(PTFS05-04-24) Alterations of the De Novo Thymidylate Biosynthesis Pathway in Mice Cause Neural Tube Defects Which Are Modified by Uracil DNA Glycosylase Deficiency and Folic Acid

Objectives: Neural tube defects (NTDs) are developmental anomalies of the brain and spinal cord resulting from incomplete neural tube closure. NTD etiology is multifactorial, with interacting genetic and environmental risk factors, including folate status. Reduced expression of serine hydroxymethyltransferase (SHMT1), a key enzyme in the nuclear de novo thymidylate biosynthesis pathway of folate-mediated one carbon metabolism, leads to sporadic folate-responsive NTDs in mice. However, it is unknown whether these NTDs occur due to dysregulated nucleotide pools leading to increased uracil in DNA, or futile cycles of DNA repair. The goal of this research was to investigate how nuclear translocation of SHMT1 for the formation of the de novo thymidylate biosynthesis metabolic complex is required for folate-dependent NTD prevention, and how dysregulated nucleotide pools, uracil misincorporation, and subsequent base excision repair (BER) processes contribute to this pathology.

Methods: To investigate this, we used mouse models with impairments in SHMT1 or uracil DNA glycosylase (UNG1/2, a BER enzyme). The following Shmt1 mouse models were used: Shmt1^–/–, which lacks SHMT1 protein, Shmt1^{Y82A,Y83F,K257Q/Y82A,Y83F, K257Q} which lacks both SHMT1 catalytic activity and folate binding capacity, but is a stable structural protein, and Shmt1^{Y83F,K257Q/Y83F,K257Q} which lacks SHMT1 catalytic activity, but retains folate binding capacity and is a stable structural protein. In addition, Ung^–/–mouse models were used to examine the consequences of disrupted BER.

Results: Our results showed that the nuclear roles of SHMT1 in folate cofactor binding, metabolic complex scaffolding, and catalytic activity all contribute to the penetrance and expressivity of the folate and SHMT1 -dependent NTD phenotype. Mouse models sensitized to NTDs by SHMT1 disruption combined with impaired BER (Shmt1^–/–, Ung^–/–) showed 4% penetrance of NTDs compared to 12% in Shmt1^–/– mice with no disruptions in BER.

Conclusions: This implicates repair of uracil in DNA, instead of its accumulation, as the causal factor behind folate-responsive NTDs. Together, these findings implicate DNA repair processes and dysregulated de novo dTMP biosynthesis in the polygenic and environmental etiology of NTDs.

Funding Sources: NIH (NICHD & NIDDK) and USDA-ARS