International Journal of

Molecular Sciences Article

Early Stimulation of TREK Channel Transcription and Activity Induced by Oxaliplatin-Dependent Cytosolic Acidification Marianna Dionisi 1 , Federico Alessandro Ruffinatti 1 , Beatrice Riva 1 , Dmitry Lim 1 , Annalisa Canta 2 , Cristina Meregalli 2 , Giulia Fumagalli 2 , Laura Monza 2 , Antonio Ferrer-Montiel 3 , Asia Fernandez-Carvajal 3 , Guido Cavaletti 2 , Armando A. Genazzani 1 and Carla Distasi 1, * 1

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Department of Pharmaceutical Sciences, University of Piemonte Orientale, Via Bovio 6, 28100 Novara, Italy; [email protected] (M.D.); [email protected] (F.A.R.); [email protected] (B.R.); [email protected] (D.L.); [email protected] (A.A.G.) Experimental Neurology Unit, School of Medicine and Surgery, University of Milano-Bicocca, Via Cadore 48, 20900 Monza, Italy; [email protected] (A.C.); [email protected] (C.M.); [email protected] (G.F.); [email protected] (L.M.); [email protected] (G.C.) Biología Molecular y Celular, Universidad Miguel Hernández de Elche, 03202 Elche (Alicante), Spain; [email protected] (A.F.-M.); [email protected] (A.F.-C.) Correspondence: [email protected]

Received: 11 September 2020; Accepted: 27 September 2020; Published: 28 September 2020







Abstract: Oxaliplatin-induced peripheral neuropathy is characterized by an acute hyperexcitability syndrome triggered/exacerbated by cold. The mechanisms underlying oxaliplatin-induced peripheral neuropathy are unclear, but the alteration of ion channel expression and activity plays a well-recognized central role. Recently, we found that oxaliplatin leads to cytosolic acidification in dorsal root ganglion (DRG) neurons. Here, we investigated the early impact of oxaliplatin on the proton-sensitive TREK potassium channels. Following a 6-h oxaliplatin treatment, both channels underwent a transcription upregulation that returned to control levels after 42 h. The overexpression of TREK channels was also observed after in vivo treatment in DRG cells from mice exposed to acute treatment with oxaliplatin. Moreover, both intracellular pH and TREK channel transcription were similarly regulated after incubation with amiloride, an inhibitor of the Na+ /H+ exchanger. In addition, we studied the role of oxaliplatin-induced acidification on channel behavior, and, as expected, we observed a robust positive modulation of TREK channel activity. Finally, we focused on the impact of this complex modulation on capsaicin-evoked neuronal activity finding a transient decrease in the average firing rate following 6 h of oxaliplatin treatment. In conclusion, the early activation of TREK genes may represent a mechanism of protection against the oxaliplatin-related perturbation of neuronal excitability. Keywords: oxaliplatin; TREK channels; neuropathic pain; pH; DRG neurons; Na+ /H+ exchanger; electrophysiology; TRPV1

1. Introduction Chemotherapy-induced peripheral neurotoxicity remains a common side effect of several anticancer agents, including vinca alkaloids, taxanes, platinum derivatives, bortezomib, and thalidomide [1]. Among these agents, oxaliplatin (OHP) induces an acute sensory neuropathy characterized by alterations of sensitivity, dysesthesias, paresthesias, and cramps, which are predominantly located in the extremities and the face and, in most patients, last for hours or even days after the first OHP injection. In addition, many OHP-treated patients who experience this acute Int. J. Mol. Sci. 2020, 21, 7164; doi:10.3390/ijms21197164

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syndrome also develop chronic sensorial symptoms that are also common to other platinum-based chemotherapies [2,3]. Currently, no effective agent for the prevention or the treatment of OHP-induced peripheral neurotoxicity (OIPN) exists, thus significantly reducing the quality of life of patients [1,4]. The pathogenesis of OIPN remains poorly understood. In this sense, OHP is known to induce hyperexcitability by remodeling the expression and/or altering the function of some molecular targets that participate in the transduction of the thermal and mechanical sensation at the peripheral level [2]. Among these, transient receptor potential (TRP) channels are generally accepted as primary receptors for the detection of both physiological and noxious temperatures [5,6]. Cumulative genetic and pharmacological evidences suggest the involvement of cold-activated TRP members in the onset of OIPN [2,7]. However, other channels are also known to modulate thermosensation. For instance, hyperpolarizing K+ two-pore domain (K2P) channels work as regulators of the excitability of primary afferent fibers, and thus of pain signaling by tuning the excitation elicited by several stimuli, including temperature [8–10] and mechanical forces [11]. In accordance with this idea, it has been proposed that all TREK-family channels (TREK-1, TREK-2, and TRAAK), which are widely expressed in dorsal root and trigeminal ganglia, are implicated in the mediation of cold hyperalgesia, cool allodynia, and mechanical hypersensitivity following the treatment with OHP [2,12]. Interestingly, recent reports have shown that therapeutically-relevant OHP concentrations induce cytosolic acidification in cultured dorsal root ganglion (DRG) neurons as well as in DRG cells from treated animals [13]. In in vitro experiments, acidification occurs after 30 min from OHP application [14] and produces a reversible hypersensitization of TRPA1 channels [13]. In the present study, we have investigated the early involvement of K2P channels, TREK-1 and TREK-2, in this context since these channels, among other stimuli, also respond to intracellular pH changes (pHi ) [15–17]. We now report that therapeutically relevant OHP concentration treatment transiently increases the expression of TREK channels, both in vitro and in vivo. Interestingly, these channels undergo an upregulation following incubation with amiloride, a nonselective inhibitor of the Na+ /H+ exchanger. Last, we show that the OHP-induced cytosolic acidification positively regulates the activity of TREK-2 channels. Overall, this complex modulation transiently reduces capsaicin-induced excitability, suggesting a possible short-term protective function of K2P. 2. Results 2.1. Effects of OHP on TREK Transcription in DRG Cells Previous data [2,18] has shown a drastic decrease in the expression of TREK channels in mice four days after treatment with a single dose of OHP, just at the peak of pain hypersensitivity [2]. To investigate the early effect of the treatment with OHP on the expression of TREK channels in DRG cells, we performed real-time quantitative PCR (RT-qPCR) both in vitro and in vivo. In in vitro experiments, the mRNA levels were measured after a 6 h treatment with OHP, i.e., immediately after removing the antineoplastic drug (OHP 6 h), and 42 h later (OHP 48 h). As shown in Figure 1A, TREK channels underwent a transient upregulation following OHP treatment. Furthermore, we also studied the effect of OHP on TRPV1 expression after 6 h of treatment and did not find any significant change (log2 FC = 0.57 ± 0.23 relative to the untreated control, p-value = 0.19, Student’s t-test), as already reported for OHP-treated neurons after 48 h from plating [13]. These data indicate a relative specificity of action for OHP in gene expression modulation. In in vivo experiments, mice were treated either with OHP twice a week for four weeks (chronic condition) or only once for 24 h (acute condition). TREK mRNA levels were measured 24 h after the last treatment. We observed that the acute treatment with OHP induced a significant increase in the mRNA levels of TREK-1 and TREK-2 channels (Figure 1B).

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Figure 1. Oxaliplatin transiently affects the transcription of TREK-1, TREK-2, and TRAAK channels. Figure 1. Oxaliplatin transiently affects the transcription of TREK-1, TREK-2, and TRAAK channels. Each bar represents the mean ± SEM of treated vs. control log2 FC of gene expression, as measured Each bar represents the mean ± SEM of treated vs. control log2FC of gene expression, as measured through real-time quantitative PCR (RT-qPCR). (A) For each channel, oxaliplatin (OHP)-induced through real-time quantitative PCR (RT-qPCR). (A) For each channel, oxaliplatin (OHP)-induced changes in mRNA level were measured in cultured dorsal root ganglion (DRG) cells after 6 h of changes in mRNA level were measured in cultured dorsal root ganglion (DRG) cells after 6 h of treatment and 42 h later (n = 2 biological replicates, each in technical triplicate). (B) mRNA expression treatment and 42 h later (n = 2 biological replicates, each in technical triplicate). (B) mRNA expression from DRG cells was measured upon in vivo acute and chronic OHP treatment and compared with the from DRG cells was measured upon in vivo acute and chronic OHP treatment and compared with untreated counterpart (n = 4 biological replicates, each in technical triplicate). In both panels, controls the untreated counterpart (n = 4 biological replicates, each in technical triplicate). In both panels, are normalized to zero. ** p-value < 0.01, *** p-value < 0.001; Bonferroni-corrected multiple t-tests. controls are normalized to zero. ** p-value < 0.01, *** p-value < 0.001; Bonferroni-corrected multiple t2.2. Effects of Amiloride on Intracellular pH and TREK Channel Transcription tests.

Riva et al. (2018) reported that 6 h of treatment with OHP was sufficient to reduce cytosolic 2.2. Effects of Amiloride on Intracellular pH and TREK Channel Transcription pH levels in DRG neurons significantly. To investigate whether the changes in intracellular pH et al. treatment (2018) reported 6 h of treatment OHP wasofsufficient to reduce cytosolic inducedRiva by OHP can bethat responsible for the with upregulation TREK mRNA in DRG cells,pH levels in DRG neurons significantly. To investigate whether the changes in intracellular pH induced we performed RT-qPCR after a 6 h treatment with amiloride, a stimulus that is known to promote + /H+ exchanger by OHP acidification treatment can responsible the upregulation ofofTREK in DRG (NHE), cells, we intracellular [19].beThis is, indeed,for a nonselective inhibitor the NamRNA RT-qPCR after a 6 h treatment with amiloride, pH a stimulus that [20,21]. is known promote an performed important class of transporters involved in the intracellular homeostasis ThetomRNA +/H+ exchanger intracellular acidification [19]. This is, indeed, a nonselective inhibitor of the Na levels of TREK channels were measured on DRG cells treated for 6 h with 0.1 and 1.0 µM amiloride. an that important class of transporters involved in the intracellular pHsignificantly homeostasisdecreased [20,21]. The We(NHE), observed the treatment with both the concentrations of amiloride mRNApH levels of TREK channels were measured on DRG cells treated for 6 h with 0.1 and 1.0 µ M neuronal and increased the transcript levels of TREK channels (Figure 2). i amiloride. We observed that the treatment with both the concentrations of amiloride significantly decreased neuronal pHi and increased the transcript levels of TREK channels (Figure 2).

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Figure 2. Effects of amiloride on DRG cells. (A) Each bar represents the mean ± SEM of treated vs. control log2 FC of gene expression, as measured through RT-qPCR. Data show a significant increase + two-pore of amiloride on DRG cells. (A) Each barinrepresents the after mean6 ± of treated vs. inFigure all the2.KEffects domain (K2P) channels considered this analysis h SEM of treatment with control log2FC of gene expression, as measured through Data show aeffects significant increase two different concentrations of amiloride (0.1 and 1 µM).RT-qPCR. The transcriptional of amiloride in allassessed the K+ two-pore considered in this analysis 6 h of treatment with were by testingdomain the log2(K2P) FC of channels each experimental condition against after its reference control value two different of amiloride (0.1 and 1 µ M). The transcriptional of amiloride were normalized to concentrations zero. In each condition, experiments were performed in n =effects 4 biological replicates assessed by testing the log2FC of each