Exclusive neuronal detection of KGDHC-specific subunits in the adult human brain cortex despite pancellular protein lysine succinylation

Arpad Dobolyi,Christos Chinopoulos,Frank Jordan,Judit Doczi,Natalia S Nemeria,Vera Adam-Vizi,Attila Ambrus,Miklos Palkovits,Attila Bago

doi:10.1007/s00429-020-02026-5

Abstract

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.

Highlights

Ketoglutarate dehydrogenase complex (KGDHC) is a multi-subunit enzyme residing in the mitochondrial matrix (Maas and Bisswanger 1990)
KGDHC is further involved in transaminations and glutamate metabolism (Cooper 2012), while its product, succinyl-CoA, can be shuttled towards heme synthesis (Atamna et al 2001; Atamna and Frey 2004; Burch et al 2018), and/or mitochondrial substrate-level phosphorylation. mSLP is almost exclusively attributed to succinyl-CoA ligase (SUCL), the enzyme following KGDHC in the citric acid cycle, catalyzing the reversible conversion of succinyl-CoA and ADP to CoASH, succinate, and ATP (Johnson et al 1998)
KGDHC consists of multiple copies of three subunits: oxoglutarate dehydrogenase (OGDH) or oxoglutarate dehydrogenase-like protein (OGDHL), dihydrolipoyl succinyltransferase (DLST), and dihydrolipoyl dehydrogenase (DLD)

Summary

Introduction

Ketoglutarate dehydrogenase complex (KGDHC) is a multi-subunit enzyme residing in the mitochondrial matrix (Maas and Bisswanger 1990). It catalyzes the irreversible decarboxylation of α-ketoglutarate (αKg) to succinyl-CoA. The complex participates in the citric acid cycle exhibiting a high flux-control coefficient in the generation of reducing equivalents (Cooney et al 1981; Sheu and Blass 1999), contributing to the maintenance of the mitochondrial redox state (Gibson et al 2000). MSLP is almost exclusively attributed to succinyl-CoA ligase (SUCL), the enzyme following KGDHC in the citric acid cycle, catalyzing the reversible conversion of succinyl-CoA and ADP (or GDP) to CoASH, succinate, and ATP (or GTP) (Johnson et al 1998). Regulation of the complex and its overall impact on energy metabolism has been extensively reviewed elsewhere (Gibson et al 2010; Starkov 2013)

Methods

Results

Conclusion