Journal: Science Advances

Article Title: TPM1 drives cytoskeleton-immunometabolism coupling and LGALS9/CD45-mediated neuroinflammatory propagation in retinitis pigmentosa

doi: 10.1126/sciadv.aea6467

Figure Lengend Snippet: ( A ) Metabolism pathway enrichment analysis of DEGs in DAM2 microglial cluster. ( B ) Lipid/cholesterol metabolism–related DEGs in DAM2. mTOR, mechanistic target of rapamycin. ( C ) Retinal section stained with Filipin (free cholesterol; magenta), Iba-1 (microglia; green), and Histone H3 (nuclei; white) antibodies and quantification of Filipin + microglia in the ONL of the retinas from rd10 retinas administrated with AAV- Cx3cr1-Tpm1 -EGFP or control vector ( n = 5 mice per group). Yellow arrowheads indicate Filipin + microglia. Scale bar, 20 μm. For image acquiring, three views in each retinal section at 100 μm (central), 1 mm (middle), and 1.8 mm (peripheral) from the optic nerve head along the dorsal and ventral directions were captured. ( D ) qPCR validation of Tpm1 and lipid metabolism regulators ( Apoe , Fabp5 , Apoc1 , Plin2 , Lypla1 , Sp1 , and Soat1 ) in primary microglia transfected with Tpm1 plasmid versus control ( n = 3 independent experiments). ( E to G ) Fluorescent colocalization analysis of lipid and cholesterol accumulation (Nile Red: lipid droplets; BODIPY C11: neutral lipids; Filipin: free cholesterol) in microglia following Tpm1 overexpression and Apoe/Fabp5 knockdown. Cell percentages of Nile Red + , BODIPY C11 + , or Filipin + microglia were quantified ( n = 3 independent experiments). Scale bars, 20 μm. ( H ) Transcriptional profiling of inflammatory mediators in dual-modified microglia ( n = 3 independent experiments). ( I ) Schematic model: Tpm1 up-regulation drives Apoe/Fabp5 -dependent lipid accumulation in microglia, promoting neuroinflammatory responses and photoreceptor degeneration in RP. Data represent means ± SEMs. Statistical significance determined using unpaired two-tailed Student’s t test (C) or one-way ANOVA with Tukey’s multiple-comparison test [(D) to (H)] (* P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001).

Article Snippet: Retinal sections were incubated with goat Iba-1 (Wako; 1:500) and rabbit Histone H3 (MCE; 1:100) in blocking buffer at 4°C overnight, followed by incubation with donkey anti-goat Alexa Fluor 488 (Invitrogen; 1:500) and donkey anti-rabbit Alexa Fluor 594 (Invitrogen; 1:500) for 2 hours.

Techniques: Staining, Control, Plasmid Preparation, Biomarker Discovery, Transfection, Over Expression, Knockdown, Modification, Two Tailed Test, Comparison