Cell metabolism is often thought of as the process of breaking down sugars to create energy, but the process also involves all the biosynthetic reactions necessary to sustain life. One of these biosynthetic processes is nucleotide synthesis. Nucleotide synthesis can be divided into two major arms: purine and pyrimidine biosynthesis. Both arms are important for DNA and RNA synthesis, but purine nucleotides are unique because they act as a form of cellular currency, powering other cellular reactions as GTP and ATP. Inosine Monophosphate Dehydrogenase 2 (IMPDH2) is an enzyme that powers the first committed step of GTP synthesis. IMPDH2 is inhibited by GTP, and the protein can form filament structures to resist GTP inhibition when cells have an increased demand for purine nucleotides.
Humans can harbor several types of IMPDH2 mutations that produce a variety of neurodevelopmental phenotypes, including low muscle tone, developmental delay, intellectual disabilities, and dystonia, a movement disorder in which muscle contractions are not regulated correctly. “Dystonia symptoms can be caused by mutations in many genes, so pinpointing the specific contributions of individual genes and working out the mechanisms by which mutations in them cause dystonia is an exciting area,” explains Dr. Andrea Wills, one of the senior authors on a recent study in PNAS exploring the role of IMPDH2 mutations during development.
Work from Dr. Andrea Wills’ and Dr. Justin Kollman’s labs at the University of Washington has shown that the mutant IMPDH2 proteins expressed by patients are hyperactive and resistant to GTP feedback inhibition. Some of these mutant proteins affected IMPDH2 filament formation, as well. Even though these studies provide some mechanistic insight into how IMPDH2 mutations impact the protein’s functions, insights into how the functional impacts translate into a developing organism were lacking. To fill this gap in knowledge, the team turned to the model organism Xenopus tropicalis, better known as the western clawed frog. These animals are widely used to model human disease, and their transparent tadpoles facilitate microscope imaging, meaning that the team would be able to examine IMPDH2 filament formation in the developing frogs. For their recent study, the team chose to interrogate the IMPDH2 variant S160del because it is associated with patient neurodevelopmental phenotypes and fails to form filaments in vitro.
The team started by expressing human versions of wild type and mutant IMPDH2 in X. tropicalis embryos. They found that the embryos that received mutant IMPDH2 had altered levels of several metabolites involved in purine biosynthesis. They also found that tadpoles that develop from these mutant embryos were unable to swim, were shorter, and had curved tails, indicating that the IMPDH2 variant produces significant morphological defects.
IMPDH2 mutations produce profound neurodevelopmental defects in humans. To see how this mutation impacts nerve development in X. tropicalis, the team next quantified the number of nerve bundles in the developing tadpoles. They found that tadpoles with the mutant IMPDH2 developed fewer nerve bundles than animals with the wild type IMPDH2. During development, somites are precursors for muscle formation and influence nerve migration patterns. The team found that somites in the mutant IMPDH2 tadpoles had poorly defined or indistinguishable boundaries. These results indicate that the IMPDH2 mutant produces profound defects in muscle formation and neurofilament distribution, which mirrors the weak muscle tone seen in patients.
After establishing that the IMPDH2 mutant produced profound defects in development, the team next investigated whether these defects could be attributed to improper rod and filament formation. Previous work from the team established that rods and filaments can be induced in vitro through treatment with mycophenolic acid (MPA), an IMPDH2 inhibitor, but whether or not this phenotype would hold in an animal model was unclear. The group treated tadpoles expressing wild type or mutant human IMPDH with MPA and used fluorescence microscopy to assess rod and filament formation. They found that the mutant IMPDH2 proteins failed to form rods and filaments after MPA treatment, confirming that the effect holds during animal development.
Alongside characterizing the variant IMPDH2 developmental effects, the team used high-resolution cryogenic electron microscopy to visualize filament structure disruption. IMPDH2 monomers can form tetramers and octamers by interacting with other monomers via the Bateman domains. The team found that the Bateman domains of IMPDH2 mutant proteins were unstable compared to those of wild type IMPDH2, concluding that this instability contributes to the impaired filament formation observed in vivo and in vitro. Next, the team chemically induced filament formation in the mutant IMPDH2 proteins and measured their sensitivity to GTP inhibition. Structural analyses showed that even though the mutant IMPDH2 proteins could form filaments, they were still resistant to GTP inhibition. In contrast, wild type IMPDH2 proteins were inhibited through filament formation alone. These results suggest that the IMPDH2 mutation impacts GTP inhibition outside of destabilizing IMPDH2 filament formation.
Overall, the team created a new model to study the S160del variant of IMPDH2 during development, finding that the mutation impairs neuromuscular development and protein filament formation and significantly impacts purine metabolite levels. “I'm very excited by the opportunity to understand how metabolite levels give cells and tissues their literal shape, as well as their function,” explains Dr. Wills, “We know that mutations in IMPDH2 can really change the amount of GMP/GTP or AMP/ATP in tissue, but we'd like to know if that's most important in the neurons or the muscle cells, or both.” In the future, the team hopes that this work paves the way to test IMPDH2 inhibitors as potential therapeutics for patients carrying these mutations.
Fred Hutch/University of Washington/Seattle Children’s Cancer Consortium Members Drs. Justin Kollman and Andrea Wills contributed to this research.
This work was supported by funding from the National Institutes of Health and a University of Washington Levinson Fellowship.
O’Neill AG, McCartney ME, Wheeler GM, Patel JH, Sanchez-Ramirez G, Kollman JM, and Wills AE. 2025. An IMPDH2 variant associated with neurodevelopmental disorder disrupts purine biosynthesis and somite organization. Proc Natl Acad Sci USA. 122(49):e2511727122. https://doi.org/10.1073/pnas.2511727122
Kelsey Woodruff is a PhD candidate in the Termini Lab at Fred Hutch Cancer Center. She studies how acute myeloid leukemia cells remodel the sugars on their membranes to reprogram cancer cell signaling. Originally from Indiana, she holds a bachelor's degree in Biochemistry from Ball State University. Outside of lab, you can find her crocheting and enjoying the Seattle summers.
For the Media