Eer-Glaucoma-Review-Libby-2015.Pdf

Experimental Eye Research xxx (2015) 1e15

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Experimental Eye Research

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Using genetic mouse models to gain insight into glaucoma: Past results and future possibilities

Kimberly A. Fernandes a, Jeffrey M. Harder b, Pete A. Williams b, Rebecca L. Rausch a, c, Amy E. Kiernan a, d, K. Saidas Nair e, Michael G. Anderson f, g, Simon W.M. John b, h, * Gareth R. Howell b, Richard T. Libby a, d, a Flaum Eye Institute, University of Rochester Medical Center, Rochester, NY, USA b The Jackson Laboratory, Bar Harbor, ME, USA c Interdepartmental Graduate Program in Neuroscience, University of Rochester Medical Center, Rochester, NY, USA d Department of Biomedical Genetics, University of Rochester Medical Center, Rochester, NY, USA e Department of Ophthalmology, University of California San Francisco, San Francisco, CA, USA f Department of Molecular Physiology and Biophysics, The University of Iowa, Iowa City, IA, USA g Center for the Prevention and Treatment of Visual Loss, Iowa City Veterans Affairs (VA) Health Care System, Iowa City, IA, USA h The Howard Hughes Medical Institute, Bar Harbor, ME, USA article info abstract

Article history: While all forms of glaucoma are characterized by a specific pattern of retinal ganglion cell death, they are Received 18 February 2015 clinically divided into several distinct subclasses, including normal tension glaucoma, primary open Received in revised form angle glaucoma, congenital glaucoma, and secondary glaucoma. For each type of glaucoma there are 16 June 2015 likely numerous molecular pathways that control susceptibility to the disease. Given this complexity, a Accepted in revised form 23 June 2015 single animal model will never precisely model all aspects of all the different types of human glaucoma. Available online xxx Therefore, multiple animal models have been utilized to study glaucoma but more are needed. Because of the powerful genetic tools available to use in the laboratory mouse, it has proven to be a highly useful Keywords: Neuroinflammation mammalian system for studying the pathophysiology of human disease. The similarity between human IOP and mouse eyes coupled with the ability to use a combination of advanced cell biological and genetic Axonal degeneration tools in mice have led to a large increase in the number of studies using mice to model specific glaucoma Trabecular meshwork phenotypes. Over the last decade, numerous new mouse models and genetic tools have emerged, Mouse genetics providing important insight into the cell biology and genetics of glaucoma. In this review, we describe Genomics available mouse genetic models that can be used to study glaucoma-relevant disease/pathobiology. Neurodegeneration Furthermore, we discuss how these models have been used to gain insights into ocular hypertension (a DBA/2J major risk factor for glaucoma) and glaucomatous retinal ganglion cell death. Finally, the potential for developing new mouse models and using advanced genetic tools and resources for studying glaucoma are discussed. © 2015 Published by Elsevier Ltd.

1. Introduction experience different stresses that trigger or contribute to cell loss (Casson et al., 2012). Age, elevated intraocular pressure (IOP; or Glaucoma is defined and unified by a distinct pattern of retinal ocular hypertension), and family history are major risk factors for ganglion cell (RGC) death. However, glaucoma is a heterogeneous glaucoma. IOP regulation is in itself complex and in most cases disorder (often termed ‘glaucomas’) with RGCs likely insulted in where IOP is a factor in glaucoma, it is not known why it becomes multiple ways. Even within a patient, different RGCs may pathologically elevated. Given this complexity, multiple models of glaucoma are required to fully understand the disease. A single animal model can never precisely model all aspects of all human * Corresponding author. Flaum Eye Institute, Department of Ophthalmology (Box glaucomas, and modeling specific aspects of the cell biology and/or 314), University of Rochester Medical Center, 601 Elmwood Ave., Rochester, NY the genetics of human glaucoma requires careful consideration. The 14642, USA. choice of the animal model should be made based on the precise E-mail address: [email protected] (R.T. Libby). http://dx.doi.org/10.1016/j.exer.2015.06.019 0014-4835/© 2015 Published by Elsevier Ltd.

Fig. 1. Glaucoma-relevant phenotypes that can be modeled and studied in mice. To study the cell biology of glaucoma, it is helpful to breakdown the disease into the individual events and/or phenotypes that can contribute to the disease. TM, trabecular meshwork; SC, Schlemm's canal; CB, ciliary body; ONH, optic nerve head; RGC, retinal ganglion cell.

Fig. 2. Tools available to study ocular disease in mice. There are many tools available to researchers using mice to study glaucoma-relevant phenotypes including: various imaging technologies, both advanced technologies which can only be used in model organisms and standard clinical imaging tools; physiological measurements to assess both anterior and posterior function; advanced cell biological analysis that can be used to probe the molecular mechanisms of glaucoma; mouse genetics, which can be used to discover new genes and molecular pathways underlying glaucoma and to critically test predicted pathways involved in glaucomatous pathophysiology; and numerous mouse models with glaucoma- relevant phenotypes that can be used to gain insight into human glaucoma. OCT, optical coherence tomography; IOP, intraocular pressure; RGC, retinal ganglion cell OMICs, genomics, proteomics and metabolomics; iPSCs, induced pluripotent stem cells; NTG, normal tension glaucoma; ASD, anterior segment dysgenesis; KI, knock in; KO, knock out; Cond, conditional allele; CRISPR, clustered regularly interspaced short palindromic repeats; TALEN, transcription activator-like effector nucleases; ZFN, xinc-ﬁnger nucleases; KOMP, knock out mouse project; EUCOMM, European Conditional Mouse Mutagenesis Program; MMRRC, Mutant Mouse Regional Resource Center; RI, recombinant inbred; CC, collaborative cross; DO, diversity outbred cross.

models can be used to examine glaucoma-relevant aspects of physiology and anatomy of glaucoma relevant structures in the anterior segment physiology. Mouse and human both have similar mouse and human anterior segment are very similar. cyclical IOP patterns (Aihara et al., 2003; Li and Liu, 2008; Liu et al., 2003; Mosaed et al., 2005; Savinova et al., 2001). Also, similar 2.2. Glaucoma relevance of the mouse retina and optic nerve head mutations lead to anterior segment disease in both mice and humans (see below). Most importantly, in terms of IOP regulation, The utility of the mouse as a model for studying ocular key physiological parameters are similar between mice and hypertension-induced RGC degeneration has been questioned in humans, including: similar mechanisms regulating aqueous humor the past primarily because of differences in the morphology and production and outflow; similar response to IOP lowering drugs; structure of the optic nerve head. The mouse optic nerve head does and no detectable washout rate in either species (e.g. Aihara et al., not have collagen beams and instead contains pores created by 2003; Boussommier-Calleja et al., 2012; Lei et al., 2011; Lindsey and optic nerve head astrocytes (this has been termed a glial lamina; Weinreb, 2002). Thus, physiological measurements show that the Howell et al., 2007). Because the optic nerve head is a key site in mouse is a good model system for studying glaucoma-relevant ocular hypertensive glaucoma, it is reasonable to assume that dif- anterior segment physiology and pathophysiology. The mouse ferences in optic nerve head morphology may limit the utility of the does however, have a proportionately larger lens than humans and mouse in modeling ocular hypertension-induced optic neuropathy. correspondingly, the shape of the anterior chamber is different in However, over the last decade, findings from several groups have mice (Schmucker and Schaeffel, 2004). With respect to glaucoma, validated the usefulness of the mouse as a model for ocular consequences of this variation have not been studied in depth, but hypertension-induced RGC death. Mouse models exhibit many would likely change the presentation of clinical features associated hallmarks thought to characterize human glaucoma, including with secondary forms of glaucoma, including iris-lens rubbing segmented axon loss, a defined insult to RGC axons at the optic (pigment dispersion syndrome) and lenticular accumulations of nerve head, early loss of axonal transport, ocular hypertension- exfoliative material (exfoliation syndrome). Overall, the basic induced abnormalities in RGC physiology, specific loss of RGCs,

Please cite this article in press as: Fernandes, K.A., et al., Using genetic mouse models to gain insight into glaucoma: Past results and future possibilities, Experimental Eye Research (2015), http://dx.doi.org/10.1016/j.exer.2015.06.019 4 K.A. Fernandes et al. / Experimental Eye Research xxx (2015) 1e15 and reduced glaucomatous damage by interventions that lower IOP relevant anterior segment disease. (Buckingham et al., 2008; Filippopoulos et al., 2006; Howell et al., DBA/2J mice are also an important resource to study the ge- 2007; Jakobs et al., 2005; Nagaraju et al., 2007; Schlamp et al., netics of ocular hypertension. C57BL/6J mice that are homozygous 2006; Schuettauf et al., 2002; Wong and Brown, 2013). Further- for the GpnmbR150X and Tyrp1b mutations develop a DBA/2J-like iris more, many molecular pathways shown to be important in human disease with severe dispersion of iris pigment (Anderson et al., glaucoma through gene expression studies have also been reported 2006). However, these mice are much less likely to develop in mouse models of ocular hypertension-induced RGC death. Thus, ocular hypertension. These results indicate that DBA/2J mice have a ocular hypertensive mouse models recapitulate key aspects of hu- greater susceptibility to IOP elevation than C57BL/6J mice. Thus, man glaucoma. genetic factors could be key mediators for how the iridocorneal angle responds to dispersed pigment in pigmentary glaucoma and 3. Glaucoma-relevant anterior segment disease other ocular hypertensive diseases. The differential response of C57BL/6J and DBA/2J mice to pigment dispersion is similar to the Over the last several decades, technical advances have greatly differing responses of people with PDS, where only some in- increased the usefulness of the mouse for studying glaucoma- dividuals with PDS develop ocular hypertension (Siddiqui et al., relevant anterior segment physiology. Intraocular pressure and 2003). The discovery of molecules or genetic pathways that aqueous outflow can now be reliably measured in the mouse segregate between DBA/2J and C57BL/6J may hold clues about (Aihara et al., 2003; Avila et al., 2001; Boussommier-Calleja and human glaucoma pathophysiology and the genetic susceptibility Overby, 2013; Cohan and Bohr, 2001; John et al., 1997; Lei et al., factors that cause/modulate glaucoma in heterogeneous 2011; Wang et al., 2005). Also, many imaging technologies used to populations. assess human anterior segment pathology are used in mice, including OCT imaging (Li et al., 2014; Nair et al., 2011), and 3.1.2. Myocilin mutant mice gonioscopy (Smith et al., 2002; Trantow et al., 2009). Advanced Mutations in the myocilin gene (MYOC) cause ocular hyperten- mouse genetic and cell biological tools are also being employed to sion and glaucoma (Menaa et al., 2011; Stone et al., 1997). In study anterior segment biology. For example, several groups humans and mice, MYOC is expressed in numerous ocular tissues, recently used genetically altered mice (conditional alleles and re- most notably in trabecular meshwork (TM) cells (for a review see; porter alleles) to study Schlemm's canal development (Aspelund Resch and Fautsch, 2009). In cultured human TM cells, MYOC is et al., 2014; Kizhatil et al., 2014; Park et al., 2014; Thomson et al., induced by glucocorticoids, oxidative stress, cell stress signaling 2014). Mouse strains also differ in IOP level, outflow rate, suscep- pathways, and potentially other unidentified factors in the aqueous tibility to steroid-induced ocular hypertension, and susceptibility to humor (Fautsch et al., 2005; Polansky et al., 1997; Tamm et al., ocular hypertension due to other anterior segment diseases. Stan- 1999). TM cells normally secrete MYOC, however mutations in dard mouse genetic tools can be used to identify the genes MYOC can prevent secretion of mutant and wild type MYOC responsible for the differences between mouse strains regarding (Jacobson et al., 2001). These data and other studies raised impor- these important glaucoma-relevant phenotypes (Anderson et al., tant questions about whether myocilin had a normal function in 2006; Boussommier-Calleja and Overby, 2013; John et al., 1997; IOP regulation and how its level and/or location of expression Savinova et al., 2001; Whitlock et al., 2010). Identifying these genes contribute to ocular hypertension. In a series of experiments, will likely provide fundamental insight into IOP regulation, opening several groups have used mouse genetics to gain a better under- up new areas of investigation and models for studying how IOP standing of the normal and pathological functions of myocilin. becomes dysregulated. Below, we describe currently available The first generation of genetic models manipulated levels of the mouse genetic models that are being used by many groups to un- mouse Myoc gene. Null mutations in mice do not result in ocular derstand anterior segment development and disease. hypertension, RGC loss, or other gross ocular abnormalities (Kim et al., 2001). There is a similar absence of glaucoma-related phe- 3.1. Ocular hypertensive mice notypes observed when Myoc is overexpressed in the mouse at levels comparable to the elevated MYOC expression seen in humans 3.1.1. DBA/2J mice with steroid-induced IOP elevation (Gould et al., 2004a). These DBA/2J mice develop a pigment dispersing iris disease that leads results helped explain genetic studies of glaucoma in human pa- to elevated IOP and RGC loss (Chang et al., 1999; John et al., 1998; tients; in humans, a likely null mutation has been identified that is Libby et al., 2005a). Mutations in two genes, Gpnmb and Tyrp1 not associated with glaucoma (Pang et al., 2002). Conversely, >90% (the mutant alleles are GpnmbR150X and Tyrp1b respectively) cause of glaucoma-related mutations are in a well-conserved functional an iris disease that has two main components: iris pigment domain of MYOC, the olfactomedin domain, and likely affect MYOC dispersion and iris stromal atrophy. The iris pigment dispersion structure (Resch and Fautsch, 2009). The results gained from ge- phenotype resembles pigment dispersion syndrome (PDS) in netic models indicated that MYOC does not have a significant humans (Anderson et al., 2002). The iris disease and subsequent function in normal IOP regulation and that mutant MYOC proteins IOP elevation can be mitigated by reconstituting the bone marrow with abnormal function (gain of function) were likely causing þ of DBA/2J mice with bone marrow from DBA/2J-Gpnmb mice ocular hypertension. (Anderson et al., 2008; Mo et al., 2003). Since bone marrow con- To determine how mutant MYOC leads to glaucoma, several tains progenitor cells of the immune system, it has been suggested groups began testing disease-specific alleles in mice. Gould et al. that the GpnmbR150X bone marrow-derived cell lineages of standard (2006) and Senatorov et al. (2006) focused on the Y437H allele, a DBA/2J mice contribute to an abnormal inflammatory response mutation that causes a severe form of juvenile open angle glau- resulting in pigment dispersion and subsequent elevation in IOP coma. Results varied, but generally, expression of mouse Myoc with (Anderson et al., 2008; Mo et al., 2003). Adaptive immune com- a mutation orthologous to Y437H affected secretion of both mutant ponents (T and B cell mediated) have been ruled out in the prop- and wild type Myoc without causing either substantially elevated agation of the iris disease (Anderson et al., 2008) and it is likely IOP or RGC loss in young or aged mice across multiple mouse driven by innate immune responses (Nair et al., 2014). Together, strains. Expression of human MYOC and MYOCY437H in the mouse these data highlight the need to further study how ocular immune lens also did not cause IOP elevation or RGC loss (Zillig et al., 2005). processes and melanosome function can contribute to glaucoma- An important finding was made when Shepard et al. used an

Please cite this article in press as: Fernandes, K.A., et al., Using genetic mouse models to gain insight into glaucoma: Past results and future possibilities, Experimental Eye Research (2015), http://dx.doi.org/10.1016/j.exer.2015.06.019 K.A. Fernandes et al. / Experimental Eye Research xxx (2015) 1e15 5 adenovirus to express human MYOCY437H in the iridocorneal angle protease, Prss56 (Nair et al., 2011). Importantly, mutations in PRSS56 and saw those eyes develop high IOP (Shepard et al., 2007). Human contribute to posterior microphthalmos or nanophthalmos in myocilin contains a targeting signal for peroxisomes that does not humans, a condition with severe reduction in ocular axial length exist in mouse myocilin (Shepard et al., 2007). This domain appears (Gal et al., 2011; Nair et al., 2011; Orr et al., 2011). As a result of this to be critical for mutant myocilin-induced toxicity in trabecular severe anatomical predisposition, some individuals go on to meshwork cells. Subsequently, three groups have reported IOP develop ACG (Gal et al., 2011; Nair et al., 2011; Orr et al., 2011). In elevation using different strategies for expressing human MYO- addition, rare variants of PRSS56 have been reported to be associ- CY437H in the mouse eye (McDowell et al., 2012; Zhou et al., 2008; ated with primary angle closure glaucoma (PACG; Jiang et al., 2013). Zode et al., 2011). Most recently, transgenic mice were generated Future studies utilizing the mutant Prss56 mouse model will pro- that express MYOCY437H under control of a CMV promoter in the vide a better understanding of ACG relevant phenotypes at a iridocorneal angle and sclera (Zode et al., 2011). Tg-MYOCY437H mice mechanistic level. Another mutant strain from an ENU-based show reduced secretion of MYOC into the aqueous humor, elevated mutagenesis screen was found to develop cataracts and high IOP IOP after 3 months of age and RGC loss after 4 months of age. Other as a result of a mutation in Tdrd7, a component of RNA granules MYOC mutations are suggested to have alternative pathogenic (Lachke et al., 2011). Interestingly, despite developing elevated IOP, mechanisms (McKay et al., 2013). Expressing these other human Tdrd7 mutants do not exhibit any gross morphological abnormal- disease-associated alleles in mice (humanized mice) will likely ities in the ocular drainage tissues and their angles are primarily provide an important method by which to distinguish the patho- open. It was hypothesized that IOP elevation in Tdrd7 mutant mice genic mechanism and test whether therapies need to be designed develops as a consequence of abnormalities in the protective stress based on specific variants. As an excellent example of this, Zode and response in the drainage tissues, in particular, oxidative stress. The colleagues showed that treating mice with a drug that reduces ER Tdrd7 mutants provide a model to study the role of RNA granules in stress significantly lessened IOP elevation in the Tg-MYOCY437H drainage tissue-specific stress responses. (Zode et al., 2011). Overall, humanized mice represent a valuable to tool to study the genetics, function, and treatment of myocilin 3.2. Developmental glaucoma models disease variants in ocular hypertension and glaucoma. Primary Congenital Glaucoma (PCG) is a less common, though 3.1.3. Other mouse genetic models for studying IOP severe subtype of glaucoma resulting from abnormal development Mouse anterior segment physiology is similar to humans and of the anterior segment. Accordingly, PCG falls under the category there is now a full range of tools to interrogate ocular fluid dy- of Anterior Segment Dysgenesis (ASD), a group of diseases char- namics in the mouse (discussed above). As a result, there are a acterized by the improper development of anterior ocular tissues. growing number of reports using both forward and reverse genetic Aberrant morphogenesis of glaucoma-relevant anterior segment approaches to study anterior segment physiology and pathophys- tissuesde.g. trabecular meshwork and Schlemm's canaldoften iology in mice. Reverse genetic approaches, specifically altering the leads to elevated IOP and ultimately, RGC death and vision loss genome, are being used to test specific hypotheses about a mole- (Fig. 1). The cellular and molecular mechanisms underlying devel- cule's role in IOP regulation (Chatterjee et al., 2014; Haddadin et al., opment of the anterior segment remain largely undefined. For 2009, 2012; Luo et al., 2014; Zhang et al., 2009). While these mu- instance, for the majority of cell types in the iridocorneal angle, tants generally have only a small increase or decrease in IOP, they including the trabecular meshwork, we do not yet understand the do provide valuable information about the molecules and pathways factors required for cell determination and commitment. Similarly, contributing to IOP homeostasis. Furthermore, this approach is for many of the genes that cause ASD, we do not know when and likely to become commonplace as more and more mouse mutants where these genes function in normal development, or how mu- with targeted alterations in specific genes are made available. A tations in them lead to ASD. Mice are ideally suited for asking basic growing number of mutant mouse alleles are being generated by developmental biological questions and assessing how mutations individual investigators and large consortiums (Schofield et al., affect tissue morphogenesis. Due to the availability of many ASD- 2012; van der Weyden et al., 2011). Liu and colleagues have made relevant mutants, the ability to genetically manipulate the several valuable Cre lines that use the Myocilin promoter to control genome in a developmentally controlled manner, and the gene expression (Liu et al., 2011). Furthermore, it is important to numerous cell biological tools available in the mouse to study develop Cre lines specific for each cell type in the anterior segment development, mice have been used extensively over the last several that contributes to IOP regulation and to make these Cre lines decades to study the molecular genetics of ASD (recently reviewed inducible, so that they can be driven at specific developmental and in depth; Ito and Walter, 2014; Reis and Semina, 2011). adult time points. Fundamental insight into the pathogenicity of human disease Forward genetic approaches in the mouse, identifying genes variants in ASD has been achieved through a combination of human based on a specific phenotype, have generated new mouse models genetics and the use of mouse models (Gould and John, 2002; as well. One example is the generation of new mutants from an N- Gould et al., 2004b; Liu and Allingham, 2011). Mice and humans ethyl-N-nitrosourea (ENU) based mutagenesis screen that was with orthologous mutations develop similar phenotypes in many designed to find mouse mutants with glaucoma-relevant anterior cases. Cloning of ASD mutations in mice has led to identifying ASD segment pathology. This approach was used to generate a mutant genes in humans (Kuo et al., 2012; McKeone et al., 2011). Addi- with cardinal features of human angle closure glaucoma (ACG) tionally, many phenotypes characteristic of ASDdciliary body and (Nair et al., 2011). In ACG, the iris is pushed against the trabecular trabecular meshwork hypoplasia, peripheral iridocorneal adhesion, meshwork (TM), thus obstructing aqueous humor outflow and buphthalmos, and elevated IOPdhave been found in both sponta- subsequently leading to elevated IOP (Quigley, 2009). Multiple neous and genetically modified mouse mutants (Chang et al., 2001; anatomical and physiological factors are thought to participate in Gould et al., 2007; Kroeber et al., 2010; Libby et al., 2003; Mao et al., the pathogenesis of ACG. Similar to patients with ACG, the mutant 2011; Sarode et al., 2014; Smith et al., 2000; Sowden, 2007; Weng mice have features that predispose them to angle closure and high et al., 2008; Zhou et al., 2013). Mutations in transcription factors IOP, including reduced ocular axial length, a relatively large lens, (PITX2, FOXC1, FOXF2, LMX1B, PAX6) are responsible for numerous and a shallow angle (Lowe, 1970; Nair et al., 2011; Quigley, 2009). In cases of ASD and PCG (Gould et al., 2004b) and mouse mutants have these mice, the causal mutation was identified in the serine been used to study the cell biology of these genes in the eye. Recent

Please cite this article in press as: Fernandes, K.A., et al., Using genetic mouse models to gain insight into glaucoma: Past results and future possibilities, Experimental Eye Research (2015), http://dx.doi.org/10.1016/j.exer.2015.06.019 6 K.A. Fernandes et al. / Experimental Eye Research xxx (2015) 1e15 work with mice carrying mutations in various cellecell signaling ability to study the mutation in the context of aging and environ- components is beginning to give us insight into the inductive mental stressors. Fortunately, there are an increasing number of events that drive anterior segment development. For instance, genetic mouse models being used to gain insight into how RGCs die BMP4, a secreted molecule belonging to the TGFb superfamily, is in both ocular hypertensive and normotensive glaucoma. believed to be necessary for proper formation of both the ciliary body and the iridocorneal angle (Chang et al., 2001; Zhou et al., 4.1. Ocular hypertensive mice 2013). Additionally, mutations in several genes influencing the extracellular matrix (e.g. CollagenIVa1, CollagenXVIIIa1, and Per- 4.1.1. DBA/2J mice oxidasin) have been shown to lead to ASD-relevant phenotypes in In DBA/2J eyes, ocular hypertension is age-related and asyn- mice (Gould et al., 2007; Mao et al., 2011; Yan et al., 2014; Ylikarppa chronous. IOP increases in the majority of eyes from 8 to 12 months et al., 2003). Recently, mouse studies demonstrated that Schlemm's of age (Libby et al., 2005a). By 10 months of age, approximately half canal develops from existing blood vessels by a newly discovered of DBA/2J eyes have significant optic nerve damage and by 12 process of vascular development with features of vasculogenesis, months, the majority of eyes have severe glaucomatous damage angiogenesis and lymphangiogenesis (Aspelund et al., 2014; (Libby et al., 2005a). Reducing IOP in DBA/2J mice pharmaceutically, Kizhatil et al., 2014; Park et al., 2014; Thomson et al., 2014). genetically, or through surgical therapy prevents significant RGC Various vascular receptors are expressed during this process damage (Anderson et al., 2006; Matsubara et al., 2006; Schuettauf including KDR (VEGFR2) and TEK (TIE2), with KDR function being et al., 2002; Wong and Brown, 2012, 2013). Thus, RGC loss in DBA/2J essential for normal development of Schlemm's canal (Kizhatil mice is dependent upon ocular hypertension. DBA/2J mice have et al., 2014). These receptors and their downstream proteins are been used to study many cell biological questions relevant to ocular excellent candidates to contribute to PCG and other developmental hypertension-induced optic nerve degeneration and RGC death. glaucomas (Kizhatil et al., 2014; Thomson et al., 2014). Together, Below, we describe a few examples where DBA/2J mice were used these results implicate many different developmental events and in combination with mouse genetic tools or pharmaceutical in- signaling pathways in the morphogenesis of anterior segment terventions to study glaucoma-relevant RGC death and optic nerve structures. On an even more basic level, these studies and others injury (see Fig. 3 for examples of genetic or pharmaceutical have identified factors involved in patterning the mammalian eye manipulation that have been used in DBA/2J mice to dissect the as well as the general time line of events in anterior segment pathological processes involved in ocular hypertension-induced morphogenesis (Cvekl and Tamm, 2004; Heavner and Pevny, 2012; RGC death). Ito and Walter, 2014; Napier and Kidson, 2007; Smith et al., 2001). There is ample evidence that RGCs die by apoptosis in human It is important to note that having any form of ASD puts children glaucoma and in various mouse models of glaucoma (Guo et al., at a 50% increased risk for developing glaucoma later in life 2005; Nickells, 1996, 1999; Nickells et al., 2008; Quigley et al., (Sowden, 2007), however it is unknown precisely why this occurs. 1995; Whitmore et al., 2005). In DBA/2J mice, several compo- It is also possible that clinically undetectable abnormalities in the nents of the Bcl2 family (major regulators of apoptotic death) are anterior segment in early childhood could significantly contribute expressed in RGCs during the window of RGC death (Harder et al., to IOP elevation later in life. Furthermore, clinical severity of ASD 2012; Libby et al., 2005c). Deficiency in the proapoptotic Bcl2 family does not appear to correlate with the development of glaucoma, member Bax, prevented RGC somal loss in DBA/2J mice but did not suggesting that clinically undetectable changes are critical for the prevent axonal degeneration (Libby et al., 2005c). Interestingly, development of ocular hypertension (Ito and Walter, 2014). ASD DBA/2J mice expressing a mutant form of FasL had an earlier onset appears to be highly susceptible to genetic modifiers in both and more severe loss of RGCs. These data suggest that modulating humans and mice (Gould et al., 2007, 2004b; Ito and Walter, 2014; FasL signaling may reduce RGC apoptosis in glaucoma and indicate Libby et al., 2003). The ability to perform precise histological that the extrinsic apoptotic pathway is capable of activating ocular analysis and measure glaucoma-relevant physiological parameters hypertension-induced RGC death (Gregory et al., 2011). During the (e.g. IOP and outflow measurements) make the mouse ideally window of RGC death in DBA/2J glaucoma, several pathological suited to study how ASD leads to ocular hypertension and to insults have been observed including axonal injury at the glial identify the morphological and genetic factors controlling this lamina, structural and functional alterations in RGCs, mitochondrial progression. dysfunction, glial dysfunction, neuroinflammation and vascular dysfunction. Along with dissociating the insults that accompany 4. Glaucoma-relevant RGC death in mice RGC degeneration from the insults that trigger RGC degeneration, determining how these cell intrinsic and cell extrinsic events finally Standard genetic tools, such as genetically modified mice, have converge on BAX activation leading to the execution of RGC death is been used extensively to study glaucoma-relevant RGC loss. Also, an area of ongoing investigation. other methods have been used in mice to test various hypotheses of Anderson, Hendricks, Quigley, and others suggested that axonal glaucoma-relevant RGC death and the effectiveness of particular injury is an important early event in human glaucoma and in pri- treatments at stopping RGC loss. Such methods include using vi- mate models of glaucoma (Anderson and Hendrickson, 1974, 1977; ruses to deliver or knockdown genes specifically in RGCs and using Quigley and Anderson, 1976; Quigley and Addicks, 1980, 1981). In microbeads or stem cells to deliver therapeutic substances DBA/2J mice, an early site of axonal injury is at the glial lamina (reviewed in Harvey et al., 2009; Lebrun-Julien and Di Polo, 2008). (Buckingham et al., 2008; Howell et al., 2007; Jakobs et al., 2005; Acute injuries in mice, such as mechanical optic nerve crush or Schlamp et al., 2006). Consistent with RGC axons being insulted intravitreally injecting substances hypothesized to injure RGCs in at the glial lamina, in Bax deficient DBA/2J mice, RGC axons sur- glaucoma have provided important cell biological information vived to the optic nerve head while the axon behind the glial lamina about RGC death; though, it is likely that these acute perturbations degenerated. Further support for a key role of axonal injury in DBA/ do not model key aspects of glaucomatous neurodegeneration. 2J mice was provided by the finding showing DBA/2J mice that Genetic models offer important advantages over other model sys- express the WldS allele, a naturally occurring mutation that slows tems for studying glaucoma-relevant RGC death as they often show axonal degeneration (Fang et al., 2005; Lyon et al., 1993; Mack et al., a greater variability and slower progression of IOP elevation (more 2001; Perry et al., 1992), were significantly protected from both akin to many human glaucomas), in addition to providing the axonal and somal degeneration compared to wild type DBA/2J mice

Fig. 3. Ocular hypertension leads to numerous events in distinct subcompartments of RGCs that contribute to RGC loss in DBA/2J mice. DBA/2J mice have been used extensively to study the pathogenesis of ocular hypertension-induced RGC death. These studies have led to the idea that different cellular compartments of RGCs respond to ocular hypertension and that targeting these events can lessen RGC loss. This diagram assumes a critical early injury occurs to RGCs axons (axonal injury) in the optic nerve head. However, it is unclear (dashed lines) whether the initial driving event is an insult directly to the axons or through extrinsic means (the gray line is used to separate these potential early events to the later important event of axonal injury). Red boxes represent either pharmaceutical or genetic interventions that blocked a speciﬁc event in the degeneration cascade and have lessened RGC loss in DBA/2J mice.

(Howell et al., 2007). Thus, axonal injury response and axonal Howell et al., 2011; Kuehn et al., 2006; Orsini et al., 2014; Ren degeneration appear to be key pathological insults for ocular and Danias, 2010; Stasi et al., 2006; Stevens et al., 2007; Tezel hypertension-induced RGC death in DBA/2J mice. It will be et al., 2010). DBA/2J mice are naturally deficient in complement important to identify the genes that control these events, as they component C5 (a key protein in the formation of the membrane could be therapeutic targets for human glaucoma. attack complex), and C5 sufficient DBA/2J show more RGC loss than The work in DBA/2J mice complements studies in human standard DBA/2J mice, suggesting C5 plays a damaging role after an glaucoma and in other animal models, showing that early changes ocular hypertensive injury. Similarly, C1qa mutant DBA/2J mice occur in multiple compartments of an RGC after an ocular hyper- have less RGC loss, suggesting that the initiating C1 complex of the tensive insult (reviewed in Almasieh et al., 2012; Morquette and Di classical arm of the complement cascade is damaging in glaucoma Polo, 2008; Whitmore et al., 2005; Yu et al., 2013). Along with (Howell et al., 2011, 2014, 2013). Interestingly, C5 seems much less axonal stress/injury, numerous studies have also shown early important than C1, as C1qa deficiency is much more protective than changes in other RGC compartments in response to ocular hyper- C5 deficiency, suggesting that substantial damaging effects of C1 tension, including changes in the soma and dendrites. Crish et al. are independent of C5 and completion of the classical arm of the (2010) showed failure of axonal transport begins in the distal complement cascade. It is unclear how decreasing complement axon and progresses toward the soma in DBA/2J mice. Early RGC activation protects RGCs, though complement has been implicated dendritic remodeling has also been described in retinas of DBA/2J in various pathological processes thought to be involved in ocular mice (Stevens et al., 2007; Williams et al., 2013). In a series of pa- hypertension-induced RGC death in DBA/2J mice, including syn- pers, Ju, Weinreb and colleagues established the potential impor- aptic remodeling and monocyte recruitment (Fig. 3; Howell et al., tance of mitochondria physiology in regulating RGC loss in DBA/2J 2011, 2013, 2012; Stevens et al., 2007). mice (Ju et al., 2009, 2010, 2008; Lee et al., 2014). Abnormalities in Neuroinflammation is often linked closely with vascular mitochondrial dynamics correlated with ocular hypertension- compromise and changes in vascular tone have been reported in induced RGC loss. Importantly, genetically or pharmacologically glaucoma (Gerber et al., 2014). Further implicating the vasculature protecting mitochondrial function also lessened glaucomatous in glaucoma is the suggestion that blood pressure may play a role in damage in DBA/2J mice. Determining whether the molecular glaucoma (Chauhan, 2008; Flammer et al., 2013; Prasanna et al., pathways responsible for these structural and functional alter- 2005, 2002, 2011; Shoshani et al., 2012). The endothelin system, ations in distinct RGC compartments interact with each other, and which controls a variety of processes including blood pressure and defining which changes are critical for RGC death should be a pri- astrocyte activation has recently been implicated in neurodegen- ority of future research. erative conditions (Jo et al., 2014). Components of the endothelin Neuroinflammation has been implicated both in human glau- system are upregulated in human and animal models of glaucoma coma (Baltmr et al., 2010; Cheung et al., 2008; Soto and Howell, (Howell et al., 2011; Krishnamoorthy et al., 2008; Prasanna et al., 2014) and in RGC loss in DBA/2J mice (Anderson et al., 2008; 2005, 2002; Rattner and Nathans, 2005). Intravitreally injected Danesh-Meyer, 2011; Fan et al., 2010; Howell et al., 2011, 2012; EDN1 or END2 peptide kills RGCs in rodents (Howell et al., 2011; Nickells et al., 2012; Zhou et al., 2005). The complement system, a Krishnamoorthy et al., 2008; Rattner and Nathans, 2005), espe- component of the innate immune system, is a key part of the cially in the presence of elevated IOP (Howell et al., 2012). Treating neuroinflammatory response and complement activation has been DBA/2J mice with endothelin receptor antagonists both slowed and reported in human glaucoma and in animal models of glaucoma lessened the severity of RGC axonal damage, suggesting that (Brennan et al., 2012; Fan et al., 2010; Harvey and Durant, 2014; endothelin signaling has a significant role in ocular hypertension-

Please cite this article in press as: Fernandes, K.A., et al., Using genetic mouse models to gain insight into glaucoma: Past results and future possibilities, Experimental Eye Research (2015), http://dx.doi.org/10.1016/j.exer.2015.06.019 8 K.A. Fernandes et al. / Experimental Eye Research xxx (2015) 1e15 induced RGC death (Howell et al., 2011, 2014). Interestingly, axonal factors involved in response to IOP elevation (Cone et al., 2010; loss in the DBA/2J mouse has been associated with monocyte entry Steinhart et al., 2012). The large number of models being used into the optic nerve head (Howell et al., 2012), further implicating and developed will allow researchers to validate results in multiple neuroinflammatory responses. An important future direction will diverse models, species, and strains within species. be to mechanistically explore the roles of neuroinflammation and vascular dysfunction in glaucoma. However, this work is 4.2. Normal tension glaucoma models confounded by the fact that components of these systems are activated in both aging, a major risk factor for glaucoma, and Normal tension glaucoma (NTG) is a subset of primary open multiple cell types, including astrocytes, RGCs, microglia and cells angle glaucoma that is characterized by RGC loss and optic neu- associated with the vasculature. Here, the mouse can play a major ropathy in the absence of detecting elevated IOP. NTG is an inter- role in determining the beneficial and/or damaging roles of specific esting type of glaucoma because it suggests that factors intrinsic to genes and cell types in aging and glaucoma using conditional alleles retina and/or optic nerve are a primary site of pathophysiology. whereby genes are ablated in particular cell types. Importantly, as Below we highlight three different mouse models of NTG, where anti-inflammatory therapies are already commonplace, the inhi- mice have been used to gain insight into mechanisms that lead to bition of glaucomatous damage using anti-inflammatory ap- RGC loss. proaches poses a tangible therapeutic option. Glia are involved in many different processes that can contribute 4.2.1. Optineurin (OPTN) to neurodegeneration, including neuroinflammation (Maragakis The OPTN E50K mutation was the first mutation identified in and Rothstein, 2006; Mrak and Griffin, 2005; Verkhratsky et al., familial normal tension glaucoma (Rezaie et al., 2002). Transgenic 2014). Microglia, Müller glia and optic nerve head astrocytes have mice overexpressing the optineurin E50K mutation (E50Ktg)were been hypothesized to play critical roles in glaucomatous neuro- made to investigate the biology of this specific mutation (Chi et al., degeneration (Chong and Martin, 2015; Seitz et al., 2013). Both 2010a). E50Ktg mice did not have elevated IOP but did have loss of retinal and optic nerve head glia have been implicated in ocular RGCs (~25% loss by 16 months of age). A caveat of the E50Ktg model hypertensive RGC death in DBA/2J mice. Astrocytes at the optic is the significant thinning of the ONL and INL and loss of other nerve head have been shown to undergo morphological changes retinal neurons, including amacrine cells, bipolar cells, and photo- prior to RGC death (Lye-Barthel et al., 2013). Also, gene expression receptors (Chi et al., 2010a), cell loss that is not generally observed studies have suggested that molecular changes in astrocytes could in glaucoma patients. The high expression levels of E50K OPTN in be involved in the pathogenesis of RGC death in DBA/2J mice the E50Ktg mice were hypothesized to result in non-specific loss of (Howell et al., 2011, 2014; Nguyen et al., 2011). Microglial activation other retinal neurons through a toxic gain of function mechanism. has been correlated with RGC death (Bosco et al., 2015) and To test this hypothesis a BAC transgenic mouse expressing human reducing microglial activation has been suggested to increase RGC E50K OPTN (BAC-hOPTNE50K) close to physiological levels was viability in DBA/2J mice (Bosco et al., 2008, 2015; Inman and made (Tseng et al., 2015). Interestingly, in these mice there was age- Horner, 2007). It will be important to functionally test specific as- related loss of RGCs without apparent loss of other retinal cell pects of glial activation in DBA/2J mice to precisely define the role of types. Thus, BAC-hOPTNE50K mice are a valuable resource for glia in the disease process. gaining insight into the molecular mechanisms of RGC death in In summary, multiple pathways, both intrinsic and extrinsic to NTG. In the mouse eye, OPTN is expressed in RGCs, as well as in the RGCs, have been suggested to contribute to ocular hypertension- cornea, trabecular meshwork, ciliary epithelium and iris (Kroeber induced RGC death in DBA/2J mice (Fig. 3). However, the initial et al., 2006) and it remains unclear why mutations in OPTN lead molecular changes that trigger neuronal injury remain unclear. to RGC loss. Optineurin functions in several physiological processes, Highlighting the complexity of glaucoma, numerous cell biological most prominently in secretory vesicle transport (Bond et al., 2011) events are suggested as key players in the disease, including axonal and autophagy (Wild et al., 2011). Determining which, if any, of injury, inflammation, glial activation, growth factor deficiency, these processes is critical for RGC death in NTG is an important mitochondrial dysfunction, and synapse withdrawal. Thus, there is future direction. still much to be learned about RGC loss in DBA/2J mice. It will be important to continue to critically test molecules and pathways 4.2.2. WD-repeat-containing protein 36 (WDR36) hypothesized to be important through functional tests, including Several studies have reported an association between WDR36 assessing their roles in specific cell types and determining critical and both NTG and POAG. Interestingly, these studies have shown molecular events. Careful functional testing of any specific pathway that WDR36 can be causative for glaucoma and be a susceptibility in multiple models (DBA/2J as well as others) will identify potential factor for developing glaucoma (Allingham et al., 2009; Hauser targets for therapies to prevent glaucomatous neurodegeneration. et al., 2006; Monemi et al., 2005). WDR36 is expressed throughout the eye, including in tissues important in glaucoma 4.1.2. Other models for studying ocular hypertension-induced RGC pathogenesis (sclera, iris, ciliary muscle, ciliary body, retina, and death optic nerve; Monemi et al., 2005). We have included WDR36 in the Along with genetic models, there are inducible models of IOP NTG section because of the data generated from mice (discussed elevation that are being used to study ocular hypertension-induced below); however, we realize that it could potentially be included in RGC death, including, microbead injections (Morgan and Tibble, the ocular hypertension discussion or in a section focusing on 2015; this issue), episcleral vein and aqueous outflow sclerosis general susceptibility factors. Several mouse mutant alleles have (Morrison et al., 2015; this issue), acute IOP elevation (Crowston been generated to study the role of WDR36 in ocular biology and et al., 2015; this issue), glucocorticoid-induced IOP elevation disease. Unfortunately, mice homozygous for a null allele of Wdr36 (Overby and Clark, 2015; this issue), and viral gene transfer to (Wdr36 / mice) die preimplantation (Gallenberger et al., 2011). þ generate TM cell dysfunction and resulting IOP elevation (Pang Wdr36 / mice do not have RGC loss at 12 months, nor do RGCs in et al., 2015; this issue). For instance, the microbead model has these mice show increased susceptibility to death when exposed to been used to test the importance of a variety of molecular pathways an excitotoxic or ocular hypertensive insult (Gallenberger et al., in ocular hypertension induced RGC death (Hu et al., 2012; Huang 2014). There have been 3 mouse alleles generated that have been et al., 2011; Ward et al., 2014) and to investigate susceptibility designed to either create an orthologous mutation to that found in

Please cite this article in press as: Fernandes, K.A., et al., Using genetic mouse models to gain insight into glaucoma: Past results and future possibilities, Experimental Eye Research (2015), http://dx.doi.org/10.1016/j.exer.2015.06.019 K.A. Fernandes et al. / Experimental Eye Research xxx (2015) 1e15 9 human glaucoma patients or to disrupt molecular function in a provide a helpful addition to the toolbox for studying NTG, espe- region of the gene with an orthologous mutation (Chi et al., 2010b). cially if the pathophysiology involves an aging component or re- Transgenic mice overexpressing one of these alleles, Wdr36 quires the interaction between different cell types. As new genes (Del605-607), had a dramatic reduction in peripheral retinal are found to cause NTG, regardless of the nature of the mutation, thickness and loss of RGCs as well as amacrine cells and bipolar mouse mutants will be important tools for determining the path- cells at 16 months of age (Chi et al., 2010b). Wdr36 (Del605-607) ophysiological mechanisms resulting from the mutations. mutant mice do not have elevated IOP and the lens, cornea, and anterior segment of these mice appear to be histologically normal. 5. The future of genetic models of glaucoma In the future, it will be important to make a conditional allele of Wdr36 so that it can be deleted selectively in adult trabecular The genomics revolution over the last twenty years is beginning meshwork, RGCs, or optic nerve head astrocytes to allow assess- to bear fruit for glaucoma research and a steady stream of genes ment of the tissue-specific function of Wdr36 in the pathogenesis of and genetic loci are being discovered that are linked or associated glaucoma. Also, it will be important to assess the effects of mutant with glaucoma. Recent large-scale genome wide association studies mouse alleles that have orthologous mutations to those found in (GWAS; Hysi et al., 2014; Springelkamp et al., 2014) have identified human glaucoma patients knocked into the mouse Wdr36 locus. genes/loci associated with glaucoma-relevant phenotypes. How- ever, in some cases, it is not clear how a gene or a specific allelic 4.2.3. Glutamate transporters variant impacts the disease. For instance, while CDKN2B-AS1,an To our knowledge, there are no hereditary studies that implicate anti-sense transcript that is transcribed on the opposite strand to alleles involved in glutamate regulation in human NTG or in any CDKN2A and CDKN2B, is strongly associated with glaucoma, the other form of glaucoma. However, elevated levels of extracellular function of CDKN2B-AS1 remains unclear (Burdon et al., 2012, 2011; glutamate cause RGC death and mice deficient in the glutamate Ng et al., 2014). In a second example, copy number variations in transporters, GLAST or EAAC1, exhibit RGC degeneration with TBK1 are associated with glaucoma, and although the function of varying time courses. GLAST deficient mice had a progressive the gene is better understood (Helgason et al., 2013; Minegishi attrition of RGCs with as much as a 50% loss by 8 months (Harada et al., 2013; Tucker et al., 2014; Weidberg and Elazar, 2011), the et al., 2007). These mice were also suggested to have other hall- reason for why copy number variations that include TBK1 impact marks of glaucomatous neurodegeneration including optic nerve glaucoma progression is less clear. cupping and degenerating axons in the optic nerve. EAAC1 deficient A major limitation of GWAS is that it generally implicates ge- mice had RGC degeneration as early as 8 weeks of age, but also have netic loci and not candidate variants. Plus, GWAS analysis cannot thinning of the inner retina (Harada et al., 2007). At 8 weeks of age, find evolutionarily recent or low frequency genetic variants there is a 20% reduction in the number of cells in the ganglion cell (Manolio et al., 2009). To address this, studies are now beginning to layer in EAAC1 deficient mice. Both GLAST and EAAC1 deficient take advantage of next generation sequencing (NGS) of human mice have normal IOP and normal iridocorneal angle morphology, samples, using targeted exome and whole genome sequencing suggesting IOP does not directly cause RGC death in these mice approaches. However, progress with these approaches will often (Harada et al., 2007). Both glutamate toxicity and oxidative stress not be straightforward and complementary animal and human have been hypothesized to contribute to RGC loss in GLAST and studies will often be necessary. This is due to both the complex EAAC1 deficient mice (Kimura et al., 2015; Namekata et al., 2013; nature of glaucoma, where variations in multiple genes contribute Semba et al., 2014). Glutamate receptor antagonists lessened RGC to the disease, and to difficulty ascribing causality to any specific death at early time points in GLAST deficient mice, indicating that variants among the large numbers of variants detected by glutamate neurotoxicity contributes to RGC degeneration in these sequencing. As with GWAS, NGS studies will likely need to involve mice (Harada et al., 2007; Namekata et al., 2013). Similarly, atten- large numbers of samples coordinated in a consortium-like manner uating oxidative stress was shown to reduce RGC loss in GLAST that has proved successful for GWAS (e.g. the Neighbor and knockout mice (Kimura et al., 2015). Collectively, the data from Neighborhood studies Burdon et al., 2011; Lu et al., 2013; animal models of glutamate transporter loss show that both RGC Springelkamp et al., 2014; Wiggs et al., 2013, 2012). The discovery intrinsic as well as extrinsic mechanisms contribute to RGC that naturally existing nucleases, first zinc fingers (Urnov et al., degeneration. It will be important in the future to determine if this 2010) then TALENS (Joung and Sander, 2013) and now CRISPRs system is involved in RGC loss in NTG in humans. (Sander and Joung, 2014) can be used to alter single bases or a few bases (‘genome editing’), means that genetically engineered mice 4.2.4. Summary of NTG models can be created in months rather than years. Further advances that Thus far, mouse models of NTG have revealed a diversity of include incorporating larger stretches of DNA into the mouse physiological processes that when disrupted in the retina can lead genome will enable insertion of human gene sequences into the to RGC degeneration independent of IOP elevation. An important mouse genome and the generation of conditional alleles and new caveat with some of the NTG mouse models is that they appear to Cre driver lines to study the temporal and spatial function(s) of have loss of other retinal neurons, which is generally thought to be genes. Thus, new genomic technologies will allow us to readily inconsistent with glaucomatous neurodegeneration. Furthermore, assess putative causal variants identified in human genetic studies there are other genetic causes of NTG that remain to be modeled. in mice. An important goal should be to incorporate genetic vari- For instance, copy number variants of TBK1 cause NTG (Awadalla ants implicated in human glaucoma into mouse models, improving et al., 2015; Ritch et al., 2014). The normal physiology and patho- utility for preclinical and translational studies. These experiments physiology of altered TBK1 expression remain to be determined. can complement genetic manipulation strategies in human tissues/ Using induced pluripotent stem cells from patients with TBK1 copy cells, including the ability to assess variants in specific cell types number variations, evidence is emerging that autophagy is derived using induced pluripotent stem cells (IPSCs; Ding et al., enhanced in these cells (Tucker et al., 2014). Mice are routinely used 2014; Tucker et al., 2014). for studying the effects of copy number variation and can easily be The mouse can play other key roles in the development of made to express different levels of specific human or mouse alleles improved therapies for glaucoma. For instance, human genetic and (Le et al., 2003; Smithies and Kim, 1994; van der Weyden and genomic studies are difficult because of the obvious limitations of Bradley, 2006). Modeling TBK1 expression level variants should working with human subjects and samples. Therefore, the mouse

Please cite this article in press as: Fernandes, K.A., et al., Using genetic mouse models to gain insight into glaucoma: Past results and future possibilities, Experimental Eye Research (2015), http://dx.doi.org/10.1016/j.exer.2015.06.019 10 K.A. Fernandes et al. / Experimental Eye Research xxx (2015) 1e15 can continue to inform us about novel genes and pathways that 31, 152e181. impact glaucoma. These experiments can include hypothesis driven Anderson, D.R., Hendrickson, A., 1974. Effect of intraocular pressure on rapid axoplasmic transport in monkey optic nerve. Invest. Ophthalmol. 13, 771e783. approaches where the importance of individual genes can be Anderson, D.R., Hendrickson, A.E., 1977. Failure of increased intracranial pressure to assessed. Alternatively, unbiased approaches should continue to be affect rapid axonal transport at the optic nerve head. Invest. Ophthalmol. Vis. e pursued where genes are mutated at random (for instance, by Sci. 16, 423 426. Anderson, M.G., Libby, R.T., Mao, M., Cosma, I.M., Wilson, L.A., Smith, R.S., John, S.W., ethylnitrosourea mutagenesis). This approach has already identi- 2006. Genetic context determines susceptibility to intraocular pressure eleva- fied novel genes for glaucoma research (e.g. Cross et al., 2014; tion in a mouse pigmentary glaucoma. BMC Biol. 4, 20. Lachke et al., 2011; Nair et al., 2011; Van Agtmael et al., 2005). Anderson, M.G., Nair, K.S., Amonoo, L.A., Mehalow, A., Trantow, C.M., Masli, S., John, S.W., 2008. GpnmbR150X allele must be present in bone marrow derived Given the genetic complexity of glaucoma, sensitized approaches cells to mediate DBA/2J glaucoma. BMC Genet. 9, 30. are also being pursued where mutagenesis results in mice carrying Anderson, M.G., Smith, R.S., Hawes, N.L., Zabaleta, A., Chang, B., Wiggs, J.L., genetic mutations that increase susceptibility for glaucoma- John, S.W., 2002. Mutations in genes encoding melanosomal proteins cause pigmentary glaucoma in DBA/2J mice. Nat. Genet. 30, 81e85. relevant phenotypes but do not directly cause glaucoma. Unbi- Aspelund, A., Tammela, T., Antila, S., Nurmi, H., Leppanen, V.M., Zarkada, G., ased genetic approaches may be greatly aided by incorporating the Stanczuk, L., Francois, M., Makinen, T., Saharinen, P., Immonen, I., Alitalo, K., latest generation of inbred and outbred mouse crosses, (collabo- 2014. The Schlemm's canal is a VEGF-C/VEGFR-3-responsive lymphatic-like e rative cross and diversity outbred cross respectively; Chesler et al., vessel. J. Clin. Invest. 124, 3975 3986. Avila, M.Y., Carre, D.A., Stone, R.A., Civan, M.M., 2001. Reliable measurement of 2008; Churchill et al., 2004, 2012). These are currently the most mouse intraocular pressure by a servo-null micropipette system. Invest. Oph- powerful strains for identifying genetic variations that impact thalmol. Vis. Sci. 42, 1841e1846. complex disease, and are ideal for mapping glaucoma-relevant Awadalla, M.S., Fingert, J.H., Roos, B.E., Chen, S., Holmes, R., Graham, S.L., Chehade, M., Galanopolous, A., Ridge, B., Souzeau, E., Zhou, T., Siggs, O.M., modifier genes. Diversity outbred mice are also ideal for assessing Hewitt, A.W., Mackey, D.A., Burdon, K.P., Craig, J.E., 2015. Copy number varia- efficacy and pharmacokinetics in the preclinical space where po- tions of TBK1 in Australian patients with primary open-angle glaucoma. Am. J. e tential new drugs can be tested in genetically unique mice under Ophthalmol. 159, 124 130 e121. Baltmr, A., Duggan, J., Nizari, S., Salt, T.E., Cordeiro, M.F., 2010. Neuroprotection in controlled environmental conditions. Finally, as the number of glaucoma e is there a future role? Exp. Eye Res. 91, 554e566. mouse strains with unique mutations and engineered manipula- Bond, L.M., Peden, A.A., Kendrick-Jones, J., Sellers, J.R., Buss, F., 2011. Myosin VI and tions relevant to glaucoma continue to grow, so will the need for its binding partner optineurin are involved in secretory vesicle fusion at the e fi plasma membrane. Mol. Biol. Cell. 22, 54 65. ef cient sharing of them amongst investigators and institutions. Bosco, A., Inman, D.M., Steele, M.R., Wu, G., Soto, I., Marsh-Armstrong, N., Several commercial and non-profit repositories for sharing mice Hubbard, W.C., Calkins, D.J., Horner, P.J., Vetter, M.L., 2008. Reduced retina exist (see Fig. 2 for some examples) and it will be critical that our microglial activation and improved optic nerve integrity with minocycline treatment in the DBA/2J mouse model of glaucoma. Invest. Ophthalmol. Vis. Sci. community remains dedicated to using them fully. 49, 1437e1446. Mice can also be used to probe some of the outstanding ques- Bosco, A., Romero, C.O., Breen, K.T., Chagovetz, A.A., Steele, M.R., Ambati, B.K., tions about complex physiological interactions that may be Vetter, M.L., 2015. Neurodegeneration severity is anticipated by early microglia important in glaucoma. For instance, it has been suggested that alterations monitored in vivo in a mouse model of chronic glaucoma. Dis. Model. Mech. 8, 443e455. both blood pressure (He et al., 2014; Moore et al., 2008) and Boussommier-Calleja, A., Bertrand, J., Woodward, D.F., Ethier, C.R., Stamer, W.D., intracranial pressure (Fleischman and Allingham, 2013) help Overby, D.R., 2012. Pharmacologic manipulation of conventional outflow facility e mediate glaucoma susceptibility. It is possible to manipulate these in ex vivo mouse eyes. Invest. Ophthalmol. Vis. Sci. 53, 5838 5845. Boussommier-Calleja, A., Overby, D.R., 2013. The influence of genetic background on traits in mice along with IOP to determine the importance of these conventional outflow facility in mice. Invest. Ophthalmol. Vis. Sci. 54, parameters in RGC susceptibility to IOP elevation (Nusbaum et al., 8251e8258. 2015; Takahashi and Smithies, 2004; Verouti et al., 2015). Also, Brennan, F.H., Anderson, A.J., Taylor, S.M., Woodruff, T.M., Ruitenberg, M.J., 2012. Complement activation in the injured central nervous system: another dual- recent work is starting to examine endophenotypes of glaucoma, edged sword? J. Neuroinflammation 9, 137. such as corneal thickness and optic disc morphology in mice Buckingham, B.P., Inman, D.M., Lambert, W., Oglesby, E., Calkins, D.J., Steele, M.R., (Harder et al., 2012; Lively et al., 2010a, 2010b; Prasov et al., 2012). Vetter, M.L., Marsh-Armstrong, N., Horner, P.J., 2008. Progressive ganglion cell degeneration precedes neuronal loss in a mouse model of glaucoma. J. Neurosci. These studies will open up avenues of research examining how 28, 2735e2744. these phenotypes affect the pathophysiology of glaucoma. Overall, Burdon, K.P., Crawford, A., Casson, R.J., Hewitt, A.W., Landers, J., Danoy, P., mice have provided important cell biological insight into Mackey, D.A., Mitchell, P., Healey, P.R., Craig, J.E., 2012. Glaucoma risk alleles at CDKN2B-AS1 are associated with lower intraocular pressure, normal-tension glaucoma-relevant phenotypes. In the future, continually inte- glaucoma, and advanced glaucoma. Ophthalmology 119, 1539e1545. grating information gained from human glaucoma studies into Burdon, K.P., Macgregor, S., Hewitt, A.W., Sharma, S., Chidlow, G., Mills, R.A., mouse models will further enhance the power of mice for studying Danoy, P., Casson, R., Viswanathan, A.C., Liu, J.Z., Landers, J., Henders, A.K., glaucoma pathophysiology. Wood, J., Souzeau, E., Crawford, A., Leo, P., Wang, J.J., Rochtchina, E., Nyholt, D.R., Martin, N.G., Montgomery, G.W., Mitchell, P., Brown, M.A., Mackey, D.A., Craig, J.E., 2011. Genome-wide association study identifies susceptibility loci for e Acknowledgments open angle glaucoma at TMCO1 and CDKN2B-AS1. Nat. Genet. 43, 574 578. Casson, R.J., Chidlow, G., Ebneter, A., Wood, J.P., Crowston, J., Goldberg, I., 2012. Translational neuroprotection research in glaucoma: a review of definitions and This work was supported by The Glaucoma Foundation (RTL, principles. Clin. Exp. Ophthalmol. 40, 350e357. GRH), The Glaucoma Research Foundation (RTL, GRH), EY018606 Chang, B., Smith, R.S., Hawes, N.L., Anderson, M.G., Zabaleta, A., Savinova, O., Roderick, T.H., Heckenlively, J.R., Davisson, M.T., John, S.W., 1999. Interacting loci (RTL), EY021525 (GRH), EY011721 (SWMJ), EY022891 (KSN), cause severe iris atrophy and glaucoma in DBA/2J mice. Nat. 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