Isotope-labeled amyloid-β does not transmit to the brain in a prion-like manner after peripheral administration (2022)
Figure 4

Figure 4 - Insoluble 13C-Lys Aβ is detectable in the brain after peripheral injection of both 13C-Lys APPtg and 13C-Lys non-tg brain extract due to incorporation of 13C-Lys

A Temporal MRM analysis of unlabeled Aβ17-28 and 13C-Lys Aβ17-28 in the soluble brain fraction after 13C-Lys APPtg brain extract i.p. injection (n = 3 mice per time point at 1-10 d, n = 4 mice per time point at 25-100 d).

B,C Comparative MRM analysis of 13C-Lys Aβ17-28 (B) and unlabeled Aβ17-28 (C) in the insoluble brain fraction after 13C-Lys-APPtg brain extract (donors labeled for 50-125 d) and 13C-Lys-non-tg brain extract (donors labeled for 10 d) i.p. injection (n = 3 mice per group and time point at 1-10 d, n = 4 mice per group and time point at 25, 50, 100 d, n = 3 mice in the APPtg extract group and n = 4 in the non-tg extract group at 75 d), right side zoom for time points 1-10 d. Unpaired two-tailed t-test * p<0.05 (P=0.0129 at 3 d, P=0.0156 at 10 d, P=0.0109 at 25 d), ** p<0.01 (P=0.0091 at 1 d, P=0.0011 at 5 d), *** p<0.001 (P=0.0006 at 7 d), 13C-Lys APPtg extract-injected vs 13C-Lys non-tg extract-injected for each time point.

D Comparative 4G8 immunoassay quantification of Aβ42 in the insoluble brain fraction after 13C-Lys APPtg or 13C-Lys non-tg brain extract i.p. injection (n = 3 mice per group and time point at 1-10 d, n = 4 mice per group and time point at 25-100 d), right side zoom for time points 1-10 d. Normalization to protein concentration.

E Representative chromatograms of unlabeled Aβ17-28 (left) and 13C-Lys Aβ17-28 (right) in the brain of an unlabeled 50-day-old APPtg mouse not injected with labeled brain extract. Note the absence of 13C-Lys Aβ17-28.

F MRM analysis of unlabeled and 13C-Lys-labeled Aβ17-28 in APPtg mouse brains after 13C-Lys-labeled diet for 1 or 3 d (n = 3 mice per time point).

G Comparative MRM analysis of 13C-Lys Aβ17-28 in the insoluble brain fraction after 13C-Lys APPtg and 13C-Lys non-tg brain extract (all donors labeled for 100 d) i.p. injection (n = 6 mice per group).

H MRM analysis of unlabeled and 13C-Lys-labeled Aβ17-28 in non-APPtg mouse brains after 13C-Lys-labeled diet for 1 d (n = 3 mice) or at 1 d after 13C-Lys-APPtg brain extract i.p. injection (n = 3 mice).

Data information: In (A-C) and (F-H), measurements in technical triplicates, normalization to 13C15N-Lys Aβ17-28 intensity as a spiked internal standard and brain weight (mg). All error bars show mean ± SD.

Table 1 - 13C-Lys-labeling of APPtg donor brains for 13C-Lys APPtg brain extract generation

Animal ID number, age at death, age during which 13C-Lys-labeled diet was fed, amount of Aβ42 per mg brain, ratio between 13C-Lys Aβ17-28 and unlabeled Aβ17-28, labeling efficiency in %, cohort for 13C-Lys APPtg brain extract injection.

Table 2 - 13C-Lys brain extract injection groups

n = number of animals, genotype of injected animals, injected brain extract, number of days during which the donors of brains used for extract generation had received the 13C-Lys diet, sacrifice time point after injection, age at death.
Figure EV1 - Seeding activity of Aβ42 fibrils extracted from APPtg brain extract

A ThT fluorescence aggregation kinetics of recombinant Aβ42 in the presence of extracted fibrils from APPtg brain extract at different concentrations from 83 nM to 1,660 nM, exhibiting a concentration-dependent seeding effect.

B ThT fluorescence aggregation kinetics in the presence of equal amounts of extract from non-tg brain extract, revealing no seeding activity but a delaying effect on the aggregation kinetics.

C, D TEM images showing seeded Aβ42 fibrils at the aggregation kinetic end points using 166 nM seeds (C), and 1,660 nM seeds (D). The fibril morphology is heterogenous.

E In vitro Aβ42 fibrils aggregated without the presence of seeds. The fibril morphology is more homogenous compared to the seeded Aβ42 fibrils with brain extract.

Data information: In (A) and (B), the same curve is shown for Aβ42 aggregated without fibril extract. In (C-E), scale bars correspond to 500 nm.