ARL3 regulates transport of prenylated and acylated proteins to photoreceptor outer segment in mouse retina

Abstract

Purpose: Based on in-vitro experiments, ARL3 (Arf-like protein 3) functions as a GDF (GDI displacement factor) for lipidated proteins. We generated photoreceptor- and retina-specific Arl3 deletions to investigate the function of ARL3 with focus on trafficking of lipidated proteins.

Methods: We generated Arl3^+/GT mice containing a EUCOMM gene trap (GT) in intron 1 of the mouse Arl3 gene. Rod- and retina-specific Arl3 conditional knockout mice were obtained by crossing with Flp-mice, followed by iCre75⁺ and Six3Cre⁺ transgenics. Photoreceptor function was measured by ERG and progress of retinal degeneration among littermate mice was ascertained by optical coherence tomography, confocal immunohistochemistry and histology.

Results: ERGs and retina histology of PN15 rod-specific knockout mice were indistinguishable from those of the WT littermates, suggesting normal photoreceptor development. OCT revealed rapid photoreceptor loss in retinas of Arl3^flox/flox; iCre75⁺ mice after PN15. At PN20, scotopic ERG a-wave amplitudes were reduced 70-80% with the photopic ERG unaffected. In retinas of 1 month-old knockout mice, scotopic ERGs were extinguished and cone ERGs were attenuated; histology showed 4-5 rows of nuclei in the ONL. The 2 month-old Arl3^flox/flox; iCre75⁺ retina was ~100 μm thinner and only one ONL row, presumably of cone nuclei, was present. Six3Cre⁺-mediated knockout of Arl3, deleting ARL3 during embryonic retina development, revealed enhanced photoreceptor degeneration: PN20 scotopic (↓80-90%) and photopic (↓70-80%) ERGs were reduced significantly, and OCT at PN15 confirmed faster retina degeneration with much thinner retina of Six3Cre⁺ knockout than iCre75⁺. Immunohistochemistry performed using retina sections of PN15 and 1 month-old rod- and retina-specific knockout mice revealed that rhodopsin, GC1 and CNGA1/3 trafficking appear normal. In contrast, prenylated PDE6 and GRK1, and acylated transducin-α mislocalize in the IS and ONL.

Conclusions: Rod- and retina-specific knockout of Arl3 revealed a rapidly-progressing rod degeneration followed by cone defects, resembling RP. Absence of ARL3 impaired the trafficking of peripheral membrane proteins, but not transmembrane proteins, consistent with its function as a GDF.