The mitochondrial genome presents some specific features, including maternal inheritance and heteroplasmy. Heteroplasmy is a condition in which at least two different mitochondrial genomes are present within the same cell. Pathogenic variants in the mt-DNA are highly recessive and usually coexist with the wild-type mt-DNA molecules. Therefore, the clinical manifestation of mt-DNA variants mainly depends on both their severity and the mutational load (heteroplasmy) of the tissues ^[1][2][2,8].

Over the last two decades, a large number of studies using different patient’s specimens and other cellular paradigms have led to in-depth investigations of the biochemical and cellular alterations caused by MT-ATP6 and MT-ATP8 variants, which are summarized in Table 1.

As shown in Table 1, the cellular dysfunctions observed for a given variant can be highly variable, and one element that contributes to this characteristic is heteroplasmy.

Consequently, as with other mt-DNA-associated diseases, a specific feature is the threshold of the percentage of mutant genome (or percentage of heteroplasmy) that must be exceeded to detect a biochemical alteration. As reported below, this defect may also depend on the variant, haplogroup, cell type, and tissue type.

The two most common variants in MT-ATP6 are m.8993T>G (p.Leu156Arg) and m.8993T>C (p.Leu156Pro), which cause a change in a highly conserved leucine residue on ATP6 ^[26][82][83][13,57,113]. These variants are the most common and are responsible for approximately 50% of reported MT-ATP6 disease cases ^[84][33]. These variants are associated with Neuropathy, Ataxia, and Retinitis Pigmentosa (NARP) or Maternal Inherited Leigh syndrome (MILS) when heteroplasmy is between 70 and 90% or greater than 90%, respectively. Furthermore, the T>G transversion usually results in a more severe clinical phenotype than the T>C transition ^{[82][83][85][86]}[13,113,114,115].

Analyses of patient specimens carrying the m.8993T>G or the m.8993T>C variant have been performed in platelets ^[26][27][57,58], lymphocytes ^{[28][29][30][31][32]}[59,60,61,62,63], muscle tissue ^[33][64], and skin fibroblasts ^{[3][4][34][35][36][37][38][39][40][41][42][43][87]}[34,35,65,66,67,68,69,70,71,72,73,74,116] (see Table 1). Several biochemical abnormalities have been identified, including a decreased ATP synthesis and oxygen consumption rate (OCR) ^{[3][26][27][28][29][30][31][34][35][36][37][38][39][40][41][42]}[34,57,58,59,60,61,62,65,66,67,68,69,70,71,72,73], often in direct correlation with the mutation load ^{[27][30][40][45]}[58,61,71,76], an alteration of the proton flux ^[30][31][32][61,62,63], and a not-fully assembled CV ^[33][64]. In human cells, these ATP synthase dysfunctions lead, as secondary effects, to a reduction in growth in stress medium ^{[3][35][38][40]}[34,66,69,71], to an increase in both mitochondrial membrane potential (MMP) ^{[31][36][39][87]}[62,67,70,116] and ROS ^{[4][31][39][43]}[35,62,70,74], as well as to an altered mitochondrial network morphology and cristae structure ^[39][42][70,73].

In cells of NARP patients carrying the m.8993T>G variant, the severe impairment of OXPHOS has been proposed as the primary pathogenic defect; instead, the increase in ROS could be the main contributor to the pathogenesis of the disease associated with the m.8993T>C variant ^[31][62].

It is worth noting that no significant effects of the m.8993T>G variant have been reported on either ATP hydrolytic activity or ATP-driven proton transport by Complex V in patient cells ^[26][36][57,67]. However, inhibition of ATP hydrolytic activity could contribute to energy preservation and survival of cells under stress conditions (oxygen shortage), leading to the collapse of the proton motive force and ATP synthase working in reverse. Incidentally, due to the heterogeneity of the membrane potential within the same mitochondrion and the possible coexistence of ATP synthase working physiologically and in reverse ^[88][89][117,118], patients might benefit from the use of a specific inhibitor of the hydrolytic activity of CV.

The different percentages of heteroplasmy and other factors, including nuclear background and the type of tissue analyzed, may contribute to the phenotypic differences observed in the analysis of patients’ specimens, as well as those observed in clinical outcomes ^[61][84][90][33,92,119]. For these reasons, transmitochondrial cybrids are widely used to validate the possible pathogenicity of a mitochondrial variant, even in homoplasmic populations, with the advantage of using a cell model with the same nuclear background ^[91][120]. In the case of the two MT-ATP6 variants, this cell model clearly demonstrated the impairment of respiration ^{[29][40][46][47][48][50][51]}[60,71,77,78,79,81,82], ATP synthesis ^{[3][35][37][40][44][45][46][47][48][49]}[34,66,68,71,75,76,77,78,79,80], mitochondrial morphology ^[52][53][83,84], and enhanced ROS production ^[47][50][54][78,81,85], confirming the milder effect of m.8993T>C compared to the T>G variant ^{[31][37][40][46][48]}[62,68,71,77,79].

Interestingly, analysis of different cybrid lines carrying the same m.8993T>G variant highlighted that the mitochondrial genome sequence, and thus the haplogroup, is a factor contributing to the variations in the observed biochemical phenotypes, ranging from normal to severe defects ^[48][79]. Moreover, clear evidence of the role of mutation load on deleterious biochemical abnormalities has been recently reported in isogenic cybrids, where the OCR reduction and the extracellular acidification rate (ECAR) increase were proportionally linked to the levels of heteroplasmy, indicating a switch toward glycolysis ^[51][82]. Metabolic remodeling induced by the m.8993T>G variant was also investigated, and both proteomics and metabolomics analysis were consistent with increased glycolysis and reductive carboxylation of glutamine to support cell survival and to maintain redox balance ^[51][82]. Accordingly, a second report showed that, in cybrids, the impaired OXPHOS activity induces compensatory energy-generating anaplerotic mechanisms where glutamine-glutamate-α-ketoglutarate metabolism sustains cell survival ^[55][86].

The deleterious mechanism hypothesized based on all these studies, especially for the m.8993T>G variant, includes defective proton transport across F_o, failure of the enzyme to couple phosphorylation of ADP on F₁ to proton flow, or alteration of the holoenzyme assembly and stability ^{[30][31][32][84]}[33,61,62,63]. Considering that these alterations have been observed in different cellular models despite not always being together, it seems reasonable that all three mechanisms contribute to the pathogenicity of the variants.

In recent years, the introduction of induced pluripotent stem cell (iPSC) technology allowed disease modeling by overcoming the difficulty of accessing clinically relevant patients’ cells or tissues, such as neurons. The generation of IPSCs requires multiple quality checks and presents the issue of heteroplasmy fluctuations due to the genetic bottleneck occurring in the reprogramming process. In addition, the mutant load can also change during differentiation or cell culture and therefore must be constantly monitored ^[92][93][121,122].

A series of patient-derived iPSCs carrying the m.8993T>G or T>C variant has been developed ^{[58][94][95][96][97]}[89,123,124,125,126], and neural progenitor cells (NPCs) and neurons have been differentiated ^[58][89]. The mutant iPSCs were able to differentiate into the three embryonic germ layers (endoderm, mesoderm, and ectoderm) ^[78][96][97][109,125,126]. However, analysis of embryoid bodies showed impaired differentiation potential in cells with a high percentage of the variant ^[96][125]. Overall, the generated cell types recapitulate the energy defects observed in other cell models and the degenerative phenotypes observed in patients ^[56][58][87,89]. Neurons, in part because of their predominantly mitochondria-dependent oxidative metabolism ^[58][78][89,109], have shown degenerative defects not detectable in other, less differentiated cells, notably ATP shortage and AMPK activation, finer neuronal fibers, and increased sensitivity to glutamate toxicity ^[58][89]. Furthermore, a study of mutant IPSCs revealed abnormalities during the three-dimensional differentiation and a defective formation of cerebral organoids, particularly in the generation of neural epithelial buds, as well as impaired corticogenesis with an altered metabolic profile ^[57][88].

In the recently released ATP synthase human structures (^[98][16], PDB id 8H9S, 8H9T, 8H9U, and 8H9V for states 1, 2, 3a, and 3b, respectively), the Leu156 residue is located on helix H5 and is buried in the core of ATP6, forming van der Waals contacts with Leu217 and Val218, located on helix H6 (Figure 13). In the four structures, the residues in the vicinity of Leu156 are the same, and there are no conformational transitions involving ATP6 in the different states of the human ATP synthase. The variant of Leu156 in arginine can damage the interaction between subunit helices H5 and H6 because of (i) the larger volume of an arginine residue with respect to a leucine and (ii) the presence of a charged side chain in a region populated only by hydrophobic residues. In other words, the presence of an arginine in position 156 can cause a divarication between helices H5 and H6 that in turn can alter the folding of ATP6. Indeed, the correct positioning of these helices is crucial for proton access to the negatively charged Glu58 in the c subunits (Figure 13).

The m.9176T>C and m.9176T>G variants, which cause a Leu217Pro and a Leu217Arg ammino acid change, are frequently found in MILS patients and were first reported in 1995 and 2001, respectively ^[68][70][99,101].

In the case of the m.9176T>G variant, a partially disassembled CV, decreased ATP synthesis, and mitochondrial respiration due to a defective OXPHOS pathway led to an increase in MMP in MILS patient-derived fibroblasts ^[40][68][71,99]. Similar observations have been described in cybrids ^[40][48][49][71,79,80] and in patient-derived muscle tissue ^[33][64]. Furthermore, analysis of this variant in yeast highlighted a severe reduction in the ATP6 protein level, suggesting that it may affect the assembly of the ATP synthase complex and cause mitochondrial and bioenergetic dysfunctions ^[67][98]. Human IPSCs have been recently generated for the m.9176T>G ^[97][126], and their use to develop neurons will be instrumental in deeply characterizing the pathogenic mechanism of this variant in a disease-target tissue.

In the first report in 1995, biochemical analysis of the m.9176T>C variant revealed no defects in ATP synthase function in patient cells with the homoplasmic variant ^[70][101]. However, further studies reported impaired ATP synthesis ^[40][69][71][71,100,102] and CV stability ^[71][102] in both human cells and mutant yeast. As for the variants at the nucleotide m.8993, the alteration caused by the T>C variant was less severe than that caused by the T>G variant ^[40][69][71,100].

In human structures, Leu217 is positioned in helix H6 of ATP6 and is part of both the interface between helices H5 and H6 and between ATP6 and the c₈-ring. While the residues in ATP6 in the vicinity of Leu217 (Leu156, Arg159, Val213, Leu216, Leu220, and Tyr221) do not change during the catalytic cycle, the residues found close to Leu217 in the c subunit are different depending on the ATP synthase state. Indeed, in state 1, Leu52 and Leu56 in the c subunit are close to Leu217 in ATP6 (Figure 24A), while in states 2 and 3a, Leu217 is in the vicinity of Leu52 and Ala55 in the c subunit (Figure 24B). Finally, in state 3b, no residue from any c subunit is in the closeness of Leu217 (Figure 24C). The variant of Leu217 in an arginine residue appears to cause two effects: (i) arginine is a larger residue with respect to leucine, causing some sort of friction between the ATP6 and the c₈-ring that is rotating during the catalytic cycle, and (ii) the arginine has a charged side chain that can form H-bonds with other residues in the vicinity, such as Tyr221 from ATP6. This newly formed H-bond can interfere with the formation of another H-bond between Tyr221 and a water molecule in the outlet proton translocation half-channel. The latter water molecule is held in the correct position by two H-bonds, the already cited one with Tyr221 and a second with Glu58 from the c subunit. On the other hand, the variant of Leu217 in a proline residue can cause some sort of effects on the folding of helix H6 downstream of the mutated residue, but—as in the case of p.Leu156Pro—the damage caused by the presence of a small hydrophobic residue in position 156 should be moderate.

The variant at nucleotide m.9185T>C (p.Leu220Pro) was first reported in 2005 ^[73][104], and functional studies revealed a moderate effect of this variant on ATP synthase functioning. Indeed, in patient cells, besides normal Complexes I-IV activity ^[73][76][77][104,107,108], a slight alteration of CV and a depolarization of the plasma membrane were reported, often only in the case of homoplasmy ^{[3][4][73][74][75][78]}[34,35,104,105,106,109]. Mild effects on ATPase function have also been observed in mutated cybrids and yeast cells ^{[3][4][72][78]}[34,35,103,109]. The evaluation of IPSCs and their patient-derived NPCs ^[78][99][109,127], in addition to the defective ATP production, allowed people to highlight mitochondrial impairment that is hidden in the other cell types. Indeed, neural cells presented a mitochondrial hyperpolarization and an alteration of mitochondrial calcium homeostasis, as evidenced by both transcriptomic and proteomic analysis ^[78][109]. All these data suggest that the variant may alter the ability of these cells to produce ATP and control MMP, causing neural impairment.

In the human ATP synthase structures, Leu220 is located on helix H6 of ATP6, just one helix turn away from Leu217, and forms van der Waals contacts with Met60, located on helix H3. Except for Glu224, the other residues found in the vicinity of Leu220 in ATP6 are all hydrophobic (Leu216, Leu217, and Tyr221). As for Leu217, Leu220 is at the interface between ATP6 and the c₈-ring, and the interacting residues from the latter depend on the ATP synthase state. Leu220 of ATP6 is in the vicinity of some residues of the c subunit: Phe47 and Ile51 in states 1 and 3b (Figure 35A) and Leu52 in states 2 and 3a (Figure 35B). As for the previously discussed cases, a variant of Leu220 in a proline residue can have some effects on the fold of helix H6 downstream of the mutated residue and, in turn, cause some problems to the ATP synthase mechanism. On the other hand, proline is a small hydrophobic residue, and no serious steric or electrostatic effects are expected.

A number of MT-ATP6 and MT-ATP8 variants reported in the literature have been reviewed by two different research groups ^[82][84][13,33], and the functional studies of cell models are reported in detail in Table 1.

The study of the m.8909T>C variant, found in a patient also carrying the pathogenic m.3243A>G variant in mt-tRNALeu (MT-TL1), reported a defect in Complex V assembly and ATP synthesis ^[18][49]. A compromised assembly of ATP synthase and a reduced OCR has been observed in fibroblasts of two patients carrying the same m.8782G>A variant, one presenting adult-onset cerebellar ataxia, chronic kidney disease, and diabetes, whereas the other had myoclonic epilepsy and cerebellar ataxia ^[14][45].

The truncating variant m.9154C>T was found in a patient with adult-onset axonal neuropathy, ataxia, and IgA nephropathy and caused alteration of Complex V assembly, mitochondrial morphology, and ultrastructure in mutated fibroblasts ^[66][97]. Interestingly, the mutation load resulted to be proportional to Complex V assembly defect in patient-derived iPSCs and responsible for impaired neurogenesis due to Notch hyperactivation and altered metabolism of mature motor neurons ^[66][97].

Other identified MT-ATP6 variants include the m.8858G>A variant in a sporadic case of NARP-MILS ^[100][128]; the m.8936T>A in a young boy with atypical mitochondrial Leigh syndrome associated with bilateral basal ganglia calcifications ^[101][129]; m.9143T>C in a patient with insulin-dependent diabetes mellitus, recurrent lactic acidosis, infections, and immunodeficiency ^[102][130]; m.9154C>T in a patient with neuropathy, cerebellar ataxia, and IgA nephropathy ^[103][131]; and m.9171A>G in a patient with mitochondrial retinopathy with atrophy ^[104][132]. The three variants m.8572G>A, the m.8578C>T and m.8812A>G were found in patients with adult-onset spinocerebellar ataxia (SCA) ^[105][133].

Regarding new variants affecting MT-ATP6, MT-ATP8, or both, m.8561C>T, which causes a defect in CV assembly, was reported in a child with early onset ataxia, psychomotor delay, and microcephaly ^[11][42], whereas functional studies have been performed for the m.8382C>T, m.8424T>C, m.8806C >G, m.8975T>C, m.9008C>G, and m.9019A>G variants ^[3][34].