Journal: bioRxiv

Article Title: Pathological TDP-43 filaments accumulate at synapses and cause synaptic dysfunction

doi: 10.64898/2026.01.27.701787

Figure Lengend Snippet: A. Representative false-coloured images of mouse primary cortical neurons expressing SypHy prior to stimulation (Rest), during stimulation with a train of 300 action potentials delivered at 10 Hz (10 Hz), following stimulation (Recovery) and during challenge with ammonium chloride (NH 4 ) buffer to reveal the total synaptic vesicle pool. Arrows indicate examples of terminals that respond to stimulation. Scale bar, 2 μm. B. Time course of relative SypHy fluorescence intensity (ΔF/F0) in mouse primary cortical neurons incubated with FTLD-TDP Type A patient brain-derived TDP-43 filaments (+ filaments) or with aged-matched control brain extracts (+ control) for 3 d, normalised to challenge with NH 4 buffer (total synaptic vesicle pool). The bar indicates the period of stimulation. n = 4 biological replicates. Means +/- SEM are shown. C. Rate of activity-dependent relative SypHy fluorescence increase (ΔF/F0) over the first 6 seconds of stimulation (stimulation triggered at 30 s). n = 4 biological replicates. Means +/- SEM are shown. Linear regression analysis was performed (lines), **p=0.0055. D. Representative traces of excitatory postsynaptic currents (EPSCs) evoked by paired presynaptic stimuli (50 ms interval) from mouse primary hippocampal neurons incubated with (+ filaments) or without (- filaments) FTLD-TDP Type A patient brain-derived TDP-43 filaments or with aged-matched control brain extracts (+ control) for 7 d. E. Plot of the ratios of the second to the first EPSC (paired-pulse ratio; PPR) from mouse primary hippocampal neurons incubated with (+ filaments) or without (- filaments) FTLD-TDP Type A patient brain-derived TDP-43 filaments or aged-matched control brain extracts (+ control) for 3-4 d (d3/4) and 6-7 d (d6/7). Each data point represents one paired recording. Data points are colour coded by biological replicate. n = 3 biological replicates. Means +/- SD are shown. A two-way ANOVA with Tukey’s multiple comparison test was performed, *p<0.05, ****p<0.0001. F. Widefield fluorescence microscopy images of mouse primary cortical neurons expressing the fluorescent calcium indicator jRGECO1a (red) over 1200 ms. Images were taken at 100 ms intervals. Scale bar, 100 μm. G. The spontaneous firing frequency calculated from (F) for mouse primary cortical neurons incubated with (+ filaments) or without (- filaments) FTLD-TDP Type A patient brain-derived TDP-43 filaments or aged-matched control brain extracts (+ control) for 9 d (d9) and 14 d (d14). Each data point represents one time-lapse recording. Data points are colour coded by biological replicate. n = 5 biological replicates. Means +/- SD are shown. A two-way ANOVA with Tukey’s multiple comparison test was performed, **p<0.01, ****p<0.0001.

Article Snippet: Cortical neurons were transfected on DIV 7 with synaptophysin-pHluorin (SypHy) using Lipofectamine 2000, at a ratio of 1 mg SypHy to 1 mL of Lipofectamine (Fisher Scientific; #11668027), as per the manufacturer’s instructions.

Techniques: Expressing, Fluorescence, Incubation, Derivative Assay, Control, Activity Assay, Comparison, Microscopy