Dystonia and spastic paraplegia are debilitating neurological conditions with distinct signs and underlying
pathophysiology. Dystonia describes a range of conditions characterized by involuntary muscle contraction,
while spastic paraplegias are characterized by progressive weakness and stiffness of the leg muscles.
Previously, a large family was published displaying an autosomal dominant pattern of progressive, age-
dependent spastic paraplegia and incomplete penetrance for generalized and focal dystonia. Genetic analysis
showed that both phenotypes were connected to a single novel missense mutation in the gene ATP5G3
(c.318C>G, p.N106K), which encodes subunit c of ATP synthase. Recently, the pathology of this variant was
confirmed by the identification of a dystonia patient from a second family who was found to be carrying the same
mutation as a de novo mutation. Further experiments demonstrated that ATP production and complex V activity
were significantly reduced in patient fibroblasts, consistent with the predicted role of ATP5G3. As additional
confirmation, experiments were also performed in Drosophila utilizing overexpression of the equivalent missense
mutation, which resulted in a significant disruption in the flies’ locomotor ability and complex V activity. Based on
these preliminary results, the central hypothesis of this proposal is that the p.N106K mutation acts in a dominant
negative manner to disrupt complex V function, leading to the dystonia and spastic paraplegia phenotype. This
hypothesis will be tested using three complementary approaches. First, experiments will be performed to
characterize a newly generated mouse model carrying a missense mutation equivalent to the pathogenic human
mutation (p.N105K) using behavioral, molecular, and electrophysiological approaches. Second, the biochemical
mechanisms of the p.N106K mutation will be elucidated using the E. coli ATP synthase system. Specifically,
mutations will generated with the equivalent “humanized” substitutions for suspected autosomal dominant
ATP5G3 mutations in the bacterial subunit c protein. The biochemical effects of each of these substitutions will
then be explored in the bacterial ATP Synthase, particularly as it relates to assembly of the c-ring, the physical
interaction of the F1 the Fo complexes, and/or proton translocation. Finally, effective therapies will be developed
for the ATP5G3N106K mutation by using CRISPR-based gene editing to inactivate the dominant p.N106K allele in
patient fibroblasts. After optimizing the editing efficiency in cell culture, the feasibility of using CRISPR-based
inactivation as an in vivo treatment approach will be evaluated using Atp5g3N105K mutant mice. The results of this
experiment will help lay the groundwork for developing CRISPR-based editing as a possible treatment for genetic
diseases caused by dominant negative mutation such as osteogenesis imperfecta, which is probably the only
option for treatment. This comprehensive characterization of these ATP5G3 mutations will yield valuable insights
into dystonia and spastic paraplegia, complex V functionality, and the pathology and treatment of autosomal
dominant genetic diseases, particularly those caused by dominant negative mutations.
