Testing mitochondrial metabolic competence by cytochrome oxidase preferential cytochemistry vs. immmunoreactivity of subunit I and IV
P. Fattoretti, C. Bertoni-Freddari, T. Casoli, G. Di Stefano, B. Giorgetti, Y. Grossi, M. Balietti
Neurobiology of Aging Laboratory, INRCA Research Department, Via Birarelli 8, 60121 Ancona Italy
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.
Key words:
Mitochondrial metabolic competence, Cytochrome oxidase, COX subunit I, COX subunit IV, COX immunohistochemistry
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