Exclusive neuronal expression of SUCLA2 in the human brain

Abstract

SUCLA2 encodes the ATP-forming b subunit (A-SUCL-b) of succinyl-CoA ligase, an enzyme of the citric acid cycle. Mutations in SUCLA2 lead to a mitochondrial disorder manifesting as encephalomyopathy with dystonia, deafness and lesions in the basal ganglia. Despite the distinct brain pathology associated with SUCLA2 mutations, the precise localization of SUCLA2 protein has never been investigated. Here, we show that immunoreactivity of A-SUCL-b in surgical human cortical tissue samples was present exclusively in neurons, identified by their morphology and visualized by double labeling with a fluorescent Nissl dye. A-SUCL-b immunoreactivity colocalized [99 % with that of the d subunit of the mitochondrial F 0 -F 1 ATP synthase. Specificity of the anti-A-SUCL-b antiserum was verified by the absence of labeling in fibroblasts from a patient with a complete deletion of SUCLA2. A-SUCL-b immunoreactivity was absent in glial cells, identified by antibodies directed against the glial markers GFAP and S100. Furthermore, in situ hybridization histochemistry demonstrated that SUCLA2 mRNA was present in Nissl-labeled neurons but not glial cells labeled with S100. Immunoreactivity of the GTP-forming b subunit (G-SUCL-b) encoded by SUCLG2, or in situ hybridization histochemistry for SUCLG2 mRNA could not be demonstrated in either neurons or astrocytes. Western blotting of post mortem brain samples revealed minor G-SUCL-b immunoreactivity that was, however, not upregulated in samples obtained from diabetic versus nondiabetic patients, as has been described for murine brain. Our work establishes that SUCLA2 is expressed exclusively in neurons in the human cerebral cortex.

Localization of SUCLA2 and SUCLG2 subunits of succinyl CoA ligase within the cerebral cortex suggests the absence of matrix substrate-level phosphorylation in glial cells of the human brain

“…In our previous study, we identified neurons and astrocytes using the markers Nissl and GFAP/S100, respectively (Dobolyi et al 2013). In the present work we extended our investigation by including the microglial marker ionized calcium-binding adapter molecule 1 (Iba1) and oligodendroglial marker myelin basic protein.…”

“…We have used a new antibody directed against the human SUCLG2 protein that does not distinguish among various isoforms (see "Materials and Methods") as in (Dobolyi et al 2013). The specificity of SUCLG2 immunoreactivity was validated using human fibroblast cells, see Fig.…”

“…This antibody was raised by immunizing rabbits with a recombinant fragment corresponding to the amino acid region 363-432 of human SUCLG2 (NP_003839). Therefore, it should not be able to distinguish among transcript variants (Dobolyi et al 2013). Cells were subsequently decorated by using the appropriate Alexa 488-linked secondary antibody (1:5,000, donkey antir a bb i t , J ac ks o n I m m un oc he m i c als E u r o pe L t d , Cambridgeshire, UK) in the presence of 1 % donkey serum.…”

“…Characteristically, in the human brain SUCLA2 is found only in the neurons, while SUCLG2 is not found either in neurons or in glial fibrillary acidic protein (GFAP)-and S100-positive astroglial cells (Dobolyi et al 2013).…”

“…Mindful of the weak immunoreactivity for SUCLG2 in Western blotting experiments from post mortem human brain samples (Dobolyi et al 2013), we sought to identify the cell type(s) harboring mitochondria with this protein. In our previous study, we identified neurons and astrocytes using the markers Nissl and GFAP/S100, respectively (Dobolyi et al 2013).…”