Dysregulation of septin cytoskeletal organization in the trabecular meshwork contributes to ocular hypertension
Rupalatha Maddala, Pallavi Gorijavolu, Levi K. Lankford, Nikolai P. Skiba, Pratap Challa, Rakesh K. Singh, K. Saidas Nair, Hélène Choquet, Ponugoti V. Rao
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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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 1

Increased levels of septin-9 and other septins in human trabecular meshwork (TM) cells treated with ocular hypertensive agents, and their distribution in the aqueous humor (AH) outflow pathway.

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Increased levels of septin-9 and other septins in human trabecular meshw...
(A–F) TM cells were treated with Dex (0.5 μM for 7 days), TGF-β2 (10 ng/mL for 24 hours), and endothelin-1 (2 μM for 24 hours). These treatments resulted in significant increases in the levels of SEPT9, SEPT11, and SEPT7 compared with control cells, as determined by immunoblot analyses. Dex effects on septin levels were examined in cytoskeletal fractions, with α-actinin used as a loading control. In contrast, the effects of TGF-β2 and endothelin-1 on septins were assessed in total cell lysates, with GAPDH serving as a loading control. These analyses were conducted using a minimum of 3 different strains of human TM cells, indicated by different colors or shapes in the histograms. Sample size: n = 4–6. *P < 0.01, **P < 0.001, ****P < 0.0001 based on Student’s t test. Data in B, D, and F are presented as mean fold changes from controls, with error bars representing ±SEM. (G) Distribution of SEPT9, SEPT7, and SEPT11 in human AH outflow pathway tissues from 70-year-old donors. The bottom right panel shows background fluorescence from a secondary antibody conjugated with a fluorophore. Scale bar: 50 μm. SC, Schlemm’s canal; US, uveoscleral; CS, corneoscleral; IW, inner wall; OW, outer wall.