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 6

Actin cytoskeleton interacts with p-Drp1S600 and mitochondria.

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Actin cytoskeleton interacts with p-Drp1S600 and mitochondria. Cultured ...
Cultured podocytes stably expressing FLAG-tagged versions of Drp1 (WT) and Drp1S600A were treated with HG (25 mM) for 48 hours. FLAG-tagged Drp1S600D was cultured under NG conditions. Cells were cross-linked, immunoprecipitated, and subjected to MS. KEGG Pathway analysis of protein interaction partners was performed. Top interaction pathways for Drp1S600 according to (A) the number of proteins and (B) enrichment score. (C) KEGG pathway analysis of the mutant proteins indicating the top pathways identified for each Drp1 mutant. (D) Podocytes stably expressing FLAG-tagged Drp1S600A or S600D in conjunction with untagged MFF or empty vector were immunoprecipitated with anti-FLAG agarose gel. The top 2 panels show recovery of MFF and Drp1 by immunoblotting (IB) following IP. Immunoblots in the bottom panels are for the WCL starting material. Different isoforms of MFF are indicated by a bracket on the right. (E) Podocytes were immunostained with Tomm20 (green) for mitochondria and rhodamine-phalloidin (red) for actin. From left to right, gray-scale image of mitochondria staining (green in merge), gray-scale images of actin staining (red in merge), and merged image. Scale bar: 25 μm. (F) Quantification of the overlap based on Mander’s coefficient for each condition. (G) Immunofluorescence staining of paraffin-embedded kidney sections. Sections were stained for total Drp1 (grayscale, green in merge) and Arp3 (grayscale, red in merge). Scale bar: 50 μm. (H) Colocalization analysis using Pearson’s correlation analysis of total Drp1 and mitochondria determined from the images represented in F. Representative images are from a sampling of 3 to5 separate cell cultures or animals. *P < 0.05, **P < 0.01, ***P < 0.001, and ****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 = 5–8/group).