease affecting Canadian newborns (1 in 3,850) and is caused by mutations in the CF transmembrane conductance regulator (CFTR) gene. This gene encodes for CFTR, a phosphorylation-dependent ion channel localized at the apical membrane. Phosphorylation of CFTR by the cyclic adenosine monophosphate (cAMP)-dependent enzyme protein kinase A activates its activity, facilitating the transport of chloride and bicarbonate ions across the epithelial membrane. CFTR contributes to ion and airway surface liquid regulation, crucial for maintaining host defenses. The inheritance of CFTR mutations leads to a variety of respiratory complications, including impaired mucociliary clearance, excessive mucus production, persistent airway infections, and heightened inflammation, ultimately causing lung damage. While there is currently no cure for CF, the development of CFTR modulators, targeting the defective CFTR protein directly, has significantly improved the quality of life for many CF patients. Despite these advancements, many patients remain unresponsive to current treatment options. It has been well-established that combination therapies outperform monotherapies, emphasizing the need for alternative or complementary therapeutic strategies for CF management. Furthermore, CFTR dysfunction extends beyond CF and has been implicated in other respiratory diseases, such as chronic obstructive pulmonary disease, which is primarily linked to tobacco smoke exposure. This Ph.D. thesis explores a complementary therapeutic approach, targeting proteins within the CFTR-containing macromolecular signaling complex to elevate intracellular cAMP levels, thereby enhancing CFTR function. We hypothesized that synergistic use of cAMP modulators, alongside CFTR modulators, will serve as an effective therapeutic strategy for CF and other respiratory diseases. Collectively, our studies highlight the potential of cAMP and CFTR modulation as a therapeutic strategy for improving the treatment of CF and other respiratory diseases, warranting further investigation, offering insights for future studies, and contributes to the ongoing pursuit of improved combination treatments. / Dissertation / Doctor of Philosophy (PhD) / Cystic fibrosis (CF) is the most common genetic condition affecting Canadian newborns, caused by inheritance of mutations in the CF transmembrane conductance regulator (CFTR) gene. These mutations result in respiratory issues, including breathlessness, excess mucus, and susceptibility to infections, causing lung damage and premature death. Despite progress in CF drug development, some patients remain unresponsive to existing drug combinations, highlighting the need for new combinations to improve the quality of life for all CF patients. CFTR function is also compromised in other respiratory diseases like chronic obstructive pulmonary disease, a lung disease that shares many characteristics with CF and is mainly caused by tobacco smoke exposure. This Ph.D. thesis explores the effectiveness of a new drug strategy targeting proteins interacting with CFTR. By investigating drugs to complement existing treatments, we aim to improve CFTR function. This research offers a promising strategy to improve treatment for CF and other respiratory diseases.

Les opioïdes sont les analgésiques les plus efficaces mais leur utilisation est limitée par la tolérance, un processus lié en partie à la désensibilisation des récepteurs. Le rôle de la présente étude était de mieux caractériser le processus de désensibilisation des récepteurs et plus particulièrement, d’étudier le rôle de la tyrosine kinase Src sur la régulation de la signalisation des récepteurs delta opioïdes. Nos résultats démontrent que l’inhibition pharmacologique avec PP2 (à faible concentration : 20- 40µM) ou encore l’inhibition moléculaire de la kinase avec de faibles concentrations d’ADN d’un mutant dominant inactif de Src (0,2µg/ml) potentialise l’amplitude et la durée de l’activation de la cascade ERK lorsqu’un agoniste, DPDPE (1µM; 5 min), se lie aux récepteurs. Nous avons également démontré que de fortes concentrations d’inhibiteurs de Src (80 et 100µM de PP2 ou 1µg/ml d’ADN du mutant dominant négatif) bloquent la cascade des MAPK suivant la stimulation de DOR par l’agoniste DPDPE. Ces observations indiquent que Src a un effet biphasique sur l’activité de ERK : l’inhibition complète de Src inhibe l’activité de la cascade MAPK alors qu’une inhibition modérée potentialise cette même cascade. Nous pensons aussi que de fortes concentrations des bloqueurs de Src interfèrent avec l’activation de ERK alors que de faibles concentrations interfèrent avec la désensibilisation des récepteurs. Cette possibilité a été testée à l’aide d’essais d’accumulation d’AMPc qui visaient à évaluer l’effet des bloqueurs de Src (PP2, 20 µM; 1h) sur la désensibilisation induite par un agoniste. L'activation de DOR par DPDPE inhibe la production d’AMPc, préalablement stimulée par du forskolin, de façon dose-dépendante. Le maximum d'inhibition observé est de 61%, mais lors d’un prétraitement au DPDPE (1 µM, 30 min) l’inhibition maximale est réduite à 72% de l’inhibition initiale observée. Cependant, un prétraitement des cellules au PP2 (20µM pendant 1 heure) avant d’effectuer la désensibilisation protège contre cette désensibilisation. L’effet protecteur des bloqueurs de Src n’entraîne pas de changement au niveau de l’internalisation des DOR mais l’altération de leur internalisation via un mutant tronqué du DOR ou via un milieu sucré hypertonique (0.4M de saccharose) réduit cette protection. Ces données suggèrent alors que l’internalisation optimale du récepteur est nécessaire pour que l’effet protecteur prenne place. Nous concluons donc que Src contribue à la désensibilisation de DOR après que l’internalisation du DOR soit survenue. / Opioids are the most effective analgesics available but their use is limited by tolerance. Tolerance is related, at least in part, to receptor desensitization. Hence, the role of the present study was to better characterize the desensitization process, in particular concerning the role of the tyrosine kinase Src on regulation of delta opioid receptor signalling. Our results show that pharmacological inhibition with PP2 (administered at low concentration: 20-40µM) or molecular inhibition of the kinase with low expression levels of a dominant negative mutant of Src (0,2µg of DNA) potentiate the magnitude and duration of agonist-dependent (DPDPE; 1µM; 5 min) activation of the ERK pathway. We also showed that higher concentrations of Src inhibitors (80 and 100µM of PP2 or 1µg/ml of dominant negative mutant DNA) block the MAPK cascade following DOR stimulation by DPDPE. These observations indicate that Src has a biphasic effect on ERK activity, respectively potentiating or inhibiting agonist stimulation of the MAPK cascade at low and high levels of Src inhibition. We reasoned that high levels of Src blockers were interfering with ERK activation mechanism while low levels of inhibition were interfering with receptor desensitization. This possibility was tested by using cAMP accumulation assays to evaluate the effect of Src blockers (PP2, 20 µM; 1h) on agonist-induced desensitization. DOR stimulation by DPDPE inhibited forskolin stimulated cAMP production in a dose dependent manner with a maximal reduction of 61%. This inhibitory response was reduced by 72% following pre-exposure to DPDPE (1 µM, 30 min), an effect that was blocked by pre-treating cells with PP2 (PP2, 20 µM; 1 h) before desensitization. The protective effect of Src blockers did not involve changes in DOR internalization but interfering with internalization by using an internalization-deficient DOR mutant or hypertonic medium (0.4M sucrose) reduced this protection, indicating the need for optimal internalization in order for the protective effect of Src blockers to take place. Based on the latter observation it was possible to conclude that Src contribution to DOR desensitization is post-endocytic.