Mutant human myocilin induces strain specific differences in ocular hypertension and optic nerve damage in mice
ABSTRACT Elevated intraocular pressure (IOP) is a causative risk factor for the development and progression of glaucoma. Glaucomatous mutations in myocilin (MYOC) damage the trabecular meshwork and elevate IOP in humans and in mice. Animal models of glaucoma are important to discover and better understand molecular pathogenic pathways and to test new glaucoma therapeutics. Although a number of different animal models of glaucoma have been developed and characterized, there are no true models of human primary open angle glaucoma (POAG). The overall goal of this work is to develop the first inducible mouse model of POAG using a human POAG relevant transgene (i.e. mutant MYOC) expression in mouse eyes to elevate IOP and cause pressure-induced damage to the optic nerve. Four mouse strains (A/J, BALB/cJ, C57BL/6J, and C3H/HeJ) were used in this study. Ad5.MYOC.Y437H (5 × 10(7) pfu) was injected intravitreally into one eye, with the uninjected contralateral eye serving as the control eye. Conscious IOP measurements were taken using a TonoLab rebound tonometer. Optic nerve damage was determined by scoring PPD stained optic nerve cross sections. Retinal ganglion cell and superior colliculus damage was assessed by Nissl stain cell counts. Intravitreal administration of viral vector Ad5.MYOC.Y437H caused a prolonged, reproducible, and statistically significant IOP elevation in BALB/cJ, A/J, and C57BL/6J mice. IOPs increased to approximately 25 mm Hg for 8 weeks (p < 0.0001). In contrast, the C3H/HeJ mouse strain was resistant to Ad5.MYOC.Y437H induced IOP elevation for the 8-week time period. IOPs were stable (12-15 mm Hg) in the uninjected control eyes. We also determined whether there were any strain differences in pressure-induced optic nerve damage. Even though IOP was similarly elevated in three of the strains tested (BALB/cJ, C57BL/6J, and A/J) only the A/J strain had considerable and significant optic nerve damage at the end of 8 weeks with optic nerve damage score of 2.64 ± 0.19 (n = 18, p < 0.001) in the injected eye. There was no statistical difference in retinal ganglion cell death or superior colliculus damage at the 8-week time point in any of the strains tested. These results demonstrate strain dependent responses to Ad5.MYOC.Y437H-induced ocular hypertension and pressure-induced optic nerve damage.
- SourceAvailable from: Monika E Danielewska[Show abstract] [Hide abstract]
ABSTRACT: Purpose To ascertain whether the incidence of ocular dicrotic pulse (ODP) increases with age, it is more pronounced in glaucomatous than in normal eyes and whether it is related to cardiovascular activity. Methods 261 subjects aged 47 to 78 years were included in the study and classified into four groups: primary open angle glaucoma (POAG), primary angle-closure glaucoma (PACG), glaucoma suspects with glaucomatous optic disc appearance (GODA) and the controls (CG). Additionally, in each group, subjects with ODP were divided into two age subgroups around the median age. A non-contact ultrasonic method was used to measure corneal indentation pulse (CIP) synchronically with the acquisition of electrocardiography (ECG) and blood pulse signals. ODP was assessed from the acquired signals that were numerically processed in a custom written program. Results ODP incidence was about 78%, 66%, 66% and 84% for CG, GODA, POAG, and PACG group, respectively. With advancing age, the ODP incidence increased for all subjects (Δ = 12%), the highest being for the PACG and POAG groups (Δ = 30%). GODA group did not show an age-related increase in the incidence of ODP. Conclusions The ocular dicrotism, measured with non-contact ultrasonic method, was found to be a common phenomenon in elderly subjects. The increased ODP incidence in PACG and POAG group may correspond to either higher stiffness of glaucoma eyes, biochemical abnormalities in eye tissues, changes in ocular hemodynamics, may reflect the effect of medications or be a combination of all those factors. The results of GODA group suggest different mechanisms governing their ocular pulse that makes them less susceptible to generating ODP and having decreased predisposition to glaucoma.PLoS ONE 07/2014; 9(7):e102814. DOI:10.1371/journal.pone.0102814 · 3.53 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: CYP1B1 has been implicated in primary congenital glaucoma with autosomal recessive mode of inheritance. Mutations in CYP1B1 have also been reported in primary open angle glaucoma (POAG) cases and suggested to act as a modifier of the disease along with Myocilin (MYOC). Earlier reports suggest that over-expression of myocilin leads to POAG pathogenesis. Taken together, we propose a functional interaction between CYP1B1 and myocilin where 17β estradiol acts as a mediator. Therefore, we hypothesize that 17β estradiol can induce MYOC expression through the putative estrogen responsive elements (EREs) located in its promoter and CYP1B1 could manipulate MYOC expression by metabolizing 17β estradiol to 4-hydroxy estradiol, thus preventing it from binding to MYOC promoter. Hence any mutation in CYP1B1 that reduces its 17β estradiol metabolizing activity might lead to MYOC upregulation, which in turn might play a role in glaucoma pathogenesis. It was observed that 17β estradiol is present in Human Trabecular Meshwork cells (HTM) and Retinal Pigment Epithelial cells (RPE) by immunoflouresence and ELISA. Also, the expression of enzymes related to estrogen biosynthesis pathway was observed in both cell lines by RT-PCR. Subsequent evaluation of the EREs in the MYOC promoter by luciferase assay, with dose and time dependent treatment of 17β estradiol, showed that the EREs are indeed active. This observation was further validated by direct binding of estrogen receptors (ER) on EREs in MYOC promoter and subsequent upregulation in MYOC level in HTM cells on 17β estradiol treatment. Interestingly, CYP1B1 mutants with less than 10% enzymatic activity were found to increase the level of endogenous myocilin in HTM cells. Thus the experimental observations are consistent with our proposed hypothesis that mutant CYP1B1, lacking the 17β estradiol metabolizing activity, can cause MYOC upregulation, which might have a potential implication in glaucoma pathogenesis.PLoS ONE 09/2012; 7(9):e45077. DOI:10.1371/journal.pone.0045077 · 3.53 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: Purpose:To characterize the morphological and functional changes in optic nerve crushed mice and evaluate electroretinogram (ERG) responses as tools to monitor retinal ganglion cell (RGC) dysfunction. Methods:Optic nerve crush (ONC) was performed unilaterally in adult BALB/cJ mice. The neuronal loss in the retinal ganglion cell layer (GCL) and superior colliculus (SC) was determined by Nissl staining. Retinal thickness was assessed by spectral-domain optical coherence tomography (SD-OCT) imaging. Retinal function was determined by pattern ERG and full-field flash ERG. Responses of pattern ERG, positive scotopic threshold response (pSTR), scotopic oscillatory potentials (OPs) and photopic negative response (PhNR) were analyzed. Results:ONC induced progressive neuronal loss in both GCL and contralateral SC starting from 7 and 28 days following ONC, respectively. A linear correlation was observed between combined thickness of nerve fiber layer (NFL), GCL and inner plexiform layer (IPL) imaged by SD-OCT and cell numbers in GCL. Only half of the normal BALB/cJ mice exhibited pattern ERG response, which was smaller and later compared to C57BL/6J mice. ONC reduced both pattern ERG and pSTR, but the reduction of pattern ERG was earlier than pSTR, preceding the anatomical cell loss in the GCL. PhNR and scotopic OPs were not affected by ONC. Conclusions:SD-OCT and ERG can be used to noninvasively monitor retinal morphological and functional changes induced by ONC. Pattern ERG and pSTR are both able to detect early RGC dysfunction, but pattern ERG exhibits higher sensitivity. Our results support the use of these tools in studies using the mouse ONC model.Investigative Ophthalmology & Visual Science 05/2014; 55(6). DOI:10.1167/iovs.14-13895 · 3.66 Impact Factor