Regulation of mitochondrial complex III activity and assembly by TRAP1 in cancer cells.

BACKGROUND: Metabolic reprogramming is an important issue in tumor biology. A recently-identified actor in this regard is the molecular chaperone TRAP1, that is considered an oncogene in several cancers for its high expression but an oncosuppressor in others with predominant oxidative metabolism. TRAP1 is mainly localized in mitochondria, where it interacts with respiratory complexes, although alternative localizations have been described, particularly on the endoplasmic reticulum, where it interacts with the translational machinery with relevant roles in protein synthesis regulation. RESULTS: Herein we show that, inside mitochondria, TRAP1 binds the complex III core component UQCRC2 and regulates complex III activity. This decreases respiration rate during basal conditions but allows sustained oxidative phosphorylation when glucose is limiting, a condition in which the direct TRAP1-UQCRC2 binding is disrupted, but not TRAP1-complex III binding. Interestingly, several complex III components and assembly factors show an inverse correlation with survival and response to platinum-based therapy in high grade serous ovarian cancers, where TRAP1 inversely correlates with stage and grade and directly correlates with survival. Accordingly, drug-resistant ovarian cancer cells show high levels of complex III components and high sensitivity to complex III inhibitory drug antimycin A. CONCLUSIONS: These results shed new light on the molecular mechanisms involved in TRAP1-dependent regulation of cancer cell metabolism and point out a potential novel target for metabolic therapy in ovarian cancer.

ZSCAN4+ mouse embryonic stem cells have an oxidative and flexible metabolic profile.

Cultured mouse embryonic stem cells are a heterogeneous population with diverse differentiation potential. In particular, the subpopulation marked by Zscan4 expression has high stem cell potency and shares with 2 cell stage preimplantation embryos both genetic and epigenetic mechanisms that orchestrate zygotic genome activation. Although embryonic de novo genome activation is known to rely on metabolites, a more extensive metabolic characterization is missing. Here we analyze the Zscan4+ mouse stem cell metabolic phenotype associated with pluripotency maintenance and cell reprogramming. We show that Zscan4+ cells have an oxidative and adaptable metabolism, which, on one hand, fuels a high bioenergetic demand and, on the other hand, provides intermediate metabolites for epigenetic reprogramming. Our findings enhance our understanding of the metastable Zscan4+ stem cell state with potential applications in regenerative medicine.

cAMP/PKA Signaling Modulates Mitochondrial Supercomplex Organization.

The oxidative phosphorylation (OXPHOS) system couples the transfer of electrons to oxygen with pumping of protons across the inner mitochondrial membrane, ensuring the ATP production. Evidence suggests that respiratory chain complexes may also assemble into supramolecular structures, called supercomplexes (SCs). The SCs appear to increase the efficiency/capacity of OXPHOS and reduce the reactive oxygen species (ROS) production, especially that which is produced by complex I. Studies suggest a mutual regulation between complex I and SCs, while SCs organization is important for complex I assembly/stability, complex I is involved in the supercomplex formation. Complex I is a pacemaker of the OXPHOS system, and it has been shown that the PKA-dependent phosphorylation of some of its subunits increases the activity of the complex, reducing the ROS production. In this work, using in ex vivo and in vitro models, we show that the activation of cAMP/PKA cascade resulted in an increase in SCs formation associated with an enhanced capacity of electron flux and ATP production rate. This is also associated with the phosphorylation of the NDUFS4 subunit of complex I. This aspect highlights the key role of complex I in cellular energy production.

Decreased amount of vimentin N-terminal truncated proteolytic products in parkin-mutant skin fibroblasts.

Vimentin, a member of cytoskeleton intermediate filaments proteins, plays a critical role in cell structure and dynamics. The present proteomic study reveals reduced amount of six different lengths, N-terminal truncated proteolytic products of vimentin, in the primary skin fibroblasts from two unrelated PD patients, as compared to control fibroblasts. The decreased amount of N-terminal truncated forms of vimentin in parkin-mutant fibroblasts, could contribute to impairment of cellular function, potentially contributing to the pathogenesis of Parkinson disease.

Segregation of dopamine and glutamate release sites in dopamine neuron axons: regulation by striatal target cells.

Dopamine (DA) is a key regulator of circuits controlling movement and motivation. A subset of midbrain DA neurons has been shown to express the vesicular glutamate transporter (VGLUT)2, underlying their capacity for glutamate release. Glutamate release is found mainly by DA neurons of the ventral tegmental area (VTA) and can be detected at terminals contacting ventral, but not dorsal, striatal neurons, suggesting the possibility that target-derived signals regulate the neurotransmitter phenotype of DA neurons. Whether glutamate can be released from the same terminals that release DA or from a special subset of axon terminals is unclear. Here, we provide in vitro and in vivo data supporting the hypothesis that DA and glutamate-releasing terminals in mice are mostly segregated and that striatal neurons regulate the cophenotype of midbrain DA neurons and the segregation of release sites. Our work unveils a fundamental feature of dual neurotransmission and plasticity of the DA system.-Fortin, G. M., Ducrot, C., Giguère, N., Kouwenhoven, W. M., Bourque, M.-J., Pacelli, C., Varaschin, R. K., Brill, M., Singh, S., Wiseman, P. W., Trudeau, L.-E. Segregation of dopamine and glutamate release sites in dopamine neuron axons: regulation by striatal target cells.

Comparative analysis of Parkinson's disease-associated genes in mice reveals altered survival and bioenergetics of Parkin-deficient dopamine neurons.

Many mutations in genes encoding proteins such as Parkin, PTEN-induced putative kinase 1 (PINK1), protein deglycase DJ-1 (DJ-1 or PARK7), leucine-rich repeat kinase 2 (LRRK2), and α-synuclein have been linked to familial forms of Parkinson's disease (PD). The consequences of these mutations, such as altered mitochondrial function and pathological protein aggregation, are starting to be better understood. However, little is known about the mechanisms explaining why alterations in such diverse cellular processes lead to the selective loss of dopamine (DA) neurons in the substantia nigra (SNc) in the brain of individuals with PD. Recent work has shown that one of the reasons for the high vulnerability of SNc DA neurons is their high basal rate of mitochondrial oxidative phosphorylation (OXPHOS), resulting from their highly complex axonal arborization. Here, we examined whether axonal growth and basal mitochondrial function are altered in SNc DA neurons from Parkin-, Pink1-, or DJ-1-KO mice. We provide evidence for increased basal OXPHOS in Parkin-KO DA neurons and for reduced survival of DA neurons that have a complex axonal arbor. The surviving smaller neurons exhibited reduced vulnerability to the DA neurotoxin and mitochondrial complex I inhibitor MPP+, and this reduction was associated with reduced expression of the DA transporter. Finally, we found that glial cells play a role in the reduced resilience of DA neurons in these mice and that WT Parkin overexpression rescues this phenotype. Our results provide critical insights into the complex relationship between mitochondrial function, axonal growth, and genetic risk factors for PD.

Lmx1a and Lmx1b regulate mitochondrial functions and survival of adult midbrain dopaminergic neurons.

The LIM-homeodomain transcription factors Lmx1a and Lmx1b play critical roles during the development of midbrain dopaminergic progenitors, but their functions in the adult brain remain poorly understood. We show here that sustained expression of Lmx1a and Lmx1b is required for the survival of adult midbrain dopaminergic neurons. Strikingly, inactivation of Lmx1a and Lmx1b recreates cellular features observed in Parkinson's disease. We found that Lmx1a/b control the expression of key genes involved in mitochondrial functions, and their ablation results in impaired respiratory chain activity, increased oxidative stress, and mitochondrial DNA damage. Lmx1a/b deficiency caused axonal pathology characterized by α-synuclein(+) inclusions, followed by a progressive loss of dopaminergic neurons. These results reveal the key role of these transcription factors beyond the early developmental stages and provide mechanistic links between mitochondrial dysfunctions, α-synuclein aggregation, and the survival of dopaminergic neurons.

Parkin Mutation Affects Clock Gene-Dependent Energy Metabolism.

Growing evidence highlights a tight connection between circadian rhythms, molecular clockworks, and mitochondrial function. In particular, mitochondrial quality control and bioenergetics have been proven to undergo circadian oscillations driven by core clock genes. Parkinson's disease (PD) is a chronic neurodegenerative disease characterized by a selective loss of dopaminergic neurons. Almost half of the autosomal recessive forms of juvenile parkinsonism have been associated with mutations in the PARK2 gene coding for parkin, shown to be involved in mitophagy-mediated mitochondrial quality control. The aim of this study was to investigate, in fibroblasts from genetic PD patients carrying parkin mutations, the interplay between mitochondrial bioenergetics and the cell autonomous circadian clock. Using two different in vitro synchronization protocols, we demonstrated that normal fibroblasts displayed rhythmic oscillations of both mitochondrial respiration and glycolytic activity. Conversely, in fibroblasts obtained from PD patients, a severe damping of the bioenergetic oscillatory patterns was observed. Analysis of the core clock genes showed deregulation of their expression patterns in PD fibroblasts, which was confirmed in induced pluripotent stem cells (iPSCs) and induced neural stem cells (iNSCs) derived thereof. The results from this study support a reciprocal interplay between the clockwork machinery and mitochondrial energy metabolism, point to a parkin-dependent mechanism of regulation, and unveil a hitherto unappreciated level of complexity in the pathophysiology of PD and eventually other neurodegenerative diseases.

Febrile temperature reprograms by redox-mediated signaling the mitochondrial metabolic phenotype in monocyte-derived dendritic cells.

Fever-like hyperthermia is known to stimulate innate and adaptive immune responses. Hyperthermia-induced immune stimulation is also accompanied with, and likely conditioned by, changes in the cell metabolism and, in particular, mitochondrial metabolism is now recognized to play a pivotal role in this context, both as energy supplier and as signaling platform. In this study we asked if challenging human monocyte-derived dendritic cells with a relatively short-time thermal shock in the fever-range, typically observed in humans, caused alterations in the mitochondrial oxidative metabolism. We found that following hyperthermic stress (3h exposure at 39°C) TNF-α-releasing dendritic cells undergo rewiring of the oxidative metabolism hallmarked by decrease of the mitochondrial respiratory activity and of the oxidative phosphorylation and increase of lactate production. Moreover, enhanced production of reactive oxygen and nitrogen species and accumulation of mitochondrial Ca2+ was consistently observed in hyperthermia-conditioned dendritic cells and exhibited a reciprocal interplay. The hyperthermia-induced impairment of the mitochondrial respiratory activity was (i) irreversible following re-conditioning of cells to normothermia, (ii) mimicked by exposing normothermic cells to the conditioned medium of the hyperthermia-challenged cells, (iii) largely prevented by antioxidant and inhibitors of the nitric oxide synthase and of the mitochondrial calcium porter, which also inhibited release of TNF-α. These observations combined with gene expression analysis support a model based on a thermally induced autocrine signaling, which rewires and sets a metabolism checkpoint linked to immune activation of dendritic cells.

Effects of Serine 129 Phosphorylation on α-Synuclein Aggregation, Membrane Association, and Internalization.

Although trace levels of phosphorylated α-synuclein (α-syn) are detectable in normal brains, nearly all α-syn accumulated within Lewy bodies in Parkinson disease brains is phosphorylated on serine 129 (Ser-129). The role of the phosphoserine residue and its effects on α-syn structure, function, and intracellular accumulation are poorly understood. Here, co-expression of α-syn and polo-like kinase 2 (PLK2), a kinase that targets Ser-129, was used to generate phosphorylated α-syn for biophysical and biological characterization. Misfolding and fibril formation of phosphorylated α-syn isoforms were detected earlier, although the fibrils remained phosphatase- and protease-sensitive. Membrane binding of α-syn monomers was differentially affected by phosphorylation depending on the Parkinson disease-linked mutation. WT α-syn binding to presynaptic membranes was not affected by phosphorylation, whereas A30P α-syn binding was greatly increased, and A53T α-syn was slightly lower, implicating distal effects of the carboxyl- on amino-terminal membrane binding. Endocytic vesicle-mediated internalization of pre-formed fibrils into non-neuronal cells and dopaminergic neurons matched the efficacy of α-syn membrane binding. Finally, the disruption of internalized vesicle membranes was enhanced by the phosphorylated α-syn isoforms, a potential means for misfolded extracellular or lumenal α-syn to access cytosolic α-syn. Our results suggest that the threshold for vesicle permeabilization is evident even at low levels of α-syn internalization and are relevant to therapeutic strategies to reduce intercellular propagation of α-syn misfolding.

Altered protein expression pattern in skin fibroblasts from parkin-mutant early-onset Parkinson's disease patients.

Parkinson's disease (PD) is the most common neurodegenerative movement disorder caused primarily by selective degeneration of the dopaminergic neurons in substantia nigra. In this work the proteomes extracted from primary fibroblasts of two unrelated, hereditary cases of PD patients, with different parkin mutations, were compared with the proteomes extracted from commercial adult normal human dermal fibroblasts (NHDF) and primary fibroblasts from the healthy mother of one of the two patients. The results show that the fibroblasts from the two different cases of parkin-mutant patients display analogous alterations in the expression level of proteins involved in different cellular functions, like cytoskeleton structure-dynamics, calcium homeostasis, oxidative stress response, protein and RNA processing.

Effect of resveratrol on mitochondrial function: implications in parkin-associated familiar Parkinson's disease.

Mitochondrial dysfunction and oxidative stress occur in Parkinson's disease (PD), but the molecular mechanisms controlling these events are not completely understood. Peroxisome proliferator-activated receptor-gamma coactivator-1α (PGC-1α) is a transcriptional coactivator known as master regulator of mitochondrial functions and oxidative metabolism. Recent studies, including one from our group, have highlighted altered PGC-1α activity and transcriptional deregulation of its target genes in PD pathogenesis suggesting it as a new potential therapeutic target. Resveratrol, a natural polyphenolic compound proved to improve mitochondrial activity through the activation of several metabolic sensors resulting in PGC-1α activation. Here we have tested in vitro the effect of resveratrol treatment on primary fibroblast cultures from two patients with early-onset PD linked to different Park2 mutations. We show that resveratrol regulates energy homeostasis through activation of AMP-activated protein kinase (AMPK) and sirtuin 1 (SIRT1) and raise of mRNA expression of a number of PGC-1α's target genes resulting in enhanced mitochondrial oxidative function, likely related to a decrease of oxidative stress and to an increase of mitochondrial biogenesis. The functional impact of resveratrol treatment encompassed an increase of complex I and citrate synthase activities, basal oxygen consumption, and mitochondrial ATP production and a decrease in lactate content, thus supporting a switch from glycolytic to oxidative metabolism. Moreover, resveratrol treatment caused an enhanced macro-autophagic flux through activation of an LC3-independent pathway. Our results, obtained in early-onset PD fibroblasts, suggest that resveratrol may have potential clinical application in selected cases of PD-affected patients.

The p66Shc protein controls redox signaling and oxidation-dependent DNA damage in human liver cells.

The p66Shc protein mediates oxidative stress-related injury in multiple tissues. Steatohepatitis is characterized by enhanced oxidative stress-mediated cell damage. The role of p66Shc in redox signaling was investigated in human liver cells and alcoholic steatohepatitis. HepG2 cells with overexpression of wild-type or mutant p66Shc, with Ser36 replacement by Ala, were obtained through infection with recombinant adenoviruses. Reactive oxygen species and oxidation-dependent DNA damage were assessed by measuring dihydroethidium oxidation and 8-hydroxy-2'-deoxyguanosine accumulation into DNA, respectively. mRNA and protein levels of signaling intermediates were evaluated in HepG2 cells and liver biopsies from control and alcoholic steatohepatitis subjects. Exposure to H2O2 increased reactive oxygen species and phosphorylation of p66Shc on Ser36 in HepG2 cells. Overexpression of p66Shc promoted reactive oxygen species synthesis and oxidation-dependent DNA damage, which were further enhanced by H2O2. p66Shc activation also resulted in increased Erk-1/2, Akt, and FoxO3a phosphorylation. Blocking of Erk-1/2 activation inhibited p66Shc phosphorylation on Ser36. Increased p66Shc expression was associated with reduced mRNA levels of antioxidant molecules, such as NF-E2-related factor 2 and its target genes. In contrast, overexpression of the phosphorylation defective p66Shc Ala36 mutant inhibited p66Shc signaling, enhanced antioxidant genes, and suppressed reactive oxygen species and oxidation-dependent DNA damage. Increased p66Shc protein levels and Akt phosphorylation were observed in liver biopsies from alcoholic steatohepatitis compared with control subjects. In human alcoholic steatohepatitis, increased hepatocyte p66Shc protein levels may enhance susceptibility to DNA damage by oxidative stress by promoting reactive oxygen species synthesis and repressing antioxidant pathways.

High OXPHOS efficiency in RA-FUdr-differentiated SH-SY5Y cells: involvement of cAMP signalling and respiratory supercomplexes.

Neurons are highly dependent on mitochondria to meet their bioenergetic needs and understanding the metabolic changes during the differentiation process is crucial in the neurodegeneration context. Several in vitro approaches have been developed to study neuronal differentiation and bioenergetic changes. The human SH-SY5Y cell line is a widely used cellular model and several differentiation protocols have been developed to induce a neuron-like phenotype including retinoic acid (RA) treatment. In this work we obtained a homogeneous functional population of neuron-like cells by a two-step differentiation protocol in which SH-SY5Y cells were treated with RA plus the mitotic inhibitor 2-deoxy-5-fluorouridine (FUdr). RA-FUdr treatment induced a neuronal phenotype characterized by increased expression of neuronal markers and electrical properties specific to excitable cells. In addition, the RA-FUdr differentiated cells showed an enrichment of long chain and unsaturated fatty acids (FA) in the acyl chain composition of cardiolipin (CL) and the bioenergetic analysis evidences a high coupled and maximal respiration associated with high mitochondrial ATP levels. Our results suggest that the observed high oxidative phosphorylation (OXPHOS) capacity may be related to the activation of the cyclic adenosine monophosphate (cAMP) pathway and the assembly of respiratory supercomplexes (SCs), highlighting the change in mitochondrial phenotype during neuronal differentiation.

Bioenergetic profile and redox tone modulate in vitro osteogenesis of human dental pulp stem cells: new perspectives for bone regeneration and repair.

BACKGROUND: Redox signaling and energy metabolism are known to be involved in controlling the balance between self-renewal and proliferation/differentiation of stem cells. In this study we investigated metabolic and redox changes occurring during in vitro human dental pulp stem cells (hDPSCs) osteoblastic (OB) differentiation and tested on them the impact of the reactive oxygen species (ROS) signaling. METHODS: hDPSCs were isolated from dental pulp and subjected to alkaline phosphatase and alizarin red staining, q-RT-PCR, and western blotting analysis of differentiation markers to assess achievement of osteogenic/odontogenic differentiation. Moreover, a combination of metabolic flux analysis and confocal cyto-imaging was used to profile the metabolic phenotype and to evaluate the redox tone of hDPSCs. RESULTS: In differentiating hDPSCs we observed the down-regulation of the mitochondrial respiratory chain complexes expression since the early phase of the process, confirmed by metabolic flux analysis, and a reduction of the basal intracellular peroxide level in its later phase. In addition, dampened glycolysis was observed, thereby indicating a lower energy-generating phenotype in differentiating hDPSCs. Treatment with the ROS scavenger Trolox, applied in the early-middle phases of the process, markedly delayed OB differentiation of hDPSCs assessed as ALP activity, Runx2 expression, mineralization capacity, expression of stemness and osteoblast marker genes (Nanog, Lin28, Dspp, Ocn) and activation of ERK1/2. In addition, the antioxidant partly prevented the inhibitory effect on cell metabolism observed following osteogenic induction. CONCLUSIONS: Altogether these results provided evidence that redox signaling, likely mediated by peroxide species, influenced the stepwise osteogenic expansion/differentiation of hDPSCs and contributed to shape its accompanying metabolic phenotype changes thus improving their efficiency in bone regeneration and repair.

Characterization of the effect of pomegranate crude extract, and its post-harvesting preservation procedures, on redox tone, cellular growth and metabolic profile of MDA-MB-231 cell line.

BACKGROUND: Pomegranate is known for its beneficial properties due to its high content in antioxidants and might constitute a natural option for preventing and treatment of different pathologies including cancer. Since mitochondria are involved in tumorigenesis through ROS production and modulation of oxidative metabolism, we investigated the biological effects of pomegranate on cellular redox state, proliferation and metabolism in the breast cancer cell line MDA-MB-231 (MDA). METHODS: MDA were treated for 24 h with graded concentration of filtered Pomegranate juice (PJ) and tested for metabolic Flux Analysis with XFe96 Extracellular Flux Analyzer, for proliferation using the xCELLigence System Real-Time Cell Analyzer and for intracellular ROS content by Confocal Microscopy Imaging. RESULTS: Cells-treatment with freshly prepared pomegranate juice (PJ) resulted in a significant reduction of the intracellular ROS content already at the lower concentration of PJ tested. Additionally, it enhanced mitochondria respiration, and decreased glycolysis at high concentrations, inhibiting at the same time cell proliferation. As pomegranate is a seasonal fruit, assessment of optimum storage conditions preserving its bio-active properties was investigated. Our results indicated that storage conditions under controlled atmosphere for 30 days was able to enhance mitochondrial respiration at the same extent than freshly extracted PJ. Conversely, freezing procedure, though retaining the antioxidant and cell-growth inhibitory property, elicited an opposite effect on the metabolic profile as compared with fresh extract. CONCLUSION: Overall, the results of our study, on the one hand, confirms the preventive/therapeutic potential of PJ, as well as of its post-harvested processing, for cancer management. On the other hand, it highlights the intrinsic difficulties in attaining mechanistic insights when a multiplicity of effects is elicited by a crude mixture of bio-active compounds.

Conditional deletion of neurexins dysregulates neurotransmission from dopamine neurons.

Midbrain dopamine (DA) neurons are key regulators of basal ganglia functions. The axonal domain of these neurons is highly complex, with a large subset of non-synaptic release sites and a smaller subset of synaptic terminals from which in addition to DA, glutamate or GABA are also released. The molecular mechanisms regulating the connectivity of DA neurons and their neurochemical identity are unknown. An emerging literature suggests that neuroligins, trans-synaptic cell adhesion molecules, regulate both DA neuron connectivity and neurotransmission. However, the contribution of their major interaction partners, neurexins (Nrxns), is unexplored. Here, we tested the hypothesis that Nrxns regulate DA neuron neurotransmission. Mice with conditional deletion of all Nrxns in DA neurons (DAT::NrxnsKO) exhibited normal basic motor functions. However, they showed an impaired locomotor response to the psychostimulant amphetamine. In line with an alteration in DA neurotransmission, decreased levels of the membrane DA transporter (DAT) and increased levels of the vesicular monoamine transporter (VMAT2) were detected in the striatum of DAT::NrxnsKO mice, along with reduced activity-dependent DA release. Strikingly, electrophysiological recordings revealed an increase of GABA co-release from DA neuron axons in the striatum of these mice. Together, these findings suggest that Nrxns act as regulators of the functional connectivity of DA neurons.

The human brain contains billions of nerve cells, known as neurons, which receive input from the outside world and process this information in the brain. Neurons communicate with each other by releasing chemical messengers from specialized structures, called axon terminals, some of which form junctions known as synapses. These messengers then generate signals in the target neurons. Based on the type of chemical they release, neurons can be classified into different types. For example, neurons releasing dopamine are considered to act as key regulators of learning, movements and motivation. Such neurons establish very large numbers of axon terminals, but very few of them form synapses. Specific sets of proteins, including neurexins and neuroligins, are thought to help regulate the activity of the connexions between these neurons. Previous research has shown that when neuroligins were removed from the neurons of worms or mice, it affected the ability of the animals to move. So far, the role of neurexins in managing the connectivity of regulatory neurons, such as those releasing dopamine, has received much less attention. To bridge this knowledge gap, Ducrot et al. explored how removing neurexins from dopamine neurons in mice affected their behaviour. The experiments revealed that eliminating neurexins did not affect their motor skills on a rotating rod, but it did reduce their movements in response to the psychostimulant amphetamine, a molecule known to enhance dopamine-associated behaviours. The cellular structure of dopamine neurons lacking neurexins was the same as in neurons containing this protein. But dopamine neurons without neurexins were slower to recycle dopamine, and they released a higher amount of the inhibitory messenger GABA. This suggests that neurexin acts as an important suppressor of GABA secretion to help regulate the signals released by dopamine neurons. These findings set the stage for further research into the role of neurexins in regulating dopamine and other populations of neurons in conditions such as Parkinson's disease, where movement and coordination are affected.

Mitochondrial defect and PGC-1α dysfunction in parkin-associated familial Parkinson's disease.

Mutations in the parkin gene are expected to play an essential role in autosomal recessive Parkinson's disease. Recent studies have established an impact of parkin mutations on mitochondrial function and autophagy. In primary skin fibroblasts from two patients affected by an early onset Parkinson's disease, we identified a hitherto unreported compound heterozygous mutation del exon2-3/del exon3 in the parkin gene, leading to the complete loss of the full-length protein. In both patients, but not in their heterozygous parental control, we observed severe ultrastructural abnormalities, mainly in mitochondria. This was associated with impaired energy metabolism, deregulated reactive oxygen species (ROS) production, resulting in lipid oxidation, and peroxisomal alteration. In view of the involvement of parkin in the mitochondrial quality control system, we have investigated upstream events in the organelles' biogenesis. The expression of the peroxisome proliferator-activated receptor gamma-coactivator 1-alpha (PGC-1α), a strong stimulator of mitochondrial biogenesis, was remarkably upregulated in both patients. However, the function of PGC-1α was blocked, as revealed by the lack of its downstream target gene induction. In conclusion, our data confirm the role of parkin in mitochondrial homeostasis and suggest a potential involvement of the PGC-1α pathway in the pathogenesis of Parkinson's disease. This article is part of a Special Issue entitled: Translating nuclear receptors from health to disease.

Myoglobin expression by alternative transcript in different mesenchymal stem cells compartments.

BACKGROUND: The metabolic phenotype of stem cells is increasingly recognized as a hallmark of their pluripotency with mitochondrial and oxygen-related metabolism playing a not completely defined role in this context. In a previous study, we reported the ectopic expression of myoglobin (MB) in bone marrow-derived hematopoietic stem/progenitor cells. Here, we have extended the analysis to mesenchymal stem cells (MSCs) isolated from different tissues. METHODS: MSCs were isolated from human placental membrane, mammary adipose tissue and dental pulp and subjected to RT-PCR, Western blotting and mass spectrometry to investigate the expression of MB. A combination of metabolic flux analysis and cyto-imaging was used to profile the metabolic phenotype and the mitochondria dynamics in the different MSCs. RESULTS: As for the hematopoietic stem/progenitor cells, the expression of Mb was largely driven by an alternative transcript with the protein occurring both in the monomer and in the dimer forms as confirmed by mass spectrometry analysis. Comparing the metabolic fluxes between neonatal placental membrane-derived and adult mammary adipose tissue-derived MSCs, we showed a significantly more active bioenergetics profile in the former that correlated with a larger co-localization of myoglobin with the mitochondrial compartment. Differences in the structure of the mitochondrial network as well as in the expression of factors controlling the organelle dynamics were also observed between neonatal and adult mesenchymal stem cells. Finally, the expression of myoglobin was found to be strongly reduced following osteogenic differentiation of dental pulp-derived MSCs, while it was upregulated following reprogramming of human fibroblasts to induce pluripotent stem cells. CONCLUSIONS: Ectopic expression of myoglobin in tissues other than muscle raises the question of understanding its function therein. Properties in addition to the canonical oxygen storage/delivery have been uncovered. Finding of Mb expressed via an alternative gene transcript in the context of different stem cells with metabolic phenotypes, its loss during differentiation and recovery in iPSCs suggest a hitherto unappreciated role of Mb in controlling the balance between aerobic metabolism and pluripotency. Understanding how Mb contributes through modulation of the mitochondrial physiology to the stem cell biology paves the way to novel perspectives in regenerative medicine as well as in cancer stem cell therapy.

Resveratrol Treatment in Human Parkin-Mutant Fibroblasts Modulates cAMP and Calcium Homeostasis Regulating the Expression of Mitochondria-Associated Membranes Resident Proteins.

Parkin plays an important role in ensuring efficient mitochondrial function and calcium homeostasis. Parkin-mutant human fibroblasts, with defective oxidative phosphorylation activity, showed high basal cAMP level likely ascribed to increased activity/expression of soluble adenylyl cyclase and/or low expression/activity of the phosphodiesterase isoform 4 and to a higher Ca2+ level. Overall, these findings support the existence, in parkin-mutant fibroblasts, of an abnormal Ca2+ and cAMP homeostasis in mitochondria. In our previous studies resveratrol treatment of parkin-mutant fibroblasts induced a partial rescue of mitochondrial functions associated with stimulation of the AMPK/SIRT1/PGC-1α pathway. In this study we provide additional evidence of the potential beneficial effects of resveratrol inducing an increase in the pre-existing high Ca2+ level and remodulation of the cAMP homeostasis in parkin-mutant fibroblasts. Consistently, we report in these fibroblasts higher expression of proteins implicated in the tethering of ER and mitochondrial contact sites along with their renormalization after resveratrol treatment. On this basis we hypothesize that resveratrol-mediated enhancement of the Ca2+ level, fine-tuned by the ER-mitochondria Ca2+ crosstalk, might modulate the pAMPK/AMPK pathway in parkin-mutant fibroblasts.