Fig 2: Enhanced phagocytosis, migration, and clustering around amyloid plaques upon PGRN deficiency AFlow cytometric analysis of phagocytic capacity in BV2wt and BV2mut cells using pHrodo green E. coli as target particles. Phagocytosis was terminated after 30 and 60 min (min) of incubation. Data are presented as mean percentage of cells positive for pHrodo uptake ± SD (n = 3).BEnhanced migration of CD68-positive, PGRN-deficient cells in an ex vivo model. Immunofluorescence analysis of cultured Grn +/+ and Grn -/- organotypic brain at 7 DIV immunostained using microglial marker CD68. Nuclei were counterstained using DAPI. Images of boxed regions in left panels are shown at higher magnifications in right panels. Scale bars: 500 µm (left panels), 250 µm (right panels).CQuantitative analysis reveals that Grn -/- microglia migrate larger distances than Grn +/+. Data are shown as mean ± SD (n = 3 independent experiments).DQuantification displays an increased number of migrating Grn -/- cells compared to Grn +/+ cells. Data are shown as mean ± SD (n = 3 independent experiments).EEnhanced microglial clustering around amyloid plaques in the absence of PGRN. Left: IBA1-stained microglial clustering around X-34-positive amyloid plaque cores in 4-month-old APPPS1/Grn +/+ mice. Right: Age-matched APPPS1/Grn -/- mice display microglial hyper-clustering around amyloid plaques compared with APPPS1/Grn +/+. Dotted white boxes indicate the area that is shown at higher magnification. Scale bars indicate 10 and 3 µm in inset.FImmunohistochemical quantification of IBA1-positive microglial cell number per plaque (APPPS1/Grn +/+ n = 4, APPPS1/Grn -/- n = 4; 4-month-old; two males and two females per genotype) normalized to APPPS1/Grn +/+ mice. Approximately 20–50 amyloid plaques were counted per mouse in 3 cortical sections to calculate the number of IBA1-positive microglia per plaque. Data are shown as mean ± SD.Data information: For statistical analysis, unpaired two-tailed Student's t-test was performed (A, C, D) between PGRN-deficient Grnmut BV2 cells or Grn -/- microglia and Grn wt BV2 cells or Grn +/+ microglia. For (F) the Mann–Whitney U-test, two-tailed analysis was used. Significance is indicated by *P < 0.05; **P < 0.01; and ***P < 0.001.Source data are available online for this figure.

Fig 3: 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 4: CSF PGRN levels across the Alzheimer's continuum Scatter plot representing the levels of CSF PGRN in healthy controls (depicted in blue) and the different stages of the Alzheimer's continuum (depicted in red). The blue and the red bars represent the mean and the standard deviation (SD). The analysis and graphs were performed excluding CSF PGRN outliers (1 “healthy control”, 1 “Preclinical AD A+/TN-”, 4 “AD CDR = 0.5” and 1 “AD CDR = 1”). Including them yielded a similar result, and CSF PGRN was still significantly higher in the “AD CDR = 1” group compared to the “healthy controls” (P = 0.001) and “Preclinical AD A+/TN-” (P = 0.0001) groups. P-values were assessed by a one-way analysis of covariance adjusted for age, gender and APOE e4, followed by Bonferroni corrected pair-wise post hoc comparisons. A: amyloid-ß biomarker status; AD: Alzheimer's disease; CDR: clinical dementia rating; CSF, cerebrospinal fluid; N, neurodegeneration biomarker status; T: tau pathology biomarker status.

Fig 5: Diagnostic accuracy of CSF PGRN as an AD biomarkerROC analysis was performed to test the accuracy to discriminate between ADAD mutation carriers and non-carriers of the DIAN study (A) and late-onset AD with mild dementia (CDR = 1) from healthy controls of the ADNI study (B). The areas under the curve (AUCs), their 95% confidence interval (CI) intervals and the P-values are reported. Optimal cut-offs were derived based on the Youden index, and the sensitivity and specificity were calculated based on these cut-offs. AD: Alzheimer's disease; ADAD: autosomal dominant Alzheimer's disease; AUC: area under the curve; CDR: clinical dementia rating; HC: healthy controls; MC: mutation carriers; NC: non-carriers.