Molecular clustering identifies complement and endothelin induction as early events in a mouse model of glaucoma
Gareth R. Howell, Danilo G. Macalinao, Gregory L. Sousa, Michael Walden, Ileana Soto, Stephen C. Kneeland, Jessica M. Barbay, Benjamin L. King, Jeffrey K. Marchant, Matthew Hibbs, Beth Stevens, Ben A. Barres, Abbot F. Clark, Richard T. Libby, Simon W.M. John
Gareth R. Howell, Danilo G. Macalinao, Gregory L. Sousa, Michael Walden, Ileana Soto, Stephen C. Kneeland, Jessica M. Barbay, Benjamin L. King, Jeffrey K. Marchant, Matthew Hibbs, Beth Stevens, Ben A. Barres, Abbot F. Clark, Richard T. Libby, Simon W.M. John
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Research Article Ophthalmology

Molecular clustering identifies complement and endothelin induction as early events in a mouse model of glaucoma

Abstract

Glaucoma is one of the most common neurodegenerative diseases. Despite this, the earliest stages of this complex disease are still unclear. This study was specifically designed to identify early stages of glaucoma in DBA/2J mice. To do this, we used genome-wide expression profiling of optic nerve head and retina and a series of computational methods. Eyes with no detectable glaucoma by conventional assays were grouped into molecularly defined stages of disease using unbiased hierarchical clustering. These stages represent a temporally ordered sequence of glaucoma states. We then determined networks and biological processes that were altered at these early stages. Early-stage expression changes included upregulation of both the complement cascade and the endothelin system, and so we tested the therapeutic value of separately inhibiting them. Mice with a mutation in complement component 1a (C1qa) were protected from glaucoma. Similarly, inhibition of the endothelin system with bosentan, an endothelin receptor antagonist, was strongly protective against glaucomatous damage. Since endothelin 2 is potently vasoconstrictive and was produced by microglia/macrophages, our data provide what we believe to be a novel link between these cell types and vascular dysfunction in glaucoma. Targeting early molecular events, such as complement and endothelin induction, may provide effective new treatments for human glaucoma.

Authors

Gareth R. Howell, Danilo G. Macalinao, Gregory L. Sousa, Michael Walden, Ileana Soto, Stephen C. Kneeland, Jessica M. Barbay, Benjamin L. King, Jeffrey K. Marchant, Matthew Hibbs, Beth Stevens, Ben A. Barres, Abbot F. Clark, Richard T. Libby, Simon W.M. John

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Figure 8

C1QA deficiency protects against DBA/2J glaucoma.

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C1QA deficiency protects against DBA/2J glaucoma. (A) Despite a delay in...
(A) Despite a delay in IOP elevation in some eyes, IOPs of C1qa WT and homozygous mutant (KO) mice overlap extensively (WT versus KO: 8.5 months, P = 0.026; 10.5 months, P = 0.34). Number of assessed eyes was at least 43 for all groups (B) The vast majority of optic nerves from C1qa mutant mice are indistinguishable from control nerves with no detectable glaucoma (NOE, green bars) at 10.5 months of age, and retain a lower glaucoma incidence than WT mice at 12 months of age. Sample sizes: 46 WT, 45 KO at 10.5 months; 62 WT, 56 KO at 12 months. P < 1 × 10–8 at each age comparing KO with WT distributions. (C) C1qa KO eyes with NOE nerve damage (n = 10) have axon number similar to that of no-glaucoma Gpnmb+ controls (n = 11, P = 0.26). For ease of comparison, axon counts for stages 1–5 are repeated from Figure 1. (D) In the ganglion cell layer (GCL), RGC somata are also spared in C1qa mutants with NOE nerve damage, but the number of RGC soma lost in KO eyes with severe optic nerve damage was similar to that in WT counterparts. Amacrine cells make up the majority of remaining GCL cells in severely affected eyes. (E) The most common optic nerve and GCL phenotypes for 12-month-old mice of each genotype are shown. Optic nerve cross sections were stained with PPD and flat mounted retinas stained with cresyl violet. Scale bar: 50 μm.