AMPKα1 knockout enhances nociceptive behaviors and spinal glutamatergic synaptic activities via production of reactive oxygen species in the spinal dorsal horn
Introduction
Adenosine monophosphate-activated protein kinase (AMPK) is a key serine/threonine kinase that controls cellular energy homeostasis (Steinberg and Kemp, 2009). AMPK is activated when the AMP/ATP ratio is high leading to a reduction in ATP consumption and the increase in ATP production (Steinberg and Kemp, 2009). Accumulating data have demonstrated that AMPK also regulates many physiological and pathological processes in the nervous system, including long-term potentiation in the hippocampus (Potter et al., 2010), aging (Douglas and Dillin, 2010), Alzheimer’s disease (Salminen et al., 2011), and stroke (Li and McCullough, 2010). Multiple studies have shown that pharmacological activation of AMPK produces potent analgesic effects in different animal pain models (Tillu et al., 2012, Price and Dussor, 2013, Taylor et al., 2013, Maixner et al., 2015). Currently, whether and how endogenous AMPK activity regulates the pain signaling system remains unclear.
Spinal central sensitization, characterized by enhanced neuronal activation in the spinal dorsal horn, is a key process underlying the genesis of pathological pain (Woolf, 2011). Enhanced glutamatergic synaptic activation in the spinal dorsal horn is a critical mechanism leading to enhanced neuronal activities in the spinal dorsal horn under pathological pain conditions (Zeng et al., 2006, Zhao et al., 2012, Yan et al., 2013). Ample evidence supports the findings that neuronal activities in the spinal dorsal horn are regulated by non-excitable glial cells (i.e., microglia and astrocytes) (Watkins et al., 2001, Watkins and Maier, 2002, Kawasaki et al., 2008). Activation of microglia and astrocytes and the subsequent increased production of inflammatory molecules, a cardinal feature of neuroinflammation, are commonly observed in animal models with inflammatory pain induced by subcutaneous injection of complete Freund’s adjuvant (Raghavendra et al., 2004) or formalin (Sweitzer et al., 1999), or animal models with neuropathic pain (Watkins et al., 2001, Watkins and Maier, 2002, Tanga et al., 2004). Pro-inflammatory cytokines like TNF-α (Kawasaki et al., 2008), interleukin-1β (IL-1β) (Kawasaki et al., 2008, Yan et al., 2013, Yan and Weng, 2013), or chemokines like CX3CL1 (Gao and Ji, 2010) can directly enhance excitatory glutamatergic synaptic activities in spinal dorsal horn sensory neurons. Activation of MAP kinases (like p-38, extracellular signal-regulated kinases (ERK)) in the spinal dorsal horn is associated with the genesis of persistent inflammatory (Ji et al., 2002) and neuropathic pain (Ji and Suter, 2007). Suppression of p-38 and ERK attenuates inflammatory pain and neuropathic pain via controlling glial activation and production of pro-inflammatory cytokines (like TNF-α, IL-1β, and IL-6), and chemokines in the spinal dorsal horn (Zhuang et al., 2005, Zhuang et al., 2007, Ji and Suter, 2007, White et al., 2007, Scholz and Woolf, 2007, Ji et al., 2009). Recent reports have demonstrated that activation of AMPK reduces the production of pro-inflammatory molecules in multiple cell types (including neutrophils (Zhao et al., 2008), macrophages (Bae et al., 2011), and astrocytes and microglia (Giri et al., 2004, Meares et al., 2013)). Our current understanding of the role of AMPK in the pain signaling system is mainly derived from studies using pharmacological activation of AMPK via different activators such as 5-Aminoimidazole-4-carboxamide ribonucleotide (AICAR), metformin, A769662, and resveratrol. Systemic or topical administration of one or more of these AMPK activators has shown to attenuate hypersensitive nociceptive behaviors in animals induced by surgical incision (Tillu et al., 2012), nerve injury (Melemedjian et al., 2011, Maixner et al., 2015), cancer chemotherapy drugs (cisplatin and paclitaxel) (Mao-Ying et al., 2014), and diabetic neuropathy (Ma et al., 2015). The role of endogenous AMPK in nociceptive processing was recently revealed by us. AMPK activities in the spinal dorsal horn were reduced in animals with partial sciatic nerve ligation. Behavioral hypersensitivity was induced when spinal AMPK is knocked down by siRNA (Maixner et al., 2015). The spinal molecular and synaptic mechanisms regulated by endogenous AMPK which alter spinal nociceptive processing remain to be determined.
Reactive oxygen species (ROS) are free radicals that are mainly generated as byproducts of cellular metabolism. ROS have functions in cellular signaling and regulation (Finkel, 1998, Rhee, 1999, Thannickal and Fanburg, 2000) and have shown to be master signaling molecules controlling inflammatory signaling cascades. ROS plays an important role in the genesis of many pathological pain conditions (Janes et al., 1822, Salvemini et al., 2011), including neuropathic pain induced by nerve injury (Park et al., 2006, Siniscalco et al., 2007) or chemotherapy (Kim et al., 2010), and inflammatory pain induced by carrageenan (Khattab, 2006), and morphine-tolerance (Muscoli et al., 2007, Doyle et al., 2010, Doyle et al., 2013). Currently, it is not fully understood about endogenous molecular signaling pathways regulating the production of ROS and the impacts of ROS on synaptic activities at the spinal dorsal horn.
AMPK has two isoforms, AMPKα1 and AMPKα2 (Hardie, 2011). Studies of different cell types have shown that AMPKα1 and AMPKα2 play different roles in physiological and pathological processes (Qin and Rodrigues, 2010, Fu et al., 2013, Ost et al., 2014). In this study, to better define the molecular signaling pathways and synaptic mechanisms by which AMPKα1 controls the pain signaling system, we utilized the Cre-LoxP system to conditionally knockout the AMPKα1 gene in the nervous system of mice. We demonstrated that AMPKα1 is imperative for maintaining normal nociception, and mice deficient for AMPKα1 exhibit mechanical allodynia and enhanced glutamatergic synaptic activities at the spinal dorsal horn. We also provide compelling evidence that the behavioral hypersensitivity and enhanced glutamatergic synaptic activities in AMPKα1 knockout mice is mediated by elevated spinal ROS.
Section snippets
Animals
Male and female mice (weight range 25–40 g) were used. FVB-Tg(GFAP-cre)25Mes/J (Zhuo et al., 2001) and Prkaa1tm1.1Sjm/J (Nakada et al., 2010) mice were purchased from Jackson Laboratories. All experiments were approved by the Institutional Animal Care and Use Committee at the University of Georgia and were fully compliant with the National Institutes of Health Guidelines for the Use and Care of Laboratory Animals.
Behavior tests
To measure mechanical sensitivity in the animals, mice were placed on a wire mesh,
Selective AMPKα1 deletion induced mechanical allodynia via enhancing spinal glutamatergic synaptic activities in the spinal dorsal horn
To determine the role of the AMPKα1 subtype in the pain signaling system, the AMPKα1 isoform in the CNS was conditionally deleted in mice through Cre-LoxP recombination (Zhuo et al., 2001). We compared mechanical thresholds of the hind paw withdrawal response between AMPKα1 knockout mice and their AMPKα1 littermate controls. We found that mechanical thresholds of the hind paw withdrawal response in AMPKα1 knockout mice were significantly (p < 0.001) lower (0.72 ± 0.09 g, n = 10) in comparison with
Discussion
Given that multiple AMPK activators like metformin and resveratrol are FDA approved or currently in clinical trials for a range of conditions such as diabetes, investigating the role of AMPK in the pain signaling system may bring a novel clinical application for this class of drugs. In this study, we for the first time revealed the spinal molecular and synaptic mechanisms by which AMPKα1 regulates nociceptive behaviors. Our study suggests that AMPKα1 may be a new target for the development of
Conclusions
We identified the spinal molecular signaling pathways and synaptic mechanisms controlled by AMPKα1. We found that increased spinal ROS levels and glutamatergic synaptic activities mediate the enhanced nociceptive behaviors in mice deficient for AMPKα1. The knockout of AMPKα1 increased ERK and p-38 activities, and the production of IL-1β, ROS, and HO-1 in the spinal dorsal horn. An intraperitoneal injection of a ROS scavenger or inducer of HO-1 attenuated mechanical allodynia in AMPKα1 knockout
Conflict of interest
The authors declare no conflict of interest.
Acknowledgments
This project was supported by the NIH RO1 grant (NS064289) to H.R.W. and in part by the National Natural Science Foundation of China (number 81300662) to X.Y.
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These authors contributed equally.