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.

Para-hydroxyphenylpyruvate inhibits the pro-inflammatory stimulation of macrophage preventing LPS-mediated nitro-oxidative unbalance and immunometabolic shift.

Targeting metabolism is emerging as a promising therapeutic strategy for modulation of the immune response in human diseases. In the presented study we used the lipopolysaccharide (LPS)-mediated activation of RAW 264.7 macrophage-like cell line as a model to investigate changes in the metabolic phenotype and to test the effect of p-hydroxyphenylpyruvate (pHPP) on it. pHPP is an intermediate of the PHE/TYR catabolic pathway, selected as analogue of the ethyl pyruvate (EP), which proved to exhibit antioxidant and anti-inflammatory activities. The results obtained show that LPS-priming of RAW 264.7 cell line to the activated M1 state resulted in up-regulation of the inducible nitric oxide synthase (iNOS) expression and consequently of NO production and in release of the pro-inflammatory cytokine IL-6. All these effects were prevented dose dependently by mM concentrations of pHPP more efficiently than EP. Respirometric and metabolic flux analysis of LPS-treated RAW 264.7 cells unveiled a marked metabolic shift consisting in downregulation of the mitochondrial oxidative phosphorylation and upregulation of aerobic glycolysis respectively. The observed respiratory failure in LPS-treated cells was accompanied with inhibition of the respiratory chain complexes I and IV and enhanced production of reactive oxygen species. Inhibition of the respiratory activity was also observed following incubation of human neonatal fibroblasts (NHDF-neo) with sera from septic patients. pHPP prevented all the observed metabolic alteration caused by LPS on RAW 264.7 or by septic sera on NHDF-neo. Moreover, we provide evidence that pHPP is an efficient reductant of cytochrome c. On the basis of the presented results a working model, linking pathogen-associated molecular patterns (PAMPs)-mediated immune response to mitochondrial oxidative metabolism, is put forward along with suggestions for its therapeutic control.

Clock-genes and mitochondrial respiratory activity: Evidence of a reciprocal interplay.

In the past few years mounting evidences have highlighted the tight correlation between circadian rhythms and metabolism. Although at the organismal level the central timekeeper is constituted by the hypothalamic suprachiasmatic nuclei practically all the peripheral tissues are equipped with autonomous oscillators made up by common molecular clockworks represented by circuits of gene expression that are organized in interconnected positive and negative feed-back loops. In this study we exploited a well-established in vitro synchronization model to investigate specifically the linkage between clock gene expression and the mitochondrial oxidative phosphorylation (OxPhos). Here we show that synchronized cells exhibit an autonomous ultradian mitochondrial respiratory activity which is abrogated by silencing the master clock gene ARNTL/BMAL1. Surprisingly, pharmacological inhibition of the mitochondrial OxPhos system resulted in dramatic deregulation of the rhythmic clock-gene expression and a similar result was attained with mtDNA depleted cells (Rho0). Our findings provide a novel level of complexity in the interlocked feedback loop controlling the interplay between cellular bioenergetics and the molecular clockwork. This article is part of a Special Issue entitled 'EBEC 2016: 19th European Bioenergetics Conference, Riva del Garda, Italy, July 2-6, 2016', edited by Prof. Paolo Bernardi.

Clock genes-dependent acetylation of complex I sets rhythmic activity of mitochondrial OxPhos.

Physiology of living beings show circadian rhythms entrained by a central timekeeper present in the hypothalamic suprachiasmatic nuclei. Nevertheless, virtually all peripheral tissues hold autonomous molecular oscillators constituted essentially by circuits of gene expression that are organized in negative and positive feed-back loops. Accumulating evidence reveals that cell metabolism is rhythmically controlled by cell-intrinsic molecular clocks and the specific pathways involved are being elucidated. Here, we show that in vitro-synchronized cultured cells exhibit BMAL1-dependent oscillation in mitochondrial respiratory activity, which occurs irrespective of the cell type tested, the protocol of synchronization used and the carbon source in the medium. We demonstrate that the rhythmic respiratory activity is associated to oscillation in cellular NAD content and clock-genes-dependent expression of NAMPT and Sirtuins 1/3 and is traceable back to the reversible acetylation of a single subunit of the mitochondrial respiratory chain Complex I. Our findings provide evidence for a new interlocked transcriptional-enzymatic feedback loop controlling the molecular interplay between cellular bioenergetics and the molecular clockwork.

A novel NHE1-centered signaling cassette drives epidermal growth factor receptor-dependent pancreatic tumor metastasis and is a target for combination therapy.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal cancers principally because of early invasion and metastasis. The epidermal growth factor receptor (EGFR) is essential for PDAC development even in the presence of Kras, but its inhibition with erlotinib gives only a modest clinical response, making the discovery of novel EGFR targets of critical interest. Here, we revealed by mining a human pancreatic gene expression database that the metastasis promoter Na(+)/H(+) exchanger (NHE1) associates with the EGFR in PDAC. In human PDAC cell lines, we confirmed that NHE1 drives both basal and EGF-stimulated three-dimensional growth and early invasion via invadopodial extracellular matrix digestion. EGF promoted the complexing of EGFR with NHE1 via the scaffolding protein Na+/H+ exchanger regulatory factor 1, engaging EGFR in a negative transregulatory loop that controls the extent and duration of EGFR oncogenic signaling and stimulates NHE1. The specificity of NHE1 for growth or invasion depends on the segregation of the transient EGFR/Na+/H+ exchanger regulatory factor 1/NHE1 signaling complex into dimeric subcomplexes in different lipid raftlike membrane domains. This signaling complex was also found in tumors developed in orthotopic mice. Importantly, the specific NHE1 inhibitor cariporide reduced both three-dimensional growth and invasion independently of PDAC subtype and synergistically sensitized these behaviors to low doses of erlotinib.