A challenge to photoreceptor cell integrity concerns the post-biosynthetic delivery of proteins from the inner, to the outer, segments over a lifetime. This application will identify: i) the roles of RP2 and ARL3 in protein transport;ii) differences in rod and cone intraflagellar transport mediated by homodimeric kinesin-II (KIF17);and iii) functions of nephrocystin-5 in photoreceptor trafficking and cell polarity.
Aim 1 A will address the physiological functions of RP2, ARL3 and the RP2/ARL3/UNC119 complex. We will generate an RP2(G2A) knockin mouse model. The RP2(G2A) knockin construct will incorporate loxP and Frt sites in intron 1 and upstream of ATG to permit, additionally, a germline knockout of RP2 which is expected to mimic XLRP in male mice.
In aim 1 B, we propose to generate a photoreceptor-specific ARL3 knockout mouse to study the role of ARL3 in rods and cones. Mutant knockin RP2, knockout RP2 and knockout ARL3 mice will elucidate the roles of RP2 and ARL3 in photoreceptor cell biology, and provide important tools for future gene therapy.
Aim 2 will focus on molecular motors which move rod phototransduction proteins to the OS by intraflagellar transport (IFT). The role of homodimeric kinesin-II (KIF17) in polarized IMCD3 cells and mouse photoreceptors will be compared and scrutinized. We will generate Kif17flox/flox mice and delete KIF17 specifcally in rod photoreceptors by mating with iCre-75 mice. Based on highly conserved IFT pathways in C. elegans, we postulate partial redundancy of KIF3 and KIF17 in rods. Therefore, we will delete both KIF3 and KIF17 in rods by generating a rod-specific KIF3/KIF17 double knockout and study the consequences.
Aim 3 will examine the NPHP5 gene that encodes nephrocystin-5, of unknown function. NPHP5 null alleles in human cause nephronophthisis, a medullary cystic kidney disease, and retinitis pigmentosa (Senior-L?ken syndrome). We will generate a rod-specific NPHP5 knockout, a mouse model that will elucidate mechanisms causative for the disease phenotype. Further, as nephrocystins are known to be involved in controlling cell polarity, we will use in-vitro renal epithelial cell cultures to probe mechanisms controlling cell polarization. Finally, AAV vectors will be designed to rescue the NPHP5-RP knockout mouse.

Public Health Relevance

We propose to generate mouse models mimicking human retinitis pigmentosa (RP). The firstaim focuses on X-linked RP (XLRP) caused by mutations in the RP2 gene; of unknown function.XLRP manifests early in life with symptoms of night blindness; visual field defects anddecreased visual function. In aim 3 we focus on the RP component of Senior-L ken syndromecaused by null alleles of nephrocystine-5. NPHP5 null alleles in human cause nephronophthisis(a medullary cystic kidney disease) and retinitis pigmentosa; the combination of which is calledSenior-L ken syndrome. Models for XLRP based on mutations in RP2; or models for Senior-L ken Syndrome currently do not exist. Once generated; they will be valuable tools foridentification of mechanisms leading to disease and for the development of gene therapyagents.We further propose to study intraflagellar transport (IFT); a pathway that delivers proteins fromthe inner segment to the outer segment of photoreceptors; passing through a connecting cilium.IFT transports cargo in the anterograde direction (toward microtubule plus ends) and returnsproducts in the retrograde direction (towards the basal body and microtubule minus ends). Ourgoal is to identify exactly which motor(s) is responsible for ciliary transport of cargo in rods.

National Institute of Health (NIH)
National Eye Institute (NEI)
Research Project (R01)
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Biology and Diseases of the Posterior Eye Study Section (BDPE)
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Neuhold, Lisa
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University of Utah
Schools of Medicine
Salt Lake City
United States
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Thomas, Sophie; Wright, Kevin J; Le Corre, St├ęphanie et al. (2014) A homozygous PDE6D mutation in Joubert syndrome impairs targeting of farnesylated INPP5E protein to the primary cilium. Hum Mutat 35:137-46
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