Effect of Cognitive Demand on Functional Visual Field Performance in Senior Drivers with Glaucoma

A grant from the One-University Open Access Fund at the University of Kansas was used to defray the author's publication fees in this Open Access journal. The Open Access Fund, administered by librarians from the KU, KU Law, and KUMC libraries, is made possible by contributions from the offices of KU Provost, KU Vice Chancellor for Research & Graduate Studies, and KUMC Vice Chancellor for Research. For more information about the Open Access Fund, please see http://library.kumc.edu/authors-fund.xml.Purpose: To investigate the effect of cognitive demand on functional visual field performance in drivers with glaucoma. Method: This study included 20 drivers with open-angle glaucoma and 13 age- and sex-matched controls. Visual field performance was evaluated under different degrees of cognitive demand: a static visual field condition (C1), dynamic visual field condition (C2), and dynamic visual field condition with active driving (C3) using an interactive, desktop driving simulator. The number of correct responses (accuracy) and response times on the visual field task were compared between groups and between conditions using Kruskal–Wallis tests. General linear models were employed to compare cognitive workload, recorded in real-time through pupillometry, between groups and conditions. Results: Adding cognitive demand (C2 and C3) to the static visual field test (C1) adversely affected accuracy and response times, in both groups (p < 0.05). However, drivers with glaucoma performed worse than did control drivers when the static condition changed to a dynamic condition [C2 vs. C1 accuracy; glaucoma: median difference (Q1–Q3) 3 (2–6.50) vs. controls: 2 (0.50–2.50); p = 0.05] and to a dynamic condition with active driving [C3 vs. C1 accuracy; glaucoma: 2 (2–6) vs. controls: 1 (0.50–2); p = 0.02]. Overall, drivers with glaucoma exhibited greater cognitive workload than controls (p = 0.02). Conclusion: Cognitive demand disproportionately affects functional visual field performance in drivers with glaucoma. Our results may inform the development of a performance-based visual field test for drivers with glaucoma

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

Sigma Receptor Ligand, (þ)-Pentazocine, Suppresses Inflammatory Responses of Retinal Microglia

PURPOSE. To evaluate the effects of the r 1 receptor (rR1) agonist, (þ)-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 (þ)-pentazocine and with or without the rR1 antagonist BD1063. Morphologic changes were assayed. Cell viability was assessed by using MTT assay. Supernatant levels of tumor necrosis factor a (TNF-a), 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 rR1 protein was expressed in retinal microglia. Incubation with LPS and/or (þ)-pentazocine did not alter cell viability or rR1 protein levels. Incubation with LPS for 24 hours induced a marked change in microglial morphology and a significant increase in secreted levels of TNF-a, IL-10, MCP-1, and NO. Pretreatment with (þ)-pentazocine inhibited the LPSinduced morphologic changes. Release of TNF-a, IL-10, MCP-1, and NO was reduced with (þ)-pentazocine. Intracellular ROS formation was suppressed with (þ)-pentazocine. Phosphorylation of extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) was reduced in the presence of (þ)-pentazocine. The rR1 antagonist BD1063 blocked the (þ)-pentazocine-mediated inhibition of LPS-induced morphologic changes. In addition, BD1063 treatment blocked (þ)-pentazocine-mediated suppression of LPS-induced TNF-a, IL-10, MCP-1, NO, and intracellular ROS release. CONCLUSIONS. Treatment with (þ)-pentazocine suppressed inflammatory responses of retinal microglia and inhibited LPS-induced activation of ERK/JNK MAPK. In neurodegenerative disease, (þ)-pentazocine may exert neuroprotective effects through manipulation of microglia

Depletion or over-expression of Sh3px1 results in dramatic changes in cell morphology

The mammalian Sorting Nexin 9 (Snx9) family consists of three paralogs: Snx9, Snx18 and Snx33. Most of the published literature to date has centered on the role of Snx9 in clathrin-mediated endocytosis (CME). Snx9 contains an Sh3 domain at its N-terminus and has been shown to interact with Dynamin and actin nucleation factors via this domain. In addition to the Sh3 domain, Snx9 also contains a C-terminal BAR domain. BAR domains are known to sense and/or induce membrane curvature. In addition to endocytosis, recent studies have implicated the Snx9 family in diverse processes such as autophagy, macropinocytosis, phagocytosis and mitosis. The Snx9 family is encoded by a single gene in Drosophila called sh3px1. In this report, we present our initial characterization of sh3px1. We found that depletion of Sh3px1 from Drosophila Schneider 2 (S2) cells resulted in defective lamellipodia formation. A similar phenotype has been reported upon depletion of Scar, the actin nucleation factor implicated in forming lamellipodia. In addition, we demonstrate that over-expression of Sh3px1 in S2 cells results in the formation of tubules as well as long protrusions. Formation of these structures required the C-terminal BAR domain as well as the adjacent Phox homology (PX) domain of Sh3px1. Furthermore, efficient protrusion formation by Sh3px1 required the actin nucleation factor Wasp. Tubules and protrusions were also generated upon over-expressing the mammalian orthologs Snx18 and Snx33 in S2 cells. By contrast, over-expressing Snx9 mostly induced long tubules

Quantitative Proteomics: TGFβ2 Signaling in Trabecular Meshwork Cells

This study expands the repertoire of proteins known to participate in TGFβ2 signaling and demonstrates common consequences of TGFβ2 signaling in normal and glaucomatous trabecular meshwork cells

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

<div><p>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.</p></div

S1R knockdown in HeLa cells and ONHAs.

<p>HeLa cells were transfected with human scrambled siRNA or with human S1R siRNA. Western blot analysis of S1R levels at 3 days (A) and 6 days (B) following transfection is shown. Quantitation of western blot results shows S1R levels normalized to GAPDH as the internal control (C, D). Results are presented as fold change of S1R levels derived from the S1R siRNA-transfected cells compared to S1R levels derived from scrambled siRNA-transfected cells. Data were analyzed using t-test. Significantly different from control *p<0.05, **p<0.01. Experiments were repeated 3 times. MTT assay was performed to assess viability at 3 days (E) and 6 days (F) after scrambled or S1R siRNA transfection. Viability was significantly decreased in S1R siRNA-transfected HeLa cells compared to scrambled siRNA-transfected cells. Transfection with scrambled siRNA did not cause significant HeLa cell death compared to non-transfection control (G, H). Data were analyzed using t-test. Significantly different from control ****p<0.0001. Experiments were performed in quadruplicate and repeated 3 times. ONHAs were transfected with rat scrambled siRNA or with rat S1R siRNA. Western blot analysis of S1R levels at 3 days (I) and 6 days (J) following transfection is shown. Quantitation of western blot results shows S1R levels normalized to GAPDH as the internal control (K, L). Results are presented as fold change of S1R levels derived from the S1R siRNA-transfected cells compared to S1R levels derived from scrambled siRNA-transfected cells. Data were analyzed using t-test. Significantly different from control: **p<0.01, ***p<0.001. Experiments were repeated 3 times. MTT assay was performed to assess viability at 3 days (M) and 6 days (N) after scrambled or S1R siRNA transfection. Viability was not significantly changed in S1R siRNA-transfected ONHAs compared to scrambled siRNA-transfected cells. Transfection with scrambled siRNA did not cause significant ONHA death compared to non-transfection control (O, P). Experiments were performed in quadruplicate and repeated 3 times.</p

Effect of PTZ on ROS generation when ONHAs exposed to H₂O₂.

<p>(A) Representative images of ONHAs treated with 100μM H₂O₂ for 24 hours in the presence or absence of PTZ (10μM, 1 hour pretreatment followed by co-treatment). ROS generation was visualized using CellROX Green reagent. Scale bar: 100μm. (B) Quantitative analysis of intracellular ROS. For each group, three coverslips were quantified, and eight images were taken from each coverslip. Mean signal intensity was quantified by ImageJ. Intracellular ROS generation increased when ONHAs were exposed to H₂O₂. The ROS generation was inhibited by PTZ. Data were analyzed using one-way ANOVA followed by Tukey-Kramer post hoc test for multiple comparisons. Significantly different from control ****P<0.0001. Significantly different between groups ####P<0.0001. Experiments were repeated 3 times.</p