Inhibition of Drp1 provides neuroprotection in vitro and in vivo

Abstract

Impaired regulation of mitochondrial dynamics, which shifts the balance towards fission, is associated with neuronal death in age-related neurodegenerative diseases, such as Alzheimer's disease or Parkinson's disease. A role for mitochondrial dynamics in acute brain injury, however, has not been elucidated to date. Here, we investigated the role of dynamin-related protein 1 (Drp1), one of the key regulators of mitochondrial fission, in neuronal cell death induced by glutamate toxicity or oxygen-glucose deprivation (OGD) in vitro, and after ischemic brain damage in vivo. Drp1 siRNA and small molecule inhibitors of Drp1 prevented mitochondrial fission, loss of mitochondrial membrane potential (MMP), and cell death induced by glutamate or tBid overexpression in immortalized hippocampal HT-22 neuronal cells. Further, Drp1 inhibitors protected primary neurons against glutamate excitotoxicity and OGD, and reduced the infarct volume in a mouse model of transient focal ischemia. Our data indicate that Drp1 translocation and associated mitochondrial fission are key features preceding the loss of MMP and neuronal cell death. Thus, inhibition of Drp1 is proposed as an efficient strategy of neuroprotection against glutamate toxicity and OGD in vitro and ischemic brain damage in vivo.

Figure 1

siRNA silencing of Drp1 attenuates glutamate-induced mitochondrial fragmentation and cell death in HT-22 cells. (a) Fluorescence photomicrographs ( × 63 objective) of mGFP-transfected and DAPI-stained HT-22 cells show that pooled Drp1 siRNA (80 nM, 48 h) prevents fission of mitochondria in glutamate-exposed (5 mM, 18 h) HT-22 cells. (b) Quantification of mitochondrial morphology of ∼500 cells per condition: category 1 (elongated tubulin-like structure), category 2 (intermediate length) and category 3 (fragmented mitochondria). ^*P<0.05 compared with glutamate 5 mM-treated cells (n=3 independent experiments, ANOVA, Bonferroni-test). (c) Silencing of Drp1 attenuates glutamate-induced cell death as determined by MTT assay. The presented data are normalized to vehicle control Lipofectamine 2000 (100%). ^***P<0.001 compared with glutamate-treated cells (n=8, ANOVA, Scheffé's-test). For statistical analysis, the experiments were independently repeated at least three times with an n=8 per treatment condition and the results are presented as mean±S.D. (d) Representative graph of real-time detection of cellular impedance by xCELLigence System (Roche) is shown. After 24 h, Drp1 siRNA-transfected HT-22 cells were seeded out for additional 24 h in 96-well E-plates with 4500 cells/well and treated with 3 mM glutamate for 20 h (n=8 per treatment condition). Glutamate-induced cell death does not differ in vehicle- or scrambled siRNA-treated cells, whereas Drp1 siRNA exerts a persistent protective effect. The cellular impedance of glutamate alone (red triangles) is obscured by the overlapping curve of scrambled siRNA+glutamate (orange dots). (e) RT-PCR analysis of Drp1 mRNA (upper panels) and western blot analysis of Drp1 protein (lower panels) in HT-22 cells pretreated with 60 and 80 nM pooled Drp1 siRNA for 48 h. RT-PCR with primers specific for glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) and anti-β-actin antibodies used as controls for respective analyses is shown. Quantification of the relative protein expression shows a reduction of 60 and 70%

Figure 2

The novel pharmacological inhibitor of Drp1, mdiviA, protects HT-22 cells against glutamate toxicity. (a) Fluorescence photomicrographs ( × 63 objective) of mGFP-transfected and DAPI-stained HT-22 cells show that Drp1 inhibitor mdiviA (75 μM) prevents fission of mitochondria in glutamate-exposed (5 mM, 18 h) HT-22 cells. (b) Quantification of mitochondrial morphology of ∼500 cells per condition: category 1 (elongated tubulin-like structure), category 2 (intermediate length) and category 3 (fragmented mitochondria). ^**P<0.01 category 1+2 mdiviA, mdiviB+glutamate compared with glutamate 5 mM-treated cells (n=3 independent experiments, ANOVA, Bonferroni-test). (c) HT-22 cells were cultured for 48 h in 96-well E-plates with 4500 cells/well and treated 24 h after seeding with 3 and 5 mM glutamate and mdiviA (75 μM) for 18 h (n=8). Representative graph of real-time detection of cellular impedance by xCELLigence System (Roche) is shown. (d) FACS analysis of HT-22 cells with n=10 000 cells per treatment condition after FITC-Annexin-V labeling to detect apoptotic cells. Exposure to glutamate (5 mM, 14 h) results in enhanced Annexin-V binding of apoptotic HT-22 cells compared with DMSO control. MdiviB (75 μM) significantly reduce glutamate-induced apoptosis. For statistical analysis, the experiments were repeated at least three times with an n=3 per treatment condition. ^***P<0.001 compared with glutamate-treated cells (n=3, ANOVA, Scheffé's-test)

Figure 3

MdiviA inhibits glutamate-induced translocation of Drp1 to the mitochondria, and prevents energy depletion and lipid peroxidation. (a) HT-22 cells were cultured for 24 h in eight-well-ibidi-plates and treated with 3 mM glutamate and mdiviA (75 μM) for 14 h. Immunostaining with specific antibody of Drp1 reveal that Drp1 (green) translocation to the mitochondria (Mitotracker Deep Red) occur after glutamate stimulus. In the magnification pictures by overlapping of the fluorescence (yellow) is shown that Drp1 interacts with the mitochondria. MdiviA inhibits the translocation of Drp1 to the mitochondria. Analysis of fluorescence intensities by Zeiss image software revealed the co-localization of Drp1 and mitochondria by overlapping of green and red fluorescence. (b) Quantification of cells with localization of Drp1 at mitochondria. Drp1 translocation (white bar) correlates with increased mitochondrial fragmentation (grey bar) after glutamate (3 mM) treatment. MdiviA inhibits the translocation of Drp1 to the mitochondria. The quantification is based on three independent experiments with an n=60–70 cells per treatment condition, which were analyzed without knowledge of the treatment history. ^***P<0.001 compared with glutamate (3 and 5 mM)-treated cells (n=3, ANOVA, Scheffé's-test). (c) Representative western blot analysis of mitochondrial and cytosolic fractions of HT-22 cells show that glutamate (3 mM) induces an increase of endogenous Drp1 protein in the mitochondrial fraction after 14 h. Treatment with mdiviA inhibit Drp1 translocation to the mitochondria. Quantitative analysis of protein amount was performed by intensity measurements of Drp1 in contrast to mitochondrial loading control Tim23. (d) Representative western blot analysis of mitochondrial and cytosolic fractions of HT-22 cells to detect the translocation of Drp1 to mitochondria after glutamate treatment in a time-dependent manner (3 mM, 8–15 h). Glutamate-treated cells in comparison with the control cells (Tim23 for mitochondria protein loading; actin for cytosolic extracts). These experiments were independently repeated three times with similar results. The representative quantification of total Drp1 in relation to the respective loading controls in the cytosolic and mitochondrial fractions confirmed that Drp1 accumulates in the glutamate-treated cells in comparison with the control cells (Tim23 for mitochondria protein loading; actin for cytosolic extracts). These experiments were independently repeated three times with similar results. (e) HT-22 cells were cultured in white 96-well plates with 7000 cells/well and treated with 3 and 5 mM glutamate and mdiviA (75 μM) at 14 h. ATP-luminescence measurements were performed according to the manufacturer's protocol. The presented data are normalized to DMSO control. For statistical analysis, the experiments were repeated at least three times with an n=8 per treatment condition. ^***P<0.001 compared with glutamate (3 and 5 mM)-treated cells (ANOVA, Scheffé's-test). (f) Fluorescence detection of lipid peroxidation by Bodipy assay, quantified by FACS analysis of three independent experiments with n=20 000 cells per treatment condition. ^***P<0.001 compared with glutamate (3 and 5 mM)-treated cells (n=3, ANOVA, Scheffé's-test)

Figure 4

Inhibition of Drp1 prevents glutamate-induced mitochondrial depolarization. (a and c) MMP was analyzed by JC-1 fluorescence: upper panels show epifluorescence photomicrographs indicating equal cellular uptake of JC-1 by green fluorescence; lower panels depict intact mitochondria exposing red fluorescence. Glutamate-treated (5 mM, 12 h) HT-22 cells show significantly reduce red fluorescence compared with controls whereas mdiviA and B (75 μM) and siRNA of Drp1 (80 nM) prevents the breakdown of the MMP as indicated by preservation of the red JC-1 fluorescence. (b and d) FACS analyses of n=3 independent experiments with 10 000 cells per treatment condition reveal a decrease of the red JC-1 fluorescence to 30% of control levels 12 h after glutamate treatment (5 mM), which has prevented mdiviA, B and siRNA of Drp1 (80 nM). Glutamate treatment was as effective as the positive damage-control CCCP, which causes a fast breakdown of the MMP. The presented data are normalized to DMSO control for mdiviA and mdiviB and vehicle control Lipofectamine 2000 for siRNA approach (100%). ^***P<0.001 compared with glutamate-treated controls (n=3, ANOVA, Scheffé's-test)

Figure 5

Inhibition of Drp1 prevents tBid-induced mitochondrial fragmentation and neuronal cell death. (a) Fluorescence photomicrographs ( × 63 objective) showed that the Drp1 inhibitors mdiviA and mdiviB (75 μM) prevent fission of mitochondria in tBid-induced toxicity after 12 h in HT-22 cells. (b) Quantification of mitochondrial morphology of ∼500 cells per condition: category 1 (elongated tubulin-like structure), category 2 (intermediate length) and category 3 (fragmented mitochondria). ^*P<0.05 compared with glutamate (5 mM)-treated cells (n=3 independent experiments, ANOVA, Bonferroni-test). (c) HT-22 cells were cultured in 24-well plates with 70 000 cells/well and tBid-induced cell death determined by MTT assay after 12 h. Treatment with mdiviA and mdiviB (75 μM) was performed 1 h before transfection. The presented data are normalized to vehicle control Lipofectamine 2000. ^***P<0.001, compared with tBid-treated cells (n=4, ANOVA, Scheffé's-test). For statistical analysis, the experiments were independently repeated at least three times with an n=4 per treatment condition and the results are presented as mean±S.D. (d) FACS analysis of HT-22 cells in n=3 independent experiments with 10 000 cells per treatment condition after FITC-Annexin-V labeling to detect apoptotic cells. Transfection of pIRES-tBid results in enhanced Annexin-V binding of apoptotic HT-22 cells compared with vehicle control Lipofectamine 2000. MdiviA and B (75 μM) significantly reduce tBid-induced cell death. ^***P<0.001 compared with tBid toxicity (n=3, ANOVA, Scheffé's-test)

Figure 6

mdivi compounds protect primary neurons against glutamate-induced excitotoxicity and OGD in vitro and attenuate ischemic brain damage in vivo. Primary rat cortical neurons (DIV 9) were exposed 4 h to OGD. After 4 h of OGD, the 1 × EBSS±glucose was replaced by NB+-medium containing glucose to all dishes and the cells were incubated for another 18–24 h in the regular oxygen-containing incubator. (a) Fluorescence microscope images ( × 40 objective) of DAPI-stained embryonic cortical neurons were obtained after 18 h of exposure to OGD. (b) Counting of ∼500 DAPI-stained neurons with a total n=5 per group displaying pycnotic nuclei after 4 h of OGD revealed that 25 μM Drp1 inhibitor mdiviA and B significantly protect neurons from hypoxic-hypoglycemic cell death. ^***P<0.001 versus untreated neurons subjected to OGD (n=5, ANOVA, Scheffé's-test). Cultures treated with 25 μM mdiviA or B contained >60% healthy nuclei, whereas all other OGD-treated groups show 75% pycnotic and/or fragmented nuclei, indicating apoptotic damage (OGD, right white column). The respective control cultures with 1 × EBSS medium + glucose contained only very few apoptotic nuclei (Control, left white column). The experiments were repeated three times and the results are presented as mean±S.D. (c) Quantification of pycnotic nuclei in percentage of four independent experiments with a total n=5 per group. Primary rat cortical neurons (DIV 9) were treated for 18 h with glutamate (30 μM) and mdiviA and B (25 μM). After 18 h, cells were fixed in 4% PFA and stained with DAPI and ∼500 nuclei were counted per treatment condition. MdiviA and B significantly reduced glutamate-induced cell death. ^***P<0.001 versus glutamate-treated neurons (n=5, ANOVA, Scheffé's-test). (d) Transient focal ischemia was induced for 45 min by a silicon-coated nylon filament that was introduced into the internal carotid artery to occlude the middle cerebral artery (MCA). Surgery was performed in deep isoflurane/N₂O anesthesia with controlled ventilation, and ischemia and reperfusion were verified by Laser-Doppler flowmetry. The infarct volume was calculated on the basis of the histomorphometric data from 12 to 15 consecutive sections (10 μM, 500 μM apart) obtained 24 h after onset of ischemia. MdiviA (3 mg/kg) and mdiviB (1 and 3 mg/kg) reduced the mean infarct volume by 34 and 30% compared with vehicle (DMSO) controls, respectively. The photomicrographs show representative pictures of cresyl-violet-stained brain slices from animals, which received vehicle or mdiviB (1 and 3 mg/kg). The infarct area remained unstained and is highlighted by dashed lines