Corinna A. Moro, Sabrina A. Sony, Latisha P. Franklin, Shirley Dong, Mia M. Peifer, Kathryn E. Wittig, Wendy Hanna-Rose
Adenylosuccinate lyase deficiency is an ultrarare congenital metabolic disorder associated with muscle weakness and neurobehavioral dysfunction. Adenylosuccinate lyase is required for de novo purine biosynthesis, acting twice in the pathway at non-sequential steps. Genetic models can contribute to our understanding of the etiology of disease phenotypes and pave the way for development of therapeutic treatments. Here, we establish the first model to specifically study neurobehavioral aspects of adenylosuccinate lyase deficiency. We show that reduction of adsl-1 function in C. elegans is associated with a novel learning phenotype in a gustatory plasticity assay. The animals maintain capacity for gustatory plasticity, evidenced by a change in their behavior in response to cue pairing. However, their behavioral output is distinct from that of control animals. We link substrate accumulation that occurs upon adsl-1 deficiency to an unexpected perturbation in tyrosine metabolism and show that a lack of tyramine mediates the behavioral changes through action on the metabotropic TYRA-2 tyramine receptor. Our studies reveal a potential for wider metabolic perturbations, beyond biosynthesis of purines, to impact behavior under conditions of adenylosuccinate lyase deficiency.
Congenital metabolic disorders (CMDs) arise from genetic mutations that result in reduction or absence of enzymatic activity, leading to phenotypic clinical manifestations. Although most CMDs are rare individually, they are collectively common and affect 1 in 1500 births . CMDs are often underdiagnosed due to the lack of metabolic testing required for diagnosis as well as the overlap of clinical manifestations with other childhood disorders leading to misdiagnosis. CMDs are frequently named after the pathways that are perturbed by loss of enzymatic activity. However, perturbation of one metabolic pathway can have implications in other, independent pathways and can lead to a global metabolic dysfunction. Our study focuses on an ultra-rare CMD in purine metabolism, adenylosuccinate lyase deficiency, that is associated with both neural and muscular deficits, leading to adverse health effects.
Materials and methods
C. elegans culture and strains
Strains were maintained on OP50 Escherichia coli under standard conditions at 20° C . The N2, MT15620 cat-2(n4547), MT14680 lgc-55(n4331), OH313 ser-2(pk1357), VC125 tyra-3(ok325), MT13113 tdc-1(n3419) and MT9455 tbh-1(n3247) strains were obtained from the Caenorhabditis Genetics Center (CGC). SSR199 tyra-2(tm1846) was obtained from Dr. Supriya Srinivasan’s lab. For all experiments, animals were synchronized as L1 larvae using hypochlorite solution. All experiments were conducted on Day 1 adults unless otherwise noted. HV854 adsl-1(tm3328/hT2) is an outcrossed strain. The allele is homozygous sterile and is balanced with hT2 that causes pharyngeal expression of GFP. Non-GFP homozygous adsl-1(tm3328) animals were picked for phenotypic analysis.
adsl-1(RNAi) animals can sense and respond to salt
Because adsl-1(RNAi) animals have locomotion deficits [9,12] we first evaluated the adsl-1(RNAi) model for suitability in movement-based behavioral assays that require intact sensory functions. C. elegans will avoid osmotic changes , and normal animals avoid sodium chloride concentrations above 200 mM . We performed an osmotic ring test using 4 M sodium chloride to test the ability of control and adsl-1(RNAi) animals to sense the osmolarity and remain in the low osmolarity environment. We also included osmotic avoidance-defective osm-9(ky10) animals as a control . osm-9(ky10) frequently cross the 4 M NaCl ring (Fig 1A). However, the response of adsl-1(RNAi) animals was indistinguishable from the control animals indicating that they can sense high sodium chloride as a noxious stimulus and will remain in areas of low osmolarity (Fig 1A).
adsl-1 reduction-of-function mutants display an altered response in a salt-aversive gustatory plasticity assay. The altered behavioral response to cue pairing of salt and starvation upon reduction of adsl-1 function is not due to the animals’ inability to learn. Instead, the animals with reduced adsl-1 function have an exaggerated avoidance response relative to the indifferent post-training response of control animals. Behavioral responses of the N2 control strain in salt-based associative learning assays vary according to specific training and assay parameters, including training times and liquid versus plate-based training experiences, and avoidance responses in salt-based associative learning assays in C. elegans have been reported (e.g., ).
Some strains were provided by the CGC, which is funded by NIH Office of Research Infrastructure Programs (P40 OD010440). Some figures were created with BioRender.com.
Citation: Moro CA, Sony SA, Franklin LP, Dong S, Peifer MM, Wittig KE, et al. (2023) Adenylosuccinate lyase deficiency affects neurobehavior via perturbations to tyramine signaling in Caenorhabditis elegans. PLoS Genet 19(9): e1010974. https://doi.org/10.1371/journal.pgen.1010974
Editor: Anne C. Hart, Brown University, UNITED STATES
Received: July 20, 2023; Accepted: September 14, 2023; Published: September 29, 2023
Copyright: © 2023 Moro et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the manuscript and it's supporting files.
Funding: We acknowledge a J. Lloyd Huck Biotechnology Mini-grant from the Huck Institutes of the Life Science for support for this work. This work was supported by NIH NINDS award #R03NS096451 to WHR. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.