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.
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.
“The primary abnormality involves the choroidal circulation, and the characteristic lesion is an inner choroidal vascular network of vessels ending in an aneurysmal bulge or outward projection, visible clinically as a reddish orange, spheroid, polyp-like structure…. The natural course of the disease often follows a remitting-relapsing course, and clinically, it is associated with chronic, multiple, recurrent serosanguineous detachments of the retinal pigment epithelium and neurosensory retina with long-term preservation of good vision.”
Optic nerve head drusen (ONHD) or optic disc drusen (ODD) occurs rarely in the population, about 1% of the population, though there seems to be a genetic association as in families with a history of ONHD, it increases to almost 3.5% of those families.
In todays Grand Rounds on Webvision, we have a classic case of ONHD with typical fundus photographs, but also red-free, autofluorescence, IR and OCT captured by James Gilman of the Moran Eye Center.
A color fundus photograph (CF) shows discreet multiple yellowish calcium deposits in the optic nerve head. The Red-Free photograph (RF) reveals clearer outlines of the drusen. The Fundus AutoFluorescence photograph (FAF) shows the highly fluorescent drusen in this patient’s optic nerve. The infrared image (IR) shows small discreet reflective bodies in the optic nerve. The OCT image (OCT) shows very dense round inclusions in the optic nerve that shadow the OCT signal and indicate the shallow depth and geographic cluster of the drusen.
We mentioned some of the outreach work that the Moran Eye Center does a few posts ago. There is now a documentary, Duk County (trailer linked in the above video) about the humanitarian expedition to South Sudan to restore vision to those who have lost it through cataracts and trachoma mediated blindness. There is also a link and article from National Geographic Adventure Blog here.
The Moran Eye Center‘s first eye intervention in South Sudan is documented in that film, but our Alan S. Crandall, MD, and Dr. Charles Weber, MD, recently completed a second major ophthalmic medical mission to South Sudan. As part of a life-changing five-year initiative, the team once again traveled to the war-torn region to provide eye surgeries to the visually impaired with a small medical team, carrying in every single supply, from Q-tips to microscopes. Through the efforts of Drs. Crandall and Weber, they helped restore sight to 325 patients with cataracts and trachoma.
After finding cataracts far more advanced than expected on their 2011 mission, the team returned in 2012 with a hand-held ultrasound machine that allows them to see into the back of the eye no matter how opaque the front of the eye is during the initial screening. “The clinic in South Sudan is quite bare, but we are able to bring all the supplies we need in order to successfully operate on patients. Last year, we operated as bats flew around our heads and relied on a generator that didn’t always work. Fortunately, there is a new operating room that better suits the needs of the patients,” said Crandall. Continue reading “Duk County And Moran Eye Center Outreach At Home And Abroad”
Choroidal neoplasms are tumors of the choroid of the eye. The most common intraocular tumors are in fact, malignant melanomas of the choroid which increase in frequency as we age. Continue reading “Choroidal Neoplasm”
The Levine lab here at the Moran Eye Center has a new publication out in the Journal of Neuroscience and even scored the cover. Specifically, the manuscript was authored by Patrick J. Gordon, Sanghee Yun, Anna M. Clark, Edwin S. Monuki, L. Charles Murtaugh, and Edward M. Levine. The Levine team explored how multipotent retinal progenitor cells (RPCs) control the ordered production of the major cell types in the mouse retina. The key finding in this manuscript is that the Lim/Homeodomain protein, Lhx2, is a progenitor-intrinsic regulator of maintenance/self-renewal, precursor production, and competence progression. They arrived at this conclusion by generating Lhx2 conditional knockout mice at multiple stages of development using an inducible Cre-driver (Hes1CreERT2) that specifically targets progenitor cells. This approach allowed them to perform day-by-day inactivations, achieving a temporal scale of gene expression control not previously reported in the retina. This is an important advance because the properties of RPCs are constantly changing, and we are now able to directly test the regulation of these properties in an appropriate temporal manner. As such, The Levine team has identified Lhx2 as an RPC-intrinsic factor that regulates maintenance, precursor fate, and competence simultaneously.
Nikko Ronquillo will be defending his dissertation on Wednesday, May 15th at 2:00pm in the Moran Eye Center auditorium on the 1st floor. Nikko’s dissertation, performed in Wolfgang Baehr’s laboratory is on the Functional and morphological studies of the NPHP5 mouse model: insights into Senior-Løken Syndrome.