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.
