Gene-environment interactions in the causation and prevention of neural tube defects

The aim of the work described in this thesis is to investigate the mechanisms underlying neural tube defects (NTDs), birth defects of the developing central nervous system. The study makes use of mouse models of NTDs, and particularly focuses on the interaction between nutritional factors and genetic risk factors in determining susceptibility to NTDs. In humans some NTDs are preventable by folic acid supplementation, but this is not fully effective so investigation of alternative strategies was a key focus. The efficacy of oral nucleotide and/or inositol supplementation was evaluated for prevention of NTDs in the curly tail (Grhl3 hypomorph) mouse, a model for Folic acid-resistant NTDs. Metabolic effects were investigated by mass spectrometry methods for analysis of folate one-carbon metabolism (FOCM) and nucleotide, nucleoside and nucleobase pools. Genetic factors influencing FOCM in curly tail embryos were investigated, focussing on expression of Mthfd1L, which encodes an enzyme of mitochondrial FOCM. Effects on downstream metabolites and the potential for rescue by supplementation with one-carbon donors was evaluated, together with mass spectrometry based analysis of the treatment. The effect of caffeine on neural tube closure was investigated in several mouse strains to test the hypothesis that caffeine may be a risk factor for NTDs. Caffeine did not interfere with neurulation, and was in fact found to prevent spina bifida in curly tail mice. This prevention was accompanied by changes in embryonic FOCM, and cellular effects were analysed. The cellular basis for prevention of NTDs by folic acid was investigate in the Splotch (Pax3 mutant) mouse. Proliferation was investigated in the cranial neuroepithelium of Splotch mutant and wild-type embryos, under standard and folic acid supplemented conditions. The molecular basis of NTDs was investigated with a focus on abnormal Sonic Hedgehog signalling and disrupted dorso-ventral patterning as potentially contributing to NTDs in Splotch mutants

Host-Microbe Co-metabolism Dictates Cancer Drug Efficacy in C. elegans

Fluoropyrimidines are the first-line treatment for colorectal cancer, but their efficacy is highly variable between patients. We queried whether gut microbes, a known source of inter-individual variability, impacted drug efficacy. Combining two tractable genetic models, the bacterium E. coli and the nematode C. elegans, we performed three-way high-throughput screens that unraveled the complexity underlying host-microbe-drug interactions. We report that microbes can bolster or suppress the effects of fluoropyrimidines through metabolic drug interconversion involving bacterial vitamin B-6, B-9, and ribonucleotide metabolism. Also, disturbances in bacterial deoxynucleotide pools amplify 5-FU-induced autophagy and cell death in host cells, an effect regulated by the nucleoside diphosphate kinase ndk-1. Our data suggest a two-way bacterial mediation of fluoropyrimidine effects on host metabolism, which contributes to drug efficacy. These findings highlight the potential therapeutic power of manipulating intestinal microbiota to ensure host metabolic health and treat disease.Peer reviewe

Host-Microbe Co-metabolism Dictates Cancer Drug Efficacy in C. elegans.

Fluoropyrimidines are the first-line treatment for colorectal cancer, but their efficacy is highly variable between patients. We queried whether gut microbes, a known source of inter-individual variability, impacted drug efficacy. Combining two tractable genetic models, the bacterium E. coli and the nematode C. elegans, we performed three-way high-throughput screens that unraveled the complexity underlying host-microbe-drug interactions. We report that microbes can bolster or suppress the effects of fluoropyrimidines through metabolic drug interconversion involving bacterial vitamin B6, B9, and ribonucleotide metabolism. Also, disturbances in bacterial deoxynucleotide pools amplify 5-FU-induced autophagy and cell death in host cells, an effect regulated by the nucleoside diphosphate kinase ndk-1. Our data suggest a two-way bacterial mediation of fluoropyrimidine effects on host metabolism, which contributes to drug efficacy. These findings highlight the potential therapeutic power of manipulating intestinal microbiota to ensure host metabolic health and treat disease

The emerging role of cranial nerves in shaping craniofacial development.

Organs and structures of the vertebrate head perform a plethora of tasks including visualization, digestion, vocalization/communication, auditory functions, and respiration in response to neuronal input. This input is primarily derived from afferent and efferent fibers of the cranial nerves (sensory and motor respectively) and efferent fibers of the cervical sympathetic trunk. Despite their essential contribution to the function and integration of processes necessary for survival, how organ innervation is established remains poorly understood. Furthermore, while it has been appreciated for some time that innervation of organs by cranial nerves is regulated in part by secreted factors and cell surface ligands expressed by those organs, whether nerves also regulate the development of facial organs is only beginning to be elucidated. This review will provide an overview of cranial nerve development in relation to the organs they innervate, and outline their known contributions to craniofacial development, thereby providing insight into how nerves may shape the organs they innervate during development. Throughout, the interaction between different cell and tissue types will be highlighted

Partitioning of One-Carbon Units in Folate and Methionine Metabolism Is Essential for Neural Tube Closure

Summary: Abnormal folate one-carbon metabolism (FOCM) is implicated in neural tube defects (NTDs), severe malformations of the nervous system. MTHFR mediates unidirectional transfer of methyl groups from the folate cycle to the methionine cycle and, therefore, represents a key nexus in partitioning one-carbon units between FOCM functional outputs. Methionine cycle inhibitors prevent neural tube closure in mouse embryos. Similarly, the inability to use glycine as a one-carbon donor to the folate cycle causes NTDs in glycine decarboxylase (Gldc)-deficient embryos. However, analysis of Mthfr-null mouse embryos shows that neither S-adenosylmethionine abundance nor neural tube closure depend on one-carbon units derived from embryonic or maternal folate cycles. Mthfr deletion or methionine treatment prevents NTDs in Gldc-null embryos by retention of one-carbon units within the folate cycle. Overall, neural tube closure depends on the activity of both the methionine and folate cycles, but transfer of one-carbon units between the cycles is not necessary. : Leung at al. find that embryonic neural tube closure depends both on the supply of one-carbon units to the folate cycle from glycine cleavage and on the methionine cycle. In contrast, transfer of one-carbon units from the folate cycle to the methionine cycle by MTHFR is dispensable. Keywords: one-carbon metabolism, folic acid, neural tube defects, spina bifida, glycine cleavage system, non-ketotic hyperglycinemia, eye, Mthfr, Gld

Neuronal-epithelial cross-talk drives acinar specification via NRG1-ERBB3-mTORC2 signaling

Acinar cells are the principal secretory units of multiple exocrine organs. A single-cell, layered, lumenized acinus forms from a large cohort of epithelial progenitors that must initiate and coordinate three cellular programs of acinar specification, namely, lineage progression, secretion, and polarization. Despite this well-known outcome, the mechanism(s) that regulate these complex programs are unknown. Here, we demonstrate that neuronal-epithelial cross-talk drives acinar specification through neuregulin (NRG1)-ERBB3-mTORC2 signaling. Using single-cell and global RNA sequencing of developing murine salivary glands, we identified NRG1-ERBB3 to precisely overlap with acinar specification during gland development. Genetic deletion of Erbb3 prevented cell lineage progression and the establishment of lumenized, secretory acini. Conversely, NRG1 treatment of isolated epithelia was sufficient to recapitulate the development of secretory acini. Mechanistically, we found that NRG1-ERBB3 regulates each developmental program through an mTORC2 signaling pathway. Thus, we reveal that a neuronal-epithelial (NRG1/ERBB3/mTORC2) mechanism orchestrates the creation of functional acini

Long-term functional regeneration of radiation-damaged salivary glands through delivery of a neurogenic hydrogel.

Salivary gland acinar cells are severely depleted after radiotherapy for head and neck cancer, leading to loss of saliva and extensive oro-digestive complications. With no regenerative therapies available, organ dysfunction is irreversible. Here, using the adult murine system, we demonstrate that radiation-damaged salivary glands can be functionally regenerated via sustained delivery of the neurogenic muscarinic receptor agonist cevimeline. We show that endogenous gland repair coincides with increased nerve activity and acinar cell division that is limited to the first week after radiation, with extensive acinar cell degeneration, dysfunction, and cholinergic denervation occurring thereafter. However, we found that mimicking cholinergic muscarinic input via sustained local delivery of a cevimeline-alginate hydrogel was sufficient to regenerate innervated acini and retain physiological saliva secretion at nonirradiated levels over the long term (>3 months). Thus, we reveal a previously unknown regenerative approach for restoring epithelial organ structure and function that has extensive implications for human patients