(A) Schematic representation of the 3 chambers in which neurons were cultured in chamber 1 (C1) chamber 2 (C2) and chamber 3 (C3) (top) or C1 and C3 (bottom). (B) α-Syn PFF was added to C1 of the microfluidic device. On day 14, α-syn-biotin PFF was detected via P-α-syn in C2 and C3 when neurons were present in all three chambers. Transmission to C3 is not detectable when neurons are not present in C2. (C) Quantification of immunofluorescent images in B. Data are the means ± SEM, n = 3. one-way ANOVA followed by Sidak’s correction. ***P < 0.001 versus C1. Power (1-β err prob) = 1. Scale bars, 100 µm. (D) Schematic of microfluidic neuron device with three chambers to separate neurons seeded in three chambers. (E)Transmission of pathologic P-α-syn from chamber 1 (C1) to chamber 2 (C2) to chamber 3 (C3) 14 days post-addition of α-syn PFF in C1. The different combinations of neurons tested in C2, listed as C1-(C2)-C3, are: WT-(WT)-WT, WT-(WT+LAG3)-WT, WT-(LAG3−/−)-WT, WT-(LAG3−/−+LAG3)-WT. Scale bar, 10 µm. (F) Quantification of panel E. Values are given as means ± SEM, n = 3. Statistical significance was determine using one-way ANOVA followed by Tukey’s correction, *P < 0.05, **P < 0.01, ***P < 0.001. Power (1-β err prob) = 1.