Research articleInteraction between extracellular ATP5A1 and LPS alleviates LPS-induced neuroinflammation in mice
Introduction
Neuroinflammation has been implicated in neurodegenerative diseases [1], such as Alzheimer’s disease (AD), Parkinson’s disease, and amyotrophic lateral sclerosis. Intracerebroventricular injection of lipopolysaccharide (LPS), a component derived from gram-negative bacteria, evokes an immunoreaction in the brain [2]. Systemic administration of LPS also induces inflammatory processes, and detrimentally affects the brain [3]. Thus, LPS has been routinely utilised in animal models of neurodegenerative disorders to evaluate the effects of neuroinflammation on disease pathogenesis. Interestingly, LPS has been found in amyloid plaques of the AD brain and is considered as an inflammatory stimulating factor in AD pathogenesis [4].
F1F0-ATP synthase, also termed complex V, was once thought to be an enzyme located exclusively in the inner mitochondrial membrane, catalyzing the synthesis of ATP under promotion of transmembrane proton motive force [5]. However, the F1F0-ATP synthase has also been found on the surface of numerous cell types [6]. Moreover, the enzyme was found in the cytoplasm in association with the HSP90 complex [7] and centromere [8], indicating that F1F0-ATP synthase has multiple functions in cells. The F1-α subunit (ATP5A1) of the enzyme is a regulatory subunit comprised of the F1 part of F1F0-ATP synthase, possessing the chaperone protein character of HSP60 [9]. Importantly, ATP5A1 not only associates with phosphorylated tau in neurofibrillary tangles [10] but is also enriches in amyloid plaques in the brains of AD[11] and APP/PS1 transgenic mice[6].
Interestingly, a recent report suggested that ATP5A1 is a lipoteichoic acid (LTA)- and LPS-binding protein, which is abundantly stored in embryos as a maternal factor capable of protecting zebrafish embryos from bacterial infection [12]. Based on findings of previous studies, we hypothesised that ATP5A1 in amyloid plaques of the AD brain might regulate LPS-induced neuroinflammation. In this study, we examined the co-localisation of LPS and ATP5A1 in amyloid plaques of AD mice, the interaction between recombinant ATP5A1(rATP5A1) and LPS in vitro, and the effect of rATP5A1 on LPS-induced neuroinflammation, tau phosphorylation, and behavioural impairment in mice.
Section snippets
Expression and purification of recombinant human ATP5A1
E. coli Rosetta (DE3) cells bearing PET30-ATP5A1 recombinant plasmids (Real Gene Bio Co.) were cultivated in LB medium containing 100 μg/mL of kanamycin at 37 °C to an OD600 of 0.5. The expression of recombinant protein was induced by 0.5 mM IPTG at 16 °C. The recombinant ATP5A1(rATP5A1) in supernatant was purified by cOmplete His-tag purification column, and then conducted by dialysis and renaturation. The collected proteins were analyzed on SDS-PAGE (Fig. S1A).
Protein binding assay in vitro
To verify the binding of LPS to
Interaction between ATP5A1 and LPS
In the present study, AD and control mice were analysed to explore the relationship between LPS and ATP5A1. A significant increase in LPS within the supernatant fraction of the cerebral cortex of AD versus control mice was shown via Western blot (Fig. 1A). Immunofluorescence results also demonstrated that levels of LPS-positive signals were increased in AD versus control mice, and that LPS-positive clusters were obvious (Fig. 1B). To demonstrate the specificity of the LPS-antibody, we showed
Discussion
The pathogenesis of neuroinflammation and related diseases indicates that effective suppression of inflammatory processes is a vital strategy for therapy [20]. It is widely believed that substances released from damaged CNS cells trigger microglial activation [21]. On the other hand, studies have also shed light on possible microglial activating signals within CNS tissue including microbial factors, serum factors, extracellular ATP, and pathological proteins such as Aβ in AD [20]. The
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgements
We thank the members of our laboratories for their assistance and for participating in discussions. And we would like to thank Editage (www.editage.cn) for English language proofreading. This study was supported by the National Natural Science Foundation of China (Grant numbers 31671041 and 81471285), the Shanghai Municipal Science and Technology Major Project (No.2018SHZDZX01), and ZJLab.
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