Pyruvate dehydrogenase complex (PDHC; EC 1.2.4.1, EC 2.3.1.12 and EC 1.6.4.3) includes 3 catalytically active mitochondrial enzymes involved in the formation of cellular energy through the tricarboxylic acid cycle and in the synthesis of ACh. We sought to determine whether immunocytochemically detected PDHC was enriched in neurons of the rat CNS, and, if so, whether the perikarya containing higher levels of PDHC immunoreactivity were differentially distributed with respect to their size or location within nuclear groups containing ACh, catecholamines or other unidentified transmitters. Under the labeling conditions used in this study, the peroxidase-antiperoxidase immunoreaction product for PDHC was detectable principally in neuronal perikarya. The intensity of immunoreactivity within perikarya was variable as judged visually and by cellular, computer-assisted densitometry. In the forebrain, the most intensely labeled perikarya were seen in the medial septal nuclei, the nuclei of the diagonal band, the nuclei basalis, the dorsal and ventral striatum, and the entorhinal cortex. More caudally, intense immunoreactivity was detected in perikarya in the supraoptic hypothalamic nuclei, reticular thalamic nuclei, lateral substantia nigra, most of the tegmental nuclei, lateral nuclei of the trapezoid body, raphe pontis and obscuris, and the caudal part of the lateral reticular nuclei. In addition, many of the motor nuclei of the cranial nerves, including the dorsal motor nuclei of the vagus and the hypoglossal nuclei, and the nucleus ambiguus contained perikarya with intense PDHC labeling. Densitometry revealed no differences in intensity of immunoreactivity in soma of varying sizes. However, the intensity of neuronal labeling for PDHC was significantly greater in several nuclear groups that were shown in adjacent sections to contain cholinergic, but not catecholaminergic, enzymes. In contrast, the primary olfactory cortex, pyramidal cell layer of the regio inferior of hippocampus, and the Purkinje cell layer of the cerebellum were regions having perikarya with intense PDHC immunoreactivity but lacking both the synthetic and the degradative enzymes for ACh. These results provide the first morphological evidence that PDHC, a general metabolic enzyme complex, is enriched in selective perikarya that are heterogeneously distributed in brain and are especially abundant in many of the regions containing cholinergic neurons. The heterogeneity of PDHC immunoreactivity suggests that certain cholinergic as well as noncholinergic nuclei may be selectively vulnerable to mitochondrial diseases involving pyruvate utilization.

Several groups have reported abnormalities of the pyruvate dehydrogenase complex (PDHC) in cultured cells or other tissues from patients with Leigh's disease (subacute necrotizing encephalomyelopathy). We therefore undertook studies to elucidate the molecular basis of the defect of PDHC in cultured skin fibroblasts from two patients with Leigh's disease. The deficit of total PDHC activity in homogenates of Leigh's disease fibroblasts could be restored by adding exogenous lipoamide dehydrogenase (LAD, E3), the third component of PDHC. The LAD in these Leigh's disease cells had a markedly reduced ability (less than 20% of normal LAD) to reconstitute with other PDHC components to form active enzyme complex. A polyclonal antibody to pig heart LAD inhibited LAD activity in control cells more efficiently than in Leigh's disease cells. Other mitochondrial enzyme activities and growth of these two Leigh's disease cells appeared normal. These results suggest that the deficiency of PDHC in these two patients with Leigh's disease was due to a structural abnormality of the LAD component of PDHC.

At least seven adult-onset neurodegenerative diseases, including Huntington's disease (HD),

A 3y/o male presented with a progressive neurologic disorder characterized by hypotonia, ophthalmoplegia and ataxia. Postmortum studies at 4 1/2y revealed Leigh's disease. Biochemical findings included elevation of blood lactate (L) (ranging from 1.9 to 7.OmM, normal mean f SD 1.0+0.4mM), pyruvate (P) (0.11 to 0.25, normal 0.11+0.03mM) and L/P ratio (14 to 35, normal 10 to 18). 0-OH-butyrate (BOH), acetoacetate and L were intermittently present in urine. Plasma amino acids were normal. CSF L (4.7mM) and P (0.24mM) were elevated. Increased urinary excretion of glycine and alanine was present. No increase in blood L or P was noted after IV glucose (100mg/kg). During an 18 hr fast blood BOH increased to 200OuM (normal 91+81vM, n=4) with little change in blood L, P, or glucose levels. Skeletal muscle biopsy (premortum) showed absent CO on histochemical staining. Fibroblasts from patients with isovaleric L d e m i a (IVA), a disorder of leucine catabolism, have deficient activity of isovaleryl-CoA dehydrogenase (IVDH), as demonstrated by Rhead and Tanakj (PNAS 77:580, 1980) with a tritium release assay using [2,3-Hlisovaleryl-CoA as substrate. They found 12% of control activity using mitochondria isolated from 5 IVA lines. However, in crude homogenatef of IVA cells they measured 50-60% of normal activity, while 12-4~]leucine oxidation by the intact cells was 1-2% of normal. While this demonstrated a specific deficiency of mitochondria1 IVDH activity in IVA cells, the magnitude of residual dehydrogenating activity could not be accurately determined. To resolve this problem, we improved the assay by using Normal ASB and the mutant enzyme in Maroteaux-Lamy syndrome (MLS) have been shown to be immunologically indistinguishable when compared using rabbit anti-ASB antiserum. In the present study a monoclonal antibody library was obtained by fusion of spleen cells from mice immunized with purified ASB and X63NSl/lAg-4 (NSl) mice myeloma cells. Seventy-six hybridoma lines with ASB antibody specificity were obtained, of which 64 were subcloned and further studied. Of these, 56 reacted with both control and mutant ASB. Five reacted only with the active form of ASB and not with either the mutant form or with partially denatured (enzymatically inactive) control ASB. Three reacted only with heat denatured or urea treated control ASB and with the mutant enzyme but not with the active form. The specificity of these antibodies toward ASB was documented using Western blotting. Several of these hybridoma lines were injected into Pristane sensitized mice, and the specific monoclonal antibodies werc purified from the ascites fluid. A solid phase radioimmunoassay was developed using IV-16-8 antibody (reacts with control and mutant ASB and has high binding affinity to polyvinyl chloride), 1251-labeled V-4-7 (reacts with both mutant and control ASB), and 1251-labeled V-18-8 (reacts specifically with active ASB). Using this assay the ratio between active and inactive ASB was determined in liver, fibroblast and leukocyte homogenates from con...