TRPV4 Regulates Calcium Homeostasis, Cytoskeletal Remodeling, Conventional Outflow and Intraocular Pressure

Krizaj glaucoma

Glaucoma is the main cause of irreversible blindness in the world. In most common types of the disease, the optic nerve is damaged by an increase in intraocular pressure (IOP) which blocks fluid drainage through canals in the eye. There is currently no cure, however, the disease can be treated by lowering IOP. Unfortunately, all IOP-lowering drugs that in the market today target the secondary drainage pathway which mediates only 5-15% of fluid outflow. Therefore, the main goal in glaucoma research has been to identify targets in the primary outflow pathway mediated through the trabecular meshwork tissue. David Krizaj’s group at the Moran Eye Institute (University of Utah School of Medicine) has done just that.

In a paper just published in Scientific Reports, they identify TRPV4, a mechanosensitive ion channel, as the main trabecular target of increased IOP. This highly collaborative project combined genetic, molecular, whole animal approaches with bioengineered nanoscaffold models of glaucoma and drug discovery to show that activation of the channel mimics the trabecular changes in glaucoma whereas elimination of the TRPV4 gene or systemic exposure to TRPV4 inhibitors protected mice from the disease. In collaboration with Glenn Prestwich’s group in Medicinal Chemistry at the University of Utah, the team synthesized new eye drops which lowered IOP to levels seen in control mice. By targeting the primary outflow pathway, this study promises to bring new, effective cures that complement current glaucoma treatment. The primary authors of the study are Dr. Dan Ryskamp, Amber Frye and Dr. Tam Phuong.

Müller Cell Metabolic Chaos During Degeneration

Muller-cell-chaos1 copy

This abstract was presented today at the 2015 Association for Research in Vision and Opthalmology (ARVO) meetings in Denver, Colorado by  Rebecca L. PfeifferBryan W. Jones and Robert E. Marc.

Purpose: Müller cells (MCs) play a critical role in glutamate (E) metabolism and carbon skeleton cycling in retina. MCs demonstrate changes in metabolism and morphology during retinal degeneration. The timing, extent, regulation, and impacts of these changes are not yet known. We evaluated metabolic phenotypes of MCs and evaluated their capacity to transport glutamate during degeneration.

Methods: Retinas were harvested from wild-type (WT) and rhodopsin Tg P347L rabbits, divided into chips mounted on filters, and incubated in Ames medium with 5 mM D-aspartate (D-Asp), D-glutamate (D-Glu), or D-glutamine (D-Gln) for 10 min at 35 deg to explore transport and metabolism. Chips were fixed in mixed aldehydes and resin embedded for computational molecular phenotyping (CMP) of a range of L- and D-amino acid markers and selected proteins including glutamine synthetase (GS) (J Comp Neurol. 464:1, 2003).

Results: CMP revealed wide variations in metabolite levels across individual MCs from Tg P347L retinas, generating chaotic patterns. GS decreased significantly while glutamine levels (Q) increased, although to varying degrees. Remarkably, E levels were variable and much higher in some MCs than normal, but did not correlate (inversely) with GS levels. Transport experiments using D-Glu, D-Asp, and D-Gln showed that alterations in MC metabolites are not the product of defective transporters, in contrast to previous reports. These results are also inconsistent with conventional models of GS-based E-Q metabolism and microenvironmental regulation of MC phenotypes.

Conclusions: These observations suggest three conclusions. (1) Although degeneration of the retina is certainly the trigger, MC phenotype changes are not a coherent response to the surrounding microenvironment but are, rather, uncoordinated individual MC responses. (2) Although GS is accepted as the primary enzyme responsible for the conversion of E to Q in the normal retina, alternative pathways appear unmasked in the degenerate state. (3) It has been previously hypothesized that MCs in retinal degenerations exhibit deficient E transport. Our experiments show no transport deficiency. This indicates that chaotic metabolite levels emerge from changes in individual MC metabolic processing.

Seminar: Cellular Response Associated to Retinal Diseases and Therapeutic Approaches

Nico Cuenca Seminar

Nicolás Cuenca, professor Department of Physiology,Genetics and Microbiology University of Alicante Spain will be delivering a seminar, Cellular Response Associated to Retinal Diseases and Therapeutic Approaches on Monday, May 11thth at Noon in the the Moran Eye Center auditorium.

 

Abstract: At the cellular and molecular level, the response to retinal injury is similar in diseases like AMD, glaucoma, diabetic retinopathy and retinitis pigmentosa. All exhibit a set of cell signals that lead to controlled cell death and retinal remodeling, inflammatory responses, oxidative stress and activation of apoptotic pathways. In our hands anti-apoptotic compounds such as TUDCA and proinsulin and an3oxidant like safranal prevent retinal degeneration in animal models. In addition using Human CNS stem cells (HuCNS-SCs) we have been able to protect photoreceptors and preserve retinal integrity in a rat model of AMD. Such approaches could potentially delay retinal degeneration in human retinal diseases.

 

Lasker/IRRF Report On Restoring Vision

Restoring Vision To The Blind

I participated in the Lasker/IRRF Initiative on Restoring Vision to the Blind in March 2014. It was a great session of research leaders working on various approaches to restore visual function lost by retinal degenerative disease. The purpose of the meeting was to identify the key issues hampering research progress and to develop innovative proposals to overcome these hurdles and accelerate research. The Initiative prepared a report of its findings that ARVO published as a special edition of its online journal Translation Vision Science and Technology. It can be viewed at http://tvstjournal.org/toc/tvst/3/7.

I am attaching the Table of Contents for the report, along with John Dowling’s introduction to give you an idea of the scope of the work discussed by participants.  If you want a pdf of the entire report, you can find it on the Lasker website at: http://www.laskerfoundation.org/programs/images/irrf_15.pdf . A print copy of the report is also available by writing to Meredith Graves as mgraves@laskerfoundation.org

Continue reading “Lasker/IRRF Report On Restoring Vision”

Seminar: How Inhibiting The Dimerization Of Vitamin A and/or Catalyzing The Reduction of Quinones May Prevent Retinal Degeneration

Ilyas W Flyer

Ilyas Washington from Columbia University Medical Center will be delivering a seminar on How Inhibiting The Dimerization Of Vitamin A and/or Catalyzing The Reduction of Quinones May Prevent Retinal Degeneration on Wednesday, February 11th, 2015 in the Moran Eye Center auditorium.

Abstract:  Many human conditions, originating from multiple genetic and environmental pressures, converge to overproduce superoxide radical anions. The bulk synthesis of cellular superoxides is believed to result from redox cycling of the quinone, coenzyme q. We have designed small molecules to control quinone redox cycling and correct superoxide fluxes as a means to prevent retinal degeneration.

Seminar: Light Damage

Jeannie Chen seminar

Jeannie Chen, Professor in the Department of Cell and Neurobiology, the Zikha Neurogenetic Institute, Keck School of Medicine, University of Southern California will present a seminar at noon on Wednesday, September 17th in the Moran Eye Center auditorium on Light Damage.

Abstract:  Excessive light exposure is known to harm the retina and exacerbates disease progression in many retinal disorders. In addition, certain genetic mutations affec6ng components in the phototransduction cascade serve as a source of “equivalent light” and cause retinal degenera6on. How light exerts its harmful effect on the retina is not well understood. Mouse models can be used to isolate distinct light damage pathways. Dr. Chen will discuss recent findings on mechanisms of retinal damage caused by environmental and genetic sources of “equivalent light”.

What It’s Like To Go Blind

This is important for scientists and non-scientists alike.  You might be surprised at how many people do not know someone who is blind or has gone through a blinding disease.  You might be further surprised at how many scientists that are engaged in vision research do not really know what its like to have gone through vision loss or have similarly interacted with someone who is going blind.  As I’ve said before in The Judgment Of Solomon post, “Every scientist studying vision and diseases affecting vision should have the opportunity to spend time with those who have lost sight.  It is important for people in the sciences to sit down and talk with those affected by the disease they study.”

The subject of this short, Mark has a cone/rod dystrophy due to a defect in the ABCA4 gene, which codes for an ATP-binding cassette transporter family.  Kris Palczewski’s group has shown that these defects ultimately cause a buildup of all trans retinal in the outer segments of the photoreceptors and leads to likely oxidative damage, cell stress and photoreceptor toxicity.  This photoreceptor toxicity then ultimately results in photoreceptor cell death and blindness.

 

The Judgment Of Solomon

I had the honor and privilege of attending a Lasker/IRRF Initiative’s plenary session on Restoring Vision to the Blind at Janelia Farm last month where Mr. Sanford D. Greenberg delivered an emotional and inspiring story of a time in his life where he lost his vision during his junior year at Columbia University.  The prospect of losing vision is absolutely and completely life altering for those affected as well as for those around the individual who has lost partial or complete sight.  Mr. Greenberg’s story in his words, reveals the raw emotion of blindness, the fear and angst as well as the compassion and love of those who travel through life alongside us.  At the end, there is also a surprise that will speak to aficionados of music and give some deeper insight into someone who has touched untold millions around the globe through their work and music.

This video is documentation of that event sent along for Webvision to share with you and the wider community by John Dowling the Chair of the Lasker/IRRF Initiative, and Janelia Farm who captured the video, the Lasker Foundation, the International Retinal Research Foundation and End Blindness by 2020.  We are grateful to Mr. Greenberg for sharing his story and allowing us to help spread his story and mission of ending blindness here on Webvision.

On a personal note: While I have friends who are blind or are going blind, Mr. Greenberg’s talk haunted me the night after I saw it, particularly because of the field of science I am engaged in.  Every scientist studying vision and diseases affecting vision should have the opportunity to spend time with those who have lost sight.  It is important for people in the sciences to sit down and talk with those affected by the disease they study.  I found this out this week after a meeting with a colleague who agreed to speak with a mutual friend who has Usher’s Syndrome.  When my colleague stated after the meeting that they had never actually sat down to talk with someone who has the disease that they study, I was initially surprised.  This is not uncommon though.  As scientists, not just in the vision sciences mind you, we obsess about the details of what we study and are absolutely driven by the work, but do not always look around and talk with people who’s diseases are the subjects of our studies.  This is fundamental to the process as it drives home the motivation for the long hours, late nights and frustrations with grant funding.  It forces an introspection and helps us to better communicate our work to a wider audience which is critical to science progress and funding.

 

 

Metabolic Changes Associated With Müller Cells In A Transgenic Rabbit Model Of Retinal Degeneration

Retina RLP

This abstract was presented today at the 2014 Association for Research in Vision and Opthalmology (ARVO) meetings in Orlando, Florida by  Rebecca L. Pfeiffer, Bryan W. Jones and Robert E. Marc.

Purpose: Müller cells play a central role in retinal metabolism via the glutamate cycle. During retinal degeneration Müller cells are among the first to demonstrate changes, reflected in alterations of metabolic signatures and morphology. The timing, extent and regulation of these changes is not fully characterized. To address this issue, we evaluated Müller cell metabolic phenotypes at multiple stages of retinal remodeling.

Methods: Samples were collected post-mortem from both WT and P347L rabbits. The retinas were then divided into fragments, fixed in buffered aldehydes, and embedded in epoxy resins. Tissues were sectioned at 200nm followed by classification with computational molecular phenotyping (CMP) using an array of small and macromolecular signatures (aspartate (D), glutamate (E), glycine (G), glutamine (Q), glutathione (J), GABA (yy), taurine (T), CRALBP, Glutamine Synthetase (GS), and GFAP). Levels of amino acid or protein were quantified by selecting a region of interest either within the Müller cell population or surrounding neurons and evaluating the intensity of the signal within that region.

Results: CMP reveals overall decreases in GS levels over the course of degeneration. Of notable importance, we saw that in regions of near complete photoreceptor loss neighboring Müller cells may express independent variation in metabolic signatures of E, Q, and GS. Also observed in these Müller cells, ratios of GS:E and GS:Q are not consistent with the ratios seen in WT retina. These results are inconsistent with the current models of both E to Q metabolism and microenvironment regulation of Müller cell phenotypes.

Conclusions: These observations indicate two conclusions. First, although the degenerate state of the retina is the likely trigger inducing Müller cells to express altered metabolic signatures, the rate at which the metabolic state changes is not purely a product of the surrounding environment, but also a stochastic change within individual Müller cells. Second, although it is commonly accepted that GS is the primary enzyme which converts Q to E as part of the glutamate cycle, in degenerate retina alternative pathways may be utilized following decrease in GS.

Support:  NIH EY02576 (RM), NIH EY015128 (RM), NSF 0941717 (RM), NIH EY014800 Vision Core (RM), RPB CDA (BWJ), Thome AMD Grant (BWJ).

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

No Slide Title

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.