Figure 4.

Enlarged early endosomes in SORL1 het Göttingen minipigs

(A) Paraffin sections of cortical brain regions from SORL1 wt (338593 and 6475) and SORL1 het (6470 and 6469) minipigs immunolabeled with Rab5 antibody to identify early endosomes, occurring as a brown signal from 3,3′-diaminobenzidine (DAB) detection of the horseradish peroxidase (HRP)-conjugated secondary antibody.

(B) High-magnification images from bright-field microscopy of early endosome compartments (arrowheads), showing an increase in Rab5-positive structures in neurons from the het compared with the wt SORL1 minipig.

(C) The mean area of Rab5-positive (early endosome) structures was measured across neurons from wt (mean area = 0.1640 μm² ± 0.009565, n = 27 cells) and het (mean area = 0.4887 μm² ± 0.002815, n = 27 cells) minipig Cx areas as depicted in (B).

(D) Immunofluorescence confocal images showing SORLA expression in cultured fibroblasts from wt (6475) and het (6469) SORL1 minipigs, demonstrating lower expression levels in cells from SORL1 het minipigs, as seen by fewer positive signals.

(E) Endosome abnormalities were also present in fibroblasts from SORL1 het minipigs, as visualized by Rab5-positive structures with increased size.

(F) The mean area of Rab5-positive (early endosome) structures was measured for cultured primary fibroblasts from wt (mean area = 0.1862 ± 0.01223 μm², n = 21 cells) and het (mean area = 0.2771 ± 0.01566 μm², n = 21 cells) SORL1 minipigs, as depicted in (E).

Scale bars equal 50 μm (A) and 10 μm (B and D). Two-tailed unpaired Student’s t test was used for all statistical analyses, with p values below 0.05 considered significantly changed. Data are expressed as mean ± SEM.