PRISM :: Browsing by Author "Altier, Christophe"

Browsing by Author "Altier, Christophe"

Now showing 1 - 20 of 30

Open Access
A Non-Canonical Function for NLRP3 and AIM2 in Kidney Diseases
(2017) Chung, Hyun Jae; Muruve, Daniel; Hollenberg, Morley; Altier, Christophe
NLRP3 and AIM2 are inflammasome-forming proteins that have been mostly studied in leukocytes. Canonical NLRP3 or AIM2 inflammasomes regulate cytokine maturation and pyroptosis via ASC and caspase-1 activation. Emerging studies demonstrate that NLRP3 or AIM2 is expressed in non-haematopoietic cells such as tubular epithelial cells (TEC) in the kidney. The central hypothesis of this thesis is that NLRP3 and AIM2 regulate host response to renal injury in the kidney. Primary mouse TEC lacking Nlrp3 displayed reduced caspase-8 activation downstream of the tumor necrosis factor (TNF) receptor and CD95. TNFα/cycloheximide treatment induced NLRP3/ASC/caspase-8 speck-like complex formation at the mitochondria during apoptosis. The assembly of NLRP3/ASC/caspase-8 specks was downstream of TNFR signaling and independent of caspase-1 or -11 activation. This data shows that NLRP3 and ASC form a conserved non-canonical platform for caspase-8 activation, independent of the inflammasome that regulates apoptosis within epithelial cells. Interestingly, AIM2 was detected primarily in podocytes in the glomerulus and distal tubules at a low level. In a mouse model of nephrotoxic serum (NTS)-mediated anti-GBM, Aim2-/- mice displayed increased glomerular cellular crescent (multilayered accumulation of activated parietal cells) formation, tubular injury and inflammation, and worse renal function compared to wild-type controls. In vitro outgrowth of podocyte lacking Aim2 was greater than wild-type and Aim2-/- podocytes did not express Nhps2 (podocin) mRNA, a podocyte maturation marker. Furthermore, AIM2 was found to augment transcriptional activity of Wilm’s tumour-1 that regulates Nphs1 expression. This data suggests that non-canonical AIM2 regulates podocyte maturation, proliferation and potentially podocyte-to-parietal cell trans-differentiation during cellular crescent formation in vivo. In a mouse model of kidney ischemia/reperfusion in vivo, a significant difference in tubular injury was not detected between wild type and Aim2-/- mice. In vitro, tubular Aim2 did not regulate caspase-8 activation during apoptosis, confirming a limited role for Aim2 in tubular cell death. However, Aim2 deficiency attenuated TGFβ-mediated Smad phosphorylation and αSMA induction. Overall, these data increase the understanding of NLRP3 and AIM2 biology in the kidney diseases and highlight overarching non-canonical roles for NLRP3 and AIM2 to regulate critical biological processes in the kidney independent of inflammasome activation
Open Access
Activation of the Transient Receptor Potential Vanilloid-1 (TRPV1) channel mediates Extracellular Signal Regulated Kinase (ERK) phosphorylation via Beta-arrestin-2 signaling
(2015-03-25) Aboushousha, Reem; Altier, Christophe
The Transient Receptor Potential Vanilloid 1 (TRPV1) channel plays a pivotal role in pain sensation and transduction under physiological and pathophysiological conditions. Recent work highlighted a possible role for β-arrestin-2, a scaffolding protein that mediates G-protein coupled receptor desensitization, in channel regulation. Interestingly, β-arrestin-2 also acts as a signaling scaffold for the MAPK (ERK1/2) pathway which was described as an important nociceptive marker. In this thesis, several experimental approaches were employed to investigate TRPV1 signaling and to characterize whether β-arrestin-2 as well as ERK play a role downstream of channel activation. The work presented here describes for the first time a unique β-arrestin-2 signaling pathway following TRPV1 channel activation. In particular, we found that calcium influx through TRPV1 channels induced translocation of β-arrestin-2 from the cytosol to the nucleus. In addition, we showed that TRPV1 activation elicited ERK phosphorylation in a β-arrestin-2-dependent manner. Our data suggest that the signaling cascade starts with calcium influx through TRPV1 channels that activates protein kinase C (PKC) and induces its translocation to the plasma membrane. The activation of PKC was necessary for ERK activation as well as β-arrestin-2 nuclear translocation. While this work is the first to describe β-arrestin-2 nuclear translocation downstream of TRPV1 stimulation, the functional relevance of this translocation is yet-to-be unveiled. Given the crucial role of TRPV1 in nociception, understanding its signaling as well as the mechanisms by which the channel is modulated may pave the way to develop a novel class of analgesics.
Open Access
AKAP79 modulation of L-type channels involves disruption of intramolecular interactions in the CaV1.2 subunit
(Landes Bioscience, 2012-05) Altier, Christophe; Dubel, Stefan J.; Barrère, Christian; Jarvis, Scott E.; Stotz, Stephanie C.; Scott, John D.; Nargeot, Joël; Zamponi, Gerald W.; Bourinet, Emmanuel
L-type voltage gated calcium channels (VGCCs) interact with a variety of proteins that modulate both their function and localization. A-Kinase Anchoring Proteins (AKAPs) facilitate L-type calcium channel phosphorylation through β adrenergic stimulation. Our previous work indicated a role of neuronal AKAP79/150 in the membrane targeting of Ca(V)1.2 L-type calcium channels, which involved a proline rich domain (PRD) in the intracellular II-III loop of the channel.(1) Here, we show that mutation of proline 857 to alanine (P857A) into the PRD does not disrupt the AKAP79-induced increase in Ca(v)1.2 membrane expression. Furthermore, deletion of two other PRDs into the carboxy terminal domain of Ca(V)1.2 did not alter the targeting role of AKAP79. In contrast, the distal carboxy terminus region of the channel directly interacts with AKAP79. This protein-protein interaction competes with a direct association of the channel II-III linker on the carboxy terminal tail and modulates membrane targeting of Ca(V)1.2. Thus, our results suggest that the effects of AKAP79 occur through relief of an autoinhibitory mechanism mediated by intramolecular interactions of Ca(v)1.2 intracellular regions.
Open Access
Analysis of GPCR/ion channel interactions
(Springer, 2011-01-01) Altier, Christophe; Zamponi, Gerald W.
Voltage-gated calcium channels are key regulators of calcium homeostasis in excitable cells. A number of cellular signaling pathways serve to fine tune calcium channel activity, including the activation of G protein-coupled receptors. Besides regulating channel activity via second messengers, GPCRs can also physically associate with calcium channels to directly regulate their functions, as well as their trafficking to and from the plasma membrane. Here we provide some methods that can be used to examine channel-receptor interactions and co-trafficking. While we focus on voltage-gated calcium channels, the techniques described herein are broadly applicable to other types of channels.
Open Access
CaV3.2 Channels and BKCa-Mediated Feedback in Vascular Smooth Muscle
(2018-06-28) Hashad, Ahmed Mohamed; Welsh, Donald Gordon; Chen, Sui Rong; Altier, Christophe; von der Weid, Pierre-Yves
The vascular T-type Ca2+ channel, CaV3.2, regulates arterial tone by triggering Ca2+ sparks and activating large conductance Ca2+-activated K+ (BKCa) channels. Despite being an integral element of an arterial feedback loop, little is known of its regulation and how key receptors and signaling pathways use this channel to influence tissue perfusion. This thesis will begin to fill key knowledge gaps, undertaking experiments that progress from individual smooth muscle cells to whole arteries, and which entail the use of patch clamp electrophysiology, Ca2+ imaging, pressure myography, immunohistochemistry, quantitative polymerase chain reaction (qPCR) and computational modeling. In initial work, perforated patch electrophysiology was used in concert with Ca2+ imaging to illustrate the coordinated interplay between CaV3.2 and two other Ca2+ permeable conductances in setting voltage-dependent Ca2+ spark production and BKCa channel activation. A second layer of experiments subsequently revealed that caveolae help couple CaV3.2 to Ca2+ sparks generation by placing this T-type Ca2+ channel in close proximity to its intracellular target, ryanodine receptors (RyR). Disruption of the structural arrangement impaired the ability of CaV3.2 to mediate BKCa-mediated feedback in intact resistance arteries. Final experiments revealed that CaV3.2 channels are targeted by common vasoactive stimuli through unique signaling pathways. Of note, was the ability of Angiotensin II to suppress CaV3.2 channel activity through the generation of reactive oxygen species (ROS) by NADPH oxidase (Nox). In summary, this thesis advances our knowledge of Ca2+ handling in vascular smooth muscle by providing new regulatory insight into CaV3.2, a T-type Ca2+ channel involved in optimizing arterial tone and tissue perfusion.
Open Access
The Cavβ subunit prevents RFP2-mediated ubiquitination and proteasomal degradation of L-type channels
(Nature, 2011-02) Altier, Christophe; You, Haitao; Chen, Lina; Walcher, Jan; Hermosilla, Tamara; García-Caballero, Agustín; Simms, Brent A.; Tedford, H. William; Zamponi, Gerald W.
It is well established that the auxiliary Cavβ subunit regulates calcium channel density in the plasma membrane, but the cellular mechanism by which this occurs has remained unclear. We found that the Cavβ subunit increased membrane expression of Cav1.2 channels by preventing the entry of the channels into the endoplasmic reticulum-associated protein degradation (ERAD) complex. Without Cavβ, Cav1.2 channels underwent robust ubiquitination by the RFP2 ubiquitin ligase and interacted with the ERAD complex proteins derlin-1 and p97, culminating in targeting of the channels to the proteasome for degradation. On treatment with the proteasomal inhibitor MG132, Cavβ-free channels were rescued from degradation and trafficked to the plasma membrane. The coexpression of Cavβ interfered with ubiquitination and targeting of the channel to the ERAD complex, thereby facilitating export from the endoplasmic reticulum and promoting expression on the cell surface. Thus, Cavββ regulates the ubiquitination and stability of the calcium channel complex.
Open Access
CCR2 receptor ligands inhibit Cav3.2 T-type calcium channels
(The American Society for Pharmacology and Experimental Therapeutics, 2010-02-01) You, Haitao; Altier, Christophe; Zamponi, Gerald W.
Monocyte chemoattractant protein-1 (MCP-1) is a cytokine known to be involved in the recruitment of monocytes to sites of injury. MCP-1 activates the chemokine (C-C motif) receptor 2 (CCR2), a seven-transmembrane helix G protein-coupled receptor that has been implicated in inflammatory pain responses. Here we show that MCP-1 mediates activation of the CCR2 receptor and inhibits coexpressed N-type calcium channels in tsA-201 cells via a voltage-dependent pathway. Moreover, MCP-1 inhibits Ca(v)3.2 calcium channels, but not other members of the Cav3 calcium channel family, with nanomolar affinity. Unlike in N-type channels, this modulation does not require CCR2 receptor activation and seems to involve a direct action of the ligand on the channel. Whole-cell T-type calcium currents in acutely dissociated dorsal root ganglia neurons are effectively inhibited by MCP-1, consistent with the notion that these cells express Ca(v)3.2. The effects of MCP-1 were eliminated by heat denaturation. Furthermore, they were sensitive to the application of the divalent metal ion chelator diethylenetriaminepentaacetic acid, suggesting the possibility that metal ions may act as a cofactor. Finally, small organic CCR2 receptor antagonists inhibit Ca(v)3.2 and other members of the T-type channel family with micromolar affinity. Our findings provide novel avenues for the design of small organic inhibitors of T-type calcium channels for the treatment of pain and other T-type channel linked disorders.
Open Access
Channeling Vision: Voltage-Gated Calcium Channels of Rods and Cones
(2018-11-27) Waldner, Derek; Stell, William K.; Bech-Hansen, N. Torben; McFarlane, Sarah; Altier, Christophe
Congenital stationary night blindness (CSNB) is a set of inherited diseases characterized by defects in neurotransmission from photoreceptors to second-order neurons in the retina. CSNB2A, specifically, is caused by mutations in Cacna1f – the pore-forming sub-unit of the voltage-gated calcium channel (VGCC), CaV1.4, which is responsible for calcium-mediated glutamate release at photoreceptor ribbon synapses. In this work, we initially expanded the characterization of the Cacna1f-KO mouse retina with a particular focus on cones using our own Cacna1fG305X mouse line. We have comprehensively characterized cone morphology and viability throughout the murine lifespan, and also verified several findings reported in an alternative Cacna1f-KO model which suggest possibility of late-stage rescue. We also identified ectopic, synapse-like cone-rod bipolar cell contacts, which have been described in no other model of retinal disease. We then sought to investigate whether late-stage rescue of retinal morphology and function is feasible in the Cacna1f-KO retina. To this end, we have designed a strategy employing transgenic mice that, with appropriate gene combinations, will allow for inducible expression of Cacna1f. We have characterized several transgenic mice relevant to these experimental aims, and provided some proof-of-concept for future experiments which may provide insight into the plausibility of therapeutic interventions. We also sought to establish an alternative model of CSNB2A in the post-embryonic chick. The relative lack of tools for genetic manipulation of this model led to us developing a novel means – avian adeno-associated viral (A3V) vectors. Using A3V vectors we were able to achieve highly efficient local transduction of photoreceptors following sub-retinal injection, thus providing us with a new tool for investigation of chicken retinal circuitry. Finally, in an attempt to replicate CSNB2A in the chicken retina, we sought to characterize VGCC expression to definitively establish the target for gene knockdown. We were able to definitively prove expression of a Cacna1f orthologue in the chicken retina, and establish its sequence and mRNA expression patterns. Unfortunately, shRNA-encoding A3Vs were unable to achieve significant knockdown, but we have established a powerful framework for future investigations in this model.
Open Access
Characterization of Different Isoforms of the K+-dependent Na+- Ca2+ Exchangers: A Look into Ca2+ Coordination and Functional Consequences of Mutations Associated with Different Genetic Diseases
(2016) Jalloul, Ali; Schnetkamp, Paul; Braun, Andrew; Altier, Christophe; Trang, Tuan; Chen, S. R. Wayne
K+-dependent Na+-Ca2+ exchangers (NCKXs) belong to the Solute Carrier 24 gene family of membrane transporters. Five different exchangers have been identified in humans and contribute to many biological processes including vision, enamel maturation, Melanocortin-4-receptor-dependent satiety, olfaction and skin pigmentation. Here, I examined the differences in cation coordination of NCKX1-4 and tested the effect of single residue substitutions in the α-repeats on Ca2+ affinity in NCKX2. In addition, I investigated the functional consequences of mutations in different NCKX genes associated with genetic diseases. I measured NCKX-mediated Ca2+ transport activity of WT and mutant NCKX proteins while manipulating external or internal ion concentrations. I concluded that the differences observed among these exchangers do not explain the variation in their tissue distribution. Also, 13 single residue substitutions significantly lowered Ca2+ affinity in NCKX2. Moreover, the functional data presented about the mutations associated with genetic diseases support that the genetic analysis describing these mutations.
Open Access
Characterizing the Immunological Role of Pulmonary Stretch Receptors
(2022-06-14) Fatehi Hassanabad, Mortaza; George Yipp, Bryan; Robert Gillrie, Mark; McDonald, Braedon; Altier, Christophe
Breathing and the resulting exchange of oxygen and carbon dioxide are normally accomplished by pressure changes within the lungs. As we inhale, chest and diaphragm muscles increase the size of our chest cavity, in turn expanding our lungs. This increase in lung volume results in a pressure gradient whereby air from higher pressure areas (i.e. the atmosphere) flows into and fills our lungs, which are at a lower pressure. Exhalation follows the reverse of this process, and so, our bodies effortlessly carry out this vital function more than 20,000 times per day. Lungs contain receptors that are highly sensitive to mechanical stimuli such as pressure changes, cyclic strain, and shear flow. Such receptors are especially relevant to the lung as it is an organ that is frequently exposed to mechanical forces during breathing. This diverse group of molecules, also known as mechanoreceptors, can be found on sensory neurons, epithelium, leukocytes, and numerous other tissues; however, their functions in the lung during infections and inflammation remain obscure. One such mechanosensitive ion channel is TRPV4 which is evolutionarily conserved across all mammalian species and has become increasingly associated with immunological function in recent years. In this body of work, we investigated how mechanoreceptors (and more specifically TRPV4) modify the pulmonary immune response during host defense and inflammation. We have found that mechanical forces affect lung architecture, capillary barrier function, and bacterial dissemination in our rodent models. Moreover, inhibiting TRPV4 using commercially available agents reduces mortality and improves clinical sickness scores during Staphylococcus aureus pneumonia. We have also observed improved immune cell viability and altered neuropeptide levels using these same compounds suggesting that there may be additional neuroimmune mechanisms at play. These findings enhance our current understanding of lung mechanoreceptors and may be useful for identifying future pharmacological interventions during bacterial pneumonia.
Open Access
Chronic morphine regulates TRPM8 channels via MOR-PKCβ signaling
(2020-04-14) Iftinca, Mircea; Basso, Lilian; Flynn, Robyn; Kwok, Charlie; Roland, Corinne; Hassan, Ahmed; Defaye, Manon; Ramachandran, Rithwik; Trang, Tuan; Altier, Christophe
Abstract Postoperative shivering and cold hypersensitivity are major side effects of acute and chronic opioid treatments respectively. TRPM8 is a cold and menthol-sensitive channel found in a subset of dorsal root ganglion (DRG) nociceptors. Deletion or inhibition of the TRPM8 channel was found to prevent the cold hyperalgesia induced by chronic administration of morphine. Here, we examined the mechanisms by which morphine was able to promote cold hypersensitivity in DRG neurons and transfected HEK cells. Mice daily injected with morphine for 5 days developed cold hyperalgesia. Treatment with morphine did not alter the expressions of cold sensitive TREK-1, TRAAK and TRPM8 in DRGs. However, TRPM8-expressing DRG neurons isolated from morphine-treated mice exhibited hyperexcitability. Sustained morphine treatment in vitro sensitized TRPM8 responsiveness to cold or menthol and reduced activation-evoked desensitization of the channel. Blocking phospholipase C (PLC) as well as protein kinase C beta (PKCβ), but not protein kinase A (PKA) or Rho-associated protein kinase (ROCK), restored channel desensitization. Identification of two PKC phosphorylation consensus sites, S1040 and S1041, in the TRPM8 and their site-directed mutation were able to prevent the MOR-induced reduction in TRPM8 desensitization. Our results show that activation of MOR by morphine 1) promotes hyperexcitability of TRPM8-expressing neurons and 2) induces a PKCβ-mediated reduction of TRPM8 desensitization. This MOR-PKCβ dependent modulation of TRPM8 may underlie the onset of cold hyperalgesia caused by repeated administration of morphine. Our findings point to TRPM8 channel and PKCβ as important targets for opioid-induced cold hypersensitivity.
Open Access
COX-talk: Communication between TRPV4 channels and prostanoids in lymphatic vessel contractile response to flow.
(2023-07) Brothers, Peter; von der Weid, Pierre-Yves; Altier, Christophe; Rinker, Kristina
The proper phasic contractility of collecting lymphatic vessels (CLVs) is critical for lymph propulsion, and it requires a tight communication between the lymphatic endothelium and surrounding smooth muscle. CLVs can sense mechanical stimuli such as pressure and shear stress generated by luminal lymph flow, which modulates their contractile function through the production of vasoactive molecules, including nitric oxide and prostanoids. Although shear stress is a vital regulator of CLV contractility, the mechanisms involved in shear sensing and the role of prostanoids in lymphatic contractile motion are not well understood. In this study, we aimed to investigate the role of the mechanosensor-transient receptor potential vanilloid 4 (TRPV4) in the crosstalk between endothelium and smooth muscle prostanoids in lymphatic vessels. Using pressure myography, we assessed the ability of rat mesenteric CLVs to respond to induced unidirectional flow by increasing the pressure gradients (1, 5, and 9 cm H2O) across the vessel. We determined the involvement of TRPV4 and cyclooxygenase (COX) through selective pharmacological inhibition of TRPV4 with GSK2193874 (GSK219), COX-1 with SC560, and COX-2 with NS398. Our data indicate that high lymph flow reduces the CLV contraction frequency in a time dependent manner. Dilatory mechanisms in response to flow are endothelial, TRPV4 channel, and COX-1-dependent. Furthermore, we found that the production of thromboxane prostanoid (TP) receptor agonists are responsible for the restoration of contraction frequency over time. Selective inhibition of the TP receptor with SQ-29,458 successfully maintains a flow-dependent contractile frequency loss in CLVs. Our findings demonstrate the expression of functional TRPV4 channels in rat mesenteric CLVs. We show that the activation of TRPV4 channels in the lymphatic endothelium induces the production of both dilatory and contractile prostanoids to regulate lymphatic pumping in response to flow. These results provide new insights into the complex regulatory mechanisms involved in the shear sensing and prostanoid-mediated crosstalk between the lymphatic endothelium and smooth muscle. Understanding the regulation of lymphatic contractility is essential for the development of therapies for lymphatic-related diseases, such as lymphedema and cancer metastasis.
Open Access
D1 receptors physically interact with N-type calcium channels to regulate channel distribution and dendritic calcium entry
(Elsevier, 2008-05-22) Kisilevsky, Alexandra E.; Mulligan, Sean J.; Altier, Christophe; Iftinca, Mircea C.; Varela, Diego L.; Tai, Chao; Chen, Lina; Hameed, Shahid; Hamid, Jawed; MacVicar, Brian Archibald; Zamponi, Gerald W.
Dopamine signaling through D1 receptors in the prefrontal cortex (PFC) plays a critical role in the maintenance of higher cognitive functions, such as working memory. At the cellular level, these functions are predicated to involve alterations in neuronal calcium levels. The dendrites of PFC neurons express D1 receptors and N-type calcium channels, yet little information exists regarding their coupling. Here, we show that D1 receptors potently inhibit N-type channels in dendrites of rat PFC neurons. Using coimmunoprecipitation, we demonstrate the existence of a D1 receptor-N-type channel signaling complex in this region, and we provide evidence for a direct receptor-channel interaction. Finally, we demonstrate the importance of this complex to receptor-channel colocalization in heterologous systems and in PFC neurons. Our data indicate that the N-type calcium channel is an important physiological target of D1 receptors and reveal a mechanism for D1 receptor-mediated regulation of cognitive function in the PFC.
Open Access
Dynamic remodeling of the ionic basis of an intrinsic inhibitory response by the phospholipid PIP2
(2020-09-22) Nicholls, Shane; Turner, Ray W.; Zamponi, Gerald W.; Altier, Christophe
The excitability of an individual neuron can be directly related to the different types of potassium channels it expresses. Potassium channels that are activated by either calcium or voltage contribute to a wide range of physiological processes and many cognitive impairments if mutated or disrupted. A key mechanism to regulate neuronal activity is to generate afterhyperpolarizations (AHPs). AHPs are brief inhibitory periods that span from a fast (ms) to slow (sec) time frame. A voltage and calcium-gated potassium channel (Kv7) and an intermediate conductance calcium-gated potassium channel (IK) in CA1 hippocampal pyramidal cells hyperpolarize the cell by generating a medium and slow AHP. The availability of at least Kv7 channels can be modulated by a phosphatidylinositol molecule (PIP2). We measured the relative activity of each potassium channel using whole-cell patch recordings in rat hippocampal tissue slices maintained in vitro and in the tsA-201 heterologous expression system. Both channels prove to be activated by calcium increases derived from a combination of Cav1.3 channels and ryanodine receptor 2. However, modulating PIP2 levels produced opposite effects on coexpressed Kv7.2/3 and IK-mediated outward currents. Super resolution microscopy of immunolabeled proteins in cultured hippocampal neurons revealed a novel close association between Kv7.2 and IK potassium channels in somatic and dendritic membranes. The ability for PIP2 to reverse the roles of Kv7.2/3 and IK channels in producing a medium/slow AHP identifies a novel mechanism by which the ionic basis of inhibitory responses can be dynamically modulated to control intrinsic excitability in CA1 pyramidal cells.
Open Access
Early Life Regulation of TRPV1+ Nociceptors by the Microbiome: Implications for Pathological Pain?
(2024-04-30) Abdullah, Nasser Salem; Altier, Christophe; Trang, Tuan; Hirota, Simon
Pain is essential for the survival and wellbeing of organisms. Dysregulation in the pain pathway leads to pathological pain in part due to poor pain management stemming from a lack of understanding of the underlying mechanisms that lead to pathological pain. Pain is initiated by specialised primary afferent neurons called nociceptors. TRPV1+ nociceptors play a central role in multiple pathological pain conditions, including inflammatory pain, where their sensitization can lead to chronic pain. In this thesis, we shed light on how TRPV1+ nociceptors participate in chronic pain and the factors that contribute to their regulation. This thesis provides two studies divided into 3 chapters. The first study (Chapter 3) is focused on the role of TRPV1+ nociceptors in initiating chronic visceral pain. The second study (Chapter 4 and 5) is focused on understanding how the early life microbiome regulates the sensitivity of TRPV1+ nociceptors. In Chapter 3, we present findings indicating that in a murine model of colitis, TRPV1+ nociceptors activate spinal microglia leading to visceral hypersensitivity, demonstrating their essential role for the transition from acute to chronic pain in the context of colitis. In Chapter 4, we investigated the role of the early life microbiome on TRPV1+ nociceptor specification and pain sensitivity using germ-free mice and then germ-free mice colonized before or after weaning. We found that a lack of microbiome in early life leads to hyposensitivity to heat and capsaicin, The hyposensitive phenotype was not due to changes in nociceptor specification, innervation, or TRPV1 expression, but it correlated with a reduction in TRPV1 trafficking to the cell membrane. In Chapter 5, we investigated the underlying mechanisms for early life microbiome induced hyposensitivity and identified that the early life microbiome regulates the sensitivity of nociceptors and the trafficking of TRPV1 through regulating mast cell derived NGF. Altogether, this study demonstrates the central role of TRPV1+ nociceptors in inducing the transition to chronic pain and the important role of the early life microbiome in regulating the sensitivity of these nociceptors through mast cell derived NGF.
Open Access
Heterodimerization of ORL1 and opioid receptors and its consequences for N-type calcium channel regulation
(The American Society for Biochemistry and Molecular Biology, Inc., 2010-01-08) You, Haitao; Hameed, Shahid; Altier, Christophe; Mezghrani, Alexandre; Bourinet, Emmanuel; Evans, Rhian M.; Zamponi, Gerald W.
We have investigated the heterodimerization of ORL1 receptors and classical members of the opioid receptor family. All three classes of opioid receptors could be co-immunoprecipitated with ORL1 receptors from both transfected tsA-201 cell lysate and rat dorsal root ganglia lysate, suggesting that these receptors can form heterodimers. Consistent with this hypothesis, in cells expressing either one of the opioid receptors together with ORL1, prolonged ORL1 receptor activation via nociceptin application resulted in internalization of the opioid receptors. Conversely, mu-, delta-, and kappa-opioid receptor activation with the appropriate ligands triggered the internalization of ORL1. The mu-opioid receptor/ORL1 receptor heterodimers were shown to associate with N-type calcium channels, with activation of mu-opioid receptors triggering N-type channel internalization, but only in the presence of ORL1. Furthermore, the formation of opioid receptor/ORL1 receptor heterodimers attenuated the ORL1 receptor-mediated inhibition of N-type channels, in part because of constitutive opioid receptor activity. Collectively, our data support the existence of heterodimers between ORL1 and classical opioid receptors, with profound implications for effectors such as N-type calcium channels.
Open Access
Increased Microglial Reactivity Alters Morphine Analgesia
(2017) Leduc-Pessah, Heather; Trang, Tuan; Pittman, Quentin; Altier, Christophe; Gilch, Sabine; Gendron, Louis
Opioids are a potent class of analgesics in the management of both moderate to severe acute pain and chronic pain. Although neuronal response to opioids is well described, we are lacking a complete characterization of the diverse interplay of neurons with other cells types in response to opioids. Microglia, the immune cells of the CNS, are key targets of opioids and their response to repeated opioid exposure is implicated in the severe side effects associated with prolonged opioid use: opioid analgesic tolerance, opioid-induced hyperalgesia and opioid use disorder. In addition, increased microglial reactivity can alter analgesic response to opioids. Thus, understanding the role of microglia in response to opioids is both critical for improving the analgesic efficacy of opioids and for interfering with the negative side effects associated with prolonged opioid use. This thesis explores the contributions of the microglial P2X7 receptor and the microglial transcription factor Runx1 in morphine analgesia and in the development of adverse effects. My over-arching hypothesis is that increased microglial reactivity diminishes the analgesic potential of opioids, such as morphine. Here I show that repeated morphine causes a potentiation in microglial P2X7R function mediated by µ-receptor activation of Src kinase. Specifically, I identified tyrosine residues 382-384 on the P2X7R C-terminal domain as a critical site of phosphorylation and found that interfering with this site attenuated the development of tolerance in rats. I also show that the microglial transcription factor Runx1 regulates microglial reactivity in vitro and in vivo and that inhibition of Runx1 causes a decline in morphine analgesia. I characterized a novel strain of microglial-specific Runx1 knock-out mice and show that Runx1 deficiency causes a reduction in acute morphine analgesia and an exacerbation of opioid tolerance, hyperalgesia, and naloxone-precipitated withdrawal. Collectively, in this thesis I found that increased microglial reactivity, through variable mechanisms causes a reduction in the antinociceptive response to morphine in rodents. In conclusion, the work of this thesis has identified novel targets (P2X7R and Runx1) for interfering with the side effects associated with prolonged opioid use and in the acute analgesic response.
Open Access
Investigating the coordination of thermoregulation by thermosensory mechanisms
(2021-11) Malik, Hannan Rauf; McFarlane, Sarah; Schnetkamp, Paul; Altier, Christophe; Thompson, Roger
Thermoregulation is a critical homeostatic process through which organisms maintain cellular and physiological integrity. Depending on how thermoregulation occurs, organisms can be categorized as either endothermic, using heat from metabolic processes for thermoregulation, and ectothermic, not relying on metabolic heat for thermoregulation. Ectotherms also link their body temperature to that of the ambient environment. Both endotherms and ectotherms utilize a superfamily of polymodal proteins known as transient receptor potential (TRP) channels to initiate the thermoregulatory response. TRPM8, a member of the TRPM subfamily, has been characterized as a transducer of cold sensor in various vertebrates through functioning as a cutaneous thermoreceptor. There is still a paucity of information on this protein, whether it is expressed in the skin of the ectotherm, Xenopus laevis and its involvement in the afferent thermoregulatory response. This thesis explores the thermoregulatory response of Xenopus laevis to cooling, as well as the expression and putative function of TRPM8. First, I demonstrate that Xenopus laevis embryos exhibit a rapid, systemic melanosome aggregation upon introduction to non-noxious cool temperatures. Second, I examine the expression of the TRPM subfamily via RT-PCR in Xenopus, and then identify the expression of TRPM8 at the protein level in the skin of this ectotherm. Lastly, I demonstrate the role of TRPM8 in the thermoregulatory response, as a TRPM8 agonist phenocopies the melanosome aggregation seen as a response in cool temperatures, while an antagonist diminishes the melanosome aggregation. My work elucidates the thermoregulatory response of Xenopus laevis, as well as the role of TRPM8 in mediating the melanosome aggregation to add to the growing literature of this ion channel in thermoregulation and transducing cold.
Open Access
L-type calcium channel β subunit modulates angiotensin II responses in cardiomyocytes
(Taylor and Francis, 2011-05) Hermosilla, Tamara; Moreno, Cristian; Itfinca, Mircea; Altier, Christophe; Armisén, Ricardo; Stutzin, Andrés; Zamponi, Gerald W.; Varela, Diego L.
Angiotensin II regulation of L-type calcium currents in cardiac muscle is controversial and the underlying signaling events are not completely understood. Moreover, the possible role of auxiliary subunit composition of the channels in Angiotensin II modulation of L-type calcium channels has not yet been explored. In this work we study the role of Ca(v)β subunits and the intracellular signaling responsible for L-type calcium current modulation by Angiotensin II. In cardiomyocytes, Angiotensin II exposure induces rapid inhibition of L-type current with a magnitude that is correlated with the rate of current inactivation. Semi-quantitative PCR of cardiomyocytes at different days of culture reveals changes in the Ca(v)β subunits expression pattern that are correlated with the rate of current inactivation and with Angiotensin II effect. Over-expression of individual b subunits in heterologous systems reveals that the magnitude of Angiotensin II inhibition is dependent on the Ca(v)β subunit isoform, with Ca(v)β(1b) containing channels being more strongly regulated. Ca(v)β(2a) containing channels were insensitive to modulation and this effect was partially due to the N-terminal palmitoylation sites of this subunit. Moreover, PLC or diacylglycerol lipase inhibition prevents the Angiotensin II effect on L-type calcium channels, while PKC inhibition with chelerythrine does not, suggesting a role of arachidonic acid in this process. Finally, we show that in intact cardiomyocytes the magnitude of calcium transients on spontaneous beating cells is modulated by Angiotensin II in a Ca(v)β subunit-dependent manner. These data demonstrate that Ca(v)β subunits alter the magnitude of inhibition of L-type current by Angiotensin II.
Open Access
Microbial dysbiosis alters serotonin signalling in a post-inflammatory murine model of visceral pain
(2024-07-10) Roth, Timothy Douglas; Sharkey, Keith; Nasser, Yasmin; Altier, Christophe
Inflammatory bowel disease (IBD) is a chronic disorder characterized by inflammation of the gastrointestinal tract, affecting a growing number of individuals worldwide. Despite achieving endoscopic remission, many IBD patients continue to experience visceral pain, suggesting underlying mechanisms beyond inflammation. One hypothesis implicates alterations in gut microbiota post-inflammation, leading to dysregulated serotonin (5-HT) signalling within the gut and heightened pain sensitivity. This thesis investigated this hypothesis using a mouse model of IBD in remission associated with visceral pain to explore changes in enterochromaffin cell populations, gene expression related to 5-HT synthesis, transport, and degradation, as well as 5-HT concentration and its metabolites. Additionally, fecal microbiota transplant (FMT) experiments were performed using stool from DSS-treated mice, alongside comparative analyses with germ-free (GF) mice, to delineate the impact of the microbiota on post-inflammatory pain in IBD and establish a baseline for gut microbiota effects on 5-HT signalling. High-performance liquid chromatography (HPLC) was utilized to assess 5-HT and 5-hydroxyindoleacetic acid (5-HIAA) tissue concentrations, and enzyme-linked immunosorbent assay (ELISA) was employed to determine 5-HT release dynamics in the gut. Our findings revealed region-specific differences in 5-HT release in the terminal ileum, proximal colon, and distal colon, suggesting localized alterations in 5-HT signalling post-inflammation. Additionally, GF mice displayed distinct patterns of altered gene expression and 5-HT/5-HIAA concentration compared to conventionally colonized counterparts, underscoring the pivotal role of gut microbiota in modulating 5-HT metabolism and signalling. FMT experiments allowed us to assess the impact of dysbiotic microbiota on post-inflammatory pain. Surprisingly, we found no significant differences in gene expression between control and DSS-treated FMT groups, suggesting resilience of the host to changes in microbiota composition. However, we observed differences in 5-HT release dynamics between FMT groups, indicating potential microbiota-driven alterations in neuronal signalling pathways. Overall, we found alterations in 5-HT signalling in the recovery model of DSS-induced colitis. These findings enhance our understanding of the pathophysiology of IBD-related pain, highlighting the complex interplay between gut microbiota and 5-HT signalling after a period of intestinal inflammation.