Journal: Biochemistry and Biophysics Reports

Article Title: Bax and caspases regulate increased production of mitochondria-derived reactive species in neuronal apoptosis: LACK of A role for depletion of cytochrome c from the mitochondrial electron transport chain

doi: 10.1016/j.bbrep.2015.09.004

Figure Lengend Snippet: A caspase inhibitor suppressed elevation of cellular ROS/RS after NGF withdrawal. (a) The broad-spectrum caspase inhibitor, BAF (Sigma or Enzyme Systems Products, Livermore, CA; 50 μM), attenuated but did not prevent increased ROS/RS levels caused by NGF deprivation. Over the period shown, this concentration of BAF prevents the death of >80% of NGF-deprived mouse sympathetic neurons . BAF was included in the culture media from the time of deprivation. n =112–356 cells for each treatment and time after withdrawal. (b) BAF decreased the number of NGF-deprived neurons having modestly elevated cellular ROS/RS (CM-H 2 DCFDA intensity) and also the number of neurons with greatly elevated ROS/RS. The single cell data shown is the same as that used for the averages in part a. The CM–H 2 DCFDA intensity of each neuron was normalized to the average intensity of NGF-maintained bax +/+ neurons receiving the same concentration of dye for the same time as the experimental cells. The fold changes are from these average values.

Article Snippet: Cultures were deprived of NGF by incubating them in culture medium containing a NGF-neutralizing antibody (Harlan Bioproducts or Abcam Inc., Cambridge, MA) and lacking NGF.

Techniques: Concentration Assay

Journal: Biochemistry and Biophysics Reports

Article Title: Bax and caspases regulate increased production of mitochondria-derived reactive species in neuronal apoptosis: LACK of A role for depletion of cytochrome c from the mitochondrial electron transport chain

doi: 10.1016/j.bbrep.2015.09.004

Figure Lengend Snippet: The concentration of cytochrome c in mitochondria was similar in bax +/+ and bax +/- neurons after NGF withdrawal. (a) Micrographs illustrating the criteria used to score cytochrome c localization by immunocytochemistry. The left panels show differential interference contrast images of neurons that had been deprived of NGF for 48 h and maintained in BAF (50 μM)-containing medium. The right panels are fluorescent micrograph of the same cells showing immunostaining for cytochrome c . Note the intense punctate staining in the top neuron in the top panel and the faint staining in the bottom one. The top neuron is representative of neurons that were scored as having retained cytochrome c in mitochondria. The bottom neuron and two of the three neurons shown in the lower panels are representative of cells scored as having released cytochrome c into the cytoplasm where it was rapidly degraded. (b) Time-courses of loss of cytochrome c (Cyt c ) immunostaining in NGF-deprived bax +/+ and bax +/- neurons. Cultures were maintained in medium containing BAF (50 μM) to prevent death. Scoring for cytochrome c status was done as in part a with fluorescence microscopy. Neurons were scored as having punctate (no cytochrome c release) or faint staining (released cytochrome c ) by a naive observer. n =3–4 cultures for each time point. c Western blots showing cytochrome c levels in cultures of neurons having the indicated genotypes. Loading control is β tubulin III (Tub) from the same cultures. Note the decrease in cytochrome c after NGF withdrawal. NGF-deprived cultures were maintained in BAF (50 μM)-containing medium for 48 h before immunoblot analysis. d Quantification of cytochrome c levels in cultures of neurons from mice having the indicated genotypes and receiving the indicated treatments. The left bars show that neurons with bax +/+ bax +/− , and bax −/− genotypes all expressed the same amount of cytochrome c when maintained in medium containing NGF. Cytochrome c concentration (density of immunoblot bands) was determined by immunoblotting and was normalized to amount of β tubulin III in the same cultures. The right bars show that cytochrome c had decreased by the same amounts in bax +/+ and bax +/- neurons at 24 or 48 h after NGF withdrawal ( p >0.1 within the time-point). There was no decrease in bax -/- cultures. Deprived cultures were maintained in BAF (50 μM)-containing medium. Stars indicate significantly different ( p <0.001) from NGF-maintained controls. n =3-6 cultures for each bar.

Article Snippet: Cultures were deprived of NGF by incubating them in culture medium containing a NGF-neutralizing antibody (Harlan Bioproducts or Abcam Inc., Cambridge, MA) and lacking NGF.

Techniques: Concentration Assay, Immunocytochemistry, Immunostaining, Staining, Fluorescence, Microscopy, Western Blot, Control

Journal: Biochemistry and Biophysics Reports

Article Title: Bax and caspases regulate increased production of mitochondria-derived reactive species in neuronal apoptosis: LACK of A role for depletion of cytochrome c from the mitochondrial electron transport chain

doi: 10.1016/j.bbrep.2015.09.004

Figure Lengend Snippet: Bax concentration determined ROS/RS levels in NGF-deprived neurons having similar cytochrome c content. (a) Decreasing bax gene dosage and, therefore amount of Bax protein expressed in neurons, decreased ROS/RS levels. Left, CM–H 2 DCFDA intensities in neurons deprived of NGF-for 24 h and having the indicated bax genotypes. Right, CM–H 2 DCFDA intensities in bax +/+ and bax +/− neurons deprived of NGF for 24-48 h and maintained alive in BAF (50 μM)-containing medium. Stars indicate significantly different ( p <0.001) from bax +/+ neurons at the same time-point. n =141–541 neurons. (b) Point plot of the single-cell CM–H 2 DCFDA data (BAF-treated) averaged in part a. Data is plotted on a semi-log scale to more clearly demonstrate the wide range of CM-H 2 DCFDA intensities.

Article Snippet: Cultures were deprived of NGF by incubating them in culture medium containing a NGF-neutralizing antibody (Harlan Bioproducts or Abcam Inc., Cambridge, MA) and lacking NGF.

Techniques: Concentration Assay

Journal: Biochemistry and Biophysics Reports

Article Title: Bax and caspases regulate increased production of mitochondria-derived reactive species in neuronal apoptosis: LACK of A role for depletion of cytochrome c from the mitochondrial electron transport chain

doi: 10.1016/j.bbrep.2015.09.004

Figure Lengend Snippet: Bax concentration regulated ROS/RS levels in neurons depleted of cytochrome c by H 2 O 2 treatment. (a) Left, average CM-H 2 DCFDA intensities of H 2 O 2 -treated neurons. Right, point plot of the single cell data averaged on the left. Cultures were exposed to H 2 O 2 (2 mM) for 30 min in medium containing NGF then deprived of NGF and maintained in BAF (50 μM)-containing medium for 48 h. They were then loaded with CM-H 2 DCFDA, and confocal microscopic imaging was done at this time. Neurons of each genotype treated in this manner loaded similar amounts of dye as determined by exposing CM–H 2 DCFDA-loaded cells to 2 mM H 2 O 2 for 30 min . Stars mean significantly different from bax +/+ cells. n =54-143 neurons. (b) Neurons treated with H 2 O 2 retained Δψ m . Confocal micrographs showing TMRM + staining in neurons maintained in NGF and ones treated with 2 mM H 2 O 2 for 30 min in NGF-containing medium followed by NGF deprivation and maintenance for 48 h in BAF (50 μM)-containing medium. Note intense punctuate TMRM + staining in cells with all three genotypes indicating retention of Δψ m by the mitochondria of each. (c) Average TMRM + staining increased above baseline values in bax −/− neurons that had been depleted of cytochrome c by H 2 O 2 -treatment. TMRM + staining decreased in NGF-deprived, BAF-saved bax +/+ and bax +/− cells by about the same amount regardless of whether they had been treated with H 2 O 2 ( p >0.1). Therefore, Bax concentration was positively related to ROS/RS levels but negatively related to Δψ m . Culture conditions were the same as in part a. Star means significantly different from bax +/+ cells. n =75–233 neurons.

Article Snippet: Cultures were deprived of NGF by incubating them in culture medium containing a NGF-neutralizing antibody (Harlan Bioproducts or Abcam Inc., Cambridge, MA) and lacking NGF.

Techniques: Concentration Assay, Imaging, Staining

Journal: Biochemistry and Biophysics Reports

Article Title: Bax and caspases regulate increased production of mitochondria-derived reactive species in neuronal apoptosis: LACK of A role for depletion of cytochrome c from the mitochondrial electron transport chain

doi: 10.1016/j.bbrep.2015.09.004

Figure Lengend Snippet: Forward electron transfer was retained in cytochrome c -depleted neurons. (a) The electron transport chain. The mitochondrial matrix is toward the bottom of the page and the intermembrane space toward the top. Solid arrows indicate direction of proton flow when electron transfer is forward. The path of forward electron transfer is indicated by the broken arrows. Rotenone (Rot) blocks electron transfer through complex I to ubiquinone (coenzyme Q). Oligomycin (Oligo) blocks proton flow through complex V (F 1 .F O -ATP synthase). Under certain conditions ( e.g ., loss of most cytochrome c ) consumption of ATP by complex V produces ADP and reverses the direction of proton movement and, thus, electron transfer. Superoxide is shown being produced at complex I. It can also be produced at complex III (not shown) . (b) The respiratory complex I inhibitor, rotenone (10 μM), decreased ROS/RS in NGF-deprived neurons maintained alive by BAF. Oligomycin (5 μg/ml), increased average CM-H 2 DCFDA intensity in NGF-deprived neurons maintained alive for 48 h in BAF-containing medium, indicating that blocking the F 1 .F O -ATP synthase increased ROS/RS levels. Left, averaged data. Right, point plot of raw data from single cells, in the same order. Neurons from bax +/+ mice were deprived of NGF for 48 h and maintained alive in BAF (50 μM)-containing medium. They then received the indicated treatments for 20 min during the time of CM-H 2 DCFDA loading. Treatments were included in the L15 medium to which cells were exposed during confocal microscopy. Neither rotenone nor oligomycin affected CM-H 2 DCFDA loading . Stars mean significantly different from cells without rotenone or oligomycin treatment ( p <0.001). n =216-580 neurons. (c) Oligomycin (5 μg/ml) increased Δψ m while rotenone (10 μM) greatly decreased it. Neurons were deprived of NGF and maintained in BAF (50 μM)-containing medium for 48 h. Treatment with oligomycin or rotenone was done during the last 20 min of incubation and also in the L15 medium the cells were exposed to for microscopy. TMRM + (10 nM) was included during the last 20 min of incubation and was also included in the L15 medium. These, and the above data, indicate that electron transfer remained in a forward direction in cytochrome c- depleted neurons. Stars mean significantly different from cells without rotenone or oligomycin treatment ( p <0.001). n =116–233 neurons.

Article Snippet: Cultures were deprived of NGF by incubating them in culture medium containing a NGF-neutralizing antibody (Harlan Bioproducts or Abcam Inc., Cambridge, MA) and lacking NGF.

Techniques: Produced, Blocking Assay, Confocal Microscopy, Incubation, Microscopy

Journal: Biochemistry and Biophysics Reports

Article Title: Bax and caspases regulate increased production of mitochondria-derived reactive species in neuronal apoptosis: LACK of A role for depletion of cytochrome c from the mitochondrial electron transport chain

doi: 10.1016/j.bbrep.2015.09.004

Figure Lengend Snippet: Higher Bax concentration caused higher ROS/RS levels but lower Δψ m in oligomycin-treated, cytochrome c- depleted neurons. a Oligomycin (5 μg/ml) increased CM-H 2 DCFDA intensity more in cytochrome c -depleted bax +/+ neurons than in bax +/- neurons ( p <0.001). Neurons were deprived of NGF and maintained alive for 48 h in BAF (50 μM)-containing medium. They then were treated with oligomycin (5 μg/ml) for 20 min during the time of CM-H 2 DCFDA loading. Oligomycin was included in the L15 medium that cells were exposed to during confocal microscopy. Right is a point-plot of the data from individual cells averaged in the bar graphs. The order is the same. Stars mean significantly different from the same cell type without oligomycin treatment ( p <0.001). n =283–587 neurons. b Oligomycin (5 μg/ml) increased Δψ m more in NGF-deprived, BAF-saved bax +/− neurons than in neurons with a bax +/+ genotype. NGF-deprived neurons were maintained in BAF (50 μM)-containing medium for 48 h. Treatment with oligomycin was done during the last 20 min of incubation and also in the L15 medium the cells were exposed to for microscopy. TMRM + (10 nM) was included during the last 20 min of incubation and was also included in the L15 medium. Stars mean significantly different from the same cell type without oligomycin treatment ( p <0.001). n =75–233 neurons.

Article Snippet: Cultures were deprived of NGF by incubating them in culture medium containing a NGF-neutralizing antibody (Harlan Bioproducts or Abcam Inc., Cambridge, MA) and lacking NGF.

Techniques: Concentration Assay, Confocal Microscopy, Incubation, Microscopy