Domenico De Rasmo

Mammalian complex I: A regulable and vulnerable pacemaker in mitochondrial respiratory function

In this paper the regulatory features of complex I of mammalian and human mitochondria are reviewed. In a variety of mitotic cell-line cultures, activation in vivo of the cAMP cascade, or direct addition of cAMP, promotes the NADH-ubiquinone oxidoreductase activity of complex I and lower the cellular level of ROS. These effects of cAMP are found to be associated with PKA-mediated serine phosphorylation in the conserved C-terminus of the subunit of complex I encoded by the nuclear gene NDUFS4. PKA mediated phosphorylation of this Ser in the C-terminus of the protein promotes its mitochondrial import and maturation. Mass-spectrometry analysis of the phosphorylation pattern of complex I subunits is also reviewed.

Occurrence of A-kinase anchor protein and associated cAMP-dependent protein kinase in the inner compartment of mammalian mitochondria.

Evidence showing the existence in the inner compartment of rat‐heart mitochondria of AKAP121 and associated PKA is presented. Immunoblotting analysis and trypsin digestion pattern show that 90% or more of mitochondrial C‐PKA, R‐PKA and AKAP121 is localized in the inner mitochondrial compartment, when prepared both from isolated mitochondria or cardiomyocyte cultures. This localization is verified by measurement of the specific catalytic activity of PKA, radiolabelling of R‐PKA by 32P‐phosphorylated C‐PKA and of AKAP by 32P‐phosphorylated R‐PKA and electron microscopy of mitochondria exposed to gold‐conjugated AKAP121 antibody.

cAMP-dependent protein kinase regulates the mitochondrial import of the nuclear encoded NDUFS4 subunit of complex I.

The subunits of complex I encoded by the mammalian nuclear genes NDUFS4 (AQDQ protein) and NDUFB11 (ESSS protein) contain serine/threonine consensus phosphorylation sequences (CPS) in their presequence, the first also in the C-terminus. We have studied the impact of PKA mediated phosphorylation on the mitochondrial import of in vitro and in vivo synthesized NDUFS4 protein. The intramitochondrial accumulation of the mature form of in vitro synthesized NDUFS4 protein, but not that of ESSS protein, was promoted by PKA and depressed by alkaline phosphatase (AP). In HeLa cells, control or transfected with the NDUFS4 cDNA construct, the mitochondrial level of mature NDUFS4 protein was promoted by 8-Br-cAMP and depressed by H89. Ser173Ala mutagenesis in the C-terminus CPS abolished the appearance in mitochondria of the mature form of NDUFS4 protein. The promoting effect of PKA on the mitochondrial accumulation of mature NDUFS4 protein appears to be due to inhibition of its retrograde diffusion into the cytosol.

The Oxidative Phosphorylation System in Mammalian Mitochondria

The chapter provides a review of the state of art of the oxidative phosphorylation system in mammalian mitochondria. The sections of the paper deal with: (i) the respiratory chain as a whole: redox centers of the chain and protonic coupling in oxidative phosphorylation (ii) atomic structure and functional mechanism of protonmotive complexes I, III, IV and V of the oxidative phosphorylation system (iii) biogenesis of oxidative phosphorylation complexes: mitochondrial import of nuclear encoded subunits, assembly of oxidative phosphorylation complexes, transcriptional factors controlling biogenesis of the complexes. This advanced knowledge of the structure, functional mechanism and biogenesis of the oxidative phosphorylation system provides a background to understand the pathological impact of genetic and acquired dysfunctions of mitochondrial oxidative phosphorylation.

cAMP response element‐binding protein (CREB) is imported into mitochondria and promotes protein synthesis

The cAMP response element‐binding protein (CREB) is a ubiquitous transcription factor in the higher eukaryotes that, once phosphorylated, promotes transcription of cAMP response element‐regulated genes. We have studied the mitochondrial import of CREB and its effect on the expression of mtDNA‐encoded proteins. [35S]Methionine‐labelled CREB, synthesized in vitro in the Rabbit Reticulocyte Lysate system using a construct of the human cDNA, was imported into the matrix of isolated rat liver mitochondria by a membrane potential and TOM complex‐dependent process. The imported CREB caused cAMP‐dependent promotion of the synthesis of mitochondrially encoded subunits of oxidative phosphorylation enzyme complexes. Thus, CREB moves from the cytosol to mitochondria, in addition to the nucleus, and, when phosphorylated by cAMP‐dependent protein kinase, promotes the expression of mitochondrial genes.

Epigallocatechin-3-gallate prevents oxidative phosphorylation deficit and promotes mitochondrial biogenesis in human cells from subjects with Down's syndrome

A critical role for mitochondrial dysfunction has been proposed in the pathogenesis of Downs syndrome (DS), a human multifactorial disorder caused by trisomy of chromosome 21, associated with mental retardation and early neurodegeneration. Previous studies from our group demonstrated in DS cells a decreased capacity of the mitochondrial ATP production system and overproduction of reactive oxygen species (ROS) in mitochondria. In this study we have tested the potential of epigallocatechin-3-gallate (EGCG) - a natural polyphenol component of green tea - to counteract the mitochondrial energy deficit found in DS cells. We found that EGCG, incubated with cultured lymphoblasts and fibroblasts from DS subjects, rescued mitochondrial complex I and ATP synthase catalytic activities, restored oxidative phosphorylation efficiency and counteracted oxidative stress. These effects were associated with EGCG-induced promotion of PKA activity, related to increased cellular levels of cAMP and PKA-dependent phosphorylation of the NDUFS4 subunit of complex I. In addition, EGCG strongly promoted mitochondrial biogenesis in DS cells, as associated with increase in Sirt1-dependent PGC-1α deacetylation, NRF-1 and T-FAM protein levels and mitochondrial DNA content. In conclusion, this study shows that EGCG is a promoting effector of oxidative phosphorylation and mitochondrial biogenesis in DS cells, acting through modulation of the cAMP/PKA- and sirtuin-dependent pathways. EGCG treatment promises thus to be a therapeutic approach to counteract mitochondrial energy deficit and oxidative stress in DS.

Respiratory chain complex I, a main regulatory target of the cAMP/PKA pathway is defective in different human diseases

In mammals, complex I (NADH‐ubiquinone oxidoreductase) of the mitochondrial respiratory chain has 31 supernumerary subunits in addition to the 14 conserved from prokaryotes to humans. Multiplicity of structural protein components, as well as of biogenesis factors, makes complex I a sensible pace‐maker of mitochondrial respiration. The work reviewed here shows that the cAMP/PKA pathway regulates the biogenesis, assembly and catalytic activity of complex I and mitochondrial oxygen superoxide production. The structural, functional and regulatory complexity of complex I, renders it particularly vulnerable to genetic and sporadic pathological factors. Complex I dysfunction has, indeed, been found, to be associated with several human diseases. Knowledge of the pathogenetic mechanisms of these diseases can help to develop new therapeutic strategies.

Complex I deficiencies in neurological disorders

Complex I is the point of entry in the mitochondrial electron transport chain for NADH reducing equivalents, and it behaves as a regulatable pacemaker of respiratory ATP production in human cells. Defects in complex I are associated with several human neurological disorders, including primary mitochondrial diseases, Parkinson disease (PD), and Down syndrome, and understanding the activity and regulation of complex I may reveal aspects of the underlying pathogenic mechanisms. Complex I is regulated by cyclic AMP (cAMP) and the protein kinase A (PKA) signal transduction pathway, and elucidating the role of the cAMP/PKA system in regulating complex I and oxygen free radical production provides new perspectives for devising therapeutic strategies for neurological diseases.

cAMP/Ca2+ response element‐binding protein plays a central role in the biogenesis of respiratory chain proteins in mammalian cells

In mammalian cells, promotion of mitochondrial biogenesis by various agents involves cAMP and Ca2+‐mediated signal transduction pathways. Recruitment of these pathways results in phosphorylation by cAMP and Ca2+‐dependent protein kinases of cAMP/Ca2+ response element‐binding protein (CREB). Phosphorylation of CREB, bound to transcriptional complexes of target genes, activates a down‐stream cascade of transcriptional complexes, which involve in sequence, the nuclear factors TORCs, PGC‐1, NRF1 and NRF2, and the mitochondrial factor mitochondrial transcriptional factor A. CREB also binds directly to the D‐loop of mitochondrial DNA and activates its expression. Activation of this network of transcriptional complexes results in concerted promotion of the expression of nuclear and mitochondrial genes encoding subunits of oxidative phosphorylation complexes.

Mitochondrial free radical overproduction due to respiratory chain impairment in the brain of a mouse model of Rett syndrome: protective effect of CNF1

Rett syndrome (RTT) is a pervasive neurodevelopmental disorder mainly caused by mutations in the X-linked MECP2 gene associated with severe intellectual disability, movement disorders, and autistic-like behaviors. Its pathogenesis remains mostly not understood and no effective therapy is available. High circulating levels of oxidative stress markers in patients and the occurrence of oxidative brain damage in MeCP2-deficient mouse models suggest the involvement of oxidative stress in RTT pathogenesis. However, the molecular mechanism and the origin of the oxidative stress have not been elucidated. Here we demonstrate that a redox imbalance arises from aberrant mitochondrial functionality in the brain of MeCP2-308 heterozygous female mice, a condition that more closely recapitulates that of RTT patients. The marked increase in the rate of hydrogen peroxide generation in the brain of RTT mice seems mainly produced by the dysfunctional complex II of the mitochondrial respiratory chain. In addition, both membrane potential generation and mitochondrial ATP synthesis are decreased in RTT mouse brains when succinate, the complex II respiratory substrate, is used as an energy source. Respiratory chain impairment is brain area specific, owing to a decrease in either cAMP-dependent phosphorylation or protein levels of specific complex subunits. Further, we investigated whether the treatment of RTT mice with the bacterial protein CNF1, previously reported to ameliorate the neurobehavioral phenotype and brain bioenergetic markers in an RTT mouse model, exerts specific effects on brain mitochondrial function and consequently on hydrogen peroxide production. In RTT brains treated with CNF1, we observed the reactivation of respiratory chain complexes, the rescue of mitochondrial functionality, and the prevention of brain hydrogen peroxide overproduction. These results provide definitive evidence of mitochondrial reactive oxygen species overproduction in RTT mouse brain and highlight CNF1 efficacy in counteracting RTT-related mitochondrial defects.