Identification of biomarkers for biochemical, pathophysiological and neurological effects of high ammonia concentration on the central nervous system in a preclinical model of neonatal hyperammonemia - Abstract Neonatal hyperammonemia (HA) from any cause, including several screenable disorders, results in brain injury leading to irreversible intellectual and developmental disabilities, and even death. Current therapies for HA are targeted at reducing blood ammonia levels; although they can prevent death from brain edema, they are inefficient at reducing or preventing brain damage. Our understanding of the mechanism of ammonia toxicity to the brain is based on experiments in cultured cells and adult animal models; HA disrupts glutamine, glutamate, and K+ metabolism in the astrocytes leading to osmotic stress and brain edema. However, it is not known how HA affects the developing brain because animal models suitable for studying molecular, biochemical, pathophysiological, and neurological effects of HA on the neonatal brain currently do not exist. We have created an animal model of inducible HA, the N-acetylglutamate synthase knockout (NAGSko) mouse. Homozygous knockout mice survive into adulthood and reproduces when treated with N-carbamylglutamate and citrulline, and develop HA when treatment is stopped. We propose to use the NAGSko mice as a model of inducible neonatal HA. Our goal is to establish biomarkers of HA that could be used in both preclinical and clinical testing of neuroprotection drugs. Our specific aims are: 1. To induce HA in neonatal NAGSko mice and determine whether associated MRS biochemical changes in their brains persist after ureagenesis has been normalized. We will induce HA in NAGSko mice at postnatal day 13 (P13) and measure metabolite differences in the brains of HA NAGSko mice and non-HA littermates during HA episode and 2 weeks post recovery from HA. Brain metabolites will be measured using proton magnetic resonance spectroscopy (1H-MRS). 2. To determine whether neonatal HA episode causes persistent abnormal astrocyte function that correlates with blood biomarkers of brain damage. We will use our novel NAGSko/ALDH1L1/GCamp5G-tdTm mice and 2-photon microscopy to monitor changes in Ca2+ signaling during neonatal HA episode and its long-term consequences on astrocytic Ca2+ signaling in awake animals, Changes in Ca2+ signaling will be correlated to serum biomarkers of brain injury S100B and NSE (neuron-specific enolase). 3. To determine whether neonatal HA episode causes persistent abnormal brain electrical activity. We will assess whether a neonatal HA episode increases frequency and severity of seizures after normalization of ureagenesis in the NAGSko mice. After validation, we plan to use EEG patterns, 1H-MRS and blood biomarkers of brain damage as biomarkers of HA in pre-clinical and clinical evaluation of drugs and therapies for the protection of the brain from ammonia toxicity. If successful, drugs that result from these trials will complement current treatment approaches and improve the outcome of patients with HA.
