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.

Store-Operated Calcium Entry In Müller Glia Is Controlled By Synergistic Activation Of TRPC And Orai Channels

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There is a new publication out in the Journal of Neuroscience (cover story) from Moran Eye Center scientists, Store-Operated Calcium Entry In Müller Glia Is Controlled By Synergistic Activation Of TRPC And Orai Channels authored by Tünde Molnár, Oleg YarishkinPeter Barabas, Anthony Iuso, Bryan William JonesRobert Marc, Tam Phuong, and David Krizaj.

Continue reading “Store-Operated Calcium Entry In Müller Glia Is Controlled By Synergistic Activation Of TRPC And Orai Channels”

Müller Cell Metabolic Chaos During Degeneration

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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.

Moran Eye Center Researcher Robert E. Marc: 2014 Paul Kayser Award In Retina Research

Our colleague and Director of Research at the University of Utah‘s Moran Eye Center, Robert E. Marc, Ph.D. has been named by the International Society for Eye Research as a recipient of the Paul Kayser International Award in Retina Research.  The award will be presented to Dr. Marc during the 2014 ISER Biennial Meeting of the Society for Eye Research in San Francisco, California on Thursday, July 24th, 2014.

The Paul Kayser International Award in Retina Research was created by the Directors of Retina Research Foundation and endowed by the Trustees of The Kayser Foundation to honor and perpetuate the memory of long-time friend and dedicated benefactor of RRF, Paul Kayser. Through this award both organizations are demonstrating the conviction they shared with Mr. Kayser that blindness caused by retinal disease is a global concern and must be addressed accordingly. It is thus the purpose of this award to foster greater awareness of the need for intensive study of the retina, its role in the visual process, and the retinal diseases that threaten and/or destroy eyesight by recognizing outstanding achievement and sustaining meritorious scientific investigations worldwide.

Dr. Marc was chosen as the recipient of this award for his lifetime body of work in retinal research, discovering the structure and function of the retina through novel technologies and approaches that have pushed our understanding of the retina forward.

Thy-1 Expressed In CNV And Increased In migrating Choroidal ECs In Human Neovascular AMD

Haibo Wang

This abstract was presented today at the 2014 Association for Research in Vision and Opthalmology (ARVO) meetings in Orlando, Florida by Haibo Wang, Yanchao Jiang and M. Elizabeth Hartnett.

Thy-1 regulates VEGF-induced choroidal endothelial cell migration 

Dept of Ophthalmology, John A. Moran Eye Center, The University of Utah-Salt Lake City. 

Purpose: Choroidal endothelial cell (CEC) activation and migration precede the development of choroidal neovascularization in neovascular AMD. Thy-1 is a cell surface protein expressed on different cells, including neurons and endothelial cells. As a glycosylphosphatidylinositol (GPI)-anchored glycoprotein, Thy-1 is located in lipid raft microdomains within the cell membrane, which brings Thy-1 into proximity of signaling molecules including cytoplasmic tyrosine kinases that can modulate adhesive and migratory events. In the retina, Thy-1 is well known as a retinal ganglion cell marker. Given the possibility that Thy-1 might be expressed in CECs, we addressed the hypothesis that upregulated Thy-1 in CECs by age-related stresses contributes to CEC migration.

Methods: Western blots of Thy-1 were determined in retinal pigment epithelial cells (RPE) and CECs. By real time quantitative PCR, Thy-1 mRNA was measured in CECs treated with vascular endothelial growth factor (VEGF) (20 ng/ml), CCL11 (100 ng/ml) or PBS for 24 hours, or in RPE/choroids from young (<40 yrs) and old (>60 yrs) donor eyes. Immunohistochemistry of Thy-1 was performed in posterior globe sections of human retina/RPE/choroids in the maculas of young and old donor eyes with or without AMD.  Colabeling with VE-cadherin was used to identify CECs. CECs transfected with Thy-1 siRNA or control siRNA were stimulated with VEGF, and CEC migration and phosphorylation of VEGF receptor 2 (VEGFR2) were measured. Statistics were performed using ANOVA.

Results:  Thy-1was highly expressed in CECs but not in RPE.  Thy-1 staining was not only detected in the retinal ganglion cell layer, but also in the choroid, and colocalized to a greater extent with VE-cadherin labeled CECs in sections from donors with AMD compared to age-matched controls without AMD.  Thy-1 mRNA was significantly increased in CECs treated with VEGF or CCL11 (p<0.05 vs. PBS) and greater in RPE/choroids from aged donor eyes (p<0.001 vs. young).  Knockdown of Thy-1 in CECs by siRNA transfection significantly inhibited VEGF-induced CEC migration (p<0.001) and VEGFR2 activation.

Conclusions: Thy-1is expressed in CECs and its expression is upregulated by stresses associated with neovascular AMD, including elderly age and increased VEGF.  Upregulated Thy-1 in CECs contributes to VEGF-induced VEGFR2 activation and CEC migration. Future studies into the potential role of Thy-1 in neovascular AMD are being considered. 

Müller Cell-derived VEGF164 Knockdown Reduces Retinal Neovascularization In A Rat Model Of Retinopathy Of Prematurity

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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  Deeksha Gambhir, Yanchao Jiang, George W. Smith, Zhihong Yang, Haibo Wang, Lori Fotheringham and M. Elizabeth Hartnett.

Full size poster available here.

Continue reading “Müller Cell-derived VEGF164 Knockdown Reduces Retinal Neovascularization In A Rat Model Of Retinopathy Of Prematurity”

A Study of PDZ Domain-Containing 7 (PDZD7) in the Mouse Retina

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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  Junhuang Zou, Tihua Zheng and Jun Yang.

Full size poster can be downloaded here.

Purpose: PDZD7 is a newly identified modifier and contributor gene of Usher syndrome (USH). In the inner ear, PDZD7 colocalizes with GPR98, an USH2C protein, at ankle links in cochlear and vestibular hair cells. Therefore, PDZD7 is proposed to be a novel component of the USH2 complex, which is composed of the three known USH2 causative proteins. In this study, we investigated PDZD7 expression and its role in the organization of the USH2 complex in the retina.

Methods: The expression of Pdzd7 was examined at the mRNA and protein levels using RT-PCR, western blotting, and immunostaining assays. A Pdzd7 knockout mouse was generated by gene trapping and characterized phenotypically by immunostaining and electroretinogram.
Results: Five Pdzd7 splice variants were identified from 19 independent RT-PCR clones in adult mouse retinas. All of them are predicted as N-terminal but not full-length Pdzd7 isoforms. At the protein level, full-length Pdzd7 was found in the mouse retina during postnatal development. However, no Pdzd7 protein expression could be detected in adulthood by either western blotting or immunostaining. Pdzd7 knockout mice showed close to normal distribution of USH2A, GPR98 and WHRN at the periciliary membrane complex in photoreceptors. The knockout mice exhibited normal ERG responses at one month of age.

Conclusions: Despite the existence of multiple splice variants, PDZD7 expression at the protein level is very low in the retina. PDZD7 is not as important as WHRN in organizing the USH2 complex in photoreceptors.

A Synaptic Basis for Small World Network Design in the ON Inner Plexiform Layer of the Rabbit Retina

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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 J Scott Lauritzen, Noah T. Nelson, Crystal L. Sigulinsky, Nathan Sherbotie, John Hoang, Rebecca L. PfeifferJames R. Anderson, Carl B. Watt, Bryan W. Jones and Robert E. Marc.

Purpose: Converging evidence suggests that large- and intermediate-scale neural networks throughout the nervous system exhibit small world’ design characterized by high local clustering of connections yet short path length between neuronal modules (Watts & Strogatz 1998 Nature; Sporns et al.2004 Trends in Cog Sci). It is suspected that this organizing principle scales to local networks (Ganmor et al. 2011 J Neurosci; Sporns 2006 BioSystems) but direct observation of synapses and local network topologies mediating small world design has not been achieved in any neuronal tissue. We sought direct evidence for synaptic and topological substrates that instantiate small world network architectures in the ON inner plexiform layer (IPL) of the rabbit retina. To test this we mined ≈ 200 ON cone bipolar cells (BCs) and ≈ 500 inhibitory amacrine cell (AC) processes in the ultrastructural rabbit retinal connectome (RC1).

Methods: BC networks in RC1 were annotated with the Viking viewer and explored via graph visualization of connectivity and 3D rendering (Anderson et al. 2011 J Microscopy). Small molecule signals embedded in RC1 e.g. GABA glycine and L-glutamate combined with morphological reconstruction and connectivity analysis allow for robust cell classification. MacNeil et al. (2004 J Comp Neurol) BC classification scheme used for clarity.

Results: Homocellular BC coupling (CBb3::CBb3 CBb4::CBb4 CBb5::CBb5) and within-class BC inhibitory networks (CBb3 → AC –| CBb3 CBb4 → AC –| CBb4 CBb5 → AC –| CBb5) in each ON IPL strata form laminar-specific functional sheets with high clustering coefficients. Heterocellular BC coupling (CBb3::CBb4 CBb4::CBb5 CBb3::CBb5) and cross-class BC inhibitory networks (CBb3 → AC –| CBb4 CBb4 → AC –| CBb3 CBb4 → AC –| CBb5 CBb5 → AC –| CBb4 CBb3 → AC –| CBb5 CBb5 → AC –| CBb3) establish short synaptic path lengths across all ON IPL laminae.

Conclusions: The retina contains a greater than expected number of synaptic hubs that multiplex parallel channels presynaptic to ganglion cells. The results validate a synaptic basis (ie. direct synaptic connectivity) and local network topology for the small world architecture indicated at larger scales providing neuroanatomical plausibility of this organization for local networks and are consistent with small world design as a fundamental organizing principle of neural networks on multiple spatial scales.

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

The Role of NMDA Receptor Activity in Retinal Ganglion Cell Dendrite Development

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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 Eerik M. Elias, Ping Wang and Ning Tian.

Full size poster available here.

Purpose: To elucidate mechanisms underlying the dendrite developmental plasticity of retinal ganglion cells, we examined the role of glutamate receptors on retinal ganglion cell dendrite elongation and filopodia elimination.

Methods: We used the JamB genetically labeled subtype of RGCs as our working model. JamB-CreER:YFP ganglion cell dendritic arbors were imaged in whole mount retina using confocal microscopy. Dendrite length, area, branching, and filopodia number were traced and measured using Neurolucida. Visual inputs were blocked by dark-rearing pups after P5. Glutamatergic activity was blocked using daily intraocular injections of AP5 and CNQX from P9 to P13 or genetic ablation of the NMDA receptor in these RGCs.

Results: To test the role of visual inputs on dendrite development, we dark-reared mice from P5 to P30 and found a modest effect on filopodia elimination in JamB RGCs. Anticipating that spontaneous glutamatergic activity in the retina may also contribute to RGC filopodia elimination, we blocked spontaneous glutamatergic activity by daily intraocular injections of AP5 and CNQX from P9 to P13. This led to an increase in filopodia density due to decreased dendrite length but no change in filopodia number. We confirmed this result by examining NMDAR knockout JamB cells (JamB-CreER:YFP:Grin1-/-). As expected, Grin1-/- JamB RGCs have decreased dendrite outgrowth like the pharmacologic blockade. However, filopodia elimination in these cells was significantly decreased as well, suggesting that NMDA and non-NMDA glutamate receptors might regulate the RGC dendritic development in a differential manner. This effect was dramatic at P13. To test if this effect persists into adulthood, we examined Grin1-/- JamB RGCs at P30 and found that they are indistinguishable from wild-type JamB RGCs, suggesting that a compensatory mechanism exists to drive dendrite elongation and filopodia elimination in the absence of the NMDA receptor.

Conclusions: Our study demonstrated that ganglion cell dendrite outgrowth and pruning of filopodia require glutamatergic activity and visual input that act via NMDA and possibly non-NMDA glutamate receptors.