Dysregulation of septin cytoskeletal organization in the trabecular meshwork contributes to ocular hypertension
Rupalatha Maddala, … , Hélène Choquet, Ponugoti V. Rao
Rupalatha Maddala, … , Hélène Choquet, Ponugoti V. Rao
Published December 6, 2024
Citation Information: JCI Insight. 2024;9(23):e179468. https://doi.org/10.1172/jci.insight.179468.
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Research Article Cell biology Ophthalmology

Dysregulation of septin cytoskeletal organization in the trabecular meshwork contributes to ocular hypertension

Abstract

Ocular hypertension, believed to result partly from increased contractile activity, cell adhesive interactions, and stiffness within the trabecular meshwork (TM), is a major risk factor for glaucoma, a leading cause of blindness. However, the identity of molecular mechanisms governing organization of actomyosin and cell adhesive interactions in the TM remains limited. Based on our previous findings, in which proteomics analyses revealed elevated levels of septins, including septin-9 in human TM cells treated with the ocular hypertensive agent dexamethasone, here, we evaluated the effects of septin-9 overexpression, deficiency, and pharmacological targeting in TM cells. These studies demonstrated a profound impact on actomyosin organization, cell adhesion, contraction, and phagocytosis. Overexpression raised intraocular pressure (IOP) in mice, while inhibition increased cell permeability. In addition, we replicated a significant association between a common variant (rs9038) in SEPT9 with IOP in the Genetic Epidemiology Research on Adult Healthy and Aging (GERA) cohort. Collectively, these data reveal a link between dysregulated septin cytoskeletal organization in the TM and increased IOP, likely due to enhanced cell contraction, adhesive interactions, and fibrotic activity. This suggests that targeting the septin cytoskeleton could offer a novel approach for lowering IOP in patients with glaucoma.

Authors

Rupalatha Maddala, Pallavi Gorijavolu, Levi K. Lankford, Nikolai P. Skiba, Pratap Challa, Rakesh K. Singh, K. Saidas Nair, Hélène Choquet, Ponugoti V. Rao

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Figure 6

Increased expression of septin-9 impairs TM cell phagocytic activity, and septin filament perturbation enhances fluid diffusion from TM cells grown in Transwells.

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Increased expression of septin-9 impairs TM cell phagocytic activity, an...
(A and B) TM cells expressing hSEPT9/eGFP exhibit impaired phagocytosis, as evidenced by reduced pHrodo uptake and fluorescence (A, red staining) and flow cytometry analysis (B), compared with control cells expressing eGFP alone. Analyses were performed on equal numbers of cells derived from 3 strains, in triplicate. FSC, forward scatter; LV, lentiviral vector. (C and D) To assess whether septin cytoskeletal reorganization affects fluid diffusion, TM cells cultured on Transwell inserts for over 1 week were treated with either the septin filament perturbator (10 μM UR214-9 for 18 hours) or the Rho kinase inhibitor (10 μM Y27632 for 2 hours). Following treatment, cells were evaluated for FITC-dextran permeability, as described in the Methods section. Treatment with UR214-9 and Y27632 led to a significant increase in dextran permeability compared with control cells, with Y27632 inducing a more rapid and robust response than UR214-9. Data are presented as mean ± SEM. After treatment, Transwell filters with TM cells were fixed, permeabilized, and stained for F-actin using phalloidin-TRITC. Representative F-actin stained images (D, right panel) show a disorganized actin cytoskeleton in cells treated with either the septin perturbator or the Rho kinase inhibitor, compared with vehicle-treated controls. n = 4–7 (using 2 strains in multiple). ****P < 0.0001 based on Student’s t test. Scale bars: 100 μm (A) and 40 μm (D).