Fig. 1 |. UNC13A and TDP-43 knockdown impair synaptic function.

a, Schematic representation of HaloTag edit at the beginning of exon 2 of TARDBP and PCR from genomic DNA indicating successful edit b, Immunofluorescence analysis of TDP-43 localization in HaloTDP iNeurons revealed nuclear localised TDP-43. Scale bar = 10 μm. c, RT-PCR analysis of UNC13A splicing after TDP-43 knockdown. d, Quantification of results in (c) n=12 biological replicates from 3 experiments. e, Western blot analysis of HaloTDP iNeurons treated with 30 nM HaloPROTAC-E for the indicated amount of time. f, Quantification of western blot in (e) n=2 biological replicates. g, Immunofluorescence labelling of synapsin and UNC13A presynaptic terminals in 4-week-old HaloTDP iNeurons grown on rat astrocytes following 2-weeks of TDP-43 depletion. Scale bar = 10 μm. h, Quantification of UNC13A intensity at synapsin positive synaptic terminals. Control n=116, TDP-43 knockdown n=115 synapses from 3 experiments. i, Miniature excitatory postsynaptic currents (mEPSCs) from control (n=12) and UNC13A knockdown (n=13) iNeurons pooled from 3 experiments. Representative traces and average traces shown. Quantification of mEPSC frequency and inter-event interval cumulative distributions. j, mEPSCs from control (n=20) and TDP-43 knockdown (n=22) HaloTDP iNeurons pooled from 3 experiments. Representative traces and average traces shown. Quantification of mEPSC frequency and inter-event interval cumulative distributions. k, Representative raster plots from D36 multi-electrode array recordings from control, UNC13A and TDP-43 knockdown conditions (30s window shown). Electrode bursts in blue and network bursts in pink. l, Time-course of network synchrony measured by the area under the normalised electrode cross correlation in control and UNC13A knockdown iNeurons. UNC13A normalised to control n=18 wells from 3 experiments. m, Time-course of network synchrony in control and TDP-43 knockdown iNeurons. TDP-43 normalised to control, n=18 wells from 3 experiments. Graphs for (d) (h) (i) and (j) represent mean ± s.e.m and independent experiments are denoted by circle, square or triangle markers. Statistics for (d) (h) (i) and (j) are two-sided Student’s t test. Statistics for L and M are ratio paired t tests. *P < 0.05; ** P <0.01; *** P <0.001; **** P < 0.0001; ns (not significant).