Journal: Annals of Clinical and Translational Neurology

Article Title: Elevated levels of extracellular vesicles in progranulin‐deficient mice and FTD‐ GRN Patients

doi: 10.1002/acn3.51242

Figure Lengend Snippet: Elevated Levels of Brain EVs in 12‐ to 13‐month‐old Grn –/– Mice. Brain EVs were isolated from wild‐type, Grn +/– , and Grn –/– littermates, and the levels of EVs in fraction 2 were compared using several methods. A, Nanoparticle tracking analysis revealed more vesicles of exosomal size in Grn –/– mice than wild‐type (RM ANOVA genotype x particle size interaction, P < 0.0001, * P < 0.05 by Dunnett’s post hoc test). B, This increase in exosome‐sized vesicles persisted when corrected for hemibrain weight in Grn –/– mice (ANOVA effect of genotype, P = 0.0133, ** P = 0.0070 by Dunnett’s post hoc test). C, Fraction 2 from Grn –/– mice also contained more total protein than wild‐type mice (ANOVA effect of genotype, P = 0.0040, ** P = 0.0021 by Dunnett’s post hoc test). Finally, fraction 2 from Grn –/– mice contained significantly more HSP‐70 (D, ANOVA effect of genotype, P = 0.0206, * P = 0.0138 by Dunnett’s post hoc test) and trended toward having higher levels of CD81 (E, ANOVA effect of genotype, P = 0.0562) and flotillin‐1 (F, ANOVA effect of genotype, P = 0.0857) than wild‐type. G, The other fractions contained undetectable levels of these proteins. All data are corrected for hemibrain weight except for the nanoparticle tracking profiles in A. n = 10–13 mice per genotype. H = brain homogenate.

Article Snippet: Nanoparticle tracking profiles (concentration by particle size) were analyzed by repeated measures ANOVA with factors of particle size and genotype (or patient group) using GraphPad Prism 8.

Techniques: Isolation

Journal: Annals of Clinical and Translational Neurology

Article Title: Elevated levels of extracellular vesicles in progranulin‐deficient mice and FTD‐ GRN Patients

doi: 10.1002/acn3.51242

Figure Lengend Snippet: Elevated Levels of EVs in Frontal Cortex of Patients with FTD‐ GRN . EVs were isolated from frozen post mortem samples of inferior frontal gyrus from controls (n = 5) and patients with FTD‐ GRN (n = 13) as shown in Figure . A, B, As with mouse brain samples, fraction 2 was enriched for EV marker proteins and total protein content. C, D, Fraction 2 from post mortem samples contained vesicles of typical EV morphology under transmission electron microscopy. E, Nanoparticle tracking analysis revealed vesicles of the size for exosomes and microvesicles, although there was not an overall difference in vesicle concentration between FTD‐ GRN patients and controls (E, RM ANOVA effect of group, P = 0.51). However, levels of the EV marker proteins HSP‐70 (F, Mann‐Whitney test, P = 0.0396) and CD81 (G, Mann‐Whitney test, P = 0.046) and were elevated in fraction 2 from FTD‐ GRN patients. Representative blots are shown in I. All data are corrected for slice weight except for the nanoparticle tracking profiles in E. Independent images of representative EVs at different magnifications are shown in C and D with 100 nm scale bars.

Article Snippet: Nanoparticle tracking profiles (concentration by particle size) were analyzed by repeated measures ANOVA with factors of particle size and genotype (or patient group) using GraphPad Prism 8.

Techniques: Isolation, Marker, Transmission Assay, Electron Microscopy, Concentration Assay, MANN-WHITNEY

Journal: Annals of Clinical and Translational Neurology

Article Title: Elevated levels of extracellular vesicles in progranulin‐deficient mice and FTD‐ GRN Patients

doi: 10.1002/acn3.51242

Figure Lengend Snippet: Elevated Levels of Plasma EVs in Patients with FTD‐ GRN . EVs were isolated by differential centrifugation of frozen plasma samples from controls (n = 8), presymptomatic GRN carriers (n = 7), and symptomatic GRN patients (n = 12). A, EV pellets contained EV marker proteins, but lacked markers for other organelles (Grp94 – ER/Golgi, cytochrome C – mitochondria, histone H3 – nucleus). Lysate from HEK‐293 cells (HEK) was used as a positive control for organelle markers. B and C Plasma EVs exhibited typical morphology under transmission electron microscopy (scale bars = 100 nm). D, Nanoparticle tracking analysis revealed that plasma from symptomatic GRN patients contained significantly more particles of exosomal size than controls and presymptomatic GRN carriers (RM ANOVA effect of group, P = 0.0447, group x size interaction, P < 0.0001, * P = 0.0415 by Dunnett’s post hoc test, E, ANOVA effect of group, P = 0.0162, * P < 0.05 by Dunnett’s post hoc test). F–I, Similarly, western blot revealed elevated levels of the EV marker proteins CD9 (F, Kruskal‐Wallis test effect of group, P = 0.0021, ** P < 0.01 by Dunn’s post hoc test) and flotillin‐1 (G, Kruskal‐Wallis test effect of group, P = 0.0182, * P < 0.05 by Dunn’s post hoc test) in symptomatic GRN patients compared to both controls and presymptomatic GRN carriers. C is an enlarged portion of B, as shown by dashed lines.

Article Snippet: Nanoparticle tracking profiles (concentration by particle size) were analyzed by repeated measures ANOVA with factors of particle size and genotype (or patient group) using GraphPad Prism 8.

Techniques: Clinical Proteomics, Isolation, Centrifugation, Marker, Positive Control, Transmission Assay, Electron Microscopy, Western Blot