Drp1S600 phosphorylation regulates mitochondrial fission and progression of nephropathy in diabetic mice
Daniel L. Galvan, Jianyin Long, Nathanael Green, Benny H. Chang, Jamie S. Lin, Paul Schumacker, Luan D. Truong, Paul Overbeek, Farhad R. Danesh
Daniel L. Galvan, Jianyin Long, Nathanael Green, Benny H. Chang, Jamie S. Lin, Paul Schumacker, Luan D. Truong, Paul Overbeek, Farhad R. Danesh
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Research Article Metabolism Nephrology

Drp1S600 phosphorylation regulates mitochondrial fission and progression of nephropathy in diabetic mice

Abstract

Phosphorylation of dynamin-related protein 1 (Drp1) represents an important regulatory mechanism for mitochondrial fission. Here, we established the role of Drp1 serine 600 (Drp1S600) phosphorylation in mitochondrial fission in vivo and assessed the functional consequences of targeted elimination of the Drp1S600 phosphorylation site in the progression of diabetic nephropathy (DN). We generated a knockin mouse in which S600 was mutated to alanine (Drp1S600A). We found that diabetic Drp1S600A mice exhibited improved biochemical and histological features of DN along with reduced mitochondrial fission and diminished mitochondrial ROS in vivo. Importantly, we observed that the effect of Drp1S600 phosphorylation on mitochondrial fission in the diabetic milieu was stimulus dependent but not cell type dependent. Mechanistically, we show that mitochondrial fission in high-glucose conditions occurs through concomitant binding of phosphorylated Drp1S600 with mitochondrial fission factor (MFF) and actin-related protein 3 (Arp3), ultimately leading to accumulation of F-actin and Drp1 on the mitochondria. Taken together, these findings establish the idea that a single phosphorylation site in Drp1 can regulate mitochondrial fission and progression of DN in vivo and highlight the stimulus-specific consequences of Drp1S600 phosphorylation in mitochondrial dynamics.

Authors

Daniel L. Galvan, Jianyin Long, Nathanael Green, Benny H. Chang, Jamie S. Lin, Paul Schumacker, Luan D. Truong, Paul Overbeek, Farhad R. Danesh

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Figure 3

Mitochondrial fission is dependent on Drp1S600 phosphorylation in the diabetic milieu in vivo.

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Mitochondrial fission is dependent on Drp1S600 phosphorylation in the di...
(A) Representative TEM images of mitochondria in podocytes. The 3 possible genotypes at the Drp1S600-knockin alleles — WT, heterozygous, and homozygous — are shown. Scale bars: 500 nm. To the right of each micrograph are tracings of mitochondria from the TEM micrographs. Changes in mitochondrial morphology were quantitated from TEM micrographs as the (B) mitochondria AR, (C) mitochondria form factor, (D) mitochondrial length, (E) mitochondrial circularity, (F) mitochondrial area, and (G) mitochondrial perimeter. (H) Immunofluorescence staining of paraffin-embedded kidney sections. Sections were stained for Drp1 (grayscale or green in merge) and Tomm20 (grayscale, or red in merge), with the merged images shown on the far right. Representative images were cropped to show the glomerular area only. Scale bar: 50 μm. (I) Colocalization of total Drp1 and mitochondria determined from the images represented in H using Pearson’s correlation coefficient. Representative images are from a sampling of 3 to 5 animals. ****P < 0.0001, by 1-way ANOVA followed by Tukey’s multiple comparisons test. Results are presented as the mean ± standard error of the mean (n = 5–8/group).