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.

Missense variant in CST3 Exerts Recessive Effect On Susceptibility to AMD


There is an interesting paper out demonstrating that CST3 exerts a recessive effect on susceptibility to AMD.  Cystatin C is a potent inhibitor of cysteine proteinases expressed by many tissues and in the eye, it is highly expressed by the retinal pigment epithelium (RPE). The team led by Luminita Paraoan recently reported data identifying a polymorphism in the cystatin C gene (CST3) that increases the risk of two major degenerative diseases, age-related macular degeneration (AMD) and Alzheimer’s disease.  Both these multifactorial diseases involve the age-related accumulation of extracellular deposits, linked to dysregulation of protein homeostasis. Since the advent of the genome-wide association study (GWAS) many SNPs have been found to be associated with these two diseases. However the SNP in CST3, which translates into an amino acid change in the leader sequence of the precursor protein, is the first identified to increase the risk of developing both diseases. Moreover the authors demonstrate that the risk associated with the mutant allele follows the same recessive model for both diseases. Thus only those individuals with two copies of the mutant cystatin allele are at elevated risk of developing both diseases.

Notable Paper: Reproducibility in Science

Reproducability in Science

This article by C. Glenn Begley and John P.A. Ioannidis is not specifically vision related, but is more generally applicable to research integrity and is well worth a read, in particular the following paragraph:

“What has shaken many in the field is not that investigators are unable to precisely reproduce an experiment. That is to be expected. What is shocking is that in many cases, the big idea or major conclusion was not confirmed simply when experi- ments were performed by the same investigators when blinded to their test samples versus control samples.2 The explanation for this was evident when the precise methodology of the experiments was reviewed. Investigators typically performed their experiments in a nonblinded fashion, so they were able to see what they were anticipating to see, and their research bias was thus able to be confirmed.18 Observer bias has long been recognized to be a problem in preclinical studies and beyond, so this result should not be surprising.19 Confirmation bias in scientific investigation unavoidably makes even the best scientists prone to try to find results or interpretations that fit their preconceived ideas and theories.20,21”


Continue reading “Notable Paper: Reproducibility in Science”

Notable Papers: An Easier Way To Make Stem Cells?

Stem Cells

We’ve talked about work out of the RIKEN Institute before here on Webvision.  The work that comes out of there is clever and often unconventional.  The latest work from the RIKEN that seems to have gotten a bit of attention is two recent papers from Haruko Obokata and colleagues, here and here that discuss almost inconceivably simple approaches to generating stem cells out of adult, differentiated blood, fat, muscle and brain mouse cells.  The approach essentially immerses the cells in an acidic solution for a half hour to induce a state of cell stress and subsequent induced pluripotency allowing them to recapitulate mature, differentiated tissues of a variety of cell types, including a reported blastocyst.  Granted, there are a variety of ways to collect and harvest stem cells, but these approaches present a variety of both ethical and logistical confounds.  So, this approach could solve a number of problems related to collecting and harvesting stem cells.

The other notable thing about this study is how elegant the screening method was.  The authors used GFP tagged Oct4 genes to note when cells had reached pluripotent status.  These GFP expressing cells were then indicators of induced pluripotency in harvested, differentiated cells that underwent stress through acid immersion.  Since only approximately 25% of the cells that underwent acid immersion survived, this approach allowed the investigators to see which of those remaining cells exhibited Oct4 gene expression revealing pluripotent stem cell status.  Other assays backed up these determinations of pluripotency including teratoma assays as well as the creation of chimeras.

If this approach proves a viable technique to generating stem cells, work in stem cell based vision rescuing therapeutics (as well as many other therapeutic applications) could be dramatically facilitated as labs exploring stem cell therapies in vision rescue are currently having to invest large efforts in using existing approved stem cell stocks or isolating stem cells through labor intensive methods.

Update 03/10/14:

The Wall St. Journal is reporting that one of the co-authors of this study said the research contained “crucial mistakes” and RIKEN is weighing whether to retract the papers.


Update 07/02/2014:

Nature has announced that the papers have been retracted.


Update 09/23/2015:

The STAP approach has been completely refuted.

Continue reading “Notable Papers: An Easier Way To Make Stem Cells?”

Notable Paper: Ras Pathway Inhibition Prevents Neovascularization By Repressing Endothelial Cell Sprouting

JCI cover

A new manuscript in the Journal of Clinical Investigation (on the cover) by Peter D. Westenskow, Toshihide Kurihara, Edith Aguilar, Elizabeth L. Scheppke, Stacey K. Moreno, Carli Wittgrove, Valentina Marchetti, Iacovos P. Michael, Sudarshan Anand, Andras Nagy, David Cheresh and Martin Friedlander at The Scripps Research Institute describes a new technique for treating aberrant growth of blood vessels in the retina from disease processes like “wet” macular degeneration and diabetic retinopathy, two leading causes of blindness.  The approach involves manipulating disease processes with short RNA strands that precisely target microRNAs (anti-microRNAs if you will) to stop the aberrant blood vessel grown without harming the existing retinal vasculature.  The results of this study showed that treatment with microRNAs “blocked aberrant vessel growth without damaging existing vasculature or neurons in three separate models of neovascular eye disease—a proof-of-principle that suggests future treatment based on the same approach may be effective in humans.”

Exciting news indeed.




Notable paper: Lhx2 Balances Progenitor Maintenance With Neurogenic Output And Promotes Competence State Progression In The Developing Retina

Levine Cover J Neurosci

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.

Notable Paper: Tuning The Electronic Absorption Of Protein-Embedded All-Trans-Retinal

Seeing red

The pigments in retinal photoreceptor cells absorb varying wavelengths of light, but the central chromophore, the molecule that actually absorbs the photon is identical.  Its ability to tune the responsiveness comes from the association with other opsins giving an organism the ability to respond from the near ultraviolet all the way to far red.  Interestingly, an article in Science by Wenjing Wang, Zahra Nossoni, Tetyana Berbasova, Camille T. Watson, Ipek Yapici, Kin Sing Stephen Lee, Chrysoula Vasileiou, James H. Geiger and Babak Borhan back in December demonstrates that you can generate “designer pigments” with a variety of applications through spectral tuning of the chromophore.

They way they went about this work was mutating human cellular retinol binding protein II (hCRPBII), a protein normally found in the gut that carries retinol.  Retinol (with an “o”) is very similar to retinal (with an “a”), but hCRPBII does not carry retinal.  So, they mutated hCRPBII so that it would bind and carry retinal (with an “a”).  At this point, the research group could start altering amino acids in the site that bound retinal to change the electrostatic charge for retinal and in doing so, created a set of pigments that absorbed pigments at differing wavelengths.  The really interesting thing is that there was no resulting substantial conformational change in retinal.  Rather, what changes is the distribution of charge that alters the responsiveness to wavelength which is kind of surprising, to me at least.  Its a pretty cool study and worth reading that may show the way to fabricating pigments with a variety of applications from transgenic work in optogenetics to labeling of specific cell classes to potential computational applications.

Image from:  Tuning the electronic absorption of protein-embedded all-trans-retinal. Science. 2012 Dec 7;338(6112):1340-3. doi: 10.1126/science.1226135.

Notable Paper: Paired-Pulse Plasticity In The Strength and Latency of Light-Evoked Lateral Inhibition to Retinal Bipolar Cell Terminals

Paired Pulse plasticity

I’ve been doing some reading in plasticity recently and found this paper in the Journal of Neuroscience by Evan Vickers, Mean-Hwan Kim, Jozsef Vigh, and Henrique von Gersdorff published last summer that looks at short term plasticity in the Inner Plexiform Layer mediating light adaptation.  Working in goldfish (Carassius auratus auratus) retina (an amazing retina), Vickers et. al. used patch clamp recordings on Mb bipolar cell terminals with paired-pulse light stimulation.  The idea was to examine and quantify plasticity in GABAergic lateral IPSCs with findings that show variation in the synaptic strength and latencies which correspond to adaptation and sensitization to surround temporal contrast.  The authors found that there are separate retinal circuitry pathways, each with differing mechanisms of plasticity that help to tune temporal response curves with glutamate release from ON bipolar cell terminals.  They conclude that “Short-term plasticity of L-IPSCs may thus influence the strength, timing, and spatial extent of amacrine and ganglion cell inhibitory surrounds”.


Notable Paper: On Cone Bipolar Cell Axonal Synapses In The OFF Inner Plexiform Layer Of The Rabbit Retina

JCN cover 2013

This paper in the Journal of Comparative Neurology by  J. Scott Lauritzen, James R. Anderson, Bryan W. Jones, Carl B. Watt, Shoeb Mohammed, John V. Hoang and Robert E. Marc is another effort out of the Marc Laboratory For Connectomics that continues to define complete neural circuits to completeness.

This paper is another elucidation of data from the first Rabbit Retinal Connectome volume (RC1) that reveals that the division between the ON and the OFF inner plexiform layer (IPL) is not structurally absolute. ON cone bipolar cells make noncanonical axonal synapses onto specific targets and receive amacrine cell synapses in the nominal OFF layer, creating novel motifs, including inhibitory crossover networks. Automated transmission electron microscopic imaging, molecular tagging, tracing, and rendering of ∼400 bipolar cells reveals axonal ribbons in 36% of ON cone bipolar cells, throughout the OFF IPL. The targets include γ-aminobutyrate (GABA)-positive amacrine cells (γACs), glycine-positive amacrine cells (GACs), and ganglion cells. Most ON cone bipolar cell axonal contacts target GACs driven by OFF cone bipolar cells, forming new architectures for generating ON–OFF amacrine cells. Many of these ON–OFF GACs target ON cone bipolar cell axons, ON γACs, and/or ON–OFF ganglion cells, representing widespread mechanisms for OFF to ON crossover inhibition. Other targets include OFF γACs presynaptic to OFF bipolar cells, forming γAC-mediated crossover motifs. ON cone bipolar cell axonal ribbons drive bistratified ON–OFF ganglion cells in the OFF layer and provide ON drive to polarity-appropriate targets such as bistratified diving ganglion cells (bsdGCs). The targeting precision of ON cone bipolar cell axonal synapses shows that this drive incidence is necessarily a joint distribution of cone bipolar cell axonal frequency and target cell trajectories through a given volume of the OFF layer. Such joint distribution sampling is likely common when targets are sparser than sources and when sources are coupled, as are ON cone bipolar cells.

Figure Above: The first Rabbit Retinal Connectome volume (RC1), constructed via automated transmission electron microscopy (ATEM) and computational molecular phenotyping (CMP), spans the mid-inner nuclear layer (INL) at section 001 to the ganglion cell layer (GCL) at section 371, shown in a mirror image below. RC1 is a short cylinder ≈ 250 μm in diameter and ≈ 30 μm high containing 341 ATEM sections and 11 intercalated CMP sections. The cylinder is capped at top and bottom with 10-section CMP series allowing molecular segmentation of cells, and an activity marker, 1-amino-4-guanidobutane (AGB), to mark cells differentially stimulated via glutamatergic synapses. ATEM section 001 is a horizontal plane section through the INL visualized with GABA.glycine.glutamate → transparency mapping and a dark gold alpha channel (ANDed taurine + glutamine channels). ATEM section 371 is a horizontal plane section through the GCL visualized with GABA.AGB.glutamate → transparency mapping.

Notable Paper: Photomechanical Responses In Drosophila Photoreceptors

This notable paper by Roger C. Hardie and Kristian Franze looks at phototransduction in the fruit fly, Drosophila melanogaster.  Drosophila melanogaster has a long history in vision research of informing our understanding of the biochemical processes involved in phototransduction going back almost 40 years to this paper.  However, the Hardie and Franze paper looks at transient receptor potential (TRP) channels, which coincidently were also first found in Drosophila in a series of papers from Montel and Rubin, Hardie and Minke and Niemeyer et. al. and have proved themselves to be fairly ubiquitous and involved in wide areas of cell function from growth cones to cellular guidance and chemotaxis.  As expected, there are also many other areas where TRP channels are involved in the retina including store operated calcium channels and functions in ganglion cell calcium modulation, spiking rate and apoptosis.

This particular paper answers a fundamental question of how light functionally opens the TRP channels.  It turns out that there is a surprising mechanical force that is created that then generates an electrical response far quicker than using standard chemical second messenger based systems seen in mammalians.  Very interesting…



Image Credit: Bbski on Wikipedia

Intrinsic Physiological Properties Of Rat Retinal Ganglion Cells With A Comparative Analysis

This paper in the Journal of Neurophysiology by Raymond C S Wong, Shaun L Cloherty, Michael R Ibbotson and Brendan J O’Brien examined properties of retinal ganglion cells types that are conserved through mammalian species by looking at 16 morphologically defined rat and cat retinal ganglion cell types.  Their work demonstrates morphologically distinct retinal ganglion cell types in rat that have homologs in cat retinal ganglion cells, but more importantly also have other intrinsic physiological features that both correlate function and also suggest variations that reflect the species individual environmental and behavioral demands. Continue reading “Intrinsic Physiological Properties Of Rat Retinal Ganglion Cells With A Comparative Analysis”

Notable Paper: Molecular Analysis Of The Amphioxus Frontal Eye…

We at Webvision have a fascination with the evolution of the eye and are always looking for interesting papers that help describe from where, how and when vision came.  This paper, Molecular analysis of the amphioxus frontal eye unravels the evolutionary origin of the retina and pigment cells of the vertebrate eye by Pavel Vopalenskya, Jiri Pergnera, Michaela Liegertova, Elia Benito-Gutierrez, Detlev Arendt, and Zbynek Kozmika attempts to answer the question of where the vertebrate eye came from.  While the amphioxus has for many years been recognized as a viable candidate for the earliest vertebrate eye, the retinal structure is different from that of other vertebrates.  Specifically, the photoreceptors of amphioxus are simple ciliated cells as opposed to the more sophisticated elaborations of structures on top of cillia in other vertebrate retinas.  This paper describes in molecular terms, gene coexpression and structural features the different cell types of amphioxus in an attempt to define neuronal circuitry.  Its a very cool paper that provides additional detail into the evolutionary origins of vision and we encourage you to have a look.

Image Credit: Hans Hillewaert from Wikipedia


Notable Paper: The Molecular Mechanism of Thermal Noise in Rod Photoreceptors


Phototransduction is the process by which photon capture by opsins in photoreceptors is transduced into a neural signal.    However, there are limits on visual sensitivity that are imposed by thermal means as opposed to the photochemical mechanisms resulting in activation of the phototransduction cascade.

The mechanism of this limit has long been a matter of debate, however this paper by Samer Gozem, Igor Schapiro, Nicolas Ferré and Massimo Olivucci demonstrates a mechanism.  Mechanistically, the authors examined the maximum absorption wavelength (λmax) and the thermal activation kinetic constant (k) of different visual pigments which indicates that the thermal and photochemical activations are related.  The authors found that rod opsin or rhodopsin possesses a transition state for thermal activation that has the same electronic structure as it does for photo-excitation.  This results in spontaneous and random signals being generated in the rod photoreceptors that impose limits on visual sensitivity.   Continue reading “Notable Paper: The Molecular Mechanism of Thermal Noise in Rod Photoreceptors”

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”