Fig 2: Systematic TPM1 functions through the FABP5/NF‐kB signal pathway. (a) Inflammation‐related DEPs in pairwise comparisons both between young and young‐HP and between aged and aged‐HP. The Y‐axes indicate the Log2 FC of co‐expressed DEPs. (b–e) qPCR analysis of Fabp5, Nfkb2, Rela, and Rel in young retinas after YMP or OMP treatment. Data are presented as mean ± SEM, n = 3 mice in each group, one‐way ANOVA with Tukey's multiple comparison test. (f–i) qPCR analysis of Fabp5, Nfkb2, Rela, and Rel in young retinas after rTPM1 protein or PBS treatment. Data are presented as mean ± SEM, n = 3 mice in each group, unpaired two‐tailed Student's t test. (j–o) Western blot analysis (j) and quantification of Adcy2, p‐PKA, PKA, FABP5, and TPM1 (k–o) proteins in BV2 cells after rTPM1 treatment following the transfection of siFABP5. Data are presented as mean ± SEM and analyzed by one‐way ANOVA with Tukey's multiple comparison test (compared with rTPM1 + siCRT, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.001; compared with rTPM1 + siFABP5, #p < 0.05, ###p < 0.001). (p) ELISA analysis of cAMP in BV2 cells after rTPM1 protein treatment following the transfection of siFABP5. Data are presented as mean ± SEM. (q–t) qPCR analysis of Fabp5, Nfkb2, Rela, and Rel in BV2 cells after rTPM1 treatment following the transfection of siFABP5. Data are presented as mean ± SEM, one‐way ANOVA with Tukey's multiple comparison test. Three independent experiments were performed. (u–v) Western blot analysis (u) and quantification of FABP5 (v) in BV2 cells after rTPM1 and H89 treatment. Data are presented as mean ± SEM. (w) Schematic depicting TPM1‐related signaling pathway. Systematic TPM1 accumulation in young‐HP, OMP‐treated or rTPM1 protein‐treated young retina upregulates endogenous TPM1 by phosphorylating PKA and by activating FABP5/NF‐kB signaling pathway, leading to elevated inflammatory responses and neuronal remodeling in retina aging process

Fig 3: Systematic TPM1 functions by phosphorylating PKA in aging retinas. (a) ELISA analysis of cAMP in BV2 cells after different treatments. Following exposure to DMSO, rTPM1, FSK, SQ, or FSK + SQ for 24 h, BV2 cells were collected to detect the protein level of cAMP. Data are presented as mean ± SEM and analyzed by one‐way ANOVA with Tukey's multiple comparison test (compared to FSK, *p < 0.05). Five independent experiments were performed. (b–e) Western blot analysis (b) and quantification of TPM1, p‐PKA, and PKA (c–e) in BV2 cells after different treatments. Data are presented as mean ±SEM and analyzed by one‐way ANOVA with Tukey's multiple comparison test (compared with rTPM1, *p < 0.05, **p < 0.01; compared with FSK, #p < 0.05, ##p < 0.01; SQ vs. FSK + SQ, $p < 0.05). Five independent experiments were performed. (f–g) Western blot analysis (f) and quantification of TPM1 (g) in BV2 cells after rTPM1 and H89 treatments. Data are presented as mean ± SEM and analyzed by one‐way ANOVA with Tukey's multiple comparison test (compared with rTPM1, **p < 0.01). (h) ELISA analysis of cAMP in young retinas after YMP or OMP treatment. Data are presented as mean ± SEM, n = 5 mice in each group. (i–n) Western blot analysis (i) and quantification of Adcy2, p‐PKA, PKA, FABP5, and TPM1 (j–n) in young retinas after YMP or OMP treatment. Data are presented as mean ± SEM, n = 4 mice in each group, one‐way ANOVA with Tukey's multiple comparison test. (o) ELISA analysis of cAMP in young retinas after rTPM1 protein or PBS treatment. Data are presented as mean ± SEM, n = 4 mice in each group. (p–u) Western blot analysis (p) and quantification of Adcy2, p‐PKA, PKA, FABP5, and TPM1 proteins (q–u) in young retinas after rTPM1 protein or PBS treatment. Data are presented as mean ± SEM, n = 5 mice in each group, unpaired two‐tailed Student's t test. (v–w) qPCR analysis of TPM1.5, TPM1.9, and TPM1.10 in young (v) or aged (w) retinas after YMP or OMP treatment. Data are presented as mean ± SEM, n = 4 mice in each group, one‐way ANOVA with Tukey's multiple comparison test

Fig 4: Systemic administration of TPM1‐specific antibody counteracts age‐related dendritic sprouting and inflammation and improves visual function in aged mice. (a) Schematic diagram of the experimental design. Anti‐TPM1 antibody or IgG isotype control (1 mg/kg) were retro‐orbitally injected into aged C57BL/6J mice (14‐month‐old) for a total of ten times, 3 days apart, and retinas were then collected for further studies at day 30. (b) Representative confocal images of RBCs (top row) and HCs (bottom row) in retinal sections from aged mice after anti‐TPM1 antibody or IgG treatment. Arrowheads indicate the aberrant dendritic sprouting of RBCs and HCs. Scale bars, 10 µm. (c–d) Quantification of the aberrant dendritic length of RBCs and HCs in aged retinas after anti‐TPM1 antibody or IgG treatment. Data were presented as mean ± SEM, n = 6, 5 mice in IgG and Anti‐TPM1 groups, unpaired two‐tailed Student's t test. (e) Retina sections from aged mice after anti‐TPM1 antibody or IgG treatment were stained with Iba‐1and CD68. Red arrowhead indicates CD68‐positive microglia, and red arrows show the dendritic extension of activated microglia. Scale bars, 20 µm. (f) Immunostaining of retinal sections from aged mice after anti‐TPM1 antibody or IgG treatment with GFAP antibody. Red arrows show activated astrocytes, and red arrowheads indicate activated Müller cells. Scale bars, 20 µm. (g) Double‐staining of whole‐mounted retinas from aged mice after anti‐TPM1 antibody or IgG treatment with Iba‐1 and CD68. Representative confocal images focus on the OPL. The boxed regions are highly magnified at the bottom showing the colocalization of CD68 with microglia. Scale bar, 50 µm. (h–i) Quantification of the numbers of Iba‐1+ and of CD68+Iba‐1+ microglial cells in the IPL and OPL of whole‐mounted aged retinas after anti‐TPM1 antibody or IgG treatment. Data are presented as mean ± SEM, n = 4 mice in each group, unpaired two‐tailed Student's t test. (j–l), ELISA analysis of IL‐1β, IL‐6, and TNF‐α in aged retinas after anti‐TPM1 antibody or IgG treatment. Data are presented as mean ± SEM, n = 5 mice in each group unpaired two‐tailed Student's t test. (m–n) ERG recordings on aged mice after anti‐TPM1 antibody or IgG treatment. Data are presented as mean ± SEM, n = 10 mice in each group, unpaired two‐tailed Student's t test

Fig 5: Analysis of differentially expressed proteins by LC‐MS/MS. (a) A heat map showing the up‐ or downregulation of 128 differentially expressed proteins (DEPs) between aged unpaired (13 months) and aged‐HP (13 months) retinas. (b) Gene ontology (GO) enrichment analysis of DEPs between aged unpaired (13 months) and aged‐HP (13 months) retinas. The Y‐axes indicate the percentage (the left axis) and number (the right axis) of DEPs. (c) KEGG pathway enrichment analysis of DEPs between aged unpaired (13 months) and aged‐HP (13 months) retinas. The numbers of DEPs in a specific pathway and corresponding p‐values are shown next to each specific bar. (d–e) A venn diagram showing upregulated (d) or downregulated (e) DEPs among young unpaired/young‐HP, aged/aged‐HP, and aged unpaired/young unpaired retinas. (f–g) Validation of TPM1 by western blot analysis (f) and quantification of TPM1 protein (g) in young unpaired, young‐HP, aged unpaired, and aged‐HP retinas 2 and 4 months after parabiosis. Data are presented as mean ± SEM (n = 5 mice in each group) and analyzed by one‐way ANOVA with Tukey's multiple comparison test (young vs. young‐HP, *p < 0.05, **p < 0.01; aged vs. aged‐HP, **p < 0.01)