Thrombospondin 1 missense alleles induce extracellular matrix protein aggregation and TM dysfunction in congenital glaucoma
Haojie Fu, … , Janey L. Wiggs, Robert J. D’Amato
Haojie Fu, … , Janey L. Wiggs, Robert J. D’Amato
Published December 1, 2022
Citation Information: J Clin Invest. 2022;132(23):e156967. https://doi.org/10.1172/JCI156967.
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Research Article Ophthalmology

Thrombospondin 1 missense alleles induce extracellular matrix protein aggregation and TM dysfunction in congenital glaucoma

Abstract

Glaucoma is a highly heritable disease that is a leading cause of blindness worldwide. Here, we identified heterozygous thrombospondin 1 (THBS1) missense alleles altering p.Arg1034, a highly evolutionarily conserved amino acid, in 3 unrelated and ethnically diverse families affected by congenital glaucoma, a severe form of glaucoma affecting children. Thbs1R1034C-mutant mice had elevated intraocular pressure (IOP), reduced ocular fluid outflow, and retinal ganglion cell loss. Histology revealed an abundant, abnormal extracellular accumulation of THBS1 with abnormal morphology of juxtacanalicular trabecular meshwork (TM), an ocular tissue critical for aqueous fluid outflow. Functional characterization showed that the THBS1 missense alleles found in affected individuals destabilized the THBS1 C-terminus, causing protein misfolding and extracellular aggregation. Analysis using a range of amino acid substitutions at position R1034 showed that the extent of aggregation was correlated with the change in protein-folding free energy caused by variations in amino acid structure. Extracellular matrix (ECM) proteins, especially fibronectin, which bind to THBS1, also accumulated within THBS1 deposits. These results show that missense variants altering THBS1 p.Arg1034 can cause elevated IOP through a mechanism involving impaired TM fluid outflow in association with accumulation of aggregated THBS1 in the ECM of juxtacanalicular meshwork with altered morphology.

Authors

Haojie Fu, Owen M. Siggs, Lachlan S.W. Knight, Sandra E. Staffieri, Jonathan B. Ruddle, Amy E. Birsner, Edward Ryan Collantes, Jamie E. Craig, Janey L. Wiggs, Robert J. D’Amato

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Figure 4

Thbs1R1034C-mutant mice show accumulation of THBS1 in TM.

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Thbs1R1034C-mutant mice show accumulation of THBS1 in TM. (A) THBS1-sta...
(A) THBS1-stained cryosections of the iridocorneal angle region from 4-month-old Thbs1R1034C homozygous mice and age-matched controls. TM was localized adjacent to the CD31-stained SC. Thbs1R1034C-mutant mice showed increased THBS1 protein expression in TM compared with WT controls. Nuclei were stained with DAPI. Scale bar: 100 μm; insets, ×2.4. (B) IF staining for THBS1 in corneal whole mounts showing an en face view of protein localization. SC is indicated by dashed lines, as revealed by CD31 staining. Scale bar: 200 μm; insets, ×0.23 (upper left in B and C). (C) Progressive THBS1 protein accumulation in TM was evident in homozygous mice, and a similar pattern of accumulated THBS1 protein was evident in ThbsR1034C heterozygous mice. Scale bar: 200 μm; insets, ×3.6 (lower right in B and C). (D) Quantification of THBS1 in TM based on fluorescence intensity (100 μm2 fields were captured for intensity measurements; n = 9 for each time point). AU, arbitrary fluorescence units. Data represent the mean ± SEM. (E) Thbs1R1034C homozygous mice did not show increased Thbs1 mRNA expression (as determined by unpaired 2-tailed Student’s t test). Relative mRNA levels were normalized to Gapdh. n = 3. Data represent the mean ± SEM. (F) Western blot of protein extracts from the corneal limbal ring containing the iridocorneal angle region. Higher levels of THBS1 protein were detected in homozygous Thbs1R1034C mice, however, there was no evidence of ER stress in the mutant mice at age 16 months of age compared with age-matched controls based on the expression of GRP94, IRE1A, and BIP.