Mitochondrial Intoxication

Mitochondrial Intoxication explores the effects toxic molecules can have upon mitochondrial physiology in the human body. Each chapter is dedicated to a specific toxicant, including pollutants, food additives, illicit and pharmaceutical drugs, and heavy metals. This book considers the implications and impact these have upon mitochondria and the diseases that can result from dysfunction and impairment in the human body. Furthermore, the book provides an overview of mitochondrial physiology and assesses the advances and challenges in testing mitochondrial toxicity. Case studies exploring mitochondrial intoxication in pregnancy and in the geriatric population are also included.

This is a comprehensive reference on the main toxicants impacting mitochondria in the human body, and the consequences this can have for health and disease.

• Features a wide range of toxicants and their effects on mitochondrial physiology
• Covers molecular markers of mitochondrial intoxication
• Includes case studies that illustrate the health impacts of toxicity upon mitochondria
• Considers future directions regarding the study of mitochondrial intoxication

• Language: English
• Publisher: Academic Press
• Release date: Nov 30, 2022
• ISBN: 9780323884631

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1: Mitochondrial physiology: An overview

Vittoria Infantino; Simona Todisco; Paolo Convertini    Department of Science, University of Basilicata, Potenza, Italy

Abstract

Mitochondria are multifaceted organelles having their own genome, although most of the mitochondrial proteins are encoded by the nuclear genome and then imported into mitochondria. Beyond the classical function in generating ATP, mitochondria regulate plenty of eukaryotic cell functions including cell death, redox status, proliferation, and differentiation. Participating in a variety of signaling pathways, a tight crosstalk between mitochondria and the rest of the cell is required. As a predictable consequence, mitochondrial dysfunctions account for countless pathological conditions such as inflammation, cancer, and metabolic diseases. Therefore deepening the mitochondrial knowledge represents an important contribution to the management of many diseases. This chapter will focus on several traits of mitochondrial physiology among which metabolism, dynamics, redox, and cell death signaling, thus opening new ways in understanding mitochondrial biology.

Keywords

Energetic metabolism; Mitochondrial dynamics; Mitochondrial biogenesis; Mitophagy; Mitochondrial apoptosis; Necroptosis; Pyroptosis; Ferroptosis; Mitochondrial redox biology

1: Introduction

Mitochondria are maternally inherited organelles bounded by two membranes: the outer mitochondrial membrane (OMM) which contains porins allowing passage of small molecules and ions (molecular mass ≤ 4–5 kDa) and the inner mitochondrial membrane (IMM) which is folded into cristae, and very impermeable. IMM architecture can be considered the responsible of the crosstalk between mitochondria and cytoplasm, of ATP synthesis by oxidative phosphorylation as well as of transport through the specific mitochondrial carriers. OMM and IMM define two separated compartments: the intermembrane space and the mitochondrial matrix containing a mitochondrial genome.

Mitochondria occupy almost all of cytoplasm of eucaryotic cells which may contain hundreds of mitochondria depending on energy requirements. Though their function is historically linked to generation of energy and intermediary metabolites for biosynthetic pathways, today we know that this view is somewhat reductive. Indeed, mitochondria act as regulators of lots of cellular functions by a myriad of signaling mechanisms. Mitochondrial communication with the rest of the cell serves at least one fundamental purpose: to prevent cells from engaging in a biological process requiring a metabolic demand when mitochondria are unable to meet it.

First of all, the presence of a mitochondrial genome (mtDNA) harboring 37 genes in humans—13 encoding mitochondrial proteins, 22 tRNAs, and 2 rRNAs needful for the mitochondrial translation—requires a bidirectional crosstalk between nuclear and mitochondrial DNA (Castegna et al., 2015). Indeed, key factors regulating mtDNA transcription and replication are nucleus-encoded proteins. More generally, if 13 subunits of the electron transport chain (ETC) complexes are mtDNA encoded, the remaining more than 1000 mitochondrial proteins are encoded by the nuclear genome and then imported into mitochondria (Calvo et al., 2016). Moreover, nuclear gene expression reprogramming and epigenetics certainly affect mitochondrial functions such as mitochondrial dynamics, biogenesis or mitochondrial respiratory activity to meet the cellular needs through the anterograde regulation (Martínez-Reyes and Chandel, 2020).

At the same time, mitochondria may control nuclear gene expression by means of a retrograde signaling, thus affecting cellular activities under both physiological and pathological conditions (Bock and Tait, 2020). A representative example of this retrograde signaling is the release of mitochondrial ROS (mtROS) in hypoxic conditions, which leads to the stabilization of hypoxia inducible factor 1 alpha (HIF-1α) and in turn to the upregulation of target genes allowing the adaptation to low oxygen (Chandel et al., 2000).

In the past decades, increasingly findings revealed mitochondria as a source of signal molecules which communicate the mitochondrial fitness not only to the nucleus but also to the rest of the cell. Notably, the just reported release of mtROS also affects the cytosolic metabolism by regulating the activity of metabolic enzymes (Nemoto et al., 2000). Moreover, mitochondria release cytochrome c induces cell death in mammalian cells (Liu et al., 1996) and regulates the evolutionarily conserved energy-sensing AMP-depending protein kinase (AMPK) activity which mediates mitochondrial dynamics and mitophagy (Toyama et al., 2016). Furthermore, mtDNA—whose regulation is much more complex than we could have imagined until a few years ago, for example because of epigenetic modifications (Iacobazzi et al., 2013)—works as a specific activator in innate immune cells (Shimada et al., 2012). Finally, emerging evidence suggests that tricarboxylic acid (TCA) cycle intermediates, beyond their essential function in biosynthesis of macromolecules such as nucleotides, lipids, and proteins, act as signal molecules regulating epigenetics and post-translational modifications of proteins or are used to produce signals whose levels are critical for cell homeostasis (Ryan and O'Neill, 2020; Infantino et al., 2011). All together these aspects suggest to look at communication with the rest of the cell to understand the mitochondrial physiology.

2: TCA cycle and oxidative phosphorylation system activity

The mitochondrial metabolism mirrors the different functions in which the mitochondria are involved such as molecular signaling with reactive oxygen species (ROS) management reactions, amino acid catabolism, lipid metabolism, heme biosynthesis, production of precursors for DNA, RNA, and protein synthesis and urea cycle (Spinelli and Haigis, 2018).

The metabolic hub of mitochondria is the TCA cycle, which links catabolism and anabolism of carbohydrates, amino acids, and fatty acids. This cycle consists of eight enzymatic reactions starting with the condensation of acetyl-CoA and oxaloacetate (OAA), catalyzed from citrate synthase, to synthetize citrate.

Acetyl-CoA comes from oxidative pyruvate decarboxylation, catalyzed by pyruvate dehydrogenase complex (PDC). Alternatively, acetyl-CoA derives from fatty acid catabolism. The citrate is converted into isocitrate from the aconitase starting the reactions that lead to the production of reducing equivalents, NADH and FADH2, and GTP. As an assembly line, the goal is the energy production to supply cell demands. The enzymes are isocitrate dehydrogenase, α-ketoglutarate dehydrogenase, succinyl-CoA synthetase, succinate dehydrogenase, fumarase, and malate dehydrogenase. For each molecule of pyruvate converted to acetyl-CoA, three molecules of NADH, one of FADH2 and one of GTP—then converted into ATP—are produced through the TCA cycle. At the end, the OAA is condensed with another molecule of acetyl-CoA to regenerate citrate and the cycle can start again (Fig. 1).

Fig. 1

Fig. 1 Mitochondrial energetic hub. TCA cycle reactions, related shuttles, and electron transport chain complexes are represented. PDC , pyruvate dehydrogenase complex; MPC , pyruvate carrier; CIC , citrate carrier; AGC , aspartate/glutamate carrier; OGC , oxoglutarate carrier; CPT1 , carnitine palmitoyltransferase 1; CPT2 , carnitine palmitoyltransferase 2; CAC , carnitine/acylcarnitine carrier; I , Complex I; II , Complex II; Q , Coenzyme Q; III , Complex III; cytc , cytochrome c ; IV , Complex IV; and V : Complex V.

Different checkpoints control the flux and guarantee the proceeding of the TCA cycle. The first checkpoint is at level of the mitochondrial PDC; the second is represented by the citrate synthase proposed under the control of ATP and NADH (Srere, 1974). Yet, the α-ketoglutarate dehydrogenase complex and malate dehydrogenase are regulated by the concentrations of NADH and ATP. This regulation suggests that cellular energy levels control the process of cellular respiration.

Interestingly, most of regulated enzymes are also involved in the biosynthesis of metabolites. Indeed, some TCA cycle intermediates supply substrates for the biosynthesis of molecules like nucleotides, heme, lipids, carbohydrates, and amino acids. For example, the succinate furnishes the building block for the heme synthesis or mitochondrial citrate, exported to the cytosol, directly provides the carbon source for fatty acids and cholesterol biosynthesis through ATP citrate lyase (ACLY). Notably, different studies indicate that TCA cycle is the best possible chemical design combining the multiple needs of the cell and represents a typical case of opportunism in molecular evolution (Linn and Srere, 1979).

When TCA cycle intermediates are subtracted for biosynthetic requires, they must be replenished to guarantee a minimum level for the cycle function; this is the anaplerosis view. Two important anaplerosis reactions are as follows: pyruvate carboxylase to synthesize OAA from pyruvate and the glutamine/glutamate metabolism via glutaminase converting glutamine to glutamate and the latter in α-ketoglutarate.

Mitochondria play another important role in the balance of redox equivalents whose compartimentalization between cytosol and mitochondria is required to maintain the cellular homeostasis and viability (Yang et al., 2007). The different NAD+/NADH ratio in the cytosol and mitochondria is sustained by shuttle systems as malate/aspartate shuttle, citrate shuttle, and glycerol-3-phosphate shuttle. The malate/aspartate shuttle transports reducing equivalents from cytosolic NADH to the mitochondrial matrix. The players involved in this shuttle are malate dehydrogenase (MDH1 and MDH2), glutamate-oxaloacetate transaminase (GOT1 and GOT2), and two mitochondrial carrier, the aspartate/glutamate carrier and the oxoglutarate carrier. Compartmentalization of reducing equivalents is crucial for survival in stress conditions, but also for promoting tumor survival and proliferation (Todisco et al., 2019).

The citrate/malate shuttle is part of the citrate pathway which, exporting mitochondrial citrate, promotes fatty acid synthesis. The citrate pathway consists of citrate synthase, the citrate/isocitrate carrier (CIC), ACLY synthesizing acetyl-CoA, for the fatty acid biosynthesis, and OAA converted, by cytosolic MDH1, in malate that come back in the mitochondria by transporting in this way the reducing equivalents. Recently, the role of the citrate pathway has been investigated in inflammation. In particular, it has been demonstrated the involvement of CIC and ACLY in modulating the synthesis of the inflammatory mediator as NO glyph_rad , ROS, and PGE2 in M1 macrophages (Infantino et al., 2019). Finally, the glycerol-3-phosphate shuttle—not the most used in humans—is composed of cytosolic and mitochondrial glycerol-3-phosphate dehydrogenase (cGPDH and mGPDH) differing from other shuttles by the involved reducing equivalents. In fact, cGPDH utilizes NADH to generate NAD+, while mGPDH employs FAD to produce FADH2 which is directly oxidized in the mitochondrial ETC. The glycerol-3-phosphate shuttle allows the reoxidation of cytosolic NADH bypassing Complex I (CI) of ETC and regulating the cytosolic glycerol-3-phosphate as a crossing point metabolite of glycolysis, lipogenesis, and oxidative phosphorylation (Mráček et al., 2013).

All mitochondrial metabolic processes depend on and sustain the oxidative phosphorylation that converts most of the energy in ATP. The reducing equivalents NADH and FADH2, produced in mitochondrial metabolism, through ETC transfer their electrons to the oxygen, the final acceptor. The electrons flow from molecules characterized by a lower reduction potential, which donate electrons, to those with a higher reduction potential, which accept electrons. The energy produced by the transfer of electrons is utilized to transfer protons from the mitochondrial matrix to the intermembrane space and create a proton gradient. The first complex of ETC is the CI (NADH dehydrogenase) consisting of NADH dehydrogenase, flavin mononucleotide (FMN), and eight iron-sulfur (Fe-S) clusters. CI oxidizes NADH to NAD+ and donates two electrons to FMN coenzyme of NDUFV1 subunit (Fig. 1). Later, electrons are transferred through Fe/S clusters at oxidized coenzyme Q that reduces to ubiquinol (QH2). This transfer of electrons creates an electrochimical proton gradient with the passage of four protons through CI from the matrix to intermembrane space. Complex II of ETC (CII, succinate dehydrogenase) is a step of TCA cycle which reduces FAD+ to FADH2, a coenzyme bound to subunit SDHA. FADH2 transfers two electrons to the Fe/S clusters present in SDHB and successively to coenzyme Q. In this process, protons are not pumped through the CII, which does not support the electrochemical proton gradient (Fig. 1). After reduction, Coenzyme Q transports the electrons to Complex III (CIII, Q-cytochrome c oxidoreductase), which in turn transfers them to cytochrome c, a soluble carrier protein.

CIII consists of cytochrome b, Rieske subunits (containing two Fe-S clusters), and cytochrome c protein that contains a heme group. Cytochrome c accepts a single electron from CIII. Therefore to transfer a pair of electrons, this process takes place in two steps (Q cycle) and simultaneously transfers four protons into the intermembrane space by contributing to form the electrochemical proton gradient (Brzezinski et al., 2021). In the cycle Q, QH2 transfers each electron to two sites on CIII: first electron is transferred to the Fe-S cluster and then cytochrome c, second electron is transferred to cytochrome b and then a Coenzyme Q bound on CIII. In this step, two protons are pumped in the intermembrane space for the proton gradient. The oxidized Coenzyme Q dissociates from CIII and can newly receive the electrons from CI and CII.

The second step of the Q cycle consists of a repeat of the first step with an oxidation of another molecule of QH2 and a complete reduction of the Coenzyme Q bound to CIII. In this step, two protons are also transferred in the intermembrane space for proton gradient. Cytochrome c is oxidized by Complex IV (CIV, cytochrome c oxidase) that transfers electrons to the oxygen, the final electron acceptor, to form one molecule of water (Fig. 1). CIV consists of two heme proteins (cytochrome a and a3) and two copper centers (CuA and CuB) (Pilet et al., 2004). Cytochrome c leaves one electron at CuA center, which in turn transfer it to heme a3 and then to CuB and heme a3. When heme a3 and CuB are reduced, one molecule of O2 is linked to form a peroxide bridge between the two groups. Finally, the reaction ends with proton transfer by mitochondrial matrix and formation of two H2O molecules. The energy associated with electron transfer is used to move four protons through the IMM in the intermembrane space for the proton gradient (Michel, 1999). Complex V (CV, ATP synthase) consists of Fo and F1 subunits and uses the electrochemical proton gradient to form ATP. Fo is an electric motor formed by the hydrophobic channel that crosses the IMM composed by a cylinder of c subunits (10–14 subunits) and a, b, d, F6 subunits, and the oligomycin sensitivity-conferring protein (Protasoni and Zeviani, 2021). These subunits can be protonated and deprotonated by H+ flow causing rotation, which affects the orientation of the F1 subunit. F1 subunit is a hydrophilic complex that contains subunits α, β, γ, δ, and ɛ, protruding into the mitochondrial matrix, and if removed from the IMM, it is a stable subcomplex. CV central axis is composed of γ, δ, and ɛ. Three copies of α and β are present and form the three catalytic sites of F1 that undergo conformational changes to catalyze the synthesis of ATP from ADP and Pi with the transfer of four protons from intermembrane space to mitochondrial matrix (Neupane et al., 2019). F1 subunit can also hydrolyzes ATP to generate a proton gradient. Notably, uncoupling proteins, as well as many compounds, inhibit the coupling between the ETC and ATP synthesis (Terada, 1990).

3: Mitochondrial dynamics: Fusion and fission

Mitochondrial homeostasis is strictly dependent on dynamics regulating mitochondrial shape, number, distribution, and functions. The interconnecting mitochondrial networks are the product of a dynamic equilibrium between fusion and fission. The movement along the microtubules and actin filaments helps to balance these processes carrying out mitochondria to areas of high metabolic needs. In cell culture, fusion and fission may also occur within seconds in the case of stress-induced fission (Meyer et al., 2017). In vivo, a mixing of mitochondria within cells takes place within an hour (Youle and van der Bliek, 2012). Notably, heterogeneous subpopulations of mitochondria have been found within cells and, in this context, fusion and fission permit subcellular specialization of mitochondria (perinuclear and axonal mitochondria) (Kowald and Kirkwood, 2011). Mitochondrial dynamics need nuclear-encoded proteins which play a critical role in regulating mitochondrial morphology, movement, and functions.

3.1: Mitochondrial fusion

Mitochondrial fusion is a process that requires fusion of OMM and IMM. In humans, three dynamin-related GTPases mediate mitochondrial fusion: Optic atrophy 1 (OPA1) and Mitofusins 1 and 2 (MFN1 and MFN2) (Wai and Langer, 2016). The last two proteins which are anchored to the OMM regulate the fusion of this mitochondrial membrane. It has been reported that while mice deficient in either Mfn1 or Mfn2 die in midgestation, Mfn2 overexpression functionally compensates for the deficiency of Mfn1 (Chen et al., 2003). Mutations in MFN2 human gene—encoding MFN2 protein—can cause neurodegenerative diseases like Charcot-Marie-Tooth neuropathy type 2A, suggesting that MFN2 activity and mitochondrial fusion are linked to the correct neuronal function (Züchner et al., 2004). Fusion of the IMM is mediated by OPA1. This protein is also involved in other critical mitochondrial functions such as maintenance of the membrane potential, cristae organization, and mtDNA preservation (Züchner et al., 2004). It is noteworthy that mutations in OPA1 human gene are associated with the dominant optic atrophy, a rare progressive and irreversible blinding disease (Alexander et al., 2000).

Fusion process, especially fusion of the inner membrane, is mitochondrial membrane potential dependent (Meyer et al., 2017). Mitochondrial fusion generates tubular mitochondria. When fusion exceeds fission, elongated and interconnected mitochondria are prevailing and form a cellular network. Such condition allows exchange of mtDNA and metabolites right through the network making the intracellular mitochondria more homogeneous (Twig and Shirihai, 2011). This process can optimize mitochondrial function and prevent the accumulation of mitochondrial mutations in aging. Furthermore, being mitochondrial fusion essential for mtDNA maintenance, defects in mitochondrial fusion cause impaired mtDNA synthesis resulting in quantitative (mtDNA depletion) and qualitative (mtDNA deletions) defects (El-Hattab et al., 2017).

After exposure to selective stresses such as ultraviolet C radiation, hyperfused mitochondria (fusion > fission) have been observed. Notably, mitochondrial fusion is a process sensitive to metabolic alterations being positively associated with nutrient deprivation, increased oxidative phosphorylation (OXPHOS) and increased ATP production. Stress-induced mitochondrial hyperfusion needs metabolically functional mitochondria which can produce more ATP leading to stress resistance (Tondera et al., 2009). For that reason, hyperfused mitochondria may represent an adaptive response against stress.

3.2: Mitochondrial fission

Mitochondrial fission needs several proteins but the best understood is the GTPase dynamin-1-like protein (DNM1L, also known as DRP1), a cytosolic protein which translocates from the cytosol to the OMM (Chan, 2012) in response to specific cellular signals. In mammals, OMM fission 1 protein (FIS1) and mitochondrial fission factor (MFF) are proposed as receptors recruiting DNM1L to the OMM (Losón et al., 2013). DNM1L mitochondrial recruitment and activity are controlled by posttranslational modifications including phosphorylation, ubiquitination, and SUMOylation. Less is understood about the mechanism underlying IMM fusion. It is unclear whether DNM1L-mediated constriction of the OMM is sufficient to simultaneously drive IMM division or additional machineries at the IMM are required, although S-OPA1 and MTP18, two IMM proteins, have been proposed to have a role in mitochondrial fission. Impaired fission results in elongated mitochondria because of unbalanced fusion. Unlike the fusion, fission may also be triggered by low membrane potential since this process does not require membrane potential (Twig et al., 2008).

Mitochondrial fission is an active process prior to mitosis when ensures into daughter cells as many mitochondria as the mother cell. Fission is used to enable transport and distribution of mitochondria. Moreover, damaged mitochondria can be generated by fragmentation of a mitochondrial network and degraded by mitophagy (El-Hattab et al., 2018).

A mutation in DNM1L gene as well as a defect of the fission of both mitochondria and peroxisomes is linked to a neonatal lethal disorder characterized by several abnormalities including optic atrophy, microcephaly, abnormal brain development, and lactic acidemia (Waterham et al., 2007). DNM1L is also substrate of Parkin E3, an E3 ubiquitin ligase, whose gene (PARK2) has been studied for its pathogenic mutations which represent one of the major causes of familial Parkinson's disease (PD). Pathogenic mutations of Parkin inhibits DNM1L ubiquitination and degradation, leading to mitochondrial fragmentation (Wang et al., 2011). Furthermore, mutant Huntingtin triggers mitochondrial fragmentation, interacts with DNM1L, and increases its enzymatic activity, suggesting a role of this protein in the pathophysiology of Huntington's disease (Song et al., 2011).

Metabolic alterations affect mitochondrial fragmentation since a reduction in MFN2 steady-state levels have been observed in rodents fed a high-fat diet. In addition, nutrient excess and impaired OXPHOS promote mitochondrial fission and increase production of ROS in a DRP1-dependent way (Yu et al., 2006). A fragmented morphology has been also observed in severe stress and cell death.

4: Mitochondrial biogenesis and mitophagy

Homeostasis of healthy cell requires a control of mitochondrial mass and function to meet the cellular energy demands. Mitochondrial homeostasis implies a dynamic equilibrium between two opposing processes: mitochondrial biogenesis that is the generation of new mitochondria and mitophagy, i.e., a mitochondria-selective autophagy which leads to the degradation of damaged mitochondria (Gatica et al., 2018). The fine regulation and the balance between these opposing processes are critical for maintaining mitochondrial fitness and cellular adaptation to metabolic shifts as well as stress signals. Indeed, imbalance between mitochondrial biogenesis and mitophagy is linked to numerous pathologic conditions (Ploumi et al., 2017). Dynamic coordination of the mitochondrial biogenesis and turnover is mainly assured by transcriptional and posttranslational regulation of key factors.

4.1: Regulation of mitochondrial biogenesis

A fine-tune regulation of the mitochondrial biogenesis involves nuclear transcription factors among which PGC1α is the master regulator. In the presence of a metabolic stress like nutrient starvation, PGC1α activation by phosphorylation via AMPK directly induces mitochondrial biogenesis (Jäger et al., 2007). PGC1α is deacetylated by SIRT1 in response to increased cellular NAD+ levels (Cantó et al., 2009). Moreover, PGC1α is target of p38 mitogen-activated kinase (p38 MAPK) and calcium/calmodulin-dependent kinase (CaMK), thus acting as a sensor of cytoplasmic calcium concentration (Abate et al., 2012). Activation of PGC1α is also induced through the transcriptional factor cAMP response element-binding protein (CREB). Following increased cAMP levels, CREB is activated through the cAMP-dependent protein kinase A (PKA) and in turn PGC1α is upregulated (Fig. 2) (Popov, 2020). Triggering of PGC1α signaling pathway stimulates the nuclear respiratory factors NRF1 and NRF2 and estrogen-related receptor-α (ERR-α) transcription factor leading to the increase in expression of mitochondrial transcription factor A (TFAM) and transcription factor B proteins (TFBs) (Fig. 2) (Gleyzer et al., 2005). The latter are the main regulators of mtDNA transcription and replication.

Fig. 2

Fig. 2 Mitochondrial homeostasis: Interplay between mitochondrial biogenesis and mitophagy. Metabolic stresses such as glucose starvation trigger the key energy stress sensor AMPK, which may promote both mitophagy and mitochondrial biogenesis via PGC1α phosphorylation (A). Moreover, AMPK—by increasing NAD + levels—boosts SIRT1 activity and in turn induces PGC1α deacetylation (B). Increased Ca ++   levels lead to PGC1α activation through CaMK-mediated phosphorylation (C). (D) Following mitochondria-toxic compounds, cAMP pathway upregulates PGC1α via CREB. (E) Protein kinase D1 acts as a sensor of mitochondrial oxidative stress (mtROS) promoting both mitochondrial biogenesis through NF-kB/PGC1β and mitophagy. (F) PGC1α positively regulates both mitochondrial biogenesis through various downstream target transcription factors (NRF1/NFR2/ERRα and in turn TFAM and TFBs) and mitophagy by upregulating the transcription TFEB transcription factor. TFEB in turn is a transcriptional activator of PGC1α, thus producing a positive feedback loop to balance between mitochondrial biogenesis and mitophagy.

4.2: Mitophagy: Molecular mechanisms

Mitophagy is an evolutionarily conserved process occurring via OMM receptors, receptor-like lipids localized at the mitochondrial membranes or receptors binding to ubiquitinated proteins of the mitochondrial surface. These targeted mechanisms drive the formation of double membrane vesicles named autophagosomes, which surround mitochondria. In mammals, autophagosomes are subsequently transported and fused with lysosomes. Here, resident acidic hydrolases degrade mitochondria, and the molecules can be recycled (Mizushima and Komatsu, 2011).

After Narendra et al. reported that impaired mitochondrial membrane potential causes degradation of damaged mitochondria via recruitment of Parkin, PTEN-induced kinase 1 (PINK1) has been identified as a key regulator of Parkin E3 following mitochondrial depolarization (Narendra et al., 2008). Currently, PINK1/Parkin-mediated mitophagy is the best-known pathway of mitochondrial degradation. In healthy mitochondria, the nuclear-encoded PINK1 is cleaved and its fragments directly bind to Parkin E3 thus blocking its recruiting and in turn inhibiting mitochondrial turnover. Loss of the mitochondrial membrane potential induces PINK1 homodimerization and autophosphorylation and finally Parkin E3 translocation to the OMM (Okatsu et al., 2012). Following Parkin E3-mediated ubiquitination of various OMM proteins, specific autophagy receptors are triggered thus leading to the autophagosome formation.

Receptors constitutively present in the OMM, among which the BCL2 interacting protein 3 (BNIP3), BCL2 like 13 (BCL2L13), and the FUN14 domain containing 1 (FUNDC1) are able to recruit the autophagic machinery to mitochondria. FUNDC1 receptor is activated under hypoxic conditions in mammalian cells. Furthermore, evidence of an interaction of FUNDC1 with both DRP1 and OPA1 strengthens the coordination between mitochondrial dynamics and mitophagy (Chen et al., 2016).

Lipid-mediated mitophagy requires the translocation of the dimeric phospholipid cardiolipin from the IMM—where is mostly localized—to the OMM in depolarized mitochondria. Here it is able to recruit mitophagic machinery (Kagan et al., 2016). The sphingolipid ceramide represents another lipid signal promoting mitophagy in damaged mitochondria since overexpression of ceramide synthase 1 as well as exogenous supplementation with ceramide trigger mitochondrial degradation and cell death in cancer. This lethal mitophagy is DRP1-dependent (Dany et al., 2016); therefore mitochondrial fission makes mitochondria susceptible to autophagosome formation.

4.3: Mitochondrial homeostasis: Coordination of mitochondrial biogenesis and mitophagy

Mitochondrial homeostasis requires a coordination of the same pathways which can affect mitochondrial mass as well as mitochondrial degradation. In this context, increased cAMP levels play a crucial role regulating both the opposing processes in response to mitochondria-specific toxic stress. Indeed, CREB upregulates the pivotal regulator of mitochondrial biogenesis PGC1α and, at the same time, cAMP pathway negatively regulates mitophagy (Cherra et al., 2010). Besides its key function in mitochondrial biogenesis, PGC1α positively regulates mitophagy by controlling gene expression of transcription factor EB (TFEB), which mediates lysosomal biogenesis. In turn, TFEB is a transcriptional activator of PGC1α gene, thus producing a positive feedback loop responsible for the balance between mitochondrial biogenesis and degradation (Settembre et al., 2011) (Fig. 2). Evidence suggests that MEK/ERK pathway while positively regulates mitochondrial mass through p38 MAPK induces both starvation- and hypoxia-induced mitophagy via MAPK1 and MAPK14 (Hirota et al., 2015). Intriguingly, AMPK promotes mitophagy through inhibition of mTOR and activation of ULK1 in response to nutrient depletion, though AMPK-mediated phosphorylation of SIRT1 activates PGC1α by deacetylation in rat hepatocytes, likely to compensate for enhanced mitochondrial turnover (Rodgers et al., 2005) (Fig. 2).

As sensor of mtROS, protein kinase D1 (PKD), induces the removal of damaged mitochondria in response to stress. Activated upon oxidative stress, PKD1 directly phosphorylates IKKβ, leading to NF-κB nuclear translocation (Tanaka et al., 2005). In response to this signaling, NF-κB upregulates SOD2 gene expression, encoding the manganese-dependent superoxide dismutase (MnSOD), which participates in detoxification (Storz et al., 2005). Moreover, NF-κB can also promote mitochondrial biogenesis via upregulation of PGC1β in skeletal muscle (Bakkar et al., 2012) (Fig. 2). Therefore the activation of PKD/NF-kB pathway in oxidative stress through mitochondrial biogenesis and mitophagy regulation likely may serve to balance two needs: cell homeostasis and survival.

4.4: Physiological functions of biogenesis and mitophagy

Mitochondrial biogenesis is mainly activated in healthy cells. Acetylcholine and resveratrol are among triggers of this process which is also induced by a lot of natural extracts through SIRT1/AMPK/PGC-1α/NRF1 pathway (Akbari-Fakhrabadi et al., 2019; Mao et al., 2019). Impaired mitochondrial biogenesis is often linked to other mitochondrial dysfunctions, such as a reduced ATP synthesis capacity leading to mitophagy, critical for cell health and reduced mtDNA/nuclear ratio. Dysregulated biogenesis is a hallmark of cell senescence and in turn of aging and is also associated with metabolic diseases such as type 2 diabetes and obesity (Hey-Mogensen and Clausen, 2017). Neurodegenerative diseases like Alzheimer's disease (AD) and PD show impaired mitochondrial biogenesis so much so that this process is emerging as a new hopeful target for treatments (Golpich et al., 2017). Finally, SIRT1/AMPK/PGC-1α/NRF1-targeted therapies seem effective to prevent cancer recurrence and distant metastasis (De Luca et al., 2015).

Active mitophagy has a physiological function and is required during development and differentiation. Elimination of mitochondria ensures erythrocyte maturation, adipocyte cell fate decision, degradation of paternal mitochondria in mammals, and is associated with longevity (Onishi et al., 2021).

Mitophagy activation upon different mitochondrial stresses such as increased mtROS promotes mitochondrial homeostasis and counteracts pathological conditions including age-related neurodegeneration, tumorigenesis, inflammation, and aging. In this context, expression of the mitophagy receptor BNIP3 decreases in cancer and is linked to cancer metastasis and chemoresistance likely due to accumulation of damaged mitochondria and elevated mtROS production (Chourasia et al., 2015). Mitophagy may also play a protective role against neurodegenerative diseases since PINK1 gene mutations have been associated with PD pathogenesis. Maybe PINK1 itself does not cause PD pathogenesis, but the correct interplay between PINK1 and Parkin promotes mitochondrial quality control and neuroprotection enabling removal of dysfunctional mitochondria (Song et al., 2017). Moreover, mitophagy is impaired in AD and overexpression of Parkin in disease mouse model lowers β-amyloid plaque levels and amyloid-induced inflammation contributing to reduce abnormalities. Stimulation of mitophagy reverses memory impairment and mitigates β-amyloid plaque formation and tau hyperphosphorylation (Fang et al., 2019). Hence mitophagy may represent a new potential target for various disease treatments.

5: Mitochondria-related cell death

Cell death is a physiological process indispensable for embryonic development as well as for normal tissue clearance. The pathogenesis of various diseases is promoted by dysregulation of cell death: its inhibition is associated with cancer and autoimmune disorders, while excessive cell death is a trait of neurodegenerative diseases, including PD and AD (Bock and Tait, 2020). Cell death occurs through a multitude of dying mechanisms with different roles in homeostasis among which apoptosis and necrosis are the best known. The extrinsic pathway, triggered by the binding of death-inducing ligands to specific membrane receptors, and the intrinsic, or mitochondrial, pathway are the main apoptotic death mechanisms. The first step of the extrinsic pathway is the activation of caspase 8. Through different ways, both pathways induce the activation of caspase 3 and caspase 7 which as executioner caspases cleave numberless target proteins generating the biochemical and morphological hallmarks of apoptosis (Julien and Wells, 2017). During the past few decades, novel forms of non-apoptotic regulated cell death including necroptosis, pyroptosis and ferroptosis have been identified (Xu et al., 2019).

Mitochondria are key organelles for cell homeostasis and their functions are tightly linked to cell fate. The crucial role in essential cellular processes and the involvement in countless cell pathways make mitochondria determinants in cell-destiny decisions. For these reasons, mitochondria represent pivotal organelles in regulating different types of cell death. Beyond their unquestionable role in apoptosis, mitochondria have also been implicated in other forms of cell death. Moreover, evidence suggests a crosstalk among these different cell death ways involving mitochondria.

5.1: Mitochondrial role in apoptosis

The mitochondrial or intrinsic pathway of apoptosis is induced by various cellular stresses such as growth-factor deprivation, viral infection, hypoxia or DNA damage. The mitochondrial pathway needs the OMM permeabilization (OMMP) in order to release signal proteins driving toward cell death. Cytochrome c, a key component of the ETC, represents a major factor released from the mitochondrial intermembrane space to the cytosol. In association with the adaptor molecule apoptotic peptidase activating factor 1 (APAF1), cytochrome c forms a complex called apoptosome (Dorstyn et al., 2018). This complex binds to and activates procaspase-9 to become the initiator caspase-9, which thereafter cleaves and activates downstream effector caspases. OMMP also releases further proapoptotic proteins such as apoptosis-inducing factor (AIF) and endonuclease G. Moving to the nucleus, AIF helps DNA fragmentation and chromatin condensation. Additional DNA fragmentation occurs as a result of endonuclease G translocation to the nucleus. The process ends when the caspase-activated DNase translocates to the nucleus and then ATP hydrolysis is required for nuclear collapse/disassembly (Toné et al., 2007). Other mitochondrial proteins are released through OMMP among which the second mitochondria-derived activator of caspase (SMAC) and OMI that inhibit X-linked inhibitor of apoptosis (XIAP), a critical member of the newly discovered family of intrinsic inhibitors of apoptosis (IAP) proteins (Bock and Tait, 2020).

Notably, the cleavage of BID—a pro-apoptotic member of the BCL-2 family—via caspase 8 indicates a crosstalk between extrinsic and mitochondrial apoptotic pathways. Caspase 8-mediated cleavage of BID strongly induces OMMP. BID symbolizes the key role of BCL-2 proteins in regulating mitochondrial apoptosis. BCL-2 family refers to three types of proteins: the anti-apoptotic proteins, proapoptotic effectors, and proapoptotic BH3-only proteins. The latter proteins are specifically activated transcriptionally—for example via p53—or post-translationally—as in the case of caspase 8-mediated cleavage of BID—upon apoptotic stress. Anti-apoptotic BCL-2 proteins avoid OMMP through BAX and BAK and BH3-only proteins in healthy cells. During mitochondrial apoptosis, activation of BAX and BAK pro-apoptotic proteins is crucial for OMMP and in turn cell death (Llambi et al., 2011). In light of the crucial role, a growing interest is emerging in targeting of mitochondrial apoptosis. A significant potential value is represented by the development of BH3-mimetics in tumor diseases. Other approaches such as small-molecule BAX activators (Lagares et al., 2017) may open new ways about targeting the mitochondrial apoptotic pathway in cancer, aging, and fibrosis.

5.2: Mitochondria in pyroptosis, necroptosis, and ferroptosis

Necroptosis is a caspase-independent pro-inflammatory form of cell death triggered by different conditions including viral infection and Toll receptor activation. However, the necroptosis following tumor necrosis factor (TNF) signaling has been best investigated. Under caspase 8 deficiency, TNF receptor leads to phosphorylation and activation of receptor interacting protein kinase 1 (RIPK1) and 3 (RIPK3) and in turn to necrosome assembly. RIPK3 phosphorylates activate mixed-lineage kinase domain-like pseudokinase (MLKL). Once activated, MLKL translocates to the plasma membrane and induces its permeabilization and the release of damage-associated molecular patterns killing the cell (Weinlich et al., 2017). Mitochondrial role in necroptosis is linked to the activation of PDC via RIPK3. Increased PDC activity heightens aerobic respiration with an enhanced production of mtROS. Cell models show that mtROS help necrosome formation together with RIPK3 activity (Yang et al., 2018). Since mtROS appear critical determinant in necroptosis, mitochondrial dysfunctions, like in aging, promote this cell death. Notably, enhanced necroptosis is linked to several inflammatory diseases and ischaemic injury, making this pathway an emerging therapeutic target.

Pyroptosis is an inflammatory type of regulated cell death driven by the inflammatory caspases 1, 4, 5, and 11. Activation of inflammasome complexes upon intracellular pathogens induces inflammatory caspases which in turn cleave proinflammatory cytokines IL-1β and IL-18. A further target of proinflammatory caspases is gasdermin D (GSDMD). Caspase-produced amino-terminal GSDMD fragments form pores and permeabilize the plasma membrane, leading to the release of mature interleukin IL-1β and IL-18 and finally resulting in pyroptotic cell death (Shi et al., 2015). Active GSDMD may also induce OMMP which therefore is not solely related to the mitochondrial apoptosis. Moreover, a crosstalk between pathways and proteins involved in mitochondrial apoptosis and pyroptosis is emerging. In this context, inflammasome induces OMMP through BID and then caspase 3 activation followed by cleavage-dependent activation of the potassium channel Pannexin 1. In this way, a potassium efflux is formed promoting inflammasome assembly (Tsuchiya et al., 2019). Moreover, mitochondrial apoptosis can foster caspase 1 activity following NLRP3 inflammasome activation.

Ferroptosis, an iron-dependent form of regulated cell death, appears as a major mechanism for cell death linked to brain damage and kidney failure, although its physiological function is not fully known. Ferroptosis helps the tumor suppressive function of p53 in cancer. Like pyroptosis, ferroptosis is a proinflammatory cell death driven by lipid peroxides which kill the cell leading to loss of cell integrity (Gao et al., 2019). Iron plays an essential role since it is required for the Fenton reaction and in turn lipid peroxidation. Usually, oxidized lipids are converted into lipid alcohols by glutathione peroxidase 4 (GPX4), which uses glutathione as a cofactor. Erastin is the first discovered and the classic inducer of iron-dependent cell death which inhibits intracellular cystine uptake resulting in glutathione depletion. Cysteine deprivation impairs GPX4 activity but may also induce glutaminolysis enhancing TCA cycle, mitochondrial respiration with a consequent increase of mtROS. Iron storage in mitochondrial ferritin and heme-containing proteins chelates iron, thus preventing iron-dependent lipid peroxidation. These iron-storing proteins are degraded under certain cell death-inducing conditions, leading to iron release. In cardiomyocytes exposed to doxorubicin or ischaemia-reperfusion, heme degradation leads to an increase in free iron and drives ferroptosis (Wang et al., 2016). Mitochondrial involvement is further supported by marked morphological changes among which mitochondrial fragmentation and cristae enlargement observed during ferroptosis (Doll et al., 2017). Moreover, tumor suppressor fumarase deficiency makes cancer cells resistant to cysteine-deprivation-induced ferroptosis. These emerging outcomes about fumarase and p53 suggest looking at ferroptosis as a natural tumor suppressive mechanism with specific physiological roles still not completely defined.

6: Mitochondrial redox biology

Although the electron and proton transfer across IMM is a highly efficient process, the mitochondrial ETC is the major intracellular source of ROS in particular of superoxide anion (O2·−) which is produced to the matrix at level of CI (Pryde and Hirst, 2011) and to the matrix and intermembrane space by CIII (Brand, 2016) and that spontaneously or catalytically lead to hydrogen peroxide (H2O2). Furthermore, other mitochondrial sites contribute to ROS production (Brand, 2010).

The toxicity of ROS is related to their chemical reactivity that causes oxidative damages to nucleic acids, lipids, and proteins, until to compromise the cellular integrity and viability. Therefore the production of mtROS is what concerns the oxidative stress theory of aging. This theory proposes that aging is caused in particular by mtROS toxicity inducing damage to mitochondrial molecules and in turn producing more ROS. Some observations corroborating this theory are the correlation between chronological age and the oxidative damage as well as mtROS levels and the loss of mitochondrial function during aging (Ku et al., 1993). Mitochondrial dysfunctions resulting from altered mtROS levels, and in turn oxidative stress, are related to countless human diseases over aging.

Recently, ROS are also considered as signaling molecules that take part in signal transduction pathways: through irreversible modification of macromolecules and different levels of H2O2, ROS are responsible for distinct responses within a cell. Thus low H2O2 levels induce expression of antioxidants, while high H2O2 levels stimulate the expression of pro-oxidants. A well-demonstrated example of this function is the modification of the redox-sensitive proteins with the oxidation of specific cysteine residues as the reversible inactivation by H2O2 of PTEN, a tumor suppressor and metabolic controller (Lee et al., 2002). Yet, ROS are involved in the regulation of apoptosis or autophagy, in the transcriptional activation, through DNA modification, and act as second messengers whose levels are controlled by synthesis and degradation that have a specific target and whose effects are reversible. Transcriptional factors as HIF-1α, FoxOs, and NF-kB are affected by mtROS (Hamanaka and Chandel, 2010) and their levels are important for the propagation of cellular signaling pathways.

For example, mtROS signaling pathway activation promotes the cellular adaptation with reduction of cellular O2 consumption and energy utilization in hypoxia. In these conditions, mitochondrial H2O2 is considered the hub of cellular response with the activation of HIFs through the stabilization of HIF-1α subunit. In normoxic conditions, HIF-α subunits suffer a proline-directed hydroxylation—carried out by a family of α-ketoglutarate-dependent prolyl hydroxylases 1, 2, and 3 (PHD 1–3)—and subsequent proteasomal degradation. During hypoxia, PHDs are inhibited allowing HIF-α subunit stabilization followed by its accumulation, heterodimerization, and translocation to the nucleus (Kaelin and Ratcliffe, 2008). All these events promote upregulation of survival genes in conditions of low oxygen, enhance glycolysis, and activate the angiogenesis in cancer cells (Semenza, 2003). Furthermore, distinct cellular responses depend on levels of H2O2, which represents an essential intracellular signal (Veal et al., 2007).

Increased ROS levels seem related to positive effects in response to damage caused by aging and open an alternative view on the ROS biology. In C. elegans, it has been demonstrated that mutations in respiratory chain complexes can extend the lifespan. In mitochondria of these mutants, increased superoxides correspond to the increased lifespan (Feng et al., 2001). Based on these considerations, a new vision proposes that ROS are produced to resolve the consequences of aging and not as cause of aging (Hekimi et al., 2011). Likely, ROS levels increase gradually to prevent aging until to a toxic level at which ROS become actors of cellular damage by enhancing the susceptibility to diseases. For these reasons, ROS homeostasis is required to extend lifespan and reduce the cellular aging. Antioxidant enzymes such as superoxide dismutase, glutathione peroxidase (GPX), and catalase as well as some antioxidant molecules like glutathione (GSH) and vitamin E (α-tocopherol) control mtROS levels. It has been reported that α-tocopherol incorporation into mitochondria helps to maintain oxidative stability of the membrane lipids (Lauridsen and Jensen, 2012). The other antioxidant systems are discussed in more detail later.

6.1: Superoxide dismutase

SOD2 gene, also known as MnSOD, encodes the manganese superoxide dismutase, a mitochondrial protein working as homotetramer. Each monomer needs a manganese ion as a cofactor (Karnati et al., 2013). Since SOD2 catalyzes the dismutation of O2·−, produced by ETC, to H2O2 with production of O2 in both mitochondrial matrix and intermembrane space, it plays a major role in mtROS detoxification (Kitada et al., 2020). Therefore SOD2-altered expression and/or activity have an inescapable effect on mitochondrial function and may underlie the development of numberless diseases (Vasconcelos et al., 2021; Steven et al., 2019; Miao and St Clair, 2009). SOD2 knockout, while does not affect embryonic development, induces death shortly after birth and dramatically reduces ETC activity in mouse (Li et al., 1995). Hence SOD2 activity is important in preserving the functionality of the complexes in the presence of oxidative stress. Overexpression of CuZnSOD does not compensate for SOD2 deficiency, suggesting its vital function as defense against oxidative stress (Copin et al., 2000). SOD2 transgenic overexpression protects ETC complexes from cancer chemotherapy-induced cardiac injury. Aconitase, a TCA cycle enzyme containing iron-sulfur centers vulnerable to oxidative damage, also requires SOD2 activity to work (Williams et al., 1998).

Another function of SOD2 is the regulation of iron handling. Previous studies suggest that decreased SOD2 activity leads to iron accumulation mostly within mitochondria (Martin et al., 2011). Therefore increasing SOD2 gene expression/activity may help the treatment of diseases linked to iron toxicity. Moreover, SOD2 overexpression protects from apoptosis avoiding mitochondrial dysfunction, loss of mitochondrial membrane potential, and the release of cytochrome c and Smac/DIABLO proteins in response to various proapoptotic agents such as ionizing radiation (Epperly et al., 2002). It has also been demonstrated a protective role of SOD2 against oxidative mtDNA damage induced by high glucose as well as UV radiation and ethanol exposure (Holley et al., 2011). SOD2 heterozygous knockout mice show a more lipid peroxidation and a significant increase in cancer incidence when compared to wild-type counterparts (Strassburger et al., 2005). In light of these reports, highlighting the essential role of SOD2 in protecting mitochondrial functions from the harmful effects of mtROS, this protein may represent a target for the treatment of diseases linked to oxidative stress including cardiovascular diseases, neurological conditions, and cancer.

6.2: Mitochondrial catalase and oxidative injury

Among the antioxidant enzymes involved in neutralizing H2O2, catalase has a main role using this nonradical ROS as its substrate. Through the catalase, two H2O2 molecules are broken down into one molecule of O2 and two molecules of H2O (Deisseroth and Dounce, 1970). Although H2O2 is not very reactive, when compared to other reactive species, it can diffuse, is relatively long lived, and is progenitor of more reactive free radicals like ·OH radical (Nandi et al., 2019). Due to the features of H2O2, catalase function becomes fundamental as scavenger of H2O2 when a large amount of ROS is present such as during inflammation, aging, and numberless diseases. Severe consequences may occur in mitochondria in the presence of high levels of H2O2 leading to peroxidation of the phospholipids and dysregulation of the mitochondrial membrane permeability. In turn these events promote cytochrome c release and proapoptotic cascade. In this context, the antioxidant activity of catalase is relevant in protecting mitochondria against H2O2-induced damages (Bai and Cederbaum, 2001). Many cells lack a specific mitochondrial catalase though a dual targeting of yeast catalase A, containing two peroxisomal targeting signals, to peroxisomes and mitochondria has been reported (Petrova et al., 2004). A high-fat diet fosters catalase expression in heart mitochondria to remove increased levels of H2O2 produced by lipid metabolism (Rindler et al., 2013). Another report provides evidence of catalase activity in purified rat liver mitochondria (Salvi et al., 2007). Despite the absence of a specific sequence for import, the mitochondrial catalase could play a central in oxidant defense. Directing catalase to mitochondria is more effective than the cytosolic enzyme in preventing H2O2-induced injury, oxidative damage, and mtDNA deletions (Arita et al., 2006). Furthermore, an extension of lifespan has been observed in mice when catalase targeted to mitochondria was overexpressed (Schriner et al., 2005). Targeted expression of mitochondrial catalase also protects against aging-associated muscle insulin resistance, doxorubicin-induced cancer, lung fibrosis, liver diseases, and aging (Lee et al., 2010; Gilliam et al., 2016; Kim et al., 2016; Shin et al., 2018). Therefore catalase significantly provides a mitochondrial protection against endogenous or exogenous H2O2, thus preventing oxidative stress-related pathological conditions.

6.3: Mitochondrial NADPH and redox homeostasis

NADP+ and NADPH levels are important to antioxidant cellular defenses since the antioxidant systems glutaredoxins (GLRXs), thioredoxin GPXs, and peroxiredoxins use NADPH as donor of reducing equivalents. GLRXs use GSH to catalyze reductions of disulfides or S-glutathionylation of proteins. The glutaredoxin system includes glutaredoxin, GSH, and glutathione reductase (GR) that utilizes NADPH. GLRX2 isoform is localized in mitochondria and is involved in the response to oxidative stress. In particular, GLRX2 plays a role in deglutathionylation of proteins such as the mitochondrial CI (Beer et al., 2004).

The thioredoxin system is composed by thioredoxin, thioredoxin reductase (TRXR), and NADPH. TRXR uses NADPH to reduce the thioredoxin, in turn able to reduce disulfide bridges in the proteins. There are two isoforms of TRXR, the cytosolic TRXR1 and the mitochondrial TRXR2. TRXR1 functions as antioxidant or to regenerate antioxidants. Little is known about TRXR2, and it is possible that it has the same effects of TRXR1. Interestingly, TRXR2 is able to reduce cytochrome c to sustain the mitochondrial respiration in the presence of CIII inhibition or dysfunction as well as to protect the mitochondria by ROS accumulation in vitro (Nalvarte et al., 2004).

Both antioxidant systems use NADPH to regenerate cellular antioxidant defenses as GSH and thioredoxin. There are many enzymes which synthesize NADP+ and NADPH. In mammals, beyond the main cytosolic source of the pentose phosphate pathway, NADPH levels are guaranteed by NADP+ derived from NAD+, via cytosolic and mitochondrial NAD+ kinase (NADK1 and NADK2, respectively) and by the NADPH synthesis. In mitochondria, five enzymes can synthesize NADPH: NNT (NAD(P) transhydrogenase), IDH2 (isocitrate dehydrogenase), ME3 (malic enzyme 3), ALDH1L2 (10-formyltetrahydrofolate dehydrogenase), and MTHFD1L (methylenetetrahydrofolate dehydrogenase 1 like) (Fernandez-Marcos and Nóbrega-Pereira, 2016).

During aging, activities of majority of NADPH forming enzymes are altered, and in particular decreased, by contributing to imbalance the [NADP+]/[NADPH] ratio also due to mitochondrial ETC dysfunction (Lenaz et al., 2000). So, the cytoplasmic and most likely also the mitochondrial [NADP+]/[NADPH] ratio pass from about 99% reduced in healthy cells to more oxidized with aging. In particular, the alteration of CI increases NADH levels, with a low activity of NADK2 (Bradshaw, 2019). At the same time, the altered function of ETC, by reducing the proton-motive force across the IMM, causes a decreased activity of mitochondrial NNT, that is driven by electrochemical proton gradient (Xiao et al., 2018).

Cytosolic and mitochondrial alteration of [NADP+]/[NADPH] ratio leads to a decrease of activities of detoxification systems, as cytoplasmic and mitochondrial GR and TRXRs, as well as a decrease of GSH/GSSG ratio. In fact, transhydrogenase functions also maintain a high GSH/GSSG ratio (Rydström, 2006). In this way, GSH, cosubstrate of GPXs for H2O2 removal, contributes to ROS detoxification.

[NADP+]/[NADPH] ratio dysregulation during aging compromises the functionality of both mitochondrial thioredoxin and glutathione systems contributing to ROS imbalance with a low GSH/GSSG ratio, an oxidation of thioredoxin, loss of activity of GPXs and peroxiredoxins as well as of other detoxification systems.

6.4: Mitochondrial glutathione

The tripeptide GSH (γ-glutamyl-cysteinyl-glycine) is ubiquitously produced in the cytosol of most mammalian cells because it represents a pivotal antioxidant defense system. Moreover, GSH modulates many other physiological processes like metabolism of xenobiotics, cell proliferation, immune function, and apoptosis. Its reduced form is more than 98% of total GSH. Following the reaction with ROS or electrophiles, GSH oxidizes to GSSG, which GR can reduces to GSH at the expense of NADPH (Forman et al., 2009).

In redox-dependent cell signaling, GSH may reversibly bind to the -SH of protein cysteinyl residues through a process called glutathionylation, producing glutathionylated proteins, thus either activating or inactivating proteins. Glutathionylation protects thiols of many transcription factors an signaling proteins from irreversible oxidation (Lu, 2013). GSH compartmentalization ensures separate redox pools: 80%–85% of the cellular GSH is cytosolic; 10%–15% mitochondrial; and a small percentage is nuclear and in the endoplasmic reticulum (Valko et al., 2007; Meredith and Reed, 1982; Hwang et al., 1992). When considering the volume of mitochondria, the concentration of mitochondrial GSH (mtGSH) is like to that of the cytosol (about 10 mM). MtGSH plays a critical role by preventing or repairing against ETC-produced ROS damage. Indeed, after SOD2 reaction, H2O2 detoxification occurs mostly through the GSH redox system which involves GPX enzyme. By using the reducing equivalents from GSH, GPX promptly reduces H2O2 to H2O Afterwards, GR reduces the oxidized GSSG to GSH, thus controlling mitochondrial GSSG levels. It is known that increased amount of GSSG promotes glutathionylation of target proteins and in turn mitochondrial dysfunction during oxidative stress (Ribas et al., 2014).

Among the eight human isoforms of GPX, five are selenoproteins, i.e., GPX1–4 and GPX6 (Brigelius-Flohé and Flohé, 2020). GPX1, the main isoform, even if shows a localization mainly cytosolic, is also present in the mitochondrial matrix (Marí et al., 2009). GPX4 plays an important role in protecting mitochondrial membranes from oxidative damage. As membrane-associated enzyme supports the reduction of hydroperoxide groups on phospholipids including cardiolipin, lipoproteins, and other lipids (Cozza et al., 2017). It has been reported that oxidized cardiolipin may affect the biophysical properties of OMM and alter OMMP (Montero et al., 2010). These events control important processes such as mitophagy and apoptosis. Therefore modulating cardiolipin redox state, mtGSH regulates critical mitochondrial functions.

7: Concluding remarks

In the past few decades, it has been increasingly uncovered the mitochondrial role in a large number of cell functions. As a consequence of this renaissance, mitochondria currently appear as active players in controlling cell fate through a tight interaction with the rest of the cell. For this reason, mitochondrial dysregulations underlie numberless pathological conditions. This fascinating perspective reveals new aspects to look in understanding the mitochondrial physiology and new potential ways for the development of treatments targeting mitochondria.

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