Fig 2: Reactive astrogliosis and Müller cell gliosis in PGRN-deficient mice retinas. (A) Expression of GFAP (red) in 6-month-old Grn+/+ and Grn-/- mice retinas were assessed with immunostaining of retinal cross-sections (a,b) and retinal flat mounts (c,d). Nuclei were stained with Hoechst 33342 (blue). GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer. Scale bars, 50 µm. (B) Quantitative analysis of the GFAP-positive area in retinal flat mounts (Grn+/+, n = 3; Grn-/-, n = 3). (C) The expression level of GFAP in the retina of Grn+/+ and Grn-/- mice were evaluated by Western blotting (Grn+/+, n = 4; Grn-/-, n = 5). Data are the means ± SEMs. * p < 0.05, ** p < 0.01 vs. Grn+/+ mice (Welch’s t-tests).

Fig 3: PGRN processing in PSAP-deficient mice. (A, B) Western blot analysis of PGRN and granulin peptides in the cortical lysates from P21 WT and Psap-/- mice. The levels of PGRN and granulin peptides (GRNs) were quantified and normalized to GAPDH. Lysates from three mice were analysed for each genotype (n = 3), Student’s t-test, *P < 0.05, **P < 0.01.

Fig 4: Severe astrogliosis in the ventral thalamus and CA3 hippocampal region of aged GrnR493X/R493X mice. a Representative hippocampal/thalamic tilescans stained for Gfap show severe astrogliosis in 18 month old Grn+/+ and GrnR493X/R493X brain (scale bar, 500 µm). b Representative Gfap immunofluorescence images of hippocampal CA3 and thalamic VPM/VPL regions in 18 month old Grn+/+ and GrnR493X/R493X brain sections (scale bar, 20 µm). c Total Gfap-positive area in CA3 (i) and VPM/VPL (ii) images were quantified and normalized to the total number of cells (DAPI, blue). n = 10 mice were used per sex/genotype (except male GrnR493X/R493X n = 8); values are shown as mean ± SEM; ns = not significant, *p < 0.05, ***p < 0.0001, Student’s t-test

Fig 5: Nlk+/– Grn+/– mice display FTLD-like neuropathological phenotypes.(A–C) Increased microglial activation in Nlk+/– Grn+/– mice compared with their littermates. Representative confocal images (A) and quantification (B and C) of Iba1 and CD68 staining from the thalamus of 1-year-old mice. (B) The number of Iba1-positive microglia was increased in the thalamus of Nlk+/– Grn+/– mice. *P < 0.05, **P < 0.01, ***P < 0.001; 1-way ANOVA with Tukey’s multiple comparisons post hoc test; F(3,18)=10.72, P = 0.0003. (C) CD68-positive vesicle volume in Iba1-positive microglia was also increased in Nlk+/– Grn+/– mice. ****P < 0.0001; 1-way ANOVA with Tukey’s multiple comparisons post hoc test; F(3,19)=82.22, P < 0.0001. (D and E) Representative images (D) and quantification (E) of autofluorescence using 488 nm excitation in the retina of 1-year-old WT, Nlk+/–, Grn+/–, and Nlk+/– Grn+/– mice. *P < 0.05, **P < 0.01; 1-way ANOVA with Tukey’s multiple comparisons post hoc test; F(3,19)=8.327, P = 0.0010. (F and G) Representative images (F) and quantification (G) of 1-year old WT, Nlk+/–, Grn+/–, and Nlk+/– Grn+/– mouse retinas stained for Brn3a (green) and DAPI (blue). ****P < 0.0001; 1-way ANOVA with Tukey’s multiple comparisons post hoc test; F(3,40)=20.52, P < 0.0001.