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K. J. Wahlin, R. Adler; Experimental Analysis of Photoreceptor Synaptogenesis: Cloning of Chicken Neuroligins, and Development of Bioassays for Their Functional Investigation. Invest. Ophthalmol. Vis. Sci. 2007;48(13):4467.
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© ARVO (1962-2015); The Authors (2016-present)
Lack of synapse formation between transplanted stem or progenitor cells and host bipolar and horizontal neurons remains as a major road block preventing photoreceptor replacement through cell transplantation in retinal degenerations. As part of a strategy towards overcoming these limitations, we have cloned several chicken homologues of the synaptogenetic molecule neuroligin (NLGN), and have established bioassays to evaluate their role in synapse formation in the chick embryo retina.
RNA was purified and cDNA synthesized from embryonic day-12 or postnatal day-1 retinas. Coding regions of NLGN genes were PCR-amplified and RACE-extended using primers designed based on published EST and genomic data. HA- tagged NLGN constructs were over-expressed by transfection in COS or primary retinal cells, which were co-cultured with low density populations of untransfected retinal cells. Immunofluorescence confocal microscopy was used to estimate synapse formation by Image J-assisted quantitation of the aggregation of pre- and post- synaptic components into "puncta".
Chicken NLGN- 1 and -3 were cloned from chick retina, showing high homology with their mammalian counterparts. Several NLGN-1 splice variants were identified, similar to known mammalian isoforms. We also identified and cloned chicken NLGN-4, which has not been reported in non-human mammals; an orthologue of mammalian NLGN-2, on the other hand, was not found in the chick. NLGN expression in transfected cells was demonstrated by ICC and Western blot. Puncta formation could be detected with antibodies against SV2, CASK, PSD95, Ribeye, vGLUT2 and GluR4, both in purified cultures and in retinal co-cultures with NLGN-transfected or control COS cells. A quantitative comparison of these experimental conditions is underway.
We have identified and cloned in the chick retina several trans-synaptic cell adhesion molecules that are linked to synapse formation elsewhere in the CNS. The established bioassays will allow evaluating their functional role, as well as that of other candidate synaptogenetic molecules.
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