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Dichloroacetate (DCA) is both an environmental contaminant and an investigational drug for diseases involving perturbed mitochondrial energetics. DCA is biotransformed to glyoxylate by maleylacetoacetate isomerase (MAAI). Previous studies have shown that DCA decreases MAAI activity in rat liver in a time- and dose-dependent manner and may target the protein for degradation in vivo. We now report that the MAAI protein is depleted in a time- and dose-dependent manner in the livers of Sprague-Dawley rats exposed to DCA. This decrease in protein expression is not mirrored by a decrease in the steady-state levels of MAAI mRNA, indicating that the depletion is exclusively a post-transcriptional event. We also investigated the pharmacokinetics of DCA in the recently developed MAAI knockout (MAAI-KO) mouse. MAAI-KO mice maintain high plasma and urine drug concentrations and do not biotransform DCA to monochloroacetate to a significant extent. Therefore, no alternative pathways for DCA clearance appear to exist in mice other than by MAAI-mediated biotransformation. DCA-nai;ve MAAI-KO mice accumulate very high levels of the tyrosine catabolites maleylacetone and succinylacetone, and DCA exposure did not significantly increase the levels of these compounds. MAAI-KO mice also have high levels of fumarylacetone and normal levels of fumarate. These results demonstrate that pharmacologic or genetic ablation of MAAI cause potentially toxic concentrations of tyrosine intermediates to accumulate in mice and perhaps in other species.

To determine the feasibility of gene transfer to correct defects in the E1alpha subunit of the pyruvate dehydrogenase (PDH) complex (PDC), we constructed rAAV vectors that expressed PDH E1alpha, either alone or with a green fluorescent protein tag, from a hybrid cytomegalovirus (CMV) enhancer/chicken beta-actin (CB) promoter. These vectors were functional in vitro, as judged by increased expression of mRNA in vector-transduced deficient cell lines and correction of the biochemical defect in PDH activity in these cells. Approximately 30% of wild-type levels of PDH activity were restored under conditions with which only about 15% of cells were transduced. These same vectors were then used in vivo to transduce neurons within the rat striatum. Gene transfer, expression, and translocation into mitochondria were observed, without any obvious untoward effects. In vivo vector-mediated PDH expression persisted for at least 1 year after injection, indicating the stability of gene transfer. These studies provide the basis for future efforts to develop a recombinant AAV (rAAV)-based gene therapy approach for the correction of PDC deficiency.

Brain cells are dependent on glucose and oxygen for energy. We investigated the effects of hypoxia, glucose deprivation, and hypoxia plus glucose deprivation on mRNA and protein levels of glucose transporter (GLUT1) and GLUT3 and 2-deoxyglucose (2-DG) uptake in primary cultures of rat neurons and astroglia. Hypoxia for 24 hours did not significantly affect cell viability but increased neuronal GLUT1 and GLUT3 mRNA up to 40-fold and fivefold, respectively, above control levels. Similar changes in GLUT1 mRNA were measured in glia. The effects of hypoxia on GLUT1 and GLUT3 mRNA were reversible. The increase in GLUT1 mRNA could be detected within 20 minutes of hypoxia and was blocked by actinomycin D. Nuclear runoff transcription assays showed that hypoxia did not alter the transcription rate of GLUT1. However, hypoxia enhanced the stability of GLUT1 mRNA in neurons (half-life [t(l/2)] > 12 hours) compared with normoxic conditions (t(1/2) approximately 10.4 hours), suggesting the existence of a posttranscriptional mechanism for the regulation of GLUT1 transcript levels. Twenty-four hours of normoxia and 1.0 mmol/L glucose increased neuronal GLUT1 mRNA less than threefold above basal, but 24 hours of glucose and oxygen deprivation increased GLUT1 over 111-fold above basal. Induction of neuronal GLUT1 mRNA was temporally associated with increased levels of GLUT1 protein and with stimulation of intracellular 2-DG accumulation. We conclude that hypoxia reversibly increases the transcript levels of GLUT1 and GLUT3 in rat brain cells and stimulates GLUT1 transcript levels by posttranscriptional mechanisms. Although glucose deprivation alone produces minimal effects on GLUT mRNA levels, hypoxia plus glucose deprivation synergize to markedly increase GLUT gene expression.

Mitochondrial transcription in the early Xenopus laevis embryo resumes several hours before active mtDNA replication, effectively decoupling mtDNA transcription and replication. This developmental feature makes Xenopus embryogenesis an appealing model system to investigate the regulation of mitochondrial transcription. Studies reported here refine our understanding of the timing, magnitude, and mechanism of this transcriptional induction event. Northern analyses of six mitochondrial mRNAs (normalized to mtDNA) reveal that transcript levels remain basal between fertilization and gastrulation and then undergo a coordinate induction, culminating in a 20-28-fold increase over egg levels by 48 h of development. Measurement of mitochondrial run-on transcription rates demonstrates a good correlation between transcription rates and transcript levels, showing that transcription itself is the primary determinant of transcript abundance. Experimental increases in mitochondrial ATP and energy charge also correlate with patterns of transcript levels and transcription rates, suggesting that developmental changes in the biochemical composition of the mitochondrial matrix could be regulating transcriptional activity. Consistent with this idea, transcriptional run-on rates in mitochondria of early embryos can be stimulated by the addition of tricarboxylic acid cycle intermediates to the run-on reaction. However, mitochondria of later stages do not show this response to the addition of metabolite. In combination, these data suggest that mitochondrial transcription is under metabolic regulation during early Xenopus embryogenesis.

Treatment of cultured human cervical carcinoma cells with the anticancer drug dequalinium (DEQ) was found to cause a delayed inhibition of cell growth. This inhibition was preceded by a loss of mitochondrial DNA (mtDNA), a decrease in cytochrome c oxidase activity, and an increase in the level of lactate, indicating that growth inhibition was due to the loss of mtDNA-encoded functions. There was a progressive two-fold loss of mtDNA following each cell division in the presence of DEQ, suggesting that this drug was acting by inhibiting some aspect of mtDNA synthesis. Furthermore, cells became resistant to the growth inhibitory and cytotoxic affects of DEQ when they were grown under conditions that bypassed the need for mtDNA-encoded functions. Resistance was not associated with significant changes in drug accumulation. These results suggest that the DEQ-induced depletion of mtDNA plays an important role in drug cytotoxicity., (Copyright 1998 Academic Press.)

Using in organello footprint analysis, we demonstrate that within human placental mitochondria there is a high level of protein-DNA binding at regularly phased intervals throughout a 500-bp region encompassing the D-loop DNA origins and two promoter regions. Comparison with in vitro DNase I protection studies indicates that this protein-DNA interaction is due to non-sequence-specific binding by human mitochondrial transcription factor A (h-mtTFA). Since h-mtTFA can bend and wrap DNA, like its yeast counterpart ABF2, a primary function of h-mtTFA appears to be specific packaging of the mitochondrial DNA control region in vivo. Intervals of protein binding coincide with the spacing of the RNA start sites and prominent D-loop DNA 5' ends, suggesting a role for phased h-mtTFA binding in defining transcription and H-strand DNA replication origins. Significant protein-DNA interaction was also observed within the human homolog of conserved sequence block 1, both in organello and in vitro, using purified h-mtTFA.