Complement and spinal cord injury: Traditional and non-traditional aspects of complement cascade function in the injured spinal cord microenvironment

Experimental Neurology 258 (2014) 35–47

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Experimental Neurology journal homepage: www.elsevier.com/locate/yexnr

Review

Complement and spinal cord injury: Traditional and non-traditional aspects of complement cascade function in the injured spinal cord microenvironment Sheri L. Peterson a,b,c,1, Aileen J. Anderson a,b,c,d,⁎ a

Sue & Bill Gross Stem Cell Center, University of California, Irvine, Irvine, CA 92697, USA Institute for Memory Impairments and Neurological Disorders, University of California, Irvine, Irvine, CA 92697, USA c Department of Anatomy & Neurobiology, University of California, Irvine, Irvine, CA 92697, USA d Department of Physical Medicine and Rehabilitation, University of California, Irvine, Irvine, CA 92697, USA b

a r t i c l e

i n f o

Article history: Received 1 October 2013 Revised 14 April 2014 Accepted 28 April 2014 Keywords: Spinal cord injury Complement C1q C3 Inflammation Scar Regeneration Guidance Axon

a b s t r a c t The pathology associated with spinal cord injury (SCI) is caused not only by primary mechanical trauma, but also by secondary responses of the injured CNS. The inflammatory response to SCI is robust and plays an important but complex role in the progression of many secondary injury-associated pathways. Although recent studies have begun to dissect the beneficial and detrimental roles for inflammatory cells and proteins after SCI, many of these neuroimmune interactions are debated, not well understood, or completely unexplored. In this regard, the complement cascade is a key component of the inflammatory response to SCI, but is largely underappreciated, and our understanding of its diverse interactions and effects in this pathological environment is limited. In this review, we discuss complement in the context of SCI, first in relation to traditional functions for complement cascade activation, and then in relation to novel roles for complement proteins in a variety of models. © 2014 Published by Elsevier Inc.

Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Traditional roles for complement cascade function . . . . . . . . . . . . . . . . . . Complement in host defense from pathogens . . . . . . . . . . . . . . . . . . Overview of pathways leading to complement activation . . . . . . . . . Effector mechanisms of complement activation and their negative regulators Summary of complement in host defense from pathogens . . . . . . . . . Complement in response to spinal cord injury . . . . . . . . . . . . . . . . . . Increased complement after SCI . . . . . . . . . . . . . . . . . . . . . Sources of complement after SCI — blood and BSB disruption. . . . . . . . Sources of complement after SCI — inflammatory cells . . . . . . . . . . . Sources of complement after SCI — CNS cells . . . . . . . . . . . . . . . Complement activation following SCI . . . . . . . . . . . . . . . . . . Complement protein interactions in the SCI microenvironment . . . . . . . Novel roles for complement in response to SCI . . . . . . . . . . . . . . . . . . . . Tissue regeneration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cell migration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cell proliferation and differentiation . . . . . . . . . . . . . . . . . . . . . .

⁎ Corresponding author at: 2026 Sue & Bill Gross Hall, 845 Health Sciences Rd, Irvine, CA 92697, USA. E-mail address: [email protected] (A.J. Anderson). 1 2101 Sue & Bill Gross Hall, 845 Health Sciences Rd, Irvine, CA 92697, USA.

http://dx.doi.org/10.1016/j.expneurol.2014.04.028 0014-4886/© 2014 Published by Elsevier Inc.

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Neuroprotection and cell survival . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Synaptic development and plasticity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Axon growth and guidance. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Complement: traditional and non-traditional roles in the context of the CNS and SCI . . . . . . . . . . . General highlights and speculations for traditional and non-traditional complement functions after SCI Role of parallel pathways . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Importance of individual complement proteins following SCI . . . . . . . . . . . . . . . . Importance of timing, source, and localization of complement following SCI . . . . . . . . . Importance of multiple roles for complement following SCI . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Introduction Complement proteins are important effectors of the host immune response to pathogens, but are also present and active after tissue injury, including CNS trauma and specifically spinal cord injury (SCI). While some experiments have investigated the effect of the complement cascade as a whole after SCI, others have begun to dissect individual roles for complement proteins and effector arms in a host of functions relevant to SCI in variety of animal models. We begin our review by summarizing the complement cascade in the standard context of inflammation, followed by a focused discussion of complement sources and activation following SCI. Next, we outline putative roles for complement proteins in SCI, and highlight examples of nontraditional roles for complement proteins in cellular functions highly relevant for SCI-related pathophysiology. These functions have been described in a variety of SCI and nonSCI models, and include tissue regeneration, cell migration, proliferation, differentiation, survival, synaptic remodeling, and axon growth. We conclude with a discussion of the influence of several key variables on nontraditional functions for complement after SCI.

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sufficient number of transmembrane pores are inserted (Edwards et al., 1983; Muller-Eberhard, 1985). Therefore, C5b-9 or terminal complement complex (TCC) is commonly called the membrane attack complex (MAC).

Traditional roles for complement cascade function Complement in host defense from pathogens Overview of pathways leading to complement activation The complement system is an enzymatic cascade consisting of over 40 proteins that participate in host defense against pathogens by recruiting inflammatory cells, marking pathogens for removal, and initiating pathogen cell lysis directly (reviewed in: (Bohana-Kashtan et al., 2004; Ehrnthaller et al., 2011; Janeway, 2001; Ricklin et al., 2010)). There are several pathways for complement activation (Fig. 1). Complement initiating proteins recognize unique molecular patterns directly on pathogen membranes or host proteins produced in response to infection or injury. The classical complement pathway is activated when complement protein C1q binds to IgM or IgG antibody:antigen complexes, the surface of a pathogen, or acute phase proteins (e.g. C-reactive protein (CRP) and pentraxin-3) (Gadjeva et al., 2008; McGrath et al., 2006; Nauta et al., 2003; Roumenina et al., 2006). This interaction results in a conformational change that induces autocatalytic cleavage in the first of two associated zymogen pairs, C1r and C1s. C1s is a substrate for C1r, with serine protease activity that cleaves both C4, and C2 if bound to C4b. Cleavage of C4 and C2 release soluble C4a (and C2a) anaphylatoxin, and create a membrane bound C3 convertase (C4b, C2b) capable of cleaving C3 into soluble C3a and membrane bound C3b. C3b can combine with C4b,C2b to form a C5 convertase (C4b,C2b, C3b), which cleaves C5 into soluble anaphylatoxin C5a and membraneassociating C5b. Further, C5b associates with C6 and C7, anchors to the membrane, and binds C8, which penetrates the lipid bilayer. Complement C9 then binds and polymerizes, resulting in creation of a transmembrane pore (Stanley et al., 1986; Whitlow et al., 1985). This pore disrupts or attacks the integrity of the cell membrane, leading to cell lysis when a

Fig. 1. Summary diagram of the traditional description of the complement cascade in inflammatory response to pathogens and to injury. Each pathway of the complement cascade (classical, lectin, alternative, and extrinsic) can be activated in the spinal cord following injury, and are likely triggered by bacterial cell membranes, antibody, acute phase proteins, degenerating myelin, DNA, coagulation cascade enzymes, and spontaneous hydrolysis. In addition to recognition/initiation, activation, and convertase-mediated cleavage by proteins outlined in green, the traditional effector functions for complement activation products are inflammatory cell recruitment (blue), opsonization (red), and direct cell lysis by the MAC (orange). C3b* of the alternative pathway denotes either C3b generated from the other pathways or C3b(H20) created upon hydrolysis, which is functionally similar to C3b.

S.L. Peterson, A.J. Anderson / Experimental Neurology 258 (2014) 35–47

The lectin complement pathway is activated by binding of soluble defense lectins, which include mannose-binding lectin (MBL) and the ficolins, to sugar residues in a particular spacing common to bacterial pathogens (Kjaer et al., 2013). Similar to the classical pathway, the lectin pathway is associated with two zymogens, MASP-1 and MASP-2, which are activated upon soluble lectin binding to cleave C4 and C2, creating the same C3 convertase and subsequent cascade as in the classical pathway. Alternative pathway complement activation can be initiated spontaneously by C3 hydrolysis or can be activated by and amplify the ongoing cascade, and begins with C3b or functionally similar C3(H2O) (Harboe et al., 2004; Pangburn et al., 1981). Complement Factor B binds to membrane bound C3b, enabling proteolytic cleavage of Factor B to release soluble Ba, creating C3b,Bb, which acts as a C3 convertase when stabilized by Factor P. Like C4b,C2b of the classical and lectin pathways, C3b,Bb produces C3a and C3b from C3, and C3b participates in the formation of the C5 convertase, C3b2,Bb. The C5 convertase C3b2,Bb, which is also analogous to C4b,C2b,C3b of the classical and lectin pathways, then cleaves C5 to C5a and C5b, and ultimately leads to MAC formation. For each of these complement activation pathways (classical, alternative, and lectin), potent effector molecules are generated from C3 and C5 cleavage by the convertases, serine proteases produced by the formation of a complex of either C4b,C2b; C3b,Bb; C4b,C2b,C3b; or C3b2,Bb. In addition, an extrinsic pathway of complement activation has been described recently, in which thrombin, a serine protease of the coagulation cascade mediates C5 cleavage and terminal complement activation (Amara et al., 2010; Huber-Lang et al., 2006; Ramos et al., 2012). Effector mechanisms of complement activation and their negative regulators A more detailed description of the initiation molecules of the complement cascade, especially C1q, as well as the molecules affected by the complement convertases, C3 and C5, is critical to understanding the catalytic production and function of complement effector proteins. Complement C1q is a 460 kDa protein of the collectin family, and is composed of 6 subunits (chains A, B, and C), each with a globular domain and a collagen-like stalk region, arranged as a bouquet of flowers (Eggleton et al., 2000; Kishore and Reid, 2000). While the globular domain of C1q binds to proteins derived from pathogens and host proteins produced in response to them, the stalk region interacts with the zymogens C1r and C1s, and subsequently with cell surface receptors on phagocytes (Eggleton et al., 2000; McGrath et al., 2006; Nauta et al., 2003; Roumenina et al., 2006). Similarly, the soluble lectins MBL and ficolin are structurally organized into homotrimers that assemble into larger oligomers with physically associated zymogens MASP-1 and MASP-2 (Kjaer et al., 2013; Turner, 1996). Complement C3 is a 185 kDa protein composed of a disulfide linked α and β chain, and is the point of convergence for activation of the three complement pathways (Bokisch et al., 1975; Janssen et al., 2005; Mastellos, 2004; Ross et al., 1982; Sahu and Lambris, 2001). The previously described functions for complement C3 are in fact mediated exclusively by C3 cleavage products, not by the whole protein. Serine protease activity of the C3 convertase (either C4b,C2b or C3b,Bb) produces both the 9 kDa soluble C3a (α chain) fragment and the larger 176 kDa C3b, with a newly exposed thioester bond for covalent adherence to cell surface carbohydrates, protein hydroxyls or amino groups. Complement C3b is quickly converted to iC3b by Factor I and Factor H, before being further processed into soluble C3c and bound C3dg, which is finally degraded to soluble C3g and precipitating C3d (another opsonizing peptide). Like C3, complement C5 is a 190 kDa protein consisting of an α and a β chain connected by disulfide bond, and its active products C5a and C5b are generated by serine protease functions of the C5 convertase (either C4b,C2b,C3b or C3b2,Bb) (Pangburn and Rawal, 2002). Complement C5a is released as a 9 kDa α chain fragment, while a newly exposed

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binding site on the 181 kDa C5b either binds C6 to nucleate membrane attack complex formation or decays into inactive C5b (Laursen et al., 2012). As noted above, generation of the C3 and C5 convertases results in the cleavage of C3 and C5 to yield both anaphylatoxins (C3a, C4a, and C5a) and opsonins (C3b and C4b). Anaphylatoxins activate and recruit inflammatory cells by initiating or increasing phagocytic activation, vascular permeability, endothelial adhesion protein concentration, expression of opsonin receptors, and migration/chemotaxis (reviewed in: (Janeway, 2001; Klos et al., 2013)). Anaphylatoxins signal through the transmembrane G protein coupled receptors C5aR and C3aR on monocytes, neutrophils, mast cells, and endothelial cells (Klos et al., 2009, 2013). A third anaphylatoxin receptor, C5L2, is likely to play a distinct role in complement C3a/C5a signaling, with reported functions in dampening the pro-inflammatory response, as well as in lipid and energy metabolism (Cui et al., 2007; Johswich and Klos, 2007; Ohno et al., 2000; Ward, 2009). Opsonins bind pathogens, marking them for removal by stimulating phagocytosis (Ricklin et al., 2010; van Lookeren Campagne et al., 2007). The complement recognition/initiation proteins C1q and MBL can also act as opsonins (Bohlson et al., 2007; Turner, 1996). Opsonin receptors are expressed on monocytes, polymorphonuclear cells (PMNs), B lymphocytes, mast cells, and dendritic cells, and include CR1, CR2, CR3 (CD11b), and CR4 (Janeway, 2001; van Lookeren Campagne et al., 2007). The autocatalytic nature of complement activation and MAC assembly makes regulatory control of this component of the innate immune response critical, because loss of or insufficient regulation can result in an attack on host tissues rather than a specific process of pathogen defense. For example, genetic mutation of complement regulatory protein C1-INH in humans causes hereditary angioedema (Davis et al., 2010; Donaldson and Evans, 1963). Accordingly, host tissues are protected from complement activation and uncontrolled positive feedback by multiple soluble and membrane-bound complement regulatory proteins (Janeway, 2001; Makrides, 1998; Sahu and Lambris, 2000; Zipfel and Skerka, 2009). Many of these inhibitors interfere with the formation or function of C3 or C5 convertases, such as CR1, DAF, MCP, C4bp, Factor H, and Factor I. For example, rapid Factor I and Factor H mediated cleavage of C3b to form iC3b quickly inactivates C3b from participation in convertases, while opsonization activity is maintained. Other regulators, such as C1-INH, act as serine protease inhibitors (serpins) at the level of C1 activation and in subsequent steps (Davis et al., 2010; Roos et al., 2002). The activity of pro-inflammatory anaphylatoxins C5a, C3a, and C4a is strongly attenuated by carboxypeptidase N mediated removal of the terminal arginine residue (“desArg”) (Huey et al., 1983; Matthews et al., 2004). Finally, MAC formation is limited by protectin (CD59), clusterin, and vitronectin. Summary of complement in host defense from pathogens In summary, complement has principally been studied in the context of inflammatory responses to pathogens. Cascade initiation by pathogen or antibody, and the effector functions of opsonization, cell recruitment, and cell lysis are all hallmarks of complement in this model. However, current literature suggests that these indications are not specific to the immune response to foreign cells, but are also active in CNS disease and injury, including SCI. In the next section, we explore what is known about complement sources, activation, and function in the context of SCI. Complement in response to spinal cord injury Increased complement after SCI A multitude of mechanisms may contribute to delayed secondary pathology caused by SCI, including apoptotic and necrotic death of neurons and oligodendrocytes, axonal injury, demyelination, excitotoxicity, ischemia, oxidative damage, and inflammation. While all of these events are likely to play important roles, inflammation has garnered increasing

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attention as understanding that the CNS is not strictly immunoprivileged has grown, and the high degree of interaction between neurons and the immune system has become increasingly appreciated. Although complement activation is usually described as an immunological response to a pathogen threat, complement may also play an important role in the inflammatory response to damaged host tissue. Complement is increased locally in the CNS within 1 d after SCI and traumatic brain injury (TBI) in rodents (Anderson et al., 2004; Tornqvist et al., 1996), and humans (Kossmann et al., 1997; Rebhun and Botvin, 1980), and persists chronically (Anderson et al., 2004; Nguyen et al., 2008). In human SCI studies, serum complement was found to be elevated in the majority of patients post-SCI (Rebhun and Botvin, 1980; Rebhun et al., 1991), along with a dampened C3amediated skin reaction to histamine injection, indicating C3/C3a consumption and complement activation (Rebhun and Botvin Madorsky, 1983). Similarly, lysis of antibody-sensitized erythrocytes decreases significantly, indicating C1q consumption and complement cascade activation in response to SCI in BUB/BnJ strain mice (Galvan et al., 2008). In parallel, experiments in rats have demonstrated increased immunoreactivity for complement proteins including C1q, FB, C4, C3, C9, MAC, Factor H, and clusterin in the gray and white matter of the spinal cord following either rhizotomy or root avulsion injury (Liu et al., 1998; Ohlsson and Havton, 2006; Tornqvist et al., 1996) and SCI (Anderson et al., 2004, 2005; Nguyen et al., 2008). Taken together, these studies demonstrate that complement is available to interact with and affect the SCI microenvironment. Next, it is important to understand both the source of complement cascade proteins and the specific interactions described for complement in the spinal cord following SCI. While complement receptors and regulatory proteins are also likely to be important in the SCI microenvironment, we focus the bulk of our discussion on components of the complement enzymatic cascade. Sources of complement after SCI — blood and BSB disruption There are several potential sources of complement cascade proteins in the spinal cord after SCI: 1) peripheral blood/serum (via hemorrhage and blood brain/spinal barrier (BSB) disruption), 2) recruited inflammatory cells (monocytes, neutrophils, lymphocytes), and 3) resident CNS astrocytes, microglia, and neurons (Fig. 2). Complement is primarily synthesized in the liver and circulates in peripheral blood plasma in an inactivated state (Morgan and Gasque, 1997). It is well established that both the rodent and primate (Goodman et al., 1976) BSB become permeable to previously impermeable molecules for several days to weeks post-SCI (Mautes et al., 2000), depending on injury severity (Beggs and Waggener, 1975), distance from the injury site (Beggs and

Waggener, 1975; Noble and Maxwell, 1983; Wells et al., 1978), and size of the molecule (Beggs and Waggener, 1975; Noble and Maxwell, 1983; Popovich et al., 1996). Specifically, studies using large molecules like HRP (40 kDa) and Evans blue albumin (68 kDa), generally report short term BSB permeability, from 1 h to 1 d until 6 h to 14 d (Beggs and Waggener, 1975; Noble and Maxwell, 1983), or 1 h to 3 d postinjury (Noble and Maxwell, 1983), respectively. However, using a much smaller 103 Da AIB tracer, BSB permeability is evident until at least 4 weeks post-injury (Popovich et al., 1996). Because complement proteins C1q, C3, and C5 circulate in human blood/serum at approximate concentrations of 100 μg/ml, 1 mg/ml, and 100 μg/ml, respectively (Pfarr et al., 2005; Sahu and Lambris, 2001; Yonemasu et al., 1978), the potential for development of a large region containing relatively high concentrations of complement proteins in the spinal cord after injury exists. However, because circulating forms of complement effector molecules range from approximately 190 kDa (C3 and C5) to 410 kDa (C1q), and the injured BSB regains its ability to limit permeability to large molecules within hours to days following injury, this source of complement is likely to be most relevant in the acute response to SCI, when large protein permeability across the BSB is maximal. Sources of complement after SCI — inflammatory cells Although hepatocyte synthesis is the primary source of complement proteins in the blood/serum, complement production by other cell types may have an important role after injury. Following SCI, innate immune responses including microglial cytokine secretion and complement anaphylatoxin production promote inflammatory cell chemotaxis and extravasation into spinal cord tissue. In this context, neutrophils (C1q, C3, C6, C7), macrophages (C1, C2, C3, C4, C5, FB) and T lymphocytes (C3, C5) can produce and secrete complement proteins (Barnum, 1995; Morgan and Gasque, 1997; Nguyen et al., 2008). In particular, PMNs recruited to the spinal cord after injury have been shown to express complement proteins C1q and C3, 30 min to 7 d post-injury (Nguyen et al., 2008). In terms of the timecourse for local inflammatory cell influx after injury, a substantial polymorphonuclear leukocyte (PMN) infiltration appears first, peaking by 1 d post-injury in rats (Beck et al., 2010; Blight, 1992; Carlson et al., 1998; Nguyen et al., 2008), or 1–3 d postinjury in humans (Fleming et al., 2006), and remaining in detectable numbers for at least 42–180 d in rats (Beck et al., 2010; Nguyen et al., 2008; Popovich et al., 1997), and until at least 10 d in humans (Fleming et al., 2006). This first wave of responders is soon followed by an increase in monocytes/microglia, starting as early as 1 d post-injury (Blight, 1992; Carlson et al., 1998; Fleming et al., 2006), and revealing an initial peak

Fig. 2. Several sources for complement proteins in the spinal cord after injury exist. These include peripheral circulating complement (in red: “blood”) via hemorrhage and BSB disruption, release from infiltrating inflammatory cells (in green: “P” for polymorphonuclear cells and “M/M” for macrophages), and local synthesis by CNS cells (in blue: “M/M” for microglia, “A” for astrocytes, “N” for neurons and “O” for oligodendrocytes). Complement is indicated by gradients of purple. Very little is known about the relative contribution of each source of complement in the spinal cord after injury over time, but predictions are depicted roughly in the diagram. The spinal cord injury epicenter is encircled with a dotted line.

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approximately 1 week (5–14 d) post-injury in rodents and humans (Beck et al., 2010; Blight, 1992; Fleming et al., 2006; Popovich et al., 1997). Similar to PMNs, macrophages/microglia also demonstrate a strong, extended secondary response, peaking at 60–90 d post-injury in rats (Beck et al., 2010). Additionally, a small but significant T lymphocyte response is observed approximately 7–9 d post-injury (Beck et al., 2010; Popovich et al., 1997), and activated B lymphocytes also accumulate in the spinal cord, where they secrete antibodies to SCI-induced antigens (Ankeny et al., 2009). Again, T cells remain elevated after the initial reported peak and trough, until at least 90–180 dpi (Beck et al., 2010). Note that the timecourse and end resolution of the cellular immune response within the spinal cord remains unclear, as both inflammatory cells and complement proteins have been shown to persist chronically in rats (Beck et al., 2010; Blight, 1992; Pruss et al., 2011) and humans (Fleming et al., 2006). Overall, these SCI cell infiltration studies demonstrate that the injured spinal cord evidences influx and activation of several inflammatory cell types with varied, overlapping, and extended timecourses. In addition to the number and timing of infiltrating inflammatory complement-producing cells, the activation state or particular subtype of cell is important for predicting the function of these cells after injury. For example, because complement functions to recruit, activate, and stimulate phagocytic cells, the particular population of macrophages affected by complement after SCI may be crucial considering the recently described pro-inflammatory (M1) and anti-inflammatory (M2) macrophage phenotypes. While some changes in M1/M2 macrophage subtype have been described in the post-SCI timecourse, this issue has only been investigated at relatively early timepoints, within 1 month post-SCI (Ankeny and Popovich, 2009; Kigerl et al., 2009). Crucially, it is not known whether the relative production of complement proteins is similar between macrophage M1/M2 and neutrophil N1/N2 phenotypes. Conversely, complement protein C1q is able to influence macrophage polarization (Benoit et al., 2012), which would be predicted to affect recovery from SCI (Kigerl et al., 2009). Although the relative contribution of cellular versus serum sources of complement in the spinal cord after SCI is largely unknown, production of complement by resident and recruited inflammatory cells may yield more specific localization and stronger local gradients with a complex timecourse post-SCI. Importantly, certain inflammatory cells are known to localize to specific spinal cord regions after injury. For example, while both display wide distribution after SCI, monocytes and microglia are abundant near the epicenter and also spread rostral and caudal in the dorsal and lateral funiculi, while neutrophils tend to concentrate in necrotic gray and white matter within and surrounding the injury (Fleming et al., 2006; Nguyen et al., 2008; Popovich et al., 1997). Additionally, neutrophils and macrophages secrete different concentrations of complement proteins (Hooshmand et al., submitted for publicationb), and distinct functions for various sources and concentrations of complement have been suggested (Klos et al., 2009).

Sources of complement after SCI — CNS cells In addition to serum and inflammatory cells, another source of complement proteins is CNS-resident cells. Although this source of complement protein production is less studied, it may be crucial for the complement response after SCI (reviewed in: (Barnum, 1995; Veerhuis et al., 2011; Woodruff et al., 2010)). The majority of complement proteins (C1, C2, C3, C4, C5, C6, C7, C8, C9, FB) can be synthesized by astrocytes (Gasque et al., 1995; Levi-Strauss and Mallat, 1987). CNS resident microglia produce C1, C3, and C4 (Gasque et al., 1995). Neurons (and to a lesser extent oligodendrocytes) also synthesize complement proteins, including classical, alternative, terminal pathway proteins (Hosokawa et al., 2003; Nataf et al., 1999, 2001; Pavlovski et al., 2012). Finally, fibroblasts, endothelial/epithelial cells, and erythrocytes also produce complement (Vastag et al., 1998), and are relevant after SCI as fibroblast infiltration into the lesion, endothelial infiltration near sites of vascular remodeling,

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and population of therapeutically implanted bridges by these cells, have been reported (Soderblom et al., 2013; Yang et al., 2009). While several sources for complement proteins in the spinal cord after injury have been described, the relative contribution of each source to complement-mediated functions remains unclear. However, timing, source, localization, and relative concentration may confer some specialization, or enhancement of specific complement function, including effects on cell loss and plasticity post-SCI. Complement activation following SCI Once complement proteins are localized to the SCI microenvironment, activation of these proteins may occur by a similar mechanism as for host defense from pathogens in the periphery. Classical complement cascade activation may be initiated following SCI by acute phase proteins, antibody, or pathogens (e.g., in the case of penetrating injuries). However, initiation of cascade activation through nontraditional ligand binding and/or alternative enzymes mediating C3 and C5 cleavage is also likely to occur after SCI. Complement C1q is known to bind to multiple ligands including not only C1r, C1s, IgG, IgM, CRP, pentraxin 3, and receptors on phagocytes, but also to DNA (Tissot et al., 2003; Van Schravendijk and Dwek, 1982), oligodendrocyte myelin glycoprotein (Omgp) (Johns and Bernard, 1997), decorin (Krumdieck et al., 1992), and β-amyloid (Jiang et al., 1994; Rogers et al., 1992). The binding domain for these diverse proteins on the large C1q macromolecule is either the collagen stalk domain, the globular head domain, or both. In particular, DNA originating from necrotic cells and Omgp liberated from damaged or dying oligodendrocytes, could each be hypothesized to play a role in complement activation in the spinal cord after injury. Similar to complement cascade initiation at the level of the C1 protein after SCI, complement activation at the level of C3 and C5 cleavage may occur by nontraditional mechanisms following injury. Aside from the complement convertases of the classical, alternative, and lectin pathways of complement activation, other serine proteases known to be present after SCI could be important complement activators. In the extrinsic pathway of complement activation, introduced above, thrombin catalyzes C5 cleavage (Amara et al., 2010; Anderson, 2014; Guo et al., 2010; Huber-Lang et al., 2006). Since thrombin is a serine protease involved in coagulation, the onset of bleeding at the site of injury makes thrombin a likely endogenous activator of complement. In addition, serine proteases such as neurosin have been identified in myelin (Blaber et al., 2002; Terayama et al., 2004), which is particularly relevant given the described association of C3 with myelin and the creation of myelin debris after trauma to the CNS. Taken together, these data suggest that several sources of complement and means of activation exist, and are likely to participate in a variety of functions post-SCI. Complement protein interactions in the SCI microenvironment While complement protein secretion by recruited and resident cells is clearly important, complement receptor expression on phagocytes and blood vessel endothelial cells (Barnum, 1995; Morgan and Gasque, 1997) cannot be overlooked, as receptor presence on these cells is critical for successful recruitment (C3aR, C5aR) and activation (C3aR, C5aR) of inflammatory cells, phagocytosis (CR3/CD11b, CR4/ CD11c, CR1/CD35), and pro-inflammatory cytokine secretion (C3aR, C5aR, C5L2). In addition, complement receptor expression on astrocytes, neurons, and oligodendrocytes (CR1, CR2, CR3, CR4, C3aR, C5aR, C5L2), may also be important in the context of SCI (Barnum, 1995; Woodruff et al., 2010). In the context of SCI, these traditional complement-receptor interactions mediate inflammatory cell infiltration, cytokine secretion, myelin clearance, and cell death, as we outline below. During an inflammatory response to infection, recruited innate immune cells lyse and phagocytose invading pathogens, while during an inflammatory response to injury, these same recruited immune cells remove apoptotic host cells and cellular debris. Accordingly, the traditional role for complement–myelin interactions has been presumed to be

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facilitation of myelin phagocytosis (Bruck and Friede, 1991; Sun et al., 2010). In this regard, complement C3 (specifically C3d, a final catabolic product of C3b) has been shown to associate with and covalently bind to myelin and myelin debris in vitro, in vivo in the spinal cord following SCI, and in several other disease models (Duce et al., 2006; Jegou et al., 2007; Sun et al., 2010), suggesting that C3 plays an important role in these events. In addition, a role for the terminal complement pathway in axonal demyelination and myelin phagocytosis has also been suggested (Bruck et al., 1995; Mead et al., 2002; Ramaglia et al., 2007). Finally, C1q has been shown to bind Omgp, a protein located in myelin with described roles in neurite outgrowth inhibition, growth cone collapse, and mitogenesis suppression (Hunt et al., 2002), suggesting that this interaction might also contribute to myelin clearance. In parallel, the traditional role for complement in cell toxicity involves C3, and specifically C3b, in formation of the C5 complement convertase and MAC, driving cell lysis (Janeway, 2001; MullerEberhard, 1985). Other described functions for complement in cell death involve complement anaphylatoxins (C5a, C3a, C4a) signaling through the G protein coupled transmembrane anaphylatoxin receptors (C3aR, C5aR) to either induce apoptosis directly, or to promote the release of inflammatory mediators (e.g. TNF-α, NOS) which injure adjacent cells (Alexander et al., 2008; Bohana-Kashtan et al., 2004; Bonifati and Kishore, 2007; Nataf et al., 1999). Recently, C5a–C5aR signaling in neurons was implicated in ischemia-induced apoptosis, which is likely to be relevant near the SCI epicenter especially after compression or contusion injuries (Pavlovski et al., 2012). As discussed above, C1q binds to a variety of CNS proteins, including Omgp, phosphotidyl serine, and DNA. As cells die by apoptosis or necrosis, the cell debris containing these proteins may be sufficient to trigger complement activation, thus augmenting local innate and adaptive immune responses throughout the injured spinal cord. A further consideration regarding the role of complement following SCI is the potential of complement proteins to interact with inflammatory and CNS cells within the injured microenvironment. Complement regulated cytokines and chemokines, including MCP-1 and IP-10, are detected in the injured spinal cord within hours after injury. Neurons, astrocytes, and microglia express C3aR and C5aR anaphylatoxin receptors. Additionally, C5a is chemotactic for both microglia and astrocytes (Armstrong et al., 1990; Yao et al., 1990) and microglia exhibit complement receptor 3 (CR3)dependent myelin phagocytosis (Bruck and Friede, 1990; van der Laan et al., 1996), suggesting that C5a and iC3b play a role in recruitment and activation of resident CNS phagocytic cells. An increase in C5a after SCI and the chemotactic effects of C5a on astrocytes may also be a novel mechanism underlying the formation of a glial scar in the injured CNS. Given that glial scar formation may be inhibitory to neuroregeneration (Fawcett and Asher, 1999; Fitch et al., 1999; McTigue et al., 2000), this could be important for both acute degenerative and long-term regenerative outcome after CNS injury. Novel roles for complement in response to SCI In sum, these data illustrate that complement influx from blood/ serum sources, synthesis by infiltrating immune and resident CNS cells, and interactions within the injured CNS microenvironment are likely to play a role after SCI. Classically, it has been assumed that widespread inflammation, e.g. complement activation, in the CNS would be detrimental (Li et al., 2009, 2010; Reynolds et al., 2004; Tei et al., 2008). In fact, C1q, FB, and C3 knock-out models have been shown to exhibit reduced histopathological damage at the injury epicenter and in some instances improved functional recovery (Galvan et al., 2008; Guo et al., 2010; Qiao et al., 2006, 2010). However, we have reported that mouse and rat C6 deficiency, and delayed antagonism of C5aR, each reduce functional recovery following SCI, suggesting a more complex function for the complement cascade (Beck et al., 2010; Hooshmand et al., submitted for publicationa). In this regard, there is evidence to suggest that complement activation can be beneficial after

CNS injury. For example, complement-induced demyelination promotes regeneration after lateral hemisection (Dyer et al., 1998), and knife cut axonal injury (Keirstead et al., 1998). Similarly, complement depletion inhibits regeneration after peripheral nerve injury (Dailey et al., 1998). Such a positive role for complement activation in regeneration may reflect the necessity of clearing cellular debris (via opsonization and mediation/stimulation of phagocytosis) and molecules inhibitory to regeneration from the area of injury in order to create a permissive environment for axonal outgrowth. In this light, Liu et al. have previously suggested that the enhanced capacity for regeneration following peripheral nerve injury may be related to enhanced complement-driven myelin clearance (Liu et al., 1998). Alternatively, recent in vitro studies suggest that the role of complement activation in the injured CNS could involve several additional mechanisms, including sub-lytic deposition of C5b-9 (Rus et al., 1996; Soane et al., 1999; Tegla et al., 2011). In the context of the proteins of the complement cascade, we suggest there are several recently identified alternative functions for these proteins in the CNS that may play a key part in the ultimate outcome from SCI (Fig. 3). Although most of this work has focused on developmental processes and various non-SCI disease models, these or closely related activities for complement may manifest following SCI. Understanding roles for complement in tissue regeneration, cell migration, cell proliferation, cell differentiation, neuroprotection, synaptic plasticity, and axon growth/guidance may provide important insight for making additional predictions about local complement interactions in the injured spinal cord.

Tissue regeneration In contrast to many organs and tissues in the body that are known to repair and regrow after injury, including the peripheral nervous system, the injured mammalian CNS demonstrates a widespread failure to regenerate. A principle goal of SCI research is to understand regeneration failure and to manipulate the cells and local environment to overcome it, facilitating improvements in functional recovery. Examination of regrowth and regeneration in other tissues and animal models may provide critical insight for SCI research. For example, complement has been implicated in the regeneration of amphibian limbs (Del RioTsonis et al., 1998; Kimura et al., 2003). Specifically, complement C3 is expressed by tissues related to limb differentiation and regeneration, including limb blastema cells and differentiating muscle cells, following mid-limb amputation in axolotls and newts. Interestingly, expression of complement C5 has also been demonstrated in newt limb regeneration, but its localization was distinct from C3, as it was detected only in the tissue of the wound epithelium. Similarly, C3 and C5 have been shown to be expressed during lens regeneration in the newt, with C3 and C5 again localized to distinct tissues (iris and lens vesicle, respectively). Along with spatial localization, the timing of complement expression also appears to be regulated, suggesting a developmental (versus immunologic) function for complement. The authors of these studies did not detect C3 or C5 expression in the normal adult limb or lens or in the developing amphibian limb. In addition, C3a has recently been demonstrated to induce regeneration of developing chick retina, reportedly through STAT-3 activation leading to IL-6, IL-8, and TNFα activity, and ultimately regulating expression of genes associated with retinal stem/progenitor cells (Haynes et al., 2013). For mammalian lens regeneration, the mechanism of C5 action in mice is likely through C5aR, as C5aR antagonist impairs lens cell proliferation as determined by BrdU incorporation (Suetsugu-Maki et al., 2011). Given the persistence of disrupted tissue, cell death, demyelination and axon regeneration failure after SCI, and the presence of complement alongside a wide variety of resident and recruited cells, these complement-mediated tissue regeneration functions might also extend to the context of damaged CNS tissue.

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Fig. 3. Functions for complement proteins in processes relevant to SCI. As indicated in the diagram, complement proteins and receptors have been implicated in peripheral tissue regeneration, and CNS cell migration, proliferation and differentiation, survival, myelin clearance, synaptic plasticity, and axon growth and guidance. Complement may exert a diverse range of affects on glial scar pathology, remyelination, and axon regeneration following SCI through these and other unidentified mechanisms. Further, these three main processes are known to interact with and influence each other to control histological and functional recovery following SCI.

On the subject of mammalian regeneration outside of the CNS, studies by Mastellos et al. (2001), Daveau et al. (2004), and Strey et al. (2003) describe a role for complement proteins C5 and C3 in mouse liver regeneration. These experiments used a toxic liver injury model, and demonstrated C5aR up-regulation with injury and growth signaling induced by C5a-C5aR interaction, as well as increased liver damage and mortality and impaired cell proliferation and regeneration in mice with genetic C5 or C3 deficiency or after pharmacological C5aR blockade (Daveau et al., 2004; Mastellos et al., 2001; Strey et al., 2003). Furthermore, intraperitoneal injection of C5, C5a, or C3a + C5a to C5 or combined C5/C3 deficient mice shortly before liver injury decreased liver pathology and increased hepatocyte proliferation (Mastellos et al., 2001; Strey et al., 2003). Based on these and subsequent studies, the mechanism for liver regeneration following mouse partial hepatectomy was proposed to be dependent on a positive-feedback loop involving injury-induced complement activation, increased IgM-mediated activation of complement, and complement induced macrophage production of IL-6 (DeAngelis et al., 2012). Cell migration Cell migration is critical for both positive and negative histological consequences following SCI. For example, the glial scar is formed from migrating astrocytes and other cells, and is known to act as a physical and chemical barrier to axon regeneration (Bradbury et al., 2002; Fawcett and Asher, 1999). However, formation of the scar may also serve to protect spared tissue by limiting the area of toxic damage (Renault-Mihara et al., 2008; Rolls et al., 2009). In addition, oligodendrocyte progenitor cell (OPC) recruitment is critical for spinal cord remyelination (Huang and Franklin, 2011; Takahashi et al., 2013).

A principle consequence of traditional complement activation is the generation of anaphylatoxins and subsequent recruitment of a local inflammatory response. As we detailed previously, complement anaphylatoxins act through C3aR and C5aR to help induce migration of phagocytic cells. Additionally, C1q exerts chemotactic activity on inflammatory cells via gC1qR and cC1qR, and a homologous molecule expressed in the leech nerve cord similarly mediates microglial migration (Tahtouh et al., 2009; Vegh et al., 2006). Outside of the traditional inflammatory response, complement proteins including C3a may also regulate normal, SDF-1a mediated, homing of hematopoietic stem cells through the C3aR (Janowska-Wieczorek et al., 2012; Reca et al., 2003), however, C3aR signaling has recently been shown to impair neutrophil migration following intestinal ischemic injury (Wu et al., 2013). Turning to CNS development, the complement anaphylatoxin receptors are transiently expressed (peak at post-natal day 12) in the neurons of the external cell granule layer (EGL) and internal granule cell layer (IGL) of the developing mouse cerebellar cortex (Benard et al., 2004). Subdural treatment with a C3aR agonist at post-natal day 12 increased the thickness of the IGL while decreasing EGL thickness, suggesting a role for C3aR in developmental neuronal cell migration from the EGL to the IGL, which was further supported by live imaging of neuron motility in cell culture (Benard et al., 2008). In addition, C3a has been shown to either positively or negatively modulate migration of mouse neural progenitor cells (NPCs) in culture, depending on the concentration of SDF-1a (low or high, respectively) (Shinjyo et al., 2009). Both C3a and C1q may also promote migration of human neural stem cells (NSCs) in culture and migration of transplanted human neural stem cells following mouse SCI (Hooshmand et al., 2014b). Finally, separate from the CNS but potentially interesting in the context of cell migration,

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C1q and gC1qR have been implicated in integrin-dependent endothelial cell adhesion and spreading (Ghebrehiwet et al., 2003). Cell proliferation and differentiation The same injury associated processes that are affected by cell migration also rely on proliferation and differentiation of migrating cell populations. Following SCI, astrocytes and other cell types proliferate and become activated as they populate the scar, and OPCs proliferate and mature during remyelination. These proliferation and differentiation dependent processes of scar formation and remyelination are crucial for determining the extent of histological and functional outcome after injury. In this regard, precursor cell proliferation was implicated in the Haynes et al. C3a-mediated chick retina regeneration study discussed above (under Tissue regeneration) (Haynes et al., 2013), and C3 has been directly implicated in several additional studies of cell proliferation and differentiation. C3 has been reported to positively regulate both basal and ischemia-induced neurogenesis in the mouse subventricular zone through a C3aR dependent, and C5/C5aR independent, mechanism (Bogestal et al., 2007; Rahpeymai et al., 2006). Through a series of culture experiments with purified C3a protein and NPCs, the same research group subsequently described a novel role for C3a in NPC differentiation into MAP2+, neurite-bearing, neurons (Shinjyo et al., 2009). Complement receptor CR2 was also reported to limit adult basal neurogenesis through interaction with C3d (Moriyama et al., 2011). In addition, C3a and C1q have been found to promote astrocytic differentiation of NSCs, both in vitro and in transplanted cells after SCI (Hooshmand et al., submitted for publicationb). Separate from the CNS but also interesting, C1q was reported to reduce proliferation in cell lines in vitro without causing cytotoxicity by signaling through the gC1qR (Ghebrehiwet et al., 1990). Beyond C3 and C1q, complement C5 has also been implicated in cell proliferation and differentiation within the CNS. The Benard et al. study, introduced above, found that subdural C5aR agonist treatment at postnatal day 12 caused increased EGL thickness without changes in IGL thickness during cerebellar development (Benard et al., 2008). This phenotype likely resulted from immature neuron proliferation in the EGL, as indicated by increased BrdU incorporation after C5aR agonist treatment, and decreased BrdU incorporation with C5aR antagonist PMX53. Further, glial differentiation from OPC to oligodendrocyte in vitro has been reported to negatively correlate with C5aR expression (Nataf et al., 2001). Also, while MAC is best known for its lytic function, sublytic levels of MAC deposition on cells has been suggested to induce proliferation in several disease models by activating various cell types to release growth factors (Cole and Morgan, 2003). This nontraditional MAC function could be particularly relevant for oligodendrocytes and axonal remyelination after SCI (Rus et al., 1996; Rutkowski et al., 2010). In line with both the proliferation and clearance hypotheses for C5 function, complement C5 deficiency in experimental autoimmune encephalomyelitis resulted in a chronically dampened remyelination response (Weerth et al., 2003). Based on the traditional immune functions of complement activation, the emerging role for complement in cell proliferation and differentiation in the CNS is intriguing and novel, but perhaps not completely unexpected. For example, returning to the immune system, C3a and C5a have been shown to promote T cell proliferation and survival through C3aR and C5aR signaling, and C1q has also been suggested to modulate T cell proliferation and differentiation through a C1q receptor-mediated mechanism (Chen et al., 1994; Raedler and Heeger, 2011; Strainic et al., 2008). These data may be relevant in the SCI microenvironment, either indirectly due to similar proliferation signaling in other cell types (e.g., astrocyte, oligodendrocyte), or directly because T cells are increased in the spinal cord both acutely and chronically following SCI (Beck et al., 2010), and have suggested roles in both exacerbating and attenuating pathology (see Bucky Jones, 2014-in this issue).

Neuroprotection and cell survival The primary mechanical trauma associated with SCI results in rapid cell death at the site of injury that includes local motor neurons, interneurons, and myelinating oligodendrocytes, and leads to an expanding zone of tissue damage near the epicenter and at distant cell bodies of injured axons over time, making neuroprotection and cell survival primary targets for therapeutic intervention after SCI (Beattie et al., 2002; Bramlett and Dietrich, 2007). In addition to the traditional role for the complement cascade in cell death after CNS injury (Pavlovski et al., 2012; J. Yang et al., 2013), complement proteins may also promote anti-apoptotic intracellular signaling and neuroprotection. First, sublytic concentrations of MAC have been shown to induce an antiapoptotic response in culture and can result in reduced cell death of oligodendrocytes in particular, which could be critical for histological and functional recovery following SCI (Soane et al., 1999). The in vitro protective effect of sublytic MAC was associated with increased BCL-2 production and inhibition of caspase-3 activation. Second, complement C1q, C3 and C5a have been demonstrated to be neuroprotective under certain stressed conditions, including Alzheimer's Disease, retinal ischemia–reperfusion, and glutamate excitotoxicity models (Benoit and Tenner, 2011; Fan and Tenner, 2004; Kuehn et al., 2008; Osaka et al., 1999). Finally, complement anaphylatoxins have been reported to activate local astrocytes and microglia to produce and secrete growth and survival factors in culture, including NGF (Heese et al., 1998; Jauneau et al., 2006; Rutkowski et al., 2010). This mechanism is in line with reports of anaphylatoxin-mediated protection and anti-apoptotic signaling in response to amyloid beta and kainic acid (or glutamate analog) neurotoxicity in culture and in vivo (Heese et al., 1998; Mukherjee and Pasinetti, 2001). Synaptic development and plasticity Another key factor in determining the histological and behavioral outcome after SCI is the connectivity and reorganization of spared and injured spinal cord circuitry. Interestingly, complement proteins C1q and C3 appear to be necessary for normal synaptic pruning in the lateral geniculate nucleus during development of the mouse visual system (Stevens et al., 2007). The mechanism for this synapse elimination function has been proposed to involve astrocyte-mediated increases in local complement C1q production by neurons during a specific developmental window, in association with phagocytosis of low-activity presynaptic terminals by microglia through activation of CR3 by C3b on tagged synapses (Schafer et al., 2012). The role of complement in synapse elimination is not specific to the dLGN or restricted to development. First, C1q KO mice display increased neocortical excitatory transmission and seizure activity (Chu et al., 2010). Second, synaptic complement localization has been described in a glaucoma disease model (Stevens et al., 2007). Importantly, synaptic stripping may occur following SCI, and synapse loss is hypothesized to affect functional recovery — either negatively by reducing signal transmission, or positively by promoting plasticity and circuit reorganization. Complement C3 does appear to play a role in synaptic stripping of spinal cord motoneurons after peripheral sciatic nerve transection, as injured C3 KO mice do not display the expected reduction in synaptic density observed with wild-type mice (Berg et al., 2012). For this PNS lesion model, in contrast to the developmental models, experiments using C1q KO mice demonstrated that C1q was not necessary for the synaptic effect (Berg et al., 2012). Axon growth and guidance Axon regeneration failure is an important target for therapeutic intervention after SCI. Therefore, understanding the mechanisms behind axon growth and the appropriate guidance of growing axons is critical. Complement proteins may play a dual role in axon growth and guidance

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following SCI. As detailed in previous sections, complement is likely to play a beneficial role in remyelination and regeneration by promoting phagocytosis of myelin and cellular debris. In contrast, one study using C3 KO mice reported an increase in neurite outgrowth in DRG and astrocyte co-culture, and an increase in neurofilament specific immunofluorescence intensity after contusion SCI in vivo, suggesting that C3 is a negative regulator of axon growth (Guo et al., 2010). Interestingly, the sciatic nerve injury study mentioned above also found increased expression of the regeneration associated gene GAP-43 in axotomized motoneurons in the genetic absence of C3 (Berg et al., 2012). Furthermore, current studies in our laboratory demonstrate that cortical neurons grown on myelin and C3 protein display exacerbated neurite outgrowth inhibition (Peterson et al., 2014a). Additionally, after SCI with sciatic nerve conditioning lesion, sensory axons in C3 KO mice penetrate further into and beyond the central injury than those in WT mice (Peterson et al., 2014a). Finally, addition of complement C1q may block the neurite outgrowth inhibition and repulsive growth cone turning functions of the myelin-associated inhibitor MAG in culture, and deficiency in C1q appears to alter the direction of sensory axon regrowth at the site of SCI (Peterson et al., 2014b). Overall, the functional diversity described here suggests that distinct roles for different complement components may be the result of specific interactions within the developing or injured microenvironment, separate from the role of these molecules in the innate immune response. Complement: traditional and non-traditional roles in the context of the CNS and SCI General highlights and speculations for traditional and non-traditional complement functions after SCI In the previous sections, we have reviewed the traditional pathways, activation, and roles of complement as a part of the innate inflammatory response. Further, we have explored emerging functions for complement in regeneration and repair, focusing on the relatively unexplored potential of these functions to play a role after CNS injury and degeneration. In the following section, we highlight several points that bring the traditional and non-traditional functions of complement proteins into an overall context for the CNS and SCI. Role of parallel pathways As discussed above, although there are discrete pathways and mechanisms of initiation of the complement cascade, in the case of SCI, it may be likely that these would all come into play in parallel due to the abundance of activators in the injured microenvironment. Additionally, while the complement cascade can be thought of in terms of a hierarchy of effector functions, there is again redundancy between the early and late parts of the cascade. For example, although early and mid-cascade complement components such as C1q, C3a, and C5a play a key effector function in inflammatory cell recruitment, terminal pathway components can also serve this function (Zhou et al., 2000). In this context, C5b-9 has been shown to contribute to inflammatory cell trafficking and extravasation across the BBB/BSB (Kilgore et al., 1998), and C6deficient rats have recently been shown to have an attenuation of EAE in correlation with reduced immune cell infiltration (Tran et al., 2002). Importance of individual complement proteins following SCI In addition, the function of individual components of the cascade must be considered separately from activation of the full cascade and from activation of the different functional arms of the cascade. As described above, there are many established roles for specific single complement proteins outside the autocatalytic assembly of a lytic pore as a part of the immune response, including activation and recruitment of inflammatory cells, opsonization and phagocytosis, and antibodymediated activation of adaptive immunity. In line with these, we suggest that single molecules represent the principal opportunity for

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complement components to serve alternative roles in the developing, mature, or injured CNS. Accordingly, recent studies strongly point to a true multiplicity of functions for single complement proteins in diverse aspects of neural regeneration and repair, including limb regeneration, stem cell migration, cell proliferation and differentiation, neuroprotection and survival, synaptic development and plasticity, and axon growth and guidance. Importance of timing, source, and localization of complement following SCI The timing, location, and source of complement proteins will likely determine whether the balance of activities initiated tips toward traditional or non-traditional roles after CNS injury or in CNS disease. Complement is often considered solely in the context of a rapid and transient response to immune challenge. Because the majority of complement synthesis takes place in the liver, and the highest levels of complement are found in blood/serum, individual complement proteins can be expected in the CNS at micromolar levels in the early phases of BBB/ BSB breakdown after an acute traumatic event. In the case of SCI, complement protein localization peaks at the injury epicenter within 24 hours (unpublished data from our laboratory). However, there is synthesis and secretion of individual complement proteins by infiltrating and resident immune cells, and resident CNS cells, which may occur over the course of days, weeks, and even months post-SCI. This type of local synthesis can be expected to result in lower levels (e.g., nM concentrations) of individual complement components within the microenvironment. Furthermore, infiltrating and resident immune cells in particular are distributed in the spinal cord both proximal and distal to the injury epicenter, with regional specificity (Fleming et al., 2006; Popovich et al., 1997). For example, neutrophils remain principally located within the area of primary damage, while infiltrating macrophages and CNS microglia extend large distances both rostrally and caudally. As a result, the potential contributions of location and source-specific effective dose to physiological activity and function of different complement proteins must be considered. Moreover, phenotypic and functional heterogeneity of immune cells at different times and locations post-SCI can be expected (Beck et al., 2010; Hawthorne and Popovich, 2011), resulting in corresponding changes in biosynthesis of multiple inflammatory proteins, including complement. Taken together, these factors suggest that the effective function of complement in traditional versus non-traditional roles within the spinal cord after injury, e.g. on myelin clearance, axonal regeneration, stem/progenitor cells, etc., will be dramatically affected by timing, location and source. The observation that delayed inhibition of the C5aR impairs recovery after SCI (Beck et al., 2010), and the reported delayed detrimental effect of C6 deficiency observed after SCI (Hooshmand et al., submitted for publicationa), support this suggestion. An additional level of specificity and control can be predicted to derive from variation in the response of CNS cells to individual complement proteins. For example, C1q has been identified as an activating protein for microglial-mediated clearance of synapses in the developing visual cortex (Schafer et al., 2012; Stevens et al., 2007); however, a parallel function in motor neuron synapses following peripheral nerve injury has been reported for C3, but not C1q (Berg et al., 2012), suggesting that multiple complement components may play similar but specialized roles in different CNS regions. In this regard, region and cell specific expression of complement receptors, including the C3aR, C5aR, C5L2, and gC1qR, or novel targets, on not only microglia and astrocytes, but also neurons and neural stem/progenitor cells, could be critical. Similarly, soluble and membrane bound complement regulatory proteins (e.g. CD35, CD46, CD55, CD59, clusterin, FH) are expressed and/or upregulated in a cell-specific manner within the CNS, and can confer cell vulnerability and/or cell resistance to MAC-mediated hemolytic lysis (Hoarau et al., 2011; Kolev et al., 2010; Koski et al., 1996; Piddlesden and Morgan, 1993; Scolding et al., 1998; Vedeler et al., 1994; Wing et al., 1992). This class of regulatory molecules may regulate the balance between conventional and alternative complement functions. Combined

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with the range of alternative functions for individual complement component proteins, the potential for cell and/or regional specificity for these molecules in developmental and regenerative plasticity suggests there may be a high degree of complexity for alternative complement functions in the CNS. Importance of multiple roles for complement following SCI Finally, the multiplicity of possible roles that the immune response, the complement cascade and individual complement proteins may play after CNS injury and disease is an important factor in the context of pharmacological target selection and target timing. There has been a strong focus on the development of complement-targeted therapeutics in recent years, with a particular focus on complement inhibitors and the treatment of age-related macular degeneration, immunorejection, cancer, and inflammatory diseases such as hemolyticuremic syndrome, paroxysmal nocturnal haemoglobinuria, and hereditary angioedema (Ricklin and Lambris, 2013; K. Yang et al., 2013). A key question, however, is whether a rush to apply therapeutic complement inhibitors to CNS trauma and disease will be fruitful. Based upon the emerging literature on non-traditional functions for complement, particularly careful analysis of route, location, and timing of administration will be necessary to assess the full effect, beneficial or detrimental, of any complement therapeutic. Moreover, promising therapeutic candidates could be missed by failure to fully consider these variables. In parallel, a key issue may be that clinical translation is ultimately dependent on the quality of the pre-clinical models employed. In particular, investigation of the role of early complement components and C5b-9 assembly following CNS trauma or in CNS disease using transgenic mouse models may have serious limitations, as the capacity for functional C5b-9 formation is low or even undetectable in hemolytic assays in a majority of mouse strains (e.g. C57Bl/6) compared with rats or humans (Galvan et al., 2008; Luchetti et al., 2010; Ong and Mattes, 1989; Osmers et al., 2006), although it should be noted that reported affects following manipulation of the terminal pathway after SCI in these mice suggest some function (Qiao et al., 2010). The impact of this relative deficiency on compensatory modulation of early complement components or other aspects of the innate and adaptive immune response is largely unknown. Conclusion In this review, we have summarized putative and hypothesized roles for complement in SCI and recovery. The functional diversity for complement interactions in the SCI microenvironment is likely due to specifics of the source of the complement, the protein or arm of the cascade involved, the available binding partners, and the timing and location of activation. Although complex, these interactions intersect to influence multiple key aspects of neurodegeneration and neuroregeneration after SCI. As highlighted throughout the review, and particularly in the final section, numerous questions regarding complement interactions after SCI remain to be addressed. Currently, little is known about the timing and concentration of complement, or the relative contribution of local complement synthesis from various cell types and blood/serum influx following SCI. In terms of functional signaling, it will be very important to understand the regulation of complement receptor expression on local CNS cells after SCI. Similarly, the subcellular localization (e.g. cell body, axons, dendrites) of these receptors may prove to be important. Furthermore, additional complement receptors could be responsible for mediating some of the SCI-relevant nontraditional functions we discussed for complement, as the mechanisms for many of these interactions are relatively unclear. While we have focused this review on predictions from the nontraditional or non-immunological roles for complement, very little is known about these functions as they relate to pathogenesis and repair after SCI. Along these lines, it will be interesting to explore the potential role for complement receptor expression

and complement interactions in host progenitor or precursor cell populations and the stem cell niche in the SCI microenvironment. Also, given the potential role of C5b-9 in cascade functions beyond cell lysis, it will be critical to re-evaluate what aspects of immunobiology are missed in assessment of SCI recovery parameters in conventional mouse models where there is minimal C5b-9 formation. For studies focused specifically on interactions of complement post-SCI, informative data will come from the conditional manipulation of individual complement components in a complement sufficient system. Finally, due to the importance of complement in animal development and the changes in microenvironment over time after SCI, the development of conditional (versus constitutive) knock-out animals may prove to be an invaluable tool for dissecting the roles for complement following experimental SCI.

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