Method to Assess the Intracellular Fate and Bioavailability of Clusterin Using Live Cell Confocal Microscopy Imaging.

Clusterin, also known as apolipoprotein J, is an ATP-independent holdase chaperone protein. Clusterin is involved in various functions including protein quality control and lipid transport. Though clusterin is secreted upon stress, the intracellular fate of clusterin after a stress response is not well understood. The protocol described here utilizes clusterin tagged to fluorescent proteins like green fluorescent protein and red fluorescent protein to understand the intracellular fate of clusterin.

Identification of the novel role of sterol regulatory element binding proteins (SREBPs) in mechanotransduction and intraocular pressure regulation.

Trabecular meshwork (TM) cells are contractile and mechanosensitive, and they aid in maintaining intraocular pressure (IOP) homeostasis. Lipids are attributed to modulating TM contractility, with poor mechanistic understanding. In this study using human TM cells, we identify the mechanosensing role of the transcription factors sterol regulatory element binding proteins (SREBPs) involved in lipogenesis. By constitutively activating SREBPs and pharmacologically inactivating SREBPs, we have mechanistically deciphered the attributes of SREBPs in regulating the contractile properties of TM. The pharmacological inhibition of SREBPs by fatostatin and molecular inactivation of SREBPs ex vivo and in vivo, respectively, results in significant IOP lowering. As a proof of concept, fatostatin significantly decreased the SREBPs responsive genes and enzymes involved in lipogenic pathways as well as the levels of the phospholipid, cholesterol, and triglyceride. Further, we show that fatostatin mitigated actin polymerization machinery and stabilization, and decreased ECM synthesis and secretion. We thus postulate that lowering lipogenesis in the TM outflow pathway can hold the key to lowering IOP by modifying the TM biomechanics.

Identification of the novel role of sterol regulatory element binding proteins (SREBPs) in mechanotransduction and intraocular pressure regulation.

Trabecular meshwork (TM) cells are highly contractile and mechanosensitive to aid in maintaining intraocular pressure (IOP) homeostasis. Lipids are attributed to modulating TM contractility with poor mechanistic understanding. In this study using human TM cells, we identify the mechanosensing role of the transcription factors sterol regulatory element binding proteins (SREBPs) involved in lipogenesis. By constitutively activating SREBPs and pharmacologically inactivating SREBPs, we have mechanistically deciphered the attributes of SREBPs in regulating the contractile properties of TM. The pharmacological inhibition of SREBPs by fatostatin and molecular inactivation of SREBPs ex vivo and in vivo respectively results in significant IOP lowering. As a proof of concept, fatostatin significantly decreased the SREBPs responsive genes and enzymes involved in lipogenic pathways as well as the levels of the phospholipid, cholesterol, and triglyceride. Further, we show that fatostatin mitigated actin polymerization machinery and stabilization, and decreased ECM synthesis and secretion. We thus postulate that lowering lipogenesis in the TM outflow pathway can hold the key to lowering IOP by modifying the TM biomechanics. Synopsis: In this study, we show the role of lipogenic transcription factors sterol regulatory element binding proteins (SREBPs) in the regulation of intraocular pressure (IOP). ( Synopsis Figure - Created using Biorender.com ) SREBPs are involved in the sensing of changes in mechanical stress on the trabecular meshwork (TM). SREBPs aid in transducing the mechanical signals to induce actin polymerization and filopodia/lamellipodia formation.SREBPs inactivation lowered genes and enzymes involved in lipogenesis and modified lipid levels in TM.SREBPs activity is a critical regulator of ECM engagement to the matrix sites.Inactivation of SCAP-SREBP pathway lowered IOP via actin relaxation and decreasing ECM production and deposition in TM outflow pathway signifying a novel relationship between SREBP activation status and achieving IOP homeostasis.

Profiling the miRNA from Exosomes of Non-Pigmented Ciliary Epithelium-Derived Identifies Key Gene Targets Relevant to Primary Open-Angle Glaucoma.

Oxidative stress (OS) on tissues is a major pathological insult leading to elevated intraocular pressure (IOP) and primary open-angle glaucoma (POAG). Aqueous humor (AH) produced by the non-pigmentary ciliary epithelium (NPCE) drains out via the trabecular meshwork (TM) outflow pathway in the anterior chamber. The exosomes are major constituents of AH, and exosomes can modulate the signaling events, as well as the responses of their target TM tissue. Despite the presence of molecular mechanisms to negate OS, oxidative damage directly, as well as indirectly, influences TM health, AH drainage, and IOP. We proposed that the expression of microRNA (miRNAs) carried by exosomes in the AH can be affected by OS, and this can modulate the pathways in target cells. To assess this, we subjected NPCE to acute and chronic OS (A-OS and C-OS), enriched miRNAs, performed miRNA microarray chip analyses, and miRNA-based gene targeting pathway prediction analysis. We found that various miRNA families, including miR27, miR199, miR23, miR130b, and miR200, changed significantly. Based on pathway prediction analysis, we found that these miRNAs can regulate the genes including Nrf2, Keap1, GSK3B, and serine/threonine-protein phosphatase2A (PP2A). We propose that OS on the NPCE exosomal miRNA cargo can modulate the functionality of the TM tissue.

Analysis of Lipid Contents in Human Trabecular Meshwork Cells by Multiple Reaction Monitoring (MRM) Profiling Lipidomics.

Lipids are among the major constituents of cells and play many important cellular functions. Lipid levels in the trabecular meshwork (TM) aqueous humor outflow pathway play an important role in the maintenance of aqueous humor drainage and intraocular pressure (IOP) homeostasis. Therefore, it is important to characterize the changes in the lipid contents in the aqueous humor outflow pathway tissues to better understand their functional significance in the maintenance of IOP. The multiple reaction monitoring (MRM)-based profiling aids in the analysis of the metabolome as a collection of functional groups and is utilized as an exploratory metabolomics and lipidomics approach. The MRM-based profiling utilizes tandem mass spectrometry experiments carried out on a commercial triple quadrupole mass spectrometer with three aligned quadrupole mass filters (Q1, Q2, and Q3). This screening methodology can be utilized for targeted lipidomics screening. This chapter focuses on the methodology for isolation and culturing of the TM cells, lipid extraction, and the MRM-based lipidomics approach with data analysis.

Multiomics analysis reveals the mechanical stress-dependent changes in trabecular meshwork cytoskeletal-extracellular matrix interactions.

Trabecular meshwork (TM) tissue is subjected to constant mechanical stress due to the ocular pulse created by the cardiac cycle. This brings about alterations in the membrane lipids and associated cell-cell adhesion and cell-extracellular matrix (ECM) interactions, triggering intracellular signaling responses to counter mechanical insults. A loss of such response can lead to elevated intraocular pressure (IOP), a major risk factor for primary open-angle glaucoma. This study is aimed to understand the changes in signaling responses by TM subjected to mechanical stretch. We utilized multiomics to perform an unbiased mRNA sequencing to identify changes in transcripts, mass spectrometry- (MS-) based quantitative proteomics for protein changes, and multiple reaction monitoring (MRM) profiling-based MS and high-performance liquid chromatography (HPLC-) based MS to characterize the lipid changes. We performed pathway analysis to obtain an integrated map of TM response to mechanical stretch. The human TM cells subjected to mechanical stretch demonstrated an upregulation of protein quality control, oxidative damage response, pro-autophagic signal, induction of anti-apoptotic, and survival signaling. We propose that mechanical stretch-induced lipid signaling via increased ceramide and sphingomyelin potentially contributes to increased TM stiffness through actin-cytoskeleton reorganization and profibrotic response. Interestingly, increased phospholipids and diacylglycerol due to mechanical stretch potentially enable cell membrane remodeling and changes in signaling pathways to alter cellular contractility. Overall, we propose the mechanistic interplay of macromolecules to bring about a concerted cellular response in TM cells to achieve mechanotransduction and IOP regulation when TM cells undergo mechanical stretch.

Cathepsin K Regulates Intraocular Pressure by Modulating Extracellular Matrix Remodeling and Actin-Bundling in the Trabecular Meshwork Outflow Pathway.

The homeostasis of extracellular matrix (ECM) and actin dynamics in the trabecular meshwork (TM) outflow pathway plays a critical role in intraocular pressure (IOP) regulation. We studied the role of cathepsin K (CTSK), a lysosomal cysteine protease and a potent collagenase, on ECM modulation and actin cytoskeleton rearrangements in the TM outflow pathway and the regulation of IOP. Initially, we found that CTSK was negatively regulated by pathological stressors known to elevate IOP. Further, inactivating CTSK using balicatib, a pharmacological cell-permeable inhibitor of CTSK, resulted in IOP elevation due to increased levels and excessive deposition of ECM-like collagen-1A in the TM outflow pathway. The loss of CTSK activity resulted in actin-bundling via fascin and vinculin reorganization and by inhibiting actin depolymerization via phospho-cofilin. Contrarily, constitutive expression of CTSK decreased ECM and increased actin depolymerization by decreasing phospho-cofilin, negatively regulated the availability of active TGFß2, and reduced the levels of alpha-smooth muscle actin (αSMA), indicating an antifibrotic action of CTSK. In conclusion, these observations, for the first time, demonstrate the significance of CTSK in IOP regulation by maintaining the ECM homeostasis and actin cytoskeleton-mediated contractile properties of the TM outflow pathway.

Hic-5 Regulates Actin Cytoskeletal Reorganization and Expression of Fibrogenic Markers and Myocilin in Trabecular Meshwork Cells.

PURPOSE: To explore the role of inducible focal adhesion (FA) protein Hic-5 in actin cytoskeletal reorganization, FA formation, fibrogenic activity, and expression of myocilin in trabecular meshwork (TM) cells. METHODS: Using primary cultures of human TM (HTM) cells, the effects of various external factors on Hic-5 protein levels, as well as the effects of recombinant Hic-5 and Hic-5 small interfering RNA (siRNA) on actin cytoskeleton, FAs, myocilin, α-smooth muscle actin (αSMA), and collagen-1 were determined by immunofluorescence and immunoblot analyses. RESULTS: Hic-5 distributes discretely to the FAs in HTM cells and throughout the TM and Schlemm's canal of the human aqueous humor (AH) outflow pathway. Transforming growth factor-ß2 (TGF-ß2), endothelin-1, lysophosphatidic acid, hydrogen peroxide, and RhoA significantly increased Hic-5 protein levels in HTM cells in association with reorganization of actin cytoskeleton and FAs. While recombinant Hic-5 induced actin stress fibers, FAs, αv integrin redistribution to the FAs, increased levels of αSMA, collagen-1, and myocilin, Hic-5 siRNA suppressed most of these responses in HTM cells. Hic-5 siRNA also suppressed TGF-ß2-induced fibrogenic activity and dexamethasone-induced myocilin expression in HTM cells. CONCLUSIONS: Taken together, these results reveal that Hic-5, whose levels were increased by various external factors implicated in elevated intraocular pressure, induces actin cytoskeletal reorganization, FAs, expression of fibrogenic markers, and myocilin in HTM cells. These characteristics of Hic-5 in TM cells indicate its importance in regulation of AH outflow through the TM in both normal and glaucomatous eyes.

Specific alterations of tyrosine hydroxylase immunopositive cells in the retina of NT-4 knock out mice.

To assess the effect of NT-4 deprivation on maturation of retinal circuitry, we investigated a mouse with targeted deletion of the gene encoding nt-4 (nt-4(-/-)). In particular, we studied neurons immunostained by an antibody recognizing tyrosine hydroxylase (TH), the rate limiting enzyme for dopamine (DA) synthesis. We found that TH immunopositive processes were altered in the retina of nt-4(-/-). Alteration of TH immunopositive processes in nt-4(-/-) mice resulted in changes of DA turnover, as assessed by high-pressure liquid chromatography measurements. These findings suggest that retinal NT-4 plays a role in the morphological maturation of dopaminergic retinal cells.

Neuronal activity regulates the developmental expression and subcellular localization of cortical BDNF mRNA isoforms in vivo.

Activity-dependent changes in BDNF expression have been implicated in developmental plasticity. Although its expression is widespread in visual cortex, developmental regulation of its different transcripts by visual experience has not been investigated. Here, we investigated the cellular expression of different BDNF transcripts in rat visual cortex during postnatal development. We found that transcripts I and II are expressed only in adults but III and IV are expressed from early postnatal stage. Total BDNF mRNA is expressed throughout the age groups. Transcripts III and IV show a differential intracellular localization, while former was detected only in cell bodies, latter is present both in cell bodies and dendritic processes. Inhibition of visual activity decreases the levels of exons, with exon IV transcript almost disappearing from dendrites. In vitro experiments also confirmed the above results, indicating activity-dependent regulation of different BDNF promoters with specific temporal and cellular patterns of expression in developing visual cortex.