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 4

Mitochondrial redox–sensitive roGFP indicates that the Drp1S600A attenuates mtROS in the kidneys of live diabetic mice.

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Mitochondrial redox–sensitive roGFP indicates that the Drp1S600A attenua...
(A) Cartoon depicting the principles of ratiometric redox-sensitive mt-roGFP. CMV-driven expression was localized to the mitochondrial matrix using a cytochrome oxidase subunit IV–signaling (COX IV–signaling) sequence. The oxidation state of the engineered thiols determines the fluorescence properties of the sensor, which shifts depending on the oxidized, disulfide-bonded glutathione/reduced glutathione (GSSG/GSH) equilibrium. Cysteine sulfhydryl groups are illustrated, HS, free sulfhydryl; SS, disulfide bonded. (B) Generation of db/dbroGFP Drp1S600A/A mice allows for monitoring of mitochondrial redox status in the kidneys of live Drp1S600-knockin mice. (C) Image of IVM setup. (D) Ratiometric changes in the fluorescence intensity obtained at the oxidized excitation wavelength (red) and the reduced excitation wavelength (green). Scale bar: 100 μm. (E) Ratiometric quantitative analysis from intravital images, in which oxidized fluorescence is placed as the numerator and reduced fluorescence as the denominator. Representative images are from a sampling of 3 animals. ****P < 0.0001, by 1-way ANOVA with Tukey’s multiple comparisons test. Results are presented as the mean ± standard error of the mean (n = 3 mice/group).