*6.3. Supercomplexes in Pathology and Aging*

In order to understand the mechanism by which a mitochondrial dysfunction can lead to failure of physiological functions, it is paradigmatic to discuss the mechanisms responsible for aging of cells and organisms. The mitochondrial theory of aging [398–400] is based on a series of assumptions linked by a causal relationship [401]: (a) mitochondrial ROS generation is the major cause of aging. (b) Mitochondrial ROS damage mitochondrial biomolecules, especially inducing mtDNA somatic mutations, mainly affecting post-mitotic tissues. (c) The mtDNA mutations alter the structure of mtDNA-encoded proteins: since these are subunits of the major OXPHOS complexes, they imply a decreased oxidative phosphorylation, which leads to bioenergetic failure, metabolic derangement and cell death.

A further consequence of the decreased electron transfer during aging is further ROS generation, so that mtDNA damage and ROS production become involved in a vicious circle [398,399], not considered by the original theory.

Even if each of these assertions is sustained by experimental evidence, there are still several controversies and the cause–effect relationships among these events are still partially obscure. The complexity of biochemical, genetic, and regulatory systems and their inter-relationships are still not fully understood and often a single linear cause to effect relationship cannot be established [402]. Barja [403] however proposes that aging is the result of several effectors, i.e., mtROS production, lipid unsaturation, autophagy, mitochondrial DNA repair and putative other events such as apoptosis, proteostasis, or telomere shortening, already considered by different classic theories of aging. The aging regulating system gathers the theories of aging, previously considered as independent and assembles them into a single unified theory.

Wallace [1] hypothesized that mitochondrial dysfunction plays a central role in a wide range of age-related disorders and cancer types. He proposes that the delayed-onset and gradual onset of the age-related diseases is due to the accumulation of somatic mutations in the mtDNAs of post-mitotic tissues. The tissue-specific pathological phenotype may

depend on the different roles and energy requirements of the various tissues. The individual varied predisposition to degenerative and cancer diseases may result from the interaction of present environment (dietary caloric intake) and ancient genetic factors (mitochondrial genetic polymorphisms). On these bases, the mitochondria make a direct connection between our environment and our genes.

Inherited and/or epigenomic variation of the mitochondrial genome determines our initial energetic capacity, but the age-related accumulation of somatic mtDNA mutations decreases the energetic capacity leading to disease [4], thus providing a unified pathophysiological and genetic mechanism for neurodegenerative diseases such as Alzheimer and Parkinson disease, metabolic diseases such as diabetes and obesity, autoimmune diseases, aging, and cancer.

Such an integrated model for the genetics and pathophysiology of complex diseases, aging, and cancer [4] is summarized in Figure 7.

**Figure 7.** Integrated mitochondrial pattern which shows the genetic and phenotypic features of the "complex" human diseases. The model integrates the genetic and pathophysiological relationships of multi-factor diseases, aging, and cancer (from Wallace [4]). See text for explanations.

Nuclear DNA variations including epigenetic changes, mtDNA variations, including ancient polymorphisms, and environmental influences including diet and calories, all impact the mitochondrial OXPHOS system. The primary defect is the reduction in energy conservation by OXPHOS, that in turn perturbs the mitochondrial biogenesis and enhances ROS production. ROS induce the progressive increase in mtDNA somatic mutations, so that the alteration of proteins in the OXPHOS complexes cause further decline of the mitochondrial function. If the mitochondrial energy production decreases below a specific tissue threshold, the bioenergetic failure can trigger cascade events which lead to cell death by apoptosis or necrosis. The clinical phenotype would result from the reduced energy storage in the tissues that require the most energy, such as brain, heart, muscle, and kidney. The symptom number and severity in these organs mirror the extent and type of the mitochondrial defect. The altered functions in mitochondria are also crucial to determine cancer initiation, promotion, and metastasis [404].

We first proposed SC disassembly as the missing link between oxidative stress and energy failure [96,405]. We proposed that an initial oxidative stress, not necessarily originating from mitochondria, and due to different possible reasons, causes dissociation of SCs. The bioenergetic consequence is the loss of electron channeling leading to random diffusion of ubiquinone, which is less efficient. A further consequence of SC disassembly would be CI dissociation with loss of electron transfer and/or proton translocation; the consequently altered electron transfer may result in further ROS generation. The possible severe metabolic and physiological consequences of SC dissociation are reported in the scheme in Figure 8 [223]. − γ

−

**Figure 8.** Scheme illustrating how the side chain (SC) organization loss may be involved in a vicious circle of oxidative stress and energy failure. Reactive oxygen species (ROS) production by CI increases due to SC disassembly. Other mitochondrial events, such as membrane phospholipid peroxidation, mtDNA damage and subsequent misassembly of the respiratory complexes with further loss of SC organization, may occur due to the increased oxidative stress, thus fueling and maintaining the vicious circle. In a dose-dependent way, ROS can also operate as molecular signals from mitochondria to the nucleus. See text for explanations.

> This model poses SC dissociation and ROS generation in a biunivocal situation. On the one hand, ROS cause dissociation of SCs, but on the other hand, SC dissociation enhances ROS generation. Clearly, these events must be tightly controlled, otherwise they might trigger a vicious circle of ROS generation. We have discussed above the hypothetic vicious circle of ROS generation and mitochondrial failure at the basis of aging and agingrelated diseases. The original mitochondrial theory of aging did not imply the existence of a vicious circle [398], nevertheless the evidence that ROS generation increases in aging is overwhelming [403].

> Alterations (dissociation) of the SCs often accompany pathological changes, however no direct proof exists that SC dissociation is the direct cause of the pathology.

> Even so, circumstantial evidence points to a vicious circle (cf. Genova et al. [405] for review).

> The major mitochondrial alterations accompanying aging and several pathologies occur at the level of CI [406,407], in line with the knowledge that this enzyme exerts the main control on respiration. Flux control analysis of respiration in coupled liver mitochondria [408] showed that the control exerted by CI is low in young rats, and becomes very high in the old ones, suggesting that aging induces CI structural changes that eventually affect the whole OXPHOS.

> Analysis of the occurrence of respiratory SCs (cf. Section 3.1) in relation to age suggested that the SC destabilization plays a key role in the development of the agingphenotype [409,410]. In mitochondria of rat cortex, a striking age-associated decline (−40%) in the CI-containing SCs, especially the SC I1III<sup>2</sup> (−58%) was reported by Frenzel et al. [411].

The so-called mutator mice are a remarkable model of premature aging [407]; they have a defect of the proof-reading function of mtDNA polymerase-γ and hence exhibit multiple mtDNA mutations that result in decreased respiration and altered assembly of respiratory complexes [412]. In these mice, the steady-state level and activity of CI is strongly lowered, possibly as a secondary effect of a decreased assembly of CIV on the stability of CI, which disrupts the SC organization [349].

The observations collected in this review locate SC assembly/disassembly in a physiological signaling network that can easily undergo alterations, leading to dramatic conditions if ROS generation becomes out of control.

We may envisage SC association/dissociation as a physiological phenomenon, as depicted by the plasticity model [64], modulated by a variety of stimuli such as the mitochondrial membrane potential and protein post-translational changes in the respiratory complexes; thus, changes in ROS production modulate the signaling pathways started by ROS. These changes are reversible and must be tightly controlled.

We propose that the primary event responsible for aging and age-related pathologies is the structural damage induced by ROS in mitochondria, as originally stated by the so-called mitochondrial theory of aging [398].

Distinct events may constitute a consecutive series which leads to pathological changes. Progressive ROS-induced damage to the mitochondrial membrane lipids and proteins was found; mtDNA mutations, although present, may not necessarily be an early phenomenon of aging development.

The ROS level may be ruled by the nutrition state and the activity of the mTOR and insulin/IGF pathways. ROS may alter mitochondrial protein structure either directly or by means of peroxidation of CL, whose damage prevents SC association [71]; in turn SC dissociation would lead to further increase in ROS generation. If maintained at low levels, ROS may induce retrograde signals which promote the induction of compensatory mechanisms that try to counteract ROS generation and the consequent damage risk (see Section 5). However, high ROS levels may cause further damage and make the signaling pathways lose their coordination (see Section 5.3). At a later stage, mtDNA mutations would make the whole process irreversible and determine the final aging phenotype.

**Author Contributions:** Writing—original draft preparation, S.N., A.P., G.L.; writing—review and editing, F.T., V.V., C.A., G.T.; supervision, A.P., G.L.; funding acquisition, S.N. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by CARISBO Foundation, grant number 2019.0534 to S.N.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

