Huntingtin Co-Isolates with Small Extracellular Vesicles from Blood Plasma of TgHD and KI-HD Pig Models of Huntington’s Disease and Human Blood Plasma

Figures

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  Figure 1. Experimental scheme.

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  Figure 2. Characterization of the morphology and size of extracellular vesicles. (A) TEM confirmed presence of spherical vesicles with diameter smaller than 200 nm in the ultracentrifugation pellets from porcine and human plasma (representative image from 20 samples analyzed by TEM is shown). Magnification 100,000×. (B) Characterization of particle size by NTA. Representative result from 46 samples analyzed by NTA is shown. Error bars (red) indicate ±1 standard error of the mean (see Table S1 for detailed data). (C) Nanoscale flow cytometry with fluorescent CFSE labelling was used to assess size of particles of biological origin in ultracentrifugation pellets from plasma. The most frequent particle size category was 110 nm. Dots represent individual samples, bars represent mean of % of total CFSE+ particles in individual genotype and size groups, error bars show standard error of the mean (see Table S1 for detailed data).

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  Figure 3. Huntingtin and exosome marker detection in 100,000× g centrifugation pellets containing extracellular vesicles, isolated from blood plasma of the transgenic Huntington´s disease pig model. (A) anti-N-terminal HTT antibody (EPR5526) was used to detect total HTT levels in plasma, 10,000× g pellets and 100,000× g pellets. Wild-type pig brain homogenate was used as positive control. Endogenous full length ~360 kDa HTT was detectable in 10,000× g and 100,000× g pellets from plasma of both wild type (wt) and transgenic (TgHD) pigs (arrow E). In addition, the ~110 kDa human HTT transgene (arrow T) and a ~70 kDa fragment (arrow F) were detectable in TgHD pig 100,000× g pellets. (B) A polyQ-specific antibody (MW1) provided the same ~110 and ~70 kDa band pattern in TgHD pig 100,000× g pellets as EPR5526, thus confirming the presence of mutant HTT (arrows). Additional polyQ-containing protein bands were detected in 100,000× g pellets (asterisks). (C) Expression of exosome markers Alix, TSG101 and CD9 were abundant in 100,000× g pellets containing extracellular vesicles, in contrast to 10,000× g pellets and original plasma (sample IDs are identical as in panel (A)).

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  Figure 4. Huntingtin and exosome marker detection in fractions from density gradient ultracentrifugation. The 100,000× g pellet from the T131 transgenic pig plasma (sample labeled “+”) was overlaid by 40% to 5% OptiPrep and separated by gradient ultracentrifugation. Seventeen fractions (1 mL each) were collected from top of the tube. The collected fractions were subjected to SDS-PAGE and western blotting to detect total huntingtin (A), mutant huntingtin (B) and exosome markers Alix, TSG101 and CD9 (C). Huntingtin appeared in fraction 13 with density 1.111–1.134 g/cm3 corresponding to exosomes. Fraction 13 was also positive to exosome markers. Arrows indicate ~360 kDa endogenous huntingtin (E), ~110 kDa huntingtin transgene (T) and ~70 kDa huntingtin fragment (F). The extracellular vesicles in fraction 13 macroscopically appeared as an opalescent band in the ultracentrifugation tube (arrow in panel (D). Representative results from 5 samples fractionated by gradient ultracentrifugation are shown (see Figures S8–S12 for full data).

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  Figure 5. Normalized intensity of total huntingtin in extracellular vesicles isolated from blood plasma of TgHD and KI-HD pig models and human plasma. EVs were isolated by ultracentrifugation and HTT forms were detected by western blot using anti-N-terminal HTT antibody recognizing both normal and mutant HTT. HTT band intensities were quantified by ImageLab software and normalized to CD9 levels to minimize variability in EV loading amount. Significantly higher total HTT content was found in vesicles from TgHD pigs compared to WT control pigs (A), while in KI-HD (B) and HD patients (C) the increase in total HTT in EVs compared to controls was not significant. The dots represent individual samples, bars show mean of the groups, and error bars indicate standard error of the mean. Statistical analysis using three-way ANOVA with genotype and age and sex as factors, followed by the post-hoc Tukey HSD test was performed for pig samples. T-test was used to compare HTT intensity between HD patient and control groups.