Date of Award
Doctor of Philosophy (PhD)
Molecular and Cellular Biology
Gerald B. Downes
Rolf O. Karlstrom
dbt, disease models, metabolism, MSUD, neurodegenerative diseases, zebrafish
Cell Biology | Molecular Biology
Locomotion in vertebrates depends on proper formation and maintenance of neuronal networks in the hind-brain and spinal cord. Malformation or loss of factors required for proper maintenance of these networks can lead to severe neurodegenerative diseases limiting or preventing locomotion. A powerful tool to investigate the genetic and cellular requirements for development and/or maintenance of these networks is a collection of zebrafish mutants with defects in motility. The zebrafish mutant quetschkommode (que) harbors a previously unknown gene defect leading to abnormal locomotor behavior. Here I show that the que mutants display a seizure-like behavior starting around four days post fertilization (dpf) that is characterized by a lack of an initial high amplitude body bend (C-bend) and simultaneous contra-lateral contractions leading to a seizure-like phenotype and paralysis. Peripheral nerve recordings show a significant increase in the number of initiated swimming bouts and overlap between left and right motor neuron activity. These data suggest that the que mutation leads to defects in nervous system function, at the level of motor neurons or central control of motor neurons. I have genetically mapped the que locus to a 0.36cM interval on chromosome 22 using meiotic mapping. I identified a splice mutation in the gene `dihydrolipoamide branched-chain transacylase E2' (dbt) as defective in que mutants. An orthologous mutation in humans lead to Maple Syrup Urine Disease (MSUD), a devastating metabolic disorder leading to seizures, mental retardation, coma and neonatal death if untreated. In zebrafish, dbt is expressed throughout early development and dbt transcripts become enriched in the hind-brain as well as in the gut and liver by 96 hpf. In MSUD patients levels of branched chain amino acids (BCAA) and their keto acids are significantly increased due to the essential role of the dbt enzyme for the BCAA metabolic pathway. The que mutation causes a significant increase of branched chain amino acids in the zebrafish mutant and a strong decrease of neurotransmitters such as glutamate and GABA as well as precursors like glutamine. I hypothesize that reduced neurotransmitter levels in que lead to the observed motility phenotype. Consistent with this hypothesis, I show a tissue specific reduction of glutamate in the hind-brain and spinal cord of que mutants. To evaluate the que mutant's potential as a vertebrate model for MSUD I performed a pilot drug screen using a selection of metabolites of the pathway as well as diet additives currently evaluated in clinical trials. Conversely, application of phenylbutyrate, one of the diet additives, had a beneficial influence on swimming abilities of que mutant embryos, while the keto acid α-ketoisocaproate (KIC), one of the elevated keto acids in human patients, decreased the percentage of larvae capable of swimming. These results help establish the zebrafish que mutant as a new model for MSUD disease that can be used to further the understanding of this disorder and to help identify therapeutic agents.
Friedrich, Timo, "Identification and Functional Characterization of the Zebrafish Gene Quetschkommode (que)" (2012). Open Access Dissertations. Paper 639.