Fig. 4

PTP1B inhibition suppresses TDP-43-induced mitochondrial dysfunction in astrocytes and spine retraction in primary cortical neurons. a–c Primary cortical neurons in XF24-well culture plates were stimulated with GFP ACM, TDP-43 ACM or TDP-43 + PTP1Bi ACM for 5 days. a Analysis of mitochondrial dysfunction in ACM-treated cells was performed by a Seahorse XF analyser to detect the basal OCR, ATP production, maximum reserve and respiratory capacity. The OCR was normalized to the total protein concentration (OD). b Quantification of the OCR, ATP production, maximum reserve, and respiratory capacity as a percentage of the basal values. Data are presented as the mean ± SEM. *p < 0.05; **p < 0.005; and N.S. not significant (one-way ANOVA). c The protein concentrations of cell lysates in each ACM-treated group were measured by a BCA protein assay kit. Data are presented as the mean ± SD. N.S. not significant (one-way ANOVA). d Representative confocal images of each ACM-treated group. Dendritic spines in the dotted circle, which is 20 μm radius from the centre of nucleus, were not analysed (panel left). Scale bars, 20 μm. Representative confocal (phalloidin-TRITC, red and anti-Map2-Alexa 594, red) and filament tracing Imaris images from randomly selected dendritic spines of each ACM-treated neuron group (right panel). Scale bars, 5 μm. The magnified image in the right panels shows filament tracing of the analysed spine. Scale bars, 2 μm. e Dendritic spine density was defined as the number of spines per 50 μm of dendrite length in the Imaris image of the right panel of Fig. 4d, which shows the average number of dendritic spines for 10 μm. Fifteen dendrites were randomly selected from four separate cortical neuron cultures per GFP ACM, TDP-43 ACM and TDP-43+PTP1Bi ACM-treated cortical neuron group. **p < 0.005, ***p < 0.001 (one-way ANOVA). f Cumulative distribution curves of spine length from cortical neurons as determined by the Kolmogorov-Smirnov (KS) test: mean spine length of GFP ACM vs. TDP-43 ACM and TDP-43 ACM vs. TDP-43 + PTP1Bi ACM. p = 0.027 and p = 0.609, respectively