Project 1. Seeking the biochemical basis of Costeff syndrome (Type III 3-methylglutaconic aciduria) through zebrafish models. Background: Costeff Syndrome is a rare disorder characterized by early-onset optic atrophy and later-onset spasticity, cerebellar ataxis, cognitive deficit and increased urinary excretion of 3-methylglutaconic acid (3MGC) and 3-methylglutaric acid (3MGA). Genetic mapping and sequencing efforts have identified two familial mutations in the OPA3 gene associated with Costeff Syndrome. The normal function of OPA3 and the biochemical basis of MGA-III remain undetermined, however it is known that 3MGC and 3MGA are derived through leucine catabolism in mitochondria and the levels of these organic acids are also influenced by the mevalonate pathway in peroxisomes. Results: We found that the zebrafish orthologue, opa3, is expressed ubiquitously during the first day of embryogenesis (from fertilization through segmentation) and is enriched in the brain from the pharyngula stage (24 hours post fertilization hpf) until at least 120 hpf. We established two genetic models of Costeff Syndrome as well as a transient (via antisense depletion) model of Type I 3-methylglutaconic aciduria). We established that one of our two Costeff models recapitulates biochemical, behavioral and morphological aspects of Costeff syndrome and we tested this model, exluding one biochemical source of elevated 3-MGA and demonstrating an increased sensitivity to inhibitors of the electron transfer chain enzyme complexes in the inner mitochondrial membrane. Project 2. Functional analysis in zebrafish of human alleles associated with holoprosencephaly. Background: Our collaborators identified a large set of SIX3 mutations in holoprosencephaly patients. SIX3 is an attractive gene for functional testing in zebrafish embryos, because it has well-defined biological effects, and also because it acts in two signaling pathways, BMP and WNT, that are important for gastrulation, the main focus of my laboratory (see Z01 HG200309-05). We accordingly established WNT-specific and BMP-specific in vivo assays in whole zebrafish embryos and functionally characterized 42 SIX3 mutations. A large fraction of these alleles displayed partial losses of function. This data adds to the diagnostic markers available to family members wishing to track their risk of having a child with holoprosencephaly. In addition, certain SIX3 alleles showing losses of function in one assay, but not the other may provide molecular clues towards understanding which structural elements of the Six3 molecule interact with which (BMP or WNT) pathway. We have similarly developed zebrafish-based assays to test the activity of holoprosencephaly-associated mutant FOXH1 alleles.
