Emergent Temporal Signaling in Human Trabecular Meshwork Cells: Role of TRPV4-TRPM4 Interactions.

Trabecular meshwork (TM) cells are phagocytic cells that employ mechanotransduction to actively regulate intraocular pressure. Similar to macrophages, they express scavenger receptors and participate in antigen presentation within the immunosuppressive milieu of the anterior eye. Changes in pressure deform and compress the TM, altering their control of aqueous humor outflow but it is not known whether transducer activation shapes temporal signaling. The present study combines electrophysiology, histochemistry and functional imaging with gene silencing and heterologous expression to gain insight into Ca2+ signaling downstream from TRPV4 (Transient Receptor Potential Vanilloid 4), a stretch-activated polymodal cation channel. Human TM cells respond to the TRPV4 agonist GSK1016790A with fluctuations in intracellular Ca2+ concentration ([Ca2+]i) and an increase in [Na+]i. [Ca2+]i oscillations coincided with monovalent cation current that was suppressed by BAPTA, Ruthenium Red and the TRPM4 (Transient Receptor Potential Melastatin 4) channel inhibitor 9-phenanthrol. TM cells expressed TRPM4 mRNA, protein at the expected 130-150 kDa and showed punctate TRPM4 immunoreactivity at the membrane surface. Genetic silencing of TRPM4 antagonized TRPV4-evoked oscillatory signaling whereas TRPV4 and TRPM4 co-expression in HEK-293 cells reconstituted the oscillations. Membrane potential recordings suggested that TRPM4-dependent oscillations require release of Ca2+ from internal stores. 9-phenanthrol did not affect the outflow facility in mouse eyes and eyes from animals lacking TRPM4 had normal intraocular pressure. Collectively, our results show that TRPV4 activity initiates dynamic calcium signaling in TM cells by stimulating TRPM4 channels and intracellular Ca2+ release. It is possible that TRPV4-TRPM4 interactions downstream from the tensile and compressive impact of intraocular pressure contribute to homeostatic regulation and pathological remodeling within the conventional outflow pathway.

Piezo1 channels mediate trabecular meshwork mechanotransduction and promote aqueous fluid outflow.

KEY POINTS: Trabecular meshwork (TM) is a highly mechanosensitive tissue in the eye that regulates intraocular pressure through the control of aqueous humour drainage. Its dysfunction underlies the progression of glaucoma but neither the mechanisms through which TM cells sense pressure nor their role in aqueous humour outflow are understood at the molecular level. We identified the Piezo1 channel as a key TM transducer of tensile stretch, shear flow and pressure. Its activation resulted in intracellular signals that altered organization of the cytoskeleton and cell-extracellular matrix contacts and modulated the trabecular component of aqueous outflow whereas another channel, TRPV4, mediated a delayed mechanoresponse. This study helps elucidate basic mechanotransduction properties that may contribute to intraocular pressure regulation in the vertebrate eye. ABSTRACT: Chronic elevations in intraocular pressure (IOP) can cause blindness by compromising the function of trabecular meshwork (TM) cells in the anterior eye, but how these cells sense and transduce pressure stimuli is poorly understood. Here, we demonstrate functional expression of two mechanically activated channels in human TM cells. Pressure-induced cell stretch evoked a rapid increase in transmembrane current that was inhibited by antagonists of the mechanogated channel Piezo1, Ruthenium Red and GsMTx4, and attenuated in Piezo1-deficient cells. The majority of TM cells exhibited a delayed stretch-activated current that was mediated independently of Piezo1 by TRPV4 (transient receptor potential cation channel, subfamily V, member 4) channels. Piezo1 functions as the principal TM transducer of physiological levels of shear stress, with both shear and the Piezo1 agonist Yoda1 increasing the number of focal cell-matrix contacts. Analysis of TM-dependent fluid drainage from the anterior eye showed significant inhibition by GsMTx4. Collectively, these results suggest that TM mechanosensitivity utilizes kinetically, regulatory and functionally distinct pressure transducers to inform the cells about force-sensing contexts. Piezo1-dependent control of shear flow sensing, calcium homeostasis, cytoskeletal dynamics and pressure-dependent outflow suggests potential for a novel therapeutic target in treating glaucoma.

Dyslipidemia modulates Müller glial sensing and transduction of ambient information.

Unesterified cholesterol controls the fluidity, permeability and electrical properties of eukaryotic cell membranes. Consequently, cholesterol levels in the retina and the brain are tightly regulated whereas depletion or oversupply caused by diet or heredity contribute to neurodegenerative diseases and vision loss. Astroglia play a central role in the biosynthesis, uptake and transport of cholesterol and also drive inflammatory signaling under hypercholesterolemic conditions associated with high-fat diet (diabetes) and neurodegenerative disease. A growing body of evidence shows that unesterified membrane cholesterol modulates the ability of glia to sense and transduce ambient information. Cholesterol-dependence of Müller glia - which function as retinal sentinels for metabolic, mechanical, osmotic and inflammatory signals - is mediated in part by transient receptor potential V4 (TRPV4) channels. Cholesterol supplementation facilitates, whereas depletion suppresses, TRPV4-mediated transduction of temperature and lipid agonists in Müller cells. Acute effects of cholesterol supplementation/depletion on plasma membrane ion channels and calcium homeostasis differ markedly from the effects of chronic dyslipidemia, possibly due to differential modulation of modality-dependent energy barriers associated with the functionality of polymodal channels embedded within lipid rafts. Understanding of cholesterol-dependence of TRP channels is thus providing insight into dyslipidemic pathologies associated with diabetic retinopathy, glaucoma and macular degeneration.

Swelling and eicosanoid metabolites differentially gate TRPV4 channels in retinal neurons and glia.

Activity-dependent shifts in ionic concentrations and water that accompany neuronal and glial activity can generate osmotic forces with biological consequences for brain physiology. Active regulation of osmotic gradients and cellular volume requires volume-sensitive ion channels. In the vertebrate retina, critical support to volume regulation is provided by Müller astroglia, but the identity of their osmosensor is unknown. Here, we identify TRPV4 channels as transducers of mouse Müller cell volume increases into physiological responses. Hypotonic stimuli induced sustained [Ca(2+)]i elevations that were inhibited by TRPV4 antagonists and absent in TRPV4(-/-) Müller cells. Glial TRPV4 signals were phospholipase A2- and cytochrome P450-dependent, characterized by slow-onset and Ca(2+) waves, and, in excess, were sufficient to induce reactive gliosis. In contrast, neurons responded to TRPV4 agonists and swelling with fast, inactivating Ca(2+) signals that were independent of phospholipase A2. Our results support a model whereby swelling and proinflammatory signals associated with arachidonic acid metabolites differentially gate TRPV4 in retinal neurons and glia, with potentially significant consequences for normal and pathological retinal function.

Dysmorphic photoreceptors in a P23H mutant rhodopsin model of retinitis pigmentosa are metabolically active and capable of regenerating to reverse retinal degeneration.

This study evaluated the capacity of Xenopus laevis retina to regenerate photoreceptor cells after cyclic light-mediated acute rod photoreceptor degeneration in a transgenic P23H mutant rhodopsin model of retinits pigmentosa. After discontinuation of cyclic light exposure, we monitored histologic progression of retinal regeneration over a 3 week recovery period. To assess their metabolomic states, contralateral eyes were processed for computational molecular phenotyping. We found that retinal degeneration in the P23H rhodopsin mutation could be partially reversed, with regeneration of rod photoreceptors recovering normal morphology (including full-length rod outer segments) by the end of the 3 week recovery period. In contrast, retinal degeneration mediated by directly induced apoptosis did not recover in the 3 week recovery period. Dystrophic rod photoreceptors with truncated rod outer segments were identified as the likely source of rod photoreceptor regeneration in the P23H retinas. These dystrophic photoreceptors remain metabolically active despite having lost most of their outer segments.

Rapid glutamate receptor 2 trafficking during retinal degeneration.

BACKGROUND: Retinal degenerations, such as age-related macular degeneration (AMD) and retinitis pigmentosa (RP), are characterized by photoreceptor loss and anomalous remodeling of the surviving retina that corrupts visual processing and poses a barrier to late-stage therapeutic interventions in particular. However, the molecular events associated with retinal remodeling remain largely unknown. Given our prior evidence of ionotropic glutamate receptor (iGluR) reprogramming in retinal degenerations, we hypothesized that the edited glutamate receptor 2 (GluR2) subunit and its trafficking may be modulated in retinal degenerations. RESULTS: Adult albino Balb/C mice were exposed to intense light for 24 h to induce light-induced retinal degeneration (LIRD). We found that prior to the onset of photoreceptor loss, protein levels of GluR2 and related trafficking proteins, including glutamate receptor-interacting protein 1 (GRIP1) and postsynaptic density protein 95 (PSD-95), were rapidly increased. LIRD triggered neuritogenesis in photoreceptor survival regions, where GluR2 and its trafficking proteins were expressed in the anomalous dendrites. Immunoprecipitation analysis showed interaction between KIF3A and GRIP1 as well as PSD-95, suggesting that KIF3A may mediate transport of GluR2 and its trafficking proteins to the novel dendrites. However, in areas of photoreceptor loss, GluR2 along with its trafficking proteins nearly vanished in retracted retinal neurites. CONCLUSIONS: All together, LIRD rapidly triggers GluR2 plasticity, which is a potential mechanism behind functionally phenotypic revisions of retinal neurons and neuritogenesis during retinal degenerations.

Proliferative reactive gliosis is compatible with glial metabolic support and neuronal function.

BACKGROUND: The response of mammalian glial cells to chronic degeneration and trauma is hypothesized to be incompatible with support of neuronal function in the central nervous system (CNS) and retina. To test this hypothesis, we developed an inducible model of proliferative reactive gliosis in the absence of degenerative stimuli by genetically inactivating the cyclin-dependent kinase inhibitor p27Kip1 (p27 or Cdkn1b) in the adult mouse and determined the outcome on retinal structure and function. RESULTS: p27-deficient Müller glia reentered the cell cycle, underwent aberrant migration, and enhanced their expression of intermediate filament proteins, all of which are characteristics of Müller glia in a reactive state. Surprisingly, neuroglial interactions, retinal electrophysiology, and visual acuity were normal. CONCLUSION: The benign outcome of proliferative reactive Müller gliosis suggests that reactive glia display context-dependent, graded and dynamic phenotypes and that reactivity in itself is not necessarily detrimental to neuronal function.

Differential response of C57BL/6J mouse and DBA/2J mouse to optic nerve crush.

BACKGROUND: Retinal ganglion cell (RGC) death is the final consequence of many blinding diseases, where there is considerable variation in the time course and severity of RGC loss. Indeed, this process appears to be influenced by a wide variety of genetic and environmental factors. In this study we explored the genetic basis for differences in ganglion cell death in two inbred strains of mice. RESULTS: We found that RGCs are more susceptible to death following optic nerve crush in C57BL/6J mice (54% survival) than in DBA/2J mice (62% survival). Using the Illumina Mouse-6 microarray, we identified 1,580 genes with significant change in expression following optic nerve crush in these two strains of mice. Our analysis of the changes occurring after optic nerve crush demonstrated that the greatest amount of change (44% of the variance) was due to the injury itself. This included changes associated with ganglion cell death, reactive gliosis, and abortive regeneration. The second pattern of gene changes (23% of the variance) was primarily related to differences in gene expressions observed between the C57BL/6J and DBA/2J mouse strains. The remaining changes in gene expression represent interactions between the effects of optic nerve crush and the genetic background of the mouse. We extracted one genetic network from this dataset that appears to be related to tissue remodeling. One of the most intriguing sets of changes included members of the crystallin family of genes, which may represent a signature of pathways modulating the susceptibility of cells to death. CONCLUSION: Differential responses to optic nerve crush between two widely used strains of mice were used to define molecular networks associated with ganglion cell death and reactive gliosis. These results form the basis for our continuing interest in the modifiers of retinal injury.

Rlbp1 promoter drives robust Müller glial GFP expression in transgenic mice.

PURPOSE: Müller glia are essential for maintaining retinal homeostasis and exhibit neuroprotective and deleterious responses during retinal degeneration. Having the ability to visualize and genetically manipulate Müller glia in vivo will facilitate a better understanding of how these cells contribute to these processes. The goal of this study was to determine whether regulatory elements of the retinaldehyde binding protein 1 (Rlbp1; formerly Cralbp) gene can drive robust Müller glial gene expression in vivo. METHODS: Transgenic mice were generated by pronuclear injection of a construct carrying a 3-kilobase (kb) region of the Rlbp1 gene and 5'-flanking sequences linked to the enhanced green fluorescent protein (GFP) cDNA. GFP expression was analyzed by immunohistology in regions of the central nervous system in which RLBP1 protein is expressed, in retinas from wild-type and retinal degeneration 1 (rd1) mice, and during retinal development. RESULTS: Three transgenic lines were generated, and the one with the strongest and most consistent GFP expression was characterized further. Müller glia displayed robust GFP expression at all postnatal developmental stages and in the rd1 retina. Onset of expression occurred by birth in retinal progenitor cells. CONCLUSIONS: Regulatory elements in a restricted region of the Rlbp1 gene are sufficient to drive GFP expression in vivo. This transgenic line provides robust GFP expression that can be used to visualize retinal progenitor cells during postnatal development and Müller glia during their differentiation and in the healthy or degenerating adult retina.

Genomic loci modulating the retinal transcriptome in wound healing.

PURPOSE: The present study predicts and tests genetic networks that modulate gene expression during the retinal wound-healing response. METHODS: Upstream modulators and target genes were defined using meta-analysis and bioinformatic approaches. Quantitative trait loci (QTLs) for retinal acute phase genes (Vazquez-Chona et al. 2005) were defined using QTL analysis of CNS gene expression (Chesler et al. 2005). Candidate modulators were defined using computational analysis of gene and motif sequences. The effect of candidate genes on wound healing was tested using animal models of gene expression. RESULTS: A network of early wound-healing genes is modulated by a locus on chromosome 12. The genetic background of the locus altered the wound-healing response of the retina. The C57BL/6 allele conferred enhanced expression of neuronal marker Thy1 and heat-shock-like crystallins, whereas the DBA/2J allele correlated with greater levels of the classic marker of retinal stress, glial fibrillary acidic protein (GFAP). Id2 and Lpin1 are candidate upstream modulators as they strongly correlated with the segregation of DBA/2J and C57BL/6 alleles, and their dosage levels correlated with the enhanced expression of survival genes (Thy1 and crystallin genes). CONCLUSION: We defined a genetic network associated with the retinal acute injury response. Using genetic linkage analysis of natural transcript variation, we identified regulatory loci and can didate modulators that control transcript levels of acute phase genes. Our results support the convergence of gene expression profiling, QTL analysis, and bioinformatics as a rational approach to discover molecular pathways controlling retinal wound healing.

Genetic networks controlling retinal injury.

PURPOSE: The present study defines genomic loci underlying coordinate changes in gene expression following retinal injury. METHODS: A group of acute phase genes expressed in diverse nervous system tissues was defined by combining microarray results from injury studies from rat retina, brain, and spinal cord. Genomic loci regulating the brain expression of acute phase genes were identified using a panel of BXD recombinant inbred (RI) mouse strains. Candidate upstream regulators within a locus were defined using single nucleotide polymorphism databases and promoter motif databases. RESULTS: The acute phase response of rat retina, brain, and spinal cord was dominated by transcription factors. Three genomic loci control transcript expression of acute phase genes in brains of BXD RI mouse strains. One locus was identified on chromosome 12 and was highly correlated with the expression of classic acute phase genes. Within the locus we identified the inhibitor of DNA binding 2 (Id2) as a candidate upstream regulator. Id2 was upregulated as an acute phase transcript in injury models of rat retina, brain, and spinal cord. CONCLUSIONS: We defined a group of transcriptional changes associated with the retinal acute injury response. Using genetic linkage analysis of natural transcript variation, we identified regulatory loci and candidate regulators that control transcript levels of acute phase genes.

Temporal changes in gene expression after injury in the rat retina.

PURPOSE: The goal of this study was to define the temporal changes in gene expression after retinal injury and to relate these changes to the inflammatory and reactive response. A specific emphasis was placed on the tetraspanin family of proteins and their relationship with markers of reactive gliosis. METHODS: Retinal tears were induced in adult rats by scraping the retina with a needle. After different survival times (4 hours, and 1, 3, 7, and 30 days), the retinas were removed, and mRNA was isolated, prepared, and hybridized to the Affymatrix RG-U34A microarray (Santa Clara, CA). Microarray results were confirmed by using RT-PCR and correlation to protein levels was determined. RESULTS: Of the 8750 genes analyzed, approximately 393 (4.5%) were differentially expressed. Clustering analysis revealed three major profiles: (1) The early response was characterized by the upregulation of transcription factors; (2) the delayed response included a high percentage of genes related to cell cycle and cell death; and (3) the late, sustained profile clustered a significant number of genes involved in retinal gliosis. The late, sustained cluster also contained the upregulated crystallin genes. The tetraspanins Cd9, Cd81, and Cd82 were also associated with the late, sustained response. CONCLUSIONS: The use of microarray technology enables definition of complex genetic changes underlying distinct phases of the cellular response to retinal injury. The early response clusters genes associate with the transcriptional regulation of the wound-healing process and cell death. Most of the genes in the late, sustained response appear to be associated with reactive gliosis.

Temporal regulation of CD81 following retinal injury in the rat.

Following retinal injury, glial cells within the retina undergo a response that is characterized by the proliferation of astrocytes, Müller cells, and retinal pigment epithelial cells. CD81, a small membrane protein known to be involved in cell proliferation, is up-regulated after injury. This study focuses on the temporal regulation of CD81, relating the expression of this protein to glial fibrillary acidic protein (GFAP), the classic marker of gliosis. CD81 levels were elevated at 7 days after injury and remained elevated at 30 days after injury; GFAP was increased at 7 days and continued to increase until 30 days post injury. This association of CD81 with glial reactivity may provide a clue to the regulation of the proliferative response following retinal injury.