Fig. 7: Altered DRP1 ISGylation in Alzheimer’s disease. | Cell Death & Disease

Fig. 7: Altered DRP1 ISGylation in Alzheimer’s disease.

From: ISGylation of DRP1 closely balances other post-translational modifications to mediate mitochondrial fission

Fig. 7

A Co-immunoprecipitation against ISG15 and DRP1 was done in human brain whole tissue lysate. Note differential ISGylation profiles of DRP1 between the samples. The input levels of DRP1, ISG15, HERC5, TRIM25 and VINCULIN in the total lysates served as loading controls. B Brain lysates from 5XFAD mice were co-immunoprecipitated and analysed as in panel A. The input levels of DRP1, ISG15, and VINCULIN served as loading controls. C SHSY5Y cells transfected with indicated constructs were treated with DMSO and 0.5 μM Aβ for 48 h. Note reduced presence of ISGylated DRP1 in samples with AICD and Aβ, as detected by co-immunoprecipitation. The input levels of DRP1, ISG15, HERC5, TRIM25 and VINCULIN were analysed. D SHSY5Y cells treated with DMSO or 1 μM Aβ for 48 h were verified for DRP1 ISGylation as in panel C. E Cytosolic and mitochondrial fractions obtained from semi-permeabilised cells, transfected with indicated constructs and treated with DMSO, 0.5 μM Aβ or 1 μM Aβ for 48 h, were immunoblotted against phospho-DRP1 (S616) and total DRP1; VINCULIN and VDAC1 served as controls for cytosolic and mitochondrial fractions, respectively. F Graph represents data from 3 independent experiments. **p ≤ 0.01 using unpaired 2-tailed Student’s t-test. Error bars,±SEM. G Cells transfected with indicated GFP-tagged constructs and treated with DMSO or 0.5 μM Aβ over indicated time periods were imaged with MitoTracker Red FM under live-cell conditions. Scale bar, 20μm. H Box plot showing quantification of mitochondrial length; ~125 cells from 3 independent experiments were analysed. The central line and the plus (+) symbol in each box show the median and mean value, respectively. ***p ≤ 0.001 using unpaired 2-tailed Student’s t-test. I Cells treated with DMSO or 1 μM Aβ for indicated time periods were imaged as in panel G. Scale bar, 20μm. J Mitochondrial length from ~120 cells were analysed and plotted from 3 independent experiments. The central line and the plus (+) symbol in each box show the median and mean value, respectively. ***p ≤ 0.001 using unpaired 2-tailed Student’s t-test.

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