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Passley Jordan Hargrove, Anand Swaroop; Developmental Delay in Outer Segment Formation Causes Photoreceptor Degeneration in Mice with a Targeted Deletion of the Guanine-Nucleotide Exchange Factor, Rabgef1. Invest. Ophthalmol. Vis. Sci. 2017;58(8):349.
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Current research predicts that one-quarter of retinal degenerations are due to defects in intracellular transport in photoreceptors; however genes required for these processes remain largely uncharacterized. Using RNA-sequencing analysis of flow-sorted photoreceptors I selected the gene Rabgef1 (a master regulator of endocytosis) for further characterization based on its increase in expression during the formation and maturation of photoreceptor outer segments and synapses. I hypothesize that loss of Rabgef1 causes defects within photoreceptor outer segments and/or synapses and results in concomitant retinal degeneration.
We performed RNA-seq on postnatal 2-28 rod and cone-like photoreceptors from C57B6/J and Nrl-/- mice respectively and selected Rabgef1 for further characterization. In-situ hybridization using RNAscope technology and two different antibodies were used to localize Rabgef1 mRNA and protein in vertebrate retina. Retinas from Rabgef1-/- and wildtype littermates were analyzed by immunohistochemistry, histology, and transmission electron microscopy. Visual function of Rabgef1-/- mice was accessed by electroretinography. Co-IP of Rabgef1 binding partners was performed using Invitrogen Dynabeads protocol. Eluted protein complexes are currently being examined using mass spectrometry.
Rabgef1 mRNA localizes primarily to photoreceptor nuclei, while protein localizes to the inner segments and OPL. Histology indicates that loss of Rabgef1 results in near-complete photoreceptor degeneration by P28. TEM shows Rabgef1-/- mice have stunted/dystrophic outer segments. IHC using retinal cell-type specific markers indicates that loss of Rabgef1 is photoreceptor specific and results in fewer synaptic contacts with bipolar cells (decreased ribeye staining) . Electroretinography of Rabgef1-/- mice (N=10, p<0.05) indicates complete loss of scotopic and photopic a and b waves upon eye opening.
My results are consistent with the hypothesis that loss of Rabgef1 leads to photoreceptor degeneration due to improper outer segment development. My data indicates that proper endocytosis of specific cargo is required for the formation of outer segments. Mass spectrometry is needed to elucidate Rabgef1 binding partners in order to understand the mechanism(s) causing photoreceptor degeneration in Rabgef1-/- mice.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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