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Towards axonal regeneration and neuroprotection in glaucoma: Rho kinase inhibitors as promising
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therapeutics
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Sarah Van de Velde1, Lies De Groef², Ingeborg Stalmans1, Lieve Moons2 and Inge Van Hove2
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Section, Department of Biology, KU Leuven, Leuven, Belgium
Laboratory of Ophthalmology, Department of Neurosciences, KU Leuven, Leuven, Belgium
Neural Circuit Development and Regeneration Research Group, Animal Physiology and Neurobiology
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Corresponding author:
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Dr. Lieve Moons
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Neural Circuit Development and Regeneration Research Group
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Animal Physiology and Neurobiology Section
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Department of Biology
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KU Leuven
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Naamsestraat 61, Box 2464
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B-3000 Leuven, Belgium
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Tel: +32-16-32.39.91
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Fax: +32-16-32.42.62
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Lieve.moons@bio.kuleuven.be
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Running title: ROCK inhibition for retinal axonal regeneration and neuroprotection
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Key words: ROCK inhibition, glaucoma, axonal regeneration, neuroprotection and ocular blood flow
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Abstract
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Due to a prolonged life expectancy worldwide, the incidence of age-related
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neurodegenerative disorders such as glaucoma is increasing. Glaucoma is the second cause of
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blindness, resulting from a slow and progressive loss of retinal ganglion cells (RGCs) and their axons.
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Up to now, intraocular pressure (IOP) reduction is the only treatment modality by which
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ophthalmologists attempt to control disease progression. However, not all patients benefit from this
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therapy, and the pathophysiology of glaucoma is not always associated with an elevated IOP. These
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limitations, together with the multifactorial etiology of glaucoma, urge the pressing medical need for
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novel and alternative treatment strategies. Such new therapies should focus on preventing or
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retarding RGC death, but also on repair of injured axons, to ultimately preserve or improve structural
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and functional connectivity. In this respect, Rho-associated coiled-coil forming protein kinase (ROCK)
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inhibitors hold a promising potential to become very prominent drugs for future glaucoma
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treatment. Their field of action in the eye does not seem to be restricted to IOP reduction by
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targeting the trabecular meshwork or improving filtration surgery outcome. Indeed, over the past
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years, important progress has been made in elucidating their ability to improve ocular blood flow, to
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prevent RGC death/increase RGC survival and to retard axonal degeneration or induce proper axonal
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regeneration. Within this review, we aim to highlight the currently known capacity of ROCK inhibition
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to promote neuroprotection and regeneration in several in vitro, ex vivo and in vivo experimental
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glaucoma models.
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Contents
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I. Introduction .......................................................................................................................................... 3
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II. Glaucoma pathophysiology ................................................................................................................. 4
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III. Novel strategies for glaucoma therapy: neuroprotection and axonal regeneration ......................... 7
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IV. The Rho-ROCK pathway ..................................................................................................................... 8
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1. Downstream effectors of ROCK: well-known cytoskeletal regulators ............................................ 9
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2. The ROCK signaling cascade and its involvement in neuronal cell death and axonal outgrowth . 10
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V. ROCK inhibition as a multi-target approach for glaucoma treatment .............................................. 12
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1. ROCK inhibitors as IOP lowering and anti-scarring agents in glaucoma ....................................... 12
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2. ROCK inhibitors as novel neuroprotective and axon regenerative agents in glaucoma ............... 14
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2.1. ROCK expression in the healthy and injured/diseased CNS ................................................... 15
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2.2. ROCK inhibition as a promising tool to induce axon regeneration in the injured visual system
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....................................................................................................................................................... 16
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2.3. ROCK inhibition as a promising tool to support neuronal survival in the injured visual system
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....................................................................................................................................................... 22
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3. ROCK inhibition as promising regulators of ocular blood flow ..................................................... 26
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V. Future perspectives: (pre-)clinical development of ROCK inhibitors for glaucoma treatment ........ 26
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VI. Conclusion ........................................................................................................................................ 28
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I. INTRODUCTION
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The term glaucoma refers to a wide range of optic neuropathies associated with
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degeneration of retinal ganglion cells (RGCs) and their respective axons, leading to slow progressive
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visual field loss (Foster et al. 2002). If not adequately treated, most types of glaucoma progress
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without obvious symptoms towards gradual loss of visual function or even blindness (Friedman et al.
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2004). Nowadays, it is considered as the second leading cause of blindness throughout the world
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(Resnikoff et al. 2008). This neurodegenerative disease mostly appears after the 4th decade of life,
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and its frequency significantly increases with age. In 2010, at least 60.5 million people suffered from
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glaucoma worldwide. This number is expected to increase up to 80 million by 2020 (Quigley and
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Broman 2006). Risk factors for glaucoma not only include age, but also family history (Wolfs et al.
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1998), gender (Rudnicka et al. 2006), ethnic background (Racette et al. 2003), severe myopia
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(Mitchell et al. 1999), disturbed cerebrospinal fluid pressure (Fleischman and Allingham 2013),
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vascular disorders (Bonomi et al. 2000) and importantly, an increased intraocular pressure (IOP)
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(Boland and Quigley 2007).
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With the currently available treatment modalities, glaucomatous visual field deterioration
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cannot be prevented, yet lowering the IOP can slow down glaucoma disease progression
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(VanVeldhuisen et al. 2000; Kass et al. 2002; Anastassiadis et al. 2011). Therefore glaucoma
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treatment is mainly directed towards lowering IOP (VanVeldhuisen et al. 2000; Kass et al. 2002). A
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sustained reduction of IOP can be achieved with topical therapy, laser therapy or surgical
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interventions (Quigley 2011). Nevertheless, signs of progression can be seen in many patients despite
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well-controlled IOP. Therefore, the development of neuroprotective strategies may be of high value
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in the future treatment of this multifactorial neurodegenerative disorder. Until now, only two
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potential neuroprotective agents have been investigated in clinical trials for glaucoma, i.e.
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memantine and brimonidine.
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Over the past few years, many studies have highlighted the important role of the Rho and
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Rho-associated coiled-coil protein kinase (ROCK) pathway in the pathogenesis and treatment of
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glaucoma. Modulation of this pathway seems to be involved in the regulation of IOP via the
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trabecular meshwork (TM) and may also serve as a potent anti-scarring agent after glaucoma
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surgery. More importantly, the amount of studies reporting ROCK inhibition as a very appealing
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therapeutic approach that confers neuroprotection and axonal regeneration, substantially increased
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over the last decade. Therefore, the purpose of this review is to summarize the current knowledge
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on the upcoming neuroprotective and axon regenerative potential of ROCK inhibitors, which may be
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of great importance in the development of a novel neuroprotective/regenerative strategy for
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glaucoma therapy.
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II. GLAUCOMA PATHOPHYSIOLOGY
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The exact mechanisms of how glaucoma develops are still not well-known, although the
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initiation and propagation of this disease is thought to be situated around the optic nerve head
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(ONH). Yet, two principal hypotheses, the mechanical and ischemic/vascular theories have been
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described (Fechtner and Weinreb 1994; Flammer et al. 2002b; Flammer and Mozaffarieh 2007)
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(Figure 1). The classical mechanical theory suggests that the development of glaucoma is a direct
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consequence of an increased IOP causing damage to the ONH (Yan et al. 1994). The IOP reflects the
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balance between aqueous humor (AH) production and outflow through either the conventional
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pathway via the TM, or the unconventional pathway via the uveoscleral route (Goel et al. 2010). IOP
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elevation above 21 mmHg due to impaired TM function, is considered as the most important
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measurable risk factor for the development of glaucoma (Weinreb and Khaw 2004) and believed to
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induce damage to the RGC axons by tissue deformation at the level of the lamina cribrosa, thereby
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leading to ONH cupping. When RGC axons exit the eye at the ONH, the lamina cribrosa provides the
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only support and protection in an otherwise firm scleral shell, making it the most vulnerable site of
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the retina/optic nerve to mechanical stress (Sigal and Ethier 2009). Cupping of the ONH in response
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to elevated IOP causes compression of RGC axons, leading to a disruption of the axoplasmic transport
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between the retina and the brain, which is essential for proper RGC survival (Flammer et al. 2002b).
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Additionally, glial cells at the ONH shift their production of extracellular matrix (ECM) components
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and increase their secretion of matrix metalloproteinases, thereby reducing mechanical support to
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nerve fibers, making them even more vulnerable to elevated IOP (Hernandez 2000; Kirwan et al.
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2004; Dahlmann-Noor et al. 2010; Quill et al. 2011; Akhter et al. 2013; De Groef et al. 2014).
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Although generally accepted for a long time, the mechanical theory fails to explain several
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features correlated with the appearance of glaucoma. Many patients (25-30%) suffer from
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glaucomatous symptoms without the appearance of an increased IOP (normal tension glaucoma
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(NTG)) and pressure reduction does not avoid glaucomatous damage in all patients with initial ocular
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hypertension. On the other hand, some patients with ocular hypertension show no damage to the
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optic nerve (Chauhan and Drance 1992; Martinez-Bello et al. 2000; Agarwal et al. 2009). These
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observations indicate that multiple other factors, unrelated to IOP, play an important role in the
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development of glaucoma. Increased incidence of glaucoma, especially NTG, is observed in patients
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with systemic vascular disorders like hypotension (Hayreh et al. 1994; Hayreh 1999), cardiovascular
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disease (Hayreh 1999), vasospastic disorders (Broadway and Drance 1998), migraine (Wang et al.
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1997), diabetes (Mitchell et al. 1997) and cerebral ischemia (Stroman et al. 1995). Therefore, the
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vascular theory considers a disturbed blood supply to the retina as the primary cause of optic
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atrophy (Flammer et al. 2002b). Insufficient vascular autoregulation to adapt ocular perfusion may
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result in a decreased retinal blood supply, causing ischemic damage (Morgan 2004). This then leads
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to an impaired perfusion of the ONH, causing ischemic injury to the retina. Notably, the mechanical
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and vascular theory are closely interlinked, because excavation of the ONH at the lamina cribrosa as
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a direct consequence of elevated IOP, also leads to kinking of the retinal blood vessels (Flammer
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1994; Flammer et al. 2002b; Flammer and Mozaffarieh 2007; Chang and Goldberg 2012b).
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It is further hypothesized that both mechanical and/or ischemic insults to RGC axons
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ultimately result in mitochondrial dysfunction leading to the induction of RGC apoptosis due to
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oxidative damage (Figure 1). Mitochondrial dysfunction causes impaired energy supply, rendering
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RGCs to exist in a lower energetic state, which results in a passive release of glutamate into the
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extracellular space. In this state, cells are still able to transmit visual information, but are more
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vulnerable to stress stimuli. Upon insult, also glial cells (microglia and astrocytes) will become
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reactive, causing a gradual rise in cytokines and other factors, such as TNF-α, glutamate,
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prostaglandins and nitric oxide. Initially, Müller glia will try to maintain a physiological balance, but
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eventually, this process will become insufficient, resulting in an increased level of these molecules,
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some of which, such as glutamate, are potentially toxic to neuronal cells (Toft-Kehler et al.2014).
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Because RGCs are already in a decreased energetic state, they are more susceptible to be affected
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and as a result, RGCs will undergo apoptosis at different rates, depending on their receptor profile
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(Osborne et al. 2001; Osborne 2009; Chrysostomou et al. 2013). Notably, next to the mechanical and
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vascular factors, inflammatory, autoimmune or even other (still unknown) factors may also
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contribute to glaucoma pathophysiology.
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III. NOVEL STRATEGIES FOR GLAUCOMA THERAPY: NEUROPROTECTION AND AXONAL REGENERATION
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Neuroprotective strategies are extensively investigated in the context of several neurological
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disorders, and form a therapeutic paradigm aiming at slowing down or preventing death of neurons.
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In the case of glaucoma, where loss of RGCs progressively manifests over many years, a
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neuroprotective therapy should enhance the survival of RGCs in the presence of chronic
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stress/injury. Several approaches for neuroprotection in glaucoma are being investigated, targeting
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neurotrophic withdrawal, excitotoxicity, oxidative stress, mitochondrial dysfunction, protein
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misfolding, etc. (Baltmr et al. 2010). Given the importance of neurotrophins, such as e.g. brain
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derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF) and nerve growth factor
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(NGF), for the survival of RGCs (Weibel et al. 1995; Ji et al. 2004b), and the reduced axoplasmic
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transport of these growth factors due to elevated IOP (Quigley et al. 2000), delivering neurotrophic
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factors to the retina has been a key target in the development of neuroprotective therapies. Indeed,
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multiple studies demonstrated the neuroprotective effect of BDNF, CNTF and NGF in animal models
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of glaucoma (Johnson et al. 2011). These neurotrophic factor supplementation therapies are now
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going into clinical trials for primary open angle glaucoma, e.g. trials with CNTF implants and NGF eye
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drops (Chang and Goldberg 2012a), although the lack of methods for sustained, safe delivery of
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biologically active neurotrophins hampers the broad clinical translation of this potential therapy.
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Alternatively, neuroprotective agents preventing excitotoxicity, i.e. RGC apoptosis due to excessive
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extracellular glutamate, have been investigated. This resulted in one of the only two clinical trials
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evaluating neuroprotective agents for the treatment of glaucoma thus far. Indeed, memantine, a N-
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methyl-D-aspartate (NMDA) receptor antagonist, prevents prolonged influx of Ca2+ ions through the
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NMDA receptor channel and as such directly protects RGCs against excitotoxicity (Danesh-Meyer
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2011). The other clinical trial, evaluating the use of the selective α2 receptor agonist brimonidine,
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was based on the observation that this IOP lowering drug may protect RGCs from glaucomatous
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neurodegeneration via a not yet known, IOP-independent mechanism (Lai et al. 2002; Dong et al.
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2008). Nevertheless, despite good preclinical results, both clinical trials failed to deliver adequate
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evidence for the applicability of memantine and brimonidine in the clinic, probably due to a too
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narrow neuroprotective window of these agents (Weinreb 2007; Osborne 2009; Krupin et al. 2011).
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Promoting survival of RGCs is a critical step in the development to a neuroprotective
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strategy. However, in glaucoma also axons of RGCs are damaged, and as such, an ideal
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neuroprotective glaucoma therapy should not only stimulate RGC survival but should also stimulate
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axon regeneration (Chang and Goldberg 2012b). Clinical applications of the latter, however, seem to
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be out of reach for now. For over three decades, the optic nerve crush (ONC) paradigm has been
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used to study axonal regeneration of the optic nerve, first by stimulation of axonal outgrowth by
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intraocular neurotrophic factor injection and peripheral nerve graft transplantation, and more
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recently, by induction of controlled intraocular inflammation via experimental lens injury or
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intravitreal zymosan injection. Moreover, manipulation of specific intracellular signaling pathways
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has led to the identification of STAT3 (signal transducer and activator of transcription 3) and mTOR
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(mammalian target of rapamycin) as central regulators in axonal outgrowth (Pernet and Schwab
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2014). Despite these advances, it remains a challenge to induce sufficient axonal regeneration in
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order to reinnervate retinal target areas in the brain, and restore visual function (de Lima et al. 2012;
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Luo et al. 2013).
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translatable to clinical practice.
Moreover, the current experimental treatments in rodents are not readily
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Of note, ‘vasoprotection’ could be added as an additional therapeutic approach to prevent
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glaucomatous neurodegeneration (Wierzbowska et al. 2010), focusing on the prevention of ischemia
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and reperfusion injury. Indeed, in a subset of patients, optic neuropathy is a consequence of vascular
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dysregulation, due to e.g. systemic circulatory disturbances and perfusion deficits of cerebral,
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retrobulbar and ocular vessels (Wierzbowska et al. 2010; Yanagi et al. 2011). Besides neuroprotective
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drugs targeting excitotoxicity, these patients would greatly benefit from therapies restoring e.g.
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vascular tone, endothelial function and neurovascular coupling (Flammer et al. 2002a; Yanagi et al.
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2011).
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IV. THE RHO-ROCK PATHWAY
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Rho is a member of the cytoplasmic Rho family of small GTP-binding molecules, which
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belongs to the Ras-superfamily of GTPases. The Rho subfamily contains 3 isoforms (RhoA, RhoB and
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RhoC) that are ubiquitously expressed. To become activated, Rho must be targeted to the membrane
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via posttranslational modification (Bishop and Hall 2000). The activity of the Rho pathway is
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regulated by signaling input from several growth factors, cytokines, mechanical stretch and the ECM,
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and as such mediated by different classes of cell surface receptors (Etienne-Manneville and Hall
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2002). Rho GTPases function as molecular switches and cycle between an inactive GDP and an active
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GTP-bound state. Activation is regulated by guanine nucleotide-exchange factors, which catalyze the
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exchange of GDP to GTP. On the other hand, GTPase activating proteins inhibit downstream signaling
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by stimulating the hydrolysis of GTP to GDP (Bos et al. 2007) (figure 2). The Rho-associated coiled-
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coil-containing protein kinases (also known as ROCK) are the first identified and most extensively
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studied downstream effectors of Rho. ROCKs are essentially distributed in the cytoplasm, but are
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translocated to the plasma membrane after RhoA activation (Matsui et al. 1996). In mammals, ROCKs
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exist as two isoforms: ROCKI and ROCKII. ROCKs contain three domains, the catalytic kinase domain,
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which is located at the N-terminus, followed by a coiled-coil region containing the Rho-binding site
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and a pleckstrin-homology domain with a cysteine-rich repeat domain at the carboxyl terminus
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(Riento et al. 2003). The two ROCK isoforms share an overall homology in their sequences of 65%.
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The highest similarities are found in their kinase domains, which are 92% identical. Both ROCK
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isoforms are ubiquitously expressed in most tissues. However, there is a significant difference in
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tissue distribution. Higher levels of ROCKII are found in brain and muscles, whereas ROCKI is
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predominately expressed in non-neuronal tissues such as liver and lungs (Nakagawa et al. 1996).
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1. Downstream effectors of ROCK: well-known cytoskeletal regulators
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Once ROCK is activated by GTP-bound Rho, it is able to phosphorylate multiple substrates,
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which almost all play a critical role in regulating cytoskeletal dynamics (Figure 2). The best-
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characterized substrate of ROCK is the cytoskeletal regulator myosin light chain (MLC).
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Phosphorylation of MLC results in stimulation of actin-myosin interactions, which play a role in
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several cytoskeletal functions like cell morphology, adhesions, motility and smooth muscle cell
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contraction. ROCK also regulates MLC activity via the inhibition of MLC phosphatase (Leung et al.
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1996; Kaneko-Kawano et al. 2012). The actin-binding kinases, LIM kinase (LIMK) 1 and 2, are other
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downstream targets of ROCK and are involved in the regulation of actin filament stabilization. Active
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ROCK phosphorylates LIMK and thereby enhances its ability to phosphorylate cofilin, an actin-binding
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and depolymerizing factor that regulates the turnover of actin filaments. Phosphorylation of cofilin
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by LIMK inactivates its activity. Therefore, the phosphorylation of LIMK by ROCK inhibits cofilin-
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mediated actin filament disassembly and leads to an increase in the number of actin filaments
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(Ohashi et al. 2000; Sumi et al. 2001). ROCK also affects the ezrin/radixin/moesin (ERM) family, a
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family of proteins that promote cross-linking of integral membrane proteins with actin filaments near
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the cell surface. Phosphorylation and activation of the ERM proteins by ROCK regulates membrane-
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actin interactions, permitting cytoskeletal reorganization and the establishment of focal adhesions
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between the actin cytoskeletal and ECM (Tsukita and Yonemura 1997; Shaw et al. 1998). Another
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ROCK effector is adducin, a filamentous-actin-binding protein which promotes actin network
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assembly beneath the cell membrane and plays a role in cell motility (Matsuoka et al. 2000).
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Intermediate filament proteins such as vimentin, glial fibrillary acidic protein (GFAP) and
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neurofilaments, are cytoskeletal structures important for maintaining cell integrity and mechanical
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strength, and their phosphorylation by ROCK causes intermediate filament disassembly and is
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needed in specific events such as cytokinesis (Riento and Ridley 2003). ROCKs are also important
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regulators of the process of inflammation, by the activation of NF-κβ, which subsequently controls
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the transcription of proinflammatory factors such as interleukins and tumor necrosis factor-α, in
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different inflammatory cells (Doe et al. 2007; He et al. 2008).
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2. The ROCK signaling cascade and its involvement in neuronal cell death and axonal outgrowth
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Several other downstream components of the ROCK pathway are highly expressed in the
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CNS, and are regulators of several neural processes, ranging from neuronal survival to axonal
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outgrowth and guidance (Schmandke and Strittmatter 2007) (Figure 2).
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ROCK is a known negative regulator of neurite extension, resulting in axon retraction and
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growth cone collapse during development and axon regeneration (Gallo 2006; Pasterkamp and
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Verhaagen 2006). Indeed, ROCK is an important regulator of collapsin response mediator protein-2
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(CRMP-2) that plays a role in semaphorin-3A-mediated repulsive axon guidance (Arimura et al. 2000).
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Other downstream targets that play a major role in neurite extension are microtubule-associated
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proteins (MAPs), such as Tau or MAP-2, which are important players in neuronal morphology by
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altering microtubule dynamics (Amano et al. 2003). One of the most recently discovered and
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interesting downstream targets of ROCK is the phosphatase and tensin homolog (PTEN). ROCK
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phosphorylates and subsequently activates PTEN, thereby negatively regulating neuronal survival
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and axonal regeneration via the PIP3/Akt/mTOR pathway (Li et al. 2005; Tonges et al. 2011).
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Neurofilaments are intermediate proteins found in high concentrations along axons of vertebrate
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neurons. Activation of these proteins by ROCK results in intermediate filament disassembly and as
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such alters axon integrity and axonal growth dynamics (Hashimoto et al. 1998; Walker et al. 2001).
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GFAP is another intermediate filament protein that is highly expressed in astrocytes and has been
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shown to be important in repair after CNS injury and the formation of a glial scar (McKeon et al.
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1991). Noteworthy, the Rho-ROCK pathway can be activated by several myelin-associated molecules
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that inhibit axonal regeneration in the CNS, such as myelin-associated glycoprotein (MAG), neurite
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outgrowth inhibitor (Nogo) and oligodendrocyte myelin glycoprotein (OMgp) (Hasegawa et al. 2004;
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Mimura et al. 2006; Kubo et al. 2008). Indeed, the inability of mature CNS neurons to regenerate
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injured axons has been attributed to a loss of an inherent growth potential of cells and to inhibitory
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signals associated with myelin and the glial scar. These myelin-associated inhibitory molecules all
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bind with high affinity the Nogo receptor (NgR) (McKerracher and Winton 2002). Interaction of this
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receptor with the extracellular domain of p75NTR is known to activate Rho GTPase, and as such
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activate ROCK, in turn resulting in neurite outgrowth inhibition and growth cone collapse (Kaplan and
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Miller 2003; Yamashita and Tohyama 2003). Chondroitin sulfate proteoglycans (CSPGs), such as
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brevican, aggrecan, versican, phosphocan and neuroglycan-2, are upregulated by reactive astrocytes
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in the glial scar, formed around the lesion site. These CSPGs constitute an important family of
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inhibitory ECM molecules (Silver and Miller 2004). It has been proposed that these CSPGs interact
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with the ROCK pathway, resulting in growth inhibition (Gungabissoon and Bamburg 2003; Monnier et
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al. 2003; Gopalakrishnan et al. 2008).
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Thus, many of the known or suggested upstream and downstream signaling molecules of the
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ROCK pathway in the CNS are related to axonal outgrowth after CNS damage, which subsequently
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implicates the major contribution of the ROCK pathway to the restricted axonal regeneration in the
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adult mammalian CNS. Blocking the Rho-ROCK signaling pathway is therefore yet another new
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strategy to promote axonal regeneration and repair after injury in the CNS.
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V. ROCK INHIBITION AS A MULTI-TARGET APPROACH FOR GLAUCOMA TREATMENT
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Given the important role of Rho and ROCK in different cellular processes, targeting this
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pathway may be of high value for various diseases. As such, inhibition of ROCK has already been
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investigated as a potential strategy for cardiovascular diseases (Shi and Wei 2013), bronchial asthma
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(Kume 2008) and cancer (Itoh et al. 1999). More recently, ROCK has also been highlighted as a
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promising target for neurological disorders, as inhibition of ROCK seems to promote neuronal cell
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survival and axonal regeneration (Mueller et al. 2005; Fujita and Yamashita 2014).
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1. ROCK inhibitors as IOP lowering and anti-scarring agents in glaucoma
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IOP lowering drugs currently in clinical use for glaucoma, suppress the production of AH or
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enhance uveoscleral outflow. None of them directly targets and/or improves conventional outflow
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via the TM. Therefore, agents that increase AH outflow by modifying the TM are promising targets
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for the development of a new IOP lowering strategy to treat most forms of glaucoma (Llobet et al.
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2003). The cells of the TM possess smooth muscle-like properties, as evidenced by the expression of
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α-smooth muscle actin (α-SMA) (de Kater et al. 1990), and their contraction-relaxation status has
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been reported to influence AH outflow facility (Wiederholt et al. 2000). Interestingly smooth muscle
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cell contraction is regulated predominately by the phosphorylation status of MLC, a main
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downstream target of ROCK.
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In vitro and ex vivo studies have reported Rho and ROCK expression in healthy and
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glaucomatous TM cells (Honjo et al. 2001a; Rao et al. 2001; Nakajima et al. 2005; Goldhagen et al.
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2012). Cellular effects and IOP-lowering efficacy of several ROCK inhibitors, such as HA-1077, H-
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1152P, K-115 Y27632, Y-39983 and AMA0076 have been previously reported (Honjo et al. 2001a;
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Honjo et al. 2001b; Rao et al. 2001; Waki et al. 2001; Tokushige et al. 2007; Lu et al. 2008; Fukunaga
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et al. 2009; Nishio et al. 2009; Isobe et al. 2014; Van de Velde et al. 2014a), and indeed unveiled that
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ROCK regulates AH outflow by directly affecting contractility and cellular organization of the TM cells
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(Honjo et al. 2001a; Rao et al. 2001). Overall, ROCK inhibition induces relaxation, actin cytoskeletal
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reorganization and loss of cell adhesions of TM cells, leading to better drainage of AH trough the TM.
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Not all patients respond adequately to anti-glaucoma medication making invasive therapies,
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such as laser trabeculoplasty and/or filtration surgery, necessary for the treatment of glaucoma (Lee
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and Higginbotham 2005). Unfortunately, filtration surgery frequently fails as the result of excessive
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postoperative wound healing and subsequent scarring, leading to poor postoperative IOP control
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and, as a consequence, progression of visual field loss (Addicks et al. 1983). During scar formation,
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the cytokine TGF-β induces a contractile response in fibroblasts, which subsequently differentiate
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into myofibroblasts. In vitro studies have shown that ROCK inhibitors might prevent this
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transdifferentiation, probably via decreasing cell contraction, which implies that ROCK inhibitors may
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reduce postoperative scarring after glaucoma filtration surgery (Meyer-ter-Vehn et al. 2006; Zhou et
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al. 2013). Indeed, Honjo et al. showed that the ROCK inhibitor Y-27632 inhibited fibroproliferation
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and collagen deposition in an animal model of glaucoma filtration surgery, leading to a prolonged
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survival of the postoperative bleb (Honjo et al. 2007).
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Thus, ROCK inhibition may possess therapeutic potential as a novel IOP lowering strategy and
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be able to reduce postoperative scarring after filtration surgery, opening new perspectives for a
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more efficient IOP control.
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2. ROCK inhibitors as novel neuroprotective and axon regenerative agents in glaucoma
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However, in NTG patients, glaucomatous damage occurs despite normal IOP levels and also
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clinical evidence indicates that IOP lowering therapy does not prevent progression in all patients
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(VanVeldhuisen et al. 2000). Since glaucomatous optic neuropathy eventually results in progressive
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loss of RGCs, it is reasonable to expect that a neuroprotective therapy may be of high value as an
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additional treatment modality for glaucoma. Such a therapy should not only directly promote RGC
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survival, but should also encourage axonal regeneration, thereby recovering functional connections
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(Chang and Goldberg 2012b). As we will discuss further in this review, there is increasing evidence
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that ROCK is upregulated during glaucomatous damage and consequently, that ROCK inhibition
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directly protects RGC survival and promotes axon regeneration. This emphasizes that ROCK inhibition
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may be a versatile strategy for the treatment of glaucoma.
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Much of the evidence concerning ROCK inhibition as a novel neuroprotective therapy has
344
been elucidated in glaucoma models (Kitaoka et al. 2004; Hirata et al. 2008; Lingor et al. 2008; Tura
345
et al. 2009; Alt et al. 2013). Furthermore, it has also been demonstrated that inhibition of ROCK limits
346
neurodegeneration in several in vitro, ex vivo and in vivo animal models for e.g. Amyotrophic Lateral
347
Sclerosis (ALS) (Tonges et al. 2014), Multiple Sclerosis (MS) (Zhang et al. ; Li et al. 2014a),
348
Huntington's (Li et al. 2009), Parkinson's (Tonges et al. 2012; Borrajo et al. 2014; Labandeira-Garcia et
349
al. 2014) and Alzheimer's disease (Mueller et al. 2005; Herskowitz et al. 2013; Song et al. 2013),
350
stroke/ischemia (Toshima et al. 2000; Satoh et al. 2001; Rikitake et al. 2005; Ding et al. 2009;
351
Koumura et al. 2011) and Spinal Muscular Atrophy (Bowerman et al. 2012; Froger et al. 2012). These
14
352
prevalent neurodegenerative disorders, which are characterized by a progressive loss of specific
353
neurons/axons, thereby leading to important dysfunctional consequences, still remain largely
354
incurable. Therefore, it is becoming increasingly clear that a therapeutic strategy with a
355
neuroprotective effect has an enormous future potential as effective treatment (Bull and Martin
356
2007; Cheung et al. 2008).
357
In the next paragraphs, this review will provide a detailed overview of the current knowledge
358
related to the potential of ROCK inhibition and the rationale why ROCK targeting seems a promising
359
tool to ensure neuronal protection and/or axonal regeneration.
360
2.1. ROCK expression in the healthy and injured/diseased CNS
361
The amount of studies describing ROCKI/II expression in the neuroretina is rather poor. In the
362
healthy rat retina, RhoA, ROCKI and ROCKII expression were reported to co-localize mainly in the
363
retinal microvessels (Arita et al. 2009). Another study could not demonstrate immunopositivity for
364
ROCKII in healthy rat retinas. However, axotomy of the rat optic nerve induced ROCKII expression,
365
but only in the RGC layer (RGCL), suggesting that ROCK might be detrimental to RGC survival and/or
366
contribute to a regeneration-inhibitory cascade (Lingor et al. 2008). Immunostainings for RhoA in rat
367
retinas also showed, after injection with the glutamate analog N-methyl-D-aspartate (NMDA), an
368
increased immunopositivity in the RGCL, the inner plexiform layer (IPL) and some somata in the inner
369
nuclear layer at 1 day post injection (1 dpi). Western blot data confirmed these findings and
370
demonstrated an upregulation of ROCKII in the NMDA-treated retina at 1 dpi, thereby suggesting a
371
commitment of Rho-ROCK signaling in NMDA/glutamate-induced retinal excitotoxicity (Kitaoka et al.
372
2004). Rho and ROCK expression in the ONH of glaucomatous patients is also significantly increased
373
as compared to healthy controls, indicating a possible involvement of the Rho-ROCK pathway in the
374
pathophysiology of optic nerve damage in glaucoma (Goldhagen et al. 2012). Clearly, additional
375
studies are necessary to investigate the cellular localization of ROCK, as well as its expression
376
patterns and activity levels in the healthy and glaucomatous retina, in order to understand the
15
377
inter/intracellular mechanisms contributing to RGC death and neurite outgrowth inhibition after
378
injury.
379
Studies in the healthy and diseased/injured brain and spinal cord reveal expression of ROCKII,
380
and to a much lower extent ROCKI, in neurons, endothelial cells and microglia (Nakagawa et al. 1996;
381
Deyts et al. 2009; Duffy et al. 2009). Moreover, treatment of PC12 cells with Nogo, a myelin-
382
associated neurite outgrowth inhibitor, did not only increase ROCKII activity, but also resulted in a
383
translocation of ROCKII towards the membrane (Alabed et al. 2006). This finding indicates that
384
ROCKII is activated by Nogo and strengthens the potential of ROCK inhibition in stimulating axonal
385
regeneration.
386
2.2. ROCK inhibition as a promising tool to induce axon regeneration in the injured visual system
387
An effective treatment to enable damaged axons to regrow beyond the lesion site and
388
reform functional connections should ideally require interventions targeting the cell-intrinsic growth
389
capacity, as well as the extrinsic inhibitory environment. In general, much progress concerning the
390
cellular and molecular mechanism underlying the failure of adult axons to regenerate, has been
391
made using studies in the rodent optic circuit. Functional recovery in the visual system implies the
392
proper regrowth of damaged axons to reinnervate the lateral geniculate nucleus and the superior
393
colliculus with sufficient numbers (Pernet et al. 2013; Murray 2014). Studies combining pten deletion
394
with either ‘suppressor of cytokine signaling 3’ (socs3) deletion, or zymosan and CPT-cAMP
395
injections, have demonstrated the potential of RGCs to reactivate their intrinsic axon growth
396
program and regenerate the full length of the optic nerve, across the optic chiasm (de Lima et al.
397
2012; Luo et al. 2013). Indeed, complete axonal regeneration to the lateral geniculate nucleus and
398
superior colliculus, along with partial recovery of visual functions, was observed upon pten deletion
399
plus CTP-cAMP and zymosan administration (de Lima et al. 2012). In an alternative approach,
400
combining pten and socs3 co-deletion with CNTF supplementation, short-distance axonal
401
regeneration to the suprachiasmatic nucleus was seen, yet no reinnervation of long-distance target
402
areas (Luo et al. 2013). Notably, adult RGCs were shown to be able to reactivate de novo axon growth
16
403
upon pten and socs3 co-deletion after injury, thereby making a future therapy to promote axon
404
repair more and more feasible. Yet, the poor number of RGC axons reaching their long-distance
405
target areas and the significant axon misguidance in the optic nerve and brain underscore the need
406
for further research efforts.
407
In vitro application of the ROCK inhibitors Y-27632, fasudil (HA-1077) and dimethylfasudil (H-
408
1152) on Ntera-2 neurons, PC12 cells and primary dorsal root ganglion cells, grown on a CSPG or
409
myelin substrate, was shown to overcome inhibition of neurite outgrowth (Fournier et al. 2003;
410
Zhang et al. 2006; Lingor et al. 2007; Cheng et al. 2008; Gopalakrishnan et al. 2008). It has also been
411
demonstrated in vivo that the amount, thickness and/or length of axons is increased after sciatic
412
nerve crush and spinal cord injury, and even locomotor activity, is improved after application of
413
ROCK inhibitors (Fournier et al. 2003; Sung et al. 2003; Hiraga et al. 2006; Cheng et al. 2008; Gunther
414
et al. 2014). Recently, inhibition of ROCK has proven to inhibit demyelination in an experimental
415
autoimmune encephalomyelitis animal model for MS (Gao et al. 2013).
416
As such, the potential of ROCK inhibitors to support axonal outgrowth and regeneration has
417
increased during the last years. Importantly, much evidence has been obtained from in vitro and ex
418
vivo studies on RGCs and retinal explants, and from experimental in vivo glaucoma models.
419
2.2.1. The neurite outgrowth-potential of ROCK inhibition in in vitro and ex vivo models for glaucoma
420
Addition of Y-27632, or the more potent Y-39983, to RGC-5 cells or postnatal rat primary
421
RGCs grown on a permissive substrate, demonstrated a clearly induced neurite outgrowth. Neurite
422
outgrowth was even more pronounced after combining Y-27632 with CNTF on these 2 cell cultures
423
(Lingor et al. 2008; Bermel et al. 2009; Tokushige et al. 2011) (Table 1). CNTF exerts its main
424
biological functions via activation of the Jak/STAT3, PI3K/Akt and MAPK/ERK signaling pathways (Park
425
et al. 2004), which are, interestingly, also activated upon ROCK inhibition (Lingor 2008, Pernet 2013,
426
Koch 2014). In general, STAT3 has been considered a central regulator of growth and regeneration of
427
adult RGCs and to promote their intrinsic growth capacity via transcription of growth-associated
428
genes and stabilization of the RGC cytoskeleton (Ng et al. 2006; Selvaraj et al. 2012; Pernet and
17
429
Schwab 2014). In contrast, STAT3 was once suggested to act as a negative regulator of neurite
430
outgrowth, proposing that Y-27632 may further increase neurite elongation in combination with
431
CNTF, by inhibiting STAT3 phosphorylation (Lingor et al. 2008). Furthermore, ROCK inhibition also
432
results in suppression of Lim kinase (LIMK) phosphorylation, which is induced upon binding of
433
myelin-derived inhibitory ligands (MAG, Nogo, OMgp) to their NgR receptor and leads to neurite
434
outgrowth inhibition and growth cone collapse (Sagawa et al. 2007). Similarly, ROCK inhibition can
435
relief the repulsive effects of glial-derived inhibitory ligands, such as CSPGs (Monnier et al. 2003).
436
Indeed, treatment of postnatal rat RGCs, grown on an inhibitory CSPG substrate, with Y-27632
437
significantly induced neurite length as compared to controls (Bermel et al. 2009). On the other hand,
438
treating adult rat retinal cells with a panel of inhibitory molecules, including Nogo-A, MAG, OMgp
439
and CSPG, prevented the stimulatory effect of Y-27632 on RGC neurite outgrowth (Ahmed et al.
440
2009) (Table 1). These, at first sight contradictory, findings might be explained by the age at which
441
the RGC cultures were obtained and might result from the suppressed 'growth activated state' of
442
adult CNS neurons, due to e.g. reduced cAMP levels, and as such, a limited regenerative capacity (Cai
443
et al. 2001). Remarkably, stimulation of adult retinal cells with Y-27632, in combination with CNTF,
444
significantly enhanced the number and length of RGC neurites, above the levels of CNTF treatment
445
alone. As such, Y-27632 does not seem to support neurite outgrowth of disinhibited adult RGCs, but
446
is able to enhance CNTF-induced neurite outgrowth (Ahmed et al. 2009). Similar findings were
447
observed when Y-27632-treated adult RGCs were supplemented with forskolin, which induces cAMP
448
levels. The combination of the three compounds enhanced neurite outgrowth of adult RGC neurites
449
to even greater levels than combined Y-27632 and forskolin treatment (Ahmed et al. 2009).
450
As the currently existing ROCK inhibitors do not distinguish between ROCKI and ROCKII, and
451
have been described to also block several other kinases and neuronal receptors (Mueller et al. 2005),
452
Koch et al. circumvented this issue by generating shRNA-expressing adeno-associated viral (AAV)
453
vectors to specifically downregulate ROCKII. Transduction of postnatal rat RGCs with AAV.ROCK2-
454
shRNA showed a trend towards longer neurites when grown on the permissive laminin substrate.
18
455
Moreover, knockdown of ROCKII enabled a full rescue of the growth-inhibiting effect of CSPG on
456
neurite outgrowth (Koch et al. 2014).
457
When embryonic rat retinal explants were applied in a stripe assay, consisting of alternate
458
laminin-coated and laminin/CSPG-coated stripes, or on a uniform laminin/CSPG substrate, axons
459
tended to avoid the CSPG substrate. Addition of Y-27632 clearly reduced this repulsive property and
460
significantly increased the amount and length of the outgrowing axons in comparison to non-treated
461
explants (Monnier et al. 2003) (Table 1). Administration of Y-27632 on adult rat retinal explants in
462
culture, dissected after optic nerve crush (ONC) (Herskowitz et al.), showed an increased number of
463
outgrowing neurites. When Y-27632 was applied together with Indolinone A, an inhibitor of cyclin
464
dependent kinase 5 (cdk5), which triggers a cascade of neurotoxic pathways in many
465
neurodegenerative disorders, an additional increase in outgrowth could be observed (Bermel et al.
466
2009). Supplementation of Y-27632 or Y-39983 also significantly induced neurite extension in adult
467
cat retinal explant studies (Sagawa et al. 2007; Ichikawa et al. 2008) (Table 1). Of note, the number of
468
extending neurites was higher when applying Y-39983, even at lower concentration than Y-27632,
469
thereby confirming the higher potency of Y-39983. Yet, no difference in neurite length could be
470
observed. Importantly, both Y-compounds also promoted the extension of glial processes in the cat
471
retinal explant assay (Sagawa et al. 2007). Notably, application of fasudil on adult cat retinal tissue
472
explants only induced glial fiber extension, while no neurite-outgrowth promoting effect was
473
observed (Ichikawa et al. 2008).
474
Using a retinal explant model, recently described by Buyens et al. (Buyens et al. 2014), our
475
group was able to demonstrate a clear neurite outgrowth-promoting effect of Y-27632 and Y-39883
476
in postnatal mouse retinal explants, respectively a 2 and 2.5 fold increase as compared to control
477
explants (Figure 3a) (Gaublomme et al. 2013). Also here, no significant neurite extension-promoting
478
effect was observed for any of the tested ROCK inhibitors (Figure 3b). Nevertheless, we also observed
479
a stimulating effect on glial process outgrowth (not shown). It is known that reactive glia, being
480
Müller glia and astrocytes, are present in the glaucomatous retina, although their exact role in the
19
481
disease pathogenesis seems to be ambiguous (Nickells 2007; Johnson and Morrison 2009). However,
482
their function as mediators of RGC survival and regeneration is being strengthened during the last
483
years and, amongst others, supported by their production of glial-derived growth factors (Muller et
484
al. 2007; Leibinger et al. 2009; Lorber et al. 2009). Next to soluble factors, also the expression of
485
membrane-bound glial derived growth factors, as well as axonal guidance molecules, contribute to
486
the, at least in part, RGC growth-promoting effect of activated retinal glia (Lorber et al. 2012).
487
These in vitro and ex vivo findings indicate that ROCK inhibitors have a strong capacity to
488
support axonal outgrowth, even in the presence of growth-inhibitory molecules, which mimics the in
489
vivo situation after axonal damage.
490
2.2.2. ROCK inhibitors promote in vivo axonal regeneration after optic nerve traumatic lesions
491
Intravitreal injection of Y-27632 or Y-39983 in the adult cat or rat, immediately after ONC,
492
promoted the number and length of regenerating axons passing the crush site, as compared to
493
crushed control eyes (Table 1). Also intravitreal injection of AAV.ROCK2-shRNA significantly increased
494
the number and length of growth associated protein 43 (GAP43)-positive neurites in the rat optic
495
nerve, after ONC, as compared to an AAV.EGFP-shRNA control vector (Koch et al. 2014). The
496
combined application of Y-27632 and the cdk5 inhibitor Indolinone A, resulted in a significantly
497
higher number, rather than an increased length, of regenerating axons beyond the ONC site, in
498
comparison to either compound alone (Bermel et al. 2009). On the other hand, administration of
499
different doses of fasudil or dimethylfasudil in a similar experimental set-up, revealed only minor
500
effects on axonal outgrowth (Lingor et al. 2007; Bermel et al. 2009) (Table 1). Repeated intravitreal
501
injections of Y-27632 in a rat sciatic nerve graft model, in which the sciatic nerve is transplanted onto
502
the optic nerve stump, did not result in additional axons in the nerve graft as compared to PBS-
503
treated eyes (Lingor et al. 2008) (Table 1). As peripheral nerves lack the major myelin-associated
504
inhibitory molecules, such as Nogo-A, this semi-permissive model allows to study the effect of
505
compounds on the intrinsic regenerative capacity of RGCs, as RGCs show a spontaneous regeneration
506
response into the graft (Thanos et al. 1997). The reported data would then suggest that inhibition of
20
507
ROCK rather promotes axon regeneration by modulating the extracellular environment at the injury
508
site than by altering the intrinsic growth capacity of RGCs. Anyway, co-application of Y-27632 and
509
CNTF enhanced the number of regenerating axons into the graft, to a greater extent than CNTF
510
alone, again indicating an advantageous interplay for these 2 molecules (Lingor et al. 2008). A
511
stronger regenerative response after combined administration of both compounds, as compared to
512
Y-27632 or CNTF treatment alone, was also observed in the (non-permissive) ONC model in the rat,
513
with a clearly more pronounced effect on the number, rather than the length, of regenerating axons
514
(Lingor et al. 2008). Importantly, also repeated injections of Y-27632 alone increased the number of
515
regenerating axons past the lesion site more than two-fold in comparison to control animals, thereby
516
reflecting the potential of ROCK inhibitors to overcome inhibitory myelin substrates. The adult rat
517
sciatic nerve graft model has also been applied to study the axon regenerative potential of Y-33983
518
(Tokushige et al. 2011) (Table 1). In comparison to the findings of Lingor et al. with Y-27632 (Lingor et
519
al. 2008), intravitreal injection and sponge application of solely Y-39983, dose-dependently increased
520
the number of RGCs with regenerating axons in peripheral nerve-transplanted retinas, as compared
521
to saline-administered eyes. Although Y-39983 is more potent than Y-27632 and might as such
522
induce axonal outgrowth to a higher extent (without CNTF), these two studies are difficult to
523
compare, as the amount and length of regenerated axons in the optic nerve was not determined in
524
the latter study (Tokushige et al. 2011).
525
Using a mouse ONC model and intravitreal injection of STAT3 cDNA-expressing AAV vectors,
526
Pernet et al. demonstrated that intracellular activation of STAT3 enhances axonal regeneration
527
(Pernet et al. 2013). However, STAT3-mediated axon elongation is characterized by massive
528
directionality and guidance problems, most likely due to the presence of growth-inhibitory molecules
529
in the optic nerve environment. Interestingly, additional intraocular delivery of Y-27632 did not only
530
result in an increased amount of outgrowing neurites (and surviving RGCs), but also counteracted the
531
Rho-LIMK-mediated misguidance effects and normalized the morphology and trajectories of the
532
regenerating axons. Intravitreal application of Y-27632 alone was not able to significantly induce
21
533
axonal regeneration. Importantly, combined delivery of Y-27632 and the Müller glia-selective AAV
534
vector ShH10.DH-CNTF, resulted in glial CNTF expression and activation of STAT3 in RGCs, eliciting a
535
more robust axonal outgrowth after ONC as compared to single ShH10.DH-CNTF or combined STAT3
536
and Y-27632 treatment (Pernet et al. 2013). These findings again support the notion that
537
combinatorial strategies will be needed. Indeed, co-injection of Y-27632 and AAV2.STAT3 raised
538
phosphorylated STAT3 (pSTAT3) levels and expression of STAT3 target genes, such as GAP43, after
539
injury (Pernet et al. 2013).
540
In summary, ROCK(II) inhibition is recently brought forward as a potential strategy to help to
541
overcome restriction of axonal regrowth in the injured CNS, such as the visual system. The amount of
542
studies on the RGC axon regenerative potential of ROCK inhibitors in animal models of glaucoma is
543
clearly increasing over the last years. As such, it seems that ROCK inhibitors might induce the number
544
of outgrowing axons, and help in a proper guidance of the axons towards their target areas, albeit in
545
combination with other neuroprotective and/or regenerative molecules.
546
2.3. ROCK inhibition as a promising tool to support neuronal survival in the injured visual system
547
Several studies highlighting the regenerative potential of ROCK inhibitors, also reported a
548
beneficial effect of ROCK inhibition on neuronal survival. However, the mechanisms responsible for
549
the neuroprotective effects of ROCK inhibitors in experimental models of neurodegenerative
550
disorders, are only gradually being elucidated over the past years. Modulation of the PTEN/Akt and
551
MAPK/ERK signaling pathways (Lingor et al. 2008; Tonges et al. 2012), a decrease in oxidative stress,
552
enhanced expression of neurotrophic factors (Li et al. 2014b) and attenuation of microglial activity
553
(Zhao et al. 2006; Suzuki et al. 2007; Borrajo et al. 2014) and CNS inflammatory responses (Ding et al.
554
2010; Villar-Cheda et al. 2012; Borrajo et al. 2014; Li et al. 2014a; Tonges et al. 2014), have all been
555
put forward as possible mechanisms underlying the observed increase in neuronal survival.
556
2.3.1. In vitro and ex vivo studies on the neuroprotective potential of ROCK inhibitors
557
Lingor et al. (2008) demonstrated that addition of Y-27632 significantly increased survival
558
and decreased apoptosis of serum-deprived RGC-5 cells and, although to a lesser extent, of
22
559
neurotrophin-deprived primary rat RGCs (Table 2). Combination of Y-27632 with CNTF in vitro,
560
resulted in a synergistic effect on RGC survival. CNTF has been repeatedly described to protect RGCs
561
in vitro and in vivo, in animal models of traumatic optic neuropathies and ocular hypertension
562
(Lehwalder et al. 1989; Mey and Thanos 1993; Weise et al. 2000; Ji et al. 2004a; Maier et al. 2004;
563
Park et al. 2004; Zhang et al. 2005). The neuroprotective effect of Y-27632 seems to be associated
564
with upregulated levels of phosphorylated MAPK, which becomes remarkably more phosphorylated
565
or activated after combined administration with CNTF, indicating that inhibition of ROCK activates
566
MAPK downstream signaling pathways that are also used by CNTF. pAkt levels were not altered after
567
Y-27632, but showed a clear trend towards upregulation after combination treatment. Addition of
568
CNTF onto RGC-5 cultures resulted in increased pSTAT3 levels. However, application of the ROCK
569
inhibitor alone, as well as the combination of both substances, did not alter or even decreased
570
pSTAT3 levels compared to CNTF treatment alone, indicating that ROCK weakens some CNTF
571
signaling pathways, while amplifying others such as MAPK and Akt pathways (Lingor et al. 2008). Also
572
Bermel et al. demonstrated increased survival of growth factor-deprived primary rat RGCs in culture
573
when supplemented with Y-27632 (Bermel et al. 2009) (Table 2). Combined application of Y-27632
574
and the cdk5 inhibitor Indolinone A promoted RGC survival to a higher extent than treatment with
575
either substance alone. Furthermore, and in accordance to the findings of Lingor et al. (Lingor et al.
576
2008), administration of Y-27632 to serum-deprived RGC-5 cells resulted in a minor increase in
577
pMAPK levels (Bermel et al. 2009). These in vitro observations indicate that, next to the MAPK
578
signaling pathway, additional survival promoting mechanisms mediate the neuroprotective effect of
579
ROCK inhibition. Importantly, the origin and nature of the RGC-5 cell line has been the subject of
580
controversy during recent years, and it is now recognized that it is in fact of mouse photoreceptor
581
origin (Krishnamoorthy et al. 2013; Sippl and Tamm 2014). Nevertheless, it is still widely used as a
582
tool to follow-up initial hypotheses that require a transformed retina cell line of neuronal origin
583
(Sippl and Tamm 2014). It is therefore imperative that findings are subsequently tested with
584
complementary, biologically relevant tools such as primary RGCs or ex vivo and in vivo models
23
585
(Krishnamoorthy et al. 2013; Sippl and Tamm 2014). Indeed, all RGC-5 data mentioned in this review
586
has been confirmed in more relevant biological backgrounds, as can readily be appreciated from
587
tables 1 and 2.
588
In addition to these cell culture studies, the effects of ROCK inhibition on RGC survival were
589
also investigated using different ex vivo models. Administration of dimethylfasudil (H-1152P) to
590
serum-deprived organotypic cultures of adult mouse retinas, significantly and dose-dependently
591
reduced cell death in the RGC layer (Tura et al. 2009) (Table 2). In addition, macro- and microglial
592
reactivity, which was dramatically elevated after serum deprivation, was clearly attenuated by
593
dimethylfasudil, and corresponded to the observed reduction in the cytokines levels produced by
594
these reactive glial cells. The neuroprotective potential of dimethylfasudil was confirmed in an ex
595
vivo model with perfused bovine retinas under hypoxia (Alt et al. 2013). Similarly, macro- and
596
microglial activities were suppressed after dimethylfasudil application, as demonstrated e.g. by the
597
preservation of quiescent, ramified microglia. These ex vivo observations suggest that inhibition of
598
ROCK is able to protect RGCs from cytotoxicity, an effect that might be mediated through
599
suppression of glial reactivity.
600
To conclude, several in vitro and ex vivo findings already point to the existence of ROCK
601
downstream signaling pathways affecting RGC survival. As ROCK inhibitors seem to possess the
602
ability to enhance neuroprotective capacities of other molecules, more in-depth insight into the
603
underlying working mechanisms and molecular players is needed. In addition, the effect of ROCK
604
inhibition on glial reactivity should be further considered to broaden our knowledge about the
605
complex actions of micro- and macroglia in neurodegeneration.
606
2.3.2. ROCK inhibitors as RGC survival-promoting molecules in animal models for glaucoma
607
Because of the involvement of RhoA in NMDA receptor signaling (Norenberg et al. 1999;
608
Nakazawa et al. 2003), a role of RhoA and ROCK in NMDA-induced retinal neurotoxicity was
609
presumed (Kitaoka et al. 2004). Co-administration of NMDA and fasudil diminished NMDA-induced
610
cell loss in the RGCL and prevented the reduction in IPL thickness, as compared to single NMDA
24
611
injected retinas, thereby proposing that inhibition of ROCK is neuroprotective against glutamate-
612
related excitotoxicity (Kitaoka et al. 2004)(Table 2). Also in a transient retinal ischemia model in rats,
613
cell loss in the RGCL and reduction of IPL thickness was diminished after intravitreal injection of Y-
614
27632 (Hirata et al. 2008). Moreover, disturbance of vascular endothelial cell alignment and
615
leukocyte infiltration was dramatically suppressed in the post-ischemic retina after Y-27632
616
administration, and might underlie the observed diminished neuronal cell death. Lingor et al.
617
demonstrated a neuroprotective role for Y-27632 after repeated intravitreal injections in the adult
618
rat eye after complete transection of the optic nerve (Lingor et al. 2008). The neuroprotective
619
capacities of dimethylfasudil were shown in an ONC model in the rat, where pretreatment with
620
dimethylfasudil reduced RGC apoptosis in a dose-dependent matter (Herskowitz et al.). Remarkably,
621
also here an additional attenuation of macro- and microglial reactivity was observed (Tura et al.
622
2009). An increased RGC survival was also observed in a mouse ONC model after daily oral treatment
623
with either fasudil or K-115, a novel ROCK inhibitor with high selectivity for ROCKII, as compared to
624
vehicle-treated mice (Yamamoto et al. 2014). Moreover, the mRNA levels of the RGC markers Thy1.2
625
and Brn3a were increased after K-115 or fasudil treatment. K-115 likely attenuates oxidative stress
626
and as such, retard RGC death and glaucoma progression, by reducing the levels of oxidized lipids,
627
reactive oxygen species and NAPDH oxidase 1, known to be involved in the ONC-induced ROS
628
production pathway (Yamamoto et al. 2014). Koch et al. (2014) showed significantly enhanced RGC
629
survival in an ONC model in rats after retinal transduction with AAV.ROCK2-shRNA. In addition,
630
axonal degeneration on the proximal side of the crush was clearly retarded after ROCKII inhibition.
631
Part of the specific ROCKII-inhibitory effects on RGC survival and axonal de- and regeneration,
632
observed after AAV.ROCK2-shRNA transduction, could probably be assigned to decreased
633
intraneuronal activity of calpain and caspase-3 and increased levels of pAkt and CRMP-2 and
634
autophagic flux (Koch et al. 2014).
635
The fact that ROCK(II) inhibition limits RGC apoptosis and supports RGC survival in various
636
animal models of glaucoma, points to its broad window of action, which might be beneficial and even
25
637
mandatory for the treatment of a complex multivariate disorder like glaucoma. Nevertheless, the
638
neuroprotective effect of ROCK inhibition is modest (~10% survival increase), and much lower in
639
comparison to e.g. CNTF of BDNF trophic support.
640
3. ROCK inhibition as promising regulators of ocular blood flow
641
Risk factors of vascular nature, leading to disturbed ocular perfusion, have been positively
642
correlated with the appearance of glaucoma. In some patients, mostly patients with NTG, vascular
643
disorders even play a predominate role. Therefore, drugs that improve ocular blood flow to the ONH
644
are promising as an additional treatment modality for glaucoma.
645
Although not studied in any detail yet, ROCK inhibitors were recently shown to affect ocular
646
blood flow. Tokushige et al. showed that topical administration of Y-39983 significantly increased
647
blood flow at the ONH in rabbit eyes (Tokushige et al. 2011). In another study, intravenous infusion
648
or topical treatment with fasudil suppressed the impairment and even improved ONH blood flow,
649
induced by a nitric oxide synthase inhibitor or endothelin-1 in rabbits (Sugiyama et al. 2011). These
650
blood flow-improving effects of ROCK inhibitors can be explained by the fact that the Rho-ROCK
651
pathway is involved in MLC regulated contraction of smooth muscle cells surrounding blood vessels
652
in the ONH. Indeed, activation of MLC is regulated by the counteracting enzymes MLC kinase and
653
MLC phosphatase and the activity of MLC phosphatase is inhibited by ROCK causing smooth muscle
654
cell contraction (Uehata et al. 1997).
655
Considering the role of vascular dysregulation and impaired ocular perfusion in the
656
pathogenesis of glaucoma, and especially in patients with NTG, these data indicate an additional
657
advantage of ROCK inhibitors as possible vasoprotective drugs for the treatment for glaucoma.
658
V. FUTURE PERSPECTIVES: (PRE-)CLINICAL DEVELOPMENT OF ROCK INHIBITORS FOR GLAUCOMA TREATMENT
659
Given the important role of ROCK signaling in numerous cellular processes, ROCK inhibitors
660
are of potential interest for the treatment of a variety of cardiovascular, inflammatory, autoimmune
661
and neurodegenerative diseases (Doe et al. 2007; Fernandes et al. 2007; Kast et al. 2007; Liao et al.
26
662
2007; LoGrasso and Feng 2009). Specifically for neurodegenerative diseases, spinal cord injury, optic
663
nerve injury, traumatic brain injury and stroke, Alzheimer’s, Parkinson’s and Huntington’s disease,
664
amyotrophic lateral sclerosis and multiple sclerosis have been named as potential indications.
665
Indeed, the improved neurological recovery after spinal cord lesion observed in a phase I/IIa clinical
666
trial with BA-210 (Cethrin, BioAxone) (Fehlings et al. 2011), illustrates that RhoA/ROCK inhibitors are
667
a rational therapeutic approach for the treatment of neurological disorders. Nevertheless, today only
668
one ROCK inhibitor, fasudil (Eril, Asahi Kasei Pharma), has been approved in Japan for the treatment
669
of cerebral vasospasm (Zhao et al. 2006; Suzuki et al. 2007). It is generally believed that this limited
670
success of clinical applications of ROCK inhibitors is due to their profound effect on blood pressure
671
(Kast et al. 2007). As a consequence, avoiding systemic exposure, recent development of ROCK
672
inhibitors has been largely restricted to topical applications, e.g. clinical trials with Y-39983
673
(Novartis), K-115 (Kowa Company), AR-12286, AR-13324 (Aerie Pharmaceuticals) and ATS907
674
(Altheos), to evaluate their IOP lowering effect. However, even when applied topically to the eye,
675
side effects such as conjunctival hyperemia are seen (Tokushige et al. 2007; Tanihara et al. 2008;
676
Mandell et al. 2011). Overall, most available ROCK inhibitors have a narrow therapeutic window, due
677
to adverse effects resulting from either their low specificity or their interference with off-target
678
ROCK-dependent cellular processes. While this limited specificity of current ROCK inhibitors, i.e.
679
inhibition of other protein kinases and lack of specificity for ROCK isoforms (Chen et al. 2011; Shi et
680
al. 2013), remains a concern, so-called ‘soft’ ROCK inhibitors (Amakem Therapeutics) have been
681
developed to overcome the second issue. These compounds, structurally related to Y-27632, are
682
locally acting drugs that are designed to be relatively stable at the site of action and undergo
683
metabolic inactivation by controlled conversion into a predictable, non-functional metabolite (Bodor
684
and Buchwald 2008). As a result, off-target activity is avoided and these compounds thus display a
685
unique pharmacokinetic profile and a superior safety profile (Boland et al. 2013). Indeed, topical
686
administration of AMA0076 potently lowered IOP in New Zealand white rabbits with minimal
687
hyperemia (Van de Velde et al. 2014b). Of note, as ROCKI and ROCKII have very distinct expression
27
688
profiles (Chen et al. 2011; Shi et al. 2013), the local mode of action of these soft ROCK inhibitors
689
implies that unwanted inhibition of the second ROCK isoform is dramatically reduced. In addition,
690
alternative pharmacological variants/classes are being generated and tested in various experimental
691
disease models, in order to improve kinase selectivity, cellular activity, microsomal stabilization and
692
pharmacokinetic properties of ROCK inhibitors (Kubo et al. 2008; Tonges et al. 2011).
693
VI. CONCLUSION
694
Since the adult mammalian CNS lacks the capacity to replace lost nerve cells or to regenerate
695
injured axons, neurodegenerative and neurotraumatic disorders seriously affect life quality and
696
human wellbeing. Also glaucoma, which is increasingly being recognized as a multifactorial disease, is
697
characterized by loss of functional RGCs, causing progressive visual field loss. IOP lowering therapies
698
still remain the mainstay of glaucoma therapy. However, considering IOP elevation as the only
699
treatable risk factor, is increasingly being challenged because some patients continue to have optic
700
nerve deterioration despite proper IOP control. This indicates that the need for complementary
701
approaches for the treatment of glaucoma is higher than ever. Considering glaucoma as a
702
neurodegenerative disease, the development of a neuroprotective strategy may provide improved
703
therapeutic options for all glaucoma patients. However, until now, and even despite good laboratory
704
evidence, no neuroprotective agent is yet available for glaucoma treatment. This could very well be
705
explained by the narrow window of their neuroprotective actions. This review summarizes
706
accumulating experimental evidence that ROCK inhibitors not only promote RGC survival, but also
707
RGC axon regeneration and ocular blood flow. Much of the progress that was recently made
708
concerning the potential of ROCK inhibition for neuroprotection and axonal regeneration has been
709
obtained from several (animal) models of glaucoma. These findings increased our knowledge on the
710
beneficial effects by which ROCK inhibitors support cell survival and axonal regrowth, and also
711
unveiled some contributing signaling pathways. In order to reach improved survival of injured
712
neurons and inhibition of repulsive environmental signaling in glaucoma, combinatorial,
713
complementary pharmacotherapies that target multiple pathogenic processes/signaling pathways
28
714
might have a higher potential than those targeting a single molecule. Therefore, it is important that
715
drug discovery work at the in vitro, ex vivo and in vivo level is being complemented by studies that
716
aim to elucidate the downstream signaling pathways by which ROCK inhibition establishes its effects.
717
Overall, to establish neuroprotection and axonal regeneration as effective treatment modalities in
718
order to improve long-term outcomes, potent, preferably locally acting, ROCK inhibitors may be a
719
novel pharmacological approach for glaucoma.
720
721
Conflict of interest
722
All authors report to have no conflict of interest.
723
724
Acknowledgments
725
The authors are financially supported by the Hercules Grant [AKUL/09/038] and national Grants from
726
the Research Council of KU Leuven [KU Leuven BOF-OT/10/033; GOA/12/008] and the Flemish
727
Institute for the promotion of scientific research (IWT and FWO).
728
29
729
Figures
730
731
Figure 1. Hypothetical cascade of events leading to RGC degeneration in glaucoma.
732
Both mechanical and/or ischemic insults may cause mitochondrial dysfunction, thereby resulting in
733
oxidative stress and subsequent RGC apoptosis due to oxidative damage. Mitochondrial dysfunction
734
compromises RGC energy supply rendering RGCs to exist in a lower energetic state. Upon insult, glial
735
cells will become reactivated causing a gradual release of growth factors and other molecules, such
736
as TNF-α, glutamate, prostaglandins (PGs) and nitric oxide (NO). Initially, Müller glia will try to
737
maintain a physiological balance, but eventually, this process will become insufficient. A gradual
738
increase in the level of these potential harmful molecules will exist and a toxic environment will be
739
established. Because RGCs are already in a decreased energetic state, they are even more susceptible
740
to further injury and as a result, also these RGCs will be triggered to death via apoptosis.
741
742
30
743
744
745
Figure 2. Downstream targets of ROCK.
746
Activation of ROCK by GTP-bound RhoA leads to phosphorylation of several substrates regulating a
747
diverse range of cellular responses. Most downstream targets of ROCKs are important cytoskeletal
748
regulators. Hence, the Rho-ROCK pathway has critical functions in cytoskeletal based activities such
749
as, cell adhesion, morphology, motility and contraction. Several other downstream effectors are
750
highly expressed in the central nervous system (CNS) (dotted lines), and are regulators of diverse
751
neural processes, ranging from neuronal survival to axon outgrowth and guidance (GDP: guanosine
752
diphosphate; GTP: guanosine triphosphate; GAP: GTP-ase activating proteins; GEF: guanine
753
nucleotide-exchange factors; ROCK: Rho-associated coiled-coil protein kinase; MLC: myosin light
754
chain; MLCP: MLC phosphatase; LIMK: LIM kinase; ERM: ezrin/radixin/moesin; MAP: microtubule-
755
associated protein; CRMP-2: collapsin response mediator protein-2; GFAP: glial fibrillary acidic
756
protein; NFs: neurofilament; PTEN: phosphatase and tensin homolog).
757
758
759
31
760
761
Figure 3. Effect of ROCK inhibitors on neurite outgrowth in mouse retinal explants.
762
(A) The neurite outgrowth-stimulating effect of Y-27632 (10µM) and Y-39983 (10µM) was
763
demonstrated in retinal explants, harvested from P3 mouse pups and immunostained for β-tubulin
764
after 3 days in culture, via automatic analysis of the immunostained neurite outgrowth area.
765
Outgrowth in control explants was set at 100%. A combinatorial treatment with the growth factors,
766
BDNF (50 ng/ml) and CNTF (10 ng/ml), was used as a positive control. Both ROCK inhibitors, but
767
mainly Y-39983, significantly promoted neurite outgrowth. (B) Neurite outgrowth extension was
768
investigated by categorizing neurite outgrowth into 4 segments, generated by successive 100 µm
769
increments in radius from the explant body edge and thus resulting in three ring segments, and an
770
outer segment covering the remaining immunolabeled neurites. BDNF/CNTF treatment clearly
771
stimulated axon extension, yet, no significant neurite-extension-promoting effect could be observed
772
for any of the ROCK inhibitors. Data are shown as mean ± SEM (n ≥ 34 for every condition, from 4
773
independent experiments; ** p < 0.01, *** p < 0.001, two-way ANOVA with Bonferroni post hoc
774
test).
32
Table 1. Overview of observed axon outgrowth/regenerative effects of ROCK inhibitors in experimental glaucoma models
Drug
Optimal
Dosage form
Model
Animal
Effects
Reference
reduced repulsive neurite outgrowth
(Monnier et al.
dose
Y-27632
50µM
E6
retinal
explants chicken
2003)
(+CSPG)
Y-27632
10-100µM
adult retinal explants
cat
Y-39983: more neurite outgrowth
(Sagawa et al.
Y-39983
3 & 10µM
adult retinal explants
cat
similar effects on neurite length
2007)
increased amount glial processes
(Sagawa et al.
2007)
Y-39983
10µM
Gel
foam
+ ONC
cat
increased amount and length of reg. axons
(Sagawa et al.
2007)
injection at crush +
IV D0 (+7)
Y-27632
1.5mM
IV D0, 3, 6
ONC
rat
increased amount of reg. axons
(Lingor et al.
dimethylfasudil
4nM-40µM;
IV D0, 3, 6
ONC
rat
no effect on axonal outgrowth
2007)
(Lingor et al.
2007)
33
Y-27632
Y-27632
10µM
RGC-5 cells
10µM
P7-8 pRGCs
enhanced neurite outgrowth
(Lingor et al.
rat
enhanced neurite outgrowth
2008)
2.9mM
IV D0, 3, 6
peri. nerve graft
rat
no effect on axon number
2.9mM
IV D0, 3, 6
ONC
rat
increased amount reg. axons
adult pRGCs
rat
no effect on neurite outgrowth in absence or (Ahmed et al.
10µM
(+ myelin)
Y-27632
30 & 100µM
adult retinal explants
presence of myelin
cat
increased amount of neurites & glia, no effect (Ichikawa et al.
on process length
fasudil
10 & 30µM
adult retinal explants
cat
2009)
2008)
increased amount of glia, not neurites, no
effect on length
Y-27632
10 & 100µM
IV D0, 7 + gel foam
ONC
cat
increased amount and length of regenerating
fasudil
10-100µM
IV D0, 7 + gel foam
ONC
cat
axons
no regenerating axons
(Ichikawa et al.
2008)
(Ichikawa et al.
2008)
(Ichikawa et al.
2008)
34
Y-27632
10µM
RGC-5 cells
10µM
P7-8 pRGCs (+ CSPG)
10µM
adult
retinal
rat
explants rat
significantly increased neurite length
(Bermel et al.
enhanced neurite outgrowth
2009)
increased amount of outgrowing axons
after in vivo ONC
3.3µg
Y-39983
Y-27632
IV D0, 3
10µM
rat
trend towards more reg. axons
P7 pRGCs
rat
extended neurite outgrowth
(Tokushige
IV D0 + sponge
peri. nerve graft
rat
increased amount regenerating axons
al. 2011)
3mM
IV D0, 7
ONC
mouse
no effect on axonal outgrowth
(Pernet et al.
P7 pRGCs
rat
enhanced neurite outgrowth
2013)
ONC
rat
increased amount of reg. axons
(Koch
IV D-14
et
2014)
Abbreviations: D: day of injection; pRGCs: primary retinal ganglion cells; ONC: optic nerve crush; RGCL: retinal ganglion cell layer, E: embryonic day; P:
postnatal day; CSPG: chondroitin sulfate proteoglycan
35
et
10µM
AAV.ROCK2shRNA
ONC
al.
Table 2. Overview of observed neuroprotective effects of ROCK inhibitors in experimental glaucoma models
Drug
Optimal dose
Y-27632
10µM
Dosage form
Model
Animal
Effects
Reference
P7-8 pRGCs (-NT)
rat
increased cell survival
(Bermel et
al. 2009)
Y-27632
100nM
IV, D0
transient retinal ischemia
rat
reduced cell loss in the RGCL
(Hirata et
increased IPL thickness
al. 2008)
diminished leukocyte infiltration & endothelial
disarrangment
Y-27632
(Lingor et
rat
increased RGC survival
al. 2008)
ONT
rat
increased RGC survival
adult retinal explant
cat
no increased RGC survival
RGC-5 (-serum)
10µM
P7-8 pRGCs (-NTs)
2.9mM
Y-39983
increased RGC survival
10µM
IV, D0,3,6
10 & 30µM
(Sagawa et
al. 2007)
Fasudil
10 & 100µM
IV, D0
NMDA excitotoxicity
rat
reduced cell loss in the RGCL
(Kitaoka et
increased IPL thickness
al. 2004)
elevated Thy-1 mRNA levels
36
Fasudil
K-115
1mg/kg
1mg/kg
oral, daily
oral, daily
ONC
mouse
ONC
mouse
increased RGC survival
increased Thy-1 mRNA levels
(Yamamot
increased RGC survival
o
reduced oxidative stress
2014)
et
al.
(Yamamot
o
et
al.
2014)
dimethylfasudil
1µM
adult retinal explant
mouse
(-serum)
1, 10 & 100µM
IV D0
ONC
rat
reduced cell death
(Tura et al.
reduced macroglia reactivity
2009)
reduced cell death in the RGCL
reduced macro/microglia activity
dimethylfasudil
1-5µM
retinal
explant
(+ calf
hypoxia)
AAV.ROCK2shRNA
IV D-14
rat
ONC
reduced cell death
(Alt et al.
reduced macro/microglia activity
2013)
increased RGC survival
(Koch et al.
2014)
37
Abbreviations: D: day of injection; pRGCs: primary retinal ganglion cells; NT: neurotrophins; P: postnatal day; ONT: optic nerve transection;
ONC: optic nerve crush; RGCL: retinal ganglion cell layer; IPL: inner plexiform layer
38
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