Fig 2: PGRN activated PI3K/Akt/GSK-3ß signalling pathway and the expression of M6PR, SORT1 and PSAP in VPA-induced rats at PND35.Representative Western blots (A, C) and densitometric quantification (B, D) of p-Akt (Ser473), Akt, p-GSK-3ß (Ser9) and GSK-3ß expressions in the cerebellum. Representative Western blots (E) and densitometric quantification (F–H) of M6PR, SORT1 and PSAP. Data are expressed as mean ± SEM. One-way ANOVA followed by Tukey’s post hoc (B, D); Student’s t test (F, H); Mann–Whitney test (G). (*P < 0.05, **P < 0.01, ***P < 0.001, sample sizes (n): n = 5/group).

Fig 3: Prenatal exposure to VPA caused the alterations of PGRN temporal expression in the cerebellum.Representative blots (A) and quantification (B) showed the expression of PGRN at four different time points. Representative images (bar = 100 µm) (C) and quantification (D) of immunofluorescence staining described that the expression of PGRN is in the same trend as Western blotting. E PGRN ELISA protein quantitation of cerebellar tissue. Data are expressed as mean ± SEM. Two-way ANOVA followed by Sidak post hoc. (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, sample sizes (n): n = 5/group for Western blotting and immunofluorescence staining. n = 4/group for ELISA).

Fig 4: r-PGRN improved the defection of behaviours, and PI3K inhibitor wortmannin blocked this effect in VPA-induced rats.Cerebellum-related behaviours: righting reflex (A), static beams (B), gait analysis (C), traction test (D). Autism-like behaviours: open field test (E), self-grooming test (F), juvenile social play (G), and the three-chamber social test (H). Data are expressed as mean ± SEM. One-way ANOVA followed by Tukey’s post hoc (B(Proportion of Trials), C(Fore, Hind, Gait), D(Traction score), E(All grid, Faeces), F, G(Social), H(10 min: Stranger1, A chamber, B chamber), H(20 min: A chamber, B chamber)); post hoc Kruskal–Wallis test for multiple comparisons (A, B(Time to Complete, Footfalls per Trial), D(Hanging time), E(Centre grid, Climbing), G(Dig, Attack), H(10 min: Object, Centre chamber), H(20 min: Stranger1, Stranger2, Centre chamber)). (ns no significant difference, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, sample sizes (n): n (CON) = 13, n (VPA + Vehicle) = 11, n (r-PGRN) = 11, n (r-PGRN + Wort) = 11).

Fig 5: (A) Cortical cells fixed at 20 days in vitro and triple stained (merge, d) with glial fibrillary acidic protein (GFAP) (green, b) and Iba-1 (red, c), and Hoechst 33342 (blue, a). The scale bar represents 100 µm; (B) the numbers of GFAP- and Iba-1-positive cells were counted. The results are expressed as the percentage of these cells among the total number of Hoechst-positive cells and as the means ± SD (n = 7 independent experiments); (C) images of triple staining (merge, d and h) with Hoechst 33342 (blue, a and e), Iba-1 (red, b and f), and PGRN (green, c and g) under Normoxia (a–d) or OGD (e–h) treatment. The scale bar represents 30 µm; (D) the numbers of Iba-1- and PGRN-positive cells under normoxia (Nor) or OGD were counted. Five images were made per experiment, and 26–100 cells were counted per image, and the average of 5 images per experiment was calculated. The results are expressed as the percentage of these cells among the total number of Hoechst-positive cells (Iba-1/Hoechst) and that of Iba-1-positive cells (PGRN/Iba-1), and as the means ± SD (n = 9 independent experiments; the total number of Hoechst-positive cells counted: 2223 under normoxia and 2244 under OGD). * Significant difference from the normoxic group (p < 0.05); (E) Effects of Normoxia or OGD exposure on the expression of PGRN mRNA in microglia. The results are expressed as the mean ratio of the Normoxia or OGD to the control group ± SD (n = 8 independent experiments). * Significant difference from the normoxic group (p < 0.05); (F,G) Effects of Normoxia or OGD exposure on the expression levels of TNF-a (F) and IL-1ß (G) mRNA in microglia. The results are expressed as the mean ratio of the Normoxia or OGD to the control group ± SD (n = 8 independent experiments). * Significant difference from the normoxic group (p < 0.05).