Purpose: Cone phototransduction and survival of cones in the human macula is essential for color vision and for visual acuity. Progressive cone degeneration in age-related macular degeneration, Stargardt disease, and recessive cone dystrophies is a major cause of blindness. Thyroid hormone (TH) signaling which regulates cell proliferation, differentiation, and apoptosis plays a central role in cone opsin expression and patterning in the retina. Here, we investigated whether TH signaling affects cone viability in inherited retinal degeneration mouse models.
Methods: Rpe65-/- mice (a model of severe Leber congenital amaurosis or LCA) and Cpfl1mice (severe recessive achromatopsia) were used to determine whether suppressing TH signaling (with anti-thyroid treatment) reduces cone death. Further, Cngb3-/- mice (moderate achromatopsia) and Gucy2e-/- mice (moderate LCA) were used to determine whether stimulating TH signaling (with triiodothyronine (T3) treatment) deteriorates cones. The serum T3 levels were analyzed by ELISA. Cone and rod survival were evaluated by examining cone density and expression levels of cone specific proteins using immunohistochemical and biochemical approaches, and by examining morphological integrity of the retinas.
Results: Cone density increased about 6-fold in Rpe65-/- and cpfl1 mice following anti-thyroid treatment and decreased about 40% inCngb3-/- and Gucy2e-/- mice following T3 treatment. Anti-thyroid treatment did not affect rod survival, manifested as unchanged outer nuclear layer (ONL) thickness and the number of nuclei in ONL. However, T3 treatment significantly reduced ONL thickness and the number of nuclei in ONL in Cngb3-/- andGucy2e-/- mice.
Conclusions: With multiple retinal degeneration mouse models, we demonstrate that TH signaling regulates photoreceptor viability in degenerating retinas. Suppressing TH signaling protects cones whereas stimulating TH signaling has a negative effect on both cones and rods. The findings of this study provide new insights into cone preservation and therapeutic interventions.
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.
Webvision is proud to announce that our colleague here at the University of Utah‘s Moran Eye Center researcher Wolfgang Baehr, Ph.D., has been named by the Association for Research in Vision and Ophthalmology (ARVO), as the 2014 recipient of the Proctor Medal —considered to be the highest honor in the world awarded to scientists working in vision research. The award will be presented to Dr. Baehr during the ARVO 2014 Annual Meeting in Orlando, Fla., scheduled for May 4 — 8. Dr. Baehr was chosen as the recipient of the Proctor Medal for a lifetime of achievements including his work in discovering mechanisms underlying retinal diseases.
P.S. Drop us a line if you’d like to celebrate with us on Monday, May 5th in Orlando, Florida.
My colleague Dr. Wolfgang Baehr at the University of Utah sends this image of friend of Webvision, Dr. Bäerbel Rohrer from Medical University, South Carolina taken last week when they ran into each other in Berlin, Germany.
Its always fun to run into friends and colleagues in different parts of the world. We will look forward to visiting with them both at ARVO 2014, down in Orlando, Florida in the next few weeks.
With the exception of a few types of cells, (acinar cells, T lymphocytes and hepatocytes), every cell in your body has a cilia. In the vision community, we are used to seeing these structures in the distal portion of the photoreceptors. The reality is that every cell in the retina has a cilium and some cells use the cilia as a means to expand a very specialized function like the photoreceptor outer segment or the hair cell or the respiratory epithelium of the lung. This particular cilia was found in an amacrine cell in a rat retina.
Cilia were thought for a long time to be vestigal organelles that are formed in development, then left over after the developmental process ended. Prachee Avasthi Crofts in the Wallace Marshall laboratory notes that “cilia are signaling centers capable of sensing a variety of extracellular stimuli: fluid flow in the kidney, odorants in olfactory neurons, and hormones in the satiety center of the brain. Motile cilia in the trachea and brain ventricles can also generate flow of mucus and cerebrospinal fluid respectively. Dysfunction in conserved ciliary structure and function therefore results in a variety of disorders (termed ciliopathies) which include polycystic kidney disease, anosmia, obesity, bronchiectasis and hydrocephalus, to name a few.
In the retina, the outer segments of photoreceptors that sense light are in fact modified sensory cilia with conserved mechanisms of formation and maintenance. Thorough characterization of phototransduction proteins that reside in the outer segment as well as rapid turnover of outer segments to recycle spent membrane and protein make this system an excellent model to study cargo transport within cilia. Furthermore, a hallmark of many pleiotropic ciliopathies is retinal degeneration that results from abnormal photoreceptor cilia function. Investigation of photoreceptor cilia dysfunction can yield much insight into generalized mechanisms of cilia-related pathogenesis and potential avenues for therapeutic intervention”.
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.
This paper by Vazquez-Chona FR, Swan A, Ferrell WD, Jiang L, Baehr W, Chien WM, Fero M, Marc RE and Levine EM addresses a long standing issue in the field of neuroscience: is the reactive phenotype of glial cells in and of itself detrimental to neural survival or function?
ARVO 2011 has come and gone. Its taken me a couple of weeks to get through the post ARVO chaos and get caught up on my own work before posting some of the snapshots, but here they are. If you’d like to post your snapshots from ARVO here, send me an email (email@example.com) and we’ll arrange to add them to the list and credit you appropriately.