Electron Transport Disturbances and Neurodegeneration: From Albert Szent-Györgyi's Concept (Szeged) till Novel Approaches to Boost Mitochondrial Bioenergetics

Impaired function of certain mitochondrial respiratory complexes has long been linked to the pathogenesis of chronic neurodegenerative disorders such as Parkinson's and Huntington's diseases. Furthermore, genetic alterations of mitochondrial genome or nuclear genes encoding proteins playing essential roles in maintaining proper mitochondrial function can lead to the development of severe systemic diseases associated with neurodegeneration and vacuolar myelinopathy. At present, all of these diseases lack effective disease modifying therapy. Following a brief commemoration of Professor Albert Szent-Györgyi, a Nobel Prize laureate who pioneered in the field of cellular respiration, antioxidant processes, and the roles of free radicals in health and disease, the present paper overviews the current knowledge on the involvement of mitochondrial dysfunction in central nervous system diseases associated with neurodegeneration including Parkinson's and Huntington's disease as well as mitochondrial encephalopathies. The review puts special focus on the involvement and the potential therapeutic relevance of peroxisome proliferator-activated receptor-gamma coactivator 1-alpha (PGC-1α), a nuclear-encoded master regulator of mitochondrial biogenesis and antioxidant responses in these disorders, the transcriptional activation of which may hold novel therapeutic value as a more system-based approach aiming to restore mitochondrial functions in neurodegenerative processes.

“The fuel of life is electron, or more exactly, the energy it takes over from photons in photosynthesis, and gives up gradually while flowing through the cellular machinery.”

This imagination originates from Professor Albert Szent-Györgyi, a Hungarian physician and biochemist, former chair of the Department of Medical Chemistry and the Department of Organic Chemistry at the University of Szeged from 1930 and 1935, respectively, until the end of World War II in 1945. His early research activities in Groningen and later in Cambridge conducted on biological combustion, cellular respiration, and energy production of plants lead to the discovery of a reducing substance called “hexuronic acid,” a substance that is able to lose and regain hydrogen atoms and capable of protecting plants from “browning,” an injury that he characterized as oxidative damage due to the excessive activity of an enzyme, peroxidase. This “antioxidant” substance was later proved by Szent-Györgyi to be equivalent with a potent antiscorbutic (antiscurvy) agent and was given the name ascorbic acid, currently widely known as vitamin C, which is most abundant in citrus fruits and paprika, an emblematic vegetable of Szeged. At this time, performing ongoing research in the field of biological respiration, Szent-Györgyi discovered and identified the catalysis of fumaric acid among other steps of the tricarboxylic acid cycle (also referred to as Szent-Györgyi–Krebs cycle, citric acid cycle), an essential component of cellular respiration that provides reducing equivalents for terminal oxidation and thereby energy production from metabolic products of dietary macromolecules. “For his discoveries in connection with the biological combustion processes, with special reference to vitamin C and the catalysis of fumaric acid,” Albert Szent-Györgyi was awarded Nobel Prize in Physiology or Medicine in 1937.

In addition to his pioneering work in muscle research—including the discovery of actin and myosin proteins and the mechanism of their joint function—as well as the discovery of vitamin P (flavanone), his subsequent research interests focused on the interactions of proteins and free radicals and their role in regulating cell division and cancer development, and he published a number of books and papers about his findings and scientific theories of bioenergetics and bioelectronics and their roles in health and disease.

Following the imaginations of our honored predecessor, this paper reviews the concepts on the role of impairments in mitochondrial respiration and subsequent excessive oxidation in degenerative central nervous system (CNS) disorders, with special attention to recent findings related to alterations in transcriptional regulation of mitochondrial biogenesis and bioenergetics, and their potential therapeutic relevance.

Mitochondria are membrane-bound intracellular organelles evolutionary originating from the endosymbiosis of an ancient aerobic alpha-proteobacterium with an early eukaryotic host cell [1]. Harboring their own maternally inherited, double-stranded, circular genome (mtDNA), supplemented by the presence of several ancillary, structural, and regulatory proteins encoded by the nuclear DNA (nDNA), mitochondria host a number of molecular processes essential for cellular life and death. These include processes related to the production of biologically utilizable energy, adaptive thermogenesis via the uncoupling of energy production, as well as the regulation of cellular calcium homeostasis, cell-cycle, and programmed cell death.

Energy production in the mitochondria is performed through the coupled function of pyruvate dehydrogenase complex (PDC), β-oxidation (processes essential in glycolytic and ketogenic metabolism, resp.), the Szent-Györgyi–Krebs cycle, and the terminal oxidation and oxidative phosphorylation (OXPHOS). While PDC, β-oxidation, and the Szent-Györgyi–Krebs cycle take place within the mitochondrial matrix, terminal oxidation and OXPHOS are linked to the function of respiratory complexes I–V (electron transport chain, ETC) embedded in the inner mitochondrial membrane. The process of energy production has been extensively reviewed elsewhere [2, 3]. Briefly, glucose is catabolized in the cytosol to yield pyruvate through multiple enzymatic steps of the glycolysis, which is then translocated into the mitochondria and metabolized to acetyl-coenzyme A (acetyl-CoA) by PDC. On the other hand, fatty acids are oxidized during the β-oxidation to acetyl-CoA entirely within the mitochondria (or in case of longer-chain fatty acids, initially within the peroxisomes). Mitochondrial acetyl-CoA subsequently enters the Szent-Györgyi–Krebs cycle to form reduced coenzyme NADH and succinate in multiple steps, which in turn provide electrons for respiratory complex I (NADH dehydrogenase) and complex II (succinate-ubiquinone oxidoreductase), respectively, using FMN and FAD as prosthetic groups, respectively. The electrons are then transported from both complexes via the mobile carrier coenzyme Q (ubiquinone) to complex III (ubiquinol-cytochrome c oxidoreductase) and flow through cytochrome c to reach complex IV (cytochrome c oxidase) to be oxidized by the final electron acceptor, oxygen.

Notably, reducing equivalents produced during the glycolysis (in form of NADH) can also be translocated to the mitochondria via the malate/aspartate shuttle and the glycerol phosphate shuttle to provide NADH and FADH2, respectively, which in turn, similarly to NADH and FADH2 produced during β-oxidation, feed the ETC at complex I and coenzyme Q, respectively. Within the ETC, respiratory complexes are arranged in an electrochemical order, corresponding to their gradually increasing redox potential and electronegativity. The flow of electrons through the respiratory complexes provides energy used to pump out protons through complexes I, III, and IV (cytochrome c oxidase) to the intermembrane space. At the end of the downstream flow of electrons, molecular respiratory oxygen as the final electron acceptor is reduced by complex IV to form water molecule in a process known as terminal oxidation. The release of protons from the matrix develops a gradient of protons between the matrix and the intermembrane space also referred to as mitochondrial membrane potential (negative inside) as well as an electrochemical gradient (alkaline inside). Being impermeable to protons, the inner membrane works as a capacitator and an electric insulator. Therefore, the electrochemical drive to equalize the concentration of protons can be satisfied by the reentry of protons through respiratory complex V (H+-ATP synthase), the subsequent activation of which leads to the formation of ATP from ADP in a process called OXPHOS. The produced ATP represents a biologically available form of electrochemical energy, serving as the main energy-provider for eukaryotic cells (Mitchell's chemiosmotic hypothesis) [4].

The energy stored in mitochondrial membrane potential (also known as “proton motive force”) can also be utilized to generate heat or to import calcium or proteins into the mitochondrion via uncoupling the transport of electrons from ATP production, which processes serve adaptive purposes. The proportion of dietary calories burnt within the mitochondrion and allocated to energy production is referred to as “coupling efficiency.”

In multicellular organisms, the ability to adaptively regulate and activate mitochondrial biogenesis and functions in response to a variety of conditions is essential to maintain energetic homeostasis and cellular viability. Several lines of evidence obtained in the past decade suggest that peroxisome proliferator-activated receptor-gamma (PPARγ) coactivator 1-alpha (PGC-1α), a nuclear-encoded coactivator of a wide range of transcriptional factors, plays a key role in the transcriptional cascade of such adaptive processes. PGC-1α-mediated coactivation of genes such as nuclear respiratory factor 1 and 2 (NRF-1, -2), PPARs, estrogen-related receptors (ERRs), and myocyte-specific enhancer factor 2C (MEF2C) leads to an increased expression of a wide range of proteins involved in mitochondrial transcription, replication, and the import and assembly of a number of nuclear-encoded respiratory complex subunits; furthermore, it boosts OXPHOS and thermoregulation in a tissue-dependent manner, enhances gluconeogenesis and fatty acid oxidation [5], and increase oxidative stress defense [6] (Figure 1). The inducing effect of physical exercise (mediated by calcineurin A-linked MEF2 activity, calcium/calmodulin-dependent protein kinase IV- (CaMKIV-) linked cyclic AMP (cAMP) response element-binding protein (CREB) activity, and a p38 mitogen-activated protein kinase- (MAPK-) linked activating transcription factor 2 (ATF-2) activity), cold exposure and starvation (mediated by catecholamine- and glucagon-induced cAMP elevation and a subsequent phosphorylation and activation of CREB by protein kinase A (PKA)) on PGC-1α expression is well documented [5].

Furthermore, energy deprivation through a high AMP/ATP ratio leads to an increased AMP-activated protein kinase (AMPK) activity and a subsequent phosphorylation of PGC-1α protein, priming PGC-1α for subsequent deacetylation and thereby activation by silent information regulator 2 homolog 1 (Sirt-1) [7, 8], the expression of which is also increased in conditions with energy shortage, such as, starvation or exercise, due to a high NAD+/NADH ratio [9]. These posttranslational modifications on PGC-1α play pivotal roles in adaptive mitochondrial biogenesis. The roles of impaired mitochondrial function and more recently a decreased function of the PGC-1α cascade in the pathogenesis of degenerative CNS disorders are of extensive research interest.

Free radicals are molecules possessing unpaired electrons in their outer orbit. This renders them highly reactive towards organic macromolecules such as carbohydrates, nucleic acids, proteins, and lipids, which suffer “injury” during such a reaction. The main routes of free radical production and subsequent toxic insults are represented in a schematic depiction in Figure 2. Under physiological conditions, the efficiency of reducing oxygen during terminal oxidation is approximately 97–99%, while 1–3% undergo incomplete reduction to superoxide (O2 •−), a highly reactive free radical. O2 •− can be transformed into hydrogen peroxide (H2O2) both spontaneously and through a reaction catalyzed by mitochondrial manganese superoxide dismutase (Mn-SOD) in the matrix. H2O2 normally undergoes degradation by glutathione peroxidase (GPX) and catalase (CAT) enzymes, yielding water. In case of an impaired function of the mitochondrial ETC, leakage of excess electrons from complexes I and III leads to a higher amount of O2 •− and subsequent H2O2 production, which when exceeding the degradative capacity of the mitochondria can be transformed into the extremely toxic hydroxyl radical (HO•), through reaction with transition metals (Fe2+ and Cu2+; Fenton reaction). HO• in turn can react with nucleic acids and phospholipids, yielding the formation of further toxic radicals and consequent severe functional impairments of the affected macromolecules. Furthermore, HO• can also react with thiol residues, yielding the formation of multiple reactive free radicals. In addition, O2 •− can also react with nitric oxide (NO•) generated from L-arginine by mitochondrial nitric oxide synthase (mtNOS) enzyme [10]; however, other sources of mitochondrial NO• associated with nitrite reductase activity under hypoxic conditions have also been identified [11–13].

The reaction of NO• with O2 •− yields the highly toxic peroxynitrite anion (ONOO−) in a reaction that is three times as fast as the dismutation of O2 •− by Mn-SOD. ONOO− can evoke injury to proteins via nitration of tyrosine, tryptophan, and phenylalanine residues by nitronium ion (NO2 +) generated through metal-peroxynitrite (ONOO−MenX) complexes. Nitration of tyrosine can also be achieved by serial reactions of further pathways involving ONOO− and NO• (see Figure 3 for detailed description). Nitrosylation of thiol, secondary amine, alcohol, and alkane residues are performed via their reaction with nitrosonium ion (NO+) or indirectly with dinitrogen trioxide (N2O3), highly reactive radicals also derived from NO•. Furthermore, NO• can directly impair the mitochondrial ETC and OXPHOS via competing with oxygen for the oxygen-binding site of complex IV [14], which leads to a further increase in free radical production.

The toxic radicals described above are known as reactive oxygen species (ROS) and reactive nitrogen species (RNS), and their damaging effects on macromolecules are referred to as oxidative and nitrative/nitrosative stress, respectively. Figure 3 presents a detailed overview on the molecular background of toxic processes related to ROS and RNS.

With mitochondrial ETC being the main source of ROS and RNS production, macromolecular components of the mitochondria are extremely exposed to injury due to oxidative/nitrative/nitrosative stress. Of note, proteins that underwent such a damage are highly susceptible to proteolytic cleavage and degradation [15]. The injury to mitochondrial respiratory complex subunits by impaired efficacy of the ETC has two main consequences: (1) it leads to decreased energy production due to impaired OXPHOS, and (2) it decreases the efficacy of terminal oxidation, which results in increased production of ROS/RNS, generating a vicious circle.

The proximity to the main source of free radical production and the relatively high proportion of coding sequences render the mitochondrial genome particularly sensitive to ROS/RNS-mediated injury [16]. Indeed, the mutation rate of mtDNA relative to nDNA is approximately 10 : 1 [17]. Furthermore, as the ability to cope with oxidative/nitrative/nitrosative stress declines with aging [18], the rate of mtDNA mutations further increases in the elderly [19].

Excessive ROS and RNS accumulation can trigger the opening of mitochondrial permeability transition pores (mtPTP), which on the one hand decreases the mitochondrial membrane potential further aggravating the initial OXHPHOS impairment, and, on the other hand, leads to the release of proapoptotic factors (including apoptosis-inducing factor, procaspase-9, and cytochrome c) from the intermembrane space to the cytosol. This is in severe cases followed by cellular death that can be either apoptotic or necrotic, depending on the severity of the initial insult and subsequent energy deprivation [20–22]. It should be noted, however, that being among the most ancient signals between mitochondria and their host cells, both ROS and RNS might have essential physiological functions under physiological conditions.

Defensive processes of the mitochondria to counteract excessive free radical production involve low molecular weight antioxidants (LMWAs), an enzymatic redox apparatus to clear ROS/RNS (e.g., SOD, CAT, GPX, and peroxiredoxin), and an nDNA-encoded repair machinery.

Ubiquinol and tocopherols represent the main groups of lipid-associated LMWAs. Since these molecules are transformed to semiquinone radicals upon reduction of toxic free radicals, the immediate restoration of antioxidant capacity is essential. This process depends on the standard redox potential of LMWAs. For example, reduction of the semiquinone form of lipid-associated tocopherol requires ascorbic acid (vitamin C, identified by Albert Szent-Györgyi) and the subsequent reduction of the produced ascorbic acid radical by glutathione. Therefore, at the end of the process, no free radicals are present. However, there is a need for the restoration of the reduced glutathione, which is mediated by the enzyme glutathione reductase (Gred). Reduced glutathione also participates in antioxidant functions associated with the activities of SOD and GPX (including the phospholipid-associated form (PHGPX) as well), and it is responsible for the detoxification of HO• and ONOO−. The proper function of this armatory requires the appropriate load of reducing equivalents (NADH + H+, NADPH + H+). The above mechanisms are depicted in Figure 3.

A number of evidence link PGC-1α to the regulation and activation of mitochondrial antioxidant responses. In a comprehensive study of St-Pierre et al. [6], the expression of PGC-1α significantly increased after H2O2 challenge in 10T1/2 cells, which effect was recapitulated by Irrcher et al. on C2C12 muscle cells [23]. This effect is in correspondence with our recent findings of significantly increased PGC-1α expression in the CNS of mice intoxicated with the neurotoxin 3-nitropropionic acid, an irreversible inhibitor or complex II [24]. Furthermore, RNAi against PGC-1α reduced the baseline expression of copper/zinc (Cu/Zn)-SOD, Mn-SOD, and GPX in 10T1/2 cells, whereas the expression of Cu/Zn-SOD, Mn-SOD and peroxisomal CAT was found reduced in the heart and brain of PGC-1α-deficient mice [6]. Similarly, overexpression of PGC-1α in C2C12 myotubes displayed increased expression of Mn-SOD and GPX in association with a decreased amount of ROS production [25]. Moreover, PGC-1α-deficient fibroblasts exhibited blunted response to ROS challenge and an increased sensitivity to oxidative stress, which was in correspondence with an increased sensitivity of PGC-1α-deficient mice to intoxication with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), an irreversible inhibitor of mitochondrial respiratory complex I, as well as to that with the excitotoxin, kainate [6].

Mitochondrial repair is now widely-acknowledged as an existing phenomenon, comprising a group of processes that aim to repair deleterious alterations in mtDNA, predominantly due to oxidative injuries. These include enzymatic apparatuses for (1) the hydrolysis of oxidized deoxyribonucleotide triphosphates to prevent mismatch errors, (2) different mechanisms of single- and double-strand break repair, (3) multiple mechanisms of base excision repair, and (4) the degradation of unrepairable mtDNA. The latter is a unique mitochondrion-specific mechanism in eukaryotes, which is enabled by the redundance of mtDNA within the organelle [26]. Certain evidence suggest that, similarly to that seen in nDNA repair, poly(ADP-ribose) polymerase-1 (PARP-1) might play central role of the epigenetic regulation of nDNA-encoded proteins involved in mtDNA repair mechanisms [27].

In case the antioxidant defense and repair systems prove insufficient to protect the organelle, severely damaged mitochondria can be sensed and degraded by a process under the regulation of PINK1 and parkin (mitophagy) [28, 29].

A number of general observations and considerations explain the special susceptibility of the CNS to suffer injuries due to mitochondrial disturbances. Indeed, the CNS has an especially high energy demand as it represents merely 2% of the total body mass and accounts for some 20% of bodily oxygen consumption [30]. Besides, unlike astrocytes, neurons store low amounts of glycogen and have a poor ability to enhance glycolysis under conditions when mitochondrial respiration is impaired [31]. Therefore, neurons depend on the constant availability of oxygen and glucose to maintain their functions. Furthermore, the CNS contains high amounts of polyunsaturated lipids, which are highly susceptible to oxidative injury by means of lipid peroxidation, and the antioxidant capacity of neurons is known to be relatively poor [32, 33]. The high sensitivity of neurons as opposed to the relative resistance of astrocytes to oxygen or glucose deprivation is well known; however, recent studies suggest that oligodendrocytes are among the most sensitive cell types within the CNS to mitochondrial stress, exceeding the vulnerability of neurons [34, 35], a feature that may have implications for the pathogenesis of characteristic myelinopathies in chronic conditions with mitochondrial dysfunction, including aging, and mitochondrial encephalopathies.

Another CNS-specific mechanism leading to an increased sensitivity to mitochondrial dysfunction is excitotoxicity due to glutamate, the major excitatory neurotransmitter in the brain [36]. In an event of energy deprivation, neurons undergo partial membrane depolarization, which removes magnesium ions that block the ionophore of N-methyl-D-aspartate-sensitive (NMDA) glutamate receptors. This leads to a persistent activation of NMDA receptors by glutamate even if it is present in physiological levels [37]. Hyperactivation of NMDA receptors results in an influx of calcium into the cytosol. The persistent increase in intracellular calcium level leads to an increased mitochondrial sequestration of calcium, which in pathological extents evokes the opening of high-conductance mtPTPs [38], resulting in mitochondrial swelling and a decreased mitochondrial membrane potential, with subsequent OXPHOS impairment and ROS overproduction [39]. These culminate in the release of proapoptotic factors eventually triggering cell death [40] by apoptotic or necrotic mechanisms, based on the severity of the event [20, 21]. It has also been postulated, however, that the mechanism and the channel (i.e., NMDA receptor) through which calcium ions get into the cell and not the calcium overload itself may play the pivotal roles in excitotoxic cell death [41]. Indeed, NMDA receptors are functionally and spatially linked to neuronal NOS (nNOS) by postsynaptic density protein of molecular weight 95 kDa (PSD-95) that synthesize NO• in a toxic amount while calcium ions enter into the cell during NMDA receptor overactivation [42]. Notably PSD-95 attaches to the NR2B subunit, which is in correspondence with the observation that glutamate excitotoxicity is predominantly mediated by NR2B subunit-containing NMDA receptors [43], which are mainly located extrasynaptically [44].

In line with these, activation of extrasynaptic NMDA receptors is regarded as neurotoxic, whereas that of the synaptic NMDA receptors appears to be neuroprotective [45, 46], indicating a role of volume transmission in NMDA receptor-mediated neurotoxicity. A central role of nNOS in excitotoxic injury was suggested by earlier studies as well [47, 48]. The role of excitotoxicity in association with mitochondrial dysfunction as well as the possible therapeutic relevance of approaches aiming to counteract glutamatergic overactivation in neurodegenerative diseases, including pharmacological manipulations with the kynurenine system, have recently been extensively reviewed [49, 50] and are not within the scope of this review.

The following subsections emphasize the relevance of mitochondrial dysfunction in the pathomechanism of degenerative CNS disorders through a detailed overview on the involvement of impaired OXPHOS and mitochondrial bioenergetics in Parkinson's disease, Huntington's disease, and mitochondrial encephalopathies, with special focus on the involvement and therapeutic relevance of PGC-1α.

Parkinson's disease (PD) is a progressive, chronic neurodegenerative disorder, the pathognomonic alterations of which include loss of dopaminergic neurons, and the presence of Lewy bodies in the substantia nigra pars compacta (SNpc), with a subsequent decrease in striatal dopamine levels [51]. Leading clinical symptoms include bradykinesia, rigidity, resting tremor, and postural instability [52, 53], eventually evolving into severe akinesia, dementia, and eventually death. The development of sporadic PD is linked to a complex interplay of genetic and environmental factors, which have multiple implications for mitochondrial involvement (Figure 4). The first implication for the role of mitochondrial dysfunction in PD came from serial cases of intoxication by the side-product of a synthetic illicit drug, MPTP, which evokes parkinsonian symptoms and recapitulates the majority of PD-related pathologies [54]. Its active metabolite 1-methyl-4-phenylpyridinium (MPP+) selectively and irreversibly impairs the function of mitochondrial complex I in dopaminergic neurons [55, 56], and since its discovery, systemic MPTP or intraventricular MPP+ intoxication became the most widely applied in vivo toxin models of PD. Similar effects can be achieved by known environmental chemicals including the herbicide paraquat and the insecticide rotenone [57]. Corresponding with the ability of complex I inhibitors to evoke parkinsonism, a decreased activity and/or expression of respiratory complex I has been detected in the SNpc [58, 59], striatum [60] frontal cortex [61], platelets [62, 63], and skeletal muscle [64, 65] of sporadic PD patients, suggesting a systemic impairment of mitochondrial functions in this disease. Less consistent reports have been published regarding the involvement of other respiratory complexes, which indicates a predominant involvement of complex I in sporadic PD.

In line with these, a decreased activity of complex I, an elevated production of ROS, an energy impairment, and an increased sensitivity to MPP+ intoxication can be detected in PD cybrids [66, 67]. The increased presence of oxidative damage has also been reported in post mortem SN of PD patients [68, 69].

In the past decades, genetic and, more recently, genome-wide association studies (GWAS) have identified over 20 loci in causative association with familial PD [70], many of them having direct implications in mitochondrial dysfunction. Among them, leucine-rich repeat kinase 2 (LRRK2) protein is known to colocalize with membrane bound intracellular structures including mitochondria [71]. Its autosomal dominantly inherited mutation, G2019S, the most frequent underlying genetic alteration in both familial and sporadic PD cases, has been associated with mitochondrial dysfunction and morphological alterations in PD tissue samples [72], abnormal mitochondrial dynamics and increased ROS production in murine primary cortical neurons [73], as well as with an increased neuronal vulnerability to rotenone and paraquat in a nematode model of PD [74].

The autosomal dominantly inherited mutation of SNCA gene (A53T) leads to mitochondrial accumulation of α-synuclein, the main constituent of Lewy bodies, resulting in the inhibition of respiratory complex I [75, 76]. The protein appears to play pivotal roles in modulating oxidative stress, as its transgenic overexpression leads to enhanced sensitivity against intoxication with paraquat and MPTP [77], whereas α-synuclein-deficiency leads to resistance against intoxication with MPTP, 3-nitropropionic acid and malonate in mice [78] (the latter two are inhibitors of complex II and serve as toxin models for Huntington's disease). Accordingly, cell lines transfected with mutant α-synuclein exhibit increased levels of oxidation products, decreased glutathione levels, and a markedly increased cell death in response to toxic insults including H2O2 and MPP+ exposure [79].