Abstract
Iba1 (ionized calcium binding adapter protein 1) is a cytoskeleton protein specific only for microglia and macrophages, where it acts as an actin-cross linking protein. Although frequently regarded as a marker of activation, its involvement in cell migration, membrane ruffling, phagocytosis or in microglia remodeling during immunological surveillance of the brain suggest that Iba1 is not a simple cytoskeleton protein, but a signaling molecule involved in specific signaling pathways. In this study we investigated if Iba1 could also represent a drug target, and tested the hypothesis that its specific silencing with customized Iba1-siRNA can modulate microglia functioning. The results showed that Iba1-silenced BV2 microglia migrate less due to reduced proliferation and cell adhesion, while their phagocytic activity and P2x7 functioning was significantly increased. Our data are the proof of concept that Iba1 protein is a new microglia target, which opens a new therapeutic avenue for modulating microglia behavior.
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References
Annovazzi L, Mellai M, Bovio E, Mazzetti S, Pollo B, Schiffer D (2018) Microglia immunophenotyping in gliomas. Oncol Lett 15(1):998–1006. https://doi.org/10.3892/ol.2017.7386
Baur A, Garber S, Peterlin BM (1994) Effects of CD45 on NF-kappa B. Implications for replication of HIV-1. J Immunol 152(3):976–983
Bianco F, Pravettoni E, Colombo A, Schenk U, Moller T, Matteoli M, Verderio C (2005) Astrocyte-derived ATP induces vesicle shedding and IL-1 beta release from microglia. J Immunol 174(11):7268–7277
Block MR, Badowski C, Millon-Fremillon A, Bouvard D, Bouin AP, Faurobert E, Gerber-Scokaert D, Planus E, Albiges-Rizo C (2008) Podosome-type adhesions and focal adhesions, so alike yet so different. Eur J Cell Biol 87(8–9):491–506. https://doi.org/10.1016/j.ejcb.2008.02.012
Bocchini V, Mazzolla R, Barluzzi R, Blasi E, Sick P, Kettenmann H (1992) An immortalized cell line expresses properties of activated microglial cells. J Neurosci Res 31(4):616–621. https://doi.org/10.1002/jnr.490310405
Borm B, Requardt RP, Herzog V, Kirfel G (2005) Membrane ruffles in cell migration: indicators of inefficient lamellipodia adhesion and compartments of actin filament reorganization. Exp Cell Res 302(1):83–95. https://doi.org/10.1016/j.yexcr.2004.08.034
Cosenza MA, Zhao ML, Si Q, Lee SC (2002) Human brain parenchymal microglia express CD14 and CD45 and are productively infected by HIV-1 in HIV-1 encephalitis. Brain Pathol 12(4):442–455
Di Virgilio F, Chiozzi P, Ferrari D, Falzoni S, Sanz JM, Morelli A, Torboli M, Bolognesi G, Baricordi OR (2001) Nucleotide receptors: an emerging family of regulatory molecules in blood cells. Blood 97(3):587–600
Eitzen UV, Egensperger R, Kosel S, Grasbon-Frodl EM, Imai Y, Bise K, Kohsaka S, Mehraein P, Graeber MB (1998) Microglia and the development of spongiform change in Creutzfeldt-Jakob disease. J Neuropathol Exp Neurol 57(3):246–256. https://doi.org/10.1097/00005072-199803000-00006
Eyo UB, Miner SA, Ahlers KE, Wu LJ, Dailey ME (2013) P2X7 receptor activation regulates microglial cell death during oxygen-glucose deprivation. Neuropharmacology 73:311–319. https://doi.org/10.1016/j.neuropharm.2013.05.032
Ford AL, Goodsall AL, Hickey WF, Sedgwick JD (1995) Normal adult ramified microglia separated from other central nervous system macrophages by flow cytometric sorting. Phenotypic differences defined and direct ex vivo antigen presentation to myelin basic protein-reactive CD4+ T cells compared. J Immunol 154(9):4309–4321
Gavegnano C, Haile WB, Hurwitz S, Tao S, Jiang Y, Schinazi RF, Tyor WR (2019) Baricitinib reverses HIV-associated neurocognitive disorders in a SCID mouse model and reservoir seeding in vitro. J Neuroinflamm 16(1):182. https://doi.org/10.1186/s12974-019-1565-6
Gehring MP, Kipper F, Nicoletti NF, Sperotto ND, Zanin R, Tamajusuku AS, Flores DG, Meurer L, Roesler R, Filho AB, Lenz G, Campos MM, Morrone FB (2015) P2X7 receptor as predictor gene for glioma radiosensitivity and median survival. Int J Biochem Cell Biol 68:92–100. https://doi.org/10.1016/j.biocel.2015.09.001
Graeber MB (2010) Changing face of microglia. Science 330(6005):783–788. https://doi.org/10.1126/science.1190929
Gu BJ, Saunders BM, Petrou S, Wiley JS (2011) P2X(7) is a scavenger receptor for apoptotic cells in the absence of its ligand, extracellular ATP. J Immunol 187(5):2365–2375. https://doi.org/10.4049/jimmunol.1101178
Hambardzumyan D, Gutmann DH, Kettenmann H (2016) The role of microglia and macrophages in glioma maintenance and progression. Nat Neurosci 19(1):20–27. https://doi.org/10.1038/nn.4185
Hanisch UK, Kettenmann H (2007) Microglia: active sensor and versatile effector cells in the normal and pathologic brain. Nat Neurosci 10(11):1387–1394. https://doi.org/10.1038/nn1997
Healy S, McMahon J, FitzGerald U (2018) Seeing the wood for the trees: towards improved quantification of glial cells in central nervous system tissue. Neural Regener Res 13(9):1520–1523. https://doi.org/10.4103/1673-5374.235222
Henn A, Lund S, Hedtjarn M, Schrattenholz A, Porzgen P, Leist M (2009) The suitability of BV2 cells as alternative model system for primary microglia cultures or for animal experiments examining brain inflammation. Altex 26(2):83–94
Horvath RJ, Nutile-McMenemy N, Alkaitis MS, Deleo JA (2008) Differential migration, LPS-induced cytokine, chemokine, and NO expression in immortalized BV-2 and HAPI cell lines and primary microglial cultures. J Neurochem 107(2):557–569. https://doi.org/10.1111/j.1471-4159.2008.05633.x
Ito D, Imai Y, Ohsawa K, Nakajima K, Fukuuchi Y, Kohsaka S (1998) Microglia-specific localisation of a novel calcium binding protein, Iba1. Brain Res Mol Brain Res 57(1):1–9
Ito D, Tanaka K, Suzuki S, Dembo T, Fukuuchi Y (2001) Enhanced expression of Iba1, ionized calcium-binding adapter molecule 1, after transient focal cerebral ischemia in rat brain. Stroke 32(5):1208–1215
Kanazawa H, Ohsawa K, Sasaki Y, Kohsaka S, Imai Y (2002) Macrophage/microglia-specific protein Iba1 enhances membrane ruffling and Rac activation via phospholipase C-gamma -dependent pathway. J Biol Chem 277(22):20026–20032. https://doi.org/10.1074/jbc.M109218200
Kaya N, Tanaka S, Koike T (2002) ATP selectively suppresses the synthesis of the inflammatory protein microglial response factor (MRF)-1 through Ca(2+) influx via P2X(7) receptors in cultured microglia. Brain Res 952(1):86–97
Kim M, Jiang LH, Wilson HL, North RA, Surprenant A (2001) Proteomic and functional evidence for a P2X7 receptor signalling complex. EMBO J 20(22):6347–6358. https://doi.org/10.1093/emboj/20.22.6347
Leon-Ponte M, Kirchhof MG, Sun T, Stephens T, Singh B, Sandhu S, Madrenas J (2005) Polycationic lipids inhibit the pro-inflammatory response to LPS. Immunol Lett 96(1):73–83. https://doi.org/10.1016/j.imlet.2004.07.019
Li K, Tan YH, Light AR, Fu KY (2013) Different peripheral tissue injury induces differential phenotypic changes of spinal activated microglia. Clin Dev Immunol 2013:901420. https://doi.org/10.1155/2013/901420
Linder S, Aepfelbacher M (2003) Podosomes: adhesion hot-spots of invasive cells. Trends Cell Biol 13(7):376–385. https://doi.org/10.1016/S0962-8924(03)00128-4
Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods 25(4):402–408. https://doi.org/10.1006/meth.2001.1262
Masliah E, Mallory M, Hansen L, Alford M, Albright T, Terry R, Shapiro P, Sundsmo M, Saitoh T (1991) Immunoreactivity of CD45, a protein phosphotyrosine phosphatase, in Alzheimer's disease. Acta Neuropathol 83(1):12–20
Maurya SK, Mishra R (2017a) Pax6 binds to promoter sequence elements associated with immunological surveillance and energy homeostasis in brain of aging mice. Ann Neurosci 24(1):20–25. https://doi.org/10.1159/000464419
Maurya SK, Mishra R (2017b) Pax6 interacts with Iba1 and shows age-associated alterations in brain of aging mice. J Chem Neuroanat 82:60–64. https://doi.org/10.1016/j.jchemneu.2017.05.002
Maurya SK, Mishra R (2018) Co-localization and interaction of Pax5 with Iba1 in brain of mice. Cell Mol Neurobiol 38(4):919–927. https://doi.org/10.1007/s10571-017-0566-1
McHugh D, Hu SS, Rimmerman N, Juknat A, Vogel Z, Walker JM, Bradshaw HB (2010) N-arachidonoyl glycine, an abundant endogenous lipid, potently drives directed cellular migration through GPR18, the putative abnormal cannabidiol receptor. BMC Neurosci 11:44. https://doi.org/10.1186/1471-2202-11-44
McHugh D, Wager-Miller J, Page J, Bradshaw HB (2012) siRNA knockdown of GPR18 receptors in BV-2 microglia attenuates N-arachidonoyl glycine-induced cell migration. J Mol Signal 7(1):10. https://doi.org/10.1186/1750-2187-7-10
Monif M, Reid CA, Powell KL, Smart ML, Williams DA (2009) The P2X7 receptor drives microglial activation and proliferation: a trophic role for P2X7R pore. J Neurosci 29(12):3781–3791. https://doi.org/10.1523/JNEUROSCI.5512-08.2009
Mori I, Imai Y, Kohsaka S, Kimura Y (2000) Upregulated expression of Iba1 molecules in the central nervous system of mice in response to neurovirulent influenza A virus infection. Microbiol Immunol 44(8):729–735
Nakamura Y (2002) Regulating factors for microglial activation. Biol Pharm Bull 25(8):945–953. https://doi.org/10.1248/bpb.25.945
Nikolakopoulou AM, Dutta R, Chen Z, Miller RH, Trapp BD (2013) Activated microglia enhance neurogenesis via trypsinogen secretion. Proc Natl Acad Sci USA 110(21):8714–8719. https://doi.org/10.1073/pnas.1218856110
Nimmerjahn A, Kirchhoff F, Helmchen F (2005) Resting microglial cells are highly dynamic surveillants of brain parenchyma in vivo. Science 308(5726):1314–1318. https://doi.org/10.1126/science.1110647
Ohsawa K, Imai Y, Kanazawa H, Sasaki Y, Kohsaka S (2000) Involvement of Iba1 in membrane ruffling and phagocytosis of macrophages/microglia. J Cell Sci 113(Pt 17):3073–3084
Ohsawa K, Imai Y, Sasaki Y, Kohsaka S (2004) Microglia/macrophage-specific protein Iba1 binds to fimbrin and enhances its actin-bundling activity. J Neurochem 88(4):844–856
Provenzano PP, Keely PJ (2011) Mechanical signaling through the cytoskeleton regulates cell proliferation by coordinated focal adhesion and Rho GTPase signaling. J Cell Sci 124(Pt 8):1195–1205. https://doi.org/10.1242/jcs.067009
Rikitake Y, Takai Y (2011) Directional cell migration: regulation by small G proteins, Nectin-like molecule-5, and afadin. Int Rev Cel Mol Biol 287:97–143. https://doi.org/10.1016/B978-0-12-386043-9.00003-7
Roesch S, Rapp C, Dettling S, Herold-Mende C (2018) When Immune cells turn bad-tumor-associated microglia/macrophages in glioma. Int J Mol Sci. https://doi.org/10.3390/ijms19020436
Romero-Sandoval A, Chai N, Nutile-McMenemy N, Deleo JA (2008) A comparison of spinal Iba1 and GFAP expression in rodent models of acute and chronic pain. Brain Res 1219:116–126. https://doi.org/10.1016/j.brainres.2008.05.004
Rosales C, Uribe-Querol E (2017) Phagocytosis: a fundamental process in immunity. Biomed Res Int 2017:9042851. https://doi.org/10.1155/2017/9042851
Roth V (2006) Doubling time computing. http://www.doubling-time.com/compute.php
Sasaki Y, Ohsawa K, Kanazawa H, Kohsaka S, Imai Y (2001) Iba1 is an actin-cross-linking protein in macrophages/microglia. Biochem Biophys Res Commun 286(2):292–297. https://doi.org/10.1006/bbrc.2001.5388
Sawano T, Tsuchihashi R, Morii E, Watanabe F, Nakane K, Inagaki S (2017) Homology analysis detects topological changes of Iba1 localization accompanied by microglial activation. Neuroscience 346:43–51. https://doi.org/10.1016/j.neuroscience.2016.12.052
Schwab JM, Frei E, Klusman I, Schnell L, Schwab ME, Schluesener HJ (2001) AIF-1 expression defines a proliferating and alert microglial/macrophage phenotype following spinal cord injury in rats. J Neuroimmunol 119(2):214–222
Siddiqui TA, Lively S, Vincent C, Schlichter LC (2012) Regulation of podosome formation, microglial migration and invasion by Ca(2+)-signaling molecules expressed in podosomes. J Neuroinflamm 9:250. https://doi.org/10.1186/1742-2094-9-250
Sigola LB, Fuentes AL, Millis LM, Vapenik J, Murira A (2016) Effects of toll-like receptor ligands on RAW 264.7 macrophage morphology and zymosan phagocytosis. Tissue Cell 48(4):389–396. https://doi.org/10.1016/j.tice.2016.04.002
Small JV, Stradal T, Vignal E, Rottner K (2002) The lamellipodium: where motility begins. Trends Cell Biol 12(3):112–120
Sogn CJ, Puchades M, Gundersen V (2013) Rare contacts between synapses and microglial processes containing high levels of Iba1 and actin—a postembedding immunogold study in the healthy rat brain. Eur J Neurosci. https://doi.org/10.1111/ejn.12213
Staniland AA, Clark AK, Wodarski R, Sasso O, Maione F, D'Acquisto F, Malcangio M (2010) Reduced inflammatory and neuropathic pain and decreased spinal microglial response in fractalkine receptor (CX3CR1) knockout mice. J Neurochem 114(4):1143–1157. https://doi.org/10.1111/j.1471-4159.2010.06837.x
Stuart LM, Bell SA, Stewart CR, Silver JM, Richard J, Goss JL, Tseng AA, Zhang A, El Khoury JB, Moore KJ (2007) CD36 signals to the actin cytoskeleton and regulates microglial migration via a p130Cas complex. J Biol Chem 282(37):27392–27401. https://doi.org/10.1074/jbc.M702887200
Svensson C, Fernaeus SZ, Part K, Reis K, Land T (2010) LPS-induced iNOS expression in Bv-2 cells is suppressed by an oxidative mechanism acting on the JNK pathway—a potential role for neuroprotection. Brain Res 1322:1–7. https://doi.org/10.1016/j.brainres.2010.01.082
Swaminathan V, Kalappurakkal JM, Mehta SB, Nordenfelt P, Moore TI, Koga N, Baker DA, Oldenbourg R, Tani T, Mayor S, Springer TA, Waterman CM (2017) Actin retrograde flow actively aligns and orients ligand-engaged integrins in focal adhesions. Proc Natl Acad Sci USA 114(40):10648–10653. https://doi.org/10.1073/pnas.1701136114
Tan J, Town T, Mullan M (2000) CD45 inhibits CD40L-induced microglial activation via negative regulation of the Src/p44/42 MAPK pathway. J Biol Chem 275(47):37224–37231. https://doi.org/10.1074/jbc.M002006200
Tanaka S, Suzuki K, Watanabe M, Matsuda A, Tone S, Koike T (1998) Upregulation of a new microglial gene, mrf-1, in response to programmed neuronal cell death and degeneration. J Neurosci 18(16):6358–6369
Taylor PR, Brown GD, Herre J, Williams DL, Willment JA, Gordon S (2004) The role of SIGNR1 and the beta-glucan receptor (dectin-1) in the nonopsonic recognition of yeast by specific macrophages. J Immunol 172(2):1157–1162. https://doi.org/10.4049/jimmunol.172.2.1157
Ton BH, Chen Q, Gaina G, Tucureanu C, Georgescu A, Strungaru C, Flonta ML, Sah D, Ristoiu V (2013) Activation profile of dorsal root ganglia Iba-1 (+) macrophages varies with the type of lesion in rats. Acta Histochem 115(8):840–850. https://doi.org/10.1016/j.acthis.2013.04.007
Vincent C, Siddiqui TA, Schlichter LC (2012) Podosomes in migrating microglia: components and matrix degradation. J Neuroinflamm 9:190. https://doi.org/10.1186/1742-2094-9-190
Yan DJ, Chen ZW (2010) 17beta-Estradiol increased the expression of daintain/AIF-1 in RAW264.7 macrophages. Biosci Biotechnol Biochem 74(10):2103–2105. https://doi.org/10.1271/bbb.100286
Yang ZF, Ho DW, Lau CK, Lam CT, Lum CT, Poon RT, Fan ST (2005) Allograft inflammatory factor-1 (AIF-1) is crucial for the survival and pro-inflammatory activity of macrophages. Int Immunol 17(11):1391–1397. https://doi.org/10.1093/intimm/dxh316
Yip PK, Kaan TK, Fenesan D, Malcangio M (2009) Rapid isolation and culture of primary microglia from adult mouse spinal cord. J Neurosci Methods 183(2):223–237. https://doi.org/10.1016/j.jneumeth.2009.07.002
Zhou J, Zhang X, Zhou Y, Wu B, Tan ZY (2019) Up-regulation of P2X7 receptors contributes to spinal microglial activation and the development of pain induced by BmK-I. Neurosci Bull. https://doi.org/10.1007/s12264-019-00345-0
Acknowledgements
We greatly appreciate Dan Cimponeriu (Genetics Department, Faculty of Biology, University of Bucharest) for the BLAST alignment, and Elena Burlacu, Mara Floare, Cornelia Dragomir and Geanina Haralambie (Department of Anatomy, Animal Physiology and Biophysics, Faculty of Biology, University of Bucharest) for technical support. The co-staining images of Iba1 and CD45 were taken by Adela Banciu and Daniel Banciu from the Department of Bioengineering and Biotechnology, Faculty of Medical Engineering, University Politehnica of Bucharest, under a Zeiss LSM 880 confocal microscope purchased as part of the Competitiveness Operational Program 2014–2020, Priority axis 1, Project No. P_36_611, MySMIS code 107066, Innovative Technologies for Materials Quality Assurance in Health, Energy and Environmental—Center for Innovative Manufacturing Solutions of Smart Biomaterials and Biomedical Surfaces—INOVABIOMED. This research was funded by the Romanian Government via UEFISCDI (Executive Unit for Higher Education, Research, Development and Innovation Funding) grant 65/2018.
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ROG and AD contributed equally to this study in RT-PCR, ICC, migration, cell adhesion and calcium imaging experiments, performed data analysis and interpretation; AF assisted in the proliferation and phagocytosis experiments; AG contributed to migration experiments; MBP contributed to P2x7 experiments; MC and GC performed Western blot experiments; CM and LS were involved in LC–MS/MS experiments and VR gave the concept and design, wrote the manuscript, provided financial support and interpreted data. All authors read and approved the final manuscript.
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10571_2020_790_MOESM1_ESM.pdf
Supplementary file1 Diagram showing the position of the nucleotide sequence inside the mRNA molecule where the antisense strand of Iba1-siRNA would bind. The total sequence represents the mRNA coding for Mus musculus allograft inflammatory factor 1 (Aif1) based on which the siRNA molecule was designed: NCBI Reference Sequence: NM_01946 (https://www.ncbi.nlm.nih.gov/nuccore/NM_019467), Gene ID:11629 (https://www.ncbi.nlm.nih.gov/gene?term=11629). In blue is indicated the translated region, in red is the exon 6 sequence and highlighted in yellow is the sequence to which the antisense strand of Iba1-siRNA would bind. The binding starts at 176 nucleotides from the ATG initiation codon (PDF 256 kb)
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Supplementary file2 Lipofectamine RNAiMAX prevents LPS-induced NO secretion by BV2 cells. In the absence of LPS, LF-RNAiMAx, sc- and Iba1-siRNA have no effect on the BV2’s NO secretion. In the presence of LPS (1 μg/ml), non-treated BV2 cells respond with a high secretion of NO, which is significantly reduced in all the conditions where LF-RNAiMAx is present. NT- non-treated; C - Control (DOCX 21 kb)
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Supplementary file3 a Diagram showing the transfection protocol with siRNA used in all the experiments. b Different concentrations of Iba1-siRNA had no cytotoxic effects on cell viability compared with control cells. By itself, LF-RNAiMAx significantly decreased cell viability compared to non-treated cells (*P < 0.05), but there was no difference compared to siRNA-treated cells (PDF 252 kb)
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Supplementary file4 Schematic drawing representing parameters which have been determined for ratiometric intracellular Ca2+ recordings. ΔF/F0 represents the ratio between the maximum fluorescence change during the stimulus (Fmax) and the baseline fluorescence before the stimulus (F0). T50 was defined as the time interval between the peak value and the time at which the signal decayed to 50% of peak amplitude. The AUC representing the area between F0 to 50% recovery of the calcium signal, is a measure of the overall amount of calcium that entered the cell. The decay slope, measured from the peak amplitude to the time at which the signal decayed to 50% of peak amplitude, is a measure of calcium kinetics during recovery period (PDF 204 kb)
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Supplementary file5 Representative images of Iba1 staining in BV2 cells treated with different concentrations of Iba1-siRNA (5, 10, 20 and 40 nM). The expression of Iba1 protein is gradually reduced by increased concentrations of Iba1-siRNA (scale bar = 20 μm) (PDF 293 kb)
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Supplementary file6 The transfection efficiency quantified with Alexa Fluor594-tagged scramble-siRNA. Graph representing the transfection efficiency quantified as the percentage of cells expressing the Alexa Fluor594-tag from the total number of cells. Five different fields were analyzed per condition, with a x40 magnification. The transfection rate was 67.10 ± 6.32 % (at 12h), 55.79 ± 5.33 % (at 24h), 25.09 ± 2.18 % (at 48h) and 10.62 ± 0.65 % (at 72h); n = 5 for all time points. The transfection rate decreased in time due to transfer of Alexa Fluor594-tagged scramble-siRNA to other cells during cell division (PDF 163 kb)
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Supplementary file7 Representative images of Iba1 and F-actin co-localization (yellow) at FAC (focal adhesion complex) level in the un-treated BV2 cells (scale bar = 10 μm) (PDF 326 kb)
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Supplementary file8 Representative images of Iba1 and CD45 staining in BV2 cells treated with 40 nM scramble- or Iba1-siRNA (scale bar = 50 μm). While there is an obvious reduction of the Iba1 expression level after Iba1-siRNA treatment, CD45 expression level was not affected by the Iba1-silencing process (PDF 333 kb)
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Gheorghe, RO., Deftu, A., Filippi, A. et al. Silencing the Cytoskeleton Protein Iba1 (Ionized Calcium Binding Adapter Protein 1) Interferes with BV2 Microglia Functioning. Cell Mol Neurobiol 40, 1011–1027 (2020). https://doi.org/10.1007/s10571-020-00790-w
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DOI: https://doi.org/10.1007/s10571-020-00790-w