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

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.


Cumulative IOP
The cumulative IOP difference for each animal was calculated according to the following equation: N is the number of IOP measurements taken per animal throughout the study (8 for Thbs1 R0134C homozygous mice and 6 for heterozygous mice over 8 weeks).

Measurement of uveoscleral outflow in mice.
Uveoscleral outflow was measured using a modification of a previously published method (1). Briefly, 2mg/ml of Fluorescein labeled dextran was prepared from 25mg/ml stock (D1822, Thermo Fisher Scientific). Six months old Thbs1 R0134C homozygous and age-matched wildtype mice were used for the experiments. The anterior chamber was perfused at 16mmHg pressure for 1 hour. Then, the choroid and sclera tissue complex were dissected, homogenized, and centrifuged at 2700g for 5 minutes. The dextran fluorescent density was determined from the supernatant by a plate reader. The exact amount of dextran was calculated based on the dextran standard that was prepared from the serial dilutions of perfused solution.

Quantification of optic nerve axons.
After enucleation, the optic nerve was cut approximately 1 mm behind the globe and fixed as described above. For degeneration axon counting, random micrographs (1000X magnification, 44.8 µm * 68.18 µm) were taken from different regions of the optic nerve. Degenerating axons were identified by one of the following structures: condensed electron opaque axoplasm, empty swollen axonal myelin sheath, myelin sheath filled with cellular debris, or destruction of the myelin sheath. Axons were counted from 4-12 micrographs for each eye and the numbers were averaged. For healthy axon counting, random micrographs (17.2 µm * 11.2 µm) were taken from different regions of the optic nerve. Axons were counted manually within the field.
The number of axons counted from 4-12 micrographs for each eye were averaged and divided by 17.2 µm * 11.2 µm to obtain the mean axon numbers per µm 2 .

Detection of apoptosis in mouse trabecular meshwork.
In situ detection of trabecular meshwork cell death used cryosections from eyes from 6 months old Thbs1 R1034C homozygous and age matched wildtype mice. TM cell death was detected based on the present of DNA breaks, which was labeled by TdT-mediated dUTP-X nick end labeling (termed TUNEL) (12156792910, Millipore Sigma). For each mouse, 6 random sections of TM tissue were selected for the labeling. The total TUNEL + TM cells were counted and analyzed.

CD47 overexpression and knockdown in COS-7 cells.
The CD47 shRNA (tagged with GFP), and the CD47 overexpression vector (cDNA tagged with GFP) lentivirus particles along with the non-targeting and empty vector controls were purchased from Horizon Discovery. The COS-7 cells were seeded in 6well plate at a confluency of 50%-60% prior to the transduction.

Detection of active TGF beta in primary trabecular meshwork cells.
Primary TM cells from Thbs1 R1034C mutants and wildtype mice were seeded in 6-well plates. High glucose media without FBS was added when the cells reached a confluency of 70%-90%. The conditioned media was collected the next day. The cellular components were removed by 0.45 um filter. The media was then concentrated by a centrifugal filter unit (Millipore Sigma, Cat no. UFC901024) before being prepared for western blot using TGF beta1 Monoclonal Antibody (Dilution,  (B) There was no significant difference in uveoscleral outflow between Thbs1 mutants and age-matched wildtype animals at 6 months of age. N=7, 9 for wildtype and homozygous, respectively, data represent the mean ± SEM. p=0.71 as determined by unpaired t-test. ns., not significant. showed similar Schlemm's canal morphology between wildtype and homozygous mice at the age of 8 months. For each group, four 20X fields on four quadrants were captured per eye (n=6, p=0.82). Scale bars: 100 μm; 20X fields comprise an area of 0.501 mm 2 . (B) There was no significant difference of lymph-(LYVE1 staining) or angiogenesis (CD31 staining) to bFGF pellet (20ng) between 4-month-old wildtype and homozygous mice. * indicates the location of pellet. The blood vessel and lymphatic vessel area were obtained from whole cornea (8 eyes from wildtype controls and 7 from homozygous, p=0.22 and 0.19, respectively). Scale bars: 200 μm. Data represent the means ± SEM. P-value was determined by Student's t test. ns., not significant.

Supplemental
Supplemental Figure 11. Cell death was not induced in Thbs1 homozygous mutants. In situ cell death was detected by TUNEL assay in 6 months old Thbs1 R1034C homozygous and age-matched wildtype mice (A). Scale bar, 100 µm. TUNEL+ cells in trabecular meshwork (TM) were counted from 6 random sections of the eye. (B) There was no significant difference in the number of dead TM cells between mutants and wildtype controls. N=7, data represent the means ± SEM. p>0.99 as determined by unpaired t-test. ns., not significant.
Supplemental Figure 12. R1034C mutation promotes the ECM deposition of TM cell-derived THBS1. Primary TM cells were isolated from both 2-month-old Thbs1 R1034C homozygous and age-matched wildtype animals. (A) Aggregation of TM cell-derived THBS1 protein was evident in mutant cells. (B) To determine the location of the aggregates, cellular components were removed by NH4OH, and the ECM was visualized through immunofluorescence staining. Compared to wildtype, mutant TM cells deposited significantly more THBS1 protein in the ECM (N=3). Notably, the mutated THBS1 protein was also co-localized with fibronectin (FN). Those findings corroborated the in vivo results from mice ( Figure 4, 5 and 8). A.U., arbitrary fluorescence units. Data represent the means ± SEM. P-value was determined by Student's t test. *** p<0.0001. Scale bars: 50 μm.