Fig 2: Reduced phagocytosis upon TREM2 deficiency in contrast to enhanced phagocytosis and clustering around amyloid plaques in the absence of PGRN AFlow cytometric analysis of phagocytic capacity in N9 Trem2 wt and N9 Trem2 mut cells using pHrodo green E. coli as target particles. Phagocytosis was terminated after 45 min of incubation. Data are presented as mean percentage of cells positive for pHrodo uptake ± SD (n = 3).BFlow cytometric analysis of phagocytic capacity of cultured primary microglia from wild-type, Grn -/-, and Trem2 -/- mice (2 months of age, male) using pHrodo green E. coli as target particles. Phagocytosis was terminated after 45 min of incubation. Left panel: Data are presented as mean percentage of CD11b+ cells positive for pHrodo uptake ± SD (three biological replicates). Right panel: Data are presented as mean percentage of CD11b+ cells positive for pHrodo uptake ± SD (two biological replicates).CPercentage IBA1-positive microglia within given plaque distance quantified from immunohistochemical stainings shown in Fig 3E. Data are normalized to APPPS1/Grn +/+ mice (n = 4; 4-month-old; two males and two females per genotype). Data are shown as mean ± SD.Data information: For statistical analysis, unpaired two-tailed Student's t-test was performed (A, B) between TREM2 or PGRN-deficient microglia against wild-type microglia, and the Mann–Whitney U-test, two-tailed analysis (C) was performed between genotypes. Significance is indicated by *P < 0.05.Source data are available online for this figure.

Fig 3: Association of CSF PGRN with mutation status, age and gender CSF PGRN is increased in mutation carriers (MC) compared to non-carriers (NC).CSF PGRN is not associated with age in either NC or MC.CSF PGRN is increased in males compared to females.CSF PGRN levels do not differ among MC participants carrying a PSEN1, PSEN2 or APP mutation.Data information: The blue or red bars in (A), (C) and (D) represent the mean and the standard deviation (SD). Group comparisons were assessed by a linear model adjusting by age, gender and APOE e4 status. The solid lines in (B) indicate the regression line for each of the groups and the 95% confidence interval (CI) calculated by a linear model adjusting by gender and APOE e4 status. The standardized regression coefficients (ß) and the P-values are also shown. In graph (B), the individual values are not shown in order to protect participants' confidentiality. All analysis and graphs are performed excluding 3 PGRN values outliers. Including the outliers in the analysis rendered similar results (Appendix Table S1). APP, amyloid precursor protein; CSF, cerebrospinal fluid; ns, non-significant; PSEN1, presenilin 1; PSEN2, presenilin 2.

Fig 4: Model of intracellular processing of PGRN into stable, lysosomal GRNs. Sortilin and other receptors target endocytosed and newly synthesized PGRN to lysosomes. Within lysosomes, PGRN is proteolytically cleaved, in part, by cysteine proteases (i.e., cathepsin L) into mature, stable GRN proteins. Ablation of sortilin results in the reduced production of GRNs. Further, lysosome dysfunction caused by alkalizing agents or TMEM106B overexpression inhibits the processing of PGRN into GRNs. FTD-GRN patients are haploinsufficient for GRNs, which may drive lysosome dysfunction leading to neurodegeneration.

Fig 5: FTD patients with a GRN mutation (FTD-GRN) are haploinsufficient for multiple GRNs. A, Primary fibroblast lysates from three control and three FTD-GRN patients were analyzed for PGRN and GRNs by immunoblot. B-D, Quantification of (B) PGRN, (C) GRN-2,3 (Sigma), and (D) GRN-4 (Novus) from the experiment in A. E, Full-blot images of soluble brain lysates from five control and five FTD-GRN patients using Sigma or Novus anti-GRN antibodies showing predominant nonspecific protein detection (denoted by *). F, The same samples from E were run on SDS-PAGE and analyzed for mature GRNs by immunoblot as outlined in Materials and Methods. Immunoblots for insoluble p62 indicate disease-associated pathology in the FTD-GRN cases. G-I, Quantification of (G) GRN-2,3 (Sigma) and (H) GRN-4 (Novus) from the experiment in F. I, Quantification of PGRN in the same soluble brain lysates from F by ELISA. All data represent the mean ± SEM; *differs from control p < 0.05; **p < 0.01; ***p < 0.001.