Abstract
Purpose :
Human mutations in the small GTPase Arl3 cause a variety of inherited retinal dystrophies that can present as either autosomal recessive or autosomal dominant; however, the underlying pathobiological mechanism of these mutations remains unexplored. Transgenic expression of constitutively active Arl3-Q71L in wild type mouse rods was shown to cause rod nuclei to be displaced to the inner nuclear layer. We hypothesized that dominant mutations in Arl3 would cause a rod nuclear migration defect leading to a possibly divergent disease mechanism.
Methods :
FLAG-tagged Arl3 constructs with either autosomal dominant or well characterized GTPase cycle mutations were exogenously expressed in mouse rod photoreceptors via in vivo electroporation. Positively expressing eyes were collected at P21 and then sectioned and immunostained with FLAG antibodies and Hoechst to visualize the location of Arl3-overexpressing rod nuclei. The 3D location of each nucleus within the nuclear layers was then graphed as a function of distance from the outer plexiform layer. In addition, the GTPase activity of each mutant was studied by overexpression in AD293 cells using GST-effector pulldowns and in vivo crosslinking with the cell membrane permeable crosslinker DSS.
Results :
Expression of the autosomal dominant mutations D67V and Y90C resulted in nuclei positioned more basally within the outer nuclear layer or even mislocalized to the inner nuclear layer, similar to the GTP-locked Q71L. We found that while Arl3-D67V behaves as a constitutively active mutant, Arl3-Y90C behaves as a fast-cycling mutant. Fast cycling Arl3-Y90C has increased binding to the guanine exchange factor, Arl13B, as well as aberrant GEF-independent Arl3 activity. Finally, we show that the Y90C-dependent nuclear migration defect can be rescued by the overexpression of Arl3 effectors or the ciliary cargo of Arl3 effectors, the disruption of Y90C-Arl13B binding, or the disruption of GTP binding to Y90C.
Conclusions :
Our results are consistent with the hypothesis that dominant mutations in Arl3 cause a defect in the migration of rod photoreceptor nuclei by disrupting the ciliary Arl3-GTP gradient. We found that either removing Arl3-GTP or restoring the ciliary Arl3-GTP gradient is sufficient to rescue the nuclear migration defect. Further studies will be necessary to find the ciliary signal underlying this phenotype.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.