Olfactomedin 1 (Olfm1) is a conserved secreted glycoprotein expressed preferentially in developing and adult neuronal tissues. To better understand functions of Olfm1, we continued characterization of functional significance of Olfm1 interactions with previously identified proteins including GluR2, Cav2.1, teneurin-4, synaptophysin, and kidins220. We used an Olfm1 mutant mouse line containing the Olfm1 gene with a deletion of exons 4 and 5, which encodes a 52 amino acid long region in the N-terminal part of the Olfm1 protein. Some of the Olfm1-interacting proteins, such as GluR2 (a subunit of the AMPA receptor) and Cav2.1 (a calcium channel 1 subunit), are membrane proteins critical for synaptic functions, while several other proteins, such as synaptophysin and synaptogyrin3, are synaptic vesicle proteins. We found that Olfm1 and GluR2 are co-localized to the synapse in cultured hippocampal neurons. Our results suggest that Olfm1 has a role on maintenance of synaptic function by interactions with synaptic receptors and channels. Since Olfm1 is actively expressed in the retinal ganglion cells of the developing and adult mouse retina, it may regulate the synaptic activity of these cells. We continued functional characterization of Olfm1 mutant line. Olfm1 knockout mice show abnormal behaviors including an increase in anxiety, abnormal social activities, and abnormal olfactory functions. They also develop an early degeneration of retinal ganglion cells and a reduced visually evoked potential. These phenotypes indicate that neuronal functions are disrupted in the brain and the retina in these mice. We observed that the development of retina and olfactory bulb of postnatal Olfm1 mutant mouse is abnormal. In the retina, the inner nuclear layer is thicker and the number of Pax6-positive cells was increased, while inner plexiform and ganglion cell layers are thinner. This may indicate the differentiation of ganglion cells and amacrine cells are abnormal. In the olfactory bulb, tyrosine hydroxylase and GAD1 positive interneurons are reduced. These developmental changes may cause abnormal adult mouse behaviors, visual and olfactory functions. We analyzed the molecular mechanism underlining the Olfm1 mutant mouse phenotype. We found that Ca2+ ion levels were higher in hippocampus, olfactory bulb, and retinal ganglion cells of Olfm1 mutant mice than corresponding levels in wild-type mice. The increase in Ca2+ concentration can contribute to neuronal death and cause early retinal ganglion cell degeneration. An increase in intracellular Ca2+ concentration led to the activation of ERK1/2, MEK1 and CaMKII in the hippocampus and olfactory bulb of Olfm1 mutant mice compared with their wild-type littermates. Excessive activation of the CaMKII and Ras-ERK pathways in the Olfm1 mutant olfactory bulb and hippocampus by elevated intracellular calcium may contribute to the abnormal behavior and olfactory activity of Olfm1 mutant mice. We also used zebrafish to study functions of olfactomedin domain-containing proteins. There are two olfm1 genes, olfm1a and olfm1b, in zebrafish. We generated a double olfm1a-/-;olfm1b-/- null zebrafish. The fertility of double olfm1 null zebrafish appeared to be normal and they grew into adulthood. Phenotype of the double olfm1 null mutant was analyzed using different tests. An optomotor response was tested by a projection of grating patterns sliding under the petri dish with zebrafish larvae. The double olfm1 null larvae followed moving grating patterns in a similar way to wild-type larvae. An optokinetic response was tested by a projection of the grating patterns on the wall of dish with larvae and recording their eye movements. The mutant larvae showed significantly less optokinetic response than the wild-type larvae. ON-OFF response to light stimulation was tested in the larvae in collaboration with Dr. Harold Burgess (NICHD). The olfm1 mutant larvae responded normally to light-OFF stimulation, while the response to light-ON stimulation was dramatically reduced in the mutant larvae compared to wild-type larvae. The electroretinogram (ERG) of the mutant and wild-type larvae was tested in collaboration with Dr. Haohua Qian (Visual Functional Core Facility, NEI) and showed slightly decreased ON response and no change in OFF response in the mutant larvae. Analysis of retina of the olfm1 double null mutant and wild-type zebrafish demonstrated that that the retinal ganglion cell and inner plexiform layers were thinner in the mutant larvae. The size of the inner nuclear layer was same in both larvae but photoreceptor layer were thicker in the mutant larvae. We concluded that olfm1 double mutant larvae have reduced visual functions and modified retinal development. To get insight into a possible molecular mechanism of the observed phenotype, we analyze the transcriptome profile of the mutant and wild-type larvae by a deep sequencing. Klf family members may regulate axon regeneration ability in vertebrates including zebrafish. In collaboration with Dr. Lijin Dong (Genetic Engineering Core Facility, NEI), we used the TALEN technology to produce Klf6 mutants. Fish eggs were injected with Klf6 TALEN mRNAs and the resulting fry were raised to adulthood to generate chimera fish with different mutations in the targeting Klf6 site. Chimera fish were produced with efficiency close to 100 percent from injected eggs. These fish were bred with wild-type fish and their larvae were genotyped to identify the exact pattern of each mutation. Several different mutants were selected to establish lines of mutant zebrafish. Homozygous Klf6 mutant fish were produced. The mutant larvae are apparently normal. They are used to study optic nerve regeneration after an optic nerve crush in the adult mutant fish.
|Tomarev, Stanislav I; Nakaya, Naoki (2009) Olfactomedin domain-containing proteins: possible mechanisms of action and functions in normal development and pathology. Mol Neurobiol 40:122-38|