Skip to main content
. 2021 Nov 10;41(45):9431–9451. doi: 10.1523/JNEUROSCI.1914-20.2021

Figure 6.

Figure 6.

P301L and R5L tau increase axonal transport pause frequency in primary rat hippocampal neurons. A, Neurons were cotransfected with an mApple-synaptophysin (mA-Syn) fusion construct and GFP, WT tau, P301L tau, or R5L tau. Axons were identified in live cells by labeling an external region of neurofascin (NF, left, cyan). After fixation cotransfection of both constructs was identified in all cells (right). Scale bar, 50 μm. B, Axonal transport of fluorescent vesicles was imaged at 28 fps for 5 min. Each panel in this image is 1 s after the previous one. Scale bar, 28 μm. The solid arrows track a vesicle moving in the anterograde direction, and the wire arrows track a vesicle moving in retrograde direction before pausing. C, Kymographs were generated and analyzed using the KymoAnalyzer macro for ImageJ. The parameters included direction, segment velocities, and pause frequency. Anterograde tracks are labeled green, retrograde tracks area labeled red, and tracks that switch direction are labeled yellow. D, The mean anterograde segment velocity was not significantly affected by expression of GFP or any of the tau proteins tested (GFP, 2.22 ± 0.22 µm/s; WT tau, 2.44 ± 0.43 µm/s; P301L, 2.49 ± 0.27 µm/s; R5L, 2.58 ± 0.28 µm/s). E, Expression of WT, P301L, or R5L tau caused a small but significant elevation in retrograde velocity (∼20%) when compared with GFP control but no significant differences were detected between the three tau proteins (GFP, 2.01 ± 0.24 µm/s; WT tau, 2.38 ± 0.28 µm/s; P301L, 2.45 ± 0.15 µm/s; R5L, 2.47 ± 0.15 µm/s). F, Expression of either P301L or R5L tau resulted in an increase of total pause frequency (number of pauses/track/s) compared with GFP and WT tau expression (GFP, 0.160 ± 0.040 pauses/s; WT tau, 0.178 ± 0.039 pauses/s; P301L, 0.326 ± 0.039 pauses/s; R5L, 0.370 ± 0.057 pauses/s). G, Anterograde pause frequency (pauses in cargo traveling in the anterograde direction) increased with P301L and R5L tau expression compared with GFP and WT tau (GFP, 0.143 ± 0.044 pauses/s; WT tau, 0.137 ± 0.043 pauses/s; P301L, 0.241 ± 0.080 pauses/s; R5L, 0.252 ± 0.067 pauses/s). H, The retrograde pause frequency also increased on P301L and R5L tau expression compared with GFP and WT tau (GFP, 0.107 ± 0.015 pauses/s; WT tau, 0.116 ± 0.046 pauses/s; P301L, 0.180 ± 0.021 pauses/s; R5L, 0.187 ± 0.043 pauses/s). The groups were compared using a one-way ANOVA with Tukey's multiple comparison test, and each data point represents an independent replicate (n = 6 for GFP, WT tau, and P301L tau and n = 5 for R5L tau; the data are the mean ± SD; *p ≤ 0.05).