Molecular Genetics Of Early Eye Development

National Eye Institute

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Abstract

Olfactomedin 1 (Olfm1), Olfm2 and Olfm3, together with Neuritin, Brorin (Vwc2) and Brorin-like (Vwc2l) proteins, are extracellular components of the AMPA receptor (AMPAR) complex. AMPARs are the major ionotropic glutamate receptors within both the brain and the retina that play a major role in synaptic formation and communication. Specific combinations of AMPAR protein constituents, mutations in the genes encoding these proteins, and differential presentation of the AMPAR heteromers at the postsynaptic density may contribute to brain and retina pathologies including glaucoma. We used Vwc2, Olfm2, and Olfm1-3 triple knockout (KO) mouse animal models to study the impact these extracellular proteins have on AMPAR kinetics and on the retina structure and function We demonstrated that the levels of the GluA1-GluA4 core subunits did not significantly change, but the levels Olfm1-3 were reduced by 40-50% in immunoprecipitates from retinal plasma membrane-enriched protein fraction of Vwc2 KO compared with wild type using a mixture of antibodies against GluA1-GluA4 subunits (in collaboration with Dr. Faklers laboratory (University of Freiburg, Germany). Reduced levels of Olfm1-3 proteins in the retina of Vwc2 KO mice may contribute to changes in retinal structure and functions, since our previous results demonstrated functional changes in Olfm1 and Olfm2 null mice. Given the overlapping expression patterns of Vwc2, Vwc2l, Gria1-4 and Olfm1-3 genes in the retina, and affinity purification of these proteins with Vwc2, we tested possible direct interactions between these proteins in vitro. We investigated the interactions between either Vwc2 or Vwc2l with the GluA2, Olfm1 and Olfm2. The GluA2 subunit was selected due to its presentation in high concentrations in the mouse retina and its critical role in regulating the permeability of Ca2+ through AMPARs. Further, we selected Olfm1 and Olfm2 because their levels decreased most dramatically in Vwc2 KO animal model. COS7 cells were co-transfected with constructs encoding FLAG-tagged Vwc2, FLAG-tagged Vwc2l, and GluA2 proteins. These experiments indicated that Olfm2 may facilitate the direct interaction between Vwc2 and GluA2. We suggest that Olfm2 may serve as a bridge mediating the interaction of Vwc2 (or Vwc2l) with GluA2. Altogether, our results identified Vwc2 as a new and potentially crucial player effecting AMPA receptor activity through interaction with the core subunits and soluble associating proteins in the retina. We demonstrated that Olfm2 KO mice exhibit structural changes in the retinal neural fiber layer and in axons and myelin within both the optic nerve and optic radiations of the visual pathway. Olfm2 KO resulted in functional defects in retinal ganglion cells as shown by pattern electroretinography. Following a monocular enucleation, a statistically significant decrease in the level of several proteins including Arc and TrkB was identified in the optic radiation and visual cortex of Olfm2 KO mice under ocular dominance plasticity at P120. Olfm2 KO mice also exhibited fear memory defects, anxiogenic behaviors, and symptoms of autism-like behavior such as reduced social interaction. TrkB stimulation reversed the altered visual functions and autism-like behavior in Olfm2 KO mice. Olfm2 may also have another unexpected non-ocular role. In collaboration with Dr. Miguel Lopezs laboratory (University of Santiago de Compostela, Santiago de Compostela, Spain) we demonstrated that a global lack of Olfm2 in mice promoted anorexia and increased energy expenditure due to elevated brown adipose tissue thermogenesis and browning of white adipose tissue. This phenotype led to resistance to high fat diet-induced obesity. Notably, virogenetic overexpression of Olfm2 in the lateral hypothalamic area induced weight gain associated with decreased brown adipose tissue thermogenesis. Overall, this evidence first identifies central Olfm2 as a new molecular actor in the regulation of whole-body energy homeostasis.