In the present study, we identified a previously unreported mechanism by which mTOR activation is involved in CCI-triggered NP. Specifically, we show that mTOR activation inhibits the ITCH-driven ubiquitinated degradation of RIP3, induces reactive activation and polarization of spinal astrocytes toward the A1 subtype, triggers neuroinflammation and generates central sensitization (summarized in Fig. 8). These findings provide robust evidence that mTOR-driven pain may involve an astrocytic-neuronal communication pathway, thus revealing the potential for the development of novel therapeutic targets.
The underlying mechanisms of NP are quite complex and involve both peripheral and central sensitization, making it refractory and unmanageable with current treatments[1]. In addition to enhancing central sensitization[11], activation of astrocytes also contributes to central nervous system (CNS) neuroinflammation[10], which in turn leads to the development and maintenance of NP[32]. It is well known that the mTOR signaling pathway is responsible for regulating transcription, translation and ribosomal biosynthesis[12], however, the mechanisms associated with its involvement in pain remain incompletely understood. Results from recent studies have shown that astrocyte activation and proliferation are mediated by mTOR[33]. In the present study, we observed that during CCI-mediated NP development, mTOR activation was mainly observed in spinal astrocytes. Moreover, a pharmacological inhibition of spinal mTOR or a specific knockdown of mTOR in astrocytes were both found to be effective in significantly alleviating nociceptive hypersensitivity. Such findings imply that astrocytic mTOR substantially contributes to CCI-induced NP.
Reactive astrocytes have been reported to be categorized into two phenotypes, C3d A1 and S100A10 A2, which exert neurotoxic and neuroprotective effects, respectively[8, 9]. Numerous signaling pathways are involved in the transformation of astrocytes from their normal state to the A1 phenotype. According to Li et al., microglial cells can produce a transformation of astrocytes to the A1 phenotype in chronic postoperative pain via controlling CXCR7/PI3K/Akt[34]. Autocrine effects may also be important in astrocyte activation, as evidenced by the fact that astrocyte activation persists significantly longer than the peak of microglial activation and, in fact, can still occur in the absence of microglia [5, 35]. In our current study we found that the proportion of C3d-positive astrocytes was elevated in the spinal cord of rats after CCI surgery and that an overexpression of mTOR, using TSC2-shRNA, further promoted the activation of type A1 astrocytes. These findings provide compelling evidence indicating a critical role for mTOR in the activation of type A1 astrocytes.
The neurotoxic capacity of A1 astrocytes has been widely discussed, but little is known about their potential function in NP[10]. It has been reported that activation of the NF-κB signaling pathway in astrocytes during CNS inflammation generates NO which, when accumulated in excess, can exert a negative impact on neurons[36]. Moreover, under conditions of chronic pain, activated astrocytes become less capable of absorbing the excessive amount of glutamate released from neurons and other astrocytes. Under normal conditions, the glutamate transporter proteins, GLT-1 and GS, are primarily responsible for mediating this uptake[11]. However, the excitotoxicity of neurons, resulting from a prevention of glutamate uptake, can induce NP[37]. In a mouse model of bone cancer pain the expression level of spinal GLT-1 steadily declined as the disease progressed[38]. Our experimental results are consistent with this finding as we observed that the expression of GS proteins in the spinal cord was significantly reduced after CCI. A reduction in these proteins may be directly responsible for the reduced pain threshold and central sensitization observed in these rats. Interestingly, a decrease of mTOR in astrocytes inhibited glutamate release within the spinal cord, which is consistent with the previously reported role of mTOR in regulating glutamate metabolism after the onset of status epilepticus[39, 40]. In addition, pain research frequently uses c-fos, a marker of neuronal activity after injurious stimuli that is mostly expressed in the nucleus of injurious sensory neurons[41]. Our results strongly suggest that knockdown of astrocytic mTOR downregulates c-fos-positive neurons.
An additional novel and significant finding resulting from this study is the revelation that the astrocytic involvement of mTOR in NP occurs via an induction of RIP3. As demonstrated in a number of studies, RIP3 is implicated in the production and maintenance of NP and inflammation and thus may represent a viable target for pain management[17, 42]. Additional evidence indicating a relationship between RIP3 and astrocytes has been provided by Fan H et al. who reported that RIP3 accumulated and persisted in reactive astrocytes for up to 2 weeks after spinal cord injury[18]. Therefore, a clear link appears to exist among RIP3, reactive astrocytes and NP when collating these findings, but few studies have been directed toward investigating this relationship. Here, we show that RIP3 was upregulated in rats subjected to CCI surgery, while an inhibition of mTOR, as achieved with RAPA, decreased RIP3 expression. Our findings that GSK872 mitigated mechanical and thermal nociceptive hypersensitivity after CCI and that neuroinflammatory responses were also greatly enhanced are supported by the study by Liang YX et al.[43]. We also found that GSK872 functioned without affecting p-p70S6K expression, implying that mTOR is an upstream regulator of RIP3. Overall, it seems reasonable to conclude that mTOR can induce astrocyte activation and produce inflammatory factors by increasing RIP3 expression.
More specifically, our findings imply that mTOR regulates RIP3 mainly at the post-transcriptional level, despite the fact that both transcriptional and post-transcriptional pathways can control RIP3 expression. In this study, with use of the UbiBrowser database[29], ITCH was identified as a candidate E3 ubiquitin ligase for RIP3. Although ITCH has been reported as an E3 ubiquitin ligase that recognizes a wide range of substrates and functions in many physiological processes[44, 45], its role in NP and effects upon RIP3 have not been described. Here, we provide the first evidence for a link between ITCH and RIP3 in astrocytes. Knockdown of ITCH decreased the ubiquitination of RIP3 and furthermore promoted the upregulation of RIP3 by mTOR overexpression. These results suggest that ITCH acts as an E3 ligase involved in the regulation of RIP3 ubiquitination by mTOR, although the exact mechanisms underlying this relationship remain to be elucidated. Two major pathways that regulate protein degradation and interact with each other include the autophagy lysosomal pathway and UPS[31, 46]. RIP3, as ubiquitinated by the E3 ligase CHIP, has been reported to be degraded by lysosomes[47]. Here, our results suggest that a selective autophagy may represent a novel mechanism involved with mediating RIP3 degradation. Amino acid starvation induces autophagy to promote RIP3 degradation, and conversely, RIP3 accumulation occurs upon inhibition of autophagy by an overexpression of mTOR. ITCH-mediated RIP3 degradation can be disrupted by CQ, but is not affected by MG132. Such findings, indicate that ITCH specifically regulates RIP3 degradation through autophagy. Thus, the results of our study suggest that the mTOR/ITCH axis regulates ubiquitination degradation of RIP3 through the autophagy pathway.