Fig 1.
Identification of Rcn1 as a microglial phagocytosis ligand.
(A) Schematics of OPD-NGS. OPD/PFC selection was performed by incubating OPD cDNA library with BV-2 microglial cells at 4°C. After washing, cells were incubated at 37°C for phagocytosis. Surface-bound unphagocytosed phages were removed by stripping with low pH isotonic buffer. Phagocytosed phages were released by cell lysis, amplified and used as input for the next round of selection. After 3 rounds of selection, the cDNA inserts of enriched clones were amplified by PCR and identified by NGS. (B) Verification of Rcn1-Phage phagocytosis by microglia. Clonal Rcn1-Phage, GFP-Phage or Control-Phage was incubated with BV-2 cells at 4°C. After washing, cells were incubated at 37°C to allow bound phages to be phagocytosed. After removal of surface-bound unphagocytosed phage, internalized phages were released by cell lysis and quantified by plaque assay (+ s.e.m., *** P<0.0001, n = 4, t-test).
Table 1.
Partial list of known or putative phagocytosis ligands identified by OPD-NGS.
Fig 2.
Rcn1 stimulates microglial phagocytosis.
(A) Expression of Rcn1-FLAG, FLAG-Tulp1 and GFP-FLAG in Neuro-2A cells was verified by Western blot using anti-FLAG mAb. (B) Rcn1 facilitates BV-2 microglial phagocytosis. Rcn1-FLAG, FLAG-Tulp1 (positive control) or GFP-FLAG (negative control) was expressed in Neuro-2A cells. Cells were treated with or without etoposide to induce apoptosis, labeled with pHrodo, incubated with BV-2 microglia for phagocytosis, and analyzed by confocal microscopy. Scale bar = 50 μm. (C) Percentage of BV-2 cells with phagocytosed cargos in (B) were quantified by ImageJ (+ s.e.m., *** P<0.0001, n = 3, t-test).
Fig 3.
Purified Rcn1 stimulates microglial phagocytosis.
(A) Purification of GST-Rcn1 and GST. Both proteins were expressed in bacteria, purified with glutathione columns and analyzed by SDS-PAGE. (B) Rcn1-facilitates microglial phagocytosis. Neuro-2A cells were treated with or without etoposide to induce apoptosis, labeled with pHrodo, and incubated with BV-2 microglia in the presence of the purified protein at indicated concentrations. Phagocytosed pHrodo signals were analyzed by confocal microscopy. Scale bar = 50 μm. (C) Percentage of BV-2 cells with phagocytosed cargos in (B) were quantified (+ s.e.m., *** P<0.0001, n = 3, t-test).
Fig 4.
Rcn1 selectively binds to the surface of apoptotic neurons but not healthy neurons.
(A) Rcn1 is a secreted protein. Rcn1-FLAG with or without the signal peptide was expressed in Neuro-2A cells. Rcn1-FLAG in the conditioned medium of the apoptotic or healthy cells was immunoprecipitated with anti-FLAG mAb and analyzed by Western blot. GFP-FLAG was a non-secretory protein control. (B) Rcn1 selectively binds to apoptotic cells. Rcn1-FLAG was expressed in Neuro-2A cells. Cell surface-bound Rcn1-FLAG was detected for apoptotic and healthy Neuro-2A cells using FITC-labeled anti-FLAG mAb and analyzed by confocal microscopy. FLAG-Tulp1 is a positive control. Apoptotic cells were labeled with propidium iodide. Scale bar = 50 μm. (C) Flow cytometry analysis of Rcn1 binding to apoptotic neurons. Lysates were prepared from cells expressing Rcn1-FLAG, FLAG-Tulp1 (positive control) or GFP-FLAG (negative control), incubated with apoptotic or healthy Neuro-2A cells and analyzed by flow cytometry using FITC anti-FLAG mAb. (D) Rcn1 binds to microglia surface. GST-Rcn1 or GST control was incubated with BV-2 cells and analyzed by flow cytometry using FITC-anti-GST antibody.
Fig 5.
Rcn1 mediates microglial engulfment via phagocytosis pathway.
Phagocytosis of apoptotic cells by BV-2 microglia was performed as in Fig 2A. Phagosome marker Rab7 was detected using anti-Rab7 antibody and FITC-labeled secondary antibody, and analyzed by confocal microscopy. The z-stack images of pHrodo and FITC are co-localized and superimposed with cognate DAPI signals and bright fields. Bar = 10 μm.
Fig 6.
Rcn1 facilitates macrophage phagocytosis.
(A) Rcn1 stimulates macrophage phagocytosis of apoptotic neurons. Healthy or apoptotic Neuro-2A cells were labeled with pHrodo, incubated with J774 macrophage cells for phagocytosis and analyzed by confocal microscopy as in Fig 2A. The z-stack images of pHrodo and DAPI are superimposed with the cognate bright fields to reveal phagocytosed cargos. (B) Percentage of macrophages with phagocytosed cargos in (A) were quantified. Bar = 50 μm (+ s.e.m., *** P<0.0001, n = 3, t-test).
Fig 7.
A cartoon model for Rcn1 to facilitate microglial phagocytosis.
Rcn1 is secreted from neurons or other cells and preferentially binds to apoptotic neurons as a bridging molecule to facilitate their clearance by microglial phagocytosis.