Molecular Genetics Of Early Eye Development

National Eye Institute

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Abstract

We analyzed a null zebrafish mutant of olfm1a and olfm1b genes (olfm1 null). Body shape and fertility of adult olfm1 null fish were normal. Retinal diameter was smaller in null mutants compared with wild-type. The thickness of the retinal ganglion cell (RGC) and inner plexiform layers was reduced in 7 days post-fertilization the larval retina. In adult olfm1 null, RGC and inner nuclear layers were oppositely thicker compared with wild-type. The optokinetic response was significantly reduced in olfm1 null as compared with wild-type larvae. The photopic flash ERG responses to light increments of olfm1 null larvae were also reduced especially in ON stimulation conditions compared with wild-type. Olfactomedin 1 (Olfm1) proteins were preferentially localized in the synaptosomes of zebrafish adult brain. In the synaptosome, Olfm1 interacted with GluR2 and SNARE complexes indicating participation of Olfm1 in both pre- and post-synaptic events. Palmitoylation of GluR2, the core subunit of the AMPA receptor complex, was reduced while carnitine palmitoyltransferase 1c (cpt1c) was increased in the brain synaptosomal membrane fraction of olfm1 null compared with wild-type fish. The levels of GluR2, SNAP25, flotillin1 and VAMP2 were markedly reduced in synaptic microdomain of the olfm1 null brain compared with wild-type, indicating that Olfm1 may regulate receptor trafficking from the intracellular compartments to the synaptic membrane microdomain partly through modification of posttranslational GluR2 modifications such as palmitoylation. In collaboration with Dr. Faklers laboratory (Institute of Physiology, University of Freiburg, Freiburg, Germany) we continue the investigation of the effects of Olfm1 and Olfm2 null mutations on the composition and activity of the AMPA receptor complex. Using shotgun proteomic screen, we identified USP19 as a protein interacting with Olfm1 in the mouse brain. Collaboration with Dr. Y. Ye (National Institute of Diabetes and Digestive and Kidney Diseases, NIH) to characterize this interaction led to the discovery of a novel protein quality control pathway that, unlike degradation-based protein quality control mechanisms, promotes protein homeostasis by exporting misfolded proteins through an unconventional protein secretion process. The CRISPR technology was used to produce zebrafish null mutations in the neuritin1a and 1b genes involved in RGC axon growth. Zebrafish with null mutations in these genes and well as with double null mutations in olfm1 and neuritin genes are currently being analyzed.