Role of the succinate/Sucnr1 signaling pathway in primary sensory neurons in the pathophysiology of paclitaxel-induced neuropathic pain

Author(s)

Francisco Isaac Fernandes Gomes

• Document type: Doctoral Thesis
• Press: Ribeirão Preto.
• Institution: Universidade de São Paulo (USP). Faculdade de Medicina de Ribeirão Preto (PCARP/BC)
• Defense date: 2024-07-04

Examining board members

Thiago Mattar Cunha; Djane Braz Duarte; Felipe Villela Gomes; Vanessa Olzon Zambelli

• Advisor: Thiago Mattar Cunha

Abstract

Neuropathic pain is a disease of the somatosensory system that can be caused by mechanical, biological, metabolic, or chemical damage. Antineoplastic agents are neurotoxic to primary sensory neurons, cause axonopathy in individuals undergoing cancer treatment, and lead to the emergence of symptoms such as allodynia and hyperalgesia to prolonged mechanical and thermal stimuli. This limits the optimal use of the appropriate dose and/or duration of antineoplastic treatment and alters the morbidity and mortality of cancer patients. Scientific evidence from the last two decades indicates that antineoplastic agents cause mitochondrial damage in primary sensory neurons by altering mitochondrial structure and function, which culminates in bioenergetic and metabolite damage. Among such evidence, the inhibitory effect of the antineoplastic agent paclitaxel on the enzymatic activity of mitochondrial complex II or succinate dehydrogenase (SDH) is observed. Therefore, we hypothesized that treatment with paclitaxel would cause stress and energy imbalance, culminating in an increase in succinate levels. This phenomenon, like other pathological contexts, would cause the activation of the succinate signaling pathway and its receptor, Sucnr1, resulting in a biological role in paclitaxel-induced neuropathic pain. Therefore, we aimed to study the role of the succinate/Sucnr1 signaling pathway in primary sensory neurons in the pathophysiology of paclitaxel-induced neuropathic pain. Our findings demonstrated that treatment with paclitaxel (8 mg/kg; i.p.) significantly increased the plasma and tissue concentration, sciatic nerve, of succinic acid, with no differences being observed in the dorsal root ganglia of the spinal cord after quantification by liquid chromatography. coupled to mass spectroscopy. Furthermore, reanalysis of RNA sequencing databases revealed that paclitaxel treatment modifies the transcriptional signature of primary sensory neurons, enriching genes related to phosphorylative oxidation, the tricarboxylic acid cycle, and the electron transport chain. Additionally, an assay of SDH enzymatic activity in the dorsal root ganglion revealed a significant reduction after treatment with paclitaxel, with the same not being observed in the sciatic nerve. To investigate the impact of the total deletion of the succinate receptor, we developed a new mutant mouse line deficient for Sucnr1. In these animals, treatment with paclitaxel did not cause the development of nociceptive responses in a similar way to that observed in its wildtype counterpart, leading to attenuation of nociceptive behavioral responses in the paclitaxel-induced neuropathic pain model. Next, it was observed that the injection of succinate (0.1 - 100 nmol) into the paw of wild-type mice reduced the mechanical withdrawal threshold in a dose-dependent manner. Furthermore, the dose of 1 nmol/paw sensitized mice to innocuous mechanical and thermal stimuli, cold and hot, however, mice with total deletion of Sucnr1 showed failure in the development of these behavioral responses. Next, we demonstrated that the inflammatory response does not contribute to the pronociceptive effect of the succinate/Sucnr1 pathway and that 10mM succinate increased the frequency of spontaneous firing of dorsal root ganglion neurons in vitro. After this, the molecular identity of the primary sensory neuron expressing Sucnr1 was determined. Reanalysis of RNA sequencing databases of sensory neurons revealed that Sucnr1 is expressed mainly by Nav1.8-negative neurons, and it was later revealed that TrkB-positive neurons carry higher expression of Sucnr1 in relation to other neuronal identities. These findings were validated by RNA scope of Sucnr1, in which we observed around 55% of TrKB+Sucnr1+ neurons. Thus, to exclude a possible functional role in nociceptors, Trpv1-positive sensory fibers were depleted with resiniferatoxin and Nav1.8-positive by using Nav1.8Cre/0; iDTRflox/0 animals treated with diphtheria toxin. Maintenance of the pronociceptive effect was observed after injection of succinate 1 nmol/paw, as well as Sucnr1 gene expression in the dorsal root ganglion of the spinal cord. Additionally, conditional depletion of Sucnr1 in Nav1.8-positive fibers by using Nav1.8Cre/0; Sucnr1flox/flox mice also did not modify the behavioral responses to succinate 1 nmol/paw or Sucnr1 gene expression in the dorsal root ganglia of the spinal cord. It was also evaluated whether nociceptors would be sensitized in vitro by 10mM succinate, however, no increase in total sodium currents was observed as a parameter of neuronal hyperexcitability. Finally, conditional deletion of Sucnr1 in Nav1.8-positive neurons, Nav1.8Cre/0; Sucnr1flox/flox did not attenuate/prevent the development of neuropathic pain induced by paclitaxel (8 mg/kg; i.p.) after analyzing behavioral responses over twenty-five days. Therefore, our findings demonstrate that paclitaxel treatment increases succinate levels, which in turn may act on Nav1.8-negative, possibly TrkB-positive, neurons, sensitizing this neuronal population to the development of nociceptive responses. (AU)

• FAPESP's process: 19/14285-3 - Role of succinate/SUCNR1 signalling pathway in sensory neurons in neuropathic pain development
• Grantee: Francisco Isaac Fernandes Gomes
• Support Opportunities: Scholarships in Brazil - Doctorate