Drp1S600 phosphorylation regulates mitochondrial fission and progression of nephropathy in diabetic mice
Daniel L. Galvan, … , Paul Overbeek, Farhad R. Danesh
Daniel L. Galvan, … , Paul Overbeek, Farhad R. Danesh
Published May 7, 2019
Citation Information: J Clin Invest. 2019;129(7):2807-2823. https://doi.org/10.1172/JCI127277.
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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 1

Generation and initial characterization of knockin mice harboring a Drp1S600A mutation.

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Generation and initial characterization of knockin mice harboring a Drp1...
(A) Diagram of the domain structure of Drp1, illustrating the S600 site at the juncture of the VD and GED. PIM1, serine/threonine protein kinase Pim-1; CaN, calcineurin; PP2A, protein phosphatase 2A; PGAM5, phosphoglycerate mutase family member 5. (B) Structure of the Drp1-targeting locus, the Drp1-targeting construct, and the conditional allele after homologous recombination. The ScaI and XhoI sites used in Southern blot analysis and the location of the 3′ and 5′ probes are indicated. FRT, flippase recognition target (yellow triangles); FLP, flippase. (C) Gross appearance of WT, homozygous, and heterozygous Drp1S600A-knockin mice, and PCR sequence from genomic DNA showing mutation of the allele in the genome. (D) Southern blot analysis of ScaI and XhoI digested genomic DNA from mice of the indicated genotypes, showing the WT (12.7-kb) and mutant (5.8-kb) bands (upper panel). PCR genotyping of Drp1S600A heterozygosity and homozygosity (lower panel). Mutant and WT products are shown. (E) Cartesian allelic discrimination plot shows the relative levels of the Drp1S600A-mutant fluorescence signal for each sample plotted on the y axis and the WT signal on the x axis. Homozygous Drp1S600A (red dots), homozygous WT (blue dots), and heterozygous (violet dots) samples are shown. The no-template control is depicted by the black circle. (F) Western blot of Drp1 protein from mice of the three S600-mutant genotypes and densitometric quantification of Drp1 normalized to GAPDH protein expression. (G) Albumin/creatinine ratio (ACR) analysis of WT, heterozygous, and homozygous Drp1S600A-knockin mice at 20 weeks of age. (H) Mitochondrial AR from podocytes from mice of the three Drp1S600A genotypes as determined from TEM images. P < 0.05, by 1-way ANOVA with Tukey’s multiple comparisons test. Data represent the mean ± standard error of the mean (n = 5–8/group).