Cellular and Organismal Ramifications of de novo Purine Synthesis Dysregulation

Purines are a class of nitrogenous bases and are essential small molecules to life. Purines are used within the cell as genetic information carriers, energy currency, signaling molecules, and cofactors for multiple processes. They are formed through de novo and salvage pathways found in cells across the phylogenetic tree. The substrates of enzymes within de novo purine synthesis are known to influence other processes within the cell, such as energy homeostasis. In humans, de novo purine synthesis disorders are rare, with around 100 people identified. These patients exhibit a range of phenotypes, with varying degrees of mental retardation, seizure activity, facial and body dysmorphic features, autistic features, respiratory failure, and congenital blindness. To date, the explanation of phenotypes associated with these disorders remains elusive and as such, no effective therapeutic has been identified. Rare disorders are often caused by a single genetic mutation and studying rare disorders can providing key insight into processes regulated by that specific enzyme. In this body of work, I use transcriptomic profiling techniques to provide cellular and organismal process characterization of a novel cellular model of de novo purine deficiency in three CRISPR generated HeLa cell lines. I examine the de novopurine synthesis enzymes ADSL, GART, and ATIC. Processes identified influenced by de novopurine dysregulation identified are focused around neural, embryonic, organ, and placental development, epithelial to mesenchymal transition, fatty acid and inflammatory response, muscle function, tumorigenesis, oxidative stress responses, as well as TGFβ/SMAD signaling among others. Metabolomic profiling was employed to bolster transcriptomic findings, with aberrations of metabolic pathways involved in energy production, vitamin B6 and B5 metabolism, oxidative stress responses, lipids, amino acids, among others. My findings highlight areas in which de novo purine synthesis enzymes influence cellular processes responsible for cellular and organismal function and represent novel avenues of continued research

Reduced purine biosynthesis in humans after their divergence from Neandertals

We analyze the metabolomes of humans, chimpanzees, and macaques in muscle, kidney and three different regions of the brain. Although several compounds in amino acid metabolism occur at either higher or lower concentrations in humans than in the other primates, metabolites downstream of adenylosuccinate lyase, which catalyzes two reactions in purine synthesis, occur at lower concentrations in humans. This enzyme carries an amino acid substitution that is present in all humans today but absent in Neandertals. By introducing the modern human substitution into the genomes of mice, as well as the ancestral, Neandertal-like substitution into the genomes of human cells, we show that this amino acid substitution contributes to much or all of the reduction of de novo synthesis of purines in humans

The CRISPR-Cas9 crADSL HeLa Transcriptome: A First Step in Establishing a Model for ADSL Deficiency and SAICAR Accumulation

Adenylosuccinate lyase (ADSL) catalyzes two steps in de novo purine synthesis (DNPS). Mutations in ADSL can result in inborn errors of metabolism characterized by developmental delay and disorder phenotypes, with no effective treatment options. Recently, SAICAR, a metabolic substrate of ADSL, has been found to have alternative roles in the cell, complicating the role of ADSL. crADSL, a CRISPR KO of ADSL in HeLa cells, was constructed to investigate DNPS and ADSL in a human cell line. Here we employ this cell line in an RNA-seq analysis to initially investigate the effect of DNPS and ADSL deficiency on the transcriptome as a first step in establishing a cellular model of ADSL deficiency. We report transcriptome changes in genes relevant to development, vascular development, muscle, and cancer biology, which provide interesting avenues for future research

Resilience to autosomal dominant Alzheimer’s disease in a Reelin-COLBOS heterozygous man

We characterized the world's second case with ascertained extreme resilience to autosomal dominant Alzheimer's disease (ADAD). Side-by-side comparisons of this male case and the previously reported female case with ADAD homozygote for the APOE3 Christchurch (APOECh) variant allowed us to discern common features. The male remained cognitively intact until 67 years of age despite carrying a PSEN1-E280A mutation. Like the APOECh carrier, he had extremely elevated amyloid plaque burden and limited entorhinal Tau tangle burden. He did not carry the APOECh variant but was heterozygous for a rare variant in RELN (H3447R, termed COLBOS after the Colombia-Boston biomarker research study), a ligand that like apolipoprotein E binds to the VLDLr and APOEr2 receptors. RELN-COLBOS is a gain-of-function variant showing stronger ability to activate its canonical protein target Dab1 and reduce human Tau phosphorylation in a knockin mouse. A genetic variant in a case protected from ADAD suggests a role for RELN signaling in resilience to dementia