PMC:3905285 / 21382-30605 JSONTXT

{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/3905285","sourcedb":"PMC","sourceid":"3905285","source_url":"http://www.ncbi.nlm.nih.gov/pmc/3905285","text":"Discussion\nWe describe here a novel disease mechanism involving lipoic acid in humans that is induced by mutations involving the LIPT1 gene. This increases the spectrum of the lipoic acid defects beyond those recently implicated in the de novo biosynthesis of lipoic acid, which include LIAS and the ancillary iron sulfur [Fe-S] cluster pathway (see above). We show that LIPT1 mutations reduce lipoylation of PDHc and α-KGDHc, and result in severely decreased PDHc and α-KGDHc enzyme activities and abnormal pyruvate utilization by polarography.\nBased on available evidence [6,7], impaired attachment of non-protein bound, AMP-activated lipoic acid on mitochondrial proteins (thus affecting a putative lipoic acid “salvage” pathway) might be responsible for PDH and α-KGDH deficiencies in this patient. However, free lipoic acid does not seem to be able to compensate for other defects in the lipoylation pathway in yeast and mouse [5,11,15], whereas we show that it can partly rescue the growth of yeasts deficient for the LIPT1 orthologue. Possible mechanisms are presented in Figure 4, which shows on the top, the putative salvage pathway of free AMP-activated lipoic acid, and on the bottom, an alternative or complementary mechanism with LIPT1 that redistributes lipoyl residues from the E2 subunit (H protein) of glycine cleavage system to the other complexes (as proposed in yeast [15]). In this mechanism, the original de novo pathway that involves LIPT2 and LIAS would act only on the H protein. Other mechanisms, such as partial redundancy between LIPT1 and LIPT2 operating either upstream of LIAS and/or in the usage of free AMP-lipoic acid as a substrate, cannot be excluded. None of these mechanisms accounts for the low branched chain amino acid levels in our patient and the greater susceptibility to BCKDHc lipoylation rescue of the patient fibroblasts (Table 1 and Figure 1C), suggesting that LIPT1 may be differentially required for BCKDHc vs α-KGDHc and PDHc (see also below).\nFigure 4 Possible pathways for lipoic acid attachment to mitochondrial α-ketodehydrogenase apoenzymes. AMP-activated forms of free lipoic acid could be a source of direct lipoylation via LIPT1 (the “lipoic acid salvage pathway”). This mechanism is at odds with some data in mice and yeast, yet it may account for apparent compensatory effects observed in the human disorders (see text). Another mechanism proposed in yeasts [15] may involve the H protein of the glycine cleavage system as a donor of lipoyl redidues to the other complexes via LIPT1. In this case, the de novo pathway involving LIPT2 and LIAS would act only on the H protein. This partly accounts for the different amino acid profiles observed in patients. The action of LIPT1 on BCKDH may be more complex than for other dehydrogenases (see text). The most striking biochemical features involved plasma amino acids that at presentation, associated increased levels of glutamine, proline with low levels of lysine and branched amino acids. Because of absent glycine increase, this profile more closely resembles the typical profile of E3 subunit/DLD deficiency rather than other genes involved in de novo biosynthesis of lipoic acid such as NFU1, LIAS, IBA57, BOLA3 (as hyperglycinemia is a hallmark of the associated biochemical phenotype). Of note, the fact that elevated glycine (in several biological fluids) was the only observed amino acid abnormality in these diseases except for NFU1 deficiency, raises the possibility that when de novo lipoate synthesis is impaired, secondary deficiency of the dehydrogenase complexes other than the glycine cleavage system may be compensated for, though only partly, by LIPT1-mediated lipoylation. This is consistent with the possibility that part of LIPT1 action may be independent of LIPT2 and LIAS, and this may be related to its ability to process free lipoate (Figure 4). In addition, the profile of our patient differed from E3 deficiency as branched chain amino acid levels were unexpectedly very low to low-normal, whereas they tend to be elevated in E3 deficiency, because of the associated BCKDH deficiency (which isolated, is responsible for maple syrup urine disease [16]). We verified that E3 activity was normal. Branched chain amino acid levels are known to be low in presence of BCKDH derepression following BKDK inactivation [17] and also in other conditions (e.g., nutritional deficiency), but none of these conditions can readily be reconciled with the proposed mechanisms of LIPT1 deficiency (Figure 4), as well as with some of our in vitro studies (Figure 1C), or the overall amino acid profile shown by our patient at diagnosis and throughout follow-up (Table 1). Tissue specific differences may be involved, because basal BCKDH activity was clearly reduced in patient fibroblasts (Figure 3D).\nOther explanations are possible and these results should be interpreted in the light that a large proportion of patients with related disorders such as E3 subunit (DLD) [18] and NFU1 deficiency have shown only transient or intermittent biochemical abnormalities (personal unpublished data), consistent with extensive heterogeneity of clinical and biochemical features in these diseases.\nBased on our enzyme assays, CO2 production by the Krebs cycle and mitochondrial respiratory chain activity in patient fibroblasts were very low compared to controls after administration of each of three substrates (glucose, butyrate and 3OHbutyrate), suggesting a defect in both the Krebs’ cycle and PDH. Interestingly, the level of 3OHbutyrate also increased in fibroblast supernatant, presumably resulting from decreased α-KGDH activity. Furthermore, 3OHbutyrate level decreased with a concomitant increase of glucose in fibroblast supernatant after LIPT1 transfection, consistent with the possibility that the cells metabolized 3OHbutyrate upon partial rescue of α-KGDH activity. The novel combination of biochemical tests presented here is a promising tool for Krebs cycle investigations, particularly for in vitro testing of potential therapeutic applications. From this test we have been able to infer that candidate dietary treatments for this metabolic disease, such as ketogenic diet, were likely detrimental. The argument was very low CO2 production after administration of C143OHbutyrate and C14butyrate.\nContrary to our patient, who had biochemical abnormalities restricted to BCKDH, PDH and α-KGDH, the phenotype of the diseases that involve lipoic acid de novo biosynthesis show numerous additional dysfunctions that are related to the [Fe-S] cluster pathway. These additional dysfunctions [5,19] affect succinate dehydrogenase and aconitase, two proteins from the tricarboxylic acid cycle, as well as the respiratory chain complexes I-III [5,19]. This may explain why in these patients, neurological symptoms were associated with multisystem abnormalities [2,3,8,10,11]. By contrast the clinical picture of our patient was limited to brain thus similar to primary PDH deficiencies. The extreme severity of the neurological defects may result from the added contribution of α-KGDH deficiency which is expected to induce a stronger energetic defect compared to isolated PDH deficiency. Differential tissue-specificity of LIPT1 function(s) vs the classical de novo lipoylation pathway might also be involved [20]. However it is of note that in our patient, biochemical clues expected from an energetic disease such as hyperlactatemia and hyperpyruvicemia, or urinary α-ketoglutarate were observed only during a metabolic attack. This intermittent and moderate biochemical presentation raises the possibility that the disease may be underdiagnosed. In fact, it was mainly the amino acid profile, partly suggestive of E3 subunit/DLD-like deficiency (see above), that prompted us to test for deficiency of α-KGDH and PDH.\nA major challenge for this new and severe enzyme cofactor disease is therapy. A treatment with lipoic acid may not be indicated as its attachment to enzyme complexes is expected to be impaired in the absence of LIPT1. However Δlip3 moderately improved growth in ethanol medium supplemented with lipoic acid, whereas activity of PDH, but not α-KGDH or BCKDH, moderately increased in patient fibroblasts cultured with lipoic acid supplementation in the medium. These preliminary results raise the possibility that lipoic acid may have therapeutic effects, though moderate and requiring further validation.\nIn conclusion, we report pathogenic LIPT1 gene mutations in humans, which alter lipoate binding in PDHc and α-KGDHc, and we provide strong evidence for the existence of a lipoylation pathway specific to PDHc and α-KGDHc in humans. This pathway is at least partly distinct from the de novo pathway and does not affect lipoylation of the glycine cleavage complex, as was proposed in yeast [[15] and Figure 4]. In addition, our data indicate that LIPT1 deficiency may affect lipoylation of BKCDHc in a partly unpredicted fashion that remains to be characterized. Further investigations are required to clarify the complex metabolism of lipoic acid cofactor in humans, and more efficient procedures should be devised for early diagnosis and prevention of metabolic decompensations.","divisions":[{"label":"Title","span":{"begin":0,"end":10}},{"label":"Figure 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