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, 129 (7), 2807-2823

Drp1S600 Phosphorylation Regulates Mitochondrial Fission and Progression of Nephropathy in Diabetic Mice

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Drp1S600 Phosphorylation Regulates Mitochondrial Fission and Progression of Nephropathy in Diabetic Mice

Daniel L Galvan et al. J Clin Invest.

Abstract

Phosphorylation of Dynamin-related protein1 (Drp1) represents an important regulatory mechanism for mitochondrial fission. Here we established the role of Drp1 Serine 600 (S600) phosphorylation on mitochondrial fission in vivo, and assessed the functional consequences of targeted elimination of the Drp1S600 phosphorylation site in 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- but not cell type-dependent. Mechanistically, we showed that mitochondrial fission in high glucose conditions occurs through concomitant binding of phospho-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 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 on mitochondrial dynamics.

Keywords: Cytoskeleton; Diabetes; Metabolism; Mitochondria; Nephrology.

Conflict of interest statement

Conflict of interest: The authors have declared that no conflict of interest exists

Figures

Figure 1
Figure 1. Generation and initial characterization of knockin mice harboring a Drp1S600A mutation.
(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).
Figure 2
Figure 2. Drp1S600A mutation protects against progression of DN.
(A) Breeding scheme to obtain diabetic Drp1S600A-mutant mice and representative images of diabetic mice of each genotype. (B) Immunostaining of glomeruli from diabetic Drp1, WT, and homozygous mutant mice. Glomeruli were stained with an antibody against the p-Drp1 (S637) site (green). Podocytes were identified with an antibody against podocin (red), and nuclei were stained with DAPI (blue). Images were acquired by confocal microscopy (original magnification, ×60). (C) ACR analysis of nondiabetic db/m mice,Drp1 WT mice, and mice of the 3 diabetic genotypes at 12, 16, and 20 weeks demonstrating a significant reduction in albuminuria in 16- and 20-week-old diabetic Drp1S600A heterozygous and homozygous mutant mice compared with diabetic db/db mice. (D) Body weight and (E) blood glucose levels at different time points for the groups described in C. (F) Representative PAS-stained images (scale bar: 50 μm), TEM images (scale bar: 500 nm), SEM images (scale bar: 10 μm), and SEM inset images (scale bar: 1 μm) of kidney glomeruli from mutant allele mice. Images are from a sampling of 3 to 5 animals. *P < 0.05, **P < 0.01, and ***P < 0.001, by 1-way ANOVA followed by Tukey’s multiple comparisons test. Results are presented as the mean ± standard error of the mean (n = 3–8/group).
Figure 3
Figure 3. Mitochondrial fission is dependent on Drp1S600 phosphorylation in the diabetic milieu in vivo.
(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).
Figure 4
Figure 4. Mitochondrial redox–sensitive roGFP indicates that the Drp1S600A attenuates mtROS in the kidneys of live diabetic mice.
(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).
Figure 5
Figure 5. Tissue-independent effect of Drp1S600A on mitochondrial fission in diabetic mice.
Hippocampal neurons and TA muscle cells from mice of different groups were fixed for histology and TEM analysis. (A) Representative TEM images. Scale bar: 500 nm. (B) Quantification of mitochondrial morphology from TEM images. (C) Immunofluorescence staining of paraffin-embedded hippocampal tissue. Left panels show mitochondria stained for Tomm20 (red). Middle panels show gray-scale images of p-Drp1S600 staining. All gray-scale images were captured with identical confocal settings, and images were manipulated simultaneously and equally for each tissue. Right panels show total Drp1 staining (green). Scale bar: 25 μm. (D) Immunofluorescence staining of TA muscle cells as performed in C. Scale bar: 25 μm. (E) Quantification of p-Drp1S600 staining intensity from sections of hippocampus and TA muscle immunostained with anti–p-Drp1S600. Representative images are from a sampling of 3 animals. ***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 = 3–5/group).
Figure 6
Figure 6. Actin cytoskeleton interacts with p-Drp1S600 and mitochondria.
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).
Figure 7
Figure 7. Drp1 binds to the Arp2/3 complex in a p-Drp1S600–dependent manner.
(A) Cultured podocytes with empty vector, FLAG-tagged WT Drp1 (WT), FLAG-tagged Drp1S600A (SA), and FLAG-tagged Drp1S600D (SD) were used. Cells were also transiently transfected with GFP-Arp3. Top panels show anti-FLAG IP material and immunoblotting against GFP and FLAG. Bottom panels show the WCLs. (B) Bacterially expressed GST, GST-Drp1S600A, GST-S600D, and GST-S600 WT proteins on GST-sepharose were mixed with purified Arp2/3 complex in the GST-pulldown assay. Coomassie staining of SDS-PAGE gel is shown on the right. Top 2 left blots show recovered materials that were immunoblotted to detect the binding of Arp2 and Arp3 to Drp1. Third blot on the left shows immunoblotting with p-Drp1S600 (p-Drp1), illustrating good mimicry of the phosphorylation epitope by the aspartate mutation. The bottom blot on the left shows immunoblotting for the total level of input Drp1 from the GST-pulldown assay. (C) Top panels show control podocyte cells cultured under HG conditions after being treated with vehicle, nontargeting (NT) shRNA, shRNA-1 against Arp3, or shRNA-2 against Arp3. Cells were fixed and stained for mitochondria with an antibody against Tomm20. Mitochondria are shown in grayscale. Bottom panels show podocytes expressing Drp1S600D cultured under NG conditions after being treated as indicated above and stained for mitochondria as before. Mitochondria are shown in grayscale. Scale bars: 25 μm. (D) Quantification of mitochondrial length and AR for native podocytes for the images shown in C (top). (E) Quantification of mitochondrial length and AR for podocytes stably expressing Drp1S600D for the images shown in C (bottom). Representative images are from a sampling of 3 to 5 separate cell cultures. ****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).

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