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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.

Mechanosensitivity of the trabecular meshwork (TM) is a key determinant of intraocular pressure (IOP) yet our understanding of the molecular mechanisms that subserve it remains in its infancy. Here, we show that mechanosensitive Piezo1 channels modulate the TM pressure response via calcium signaling and dynamics of the conventional outflow pathway. Pressure steps evoked fast, inactivating cation currents and calcium signals that were inhibited by Ruthenium Red, GsMTx4 and Piezo1 shRNA. Piezo1 expression was confirmed by transcript and protein analysis, and by visualizing Yoda1-mediated currents and [Ca2+]i elevations in primary human TM cells. Piezo1 activation was obligatory for transduction of physiological shear stress and was coupled to reorganization of F-actin cytoskeleton and focal adhesions. The importance of Piezo1 channels as pressure sensors was shown by the GsMTx4 -dependence of the pressure-evoked current and conventional outflow function. We also demonstrate that Piezo1 collaborates with the stretch-activated TRPV4 channel, which mediated slow, delayed currents to pressure steps. 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 a novel potential therapeutic target for treating glaucoma.Significance StatementTrabecular meshwork (TM) is a highly mechanosensitive tissue in the eye that regulates intraocular pressure through the control of aqueous humor drainage. Its dysfunction underlies the progression of glaucoma but neither the mechanisms through which TM cells sense pressure nor their role in aqueous humor 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. These findings provide a new mechanistic framework for trabecular mechanotransduction and its role in the regulation of fast fluctuations in ocular pressure, as well as chronic remodeling of TM architecture that epitomizes glaucoma.

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 Muller 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 Muller 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.

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 [Ca2+]ielevations that were inhibited by TRPV4 antagonists and absent inTRPV4−/−Müller cells. Glial TRPV4 signals were phospholipase A2- and cytochrome P450-dependent, characterized by slow-onset and Ca2+waves, and, in excess, were sufficient to induce reactive gliosis. In contrast, neurons responded to TRPV4 agonists and swelling with fast, inactivating Ca2+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.

This study evaluated the capacity ofXenopus laevisretina 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.

Defining the master regulators of retinal wound-healing response is the holy grail of transcriptome-wide analyses. To predict regulatory networks, we integrated transcriptome-wide changes with genetic linkage analysis and bioinformatics. Our studies yielded three complementary insights. First, groups of functionally related genes underlie the early, delayed, and sustained responses of wound healing. For example, transcriptional factor upregulation define the early response, whereas glial reactive markers and crystallin family upregulation define the sustained response. Second, expression of a subset of neural development, proliferation, and cell survival genes displayed genetic linkage to a locus that includes positional candidate genes Id2 and Lpin1. Third, mice with a nonfunctional Lipin 1 protein displayed increased ganglion cell survival and crystallin expression after optic nerve crush. The Crystallin Network represents one of the first modulatory mechanisms predicted from expression data of injured retina, expression genetics, and bioinformatics.

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 p27 Kip1 (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.

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.

Purpose Muller glia are essential for maintaining retinal homeostasis and exhibit neuroprotective and deleterious responses during retinal degeneration. Having the ability to visualize and genetically manipulate Muller 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 Muller 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. Muller 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 Muller glia during their differentiation and in the healthy or degenerating adult retina.

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 candidate 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.