Differential Loss of Bidirectional Axonal Transport with Structural Persistence Within the Same Optic Projection of the DBA/2J Glaucomatous Mouse

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Differential Loss of Bidirectional Axonal Transport with Structural Persistence Within The Same Optic Projection of the DBA/2J Glaucomatous Mouse

A Thesis

Presented in partial fulfillment of the requirements for the degree of Masters of Science in the College of Graduate Studies of Northeast Ohio Medical University

Matthew A. Smith B.A.

Integrated Pharmaceutical Medicine Northeast Ohio Medical University

2014

Thesis Committee: Dr. Samuel D. Crish (advisor) Dr. Christine Crish Dr. Denise Inman

Abstract

Glaucoma, the second leading cause of blindness worldwide, involves the degeneration of retinal ganglion cell bodies and their axons resulting in progressive vision loss. Much like other neurodegenerations, deficits in axonal transport are an early manifestation in the pathological progression of glaucoma. Previous studies suggest that anterograde and retrograde transport are differentially challenged and pre-degenerative in glaucoma, yet, both forms of transport have never been assessed within the same animal. We used a modified surgical procedure to assess retrograde transport while preserving the structure of the superior colliculus (SC) for both anterograde transport and immunohistochemical analysis in the same optic projection. Our findings demonstrate a 3-fold greater reduction in anterograde transport compared to retrograde transport in 9-10 month old animals.

Retrograde transport remained largely intact until 13 months of age, where a reduction similar to anterograde transport was observed. Additionally, immunohistochemical staining revealed that retinal ganglion cell (RGC) axons remained intact until 13 months despite these early transport deficits. Together these data support that the RGC axonal projection remains at least semi-functional and structurally intact after anterograde transport loss in glaucoma. This pre-degenerative loss of transport may provide a target for innovative treatments aimed at restoring function in glaucomatous axons that still remain largely intact.

Acknowledgments

I would like to thank first and foremost my family for their love and support not only over the last two years, but throughout my life and academic career. I may never be able to repay the extent of everything they have done to guide my success in any of my endeavors, but I certainly will do my best. Secondly, I would like to acknowledge Dr. Inman for the tremendous steps she has taken to push the Integrated Pharmaceutical Medicine program forward and for being both a constant and patient mentor through this project. Your many insights have been indispensable. Additionally, I would like to thank my fellow graduate students Gina and Lucy for providing reassurance and comedic relief along this journey.

Lastly, I would like to extend my greatest appreciation to Dr. Sam Crish and Dr. Christine

Crish who have played and continue to play the most essential role in my development as a student and as a researcher. I am extraordinarily grateful for their mentorship that has reignited my passion for neuroscientific research, yet, has humbled me to know that I still have much more to learn. Most of all, the truly unique dynamic of the lab is a constant reminder that regardless of the path you take, what is most important, is that you enjoy doing it.

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Vita

Research Technician, Northeast Ohio Medical University………………………………………….…2012 Teaching Assistant, Brain Mind and Behavior……………………………………………………….…..…2014

Fields of Study

Major Field: Integrated Pharmaceutical Medicine Minor Field: Neurobiology

Table of Contents

Abstract ...... ii

Acknowledgements...... iii

Vita ...... iv

List of Figures ...... viii

General Introduction ...... 1

Anatomical Orientation of the Eye ...... 1

Primary Visual Pathway ...... 4

Axonal Transport...... 5

Intraocular Pressure ...... 7

Neurobiological Deficits in Glaucoma...... 8

Transport Deficits in Glaucoma ...... 10

Molecular Motor Implications ...... 10

Metabolic Implications...... 11

Cytoskeletal Implications...... 12

Introduction to Methods ...... 15

Research Goals ...... 15

Mouse Model of Glaucoma...... 15

Neuronal Tract Tracing...... 16

Pilot Trials...... 18

Methods...... 19

Animals ...... 19

Anterograde Transport Labeling (intravitreal injections) ...... 21

Retrograde Transport Labeling (intracranial injections into SC) ...... 21

Tissue Collection and Preparation ...... 22

Immunohistochemistry for Structural Marker...... 22

Microscopy ...... 23

Measurement of Retrograde Transport...... 23

Measurement of Anterograde Transport and Structure ...... 24

Statistical Analysis and Variables...... 24

Results ...... 26

FG Density Does Not Differ by Strain or Age in Control Mice...... 26

Bidirectional Tracing Main Effects and Interaction Results ...... 27

Bidirectional Tracing Does Not Differ Between D2G Controls and 3 mo. DBA/2J.... 29

Anterograde Tracing in the SC is Significantly Reduced at 9-months in DBA/2J ..... 29

Structure of SC Remains Intact Until 13 months of Age in DBA/2J ...... 30

FG Density and Percent Are of Intact FG in Retina Are Correlated...... 31

Retrograde Tracing in the Retina is Reduced at 9-months ...... 35

Structural Integrity of the SC is Maintained Beyond Retrograde Transport Loss .... 35

Discussion ...... 39

Structural Persistence of the SC...... 40

Differential Transport Loss in Glaucoma ...... 40

Molecular Motor Modification and Transport Deficits...... 41

Metabolic Dysfunction and Transport Deficits...... 42

Cytoskeletal Alterations and Transport Deficits ...... 43

Conclusion...... 45

References...... 46

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List of Figures

Figure 1: Anatomical Depiction of the Retina...... 3

Figure 2: Visual Pathway ...... 4

Figure 3: Neuronal Tract Tracing Scheme...... 17

Figure 4: Fluorogold Density in Controls...... 27

Figure 5: Whole Mount SC and Retina ...... 28

Figure 6: Differential Transport Loss with Structural Persistence...... 30

Figure 7: Percent Area Fraction of Intact Label Across Age Groups ...... 32

Figure 8: Differential Loss of Transport...... 34

Figure 9: Structural Persistence After Differential Transport Loss in the DBA/2J ...... 36

Figure 10: Progression of Transport Loss...... 37

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General Introduction

In 2010, a national survey conducted by Harris Interactive claimed that 82 percent of individuals fear loss of vision more than any other sense. It is estimated that by the year

2020, this fear will become a reality for over 80 million people worldwide, as this is the projected number that will develop some form of glaucoma (Quigley & Broman, 2006).

Glaucoma is a group of neurodegenerative conditions characterized by the dysfunction and degeneration of the 1.5 million retinal ganglion cells (RGCs) and their axons that provide a direct connection between the eye and the brain, giving rise to sight. Before delving into understanding what is ultimately blinding in glaucoma, it is necessary to first discuss the components and mechanisms that make normal vision possible.

Anatomical Orientation of the Eye

The eye is a fluid-filled sphere constructed from three layers of tissue. The first layer, the outer fibrous tunic, consists of the sclera and the cornea. The sclera, commonly known as the white of the eye, allows attachment of the innervating eye muscles that rotate the eye. The cornea is the most anterior structure of the eye and serves as a fixed lens that initially bends incoming light allowing it to enter into the eye (DelMonte & Kim, 2011; Knop

& Knop, 2007; National Eye Institute, 2013; Pansky & Allen, 1980; Purves, 2012).

The second layer of the eye, known as the uveal tract, is composed of three continuous structures: the choroid, the ciliary body, and the iris. The highly vascularized choroid is the largest component of the uveal tract and is a richly pigmented structure that absorbs stray light. The ciliary body extends from the choroid and contains the ciliary muscles for fine

1 focusing of the lens and a vascular component that produces fluid that fills the anterior chamber of the eye. The ciliary body, in combination with a ring of fibrous tissue

(suspensory ligament), anchors to the lens allowing it to suspend in place. The lens is a transparent crystalline structure that alters its shape to refract light at various distances to be focused onto the retina. The amount of light allowed to be refracted though the lens is regulated by the iris, a thin, muscular diaphragm that can be seen through the cornea. It accomplishes this task by the use of two sets of muscles with opposing actions that adjust the size of the pupil (Kiel et al., 2011; Koretz & Handelman, 1988, Kuszak et al., 2006;

McMenamin, 1999; Pansky & Allen, 1980; Purves, 2012).

Light passes through two distinct fluid environments before reaching the retina. The anterior chamber, designated as the enclosure between the cornea and the lens, is filled by a clear, alkaline, watery solution that is secreted by the ciliary processes in the posterior chamber of the eye. The space from the rear of the lens to the surface of the retina is known as the posterior chamber and contains a viscous, gelatinous substance called the vitreous humor. Accounting for 80% of the total volume of the eye, the vitreous humor is required for the eye to maintain its shape. Additionally, the vitreous humor contains phagocytic cells that remove blood and other debris that could interfere with light transmission. As it will be described later, the dynamic equilibrium between the production and drainage of the aqueous fluid can be compromised, producing changes in eye pressure that characterize many glaucomas, but, do not always lead to vision loss (Goel et al., 2010; Hodgson, 1941;

Kiel et al., 2011; Purves, 2012; Pansky & Allen, 1980).

The third and innermost layer of the eye, the retina, is an eight-layered structure consisting of six classes of neurons and is part of the central nervous system. These cells consist of photosensitive receptors called rods and cones, bipolar cells, ganglion cells,

2 horizontal cells, and amacrine cells (Boycott & Dowling, 1969; Kolb, 2005) and are organized as shown in Figure 1.

Figure 1. Anatomical Depiction of the Retina (Crish, 2014; unpublished)

A single human retina contains about 120 million rods, 7 million cones, and 1.2 million ganglion cells (Anderson & Quigley, 1992; Dowling, 2012; Pansky & Allen, 1980).

Rods are most abundant in the periphery of the retina and are absent in the fovea (center of retina), whereas, cones are concentrated at the fovea. Rods are stimulated during low light conditions. Contrarily, cones are stimulated by high illumination, and are responsible for fine detailed vision (Tessier-Lavigne, 2000). Photoreceptor cells synapse onto bipolar cells within the outer plexiform layer of the retina. Bipolar cells receive multiple synaptic inputs from rod photoreceptors, while only some cone photoreceptors synapse on one bipolar cell.

In turn, many bipolar cells synapse onto ganglion cells that are the projection neurons of the 3 retina (Hildebrand & Fielder, 2011; Nelson & Connaughton, 2007). Additionally, horizontal cell processes aid in the modulation of photoreceptors inputs that are thought to maintain the visual system’s sensitivity to contrast. Amacrine cells whose processes are post-synaptic to bipolar cell terminals and presynaptic to retinal ganglion cell dendrites are thought to contribute inhibitory signals to bipolar cells and retinal ganglion cells (Masland, 2012;

Ogden, 1983).

The Primary Visual Pathway in the Brain

Ganglion cell axons converge at the optic disc and then exit the eye to form the optic nerve. The axons in the optic nerve from each eye project posteriorly and intersect at the optic chiasm. In humans, around 60% of the axons cross in the chiasm before entering the brain while 40% project ipsilaterally, however, in rodents 85-95% of RGC axons decussate while the remainder do not (Crish et al., 2011; Levkovitch-Verbin, 2004) (see Figure 2).

Figure 2. Visual Pathway (Reprinted from Gray’s Anatomy 20th edition , within the public domain). RGC axons from the nasal retina (blue) and the temporal retina (red) converge to 4 form the optic nerve. The majority, but not all of the axons from each eye will decussate beforing entering the brain, this decessation is known as the optic chiasm. Once past the chiasm, retinal ganglion axons from each eye form the optic tract. In the mouse, the majority of ganglion cell axons in the optic tract project to the midbrain where they invervate the superficial layer of the superior colliculus. In humans, RGC axons project primairly to the lateral geniculate nucleus of the the dorsal thalamus within the diencephalon. Neuons in these areas send their axons through the internal capsule eventually terminating in the primary visual cortex (Pansky and Allen, 1980).

As the optic nerve enters the brain, it becomes known as the optic tract and projects to several structures in the diencephalon and midbrain. In humans, the major target of these projections to the diencephalon is the lateral geniculate nucleus of the thalamus (LGN); however, in rodents this is the superior colliculus (SC). This is an important species-specific anatomical difference with regard to evaluating research studies using rodent models to examine glaucoma and visual defects. The superficial layer of the SC receives input mainly from the retina; beyond the superficial layer the SC primarily receives descending sensory and motor inputs from cortical areas to direct eye movement. Other structures to which

RGCs project include the pretectum, which mediates pupillary reflexes and the suprachiasmic nucleus of the hypothalamus for regulation of circadian rhythm.

Axonal Transport

Aside from the static structural components that comprise the visual pathway, important dynamic processes take place within the cells and axons of retinal ganglion cells that contribute to vision.

Axonal transport is the cellular process responsible for the movement of synaptic vesicles, mitochondria, lipids and proteins through the axon, to and from the cell body, and to the synaptic boutons of the neuron (Leopold et al., 1992; Morfini et al., 2006a). Therefore, axonal transport not only mediates trafficking of neurotransmitter-filled synaptic vesicles to the bouton required for neurotransmission, but also meets the high metabolic and structural demand of the neuron by trafficking mitochondria, lipids, proteins, and growth factors to ensure proper health and function of the cell (Morfini et al., 2009a).

Transport can be classified based on the direction by which the molecules and proteins are being trafficked. Anterograde transport refers to the trafficking of elements from the cell body to the axon bouton. Conversely, transport from the bouton back to the cell body is referred as retrograde transport. In relation to the eye and visual system, anterograde transport defines transport from the retina (where RGC bodies are located) to the brain where axons input into the superficial layer of the SC. In turn, retrograde transport entails a pathway of transport returning to the retina from brain structures such as the SC (see Figure 2 & 3). Both forms of transport use molecular motors that attach and travel along the extensive microtubule tracts that run the length of the axon. Anterograde transport specifically relies upon the family of molecular motor proteins called kinesins, whereas retrograde transport uses the dynein motor proteins (Elluru et al., 1995; Susalka &

Pfister, 2000). Anterograde transport mechanisms carry cargo as fast as 50-400 mm/day faster than retrograde transport that typically moves 100-200 mm/day (Yu et al., 2013).

Deficits in specific mechanisms of axonal transport have been demonstrated in neurodegenerative disorders such as Alzheimer’s and Parkinson’s diseases, and amyotropic lateral sclerosis (ALS)(Coleman, 2005; Morfini et al., 2009b; Stokin et al., 2005), but most

6 recently, and most important to the current research, in glaucoma (Buckingham et al., 2008;

Crish et al., 2010).

Intraocular Pressure

Glaucoma is the second leading cause of blindness worldwide behind cataracts—a treatable condition—thus making glaucoma the leading cause of irreversible blindness in the world (Quigley & Broman, 2006).

For years, physicians and researchers viewed glaucoma as an eye disease because glaucoma is often, but not always, associated with elevated intraocular pressure (IOP)

(Anderson, 1989; Gramer & Tausch, 1995; Heijl et al., 2002; Mittag et al., 2000; Nargarju et al., 2007; Sommer, 1991). Intraocular pressure is maintained by a precise balance between the production and outflow of aqueous humor within the anterior chamber of the eye.

Produced by the ciliary body, this fluid travels around the lens and through the pupil to fill the anterior chamber (Tormey, 1966). Aqueous fluid drainage from the anterior chamber is accomplished by a specialized meshwork of cells referred to as the trabecular meshwork.

The trabecular meshwork lies within an angle formed by the junction of the iris and the cornea, known as the irido-corneal angle. After filtration, fluid enters the Canal of Schlemm, a duct that eventually empties aqueous humor into veins under the conjunctiva where the fluid is absorbed into the bloodstream (Flocks, 1956; Gong et al., 1996).

If production of aqueous humor occurs beyond the rate that it can be adequately drained, elevation in ocular pressure can occur, exceeding the average normal pressure range of 12-22 mmHG (Weinreb & Khaw, 2004; Young et al., 2009). Traditionally, presentation of abnormal ocular pressure where irido-corneal angle morphology is normal is classified as primary open-angle glaucoma. In other circumstances where the irido- corneal angle becomes structurally compromised due to eye trauma, distortions of the iris,

7 or congenital abnormalities, IOP is suddenly elevated and this is known as angle-closure glaucoma (Goel et al., 2010; Quigley, 2011; Weinreb & Khaw, 2004).

However, nearly 50% of patients with diagnosed glaucomatous pathology have normal ocular pressure. Furthermore, it had been previously estimated that between 3-6 million people in the United States have elevated IOP, but present without detectable glaucomatous damage on clinical tests (Leibowitz et al., 1980; Shields, 2008). Even so, IOP lowering strategies have and remain a staple for the treatment of glaucoma despite the estimate that 50% of glaucoma patients, when treated with an IOP-lowering regimen, continue to lose vision due to optic nerve degeneration (Leske et al., 2003). This may be partly due to poor compliance, since in order to achieve therapeutic efficacy these topical drugs have to be applied multiple times a day and can be irritating to the eye. However, the most probable reason for low treatment efficacy may be attributed to the fact that diagnoses of glaucoma are often made at a late stage of the disease as patients in the early stages of the disease do not present with noticeable symptoms (Quigley, 2011). By the time patients become symptomatic with noticeable loss of vision, the optic nerve has already begun to degenerate and treatments reducing IOP are largely ineffective (Leske et al. 2003).

Therefore, addressing the neurobiological events that precede and contribute to degeneration of the optic nerve is a much more complete and effective treatment for the disease.

Neurobiological Deficits in Glaucoma

Despite the multiple known pathological events that occur through the progression of the disease, the ultimate initiating factor that sets the disease in motion remains unclear and might also vary from person to person. What is clear is that the progression ends by the selective caspase-dependent, mitochondrial-mediated apoptosis of retinal ganglion cell

8 bodies in the retina (Huang et al., 2005a; Kerrigan et al., 1997; McKinnon et al., 2002; Qu et al., 2010; Quigley, 1999; Tatton et al., 2001; Tahzib et al., 2004; Waldmeier & Tatton, 2004).

Early research postulated that the mechanism responsible for initiating RGC apoptosis was obstruction of axonal transport at the ONH, which was thought to be prompted by pressure induced compression of the un-myelinated axons by the cribriform plates as they exited the eye (Mabuchi et al., 2004; Osborne et al., 2004; Schlamp et al., 2006; Quigley, 1999). This led many researchers to focus solely on finding ways to prevent or halt apoptosis in the retina.

However, a study by Libby et al. (2005), showed that deletion of the pro-apoptotic gene BAX in a mouse model of glaucoma halted apoptosis of the RGC soma, but RGC axon loss continued. This evidence suggested that different neuronal compartments such as the RGC cell body and the RGC axons were affected differently in glaucoma. This raises the question of what changes are occurring in the axons independently of caspase-dependent cell death mechanisms.

In other neurodegenerative conditions such as Alzheimer’s and Parkinson’s diseases, and ALS, axonal defects prompting deficits in axonal transport are early manifestations often occurring before traditional pathological signs are observed (Coleman et al., 2005; Morfini et al., 2009a; Stokin et al., 2005). Amongst these conditions, axonal defects appear as large spheroids and varicosities in areas of the axon and are suggested to be result of intra-axonal abnormalities such as molecular motors modification, disruption of cytoskeletal structures, and metabolic dysfunction (Conforti et al., 2007; Cuchillo-Ibanez et al., 2008; Fisher et al., 2004). These intra-axonal aberrations have been suggested to both directly and indirectly alter motor proteins and cytoskeletal structures such as the microtubule tracts that drive cargo transport through the axon.

In animal models of glaucoma, early signs of these axon spheroids and varicosities are seen in the distal projection sites of the brain (Crish et al., 2010). Furthermore, dendritic field remodeling including loss of arborization and abnormal morphologies parallel the early occurrence of axon varicosities, but most importantly are seen to precede cell body loss in the retina (Jakobs et al., 2006). These distal axonal dystrophies coincide with loss of axon transport that first appears in distal sites where RGC axons terminate in the SC. The progression of transport loss beginning in the SC eventually progresses to more anterior

RGC brain targets, to the optic nerves, and finally to the retina, where apoptosis eventually occurs. When degeneration of the optic nerve finally occurs, it progresses in similar fashion to the aforementioned loss of transport, spreading along the nerve in a distal to proximal manner affecting axon segments in the distal projection first. (Crish et al., 2010).

Transport Deficits in Glaucoma

Additional reports have shown that anterograde (Crish et al., 2010) and retrograde

(Buckingham et al., 2008) transport in the primary retinal projection are disrupted differently in glaucoma. In the DBA/2J mouse model of glaucoma, anterograde transport is challenged considerably with deficits beginning at 8-9 months and complete failure of transport to the SC occurring at 11-12 months. Conversely, retrograde transport from the

SC to the retina in the DBA2/J mouse is maintained at 20-30% capacity up to 18 months of age (Buckingham et al., 2008; Danias et al., 2003). Several explanations exist for why these two axonal transport mechanisms are affected differently.

Molecular Motor Implications

Phosphorylation of molecular transport motors represents a major mechanism in controlling transport dynamics (Donellan, 2002; Hollenback, 1990; Morfini et al., 2001;

Morfini et al., 2004). Molecular motors are phosphorylated by several kinases including

GSK3 (Morfini et al., 2002a) and Cdk5 (Morfini et al., 2004). Under normal physiological conditions these kinases directly or indirectly modify specific subunits of the molecular motors to regulate microtubule binding and cargo attachment necessitating transport

(Morfini et al., 2009a). However, it has been suggested that differential alterations in axonal transport could be a result of the abnormal activation of these kinases (GSK3, Cdk5, JNK) causing aberrant motor protein phosphorylation (Morfini et al., 2002a, 2004, 2005, 2006b,

2007; Wagey & Krieger, 1998). Aberrant phosphorylation of transport motors could potentially interfere with cargo docking and/or motor attachment and movement along the microtubule, thus prohibiting delivery of mitochondria or trophic factors that are essential for both function and survival of the optic nerve. Depending on the kinases involved or the susceptibility to modification of the motor proteins, transport could be differentially affected.

Metabolic Implications

Like most neurons in the CNS, RGCs require free energy to meet the high-energy demands required for proper function and survival. Adenosine triphosphate (ATP) molecules are the predominate form of energy necessary for intra-cellular/intra-axonal RGC mechanisms, but intracellular diffusion of ATP is limited (Brady & Morfini, 2010).

Therefore, RGC function and survival rely heavily on the distribution of mitochondria along the RGC projection. Mitochondria are distributed asymmetrically along the optic projection, primarily localizing in areas that have high local energy demands such as around the nucleus, the optic nerve head where the axons are unmyelinated, and at the terminals where synaptic transmission occurs (Andrews et al., 1999; Bristow et al., 2002; Yu et al.,

2013; Valverde, 1973). In order to meet these regional energy demands, mitochondria rely on fast axonal transport along microtubules by kinesin and dynein molecular motors that

11 are guided by the changing ATP gradients in these regions. That is, mitochondria move toward regions with high ATP demand and depart when that demand decreases (Whitmore et al., 2005). Several inherited disorders of mitochondrial dysfunction have been shown to affect the optic nerve (Carelli et al., 2002). Given that mitochondria are the major source of

ATP and their transport by kinesin and dynein is dependent on ATPase activity (Ochs &

Hollingsworth, 1971), it is possible that energy depletion due to mitochondrial dysfunction has the potential to disrupt axonal transport.

Cytoskeletal Implications

Predegenerative occurrence of spheroids and swellings along axons have been noted in several diseases including glaucoma and can be attributed to aberrant cytoskeletal elements within the axon (Coleman et al., 2005; Crish et al. 2010; Fischer et al., 2004; Stokin et al., 2005) Regions of disorganized cytoskeleton along the axon ultimately serve as “road blocks” that can prohibit molecular motor movement in either direction past the site of the insult. This would result in differential transport deficits due to dynein having greater lateral and bidirectional movement across microtubule tracts. This ability would presumably allow it to more easily bypass areas of disorganized cytoskeleton within the axon (Shea, 2008; Wang, Khan, & Sheetz, 1995). This unique ability attributed to dynein and not kinesin may allow for mitochondria and trophic factor delivery to the retina to be maintained while delivery of these factors to the distal projection of the optic nerve is lost.

This provides a potential explanation to why both transport loss and degeneration of the optic nerve progress in distal to proximal manner in glaucoma (Crish et al., 2010, Crish &

Calkins, 2011).

Further explanation of the cause of these cytoskeletal changes is needed, but one prominent theory indicates dysregulation of cellular Ca2+ homeostasis (Crish & Calkins,

2011). Increased influx of extracellular Ca2+ into the cell is a common component of axonopathy across neurodegenerative conditions and can trigger enzyme mediated cytoskeletal degradation (Coleman, 2005). Under normal conditions, the concentration of cytosolic Ca2+ is roughly 10,000 times lower than that in the extracellular space (Hernandez-

Fonseca & Massieu, 2005). Breakdown of Ca2+ homeostasis can initiate a series of cytoplasmic processes that promote caspase-dependent neuronal cell death (Bredesen et al., 2006). RGCs utilize a variety of cation channels that conduct Ca2+, for processing excitatory signals from bipolar neurons. Furthermore, they express a number of additional pressure-related membrane channels such as the vanilloid transient receptor potential

(TRPV) channels. TRPV1 and TRPV4 channel expression increases in RGCs with elevated

IOP and has been suggested to contribute to increase intracellular Ca2+ and retinal ganglion cell death (Ryskamp, 2011; Sappington et al., 2009). IOP elevations can also promote cleavage of calcineurin, a Ca2+-dependent protein phosphatase, and inhibiting calcineurin systemically inhibits pressure-induced RGC axon loss in the optic nerve (Huang et al., 2005).

Relevant to transport deficits and axonopathy, evidence suggests that calpains are activated in glaucoma (Huang et al., 2010). Calpains are Ca2+-dependent proteases that have been implicated in a number of neurodegenerative conditions that act directly on cytoskeletal components (Lu et al., 2000; Jourdi et al., 2005; Vosler et al., 2008). Calcium- activated calpains break down the structural proteins alpha-II-spectrin and heavy chain neurofilaments (Siman et al., 1984; Chan & Mattson, 1999) and can activate kinases such as cyclin dependent kinase-5 (Lee et al., 2000), extracellular signal regulated kinases

(Veeraana et al., 2004), and stress activated protein kinases (Goni-Oliver et al., 2007). These kinases can go on to phosphorylate neurofilaments and the microtubule associated protein

Tau, thus slowing or blocking axonal transport by interfering with molecular motor

13 attachment to the microtubule tract. (Crish & Calkins, 2011; Shea et al., 2004, Shea and

Chan, 2008).

Introduction to Methods

Research Goals

Correcting the loss of transport and/or axonal dystrophy in the primary visual projection is an easier problem to address than the far more drastic approach focused on rewiring long axon segments, or replacing cell bodies within the retina and optic nerve projection. However, a significant gap in the literature exists regarding axonal transport deficits in glaucoma. A direct assessment of anterograde versus retrograde transport within the same animal has not been conducted. Although each process has been looked at individually in separate studies, given the differences in methodologies and ages tested, this is not sufficient to confirm a true time course of events in glaucoma. If anterograde transport defects precede retrograde loss in the same animal, then not only do RGC axonal elements persist after anterograde transport loss in glaucoma (Crish et al., 2010), but there is an intact, semi-functional axon, suggesting some very specific mechanisms of transport blockade (e.g. subtle cytoskeletal changes and/or motor protein dysfunction) that would provide attractive targets for intervention. The purpose of my thesis research was to compare both anterograde and retrograde axonal transport in a rodent glaucoma model to investigate possible mechanisms of transport blockade that contribute to pathology.

Mouse Model of Glaucoma

The DBA/2J mouse is a model of glaucoma secondary to iris pigment dispersion disease, characterized by chronic, age-related degeneration of visual structures similar to the human presentation of the disorder. DBA/2J mice have mutations in both their Tyrp1

15 and Gpnmb genes. In particular, the Gpnmb gene is truncated and as a result has aberrant function that initiates degeneration of the iris as the mouse ages (John et al., 1998). Pigment cells that break free from the degenerating iris can disperse throughout the anterior chamber of the eye and accumulate in the irido-corneal angle, blocking aqueous humor drainage, and thereby, elevating the animals’ IOP. For these reasons the animal model is widely used throughout glaucoma research and is the model we used to conduct the bidirectional transport study described in this thesis. Comparatively, the DBA/2J-Gpnmb+

(D2G) mouse shares the same background as the DBA/2J, however, they express a functioning wild type Gpnmb+ allele that prevents them from developing glaucomatous pathology, therefore, they serve as a non-glaucomatous control (Porciatti et al., 2010).

Neuronal Tract Tracing

The most common approach for examining axonal transport in glaucomatous animals is neuronal tract tracing that incorporates the use of tracers that, when applied, are readily taken up into neuronal cell bodies and transported along the entire projection to the site of termination. For anterograde transport assessment, the neuronal tracer cholera toxin beta (CTB), conjugated to a fluorophore, is injected into the posterior chamber of the eye where it is taken up into the retinal ganglion cell bodies and transported to the SC (see

Figure 3). When transport is compromised, this is represented in the SC as areas devoid of

CTB label, equated to missing pieces of a pie. Conversely, retrogradely-transported neuronal tracers such as fluorogold (FG) are applied to the SC for retrograde tracing to the retina with a lack of FG labeled cells in the retina most often interpreted as missing RGC’s (Mittag et al., 2000; Vidal-Sanz et al., 2001; Danias et al., 2003; Filippopolous et al., 2006)(see Figure

3).

Figure 3. Neuronal Tract Tracing Scheme. Cholera Toxin-beta (CTB) (green) when injected into the vitreal chamber of the eye (green syringe) is taken up by RGC bodies in the retina

(RET) via GM1-mediated endocytosis. Once in the cell, CTB is moved to the Golgi apparatus where it is packaged into vesicles in preparation for transport. The delivery of CTB-filled vesicles from the cell body to the terminal known as anterograde transport (green arrow) is accomplished by attachment to the molecular motor kinesin. Kinesin bound with vesicular

CTB move via ATP hydrolysis along microtubules that extend the entire length of the axon.

Once CTB reaches the axon terminals in the superior colliculus (SC)(hexagon) it is released from kinesin and its journey is complete. Using fluorescent microcopy, CTB that has been successfully delivered from the retina can be seen as green label across the breadth of the superficial layer of the SC (2). Comparatively, injection (blue syringe) of Fluorogold (blue) to the superficial surface of the SC is endocytosed into axon terminals and delivered to the

17 eye via retrograde transport (blue arrow). Vesicular FG is carried along the microtubule tract by the molecular motor dynein. Dynein also utilizes ATP hydrolysis to propel along the distance of the axon, eventually being released at the cell body of ganglion cell. Fluorescent microscopy of the retina will reveal FG-positive cells (1) evenly and densely distributed throughout the retina indicating effective retrograde transport.

Pilot Trials

The major hurdle for investigating bidirectional transport tracing in the same animal is methodological in nature. FG application in the brain has been performed using one or more large injections into the SC followed by intracranial insertion of FG-soaked gelfoam for additional tracer exposure (Buckingham et al., 2008; Soto et al., 2008). This method results in extensive damage to the SC, precluding analysis of RGC projection persistence, anterograde transport levels, or FG exposure in this structure. In order to optimize this method so as to perform retrograde tracing in animals while preserving the structure of the SC for analysis, we incorporated modifications of each independent tracer- injection procedure in order to achieve both anterograde and retrograde tracing in each animal. This involved varying the number, size, angle, and volume of the injections, modifying the craniotomy and injection procedures, as well as adjusting the fluorogold concentration. The first method we tested included the direct application of 1.5 μl of 5% FG directly onto the brain surface thorough a bilateral craniotomy. This method initially provided complete uptake and transport to the retina while leaving the SC intact for histological and immunochemical analysis. However, the topical application proved extremely variable and the penetration of FG into the tissue was poor resulting in faint levels of label in the retina.

The next attempt we made was directed at the penetration problems of the tracer, which utilized the same procedure as before with the exception that the FG be dissolved in dimethyl sulfoxide (DMSO). DMSO is a polar aprotic solvent commonly used as a drug delivery vehicle because it readily crosses most tissue membranes, including the blood brain barrier (Pardridge, 2005). For this reason we used DMSO to increase the penetrance of FG into the SC. This proved to be a successful correction of the previous penetration issue as evidenced by brighter, fuller label in the retina. However, complete retinal coverage was often lacking in the controls where we did not expect a deficit to be present and the SC often showed damage that did not seem to be a result of direct injection. The unlabeled sectors in the retina seemed to be a result of a lack of tracer application across the entire surface of the SC. We tested numerous procedural modifications involving tracer delivery systems and refinements in the craniotomy technique used to expose the SC. After significant procedural testing and technique refinement, we developed a protocol that allowed us to administer our tracer to multiple aspects of the SC while being as minimally invasive as possible. This involved performing three craniotomies surrounding the perimeter of the SC, which allowed greater access and injection diffusion across the entire SC surface. To ensure the structural integrity of the SC, we lowered the tracer injection syringe at pre-determined angle to a depth just below the collicular surface. The injection was administered steadily over the course of a minute to reduce any trauma to SC or surrounding area. We were able to take this newly validated approach for retrograde tracing—along with intraocular injections of CTB tracer prior to the craniotomy surgeries, to make a direct comparison of the temporal differences in anterograde vs. retrograde transport deficits and how they relate to loss of RGC axon/terminal structure.

Methods

Animals

This study used mixed-sex DBA/2J (n=27; 6-9 per age group), D2-Gpnmb+ (n=8), and

C57BL/6J (n=7) mice of different ages. The DBA/2J mouse had two loss-of-function mutations and is characterized by chronic, age-related course of degeneration of visual structures that mimics the human presentation of the glaucoma (John et al., 1998). D2-

Gpnmb+ (D2G) mice have the same background as the DBA/2J; however, they express a functioning wild type Gpnmb+ allele that prevents them from developing glaucomatous pathology (Porciatti et al., 2010). Given the recent development of D2G mice as controls for glaucoma, we have also used C57BL/6J (C57) that have historically been used as controls in mouse glaucoma studies (Crish et al., 2010). All animals were originally obtained from The

Jackson Laboratory (Bar Harbor, ME) and then housed and aged in the Comparative

Medicine Unit at Northeast Ohio Medical University to the following time points. In the

DBA/2J model, we used 3 month old mice representing the pre-glaucomatous ages, 9-10 month old mice representing ages at the onset of glaucomatous pathology where anterograde transport deficits and mild axonopathy are evident, 11-12 month old mice representing increasing transport deficits and axonopathy, and 13 months where retrograde transport deficits along with axon and cell body loss is evident (Buckingham et al., 2008; Crish et al., 2010). For controls, we used a convenience sample of a variety of C57s ranging from 4-17 months old and 9-12 month old D2Gs. Mice were maintained in a 12 h light/dark cycle with standard rodent chow available ad libitum. The Northeast Ohio

Medical University Institutional Animal Care and Use Committee approved all experimental procedures.

Anterograde Transport Labeling (intravitreal injections)

Mice were placed prone in a stereotaxic device (Stoelting, Wood Dale, IL) equipped with nose cone set to deliver 2.5 % isoflurane at 0.8 L/min. 1.5 µl injections of 1% cholera toxin subunit B (CTB) conjugated to Alexa Fluor-488 (CTB; Invitrogen, Carlsbad Ca) in sterile phosphate buffered saline (PBS) were administered posterior to the ora serrata of the eye into the vitreal chamber using a 33-G needle attached to a 25 µl Hamilton Syringe.

Animals remained anesthetized in the stereotaxic device for immediate surgical preparation for the retrograde labeling procedure.

Retrograde Transport Labeling (intracranial injections into SC)

Surgical sites on the dorsal surface of the skull for mice were cleaned and prepared for the craniotomy procedure. Craniotomies (each 1 mm2) to expose the surface of the brain were drilled into each side of the skull using a 0.5 mm burr attached to a micro-motor drill.

Two injection sites per hemisphere were used to administer fluorogold to the entire rostral- caudal span of the SC (see Figure 5) and were delivered at the following stereotaxic coordinates (each relative to Bregma and midline suture skull landmarks): -4.0 mm anterior-posterior, +/-0.5 mm medial lateral; and -4.4 mm anterior-posterior, +/- 0.5 mm medial lateral. At each craniotomy site, a 25 µl Hamilton syringe equipped with a 33G needle was lowered (via stereotaxic device) at a 45 degree angle to a depth of 0.5 mm below the skull surface. This approach differs from previous ones in that it leaves minimal damage to the injection site within the SC, allowing analysis of intact anterograde transport labeling from the retina. Once syringe was in position, 1 µl of 3% fluorogold (Fluoro-Gold,

Flourochrome; Denver, CO in 10% DMSO/PBS) was injected. After each injection was

21 complete, the needle was retracted over 30 seconds. Sterile bone wax was used to seal each of the skull openings and the incision site was closed with tissue adhesive (Vetbond; 3M,

USA). Animals were allowed to recover for seventy-two hours before sacrifice.

Tissue Collection and Preparation

Seventy-two hours after injections, subjects received a single intraperitoneal injection of 120 mg/kg sodium pentobarbital and animals were transcardially perfused with 4% paraformaldehyde in PBS solution. The brains, optic nerve, and eyes were harvested and retinas were dissected from the eyes. Retinas were prepared as flattened whole-mounts: four radial cuts were made in the surface and retina were mounted nerve fiber layer side up on slides (see Figure 5), covered with mounting media and coverslipped.

Immunohistochemistry for Structural Marker

In order to assess whether cell loss was driving deficiencies in transport from the

SC, we stained for estrogen-related receptor beta (ERRβ), which labels all components of retinal ganglion cells including their projection to the SC (Real et al., 2008; Crish et al.,

2010). Using a freezing sliding microtome, 50 µm coronal serial slices through the superior colliculus were produced. Every third section of the serial SC slices was added to a 96-well plate containing 200 µl (per well) block solution (5 % donkey serum, 1% Trition-X 100 in

1x PBS) for 2 hours at room temperature. Sections were then incubated in primary antibody solution containing rabbit polyclonal antibody ERRβ (1:500, Sigma) in 3% donkey serum,

1% Triton-X 100 in 1x PBS for 72 hours at 40C. Sections were then removed and washed 3x for 10 minutes. Sections were then incubated in Alexa Fluor-594 conjugated secondary antibody (1:200, Invitrogen) overnight at 40C. Sections were rinsed 3x 10minutes in 1x PBS before being mounted on microscope slides and coverslipped.

Microscopy

Retina and sections containing superior colliculus were photographed with a Zeiss

AxioZoom V16 fluorescent microscope equipped with a digital high-resolution camera and a computer guided motorized stage. Retinal whole-mount reconstructions were obtained by multi-frame acquisitions captured side-by-side with 10% overlap between them with a 3.6 x/0.50 objective (Plan-Neofluar, Zeiss, Jena, Germany) at a final magnification of 160x. Each frame was Z-stacked at an interval 4-8 µm producing a stack between 5-10 images. The scan area was defined to cover the whole retina completely (see Figure 5). This area indicated by frames in columns by frames in rows (m x n) usually consisted of 120 fields for each retina.

Superior colliculi from each animal were imaged at 80x magnification under multiple channels to capture CTB and ERRβ label and FG injection sites (see Figure 6).

Measurement of Retrograde Transport

For analysis, Z-stack retina images were flattened into a single layer TIFF using extended depth of field (EDF) software in Zen (Zeiss; Jena, Germany). ImagePro (Media

Cybernetics; Rockville, MD) software was used to count FG-labeled RGCs in the retina using subroutines similar to ones published by Salinas-Navarro et al. (2009). In brief, we applied a sequence of 2-D filters to each retinal image that: a) defined and separated individual cells, b) established criteria for cell size boundaries and roundness, and c) removed artifacts/noise. In order to calculate density of FG-labeled RGCs in each retina, we used the

ImagePro software to measure the area of each retina. We divided the total number of FG positive RGCs counted in each retina by the total area of that retina to yield a density measure of cells/mm2. To quantify sectorial loss of FG labeling in retina (see Figure 5,6,7) characteristic of glaucomatous pathology, we measured the area of sections within the retina where FG+ cells were absent (see Figure 5). We subtracted these areas from the total

23 area of the retina to produce a value that represented the total area of intact FG and then divided the total area of intact FG by the total retinal area to create the variable “percent area intact” for analysis of intact retrograde tracing. This variable was created to allow direct comparison with deficits in the retinotopic collicular map.

Measurement of Anterograde Transport and Structure

We quantified CTB and ERRβ signal density in multiple slices of the SC using a custom-written macro for NIH ImageJ (Rasband 1997-2007). The macro quantified the number of pixels within bins of customizable width that are brighter than tissue in the unstained region outside user-selected the region of interest (ROI) in each slice. The primary data output from the macro is the normalized intensity of each bin, calculated as the number of pixels above the intensity threshold, expressed as proportion of the total number of pixels in the bin. Additional data include the position and width of each bin, the absolute number of labeled pixels (e.g., those above the brightness threshold) and the total number of pixels in the bin.

Statistical Analysis and Variables

We used two-way between-subjects factorial analyses of variance (ANOVA) with post-hoc Fisher’s LSD tests to determine differences in transport between strains/ages. To compare differences in magnitude between anterograde, retrograde, and structural label within subjects for each age group, we used paired t-tests to delineate the effects of these planned comparisons. We also used one-way ANOVA to assess control data and Pearson’s correlations for additional description measures. Retrograde transport was defined as either FG+ cell density in retina or percent area of intact FG staining in retina. Anterograde transport was defined as percent area fraction of intact CTB label in the SC contralateral to the retina analyzed for retrograde transport. Additionally, the structural intactness of each

SC was measured by percent area fraction of intact ERRβ label. Retrograde and anterograde transport were compared within the same projection. Each retina and corresponding contralateral SC set were analyzed separately within each animal, as glaucomatous pathology differentially affects each eye thus necessitating the analysis of each projection as an independent measure (Schlamp et al., 2006; Crish et al., 2010).

Results

FG density does not differ by strain or age in C57BL/6J and D2.Gpnmb+ control mice

FG-positive cell density in retina from control strains were compared across age groups to determine if the FG distribution pattern in D2G mice matched that of C57s and if there was similarity between younger and older controls. This is the first FG tracing analysis done in the D2G strain, therefore, it was important to validate if they could serve as an effective control. We compared the density of FG+ cells in the retina across 4, 10, and 17- month-old C57 mice and 9, 11, and 12-month-old D2G mice. Results of an ANOVA indicated no significant differences between ages or the two strains, (F4, 14 = 0.935, P = 0.482, see

Figure 4). This validated our use of D2G control mice as comparable (and more background relevant) substitute for the C57 control strain. For the remaining analyses, we pooled the

D2G control data across ages where we typically see glaucomatous pathology in the DBA/2J

(9-12 months old) to form a single control group for this strain.

Figure 4. Fluorogold (FG) density in control mice. Density of positively labeled FG cells (x- axis) in the retina of control strains C57BL/6J (C57) and DBA/2J.Gpnmb+ (D2G) were compared across ages (4, 9, 10, 11, 12, and 17 months) and strains. No significant differences in FG-positive cell densities were observed across 4, 10, and 17-month old C57 retinas or across 9, 11, and 12-month-old D2G retina. Comparison across each strain indicated no significant difference in FG-positive cell densities between similarly aged C57 and D2G retinas.

Bidirectional tracing main effect and interaction results

The following abbreviations will be used to label the groups represented by our data: D2G (D2-Gpnmb+ control), D3 (3-month old DBA/2J), D9-10 (9-10 month old DBA/2J),

D11-12 (11-12 month old DBA/2J), and D13 (13-month DBA/2J). In order to compare intact

27 retrograde transport (FG), anterograde transport (CTB), and structure (ERRβ) for each group of mice, as well as compare how these values changed as a function of age in the

DBA/2J mice, we used a 3 (CTB, FG, ERRβ) by 5 (D2G, D3, D9-10, D11-12, D13) factorial

ANOVA to analyze data. Significant main effects for label (F2, 104 = 29.98, P < 0.001) and strain/age (F4, 104 = 91.57, P < 0.001) were shown. A significant interaction between type of label and animal group qualified these main effects (F8, 104 = 8.64, P < 0.001). Post-hoc

Fisher’s Least Significant Difference (LSD) tests were used to define the significant pairwise comparisons as described below.

Figure 5. Whole mount superior colliculus (SC) and retina. (TOP) Whole mount SC images from a pre-glaucomatous 3mo. DBA2/J showing normal anterograde transport to the SC following bidirectional injection of CTB (green), as well as, a successful FG injection (blue)

28 with complete coverage of the SC without damage. (MIDDLE) In comparison, a 9-mo. glaucomatous SC shows complete absence of CTB label (green) in left SC (dotted lines) with a slight CTB deficit in the right SC (dotted lines). Confirmed FG injection across the SC without damage is also apparent. (BOTTOM) Grayscale outlines of retina whole mounts from an 11-month non-glaucomatous animal (D2G) compared with a 12-month and 13- month glaucomatous DBA2/J. Retina from non-glaucomatous animals shows complete coverage of FG-positive RGCs (white dots) throughout. Comparatively, a 12-month glaucomatous retina shows sectors of missing or void of FG-positive RGCs indicating loss of retrograde transport within those specific retinal regions. Additionally, a 13-month DBA/2J retina is entirely devoid of FG-positive RGCs. (Scale bar: C-E=500 µm. FG, Fluorogold.)

Bidirectional tracing does not differ between D2G controls and 3-month old DBA/2J mice

As anticipated, there were no significant differences in intact anterograde (P =

0.808), retrograde (P= 0.8310), or structural label (P= 0.943) between D2G controls and D3

(pre-glaucomatous) mice (see Figure 8). Pairwise comparisons between the different

DBA/2J age groups are reported below.

Anterograde tracing (CTB) in SC is significantly reduced at 9-months of age in DBA/2J mice

Glaucomatous DBA/2J (D9 and older animals) showed marked reductions in CTB label in the SC compared to the D3 group (see Figure 5). The D3 group had significantly more intact label than D9-10, D11-12 and D13 (Fishers LSD P < 0.001 in all three comparisons; see Figure 7, panel A; Figure 8). There were no differences in CTB label

29 between the three oldest DBA/2J groups (Fisher’s LSD: D9-10 vs D11-12 P = 0.545; D9-10 vs D13 P = 0.098; D11-12 vs. D13 P = 0.219; see Figure 7, panel A; Figure 8).

Structure of SC (Errβ) remains intact until 13 months of age in DBA/2J

Percent intact ERRβ in the SC did not differ between D3 and D9-10 (Fishers LSD P =

0.978), D3 and D11-12 groups (P = 0.088), and D9-10 and D11-12 groups (P = 0.094; see

Figure 7, panel B; Figure 8). However, there was a significant reduction in ERRβ in the 13- month-old DBA/2J mice in contrast to D3, D9-10, and D11-12 groups (Fishers LSD P < 0.001 in all comparisons; see Figure 7, panel B; Figure 8).

Figure 6. Differential transport loss with structural persistence. Coronal SC sections with corresponding retina flat mounts (high magnification) for (A) a non-glaucomatous 12 mo.

D2.Gpnmb (D2G) compared to (B) 12 month and (C) 13 month glaucomatous DBA2/J after bilateral intravitreal CTB (green) and intracranial FG (blue) injections and post-mortem immunohistochemistry for ERRβ (red). (A) Twelve-month D2G SC shows complete presence of CTB, FG, ERRβ label, indicative of normal anterograde transport (CTB), RGC axon terminals (ERRβ) and confirmed non-damaging FG injection. High magnification of

30 corresponding retina shows efficient CTB uptake into RGC axons and astrocytes along with the presence of FG+ RGCs, indicating normal retrograde transport (FG) and effective intravitreal injection (CTB) (B) Twelve-month DBA/2J shows CTB labeled RGC axons in the retina, but shows a 90% reitinotopic deficit in CTB label with complete ERRβ in the SC.

Furthermore, FG-positive labeled RGCs in the retina of the same projection shows no noticeable deficit. (C) A 13 mo. DBA/2J shows complete absence of CTB label within the SC, but CTB-positive labeled axons in the retina. Both ERRβ labeled axon terminals in the same

SC section and FG-positive RGCs in the corresponding retina persist without observable deficits. (Scale: 200µm for all sections)

FG density and percent area of intact FG in retina are correlated

Other studies of retrograde transport use FG+ cell density in the retina as the primary variable of intact transport (Buckingham et al., 2008). However, to perform within animal comparisons between retrograde and anterograde transport, we needed to establish a variable that allowed us to compare FG measurements in the retina with percent area fraction of CTB and Errβ in the SC (see Figure 6). Therefore, we quantified the percent area of intact FG label in the retina as well as FG+ cell density in the same retina for all our mice and found that these variables were highly correlated with each other (Pearson’s r = .80, P <

0.0001; see Figure 7, panels C&D). This validated our use of the percent area intact FG in the retina as our retrograde transport variable for comparison with the anterograde transport and structural variables.

Figure 7. Percent area fraction of label across age groups. (A, B, C) Percent area fraction of mean CTB (anterograde) and ERRβ (structure) label within the SC, along with the mean percent area fraction of FG-positive RGCs in the corresponding retina of the DBA/2J were compared across several age groups (3, 9-10, 11-12, and 13 months) and to non- glaucomatous controls (D2G). (A) No significant difference in CTB label was found between

3-month pre-glaucomatous DBA/2J mice and D2G non-glaucomatous controls. The percent of CTB label in the SC of 9-10 month, 11-12 month, and 13 month DBA/2J mice was significantly reduced compared to the 3-month DBA/2J mice and non-glaucomatous controls (P < 0.001). No differences in the percent of intact CTB label were observed across

32 the 9-10, 11-12, and 13-month DBA/2J mice (B) Percent area fraction of intact ERRβ across

3-month, 9-10 month, and 11-12 month DBA/2J mice did not differ from each other or from the D2G controls. However, 13-month DBA/2J mice showed significant lower percentage of intact ERRβ label from all other DBA/2J age groups and D2G controls. (C, D) In order to validate the use of the percent area intact FG in the retina as our retrograde transport variable for comparison with the anterograde transport and structural variables, percent area of intact FG label in the retina as well as FG+ cell density in the same retina were conducted and compared for similar trends. (C) Percent area intact FG in the retinas for 3- month DBA2/2J mice compared to D2G controls showed no significant difference. This lack of difference was consistent with the FG density (D) in the retina for the same groups. (C)

The percent intact FG in the retinas of 9-10 and 11-12 month DBA/2J mice were significantly reduced compared to the retinas of both the 3-month DBA/2J and D2G (p <

0.001). The retinas of 13 month old DBA/2J mice showed the smallest percent area of intact

FG that was significant across all other groups. These effects in percent area intact FG across each age group correlated with the effects seen in retinal FG-density for the same groups

(D)(r= 0.08, P < 0.001). Asterisks and brackets indicate statistically significant differences between specific groups.

Figure 8. Differential loss of transport. Post-hoc paired t-tests were used to compare the relative loss of CTB, FG, and ERRβ (means) within each subject by age group. For both D2G and 3-month DBA/2J mice no measurable difference in percent area intact CTB (green),

ERRβ (red), and FG (blue) was observed. The percent of CTB label in the SC was significantly less intact than FG in the retina at 9-10 months (P= 0.03) and 11-12 months

(P=0.02) in the DBA/2J. Percent area of ERRβ label is significantly greater at 9-10 and 11-12 months compared to both CTB and FG label in the DBA2/J. There was no differences in the magnitude of intact CTB, FG, or ERRβ label at 13-months. In toto, while both CTB and FG are reduced in DBA/2J at 9-10 months and 11-12 months, FG remains more intact than CTB until 13 months. ERRβ persists most intact with no significant label reduction before 13

34 months. (Asterisks and brackets indicate statistically significant differences between subject groups)(n=8 for D2G; n=6 for 3mo. DBA; n=6 for 9-10mo. DBA; n=9 for 11-12 mo.

DBA; n=9 for 13 mo. DBA)

Retrograde tracing (FG) in the retina is reduced at 9-months but remains more intact than anterograde tracing in SC until 13-months

In contrast to our pre-glaucomatous D3 mice, retrograde transport was significantly reduced in the D9-10 group (Fishers LSD P = 0.02), D11-12 group (P < 0.001), and D13 group (P < 0.001) (see Figure 9). Intact retrograde transport levels were similar between the D9-10 and D11-12 groups (P = .312), but were significantly reduced in the D13 age group (Fishers LSD P< 0.001 in both sets of comparisons, see Figure 7, panel C & D).

Paired t-tests were used to compare differences in the magnitude of intact CTB, FG, and ERRβ within each subject by age group. In both the D2G control and D3 groups, there were no measurable differences in the magnitude of percent label between anterograde, retrograde, and structural markers. However, in the D9-10 group, the magnitude of anterograde transport loss was significantly greater than retrograde transport, t5 = 2.35, P =

0.03. This effect was echoed in the D11-12 group, t8 = 3.059, P = 0.02 (see Figure 8).

Structural integrity of the SC is maintained beyond retrograde transport loss

There was greater percent area fraction of intact collicular ERRβ label than retinal

FG coverage at D9-10 (t5= -3.41, P = 0.02) and D11-12 (t8= 3.85, P = 0.005)(see Figure 9), indicating that structure of the SC remained intact after both forms of transport showed significant deficits.

By D13, both forms of transport showed over 90% deficits in intact label with a corresponding 86% reduction in ERRβ.

Figure 9. Structural persistence after differential transport loss in the DBA/2J. Scatter plots compare the percent area of intact CTB and ERRβ in the SC to percent area FG in the retina for all individual cases within species and age groups. The use of a unity line (slope=1) in these figures represents a proportional equivalent relationship between two variables for cases that fall directly on the line. For instance, if anterograde and retrograde transport were proportionally reduced with increasing age in the DBA/2J, data points corresponding to percent area intact CTB and percent area intact FG will fall directly along the line with older animals clustered at the bottom of the slope and younger aged DBA/2J and D2G

36 controls at the top of the slope. (TOP) Individual cases for comparison of CTB (y-axis) label versus retrograde (FG; x-axis) tracing. The majority of 9-10 mo. old and 11-12 mo. old

DBA/2J cases fall well below the unity line, indicating a disproportionate relationship between CTB and FG transport loss, where CTB transport is more diminished than retrograde transport that remains more intact across these age groups. (BOTTOM)

Individual cases for comparison of ERRβ (y-axis) label versus retrograde (FG; x-axis) tracing. Unlike the CTB data, the majority of 9-10 and 11-12 aged DBA/2J cases fall above the unity line, indicating that percent area of ERRβ in the SC of these cases remains more intact than the percent area of FG in the retina across multiple ages.

Figure 10. Progression of transport loss. The percent area loss of anterograde (CTB in the

SC), retrograde (FG in the retina), and structure (ERRβ in the SC) was compared across

DBA/2J age groups. Percent loss is determined by subtracting the mean value at each age group by the mean value at the origin (D3) and then dividing this difference by the origin

37 value * 100. Anterograde transport experiences a greater magnitude of loss from 3 months to 9-10 months compared to retrograde transport. Though the magnitude of loss was greater for anterograde transport, both anterograde and retrograde transport across 3, 9-

10, and 11-12 age groups were reduced while no measurable loss was seen in the SC structure (ERRβ). By 13 months, both forms of transport showed over 90% loss in intact label with a corresponding 86% reduction in ERRβ.

Discussion

Our results are consistent with other studies that have examined these forms of transport separately in rodent glaucoma models. In the DBA/2J mouse, anterograde transport deficits have been shown at 8-10 months of age with pervasive loss by 12-months, while RGC axons and presynaptic terminals in the SC persist up to 18 months (Crish et al.,

2010; 2013). While these findings indicate that axonal transport deficits are pre- degenerative and may be differentially impaired in glaucoma, both forms of transport have never been assessed within the same animal—primarily due to technical limitations of the surgeries used for FG application. Historically, FG application required one or more large injections into the SC followed by intracranial insertion of FG-soaked pledgets for additional tracer exposure (Buckingham et al., 2008; Soto et al; 2008). This method resulted in extensive damage to the SC, precluding analysis of the RGC projection, anterograde transport levels, or FG exposure in this structure. Therefore, we modified our surgical methodology in order to achieve successful retrograde tracing to the retina while preserving the structure of the SC for anterograde transport analysis (see Figure 5 & 6).

Using bidirectional tract tracing of the central visual pathway within the same subject, we found that severe deficits in anterograde transport appeared earlier than severe deficits in retrograde transport and loss of RGC structure in the SC (see Figure 10). Anterograde transport was reduced by almost 70% at 9-10 months of age, a 3-fold greater reduction than was seen for retrograde transport at the same age range, which was reduced only by

23%(see Figure 10). Retrograde transport remained largely intact until 13-months where it showed a near-maximal 92% loss in label comparable to (and statistically indistinguishable from) anterograde loss (see Figure 10)—a finding consistent with other published reports that assessed retrograde transport in DBA/2J (Buckingham et al., 2008). This would indicate that the RGC axonal projection remains at least semi-functional after anterograde transport loss in glaucoma.

Structural Persistence of the SC

Structural persistence of the SC (as measured by intact ERRβ) was maintained in glaucomatous mice from 9 to 12 months of age and significant decreases in ERRβ label were only first measured at 13-months of age (see Figure 6, Figure 7, panel B, Figure 8, Figure 9).

Contrasting with both anterograde and retrograde labels that first showed significant reductions in the 9-10 month age group, our findings suggest that transport deficits are likely due to physiological or functional abnormalities as opposed to structural loss. Our results did differ from previous reports of Crish et al. (2010) in that our label for intact SC was significantly reduced at 13-months, whereas significant reductions in ERRβ did not appear in their study until 15-22 months. However, there is a significant amount of variability in structural persistence beyond 12-months of age (Crish et al., 2010) and it is possible that our smaller sample size of bidirectionally-traced animals captured more extreme deficit in ERRβ at 13-months.

Differential Transport Loss in Glaucoma

Ultimately, despite the deficit variation across each mode of transport, all transport eventually fails (see Figure 8); consequently this impaired movement of cargo along the axon, which eventually initiates several degenerative mechanisms including distal

Wallerian degeneration and apoptosis due to neurotrophin deprivation (Coleman, 2005).

Therefore, in the interest of preventing the eventual degeneration of RGC architecture, current and future objectives may want to address causative nature of these transport deficits.

Given the current literature, there is a large number of possibilities that may negatively affect proper transport function, including modification of molecular motors, cytoskeletal disruption, and metabolic dysfunction (Coleman et al., 2005; Shea et al., 2008).

As characterized in other well-known degenerative conditions, these changes can often affect very specific intra-axonal components necessary for transport within different compartments of the axon (Shea et al., 2008).

Molecular Motor Modification and Transport Deficits

Axonal transport described in its most basic form is the movement of protein cargo attached to a molecular motor traveling along a microtubule tract. Under normal physiological conditions, various protein kinases such as GSK3β, JNK, Cdk5, and PKC mediate molecular motor activity within the axon. However, the dysregulation of these kinases have been implicated in several age-related neurodegenerations to cause differential axonal transport impairment leading to apoptosis (Morfini et al., 2002,

2004,2005,2006b,2007; Wagey & Krieger, 1998). For instance, an increase and/or sustained activity of GSK3β has been shown to inhibit kinesin-mediated fast axonal transport in neurons. Though it remains unclear how dysregulation of GSK3β is initiated, it is clear that GSK3β directly phosphorylates kinesin to regulate its motility. Under pathological conditions where the activity of GSK3β is aberrant, both detachment of cargo from the motor and decreased motility of the motor are suggested to occur (Morfini et al.,

2002).

Additionally, stressor activation of the JNK-isoform JNK3 has been shown to phosphorylate both kinesin and dynein motors, preventing microtubule binding (Morfini et al., 2006; Morfini et al., 2009b), while cleavage of PKCδ by Caspase-3 as a result of CDK5 activation has also been shown to prevent axonal transport (Morfini et al., 2007).

Interestingly, it is important to note that several isoforms exist for each of the kinases described and not all act affect transport equally. While JNK3 affects both anterograde and retrograde transport, JNK2 like GSK3β impairs anterograde transport, but not retrograde transport (Morfini et al., 2006; Morfini et al., 2009b). Therefore, the contrasting selectivity across kinases and their isoforms provides a compelling hypothesis to the differential impairment of axonal transport observed in our study.

Metabolic Dysfunction and Transport Deficits

Retinal ganglion cells relay an immense amount of visual information over a great distance from the retina to the brain. In order to cover the distance, kinesin and dynein are dependent on ATP to effectively transport cargo (Carelli et al., 2002). Mitochondria are the major sources of ATP in RGC neurons, however, mitochondrial biogenesis occurs only in the retinal ganglion cell body; therefore, in order to meet the high-energy demands of other compartments, transport is required. Thus, it is possible that energy depletion due to mitochondria dysregulation could impair axonal transport (Ochs and Hollingsworth, 1971).

The co-occurrence of abnormal mitochondrial morphology and features (accumulation of debris and vesicles, microtubule reduction) suggesting transport deficits have been observed across several other optic neuropathies (Carelli et al., 2002; Carrelli et al., 1999).

Several factors can alter mitochondrial function including CDK5 (Cheung & Ip, 2004) and reactive-oxygen species (ROS) accumulation. ROS can impair mitochondrial respiration and depolarize its membrane (Ward et al., 2000). Given that these factors could selectively

42 target mitochondria within a specific compartment of the RGC neuron, depending on the compartment affected, differential transport impairment could result.

Cytoskeletal Alterations and Transport Deficits

Breakdown or modification of cytoskeletal elements are common pathologies in neurological disorders such as Alzheimer’s Disease and ALS and can negatively affect neuron structure and function in terms of cellular integrity, axonal transport, and signaling

(Coleman, 2005; Conforti et al., 2007; Shea & Chan, 2008, Cuchillo-Ibanez et al., 2008;

Morfini et al., 2009b; Shea et al., 2009). It is possible that disruptions in axonal transport shown in our current studies may result from intra-axonal blockades caused by aberrant cytoskeletal organization/breakdown and may reflect pre-degenerative changes occurring in glaucoma (see Figure 8). Calpains are Ca2+ -dependent proteases implicated in a number of neurodegenerative diseases (Huang et al., 2008) that can directly break down alpha-II- spectrin structural proteins and heavy chain neurofilaments (Siman et al., 1984; Chan &

Mattson, 1999). Calpain also activates CDK5 and Errk-1,2, both of which can phosphorylate tau (Cheung & Ip, 2004; Veeranna et al., 2004). In the normal brain, tau is utilized in neurons for stabilization of the microtubule tracts. However, hyperphosphorylation and aggregation of tau has been shown to reduce axonal transport, both anterograde and retrograde, in the optic nerve of transgenic tau mice (Bull et al., 2012). It has been suggested that this specific “hyperphosphorylation” of tau may destabilize microtubules, thereby preventing motor binding, thus, slowing or blocking transport (Shea & Chan, 2008). Even so, these aforementioned blockades may be more easily bypassed by dynein given its ability for both bidirectional and lateral movement along the microtubule tract (Shea et al., 2004).

It is possible that this unique ability attributed to dynein and not kinesin may reflect why a

43 greater magnitude loss for anterograde transport is observed earlier than retrograde transport (see Figure 9).

Conclusion

Glaucoma is a multi-faceted condition that shares many pathological traits to other complex neurodegenerations. This work establishes that predegenerative differential impairment of axonal transport is a major hallmark in the progression of the disease. In addition, we reviewed several pathological processes that could potentially account for these observed deficits in transport. The implication of this work may open the door to innovative treatments that specifically target these processes in order to restore function and substructure in glaucomatous axons that still maintain largely intact.

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