Chondroitin Sulfate Proteoglycan Modulation as a Potential Therapy for Traumatic Injury to the Central Nervous System Maximilian T. Rosenfeld Advisor: Travis E. Brown, Ph.D. School of Pharmacy, University of Wyoming Abstract Compared to the peripheral nervous system (PNS), the central nervous system (CNS) is limited in its ability to regenerate, resulting in chronic deficits that are difficult to treat. Elements of mild plasticity that contribute to functional recovery are observed after injury. Therefore, strategies to enhance plasticity after injury to promote repair and regeneration are key, and this can be accomplished through the modulation of the extracellular matrix (ECM) in the brain and spinal cord. One such strategy utilizes the bacterial enzyme chondroitinase ABC (ChABC) to degrade inhibitory chondroitin sulfate proteoglycans (CSPGs) to produce a growth permissive environment for neurons and their regenerating axons. This review will discuss the pathology behind CNS injury, the role of CSPGs as inhibitors, and the efficacy of modulating CSPGs to promote restorative plasticity and functional recovery after CNS injury. 1. Introduction Humans have known the limited capacity of the CNS to repair itself since Ancient Egypt (3,000- 2500 BCE) (Eltorai, 2003). Damage to the brain and spinal cord can result in permanent sensory, motor, cognitive, and autonomic deficits. Traumatic Brain Injury (TBI) is a significant public health concern commonly caused by falls, motor vehicle accidents, assaults, work-related accidents, and sports concussions (Gardner and Zafonte, 2016). TBI accounts for 52,000 deaths in the United States (Faul et al., 2010), comprises 1.4% of all emergency department visits, and 4.8% of injuries presented in the emergency department. Of all injury-related hospitalizations TBI constituted 15.1% of those injuries (Faul et al., 2010), which shows the significant prevalence of the issue. Sex and age are significant risk factors; males are almost three times as likely to have a TBI than females and persons over the age of 65 and under the age of 14 are at increased risk (Gardner and Zafonte, 2016). Not everyone fully recovers from TBI with some patients requiring life long, daily support. The volume of TBI cases in combination with the cost of long-term rehabilitation/support makes TBI a major financial burden, estimated to be between 9 to 10 billion dollars (Gardner and Zafonte, 2016). Spinal cord injury (SCI) similarly represents a major public health issue due to the associated long-term consequences. The global incidence of SCI was 8 to 246 cases per million per year, with a trend towards increasing rates (Furlan et al., 2013). Despite the adverse socioeconomic impact of CNS injuries, the treatment standard is still rehabilitation; no effective therapies have been developed to restore high levels of function (Wu et al., 2017; Quraishe, Forbes, and Andrews 2018; Grève and Zink, 2009; George and Geller, 2018). Effective treatment of CNS injuries is quickly becoming more heavily researched, in part due to the increase in CNS injury and TBI related deaths. Chondroitin sulfate proteoglycans (CSPGs) are of particular interest as a therapeutic target. CSPGs are a well-studied family of extracellular matrix molecules that are involved in the development of the nervous system, modulation of synaptic connections, and restriction of plasticity following injury to the CNS (Bartus et al., 2012). Upregulation of CSPG expression after injury is thought to be a protective mechanism to limit the spread of damage but is also central to regenerative failure due to their inhibitory properties (Bartus et al., 2012; Faulkner et al., 2004). Promising therapies utilizing CSPGs include ChABC administration, ChABC administration in combination with a peripheral nerve graft (PNG), and arylsulfatase B (ARSB) administration. However, there is still room for improvement due to ChABC’s chemical instability and possible immunogenicity (Yoo et al., 2013). The following sections will dig into the complex pathologies implicated in CNS injury and how ECM modulators can be used as a rehabilitation tactic for CNS injury. 2. Injury to the CNS Phase 1: Cell death and inflammation The progression of a traumatic injury to the CNS happens in three phases: cell death and inflammation, cell proliferation for tissue replacement, and tissue remodeling (Burda and Sofroniew, 2014). The first phase of injury begins with an insult to the CNS that initially causes acute local cell death and compromises the vasculature leading to an almost immediate hemostasis response to stop the bleeding. Similar to injury in the periphery, formation of platelet plugs are followed by a fibrin clot. The compromise of the blood brain barrier (BBB) is due to the traumatic or ischemic injury that destroys the endothelial cells that line the BBB; these endothelial cells directly regulate vascular permeability (Burda and Sofroniew, 2014). Vascular permeability can be further increased through molecular signaling of the immune response. Specifically, the pro-inflammatory cytokine interleukin-1β (IL-1β) is a mediator of increasing vascular permeability. Insult to the CNS will generate large amounts of damage associated molecular patterns (DAMPs) and pathogen associated molecular patterns (PAMPs) that stimulate microglia to release IL-1β. In turn, this IL-1β stimulates nearby reactive astrocytes to release vascular endothelial growth factor (VEGF) and local oligodendrocyte precursor cells (OPC) expressing neuron-glial antigen 2 protein (NG2) to release matrix metalloproteinases (MMPs), specifically MMP-9. VEGF and MMP-9 will then modulate tight junctions between endothelial cells to increase permeability (Argaw et al., 2009, 2012; Seo et al., 2013). The now damaged BBB leaks molecules that do not belong in the CNS. For example, thrombin is an essential protease for clotting but when exposed to the CNS, promotes neuronal death and inhibits plasticity (Popovich and Longbrake, 2008). Thrombin can also bind protease activated receptors on microglia that cause them to become hyperactive and neurotoxic (Suo et al., 2002). Other unwanted molecules such as antibodies can also cross the damaged BBB and recruit the complement system, microglia, and macrophages to kill neurons and oligodendrocytes, which leads to further axonal injury and demyelination (Kadota et al., 2000; Stahel, Morganti-Kossmann, and Kossmann 1998; Leinhase et al., 2006). The wave of inflammatory cytokines, chemokines, and growth factors released from the formation of the clot along with activated microglia and astrocytes attracts more leukocytes, further enhancing capillary permeability, and upregulating endothelial adhesion molecules. This amplification causes a massive influx of leukocytes that monitor pathogens, remove debris, and mediate wound repair. It should be mentioned that CNS intrinsic microglia are very fast acting and are recruited to the lesion almost immediately to phagocytose debris and recruit more cells. Additionally, NG2-OPCs also quickly migrate to the site, differentiating into mature oligodendrocytes to remyelinate axons (Quraishe, Forbes, and Andrews 2018; Burda and Sofroniew, 2014). Phase 2: Tissue replacement The second phase in CNS injury is characterized by the local migration and proliferation of cells that repair and replace damaged tissue (Burda and Sofroniew, 2014). At this point, the injury is made up of the lesion core and the glial/astrocytic scar. The nonneural lesion core will be primarily comprised of perivascular fibroblasts, pericytes (Göritz et al. 2011; Soderblom et al. 2013) and proliferating endothelial progenitor cells (Burda and Sofroniew, 2014). Proliferation of certain cell types reflect classic wound responses, such as endothelial cells for neovascularization (Casella et al., 2002), while the proliferation of astrocytes is specific to the CNS. The newly proliferated reactive astrocytes are the main component of the scar that serve to restore homeostasis, repair and confine the lesion core (Burda and Sofroniew, 2014; Sun and Jakobs, 2012). The glial scar is crucial for recovery through suppression of a pro-inflammatory environment. For example, blocking scar synthesis keeps the BBB permeable and results in an increase of infiltrating leukocytes and other non-CNS elements that promote inflammation, resulting in a larger lesion and increased motor deficits (Faulkner et al., 2004; Burnside and Bradbury, 2014). Phase 3: Tissue Remodeling Tissue remodeling constitutes the third phase and includes processes that happen over a period of weeks to months. In 2-3 weeks after an acute insult, the lesion core becomes surrounded by a mature astrocyte scar (Wanner et al., 2013) while the gradual contraction of the scar in the peri-lesion area can happen over the course of 6 months, with fibrotic/astrocyte scars persisting for years (Burda and Sofroniew, 2014). Despite the important protective effects, the chronic glial scar is largely detrimental to tissue regeneration (Cregg et al., 2014; Bartus et al., 2012). Failure to regenerate axons is a result of the dense, interlocking organization of reactive astrocytes that block growth cones from advancing, and the upregulation in inhibitory ECM molecules, particularly chondroitin sulfate proteoglycans (CSPGs) (Reier, Stensaas, and Guth 1996; Burnside and Bradbury, 2014). Astrocytes synthesize and deposit CSPGs within 24 hours postinjury, with these CSPGs persisting in high concentrations within the lesion for months (Jones, Margolis, and Tuszynski 2003; McKeon, Jurynec, and Buck 1999; Tang, Davies, and Davies 2003). CSPGs are known to restrict plasticity at the lesion site by inhibiting sprouting and reorganization (Bartus et al., 2012), and are inhibitory to growing axons in culture (Snow et al., 1990). Interestingly, CSPGs synthesized by astrocytes within 24 hours postinjury are still high in concentration throughout the lesion months later (Cregg et al., 2014) suggesting a role in a persistent, long lasting scar. 3. Chondroitin Sulfate Proteoglycans CSPGs are a versatile family of inhibitory ECM molecules involved in many processes, especially in the developing CNS. There are many types of CSPGs, but all contain a core protein covalently bonded to negatively charged polysaccharide chains called chondroitin sulfate glycosaminoglycans (CS-GAG) (Kjellén and Lindahl 1991). The sugar residues of these chains can be mono- or disulfated resulting in different sulfation patterns that determine how CS-GAGs will interact with other elements (Bartus et al., 2012). When axonal growth cones encounter CSPGs they collapse and retract. Specifically, the most inhibitory CS-GAG, chondroitin-4-sulfate (C4S), is dominant in the adult brain and provides a repelling guidance cue for developing neurons and is upregulated in lesions after SCI and TBI in mice (Wang et al., 2008; Yi et al., 2012). In addition to inhibiting axon growth and regeneration, CSPGs also exert inhibitory effects on oligodendrocyte progenitor cells (OPCs). In vitro animal studies discovered that OPC processes that come into contact with CSPG rich surfaces would retract, similar to when axonal growth cones meet CSPGs (Siebert and Osterhout, 2011; Lau et al., 2012; Pendleton et al., 2013). These findings provide strong evidence that expression of CSPGs at CNS lesions and glial scars inhibits axonal regeneration and remyelination. Perineuronal Nets In the adult CNS, CSPGs are concentrated at synapses in structures called perineuronal nets (PNNs) which have many functions, one of which is to stabilize synaptic connections by restricting plasticity (Bartus et al., 2012). PNNs are specialized exctracellular matrix structures that assemble in a lattice-like structure surrounding the soma and dendrites of neurons. PNNs are known to restrict plasticity by inhibiting dendrite growth, restricting movement of receptors at synapses, and acting as a scaffold for molecules that inhibit synaptic formation (Quraishe, Forbes, and Andrews 2018; Sorg et al., 2016). PNNs are also thought to protect neurons that are prone to higher levels of oxidative stress (Cabungcal et al., 2013) and are implicated in neurological diseases, recovery from SCI, learning and memory, and addiction (Sorg et al., 2016; Irvine and Kwok, 2018; Gogolla et al. 2009; Vazquez-Sanroman et al. 2015). The majority of neurons in the spinal cord are surrounded by PNNs (Irvine and Kwok, 2018), suggesting a promising therapeutic target for recovering from CNS injury, at least in the spinal cord (Bartus et al., 2012). In the mouse brain, PNNs were greatly reduced in the areas adjacent to the lesion core, most likely through the action of MMPs (Yi et al., 2012). If the reduction in PNNs is a regenerative response to TBI, then further reducing PNNs to stimulate plasticity could be an effective strategy in enhancing CNS repair (George and Geller, 2018). 4. CSPG Modulation Because CSPGs are inhibitory and detrimental to regeneration, modifying the extracellular environment has gained increasing attention in research. Many in vivo studies have shown that treating CNS lesions with the bacterial enzyme chondroitinase ABC significantly increases axonal sprouting, growth, and plasticity along with improvements in functional recovery. (Siebert, Conta Steencken, and Osterhout 2014; Zuo et al., 1998; Yick et al., 2000; Bradbury et al., 2002; Yick et al., 2003; Barrit et al., 2006; Tom, Kadakia, et al. 2009; Soleman et al., 2012; Hill et al., 2012; Harris et al., 2010). ChABC catalyzes the digestion of CSPGs through the breakdown of the bond between the GAG side chains and the core protein (Yamagata et al., 1968). ChABC promoted regeneration of axons towards their original targets after an experimentally induced nigrostriatal lesion in adult rats (Moon et al., 2001). In a dorsal column crush injury model, ChABC promoted locomotor and proprioceptive recovery following injury through connections formed by corticospinal tract (CST) axons caudal to the injury (Bradbury et al., 2002). Yick et al. (2003) demonstrated that application of ChABC infused gel-foam promoted regeneration of Clarke’s nucleus neurons in the dorsal column beyond the lesion scar. Another in vivo study revealed remarkable long-distance axon regeneration in the dorsal root entry zone (DREZ) to the spinal cord using a combination therapy of ChABC mediated digestion of CSPGs and zymosan, an inflammatory activator of macrophages (Steinmetz et al., 2005). The DREZ is the transition between the CNS and PNS where regenerating axons are unable to enter the CNS under normal conditions (Golding, Shewan, and Cohen 1997). Caggiano et al. (2005) gave rats continuous ChABC treatment for two weeks using multiple infusions with a spinal catheter and found that rats with severe compression SCI regained bladder and hindlimb function. Studies show similar anatomical reorganization following ChABC treatment after TBI or stroke, with evidence of CSPG and PNN degradation, axonal sprouting, and growth around the distal middle cerebral artery (MCA) after stroke, and in the cervical spine after TBI (Soleman et al., 2012; Hill et al., 2012; Harris et al., 2010). Despite evidence of anatomical recovery, functional recovery varies with stroke and TBI in these studies. This suggests a more complicated mechanism is at play, but it does not necessarily mean ChABC treatment is not effective for TBI. For example, mice were given intracerebroventricular (ICV) administration of ChABC following TBI to reduce acute edema. Nearly half of the edema caused by a controlled cortical impact was reduced, indicating that this treatment may be useful in preventing chronic intracranial hypertension, which can lead to severe morbidity or death (Finan et al., 2016). The uninjured control group was not affected, suggesting that ChABC treatment is selective to injured tissue, despite the global delivery system used (Finan et al., 2016). Regeneration of axons is often attributed to the functional recovery observed, however a study that treated dorsal column injury with ChABC found that the mechanism contributing to functional recovery is in part due to the reorganization of spared neurons and pathways. This includes aberrant sprouting of the rubrospinal tract into grey matter, or an enhancement of spontaneous CST plasticity (Garcia-Alias et al., 2008). Bradbury et al. (2002) suggested that although CST regeneration had occurred, functional improvement was also because of sprouting of CST axons rostral to or at the injury site and sprouting from spared components of the dorsal column. These findings indicate that the effects of ChABC on increasing plasticity are just as important as its effects on regeneration, if not more so when attempting to restore function (Bartus et al., 2012). ChABC combined with Peripheral Nerve Graft While ChABC as a single therapy has shown promising results (Caggiano et al., 2005), administrating ChABC in combination with a peripheral nerve graft (PNG) has the potential to produce superior results than ChABC treatments alone (Tom, Sandrow-Feinberg, et al. 2009). The PNG serves as a bridge to fill the space left behind from the lesion (Lee et al., 2013). One study demonstrated that ChABC administered after cervical hemisection at C3 in adult rats prompted significant axonal regeneration and functional motor recovery (Houle et al., 2006). These researchers found that axonal regeneration from the distal end of a PNG bypassed the C3 hemisection back into the spinal cord at C5. The resulting functional recovery in these rats was exhibited by improvements in control of their forelimbs as well as improvement in balance and weight bearing on a horizontal rope (Houle et al., 2006). In a more severe model of SCI, adult rats underwent complete spinal cord transection in the thoracic region (T8) followed by implantation of multiple PNGs covered in an acidic fibroblast growth factor (aFGF)-fibrin matrix “glue” mixture in addition to ChABC microinjection to the graft and at the graft/spinal cord interface. The PNG+aFGF+ChABC treatment resulted in restoration of supraspinal control of bladder function, through observed regeneration of 5-hydroxytryptamine (5-HT) and tyrosine hydroxylase (TH) positive fibers (these are essential to controlling bladder function) into the PNG and across the graft/cord interface caudally. Surprisingly, these fibers regenerated far beyond the graft into the lumbar area of the spinal cord (Lee et al., 2013). These findings present a real possibility for repairing more severe cases of CNS injury in the spinal cord. Sustaining Delivery of ChABC Sustaining enzyme activity for longer lasting administration has been a small issue in improving ChABC treatment efficacy for TBI and SCI. Enzyme activity is temporally limited and after a single ChABC injection, enzymatic activity is significantly reduced within 5-10 days due to factors like temperature (Lin et al., 2008; Tester, Plaas, and Howland 2007; Yoo et al., 2013). Implanted intrathecal osmotic mini-pumps and repeated intrathecal injections have been used to administer drugs in management of SCI, but these methods have shown to induce scar formation, impede drug infusion, and sometimes cause direct damage to the spinal cord (Jones and Tuszynski, 2001; Zhang et al. 2010). Tom and Houle (2008) overcame these technical issues by microinjection of ChABC rostrally and caudally to the lesion in association with a PNG. Results were similar to their previous study (Houle et al., 2006) while also finding almost no inflammation was associated with microinjection despite repeated injections, providing an excellent method to treat the injured CNS with ChABC to promote axonal regeneration. ARSB ChABC treatment has shown promising results for TBI and SCI, as well a myriad of other conditions (Muir et al., 2019). Unfortunately, ChABC is somewhat limited by its unknown immunogenicity and chemical instability in vivo which is possibly why it has only been tested in few human clinical trials (Muir et al., 2019). A phase III clinical trial in humans demonstrated no long-lasting adverse effects from ChABC administration to treat lumbar disc herniations; however, these injections were delivered to intervertebral discs only, and not in other areas like the brain where a different, potentially adverse immune reaction could occur. (Chiba et al., 2018). Therefore, the immunogenicity of ChABC will need to be further assessed in humans specifically before a treatment can be developed for TBI and SCI in humans. To avoid these issues, a promising alternative was presented by Yoo et al., 2013. Aside from the fact that ARSB degrades CSPGs, the researchers chose to use the ARSB enzyme because it is already approved to treat patients with mucopolysaccharidosis type VI, a rare genetic disease in which the body is unable to break down glycosaminoglycans due to a lack of mucopolysaccharidase enzymes (Harmatz et al., 2004), and because its enzymatic activity is optimal at a more acidic pH that is characteristic of the CNS injury environment. The authors discovered that ARSB retained half of its enzymatic activity after five days, while ChABC lost 90% of its activity after 24 hours. Results showed that a one-time injection of human derived ARSB after compression SCI in mice eliminated immunoactivity of C4S GAGs, which were previously mentioned as the major inhibitor to axon regeneration in SCI (Wang et al., 2008, Yi et al., 2012), and this led to improved locomotor recovery. Interestingly, there was no significant difference in functional recovery between ARSB treated mice and ChABC treated mice (Yoo et al., 2013). Based on these results, human ARSB may be more suited than ChABC for use in mammals. 6. Summary Understanding plasticity in the CNS is critical to the development of successful treatments. The ECM is well known for its structural, supportive functions yet it is very much a part of other processes such as axon guidance and synaptic plasticity. Specifically, chondroitin sulfate proteoglycans play a large part in these processes as evidenced by their upregulation and altered sulfation patterns following injury. Modification of the ECM using ChABC or ARSB presents a strong approach to overcoming CSPG-mediated inhibition on axon and neuron growth. However, ChABC presents two main limitations: its lack of chemical stability and its unknown immunogenicity in humans. To advance the likelihood that ChABC administration for treatment of CNS injuries will be tested in clinical trials, future investigations should concentrate on improving the chemical stability of ChABC. If this is not viable then a safe route for multiple infusions must be elucidated. Furthermore, the compatibility of the human immune system needs to be addressed to ensure there are no adverse effects associated with administration into the sensitive, regulated environment of the CNS. The promising ARSB alternative enzyme should be tested in a variety of CNS injury models to confirm its effectiveness with both TBI and SCI, as well as penetrating and contusive injuries. ChABC has proven to be quite effective when used in conjunction with a peripheral nerve graft, so testing ARSB in combination treatments should be explored. Moving forward, it is very unlikely that a single therapy will be successful in promoting significant restoration of neural tissue and functional recovery. 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