Dock GEFs and their therapeutic potential: Neuroprotection and axon regeneration

Progress in Retinal and Eye Research xxx (2014) 1e16

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Progress in Retinal and Eye Research journal homepage: www.elsevier.com/locate/prer

Dock GEFs and their therapeutic potential: Neuroprotection and axon regeneration Kazuhiko Namekata 1, Atsuko Kimura 1, Kazuto Kawamura 1, Chikako Harada 1, Takayuki Harada*, 1 Visual Research Project, Tokyo Metropolitan Institute of Medical Science, 2-1-6 Kamikitazawa, Setagaya-ku, Tokyo 156-8506, Japan

a r t i c l e i n f o

a b s t r a c t

Article history: Available online xxx

The dedicator of cytokinesis (Dock) family is composed of atypical guanine exchange factors (GEFs) that activate the Rho GTPases Rac1 and Cdc42. Rho GTPases are best documented for their roles in actin polymerization and they regulate important cellular functions, including morphogenesis, migration, neuronal development, and cell division and adhesion. To date, 11 Dock family members have been identified and their roles have been reported in diverse contexts. There has been increasing interest in elucidating the roles of Dock proteins in recent years and studies have revealed that they are potential therapeutic targets for various diseases, including glaucoma, Alzheimer's disease, cancer, attention deficit hyperactivity disorder and combined immunodeficiency. Among the Dock proteins, Dock3 is predominantly expressed in the central nervous system and recent studies have revealed that Dock3 plays a role in protecting retinal ganglion cells from neurotoxicity and oxidative stress as well as in promoting optic nerve regeneration. In this review, we discuss the current understanding of the 11 Dock GEFs and their therapeutic potential, with a particular focus on Dock3 as a novel target for the treatment of glaucoma and other neurodegenerative diseases. © 2014 Elsevier Ltd. All rights reserved.

Keywords: Dock3 Guanine exchange factors Glaucoma Neuroprotection Axon regeneration Optic nerve Retinal ganglion cells

Contents 1. 2.

3.

4.

5.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The Dock Family . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Molecular structures of the Dock proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. The Dock Family members . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.1. Dock-A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.2. Dock-B . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.3. Dock-C . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.4. Dock-D . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. The Dock Family and Elmo . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Dock3 and neurodegeneration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Dock3 and Alzheimer's disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Dock3 and glaucoma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Dock3 in RGC protection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. Glutamate excitotoxicity and RGC death . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. Dock3 controls cell-surface expression of NMDA receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Role of Dock3 in optic nerve regeneration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00

* Corresponding author. Tel.: þ81 3 6834 2338. E-mail address: [email protected] (T. Harada). 1 Percentage of work contributed by each author in the production of the manuscript is as follows: Namekata: 30%; Kimura: 30%; Kawamura: 10%; Harada C: 5%; Harada T: 25%. http://dx.doi.org/10.1016/j.preteyeres.2014.06.005 1350-9462/© 2014 Elsevier Ltd. All rights reserved.

Please cite this article in press as: Namekata, K., et al., Dock GEFs and their therapeutic potential: Neuroprotection and axon regeneration, Progress in Retinal and Eye Research (2014), http://dx.doi.org/10.1016/j.preteyeres.2014.06.005

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K. Namekata et al. / Progress in Retinal and Eye Research xxx (2014) 1e16

5.1. 5.2.

6.

Recent progress in optic nerve regeneration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Dock3 in axon regeneration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2.1. Regulation of actin filament dynamics by Dock3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2.2. Regulation of microtubule assembly by Dock3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions and future directions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Uncited reference . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction The dedicator of cytokinesis (Dock) proteins belong to a family of atypical guanine exchange factors (GEFs) that regulate the activation of Rho GTPases. The first Dock protein was discovered in 1996 as a major CT10 regulator of kinase (CRK)-binding protein; to ^ te  and Vuori, date, 11 members (Dock1-11) have been identified (Co 2007; Hasegawa et al., 1996; Meller et al., 2005) (Table 1). Dock proteins exhibit differential expression patterns and each plays important roles in cellular function, including in cell migration, phagocytosis, lymphocyte migration, neuronal polarization, neu^ te  and Vuori, 2007; Laurin roprotection and neuroregeneration (Co ^ te , 2014; Namekata et al., 2010, 2012b). Dock proteins have and Co been associated with a number of diseases; for example, Dock1 (also known as Dock180), the best-studied and archetypal Dock protein, plays a significant role in metastatic breast cancers and glioma tumorigenesis (Feng et al., 2011, 2012), Dock3 is associated with Alzheimer's disease and attention deficit hyperactivity disorder (ADHD) (de Silva et al., 2003; Kashiwa et al., 2000), and Dock8 deficiency has been observed in patients with combined immunodeficiency (Zhang et al., 2009). Accordingly, there has been increasing interest in elucidating the roles of Dock proteins in disease, and the functional diversity of Dock proteins is an attractive area for research. Dock proteins typically regulate important cellular functions through activation of the Rho GTPases Rac1 and/or Cdc42. GTPases are proteins that bind GTP and hydrolyze it to GDP (Fig. 1). The GTPbound state is the active state, in which the GTPase transduces signals to effectors, while the GDP-bound state is the inactive state. However, no obvious rules forbid a GTPase cycle in which a GDP-

00 00 00 00 00 00 00 00

bound protein serves as the active form and the GTP-bound form the inactive form (Bourne et al., 1990). GEFs are one of the main regulators for GTPase activation, together with GTPase-activating proteins (GAPs) (Rossman et al., 2005). GEFs facilitate the exchange of GDP for GTP and thus activate GTPases, while GAPs accelerate the rate of GTP hydrolysis, resulting in inactivation of GTPases. Among the large group of GTPases, small GTPases are monomeric proteins with a molecular weight of approximately 21 kDa. Based on primary amino acid sequences and biochemical properties, small GTPases are divided into five subfamilies: Ras, Rho, Rab, Sar1/Arf and Ran. The best known family is the Ras superfamily, which became a popular topic of research because mutations in mammalian Ras genes cause neoplastic transformation (Bourne et al., 1990; Cox and Der, 2010; Karnoub and Weinberg, 2008). The mammalian Rho subfamily GTPases are best documented for their roles in actin dynamics and they regulate a variety of cellular processes, including morphogenesis, migration, neuronal development, and cell division and adhesion (Cook et al., 2013; Hall and Lalli, 2010; Heasman and Ridley, 2008). Dock proteins activate Rho GTPases and are thus important factors for the regulation of diverse signal transduction pathways that are associated with a variety of human diseases (Table 1). 2. The Dock Family To date, two distinct families of GEFs for Rho GTPases have been identified based on the features of their catalytic domains: the classical Dbl family GEFs and the atypical Dock family GEFs. The Dbl family GEFs share a ~200 amino acid catalytic Dbl homology (DH)

Table 1 The Dock family GEFs and disease. GEF activity

Dock

Expression

Cellular function

Reference

Disease

Reference

Rac1

Dock1

Ubiquitous

Axon guidance, myoblast fusion

Cancer

Feng et al., 2011

Dock2

Lymphocyte

Alzheimer's disease

Cimino et al., 2009

Dock3

CNS

Lymphocyte migration Immunological synapse formation Axon regeneration and neuroprotection

Katoh and Negishi 2003 Laurin et al., 2008 Li et al., 2008 Fukui et al., 2001 Le Floc'h et al., 2013 Namekata et al., 2010 Namekata et al. 2012a, 2012b Namekata et al., 2013

Chen et al., 2001 de Silva et al., 2003

Dock4

CNS, lung

Dendrite development

Ueda et al., 2013

Dock5

Ubiquitous

Rac1/ Cdc42

Dock6 Dock7

Ubiquitous Ubiquitous

Shaheen et al., 2011

Dock8

Lymphocyte

Dock9 Dock10 Dock11

Ubiquitous Ubiquitous Lymphocyte

Laurin et al., 2008 Ogawa et al., 2014 Miyamoto et al., 2007 Yang et al., 2012 Yamauchi et al., 2008 Randall et al., 2009 Harada et al., 2012 Kuramoto et al., 2009 Gadea et al., 2008 Sakabe et al., 2012

Adams-Oliver syndrome

Cdc42

Myoblast fusion Mast cell degranulation Axon outgrowth Neurogenesis, Schwann-cell migration T-cell and B-cell development Dendritic cell migration Dendrite development Melanoma-cell invasion Lymphocyte migration

Alzheimer's disease Attention deficit hyperactivity disorder (ADHD) Autism and dyslexia Schizophrenia Parkinson's disease

Mental retardation Immunodeficiency Bipolar disorder Autism

Griggs et al., 2008 Zhang et al., 2009 Detera-Wadleigh et al., 2007 Nava et al., 2014

Pagnamenta et al., 2010 Alkelai et al., 2012 Pankratz et al., 2012

Please cite this article in press as: Namekata, K., et al., Dock GEFs and their therapeutic potential: Neuroprotection and axon regeneration, Progress in Retinal and Eye Research (2014), http://dx.doi.org/10.1016/j.preteyeres.2014.06.005

K. Namekata et al. / Progress in Retinal and Eye Research xxx (2014) 1e16

Fig. 1. The Rho GTPase activation cycle. Rho GTPases are activated by guanine nucleotide exchange factors (GEFs) and inactivated by GTPase activating proteins (GAPs). GEFs receive signals upon activation of relevant receptors and promote conversion of the Rho GTPase state from the inactive form to the active form. Activated Rho GTPases stimulate cellular dynamics, such as actin rearrangement, gene expression, vesicle trafficking and morphogenesis.

domain and a regulatory ~100 amino acid pleckstrin homology (PH) domain that is immediately adjacent to the DH domain (Fig. 2). There are 70 human Dbl family GEFs, many of which have conserved orthologs found in all vertebrate species and some invertebrates, such as Drosophila, Caenorhabditis elegans, Saccharomyces cerevisiae and Schizosaccharomyces pombe (Rossman et al., 2005). Accumulating evidence, including those from mice with particular Dbl GEF deficiencies, indicates that Dbl GEFs are involved in several biological functions, such as long-term potentiation (LTP), spine development, and T-cell and B-cell development (Schwechter et al.,. 2013; Tolias et al., 2005; Miletic et al., 2006). On the other hand, the atypical Dock family GEFs contain Dock homology region-1 (DHR-1) and DHR-2 domains, but not the DHePH domain (Fig. 2). The Dock family GEFs have been identified across multiple species, including many eukaryotes, such as mice and humans, Arabidopsis in plants and Neurospora in fungi (Meller et al., 2005). To date, 11 Dock proteins have been identified in humans. Based on the sequence homology, they are divided into four subfamilies: Dock-A (Dock1, 2 and 5), Dock-B (Dock3 and 4), Dock-C (Dock6, 7 and 8) and Dock-D (Dock9, 10 and 11) (Fig. 3). 2.1. Molecular structures of the Dock proteins Dock proteins all possess the conserved DHR-1 domain, which precedes the DHR-2 domain in their structure. Although the DHR-1 domain lacks GEF activity for Rho GTPases, it has an important role in delivering the Dock GEF activity to sites that require it. The DHR1 domain is approximately 160e190 amino acids in length and

Fig. 2. The catalytic domains of Dbl family GEFs and Dock family GEFs. The catalytic domain for GEF activity of Dbl family GEFs is different from that of Dock family GEFs. It resides on the DH domain in Dbl family GEFs and the DHR-2 domain in Dock family GEFs.

3

Fig. 3. The domain structure of Dock family GEFs. All Dock family GEFs have DHR-1 and DHR-2 domains. The Dock-A (Dock1, Dock2 and Dock5) and Dock-B (Dock3 and Dock4) subfamilies possess an SH3 domain at the N-terminus and a proline rich (Prich) region at the C-terminus. The Dock-C (Dock6, Dock7 and Dock8) and Dock-D (Dock9, Dock10 and Dock11) subfamilies possess neither the SH3 domain nor the Prich region, but the Dock-D subfamily proteins possess a PH domain at the N-terminus.

mediates binding with phospholipids, thus controlling the membrane localization of Dock proteins (Table 2). The crystal structure of the Dock1 DHR-1 domain has provided vital information on the structure-function relationship of the Dock protein family (Premkumar et al., 2010). The main function of the DHR-1 domain is to bind phosphatidylinositol (3,4,5)-triphosphate (PIP3 or PtdIns(3,4,5)P3), a phosphatidylinositol 3-kinases (PI3K or PtdIns 3-kinase) reaction product, at the plasma membrane, and this binding results in initiation of membrane protrusion and cell ^ te  et al., 2005). Further analysis revealed that migration (Co all Dock proteins act as interactors of phosphorylated forms of phosphatidylinositols, known as phosphoinositides (PIPs), and that Dock 6, 7 and 8 preferentially bind to phosphatidylinositol (4,5)-bisphosphate (PIP2 or PtdIns(4,5)P2) and to a lesser extent to PIP3 (Jungmichel et al., 2014). The interaction of the DHR-1 domain with PIP3 leads to relocalization of Dock proteins to the plasma membrane of the leading edge, where various signal transduction pathways can be initiated. Supporting this model, a mutant Dock1 protein lacking the DHR-1 domain failed to promote cell migration, although it was capable of inducing Rac1 GTP loading. In addition, a chimeric form of Dock1, in which the DHR-1 domain was replaced by a canonical PIP3-binding PH domain, was fully functional, suggesting that the coupling of PIP3 to DHR-1 regulates ^ te  relocalization of Dock proteins to the plasma membrane (Co et al., 2005). These observations suggest that PI3K-Dock signaling is essential for directional cell movement. The intracellular localization of Dock proteins and their respective effector molecules present in different subcellular components seem to be important factors for transducing the downstream effects of Rho GTPase activation. The GEF activities of Dock proteins are mediated by the DHR-2 domain. The DHR-2 domain is approximately 400e470 amino acids in length and has been shown to be necessary and sufficient for activating Rho GTPases (Table 2). Structural analysis of Dock9 DHR-2 domains provided detailed information on the structureeactivity relationship and indicated that the DHR-2 domain confers differential specificity towards either Rac1 or Cdc42 (Yang et al., 2009). The DHR-2 domain can be further divided into the helix-rich DHR-2 N-terminus (Dock1 amino acid residues 1178e1334) and DHR-2 C-terminus (Dock1 amino acid residues 1335e1657) (Wu et al., 2011). It has been reported that the DHR-2

Please cite this article in press as: Namekata, K., et al., Dock GEFs and their therapeutic potential: Neuroprotection and axon regeneration, Progress in Retinal and Eye Research (2014), http://dx.doi.org/10.1016/j.preteyeres.2014.06.005

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Table 2 Domains of Dock family GEFs.

Dock1 Dock 2 Dock 3 Dock 4 Dock 5 Dock 6 Dock 7 Dock 8 Dock 9 Dock 10 Dock 11

Full length (amino acids)

SH3 domain Position

Length

DHR-1 domain Position

Length

Position

DHR-2 domain Length

PH domain Position

Length

1865 1830 2030 1966 1870 2047 2140 2099 2069 2186 2073

9e70 8e69 6e67 6e67 8e69 e e e e e e

62 62 62 62 62 e e e e e e

425e609 423e607 421e599 401e574 443e627 548e714 561e727 560e729 640e818 672e850 640e818

185 185 179 174 185 167 167 170 179 179 179

1207e1617 1211e1622 1228e1635 1190e1596 1231e1642 1587e2023 1678e2114 1632e2066 1605e2069 1690e2150 1609e2023

411 412 408 407 412 437 437 435 465 461 428

e e e e e e e e 174e281 181e290 165e272

e e e e e e e e 108 110 108

The information is obtained from the neXtProt database (http://www.nextprot.org/db/).

C-terminus alone is capable of binding Rac1 and is believed to fully account for the GEF activity of Dock1. Interestingly, the DHR-2 domain plays an additional role as an interacting domain for dimerization. Homodimerization of Dock proteins through the DHR-2 domain has been reported for Dock2 and Dock9, and Dock2 homodimerization is necessary for Rac1 activation and lymphocyte migration, demonstrating the functional significance of this process (Meller et al., 2004; Terasawa et al., 2012). Structural analyses support these data and revealed that the DHR-2 domains of Dock2 and Dock9 share similar homodimer interface residues (Kulkarni et al., 2011). These observations suggest the conserved nature of Dock proteins and the possibility that other Dock proteins form homodimers. In addition to homodimerization, Dock1-Dock5 heterodimerization has been reported (Patel et al., 2011). Although it is unknown if the DHR-2 region mediates the interaction between Dock1 and Dock5, this line of evidence suggests the possibility that Dock proteins could form homo- or/ and hetero-oligomers to potentiate Rho GTPase activation in a localized area where only limited amounts of Dock proteins and Rho GTPases are available. Considering that activation of Rac1 and Cdc42 occurs during the formation of membrane protrusions (Hall, 1998), Dock protein hetero-oligomers have the potential to exert substantial structural effects on cells via activation of multiple Rho GTPases. Furthermore, the DHR-2 domain has been reported to play a role in an autoinhibitory mechanism. In Dock-A and Dock-B, the DHR-2 domain can interact with the SH3 domain located at the N-terminus, resulting in inhibition of Rac1-binding and GEF activities (Lu et al., 2005) (Fig. 3). Thus, the GEF activities of the DHR-2 domain may be tightly regulated by two molecular mechanisms: enhancement via homo/hetero-dimerization and inhibition via binding to the SH3 domain. The lack of an SH3 domain in Dock-C and Dock-D implies that their inhibitory mechanisms are different from those of Dock-A and Dock-B. In the next section, the expression patterns and functions of individual Dock proteins are described. 2.2. The Dock Family members 2.2.1. Dock-A The Dock-A subfamily contains Dock1, 2 and 5, and these proteins activate Rac1. Dock1 was the first Dock protein to be identified and is the archetypal Dock protein. Dock1 is ubiquitously distributed with relatively higher expression levels in the placenta, lungs, kidneys, pancreas and ovaries compared with the thymus, testes and colon (Hasegawa et al., 1996). Dock1 is generally a cytoplasmic protein, but multiple stimuli, such as epidermal growth factor (EGF) and active RhoG, a small GTP-binding protein, can alter its subcellular distribution to a membrane-bound form (Hasegawa et al.,

1996; Katoh and Negishi, 2003). Dock1 has an essential role in embryonic development, as seen in Dock1 knockout (KO) mouse embryos that display a dramatic reduction in all skeletal muscle tissues (Laurin et al., 2008). This defect is due to a strong deficiency in myoblast fusion, the first stage of myogenesis. Myogenesis is the formation of muscle tissue from myoblasts, which are embryonic progenitor cells that form muscle cells. The first stage of myogenesis occurs when myoblasts fuse together and form multinucleated muscle fibers known as myotubes, and this process requires Dock1. A recent study revealed that Dock1-meditated myogenesis is regulated by a G-protein coupled receptor brainspecific angiogenesis inhibitor (BAI3), which binds to engulfment and cell motility (Elmo), and the formation of Dock1/Elmo/BAI3 complex is essential for myoblast fusion (Hamoud et al., 2014). The related BAI family protein BAI1 has previously been reported to regulate Dock1-mediated internalization of apoptotic cells (Park et al., 2007), and more recently, it was reported that it also promotes myoblast fusion via the ELMO/Dock1/Rac1 pathway (Hochreiter-Hufford et al., 2013). Interestingly, the BAI3 gene is associated with schizophrenia (DeRosse et al., 2008), suggesting the possibility that Dock1 may also be associated with this neurological disorder. In addition to myoblast formation in vivo, Dock1 also plays a significant role in metastatic breast cancers and in glioma tumorigenesis (Feng et al., 2011, 2012; Laurin et al., 2013). In cancer cells, Dock1 function is regulated by its phosphorylation state; phosphorylation at Y722, Y1811, or S1250 increases its GEF activity for Rac1 and phosphorylation rates at these sites are increased in cancer cells, suggesting that targeting particular phosphorylation sites may have therapeutic potential. In the CNS development, axon guidance is a critical event that must be regulated tightly for correct formation of synaptic connections. Recent studies revealed that Dock1 deficiency induces impairment in axon outgrowth and abnormal axon reorientation toward the guidance factor (Li et al., 2008). They showed that Dock1 mediates axon guidance by a signal transduction involving netrins, a family of axon guidance cues, and promotes neurite outgrowth. Taken together, accumulated evidence suggest that Dock1 is involved in a broad spectrum of biological functions including myoblast fusion, tumorigenesis and CNS development in vivo. Dock2 is predominantly expressed in hematopoietic cells and also found in microglia. Dock2 has been shown to be indispensable for lymphocyte chemotaxis, and plays an important role in immune systems (Fukui et al., 2001; Sanui et al., 2003). Dock2 KO mice exhibit defects in migration of T and B lymphocytes but not of monocytes in response to chemokines. In addition, Dock2 deficiency exhibits T lymphocytopenia, atrophy of lymphoid follicles and loss of marginal-zone B cells. These effects are believed to be a result of diminished Rac1 activation. Recently, it has been reported

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that Dock2 is required for PIP3-mediated immunological synapse formations that contain a ring of filamentous actin that promotes adhesion and facilitates the directional secretion of cytokines (Le Floc'h et al., 2013). Furthermore, natural killer cells, which play an important role in protective immune responses against viral infection and tumor progression, that lacked Dock2 failed to effectively kill leukemia cells in vitro and MHC class Iedeficient bone marrow cells in vivo (Sakai et al., 2013). These observations suggest that Dock2 plays critical roles in adaptive immunity. Like Dock1, over 20 mutations in Dock2 were also detected in human esophageal adenocarcinoma cells (Dulak et al., 2013), suggesting that the aberrant Rac1 activation by mutant Dock2 is also involved in esophageal adenocarcinoma tumogenesis. Therefore, Dock2 may be involved in tumogenesis in addition to its key roles in immune responses. However, it is not known whether Dock2 is phosphorylated under pathological conditions, like Dock1, or if in fact there is a phosphorylation site in the Dock2 molecule. In addition to Dock3, Dock2 may also play a critical role in Alzheimer's disease pathology and it raises an interesting possibility that Dock2 may be a therapeutic target for Alzheimer's disease. (Cimino et al., 2013). Dock2 is upregulated in human Alzheimer's disease brains compared with age-matched controls. Dock2 is associated with the prostaglandin signaling pathway via the prostaglandin E2 receptor (EP2) (Cimino et al., 2009); EP2 has been shown to regulate neuroinflammation and reduce the Ab plaque burden in a mouse model of Alzheimer's disease (Liang et al., 2005). These observations suggest a role for Dock2 in regulation of microglial innate immune function that is associated with the pathogenesis of Alzheimer's disease. Dock5 is expressed in various tissues including the heart and lens. In the rupture of murine lens cataracts, a mouse model of hereditary cataracts, the Dock5 protein expression is significantly reduced, suggesting that Dock5 is involved in maintenance of lens integrity (Iida et al., 1997; Omi et al., 2008). Unlike Dock1, Dock5 is dispensable for normal mouse embryogenesis and studies on Dock1þ/:Dock5þ/ animals have shown that Dock5 also plays a role in myoblast fusion during muscle development in mammals (Laurin et al., 2008). Dock5 has also been reported to regulate osteoclast function and is essential for bone resorption by osteoclasts, indicating that Dock5 may be a target for anti-osteoporotic therapy (Vives et al., 2011). Recently, it has been reported that Dock5 regulates mast cell degranulation (Ogawa et al., 2014). Interestingly, Dock5 GEF activity is not required for this process but instead, it forms a complex with Nck2, Akt and glycogen synthase kinase-3b (GSK-3b). The association of Dock5 with these molecules induces phosphorylation and thus inactivation of GSK-3b, which regulates mast cell degranulation. Therefore, Dock5 indirectly regulates microtubule dynamics that are required for mast cell degranulation. 2.2.2. Dock-B The Dock-B subfamily contains Dock3 and Dock4, and these proteins activate Rac1. Dock3 was originally identified as a binding partner of presenilin1, a protein that is mutated in multiple cases of familial Alzheimer's disease (Kashiwa et al., 2000). Dock3 was initially known as a presenilin binding protein (PBP) and then as modifier of cell adhesion (MOCA) (Chen et al., 2002; Kashiwa et al., 2000). Further investigations revealed that MOCA was a GEF that belonged to the Dock family and it is now commonly known as Dock3. Dock3 is a cytoplasmic protein that specifically activates Rac1 and is mainly expressed in the brain, spinal cord and retina (Namekata et al., 2004, 2010). It is the only Dock protein that shows high tissue specificity to the central nervous system (CNS); however, ubiquitously expressed Dock proteins, such as Dock1, are also expressed in neural tissues and they may serve similar functions as

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Dock3 in the CNS. Dock3 shares 40% sequence homology with Dock1 (Kashiwa et al., 2000). In humans, mutations in the Dock3 gene have been identified in subjects presenting with ADHD-like phenotypes (de Silva et al., 2003). However, the prominent phenotypes of Dock3 KO mice are axon degeneration and sensorimotor impairments, although ADHD-like phenotypes cannot be formally excluded (Chen et al., 2009). Dock3 has been associated with Alzheimer's disease, and recent studies have demonstrated diverse roles of Dock3 in neural development, with some speculation on its role in synaptic plasticity and LTP. Details on the roles of Dock3 in neurodegeneration, neuroprotection and neuroregeneration are discussed later. A recent study reported that a bioinformatics approach and microarray assay identified Dock3 to be a target of microRNA-486 (miR-486) and that the expression of Dock3 was increased in Duchenne muscular dystrophy (DMD) muscle biopsies (Alexander et al., 2014). In this study, they demonstrated that miR486 regulates Dock3 expression and that the Dock3 expression level is elevated in myoblasts from DMD patients and in the muscles of dystrophic mice (Dmdmdx-5Cv) at the timepoints that coincide with stages of muscle regeneration and myoblast fusion. These observations suggest that Dock3 may play a role in muscle regeneration. Further studies are required to determine if the reported Dock3 pathway applies to tissues other than the dystrophic muscles. Dock4 is expressed in the lung and is also highly expressed in the brain. It activates Rac1 and Rap1, and is associated with several neurological disorders, including autism, dyslexia and schizophrenia (Alkelai et al., 2012; Eguchi et al., 2013; Pagnamenta et al., 2010; Yajnik et al., 2003). A recent study showed that Dock4 is concentrated in the dendritic spines in hippocampal neurons and regulates formation of dendritic spines in cooperation with cortactin, which is an actin-binding protein (Ueda et al., 2013). Furthermore, Dock4 is also involved in organization of cellular morphology in neurons, similar to Dock3 (Xiao et al., 2013), suggesting that Dock4 may also be useful for neuroregenerative therapy. Dock4 was originally identified by a screening process as a gene that is disrupted during tumorigenesis in a mouse tumor model, and Dock4 mutations are found in prostate and ovarian cancer cell lines (Yajnik et al., 2003). However, Dock4 increases cell migration and formation of protrusions in invading breast cancer cells via Rac1 activation (Hiramoto-Yamaki et al., 2010). Dock4 is phosphorylated by GSK-3b, which enhances Wnt-induced Rac1 activation (Upadhyay et al., 2008). This signaling pathway may play important roles in regulating stem cell function in hematopoiesis (Zhou et al., 2011). Based on the proto-oncogenic effects of Wnt in human cancer, Wnt-Dock4 signaling may be a novel target for modulating tumorigenesis. Interestingly, Sponge, the Drosophila ortholog of human Dock-B, is expressed abundantly in the Drosophila CNS and is required for CNS development as well as Myoblast city (Mbc), the Drosophila ortholog of human Dock1 (Biersmith et al., 2011). Consistently, specific knockdown of Sponge in eye imaginal discs induced abnormal eye morphology in the adult fly (Eguchi et al., 2013). These observations suggest that the Dock-B subfamily proteins play important roles in the development of the mammalian CNS, including the visual system. 2.2.3. Dock-C The Dock-C subfamily includes Dock6, 7 and 8, and these members possess neither the SH3 domain nor the proline rich region, unlike the Dock-A and -B subfamilies (Fig. 3). Dock6 and 7 activate both Rac1 and Cdc42, whereas Dock8 selectively activates Cdc42 (Harada et al., 2012; Miyamoto et al., 2007; Yamauchi et al., 2008). Dock6 is phosphorylated by Akt and dephosphorylated by protein phosphatase 2A (PP2A) (Miyamoto et al., 2013).

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Interestingly, Dock6 phosphorylation by Akt results in inhibition of axon growth, but increases branching, and PP2A-induced dephosphorylation that activates the GEF activity stimulates axon extension, but not branching in the peripheral nervous system (PNS). In addition, human Dock6 mutations have been found in AdamsOliver syndrome, an autosomal-dominant or -recessive disorder characterized by cutis aplasia congenita and terminal transverse limb defects (Shaheen et al., 2011). Dock7 was initially identified as a Rac1 activator that is highly expressed in various regions of the developing rodent brain, including hippocampus and cortex, and has been shown to control axon formation and myelination through activation of both Rac1 and Cdc42 (Watabe-Uchida et al., 2006). In radial glial progenitor cells, Dock7 knockdown impedes neuronal differentiation and maintains cells as cycling progenitors. In contrast, Dock7 overexpression promotes differentiation of radial glial progenitor cells to neurons by interacting with the centrosome-associated protein transforming acidic coiled coil-containing protein 3 (TACC3) (Yang et al., 2012). In the PNS, Dock7 has been shown to act as an intracellular substrate for ErbB2, a proto-oncogene encoding a receptorlike glycoprotein, to promote Schwann cell migration (Yamauchi et al., 2008). In addition, mice with Dock7 mutations have generalized hypopigmentation, indicating that Dock7 plays an important role in pigmentation and may contribute to melanocyte ontogeny and function (Blasius et al., 2009). Recently, Dock8 has gained attention due to the discovery of a combined immunodeficiency syndrome caused by Dock8 mutations in humans (Zhang et al., 2009). High-resolution oligonucleotide array-based comparative genomic hybridization revealed large deletions in the Dock8 gene in patients who had been previously diagnosed with autosomal recessive hyper-IgE syndrome or unknown combined immunodeficiency disorders (Engelhardt et al., 2009). Dock8 deficient patients suffer from recurrent viral and bacterial infections and malignancies, which can lead to early death. Interestingly, Dock8 mutations were found in a cohort of mostly Turkish patients from consanguineous families, indicating a susceptibility trait (Sanal et al., 2012). Accumulating evidence indicates that Dock8 deficiency causes defects in T cell memory persistence and recall as well as maturation and activation of B cells, in which Dock8 was found to mediate the Toll-like receptor (TLR)-MyD88 signaling pathway required for TLR9-driven B cell activation (Jabara et al., 2012; Randall et al., 2009). Furthermore, in humans and mice, Dock8 plays a key role in survival and function of CD8þ T cells (Randall et al., 2011). Studies on Dock8 KO mice demonstrated that Dock8 also plays a critical role in dendritic cell migration (Harada et al., 2012) and that natural killer T (NKT) cell development is impaired indicating that Dock8 is essential for NKT survival and function (Crawford et al., 2013). Moreover, Dock8 promotes migration of thymocytes by binding to phosphorylated Mob1, a substrate of Mst1 and Mst2 kinases that are important regulators of T cells (Mou et al., 2012). Other interacting proteins of Dock8 have been reported as talin and Wiskott-Aldrich syndrome protein (WASP) (Ham et al., 2013). Dock8 forms a complex with the Cdc42 effector WASP as well as the key integrin regulator talin, recruits these interacting molecules to the membrane and thereby regulates F-actin and integrin-mediated adhesion in NK cells. The functional significance of this interaction is partly shown by the fact that WASP deficiency exhibits impaired immune cells like Dock8 deficiency (Thrasher and Burns, 2010). Taken together, these findings suggest that Dock8 plays a pivotal role in immunity and is a potential therapeutic target for immunological diseases. Furthermore, a recent study reported that Dock8 is a downstream target for the methyl-CpG-binding protein 2 whose mutations is thought to be etiologically relevant in Rett syndrome, a genetic neurodevelopmental disorder, indicating that the role of Dock8 extends

beyond the immune system (Orlic-Milacic et al., 2014). The discovery that a genetic mutation of Dock proteins is associated with a hereditary disease is novel and offers an innovative therapeutic approach. 2.2.4. Dock-D The Dock-D subfamily includes Dock9, 10 and 11, also known as Zizimin1, Zizimin3 and Zizimin2, respectively. These proteins all activate Cdc42 and possess N-terminal PH domains but do not contain the SH3 domain or the proline-rich region. Dock9 was originally identified as a Cdc42-activating protein in fibroblasts and is expressed in the brain, heart, skeletal muscle, kidney, placenta and lung (Meller et al., 2002). Dock9 is highly expressed in the developing brain and overexpression of Dock9 promotes dendrite growth in hippocampal neurons through activation of Cdc42 (Kuramoto et al., 2009), suggesting an important role of Dock9 during development and that it may be a target for neuroregenerative therapy. In addition, knockdown of Dock9 disrupts pseudopodia formation and invasion of glioblastoma cells, indicating that Dock9 regulates invasiveness of glioblastoma cells (Hirata et al., 2012). Dock9 forms a homodimer via the DHR-2 domain, similar to Dock2 (Meller et al., 2004). Structural analysis identified a nucleotide sensor region in the DHR2 domain that contributes to release of GDP and then discharge of the activated GTP-bound Cdc42 (Yang et al., 2009). Dock9 mutations have been found in patients with keratoconus, typically a bilateral, noninflammatory, progressive corneal disorder, which can cause loss of vision (Czugala et al., 2012). The functions of Dock10 and Dock11 in vivo are not well understood. Dock10 expression is widely distributed in mice, overlapping with the expression patterns of both Dock9 and Dock11, and it appears to bind to Cdc42 with lower affinity (Nishikimi et al., 2005). Dock10 has been shown to inhibit neurite protrusion and retraction in vitro (Pertz et al., 2008) and it mediates amoeboid invasion of melanoma cells through Cdc42 activation (Gadea et al., 2008). These findings raise possibilities that Dock10 may be a therapeutic target for neurodegenerative diseases and/or cancer. Moreover, the Dock10 gene is induced by interleukin-4 (IL-4) in chronic lymphocytic leukemias, indicating a role for Dock10 in IL-4induced B-cell activation (Yelo et al., 2008). IL-4 has a protective role in chronic lymphocytic leukemic B cells （Dancescu et al., 1992）, and so, it is possible that Dock10 mediates the protective effects of IL-4. A recent study revealed that a deletion of the entire Dock10 gene is associated with autism spectrum disorders (Nava et al., 2014). It is interesting to examine if autosomal recessive mutations in Dock10 is a causative factor of autism spectrum disorders. Dock11 has a high level of amino acid sequence resemblance to Dock9, but their tissue distribution in mice is different; Dock9 is predominantly expressed in non-hematopoietic cells, whereas Dock11 is predominantly expressed in lymphocytes (Meller et al., 2002; Nishikimi et al., 2005). It has been reported that the Fcg receptor and TLR4 induce upregulation of Dock11 and stimulate filopodial formation through activation of Cdc42 in bone marrowderived dendritic cells, suggesting a role of Dock11 in the immune system (Sakabe et al., 2012). Interestingly, Dock11 has been shown to mediate a positive feedback activation of Cdc42 (Lin et al., 2006), and future studies should investigate if this type of activation is beneficial or detrimental to Dock11 function. 2.3. The Dock Family and Elmo An important effector of Dock-A and Dock-B subfamily members is engulfment and cell motility (Elmo). Elmo forms a complex with Dock proteins and regulates Rac1, which is important for Rac1-

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mediated phagocytosis and cell migration (Gumienny et al., 2001; ^te , 2014: Patel et al., Hanawa-Suetsugu et al., 2012; Laurin and Co 2010). Elmo has been shown to bind to the SH3 domain of Dock1 and releases autoinhibition of the Dock1 GEF activity while sterically allowing Rac1 access that contributes to the GEF activity of the Dock1/Elmo complex (Lu et al., 2005). Further studies have revealed that disruption of the Dock1/Elmo complex formation does not affect the Dock1 GEF activity, but significantly impairs Rac1 signaling and fails to promote cytoskeleton modeling, indicating that Elmo has an additional role in Rac1 signaling (Komander et al., 2008). Dock1e4 forms a complex with Elmo in the cytoplasm, and this interaction has been shown to be required for the biological function of Dock proteins (Grimsley et al., 2004; Namekata et al., 2012b). The association of Dock5 and Elmo has not been confirmed yet, but a sequence homology analysis indicates that Dock5 may also associate with Elmo. Recently, it has been reported that the stimulation of the G-protein-coupled receptor Cxcr4 activates the Gai2 subunit, which associates with the Elmo/Dock1 complex and the whole complex is translocated from the cytosol to the plasma membrane to activate Rac proteins during breast cancer metastasis (Li et al., 2013). This process stimulates actin polymerization in breast cancer cells, suggesting Elmo may be a therapeutic target for cancer. In addition, it has been shown that Elmo suppresses ubiquitylation of Dock1 (Makino et al., 2006) and also of Dock2 (Stevenson et al., 2014), indicating an important role of Elmo in regulating the expression levels of Dock proteins. Elmo also interacts with RhoG in a GTP-dependent manner, forms a ternary complex with Dock1 at the plasma membrane and induces RhoGdependent Rac1 activation (Katoh and Negishi, 2003). Similarly, Dock3 forms a ternary complex with Elmo and RhoG, and promotes neurite outgrowth (Namekata et al., 2012a, 2012b), which is discussed later. 3. Dock3 and neurodegeneration 3.1. Dock3 and Alzheimer's disease Alzheimer's disease is the most common form of dementia and is characterized by progressive neurodegeneration. Several lines of evidence suggest that the pathogenesis of Alzheimer's disease is associated with progressive accumulation of the amyloid-b (Ab) protein, the main component of senile plaques in the Alzheimer's disease brain, which is generated from the proteolysis of the Ab precursor protein (APP). Dock3 binds to presenilin1, which is associated with g-secretase activity in APP processing (De Strooper et al., 2012), and acts downstream of both APP- and presenilinmediated intracellular signals (Tachi et al., 2012). Although the functional significance of the interaction between Dock3 and presenilin1 in Alzheimer's disease remains unknown, Dock3 overexpression suppresses Ab protein secretion by regulating APP metabolism (Chen et al., 2002). In addition, Dock3 expression is decreased in the soluble fraction of extracts from Alzheimer's disease brains compared with those from age-matched controls (Kashiwa et al., 2000). Based on these findings, one speculates that Dock3 binds to presenilin1, inhibits Ab protein accumulation and therefore, increasing Dock3 interaction with presenilin1 may be a therapeutic target for Alzheimer's disease. Indeed, it was hypothesized that Dock3 KO mice might display accelerated pathogenesis of Alzheimer's disease; however, they did not show alterations in senile plaques or NFT formation (Chen et al., 2009). This lack of pathological phenotype was thought to be due to compensatory mechanisms involving other Dock proteins expressed in the brain, such as Dock1 and Dock4, as they share the same catalytic domain (Namekata et al., 2010). On the other hand, there is a report demonstrating that Dock3 enhances Tau phosphorylation at

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Ser199, one of the abnormal phosphorylation sites in Alzheimer's disease (Chen et al., 2001). In addition, a study reported that Dock3 acts downstream of the APP-mediated signaling pathway and promotes neuronal cell death (Tachi et al., 2012). These findings suggest that Dock3 is associated with the pathogenesis of Alzheimer's disease; however, the majority of evidence points to the therapeutic role of Dock3 at present. Research to elucidate the role of Dock3 in Alzheimer's disease continues to progress. 3.2. Dock3 and glaucoma DBA/2J mice develop a form of glaucoma characterized by a pigment-dispersing iris disease that aberrantly deposits pigment throughout the anterior chamber, including the drainage structures of the eye. As a consequence, intraocular pressure (IOP) becomes elevated and glaucomatous retinal ganglion cell (RGC) degeneration ensues (Anderson et al., 2006). The research group led by Simon John has assembled a comprehensive database of gene expression changes during glaucoma progression in DBA/2J mice (http://glaucomadb.jax.org/glaucoma). According to the information provided by this database, Dock3 expression levels were decreased, and 7 of the 11 Dock proteins underwent significant changes in gene expression in the optic nerve head in at least 3 of the 5 stages of glaucoma progression. Among these 7 Dock proteins, Dock3 and Dock1 expression levels were significantly downregulated, whereas expression levels of Dock2, 10 and 11, all of which are known to function in the immune system, were upregulated. These observations suggest that glaucomatous injury differentially affects Dock proteins and imply that the immune response to glaucomatous injury may play a role in slowing or accelerating the rate of disease progression. Normal tension glaucoma (NTG) is a subset of primary openangle glaucoma (POAG) that shows statistically normal IOP, but exhibits glaucomatous optic neuropathy and relevant visual field defects. Several population studies have suggested that NTG represents 20e90% of all POAG, with percentages appearing to vary according to race (Bonomi et al., 1998; Iwase et al., 2004; Klein et al., 1992). Although there are several inherited and experimentally induced animal models of high IOP glaucoma, including DBA/2J mice, mouse models of NTG have only recently become available. To investigate the mechanisms of neurodegeneration in NTG and discover therapeutic strategies directed at IOP-independent mechanisms of RGC loss, Harada et al. (Harada et al., 2007) generated an animal model of NTG by deleting the glutamate/aspartate transporter (GLAST) or excitatory amino acid carrier 1 (EAAC1) gene. GLAST is expressed in Müller glial cells and EAAC1 is mainly expressed in RGCs and amacrine cells. These two glutamate transporters clear excessive glutamate from the synapse, thus preventing excitotoxic effects on retinal neurons including RGCs (Harada et al., 1998, 2007). In addition to glutamate neurotoxicity, oxidative stress is recognized as a common pathologic pathway in glaucoma; for example, reduction in glutathione, a major antioxidant in the retina, was reported in the plasma of human glaucoma patients (Gherghel et al., 2013). Glutathione is a tripeptide of glutamate, cysteine and glycine found in the retina and it is primarily produced in Müller glial cells, but it is also produced in RGCs. Consistently, decreased glutamate uptake into Müller glial cells leads to reduced production of glutathione in GLAST KO mice (Harada et al., 2007). In addition, cultured RGCs from EAAC1 KO mice were more susceptible to H2O2 compared with those from WT mice (Harada et al., 2007). Thus, both GLAST and EAAC1 KO mice exhibit key features of glaucomatous pathology, including RGC loss and optic nerve atrophy due to glutamate neurotoxicity and increased oxidative stress (Semba et al., 2014a; 2014b). Interestingly, Dock3 protects RGCs in GLAST KO mice by suppressing these

Please cite this article in press as: Namekata, K., et al., Dock GEFs and their therapeutic potential: Neuroprotection and axon regeneration, Progress in Retinal and Eye Research (2014), http://dx.doi.org/10.1016/j.preteyeres.2014.06.005

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two causes of cell death (Namekata et al., 2013) via mechanisms discussed in detail later. The findings that Dock3 overexpression can suppress RGC loss in an animal model of NTG indicate that Dock3 gene therapy may be effective for treatment of NTG. One of the innovative therapeutic targets for glaucoma may be apoptosis signal-regulating kinase 1 (ASK1), a member of the mitogen-activated protein 3 kinases. ASK1 has key roles in human diseases closely related to the dysfunction of cellular responses to oxidative stress and endoplasmic reticulum stressors, and ASK1 deficiency demonstrated reduced RGC loss in the retina after ischemic injury (Harada et al., 2006). The deletion of ASK1 gene from GLAST KO mice (GLAST/:ASK1/) revealed that ASK1 is associated with progressive RGC loss, glaucomatous optic nerve degeneration and visual disturbances in GLAST KO mice (Harada et al., 2010). Interestingly, inhibition of p38, the downstream effector of ASK1, protects RGCs following optic nerve injury (ONI) (Katome et al., 2013). Therefore, combination of Dock3 overexpression and inhibition of ASK1 signaling may offer a novel therapeutic strategy for glaucoma management. Future gene expression analyses in GLAST and EAAC1 KO mice, similar to those performed in DBA/2J mice described above, would provide important information, and a comparison of gene expression changes between these two mouse models may reveal target genes common to IOP-dependent and -independent glaucoma. Recently, ROCK inhibitors have been emerging as a novel class of drugs for glaucoma (Inoue and Tanihara, 2013; Wang and Chang, 2014). The ROCK inhibitor K-115 has successfully completed the Phase II trial for POAG and ocular hypertension (Tanihara et al., 2013). ROCK is an effector molecule of the Rho GTPase RhoA and one of the roles of the ROCK signaling pathway is to promote cell contractility. The ROCK inhibitors stimulate actin motility and increase aqueous humor drainage through the trabecular meshwork and thus lower IOP, which is an effective treatment for glaucoma. As RhoA signaling has an opposing effect to Rac1 signaling, it is possible that enhancing Rac1 signaling by Dock3 may provide similar results to ROCK inhibitors, suggesting that stimulation of the Dock3 signaling may lower IOP and may also be a potential therapeutic target for IOP-dependent glaucoma. 4. Dock3 in RGC protection

neuroprotective effects in the rat ischemic retina (Li et al., 2014b) supporting the notion that NMDA receptor antagonists may be a candidate for glaucoma treatment. NMDA receptors are tetramers that form functional receptor channels. Two GluN1 subunits, which bind the co-agonist glycine, must combine with at least two GluN2 (A-D) subunits, which bind glutamate (Paoletti et al., 2013). GluN2B-containing receptors have been shown to mediate prolonged channel opening and greater overall calcium current per event (Sobczyk et al., 2005; Yashiro and Philpot, 2008). A recent report shows an increased expression of GluN2B in DBA/2J mice (Dong et al., 2013). Similarly, pharmacological inhibition of GluN2B protected RGCs in GLAST KO mice (Bai et al., 2013a). These data indicate that inhibition of GluN2B activity may be a promising therapeutic approach for treatment of retinal diseases such as glaucoma. 4.2. Dock3 controls cell-surface expression of NMDA receptors Recent studies have shown that Dock3 directly interacts with the intracellular C-terminus of both GluN2B and GluN2D subunits (Fig. 4), and overexpression of Dock3 inhibits the NMDA receptor activities, thus promoting RGC survival (Bai et al., 2013b; Namekata et al., 2013). In mice overexpressing Dock3, the retinal GluN2B expression was similar to controls; however, following intraocular NMDA injection, their retinal GluN2B expression was significantly reduced compared with controls. Similarly, Dock3 reduces the surface expression of GluN2D-containing NMDA receptors in vitro. GluN2B has been implicated in various conditions including traumatic brain injury, pain, Parkinson's disease, major depression and Alzheimer's disease, and several selective antagonists have entered clinical trials (Mony et al., 2009; Nutt et al., 2008; Pochwat et al., 2014; Yurkewicz et al., 2005), demonstrating manipulation of GluN2B activity has great therapeutic potential. The C-terminus of GluN2B has been well characterized, and the intracellular C-terminal domain of GluN2B is known to regulate the internalization and degradation of GluN2B-containing NMDA receptors (Roche et al., 2001). GluN2B interacts with the postsynaptic density protein 95 (PSD95), and this NMDA receptor-PSD95 interaction is required for the stabilization of the NMDA receptor complex at the plasma membrane and excitotoxic downstream signaling. At

4.1. Glutamate excitotoxicity and RGC death Glutamate is the major excitatory neurotransmitter in the CNS and plays an essential role during neural development and synaptic plasticity through both ionotropic (ligand-gated) and metabotropic (G-protein-coupled) receptors. N-methyl-D-aspartate (NMDA) receptors, one of the ionotropic glutamate receptors, have fundamental roles in both physiological and pathological processes in the mammalian CNS (Hardingham and Bading, 2010; Paoletti et al., 2013). Overactivation of NMDA receptors is thought to be a key contributing factor in the pathophysiology of many CNS disorders, including Alzheimer's disease and Huntington disease (Ittner et al., 2010; Shankar et al., 2007; Zeron et al., 2002). As mentioned in the previous section, glutamate excitotoxicity may also be implicated in the degeneration of the RGCs and optic nerves observed under pathological conditions, including glaucoma. Indeed, the NMDA receptor antagonist memantine suppresses RGC loss in GLAST KO mice (Harada et al., 2007) and increases RGC survival and diminishes retinal degeneration in DBA/2J mice (Atorf et al., 2013; Ju et al., 2009). In addition, although the clinical trial to validate memantine as a neuroprotectant for glaucoma was unsuccessful, it is suggested that it may still be a therapeutic candidate for glaucoma in combination with other drugs (Osborne, 2009). Another NMDA receptor antagonist bis(7)-tacrine has exhibited

Fig. 4. Dock3-binding proteins. The PH domain in Elmo binds to the SH3 domain in Dock3. The WAVE homology domain (WHD) in WAVE and phosphatidylinositol (3,4,5)triphosphate (PIP3) bind to the DHR-1 domain. The amino acid residues 796-1154, which include part of the DHR-2 domain, bind to the intracellular C-terminal domain (CTD) of NMDA receptor subunits. Rac1 binds to the DHR-2 domain. The GSK-3bbinding domain is located at the amino acid residues 1628-1777, adjacent to the DHR-2 domain, but at the opposite end of the binding domain for the NMDA receptor subunits. The SH3 domain in Fyn binds to the proline rich (P-rich) region.

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postsynaptic sites, GluN2B is phosphorylated at Tyr1472 by the tyrosine kinase Fyn, which stabilizes the NMDA receptor complex at the plasma membrane. Together with the evidence that Dock3 directly interacts with Fyn (Namekata et al., 2010), these results suggest that Dock3 may play a part in the process of GluN2B internalization, thereby protecting RGCs from excitotoxicity (Fig. 5). Indeed, when Dock3 is overexpressed in GLAST KO mice, an NTG model that shows increased glutamate neurotoxicity, Tyr1472 phosphorylation was reduced, and RGC loss was ameliorated (Namekata et al., 2013). These results suggest that a Fyn-Dock3GluN2B complex may be formed and this formation inhibits Tyr1472 phosphorylation thereby destabilizes the NMDA receptor complex and suppresses excitotoxic damage in GLAST KO mice. Functionally, GluN2B has been implicated in many forms of synaptic plasticity related to the physiology of striatal neurons and the pathogenesis of various neurological disorders. Indeed, Ab and tau proteins promote Fyn-mediated GluN2B stabilization at the plasma membrane, resulting in enhanced NMDA receptormediated neurodegeneration in the brain of a mouse model of Alzheimer's disease (Amadoro et al., 2006; Ittner et al., 2010). It is possible that Dock3 inhibits the Fyn-mediated GluN2B stabilization at the plasma membrane and exerts neuroprotective effects on the Alzheimer's disease brain. Taken together, the Dock3GluN2B interaction may play an important role in regulating NMDA receptor function and thus excitotoxicity.

Fig. 5. Dock3 suppresses glutamate-induced excitotoxicity. Glutamate receptors are required for synaptic function under normal conditions (top). Excess glutamate causes prolonged stimulation of glutamate receptors and induces excitotoxic neuronal death (middle). Dock3 overexpression suppresses glutamate-induced excitotoxic neurodegeneration by reducing NMDA receptor expression (bottom).

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5. Role of Dock3 in optic nerve regeneration 5.1. Recent progress in optic nerve regeneration The axons of RGCs bundle together to form the optic nerve. Similar to other CNS axons, the optic nerve has a very limited regenerative capacity. Therefore, damage to the optic nerve or RGCs can lead to permanent vision loss and cause devastating effects on quality of life. However, recent advances in research have shed light not only on various ways to encourage optic nerve regeneration, but also on the functional recovery of an injured optic nerve. There are a number of factors that are known to hinder axon regeneration. Following injury to the adult CNS tissues, molecules that are major suppressors of regenerative axon growth and sprouting are secreted. These molecules include myelin-associated inhibitor molecules, and inhibition of these molecules by destroying myelin either by X-radiation (Savio and Schwab, 1990) or immunological approaches to neutralize the molecules has resulted in successful CNS axon regeneration (Wahl and Schwab, 2014). In addition, a glial scar, primarily consisting of reactive astrocytes and microglia, is formed following injury. This acts as a physical and likely biochemical barrier for regenerating CNS axons, and reducing the amount of chondroitin sulfate proteoglycan, the main component of the glial scar, by enzymatic digestion or other means has been shown to enhance regeneration (Soleman et al., 2013). Meanwhile, there are molecules that can promote neuronal regeneration, such as neurotrophic factors, inflammation molecules, growth-promoting extracellular matrix molecules and cell adhesion molecules. Recent studies have shown that both locally available growth regulatory molecules and intracellular signaling molecules greatly influence injury-induced neuronal growth (Hammarlund et al., 2009; Watkins et al., 2013). Furthermore, stimulation or inhibition of some intracellular signaling pathways play major roles in injury-induced neuronal growth and regeneration. For example, the phosphatase and tensin homologue (PTEN), a tumor suppressor gene, is a well-known negative regulator of cell growth and migration in most cell types. PTEN deletion in mice produces striking regeneration capacity in the adult optic nerve and synergistically promotes robust axon regeneration under conditions that also stimulate regeneration, such as deletion of the suppressor of cytokine signaling 3 (SOCS) gene (Park et al., 2008; Sun et al., 2011). Therefore, successful long-distance optic nerve regeneration may be achieved by a combination of lowering inhibitory cues, increasing environmental growth-promoting cues and activation of cell growth programs. Recently, Larry Benowitz's group has identified important regulators of axon regeneration and successfully achieved full-length axon regeneration in mice following ONI by combining three treatments (de Lima et al., 2012; Kurimoto et al., 2010; Yin et al., 2006). Application of zymosan, which induces inflammatory responses, and cyclic AMP combined with PTEN gene deletion enabled optic nerve fibers to regrow to their full length to innervate visual brain areas, including the dorsal lateral geniculate nucleus and superior colliculus. Behavioral studies, such as the visual cliff test, optomotor response, circadian photoentrainment and pupillary light reflex, showed partial recovery of visually guided behaviors, suggesting the formation of functional synapses in visual centers of the brain. Although there are some limitations that prevent immediate translation of this strategy to clinical settings, these findings raise hope that the eyesight lost following traumatic or glaucomatous injury can be restored. 5.2. Dock3 in axon regeneration Dock3 has previously been shown to play a role in neurite extension and in maintaining the functional integrity of axons

Please cite this article in press as: Namekata, K., et al., Dock GEFs and their therapeutic potential: Neuroprotection and axon regeneration, Progress in Retinal and Eye Research (2014), http://dx.doi.org/10.1016/j.preteyeres.2014.06.005

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(Chen et al., 2009). Recently, we revealed that overexpression of Dock3 induces optic nerve regeneration by stimulating cytoskeletal remodeling via two signaling pathways: the actin polymerization pathway (Namekata et al., 2010, 2012b) and the microtubule assembly pathway (Namekata et al., 2012a) (Fig. 6). The molecular dynamics in the growth cone is critical for axon elongation. The growth cone is composed of two cytoskeletal filaments: actin that is located in the peripheral region and microtubules in the central region that generally extends to the axons. Actin monomers undergo continuous polymerization and deploymerization, and the balance of these two processes determines the extent of elongation. As mentioned previously, Rho GTPases regulate actin motility, and RhoA is involved in growth cone retraction whilst Rac1 and Cdc42 promote neurite outgrowth by formation of lamellipodia and filopodia respectively (Kozma et al., 1997; Nobes and Hall, 1995). Pharmacological inhibition of RhoA (e.g. with C3 transferase) or its downstream effector ROCK (e.g. with Y27632) overcomes the intrinsic inhibitory environment in the adult CNS and promotes axon regeneration (Bertrand et al., 2005; Fournier et al., 2003; Kubo et al., 2007), suggesting that manipulation of the actin dynamics through Rho GTPases can be effective for neuroregenerative therapy. Actin is not the only molecule important for axon outgrowth. Microtubules polymerization is also required for successful axon elongation. The clinically established anticancer drug paclitaxel, also known as Taxol, directly stimulates microtubule polymerization at a low dose and has been shown to promote axonal growth and regeneration (Hellal et al., 2011; Sengottuvel and Fischer, 2011; Sengottuvel et al., 2011). Interestingly, taxol improves the motility of the growth cones as well as decreases the inhibitory signaling, such as NgR signaling via Nogo, MAG and OMgp, and glial scar formation thereby providing optimal conditions for axon regeneration (Geraldo and Gordon-Weeks, 2009; Sengottuvel et al., 2011). These data indicate that stimulation of microtubule dynamics is a promising target for neuroregeneration therapy. Dock3 stimulates both of the cytoskeletal dynamics in the growth cone during axon regeneration. As the phosphorylation status of Dock6 determines whether it promotes axon extension or branching in sensory neurons (Miyamoto et al., 2013), it would be interesting to examine the phosphorylation status of Dock3 during neuroregeneration and identify the kinases for Dock3. In the

Fig. 6. Overexpression of Dock3 promotes optic nerve regeneration. The optic nerve was transected in wild-type mice (WT) and mice with Dock3 overexpression (Dock3 Tg). Regenerating axons in the optic nerve proximal to the injury site (dotted line) were immunostained with GAP-43 antibody 2 weeks after surgery. Arrowheads indicate regenerating axons. (Scale bar, 50 mm). Reproduced with permission from Namekata et al. (Proc. Natl. Acad. Sci. U. S. A. 107: 7586-7591, 2010).

following sections, the underlying mechanisms for Dock3mediated optic nerve regeneration are discussed. 5.2.1. Regulation of actin filament dynamics by Dock3 As previously mentioned, Dock3 activates the Rho GTPase Rac1. Activation of Rac1 leads to stimulation of actin polymerization, which promotes neurite outgrowth (Neubrand et al., 2010; Picard et al., 2009; Waters et al., 2008). Therefore, elucidating the detailed components of the Dock3-mediated actin polymerization pathway may provide a novel strategy for promoting axon regeneration. In addition, little is known regarding whether GEFs are only involved in the activation of Rho GTPases or are also actively involved in recruiting downstream effectors. Dock3 has been shown to play a role in plasma membrane recruitment of the WASP family verprolin-homologous (WAVE) complex, an important downstream effector of Rac1, in addition to activation of Rac1 (Namekata et al., 2010). WAVE proteins are constitutively associated with four other proteins, Sra1/Cyfip1, Nap1/Hem-2, ABI1/2 and HSPC300, and they function as a complex (Takenawa and Suetsugu, 2007). The WAVE complex stimulates the Arp2/3 complex to promote actin nucleation and cellular actin dynamics. It is intrinsically inactive and is activated by binding to active Rac1 or by phosphorylation. Dock3 binds to the WAVE protein through the WAVE homology domain (WHD) (Fig. 4), which mediates direct binding with the WAVE complex proteins ABI1/2 and HSPC300 (Takenawa and Suetsugu, 2007). WAVE has been implicated in microglial phagocytosis of Ab42 and tumor progression (Kitamura et al., 2003; Sakthivel et al., 2012), suggesting that manipulation of WAVE regulation by Dock3 may have therapeutic potential for Alzheimer's disease and cancer. It would be interesting to investigate whether Dock3 affects the efficiency of the WAVE complex formation directly or alters the activity of the WAVE complex through binding during stimulation of axon regeneration. The association between Dock3 and WAVE suggests a novel mechanism for the membrane recruitment of Dock3. The tyrosine kinase Fyn, which is activated upon stimulation with brain-derived neurotrophic factor (BDNF), interacts with Dock3. Active Fyn resides at the membrane, thus concentrating Dock3 at the plasma membrane, where it is capable of activating Rac1. During this membrane recruitment of Dock3, the WAVE complex is carried as a cargo to membrane sites. At the membrane, Dock3 more efficiently activates Rac1 and also undergoes phosphorylation by an as yet unidentified kinase. Phosphorylated Dock3 fails to bind WAVE, indicating release of WAVE at the membrane. This release may enable active Rac1 to more efficiently interact with WAVE, leading to enhanced axon regeneration (Fig. 7, middle). The fact that the domain critical for the GEF catalytic activity is conserved among Dock1, 2, 3 and 4 (Namekata et al., 2010) suggests that this mechanism may also apply to the other Dock proteins, although specific tissue expressions may limit the beneficial effects. These findings provide a framework for understanding how GEFs can manipulate the direction of downstream signaling of GTPases. Furthermore, the association of Dock3 with its binding partner Elmo (Fig. 4) results in the formation of a ternary complex with activated RhoG upon BDNF stimulation (Namekata et al., 2012b). The formation of this ternary complex allows the Dock3-Elmo complex to be translocated to the plasma membrane, where Dock3 can be phosphorylated and Rac1 is efficiently activated, leading to neurite outgrowth (Fig. 7, left). These findings indicate the importance of Dock3 phosphorylation as a result of the membrane recruitment of Dock3 in axon regeneration. This membrane recruitment of Dock3 is initiated by BDNF stimulation and induced by at least two pathways (Fyn- and RhoG-mediated), indicating the significance of Dock3 localization in cellular dynamics during optic nerve regeneration.

Please cite this article in press as: Namekata, K., et al., Dock GEFs and their therapeutic potential: Neuroprotection and axon regeneration, Progress in Retinal and Eye Research (2014), http://dx.doi.org/10.1016/j.preteyeres.2014.06.005

K. Namekata et al. / Progress in Retinal and Eye Research xxx (2014) 1e16

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Fig. 7. The Dock3 signaling pathway in optic nerve regeneration. In the RhoG-Elmo pathway, Dock3 is recruited to the plasma membrane by the formation of a RhoG-Elmo-Dock3 complex, leading to translocation of WAVE and Rac1 activation. This signaling pathway stimulates actin dynamics (left). In the TrkB-Fyn pathway, Dock3 promotes both actin dynamics and microtubule dynamics. Recruitment of Dock3 to the plasma membrane by BDNF stimulation induces activation of Rac1/WAVE signaling and promotes actin dynamics (middle). GSK-3b is translocated to the plasma membrane by Dock3, where it is inactivated, leading to stimulation of microtubule dynamics via CRMP-2 and APC (right).

5.2.2. Regulation of microtubule assembly by Dock3 One of the observed features in injured CNS axons is the depolymerization of microtubules at the axon stump, which removes protrusive activity from microtubules and hampers axon growth. Therefore, promoting microtubule assembly is a way to

increase axon regeneration. For example, GSK-3b is a well-known regulator of axon growth (Hur and Zhou, 2010), and specific substrates of GSK-3b, such as collapsin response mediator protein-2 (CRMP-2) and adenomatous polyposis coli (APC), have been identified as important mediators of axonal microtubule regulation

Fig. 8. Dock3 modulates GSK-3b-mediated microtubule dynamics in regenerating axon. In the growth cone, GSK-3b phosphorylates CRMP-2 and APC, and thus inhibits microtubule stabilization, resulting in suppression of axon regeneration. BDNF stimulation induces formation of a Dock3-GSK-3b complex at the plasma membrane. GSK-3b is then phosphorylated by Akt near the plasma membrane and inactivated, leading to disinhibition of CRMP-2 and APC. This event can enhance axon regeneration through stabilization of microtubules without Dock3 GEF activity.

Please cite this article in press as: Namekata, K., et al., Dock GEFs and their therapeutic potential: Neuroprotection and axon regeneration, Progress in Retinal and Eye Research (2014), http://dx.doi.org/10.1016/j.preteyeres.2014.06.005

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(Koester et al., 2007; Yokota et al., 2009; Yoshimura et al., 2005). In neurons, CRMPs are expressed in the lamellipodia and filopodia of growth cones, the shafts of axons and cell bodies (Yoshimura et al., 2005). The importance of CRMPs in axonal formation is highlighted by the evidence that axonal outgrowth is arrested when CRMPs are phosphorylated by certain kinases, such as GSK-3b, because this phosphorylation causes the dissociation of CRMPs from microtubules (Eickholt et al., 2002; Yoshimura et al., 2005). In addition, APC, which promotes microtubule assembly at the growth cone, is another downstream target of GSK-3b (Koester et al., 2007; Purro et al., 2008; Zhou et al., 2004), indicating the importance of GSK3b in this dynamic process. In other words, phosphorylated GSK-3b, the inactive form, induces binding of CRMP-2 and APC to tubulin dimers, promoting microtubule polymerization and inducing axonal outgrowth (Fig. 8). Recently, it was revealed that Dock3 binds to GSK-3b (Fig. 4) and enhances BDNF-dependent axonal outgrowth by inactivating GSK3b (Namekata et al., 2012b). Upon BDNF stimulation, Dock3 promotes membrane recruitment of GSK-3b and enhances GSK-3b phosphorylation at the plasma membrane, leading to GSK-3b inactivation, which enhances CRMP-2/APC-dependent microtubule stabilization (Fig. 7, right; Fig. 8). These findings are fascinating because the interaction between Dock3 and GSK-3b is independent of the GEF catalytic activity, but it can also stimulate axon regeneration. Interestingly, GSK-3b is a target for lithium-induced neuroprotection against excitotoxicity in neuronal cultures and animal models of ischemic stroke (Ren et al., 2003). GSK-3b activity has been associated with psychiatric, neurodegenerative and other diseases, including bipolar disorder, depression, schizophrenia, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, type II diabetes and cancer (Cole, 2013; Gao et al., 2011; Li et al., 2014a; Maes et al., 2012; McCubrey et al., 2014; Medina and Avila, 2014; Palomo et al., 2011; Valvezan and Klein, 2012). It has become increasingly apparent that GSK-3b may be a common therapeutic target for treatment of multiple conditions. Thus, the association of Dock3 and GSK-3b may be a novel and effective target for both neuroprotection and neuroregeneration therapy, but its therapeutic potential could extend even further. 6. Conclusions and future directions It has been nearly 20 years since the discovery of the first Dock protein, and significant advances in understanding the nature of the Dock proteins have been achieved. The functional diversity of Dock proteins suggests that these proteins play important roles in a variety of tissues and processes, and the concept that GEFs may offer novel therapeutic strategies for neurodegenerative diseases, immunological diseases, cancer and perhaps additional conditions is attractive. Dock3, expressed abundantly in neural tissues, provides neuroprotective effects and strikingly also stimulates optic nerve regeneration. We are currently investigating various approaches for glaucoma management, including Dock3 overexpression in combination with trophic factors, ASK1 inhibitors and GLAST or EAAC1 overexpression. For this purpose, we are using a transgenic mouse technology as well as Dock3 viral vectors. Based on our current knowledge, the combinatory approach appears to be the most effective method for the treatment of complex diseases, such as glaucoma and Alzheimer's disease. Acknowledgments This study was supported by the Ministry of Education, Culture, Sports, Science and Technology of Japan (KN, AK, CH, TH) and the Funding Program for Next Generation World-Leading Researchers (NEXT Program) (TH).

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