*2.1. Mitochondrial Gene Mutations of ATP Synthase*

The better characterized ATP synthase diseases are caused by mutations in the mtDNA *ATP6* and *ATP8* genes [28], encoding for the human a and A6L subunits, respectively. The open reading frame of these two subunits overlap for 46 nucleotides; thus, changes occurring in this region can affect the expression of both subunits [44–48]. The majority mutations that cause defects of ATP synthase involve the *ATP6* gene. We focused our

attention here on ATP synthase mutations, at the level of the *ATP6* gene, leading to altered enzyme functions that have been characterized, while we refer to other detailed reviews [11,28,60,61] and www.mitomap.org (accessed on 7 April 2021) for updated lists for the other *ATP6* mutations.

The most common of these mutations are the m.8993T>G/C (p.Leu156Arg/Pro) and the m.9176T>G/C (p.Leu217Arg/Pro) substitutions, which cause different clinical phenotypes varying from NARP to MILS, depending on mtDNA heteroplasmy [28]. These four mutations compromise mitochondrial ATP production with different degrees of severity and have also been modeled in yeast, in order to better clarify their role in the ATP synthase activity and assembly [34,35,62,63]. Not surprisingly, mutations in the yeast mitochondrial *ATP6* gene impair the ATP synthase function, since the a subunit is involved in the formation of the proton channel at the interface with the c-ring, which is formed in the F<sup>o</sup> sector of the enzyme and is fundamental for the catalytic activity [15,64]. Another common mutation is the m.9035T>C, reported in studies of large patient cohorts [36,65,66]. Although a high level of heteroplasmy in patients has been shown to be required to develop a phenotype [36,66], the m.9035T>C mutation causes lower ATP levels, decreased ATP hydrolysis and increased reactive oxygen species (ROS) in patient tissues [42].

As clearly reported, the mtDNA 8993T>G mutation is associated with a more severe NARP/MILS clinical phenotype than the 8993T>C mutation [28]. Biochemical studies aimed at elucidating the pathogenic mechanisms of the two mutations showed that, in NARP/MILS patient cells harboring a high mutant load (>80%), the ATP synthase activity was drastically reduced (about 70%) and only slightly affected (about 20%) compared to the controls, when the mutations were the 8993T>G and 8993T>C, respectively [29–31]. Although, both mutations lead to cellular energy deficiency and increased ROS levels, the latter was reported as a major contributor to the pathogenesis of the NARP/MILS associated to the 8993T>C mutation [30]. In addition, a high percentage of 8993T>G mutation did not significantly affect either the ATP hydrolytic activity or the ATP-driven proton transport in mitochondria of patient cells, excluding that the mutation affects the assembly of the ATP synthase complex [32,33]. However, biochemical analyses in NARP/MILS lymphocytes revealed that the Leu156Arg mutant a subunit slightly affected the proton translocation through the enzyme, suggesting that the coupling between proton translocation through F<sup>o</sup> and ATP synthesis on F<sup>1</sup> was altered in the mutant ATP synthase complex [29]. These studies also suggested a close relationship of biochemical defect and tissue heteroplasmy. In addition, the clinical phenotype associated to mutations at both 8993 and 9176 nucleotides was found to be worsened by defects in respiratory complex function and assembly [67]. These findings elucidate plausible factors that might contribute to the difference in severity of the clinical phenotype associated with MILS and NARP, which the alteration in ATP synthase alone was unable to explain. The rescue of the energy deficiency that characterizes the cells of NARP and MILS patients has been positively targeted by both genetic and biochemical approaches providing different tools for the development of therapeutic strategies for patient treatment [68–70].

The m.9185T>C or m.9191T>C mutations of *ATP6* are variants of the early described NARP-MILS clinical spectrum. In both cases, leucine is changed into proline, at position 220 or 222 in humans, respectively, near the C-terminus of the protein. The first mutation was reported in many patients with a mild clinical phenotype [37,38] and was associated with decreased Mg2+-ATPase activity in isolated muscle mitochondria but normal respiratory chain enzyme activity [37,38]. The second mutation was instead discovered in a two-yearold patient who died presenting a severe clinical phenotype. This second mutation caused a severe reduction in Mg2+-ATPase activity accompanied by a decrease in the mitochondrial respiration rate, indicating a possible reduction also in ATP synthesis [53].

The *Saccharomyces cerevisiae* yeast equivalent of the m.9185T>C mutation (p.Ser250Pro, corresponding to human p.Leu220Pro) partially impaired the yeast ATP synthase activity with a 30% decrease in mitochondrial ATP production without any evidence of a proton leak [39]. The equivalent of the human m.9191T>C mutation (p.Leu252Pro in *S. cerevisiae*)

instead caused a more severe dysfunction in terms of a >95% decrease in the ATP synthesis rate accompanied by a defective ATP synthase assembly. Subcomplexes of the ATP synthase and free F<sup>1</sup> were detected by BN-PAGE analysis, suggesting that the mutant a subunit was not stably incorporated in the enzyme complex and, therefore, degraded. Since the proline amino acid is indeed a well-known α-helix breaker residue, a possible explanation of the described impaired ATP synthase assembly might reside in the fact that this mutation prevents the correctly folded structure of the a subunit and alters its proper interaction with the c-ring [39].

Another less frequent mutation is the *ATP6* m.8969G>A transition, which leads to the replacement of a highly conserved serine residue at position 148 of the human sequence with asparagine. This mutation was found in a six-year old male with Mitochondrial Myopathy, Lactic Acidosis and Sideroblastic Anemia (MLASA) [40], and in a 14-year old female with a severe nephropathy, carrying a high mutation level (>89%) in the kidney [57]. Biochemical investigations of mutant yeast and human cells revealed a decreased basal and oligomycin-sensitive respiration [40,41], indicating that the substitution of this serine into an asparagine severely compromised the ATP synthase activity, with a block of the proton transfer through the F<sup>o</sup> [41]. Later on, it was shown that these detrimental consequences are caused by the amino acid substitution for asparagine. According to the authors, the asparagine (Asn175 in yeast), which replaces the serine of the normal sequence, binds (with the hydrogen bond) and neutralizes the nearby glutamate (Glu172 in yeast), which is critical for the proton flux in yeast ATP synthase [42].

A novel frameshift mutation (m.8611\_8612insC) in the *ATP6* gene was discovered in 2017 in a patient with ataxia and encephalopathy symptoms [43]. A biochemical analysis revealed impaired assembly and accumulation of subcomplexes of ATP synthase, decrease in the enzyme activity and altered mitochondrial ultrastructure with aberrant cristae formation. All these features were attributed to an aberrant a subunit translation, with the consequent formation of a truncated form [43].

Mutations in the *ATP8* gene are less common. The mutations that cause ATP synthase deficiency are those occurring in the overlapping region of the *ATP6* and *ATP8* genes, thus interfering with the synthesis of both subunits. Two different nucleotide substitutions, m.8528T>C and m.8529G>A, both affecting the first amino acid residue in the human a subunit, and the amino acid residue in position 55 of the human A6L subunit (m.8528T>C (a subunit p.Met1Thr and A6L subunit p.Trp55Arg), m.8529G>A (a subunit p.Met1Ile and A6L subunit p.Trp55\*)), were identified in patients suffering from severe cardiomyopathies [44–46]. The other two mutations, occurring in the overlapping region in the nucleotide m.8561 caused amino acid residue changes in position 12 and 66 of the human a and A6L subunits, respectively (m.8561C>G (a subunit p.Pro12Arg and A6L subunit p.Pro66Ala) and m.8561C>T (a subunit p.Pro12Arg and A6L subunit p.Pro66Ser)). These mutations were detected in individuals who had also early-onset ataxia and severe neurological signs [47,48]. In all these reported cases, the ATP synthase deficiency was due to an altered enzyme assembly that causes a consequent increase in the F<sup>1</sup> subcomplex [46–48] and a decrease in ATP production [44–48].
