Fig. 5: HSV-1 promotes the release of p-tau from infected neurons and the accumulation of p-tau in uninfected neurons adjacent to infection.
ReNcell VM cultures were infected with HSV-1 for 24 hours to characterize p-tau’s extracellular release and changes in proximity to viral infection. a−d, 3.5−4-week-old 3D ReNcell VM cultures were infected with serial dilutions of HSV-1 for 24 hours, and cell media and cell lysates were analyzed by MSD Multi-Spot Phospho (Thr 231)/Total Tau Assay for soluble p-tau and total tau. a, Comparison of cell media soluble p-tau at different viral loads (F3, 54 = 7.521 ((uninfected versus 2.55, ***P = 0.0005), (uninfected versus 5.10, ***P = 0.0004)). b, Comparison of cell media soluble total tau at different viral loads (F3, 55 = 10.49, **P = 0.0011). c, Comparison of the ratio between cell media p-tau and total tau at different viral loads (F3, 54 = 19.94, ****P < 0.0001). d, Comparison of the ratio between cell lysate soluble p-tau and soluble total tau at different viral loads (F3, 96 = 202.5, ****P < 0.0001). e, 2D ReNcell VM cultures in microfluidic devices were infected in the left chamber with HSV-1 for 48 hours, immunoprobed with anti-p-tau (PHF1) labeled with a fluorescent secondary antibody and analyzed for neurons (GFP), HSV-1 (RFP) and p-tau (647) fluorescence by confocal microscopy. Fluorescence signals for ReNcell VM, HSV-1 and p-tau were imaged for infected and uninfected conditions. f, GA3 analysis of HSV-1-positive neurons compared intracellular p-tau fluorescence among infected neurons (triangles), uninfected neurons proximal to infected neurons (*) and uninfected neurons not proximal to infected neurons (arrows) (F2, 3,288 = 45.35, ****P < 0.0001). g, Individual neuronal p-tau fluorescence intensity was compared to total proximal HSV-1−RFP fluorescence intensity (R2 = 0.7912). Box plots are representative of ±s.e.m. ((a−c, n = 13), (d, n = 25)) depicting median and interquartile range, with whiskers denoting variability according to Tukey’s method. Statistical mean comparisons were calculated by one-way ANOVA using Dunnett’s multiple comparisons test (a−d), one-way ANOVA using Tukey’s multiple comparisons test (f) and simple linear regression (g).
