E50K mutation are not reported. Development of a mouse model either with the E50K mutation knocked into the mouse Optn locus or with the mutant human gene will be important for investigating the role of this gene in POAG.

WDR36

The WD40 repeat domain 36 gene (WDR36) was recently implicated in POAG (Monemi et al.,

2005). Mutations in WDR36 do not always cause glaucoma, and current data suggest that this gene primarily modifies the severity of glaucoma induced by other glaucoma genes (Hauser et al., 2006). Currently, no mouse models with mutations in the WDR36 gene exist.

Strategies for developing new models of POAG

As discussed above, mice are useful for modeling the effects of POAG genes, but there is still a need

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for POAG models with high IOP and glaucomatous neurodegeneration. A number of strategies are being used to develop these models. In addition to modeling the effects of mutations in the MYOC, OPTN, and WDR36 genes alone, mice can be used to model and understand the combined effects of these mutant genes. Mice with combinations of these mutations may also provide valuable models of glaucomatous neurodegeneration.

A complementary strategy for producing POAG models is to alter genes whose known function suggests that they may cause glaucoma when mutant. The more we understand about the mechanisms of POAG, the smarter we can be in predicting candidate genes/pathways that may be worth perturbing to produce additional models. Candidate genes can be altered so that they either produce no proteins or produce specific mutant proteins. As discussed for myocilin, disease pathogenesis can require specific mutant proteins and even the presence of mutations in the human protein. A pitfall of the candidate gene approach is that it requires knowledge of gene function and depends on a priori assumptions about pathogenic mechanisms. Since the mechanisms are poorly understood, the candidate gene approach is often unsuccessful.

Another valuable strategy is to induce novel mutations by random mutagenesis of the mouse genome. Commonly, the genomes of founder males are mutagenized with chemical agents such as ethyl nitrosourea (ENU) and breeding and screening strategies are developed to uncover phenotypes of interest in mutant offspring (John et al., 1999; Thaung et al., 2002; O’Brien and Frankel, 2004). An ENU mutagenesis is also underway in our laboratory and is producing a series of new mouse lines with POAG relevant phenotypes. This approach requires no previous assumptions or mechanistic knowledge to identify glaucoma genes and is a powerful tool for producing new models.

Developmental glaucoma

Developmental glaucomas are caused by genes involved in ocular development. Many developmental glaucomas involve obvious dysgenesis of readily visible anterior chamber structures (anterior segement dysgenesis, ASD) such as the iris and

pupil. In others (primary congenital glaucoma, PCG), the defects are subtle involving abnormal development of SC and TM drainage structures.

The mouse provides an important model system for deciphering the molecular cascades involved in ASD and developmental glaucoma (Gould and John, 2002). A number of mutant genes cause ASD (reviewed in Gould et al., 2004b), and some were first discovered to do so in mice. For example, mutation of a basement membrane collagen gene, Col4a1, was recently shown to cause severe ASD in mice (Van Agtmael et al., 2005; Gould et al., 2007). Soon afterwards, Sibon and colleagues found that mutations in the human orthologue of Col4a1 are associated with Axenfeld-Rieger syndrome, a form of ASD that is often associated with glaucoma (Sibon et al., 2007).

Although some mutant genes that cause ASDassociated phenotypes also induced high IOP in mice (e.g., Col4a1 and Bmp4), some have not been shown to do so. Even in cases without high IOP, mouse mutants provide valuable models for investigating ASD pathways. Additionally, breeding mutations that are not associated with high IOP into different mouse strains can uncover new high IOP phenotypes. This strategy also enables the identification of modifier genes and pathways that interact with the known mutant genes to determine whether or not IOP becomes elevated (Gould and John, 2002). The mouse is a powerful mammalian model for identifying modifier genes and unraveling complex genetic interactions. Recently, we found that genetic deficiency of tyrosinase increased the severity of the pathology of Foxc1 and Cyp1b1 mutations in mice (Libby et al., 2003), and that a pathway involving l-DOPA can be targeted with therapeutic benefit.

In addition to allowing studies of IOP elevation, ASD models may also provide new and early onset models of glaucomatous neurodegeneration. Although no robust models of glaucomatous neurodegeneration are reported, there are promising leads. For example, Col4A1 mutants are reported to suffer optic nerve excavation (Van Agtmael et al., 2005) and we have confirmed this on some genetic backgrounds (unpublished data). However, further studies are needed to determine glaucoma frequency and whether or not these mutants will become a tractable model. On some

but not all genetic backgrounds, Col4A1 and Bmp4 mutations result in developmental abnormalities of the optic nerve, which complicates their use as glaucoma models but provides a valuable opportunity to study genes that impact the health and development of RGCs. Again, mice will allow the identification of important modifier genes and the characterization of genetic interactions that modulate susceptibility to glaucoma.

Pigmentary glaucoma

Pigmentary glaucoma (PG) results secondarily to the dispersion of iris pigment into the anterior chamber of the eye. Dispersed pigment enters the drainage structures, causing damage that leads to IOP elevation and glaucomatous neurodegeneration. Forms of PG are reported in DBA/2J, DBA/ 2Nnia, and AKXD-28/TyJ mice (Sheldon et al., 1995; John et al., 1998; Anderson et al., 2001). DBA/2J mice have mutations in two genes that induce the pigment dispersion, the b mutation in the tyrosinase-related protein 1 gene (Tyrp1b) and

a stop-codon mutation in the glycoprotein (transmembrane) nmb gene (GpnmbR150X) (Chang et al.,

1999; Anderson et al., 2002). The pigment dispersion is a consequence of both melanosomal toxicity and abnormal ocular immunity (Anderson et al., 2002; Mo et al., 2003; John, 2005). The glaucoma in DBA/2J mice has hallmarks of human glaucoma, including an age-related variable progression of optic nerve atrophy in response to elevated IOP, a regional pattern of RGC death, and optic nerve excavation. Importantly, at least in our colony, damage appears limited to RGCs and the high pressure results in direct axon damage within the lamina of the optic nerve (Danias et al., 2003; Jakobs et al., 2005; Schlamp et al., 2006; Howell et al., 2007), as discussed below. DBA/2J mice have become the most widely used mouse model to decipher mechanisms of glaucomatous neurodegeneration and for developing new neuroprotective strategies.

Experimentally induced models of glaucoma

Experimentally induced models have the advantage that IOP can be elevated conveniently and

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experiments conducted over a short time frame. IOP can be artificially elevated by damaging the drainage structures or the blood vessels into which they drain (reviewed in Pang and Clark, 2007). Evaluation of retinas following sustained IOP elevation (4–12 weeks) has indicated increased RGC apoptosis, decreased optic nerve cross-sec- tional area and axonal density (Gross et al., 2003; Grozdanic et al., 2003), preferential loss of superior axons in the optic nerve (Mabuchi et al., 2004), and sustained ERG deficits (Grozdanic et al., 2003).

Although these induced models are valuable and can provide important insights (Nakazawa et al., 2006), there is still phenotypic variability and not all groups have been able to reproduce the procedures. Additionally, due to the sudden nature of IOP elevation following induced ocular trauma, there may be differences in the neurodegenerative and remodeling mechanisms compared to the naturally occurring inherited glaucomas. Optic nerve excavation – a hallmark of glaucoma – has not been reported for any of these models. For each of these models, it is not clear if the lack of reported optic nerve excavation is due to differences in disease mechanisms, due to the strain backgrounds used, or simply because detailed optic nerve head evaluation is needed.

In addition to pressure-induced neurodegeneration, direct neuronal injury can provide important new information. Optic nerve crush is an experimentally induced model for direct optic nerve and axon injury, and apoptotic RGC death (Li et al., 1999). RGC loss after controlled optic nerve crush occurs over 3 weeks (Li et al., 1999). Although crush provides a robust and rapid system for evaluating potential role(s) of individual genes in RGC death and optic nerve degeneration, it is a more severe insult than glaucoma. There are undoubtedly important common pathogenic mechanisms between RGC death in crush and in glaucoma, but there are also differences. For example, a radiation treatment prevents glaucomatous RGC death and associated optic nerve degeneration, but does not appear to protect against crush-induced damage in DBA/2J mice (Anderson et al., 2005 and unpublished observations). Similarly, cleavage of the autoinhibitory domain of the protein phosphatase calcineurin