Journal: Journal of neurochemistry

Article Title: Mutant Huntingtin Fails to Directly Impair Brain Mitochondria

doi: 10.1111/jnc.14852

Figure Lengend Snippet: In A, mHtt was detected with mHtt-specific monoclonal antibody 1C2 as a single 350 kDa band in brain cytosolic fraction from YAC128 but not WT mice. Here and in other panels (B-D), MEK1/2 was used as a cytosolic marker, indicating the absence of cytosolic contamination in mitochondrial fraction, and Complex II, 70 kDa subunit (CII) was used as a mitochondrial marker as control for loading. In B, mHtt enrichment in brain cytosolic fraction from YAC128 mice after concentrating with Spin-XR UF 500 100k MWCO concentrators (Sigma). In C, mHtt presence in brain mitochondrial fraction from YAC128 mice (YAC) and binding of mHtt to WT mitochondria. WT mitochondria were incubated with either 20 or 80μg of protein of concentrated YAC128 brain cytosolic fraction for 5 minutes and then washed out three times. In D, enrichment of YAC128 mitochondria with mHtt following incubation with either 80 or 120μg of protein of concentrated YAC128 brain cytosolic fraction for 5 or 15 minutes. In E, the statistical summary of densitometry of mHtt bands obtained with 1C2 antibody in cytosolic fractions before and after concentrating procedure. Data are mean±SD, n=4 separate independent mitochondrial preparations, **p<0.01 comparing the level of mHtt in YAC128 cytosolic fraction before and after concentrating procedure. In F and G, the statistical summaries of densitometry of mHtt bands obtained with 1C2 antibody in mitochondrial samples before and after treatment with concentrated mHtt-containing cytosolic fraction. Mutant Htt band intensities were normalized to respective CII signals. Data are mean±SD, n=4 separate independent mitochondrial preparations, *p<0.05 comparing the level of mHtt bound to WT mitochondria treated with 80μg of concentrated mHtt-containing cytosolic fraction and to untreated YAC128 mitochondria. **p<0.01 comparing the level of mHtt bound to WT mitochondria treated with either 20 or 80μg of concentrated mHtt-containing cytosolic fraction (F); **p<0.01 comparing the level of mHtt bound to untreated YAC128 mitochondria and YAC128 mitochondria treated with either 80 or 120μg of concentrated mHtt-containing cytosolic fraction (G).

Article Snippet: Wild-type FVB/NJ (RRID: IMSR_JAX:001800) mice and transgenic HD YAC128 (RRID: IMSR_JAX:004938) mice of both sexes were used in this study.

Techniques: Marker, Control, Binding Assay, Incubation, Mutagenesis

Journal: Journal of neurochemistry

Article Title: Mutant Huntingtin Fails to Directly Impair Brain Mitochondria

doi: 10.1111/jnc.14852

Figure Lengend Snippet: In A and C, representative traces of oxygen consumption by mitochondria treated with either vehicle (green traces), WT concentrated cytosolic fraction (WT cf, blue traces), or YAC128 concentrated cytosolic fraction (HD cf, red traces). Where indicated, ADP (250μM in A, 150μM in C), 2,4-dinitrophenol (2,4-DNP, 60μM), and vehicle (10μL of Isolation Medium 1), or WT or YAC128 cytosolic fractions (WT cf or HD cf, 80μg of protein) were added to mitochondria. The incubation medium was supplemented with either 3 mM pyruvate (pyr) and 1 mM malate (mal) (A) or 3 mM succinate (succ) and 3 mM glutamate (glu) (C). In B and D, the statistical summary of respiratory rates of mitochondria incubated with either pyruvate plus malate (B) or succinate plus glutamate (D). Data are mean±SD, n=5 separate independent mitochondrial preparations.

Article Snippet: Wild-type FVB/NJ (RRID: IMSR_JAX:001800) mice and transgenic HD YAC128 (RRID: IMSR_JAX:004938) mice of both sexes were used in this study.

Techniques: Isolation, Incubation

Journal: Journal of neurochemistry

Article Title: Mutant Huntingtin Fails to Directly Impair Brain Mitochondria

doi: 10.1111/jnc.14852

Figure Lengend Snippet: In A and C, representative traces of oxygen consumption by mitochondria treated with either WT concentrated cytosolic fraction (WT cf, blue traces) or YAC128 concentrated cytosolic fraction (HD cf, red traces). Where indicated, ADP (250μM in A, 150μM in C), 2,4-dinitrophenol (2,4-DNP, 60μM), and WT or YAC128 cytosolic fractions (WT cf or HD cf, 80μg of protein) were added to mitochondria. The incubation medium was supplemented with either 3 mM pyruvate (pyr) and 1 mM malate (mal) (A) or 3 mM succinate (succ) and 3 mM glutamate (glu) (C). In B and D, the statistical summary of respiratory rates of mitochondria incubated with either pyruvate plus malate (B) or succinate plus glutamate (D). Data are mean±SD, n=5 separate independent mitochondrial preparations.

Article Snippet: Wild-type FVB/NJ (RRID: IMSR_JAX:001800) mice and transgenic HD YAC128 (RRID: IMSR_JAX:004938) mice of both sexes were used in this study.

Techniques: Incubation

Journal: Journal of neurochemistry

Article Title: Mutant Huntingtin Fails to Directly Impair Brain Mitochondria

doi: 10.1111/jnc.14852

Figure Lengend Snippet: In A-C and E-G, representative TPP+ traces indicative of changes in mitochondrial membrane potential (Δψ) in response to 250μM ADP and 60μM 2,4-dinitrophenol following addition of vehicle (10μL of Isolation Medium 1, green traces) (A, E), or cytosolic fractions from either WT (WT cf, blue traces) (B, F) or YAC128 (HD cf, red traces) (C, G) mice. In A-C, the standard incubation medium was supplemented with 3 mM pyruvate (pyr) and 1 mM malate (mal). In E-G, the standard incubation medium was supplemented with 3 mM succinate (succ) and 3 mM glutamate (glu). In D and H, statistical summaries of mitochondrial membrane potential measurements taken 30 seconds prior to ADP addition in the standard incubation medium containing pyruvate plus malate or succinate plus glutamate, respectively. Data are mean±SD, n=4 separate independent mitochondrial preparations.

Article Snippet: Wild-type FVB/NJ (RRID: IMSR_JAX:001800) mice and transgenic HD YAC128 (RRID: IMSR_JAX:004938) mice of both sexes were used in this study.

Techniques: Membrane, Isolation, Incubation

Journal: Journal of neurochemistry

Article Title: Mutant Huntingtin Fails to Directly Impair Brain Mitochondria

doi: 10.1111/jnc.14852

Figure Lengend Snippet: In A-D, representative TPP+ traces indicative of changes in mitochondrial membrane potential (Δψ) in response to 250μM ADP and 60μM 2,4-dinitrophenol following addition of brain cytosolic fractions from either WT (WT cf, blue traces) (A, C), or YAC128 (HD cf, red traces) (B, D) mice. In A-B, the standard incubation medium was supplemented with 3 mM pyruvate (pyr) and 1 mM malate (mal). In C-D, the standard incubation medium was supplemented with 3 mM succinate (succ) and 3 mM glutamate (glu). In E and F, statistical summaries of mitochondrial membrane potential measured 30 seconds prior to ADP addition in the standard incubation medium containing pyruvate plus malate or succinate plus glutamate, respectively. Data are mean±SD, n=4 separate independent mitochondrial preparations.

Article Snippet: Wild-type FVB/NJ (RRID: IMSR_JAX:001800) mice and transgenic HD YAC128 (RRID: IMSR_JAX:004938) mice of both sexes were used in this study.

Techniques: Membrane, Incubation

Journal: Journal of neurochemistry

Article Title: Mutant Huntingtin Fails to Directly Impair Brain Mitochondria

doi: 10.1111/jnc.14852

Figure Lengend Snippet: In A and B, H2O2 production by brain mitochondria isolated from WT mice before and after the addition of vehicle (10μL of Isolation Medium 1, green traces) or brain cytosolic fractions from either WT (WT cf, blue traces) or YAC128 (HD cf, red traces) mice. H2O2 production was assessed using an Amplex Red assay (Molecular Probes) with mitochondria incubated in the standard incubation medium supplemented with either 3 mM pyruvate (pyr) and 1 mM malate (mal) (A) or 3 mM succinate (succ) and 3 mM glutamate (glu) (B). In C and D, statistical summaries of mitochondrial H2O2 production rate. Data are mean±SD, n=4 separate independent mitochondrial preparations.

Article Snippet: Wild-type FVB/NJ (RRID: IMSR_JAX:001800) mice and transgenic HD YAC128 (RRID: IMSR_JAX:004938) mice of both sexes were used in this study.

Techniques: Isolation, Amplex Red Assay, Incubation

Journal: Journal of neurochemistry

Article Title: Mutant Huntingtin Fails to Directly Impair Brain Mitochondria

doi: 10.1111/jnc.14852

Figure Lengend Snippet: In A and B, H2O2 production by brain mitochondria isolated from YAC128 mice before and after the addition of brain cytosolic fractions from either WT (WT cf, blue traces) or YAC128 (HD cf, red traces) mice. H2O2 production was assessed using an Amplex Red assay (Molecular Probes) with mitochondria incubated in the standard incubation medium supplemented with either 3 mM pyruvate (pyr) and 1 mM malate (mal) (A) or 3 mM succinate (succ) and 3 mM glutamate (glu) (B). In C and D, statistical summaries of mitochondrial H2O2 production rate. Data are mean±SD, n=4 separate independent mitochondrial preparations.

Article Snippet: Wild-type FVB/NJ (RRID: IMSR_JAX:001800) mice and transgenic HD YAC128 (RRID: IMSR_JAX:004938) mice of both sexes were used in this study.

Techniques: Isolation, Amplex Red Assay, Incubation

Journal: Journal of neurochemistry

Article Title: Mutant Huntingtin Fails to Directly Impair Brain Mitochondria

doi: 10.1111/jnc.14852

Figure Lengend Snippet: In A and C, Ca2+ uptake capacity of brain mitochondria from either WT or YAC128 mice, respectively, following addition of cytosolic fractions from either WT (WT cf, blue traces) or YAC128 (HD cf, red traces) mice. The standard incubation medium was supplemented with 0.1 mM ADP and 1μM oligomycin (Chalmers and Nicholls 2003). In A, control with vehicle (10μL of Isolation Medium 1, green trace) is shown. Ca2+ uptake was assessed with a miniature Ca2+-selective electrode as a decrease in Ca2+ concentration in the standard incubation medium supplemented with 3 mM pyruvate (pyr) and 1 mM malate (mal). In B and D, statistical summaries of Ca2+ uptake capacity of brain mitochondria from WT and YAC128 mice, respectively. Data are mean±SD, n=5 separate independent mitochondrial preparations.

Article Snippet: Wild-type FVB/NJ (RRID: IMSR_JAX:001800) mice and transgenic HD YAC128 (RRID: IMSR_JAX:004938) mice of both sexes were used in this study.

Techniques: Incubation, Control, Isolation, Concentration Assay