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.

Interesting Article: Mechanism of RPE Cell Death in α-Crystallin Deficient Mice: A Novel and Critical Role for MRP1-Mediated GSH Efflux

RPE

I ran across an interesting paper in PLOS One published back in March of 2012 by Parameswaran G. Sreekumar, Christine Spee, Stephen J. Ryan, Susan P. C. Cole, Ram Kannan and David R. Hinton.  This manuscript looks at a mechanism of retinal pigment epithelium (RPE) cell death with notable findings identifying therapeutic targets for disorders that involve the RPE cells.

The authors tested whether α-crystallin has a protective effect that is influenced by changes in glutathione (GSH) content while exploring the mechanism of glutathione efflux from the cells.  Interestingly, they found that the multiple multidrug resistance proteins (MRP) were expressed in RPE, particularly MRP1 and that it is MRP1 that mediates GSH (reduced form) and GSSG (oxidized form) efflux from the RPE cells.  In addition, they noted that inhibition of MRP1 makes RPE cells resistant to oxidative stress induced cell death pathways and conversely, over expression of MRP1 renders them more susceptible to oxidatively induced cell death pathways. Finally the authors note that α-crystallin’s antiapoptotic function is mediated by both GSH and MRP1.

Continue reading “Interesting Article: Mechanism of RPE Cell Death in α-Crystallin Deficient Mice: A Novel and Critical Role for MRP1-Mediated GSH Efflux”

Interesting: Gene Regulator, Onecut1 Important To Retinal Development And Integrity

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This paper, Onecut1 Is Essential for Horizontal Cell Genesis and Retinal Integrity in the Journal of Neuroscience by authors Fuguo Wu, Renzhong Li, Yumiko Umino, Tadeusz J. Kaczynski, Darshan Sapkota, Shengguo Li, Mengqing Xiang, Steven J. Fliesler, David M. Sherry, Maureen Gannon, Eduardo Solessio, and Xiuqian Mu describes the gene regulator Onecut1 as being the key to healthy retinal development and good vision in adulthood.

Essentially, Onecut1 is critical for the formation of horizontal cells, but of fundamental importance to retinal degenerative research, this work implies that horizontal cells might be necessary for the survival of photoreceptor cells.  Of course we have known for some years that horizontal cells are some of the very first cells to respond to retinal degeneration by extensively remodeling, but this is an interesting result that suggests a direct dependence of photoreceptors on the horizontal cells themselves for survival.

The Cilium

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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”.

In the retina, the applications being explored by a number of labs including Jun Yang’s laboratory here at the Moran Eye Center and by a recent student who’s work on Senior-Loken Syndrome in Wolfgang Baehr’s laboratory.  This is in addition to a number of labs throughout the world including Joe Besharse at the University of Wisconsin Madison, and Uwe Wolfrum at the University of MainzDavid S. Williams, University of California Los AngelesMarius Ueffing, University of TübingenEric A. Pierce, Harvard Medical School, Gregory J. Pazour, University of Massachusetts Medical School, Nicholas Katsanis, Duke University, USA,  Tiansen Li, NEI and many others.

 

Interesting paper: Characterization Of Neurite Outgrowth And Ectopic Synaptogenesis In Response To Photoreceptor Dysfunction

Gene heat map

Authors Stylianos Michalakis, Karin Schäferhoff, Isabella Spiwoks-Becker, Nawal Zabouri, Susanne Koch, Fred Koch, Michael Bonin, Martin Biel, and Silke Haverkamp have a new paper out that looks at the earliest gene microarray analysis results associated with neurite outgrowth in the degenerate retina.  The title is a overly broad, but the results focusing on gene expression changes in the A3B1 mouse retina (a CNGA3/CNGB1 double-knockout) are intriguing, particularly their proposal that Tp53, Smad and Stat3 signaling contribute to synaptic plasticity at least. Continue reading “Interesting paper: Characterization Of Neurite Outgrowth And Ectopic Synaptogenesis In Response To Photoreceptor Dysfunction”

Activation of Survival Pathways In The Degenerating Retina of rd10 Mice

Survival rd10

An interesting article was published  in Experimental Eye Research by Marijana Samardzija, Hedwig Wariwoda, Cornelia Imsand, Philipp Huber, Severin R. Heynen, Andrea Gubler and Christian Grimm that examines survival pathways that are induced in the retinas of rd10 mice.  Dynamics of retinal degeneration in the rd10 mouse was also examined including an analysis of retinal vasculature and kinetics.  The study is fairly comprehensive including crude anatomical approaches, biochemistry, real-time PCR, Western blotting and immunofluorescence.  They recapitulate some of the studies that have examined development of the rd10 mouse up to pnd15, but then explored the phases of initial retinal degeneration and explored survival mechanisms and pathways (Lif, Edn2, Fgf2, Mt1, Mt2, p-JAK2, CASP1 and GFAP) in the cells that remain.  It would have been interesting to follow these results at later stages of degeneration.  The authors mention remodeling, but only in passing which was too bad as there are some really interesting aspects of cell survival there.  Regardless, its an interesting paper worthy of having a look at.

 

Interesting Paper: Gene Expression Changes Within Müller Glial Cells in Retinitis Pigmentosa

Retinal degenerations are accompanied by retinal remodeling events.  These events alter the structure and function of the retina and involve to a large extent, Müller cells which seem to serve as pathways for neuronal migration.  This paper by Karin Roesch, Michael B. Stadler and Constance L. Cepko looks at gene expression changes in the Müller cells, one of the glial cells of the retina as the rd1 mouse retina degenerates.

While the paper is not terribly conclusive in its definition of genes or pathways involved, (partially I suspect because of the limited time points examined and the late point in the examinations), this paper does however point in a direction that is useful to the retinal degeneration community.  Specifically, Müller cells are fundamentally involved in the remodeling process.  Intervening there is an opportunity to arrest or slow down the retinal remodeling process to allow for interventions and understanding which genes are involved is a good first step.

Notable Paper: Partial Rescue of Retinal Function in Chronically Hypoglycemic Mice

This paper by Yumiko Umino, Nicolas Cuenca, Drew Everhart, Laura Fernandez-Sanchez, Robert B. Barlow and Eduardo Solessio examined late onset retinal degeneration in a model of diabetic retinopathy.  Specifically, this manuscript attempted to examine the impact on retinas from the Gcgr knockout mice with long term high dietary glucose to see if that rescues retinal structure and physiology in the aged animal.  Interestingly, prolonged exposure to to the diet induced euglycemia did improve retinal function, but did not result in re-restablishement of synaptic connectivity lost in hypoglycemia.  The curious part about this is that there seems to be an ability to maintain metabolic status in these animals over long periods of time in spite of the loss of the synaptic connectivity.

Notable Paper: Receptor interacting protein kinase mediates necrotic cone but not rod cell death in a mouse model of inherited degeneration

How photoreceptor cells go through the process of cell death has been an outstanding question.  The authors of this paper by Yusuke Murakami, Hidetaka Matsumoto, Miin Roh, Jun Suzuki, Toshio Hisatomi, Yasuhiro Ikeda, Joan W. Miller, and Demetrios G. Vavvas have further defined the process and identified the receptor interacting protein kinase (RIP) pathway as a possible target for intervention in patients with retinitis pigmentosa (RP).  The authors used the rd10 mouse model, a mouse model of retinitis pigmentosa to examine the cell death process.  They defined RIP kinase as a mediator of necrotic cell death in cones.  RIP3, has been defined as they key regulator of programmed necrosis and its expression was elevated in rd10 retinas during cone photoreceptor death and not rod photoreceptor death.  Furthermore, the cone photoreceptor cell death was rescued by RIP3 deficiency and by pharmacological treatment with RIPkinase inhibitors. Continue reading “Notable Paper: Receptor interacting protein kinase mediates necrotic cone but not rod cell death in a mouse model of inherited degeneration”

The Rd8 mutation of the Crb1 gene is present in vendor lines of C57BL/6N mice and embryonic stem cells

This is an important issue for anyone involved in using murine models of retinal degeneration.  It turns out that contamination of Rd8 mutation in the B6 mice is more wide spread than the C57BL/6N mice.  Labs worldwide are going to have to reassess their data due to this mutation and all reviewers will ask about this in the immediate future.  The genotyping analysis of a variety of vendor lines is described in this paper by Mary J. Mattapallil, Eric F. Wawrousek, Chi-Chao Chan, Hui Zhao, Jayeeta Roychoudhury, Thomas A. Ferguson, and Rachel R. Caspi.  The take home message is that the rd8 mutation is in the C57BL/6N strain which is used worldwide to produce transgenic and knockout models.  The implications for non-vision labs are not as clear, but for vision labs, substantial disease can be present unrelated to another specific disease gene and will need to be accounted for.

Undersized Dendritic Arborizations in Retinal Ganglion Cells of the rd1 Mutant Mouse: A Paradigm of Early Onset Photoreceptor Degeneration

This paper by Devid Damiani, Elena Novelli, Francesca Mazzoni and Enrica Strettoi documents continued negative plasticity in retina by examining ganglion cells in the rd1 mouse.  The rd1 mouse is one of many models of retinal degenerative disease, in this case as an autosomal recessive retinal degenerative disease.  This work gets at the remodeling issue in retinal degenerative diseaseby examining the last cells in the chain of retinal cells that process information before sending it out to the brain and other CNS centers for further processing.   Continue reading “Undersized Dendritic Arborizations in Retinal Ganglion Cells of the rd1 Mutant Mouse: A Paradigm of Early Onset Photoreceptor Degeneration”

Functional and Anatomical Remodeling in Human Retinal Detachment

This manuscript by Clairton F de Souza, Michael Kalloniatis, Philip J Polkinghorne, Charles N J McGhee and Monica L Acosta examined retinal remodeling in response to a form of retinal detachment.  Rhegmatogenous retinal detachment.  The authors describe the changes observed and note that retinal plasticity is acute and likely occurs quickly enough that it may explain persistent vision loss post-reattachment.  They also later conclude that retinal detachment, particularly with macular involvement is an emergent condition which is a fundamentally important conclusion.

Continue reading “Functional and Anatomical Remodeling in Human Retinal Detachment”