Fig 2: Treatment by recombinant TPM1 protein induces age-related alternations in young mice. (a) Schematic diagram of the experimental design. Young C57BL/6J mice (3-month-old) received a total of ten retro-orbital injections of recombined TPM1 protein (200 µg/kg), 3 days apart, and retinas were collected for further studies at day 30. (b) Representative confocal images of RBCs (top row) and HCs (bottom row) in retinal sections from young mice after rTPM1 protein or PBS 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 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. (e) Retina sections stained with Iba-1and CD68. White arrows show CD68-negative microglia, red arrowhead indicates CD68-positive microglia, and red arrow shows the dendritic extension of activated microglia to the ONL. Scale bars, 20 µm. (f) Retinal sections stained with GFAP antibody. White arrow shows resting astrocytes, red arrows indicate activated astrocytes, and red arrowheads show activated Müller cells. Scale bars, 20 µm. (g) Double-staining of whole-mounted retinas from young mice after rTPM1 protein or PBS treatment with Iba-1 and CD68 antibodies. 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 retinas. 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-1ß, IL-6, and TNF-a in retinas from young mice after rTPM1 protein or PBS treatment. Data are presented as mean ± SEM, n = 4 mice in each group, unpaired two-tailed Student's t test. (m–n) ERG responses on young mice after rTPM1 protein or PBS treatment. Data are presented as mean ± SEM, n = 11 mice in each group, unpaired two-tailed Student's t test

Fig 3: TPM1 upregulation and age-related retinal alternations in mouse models of Alzheimer's disease. (a–b) Western blot analysis (a) and quantification of TPM1 (b) in C57BL/6J (WT) and 5xFAD retinas. Data are presented as mean ± SEM, n = 5 mice in each group, unpaired two-tailed Student's t test. (c) Retina sections from WT and 5xFAD retinas were stained with Iba-1 and CD68. White arrows indicate CD68- microglia. Red arrowhead shows CD68+ microglia, and red arrow indicates the dendritic extension of activated microglia. Scale bars, 20 µm. (d) Retina sections from WT and 5xFAD retinas were stained with GFAP. White arrow shows resting astrocytes. Red arrows show activated astrocytes, and red arrowhead indicates activated Müller cells. Scale bars, 20 µm. (e) Confocal images of RBCs (top row) and HCs (bottom row) in retinal sections from WT and 5xFAD mice. Arrowheads indicate the aberrant sprouting of RBCs and HCs. Scale bars, 10 µm. (f-g) Quantification of the aberrant dendritic length of RBCs and HCs. Data are presented as mean ± SEM, n = 4 mice in each group, unpaired two-tailed Student's t test. (h–i) Western blot analysis (h) and quantification of TPM1 (i) in WT and TgCRND8 retinas. Data are presented as mean ± SEM, n = 5 mice in each group, unpaired two-tailed Student's t test. (j) Retina sections from WT and TgCRND8 retinas were stained with Iba-1 and CD68. White arrow indicates CD68- microglia. Red arrowheads show CD68+ microglia, and red arrows indicate the dendritic extension of activated microglia. Scale bars, 20 µm. (k) Retina sections from WT and TgCRND8 retinas were stained with GFAP. White arrow shows the resting astrocytes. Red arrows show activated astrocytes, and red arrowheads indicate activated Müller cells. Scale bars, 20 µm. (l) Confocal images of RBCs (top row) and HCs (bottom row) in retinal sections from WT and TgCRND8 mice. Arrowheads indicate the aberrant dendritic sprouting of RBCs and HCs. Scale bars, 10 µm. (m–n) Quantification of the aberrant dendritic length of RBCs and HCs. Data are presented as mean ± SEM, n = 4 mice in each group, unpaired two-tailed Student's t test

Fig 4: 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)

Fig 5: Gene expression of 10 genes (MYC, VEGFA, ACTA2, CDH1, CCNB1, FLNA, MCM5, MAD2L1, TAGLN and TPM1) based on GEPIA (*p < 0.05).