We first determined whether mitochondrial dysfunction is evident in fibroblasts of ALS patients carrying pathogenic mutations in SOD1 (I113T), in FUS1 (fused in sarcoma; R521G) or in TDP43 (TAR DNA‐binding protein 43; G289S) genes. As fibroblasts have a mainly glycolytic metabolism, they were cultured in galactose‐containing medium for 48 h, to induce dependence on oxidative phosphorylation (OX‐PHOS) for ATP production (Aguer et al, 2011). In fibroblasts derived from ALS patients, the mitochondrial network was fragmented as compared with fibroblasts from healthy subjects (control), with prevalence of round‐shaped mitochondria or sphere‐like clusters (Fig 1A). To quantify the mitochondrial structure change, we utilized automated image analysis and examined the effects of these ALS mutations on mitochondrial morphology. ALS patient‐derived fibroblasts carrying any one of these three mutations showed a ~50% decrease in mitochondrial interconnectivity score (1.01 vs. 0.48 for control and ALS, respectively; P < 0.0001) and elongation scores (1.54 vs. 0.77 for control and ALS, respectively; P < 0.0001) (Fig 1B and C). We next determined if this mitochondrial fragmentation was mediated by Drp1/Fis1 interaction, using P110, a heptapeptide conjugated to TAT47–57 (TAT, for intracellular delivery) that selectively inhibits the interaction between Drp1 and Fis1, one of its adaptor proteins on the mitochondria (Qi et al, 2013). Treatment with P110 (1 μM/day for 2 days) significantly improved mitochondrial structure and improved the interconnectivity (from 0.48 to 1.32 following P110 treatment; P < 0.0001) (Fig 1A–C). Furthermore, to confirm that Fis1 is critical for the mitochondrial structural changes observed in ALS patient‐derived fibroblasts, we transiently knocked down the expression Fis1 (Fig EV1A) and observed a significant recovery in mitochondrial structure as measured by previously described methods (Figs 1A and EV1B and C).