P. Fattoretti, C. Bertoni-Freddari, T. Casoli, G. Di Stefano, B. Giorgetti, Y. Grossi, M. Balietti

Cytochrome oxidase (COX), complex IV of the mitochondrial respiratory chain, is composed of 13 subunits: three encoded by mitochonmdrial DNA (mtDNA) and ten encoded by nuclear DNA. The coordinated expression of both genomes constitutes an early step in the synthesis of the COX enzyme complex, conceivably quantitative estimations of the level of expression of COX subunits may provide clues to identifying precocious alterations of mitochondrial dysfunction. On the other hand, phenotypically evident changes in the mitochondrial capacity to provide adequate amounts of ATP (a term also referred to as mitochondrial metabolic competence: MMC) may be due to significant changes occurring in mitochondria considered as functional units of the cellular bioenergetic machinery. To seek age-related signs of early mitochondrial damage in adult and old rats, we measured the activity of COX of synaptic mitochondria by a preferential cytochemical method and then compared these data with the levels of immunohistochemically evidenced mitochondrial and nuclear encoded subunits. COX activity within mitochondria was evidenced by the diaminobenzidine (DAB) procedure that enables the fine localization of the cytochemical precipitate at the site where the enzyme is located, i.e. the mitochondrial inner membrane. The ratio (R) between the overall area of the DAB-COX deposits and the area of the mitochondrion was estimated by a computer-assisted image analysis system. From a functional standpoint, R may be considered as the fraction of the mitochondrial area directly involved in cellular respiration. The levels of mtDNA encoded subunit I and of nuclear encoded subunit IV were measured by quantitative immunohistochemistry. In the hippocampus (dentate gyrus distal dendrites) and cerebellum (granular layer of the cerebellar cortex) of old rats R was significantly decreased, while the levels of the two investigated subunits were higher, at a not significant extent, in the granular and molecular layer of the cerebellar cortex. In the same hippocampal area of old animals the levels of the two subunits were significantly higher. The present findings support that gene expression of COX subunit I and IV appears not to be involved in the currently reported age-related mitochondrial functional decay as also confirmed by the present significant reduction of R. This apparent discrepancy between our present results finds a tenable explanation by taking into account that COX activity relies on the overall balance of several determinants (e.g. assembly factors and the physico-chemical condition of the mitochondrial membranes) that can be differently affected by aging.

Keywords (Optional): 
Mitochondrial metabolic competence
Cytochrome oxidase
COX subunit I
COX subunit IV
COX immunohistochemistry