Proteomic analysis uncovers clusterin-mediated disruption of actin-based contractile machinery in the trabecular meshwork to lower intraocular pressure.

Glaucoma, a major cause of blindness, is characterized by elevated intraocular pressure (IOP) due to improper drainage of aqueous humor via the trabecular meshwork (TM) outflow pathway. Our recent work identified that loss of clusterin resulted in elevated IOP. This study delves deeper to elucidate the role of clusterin in IOP regulation. Employing an ex vivo human anterior segment perfusion model, we established that constitutive expression and secretion as well as exogenous addition of clusterin can significantly lower IOP. Interestingly, clusterin significantly lowered transforming growth factor ß2 (TGFß2)-induced IOP elevation. This effect was linked to the suppression of extracellular matrix (ECM) deposition and, highlighting the crucial role of clusterin in maintaining ECM equilibrium. A comprehensive global proteomic approach revealed the broad impact of clusterin on TM cell structure and function by identifying alterations in protein expression related to cytoskeletal organization, protein processing, and cellular mechanics, following clusterin induction. These findings underscore the beneficial modulation of TM cell structure and functionality by clusterin. Specifically, clusterin influences the actin-cytoskeleton and focal adhesion dynamics, which are instrumental in cell contractility and adhesion processes. Additionally, it suppresses the activity of proteins critical in TGFß2, G-protein, and JAK-STAT signaling pathways, which are vital for the regulation of ocular pressure. By delineating these targeted effects of clusterin within the TM outflow pathway, our findings pave the way for novel treatment strategies aimed at mitigating the progression of ocular hypertension and glaucoma through targeted molecular interventions.

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.

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.

Novel insight into the role of clusterin on intraocular pressure regulation by modifying actin polymerization and extracellular matrix remodeling in the trabecular meshwork.

This study provides comprehensive mechanistic evidence for the role of clusterin, a stress-response secretory chaperone protein, in the modulation of intraocular pressure (IOP) by regulating the trabecular meshwork (TM) actin cytoskeleton and the extracellular matrix (ECM). The pathological stressors on TM known to elevate IOP significantly lowered clusterin protein levels indicating stress-related clusterin function loss. Small interfering RNA-mediated clusterin loss in human TM cells in vitro induced actin polymerization and stabilization via protein kinase D1, serine/threonine-protein kinase N2 (PRK2), and LIM kinase 1 (LIMK1), and the recruitment and activation of adhesome proteins including paxillin, vinculin, and integrin αV and ß5. A complete loss of clusterin as seen in clusterin knockout mice (Clu-/- ) led to significant IOP elevation at postnatal Day 70. Contrarily, constitutive clusterin expression using adenovirus (AdCLU) in HTM cells resulted in the loss of actin polymerization via decreased PRK2, and LIMK1 and negative regulation of integrin αV and ß5. Furthermore, we found that AdCLU treatment in HTM cells significantly decreased the ECM protein expression and distribution by significantly increasing matrix metalloprotease 2 (MMP2) activity and lowering the levels of pro-fibrotic proteins such as transforming growth factor-ß2 (TGFß2), thrombospondin-1 (TSP-1), and plasminogen activator inhibitor-1 (PAI-1). Finally, we found that HTM cells supplemented with recombinant human clusterin attenuated the pro-fibrotic effects of TGFß2. For the first time this study demonstrates the importance of clusterin in the regulation of TM actin cytoskeleton - ECM interactions and the maintenance of IOP, thus making clusterin an interesting target to reverse elevated IOP.

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.

Peri-natal growth retardation rate and fat mass accumulation in mice lacking Dip2A is dependent on the dietary composition.

Disco-interacting protein 2 is a highly conserved three-domain protein with two tandem Adenylate-forming domains. It is proposed to influence the processes involved in neuronal development by influencing lipid metabolism and remains to be characterized. In this study, we show that Disco-interacting protein 2a null mice do not exhibit overt phenotype defects. However, the body composition differences were observed in these mice under different dietary regimens. The neutral lipid composition of two different diets was characterized, and it was observed that the new-born mice grow relatively slower than the wild-type mice with delayed appearance of features such as dentition when fed with high-triacylglycerol NIN-formulation diet. The high-diacylglycerol Safe-formulation diet was found to accumulate more fat mass in mice than those fed with high-triacylglycerol NIN-formulation diet beyond 10 months. These findings point to a proposed relationship between dietary components (particularly the lipid composition) and body composition along with the growth of neonates in mice lacking the gene Disco-interacting protein 2a.

Bisphenol A exposure under metabolic stress induces accelerated cellular senescence in vivo in a p53 independent manner.

Senescence is an irreversible process that is a characteristic of age-associated disease like Type 2 diabetes (T2D). Bisphenol-A (BPA), one of the most common endocrine disruptor chemicals, received special attention in the development of insulin resistance and T2D. To understand the role played by BPA in cellular senescence under metabolic stress, zebrafish embryos were exposed to BPA in the absence and presence of hyperglycaemia. Transcriptional levels of the senescence markers p15, p53, Rb1 and ß-galactosidase were increased when BPA was combined with metabolic stress. In addition, zebrafish embryos that were exposed to combination of hyperglycaemia and BPA exhibited increased levels of apoptosis. However, cellular senescence remained induced by a combination of hyperglycaemia and BPA exposure even in the absence of a translated p53 protein suggesting that senescence is primarily independent of it but dependent on the p15-Rb1 pathway under our experimental conditions. To confirm that our results hold true in adult mammalian tissues, we validated our embryonic experiments in an adult mammalian metabolic model of skeletal muscle cells. Our work reveals a novel and unique converging role of senescence and apoptosis axis contributing to glucose dyshomeostasis. Thus, we conclude that BPA exposure can exacerbate existing metabolic stress to increase cellular senescence that leads to aggravation of disease phenotype in age-associated diseases like type 2 diabetes.

Novel insights of elevated systemic levels of bisphenol-A (BPA) linked to poor glycemic control, accelerated cellular senescence and insulin resistance in patients with type 2 diabetes.

There is a striking interaction of genes and environment in the etiology of type 2 diabetes mellitus (T2DM). While endocrine disrupting chemicals (EDCs) like bisphenol-A (BPA) have received special attention for their mechanistic role in metabolic disruption, there is a lack of clinically relevant data on BPA levels in Asian Indians, a population which is more susceptible to type 2 diabetes mellitus (T2DM) and cardiovascular diseases. Therefore, we measured systemic levels of BPA in patients with T2DM compared to individuals with normal glucose tolerance (n = 30 each). Serum BPA levels were estimated using ELISA kit, and biochemical determinations were done by standard protocols. Peripheral blood mononuclear cells (PBMCs) were used to profile the gene expression alterations with special reference to inflammation, estrogen receptors, and cellular senescence in these subjects. Serum levels of BPA were significantly higher in patients with T2DM compared to control individuals and positively correlated to poor glycemic control and insulin resistance. Patients with T2DM exhibited significantly elevated mRNA levels of senescence (GLB1, p16, p21, and p53) and inflammatory (IL6 and TNF-α) markers, shortened telomeres as well as elevated levels of estrogen-related receptor gamma (ERRγ), a recently identified receptor for BPA. BPA levels were positively correlated to senescence indicators, inflammatory markers and ERRγ and negatively correlated to telomere length. Our study is the first data in the clinical diabetes setting to demonstrate an association of increased BPA levels with cellular senescence, proinflammation, poor glycemic control, insulin resistance, and shortened telomeres in patients with T2DM.