Dystonia in ATP Synthase Defects: Reconnecting Mitochondria and Dopamine

Four decades of research highlighted a significant role for mitochondrial dysfunction in the pathogenesis of movement disorders. A first milestone was set by the discovery that the neurotoxin N-methyl-4-phenylpyridinium (MPP+), an apoptosis-inducing mitochondrial complex I inhibitor, induces parkinsonism by selectively damaging the substantia nigra.1, 2 This report sparked intensive investigations on the role of mitochondria in Parkinson's disease (PD),3 fueled by the recognition of mitochondrial functions for several PD-associated genes.2 Sequencing of the mitochondrial genome was completed in 1981.4 Reports linking mitochondrial DNA (mtDNA) variants to neurological phenotypes rapidly followed. Notably, two early works identified dystonia as a prominent feature of distinct mitochondrial syndromes,5, 6 leading to the speculation that “movement disorders might have a mitochondrial etiology”.7 The association between mitochondrial diseases and various movement disorders has been confirmed by several case reports and few larger series.8-11 Nonetheless, systematic explorations of the underlying pathophysiological mechanisms are scarce. Recently, independent studies elucidated a direct causal relationship between isolated dystonia and variants in genes encoding components of the mitochondrial ATP synthase (ATPase).12, 13 These remarkable findings encouraged us to reappraise the “mitochondria-movement disorder connection”7 from a genetics-driven perspective. We posit that mild energetic failure caused by specific ATPase variants may influence the synaptic activity of several nodes of the motor circuits involved in dystonia pathogenesis. ATPase activity modulation driven by other genetic or environmental stressors may play a role in the functional network alterations seen in more common idiopathic dystonias. Unique among the cellular organelles, mitochondria underlie a dual genomic control by both autochthonous mtDNA and the nuclear genome.14, 15 MtDNA encodes 13 subunits of the enzymatic complexes governing oxidative phosphorylation (OXPHOS) and 24 mitochondrial specific RNAs.4 Further >1000 mitochondrial proteins are nuclear-encoded.16 They are involved not only directly in OXPHOS, but also in respiratory chain-complex assembly, mtDNA replication, expression, and repair, as well as in other metabolic pathways.15 Defects in any of these processes may result in a mitochondrial phenotype. Dystonia is a recurrently observed manifestation of mitochondrial diseases, particularly in pediatric cohorts.8-11, 17 In this setting, dystonia usually occurs within multisystemic phenotypes such as Leigh syndrome, a progressive encephalopathy associated with basal-ganglia necrotizing lesions.18 Leigh syndrome presents in childhood, triggered by infection or other illnesses after an initial period of normal development.19 Following metabolic crises, affected children develop various neurological symptoms including developmental regression, ataxia, and dystonia. Variants in several other mitochondrial genes (eg, PMPCA) present with a wide clinical spectrum ranging from relatively mild dystonic phenotypes to severe epileptic encephalopathies with movement disorders.20 Additionally, dystonia is a key feature of rare mitochondrial syndromes such as Mohr-Tranebjaerg disease, along with deafness and optic atrophy.6 Neurodegenerative diseases with a mitochondrial background may also feature dystonia as a part of their phenotype. In these cases, dystonia can precede other manifestations for several years21 or outlast further more typical neurological signs.22 Together, dystonia occurs in a variety of genetic defects linked to mitochondrial dysfunction, therefore, representing a rather non-specific manifestation, fitting the older definition of “secondary” dystonia within a broader phenotype. Isolated dystonia as clinical presentation of mitochondrial diseases is exceptional.21 OXPHOS is orchestrated by five multiprotein complexes located in the inner mitochondrial membrane. The first four complexes (I–IV), known as the respiratory chain, generate a transmembrane proton gradient by transferring electrons to molecular oxygen.23 The fifth complex, referred to as ATPase, funnels this chemical force in the production of ATP.24 ATPase consists of a globular F1 head made of three αβ dimers and a membrane-embedded Fo subcomplex formed by a pore and a ring of multiple c subunits.25, 26 These two moieties are connected by a peripheral stalk and a central stalk. This peculiar structure enables ATP synthesis via a “chemomechanical” coupling.27 Indeed, the proton influx through the Fo pore drives the rotation of the c-ring, which is transmitted via the central stalk to the catalytic F1 sector, where the reaction ADP + Pi → ATP occurs.28 Dimeric ATPase complexes assembly at the inner mitochondrial membrane in a modular fashion contributing to the shaping of mitochondrial cristae.29 Only two of the 17 ATPase subunits are coded by mtDNA (MT-ATP6 and MT-ATP8),30 whereas the remaining ones are nuclear-encoded.31 ATPase defects are underrepresented among mitochondrial disease etiologies.31 Beyond possible epidemiological considerations, this issue may be historically related to earlier difficulties in assaying complex V activity.32, 33 The group of Anita Harding in 1990 reported the first association between ATPase and human disease, by describing a MT-ATP6 variant in a syndrome encompassing developmental delay, dementia, ataxia, and neuropathy.34 The first disease-causing variant in a nuclear gene for ATPase, ATP5F1E, was reported 20 years later.35 Most ATPase-deficiency syndromes are clinically severe as expected by the cardinal role of the enzyme. MT-ATP6 variants have been associated with Leigh syndrome.36 Early-onset metabolic encephalopathies are also the main presentation of biallelic variants in genes encoding nuclear subunits (ATP5F1A, ATP5F1D, and ATP5MK).37-40 In these settings, dystonia41 and other movement disorders may be noticed after a stabilization of severe metabolic crises. An isolated ATPase-related movement disorder phenotype was observed for the first time in association with a heterozygous variant in ATP5MC3, which codes for the Fo ring c-subunit.42 Neilson and colleagues42 described a missense variant (p.Asn106Lys) in an autosomal dominant pedigree in which several relatives displayed either childhood-onset generalized dystonia or adult-onset spastic paraplegia. Several members also displayed isolated focal limb or neck dystonia. Notably, the same p.Asn106Lys variant was found as a de novo event in a nonrelated 11-year-old boy suffering from isolated upper-limb dystonia.42 Following this report, further observations expanded the association between ATPase defects and dystonia. Zech et al13 described a heterozygous ATP5MC3 missense variant in a 15-year-old girl with developmental delay and dystonia. The same group confirmed the role of ATP5F1E as a mitochondrial disease gene by describing homozygous variants in independent patients who developed limb/generalized dystonia after the stabilization of metabolic crises. Furthermore, they expanded the inheritance pattern and phenotypes associated with variants in ATP5F1A, the gene encoding the α-subunit of the globular F1 subcomplex.13 Biallelic ATP5F1A defects were previously described in two infants with fatal neonatal encephalopathy.13 Zech et al13 reported de novo heterozygous ATP5F1A variants in two non-related children with a dystonic cerebral-palsy phenotype and in a third proband with failure-to-thrive and lactic acidosis in the first months of life, who subsequently recovered and had no persistent neurologic phenotype. Finally, they individuated ATP5PO, encoding the peripheral stalk subunit OSCP, as a new gene linked to epileptic encephalopathy and dystonia.13 The latest association between ATPase defects and dystonia was described in two families with heterozygous missense variants in ATP5F1B, coding for the β-subunit of the F1 catalytic sector. In affected individuals, age at dystonia onset ranged from infancy to adolescence. Dystonia began in a limb and showed a different rate of progression, but all affected individuals retained independent gait and showed no additional neurological features.12 Non-affected relatives also carried the ATP5F1B variant, pointing to a reduced penetrance mechanism. Intriguingly, ATP5F1B had been associated with human disease in a single previous report. Here, a de novo missense variant affecting a nearby amino acid was the cause of a severe congenital mitochondrial uncoupling syndrome.43 Table 1 summarizes ATPase defects associated with dystonia. Several mechanisms drive the pathogenicity of ATPase variants.44 A constant biochemical feature is the reduction of ATPase enzymatic activity, whose extent may correlate with phenotypic severity.13, 42 In metabolic encephalopathies caused by biallelic variants, the protein levels of ATPase are invariably reduced.37, 38, 40 This finding is accompanied by alterations of cristae morphology.37, 40 Conversely, ATPase levels as well as gross mitochondrial morphology are mostly preserved in dominant ATPase disorders.12, 13 In this setting, the reported missense variants mostly alter highly conserved amino acids in interhelical or intersubunit contact areas of the affected proteins, suggesting a possible dominant negative effect of the mutant subunits. Interestingly, in ATP5F1B-related isolated dystonia, ATPase levels are even slightly increased and assemble in larger oligomeric structures.12 This phenomenon might reflect an attempt of functional compensation, because dimerization and oligomerization regulate ATPase enzymatic efficiency.45 Notably, ATP5F1A and ATP5F1B display high predicted constraints against both missense and loss-of-function variants (see Table 1). Therefore, it cannot be ruled out that haploinsufficiency may also be a pathogenic mechanism in some ATPase defect-related disorders, although direct evidence is currently lacking. Subunits coded by ATP5F1A, ATP5F1B, and ATP5MC3 are present in multiple copies in the functioning ATPase. The relative expression of the mutated gene likely contributes to the variable clinical expression and incomplete penetrance of milder phenotypes. Differential expression of mutated genes as well as of subunit homologues may be in turn shaped by stressors and other environmental factors. Oxygen consumption rate is variably reduced in severe recessive phenotypes,13 whereas it is normal in ATP5F1B-related isolated dystonia.12 The opposite alteration, namely a significant increase in oxygen consumption rate is the hallmark of the allelic ATP5F1B-associated uncoupling syndrome.43 This is the consequence of a proton gradient dissipation without parallel generation of ATP through defective ATPase.43 Genetic, imaging, and functional studies46, 47 highlighted the involvement of multiple pathways in the pathophysiology of dystonia. Several of these biological processes eventually converge in a dysfunction of basal ganglia and dopaminergic neurotransmission. Notably, dystonia in Leigh syndrome is accompanied by striatal lesions.19 An obvious structural damage is, however, missing in ATPase defect-related isolated dystonia.12 The net biological consequences of ATPase defects may quite differ from those of respiratory chain defects (for an overview of associated clinical syndromes see Goldstein et al and Zeviani and Viscomi).32, 48 In dopaminergic cells, complex I–IV defects impair ATP production, increase reactive oxygen species (ROS) formation, and induce cell death. A selective functional inhibition of ATPase results in ATP depletion, but not in enhanced ROS production or apoptosis.49 A slightly less efficient energy supply may produce a relevant functional disturbance in susceptible networks. Among the monoaminergic systems, a peculiarity of the dopaminergic neurons resides in the intrinsic cytotoxicity of dopamine, because its metabolism via monoamine oxidase may produce ROS.50, 51 Graves et al51 showed that in the dopaminergic terminals, radicals produced by monoamine oxidase do not increase oxidative stress, but are shuttled in the respiratory chain entering at complex IV as electron donor and contribute to ATP production (see Fig. 1). Here, the application of the Fo subunit inhibitor oligomycin induces dropping of dopamine release. This ROS recycling system may on one hand limit autoxidation and on the other hand drive the additional ATP generation needed for the phasic firing of dopaminergic neurons.51, 52 This tenuous balance may be easily disrupted (eg, if ATPase efficiency decreases). This hypothesis might be tested experimentally by expressing a known pathogenic ATPase variant in the same setting (see Fig. 1) and in turn by exploring the link with a dystonic phenotype (eg, in a murine model). Indeed, a semicritical dopaminergic deficit may clinically manifest as dystonia. Interestingly, in a murine model of dopa-responsive dystonia, several mitochondrial proteins were upregulated in the striatum, including the key F1-rotor subunit ATP5F1C.53 This event could represent a compensatory mechanism to defective dopamine biogenesis and inefficient dopaminergic firing. From a broader perspective, mild energetic failure caused by specific ATPase variants may result in widespread functional changes in several other neuronal circuits (striatal, cortical, and cerebellar) known to be involved in the pathogenesis of dystonia.46, 47 In this setting, fluctuations in energy supply may directly influence the synaptic activity or lead to compensatory changes that eventually result in abnormal synaptic plasticity and aberrant neuronal firing mechanisms, which have been linked to the pathogenesis of dystonia in several animal models.46, 47 The synaptic impairment in multiple pathways might explain a lacking response to levodopa therapy in ATP5F1B-related isolated dystonia.12 It furthermore highlights the unmet need for an overarching therapeutic target with broader modulatory effect on synaptic activity in dystonia-relevant circuits. Several classical forms of monogenic dystonia display reduced penetrance. Affected subjects become symptomatic early in life, whereas asymptomatic carriers are not expected to convert beyond the third decade (eg, in TOR1A-related dystonia).54 These peculiarities suggest a relevant role for extragenetic stressors in the pathophysiology of dystonia. According to the second-hit hypothesis, the “first” genetic alteration underlies maladaptive synaptic plasticity and requires additional genetic or environmental factors to become clinically manifest.55 Alternatively, a compensation via endogenous protection factors may play a role.55 The interplay between genetics and stressors may be especially detrimental in a defined time-window of neurodevelopment. This may explain the age-dependent susceptibility to develop a movement disorder phenotype as well as its specific manifestation (eg, dystonia or spastic paraplegia in childhood- vs. adult-onset ATP5MC3-related disease). ATPase defect-related isolated dystonia recapitulates the incomplete penetrance and age-dependent onset of other monogenic dystonias. This suggests that additional modifiers may shape the net biological effect of otherwise mild or potentially compensated ATPase defects. In respect to autosomal recessive ATPase defect-related disorders, it may also be interesting to explore the presence of an endophenotype in heterozygous carriers.56 Two phenotypic clusters associated with ATPase defects are emerging. Recessive forms underlie multisystemic phenotypes in which non-progressive dystonia becomes evident after an early period of metabolic crises or worsening encephalopathy. Some dominant pedigrees feature isolated dystonia with incomplete penetrance. The cardinal role of ATPase in energy metabolism makes clear that even slight disturbances entail relevant functional alterations. Therefore, the association of ATPase defects with early-onset severe encephalopathies and uncoupling syndromes is not surprising. In these complex pictures, the detection rate of dystonia may be poor.57 We posit that ATPase defects underlie a much larger number of phenotypes also featuring dystonia, warranting reverse phenotyping in diagnosed cases. Monogenic disorders offer unique insights into the pathophysiology of phenotypically related sporadic conditions. The history of neurogenetics in PD exemplifies this notion. The discovery of α-synuclein variants in familial PD58 preceded the protein identification in Lewy bodies,59 and thereafter, contributed to its recognition as central etiological substrate in sporadic PD. Such strong overarching paradigm is missing in dystonia. The recent delineation of ATPase-related dystonias reawakes our endeavor to fulfill this unmet goal. Indeed, the exceptional association of certain ATPase defects with an exquisite dystonia phenotype with incomplete penetrance highlights the vulnerability of specific neuronal circuitries to this event and the likely role of cofactors. Dissecting the molecular basis of these ATPase defects may, therefore, help to shed light on the pathophysiology of common idiopathic dystonias. (1) Research project: A. Conception, B. Organization, C. Execution; (2) Statistical Analysis: A. Design, B. Execution, C. Review and Critique; (3) Manuscript: A. Writing of the First Draft, B. Review and Critique. E.I. 1A, 3A S.B. 3B N.E.M. 1A, 3B D.G. 3B H.P. 1A, 3B J.W. 3B M.Z. 1A, 3A, 3B The authors report no financial disclosures. E.I. is supported by the European Joint Programme on Rare Diseases (EJP-RD WP17 research mobility fellowship). J.W. and M.Z. receive research support from the German Research Foundation (DFG 458949627; WI 1820/14-1; ZE 1213/2-1). H.P. acknowledges grant support from the German Federal Ministry of Education and Research (BMBF, Bonn, Germany) awarded to the German Network for Mitochondrial Disorders (mitoNET, 01GM1906A). M.Z. acknowledges grant support by the European Joint Programme on Rare Diseases (EJP RD Joint Transnational Call 2022), and the German Federal Ministry of Education and Research (BMBF, Bonn, Germany), awarded to the project PreDYT (Predictive biomarkers in dystonia, 01GM2302), by the Federal Ministry of Education and Research (BMBF) and the Free State of Bavaria under the Excellence Strategy of the Federal Government and the Länder, as well as by the Technical University of Munich-Institute for Advanced Study. D.G. acknowledges grant support from the Italian Ministry of Health and Fondazione Mariani. Open Access funding enabled and organized by Projekt DEAL. Not applicable.