Browsing by Author "Ramachandran, Rithwik"

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    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.
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    Limiting Autoimmune Neuroinflammation using Novel T Cell Suppressants & Investigating Overlapping Lysosomal Reductase Function in Macrophages and Osteoclasts
    (2019-07-19) Ewanchuk, Benjamin Walter; Yates, Robin M.; Ramachandran, Rithwik; Ousman, Shalina S.; Riabowol, Karl T.
    Aberrant activation of adaptive immune cells can culminate in autoimmune diseases such as multiple sclerosis (MS). Recently, potent anti-inflammatory properties for the cooling compound, icilin, and its receptor target, Transient Receptor Potential Melastatin-8 (TRPM8), were characterized in the context of inflammatory colitis—the first half of this dissertation describes an attempt to repurpose the anti-inflammatory qualities of icilin and TRPM8 for the treatment of lymphocyte-mediated neuroinflammation. We found icilin treatment strongly attenuated experimental autoimmune encephalomyelitis (EAE), a murine model of MS, via an unexpected TRPM8-independent mechanism. Icilin inhibited the proliferation, polarization and downstream effector function of CD4+ T cells, suggesting a promising drug for limiting neuroinflammation. Citing the advantageous pharmacodynamics of icilin, we additionally screened a library of icilin-related analogues for anti-proliferative character. We identified several lead compounds with improved anti-proliferative properties compared to icilin in vitro and preliminary efficacy in vivo limiting EAE severity. Collectively, this work characterizes a new class of T cell suppressants while emphasizing clear off-target effects for the cooling drug icilin beyond TRP channel activation. Antigen presenting cells such as macrophages process phagocytosed cargo within the highly degradative phagolysosome, facilitating antigenic stimulation of T cells and adaptive immunity. The redox microenvironment of the phagolysosomal lumen regulates several phagolysosomal biochemistries, including proteolysis and disulfide reduction. The lysosomal enzyme γ-interferon-inducible lysosomal thiol reductase (GILT) directly catalyzes disulfide reduction and enhances proteolysis by thiol-dependent cysteine cathepsins within the phagolysosome—while clearly implicated in antigen processing, secondary roles for GILT remain largely undefined. The second half of this dissertation establishes a role for GILT in osteoclasts, bone resorbing cells derived from macrophages. GILT expression was highly upregulated in osteoclasts in response to osteoclastogenic and inflammatory cytokines, and GILT-deficient mice were discovered to be osteopetrotic. In vitro, GILT-deficient osteoclasts demonstrated a reduced capacity to resorb bone. GILT not only directly reduced disulfides within bone matrix structural proteins, but also enhanced the activity of cathepsin K, the prototypical osteoclast collagenase. Thus, this work reveals a novel, non-immunological role for GILT in osteoclast function and bone turnover.
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    Natural-Product-Derived Transient Receptor Potential Melastatin 8 (TRPM8) Channel Modulators
    (2016-01) LeGay, Christina M; Gorobets, Evgueni; Iftinca, Mircea; Ramachandran, Rithwik; Altier, Christophe; Derksen, Darren J
    A library of novel structural hybrids of menthol and cubebol was tested for each derivative's ability to interact with the transient receptor potential subfamily melastatin member 8 (TRPM8) channel. This structure-activity relationship study revealed three potent modulators of the TRPM8 ion channel: a novel agonist (4) with an EC50 value of 11 ± 1 μM, an antagonist (15) with an IC50 value of 2 ± 1 μM, and an allosteric modulator (21) that minimized channel desensitization toward menthol. Each of these novel exocyclic olefin analogues of menthol is readily accessible by synthesis and was tested using Ca(2+) assays and electrophysiology.