Friess et al. Mol Brain (2021) 14:87 https://doi.org/10.1186/s13041-021-00794-7 RESEARCH Open Access Atg7 defciency in microglia drives an altered transcriptomic profle associated with an impaired neuroinfammatory response Lara Friess, Mathilde Cheray, Lily Keane, Kathleen Grabert and Bertrand Joseph* Abstract Microglia, resident immunocompetent cells of the central nervous system, can display a range of reaction states and thereby exhibit distinct biological functions across development, adulthood and under disease conditions. Distinct gene expression profles are reported to defne each of these microglial reaction states. Hence, the identifcation of modulators of selective microglial transcriptomic signature, which have the potential to regulate unique microglial function has gained interest. Here, we report the identifcation of ATG7 (Autophagy-related 7) as a selective modula- tor of an NF-κB-dependent transcriptional program controlling the pro-infammatory response of microglia. We also uncover that microglial Atg7-defciency was associated with reduced microglia-mediated neurotoxicity, and thus a loss of biological function associated with the pro-infammatory microglial reactive state. Further, we show that Atg7- defciency in microglia did not impact on their ability to respond to alternative stimulus, such as one driving them towards an anti-infammatory/tumor supportive phenotype. The identifcation of distinct regulators, such as Atg7, controlling specifc microglial transcriptional programs could lead to developing novel therapeutic strategies aim- ing to manipulate selected microglial phenotypes, instead of the whole microglial population with is associated with several pitfalls. Keywords: Atg7, Infammatory response, Microglia, Neurotoxicity, NF-κB, Transcriptome Introduction in [4, 5]). However, microglia can also become over- Microglia are the resident immunocompetent cells activated or deregulated in the context of brain injury, of the central nervous system, reported to populate neurodegenerative and proliferative disorders, and the brain during early embryonic development and to thereby could contribute to disease severity. Undeni- derive from myeloid precursors originating from the ably, activation of microglia and infammation-medi- yolk sac in mouse [1]. Accumulating data demonstrates ated neurotoxicity are suggested to play a decisive role a vast array of functions for these myeloid cells across in the pathogenesis of several neurodegenerative dis- development, adulthood and under disease conditions orders like Alzheimer’s disease, Parkinson’s disease, (reviewed in [2, 3]). Adequate microglial functions are Huntington’s disease, as well as amyotrophic lateral crucial throughout life for neurogenesis, neuronal cir- sclerosis [6, 7]. Uncontrolled microglia will also lead to cuit shaping, vascular formation and remodeling, and increased tumor cell migration and invasion in the case overall maintenance of brain homeostasis (reviewed of primary and metastatic brain tumors [8–10]. Tus, it is now recognized that microglia can display a wide range of reaction states based on stimuli they perceive *Correspondence: [email protected] and thereby exert distinct, sometime contrasting, func- Toxicology Unit, Institute of Environmental Medicine, Karolinska Institutet, Stockholm, Sweden tions. Another consolidating view in the feld is that © The Author(s) 2021. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. 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Mol Brain (2021) 14:87 Page 2 of 21 the numerous functions of microglia would be fulflled Materials and methods through their reaction toward multiple phenotypes, Reagents and antibodies each associated with a distinct transcriptomic signature Reagents used in this study include baflomycin A1 (reviewed in [11, 12]). In fact, the number of distinct (BafA1, Santa Cruz Biotechnology, sc-201550), DAPI gene expression profles used to defne either various (Invitrogen, P36931), Hoechst 33342 (Invitrogen, H3570), microglial reaction states, or transcriptomes associ- IL-4 (Peprotech, 214-14), LPS (Sigma, L2654), Phalloi- ated to diseases are accumulating [13–23]. As a result, din Alexa Fluor™ 594 (Invitrogen, A12381), ProLong identifying potential modulators of these distinct Gold antifade mountant with DAPI (Life technologies, microglial transcriptomic profles, which are both asso- P36935) and Torin1 (Tocris, 4247). Primary antibod- ciated with unique functions and linked to diseases, ies were used according to manufacturer’s instructions. has gained increased interest in the microglia feld. We A list of primary and secondary antibodies used in this brought our attention to ATG7 (Autophagy related 7) study is shown in Table 1. encoded by the Atg7 gene, considering reports indi- cating involvement of this protein in the regulation of Generation of Atg7 knockdown BV2 cells transcription as well as cell phenotypes. Of interest, Te shAtg7 BV2 and shCtrl BV2 cell lines were a gen- microglial Atg7 defciency has been reported to impact erous gift from Dr. Seong-Woon Yu (Daegu Gyeo- on several animal models of diseases, including autism ngbuk Institute of Science and Technology, Republic spectrum disorders [24], multiple sclerosis [25], Alzhei- of Korea) and their generation has been previously mer’s disease [26] and seizures [27]. Te encoded pro- described [36]. Briefy, shCtrl and shAtg7 lentiviruses tein is an E1-like activating enzyme, which is known for (TRCN0000092164, Sigma-Aldrich) were produced fol- its essential role in macroautophagy (hereafter referred lowing published protocol [37]. BV2 cells were infected to as autophagy), where it activates ATG12 for its con- with the lentiviruses in the presence of hexadimethrine jugation with ATG5 as well as the MAP1LC3/ATG8 bromide (8 µg/ml) for 24 h and then the medium was family proteins for their conjugation with phosphati- replaced with fresh culture medium. After 72 h, cells dylethanolamine (reviewed in [28]). Worth a note, the were selected using puromycin (8 µg/ml) for 2–3 days, expression of ATG5 in microglia is reported to not be and puromycin-resistant cells were re-seeded and grown essential to autoimmune neuroinfammation response in fresh culture medium. in mice [29]. However, independently of its E1-like enzymatic activ- Cell culture and treatment ity, ATG7 is reported to bind to the transcription factor BV2 shCtrl and shAtg7 cells, glioma C6 (ATCC® CCL- p53 and thereby regulates the expression of target genes 107™) and neuronal MN9D (gift from Dr. Alfred Heller, such as CDKN1A, which encodes for the cyclin-depend- ent kinase (CDK) inhibitor p21. Hence, ATG7 is thought to modulate p53-dependent cell cycle control [30]. ATG7 is also suggested to bind to another transcription factor, Table 1 List of antibodies FOXO1 (Forkhead box protein O1), when present in the Antibodies Companies cytoplasm and is acetylated [31]. In bladder cancer cells Primary antibodies ATG7 is reported to indirectly repress the expression ACTB, mouse monoclonal antibody Sigma-Aldrich (A-3853) of FOXO1, which controls the expression of CDKN1B APOE, rabbit monoclonal antibody Abcam (ab183597) gene encoding for a second CDK inhibitor p27 [32]. One ARG1, rabbit polyclonal antibody Abcam (ab91279) additional example of a transcription factor proposed ATG7, rabbit polyclonal antibody Abcam (ab133528) to be regulated by ATG7 and of particular interest for GAPDH, mouse monoclonal antibody Millipore (CB1001) the present study, is nuclear factor of kappa B (NF-κB). LAMIN A/C, mouse monoclonal antibody Sigma-Aldrich (SAB4200236) In fact, tumor necrosis factor α-induced NF-κB nuclear LC3B, rabbit polyclonal antibody Sigma-Aldrich (L7543) translocation was found to be abolished in Atg7-def- NOS2, rabbit polyclonal antibody Cell signaling (13120) cient [33]. Worth a note, the non-autophagy functions P65, NF-κB subunit, rabbit polyclonal Santa Cruz (sc-372) of the molecular players in the autophagy machinery has antibody attracted increased attention in recent years (reviewed in Secondary antibodies [34, 35]). In the present study, we investigated whether ­IRDye® 800CW Goat anti-Mouse IgG Li-COR (926-32210) Atg7 defciency could impact the ability of microglial cells ­IRDye® 680RD Goat anti-Rabbit IgG Li-COR (926-68071) to respond to stimuli in their microenvironment and to Donkey anti-Rabbit IgG, Alexa Fluor 488 Invitrogen (A21206) acquire distinct activation states associated with unique List of primary and secondary antibodies used in this study, the respective biological functions.