Ординатура / Офтальмология / Английские материалы / Eye, Retina, and Visual System of the Mouse_Chalupa, Williams_2008

In this chapter, we highlight important advances that have been made using mouse models to understand the pathogenesis of glaucoma. We also provide insights into how findings in the mouse are likely to affect both our understanding of glaucoma and the development of new therapies. Comprehensive reviews relevant to glaucoma studies in mice are available elsewhere ( John et al., 1999; Goldblum and Mittag, 2002; Gould, Smith, et al., 2004; John, 2005; Libby et al., 2005b; Lindsey and Weinreb, 2005; Weinreb and Lindsey, 2005).

An introduction to glaucoma

Glaucoma is a group of genetically heterogeneous diseases characterized by the death of retinal ganglion cells (RGCs), specific visual field deficits, and optic nerve atrophy. It is a major cause of blindness worldwide, with an estimated 70 million people affected (Quigley, 1996). Glaucoma is frequently associated with elevated intraocular pressure (IOP) (Ritch et al., 1996). High IOP and increasing age are strong risk factors for developing the disease. Nevertheless, high IOP is neither necessary to cause glaucoma—some individuals develop glaucoma despite IOPs in the normal range (Ritch et al., 1996)—nor, by itself, sufficient to cause glaucoma. Many individuals with high IOP do not develop glaucomatous visual loss (Leske, 1983). There are profound interpatient differences in the rate of glaucoma progression and the response to treatment. This phenomenon points to the existence of individual susceptibility factors that determine both the magnitude of IOP that is harmful to each individual and the ultimate severity and speed of visual damage (Libby et al., 2005b).

Many forms of glaucoma have a genetic component. Although a number of glaucoma genes have been identified, we are far from understanding the genetic or molecular etiology of glaucoma susceptibility ( John, 2005; Libby et al., 2005b). Characterizing the genetic factors that contribute to glaucoma should facilitate the early identification of individuals who are at increased risk for disease and should be

monitored closely. Understanding the molecular mechanisms that kill RGCs and the role elevated IOP and other risk factors play in these processes is important for designing new treatments to prevent vision loss.

The mouse as a model system in which to study glaucoma

The mouse is an ideal mammalian model for deciphering the complex genetic interactions that underlie human glaucoma susceptibility ( John et al., 1999). Glaucomatous mouse strains often develop glaucoma with a similar age-related progression as people do, and they do so in a relatively short span of time (within 1–2 years). It is possible to generate new models by mutating mouse orthologues of human glaucoma genes or by introducing known human mutant alleles into mice. This facility allows functional studies to be conducted in a highly controlled experimental setting. The burgeoning array of existing transgenic and gene-targeted alleles can also be exploited to study mechanisms of glaucoma. Studies in mice allow strain-specific genetic modifiers of the disease to be characterized. Last, mutagenesis screens can be performed to identify new genes and mechanisms that cause glaucoma. With the development of reliable means of measuring IOP and the availabily of models of both experimentally induced and inherited glaucoma, the mouse glaucoma model is poised to be a key player in revolutionizing our understanding of the molecular and cellular mechanisms of glaucoma susceptibility, initiation, and progression.

Humans and mice have comparable glaucoma-relevant structures

The aqueous drainage (outflow) structures of the eye play an important role in IOP elevation. Careful analysis of the aqueous humor drainage pathway and its role in controlling IOP is a major goal for glaucoma research. The mouse is well suited to these studies because its aqueous drainage structures are similar to those in humans. The two types of outflow pathways present in humans, conventional and

uveoscleral, exist in mice (see John et al., 1999; Lindsey and Weinreb, 2002). Both species have an endothelial-lined canal of Schlemm and a trabecular meshwork (TM) consisting of layers of well-organized trabecular beams covered with endothelial-like trabecular cells. The organizational similarity extends to drainage structure development and the genes that influence it (Gould, Smith, et al., 2004). The biggest anatomical difference between mice and humans is that mice have a poorly developed ciliary muscle. Nevertheless, the prostaglandin analogue Latanaprost (which decreases ciliary muscle–mediated resistance to outflow) lowers mouse IOP as it does in humans, and the effects of adenosine receptors on IOP are similar in the two species. The documented similarities between mice and humans in drainage structure anatomy, in functional responses to drugs that inhibit aqueous production and facilitate outflow, and in values for various outflow parameters indicate that mice represent very suitable models for studying IOP and its glaucoma-associated elevation.

The neural retina is the most complex structure of the eye and is considered to be an extension of the brain. Humans and mice have essentially the same neural retinal structures. The human eye has approximately 1.2–1.5 million RGCs, as opposed to 40,000–80,000 in mice (Strom and Williams, 1998). The common denominator in human glaucomas is the loss of RGCs along with their axons in the optic nerve and a specific pattern of optic nerve atrophy called excavation (Shields, 1997), and these same changes occur in the mouse.

Accurate intraocular pressure measurements can be generated in mice

Measuring IOP accurately and precisely is fundamental to the study of glaucoma. In humans, IOP is measured by a noninvasive technique known as tonometry. The mouse eye is only 3 mm in diameter (eight times smaller than the human eye), and this makes it more challenging to measure IOP. Noninvasive tonometric devices are available for use in mouse eyes, and two newer tonometric methods have recently been assessed (Filippopoulos et al., 2006). The impact-rebound (I/R) tonometer functions on the dynamic principle of probe deceleration following contact with the cornea (Danias et al., 2003a). In contrast, optical interferometry tonometry (OIT) is based on a static principle whereby an applied force applanates a specific area of the cornea (Ahmed et al., 2003). Only the OIT and I/R tonometers have been directly compared with a more accurate, invasive measuring method in the same eye in vivo (Filippopoulos et al., 2006; Morris et al., 2006), and so these are the best validated instruments. We and others could not obtain accurate or reliable readings using the Tono-Pen tonometer (John et al., 1997; Reitsamer et al., 2004; Dalke

et al., 2005; Pease et al., 2006). A commercial version of the I/R tonometer recently became available and gives accurate group means (Wang et al., 2005) but awaits direct in vivo comparison with an invasive method. Noninvasive methods have the advantage of allowing many repeated measurements but are not as accurate as invasive methods (even on human eyes), and must be used with great care. Differences in user technique, differences in normal corneal properties between mouse strains, and the presence of any corneal abnormalities alter the measured IOP. These factors can render these instruments substantially inaccurate. Thus, it is worth mentioning that the commonly used glaucomatous mouse strain (DBA/2J) has keratopathy at a young age and develops corneal calcification with increasing age.

Invasive methods for measuring IOP in the mouse entail inserting a cannula into the eye. They are widely accepted to be the only true measure of IOP in mice and other species. In one system, a very fine fluid-filled glass microneedle is inserted into the anterior chamber. The needle is connected to a pressure transducer and the pressure reading is monitored on a computer ( John et al., 1997; Savinova et al., 2001). Since we developed this method, it has been successfully adopted by other groups. Another system available is an adaptation of the servo-null micropipette system (SNMS), which was developed for measuring pressure in structures smaller than 25 μm (Avila et al., 2001). Cannulation methods have the disadvantage of penetrating the eye, and thus limiting the number of reasonable longitudinal readings. Nevertheless, because large numbers of genetically identical mice of different ages can be evaluated, an accurate record of IOP for a given population or strain can be achieved. It is also possible to study the variability of response and the effects of systematic manipulations. Invasive methods require general anesthesia, which can alter IOP. With appropriate care and attention to anesthetic mix, dose, and environment, however, anesthetic effects can be avoided (Savinova et al., 2001).

Mouse models of glaucoma

Mouse models relevant to high IOP and glaucoma can be divided into two classes, experimentally induced and inherited. Experimentally induced models have the advantage that IOP can be elevated conveniently and experiments can be conducted over a short time frame, and these models are therefore a valuable resource. However, they may have at least some mechanistic differences from inherited glaucoma. IOP-independent toxic effects on RGCs induced by the IOP-elevating procedure have not been carefully controlled in most cases. Not all investigators have readily reproduced reliably or consistently elevations in IOP, and so consultation on technical details with the appropriate groups is advised. Although experiments involving inherited forms of glaucoma are more time-consuming, the outcomes are more

likely to accurately model human glaucoma, which has a strong genetic component. Optic nerve head excavation, a hallmark of human glaucoma, is reported only for the inherited models.

Experimentally Induced Glaucoma Translimbal laser photocoagulation is used to damage the drainage structures themselves or the blood vessels into which they drain. The net effect is to reduce aqueous outflow and induce ocular hypertension. Single-session treatments are reported to elevate IOP for up to 12 weeks. Although some studies have shown laser treatment alone to be sufficient to increase IOP (Gross et al., 2003), others have coupled laser treatment with preflattening of the anterior chamber (by aqueous aspiration) to appose the cornea, iris, and drainage structures during laser use (Aihara et al., 2003a).

Evaluation of retinas following sustained IOP elevation (4–12 weeks) has indicated increased RGC apoptosis (Gross et al., 2003; Grozdanic et al., 2003), decreased optic nerve cross-sectional 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). A number of complications from these procedures do occur, such as corneal edema and opacity, hyphema, and cataracts, but these appear to be short-lived.

The injection of hyperosmotic saline into the limbus is an alternative means of increasing aqueous outflow resistance and hence IOP. This is an established method for use in the rat (Morrison et al., 1997) but is only preliminarily reported (in abstract form) in the mouse (McKinnon et al., 2003). Eight weeks after injection of 2.0M NaCl into the limbus of C57BL/6 mice, IOP was elevated and an average of 20% RGC axonal loss was observed. In this initial study, the animal numbers were low, so, although the results are promising, further testing is needed. The real benefit of these experimentally induced glaucoma models is the ability to induce chronic elevated IOP in genetically manipulated mice at will. As more mouse mutants become available, these models should help shed light on the risk factors that predispose to RGC death following sustained chronic ocular hypertension.

Models of Inherited Glaucoma Because glaucoma represents a set of heterogeneous disorders, in this section different inherited glaucoma models are discussed in relation to pertinent types of human glaucoma.

Primary open-angle glaucoma. Primary open-angle glaucoma (POAG) is the most common clinically defined subset of glaucoma. The term open-angle is used because the angle and drainage routes are clinically observed to be unimpeded. Based on existing genetic mapping studies, there are

at least 10 loci implicated in initiating POAG, and several genes known to contribute to the disease have been identified (see Libby et al., 2005a). The two best characterized are myocilin (GLC1A) and optineurin (GLC1E).

Myocilin (MYOC) was the first identified POAG gene. Mutations exist in approximately 3%–5% of late-onset POAG patients and up to 30% of juvenile open-angle glaucoma patients (JOAG, an earlier and more severe form of POAG). Myocilin is found in many ocular tissues, including the aqueous humor, TM, ciliary body, and RGCs. Despite its discovery nearly 10 years ago, its function is still unclear. Most of the 70 MYOC mutations so far identified in human glaucoma patients occur in a region with homology to the extracellular matrix protein olfactomedin (Kanagavalli et al., 2004).

The disease-causing mutations are believed to act through a gain-of-function mechanism. In both people and mice with null alleles, glaucoma does not develop (Kim et al., 2001). In addition, neither 15-fold overexpression of mouse MYOC (Gould, Miceli-Libby, et al., 2004) nor elevated expression of human MYOC from the mouse lens (Zillig et al., 2005) leads to glaucoma in mice. In experiments using cultured human TM cells, MYOC containing a Pro370Leu mutation accumulates in cells and stimulates the unfolded protein response, ER stress, and cell death when cells are grown at normal body temperature. However, when cells are grown at a lower temperature that promotes protein folding and secretion, the cells survive (Liu and Vollrath, 2004). These data strongly suggest that the accumulation of misfolded proteins is pathogenic.

In one in vivo mouse study by Gould et al. (2006), expression of a Myoc allele (Tyr423His), equivalent to a severe disease-associated human mutation (Tyr437His), led to the accumulation of mutant protein within the iridocorneal angle of the eye but did not activate ER stress or lead to elevated IOP and glaucoma. Several mouse strains were used, including CBA/CaJ, AKR/J, BALB/cJ, and C57BL/6J, and all demonstrated consistent, nonpathological phenotypes. In humans, the equivalent mutation is associated with the development of aggressive glaucoma, so the lack of disease progression in the mouse is surprising. There is just 82% identity between the mouse and human MYOC protein. The mouse protein may contain sequences that make it unable to induce the unfolded protein response and ER stress. Alternatively, activation of the unfolded protein response may not occur in vivo in mice or in humans. Abnormally accumulating mutant proteins that form insoluble polymeric aggregates do not always activate the unfolded protein response, and to date, activation of the response by mutant MYOC has been observed only in vitro. The production of mice that express the mutant version of the human protein in the iridocorneal angle should provide a useful means of testing these possibilities.

In a separate in vivo study by Senatorov et al. (2006), the same Tyr423His mutation also led to MYOC accumulation in the TM. In this study, a modest increase (2 mm Hg) in IOP was observed, and apoptotic RGC loss and axonal degeneration occurred with age. Optic nerve head excavation was not demonstrated, however. Additionally, the reported elevation of IOP was minor, far below the typical values induced by glaucomatous MYOC mutations. Thus, it remains unclear whether the mice truly had a form of glaucoma or whether the transgene insertion site or the level of transgene expression directly induced the RGC death. The slightly elevated IOP in this study may also reflect a greater expression level of the mutant Myoc than in the study by Gould et al., which used a knock-in strategy to introduce the mutation into the endogenous Myoc gene. Also, it is possible that the divergent results from these two studies may relate to genetic background. Senatorov et al. used a C57BL/6Hsd background rather than C57BL/6J background that was used in the study by Gould et al. RGC loss has been reported in the parental nontransgenic Hsd substrain beginning at 12–15 months, and to be as high as 46% by 18 months of age (Danias et al., 2003b).

Several potential modifier genes have been identified that may affect MYOC function. In one study, a specific MYOC mutation was associated with POAG, but the age at onset was substantially younger when patients were also heterozygous for a mutation in another glaucoma gene, CYP1B1 (Vincent et al., 2002). The CYP1B1 allele was suggested to modify the glaucoma phenotype. It is not clear, however, whether heterozygosity for the mutation truly affects the phenotype or whether it simply acts as a marker for a linked modifier gene. Similarly, an allele of optineurin was proposed to exacerbate MYOC-associated phenotypes, (Willoughby et al., 2004), but further examination is needed. These studies highlight the complexity of POAG and suggest that phenotypic differences observed between patients may be affected by modifier genes. The future analysis of mouse models with mutations in the human MYOC gene may facilitate the characterization of modifier genes relevant to the severity of glaucoma in different patients.

Optineurin (OPTN) has been associated with POAG and normal-tension glaucoma (NTG). Mutations in OPTN have been shown to cause a dominant NTG in humans (Rezaie et al., 2002). OPTN is expressed throughout the eye, including the TM, canal of Schlemm, ciliary epithelium, retina, and optic nerve. It is also expressed in neuronal and glial cells of the retina and optic nerve. Although the function of OPTN is unclear, it has been shown in vitro to interfere with TNFα-mediated apoptosis and thus could directly affect RGC survival. In a recent in vivo study, however, OPTN ectopically expressed in lens cells of transgenic mice was unable to modulate apoptosis (Kroeber et

al., 2006). This study also provided evidence for a cytoplasmic localization for OPTN rather than extracellular as reported by Rezaie et al. (2002). Thus, further characterization studies of this protein and its functions are clearly needed.

A number of OPTN sequence variants have been identified in glaucoma patients. Although the majority of these are associated with NTG, a significant number appear to be associated with POAG. However, a clear role in glaucoma has not been definitively proved for many of the alleles reported. Many of the known OPTN variants identified in glaucoma patients are also found in controls. Either these alleles are not important for glaucoma or other factors must interact with them to induce disease (Libby et al., 2005b). One possibility is that OPTN mutations do not induce glaucoma unless the genetic context is permissive; this would explain why mutations are also present in controls. This complexity can confound genotype to phenotype associations and makes it difficult to define alleles that truly contribute to disease. For these reasons, animal experiments are needed as an important complement to human studies. For instance, it will be useful to generate mice with the E50K mutation, which is clearly a disease-associated allele and leads to a severe form of NTG. A potentially useful mouse resource exists since the OPTN orthologue (Optn) of strain C57BL/6J has a glutamine (Q) at residue 552 and a lysine

(K) at residue 98 (orthologous to the POAG-associated variants R545Q and M98K, respectively). Although we have not found age-related glaucoma in C57BL/6J mice, a related mouse strain, C57BL/6Hsd, is reported to lose almost 50% of its RGCs during aging but does not appear to have elevated IOP (Danias et al., 2003b). This suggests that C57BL/ Hsd may be a useful model for studying NTG.

Collagen I. Although the iridocorneal angle remains open in POAG, extracellular alterations that develop coincident with disease progression are commonly reported. It has long been suggested that extracellular matrix (ECM) components of the ocular drainage pathways are crucial determinants of resistance to aqueous humor outflow (Scott and Wirtz, 1996). It is of interest that mice harboring a targeted mutation in the α1 subunit of collagen I (COL1A1) develop elevated IOP and that by 1 year of age lose almost 25% of their RGC axons (Aihara et al., 2003b). This mutation substitutes 5 amino acids adjacent to the MMP-1 cleavage site and completely blocks MMP-1 hydrolysis (Wu et al., 1990). These results implicate an association between fibrillar collagen turnover and IOP regulation. We have not reproduced these findings in our laboratory, however, and so environmental differences or genetic complexity must affect the phenotype. With further characterization, this will be an exciting new model with relevance to IOP elevation and RGC loss in POAG.

Developmental glaucoma. Developmental glaucoma refers to the subset of glaucomas associated with anterior segment dysgenesis. Many relevant genes have been described (see Gould, Smith, et al., 2004). Although the phenotypes often exhibit autosomal dominant or recessive inheritance, variable expressivity and incomplete penetrance point to a multifactorial etiology (see Gould and John, 2002). Many of the conditions involve obvious dysgenesis of readily visible anterior chamber structures such as the iris and pupil. In others (primary congenital glaucoma), the defects are subtle, involving abnormal development of canal of Schlemm and TM drainage structures of the iridocorneal angle.

Primary congenital glaucoma. Primary congenital glaucoma (PCG) is a severe form of early-onset glaucoma. Many PCG cases are caused by recessive mutations in the CYP1B1 gene (Libby et al., 2005b). Striking phenotypic differences exist between individuals with CYP1B1 mutations, suggesting the involvement of modifier gene(s). Consistent with this human phenotypic variability, Cyp1b1 mutant mice have focal angle abnormalities similar to those observed in PCG patients but do not develop high IOP or glaucoma. Motivated by these observations, studies have identified a modifier gene that alters the phenotype in Cyp1b1 mutant mice (Libby et al., 2003). A null allele of the tyrosinase gene (Tyr) was identified as an enhancer of angle dysgenesis. Cyp1b1-deficient mice that are also deficient for Tyr have more severe angle malformations than do mice carrying the Cyp1b1 mutation alone (Libby et al., 2003). Tyr also modified the phenotype of Foxc1-deficient mice, another gene whose orthologue causes human glaucoma. Tyrosinase produces levodopa, and it was demonstrated that administration of levodopa in the drinking water of pregnant mice deficient in both CYP1B1 and tyrosinase substantially alleviated the developmental abnormalities (Libby et al., 2003).

These experiments raise the possibility that mutations in multiple genes contributing to developmental glaucomas affect dopa levels. Dopa levels may be altered in the neural crest cells from which the angle structures and iris stroma derive. Tyrosine hydroxylase (TH) is the major enzyme responsible for producing dopa from tyrosine. Remarkably, a number of the genes that are known to cause anterior segment dysgenesis and glaucoma in humans or mice are known to promote either TH-expression or the proliferation of TH-expressing neural crest cells during development (Gould, Smith, et al., 2004). These findings demonstrate the utility of mice for defining multifactorial genetic interactions and for defining new pathways that are relevant to glaucoma.

Pigmentary glaucoma. Pigment dispersion syndrome (PDS) is a common condition that often progresses to pigmentary glaucoma. PDS involves focal iris pigment epithelial atrophy

and dispersal of liberated pigment onto anterior chamber structures and into the ocular drainage structures. Clinically, there is a radial slit-like pattern of iris transillumination. Various mechanisms have been suggested to account for the pigment dispersion, including iris rubbing against the zonules or lens, developmental defects, inherited disease of the pigment epithelium, and hypovascularity of the iris (Ritch et al., 1996; Shields, 1997), but the molecular mechanisms of pigment dispersion are not conclusively established. Although autosomal dominant inheritance has been reported, the majority of PDS cases do not exhibit clear Mendelian inheritance and likely have a multifactorial etiology.

DBA/2J mice develop a pigmentary form of glaucoma characterized by a pigment liberating iris disease, increased IOP, and optic nerve degeneration. The degree of pigment dispersion and iris destruction in DBA/2J mice is much greater than that observed in human PDS patients. This is likely explained by the discovery that DBA/2J mice are mutant for two genes that can independently cause disease but when inherited together interact to cause the severe DBA/2J phenotype (John et al., 1998). The DBA/2J disease is induced by the b allele of tyrosinase-related protein 1 gene (Tyrp1b) and a stop codon mutation in the glycoprotein (transmembrane) nmb gene (GpnmbR150X). While mice homozygous for both Tyrp1b and GpnmbR150X develop severe iris disease, single homozygotes are more mildly affected and have distinct phenotypes (figure 39.1) (Chang et al., 1999; Anderson et al., 2002). Mice homozygous for Tyrp1b develop an iris stromal atrophy phenotype, whereas mice homozygous for GpnmbR150X develop an iris pigment dispersion phenotype involving deterioration of the iris pigment epithelium.

The etiology of DBA/2J pigment dispersion lies with dysfunction of pigmented iris cells. Under normal conditions, potentially toxic intermediates generated during melanin production are sequestered inside melanosomes. The Gpnmb and Tyrp1 gene products are melanosome components, and their mutations perturb melanosome structure and appear to allow these toxic molecules to escape, damaging the cells that contain them (Chang et al., 1999; Anderson et al., 2002, 2006; Libby et al., 2005b).

In addition to being present in iris cells, Gpnmb is also present in dendritic cells. Insofar as dendritic cells are normally present in the iris and ocular outflow pathway (McMenamin, 1999; McMenamin and Holthouse, 1992), the Gpnmb mutation may alter dendritic cell function(s) and promote iris disease through ocular immune abnormalities. DBA/2J eyes have deficiencies in some aspects of immune privilege before overt pigment dispersion (Mo et al., 2003). For example, they are deficient in anterior chamber–associated immune deviation (ACAID), an active physiological process that acts to suppress pro-inflammatory responses to antigens that are first detected in the eye (Mo et al., 2003). Although

Figure 39.1 Melanosomes contribute to DBA/2J pigment dispersion. A–D, Mutations in genes encoding the melanosomal proteins TYRP1 and GPNMB cause iris disease. DBA/2J mice that are homozygous mutant for both genes have the most severe disease (D). All images are from 24-month-old mice. E, Evidence of an immune contribution to DBA/2J pigment dispersion. Bone marrow genotype has an important effect on the phenotype. See

DBA/2J eyes lack clinical signs of overt inflammation, a chronic but mild form of inflammation does attack the iris (Mo et al., 2003). Furthermore, reconstituting the immune system of DBA/2J mice with cells that express wild-type Gpnmb (through bone marrow transfer) substantially alleviates the pigment dispersion (Mo et al., 2003) (figure 39.1E ). Of note, a Gpnmb mutant immune system is not sufficient to induce the disease in otherwise wild-type mice (Mo et al., 2003). Overall, these experiments suggest that DBA/2J iris damage is initiated by melanosome toxicity and that inadequate immune suppression resulting from a susceptible immune genotype then allows an inflammatory response to propagate the disease.

In many human cases, PDS progresses to high IOP and causes pigmentary glaucoma. However, a significant number of PDS patients do not progress to high IOP. This implies that factors in addition to direct obstruction of the drainage structures by pigment are necessary to cause sustained IOP elevation. It is likely that a genetic susceptibility of the drainage tissues to a pigment/cell debris–induced pathology is needed for glaucoma progression. Mouse experiments provide strong evidence for such inherited susceptibility. The DBA/2J mutations have now been introduced into a genetically different strain background, C57BL/6J. On this

color plate 32. (Modified from John, 2005. A–D, Reproduced from Mo et al., 2003 [ Journal of Experimental Medicine, 2003, 197:1335– 1344. Copyright 2003, Rockefeller University Press]. E, Reproduced from John, 2005 [Investigative Ophthalmology and Visual Science, 2005, 46:2650–2661. Copyright 2005, Association for Research in Vision and Ophthalmology].)

new background these mutations induce the iris disease, but there is rarely progression to high IOP or glaucoma (Anderson et al., 2006). This strongly suggests that genetic susceptibility factors determine the likelihood of pigment dispersion progressing to elevated IOP. The DBA/2J and double mutant C57BL/6J strains represent powerful tools for understanding the pathways conferring susceptibility or resistance to IOP elevation.

TM alteration in DBA/2J. By the time of overt pigment liberation, large aggregates of the protein cochlin have accumulated in the TM of DBA/2J mice (Bhattacharya et al., 2005a). Interestingly, cochlin is reported to progressively accumulate in the TM of human POAG patients as well. In one study, Western analyses detected cochlin in 20 out of 20 TM samples from patients with POAG (irrespective of source, fresh trabeculectomy or cadaver glaucomatous tissue) and in 0 out of 20 TM samples from normal control tissue donors (Bhattacharya et al., 2005b). In both mice and humans, cochlin accumulates as large aggregates in acellular regions and is often associated with mucopolysaccharide deposits. Cochlin is the major noncollagenous ECM protein in the inner ear, and mutations in the cochlin gene (Coch) are pathogenic for the sensorineurial deafness and vestibular

disorder, DFNA9. Although its function in the ear is still unknown, cochlin aggregates may alter ECM function or turnover in the TM. Cochlin possesses two von Willebrand factor A (vWFA)-like domains, and as cochlin accumulates, these domains may interfere with similar vWFA-like domains that are abundant in ECM proteins present in the TM. Interestingly, both DBA/2J mice and human POAG patients show a concomitant decrease in type II collagen in parallel with cochlin accumulation. Currently, a cochlin knockout allele is being crossed to DBA/2J, and this mouse model should help determine if cochlin has a role in the IOP elevation of these mice; it may shed light on a possible role in human POAG as well (Bhattacharya et al., 2005a).

Glaucoma and neurodegeneration

The mechanisms leading to RGC death in glaucoma in response to an IOP insult are not clearly understood. Proposed theories include ischemia, excitotoxicity, autoimmunity, axonal injury, and glial activation (figure 39.2) (see Libby et al., 2005b). A combination of these insults may be active in any one patient. The relative significance of each insult likely varies between patients depending on the kinetics and magnitude of IOP elevation, the individual constellation of risk factors in the optic nerve and retina, and the effects of environment and lifestyle.

In this section we describe how the DBA/2J model is being used to understand the mechanisms involved in glaucomatous optic neuropathy. DBA/2J glaucoma shows hallmarks of human glaucoma, including age-related variable progression of optic nerve atrophy in response to elevated IOP, asynchrony, and optic nerve excavation. (A second

substrain of DBA/2, DBA/2NNia, has also been used, and in terms of glaucoma progression is considered essentially the same as DBA/2J.)

Regional Patterns of Retinal Ganglion Cell Death A diagnostic feature of human glaucoma is the occurrence of focal visual defects due to region-specific loss or impairment of RGCs. The most reliable of these are arcuate defects or scotomas measured on visual field tests (Shields, 1997). The arcuate nerve fibers originate in the temporal region of the retina and arch above or below the fovea to the optic nerve head. The molecular mechanisms inducing these defects are not well defined. In the mouse, the RGC axons do not curve across the retinal surface but radiate straight toward the optic nerve. For this reason, equivalent regional damage occurs in mice. In DBA/2 retinas with glaucoma, RGCs and their axons are lost in fan-shaped or patchy regions (figure 39.3) (Danias et al., 2003b; Jakobs et al., 2005; Schlamp et al., 2006). These fan-shaped regions of axon loss are likely to be analogous to the arcuate scotomas seen in human glaucoma.

Understanding regional patterns of retinal ganglion cell death

The link between a generalized insult, high IOP, and specific, regional deficits of RGCs in many patients is unclear. The lamina cribrosa (LC) has long been considered an important site of early damage in glaucoma (Quigley et al., 1983). In primates and humans, the LC consists of fenestrated plates of ECM covered with astrocytes. The LC is located in the optic nerve and the RGC axons pass through

Figure 39.2 Diverse insults may contribute to retinal ganglion cell (RGC) death in glaucoma. A number of commonly proposed damaging factors are shown. An array of these factors may conspire to cause glaucoma in an individual, and genetic differences will determine susceptibility or resistance to each damaging mecha-

nism. The relative importance of specific damaging processes may differ between patients. (Reproduced from Libby et al., 2005b. Reprinted with permission from the Annual Review of Genomics and Human Genetics, vol. 6, copyright © 2005 by Annual Reviews, www. annualreviews.org.)

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Figure 39.3 Whole-mounted glaucomatous retina showing fanshaped patterns of RGC loss and survival. A, High-resolution survey of a whole-mounted retina from a moderately affected eye stained for axons (green, Smi32) and amacrine cells (red, ChAT) and nuclei counterstained with TOPRO (blue). The axon bundles entering the optic nerve head are markedly reduced and show sectors of relatively high axon densities alternating with sectors with almost no persisting axons. Scale bar = 500 μm. B–E, High-power views of the boxed areas outlined in A. Scale bar = 100 μm. See color plate 33. (Reproduced from Jakobs et al., 2005 [Journal of Cell Biology, 2005, 171:313–325. Copyright © 2005, Rockefeller University Press].)

the spaces in the plates as they exit the eye. Mechanical distortion of the ECM plates of the lamina by high IOP is suggested to locally damage RGC axons in glaucoma (Quigley et al., 1983). Local damage of axon bundles would explain the regional patterns of RGC dysfunction and loss.

Although an attractive hypothesis, mechanical injury of axons by the ECM plates of the lamina is not proved. Evidence supporting this mechanical hypothesis includes reports of the first identified site of RGC damage as the axon segment in the LC (shown in both human glaucoma and primate models) (Quigley, 1995). However, identifying the first part of a neuron to degenerate does not necessarily implicate that region as the site of primary insult to the neuron. A primary insult to one region of a neuron may result in degeneration of a secondary region, especially if the secondary region has greater metabolic needs or has greater sensitivity to damage. Additionally, it is debatable whether the sometimes small pressure increases in some glaucoma patients are sufficient to cause mechanical distortion of the LC; furthermore, high IOP is not detected in all glaucoma patients. Since mice develop glaucoma but are suggested to lack a lamina with robust ECM plates (May and LutjenDrecoll, 2002), further studies evaluating the role of the LC are needed. Evidence is growing to suggest that mice do not have an ECM-based LC but instead have a cellular lamina (John, 2005) formed by astrocytes (May and Lutjen-Drecoll, 2002; Petros et al., 2006; Schlamp et al., 2006). Nevertheless, some mouse studies suggest that the RGC axons are damaged early, prior to the death of RGCs ( Jakobs et al., 2005; Schlamp et al., 2006; May and Mittag, 2006).

Evidence from experiments with DBA/2 supports the theory that the optic nerve is a site of early damage in glaucoma. Semithin sections through the optic nerve head region of DBA/2NNia mice with mild glaucoma revealed focal degeneration around the entrance of the central retinal artery into the eye at the optic nerve head, close to the retinal side of the cellular lamina. It was concluded that axons in this region may be preferentially at risk from early axon damage in glaucoma (May and Mittag, 2006). In contrast, a second study using DBA/2J mice showed that degeneration was first observed in axons proximal (with respect to the brain) to the cellular lamina and continued in a retrograde direction toward the cell body (Schlamp et al., 2006). More experiments are needed to resolve these differences and to establish the role specific components of the lamina may have in glaucoma.

Retinal ganglion cell soma death and axon degeneration

Although the distinctions are not always clear-cut, neurons can degenerate through different processes, including apoptosis, necrosis, and autophagy. It is also clear that within a single neuron, degeneration can occur by different mecha-

nisms in separate compartments (e.g., soma and axon) (see John, 2005). RGCs die by apoptosis in experimentally induced and inherited models of glaucoma (Huang et al., 2005; Reichstein et al., 2007). Huang et al. showed that calcineurin, a Ca2+ calmodulin-dependent protein phosphatase, participates in the molecular events leading to apoptosis of RGCs after experimentally induced IOP elevation (Huang et al., 2005). Of note, genetically ablating the function of BAX, a proapoptotic molecule, in DBA/2J mice prevents the death of essentially all RGC somata (figure 39.4) (Libby et al., 2005c). The axons in these mice still degenerate but at a slower rate than axons in DBA/2J mice that retain BAX function. This study uncoupled RGC soma death (completely BAX dependent) from axon degeneration (occurs without BAX). Interestingly, survival of RGC soma was seen in DBA/2J mice with only one functioning copy of Bax (D2. Bax+/−). This heterozygous deficiency mimics the quantitative variation of BAX levels that are known to occur in the human population (see Libby et al., 2005c). Thus, these data suggest that BAX is a reasonable candidate gene to assess as a modulator of susceptibility of RGC death in human glaucoma.

Future prospects for new glaucoma therapies

Now that mouse studies have uncovered mechanisms involved in the pathogenesis of glaucoma, an important goal is to apply this knowledge to the development of new therapies to treat glaucoma in humans. Most available treatments endeavor to reduce IOP levels. However, glaucoma can

occur without an elevated IOP, and symptoms may become evident only after RGC death is well under way. New treatments need to be developed that directly target RGC death and optic nerve degeneration.

Neuroprotection Many groups are now using inherited and induced models to study neuroprotection relevant to glaucoma. Over the next several years, we anticipate many important advances. Because of disease complexity, it is important to use large numbers of mice when using the inherited models (see Libby et al., 2005b). Here we highlight two of our studies in DBA/2J mice that had a large effect on disease outcome.

The protective effect of BAX deficiency in DBA/2J mice raises the possibility that BAX inhibitors may have a similar protective effect in humans. One can imagine that such treatments might block or delay RGC soma death until regenerative treatments can be developed to regrow axons. Alternatively, combination therapies may be developed involving both BAX inhibitors and drugs that target axon degeneration. These ideas can be tested first in mice.

A radiation-based neuroprotective treatment has been discovered that completely prevents glaucomatous damage in DBA/2J mice. This treatment, discovered serendipitously, entails administering a dose of lethal radiation to mice in combination with a syngeneic bone marrow transplant (figure 39.5) (Anderson et al., 2005). The treatment is administered to young mice and remains protective until old age. This protection appears to exist in humans as well, since the incidence of glaucoma is lower in populations exposed to

Figure 39.4 Bax deficiency prevents glaucomatous RGC death but is not required for optic nerve degeneration. RGC layers (A, C, E, and G) and optic nerves (B, D, F, and H) were analyzed in Bax-sufficient and Bax-deficient DBA/2J mice (genotype indi-

cated). Mice were analyzed at preglaucomatous (A–D) and severe glaucomatous (E–H) stages. Bax-deficient DBA/2J mice had severe optic nerve damage but no RGC death. See color plate 34. (Modified from Libby et al., 2005c.)