Elsevier

Neuroscience

Volume 88, Issue 4, February 1999, Pages 1137-1151
Neuroscience

Postembedding immunogold labelling reveals subcellular localization and pathway-specific enrichment of phosphate activated glutaminase in rat cerebellum

https://doi.org/10.1016/S0306-4522(98)00298-XGet rights and content

Abstract

Phosphate activated glutaminase is a key enzyme in glutamate synthesis. Here we have employed a quantitative and high resolution immunogold procedure to analyse the cellular and subcellular expression of this enzyme in the cerebellar cortex. Three main issues were addressed. First, is phosphate activated glutaminase exclusively or predominantly a mitochondrial enzyme, as biochemical data suggest? Second, to what extent is the mitochondrial content of glutaminase dependent on cell type and transmitter identity? Third, can individual neurons maintain a subcellular segregation of mitochondria with different glutaminase content? An attempt was also made to disclose the intramitochondrial localization of glutaminase, and to correlate the content of this enzyme with that of glutamate and glutamine in the same mitochondria (by use of triple labelling). Antisera to the N- and C-termini of glutaminase revealed strong labelling of the putatively glutamatergic mossy fibre terminals. The vast majority of gold particles (∼96%) was associated with the mitochondria. Equally high labelling intensities were found in mitochondria of perikarya and dendrites in the pontine nuclei, a major source of mossy fibres. The level of glutaminase immunoreactivity in parallel and climbing fibres (which like the mossy fibres are thought to use glutamate as transmitter) was only about 20% of that in mossy fibres, and similar to that in non-glutamatergic neurons (Purkinje and Golgi cells). Glial cell mitochondria were devoid of specific glutaminase labelling and revealed a much lower glutamate:glutamine ratio than did the mitochondria of mossy fibres. As to the submitochondrial localization of glutaminase, immunogold particles were often found to be aligned with the cristae, suggesting an association of the enzyme with the inner mitochondrial membrane. However, the existence of a glutaminase pool in the mitochondrial matrix could not be excluded. The outer mitochondrial membrane was unlabelled.

The present study provides quantitative evidence for a substantial heterogeneity in the mitochondrial content of glutaminase. This heterogeneity applies not only to neurons with different transmitter signatures, but also to different categories of glutamatergic pathways. In terms of the routes involved the synthesis of transmitter glutamate may be less uniform than previously expected.

Section snippets

Immunogens

The antisera to PAG were raised against cysteine conjugated peptides (purity ∼80%; The Centre of Biotechnology, Oslo, Norway) corresponding to the N- and C-terminus (Cys-SEILQELGKGG, amino acids 77–87; Cys-TVHKNLDGLL, amino acids 665–674) of the rat enzyme.[50]The peptides were coupled via the cysteine to keyhole limpet hemocyanine (KLH; Sigma), using m-maleido-benzoyl-N-hydroxysuccinimide ester (MBS; Pierce, Rockford, IL, U.S.A.) as a coupling agent. For each peptide 1.4 mg MBS (dissolved in

Immunoblotting

SDS–PAGE and subsequent immunoblotting of tissue from cortex, hippocampus, striatum, cerebellum and brainstem revealed only two bands (Fig. 1) corresponding to the 65,000 and 68,000 mol. wt bands observed by Haser et al.[15]

Immunogold analysis of phosphate activated glutaminase

Profiles labelled with the PAG antisera could be divided into three groups based on labelling intensity (Fig. 2). The mossy fibres were the only type of structure in the cerebellar cortex that consistently showed strong labelling with the PAG antisera (Fig. 2, Fig. 3). The

Subcellular localization of phosphate activated glutaminase

With the present postembedding immunogold approach, only epitopes expressed at the surface of the sections are available for immunolabelling. This implies that all pools of PAG should stand an equal chance of being detected, irrespective of their intracellular localization.[30]Previous immunocytochemical analyses of PAG have been based on pre-embedding labelled material. In such material the relationship between antigen concentration and signal strength is less well defined, due to the

Conclusion

Previous immunoperoxidase analyses have revealed the presence of PAG in mitochondria of perikarya and dendrites.[2]We have presently shown, by use of a quantitative postembedding technique, that PAG immunoreactivity also occurs in nerve terminal mitochondria. Mossy fibres were found to exhibit a five-fold enrichment in PAG immunolabelling, compared to parallel and climbing fibres. These results point to differences among glutamatergic fibres, in regard to the properties of PAG, or with respect

Acknowledgements

The expert technical assistance of Bjørg Riber, Karen Marie Gujord, Hilde Raanaas, Gunnar Lothe, Carina Knudsen and Thorolf Nordby is gratefully acknowledged. This study was supported by the Jahre Fund, the Nansen Fund, the Laerdal Foundation for Acute Medicine, the Norwegian Research Council, the Sasakawa Foundation, and an EU Biomed grant (BMH4-96-0851).

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