Mitochondria and Cystic Fibrosis Transmembrane Conductance Regulator dialogue: some news.

Cystic fibrosis is a progressive, genetic disease that causes persistent lung infections and limits the ability to breathe over time. It results from different possible mutations in the CFTR gene, which encodes the CFTR chloride channel, a protein that controls the movement of salt and water in and out of your body's cells. It follows that the abnormal channel function of the expressed protein on the secretory cell membrane determines the clinical phenotype in its classical form. Novel and more recent studies on mitochondrial bioenergetics aiming to rediscover a possible role of mitochondria in this disease provide a springboard for upcoming research to further understand the molecular mechanisms responsible for the involvement of mitochondria in CF and identify the protein/s primarily responsible for the F508del-CFTR-dependent mitochondrial alterations. Here, we review these CFTR-driven mitochondrial defects, thus revealing potential new targets for therapy.


Introduction
Worldwide the research on mitochondria is feverish because they are considered decisive for the role played in both health and disease. Research on mitochondria rages because if we learn to know them better, to protect them and treat them, maybe we can live longer and in greater health. Every single human cell contains thousands of mitochondria which fulfil a multitude of essential cellular functions, ultimately reciprocally dependent on the production of ATP and reactive oxygen species (ROS) 1 . The production of ATP over ROS (or vice versa) ultimately depends on metabolite availability and oxidation, electron transfer through the respiratory chain, electron chemical proton gradient (ΔμH+) formation, ADP availability, adenine nucleotide translocator (ANT) activity and mitochondrial structure and morphology [2][3][4] . If maintained at low enough concentrations, ROS serve as signalling molecules; in excess, ROS are detrimental. Therefore, by controlling the types and levels of ROS, mitochondria play a major role in cellular redox homeostasis. It is clear that when mitochondria fail, less and less energy is generated within the cell; cell injury and even cell death follow. The dysregulation of redox balance can lead to acute and/or long-term oxidative or reductive stresses that are associated with many abnormalities in disease processes, including Cystic Fibrosis (CF) [5][6] . CF, a lethal autosomal recessive disorder, is classically characterized by obstructive lung disease, pancreatic insufficiency and elevated sweat chloride values 7 . These manifestations are the consequence of abnormal epithelial cell transport of electrolytes caused by dysfunction of the CF transmembrane conductance regulator (CFTR).
Although the first discoveries on mitochondria function in CF date back to over 30 years ago 8 , to date still little is known about the role of mitochondria in this insidious disease. This scarcity of information is due primarily to the fact that since CF is caused by mutations in the gene encoding the CFTR protein, major research is essentially centred on this mutated protein [9][10] .
Here we review the CFTR-driven mitochondrial defects -with particular attention to functional ones -including those reported in more recent studies, which overall constitutes an extremely necessary and essential starting point to identify areas needed for further research aimed at understanding the molecular mechanisms responsible for the involvement of mitochondria in CF. Future perspectives are also considered.

Cystic Fibrosis. Brief description
CF is a relatively common genetic disorder in the Caucasian population: people with CF tend to have a shorter-than-normal life span. In the 1950s, many children with CF died before attending elementary school; now, many CF adults reach middle age and are predicted to get older in view of advances in medical management.
CF is caused by various mutations in a gene on chromosome 7 encoding the CFTR protein, which functions as a chloride channel within a number of epithelial tissues [11][12][13] . Mutations of the CFTR gene reduce the channel function of the protein leading to an altered fluid and electrolyte composition of secretions, thereby resulting in their increased viscosity which is responsible for progressive obstruction and fibrosis of various organs 14 . The most frequent mutation associated with CF is the deletion of phenylalanine 508 of the CFTR (F508del-CFTR) 13 . The mutated CFTR channel is not fully Figure 1: CFTR function in cell. In A, normal CFTR channel protein reaches the membrane and mediates Chloride (Cl-) flux across the plasma membrane; in B, mutated CFTR channel protein (F508del-) doesn't reach the plasma membrane, then preventing Cl-ion movement; in C, Corrector rescued F508del-CFTR reaches the plasma membrane, where it facilitates Cl-ion movement, albeit with lower efficiency with respect to the control, i.e. A. glycosylated thus impairing the achievement of the plasma membrane and consequently its function in epithelial cells ( Figure 1). In addition to chloride, the CFTR channel is able to transport bicarbonate 6 and also glutathione (GSH), so participating to its homeostasis 15 . Extensive research on the CFTR mutation has shed light on the ways in which this gene is vital to normal human development.
As CF is a multi-system disease, an aggressive combination of specific therapies is required to treat the disease symptoms. Surprisingly, a novel drug class, i.e. CFTR corrector, widely used in laboratory research, could revolutionise CF treatment, together with respiratory and nutritional therapies. The fundamental premise of CFTR corrector therapy for CF is that by addressing the basic defect underlying CF will result in clinical benefit 16 . CFTR correctors are principally targeted at F508del cellular misprocessing. In particular, they do not correct errors in the CFTR gene itself (as with gene therapy), but rather, errors that occur from transcription onwards, i.e. they are able to overcome the inefficient folding of the mutant protein defect and to improve F508del trafficking, thus allowing chloride to move properly in and out of cells lining the lungs and other organs ( Figure 1).

Early and latest studies concerning mitochondrial alterations in CF
Even if to date there have been very few studies on the possible functional link between mitochondrial defects and CF [5][6][7][8][17][18][19][20][21][22][23][24] , the first suspicions that mitochondrial lesions occur in CF dates back to 1979 with weak hints of Complex I (mtCx-I) impairment 7,[17][18][19][20]25 . Using cultured fibroblast cells from CF and control patients, Shapiro and collaborators have reported that CF cells showed more oxygen consumption and were more sensitive to the mtCx-I inhibitor rotenone than normal cells 8 . In the same time, altered optimal pH and Km values for the NADH dehydrogenase, the enzyme of mtCx-I, were also found 8 . Later, Santa-Coloma's group found a CFTR-mediated down-regulation of MT-ND4 in CF, a gene encoding for ND4, a mtCx-I subunit essential for its assembly and activity 6,26 , thus endorsing the hypothesis that the activity of mtCx-I may be reduced in CF cells. Further, more recently, Santa-Coloma's group observed that autocrine IL-1β has a major role in the reduction of the mCx-I activity observed in cultured CF cells or cells with impaired CFTR activity (IB3-1, Caco-2/pRS26 cells). Interestingly, the IKK inhibitor III, the IL-1β blocking Ab or the IL-1RN, were able to fully restore the mCx-I activity, and to produce a significant reduction in the ROS levels, to near basal values for cellular ROS levels 27 .
Altered kinetics also for cytochrome c oxidase (COX) resulting in increased Km have been reported in CF fibroblast by Lenaz's group 28 . Later, Antigny et al. 22 observed that the mitochondria network is fragmented in F508del-CFTR cells and the mitochondrial Ca 2+ uptake is reduced, compared to healthy cells, proposing that these defects are the consequence of mitochondrial membrane depolarization leading to a deficient mitochondrial Ca 2+ uptake.
Another very interesting aspect -that requires insights -is that on the role of mitochondria in decoding the Ca 2+ signal that regulates their activity and determines their cellular fate. Rimessi et al. 29 showed the supplementary role of mitochondria as drivers of the P. aeruginosa-dependent inflammatory process in CF cells and demonstrate that mitochondria are critical targets of the 'proinflammatory' Ca 2+ signal, thus considering mitochondrial Ca 2+ signalling to have a critical role in inflammasome induction, NLRP3 recruitment and the exacerbated P. aeruginosa-dependent inflammatory response in CF cells.
This paucity of data essentially concerning the involvement of disrupted mitochondrial function in CF led us to more fully explore mitochondrial bioenergetics in cells homozygous for F508del CFTR with an added value, never before investigated, that of determining if the CFTR corrector mediated rescue of F508del-CFTR could involve corrections in the bioenergetics dynamics. So, it is of a few months ago the paper by Atlante et al. 30 , showing that some steps of oxidative phosphorylation (OXPHOS), such as ANT-dependent ADP/ATP exchange, oxygen consumption, mitochondrial membrane potential (∆ψ) generation and both mtCx-I and COX activities are impaired in CF cells while both ROS production and mitochondrial membrane lipid peroxidation increase ( Figure 2). Importantly, treatment with the CFTR correctors, i.e. VX-809 and TMA [31][32] , which increase the amount of functional CFTR at the cell surface and per se are without any direct effect on the main mitochondrial activities, partially restored the mitochondrial function in CF cells. Doubtless, one of the outstanding problems in the role played by mitochondria in disease processes concerns cellular ATP production, mostly by OXPHOS, and its utilization. ATP reaches the cytosol, via the ANT, and becomes available for various cellular processes. A decrease (about 50%) in the transport efficiency of the ANT occurs in CF cells, strongly prevented in the cells treated with the CFTR correctors 30 . As ANTdependent ATP availability in the cytosol regulates CFTR channel activity gating 33 , it follows that the activity of the ADP/ATP translocator, in concert with cytosolic enzymes that consume ATP, is critical in CF by playing a decisive role in the maintenance of cytosolic ATP levels.
Furthermore, confirming previous tips 5-8, 17-20 , Atlante et al. have shown that mitochondria from CF cells are defective in NADH utilization by mtCx-I, measured both as mitochondrial oxygen consumption and Δψ generation 30 . It is known that when a mitochondrial defect is attributed to mtCx-I, it can arise from i) a loss of Complex I activity, ii) an increase in its ROS production or iii) both, thus attributing to mtCx-I a role in inducing oxidative stress. Thus, in this respect, an innovative finding was highlighted: the rotenone-sensitive mtCx-I -dependent ROS production increases in CF, thus suggesting that the mitochondrial defect attributed to mtCx-I arises from both a loss of activity and an increase in ROS production ( Figure 2). Concerning this, Kelly Aubert 5 observed that the decrease in the mtCx-I activity of CF cells and CFTR knockout mice was reverted to healthy values by treating cells with GSH monoethylester, a membrane permeable analogue of GSH proved to be able in increasing mitochondrial GSH (mGSH) levels in several cellular models [34][35][36] . Further, consistently with this, the existence of mitochondrial oxidative stress in lungs from CFTR-knockout mice has been confirmed by both an increase in the oxidation of mitochondrial DNA and a loss of aconitase activity 37 .
Importantly, in addition to the defect of mtCx-I, Atlante et al. 30 also observed a functional defect in Complex IV (i.e. COX), confirming the finding 28 of about 30years ago, according which there was a significant increase of the Km of COX for cytochrome c in CF fibroblasts, but this without providing the possible mechanism for the change in kinetics. The activity of COX, a multi-subunit protein complex embedded in the inner mitochondrial membrane, is strongly dependent on the membrane lipid environment. So, since mitochondrial ROS production and membrane lipid peroxidation increase in CF cells, Atlante et al. 30 have suggested that ROS-mediated damage of the membrane microenvironment may be responsible for inhibition of Complex IV. A note very important and exciting in this context is the finding that treatment of CF cells with the CFTR correctors partially restores the mitochondrial function as well it reduces the extent of membrane lipid peroxidation in CF cells (Figure 2 ).

Future Perspectives
The novel information on mitochondrial bioenergetics in CF reported in Atlante et al. 30 , together with the restorative action provided by the CFTR correctors, strongly suggest the profound interaction dynamics between CFTR, mitochondrial (patho)physiology and ROS.
Currently, we cannot provide any molecular mechanism underlying how CFTR dysfunction affects so many parameters of mitochondrial function, let alone how corrector-induced increased CFTR cell surface expression is able to repair these mitochondrial dysfunctions. However, we are confident and with clear ideas on how to proceed in research concerning the existing dialogue between mitochondria and CFTR in CF.
Here, we briefly design the research issues on which we're already working: i) one of these regards GSH; ii) the other, the ANT.
i. As a drop of GSH levels in lung epithelial lining fluid has been reported in CF 38,39 , we are putting in the spotlight the GSH level, both mitochondrial and cytosolic forms, the GSH-dependent enzymes and all that concerns it in-order-to establish and understand the link between mitochondrial impairment, low GSH levels and defective F508del-CFTR. The reason for this is because cell redox balance within a cell -mainly influenced by the ratio between reduced GSH and its oxidized formis regulated by complex processes, which converge exclusively upon mitochondria. Concerning this, that a link exists between GSH and mitochondria is demonstrated by the fact that a decrease in the mtCx-I activity of CF cells and CFTR knockout mice was reverted to control values by treating cells with GSH monoethylester 5 , a membrane permeable analogue of GSH proved to be effective in increasing mGSH levels in several cellular models [34][35][36] .
ii. In view of the fact that mitochondrial ANT-dependent ATP availability in the cytosol serves to gate CFTR channel activity, it follows that the activity of ANT is critical in CF. In this regard, we cannot exclude that the ANT impairment occurring in CF cells 30 is due i) to ROS-dependent oxidation of thiol group/s of the carrier, present at the active site of ANT and oriented toward the external hydrophilic phase, and/or ii) to changes in mitochondria configuration from the orthodox to a condensed form, an alteration that may modify thiol groups of membrane protein/s against oxidation by ROS. This consistently with the observation that ROS can regulate protein function thorough modification/s of specific thiol(s) of a protein or of its surrounding lipid environment 40 . So, that being said, the possibility that thiol groups on the matrix surface of the ANT may trigger the mechanism responsible of the mitochondrial permeability transition pore opening in CF cannot be excluded.
Needless to say that further exploration of mechanism underlying mitochondrial function in CF may open new possible avenues for therapeutics. Will see some good!

Schematic representation of the mitochondrial respiratory chain
Oxidation of respiratory substrates leads to reduction of coenzymes to NADH and FADH 2 . Ubiquinone (Q) shuttles electrons from both Complexes I and II to Complex III, while cytochrome c (cyt c) shuttles electrons from Complex III to Complex IV. Molecular oxygen (O 2 ) is the terminal electron acceptor. Protons (2H + ) are pumped out by Complexes I, III and IV creating a proton electrochemical potential gradient. The resulting proton gradient drives both the exchange of ADP/ATP via the ANT and the synthesis of ATP from ADP and inorganic phosphate (Pi) by Complex V.

Mitochondrial targets in CF are marked with X
i) ANT and Complex I are inhibited; Δψ generation decreases; ii) production of ROS at Complex I is exacerbated; iii) COX is inhibited through ROS-induced damage to mitochondrial lipids, such as cardiolipin. In particular, the mitochondrial defect attributed to Complex I arises from both a loss of activity and an increase in ROS production; whereas Complex IV is impaired through Complex I-dependent ROS production and membrane lipid peroxidation in CF cells.

Main abbreviations
I, Complex I or NADH-ubiquinone oxidoreductase; II, Complex II or succinate-ubiquinone oxidoreductase; III, Complex III or ubiquinone-cytochrome-c oxidoreductase; IV, Complex IV or cytochrome-c oxidase; V, Complex V or FoF1 ATP synthase; ANT, adenine nucleotide translocator; MIM, mitochondrial inner membrane.