Fig 2: Elastase-mediated removal of C-terminal motif of PGRN blocks PGRN endocytosis by SORT1. (A) A synthetic PGRN574–593 peptide was enzymatically cleaved by recombinant elastase (EL) in a time-dependent manner as analyzed by MS-MALDI analysis. The PGRN574–593 peptide with a molecular weight of 2430 Da was processed into a product with 1806Da indicating the last five residues were removed by EL as shown schematically in (B). (C) WT or EL-site-mutated A588G-rPGRN protein in cell culture media was set up to react with EL in vitro. After reaction, the media was used as input to carry out a cellular endocytosis assay in M17 cells overexpressing SORT1. PGRN endocytosed in cells was detected in cell lysate after treatment (uptake). Unlike WT rPGRN, A588G-rPGRN was more resistant to EL activity as shown by the amount of residual full-length protein in the medium (input), which was endocytosed by cells to a similar extent compared with no EL control. Immunocytofluorescence analysis showed that only full-length rPGRN (FL) (E) but not the carboxyl-terminal-truncated PGRN1-588 (F) was significantly endocytosed by M17SORT1 cells. (D) The untreated M17SORT1 control was included. PGRN and SORT1 were labeled in red and green, respectively.

Fig 3: Schematic diagram summarizing the strategies applied to inhibit SORT1-mediated endocytosis in the current study. (A) Under normal conditions, extracellular PGRN interacts with the β-propeller tunnel structure of SORT1 using its C-terminal end binding motif as shown in red color. SORT1 facilitates endocytosis of exPGRN and directs it to the endolysosomal pathway for degradation. (B) High-affinity SORT1 ligands such as NTS or the PGRN(588–593) peptide competitively limits the access of exPGRN to SORT1-binding sites, thereby inhibiting PGRN endocytosis. (C) To improve target specificity, we have also identified a small-molecule binder, BVFP, targeting the PGRN(588–593) motif that is essential for PGRN–SORT1 interaction. We demonstrated that pretreatment of BVFP to rPGRN significantly reduced the amount of rPGRN captured by SORT1 in vitro and inhibited SORT1-mediated rPGRN endocytosis. (D) Suppressors of SORT1 expression, such as MPEP, reduce SORT1-mediated endocytosis, thereby increasing extracellular PGRN levels. The above-mentioned strategies, used alone or in combination with others, are potential avenues for discovery of SORT1-dependent PGRN enhancers for the treatment of FTD-GRN.

Fig 4: Sort1 deficiency impaired WAT formation in female Ldlr−/− mice fed a HF/HC diet. (a) Representative hematoxylin and eosin stained sections (example of a white adipocyte outlined by dashed red line; scale bars indicate 100 μm), quantified adipocyte size distribution, and average size of white adipose tissue (WAT) adipocytes in female Ldlr−/−Sort1+/+ and Ldlr−/−Sort1−/− mice fed a high-fat/cholesterol (HF/HC) diet for 15 weeks; (n = 5–6 mice/group). (b) Female mice on a HF/HC diet for 15 weeks mRNA levels of genes related to white adipocyte formation in WAT, and in (c) primary white adipocytes differentiated from female mouse WAT; (n = 3–6 mice/group). (d) WAT Adipoq mRNA levels (n = 5–6 mice/group), and (e) plasma Adiponectin protein concentration in female Ldlr−/−Sort1+/+ and Ldlr−/−Sort1−/− mice fed a HF/HC diet for 15 weeks; (n = 10 mice/group). *P < 0.05, **P < 0.01, ***P < 0.001 versus Ldlr−/−Sort1+/+, analyzed by t-test; values are presented as mean ± SEM.

Fig 5: Sort1 deficiency reduced body and WAT weight in female Ldlr−/− mice. (a) Body weight in grams (g), (b) body weight gain, and (c) WAT weight ratio for Ldlr−/−Sort1+/+ and Ldlr−/−Sort1−/− mice after 15 weeks (15w) on normal chow (NC) or high-fat/cholesterol diet (HF/HC); (n = 5–10 mice/group). (d) Confirmation of Sortilin protein deficiency in 15-week HF/HC-diet fed female Sort1−/− mice WAT and brown adipose tissue (BAT); (n = 5 mice/group). GAPDH loading controls were run on the same blot, and the membrane was cut prior to incubating with primary antibody. Full-length blots are presented in Supplemental Fig. S4a and b. *P < 0.05, **P < 0.01, ***P < 0.001 versus sex and diet matched Ldlr−/−Sort1+/+ mice, analyzed by t-test; values are presented as mean ± SEM.