Mice deficient in dihydrolipoyl succinyl transferase show increased vulnerability to mitochondrial toxins

Abstract

The activity of a key mitochondrial tricarboxylic acid cycle enzyme, alpha-ketoglutarate dehydrogenase complex (KGDHC), declines in many neurodegenerative diseases. KGDHC consists of three subunits. The dihydrolipoyl succinyl transferase (DLST) component is unique to KGDHC. DLST(+/-) mice showed reduced mRNA and protein levels and decreased brain mitochondrial KGDHC activity. Neurotoxic effects of mitochondrial toxins were exacerbated in DLST(+/-) mice. MPTP produced a significantly greater reduction of striatal dopamine and tyrosine hydroxylase-positive neurons in the substantia nigra pars compacta of DLST(+/-) mice. DLST deficiency enhanced the severity of lipid peroxidation in the substantia nigra after MPTP treatment. Striatal lesions induced by either malonate or 3-nitropropionic acid (3-NP) were significantly larger in DLST(+/-) mice than in wildtype controls. DLST deficiency enhanced the 3-NP inhibition of mitochondria enzymes, and 3-NP induced protein and DNA oxidations. These observations support the hypothesis that reductions in KGDHC may impair the adaptability of the brain and contribute to the pathogenesis of neurodegenerative diseases.

Figure 1

Gene expression and protein levels of KGDHC subunits in DLST^+/- mice. A. Brain homogenates were subjected to quantitative real-time PCR to assess changes in the mRNA level of ogdh , dlst and dld subunits of KGDHC. mRNA for β-2-microglobulin (β2m) was measured in the same sample as an internal control. Values represent means ± SEM of fold changes over DLST^+/+ mice from at least two independent experiments done in triplicate after normalization to β2m. * P < 0.05. B. Total protein was isolated from brains of mice and subjected to SDS-PAGE followed by Western blotting probed with antibodies against OGDH, DLST or DLD. β-actin immunoreactivity was used as an internal control. Representative blots are shown for three subunits and β-actin. C. Quantitation of protein levels of OGDH, DLST and DLD. Values represent means ± SEM of relative densities of the subunit from three independent experiments after normalization to β-actin. * P < 0.05.

Figure 2

KGDHC activity was reduced in brains of DLST^+/- mice. A. Enzymatic activity was measured in whole brain homogenates (6 week old mice). DLST^+/- brains showed significantly lower activity. B. Nitroblue tetrazolium (NBT) staining density, measured in brain sections by in situ activity staining, was lower in each region of DLST^+/- brains. Abbreviations; Cb, cerebellum; Ctx, neocortex; HB, hindbrain; Hc, hippocampus; HT, hypothalamus; MB, midbrain; OB, olfactory bulb; St, striatum; Th, thalamus. Data for each group were done in at least three brains, in triplicate. * P < 0.05. All values represent means ± SEM.

Figure 3

KGDHC activity during development. Specific enzymatic activity was measured in triplicate. Values represent means ± SEM. * P < 0.05. n = 3-9. No significant increase in the activity after P30.

Figure 4

Enzyme activities in mitochondria of DLST deficient mice. A. The activity of mitochondria KGDHC isolated from DLST^+/- brains was half of that from DLST^+/+ brains. Other evaluated mitochondrial enzymes activities were similar in DLST^+/- and DLST^+/+ mitochondria. Values are means ± SEM. * p < 0.05. n = 12. B. Immunoblotting of MnSOD, GR, and GPx1 proteins in isolated mitochondria shows no difference between DLST^+/- and DLST^+/+. C-I, respiratory chain Complex I; COX, cytochrome oxidase; MDH, malate dehydrogenase; PDHC, pyruvate dehydrogenase complex; KGDHC, α-ketoglutarate dehydrogenase complex; ME, malic enzyme; GR, glutathione reductase; GPx1, mitochondrial glutathione peroxidase 1; MnSOD, manganese superoxide dismutase.

Figure 5

DLST deficiency enhances MPTP toxicity. A. DLST deficiency enhances MPTP-induced dopamine depletion in the striatum. *** p < 0.001, versus wildtype/PBS; * p < 0.05, versus wildtype/MPTP. B. Tyrosine hydroxylase (TH) immunoreactivity in the substantia nigra pars compacta (SNpc). Boxed areas on top are magnified in lower panel of photomicrographs. C. Stereological analysis of TH-immunoreactive (TH+) neurons in SNpc showing exacerbation of MPTP-induced TH+ neuron loss in the SNpc of DLST^+/- mice. Values are means ± SEM. * p < 0.05, versus wildtype/MPTP; ** p < 0.01 versus wildtype/PBS. D. Malondialdehyde (MDA) immunoreactivity in the SNpc. Boxed areas on top are magnified in lower panel of photomicrographs. E. Stereological analysis of MDA-immunoreactive neurons in SNpc showing increased number of intensely stained neurons in MPTP-treated DLST-deficient mice compared to MPTP-treated wildtype control. Values are means ± SEM. ** p < 0.01 versus wildtype/MPTP. n = 10-12. I, Wildtype/PBS; II, DLST^+/-/PBS; III, Wildtype/MPTP; IV, DLST^+/-/MPTP. F. Confocal images of combined immunofluorescence of TH (green) and MDA (red) antisera showing co-existence (yellow) in neurons in the SNpc of a DLST^+/- mouse treated with MPTP. Arrows show a TH-positive neuron with increased MDA immunoreactivity. Point bars show MDA immunostaining in a TH-negative cell. Arrowheads show a TH-immunoreactive neuron without MDA-immunoreactivity.

Figure 6

DLST-deficiency enhances malonate toxicity. A. NeuN-stained sections through the caudate putamen lesioned with malonate show an exacerbation of the lesion in DLST^+/- mice. Broken lines show demarcation of the areas of NeuN-stained neuron loss. Boxed areas in the photomicrographs are magnified in the lower panel showing the severity of loss of NeuN-labeled neurons. B. Lesion volume analysis using stereological cavalieri method shows an increase of lesion volumes in DLST^+/- mice. Values are means ± SEM. * p < 0.05. n = 10-11. C. CD40L immunoreactivity in the caudate putamen lesioned with malonate. There is marked proliferation and hypertrophy of CD40L-labeled astrocytes in DLST^+/- mice. Boxed areas in the photomicrographs are magnified in the lower panel. ec= external capsule, LV= lateral ventricle, CPu= caudate putamen.

Figure 7

DLST-deficiency enhances 3-NP toxicity. A. Motor function in 3NP lesioned mice when suspended by the tail. After 5 days of 3-NP intoxication, the DLST^+/- mouse exhibited sustained flexure of both forelimbs and hindlimbs, while the wildtype mouse showed less severe symptoms. B. Effect of 3-NP treatment on the activity of mitochondrial enzymes in striata of DLST^+/- mice and their wild type littermates (see results for interpretation). * p < 0.05 compared with its respective control; # p < 0.05 compared to wildtype/3-NP. n = 3. C. NeuN-stained sections through the caudate putamen lesioned with 3-NP show an exacerbation of lesion in DLST^+/- mice. Broken lines show demarcation of the areas of NeuN-stained neuron loss. Boxed areas in the photomicrographs are magnified in the lower panel to show the extent of loss of NeuN-immunoreactive neurons. ec=external capsule, LV=lateral ventricle, CPu=caudate putamen; acp=anterior commissure, posterior part. D. Lesion volumes obtained by stereological cavalieri method show an increase of lesion volumes in DLST^+/- mice. Values are means ± SEM, * p < 0.05. n = 8. E. Representative photomicrographs of nitrotyrosine immunoreactivity in the cortex. Insets show the staining in neurons. F. Representative photomicrographs of 8-hydroxy-2-deoxyguanosine immunoreactivity in the cortex. Insets show the staining in neuronal nuclei. n=5 in each group. cg=cingulum. I, Wildtype/PBS; II, DLST^+/-/PBS; III, Wildtype/3-NP; IV, DLST^+/-/3-NP. C-I, respiratory chain Complex I; C-II, Complex II; CS, citrate synthase; KGDHC, α-ketoglutarate dehydrogenase complex.