The AMPK pathway triggers autophagy during CSF1-induced microglial activation and may be implicated in inducing neuropathic pain
Graphical abstract
AMPK pathway triggers autophagy during CSF1-induced microglia activation and increased neuropathic pain sensitization. The interaction between CSF1 and CSFR1 in microglia mediates the microglial proliferation and activation via AMPK pathway. Consequently, AMPK pathway triggers autophagy during microglia activation and subsequently enhances the production of TNF-α and BDNF in microglia. Finally, TNF-α via acting on TNF-α receptors, and BDNF via acting on TrkB receptors, promotes neuronal hyperactivity in dorsal horn, which contributes to central sensitization and behavioral hypersensitivity.
Introduction
Neuropathic pain is related to disorders of the central nervous systems and causes a huge socioeconomic burden on society (Haanpaa et al., 2011). For its unclear and complex pathogenesis, it has been to be one of the most challenging clinical problems (Centre for Clinical Practice at NICE UK., 2013). Therefore, there is an urgent need to develop safe and more effective treatments for neuropathic pain.
Microglia are the resident macrophages cells in the central nervous systems that respond to adverse physiological conditions (Hunt et al., 2001). Findings have demonstrated that the activation of microglia was implicated in the development of neuropathic pain by directly enhancing the excitability of pain-transmitting neurons (Lu et al., 2009; Kawasaki et al., 2008). A recent study demonstrated that injury to the peripheral nerves released colony stimulating factor 1 (CSF1) from primary sensory neurons, which subsequently promoted spinal microglial activity and induced neuropathic pain (Brifault et al., 2019). A tracer study further confirmed the CSF1 mediated microglial activation and proliferation, which facilitates nociceptive output and contributes to chronic neuropathic pain (Tang et al., 2018). Thus, it is increasingly recognized that the regulation of neuropathic pain by microglia can be caused by CSF1. Yet, how CSF1 modulates microglial metabolism and further regulate the neuropathic pain remains unknown.
Autophagy is a cellular material degradation process that mediates and maintains cellular metabolism (Voigt and Poggeler, 2013). Although researches have revealed that autophagy exhibited a profound impact on the homeostasis of microglia (Deretic and Klionsky, 2018), it is still puzzling that whether autophagy implicated in microglia-mediated neuropathic pain. Interestingly, the role of CSF1 in modulation of autophagy has been fully demonstrated in monocyte proliferation and differentiation (Obba et al., 2015). As microglia is originated from macrophage precursors and kept highly homologous to the monocyte (Herz et al., 2017), we speculate that autophagy involves in CSF1 mediated microglial activation and subsequently induced the pain processes, however, the specific molecular mechanisms remain largely unexplored.
Adenosine 5′-monophosphate activated protein kinase (AMPK) is a heterotrimeric conventional serine/threonine kinase that activates autophagy by directly or indirectly activating unc-51 like autophagy activating kinase 1 (ULK1) (Hindupur et al., 2015; Kim et al., 2011). Studies have revealed that AMPK signaling mediated lysosomal biogenesis and remodeling, which was responsible for digesting cargo acquired during autophagy (Carroll and Dunlop, 2017). Moreover, study highlighted the essential role of AMPK-mediated autophagy during differentiation of monocytes (Obba et al., 2015). However, the relationships among CSF1 and AMPK-autophagy in regulating the activity of microglia during neuropathic pain development remain unknown. In the present study, we demonstrated that CSF1 promoted microglial activation and companied with increased microglial autophagic level. An increasing CSF1 level in the central nervous system can mimic and cause pain syndromes by up-regulation of AMPK-dependent autophagy, thus offering a new target for therapy of neuropathic pain.
Section snippets
Ethical approval for studies
The study was approved by the Committee for the Protection of Animal Care Committee at the Hunan Children's Hospital.
Primary microglial cell culture
Primary microglia was isolated from mixed glial cultures that were obtained from 9 to 10 brains from postnatal day 1–3 C57/BL6 mice (SIPPR Bk Laboratory Animal Co. Ltd., China). Briefly, the primary culture of glial cells was obtained from the cerebral cortex which was dissected in Ca2+- and Mg2+- free HBSS (Life Technologies, CN) and incubated in a 0.125% trypsin (Gibco, USA)
CSF1-induced activation of microglia is related to the induction of AMPK in vitro
It has been widely proved that cyclinD1 activates cell-cycle progression and can be a good marker to reflect cellular proliferation (Liu et al., 2019; Wei et al., 2019; Liang et al., 2019). When stimulated with CSF1, microglia was observed by microscope and exhibited an obvious increase within 24 h (Fig. 1A). To confirm the degree of microglial proliferation, we further assessed the cell proliferation marker cyclinD1 and results showed that microglia proliferation started at 1 h and peaked at
Discussion
Microglial autophagy is suggested to be involved in modulating homeostasis in the central nervous system, CSF1 is considered as a trigger for microglial activation and the induction of neuropathic pain. Nevertheless, the molecular mechanisms that link the microglial autophagy to the induction of neuropathic pain remained ill defined. Using pharmacological and siRNA approaches, we demonstrated CSF1 increased microglial proliferation and activity, which was accompanied by the induction of
Conclusion
Taken together, our findings decipher the signaling pathways involved in CSF1-mediated induction of autophagy and microglial activation, as well as provide a new research direction for future therapeutic interventions through CSF1-induced microglial activation in the context of neuropathic pain.
Funding
This study was partially supported by Natural Science Foundation of Hunan Province (No. 2020JJ5282), 2019 Annual Scientific Research Project of Health and Family Planning Commission of Hunan Province (No. B2019017), 2020 Annual Scientific Research Project of Hunan Provincial Health and Family Planning Commission (No.20200390), and Clinical Research Center for Limb Deformity of Children in Hunan Province (2019SK4006).
Declaration of Competing Interest
The authors have no conflicts of interest to declare.
Acknowledgements
The authors thank Ms. Wanli Dong and Weiran Ye for their kind help during the preparation of the manuscript.
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Ge Yang and Qian Tan contributed equally.