Volume 22, Issue 1, Pages 136-148 (January 2018) Prion-like Propagation of α-Synuclein Is Regulated by the FcγRIIB-SHP-1/2 Signaling Pathway in Neurons  Yu Ree Choi, Seon-Heui Cha, Seo-Jun Kang, Jae-Bong Kim, Ilo Jou, Sang Myun Park  Cell Reports  Volume 22, Issue 1, Pages 136-148 (January 2018) DOI: 10.1016/j.celrep.2017.12.009 Copyright © 2017 The Author(s) Terms and Conditions

Cell Reports 2018 22, 136-148DOI: (10.1016/j.celrep.2017.12.009) Copyright © 2017 The Author(s) Terms and Conditions

Figure 1 α-Syn Fibrils Bind to Human FcγRIIB (A) Diagram for mouse FcγRIIB (mFcγRIIB), hFcγRIIB, and hFcγRIIB deletion mutants. (B) 293T cells were transfected with hFcγRIIB and hFcγRIIB deletion mutants, and then a western blot was performed with anti-myc antibody. (C) 293T cells were transfected with mFcγRIIB, hFcγRIIB, and hFcγRIIB deletion mutants. The cells were incubated with 1 μM α-syn fibrils for 10 min and then immunostained with anti-myc- and anti-α-syn antibodies. The samples were observed under a confocal microscope, and the intensity was analyzed. Blue indicates DAPI staining. The scale bar indicates 20 μm. The values obtained are from three independent experiments. ∗∗p < 0.01. (D and E) 293T cells were transfected with hFcγRIIB and hFcγRIIB deletion mutants. The cells were incubated with 1 μM α-syn fibrils (D) and 1 μM α-syn monomer (E) for 30 min, co-immunoprecipitation was performed with anti-myc antibody as described in the Experimental Procedures, and then a western blot was performed. Cell Reports 2018 22, 136-148DOI: (10.1016/j.celrep.2017.12.009) Copyright © 2017 The Author(s) Terms and Conditions

Figure 2 FcγRIIB Signaling Is Involved in Cell-to-Cell Transmission of α-Syn (A) Primary cortical neurons were incubated with 1 μM α-syn fibrils for 10 min and then fixed and immunostained with anti-Tuj-1, a marker for neurons, and anti-α-syn antibodies. (B) Western blot was performed using lysates of non-targeting (NT) and FcγRIIB KD #1 and #2 SH-SY5Y cells. (C) NT and FcγRIIB KD #1 and #2 SH-SY5Y cells were incubated with 1 μM α-syn fibrils for 20 min and then fixed and immunostained with the anti α-syn antibody. The intensity was analyzed. The values obtained are from three independent experiments. (D) Diagram of the dual chamber system. (E) NT and FcγRIIB KD #1 and #2 SH-SY5Y cells were cocultured with differentiated α-syn overexpressing SH-SY5Y cells for 12 hr using the dual chamber system. The cells were immunostained with the anti-α-syn antibody. The intensity was analyzed. (F) Rat primary cortical neurons were infected with shRNA lentivirus for NT and FcγRIIB 11 days after plating. After 2 days of infection, the cells were lysed, and a western blot was performed. (G) The cells were co-cultured with differentiated α-syn OE SH-SY5Y cells for 24 hr. The cells were immunostained with anti-human α-syn antibody and observed under a confocal microscope. (H) SH-SY5Y cells were treated with 0.5 mg/mL IC and then were co-cultured with differentiated α-syn OE SH-SY5Y cells for 12 hr. The cells were immunostained with the anti α-syn antibody. The intensity was analyzed. The values obtained are from three independent experiments. Blue indicates DAPI staining. All scale bars indicate 20 μm. ∗∗p < 0.01 against NT or control cells. Cell Reports 2018 22, 136-148DOI: (10.1016/j.celrep.2017.12.009) Copyright © 2017 The Author(s) Terms and Conditions

Figure 3 α-Syn Fibrils Induce Phosphorylation of SHP-1 and SHP-2 by Interacting with FcγRIIB (A) SH-SY5Y cells were transfected with WT FcγRIIB or the Y273F FcγRIIB mutant. After 1 day, the cells were cocultured with differentiated α-syn OE SH-SY5Y cells for 12 h and then fixed and immunostained with anti-myc and anti-α-syn antibodies. The intensity was analyzed. The values obtained are from three independent experiments. Blue indicates DAPI staining. The scale bar indicates 20 μm. ∗∗p < 0.01 against mock cells. (B–D) SH-SY5Y cells (B), primary cortical neurons (C), and FcγRIIB KD SHSY5Y cells (D) were treated with 1 μM α-syn fibrils for 10 min. (E) SH-SY5Y cells were transfected with WT FcγRIIB or the Y273F FcγRIIB mutant. After 1 day, the cells were incubated with 1 μM α-syn fibrils for 10 min. The cells were lysed, and a western blot was performed with the indicated antibodies. Data are representative of three independent experiments. ∗p < 0.05, ∗∗p < 0.01 against control cells. Cell Reports 2018 22, 136-148DOI: (10.1016/j.celrep.2017.12.009) Copyright © 2017 The Author(s) Terms and Conditions

Figure 4 SHP-1/2 Expressed in Neurons Mediate Cell-to-Cell Transmission of α-Syn (A) SH-SY5Y cells were co-cultured with differentiated α-syn OE SH-SY5Y cells for 12 hr in the presence of 20 μM NSC87877, 10 μM sodium stibogluconate (SSG), or 1 μM PHPS-1 for 12 hr. The cells were then fixed and immunostained with the anti-α-syn antibody. The intensity was analyzed. The values obtained are from three independent experiments. ∗∗p < 0.01 against control. (B) A western blot was performed using lysates of NT and SHP-1 KD #1 and #2 SH-SY5Y cells. (C) NT and SHP-1 KD #1 and #2 SH-SY5Y cells were cocultured with differentiated α-syn OE SH-SY5Y cells for 12 hr and then fixed and immunostained with the anti-α-syn antibody. The values obtained are from three independent experiments. ∗∗p < 0.01 against NT cells. (D) SH-SY5Y cells were transfected with SHP-1 WT-EGFP, SHP-1 C452S-EGFP, and SHP-1ΔN-EGFP. After 1 day, the cells were cocultured with differentiated α-syn OE SH-SY5Y cells for 12 hr and then fixed and immunostained with anti-α-syn antibody. The values obtained are from three independent experiments. ∗∗p < 0.01 against untransfected cells in the same fields. (E) A western blot was performed using lysates of NT and SHP-2 KD #1 and #2 SH-SY5Y cells. (F) NT and SHP-2 KD #1 and #2 SH-SY5Y cells were cocultured with differentiated α-syn OE SH-SY5Y cells for 12 hr and then fixed and immunostained with anti-α-syn antibody. The values obtained are from three independent experiments. ∗∗p < 0.01 against NT cells. (G) SH-SY5Y cells were transfected with SHP-2 WT-EGFP, SHP-2 D61A-EGFP, SHP-2 E76A-EGFP, and SHP-2 C459S-EGFP. After 1 day, the cells were cocultured with differentiated α-syn OE SH-SY5Y cells for 12 hr and then fixed and immunostained with the anti-α-syn antibody. The values obtained are from three independent experiments. ∗∗p < 0.01 against untransfected cells in the same fields. (H) FcγRIIB KD SH-SY5Y cells were transfected with ΔN SHP-1-GFP and E76A SHP-2-GFP. After 1 day, the cells were cocultured with differentiated α-syn OE SH-SY5Y cells for 12 hr. The cells were then fixed and immunostained with anti-α-syn antibody. The intensity was analyzed. The values obtained are from three independent experiments. ∗∗p < 0.01 against untransfected cells in the same fields. Blue indicates DAPI staining. All scale bars indicate 20 μm. Cell Reports 2018 22, 136-148DOI: (10.1016/j.celrep.2017.12.009) Copyright © 2017 The Author(s) Terms and Conditions

Figure 5 FcγRIIB-SHP-1/2 Downstream Signaling Is Involved in Lipid Raft-Dependent Endocytosis (A–D) NT (A), FcγRIIB KD (B), SHP-1 KD (C), and SHP-2 KD SHSY5Y (D) cells were incubated with 1 μM α-syn fibrils and 0.5 mg/mL IC for 10 min, and then these cells were further incubated with 50 nM BODIPY FL C5-LacCer and 2.5 μg/mL rhodamine-conjugated transferrin for 20 min. The cells were then fixed and observed under confocal microscopy. Blue indicates DAPI staining. The scale bar indicates 20 μm. (E and F) The LacCer (E) and transferrin (F) intensities were analyzed. The values obtained are from five independent experiments. ∗∗p < 0.01 against control. Cell Reports 2018 22, 136-148DOI: (10.1016/j.celrep.2017.12.009) Copyright © 2017 The Author(s) Terms and Conditions

Figure 6 Generation of an In Vitro Model System to Quantify the Formation of Lewy Body-like Inclusions by Cell-to-Cell Transmission of α-Syn (A) Diagram of a coculture system using A53T α-syn-EGFP and A53T α-syn-mCherry SH-SY5Y cells. (B–D) WT α-syn-EGFP (B), A53T α-syn-EGFP (C), EGFP (D), WT α-syn-mCherry (B), A53T α-syn-mCherry (C), or mCherry SH-SY5Y (D) cells were cocultured for the indicated times in the presence of 50 μM RA. Then the samples were observed under a confocal microscope, and the number of cells containing yellow puncta was analyzed. Arrows indicate puncta containing both WT α-syn-EGFP, A53T α-syn-EGFP, or EGFP and WT α-syn-mCherry, A53T α-syn-mCherry, or mCherry, respectively (yellow puncta). In the graphs, green indicates the number of WT α-syn-EGFP, A53T α-syn-EGFP, or EGFP SH-SY5Y cells containing yellow puncta, and red indicates the number of WT α-syn-mCherry, A53T α-syn-mCherry, mCherry SH-SY5Y cells containing yellow puncta. The values obtained are from three independent experiments. (E) A53T α-syn-EGFP and A53T α-syn-mCherry SH-SY5Y cells were cocultured for 5 days in the presence of 50 μM RA. The cells were fixed and immunostained with anti-pSer129 α-syn antibody (violet). Arrows indicate puncta containing both WT α-syn-EGFP and WT α-syn-mCherry and costained with the anti-pSer129 α-syn antibody. Blue indicates DAPI staining. The scale bar indicates 20 μm. Cell Reports 2018 22, 136-148DOI: (10.1016/j.celrep.2017.12.009) Copyright © 2017 The Author(s) Terms and Conditions

Figure 7 The FcγRIIB-SHP-1/2 Signaling Pathway Mediates the Formation of Lewy Body-like Inclusions Induced by Cell-to-Cell Transmission of α-Syn (A) The cells were cocultured for 5 days in the presence of 50 μM RA. Then the samples were observed under a confocal microscope, and the number of cells containing yellow dots was analyzed. Arrows indicate puncta containing both A53T α-syn-EGFP and A53T α-syn-mCherry (yellow puncta). Blue indicates DAPI staining. The scale bar indicates 20 μm. (B) A53T α-syn-EGFP and A53T α-syn-mCherry SH-SY5Y cells were cocultured for 5 days in the presence of 20 μM NSC87877, 10 μM SSG, and 1 μM PHPS-1, respectively, with 50 μM RA. Then the samples were observed under a confocal microscope, and the number of cells containing yellow puncta was analyzed. (C) A53T α-syn-EGFP/SHP-1 KD #1 or #2 and A53T α-syn-mCherry SH-SY5Y cells were cocultured in the presence of 50 μM RA for 5 days. Then the samples were observed under a confocal microscope, and the number of cells containing yellow puncta was analyzed. (D) A53T α-syn-EGFP/SHP-2 KD #1 or #2 and A53T α-syn-mCherry SH-SY5Y cells were cocultured in the presence of 50 μM RA for 5 days. Then the samples were observed under a confocal microscope, and the number of cells containing yellow puncta was analyzed. The values obtained are from three independent experiments. ∗p < 0.05, ∗∗p < 0.01 against NT or control cells. Cell Reports 2018 22, 136-148DOI: (10.1016/j.celrep.2017.12.009) Copyright © 2017 The Author(s) Terms and Conditions