Drp1 levels constitutively regulate mitochondrial dynamics and cell survival in cortical neurons - PubMed (original) (raw)

Comparative Study

Drp1 levels constitutively regulate mitochondrial dynamics and cell survival in cortical neurons

Takuma Uo et al. Exp Neurol. 2009 Aug.

Abstract

Mitochondria exist as dynamic networks that are constantly remodeled through the opposing actions of fusion and fission proteins. Changes in the expression of these proteins alter mitochondrial shape and size, and may promote or inhibit the propagation of apoptotic signals. Using mitochondrially targeted EGFP or DsRed2 to identify mitochondria, we observed a short, distinctly tubular mitochondrial morphology in postnatal cortical neurons in culture and in retinal ganglion cells in vivo, whereas longer, highly interconnected mitochondrial networks were detected in cortical astrocytes in vitro and non-neuronal cells in the retina in vivo. Differential expression patterns of fusion and fission proteins, in part, appear to determine these morphological differences as neurons expressed markedly high levels of Drp1 and OPA1 proteins compared to non-neuronal cells. This finding was corroborated using optic tissue samples. Moreover, cortical neurons expressed several splice variants of Drp1 including a neuron-specific isoform which incorporates exon 3. Knockdown or dominant-negative interference of endogenous Drp1 significantly increased mitochondrial length in both neurons and non-neuronal cells, but caused cell death only in cortical neurons. Conversely, depletion of the fusion protein, Mfn2, but not Mfn1, caused extensive mitochondrial fission and cell death. Thus, Drp1 and Mfn2 in normal cortical neurons not only regulate mitochondrial morphology, but are also required for cell survival. The present findings point to unique patterns of Drp1 expression and selective vulnerability to reduced levels of Drp1 expression/activity in neurons, and demonstrate that the regulation of mitochondrial dynamics must be tightly regulated in neurons.

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Figures

Figure 1. Mitochondrial morphology in neurons and non-neuronal cells in vitro and in vivo

A) Primary cultures of (a) postnatal mouse cortical neurons, (b) postnatal mouse cortical astrocytes and (c) mouse embryonic fibroblasts (MEF) were infected with lentivirus expressing mitochondrially targeted EGFP (10 MOI) or DsRed2 (10 MOI) 24 hr after plating. Cells were fixed 48 hr after infection. Neurons in (a) were immunostained for β-tubulin III (TuJ1 antigen), a neuronal marker. The gamma factor for (a) was set at 0.4 using Slidebook software in order to bring up the MitoDsRed2 fluorescence intensity in the neurites. An inset using a lower fluorescence intensity depicts mitochondrial morphology in the soma of neurons. Scale bar (a–c) = 10 μm. The images are representative of four independent experiments. B) Mito-EGFP lentivirus (5×103 TU) was injected into the posterior chamber of the eye where it was taken up by several cell types. (a) NeuN-positive retinal ganglion cells expressing Mito-EGFP show small, punctate or tubular mitochondria well-dispersed throughout the neuronal architecture including the cell soma. An exception is seen in the axon initial segment where mitochondria tend to aggregate and form clusters of EGFP-positive mitochondria (arrowheads: see Supplemental Figure 2 for more detail). (b) Retinal ganglion cell axons immunopositive for neurofilament-200 (NF-H) chain, an axonal marker, show a wide range of EGFP-positive mitochondrial morphology. The larger puncta may represent individual or clusters of mitochondria. (c) NeuN-negative cells present in the nerve fiber layer, above the retinal ganglion cells, also display highly elongated mitochondria. (d) A NeuN-negative cell in the retinal nerve fiber layer has large numbers of elongated mitochondria. (e) Another NeuN-negative cell, possibly a macrophage, also has a large concentration of EGFP-positive elongated mitochondria. The images are representative of three independent experiments. Scale bars: 20 μm in (a), 10 μm in (b–e). Note: in (d) and (e), neurofilament-200 staining is also presented in red, not in blue, together with NeuN staining.

Figure 2. Comparative analysis of expression of mitochondrial fusion/fission proteins among various cultured cells and tissues

(A) Protein samples were prepared from primary neuronal cultures derived from embryonic day 14.5 telencephalon (E14.5 Tel), postnatal day 0 cortex (P0 Ctx) or hippocampus (P0 Hipp), astrocyte cultures from postnatal day 0 cortex (P0 Ctx A) and mouse embryonic fibroblasts (MEF). Expression of mitochondrial fusion (Mfn1, Mfn2 and OPA1) and fission (Drp1) GTPase proteins in each sample was evaluated by Western blot analysis. The intensity of each band corresponding to the specified protein was quantitated using ImageJ 1.41o software (National Institutes of Health, Bethesda, MD) and normalized against actin. For OPA1, the summed intensity of two major bands (indicated by arrows) was used for quantitation. The value for the respective protein in each sample was then normalized to that in postnatal cortical neuronal extracts. Similar results were obtained for P0 Ctx N and MEF in at least three independent experiments. (B) Protein samples were prepared from retina, optic nerve and superior colliculus dissected out from 6 month old C57BL/6 mice and subjected to Western blot analysis as described in (A). The data is representative of two independent experiments.

Figure 3. Analysis of expression of Drp1 splicing variants in mouse cells

(A) Schematic representation of genomic organization, transcript and protein of mouse Drp1. Numbering of exons is based on the sequence of transcript variant “a” (GenBank accession No., NM152816). shRNA target sequence resides in the region corresponding to exon 11, whereas the peptide sequence used to raise the Drp1 antibody we used encompasses the region corresponding to exon 18–20. Thus, all of the splicing variants and the respective proteins can be targeted by our shRNA sequence and recognized by the antibody used in this study, respectively. (B) Total RNA was prepared from adult spinal cord neural progenitor cells (aSPC), primary neuronal cultures derived from postnatal day 0 cortex (P0 Ctx N), astrocyte cultures from postnatal day 0 cortex (P0 Ctx A) and mouse embryonic fibroblasts (MEF). Each sample was subjected to RT-PCR analysis using primers P1 and P2 for amplification of exon 2–5. The resultant reaction mixtures were resolved on 2.0 % agarose gels with markers (100 bp PCR Molecular Ruler, Bio-Rad. Hercules, CA). The structure of each splicing variant was confirmed by direct sequencing. (C) Total RNA was prepared and subjected to RT-PCR analysis using primers P3 and P4 for amplification of exon 14–19, followed by agarose gel analysis as described above in (B). The aSPC and other samples were applied to the same agarose gel. The resultant photo images, which contained unrelated samples, were cut and rearranged to facilitate the comparison. (D) Protein samples prepared as described in Fig. 2A were subjected to SDS polyacrylamide gel electrophoresis using a constant 7.5% gel followed by Western blot analysis. Drp1 protein from MEF migrated faster than those from P0 cortical neurons (P0 Ctx N) and astrocytes (P0 Ctx A). (E) HEK 293 FT cells were transiently transfected with plasmids harboring FLAG-tagged human Drp1 cDNA with all combinations of inclusion/exclusion of exon 16 and 17. All of these cDNAs lack exon 3. Protein extracts from transfectants were subjected to Western blot using a constant SDS 7.5% polyacrylamide gel to detect FLAG-tagged proteins. The DNA sequences corresponding to exon 16 and 17 encode 26 and 11 amino acids, respectively. Exclusion of exon 16 yielded a faster migrating form as expected, whereas inclusion or exclusion of exon 17 did not provide separable migration patterns under these conditions. Since neuronal Drp1 is expected to be predominantly the form that includes both exon 16 and 17 (C), exclusion of exon 16, not exon 17, can explain the faster moving Drp1 from MEF relative to the neuronal Drp1. (F) FLAG-tagged human Drp1 cDNAs containing both exon 16 and 17 with/without exon 3 were used in the same way as described in (E). The DNA sequence corresponding to exon 3 encodes 13 amino acids. Inclusion or exclusion of exon 3 did not provide separable migration patterns under these conditions.

Figure 4. Drp1 variants display similar properties in binding to Bcl-xL, SUMOylation and intracellular localization

(A) Schematic representation of the Drp1 constructs used for (B)–(D). Note the variations in the inclusion/exclusion of exon 3 and exon 16/17. (B) HEK 293 FT cells were transiently co-transfected with a MYC-tagged Bcl-xL plasmid and one of the Drp1 variant plasmids, as shown in (A), as indicated by the numbers. Lysates were prepared 24 hr after transfection and subjected to immunoprecipitation (IP) with anti-Bcl-xL antibody followed by Western blot analysis (WB) using anti-FLAG and anti-MYC tag antibodies to detect the exogenously expressed Drp1 and Bcl-xL proteins, respectively. The positions of IgG heavy chains (IgG H) are also indicated. Normal rabbit IgG was used as a negative control for the IP. (C) HEK 293 FT cells were transiently transfected with one of the Drp1 variant plasmids, as shown in (A), as indicated by the numbers. Lysates were prepared 24 hr after transfection and were subjected to IP with anti-FLAG antibody followed by Western blot analysis (WB) using anti-SUMO-1 to detect SUMOylated Drp1. The bands recognized by SUMO-1 antibody were also detected by FLAG antibody (data not shown). (D) HEK 293 FT cells and (E) postnatal cortical neurons were transiently transfected with one of the plasmids shown in (A). Intracellular distribution of the exogenously expressed Drp1 was determined by FLAG immunostaining 24 (D) and 48 hr (E) after transfection. Mitochondria in HEK 293 FT cells were visualized by co-transfection with the Mito-EGFP plasmid. Upper and lower panels show the distribution of each splice variant in the soma and neurites in the same cell, respectively (E).

Figure 5. Drp1 shRNA reduces Drp1 protein levels and induces neuronal cell death

(A) Primary cultures of postnatal mouse cortical neurons were infected with control shRNA (10 MOI) or Drp1 shRNA (10 MOI) lentivirus 24 hr after plating. Cells were fixed five days after infection and immunostained for Drp1 or β-tubulin III (TuJ1 antigen). The data is representative of three independent experiments. (B) Primary cultures of postnatal mouse cortical neurons were infected as described in (A) above. Five days later protein extracts were prepared as described in the methods and expression of mitochondrial fission (Drp1) and fusion (OPA1) proteins was evaluated by Western blot analysis. Drp1 expression levels were quantitated and normalized against actin as described in Fig. 2A and presented as values relative to the level in control shRNA. The data is representative of two independent experiments. No, no infection; Cont, control shRNA lentivirus; Drp1, Drp1 shRNA lentivirus (C) Primary cultures of postnatal mouse cortical neurons were infected as described in (A) above and five day later the cells were fixed, immunostained for Drp1 and stained with Hoechst 33258 to depict nuclear morphology. The data is representative of three independent experiments. (D) The percentage of damaged neurons displaying shrunken, condensed nuclei based on Hoechst 33258 DNA staining was determined five days after infection in ten random fields per condition (approximately 1500 cells counted in total per condition). The data is representative of three independent experiments. *, significantly different from all other conditions (p < 0.0005, one-way ANOVA using Tukey’s post hoc test). (E) Primary cultures of postnatal mouse cortical neurons were co-infected with Mito-DsRed2 lentivirus (10 MOI) and either control shRNA or Drp1 shRNA lentivirus (10 MOI) that co-express EGFP 24 hr after plating. Cells were fixed five days after infection and immunostained for β-tubulin III (TuJ1 antigen), a neuronal marker. EGFP fluorescence represents neurons expressing control/Drp1 shRNA. Representative data are shown from three independent experiments.

Figure 6. Dominant negative Drp1 induces neuronal cell death

Primary cultures of postnatal mouse cortical neurons were infected with Drp1WT (10 MOI) or Drp1K38A (10 MOI) lentivirus 24 hr after plating. The lentivirus expressing either form of Drp1 also co-expressed EGFP from an internal ribosomal entry site. Cells were fixed five days after infection and immunostained for β-tubulin III (TuJ1 antigen), a neuronal marker. Representative data are shown from four independent experiments.

Figure 7. Drp1 shRNA reduces Drp1 protein levels and increases mitochondrial fusion but does not cause cell death in mouse embryonic fibroblasts

(A) MEF were co-infected with Mito-DsRed2 lentivirus (2 MOI) and with either control lentivirus expressing EGFP (10 MOI) or dominant negative Drp1K38A lentivirus co-expressing EGFP (10 MOI) 24 hr after plating. Cells were fixed five days after infection and stained with Hoechst 33258 to depict nuclear morphology. Under the infection conditions used, the infection efficiency with Drp1K38A lentivirus and its control virus was 100% (EGFP fluorescence not shown). Representative data are shown from two independent experiments. (B) MEF were infected with lentivirus expressing control shRNA (10 MOI) or Drp1 shRNA (10 MOI) 24 hr after plating. Expression of mitochondrial fission (Drp1) and fusion (OPA1) proteins in each sample was evaluated five days after infection by Western blot analysis. The intensity of Drp1 bands was quantitated and normalized against β-actin as described in Figure 2A and presented as values relative to the level in control shRNA. Representative data are shown from two independent experiments. (C) MEF were co-infected with Mito-EGFP lentivirus (2 MOI) and either control shRNA or Drp1 shRNA lentivirus (10 MOI) 24 hr after plating. Cells were fixed five days after infection and stained with Hoechst 33258 to depict nuclear morphology. The lentivirus used in this study did not express an infection marker (EGFP), so viability was judged in those cells displaying a high degree of mitochondrial fusion that was never observed in naïve MEF expressing only Mito-EGFP or those co-infected with Mito-EGFP and control shRNA lentivirus. No evidence of damage or cell death was observed in cells expressing extensive mitochondrial fusion even five days after infection based on the morphology of Hoechst stained nuclei. Representative data are shown from two independent experiments.

Figure 8. Mfn2 knockdown promotes extensive mitochondrial fission and loss of neuronal viability

(A) Primary cultures of postnatal mouse cortical neurons were used without infection or infected with control shRNA, Mfn1 shRNA or Mfn2 shRNA lentivirus (10 MOI) 24 hr after plating. Neurons were treated with camptothecin (CPT: 5 μM) or DMSO (control) 48 after infection. Protein extracts were prepared, as described in the methods, 12 hr after treatment when neurons are committed to undergo apoptosis in a p53- and Bax-dependent manner. Expression of mitochondrial fusion proteins, Mfn1 and Mfn2, was evaluated by Western blot analysis. (B) Neurons were co-infected with Mito-DsRed2 lentivirus (2 MOI) and either Mfn1 shRNA or Mfn2 shRNA lentivirus (10 MOI) that co-expresses EGFP 24 hr after plating. Cells were fixed two days after infection and immunostained for β-tubulin III, a neuronal marker (TuJ1 antigen). Representative data are shown from five independent experiments. (C) and (D) Neurons were co-infected with Mito-DsRed2 lentivirus (2 MOI) and either control shRNA, Mfn1 shRNA or Mfn2 shRNA lentivirus (10 MOI) 24 hr after plating. Cells were fixed two days after infection and stained with Hoechst 33258 to depict nuclear morphology. Infected cells and neurons were identified based on the fluorescence of EGFP, co-expressed by the shRNA lentivirus used, and β-tubulin III (TuJ1) immunoreactivity, respectively. The fraction (%) of EGFP+/Tuj1+ neurons displaying (C) mitochondrial fragmentation (mitochondria less than 2μm in size) or (D) cell death (nuclear condensation and fragmentation based on Hoechst staining) was determined in three independent experiments by an observer that was blind to the treatments. The data reflects the average ± S.D. and is representative of three independent experiments. *, significantly different from all other conditions (p < 0.0005, one-way ANOVA using Tukey’s post hoc test).

References

1. Akepati VR, Muller EC, Otto A, Strauss HM, Portwich M, Alexander C. Characterization of OPA1 isoforms isolated from mouse tissues. J Neurochem. 2008;106:372–383. - PubMed
1. Alexander C, Votruba M, Pesch UE, Thiselton DL, Mayer S, Moore A, Rodriguez M, Kellner U, Leo-Kottler B, Auburger G, Bhattacharya SS, Wissinger B. OPA1, encoding a dynamin-related GTPase, is mutated in autosomal dominant optic atrophy linked to chromosome 3q28. Nat Genet. 2000;26:211–215. - PubMed
1. Barsoum MJ, Yuan H, Gerencser AA, Liot G, Kushnareva Y, Graber S, Kovacs I, Lee WD, Waggoner J, Cui J, White AD, Bossy B, Martinou JC, Youle RJ, Lipton SA, Ellisman MH, Perkins GA, Bossy-Wetzel E. Nitric oxide-induced mitochondrial fission is regulated by dynamin-related GTPases in neurons. Embo J. 2006;25:3900–3911. - PMC - PubMed
1. Cassidy-Stone A, Chipuk JE, Ingerman E, Song C, Yoo C, Kuwana T, Kurth MJ, Shaw JT, Hinshaw JE, Green DR, Nunnari J. Chemical inhibition of the mitochondrial division dynamin reveals its role in Bax/Bak-dependent mitochondrial outer membrane permeabilization. Dev Cell. 2008;14:193–204. - PMC - PubMed
1. Chan PH. Mitochondria and neuronal death/survival signaling pathways in cerebral ischemia. Neurochem Res. 2004;29:1943–1949. - PubMed

Drp1 levels constitutively regulate mitochondrial dynamics and cell survival in cortical neurons - PubMed (original) (raw)