Fig. 1

mGluR5 interacted with α-syn in microglia. a Interaction of mGluR5 and α-syn detected by immunoprecipitation with antibodies against myc and FLAG in co-transfected HEK293T cells. b HEK293T cells were co-transfected with myc-α-syn, and FLAG-mGluR5 or FLAG-GFP-mGluR3 plasmids; the specificity of mGluR5 interaction with α-syn was detected by co-immunoprecipitation (left). HEK293T cells were co-transfected with FLAG-mGluR5, and full-length myc-α-syn, myc-α-syn N, or myc-α-syn delN plasmids; mGluR5 interaction with α-syn truncations was detected by co-immunoprecipitation (right). c FLAG-mGluR5 or FLAG-GFP-mGluR3 overexpressed HEK293T cells were cultured with the collected medium from myc-α-syn-overexpressed HEK293T cells for 12 h, followed by co-immunoprecipitation. d Primary microglia were treated with 1 μM recombinant α-syn protein for 24 h, followed by co-immunoprecipitation. e BV2 cells were transfected with myc-α-syn for 24 h followed by CHPG (150 μM) or MTEP (100 μM) treatment for 24 h. Co-immunoprecipitation was performed to detect the interaction between mGluR5 and α-syn (left). Quantification of immunoprecipited mGluR5 level was normalized to IgG, and protein levels of mGluR5 and α-syn in cell lysates were normalized to β-tubulin (right). Cells transfected with vector without CHPG treatment served as control. f Immunofluorescence analysis was conducted from HEK293T cells co-transfected with myc-α-syn and FLAG-mGluR5 for 48 h (top), or BV2 cells transfected with myc-α-syn for 48 h (bottom). Cells were fixed and stained with anti-mGluR5 (red) and anti-α-syn (green), and nuclei were stained with DAPI. Arrowheads indicate examples of co-localization. Scale bar, 10 μm. g Brain lysates from LPS-induced rat PD model were immunoprecipitated with control mouse IgG or anti-α-syn, and the coprecipitated proteins were analyzed by immunoblotting with anti-mGluR5 (top). Quantification of protein levels of mGluR5 and α-syn in lysates were normalized to GAPDH and represented as the fold different ratio of the sham group. Immunoprecipited mGluR5 level was normalized to IgG and represented as the fold different ratio (lesioned/intact) of the sham group (bottom). h Immunofluorescent co-stainings with anti-mGluR5 (red) and anti-α-syn (green) were conducted with brain sections of SN in sham group (top) and LPS group (bottom). Microglia were stained with anti-Iba-1 (gray). Arrowheads indicate examples of co-localization. Scale bar, 10 μm. i Structural view of the representative mGluR5-α-syn complex predicted by protein-protein docking. The left showed the complex structure, and mGluR5 was colored in green and α-syn was colored in red. The right showed the predicted key point residues from the region of mGluR5 and the α-syn in each part. The residues in mGluR5 were colored in black, and the residues in α-syn were colored in red. Hydrogen bonds were shown as black dashed lines, and salt bridges were shown as red dashed lines, also represented the stronger interaction between five key point residues on intracellular C-terminal domain of mGluR5 and four key point residues from C-terminal region of α-syn indicated above. Carbon atoms, oxygen atoms, and nitrogen atoms for both mGluR5 and α-syn were colored in green, red, and blue, respectively. Ribbon were colored green for mGluR5 and colored red for α-syn. Data shown in this figure represent mean ± SEM (e, n ≥ 3) and mean ± SD (g, n ≥ 6). The statistical significance was determined by one-way ANOVA followed by Dunnett’s test (e). A comparison of mGluR5 expression in co-immunoprecipitation studies between sham group and LPS group using Student’s t test (g). ^**p < 0.01 and ^***p < 0.001 versus control (ctr) or sham group; ^#p < 0.05 and ^##p < 0.01 versus α-synuclein (α-syn) transfection; ns, not significant