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Silencing the Cytoskeleton Protein Iba1 (Ionized Calcium Binding Adapter Protein 1) Interferes with BV2 Microglia Functioning

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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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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.

Author information

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.

Correspondence to Violeta Ristoiu.

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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)

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)

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)

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)

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)

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)

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)

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, R., 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 (2020). https://doi.org/10.1007/s10571-020-00790-w

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Keywords

  • Iba1 protein
  • BV2 microglia
  • siRNA
  • Migration
  • Proliferation
  • Phagocytosis