The present study shows that in isolated mitochondria and myoblast cultures depletion of cAMP, induced by sAC inhibition, depresses both ATP synthesis and hydrolysis by the FOF1 ATP synthase (complex V) of the oxidative phosphorylation system (OXPHOS). These effects are accompanied by the decrease of the respiratory membrane potential, decreased level of FOF1 connecting subunits and depressed oligomerization of the complex. All these effects of sAC inhibition are prevented by the addition of the membrane-permeant 8-Br-cAMP. These results show, for the first time, that cAMP promotes ATP production by complex V and prevents, at the same time, its detour to a mitochondrial membrane leak conductance, which is involved in cell death.

A critical role for mitochondrial dysfunction has been proposed in the pathogenesis of Down's 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-1? 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.

Nephropathic cystinosis (NC) is a rare disease caused by mutations in the CTNS gene encoding for cystinosin, a lysosomal transmembrane cystine/H+ symporter, which promotes the efflux of cystine from lysosomes to cytosol. NC is the most frequent cause of Fanconi syndrome (FS) in young children, the molecular basis of which is not well established. Proximal tubular cells have very high metabolic rate due to the active transport of many solutes. Not surprisingly, mitochondrial disorders are often characterized by FS. A similar mechanism may also apply to NC. Because cAMP has regulatory properties on mitochondrial function, we have analyzed cAMP levels and mitochondrial targets in CTNS-/- conditionally immortalized proximal tubular epithelial cells (ciPTEC) carrying the classical homozygous 57-kb deletion (delCTNS-/-) or with compound heterozygous loss-of-function mutations (mutCTNS-/-). Compared to wild-type cells, cystinotic cells had significantly lower mitochondrial cAMP levels (delCTNS-/- ciPTEC by 56% ± 10.5, P < 0.0001; mutCTNS-/- by 26% ± 4.3, P < 0.001), complex I and V activities, mitochondrial membrane potential, and SIRT3 protein levels, which were associated with increased mitochondrial fragmentation. Reduction of complex I and V activities was associated with lower expression of part of their subunits. Treatment with the non-hydrolysable cAMP analog 8-Br-cAMP restored mitochondrial potential and corrected mitochondria morphology. Treatment with cysteamine, which reduces the intra-lysosomal cystine, was able to restore mitochondrial cAMP levels, as well as most other abnormal mitochondrial findings. These observations were validated in CTNS-silenced HK-2 cells, indicating a pivotal role of mitochondrial cAMP in the proximal tubular dysfunction observed in NC.

A strong correlation between oxidative stress (OS) and Rett syndrome (RTT), a rare neurodevelopmental disorder affecting females in the 95% of the cases, has been well documented although the source of OS and the effect of a redox imbalance in this pathology has not been yet investigated. Using freshly isolated skin fibroblasts from RTT patients and healthy subjects, we have demonstrated in RTT cells high levels of H2O2 and HNE protein adducts. These findings correlated with the constitutive activation of NADPH-oxidase (NOX) and that was prevented by a NOX inhibitor and iron chelator pre-treatment, showing its direct involvement. In parallel, we demonstrated an increase in mitochondrial oxidant production, altered mitochondrial biogenesis and impaired proteasome activity in RTT samples. Further, we found that the key cellular defensive enzymes: glutathione peroxidase, superoxide dismutase and thioredoxin reductases activities were also significantly lower in RTT. Taken all together, our findings suggest that the systemic OS levels in RTT can be a consequence of both: increased endogenous oxidants as well as altered mitochondrial biogenesis with a decreased activity of defensive enzymes that leads to posttranslational oxidant protein modification and a proteasome activity impairment.

Functional and structural damages to mitochondria have been critically associated with the pathogenesis of Down syndrome (DS), a human multifactorial disease caused by trisomy of chromosome 21 and associated with neurodevelopmental delay, intellectual disability and early neurodegeneration. Recently, we demonstrated in neural progenitor cells (NPCs) isolated from the hippocampus of Ts65Dn mice -a widely used model of DS - a severe impairment of mitochondrial bioenergetics and biogenesis and reduced NPC proliferation. Here we further investigated the origin of mitochondrial dysfunction in DS and explored a possible mechanistic link among alteration of mitochondrial dynamics, mitochondrial dysfunctions and defective neurogenesis in DS. We first analyzed mitochondrial network and structure by both confocal and transmission electron microscopy as well as by evaluating the levels of key proteins involved in the fission and fusion machinery. We found a fragmentation of mitochondria due to an increase in mitochondrial fission associated with an up-regulation of dynamin-related protein 1 (Drpl), and a decrease in mitochondrial fusion associated with a down-regulation of mitofusin 2 (Mnf2) and increased proteolysis of optic atrophy 1 (Opal). Next, using the well-known neuroprotective agent mitochondrial division inhibitor 1 (Mdivi-1), we assessed whether the inhibition of mitochondrial fission might reverse alteration of mitochondrial dynamics and mitochondrial dysfunctions in DS neural progenitors cells. We demonstrate here for the first time, that Mdivi-1 restores mitochondrial network organization, mitochondrial energy production and ultimately improves proliferation and neuronal differentiation of NPCs. This research paves the way for the discovery of new therapeutic tools in managing some DS-associated clinical manifestations.

In mammalian cells the nuclear-encoded subunits of complex I are imported into mitochondria, where they are assembled with mt-DNA encoded subunits in the complex, or exchanged with pre-existing copies in the complex. The present work shows that in fibroblast cultures inhibition by KH7 of cAMP production in the mitochondrial matrix by soluble adenylyl cyclase (sAC) results in decreased amounts of free non-incorporated nuclear-encoded NDUFS4, NDUFV2 and NDUFA9 subunits of the catalytic moiety and inhibition of the activity of complex I. Addition of permeant 8-Br-cAMP prevents this effect of KH7. KH7 inhibits accumulation in isolated rat-liver mitochondria and incorporation in complex I of "in vitro" produced, radiolabeled NDUFS4 and NDUFV2 subunits. 8-Br-cAMP prevents also this effect of KH7. Use of protease inhibitors shows that intramitochondrial cAMP exerts this positive effect on complex I by preventing digestion of nuclear-encoded subunits by mitochondrial protease(s), whose activity is promoted by KH7 and H89, an inhibitor of PKA.

Multiple Mitochondrial Dysfunction Syndromes (MMDS) comprise a group of severe autosomal recessive diseases characterized by impaired respiration and lipoic acid metabolism, resulting in infantile-onset mitochondrial encephalopathy, non-ketotic hyperglycinemia, myopathy, lactic acidosis and early death. Four different MMDS have been analyzed in detail according to the genes involved in the disease, MMDS1 (NFU1), MMDS2 (BOLA3), MMDS3 (IBA57), and MMDS4 (ISCA2). MMDS5 has recently been described in a clinical case report of patients carrying a mutation in ISCA1, but with no further functional analysis. ISCA1 encodes a mitochondrial protein essential for the assembly of [4Fe-4S] clusters in key metabolic and respiratory enzymes. Here, we describe a patient with a severe early onset leukodystrophy, multiple defects of respiratory complexes, and a severe impairment of lipoic acid synthesis. A homozygous missense mutation in ISCA1 (c.29T>G; p.V10G) identified by targeted MitoExome sequencing resulted in dramatic reduction of ISCA1 protein level. The mutation located in the uncleaved presequence severely affected both mitochondrial import and stability of ISCA1. Down-regulation of ISCA1 in HeLa cells by RNAi impaired the biogenesis of mitochondrial [4Fe-4S] proteins, yet could be complemented by expression of wild-type ISCA1. In contrast, the ISCA1 p.V10G mutant protein only partially complemented the defects, closely resembling the biochemical phenotypes observed for ISCA1 patient fibroblasts. Collectively, our comprehensive clinical and biochemical investigations show that the ISCA1 p.V10G mutation functionally impaired mitochondrial [4Fe-4S] protein assembly and hence was causative for the observed clinical defects.

Mitochondria play a pivotal role in cellular energy-generating processes and are considered master regulators of cell life and death fate. Mitochondrial function integrates signalling networks in several metabolic pathways controlling neurogenesis and neuroplasticity. Indeed, dysfunctional mitochondria and mitochondrial-dependent activation of intracellular stress cascades are critical initiating events in many human neurodegenerative or neurodevelopmental diseases including Down syndrome (DS). It is well established that trisomy of human chromosome 21 can cause DS. DS is associated with neurodevelopmental delay, intellectual disability and early neurodegeneration. Recently, molecular mechanisms responsible for mitochondrial damage and energy deficits have been identified and characterized in several DS-derived human cells and animal models of DS. Therefore, therapeutic strategies targeting mitochondria could have great potential for new treatment regimens in DS. The purpose of this review is to highlight recent studies concerning mitochondrial impairment in DS, focusing on alterations of the molecular pathways controlling mitochondrial function. We will also discuss the effects and molecular mechanisms of naturally occurring and chemically synthetized drugs that exert neuroprotective effects through modulation of mitochondrial function and attenuation of oxidative stress. These compounds might represent novel therapeutic tools for the modulation of energy deficits in DS.

Mitochondria, responding to a wide variety of signals, including oxidative stress, are criticalin regulating apoptosis that plays a key role in the pathogenesis of a variety of cardiovasculardiseases. A number of mitochondrial proteins and pathways have been found to be involvedin the mitochondrial dependent apoptosis mechanism, such as optic atrophy 1 (OPA1), sirtuin3 (Sirt3), deacetylase enzyme and cAMP signal. In the present work we report a networkamong OPA1, Sirt3 and cAMP in ROS-dependent apoptosis. Rat myoblastic H9c2 cell lines,were treated with tert-butyl hydroperoxide (t-BHP) to induce oxidative stress-dependentapoptosis. FRET analysis revealed a selective decrease of mitochondrial cAMP in response tot-BHP treatment. This was associated with a decrease of Sirt3 protein level and proteolyticprocessing of OPA1. Pretreatment of cells with permeant analogous of cAMP (8-Br-cAMP)protected the cell from apoptosis preventing all these events. Using H89, inhibitor of theprotein kinase A (PKA), and protease inhibitors, evidences have been obtained that ROSdependentapoptosis is associated with an alteration of mitochondrial cAMP/PKA signal thatcauses degradation/proteolysis of Sirt3 that, in turn, promotes acetylation and proteolyticprocessing of OPA1.

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.

The serine-threonine protein phosphatase 2A (PP2A) regulates multiple cell signaling cascades and its inactivation by viral oncoproteins, mutation of specific structural subunits or upregulation of the cellular endogenous inhibitors may contribute to malignant transformation by regulating specific phosphorylation events. Pharmacological modulation of PP2A activity is becoming an attractive strategy for cancer treatment. Some compounds targeting PP2A are able to induce PP2A reactivation and subsequent cell death in several types of cancer.METHODS:We undertook a search of bibliographic databases for peer-reviewed articles focusing on the main item of the review. We selected articles published in indexed journals. The quality of retrieved papers was appraised using the standard bibliometric indicators.RESULTS:One hundred and fourteen papers were included in the review. Twenty-seven papers gave an overview of structure and physiological role of PP2A. Twenty-five papers outlined the role of PP2A in tumor suppression. Forty papers analyzed the mechanism involved in PP2A reactivation by synthetic compounds, and twenty-two papers outlined the capability of natural compounds of restoring PP2A activity and how this could be beneficial.CONCLUSIONS:Findings analyzed in this review underline the central role of PP2A as a regulator of cell growth and survival, hence its function as tumor suppressor. The discovery that some compounds, either synthetic or natural, are capable of reactivating PP2A opens up new perspectives for future strategies to fully exploit therapeutic potential in human cancer. Thus, this review could also be of particular interest to pharmaceutical or biotechnology companies for drug design and targeted delivery.

A study is presented on the expression of mitochondrial oxidative phosphorylation complexes in exponentially growing and serum-starved, quiescent human fibroblast cultures. The functional levels of respiratory complexes I and III and complex V (adenosine triphosphate (ATP) synthase) were found to be severely depressed in serum-starved fibroblasts. The depression of oxidative phosphorylation system (OXPHOS) complexes was associated with reduced levels of peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1?) and the down-stream nuclear respiratory factor 1 (NRF1) and mitochondrial transcription factors (TFAM). In serum-starved fibroblasts decrease of the catalytic activity of AMP cyclic dependent protein kinase (PKA) and phosphorylation of cAMP response element-binding protein (CREB), the transcription coactivator of the PGC-1? gene, was found. Hydroxytyrosol prevented the decline in the expression of the PGC-1? transcription cascade of OXPHOS complexes in serum-starved fibroblast cultures. The positive effect of HT was associated with activation of PKA and CREB phosphorylation. These results show involvement of PKA, CREB and PGC-1? in the regulation of OXPHOS in cell transition from the replicating to the quiescent state. © 2014 Elsevier B.V.

Objectives: Different nuclear genes are thought to be involved in the regulation of the complex phenotype of metabolic syndrome (MS) and their number is increasing. A mutation in mitochondrial DNA (mtDNA), T4291C in transfer RNA isoleucine (tRNAile), has been associated with MS in a large American family. In addition, a mtDNA T16189C variant, already known to be associated with insulin resistance and type 2 diabetes mellitus in Caucasians, seems to underlie susceptibility to MS in the Chinese population. Our aim was to verify the T4291C and T16189C variants in subjects affected by different phenotypes of MS. Methods: Seventy patients with MS and 35 healthy individuals were investigated for the presence of the mtDNA variants by polymerase chain reaction-restriction fragment length polymorphism analysis. Results: The T4291C variant was absent in patients and in controls. The T16189C variant was more frequent in patients with MS than in control subjects (21.4% versus 5.7%, P < 0.04) and was associated with hypertension (P = 0.01), waist circumference (P = 0.02), body mass index (P = 0.009), visceral fat thickness (P = 0.04), homeostasis model assessment (P = 0.03), and the number of MS diagnostic criteria (P = 0.01). Conclusion: The mtDNA T16189C variant is associated with MS and its different clinical expressions. Prospective studies are warranted to establish the clinical relevance of this association. © 2011 Elsevier Inc.

Mitochondrial dysfunctions critically impair nervous system development and are potentially involved in the pathogenesis of various neurodevelopmental disorders, including Down syndrome (DS), the most common genetic cause of intellectual disability. Previous studies from our group demonstrated impaired mitochondrial activity in peripheral cells from DS subjects and the efficacy of epigallocatechin-3-gallate (EGCG) - a natural polyphenol major component of green tea - to counteract the mitochondrial energy deficit. In this study, to gain insight into the possible role of mitochondria in DS intellectual disability, mitochondrial functions were analyzed in neural progenitor cells (NPCs) isolated from the hippocampus of Ts65Dn mice, a widely used model of DS which recapitulates many major brain structural and functional phenotypes of the syndrome, including impaired hippocampal neurogenesis. We found that, during NPC proliferation, mitochondrial bioenergetics and mitochondrial biogenic program were strongly compromised in Ts65Dn cells, but not associated with free radical accumulation. These data point to a central role of mitochondrial dysfunction as an inherent feature of DS and not as a consequence of cell oxidative stress. Further, we disclose that, besides EGCG, also the natural polyphenol resveratrol, which displays a neuroprotective action in various human diseases but never tested in DS, restores oxidative phosphorylation efficiency and mitochondrial biogenesis, and improves proliferation of NPCs. These effects were associated with the activation of PGC-1?/Sirt1/AMPK axis by both polyphenols. This research paves the way for using nutraceuticals as a potential therapeutic tool in preventing or managing some energy deficit-associated DS clinical manifestations.

While aberrant cancer cell growth is frequently associated with altered biochemical metabolism, normal mitochondrialfunctions are usually preserved and necessary for full malignant transformation. The transcription factor FoxO3A is akey determinant of cancer cell homeostasis, playing a dual role in survival/death response to metabolic stress andcancer therapeutics. We recently described a novel mitochondrial arm of the AMPK-FoxO3A axis in normal cells uponnutrient shortage. Here, we show that in metabolically stressed cancer cells, FoxO3A is recruited to the mitochondriathrough activation of MEK/ERK and AMPK, which phosphorylate serine 12 and 30, respectively, on FoxO3A N-terminaldomain. Subsequently, FoxO3A is imported and cleaved to reach mitochondrial DNA, where it activates expression ofthe mitochondrial genome to support mitochondrial metabolism. Using FoxO3A-/- cancer cells generated with theCRISPR/Cas9 genome editing system and reconstituted with FoxO3A mutants being impaired in their nuclear ormitochondrial subcellular localization, we show that mitochondrial FoxO3A promotes survival in response tometabolic stress. In cancer cells treated with chemotherapeutic agents, accumulation of FoxO3A into themitochondria promoted survival in a MEK/ERK-dependent manner, while mitochondrial FoxO3A was required forapoptosis induction by metformin. Elucidation of FoxO3A mitochondrial vs. nuclear functions in cancer cellhomeostasis might help devise novel therapeutic strategies to selectively disable FoxO3A prosurvival activity.