Molecules and Cells The Effect of Annexin A1 as a Potential New Therapeutic Target on Neuronal Damage by Activated Microglia Ji-Eun You, Se-Hwa Jung, and Pyung-Hwan Kim* Department of Biomedical Laboratory Science, Konyang University, Daejeon 35365, Korea *Correspondence: [email protected] https://doi.org/10.14348/molcells.2021.0020 www.molcells.org Brain disease is known to cause irrevocable and fatal loss of INTRODUCTION biological function once damaged. One of various causes of its development is damage to neuron cells caused by Recent trends in the study of neurodegenerative diseases hyperactivated microglia, which function as immune cells in such as Alzheimer’s disease (AD) and Parkinson’s disease (PD) brain. Among the genes expressed in microglia stimulated focus on the role and function of microglia and the relation by various antigens, annexin A1 (ANXA1) is expressed in with neuron (Arends et al., 2000; Perry et al., 2010). The the early phase of the inflammatory response and plays an main causes of this diseases are the high susceptibility of neu- important role in controlling the immune response. In this rons to inflammatory damage, and the main role of microglia study, we assessed whether ANXA1 can be a therapeutic as mediators of the inflammatory process (Chan et al., 2001; target gene for the initial reduction of the immune response Magnus et al., 2001). Various omics data suggest about the induced by microglia to minimize neuronal damage. To key role of microglia in the pathogenesis of brain diseases address this, mouse-origin microglial cells were stimulated such as AD (Lewcock et al., 2020). There are evidences that to mimic an immune response by lipopolysaccharide (LPS) neurodegenerative diseases, which are caused by misfolding treatment. The LPS treatment caused activation of ANXA1 and aggregation of proteins such as β-amyloid and tau tan- gene and expression of inflammatory cytokines. To assess gles, could cause dementia (Ashrafian et al., 2020; Binder the biological function in microglia by the downregulation et al., 2005). Among brain disorders, neuroinflammation is of ANXA1 gene, cells were treated with short hairpin RNA- a defense mechanism that initially protects the brain by re- ANXA1. Downregulated ANXA1 affected the function of moving or inhibiting diverse pathogens (Picca et al., 2020). mitochondria in the microglia and showed reduced neuronal This inflammatory response can have beneficial effects that damage when compared to the control group in the co- activated microglia by pathogens involve immune regula- culture system. Taken together, our results showed that tion or injury resolution by acquiring phenotypes as well as ANXA1 could be used as a potential therapeutic target for participate in cell cytotoxic. However, when inflammatory re- inflammation-related neurodegenerative diseases. sponses continue, it become chronic inflammatory which are detrimental, and they inhibit regeneration as well as involve Keywords: annexin A1, brain diseases, immune response, microglia and astrocytes and can lead to neurodegenerative microglia, neural damage diseases (Scheiblich et al., 2020). Microglia, which are immune cells in the central nervous Received 29 January, 2021; revised 25 February, 2021; accepted 15 March, 2021; published online 22 April, 2021 eISSN: 0219-1032 ©The Korean Society for Molecular and Cellular Biology. All rights reserved. cc This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-sa/3.0/. Mol. Cells 2021; 44(4): 195-206 195 Biological Function of ANXA1 in Microglia Activated by LPS Ji-Eun You et al. system (CNS) (Fan et al., 2017), responds to endogenous stimuli generated following infection or injury. Upon appro- priate stimulation, classically activated microglia trigger the LPS treatment Cytokine expression :IL-6, TNF-α first line of host defense against pathogens, leading to a neu- roprotective role (Graeber, 2010; Nimmerjahn et al., 2005). However, chronic activation of microglia leads to potential Damaged neuron cytotoxicity (Franco and Fernandez-Suarez, 2015; Pascual et al., 2012), such as pro-inflammatory cytokines and reactive Microglia ActivatedActivated microgliamicroglia oxygen intermediates, which can cause various CNS disor- Knock-down of ders such as pain, neurodegeneration, and stroke that carry ANXA1 actual or potential risks (Dai et al., 2015). Consequently, the activated microglia in the brain attack the neurons, leading Healthy neuron to dementia due to the damaged neurons (Eikelenboom and Fig. 1. Schematic representation of the proposed role in the Veerhuis, 1996; Shaji et al., 2018). control of microglial phagocytosis. Microglia is activated by LPS Currently, active research in various fields has been con- treatment, and in the CNS, the activated microglia damage the ducted to find effective treatments, including gene therapy neuron. (Nilsson et al., 2010), stem cell-based cell therapy (Duncan and Valenzuela, 2017), and drug development. Among them, risperidone is the only treatment currently approved ride (LPS)-stimulated microglia. We evaluated whether the to treat agitation and aggression in patients with AD. Risper- neuronal damage was reduced when ANXA1, expressed in idone is a clinically preferred drug, because it has fewer side the microglia activated by antigens, was substantially down- effects as compared to other drugs, and it effectively reduces regulated (Fig. 1). the symptoms and works against the negative symptoms by blocking dopamine receptors (Katz et al., 1999). Although MATERIALS AND METHODS several other drugs such as citalopram, aripiprazole, and quetiapine are used, they are not recommended because of Cell cultures the greater risk of side effects than positive effects (Dai et BV2 microglial cells and SH-SY5Y neuron cell lines were pro- al., 2015). Despite various treatments, including β-amyloid- vided by professor Seung-Ju Yang of Konyang University. Two inhibiting drugs, and studies conducted for dementia, no cell lines were incubated at 37°C and 5% CO2 in Dulbecco’s fundamental treatment or significant therapeutic effect has modified Eagle medium with high glucose (HyClone, USA) been reported. Therefore, in addition to the existing treat- containing 10% fetal bovine serum (HyClone) and 1% peni- ment methods, the development and approaches of new cillin G-streptomycin solution (Gibco, USA). treatments that can cure the fundamental causes is required. To address this issue, we focused on genetic material RNA extraction and relative quantitative real-time poly- related to immune response as a way to treat the disease merase chain reaction or conventional polymerase chain at its source in order to slow it down. Among the genes ex- reaction pressed in microglia stimulated by various antigens, annexin Total RNA was isolated from BV2 microglial cells using Trizol A1 (ANXA1) was considered as a candidate genetic material reagent (Invitrogen, USA) by following the manufacturer’s involved in inflammatory reactions that causes microglia to instructions. It was quantified using NanoDropTM One (Ther- damage nerve cells. ANXA1 is a protein that is expressed and mo Fisher Scientific, USA), and the reverse transcription-poly- activated in the early phase of the inflammatory response and merase chain reaction (RT-PCR) assay was performed with plays an important role in controlling the immune response DiaStar 2X RT Pre-Mix (Solgent, Korea) on T100 (Bio-Rad, (McArthur et al., 2010). It exists in the plasma membrane, USA). The RT-PCR experiment using primers for the detec- extracellular region, and cytosol of cells (Han et al., 2020). tion of each gene was performed as per the manufacturer’s Moreover, several studies have reported that the expression PCR conditions. The products were electrophoresed in 2% of ANXA1 is found in glial cells (specifically in microglia) of agarose gels (Agarose, Molecular Biology Grade; Vivantis the normal adult human and rodent brain (Buckingham et Technologies, Malaysia) prepared in 1X tris-acetate-ethylene- al., 2006; Dreier et al., 1998). Thus, ANXA1 is observed to diaminetetraacetic acid buffer (Biosesang, Korea). Gel images be constitutively expressed in cells related with the innate were obtained using a Fusion SL (Vilber Lourmat, France). immune stage of the normal brain (Solito et al., 2008). Al- Real-time PCR was conducted in a CFX96TM real-time system though ANXA1 is controversial because it is responsible for employing the SolgTM 2X RT-PCR Smart mix (Solgent). Table 1 the functions of pro-inflammatory and anti-inflammatory shows the primer sequences used in this study. in the CNS through various studies, enhanced ANXA1 ex- pression has been found in brain lesions of patients with PD Transfection of annexin A1 short hairpin RNA to BV2 mi- (Solito et al., 2008), and AD, which may specifically act in the croglial cells inflammatory phase before dementia occurs. ANXA1 short hairpin RNA (shRNA) was constructed using Using this characteristic of ANXA1, we hypothesized that the same sequences (NM_010730) (Supplementary Fig. S1). ANXA1 would engage in early immune responses and affect To establish the conditions of ANXA1 knockdown (Kd) and neuronal damage in the immune response by lipopolysaccha- scrambles (Scr), a total of 1 × 105 BV2 cells were transfected 196 Mol. Cells 2021; 44(4): 195-206 Biological Function of ANXA1 in Microglia Activated by