Exclusive neuronal expression of SUCLA2 in the human brain

SUCLA2 encodes the ATP-forming subunit (A-SUCL-) 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- 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- immunoreactivity co-localized >99% with that of the d subunit of the mitochondrial F0-F1 ATP synthase. Specificity of the anti-A-SUCL- antiserum was verified by the absence of labeling in fibroblasts from a patient with a complete deletion of SUCLA2. A-SUCL- 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 subunit (G-SUCL-) 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- immunoreactivity that was however, not upregulated in samples obtained from diabetic versus non-diabetic patients, as has been described for murine brain. Our work establishes that SUCLA2 is expressed exclusively in neurons in the human cerebral cortex

Parathyroid hormone 2 receptor and its endogenous ligand tuberoinfundibular peptide of 39 residues are concentrated in endocrine, viscerosensory and auditory brain regions in macaque and human

Parathyroid hormone receptor 2 (PTH2R) and its ligand, tuberoinfundibular peptide of 39 residues (TIP39) constitute a neuromodulator system implicated in endocrine and nociceptive regulation. We now describe the presence and distribution of the PTH2R and TIP39 in the brain of primates using a range of tissues and ages from macaque and human brain. In situ hybridization histochemistry of TIP39 mRNA, studied in young macaque brain, due to its possible decline beyond late postnatal ages, was present only in the thalamic subparafascicular area and the pontine medial paralemniscal nucleus. In contrast, in situ hybridization histochemistry in macaque identified high levels of PTH2R expression in the central amygdaloid nucleus, medial preoptic area, hypothalamic paraventricular and periventricular nuclei, medial geniculate, and the pontine tegmentum. PTH2R mRNA was also detected in several human brain areas by RT-PCR. The distribution of PTH2R-immunoreactive fibers in human, determined by immunocytochemistry, was similar to that in rodents, including dense fiber networks in the medial preoptic area, hypothalamic paraventricular, periventricular and infundibular (arcuate) nuclei, lateral hypothalamic area, median eminence, thalamic paraventricular nucleus, periaqueductal gray, lateral parabrachial nucleus, nucleus of the solitary tract, sensory trigeminal nuclei, medullary dorsal reticular nucleus, and dorsal horn of the spinal cord. Colocalization suggested that PTH2R fibers are glutamatergic, and that TIP39 may directly influence hypophysiotropic somatostatin containing and indirectly influence corticotropin releasing-hormone containing neurons. The results demonstrate that TIP39 and the PTH2R are expressed in the brain of primates in locations that suggest involvement in regulation of fear, anxiety, reproductive behaviors, release of pituitary hormones, and nociception. Published by Elsevier Ltd on behalf of IBRO

Paralemniscal TIP39 is induced in rat dams and may participate in maternal functions

The paralemniscal area, situated between the pontine reticular formation and the lateral lemniscus in the pontomesencephalic tegmentum contains some tuberoinfundibular peptide of 39 residues (TIP39)-expressing neurons. In the present study, we measured a 4 times increase in the level of TIP39 mRNA in the paralemniscal area of lactating mothers as opposed to nulliparous females and mothers deprived of pups using real-time RT-PCR. In situ hybridization histochemistry and immunolabeling demonstrated that the induction of TIP39 in mothers takes place within the medial paralemniscal nucleus, a cytoarchitectonically distinct part of the paralemniscal area, and that the increase in TIP39 mRNA levels translates into elevated peptide levels in dams. The paralemniscal area has been implicated in maternal control as well as in pain perception. To establish the function of induced TIP39, we investigated the activation of TIP39 neurons in response to pup exposure as maternal, and formalin injection as noxious stimulus. Both stimuli elicited c-fos expression in the paralemniscal area. Subsequent double labeling demonstrated that 95% of neurons expressing Fos in response to pup exposure also contained TIP39 immunoreactivity and 91% of TIP39 neurons showed c-fos activation by pup exposure. In contrast, formalin-induced Fos does not co-localize with TIP39. Instead, most formalin-activated neurons are situated medial to the TIP39 cell group. Our data indicate that paralemniscal neurons may be involved in the processing of maternal and nociceptive information. However, two different groups of paralemniscal neurons participate in the two functions. In particular, TIP39 neurons may participate in the control of maternal functions

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.

We have recently shown that the ATP-forming SUCLA2 subunit of succinyl-CoA ligase, an enzyme of the citric acid cycle, is exclusively expressed in neurons of the human cerebral cortex; GFAP- and S100-positive astroglial cells did not exhibit immunohistoreactivity or in situ hybridization reactivity for either SUCLA2 or the GTP-forming SUCLG2. However, Western blotting of post mortem samples revealed a minor SUCLG2 immunoreactivity. In the present work we sought to identify the cell type(s) harboring SUCLG2 in paraformaldehyde-fixed, free-floating surgical human cortical tissue samples. Specificity of SUCLG2 antiserum was supported by co-localization with mitotracker orange staining of paraformaldehyde-fixed human fibroblast cultures, delineating the mitochondrial network. In human cortical tissue samples, microglia and oligodendroglia were identified by antibodies directed against Iba1 and myelin basic protein, respectively. Double immunofluorescence for SUCLG2 and Iba1 or myelin basic protein exhibited no co-staining; instead, SUCLG2 appeared to outline the cerebral microvasculature. In accordance to our previous work there was no co-localization of SUCLA2 immunoreactivity with either Iba1 or myelin basic protein. We conclude that SUCLG2 exist only in cells forming the vasculature or its contents in the human brain. The absence of SUCLA2 and SUCLG2 in human glia is in compliance with the presence of alternative pathways occurring in these cells, namely the GABA shunt and ketone body metabolism which do not require succinyl CoA ligase activity, and glutamate dehydrogenase 1, an enzyme exhibiting exquisite sensitivity to inhibition by GTP