Expression of Gls and Gls2 glutaminase isoforms in astrocytes

Abstract

The expression of glutaminase in glial cells has been a controversial issue and matter of debate for many years. Actually, glu-taminase is essentially considered as a neuronal marker in brain. Astrocytes are endowed with efficient and high capacitytransport systems to recapture synaptic glutamate which seems to be consistent with the absence of glutaminase in theseglial cells. In this work, a comprehensive study was devised to elucidate expression of glutaminase in neuroglia and, moreconcretely, in astrocytes. Immunocytochemistry in rat and human brain tissues employing isoform-specific antibodies revealedexpression of both Gls and Gls2 glutaminase isozymes in glutamatergic and GABAergic neuronal populations as well as inastrocytes. Nevertheless, there was a different subcellular distribution: Gls isoform was always present in mitochondria whileGls2 appeared in two different locations, mitochondria and nucleus. Confocal microscopy and double immunofluorescencelabeling in cultured astrocytes confirmed the same pattern previously seen in brain tissue samples. Astrocytic glutaminaseexpression was also assessed at the mRNA level, real-time quantitative RT-PCR detected transcripts of four glutaminase iso-zymes but with marked differences on their absolute copy number: the predominance of Gls isoforms over Gls2 transcriptswas remarkable (ratio of 144:1). Finally, we proved that astrocytic glutaminase proteins possess enzymatic activity by in situactivity staining: concrete populations of astrocytes were labeled in the cortex, cerebellum and hippocampus of rat braindemonstrating functional catalytic activity. These results are relevant for the stoichiometry of the Glu/Gln cycle at the tripartitesynapse and suggest novel functions for these classical metabolic enzymes