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The ketoglutarate dehydrogenase complex (KGDHC) consists of three different subunits encoded by OGDH (or OGDHL), DLST, and DLD, combined in different stoichiometries. DLD subunit is shared between KGDHC and pyruvate dehydrogenase complex, branched-chain alpha-keto acid dehydrogenase complex, and the glycine cleavage system. Despite KGDHC’s implication in neurodegenerative diseases, cell-specific localization of its subunits in the adult human brain has never been investigated. Here, we show that immunoreactivity of all known isoforms of OGDHL, OGDH, and DLST was detected exclusively in neurons of surgical human cortical tissue samples identified by their morphology and visualized by double labeling with fluorescent Nissl, while being absent from glia expressing GFAP, Aldhl1, myelin basic protein, Olig2, or IBA1. In contrast, DLD immunoreactivity was evident in both neurons and glia. Specificity of anti-KGDHC subunits antisera was verified by a decrease in staining of siRNA-treated human cancer cell lines directed against the respective coding gene products; furthermore, immunoreactivity of KGDHC subunits in human fibroblasts co-localized > 99% with mitotracker orange, while western blotting of 63 post-mortem brain samples and purified recombinant proteins afforded further assurance regarding antisera monospecificity. KGDHC subunit immunoreactivity correlated with data from the Human Protein Atlas as well as RNA-Seq data from the Allen Brain Atlas corresponding to genes coding for KGDHC components. Protein lysine succinylation, however, was immunohistochemically evident in all cortical cells; this was unexpected, because this posttranslational modification requires succinyl-CoA, the product of KGDHC. In view of the fact that glia of the human brain cortex lack succinate-CoA ligase, an enzyme producing succinyl-CoA when operating in reverse, protein lysine succinylation in these cells must exclusively rely on propionate and/or ketone body metabolism or some other yet to be discovered pathway encompassing succinyl-CoA. Electronic supplementary material The online version of this article (10.1007/s00429-020-02026-5) contains supplementary material, which is available to authorized users.

The activity of mitochondrial alpha-ketoglutarate dehydrogenase complex (KGDHC) is severely reduced in human pathologies where oxidative stress is traditionally thought to play an important role, such as familial and sporadic forms of Alzheimer's disease and other age-related neurodegenerative diseases. This minireview is focused on substantial data that were accumulated over the last 2 decades to support the concept that KGDHC can be a primary mitochondrial target of oxidative stress and at the same time a key contributor to it by producing reactive oxygen species. This article is part of a Special Issue entitled 'Mitochondrial function and dysfunction in neurodegeneration'.

4-hydroxynonenal (HNE), a product of omega-6 polyunsaturated fatty acid peroxidation, impairs mitochondrial respiration in vitro by adducting the alpha-ketoglutarate dehydrogenase complex (KGDC) and inhibiting its activity. The present study seeks to define whether aging increases HNE adduction to rat heart KGDC, and whether such adduction impacts KGDC activity. We found that hearts from old rats exhibit significantly (por =0.01) higher HNE-modified mitochondrial proteins when compared with those from young rats. Among these proteins, dihydrolipoamide succinyltransferase, the E2k component of KGDC, was most markedly modified (por =0.01) by HNE with age. As opposed to that seen in vitro, no significant change in electrophoretic mobility or impairment in enzyme activity was observed. On the contrary, KGDC activity increased onefold (por =0.01) in old rats, suggesting that the aging myocardium is not affected by HNE adduction or compensates for such damage. Further analysis revealed that heightened KGDC activity was not due to increased protein content or gene expression, but correlates with a lower Km for alpha-ketoglutarate. Thus, contrary to that observed in vitro, the measurement of HNE-KGDC adduct in rat heart is more relevant as a marker of age-related protein oxidation than a factor of mitochondrial dysfunction.

The activity of a key mitochondrial enzyme, the alpha-ketoglutarate dehydrogenase complex (KGDHC), declines in the brains of patients with neurodegenerative diseases such as Alzheimer's disease, as well as in thiamine-deficient (TD) animals. The decreased activity often occurs without a reduction in enzyme protein, which negates the use of immunocytochemistry to study cellular or regional changes in enzyme activity within the brain. To overcome this limitation, an activity staining method using nitroblue tetrazolium was developed. The histochemical activity staining was standardized in cultured cells. The assay was linear with time and was highly specific for KGDHC. The dark-blue reaction product (formazan) formed a pattern that was consistent with mitochondrial localization. Treatment of the cultured cells with both reversible and irreversible inhibitors decreased formazan production, whereas conventional enzyme assays on cell lysates only revealed loss of KGDHC activity with irreversible inhibitors. The activity staining was also linear with time and highly specific for KGDHC activity in mouse brain sections. Staining occurred throughout the brain, and discrete neuronal populations exhibited particularly intense staining. The pattern of staining differed markedly from the distribution of KGDHC protein by immunocytochemistry. Generalized decreases in the intensity of activity staining that occurred in the TD brains compared to controls were comparable with the loss of KGDHC activity by conventional enzyme assay. Thus, the present study introduces a new histochemical method to measure KGDHC activity at the cellular and regional level, which will be useful to determine changes of in situ enzyme activity.

Succinylphosphonate (SP) is a powerful inhibitor or α-ketoglutarate dehydrogenase (KGD). Methylation of the phosphonate reduces its inhibitory effect. The complex of KGD with SP undergoes a kinetically slow transition similar to the process observed during catalysis. α-Ketoglutarate binds to the enzyme—inhibitor complex, preventing its isomerisation.

The burst of product accumulation during the KGD reaction was investigated. It has been shown not to be the obligatory feature of catalysis, but appears when increasing the enzyme saturation by KG. Structural analogues of KG and the SH-group modification suppress the initial burst without preventing catalysis. The results obtained are in favour of the existence of the regulatory site for binding KG and its structural analogues essential for hysteretic properties of KGD.

An α-ketoglutarate dehydrogenase-deficient mutant of Bradyrhizobium japonicum, the endosymbiont of soybean, is delayed in early symbiotic development, but eventually forms normal, acetylene-reducing bacteroids. This result suggests that B. japonicum bacteroids are not as dependent on the energy-yielding functions of the TCA cycle as previously believed, and raises the possibility that other routes for malate and succinate catabolism could be important during symbiotic nitrogen fixation.

Reducing 2 SH-groups of KGD by dihydrolipoate leads to cooperativity in substrate binding. Cooperative properties of KGD in the KGD complex are modulated by NADH. Physiological significance of these observations is discussed.

Since ancient times, a creative tension has existed between two viewpoints on illness. One, associated with the Platonic physicians of the island of Cnos, looks on diseases as specific entities which can be classified like plants. From this viewpoint, the specific patient in the examining room is a more or less close copy of an idealized patient. In modern times, this view was developed with particular strength by Sydenham in London in the 1600s. The second viewpoint, associated with the Hippocratic physicians of the island of Cos, views illness as occurring when an organism can no longer remain in balance with the challenges of its environment. In modern times, this view was put forward with particular clarity by Claude Bernard in Paris in the 1800s. He emphasized that an illness occurs when living organisms cannot maintain their “milieu interne” in the face of the challenges of their environment.

alpha-Ketoglutarate dehydrogenase was inactivated irreversibly and completely during oxidation of alpha-ketoadipic acid. The inactivation was revealed both in the model system with ferricyanide and in the overall reaction catalyzed by the alpha-ketoglutarate dehydrogenase complex. Neither substrate depletion nor product accumulation induced the inactivation. The results obtained were compared with recent data on the enzyme inactivation during oxidation of alpha-ketoglutaric acid. The differences in the inactivation kinetics observed with the two substrates of the enzyme were analyzed. They seem not to reflect the different mechanisms of the inactivation, but, rather, depend on the changes in the rates of the individual stages of the process.

Activities of pyruvate decarboxylase (PDC), alpha-ketoglutarate decarboxylase (KGDC) and both the pyruvate and alpha-ketoglutarate dehydrogenase complexes (PDH complex and KGDH complex) were measured, and kinetic properties of PDC were studied in fibroblasts derived from normal individuals and from a 2-yr-old girl with congenital lactic acidemia and severe retardation of growth and development. The activities of PDC, KGDC, PDH complex, and KGDH complex in the patient were 1.12 +/- 0.12, 2.33 +/- 0.42, 9.00 +/- 0.50, and 16.46 +/- 1.57 and in controls 3.10 +/- 0.16, 5.36 +/- 0.56, 24.13 +/- 1.61 and 44.95 +/- 3.72 nmole/mg protein/hr. The optimum pH (6.0) and Michaelis constants (Km) for pyruvate of PDC (1.0-1.6 X 10(-5) M) were similar in fibroblasts of the patient and controls. PDC activity was more sensitive to denaturation by heat in the fibroblasts of the patient than those from controls, while heat denaturation curves of KGDC were similar in the patient and control. Higher concentrations of thiamine pyrophosphate (TPP) were required to protect PDC from heat denaturation in the patient. TPP was more easily removed from PDC in the patient than in the control by washing the fibroblasts with alkaline buffer. These results suggest that the PDC enzyme of the patient is in an altered molecular form, to which TPP is loosely bound. This particular constellation of abnormalities has not previously been reported in patients with lactic acidemia.

Radiochemical methods are described in detail to measure the activities of the pyruvate dehydrogenase complex and of the ketoglutarate dehydrogenase complex in platelet-enriched fractions. Determinations can be completed in one day with as little as 5 ml of venous blood. Activities are proportional to the length of the incubation and the amount of tissue protein added, show appropriate dependence on added cofactors, are stable for up to 2 days at −20°C, and do not appear to be affected by diet. The pyruvate dehydrogenase complex appears to be fully activated (dephosphorylated) in these preparations. Activities were comparable in platelet-enriched fractions from 25 normal subjects and 25 patients with a variety of neurological and psychiatric diagnoses. Mean values (± S.E.M.) for these 50 individuals were 169 ± 9 pmol/min per mg protein for the pyruvate dehydrogenase complex and 535 ± 27 pmol/ min per mg protein for the ketoglutarate dehydrogenase complex. These values are comparable to those found in cultured skin fibroblasts with similar techniques. Deficient pyruvate dehydrogenase activity (19 ± 6 and 11 ± 4 pmol/min per mg protein) was demonstrated in platelet-enriched preparations from two brothers whose fibroblasts had previously been shown to be deficient in pyruvate dehydrogenase and who responded to a ketogenic diet. Experimental details critical to obtaining reproducible results with these methods are stressed (notably the crucial importance of maintaining the purity of the radioactive substrates). These techniques allow identification of patients with pyruvate dehydrogenase deficiencies within one day without requiring liver or muscle biopsy.

Two classes of mutant lacking α-ketoglutarate dehydrogenase complex activity were detected by biochemical analysis of strains of Escherichia coli requiring succinate for aerobic growth on glucose minimal medium. One class, designated sucA, lacked the α-ketoglutarate decarboxylase component (E1) whereas the other class, sucB, lacked the dihydrolipoyl transsuccinylase component (E2). Studies with mixed cell-free extracts showed that the overall dehydrogenase activity could be reconstituted from several pairs of sucA plus sucB mutants but not from mixtures of mutants of the same class. Transduction analysis with phage P1 indicated close linkage between the two genes and their frequencies of cotransduction with gal were similar. The order of the two genes was also established as sucA (E1)-sucB(E2)...gal by reciprocal three-point crosses with several pairs of mutants.