Дисертації з теми "TRPM2 and TRPM7"

Nowadays, new spinal cord injury (SCI) cases are frequently due to non traumatic causes, especially vascular disorders. A prerequisite to developing mechanism-based neuroprotective strategies for acute SCI is a full understanding of the early pathophysiological changes to prevent later disability and paralysis. The immediate damage spreads from the initial site through excitotoxicity and metabolic dysfunction (ischemia, free radicals and neuroinflammation) to surrounding tissue (secondary damage). Using an in vitro neonatal rat spinal cord model, an experimental protocol (pathological medium, PM) has been developed to mimic the profound metabolic perturbation (hypoxia, aglycemia, oxidative stress, acidosis, toxic free radicals) occurring in vivo after ischemic SCI, a condition surprisingly worsened by extracellular Mg2+ (1 mM). The current study sought to identify the cells affected by PM (with Mg2+), and the associated molecular death pathways in the spinal lumbar region which contains the locomotor networks. The results indicated that 1 h PM+Mg2+ application induced delayed pyknosis chiefly in the spinal white matter via overactivation of poly (ADP-ribose) polymerase 1 (PARP1), suggesting cell death mediated by the process of parthanatos and also via caspase 3-dependent apoptosis. Grey matter damage was less intense and concentrated in dorsal horn neurons and motoneurons which became nuclearimmunoreactive for the mitochondrial apoptosis-inducing factor. Moreover, TRPM2 channel expression was enhanced 24 h later in dorsal horn and motoneurons, while TRPM7 channel expression concomitantly decreased. Conversely, TRPM7 expression grew earlier (3 h) in white matter cells, while TRPM2 remained undetectable. Our results show that extracellular Mg2+ amplified the white matter cell death via parthanatos and apoptosis, and motoneuronal degeneration via PARP1-dependent pathways with distinct changes in their TRPM expression. In fact, the PARP-1 inhibitor PJ34, when applied 30 min after the moderate excitotoxic insult, could protect spinal networks controlling locomotion in more than 50 % of preparations. Interestingly, the drug per se strongly increased spontaneous network discharges without cell damage. Glutamate ionotropic receptor blockers suppressed this phenomenon reversibly. Our results suggest that pharmacological inhibition of PARP-1 could prevent damage to the locomotor networks if this procedure had been implemented early after the initial lesion and when the lesion was limited. PJ34 had also a positive effect on PM+Mg2+ treated spinal cords, especially in the white matter after 24 h, both alone or administered together with caspase-3 inhibitor. The neonatal rat in vitro SCI model was also useful to study the activation of endogenous spinal stem cells. We identified the ATF3 transcription factor as a novel dynamic marker for ependymal stem/progenitor cells (nestin, vimentin and SOX2 positive) located around the central canal of the neonatal or adult rat spinal cord. While quiescent ependymal cells showed cytoplasmic ATF3 expression, over 6-24 h in vitro these cells mobilized and acquired intense nuclear ATF3 staining. The migration of ATF3-nuclear positive cells preceded the strong proliferation of ependymal cells occurring after 24 h in vitro. Pharmacological inhibition of MAPK-p38 and JNK/c-Jun, upstream effectors of ATF3 activation, prevented the mobilization of ATF3 nuclear-positive cells. Excitotoxicity or ischemia-like conditions did not enhance migration of ependymal cells at 24 h. ATF3 is, therefore, suggested as a new biomarker of activated migrating stem cells in the rat spinal cord in vitro that represents an advantageous tool to study basic properties of endogenous stem cells.

Primary afferent neurons are equipped with sensory transduction channels which allow the conversion of physical and chemical stimuli into electrical signals. TRP channels are a heterogeneous superfamily of largely non-selective cation channels, which have been implicated in a myriad of sensory transduction mechanisms from the detection of temperature to the sensation of touch. Many TRP channels are key targets for the study of pain physiology due to their polymodal activation and expression in small diameter, unmyelinated sensory fibres. The aim of my project was to examine the roles of TRP channels in sensory transduction mechanisms. Three results chapters focusing on three different TRP channels are presented. A novel role for the established cold thermosensor, TRPM8, as a cellular osmosensor was determined. The studies presented establish that TRPM8 is activated by increases in extracellular osmolality and is partially activated at normal physiological osmolalities. Cool temperatures increase the sensitivity of TRPM8 to osmotic stimuli and activation of phospholipase enzymes modulates activation of TRPM8 by hyperosmotic solutions. TRPM8 is expressed within sensory neurons where it functions as the chief detector of increased osmolality in addition to a molecular sensor of cold sensations. The role of TRPM3 as a candidate heat transduction channel is examined. The findings presented demonstrate that recombinantly expressed TRPM3 channels are heat-sensitive and mice lacking functional TRPM3 channels lose a population of heat-activated neurons and have impaired behavioural responses to noxious heat. Moreover, modulation of TRPM3 by intracellular pathways downstream of G-protein coupled receptor activation has been determined. Activation of TRPM3 in sensory neurons is shown to be robustly inhibited by morphine in a predominantly mu-opioid receptor and Gi dependent mechanism. Additionally the role of TRPM3 in several pain states is examined. Finally, this thesis reports on the characterisation of a medium-throughput CGRP release assay for examining activation of TRPA1 natively expressed on the central terminals of dorsal root ganglion neurons. Activation of TRPA1 expressed on spinal cord synaptosomes by a selection of agonists evokes a concentration-dependent release of CGRP which is inhibited by TRPA1 antagonists. The VGCC subtypes important for TRPA1 and depolarisation-induced CGRP release are examined.

A resistência dos tecidos à ação da insulina é uma das principais complicações do excesso de peso. O aumento da gordura corporal, decorrente do consumo excessivo de nutrientes, é acompanhado por um quadro de inflamação crônica de baixa intensidade que está relacionado com a fisiopatologia da resistência à insulina. O magnésio (Mg) é um mineral envolvido com diversos processos fisiológicos e bioquímicos, especialmente aqueles relacionados ao metabolismo energético e ao controle glicêmico. Apesar de a deficiência deste mineral estar relacionada com condições pré-diabéticas, não está claro se a inadequação dietética promove alterações na sensibilidade à insulina e/ou se condições de resistência à insulina causam distúrbios na homeostase de Mg. O objetivo deste trabalho foi investigar os efeitos da restrição dietética de Mg e sua associação com o excesso de lipídios sobre a homeostase do mineral e a sensibilidade à insulina. Ratos Wistar, machos, com peso entre 97-123 g, permaneceram em gaiolas individuais por 24 semanas. Os animais receberam rações normolipídicas (CON, 7% de lipídios) ou hiperlipídicas (HL, 32% de lipídios), adequadas (CON e HL Mg; 500 mg de Mg/kg de ração; n = 6 para cada grupo) ou com restrição de Mg (Mg[50] e HL Mg[50]; 50 mg de Mg/kg de ração; n = 6 para cada grupo). O consumo da dieta HL promoveu maior acúmulo de tecido adiposo e maior ganho de peso corporal (p < 0,05). Os animais que consumiram rações com restrição de Mg apresentaram hipomagnesemia (p<0,01), menor excreção urinária (p < 0,01) e fecal (p < 0,001) de Mg e menor concentração óssea desse mineral (p < 0,001). No entanto, não foram observadas alterações no Mg muscular (p > 0,05). O grupo HL Mg[50] apresentou maior concentração de Mg no eritrócito quando comparado aos outros grupos. A restrição dietética de Mg, isoladamente, não promoveu alterações na sensibilidade à insulina (avaliada pelo teste de tolerância à insulina). Quando associada à dieta hiperlipídica, resultou em aumento da glicemia de jejum e em redução da sensibilidade à insulina, após 16 semanas (p < 0,01). Em nível molecular, a fosforilação da proteína quinase B (Akt) no músculo e no fígado foi significantemente menor no grupo HL Mg[50] (p < 0,05). A restrição dietética de Mg induziu ao aumento do conteúdo proteico dos canais TRPM6 e TRPM7 no rim, independentemente da sensibilidade à insulina. Os resultados deste estudo apontam que a deficiência de Mg tende a agravar as repercussões metabólicas do consumo de dietas hiperlipídicas na sensibilidade à insulina e que a resistência à insulina altera a compartimentalização do Mg.
Insulin resistance is one of the main complications of overweight. Increase body fat, due to excessive consumption of nutrients is accompanied by a chronic low-grade inflammation related to insulin resistance pathophysiology. Magnesium (Mg) is a mineral involved in many physiological and biochemical processes, especially those related to energy metabolism and glycemic control. Although Mg deficiency is related to pre-diabetic conditions, it is unclear whether dietary inadequacy promotes changes in insulin sensitivity and/or if conditions of insulin resistance cause disturbances in Mg homeostasis. This work aimed to investigate the effects of dietary Mg restriction and its association with high-fat diet on mineral homeostasis and insulin sensitivity. Male Wistar rat (97-123 g) remained in individual cages for 24 weeks. Animals received normolipid diet (CON, 7% lipid) or high-fat diet (HF, 32% lipid), adequate (CON and HF, 500 mg Mg / kg diet, n = 6 for each group) or Mg restricted (Mg[50] and HF Mg[50], 50 mg of Mg / kg of diet, n = 6 for each group). High-fat diet promoted a greater adipose tissue excess and body weight gain (p<0.05). Animals with Mg restricted diet had hypomagnesemia (p<0.01), lower Mg urinary (p<0.01) and faecal loss (p<0.001) and lower bone Mg concentration (p<0.001). However, no changes were observed in muscle Mg (p>0.05). HF Mg[50] group presented higher concentration of erythrocyte Mg when compared to the other groups. Singly, dietary Mg restriction did not induce changes in insulin sensitivity (as assessed by the insulin tolerance test). When associated with high-fat diet, dietary Mg restriction resulted in higher fasting glycemia and lower insulin sensitivity after 16 weeks (p<0.01). At the molecular level, protein kinase B (Akt) phosphorylation in muscle and liver was significantly lower in HFMg [50] group (p<0.05). Dietary Mg restriction induced increased protein content of renal TRPM6 and TRPM7 channels, regardless of insulin sensitivity. The results of this study indicate that Mg deficiency worsens metabolic effects of high-fat diet on insulin sensitivity. In addition, insulin resistance changes Mg compartmentalization.

ABSTRACTTRPM7 (transient receptor potential melastatin), a member of the large TRP ion channel family, is ubiquitously expressed in cells and is constitutively active. It is comprised of six transmembrane domains that assemble into tetramers to form a central Mg2+ and Ca2+ permeable pore. TRPM7 and its homologue TRPM6 are some of the only channels known to carry Mg2+. Hypertension, a cardiovascular condition linked to low levels of intracellular Mg2+ is also associated with high levels of aldosterone. Previous results have demonstrated that aldosterone increases mRNA levels of TRPM7 whereas protein levels decreased in VSMCs. To understand if TRPM7 may be implicated in hypertension, we questioned whether aldosterone could regulate TRPM7 currents, and inquired for a possible underlying mechanism. Whole-cell patch clamp studies were conducted in inducible HEK-293 cells, stably expressing wild type TRPM7. We found that TRPM7 currents are enhanced after overnight stimulation with aldosterone compared to non-stimulated cells. When the mineralocorticoid receptor (hMR) is transfected two days prior aldosterone stimulation, this response is further increased. The introduction of 10mM BAPTA, a Ca2+ chelator, to the intracellular medium doubled the TRPM7 current in WT cells and also increased the response to aldosterone in cells transfected with the hMR receptor. Surprisingly, protein levels of TRPM7 do not vary, suggesting a redistribution of already existing channels to the membrane. SGK-1, a serine threonine kinase was suggested as a possible mediator of the response. In fact, when a specific blocker to SGK-1 is applied onto the cells, both current and total protein levels of TRPM7 are significantly decreased. Overall, these results demonstrate that aldosterone can regulate TRPM7 through an increase in total current. This response appears to be mediated by SGK-1 in a calcium sensitive manner.
RÉSUMÉTRPM7 (transient receptor potential melastatin), membre de la large famille des canaux ioniques des TRP, est exprimée de façon omniprésente dans toutes les cellules, et est active de façon constitutive. TRPM7 est composée de six domaines transmembranaires qui s'assemblent en tétramères pour former un pore central, perméable aux ions Mg2+ et Ca2+. TRPM7, et son homologue TRPM6, sont les seuls canaux ioniques connus pour le transport du Mg2+. L'hypertension, une maladie cardiovasculaire associée à de faibles niveaux en Mg2+ intracellulaire est aussi liée à de niveaux élevés d'aldosterone. Des recherches antérieures ont démontré que l'aldosterone augmente les niveaux d'ARNm de TRPM7 tandis que la quantité de protéines diminue dans les cellules vasculaires lisses du muscle. Afin de comprendre si TRPM7 peut être impliquée dans l'hypertension, nous nous sommes demandés si l'aldosterone pouvait réguler les courants associés à TRPM7, et si nous pouvions définir un mécanisme d'action qui pourrait expliquer une telle régulation. La technique du patch clamp a été utilisée sur des cellules HEK-293 inductibles exprimant de façon stable le phénotype humain de TRPM7. Nous avons trouvé que les courants de TRPM7 sont augmentés après une stimulation de nuit avec de l'aldosterone, comparé à des cellules non stimulées. Lorsque le récepteur humain mineralocorticoid (hMR) est transfecté deux jours avant la stimulation par l'aldosterone, la réponse en courant est rehaussée. L'ajout de 10mM de BAPTA, un chélateur du Ca2+, dans la solution intracellulaire permet de doubler la réponse en courant dans ces cellules, ainsi que d'augmenter la réponse à l'aldosterone dans les cellules transfectées avec le récepteur hMR. Etonnamment, les niveaux de protéines de TRPM7 ne sont pas affectés, suggérant une redistribution des canaux ioniques déjà existants à la membrane. SGK-1, une kinase membre de la famille des serine-threonines a été proposée comme un possible médiateur de la réponse a l'aldosterone. En effet, après l'application d'un bloquer spécifique pour le SGK-1, une diminution des courants ainsi que de la quantité de protéines associées à TRPM7 a été observée. De façon générale, ces résultats démontrent que l'aldosterone est capable de réguler TRPM7 à travers une augmentation des courants. Cette réponse, qui semble être sous l'influence de SGK-1, utilise un mécanisme sensible aux niveaux de calcium intracellulaire..

Melastatin-related transient receptor potential 2 (TRPM2) channel is a Ca2+-permeable cation channel that is gated by ADP-ribose (ADPR) and also activated by reactive oxygen species (ROS) such as H2O2. TRPM2 channel are shown to be critically involved in several physiological and pathological cell processes. Previous studies have reported inhibition of the human TRPM2 channel by extracellular acidic pH. However, the underlying mechanism is not fully understood. In the present study, I performed patch-clamp recordings to examine the effect of extracellular acidic pH on ADPR-induced currents in HEK293 cells heterogeneously expressing human TRPM2 (hTRPM2) or mouse TRPM2 (mTRPM2) channels. The results showed that the inhibition was substantially reversible upon brief exposure to acidic pH but became irreversible after prolonged exposure, supporting the mechanism in which protons bind to and inhibit the open TRPM2 channel and the proton-binding induces further conformational changes leading to channel inactivation. Furthermore, the mTRPM2 channel exhibited a lower sensitivity to, and slower kinetics of, inhibition, than the hTRPM2 channel. A residue in the pore region (His-995 in hTRPM2 and Gln-992 in mTRPM2) had a crucial role in determining such species differences. The pharmacology of the TRPM2 channel is poor, with no specific inhibitor. Here, I examined the effects of 48 hit compounds identified from screening chemical libraries on hTRPM2 channels expressed in HEK293 cells. Four compounds inhibited H2O2-induced Ca2+-response with a micromolar to submicromolar potency and abolished ADPR-induced currents at 10 μM, indicating that they act as TRPM2 channel inhibitors. The TRPM2 channel was reported to be functionally expressed in macrophage cells, but its role in mediating ROS-induced Ca2+ signalling and cell death is largely unclear. This study examined the contribution and mechanism of the TRPM2 channel in H2O2-induced Ca2+-responses and cell death in RAW264.7 and differentiated THP-1 macrophage cells and peritoneal macrophage cells isolated from TRPM2+/+ and TRPM2-/- mice. The results showed that TRPM2 channels operated as cell surface Ca2+-permeable channels and constituted the principal Ca2+ signalling mechanism, but played a limited role in cell death. In summary, the results from my study provided useful information to advance the understanding of the pharmacology and functional roles of the TRPM2 channels.

TRPM7 (Transient Receptor Potential Melastatin-like 7) is an ion channel necessary for the proper development of many cell types. Insight into the precise role of the channel in different cells has been hampered by the lethality of knocking out the gene in model organisms such as the mouse. Here I examine a zebrafish that has a loss-of-function mutation in the gene encoding Trpm7. First, I show that trpm7 is important for the function of developing dopaminergic neurons in the zebrafish. Second, I examine the interaction between trpm7 and the related gene vmat2 in order to develop a cellular mechanism of trpm7 function in presynaptic dopaminergic neurons. Finally, I investigate the necessity of the kinase and ion channel domains of trpm7 in their ability to promote pigmentation in melanophores as a model cell type. Based on the results from these experiments and observations from other researchers, I form a new hypothesis for Trpm7 function in protein sorting. These studies provide a detailed and novel analysis of the function of an ion channel that is necessary for life.

Transient receptor potential melastatin-related 2 (TRPM2) channel is gated by ADP-ribose (ADPR) and potently activated by reactive oxygen species (ROS) through stimulating ADPR-generating mechanisms. Recent studies provide evidence to show a crucial role for TRPM2 in neuronal death and cognitive impairment associated with ischemic stroke and Alzheimer’s disease. However, the underlying mechanisms are poorly understood. Studies described in this thesis adopted genetic and pharmacological interventions, in conjunction with immunofluorescent and live cell imaging, to investigate TRPM2-dependent cell death induced by H2O2 and the 42-residue of amyloid β (Aβ42) in cultured hippocampal neurons. H2O2 and Aβ42 induced significant neuronal death, which was reduced or prevented by TRPM2 knock-out (TRPM2-KO), TRPM2 channel inhibitors, or Zn2+ chelator TPEN. H2O2 and Aβ42 induced intracellular Zn2+ increase, lysosomal dysfunction and Zn2+ release, mitochondrial Zn2+ accumulation, dysfunction and ROS generation. Bafilomycin A1-induced lysosomal dysfunction also resulted in mitochondrial Zn2+ accumulation and ROS generation. These events were abolished by TRPM2-KO or suppressed by inhibiting poly(ADP-ribose) polymerase-1 (PARP-1) or TRPM2 channel. Immunofluorescent imaging suggests mitochondrial localization of TRPM2. ADPR enhanced Zn2+ accumulation in isolated mitochondria from wild-type (WT) but not TRPM2-KO neurons. Finally, the inhibition of protein kinase C (PKC) and NADPH oxidases (NOX), particularly NOX1/4, suppressed H2O2/Aβ42-induced neuronal death and Aβ42-induced intracellular Zn2+ increase, lysosomal and mitochondrial dysfunction, and mitochondrial ROS generation. The inhibition of the proline-rich tyrosine kinase 2 (Pyk2) and the downstream MEK/ERK kinases protected against Aβ42-induced neuronal death. Taken together, these results provide evidence to support a vicious positive feedback signalling loop that drives hippocampal neuronal death in response to ROS and Aβ42, in which the TRPM2 channel in mitochondria integrates multiple mechanisms comprising PKC/NOX-mediated ROS generation, lysosomal dysfunction and Zn2+ release, mitochondrial Zn2+ accumulation, mitochondrial dysfunction and ROS generation. In addition, the Pyk2-MEK-ERK signalling pathway is critically involved in Aβ42-induced TRPM2-dependent neuronal death. These findings provide novel insights into the mechanisms underlying neuronal death and cognitive impairment related to ischemic stroke and AD.

Microglial cell plays a key role in neuroinflammation induced by diverse danger associated molecular patterns (DAMP) molecules, such as Zn2+, Aβ42 and TNF-α, and strongly implicated in neurodegenerative diseases. The molecular mechanisms for neuroinflammation are however not fully defined. Reactive oxygen species (ROS) production is critical in DAMP-induced microglial cell activation and cytokine production. Studies presented in this thesis aimed to investigate, using immunocytochemistry, single cell imaging, cell death and ELISA assays in combination with genetic and pharmacological interventions, the role of ROS-sensitive TRPM2 channel in cell death, cell activation and production of TNF-α in primary microglial cells in response to Zn2+, Aβ42 and TNF-α as well as H2O2. H2O2 (10-300 mM) and Zn2+ (10-300 mM) induced concentration-dependent increases in the intracellular Ca2+ concentration ([Ca2+]i) via Ca2+ influx, which were prevented by TRPM2 knockout (TRPM2-KO) or treatment with TRPM2 inhibitor 2-APB or PARP inhibitor PJ34. Pathological concentrations of H2O2 (100-300 mM) and Zn2+ (100-300 mM) induced substantial cell death that was ablated by TRPM2-KO and treatment with 2-APB or PJ34. Zn2+ also induced ROS production and PARP-1 activation. All these Zn2+-induced effects were suppressed by treatment with PKC inhibitor chelerythrine, NOX inhibitors DPI, GKT137831 or Phox-I2. Zn2+-induced PARP-1 stimulation, increase in the [Ca2+]i and cell death were also inhibited by PYK2 inhibitor PF431396 or MEK/ERK inhibitor U0126. Exposure to Aβ42 (30-300 nM) and TNF-α (10-100 ng/ml) resulted in concentration-dependent TRPM2-mediated Ca2+ influx and increases in the [Ca2+]i, microglial cell activation and TNF-α production. Aβ42 and TNF-α stimulated ROS production and PARP-1 activation. These effects induced by Aβ42 or TNF-α were suppressed by inhibiting PKC and NOX. Moreover, Aβ42/TNF-α induced PARP-1 activation, increase in the [Ca2+]i, microglial cell activation and TNF-α production were attenuated by inhibiting PYK2 and MEK/ERK. In summary, studies provide strong evidence to reveal a critical role for the TRPM2 channel in Ca2+ signalling in microglial cells induced by Zn2+, Aβ42 and TNF-α. TRPM2 channel activation by Zn2+, Aβ42 and TNF-α depends on PKC/NOX-mediated ROS production and PARP-1 activation and is additionally enhanced by the PYK2-MEK-ERK signalling pathway. Such mechanisms are critically involved in cell death in response to Zn2+, or microglial cell activation and TNF-α production in response to Aβ42 and TNF-α. These findings provide novel insights into the role of microglial cells in neuroinflammation and in the pathogenesis of neurodegenerative diseases.

While oxidative stress and dysregulation of Ca2+ homeostasis in vascular signalling are hallmarks of hypertension-associated vascular injury, downstream molecular mechanisms that govern reactive oxygen species (ROS)-dependent regulation of ion channels in hypertension are complex and have not yet been fully elucidated. Activation of poly(ADPribose)-polymerase 1 (PARP1) in response to oxidative stress and DNA damage leads to subsequent activation of transient receptor potential melastatin 2 (TRPM2), an ion channel that regulates Na+ and Ca2+ influx. Considering that ROS generation and Ca2+ influx are increased in hypertension we hypothesised that redox regulation of PARP1-TRPM2 may play a role in vascular injury and target organ-damage associated with hypertension. The proof of concept was tested in a TRPM2-overexpressing human embryonic kidney cell line and the relationship between ROS, PARP1 and TRPM2 was studied in vitro, in human vascular smooth muscle cells and endothelial cells. The regulatory role of PARP1 and TRPM2 on vascular function was assessed in isolated resistance arteries from LinA3 mice, a transgenic model expressing human renin gene resulting in chronic hypertension. Finally, the effects of pharmacological inhibition of PARP1 on blood pressure, target organ damage and cellular signalling were evaluated in vivo in hypertensive LinA3 mice. To our knowledge, findings from this study provide the first evidence in clinically-relevant models, that the redox-sensitive PARP1-TRPM2 pathway regulates vascular contraction in the context of hypertension. This is supported by the following findings: i) The in vitro studies demonstrated that PARP1 and TRPM2 facilitate Ang II and oxidant-dependent activation of a pro-contractile protein MLC20 and partially reduce the anti-contractile activity of MYPT1. ii) The ex vivo experiments on mesenteric resistance arteries from LinA3 mice confirmed that the PARP1-TRPM2 pathway exacerbates vascular hypercontractility of the arteries isolated from hypertensive mice. iii) In vivo inhibition of PARP1 had significant effects on renal and cardiac Akt/PKB-dependent signalling, leading to upregulation of prosurvival and anti-apoptotic proteins, effects that were independent of blood pressure lowering. Overall, the studies presented in this thesis highlight a novel pathway linking ROS to vascular signalling pathways through PARP1 and TRPM2. Dysregulation of this system, in the context of oxidative stress in hypertension, may play a role in hypertension-associated vascular injury and target organ damage. While the present studies have opened the field, further investigations to unambiguously prove the importance of the ROS-PARP1-TRPM2-Ca2+ axis in hypertension are required.

For many ion channels there are few, if any, pharmacological agents, and even fewer showing specificity. In this study, a set of pharmacological tools were developed to investigate TRPM3, a widely expressed transient receptor potential (TRP) channel for which no functional role has yet been identified. Human TRPM3 was first expressed in HEK 293 cells and shown to be activated by hypo-osmotic challenge or sphingosine, consistent with previous reports. In addition, TRPM3 was activated by pregnenolone sulphate. Hydrophobicity analysis of the TRPM3 amino acid sequence revealed a short and reasonably unique peptide in the 3rd extracellular loop (E3) region, to which polyclonal antiserum (TM3E3) was produced. Extracellular application of TM3E3 inhibited TRPM3 function with a high degree of specificity, having no effect on TRPM2 or example members of other sub-types of mammalian TRP, TRPC5 or TRPV4. The data validate E3-targeting as an approach for production of isoform-specific channel blockers and reveal a specific agent for blocking TRPM3. The cellular and tissue functions of TRPM3 were also investigated. RT-PCR and immunocytochemistry demonstrated TRPM3 expression in human saphenous vein smooth muscle cells, where sphingosine- and pregnenolone sulphate-induced calcium responses were also apparent. These calcium responses could be selectively blocked by TM3E3. Furthermore, TRPM3 activators inhibited matrix metalloproteinase and interleukin-6 secretion, indicating a protective function for TRPM3 in vascular smooth muscle cells. Medium throughput screening systems were employed to screen a library of compounds for further TRPM3 modulators with vascular relevance. Cholesterol, antidepressants, antipsychotics, calmodulin inhibitors, and PIP2 all inhibited TRPM3, whereas nifedipine and elevated temperature activated the channel. TRPM3 appears to be regulated by a large number of different chemicals and mechanisms. In summary, TRPM3 has constitutive, protective, activity which can be suppressed by a multitude of compounds, including known vascular disease factors such as cholesterol.

La mucoviscidose est une maladie causée par des mutations du gène cftr entraînant des défauts importants de la protéine CFTR. La mutation la plus fréquente (F508del) est caractérisée par un repliement incorrect conduisant à la rétention de la protéine dans le RE.L’accumulation de CFTR-F508del dans le RE, l’inflammation et les infections vont déclencher un stress du RE dans les cellules épithéliales ainsi que l’UPR. Cette dernière est une réponse adaptative déclenchée par le stress du RE et permet de rétablir l’homéostasie de ce compartiment. L’UPR est constituée de trois voies majeures dont l’une d’entre elles est activée dans les cellules exprimant un CFTR-F508del. Il s’agit de la voie ATF6 qui est de plus responsable de la répression transcriptionnelle du CFTR, ce qui en fait une cible thérapeutique potentielle. Nous avons montré que son inhibition conduit à l’amélioration de la fonction duCFTR-F508del et à l’augmentation de sa présence à la membrane des cellules.Nous nous sommes également intéressés au Mg2+ et au TRPM7, le régulateur principal de la [Mg2+]i dans les cellules. Nous avons émis l’hypothèse que TRPM7 était en partie responsable de l’activation d’ATF6 dans les cellules exprimant un CFTR-F508del. Le but de cette seconde partie du projet était donc tout d’abord d’étudier la relation existante entre le Mg2+, TRPM7 et le CFTR. Nous avons montré qu’il existait des différences de [Mg2+]i selon le type de mutation du CFTR exprimé par les cellules. Ces différences sont en partie dues à un défaut d’activation de TRPM7, lui-même probablement lié à un défaut du CFTR. En augmentant l’activité de TRPM7 par du Naltriben, nous avons pu montrer un effet potentialisant sur leCFTR-G551D
Cystic fibrosis is caused by mutations in the cftr gene resulting in several defaults on the CFTR protein. The most frequent mutation is F508del which is characterized by an incorrect folding causing its retention within the ER. CFTR-F508del protein accumulation in the ER, inflammation and infections will trigger the ER stress in epithelial cells, as well as UPR. UPR constitutes an adaptive response of the ER in order to restore ER’s homeostasis. UPR consists in three major pathways. Among them, one is activated in cells expressing CFTR-F508del protein. The ATF6 pathway of UPR is responsible of the transcriptional repression of CFTR, which makes of it a potential therapeutic target. We showed that the inhibition of ATF6 leads to the improvement of CFTR-508del function, as well as its increased presence in the cellular membrane. We were also interested in Mg2+ and TRPM7, the main regulator of [Mg2+]i. We suspected that TRPM7 is, at least in part, responsible for the activation of ATF6 in cells expressing the mutant CFTR-F508del. Thus, the second part of my work was focused on the study of the relationship between Mg2+, TRPM7 and CFTR. We showed the existence of [Mg2+]I differences according to CFTR mutant expressed in cells. These differences are the result of an altered TRPM7 activation, probably in link with the mutated CFTR’s malfunction. We proved that increasing TRPM7 activity by Naltriben treatment potentiates CFTR-G551D

TRPM3 (melastatin-related transient receptor potential 3) is a calcium-permeable nonselective cation channel that is expressed in various tissues, including insulin-secreting β-cells and a subset of sensory neurons from trigeminal and dorsal root ganglia (DRG). TRPM3 can be activated by the neurosteroid pregnenolone sulphate (PregS) or heat. TRPM3α2 mice display an impaired sensation of noxious heat and inflammatory thermal hyperalgesia. A calcium-based screening of a compound library identified four natural compounds as TRPM3 blockers. Three of the natural compounds belong to the citrus fruit flavanones (hesperetin, eriodictyol and naringenin), the forth compound is a deoxybenzoin that can be synthesized from an isoflavone of the root of Ononis spinosa (ononetin). The IC50 for the substances ranged from upper nanomolar to lower micromolar concentrations. Electrophysiological whole-cell measurements as well as calcium measurements confirmed the potency of the compounds to block TRPM3 in DRG neurones. To further improve the potency and the selectivity of TRPM3 block and to identify the pharmacophore within the flavanone structure, we conducted a hit optimisation procedure by re-screening a focussed library. The library composed of several flavanones with different substitutions on relevant chemical positions and of representatives from different flavonoid subgroups. Within this secondary screen, we identified isosakuranetin and liquiritigenin as active blockers of PregS-induced Ca2+ entry through TRPM3. Isosakuranetin, a flavanone that can be found in blood oranges and grapefruits, displayed an IC50 of 50 nM, and is the most potent inhibitor of TRPM3 identified so far. The novel compounds exhibited a marked specificity for TRPM3 compared with other thermosensitive TRP channels, and blocked PregS-induced [Ca2+]i signals and ionic currents in freshly isolated DRG neurones. Furthermore, isosakuranetin and hesperetin reduced the sensitivity of mice to noxious heat and PregS-induced chemical pain. Since the physiological functions of TRPM3 channels are still poorly defined, the development and validation of potent and selective blockers is expected to contribute to clarifying the role of TRPM3 in vivo. Considering the involvement of TRPM3 in nociception, TRPM3 blockers may represent a novel concept for analgesic treatment.

L'adénocarcinome canalaire pancréatique (ACP) est le type de cancer le plus fréquent touchant le pancréas exocrine. Il est caractérisé par un phénotype métastatique et chimio-résistant pour lequel il n'existe aucun marqueur diagnostic, ni de traitement efficace. Les projections pour 2030 montrent que ce cancer pourrait devenir la deuxième cause de mortalité. Il y a un besoin urgent de mieux comprendre comment progresse l'ACP. La dissémination métastatique dépend de plusieurs mécanismes cellulaires dont l'invasion du stroma par les cellules cancéreuses. Nous avons montré récemment que le canal transmembranaire TRPM7 est surexprimé dans l'ACP et régule la migration cellulaire. Le but de ce travail est d'évaluer le rôle de TRPM7 dans l'invasion et de mettre en évidence les mécanismes moléculaires impliqués dans les cellules cancéreuses d'ACP et également dans les cellules non-cancéreuses. TRPM7 régule l'invasion basale dans les cellules cancéreuses pancréatiques via l'entrée constitutive de magnésium et la régulation de la sécrétion de MMP-2, uPA et Hsp90α. TRPM7 interagit directement avec Hsp90α et sa kinase participe à la phosphorylation des résidus sérines. De plus, l'expression de TRPM7 dans les métastases est corrélée à celle dans la tumeur primaire. Dans les cellules non cancéreuses, TRPM7 n'est pas impliqué dans l'invasion basale mais sa surexpression (par transfections de plasmides ou induite par une exposition au cadmium, un polluant probablement carcinogène) entraine la transformation des cellules vers un phénotype invasif. TRPM7 est impliqué dans la modification de l'homéostasie magnésique majoritairement, dans la modification de morphologie cellulaire et la transition épithélio-mésenchymateuse. Pour conclure, nos résultats apportent de nouvelles connaissances sur le rôle TRPM7 en tant que régulateur de l'invasion basale dans l'ACP et initiateur dans l'acquisition du phénotype invasif des cellules non-cancéreuses
Pancreatic ductal adenocarcinoma (PDAC) is the most common form of pancreatic cancer. It is characterized by a metastatic and chemoresistant phenotype for which there is no diagnostic marker or effective treatment. It may become the second leading cause of cancer-related death by 2030. There is an urgent need to better understand PDAC progression. Metastatic spread depends on several cellular mechanisms, including the invasion of stroma by cancer cells. We have recently shown that the transmembrane channel TRPM7 (Transient Receptor Potential Melastatin related 7) is overexpressed in PDAC and regulates cell migration. The aim of this work is to evaluate TRPM7 implication in invasion and to highlight the molecular mechanisms in PDAC and non-cancer pancreatic cells. TRPM7 channel regulates basal cell invasion, MMP-2, uPA and Hsp90α secretion in human pancreatic cancer cell lines through constitutive magnesium entry. TRPM7 interacts directly with Hsp90α and it contributes to the phosphorylation of serine residues. Magnesium could participate by activating TRPM7 kinase or by modifying Hsp90α conformation. Moreover, TRPM7 expression in metastatic lymph nodes is correlated to its expressionin primary tumor. In non-cancer cells, TRPM7 is not implicated in basal cell invasion but its overexpression (through plasmid orchronic treatment with cadmium, known as probable carcinogen pollutant) induces invasive phenotype transition. TRPM7 is mainly involved in magnesium homeostasis variation, in cellular morphology modification and mesenchymal transition. In conclusion, our results provide new insights into the key role of TRPM7 in both regulation of basal cell invasion in ACP and initiation of invasive phenotype acquisition in non-cancer epithelial cells

Le cancer du pancréas est l’un des cancers les plus meurtriers dans le monde. C’est de plus un cancer extrêmement agressif avec un faible pronostic vital. Nous avons démontré une augmentation de l’expression du canal TRPM7 dans les tissus tumoraux d’ACP par rapport aux tissus pancréatiques sains. De plus, le marquage est plus fort dans les cancers indifférenciés et la surexpression de TRPM7 dans les tumeurs est associée à une diminution de la survie des patients. Les résultats sur les lignées montrent par la technique de patch-clamp la présence d’un courant cationique non-sélectif activé en l’absence de Mg2+ intracellulaire. L’inhibition par ARN interférent du canal TRPM7 entraîne une diminution de la migration dans la lignée moyennement différenciée BxPC-3. Cette diminution est restaurée par une supplémentation en Mg2+. Dans la lignée indifférenciée Panc-1 l’inhibition de l’expression de TRPM7 entraîne une diminution de l’invasion, indépendante du Mg2+. TRPM7 est un chanzyme et possède donc un domaine kinase. L’influence de l’homéostasie ionique dans le cancer est connue mais le rôle de la kinase reste à déterminer. Nos résultats préliminaires pour la surexpression dans les lignées d’ACP du canal TRPM7 sauvage ou de son mutant, sans domaine kinase, montrent un rôle différent en fonction des lignées. Ce canal peut donc être un marqueur biologique potentiellement intéressant dans l’adénocarcinome canalaire pancréatique. Mais son rôle dans le développement de l’adénocarcinome pancréatique est encore loin d’être élucidé
Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive cancers leading to metastasis and low survival rates. We showed that the Transient Receptor Potential Melastatin-related 7 (TRPM7) channel expression is higher in human PDAC tissues compared to exocrine pancreatic tissues form healthy donors. TRPM7 expression is upregulated in undifferentiated, tumours and the patient’s overall survival was inversely correlated to the TRPM7 expression. Role of TRPM7 was also assessed in the moderately differentiated BxPC-3 and in the more undifferentiated Panc-1 human PDAC cell lines. In both cell lines, a Magnesium-Inhibited Cation (MIC) current was recorded by patch-clamp. While TRPM7 silencing decreased BxPC-3 cell migration, which is restored by the Mg2+ supplementation, it reduced Panc-1 cell invasiveness in a Mg2+ independent manner. TRPM7 belongs to the chanzyme family of ion channels and is fused with a kinase domain. Although the role of ion homeostasis is now well known in cancer, the function of the kinase domain of TRPM7 is not well understood. Our preliminary data shows that overexpression of wt-TRPM7 or TRPM7 mutant lacking the kinase domain induces different roles regarding the cell line used. This data suggests that TRPM7 regulates pancreatic cancer cell migration and invasion by regulating magnesium homeostasis and/or kinase function. Thus, TRPM7 could represent a promising biomarker of PDAC but its role in pancreatic carcinogenesis needs further investigation

Les cellules dendritiques (DC) sont des cellules centrales du système immunitaire. Elles activent les lymphocytes et permettent l'orientation de la réponse immune adaptative. Pour cela, les DC doivent maturer et migrer vers les organes lymphoïdes secondaires, lieus de la mise en place d'une réponse lymphocytaire spécifique de l'agent infectieux. Le calcium est un second messager ubiquitaire régulant de nombreuses fonctions cellulaires dont la migration. Cependant, le rôle du calcium dans la biologie des cellules dendritiques a été relativement peu étudié. Nous montrons que le canal ionique TRPM4 est un acteur majeur de la régulation de l'homéostasie du calcium des DC après activation. En effet, l'absence de TRPM4 dans les DC induit une surcharge calcique après activation bactérienne ce qui affecte fortement la migration des DC sans affecter leur maturation. Nous avons observé qu'une surcharge calcique entraînait une diminution de l'expression de la PLC-p2 ce qui est corrélé à une absence de réponse lors d'une seconde stimulation calcique. Ainsi, ces travaux nous ont permis de montrer que TRPM4 est essentiel à la régulation de la migration et non de la maturation des DC renforçant l'idée selon laquelle ces deux entités biologiques sont régulées différemment. Un substrat artificiel des serines protéine-kinases F a-caséine et une protéine cytoplasmique de poids moléculaire de 65 kDa et de pH isoélectrique de 6,8
Dendritic cells (DC) are central cells in immune System. DCs lead to lymphocyte activation and control adaptative immune response. To do so, DCs have to maturate and migrate toward secondary lymphoid organs where they initiate pathogen-specific lymphocyte responses. Calcium is an ubiquitous second messenger controlling a variety of cellular functions such as migration. However, the role of calcium in dendritic cells biology is poorly understood. We show that the ionic channel TRPM4 has a crucial role in calcium homeostasis in DC during stimulation. The lack of TRPM4 in DC leads to calcium overload after bacterial stimulation and dramatically decrease their migratory capacities but without affecting their maturation. We observed that a calcium overload leads to a decrease of the PLC-p2 expression which is correlated with an absence of a subsequent signalling response. Thus, this work shows the key rôle of TRPM4 in the migration but not the maturation of DC, emphasizing that these two cellular events are regulated differently

L'expression et/ou l'activation des canaux ioniques, protéines transmembranaires qui contrôlent de nombreuses fonctions cellulaires, sont altérées dans le cancer. Nous avons déjà montré l'implication des canaux TRPM7 (Transient Receptor Potential Melastatin-related 7) dans la migration et l'invasion des cellules d’ACP (Rybarczyk et al. 2012 et 2017). Par contre, son expression et son rôle dans les cellules stellaires pancréatiques sont inconnus. Au cours de ce travail nous avons démontré que le canal TRPM7 est impliqué dans la prolifération et le maintien de l'homéostasie magnésique dans les CSP. Le canal régule la prolifération cellulaire par l'activation magnésium dépendante de la voie PI3K/AKT permettant la répression de P53 et GSK3, la synthèse de CDK2 et PCNA, et la transition G1-S du cycle cellulaire. Nous avons également démontré que le canal TRPM7 régule l'expression de l'α-SMA, principal marqueur d'activation des CSP, en modulant l'activation de la voie ERK de façon dépendante du magnésium. Enfin nos résultats indiquent que l'expression du canal TRPM7 varie en fonction du niveau d'activation des CSP. L'ensemble de nos résultats suggèrent que le canal TRPM7 pourrait constituer un biomarqueur important concernant l'activation et la prolifération des CSP
Pancreatic Ductal Adenocarcinoma (PDAC) is one of the deadliest cancers with a 5 years survival rate under 5%. PDAC development and aggressivness involve pancreatic stellate cells (PSC) activation occurring during desmoplasia. PSC activation induces α-SMA expression, increases proliferative capacities of the CSP, and leads to cancer growth and metastasis. Ion channels are involved in many cellular mechanisms including cell migration, invasion and proliferation. We previously described the role of TRPM7 in PDAC cell invasion and migration (Rybarczyk et al. 2012 and 2017) but its role in PSC remains unknown. Here we show that TRPM7 is involved in magnesium homeostasis and PSC cell proliferation throughPI3K/AKT pathway activation by magnesium. TRPM7 mediated magnesium entry inducing PI3K/AKT pathway activation and leading to P53 and GSK3 inhibition, and CDK2 and PCNA upregulation inducing G1 to S transition. Our results show that TRPM7 is also involved in α-SMA expression by regulating ERK activation in a magnesium dependent manner. PSC activation also regulates TRPM7 expression. Taking together our results show TRPM7 as a potential biomarker for CSP activation and proliferation in PDAC

Mitochondria play a central role in oxidative stress-induced cell death. By increasing the production of reactive oxygen species, such as H2O2, oxidative stress causes mitochondrial fragmentation and apoptosis. It was hypothesised that Transient Receptor Potential Melastatin 2 (TRPM2) channels play a role in mitochondrial fragmentation and cell death. The rationale behind this hypothesis was the published evidence that oxidative stress stimulates TRPM2 channels, resulting in an increase in the cytosolic levels of Ca2+ and Zn2+, and that both these ions are detrimental to mitochondrial health and cell survival. To test the hypothesis, human umbilical vein endothelial cells (HUVECs) and endothelial cells isolated from wild-type and TRPM2 knock-out mice were used. TRPM2 actions were suppressed using pharmacological agents and small interfering RNA (siRNA). Fluorescent reporters were used to examine changes in intracellular ion distribution and organelle morphology. Molecular biology, biochemical and imaging techniques were used to examine the dynamics of ions and organelles. Exposure of HUVECs to H2O2 or high glucose stress led to TRPM2 activation, resulting in extracellular Ca2+ entry, lysosomal membrane permeability (LMP) and the release of lysosomal free Zn2+. Unexpectedly, this was accompanied by the accumulation of Zn2+ in the mitochondria. The rise in mitochondrial Zn2+ led to extensive mitochondrial fragmentation, mitochondrial outer membrane permeabilisation (MMP) and cell death. Silencing of TRPM2 channels with siRNA prevented intracellular Zn2+ redistribution, mitochondrial fragmentation and cell death. Endothelial cells derived from TRPM2 knock-out mice were resistant to oxidative stress-induced mitochondrial fragmentation. Biochemical and immunostaining experiments revealed an unexpected presence of TRPM2 channels in mitochondria, where they mediated mitochondrial Zn2+ uptake. Accumulation of Zn2+ in the mitochondria led to mitochondrial fragmentation by promoting the recruitment of cytoplasmic Drp1, an enzyme responsible for mitochondrial fission. Taken together, the results of this thesis revealed a novel mechanism for how oxidative stress can cause excessive mitochondrial fragmentation and cell death: the mechanism involves activation of TRPM2 channels leading to increased Ca2+ entry, LMP and release of lysosomal Zn2+; Zn2+ thus released is taken up by the mitochondria, leading to Drp1 recruitment, mitochondrial fragmentation and finally cell death. Since mitochondrial fragmentation is associated with several age-related chronic illnesses, including neuronal (Alzheimer’s, Parkinson’s), cardiovascular (atherosclerosis, myocardial infarction) and metabolic/inflammatory (diabetes) disorders, these results suggest that the TRPM2 channel is a novel target that could be explored for therapeutic intervention of age-related illnesses.

BACKGROUND: Different strategies to boost cellular NAD+ levels, such as supplementation of NAD+ precursors, or inhibition of NAD+ consumption, are currently under investigation as promising means to promote healthy aging and ameliorate dysfunctional metabolism. CD38 is a NAD+-dependent enzyme that converts NAD+ to different Ca2+-active second messengers, involved in the regulation of different signaling pathways, cell functions and metabolism. TRPM2, is an ion channel that allows Ca2+ influx from the extracellular space toward the cytosol, and is gated by ADPR, one of the molecules produced from the NAD+ degradation. In the context of systemic energy metabolism, brown adipocytes, the parenchymal cells of brown adipose tissue (BAT) as well as beige adipocytes that emerge in white adipose tissue (WAT) depots in response to catabolic conditions, are important to maintain metabolic homeostasis, together with liver. HYPOTHESIS: We aim to understand the functional relevance of CD38 and TRPM2 in the regulation of energy metabolism and NAD(P)(H) levels in BAT, WAT and liver during thermogenesis. METHODS: We used wild type, Cd38-/- and Trpm2-/- mice, exposed to cold temperatures and BAT, WAT and liver were collected. We evaluated mRNA levels by RT-PCR, proteins/enzymes levels by Western blot, FACS analysis and enzymatic activities. NAD(P)(H) levels were determined with cycling assays. Furthermore, we performed a set of in vivo experiments in which O2 consumption, CO2 production and energy expenditure were measured in mice upon thermogenic stimulation. RESULTS: We confirmed that CD38 is a major NAD+-consumer in BAT, WAT and liver: increased NAD+ levels were observed in these tissues from Cd38-/- compared with wild type mice. Interestingly, during cold exposure, a marked downregulation of CD38 expression (as detected at the mRNA, protein and enzymatic level) occurred in BAT, WAT and in liver of wild type mice. As a consequence of CD38 downregulation, an increase in NAD+ levels occurred in BAT. Instead, in WAT, CD38 downregulation was accompanied by a strong increase in NADP(H) levels, likely as a consequence of increased NADK, G6PD and malic enzyme activities. In liver, CD38 downregulation was paralleled by increased NAD(H) levels. NADK activity and NADP+ levels were not significantly modified in liver during cold-exposure. Notably, a marked decrease of NADPH level occurred in liver from both wild type and Cd38-/- mice exposed to a cold temperature, possibly as a consequence of the observed downregulation of the hepatic G6PD activity. Saving G6P from the PPP is in line with the increased activity of the enzyme glucose-6-phosphatase in liver of wild type, but not Cd38-/-, cold-exposed mice, with up-regulated gluconeogenesis. When Cd38-/- mice were kept at 6°C, higher levels of Ucp1 and Pgc-1 in BAT and WAT were revealed, compared with wild type mice. Conversely, when Trpm2-/- mice were exposed to cold temperature, lower levels of these two browning marker genes were detected, compared with wild type mice. In line with this, mice lacking Trpm2 displayed lower respiration rate and energy expenditure, when thermogenesis was induced by cold exposure and CL316,243 (a specific compound triggering adipose tissue activation). Interestingly, during cold exposure, a marked Trpm2 overexpression was observed in WAT and BAT of wild type mice. In addition, ADPR levels and mono/poly-ADPR hydrolases expression were higher in mice exposed to cold, in comparison with wild type mice. CONCLUSION: Taken together, these results demonstrate that CD38, by modulating cellular NAD(P)+ levels, is involved in the regulation of thermogenic responses in cold-activated BAT and WAT. Indeed, CD38 inhibition is being investigated as a possible strategy to ameliorate dysfunctional metabolism, by boosting NAD+ levels and sirtuins’ activity. In addition, TRPM2 plays a pivotal role in BAT and WAT activation. TRPM2 gating, is likely due to alternative pathways that do not include CD38 activity.

To identify novel analgesic strategies for chronic pain, we investigated the phenomenon of analgesia produced by cutaneous cooling. The recent identification of specific cold sensory receptors has allowed, for the first time, investigation of the molecular mechanism underlying cooling-induced analgesia. We have shown that the cold-and-menthol receptor. TRPM8, is critically involved in cooling-induced analgesia. Activation of TRPM8 in a subpopulation of sensory afferents (by either cutaneous or intrathecal application of pharmacological agents or by modest cooling) elicits analgesia in neuropathic and other chronic pain models in rats, and inhibits the characteristic sensitisation of dorsal horn neurons that occurs ipsilateral to nerve injury. This analgesia is abolished following antisense knockdown of the TRPM8 receptor. In contrast, activation of the related putative cold-receptor TRPA1 produces hyperalgesia in naïve and neuropathic rats. TRPM8 expression was observed in small diameter sensory neurons in dorsal root ganglia and on afferent terminals in the spinal cord, with increases in specific subsets of sensory neurons following nerve injury. We further found that the central mechanism of TRPM8-mediated analgesia is mediated through inhibitory Group I/III metabotropic glutamate receptors, and is opioid-independent. These results identify TRPM8 as an essential molecular mediator of cooling-induced analgesia. We propose a novel analgesic axis in which activation of TRPM8-expressing afferents by innocuous cooling or chemical ligands leads to activation of inhibitory Group II/III metabotropic glutamate receptors in the spinal cord, which then exert inhibition over nociceptive inputs. These findings suggest that both TRPM8 and the inhibitory metabotropic glutamate receptors are promising targets for the development of novel analgesics for the treatment of neuropathic pain states.

Le muscle cardiaque est un organe qui s'adapte à différents stress hémodynamiques en activant la synthèse protéique et en augmentant la taille des cardiomyocytes, résultant sur le développement d'une hypertrophie cardiaque. L'objectif de cette thèse est d'étudier le rôle potentiel du canal TRPM4 dans différents types d'hypertrophie cardiaque. Une altération du Ca2+ diastolique est à l'origine du signal initial activant les voies de signalisation d'une hypertrophie cardiaque délétère de type pathologique telle que la voie de la calcineurine-NFAT et la ré-expression de gènes fœtaux. Cette hypertrophie est alors compensatrice et vise à préserver la fonction de pompe du myocarde. Cette altération peut être conduite par divers stimulis tels qu'une augmentation de l'angiotensine II ou par des pathologies cardiovasculaires telles que l'infarctus du myocarde et l'hypertension. Cependant, une hypertrophie cardiaque bénéfique est également décrite dans la littérature, notamment lors des stades de développement du myocarde lors de l'embryogénèse ou en encore en réponse à une activité physique modérée régulière. Elle se caractérise par l'activation d'une toute autre voie de signalisation qu'est la voie de l'IGF-1-PI3K-Akt engendrée par une augmentation du taux de facteur de croissance qu'est l'insulin growth factor-1. Ces voies de signalisation ont été largement décrites dans la littérature et s'entrecroisent. Le canal TRPM4 est un canal cationique non sélectif perméable de manière égale aux ions Na+ et au K+, imperméables au Ca2+, mais activé par le Ca2+ intracellulaire. Dans le système immunitaire, il régule négativement l'entrée de Ca2+ et ce canal apparaît donc impliqué dans de nombreuses fonctions cellulaires dépendantes du Ca2+ dans différents types cellulaires. Par l'utilisation de deux modèles d'hypertrophie cardiaque, un physiologique généré par quatre semaines d'entraînement en endurance et un pathologique suite à un infarctus du myocarde induit par la ligature de l'artère coronaire gauche sur des souris wild-type et knock-out (KO) pour le canal TRPM4, nous avons mis en évidence une augmentation d'expression fonctionnelle du canal TRPM4 au sein du ventricule gauche associée à une régulation négative d'entrée de Ca2+. Le canal TRPM4 étant un régulateur de l'homéostasie calcique des cardiomyocytes, son expression fonctionelle après l'infarctus du myocarde ainsi que l'entraînement favorise l'activation de la voie de l'IGF-1-PI3K-Akt et prévient partiellement l'activation de la voie de la Calcineurine-NFAT et le développement d'une hypertrophie cardiaque pathologique, notamment dans le modèle d'infarctus du myocarde. En effet, en absence d'expression du canal, l'entrée de Ca2+ n'étant plus régulée, la voie de la Calcineurin-NFAT est favorisée. Mots clés : TRPM4, hypertrophie cardiaque, entraînement, IGF-1-PI3K-Akt, Calcineurine
Abstract: Cardiac muscle is an organ that adapts to different hemodynamic stress by activating protein synthesis and increasing cardiomyocytes size, resulting in cardiac hypertrophy. The objective of this PhD is to study the potential role of TRPM4 channel in different types of cardiac hypertrophy. Impaired diastolic Ca2+ is responsible for the initial signal activating signaling pathways in a deleterious cardiac hypertrophy pathological type such as Calcineurin-NFAT pathway and the re-expression of fetal genes. This hypertrophy is first compensatory and preserves the myocardial pump function. This alteration can be carried out by various stimuli such as increased angiotensin II or by cardiovascular diseases such as myocardial infarction and hypertension.However, a beneficial cardiac hypertrophy is also described in the literature, especially during development stages during embryogenesis or even in response to regular moderate physical activity. It is characterized by the activation one different signaling pathway, the IGF-1 - PI3K –Akt, generated by an increase in growth factor levels that is the insulin growth factor -1. These signaling pathways have been widely described in the literature and cross-talking. TRPM4 channel is a nonselective cation channel permeable equally to Na+ and K+, impermeable to Ca2+ but activated by the intracellular Ca2+. In the immune system, it downregulates Ca2+ entry and therefore appears to be involve in many Ca2+-dependent cellular functions in different cell types. By the use of two models of cardiac hypertrophy, a physiological generated by four weeks of training in endurance and pathological after myocardial infarction induced by ligation of the left coronary artery on wild-type and knockout mice -out (KO) for TRPM4 channel, we have demonstrated a functional expression increased TRPM4 channel within the left ventricle associated with down-regulation of Ca2 + entry. TRPM4 the channel being a regulator of calcium homeostasis in cardiomyocytes functional expression after myocardial infarction as well as the drive promotes the activation of the pathway of IGF-1-PI3K-Akt and partially prevents the pathway activation of the NFAT-calcineurin and the development of pathological cardiac hypertrophy, in particular myocardial infarction model. Indeed, in the absence of expression of the channel, the Ca2 + is not regulated, the path of Calcineurin-NFAT is favored. Keywords: TRPM4, cardiac hypertrophy, training, IGF-1-PI3K-Akt, calcineurin

For every 200 births in the UK, one will end in a stillbirth. Stillbirth is classified as a baby born dead after 24 weeks gestation. Mutations in genes that cause ion channelopathies are known to cause sudden cardiac death in adults and children. Prenatal diagnosis of LQT has been possible for decades, creating a disease spectrum where channelopathies may fatally influence pregnancy. We sequenced 35 candidate genes in 70 unexplained stillbirth cases. Thirty-nine cases harboured a predicted damaging protein missense variant. Two novel and two rare variants were observed in the transient receptor potential melastatin 7 (TRPM7) gene and five rare genetic variants were found in A-kinase anchor protein 9 (AKAP9). The aim of this PhD was to perform functional studies of these variants in TRPM7 and AKAP9. TRPM7 is an ion channel indispensable for mouse cardiogenesis. Two TRPM7 variants (p.G179V and p.T860M) showed significantly reduced current compared to wild-type channels. Conversely, cells expressing p.R494Q TRPM7, had a significant increase in current compared to WT channels, but only in CHO-K1 cells. Western blot analyses failed to detect full length TRPM7 in cells transfected with either p.G179V or p.T860M compared to wild-type expressing cells. Proteosomal inhibition using MG132 produced a small but visible band in p.T860M transfected cells. Expression of TRPM7 in iPSC-derived cardiomyocytes increases during cell maturation, and TRPM7-like current was measured in 20-23 day old cardiomyocytes. AKAP9 is required to couple adrenergic stimulation in the heart with faster cardiac repolarisation. Cells expressing WT AKAP9 alongside the KCNQ1/KCNE1 potassium channel responded to β-adrenergic stimulation, however those transfected with p.A3043T AKAP9 did not respond to treatment with forskolin. Our analyses supports two deleterious variants in TRPM7 and one in AKAP9 in unexplained stillbirth cases. These heterozygous variants could lead to haploinsufficiency and may be a cause of stillbirth.

La protéine TRPM4 porte un courant cationique non-sélectif activé par le calcium intracellulaire (NSCCa). Au niveau cardiaque, il est présent sur l’oreillette (humain, rat et souris) et dans le noeud sinusal (souris), mais peu exprimé au niveau ventriculaire. Toutefois son expression ventriculaire est augmentée en cas d’hypertrophie cardiaque. Malgré une cartographie bien établie l’implication de TRPM4 dans l’activité électrique cardiaque était jusqu’à aujourd’hui inconnue. Ce travail de thèse a eu pour objectif d’évaluer l’implication du canal TRPM4 dans l’activité cardiaque, en utilisant des techniques d’électrophysiologie. Nous avons combiné une approche pharmacologique, utilisant les inhibiteurs du TRPM4 et une approche de transgénèse, en utilisant des souris invalidées pour le gène TRPM4. Nous avons observé que le canal TRPM4 est impliqué dans la durée du potentiel d’action de l’oreillette de souris, puisque son inhibition diminue la durée du PA, et que les souris invalidées pour le gène TRPM4 présentent un PA plus court que les souris sauvages. Le canal participe également à certaines formes d’arythmies ventriculaires. Nous avons développé un modèle d’arythmies d’hypoxie-réoxygénation sur du ventricule de souris, où l’application des inhibiteurs du TRPM4 conduit à la disparition de ces arythmies. Enfin nous avons établi un lien entre des mutations du gène TRPM4 et le syndrome de Brugada. En particulier, une mutation K914X aboutissant à un canal non fonctionnel, a été identifiée chez un malade atteint du syndrome de Brugada. L’ensemble de nos travaux identifiant, le TRPM4 comme une nouvelle cible pharmacologique dans la prévention des troubles du rythme cardiaque
The TRPM4 protein, is a member of the larger family "Transient Receptor Potentiel" channels, and supports a nonselective cationique current activated by intracellular calcium (NSCCa). TRPM4 presents a large tissular distribution. In the heart, it is present in the atrium (human, rat and mouse) and sinus node (mouse), but it is only slightly expressed in the ventricle. However its ventricular expression is increased in case of cardiac hypertrophy (SHR rat). While TRPM4 cardiac mapping is well established, its implication in the cardiac activity was still unknown. Our work had for objective to estimate the implication of the TRPM4 channel in the cardiac activity, by using electrophysiological technics (intracellular microelectrode and patch-clamp). We combined a pharmacological approach, using two inhibitors of the TRPM4 (the acid flufénamique and 9-phénanthrol) and an approach of transgenesis, by using mice invalidated for the TRPM4 gene. We observed that TRPM is implied in the mice atrial action potential duration (APD), because its inhibition decreases the APD, and TRPM4-/-transgenics mice present a shorter AP than TRPM4+/+ mice. TRPM4 channel also participates in ventricular arrhythmias. We developed a model of hypoxia-reoxygenation to produce arrhythmias. The application of TRPM4 inhibitors eliminates these arrhythmias. Finally we established a link between TRPM4 mutations and Brugada syndrome. In particular, the mutation K914X giving an unfunctional channel, was identified in a patient affected by Brugada Syndrome. Our study identifies, TRPM4 as a new promising pharmacological target in the prevention of cardiac electrical disturbance

Even though it is confirmed that ion channels are at the centre of many diseases, approved drugs are only available for small percentage of these proteins, and yet many pathologically important ion channels like transient receptor potential (TRP) cation channels remain without approved drugs. One reason could be the time-consuming and expensive process in drug discovery. Which has high possibility of failure in any step even after approval and marketing. Therefore, repurposing approved drugs might be considered as a solution and may offer an accelerated procedure in finding new treatments for patients. For the present research we selected TRPM8 ion channel as a neglected target despite growing number of studies regarding its association with numerous diseases. In this project we have first identified potent antagonists for TRPM8 ion channel among approved drugs, by using mainly the automated patch clamp device IonFlux 16. Such device allowed us to screen blocking potency of drugs against TRPM8 ion channel in time efficient way. Our approach consisted of using ligand-based virtual screening method, to optimize our screening by identifying candidates for further screening. We also studied possible interactions of identified drugs with antagonist binding site on TRPM8 channel by molecular docking. Furthermore, we have evaluated the effects of identified antagonists against different types of pancreatic ductal adenocarcinoma (PDAC) cells. We were able to identify four drugs with IC50 lower than 50 µM including propranolol, propafenone, carvedilol and nebivolol. Among them nebivolol with IC50 = 0.97± 0.15 µM and carvedilol with IC50 = 9.1 ± 0.6 µM were the most potent blockers. Studying the interactions of identified drugs with known binding site of TRPM8 by molecular docking, revealed high possibility of direct binding of nebivolol to binding site of TRPM8. Nebivolol was the most cytotoxic drug against PDACs, but it was also toxic against non-cancerous HEK-293 cells. While carvedilol had cytotoxic against PDACs, interestingly it wasn’t cytotoxic against HEK-293 cells. Result of these study will provide promising candidates for drug repurposing and will propose promising lead compound in drug discovery for new antagonists of TRPM8 ion channel. Also, our method of approach for identifying candidate drugs as agonist or antagonist could be applied for other ion channels.

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior
A Riparina IV, uma alcamida sintetizada de Aniba riparia, foi testada em modelos animais padronizados de dor, bem como os possÃveis mecanismos de aÃÃo envolvidos. Foram utilizados camundongos Swiss (20-30g), e a Riparina IV foi administrada de forma aguda em todos os testes, nas doses de 25 e 50 mg/kg, por via oral. Foram utilizados os testes de contorÃÃes abdominais induzidas por Ãcido acÃtico; placa quente; teste da formalina; hipernocicepÃÃo mecÃnica induzida pela carragenina; teste da nocicepÃÃo induzida por capsaicina, cinamaldeÃdo, mentol; teste da nocicepÃÃo induzida por glutamato, bem como em modelos comportamentais que permitam excluir a possibilidade de uma atividade relaxante muscular ou induzir resultados falso-positivos nos modelos anteriores, tais como testes do campo aberto e rota Rod. Os resultados demonstraram que a Riparina IV possui uma atividade antinociceptiva no modelo de nocicepÃÃo visceral induzida por Ãcido acÃtico. A Riparina IV nÃo demonstrou atividade no modelo de nocicepÃÃo tÃrmica da placa quente. O prÃ-tratamento com a Riparina IV reduziu significativamente a nocicepÃÃo inflamatÃria induzida pela segunda fase da formalina, porÃm nÃo alterou a nocicepÃÃo neurogÃnica induzida pela primeira fase do teste da formalina. Os animais prÃ-tratados com a Riparina IV tambÃm exibiram uma reduÃÃo significativa na hipernocicepÃÃo mecÃnica induzida pela carragenina. Em relaÃÃo à participaÃÃo dos receptores de potencial transitÃrio (TRP), a Riparina IV demonstrou atividade nos modelos de nocicepÃÃo induzida pela administraÃÃo de capsaicina e mentol, porÃm nÃo apresentou atividade na nocicepÃÃo induzida por cinamaldeÃdo. TambÃm reduziu a nocicepÃÃo induzida pela administraÃÃo intraplantar de glutamato. Para o estudo dos mecanismos de aÃÃo da Riparina IV foi utilizada somente a dose de 50 mg/kg da substÃncia. Na avaliaÃÃo da participaÃÃo dos canais de potÃssio ATP-dependentes, o prÃ-tratamento com glibenclamida nÃo foi capaz de reverter a aÃÃo antinociceptiva da Riparina IV, descartando-se o seu envolvimento; da mesma forma, o prÃ-tratamento com ioimbina, um antagonista α2-adrenÃrgico, e pCPA, um depletor das reservas de serotonina, tambÃm nÃo foram capazes de reverter tal aÃÃo, nÃo havendo envolvimento com o mecanismo de aÃÃo da Riparina IV. O prÃ-tratamento com L-arginina, um precursor do Ãxido nÃtrico, reverteu a aÃÃo antinociceptiva da Riparina IV, sugerindo, em parte, a participaÃÃo da via do Ãxido nÃtrico no seu mecanismo de aÃÃo. Os resultados mostraram que essa substÃncia nÃo alterou a atividade locomotora no teste do campo aberto, nem diminuiu o nÃmero de quedas no teste do rota Rod, descartando a possibilidade de haver sedaÃÃo ou incoordenaÃÃo motora por parte da Riparina IV. Em sÃntese, os resultados demonstraram que a Riparina IV possui uma atividade em modelos animais de nocicepÃÃo, possivelmente envolvendo os receptores TRPV1, TRPM8, glutamatÃrgicos e a via do Ãxido nÃtrico.
Riparin IV, an alkamide synthesized from Aniba riparia, was tested in standard animal models of pain, as well as the possible mechanisms of action involved. It was used Swiss mice (20-30g), and Riparin IV was administred acutely in all tests, at the doses of 25 and 50 mg/kg, by gavage. It was used the tests of abdominal writhing induced by acetic acid, hot plate test, formalin test, mechanical hypernociception induced by carrageenan, nociception test induced by capsaicin, cinnamaldehyde and menthol, nociception test induced by glutamate, as well as models of behavior that ruled out the possibility of a muscle relaxing activity or induce false-positive results in previous models, such as the open field test and the rota Rod test. The results showed that Riparin IV has an antinociceptive activity at the model of visceral nociception induced by acetic acid. Riparin IV did not show any activity at the hot plate thermal nociception model. Pretreatment with Riparin IV reduced significantly the inflammatory nociception induced at the second phase of formalin test, but did not alter the neurogenic nociception induced at the first phase of formalin test. The animals pretreated with Riparin IV also exhibited a significant reduction at the mechanical hypernociception induced by carrageenan. Related to the participation of the Transient Potential Receptors (TRP), Riparin IV showed an activity at the models of nociception induced by capsaicin and menthol, but did not show any activity at the nociception induced by cinnamaldehyde. Also reduced the nociception induced by administration of glutamate at the rind paw. To study the mechanisms of action of Riparin IV, it was used only the dose of 50 mg/kg of the substance. At the evaluation of participation of the ATP-dependent potassium channels, pretreatment with glibenclamide was not able to reverse the antinociceptive action of Riparin IV, discharging its involvment; at the same way, pretreatment with yohimbine, an a2-adrenergic antagonist, and pCPA, a depletor of the serotonin reservations, were not able of reverse such action, not having any involvement with the mechanism of action of Riparin IV. Pretreatment with L-arginine, a precursor of Nitric Oxide, reversed the antinociceptive action of Riparin IV, suggesting, in part, the participation of nitric oxide pathway at the mechanism of action. The results showed that this substance did not alter the locomotor activity at the open field test, neither diminished the number of falls at the rota Rod test, discharging the possibility of sedation or incoordination by Riparin IV. In summary, the results showed that Riparin IV has an action in animal models of nociception, possibly involving the receptors TRPV1, TRPM8, glutamatergic receptors and the nitric oxide pathway.

Chronic neuropathic pain, resulting from dysfunction of the nervous system, is a clinical concern in both humans and animal patients. Neuropathic pain is characterised by spontaneous pain, hypersensitivity, manifested as hyperalgesia and allodynia, and refractoriness to conventional analgesics such as non-steroidal anti-inflammatory drugs, thus representing an unmet therapeutic need. Equine laminitis is a disease that involves the disruption of the dermoepidermal junction within the hoof, leading to severe pain and lameness, with poor responsiveness to anti-inflammatory therapy. We developed a Quantitative Sensory Testing method, using a novel hydraulically-powered feedbackcontrolled hoof tester, in order to provide an objective tool for the assessment of mechanical hyperalgesia in laminitic horses. Hoof Compression Thresholds of laminitic horses were significantly lower than those of normal horses and variance component analysis of the data confirmed the reliability of the method. In order to investigate mechanisms underlying laminitis pain, we performed histological studies of peripheral nerves innervating the hoof. Electron micrographic analysis of the digital nerve of laminitic horses revealed a significant reduction in the number of unmyelinated and myelinated fibres together with abnormal morphology. Additionally, cell bodies of sensory neurons innervating the hoof in cervical C8 dorsal root ganglia showed an upregulated expression of the nerve injury marker activating transcription factor-3 (ATF3), neuropeptide Y (NPY), and the TRPM8 channel; each of which has been associated with laboratory models of neuropathic pain. Previous work has shown that, in a rodent model of neuropathic pain, the TRPM8 channel is upregulated in sensory neurons and its activation by cool temperature, menthol or icilin leads to reversal of the hypersensitive pain state. Further investigation of TRPM8-channel mediated analgesia was aimed at uncovering the molecular mechanisms involved in the activation of this system in sensitised states. It was hypothesised that serotonin, released following inflammation and nerve damage, can enhance TRPM8 channel activity through peripheral 5-HT1B receptors. Calcium fluorometry carried out in HEK293 cells transfected with the TRPM8 channel and the 5-HT1B receptor revealed that coadministration of a 5-HT1B receptor agonist facilitated the activation of the TRPM8 channel by icilin. Moreover, it appears that this effect is mediated through phospholipase D1 (PLD1), possibly leading to increased production of phosphatidylinositol (4,5-) bisphosphate (PIP2), a known positive modulator of TRPM8 channel activity. In vitro co-immunoprecipitation studies suggested that the TRPM8 channel, the 5-HT1B receptor and PLD1 physically interact with each other, further providing a molecular basis for their functional co-operation. Calcium imaging carried out in cultured rat DRG cells showed that the 5-HT1B receptor-mediated enhancement of icilin responses at the TRPM8 channel also occurs in sensory cells and is reversed by inhibition of PLD1. Moreover, TRPM8 and the 5-HT1B receptor appear to be physically associated in vivo as shown by their co-immunoprecipitation from spinal cord homogenates. Assessment of nociceptive behavioural reflexes following intrathecal injection of selective pharmacological agents provided further support for the idea of 5-HT1B receptor facilitation of TRPM8 channel responses in vivo. In addition to providing novel evidence of a neuropathic component to equine laminitis and validation of a novel QST method for pain assessment in horses, this study reveals for the first time a physical and functional interaction between the 5-HT1B receptor and the TRPM8 channel.

Oral Biology
M.S.
A major physiological risk factor of temporomandibular disorders (TMD) is sensitization of peripheral and central nervous system pain processing pathways. Calcium channel, voltage-dependent, alpha-2/delta subunit-1 (CACNA2D1) has a crucial role in relaying nociceptive information in the spinal dorsal horn. Up-regulation of CACNA2D1 results in abnormal excitatory synapse formation and enhanced presynaptic excitatory neurotransmitter release. Blocking CACNA2D1 with gabapentinoid-class drugs relieves orofacial hypersensitivity. Drs. Foley, Horton, and Sciote previously reported that in a small sample group (n=12), CACNA2D1 expression was greater in males than females, but increased in women with TMD. The objectives of this study are to corroborate these data and investigate expression patterns of other ion channel and conducting system genes. Additionally, since the null polymorphism ACTN3-577XX associates with muscle fiber microdamage during eccentric contraction, we tested for possible gene associations with ACTN3-R577XX genotypes. Masseter muscle samples came from human subjects (n=23 male; 48 female) with malocclusions undergoing orthognathic surgery. This population had skeletal disharmony of the jaws and thus was prone to eccentric contraction. Three males and eighteen females were diagnosed with localized masticatory myalgia. Muscle total RNA was isolated and CACNA2D1, CACNA1S, GABARAP, and TRPM7 expression was quantified using RT-PCR. Expression of these genes were compared based on TMD status and various characteristics that may influence TMD including: sex, age, facial symmetry, sagittal dimension, vertical dimension, ACTN3-577 genotype and fiber type. CACNA2D1 expression differed significantly between sexes, overall (p<0.02), and without TMD (p=0.001). Women with (n=13) and without (n=23) TMD differed significantly (p<0.03). CACNA2D1 expression was also significantly higher (p=0.031) in subjects below age 25. Similarly, GABARAP expression was significantly higher (p=0.001) for patients younger than 25 and for patients less than or equal to age 18 (p=0.013). Otherwise, CACNA1S, TRPM7 and GABARAP differences were not significant. GABARAP expression differed, but not significantly by sex and for the ACTN3-577XX-null genotype. In a population of malocclusion patients, masseter muscle CACNA2D1 expression is significantly higher than CACNA1S, TRPM7, and GABARAP. CACNA2D1 expression is greater in males than females without TMD. However, CACNA2D1 expression increases significantly in females with TMD-associated myalgia. This may support evidence for calcium channel regulation of nociception differences seen between sexes in TMD. It was also found that expression of CACNA2D1 and GABARAP is significantly higher in younger subjects. Additionally, observations presented here suggest potential influence of ACTN3-null condition on function of GABARAP.
Temple University--Theses

Myalgic Encephalomyelitis/ Chronic Fatigue Syndrome (ME/CFS) is a disabling condition characterised by unexplained chronic fatigue that is associated with immune, neurological (including autonomic), musculoskeletal, cardiovascular and gastrointestinal symptoms [1, 2]. Currently, accurate diagnosis remains challenging in the absence of a clinical or laboratory test. Although the aetiology of ME/CFS remains undefined, a significant reduction in natural killer (NK) cell cytotoxicity is consistently reported in ME/CFS patients compared with healthy controls (HC) [3-6]. NK cells are effector lymphocytes of the innate immune system principally responsible for recognising and responding to pathogen invasion [7]. Approximately 90% of peripheral NK cells are CD56DimCD16+ which are highly cytotoxic and kill infected, tumour or ‘missing self’ cells through cytotoxic processes [8]. Conversely, the CD56BrightCD16Dim/- subset is responsible for immunosurveillance and cytokine production [9]. Importantly, NK cells require calcium (Ca2+) to regulate various cellular functions, such as cell differentiation, cell division, apoptosis, transcription, and cytotoxicity [10]. Transient Receptor Potential (TRP) channels are a group of unique ion channels whereby majority are highly selective to Ca2+ [11]. Functionally, TRP ion channels regulate “threat” stimuli, such as pain, thermosensation, mechanosensation, pathogens, and chemicals, via sensory transduction pathways. TRPM2 and TRPM3 are cation channels ubiquitously expressed throughout the human body and expressed in almost all cell types, such as NK cells. Both TRPM members are highly permeable to Ca2+, therefore are critical regulators of Ca2+-dependent pathways, such as NK cell cytotoxicity. TRPM2 is activated by adenine dinucleotides (ADPR, cADPR, NAADP, ꞵ-NAD), reactive oxygen species (hydrogen peroxide and OH-), and intracellular ([Ca2+]i). Conversely, TRPM3 is potently activated by neural steroids, such as pregnenolone sulphate (PregS) and nifedipine. Upon stimulation, TRPM2 and TRPM3 ion channels trigger a rapid influx of Ca2+ influx and rise in [Ca2+]i, which subsequently facilitates intracellular pathways for Ca2+ release from cellular organelles. Recently, TRPM2 has emerged as a key receptor in mediating Ca2+-induced anti-tumour activity in mouse NK cells via synergistic activation with CD38. CD38 generates Ca2+ mobilizing secondary messengers, such as ADPR, to activate TRPM2. Rah et al., determined inhibition of sustained tumour-induced Ca2+ signals and degranulation in mouse NK cells following 8-bromoadenosine diphosphoribose (8-Br-ADPR) treatment [12]. Moreover, N6-Benzoyladenosine-3′,5′-cyclic monophosphate (N6-Bnz-cAMP) significantly increased intracellular ADPR, however was inhibited in Ca2+-free conditions [12]. Currently, no in vitro studies have examined the phenotype and function of TRPM2 ion channels on human NK cell subsets, notably in ME/CFS research. Conversely, five single nucleotide polymorphisms in NK cells associated with TRPM3 have been previously identified in ME/CFS patients [13]. Reduced TRPM3 surface expression and impaired Ca2+ influx has furthermore been identified on NK cells in ME/CFS patients [14]. Given TRPM2 and TRPM3 are both critical regulators for Ca2+ signalling in NK cells, the overall aim of this thesis was to investigate the role of TRPM2 and TRPM3 in mediating NK cell cytotoxicity to identify a potential mechanism of reduced NK cell cytotoxic activity in ME/CFS patients. Study one aimed to develop an in vitro methodology to characterise TRPM2 and CD38 surface expression on NK cell subsets using an antibody that has not been previously used with flow cytometry. Applying this optimised methodology, study two aimed to quantify TRPM2 and CD38 surface expression on NK cell subsets at baseline and post in vitro drug treatments (N6-Bnz-cAMP and 8-Br-ADPR) in ME/CFS patients and HC. NK cell cytotoxicity was furthermore measured at baseline and post in vitro drug treatments (N6-Bnz-cAMP and 8-Br-ADPR) between groups. Lastly, study three aimed to examine the clinical presentation in a moderate-severe ME/CFS group, as well as measure NK cell cytotoxicity post in vitro drug treatment with TRPM3 agonists, PregS, nifedipine and ononetin, in ME/CFS patients and HC. Age and sex matched HC were included in study one. Age and sex-matched ME/CFS patients meeting the Canadian Consensus Criteria (CCC) and HC were included in studies two and three. All participants donated 85ml of whole blood and peripheral NK cells were isolated. TRPM2 and CD38 surface expression was measured on CD56DimCD16+ and CD56BrightCD16Dim/- subsets, as well as NK cell cytotoxicity at baseline and post in vitro drug treatments by flow cytometry. Drug treatments included: interleukin-2, N6-Bnz-cAMP, 8-Br-ADPR, PregS, nifedipine and ononetin. Study one determined 1:50 as the optimal primary TRPM2 antibody dilution following a two-hour incubation period. TRPM2 surface expression with and without CD38 co-expression significantly increased between 1:300 and 1:50 primary TRPM2 antibody dilutions following a two-hour incubation period on both CD56DimCD16+ and CD56BrightCD16Dim/- NK cell subsets. On the CD56DimCD16+ subset only, TRPM2 and CD38 surface expression also significantly increased at 1:50 compared with 1:100. Moreover, TRPM2 surface expression significantly decreased between 1:50 and 1:5 TRPM2 antibody dilution following a two-hour incubation period. This significant decrease highlights the high-dose hook effect, whereby the highly concentrated 1:5 antibody dilution saturated both capture and detection TRPM2 antibodies. Study two identified a significant overexpression of the TRPM2 ion channel on NK cell subsets in ME/CFS patients compared with HC. No significant differences in NK cell cytotoxicity were observed between or within groups post N6-Bnz-cAMP and 8-Br-ADPR drug treatments. Lastly, study three revealed no signficiant differences in NK cell cytotoxicity post PregS and nifedipine drug treatments, as well as subsequent blocking with ononentin in both groups. In both Study 2 and Study 3, viral infections and various clinical ME/CFS symptoms were significantly associated with reduced NK cell cytotoxicity in ME/CFS patients. Associations included: pain, cognitive difficulties, sleep disturbances, sensory impairments, thermostatic instability and gastrointestinal disturbances, possibly involving TRPM2 and TRPM3. In conclusion, the results of this thesis are the first to develop a novel and optimal in vitro methodology to measure TRPM2 and CD38 surface expression on human NK cell subsets using flow cytometry. This thesis is also the first to report overexpressed TRPM2 ion channels on NK cell subsets in ME/CFS patients. Oxidative stress induced by viral infections is hypothesised to cause this overexpression in TRPM2 ion channels in ME/CFS patients as previously reported. Overexpressed TRPM2 ion channels may cause mitochondrial dysfunction, cellular death, DNA damage, and disruption to MAPK pathways following uncontrolled increases in [Ca2+]i. Collectively, these processes interfere with downstream Ca2+-dependent pathways, such as NK cell cytotoxicity, which was found to be significantly reduced at baseline in ME/CFS patients compared to HC. The drug-treated NK cell cytotoxicity results may reflect the sensitivity of the cytotoxic assay to capture the TRPM2 and TRPM3 drug-modulatory effects on NK cell cytotoxicity. Given the drugs were incubated in the media and cells for more than 24 hours, the TRPM2 and TRPM3 ion channels may have undergone repetitive activation and inhibition cycles. Consequently, this may have activated and caused sustained long-term Ca2+-dependent pathways which may have potentially resulted in disrupted gene expression, irreversible cellular death and the development of NK cell hyporesponsiveness. Furthermore, a primary limitation with TRPM2 is the lack of potent and specific pharmacological tools. Consequently, the TRPM2 signals may have been reduced or lost, subsequently modulating activation of downstream NK cell cytotoxic processes, such as the extracellular signal-regulated protein kinase 1/2 and mitogen-activated protein kinase pathways. Taken together, this thesis warrants the identification of additional experiments with a more appropriate time-sensitivity to capture the pharmacological effects of specific cellular mechanisms of interest, as well as the identification and development of more potent, specific, and non-toxic pharmacological tools targeting TRPM2. Additional rationales include co-localisation of between TRPM2 and CD38 and the involvement of TRPM2 and TRPM3 spliced isoforms. A common feature shared amongst the significant associations between reduced NK cell cytotoxicity clinical ME/CFS symptoms is the high expression and functional activity of TRPM2 and TRPM3 in the CNS, which functions as the control centre for these physiological systems. However, additional quantitative tests examining these clinical functions, such as nociceptive pain and thermoregulation, are required to definitvely associate the possible roles of TRPM2 and TRPM3 activity and the unique clinical presentation of ME/CFS. Interestingly, positive correlations were determined between reduced NK cell cytotoxicity and overexpressed TRPM2 ion channels on both NK cell subsets within the ME/CFS group. ROC analyses also revealed diagnostic potential for reduced NK cell cytotoxicity and overexpressed TRPM2 ion channels in ME/CFS patients. Collectively, these results highlight a relationship between TRPM2 and reduced NK cell cytotoxicity in ME/CFS. Therefore, furthur investigations in this vital area of ME/CFS research will assist in the validation of TRPM2 and TRPM3 as potential biological markers to further understand the unique pathomechanism of ME/CFS and facilitate the development of targeted therapeutic interventions to improve the quality of life of ME/CFS patients.
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Medical Science
Griffith Health
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