Autophagy plays a critical role in the maintenance of cellular homeostasis by degrading proteins, lipids and organelles. Autophagy is activated in response to stress, but its regulation in the ...context of other stress response pathways, such as those mediated by heat shock factor 1 (HSF1) and nuclear factor-erythroid 2 p45-related factor 2 (NRF2), is not well understood. We found that the Michael acceptor bis(2-hydoxybenzylidene)acetone (HBB2), a dual activator of NRF2 and HSF1, protects against the development of UV irradiation-mediated cutaneous squamous cell carcinoma in mice. We further show that HBB2 is an inducer of autophagy. In cells, HBB2 increases the levels of the autophagy-cargo protein p62/sequestosome 1, and the lipidated form of microtubule-associated protein light chain 3 isoform B. Activation of autophagy by HBB2 is impaired in NRF2-deficient cells, which have reduced autophagic flux and low basal and induced levels of p62. Conversely, HSF1-deficient cells have increased autophagic flux under both basal as well as HBB2-induced conditions, accompanied by increased p62 levels. Our findings suggest that NRF2 and HSF1 have opposing roles during autophagy, and illustrate the existence of tight mechanistic links between the cellular stress responses.
TREK2 (KCNK10, K2P10.1) is a two-pore domain potassium (K2P) channel and a potential target for the treatment of pain. Like the majority of the K2P superfamily, there is currently a lack of useful ...pharmacological tools to study TREK2. Here we present a strategy for identifying novel TREK2 activators. A cell-based thallium flux assay was developed and used to screen a library of drug-like molecules, from which we identified the CysLT1 antagonist Pranlukast as a novel activator of TREK2. This compound was selective for TREK2 versus TREK1 and showed no activity at TRAAK. Pranlukast was also screened against other members of the K2P superfamily. Several close analogues of Pranlukast and other CysLT1 antagonists were also tested for their ability to activate K2P channels. Consistent with previous work, structure activity relationships showed that subtle structural changes to these analogues completely attenuated the activation of TREK2, whereas for TREK1, analogues moved from activators to inhibitors. Pranlukast’s activity was also confirmed using whole-cell patch clamp electrophysiology. Studies using mutant forms of TREK2 suggest Pranlukast does not bind in the K2P modulator pocket or the BL-1249 binding site. Pranlukast therefore represents a novel tool by which to study the mechanism of TREK2 activation.
•Two-pore domain potassium channels (K2Ps) carry background (or leak) potassium current.•Lack of specific, pharmacological tools which target K2Ps.•TREK2 is a potential target for the treatment of pain.•Developed an assay to screen compounds and identify novel activators of TREK2.•Pranlukast is a novel activator of TREK2.
Despite the prevalence of pain originating from the teeth (dental, or odontogenic, pain) and its impact on patients' lives, the cellular and molecular mechanisms of odontogenic pain are still not ...completely understood. The teeth are unique structures, innervated by sensory neurons that originate in the trigeminal ganglia. Factors that increase the sensitivity of these neurons, such as inflammatory cytokines, may contribute to odontogenic pain associated with dental pulp inflammation. To address this, a calcium imaging-based mouse trigeminal ganglion (TG) neuron sensitisation assay was established as part of this project. Short-term exposure to tumour necrosis factor alpha (TNFα), but not to interleukin 1 beta (IL-1β), was found to sensitise the transient receptor potential (TRP) ion channels TRPA1 and TRPV1 on TG neurons. This assay provides the opportunity to test other molecules relevant to odontogenic pain for their ability to sensitise TG neurons. Previous research suggests that initiation of dentine hypersensitivity, a type of odontogenic pain evoked by thermal, osmotic, chemical, evaporative, or mechanical stimuli to the exposed dentine, might involve both neurons and odontoblasts, the specialised mineralising cells in the teeth. In this project, mouse odontoblast-like (OB) cells were used as a cellular model to study the sensory function of odontoblasts in vitro. OB cells were demonstrated to express functional TRPV4 ion channels, known to be involved in thermal, osmotic, and mechanosensation. TRPV4 activation was also found to stimulate ATP release from OB cells, highlighting a potential means for odontoblast communication with dental afferent fibres. 3 In addition, this study was the first to demonstrate the odontoblast ability to detect biologically relevant acids. Both an acid-induced increase in OB cell intracellular calcium concentrations and subsequent ATP release were detected. These processes were found to be independent of TRPV4 activity. Finally, to address the ability of odontoblasts to transmit sensory information to adjacent neurons, a co-culture approach was used to directly study intercellular communication between mouse OB cells and TG neurons. Pharmacological activation of TRPV4 was chosen as an odontoblast-selective stimulus, based on the functional expression of TRPV4 ion channels in OB cells, but not in mouse TG neurons. Blocking purinergic signalling disrupted the interaction between stimulated OB cells and TG neurons, demonstrating the importance of ATP as a primary mediator. Taken together, the findings described in this thesis support the ability of odontoblasts to detect physiologically-relevant stimuli and respond with a release of ATP to modulate the activity of adjacent sensory neurons. Thus, this interaction may play an important role in the initiation of odontogenic pain in vivo.
Activation of neuronal nitric oxide synthase, and consequent production of nitric oxide (NO), contributes to spinal hyperexcitability and enhanced pain sensation. All NOS isoforms are inhibited ...endogenously by asymmetric dimethylarginine, which itself is metabolised by dimethylarginine dimethylaminohydrolase (DDAH). Inhibition of DDAH can indirectly attenuate NO production by elevating asymmetric dimethylarginine concentrations. Here, we show that the DDAH-1 isoform is constitutively active in the nervous system, specifically in the spinal dorsal horn. DDAH-1 was found to be expressed in sensory neurons within both the dorsal root ganglia and spinal dorsal horn; L-291 (NG-2-Methoxyethyl-L-arginine methyl ester), a DDAH-1 inhibitor, reduced NO synthesis in cultured dorsal root ganglia neurons. Spinal application of L-291 decreased N-methyl-D-aspartate-dependent postdischarge and windup of dorsal horn sensory neurons--2 measures of spinal hyperexcitability. Finally, spinal application of L-291 reduced both neuronal and behavioral measures of formalin-induced central sensitization. Thus, DDAH-1 may be a potential therapeutic target in neuronal disorders, such as chronic pain, where elevated NO is a contributing factor.
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