Fig 2: Lysosomal changes caused by PGRN deficiency in the sPLA2-IIA expressing background.(A, B) Immunostaining of Cathepsin D (CathD), LAMP1, and IBA1 in brain sections from 2-month-old Pla2g2a+/+ and Pla2g2a+/+ Grn−/− mice. Representative images from the cortex were shown. CathD and LAMP1 signals in IBA1-positive microglia were quantified. 3 sections per mouse brain and 3 mouse brains were used for each genotype. 10–12 cells per section were analyzed. Scale bar =10 µm. n=3, **, p<0.01. (C) Immunostaining of CathD, GFAP, and NeuN in brain sections from 2-month-old Pla2g2a+/+ and Pla2g2a+/+ Grn−/− mice. Representative images from the cortex were shown. Similar results were obtained from 3 mouse brains. Scale bar=8µm

Fig 3: The levels of sPLA2-IIA are upregulated in FTLD patients with GRN mutations.(a) sPLA2-IIA levels were measured in the lysates from postmortem human brain tissues from healthy controls and FTLD with GRN mutations or Tau aggregation using ELISA. Data are presented as mean ± SEM from 4–6 cases per group (n=4–6). *, p<0.05, Student’s t-test. (b) Anterior orbital gyrus tissues from healthy controls and FTLD-GRN patients were immunostained with anti-sPLA2-IIA and GFAP antibodies. Representative confocal images from 3 different cases for each genotype were shown (Scale bar=10 µm).

Fig 4: Gene expression changes caused by PGRN deficiency in the sPLA2-IIA expressing background.(A, B) Total RNAs were extracted from the cortex of 5-month-old Pla2g2a+/+ and Pla2g2a+/+Grn−/− mice and the RNA-seq was performed to analyze gene expression changes (n=3). Differential expressed genes (DEGs) with FDR P ≤ 0.05, absolute Log2FC ≥ 0.5 between Pla2g2a+/+ and Pla2g2a+/+Grn−/− groups are plotted using Heatmapper (A; Upregulated in Grn−/−; B: downregulated in Grn−/−). Inflammation genes and lysosomal genes are highlighted in red and green, respectively. (C) GSEA pathway analysis of the upregulated pathways with NES (normalized enrichment score)>1.0, NOM p-val (nominal p-value)<0.05, and FDR q-val (false discovery rate q value)<0.25 in RNA samples from the cortex of Pla2g2a+/+ Grn−/− mice compared to these from Pla2g2a+/+ mice.

Fig 5: Physical interactions between PGRN and sPLA2-IIA.(A) Schematic illustration of the SILAC experiment searching for PGRN interactors in HEPG2 cells. Proteins with more than 4 peptides and p value <0.01 and Log2 (GFP/GFP-PGRN) ratio <−1 were labeled in green. (B) Human PGRN and sPLA2-IIA interact when overexpressed in HEK293T cells. Human sPLA2-IIA-myc and human PGRN expressing constructs were transfected into HEK293T cells as indicated. Lysates and conditioned medium were prepared 3 days later and immunoprecipitated with anti-Myc antibodies. (C) Mouse-sPLA2-IIA-myc and FLAG-mouse-PGRN expressing constructs were transfected into HEK293T cells as indicated. Lysates and conditioned medium were prepared 3 days later and immunoprecipitated with anti-Myc antibodies. (D) Human PGRN interacts with human sPLA2-IIA through granulin F and G. Human-sPLA2-IIA-myc and GFP-PGRN / granulin constructs were transfected into HEK293T cells as indicated. Lysates were prepared 2 days later and immunoprecipitated with GFP-Trap. (E) Diagram of PGRN structure. (F) Deletion of granulin F and G reduces the interaction between PGRN and sPLA2-IIA. Untagged human sPLA2-IIA and Flag-PGRN / Flag-PGRNΔGF constructs were transfected into HEK293T cells as indicated. Lysates and conditioned medium were collected 2 days later and immunoprecipitated with anti-FLAG antibodies.