Defining the ligand-dependent proximatome of the sigma 1 receptor.

Sigma 1 Receptor (S1R) is a therapeutic target for a wide spectrum of pathological conditions ranging from neurodegenerative diseases to cancer and COVID-19. S1R is ubiquitously expressed throughout the visceral organs, nervous, immune and cardiovascular systems. It is proposed to function as a ligand-dependent molecular chaperone that modulates multiple intracellular signaling pathways. The purpose of this study was to define the S1R proximatome under native conditions and upon binding to well-characterized ligands. This was accomplished by fusing the biotin ligase, Apex2, to the C terminus of S1R. Cells stably expressing S1R-Apex or a GFP-Apex control were used to map proximal proteins. Biotinylated proteins were labeled under native conditions and in a ligand dependent manner, then purified and identified using quantitative mass spectrometry. Under native conditions, S1R biotinylates over 200 novel proteins, many of which localize within the endomembrane system (endoplasmic reticulum, Golgi, secretory vesicles) and function within the secretory pathway. Under conditions of cellular exposure to either S1R agonist or antagonist, results show enrichment of proteins integral to secretion, extracellular matrix formation, and cholesterol biosynthesis. Notably, Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9) displays increased binding to S1R under conditions of treatment with Haloperidol, a well-known S1R antagonist; whereas Low density lipoprotein receptor (LDLR) binds more efficiently to S1R upon treatment with (+)-Pentazocine ((+)-PTZ), a classical S1R agonist. Furthermore, we demonstrate that the ligand bound state of S1R correlates with specific changes to the cellular secretome. Our results are consistent with the postulated role of S1R as an intracellular chaperone and further suggest important and novel functionalities related to secretion and cholesterol metabolism.

Identification of Estrogen Signaling in a Prioritization Study of Intraocular Pressure-Associated Genes.

Elevated intraocular pressure (IOP) is the only modifiable risk factor for primary open-angle glaucoma (POAG). Herein we sought to prioritize a set of previously identified IOP-associated genes using novel and previously published datasets. We identified several genes for future study, including several involved in cytoskeletal/extracellular matrix reorganization, cell adhesion, angiogenesis, and TGF-ß signaling. Our differential correlation analysis of IOP-associated genes identified 295 pairs of 201 genes with differential correlation. Pathway analysis identified ß-estradiol as the top upstream regulator of these genes with ESR1 mediating 25 interactions. Several genes (i.e., EFEMP1, FOXC1, and SPTBN1) regulated by ß-estradiol/ESR1 were highly expressed in non-glaucomatous human trabecular meshwork (TM) or Schlemm's canal (SC) cells and specifically expressed in TM/SC cell clusters defined by single-cell RNA-sequencing. We confirmed ESR1 gene and protein expression in human TM cells and TM/SC tissue with quantitative real-time PCR and immunofluorescence, respectively. 17ß-estradiol was identified in bovine, porcine, and human aqueous humor (AH) using ELISA. In conclusion, we have identified estrogen receptor signaling as a key modulator of several IOP-associated genes. The expression of ESR1 and these IOP-associated genes in TM/SC tissue and the presence of 17ß-estradiol in AH supports a role for estrogen signaling in IOP regulation.

Mechanosensitive channel inhibition attenuates TGFß2-induced actin cytoskeletal remodeling and reactivity in mouse optic nerve head astrocytes.

Astrocytes within the optic nerve head undergo actin cytoskeletal rearrangement early in glaucoma, which coincides with astrocyte reactivity and extracellular matrix (ECM) deposition. Elevated transforming growth factor beta 2 (TGFß2) levels within astrocytes have been described in glaucoma, and TGFß signaling induces actin cytoskeletal remodeling and ECM deposition in many tissues. A key mechanism by which astrocytes sense and respond to external stimuli is via mechanosensitive ion channels. Here, we tested the hypothesis that inhibition of mechanosensitive channels will attenuate TGFß2-mediated optic nerve head astrocyte actin cytoskeletal remodeling, reactivity, and ECM deposition. Primary optic nerve head astrocytes were isolated from C57BL/6J mice and cell purity was confirmed by immunostaining. Astrocytes were treated with vehicle control, TGFß2 (5 ng/ml), GsMTx4 (a mechanosensitive channel inhibitor; 500 nM), or TGFß2 (5 ng/ml) + GsMTx4 (500 nM) for 48 h. FITC-phalloidin staining was used to assess the formation of f-actin stress fibers and to quantify the presence of crosslinked actin networks (CLANs). Cell reactivity was determined by immunostaining and immunoblotting for GFAP. Levels of fibronectin and collagen IV deposition were also quantified. Primary optic nerve head astrocytes were positive for the astrocyte marker GFAP and negative for markers for microglia (F4/80) and oligodendrocytes (OSP1). Significantly increased %CLAN-positive cells were observed after 48-h treatment with TGFß2 vs. control in a dose-dependent manner. Co-treatment with GsMTx4 significantly decreased %CLAN-positive cells vs. TGFß2 treatment and the presence of f-actin stress fibers. TGFß2 treatment significantly increased GFAP, fibronectin, and collagen IV levels, and GsMTx4 co-treatment ameliorated GFAP immunoreactivity. Our data suggest inhibition of mechanosensitive channel activity as a potential therapeutic strategy to modulate actin cytoskeletal remodeling within the optic nerve head in glaucoma.

Survey of Microinvasive Glaucoma Surgery and Other Glaucoma Surgical Experience among United States Ophthalmology Residency Programs.

Purpose The aim of the study is to assess the state of glaucoma surgical training in United States ophthalmology residency programs, including experience with microinvasive glaucoma surgery (MIGS). Design The design of the study is anonymous, internet-based national survey. Participants Current United States ophthalmology residents of residency programs accredited by the Accreditation Council for Graduate Medical Education (ACGME). Methods An anonymous survey link was emailed to all 120 accredited United States ophthalmology residency programs inviting residents to participate in an assessment of residency glaucoma surgical experience. Survey responses were collected between January 21, 2019 and March 4, 2019 and analyzed using descriptive statistics. Main Outcome Measures The main outcomes of the study are demographic information, practice intentions, and anticipated primary surgical experience with ACGME-required glaucoma procedures and MIGS procedures, as self-reported by U.S. ophthalmology residents. Results Of the estimated 1,479 U.S. ophthalmology residents, 161 residents participated (10.9%). A total of 118 residents (73.2%) reported any degree of anticipated MIGS primary surgical experience during residency, with the iStent being the most familiar technique. The likelihood of any anticipated MIGS experience during residency was not significantly different by geographic region ( p = 0.16), however, anticipated volume varied significantly ( p = 0.037). Of the 113 respondents who reported an intention to manage glaucoma surgically in their eventual practice, 25 (22.1%) reported no anticipated primary MIGS experience during residency. 73.3% of residents anticipating MIGS experience anticipated 0 to 10 cases, with 42.9% anticipating less than 5 cases as primary surgeon. Conclusion MIGs are not a required component of the glaucoma surgical curriculum for U.S. ophthalmology residents. Although the majority of ophthalmology residents surveyed intend to manage glaucoma surgically in eventual practice, most receive minimal experience with these novel techniques during residency. Surgical training is variable by geographic region.

Hyperhomocysteinemia-induced death of retinal ganglion cells: The role of Müller glial cells and NRF2.

Hyperhomocysteinemia (Hhcy), or increased levels of the excitatory amino acid homocysteine (Hcy), is implicated in glaucoma, a disease characterized by increased oxidative stress and loss of retinal ganglion cells (RGCs). Whether Hhcy is causative or merely a biomarker for RGC loss in glaucoma is unknown. Here we analyzed the role of NRF2, a master regulator of the antioxidant response, in Hhcy-induced RGC death in vivo and in vitro. By crossing Nrf2-/- mice and two mouse models of chronic Hhcy (Cbs+/- and Mthfr+/- mice), we generated Cbs+/-Nrf2-/- and Mthfr+/-Nrf2-/- mice and performed systematic analysis of retinal architecture and visual acuity followed by assessment of retinal morphometry and gliosis. We observed significant reduction of inner retinal layer thickness and reduced visual acuity in Hhcy mice lacking NRF2. These functional deficits were accompanied by fewer RGCs and increased gliosis. Given the key role of Müller glial cells in maintaining RGCs, we established an ex-vivo indirect co-culture system using primary RGCs and Müller cells. Hhcy-exposure decreased RGC viability, which was abrogated when cells were indirectly cultured with wildtype (WT) Müller cells, but not with Nrf2-/- Müller cells. Exposure of WT Müller cells to Hhcy yielded a robust mitochondrial and glycolytic response, which was not observed in Nrf2-/- Müller cells. Taken together, the in vivo and in vitro data suggest that deleterious effects of Hhcy on RGCs are likely dependent upon the health of retinal glial cells and the availability of an intact retinal antioxidant response mechanism.

Excess homocysteine upregulates the NRF2-antioxidant pathway in retinal Müller glial cells.

This study evaluated the effects of elevated homocysteine (Hcy) on the oxidative stress response in retinal Müller glial cells. Elevated Hcy has been implicated in retinal diseases including glaucoma and optic neuropathy, which are characterized by retinal ganglion cell (RGC) loss. To understand the mechanisms of Hcy-induced RGC loss, in vitro and in vivo models have been utilized. In vitro isolated RGCs are quite sensitive to elevated Hcy levels, while in vivo murine models of hyperhomocysteinemia (HHcy) demonstrate a more modest RGC loss (∼20%) over a period of many months. This differential response to Hcy between isolated cells and the intact retina suggests that the retinal milieu invokes mechanisms that buffer excess Hcy. Oxidative stress has been implicated as a mechanism of Hcy-induced neuron loss and NRF2 is a transcription factor that plays a major role in regulating cytoprotective responses to oxidative stress. In the present study we investigated whether HHcy upregulates NRF2-mediated stress responses in Müller cells, the chief retinal glial cell responsible for providing trophic support to retinal neurons. Primary Müller cells were exposed to L-Hcy-thiolactone [50µM-10mM] and assessed for viability, reactive oxygen species (ROS), and glutathione (GSH) levels. Gene/protein levels of Nrf2 and levels of NRF2-regulated antioxidants (NQO1, CAT, SOD2, HMOX1, GPX1) were assessed in Hcy-exposed Müller cells. Unlike isolated RGCs, isolated Müller cells are viable over a wide range of Hcy concentrations [50 µM - 1 mM]. Moreover, when exposed to elevated Hcy, Müller cells demonstrate decreased oxidative stress and decreased ROS levels. GSH levels increased by ∼20% within 24 h exposure to Hcy. Molecular analyses revealed 2-fold increase in Nrf2 expression. Expression of antioxidant genes Nqo1, Cat, Sod2, Hmox1, Gpx1 increased significantly. The consequences of Hcy exposure were evaluated also in Müller cells harvested from Nrf2-/- mice. In contrast to WT Müller cells, in which oxidative stress decreased upon exposure to Hcy, the Nrf2-/- Müller cells showed a significant increase in oxidative stress. Our data suggest that at least during early stages of Hhcy, a cytoprotective response may be in place, mediated in part by NRF2 in Müller cells.

Proteomic Alterations in Aqueous Humor From Patients With Primary Open Angle Glaucoma.

Purpose: Primary open angle glaucoma (POAG) is the most prevalent form of glaucoma, accounting for approximately 90% of all cases. The aqueous humor (AH), a biological fluid in the anterior and posterior chambers of the eye, is involved in a multitude of functions including the maintenance of IOP and ocular homeostasis. This fluid is very close to the pathologic site and is also known to have a significant role in glaucoma pathogenesis. The purpose of this study was to identify proteomic alterations in AH from patients with POAG. Methods: AH samples were extracted from 47 patients undergoing cataract surgery (controls: n = 32; POAG: n = 15). Proteomic analysis of the digested samples was accomplished by liquid-chromatography-mass spectrometry. The identified proteins were evaluated using a variety of statistical and bioinformatics methods. Results: A total of 33 proteins were significantly altered in POAG subjects compared with the controls. The most abundant proteins in POAG subjects are IGKC (13.56-fold), ITIH4 (4.1-fold), APOC3 (3.36-fold), IDH3A (3.11-fold), LOC105369216 (2.98-fold). SERPINF2 (2.94-fold), NPC2 (2.88-fold), SUCLG2 (2.70-fold), KIAA0100 (2.29-fold), CNOT4 (2.23-fold), AQP4 (2.11-fold), COL18A1 (2.08-fold), NWD1 (2.07-fold), and TMEM120B (2.06-fold). A significant increasing trend in the odds ratios of having POAG was observed with increased levels of these proteins. Conclusion: Proteins identified in this study are implicated in signaling, glycosylation, immune response, molecular transport, and lipid metabolism. The identified candidate proteins may be potential biomarkers associated with POAG development and may lead to more insight in understanding the mechanisms underlying the pathogenesis of this disease.

Sigma 1 receptor regulates ERK activation and promotes survival of optic nerve head astrocytes.

The sigma 1 receptor (S1R) is a unique transmembrane protein that has been shown to regulate neuronal differentiation and cellular survival. It is expressed within several cell types throughout the nervous system and visceral organs, including neurons and glia within the eye. S1R ligands are therapeutic targets for diseases ranging from neurodegenerative conditions to neoplastic disorders. However, effects of S1R activation and inhibition within glia cells are not well characterized. Within the eye, the astrocytes at the optic nerve head are crucial to the health and survival of the neurons that send visual information to the brain. In this study, we used the S1R-specific agonist, (+)-pentazocine, to evaluate S1R activation within optic nerve head-derived astrocytes (ONHAs). Treatment of ONHAs with (+)-pentazocine attenuated the level and duration of stress-induced ERK phosphorylation following oxidative stress exposure and promoted survival of ONHAs. These effects were specific to S1R activation because they were not observed in ONHAs that were depleted of S1R using siRNA-mediated knockdown. Collectively, our results suggest that S1R activation suppresses ERK1/2 phosphorylation and protects ONHAs from oxidative stress-induced death.

Efficient Endocytic Uptake and Maturation in Drosophila Oocytes Requires Dynamitin/p50.

Dynactin is a multi-subunit complex that functions as a regulator of the Dynein motor. A central component of this complex is Dynamitin/p50 (Dmn). Dmn is required for endosome motility in mammalian cell lines. However, the extent to which Dmn participates in the sorting of cargo via the endosomal system is unknown. In this study, we examined the endocytic role of Dmn using the Drosophila melanogaster oocyte as a model. Yolk proteins are internalized into the oocyte via clathrin-mediated endocytosis, trafficked through the endocytic pathway, and stored in condensed yolk granules. Oocytes that were depleted of Dmn contained fewer yolk granules than controls. In addition, these oocytes accumulated numerous endocytic intermediate structures. Particularly prominent were enlarged endosomes that were relatively devoid of Yolk proteins. Ultrastructural and genetic analyses indicate that the endocytic intermediates are produced downstream of Rab5. Similar phenotypes were observed upon depleting Dynein heavy chain (Dhc) or Lis1. Dhc is the motor subunit of the Dynein complex and Lis1 is a regulator of Dynein activity. We therefore propose that Dmn performs its function in endocytosis via the Dynein motor. Consistent with a role for Dynein in endocytosis, the motor colocalized with the endocytic machinery at the oocyte cortex in an endocytosis-dependent manner. Our results suggest a model whereby endocytic activity recruits Dynein to the oocyte cortex. The motor along with its regulators, Dynactin and Lis1, functions to ensure efficient endocytic uptake and maturation.

Sigma receptor ligand, (+)-pentazocine, suppresses inflammatory responses of retinal microglia.

PURPOSE: To evaluate the effects of the σ 1 receptor (σR1) agonist, (+)-pentazocine, on lipopolysaccharide (LPS)-induced inflammatory changes in retinal microglia cells. METHODS: Retinal microglia cells were isolated from Sprague-Dawley rat pups. Cells were treated with LPS with or without (+)-pentazocine and with or without the σR1 antagonist BD1063. Morphologic changes were assayed. Cell viability was assessed by using MTT assay. Supernatant levels of tumor necrosis factor α (TNF-α), interleukin 10, (IL-10), monocyte chemoattractant protein-1 (MCP-1), and nitric oxide (NO) were determined. Reactive oxygen species (ROS) formation was assayed, and levels of mitogen-activated protein kinases (MAPKs) were analyzed by using Western blot. RESULTS: The σR1 protein was expressed in retinal microglia. Incubation with LPS and/or (+)-pentazocine did not alter cell viability or σR1 protein levels. Incubation with LPS for 24 hours induced a marked change in microglial morphology and a significant increase in secreted levels of TNF-α, IL-10, MCP-1, and NO. Pretreatment with (+)-pentazocine inhibited the LPS-induced morphologic changes. Release of TNF-α, IL-10, MCP-1, and NO was reduced with (+)-pentazocine. Intracellular ROS formation was suppressed with (+)-pentazocine. Phosphorylation of extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) was reduced in the presence of (+)-pentazocine. The σR1 antagonist BD1063 blocked the (+)-pentazocine-mediated inhibition of LPS-induced morphologic changes. In addition, BD1063 treatment blocked (+)-pentazocine-mediated suppression of LPS-induced TNF-α, IL-10, MCP-1, NO, and intracellular ROS release. CONCLUSIONS: Treatment with (+)-pentazocine suppressed inflammatory responses of retinal microglia and inhibited LPS-induced activation of ERK/JNK MAPK. In neurodegenerative disease, (+)-pentazocine may exert neuroprotective effects through manipulation of microglia.

Proteomic similarities in steroid responsiveness in normal and glaucomatous trabecular meshwork cells.

PURPOSE: Glucocorticoids (GCs) are common anti-inflammatory agents that can cause ocular hypertension and secondary glaucoma as a consequence of impaired aqueous humor outflow through the trabecular meshwork (TM). Mechanisms of GC-signaling are complex and poorly understood. To better understand GC-signaling in the eye, we tested the hypothesis that common mechanisms of steroid responsiveness exist in TM cells from normal and glaucomatous donors. METHODS: Four primary cultures of human TM cells from normal and glaucomatous donors were treated with or without dexamethasone (Dex) for 10 days, then cellular proteins were extracted, identified and quantified by liquid chromatography tandem mass spectrometry (LC MS/MS) iTRAQ (isobaric tags for relative and absolute quantitation) technology. RESULTS: A total of 718 proteins were quantified. Dex-treatment significantly altered the abundance of 40 proteins in ≥3 cell samples, 37 of which have not previously been associated with GC-signaling in TM cells. Most steroid responsive proteins were changed in all four TM cells analyzed, both normal and glaucomatous. GC-induced proteomic changes support remodeling of the extracellular matrix, disorganization of the cytoskeleton/cell-cell interactions, and mitochondrial dysfunction. Such physiologic consequences appear common to those induced in TM cells by transforming growth factor-ß(2), another putative contributor to ocular hypertension and glaucoma pathology. CONCLUSIONS: The results expand the repertoire of TM proteins involved in GC-signaling, demonstrate common consequences of GC-signaling in normal and glaucomatous TM cells, and reveal similarities in proteomic changes induced by steroids and TGFß(2) in normal and glaucomatous TM cells. Finally, the data contributes to a TM quantitative proteomic database.

Quantitative proteomics: TGFß2 signaling in trabecular meshwork cells.

PURPOSE: Transforming growth factor beta 2 (TGFß2) is often elevated in the aqueous humor (AH) and trabecular meshwork (TM) of patients with primary open-angle glaucoma (POAG) and appears to contribute to POAG pathogenesis. To better understand TGFß2 signaling in the eye, TGFß2-induced proteomic changes were identified in cells cultured from the TM, a tissue involved in intraocular pressure (IOP) elevation in glaucoma. METHODS: Primary cultures of human TM cells from four donors were treated with or without TGFß2 (5 ng/mL) for 48 hours; then cellular protein was analyzed by liquid chromatography-mass spectrometry iTRAQ (isobaric tags for relative and absolute quantitation) technology. RESULTS: A total of 853 proteins were quantified. TGFß2 treatment significantly altered the abundance of 47 proteins, 40 of which have not previously been associated with TGFß2 signaling in the eye. More than half the 30 elevated proteins support growing evidence that TGFß2 induces extracellular matrix remodeling and abnormal cytoskeletal interactions in the TM. The levels of 17 proteins were reduced, including four cytoskeletal and six regulatory proteins. Both elevated and decreased regulatory proteins implicate TGFß2-altered processes involving transcription, translation, and the glutamate/glutamine cycle. Altered levels of eight mitochondrial proteins support TGFß2-induced mitochondrial dysfunction in the TM that in POAG could contribute to oxidative damage in the AH outflow pathway, TM senescence, and elevated IOP. CONCLUSIONS: The results expand the repertoire of proteins known to participate in TGFß2 signaling, provide new molecular insight into POAG, and establish a quantitative proteomics database for the TM that includes candidate glaucoma biomarkers for future validation studies.

What can patients expect from cataract surgery?

Cataract surgery has evolved into an outpatient procedure that requires minimal anesthesia and significantly improves visual function for about 90% of patients. With the help of their primary care physician and ophthalmologist, patients can decide when cataract surgery is appropriate for them. In addition, patients may have a choice about the type of synthetic lens implant that fits their visual needs.