Retinal ganglion cell dendrite pathology and synapse loss

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Retinal ganglion cell dendrite pathology and synapse loss: implications for glaucoma Jessica Agostinone*,†, Adriana Di Polo*,†,1 *Department of Neuroscience, University of Montreal, Montreal, QC, Canada Centre de Recherche du Centre Hospitalier de l’Universite´ de Montre´al (CRCHUM), University of Montreal, Montreal, QC, Canada 1 Corresponding author: Tel.: 514-890-8000; Ext. 31280; Fax: 514-412-7936, e-mail address: [email protected]

†

Abstract Dendrites are exquisitely specialized cellular compartments that critically influence how neurons collect and process information. Retinal ganglion cell (RGC) dendrites receive synaptic inputs from bipolar and amacrine cells, thus allowing cell-to-cell communication and flow of visual information. In glaucoma, damage to RGC axons results in progressive neurodegeneration and vision loss. Recent data indicate that axonal injury triggers rapid structural alterations in RGC dendritic arbors, prior to manifest axonal loss, which lead to synaptic rearrangements and functional deficits. Here, we provide an update on recent work addressing the role of RGC dendritic degeneration in models of acute and chronic optic nerve damage as well as novel mechanisms that regulate RGC dendrite stability. A better understanding of how defects in RGC dendrites contribute to neurodegeneration in glaucoma might provide new insights into disease onset and progression, while informing the development of novel therapies to prevent vision loss.

Keywords Retinal ganglion cell, Dendrite, Synapse, Axonal injury, Ocular hypertension, Glaucoma

1 DOES DENDRITIC PATHOLOGY CONTRIBUTE TO VISION LOSS IN GLAUCOMA? During normal visual processing, retinal ganglion cell (RGC) dendrites receive synaptic inputs from bipolar and amacrine cells in the inner plexiform layer. This information is integrated, processed, and sent via RGC axons in the optic nerve to visual centers in the brain (Masland, 2012). The structural integrity of dendrites is essential

Progress in Brain Research, ISSN 0079-6123, http://dx.doi.org/10.1016/bs.pbr.2015.04.012 © 2015 Elsevier B.V. All rights reserved.

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for vision. Dendrites allow communication between RGCs and other retinal neurons via synapses. Furthermore, dendrites integrate and propagate input signals to the RGC soma before action potentials can be generated at the axon hillock, a prerequisite for successful transmission of visual information. In glaucoma, progressive RGC degeneration results in irreversible vision loss. The optic nerve head (ONH) has been identified as a critical point of initial axonal damage in glaucoma. The arcuate pattern of RGC and visual field loss correlates with the topographic organization of axons within the ONH and alterations in the lamina cribrosa (Burgoyne et al., 2005; Nickells et al., 2012). Importantly, it is increasingly recognized that injury to RGC axons triggers rapid changes in dendrites. Pathological changes in RGC dendritic arbors include branch retraction, reduced complexity, and synapse loss (Della Santina et al., 2013; Liu et al., 2011; Morgan, 2012). Furthermore, high intraocular pressure (IOP), a major risk factor in glaucoma, has been recently shown to dramatically alter retinal function before irreversible structural damage in the optic nerve occurs, suggesting early synaptic defects (Frankfort et al., 2013; Pang et al., 2015). In the hypothetical scenario in which pathological changes in the ONH could be halted or repaired, visual impairment might still persist if functional connections between RGCs and their presynaptic targets in the retina are not restored. Dendritic retraction and loss of synapses might prevent cell–cell connectivity and neurotransmission thereby contributing to RGC death and permanent visual deficits (Fig. 1). Although our understanding of how alterations in RGC dendrites contribute to neurodegeneration is still in its infancy, this expanding field of investigation might provide new insights into disease onset and progression, while informing the development of new therapies to prevent vision loss in glaucoma and other optic neuropathies. In this review, we aim to provide an update on RGC dendrite pathology and key factors that might influence this response with a focus on data derived from in vivo models of optic nerve damage. We also highlight areas where scarcity of information prevails and discuss the challenges to fully understand the contribution of dendritic and synaptic defects in glaucoma.

2 MORPHOLOGICAL DIVERSITY OF RGC DENDRITES: AN EMBARRASSMENT OF RICHES Newly born RGCs initiate an axon that grows along the nerve fiber layer lining the innermost surface of the retina, then exits the eye via the ONH, and extends into the optic nerve proper to reach targets in the brain (Bao, 2008; Haupt and Huber, 2008). As RGC axons enter the brain, dendrites begin to grow from the cell soma extending through the inner plexiform layer to form synapses with bipolar and amacrine cell processes (Choi et al., 2010; Holt, 1989). Primary dendrites then branch to produce higher order dendrites with no initial preference as to cell polarity, but the expanding dendritic arbor is eventually sculpted to occupy only the apical side of the RGCs (Choi et al., 2010). Dendrites are extremely dynamic during development, extending

ARTICLE IN PRESS 2 Morphological diversity of RGC dendrites: An embarrassment of riches

FIGURE 1 The role of RGC dendritic pathology in glaucoma: a model. (A) In healthy RGCs, dendrites are fully arborized within the inner plexiform layer (IPL) and establish synaptic contacts with bipolar and amacrine cells allowing neurotransmission. (B) Stress or damage at the level of the optic nerve head (ONH), believed to be the initial site of injury to RGC axons, causes rapid dendritic alterations including process retraction and synapse loss. (C) A hypothetical scenario in which pathological changes in the ONH are halted or repaired but visual impairment persists due to lack of functional connections between RGCs and their presynaptic counterparts. PS: photoreceptor segments; ONL: outer nuclear layer; OPL: outer plexiform layer; INL: inner nuclear layer; IPL: inner plexiform layer; GCL: ganglion cell layer; ONH: optic nerve head; ON: optic nerve.

and retracting actively, and the final arbor morphology is achieved through a dynamic process of branch addition and elimination (Cohen-Cory and Lom, 2004; Wong and Ghosh, 2002). To achieve functional specificity, RGC dendrites must be confined to specific strata within the inner plexiform layer in order to synapse with a small group of amacrine and bipolar neurons (Wassle, 2004). The mechanisms by which RGC dendrites become confined to specific lamina are still an open question, but both extrinsic molecular cues and activity-dependent refinement have been proposed (Atkinson-Leadbeater and McFarlane, 2011; Tian, 2011). The end result of this complex developmental process is a staggering diversity of RGC dendritic arbor shapes and sizes. Depending on the study, RGCs have been classified into 11–22 different subtypes based on dendritic arbor morphology and

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synaptic connections in mice (Badea and Nathans, 2004; Coombs et al., 2006; Kong et al., 2005; Sun et al., 2002b; Volgyi et al., 2009), rats (Huxlin and Goodchild, 1997; Sun et al., 2002a), rabbits (Rockhill et al., 2002), cats (Citron et al., 1988; Hochstein and Shapley, 1976), and primates (Dacey, 1993a,b; Peichl, 1991). In spite of this large morphological diversity, the few identified molecular markers for discrete RGC subtypes include neurofilament-H (NF-H, SMI32), melanopsin, and junctional adhesion molecule B (Coombs et al., 2006; Kim et al., 2008; Lin et al., 2004). Several recent studies have capitalized on the use of transgenic mice that selectively express yellow fluorescent protein (YFP) in RGCs under control of the Thy1 promoter (Thy1-YFPH) (Feng et al., 2000), in which all RGC subtypes are found (Coombs et al., 2006). In this mouse strain, RGC-specific YFP expression is detected in a small number of RGCs, thus allowing visualization of individual dendritic arbors without interference from overlapping dendrites in neighboring neurons. More recently, mice carrying other genetically driven markers show promise as a tool to identify RGC subtypes for dendritic arbor analysis (El-Danaf and Huberman, 2015; Kay et al., 2011; Su¨mbu¨l et al., 2014; Zhang et al., 2012), but electrophysiological testing is needed to verify these classification systems. It has long been proposed that RGC subtypes may differ in their vulnerability to axonal damage. Early studies in the feline retina demonstrated that a cells endowed with large somata and dendritic arbors undergo a significant decrease in dendritic field size after axotomy, while b cells with medium-sized somata and more compact dendritic trees are less affected (Weber and Harman, 2008). Recent work supporting the idea that the dendrites of distinct RGC subtypes are differentially affected depending on the onset, duration of injury, and arbor stratification (Della Santina et al., 2013; El-Danaf and Huberman, 2015; Feng et al., 2013; Morquette et al., 2014) will be described below. Furthermore, the magnitude of some neuroprotective strategies differs substantially among different RGC classes (Lindsey et al., 2015). Although new and potentially useful methods to classify and analyze RGCs in disease are emerging (Su¨mbu¨l et al., 2014; Tribble et al., 2014), at present there is no agreement on a universal classification system that would allow reliable comparison of data sets from different studies. Regardless of methodological issues, it is clear that the structural and functional diversity of RGCs needs careful consideration when studying pathological changes in dendrites as this variability may confound data interpretation. Thus, to reach a consensus regarding RGC subtype classification will be an important step to make headway in our understanding of how cell-specific changes contribute to neurodegeneration in glaucoma.

3 AXONAL INJURY TRIGGERS PATHOLOGICAL CHANGES IN RGC DENDRITES Changes in RGC dendrites have been historically perceived as anecdotal or intriguing at most. Indeed, very few studies on RGC dendritic alterations in models of optic nerve damage were published before 2010. Among these were the pioneering work

ARTICLE IN PRESS 3 Axonal injury triggers pathological changes in RGC dendrites

by Weber and colleagues describing early structural abnormalities in RGC dendrites in a nonhuman primate model of chronic IOP elevation (Weber et al., 1998). In this study, parasol cells in glaucomatous eyes showed a significant reduction in dendritic field size compared to control subjects. Morphological changes in RGC dendrites were detected slightly prior to axonal thinning or soma shrinkage, suggesting that dendritic abnormalities precede degeneration of other ganglion cell compartments. Importantly, this pattern of dendritic damage correlated with significant functional deficits in affected RGCs characterized by reduced spatial–temporal response properties (Weber and Harman, 2005). Subsequently, other groups confirmed a decrease in dendritic area and number of branches in feline (Shou et al., 2003) and rat (Morgan et al., 2006) ocular hypertension models. The landscape of the field, however, is rapidly changing and in recent years there has been a substantial increase in information confirming and expanding our understanding of RGC dendritic pathology in glaucoma. In the next sections, we will discuss current work stemming from models of acute optic nerve injury, ocular hypertension, and other neurodegenerative diseases.

3.1 ACUTE AXONAL INJURY A valuable approach to study the onset and time-course of dendritic changes in injured RGCs has been the use of acute models of axonal damage such as optic nerve crush or cut (axotomy). The advantages of acute lesions include a well-defined onset of cellular loss, a demarcated time window to investigate early morphological changes in dendrites, and less variability among experimental animals. Using Thy1-YFP mice subjected to either optic nerve crush or axotomy, recent studies have demonstrated injury-induced RGC dendritic shrinkage (Kalesnykas et al., 2012; Leung et al., 2011; Lindsey et al., 2015; Morquette et al., 2015). Real-time confocal scanning laser ophthalmoscope (CSLO) imaging was used to show that optic nerve crush led to dendritic retraction preceding axon and soma loss in YFP-labeled RGCs (Leung et al., 2011). Although the ability to monitor the same RGC over time is powerful, the level of resolution of fine dendrites using CSLO is not yet on par with conventional confocal microscopy and three-dimensional reconstruction of dendritic arbors (Badea and Nathans, 2004; Coombs et al., 2006; Sun et al., 2002b). As a result, fewer morphological RGC groups, each likely to comprise several of the traditional subtypes, have been described using CSLO. For example, RGCs in Thy1-YFP mice were classified into six groups with CSLO (Leung et al., 2011; Lindsey et al., 2015) compared to 14 clusters described using standard techniques (Coombs et al., 2006). Among the six RGC subtypes, cells with larger arbors, longer branches, and located at larger distances from the optic disc had slower rates of dendritic shrinkage and a higher probability of survival (Leung et al., 2011). Another study using the Thy1-YFP mouse strain and crush injury model reported a modest reduction in the length and complexity of RGC dendrites primarily in the nasal retinal quadrant (Kalesnykas et al., 2012). RGCs were classified only as a function of fluorescence intensity, as dim or bright YFP cells, which might not be

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sensitive enough to accurately discriminate the most vulnerable RGC subtypes from more resistant neurons. More recently, the structure of RGC dendritic arbors was investigated at 3 days after axotomy, a time when no change in the intensity or number of YFP-labeled RGCs was observed and prior to overt axonal or soma loss (Morquette et al., 2015). This study focused on RGCs expressing NF-H, also known as SMI32, a marker that labels several structural classes identified by cluster analysis and comprising a substantial RGC population (Coombs et al., 2006; Lin et al., 2004). NF-H-positive RGCs, endowed with medium- to large-sized dendritic arbors, underwent substantial atrophy and loss of arbor complexity at 3 days after axotomy. Dendritic tree shrinkage was observed in all retinal quadrants independently of retinal eccentricity (Morquette et al., 2015). In this study, whole-cell electrophysiological recordings demonstrated that, as early as 3 days after axotomy, RGCs displayed increased firing rates characteristic of hyperexcitable neurons. This finding is consistent with a recent study showing a significant increase in spontaneous RGC firing of action potentials in a mouse model of microbead-induced ocular hypertension (Ward et al., 2014). The excitability of a neuron is the result of a balance between several factors including resting potential, input resistance, and soma size. The observed dendritic abnormalities were not accompanied by detectable changes in any of these parameters (Morquette et al., 2015); therefore, it is unlikely that axotomy-induced hyperexcitability is the result of changes in intrinsic membrane properties or soma size. These data suggest that functional deficits most likely derive from injuryinduced changes in dendritic integrity and rearrangement of synaptic inputs, with hyperexcitability resulting from an increase in excitatory connections or a decrease in inhibitory synapses.

3.2 OCULAR HYPERTENSION MODELS It is widely accepted that current animal models of glaucoma have pros and cons; however, sufficient refinement of these methodologies has allowed analysis of RGC dendrite pathology induced by ocular hypertension (Della Santina et al., 2013; El-Danaf and Huberman, 2015; Feng et al., 2013; Kalesnykas et al., 2012; Li et al., 2011; Liu et al., 2014; Williams et al., 2013a). To investigate early dendritic damage, Thy1-YFP mice were subjected to acute IOP elevation leading to retinal ischemia. CSLO imaging revealed progressive loss of RGC dendrites when high IOP was sustained for 90 or 120 min, while ocular hypertension for 30 or 60 min had no effect (Li et al., 2011). At the other end of the spectrum, Williams et al. asked whether chronic exposure to elevated IOP led to morphological alterations in RGC dendrites using DBA/2J mice, a model of spontaneous pigmentary glaucoma, which develop high IOP and RGC loss with age (Anderson et al., 2002; Chang et al., 1999). To visualize dendritic structure, DBA/2J mice were crossed with Thy1-YFP mice and analyzed between 9.5 and 11 months of age. While no significant changes in dendritic structure were detected when YFP was used as marker, a reduction in arbor field area and complexity was revealed when RGCs were visualized by DiOlistic

ARTICLE IN PRESS 3 Axonal injury triggers pathological changes in RGC dendrites

labeling (Williams et al., 2013a). Thy1 gene expression is downregulated following RGC injury (Huang et al., 2006; Schlamp et al., 2001); therefore, reduced Thy1driven YFP levels might preclude characterization of long-term dendritic changes. Alternatively, DiOlistic labeling can be used for long-term studies and analysis of RGC dendrites in species other than mice. For example, in rats with elevated IOP induced by injection of hypertonic saline solution into an episcleral vein (Morrison model) (Morrison et al., 1997), DiOlistic labeling was effective to show substantial RGC dendritic arbor retraction as early as 1 week after high IOP induction (Liu et al., 2014). These changes were followed by retraction of dendrites in target neurons of the superior colliculus and lateral geniculate nucleus (Liu et al., 2014), confirming that structural alterations of dendritic processes in glaucoma occur throughout the visual system as previously shown in primates (Gupta et al., 2007; Ly et al., 2011). Recent studies have tackled the important issue of differential vulnerability of RGC dendrite degeneration and the timing of functional deficits in ocular hypertension models. Feng et al. (2013) used laser photocoagulation to elevate IOP in Thy1YFP mice and classified RGCs into three subtypes: On, Off, and On–Off based on their dendritic lamination pattern in the inner plexiform layer. At 2 months after laser treatment, On cells exhibited a decrease in dendritic field area, while there was no significant changes in On–Off cells (Feng et al., 2013). An elegant study by Della Santina et al. (2013) used Thy1-YFP mice to induce IOP elevation by polystyrene microbead injection (Sappington et al., 2010) and performed multielectrode array recordings to identify four major functional RGC subtypes, namely, On (sustained or transient) and Off (sustained or transient). The light response of three RGC subtypes, On (sustained) and Off (sustained and transient), was reduced at 2 weeks after microbead injection before pronounced dendritic changes were detected (Della Santina et al., 2013). More recently, the microbead occlusion model was applied to transgenic mouse lines that express green fluorescent protein (GFP) in selective RGC subtypes to investigate dendritic changes as early as 1 week after high IOP induction (El-Danaf and Huberman, 2015). This study reported that among the four subtypes examined, those RGCs that stratified within the Off sublamina of the inner plexiform layer were among the first to undergo arbor shrinkage, asymmetry, and death, whereas dendrites stratified within the On sublamina exhibited no change. This observation is significant because while both Off (transient) and On (direction selective) RGCs have very large dendritic arbors, only Off (transient) cells undergo significant dendrite shrinkage, ruling out field size as a vulnerability factor. Intriguingly, in anterior-preferring On–Off bistratified RGCs, the reduction of dendritic arbors in the Off sublamina was accompanied by expansion of arbors in the On sublamina, which can be interpreted as compensation for the loss of Off dendrites and might explain previous reports showing arbor size increase (Ahmed et al., 2001; Kalesnykas et al., 2012). Moreover, a reduced number of dendritic branches was observed in melanopsin-positive intrinsically photosensitive RGCs, which are

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functionally On-type cells but stratify primarily in the Off sublamina (El-Danaf and Huberman, 2015). Together, these studies put forward the interesting hypothesis that RGCs with dendritic arbor stratification in the Off sublamina, located in the outer half of the inner plexiform layer toward the sclera, are more vulnerable to glaucomatous damage (Della Santina et al., 2013; El-Danaf and Huberman, 2015). The observation that the electrophysiological response of RGCs decreases before dendritic arbor shrinkage is detected correlates with findings showing that key components of synaptic complexes or activity in the inner plexiform layer are rapidly altered by ocular hypertension (Della Santina et al., 2013; Fu et al., 2009; Park et al., 2014; Ward et al., 2014; Weitlauf et al., 2014). Collectively, these studies suggest a model in which alterations of functional synapses precede morphological changes, including dendritic retraction. Hence, synaptic changes might be among the earliest signs of RGC dysfunction and neurodegeneration in glaucoma.

3.3 RGC DENDRITIC CHANGES IN OTHER NEURODEGENERATIVE DISEASES There is substantial evidence that dendritic abnormalities and loss of synapses are prominent features of neurodegenerative diseases including Alzheimer’s disease (AD), amyotrophic lateral sclerosis, and Parkinson’s disease (Cochran et al., 2014; D’Ambrosi et al., 2014). Among these, AD and glaucoma have been proposed to share common neurodegenerative pathways. Visual deficits are found in AD patients, including difficulty reading, impaired depth perception, abnormal color recognition, and spatial contrast sensitivity (Cronin-Golomb, 1995; Cronin-Golomb et al., 1991; Gilmore and Whitehouse, 1995; Guo et al., 2010; Hutton et al., 1993; Jackson and Owsley, 2003; Katz and Rimmer, 1989; Lee and Martin, 2004; Mendez et al., 1996). Loss of RGCs has been documented in AD (Blanks et al., 1989, 1996; Hinton et al., 1986) and, although controversial (Curcio and Drucker, 1993), it might contribute to defects in contrast sensitivity and motion perception in AD patients (Hinton et al., 1986; Jackson and Owsley, 2003; Katz and Rimmer, 1989; Lee and Martin, 2004). Imaging studies using optical coherence tomography have reported thinning of the retinal nerve fiber layer in individuals affected by AD (Danesh-Meyer et al., 2006; Paquet et al., 2007; Valenti, 2007). To address whether RGC dendrites are altered in AD, a recent study investigated their morphology in 14-month-old transgenic mice expressing the APP(SWE) (amyloid precursor protein-Swedish mutation Tg2576) (Williams et al., 2013b). Tg2576 mice exhibit increased APP levels in the brain and develop amyloid deposits and behavioral deficits as they age (Irizarry et al., 1997). Using DiOlistic labeling, the authors demonstrated a significant reduction in RGC dendritic arbor size and complexity in Tg2576 mice, in the absence of RGC loss (Williams et al., 2013b). Therefore, imaging changes in RGC dendrites might serve as a complementary tool to assess AD onset and progression.

ARTICLE IN PRESS 4 Mechanisms that regulate dendrite and synapse stability

4 MECHANISMS THAT REGULATE DENDRITE AND SYNAPSE STABILITY Despite the fact that dendritic and synaptic defects are likely to have devastating consequences on neuronal function and survival, the mechanisms that regulate RGC dendrite degeneration in glaucoma are vastly unknown. A better understanding of the molecular pathways that regulate dendritic stability or loss is critical for the development of targeted therapies to maintain or enhance RGC connectivity and function. The next sections describe new findings on molecular pathways that regulate RGC dendrite dynamics and stability.

4.1 THE RHO-FAMILY OF GTPASES The dendrite cytoskeleton consists of a packed network of microtubules that provides structural stability and serves as a frame for the transport of organelles and membrane trafficking (Conde and Caceres, 2009). Dendritic spines are enriched in filamentous actin (F-actin), which regulates spine shape and stability as well as the organization of postsynaptic proteins (Star et al., 2002). Although RGCs lose dendritic spines as they mature and only spine-like processes remain in a subset of RGCs (Coombs et al., 2006; Dacey, 1993a; Wong, 1990), their dendrite and synapse stability are likely to rely on fine regulation of cytoskeletal dynamics (Hotulainen and Hoogenraad, 2010; Koleske, 2013; Star et al., 2002). The Rho-family of GTPases mediates many critical cellular processes including the organization of the microtubule and actin cytoskeletons (Govek et al., 2005). The most extensively studied members, RhoA, Rac1, and Cdc42, have been implicated in the regulation of dendritic arborization, spine morphogenesis, growth cone development, axon guidance, and neuronal survival (Hall and Lalli, 2010; Stankiewicz and Linseman, 2014; Tolias et al., 2011). Using embryonic chick retina, Wong et al. (2000) demonstrated that rapid RGC dendritic motility during development is an actin-dependent process regulated by Rho-GTPases. Using gain- and lossof-function experiments, this group demonstrated a reciprocal role of Rac1 and RhoA, with Rac1 stimulating dendrite turnover rate and RhoA decreasing the rate and extent of dendrite motility (Wong et al., 2000). RhoA and its downstream effector Rho kinase (ROCK) have been extensively studied in the context of axonal growth. Their activation results in growth cone collapse and axon regeneration failure (McKerracher et al., 2012; Yamashita and Fujita, 2014). RhoA and ROCK inhibition has been shown to promote RGC survival and axon growth (Bertrand et al., 2005; Lehmann et al., 1999; Lingor et al., 2008), but their role on RGC dendritic growth and stability are poorly understood. A study examined the effect of RhoA inhibition using BA-210, a cell-permeable RhoA inhibitor (Winton et al., 2002), on axon regeneration and dendritic morphology after optic nerve crush in rats (Drummond et al., 2014). Analysis of RGC dendritic arbors visualized by intracellular injection of Lucifer Yellow demonstrated that a combination of BA-210 with

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ciliary neurotrophic factor and cell-permeable cAMP promoted RGC axon regeneration but, intriguingly, resulted in abnormal dendritic arborization including long aberrant processes (Drummond et al., 2014). Another recent study demonstrated that intravitreal injection of cell-permeable constitutively active Rac1 protein preserved RGC dendritic morphology at 15 days after optic nerve crush in Thy1-YFP mice (Lorenzetto et al., 2013). In the same study, Rac1 activation was shown to promote RGC survival and axonal regrowth past the crush lesion site. Collectively, these studies suggest that Rho-GTPases contribute to the regulation of RGC dendritic arbor dynamics during development and after acute crush injury, but additional work is required to determine whether these proteins play a role in glaucoma.

4.2 THE MAMMALIAN TARGET OF RAPAMYCIN PATHWAY The mammalian target of rapamycin (mTOR) kinase is well known for its function in the growth and proliferation of nonneuronal cells (Zoncu et al., 2011), but has attracted recent attention due to emerging roles in the nervous system (Jaworski and Sheng, 2006; Swiech et al., 2008). mTOR regulates protein translation and is a key signal integrator for a variety of extracellular stimuli including growth factors, neurotransmitters, energy, and stress (Lipton and Sahin, 2014). mTOR is stimulated by the availability of nutrients, while it is inhibited by cellular stressors such as hypoxia, inflammation, or low ATP levels (Cam and Houghton, 2011; Frost and Lang, 2011; Zoncu et al., 2011). mTOR has been identified as a critical component of dendritic tree development (Gao et al., 2000; Jaworski et al., 2005; Kumar et al., 2005; Kwon et al., 2006). For example, a substantial reduction in the number of dendritic branches and arbor shrinkage was observed in developing hippocampal neurons treated with rapamycin, a specific inhibitor of the mTOR complex 1 (mTORC1), or short interference RNA (siRNA) against mTOR ( Jaworski et al., 2005; Kumar et al., 2005). Recent work supports a role for mTOR in RGC axon regeneration (Leibinger et al., 2012; Morgan-Warren et al., 2013; Park et al., 2010). In addition, mTOR has been recently implicated in the regulation of dendritic spine morphology, synaptogenesis, and synaptic plasticity (Hoeffer and Klann, 2010; Li et al., 2010). A recent study investigated the role of mTOR in the regulation of RGC dendrite pathology after axonal injury (Morquette et al., 2015). Thy1-YFP mice subjected to optic nerve axotomy were used to demonstrate rapid retraction of RGC dendrites accompanied by downregulation of mTOR function in these neurons but not in other retinal cells. Importantly, axonal injury led to upregulation of the stress-induced protein Regulated in development and DNA damage response 2 (REDD2 or RTP801L), a potent inhibitor of mTOR. REDD2 knockdown using targeted siRNA restored mTOR function in injured RGCs and fully rescued their dendritic arbors, increasing dendritic length, field area, and branch complexity. REDD2 depletion also abrogated pathological RGC hyperexcitability, restoring light-triggered responses, and extending cell survival after optic nerve lesion (Morquette et al., 2015). Rapamycin treatment obliterated the effect of REDD2 knockdown indicating that dendritic rescue was mediated by mTORC1. These data support a model in which axonal

ARTICLE IN PRESS 5 Conclusions and future directions

injury-induced upregulation of the stress-response protein REDD2 leads to mTOR inhibition triggering early dendritic arbor retraction, neuronal dysfunction, and subsequent death of adult RGCs. Future work is required to determine whether mTOR plays a role in RGC dendrite stability in models of ocular hypertension.

5 CONCLUSIONS AND FUTURE DIRECTIONS In recent years, there has been a tangible increase in the number of studies investigating RGC dendritic abnormalities triggered by axonal injury. Emerging data indicate that damage to RGC axons, by ocular hypertension or acute insults, triggers rapid, and marked dendritic modifications. Most studies agree that the primary changes involve dendritic retraction, characterized by a reduction in dendritic field area, process length, and arbor complexity. The consensus is that these dendritic abnormalities occur soon after injury and prior to overt loss of RGC axons, thus identifying dendrite pathology as an early sign of neurodegeneration. Importantly, recent data put forward the tantalizing hypothesis that damage to RGC axons triggers stress signals that cause loss of synapses and functional deficits before structural changes in dendrites can be detected (Della Santina et al., 2013). If confirmed, this observation opens the possibility that synaptic rearrangements underlie early RGC dysfunction and visual deficits in glaucoma. Most studies agree that not all RGC subtypes are affected equally by optic nerve injury. However, the question regarding which RGC subtype is more susceptible to neurodegeneration based on dendritic morphology is still open for discussion. A major road block has been the lack of a unifying classification system used consistently by investigators that would allow comparison among data sets obtained from different injury paradigms and animal species. The availability of transgenic mice strains expressing genetically encoded markers is extremely promising; however, their utility is limited to mouse models. Nonetheless, the abundance of recent data generated with these tools, and covered in this review, is a good sign of progress in the field. An interesting new observation challenges the prevalent idea that larger RGCs are more susceptible to neurodegeneration in glaucoma. Indeed, using transgenic markers, recent studies showed that RGCs with dendrites stratified in the Off sublamina of the inner plexiform layer appear to be more sensitive to dendritic defects and neurodegeneration (El-Danaf and Huberman, 2015). Although progress has been made regarding the description of dendritic changes after injury and the RGC subtypes affected, the field is still lagging behind in our understanding of molecular mechanisms that regulate RGC dendrite and synapse stability and how these are disrupted in disease. Recent work provides insights into the role of mTOR and the family of Rho-GTPases (Drummond et al., 2014; Lorenzetto et al., 2013; Morquette et al., 2015). mTOR is an interesting candidate because many of the stress signals proposed to contribute to glaucomatous damage converge on this kinase. Nevertheless, a better understanding of the molecular mechanisms leading to RGC dendrite degeneration during ocular hypertension is important. This knowledge

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will likely be useful for the development of strategies that maintain or promote the stability of dendrites and synapses, allowing RGC connectivity, to preserve visual function in glaucoma.

ACKNOWLEDGMENTS This work was supported by grants to A.D.P. from the Canadian Institutes of Health Research (CIHR). We thank Dr. Timothy E. Kennedy for comments on the manuscript, and Mr. James Correia for assistance with the figure.

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