Inhibition of AGEs/RAGE/Rho/ROCK pathway suppresses non-specific neuroinflammation by regulating BV2 microglial M1/M2 polarization through the NF-κB pathway
Graphical abstract
Introduction
Alzheimer's disease (AD), a chronic neurodegenerative disease that is the most common cause of dementia in the elderly (Goedert, 2015). It is characterized by the amyloid-β (Aβ) peptides deposition and the formation of tangles of the microtubule associated protein tau (Jr et al., 2013). Besides these hallmarks, growing numbers of literature has linked neuroinflammation with AD, which is characterized by hyperactive microglia and reactive oxygen species (ROS) (Heppner et al., 2015). Neuroinflammation in AD brain is not a specific inflammation caused by particular pathogen. It is a chronic non-specific inflammatory response induces by sustain activation of microglia, astrocytes and other immune cells. Such over-activation of glial cells and chronic inflammation has been classified as a detrimental process due to the non-specific nature of the innate immune system, without causing symptoms of redness, swelling, heat, and pain (Lull & Block, 2010).
Microglia, the resident macrophages of brain (Perry & Teeling, 2013), are considered to be pivotal players in inflammatory responses in neurodegenerative diseases, including AD (Mandrekar-Colucci & Landreth, 2010). Microglia can be polarized by specific cytokines and chemical compounds into a cytotoxic (pro-inflammatory) M1 or pro-repair (anti-inflammatory) M2 states in different physiological environment (Hu et al., 2014). Broad inhibition of inflammation would not be desirable because of the beneficial immune responses induced by inflammation that may limit disease (Wysscoray, 2006). With the development of AD, activated microglia will secrete tumor necrosis factor-α (TNF-α), Interferon-γ (IFN-γ) and ROS and then lead to the damage of cholinergic neurons.
NOD-like receptors family members, NLRP1, NLRP3, NLRC4, have been identified as being capable of forming inflammasomes, multiprotein complexes that activate caspase-1, which leads to the processing and secretion of pro-inflammatory cytokines interleukin-1β (IL-1β) and IL-18 (Latz et al., 2013). Among NLRs, NLRP3 is so far the best described inflammasome (Alfonso-Loeches et al., 2014). More and more evidence suggests that inflammasomes play a pivotal role in AD pathogenesis. Therefore, modulating NLRP3 inflammasome activation may be a novel therapeutic strategy for AD (Liu & Chan, 2014).
Advanced glycation end products (AGEs) are a heterogeneous group of complex compounds that are formed irreversibly in serum and tissues via a chain of non-enzymatic chemical reactions (Vlassara et al., 2008). AGEs modification and resulting cross-linking of protein deposits were observed to occur in both plaques and tangles (Younessi & Yoonessi, 2011). The interaction of AGEs with the receptor of AGEs (RAGE), resulting in the activation of nuclear factor-κB (NF-κB). NF-κB in turn upregulates RAGE expression and plays a pivotal role in non-specific inflammation, apoptosis and oxidative stress (Lu et al., 2013). FPS-ZM1 is a high-affinity RAGE-specific blocker, which targets the V-type domain of RAGE preventing Aβ binding and RAGE activation. FPS-ZM1 treatment was found to inhibit Aβ production and inflammation in AD mice (Deane et al., 2012).
Rho-kinase (ROCK) has been shown to be involved in many cellular functions such as cell contraction, adhesion, migration, and neuronal growth cone guidance (Ohsawa et al., 2016). Rho-kinase activated by the GTP-bound form of Rho not only phosphorylates myosin light chain (MLC) directly, but also inhibits the dephosphorylation of phosphorylated MLC by inactivating MLC phosphatase (MLCP) (Mali, 2011). Therefore, the phosphorylation level of MLC can serve as an index of ROCK activity. Fasudil, a selective ROCK inhibitor that targets ATP-dependent kinase domains, has been clinically applied since 1995 for the treatment of subarachnoid hemorrhage (SAH) in Japan (Chen et al., 2013). Fasudil-application rescued spatial learning and memory deficits as well as apoptosis phenotype in the AD-rat hippocampus (Hensel et al., 2015).
The activation of AGEs/RAGE in neuronal cells promotes the transportation of Aβ (Wang et al., 2013, Walker et al., 2015), and Rho/ROCK can modulate the formation of Aβ (Sonkar et al., 2014). AGEs/RAGE and Rho/ROCK are both involved in the development of non-specific inflammation. However, there are few reports about their effects on neuroinflammation. Therefore, the present study was designed to investigate mechanisms of AGEs/RAGE/Rho/ROCK pathway underlying the non-specific inflammation and microglial polarization in AD.
Section snippets
Chemicals and reagents
Dihydroethidium (DHE), dimethyl sulfoxide (DMSO), fasudil, FPS-ZM1 was purchased from Sigma-Aldrich (St. Louis, MO, USA). Fetal bovine serum (FBS) and Dulbecco's modified Eagle's medium (DMEM) were purchased from Gibco (Carlsbad, CA). AGEs stock solution (20 mg/mL) was dissolved in water and stored at − 20 °C. Primary antibodies were as follows: Rabbit polyclonal antibodies against inducible nitric oxide synthase (iNOS), Anti-Myosin Light Chain 2 (MLC), cyclooxygenase-2 (COX-2), CD206 (1:1000,
AGEs induced ROCK pathway activation via RAGE in BV2 cells
ROCK elevated MLC phosphorylation via the inhibition of myosin light chain phosphatase (MLCP), hence, phosphorylated MLC is considered to represent that of activated ROCK. To explore the dose effects of AGEs on ROCK pathway, BV2 cells were treated with 250–1000 μg/mL AGEs. As shown in Fig. 1a, AGEs could increase the level of MLC phosphorylation in a concentration dependent manner. Pretreatment with 50 μM fasudil or 0.5 μM RAGE inhibitor FPS-ZM1 inhibited AGEs-induced phosphorylation of MLC.
Inhibition of RAGE/ROCK decreased AGEs-induced activation of BV2 cells
We
Discussion
AGEs/RAGE and Rho/ROCK modification may explain many of the pathological and biochemical features of AD such as extensive protein cross-linking, oxidative stress, and inflammatory response (Zhu et al., 2015, Lv et al., 2015). However, little is known about their effects on neuroinflammation in microglia cell. In this study, we investigated the AGEs/RAGE/Rho/ROCK pathway underlying the non-specific inflammation and microglial polarization in AD. AGEs (500 μg/mL) activated BV2 cells (Fig. 2), then
Conflicts of interest
The authors have no conflicts of interest.
Acknowledgments
This study was supported by Guangdong Provincial International Cooperation Project of Science & Technology (No. 2013B051000038), National Natural Science Foundation of China (No. 31371070 and No. 81671264), Science and Technology Project of Guangdong Province (2016A070712004) and the Fundamental Research Funds for the Central Universities (No.15ykjc08b) to R. Pi.
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Contributed equally to this work.