Retinitis Pigmentosa 2 Protein Regulates Transport Of Isoprenylated Proteins To Photoreceptor Outer Segments

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This abstract was presented today at the 2014 Association for Research in Vision and Opthalmology (ARVO) meetings in Orlando, Florida by Houbin Zhang, Li Jiang, Christin Hanke and Wolfgang Baehr.

Full size poster can be downloaded here.

Purpose: X-linked retinitis pigmentosa (XLRP) is a devastating form of retinal degeneration, manifesting early in life with symptoms of night blindness, visual field defects, and decreased visual function. In-vitro, RP2 functions as a GAP for the small GTPase ARL3, a GDI displacement factor (GDF). Mutations in the Rp2 gene account for approximately one quarter of all XLRPs. The purpose of this study was to investigate the consequences of RP2 deletion and identify mechanisms causative of XLRP.

Methods: Intracellular localization of RP2 in photoreceptors was determined by neonatal electroporation of an RP2-EGFP expression vector. An Rp2 knockout mouse was generated using a EUCOMM ES cell line containing a gene trap in intron 1. The knockout mice were characterized by Western blot, immunocytochemistry, and electroretinography (ERG).

Results: RP2-eGFP was localized to the plasma membrane of inner segments, axons and synaptic termini in photoreceptors, but not in outer segments. The Rp2 gene knockout mice were viable and developed normally. Ablation of Rp2 gene expression led to slowly progressing degeneration of cone and rod photoreceptors as indicated by ERG recordings. Scotopic a-wave and photopic b wave amplitudes were reduced as early as one month of age in the knockout mice. The Rp2Y/- ERG amplitudes were further reduced at 6 months of age. Trafficking of transmembrane phototransduction proteins, including cone opsins, to Rp2Y/- photoreceptors outer segments was normal up to 14 months of age. While targeting of transducin α and βγ to the Rp2Y/- outer segments was not affected in the knockout, transport of rod and cone PDE6 as well as GRK1 to outer segments was impeded.

Conclusions: RP2 is distributed to plasma membrane of inner segments and synaptic termini in photoreceptors. RP2 is not essential for trafficking cone opsins and transducin to photoreceptor outer segments, but regulates transport of isoprenylated proteins to photoreceptor outer segments. Our results suggest that RP2/ARL3 may allosterically release prenylated proteins from their soluble complex with PDE6D and unload them to donor membranes (e.g., TGN vesicles). ). In the Rp2 knockout, this process is impeded.

Arl3 Rod-Specific Knockout Displays RP-Like Photoreceptor Degeneration

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This abstract was presented today at the 2014 Association for Research in Vision and Opthalmology (ARVO) meetings in Orlando, Florida by Christin Hanke, Houbin Zhang, Cecilia D. Gerstner, Jeanne M. Frederick AND Wolfgang Baehr.

Full size poster can be downloaded here.

Purpose: Arf-like protein 3 (Arl3) localizes predominantly in the photoreceptor inner segment. Germline Arl3 knockout mice do not survive beyond PN 21 and display multiple organ ciliary defects as well as retinal regeneration (Schrick et al., (2006). Am. J. Pathol. 168, 1288-1298). We therefore generated rod-specific Arl3 knockouts to elucidate the role of Arl3 in transport of photoreceptor membrane-associated proteins.

Methods: Knockouts containing a gene trap in intron 1 of the Arl3 gene were generated using a EUCOMM cell line. Breeding with Flp mice, followed by mating with iCre75+ mice, generated rod-specific knockouts. Photoreceptor function and retina morphology of wild-type (WT) and mutant mice were analyzed by confocal microscopy, ERG and immunohistochemistry. An Arl3-specific polyclonal antibody (Ab) was generated using a full-length recombinant Arl3 polypeptide expressed in bacteria.

Results: Western blot of WT retina with anti-Arl3-Ab identified a 20 kDa protein, which was significantly reduced in two month-old mutant (Arl3flox/flox;iCre75+) retina. Immunohistochemistry revealed Arl3 localization predominantly in the inner segments of WT photoreceptor cells. Arl3 immunoreactivity was absent in homozygous rod knockouts, but still present in cones and the inner retina. Scotopic and photopic ERGs of rod knockout and WT mice at PN15 had comparable amplitudes suggesting normal phototransduction. Retina histology of PN15 knockout mice was comparable to WT. One month-old Arl3flox/flox;iCre75+ mice showed reduced (80-90%) scotopic, but normal photopic ERG responses. In retinas of two month-old knockout mice, scotopic ERGs were extinguished, whereas cone ERGs were highly attenuated. Retinas of one month-old homozygous knockout mice had 4-5 rows of nuclei in the ONL, and only one row in two month-old mice. Immunohistochemistry of PN 15 and one month-old retina sections revealed that rhodopsin transport, as shown by rho1D4 labeling of ROS, is normal. Rhodopsin was undetectable in two month-old conditional knockout mice due to complete photoreceptor degeneration.

Conclusions: Rod-specific knockout of Arl3 revealed rapidly progressing photoreceptor degeneration, with knockout mice being completely blind at two months of age. Outer segment development appeared to be unimpaired by Arl3 deletion and rod photoreceptor function was normal at P14.

Notable Paper: Long-term RNA interference gene therapy in a dominant retinitis pigmentosa mouse model

Hereditary retinal dystrophies (retinitis pigmentosa, Leber congenital amaurosis, cone-rod dystrophies, macular degeneration) are characterized by loss of visual function, sometimes starting during early childhood, other times in late adulthood.  About 30% of these dystrophies are inherited in an autosomal dominant fashion (RetNet), caused by gain-of-function mutant alleles which encode a malignant form of a normal protein. Continue reading “Notable Paper: Long-term RNA interference gene therapy in a dominant retinitis pigmentosa mouse model”