Journal: Cell Death & Disease

Article Title: Mitochondrial dysfunction in an Opa1 Q285STOP mouse model of dominant optic atrophy results from Opa1 haploinsufficiency

doi: 10.1038/cddis.2016.160

Figure Lengend Snippet: Opa1 protein level and mitochondrial morphology in MEFs isolated from the Opa1 Q285STOP mouse. ( a ) Opa1 protein level is reduced in Opa1 Q285STOP MEFs. Cytochrome c (Cyt c ) is shown as a loading control. ( b ) Mitochondrial morphology in WT and Opa1 Q285STOP MEFs. Scale bar, 20 μ m. ( c ) Representative images of different mitochondrial phenotypes: elongated, mixed (heterogeneous), and fragmented mitochondria. Scale bar, 20 μ m. ( d ) Quantification of the different phenotypes in WT and mutant MEFs. Data are means±S.E.M. from six independent cell cultures/plates combined from two independent preparations of MEFs

Article Snippet: Immortalized opa1 -null and control MEFs were obtained from ATCC (Manassas, VA, USA).

Techniques: Isolation, Control, Mutagenesis

Journal: Cell Death & Disease

Article Title: Mitochondrial dysfunction in an Opa1 Q285STOP mouse model of dominant optic atrophy results from Opa1 haploinsufficiency

doi: 10.1038/cddis.2016.160

Figure Lengend Snippet: Delayed impairment in bioenergetic function and decreased expression of Complex IV in Opa1 Q285STOP MEFs. ( a and b ) Assessment of the mitochondrial membrane potential by TMRE staining and flow cytometry: ( a ) A significant decline in mitochondrial membrane potential is observed in the Opa1 mutant MEFs (blue lines) at passage 6 but not at passage 2. Right panel shows the effect of FCCP (1 μ M) that was used as a control for weak TMRE staining (low membrane potential). ( b ) Quantification of the TMRE staining data. Data shown are percentage of cells with weak TMRE staining. Error bars are S.D. from three to four independent measurements. ( c and d ) Cellular respiration (OCR) was measured in intact MEFs using Seahorse Biosciences Inc. technology. ( c ) Representative results obtained for WT and Opa1 mutant MEFs at different passages. Output panels of the Seahorse XF24 Flux Analyzer are shown, each representing ‘raw' data from an individual 24-well plate. Vertical lines indicate timing of injection of oligomycin (2 μ g/ml) and sequential injections of FCCP (600 nM) to induce resting (State 4) and maximal (state 3u) respiration, respectively. Data are means±S.E. from five to eight replicate wells. OCRs were significantly reduced in Opa1 mutant MEFs at passage 7 (left panel). Opa1-null MEFs exhibited a nearly complete loss of respiration and were used as a positive control (right panel). ( d ) Summary of respiration measurements (seven experiments similar to one shown in ( c ) were performed and averaged). Basal (before additions), State 4 (resting, oligomycin-inhibited) and state 3 u (maximal FCCP-induced) OCR in WT and late-passage (between 7 and 10) mutant MEFs are shown. Data are means±S.E. from seven independent cell cultures/plates. ( e ) Western blot analysis of ETC subunits in whole- cell lysates prepared from WT and Opa1 mutant MEFs. Some blots also include samples from Opa1-null MEFs for comparison. The levels of Complex IV subunits (COX I and COX II) were reduced in Opa1 mutant MEFs while there were no decreases in the levels of Complex I subunits (ND4, NDUFB8, and NDUFS7). The levels of COX I subunit are shown on two immunoblots (from two independent cell lysate preparations). VDAC, Hsp60 or Tom20 remained unchanged in Opa1 mutant MEFs. The membranes from each individual blot were probed with an Opa1 antibody to verify the partial loss of Opa1 in mutant samples (see also ). ( f and g ) Complex IV activity measured in MEFs permeabilized with perfringolysin O (PFO). ( f ) Results of a representative experiment shown as the Seahorse XF e 96 Flux Analyzer output panel. Vertical lines indicate following injections: 1 μ M antimycin A (Ant A); 2.5 mM TMPD and 5 mM ascorbate (TMPD); 20 mM NaN 3 (azide). ( g ) Summary results of three experiments. Complex IV activity was measured as OCR in the presence of TMPD (as shown on panel f). Note that this reaction was almost completely inhibited by addition of NaN 3 (‘+ azide'). Data are mean±S.E.M. ***, **, and * statistically significant differences with P <0.001, P <0.01, and P <0.05, respectively

Article Snippet: Immortalized opa1 -null and control MEFs were obtained from ATCC (Manassas, VA, USA).

Techniques: Expressing, Membrane, Staining, Flow Cytometry, Mutagenesis, Control, Injection, Positive Control, Western Blot, Comparison, Activity Assay

Journal: Cell Death & Disease

Article Title: Mitochondrial dysfunction in an Opa1 Q285STOP mouse model of dominant optic atrophy results from Opa1 haploinsufficiency

doi: 10.1038/cddis.2016.160

Figure Lengend Snippet: Mutated Opa1 is not expressed at detectable levels in Opa1 Q285STOP mice. ( a ) Samples of indicated tissues derived from the Opa1 Q285STOP mice were probed for the presence of Opa1 1–285 protein by western blot analysis using Opa1 antisera generated against an N-terminal Opa1 polypeptide. As a positive control for the specific antisera reactivity, samples of HeLa cells with doxycycline-inducible expression of Opa1 1–289 (also shown in panel c) or the recombinant Opa1 polypeptide (rOpa1 114–289 ) were loaded on the gels, where indicated. As a negative control for antibody crossreactivity, a sample of Opa1-null cells was added, where indicated. Samples were loaded at 30–50 μ g per lane. Endogenous truncated Opa1 was not detected in any of the samples tested (including isolated liver mitochondria), whereas WT Opa1 and inducibly expressed Opa1 1–289 were readily detected by the Opa1 antisera. ( b ) Quantification of Opa1 protein level confirms its ~50% reduction in all mutant samples. ( c ) Doxycycline-induced Opa1 1–289 expression in HeLa cells one day (second lane) or 2 days (last lane) after the addition of doxycycline. Whole-cell lysates were loaded at 30 μ g per lane. ( d ) Proteasomal inhibition by MG132 does not lead to accumulation of endogenous Opa1 1–285 . MEFs were pretreated with 2 μ M MG132 for 24 h before cell lysate preparation. (Last three lanes on the blot contained the recombinant Opa1 polypeptide loaded at indicated amounts.) The membrane was reprobed with ubiquitin antibody to confirm accumulation of ubiquinated proteins and VDAC antibody for a loading control

Article Snippet: Immortalized opa1 -null and control MEFs were obtained from ATCC (Manassas, VA, USA).

Techniques: Derivative Assay, Western Blot, Generated, Positive Control, Expressing, Recombinant, Negative Control, Isolation, Mutagenesis, Inhibition, Membrane, Ubiquitin Proteomics, Control

Journal: Cell Death & Disease

Article Title: Mitochondrial dysfunction in an Opa1 Q285STOP mouse model of dominant optic atrophy results from Opa1 haploinsufficiency

doi: 10.1038/cddis.2016.160

Figure Lengend Snippet: Differential effects of apoptogens on WT and Opa1 Q285STOP MEFs. ( a-d ) Maximal respiratory capacity of intact cells (state 3u) treated with various apoptogens was determined in experiments similar to one shown in . ( a ) 300 μ M etoposide (Etop), 1 μ M actinomycin D (Actin D), 1 μ M thapsigargin (Thaps) and 2 μ g/ml tunicamycin (Tunic) were added 24 h before the measurements, and STS (1 μ M) was added for 5 h. All incubations were performed in the presence of 20 μ M Q-VD. ( b ) Relative change in maximal respiratory capacity was calculated from data plotted in ( a ). OCRs of apoptogen-treated WT and mutant MEFs were normalized to OCRs of respective untreated (either WT or mutant) cells in each experiment and averaged. Data shown are means±S.E., n =3–7. ( c and d ) Time course of the effect of ER stressors on respiration. WT and Opa1 mutant MEFs were treated with thapsigargin ( c ) or tunicamycin ( d ) for 1, 6, and 24 h, and respective OCRs (averaged from five replicate wells for each time point) were normalized to untreated cells at time 0 h. ( e ) Representative images of WT and Opa1 mutant MEFs treated with indicated apoptogens (as in a ) and immunostained for active Bax (red). Cells were also immunostained for Tim23 or cytochrome c (green) as indicated. Nuclei were stained with Hoechst 33342 (blue). Some confocal images are shown with phase contrast to demonstrate cell morphology. Arrows indicate Bax-positive (apoptotic) cells. Scale bar, 20 μ m. Bax-positive cells were virtually absent from untreated samples. ( f ) A fold change in the number of apoptotic Opa1 mutant cells relative to the WT. Statistical analysis was performed using one-way ANOVA with post hoc Newman–Keuls multiple comparison test. Significant differences between effects of apoptogens are indicated (** P <0.01). Effects of apoptogens (STS, thapsigargin, and tunicamycin) compared with control are also statistically significant ( P <0.05). ( g ) Percentage of cells with activated Bax among WT and Opa1 mutant MEFs treated with thapsigargin, tunicamycin, or etoposide (as in a ). Data are means±S.E. from three independent experiments. (For STS, the numbers were variable between experiments and only normalized data are shown in panel f)

Article Snippet: Immortalized opa1 -null and control MEFs were obtained from ATCC (Manassas, VA, USA).

Techniques: Mutagenesis, Staining, Comparison, Control

Journal: International Journal of Molecular Sciences

Article Title: Reduced VDAC1, Maintained Mitochondrial Dynamics and Enhanced Mitochondrial Biogenesis in a Transgenic Tau Mouse Model of Alzheimer’s Disease

doi: 10.3390/ijms23158561

Figure Lengend Snippet: Western blot, immunofluorescence, and quantification analysis of proteins regulating mitochondrial dynamics in 6-month-old WT, VDAC1 +/− , TAU, and VDAC1 +/− /TAU mice. ( A ) Representative immunoblots. ( B ) Quantitative densitometry study of mitochondrial dynamics found that the fission of DRP1 (* p < 0.05) and FIS1 (** p < 0.01) was dramatically decreased, whereas the fusion of MFN1, MFN2, and OPA1 (* p < 0.05) was significantly enhanced in VDAC1 +/− /TAU animals compared to TAU mice. Forty micrograms (g) of total protein were put into each lane. The loading control was carried out with the help of the housekeeping protein beta-actin. Data are from three independent experiments showed similar results (N = 3). Three animals were randomly selected from each group/genotype for immunoblotting and immunofluorescence analyses from a total of ten animals studied for behavioral phenotype . ( C ) Representative immunofluorescence images of 10-micron coronal sections (10×). ( D ) Fluorescence intensity analysis of mitochondrial dynamics-DRP1 (**** p < 0.0001), FIS1 (**** p < 0.0001) (fission) was significantly decreased, MFN1 (*** p < 0.001), MFN2 (**** p < 0.0001) and OPA1 (**** p < 0.0001) (fusion) were significantly increased in VDAC1 +/− /TAU mice compared to TAU mice. The data are from three separate experiments, all of which yielded comparable findings (N = 3), and each mouse was exposed to 10–15 fields. Scale bar: 200 μm. The results were presented as the mean accompanied by the standard error of the mean; ns denotes that the difference did not reach statistical significance; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; one-way ANOVA followed by Turkey’s test for multiple comparisons.

Article Snippet: The processes of mitochondrial fission proteins: DRP1 (12957-1-AP, Rabbit Polyclonal 1:1000; Protein Tech Group, Rosemont, IL, USA), FIS1 (NB100-56646, Rabbit Polyclonal 1:500; Novus Biologicals, Littleton, CO, USA), fusion proteins: MFN1: (13798-1-AP, Rabbit Polyclonal 1:500; Protein Tech Group), MFN2 (9482, Rabbit Polyclonal 1:1000; Cell Signaling Technology, Danvers, MA, USA), OPA1 (NBP2-59770, Rabbit Polyclonal 1:1000; Novus Biologicals) and biogenesis proteins: PGC1A (NBP1-04676, Rabbit Polyclonal 1:1000; Novus Biologicals), NRF1 (46743, Rabbit Polyclonal 1:1000; Cell Signaling Technology), NRF2 (NBP1-32822, Rabbit Polyclonal 1:1000; Novus Biologicals), TFAM (ab131607, Rabbit Polyclonal 1:2000; Abcam, Waltham, MA, USA) were investigated using immunoblotting.

Techniques: Western Blot, Immunofluorescence, Control, Fluorescence

Journal: Human Molecular Genetics

Article Title: Loss of mitochondrial peptidase Clpp leads to infertility, hearing loss plus growth retardation via accumulation of CLPX, mtDNA and inflammatory factors

doi: 10.1093/hmg/ddt338

Figure Lengend Snippet: Most Clpp -/- tissues show up-regulation of mitochondrial chaperones

Article Snippet: Opa1 , Mm00453879_m1 , 1.5× P = 0.001 , ns , ns , ns.

Techniques: Western Blot

Journal: Human Molecular Genetics

Article Title: Loss of mitochondrial peptidase Clpp leads to infertility, hearing loss plus growth retardation via accumulation of CLPX, mtDNA and inflammatory factors

doi: 10.1093/hmg/ddt338

Figure Lengend Snippet: Clpp -/- testis shows massive affection of transcript levels, while most tissues show moderate anomalies

Article Snippet: Opa1 , Mm00453879_m1 , 1.5× P = 0.001 , ns , ns , ns.

Techniques: TaqMan Assay