Neuroprotection from Hyperammonemia

Feldman B1, Tuchman M2, Caldovic L2
Eunice Kennedy Shriver National Institute of Child Health and Human Development, Bethesda MD, USA;
Children’s National Medical Center, Washington DC, USA
Hyperammonemia has traditionally been studied in the context of urea cycle defects and liver failure.
However it is the exposure of the brain to elevated ammonia that leads to a wide range of neurocognitive deficits, intellectual disabilities and in the most severe cases, coma and death. Although the
brain is damaged by hyperammonemia, current treatments focus almost exclusively on reducing
ammonia levels through the activation of alternative liver pathways for ammonia disposal or liver
transplantation. Our aim is to discover novel drugs that protect the brain from elevated levels of blood
Ammonia is toxic to all fish and its effects appear to be similar in fish and mammals. Although
fish have a complete urea cycle, hyperammonemia can be induced simply by immersing them in water
with elevated concentration of ammonia, which is transferred from the water into the blood. In fish
hyperammonemia manifests with neurological similarities to humans including hyperventilation,
lethargy, convulsions, coma, and death.
We developed a zebrafish model of hyperammonemia by immersing 4 days old (dpf) fish in
water containing ammonium acetate (AmAc). The LD50 for AmAc in 4 dpf zebrafish was determined by
exposing them to increasing concentrations of either AmAc or sodium acetate (control) and monitoring
for the cessation of heartbeat as the endpoint. The ability of small molecules to protect 4 dpf zebrafish
from ammonia toxicity was determined by exposing them to collections of small molecules dissolved in
10% DMSO followed by addition of AmAc and monitoring whether small molecules prolong survival in
AmAc. A screen of NIH Clinical Collections 1 & 2 of 727 compounds with known safety profiles is
currently under way.
When 4 dpf zebrafish were exposed to increasing concentrations of AmAc, 50% of 4 dpf
zebrafish died within 3 hr when exposed to 3 mM AmAc whereas 4 mM AmAc was 100% lethal.
Therefore, we used 4 dpf zebrafish exposed to 4 mM AmAc to screen for drugs that increase their
survival. We then tested whether glutamine synthetase inhibitor methionine sulfoximine (MSO) and/or
NMDA receptor antagonists MK-801, memantine and ketamine, which are known to protect the
mammalian brain from hyperammonemia prolong the survival of 4 dpf zebrafish exposed to lethal dose
of AmAc. Treatment with MSO, MK-801, memntine or ketamine prolonged the lives of 4 dpf fish
exposed to a lethal dose of AmAc. Treatment with the combination of MSO and an NMDA receptor
antagonist was more effective than either drug alone. These results demonstrate that zebrafish can be
used in a high throughput screen to select ammonia-neuroprotective agents. If successful, drugs that
result from this screen would complement current treatment approaches to improve the outcome of
patients with hyperammonemia.